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Exploring the potential reservoirs of Pseudomonas spp. bacteria at meat processing factories and poultry farms
Abstract
One of the microorganisms that cause spoilage of meat during its storage is the bacteria Pseudomonas. To prevent contamination of the finished products with these bacteria, it is important to find the places at the enterprise where they aggregate. Within the framework of this study, the objects and premises of the production facilities at meat processing factories and poultry farms were explored to detect their contamination with bacteria of Pseudomonas spp. The potential reservoirs of those bacteria were defined at these plants. In addition, the species diversity of Pseudomonas was established at the production facilities environment at the enterprises. 27 production facilities environments (structures, equipment, package containers) were examined for the presence of bacteria with the method of washings. The samples were examined to detect Pseudomonas bacteria, with their subsequent identification with the method of time-of-flight mass spectrometry MALDI-Tof-MS. 487 strains of bacteria of the genus Pseudomonas were isolated, which strains are represented by 47 species. As a result of the study it was found that all 27 production facilities were contaminated with various species of Pseudomonas. From two to fourteen species of Pseudomonas bacteria were detected at all facilities. 12 facilities of the enterprise for slaughter and processing of broiler chickens were contaminated with Pseudomonas gessardii. Pseudomonas bacteria spp. (identification is traced down only to its genus) were found at 10 objects. Pseudomonas tolaasii and Pseudomonas brenneri were found at 9 and 8 objects, respectively. The surfaces of 6 objects demonstrated contamination with Pseudomonas chlororaphis ssp chlororaphis and Pseudomonas koreensis. Other Pseudomonas species were found at 1–5 sites. Pseudomonas fluorescens were detected at 8 pork processing plant sites, Pseudomonas gessardii were found at 5 sites. 4 sites were contaminated with Pseudomonas chlororaphis ssp. chlororaphis and Pseudomonas koreensis, 3 objects contained Pseudomonas tolaasii, Pseudomonas spp., Pseudomonas rhodesiae, Pseudomonas libanensis and Pseudomonas extremorientalis. The remaining species of Pseudomonas were found at one or two sites in the territory of the pork processing plant. It was found that all production environment sites, regardless of their distance from the raw materials and the finished products, were contaminated with Pseudomonas bacteria. At the same time, the sites that had no contact with the food products showed wider diversity of Pseudomonas species than in the places where the contact took place. Thus, all the explored objects of the production environment at the pork processing enterprises and the facilities for slaughter and processing of broiler chickens are the potential reservoirs of Pseudomonas bacteria.
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Pseudomonas are among psychrophilic bacteria and have been reported in various studies as the predominant spoilage bacterial in slaughter carcass. The aim of this study was to track Pseudomonas spp. from different stages of the slaughter line, slaughterhouse environment and equipment and drop in the packaging. Characteristics such as pigment production, movement pattern and biofilm formation capability of the isolates were also determined. For this purpose, 108 samples were sampled from three industrial poultry slaughterhouses in Tabriz. According to the results, the highest contamination was detected in the samples of the floors, abdominal cavity of carcass and drip samples, respectively. The lowest contamination was observed in the samples related to drinking water, live chicken breast swab and scalder, respectively. The average movement of the Swimming type was significantly (p ≤ 0.001) higher than the two types of Swarming and Twitching movements. And in terms of pigment production, the dominant color was green. Moreover, most of the isolates were able to form biofilms and about 30% of the isolates had moderate and strong ability to produce biofilms. In conclusion, most of the Pseudomonas spp. contamination occurs through different parts of the slaughter line and also the equipment and environment of the slaughterhouse. Due to the biofilm production capacity of Pseudomonas isolates, the issue of proper and more effective washing and disinfection of the slaughter line and equipment is of particular importance. Conflict of interest: None declared.
