Antimicrobial resistance profiles and clonal relatedness of canine and feline Escherichia coli pathogens expressing multidrug resistance in the United States.

BACKGROUND: Antimicrobial resistance is increasing among Escherichia coli isolates associated with spontaneous infection in dogs and cats. OBJECTIVES: To describe E. coli resistance phenotypes and clonal relatedness and their regional prevalence. ANIMALS: Isolates of E. coli (n = 376) collected from dogs and cats in the United States between May and September 2005. METHODS: Isolates submitted from the South, West, Northeast, and Midwest regions of the United States were prospectively studied. Phenotype was based on E-test susceptibility to 7 antimicrobials. Isolates were classified as no (NDR), single (SDR), or multidrug resistance (MDR). Clonal relatedness was determined by pulsed-field gel electrophoresis (PFGE). RESULTS: One hundred and ninety-three (51%) isolates expressed resistance to at least 1 drug, yielding 42 phenotypes. SDR isolates (n = 84; 44%, 8 phenotypes), expressed resistance most commonly to amoxicillin (30%, n = 25) and least commonly to cefpodoxime (1%, n = 1). MDR isolates (n = 109; 56%, 31 phenotypes) were resistant to amoxicillin (96%, n = 105), amoxicillin-clavulanate (85%, n = 93), and enrofloxacin (64%, n = 70); 18% (n = 20) were resistant to all drugs tested. The frequency of MDR did not differ regionally (P = .066). MDR minimum inhibitory concentrations (MICs) were 6-fold higher than SDR MICs (P < .0001). Dendrograms of 91 isolates representing 25 phenotypes revealed 62 different PFGE profiles. CONCLUSIONS AND CLINICAL IMPORTANCE: E. coli strains spontaneously infecting dogs and cats are genetically and phenotypically diverse. Given the current prevalence of MDR among clinical isolates of E. coli in United States, implementation of a robust surveillance program is warranted.

DNA identification and characterization of Campylobacter jejuni and Campylobacter coli isolated from caecal samples of chickens in Grenada.

AIMS: To speciate Campylobacter strains from the caeca of chickens in Grenada using PCR and to evaluate DNA-based typing methods for the characterization of these isolates. METHODS AND RESULTS: Isolates were speciated with two multiplex PCR assays and were typed with flaA-RFLP, pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). Results confirmed that Campylobacter coli strains were more predominant than Campylobacter jejuni strains. From 56 isolates, 18 were misidentified using biochemical tests. PFGE typing gave the highest discriminatory power among the methods used (Simpson's index of diversity, D=0.9061). However, the combination of flaA-RFLP, PFGE and MLST results gave the highest discrimination for subtyping of these isolates (D=0.9857). A band position tolerance of 4% in BioNumerics was the most appropriate for the analysis of this database. MLST profiles were generally concordant with PFGE and/or flaA-RFLP types. Several isolates exhibited new MLST sequence types (STs), and 43 of the 49 Camp. coli strains belonged to the ST-828 clonal complex. CONCLUSIONS: Campylobacter coli was the most prevalent species isolated from broilers and layers in Grenada, and a combination of restriction and sequence methods was most appropriate for the typing of Camp. coli isolates. Campylobacter coli STs clustered with described poultry-associated Camp. coli STs by phylogenetic analysis. SIGNIFICANCE AND IMPACT OF THE STUDY: Further studies to understand the predominance of Camp. coli within Campylobacter spp. from chickens in Grenada may help elucidate the epidemiology of these pathogens in chickens.

Development of a polymerase chain reaction assay for specific identification of Clostridium colinum.

Clostridium colinum is the causative agent of ulcerative enteritis, a serious disease of the bobwhite quail (Colinus virginianus) and sporadically of young chickens. The aim of the present study was to develop a polymerase chain reaction (PCR) assay specific for C. colinum identification. The 16S rDNA sequence of C. colinum was analysed and two species-specific primers were designed. The specificity of these primers was tested with closely related Clostridium species and the expected amplified product (935 base pairs) was observed only with DNA from samples containing C. colinum. Results from performing PCR assays on faecal samples from quails spiked with different concentrations of C. colinum, showed that the detection limit of the assay was 1.6 x 10(4) colony-forming units per gram of faecal material. This PCR assay can be used in diagnostic laboratories to confirm the presence of C. colinum in pure cultures and could be used to screen enriched samples or faecal samples for the presence of this pathogen.

Molecular typing, serotyping and cytotoxicity testing of Campylobacter jejuni strains isolated from commercial broilers in Puerto Rico.

