A comparison of three molecular typing methods for the discrimination of Salmonella enterica serovar Infantis

Seventy-six epidemiologically unrelated Salmonella enterica serovar Infantis (S. Infantis) isolates were typed by pulsed-field gel electrophoresis (PFGE), multiple amplification of phage loci typing (MAPLT) and multiple-locus variable-number tandem-repeat analysis (MLVA). PFGE, using the restriction endonuclease XbaI, generated 23 different profiles for the 76 isolates (DI=0.848). MAPLT was undertaken using a combination of 11 primer sets based on bacteriophage sequences and generated 28 different profiles (DI=0.938). By contrast, MLVA only produced nine profiles (DI=0.668) with 13 different primer sets, including the five primer sets routinely used for S. Typhimurium typing. Reducing the number of MAPLT primer sets to four still provided a diversity index of 0.838. All three typing methods revealed two distinct lineages of S. Infantis, with most isolates demonstrating genetic traits of either lineage but not both. The results demonstrate that MAPLT can potentially provide greater discrimination and separation of S. Infantis isolates than both PFGE and MLVA. Furthermore, MAPLT data can be generated much more rapidly and with reduced labour input than PFGE and without the need for expensive PFGE electrophoresis equipment, nor does it require capillary sequencing of PCR fragments to accurately determine PCR fragment lengths as is the case with MLVA.     

Clonal expansion may account for high levels of quinolone resistance in Salmonella enterica serovar enteritidis

We have observed a high incidence of isolated nalidixic acid resistance in Salmonella enterica serovar Enteritidis isolates in Ireland, particularly isolates of phage type 1 (PT1). A group of nalidixic acid-resistant (n = 22) and nalidixic acid-susceptible (n = 28) isolates of serovar Enteritidis from multiple sites in Ireland were selected. Isolates were typed by pulsed-field gel electrophoresis (PFGE) with XbaI, and the MICs for nalidixic acid and ciprofloxacin were determined. Mutations associated with nalidixic acid resistance in clinical isolates and laboratory mutants of serovar Enteritidis and 32 nalidixic acid-resistant isolates of 15 other salmonella serovars were identified. PFGE had limited discriminatory power. A specific point mutation (G246T) associated with amino acid substitution Asp87Tyr in the quinolone resistance determining region of the gyrA gene accounted for 95% of all mutations in serovar Enteritidis and for all mutations in PT1 isolates. Greater diversity of mutations was observed among all non-Enteritidis salmonella serovars studied. Rates of nalidixic acid resistance in serovar Enteritidis may predominantly reflect clonal expansion after infrequent mutation or selection events.     

Phenotypic evidence for inducible multiple antimicrobial resistance in Salmonella choleraesuis

Multiple antimicrobial resistance (MAR) in Salmonella choleraesuis is becoming a major concern. It has been demonstrated that a MAR phenotype can be induced in Escherichia coli and other members of the Enterobacteriaceae by exposing the isolates to salicylates, various antimicrobials, or organic solvents used to combat and control bacterial infection. Therefore the purpose of the present study was to determine whether this marA-associated MAR-phenotype is inducible in S. choleraesuis. Isolates used in the present study were able to withstand toxic effects of the organic solvent cyclohexane naturally, or following exposure to the inducing compounds salicylate, tetracycline, or chloramphenicol. All isolates possessed fragments of marA with the predicted size of 408 bp when amplified using marA-specific primers by PCR. The resulting PCR products that were sequenced revealed that amplified S. choleraesuis marA was 99% and 85% homologous to the published Salmonella typhimurium and E. coli marA sequences respectively. Minimum inhibitory concentrations of tetracycline (P<0.08), chloramphenicol (P<0.001), rifampin (P<0.08), and nalidixic acid (P<0.001) against cyclohexane-tolerant mutants were significantly increased when compared with wild-type S. choleraesuis. Northern hybridization signals for both marA and acrB were increased in the induced isolates when compared to uninduced controls while soxS expression did not change between induced and uninduced cultures. The results suggest that marA is present in S. choleraesuis and a MAR-phenotype is inducible in S. choleraesuis presumably due to the overexpression of marA and acrB and not to the overexpression of soxS.     

