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Application of Random Amplified Polymorphic DNA analysis to differentiate strains of Salmonella typhi and other Salmonella species
Abstract
A Random Amplified Polymorphic DNA (RAPD) fingerprinting method was developed to differentiate isolates of Salmonella serotype typhi (S. typhi) and other Salmonella isolates. A panel of five primers was used to examine 63 isolates of Salm. typhi, including 56 strains isolated in Taiwan and seven strains obtained abroad. Twenty-one RAPD types were revealed using the RAPD fingerprinting method. An RAPD with primer 6032 yielded a polymorphism in a 350 bp fragment that differentiated the attenuated vaccine strain Salm. typhi Ty21a from the rest of the Salm. typhi strains. Strains of Salm. typhi were divided into five types with primer D14307. Primer D14307 also proved capable of discrimination among 65 other Salmonella isolates representing 42 different serotypes. The bacterial DNA used in this RAPD protocol was obtained using a commercially available DNA extraction kit (GeneReleaser). The DNA of various strains of Salmonella from this simple extraction procedure could be discriminated within a few hours using the RAPD technique.
... Oxytetracyclines were the second group of tetracyclines to be discovered. They were first identified in 1950 as being produced by the bacteria Streptomyces rimosus (43), and are used against a broad range of bacteria as a bacteriostatic antimicrobial through binding to the 30S ribosomal subunit of prokaryotes (50,53,110). Oxytetracycline has been used in livestock feed as a prophylactic against infection and as a growth promoter (23,41). ...
... coli O157 responds to catecholamines by increased expression of shiga-toxin [41], exalted chemotaxis, and adherence to eukaryotic cells [42,43], enhanced attachment and effacement (A/E) lesions [44], attachment to murine cecal mucosa [43], and increased flagella expression and motility [45]. In Salmonella, catecholamines have been implicated to enhance motility and colonization in the GI tract of pigs [46]. ...
... coli O157 responds to catecholamines by increased expression of shiga-toxin [41], exalted chemotaxis, and adherence to eukaryotic cells [42,43], enhanced attachment and effacement (A/E) lesions [44], attachment to murine cecal mucosa [43], and increased flagella expression and motility [45]. In Salmonella, catecholamines have been implicated to enhance motility and colonization in the GI tract of pigs [46]. ...
The ability of pathogenic bacteria to sense and respond to periods of host stress is critical to their lifestyle. Adrenaline and norepinephrine are catecholamines that mediate acute host stress in vertebrates and invertebrates. Catecholamines are also used as environmental cues to enhance growth, motility and virulence of bacterial pathogens via specific binding receptors. Incidence of multidrug resistant and highly virulent bacterial pathogens is on the rise, and majority of the genes for antimicrobial resistance (AMR) and virulence are carried on horizontally transferable genetic elements. Conjugation machinery offers an efficient method for acquisition of AMR and virulence genes, which may be responsible for propelling the evolution of pathogenic bacteria. Here we show that norepinephrine (NE) at physiological concentrations enhances horizontal gene transfer (HGT) efficiencies of a conjugative plasmid from a clinical strain of Salmonella Typhimurium to an Escherichia coli recipient in vitro. Expressions of plasmid encoded transfer (tra) genes necessary for conjugation were also significantly upregulated in the presence of NE. Phentolamine, an α-adrenergic receptor antagonist, negated the effects of NE on conjugation more strongly than propranolol, a β-adrenergic receptor antagonist. This study for the first time provides evidence that innate mediators of acute host stress may influence evolution and adaptation of bacterial pathogens.
... Despite the use of commercial kits for DNA extraction, varying DNA template concentrations in this study did not produce any significant result (not shown). Our observations were similar to the work of Shangkuan and Lin [36] who reported that DNA template concentrations over a wide range had no effect to RAPD-PCR band profiles. ...
... intercept that measures the are independent variables (DNA DNA polymerase concentrations, , β35 , β36 , β 46 and are quadratic coefficients. In addition, the statistical model representing the predicted number of bands as a function of the independent the region under investigation is expressed by the following quadratic equation in coded form: ...
... This contradictory observation revealed that RAPD profiles may be used as complementary epidemiological tool in association with other methods of strain differentiation rather than to identify any marker of virulence. Similar observations have been reported earlier on other Salmonella serovars (Laconcha et al., 1998 and Shangkuan and Lin, 1999). Of the 3 primers used in the study, ...
... This contradictory observation revealed that RAPD profiles may be used as complementary epidemiological tool in association with other methods of strain differentiation rather than to identify any marker of virulence. Similar observations have been reported earlier on other Salmonella serovars (Laconcha et al., 1998 and Shangkuan and Lin, 1999). Of the 3 primers used in the study, ...
A study on 22 Salmonella enterica serovar Paratyphi B (5. paratyphi B) var 'Java' (SPVJ) and 13 classical S. enterica serovar Paratyphi B (CSPB) strains isolated from poultry meat (12 SPVJ), fish (9 SPVJ, 11 CSPB), milk products (1 CSPB, 1 SPVJ) and pork (1 CSPB) revealed that all strains except one CSPB strain of fish origin, were lethal to mice on intraperitoneal inoculation with 108 organisms from overnight grown culture. Only 5 strains revealed presence of a heavy plasmid (>35.8 MDa ) however, 35.8 MDa plasmid was common in all isolates. None of the isolates produced lecithinase, gelatinase, casienase, phospholipase A or C but many of the isolates produced DNase. Variable results were observed in salt aggregation assay with indices ranging from 0.6-2.2 M. All produced brick red colonies on Congo Red dye agar in >24 h of incubation without any effect of 0.15% bile salt addition in the medium. All strains haemolysed washed erythrocytes of horse, guinea pig and human blood group B. The best haemolysis was evident on blood agar having horse or guinea pig erythrocytes. No correlation could be established between haemolysis of RBC of different origin and mouse lethality. Multiple drug resistance (MDR) pattern of S. Paratyphi B isolates revealed their resistance to dicloxacillin, cefazolin, colistin sulphate and ceftazidime and sensitiveness to augmentin, ciprofloxacin, nalidixic acid, enrofloxacin, tetracyclin, lomefioxacin, crystal violet, acriflavin and mercuric chloride. Other frequently resisted drugs were nitrofurantoin, streptomycin and cephalexin. Though different in vitro virulence markers have no or little correlation with mouse-lethality, but can be used for epidemiological typing during paratyphoid outbreaks of food origin, particularly haemolysin production and DNase test appears to be of immense value.
... This contradictory observation revealed that RAPD profiles may be used as complementary epidemiological tool in association with other methods of strain differentiation rather than to identify any marker of virulence. Similar observations have been reported earlier on other Salmonella serovars (Laconcha et al., 1998 and Shangkuan and Lin, 1999). Of the 3 primers used in the study, ...
... This contradictory observation revealed that RAPD profiles may be used as complementary epidemiological tool in association with other methods of strain differentiation rather than to identify any marker of virulence. Similar observations have been reported earlier on other Salmonella serovars (Laconcha et al., 1998 and Shangkuan and Lin, 1999). Of the 3 primers used in the study, ...
In this study on virulence marker of 24 S. Paratyphi B var Java (SPVJ)and 16 classical S. Paratyphi B (CSPB) strains isolated from various places, attempts were made to determine relationship between mouse lethality potential and different virulence markers. All of the 24 isolates of S. Paratyphi B var java (SPVJ) had about 85 Kb plasmid while 4 had an additional heavy plasmid. All isolates from poultry shop or meat shop produced DNase. None of the isolate produced lecithinase, gelatinase or phospholipase A, but produced variable results in salt aggregation assay with indices ranging from 0.6 – 2.2 M. All produced brick red colonies in congo red agar in >24 hrs without any effect of bile salt addition in the medium. All isolates haemolysed washed erythrocytes of horse, guinea pig and human blood group B. The best haemolysis zone was evident on horse and guinea pig erythrocytes. All 24 isolates of S. Paratyphi B var java could be grouped into 10 haemolysin (Hly) types. The strains unable to haemolyse goat, sheep, rabbit, human O group erythrocytes possessed a heavy plasmid and were all from Mumbai. Among 16 S.Paratyphi B (CSPB), 6 haemolytic patterns were evident and all except an isolate from Hyderabad pig, were pathogenic to mouse. Though all had a single ~ 35.8 MDa plasmid, none except the non-pathogenic one had 2 plasmids. All but five (one each from Kolkata fish, sewage, Mathura sewage and human patient at Bareilly) isolates produced DNase. Congo red dye binding assay was inconclusive and had no correlation to other virulence markers similar to salt aggregation indices ranging between 0.8 – 1.8 M. None of the six 100 % lethal acutely pathogenic isolates (4 CSPB and 2 SPVJ) haemolysed all types of erythrocytes, while none of those which haemolysed all types of erythrocytes could induce 100% mortality in mice even after 7 days of observation. The different virulence markers though have no or little relation to virulence can be used for epidemiological typing of the isolates during outbreaks, particularly haemolysis pattern and DNase test appears to be of immense value.
... There are only few studies that used RAPD-PCR to predict Salmonella serovars; however, the results are rather contradictory and serovar-prediction accuracy ranged from 0 to 100% (Burr et al., 1998;De Cesare et al., 2001;Rizzi et al., 2005;Shangkuan & Lin, 1998;Soto et al., 1999) (Table 1), which may reflect the typically poor reproducibility of RAPD-PCR. In two of the studies (Burr et al., 1998;Rizzi et al., 2005) we identified a 0% serovar-prediction accuracy; in both studies RAPD-PCR produced identical patterns for different serovars (Table 1). ...
... In two of the studies (Burr et al., 1998;Rizzi et al., 2005) we identified a 0% serovar-prediction accuracy; in both studies RAPD-PCR produced identical patterns for different serovars (Table 1). Conversely, in three other studies RAPD-PCR was sensitive enough to predict Salmonella serovars and the RAPD-PCR serovar-prediction accuracy ranged from 91 to 100% (De Cesare et al., 2001;Shangkuan & Lin, 1998;Soto et al., 1999) (Table 1). While in some studies RAPD-PCR methods have shown high discriminatory power, a major drawback is the lack of reproducibility. ...
Abstract Salmonella is a diverse foodborne pathogen, which has more than 2600 recognized serovars. Classification of Salmonella isolates into serovars is essential for surveillance and epidemiological investigations; however, determination of Salmonella serovars, by traditional serotyping, has some important limitations (e.g. labor intensive, time consuming). To overcome these limitations, multiple methods have been investigated to develop molecular serotyping schemes. Currently, molecular methods to predict Salmonella serovars include (i) molecular subtyping methods (e.g. PFGE, MLST), (ii) classification using serovar-specific genomic markers and (iii) direct methods, which identify genes encoding antigens or biosynthesis of antigens used for serotyping. Here, we reviewed reported methodologies for Salmonella molecular serotyping and determined the "serovar-prediction accuracy", as the percentage of isolates for which the serovar was correctly classified by a given method. Serovar-prediction accuracy ranged from 0 to 100%, 51 to 100% and 33 to 100% for molecular subtyping, serovar-specific genomic markers and direct methods, respectively. Major limitations of available schemes are errors in predicting closely related serovars (e.g. Typhimurium and 4,5,12:i:-), and polyphyletic serovars (e.g. Newport, Saintpaul). The high diversity of Salmonella serovars represents a considerable challenge for molecular serotyping approaches. With the recent improvement in sequencing technologies, full genome sequencing could be developed into a promising molecular approach to serotype Salmonella.
... These loci were chosen for their ability to give unique results and so to differentiate the clinically most common serotypes of Salmonella. The biological functions and phylogenetic implications of these loci have been previously discussed (Shangkuan and Lin 1998;Chan et al. 2003;Porwollik et al. 2002Porwollik et al. , 2004. ...