AIMS: Thirty Campylobacter jejuni strains isolated from fecal samples (n = 94; 32%) from 13 positive farms (n = 17; 76%) from commercial broiler chickens in Puerto Rico were analysed by molecular methods. METHODS AND RESULTS: Isolates were identified with multiplex polymerase chain reaction assays, tested for their antimicrobial susceptibility and characterized with pulsed-field gel electrophoresis (PFGE), multilocus sequence typing (MLST), serotyping and bacterial cytotoxicity in mammalian cells. Isolates exhibited high resistance to vancomycin (minimum inhibitory concentration, MIC of >256 microg ml(-1)) and trimethoprim (MIC of >32 microg ml(-1)); few were resistant to clindamycin (MIC(90) 4 microg ml(-1)), erythromycin (MIC(90) 8 microg ml(-1)) and tetracycline (MIC(90) 8 microg ml(-1)); but none was resistant to azithromycin (MIC(90) 4 microg ml(-1)), ciprofloxacin (MIC(90) 1 microg ml(-1)) or gentamycin (MIC(90) 4 microg ml(-1)). Most strains restricted with SmaI, but a combination of SmaI-KpnI digestion was more discriminatory. MLST analysis yielded four sequence types (ST), and ST-2624 was the predominant one. Phylogenetic analysis revealed a high degree of recombination for glnA and pgm genes. The predominant serotypes were O:3 and O:5. Most strains had lowest cytotoxicity potential with Caco-2 cells, medium cytotoxicity with INT-407 and Hep-2 cells and high cytotoxicity with CHO cells. CONCLUSION: A low degree of antimicrobial resistance, 13 PFGE profiles, 4 ST and a large variability in cytotoxicity assays were found for these strains. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first characterization of C. jejuni strains isolated from broilers in Puerto Rico. The genetic diversity of these strains suggests that several techniques are needed for strain characterization.

Evaluation of different plate media for direct cultivation of Campylobacter species from live broilers.

Accurate identification and optimal culturing procedures for Campylobacter spp. from live broilers are needed for epidemiological studies. Because there is no standardized protocol, we designed and conducted studies to evaluate different selective media for the culturing and isolation of Campylobacter spp. from cecal and fecal samples obtained from battery-reared and commercial broilers. Five media selective for Campylobacter were evaluated: Campylobacter agar base, Campylobacter, Campy-Line, modified Campy-Cefex, and modified charcoal cefoperazone deoxycholate agar. With contaminated broilers reared in battery cages, Campylobacter agar base, Campylobacter, modified Campy-Cefex, and modified charcoal cefoperazone deoxycholate agar revealed similar isolation rates (P > 0.05), whereas Campy-Line showed a lower efficacy (P < 0.05). With commercial live broilers, modified Campy-Cefex agar was more consistent for the isolation of Campylobacter from feces, whereas modified Campy-Cefex and modified charcoal cefoperazone deoxycholate agar showed similar isolation rates from cecal samples. Campy-Line agar showed a lower identification rate (P < 0.05) for both fecal and cecal samples. A multiplex PCR assay used for identification showed that Campylobacter jejuni and Campylobacter coli DNA was present in the samples. Pulsed field gel electrophoresis restriction profiles differed among samples collected from different commercial farms but were similar for isolates from the same farm, suggesting clonal differences. No variation was seen in pulsed field gel electrophoresis patterns among isolates cultured on different media. Our data suggest that the choice of plate medium may influence the efficiency of isolating Campylobacter spp. from broiler chickens by direct plating from fecal or cecal samples.

High content, size and distribution of single-stranded DNA in the mitochondria of Chenopodium album (L.).

Mitochondrial (mt) DNA of higher plants is unique in its large size and complexity. We report here a hitherto unknown feature, the presence of large quantities of single-stranded (ss) DNA. About 2.0-8.5% of the chromosomal mtDNA from a suspension culture (depending on the growth stage) and 6.5% of the chromosomal mtDNA from whole plants of Chenopodium album were found to be in ss form by dot-blot hybridization after neutral transfer. Similar amounts of ss mtDNA were observed by binding of the single-strand binding (SSB) protein of Escherichia coli under the electron microscope. Significantly less ssDNA was found in plastids of C. album and in E. coli cells. We observed ss regions between 100 and 22,800 bases distributed in the mt genome spaced from 0.5-100 kb apart. After pulsed-field gel electrophoresis (PFGE), the well-bound fraction of mtDNA (found to consist of circular, sigma-shaped and rosette-like molecules), contained the major part of ssDNA as opposed to the migrating linear molecules. Digestion of mtDNA by ss-specific nucleases followed by PFGE mobilized all well-bound DNA and correspondingly increased the quantity of migrating linear DNA molecules. The implications of ssDNA for the structural organization on plant mt genomes are discussed.

Specific identification of Campylobacter fetus by PCR targeting variable regions of the 16S rDNA.

Campylobacter fetus is recognized as a human and animal pathogen. The isolation and differentiation of C. fetus in diagnostic laboratories is hindered by its relatively slow growth and lack of distinguishing biochemical characteristics. We developed a fast, reliable PCR assay that specifically amplifies a 554-bp segment of the 16S rDNA from C. fetus. Fifty-two ATCC reference strains and 255 bacterial field isolates comprising the genera Campylobacter, Arcobacter, Helicobacter, Escherichia, Listeria, Salmonella, and Wolinella were evaluated using this PCR protocol. Only C. fetus strains were amplified. Sequence analysis of amplicons from ATCC and field strains of C. fetus confirmed the presence of the target DNA fragment. The detection limit of the technique was 5.9 x 10(3) CFU/ml. This PCR assay can yield reliable detection of C. fetus within 3 h after isolation of presumptive colonies on agar plates.

