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.     

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.     

Antimicrobial resistance, integrons and plasmid replicon typing in multiresistant clinical Escherichia coli strains from Enugu State, Nigeria

Eleven multiresistant Escherichia coli strains of animal and human origin were assayed for the presence of antimicrobial resistance genes, integrons and associated gene cassettes, as well as plasmid content. Ciprofloxacin-resistant strains were screened for amino acid changes in GyrA and ParC proteins. The E. coli strains were found to harbor a variety of genes including cmlA, aac (3)-II, aac (3)-IV, aadA, strA-strB, tet (A), tet (B), bla(TEM), sul1, sul2 and sul3. Four of the eight int I1-positive strains were also positive for qacE Δ1 -sul1 region and the following gene cassettes were detected: dfrA7, dfrA12 + orfF + aadA2 and bla(OXA1)+ aadA1. Five strains contained class 1 integrons lacking the qacE Δ1 -sul1 region and they showed a single type of gene cassette arrangement (estX + psp + aadA2 + cmlA + aadA1 + qacH + IS440 + sul3). The two int I2-positive strains carried the same type of gene cassette arrangement (dfrA1 + sat + aadA1). The seven ciprofloxacin-resistant E. coli strains exhibited a Ser-83-Leu substitution in GyrA protein and a Ser-80-Ile substitution in ParC protein; six of these strains presented an additional substitution in GyrA (Asp-87-Gly or Asp-87-Asn) and one strain in ParC (Glu-84-Gly). Eight different plasmid-replicon-types were detected among the 11 E. coli strains, IncF being the most frequent one detected, found in nine strains; other plasmid replicon types detected were IncX, IncI1, IncY, IncW, IncFIC, IncB/O, and IncK. Antimicrobial resistance in the E. coli strains studied was mediated by a variety of genes, some of them included in integrons, as well as by mutations gyr A and par C genes.     

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.     

Heterogeneity among virulence and antimicrobial resistance gene profiles of extraintestinal Escherichia coli isolates of animal and human origin

Extraintestinal pathogenic Escherichia coli (ExPEC) isolates collected from different infected animals and from human patients with extraintestinal infections in 2001 were characterized for their phenotypic and genotypic antimicrobial resistance profiles, genotypes, and key virulence factors. Among the 10 antimicrobial agents tested, resistance to ampicillin, tetracycline, and sulfonamides was most frequent. Multiresistant strains were found in both the animal and the human groups of isolates. Resistance gene distribution was assessed by colony hybridization. Similar antibiotic resistance patterns could be observed in the animal and the human isolates. Although some resistance genes, such as bla(TEM), sulI, and sulII, were equally represented in the animal and human ExPEC isolates, differences in the distributions of tetracycline [tet(D)], chloramphenicol (catI, catIII, and floR), and trimethoprim (dhfrI, dhfrV, dhfrVII, and dhfrXIII) resistance genes were observed between the animal and the human isolates. Approximately one-third of the ExPEC isolates possessed a class 1 integron. The four major different variable regions of the class 1 integron contained aminoglycoside (aadA1, aadA2, aadA5, and aadA6) and/or trimethoprim (dhfrIb, dhfrXII, and dhfrXVII) resistance genes. The ExPEC strains belonged to different phylogenetic groups, depending on their host origin. Strains isolated from animal tissues belonged to either a commensal group (group A or B1) or a virulent group (group B2 or D), while the majority of the human isolates belonged to a virulent group (group B2 or D). Although the limited number of isolates evaluated in the present study prevents firm epidemiological conclusions from being made, on a more global scale, these data demonstrate that extraintestinal isolates of E. coli can possess relatively distinct intra- and intergroup resistance gene profiles, with animal isolates presenting a more heterogeneous group than human isolates.     

