Frieda Jørgensen

Sources of Campylobacter spp. Colonizing Housed Broiler Flocks during Rearing

ABSTRACT The study aimed to identify sources of campylobacter in 10 housed broiler flocks from three United Kingdom poultry companies. Samples from (i) the breeder flocks, which supplied the broilers, (ii) cleaned and disinfected houses prior to chick placement, (iii) the chickens, and (iv) the environments inside and outside the broiler houses during rearing were examined. Samples were collected at frequent intervals and examined for Campylobacter spp. Characterization of the isolates using multilocus sequence typing (MLST), serotyping, phage typing, and flaA restriction fragment length polymorphism typing was performed. Seven flocks became colonized during the growing period. Campylobacter spp. were detected in the environment surrounding the broiler house, prior to as well as during flock colonization, for six of these flocks. On two occasions, isolates detected in a puddle just prior to the birds being placed were indistinguishable from those colonizing the birds. Once flocks were colonized, indistinguishable strains of campylobacter were found in the feed and water and in the air of the broiler house. Campylobacter spp. were also detected in the air up to 30 m downstream of the broiler house, which raises the issue of the role of airborne transmission in the spread of campylobacter. At any time during rearing, broiler flocks were colonized by only one or two types determined by MLST but these changed, with some strains superseding others. In conclusion, the study provided strong evidence for the environment as a source of campylobacters colonizing housed broiler flocks. It also demonstrated colonization by successive campylobacter types determined by MLST during the life of a flock.

Impact of transport crate reuse and of catching and processing on Campylobacter and Salmonella contamination of broiler chickens.

ABSTRACT The influence of transport, catching, and processing on contamination of broiler chickens with Salmonella and Campylobacter was investigated. Transport crates were reused with high frequency and were often still contaminated with Salmonella and Campylobacter when they arrived at the farm despite the fact that they were washed at the factory, and thus they were a potential route of infection. These organisms contaminated the feathers of previously Campylobacter- and Salmonella-negative birds going to the processing plant and were isolated from processed carcasses, albeit at a low frequency. The Campylobacter types which were the predominant organisms on the live birds when they arrived at the processing plant were not necessarily the types that were most frequently isolated from processed carcasses. This finding may reflect cross-contamination that occurred during processing or differences in the tolerance of the strains to the hostile environments that the bacteria experienced. The process of catching and putting the birds in crates significantly increased the chance of contamination with Campylobacter (P < 0.001).

Effect of Challenge Temperature and Solute Type on Heat Tolerance of Salmonella Serovars at Low Water Activity

ABSTRACT Salmonella spp. are reported to have an increased heat tolerance at low water activity (aw; measured by relative vapor pressure [rvp]), achieved either by drying or by incorporating solutes. Much of the published data, however, cover only a narrow treatment range and have been analyzed by assuming first-order death kinetics. In this study, the death ofSalmonella enterica serovar Typhimurium DT104 when exposed to 54 combinations of temperature (55 to 80°C) and aw (rvp 0.65 to 0.90, reduced using glucose-fructose) was investigated. The Weibull model (LogS = −btn) was used to describe microbial inactivation, and surface response models were developed to predict death rates for serovar Typhimurium at all points within the design surface. The models were evaluated with data generated by using six different Salmonella strains in place of serovar Typhimurium DT104 strain 30, two different solutes in place of glucose-fructose to reduce aw, or six low-awfoods artificially contaminated with Salmonella in place of the sugar broths. The data demonstrate that, at temperatures of ≥70°C, Salmonella cells at low aw were more heat tolerant than those at a higher aw but below 65°C the reverse was true. The same patterns were generated when sucrose (rvp 0.80 compared with 0.90) or NaCl (0.75 compared with 0.90) was used to reduce aw, but the extent of the protection afforded varied with solute type. The predictions of thermal death rates in the low-aw foods were usually fail-safe, but the few exceptions highlight the importance of validating models with specific foods that may have additional factors affecting survival.

Survival and Filamentation of Salmonella enterica Serovar Enteritidis PT4 and Salmonella enterica Serovar Typhimurium DT104 at Low Water Activity

