Comparison of multilocus sequence typing, pulsed-field gel electrophoresis, and antimicrobial susceptibility typing for characterization of Salmonella enterica serotype Newport isolates

In the United States, multidrug-resistant phenotypes of Salmonella enterica serotype Newport (commonly referred to as MDR-AmpC) have emerged in animals and humans and have become a major public health problem. Although pulsed-field gel electrophoresis (PFGE) is the current “gold standard” typing method for Salmonella, multilocus sequence typing (MLST) may be more relevant to investigations exploring evolutionary and population biology relationships. In this study, 81 Salmonella enterica serotype Newport isolates from humans, food animals, and retail foods were examined for antimicrobial susceptibility and characterized using PFGE and MLST of seven genes, aroC, dnaN, hemD, hisD, purE, sucA, and thrA. Forty-nine percent of the isolates were resistant to nine or more of the tested antimicrobials. Salmonella isolates displayed resistance most often to sulfamethoxazole (57%), streptomycin (56%), tetracycline (56%), ampicillin (52%), and ceftiofur (49%) and, to a lesser extent, to kanamycin (19%), trimethoprim-sulfamethoxazole (17%), and gentamicin (11%). A total of 43 PFGE patterns were generated using XbaI, indicating a genetically diverse population. The largest PFGE cluster contained isolates from clinically ill swine, cattle, and humans. MLST resulted in 12 sequence types (STs), with one type encompassing 62% of the strains. Ten new sequence types and one novel allele type were identified. Furthermore, MLST typing showed that strains closely related by PFGE clustered in major STs, whereas more distantly related strains were separated into two clusters by PFGE. The results of this study demonstrated that the MLST scheme employed here clustered S. enterica serovar Newport isolates in distinct molecular populations, and strain discrimination was enhanced by combining PFGE, antimicrobial susceptibility, and MLST results.     

Antimicrobial use and outcomes in patients with multidrug-resistant and pansusceptible Salmonella Newport infections, 2002-2003

Multidrug-resistant Salmonella Newport with decreased susceptibility to ceftriaxone (MDR-AmpC) is becoming increasingly common in its food animal reservoirs and in humans. Few data exist on rates of antimicrobial use or differences in clinical outcomes in persons infected with MDR-AmpC or other Salmonella strains. We conducted a case-comparison analysis of data from a multistate population-based case-control study to identify antimicrobial treatment choices and differences in clinical outcomes in those infected with MDRAmpC compared to pansusceptible S. Newport. Of isolates from 215 laboratory-confirmed S. Newport cases, 54 (25%) were MDR-AmpC, 146 (68%) were pansusceptible, and 15 (7%) had other resistance patterns; 146 (68%) patients with S. Newport were treated with antimicrobial agents and 66 (33%) were hospitalized. Over two-thirds of cases at low-risk for serious complications received antimicrobial therapy, most commonly with fluoroquinolones, to which this strain was susceptible. There were no significant differences in symptoms, hospitalization, duration of illness, or other outcomes between the persons infected with MDR-AmpC and pansusceptible S. Newport. Although currently prevalent MDR-AmpC S. Newport strains remains susceptible to the antimicrobial most commonly prescribed for it, continued efforts to reduce unnecessary use of antimicrobial agents in food animals and humans are critical to prevent further development of resistance to quinolones and cephalosporins, which is likely to lead to substantial adverse outcomes.     

Multidrug resistance in Salmonella enterica serotype Typhimurium from humans in France (1993 to 2003)

