Characterization of Listeria monocytogenes isolates in import food products of China from 8 provinces between 2005 and 2007

A total of 48 Listeria monocytogenes isolates of different import food products from 8 provinces between 2005 and 2008 were characterized. The serotype and virulence were confirmed for each strain and molecular subtyping were analyzed by multilocus sequence typing (MLST). Twenty five strains were assigned to serotype 1/2a, and 11 isolates to serotype 1/2b, serotype 4b were found in 7 isolate, and the remaining 5 strains were grouped into serotypes 1/2c, 4a, and 4e. Molecular subtyping schemes found thirty two sequence types (STs) among these isolates and the majority of L. monocytogenes strains belonged to lineage II (56%), followed by lineage I (38%), lineage III (6%). Two molecular subtype clusters, cluster A included all isolates of lineage II, while cluster B contained the isolates of lineages I and lineages III. Two L. monocytogenes strains were not grouped in either of the two clusters. Fifty three isolates were as virulent as L. monocytogenes reference strain EGD in mouse virulence assay, while the isolates 22213 and 22265 had low pathogenicity. These results provide the first molecular insight into the L. monocytogenes strains isolated from import food products of 8 provinces in China and indicate the potential risk to cause human disease if intake by contaminated foods. MLST could be used as a routine subtyping method of L. monocytogenes isolates. In China, inspection and quarantine strategies of imported foods should be strengthened. PRACTICAL APPLICATION: There is a potential risk of listeriosis in China and routine subtyping of L. monocytogenes isolates is important. It is necessary for food hygiene management to strengthen the supervision of imported foods.     

Virulence characterization and genotypic analyses of Listeria monocytogenes isolates from food and processing environments in eastern China

In this study, twenty L. monocytogenes food-related isolates collected from eastern China Zhejiang province were compared by in vivo LD50 assays as well as in vitro cytopathic plaque forming assay. Nineteen L. monocytogenes isolates (19/20) were as virulent as reference strain 10403S, while the isolate M4 had low pathogenicity. The unique isolate M4 fell into lineage III based on the partial nucleotide variations of actA, while the other isolates belonged to the more common lineages I and II. L. monocytogenes isolates were grouped in 17 to 19 subtypes using pulsed-field gel electrophoresis (PFGE) with SmaI digestion, and multilocus sequence typing (MLST) based on three virulence genes (actA, inlA and inlB) and four housekeeping genes (betL, dat, recA and sigB). The virulence genes based MLST had better discriminatory power than that targeting the housekeeping genes (0.990 vs 0.895), similar to PFGE (0.976). An isolate from the processing desk was found having the same pulsotype as the two isolates from final shrimp products in the same plant, indicating that process contamination could be the source of Listeria contamination.     

Genetic relatedness between Listeria monocytogenes isolates from seafood and humans using PFGE and REP-PCR

Listeria monocytogenes has been isolated from catfish and various non-catfish seafoods. Despite progress that was made to understand the relationship between L. monocytogenes isolated from seafood and humans, no study has emphasized the genetic relatedness between catfish and non-catfish seafood and human isolates. The objectives of this study were to (1) investigate the genetic relationship between L. monocytogenes isolates from catfish, non-catfish seafood and humans using pulsed-field gel electrophoresis (PFGE) and repetitive extragenic palindromic element-based PCR (REP-PCR) linking with serotypes; and (2) compare the distinct capabilities of these methods. The genetic relationship among 36 catfish, 35 non-catfish seafood, and 57 human isolates were analyzed using Bionumerics software. Among a total of 128 isolates, only two subtypes by PFGE with ApaI-digestion, one subtype by PFGE with AscI-digestion and three subtypes by REP-PCR were shared by non-catfish seafood or catfish with human isolates. Although most of the non-catfish seafood and catfish isolates were genetically distinct from human isolates, the human isolates used in this study may not be truly representative of all clinical isolates. Serotype 4b was dominant in human isolates, whereas serotypes 3b, 1/2a, and 1/2b and serotypes 4b, 1/2a and 1/2b were commonly found in catfish and non-catfish seafood, respectively. Furthermore, 97, 87 and 94 subtypes of L. monocytogenes were revealed using the ApaI-digestion, the AscI-digestion, and the REP-PCR, respectively. Their respective discriminatory indexes were 0.994, 0.990 and 0.994. Distinct genetic groups based on the difference of flagella antigens were observed in all three methods. The study suggests that the REP-PCR possesses a similar discriminatory ability as the PFGE for subtyping L. monocytogenes. Therefore, the REP-PCR that is rapid and less expensive could be considered as an alternative method for subtyping L. monocytogenes.     

