Selection and transfer of an IncI1-tet(A) plasmid of Escherichia coli in an ex vivo model of the porcine caecum at doxycycline concentrations caused by crosscontaminated feed.

AIMS: The aim of this study was to investigate the effect of subtherapeutic intestinal doxycycline (DOX) concentrations (4 and 1 mg l-1 ), caused by cross-contamination of feed, on the enrichment of a DOX-resistant commensal Escherichia coli and its resistance plasmid in an ex vivo model of the porcine caecum. METHODS AND RESULTS: A DOX-resistant, tet(A)-carrying, porcine commensal E. coli strain (EC 682) was cultivated for 6 days in the porcine caecum model under different conditions (0, 1 and 4 mg l-1 DOX). EC 682, other coliforms and anaerobic bacteria were enumerated daily. A selection of isolated DOX-resistant coliforms (n = 454) was characterized by rep-PCR clustering, PCR assays (Inc1 and tet(A)) and micro broth dilution susceptibility tests (Sensititre). Both 1 and 4 mg l-1 DOX-enriched medium had a significantly higher selective effect on EC 682 and other resistant coliforms than medium without DOX. Transconjugants of EC 682 were isolated more frequently in the presence of 1 and 4 mg l-1 DOX compared to medium without DOX. CONCLUSIONS: Subtherapeutic intestinal DOX concentrations have the potential to select for DOX-resistant E. coli, and promote the selection of transconjugants in a porcine caecum model. SIGNIFICANCE AND IMPACT OF THE STUDY: Cross-contamination of feed with antimicrobials such as DOX likely promotes the spread of antimicrobial resistance. Therefore, it is important to develop or fine-tune guidelines for the safe use of antimicrobials in animal feed and its storage.

Identification of a novel plasmid-associated spectinomycin adenyltransferase gene spd in methicillin-resistant Staphylococcus aureus ST398 isolated from animal and human sources.

OBJECTIVES: Previously described methicillin-resistant Staphylococcus aureus (MRSA) ST398 strains revealed a high frequency of phenotypic resistance to spectinomycin. However, only a few were found to carry the spc resistance determinant. The aim of this study was to identify the genetic mechanism of spectinomycin resistance among spc-negative MRSA ST398 strains. METHODS: Nine spectinomycin-resistant, but spc-negative, MRSA ST398 strains were analysed. The strains were screened for carriage of the spw gene and tested for the presence of transferrable spectinomycin resistance. Plasmid DNA was isolated from all strains and used in transformation assays. The plasmid identified as mediating resistance to spectinomycin was fully sequenced. The function of the novel spectinomycin resistance gene was confirmed by restriction digest inactivation and its distribution was determined using a PCR assay. RESULTS: A single MRSA ST398 strain was spw positive. The remaining strains carried a plasmid that mediated resistance to spectinomycin. Sequence analysis of a single plasmid, termed pDJ91S, revealed that it was 3928 bp in size and contained three open reading frames: a novel spectinomycin resistance gene, designated spd, as well as a repN gene and a rec gene. The XmnI digest inactivation of the spd gene resulted in a 4-fold decrease in spectinomycin MIC. The spd gene was detected in seven other spectinomycin-resistant MRSA ST398 strains that carried a plasmid comparable in size to pDJ91S. CONCLUSIONS: A novel gene, designated spd, that confers resistance to spectinomycin has been identified on a small plasmid in MRSA ST398.

High seroprevalence of respiratory pathogens in hobby poultry.

Seroprevalence studies on respiratory pathogens have been done extensively in commercial laying hens, broilers, and, to a lesser extent, backyard poultry. In Europe, seroprevalence studies in backyard and fancy breed poultry flocks are scarce and limited to a few pathogens, such as Mycoplasma gallisepticum (MG); others, such as Ornithobacterium rhinotracheale (ORT), are missing. A commercial ELISA for detection of antibodies against six selected pathogens was performed on 460 serum samples from chickens across Flanders. Anti-ORT antibodies were, by far, the most prevalent, with a prevalence of 95.4%. Infectious bronchitis virus, Mycoplasma synoviae, and avian metapneumovirus antibodies were found in 75.6%, 76.3%, and 63.5% of the animals, respectively. Antibodies against MG and infectious laryngotracheitis virus were found in 36.7% and 30% of the animals, respectively. These data demonstrate the high seroprevalence of respiratory pathogens among hobby poultry; therefore, it is possible that this group could act as a reservoir for commercially kept poultry.