Pseudomonas aeruginosa is one of the leading invasive agents of human pulmonary infection, especially in patients with compromised immunity. Prior studies have used various in vitro models to establish P. aeruginosa infection and to analyze transcriptomic profiles of either the host or pathogen, and yet how much those works are relevant to the genuine human airway still raises doubts. In this study, we cultured and differentiated human airway organoids (HAOs) that recapitulate, to a large extent, the histological and physiological features of the native human mucociliary epithelium. HAOs were then employed as a host model to monitor P. aeruginosa biofilm development. Through dual-species transcriptome sequencing (RNA-seq) analyses, we found that quorum sensing (QS) and several associated protein secretion systems were significantly upregulated in HAO-associated bacteria. Cocultures of HAOs and QS-defective mutants further validated the role of QS in the maintenance of a robust biofilm and disruption of host tissue. Simultaneously, the expression magnitude of multiple inflammation-associated signaling pathways was higher in the QS mutant-infected HAOs, suggesting that QS promotes immune evasion at the transcriptional level. Altogether, modeling infection of HAOs by P. aeruginosa captured several crucial facets in host responses and bacterial pathogenesis, with QS being the most dominant virulence pathway showing profound effects on both bacterial biofilm and host immune responses. Our results revealed that HAOs are an optimal model for studying the interaction between the airway epithelium and bacterial pathogens. IMPORTANCE Human airway organoids (HAOs) are an organotypic model of human airway mucociliary epithelium. The HAOs can closely resemble their origin organ in terms of epithelium architecture and physiological function. Accumulating studies have revealed the great values of the HAO cultures in host-pathogen interaction research. In this study, HAOs were used as a host model to grow Pseudomonas aeruginosa biofilm, which is one of the most common pathogens found in pulmonary infection cases. Dual transcriptome sequencing (RNA-seq) analyses showed that the cocultures have changed the gene expression pattern of both sides significantly and simultaneously. Bacterial quorum sensing (QS), the most upregulated pathway, contributed greatly to biofilm formation, disruption of barrier function, and subversion of host immune responses. Our study therefore provides a global insight into the transcriptomic responses of both P. aeruginosa and human airway epithelium.
The purpose of this study was to investigate the abundance and distribution of psychrophilic microorganisms associated with spoilage in beef slaughterhouse environments after cleaning. The processing lines and equipment used in slaughtering and boning were swabbed, and the microbial count was determined using a TSA and MRS medium and Chromocult® Coliform agar incubated at 15ºC and 37ºC, respectively. As a result, the brisket saw (handle side) and trolley hook were the most heavily contaminated with microorganisms, with each having a microbial adhesion rate of 66.7%. The microbial adhesion rates of the apron and milling cutter (edge side) were 50%, respectively, and those of the foot cutter (edge and handle side), splitting saw (edge side), and knife (handle side) were 33.3%, respectively. Next, four colonies were randomly isolated from the petri dish used for the bacterial count measurement to identify the predominant microbial species of the microorganisms attached to each equipment. As a result of Sanger sequencing analysis, yeasts such as Candida zeylanoides and Rhodotorula sp. and bacteria including Pseudomonas sp. and Rhodococcus sp. were identified from the equipment used in the slaughtering line, and it was assumed that these microorganisms were of environmental origin. In contrast, only Pseudomonas sp. and Candida zeylanoides were isolated from the boning line. Despite the use of cleaning operations, this study identified some equipment was contaminated with microorganisms. Since this equipment frequently comes into direct contact with the carcass, it is critical to thoroughly remove the microorganisms through accurate cleaning to prevent the spread of microbial contamination on the carcasses.
Safe and hygienic water distribution is essential for maintaining product quality and safety. It is known that biofilms alter the appearance and microbial quality of water along the distribution chain. Yet, biofilms in water hoses throughout the food processing environment have not been investigated in detail. Here, microbial communities from water hoses and other environmental sites in contact with water, in addition to the source water itself, were studied in the meat processing environment. Biofilms were present in all water hoses as determined by the presence of bacterial DNA and biofilm matrix components (carbohydrates, extracellular DNA, and proteins). The microbial community of the biofilms was dominated by Proteobacteria , represented mainly by Comamonadaceae and Pseudoxanthomonas . Moreover, genera that are associated with an intracellular lifestyle (e.g., Neochlamydia and Legionella ) were present. Overall, the microbial community of biofilms was less diverse than the water microbial community, while those from the different sample sites were distinct from each other. Indeed, only a few phyla were shared between the water hose biofilm and the source water or associated environmental samples. This study provides first insights towards understanding the microbiota of water hose biofilms in the food processing environment.
Background
The microorganisms that inhabit food processing environments (FPE) can strongly influence the associated food quality and safety. In particular, the possibility that FPE may act as a reservoir of antibiotic-resistant microorganisms, and a hotspot for the transmission of antibiotic resistance genes (ARGs) is a concern in meat processing plants. Here, we monitor microbial succession and resistome dynamics relating to FPE through a detailed analysis of a newly opened pork cutting plant over 1.5 years of activity.
Results
We identified a relatively restricted principal microbiota dominated by Pseudomonas during the first 2 months, while a higher taxonomic diversity, an increased representation of other taxa (e.g., Acinetobacter, Psychrobacter), and a certain degree of microbiome specialization on different surfaces was recorded later on. An increase in total abundance, alpha diversity, and β-dispersion of ARGs, which were predominantly assigned to Acinetobacter and associated with resistance to certain antimicrobials frequently used on pig farms of the region, was detected over time. Moreover, a sharp increase in the occurrence of extended-spectrum β-lactamase-producing Enterobacteriaceae and vancomycin-resistant Enterococcaceae was observed when cutting activities started. ARGs associated with resistance to β-lactams, tetracyclines, aminoglycosides, and sulphonamides frequently co-occurred, and mobile genetic elements (i.e., plasmids, integrons) and lateral gene transfer events were mainly detected at the later sampling times in drains.