Typing of Listeria monocytogenes isolates originating from the food processing industry with automated ribotyping and pulsed-field gel electrophoresis

A total of 486 Listeria monocytogenes isolates originating from 17 Finnish food processing plants (representing meat, poultry, fish, and dairy production) were collected and typed by automated ribotyping using EcoRI as the restriction enzyme. The isolates were divided into 16 different ribotypes (RTs). Some of these isolates (121), representing all EcoRI types and 16 food plants, were subjected to ribotyping with the PvuII enzyme, to pulsed-field gel electrophoresis (PFGE) typing with AscI and SmaI restriction enzymes, and to serotyping with O-antigen antisera. Nineteen ribotypes were generated with PvuII, 42 macrorestriction patterns were generated with AscI and 24 with SmaI, and three serotypes were generated with antisera. When the results were combined, the overall number of RTs was 23, and that of the PFGE types was 46. Thus, the overall discrimination power of PFGE was higher (discrimination index [DI] 0.966) than that of ribotyping (DI 0.906). The most common serotype (90.1% of the isolates) was 1/2, and isolates of serotype 4 (3.3%) were rare. There was no connection between food sectors and RTs or PFGE types, but PFGE indicated the single plants (78.3% of the types) better than ribotyping (56.5%). On the basis of its automation and on the availability of identification databases, automated ribotyping had some advantages over PFGE. Overall, automated ribotyping can be considered a practical and rapid tool when Listeria contamination is suspected and when screening a large number of isolates is necessary, e.g., when tracing contamination sources. However, in cases of outbreaks, the identical patterns must be confirmed by PFGE, which is a more discriminatory method.     

进出口食品中不同血清型沙门氏菌PFGE和 MLST分型比较研究

目的：通过PFGE和MLST两种方法对进出口食品中不同血清型沙门氏菌的分辨力和应用价值进行了比较研究。方法：采用PFGE和MLST两种方法对进出口食品中分离得到的鼠伤寒及肠炎两种血清型共30株沙门氏菌进行分型研究。结果：两种血清型共30株沙门氏菌经PFGE分型可得到23种型别,DI值为0.9563;鼠伤寒和肠炎两种血清型菌分别经PFGE分型,DI值分别为1.000和0.9048。MLST对30株沙门氏菌分型得到4种ST型,DI值为0.5793;鼠伤寒和肠炎两种血清型菌分别经MLST分型,DI值分别为0.2571和0.1333。结论：在分辨力方面,PFGE更适合于同一沙门氏菌血清型的分型,MLST适合于不同沙门氏菌血清型间的分型。MLST在替代、补充血清型分型方面有潜在的优势,PFGE在溯源研究方面似乎更胜一筹。     

Subtyping of Listeria monocytogenes at the haplotype level by Fourier transform infrared (FT-IR) spectroscopy and multivariate statistical analysis

Listeria monocytogenes is a widespread foodborne pathogen that represents a major concern with respect to food safety. Rapid identification of this bacterium at a subspecies level is important to trace back an outbreak and improve risk-based inspection programs. A method for subtyping L. monocytogenes at the serotype and haplotype levels was developed using Fourier transform infrared (FT-IR) reflectance microscopy. Thirty strains of L. monocytogenes belonging to four different PCR serotypes (1/2a, 1/2b, 4b, and 4c) that had previously been characterized by Multilocus genotyping (MLGT) and Pulsed field gel electrophoresis (PFGE) assays were used in this study. The FT-IR based identification and classification was compared to the known MLGT and PFGE subtyping of the L. monocytogenes. Canonical variate analysis (CVA) of the spectra resulted in 96.6% correct identification of L. monocytogenes at the serotype level. Hierarchical cluster analysis (HCA) and CVA of the spectra showed 91.7% correct identification of strains at the haplotype level consistent with their MLGT groupings. FT-IR spectra of strains were also differentiated correctly in accordance with their PFGE haplotyping. Additionally, by using HCA of FT-IR spectra, each bacterium was differentiated at the strain level. Starting from a pure culture, this method enabled classification of L. monocytogenes at the serotype, haplotype, and/or strain level within 18 h, which is faster and potentially less expensive than the molecular methods and previous FT-IR methods. This is the first report of the identification of L. monocytogenes at the haplotype level using FT-IR.     

