Geographical variation in antibiotic resistance profiles of Escherichia coli isolated from swine, poultry, beef and dairy cattle farm water retention ponds in Florida

AIMS: The aim of this study was to assess geographical variation in multiple antibiotic resistance (MAR) profiles of livestock Escherichia coli as well as to evaluate the ability of MAR profiles to differentiate sources of faecal pollution. METHODS AND RESULTS: More than 2000 E. coli isolates were collected from water retention ponds and manure of swine, poultry, beef and dairy farms in south, central and north Florida, and analysed for MAR using nine antibiotics. There were significant differences in antibiotic resistance of E. coli by season and livestock type for more than one antibiotic, but regional differences were significant only for ampicillin. Over the three regions, discriminant analysis using MAR profiles correctly classified 27% of swine, 49% of poultry, 56% of beef and 51% of dairy isolates. CONCLUSIONS: Regional variations in MAR combined with moderate discrimination success suggest that MAR profiles of E. coli may only be marginally successful in identifying sources of faecal pollution. SIGNIFICANCE AND IMPACT OF THE STUDY: This study demonstrates the existence of regional and seasonal differences in MAR profiles as well as the limited ability of MAR profiles to discriminate among livestock sources.     

Antimicrobial resistance of Escherichia coli O157 isolated from humans, cattle, swine, and food

A total of 361 Escherichia coli O157 isolates, recovered from humans, cattle, swine, and food during the years 1985 to 2000, were examined to better understand the prevalence of antimicrobial resistance among these organisms. Based on broth microdilution results, 220 (61%) of the isolates were susceptible to all 13 antimicrobials tested. Ninety-nine (27%) of the isolates, however, were resistant to tetracycline, 93 (26%) were resistant to sulfamethoxazole, 61 (17%) were resistant to cephalothin, and 48 (13%) were resistant to ampicillin. Highest frequencies of resistance occurred among swine isolates (n = 70), where 52 (74%) were resistant to sulfamethoxazole, 50 (71%) were resistant to tetracycline, 38 (54%) were resistant to cephalothin, and 17 (24%) were resistant to ampicillin. Based on the presence of Shiga toxin genes as determined by PCR, 210 (58%) of the isolates were identified as Shiga toxin-producing E. coli (STEC). Among these, resistance was generally low, yet 21 (10%) were resistant to sulfamethoxazole and 19 (9%) were resistant to tetracycline. Based on latex agglutination, 189 (52%) of the isolates were identified as E. coli O157:H7, among which 19 (10%) were resistant to sulfamethoxazole and 16 (8%) were resistant to tetracycline. The data suggest that selection pressure imposed by the use of tetracycline derivatives, sulfa drugs, cephalosporins, and penicillins, whether therapeutically in human and veterinary medicine or as prophylaxis in the animal production environment, is a key driving force in the selection of antimicrobial resistance in STEC and non-STEC O157.     

Variable within- and between-herd diversity of CTX-M cephalosporinase-bearing Escherichia coli isolates from dairy cattle

bla(CTX-M) beta-lactamases confer resistance to critically important cephalosporin drugs. Recovered from both hospital- and community-acquired infections, bla(CTX-M) was first reported in U.S. livestock in 2010. It has been hypothesized that veterinary use of cephalosporins in livestock populations may lead to the dissemination of beta-lactamase-encoding genes. Therefore, our objectives were to estimate the frequency and distribution of coliform bacteria harboring bla(CTX-M) in the fecal flora of Ohio dairy cattle populations. In addition, we characterized the CTX-M alleles carried by the isolates, their plasmidic contexts, and the genetic diversity of the bacterial isolates themselves. We also evaluated the association between ceftiofur use and the likelihood of recovering cephalosporinase-producing bacteria. Thirty fresh fecal samples and owner-reported ceftiofur use data were collected from each of 25 Ohio dairy farms. Fecal samples (n = 747) yielded 70 bla(CTX-M)-positive Escherichia coli isolates from 5/25 herds, 715 bla(CMY-2) E. coli isolates from 25/25 herds, and 274 Salmonella spp. from 20/25 herds. The within-herd prevalence among bla(CTX-M)-positive herds ranged from 3.3 to 100% of samples. Multiple pulsed-field gel electrophoresis (PFGE) patterns, plasmid replicon types, and CTX-M genes were detected. Plasmids with CTX-M-1, -15, and -14 alleles were clonal by restriction fragment length polymorphism (RFLP) within herds, and specific plasmid incompatibility group markers were consistently associated with each bla(CTX-M) allele. PFGE of total bacterial DNA showed similar within-herd clustering, with the exception of one herd, which revealed at least 6 different PFGE signatures. We were unable to detect an association between owner-reported ceftiofur use and the probability of recovering E. coli carrying bla(CTX-M) or bla(CMY-2).     