Conclusions
The observations made suggest that pig carcasses were a source of resistant bacteria that then colonized FPE and that drains, together with some food-contact surfaces, such as equipment and table surfaces, represented a reservoir for the spread of ARGs in the meat processing facility.
9rXSuE523Ksyv5BLWfnbxPVideo Abstract
Zoonotic diseases are diseases that are transmitted from animals to humans and vice versa. Pseudomonas aeruginosa (P. aeruginosa) is a pathogen with zoonotic nature. Commercial poultry could be infected with P. aeruginosa, especially at young ages with great losses. Infection of embryos with P. aeruginosa induced death in the shell, while infection of chicks led to septicemia, respiratory and enteric infections, and high mortality. Humans are also highly susceptible to P. aeruginosa infection, and the disease is associated with severe lung damage, especially in immunocompromised patients. Chicken carcass and related poultry retail products play an important role in the transmission of P. aeruginosa to humans, especially after processing in abattoirs. Treatment of P. aeruginosa infection is extremely difficult due to continuous development of antibiotic resistance. The transfer of antibiotic-resistant genes from poultry products to humans creates an additional public health problem. Accordingly, this study focused on avian pseudomonad, especially P. aeruginosa, with respect to infection of poultry, transmission to humans, and treatment and antibiotic resistance.
Driven in part by its metabolic versatility, high intrinsic antibiotic resistance, and a large repertoire of virulence factors, Pseudomonas aeruginosa is expertly adapted to thrive in a wide variety of environments, and in the process, making it a notorious opportunistic pathogen. Apart from the extensively studied chronic infection in the lungs of people with cystic fibrosis (CF), P. aeruginosa also causes multiple serious infections encompassing essentially all organs of the human body, among others, lung infection in patients with chronic obstructive pulmonary disease, primary ciliary dyskinesia and ventilator-associated pneumonia; bacteremia and sepsis; soft tissue infection in burns, open wounds and postsurgery patients; urinary tract infection; diabetic foot ulcers; chronic suppurative otitis media and otitis externa; and keratitis associated with extended contact lens use. Although well characterized in the context of CF, pathogenic processes mediated by various P. aeruginosa virulence factors in other organ systems remain poorly understood. In this review, we use an organ system-based approach to provide a synopsis of disease mechanisms exerted by P. aeruginosa virulence determinants that contribute to its success as a versatile pathogen.
Chickens play host to a diverse community of microorganisms which constitute the microflora of the live bird. Factors such as diet, genetics and immune system activity affect this complex population within the bird, while external influences including weather and exposure to other animals alter the development of the microbiome. Bacteria from these settings including Campylobacter and Salmonella play an important role in the quality and safety of end-products from these birds. Further steps, including washing and chilling, within the production cycle aim to control the proliferation of these microbes as well as those which cause product spoilage. These steps impose specific selective pressures upon the microflora of the meat product. Within the next decade, it is forecast that poultry meat, particularly chicken will become the most consumed meat globally. However, as poultry meat is a frequently cited reservoir of zoonotic disease, understanding the development of its microflora is key to controlling the proliferation of important spoilage and pathogenic bacterial groups present on the bird. Whilst several excellent reviews exist detailing the microbiome of poultry during primary production, others focus on fate of important poultry pathogens such as Campylobacter and Salmonella spp. At farm and retail level, and yet others describe the evolution of spoilage microbes during spoilage. This review seeks to provide the poultry industry and research scientists unfamiliar with food technology process with a holistic overview of the key changes to the microflora of broiler chickens at each stage of the production and retail cycle.
Psychrotolerant Pseudomonas spp. are among the most common spoilage agents in fresh, soft and semi-soft cheeses; therefore, hurdles inhibiting their growth are in strong demand by producers. This study aimed to establish Minimal Inhibiting Concentrations (MICs) of lactic and acetic acid towards P. fluorescens and to evaluate the efficacy of a cheese surface treatment with these two organic acids. MICs were determined in Brain Heart Infusion broth at 30 °C: the inhibition was achieved at a concentration of 49.96 mM and 44.40 mM of acetic and lactic acid, respectively. Two series of inhibition tests were performed on fresh “Primo sale” cheese, inoculated with P. brenneri MGM3, then dipped into different acid solutions (acetic acid: 49.96, 99.92 and 149.88 mM; lactic acid: 44.40, 88.80 and 133.20 mM) and stored at 6 °C. P. brenneri MGM3 were enumerated, including a control series. A significantly lower growth was revealed at the highest concentrations tested, both for acetic (p < 0.01) and lactic acid (p < 0.05) if compared to control samples. A conditioning of “Primo sale” surface with organic acid solutions could be a useful hurdle for Pseudomonas inhibition and shelf-life extension; it should be applied in combination with other mild interventions to fight spoilage and maintain the original product characteristics.
