Bacteriophage Therapy To Reduce Campylobacter jejuni Colonization of Broiler Chickens

Colonization of broiler chickens by the enteric pathogen Campylobacter jejuni is widespread and difficult to prevent. Bacteriophage therapy is one possible means by which this colonization could be controlled, thus limiting the entry of campylobacters into the human food chain. Prior to evaluating the efficacy of phage therapy, experimental models of Campylobacter colonization of broiler chickens were established by using low-passage C. jejuni isolates HPC5 and GIIC8 from United Kingdom broiler flocks. The screening of 53 lytic bacteriophage isolates against a panel of 50 Campylobacter isolates from broiler chickens and 80 strains isolated after human infection identified two phage candidates with broad host lysis. These phages, CP8 and CP34, were orally administered in antacid suspension, at different dosages, to 25-day-old broiler chickens experimentally colonized with the C. jejuni broiler isolates. Phage treatment of C. jejuni-colonized birds resulted in Campylobacter counts falling between 0.5 and 5 log₁₀ CFU/g of cecal contents compared to untreated controls over a 5-day period postadministration. These reductions were dependent on the phage-Campylobacter combination, the dose of phage applied, and the time elapsed after administration. Campylobacters resistant to bacteriophage infection were recovered from phage-treated chickens at a frequency of <4%. These resistant types were compromised in their ability to colonize experimental chickens and rapidly reverted to a phage-sensitive phenotype in vivo. The selection of appropriate phage and their dose optimization are key elements for the success of phage therapy to reduce campylobacters in broiler chickens.     

Correlation of Campylobacter Bacteriophage with Reduced Presence of Hosts in Broiler Chicken Ceca

Campylobacter jejuni and Campylobacter-specific bacteriophage were enumerated from broiler chicken ceca selected from 90 United Kingdom flocks (n = 205). C. jejuni counts in the presence of bacteriophage (mean log₁₀ 5.1 CFU/g) were associated with a significant (P < 0.001) reduction compared to samples with Campylobacter alone (mean log₁₀ 6.9 CFU/g).     

Impact of a Single Phage and a Phage Cocktail Application in Broilers on Reduction of Campylobacter jejuni and Development of Resistance

Campylobacteriosis is currently the most frequent foodborne zoonosis in many countries. One main source is poultry. The aim of this study was to enhance the knowledge about the potential of bacteriophages in reducing colonization of broilers with Campylobacter , as there are only a few in vivo studies published. Commercial broilers were inoculated with 10⁴ CFU/bird of a Campylobacter jejuni field strain. Groups of 88 birds each were subsequently treated with a single phage or a four-phage cocktail (10⁷ PFU/bird in CaCO₃ buffered SM-Buffer). Control birds received the solvent only. Afterwards, subgroups of eleven birds each were examined for their loads with phages and Campylobacter on day 1, 3, 7, 14, 21, 28, 35 and 42 after phage application. The susceptibility of the Campylobacter population to phage infection was determined using ten isolates per bird. In total 4180 re-isolates were examined. The study demonstrated that the deployed phages persisted over the whole investigation period. The Campylobacter load was permanently reduced by the phage-cocktail as well as by the single phage. The reduction was significant between one and four weeks after treatment and reached a maximum of log₁₀ 2.8 CFU/g cecal contents. Phage resistance rates of initially up to 43% in the single phage treated group and 24% in the cocktail treated group later stabilized at low levels. The occurrence of phage resistance influenced but did not override the Campylobacter reducing effect. Regarding the reduction potential, the cocktail treatment had only a small advantage over the singe phage treatment directly after phage administration. However, the cocktail moderated and delayed the emergence of phage resistance.     

