Detection of multidrug-resistant Salmonella typhimurium DT104 by multiplex polymerase chain reaction

Salmonella typhimurium definitive type 104 (DT104) is a virulent pathogen for humans and animals with many strains having multiple drug resistance characteristics. The organism typically carries resistance to ampicillin, chloramphenicol, florfenicol, streptomycin, sulfonamides, and tetracycline (ACSSuT-resistant). A multiplex PCR method was developed to simultaneously amplify four genes, florfenicol (flo(st)), virulence (spvC), invasion (invA), and integron (int) from S. typhimurium DT104 (ACSSuT-type). Twenty-two ACSSuT-resistant DT104 isolates in our collection gave 100% positive reactions to this PCR assay by amplifying 584-, 392-, 321- and 265-bp PCR products, using primers specific to the respective target genes. One Salmonella strain DT23, ACSSuT-resistant, phage type 711 failed to amplify the 584-bp fragment, indicating that this method is specific for DT104-type ACSSuT-resistant S. typhimurium strains. One clinical and one bovine ASSuT-resistant strains that were sensitive to chloramphenicol and florfenicol did not yield a 584-bp fragment, indicating the absence of the flo(st) gene. This method will be useful for rapid identification of ACSSuT-type DT104 strains from clinical, food and environmental samples.     

Prevalence and genetic properties of Salmonella enterica serovar typhimurium definitive phage type 104 isolated from Rattus norvegicus and Rattus rattus house rats in Yokohama City, Japan

Salmonella enterica serovar Typhimurium was isolated from the intestinal contents of Rattus rattus and Rattus norvegicus house rats captured at two buildings, designated buildings J and YS, in Yokohama City, Japan. From October 1997 to September 1998, 52 of 339 (15.3%) house rats were found to carry Salmonella serovar Typhimurium definitive phage type 104 (DT104). In building J, 26 of 161 (16.1%) house rats carried DT104 over the 1-year study period, compared to 26 of 178 (14.6%) rats in building YS. The isolation rates of DT104 from R. rattus and R. norvegicus were similar in the two buildings. Most DT104 strains from building J (24 of 26) showed resistance to ampicillin, chloramphenicol, streptomycin, sulfisoxazole, and tetracycline and contained both the 1.0- and 1.2-kbp integrons, carrying genes pse1, pasppflo-like, aadA2, sulI, and tet(G). All DT104 strains from building YS were resistant to ampicillin and sulfisoxazole, and had the 1.2-kbp integron carrying pse1 and sulI. Cluster analysis of pulsed-field gel electrophoresis patterns of BlnI-digested DT104 DNAs showed that 22 of 26 DT104 strains from building J and 24 of 26 strains from building YS could be grouped into separate clusters each specific for the building origin. These results indicated that DT104 strains were prevalent in house rat colonies in each building and suggest that house rats may play an important role in the epidemiology of DT104.     

Molecular characterization of Irish Salmonella enterica serotype typhimurium: detection of class I integrons and assessment of genetic relationships by DNA amplification fingerprinting

Salmonella enterica is among the principal etiological agents of food-borne illness in humans. Increasing antimicrobial resistance in S. enterica is a cause for worldwide concern. There is concern at present in relation to the increasing incidence of human infection with antimicrobial agent-resistant strains of S. enterica serotype Typhimurium, in particular of phage type DT104. Integrons appear to play an important role in the dissemination of antimicrobial resistance genes in many Enterobacteriaceae including S. enterica. In this study the antimicrobial susceptibilities and phage types of 74 randomly collected strains of S. enterica serotype Typhimurium from the Cork region of southern Ireland, obtained from human, animal (clinical), and food sources, were determined. Each strain was examined for integrons and typed by DNA amplification fingerprinting (DAF). Phage type DT104 predominated (n = 48). Phage types DT104b (n = 3), -193 (n = 9), -195 (n = 6), -208 (n = 3), -204a (n = 2), PT U302 (n = 1), and two nontypeable strains accounted for the remainder. All S. enterica serotype Typhimurium DT104 strains were resistant to ampicillin, chloramphenicol, streptomycin, Sulfonamide Duplex, and tetracycline, and one strain was additionally resistant to trimethoprim. All DT104 strains but one were of a uniform DAF type (designated DAF-I) and showed a uniform pattern of integrons (designated IP-I). The DT104b and PT U302 strains also exhibited the same resistance phenotype, and both had the DAF-I and IP-I patterns. The DAF-I pattern was also observed in a single DT193 strain in which no integrons were detectable. Greater diversity of antibiograms and DAF and IP patterns among non-DT104 phage types was observed. These data indicate a remarkable degree of homogeneity at a molecular level among contemporary isolates of S. enterica serotype Typhimurium DT104 from animal, human, and food sources in this region.     