Proximity-dependent inhibition in Escherichia coli isolates from cattle

We describe a novel proximity-dependent inhibition phenotype of Escherichia coli that is expressed when strains are cocultured in defined minimal media. When cocultures of "inhibitor" and "target" strains approached a transition between logarithmic and stationary growth, target strain populations rapidly declined >4 log CFU per ml over a 2-h period. Inhibited strains were not affected by exposure to conditioned media from inhibitor and target strain cocultures or when the inhibitor and target strains were incubated in shared media but physically separated by a 0.4-μm-pore-size membrane. There was no evidence of lytic phage or extracellular bacteriocin involvement, unless the latter was only present at effective concentrations within immediate proximity of the inhibited cells. The inhibitory activity observed in this study was effective against a diversity of E. coli strains, including enterohemorrhagic E. coli serotype O157:H7, enterotoxigenic E. coli expressing F5 (K99) and F4 (K88) fimbriae, multidrug-resistant E. coli, and commensal E. coli. The decline in counts of target strains in coculture averaged 4.8 log CFU/ml (95% confidence interval, 4.0 to 5.5) compared to their monoculture counts. Coculture of two inhibitor strains showed mutual immunity to inhibition. These results suggest that proximity-dependent inhibition can be used by bacteria to gain a numerical advantage when populations are entering stationary phase, thus setting the stage for a competitive advantage when growth conditions improve.     

The seasonal variation of thermophilic campylobacters in beef cattle, dairy cattle and calves

The epidemiology of clinical cases of campylobacter in temperate climates shows a striking seasonality. In the search for a seasonal environmental reservoir changes in the carriage rate and population size of campylobacters in bovine hosts with time have been measured. Most probable number (MPN) methodology was used to enumerate thermophilic campylobacters in samples taken from the small intestines of beef cattle at slaughter and the fresh faeces of four dairy herds and new-born calves. Statistical analyses revealed significant evidence for seasonal periodicity in the data from dairy herds ( P = 0·044). Not only was there a departure from constancy within a 12-month interval but these data revealed a true seasonality, that is, the same periodicity in numbers from one year to the next. Each herd had two peaks per year, in approximately spring and autumn. Peaks coincided in herds on neighbouring farms but those on farms in the north preceded those on farms in the south by 2 and 1 months, respectively ( P = 0·0057). Intestinal carriage by beef cattle at slaughter was 89·4% ( n = 360) with an average MPN campylobacters per gram fresh weight (MPN gfw⁻¹) of 6·1 × 10². Average MPN gfw⁻¹ in faeces from the dairy herds and calves were 69·9 ( S.D. 3) and 3·3 × 10⁴ ( S.D. 1·7 × 10²). There was no evidence of seasonal periodicity in the size of the campylobacter population in beef cattle at slaughter. Calves were campylobacter free at birth but became colonized within a few days.     

