Identification and characterization of integron-mediated antibiotic resistance among Shiga toxin-producing Escherichia coli isolates

A total of 50 isolates of Shiga toxin-producing Escherichia coli (STEC), including 29 O157:H7 and 21 non-O157 STEC strains, were analyzed for antimicrobial susceptibilities and the presence of class 1 integrons. Seventy-eight (n = 39) percent of the isolates exhibited resistance to two or more antimicrobial classes. Multiple resistance to streptomycin, sulfamethoxazole, and tetracycline was most often observed. Class 1 integrons were identified among nine STEC isolates, including serotypes O157:H7, O111:H11, O111:H8, O111:NM, O103:H2, O45:H2, O26:H11, and O5:NM. The majority of the amplified integron fragments were 1 kb in size with the exception of one E. coli O111:H8 isolate which possessed a 2-kb amplicon. DNA sequence analysis revealed that the integrons identified within the O111:H11, O111:NM, O45:H2, and O26:H11 isolates contained the aadA gene encoding resistance to streptomycin and spectinomycin. Integrons identified among the O157:H7 and O103:H2 isolates also possessed a similar aadA gene. However, DNA sequencing revealed only 86 and 88% homology, respectively. The 2-kb integron of the E. coli O111:H8 isolate contained three genes, dfrXII, aadA2, and a gene of unknown function, orfF, which were 86, 100, and 100% homologous, respectively, to previously reported gene cassettes identified in integrons found in Citrobacter freundii and Klebsiella pneumoniae. Furthermore, integrons identified among the O157:H7 and O111:NM strains were transferable via conjugation to another strain of E. coli O157:H7 and to several strains of Hafnia alvei. To our knowledge, this is the first report of integrons and antibiotic resistance gene cassettes in STEC, in particular E. coli O157:H7.     

Functional characteristics of plasmids of Shigella sonnei 47 strains

The phenotypic characteristics of Shigella sonnei strain 47 containing 7 plasmids of low molecular weight and 2 plasmids 60-100 Md large have been studied. The strains of Escherichia coli containing the single plasmids or plasmid groups from Shigella sonnei have been obtained by transformation and conjugation. The comparison of phenotypes of the obtained strains has helped to find the plasmid location of the determinants for streptomycin resistance (P7), genes for colicinogenicity and colicin immunity (P5), the enzymes of host cell specificity system Sso47I (P6), Sso47II (P4), and the genes for the conjugative DNA transfer (P9). Escherichia coli strains producing individual restriction enzymes SsoI and SsoII have been isolated.     

Antimicrobial resistance in generic Escherichia coli isolates from wild small mammals living in swine farm, residential, landfill, and natural environments in southern Ontario, Canada

To assess the impacts of different types of human activity on the development of resistant bacteria in the feces of wild small mammals, we compared the prevalences and patterns of antimicrobial resistance and resistance genes in generic Escherichia coli and Salmonella enterica isolates from fecal samples collected from wild small mammals living in four environments: swine farms, residential areas, landfills, and natural habitats. Resistance to antimicrobials was observed in E. coli isolates from animals in all environments: 25/52 (48%) animals trapped at swine farms, 6/69 (9%) animals trapped in residential areas, 3/20 (15%) animals trapped at landfills, and 1/22 (5%) animals trapped in natural habitats. Animals trapped on farms were significantly more likely to carry E. coli isolates with resistance to tetracycline, ampicillin, sulfisoxazole, and streptomycin than animals trapped in residential areas. The resistance genes sul2, aadA, and tet(A) were significantly more likely to be detected in E. coli isolates from animals trapped on farms than from those trapped in residential areas. Three S. enterica serotypes (Give, Typhimurium, and Newport) were recovered from the feces of 4/302 (1%) wild small mammals. All Salmonella isolates were pansusceptible. Our results show that swine farm origin is significantly associated with the presence of resistant bacteria and resistance genes in wild small mammals in southern Ontario, Canada. However, resistant fecal bacteria were found in small mammals living in all environments studied, indicating that environmental exposure to antimicrobials, antimicrobial residues, resistant bacteria, or resistance genes is widespread.     