The Pseudomonas syringae complex comprises different genetic groups that include strains from both agricultural and environmental habitats. This complex group has been used for decades as a “hodgepodge,” including many taxonomically related species. More than 60 pathovars of P. syringae have been described based on distinct host ranges and disease symptoms they cause. These pathovars cause disease relying on an array of virulence mechanisms. However, P. syringae pv. syringae (Pss) is the most polyphagous bacterium in the P. syringae complex, based on its wide host range, that primarily affects woody and herbaceous host plants. In early 1990s, bacterial apical necrosis (BAN) of mango trees, a critical disease elicited by Pss in Southern Spain was described for the first time. Pss exhibits important epiphytic traits and virulence factors, which may promote its survival and pathogenicity in mango trees and in other plant hosts. Over more than two decades, Pss strains isolated from mango trees have been comprehensively investigated to elucidate the mechanisms that governs their epiphytic and pathogenic lifestyles. In particular, the vast majority of Pss strains isolated from mango trees produce an antimetabolite toxin, called mangotoxin, whose leading role in virulence has been clearly demonstrated. Moreover, phenotypic, genetic and phylogenetic approaches support that Pss strains producers of BAN symptoms on mango trees all belong to a single phylotype within phylogroup 2, are adapted to the mango host, and produce mangotoxin. Remarkably, a genome sequencing project of the Pss model strain UMAF0158 revealed the presence of other factors that may play major roles in its different lifestyles, such as the presence of two different type III secretion systems, two type VI secretion systems and an operon for cellulose biosynthesis. The role of cellulose in increasing mango leaf colonization and biofilm formation, and impairing virulence of Pss, suggests that cellulose may play a pivotal role with regards to the balance of its different lifestyles. In addition, 62-kb plasmids belonging to the pPT23A-family of plasmids (PFPs) have been strongly associated with Pss strains that inhabit mango trees. Further, complete sequence and comparative genomic analyses revealed major roles of PFPs in detoxification of copper compounds and ultraviolet radiation resistance, both improving the epiphytic lifestyle of Pss on mango surfaces. Hence, in this review we summarize the research that has been conducted on Pss by our research group to elucidate the molecular mechanisms that underpin the epiphytic and pathogenic lifestyle on mango trees. Finally, future directions in this particular plant–pathogen story are discussed.
Safe drinking water is delivered to the consumer through kilometres of pipes. These pipes are lined with biofilm, which is thought to affect water quality by releasing bacteria into the drinking water. This study describes the number of cells released from this biofilm, their cellular characteristics, and their identity as they shaped a drinking water microbiome. Installation of ultrafiltration (UF) at full scale in Varberg, Sweden reduced the total cell count to 1.5 × 103 ± 0.5 × 103 cells mL−1 in water leaving the treatment plant. This removed a limitation of both flow cytometry and 16S rRNA amplicon sequencing, which have difficulties in resolving small changes against a high background cell count. Following installation, 58% of the bacteria in the distributed water originated from the pipe biofilm, in contrast to before, when 99.5% of the cells originated from the treatment plant, showing that UF shifts the origin of the drinking water microbiome. The number of bacteria released from the biofilm into the distributed water was 2.1 × 103 ± 1.3 × 103 cells mL−1 and the percentage of HNA (high nucleic acid) content bacteria and intact cells increased as it moved through the distribution system. DESeq2 analysis of 16S rRNA amplicon reads showed increases in 29 operational taxonomic units (OTUs), including genera identified as Sphingomonas, Nitrospira, Mycobacterium, and Hyphomicrobium. This study demonstrated that, due to the installation of UF, the bacteria entering a drinking water microbiome from a pipe biofilm could be both quantitated and described.
This study investigated the psychrotrophic bacteria isolated from chicken meat to characterize their microbial composition during refrigerated storage. The bacterial community was identified by the Illumina MiSeq method based on bacterial DNA extracted from spoiled chicken meat. Molecular identification of the isolated psychrotrophic bacteria was carried out using 16S rDNA sequencing and their putrefactive potential was investigated by the growth at low temperature as well as their proteolytic activities in chicken meat. From the Illumina sequencing, a total of 187,671 reads were obtained from 12 chicken samples. Regardless of the type of chicken meat (i.e., whole meat and chicken breast) and storage temperatures (4°C and 10°C), Pseudomonas weihenstephanensis and Pseudomonas congelans were the most prominent bacterial species. Serratia spp. and Acinetobacter spp. were prominent in chicken breast and whole chicken meat, respectively. The 118 isolated strains of psychrotrophic bacteria comprised Pseudomonas spp. (58.48%), Serratia spp. (10.17%), and Morganella spp. (6.78%). All isolates grew well at 10°C and they induced different proteolytic activities depending on the species and strains. Parallel analysis of the next generation sequencing and culture dependent approach provides in-depth information on the biodiversity of the spoilage microbiota in chicken meat. Further study is needed to develop better preservation methods against these spoilage bacteria.