Antimicrobial resistance patterns of Shiga toxin-producing Escherichia coli O157:H7 and O157:H7- from different origins

Shiga toxin-producing Escherichia coli (STEC) serotypes including O157:H7 (n = 129) from dairy cows, cull dairy cow feces, cider, salami, human feces, ground beef, bulk tank milk, bovine feces, and lettuce; and O157:H7- (n = 24) isolated from bovine dairy and bovine feedlot cows were evaluated for antimicrobial resistance against 26 antimicrobials and the presence of antimicrobial resistance genes (tetA, tetB, tetC, tetD, tetE, tetG, floR, cmlA, strA, strB, sulI, sulII, and ampC). All E. coli exhibited resistance to five or more antimicrobial agents, and the majority of isolates carried one or more target antimicrobial resistance gene(s) in different combinations. The majority of E. coli showed resistance to ampicillin, aztreonam, cefaclor, cephalothin, cinoxacin, and nalidixic acid, and all isolates were susceptible to chloramphenicol and florfenicol. Many STEC O157:H7 and O157:H7-isolates were susceptible to amikacin, carbenicillin, ceftriaxone, cefuroxime, ciprofloxacin, fosfomycin, moxalactam, norfloxacin, streptomycin, tobramycin, trimethoprim, and tetracycline. The majority of STEC O157:H7 (79.8%) and O157:H7- (91.7%) carried one or more antimicrobial resistance gene(s) regardless of whether phenotypically resistant or susceptible. Four tetracycline resistant STEC O157:H7 isolates carried both tetA and tetC. Other tetracycline resistance genes (tetB, tetD, tetE, and tetG) were not detected in any of the isolates. Among nine streptomycin resistant STEC O157:H7 isolates, eight carried strA-strB along with aadA, whereas the other isolate carried aadA alone. However, the majority of tetracycline and streptomycin susceptible STEC isolates also carried tetA and aadA genes, respectively. Most ampicillin resistant E. coli of both serotypes carried ampC genes. Among sulfonamide resistance genes, sulII was detected only in STEC O157:H7 (4 of 80 sulfonamide-resistant isolates) and sulI was detected in O157:H7- (1 of 16 sulfonamide resistant isolates). The emergence and dissemination of multidrug resistance in STEC can serve as a reservoir for different antimicrobial resistance genes. Dissemination of antimicrobial resistance genes to commensal and pathogenic bacteria could occur through any one of the horizontal gene transfer mechanisms adopted by the bacteria.     

Molecular characterization of multidrug-resistant Salmonella enterica subsp. enterica serovar Typhimurium isolates from swine

As part of a longitudinal study of antimicrobial resistance among salmonellae isolated from swine, we studied 484 Salmonella enterica subsp. enterica serovar Typhimurium (including serovar Typhimurium var. Copenhagen) isolates. We found two common pentaresistant phenotypes. The first was resistance to ampicillin, chloramphenicol, streptomycin, sulfamethoxazole, and tetracycline (the AmCmStSuTe phenotype; 36.2% of all isolates), mainly of the definitive type 104 (DT104) phage type (180 of 187 isolates). The second was resistance to ampicillin, kanamycin, streptomycin, sulfamethoxazole, and tetracycline (the AmKmStSuTe phenotype; 44.6% of all isolates), most commonly of the DT193 phage type (77 of 165 isolates), which represents an unusual resistance pattern for DT193 isolates. We analyzed 64 representative isolates by amplified fragment length polymorphism (AFLP) analysis, which revealed DNA fingerprint similarities that correlated with both resistance patterns and phage types. To investigate the genetic basis for resistance among DT193 isolates, we characterized three AmKmStSuTe pentaresistant strains and one hexaresistant strain, which also expressed resistance to gentamicin (Gm phenotype), all of which had similar DNA fingerprints and all of which were collected during the same sampling. We found that the genes encoding the pentaresistance pattern were different from those from isolates of the DT104 phage type. We also found that all strains encoded all of their resistance genes on plasmids, unlike the chromosomally encoded genes of DT104 isolates, which could be transferred to Escherichia coli via conjugation, but that the plasmid compositions varied among the isolates. Two strains (strains UT08 and UT12) had a single, identical plasmid carrying bla(TEM) (which encodes ampicillin resistance), aphA1-Iab (which encodes kanamycin resistance), strA and strB (which encode streptomycin resistance), class B tetA (which encodes tetracycline resistance), and an unidentified sulfamethoxazole resistance allele. The third pentaresistant strain (strain UT20) was capable of transferring by conjugation two distinct resistance patterns, AmKmStSuTe and KmStSuTe, but the genes were carried on plasmids with slightly different restriction patterns (differing by a single band of 15 kb). The hexaresistant strain (strain UT30) had the same plasmid as strains UT08 and UT12, but it also carried a second plasmid that conferred the AmKmStSuGm phenotype. The second plasmid harbored the gentamicin resistance methylase (grm), which has not previously been reported in food-borne pathogenic bacteria. It also carried the sul1 gene for sulfamethoxazole resistance and a 1-kb class I integron bearing aadA for streptomycin resistance. We also characterized isolates of the DT104 phage type. We found a number of isolates that expressed resistance only to streptomycin and sulfamethoxazole (the StSu phenotype; 8.3% of serovar Typhimurium var. Copenhagen strains) but that had AFLP DNA fingerprints similar or identical to those of strains with genes encoding the typical AmCmStSuTe pentaresistance phenotype of DT104. These atypical StSu DT104 isolates were predominantly cultured from environmental samples and were found to carry only one class I integron of 1.0 kb, in contrast to the typical two integrons (InC and InD) of 1.0 and 1.2 kb, respectively, of the pentaresistant DT104 isolates. Our findings show the widespread existence of multidrug-resistant Salmonella strains and the diversity of multidrug resistance among epidemiologically related strains. The presence of resistance genes on conjugative plasmids and duplicate genes on multiple plasmids could have implications for the spread of resistance factors and for the stability of multidrug resistance among Salmonella serovar Typhimurium isolates.     

