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Enterobacter sakazakii in food and beverages (other than infant formula and milk powder)
Abstract
The ubiqitous microorganism Enterobacter sakazakii is a rare contaminant of infant formula and may cause severe systemic infection in neonates. So far, other food is not known to cause E. sakazakii-infections. The scarce information about the ecology of E. sakazakii and the uncertainty concerning the source of infection in children and adults warrant a summary of the current knowledge about the presence of this opportunistic microorganism in food other than infant formula. This review systematizes publications on the presence of E. sakazakii in food and beverages until June 2006. Food other than infant formula has been rarely investigated for the presence of E. sakazakii. Nevertheless, this microorganism could be isolated from a wide spectrum of food and food ingredients. E. sakazakii was isolated from plant food and food ingredients like cereal, fruit and vegetables, legume products, herbs and spices as well as from animal food sources like milk, meat and fish and products made from these foods. The spectrum of E. sakazakii-contaminated food covers both raw and processed food. The kind of processing of E. sakazakii-contaminated food was not restricted to dry products. Fresh, frozen, ready-to-eat, fermented and cooked food products as well as beverages and water suitable for the preparation of food, were found to be contaminated by E. sakazakii. Although E. sakazakii-contaminated food do not have general public health significance, measures for prevention should consider the presence of E. sakazakii in food, food ingredients, their processing and preparation as possible source of contamination, colonization or infection.
... Given the ubiquity of Cronobacter sakazakii animate environment (animals, man) and inanimate environment (plants, soil, water), it is not surprising that C. sakazakii is detected in many foods and food products of animal and vegetable origin [35]. ...
... Oral and intestinal colonization with C. sakazakii may be associated with the ingestion of contaminated foods. As C. sakazakii is an opportunistic pathogen, the patient's colonization flora is the most probable source of infection under circumstances of immunosuppression and severe underlying diseases in patients after the neonatal period [35,37]. C. sakazakii has been isolated from plant food and food ingredients like cereal, fruit and vegetables, legume products, herbs and spices as well as animal food sources like milk, meat, and fish and products made from these foods. ...
... C. sakazakii has been isolated from plant food and food ingredients like cereal, fruit and vegetables, legume products, herbs and spices as well as animal food sources like milk, meat, and fish and products made from these foods. The spectrum of C. sakazakii-contaminated food covers both raw and processed foods [35]. Food and food ingredients may be contaminated with C. sakazakii under hygiene mismanagement by contaminated insects and rats. ...
Aims: Cronobacter sakazakii (CS) is a member of the Enterobacteriaceae family. It is a genomically heterogeneous, motile, Gram-negative bacillus. It is also an emergent foodborne pathogen associated with the ingestion of infant formula milk that can cause neonatal sepsis, necrotizing enterocolitis, and meningitis. This review is focused on the newest information about the bacterial characteristics of C. sakazakii and human infections causing by this pathogenic bacterium. Methods & Materials: We searched medical databases such as ISI Web of Science, PubMed, Scopus, and other websites. Findings: Cronobacter sakazakii acts as a microbiological hazard in the infant food chain, with historic high mortality in neonates. The International Commission for Microbiological Specifications for Foods has categorized C. sakazakii as a severe hazard bacterium for some individuals, with long duration, substantial chronic sequelae, or life-threatening complications. Although the incidence of C. sakazakii infection is low, the prognosis of the disease is poor, and infection is associated with significant morbidity and mortality. Powdered Infant Formula (PIF) milk products contaminated with C. sakazakii have been epidemiologically linked to several clinical cases. Premature infants, low-birth-weight ones, and patients hospitalized in the Neonatal Intensive Care Units (NICUs) are more at infection risk than older infants. Conclusion: We recommend focusing on simple preventative strategies such as the promotion of breast milk feeding, the inclusion of warnings on the powder infant formula packages that may be contaminated with C. sakazakii, and abstinence from the practice of re-warming of reconstituted formula. Reconstituted dairy products should be avoided in adult immunosuppressed populations. Appropriate barrier precautions should be observed in NICU and intensive care unit settings, where the spread of infection may be more prevalent.
... PIF was thought to be the source of neonatal/infantile infections. However, it is clear now that contamination of reconstituted PIF can occur intrinsically and extrinsically, although the main reservoir(s) and routes(s) of contamination have yet to be determined [18][19][20]. Jason reported surveillance data on 82 Cronobacter cases (between 1958 and 2010) and showed that these infants became ill (defined here as a confirmed culture-positive case of septicemia or meningitis) after ingesting breast milk exclusively (without consumption of PIF, FUF, or powdered human milk fortifiers) prior to illness onset [11]. Friedemann had also reported similar observations [20]. ...
... Jason reported surveillance data on 82 Cronobacter cases (between 1958 and 2010) and showed that these infants became ill (defined here as a confirmed culture-positive case of septicemia or meningitis) after ingesting breast milk exclusively (without consumption of PIF, FUF, or powdered human milk fortifiers) prior to illness onset [11]. Friedemann had also reported similar observations [20]. To underscore this point, Bowen et al. [21] and McMullan [22] recently reported infantile cases of C. sakazakii septicemia/meningitis where these infants only consumed expressed maternal milk (EMM) during the first weeks after birth. ...
... Two such systems are the National Notifiable Diseases Surveillance System, which is maintained by the Centers for Disease Control and Prevention (Available online: https://wwwn.cdc.gov/nndss/data-and-statistics.html, last accessed 1/17/2020) and the European Centre for Disease Prevention and Control's Surveillance System (Available online: TESSy, https://ecdc.europa.eu/en/publications-data/european-surveillancesystem-tessy, last accessed 1/17/2020) [20,155]. Currently, Minnesota is the only state in the USA that does this. ...
Cronobacter species are considered an opportunistic group of foodborne pathogenic bacteria capable of causing both intestinal and systemic human disease. This review describes common virulence themes shared among the seven Cronobacter species and describes multiple exoproteins secreted by Cronobacter, many of which are bacterial toxins that may play a role in human disease. The review will particularly concentrate on the virulence factors secreted by C. sakazakii, C. malonaticus, and C. turicensis, which are the primary human pathogens of interest. It has been discovered that various species-specific virulence factors adversely affect a wide range of eukaryotic cell processes including protein synthesis, cell division, and ion secretion. Many of these factors are toxins which have been shown to also modulate the host immune response. These factors are encoded on a variety of mobile genetic elements such as plasmids and transposons; this genomic plasticity implies ongoing re-assortment of virulence factor genes which has complicated our efforts to categorize Cronobacter into sharply defined genomic pathotypes.
... As reported by numbers of scholars this bacterium has been isolated from different samples such as clinical specimens, environmental, foodstuffs and food processing environments [46,47]. Cronobacter sakazakii has been isolated from a wide range of environmental sources and several foods of animal and plant origins such as cereal, fruit and vegetables, legume products, herbs, spices, milk, meat, fish, infant milk formula, milk powder, herbal teas, cheese products, and even isolated from environmental samples such as in dust [8,13,28,44,[48][49][50]. Cronobacter sakazakii was also identified from the mouths of stroke patients [51]. ...
... For instance, in a neonatal intensive care unit Cronobacter sakazakii infections had occurred in 1994 in France and 17 neonates were infected and showed enterocolitis, septicemia, and meningitis with different prevalence [4]. Cronobacter sakazakii is ubiquitous [48], but it is profoundly linked with infant formula powders which are implicated in infant infections [52]. Intrinsic contamination reports are coming up frequently that indicates the product is not sterile. ...
... Cronobacter (previously known as Enterobacter sakazakii) is a genus consisting of rod shaped, motile Gram-negative bacterial pathogens belonging to the family of Enterobacteriaceae (Kucerova et al. 2010); see Figure 1 Panel A. The well-known bacterial pathogens E. coli and Salmonella belong to the same family as well, however Cronobacter is more closely related to the genera of Enterobacter and Citrobacter (Friedemann, 2007); see Figure 1 Panel B. In fact, before the revolutionary taxonomic revision in 2007, some isolates of Enterobacter hormaechei and Enterobacter ludwigii were wrongly recognized as Cronobacter, which led to many misunderstandings and confusions in the literature (Iversen et al. 2008;Joseph el al. 2012). ...
... The original source of this bacterium is thought to be from plant materials, such as rice, spices, wheat etc. (Iversen and Forsythe, 2003). Cronobacter was isolated from herbs, spices, products of cheese, and meat (Iversen and Forsythe, 2004;Friedemann, 2007;Baumgartner et al. 2009). Agogue and coworkers (2005) isolated Cronobacter from marine environment. ...
C. sakazakii CC4 strains were frequently isolated from powder infant formula (PIF) and predominantly linked to neonatal meningitis. However, no clear explanation exists for the predominant linkage of C. sakazakii CC4 to neonatal meningitis. Therefore, it is important to fully understand this species and identify any virulence or environmental fitness associated traits specific to CC4 isolates. Physiological assays which include introducing the bacterial strains to specific conditions such as dryness, acidity, and antimicrobial resistance were performed to differentiate between CC4 and non-CC4 isolates. The C. sakazakii isolates were also tested for their motility rates, ability to lyse erythrocytes, toleration to dry heat, as well as capsule and biofilm formation.
... Cronobacter sakazakii (formerly known as Enterobacter sakazakii) is an emerging foodborne pathogen that can adhere tightly to the surface of packaging materials and cause lifethreatening symptoms (Chauhan et al., 2020;Parra-Flores et al., 2021). This species of bacteria has strong propensity of forming biofilm, which constitutes the major virulence of C. sakazakii to survive in a broad range of food and food ingredients, such as dairy product, cereal, meats and drinking water (Friedemann, 2007;Harouna et al., 2020;Hong et al., 2022). Of importance, powdered infant formula (PIF) is long considered as the major source for C. sakazakii-related risks, while being defined as a category A pathogen of relevance to PIF by FAO/WHO (Gan et al., 2021). ...
Cronobacter sakazakii is an opportunistic foodborne pathogen primarily found in powdered infant formula (PIF). To date, it remains challenging to control the growth of this ubiquitous bacterium. Herein, antimicrobial photodynamic inactivation (aPDI) was first employed to inactivate C. sakazakii. Through 460 nm light irradiation coupled with hypocrellin B, the survival rate of C. sakazakii was diminished by 3~4 log. The photokilling effect was mediated by the attenuated membrane integrity, as evidenced by PI staining. Besides, scanning electron microscopy showed the deformed and aggregated cell cluster, and intracellular ROS was augmented by 2~3 folds when light doses increase. In addition to planktonic cells, the biofilm formation of C. sakazakii was also affected, showing an OD590nm decline from 0.85 to 0.25. In terms of molecular aspects, a two-component system called CpxRA, along with their target genes, was deregulated during illumination. Using the knock-out strain of ΔCpxA, the bacterial viability was reduced by 2 log under aPDI, a wider gap than the wildtype strain. Based on the promoted expression of CpxR and OmpC, aPDI is likely to play its part through attenuating the function of CpxRA-OmpC pathway. Finally, the aPDI system was applied to PIF, and C. sakazakii was inactivated under various desiccated or heated storage conditions. Collectively, aPDI serves as an alternative approach to decontaminate C. sakazakii, providing a new strategy to reduce the health risks caused by this prevalent foodborne pathogen.
... Despite global research to determine C. sakazakii prevalence, its epidemiology remains incomplete and poorly understood [42]. Given that C. sakazakii is ubiquitous in animate (man, animals) and inanimate (soil, water, plants) environments, it is not unusual that C. sakazakii has been isolated from a variety of foods as well as animal and vegetable sources of food products [43], though little is known about Cronobacter existence in their various environments [44]. The most common food implicated in Cronobacter infection globally is PIF [12]. ...
Cronobacter sakazakii is an emerging and opportunistic foodborne pathogen that causes severe infantile diseases, including meningitis, necrotizing enterocolitis, and septicemia. It has been reported in numerous countries around the world, including those in Africa. Although it has been isolated from food, environmental and clinical samples across Africa, the most implicated source of the C. sakazakii infection outbreaks across the globe has been the consumption of contaminated powdered infant formula. Cronobacter has many unique characteristics that contribute to its survival in harsh environments and transmission along the food chain from production to consumption. A potential foodborne disease outbreak caused by C. sakazakii can increase the overall foodborne disease burden and hinder any progress in managing the overly strained public health situation in Africa. This article presents an insight into the occurrence and prevalence of C. sakazakii infection in Africa's food environment, pointing out the transmission route along the food chain and its accompanying food safety concerns. This paper advocates for strict compliance with food safety and control measures to prevent its spread in African countries.
... It is also found in several types of foods, including dried foods, meats, vegetables, infant milk powder, cereal formula and other dairy products [4,5]. Many cases of meningitis, sepsis, or necrotizing enterocolitis have been recorded in the neonatal intensive care unit due to the survival of this microorganism in reconstituted infant milk formula (RIMF) and nutrient preparation equipment [6]. ...
Background: Cronobacter sakazakii is an emerging pathogen shown to be responsible for many neonatal outbreaks with high mortality rate and is remarkably known to resist desiccation and survive in powder infant formula (PIF) for extended period of time. C. pulveris is also important as a newly developed foodborne pathogen but there is not yet enough published data on its surviving behavior. Reconstituted infant milk formula (RIMF) usually stated as the main vehicle associated with neonatal Cronobacter infections.
Aim: The aim of this study was to investigate the survival of Cronobacter spp. (C. sakazakii and C. pulveris) in RIMF at various storage temperatures.
Methods: The reconstituted formula was inoculated with five C. sakazakii isolates and four C. pulveris isolates separately and stored at room and refrigeration temperatures for 2, 4, 8, 24, 48, 72, and 96 h.
Results: The results showed that C. sakazakii and C. pulveris were able to grow and multiply in RIMF at room temperature as the storage time increases. At 4ºC, population of CP4, CP2, and CS4 were remained as the initial levels until the end of storage period. Whereas CS1, CS3, CP1 and CP3 were not detected at 4°C after 72, 24, 72 and 8 h respectively. However, the viable count of CS5 and CS6 had increased by about 1 log at 4ºC after 8 h.
Conclusion: This study demonstrated the significant diverse in behavior between the examined isolates in RIMF at room and refrigeration temperatures as highlighted. Furthermore, these results may improve understanding of C. sakazakii and C. pulveris surviving strategies which may lead to create an effective control of Cronobacter infections.
