Molecular characterization and antibiotic resistance profiling of Campylobacter isolated from cattle in Polish slaughterhouses

A total of 812 samples from bovine hides and the corresponding carcasses collected at the slaughterhouse level in the eastern part of Poland were examined for the presence of Campylobacter jejuni and Campylobacter coli. Recovered isolates were confirmed using species-specific PCR, characterized by the presence of 11 putative virulence genes and antimicrobial susceptibility was determined using a microbroth dilution method. Furthermore, the genotypic relatedness of the isolates was determined by PFGE profiling and virulence pattern cluster analysis. The prevalence of Campylobacter was 25.6% and 2.7% in bovine hide and carcass samples, respectively. The presence of virulence markers varied between C. jejuni and C. coli species however, the majority of strains possessed the cadF, flhA, flaA genes, irrespective of the bacterial species and origin. The lower number of the strains was positive for the invasive associated markers – virB11 and wlaN. Antibiotic profiling showed that campylobacters were most frequently resistant to quinolones and fluoroquinolones (nalidixic acid and ciprofloxacin, 38.3% of each, respectively) followed by streptomycin (24.3%) and tetracycline (20.9%). Resistance to erythromycin and gentamicin was demonstrated in 4.3% and 2.6% of strains, respectively. Comparisons of the PFGE and virulence marker profiles of the isolates reflected the high genetic diversity of Campylobacter tested. Moreover, a poor correlation between the PFGE type, pathogenic gene marker and antimicrobial resistance patterns was observed.     

Biofilm formation by Campylobacter jejuni in controlled mixed-microbial populations

This study was to screen the ability of biofilm formation by Campylobacter jejuni strains found in New Zealand, and investigate the biofilm growth of C. jejuni in a controlled mixed-microbial population that includes five different bacteria. The ability of C. jejuni to form a biofilm in monoculture and mixed-microbial populations was measured in a laboratory assay using a microtiter plate screening assay. The optical density of the biofilm and cell growth from mixed-microbial populations was converted to a Biofilm Formation Index (BFI). This index was used to standardize the biofilm formation in the mixed-microbial populations. High BFI was observed for Enterococcus faecalis (2.30) and Staphylococcus simulans (3.75) when they were grown with C. jejuni multilocus sequence type ST-474: a dominant poultry and human-associated type in New Zealand. C. jejuni cells were recovered from most of the biofilms containing E. faecalis and/or S. simulans. These results suggest that E. faecalis and S. simulans may play a role in biofilm formation in the poultry environment as both of these microorganisms are found in poultry processing environments and were able to form a biofilm in association with C. jejuni under microaerobic conditions. Understanding the relationships among C. jejuni, E. faecalis and S. simulans in poultry processing plants and farms may help in the design of strategies to reduce the reservoir of contamination of these bacteria and reduce the incidence of campylobacteriosis.     

Campylobacter contamination of broiler caeca and carcasses at the slaughterhouse and correlation with Salmonella contamination

In order to estimate the prevalence of Campylobacter spp. and Salmonella spp. on broiler chicken carcasses and the prevalence of Campylobacter spp. in caeca, 58 French slaughterhouses were investigated in 2008. Enumeration of Campylobacter spp. was also performed in order to study the relation between caeca and carcass contamination. A pool of 10 caeca and one carcass were collected from 425 different batches over a 12-month period in 2008. Salmonella was isolated on 32 carcasses leading to a prevalence of 7.5% ([5.0-10.0]_95%CI). The prevalence of Campylobacter was 77.2% ([73.2-81.2]_95%CI) in caeca and 87.5% ([84.4-90.7]_95%CI) on carcasses. No significant correlation was found between Campylobacter and Salmonella. Positive values of Campylobacter were normally distributed and the average level was 8.05 log₁₀ cfu/g ([7.94-8.16]_95%CI) in caeca and 2.39 cfu/g ([2.30-2.48]_95%CI) on carcasses. A positive correlation (r = 0.59) was found between the mean of Campylobacter in caeca and on carcasses (p < 0.001). Thus, carcasses from batches with Campylobacter-positive caeca had significantly (p < 0.001) higher numbers of Campylobacter per gram than batches with negative caeca. These results show that Campylobacter can be present in both matrices and reduction in caeca could be a possible way to reduce the amount of bacteria on carcasses. Of the 2504 identifications performed, 3 species of Campylobacter (Campylobacter jejuni, Campylobacter coli and Campylobacter lari) were identified. The main species recovered were C. jejuni and C. coli, which were isolated in 55.3% and 44.5% of positive samples, respectively. These two species were equally represented in caeca but C. jejuni was the most frequently isolated on carcasses with 57.1% and 42.5% of positive carcasses for C. jejuni and C. coli, respectively. This study underlines that target a reduction of Campylobacter on final products requires a decrease of contamination in caeca.     