Resistance patterns of non-O157 Shiga toxin-producing Escherichia coli (STEC) strains isolated from animals, food and asymptomatic human carriers in Switzerland

AIMS: The objective of the present study was to determine the resistance patterns of non-O157 Shiga toxin-producing Escherichia coli (STEC) strains isolated from different sources in Switzerland during the period 1997-99 as an epidemiological marker. METHODS AND RESULTS: The disk diffusion method was used to test 82 non-O157 STEC strains for susceptibility to 13 antibiotics. Ten strains were resistant to one and 20 strains to two and more antibiotics. The most frequent resistance types were streptomycin (14 strains), cephalothin (14 strains), sulfamethoxazole (14 strains) and tetracycline (14 strains). Three O100:H- STEC strains isolated from healthy slaughter pigs were resistant to eight antibiotics: streptomycin, sulfamethoxazole, trimethoprim, tetracycline, ampicillin, chloramphenicol, neomycin and gentamicin. CONCLUSION: Periodic surveillance of the antibiotic susceptibilities would be an important measure in detecting emergence and spread of resistance. SIGNIFICANCE AND IMPACT OF THE STUDY: Antibiotic susceptibility testing can be a useful tool for typing strains and should be used in combination with other phenotypic and genotypic methods.     

Phenotypic and genotypic characterization of sorbitol-negative or slow-fermenting (suspected O157) Escherichia coli isolated from milk samples in Lombardy region

AIMS: To investigate phenotypic and genotypic aspects of sorbitol-negative or slow-fermenting Escherichia coli, suspected to belong to O157 serogroup, isolated in Italy. METHODS AND RESULTS: Milk samples originating from goats and cows were screened for the presence of E. coli O157 with cultural methods. Sorbitol-negative or slow-fermenting strains were subjected to phenotypic characterization, antibiotic resistance profiles, PCR reactions for detection of toxins (stx(1) and stx(2)) and intimin (eae(GEN) and eae(O157)) genes and clustering by pulsed field gel electrophoresis (PFGE). Only one strain revealed to be O157. Susceptibility to 11 antibiotics highlighted the high resistance to tetracycline (50%), sulfonamide and streptomycin (33%). The stx(2) gene was detected in two strains; only the strain identified as O157 exhibited an amplicon for both eae genes. PFGE identified seven distinct XbaI macrorestriction patterns at a similarity level of 41%. CONCLUSIONS: The use of sorbitol fermentation as cultural method is not sufficient for STEC discrimination while PCR assay proved to be a valuable method. SIGNIFICANCE AND IMPACT OF THE STUDY: The study reports presence of Shiga toxin-producing E. coli in raw milk, signalling a potential risk for humans.     

Enumeration and antibiotic resistance patterns of fecal indicator organisms isolated from migratory Canada geese (Branta canadensis)

Thermotolerant fecal indicator organisms carried by migratory waterfowl may serve as reservoirs of antibiotic resistance. To determine the extent to which such antibiotic resistance markers were present in migratory Canada geese (Branta canadensis) on the Maryland Eastern Shore, we isolated Enterococcus spp. and Escherichia coli from fresh feces and examined the antibiotic resistance profiles of these bacteria. Samples were obtained in October 2002, January 2003, and March 2003. Thermotolerant E. coli counts ranged from 0 to 1.0x10(7) colony forming units (CFU)/0.1g (g-1) wet weight of feces, whereas Enterococcus spp. counts ranged from 1.0x10(2)-1.0x10(7) CFU g-1 wet weight of feces. Primary isolates of each indicator organism were tested against a panel of 10 antibiotics. Greater than 95% of E. coli isolates were resistant to penicillin G, ampicillin, cephalothin, and sulfathiazole; no E. coli were resistant to ciprofloxacin. Enterococcal isolates showed highest resistance to cephalothin, streptomycin, and sulfathiazole; no enterococci were resistant to chloramphenicol. The tetracyclines, streptomycin, and gentamycin provided the greatest discrimination among E. coli isolates; chlortetracycline, cephalothin, and gentamycin resistance patterns provided the greatest discrimination between enterococcal strains. Multiple antibiotic resistance (MAR) profiles were calculated: fall (E. coli=0.499; enterococci=0.234), winter (E. coli=0.487; enterococci=0.389), and spring (E. coli=0.489; enterococci=0.348). E. faecalis and E. faecium, which are recognized human nosocomial pathogens, were cultured from winter (44 and 56%, respectively) and spring (13 and 31%, respectively) fecal samples.     