Direct Real-Time PCR Quantification of Campylobacter jejuni in Chicken Fecal and Cecal Samples by Integrated Cell Concentration and DNA Purification

Campylobacter jejuni is a major cause of diarrheal disease and food-borne gastroenteritis. The main reservoir of C. jejuni in poultry is the cecum, with an estimated content of 6 to 8 log₁₀ CFU/g. If a flock is infected with C. jejuni, the majority of the birds in that flock will harbor the bacterium. Diagnostics at the flock level could thus be an important control point. The aim of the work presented here was to develop a complete quantitative PCR-based detection assay for C. jejuni obtained directly from cecal contents and fecal samples. We applied an approach in which the same paramagnetic beads were used both for cell isolation and for DNA purification. This integrated approach enabled both fully automated and quantitative sample preparation and a DNA extraction method. We developed a complete quantitative diagnostic assay through the combination of the sample preparation approach and real-time 5′-nuclease PCR. The assay was evaluated both by spiking the samples with C. jejuni and through the detection of C. jejuni in naturally colonized chickens. Detection limits between 2 and 25 CFU per PCR and a quantitative range of >4 log₁₀ were obtained for spiked fecal and cecal samples. Thirty-one different poultry flocks were screened for naturally colonized chickens. A total of 262 (204 fecal and 58 cecal) samples were analyzed. Nineteen of the flocks were Campylobacter positive, whereas 12 were negative. Two of the flocks contained Campylobacter species other than C. jejuni. There was a large difference in the C. jejuni content, ranging from 4 to 8 log₁₀ CFU/g of fecal or cecal material, for the different flocks tested. Some issues that have not yet promoted much attention are the prequantitative differences in the ability of C. jejuni to colonize poultry and the importance of these differences for causing human disease through food contamination. Understanding the colonization kinetics in poultry is therefore of great importance for controlling human infections by this bacterium.     

Reduction of Campylobacter jejuni in Broiler Chicken by Successive Application of Group II and Group III Phages

Background

Bacteriophage treatment is a promising tool to reduce Campylobacter in chickens. Several studies have been published where group II or group III phages were successfully applied. However, these two groups of phages are different regarding their host ranges and host cell receptors. Therefore, a concerted activity of group II and group III phages might enhance the efficacy of a treatment and decrease the number of resistant bacteria.

Results

In this study we have compared the lytic properties of some group II and group III phages and analysed the suitability of various phages for a reduction of C. jejuni in broiler chickens. We show that group II and group III phages exhibit different kinetics of infection. Two group III and one group II phage were selected for animal experiments and administered in different combinations to three groups of chickens, each containing ten birds. While group III phage CP14 alone reduced Campylobacter counts by more than 1 log₁₀ unit, the concomitant administration of a second group III phage (CP81) did not yield any reduction, probably due to the development of resistance induced by this phage. One group of chickens received phage CP14 and, 24 hours later, group II phage CP68. In this group of animals, Campylobacter counts were reduced by more than 3 log₁₀ units.

Conclusion

The experiments illustrated that Campylobacter phage cocktails have to be carefully composed to achieve the best results.     

Concurrent Quantitation of Total Campylobacter and Total Ciprofloxacin-Resistant Campylobacter Loads in Rinses from Retail Raw Chicken Carcasses from 2001 to 2003 by Direct Plating at 42°C

This is the first report on the use of a normally lethal dose of ciprofloxacin in a Campylobacter agar medium to kill all ciprofloxacin-sensitive Campylobacter spp. but allow the selective isolation and quantitation of naturally occurring presumptive ciprofloxacin-resistant Campylobacter CFU in rinses from retail raw chicken carcasses (RTCC). Thermophilic-group total Campylobacter CFU and total ciprofloxacin-resistant Campylobacter CFU (irrespective of species) were concurrently quantified in rinses from RTCC by direct plating of centrifuged pellets from 10 or 50 ml out of 400-ml rinse subsamples concurrently on Campylobacter agar and ciprofloxacin-containing Campylobacter agar at 42°C (detection limit = 0.90 log₁₀ CFU/carcass). For 2001, 2002, and 2003, countable Campylobacter CFU were recovered from 85%, 96%, and 57% of RTCC, while countable ciprofloxacin-resistant Campylobacter CFU were recovered from 60%, 59%, and 17.5% of RTCC, respectively. Total Campylobacter CFU loads in RTCC rinses ranged from 0.90 to 4.52, 0.90 to 4.58, and 0.90 to 4.48 log₁₀ CFU/carcass in 2001, 2002, and 2003, respectively. Total ciprofloxacin-resistant Campylobacter CFU loads in RTCC rinses ranged from 0.90 to 4.06, 0.90 to 3.95, and 0.90 to 3.04 log₁₀ CFU/carcass in 2001, 2002, and 2003, respectively. Overall, total Campylobacter loads of 0.90 to 2.0, 2 to 3, 3 to 4, 4 to 5 log₁₀ CFU/carcass, respectively, were recovered from 16%, 32%, 26%, and 5% of RTCC tested over the 2-year sampling period. For the same period, total ciprofloxacin-resistant Campylobacter loads of 0.90 to 2.0, 2 to 3, 3 to 4, and 4 to 5 log₁₀ CFU/carcass, respectively, were recovered from 24%, 11%, 7%, and 0.2% of RTCC tested. There was a steady decline in total Campylobacter and total ciprofloxacin-resistant Campylobacter loads in RTCC rinses from 2001/2002 to 2003.     