The aim of this study was to determine the distribution of the antimicrobial resistance phenotypes (R types), the phage types and XbaI-pulsed-field gel electrophoresis (PFGE) types, the genes coding for resistance to beta-lactams and to quinolones, and the class 1 integrons among a representative sample of Salmonella enterica serotype Typhimurium isolates collected from humans in 2002 through the French National Reference Center for Salmonella (NRC-Salm) network. The trends in the evolution of antimicrobial resistance of serotype Typhimurium were reviewed by using NRC-Salm data from 1993, 1997, 2000, and 2003. In 2002, 3,998 isolates of serotype Typhimurium were registered at the NRC-Salm among 11,775 serotyped S. enterica isolates (34%). The most common multiple antibiotic resistance pattern was resistance to amoxicillin, chloramphenicol, streptomycin and spectinomycin, sulfonamides, and tetracycline (ACSSpSuTe R type), with 156 isolates (48.8%). One isolate resistant to extended-spectrum cephalosporins due to the production of TEM-52 extended-spectrum beta-lactamase was detected (0.3%), and one multidrug-resistant isolate was highly resistant to ciprofloxacin (MIC > 32 mg/liter). We found that 57.2% of the isolates tested belonged to the DT104 clone. The main resistance pattern of DT104 isolates was R type ACSSpSuTe (83.2%). However, evolutionary changes have occurred within DT104, involving both loss (variants of Salmonella genomic island 1) and acquisition of genes for drug resistance to trimethoprim or to quinolones. PFGE profile X1 was the most prevalent (74.5%) among DT104 isolates, indicating the need to use a more discriminatory subtyping method for such isolates. Global data from the NRC-Salm suggested that DT104 was the main cause of multidrug resistance in serotype Typhimurium from humans from at least 1997 to 2003, with a roughly stable prevalence during this period.     

Molecular epidemiology of sequential outbreaks of Acinetobacter baumannii in an intensive care unit shows the emergence of carbapenem resistance

The molecular epidemiology of multidrug-resistant Acinetobacter baumannii was investigated in the medical-surgical intensive care unit (ICU) of a university hospital in Italy during two window periods in which two sequential A. baumannii epidemics occurred. Genotype analysis by pulsed-field gel electrophoresis (PFGE) of A. baumannii isolates from 131 patients identified nine distinct PFGE patterns. Of these, PFGE clones B and I predominated and occurred sequentially during the two epidemics. A. baumannii epidemic clones showed a multidrug-resistant antibiotype, being clone B resistant to all antimicrobials tested except the carbapenems and clone I resistant to all antimicrobials except ampicillin-sulbactam and gentamicin. Type 1 integrons of 2.5 and 2.2 kb were amplified from the chromosomal DNA of epidemic PFGE clones B and I, respectively, but not from the chromosomal DNA of the nonepidemic clones. Nucleotide analysis of clone B integron identified four gene cassettes: aacC1, which confers resistance to gentamicin; two open reading frames (ORFs) coding for unknown products; and aadA1a, which confers resistance to spectinomycin and streptomycin. The integron of clone I contained three gene cassettes: aacA4, which confers resistance to amikacin, netilmicin, and tobramycin; an unknown ORF; and bla(OXA-20), which codes for a class D beta-lactamase that confers resistance to amoxicillin, ticarcillin, oxacillin, and cloxacillin. Also, the bla(IMP) allele was amplified from chromosomal DNA of A. baumannii strains of PFGE type I. Class 1 integrons carrying antimicrobial resistance genes and bla(IMP) allele in A. baumannii epidemic strains correlated with the high use rates of broad-spectrum cephalosporins, carbapenems, and aminoglycosides in the ICU during the study period.     

Characterization of isolates of Salmonella enterica serovar Stanley, a serovar endemic to Asia and associated with travel

Salmonella enterica serovar Stanley (S. Stanley) is a common serovar in Southeast Asia and was the second most common serovar implicated in human salmonellosis in Thailand in the years 2002 to 2007. In contrast, this serovar is relatively uncommon in Europe. The objective of this study was to characterize a collection of S. Stanley strains isolated from Thai (n = 62), Danish (n = 39), and French (n = 24) patients to gain a broader understanding of the genetic diversity, population dynamics, and susceptibility to antimicrobials. All isolates were characterized by pulsed-field gel electrophoresis and antimicrobial susceptibility testing. The molecular mechanisms of resistance to extended-spectrum cephalosporins and plasmid-mediated resistance to quinolones were characterized by PCR and sequencing. Plasmid profiling, replicon typing, and microarray analysis were used to characterize the genetic mechanisms of antimicrobial resistance in 10 extended-spectrum cephalosporinase-producing isolates. Considerable genetic diversity was observed among the isolates characterized with 91 unique XbaI pulsed-field gel electrophoresis (PFGE) patterns, including 17 distinct clusters consisting of two to seven indistinguishable isolates. We found some of the S. Stanley isolates isolated from patients in Europe were acquired during travel to Southeast Asia, including Thailand. The presence of multiple plasmid lineages carrying the extended-spectrum cephalosporinase-encoding bla(CMY-2) gene in S. Stanley isolates from the central part of Thailand was confirmed. Our results emphasize that Thai authorities, as well as authorities in other countries lacking prudent use of antimicrobials, should improve the ongoing efforts to regulate antimicrobial use in agriculture and in clinical settings to limit the spread of multidrug-resistant Salmonella isolates and plasmids among humans and pigs in Thailand and abroad.     

