Transfer of antibiotic resistance between commensal and pathogenic members of the Enterobacteriaceae under ileal conditions

AIM: To determine the rate of antibiotic resistance transmission between commensal and pathogenic representatives of the Enterobacteriaceae. METHODS AND RESULTS: Through the use of a validated in vitro simulation of the porcine ileum, the transmission of antibiotic resistance was detected between commensal Escherichia coli, E. coli O157 and Salmonella spp. Countable transconjugant populations arose readily and, in one example, proved capable of indefinite persistence. CONCLUSIONS: Genetic material conferring antibiotic resistance is readily transmissible between members of the Enterobacteriaceae under ileal conditions. Recipient phenotype influences the persistence of multi-resistant transconjugants. SIGNIFICANCE AND IMPACT OF THE STUDY: The observation that the conjugal transmission of antibiotic resistance is commonplace under ileal conditions impacts primarily on the risk of food contamination by multi-resistant bacteria. The establishment of a multi-resistant transconjugant population as a dominant member of the microflora maintains a genetic reservoir of antimicrobial resistance.     

Occurrence, virulence characteristics and antimicrobial resistance of Escherichia coli O157 in slaughtered cattle and diarrhoeic calves in West Bengal, India

AIMS: (i) To study the occurrence of Escherichia coli serotype O157 in cattle stool in West Bengal, India, and (ii) the virulence properties and antimicrobial resistance of the E. coli isolates. METHODS AND RESULTS: Following enrichment in modified EC broth and plating onto HiCrome MS.O157 agar, a total of 14 strains of E. coli serotype O157 was isolated from faecal samples from two (2.04%) slaughtered cattle and six (7.59%) diarrhoeic calves. By multiplex PCR, Shiga toxin genes were detected in all the isolates. The enterohaemolysin phenotype was found in all, but one strain. Among 14 strains, ten were resistant to at least one of the antimicrobial agents tested. Multiple antibiotic resistance was frequent. CONCLUSIONS: The study showed that occurrence of Shiga toxin-producing and multiple antibiotic-resistant E. coli O157 among cattle population in this region of India is significant. SIGNIFICANCE AND IMPACT OF THE STUDY: Considering routine human contacts with cattle, a large human population in this region may be at risk for exposure to Shiga toxin-producing E. coli O157.     

Antibiotic resistant Escherichia coli and Salmonella in Russian rooks (Corvus frugilegus) wintering in the Czech Republic

AIMS: To characterize antibiotic resistant Escherichia coli and Salmonella isolates in rooks wintering in the Czech Republic. METHODS AND RESULTS: Three hundred and sixty-three faeces samples from rooks were examined for antibiotic resistant Escherichia coli and Salmonella. Altogether 13.7%E. coli isolates were resistant to antimicrobial agents tested. The dominant type of resistance was to tetracycline. Resistant E. coli isolates were examined for antibiotic resistance genes and class 1 integrons. Five of 29 antibiotic resistant isolates possessed the int1 gene. Nine Salmonella isolates (2.5%) were found in rook faeces. All the isolates belonged to serotype Salmonella enterica serovar Enteritidis phage type PT8 and PT23. CONCLUSIONS: The study suggests that rooks can be infected by antibiotic resistant E. coli and Salmonella isolates, probably reflecting the presence of such isolates in their sources of food and/or water in the environment. SIGNIFICANCE AND IMPACT OF THE STUDY: Rooks can serve as reservoirs and vectors of antibiotic resistant E. coli and Salmonella isolates and potentially transmit these isolates over long distances.     

Abundance and characteristics of the recreational water quality indicator bacteria Escherichia coli and enterococci in gull faeces

AIMS: To evaluate the numbers and selected phenotypic and genotypic characteristics of the faecal indicator bacteria Escherichia coli and enterococci in gull faeces at representative Great Lakes swimming beaches in the United States. METHODS AND RESULTS: E. coli and enterococci were enumerated in gull faeces by membrane filtration. E. coli genotypes (rep-PCR genomic profiles) and E. coli (Vitek GNI+) and enterococci (API rapid ID 32 Strep and resistance to streptomycin, gentamicin, vancomycin, tetracycline and ampicillin) phenotypes were determined for isolates obtained from gull faeces both early and late in the swimming season. Identical E. coli genotypes were obtained only from single gull faecal samples but most faecal samples yielded more than one genotype (median of eight genotypes for samples with 10 isolates). E. coli isolates from the same site that clustered at >/=85% similarity were from the same sampling date and shared phenotypic characteristics, and at this similarity level there was population overlap between the two geographically isolated beach sites. Enterococcus API(R) profiles varied with sampling date. Gull enterococci displayed wide variation in antibiotic resistance patterns, and high-level resistance to some antibiotics. CONCLUSIONS: Gull faeces could be a major contributor of E. coli (10(5)-10(9) CFU g(-1)) and enterococci (10(4)-10(8) CFU g(-)1) to Great Lakes recreational waters. E. coli and enterococci in gull faeces are highly variable with respect to their genotypic and phenotypic characteristics and may exhibit temporal or geographic trends in these features. SIGNIFICANCE AND IMPACT OF THE STUDY: The high degree of variation in genotypic or phenotypic characteristics of E. coli or enterococci populations within gull hosts will require extensive sampling for adequate characterization, and will influence methods that use these characteristics to determine faecal contamination sources for recreational waters.     