Application of direct-fed microbial bacteria and fructooligosaccharides for salmonella control in broilers during feed withdrawal.

Providing direct-fed-microbial (DFM) bacteria and fructooligosaccharides (FOS) for the control of potential escalation of Salmonella colonization during simulated feed withdrawal and confinement was assessed. Eight hundred and eighty broilers (16 pens; 55 chicks per pen) were reared to 6 wk of age. Chicks were sprayed with a solution containing 10(6) nalidixic-acid resistant Salmonella typhimuriumNR cells per milliliter on the 2nd d after hatching. Because this first challenge did not yield a high infection rate, chickens were rechallenged per Os at Day 18 by providing water containing 10(7) cells of S. typhimuriumNR per milliliter. At 3 and 5 wk of age, 10 birds per pen were euthanatized and cecal Salmonella were quantified (log colony-forming units per gram). Feed was removed from all pens at 6 wk, and pens were randomly assigned to be either the treatment group or the control group. The treatment groups were provided a DFM (mixture of nine bacteria) and FOS 50 (R) (10%) in the drinking water. The control groups received drinking water only. After 6 h of feed withdrawal, chickens were cooped (eight per coop) and held 10 h. Immediately after confinement, 10 chickens were used for cecal enumeration of S. typhimuriumNR. Salmonella colonization declined from 99% at 3 wk to 44% at 5 wk. After feed withdrawal, application of the treatment, and confinement, 11 and 14% of the treated and control groups, respectively, yielded S. typhimuriumNR by direct plating from ceca (3.87 and 3.75 log 10 cfu/g, respectively). No difference (P > 0.05) in Salmonella colonization occurred between the treated and the control groups; however, enrichment of ceca (incubation in nutrient broth at 37 C for 24 h) yielded a higher incidence of S. typhimuriumNR in the control groups (32% in the treated vs 51% in the control). Ceca weights were greater in the treated group (P < 0.05). Simulated feed withdrawal and confinement did not escalate Salmonella colonization in the chicken ceca.

In vitro fructooligosaccharide utilization and inhibition of Salmonella spp. by selected bacteria.

In vitro experiments were conducted to determine: 1) inhibitory capacities of potential direct-fed microbial bacteria against Salmonella serotypes; and 2) the ability of Bifidobacterium bifidum, Enterococcus faecium, Lactobacillus casei, Lactococcus lactis, Pediococcus sp., and Salmonella spp. to grow in media containing fructooligosaccharides (FOS-50 or FOS pure formulation) as the only carbohydrate source. Thirteen bacteria (two strains of Bacillus coagulans, Bacillus licheniformis, Bacillus subtilis, B. bifidum, E. faecium, two strains of Lactobacillus acidophilus, L. casei, Pediococcus sp., Propionibacterium acidopropionici, P. jensenii, and Propionibacterium sp.) were tested for inhibition of six Salmonella serotypes (S. california, S. enteritidis, S. heidelberg, S. mission, S. senftenberg, and S. typhimurium) using a spot-the-lawn technique. Bifidobacterium bifidum, E. faecium, all lactobacilli, and Pediococcus sp. clearly inhibited growth of all Salmonella serotypes. In the growth experiments, E. faecium, L. lactis, and Pediococcus sp. grew in media with either FOS-50 or the pure formulation of FOS as the sole carbohydrate source. All tested Salmonella serotypes utilized FOS-50 for growth; however growth varied among the serotypes. In contrast, none of the Salmonella serotypes grew in media containing the pure formulation of FOS as the only carbohydrate source.

Incidence of campylobacters in the intestine of avian species in Alabama.

Avian species necropsied at the C. S. Roberts Veterinary Diagnostic Laboratory, Auburn, Alabama, from December 1993 until May 1994 were examined for the incidence of intestinal campylobacters. Ninety-one intestinal swabs, representing 66 separate cases and 17 different avian species, were collected and placed into Cary-Blair transport medium. Selective enrichment and culture media were used for initial isolation of Campylobacter spp. Presumptive colonies were identified as Campylobacter spp. by phase-contrast microscopy and Gram stain, and they were confirmed by serological latex agglutination. Campylobacter spp. were isolated in 18 (19.7%) of the 66 cases. From the remainder of the cases, 13 (15%) yielded presumptive colonies on Campy-Cefex agar; however, they were not confirmed serologically as Campylobacter spp. Use of Cary-Blair transport medium held in refrigeration for up to 24 days did not hinder the determination of campylobacters in intestinal samples. A variety of avian species, including chicken, emu, hawk, ostrich, and parrot, harbored commensal campylobacters and therefore should be considered potential reservoirs.