Clonal Expansion May Account for High Levels of Quinolone Resistance in Salmonella enterica Serovar Enteritidis

We have observed a high incidence of isolated nalidixic acid resistance in Salmonella enterica serovar Enteritidis isolates in Ireland, particularly isolates of phage type 1 (PT1). A group of nalidixic acid-resistant (n = 22) and nalidixic acid-susceptible (n = 28) isolates of serovar Enteritidis from multiple sites in Ireland were selected. Isolates were typed by pulsed-field gel electrophoresis (PFGE) with XbaI, and the MICs for nalidixic acid and ciprofloxacin were determined. Mutations associated with nalidixic acid resistance in clinical isolates and laboratory mutants of serovar Enteritidis and 32 nalidixic acid-resistant isolates of 15 other salmonella serovars were identified. PFGE had limited discriminatory power. A specific point mutation (G246T) associated with amino acid substitution Asp87Tyr in the quinolone resistance determining region of the gyrA gene accounted for 95% of all mutations in serovar Enteritidis and for all mutations in PT1 isolates. Greater diversity of mutations was observed among all non-Enteritidis salmonella serovars studied. Rates of nalidixic acid resistance in serovar Enteritidis may predominantly reflect clonal expansion after infrequent mutation or selection events.     

Genetic diversity of human isolates of Salmonella enterica serovar Enteritidis in Malaysia

AIMS: The study was undertaken to determine clonal relationship and genetic diversity of the human strains of Salmonella enterica serovar Enteritidis isolated from 1995 to 2002 from different parts of Malaysia. METHODS AND RESULTS: Antimicrobial susceptibility test, plasmid profiling and pulsed-field gel electrophoresis were applied to analyse 65 human isolates of S. Enteritidis obtained over an eight year period from different parts of Malaysia. Four nonhuman isolates were included for comparison. A total of 14 distinct XbaI-pulsed-field profiles (PFPs) were observed, although a single PFP X1 was predominant and this particular clone was found to be endemic in Malaysia. The incidence of drug resistant S. Enteritidis remained relatively low with only 37% of the strains analysed being resistant to one or more antimicrobial agents. All except one resistant strain carried at least one plasmid ranging in size from 3.7 to 62 MDa giving nine plasmid profiles. The three isolates from raw milk and one from well-water had similar PFPs to that of the human isolates. CONCLUSIONS: Salmonella Enteritidis strains were more diverse than was previously thought. Fourteen subtypes were noted although one predominant clone persisted in Malaysia. The combination of pulsed-field gel electrophoresis, plasmid profiling and antibiograms provided additional discrimination to the highly clonal strains of S. Enteritidis. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report to assess the genotypes of the predominant clinical S. Enteritidis in different parts of the country. As S. Enteritidis is highly endemic in Malaysia, the data generated would be useful for tracing the source during outbreaks of gastroenteritis in the study area.     

Long-term prevalence of antimicrobial-resistant Salmonella enterica subspecies enterica Serovar infantis in the broiler chicken industry in Japan

Eleven broiler isolates of Salmonella Infantis obtained between 1989 and 1998 were examined for antimicrobial susceptibility and pulse field gel electrophoresis (PFGE) profiles. Seven strains of S. Infantis isolated after 1993 harbored similar antimicrobial susceptibilities to the recent isolates between 2001 and 2003. In comparison of PFGE profile with 22 isolates obtained from 22 apparently healthy broiler chickens between 2001 and 2003, the predominant cluster included the seven strains isolated after 1993. We could not clarify the reasons why the serovar has been prevalent in the broiler industry for a long time, but current antimicrobial usage is not always linked to its prevalence.     