Risk factors for ceftiofur resistance in Escherichia coli from Belgian broilers.

A cross-sectional study on 32 different Belgian broiler farms was performed in 2007 and 2008 to identify risk factors for ceftiofur resistance in Escherichia coli. On each farm, one E. coli colony was isolated from 30 random birds. Following susceptibility testing of 14 antimicrobials, an on-farm questionnaire was used to obtain information on risk factors. Using a multilevel logistic regression model two factors were identified at the animal level: resistance to amoxicillin and to trimethoprim-sulfonamide. On the farm level, besides antimicrobial use, seven management factors were found to be associated with the occurrence of ceftiofur resistance in E. coli from broilers: poor hygienic condition of the medicinal treatment reservoir, no acidification of drinking water, more than three feed changes during the production cycle, hatchery of origin, breed, litter material used, and treatment with amoxicillin. This study confirms that not only on-farm antimicrobial therapy, but also management- and hatchery-related factors influence the occurrence of antimicrobial resistance.

In situ ESBL conjugation from avian to human Escherichia coli during cefotaxime administration.

AIMS: The behaviour of an Escherichia coli isolate of broiler origin harbouring a bla(TEM-52) -carrying plasmid (lactose-negative mutant of B1-54, IncII group) was studied in an in situ continuous flow culture system, simulating the human caecum and the ascending colon during cefotaxime administration. METHODS AND RESULTS: Fresh faeces from a healthy volunteer, negative for cephalosporin-resistant E. coli, were selected to prepare inocula. The microbiota was monitored by plating on diverse selective media, and a shift in the populations of bacteria was examined by 16S rDNA PCR denaturing gradient gel electrophoresis. Escherichia coli transconjugants were verified by plasmid and pulsed-field gel electrophoresis profiles (PFGE). The avian extended-spectrum ß-lactamase-positive E. coli was able to proliferate without selective pressure of cefotaxime, and E. coli transconjugants of human origin were detected 24 h after inoculation of the donor strain. Upon administration of cefotaxime to the fresh medium, an increase in the population size of E. coli B1-54 and the transconjugants was observed. PFGE and plasmid analysis revealed a limited number of human E. coli clones receptive for the bla(TEM-52) -carrying plasmid. CONCLUSIONS: These observations provide evidence of the maintenance of an E. coli strain of poultry origin and the horizontal gene transfer in the human commensal bowel microbiota even without antimicrobial treatment. SIGNIFICANCE AND IMPACT OF THE STUDY: The fact that an E. coli strain of poultry origin might establish itself and transfer its bla gene to commensal human E. coli raises public health concerns.

Methicillin-resistant Staphylococcus aureus (MRSA) in food production animals.

Until recently, reports on methicillin-resistant Staphylococcus aureus (MRSA) in food production animals were mainly limited to occasional detections in dairy cattle mastitis. However, since 2005 a MRSA clone, CC398, has been reported colonizing pigs, veal calves and broiler chickens and infecting dairy cows. Many aspects of its prevalence in pigs remain unclear. In other livestock, colonizing capacity and reservoir status still require elucidation. MRSA CC398 has also been detected in meat, but, as for other MRSA, the risk this poses is somewhat unclear. Currently, the most worrying aspect of MRSA CC398 appears to be its capacity to spread to humans. This might complicate MRSA control measures in human healthcare, urging research into risk factors and transmission routes. Although infections with MRSA CC398 are much less reported than carriage, more investigation into its pathogenic potential is required. Moreover, the origin and evolution of this clone remain unknown.

Comparison of fingerprinting methods for typing methicillin-resistant Staphylococcus aureus sequence type 398.

