Kevina McGill

Quantitative Investigation of the Effects of Chemical Decontamination Procedures on the Microbiological Status of Broiler Carcasses during Processing

The effects of elevated chlorine concentrations (25 ppm) added to water in the final carcass washing equipment on total viable counts (TVCs 22 degrees C) and Escherichia coli and Enterobacteriaceae levels on poultry carcasses were investigated. Mean TVC counts on neck skin samples were significantly reduced when pre-evisceration and postwash samples were compared with log10 4.98 to 4.52 CFU/g recovered, respectively (P < or = 0.05). No significant reductions in TVC counts were observed in control samples at corresponding sampling points subjected to wash water containing 1 to 2 ppm chlorine. E. coli and Enterobacteriaceae counts were not significantly altered following final carcass washing in the processing plant. A second trial assessed the microbial decontamination capabilities of sodium triphosphate (TSP) on broiler carcasses. Neck skin samples from carcasses were obtained before final washing (control), following a 15-s dip in potable water and after dipping in a 10% TSP solution (pH 12) for 15 s. Reductions in E. coli and Enterobacteriaceae counts were all statistically significant for both water and TSP-treated samples when compared with corresponding controls (P < or = 0.01). The TSP treatment resulted in higher reductions of log10 1.95 and 1.86/g for E. coli and Enterobacteriaceae, respectively. In contrast, reductions of log10 0.37 and 0.3 l/g were observed for E. coli and Enterobacteriaceae counts when water-dipped carcasses were compared with corresponding controls. Significantly, Salmonella was not detected in any of the TSP-treated carcasses, while log10 1.92 and 1.04/g were found in control and water-dipped samples, respectively. Thermophilic Campylobacter counts were significantly lower in both treatment groups when compared with corresponding controlsresulting in log10 0.55 and 1.71/g reductions for water- and TSP-dipped carcasses, respectively (P < or = 0.01).

Distribution and Prevalence of Airborne Microorganisms in Three Commercial Poultry Processing Plants

Airborne microbial contaminants and indicator organisms were monitored within three poultry processing plants (plants A, B, and C). In total, 15 cubic feet (c.f.) of air was sampled per location during 15 visits to each plant and quantitatively analyzed for total mesophilic and psychrophilic aerobic counts, thermophilic campylobacters, Escherichia coli, and Enterobacteriaceae. The prevalence of Salmonella spp. in air samples was also evaluated. Significant reductions in total aerobic counts were observed between defeathering and evisceration areas of the three plants (P < 0.05). Mesophilic plate counts were highest in the defeathering areas of all plants compared to equivalent psychrophilic plate counts. Enterobacteriaceae counts were highest in the defeathering areas of all three plants with counts of log10 1.63, 1.53, and 1.18 CFU/15 c.f. recovered in plants A, B, and C, respectively. E. coli enumerated from air samples in the defeathering areas exhibited a similar trend to those obtained for Enterobacteriaceae with log10 1.67, 1.58, and 1.18 CFU for plants A, B, and C, respectively. Thermophilic campylobacters were most frequently isolated from samples in the defeathering areas followed by the evisceration areas. The highest mean counts of the organism were observed in plant A at 21 CFU/15 c.f. sample with plants B and C at 9 and 8 CFU/sample, respectively. With the exception of low levels of Enterobacteriaceae recovered from samples in the on-line air chill in plant A, E. coli, Enterobacteriaceae, or Campylobacter spp. were not isolated from samples in postevisceration sites in any of the plants examined. Salmonella spp. were not recovered from any samples during the course of the investigation.

Prevalence of thermophilic Campylobacter species in cats and dogs in two animal shelters in Ireland

Rectal swabs or faecal samples for the isolation of Campylobacter species were taken from 120 dogs and cats in an animal shelter in which only one kitten showed signs of gastrointestinal disease, and rectal swabs were taken from 46 dogs, 22 of which showed signs of gastrointestinal disease, in another shelter. At the first shelter, the swabs from 24 of 47 dogs (51.1 per cent) and 36 of 48 cats (75 per cent) yielded a Campylobacter species. The rate of isolation was significantly higher from dogs and cats less than six months old, and significantly higher from cats than from dogs (P≤0·05). At the second shelter Campylobacter species were isolated from 40 of 46 dogs (87 per cent), but there was no significant difference between the age groups. Campylobacter species were isolated from 19 (86·4 per cent) of the 22 dogs with signs of gastrointestinal disease and from 21 (87·5 per cent) of the 24 unaffected dogs. Several culture methods were applied to the samples collected from both shelters, and the combination significantly increased the recovery of Campylobacter species.