Chronological study of antibiotic resistances and their relevant genes in Korean avian pathogenic Escherichia coli isolates

Antibiograms and relevant genotypes of Korean avian pathogenic Escherichia coli (APEC) isolates (n = 101) recovered between 1985 and 2005 were assessed via disc diffusion test, PCR, restriction enzyme analysis, and sequencing. These isolates were highly resistant to tetracycline (84.2%), streptomycin (84.2%), enrofloxacin (71.3%), and ampicillin (67.3%), and most of the tetracycline, streptomycin, enrofloxacin, and ampicillin resistances were associated with tetA and/or tetB, aadA and/or strA-strB, mutations in gyrA and/or parC, and TEM, respectively. Class 1 integrons were detected in 40 isolates (39.6%), and a variety of gene cassettes conferring streptomycin (aadA), gentamicin (aadB), and trimethoprim (dfr) resistances were identified: aadA1a (27.5%), dfrV-orfD (2.5%), aadB-aadA1a (2.5%), dfrI-aadA1a (47.5%), dfrXVII-aadA5 (12.5%), and dfrXII-orfF-aadA2 (7.5%). In addition, several types of common promoters (P(ant)) of the gene cassettes (hybrid P1, weak P1, or weak P1 plus P2) and single-nucleotide polymorphisms in aadA1a were identified. The results of a chronological analysis demonstrated significant and continuous increases in the frequencies of resistances to several antibiotics (tetracycline, streptomycin, enrofloxacin, ampicillin, and trimethoprim-sulfamethoxazole) and of the relevant resistance genes (tetA, strA-strB, and TEM), mutations in gyrA and parC, and multidrug-resistant APEC strains during the period 2000 to 2005.     

Streptomycin and chloramphenicol resistance genes in Escherichia coli isolates from cattle, pigs, and chicken in Kenya

The aims of this study were to determine the genetic basis of streptomycin and chloramphenicol resistance in 30 Escherichia coli isolates from food animals in Kenya and the role of plasmids in the spread of the resistance. Seven of the 29 streptomycin-resistant isolates harbored both the strA and strB genes. Twenty-one of isolates had the strA, strB, and aadA1 genes. The strA gene was disrupted by a functional trimethoprim gene, dfrA14 in 10 of the 21 isolates harboring the three streptomycin resistance genes. Physical linkage of intact strA and sul2 genes was found in two different plasmids from four isolates. Linkage of cassette-borne aadA1 and dfrA1 genes in class 1 integrons was found in two of the isolates. Chloramphenicol resistance was due to the gene catA1 in all the chloramphenicol resistant isolates. The strB, strA, and catA1 genes were transferable by conjugation and this points to the significance of conjugative resistance plasmids in the spread and persistence of streptomycin and chloramphenicol resistance in food animals in Kenya.     