Baseline Data from a Belgium-Wide Survey of Campylobacter Species Contamination in Chicken Meat Preparations and Considerations for a Reliable Monitoring Program

From February to November 2007, chicken meat preparations (n = 656) were sampled at 11 processing companies across Belgium. All samples were tested for Campylobacter by enrichment culture and by direct plating according to standard methods. Almost half (48.02%) of the samples were positive for Campylobacter spp. The mean Campylobacter count was 1.68 log₁₀ CFU/g with a standard deviation of ± 0.64 log₁₀ CFU/g. The study revealed a statistically significant variation in Campylobacter contamination levels between companies; processors with a wider frequency distribution range of Campylobacter counts provided chicken meat preparations with higher Campylobacter incidences and concentrations. There was no significant difference between the counts of Campylobacter spp. in various preparation types. However, the Campylobacter counts and incidences in chicken wings were the highest and portioned-form products (legs, wings, and breasts) showed a higher probability of being Campylobacter positive compared to minced-form products (sausages, burgers, and minced meat). The proportion of Campylobacter-positive samples was significantly higher in July than in other months. Recovery of Campylobacter spp. recovery by direct plating was higher (41.0%) compared to detection after enrichment (24.2%). Statistical modeling of the survey data showed that the likelihood of obtaining a positive result by enrichment culture increases with an increase in the Campylobacter concentration in the sample. In the present study, we provide the first enumeration data on Campylobacter contamination in Belgian chicken meat preparations and address proposals for improving Campylobacter monitoring programs.     

Effectiveness of phages in the decontamination of Listeria monocytogenes adhered to clean stainless steel, stainless steel coated with fish protein, and as a biofilm

Listeria monocytogenes is a food-borne pathogen which causes listeriosis and is difficult to eradicate from seafood processing environments; therefore, more effective control methods need to be developed. This study investigated the effectiveness of three bacteriophages (LiMN4L, LiMN4p and LiMN17), individually or as a three-phage cocktail at ≈9 log₁₀ PFU/ml, in the lysis of three seafood-borne L. monocytogenes strains (19CO9, 19DO3 and 19EO3) adhered to a fish broth layer on stainless steel coupon (FBSSC) and clean stainless steel coupon (SSC), in 7-day biofilm, and dislodged biofilm cells at 15 ± 1 °C. Single phage treatments (LiMN4L, LiMN4p or LiMN17) decreased bacterial cells adhered to FBSSC and SSC by ≈3–4.5 log units. Phage cocktail reduced the cells on both surfaces (≈3.8–4.5 and 4.6–5.4 log₁₀ CFU/cm², respectively), to less than detectable levels after ≈75 min (detection limit = 0.9 log₁₀ CFU/cm²). The phage cocktail at ≈5.8, 6.5 and 7.5 log₁₀ PFU/cm² eliminated Listeria contamination (≈1.5–1.7 log₁₀ CFU/cm²) on SSC in ≈15 min. One-hour phage treatments (LiMN4p, LiMN4L and cocktail) in three consecutive applications resulted in a decrease of 7-day L. monocytogenes biofilms (≈4 log₁₀ CFU/cm²) by ≈2–3 log units. Single phage treatments reduced dislodged biofilm cells of each L. monocytogenes strain by ≈5 log₁₀ CFU/ml in 1 h. The three phages were effective in controlling L. monocytogenes on stainless steel either clean or soiled with fish proteins which is likely to occur in seafood processing environments. Phages were more effective on biofilm cells dislodged from the surface compared with undisturbed biofilm cells. Therefore, for short-term phage treatments of biofilm it should be considered that some disruption of the biofilm cells from the surface prior to phage application will be required.     