Stx genotypes and antimicrobial resistance profiles of Shiga toxin-producing Escherichia coli strains isolated from human infections, cattle and foods in Brazil

A total of 107 Shiga toxin-producing Escherichia coli strains (STEC) isolated from different origins in S?o Paulo, Brazil, and belonging to different serotypes were characterized regarding stx subtypes and susceptibility to antimicrobial agents. Most of the human STEC strains harbored stx1 (85.7%), while stx2, associated or not to stx1, was identified preferentially in the animal and food strains. None of the STEC strains carried stx1c. Some genotypes occurred exclusively among strains of bovine origin as stx2c, stx1+2+2c (16.5% each), and stx2d (0.9%), whereas stx2+2c2vha) was only identified among the O157:H7 human strains. Moreover, the stx(2c2vhb) subtype was found more frequently among bovine than human strains (39% vs. 4.8%). The highest frequencies of susceptibility to antimicrobial agents were observed among bovine (87%) and food (100%) STEC strains, while 47.6% of the human isolates were resistant to at least one drug. Multiresistance occurred among O111 STEC strains from human and bovine origin. The antimicrobials to which resistance was most frequently observed were tetracycline (90%) and streptomycin (75%) among human strains, and also sulphazotrin (88%) in animal strains. A few serotypes were commonly identified among STEC strains isolated from diverse sources in Brazil, but in general the strains presented distinct stx subtypes and/or antimicrobial resistance profiles.     

Transfer of multiple drug resistance plasmids between bacteria of diverse origins in natural microenvironments

Plasmids harboring multiple antimicrobial-resistance determinants (R plasmids) were transferred in simulated natural microenvironments from various bacterial pathogens of human, animal, or fish origin to susceptible strains isolated from a different ecological niche. R plasmids in a strain of the human pathogen Vibrio cholerae O1 E1 Tor and a bovine Escherichia coli strain were conjugated to a susceptible strain of the fish pathogenic bacterium Aeromonas salmonicida subsp. salmonicida in marine water. Conjugations of R plasmids between a resistant bovine pathogenic E. coli strain and a susceptible E. coli strain of human origin were performed on a hand towel contaminated with milk from a cow with mastitis. A similar conjugation event between a resistant porcine pathogenic E. coli strain of human origin was studied in minced meat on a cutting board. Conjugation of R plasmids between a resistant strain of the fish pathogenic bacterium A. salmonicida subsp. salmonicida and a susceptible E. coli strain of human origin was performed in raw salmon on a cutting board. R plasmids in a strain of A. salmonicida subsp. salmonicida and a human pathogenic E. coli strain were conjugated to a susceptible porcine E. coli strain in porcine feces. Transfer of the different R plasmids was confirmed by plasmid profile analyses and determination of the resistance pattern of the transconjugants. The different R plasmids were transferred equally well under simulated natural conditions and under controlled laboratory conditions, with median conjugation frequencies ranging from 3 x 10(-6) to 8 x 10(-3). The present study demonstrates that conjugation and transfer of R plasmids is a phenomenon that belongs to the environment and can occur between bacterial strains of human, animal, and fish origins that are unrelated either evolutionarily or ecologically even in the absence of antibiotics. Consequently, the contamination of the environment with bacterial pathogens resistant to antimicrobial agents is a real threat not only as a source of disease but also as a source from which R plasmids can easily spread to other pathogens of diverse origins.     

Transfer of multiple drug resistance plasmids between bacteria of diverse origins in natural microenvironments.

Plasmids harboring multiple antimicrobial-resistance determinants (R plasmids) were transferred in simulated natural microenvironments from various bacterial pathogens of human, animal, or fish origin to susceptible strains isolated from a different ecological niche. R plasmids in a strain of the human pathogen Vibrio cholerae O1 E1 Tor and a bovine Escherichia coli strain were conjugated to a susceptible strain of the fish pathogenic bacterium Aeromonas salmonicida subsp. salmonicida in marine water. Conjugations of R plasmids between a resistant bovine pathogenic E. coli strain and a susceptible E. coli strain of human origin were performed on a hand towel contaminated with milk from a cow with mastitis. A similar conjugation event between a resistant porcine pathogenic E. coli strain of human origin was studied in minced meat on a cutting board. Conjugation of R plasmids between a resistant strain of the fish pathogenic bacterium A. salmonicida subsp. salmonicida and a susceptible E. coli strain of human origin was performed in raw salmon on a cutting board. R plasmids in a strain of A. salmonicida subsp. salmonicida and a human pathogenic E. coli strain were conjugated to a susceptible porcine E. coli strain in porcine feces. Transfer of the different R plasmids was confirmed by plasmid profile analyses and determination of the resistance pattern of the transconjugants. The different R plasmids were transferred equally well under simulated natural conditions and under controlled laboratory conditions, with median conjugation frequencies ranging from 3 x 10(-6) to 8 x 10(-3). The present study demonstrates that conjugation and transfer of R plasmids is a phenomenon that belongs to the environment and can occur between bacterial strains of human, animal, and fish origins that are unrelated either evolutionarily or ecologically even in the absence of antibiotics. Consequently, the contamination of the environment with bacterial pathogens resistant to antimicrobial agents is a real threat not only as a source of disease but also as a source from which R plasmids can easily spread to other pathogens of diverse origins.     