Antimicrobial resistances of extraintestinal pathogenic Escherichia coli isolates from swine in China

Antibiograms and relevant genotypes of porcine extraintestinal pathogenic Escherichia coli (ExPEC) isolates (n = 315) recovered between 2004 and 2007 in China were assessed. Among the 14 antimicrobials tested, the most prevalent resistance was to ampicillin, trimethoprim, sulfadimidine, tetracycline, neomycin, streptomycin, kanamycin, ciprofloxacin and ofloxacin (ranging from 81.9 to 100%). Forty-six multiresistant patterns were found. For each antimicrobial agent, ampicillin resistance was primarily mediated by bla_TEM, streptomycin resistance by strA and strB, kanamycin/neomycin resistance by aphA1, gentamicin resistance by aac(3)-IV, quinolones resistance by mutations in gyrA, tetracycline resistance by tet(A), tet(B) and tet(G), trimethoprim resistance by dfrA7, dfrA12 and dfrA13, and sulfadimidine resistance by sul1 and sul2. Both bla_TEM-1 and bla_CTX-M-14 were found in two ESBLs-producing isolates. Strains that harbored several genes that conferred resistance to the same antimicrobial agent were often significantly more multiresistant than others. Class 1 integrons were identified in 86 (27.3%) ExPEC isolates, which harbored dfrA14, aadA2, aadA22, dfrA17, aadA5, dfrA17-aadA2, dfrA1-aadA1, dfrA12-aadA2, dfrA17-aadA5 gene cassettes in five major different variable regions, conferring resistance to trimethoprim and aminoglycosides. These results provide novel insights into the epidemiological characteristics of porcine ExPEC strains in China, and suggest the need for the prudent use of antimicrobial agents in food animals.     

Persistence of Escherichia coli clones and phenotypic and genotypic antibiotic resistance in recurrent urinary tract infections in childhood

We assessed the clonality of consecutive Escherichia coli isolates during the course of recurrent urinary tract infections (RUTI) in childhood in order to compare clonality with phenotypic antibiotic resistance patterns, the presence of integrons, and the presence of the sul1, sul2, and sul3 genes. Altogether, 78 urinary E. coli isolates from 27 children, who experienced recurrences during a 1-year follow-up after the first attack of acute pyelonephritis, were investigated. The MICs of sulfamethoxazole, trimethoprim-sulfamethoxazole (SXT), ampicillin, cefuroxime, cefotaxime, and gentamicin and the presence or absence of the intI gene for class 1 integrons and the sulfamethoxazole resistance-encoding genes sul1, sul2, and sul3 were determined. All E. coli strains were genotyped by pulsed-field gel electrophoresis. There were no significant differences in the prevalences of resistance to beta-lactams and SXT between initial and consecutive E. coli isolates (41 versus 45% and 41 versus 29%, respectively). However, the E. coli strains obtained after SXT administration more frequently carried two or more sul genes than the nonexposed strains (9/21 [43%] versus 11/57 [19%], respectively; P = 0.044). In 78% of the patients, the recurrence of unique clonal E. coli strains alone or combined with individual strains was detected. Phenotypic resistance and the occurrence of sul genes were more stable in clonal strains than in individual strains (odds ratios, 8.7 [95% confidence interval {95% CI}, 1.8 to 40.8] and 4.4 [95% CI, 1.1 to 17.7], respectively). Thus, in children with RUTIs, the majority of E. coli strains from consecutive episodes are unique persisting clones, with rare increases in the initially high antimicrobial resistance, the presence of sul genes, and the presence of integrons.     