ABSTRACT In this study we investigated the long-term survival of and morphological changes in Salmonella strains at low water activity (aw). Salmonella enterica serovar Enteritidis PT4 and Salmonella enterica serovar Typhimurium DT104 survived at low aw for long periods, but minimum humectant concentrations of 8% NaCl (aw, 0.95), 96% sucrose (aw, 0.94), and 32% glycerol (aw, 0.92) were bactericidal under most conditions. Salmonella rpoS mutants were usually more sensitive to bactericidal levels of NaCl, sucrose, and glycerol. At a lethal aw, incubation at 37°C resulted in more rapid loss of viability than incubation at 21°C. At aw values of 0.93 to 0.98, strains of S. enterica serovar Enteritidis and S. enterica serovar Typhimurium formed filaments, some of which were at least 200 μm long. Filamentation was independent of rpoS expression. When the preparations were returned to high-aw conditions, the filaments formed septa, and division was complete within approximately 2 to 3 h. The variable survival ofSalmonella strains at low aw highlights the importance of strain choice when researchers produce modelling data to simulate worst-case scenarios or conduct risk assessments based on laboratory data. The continued increase in Salmonellabiomass at low aw (without a concomitant increase in microbial count) would not have been detected by traditional microbiological enumeration tests if the tests had been performed immediately after low-aw storage. If Salmonellastrains form filaments in food products that have low awvalues (0.92 to 0.98), there are significant implications for public health and for designing methods for microbiological monitoring.

The survival of foodborne pathogens during domestic washing-up and subsequent transfer onto washing-up sponges, kitchen surfaces and food.

In this study, the survival of Salmonella, Campylobacter and Escherichia coli O157: H7, when exposed to a range of constant temperatures (47-60 degrees C), in hard or soft water, in the presence/absence of detergent (0-0.3%) and organic matter, and during drying, was investigated. Further experiments used a washing-up process simulation, where soiled dishes contaminated with bacteria were washed in a bowl of warm water containing detergent. In addition, this study considered the risk of bacterial transfer onto (1) sterile dishes and sponges via contaminated water, (2) kitchen surfaces wiped with a contaminated sponge, (3) items placed in direct contact with a contaminated kitchen surface, (4) food placed on a contaminated dish or (5) dishes from contaminated food. A proportion of dishes remained contaminated with all pathogen types after a typical washing-up. Water hardness did not appear to affect survival. E. coli, and to a lesser extent Salmonella, survived towel- or air-drying on dishes and after towel-drying the cloth became contaminated on every occasion, regardless of the test organism. A proportion of sterile dishes washed after contaminated dishes became contaminated with pathogens but transfer from dishes onto food was rare. Washing-up sponges frequently became contaminated with pathogens. The results of this study highlight the potential for survival and cross contamination of food borne pathogens in the kitchen environment.

RpoS-dependent stress tolerance in Pseudomonas aeruginosa.

Pseudomonas aeruginosa is able to persist during feast and famine in many different environments including soil, water, plants, animals and humans. The alternative sigma factor encoded by the rpoS gene is known to be important for survival under stressful conditions in several other bacterial species. To determine if the P. aeruginosa RpoS protein plays a similar role in stationary-phase-mediated resistance, an rpoS mutant was constructed and survival during exposure to hydrogen peroxide, high temperature, hyperosmolarity, low pH and ethanol was investigated. Disruption of the rpoS gene resulted in a two- to threefold increase in the rate of kill of stationary-phase cells. The rpoS mutant also survived less well than the parental strain during the initial phase of carbon or phosphate-carbon starvation. However, after 25 d starvation the remaining population of culturable cells was not significantly different. Stationary-phase cells of the RpoS-negative strain were much more stress resistant than exponentially growing RpoS-positive cells, suggesting that factors other than the RpoS protein must be associated with stationary-phase stress tolerance in P. aeruginosa. Comparison of two-dimensional PAGE of the rpoS mutant and the parental strain showed four major modifications of protein patterns associated with the rpoS mutation.

Incidence and Mechanism of Ciprofloxacin Resistance in Campylobacter spp. Isolated from Commercial Poultry Flocks in the United Kingdom before, during, and after Fluoroquinolone Treatment

ABSTRACT Five commercial broiler flocks were treated with a fluoroquinolone for a clinically relevant infection. Fresh feces from individual chickens and environmental samples were cultured for campylobacters before, during, and weekly posttreatment until slaughter. Both Campylobacter jejuni and C. coli were isolated during all treatment phases. An increased proportion of quinolone-resistant strains was seen during treatment, and these strains persisted posttreatment. One quinolone-resistant isolate of each species, each serotype, and each phage type from each sample at all treatment phases was examined for its phenotype and mechanism of resistance. Two resistant phenotypes were isolated: Nalr Cipr and Nalr Cips. The majority (269 of 290) of fluoroquinolone-resistant isolates, whether they were C. jejuni or C. coli, had a mutation in gyrA that resulted in the substitution Thr-86→Ile. The other gyrA mutations detected were Thr-86→Ala (n = 17) and Asp-90→Asn (n = 10). The genotypic variation, based on the silent mutations in gyrA identified by the denaturing high-performance liquid chromatography pattern and DNA sequencing, was used to supplement typing data and provided evidence for both the spread of preexisting resistant strains and the selection of spontaneous resistant mutants in treated flocks. Multidrug resistance was significantly (P < 0.01) associated with resistance to ciprofloxacin. Twenty-five percent (73 of 290) of ciprofloxacin-resistant isolates but only 13% (24 of 179) of susceptible isolates were resistant to three or more unrelated antimicrobial agents. In conclusion, quinolone-resistant campylobacters were isolated from commercial chicken flocks in high numbers following therapy with a veterinary fluoroquinolone. Most ciprofloxacin-resistant isolates had the GyrA substitution Thr-86→Ile. Resistant isolates were isolated from the feces of some flocks up to the point of slaughter, which may have consequences for public health.