Comparison of Salmonella enterica serotype Infantis isolates from a veterinary teaching hospital

AIMS: To compare Salmonella enterica serotype Infantis isolates obtained from patients or the environment of a veterinary teaching hospital over a period of 9 years following a nosocomial outbreak to determine whether isolates were epidemiologically related or represented unrelated introductions into the hospital environment. METHODS AND RESULTS: Fifty-six S. Infantis isolates were compared based on their phenotypic (antimicrobial drug [AMD] susceptibility pattern) and genotypic (pulsed-field gel electrophoresis [PFGE] pattern and presence of integrons) characteristics. Epidemiologically unrelated S. Infantis isolates clustered separately from all but two of the hospital isolates, and several isolates from different years and various sources were indistinguishable from each other in cluster analysis of two-enzyme PFGE results. A high percentage of isolates (80.3%) were resistant to at least one AMD, with 67.8% showing resistance to >5 AMD. The majority (74.1%) of isolates tested contained type 1 integrons. CONCLUSION: Results strongly suggest that there was nosocomial transmission of S. Infantis during the initial outbreak, and that contamination arising from this outbreak persisted across years despite rigorous hygiene and biosecurity precautions and may have led to subsequent nosocomial infections. SIGNIFICANCE AND IMPACT OF THE STUDY: Evidence of persistence and transmission of Salmonella clones across years, even in the face of rigorous preventive measures, has important implications for other facilities that have experienced outbreaks of Salmonella infections.     

Salmonella choleraesuis proteins and their relation to proteins from bacteria of heterologous sero-groups.

A diversity of proteins was identified in the material isolated from S. choleraesuis with the help of sera prepared in rabbits with this material. The sera displayed, in agar-gel diffusions, numerous superimposed precipitation lines against proteins from: Salmonellae, Shigellae and E. coli. In contrast to proteins from S. paratyphi C, sharing identical identical 'O' 'factors, the serological activity of the S. choleraesuis proteins was impaired by heating. The immunochemical analysis of the sera before and after exhaustive absorptions with heterologous proteins exhibited a stronger relation of S. choleraesuis with S. thyphimiurium and S. Newport than with S. paratyphi C. The antibodies induced against free proteins with S. paratyphi C specificity, present in the mosaic of proteins isolated from S. choleraesuis, were removed by the respective absorption without substantial modifications of the homologous precipitation. In contrast, the absorption of the serum with proteins from either S. newport or S. typhimurium removed almost all the homologous induced antibodies. The strong relations found among species belonging to different serogroups underline the non-conformity of the empirical established serofactors.     

Investigation of clonal distribution and persistence of Salmonella Senftenberg in the marine environment and identification of potential sources of contamination

Salmonella Senftenberg was detected in the coastal areas of Galicia (NW Spain) in 1998, where it remained the predominant serovar for the next four years. Although the overall incidence of this serovar in the zone was lower than 1%, contamination by Salmonella serovar Senftenberg was located in very specific areas of the Ría de Arousa, where it persisted for more than five years. A total of 60 Salmonella serovar Senftenberg isolates, originating from surveillance activities in marine environments, was subjected to molecular characterization by pulsed-field gel electrophoresis (PFGE). PFGE analysis of the marine isolates allowed the differentiation of three main PFGE types, which contained the majority of the isolates, each type showing a specific spatial distribution in the coastal waters. The most prevalent pulse types persisted for more than four years, emphasizing their capacity to adapt and survive in marine environments. Using PFGE analysis, marine isolates were compared with Salmonella serovar Senftenberg isolates from neighbouring mussel-processing facilities and to other epidemiologically unrelated isolates from human, animal and feed sources. Comparison of the restriction patterns showed that indistinguishable PFGE types were present in the isolates from mussel-processing facilities and their surrounding marine areas, suggesting that the mussel processing is the main source for contamination with Salmonella Senftenberg in these marine environments. A molecular fingerprinting relationship was established between three shellfish isolates and a human isolate, which could be considered as preliminary evidence of infection caused by Salmonella Senftenberg associated with molluscan consumption.     