Surface hygiene is commonly measured as a part of the quality system of food processing plants, but as the bacteria present are commonly not identified, their roles for food quality and safety are not known. Here, we review the identity of residential bacteria and characteristics relevant for survival and growth in the food industry along with potential implications for food safety and quality. Sampling after cleaning and disinfection increases the likelihood of targeting residential bacteria. The increasing use of sequencing technologies to identify bacteria has improved knowledge about the bacteria present in food premises. Overall, nonpathogenic Gram-negative bacteria, especially Pseudomonas spp., followed by Enterobacteriaceae and Acinetobacter spp. dominate on food processing surfaces. Pseudomonas spp. persistence is likely due to growth at low temperatures, biofilm formation, tolerance to biocides, and low growth requirements. Gram-positive bacteria are most frequently found in dairies and in dry production environments. The residential bacteria may end up in the final products through cross-contamination and may affect food quality. Such effects can be negative and lead to spoilage, but the bacteria may also contribute positively, as through spontaneous fermentation. Pathogenic bacteria present in food processing environments may interact with residential bacteria, resulting in both inhibitory and stimulatory effects on pathogens in multispecies biofilms. The residential bacterial population, or bacteriota, does not seem to be an important source for the transfer of antibiotic resistance genes to humans, but more knowledge is needed to verify this. If residential bacteria occur in high numbers, they may influence processes such as membrane filtration and corrosion.
Aims:
To identify production and processing practices that might reduce Campylobacter numbers contaminating chicken broiler carcasses.
Methods and results:
Numbers of campylobacters were determined on carcass neck skins after processing or in broiler house litter samples. Supplementary information that described farm layouts, farming conditions for individual flocks, the slaughterhouse layouts and operating conditions inside plants was collected, matched with each Campylobacter test result. Statistical models predicting the numbers of campylobacters on neck skins and in litter were constructed. Carcass microbial contamination was more strongly influenced by on-farm production practices compared with slaughterhouse activities. We observed correlations between the chilling, washing and de-feathering stages of processing and numbers of campylobacters on carcasses. There were factors on farm that also correlated with numbers of campylobacters in litter. These included bird gender, the exclusion of dogs from houses, beetle presence in the house litter and the materials used to construct the house frame.
Conclusions:
Changes to farming practices have greater potential for reducing chicken carcass microbial contamination compared with processing interventions.
Significance and impact of the study:
Routine commercial practices were identified that were correlated with lowered numbers of campylobacters. Consequently, these practices are likely to be both cost-effective and suitable for adoption into established farms and commercial processing. This article is protected by copyright. All rights reserved.
The purpose of this study was to 1) identify microbial compositional changes on chicken carcasses during processing, 2) determine the antimicrobial efficacy of peracetic acid (PAA) and Amplon (blend of sulfuric acid and sodium sulfate) at a poultry processing pilot plant scale, and 3) compare microbial communities between chicken carcass rinsates and recovered bacteria from media. Birds were collected from each processing step and rinsates were applied to estimate aerobic plate count (APC) and Campylobacter as well as Salmonella prevalence. Microbiome sequencing was utilized to identify microbial population changes over processing and antimicrobial treatments. Only the PAA treatment exhibited significant reduction of APC at the post chilling step while both Amplon and PAA yielded detectable Campylobacter reductions at all steps. Based on microbiome sequencing, Firmicutes were the predominant bacterial group at the phyla level with over 50% frequency in all steps while the relative abundance of Proteobacteria decreased as processing progressed. Overall microbiota between rinsate and APC plate microbial populations revealed generally similar patterns at the phyla level but they were different at the genus level. Both antimicrobials appeared to be effective on reducing problematic bacteria and microbiome can be utilized to identify optimal indicator microorganisms for enhancing product quality.
Refrigerated food processing facilities are specific man-made niches likely to harbor cold-tolerant bacteria. To characterize this type of microbiota and study the link between processing plant and product microbiomes, we followed and compared microbiota associated with the raw materials and processing stages of a vacuum-packaged, cooked sausage product affected by a prolonged quality fluctuation with occasional spoilage manifestations during shelf life. A total of 195 samples were subjected to culturing and amplicon sequence analyses. Abundant mesophilic psychrotrophs were detected within the microbiomes throughout the different compartments of the production plant environment. However, each of the main genera of food safety and quality interest, e.g. Leuconostoc, Brochothrix and Yersinia, had their own characteristic pattern of contamination. Bacteria from the genus Leuconostoc, commonly causing spoilage of cold-stored, modified-atmosphere-packaged foods, were detected in high abundance (up to >98%) in the sausages studied. The same operational taxonomic units (OTUs) were, however, detected in lower abundances in raw meat and emulsion (average relative abundance 2%±5%), as well as on the processing plant surfaces (<4%). A completely different abundance profile was found for OTUs phylogenetically close to the species Yersinia pseudotuberculosis. These OTUs were detected in high abundance (up to 28%) on the processing plant surfaces, but to a lesser extent (<1%) in raw meat, sausage emulsion and sausages. The fact that Yersinia-like OTUs were found on the surfaces of a high-hygiene packaging compartment raises food safety concerns related to their resilient existence on surfaces.