... So easier and simpler methods were needed for identification of Salmonella serovars. Recently, different molecular techniques were developed for detection of Salmonella serovars such as pulsed-field gel electrophoresis (PFGE) (Uzzau et al. 1999;Torpdahl and Ahrens 2004), ribotyping (Altwegg et al. 1989;Pang et al. 1992), and random amplification of polymorphic DNA (RAPD) (Shangkuan and Lin 1998), as well as the recent development of amplified fragment length polymorphism (AFLP) (Nair et al. 2000) and the real-time multiplex PCR (O'Regan et al. 2008). ...
In Tunisia, Salmonella is the most common bacterial agent responsible for childhood diarrhoea. Currently, isolation of the bacterium by microbiological
and biochemical methods and confirmation of the serotype by serological method remain as the "gold standard". This study aimed
to differentiate among the most common serotypes of Salmonella spp. via two rapid five-plex PCRs assay (MPCR) to evaluate the molecular serotyping method compared with the gold standard
serotyping technique. The two five-plex PCRs assays were designed for the simultaneous detection of six genetic loci from
Salmonella enterica serovar Typhimurium and four from S. enterica serovar Typhi. Sixty-one Tunisian strains (46 collected from patients and 15 from food) were isolated during the period 2002–2007.
The STM and STY primers were able to discriminate all tested Salmonella serotypes that represent the most common clinical and food strains of S. enterica subsp. enterica in our laboratory. All strains belonged to 19 different serotypes: 15 serotypes gave unique amplification patterns compared
each other and the other 4 serotypes were grouped into two pairs that gave the same molecular profile. We resolved this problem
through the addition of a monoplex PCR. Salmonella typhimurium ATCC 14028 consistently produced the same molecular profile as S. typhimurium laboratory isolates. Interestingly, seven strains of Anatum serovar produced two different PCR profiles with these primers:
five strains had the same amplification pattern STM 2,4,5 and STY 2; however, two strains had another molecular profile STM
2,3,5 and STY 2; so the reproducibility of this method was reduced to 93%. The MPCR system is a rapid, specific, and cost-effective
molecular method that gas been proved to have efficient discrimination in serotyping of the most common isolates of S. enterica subsp. enterica.
Keywords
Salmonella spp.-Serotypes-Somatic O and flagellar H antigens-Molecular serotyping
... RAPD is a PCR based method where arbitrary sets of random primers that can bind to the genome at multiple sites are used for the amplification. Amplicons of variable size are amplified based on the proximity of pairs of such sites and the fragments are separated by agarose gel electrophoresis (Hilton et al., 1997; Shangkuan and Lin, 1998). RAPD is easy to use, low cost and provides rapid output. ...
... RAPD is easy to use, low cost and provides rapid output. The method has been used for strain discrimination in various subspecies enterica serovars, including Typhi (Shangkuan and Lin, 1998; Liebana, 2002; Yan et al., 2003). Low stringency conditions are used for PCR amplification in RAPD and hence, minor quantitative differences in PCR components can noticeably alter the banding patterns introducing batch to batch variation (Foley et al., 2007). ...
Serovare von Salmonella enterica subspecies enterica sind im allgemeinen pathogen für Mensch und andere Säugetiere. In dieser Arbeit habe ich anhand eines “Multilocus Sequences Typing” Typisierungsschemas die Populationsstruktur einer der am häufigsten auftretenden Serovaren dieser Subspecies, das aus Menschen und Schlachttieren isolierte Serovar Newport charakterisiert. Dieses Schema wurde auch für die Charakterisierung von Isolaten derselben Subspecies aus humanen Dauerträgern und Reptilien verwandt, um zu bestimmen, ob Isolate aus diesen Quellen sich in ihrer Populationstruktur von denjenigen unterscheiden, die aus anderen Quellen isoliert wurden. Multilocus Sequences Typing ist eine weitgehend für die Untersuchung der Evolution und Populationsstruktur von einen breiten Spektrum von Organismen verwendete Technik. 400 - 600 bp lange Fragmente von 7 Haushaltsgenen wurden sequenziert, und jede einzelne Sequenz jedes einzelnen Gens wurde eine Allelnummer zugeordnet. Jede einzelne Allelkombination wurde einem Sequenztyp zugeordnet. Die so gewonnenen Daten wurden weiter analysiert. Drei “Lineages”, Newport-I, Newport-II und Newport-III, wurden innerhalb dieses Serovars identifiziert, die jeweils aus Menschen in Europa, Tieren und Menschen in Nordamerika isoliert wurden. Der Multiresistenz-Phänotyp wurde häufiger in Newport II gefunden, während die meisten Newport III Isolate pan-sensitiv waren. Verglichen mit anderen Serovaren war die Anzahl von “Lineages” innerhalb Newport höher als bei Enteritidis, Kentucky und Typhimurium, aber niedriger als bei Paratyphi B. Das heisst, die Serovare von S. enterica subspecies enterica variieren stark in ihrer Populationsstruktur. Die Sequenztypen in Isolaten aus humanen Dauerträgern waren im allgemeinen am häufigsten in Isolaten von klinischen Patienten und Tieren vorhanden. In der Mehrheit der Serovaren waren die meisten Isolate aus Patienten und Tieren genetisch identisch mit solchen, die aus gesunden Trägern isoliert wurden. Die genetische Variabilität war zwischen Isolaten aus diesen Quellen vergleichbar. Diese Ergebnissen deuten daraufhin, dass Salmonellen aus Dauerträgern sowie Isolate aus Patienten und Tieren derselben Population angehören. Die meisten Serovare aus Reptilienisolaten waren genetisch identisch mit denen von Menschen und warmblütigen Tieren. In den Serovaren Bovismorbificans, Decatur, Miami und Oranienburg hingegen waren die meisten Isolate aus Reptilien genetisch anders als Isolate aus anderen Wirten. Allerdings wurden nur wenige Isolate der Serovaren Bovismorbificans, Decatur und Miami aus Reptilien und nur wenige Isolate der Serovaren Oranienburg aus anderen Quellen getestet; eine grössere Anzahl von Isolaten müsste daher untersucht werden, um festzustellen ob diese genetischen Unterschiede statistich signifikant sind oder nicht. Serovars of Salmonella enterica subspecies enterica are generally pathogenic to humans and other mammals. In this study, I examined the population structure of one of the most common serovars of this subspecies isolated from humans and food animals, serovar Newport, using a multilocus sequence typing scheme. This scheme was also used to analyze isolates of this subspecies from chronic human carriers and reptiles to determine whether isolates from these sources represent distinct populations than those from other hosts. Multilocus sequence typing has extensively been used to study evolution and population structure of a wide range of organisms. 400-600 bp fragments of 7 housekeeping genes were sequenced and every unique sequence of each gene fragment was given a distinct allele number. Each unique combination of alleles was assigned a distinct sequence type number. The data were used in further analyses. Three lineages, namely Newport-I, Newport-II and Newport-III were identified within serovar Newport which were associated to European humans, animals and humans in North America, respectively. Multidrug resistance phenotypes were most common in Newport-II whereas most isolates in Newport-III were pan-susceptible. When compared to other serovars, the numbers of lineages within Newport were higher than for Enteritidis, Kentucky and Typhimurium but lower than for Paratyphi B. Therefore, serovars of S. enterica subspecies enterica vary greatly in their population structures. The sequence types observed for isolates from chronic human carriers were generally the most common among human-clinical and animal isolates. Most isolates from non-carrier humans plus animals were genetically identical to the carried isolates within most serovars. Genetic diversity was also comparable between isolates from these sources. These results suggest that salmonellae from chronic human carriers belong to the same population as isolates from non-carrier humans and animals. For most serovars, most isolates from reptiles were genetically identical to those from humans or other warm blooded animals. However, in serovars Bovismorbificans, Decatur, Miami and Oranienburg, most reptile isolates were genetically distinct from isolates from other hosts. Only few reptile isolates were tested from Bovismorbificans, Decatur and Miami and only few non-reptile isolates were tested from Oranienburg, and in larger numbers of such isolates would be needed to determine whether these differences are statistically significant.
... Genotyping methods are less subject to natural variation, though various factors may be responsible for genetic variants such as insertions or deletions of DNA into the chromosome, the gain or loss of the extra chromosomal DNA, and random mutations that may create or eliminate restriction sites . There is currently no gold standard typing system available for Salmonella fingerprinting, however, the combination of different genotyping methods such as plasmid profile analysis, ribotyping, characterization of virulence factors in Salmonella serovars, enterobacterial repetitive intergenic consensus sequence analysis (ERIC-PCR), random amplified polymorphic DNA (RAPD) and pulsed field gel electrophoresis methods have been evaluated for more precise subtyping of Salmonella serovars (Mohand et al., 1999;Lagatolla et al., 1996;Shangkuan and Lin, 1998). ...
The prevalence of enteritis and its accompanying diarrheal and other health challenges linked to infections with Salmonella has continuously plagued sub Saharan Africa. In Nigeria, typhoid fever is among the major widespread diseases affecting both young and old as a result of many interrelated factors such as inadequate sanitaion, indiscriminate use of antibiotics and fecal contamination of water sources. Morbidity associated with illness due to Salmonella continues to increase with untold fatal consequences, often resulting in death. An accurate figure of cases is difficult to arrive at because only large outbreaks are mostly investigated whereas sporadic cases are under-reported. A vast majority of rural dwellers in Africa often resort to self-medication or seek no treatment at all, hence serving as carries of this disease. Non typhoidal cases of salmonellosis account for about 1.3 billion cases with 3 million deaths annually. Given the magnitude of the economic losses incurred by African nations in the battle against salmonella and salmonellosis, this article takes a critical look at the genus Salmonella , its morphology, isolation, physiological and biochemical characteristics, typing methods, methods of detection, virulence factor, epidemiology and methods of spread within the environment.
... and their serotypes. These include real-time PCR (Hoorfar et al. 2000), multiplex PCR (Alvarez et al. 2004), ribotyping, sequence analysis (Mortimer et al. 2004), protein analysis (Cai et al. 2005), mass spectrometry (Wilkes et al. 2005), ribosomal DNA intergenic spacer amplification, IS200 analysis (Ezquerra et al. 1993;Uzzau et al. 1999) and random amplification of DNA polymorphism (Shangkuan and Lin 1998). Multiplex qPCR has proven particularly sensitive, rapid, easy to perform and able to detect a variety of Salmonella spp. ...
• The aim of this study was to develop a multiplex quantitative polymerase chain reaction (qPCR) based molecular diagnostic kit for rapid diagnosis of Salmonella enterica serovar Enteritidis and S. enterica serovar Typhimurium serotypes, which are frequently isolated worldwide from poultry samples.
• Detection and discrimination of S. enteritidis and S. typhimurium were performed by targeting the sdf and the STM4492 (putative cytoplasmic protein) gene, respectively. The invA (invasion protein) gene was used to detect Salmonella spp. as a target gene, since it is considered a standard. In this study, a total of 200 bacterial strains (178 Salmonella spp. strains and 22 other genera) were used to test the specificity and sensitivity of the developed kit. The limit of detection (LOD) of the assays was determined to be 10⁰-10¹ cfu/25g from chicken meat samples artificially contaminated by litter and 10⁰-10¹ cfu/ml for cloacal swab samples.
• The multiplex qPCR results were 100% compatible with conventional serotyping results while the specificity and sensitivity values were 100%. These findings indicated that the newly developed multiplex qPCR technique can provide an alternative method to conventional serotyping of S. enteritidis and S. typhimurium in laboratories lacking adequate infrastructure.
... and their serotypes. These include real-time PCR (Hoorfar et al. 2000), multiplex PCR (Alvarez et al. 2004), ribotyping, sequence analysis (Mortimer et al. 2004), protein analysis (Cai et al. 2005), mass spectrometry (Wilkes et al. 2005), ribosomal DNA intergenic spacer amplification, IS200 analysis (Ezquerra et al. 1993;Uzzau et al. 1999) and random amplification of DNA polymorphism (Shangkuan and Lin 1998). Multiplex qPCR has proven particularly sensitive, rapid, easy to perform and able to detect a variety of Salmonella spp. ...