Effect of the growth promoter avilamycin on emergence and persistence of antimicrobial resistance in enteric bacteria in the pig

AIM: To assess the effect of the growth promoter avilamycin on emergence and persistence of resistance in enteric bacteria in the pig. METHODS AND RESULTS: Pigs (treated with avilamycin for 3 months and controls) were challenged with multi-resistant Salmonella Typhimurium DT104 and faecal counts were performed for enterococci, Escherichia coli, S. Typhimurium and Campylobacter (before, during and 5 weeks post-treatment). Representative isolates were tested for antibiotic resistance and for the presence of resistance genes. Avilamycin-resistant Enterococci faecalis (speciated by PCR) were isolated from the treated pigs and continued to be detected for the first week after treatment had ceased. The avilamycin-resistance gene was characterized by PCR as the emtA gene and speciation by PCR. MIC profiling confirmed that more than one strain of Ent. faecalis carried this gene. There was no evidence of increased antimicrobial resistance in the E. coli, Salmonella and Campylobacter populations, although there was a higher incidence of tetB positive E. coli in the treated pigs than the controls. CONCLUSION: Although avilamycin selects for resistance in the native enterococci population of the pig, no resistant isolates were detected beyond 1 week post-treatment. This suggests that resistant isolates were unable to persist once selective pressure was removed and were out-competed by the sensitive microflora. SIGNIFICANCE AND IMPACT OF THE STUDY: Our data suggest the risk of resistant isolates becoming carcass contaminants and infecting humans could be minimized by introducing a withdrawal period after using avilamycin and prior to slaughter.     

Effect of subtherapeutic concentrations of tylosin on the inhibitory stringency of a mixed anaerobe continuous-flow culture of chicken microflora against Escherichia coli O157:H7

AIMS: The aim of this study was twofold: first to determine the effect of subtherapeutic concentrations of tylosin, a macrolide antibiotic used for growth promotion, on a mixed anaerobic continuous-flow fermentation culture of chicken gastrointestinal microorganisms (CCF) and secondly, to determine if these concentrations would allow persistence of Escherichia coli O157:H7 in CCF. METHODS AND RESULTS: CCF was treated with tylosin at 10.0, 20.0 and 40.0 microg ml(-1). Tylosin treatment resulted in a significant (P < 0.0001) decrease in total volatile fatty acids (VFAs) from a mean concentration of 101 +/- 10.8 micromol ml(-1) in control cultures to 32.0 +/- 6.3 and 40.2 +/- 9.6 micromol ml(-1) in 10 and 40 microg ml(-1) treated cultures, respectively. Untreated CCF challenged with E. coli O157:H7 cleared the challenge microorganism in 7 days at a rate of 0.96 log10 CFU ml(-1) day(-1). In contrast, E. coli O157:H7 persisted in all tylosin treated cultures. CONCLUSIONS: In the presence of tylosin, E. coli O157:H7 was able to persist in the CCF culture. The significant decrease in the production of VFAs may have been a contributing factor. SIGNIFICANCE AND IMPACT OF THE STUDY: The use of low-level, growth-promoting antimicrobials may compromise the ability of normal microflora that serve as a natural host defence against infection.     