This study evaluates the multiple-locus variable-number tandem-repeat assay (MLVA) and pulsed-field gel electrophoresis (PFGE) when using restriction enzymes BstZI, SacII, and ApaI to fingerprint a diverse collection of methicillin (meticillin)-resistant Staphylococcus aureus (MRSA) sequence type 398 (ST398) isolates. These isolates had been characterized previously by multilocus sequence typing, spa typing, and staphylococcal cassette chromosome mec (SCCmec) typing. Typeability and discriminatory power were analyzed, and the concordance between the various methods was determined. All MRSA ST398 isolates were typeable by the MLVA and PFGE using BstZI, SacII, and ApaI. With each method, the MRSA ST398 isolates formed a separate group from the two non-ST398 MRSA strains. PFGE, performed with any of the three restriction enzymes, had the most discriminatory power, followed by MLVA, spa typing, and SCCmec typing. The MLVA showed the highest concordance with PFGE using ApaI and spa typing. As further expressed by the Wallace coefficient, the MLVA type was poorly predicted by spa typing, whereas the spa type was well predicted by MLVA. PFGE, using a combination of all three restriction enzymes, had the highest concordance with the MLVA but had a low probability of being predicted by MLVA. PFGE, using a combination of all three restriction enzymes, was able to predict SCCmec type and MLVA type completely and had a high probability of predicting spa type. Both the MLVA and PFGE could be used to discriminate among the MRSA ST398 isolates. Although the MLVA is a faster technique, PFGE had more discriminatory power than the MLVA, especially when a combination of restriction enzymes was used.

Heavy metal resistance in bacteria from animals.

Resistance to metals and antimicrobials is a natural phenomenon that existed long before humans started to use these products for veterinary and human medicine. Bacteria carry diverse metal resistance genes, often harboured alongside antimicrobial resistance genes on plasmids or other mobile genetic elements. In this review we summarize the current knowledge about metal resistance genes in bacteria and we discuss their current use in the animal husbandry.

Resistance mechanism against fluoroquinolones in Mycoplasma hyopneumoniae field isolates.

The quinolone resistance-determining regions (QRDR) of gyrA, gyrB, parC, and parE of ten Mycoplasma hyopneumoniae field isolates that were either sensitive (5) or resistant (5) to the fluoroquinolones flumequine and enrofloxacin were characterized. In all five resistant isolates, one point mutation (C --> A) in parC was found, resulting in an amino acid change from serine to tyrosine at position 80 (Escherichia coli numbering). For four of these isolates, this was the only mutation found. These isolates had a minimum inhibitory concentration (MIC) of enrofloxacin of 0.5 microg/ml, whereas for sensitive isolates the MIC of enrofloxacin was < or =0.06 microg/ml. One resistant isolate (Mh 20) had an extra mutation (C --> T) in gyrA resulting in an amino acid change from alanine to valine at position 83 (E. coli numbering), leading to a further increase in the MIC of enrofloxacin (>1 microg/ml). No mutations resulting in an amino acid change were detected in the QRDR of the gyrB and parE genes of the selected isolates. This is the first description of the mechanism of stepwise resistance against fluoroquinolones in M. hyopneumoniae.

Protein variability among Mycoplasma hyopneumoniae isolates.

Sodium-dodecyl-sulphate polyacrylamide gel electrophoresis (SDS-PAGE) was used to study the protein variability of Mycoplasma hyopneumoniae isolates. Fifty-six M. hyopneumoniae isolates from 6 different countries and 37 different herds were used. From eight herds, more than one isolate was available. All SDS-PAGE patterns of isolates originating from different herds were clearly divergent. Intra-species protein variability was quantified using the reference strain J and seven field strains all obtained from different herds and classified according to virulence. Between the field strains, a variability of 25% was found, while the culture-adapted strain J was clearly divergent and showed 30% variability with the field strains. No clustering according to virulence was obtained, but a protein band of about 181kDa was present in the two highly virulent isolates whereas this protein band was absent in the moderately and low virulent isolates. Protein patterns of isolates derived from different animals from the same herd, were identical or differed in only a few protein bands. This study clearly indicates that, in agreement with previous studies on genomic diversity of M. hyopneumoniae isolates, proteomic variability within the species is high. Our study did not find clear evidence that more than one M. hyopneumoniae isolate circulates within a herd at a specific time point. The minor differences found between M. hyopneumoniae isolates from the same herd might reflect the organism's ability to alter its proteomic expression profile under field conditions.

Nosocomial Intravascular Catheter Infections with Extended-spectrum Beta-lactamase-producing Escherichia coli in Calves after Strain Introduction from a Commercial Herd.

