Molecular typing of Salmonella enterica serovar 4,[5],12:i:- isolates from humans,animals and river water in Japan by multilocus variable-number tandem repeat analysis and pulsed-field gel electrophoresis

Fifty-one Salmonella enterica serovar 4,[5],12:i:- (S. 4, [5],12:i:-) isolates (14 human strains, 34 animal strains and 3 river water strains) which are assumed to be monophasic variants of S. Typhimurium were analyzed using pulsed-field gel electrophoresis (PFGE) and multiple-locus variable-number tandem repeat analysis (MLVA) in order to investigate their genetic diversities and relationships. PFGE, MLVA and combination of them identified 28, 27 and 34 profiles (Simpson’s diversity indices [DI]=0.94, 0.96 and 0.97), respectively. No correlations were detected between MLVA clustering and PFGE clustering or phage typing. These results suggested that S. 4,[5],12:i:- originated from multiple S. Typhimurium ancestors. Two cattle and one pig isolates showing identical phage types as well as PFGE and MLVA profiles to human isolates S. 4,[5],12:i:- suggested the existence of the links between human infections and animal reservoirs.     

Characterization of Salmonella enterica serovar Typhimurium and Salmonella enterica serovar 4,[5],12:i:- isolates from pigs presenting with diarrhea in Korea

Between 2011 and 2012, a total of 896 pig fecal samples were collected from nine provinces in Korea, and 50 salmonella enterica susp. enterica serovar Typhimurium (S. Typhimurium) was isolated. The characteristics of the 50 strains were analyzed, and 4 strains were identified as Salmonella enterica subsp. enterica serovar 4,[5],12:i:-. Salmonella 4,[5],12:i:- could not be distinguished from S. Typhimurium through phage typing, antimicrobial resistance testing or multiple-locus variable-number tandem repeat analysis (MLVA). However, among the four Salmonella 4,[5],12:i:- strains, one (KVCC-BA1400078) was identified as a Salmonella 4,[5],12:i:- clone isolated from humans in the United States, and another (KVCC-BA1400080) was identified as DT193, which has been primarily isolated from humans and animals in European countries. The presence of Salmonella 4,[5],12:i:- in Korea poses a significant threat of horizontal transfer between pigs and humans.     

Investigation and characterization of the frozen feeder rodent industry in Texas following a multi-state Salmonella Typhimurium outbreak associated with frozen vacuum-packed rodents

A multi-state outbreak investigation of Salmonella Typhimurim cases associated with pet snakes and the frozen vacuum-packed rodents used to feed them identified a Texas frozen feeder rodent facility (Supplier A) as the source of the Salmonella-infected frozen rodents. Texas authorities collected samples directly from Supplier A. Seven Salmonella-positive samples out of 49 environmental swabs were found and one adult mouse out of 88 frozen feeder rodents was Salmonella-positive by culture. No Salmonella strains were isolated from rodent feeds. The pulsed-field gel electrophoresis (PFGE) subtype patterns of S. Typhimurium isolates from feeder rodent and environment samples were indistinguishable from the outbreak strain isolated from humans. A follow-up investigation was performed on all additional feeder rodent facilities identified in Texas. Salmonella was isolated at one of four facilities; seven of 100 rodent samples were positive for Salmonella at this facility. The serotype S. I 4,[5],12:i:- was isolated from seven feeder rodent samples, and PFGE patterns of the seven isolates were indistinguishable. As observed in the initial outbreak investigation, no Salmonella were cultured from rodent feeds at any of the facilities. The feeder rodent industry is an insufficiently recognized industry in the United States. Outbreak investigation and testing of additional feeder rodent facilities in Texas indicate that further evaluation of feeder rodent facilities as a source of Salmonella for pet snakes and humans is warranted.     

Report on Salmonella isolates submitted to the German National Reference Laboratory for running analyses and tests on zoonoses (Salmonella) in the year 2009

The present report deals with Salmonella strains received at the German National Reference Laboratory for Salmonella (NRL-Salm) for routine diagnostic in the year 2009. Hence, the working group continues the previous report from Friedrich et al. (2010) about the documentation on the serovar distribution of Salmonella received at the NRL-Salm in the years 2004-2008. As in the recent years, most of the Salmonella strains originated from livestock and food. In the year 2009 the NRL-Salm received 4765 isolates, most of them (85,1 %) were routine diagnostic. Salmonella ser. Typhimurium, its monophasic variant S. enterica subspecies enterica serovar 1,4,[5],12:i:- and Salmonella ser. Enteritidis were the most prevalent serovars. The number of S. enterica subsp. enterica serovar 1,4,[5],12:i:- isolates increased in 2009, in comparison to the years 2004-2008, and became the second most prevalent serovar serotyped at the NRL-Salm.     