Longitudinal study of antimicrobial resistance among Escherichia coli isolates from integrated multisite cohorts of humans and swine

In a 3-year longitudinal study, we examined the relationship between the seasonal prevalence of antimicrobial-resistant (AR) Escherichia coli isolates from human wastewater and swine fecal samples and the following risk factors: the host species, the production type (swine), the vocation (human swine workers, non-swine workers, and slaughter plant workers), and the season, in a multisite, vertically integrated swine and human population representative of a closed agri-food system. Human and swine E. coli (n = 4,048 and 3,429, respectively) isolates from wastewater and fecal samples were tested for antimicrobial susceptibility, using the Sensititre broth microdilution system. There were significant (P < 0.05) differences among AR E. coli prevalence levels of (i) the host species, in which swine isolates were at higher risk for resistance to tetracycline, kanamycin, ceftiofur, gentamicin, streptomycin, chloramphenicol, sulfisoxazole, and ampicillin; (ii) the swine production group, in which purchased boars, nursery piglets, and breeding boars isolates had a higher risk of resistance to streptomycin and tetracycline; and iii) the vocation cohorts, in which swine worker cohort isolates exhibited lower sulfisoxazole and cefoxitin prevalence than the non-swine worker cohorts, while the slaughter plant worker cohort isolates exhibited elevated cefoxitin prevalence compared to that of non-swine workers. While a high variability was observed among seasonal samples over the 3-year period, no significant temporal trends were apparent. There were significant differences in the prevalence levels of multidrug-resistant isolates between host species, with swine at a higher risk of carrying multidrug-resistant strains than humans. Considering vocation, slaughter plant workers were at higher risk of exhibiting multidrug-resistant E. coli than non-swine workers.     

Molecular epidemiology of ceftiofur-resistant Escherichia coli isolates from dairy calves

Healthy calves (n = 96, 1 to 9 weeks old) from a dairy herd in central Pennsylvania were examined each month over a five-month period for fecal shedding of ceftiofur-resistant gram-negative bacteria. Ceftiofur-resistant Escherichia coli isolates (n = 122) were characterized by antimicrobial resistance (disk diffusion and MIC), serotype, pulsed-field gel electrophoresis subtypes, beta-lactamase genes, and virulence genes. Antibiotic disk diffusion assays showed that the isolates were resistant to ampicillin (100%), ceftiofur (100%), chloramphenicol (94%), florfenicol (93%), gentamicin (89%), spectinomycin (72%), tetracycline (98%), ticarcillin (99%), and ticarcillin-clavulanic acid (99%). All isolates were multidrug resistant and displayed elevated MICs. The E. coli isolates belonged to 42 serotypes, of which O8:H25 was the predominant serotype (49.2%). Pulsed-field gel electrophoresis classified the E. coli isolates into 27 profiles. Cluster analysis showed that 77 isolates (63.1%) belonged to one unique group. The prevalence of pathogenic E. coli was low (8%). A total of 117 ceftiofur-resistant E. coli isolates (96%) possessed the bla(CMY2) gene. Based on phenotypic and genotypic characterization, the ceftiofur-resistant E. coli isolates belonged to 59 clonal types. There was no significant relationship between calf age and clonal type. The findings of this study revealed that healthy dairy calves were rapidly colonized by antibiotic-resistant strains of E. coli shortly after birth. The high prevalence of multidrug-resistant nonpathogenic E. coli in calves could be a significant source of resistance genes to other bacteria that share the same environment.     

Antimicrobial resistance of Escherichia coli isolates from broiler chickens and humans

Background

Inguinal hernias are usually caused by a congenital defect, which occurs as a weakness of the inguinal canal. Porcine β-glucuronidase gene (GUSB) was chosen as functional candidate gene because of its involvement in degradation of hyaluronan within gubernacular tissue during descent of testes. Since a genome-wide linkage analysis approach has shown evidence that two regions on porcine chromosome 3 (SSC 3) are involved in the inheritance of hernia inguinalis/scrotalis in German pig breeds, GUSB also attained status as a positional candidate gene by its localization within a hernia-associated chromosomal region.

Results

A contig spanning 17,157 bp, which contains the entire GUSB, was assembled. Comparative sequence analyses were conducted for the GUSB gene locus. Single nucleotide polymorphisms (SNPs) located within the coding region of GUSB were genotyped in 512 animals. Results of transmission disequilibrium test (TDT) for two out of a total of five detected SNPs gave no significant association with the outcome of hernia in pigs.

