Rapid identification of Salmonella enterica serovars, Typhimurium, Choleraesuis, Infantis, Hadar, Enteritidis, Dublin and Gallinarum, by multiplex PCR

Salmonella enterica subsp. enterica poses a threat to both human and animal health, with more than 2500 serovars having been reported to date. Salmonella serovars are identified by slide and tube agglutination tests using O and H antigen-specific anti-sera, although this procedure is both labor intensive and time consuming. Establishment of a method for rapid screening of the major Salmonella serovars is therefore required. We have established multiplex polymerase chain reaction (m-PCR) assays for identification of seven serovars of Salmonella, i.e., Typhimurium, Choleraesuis, Infantis, Hadar, Enteritidis, Dublin and Gallinarum. Three serovar-specific genomic regions (SSGRs) of each serovar were selected using an approach in comparative genomics. The Salmonella-specific invA gene was used to confirm the genetic background of the organisms. The isolates tested were identified as a target serovar when the three selected SSGRs and invA were all positive for amplification. The specificity of each m-PCR assay was investigated using 118 serovars of Salmonella and 12 species of non-Salmonella strains. Although a small number of false-positive results were observed in the m-PCR assays used to identify Typhimurium, Choleraesuis, Enteritidis and Dublin for closely related serovars, false-negative results were not observed in any assays. These assays had sufficient specificity to identify the seven Salmonella serovars, and therefore, have the potential for use as rapid screening methods.     

Comparison of a semi-automated rep-PCR system and multilocus sequence typing for differentiation of Salmonella enterica isolates

The accurate sub-typing of Salmonella enterica isolates is essential for epidemiological investigations and surveillance of Salmonella infections. Salmonella isolates are currently identified using the Kauffman-White serotyping scheme. Multilocus sequence typing (MLST) schemes have been developed for the major bacterial pathogens including Salmonella and have assisted in understanding the molecular epidemiology and population biology of these organisms. Recently, the DiversiLab rep-PCR system has been developed using micro-fluidic chips to provide standardized, semi-automated fingerprinting for pathogens including S. enterica. In the current study, 71 isolates of S. enterica, representing 21 serovars, were analyzed using MLST and the DiversiLab rep-PCR system. MLST was able to identify 31 sequence types (STs), while the DiversiLab system revealed 38 DiversiLab types (DTs). The rep-PCR distinguished isolates of different serovars and showed greater discriminatory power (0.95) than MLST typing (0.89). Rep-PCR exhibited 92% concordance with MLST and 90% with serotyping, while the concordance level of MLST typing with serotyping was 96%, representing a strong association. Comparison of rep-PCR profiles with those held in an online library database led to the accurate prediction of serovar in 63% of cases and resulted in inaccurate predictions for 10% of profiles. MLST and the rep-PCR system may provide useful additional informative techniques for the molecular identification of S. enterica. We conclude that the DiversiLab rep-PCR system may provide a rapid (less than 4 h) and standardized method for sub-typing isolates of S. enterica.     

Uptake and Replication of Salmonella enterica in Acanthamoeba rhysodes

The ability of salmonellae to become internalized and to survive and replicate in amoebae was evaluated by using three separate serovars of Salmonella enterica and five different isolates of axenic Acanthamoeba spp. In gentamicin protection assays, Salmonella enterica serovar Dublin was internalized more efficiently than Salmonella enterica serovar Enteritidis or Salmonella enterica serovar Typhimurium in all of the amoeba isolates tested. The bacteria appeared to be most efficiently internalized by Acanthamoeba rhysodes. Variations in bacterial growth conditions affected internalization efficiency, but this effect was not altered by inactivation of hilA, a key regulator in the expression of the invasion-associated Salmonella pathogenicity island 1. Microscopy of infected A. rhysodes revealed that S. enterica resided within vacuoles. Prolonged incubation resulted in a loss of intracellular bacteria associated with morphological changes and loss of amoebae. In part, these alterations were associated with hilA and the Salmonella virulence plasmid. The data show that Acanthamoeba spp. can differentiate between different serovars of salmonellae and that internalization is associated with cytotoxic effects mediated by defined Salmonella virulence loci.     