Salmonella enterica serotypes are identified according to the Kauffman-White scheme based on the somatic O antigen and flagellar H antigen which are present in the cell wall of Salmonella. The aim of this study is to develop a multiplex quantitative real time polymerase chain reaction (multiplex-qPCR) based molecular diagnostic kit for the rapid diagnosis of Salmonella Enteritidis and Salmonella Typhimurium serotypes, which are frequently isolated in the world. In this study, a total of 175 (30 S. Enteritidis, 35 S. Typhimurium, 55 S. Infantis, 22 S. Mbandaka, 12 S. Liverpool, 4 S. Newport, 17 S. Virchow strains) were used to test the specificity and sensitivity of the developed kit while Escherichia coli ATCC (25922), Pasteurella multocida, Avibacterium paragallinarum, Staphylococcus aureus, Streptococcus pneumoniae, Pseudomonas aeruginosa, Staphylococcus epidermidis, Listeria monocytogenes, S. Mbandka, S. Hadar, S. II, S. Kentucky and S. Havana strains were used to optimize the method. The multiplex-PCR results were 100% compatible with the conventional serotyping results, and the specificity and sensitive ratio was 100%. It is assumed that the newly developed multiplex qPCR technique could be used as an alternative method to conventional serotyping in laboratories which do not have adequate infrastructure.
... Molecular detection methods have been studied to replace or supplement traditional serotyping methods, such as ribose typing, RAPD molecular marker technology [37], IS200 typing technology [38] analysis. With the continuous deepening of research, PCR and other nucleic acid-based molecular diagnostic techniques have been widely used in biological classification and identification, which greatly shortens the laboratory testing time, is easy to standardize, and does not require high staffing, to a certain extent, to make up for the shortcomings of traditional identification and typing methods [39]. ...
Salmonella is a zoonotic disease widely spread in the environment, causing serious economic losses to the world. Objective: To improve people's understanding of Salmonella disease and reduce economic losses. Methods: Based on the recent research progress, the biochemical characteristics, clinical manifestations, laboratory detection methods and preventive measures of bacteria were discussed in this paper. Conclusion: Salmonella infection in China is becoming more and more serious, which has caused great harm to pig industry and even human beings.
... Molecular detection methods have been studied to replace or supplement traditional serotyping methods, such as ribose typing, RAPD molecular marker technology [37], IS200 typing technology [38] analysis. With the continuous deepening of research, PCR and other nucleic acid-based molecular diagnostic techniques have been widely used in biological classification and identification, which greatly shortens the laboratory testing time, is easy to standardize, and does not require high staffing, to a certain extent, to make up for the shortcomings of traditional identification and typing methods [39]. ...
... Bacterial typing by means of PCR fingerprinting is a well-established concept. It may be accomplished using primers directed at repetitive sequences that are interspersed throughout the genome, as in the case of the Repetitive Extragenic Palindromic (REP) sequence (1), the Enterobacterial Repetitive Intergenic Consensus (ERIC) sequence (1,2), or the BOX elements (3); it may also employ primers that anneal randomly in several locations along the genome, such as Random Amplified Polymorphic DNA (RAPD) (4)(5)(6)(7)(8) and Arbitrarily Primed PCR (AP-PCR) (9,10). When it comes to identifying human individuals through molecular means, primers that amplify hypervariable genomic regions, such as variable number tandem repeats (VNTR) and short tandem repeats (STR), are used (11). ...
DNA fingerprinting is a major tool in identifying individuals and in evidence matching. However, this technique can be difficult to reproduce in practical classes. Here, we report on distinct PCR profiles obtained when amplifying saliva DNA of a score of distinct individuals with Random Amplified Polymorphic DNA (RAPD)-PCR primer BOXA1R. The RAPD-PCR method is simple and efficient for discrimination between bacterial strains and is used in this instance to obtain personalized fingerprints of each individual’s oral microbiota.
We present real results with undergraduate students confirming that this procedure is easily feasible in practical classes. Based on the results presented, we suggest a laboratory activity for undergraduate Molecular Biology or Microbiology students.
... Two years later, a RAPD method was developed to differentiate strains of Salmonella Typhi and other Salmonella spp. (104). Fiveprimer RAPD successfully differentiated several strains of Salmonella Typhi into 21 distinct RAPD profiles. ...
Molecular Tools To Study Preharvest Food Safety Challenges, Page 1 of 2
Abstract
Preharvest food safety research and activities have advanced over time with the recognition of the importance and complicated nature of the preharvest phase of food production. In developed nations, implementation of preharvest food safety procedures along with strict monitoring and containment at various postharvest stages such as slaughter, processing, storage, and distribution have remarkably reduced the burden of foodborne pathogens in humans. Early detection and adequate surveillance of pathogens at the preharvest stage is of the utmost importance to ensure a safe meat supply. There is an urgent need to develop rapid, cost-effective, and point-of-care diagnostics which could be used at the preharvest stage and would complement postmortem and other quality checks performed at the postharvest stage. With newer methods and technologies, more efforts need to be directed toward developing rapid, sensitive, and specific methods for detection or screening of foodborne pathogens at the preharvest stage. In this review, we will discuss the molecular methods available for detection and molecular typing of bacterial foodborne pathogens at the farm. Such methods include conventional techniques such as endpoint PCR, real-time PCR, DNA microarray, and more advanced techniques such as matrix-assisted layer desorption ionization–time of flight mass spectrometry and whole-genome sequencing.
... PFGE and RAPD is used as a molecular tool for determination of relatedness among the S. Typhi strains and tracing the source of outbreak [9,10,11]. ...
... E-mail: jimohtijani@futminna.edu.ng. that affects over 16 million people in the world annually (Shangkuan and Lin, 1998;Parkhill et al., 2001;Ganai et al., 2009). It is a multiple drugs resistant bacterium (Cristina and Claudia, 1994). ...
... Over the last 20 years, alternative strategies to replace or complement traditional serotyping methods have been proposed. These include ribotyping (12), ribosomal DNA intergenic spacer amplification (22), random amplification of DNA polymorphism (35), IS200 analysis (13,38), real-time PCR (21), PCR-single-strand conformation polymorphism analysis (27), amplified fragment length polymorphism (37), sequence analysis (26), multiplex PCR (2), and DNA microarrays (8,31). Other laboratories have taken protein-based approaches to type Salmonella enterica by methods including protein arrays (7) and mass spectrometry (40). ...
... The use of DNA-based typing methods is becoming increasingly important in epidemiological survey and differentiation of Salmonella species. Some of these methods include pulsed field gel electrophoresis (PFGE) (Mohand et al., 1999;Kubota et al., 2005), PCR ribotyping (Lagatolla et al., 1996), automated nuclease PCR assay (Hoorfar et al., 2000) and random amplification of polymorphic DNA (RAPD) (Shangkuan and Lin, 1998;Smith et al., 2011). RAPD-PCR does not require any specific knowledge of the target DNA sequence, making it a flexible and powerful tool with general applicability. ...
The objective of this investigation was to isolate and identify Salmonella serovars present in wastewater from the University of Nigeria, Nsukka (UNN) wastewater treatment plant and to evaluate the sensitivity and precision of different microbial typing methods (conventional and molecular), in identifying and characterizing Salmonella species. A total of 100 suspected Salmonella colonies on selective media (Salmonella-Shigella agar and MacConkey Agar) were subjected to biochemical testing. A total of 12 biochemically typical Salmonella isolates were identified and further characterized. Serotyping analysis further identified 3 (25%) of the isolates as Salmonella enterica serovar Limete. Salmonella specific (16S) polymerase chain reaction (PCR) assay validated the result obtained by serotyping, although 2 of the isolates could not be serotyped and were identified as rough strains. PCR assay produced positive amplifications of 574 bp of the 16S rRNA gene specific for Salmonella, while non-Salmonella serovars were negative (100%). Random amplified polymorphic DNA (RAPD-PCR) analysis revealed the genetic relatedness of Salmonella serovars isolated from wastewater. Primers 787 and RAPD2 identified 4 RAPD-binding patterns, while primer 1254 did not give any discriminatory pattern. Molecular analyses (16S PCR and RAPD) showed discriminatory power, reproducibility, easy interpretation and performance. It is therefore a promising alternative method for typing Salmonella species.
... Molecular methods have been applied in the identification of Salmonella and were found to be very useful in differentiating between species. These methods include pulsed field gel electrophoresis (PFGE) (Mohand et al., 1999;Nair et al., 1994), IS200 fingerprinting (Ezquerra et al., 1993), PCR ribotyping (Lagatolla et al., 1996), ribosomal DNA intergenic spacer amplification and heteroduplex analysis (Jensen and Hubner, 1996), amplification fragment length polymorphism (Aarts et al., 1998;Nair et al., 2000), automated nuclease PCR assay (Hoorfar et al., 2000) and random amplification of polymorphic DNA (RAPD) (Shangkuan and Lin, 1998). Since all molecular techniques are based on variability of microbial chromosomes or plasmids, then DNA sequencing would appear to be the best approach for differentiating subtypes (Liebana et al., 2001) Many oligonucleotide primer sets have been described for the detection of Salmonella using the PCR technique. ...
... This method is different from previous PCR in the use of a single primer instead of two, and used low annealing temperature. The primers that work for some bacteria may fail for others (Shangkuan and Lin, 1998) and because of that, the screening process need to determine the appropriate primers. In the present study, ten primers (50% G+C) were screened and two primers (Gold Oligo OPAR3 and Gold Oligo OPAR8; New England Biolabs Inc.) were chosen because they yielded more clearer band patterns than those obtained with other primers. ...
In this study, RAPD-PCR and ERIC-PCR were used to study the epidemiology of V. parahaemolyticus isolated from cockles in Padang, Indonesia. The Gold Oligo OPAR3 primer produced bands ranged from 1-8 with sizes from 0.2 - 5.0 kb and the Gold Oligo OPAR8 primer produced 1-7 bands with sizes 0.7 - 1.5 kb. Both primers produced twenty five RAPD patterns with a few isolates failed to produce any products. Based on phylogenetic dendrogram, all the isolates can be divided into 6 major clusters with similarity between 0 to 52%. For the ERIC primer, it produced bands ranged from 3-15 with sizes from 0.1 - 5.0 kb and twenty seven different ERIC patterns. Construction of the phylogenetic dendogram showed the isolates can be divided into 4 major clusters with similarity between 56 to 86%. The high diversity of both processes may be due to the multiple contamination sources of V. parahaemolyticus.
... Genotyping methods are less subject to natural variation, though various factors may be responsible for genetic variants such as insertions or deletions of DNA into the chromosome, the gain or loss of the extra chromosomal DNA, and random mutations that may create or eliminate restriction sites. There is currently no gold standard typing system available for Salmonella fingerprinting, however, the combination of different genotyping methods such as plasmid profile analysis, ribotyping, characterization of virulence factors in Salmonella serovars, enterobacterial repetitive intergenic consensus sequence analysis (ERIC-PCR), random amplified polymorphic DNA (RAPD) and pulsed field gel electrophoresis methods have been evaluated for more precise subtyping of Salmonella serovars(Mohand et al., 1999;Lagatolla et al., 1996;Shangkuan and Lin, 1998). ...
The prevalence of enteritis and its accompanying diarrheal and other health challenges linked to infections with Salmonella has continuously plagued sub Saharan Africa. In Nigeria, typhoid fever is among the major widespread diseases affecting both young and old as a result of many interrelated factors such as inadequate sanitaion, indiscriminate use of antibiotics and fecal contamination of water sources. Morbidity associated with illness due to Salmonella continues to increase with untold fatal consequences, often resulting in death. An accurate figure of cases is difficult to arrive at because only large outbreaks are mostly investigated whereas sporadic cases are under-reported. A vast majority of rural dwellers in Africa often resort to self-medication or seek no treatment at all, hence serving as carries of this disease. Non typhoidal cases of salmonellosis account for about 1.3 billion cases with 3 million deaths annually. Given the magnitude of the economic losses incurred by African nations in the battle against salmonella and salmonellosis, this article takes a critical look at the genus Salmonella, its morphology, isolation, physiological and biochemical characteristics, typing methods, methods of detection, virulence factor, epidemiology and methods of spread within the environment.