Conclusion

On the basis of our studies we are able to exclude the two analyzed SNPs within the porcine GUSB gene as causative for the transmission of inguinal hernia.     

Longitudinal study of Finnish Campylobacter jejuni and C. coli isolates from humans, using multilocus sequence typing, including comparison with epidemiological data and isolates from poultry and cattle

We describe a study on the application of multilocus sequence typing for the analysis of Campylobacter jejuni and C. coli isolates from human domestically acquired infections in the Helsinki-Uusimaa area of Finland in 1996, 2002, and 2003. In addition, isolates from poultry meat and fecal samples of cattle from the seasonal peak (July to September) in 2003 were included in the study. In total, 361 Finnish C. jejuni and C. coli strains were typed. Sequence type 45 (ST-45) (45%), ST-21 (21%), and ST-677 (11%) clonal complexes were the most prevalent. The ST-45 and ST-677 complexes were overrepresented in comparison with previous studies. The longitudinal study revealed an association between C. coli (ST-828 complex) infection and elderly patients (>/=60 years). Analysis of exposure factors, determined by a previous case-control study conducted during the seasonal peak in 2002, revealed that the ST-48 complex was significantly (P < 0.05) associated with the tasting or eating of raw minced meat. New and unassigned STs were associated with swimming in natural bodies of water, whereas the ST-677 complex was related to drinking nonchlorinated water from a small water plant or water from natural sources. The ST-45 complex was associated with contact with pet cats and dogs. In 2003, ST-45 occurrence was significantly associated with poultry whereas ST-50 was associated with isolates from humans. In contrast, ST-53, ST-58, ST-61, and ST-883 were significantly associated with isolates from cattle. Further studies are needed to reveal the significance of the observed associations.     

Antimicrobial resistance and virulence genes of Escherichia coli isolates from swine in Ontario

A total of 318 Escherichia coli isolates obtained from diarrheic and healthy pigs in Ontario from 2001 to 2003 were examined for their susceptibility to 19 antimicrobial agents. They were tested by PCR for the presence of resistance genes for tetracycline, streptomycin, sulfonamides, and apramycin and of 12 common virulence genes of porcine E. coli. Antimicrobial resistance frequency among E. coli isolates from swine in Ontario was moderate in comparison with other countries and was higher in isolates from pigs with diarrhea than in isolates from healthy finisher pigs. Resistance profiles suggest that cephamycinases may be produced by > or = 8% of enterotoxigenic E. coli (ETEC). Resistance to quinolones was detected only in enterotoxigenic E. coli (< or = 3%). The presence of sul3 was demonstrated for the first time in Canada in porcine E. coli isolates. Associations were observed among tetA, sul1, aadA, and aac(3)IV and among tetB, sul2, and strA/strB, with a strong negative association between tetA and tetB. The paa and sepA genes were detected in 92% of porcine ETEC, and strong statistical associations due to colocation on a large plasmid were observed between tetA, estA, paa, and sepA. Due at least in part to gene linkages, the distribution of resistance genes was very different between ETEC isolates and other porcine E. coli isolates. This demonstrates that antimicrobial resistance epidemiology differs significantly between pathogenic and commensal E. coli isolates. These results may have important implications with regards to the spread and persistence of resistance and virulence genes in bacterial populations and to the prudent use of antimicrobial agents.     

Extended-spectrum beta-lactamase CTX-M-1 in Escherichia coli isolates from healthy poultry in France

Genes encoding extended-spectrum beta-lactamase CTX-M-1 were detected in 12 Escherichia coli isolates recovered over a 7-month period from the ceca of healthy poultry in seven districts in France in 2005. Eleven of those strains were not clonally related and had a bla(CTX-M-1) gene located on transferable plasmids of different sizes and structures.     