Detection and characterisation of integrons in Salmonella enterica serotype enteritidis

Integrons have been widely described among the Enterobacteriaceae including strains of multi-resistant Salmonella enterica serotype Typhimurium DT104; however, information with respect to the presence of integrons among S. enterica serotype Enteritidis strains is limited. Multi-resistant isolates of Enteritidis were screened for the presence of integrons using a PCR protocol. One integron was detected in all isolates that were resistant to sulfonamide and streptomycin. Characterisation of these isolates indicated an integron which ranged in size between 1000 and 2000 bp and which harboured a gene cassette encoding the ant(3")-Ia gene specifying streptomycin and spectinomycin resistance. Further studies revealed the integrons to be located on large conjugative plasmids. This appears to be the first report of plasmid-borne integrons in Enteritidis.     

Molecular Characterization of Irish Salmonella enterica Serotype Typhimurium: Detection of Class I Integrons and Assessment of Genetic Relationships by DNA Amplification Fingerprinting

Salmonella enterica is among the principal etiological agents of food-borne illness in humans. Increasing antimicrobial resistance in S. enterica is a cause for worldwide concern. There is concern at present in relation to the increasing incidence of human infection with antimicrobial agent-resistant strains of S. enterica serotype Typhimurium, in particular of phage type DT104. Integrons appear to play an important role in the dissemination of antimicrobial resistance genes in many Enterobacteriaceae including S. enterica. In this study the antimicrobial susceptibilities and phage types of 74 randomly collected strains of S. enterica serotype Typhimurium from the Cork region of southern Ireland, obtained from human, animal (clinical), and food sources, were determined. Each strain was examined for integrons and typed by DNA amplification fingerprinting (DAF). Phage type DT104 predominated (n = 48). Phage types DT104b (n = 3), -193 (n = 9), -195 (n = 6), -208 (n = 3), -204a (n = 2), PT U302 (n = 1), and two nontypeable strains accounted for the remainder. All S. enterica serotype Typhimurium DT104 strains were resistant to ampicillin, chloramphenicol, streptomycin, Sulfonamide Duplex, and tetracycline, and one strain was additionally resistant to trimethoprim. All DT104 strains but one were of a uniform DAF type (designated DAF-I) and showed a uniform pattern of integrons (designated IP-I). The DT104b and PT U302 strains also exhibited the same resistance phenotype, and both had the DAF-I and IP-I patterns. The DAF-I pattern was also observed in a single DT193 strain in which no integrons were detectable. Greater diversity of antibiograms and DAF and IP patterns among non-DT104 phage types was observed. These data indicate a remarkable degree of homogeneity at a molecular level among contemporary isolates of S. enterica serotype Typhimurium DT104 from animal, human, and food sources in this region.     

Prevalence and Genetic Properties of Salmonella enterica Serovar Typhimurium Definitive Phage Type 104 Isolated from Rattus norvegicus and Rattus rattus House Rats in Yokohama City, Japan