... Salmonella is a Gram-negative bacteria and another food-born pathogen producing infectious diseases such as salmonellosis, typhoid fever, or other problems. Typhoid fever caused by S. typhi leads to 600,000 deaths annually , most cases occurring in South Asia, Africa, and South America [3] . Generally, infectious dosages by pathogenic bacteria such as Salmonella and E. coli are as low as 10 cells/mL [4] , thus effective and sensitive methods for screening these microorganisms are essential. ...
This review intends to present an overview of methods currently under development for the specific and sensitive detection of pathogenic bacteria that exist in a variety of human environments. Bacteria continue to be a major health threat in general, and much effort is being deployed to counteract this problem. In a first instance, current and efficient techniques in use for the detection of bacteria are described. In a second instance, this review serves to compare the more conventional techniques to emerging technologies for the direct (non-labelled) detection of bacteria (referred to as “biosensors”). These approaches are mainly optical, piezoelectric, and electro-chemical in nature. They are cost-effective, quite sensitive, and potentially portable for rapid on-site/real-time detection, and rapid prevention. These devices are comprised of specific chemical/ biochemical probes immobilized onto physical transducers. This work also presents comparisons between the efficiencies (assay time and sensitivity) of various techniques being employed.
... PCR fingerprinting directly interferes in the epidemiological study success and orientation, suggesting that it must be carefully and rigorous made in order to select the more efficient to discriminate Salmonella isolates, since some primers are more efficient to discriminate specific serotypes than others. This concern was already expressed by Lin et al. (4) , Burr et al. (36) and Shangkuan and Lin (37) . ...
AbSTRACT Effective epidemiological surveillance and control of Salmonella sp. requires accurate and expeditious genetic typing methods. In the present study, rapid PCR-based methods (ERIC-PCR, M13-PCR and RAPDs) were applied to 73 Salmonella sp. isolates, and the results compared with those previously obtained by RFLP-PFGE (Salmonella gold standard genotyping method), in order to evaluate their discriminatory ability. Results were very diverse among the primers used and, for each primer, the performance level was variable among the different serotypes. ERIC-PCR and RAPD with OPC19 was inefficient for Salmonella sp. discrimination beyond the serotype level. In opposite, M13-PCR, OPC15-RAPD and OPB17-RAPD allowed intraserotype discrimination that, in general, were less discriminative than RFLP-PFGE, indicating that should not be used as a unique typing method in epidemiological studies. Nevertheless, in particular situations, these PCR methods, which are faster and less expensive than RFLP-PFGE, could offers an attractive choice as a preliminary screening of the isolates to reduce the number of suspicious isolates that should be subsequently typed with a more discriminative and accurate methods such as RFLP-PFGE.
... Random amplified polymorphic DNA (RAPD) is the first PCR based molecular marker technique developed [167] and then applied to SNP detection for various organisms [168,169]. Random and variable sequences of genome PCR products are generated by random primer sets and produce a pattern of bands resulting from electrophoresis. Presence/absence of certain size product fragments indicates individual variety ( Figure 3B). ...
DNA mutations are the cause of many human diseases and they are the reason for natural differences among individuals by affecting the structure, function, interactions, and other properties of DNA and expressed proteins. The ability to predict whether a given mutation is disease-causing or harmless is of great importance for the early detection of patients with a high risk of developing a particular disease and would pave the way for personalized medicine and diagnostics. Here we review existing methods and techniques to study and predict the effects of DNA mutations from three different perspectives: in silico, in vitro and in vivo. It is emphasized that the problem is complicated and successful detection of a pathogenic mutation frequently requires a combination of several methods and a knowledge of the biological phenomena associated with the corresponding macromolecules.
... isolates. However, they are in agreement with those of Blixt et al. (2003) and Shangkuan and Liu (1998) stating that primers efficient for some bacteria may fail for others. Thus, the selection of an appropriate primer is an important factor in RAPD analysis. ...
Salmonellosis is one of the most common causes of food-borne infection worldwide. In the last decade, Salmonella
enterica serovar Kentucky has shown an increase in different parts of the world with the concurrent emergence of multidrug-resistant isolates. These drug-resistant types spread from Africa and the Middle East to Europe and Asia. Although S. Kentucky serovar is of potential human relevance, there is currently no standardized fingerprinting method for it, in Tunisia. In the present study, a collection of 57 Salmonella Kentucky isolates were analyzed using plasmid profiling, pulsed-field gel electrophoresis (PFGE), ribotyping, enterobacterial repetitive intergenic consensus (ERIC) fingerprinting, and Random Amplification of Polymorphic DNA. Plasmid profiling showed a discriminatory index (D) of 0.290, and only 9 out of 57 (16 %) isolates carried plasmids, which represents a limitation of this technique. Fingerprinting of genomic DNA by PFGE and ribotyping produced 4 and 5 patterns, respectively. Distinct PFGE patterns (SX1, SX2, SX3, and SX4) were generated for only 28 strains out of 57 (49.1 %) with a D value of 0.647. RAPD fingerprinting with primers RAPD1 and RAPD2 produced 4 and 20 patterns, respectively. ERIC fingerprinting revealed 14 different patterns with a high discriminatory index (D) of 0.903. When the methods were combined, the best combination of two methods was ERIC-2 with RAPD2. These results indicates that a single method cannot be relied upon for discriminating between S. Kentucky strains, and a combination of typing methods such ERIC2 and RAPD2 allows further discrimination.
... The worldwide rise of S. Enteritidis nowadays constitutes a public health concern; it is a challenge to epidemiologist and clinical staff alike (22,24) . The main approach taken to control the dissemination of salmonellosis is to employ molecular techniques. ...
BACKGROUND: The approach generally employed in leading laboratories worldwide to identify strains of Salmonella is to detect epidemiological markers by serotyping and molecular techniques. These molecular methods are important in sanitary surveillance and lead to the source of infection. OBJECTIVES: The aim of the present study is to characterize Salmonella Enteritidis strains by ribotyping. MATERIAL AND METHODS: Thirty-eight strains of S. Enteritidis were isolated from in patients at the university hospital of Universidade de São Paulo, Ribeirão Preto, SP, Brazil, between 1996 and 1998. These strains were isolated from stools (31 samples), blood (4 samples) and other body fluids (3 samples), using routine bacteriological methods, and were serotyped and ribotyped. RESULTS: The 38 strains of serotype S. Enteritidis were shown by the ribotyping to belong to two ribotypes: A (94.7% of the samples) and B (5.3%). DISCUSSION AND CONCLUSION: These results suggest that the majority of patients (94.7%) were infected by the same strain. This strain could be endemic in the Ribeirão Preto community, or these patients may have been exposed to a common source of infection.
... This method is different from previous PCR in the use of a single primer instead of two, and used low annealing temperature. The primers that work for some bacteria may fail for others (Shangkuan and Lin, 1998) and because of that, the screening process need to determine the appropriate primers. In the present study, ten primers (50% G+C) were screened and two primers (Gold Oligo OPAR3 and Gold Oligo OPAR8; New England Biolabs Inc.) were chosen because they yielded more clearer band patterns than those obtained with other primers. ...
In this study, RAPD-PCR and ERIC-PCR were used to study the epidemiology of V. parahaemolyticus
isolated from cockles in Padang, Indonesia. The Gold Oligo OPAR3 primer produced bands ranged from 1-8
with sizes from 0.2 – 5.0 kb and the Gold Oligo OPAR8 primer produced 1-7 bands with sizes 0.7 – 1.5 kb.
Both primers produced twenty five RAPD patterns with a few isolates failed to produce any products. Based
on phylogenetic dendrogram, all the isolates can be divided into 6 major clusters with similarity between 0 to
52%. For the ERIC primer, it produced bands ranged from 3-15 with sizes from 0.1 – 5.0 kb and twenty seven
different ERIC patterns. Construction of the phylogenetic dendogram showed the isolates can be divided into
4 major clusters with similarity between 56 to 86%. The high diversity of both processes may be due to the
multiple contamination sources of V. parahaemolyticus.
... The ability to characterize and subtype S. typhi isolates is important from an epidemiological perspective in order to trace and control the dissemination of this infectious agent. Recently, many DNA-based approaches have been utilized in the molecular subtyping of S. typhi, including ribotyping (Ling et al., 2000), pulsed-field gel electrophoresis (PFGE) (Thong et al., 2002), IS200 (Navarro et al., 1996), RAPD (Shangkuan and Lin, 1998) and amplified fragment length polymorphism (AFLP) (Nair et al., 2000). MDR S.typhi from Vellore, India (Shanahan et al., 1998), Bangladesh (Hermans et al., 1996), Pakistan (Thong et al., 2000) and Vietnam (Connerton et al., 2000) showed limited genetic diversity. ...
Sixty-three isolates of Salmonella enterica serotype Typhi (S.typhi) from sporadic cases of typhoid fever obtained from Malaysia (n=6), Vietnam (n=13), India (n=8) and Pakistan (n=36) were characterized by phage typing, drug-susceptibility testing, amplified fragment length polymorphism (AFLP) and pulsed-field gel electrophoresis (PFGE) with restriction endonuclease, XbaI. The strains analyzed were mostly resistant to ampicillin, co-trimoxazole, tetracycline and chloramphenicol. These strains were represented by phage types, E1, E9, 46, J1, VNS and UVS. PFGE analysis showed that multidrug resistant (MDR) strains isolated in different countries and at different periods were genetically very homogenous where 90% of the strains analyzed had very closely related XbaI patterns. AFLP was able to subtype 42 clonally related MDR strains (represented by 8 PFGE patterns) into 16 profiles. Cluster analysis based on the AFLP and PFGE data using unweighted pair group mean averages could differentiate MDR strains from different geographical region and that the drug-sensitive strains were in a distinct cluster. The clustering of MDR strains from each country and the presence of a dominant XbaI-PFGE pattern indicated that the MDR S.typhi had probably been spread clonally in these countries. AFLP is clearly more discriminative than PFGE in differentiating the MDR S.typhi, hence providing an alternative, sensitive method for detailed analysis of the multidrug resistant strains.
... The RAPD method has been widely used in research to study population genetic structure [3], mutagenesis [4], genetic mapping [5], and phylogenetics [6,7] for a variety of species. Of particular interest to students of microbiology are applications of RAPD PCR in bacterial epidemiology [8,9], parasitology [10,11], and virology [12]. ...
There continues to be a need for authentic and engaging undergraduate teaching laboratory investigations that reflect modern molecular biological research methods. We adapted random amplified polymorphic DNA (RAPD) analysis for the microbiology laboratory as a practical activity that allowed students to generate and analyze data for the identification of bacterial unknowns. The activity gave students an appreciation of the value of applying a molecular biological method to a discipline-specific question.
The abbreviation used is: RAPD, random amplified polymorphic DNA.
... The selection of an appropriate primer and optimization of PCR conditions are the important factor in RAPD analysis (Blixt et al., 2003). The primers that work for some bacteria may fail for others and because of that, the screening process needs to determine the appropriate primers (Shangkuan & Lin, 1998). RAPD uses oligonucleotide (9 to 10 bases in length) primers with arbitrary sequence, which hybridize with sufficient affinity to chromosomal DNA sequences at low annealing temperatures such that they can be used to initiate amplification of regions of the bacterial genome. ...
... Molecular methods have been developed to identify Salmonella isolates based on the detection of DNA or RNA polymorphisms (Esteban et al., 1993;Mortimer et al., 2004;Nair et al., 2002;Porwollik et al., 2004;Shangkuan and Lin, 1998;Torpdahl and Ahrens, 2004); however, these methods are not at all comparable with serotyping. Current study developed 2 multiplex polymerase chain reactions (M-PCRs) such that the first reaction identifies the serogroup; once the serogroup is known, a second reaction is then conducted for serotype identification, a strategy that differs from those presented in previous publications (Agron et al., 2001;Alvarez et al., 2004;Echeita et al., 2002;Herrera-Leon et al., 2004;Herrera-León et al., 2007;Luk et al., 1993). ...