PCR Detection of Salmonella enterica Serotype Montevideo in and on Raw Tomatoes Using Primers Derived from hilA

Salmonellae have been some of the most frequently reported etiological agents in fresh-produce-associated outbreaks of human infections in recent years. PCR assays using four innovative pairs of primers derived from hilA and sirA, positive regulators of Salmonella invasive genes, were developed to identify Salmonella enterica serotype Montevideo on and in tomatoes. Based on examination of 83 Salmonella strains and 22 non-Salmonella strains, we concluded that a pair of hilA primers detects Salmonella specifically. The detection limits of the PCR assay were 10¹ and 10⁰ CFU/ml after enrichment at 37°C for 6 and 9 h, respectively. When the assay was validated by detecting S. enterica serotype Montevideo in and on artificially inoculated tomatoes, 10² and 10¹ CFU/g were detected, respectively, after enrichment for 6 h at 37°C. Our results suggest that the hilA-based PCR assay is sensitive and specific, and can be used for rapid detection of Salmonellae in or on fresh produce.     

Comparison of Salmonella enterica Serovar Typhimurium LT2 and Non-LT2 Salmonella Genomic Sequences, and Genotyping of Salmonellae by Using PCR

Genes of Salmonella enterica serovar Typhimurium LT2 expected to be specifically present in Salmonella were selected using the Basic Local Alignment Search Tool (BLAST) program. The 152 selected genes were compared with 11 genomic sequences of Salmonella serovars, including Salmonella enterica subsp. I and IIIb and Salmonella bongori (V), and were clustered into 17 groups by their comparison patterns. A total of 38 primer pairs were constructed to represent each of the 17 groups, and PCR was performed with various Salmonella subspecies including Salmonella enterica subsp. I, II, IIIa, IIIb, IV, VI, and V to evaluate a comprehensive DNA-based scheme for identification of Salmonella subspecies and the major disease-causing Salmonella serovars. Analysis of PCR results showed that Salmonella enterica subsp. I was critically divided from other subspecies, and Salmonella strains belonging to S. enterica subsp. I were clustered based on their serovars. In addition, genotypic relationships within S. enterica subsp. I by PCR results were investigated. Also, Salmonella signature genes, Salmonella enterica serovar Typhimurium signature genes, and Salmonella enterica subsp. I signature genes were demonstrated based on their PCR results. The described PCR method suggests a rapid and convenient method for identification of Salmonella serovars that can be used by nonspecialized laboratories. Genome sequence comparison can be a useful tool in epidemiologic and taxonomic studies of Salmonella.     

Optimal swab processing recovery method for detection of bioterrorism-related Francisella tularensis by real-time PCR

Francisella tularensis, the etiological agent of tularemia, is regarded as a potential bioterrorism agent. The advent of bioterrorism has heightened awareness of the need for validated methods for processing environmental samples. In this study we determined the optimal method for processing environmental swabs for the recovery and subsequent detection of F. tularensis by the use of real-time PCR assays. Four swab processing recovery methods were compared: heat, sonication, vortexing, and the Swab Extraction Tube System (SETS). These methods were evaluated using cotton, foam, polyester and rayon swabs spiked with six pathogenic strains of F. tularensis. Real-time PCR analysis using a multi-target 5′nuclease assay for F. tularensis showed that the use of the SETS method resulted in the best limit of detection when evaluated using multiple strains of F. tularensis. We demonstrated also that the efficiency of F. tularensis recovery from swab specimens was not equivalent for all swab processing methodologies and, thus, that this variable can affect real-time PCR assay sensitivity. The effectiveness of the SETS method was independent of the automated DNA extraction method and real-time PCR platforms used. In conclusion, diagnostic laboratories can now potentially incorporate the SETS method into specimen processing protocols for the rapid and efficient detection of F. tularensis by real-time PCR during laboratory bioterrorism-related investigations.     

An Allelotyping PCR for Identifying Salmonella enterica serovars Enteritidis, Hadar, Heidelberg, and Typhimurium