Key words: Cronobacter spp.; C. sakazakii; C. pulveris; survival; RIMF.
... Contaminated powdered milk and powdered infant formula have been predominantly linked to C. sakazakii infections in neonates and infants. C. sakazakii has also been isolated from several foods of both animal and plant origin and from a wide range of environmental sources (10,27), raising food safety concerns to immunocompromised individuals, both the very young and the elderly. The use of different food processing technologies to inactivate C. sakazakii in or on foods and food contact surfaces has been the subject of several studies and reviews (18,19,(50)(51)(52)(53)(54). ...
A study was undertaken to model the UV-C inactivation kinetics and determine the fluences required for the incremental inactivation of several strains of Cronobacter spp. suspended in clear phosphate-buffered saline (PBS). In total, 13 strains of Cronobacter spp. were individually suspended in PBS and treated with UV-C doses of 0, 2, 4, 6, 8, and 10 mJ cm−2 with a collimated beam device emitting UV-C at 253.7 nm. The log reduction from each treatment was identified using the plate count method and plotted against the UV-C dose and then curve fitted using several mathematical models. The UV-C dose required for incremental inactivation of each isolate was determined using both linear and nonlinear regression. For the 13 strains tested, a UV-C dose of 10 mJ cm−2 inactivated between 3.66 ± 0.101 and 5.04 ± 0.465 log CFU mL−1. The survival behavior of all strains was best fitted to the Weibull+tail model, with correlation coefficients between 97.17 and 99.71%, and was used to determine the fluences required for incremental inactivation. The UV-C fluences needed to inactivate 1 log (D10-value) of Cronobacter spp. in buffer were between 3.53 and 5.50 mJ cm−2, whereas a fluence greater than 6.57 mJ cm−2 was required to achieve a 4-log inactivation. A clear understanding of the UV-C dose-response of several strains of Cronobacter spp. lays the foundation to design effective UV-based disinfection systems.
HIGHLIGHTS
... The Cronobacter genus has been isolated from a wide range of food, including PIF, milk powder and dry powdered foods Friedemann, 2007). The Cronobacter genus, in particular C sakazakii strains have been associated with serious neonatal infections, whereas C. malonaticus strains have been associated with infections of immunocompromised adult patient. ...
The genus Cronobacter includes food-borne pathogens causing neonatal infections such as
meningitis, and necrotizing enterocolitis as well as bacteraemia in immunocompromised adults.
Understanding the molecular characterisation, clonality and phenotypic diversity of Cronobacter
species is essential to reduce the source of microbial contamination of powdered infant formula
(PIF) and other food. Therefore, an improved understanding of the diversity of the genus is
warranted.
In the first part of the study, the 7-loci Multilocus sequence typing (MLST) scheme was applied to
investigate the diversity of Cronobacter spp. isolated from food and environmental sources.
Twenty-six strains that had not previously been profiled were divided into 21 sequence type (STs),
and 9 new STs were identified, which had not been previously reported. Cronobacter strains
isolated from food and environmental sources were highly diverse with respect to their ST,
particularly those from different sources of food.
This study is the first to describe the development and application of variable number tandem
repeat analysis (VNTRA) typing method for C. sakazakii ST4 strains. Nineteen C. sakazakii ST4
strains, which were widely distributed geographically, temporally and origin of source were profiled.
These strains were divided into 15 distinct groups based on the number of tandem repeats of 6
VNTR loci. It was concluded that VNTRA profiling could contribute to further understanding of C.
sakazakii ST4 diversity and tracking of infection sources. Of particular interest in this research was
the finding that the analysis of the lipopolysaccharide (LPS) profiling using BioNumerics software,
(version 7.1) showed great ability to discriminate between strains within the same serotype.
Furthermore, the present study developed a multiplex PCR assay targeting capsular polysaccharide
genes such as kpsS (K1 and K2) and galE (CA1 and CA2) for the specific detection and rapid
identification of K-capsule type and colanic acid type respectively.
Another important finding was that a strong correlation between the amount of mucoid production,
type and ratio of monosaccharides production and type of O-antigen serotype. This study indicated
also that rhamnose is the main sugar in C. sakazakii serotype O:2 strain. The most interesting
observation was that C. sakazakii strains with serotype O:1 and O:4 had high numbers of
sublethally injured cells after desiccation, while strains with serotype O:2 and O:3 showed low
numbers of sublethally injured cells. C. sakazakii strains showed a higher survival rate after the
exposure to drying (90 days) than other Cronobacter species. Strains (6 of 54) containing the
thermotolerance genomic island tended to survive better at 58°C than other strains. In general, C.
sakazakii strains were much more resistant than other Cronobacter species to environmental
stresses such as desiccation, long-term drying and heat. This might explain why C. sakazakii strains
are associated with PIF, milk powder and dry powdered foods, and more frequently isolated than
other Cronobacter species.
This study analysed multiple methods for typing Cronobacter at the species and strain level, and
showed different discriminatory powers. The present study recommended that using a
combination of genomic cluster, chromogenic agar (DFI), and sialic acid, malonate, indole and
inositol utilisation tests can be useful tools to distinguish the seven species of Cronobacter,
distinguish pathogenic C. ST7 from other C. malonaticus STs, as well as to characterize and
distinguish C. dublinensis strains to the subspecies level. The present study is an important
contribution to the understanding of the diversity and characteristics of the Cronobacter genus
xi
using different typing methods, which is essential to reduce the risk of contaminations for the food
products, in particular baby food such as PIF, milk powder and weaning food.
... C. sakazakii infections observed in these individuals have been epidemiologically linked to consumption of intrinsically and extrinsically contaminated lots of reconstituted powdered infant (PIF) and follow up formulas; thus, contamination of such products is a challenging task for both infant formula manufacturers and caretakers [7][8][9][10]. Another trend that both clinicians and public health scientists must recognize is that unsafe personal hygiene breast-feeding practices, such as the use of contaminated personalized breast pumps, may also lead to infantile infections such as septicemia and meningitis [11][12][13][14]. ...
Background
Cronobacter sakazakii is a foodborne pathogen that causes septicemia, meningitis, and necrotizing enterocolitis in neonates and infants. The current research details the full genome sequences of two extremely persistent C. sakazakii strains (H322 and GK1025B) isolated from powdered infant formula (PIF) manufacturing settings. In addition, the genetic attributes associated with five plasmids, pH322_1, pH322_2, pGK1025B_1, pGK1025B_2, and pGK1025B_3 are described.
Materials and Methods
Using PacBio single-molecule real-time (SMRT ® ) sequencing technology, whole genome sequence (WGS) assemblies of C. sakazakii H322 [Sequence type (ST)83, clonal complex [CC] 83) and GK1025B (ST64, CC64) were generated. Plasmids, also sequenced, were aligned with phylogenetically related episomes to determine, and identify conserved and missing genomic regions.
Results
A truncated ~ 13 Kbp type 6 secretion system (T6SS) gene cluster harbored on virulence plasmids pH322_2 and pGK1025B_2, and a second large deletion (~ 6 Kbp) on pH322_2, which included genes for a tyrosine-type recombinase/integrase, a hypothetical protein, and a phospholipase D was identified. Within the T6SS of pH322_2 and pGK1025B_2, an arsenic resistance operon was identified which is in common with that of plasmids pSP291_1 and pESA3. In addition, PHASTER analysis identified an intact 96.9 Kbp Salmonella SSU5 prophage gene cluster in pH322_1 and pGK1025B_1 and showed that these two plasmids were phylogenetically related to C. sakazakii plasmids: pCS1, pCsa767a, pCsaC757b, pCsaC105731a. Plasmid pGK1025B_3 was identified as a novel conjugative Cronobacter plasmid. Furthermore, WGS analysis identified a ~ 16.4 Kbp type 4 secretion system gene cluster harbored on pGK1025B_3, which contained a phospholipase D gene, a key virulence factor in several host–pathogen diseases.
Conclusion
These data provide high resolution information on C. sakazakii genomes and emphasizes the need for furthering surveillance studies to link genotype to phenotype of strains from previous investigations. These results provide baseline data necessary for future in-depth investigations of C. sakazakii that colonize PIF manufacturing facility settings and genomic analyses of these two C. sakazakii strains and five associated plasmids will contribute to a better understanding of this pathogen's survival and persistence within various “built environments” like PIF manufacturing facilities.
... Furthermore, several documented cases of Cronobacter infections are linked with consumption of infant formula in neonatal intensive care (Himelright et al., 2002). C. sakazakii is an opportunistic foodborne pathogen that is isolated from various food sources like dairy-based food, dried meat, vegetable, and milk powders (Beuchat et al., 2009;Friedemann, 2007). It not only survives at room temperature for up to one year in milk powder (Hurrell et al., 2009) but can also multiply at a lower refrigeration temperature such as 5.5 • C (Nair et al., 2004). ...
Cronobacter and Pseudomonas spp. are pathogenic organisms that are associated with neonatal morbidities such as bacteremia, sepsis, necrotizing enterocolitis (NEC), and infant meningitis. Lactoferrin (Lf) is a known bactericidal component of fresh human milk. Infants receiving formula or pasteurized donor milk however may not be receiving lactoferrin at same concentration as mother's own milk, so it is beneficial to understand an exogenous role for dairy bovine lactoferrin (bLf). Our aim was to investigate the potential for bLf to provide a novel way to inhibit the growth of these harmful organisms in infant feeding. Subinhibitory doses, Disc-diffusion assay, Minimum Inhibitory Concentrations (MIC), Minimum Bactericidal Concentrations (MBC), and challenge experiments in powder formula were used to investigate the effects of bLf on sepsis-causing bacteria such as C. muytjensii (ATCC-51329), C. sakazakii (ATCC-12868), C. sakazakii (ATCC-9027), P. aeruginosa (ATCC-19429), and P. aeruginosa (ATCC-9027). MIC and MBC of bLf in the range of 12.5–31.25 μM were sufficient to achieve a 3 log reduction of Cronobacter in nutrient broth (P < 0.0001). Similarly, minimum concentrations of 43.75–118.75 μM bLf against Pseudomonas (P < 0.0001) were demonstrated. After bactericidal concentrations were determined, higher resistant strains such as C. sakazakii (ATCC-12868) and P. aeruginosa (ATCC-19429) were chosen as ideal candidates for the bLf supplementation challenge studies. The bLf reduced the C. sakazakii (ATCC-12868) by 3.3 log at 35 °C, 2.8 log at 23 °C, and 1.9 log at 4 °C (P < 0.0001), whereas P. aeruginosa (ATCC-19429) showed 2.4 log reduction at 35 °C, 2.8 log at 23 °C (P < 0.0001), and 1.8 log at 4 °C (P < 0.01). Results indicated that bLf could effectively inactivate the sepsis-causing organisms in reconstituted infant formula and in bacterial growth media. Fortification of infant formula with bLf may address the problems of spoilage/contamination issues and provide a safety measure for feeding infants without mother's own milk.
... were isolated from 20% of powdered infant formulae in Korea (Lee et al, 2012). Understanding the transmission routes (e.g., raw materials, or environmental) and vehicles (e.g., powdered infant formula, or cereals) of sporadic Cronobacter outbreaks is of public health importance (Friedemann, 2007). ...
It is documented that Cronobacter spp.(formely named as Enterobacter sakazakii ) can live in the powdered infant formula (PIF) for a long period, and special concerns have been raised about the safety of powdered infant formula (PIF) contaminated by Cronobacter spp with the globalization of the food supply. A method comparison study between the Chinese standard method GB 4789.40–2016 (reference method) and the European standard method EN ISO 22964:2017 (alternative method) for detecting Cronobacter spp. in PIF was performed according to ISO 16140-2 2016. A total of 576 blind-coded test portions of the product spiked with the test strain at 3 different contamination levels were analyzed by 12 participating laboratories. The results showed that the sensitivity of GB 4789.40–2016 was 80% and 81.16% at the low and high contamination levels, respectively, while those of ISO 22964:2017 were 90% and 97.10%, respectively. The Relative Levels of Detection (RLODs) were below the acceptance limit (AL) regardless of the contamination level. The results were also analyzed using the probability of detection (POD) model proposed in the AOAC guidelines, and showed no statistically significant difference between the alternative and reference methods. The ISO 22964:2017 method was considered equivalent to the GB 4789.40–2016 method for the detection of C. sakazakii in PIF.
... sakazakii; Cs) is a common Gram-negative foodborne pathogen. This pathogen is highly pathogenic to infants (bacteraemia, necrotizing enterocolitis and neonatal meningitis) and adults with weakened immunity (conjunctivitis, urosepsis and sepsis), especially the case fatality rate for infants is up to 80% (Friedemann, 2007;Patrick et al., 2014;Forsythe, 2018). Previous studies have reported that C. sakazakii can be detected in foods such as formula milk powder, vegetables, cereals, meat products and aquatic products and in production environments (Fei et al., 2018;Forsythe, 2018;Li et al., 2020). ...
Cronobacter sakazakii could enter a growth‐arrested state when exposed to ampicillin. Growth‐arrested bacteria can become more tolerant to a wide range of antibiotics, posing a serious threat to food safety and human health. The aim of this study was to investigate the tolerance of growth‐arrested C. sakazakii to a variety of antibiotics and to explore the underlying mechanisms responsible for the variations in antibiotic tolerance levels. The results of the study, as tested by flat colony counting method as well as PMAxx‐qPCR, showed that growth‐arrested C. sakazakii had higher tolerance to four different antibiotics. Results validated by RT‐qPCR indicated that almost all the changes in the expression of antibiotic resistance genes in growth‐arrested cells showed a tendency to promote antibiotic tolerance compared to culturable cells, with significant upregulation of expression of the multidrug efflux pump genes in particular. Furthermore, the up‐regulated gene expression of relA and rpoS and insignificant changes in gene expression of spoT suggested that the stringent response could positively regulate the multidrug efflux pumps of growth‐arrested cells. Growth‐arrested C. sakazakii pose a potential source of contamination and should be taken into account when monitoring of food safety.
... Cronobacter spp. have also been isolated from various cereal-based foods (Akineden et al., 2017;Friedemann, 2007;Lou et al., 2019;Silva et al., 2019), but little information about the frequency in PCF is available. In our study, colony forming units of Cronobacter spp. ...