To improve limitations of Salmonella serotyping, 2 multiplex polymerase chain reaction (M-PCR) were developed using a strategy that identifies first the genes encoding serogroups (rfbJ, wzx). According to the serogroup determined, a second M-PCR identifies serotype (fliC, fljB, wcdB, and sdf-I sequence). Standardization and evaluation of both M-PCRs were carried out.
... DNA-based approaches have recently been utilized in the molecular typing of Salmonella serovars and to provide better epidemiological tools to identify sources of foodborne illness. Among these are pulsed-field gel electrophoresis (PFGE) (Thong et al. 1995(Thong et al. , 1996Kariuki et al. 1999;Liebana et al. 2001), RAPD-PCR (Shangkuan and Lin 1998), ribotyping (Altwegg et al. 1989), IS200 fingerprinting (Navarro et al. 1996;Old et al. 1999;Millemann et al. 2000), repetitive element PCR (rep-PCR) (Johnson et al. 2001). Multilocus enzyme electrophoresis (MLEE) in combination with DNA sequence analysis of housekeeping and invasion genes (Boyd et al. 1996), as well as high-resolution methods such as amplified fragment length polymorphism (AFLP; Nair et al. 2000), have also been utilized to assess molecular genetic relationships of the salmonellae. ...
The intent of this study is to exploit both the genetic diversity and conservation demonstrated between the Salmonella enterica ssp. enterica serovar Typhi CT18 and Salm. enterica ssp. enterica serovar Typhimurium LT2 genomes by utilizing amplicon length polymorphism (ALP) to detect and differentiate various Salmonella strains.
Methods of ALP-PCR analysis were developed based on identifying DNA sequence deletions within highly homologous regions of the Salm. Typhi CT18 and Salm. Typhimurium LT2 genomes. This study describes the application of genome-based ALP-PCR using primer pairs designed to detect genomic differences present within both Salmonella genomes and evaluated against a reference set of Salmonella strains.
This study defines a collection of primer sequences broadly distributed along the Salmonella genome that can differentiate various Salmonella strains.
Genome-based ALP-PCR provides a useful and powerful analytical method to evaluate variability within a group of Salmonella strains independent of serological, phenotypic or other molecular approaches.
Advancements in the survival mechanisms of foodborne pathogens have confronted several challenges for the storage and preservation of food products, which, when consumed, results in unwellness or sickness. One of the significant challenges in the field of food science and technology is the accuracy of the detection of foodborne pathogens. The detection of foodborne pathogens involves different processes, some of which are based on the detection by using the microorganism’s genetic material (DNA/RNA) or by using the specific enzyme or peptide sequence. Modern microbiology and biotechnology offering FBP detection techniques involve nucleotide-based methods (e.g., PCR, AFLP, RFLP, and FISH), signal-based methods (e.g., biosensors), and immunological methods (ELISA and MALDI-TOF/MS). The application of nanotechnology and bioinformatics is gaining popularity in the recent past to improve the accuracy in identification of bacterial toxins and viruses. Different molecular identification methods applied to detect different food pathogens, challenges, and prospects have been discussed in this chapter.
Imaging-based total bacterial count and type identification of bacteria play crucial rules in clinical diagnostics, public health, biological and medical science, environmental protection. Herein, we designed and synthesized a series of tetraphenylethenes (TPEs) functionalized with one or two aldehyde, carboxylic acid and quaternary ammonium groups, which were successfully used as fluorescent materials for rapid and efficient staining of 8 kinds of representative bacterial species, including pathogenic bacteria Vibrio cholera, Klebsiella pneumoniae, Listeria monocytogenes and potential bioterrorism agent Yersinia pestis. By comparing the fluorescence intensity changes of the AIE materials before and after bacteria incubation, the sensing mechanisms (electrostatic versus hydrophobic interactions) were simply discussed. Moreover, the designed AIE materials were successfully used as an efficient artificial tongue for bacteria discrimination and all of the bacteria tested were identified via linear discriminant analysis (LDA). Our current work provided a general method for simultaneous broad-spectrum bacterial imaging and species discrimination, which is helpful for bacteria surveillance in many fields.
Salmonella is recognized as a major cause of food poisoning worldwide. Because of the importance of this organism as a pathogen, it is needed to diagnosis and detect by molecular methods. In the present study we evaluated the possibility of differentiating Iranian Salmonella isolates at the serotype level on the basis of the polymorphism of the 16S-23S rRNA intergenic spacer region. So, seventy-one Salmonella isolates corresponding to different serotype/serogroups from Iranian patients were analyzed in this study. Genomic DNA was extracted by phenol / chloroform method and then evaluated by PCR-Ribotyping technique. The PCR-Ribotyping method could categorize 71 available strains in four distinct classes. The results show that a high degree of genotypic homogeneity in s. enterica ser. Enteritidis. This study indicated that the PCR-Ribotyping technique has acceptable power in distinct but it cannot distinguish different serotypes of Salmonella from each other
The ability of Aeribacillus pallidus E334 to produce pellicle and form a biofilm was studied. Optimal biofilm formation occurred at 60 °C, pH 7.5 and 1.5% NaCl. Extra polymeric substances (EPS) were composed of proteins and eDNA (21.4 kb). E334 formed biofilm on many surfaces, but mostly preferred polypropylene and glass. Using CLSM analysis, the network-like structure of the EPS was observed. The A. pallidus biofilm had a novel eDNA content. DNaseI susceptibility (86.8% removal) of eDNA revealed its importance in mature biofilms, but the purified eDNA was resistant to DNaseI, probably due to its extended folding outside the matrix. Among 15 cleaning agents, biofilms could be removed with alkaline protease and sodium dodecyl sulphate (SDS). The removal of cells from polypropylene and biomass on glass was achieved with combined SDS/alkaline protease treatment. Strong A. pallidus biofilms could cause risks for industrial processes and abiotic surfaces must be taken into consideration in terms of sanitation procedures.
The objectives of the present work were: i) to assess the rates of antibiotic resistance of Salmonella Typhimurium isolates from various regions of Greece, during the period 1989-1997, ii) to investigate the genotypic variability and relationships among isolates belonging to various resistance phenotypes and sources of origin, iii) to study the mechanisms of resistance of multirésistant isolates, and iv) to investigate the presence of extended spectrum ß-lactamases in ß-lactam-resistant isolates..
Initially, the resistance rates of 328 S. Typhimurium isolates to seventeen antibiotics of various classes were determined. Resistance rates to the majority of antibiotics had significantly increased during the period 1989-1996, while, during the last year of the study, the resistance rates seemed to remain stable or decline. The most frequent among the 26 observed resistance phenotypes was streptomycin/ sulfonamides. Furthermore, since 1993, a significant increase in the number of multi-resistant isolates (resistance to three or more antibiotics of different classes) was observed, particularly those with the “core” pattern of resistance to ampicillin, chloramphenicol, streptomycin, sulfonamides and tetracycline (ACSSuT-isolates), which were compatible with the multirésistant phage type DTI04.
Fourteen ACSSuT-isolates were selected for further analysis by pulsed field gel electrophoresis (PFGE), and for investigation of their mechanisms of resistance to antibiotics by the polymerase chain reaction. Eleven of the 14 isolates belonged to the phage type DTI04, two to the closely related phage type DTI04b, and one to phage type DTI 93. PFGE discerned six distinct types, A-F. Six isolates belonged to PFGE type A, while the remaining belonged to the PFGE types B (two isolates), C (two), D (two), E (one) and F (one). A variety of type I intégrons were detected among the DTI 04 isolates. The majority harbored the antibiotic resistance genes pse-1 and aadA, which encoded resistance to ampicillin and streptomycin, respectively. Some remaining non-integron associated isolates possessed tern-1, instead of pse-1.
The chromosomal fingerprints of 231 isolates belonging to the various resistance phenotypes, were generated by PFGE. Twenty-six distinct PFGE types were detected. A total of 78.5% of the isolates belonged to the three PFGE types A, B and C. These occurred across the entire range of resistance phenotypes and sources of origin, contrary to, for example, type L, which was present only among susceptible isolates, or type O, present only among non-human isolates. PFGE type A, major among detected types, was more frequent among multi-resistant isolates compared to the
susceptible or to streptomycin and/or sulfonamides resistant isolates. Conversely, PFGE types B and C were more common among the susceptible and the streptomycin and/or or sulfonamides resistant isolates. Seven PFGE types were unique for isolates not directly involved in the human food chain, but originated for example, from pets or the environment. In general, there was greater genetic variability among isolates of non-human origin compared to those of human origin.
Finally, resistance to third generation cephalosporins (cefotaxime and ceftriaxone) and also to the amoxycillin/clavulanate combination was observed in a clinical isolate of 1996. In this isolate, an enzyme with isoelectric point 8.4 was found, and its corresponding gene was sequenced. Nucleotide sequencing determined that this β- lactamase belonged to the CTX-M family, and was nominated CTX-M-6.
In conclusion, increasing resistant rates to most antibiotics were observed, which remained stable or declined during the last year of the study. At least six genotypically distinct clones with differing chromosomal backgrounds and mechanisms of resistance to antibiotics were involved in multi-drug resistant DTI04. Although the majority of the examined population was composed of three genomic types, some minor types were related to specific resistance phenotypes or isolates from a specific source, whilst the detection of a resistance gene which encoded for an extended spectrum ß-lactamase, located on a small size transferable plasmid makes possible its fiirther dissemination among other salmonella serotypes or enterobacteriae species.
Background & Objective: Molecular typing is an important tool in surveillance and outbreak investigations of human
Salmonella infections. In this study, Subtyping of Salmonella Paratyphi B and C isolates derived from Iranian patients was
carried out by RAPD-PCR to assess the extent of genetic diversity of these isolates.
Materials & Methods: Fourteen Salmonella isolates including 6 strains of Salmonella paratyphi B and 8 strains of Salmonella
paratyphi C were characterized using RAPD-PCR. Two arbitrary primers, namely OPP-16 and P1254 were used
for RAPD analysis and the dendrograms were constructed with NTsys 2.0 computer software.
Results: Both primers showed high discriminatory power in differentiating of the related strains of Salmonella. The dendrograms
constructed based on RAPD-PCR profiles (with both primers) involving 14 salmonella strains revealed 4 distinct
patterns, indicating that these isolates are genetically heterogeneous. Furthermore, a good correlation was not observed
between the serotype and the molecular profiles obtained from RAPD data of the Salmonella isolates.
Conclusion: The findings of the present study verify the usefulness of RAPD-PCR in characterizing and comparing strains
of Salmonella Paratyphi B and C.
Salmonella is a mesophilic bacterium that share common characteristics with Enterobacteriaceae. Two species are described: Salmonella enterica and Salmonella bongori. Beside the fact that the infection in host cells requires type III secretion-systems, little is known, at present, about virulence mechanisms. Among the cultural detection methods, the use of semi-solid media seems more efficient than the others for Salmonella recovery. The techniques based on genetic amplification may be useful in order to further characterize the isolated strains. Salmonella can be isolated from the intestine of numerous animal species and its survival in the surroundings may be quite long. Several serotypes may cause clinical salmonellosis while others may be responsible for animal species of a carrier state. In this abstract, the influence of swine production system will be developed. The sustainable and ongoing surveillance are justified by the fact that Salmonella leads to numerous foodborne cases and outbreaks and is responsible of important economic and social costs. This surveillance aims to improve the sanitary quality of food from "farm to fork". The preventive methods available for the pre-harvest production step and for the slaughterhouse will be also evoked.