Current commercial PCRs tests for identifying Salmonella target genes unique to this genus. However, there are two species, six subspecies, and over 2,500 different Salmonella serovars, and not all are equal in their significance to public health. For example, finding S. enterica subspecies IIIa Arizona on a table egg layer farm is insignificant compared to the isolation of S. enterica subspecies I serovar Enteritidis, the leading cause of salmonellosis linked to the consumption of table eggs. Serovars are identified based on antigenic differences in lipopolysaccharide (LPS)(O antigen) and flagellin (H1 and H2 antigens). These antigenic differences are the outward appearance of the diversity of genes and gene alleles associated with this phenotype.We have developed an allelotyping, multiplex PCR that keys on genetic differences between four major S. enterica subspecies I serovars found in poultry and associated with significant human disease in the US. The PCR primer pairs were targeted to key genes or sequences unique to a specific Salmonella serovar and designed to produce an amplicon with size specific for that gene or allele. Salmonella serovar is assigned to an isolate based on the combination of PCR test results for specific LPS and flagellin gene alleles. The multiplex PCRs described in this article are specific for the detection of S. enterica subspecies I serovars Enteritidis, Hadar, Heidelberg, and Typhimurium.Here we demonstrate how to use the multiplex PCRs to identify serovar for a Salmonella isolate.     

Genome-Wide Methylation Patterns in Salmonella enterica Subsp. enterica Serovars

The methylation of DNA bases plays an important role in numerous biological processes including development, gene expression, and DNA replication. Salmonella is an important foodborne pathogen, and methylation in Salmonella is implicated in virulence. Using single molecule real-time (SMRT) DNA-sequencing, we sequenced and assembled the complete genomes of eleven Salmonella enterica isolates from nine different serovars, and analysed the whole-genome methylation patterns of each genome. We describe 16 distinct N⁶-methyladenine (m6A) methylated motifs, one N⁴-methylcytosine (m4C) motif, and one combined m6A-m4C motif. Eight of these motifs are novel, i.e., they have not been previously described. We also identified the methyltransferases (MTases) associated with 13 of the motifs. Some motifs are conserved across all Salmonella serovars tested, while others were found only in a subset of serovars. Eight of the nine serovars contained a unique methylated motif that was not found in any other serovar (most of these motifs were part of Type I restriction modification systems), indicating the high diversity of methylation patterns present in Salmonella.     

Development of a real-time SYBRGreen PCR assay for rapid detection of acquired AmpC in Enterobacteriaceae

Introduction

Acquired AmpC enzymes, classified as miscellaneous extended-spectrum β-lactamase (ESBL_M) enzymes according to a recently proposed β-lactamase classification, are increasing according to several publications. Simple and rapid methods for detection of ESBL_M are needed for appropriate infection control. A gel-based multiplex PCR method for acquired bla_AmpC detection and subtype classification has been available for several years. Here, we describe a modification of the protocol to suit real-time PCR platforms and to include novel genotypes.

Material and methods

Clinical isolates with clavulanic acid non-reversible non-susceptibility to extended-spectrum cephalosporins were subjected to combination disk testing with cefoxitin +/− cloxacillin at Malmö University Hospital. Phenotypical AmpC production was defined as cloxacillin reversible cefoxitin resistance. In this study 51 phenotypical AmpC-producing isolates, were subjected to the acquired bla_AmpC real-time PCR assay. The acquired blaAmpC positive isolates were further characterized by DNA sequencing of the acquired AmpC encoding gene, Pulsed-Field Gel Electrophoresis (PFGE) and PCR-based replicon typing.

Results and discussion

The real-time PCR assay was able to detect and sub-classify all acquired bla_AmpC genes described to date. The assay can be performed in less than 3 h, including pre-PCR preparations. Analysis of the isolate collection resulted in 18 of 51 phenotypical AmpC-producing isolates being positive in the acquired bla_AmpC real-time multiplex PCR assay; 17 of subtype CIT and one DHA. Sequence analysis identified 16 isolates as blaCMY-2, one as blaCMY-16 and one as blaDHA-1. Detected plasmid replicon types were I1 and B/O. Two of the E. coli isolates were identical according to PFGE and the others were unrelated.     

Rapid Detection of Campylobacter coli, C. jejuni, and Salmonella enterica on Poultry Carcasses by Using PCR-Enzyme-Linked Immunosorbent Assay