Prevalence of thermophilic Campylobacter species in household cats and dogs in Ireland

Rectal swabs were collected from 147 household dogs and 35 household cats, including healthy animals, animals with gastrointestinal signs and animals with a variety of medical and surgical conditions. A combination of selective culture methods was used to optimise the recovery of Campylobacter species, and a PCR was used to confirm their isolation and to identify the species. The overall prevalence of Campylobacter species was 42·9 per cent in the cats and 41·5 per cent in the dogs. Campylobacter upsaliensis was the species most commonly isolated from the dogs and cats, and Campylobacter jejuni was the second most commonly isolated. Particularly high prevalences were detected in the few cats and dogs with diarrhoea, and in the cats and dogs that were six months old or younger.

A comparison of different culture methods for the recovery of Campylobacter species from pets.

Five culture methods for the recovery of Campylobacter species (spp.) were evaluated on 361 rectal swabs collected from cats and dogs in Ireland. Speciation using PCR methods was performed on all isolates to assess the sensitivity of each culture method for isolation of Campylobacter spp., and to establish the prevalence of Campylobacter jejuni, C. coli, C. upsaliensis, C. lari and C. helveticus. Overall 163 of 361 (45.2%) samples were confirmed Campylobacter spp. positive. Direct plating onto modified charcoal cefoperazone deoxycholate agar (mCCDA) with cefoperazone, amphotericin and teicoplanin (CAT) selective supplement yielded a significantly higher prevalence of Campylobacter spp. (33.0%) than each of the other four methods (P ≤ 0.05). This method was also the most sensitive method for isolation of C. upsaliensis compared with any of the other four methods used in the current study (P ≤ 0.05). A direct plating method onto mCCDA agar with CCDA selective supplement and a filtration method onto blood agar after pre‐enrichment in CAT supplemented broth yielded lower Campylobacter spp. prevalences of 19.7% and 17.5% respectively. A filtration method onto CAT agar and pre‐enrichment in Preston broth before plating onto mCCDA agar were less sensitive for the isolation of Campylobacter spp. Speciation results of Campylobacter isolates revealed the majority of Campylobacter isolates were C. upsaliensis (50.0%) and C. jejuni (41.9%). A small number of isolates were C. coli (2.6%), C. lari (1.5%) and C. helveticus (1.1%). The overall detection of Campylobacter spp. in the 361 pets sampled was significantly increased by using a combination of isolation methods (P ≤ 0.05), producing a more accurate determination of the prevalence of Campylobacter spp. in pets in Ireland and of the actual Campylobacter species. As the majority of Campylobacter spp. were recovered by direct plating onto mCCDA agar with CAT supplement, this method is the method of choice if only a single method is selected for isolation of the most common Campylobacter spp. detected in pets and humans.

Comparison of disc diffusion and epsilometer (E-test) testing techniques to determine antimicrobial susceptibiliy of Campylobacter isolates of food and human clinical origin

The antibiotic resistance profiles of 75 Campylobacter isolates of food and human clinical origin was determined by two agar diffusion susceptibility methods; disc diffusion and epsilometer-test (E-test). The most common therapeutic antimicrobials, erythromycin, ciprofloxacin and tetracycline were studied, along with chloramphenicol, ampicillin and naladixic acid. The resistance observed for each antimicrobial, as determined by both of methods, were statistically compared using Fisher two-tailed analysis. Of the six antimicrobials studied only two were shown to have statistically different patterns when resistance was compared by disc diffusion and E-test. The percentage of isolates resistant to clinically relevant antimicrobials using both techniques ranged from 6.6 to 21.3% for erythromycin, 25.3-26.6% for tetracycline and 33.3-36.0% for ciprofloxacin. The prevalence of multi-drug resistant (MDR) campylobacters (isolates resistant to 2 or more antimicrobials) for both disc diffusion and E-test was 44%. It can be concluded that, for four of the six antimicrobials assessed, antimicrobial resistance prevalences could be equally determined by either of the methods studied.

Genetic diversity among Campylobacter jejuni isolates from pets in Ireland.