Pulsed field gel electrophoresis for animal Salmonella enterica serovar Typhimurium isolates in Taiwan

Salmonella enterica subspecies enterica serovar Typhimurium is a common pathogen for humans and animals. In order to trace the clonal relationship and to find the circulating strains between human and animal isolates, chromosomal DNAs from 87 serovar Typhimurium strains isolated from animals (pigs were the majority) were subjected to XbaI and SpeI digestion and pulsed field gel electrophoresis (PFGE). For the 87 animal isolates, 38 PFGE pattern combinations were obtained. As the subtyping results from animal isolates were compared with those from the 45 human isolates, it was found that 14 of the animal isolates and 13 of the human isolates shared a common PFGE pattern combination, i.e., pattern XgSf (or called X5S4). When these human and animal isolates were subjected to antibiotic susceptibility test using 11 antibiotics, it was found that strains of pattern XgSf (X5S4) belong to a common antibiogram pattern which is tetracycline, gentamicin, ampicillin, streptomycin and chloramphenicol resistant. Since most of the animal and human strains in pattern XgSf were originally isolated from various areas over different years, strains of this PFGE pattern may be the most epidemic strains which circulating between human and animal sources.     

Detection and identification of Salmonella Typhimurium in bovine diarrhoeic fecal samples by immunomagnetic separation and multiplex PCR assay

The aim of this study was to use the immunomagnetic separation (IMS) test plus a multiplex polymerase chain reaction (m-PCR) assay to detect Salmonella at genus level and also for the identification of Salmonella enterica serovar Typhimurium in bovine diarrhoeic fecal samples. In all, 400 bovine diarrhoeic fecal specimens were examined by conventional bacterial culture, IMS, and m-PCR. For m-PCR assay, four set primers were selected: 139-141, specific for inv-A gene of Salmonella spp and the RfbJ, FliC and FljB, specific for the rfbJ, FliC and fljB genes of Salmonella Typhimurium or other Salmonella serovars with similar antigenic properties. Thirty-three (8.25%) out of the 400 fecal samples were culture positive for Salmonella serovars. Of these, 66.7% (22 of 33) were Salmonella enterica serovar Typhimurium, and 9.1% (three of 33) were serovar Dublin. In the IMS + m-PCR, four amplified product (663, 526, 284 and 183 bp) were found in all specimens that had serovar Typhimurium (4,5,12:i:1,2), they corresponded, respectively, to the rfbJ, fljB, inv-A and Flic genes of this serovar. In serovar Dublin (1,9,12:g,p:-), Georgia (6,7:b:e,n,z(15)) and, Enteritidis (1,9,12;g,m:-) only one PCR product (284 bp) was amplified from the inv-A gene. In serovars Augustenborg (6,7:i:1,2) and Lindenburg (6,8:i:1,2) three positive bands (526, 284 and 183 bp) were amplified corresponding to the fljB, inv-A and Flic genes, respectively. In serovar Virchow (6,7:r:1,2) two amplified products (284 and 526 bp) from the inv-A and FliC genes were observed. In serovar Gloucster (1,4,12(27):i:1,w) three fragments (183, 284 and 663) from the FliC, inv-A and, rfbJ genes respectively, were observed. In the positive control as expected, four PCR products were amplified corresponding to the FliC, inv-A, fljB and rfbJ genes, respectively. In conclusion, the results of this study showed that detection of Salmonella at genus level with universal ST139-141 primers and identification of Salmonella Typhimurium by using specific primers of O4, H(2):1, 2 and H(1) antigens can potentially permit to more readily evaluate fecal and other types of samples for the presence of these organisms. Compared to bacteriological culture the combination of IMS and m-PCR resulted a faster method for the detection and identification of Salmonella at genus and serovar level by using of universal and specific primers.     