Phenotypical characteristics of Shiga-like toxin Escherichia coli isolated from sheep dairy products

AIMS: To analyse phenotypical characteristics of Shiga toxin-producing Escherichia coli (STEC) strains from ovine origin. METHODS AND RESULTS: A total of 13 STEC strains (eight O157 and five non-O157) isolated from sheep dairy products were used in this study. Biochemical traits, motility, haemolytic activity, resistance to tellurite-cefixime, maximum growth temperature and antibiotic resistance were determined. The STEC strains were grouped into nine biochemical and physiological biotypes (five for the O157 and four for the non-O157 strains). All STEC strains showed resistance to bacitracin, cloxacilin, penicillin and tylosin. CONCLUSIONS: Different biotypes and antibiotic resistance patterns of STEC isolated from sheep dairy products were observed. SIGNIFICANCE AND IMPACT OF THE STUDY: This work will be a contribution to the better characterization of STEC isolated from sheep dairy products, which have, to date, been scarcely studied, and to the better understanding of the risks associated with its consumption.     

Long-term survival of shiga toxin-producing Escherichia coli O26, O111, and O157 in bovine feces.

Cattle are an important reservoir of Shiga toxin-producing Escherichia coli (STEC) O26, O111, and O157. The fate of these pathogens in bovine feces at 5, 15, and 25 degrees C was examined. The feces of a cow naturally infected with STEC O26:H11 and two STEC-free cows were studied. STEC O26, O111, and O157 were inoculated into bovine feces at 10(1), 10(3), and 10(5) CFU/g. All three pathogens survived at 5 and 25 degrees C for 1 to 4 weeks and at 15 degrees C for 1 to 8 weeks when inoculated at the low concentration. On samples inoculated with the middle and high concentrations, O26, O111, and O157 survived at 25 degrees C for 3 to 12 weeks, at 15 degrees C for 1 to 18 weeks, and at 5 degrees C for 2 to 14 weeks, respectively. Therefore, these pathogens can survive in feces for a long time, especially at 15 degrees C. The surprising long-term survival of STEC O26, O111, and O157 in bovine feces shows that such feces are a potential vehicle for transmitting not only O157 but also O26 and O111 to cattle, food, and the environment. Appropriate handling of bovine feces is emphasized.     

A multiresistant clone of Shiga toxin-producing Escherichia coli O118:[H16] is spread in cattle and humans over different European countries

Multiresistant Shiga toxin-producing Escherichia coli (STEC) O118:H16 and O118 nonmotile strains (designated O118:[H16]) were detected by examination of 171 STEC isolates for their antimicrobial sensitivity. Of 48 STEC O118:[H16] strains, 98% were resistant to sulfonamide, 96% were resistant to streptomycin, 79% were resistant to kanamycin, 75% were resistant to tetracycline, 67% were resistant to ampicillin, 60% were resistant to chloramphenicol, 48% were resistant to trimethoprim, and 10% each were resistant to gentamicin and nalidixic acid. Nalidixic acid resistance and reduced susceptibility to ciprofloxacin were associated with the mutation gyrA(LEU-83). The STEC O118:[H16] strains were found to belong to a single genetic clone as investigated by multilocus enzyme electrophoresis and by multilocus sequence analysis of E. coli housekeeping genes. The STEC O118:[H16] strains originated from humans and cattle and were isolated in seven different countries of Europe between 1986 and 1999. Strains showing multiresistance to up to eight different antimicrobials predominated among the more recent STEC O118:[H16] strains. The genes in parentheses were associated with resistance to kanamycin (aphA1-Ia), chloramphenicol (catA1), tetracycline [tet(A)], and ampicillin (bla(TEM-1)). Class 1 integrons containing sulI (sulfonamide resistance), aadA1a (streptomycin resistance), or dfrA1 (trimethoprim resistance)-aadA1a gene cassettes were detected in 28 strains. The bla(TEM-1b) gene was present in 18 of 21 strains that were examined by nucleotide sequencing. Class 1 integrons and bla(TEM) genes were localized on plasmids and/or on the chromosome in different STEC O118:[H16] strains. Hybridization of XbaI-digested chromosomal DNA separated by pulsed-field gel electrophoresis revealed that bla(TEM) genes were integrated at different positions in the chromosome of STEC O118:[H16] strains that could have occurred by Tn2 insertion. Our data suggest that strains belonging to the STEC O118:[H16] clonal group have a characteristic propensity for acquisition and maintenance of resistance determinants, thus contrasting to STEC belonging to other serotypes.     