Characterization of AmpC-mediated resistance in clinical Salmonella isolates recovered from humans during the period 1992 to 2003 in England and Wales

The increase in AmpC-mediated resistance in salmonellae constitutes a serious public health concern, since these enzymes confer resistance to a wide range of beta-lactams. One hundred six isolates were selected from 278,308 Salmonella isolates based on resistance to ampicillin and cephalosporins and were subjected to further characterization. Nine isolates had a cefoxitin inhibition diameter < or = 17 mm and were proven to be AmpC positive by multiplex PCR. Sequence analysis revealed the presence of bla(DHA-1), bla(CMY-2), and bla(CMY-4) genes. All nine isolates presented different pulsed-field gel electrophoresis restriction profiles. The AmpC genetic determinants were present in transferable plasmids of around 11, 42, 70, 98, and 99 MDa. A combination of size and restriction fragment length polymorphism (RFLP) analysis showed that all the bla(CMY) plasmids investigated in our study were different, which suggests that bla(CMY) may be located in different plasmid environments. Some United Kingdom isolates linked to foreign travel showed RFLP plasmid patterns consistent with plasmids previously seen in the United States, which suggests that bla(CMY-2) has also been disseminated through plasmid transfer. The fact that two of the domestically acquired United Kingdom isolates presented previously unseen RFLP plasmid patterns could indicate that these strains have followed routes different from those prevalent in North America or other parts of the world. This study represents the first report of bla(CMY) genes in Salmonella isolates in the United Kingdom and the first report of CMY-4 in Salmonella enterica serotype Senftenberg worldwide.     

Characterization of multidrug-resistant Escherichia coli isolates associated with nosocomial infections in dogs

Multidrug-resistant opportunistic pathogens have become endemic to the veterinary hospital environment. Escherichia coli isolates resistant to 12 antibiotics were isolated from two dogs that were housed in the intensive care unit at The University of Georgia Veterinary Teaching Hospital within 48 h of each other. Review of 21 retrospective and prospective hospital-acquired E. coli infections revealed that the isolates had similar antibiotic resistance profiles, characterized by resistance to most cephalosporins, beta-lactams, and the beta-lactamase inhibitor clavulanic acid as well as resistance to tetracycline, spectinomycin, sulfonamides, chloramphenicol, and gentamicin. E. coli isolates with similar resistance profiles were also isolated from the environment in the intensive care unit and surgery wards. Multiple E. coli genetic types were endemic to the hospital environment, with the pulsed-field gel electrophoresis fingerprint identified among E. coli isolates from diseased animals and the hospital environment matching. The extended-spectrum cephalosporin resistance in these nosocomial E. coli isolates was attributed to the cephamycinase-encoding gene, bla(CMY2). Chloramphenicol resistance was due in part to the dissemination of the florfenicol resistance gene, flo, among these isolates. Resistance encoded by both genes was self-transmissible. Although bla(CMY2) and flo were common to the polyclonal, nosocomial E. coli isolates, there was considerable diversity in the genetic compositions of class 1 integrons, especially among isolates belonging to the same genetic type. Two or more integrons were generally present in these isolates. The gene cassettes present within each integron ranged in size from 0.6 to 2.4 kb, although a 1.7-kb gene cassette was the most prevalent. The 1.7-kb gene cassette contained spectinomycin resistance gene aadA5 and trimethoprim resistance gene dfrA17.     