High-resolution genotyping of Listeria monocytogenes by fluorescent amplified fragment length polymorphism analysis compared to pulsed-field gel electrophoresis, random amplified polymorphic DNA analysis, ribotyping, and PCR-restriction fragment length polymorphism analysis

The purpose of this study was to evaluate fluorescent amplified fragment length polymorphism (AFLP) analysis for the inter- and intraspecies differentiation of a collection of 96 strains of Listeria monocytogenes and 10 non-L. monocytogenes strains representing six other Listeria species of different origin. The AFLP technique was compared with three other molecular typing methods--ribotyping, random amplified polymorphic DNA analysis (RAPD), and pulsed-field gel electrophoresis (PFGE)--in terms of discriminatory ability. PCR-restriction fragment length polymorphism was included for virulence gene allele characterization. The 96 L. monocytogenes strains were divided into two major clusters by AFLP fingerprinting at a similarity level of 82% in concordance with the results of PFGE, RAPD, and ribotyping. One main cluster consisted of all of the 24 L. monocytogenes hly allele 1 strains, while another main cluster consisted of all of the 72 L. monocytogenes hly allele 2 strains. This indicates the existence of two distinct phylogenetic divisions. Isolates of the remaining Listeria species were not included in the clusters. AFLP, PFGE, and RAPD typing were highly discriminatory methods, with discrimination (D) indices of 0.974, 0.969, and 0.954, respectively, whereas ribotyping had a lower D index of 0.874. AFLP, PFGE, and RAPD typing showed some level of agreement in terms of strain grouping and differentiation. However, all three methods subdivided types of strains grouped by the other methods. Isolates with identical DNA profiles were distributed across the spectrum of origin. It was not possible to associate certain types with specific food sectors or clinical cases, which is indicative of the spread of L. monocytogenes clones across species. Overall, AFLP fingerprinting was suitable for the high-resolution genotyping of L. monocytogenes and had an equally high or higher differentiation power compared to PFGE or RAPD typing.     

Application of Random Amplified Polymorphic DNA (RAPD) and Pulsed-Field Gel Electrophoresis (PFGE) Analysis to Listeria monocytogenes: A Review of Methodology and Results

Random amplified polymorphic DNA (RAPD) and pulsed-field gel electrophoresis (PFGE) analyses have been found to be powerful molecular methods for differentiating isolates of a given bacterial species. When applied to Listeria monocytogenes, both methods have been found highly effective in tracking isolates involved in food borne outbreaks of listeriosis and in identifying routes of contamination in food processing plants. Among the two methods, PFGE is considered somewhat superior in discriminatory power. However, the use of two or more independent random primers with RAPD is considered to result in a level of discrimination equal to that of PFGE. When results from both methods are combined, a maximum level of discrimination that exceeds that obtained with both methods independently can be achieved. Individually, both methods far exceed the discriminatory power of serotyping and phage typing of L. monocytogenes strains in that serotypes 1/2a, 1/2b, and 4b, represent over 90% of all human isolates, and phage typing at times has allowed typing of no more than about 50% of isolates. In addition, both RAPD and PFGE on occasion have been found to be superior to ribotyping, multilocus enzyme electrophoresis, and restriction enzyme analysis of L. monocytogenes isolates.     