This study investigated several food safety criteria in 38 different commercial products of processed cereal‐based foods (PCF) from the German market. Microbiological assessment, followed by 16S RNA gene sequencing of suspect colonies, included aerobic mesophilic bacteria, moulds, Enterobacteriaceae, Cronobacter spp., and presumptive Bacillus cereus. Mycotoxin analyses were performed by enzyme immunoassays for deoxynivalenol (DON), zearalenone (ZEN), T‐2/HT‐2 toxins (T‐2/HT‐2; oat containing products only), ergot alkaloids (EA), and alternariol (AOH). No violative result above existing European Union regulations or international guidelines was obtained. Most samples had very low aerobic mesophilic cell counts (<2.0 × 101 CFU/g), the maximum was 9.6 × 102 CFU/g. A few samples contained low numbers of opportunistic pathogens, most notably Cronobacter sakazakii, Acinetobacter spp., Pantoea spp., and enterotoxigenic Bacillus wiedmannii. Levels of mycotoxin contamination were very low, well below European Union maximum limits. DON was found in 10 samples, at levels of 9–35 µg/kg. T‐2/HT‐2 were found in all 15 oat‐based products (1–8 µg/kg). All samples were negative for ZEN and EA. A high number (n = 25) of samples yielded weakly positive results for the nonregulated AOH (0.4–2 µg/kg), but just three samples exceeded a level of 1 µg/kg. No relationship between cereal composition and analytical findings for microbiological parameters and mycotoxins could be found. As long as PCF meals are freshly prepared and consumed immediately after preparation, the risk from sporadically occurring opportunistic bacteria appears to be minimal.
... In the present study, we focused on C. sakazakii, which has long been a concern with regard to the safety of powdered infant formula, as a representative desiccationtolerant bacterium (20)(21)(22)(23)(24)(25)(26)(27). The objectives of this study were to determine and compare the mechanical T g of dried C. sakazakii cells prepared by different drying methods and with different a w levels, using a thermomechanical technique. ...
ABSTRACT To investigate the mechanism of adaptation of Cronobacter sakazakii to desiccation stress, the present study focused on the glass transition phenomenon of dried bacterial cells, using a thermomechanical technique. The mechanical glass transition temperature (Tg) of dried C. sakazakii cells per se, prepared by different drying methods (air drying and freeze-drying) and with different water activity (aw) levels (0.43, 0.57, 0.75, and 0.87), were determined. In addition, we investigated the survival of two strains of C. sakazakii (JCM 1233 and JCM 2127) prepared by different drying methods under different storage temperatures (4, 25, and 42°C) and aw conditions (0.43 and 0.87). While the Tg of the air-dried C. sakazakii cells increased as the aw decreased, the freeze-dried C. sakazakii cells showed an unclear aw dependency of the Tg. Air-dried C. sakazakii cells showed a higher Tg than freeze-dried C. sakazakii cells at an aw of 20°C, the dried C. sakazakii cells survived stably regardless of the drying method. In contrast, when the difference between the Tg and storage temperature was reduced to
... Namely, these procedures took up at least 5 days to obtain a result [1][2][3]. Therefore, in order to isolate and identify E. sakazakii (Cronobacter spp.) rapidly, various studies have been conducted, and various attempts to inhibit the growth of E. sakazakii (Cronobacter spp.) had also been conducted [1,[5][6][7]. Fortunately, Real-Time PCR could provide the rapid-accurate-quantitative analysis for confirming various food-borne pathogens [8][9][10]. The format was developed for detecting E. sakazakii (Cronobacter spp.) in dried infant formula using by applying the fluorogenic 5' nuclease assay (TaqMan ® ) with ABI Prism 7000 [10]. ...
... Cronobacter sakazakii are facultative, anaerobic Gramnegative bacteria that are present in various foods and raw materials [1,2]. In recent years, a new foodborne pathogen, C. sakazakii, has been commonly found in formula milk powder and is associated with several infectious diseases, including meningitis, necrotizing enterocolitis, and sepsis [3,4]. ...
The genome of a Cronobacter sakazakii M1 phage named PF-CE2 was characterized in this work, and a new species named "Cronobacter virus PF-CE2", in the genus Pseudotevenvirus of the subfamily Tevenvirinae of the family Myoviridae is proposed. The Gp190 gene of phage PF-CE2 is predicted to encode a bacteriophage-borne glycanase that is capable of degrading fucose-containing exopolysaccharides produced by C. sakazakii M1. Furthermore, we propose changing the taxonomic status of eight additional phages based on nucleotide sequence comparisons. This work provides a theoretical basis for subsequent heterologous expression of the phage PF-CE2 glycanase and provides an important reference for the preservation and sharing of these phages.
... Cronobacter sakazakii (Band 14) is a bacterial species that can be easily found from various sources in the environment; from insects [65] to plants such as wheat, rice, spices [66], and household foods [67]. The presence of Cronobacter sakazakii in various environments indicates that this species has developed many features that enhance its survival in difficult environments such as (1) resistance to UV radiation, (2) the ability to attach to various surfaces due to fimbria formation, (3) biofilm formation, and (4) the ability to resist drying [68]. ...
A hybrid treatment system of multimedia-sequencing batch biofilm reactor (MM-SBBR; B) and three control bioreactors (C, D, E) processing synthetic wastewater containing pentachlorophenol (PCP), were used to study the effectiveness of PCP removal and the impact of high-levels of PCP (100 mg L − 1) on bacterial community compositions. Another MM-SBBR (A) treating real effluent of the recycled paper mill industry containing PCP was also operated and compared. The MM-SBBR (B) achieved a high chemical oxygen demand (COD), ammo-niacal nitrogen (NH 3-N), and PCP removal efficiencies, reaching 97%, 93% and 99%, respectively, which were attributed to the enrichment of high PCP resistant bacteria in this reactor. It was found that the number of bacterial species was the highest (10 species) in Bioreactor B when compared to the other bioreactors (Bioreactor A, C, D, and E). Proteobacteria phylum (9 species) was found dominated throughout the study, suggesting that the combination MM with granular activated carbon (GAC) and plastic media encouraged the growth of various bacterial species that were resistant to high-levels of PCP and thus enhancing the removal of PCP. It can be concluded that the hybrid treatment system of multimedia (MM) and SBBR is highly capable of removing PCP from water contaminated with PCP due to the diversity presence of PCP resistant bacteria in this bioreactor. MM-SBBR could be the effective treatment method for wastewater containing PCP to improve water quality and ensure sustainability.
... Many researchers have focused on the control of Cronobacter spp. in powdered infant formulas [20][21][22]. These microorganisms have also been isolated from different food products: cereals [23][24][25], fruits and vegetables [26][27][28][29], herbs and spices [30,31], milk products [32,33], meat [34,35], fish and products made from them [36] and ready-to-eat products [37]. ...
Cronobacter genus bacteria are food-borne pathogens. Foods contaminated with Cronobacter spp. may pose a risk to infants or immunocompromised adults. The aim of this study was to determine the microbiological quality of nuts, seeds and dried fruits with special emphasis on the occurrence of Cronobacter spp. Analyses were carried out on 64 samples of commercial nuts (20 samples), dried fruits (24), candied fruits (8), seeds (4), and mixes of seeds, dried fruits and nuts (8). The samples were tested for the total plate count of bacteria (TPC), counts of yeasts and molds, and the occurrence of Cronobacter spp. Cronobacter isolates were identified and differentiated by PCR-RFLP (Polymerase Chain Reaction - Restriction Fragments Length Polymorphism) and RAPD-PCR (Random Amplified Polymorphic DNA by PCR) analysis. TPC, and yeasts and molds were not detected in 0.1 g of 23.4%, 89.1%, and 32.8% of the analyzed samples. In the remaining samples, TPC were in the range of 1.2–5.3 log CFU g−1. The presence/absence of Cronobacter species was detected in 12 (18.8%) samples of: nuts (10 samples), and mixes (2 samples). The 12 strains of Cronobacter spp. included: C. sakazakii (3 strains), C. malonaticus (5), and C. turicensis (4). The results of this study contribute to the determination of the presence and species identification of Cronobacter spp. in products of plant origin intended for direct consumption.
... are universal and have been isolated from various types of foods of animal or vegetable origin, including ready-to-eat, fermented, and cooked food products. In addition, beverages and water, owing to the steps involved in their processing and preparation could be other possible sources of contamination, colonization or infection (Friedemann 2007;Iversen and Forsythe 2003). ...
The goal was to identify the biofilm-forming ability of Cronobacter sakazakii on surfaces of stainless steel (SS) and silicone rubber (SR) in contact with infant formula milk. Two representative bacteriophages (PBES04 and PBES19) were used to control the growth of C. sakazakii as well as its biofilm forming ability on either SS or SR surfaces. Bacterial growth was confirmed at 20 °C when PBES04 and PBES19 were used, whereas C. sakazakii was not normally detected in infant formula milk treated with both bacteriophages for 6 h. In an additional biofilm reduction experiment, the biofilm on SS or SR surfaces were reduced by 3.07 and 1.92 log CFU cm⁻², respectively after PBES04 treatment, and 3.06 and 2.14 log CFU cm⁻², respectively, after PBES19 treatment. These results demonstrate that bacteriophages can be effective in inactivating C. sakazakii in biofilms which could potentially increase food safety in commercial facilities.
... C. sakazakii has been reported in the human gastrointestinal tract, domestic and nosocomial settings, laboratory samples, and food items [9][10][11][12][13]. Bacterial contamination of infant formula milk was thought accountable for outbreaks in the neonatal population [14][15][16][17][18][19]. ...
Introduction
Cronobacter sakazakii is an opportunistic Gram-negative, rod-shaped bacterium which may be a causative agent of meningitis in premature infants and enterocolitis and bacteremia in neonates and adults. While there have been multiple cases of C. sakazakii infections, there have been no acute cholangitis cases reported in humans.
Case presentation
An 81-year-old male with a past medical history of basal cell carcinoma, alcoholic liver cirrhosis, transjugular intrahepatic portosystemic shunt procedure, complicated by staphylococcus bacteremia, pituitary tumor, glaucoma, and hypothyroidism presented to the emergency room with the complaint of diffuse and generalized 10/10 abdominal pain of 1 day’s duration. There was a concern for pancreatitis, acute cholangitis, and possible cholecystitis, and the patient underwent a percutaneous cholecystostomy tube placement. Blood cultures from admission and biliary fluid cultures both grew C. sakazakii. The patient was treated with a carbapenem and clinically improved.
Conclusions
The case study described a patient with multiple medical comorbidities that presented with C. sakazakii bacteremia and cholangitis. While this bacterium has been implicated in other infections, we believe this is the first time the bacteria is being documented to have caused acute cholangitis.
... Previously referred to as 'yellow pigmented E. cloacae', Enterobacter sakazakii (Cronobacter spp.) was reclassified based on differences from E. cloacae in DNA relatedness, pigment production and biochemical reactions [1][2][3][4][5]. E. sakazakii (Cronobacter spp.) caused a severe form of neonatal meningitis with a high mortality rate [2]. ...
... Since the organism is not part of the normal animal and human gut biota, it is probable that soil, water and vegetables are the principal sources of Cronobacter spp. It is frequently isolated from infant milk formulae, herbs, spices, cheese, diverse plant-based food products, rice and other cereals seeds, and fermented products of plant origin and meat [7,15,24,27]. Cronobacter spp. has been reported from dried fish samples, shrimp, anchovy, and brown seaweed [3,18]. ...
In this study, we investigated the incidence of Cronobacter spp. in seafood collected from retail fish markets of Mumbai, India. A total of 50 samples comprising fresh finfish (n = 32), shellfish (n = 6), dried fish (n = 9) and water (n = 3) were analyzed for Cronobacter spp. by selective enrichment, isolation and biochemical tests. Of 145 isolates presumptively identified as Cronobacter spp. by biochemical tests, 37 were confirmed as Cronobacter spp. by Polymerase Chain Reaction (PCR) specific to the internal transcribed spacer (ITS) regions. Based on the partial ITS gene sequence analysis, 35 isolates were identified as Cronobacter malonaticus and two as Cronobacter sakazakii. The highest incidence of Cronobacter spp. was in dried fish (55.6%), followed by shellfish (33.3%). The virulence gene ompA was detected in two Cronobacter sakazakii isolates. This is the first report of the incidence of Cronobacter spp. in fresh and dried seafood from India, which highlights the need to focus on this emerging pathogen in tropical seafood.
... Cronobacter sakazakii, a gram-negative, facultatively anaerobic, motile emerging food-borne pathogen, can adhere tightly to the surface of equipment, packaging materials, and utensils due to its strong ability for biofilm formation, which is believed to be one of the important reasons for the contamination of food by this pathogen (Friedemann, 2007;Amalaradjou and Venkitanarayanan, 2011). Importantly, C. sakazakii can cause some serious life-threatening diseases including sepsis, meningitis, and necrotizing enterocolitis in infants and adults with immunodeficiency, with a 40-80% mortality rate (Hunter and Bean, 2013). ...
Cronobacter sakazakii is an opportunistic food-borne pathogen that endangers the health of neonates and infants. This study aims to elucidate the antibacterial activity and mechanism of Chrysanthemum buds crude extract (CBCE) against C. sakazakii and its application as a natural disinfectant. The antibacterial activity was evaluated by the determination of the diameter of inhibition zone (DIZ), minimum inhibitory concentration (MIC), and minimum bactericide concentration (MBC). The antibacterial mechanism was explored based on the changes of growth curve assay, intracellular ATP concentration, membrane potential, intracellular pH (pH in ), content of soluble protein and nucleic acid, and cell morphology. Finally, the inactivation effects of CBCE against C. sakazakii in biofilm on stainless steel tube, tinplate, glass, and polystyrene were evaluated. The results showed that the DIZ, MIC, and MBC of CBCE against C. sakazakii were 14.55 ± 0.44–14.84 ± 0.38 mm, 10 mg/mL, and 20 mg/mL, respectively. In the process of CBCE acting on C. sakazakii , the logarithmic growth phase of the tested bacteria disappeared, and the concentrations of intracellular ATP, pH in , bacterial protein, and nucleic acid were reduced. Meanwhile, CBCE caused the cell membrane depolarization and leakage of cytoplasm of C. sakazakii . In addition, about 6.5 log CFU/mL of viable C. sakazakii in biofilm on stainless steel tube, tinplate, glass, and polystyrene could be inactivated after treatment with 1 MIC of CBCE for 30 min at 25°C. These findings reveal the antibacterial activity and mechanism of CBCE against C. sakazakii and provide a possibility of using a natural disinfectant to kill C. sakazakii in the production environment, packaging materials, and utensils.