PorA specific primers for the identification of Campylobacter species in food and clinical samples

Campylobacteriosis is a public health problem with considerable socio-economic impact. As the European Food Safety Authority has emphasized the importance of a surveillance programme for campylobacteriosis, the aim of the present study was the optimization of a specific and sensitive PCR protocol able to detect Campylobacter species responsible for gastrointestinal infections. Raw poultry meat samples were analysed for the presence of Campylobacter sp., by plating onto mCCD (Modified Charcoal-Cefoperazone-Deoxycholate) Agar and Campylobacter Selective Preston Agar and using four sets of species-specific primers for Campylobacter jejuni, Campylobacter coli, Campylobacter upsaliensis and Campylobacter lari designed to bridge the porA gene. The resulting primers demonstrated a sensitivity of 0.01 ng/μl for the C. coli-specific, C. lari-specific, and C. upsaliensis-specific primer sets and 0.5 ng/μl for the C. jejuni-specific primer sets using DNA from pure cultures. Non-specific amplification of non-target DNA was not observed indicating excellent specificity. The primers were useful for the analyses of poultry meat samples both for direct plating onto mCCDA, and for DNA extracted directly from the cells grown for 48 h in Preston enrichment broth. The sets of primers were also useful when used for species identification of human isolates.     

Campylobacter jejuni: A Review of its Characteristics,Pathogenicity, Ecology,Distribution, Subspecies Characterization and Molecular Methods of Detection

Campylobacter jejuni is considered to be the leading cause of enteric illness in the United States and other industrialized nations, causing mild to severe symptoms including serious infections of the extremities and permanent neurological symptoms. The organism is a cytochrome oxidase positive, microaerophilic, curved Gram-negative rod exhibiting corkscrew motility and is carried in the intestine of many wild and domestic animals, particularly avian species including poultry, where the intestine is colonized resulting in healthy animals as carriers. Wildlife have long been considered an infectious reservoir for campylobacters because of their close association with and contamination of surface waters. This review deals with the areas of: phenotypic characteristics of C. jejuni and related human pathogenic species of Campylobacter, their ecological distribution, virulence factors, isolation of C. jejuni from foods, serotyping of Campylobacter isolates, bacteriophage typing, molecular methods of detecting and typing campylobacters, the viable but nonculturable state of campylobacters, the coccoid form of C. jejuni and immunomagnetic capture of C. jejuni.     

In vitro antimicrobial activity of essential oil from endemic Origanum minutiflorum on ciprofloxacin-resistant Campylobacter spp.

This study evaluated the antimicrobial activities of an essential oil of Origanum minutiflorum (O. Schwarz and P.H. Davis) against ciprofloxacin-resistant Campylobacter spp., by broth microdilution and agar well-diffusion methods. Moreover, O. minutiflorum oil was analyzed by gas chromatography/mass spectrometry (GC/MS). Twenty-nine components were identified, representing 98.7 of the oil. The oil yield from the plants was 4.0–4.4% v/w. The major components of O. minutiflorum oil were carvacrol (73.9%) and p-cymene (7.20%). The oil has lower contents of carvacrol methyl ether (0.05%), heptadecanol (0.06%) and carvacryl acetate (0.06%). Twenty-one Campylobacter spp. (12 C. jejuni, 5 C. lari and 4 C. coli) strains using in this study were selected among 300 isolates according to their resistance to ciprofloxacin. The minimum inhibitory concentration (MIC) values for bacterial strains, which were sensitive to the essential oil of O. minutiflorum, were in the range of 7.8–800 μg/ml. The essential oil obtained showed strong antimicrobial activity against all of the tested ciprofloxacin-resistance Campylobacter spp. These results suggest that the essential of O. minutiflorum may be used as a natural preservative in food against food-born disease, such as Campylobacteriosis.     

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.     