Contamination of retail poultry by Campylobacter spp. and Salmonella enterica is a significant source of human diarrheal disease. Isolation and identification of these microorganisms require a series of biochemical and serological tests. In this study, Campylobacter ceuE and Salmonella invA genes were used to design probes in PCR-enzyme-linked immunosorbent assay (ELISA), as an alternative to conventional bacteriological methodology, for the rapid detection of Campylobacter jejuni, Campylobacter coli, and S. enterica from poultry samples. With PCR-ELISA (40 cycles), the detection limits for Salmonella and Campylobacter were 2 × 10² and 4 × 10¹ CFU/ml, respectively. ELISA increased the sensitivity of the conventional PCR method by 100- to 1,000-fold. DNA was extracted from carcass rinses and tetrathionate enrichments and used in PCR-ELISA for the detection of Campylobacter and S. enterica, respectively. With PCR-ELISA, Salmonella was detected in 20 of 120 (17%) chicken carcass rinses examined, without the inclusion of an enrichment step. Significant correlation was observed between PCR-ELISA and cultural methods (kappa = 0.83; chi-square test, P < 0.001) with only one false negative (1.67%) and four false positives (6.67%) when PCR-ELISA was used to screen 60 tetrathionate enrichment cultures for Salmonella. With PCR-ELISA, we observed a positive correlation between the ELISA absorbance (optical density at 405 nm) and the campylobacter cell number in carcass rinse, as determined by standard culture methods. Overall, PCR-ELISA is a rapid and cost-effective approach for the detection and enumeration of Salmonella and Campylobacter bacteria on poultry.     

Diagnostic Real-Time PCR for Detection of Salmonella in Food

A robust 5′ nuclease (TaqMan) real-time PCR was developed and validated in-house for the specific detection of Salmonella in food. The assay used specifically designed primers and a probe target within the ttrRSBCA locus, which is located near the Salmonella pathogenicity island 2 at centisome 30.5. It is required for tetrathionate respiration in Salmonella. The assay correctly identified all 110 Salmonella strains and 87 non-Salmonella strains tested. An internal amplification control, which is coamplified with the same primers as the Salmonella DNA, was also included in the assay. The detection probabilities were 70% when a Salmonella cell suspension containing 10³ CFU/ml was used as a template in the PCR (5 CFU per reaction) and 100% when a suspension of 10⁴ CFU/ml was used. A pre-PCR sample preparation protocol including a preenrichment step in buffered peptone water followed by DNA extraction-purification was applied when 110 various food samples (chicken rinses, minced meat, fish, and raw milk) were investigated for Salmonella. The diagnostic accuracy was shown to be 100% compared to the traditional culture method. The overall analysis time of the PCR method was approximately 24 h, in contrast to 4 to 5 days of analysis time for the traditional culture method. This methodology can contribute to meeting the increasing demand of quality assurance laboratories for standard diagnostic methods. Studies are planned to assess the interlaboratory performance of this diagnostic PCR method.     

Identification by Subtractive Hybridization of Sequences Specific for Salmonella enterica Serovar Enteritidis

Salmonella enterica serovar Enteritidis, a major cause of food poisoning, can be transmitted to humans through intact chicken eggs when the contents have not been thoroughly cooked. Infection in chickens is asymptomatic; therefore, simple, sensitive, and specific detection methods are crucial for efforts to limit human exposure. Suppression subtractive hybridization was used to isolate DNA restriction fragments present in Salmonella serovar Enteritidis but absent in other bacteria found in poultry environments. Oligonucleotide primers to candidate regions were used in polymerase chain reactions to test 73 non-Enteritidis S. enterica isolates comprising 34 different serovars, including Dublin and Pullorum, two very close relatives of Enteritidis. A primer pair to one Salmonella difference fragment (termed Sdf I) clearly distinguished serovar Enteritidis from all other serovars tested, while two other primer pairs only identified a few non-Enteritidis strains. These primer pairs were also useful for the detection of a diverse collection of clinical and environmental Salmonella serovar Enteritidis isolates. In addition, five bacterial genera commonly found with Salmonella serovar Enteritidis were not detected. By treating total DNA with an exonuclease that degrades sheared chromosomal DNA but not intact circular plasmid DNA, it was shown that Sdf I is located on the chromosome. The Sdf I primers were used to screen a Salmonella serovar Enteritidis genomic library and a unique 4,060-bp region was defined. These results provide a basis for developing a rapid, sensitive, and highly specific detection system for Salmonella serovar Enteritidis and provide sequence information that may be relevant to the unique characteristics of this serovar.     