Antimicrobial resistance, virulence-associated genes, and pulsed-field gel electrophoresis profiles of Salmonella enterica subsp. enterica serovar Typhimurium isolated from piglets with diarrhea in Korea

Salmonella enterica subsp. enterica serovar Typhimurium was isolated from diarrheic piglets in 2 periods, 2000-2001 (n = 25) and 2005-2006 (n = 17). To compare the characteristics of the isolates collected during the 2 periods, all isolates were tested for antimicrobial resistance, the presence of virulence genes, and pulsed-field gel electrophoresis (PFGE) patterns. All 42 isolates were resistant to at least 1 of the 20 antimicrobials tested, and 39 (93%) were resistant to 2 or more antimicrobials. One isolate was resistant to 12 antimicrobials. Profiles of antimicrobial resistance revealed 20 resistance types. Several isolates were also resistant to quinolones and expanded-spectrum cephalosporins. Ten isolates (24%) were resistant to ampicillin, chloramphenicol, streptomycin, sulfonamides, and tetracycline (ACSSuT); only one isolate had been isolated in 2000-2001, indicating that this type of resistance has rapidly disseminated. Polymerase chain reaction (PCR) assays revealed that all the isolates carried invA. Among the 25 strains isolated in 2000-2001, all carried the sipA, sopA, sopD, sopE2, and ssaR genes, and 96% carried sopB and sifA. Among the 17 strains isolated in 2005-2006, all carried sifA, and approximately 90% carried sipA, sopA, sopB, sopD, sopE2, and ssaR. However, only 6 (14%) of the 42 isolates carried spvC. By PFGE analysis, all 42 strains were classified into 4 major clusters, basically by collection period. The genetic similarity according to PFGE suggests that the strains isolated from diarrheic piglets of this region within the same period may be closely related.     

Distribution of Salmonella Serovars and Antimicrobial Susceptibility from Poultry and Swine Farms in Central Vietnam

This study was conducted to estimate the prevalence of Salmonella spp. and their antimicrobial susceptibilities on poultry and swine farms, sampled in 2 regions in Central Vietnam. A total of 67 poultry farms and 46 swine farms were sampled in a period of 5 months (from September 2012 to January 2013). Salmonella spp. was prevalent in 46.3% and 71.7% of poultry and swine farms, respectively. Altogether, 99 non‐typhoidal Salmonella were isolated and the most common serovars were Salmonella Weltevreden (19%), followed by Salmonella Typhimurium (12%) and Salmonella 4,[5],12:i:‐ (11%). Overall, 71 of 99 (72%) Salmonella isolates were resistant to at least one of the 14 antimicrobial agents tested. Both in poultry and swine farms, high levels of resistance were observed for ampicillin, chloramphenicol, ciprofloxacin, sulphamethoxazole and tetracycline. The presence of Salmonella isolates from poultry and swine farms which were resistant to different classes of antimicrobials suggests that alternative control measures to antimicrobials should be implemented. Moreover, an effective policy should be promoted to encourage a prudent use of these agents in animal farming in Vietnam.     

An epidemiological study of Salmonella enterica serovar 4, 12:b:- in broiler chickens in Denmark

Epidemiological investigations of isolates of Salmonella enterica serovar 4, 12:b:- were carried out to establish particular molecular markers to assign isolates to a common origin. Plasmid profiling demonstrated that over 50% of 291 isolates, obtained between 1991 and 1996, were plasmid-free. The remaining isolates exhibited a common trend in plasmid content of 105 and 2kb. Although no specific correlation to any particular source within the poultry industry was discernible using plasmid analysis, there were indications of clonality with local divergence. Ribotyping with EcoRI demonstrated limited discriminative potential as 96% of the isolates expressed a common profile. Ribotyping with HindIII failed to further differentiate the isolates. IS200 (PstI) typing and PFGE (NotI and XbaI) afforded some degree of further discrimination with selected isolates. Each technique produced four profiles, but dominant profiles were also apparent. Eighteen variables were selected for multivariate logistic regression analysis in order to identify risk areas associated with broiler flocks within the industry. An increased risk for S. 4, 12:b:- infection was only associated with the feedmills used. Random effects at the house and/or farm level were also found to be statistically significant. Of the 16 feedmills associated with the isolation of 4, 12:b:-, six were deemed to be significant risk factors.     