Differentiation of faecal Escherichia coli from humans and animals by multiple antibiotic resistance analysis

AIMS: Multiple antibiotic resistance (MAR) was performed on 128 Escherichia coli isolates, recovered from faecal samples of humans and animals (cattle, goat, sheep) to determine and compare their antibiotic resistance patterns and to evaluate them statistically in order to specify the source of the faecal material. METHODS AND RESULTS: Disk diffusion method was applied with a selection of antibiotics. Statistical approach was performed with hierarchical cluster analysis (CA), discriminant analysis (DA) and principal component analysis. Comparing human and animal isolates there was significant difference in levels of resistance to all antibiotics tested (P<0.05) with 46 and 24 distinct resistance patterns for human and animal isolates respectively. CA and DA separated human and animal isolates with a high average rate of correct classification (99.2%), when all animal isolates were pooled together. CONCLUSIONS: MAR analysis compared with appropriate statistical evaluation may provide a useful tool for differentiating the human or animal origin of E. coli isolates derived from environmental samples. Subsequently, determination of the source of faecal pollution becomes possible. SIGNIFICANCE AND IMPACT OF THE STUDY: Determining the source of faecal pollution enables the prediction of possible risk for public health and the application of appropriate management plans for prevention of further contamination.     

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.     

Variation in resistance to high hydrostatic pressure and rpoS heterogeneity in natural isolates of Escherichia coli O157:H7

Several natural isolates of Escherichia coli O157:H7 have previously been shown to exhibit stationary-phase-dependent variation in their resistance to inactivation by high hydrostatic pressure. In this report we demonstrate that loss of the stationary-phase-inducible sigma factor RpoS resulted in decreased resistance to pressure in E. coli O157:H7 and in a commensal strain. Furthermore, variation in the RpoS activity of the natural isolates of O157:H7 correlated with the pressure resistance of those strains. Heterogeneity was noted in the rpoS alleles of the natural isolates that may explain the differences in RpoS activity. These results are consistent with a role for rpoS in mediating resistance to high hydrostatic pressure in E. coli O157:H7.     

Temporal dynamics and impact of manure storage on antibiotic resistance patterns and population structure of Escherichia coli isolates from a commercial swine farm

Many confined-livestock farms store their wastes for several months prior to use as a fertilizer. Storing manure for extended periods could significantly bias the composition of enteric bacterial populations subsequently released into the environment. Here, we compared populations of Escherichia coli isolated from fresh feces and from the manure-holding tank (stored manure) of a commercial swine farm, each sampled monthly for 6 months. The 4,668 confirmed E. coli isolates were evaluated for resistance to amikacin, ampicillin, cephalothin, chloramphenicol, kanamycin, nalidixic acid, streptomycin, sulfamethoxazole, tetracycline, trimethoprim, and trimethoprim plus sulfamethoxazole. A subset of 1,687 isolates was fingerprinted by repetitive extragenic palindromic PCR (rep-PCR) with the BOXA1R primer to evaluate the diversity and the population structure of the collection. The population in the stored manure was generally more diverse than that in the fresh feces. Half of the genotypes detected in the stored manure were never detected in the fresh fecal material, and only 16% were detected only in the fresh feces. But the majority of the isolates (84%) were assigned to the 34% of genotypes shared between the two environments. The structure of the E. coli population showed important monthly variations both in the extent and distribution of the diversity of the observed genotypes. The frequency of detection of resistance to specific antibiotics was not significantly different between the two collections and varied importantly between monthly samples. Resistance to multiple antibiotics was much more temporally dynamic in the fresh feces than in the stored manure. There was no relationship between the distribution of rep-PCR fingerprints and the distribution of antibiotic resistance profiles, suggesting that specific antibiotic resistance determinants were dynamically distributed within the population.     

rpoS Mutations and Loss of General Stress Resistance in Escherichia coli Populations as a Consequence of Conflict between Competing Stress Responses

The general stress resistance of Escherichia coli is controlled by the RpoS sigma factor (phi(S)), but mutations in rpoS are surprisingly common in natural and laboratory populations. Evidence for the selective advantage of losing rpoS was obtained from experiments with nutrient-limited bacteria at different growth rates. Wild-type bacteria were rapidly displaced by rpoS mutants in both glucose- and nitrogen-limited chemostat populations. Nutrient limitation led to selection and sweeps of rpoS null mutations and loss of general stress resistance. The rate of takeover by rpoS mutants was most rapid (within 10 generations of culture) in slower-growing populations that initially express higher phi(S) levels. Competition for core RNA polymerase is the likeliest explanation for reduced expression from distinct promoters dependent on phi(70) and involved in the hunger response to nutrient limitation. Indeed, the mutation of rpoS led to significantly higher expression of genes contributing to the high-affinity glucose scavenging system required for the hunger response. Hence, rpoS polymorphism in E. coli populations may be viewed as the result of competition between the hunger response, which requires sigma factors other than phi(S) for expression, and the maintenance of the ability to withstand external stresses. The extent of external stress significantly influences the spread of rpoS mutations. When acid stress was simultaneously applied to glucose-limited cultures, both the phenotype and frequency of rpoS mutations were attenuated in line with the level of stress. The conflict between the hunger response and maintenance of stress resistance is a potential weakness in bacterial regulation.     