Characterization of antimicrobial resistance among Escherichia coli O111 isolates of animal and human origin

Fifty isolates of Escherichia coli serogroup O111 recovered from humans and various animal species over a 24-year period (1976-1999) were examined for typical virulence-associated factors and susceptibilities to antimicrobials of human and veterinary significance. Nine H (flagellar) types were identified including nonmotile (n = 24), 32 (n = 12), negative (n = 5), and 56 (n = 3). Thirty-five (70%) isolates possessed at least one Shiga-toxin-producing E. coli (STEC)-associated virulence determinants (eae, stxl, stx2, hlyA) via PCR analysis. Of these 35 isolates, 20 possessed eae, stxl, and hlyA genes, whereas three isolates possessed eae, stxl, stx2, and hylA genes. Multiple antibiotic resistance was observed in 70% of the 50 E. coli O111 isolates. The majority of isolates displayed resistance to streptomycin, sulfamethoxazole, tetracycline, and kanamycin. Bacterial resistance to ampicillin, gentamicin, chloramphenicol, trimethoprim and apramycin was also observed. Integrons were identified in 23 (46%) of the E. coli isolates assayed, with a 1-kb amplicon being most frequently observed. DNA sequencing of these integrons revealed the presence of the aadA gene, encoding resistance to streptomycin. Two integrons of 1.5 and 2 kb contained the aadA2 and either dfrI or dfrXII genes, encoding resistance to streptomycin and trimethoprim, respectively. Integrons were also identified from isolates dating back to 1982. Isolates were further genetically characterized via ribotyping, which identified 15 distinct ribogroups, with 62% of isolates clustering into four major ribogroups. Certain riboprint patterns from different animal species, including humans, were observed in isolates spanning the 24-year collection period, suggesting the dissemination of specialized pathogenic O111 clones.     

Occurrence of plasmids and antibiotic resistance among Campylobacter jejuni and Campylobacter coli isolated from healthy and diarrheic animals.

Serologically defined strains of Campylobacter jejuni and Campylobacter coli from healthy and diarrheic animals were examined for the occurrence of plasmid DNA in association with the antibiotic susceptibility of the bacterial host and the health status of the animal host. Of all campylobacter organisms surveyed, 53% (116 of 200) contained plasmid DNA. A plasmid occurrence rate of 73.8% was obtained for C. coli from healthy pigs, contrasted by lower plasmid occurrence rates for C. coli from diarrheic pigs (30%) and from all diarrheic animals (21.4%). For C. jejuni, in contrast, only 13.6% of healthy cattle contained plasmid DNA, contrasted by a higher plasmid occurrence rate of 31.2% from diarrheic cattle. A high plasmid occurrence rate of 75.8% was observed for C. jejuni from healthy chickens. Campylobacter plasmids ranged in size from less than or equal to 1 to 86 megadaltons. Antibiotic susceptibility for 52 animal isolates (excluding chickens) indicated that most isolates were susceptible to kanamycin, erythromycin, gentamicin, tetracycline, and compound sulfonamide, whereas few were susceptible to bacitracin (19.2%); approximately half were susceptible to ampicillin (55.8%) and streptomycin (51.9%), and no isolates were susceptible to penicillin G. More isolates containing plasmids were resistant to ampicillin, tetracycline, and gentamicin than were isolates not carrying plasmids, there being a statistically significant difference for tetracycline and gentamicin, which suggested that these two antibiotics were probably plasmid mediated. The antibiotic susceptibility patterns of 21 chicken isolates of C. jejuni, by contrast, were different in that most were susceptible to ampicillin in addition to kanamycin, erythromycin, and gentamicin, whereas few wer susceptible to compound sulfonamide, streptomycin, and tetracycline in addition to penicillin G and bacitracin. A 30- or 39-megadalton plasmid, or both, common to many of the chicken isolates was usually associated with tetracycline resistance.     

pCERC1, a small, globally disseminated plasmid carrying the dfrA14 cassette in the strA gene of the sul2-strA-strB gene cluster