Simultaneous Detection and Differentiation of Campylobacter jejuni, C. coli, and C. lari in Chickens Using a Multiplex Real-Time PCR Assay

A multiplex real-time PCR (qPCR) assay was developed for simultaneous detection and differentiation of the three most important Campylobacter species in chickens. Three novel sets of PCR primers and TaqMan probes were designed to amplify the unique DNA sequences within the hipO, cdtA, and pepT genes which are specific to Campylobacter jejuni, Campylobacter coli, and Campylobacter lari, respectively. To avoid competition in the multiple target amplifications, the concentrations of primers and probes were optimized. By using the optimized qPCR conditions together with a minor-groove binding probe of pepT, amplification efficiency greater than 92% and detection sensitivity of 38 genome copies/reaction have been achieved for all three targets. The assay was highly specific for C. jejuni, C. coli, and C. lari with testing of 33 Campylobacter strains and 20 non-Campylobacter strains. In chicken samples spiked with known quantities of Campylobacter cells, the assay was able to detect 1 CFU/g after a 24-h enrichment. Application of the assay in food was further evaluated using 21 fresh chicken samples obtained from local supermarkets. The results revealed that, after a 24-h or 48-h enrichment, 14 samples (66.7%) were positive for C. jejuni, five samples (23.8%) were positive for C. coli, and none of the samples was contaminated by C. lari. Taken together, the multiplex qPCR assay combined with an enrichment step is a sensitive, species-specific, and non-labor-intensive method suitable for rapid detection of C. jejuni, C. coli, and C. lari in chicken samples.     

Occurrence of multidrug resistance in Campylobacter from Ivorian poultry and analysis of bacterial response to acid shock

The level of antibiotic multiresistance of Campylobacter strains from chicken was evaluated and responses to the bactericidal effects of organic acids were analyzed. Strains (76) isolated from chicken were analyzed for resistance to antibiotics and acid shock. A high strain resistance to fluoroquinolones (ciprofloxacin and nalidixic acid) was observed, with cross resistance to both drugs in 41% of strains. A low resistance was observed for amoxicillin, erythromycin, and gentamicin. Campylobacter jejuni was the most resistant species. Antibacterial activities against multiresistant Campylobacter strains were observed using acetic, citric, and ascorbic acids at minimum bactericidal concentrations (MBC) ranging from 0.3 to 3 mg/mL. Acetic acid was the most efficient acid with the lowest MBC value. However, a contact time of 4 h was required for an efficient effect against Campylobacter contaminated chicken skin. Using organic acids in the poultry production chain can reduce propagation of antibiotic multiresistant strains of Campylobacter.     

The temporal,PFGE and resistance pattern associations suggest that poultry products are only a minor source of human infections in Western Finland

In order to compare human and retail poultry meat thermophilic Campylobacter isolates originating in a regional area in Western Finland, minimum inhibitory concentration (MICs) for six antimicrobials (96 isolates) and pulsed-field gel electrophoresis (PFGE) (102 isolates) were analysed. Campylobacter spp. were detected in 10.5% out of 305 fresh poultry products studied; 29 (90.5%) isolates were identified as Campylobacter jejuni. Among the 70 human isolates, 66 (94.3%) isolates were identified as C. jejuni. Only one C. jejuni domestic poultry isolate showed resistance (ampicillin), whereas domestic human C. jejuni isolates were more commonly resistant to ciprofloxacin, nalidixic acid, ampicillin and tetracycline. The resistance in foreign human isolates was significantly more common than among domestic isolates. PFGE analysis with KpnI restriction enzyme resulted in 59 different PFGE types among the poultry and human isolates. Three types were detected first in poultry meat and thereafter during the following month in domestic human samples, whereas the other conjoint types were detected only after many months. This study suggests that poultry products play only a minor role in human campylobacteriosis in the study area and that the resistance found in domestic human isolates is not likely related to retail poultry meat products.     

Quantitative analysis of viable,stressed and dead cells of Campylobacter jejuni strain 81-176