Shiga-toxigenic Escherichia coli O157 and non-Shiga-toxigenic E. coli O157 respond differently to culture and isolation from naturally contaminated bovine faeces

AIM: To quantify the effect of enrichment, immunomagnetic separation (IMS), and selective plating procedures on isolation of Shiga-toxigenic Escherichia coli O157 (STEC O157) and non-Shiga-toxigenic Escherichia coli O157 (non-STEC O157) from naturally contaminated bovine faeces. METHODS AND RESULTS: Two broth enrichment times, two IMS strategies, and two selective plating media were evaluated. STEC O157 and non-STEC O157 strains were often isolated from the same faecal specimen and responded differently to the isolation protocols. A large-volume IMS system was more sensitive than a conventional small-volume IMS method, but was also more expensive. STEC O157 was more frequently isolated from 6 h enriched broth and ChromAgar plates containing 0.63 mg l(-1) potassium tellurite (TCA). Non-STEC O157 was more frequently isolated from un-enriched broth and ChromAgar plates without tellurite (CA). CONCLUSIONS: The combination of 6-h enrichment in Gram-negative broth containing vancomycin, cefixime and cefsuludin, large volume IMS and selective plating on TCA maximized STEC O157 recovery from naturally contaminated cattle faecal specimens. SIGNIFICANCE AND IMPACT OF THE STUDY: The pairing of proper enrichment with a specific plating procedure is key for STEC O157 recovery from naturally contaminated bovine faeces. Incorporating tellurite into an E. coli O157 detection strategy may select for the subset of E. coli O157 that contains the Shiga-toxin genes.     

Preliminary characterization of a food-borne multiple-antibiotic-resistant Salmonella typhimurium strain.

Plasmid characterization studies were conducted on a Salmonella typhimurium strain isolated from pasteurized milk and from a symptomatic patient during the 1985 Illinois salmonellosis outbreak. This strain (Hf) was reported to possess an unusual plasmid profile which distinguished it from all Salmonella strains isolated in the United States prior to 1984. Antibiotic susceptibility testing revealed that the strain was resistant to tetracycline, erythromycin, clindamycin, sulfisoxazole, sulfadiazene, triple sulfa, cefoperazone, streptomycin, mezlocillin, piperacillin, carbenicillin, penicillin, ampicillin, and kanamycin. Plasmid analysis revealed that the strain possessed four plasmids with sizes of approximately 158, 98, 10.2, and 6.0 kilobase pairs (kb). Successive transfer at 43 degrees C led to increased antibiotic sensitivity in 75.5% of the isolates screened. Electroporation and calcium chloride treatment were each used to transform plasmid-free Escherichia coli strains with the plasmid pool from S. typhimurium Hf. Plasmids introduced by transformation ranged in size from 4.4 to 23.2 kb and correlated with resistance to penicillin G, ampicillin, carbenicillin, cephalothin, cefoperazone, cefamandole, mezlocillin, piperacillin, and in some cases, tetracycline and kanamycin. DNA-DNA hybridization experiments localized these resistance genes to a highly duplicated 6.3-kb fragment of the total EcoRI restriction digest of the S. typhimurium Hf plasmid pool.     