Development and application of Multiple-Locus Variable number of tandem repeat Analysis (MLVA) to subtype a collection of Listeria monocytogenes

In this study we report the development and application of a Multiple-Locus Variable number of tandem repeat Analysis (MLVA) strategy for subtyping Listeria monocytogenes. Genome profiles of a collection of forty-five food-borne L. monocytogenes isolates were compared using MLVA. These isolates were obtained as part of an active surveillance programme of foods in the south-east region of Ireland. MLVA successfully discriminated amongst the isolates. The method was easy to perform, relatively fast and could be deployed in any molecular laboratory with basic laboratory equipment. This approach is a valuable tool, which has the capability to provide comparable results when compared with other more established typing methods, including pulsed-field gel electrophoresis (PFGE).     

International Life Sciences Institute North America Listeria monocytogenes strain collection: development of standard Listeria monocytogenes strain sets for research and validation studies

Research and development efforts on bacterial foodborne pathogens, including the development of novel detection and subtyping methods, as well as validation studies for intervention strategies can greatly be enhanced through the availability and use of standardized strain collections. These types of strain collections are available for some foodborne pathogens, such as Salmonella and Escherichia coli. We have developed a standard Listeria monocytogenes strain collection that has not been previously available. The strain collection includes (i) a diversity set of 25 isolates chosen to represent a genetically diverse set of L. monocytogenes isolates as well as a single hemolytic Listeria innocua strain and (ii) an outbreak set, which includes 21 human and food isolates from nine major human listeriosis outbreaks that occurred between 1981 and 2002. The diversity set represents all three genetic L. monocytogenes lineages (I, n = 9; II, n = 9; and III, n = 6) as well as nine different serotypes. Molecular subtyping by EcoRI automated ribotyping and pulsed-field gel electrophoresis (PFGE) with AscI and ApaI separated the 25 isolates in the diversity set into 23 ribotypes and 25 PFGE types, confirming that this isolate set represents considerable genetic diversity. Molecular subtyping of isolates in the outbreak set confirmed that human and food isolates were identical by ribotype and PFGE, except for human and food isolates for two outbreaks, which displayed related but distinct PFGE patterns. Subtype and source data for all isolates in this strain collection are available on the Internet and are linked to the PathogenTracker database (www.pathogentracker.com), which allows the addition of new, relevant information on these isolates, including links to publications that have used isolates from this collection. We have thus developed a core L. monocytogenes strain collection, which will provide a resource for L. monocytogenes research and development efforts with centralized Internet-based data curation and integration.     

Phenotypic Characterization and Genotypic Subtyping of Salmonella enterica Serovars Enteritidis and Gallinarum Isolated from Human and Poultry-Related Samples

This study aimed to assess the serotypes, antibiotic susceptibility, and molecular subtyping of Salmonella enterica isolated from food products and human patients with gastroenteritis. A total of 59 isolates were investigated, and the results revealed a predominance of S. Enteritidis with 57.63% (34/59) S. Gallinarum held second with 15.62% (5/32) of total food borne. While, isolates from humans showed 18.51% (5/27) of S. Typhimurium. High level of resistance to nalidixic acid was noted among food strains and 35.29% of human isolates were found to be multidrug resistant. Eight representative isolates were subtyping using three molecular approaches, ribotyping, Multilocus sequence typing (MLST) and Multiple-locus variable number tandem repeat analysis (MLVA). MLST showed three sequence types corresponding to two clonal complexes, (ST-78, CC-4) for S. Gallinarum, (ST-11, CC-4) for S. Enteritidis and (S-367, CC-37) for S. Cerro. While, MLVA generated six different profiles targeting nine loci for S. Enteritidis and S. Gallinarum.     

Traceback identification of an ingredient (pork dewlap) as the possible source of Listeria monocytogenes serotype 4b contamination in raw chicken products