Bacteriophage Therapy To Reduce Salmonella Colonization of Broiler Chickens

Acute enteric infections caused by salmonellas remain a major public health burden worldwide. Poultry, particularly chickens, are known to be the main reservoir for this zoonotic pathogen. Although some progress has been made in reducing Salmonella colonization of broiler chickens by using biosecurity and antimicrobials, it still remains a considerable problem. The use of host-specific bacteriophages as a biocontrol is one possible intervention by which Salmonella colonization could be reduced. A total of 232 Salmonella bacteriophages were isolated from poultry farms, abattoirs, and wastewater in 2004 and 2005. Three phages exhibiting the broadest host ranges against Salmonella enterica serotypes Enteritidis, Hadar, and Typhimurium were characterized further by determining their morphology and lytic activity in vitro. These phages were then administered in antacid suspension to birds experimentally colonized with specific Salmonella host strains. The first phage reduced S. enterica serotype Enteritidis cecal colonization by ≥4.2 log₁₀ CFU within 24 h compared with controls. Administration of the second phage reduced S. enterica serotype Typhimurium by ≥2.19 log₁₀ CFU within 24 h. The third bacteriophage was ineffective at reducing S. enterica serotype Hadar colonization. Bacteriophage resistance occurred at a frequency commensurate with the titer of phage being administered, with larger phage titers resulting in a greater proportion of resistant salmonellas. The selection of appropriate bacteriophages and optimization of both the timing and method of phage delivery are key factors in the successful phage-mediated control of salmonellas in broiler chickens.     

Enumeration and Diversity of Campylobacters and Bacteriophages Isolated during the Rearing Cycles of Free-Range and Organic Chickens

Campylobacters and Campylobacter-specific bacteriophages were isolated and enumerated during the rearing cycle of free-range (56 days) and organic chickens (73 days) at 3-day intervals from hatching until slaughter. In both flocks Campylobacter jejuni was the initial colonizer but Campylobacter coli was detected more frequently from 5 weeks of age. The diversity of the Campylobacter isolates was examined by pulsed-field gel electrophoresis of SmaI-digested genomic DNA and antimicrobial resistance typing. Bacteriophages were isolated from 51% (19 of 37 birds) of Campylobacter-positive organic birds (log₁₀ 2.5 to log₁₀ 5.7 PFU/g of cecal contents). The bacteriophages were all typical group III Campylobacter bacteriophages in terms of genomic size but could be characterized in terms of their host range and placed into five different groups. In contrast to the organic birds, anti-Campylobacter activity (bacteriocin-like) was observed in 26% (10 of 38 birds) of Campylobacter-positive free-range birds, and only one bacteriophage was isolated. Appearance of either bacteriophages or anti-Campylobacter activity was associated with changes in the levels of colonization and the predominant genotypes and species isolated. The frequency and potential influence of naturally occurring bacteriophages and/or inhibitory substances on the diversity and fluctuations of populations of campylobacters have not previously been reported in either free-range or organic chickens.     

Enumeration of Escherichia coli Cells on Chicken Carcasses as a Potential Measure of Microbial Process Control in a Random Selection of Slaughter Establishments in the United States