... Cronobacter spp. have been detected in various food types such as meat and meat products, rice, other cereals, poultry, milk and dairy products, infant formula, herbs, seasonings and spices [89][90][91][92][93][94][95][96]. Members have also been isolated from fresh cut, whole as well as minimally processed vegetables or salads [97-99,80 ,100,101] (Table 1). ...
Emerging pathogens, which although have been periodically detected in produce items, are comparatively rarely implicated in large outbreaks. Many of these pathogens are inhabitants of the natural environment and may be major potential sources of contamination for fruits and vegetables. This overview examines the growing epidemiological relevance of three of such emerging pathogens; Arcobacter spp., Helicobacter pylori and Cronobacter sakazakii and the recent status of the scientific literature on their potential for transmission to humans via the consumption of fruits and vegetables. There appears to be a potentially important, yet overlooked exposure risk for humans via produce consumption. Certain crucial research gaps such as the need to optimize detection approaches for the swift and accurate isolation of these agents from produce items has been identified. To establish comprehensive microbiological criteria for produce safety, it is important to characterize all associated potential human pathogens.
... C. sakazakii is an emerging foodborne pathogen that has been isolated from a wide range of food products of animal and vegetal origin, as well as from other sources, such as water and soil (Friedemann, 2007;Li et al., 2014;Shaker et al., 2007;Yao et al., 2016). Surveillance studies have detected C. sakazakii in a variety of different environments, including households, livestock facilities, and food production operations, particularly in powdered infant formula milk (PIFM) manufacturing facilities (Kandhai et al., 2004;Mullane et al., 2008;Müller et al., 2013). ...
Bovine lactoferrin (bLF) is an iron-binding glycoprotein used in functional and therapeutic products due to its biological properties, the most important being its antimicrobial activity. In this study, hydrolysates of bovine lactoferrin (bLFH) obtained with pepsin, chymosin and microbial rennet were assayed against Cronobacter sakazakii (104 CFU/mL) in different media: phosphate buffered saline (PBS), bovine skim milk and whey, and reconstituted powdered infant formula (PIFM). The results obtained have shown that hydrolysis of bLF enhances its antibacterial activity against C. sakazakii. The three types of bLFH dissolved in PBS reduced C. sakazakii growth from a concentration of 0.1 mg/mL and inhibited it completely above 0.5 mg/mL, after 4 and 8 h of incubation at 37 °C. The three bLFH (1 and 2 mg/mL) did not show any antibacterial activity in skim milk, whey and reconstituted PIFM after 8 h of incubation at 37 °C. However, C. sakazakii growth was completely inhibited in whey when pepsin and chymosin bLFH (2 mg/mL) were combined with undigested bLF (2 mg/mL), after 8 h of incubation at 37 °C. On the other hand, the combination of any of the three hydrolysates with bLF showed very low activity in skim milk and practically no activity in reconstituted PIFM. Furthermore, the effect of temperature after reconstitution (4, 23 and 37 °C), on the antibacterial activity of bLF (2.5 and 5 mg/mL) in reconstituted PIFM contaminated with C. sakazakii (10-102 CFU/mL) was also investigated. bLF at 5 mg/mL significantly reduced (p < .05) the proliferation of C. sakazakii in reconstituted PIFM at 37 °C until 2 h. C. sakazakii did not grow at 4 °C for 6 days in reconstituted PIFM with or without bLF. The effect of microwave heating (450, 550 and 650 W for 5, 10 and 15 s) on the antibacterial activity and stability of bLF (2.5 mg/mL) in reconstituted PIFM contaminated with C. sakazakii (10-102 CFU/mL) was also studied. The antibacterial activity of bLF was maintained after treatments at 450 and 550 W for 5 s, which kept 94 and 89% of bLF immunoreactivity, respectively. Moreover, microwave treatments of reconstituted PIFM with or without bLF, at 650 W for 5 s, and at 450, 550 and 650 W for 10 and 15 s, completely inactivated C. sakazakii.
... Jason reported surveillance information on over 80 infant Cronobacter cases (which occurred between 1958 and 2010 and is defined here as a confirmed culture-positive case of septicemia or meningitis) where infants exclusively ingested breast milk, without consumption of a commercially manufactured PIF product, prior to illness onset [17]. Friedemann had also reported similar earlier observations [26]. More recently, Bowen et al. [27] and McMullan et al. [28] described cases involving infantile C. sakazakii septicemia/meningitis infections where infants had only consumed expressed maternal milk (EMM) during the first weeks after birth. ...
Cronobacter species are a group of foodborne pathogenic bacteria that cause both intestinal and systemic human disease in individuals of all age groups. Little is known about the mechanisms that Cronobacter employ to survive and persist in foods and other environments. Toxin–antitoxin (TA) genes are thought to play a role in bacterial stress physiology, as well as in the stabilization of horizontally-acquired re-combinatorial elements such as plasmids, phage, and transposons. TA systems have been implicated in the formation of a persistence phenotype in some bacterial species including Escherichia coli and Salmonella. This project’s goal was to understand the phylogenetic relatedness among TA genes present in Cronobacter. Preliminary studies showed that two typical toxin genes, fic and hipA followed species evolutionary lines. A local database of 22 TA homologs was created for Cronobacter sakazakii and a Python version 3 shell script was generated to extract TA FASTA sequences present in 234 C. sakazakii genomes previously sequenced as part of Center for Food Safety and Applied Nutrition’s (CFSAN) GenomeTrakr project. BLAST analysis showed that not every C. sakazakii strain possessed all twenty-two TA loci. Interestingly, some strains contained either a toxin or an antitoxin component, but not both. Five common toxin genes: ESA_00258 (parDE toxin-antitoxin family), ESA_00804 (relBE family), ESA_01887 (relBE family), ESA_03838 (relBE family), and ESA_04273 (YhfG-Fic family) were selected for PCR analysis and the primers were designed to detect these genes. PCR analysis showed that 55 of 63 strains possessed three of these genes Sequence analysis identified homologs of the target genes and some of the strains were PCR-negative for one or more of the genes, pointing to potential nucleotide polymorphisms in those loci or that these toxin genes were absent. Phylogenetic studies using a Cronobacter pan genomic microarray showed that for the most part TAs follow species evolutionary lines except for a few toxin genes possessed by some C. malonaticus and C. universalis strains; this demonstrates that some TA orthologues share a common phylogeny. Within the C. sakazakii strains, the prevalence and distribution of these TA homologs by C. sakazakii strain BAA-894 (a powdered infant formula isolate) followed sequence-type evolutionary lineages. Understanding the phylogeny of TAs among the Cronobacter species is essential to design future studies to realize the physiological mechanisms and roles for TAs in stress adaptation and persistence of Cronobacter within food matrices and food processing environments.
... The sources of C. sakazakii and its vehicles of transmission are not clearly documented. It is distributed and frequently contaminated in the environment [25], plant materials [26], powdered infant formulas [27], cereal foods [28], fermented beverages [29], fruits, and vegetables [26]. In particular, contamination on powdered infant formula occurs more easily because it is a non-sterilized product. ...
Cronobacter sakazakii (Cz) infections linked with powdered milk/flour (PMF) are on the increase in recent times. The current study aimed at assessing worldwide and regional prevalence of Cz in PMF. Cz-PMF-directed data were conscientiously mined in four mega-databases via topic-field driven PRISMA protocol without any restriction. Bivariate analysis of datasets was conducted and then fitted to random-intercept logistic mixed-effects regressions with leave-one-study-out-cross-validation (LOSOCV). Small-study effects were assayed via Egger’s regression tests. Contributing factors to Cz contamination/detection in PMF were determined using 1000-permutation-bootstrapped meta-regressions. A total of 3761 records were found out of which 68 studies were included. Sample-size showed considerable correlation with Cz positivity (r = 0.75, p = 2.5e−17), Milkprod2020 (r = 0.33, p = 1.820e−03), and SuDI (r = − 0.30, p = 4.11e−03). The global prevalence of Cz in PMF was 8.39% (95%CI 6.06–11.51, PI: 0.46–64.35) with LOSOCV value of 7.66% (6.39–9.15; PI: 3.10–17.70). Cz prevalence in PMF varies significantly (p < 0.05) with detection methods, DNA extraction method, across continents, WHO regions, and world bank regions. Nation, detection method, world bank region, WHO region, and sample size explained 53.88%, 19.62%, 19.03%, 15.63%, and 9.22% of the true differences in the Cz prevalence in PMF, respectively. In conclusion, the results indicated that national will power in the monitoring and surveillance of Cz in PMF matched with adequate sample size and appropriate detection methods will go a long way in preventing Cz contamination and infections.
The climate crisis is an emerging global challenge that poses potential risks to breastfeeding practices and outcomes. There are multifaceted effects of climate change affecting the breastfeeding dyad across environmental, societal, and human health dimensions. Breastfeeding support in the face of climate change will require solutions at the structural level—healthcare, community, and workplace settings—and at the mother-infant dyad level. Breastfeeding can additionally be an adaptive response to crisis situations and can mitigate some of the environmental challenges associated with climate change. Despite the undeniable significance of climate change on breastfeeding (and vice versa), our perspective as experts in the field is that this topic has not been systematically addressed. Although we highlight some of the challenges, potential solutions, and co-benefits of breastfeeding in the context of climate change, there are numerous issues that could be further explored and necessitate additional preparedness planning.
Cronobater spp. (E. sakazakii) é considerada um micro-organismo oportunista que vem ganhando atenção de autoridades de Saúde Pública, pelo crescente número de surtos de infecção em recém–nascidos e lactentes. A bactéria está associada a casos raros, com alta taxa de mortalidade, podendo causar meningites, enterocolite necrosante e septicemia. Cronobacter spp. tem ampla disseminação, porém apenas as fórmulas lácteas infantis em pó foram, epidemiologicamente, associadas às doenças causadas por esse agente. No presente estudo foi avaliada a ocorrência de Cronobacter spp. em alimentos destinados às crianças de 0-36 meses de idade, adquiridos em lactário de um hospital público do município de São Paulo. Vinte e seis amostras de fórmulas reconstituídas e 24 produtos em pó foram analisados segundo a metodologia da ISO. Cronobacter spp. foi detectada em uma amostra (3,8%) reconstituída de alimento infantil à base de farinha de milho e em quatro desse produto em pó (16,7%). A bactéria não foi detectada nas fórmulas infantis destinadas às crianças de 0-6 meses, contudo sua presença em outros alimentos infantis pode contribuir para a contaminação do ambiente e dos utensílios dos lactários por meio da contaminação cruzada.
Cronobacter sakazakii, as a most important foodborne pathogen in powdered infant formula (PIF), can cause diseases with high mortality in infants and young children, and has become a key monitoring target in dairy industry. In this study, a visual detection strategy based on DNAzyme and asymmetry recombinase polymerase amplification (aRPA) was developed for monitoring C. sakazakii in PIF. A large amount of single-stranded DNA (ssDNA) rich of guanine (G) was produced in this process and ssDNA could bend and fold into intramolecular parallel G-quadruplex in the presence of potassium chloride. The complex of G-quadruplex and hemin, described as DNAzyme, has strong peroxidase-like activity and catalyze the reaction of H2O2 and 3,3′,5,5′-tetramethylbenzidine (TMB), making the color of solution blue. To our knowledge, this is the first attempt in the detection of C. sakazakii based on aRPA and DNAzyme. The limits of detection (LOD) of this method for C. sakazakii were as low as 2.2 CFU mL⁻¹ in pure culture and 5.4 × 10¹ CFU g⁻¹ in artificially contaminated PIF, respectively. Besides, good stability of the method was shown in the on-site simulation evaluation, which was realized by only warm water, indicating that such strategy may have bright application prospects for field test without heating equipment.
Cronobacter species are considered an opportunistic group of foodborne pathogenic bacteria capable of causing both intestinal and systemic human disease. The genus has undergone a significant adjustment since 2008 when it was reclassified to include its current seven species, and undoubtedly, this taxonomic outlook will continue to evolve as whole-genome sequencing is used to understand the phylogenetic relatedness of its members and those of phylogenetically related species. There are common themes of virulence shared among Cronobacter such as possession of a virulence plasmid that encodes for shared factors such as iron acquisition loci (ferric ABC transporter and siderophore gene clusters). Powdered infant formula (PIF) is thought to be the source of neonatal/infantile infections. However, it is clear now that contamination of reconstituted and temperature-abused PIF can occur intrinsically and extrinsically, although the main reservoir(s) and routes(s) of contamination have yet to be determined. Cronobacter species are now considered to be a group of pathogens with notable versatility in their ability to cause human disease in all age groups. In summary, combining a better epidemiological reporting system with a more comprehensive understanding of virulence would lead to improved patient care, enabling better clinical outcomes.
The genome of Cronobacter sakazakii M1 phage named PF-CE2 was characterized in this work. And a new species named Cronobacter virus PF-CE2, in the Escherichia virus RB16 genus of the subfamily Tevenvirinae of the family Myoviridae was established. The Gp190 gene of phage PF-CE2 was first proposed to encode a bacteriophage-borne glycanase, which is capable of degrading fucose-containing exopolysaccharides produced by C. sakazakii M1. Further, the taxonomic status of eight additional phages was modified according to average nucleotide identity analysis. This finding provides a theoretical basis for subsequent heterologous expression of the phage PF-CE2 glycanase and provides an important reference for the preservation and sharing of these phages.
Herein, we propose the light scattering intensity of silver and gold alloy as signal transducer to enhance the sensitivity and dynamic linearity of immunoassay for quantitative detection of Cronobacter muytjensii (C. muytjensii) in powered infant formula (PIF). Silver and gold alloy, namely, silver-coated gold nanocomposite ([email protected]), was obtained by growing silver on the surface of multi-dendritic colloidal gold. The silver growth was triggered by the hydrolysis of urease to urea in the presence of silver nitrate and glucose. Owing to integrating the advantages of light scattering of [email protected] and enzyme-catalyzed mediated signal amplification, the developed light scattering based immunoassay shows high sensitivity for detection of C. muytjensii, with a detection limit of 51 CFU/mL, which is three orders of magnitude lower than that of conventional enzyme-linked immunosorbent assay (ELISA, 6.39 × 10⁴ CFU/mL). In addition, the proposed method also exhibits wide dynamic curve from 3.4 × 10² CFU/mL to 3.4 × 10⁹ CFU/mL for quantitative detection of C. muytjensii, and is significantly superior conventional ELISA (3.4 × 10⁵ CFU/mL to 3.4 × 10⁹ CFU/mL). The accuracy and precision of the proposed immunoassy were evaluated by analysis of C. muytjensii-fortified PIF samples. The average recoveries of the intra- and inter-assays are within 87.90%–107.91%, and the coefficient of variation ranges from 2.72% to 9.32%. This finding indicates an acceptable accuracy for quantitative detection of C. muytjensii in real PIF samples.