In surveys conducted on finished product samples from a single poultry processing plant in Spain, Listeria monocytogenes was found in 14 different uncooked products. To track contamination patterns, 77 L. monocytogenes isolates were characterized by PCR-based serotyping, pulsed-field gel electrophoresis (PFGE) restriction analysis, and PCR-based allelic analysis of the virulence gene actA. Serotyping revealed that 12 isolates (15.6%) were of the L. monocytogenes serotype 4b complex (serotype 4b or the closely related serotypes 4d and 4e). A combination of endonucleases AscI and ApaI PFGE patterns yielded 15 different pulsotypes among all 77 tested isolates. All the serotype 4b isolates belonged to one pulsotype. Sequencing of the actA gene confirmed that all serotype 4b isolates corresponded to the same allelic subtype. The subtype was recovered from five product types, but its presence was not correlated with the production line or the date of isolation, suggesting a possible association of this strain with a common ingredient. This traceback investigation established that pork dewlap, an ingredient common to all the products contaminated with this strain, was the most probable source of L. monocytogenes 4b. The same 4b strain was isolated from four samples of pork dewlap from one specific supplier. After replacement of this contaminated ingredient in the fresh products, this strain of L. monocytogenes serotype 4b was not detected. This study confirms the effectiveness of molecular subtyping to control contamination by specific strains of L. monocytogenes and the importance of testing the different ingredients added to the food products.     

An enhanced discriminatory pulsed-field gel electrophoresis scheme for subtyping Salmonella serotypes Heidelberg, Kentucky, SaintPaul, and Hadar

Conventional pulsed-field gel electrophoresis (PFGE) protocols, used extensively as a successful approach for subtyping many salmonellae, may be inadequate for discriminating strains sharing levels of homogeneity within the same serotype. Four additional restriction enzymes (SpeI, PacI, SfiI, and NotI), in addition to XbaI and BlnI, were used in PFGE typing of 33 Salmonella Heidelberg, 27 Salmonella Kentucky, 27 Salmonella SaintPaul, and 27 Salmonella Hadar isolates that were recovered from poultry and porcine retail meats from different states of the United States. A dendrogram derived from the combined analysis of six enzymes was highly discriminatory with a Simpson index of diversity value of over 0.950. The ratio of nodes to isolates was more than 0.75 with an average of fewer than three isolates in each polytomy for all four serotypes. Two three-enzyme combinations, SpeI/NotI/SfiI for Salmonella Heidelberg and Salmonella Hadar, and SpeI/BlnI/SfiI for Salmonella Kentucky and Salmonella SaintPaul, were found to have comparable discriminatory abilities of differentiating isolates of these Salmonella serotypes with the six-enzyme combination. The enhanced discriminatory PFGE-based subtyping scheme can be used effectively for the differentiation of strains of the four Salmonella serotypes. The findings also highlight PFGE analysis as a continued essential and informative subtyping method for source tracking and outbreak investigations of these and other Salmonella pathogens.     

Evaluation of <Emphasis Type="Italic">flaA</Emphasis> Short Variable Region Sequencing,Multilocus Sequence Typing,and Fourier Transform Infrared Spectroscopy for Discrimination between <Emphasis Type="Italic">Campylobacter jejuni</Emphasis> Strains

Discriminatory and robust typing methods are needed to improve the understanding of the dynamics of food-borne Campylobacter infections and epidemiology in primary animal production. To evaluate the strain discriminatory potential of typing methods, flaA short variable region (SVR) sequencing and Fourier transform infrared (FTIR) spectroscopy were applied on a collection of 102 epidemiologically related and unrelated Campylobacter jejuni strains. Previous application of FTIR spectroscopy for subtyping of Campylobacter has been limited. A subset of isolates, initially discriminated by flaA SVR sequencing, were further subjected to multilocus sequence typing (MLST). It was found that flaA SVR sequencing had a slightly higher discriminatory power than FTIR spectroscopy, based on the Simpson diversity index. The clustering of strains indicated that FTIR spectroscopy is indeed a suitable method for discrimination of Campylobacter. The isolates were assigned to six clusters based on flaA SVR sequences and nine clusters based on the FTIR spectroscopy profiles. Furthermore, the cluster analysis of flaA SVR sequences, MLST, and FTIR spectroscopy profiles showed a high degree of congruence, assigning the isolates to similar cluster structures. In conclusion, FTIR spectroscopy can be applied for subtyping of Campylobacter, and the high discriminatory potential of both flaA SVR sequencing and FTIR spectroscopy render them suitable screening methods for large numbers of strains.     