Extended spectrum beta-lactamase SHV-12-producing Salmonella from poultry

Salmonella strains isolated from poultry and poultry products over the period 2005-2006 have been investigated in order to ascertain the presence of extended spectrum cephalosporins (ESC) resistance. Twelve (ESC)-resistant isolates (n=1 S. Enteritidis, n=1 S. Braenderup and n=10 S. Livingstone) were characterized as SHV-12-positive. The multi-drug resistant S. Livingstone SHV-12-producing isolates, untypeable by pulsed-field gel electrophoresis (PFGE), showed a clonal relationship by random amplified polymorphic DNA (RAPD) analysis. The SHV-12 beta-lactamase is reported for the first time in Salmonella enterica strains isolated from poultry in Italy. The results suggest poultry as a source of Salmonella carrying extended spectrum beta-lactamases (ESBLs) genes and highlights the need of monitoring animal productions to prevent spreading of (ESC)-resistant strains.     

Changes in the prevalence of Salmonella serovars associated swine production and correlations of avian,bovine and swine‐associated serovars with human‐associated serovars in the United States (1997–2015)

As Salmonella enterica is an important pathogen of food animals, surveillance programmes for S. enterica serovars have existed for many years in the United States. Surveillance programmes serve many purposes, one of which is to evaluate alterations in the prevalence of serovars that may signal changes in the ecology of the target organism. The primary aim of this study was to evaluate changes in the proportion of S. enterica serovars isolated from swine over a near 20‐year observation period (1997–2015) using four longitudinal data sets from different food animal species. The secondary aim was to evaluate correlations between changes in S. enterica serovars frequently recovered from food animals and changes in S. enterica serovars associated with disease in humans. We found decreasing proportions of S. enterica serovar Typhimurium, serovar Derby and serovar Heidelberg and increasing proportions of S. enterica serovar 4,[5],12:i:‐, serovar Infantis and serovar Johannesburg in swine over time. We also found positive correlations for the yearly changes in S. enterica serovar 4,[5],12:i:‐, serovar Anatum and serovar Johannesburg between swine and human data; in S. enterica Worthington between avian and human data; and in S. enterica serovar 4,[5],12:i:‐ between bovine and human data. We found negative correlations for the yearly changes in S. enterica serovar 4,[5],12:i:‐ and serovar Johannesburg between avian and human data.     

Changes of multi-drug resistance pattern in Salmonella enterica subspecies enterica serovar typhimurium isolates from food-producing animals in Japan

A total of 153 isolates of Salmonella Typhimurium derived from food-producing animals in Japan between 2002 and 2005 were investigated for antimicrobial resistance and phage types related to definitive phage type 104 (DT104). The predominant resistance type was resistance to ampicillin, dihydrostreptomycin, kanamycin, and oxytetracycline in bovine (45.2%, 48/104) and resistance to dihydrostreptomycin and oxytetracycline in porcine isolates (58.7%, 27/48). DT104-related phage type was found in 32 of 104 bovine isolates, two of 48 porcine isolates, and one of eight isolates from poultry, showing that the proportion of the phage type in S. Typhimurium isolates from cattle and pigs significantly (P<0.01) decreased from 71.9% and 31.4% in 1999-2001 to 30.8% and 4.1% in 2002-2005, respectively.     

Molecular typing of monophasic Salmonella 4,[5]:i:- strains isolated in Belgium (2008–2011)

To assess the distribution of Salmonella 4,[5]:i:- subtypes in the Belgian food chain and compare it to the subtypes associated with human infections, a molecular assessment was initiated. Two hundred fifty-three Salmonella isolates serotyped as 4,[5]:i:- during the period 2008–2011 in Belgium and originating from animal productions, food or human clinical samples were analysed by a specific duplex PCR. One hundred ninety-four isolates (76.7%) fit the profile of a S. Typhimurium monophasic variant as defined by the European Food Safety Authority. The other isolates possessed but did not express the phase II flagellin gene (23.3%). Multiple Locus Variable Number of Tandem Repeats Analysis (MLVA) revealed many but closely related profiles in the fljB-negative S. Typhimurium monophasic variant isolates. Some MLVA types were associated with both human and animal isolates but no unique source of human contamination could be demonstrated.     