The presence of Cronobacter sakazakii must be controlled in infant powder plants, because it may cause infectious disease in infants, with high mortality. Testing for C. sakazakii in powdered infant formula should be performed before delivery, and it requires rapid and specific detection methods. In this study, we established a surface-enhanced Raman scattering (SERS) immunochromatographic test strip for the quantitative determination of C. sakazakii in powdered infant formula. Monoclonal antibodies for C. sakazakii were labeled with p-aminothiophenol-bound colloidal gold nanoparticles. Color change in the test line indicated the presence of C. sakazakii. A highly sensitive and quantitative test method was developed based on the Raman signal produced by the p-aminothiophenol bonding on gold nanoparticles. The SERS immunochromatographic test strip assay required a short analysis time (12 min) and exhibited a linearity range from 10² to 10⁷ cfu/mL. The limit of detection was 201 cfu/mL without preculture. The SERS immunochromatographic test strip assay is a promising tool for the simple and rapid quantitative analysis of C. sakazakii and other pathogenic bacteria.
Thymol has a broad-spectrum antibacterial effect, but few studies have elucidated the antibacterial mechanism of thymol on Enterobacter sakazakii to date. This study aimed to uncover its antimicrobial mechanism against E. sakazakii. The minimum inhibitory concentration (MIC) of thymol was determined using the broth microdilution. Membrane potential, intracellular ATP concentrations, and intracellular pH (pHi) were measured, and the membrane damage was observed using a confocal laser scanning microscopy (CLSM) and a field emission gun scanning electron microscope (FEGSEM) to discuss the antibacterial effect of thymol. The MIC of thymol against E. sakazakii BNCC 186088 was 1.25 mg/mL. Cell membrane depolarization, decreased intracellular ATP concentrations, and lower pHi were observed after treatment with thymol, which indicates broken cell membranes and disrupted intracellular homeostasis. These results suggest that thymol has the potential to prevent bacterial contamination by E. sakazakii in the food industry.
Here a triple functional sensing chip was created for L. monocytogenes detection by integrating three biomarkers (Listeriolysin O (LLO) at protein level, hly gene at genetic level, and acetoin at metabolic level). Liposome encapsulated catechol was used for LLO detection via LLO pore-forming ability. hly gene was specifically captured by using a thiolated capture probe on nanoporous gold (NPG). As an electroactive label, methylene blue was embedded in double-stranded structures to generate an electrochemical signal for hly detection. Combined with the electrocatalysis of NADH by NPG, the acetoin detection was achieved by measuring the consumption of NADH as a cofactor under acetoin reductase catalysis. Importantly, the L. monocytogenes detection results obtained by detecting three biomarkers using the chip can be mutually verified, which reduces the probability of false positives based on a single marker. Moreover, the detection time was reduced to about 90 min, making it a rapid and reliable tool for L. monocytogenes detection.
Cronobacter spp. are opportunistic pathogens that cause serious infections, especially in infants, elderly, and immunocompromised people. Dehydrated infant foods are the main vehicle associated with infections caused by these bacteria. Thus, this study aims to investigate the occurrence of Cronobacter spp. in 152 commercial dehydrated infant formulas (77 samples) and dehydrated infant cereals (75 samples), as well as characterize the isolates. Polymerase Chain Reaction (PCR) and Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF/MS) methods for isolate identification were used, and their results compared. Furthermore, the susceptibility to 11 antibiotics was tested, and DNA sequencing of one isolate with multi-drug resistance was analyzed. No contamination in the infant formula samples was found, whereas 17.33% (13/75) of the infant cereal samples presented contamination with Cronobacter sakazakii. The identification results by PCR and MALDI-TOF/MS were divergent for some isolates. The antimicrobial resistance results showed a high incidence of resistance to cefazolin (94.4%) besides resistance to amoxicillin (9.45%), cefpodoxime (5.55%), streptomycin (1.35%), and trimethoprim/sulfamethoxazole (1.35%). Whole genome sequencing of one multi-drug resistant isolate showed six genes associated with antimicrobial resistance and an 82% possibility of being a human pathogen based on the presence of virulence factors. The presence of Cronobacter spp. in infant foods represents a risk for the infant’s health. Moreover, the presence of a pathogenic multi-drug resistant isolate in infant’s food reinforces the necessity of improving food safety policies to protect young children.
Cronobacter sakazakii is a typical food-borne bacterium that causes clinical symptoms including necrotizing enterocolitis, bacteremia, and meningitis with a mortality rate of 40%–80%. In this study, a pair of split G-rich DNA probes was assembled into the DNAzyme capable of mimicking peroxidase. The assembly was utilized to provide a simple, low-cost, highly specific, and sensitive method for rapid detection of viable C. sakazakii. In the absence of viable C. sakazakii, the two split G-rich DNA probes formed DNAzyme, mimicked the activity of peroxidase and displayed colorimetric signals to indicate detection results. In the presence of viable C. sakazakii, specific recognition by the aptamer results in the destruction of the DNAzyme-aptamer complex, and subsequently the colorimetric signal is “turned-off” due to the decrease in DNAzyme bioactivity. Under the optimal reaction conditions, there is a linear relationship between absorption intensity at 418 nm and logarithmic concentration of C. sakazakii in the range of 2–1200 CFU/mL (R² = 0.9839), with a detection limit of 1.2 CFU/mL. This is the highest sensitivity ever reported for viable C. sakazakii detection. Artificially contaminated samples were assayed by this novel sensor, and numerous advantages were discovered, including ultra-low sensitivity, low-cost, simple operation, and good performance, compared to existing methods. We believe the DNAzyme shows great potential for food safety applications.
Cronobacter sakazakii is an important opportunistic food-borne pathogen, and it can cause severe diseases with main symptoms including neonatal meningitis, necrotizing enterocolitis, and sepsis. For the achievement of practical and convenient detection of viable C. sakazakii, a simple and robust strategy based on the cascade signal amplification of RT-PCR triggering G-quadruplex DNAzyme catalyzed reaction was firstly used to develop the effective and sensitive DNAzyme electrochemical assay. Without viable C. sakazakii in the samples there is no any RT-PCR and DNAzyme products, which can cause a weak electrochemical response. Once viable C. sakazakii exists in the samples, an obvious enhancement of the electrochemical response can be achieved after target signal is amplified by RT-PCR and the resulting DNAzyme, which catalyze the oxidation of 3,3',5,5'-tetramethylbenzidine (TMB) by H2O2 with the assistance of the cofactor hemin. Our novel assay can be detected in the range of 2.4×107CFU/ mL to 3.84×104CFU/ mL (R2=0.9863), with a detection limit of 5.01×102 CFU/mL. Through 15 real samples assay, electrochemical detection assay had the same results as conventional detection methods. Therefore, detection of viable C. sakazakii based on G-quadruplex DNAzyme electrochemical assay with RT-PCR demonstrates the significant advantages of high sensitivity, low cost and simple manipulation over existing approaches and offers the opportunity for potential application in pathogen detection.
Cronobacter sakazakii (C. sakazakii) is an opportunistic foodborne pathogen in infant formula. This study was designed to explore the inhibitory effect of TGML on C. sakazakii in reconstituted infant formula (RIF). Firstly, the growth curve of C. sakazakii in RIF treated by TGML and the effect of different temperatures (4, 10, 21, 30 and 37 °C), pH values (5, 6, 7, 8 and 9) and ionic strengths (25, 50, 100, 200, 400 and 800 mM) on its activity were assessed. The results showed that the inhibitory effect of TGML on C. sakazakii was dose-dependent, and 1, 2 and 5 μg/mL TGML delayed the visible growth of pathogen by 4, 12 and 24 h, respectively. Storage temperature above or below room temperature enhanced the bioactivity of TGML. And a decrease in pH also increased the antibacterial effect of TGML. However, the effect of ionic strength on its activity was not obvious. Subsequently, the antibacterial effect of TGML in physiological gastric acid and simulated gastric juice in vitro was further explored. We found that only 5 μg/mL TGML could inhibit the growth of pathogen below the infectious dose (10,000 CFU in total) in simulated gastric juice during the whole gastric emptying period (3.5-21 h), weaker than its antibacterial effect in physiological gastric acid and room temperature culture. Finally, the effect of TGML and the above environmental factors on the color and aroma of infant milk was evaluated by a 12-person panel. The results revealed that TGML did not affect the sensory flavor of milk, and the color and odor scores of infant milk under different environmental conditions did not show any significant differences. Therefore, it is concluded that TGML has a good inhibitory effect on C. sakazakii in RIF and a high sensory acceptability for consumers. Adjusting the temperature or lowering the pH enhances its bacteriostatic activity. However, the presence of infant gastric juice can impair the bioactivity of TGML. Overall, this study will provide some new ideas for controlling and eliminating the potential risk of C. sakazakii infection during infant feeding.
Plate counts and pH measurements were performed on tofu from different aged lots. The pH declined from to 5.8 to 5.2 with age. Aerobic plate counts of 1- and 30-d-old samples were approximately 106 CFU/g. Older samples had counts of about 108 CFU/g. Anaerobic counts rose from 106 CFU/g in the 1-d-old lot to a high of 109 CFU/g in the 30-d-old lot. The major species from the different age lots were lactic acid bacteria, enteric bacteria, and Pseudomonas species. Representatives from each of these groups were inoculated into autoclaved tofu and incubated at 5°C for 23 d. CFU/ml and pH of both water and cake were measured. Turbidity, mg protein per ml, and mg NH4+ per mi of the water were measured. All species tested increased in numbers in the tofu and caused changes in at least some of the characteristics measured. Samples were taken during the manufacture of tofu, and all organisms found in each sample were characterized. All organisms that were shown capable of causing spoilage in tofu were present in la...
The O-specific polysaccharide from Enterobacter sakazakii cell was isolated and structurally characterized. Lipopolysaccharide (LPS) was obtained from cell mass by hot phenol-water extraction procedure. Mild acid hydrolysis followed by gel filtration provided pure O-antigen (OPS). Two-stage sugar analysis detected tyvelose, rhamnose and galactose in the molar ratio of 1:1:2, and their linkages were established by means of methylation analysis. Sugar configurations, D or L, were determined by gas-liquid chromatography on an achiral liquid phase for (S)-(+)-2-butyl glycosides. D configuration was determined for galactose and 3,6-dideoxy-mannose (tyvelose), but L for rhamnose. Repeating unit structure was deduced by analysis of 1H and 13C NMR spectra. 1H and 13C NMR resonances have been assigned by homonuclear (COSY, TOCSY) and heteronuclear (HSQC, HMBC) correlations spectra. Anomeric configurations were determined from anomeric proton chemical shifts and 3JH1-H2 and JC-H coupling constants. Sugar sequences were established from comparisons of specific carbon chemical shifts with those in literature, two-dimensional nuclear Overhauser effect spectroscopy (NOESY), and heteronuclear multiple-bond correlation experiments (HMBC). The repeating unit structure of Enterobacter sakazakii was found to be as: (Equation Presented).
This study was conducted to determine bacterial species of Enterobacteriaceae in adults of lesser mealworm, A. diaperinus and make the correlation of the bacterial species in the insects and in the litter that infest poultry brooder houses in West of Parana State, Brazil. In the first experiment, the adults were collected in 14 poultry houses. In the second experiment the insects and the litter were collected in 12 poultry houses. The adults were anaesthetized with eter, macerated in sterile saline solution and the litter material was taken off by swab. The enrichment broth was plated on the BHI and in Rappaport-Vassiliadis and Tetrationate. The agar MacConkey, agar Salmonella-Shigella and agar brilliant green was used as plating media for isolating the enterobacteria. The enterobacteria isolated in adults of A. diaperinus were: Proteus Vulgaris, P. mirabilis, Escherichia Coli, Enterobacter spp., E. agglomerans, E. gergoviae, E. sakasakii, Citrobacter Diversus and Klebsiella Pneumoniae. In the litter were found Proteus Vulgaris, P. mirabilis, Escherichia Coli, Enterobacter Agglomerans. There was no Salmonella spp . found in the insect and the litter and P. vulgaris was predominant. E. coli was found in the poultry houses, in the litter and the insects and the bacteria are responsible for dissemination of colibacilose.
Enterobacter sakazakii has been implicated in a severe form of neonatal meningitis. Although studies have failed to identify an environmental source for the organism, dried infant formula has been implicated in outbreaks and sporadic cases of E. sakazakii meningitis. The high mortality rate (50 to 75%), the severity of the infection in infants, and the lack of information on the incidence, survival, and growth of E. sakazakii in foods led to this study. Experiments were undertaken to determine the incidence of E. sakazakii in dried infant formula, the temperature range for growth, and the growth characteristics of E. sakazakii in reconstituted dried infant formula. Strains of E. sakazakii were isolated from dried infant formula available on the Canadian retail market. The prevalence varied from 0 to 12% in samples from five different companies. For both clinical and food isolates, minimum growth temperatures of 5.5 to 8.0°C were observed by using a temperature-gradient incubator. The potential growth of E. sakazakii was followed by using a mixture of food and clinical isolates in three different formulas incubated at 4, 10, and 23°C. Average generation times were 40 min at 23°C and 4.98 hat 10°C. E. sakazakii strains did not grow at 4°C and began to die off during storage at this temperature. The results of this study stress the importance of using aseptic methods and proper temperature control in the preparation, use, and storage of dried infant formula.