Copyright © 2015, American Society for Microbiology. All Rights Reserved.
The microbiological quality of beef and meat products is strongly influenced by the conditions of hygiene prevailing during their production and handling. Without proper hygienic control, the environment in slaughterhouses and butcher shops can act as an important source of microbiological contamination. To identify the main points of microbiological contamination in the beef processing chain, 443 samples of equipment, installations and products were collected from I I establishments (I slaughterhouse and 10 butcher shops) located in the state of Parana, Brazil. The microbiological quality of all the samples was evaluated using Petri dishes to obtain counts of mesophilic aerobes (AC), total coliforms, Escherichia colt (EC), yeasts and molds (YM). The main contamination points identified in butcher shops, in decreasing order, were stainless steel boxes, beef tenderizers, grinders, knives, mixers, sausage stuffers, plastic boxes, floors and drains. In the slaughterhouse, these points were sausage stuffers, platforms, floors and drains. The most severely contaminated products were fresh sausages and ground beef. This information about the main points of microbiological contamination in the beef processing chain is expected to aid professionals responsible for hygiene in similar establishments to set up proper hygienic procedures to prevent or reduce microbiological contamination of beef and meat products.
Biofilms are described as a matrix of microorganisms which have adhered to and colonized a surface. Once formed, biofilms are difficult to remove and may be a source of contamination in food-processing environments. In this study, stainless-steel chips were fixed to surfaces adjacent to food-contact surfaces and cast-iron chips were suspended in the floor drains of four meat-processing plants. Biofilm formation was quantified by staining the attached cells and viewing them under epifluorescence microscopy. The stainless-steel and cast-iron chips removed from the plant environment showed some attached microorganisms. Floor drains appeared to provide an excellent environment for the formation of biofilms. Pseudomonas, Klebsiella, Aeromonas, and Hafnia species were identified as gram-negative microorganisms associated with the test surfaces.
Understanding the bacterial community profile through poultry processing could help the industry to produce better poultry products. In this study, 10 chicken carcasses were randomly sampled from before and after scalding, before and after immersion chilling, and after air chilling each through a modern commercial processing line, along with the contents of 10 caeca. The sampled processing line effectively reduced the bacterial counts by > 4.6 Log10 CFU/ml for each of Total Viable Counts, Escherichia coli and Campylobacter. However, the metagenomics results suggested that Lactobacillus, Staphylococcus and unclassified Lachnospiraceae persisted at all sampling stages. Pseudomonas, Paeniglutamicibacter, Chryseobacterium and Pseudarthrobacter comprised 47.2% in the bacterial community on samples after air chilling compared to 0.3% on samples after immersion chilling, whereas TVCs were the same. Overall, the current interventions of the investigated poultry processing line were unable to eliminate persistence of certain foodborne pathogens, despite a significant reduction of the overall bacterial counts. Chilling is an important controlling point in contamination/cross-contamination, particularly extended air chilling. Lastly, the large presence of Pseudomonas on chickens after air chilling may lead to downstream spoilage related issues, which needs more investigation to explore quantitatively the effect on the shelf life of poultry products.
Microbial spoilage of meat during chilled aerobic storage causes significant financial losses to the industry. Even with modern day preservation techniques, spoilage remains an unsolved problem. Spoilage of meat is a complex process that involves the activity of endogenous enzymes and microorganisms. Psychrotrophic Pseudomonas species are the key microorganisms that cause spoilage in aerobically stored chilled meat. Spoilage pseudomonads are highly robust and able to withstand stressful environmental conditions that would otherwise inhibit the growth of other spoilage organisms. In order to implement efficient control measures, and to minimize spoilage, a thorough understanding of the characteristics of spoilage pseudomonads is essential. This review focuses on the spoilage process and the key metabolic attributes of the main psychrotrophic spoilage Pseudomonas species to explain their predominance on meat over other psychrotrophic bacteria. This review also highlights less studied, but important, characteristics of psychrotrophic pseudomonads such as biofilm formation and quorum sensing in the context of meat spoilage. The importance of the use of model systems that are closely applicable to the food industry is also discussed in detail.