Antimicrobial resistance and serotype prevalence of Salmonella isolated from dairy cattle in the southwestern United States

Mature dairy cattle were sampled over a 2-year period (2001-2002) on six farms in New Mexico and Texas. Fecal samples (n = 1560) were collected via rectal palpation and cultured for Salmonella, and one isolate from each positive sample was serotyped. Three isolates of each serotype, with the exception of Salmonella Newport (n = 12), were examined for susceptibility to 17 antimicrobial agents. Twenty-two different serotypes were identified from a total of 393 Salmonella isolates. Montevideo was the predominant serotype (27%) followed by Mbandaka (15%), Senftenberg (11.4%), Newport (6.4%), Anatum (4.8%), and Give (4.8%). Salmonella Typhimurium and Dublin, two frequently reported serotypes, accounted for only 1% of the observed serotypes in this study. Sixty-four percent of the serotypes were susceptible to all 17 antimicrobials, 14% were resistant to a single agent, and 22% were multiresistant (2-11 types of resistance). All isolates tested were susceptible to amikacin, apramycin, imipenem, ceftriaxone, nalidixic acid, and ciprofloxacin. The most frequent types of resistance were to sulfamethoxazole, tetracycline, streptomycin, kanamycin, chloramphenicol, and ampicillin (ranging from 8.9 to 22.4%). Serotypes demonstrating multiple resistance included Dublin and Give (resistant to three or more antibiotics), Typhimurium (resistant to five antibiotics), and Newport (four and two isolates resistant to six and nine antibiotics, respectively). Class 1 integrons were present in only two Salmonella Dublin isolates and one Salmonella Newport isolate. The most prevalent resistance patterns observed in this study were toward antimicrobial agents commonly used in cattle, while all Salmonella isolates were susceptible to ceftriaxone and ciprofloxacin, antibiotics used in human medicine.     

Molecular characterization and occurrence of extended-spectrum beta-lactamase resistance genes among Salmonella enterica serovar Corvallis from Thailand, Bulgaria, and Denmark

Fifty nine Salmonella Corvallis isolates from humans and food products in Bulgaria, Denmark, and Thailand were examined for antimicrobial susceptibility and characterized by pulsed-field gel electrophoresis (PFGE). Cephalosporin-resistant isolates were examined for the presence of genes encoding beta-lactamases by PCR and sequencing. Ten different PFGE types were observed. One type (30 isolates) was recovered in all three countries; three types were found only in Bulgaria, two only in Denmark, two only in Thailand, and two both in Denmark and Thailand. Ten isolates were susceptible to all antimicrobial agents tested, whereas 41 were resistant to three or more antimicrobials. Most resistance was observed among the isolates from Bulgaria. Of the 25 isolates from Bulgaria, 20 displayed resistance to ampicillin and the cephalosporins ceftiofur and cephalothin. All 20 isolates tested negative for bla (CMY-1), bla (CMY-2), and bla (ACC), but positive for bla (SHV), of which five were sequenced to bla (SHV-2). Plasmid profiling and hybridization revealed that the bla (SHV) gene was located on plasmids of approximately 70 kb. Five plasmid profiles were found among these 20 isolates. The plasmid profiling confirmed the PFGE-type and was able to further subdivide the strains. Seventeen of these 20 isolates contained also bla (TEM), of which nine representatives were sequenced to bla (TEM-1B), or bla (TEM-1H). One isolate contained bla (CTX-M-15), bla (SHV-2), and bla (TEM-1H), with the bla (CTX-M-15), and bla (TEM-1H) genes located on a 63-kb transferable plasmid. This study showed a high frequency of resistance among S. Corvallis isolated from humans and food products in Bulgaria, with a lower frequency in Thailand and Denmark. The clonal relatedness among the isolates from three countries could indicate a recent spread of this serovar.     