We evaluated the TaqMan Salmonella amplification/detection kit from PE Applied Biosystems, which uses a polymerase chain reaction (PCR) assay for rapid detection of Salmonella in food samples. This system uses the 5 nuclease activity of Taq DNA polymerase, which digests an internal fluorogenic probe to monitor the amplification of the target gene. The system's sensitivity and specificity were evaluated using 42 serotypes of 68 Salmonella strains isolated from fecal samples from patients in Tokyo, Japan, and 39 non-Salmonella strains in 22 genera. There were no false-negative or false-positive results. This PCR assay can detect 3 CFU per PCR tube of Salmonella in pure culture (120 CFU/ml of TSB culture). PCR signals were attenuated with artificially contaminated shrimp, but a similar detection limit was obtained. TaqMan's performance was tested with 100 meat and chicken samples purchased from stores in Tokyo. Overall, two of the DNA extraction protocols (the Chelex and EnviroAmp methods) worked equally well, with some exceptions. Of the 100 samples analyzed, 10 were positive for Salmonella with both conventional culture methods and the kit and 89 were negative with both. One sample was negative by the culture method but positive by the kit assay. These results indicate that TaqMan is a reliable and rapid method for Salmonella analysis in the food industry. With this system, food samples can be analyzed for Salmonella in less than 20 h.
In late 1995, fecal coliforms were detected in iced tea obtained from several restaurants in the U.S. On the basis of fecal coliform results, the news media inaccurately and sensationally accused the tea industry of marketing tea containing feces. We analyzed 11 iced-tea samples obtained from fast-food restaurants and 25 dry leaf-tea samples purchased from retail grocers for the presence of coliforms and fecal coliforms. All samples of iced tea contained coliforms and fecal coliforms; most probable numbers of coliforms in iced tea ranged from 210 to >1,100/ml, whereas those of fecal coliforms were 15 to >1,100/ml. Twenty-three of twenty-five leaf-tea samples contained coliforms and fecal coliforms; ranges for positive samples were 3 to 1,100/g and 3 to 460/g, respectively. Two Klebsiella species and three Enterobacter species were isolated from iced tea. Only Klebsiella pneumoniae (93.8% of isolates) and Enterobacter cloacae (6.2%) were isolated from leaf tea. Escherichia coli was not isolated from any of the iced-tea or leaf-tea samples analyzed. The D55°c values of two isolates of K. pneumoniae and three isolates of E. cloacae from leaf tea, when heated in steeped tea, ranged from 3.75 to 5.08 min. Initial populations of up to 5.70 log CFU/ml were reduced to <10 CFU/ml within 5 min at 65°C. While 23 of 25 (92%) of the leaf-tea samples analyzed contained fecal coliforms, as defined by standard methodology, there is no evidence that leaf tea represents a health hazard. The expected presence in leaf tea of Klebsiella and Enterobacter species, which test positive for the fecal coliform group, should not automatically be construed as indicators of fecal contamination nor as an imminent threat to human health. The presence of coliforms and fecal coliforms in iced tea indicates post-steeping contamination caused by poor sanitation practices in restaurants at which the iced tea was purchased.
Sobia is a traditional fermented beverage prepared from wheat and malt flours in the Western province of Saudi Arabia. Fourteen samples of sobia were collected from Makkah Al-Mukarrmah (Western province) and from Riyadh (Central province). Samples were examined microbiologically for total bacterial counts, lactic acid bacteria, yeasts and moulds as well as coliforms. Titratable acidity and pH were also analysed. The results revealed that sobia samples had high total bacterial and lactic acid bacteria counts that ranged from 4.17 to 8.09 log c.f.u./ml and from 4.01 to 8.19 log c.f.u./ml respectively. Yeasts and moulds counts ranged from 3.96 to 5.87 log c.f.u./ml. The coliform count was from 0.67 to 3.84 log c.f.u./ml. Acidity expressed as percent lactic acid ranged from 0.04 to 0.30%, while the pH of the samples ranged from 3.37 to 5.53. Identification of microorganisms in sobia revealed the presence of lactic acid bacteria, coliforms, yeasts and moulds. Lactic acid bacteria consisted of Lactobacillus cellobiosus (26.8%), L. buchneri (17.9%), L. plantarum (8.9%), L. brevis (23.2%), L. delbrueckii. delbrueckii (8.9%), Leuconostoc lactis (8.9%), and Pediococcus pentosaceus (5.4%). Coliforms consisted of Klebsiella pneumoniae (25.8%), Enterobacter aerogenes (6.1%), E. sakazakii (33.3%), E. cloacae (19.7%), and Serratia liquefaciens (3%). Yeasts comprised Saccharomyces cerevisiae (27.6%), Candida tropicalis (26.3%), C. ciferrii (13.1%), C. guilliermondii (13.1%), C. lipolytica (6.58%), Kloeckera japonica (13.1%), and Rhodotorula rubra (1.3%). All moulds were Penicillium spp.
Endophytic bacteria are ubiquitous in most plant species influencing the host fitness by disease suppression, contaminant degradation, and plant growth promotion. This endophytic bacterial community may be affected by crop management such as the use of chemical compounds. For instance, application of glyphosate herbicide is common mainly due to the use of glyphosate-resistant transgenic plants. In this case, the bacterial equilibrium in plant–endophyte interaction could be shifted because some microbial groups are able to use glyphosate as a source of energy and nutrients, whereas this herbicide may be toxic to other groups. Therefore, the aim of this work was to study cultivable and noncultivable endophytic bacterial populations from soybean (Glycine max) plants cultivated in soil with and without glyphosate application (pre-planting). The cultivable endophytic bacterial community recovered from soybean leaves, stems, and roots included Acinetobacter calcoaceticus, A. junii, Burkholderiasp., B. gladioli, Enterobacter sakazaki, Klebsiella pneumoniae, Pseudomonas oryzihabitans, P. straminea, Ralstonia pickettii,and Sphingomonassp. The DGGE (Denaturing Gradient Gel Electrophoresis) analysis from soybean roots revealed some groups not observed by isolation that were exclusive for plants cultivated in soil with pre-planting glyphosate application, such as Herbaspirillum sp., and other groups in plants that were cultivated in soil without glyphosate, such as Xanthomonas sp. and Stenotrophomonas maltophilia. Furthermore, only two bacterial species were recovered from soybean plants by glyphosate enrichment isolation. They were Pseudomonas oryzihabitans and Burkholderia gladioliwhich showed different sensibility profiles to the glyphosate. These results suggest that the application at pre-planting of the glyphosate herbicide may interfere with the endophytic bacterial communityrsquos equilibrium. This community is composed of different species with the capacity for plant growth promotion and biological control that may be affected. However, the evaluation of this treatment in plant production should be carried out by long-term experiments in field conditions.
A precise 5' nuclease (TaqMan) real-time PCR was developed and validated in house for the specific detection of Enterobacter sakazakii isolates. Specifically designed nonpatented primers and a hydrolysis (TaqMan) probe were used to target the 16S rRNA gene. All 27 E. sakazakii and 141 non-E. sakazakii strains tested with the real-time PCR were identified correctly. To monitor false-negative results, an internal amplification control was coamplified with the same primers used for the E. sakazakii DNA. The detection probability of the assay was 56% when an E. sakazakii cell suspension containing 10(2) CFU/ml was used as template in the PCR (0.5 CFU per reaction) and 100% with a 10(3) CFU/ml suspension. This PCR assay should be very useful for the diagnostic detection of E. sakazakii in foods, especially powdered infant formula, after cultural enrichment.
O objetivo deste trabalho foi realizar o isolamento de bactérias da família Enterobacteriaceae em adultos de A. diaperinus, buscando uma correlação entre as bactérias presentes no inseto e na cama, em aviários para produção de frangos de corte no oeste do Paraná, Brasil. No primeiro experimento, insetos adultos foram coletados em 14 granjas. No segundo experimento, foram coletados insetos e material da cama de 12 diferentes aviários. Os adultos foram anestesiados com éter, macerados em solução salina e o material da cama colhido por "swab" de arrasto. O enriquecimento não seletivo foi feito com caldo BHI e o seletivo com Rappaport-Vassiliadis e Tetrationato. Os meios de cultivo para plaqueamento foram o ágar MacConkey, ágar Salmonella-Shigella e ágar verde-brilhante. As enterobactérias isoladas em adultos de A. diaperinus foram: Proteus Vulgaris, P. mirabilis, Escherichia Coli, Enterobacter spp., E. agglomerans, E. gergoviae, E. sakasakii, Citrobacter Diversus e Klebsiella Pneumoniae, enquanto que na cama foram encontrados Proteus Vulgaris, P. mirabilis, Escherichia Coli, Enterobacter Agglomerans. Não foram isoladas Salmonella spp. do inseto nem da cama e P. vulgaris foi a predominante. E. coli foi freqüente nas granjas, tanto na cama como nos insetos e contribuem na disseminação da colibacilose em aviários.
ABSTRACT During the 1995 wet season, harvested rice seed was collected from farmers' fields at different locations in Iloilo, Philippines. Bacterial isolations from crushed seed yielded 428 isolates. The isolates were characterized by BOX-polymerase chain reaction fingerprinting of total genomic DNA and represented 151 fingerprint types (FPT). Most FPTs were found on a single occasion, although matching fingerprints for isolates from different samples also were found. Identifications were made by cellular fatty acid methyl ester analysis and additional use of Biolog GN/GP MicroPlates and API 20E/50CHE systems. The predominant bacteria were Enterobacteriaceae (25%), Bacillus spp. (22%), and Pseu-domonas spp. (14%). Other bacteria regularly present were identified as Xanthomonas spp., Cellulomonas flavigena, and Clavibacter michiganense. Of the total number of isolated bacteria, 4% exhibited in vitro antifungal activity against Rhizoctonia solani or Pyricularia grisea. Two percent of isolates were pathogens identified as Burkholderia glumae and Burkholderia gladioli. Five percent of isolates induced sheath necrosis on only 50 to 90% of inoculated plants and were related to Bacillus pumilus, Paenibacillus spp., Pseudomonas spp., and Pantoea spp.
Escherichia coli was isolated from the body surface, alimentary canal and excreta of the Musca domestica (Linnaeus), and the alimentary canal of Stomoxys calcitrans (Linnaeus) and Phaenicia sericata (Meigen) collected at a dairy farm in Tokushima Prefecture in 1997. We detected E. coli O157 : H7 in the excreta and other pathogenic bacteria like Serratia marcescens, Enterobacter sakazakii and Pseudomonas fluorescens in the alimentary canal of M. domestica using the immunomagnetic separation method, polymerase chain reaction method and rapid identification kits. The isolated bacteria were resistant to more than three antibiotics. Escherichia coli O157 : H7 from human and E. coli O157 : H7 from M. domestica had similar susceptibility against tested antibiotics. These facts suggest that houseflies or other flies would be possible mechanical vectors to transmit the pathogens like enterohemorrhagic colitis or enteritis, although general sanitary conditions seem to be clearly improved in Japan. This is the first report of E. coli O157 : H7 in the excreta of houseflies collected at a dairy farm field.
A total of 256 coliform strains (132 from central, 124 from decentralized water supplies) as defined by the German drinking water regulation were characterized by the API 20E-system and by their susceptibility to 15 antimicrobial agents. Alltogether 22 different species from 10 genera could be differentiated, whereas 14 strains were unidentifiable by the applied system. The frequency distribution of the strains isolated from central water supplies yielded 48.5 % for Citrobacter freundii, 17.4 % for Klebsiella pneumoniae, 6.8 % for Buttiauxella agrestis and 6.1 % for C. diversus. In decentralized water supplies C. freundii was found in 33.9 %, K. pneumoniae in 14.5 %, Serratia fonticola in 8.9 % and K. oxytoca in 6.5 %.
About one third (73 strains) were fully susceptible to all used antibiotics. 42 strains, however, revealed complete or moderate resistance against three or more agents (multiresistant strains). Within this group a seasonal variation was evident. The proportion of multiresistant isolates was found to be relatively high in the first half of the year (21.4 % in January- March and 29.4 % in April-June) and relatively low in the second half-year (7.9 % in July-September and 11.3 % in October-December). This different distribution may indicate a conservation of R-factor bearing bacteria in the environment.
Enterobacter sakazakii, designated as an unique microbial species in 1980 may cause bacteremia necrotizing enterocolitis and infant meningitis. The distribution and the thermostability of E. sakazakii in unprocessed ready-to-eat (RTE) agricultural products of 252 and in 25 powdered infant formulas (PIF) were analyzed. Eighty one, 50, 43 and 47% of brown rice, pumpkin, potato, and carrot samples, respectively, had aerobic plate counts (ARC) in the range of 5 log CFU/g or more. Almost all the other products sampled had APC of approximately 2 log CFU/g. Fifty three, 75, 67, and 68% of banana, pumpkin, soybean, and carrot had Enterobacteriaceae counts approximating 3 log CFU/g. Sixty six percent of the brown rice tested had Enterobacteriaceae counts approximating 5-6 log CFU/g. E. sakazakii was isolated from 3/25(12%), 4/23(17%), 1/24(4%), and 1/27(4%) of PIF, brown rice, laver, and tomato samples, respectively. D-values were 3.52-4.79 min at 60 and D-60-values were similar as the isolates reported. Thermal inactivation of four thermovariant E. sakazakii strains during the rehydration of PIF with hot water were investigated. At 50 C, the levels of E. sakazakii decreased one log CFU/g for 4-6 min and thereafter the levels remained stable for 20 min. At 60 C, inactivation by about 2 log CFU/g occurred for 20 min. Therefore, the unprocessed agricultural products might be a source of contamination for PIF when used as an ingredient after drying and pulverization. Rehydration of PIF for infant feeding with a water temperature of 60 degrees C rather than 50 degrees C, as recommended by the manufacturers, may be helpful in the reduction of potential E. sakazakii risk.
29 strains of endophyte associated diazotrophs isolated from rice (Oryza sativa L.) "Yuefu" plant were selected by in vitro acetylene reduced activity and 15N2-fixing activity determination. They were identificated into 14 species of 9 genera: Agrobacterium tumefaciens (Smith et Townsend) Conn, A. radiobacter (Beijerinck et van Delden) Conn; Akaligenes piechaudii Kiredjian et al., Al. denitrificans (Leifson et Hagh) Ruger et Tan; Bacillus sphaericus Meyer et Neide, B. licheniformis Weigmann Chester, B. cereus Frankland et Frankland; Chryseomonas luteola (Kodama, Kirmura et Komagata) Holmes et al.; Enterobacter cloacae (Jordan) Hormaeche et Edwards, E. sakazakii Famer et al.; E. agglomerans (Beijerinck) Ewing et Fife; Pseudomonas pseudoalcaligenes Stanier, P. akaligenes Monias, P. putida Biotype A Evans and Aeromonas Kluyver et van Niel, Serratia Bizio, Staphyloccocus Rosenbach, Xanthomonas Dowson. Among them C. luteola, E. sakazakii, E. agglomerans, P. pseudoalcaligenes have not been previously reported as diazotrophs. Studying on the distribution of endophytic dizoatrophs in rice seeds and rice plants has demonstrated that the diversity of endophytic associated diazotrophs in roots was more than that in other organs of rice plant. Endophytic associative diazotroph E. cloacae not only occurred on the surface but also occurred intercellularly in R48 rice roots as observed with scanning electron microscopy and transmission microscopy.