Genotypic-phenotypic discrepancies between antibiotic resistance characteristics of Escherichia coli isolates from calves in management settings with high and low antibiotic use

We hypothesized that bacterial populations growing in the absence of antibiotics will accumulate more resistance gene mutations than bacterial populations growing in the presence of antibiotics. If this is so, the prevalence of dysfunctional resistance genes (resistance pseudogenes) could provide a measure of the level of antibiotic exposure present in a given environment. As a proof-of-concept test, we assayed field strains of Escherichia coli for their resistance genotypes using a resistance gene microarray and further characterized isolates that had resistance phenotype-genotype discrepancies. We found a small but significant association between the prevalence of isolates with resistance pseudogenes and the lower antibiotic use environment of a beef cow-calf operation versus a higher antibiotic use dairy calf ranch (Fisher's exact test, P = 0.044). Other significant findings include a very strong association between the dairy calf ranch isolates and phenotypes unexplained by well-known resistance genes (Fisher's exact test, P < 0.0001). Two novel resistance genes were discovered in E. coli isolates from the dairy calf ranch, one associated with resistance to aminoglycosides and one associated with resistance to trimethoprim. In addition, isolates resistant to expanded-spectrum cephalosporins but negative for bla(CMY-2) had mutations in the promoter regions of the chromosomal E. coli ampC gene consistent with reported overexpression of native AmpC beta-lactamase. Similar mutations in hospital E. coli isolates have been reported worldwide. Prevalence or rates of E. coli ampC promoter mutations may be used as a marker for high expanded-spectrum cephalosporin use environments.     

Anaerobic transfer of antibiotic resistance from Pseudomonas aeruginosa

Pseudomonas aeruginosa, an opportunistic pathogen that often initiates infections from a reservoir in the intestinal tract, may donate or acquire antibiotic resistance in an anaerobic environment. Only by including nitrate and nitrite in media could antibiotic-resistant and -sensitive strains of P. aeruginosa be cultured in a glove box isolator. These anaerobically grown cells remained sensitive to lytic phage isolated from sewage. After incubation with a phage lysate derived from P. aeruginosa 1822, anaerobic transfer of antibiotic resistance to recipients P. aeruginosa PS8EtBr and PS8EtBrR occurred at frequencies of 6.2 x 10(-9) and 5.0 x 10(-8) cells per plaque-forming unit, respectively. In experiments performed outside the isolator, transfer frequencies to PS8EtBr and PS8EtBrR were higher, 1.3 x 10(-7) and 6.5 x 10(-8) cells per plaque-forming unit, respectively. When P. aeruginosa 1822 was incubated aerobically with Escherichia coli B in medium containing nitrate and nitrite, the maximum concentration of carbenicillin-resistant E. coli B reached 25% of the total E. coli B population. This percentage declined to 0.01% of the total E. coli B population when anaerobically grown P. aeruginosa 1822 and E. coli B were combined and incubated in the glove box isolator. The highest concentration of the recipient population converted to antibiotic resistance occurred after 24 h of aerobic incubation, when an initially high donor/recipient ratio (>15) of cells was mixed. These data indicate that transfer of antibiotic resistance either by transduction between Pseudomonas spp. or by conjugation between Pseudomonas sp. and E. coli occurs under strict anaerobic conditions, although at lower frequencies than under aerobic conditions.     

Adaptation to flavomycin in the ruminal bacterium, Prevotella bryantii

Aims:  The recent EU ban of growth-promoting antibiotics in animal production was based on fears concerning antibiotic resistance being transmitted to human pathogens. This paper explores the adaptation mechanism of a common ruminal bacterium, Prevotella bryantii , to one of the banned compounds, flavomycin (flavophospholipol).
Methods and Results:  Growth in the presence of flavomycin (2 and 20  μ g ml⁻¹) was characterized by a concentration-dependent increase in the length of the lag phase, which decreased after previous flavomycin exposure. From growth patterns on solid medium, decreased sensitivity appeared to be due to a whole-population adaptation. Proteomic analysis indicated upregulation of three native proteins occurred following flavomycin adaptation. Further analysis of two of these proteins resulted in no database matches, suggesting that they may be species-specific. Flavomycin adaptation also resulted in co-adaptation to bacitracin and vancomycin.
Conclusions:  Adaptation of P. bryantii to flavomycin, which also resulted in co-adaptation to bacitracin and vancomycin, may involve an increased availability of undecaprenyl pyrophosphate.
Significance and Impact of the Study:  The use of flavomycin, and similar growth-promoting antibiotics, in animal production may prompt adaptive responses in ruminal bacteria which can significantly change their antibiotic sensitivity.     