Analysis of antimicrobial resistance genes detected in multidrug-resistant Salmonella enterica serovar Typhimurium isolated from food animals

Multidrug-resistant (MDR) Salmonella enterica serovar Typhimurium is the most prevalent penta-resistant serovar isolated from animals by the U.S. National Antimicrobial Resistance Monitoring System. Penta-resistant isolates are often resistant to ampicillin, chloramphenicol, streptomycin, sulfamethoxazole, and tetracycline. To investigate MDR in Salmonella Typhimurium (including variant 5-), one isolate each from cattle, poultry, and swine with at least the ampicillin, chloramphenicol, streptomycin, sulfamethoxazole, and tetracycline phenotype were selected for each year from 1997 to 2007 (n = 33) for microarray analysis of antimicrobial resistance, incompatibility IncA/C, and HI1 plasmid genes. Cluster analysis based on these data separated 31 of the isolates into two groups A and B (15 and 16 isolates, respectively). Isolates in group A were phage type DT104 or U302 and were mostly swine isolates (7/15). Genes detected included intI1, bla(PSE-1), floR, aadA, sulI, tet(G), and tetR, which are often found in Salmonella Genomic Island I. Isolates in group B had numerous IncA/C plasmid genes detected and were mostly cattle isolates (9/16). Genes detected included bla(CMY-2), floR, aac(3), aadA, aphA1, strA, strB, sulI, sulII, dfrA, dhf, tet(A)(B)(C)(D), and tetR, which are often found on MDR-AmpC IncA/C plasmids. The IncA/C replicon was also detected in all group B isolates. The two remaining isolates did not cluster with any others and both had many HI1 plasmid genes detected. Linkage disequilibrium analysis detected significant associations between plasmid replicon type, phage type, and animal source. These data suggest that MDR in Salmonella Typhimurium is associated with DT104/Salmonella Genomic Island I or IncA/C MDR-AmpC encoding plasmids and these genetic elements have persisted throughout the study period.     

Class 1 integrons and Salmonella genomic island 1 among Salmonella enterica isolated from poultry and swine

Two hundred eleven Salmonella enterica strains representing 35 serotypes isolated from healthy poultry (n=103) and swine (n=108) were used in this study. The occurrence and characteristics of class 1 integrons were investigated. Salmonella genomic islands (SGIs) and the horizontal transfer of integrons were assessed. One hundred eighty-six isolates (88%) were resistant to at least one antimicrobial and 140 isolates (66%) were multidrug resistant. The intI1 gene was present in 54 isolates (25.6%), of which 33 (15.6%) carried gene cassettes with sizes ranging from 0.7 to 2.3 kb. Sequence analysis revealed 11 distinct integron profiles in which resistance genes bla(PSE-1), dfrA1, dfrA12, aadA2, aadA4a, and silB were present. The gene cassette array dfrA12-aadA2 was the most prevalent among the isolates whereas most integrons were located on conjugative plasmids. SGI1 variants (SGI1-A and -F) were present in nine isolates belonging to serovars Albany, Emek, Kedougou, and Kingston.     

Emergence of extended-spectrum-beta-lactamase (CTX-M-9)-producing multiresistant strains of Salmonella enterica serotype Virchow in poultry and humans in France

During 2002 to 2003, eight Salmonella enterica serotype Virchow poultry and poultry product isolates from various sources (chicken farms, poultry slaughterhouse, or retail store) and one S. enterica rough strain isolated from human feces were found to produce extended-spectrum beta-lactamase CTX-M-9. Poultry and poultry product isolates were recovered from different locations in the southwest of France. The human rough isolate had sequences of flagellin genes (fliC and fljB) typical of serotype Virchow and ribotyping and pulsed-field gel electrophoresis (PFGE) patterns closely similar to those of serotype Virchow strains. PFGE confirmed the clonal relationship between the poultry isolates, while the human isolate displayed a pattern with 94% homology. The bla(CTX-M-9) gene was located on a conjugative plasmid and was shown to be linked to orf513. Plasmid profiling found a very similar EcoRI restriction pattern in six transconjugants studied, including transconjugants obtained from the human isolate. A single hatchery, supplying chicks to the six farms, was identified. Emergence of extended-spectrum beta-lactamase-producing S. enterica strains in food animals is a major concern, as such strains could disseminate on a large scale and lead to antibiotic therapy difficulties.     