A total of 32 clinical strains of Vibrio cholerae, including members of the 01 and 0139 serogroup were collected from Klang, Selangor; Penang Island; Samarahan, Sarawak and Miri, Sarawak in Malaysia. In general, all the isolates except the 0139 serotype expressed low resistance to all the antibiotics tested with their Multiple Antibiotic Resistance (MAR) indices ranged from 0.10 to 0.48. The presence of ctx gene that encoded the cholera toxin was confirmed in all these clinical isolates by polymerase chain reaction. The results from the RAPD-PCR were analyzed using the RAPDistance software (Version 1.04). From the dendrogram generated, two main groups were observed which were subdivided into two clusters each. The Selangor's isolates and the 0139 Penang's isolates formed one group whereas the Samarahan, Sarawak isolates and the Miri, Sarawak isolates made up the other group, thus delineating their different sources of origin based on their geographical location.
Different PCR-based DNA fingerprinting techniques were evaluated for identification and discrimination of bacterial strains. Fifteen strains of Salmonella enterica subspecies enterica serotypes Typhi (10), Paratyphi A (1) and Typhimurium (3) collected over a period of 15 years from stool, blood, and urine samples and river Ganga were tested. All the strains were analysed by restriction analysis of the amplified 16s rDNA (ARDRA), random amplified polymorphic DNA (RAPD) and BOX-PCR methods. In ARDRA, strains belonging to the same species were identified by identical fingerprints; RAPD on the other hand, divided Salmonella into 9 different groups. Two ciprofloxacin (CIP)-resistant strains showed similar PCR fingerprints which were different from the rest of the 12 strains. In BOX-PCR, all the strains of Salmonella showed 6 different groups which showed the presence of a common band. Serotype Typhimurium could be placed in the same group by BOX-PCR. CIP-resistant strains also produced one characteristic extra band by BOX-PCR. It was observed that RAPD had higher discriminatory power than BOX-PCR and was a simple and rapid technique for use in epidemiological studies of isolates belonging to S. enterica.
Publisher Summary
This chapter focuses on the measurement of the microbiological contamination in fruit and vegetables. Foodborne diseases are among the most serious public health concerns all over the world and are a major cause of morbidity. This threat has been increasing with global trade and travel over the past decades, affecting both industrialized and developing countries. More than 200 known diseases are transmitted through food, with symptoms ranging from mild gastroenteritis to life-threatening syndromes, with the possibility of chronic complications or disability. More than 40 different foodborne pathogens are known to cause human illness, among which over 90% of confirmed foodborne human illness cases and deaths caused by foodborne pathogens reported to the Center for Disease Control and Prevention (CDC) have been attributed to bacteria, the rest being due to fungi, parasites, and viruses. In consequence, microbiological quality control programs are being increasingly applied throughout the food production chain in order to minimize the risk of infection for the consumer.
Globalization is a primary factor aiding spread of emerging infectious diseases. Thus, detection of pathogens is more critical than in the past, as recent outbreaks of SARS along with emergence of antibiotic-resistant microorganisms have demonstrated. Owing to the increasing need for detection of infectious agents in the field, and in third world countries, field operability of detection systems is becoming key for surveillance in endemic regions of the world. As such this chapter presents currently available technologies that can be employed in such portable detection systems to help meet the emerging disease challenges of the coming years.
Keywords:
viruses;
field operable;
biosensor;
surveillance;
tropical;
emerging diseases;
biochips, immunosensor;
pathogen detection;
BioPen
This article examines the role of nucleic acid analysis in clinical chemistry. It is a broad-based article and begins with the background knowledge which will be needed to appreciate the succeeding sections. In the Introduction, structure and function of nucleic acids, hybridization, probe labeling, hybridization protection, polymerase chain reaction (PCR) and sequencing are discussed. Major sections are devoted to microbiological analysis (with particular emphasis on bacteriology), genetic diseases and screening [discussions are provided for cystic fibrosis (CF), heriditary fructose intolerance (HFI) and hemochromatosis along with an introduction to the Human Genome Project and single nucleotide polymorphisms (SNP)], other areas of human medicine (oncology, colorectal cancer and Alzheimer's disease (AD) are considered) and the biosensor approach. A discussion of interesting current and future developments, not only in nucleic acid analysis, but also in gene therapy, concludes the article.
A simple and efficient method has been developed for isolating genomic DNA from single specimens of extremely small metazoan animals, e.g. rotifers (Rotifera: Monogononta). The amount and quality of the extracted DNA allows the Random Amplified Polymorphic DNA Polymerase Chain Reaction (RAPD-PCR) to be employed with up to three random primers per specimen. Reproducibility of RAPD-fingerprints has been checked and verified. Exemplarily, RAPD patterns of three Brachionus species (B. angularis, B. calyciflorus, B. urceolaris) amplified with the primer OPA 07 are demonstrated.
In most developed countries a sharp increase in foodborne intoxications occurs since the last decade. Amongst the bacterial pathogens, the incidence rate is highest for Campylobacter jejuni and Salmonella spp. Nucleid acid based identification and detection methods have been developed for nearly all bacterial pathogens, based on probes in hybridisation assays or primers in PCR, NASBA or RT-PCR assays. As targets for molecular identification, virulence genes, the rRNA gene region or other specific sequences can be used and several commercialised systems are already available. For the (direct) detection of pathogens in food products, several problems may be encountered: PCR inhibition by food components, contamination in sensitive PCR assays, detection of living as well as dead cells. The latter problem can be solved by using mRNA as amplification target, but for routine applications the combination of a short culturing period with a less sensitive PCR is more suitable. Direct quantification of pathogens is possible with quantitative competitive PCR using an internal standard or with kinetic quantitative PCR (TaqMan or LightCycler commercial system).
Salmonella enterica serotype gallinarum biotype gallinarum and biotype pullorum are non-motile and pathogenic avian strains. Biotype gallinarum causes fowl typhoid and biotype pullorum is the cause of pullorum disease in chickens. The two biotypes could be differentiated based on biochemical characteristics. However, conventional culture and biochemical assays are time-consuming, laborious and need sterile laboratory practices. Although the two biotypes, gallinarum and pullorum are non-motile, they possess the phase 1 flagellin C gene. The variable regions of the flagellin C gene from 41 biotype pullorum and 52 biotype gallinarum were amplified by colony-PCR and analyzed by single strand conformational polymorphism (SSCP) method. Differences in SSCP electrophoretic patterns were confirmed by nucleotide sequencing. In addition, PCR-RFLP with Hinp1I was also successfully applied to differentiate the two biotypes. These results suggested that the variable regions of fliC could be used as a genetic marker to differentiate biotype gallinarum from biotype pullorum.
RESUMEN Bacillus cereus es un bacilo Gram positivo esporulado contaminante habitual de alimentos tanto frescos como procesados. La detección y cuantificación de este patógeno de alimentos se realiza habi-tualmente por técnicas culturales. Dichas técnicas consumen mucho tiempo y, en ocasiones, llevan a identificaciones erróneas. Como alternativa, las técnicas moleculares basadas en la PCR tienen como ventaja una mayor rapidez y seguridad en la identificación del microorganismo, e incluso permiten su cuantificación mediante PCR a tiempo real (Q-PCR). En este estudio se han desarrollado diferentes métodos para el control de este microorganismo en alimentos, basados en la técnica de la PCR. A partir de una colección de cepas de referencia y aislados de alimentos, identificados previamente por métodos fenotípicos, (ISO 7932 y API-50CH/B) y genotípicos, (ISR-PCR y RAPD), se determinó la presencia de genes relacionados con factores de virulencia mediante PCR convencional. El gen pc-plc se detectó en un mayor porcentaje en las cepas del grupo B. cereus, y dado que se encuentra en una única copia por genoma, fue seleccionado como gen diana. Se diseñaron 4 oligonucleótidos para el desarrollo de procedimientos de Q-PCR en modo SYBR Green y Taqman. Se optimizaron los parámetros de la reacción de cuantificación, y se evaluó su especificidad para el grupo B. cereus, resultando más adecuado el procedimiento de SYBR Green Q-PCR. A continuación se ensayó la capacidad para la detección y cuantificación en alimentos contaminados artificialmente: huevo líquido y leche en polvo infantil. Como resultado el sistema desarrollado ha demostrado su capacidad para la cuantificación de B. cereus entre 105 y 100 ufc/ml a partir de la matriz ensayada, que corresponde al mismo nivel que el método de referencia por recuento en placa. Dado que B. cereus puede estar presente en alimentos tanto en forma vegetativa como esporulada, para su detección por PCR se abordó, asimismo el desarrollo de un protocolo para asegurar la liberación del DNA a partir de esporas. Se partió de suspensiones calibradas de esporas que fueron sometidas a diferentes tratamientos: a) térmicos y b) adición de germinadores en diferentes combinaciones. Tras los tratamientos, se realizó la extracción de DNA con DNeasy Tissue kit (Qiagen), se cuantificó y se evaluó el rendimiento espectrofluorimétricamente (PicoGreen, Invitrogen) y mediante PCR convencional y SYBR Green Q-PCR. El tratamiento de germinación con 0,5 mM L-alanina/inosina resultó el más eficiente en suspensiones concentradas de esporas. Sin embargo, cuando se ensayó en alimentos inoculados (104 - 100 esporas/ml), la extracción con DNeasy Tissue Kit, sin necesidad de tratamientos previos, resultó satisfactorio. Por último, se realizó una aproximación cuantitativa para la detección de formas viables de B. cereus. Para ello se desarrolló un procedimiento de PCR a tiempo real con transcripción inversa (Q-RT-PCR) utilizando como diana el RNA mensajero del gen pc-plc, como indicador del estado de viabilidad celular. El nivel de sensibilidad obtenido por Q-RT-PCR fue de 30 células/reacción a partir de suspensiones celulares, y de 847 células/reacción tras su aplicación en huevo líquido. Los procedimientos de Q-PCR y Q-RT-PCR desarrollados en este estudio han mostrado ser eficientes para detectar la presencia de las especies del grupo B. cereus en alimentos, al menos al mismo nivel que el método de referencia, permitiendo además la cuantificación incluso de formas viables. __________________________________________________________________________________________________ Bacillus cereus is a rod-shaped, Gram-positive sporeforming bacterium widely recognized as a food poisoning organism. Detection of this pathogen in food is usually carried out by culture techniques that are time consuming and frequently lead to misidentifications. As an alternative, molecular genetic techniques allow rapid, sensitive and accurate detection. In the present work, PCR based systems were applied for a rapid and unequivocal detection of B. cereus group which would be very valuable in food safety assessment. The presence of virulence genes was tested by PCR amplification in a collection of reference strains and food isolates that had been phenotypically and genotypically characterized. The pc-plc gene showed the highest prevalence among the tested strains and five oligonucleotides were designed in order to develop SYBR Green and TaqMan real-time PCR (Q-PCR) procedures. The SYBR Green Q-PCR was applied on spiked liquid egg and dried infant formulae showing suitable quantification results in a range of 105 to 100 ufc/ml, and high degrees of correspondence between Q-PCR assays and plate counts. To assess detection of B. cereus spores by PCR procedures, several DNA isolation methods were tested on spore suspensions: i) heat treatment and ii) germination triggered by nutrient germinants followed by DNA isolation (DNeasy tissue kit, Qiagen). Recovery of DNA was evaluated by PCR, Q-PCR, and PicoGreen fluorescence (Invitrogen). Germination with L-alanine 0.5 mM/inosine 0.5 mM rendered the maximum rate of DNA at 108 spores/ml. However, DNA extraction without any modifications rendered similar DNA recovery on 105 to 100 spores/ml range. Spore detection was established in 3-4 spores/reaction using both spore suspensions and spiked food samples. Besides pathogen detection, cell viability is important in food safety. Thus a one-step Q-RT-PCR detection system, was established to investigate the presence of metabolically active cells. By using this approach, 30 cells/reaction and 847 cells/reaction were detected in cell suspensions and spiked liquid egg samples, respectively. The developed Q-PCR and Q-RT-PCR systems proved to be highly sensitive and specific for the rapid and accurate detection and quantification of species of the B. cereus group in food, and constitute a promising tool in the detection of B. cereus including spores as well as viable cells.