Molecular characterization of multidrug-resistant Escherichia coli isolates from Irish cattle farms

This study describes the genotypic characteristics of a collection of 100 multidrug-resistant (MDR) Escherichia coli strains recovered from cattle and the farm environment in Ireland in 2007. The most prevalent antimicrobial resistance identified was to streptomycin (100%), followed by tetracycline (99%), sulfonamides (98%), ampicillin (82%), and neomycin (62%). Resistance was mediated predominantly by strA-strB (92%), tetA (67%), sul2 (90%), bla(TEM) (79%), and aphA1 (63%) gene markers, respectively. Twenty-seven isolates harbored a class 1 integrase (intI1), while qacEΔ1 and sul1 markers were identified in 25 and 26 isolates, respectively. The variable regions of these integrons contained aminoglycoside, trimethoprim, and β-lactam resistance determinants (aadA12, aadB-aadA1, bla(OXA-30)-aadA1, dfrA1-aadA1, dfrA7). Class 2 integrons were identified less frequently (4%) and contained the gene cassette array dfrA1-sat1-aadA1. Resistance to ampicillin, neomycin, streptomycin, sulfonamide, and tetracycline was associated with transferable high-molecular-weight plasmids, as demonstrated by conjugation assays. A panel of virulence markers was screened for by PCR, and genes identified included vt1, K5 in 2 isolates, papC in 10 isolates, and PAI IV(536) in 37 isolates. MDR commensal E. coli isolates from Irish cattle displayed considerable diversity with respect to the genes identified. Our findings highlight the importance of the commensal microflora of food-producing animals as a reservoir of transferable MDR.     

Prevalence, antibiotic susceptibility, and diversity of Escherichia coli O157:H7 isolates from a longitudinal study of beef cattle feedlots

Prevalence, antibiotic susceptibility, and genetic diversity were determined for Escherichia coli O157:H7 isolated over 11 months from four beef cattle feedlots in southwest Kansas. From the fecal pat (17,050) and environmental (7,134) samples collected, 57 isolates of E. coli O157:H7 were identified by use of bacterial culture and latex agglutination (C/LA). PCR showed that 26 isolates were eaeA gene positive. Escherichia coli O157:H7 was identified in at least one of the four feedlots in 14 of the 16 collections by C/LA and in 9 of 16 collections by PCR, but consecutive positive collections at a single feedlot were rare. Overall prevalence in fecal pat samples was low (0.26% by C/LA, and 0.08% by PCR). No detectable differences in prevalence or antibiotic resistance were found between isolates collected from home pens and those from hospital pens, where antibiotic use is high. Resistant isolates were found for six of the eight antibiotics that could be used to treat E. coli infections in food animals, but few isolates were multidrug resistant. The high diversity of isolates as measured by random amplification of polymorphic DNA and other characteristics indicates that the majority of isolates were unique and did not persist at a feedlot, but probably originated from incoming cattle. The most surprising finding was the low frequency of virulence markers among E. coli isolates identified initially by C/LA as E. coli O157:H7. These results demonstrate that better ways of screening and confirming E. coli O157:H7 isolates are required for accurate determination of prevalence.     

Multiple antibiotic resistance patterns of Escherichia coli isolates from swine farms.

Antibiotic resistance of Escherichia coli from sows and pigs was determined to compare patterns between pigs of various ages and degrees of antibiotic use. Resistance patterns differed between farm types and pigs of differing ages, indicating that pig age and degree of antibiotic use affect resistance of fecal E. coli.     

Potential pathogenicity and host range of extended-spectrum beta-lactamase-producing Escherichia coli isolates from healthy poultry

Thirty of 33 epidemiologically unrelated extended-spectrum β-lactamase (ESBL)-producing Escherichia coli isolates from healthy poultry lacked the virulence genes commonly associated with human-pathogenic strains. The main zoonotic risk is associated with the broad host range of avian E. coli belonging to sequence type complex 10 and of IncN and IncI1 plasmids carrying bla(CTX-M) or bla(SHV).     