Influence of antibiotic selection on genetic composition of Escherichia coli populations from conventional and organic dairy farms

The widespread agricultural use of antimicrobials has long been considered a crucial influence on the prevalence of resistant genes and bacterial strains. It has been suggested that antibiotic applications in agricultural settings are a driving force for the development of antimicrobial resistance, and epidemiologic evidence supports the view that there is a direct link between resistant human pathogens, retail produce, farm animals, and farm environments. Despite such concerns, little is understood about the population processes underlying the emergence and spread of antibiotic resistance and the reversibility of resistance when antibiotic selective pressure is removed. In this study, hierarchical log-linear modeling was used to assess the association between farm type (conventional versus organic), age of cattle (calf versus cow), bacterial phenotype (resistant versus susceptible), and the genetic composition of Escherichia coli populations (E. coli Reference Collection [ECOR] phylogroup A, B1, B2, or D) among 678 susceptible and resistant strains from a previously published study of 60 matched dairy farms (30 conventional and 30 organic) in Wisconsin. The analysis provides evidence for clonal resistance (ampicillin resistance) and genetic hitchhiking (tetracycline resistance [Tet(r)]), estimated the rate of compositional change from conventional farming to organic farming (mean, 8 years; range, 3 to 15 years), and discovered a significant association between low multidrug resistance, organic farms, and strains of the numerically dominant phylogroup B1. These data suggest that organic farming practices not only change the frequency of resistant strains but also impact the overall population genetic composition of the resident E. coli flora. In addition, the results support the hypothesis that the current prevalence of Tet(r) loci on dairy farms has little to do with the use of this antibiotic.     

Escherichia coli O157:H7 becomes resistant to sodium chlorate in pure culture, but not in mixed culture or in vivo

AIMS: Chlorate kills Escherichia coli O157:H7 and may be an effective feed additive for use in food animals. This study was designed to determine if development of chlorate-resistant E. coli O157:H7 strains was likely. METHODS AND RESULTS: Escherichia coli O157:H7 was chlorate-sensitive, but became chlorate-resistant in pure batch culture and in sterilized faecal fluid; it was killed in mixed culture and did not become resistant. Addition of chlorate to continuous pure cultures caused chlorate resistance, but chlorate addition to mixed continuous culture eliminated E. coli O157:H7 and no resistance occurred. Piglets challenged with E. coli O157:H7 were treated with chlorate; populations were reduced and colonies were always chlorate-sensitive. CONCLUSION: Chlorate-resistant E. coli O157:H7 can be selected in pure, but not mixed culture, and results suggest that terminal chlorate feeding will not select for chlorate-resistance in vivo. SIGNIFICANCE AND IMPACT OF THE STUDY: Chlorate can reduce food-borne pathogens prior to harvest, but development of resistance does not appear likely in vivo.     

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.     

Cross-resistance to antibiotics of Escherichia coli adapted to benzalkonium chloride or exposed to stress-inducers

AIMS: To study the effects of adaptation and stress on the resistance to benzalkonium chloride (BC) and cross-resistance to antibiotics in Escherichia coli. METHODS AND RESULTS: Precultivation of E. coli ATCC 11775 and E. coli DSM 682 in the presence of subinhibitory concentrations of BC or stress inducers (salicylate, chenodeoxycholate and methyl viologen) resulted in higher minimum inhibitory concentration (MIC) of BC and chloramphenicol (CHL). Adaptation to growth in sixfold of the initial MIC of BC resulted in stable BC resistance and enhanced tolerance to several antibiotics and ethidium bromide (EtBr). The MIC of CHL increased more than 10-fold for both strains. Enhanced efflux of EtBr in adapted E. coli ATCC 11775 indicated that the observed resistance was due to efflux. Changes in outer membrane protein profiles were detected in the BC-adapted cells. There were no indications of lower membrane permeability to BC. CONCLUSIONS: Induction of stress response or gradual adaptation to BC or CHL results in acquired cross-tolerance between BC and antibiotics in E. coli. Enhanced efflux was one of the observed differences in adapted cells. SIGNIFICANCE AND IMPACT OF THE STUDY: Provided not taking due precautions, extensive use of disinfectants could lead to emergence of antibiotic-resistant isolates.