A multi-state Salmonella Typhimurium outbreak associated with frozen vacuum-packed rodents used to feed snakes

From December 2005 through January 2006, the Minnesota Department of Health (MDH) identified four human clinical isolates of Salmonella Typhimurium that were indistinguishable by pulsed-field gel electrophoresis (PFGE). During routine interviews, three of the cases reported attending the same junior high school and two handled snakes in the science classroom. MDH collected environmental samples from the school's science classroom for Salmonella culturing; these included environmental samples and frozen vacuum-packed mice purchased over the internet to feed the classroom snakes. Through PulseNet, a national molecular subtyping surveillance network for enteric bacteria, 21 human S. Typhimurium isolates with indistinguishable PFGE patterns were identified in the United States since December 2005. Each state determined whether these human cases had recent exposure to snakes fed vacuum-packed rodents. Texas state officials conducted tracebacks of the vacuum-packed mice and collected samples at the breeding facility. Nineteen of 21 cases were interviewed, and seven reported contact with frozen vacuum-packed rodents from the same internet-based supplier in Texas. In Minnesota, the outbreak PFGE subtype of S. Typhimurium was isolated from the snakes, frozen feed rodents, and the classroom environment. Three human cases were identified in Michigan, Pennsylvania, and Wyoming. The outbreak PFGE subtype of S. Typhimurium was isolated from the Pennsylvania case's frozen rodents and the Michigan case's pet snake. The outbreak PFGE subtype of S. Typhimurium was also isolated from the supplier's rodent facility. This was a S. Typhimurium outbreak associated with frozen rodents. Human transmission likely occurred through direct contact with snakes and contaminated environmental surfaces. This report represents the second recent multi-state salmonellosis outbreak associated with commercially distributed rodents. Stronger oversight of the commercial rodent industry is warranted.     

Dynamics of Salmonella contamination in a commercial quail operation.

Control of carcass contamination requires knowledge of the source and dynamics of spread of Salmonella in commercial poultry production. We examined Salmonella contamination at a U.S. commercial quail operation. Pulsed-field gel electrophoresis (PFGE) was used to type isolates in order to trace Salmonella throughout this production environment. During a 6-mo survey, Salmonella serotypes hadar, typhimurium, typhimurium variant Copenhagen, and paratyphi were encountered within this poultry operation. Ninety-four percent of the Salmonella isolated from breeder and production houses and from carcass rinses belonged to Salmonella serotypes typhimurium variant Copenhagen and hadar. There were six distinct S. typhimurium variant Copenhagen genetic types, as identified by PFGE, present within this particular poultry operation. Seventy-nine percent of S. typhimurium variant Copenhagen identified from the environment of the breeder and production houses produced the same PFGE pattern. Thirty-eight percent of S. typhimurium Copenhagen isolated from carcass rinses and the breeder house shared the same PFGE DNA pattern. This study demonstrates the transmission of salmonellae throughout this production environment, from the breeders to their progeny and to the birds ultimately processed for human consumption.     

Evaluation of molecular typing methods for Salmonella enterica serovar Typhimurium DT104 isolated in Germany from healthy pigs

The discriminatory power of four different DNA based typing methods was tested for the molecular subtyping of Salmonella Typhimurium phage type DT104 isolates. German DT104 strains (n = 133) originating from slaughter pigs were analysed by plasmid profiling, and 32 of them by pulsed-field gel electrophoresis (PFGE) using the restriction enzymes XbaI, SpeI or BlnI, random amplification of polymorphic DNA (RAPD) using 13 different primers and IS200 typing. A resulting subtyping scheme was obtained which is based on the most discriminatory power of the individual methods i.e. plasmid profiling and PFGE with all three enzymes. The index of discrimination obtained by the subtyping scheme was 0.909 closely approaching the maximum value of one. Although minor differences occurred in the molecular DNA pattern of single DT104 strains, a dominating subtyping pattern was observed confirming other studies which showed, that S. Typhimurium DT104 isolates are highly clonal.     