Salmonella enterica serovar Typhimurium was isolated from the intestinal contents of Rattus rattus and Rattus norvegicus house rats captured at two buildings, designated buildings J and YS, in Yokohama City, Japan. From October 1997 to September 1998, 52 of 339 (15.3%) house rats were found to carry Salmonella serovar Typhimurium definitive phage type 104 (DT104). In building J, 26 of 161 (16.1%) house rats carried DT104 over the 1-year study period, compared to 26 of 178 (14.6%) rats in building YS. The isolation rates of DT104 from R. rattus and R. norvegicus were similar in the two buildings. Most DT104 strains from building J (24 of 26) showed resistance to ampicillin, chloramphenicol, streptomycin, sulfisoxazole, and tetracycline and contained both the 1.0- and 1.2-kbp integrons, carrying genes pse1, pasppflo-like, aadA2, sulI, and tet(G). All DT104 strains from building YS were resistant to ampicillin and sulfisoxazole, and had the 1.2-kbp integron carrying pse1 and sulI. Cluster analysis of pulsed-field gel electrophoresis patterns of BlnI-digested DT104 DNAs showed that 22 of 26 DT104 strains from building J and 24 of 26 strains from building YS could be grouped into separate clusters each specific for the building origin. These results indicated that DT104 strains were prevalent in house rat colonies in each building and suggest that house rats may play an important role in the epidemiology of DT104.     

Characterization and chromosomal mapping of antimicrobial resistance genes in Salmonella enterica serotype typhimurium

Two hundred and twenty-six Salmonella enterica serotype Typhimurium isolates were examined for the presence of integron-associated gene cassettes. All but two of the non-DT104 isolates, together with DT104 isolates, contained gene cassettes. Amplicons of 1.5 kbp each were found in two non-DT104 isolates, encoding a dhfrI gene (trimethoprim resistance) linked to an aadA gene (streptomycin and spectinomycin resistance), by site-specific recombination. DT104 isolates of resistance (R) type ACSSuT possessed the recently described 1.0- and 1.2-kbp gene cassettes. Macrorestriction analysis with XbaI and DNA probing mapped ant(3")-1a, bla(PSE-1), and dhfrI genes to large multiresistant gene clusters in a DT170a isolate and a DT193 isolate. In contrast, all DT104 isolates (R-type ACSSuT) possessed a conserved 10-kbp Xba1 DNA fragment. Our study highlights the occurrence of integrons (and antimicrobial resistance determinants) among serotype Typhimurium isolates other than DT104. Larger and previously unrecognized multiresistance gene clusters were identified in these isolates by DNA probing.     

Transduction of multiple drug resistance of Salmonella enterica serovar typhimurium DT104

Epidemic strain Salmonella typhimurium DT104 is characterized by various multiresistance patterns. At least some of the resistance genes are organized as integrons. Resistance genes of DT104 isolates can be efficiently transduced by P22-like phage ES18 and by phage PDT17 which is released by all DT104 isolates so far analyzed. Cotransduction tests demonstrate that the resistance genes, although not organized in a unique integron, are tightly clustered on the Salmonella chromosome. The spread of resistance genes in this strain by generalized transduction is discussed.     

Phenotypic and Molecular Characterization of Human <Emphasis Type="Italic">Salmonella enterica</Emphasis> serovar 4,[5],12:i:- Isolates in Slovakia

Forty-three epidemiologically unrelated emerging Salmonella enterica subsp. enterica serovar 4,[5],12:i:- strains isolated during the period 2009–2010 in Slovakia were characterized by phenotypic and genotypic methods. Thirty-one isolates (72.1%) expressed resistance to ampicillin, streptomycin, sulfizoxazole, and tetracycline [R-type ASSuT]. The majority of the strains belonged to both definitive phage types DT193 (30.2%) and U311 (27.9%). Other phage types identified were U302 (6.9%), DT18 (4.7%), and DT194 (2.3%). Twelve strains (27.9%) were not typeable. Pulsed-field gel electrophoresis analysis identified three closely related major banding profiles (X1, X1a, and X2), suggesting the similarity and close epidemiological relationship between S. enterica serovar 4,[5],12:i:- strains. In two isolates with R-type ASSuT, phage type NT and in one isolate with R-type ACROSSuSxTTTMPNA, phage type DT193 class 1 integrons were found encoding bla_PSE-1 and dfrA, aadA1, respectively.     