An outbreak of intravascular catheter-related infections by extended-spectrum ß-lactamase (ESBL)-producing Escherichia coli in calves in an animal teaching hospital is reported. Pulsed-field gel electrophoresis was used for strain typing to determine the origin and dissemination of these strains. All 19 strains harboured the blaCTX-M-14, and six strains also overexpressed their chromosomal AmpC gene. Evidence on the introduction of the strain from a beef herd, experiencing neonatal diarrhoea and increased mortality, to the clinic through admission of diarrhoeic calves was provided. Strains isolated from phlebitis cases from other herds up to 5 months later showed a high similarity with the initial strain, suggesting that the strain had become nosocomial. The catheter infections with ESBL/AmpC-producing E. coli resulted in a prolonged hospitalization, increased anti-microbial use and mortality. This report points towards the potential dangers of the emergence of ESBL/AmpC-producing bacteria in susceptible food animals and warns farmers and veterinarians for the facility by which they are introduced into another environment.

Effect of avilamycin fed to chickens on E. faecium counts and on the selection of avilamycin-resistant E. faecium populations.

This study was conducted to investigate the effect of avilamycin used as a growth promoter on the number of E. faecium and on avilamycin-resistant E. faecium in the intestines of broilers over time. Avilamycin was added at 13.6 ppm to the feed of chickens during 28 days or during a typical growth period of 42 days; a nonmedicated group was included. Three hundred twenty-four Ross broiler chickens were equally distributed over the different groups in a treatment trial and kept in three isolation rooms. In each room, two replicates of the three experimental groups were kept in separate pens. At various time points, samples from different intestinal compartments or the feces were serially diluted and plated on avilamycin-supplemented and on unsupplemented Slanetz and Bartley (SL) media, and E. faecium counts were recorded. Only in the feces and only on the last sampling day was a significant decrease noted in the E. faecium counts in chickens treated with avilamycin for 42 days. Intermediate resistant (MIC 4-8 microg/ml) and resistant strains (MIC>or=16 microg/ml) were isolated from all groups, and there was a rise in prevalence over time. Pulsed-field gel electrophoresis profiles of these strains indicated clonal spread from one pen to another within the same room. The ratio between the counts of E. faecium isolated on antibiotic-supplemented to unsupplemented plates was significantly higher at the end of the trial in the feces samples from the group fed avilamycin for 42 days compared to the other groups, indicating a selective effect of avilamycin.

Virulence-associated traits in avian Escherichia coli: comparison between isolates from colibacillosis-affected and clinically healthy layer flocks.

Colibacillosis appears to be of increasing importance in layer flocks. The aim of this study was to determine characteristics of avian pathogenic Escherichia coli associated with the occurrence of colibacillosis outbreaks at flock level. Forty E. coli strains originating from layers from healthy flocks ('control isolates'), consisting of 25 caecal and 15 extra-intestinal isolates, were compared with 40 strains isolated from layers originating from colibacillosis-affected flocks ('outbreak isolates'), consisting of 20 caecal and 20 extra-intestinal isolates. The examined characteristics were adhesins, invasivity in T84 cell culture, serum resistance, iron uptake, colicin production, and toxinogenicity. The following traits were significantly more often detected in the outbreak isolates than in the control isolates: tsh, iss, iucA, iutA, irp2, fyuA, iroC, cvaC, colicin and colicin V production. A comparison of the extra-intestinal outbreak isolates and the caecal control isolates yielded the same results as when the caecal isolates, extra-intestinal isolates and total number of isolates of the outbreak and the control group were compared. When comparing the caecal and extra-intestinal isolates within the control and within the outbreak group, no significant differences were detected. The O78 and O2 groups showed significant differences with other O-types and NT strains for prevalence of most of the same characteristics. The combination of type 1 fimbriae, tsh, serum resistance, iss, traT, iucA, fyuA, iroC and colicin or colicin V production was significantly more often present in extra-intestinal outbreak isolates than in extra-intestinal control isolates. Only the combination of serum resistance, fyuA and colicin production was present in all outbreak isolates, with a significantly lower prevalence in the control isolates. None of the characteristics or combinations examined were exclusive to the outbreak isolates.

Characterization of avian Chlamydia psittaci strains using omp1 restriction mapping and serovar-specific monoclonal antibodies.