The major aim of the study was to establish the routes via which spoilage associated psychrotrophic bacteria contaminate poultry products at a large processing plant located in Belgium. Environmental samples were collected consisting of samples of air and swabs of food contact surfaces. Product samples were also collected consisting of modified atmosphere packaged (MAP) chicken wings and legs, which were analyzed microbiologically on the same day they were produced as well as after their sell-by date. Psychrotrophic bacteria from these samples were subsequently clustered and identified by means of MALDI-TOF MS and 16S rRNA gene sequencing. Carnobacterium maltaromaticum was determined to dominate the spoilage flora of both wings and legs. Other psychrotrophic bacteria able to grow on MRS which were identified on expired wings and legs included Carnobacterium divergens, Brocothrix thermosphacta, Lactobacillus curvatus, and Lactobacillus brevis. These were determined to arise from food contact surfaces such as cutting blades, leg hooks, Ertalon and polyurethane conveyor belts, working tables, and the hands of the operators. Importantly, it was determined that cleaning and disinfection was largely inadequate. Air was also determined to be an important vector of psychrotrophic bacteria in the processing environment, potentially contaminating the products directly or indirectly.
Microorganisms play an important role in the spoilage of chilled chicken. In this study, a total of 53 isolates, belonging to 7 species of 3 genera, were isolated using a selective medium based on the capacity to spoil chicken juice. Four isolates, namely Aeromonas salmonicida 35, Pseudomonas fluorescens H5, Pseudomonas fragi H8 and Serratia liquefaciens 17, were further characterized to assess their proteolytic activities in vitro using meat protein extracts and to evaluate their spoilage potential in situ. The in vitro studies showed that A. salmonicida 35 displayed the strongest proteolytic activity against both sarcoplasmic and myofibrillar proteins. However, the major spoilage isolate in situ was P. fragi H8, which exhibited a fast growth rate, slime formation and increased pH and total volatile basic nitrogen (TVBN) on chicken breast fillets. The relative amounts of volatile organic compounds (VOCs) originating from the microorganisms, including alcohols, aldehydes, ketones and several sulfur compounds, increased during storage. In sum, this study demonstrated the characteristics of 4 potential spoilage bacteria on chilled yellow-feather chicken and provides a simple and convenient method to assess spoilage bacteria during quality management.
Listeria monocytogenes is a major concern for the meat processing industry because many listeriosis outbreaks have been linked to meat product consumption. The aim of this study was to elucidate L. monocytogenes diversity and distribution across different Spanish meat processing plants. L. monocytogenes isolates (N = 106) collected from food contact surfaces of meat processing plants and meat products were serotyped and then characterised by multilocus sequence typing (MLST). The isolates were serotyped as 1/2a (36.8%), 1/2c (34%), 1/2b (17.9%) and 4b (11.3%). MLST identified ST9 as the most predominant allelic profile (33% of isolates) followed by ST121 (16%), both of which were detected from several processing plants and meat products sampled in different years, suggesting that those STs are highly adapted to the meat processing environment. Food contact surfaces during processing were established as an important source of L. monocytogenes in meat products because the same STs were obtained in isolates recovered from surfaces and products. L. monocytogenes was recovered after cleaning and disinfection procedures in two processing plants, highlighting the importance of thorough cleaning and disinfection procedures. Epidemic clone (EC) marker ECI was identified in 8.5%, ECIII was identified in 2.8%, and ECV was identified in 7.5% of the 106 isolates. Furthermore, a selection of presumably unrelated ST9 isolates was analysed by multi-virulence-locus sequence typing (MVLST). Most ST9 isolates had the same virulence type (VT11), confirming the clonal origin of ST9 isolates; however, one ST9 isolate was assigned to a new VT (VT95). Consequently, MLST is a reliable tool for identification of contamination routes and niches in processing plants, and MVLST clearly differentiates EC strains, which both contribute to the improvement of L. monocytogenes control programs in the meat industry.
Ninety three isolates of Brochothrix spp. from raw meat were analysed by PCR-based molecular fingerprinting and identified by species-specific Real-Time (RTi)-PCR and 16S rRNA gene sequencing. Thirty three strains were characterized for their growth capability in sarcoplasmic extract with and without glucose, for in vitro lipolytic activity, in vitro and in situ (in beef) proteolytic activity and for amino-decarboxylase activity. Moreover, spoilage potential of seven selected strains in sterile and non-sterile meat was investigated by analyzing the release of volatile organic compounds (VOCs) during storage in air. All the strains analysed were unable to hydrolyze sarcoplasmic proteins in vitro and in situ and they did not show lipolytic activity at 4 °C or 20 °C. Almost all were able to grow in the presence of sarcoplasmic extract with glucose and produced histamine. The release of VOCs by each strain in sterile and non sterile beef stored at 4 °C in air was evaluated by HS-SPME-GC/MS analysis. Acetoin and 1-octen-3-ol and 3-methyl-1-butanol were the major compounds isolated from sterile and non-sterile meat samples inoculated with single strains of Brochothrix (B.) thermosphacta and high concentration of acetoin was found in all inoculated meat samples. The role of B. thermosphacta as meat spoiler does not seem to be influenced by indigenous microbiota of meat while its development in meat is associated to a significant increase (P < 0.05) of acetoin and other compounds recognized as important contributors to the spoilage of meat and meat products.