江苏部分地区食源性和人源沙门氏菌的多重耐药性研究

从江苏省部分地区收集了117个沙门氏菌分离株,其中食物源和人源菌株分别有81株和36株。16种抗生素敏感性试验表明,有111个分离株对2种或2种以上的抗生素有耐药性,人源沙门氏菌分离株的抗生素耐药率比食物源的高,单一抗生素以链霉素耐药率(92.3%,108/117)最高。对5种或5种以上抗生素耐药的分离株有59株(50.4%),其中对特定六种抗生素:氨苄青霉素、氯霉素、链霉素、磺胺、四环素和卡那霉素耐药(ACSSuTK,R型)的菌株有12株。设计18对耐药基因和I类整合子保守区的引物,对36株有不同来源和耐药特征的多重耐药菌株进行耐药基因和I类整合子的检测,PCR扩增结果与抗生素敏感性表型一致。有30株细菌携带有I类整合子,大小为0.3、0.6、1.0、1.2和1.6kb,其中1.6kb(aadA5-dfr17)大小的整合子在25株细菌中分布(24/36)。接合试验表明,氨苄青霉素、氯霉素、链霉素、甲氧苄氨嘧啶和四环素的耐药特性是由接合性质粒携带。结果显示,耐药基因多数由I类整合子和质粒携带,可以通过接合试验发生转移,可移动的DNA成分可能在耐药特性的转移和分布中起到重要作用。     

Characterization and Chromosomal Mapping of Antimicrobial Resistance Genes in Salmonella enterica Serotype Typhimurium

Two hundred and twenty-six Salmonella enterica serotype Typhimurium isolates were examined for the presence of integron-associated gene cassettes. All but two of the non-DT104 isolates, together with DT104 isolates, contained gene cassettes. Amplicons of 1.5 kbp each were found in two non-DT104 isolates, encoding a dhfrI gene (trimethoprim resistance) linked to an aadA gene (streptomycin and spectinomycin resistance), by site-specific recombination. DT104 isolates of resistance (R) type ACSSuT possessed the recently described 1.0- and 1.2-kbp gene cassettes. Macrorestriction analysis with XbaI and DNA probing mapped ant(3")-1a, bla_PSE-1, and dhfrI genes to large multiresistant gene clusters in a DT170a isolate and a DT193 isolate. In contrast, all DT104 isolates (R-type ACSSuT) possessed a conserved 10-kbp Xba1 DNA fragment. Our study highlights the occurrence of integrons (and antimicrobial resistance determinants) among serotype Typhimurium isolates other than DT104. Larger and previously unrecognized multiresistance gene clusters were identified in these isolates by DNA probing.     

Antimicrobial resistance of Salmonella spp. from pigs at slaughter in Spain in 1993 and 2001

AIM: To compare the incidence of antimicrobial resistance among Salmonella serotypes isolated in a pig slaughterhouse in Zaragoza (Spain) during 1993 and 2001. METHODS AND RESULTS: A total of 168 isolates representing 10 serotypes were examined by disc diffusion method using 17 antibiotics. Data showed that the majority of the strains were resistant to streptomycin (97%), sulfadiazine (93.4%) and tetracycline (83.3%). A large proportion of the collection was multidrug resistant (MDR, resistance to four or more antibiotics) with a greater incidence in 2001. The findings imply an increasing incidence of MDR amongst S. Typhimurium, and all S. Typhimurium-definitive phage type (DT) 104 isolates were resistant to ampicillin, chloramphenicol, streptomycin, sulphonamide and tetracycline (R-ACSSuT). This resistance phenotype had spread among other phage and serotypes. Salmonella Ohio was also a MDR serotype and this is not a serotype normally associated with drug resistance. CONCLUSIONS: A large proportion of the strains were MDR and this showed that pork products could be a potential vehicle of MDR Salmonella food-borne infections. SIGNIFICANCE AND IMPACT OF THE STUDY: The findings may have significant public health consequences and could contribute to the development of useful practices aimed at limiting the transmission of MDR Salmonella serotypes through the food chain.     