Prevalence and Subtypes of Ciprofloxacin-Resistant Campylobacter spp. in Commercial Poultry Flocks before, during, and after Treatment with Fluoroquinolones

ABSTRACT Five commercial broiler chicken flocks were treated with either difloxacin or enrofloxacin for a clinically relevant infection, as instructed by a veterinarian. Campylobacters were isolated from individual fecal samples and from samples associated with the broiler environment before, during, and after treatment. Ciprofloxacin-resistant Campylobacter jejuni and/or C. coli strains were detected pretreatment in four flocks, but they constituted a very small proportion of the campylobacters present. When the broilers were treated with a fluoroquinolone, a rapid increase in the proportion of ciprofloxacin-resistant campylobacters was observed. During treatment nearly 100% of campylobacters were resistant, and in some flocks a high proportion of resistant strains persisted for up to 4 weeks after treatment. Prior to treatment a variety of campylobacter subtypes were present. During and after treatment considerable changes in both species and subtype prevalence were observed, but no single fluoroquinolone-resistant clone became dominant. Instead, resistant C. coli strains or a mixture of resistant C. coli and C. jejuni strains became dominant, whereas susceptible C. jejuni strains had usually been dominant prior to treatment. The resistant subtypes which emerged and became dominant were not always the same as those detected pretreatment. The persistence of resistant strains for up to 4 weeks posttreatment has important implications for any strategy designed to avoid the introduction of such strains into the food chain.

Influence of season and geography on Campylobacter jejuni and C. coli subtypes in housed broiler flocks reared in Great Britain.

ABSTRACT Geographical and seasonal variation in the incidence and prevalence of Campylobacter jejuni and C. coli in housed broiler flocks reared in Great Britain in 2004 to 2006 was investigated in this study. Ceca (30) from 797 flocks, not subject to prior partial depopulation and reared on 211 farms, were examined individually for the presence of Campylobacter spp. The best-fitting climatic factors explained approximately 46% of the prevalence of Campylobacter-colonized flocks at slaughter and consisted of a combination of temperature at slaughter, number of sunshine hours in placement month, and millimeters of rainfall in placement month. Positive flocks were more likely to be slaughtered between June and November than during the rest of the year and to be reared in northern Great Britain than in central or southern Great Britain. C. jejuni was identified in approximately 90% of flocks, and C. coli was present in 10% of flocks. The most common clonal complexes identified in 226 isolates typed by multilocus sequence typing (MLST) were ST-45, ST-21, ST-574, ST-443, and ST-828. Flocks slaughtered at the same time were more likely to have similar complexes, and ST-45 had a seasonal pattern, with the highest prevalence in June, and was also more likely to be present in flocks reared in northern Great Britain.

The prevalence and number of Salmonella in sausages and their destruction by frying, grilling or barbecuing

Aims: To determine the prevalence and number of Salmonella and Campylobacter in sausages and to evaluate their destruction during cooking. 
Methods and Results: One hundred and sixty‐two packs of uncooked economy or catering sausages, comprising 53 packs of frozen and 109 of chilled sausages, were purchased in Devon between March and July 2000. All were tested for the presence of Salmonella and 51 packs of chilled sausages were also examined for the presence of Campylobacter spp. To investigate the heat tolerance of Salmonella enterica serovar Typhimurium DT104 in sausage‐meat, chilled, handmade and frozen sausages were inoculated with approx. 1·5 × 104 bacterial cells per sausage (∼300 cfu g−1) and then cooked by frying, grilling or barbecuing. The levels of creatinine kinase, lactate dehydrogenase and alkaline phosphatase in uncooked and cooked sausages were measured to evaluate their potential as indicators of adequate cooking and, therefore, pathogen elimination. Salmonella were detected in 7·5% of frozen and 9·1% of the chilled sausages (8·6% overall) but Campylobacter spp. were not isolated. After cooking, a visual assessment suggested that all of the sausages were thoroughly cooked. Despite this, barbecuing and frying sometimes allowed Salmonella cells to survive and the temperature profiles during cooking indicated that the lethal range was sometimes not reached. The enzyme levels tested were not reliable indicators of the inactivation of bacterial pathogens because Salmonella were sometimes isolated from sausages with low values of all three enzymes. 
Conclusions:Salmonella spp. are present in a significant proportion of sausages and are not always killed during the cooking process. 
Significance and Impact of the Study: These findings have clear implications for public health.