To evaluate whether the number of Escherichia coli bacteria in carcass rinses from chicken slaughter establishments could be monitored for the purpose of microbial process control, we drew a random sample from 20 of 127 large USDA-inspected operations. In 2005, every 3 months, two sets of 10 carcass rinses, 100 ml each, were collected from establishments, netting 80 sample sets from the rehang and postchill stages. E. coli and Campylobacter numbers and Salmonella prevalence were measured. Mixed-effect models were used to estimate variance of mean log₁₀ E. coli cell numbers of 10-carcass rinse sample sets. Relationships between E. coli and Campylobacter and Salmonella were examined. For 10-carcass rinse sets, at both the rehang and postchill stages the mean log₁₀ E. coli CFU/ml fit the logistic distribution better than the normal distribution. The rehang overall mean log₁₀ E. coli was 3.3 CFU/ml, with a within-sample set standard deviation of 0.6 CFU/ml. The overall postchill mean log₁₀ E. coli was 0.8 CFU/ml, with 13 establishments having mean log₁₀ E. coli CFU/ml values of less than 1.0 and 7 having mean values of 1.2 or more. At the midpoint separating these establishments, a mean log₁₀ E. coli CFU/ml of 1.1, the within-sample set standard deviation was 0.5 CFU/ml, with smaller standard deviations as means increased. Postchill sample sets with mean log₁₀ E. coli counts less than or equal to 1.1 CFU/ml had lower overall prevalence of Salmonella and mean log₁₀ Campylobacter CFU/ml than sample sets with higher means. These findings regarding reductions in E. coli numbers provide insight relevant to microbial process control.Regulatory food microbiology standards are defined and enforced with the intent of protecting public health and maintaining consumer confidence in the safety of the food supply. Resource demands (22) and legal constraints (21) have hindered the U.S. Department of Agriculture (USDA) from enforcing its current Salmonella performance standard (3). For this reason, in 2004 the USDA requested guidance from its national scientific advisory committee on the possible use of E. coli numbers to monitor sanitary conditions during poultry slaughter (12). The committee acknowledged that, if valid, such a performance standard could facilitate inspection of slaughter processing establishments. The committee recommended studies to define how E. coli numbers vary in poultry carcass rinses during poultry processing by processing stage, time of year, and geographic region and with respect to food-borne pathogens.The widespread presence and high numbers of generic E. coli bacteria on poultry entering the slaughter establishment (2, 5, 14) are suitable characteristics for an indicator organism used to monitor microbial control processes. The ease and lower cost (5, 13) of E. coli enumeration also allow more observations than can be made when comparable resources are allocated for Campylobacter or Salmonella testing (15).Regulatory agencies and food manufacturers have recognized the potential utility of E. coli numbers as a measure of slaughter process control. For example, USDA''s hazard analysis and critical control point rule (3) specifies two criteria for evaluating process control: establishments are to maintain less than 100 CFU of E. coli per ml in 80% of poultry carcass rinses and never exceed 1,000 CFU/ml. Surveys have been performed to define precise E. coli performance criteria for poultry (5), to monitor microbial reduction during slaughter processing (6), and to validate interventions to reduce microbial numbers on poultry (20).If generic E. coli numbers on poultry carcasses fit a parametric distribution, with a predictable mean and standard deviation, then carcasses could be monitored using a statistical process control plan. For example, if E. coli numbers decrease by an acceptable amount during processing to a reasonable level, then the process could be considered to be under control. Or a plan could be designed to monitor for acceptable occurrences of small, medium, and large deviations above a target E. coli number (7). If relationships were found between E. coli and Campylobacter numbers during chicken slaughter as well as Salmonella prevalence, they would further support the use of E. coli numbers as a measure of process control.This study of a random sample of 20 large chicken slaughter operations located throughout the United States measured microbial numbers at two processing line locations. Once a quarter, 10 carcass rinse samples were collected from both the post-feather-pick (rehang) and postchill locations. Rinses were examined to estimate mean Salmonella prevalence and E. coli and Campylobacter numbers by location within establishments. The primary objective was to assess whether the reduction in E. coli numbers between the rehang and postchill stages or numbers at the postchill location might have utility as a measure of process control during chicken slaughter. A related objective was to estimate values of parameters that could be used to design statistical process control plans (7).     

Influence of Process Parameter on Campylobacter spp. Counts on Poultry Meat in a Slaughterhouse Environment

Campylobacter spp. are the most important food-borne pathogens in broilers. Exposure of the consumer can be influenced by the reduction of contaminated broiler meat at various steps along the production line. This study was performed at a poultry slaughterhouse in Germany. Steps within the slaughter process were defined by the slaughterhouse quality control for potential Campylobacter reduction. Their impact was tested for two process variations. The first process variation was the increase of the temperature of the scalding water from 53.0 to 53.9 °C. The second step was the application of an additional outside sprayer which was placed after plucking. The increase of the scalding water temperature was the most effective measure (>2 log reduction), but resulted in defects to the broiler skin. This would limit marketing of fresh broiler meat with skin. The additional water spray after plucking had no additional effect. In fact, numbers of Campylobacter were lower before introduction of the sprayer. In conclusion, modifications of the processing technology have to be evaluated carefully, but can have additional effects for Campylobacter reduction.     