Enterobacteriaceae are considered to be the main cause of spoilage in plant products. Especially ready-to-eat vegetable salads are highly contaminated with these bacteria. Already the raw products used for the production of mixed salads show high microbiological counts whilst no significant reduction is achieved by technical means during processing. Some Enterobacteriaceae, for example Enterobacter sakazakii, represent a serious health risk for weak and elderly persons, infants, immunocompromised persons and those with chronical illness. For this reason, reliable identification of this species is most important. Different methods are available for the phenotypic identification of Enterobacter sakazakii. In this study, three commercial systems for the biochemical identification of Enterobacteriaceae were used: the Api® 20E-System (bioMérieux® Deutschland GmbH), the Microbact™ 24E-System (OXOID GmbH Deutschland) and the BiologSystem (Biolog Inc. USA). The objective of this study was a comparative identification of 109 isolates of Enterobacteriaceae from 72 samples of mixed salads, packed in plastic bags or trays, as well as the assessment of the systems used for identification. The occurrence and frequency of detection of Enterobacter sakazakii in ready-to-eat salads was to be determined. 19 strains of the 109 isolates were identified as Enterobacter sakazakii with the Api 20E-System. However, these identifications could not be confirmed by the other systems. Subsequently, 18 of the 19 strains were examined by additional tests. The proof of pigment formation at 25°C on tryptone-soy-agar did not bring any clarity. A final identification was achieved only with the BAX® Detection System (DuPont Qualicon) and the special testkit for Enterobacter sakazakii, showing that none of the 18 strains belonged to Enterobacter sakazakii.
Objectives. To determine growth behaviour of pathogens and spoilage micro-organisms in infant formulas after reconstitution, and comparison of the bacteriostatic effect of acidified formulas obtained through fermentation or by direct addition of lactic acid. Design. Four commercially available infant formulas were deliberately contaminated with eight different pathogens and stored at 4, 25 or 37°C. Growth of the micro-organisms was followed by enumeration after 0, 3 and 6 hours. In a second challenge test the fate of pathogens added to a fermented infant formula was compared with that of pathogens in a non-fermented acidified formula. Results. After a lag phase of a few hours, most of the examined micro-organisms grew well in the pH-neutral products at 37°C. At 25°C growth was clearly retarded and at 4°C no significant growth was detected within 6 hours. Fermented formula exerted an inhibitory effect on all micro-organisms. The same effect was observed with a non-fermented acidified formula. Conclusions. Because pH-neutral reconstituted infant formulas may support rapid growth of many undesirable micro-organisms, including pathogens, utmost care should be taken to prevent contamination, and storage under conditions favouring growth of pathogens should be avoided. Alternatively, prevention of rapid microbial proliferation in infant formulas may be achieved by acidification, either through fermentation or by direct addition of lactic acid, provided that the pH is lower than 5.0.
Enterobacter sakazakii is the name proposed for the organism previously know as 'yellow-pigmented Enterobacter cloacae.' The type strain (holotype) of this species is ATCC 29544. The proposed change in the classification of this organism is based on differences between E. cloacae and E. sakazakii in deoxyribonucleic acid (DNA)-DNA hybridization, biochemical reactions, pigment production, and antibiotic susceptibility. By DNA hybridization, E. sakazakii was about 50% related to E. cloacae, citrobacter diversus ('Citrobacter intermedius' biotype b), and 'Citrobacter amalonaticus' ('Citrobacter intermedius' biotype a). The new species was placed in Enterobacter rather than Citrobacter because of its closer phenotypic and DNA similarity to E. cloacae, the type species of the genus Enterobacter, and because it was only 41% related by DNA hybridization to Citrobacter freundii, the types species of Citrobacter. E. sakazakii had biochemical reactions very similar to those of E. cloacae but was D-sorbitol negative and positive for extracellular deoxyribonuclease at 2 to 7 days and produced yellow-pigmented colonies. E. sakazakii had larger zones of inhibition around ampicillin and cephalothin antibiotic disks, which also helps to differentiate it from E. cloacae. E. sakazakii grew on the nonselective (but differential) plating media commonly used in enteric bacteriology, but its plating efficiency was reduced on more inhibitory enteric plating media. It has been isolated from human clinical specimens such as sputum, feces, and wounds, where it is probably only a colonizer and not clinically significant. However, it is also a documented, although rare, cause of neonatal meningitis. Other sources have included food, a physician's stethoscope, and an uninoculated blood culture bottle.
Bacterial counts on the liquid phase of an anaerobic, fixed-bed digester, treating a deproteinated, prefermented cheese whey substrate, were conducted on two different media under aerobic and facultative conditions. Average counts of 16.6×10(6) and 26.5×10(6) ml(-1) were obtained on the two media, with the nutritionally poorer of the two media giving the highest average count. Seventy-five isolates from both media, incubated aerobically as well as in anaerobic jars, were obtained. These isolates as well as 13 reference strains were phenotypically characterized. The similarities between cultures were calculated using the similarity coefficient of Sokal and Michener [16]. The organisms were clustered using the unweighted pair group method, and the results presented as a simplified dendrogram. The isolates could be divided into 3 main groups: gram-negative fermentative rods, mainlyEnterobacter, Klebsiella, andCitrobacter, withKlebsiella as the predominant genus; gram-positive bacteria, mainly enterococci; and gram-negative nonfermentive rods of the generaPseudomonas, Alcaligenes, andAcinetobacter. All the enterobacteria and enterococci were able to produce acetic acid, an intermediate in methanogenesis.
The mesophilic aerobic total microbial cell numbers as well as the cell counts of Enterobacteriaceae and pseudomonads were determinated in 52 samples of fresh ready-to-serve salads within a storage period or four days. Thereby the influence of two types of commercial cooling systems on the microbial development on the salads was observed. After evaluation of the thermographic and hygrographic data, both systems (the closed as well as the open) appeared to be suitable for storage of these perishable MPF products. Yet the recommended DGHM storage temperature (+6 degrees C) was not maintained throughout in the two systems. Initial cell counts of all samples ranged between 1.8 x 10(5) and 6.3 x 10(6) cfu/g. In 15 samples (18.8 %) stored at temperatures between 10 degrees C and 14 degrees C the total cell counts exceeded or at least reached the DGHM upper value of 5.0 x 10(7) cfu/g within the experimental period of storage. A larger distance between the samples and the radiator lead to a faster increase of the cell counts. The Enterobacteriaceae and pseudomonads were found to represent up to 95.5 % of the total cell counts. With few exceptions (Erwinia, Sphingomonas, Rahnella, Serratia and Pasteurella) all identified Enterobacteriaceae belonged to the ZKBS risk group 2 (ZKBS, 1995). In two samples even Klebsiella spp. were found.
Enterobacteriaceae are considered to be the cause of spoilage in plant products. Especially ready-to-eat vegetable salads are highly contaminated with these bacteria. Even the raw products used for the production of mixed salads show high microbiological counts whilst no significant reduction is achieved by technical means during processing.
Some Enterobacteriaceae, for example Enterobacter sakazakii, represent a serious health risk for weak and elderly persons, infants, immunocompromised and persons with chronical illness. For this reason, reliable identification of this species is most important.
Different methods are available for the phenotypic identification of Enterobacter sakazakii. In this study, three commercial systems for the biochemical identification of Enterobacteriaceae were used: the Api® 20E-System (bioMérieux® Deutschland GmbH), the Microbact™ 24E-System (OXOID GmbH Deutschland) and the Biolog-System (Biolog, Inc. USA).
The objective of this study was a comparative identification of 109 isolates of Enterobacteriaceae from 72 samples of mixed salads, packed in plastic bags or trays, as well as the assessment of the systems used for identification. The occurrence and frequency of detection of Enterobacter sakazakii in ready-to-eat salads had to be determined.
19 strains of the 109 isolates were identified as Enterobacter sakazakii with the Api 20E-System. However, these identifications could not be confirmed by the other systems. Subsequently, 18 of the 19 strains were examined by additional tests. The proof of pigment formation at 25 °C on tryptone-soy-agar did not bring any clarity. A final solution was achieved only with the BAX® Detection System (DuPont Qualicon) and the special test-kit for Enterobacter sakazakii, showing that none of the 18 strains belonged to Enterobacter sakazakii.
Interactions between the natural background microflora of shredded lettuce and Listeria innocua (in lieu of Listeria monocytogenes) were studied. The effect of increasing the initial size of indigenous populations (from 103 to 106–107 CFU g−1) was tested for its ability to reduce L. innocua growth on shredded lettuce. Co-culture experiments were performed in model media, where bacterial isolates from the indigenous microflora were tested for possible inhibitory effects. Varying the size of the indigenous populations had no effect on L. innocua survival or growth. However, interactions with individual species and mixed populations from lettuce did affect the survival and growth of L. innocua in model media. In general, mixed populations diminished L. innocua growth. In the undiluted lettuce medium, the various species tested individually either reduced or did not affect the growth of L. innocua. However, when the medium was diluted, some species extended the survival of L. innocua. Competition between the indigenous microflora and L. innocua resided mostly with the Enterobacter spp. and not with the pseudomonads. Enterobacter cloacae was particularly effective in reducing L. innocua growth. Lactic acid bacteria also reduced L. innocua growth in undiluted media. It is concluded that interactions with the natural background microflora may play an important role in determining the dynamics of Listeria populations on shredded lettuce.
Cowpea (Vigna unguiculata) paste used to prepare akara was collected from three Nigerian marketplaces and analyzed to determine populations and predominant types of bacteria, yeasts, and molds. Total aerobic microbial populations were initially high (106-108/g) and increased after 12 hr incubation at 30–32°C to ca. 109/g. Initial coliform populations were 102-104/g and decreased slightly between 6 hr and 12 hr; yeast/mold populations remained constant at 104-105/g. The average initial pH of 6.0 declined to 5.1 and 4.5 after 6 hr and 12 hr incubation, respectively. Predominant bacteria consisted of Enterobacter, Klebsiella, and Lactobacillus species; Candida species and Aspergillus mger were the predominant fungi isolated from the pastes.
Seven bacteria were isolated from spoiled tofu and identified as Bacillus sp. (S08), B. megaterium (S10), B. cereus (S17, S27, S28, S32), and Enterobacter sakazakii (S35). In a paper disc test with 6 chitosans and 6 chitosan oligomers of different molecular weights, chitosans showed higher antimicrobial activity than did chitosan oligomers at a 0.1% concentration. Results of inhibitory effects of 6 chitosans on growth of Bacillus sp. (S08) failed to detect viable cells after incubation for 24 hrs at 37 C, even at 0.02% concentration. With B. megaterium (S10) and B. cereus (S27), a 3 to 4 log cycle reduction was found in the chitosan-treated group. The growth of Enterobacter sakazakii (S35) was completely suppressed in the presence of 0.04% chitosan except for 1 chitosan product. The minimum inhibitory concentration of chitosan differed with products and isolates, ranging from 0.005% to above 0.1%.
ABSTRACTA new method was developed to rapidly monitor the Enterobacter sakazakii viable counts in reconstituted infant milk formula. Ninety six–well microtiter plates were used to perform a miniaturized 10-tube most-probable-number (MPN) enumeration protocol for E. sakazakii. This procedure is based on fluorogenic detection as a result of the growth and α-glucosidase production from E. sakazakii in broth containing 4-methylumbelliferyl-α-D-glucoside (OK medium). The correlation between E. sakazakii counts by conventional plating and microtiter MPN methods was highly agreeable in 0.2% peptone water (R2 = 0.94) and reconstituted infant milk formula (R2 = 0.91). From these results, the new rapid method can quickly monitor E. sakazakii counts in infant milk formula with a high correlation with the conventional plating method. The results indicate that the microtiter MPN method required much shorter incubation times (<10 h) than the conventional plating method (<24 h) and provided a more economical way to monitor E. sakazakii, using selective and differential medium in end point determinations.
The microbiological quality and heterotrophic bacterial populations of 26 thermal mineral water springs in Spain were studied. In most of the springs the number of viable aerobes was less than 103 cfu ml-1 and the number of sporulated bacteria less than 102 cfu ml-1. No significant differences were foundin the counts obtained with Plate Count Agar (PCA) and PCA diluted 1 : 10 and incubated at 22°, 37° and 45°C. Total coliforms were found in 14 springs, faecal streptococci in three, spores of sulphite-reducing Clostridium and Pseudomonas aeruginosa in seven. Neither Escherichia coli nor Staphylococcus aureus were found. A total of 665 strains were isolated and 85·4% of these identified; 329 were Gram-positive and 239 were Gram-negative. The genera most prevalent present in the springs were Pseudomonas (in 92.3%), Bacillus (65.4%), Enterobacter, Micrococcus and Staphylococcus (50%), Acinetobacter (42.3%), Arthrobacter (38.4%), Clostridium (27%) and Xanthomonas (23%). Gram-negative bacteria predominated in the mesothermal springs and Gram-positive bacteria in the hyper- and hypothermal springs. The most common Gram-negative rod species isolated were Ps. fluorescens, Ps. aeruginosa, Ps. putida, Ent. agglomerans, Ent. sakazakii, Ac. calcoaceticus and Ent. amnigenus.
Aims: To study the growth, thermotolerance and biofilm formation of the emergent pathogen Enterobacter sakazakii in infant formula milk (IFM).
Methods and Results: The temperature range, death kinetics and biofilm formation of E. sakazakii were determined using impedance microbiology and conventional methods. In IFM the organism grew as low as 6°C and optimally at 37–43°C. In faecal coliform tests, 23% of strains (n = 70) produced gas from lauryl sulphate broth (LSB) at 44°C after 48 h incubation. Three strains failed to grow in LSB at any of the temperatures. The D-value of cells suspended in IFM was determined between 54 and 62°C. The resultant z-value was 5·7°C. The organism was able to adhere and grow on latex, polycarbonate, silicon and to a lesser extent stainless steel.