Commensal Escherichia coli from healthy adult humans were screened for antibiotic resistance genes. Two unrelated strains contained the sul2 sulphonamide resistance gene and strAB streptomyicn resistance genes with the dfrA14 trimethoprim resistance gene cassette in the strA gene and conferred resistance to trimethoprim and sulphamethoxazole. A 6.8 kb plasmid, pCERC1, that contains these resistance genes was recovered and sequenced. Deletions were constructed, and the pCERC1 replication region was confined to a 1 kb segment carrying genes for RNAs that are closely related to the ColE1 replication initiation RNAs. Polymerase chain reaction assays, developed to detect the sul2-strA-strB gene cluster in this context, identified a streptomycin and sulphonamide resistance plasmid, pCERC2, identical to pCERC1 without the dfrA14 cassette in two further E. coli isolates. Bioinformatic analysis revealed plasmids similar to pCERC1 and two more members of this family. One, the probable progenitor, carries only the sul2 gene adjacent to the small mobile element CR2. The other has a variant resistance gene cluster that has evolved from pCERC2 via acquisition of the tet(A) tetracycline resistance determinant. pCERC1 and pCERC2 have been detected in many countries, indicating a global distribution and appear to have been circulating in Gram-negative bacteria for more than 25 years.     

Genetic basis of antimicrobial drug resistance in clinical isolates of Salmonella enterica serotype Hadar from a Spanish region

The genetic bases of antimicrobial drug resistance (R) of 79 Salmonella enterica serotype Hadar clinical isolates (recovered during 1995-2001 in a Spanish region) was investigated. The isolates showed a limited genomic variation, as demonstrated by PFGE analysis using XbaI (three profiles, S>or=0.77) and BlnI (seven profiles, S>or=0.49; with 95% of the isolates falling into two clusters, S>or=0.75). Thirteen R-profiles, ranging from susceptible to multidrug resistant, were recognized. All susceptible isolates (14%) were recovered before or during 1998, when multidrug resistance (MDR) was still uncommon (20% from 1995-1998). In later years, the percentage of MDR increased considerably (92% in 2001). Resistance to nalidixic acid, tetracycline, streptomycin and ampicillin-cefalotin, encoded by gyrA-Asp87/Asn, tet(A), strA/B, and bla (TEM) genes, respectively, were the most common, appearing together in 38% of the isolates. In all tetracycline- and streptomycin-resistant isolates, strA/B and tet(A) were chromosomally located, whereas bla (TEM) was plasmid-born. Five different bla (TEM) plasmids (pUO-ShR1 to pUO-ShR5, of about 9.4, 23, 30, 45, and 95 kb, respectively) were identified. pUO-ShR3 and pUO-ShR5 harbored additional R-genes: [dfrA1] and [acc(3)IV-strA/B], respectively. pUO-Sh2, pUO-Sh3, pUO-ShR4, and pUO-Sh5 were self-transferable, and the latter could also mobilize pUOShR1. The reported data constitute a useful background for further epidemiological studies of MDR in S. Hadar.     

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.     

Characterization of Salmonella enterica serotype newport isolated from humans and food animals

Salmonella enterica serotype Newport isolates resistant to at least nine antimicrobials (including extended-spectrum cephalosporins), known as serotype Newport MDR-AmpC isolates, have been rapidly emerging as pathogens in both animals and humans throughout the United States. Resistance to extended-spectrum cephalosporins is associated with clinical failures, including death, in patients with systemic infections. In this study, 87 Salmonella serotype Newport strains were characterized by pulsed-field gel electrophoresis (PFGE) and antimicrobial susceptibility testing and examined for the presence of class 1 integrons and bla(CMY) genes. Thirty-five PFGE patterns were observed with XbaI, and three of these patterns were indistinguishable among isolates from humans and animals. Fifty-three (60%) Salmonella serotype Newport isolates were identified as serotype Newport MDR-AmpC, including 16 (53%) of 30 human isolates, 27 (93%) of 29 cattle isolates, 7 (70%) of 10 swine isolates, and 3 (30%) of 10 chicken isolates. However, 28 (32%) Salmonella serotype Newport isolates were susceptible to all 16 antimicrobials tested. The bla(CMY) gene was present in all serotype Newport MDR-AmpC isolates. Furthermore, the plasmid-mediated bla(CMY) gene was transferable via conjugation to an Escherichia coli strain. The transconjugant showed the MDR-AmpC resistance profile. Thirty-five (40%) of the isolates possessed class 1 integrons. Sequence analyses of the integrons showed that they contained aadA, which confers resistance to streptomycin, or aadA and dhfr, which confer resistance to trimethoprim-sulfamethoxazole. One integron from a swine isolate contained the sat-1 gene, which encodes resistance to streptothricin, an antimicrobial agent that has never been approved for use in the United States. In conclusion, Salmonella serotype Newport MDR-AmpC was commonly identified among Salmonella serotype Newport isolates recovered from humans and food animals. These findings support the possibility of transmission of this organism to humans through the food chain.     