Carriage and Fecal Counts of Cefotaxime M-Producing Escherichia coli in Pigs: a Longitudinal Study

Current knowledge on extended-spectrum beta-lactamases (ESBLs) in animals is based largely on cross-sectional studies and qualitative data. The aim of this longitudinal study was to elucidate carriage proportions and fecal counts of ESBL-producing Escherichia coli in pigs during the production cycle. At each of three ESBL-positive single-sited farrow-to-finisher pig farms (farms A, B, and C) included in the study, individual fecal samples were taken from 17 to 20 sows 1 week before farrowing and from 2 piglets of each sow''s litter four times from birth to slaughter (as piglets, weaners, and finishers). Cefotaxime (CTX)-resistant coliforms in feces were counted on MacConkey agar containing 2 μg/ml CTX and characterized for the presence of ESBL-encoding genes by PCR and sequencing. CTX-M-positive pigs were detected in all age groups at farms A (bla_CTX-M-9 group, compatible with bla_CTX-M-14/17) and B (bla_CTX-M-1 group, compatible with bla_CTX-M-1/61), whereas only three weaners were positive at farm C (bla_CTX-M-1 group, compatible with bla_CTX-M-1/61). A significant decrease in carriage was detected during the production cycle, with on average 50% carriage immediately after birth, 58% just before weaning, 29% during weaning, and 12% during finishing. The observed reduction in numbers of CTX-M-positive pigs was accompanied by a significant reduction in mean fecal counts of CTX-resistant coliforms from ∼10⁷ CFU/g in piglets to ∼10³ CFU/g in finishers (P < 0.001). These findings provide novel information about the epidemiology of ESBLs at the farm level and have important implications for assessments of risks of meat contamination during slaughter.     

Control of Listeria spp. by Competitive-Exclusion Bacteria in Floor Drains of a Poultry Processing Plant

In previous studies workers determined that two lactic acid bacterium isolates, Lactococcus lactis subsp. lactis C-1-92 and Enterococcus durans 152 (competitive-exclusion bacteria [CE]), which were originally obtained from biofilms in floor drains, are bactericidal to Listeria monocytogenes or inhibit the growth of L. monocytogenes both in vitro and in biofilms at 4 to 37°C. We evaluated the efficacy of these isolates for reducing Listeria spp. contamination of floor drains of a plant in which fresh poultry is processed. Baseline assays revealed that the mean numbers of Listeria sp. cells in floor drains sampled on six different dates (at approximately biweekly intervals) were 7.5 log₁₀ CFU/100 cm² for drain 8, 4.9 log₁₀ CFU/100 cm² for drain 3, 4.4 log₁₀ CFU/100 cm² for drain 2, 4.1 log₁₀ CFU/100 cm² for drain 4, 3.7 log₁₀ CFU/100 cm² for drain 1, and 3.6 log₁₀ CFU/100 cm² for drain 6. The drains were then treated with 10⁷ CE/ml in an enzyme-foam-based cleaning agent four times in 1 week and twice a week for the following 3 weeks. In samples collected 1 week after CE treatments were applied Listeria sp. cells were not detectable (samples were negative as determined by selective enrichment culture) for drains 4 and 6 (reductions of 4.1 and 3.6 log₁₀ CFU/100 cm², respectively), and the mean numbers of Listeria sp. cells were 3.7 log₁₀ CFU/100 cm² for drain 8 (a reduction of 3.8 log₁₀ CFU/100 cm²), <1.7 log₁₀ CFU/100 cm² for drain 1 (detectable only by selective enrichment culture; a reduction of 3.3 log₁₀ CFU/100 cm²), and 2.6 log₁₀ CFU/100 cm² for drain 3 (a reduction of 2.3 log₁₀ CFU/100 cm²). However, the aerobic plate counts for samples collected from floor drains before, during, and after CE treatment remained approximately the same. The results indicate that application of the two CE can greatly reduce the number of Listeria sp. cells in floor drains at 3 to 26°C in a facility in which fresh poultry is processed.     