Comparison of methods for differentiation of Salmonella enterica serovar Enteritidis phage type 4 isolates

OBJECTIVE: To compare molecular typing methods for the differentiation of Salmonella enterica serovar Enteritidis phage type (PT) 4 isolates that allowed for the determination of their genetic relatedness. SAMPLE POPULATION: 27 Salmonella Enteritidis PT 4 strains isolated in the United States and Europe. PROCEDURE: Several molecular typing methods were performed to assess their ability to genetically differentiate among Salmonella Enteritidis PT 4 isolates. Results of pulse-field gel electrophoresis (PFGE), repetitive polymerase chain reaction (PCR) assay, 16S rRNA gene sequencing, random amplification of polymorphic DNA (RAPD), PCR-restriction fragment length polymorphism of 16S rRNA, and antimicrobial susceptibility were evaluated. RESULTS: Compared with results for other techniques, results for the RAPD typing method with the RAPD1 primer reveal that it was the most discriminatory fingerprinting technique, and it allowed us to cluster Salmonella Enteritidis PT 4 isolates on the basis of their genetic similarity. CONCLUSIONS AND CLINICAL RELEVANCE: This study revealed the value of RAPD with the RAPD1 primer as a tool for epidemiologic investigations of Salmonella Enteritidis PT 4. It can be used in conjunction with PFGE and phage typing to determine the genetic relatedness of Salmonella Enteritidis isolates involved in outbreaks of disease. A reliable and highly discriminatory method for epidemiologic investigations is critical to allow investigators to identify the source of infections and consequently prevent the spread of Salmonella Enteritidis PT 4.     

Assessment of antibiotic resistance phenotype and integrons in Salmonella enterica serovar Typhimurium isolated from swine

Salmonella enterica serovar Typhimurium (S. Typhimurium) isolated and identified from swine were subjected for the analysis of antibiotic resistance pattern and clinically important class 1 and 2 integrons. In addition, S. Typhimurium isolates exhibiting ampicillin, chloramphenicol, streptomycin, sulfamethoxazole, tetracycline and florfenicol (ACSSuTF) resistance pattern as described in most Salmonella enterica serotype Typhimurium definitive type 104 (DT104) were characterized by polymerase chain reaction. All the isolates were resistant to more than four antibiotics and showed the highest resistance to streptomycin (94.1%), followed by tetracycline (90.1%), ampicillin (64.7%), chloramphenicol (56.8%) and gentamicin (54.9%). MIC value for the ten isolates ranged between 0.125-2 mug/ml for ciprofloxacin. Among the beta-lactams used, only one of the isolate exhibited resistance to ceftiofur (MIC 8 microg/ml). Sixty eight percent of these multi drug resistance (MDR) S. Typhimurium isolates carried clinically important class 1 integron with 1kb (aadA) and/or 2kb (dhfrXII-orfF-aadA2) resistance gene cassettes. This study reports the increasing trend of multi drug resistance (MDR) S. Typhimurium with clinically important class 1 integron in pigs. In addition, emergence of the ACSSuTF-type resistance in S. Typhimurium PT other than DT104 may limit the use of resistance gene markers in its detection methods by PCR.     

Class 1 integrons in Dutch Salmonella enterica serovar Dublin isolates from clinical cases of bovine salmonellosis

Fifty-nine Salmonella enterica serovar Dublin (Salmonella Dublin) isolates from clinical cases of bovine salmonellosis between 1993 and 2004 were tested for their susceptibility to 15 antimicrobial agents and the presence of class 1 integrons. Integrons were further analyzed by conserved segment PCR-RFLP. DNA sequencing was used to identify the inserted gene cassette. Twelve (20.3%) isolates were multidrug-resistant. A combination of resistance against chloramphenicol, streptomycin and sulphonamides was the most common phenotype observed. Multidrug-resistance (MDR) was found to be strongly associated with the presence of integrons, since a class 1 integron with the aadA1 gene cassette encoding resistance to streptomycin and spectinomycin was found in all 12 multidrug-resistant isolates. The presence of the aadA1 gene in Salmonella Dublin has not been reported before. None of the integron carrying Salmonella Dublin isolates could transfer its antimicrobial resistance to E. coli K12 by conjugation. Analysis of plasmid profiles and pulsed field gel electrophoresis (PFGE) patterns showed at least some clonality among the Salmonella Dublin isolates, but 11 different types could be distinguished based on both XbaI and BlnI-PFGE patterns. Thus, the Dutch Salmonella Dublin strains were closely related but not clonal.