Characterization of antimicrobial susceptibility and virulence genes of Salmonella serovars collected at a commercial turkey processing plant

Aims:  To determine the antimicrobial susceptibility profiles, distribution of class 1 integrons, virulence genes and genes encoding resistance to tetracycline ( tetA , tetC , tetD and tetE ) and streptomycin ( strA , strB and aadA1 ) in Salmonella recovered from turkeys.
Methods and Results:  The antimicrobial susceptibility of 80 isolates was determined using National Antimicrobial Resistance Monitoring System. The distribution of resistance genes, class 1 integrons and virulence genes was determined using PCR. Resistances to tetracycline (76·3%) and streptomycin (40%) were common. Sixty-two (77·5%) isolates displayed resistance against one or more antimicrobials and 33 were multi-drug resistant. tetA was detected in 72·5% of the isolates, while tetC , tetD and tetE were not detected. The strA and strB genes were detected in 73·8% of the isolates. Two isolates possessed class 1 integrons of 1 kb in size, containing the aadA1 gene conferring resistance to streptomycin and spectinomycin. Fourteen of the virulence genes were detected in over 80% of the isolates.
Conclusions:  This study shows that continuous use of tetracycline and streptomycin in poultry production selects for resistant strains. The Salmonella isolates recovered possess significant ability to cause human illness.
Significance and Impact of the Study:  Information from this study can be employed in guiding future strategies for the use of antimicrobials in poultry production.     

Characterisation of integrons and antibiotic resistance genes in Danish multiresistant Salmonella enterica Typhimurium DT104

The presence and genetic content of integrons was investigated in eight Salmonella enterica Typhimurium DT104 isolates from different pig herds in Denmark. Two different integrons were identified using PCR and sequencing. Each of the integrons carried a single resistance cassette in addition to the sul1 and qacEΔ1 genes characteristic of integrons. The first integron encoded the ant (3″)-Ia gene that specified resistance to spectinomycin and streptomycin. The second contained the pse-1 β-lactamase gene. All the multiresistant strains contained both integrons. The presence of these two integrons did not account for the total phenotypic resistance of all the isolates and does not exclude the presence of other mobile DNA elements.     

Isolation of multidrug-resistant Salmonella typhimurium DT104 from swine in Korea

We report the isolation of Salmonella enterica serotype Typhimurium phage type DT104 (CCARM 8104) from swine in Korea. The CCARM 8104 isolate was resistant to nalidixic acid and showed reduced susceptibility to quinolones. The CCARM 8104 isolate had a missense mutation, Asp87Asn, in the quinolone resistance-determining region in gyrA and produced PSE-1. The CCARM 8104 isolate carried two different class 1 integrons, and the PSE-1 beta-lactamase gene was inserted into a 1,200 bp class 1 integron. The presence of DT104 with pse-1 in an integron located in a plasmid and reduced susceptibility to quinolone in swine pose a significant threat of possible horizontal spread between swine and humans.     

Characterisation of integrons and antibiotic resistance genes in Danish multiresistant Salmonella enterica Typhimurium DT104

The presence and genetic content of integrons was investigated in eight Salmonella enteritica Typhimurium DT104 isolates from different pig herds in Denmark. Two different integrons were identified using PCR and sequencing. Each of the integrons carried a single resistance cassette in addition to the sul1 and qacEΔ1 genes characteristic of integrons. The first integron encoded the ant (3″)-Ia gene that specified resistance to spectinomycin and streptomycin. The second contained the pse-1 β-lactamase gene. All the multiresistant strains contained both integrons. The presence of these two integrons did not account for the total phenotypic resistance of all the isolates and does not exclude the presence of other mobile DNA elements.     

Molecular epidemiology of Salmonella enterica serovar typhimurium isolates from cattle in hokkaido, Japan: evidence of clonal replacement and characterization of the disseminated clone