Genetic and phenotypic variability among Salmonella enterica serovar Typhimurium isolates from California dairy cattle and humans

Fifty-six human and 24 adult dairy cattle isolates of Salmonella enterica serovar Typhimurium from a single county in California were compared using ribotyping, insertion sequence typing (IS200), pulsed-field gel electrophoresis, plasmid typing, phage typing, and antimicrobial resistance testing. The majority of the isolates fell into one of two groups which were phage types DT104 and DT193. Combining the information from all typing methods, a total of 45 different "clusters" were defined, with 35 of those including only a single isolate. The library of isolates had a high degree of variability, but antibiotic resistance and plasmid typing each defined single clusters in which human or bovine isolates predominated (chi2, P < 0.05).     

Genomic relationship of Salmonella enterica serovar typhimurium DT104 isolates from Korea and the United States

Salmonella enterica serovar Typhimurium DT104 (Salmonella Typhimurium DT104 or DT104) has been emerging as a common pathogen for human in Korea since 1997. In order to compare the genomic relationship and to search for the dominant strains in Korea, we conducted pulsed-field gel electrophoresis (PFGE) and IS200 fingerprinting of 25 epidemiological unrelated isolates from human and animals from Korea and cattle from America. Two Salmonella Typhimurium DT104 isolates from human in Korea and all 8 isolates from American cattle had indistinguishable patterns from the PFGE and IS200 fingerprinting but multidrug-resistant Salmonella Typhimurium isolates, including DT104, from Korean animals had diverse genetic patterns. The data suggest that a dominant DT104 strain might have circulated between Korean and American cattle and that it had a high level of clonality.     

Population structure and evolution of the Bacillus cereus group

Representative strains of the Bacillus cereus group of bacteria, including Bacillus anthracis (11 isolates), B. cereus (38 isolates), Bacillus mycoides (1 isolate), Bacillus thuringiensis (53 isolates from 17 serovars), and Bacillus weihenstephanensis (2 isolates) were assigned to 59 sequence types (STs) derived from the nucleotide sequences of seven alleles, glpF, gmk, ilvD, pta, pur, pycA, and tpi. Comparisons of the maximum likelihood (ML) tree of the concatenated sequences with individual gene trees showed more congruence than expected by chance, indicating a generally clonal structure to the population. The STs followed two major lines of descent. Clade 1 comprised B. anthracis strains, numerous B. cereus strains, and rare B. thuringiensis strains, while clade 2 included the majority of the B. thuringiensis strains together with some B. cereus strains. Other species were allocated to a third, heterogeneous clade. The ML trees and split decomposition analysis were used to assign STs to eight lineages within clades 1 and 2. These lineages were defined by bootstrap analysis and by a preponderance of fixed differences over shared polymorphisms among the STs. Lineages were named with reference to existing designations: Anthracis, Cereus I, Cereus II, Cereus III, Kurstaki, Sotto, Thuringiensis, and Tolworthi. Strains from some B. thuringiensis serovars were wholly or largely assigned to a single ST, for example, serovar aizawai isolates were assigned to ST-15, serovar kenyae isolates were assigned to ST-13, and serovar tolworthi isolates were assigned to ST-23, while other serovars, such as serovar canadensis, were genetically heterogeneous. We suggest a revision of the nomenclature in which the lineage and clone are recognized through name and ST designations in accordance with the clonal structure of the population.     

Features of Salmonella serovars among food handlers in Kyushu, Japan

Salmonella were isolated from 106 (0.032%) of 331,644 fecal samples from food handlers, and from 144 of 11,478 fecal samples from symptomatic patients in Japan to determine the incidence and features of Salmonella serovars among food handlers. S. enterica subspecies enterica serovar Infantis (S. serovar Infantis) was the dominant serovar (accounting for 48.1%), followed by S. serovar Corvallis, which showed poor genetic diversity, and S. serovar Enteritidis among food handlers. The former two serovars were not dominant among symptomatic patients. The present study demonstrates the need for education on the sanitary handling of chicken eggs and chicken meat, which are possible infectious sources of these Salmonella serovars.     