High-level genotypic variation and antibiotic sensitivity among Escherichia coli O157 strains isolated from two Scottish beef cattle farms

Escherichia coli O157:H7 is a human pathogen that is carried and transmitted by cattle. Scotland is known to have one of the highest rates of E. coli O157 human infections in the world. Two hundred ninety-three isolates were obtained from naturally infected cattle and the environment on two farms in the Scottish Highlands. The isolates were typed by pulsed-field gel electrophoresis (PFGE) with XbaI restriction endonuclease enzyme, and 19 different variations in patterns were found. There was considerable genomic diversity within the E. coli O157 population on the two farms. The PFGE pattern of one of the observed subtypes matched exactly with that of a strain obtained from a Scottish patient with hemolytic-uremic syndrome. To examine the stability of an individual E. coli O157 strain, continuous subculturing of a strain was performed 110 times. No variation from the original PFGE pattern was observed. We found three indistinguishable subtypes of E. coli O157 on both study farms, suggesting common sources of infection. We also examined the antibiotic resistance of the isolated strains. Phenotypic studies demonstrated resistance of the strains to sulfamethoxazole (100%), chloramphenicol (3.07%), and at a lower rate, other antibiotics, indicating the preservation of antibiotic sensitivity in a rapidly changing population of E. coli O157.     

Speciating Campylobacter jejuni and Campylobacter coli isolates from poultry and humans using six PCR-based assays

Six previously published polymerase chain reaction (PCR) assays each targeting different genes were used to speciate 116 isolates previously identified as Campylobacter jejuni using routine microbiological techniques. Of the 116 isolates, 84 were of poultry origin and 32 of human origin. The six PCR assays confirmed the species identities of 31 of 32 (97%) human isolates and 56 of 84 (67%) poultry isolates as C. jejuni. Twenty eight of 84 (33%) poultry isolates were identified as Campylobacter coli and the remaining human isolate was tentatively identified as Campylobacter upsaliensis based on the degree of similarity of 16S rRNA gene sequences. Four of six published PCR assays showed 100% concordance in their ability to speciate 113 of the 116 (97.4%) isolates; two assays failed to generate a PCR product with four to 10 isolates. A C. coli-specific PCR identified all 28 hippuricase gene (hipO)-negative poultry isolates as C. coli although three isolates confirmed to be C. jejuni by the remaining five assays were also positive in this assay. A PCR-restriction fragment length polymorphism assay based on the 16S rRNA gene was developed, which contrary to the results of the six PCR-based assays, identified 28 of 29 hipO-negative isolates as C. jejuni. DNA sequence analysis of 16S rRNA genes from four hipO-negative poultry isolates showed they were almost identical to the C. jejuni type strain 16S rRNA sequences ATCC43431 and ATCC33560 indicating that assays reliant on 16S rRNA sequence may not be suitable for the differentiation of these two species.     

Comparison of genomic profiles of Escherichia coli isolates from urinary tract infections

Formally included in the larger category of extraintestinal pathogenic Escherichia coli (ExPEC), the uropathogenic E. coli remains the most frequent cause of urinary tract infection (UTI), an important endemic health problem. The genomic DNA of E. coli urinary isolates from adults diagnosed with urinary tract infections and of E. coli fecal isolates from healthy subjects was analysed by PCR for the presence of virulence factor encoding genes pap, sfa/foc, afa, hly and cnf and by field inversion gel electrophoresis (FIGE) fingerprinting of XbaI DNA macrorestriction fragments. The aim was to obtain more detailed microbiological data regarding the community circulating strains in respect of their virulence potential and genetic relatedness. Almost 70% of the urinary strains carried at least one of the target virulence genes, and only 35.5% of the fecal E. coli strains were positive in the PCR screening. Taking into account the virulence genotypes exhibited, a part of the strains isolated from the urinary tract could be defined as belonging to the ExPEC pathotype. A unique FIGE profile was obtained for each of the selected isolates and the dendrogram generated by Taxotron software package analysis suggested a polyclonal population of potential uropathogenic strains clustered into 14 groups of only 60% similarity. For better understanding the epidemiology of UTIs, diseases commonly caused by such a heterogeneous species like E. coli, molecular analysis methods could be essential due to their increased power of identification and fingerprinting.