Colonisation of a Phage Susceptible Campylobacter jejuni Population in Two Phage Positive Broiler Flocks

The pathogens Campylobacter jejuni and Campylobacter coli are commensals in the poultry intestine and campylobacteriosis is one of the most frequent foodborne diseases in developed and developing countries. Phages were identified to be effective in reducing intestinal Campylobacter load and this was evaluated, in the first field trials which were recently carried out. The aim of this study was to further investigate Campylobacter population dynamics during phage application on a commercial broiler farm. This study determines the superiority in colonisation of a Campylobacter type found in a field trial that was susceptible to phages in in vitro tests. The colonisation factors, i.e. motility and gamma glutamyl transferase activity, were increased in this type. The clustering in phylogenetic comparisons of MALDI-TOF spectra did not match the ST, biochemical phenotype and phage susceptibility. Occurrence of Campylobacter jejuni strains and phage susceptibility types with different colonisation potential seem to play a very important role in the success of phage therapy in commercial broiler houses. Thus, mechanisms of both, phage susceptibility and Campylobacter colonisation should be further investigated and considered when composing phage cocktails.     

Reduction of Escherichia coli O157:H7 Populations in Cattle by Addition of Colicin E7-Producing E. coli to Feed

A cattle trial using artificially inoculated calves was conducted to determine the effect of the addition of colicinogenic Escherichia coli strains capable of producing colicin E7 (a 61-kDa DNase) to feed on the fecal shedding of serotype O157:H7. The experiment was divided into three periods. In period 1, which lasted 24 days, six calves were used as controls, and eight calves received 10⁷ CFU of E. coli (a mixture of eight colicinogenic E. coli strains) per g of feed. Both groups were orally inoculated with nalidixic acid-resistant E. coli O157:H7 strains 7 days after the treatment started. In periods 2 and 3, the treatment and control groups were switched, and the colicinogenic E. coli dose was increased 10-fold. During period 3, which lasted as long as period 1, both groups were reinoculated with E. coli O157:H7. The numbers of E. coli O157:H7 were consistently greater in the control groups during the three periods, but comparisons within each time period determined a statistically significant (P < 0.05) difference only at day 21 of period 1. However, when the daily average counts were compared between the period 1 control group and the period 3 treatment group that included the same six animals, an overall reduction of 1.1 log₁₀ CFU/g was observed, with a maximum decrease of 1.8 log₁₀ CFU/g at day 21 (overall statistical significance, P = 0.001). Serotype O157:H7 was detected in 44% of the treatment group's intestinal tissue samples and in 64% of those from the control group (P < 0.04). These results indicated that the daily addition of 10⁸ CFU of colicin E7-producing E. coli per gram of feed could reduce the fecal shedding of serotype O157:H7.     

Microbiological Quality of Bagged Cut Spinach and Lettuce Mixes

Analysis of 100 bagged lettuce and spinach samples showed mean total bacterial counts of 7.0 log₁₀ CFU/g and a broad range of <4 to 8.3 log₁₀ CFU/g. Most probable numbers (MPN) of ≥11,000 /g coliforms were found in 55 samples, and generic Escherichia coli bacteria were detected in 16 samples, but no E. coli count exceeded 10 MPN/g.     

Survival of Cold-Stressed Campylobacter jejuni on Ground Chicken and Chicken Skin during Frozen Storage

Campylobacter jejuni is prevalent in poultry, but the effect of combined refrigerated and frozen storage on its survival, conditions relevant to poultry processing and storage, has not been evaluated. Therefore, the effects of refrigeration at 4°C, freezing at −20°C, and a combination of refrigeration and freezing on the survival of C. jejuni in ground chicken and on chicken skin were examined. Samples were enumerated using tryptic soy agar containing sheep's blood and modified cefoperazone charcoal deoxycholate agar. Refrigerated storage alone for 3 to 7 days produced a reduction in cell counts of 0.34 to 0.81 log₁₀ CFU/g in ground chicken and a reduction in cell counts of 0.31 to 0.63 log₁₀ CFU/g on chicken skin. Declines were comparable for each sample type using either plating medium. Frozen storage, alone and with prerefrigeration, produced a reduction in cell counts of 0.56 to 1.57 log₁₀ CFU/g in ground chicken and a reduction in cell counts of 1.38 to 3.39 log₁₀ CFU/g on chicken skin over a 2-week period. The recovery of C. jejuni following freezing was similar on both plating media. The survival following frozen storage was greater in ground chicken than on chicken skin with or without prerefrigeration. Cell counts after freezing were lower on chicken skin samples that had been prerefrigerated for 7 days than in those that had been prerefrigerated for 0, 1, or 3 days. This was not observed for ground chicken samples, possibly due to their composition. C. jejuni survived storage at 4 and −20°C with either sample type. This study indicates that, individually or in combination, refrigeration and freezing are not a substitute for safe handling and proper cooking of poultry.     