Integrons, β‐lactamase and qnr genes in multidrug resistant clinical isolates of Proteus mirabilis and P. vulgaris

Thirty‐three isolates of Proteus mirabilis and two P. vulgaris were examined for their antimicrobial resistance, the presence of integrons with regard to gene cassette content, and genetic determinants of β‐lactam and low‐level quinolone resistance. Integrons were detected in 23 (69.7%) P. mirabilis isolates; six (18.2%) of them had class 1 integrons, 11 (33.3%) possessed class 2 integrons and six (18.2%) carried integrons of both classes. One P. vulgaris strain possessed class 1 and class 2 integrons. The presence of integrons was associated with increased frequency of resistance to gentamicin, ciprofloxacin, sulfamethoxazole and co‐trimoxazole. Moreover, integron presence was associated with increased resistance range in terms of both the number of antimicrobials and the number of classes of antimicrobials to which a strain was resistant. Class 1 integrons contained aadA1, aadB‐aadA1, dfrA1‐aadA1, bla_PSE‐1‐aadA1 and aacA4‐orfA‐orfB‐aadA1 gene cassette arrays, whereas all class 2 integrons had a dfrA1‐sat2‐aada1 array. β‐lactamase genes not associated with integrons comprised bla_TEM‐2, bla_DHA‐1 and bla_CMY‐15. Plasmid‐mediated fluoroquinolone resistance was determined by qnrD and qnrS1 genes. This is the first report of P. vulgaris strains harbouring qnrD genes in Europe.     

Association between handling of pet treats and infection with Salmonella enterica serotype newport expressing the AmpC beta-lactamase, CMY-2

Resistance to the extended-spectrum cephalosporins can occur in Salmonella species via the production of extended-spectrum and AmpC beta-lactamases. We describe human infections with Salmonella enterica serotype Newport phage type 14 strains resistant to ceftazidime (CAZ) and cefoxitin (FOX) related to the handling of pet treats containing dried beef. These strains were isolated from five patients in Calgary, Alberta, Canada, during 2002 and were compared to a strain cultured from a commercial pet treat present at the property of one of the patients. The strains were resistant to FOX, CAZ, cefpodoxime, ampicillin, and chloramphenicol; intermediate resistant to ceftriaxone and cefotaxime; and sensitive to the aminoglycosides, ciprofloxacin, cefepime, and imipenem. Isoelectric focusing, multiplex PCR, and sequencing of the amplicons showed that all strains produced the plasmid-encoded AmpC beta-lactamase, CMY-2. Restriction analysis of plasmid DNA following transformation demonstrated that bla(CMY-2) was encoded on an approximately 140-kb plasmid. Pulsed-field gel electrophoresis showed the human and pet treat Salmonella strains to be highly related. This study is the first to implicate the transfer of multidrug-resistant Salmonella species through the handling of commercial pet treats containing animal products. In addition to documenting the first cases of human infection caused by CMY-2-producing S. enterica serotype Newport strains in Canada, this study illustrates the necessity of rapid and accurate laboratory-based surveillance in the identification of novel types of antimicrobial resistance.     

Prevalence and characterization of extended-spectrum beta-lactamases-producing Salmonella enterica isolates in Saragossa, Spain (2001-2008)

We analyzed the prevalence of resistance to extended-spectrum cephalosporins (ESCs) among clinical strains of Salmonella enterica collected by the Laboratory of Clinical Microbiology in the University Clinical Hospital Lozano Blesa in the region of Aragón (Spain), for which very few epidemiological information exists. A total of 2,092 strains of S. enterica were identified in stool samples from patients with gastroenteritis. Five isolates showed an extended-spectrum beta-lactamase (ESBL) phenotype: four isolates of S. enterica serotype Virchow harbored the ESBL-encoding bla(CTX-M-9) gene and an isolate of serotype Enteritidis carried a bla(CTX-M-1) gene, which, to the best of our knowledge, is described here for the first time in this serotype of S. enterica. The five ESC-resistant isolates were also resistant to spectinomycin, streptomycin, kanamycin, sulfonamides, tetracycline, and trimethoprim as well as to nalidixic acid. The ESBL isolate of serotype Enteritidis, however, remained susceptible to kanamycin and nalidixic acid. A class 1 integron of 1.5?kb was detected for the four serotype Virchow isolates with the gene cassette dfrA16-aadA2. The bla(CTX-M-9) gene was carried by an ～300-kb IncHI2 conjugative plasmid in the case of the S. enterica serotype Virchow isolates. The bla(CTX-M-1) gene was carried by an ～100-kb IncI1-N conjugative plasmid for the serotype Enteritidis ESC-resistant isolate. All the four ESC-resistant strains of S. enterica serotype Virchow clustered together in a XbaI pulsed-field gel electrophoresis, which also revealed a strong similarity between them and some pulsotypes of S. enterica serotype Virchow from France.     