In order to investigate the prevalence of resistant bacteria to biocides and/or antibiotics throughout meat chain production from sacrifice till end of production line, samples from various surfaces of a goat and lamb slaughterhouse representative of the region were analyzed by the culture dependent approach. Resistant Psychrotrophs (n=255 strains), Pseudomonas sp. (n=166 strains), E. coli (n=23 strains), Staphylococcus sp. (n=17 strains) and LAB (n=82 represented mainly by Lactobacillus sp.) were isolated. Resistant psychrotrophs and pseudomonads (47 and 29%, respectively) to different antimicrobials were frequently detected in almost all areas of meat processing plant regardless the antimicrobial used, although there was a clear shift in the spectrum of other bacterial groups and for this aim such resistance was determined according to several parameters: antimicrobial tested, sampling zone and the bacterial group. Correlation of different parameters was done using a statistical tool "Principal component analysis" (PCA) which determined that quaternary ammonium compounds and hexadecylpyridinium were the most relevant biocides for resistance in Pseudomonas sp., while ciprofloxacin and hexachlorophene were more relevant for psychrotrophs, LAB, and in lesser extent Staphylococcus sp. and Escherichia coli. On the other hand, PCA of sampling zones determined that sacrifice room (SR) and cutting room (CR) considered as main source of antibiotic and/or biocide resistant bacteria showed an opposite behaviour concerning relevance of antimicrobials to determine resistance being hexadecylpyridinium, cetrimide and chlorhexidine the most relevant in CR, while hexachlorophene, oxonia 6P and PHMG the most relevant in SR. In conclusion, rotational use of the relevant biocides as disinfectants in CR and SR is recommended in an environment which is frequently disinfected.
To understand why Listeria monocytogenes may persist in food industry equipment and premises, notably at low temperature, scientific studies have so far focused on adhesion potential, biofilm forming ability, resistance to desiccation, acid and heat, tolerance to increased sublethal concentration of disinfectants or resistance to lethal concentrations. Evidence from studies in processing plants shows that the factors associated with the presence of L. monocytogenes are those that favor growth. Interestingly, most conditions promoting bacterial growth were shown, in laboratory assays, to decrease adhesion of L. monocytogenes cells. Good growth conditions can be found in so-called harborage sites, i.e. shelters due to unhygienic design of equipment and premises or unhygienic or damaged materials. These sites are hard to eliminate. A conceptual model of persistence/no persistence based on the relative weight of growth vs. outcome of cleaning and disinfection is suggested. It shows that a minimum initial bacterial load is necessary for bacteria to persist in a harborage site and that when a low initial bacterial charge is applied, early cleaning and disinfection is the only way to avoid persistence. We conclude by proposing that there are no strains of L. monocytogenes with unique properties that lead to persistence, but harborage sites in food industry premises and equipment where L. monocytogenes can persist.
Doctor of Technical Sciences
- K Yuliya
- Yushina
Yuliya K. Yushina, Doctor of Technical Sciences, Head of the Laboratory of Hygiene of Production and Microbiology,
V. M. Gorbatov Federal Research Center for Food Systems. 26, Talalikhin str., 109316, Moscow. Tel.: +7-495-676-95-11 (410),
E-mail: yu.yushina@fncps.ru
ORCID: http://orcid.org/0000-0001-9265-5511
E-mail: a.semenova@fncps
- Anastasia A Semenova
Anastasia A. Semenova, Doctor of Technical Sciences, Professor, Deputy Director, V. M. Gorbatov Federal Research Center for
Food Systems. 26, Talalikhin str., 109316, Moscow, Russia. Tel.: +7-495-676-95-11 (105), E-mail: a.semenova@fncps.ru
ORCID: https://orcid.org/0000-0002-4372-6448
Candidate of Technical Sciences, Researcher Laboratory of Hygiene of Production and Microbiology
- Maria A Grudistova
Maria A. Grudistova, Candidate of Technical Sciences, Researcher Laboratory of Hygiene of Production and Microbiology,
V. M. Gorbatov Federal Research Center for Food Systems. 26, Talalikhin str., 109316, Moscow, Russia. Tel.: +7-495-676-95-11
(404), E-mail: m.grudistova@fncps.ru
ORCID: https://orcid.org/0000-0002-8581-2379