Elimination of Escherichia coli O157:H7 from Fermented Dry Sausages at an Organoleptically Acceptable Level of Microencapsulated Allyl Isothiocyanate

Four sausage batters (17.59% beef, 60.67% pork, and 17.59% pork fat) were inoculated with two commercial starter culture organisms (>7 log₁₀ CFU/g Pediococcus pentosaceus and 6 log₁₀ CFU/g Staphylococcus carnosus) and a five-strain cocktail of nonpathogenic variants of Escherichia coli O157:H7 to yield 6 to 7 log₁₀ CFU/g. Microencapsulated allyl isothiocyanate (AIT) was added to three batters at 500, 750, or 1,000 ppm to determine its antimicrobial effects. For sensory analysis, separate batches with starter cultures and 0, 500, or 750 ppm microencapsulated AIT were produced. Sausages were fermented at ≤26°C and 88% relative humidity (RH) for 72 h. Subsequently sausages were dried at 75% RH and 13°C for at least 25 days. The water activity (a_w), pH, and levels of starter cultures, E. coli O157:H7, and total bacteria were monitored during fermentation and drying. All sausages showed changes in the initial pH from 5.57 to 4.89 and in a_w from 0.96 to 0.89 by the end of fermentation and drying, respectively. Starter culture numbers were reduced during sausage maturation, but there was no effect of AIT on meat pH reduction. E. coli O157:H7 was reduced by 6.5 log₁₀ CFU/g in sausages containing 750 and 1,000 ppm AIT after 21 and 16 days of processing, respectively. E. coli O157:H7 numbers were reduced by 4.75 log₁₀ CFU/g after 28 days of processing in treatments with 500 ppm AIT, and the organism was not recovered from this treatment beyond 40 days. During sensory evaluation, sausages containing 500 ppm AIT were considered acceptable although slightly spicy by panelists.     

Bacteriophage-Mediated Dispersal of Campylobacter jejuni Biofilms

Bacteria in their natural environments frequently exist as mixed surface-associated communities, protected by extracellular material, termed biofilms. Biofilms formed by the human pathogen Campylobacter jejuni may arise in the gastrointestinal tract of animals but also in water pipes and other industrial situations, leading to their possible transmission into the human food chain either directly or via farm animals. Bacteriophages are natural predators of bacteria that usually kill their prey by cell lysis and have potential application for the biocontrol and dispersal of target bacteria in biofilms. The effects of virulent Campylobacter specific-bacteriophages CP8 and CP30 on C. jejuni biofilms formed on glass by strains NCTC 11168 and PT14 at 37°C under microaerobic conditions were investigated. Independent bacteriophage treatments (n ≥ 3) led to 1 to 3 log₁₀ CFU/cm² reductions in the viable count 24 h postinfection compared with control levels. In contrast, bacteriophages applied under these conditions effected a reduction of less than 1 log₁₀ CFU/ml in planktonic cells. Resistance to bacteriophage in bacteria surviving bacteriophage treatment of C. jejuni NCTC 11168 biofilms was 84% and 90% for CP8 and CP30, respectively, whereas bacteriophage resistance was not found in similarly recovered C. jejuni PT14 cells. Dispersal of the biofilm matrix by bacteriophage was demonstrated by crystal violet staining and transmission electron microscopy. Bacteriophage may play an important role in the control of attachment and biofilm formation by Campylobacter in situations where biofilms occur in nature, and they have the potential for application in industrial situations leading to improvements in food safety.