SMM-system: A mining tool to identify specific markers in Salmonella enterica

This report presents SMM-system, a software package that implements various personalized pre- and post-BLASTN tasks for mining specific markers of microbial pathogens. The main functionalities of SMM-system are summarized as follows: (i) converting multi-FASTA file, (ii) cutting interesting genomic sequence, (iii) automatic high-throughput BLASTN searches, and (iv) screening target sequences. The utility of SMM-system was demonstrated by using it to identify 214 Salmonella enterica-specific protein-coding sequences (CDSs). Eighteen primer pairs were designed based on eighteen S. enterica-specific CDSs, respectively. Seven of these primer pairs were validated with PCR assay, which showed 100% inclusivity for the 101 S. enterica genomes and 100% exclusivity of 30 non-S. enterica genomes. Three specific primer pairs were chosen to develop a multiplex PCR assay, which generated specific amplicons with a size of 180 bp (SC1286), 238 bp (SC1598) and 405 bp (SC4361), respectively. This study demonstrates that SMM-system is a high-throughput specific marker generation tool that can be used to identify genus-, species-, serogroup- and even serovar-specific DNA sequences of microbial pathogens, which has a potential to be applied in food industries, diagnostics and taxonomic studies. SMM-system is freely available and can be downloaded from http://foodsafety.sjtu.edu.cn/SMM-system.html.     

Development of multiplex PCR assay for rapid detection of Riemerella anatipestifer, Escherichia coli, and Salmonella enterica simultaneously from ducks

Three pathogens, Riemerella anatipestifer, Escherichia coli, and Salmonella enterica, are leading causes of bacterial fibrinous pericarditis and perihepatitis in ducks in China and worldwide. It is difficult to differentiate these pathogens when obtaining a diagnosis on clinical signs and pathological changes. The aim of this research was to develop a multiplex polymerase chain reaction (m-PCR) that could discriminate R. anatipestifer, E. coli, and S. enterica rapidly in field isolates, or detect the three bacteria in clinical samples from diseased ducks. We selected the DnaB helicase (dnaB) gene of R. anatipestifer, alkaline phosphatase (phoA) gene of E. coli and invasion protein (invA) gene of S. enterica as target genes. In optimized conditions, the limitation of detection was approximately 10³ colony forming units (CFU) of each of these three bacterial pathogens per PCR reaction tube. The m-PCR method showed specific amplification of respective genes from R. anatipestifer, E. coli, and S. enterica. Using the m-PCR system, bacterial strains isolated from diseased ducks in our laboratory were categorized successfully, and the pathogens could also be detected in clinical samples from diseased ducks. Therefore, the m-PCR system could distinguish the three pathogens simultaneously, for identification, routine molecular diagnosis and epidemiology, in a single reaction.     

A real-time PCR diagnostic method for detection of Naegleria fowleri

Naegleria fowleri is a free-living amoeba that can cause primary amoebic meningoencephalitis (PAM). While, traditional methods for diagnosing PAM still rely on culture, more current laboratory diagnoses exist based on conventional PCR methods; however, only a few real-time PCR processes have been described as yet. Here, we describe a real-time PCR-based diagnostic method using hybridization fluorescent labelled probes, with a LightCycler instrument and accompanying software (Roche), targeting the Naegleria fowleriMp2Cl5 gene sequence.Using this method, no cross reactivity with other tested epidemiologically relevant prokaryotic and eukaryotic organisms was found. The reaction detection limit was 1 copy of the Mp2Cl5 DNA sequence. This assay could become useful in the rapid laboratory diagnostic assessment of the presence or absence of Naegleria fowleri.     

A multiplex PCR assay to diagnose and quantify Nosema infections in honey bees (Apis mellifera)

Correct identification of the microsporidia, Nosema apis and Nosema ceranae, is key to the study and control of Nosema disease of honey bees (Apis mellifera). A rapid DNA extraction method combined with multiplex PCR to amplify the 16S rRNA gene with species-specific primers was compared with a previously published assay requiring spore-germination buffer and a DNA extraction kit. When the spore germination-extraction kit method was used, 10 or more bees were required to detect the pathogens, whereas the new extraction method made it possible to detect the pathogens in single bees. Approx. 4-8 times better detection of N. ceranae was found with the new method compared to the spore germination-extraction kit method. In addition, the time and cost required to process samples was lower with the proposed method compared to using a kit. Using the new DNA extraction method, a spore quantification procedure was developed using a triplex PCR involving co-amplifying the N. apis and N. ceranae 16S rRNA gene with the ribosomal protein gene, RpS5, from the honey bee. The accuracy of this semi-quantitative PCR was determined by comparing the relative band intensities to the number of spores per bee determined by microscopy for 23 samples, and a high correlation (R² = 0.95) was observed. This method of Nosema spore quantification revealed that spore numbers as low as 100 spores/bee could be detected by PCR. The new semi-quantitative triplex PCR assay is more sensitive, economical, rapid, simple, and reliable than previously published standard PCR-based methods for detection of Nosema and will be useful in laboratories where real-time PCR is not available.     