产志贺毒素大肠埃希菌的分子生物学鉴定和耐药性分析

为了解产志贺毒素大肠埃希菌 (Shigatoxin producingEscherichiacoli ,STEC)stx1,stx2 ,eaeA ,hlyA 4种毒力基因的分布情况 ,以及分离株对 18种抗生素的敏感性 ,采用多重PCR(multiplexPCR ,mPCR)法对分离株进行毒力基因的分子生物学鉴定 ;用WHO推荐的K B法对分离株进行抗生素的敏感性测定。产志贺毒素的大肠埃希菌共有 4 6株 ,其中 2种毒素均产生的有 2 2株 (4 7.8% ) ;单纯产生stx1的有 16株 (36 .9% ) ,stx2 的有 8株 (17.4 % ) ;4种毒力基因均存在的有 19株 (4 1.3% ) ,血清型为O15 7∶H7,而非O15 7∶H7血清型的菌株 (2 3/46 )中 ,4种毒力基因同时存在的仅有 3株 (6 .6 % ) ,但有 13株 (5 6 .9% )hlyA基因阳性。全部STEC对复方新诺明耐药 ,对链霉素耐药率为 2 8.3% ,氨苄西林为 30 .4 % ,红霉素为 6 9.6 % ,而且有 5株对至少 4种以上抗生素多重耐药 ,耐药谱为复方新诺明 链霉素 红霉素 氨苄西林。非O15 7型STEC耐药菌次为 12 2 ,而O15 7型为 6 3。可见 ,mPCR法可以快速检测STEC特征性毒力基因 ,以判定其致病性能。非O15 7型STEC对抗生素较易形成耐药性。     

Plasmid-mediated conjugative transfer of Klebsiella sp. rcs genesable to induce colanic acid capsular polysaccharide biosynthesis in Escherichia coli

Regulation of capsular biosynthesis (rcs) genes, encoding the ability to induce the production of a colanic acid polysaccharide capsule, were transferred to Escherichia coli by conjugation with Klebsiella pneumoniae (aerogenes) of capsular serotype K36. Transfer was mediated by a 58.4-MDa conjugative plasmid of incompatibility group IncM, which carried a copy of Tn7 (specifying resistance to trimethoprim and streptomycin) together with determinants for several further resistances. This plasmid did not carry the rcs genes itself, but mediated the conjugative recA-dependent transfer of part of the Klebsiella chromosome to E. coli. Once resident in E. coli, the rcs gene(s) could not be mobilised to other strains of E. coli, and the mobilising plasmid could be cured from capsulate transconjugants without loss of the ability to produce colanic acid. All such cured transconjugants contained an insertion of Tn7 in the chromosome, suggesting that the transposon might be involved in mobilisation of the rcs genes from Klebsiella sp. to E. coli. These findings explain previous observations that the ability to manufacture capsular polysaccharide could be transferred by plasmids between Klebsiella sp. and E. coli.     

Preliminary characterization of a food-borne multiple-antibiotic-resistant Salmonella typhimurium strain

Plasmid characterization studies were conducted on a Salmonella typhimurium strain isolated from pasteurized milk and from a symptomatic patient during the 1985 Illinois salmonellosis outbreak. This strain (Hf) was reported to possess an unusual plasmid profile which distinguished it from all Salmonella strains isolated in the United States prior to 1984. Antibiotic susceptibility testing revealed that the strain was resistant to tetracycline, erythromycin, clindamycin, sulfisoxazole, sulfadiazene, triple sulfa, cefoperazone, streptomycin, mezlocillin, piperacillin, carbenicillin, penicillin, ampicillin, and kanamycin. Plasmid analysis revealed that the strain possessed four plasmids with sizes of approximately 158, 98, 10.2, and 6.0 kilobase pairs (kb). Successive transfer at 43 degrees C led to increased antibiotic sensitivity in 75.5% of the isolates screened. Electroporation and calcium chloride treatment were each used to transform plasmid-free Escherichia coli strains with the plasmid pool from S. typhimurium Hf. Plasmids introduced by transformation ranged in size from 4.4 to 23.2 kb and correlated with resistance to penicillin G, ampicillin, carbenicillin, cephalothin, cefoperazone, cefamandole, mezlocillin, piperacillin, and in some cases, tetracycline and kanamycin. DNA-DNA hybridization experiments localized these resistance genes to a highly duplicated 6.3-kb fragment of the total EcoRI restriction digest of the S. typhimurium Hf plasmid pool.     