Monitoring transmission routes of Listeria spp. in smoked salmon production with repetitive element sequence-based PCR techniques

Various techniques have been used for tracing the transmission routes of Listeria species and for the assessment of hygiene standards in food processing plants. The potential of repetitive element sequence-based PCR (Rep-PCR) methods (GTG? and REPI + II) for the typing of Listeria isolates (n = 116), including Listeria monocytogenes (n = 46), was evaluated in a particular situation arising from the relocation of a company producing cold-smoked salmon. Pulsed-field gel electrophoresis (PFGE) using three restriction enzymes (ApaI, AscI, and SmaI) was used for comparison. Identical transmission scenarios among two companies could be identified by cluster analysis of L. monocytogenes isolates that were indistinguishable by both Rep-PCR and PFGE. The calculated diversity index (DI) indicates that Rep-PCR subtyping of Listeria species with primer sets GTG? and REPI + II has a lower discrimination power than does PFGE. When concatenated Rep-PCR cluster analysis was used, the DI increased from 0.934 (REPI + II) and 0.923 (GTG?) to 0.956. The discrimination power of this method was similar to that of PFGE typing based on restriction enzyme Apa I (DI = 0.955). Listeria welshimeri may be useful as an indicator for monitoring smoked salmon processing environments. Rep-PCR meets the expectations of a reasonable, fast, and low-cost molecular subtyping method for the routine monitoring of Listeria species. The discriminatory power as characterized by the DI sufficiently quantifies the probability of unrelated isolates being characterized as different subtypes. Therefore, Rep-PCR typing based on two primer systems (GTG? and REPI + II) may be a useful tool for monitoring industrial hygiene.     

Comparison of antibiotic susceptibility profiles and molecular typing patterns of clinical and environmental Salmonella enterica serotype Newport

The genus Salmonella is composed of more than 2,400 serotypes, many of which cause enteric diseases in humans and animals. Several Salmonella serotypes are multidrug resistant, and there is evidence of the clonal spread of these strains from animals to humans. Salmonella enterica serotype Newport is one of the serotypes that increasingly present a multidrug-resistant phenotype. Source tracking and antibiotic resistance testing are important considerations for identifying the outbreak strain. The first goal of this study was to examine the antibiotic susceptibility patterns of clinical and environmental Salmonella Newport isolates from various geographic locations and to compare the discriminatory ability of two DNA fingerprinting techniques. The second goal was to determine whether the antibiotic resistance profiles and typing patterns correlated. Thirty Salmonella Newport isolates, including environmental and human clinical strains, were subjected to pulsed-field gel electrophoresis (PFGE), ribotyping, and antibiotic susceptibility testing. Eighty percent of the isolates showed total or intermediate resistance to one or more drugs; 75% of the isolates were multidrug resistant. Ribotyping with the EcoRI enzyme and PFGE with the XbaI enzyme each divided the isolates into 14 groups. Cluster analysis based on antibiotic susceptibility patterns generated 23 profiles. The susceptible and resistant isolates were not differentiated on the basis of either of the molecular typing techniques. Hence, no correlation was observed between the antibiotic resistance profiles and the DNA subtyping patterns. In conclusion, ribotyping is as discriminatory as PFGE and, when used in combination with antibiotic resistance profiles, provides a powerful tool for the source tracking of Salmonella Newport.     

1例急性淋巴细胞白血病患者感染单核细胞增生李斯特菌病原学及分子分型特征分析

目的对北京市1名急性淋巴细胞白血病患者感染单核细胞增生李斯特菌病例进行病因溯源,对分离到的单核细胞增生李斯特菌进行血清学分型、耐药及分子分型研究。方法对患者不同时期外周血分离的2株单核细胞增生李斯特菌和1株环境涂抹样品单核细胞增生李斯特菌分离株进行血清学分型、耐药性分析、脉冲场凝胶电泳(PFGE)和多位点序列分析(MLST)。结果 3株单核细胞增生李斯特菌均为1/2a-3a血清型,耐药结果一致,均对青霉素、氨苄西林、复方新诺明、美罗培南及红霉素敏感,3株菌的PFGE带型一致,MLST型别均为ST155。结论本研究中患者生活环境中存在单核细胞增生李斯特菌的污染情况,高度怀疑患者感染单核细胞增生李斯特菌与其生活环境中分离到的菌株为同一来源。     