Detection of Salmonella enterica serovar Typhimurium by selective amplification of fliC, fljB, iroB, invA, rfbJ, STM2755, STM4497 genes by polymerase chain reaction in a monoplex and multiplex format

The aim of the work was to specifically differentiate S. typhimurium from other closely related Salmonella serovars by monoplex or multiplex PCR and to detect it from water and food samples. Genes targeted were invA, iroB, STM4497, STM2755, fliC, fljB and rfbJ and evaluated on 58 Salmonella standard serovars/strains including 9 S. typhimurium strains, 7 suspected Salmonella isolates and 8 other organisms as negative controls. Both invA and iroB showed a uniform amplification with all serovars of S. enterica group. STM2755 and STM4497 gene based PCR’s specifically exhibited amplification in all the nine confirmed S. typhimurium strains. The rfbJ PCR produced amplification with confirmed S. typhimurium strains, in addition showed reaction with S. abony. Both STM4497, STM2755 PCR’s and rfbJ could identify two of the seven biochemically suspected Salmonella isolates that were later confirmed to be S. typhimurium on the basis of sequence data. PCR for fliC genes had amplification exhibited by a large number of serovars of the S. enterica group, including S. typhimurium strains but not to S. brunei, S. newporti, S. abony and S. weltevreden. fljB was detected in all strains of S. enterica and E. coli with the exception of S. typhi. fljB and fliC were amplified in 6/7 and 5/7 presumptive Salmonella isolates. The same PCR’s were converted into two multiplex formats for simultaneous identification of the Salmonella genus, S. enterica group and S. typhimurium as a species. The first multiplex set comprised on invA, iroB, STM4497, STM2755 and the IAC. The second multiplex set comprised of invA, iroB, fljB, fliC, rfbJ along with IAC. The detection limit for S. typhimurium in the two multiplex PCR sets was in the range of 350–400 cfu/PCR reaction and that of DNA around 2 pg. The multiplex PCR (format 1) was first evaluated on spiked water, chicken and mutton samples and the detection limit for S. typhimurium was in the range of 100 cfu/100 ml, <60 and <50 cfu/gm, respectively. Further evaluation of multiplex PCR (format 1) was undertaken on 50 natural samples of chicken, eggs, litter, soil etc. and the comparison done with conventional culture isolation and identification procedure. The multiplex PCR could identify the presence of Salmonellla in three samples and the same three samples also yielded Salmonella by the conventional method. Therefore, the presently described multiplex PCR can serve as an alternative to the tedious time-consuming procedure of Salmonella culture and identification in food safety laboratories.     

Validation of a same-day real-time PCR method for screening of meat and carcass swabs for Salmonella

Background  

One of the major sources of human Salmonella infections is meat. Therefore, efficient and rapid monitoring of Salmonella in the meat production chain is necessary. Validation of alternative methods is needed to prove that the performance is equal to established methods. Very few of the published PCR methods for Salmonella have been validated in collaborative studies. This study describes a validation including comparative and collaborative trials, based on the recommendations from the Nordic organization for validation of alternative microbiological methods (NordVal) of a same-day, non-commercial real-time PCR method for detection of Salmonella in meat and carcass swabs.     

Characterization of Salmonella spp. isolated from patients below 3 years old with acute diarrhoea

Fifteen strains of Salmonella were isolated from children with clinically diagnosed diarrhoea aged below 3 years old, who had been admitted to K7 ward, Pediatric Institute, Kuala Lumpur Hospital. The isolates were tested for their susceptibility to a range of antimicrobial agents, and typed by serological tests and randomly amplified polymorphic DNA (RAPD) fingerprinting. All the strains had a similar pattern of antimicrobial susceptibility, where they were susceptible to a wide range of antimicrobial agents. The serological test has typed them into three serovars, which were identified as Salmonella enterica ser. Akanji, Salmonella enterica ser. Hindmarch and Salmonella enterica ser. Richmond. In contrast, the RAPD fingerprinting classed them into two major clusters, cluster 1 consisting of 12 strains of Salmonella and cluster 2 consisting of three strains of Salmonella.