Salmonella enterica is an important enteric pathogen consisting of many serovars that can cause severe clinical diseases in animals and humans. Rapid identification of Salmonella isolates is especially important for epidemiologic monitoring and controlling outbreaks of disease. Although immunologic and DNA-based serovar identification methods are available for rapid identification of isolates, they are time consuming or costly or both. In the current study, 2 molecular methods for identification of Salmonella serovars were developed and validated. A 70-mer oligonucleotide spotted microarray was developed that consisted of probes that detected genes responsible for genetic variation among isolates of Salmonella that can be used for serotyping. A multiplex polymerase chain reaction (PCR) assay was also developed, which is capable of identifying 42 serovars, thus providing a valuable prediction of the pathogenicity of the isolates by detecting the presence of virulence genes sseL, invA, and spvC. The gene spvC was the best predictor of pathogenicity. In a blind study, traditional serologic methods were correlated at 93.3% with the microarray-based method and 100% with the multiplex PCR-based serovar determination.
The RAPD (or AP-PCR) DNA fingerprinting method was used to distinguish among clinical isolates of Helicobacter pylori, a bacterium whose long term carriage is associated with gastritis, peptic ulcers and gastric carcinomas. This method uses
arbitrarily chosen oligonucleotides to prime DNA synthesis from genomlc sites to which they are fortuitously matched, or almost
matched. Most 10-nt primers with ss 60% G + C yielded strain-specific arrays of up to 15 prominent fragments, as did most
longer (≥ 17-nt) primers, whereas most 10-nt primers with 50% G + C did not. Each of 64 Independent H.pylori isolates, 60 of which were from patients in the same hospital, was distinguishable with a single RAPD primer, which suggests
a high level of DNA sequence diversity within this species. In contrast, isolates from initial and followup biopsies were
indistinguishable in each of three cases tested.
Characterization of 169 strains of Salmonella typhi of phage types C 1 , C 4 , D 1 and D 9 isolated in 1975–88 was carried out by rDNA gene restriction pattern analysis. Twenty-four isolates had been recovered during four large waterbone outbreaks in the last 20 years in Sicily; 145 strains, isolated from apparently sporadic cases of infection in Southern Italy in the same period of time, were also examined.
Application of rRNA–DNA hybridization technique after digestion of chromosomal DNA with Cla I showed the identity of patterns of the epidemic strains of phage types C 1 and D 1 , confirming attribution of the outbreaks to single bacterial clones. Patterns of the two available strains of lysotype D 9 were slightly different, whilst the 12 epidemic strains of phage type C 4 could be assigned to two distinct patterns scarcely related to each other and, consequently, to two different clones. A considerable heterogeneity was detected among all apparently sporadic isolates of the four phage types under study.
This fingerprinting method appears a reliable tool to complement phage typing in characterizing isolates of S. typhi. In particular, epidemiological features of spread of this salmonella serovar in areas, where simultaneous circulation of indigenous and imported strains occurs, can be elucidated.
Multilocus enzyme electrophoresis was employed to measure chromosomal genotypic diversity and evolutionary relationships among 761 isolates of the serovars Salmonella typhi, S. paratyphi A, S. paratyphi B, S. paratyphi C, and S. sendai, which are human-adapted agents of enteric fever, and S. miami and S. java, which are serotypically similar to S. sendai and S. paratyphi B, respectively, but cause gastroenteritis in both humans and animals. To determine the phylogenetic positions of the clones of these forms within the context of the salmonellae of subspecies I, comparative data for 22 other common serovars were utilized. Except for S. paratyphi A and S. sendai, the analysis revealed no close phylogenetic relationships among clones of different human-adapted serovars, which implies convergence in host adaptation and virulence factors. Clones of S. miami are not allied with those of S. sendai or S. paratyphi A, being, instead, closely related to strains of S. panama. Clones of S. paratyphi B and S. java belong to a large phylogenetic complex that includes clones of S. typhimurium, S. heidelberg, S. saintpaul, and S. muenchen. Most strains of S. paratyphi B belong to a globally distributed clone that is highly polymorphic in biotype, bacteriophage type, and several other characters, whereas strains of S. java represent seven diverse lineages. The flagellar monophasic forms of S. java are genotypically more similar to clones of S. typhimurium than to other clones of S. java or S. paratyphi B. Clones of S. paratyphi C are related to those of S. choleraesuis. DNA probing with a segment of the viaB region specific for the Vi capsular antigen genes indicated that the frequent failure of isolates of S. paratyphi C to express Vi antigen is almost entirely attributable to regulatory processes rather than to an absence of the structural determinant genes themselves. Two clones of S. typhisuis are related to those of S. choleraesuis and S. paratyphi C, but a third clone is not. Although the clones of S. decatur and S. choleraesuis are serologically and biochemically similar, they are genotypically very distinct. Two clones of S. typhi were distinguished, one globally distributed and another apparently confined to Africa; both clones are distantly related to those of all other serovars studied.
Simple and reproducible fingerprints of complex genomes can be generated using single arbitrarily chosen primers and the polymerase
chain reaction (PCR). No prior sequence information is required. The method, arbitrarily primed PCR (AP-PCR), involves two
cycles of low stringency amplification followed by PCR at higher stringency. We show that strains can be distinguished by
comparing polymorphisms in genomic fingerprints. The generality of the method Is demonstrated by application to twenty four
strains from five species of Staphylococcus, eleven strains of Streptococcus pyogenes and three varieties of Oryza sativa (rice).
To date, epidemiologic associations among strains of Salmonella typhi are based exclusively on phage typing, which may be of limited value if a common phage type is involved. Analysis of ribosomal
RNA gene restriction patterns allows separation of most independently isolated strains of identical phage types. The sensitivity
of the method is dependent on the restriction enzymes used to digest chromosomal DNA. It was highest for PstI, which separated 16 of 20 strains that belonged to 8 phage types including 3 untypable strains. Three strains differed in
their phage types but had identical ribosomal RNA gene restriction patterns. Also, two pairs of strains indistinguishable
by phage typing exhibited identical patterns; however, two of these strains were expected to be identical because they were
isolated from two patients who were likely exposed to the same source. Ribosomal RNA gene restriction patterns appear to be
stable. Thus, the method may complement phage typing and aid in further differentiation of strains.
Shigella sonnei is a major cause of diarrheal disease in developed as well as in developing countries. Epidemiologic studies of this organism have been limited by the lack of a simple and effective method for comparing strains. In this study, we have compared different molecular typing methods, i.e., plasmid profile analysis, restriction endonuclease analysis of plasmids, rRNA gene restriction analysis (ribotyping), pulsed-field gel electrophoresis (PFGE), and enterobacterial repetitive intergenic consensus (ERIC) sequence-based PCR (ERIC-PCR) for typing 20 clinical isolates of S. sonnei collected from six incidents of infection. PFGE and ERIC-PCR fingerprintings had the highest discriminatory power for discrimination of epidemiologically related isolates from epidemiologically unrelated strains of S. sonnei, and both gave seven distinct strain types among these isolates and the type strain of the species. Plasmid study and ribotyping produced only six and typing techniques demonstrated two distinct patterns, respectively, among these strains. All of these molecular an identical fingerprint for eight temporally related sporadic isolates. It is possible that these temporally related isolates belonged to a single bacterial clone and circulated obscurely through the community. Our results indicate that the ERIC-PCR technique represents a rapid and simple means for typing S. sonnei with a level of discrimination equivalent to that of PFGE but greater than those of plasmid profile analysis, restriction endonuclease analysis of plasmids, and ribotyping.
Live attenuated vaccines, developed with molecular genetical techniques, require new approaches for their quality control. To develop novel quality control tests that enhanced and extended existing procedures, the attenuated vaccine strain Salmonella typhi Ty21a and its parent strain Ty2 were characterized by pulsed-field gel electrophoresis (PFGE) and direct nucleotide sequence analysis. Mutant and parent strains were distinguished using fingerprints generated by the resolution on PFGE of chromosomal DNA digested with each of the enzymes SfiI, SpeI or XbaI. These fingerprints were stable through multiple in vitro passages of the vaccine strain and were identical from one batch of vaccine to another. It was also possible to distinguish between the mutant and parent strains by direct nucleotide sequence analysis of the galE gene. This analysis identified two base changes in the gene from strain Ty21a: a single base deletion causing a frameshift that would result in a truncated gene product, accounting for the galE phenotype; and a transition that eliminated an AluI restriction site. The consequent change in the AluI fingerprint of the galE gene in strain Ty21a provided a rapid, PCR-based alternative to the use of differential media or biochemical assays for the identification of the vaccine strain.
Pulsed-field gel electrophoresis (PFGE) revealed that multiple genetic variants of Salmonella typhi are simultaneously present in Southeast Asia and are associated with sporadic cases of typhoid fever and occasional outbreaks. Comparative analysis of PFGE patterns also suggested that considerable genetic diversity exists among S. typhi strains and that some PFGE patterns are shared between isolates obtained from Malaysia, Indonesia, and Thailand, implying movement of these strains within these regions of Southeast Asia, where they are endemic.
Pulsed-field gel electrophoresis (PFGE) was used to compare and analyze 158 isolates of Salmonella typhi from five well-defined outbreaks of typhoid fever in Malaysia and also isolates involved in sporadic cases of typhoid fever occurring during the same period. Digestion of chromosomal DNAs from these S. typhi isolates with the restriction endonucleases XbaI (5'-TCTAGA-3'), SpeI (5'-ACTAGT-3'), and AvrII (5'-CCTAGG-3') and then PFGE produced restriction endonuclease analysis (REA) patterns consisting of 11 to 24 DNA fragments ranging in size from 20 to 630 kbp. Analysis of the REA patterns generated by PFGE after digestion with XbaI and SpeI indicated that the S. typhi isolates obtained from sporadic cases of infection were much more heterogeneous (at least 13 different REA patterns were detected; Dice coefficient, between 0.73 and 1.0) than those obtained during outbreaks of typhoid fever. The clonal nature and the close genetic identities of isolates from outbreaks in Alor Setar, Penang, Kota Kinabalu, Johor Bahru, and Kota Bahru were suggested by the fact that only a limited number of REA patterns, which mostly differed by only a single band, were detected (one to four patterns; Dice coefficient, between 0.82 and 1.0), although a different pattern was associated with each of these outbreaks. Comparison of REA patterns with ribotyping for 18 S. typhi isolates involved in sporadic cases of infection showed a good correlation, in that 72% of the isolates were in the same group. There was no clear correlation of phage types with a specific REA pattern. We conclude that PFGE of s. typhi chromosomal DNA digested with infrequently cutting restriction endonucleases is a useful method for comparing and differentiating S. typhi isolates for epidemiological purposes.
The genomic DNA of 39 strains of Salmonella typhi isolated from local residents and patients who had visited countries in the Asian region was analysed for restriction fragment length polymorphisms (RFLP). Pulsed-field gel electrophoretic (PFGE) analysis of Xba I- and Spe I-generated genomic restriction fragments established 22 PFGE types whereas phage typing differentiated the 39 isolates into 9 distinct phage types. This study showed that PFGE is more discriminatory than phage typing as it is capable of subtyping S. typhi strains of the same phage types. Genetic relatedness among the isolates was determined. Seven major clusters were identified at SABs of > 0.80 and the remaining 13 isolates were distributed into minor clusters which were related at SABs of less than 0.80. In conclusion, PFGE analysis in conjunction with distance matrix analysis served as a useful tool for delineating common S. typhi phage types of diverse origins from different geographical locales and separated in time.
