Cyril Carroll

Environmental survival mechanisms of the foodborne pathogen Campylobacter jejuni

Campylobacter spp. continue to be the greatest cause of bacterial gastrointestinal infections in humans worldwide. They encounter many stresses in the host intestinal tract, on foods and in the environment. However, in common with other enteric bacteria, they have developed survival mechanisms to overcome these stresses. Many of the survival mechanisms used by Campylobacter spp. differ from those used by other bacteria, such as Escherichia coli and Salmonella spp. This review summarizes the mechanisms by which Campylobacter spp. adapt to stress conditions and thereby increase their ability to survive on food and in the environment.

Evaluation of Culture Methods and a DNA Probe-Based PCR Assay for Detection of Campylobacter Species in Clinical Specimens of Feces

ABSTRACT Campylobacter species are the leading agents of bacterial gastroenteritis in developed countries. In this study 320 specimens of feces from patients with symptoms of acute gastroenteritis were cultured for Campylobacter species by direct plating on modified charcoal cefoperazone deoxycholate agar and by enrichment in modified Preston broth, with or without blood added, for 48 h at 37°C prior to plating. A 16S/23S PCR/DNA probe membrane-based colorimetric detection assay was evaluated on a subset of the feces (n = 127), including 18 culture-positive and 109 culture-negative specimens. DNA was extracted directly from the fecal specimens by using the QIAamp DNA stool Minikit for the DNA probe-based PCR assay (PCR/DNA probe assay). A second PCR/DNA probe assay based on the 16S rRNA gene in Campylobacter spp. was applied to all specimens that were culture negative, PCR/DNA positive on initial analysis. Campylobacter species were cultured in 20 of the 320 specimens. The 16S/23S PCR/DNA probe assay detected campylobacter DNA in 17 of 18 (94% sensitivity) culture-positive specimens and in 41 (38%) culture-negative specimens. The presence of campylobacter DNA in 35 of 41 culture-negative specimens was confirmed by the 16S PCR/DNA probe assay. DNA sequence analysis of seven 16S/23S PCR products and five 16S PCR products amplified from a selection of these specimens confirmed the presence of campylobacter DNA and more specifically Campylobacter jejuni, C. concisus, C. curvus, and C. gracilis DNA in these specimens. The molecular assays described in this study are rapid methods for the detection and identification of Campylobacter species in fecal specimens. The finding of Campylobacter spp. DNA in a large number of specimens of feces from patients with no other identified cause of diarrhea may suggest that Campylobacter spp. other than C. jejuni and C. coli may account for a proportion of cases of acute gastroenteritis in which no etiological agent is currently identified.

Induction of an adaptive tolerance response in the foodborne pathogen, Campylobacter jejuni

In this study we aimed to determine if Campylobacter had the ability to induce an adaptive tolerance response (ATR) to acid and/or aerobic conditions. Campylobacter jejuni CI 120 was grown to the appropriate phase in Brucella broth under microaerobic conditions. Cells were initially adapted to a mild stress (pH 5.5) for 5 h prior to challenge at pH 4.5, a lethal pH. Survival was examined by determining the numbers of viable cells on Campylobacter blood free selective agar base. Stationary phase cells adapted at pH 5.5 induced an ATR that enabled a 100-fold greater survival compared to an uninduced culture. Aerobic adaptation also protected the cells against acid challenge. The cross protection provided a 500-fold increase in survival when compared to unadapted cells. The incorporation of chloramphenicol during the induction period eliminated the ATR and resulted in death kinetics similar to an uninduced culture. These data suggest that Campylobacter spp. have the ability to induce an ATR to sublethal treatments, which increased their ability to withstand subsequent stresses.

Antimicrobial resistance of Campylobacter jejuni and Campylobacter coli isolates from broiler chickens isolated at an Irish poultry processing plant.

Aims: The antibiotic susceptibility of Campylobacter jejuni and Campylobacter coli isolates from broiler chickens were determined in order to evaluate the level of antibiotic resistance of Campylobacter species in the Irish poultry industry.