Prevalence and fingerprinting of Listeria monocytogenes strains isolated from raw whole milk in farm bulk tanks and in dairy plant receiving tanks

The incidence of Listeria species in raw whole milk from farm bulk tanks and from raw milk in storage at a Swedish dairy plant was studied. Listeria monocytogenes was found in 1.0% and Listeria innocua was found in 2.3% of the 294 farm bulk tank (farm tank) milk specimens. One farm tank specimen contained 60 CFU of L. monocytogenes ml(-1). L. monocytogenes was detected in 19.6% and L. innocua was detected in 8.5% of the milk specimens from the silo receiving tanks at the dairy (dairy silos). More dairy silo specimens were positive for both Listeria species during winter than during summer. Restriction enzyme analysis and pulsed-field gel electrophoresis were applied to 65 isolates of L. monocytogenes, resulting in 16 different clonal types. Two clonal types were shared by the farm tank milk and the dairy silo milk. All except one clonal type belonged to serovar 1/2a. In the dairy silo milk five clonal types were found more frequently and for a longer period than the others. No Listeria species were found in any other samples from the plant.     

Natural transmission of Salmonella choleraesuis in swine.

This experiment was designed to study the natural transmission of Salmonella choleraesuis in swine. Forty pigs were divided into three groups. Group 1 (n = 12) was challenged with 10(8) CFU of S. choleraesuis per ml by intranasal inoculation. One day postinoculation (p.i.), group 2 (n = 24) was commingled with group 1. Group 3 (n = 4) served as uninoculated controls. Serum samples were collected weekly. Blastogenesis assays and necropsies were performed at 1, 2, 4, 6, 9, and 12 weeks p.i., and 16 tissue samples per pig were collected and cultured. Environmental (pooled feces from the pen floor) levels of S. choleraesuis were 2.61 log10 CFU/g of feces at 24 h p.i. (immediately prior to commingling). Severe clinical signs were observed in groups 1 and 2. The results indicated that at least 16% of group 2 pigs were shedding S. choleraesuis within 24 h of commingling. At 1 week p.i., 32 of 32 group 1 and 39 of 62 group 2 tissue samples were positive for S. choleraesuis. Only 3 of 12 group 2 pigs were positive at 6, 9, and 12 weeks (1 pig for each week), indicating that only a small proportion of infected swine become long-term carriers. At 12 weeks p.i., only the colon and colonic lymph node samples of one pig from group 2 were positive. Humoral, mucosal, and cellular immune responses were similar between groups 1 and 2. These data demonstrate that a few pigs shedding low levels of Salmonella organisms before slaughter can result in rapid transmission and subsequent shedding by many swine.     

Nontyphoidal salmonellae in United Kingdom badgers: prevalence and spatial distribution

Eighteen (72%) of 25 badger social groups were found to excrete Salmonella enterica serovar Ried, S. enterica serovar Binza, S. enterica serovar Agama, or S. enterica serovar Lomita. Each serovar was susceptible to a panel of antimicrobials. Based on results of pulsed-field gel electrophoresis, the S. enterica serovar Agama and S. enterica serovar Binza isolates were very similar, but two clones each of S. enterica serovar Lomita and S. enterica serovar Ried were found. Badgers excreting S. enterica serovar Agama were spatially clustered.     