Involvement of surface polysaccharides in the organic acid resistance of Shiga Toxin-producing Escherichia coli O157:H7

In general, wild Escherichia coli strains can grow effectively under moderately acidic organic acid-rich conditions. We found that the Shiga Toxin-producing E. coli (STEC) O157:H7 NGY9 grows more quickly than a K-12 strain in Luria-Bertani (LB)-2-morpholinoethanesulphonic acid (MES) broth supplemented with acetic acid (pH 5.4). Hypothesizing that the resistance of STEC O157:H7 to acetic acid is as a result of a mechanism(s) other than those known, we screened for STEC mutants sensitive to acetic acid. NGY9 was subjected to mini-Tn5 mutagenesis and, from 50,000 colonies, five mutants that showed a clear acetic acid-sensitive phenotype were isolated. The insertion of mini-Tn5 in three mutants occurred at the fcl, wecA (rfe) and wecB (rffE) genes and caused loss of surface O-polysaccharide, loss of both O-polysaccharide and enterobacterial common antigen (ECA) and loss of ECA respectively. The other two mutants showed inactivation of the waaG (rfaG) gene but at different positions that caused a deep rough mutant with loss of the outer core oligosaccharide of lipopolysaccharide (LPS) as well as phenotypic loss of O-polysaccharide and ECA. With the introduction of plasmids carrying the fcl, wecA, wecB and waaG genes, respectively, all mutants were complemented in their production of O-polysaccharide and ECA, and normal growth was restored in organic acid-rich culture conditions. We also found that the growth of Salmonella LPS mutants Ra, Rb1, Rc, Rd1, Rd2 and Re was suppressed in the presence of acetic acid compared with that of the parents. These results suggest that the full expression of LPS (including O-polysaccharide) and ECA is indispensable to the resistance against acetic acid and other short chain fatty acids in STEC O157:H7 and Salmonella. To the best of our knowledge, this is a newly identified physiological role for O-polysaccharide and ECA as well as an acid resistance mechanism.     

Appearance of enterotoxigenic Escherichia coli in piglets with diarrhea in connection with feed changes

Twelve weaned piglet (3-week-old) were divided into three groups according to time of feed change and observed for diarrhea during the time they were 3 to 8 weeks of age. A total of 553 strains of Escherichia coli were isolated from rectal fecal samples and examined for heat-labile (LT) and heat-stable (ST) enterotoxins, pilus antigens (K88, K99, and 987P), hemolysin (Hly), raffinose utilization (Raf) and drug resistance. Enterotoxins and/or hemolytic E. coli strains appeared in the rectal feces of 5- to 6-week-old piglets with diarrhea in connection with feed change and changing temperatures. Most of the isolates showed multiple drug resistance to sulfonamides (Sa), streptomycin (Sp), ampicillin, and/or mercury. Enterotoxigenic E. coli isolates represented four phenotypes: K88+.LT+.Hly+.Raf+.(SaSmCpTcKmSp) (12 strains), K99+.ST+.Raf+.(TcKm) (7 strains), ST+.Raf+.(TcKm) (7 strains), and ST.+(SaSmKm) (25 strains). The drug resistance determinants were transferable concurrently and some of them mobilized the determinants for K88, LT, Hly, and Raf to an E. coli C strain.     

Characterization of antimicrobial resistant Escherichia coli isolated from processed bison carcasses

AIM: To determine the phenotypic and genotypic antimicrobial susceptibility profiles of Escherichia coli from bison carcasses. METHODS AND MATERIALS: The antimicrobial resistance of 138 E. coli isolates recovered from processed bison carcasses was determined by using the National Antimicrobial Resistance Monitoring System panels, polymerase chain reaction assays, plasmid analysis and conjugation studies. RESULTS: Resistance to 14 of the 16 antimicrobials was observed. Twenty-three (16.7%) isolates displayed resistance to at least one antimicrobial agent. The most prevalent resistances were to tetracycline (13.0%), sulfamethoxazole (7.9%) and streptomycin (5.8%). No resistance was observed to amikacin and ciprofloxacin. Further analysis of 23 antimicrobial-resistant E. coli isolates showed the presence of resistance genes corresponding to their phenotypic profiles. Results of conjugation studies carried out showed most isolates tested were able to transfer their resistance to recipients. CONCLUSION: This study indicated that multidrug-resistant E. coli isolates are present in bison. However, the resistance rate is lower than that reported in other meat species. SIGNIFICANCE AND IMPACT OF THE STUDY: The beneficial effects of antimicrobial-free feeding practice in bison may be promoting a reduction in the prevalence of antimicrobial resistance in commensal flora of bison.