The molecular epidemiology of 545 Salmonella enterica serovar Typhimurium isolates collected between 1977 and 2009 from cattle in Hokkaido, Japan, was investigated using pulsed-field gel electrophoresis (PFGE). Nine main clusters were identified from 116 PFGE patterns. Cluster I comprised 248 isolates, 243 of which possessed a sequence specific to definitive phage type 104 (DT104) or U302. The cluster I isolates were dominant in 1993 to 2003, but their numbers declined beginning in 2004. Beginning in 2002, an increase was observed in the number of cluster VII isolates, consisting of 21 PFGE patterns comprising 165 isolates. A total of 116 isolates representative of the 116 PFGE profiles were analyzed by multilocus variable-number tandem-repeat analysis (MLVA). Other than two drug-sensitive isolates, 19 isolates within cluster VII were classified in the same cluster by MLVA. Among the cluster VII isolates, an antibiotic resistance type showing resistance to ampicillin, chloramphenicol, streptomycin, sulfonamides, tetracycline, kanamycin, cefazolin, and sulfamethoxazole-trimethoprim and a resistance type showing resistance to ampicillin, streptomycin, sulfonamides, tetracycline, and kanamycin were found in 23 and 125 isolates, respectively. In the 19 isolates representative of cluster VII, the bla(TEM-1) gene was found on a Salmonella serotype Typhimurium virulence plasmid, which was transferred to Escherichia coli by electroporation along with resistance to two to four other antimicrobials. Genomic analysis by subtractive hybridization and plasmid analysis suggested that the bla(TEM-1)-carrying virulence plasmid has a mosaic structure composed of elements of different origin. These results indicate an emerging multidrug-resistant S. Typhimurium clone carrying a virulence-resistance plasmid among cattle in Hokkaido, Japan.     

Evaluation of methods for the identification of Salmonella enterica serotype Typhimurium DT104 from poultry environmental samples

An increase in the prevalence of Salmonella enterica serotype Typhimurium DT104 has been reported worldwide. This study examined the prevalence of this microorganism in poultry environmental samples from commercial layer flocks and pullet environments as well as the sensitivity and specificity of a PCR-based method, and multiple antibiotic resistance profile of Salmonella serogroup B isolates in relation to the serotype and phagetype reference method for the identification of Salmonella Typhimurium DT104. A total of 435 Salmonella isolates were obtained from poultry house environmental samples tested during a 20-month period representing a prevalence of 5.5%. Of these, 313 (72%) isolates were identified as Salmonella serogroup B isolates. These isolates were tested by a PCR-based assay, and for resistance to five antibiotics: ampicillin, chloramphenicol, streptomycin, sulfonamides, and tetracycline (ACSSuT) for the rapid identification of Salmonella Typhimurium DT104. Upon comparing the antibiotic resistance and PCR results with serotype and phage type data, the sensitivity and specificity for the identification of Salmonella Typhimurium DT104 of both methods were found to be 100%, and 99.6%, respectively. Both methods can be completed within 24 h after obtaining an isolate, while serotyping and phagetyping required more than 5 days to complete.     

The genetics of Salmonella genomic island 1

Multidrug-resistant Salmonella enterica serovar Typhimurium phage type DT104, resistant to ampicillin, chloramphenicol/florfenicol, streptomycin, sulfonamides, and tetracycline, has disseminated worldwide. The resistance genes reside on the 43-kb Salmonella genomic island 1 (SGI1), which is transferable. Drug-resistant variants of SGI1 have been identified in numerous serotypes. Strains harboring SGI1 may be more virulent and have a tendency to rapidly disseminate.     

Identification of novel Salmonella enterica serovar Typhimurium DT104-specific prophage and nonprophage chromosomal sequences among serovar Typhimurium isolates by genomic subtractive hybridization

Genomic subtractive hybridization was performed between Salmonella enterica serovar Typhimurium LT2 and DT104 to search for novel Salmonella serovar Typhimurium DT104-specific sequences. The subtraction resulted mainly in the isolation of DNA fragments with sequence similarity to phages. Two fragments identified were associated with possible virulence factors. One fragment was identical to irsA of Salmonella serovar Typhimurium ATCC 14028, which is suggested to be involved in macrophage survival. The other fragment was homologous to HldD, an Escherichia coli O157:H7 lipopolysaccharide assembly-related protein. Five selected DNA fragments-irsA, the HldD homologue, and three fragments with sequence similarity to prophages-were tested for their presence in 17 Salmonella serovar Typhimurium DT104 isolates and 27 non-DT104 isolates by PCR. All five selected DNA fragments were Salmonella serovar Typhimurium DT104 specific among the serovar Typhimurium isolates tested. These DNA fragments can be useful for better detection and typing of Salmonella serovar Typhimurium DT104.