猪霍乱沙门菌载体介导猪瘟病毒DNA免疫研究

构建了猪瘟病毒(CSFV)主要保护性抗原E2基因的真核表达质粒pVAXE2。将其电转化猪霍乱沙门氏菌C500疫苗株,得到了携带pVAXE2质粒的猪霍乱沙门氏菌工程菌株S.C500/pVAXE2,对该菌株的特征、培养特性和生化特性进行了鉴定。分别用1×10⁸CFU、2×10⁹CFU S.C500/pVAXE2经口服或肌肉注射免疫小鼠和家兔,间接ELISA检测免疫动物的特异性抗体,在第三次免疫后2周用20ID50猪瘟兔化弱毒和致死量猪霍乱沙门氏菌强毒株对免疫兔进行攻击。结果表明,S.C500/pVAXE2保持了猪霍乱沙门氏菌原有形态特征、培养特性和生化特性,免疫鼠和兔都产生了抗CSFV和猪霍乱沙门菌的ELISA抗体,免疫家兔能抵抗猪瘟兔化弱毒株和猪霍乱沙门氏菌强毒株的攻击。显示了以S.C500为DNA运输载体构建二联或多联猪用疫苗的可行性。     

Genetic and Phenotypic Variability among Salmonella enterica Serovar Typhimurium Isolates from California Dairy Cattle and Humans

Fifty-six human and 24 adult dairy cattle isolates of Salmonella enterica serovar Typhimurium from a single county in California were compared using ribotyping, insertion sequence typing (IS200), pulsed-field gel electrophoresis, plasmid typing, phage typing, and antimicrobial resistance testing. The majority of the isolates fell into one of two groups which were phage types DT104 and DT193. Combining the information from all typing methods, a total of 45 different “clusters” were defined, with 35 of those including only a single isolate. The library of isolates had a high degree of variability, but antibiotic resistance and plasmid typing each defined single clusters in which human or bovine isolates predominated (χ², P < 0.05).     

Molecular analysis of Leptospira spp. isolated from humans by restriction fragment length polymorphism, real-time PCR and pulsed-field gel electrophoresis

A total of 17 Leptospira clinical strains isolated from humans in Croatia were serologically and genetically analysed. For serovar identification, the microscopic agglutination test (MAT) and pulsed-field gel electrophoresis (PFGE) were used. To identify isolates on genomic species level, PCR-based restriction fragment length polymorphism (RFLP) and real-time PCR were performed. MAT revealed the following serogroup affinities: Grippotyphosa (seven isolates), Icterohaemorrhagiae (eight isolates) and Javanica (two isolates). RFLP of PCR products from a 331-bp-long fragment of rrs (16S rRNA gene) digested with endonucleases MnlI and DdeI and real-time PCR revealed three Leptospira genomic species. Grippotyphosa isolates belonged to Leptospira kirschneri , Icterohaemorrhagiae isolates to Leptospira interrogans and Javanica isolates to Leptospira borgpetersenii . Genomic DNA from 17 leptospiral isolates was digested with NotI and SgrAI restriction enzymes and analysed by PFGE. Results showed that seven isolates have the same binding pattern to serovar Grippotyphosa, eight isolates to serovar Icterohaemorrhagiae and two isolates to serovar Poi. Results demonstrate the diversity of leptospires circulating in Croatia. We point out the usefulness of a combination of PFGE, RFLP and real-time PCR as appropriate molecular methods in molecular analysis of leptospires.     

Molecular analyses of Salmonella enterica isolates from fish feed factories and fish feed ingredients

Isolates of the most commonly observed salmonella serovars in Norwegian fish feed factories from 1998 to 2000 (Salmonella enterica serovar Agona, S. enterica serovar Montevideo, S. enterica serovar Senftenberg, and S. enterica serovar Kentucky) were studied by pulsed-field gel electrophoresis (PFGE) and plasmid profile analysis and compared to isolates of the same serovars from fish feed ingredients, humans, and other sources (a total of 112 isolates). Within each serovar, a variety of distinct PFGE types (with similarity levels less than 90%) were observed in the feed ingredients and other sources, while only two distinct types of each serovar were identified in the factories. The combined results of PFGE and plasmid analyses showed that each factory harbored only a few S. enterica clones. Some of these clones persisted for at least 3 years in the factories, indicating that there was long-lasting contamination probably due to inadequate decontamination procedures.