Seventy-eight Salmonella typhi strains isolated in 1994 and 1995 from patients living in Dhaka, Bangladesh, were subjected to phage typing, ribotyping, IS200 fingerprinting, and PCR fingerprinting. The collection displayed a high degree of genetic homogeneity, because restricted numbers of phage types and DNA fingerprints were observed. A significant number of the S. typhi strains (67%) were demonstrated to be multiple drug resistant (MDR). The vast majority of the MDR strains were resistant to chloramphenicol, ampicillin, trimethoprim, streptomycin, sulfamethoxazole, and tetracycline (R type CATmSSuT), a resistance phenotype that has also frequently been observed in India. Only two strains displayed a distinct MDR phenotype, R type AT-mSSuT. Pulsed-field gel electrophoresis demonstrated the presence of large plasmids exclusively in the MDR strains of both R types. The plasmids present in the S. typhi strains of R type CATmSSuT could be conjugated to Escherichia coli and resulted in the complete transfer of the MDR phenotype. PCR fingerprinting allowed discrimination of MDR and susceptible strains. The DNA fragments enabling discrimination of MDR and susceptible S. typhi strains by PCR were useful genetic markers for identifying MDR encoded by large plasmids of the H1 incompatibility group.
The efficiencies of different tests for epidemiological markers--phage typing, ribotyping, IS200 typing, and pulsed-field gel electrophoresis (PFGE)--were evaluated for strains from sporadic cases of typhoid fever and a well-defined outbreak. Ribotyping and PFGE proved to be the most discriminating. Both detected two different patterns among outbreak-associated strains.
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Random amplification of polymorphic DNA (RAPD) was evaluated for its ability to differentiate Salmonella strains from various sources. Under defined conditions RAPD using a 10-mer primer (1254) produced a series of amplification products able to reproducibly distinguish strains representing 20 different serotypes of Salmonella. Primer 1254 also proved capable of discrimination between some but not all isolates of Salm. ser. Enteritidis and Salm. ser. Typhimurium, phage typing proving to be most discriminatory for the latter serotype. Cloning of fragments into a vector allowed sequencing and database searching for identification of fragments and an indication of criteria for primer template interaction in RAPD. Southern blotting using a digoxigenin-labelled probe allowed identification of related bands between RAPD profiles. These observations demonstrate the potential of rapid molecular typing by RAPD for the genomic typing of Salmonella strains.
Randomly amplified polymorphic DNA (RAPD) and arbitrarily primed PCR (AP-PCR) represent novel DNA polymorphism assays that involve the amplification of random DNA segments using PCR and oligonucleotide primers of arbitrary sequence. Products defining the polymorphisms exhibit Mendelian inheritance and thus possess tremendous potential utility as genetic markers in a diverse array of scientific disciplines. Amplification profiles for specific oligonucleotide primers are highly dependent on the specific conditions of the reaction; banding patterns may thus vary extensively because of inconsistencies in a number of reaction parameters. Artifactual variation represents a potential problem in surveys of genetic variation in natural populations and must be discriminated from true polymorphism for the applications of RAPD to be both accurate and reliable.
An outbreak of typhoid fever in Zurich, Switzerland, which involved seven customers and three employees of a city mall restaurant, was investigated by comparing three molecular typing methods: pulsed-field gel electrophoresis (PFGE), ribosomal RNA gene restriction patterns (ribotyping), and random amplified polymorphic DNA (RAPD). Both PFGE and ribotyping identified two molecular patterns among the outbreak-related isolates which differed in one band: these isolates were considered clonally related and differed clearly from other unrelated S. typhi strains. RAPD could not distinguish among outbreak isolates and control strains.
We present a method that permits the rapid generation of DNA probes for eubacteria. In the procedure the variable regions for the 16s rRNA genes are amplified using polymerase chain reaction (PCR) technology and primers based on the conserved regions of these genes. Following sequencing of the variable regions, a choice is possible for a probe specific for that organism. No knowledge of the molecular biology of the microorganism is required prior to the application of this approach. The generality of the method is shown using Salmonella typhimurium, Staphylococcus aureus, Clostridium perfringens, Klebsiella pneumoniae, Pseudomonas fluorescens, Aeromonas salmonicida and Mycobacterium bovis. A. salmonicida was examined in detail and a DNA probe was prepared that distinguishes it from other Aeromonas species.
Molecular genetic maps are commonly constructed by analyzing the segregation of restriction fragment length polymorphisms
(RFLPs) among the progeny of a sexual cross. Here we describe a new DNA polymorphism assay based on the amplification of random
DNA segments with single primers of arbitrary nucleotide sequence. These polymorphisms, simply detected as DNA segments which
amplify from one parent but not the other, are inherited in a Mendellan fashion and can be used to construct genetic maps
in a variety of species. We suggest that these polymorphisms be called RAPD markers, after Random Amplified Polymorphic DNA.
Crude cell extracts of 26 isolates of Salmonella serotype typhi (S. typhi) and 48 other Salmonella isolates representing 28 serotypes and seven DNA hybridization subgroups were analyzed for electrophoretic variants of 24 metabolic enzymes by starch gel electrophoresis. All strains of S. typhi had identical isoenzyme patterns, indicating that they were a single clone. All of the enzymes detected in the remaining strains were polymorphic, and the degree of genetic variation was quite high. The average number of alleles per enzyme locus was 4.7, and the mean genetic diversity per locus was 0.556. Thirty-two distinct allele profiles, or electrophoretic types (ETs), were found in these 48 strains of Salmonella serotypes other than S. typhi. Analysis of the genetic relationships of the ETs to each other showed that, with one exception, the ETs formed subgroups that were consistent with the subgroupings based on DNA hybridization studies. ET profiles were not always linked to specific serologic patterns. These data show that multilocus enzyme electrophoresis has a potential application in epidemiologic and taxonomic studies of salmonellae, although it is not differential for S. typhi. We also propose a new species, Salmonella bongori comb. nov., a new combination base on the elevation of Salmonella choleraesuis subsp. bongori to the level of species.
From 1974 through 1981, 4,089 isolates of Salmonella typhi were phage typed at the Centers for Disease Control and nine regional laboratories in the United States. The most prevalent types were degraded Vi (27%), E1 (20.6%), A (9.8%), C1 (5.7%), untypable Vi (3.5%), W form (3.5%), D1 (3.4%), B3 (3.4%), and F1 (2.4%). There were less than 2% of each of the remaining types. The distribution of phage types for this time period was similar to that seen in the periods 1966-1969 and 1970-1973, except that phage type B3 was one of the 10 most prevalent types in 1974-1981 but was not seen in 1966-1973. Phage typing is presently the most valuable laboratory tool for differentiation of strains of S. typhi in epidemiological studies.
Seventy-seven Salmonella isolates comprising 61 different serotypes were subjected to polymerase chain reaction (PCR) fingerprinting using two primersets. Primerset L1/G1, amplifying the spacer regions between the 16S and 23S rRNA genes, resulted in simple PCR fingerprints. However, in some cases PCR amplification of different Salmonella serotypes with primerset L1/G1 resulted in identical fingerprint profiles. Fingerprints obtained with the ERIC primerset, that matches the enterobacterial repetitive intergenic consensus sequence, were more complicated but were serotype-specific. Consequently, fingerprinting with the ERIC primerset is applicable for typing Salmonella up to the serotype level. Fingerprinting with the L1 and G1 primers requires an additional treatment of the amplification product for accurate typing of salmonellas. Phage typing is not possible with either primerset.
Randomly amplified polymorphic DNA (RAPD) and arbitrarily primed PCR (AP-PCR) represent novel DNA polymorphism assays that involve the amplification of random DNA segments using PCR and oligonucleotide primers of arbitrary sequence. Products defining the polymorphisms exhibit Mendelian inheritance and thus possess tremendous potential utility as genetic markers in a diverse array of scientific disciplines. Amplification profiles for specific oligonucleotide primers are highly dependent on the specific conditions of the reaction; banding patterns may thus vary extensively because of inconsistencies in a number of reaction parameters. Artifactual variation represents a potential problem in surveys of genetic variation in natural populations and must be discriminated from true polymorphism for the applications of RAPD to be both accurate and reliable.
A random amplified polymorphic DNA (RAPD) fingerprinting method has been developed to differentiate Salmonella enteritidis isolates. A total of 65 arbitrary primers were screened with S. enteritidis isolates of different phage types. This allowed selection of a panel of primers capable of detecting DNA polymorphisms among S. enteritidis isolates. This panel was used to examine a panel of 29 isolates of S. enteritidis which had been previously characterized by other subtyping methods, including phage typing (PT) (n = 7), ribotyping (RT) (n = 13), and pulsed-field gel electrophoresis (PFGE). Applied collectively, these three methods resolved the collection into 20 different subtypes. However, by the RAPD fingerprinting method alone, 14 RAPD subtypes were revealed. Eight isolates of S. enteritidis phage type 8 that failed to be discriminated by other typing methods (PT, RT, and PFGE) were resolved into three different subtypes by RAPD analysis. In contrast, isolates that were derived from the same sources were not differentiated by any of the subtyping methods employed, including PT, RT, PFGE, and RAPD analysis. This RAPD approach to S. enteritidis subtyping provided more discriminatory power than did any of several other subtyping methods applied individually. Once the challenging step of primer identification was accomplished, determinations of the appropriate concentrations of arbitrary primer, DNA template, and MG2+ ion were also necessary for optimal discriminatory power. The bacterial DNA used in this RAPD protocol was obtained by boiling the bacterial sample. This simple procedure yielded DNA that produced fingerprint patterns as consistent as those obtained from phenol-chloroform-extracted DNA. Clearly, when appropriately constituted primer sets are identified and employed, RAPD analysis provides a simple, rapid, and powerful subtyping method for S. enteritidis.
Random amplification of polymorphic DNA (RAPD) was evaluated for its ability to differentiate Salmonella strains from various sources. Under defined conditions RAPD using a 10-mer primer (1254) produced a series of amplification products able to reproducibly distinguish strains representing 20 different serotypes of Salmonella. Primer 1254 also proved capable of discrimination between some but not all isolates of Salm. ser. Enteritidis and Salm. ser. Typhimurium, phage typing proving to be most discriminatory for the latter serotype. Cloning of fragments into a vector allowed sequencing and database searching for identification of fragments and an indication of criteria for primer template interaction in RAPD. Southern blotting using a digoxigenin-labelled probe allowed identification of related bands between RAPD profiles. These observations demonstrate the potential of rapid molecular typing by RAPD for the genomic typing of Salmonella strains.
Random amplification of polymorphic DNA (RAPD) is proving to be a useful technique in studying the epidemiology of micro-organisms. The technique can be troublesome and time consuming to establish due to the essentially empirical approach to optimization. By standardization of certain parameters and use of a commercially available PCR buffer optimization kit, a particularly promising primer was identified and RAPD conditions for a highly discriminatory and reproducible characterization of Salmonella isolates was achieved. In addition, a technique to obtain reproducible RAPD fingerprints of Salmonella isolates without the need to purify genomic DNA is described.
The rRNA gene restriction patterns and the polymerase chain reaction (PCR) fingerprinting types of 53 Vibrio cholerae O1 isolates were studied. Five and eight patterns were observed from 27 toxigenic and 26 non-toxigenic O1 isolates after BglI cleavage. PCR fingerprinting with three primer sets aimed at enterobacterial repetitive intergenic consensus (ERIC) sequences, ERIC-related sequences in V. cholerae, another kind of repeated sequences in V. cholerae (VCR) and arbitrary sequences divided the same strains into seven and 10 PCR types, respectively. Eight ribotypes had unique PCR patterns. PCR fingerprinting identified more than one pattern among isolates within each of the remaining ribotypes. However, ribotyping was able to differentiate the same PCR types in one case. A single ribotype and a single PCR pattern were found in toxigenic O1 strains isolated in Taiwan from imported food and imported cases of cholera between 1993 and 1995. Typing of V. cholerae O1 by PCR fingerprinting correlated well with ribotyping, but was more discriminating. PCR assay provides a rapid and simple means of typing these strains for epidemiological studies.
III and Regional Centers for Salmonella typhi Bacteriophage Typing in the United States (1983) Bacteriophage types of Salmonella typhi in the United States from
- F W Hickman-Brenner
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