Identification of a novel stress resistance mechanism in Campylobacter jejuni.

Aim: To study stress resistance mechanisms in Campylobacter spp.

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.

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.

Pulsed Field Gel Electrophoresis typing of human and retail foodstuff Campylobacters: An Irish perspective

Campylobacter enteritis is a zoonosis, an infectious disease transmissible under normal conditions from vertebrate animals to man, presenting a major global public health burden. In this study, Pulsed Field Gel Electrophoresis (PFGE) was employed to identify common genotypes in a collection of 600 Campylobacter isolates in order to investigate if profiles obtained from retail samples of foodstuffs matched genotypes causing illness in the community in Ireland. The Campylobacters were isolated from retail foodstuffs, and cases of gastroenteritis, over the same 20-month period in three population centres in Ireland. The major observation made was of a high level of PFGE-genotype heterogeneity; 236 SmaI discrete genotypes were found in 507 strains successfully analysed. Analysis of the PFGE profiles revealed 22 common profiles amongst food isolates and those causing enteritis in humans. These cojoint PFGE genotypes indicate that 56 (38%) of the human clinical isolates are genetically related to 129 (36%) of the food isolates. The identification of these recurrent PFGE types, in the sampled Campylobacter coli and Campylobacter jejuni populations, indicates that a high proportion of Campylobacter isolates found in foods of animal origin also occur in patients with symptoms of enteritis. This data adds weight to the epidemiological hypothesis that a high proportion of human Campylobacter cases are contracted via the handling and consumption of contaminated foodstuffs, in particular poultry.

The effect of wind speed and direction on the distribution of sewage-associated bacteria.

A study of the relationship between wind and the distribution of sewage‐associated bacteria was undertaken at a location where the sewage was discharged into the sea adjacent to the mouth of a river. The numbers of presumptive Escherichia coli were determined in 149 sea‐water samples taken from three locations at distances of 1·9, 2·4 and 4·3 km from the outfall. On each sampling occasion, data on the wind speed and direction in the 3 h prior to collection of the samples were also collected. Analysis of these data demonstrated a significant role for wind speed and direction. With respect to wind direction, the numbers of presumptive E. coli present in a sample were significantly higher when the sample site lay downwind of the outfall. Wind speed was shown to have an influence on the numbers of presumptive E. coli only when the sample site was downwind of the outfall. In an analysis of 61 samples, an inverse correlation (r2 = 0·73) between salinity and log presumptive E. coli numbers was demonstrated. These data demonstrate that wind speed and direction at the time of sampling significantly influence the numbers of presumptive E. coli detected in any sea‐water sample. It is argued that failure to pay sufficient attention to these parameters in the design of monitoring programmes may result in the generation of data that could provide a seriously distorted picture of the microbiological status of a water body.

Genotypic characterisation and cluster analysis of Campylobacter jejuni isolates from domestic pets, human clinical cases and retail food.

The genetic similarity of Campylobacter jejuni isolates from pets, compared to human clinical cases and retail food isolates collected in Ireland over 2001-2006 was investigated by cluster analysis of pulsed-field gel electrophoresis (PFGE) fingerprinting profiles. Comparison of the PFGE profiles of 60 pet isolates and 109 human isolates revealed that seven (4.1%) profiles were grouped in clusters including at least one human and one pet C. jejuni isolate. In total six (1.6%) of 60 pet and 310 food profiles were in clusters with at least one food and one pet C. jejuni isolate. The detection of only a small number of genetically indistinguishable isolates by PFGE profile cluster analysis from pets and from humans with enteritis in this study suggests that pets are unlikely to be an important reservoir for human campylobacteriosis in Ireland. However, genetically indistinguishable isolates were detected and C. jejuni from pets may circulate and may contribute to clinical infections in humans. In addition, contaminated food fed to pets may be a potential source of Campylobacter infection in pets, which may subsequently pose a risk to humans.