Prevalence of plasmid-mediated quinolone resistance genes in uropathogenic Escherichia coli isolated in a teaching hospital of northern Italy

A retrospective study was conducted to determine the prevalence of plasmid-mediated quinolone resistance (PMQR) determinants in uropathogenic Escherichia coli isolated from inpatients and outpatients in a teaching hospital of northern Italy. The presence of qnrA, qnrB, qnrS, aac(6')-Ib-cr, and qepA was evaluated in 76 and 72 nalidixic acid-resistant E. coli, isolated in 2004 and 2006, respectively. Positivity for the aac(6')-Ib-cr gene was demonstrated in 3 of 76 (3.9%) and 8 of 72 (11%) isolates, respectively; no other PMQR determinant was found. All aac(6')-Ib-cr-positive strains also showed two point mutations in the gyrA and parC genes. Most aac(6')-Ib-cr-positive isolates demonstrated the contemporary presence of bla(CTX-M-15), bla(OXA-1/30), and bla(TEM-1) genes and 4/11 harbored a class 1 integron with a dfrA17-aadA5 gene cassette arrangement. Interestingly, all aac(6')-Ib-cr-positive isolates belonged to B2 phylogenetic group, O25b antigen type, multi locus sequence type 131, and to a cluster of approximately 70% similarity level by pulsed-field gel electrophoresis (PFGE). These findings suggest the circulation of the previously described intercontinentally spreading E. coli O25:H4-ST131 clone in our geographical area since 2004. Hybridization studies of the PFGE profiles showed the aac(6')-Ib-cr gene to be associated with different molecular weight bands (40-350 kb) and interestingly aac(6')-Ib-cr chromosomal integration was demonstrated in one strain by I-Ceu I method. This represents the first report to investigate the presence and diffusion of PMQR determinants in northern Italy and to describe aac(6')-Ib-cr chromosomal integration in E. coli.     

Presence and mechanism of antimicrobial resistance among enterococci from cats and dogs

The presence and mechanism of acquired resistance to erythromycin, tylosin, lincomycin, quinupristin/dalfopristin, tetracycline, chloramphenicol, gentamicin, kanamycin, and vancomycin were determined in 97 and 104 enterococci isolated from rectal swabs of cats and dogs, respectively. Eleven feline and three canine enterococcal isolates contained the aac(6')-Ie-aph(2')-Ia gene encoding high-level resistance to gentamicin, an antibiotic often used for treating enterococcal infections in humans. The combination of erm(B) and vat(E) genes encoding resistance to streptogramins was detected in one canine quinupristin/dalfopristin-resistant Enterococcus faecium isolate. Four quinupristin/dalfopristin-resistant enterococci only contained the erm(B) gene. Cross resistance against macrolides and lincosamides (30%) and resistance against tetracyclines (55%) was found to be widely distributed among enterococci from pets. In all of the feline and in 93% of the canine macrolide and lincosamide-resistant isolates, this resistance was encoded by the erm(B) gene. tet(M) was the most prevalent tetracycline resistance gene. It was detected in 91% of the feline and 86% of the canine tetracycline- resistant enterococci. A high occurrence of the Tn916/Tn1545 transposon family was found among these tet(M)-positive isolates. Enterococci from pet animals with resistance against vancomycin were not found. This study shows that enterococci from the intestinal microbiota of cats and dogs may act as a reservoir of resistance genes for animal or human pathogens.