Campylobacter jejuni isolates obtained from pets housed in shelters and in private households were subtyped by fla typing (using DdeI and HinfI restriction enzymes) and pulsed-field gel electrophoresis (PFGE) techniques. Composite fla cluster analysis on 78 C jejuni isolates was more discriminative than either single fla typing technique with 39.7 per cent single isolate patterns. PFGE on 52 C jejuni isolates revealed 53.8 per cent single isolate patterns and was the most discriminative method applied. A database of C jejuni subtyping profiles from pets in Ireland was assembled. The presence of genetic heterogeneity detected in the C jejuni subtypes suggests that pets can acquire the organisms from multiple potential sources. In addition, heterogeneity was detected in the C jejuni subtypes obtained by different culture methods within the same pet. There was a link between isolates from dogs in close contact in the same environment, confirming that this is a potential route of infection, and clusters were detected containing both cat and dog C jejuni isolates, suggesting possible interspecies transmission.

Antibiotic resistance of retail food and human Campylobacter isolates on the island of Ireland from 2001-2002.

The antimicrobial resistance profiles of Campylobacter isolates recovered from a range of retail food samples (n=374) and humans (n=314) to eight antimicrobial compounds were investigated. High levels of resistance in food C. jejuni isolates were observed for ceftiofur (58%), ampicillin (25%) and nalidixic acid (17%) with lower levels observed for streptomycin (7.9%) and chloramphenicol (8.3%). A total of 80% of human C. jejuni isolates were resistant to ceftiofur, while 17% showed resistance to ampicillin and nalidixic acid, 8.6% to streptomycin and 4.1% to chloramphenicol. Resistance to clinically relevant antimicrobials such as erythromycin, ciprofloxacin and tetracycline was 6.7, 12, and 15% respectively for all food isolates and was similar to corresponding resistance prevalences observed for human isolates, where 6.4, 12 and 13% respectively were found to be resistant. Comparisons of C. jejuni isolates in each location showed a high degree of similarity although some regional variations did exist. Comparison of total C. jejuni and C. coli populations showed minor differences, with C. jejuni isolates more resistant to ampicillin and ceftiofur. Multidrug resistance patterns showed some profiles common to human and clinical isolates.

Antimicrobial resistance profiles and mechanisms of resistance in Campylobacter jejuni isolates from pets.

The presence of antimicrobial resistance in 51 Campylobacter jejuni isolates obtained from cats and dogs was determined by E-testing. Resistance to nalidixic acid (37.3% of isolates), ciprofloxacin (19.6%), tetracycline (13.7%), ampicillin (13.7%), erythromycin (11.8%), and chloramphenicol (5.9%) was detected. Resistance to two antimicrobials or more was present in 31.4% of isolates, and one isolate was resistant to all six antimicrobials. Of the isolates with ciprofloxacin and/or nalidixic acid resistance, 54.5% had the gyrA substitution Thr-86-Ile on sequencing. The tet o gene was detected in 75.0% isolates with high-level resistance to tetracycline. With the observed antimicrobial resistance in C. jejuni isolates from pets in this study, and the detection of identical mechanisms for quinolone and tetracycline resistance in pets and humans, pets should be considered a potential source of (multi)resistant C. jejuni infections in humans.

Restoring the selectivity of modified charcoal cefoperazone deoxycholate agar for the isolation of Campylobacter species using tazobactam, a β-lactamase inhibitor.

Extended spectrum β-lactamase (ESBL) producing Escherichia coli have emerged as a contaminant on modified charcoal cefoperazone deoxycholate agar (mCCDA) when attempting to selectively isolate Campylobacter spp. from poultry. E. coli are particularly problematic given their ability to grow under microaerophilic conditions and have been shown to outcompete Campylobacter species making Campylobacter detection or enumeration difficult. This paper recommends a novel method for restoring the selectivity of mCCDA using tazobactam, a β-lactamase inhibitor. The method significantly inhibited ESBL E. coli growth in spiked or naturally contaminated broiler caecal samples (p≤0.01) when compared to conventional mCCDA. This effect was seen at concentrations as low as 1mg/L tazobactam. TmCCDA(1) was found to inhibit up to 8 log10 CFU/mL of ESBL E. coli in mixed pure cultures and 7.5 log10 CFU/mL in caecal samples. Furthermore TmCCDA concentrations up to 10 mg/L had no statistically significant inhibitory effect (p≥0.05) on the recovery of a panel of 27 Campylobacter jejuni and 5 Campylobacter coli isolates when compared to conventional mCCDA. From this study it is suggested that tazobactam, which is more chemically stable than clavulanic acid or sulbactam, is more suitable for restoring the selectivity of mCCDA for the detection or isolation of campylobacters.