Conclusions: Enterobacter sakazakii was able to grow at refrigeration temperatures and on infant-feeding equipment. The thermotolerance of the organism was similar to other Enterobacteriaceae and should be killed during standard pasteurization treatment.
Significance and Impact of the Study: Enterobacter sakazakii has been associated with infant meningitis through consumption of contaminated IFM. Enterobacter sakazakii is able to grow in IFM during storage at refrigeration temperatures and attach to infant-feeding equipment, which may become reservoirs of infection.
The antibacterial efficacy of the methanol extract of the aerial parts of Seseli libanotis by using disc diffusion assay against 107 strains from 52 bacterial species and the chemical composition of the essential
oil using GC/MS analysis were studied for the first time. The minimum inhibitory concentrations (MIC) of single compounds
were determined by the microbroth dilution method. Gas chromatography–mass spectrophotometry analyses allowed 13 compounds
to be determined; the main constituents of the essential oil of aerial parts of Seseli libanotis were trans-caryophyllene (20.39%), spathulenol (11.89%), (−)-caryophyllene oxide (11.47%), euasarone (10.66%) and delta-cadinene (9.16%).
The methanol extract of Seseli libanotis had a broad-spectrum antibacterial activity (>14mm inhibition zone in diameter) in particular against Bacillus cereus, Bacillus dipsauri, Bacillus lentimorbus, Bacillus sphaericus, Bacillus subtilis, Corynebacterium ammoniagenes,
Kocuria rosea, Neisseria subflava and Micrococcus lylae. These inhibitory effects are interesting in relation to the prevention of microbial contamination in foods.
Histamine and tyramine-forming micro-organisms were studied in 44 samples of different varieties of Spanish cheeses. Isolates (694) were obtained and tested for histidine and tyrosine-decarboxylase activity. Tyramine-forming activity was more frequent in the microorganisms isolated from most of the selective culture media except in those for enterobacterias and moulds. Tyramine-forming identified isolates were mostly Gram positive bacteria (enterococci and some lactic acid bacteria), whereas histamine was formed mainly by enterobacteria, but by some lactic acid bacteria as well. Most of the tyramine-forming lactic acid bacteria strains were isolated from cheeses containing the highest tyramine amounts. Some of these cheeses also showed the highest counts of enterococci. However, histamine-forming lactic acid bacteria were isolated from samples generally containing less than 100 mg/kg of histamine. The amount of tyramine found in the samples was significantly higher than histamine. One sample of Cabrales cheese (a mould-matured cheese) presented the highest amounts of both histamine (928 mg/kg) and tyramine (1807 mg/kg).
A new isolated bacterium, identified asEnterobacter sakazakii, produces a highly viscous, anionic heteropolysaccharide. Optimum conditions for production, preliminary results about the composition and also comparative rheological measurements with xanthan andA. viscosus-polysaccharide are described.
Sugar beet seeds disinfected with the carbofuran-containing insecticide adifur and the fungicide tachygaren by seed-producing firms were found to be abundantly populated with bacterial microflora. The bacteria isolated from the seed surface were identified to a species level. The selection of bacteria with respect to pesticide resistance may lead to the obtaining of agronomically useful bacterial strains.
The side effects of sulfonylurea and imidazolinone herbicides on plant-associated bacteria were investigated under pure culture conditions. Eighteen isolates, belonging to the genera Azotobacter, Azospirillum, Bacillus, Enterobacter Pseudomonas and Serratia, were exposed to four active compounds at concentration ranges similar to those in field soil. The sulfonylureas chlorsulfuron and rimsulfuron inhibited the growth of one of two Azospirillum and one of four Pseudomonas strains, while the imidazolinones imazapyr and imazethapyr were effective on two out of five Bacillus isolates. Surfactants in commercial formulation significantly enhanced rimsulfuron toxicity. With the exception of one Azospirillum strain, the differential tolerance of rhizobacteria to these herbicides was related to a differential sensitivity of their target, the activity of the first enzyme in branched-chain amino acid biosynthesis, acetohydroxyacid synthase (AHAS).
Greenhouse pot studies were performed to assess the occurrence of inhibitory effects on bacterial growth in field conditions. Maize seedlings were bacterized with the two strains which had shown in vitro sensitivity to sulfonylureas. Following the application to the soil of a commercial formulation of rimsulfuron at rates of 0, 0.2 and 0.5 μmol a.i. kg−1, significative differences in the resulting degree of bacterial root colonization were found. Moreover, upon co-inoculation with two strains, one tolerant and one sensitive to the herbicide, the presence of rimsulfuron significantly enhanced root occupancy by resistant bacteria, suggesting that shifts in the microbial community structure of crop rhizosphere could indeed result as a consequence of weed control by AHAS inhibitors.
The presence of Enterobacter sakazakii and other Enterobacteriaceae was surveyed in 82 powdered infant formula milk (IFM) and 404 other food products. The presence of Ent. sakazakii was detected using the conventional method (growth on violet red bile glucose agar plus yellow pigment production on TSA) and a new chromogenic medium (Druggan–Forsythe–Iversen agar, DFI) which enables results to be obtained 2 days earlier than the conventional method. Ent. sakazakii was isolated from 2/82 powdered IFM, 5/49 dried infant foods, 3/72 milk powder, 2/62 cheese products and various dry food ingredients, especially herbs and spices (40/122). Ent. sakazakii was isolated from 67 samples using the DFI medium, however only 19 of the samples were positive following the conventional method. The largest difference in isolation between the two methods was with dry food ingredients.Although Enterobacteriaceae were enumerated from one powdered IFM sample (Klebsiella ozaenae, 200 cfu/g), 7/82 had detectable Enterobacteriaceae after enrichment in EE broth. Using the ISO 6579 2002 method and immuno-magnetic separation technique no Salmonella serovars were isolated from powdered IFM, dried infant foods or milk samples. Therefore hygienic production of powdered IFM and milk production as monitored by control of Salmonella and enumeration of Enterobacteriaceae did not control Ent. sakazakii.
A total of 370 samples including lettuce, meats (beef, pork and chicken) and Spanish potato omelette from restaurants were studied to evaluate the incidence of Escherichia coli, E. coli O157:H7, Staphylococcus aureus, Salmonella spp., Yersinia enterocolitica, enterococci and some micro-organisms that can cause spoilage or can be used as indicators for food safety. Escherichia coli and enterococci were harboured with the highest incidence in lettuce, whereas incidence of Staphylococcus aureus was higher in meat than in the other foods studied. Enterobacter cloacae and Klebsiella pneumoniae were isolated from the three food groups. Chryseomonas luteola, Enterobacter sakazakii, Klebsiella ozaenae, Moraxella spp. and Serratia odorifera were isolated from lettuce, whilstProvidencia spp. were detected only in beef. Salmonella spp., E. coli O157:H7 or Yersinia enterocolitica were not isolated from any of the raw or ‘ready-to-eat’ samples analysed.
Enterobacter sakazakii infections in neonates cause bacteraemia, necrotizing enterocolitis (NEC) and infant meningitis. Where the source of an outbreak was traced to infant formula milk powder the levels of Enterobacteriacae were below the statutary limit. In order to determine whether a full risk assessment of E. sakazakii in IFM is required, a risk profile is necessary summarizing our knowledge to date. The risk profile presented here includes hazard identification, exposure assessment and hazard characterisation which are parts of a microbiological risk assessment (MRA), as well as risk management. In addition current detection methods are described.
A specific and senstive modification of the most-probable-number (MPN) technique by addition of 4-methylumbelliferyl-β-D-glucuronide (MUG) to both presumptive and confirmatory media was performed. The use of this modification allows the precise determination of Escherichia coli from marine samples (seawater, sediment and shellfish) within 7 days compared to 10–12 days required by using of the standard methodology. No false-positive isolates for fluorescence reaction have been observed, although one E. coli strain fluorescent-positive on agar was isolated from nonfluorescent tubes. Klebsiella pneumoniae was the species most frequently detected from tubes with gas and fluorescence production and typical morphology on mFC agar, but failed on nutrient-MUG agar.Using a unifactorial variance analysis of the mean, fluorescence in tube and on agar has been determined as the factor which allows the detection of E. coli presence in the three sample types with high accuracy. The incorporation of MUG to the selective broth in the confirmatory test has been shown to avoid false-positive results due to shellfish tissue β-glucuronidase activity.Specificity of the MUG-MPN method was high (Se = 0.94 and Sp = 0.79). The results obtained using laboratory strains showed that although E. coli, Citrobacter freundii, Enterobacter cloacae and K. pneumoniae produce acid and gas in MacConkey-purple broth, β-glucuronidase activity in this selective broth is specific to E. coli.
The fermentation of milk inoculated with bacteria was monitored amperometrically using an l-lactate biosensor and a flow-injection system. This approach was compared with the measurement of pH. The effects of Enterococcus faecalis, Bacillus coagulans, Enterobacter sakazakii, Staphylococcus aureus, and Bacillus sphericus were investigated. The most promising results were obtained for Staphylococcus aureus, whose fermentation increases milk pH.
This article has been retracted at the request of the chief editor and authors.Reason: The article, “A medium for the presumptive detection of Enterobacter sakazakii in infant formula,” Food Microbiology 21:527 (2004), was submitted for publication with a wrongful claim of authorship. The researchers who did the work, having written an internal report before departing from the institution where the work was done, were not consulted when a manuscript reporting their data was submitted for publication. Although listed as authors, they knew nothing about the submission or acceptance of the article until after its publication. Their disagreement with certain interpretations of the data underscores the fact that the manuscript was improperly submitted.
Antibiotic-resistant bacteria or their corresponding resistance determinants are known to spread from animals to humans via the food chain. We screened 20 vegetable foods for antibiotic-resistant coliform bacteria and enterococci. Isolates were directly selected on antibiotic-containing selective agar (color detection). Thirteen "common vegetables" (tomato, mushrooms, salad) possessed 10(4)-10(7) cfu/g vegetable of coliform bacteria including only few antibiotic-resistant variants (0-10(5) cfu/g). All seven sprout samples showed a some orders of magnitude higher contamination with coliform bacteria (10(7)-10(9) cfu/g) including a remarkable amount of resistant isolates (up to 10(7) cfu/g). Multiple resistances (up to 9) in single isolates were more common in sprout isolates. Resistant bacteria did not originate from sprout seeds. The most common genera among 92 isolates were: 25 Enterobacter spp. (19 E. cloacae), 22 Citrobacter spp. (8 C. freundii), and 21 Klebsiella spp. (9 K. pneumoniae). Most common resistance phenotypes were: tetracycline (43%), streptomycin (37%), kanamycin (26%), chloramphenicol (29%), co-trimoxazol (9%), and gentamicin (4%). The four gentamicin-resistant isolates were investigated in molecular details. Only three (chloramphenicol) resistant, typical plant-associated enterococci were isolated from overnight enrichment cultures. In conclusion, a contribution of sprouts contaminated with multiresistant, Gram-negative enterobacteria to a common gene pool among human commensal and pathogenic bacteria cannot be excluded.
Minimum inhibitory concentrations were determined for selected antimicrobial agents against 872 bacteria isolated from intramammary infections in heifers in New Zealand (n = 401) and Denmark (n = 471). These values were reported in micrograms per milliliters. Antimicrobial agents tested against isolates from New Zealand were penicillin, cloxacillin, cephapirin, ceftiofur, novobiocin, enrofloxacin, erythromycin, and pirlimycin. The minimum inhibitory concentrations that inhibit 90% of the strains tested for these antimicrobial agents with Staphylococcus aureus were 4.0, 0.5, 0.5, 2.0, 1.0, 0.25, 0.5, and 1.0, respectively. The minimum inhibitory concentration values that inhibit 90% of the strains tested against the Staphylococcus spp. ranged from 0.5 to 1.0 for all antimicrobics. The minimum inhibitory concentrations against streptococci were < or = 0.06, 0.5, 0.13, 0.13, 4.0, 1.0, 0.13, and < or = 0.06, respectively. Antimicrobial agents tested against isolates from Denmark included penicillin, ampicillin, oxacillin, cephalothin, ceftiofur, penicillin plus novobiocin, erythromycin, and pirlimycin. Against S. aureus, the minimum inhibitory concentrations were 0.13, 0.5, 0.5, 0.5, 1.0, 0.25, 0.5, and 0.5, respectively. The minimum inhibitory concentrations against Staphylococcus spp. were 0.25, 0.25, 0.5, 0.5, 1.0, < or = 0.06, 0.13, 1.0, and 0.5, respectively. The minimum inhibitory concentrations against the streptococci were < or = 0.06, 0.13, 0.5, 0.5, 1.0, < or = 0.06, 0.13, 0.5, and 0.5, respectively. Minimum inhibitory concentration values for staphylococci from New Zealand and Denmark were similar to values reported for US isolates. Streptococci from New Zealand and Denmark had lower minimum inhibitory concentration values than did US isolates. Only ceftiofur and enrofloxacin were active against the Gram-negative bacilli.
Thesis (M.A.)--Indiana University, 1989. Vita. Includes bibliographical references (leaves 55-56).
Two unrelated hospital outbreaks of Enterobacter sakazakii, involving meningitis, bacteremia, and colonization of neonates, were investigated. In each of these outbreaks, E. sakazakii was isolated from both patients and dried infant formula. In previous outbreaks, the source and mode of transmission of E. sakazakii in neonatal infections was not determined. In this study, we used a combination of typing methods (plasmid analysis, antibiograms, chromosomal restriction endonuclease analysis, ribotyping, and multilocus enzyme electrophoresis) to evaluate the isolates from each outbreak as to their relatedness. The typing results differed among outbreaks, but in each one, patient and formula isolates shared the same typing pattern. The only exceptions were disk antibiograms, which often varied among colonies selected from each of the isolates. Plasmid analysis, chromosomal restriction endonuclease analysis, ribotyping, and multilocus enzyme electrophoresis all were effective as epidemiological typing methods for E. sakazakii, especially when used in combination. By using this typing scheme, we have confirmed that E. sakazakii from intrinsically contaminated dried infant formula was the source of neonatal infection.