In the present study, 60 avian Chlamydia psittaci isolates were characterized using restriction fragment length polymorphism as well as serovar-specific monoclonal antibodies, enabling a comparison between the two characterization methods. Sixty avian C. psittaci isolates were characterized by Alul restriction mapping of the major outer membrane protein gene omp1 obtained after amplification by the polymerase chain reaction. The 60 avian C. psittaci strains were also characterized using serovar-specific monoclonal antibodies in a microimmunofluorescence test. Digestion of 60 avian C. psittaci omp1 amplicons by Alul generated 5 of the 6 known distinct restriction patterns (A, B, D, E and F). Restriction pattern C was not observed. Serotyping revealed 4 avian C. psittaci serovars (A, B, C and D). None of the 60 isolates was typed as serovar E. AluI restriction patterns A, B, D and E corresponded in 98% of the cases to serovars A, B, C and D, respectively. One isolate, classified as serovar A, generated restriction pattern F instead of A. Genotyping enabled a more precise differentiation of avian C. psittaci serovar A strains. Serovar A strains were divided into two groups according to their Alul restriction pattern (A or F). For epidemiological studies, genotyping can thus be a highly valuable alternative to serotyping, especially when applied directly to the clinical samples.

Incomplete cross resistance against ionophores in Enterococcus faecium and Enterococcus faecalis strains from pigs and poultry.

Thirty-two Enterococcus faecium strains and 33 Enterococcus faecalis strains were tested for their susceptibility to the ionophore antibiotics salinomycin, narasin, monensin, and lasalocid. Enterococcal strains originated from poultry in which these products are in use as coccidiostats, and from pigs in which these products are allowed as growth promoters. Resistance against salinomycin and narasin in enterococci was frequent among poultry strains, whereas in pig strains, resistance was less common. No resistance was found against monensin and lasalocid. Full cross resistance between salinomycin and narasin was evident. There was no cross resistance between these two ionophores and monensin and lasalocid.

Comparison of direct and enrichment methods for the selective isolation of vancomycin-resistant enterococci from feces of pigs and poultry.

Isolation results of vancomycin-resistant enterococci (VRE) of fecal samples from pigs and broiler and layer chickens obtained with two vancomycin-supplemented enrichment media, kanamycin aesculin azide (KAA) broth and Enterococcosel (ECC) broth, and three isolation media, KAA agar, ECC agar, and Slanetz and Bartley (SL) agar, were compared. Direct isolation on vancomycin-containing agar plates was not efficient in swine and layer chickens, which had only low numbers of VRE. In broilers chickens, the VRE content of the samples was high, and SL as well as ECC were found to perform better than KAA agar. The same three agar media were used as selective plating media after 1 and 2 days incubation of the samples in KAA and ECC enrichment broths. Sensitivities of the 12 different enrichment-plate combinations tested ranged from 0 to 81% in layer chickens and from 5 to 44% in samples from pigs. In the high prevalence type of samples from broilers, sensitivities still varied substantially from 52 to 78%. Incubating vancomycin-containing enrichment broths for 2 days compared with 1 day was favorable for the isolation of vancomycin-resistant Enterococcus faecalis, E. gallinarum, and E. casseliflavus but not for E. faecium and E. hirae/E. durans. ECC broth and ECC plates yielded the highest number of E. gallinarum and E. casseliflavus. In layer as well as in broiler chickens, ECC broth incubated for 2 days and plated on ECC agar was the most sensitive method. In pigs, however, KAA broth incubated for 2 days and plated on ECC medium yielded the highest number of VRE.

In vitro susceptibility of Enterococcus faecium isolated from food to growth-promoting and therapeutic antibiotics.

A total of 76 E. faecium strains, isolated at retail level from raw poultry meat, cheese, raw pork, and preparations of cheese and raw pork, were tested for their susceptibility and resistance to growth-promoting antibacterials used in animals and antibiotics used therapeutically in humans. All strains were uniformly susceptible to the growth promoters bambermycin and avilamycin. Resistance against bacitracin, virginiamycin and narasin was high among strains from poultry meat. With tylosin, a macrolide antibiotic used therapeutically and for growth promotion, resistance was mainly detected in strains originating from poultry meat, though also in some strains from pork and from pork and cheese preparations. The therapeutic antibiotic dalfopristin/quinupristin did not show full cross-resistance with the growth-promoting antibiotic virginiamycin. With dalfopristin/quinupristin two different levels of resistance were found. Only one E. faecium strain isolated from poultry was resistant to the glycopeptides avoparcin and vancomycin. Only one poultry meat strain was highly resistant to ampicillin. However, nearly all poultry meat strains showed decreased sensitivity. Only 3 out of 24 poultry strains were susceptible to minocycline, while all strains from other origins were susceptible to this tetracycline antibiotic. High-level streptomycin resistance was seen in strains of all origins, though infrequently. High-level gentamicin resistance was not found.