Variation in clonality and antibiotic-resistance genes among multiresistant Salmonella enterica serotype typhimurium phage-type U302 (MR U302) from humans, animals, and foods

Since 1990 multiresistant (MR) Salmonella enterica serotype Typhimurium definitive phage-type (DT) 104 (MR DT104) and closely related phage types have emerged as a worldwide health problem in humans and food animals. In this study the presence of the blaCARB-2 (ampicillin), cmlA (chloramphenicol), aadA2 (streptomycin/spectinomycin), sul1 (sulphonamide), and tetG (tetracycline) resistance genes in isolates of one such phage type, U302, have been determined. In addition blaTEM primers have been used for the detection of TEM-type beta-lactamases. Isolates have also been characterized by plasmid profile and pulsed field gel electrophoresis (PFGE). Thirty-three of 39 isolates were positive for blaCARB-2, cmlA, aadA2, sul1 and tetG, four for blaTEM, aadA2 and sul1, one for aadA2 and sul1, and one for blaTEM only. blaTEM-mediated ampicillin resistance was transferred to Escherichia coli K12 from three isolates along with other resistance markers, including resistance to chloramphenicol, streptomycin, spectinomycin, sulphonamides, and tetracyclines. Strains carried up to 6 plasmids and 34 plasmid profiles were identified. Although the majority of strains (33/39) produced a PFGE profile identical to that predominant in MR DT104, six different patterns were generated demonstrating the presence of various clones within MR U302. The results show that the majority of the MR U302 strains studied possessed the same antibiotic resistance genes as MR DT104. However, isolates with distinctive PFGE patterns can have different mechanisms of resistance to ampicillin, chloramphenicol, streptomycin, sulphonamides, and tetracyclines. Such resistance genes may be borne on transmissible plasmids.     

Detection of a bla(SHV) extended-spectrum {beta}-lactamase in Salmonella enterica serovar Newport MDR-AmpC

Salmonella enterica serovar Newport MDR-AmpC expressing TEM-1b and extended-spectrum beta-lactamase SHV-12 was isolated from affected animals during an outbreak of salmonellosis that led to a 3-month closure of one of the largest equine hospitals in the United States.     

Analysis of antimicrobial resistance genes detected in multiple-drug-resistant Escherichia coli isolates from broiler chicken carcasses

Multi-drug-resistant (MDR) bacteria in food animals are a potential problem in both animal and human health. In this study, MDR commensal Escherichia coli isolates from poultry were examined. Thirty-two E. coli isolates from broiler carcass rinses were selected based on their resistance to aminoglycosides, β-lactams, chloramphenicols, tetracyclines, and sulfonamide antimicrobials. Microarray analysis for the presence of antimicrobial resistance and plasmid genes identified aminoglycoside [aac(6), aac(3), aadA, aph, strA, and strB], β-lactam (bla(AmpC), bla(TEM), bla(CMY), and bla(PSE-1)), chloramphenicol (cat, flo, and cmlA), sulfamethoxazole (sulI and sulII), tetracycline [tet(A), tet(C), tet(D), and tetR], and trimethoprim (dfrA) resistance genes. IncA/C plasmid core genes were detected in 27 isolates, while IncHI1 plasmid genes were detected in one isolate, indicating the likely presence of these plasmids. PCR assays for 18 plasmid replicon types often associated with MDR in Enterobacteriaceae also detected one or more replicon types in all 32 isolates. Class I integrons were investigated by PCR amplification of the integrase I gene, intI1, and the cassette region flanked by conserved sequences. Twenty-five isolates were positive for the intI1 gene, and class I integrons ranging in size from ~1,000 to 3,300 bp were identified in 19 of them. The presence of class I integrons, IncA/C plasmid genes, and MDR-associated plasmid replicons in the isolates indicates the importance of these genetic elements in the accumulation and potential spread of antimicrobial resistance genes in the microbial community associated with poultry.