Comparison of genotyping methods by application to Salmonella livingstone strains associated with an outbreak of human salmonellosis

During 2000 and 2001, an outbreak of human salmonellosis occurred in Sweden and Norway, caused by Salmonella livingstone. In this study, the genotypic differences between three strains obtained from food sources during the outbreak, two human strains and 27 more or less unrelated strains were analysed, using the three methods; automated ribotyping, pulsed field gel electrophoresis (PFGE) and randomly amplified polymorphic DNA (RAPD). Each method was evaluated regarding its discriminatory ability, reproducibility and typeability. Simpson's discriminatory index calculated for each method was 0.556 for automated ribotyping, 0.766 for PFGE and 0.236 for RAPD. The reproducibility, defined as the minimum similarity between individual replicates in a cluster analysis, was 96% for automated ribotyping and PFGE, and 90% for RAPD. All the strains were typeable with each method. When combining results for the three genotyping methods, it was found that RAPD did not increase the discriminatory index and was therefore excluded from further analysis. Using a combination of the results obtained from ribotyping and PFGE (D=0.855), two strains that had been isolated from feed factories during 1998 were shown to be identical to the outbreak strain, indicating a possible route of contamination due to a clone of Salmonella livingstone persisting in feed producing facilities. No connection to poultry was established.     

Characterization of Listeria monocytogenes isolated from retail foods

Listeria monocytogenes isolates recovered from retail ready-to-eat (RTE) meats, raw chickens and fresh produce were characterized by serogroup identification using PCR, genotyping using pulsed-field gel electrophoresis (PFGE) and antimicrobial susceptibility testing. Five L. monocytogenes serogroups were identified. Of the 167 isolates 68 (41%) belonged to serogroup 1/2b, 3b; 53 (32%) belonged to serogroup 4b, 4d, 4e; 43 (26%) belonged to serogroup 1/2a, 3a; 2 (1.2%) belonged to serogroup 1/2c, 3c; and 1 (0.6%) belonged to serogroup 4a, 4c. PFGE generated 120 patterns which correlated well with PCR serogrouping. Most L. monocytogenes isolates were resistant to sulfonamide (73%) and some were resistant to tetracycline (8.4%) and ciprofloxacin (1.8%). Tetracycline resistance was conjugatively transferable and the tet(M) gene was identified in 14 tetracycline-resistant isolates as well as their transconjugants. These findings indicate that L. monocytogenes present in food were diverse, and that resistance to one or more antibiotics among these isolates was common. In addition, the presence of potential serotype 4b in all food categories is of public health concern, as serotype 4b has been the serotype most frequently associated with human listeriosis.     

Antimicrobial resistance and molecular subtyping of Campylobacter jejuni and Campylobacter coli from retail meats

Campylobacter isolates (n = 297; 202 C. jejuni and 95 C. coli isolates) recovered from 2,513 retail meat samples (chicken breasts, ground turkey, ground beef, and pork chops) were examined for antimicrobial susceptibility. The isolates were further analyzed for genetic relatedness by pulsed-field gel electrophoresis (PFGE) using SmaI and KpnI restriction enzymes, and a subset of isolates (n = 174) were subtyped by multilocus sequence typing (MLST). The resistance most frequently observed was that to doxycycline (27.6%), followed by ciprofloxacin (13.8%) and erythromycin (6.4%). All isolates were susceptible to gentamicin and meropenem. C. coli showed higher resistance to doxycycline than did C. jejuni (42.1 versus 20.8%) and lower resistance to ciprofloxacin than did C. jejuni (10.5 versus 15.3%). Erythromycin resistance was only observed in C. coli. PFGE using SmaI plus KpnI digestion generated 168 clusters from 297 isolates: 115 from C. jejuni and 53 from C. coli. MLST revealed 44 sequence types (STs) under 10 clonal complexes from 120 C. jejuni and 27 STs under two clonal complexes from 54 C. coli. There was a positive association between PFGE and STs; however, PFGE showed greater discriminatory power than MLST. Subtyping data did not correlate with antimicrobial resistance phenotypes.