Campylobacter jejuni is an important foodborne gastrointestinal pathogen and highly sensitive to environmental stresses. Research has shown that changes in culturability, cell morphology, and viability occur when C. jejuni cells are subjected to stresses. In this study, real-time PCR, ethidium monoazide (EMA) in combination with real-time PCR (EMA-PCR), BacLight bacterial viability staining, and agar plate counting methods were used to quantitatively analyze viable, stressed, and dead C. jejuni strain 81-176. The real-time PCR assay provides highly sensitive and specific quantification of total genome copies of C. jejuni culture in different growth phases. Our results also reveal that real-time PCR can be used for direct quantification of Campylobacter genome release into Phosphate Buffered Saline (PBS) as an indicator of cell lysis. Using EMA-PCR, we obtained a dynamic range of greater than 3 logs for differentiating viable vs. dead cells. The viability and morphological characteristics of the stressed cells after one-week incubation at 25 °C, in air, and under nutrient-poor conditions were investigated. Our results indicated that, over 99% of the stressed cells were converted from the spiral to the coccoid form and became non-culturable. However, more than 96% of the coccoid cells retained their membrane integrity as suggested by both the BacLight staining and EMA-PCR analyses. Thus, to detect C. jejuni under stress conditions, conventional culturing method in conjunction with EMA-PCR or BacLight staining might be a more appropriate approach.     

A New Multiplexed Real-Time PCR Assay to Detect <Emphasis Type="Italic">Campylobacter jejuni</Emphasis>, <Emphasis Type="Italic">C. coli</Emphasis>, <Emphasis Type="Italic">C. lari</Emphasis>, and <Emphasis Type="Italic">C. upsaliensis</Emphasis>

A new rapid method based on real-time PCR was developed to detect four thermophilic Campylobacter species (Campylobacter jejuni, Campylobacter coli, Campylobacter lari, and Campylobacter upsaliensis) in food samples. The assay targeted the bipA gene for C. upsaliensis and C. lari, whereas the gene encoding the ATP-binding protein CJE0832 was used to detect C. coli and C. jejuni. These genes were chosen for this assay due to their low variability and mutation rate at a species level. The multiplex PCR showed 100% inclusivity for all 25 thermophilic Campylobacter strains tested and 100% exclusivity for 38 non-targeted strains belonging to closely related species. The newly developed real-time PCR could detect down to 10² genomes/reaction and displayed efficiency above 97% for all species except for C. upsaliensis (90.1%). The method proved to be a reliable tool for food analysis, showing 100% sensitivity, 96% efficiency, and 92.45% specificity when validated against the gold standard method UNE-EN ISO 10272:2006 using 200 diverse food samples (meat, fish, fruits and vegetables, and raw milk). In artificially spiked samples, the detection limit of the method was 10 cfu/g in salad, 5 cfu/g in turkey meat, and 1 cfu/g in the rest of meat samples tested. Consequently, the newly designed molecular tool represents a quick and safe alternative to obtain reliable results concerning the presence/absence of the main thermophilic Campylobacter in any food sample.     

PREVALENCE OF THERMOPHILIC CAMPYLOBACTER SPP. IN RETAIL CHICKEN MEAT IN ANKARA

From May to August 2004, 127 samples of chicken meat for sale on the retail market in Ankara were analyzed for the prevalence of thermophilic Campylobacter spp. Campylobacter spp. were isolated from 83.4% of the samples analyzed. Campylobacter jejuni was found in 74.8% of all samples. A total of 364 thermophilic Campylobacter strains were isolated and the species distribution among these strains was 70.1% C. jejuni, 21.1% Campylobacter coli and 8.6% Campylobacter lari. The results obtained from the study indicate that hygienic and technical compliance is needed in all stages of poultry processing to reduce contamination and to prevent public health hazard. An integrated HACCP plan must be applied from the farm to the table for the prevention of C. jejuni infections.     

A survey on the incidence of Campylobacter spp. and the development of a surface adhesion polymerase chain reaction (SA-PCR) assay for the detection of Campylobacter jejuni in retail meat products

A rapid and sensitive method for the detection of Campylobacter in meat products was developed.Campylobacter were isolated from a range of Irish retail meats (n=80) and poultry (n=100) samples by direct plating on Campylobacter Selective Agar (CCDA, Oxoid). A total of 30·5% of samples tested positive forCampylobacter and Campylobacter jejuni was the most commonly identified species. The pathogen was detected in 39 (65%) poultry, 12 (60%) offal and 4 (20%) minced beef samples examined. Estimates derived from direct plate counts ranged from log₁₀0·7 to log₁₀2·75 cfu g⁻¹. The data from this study was used in the development of a rapid and sensitive method for the detection of Campylobacter in poultry. A rapid method was developed based on an initial sample enrichment for 24 h in Campylobacter Enrichment Broth (CEB), recovery of the pathogen from the enriched sample by surface adhesion onto a polycarbonate membrane, phenol:chloroform extraction of DNA from the adherent bacteria, and PCR analysis using primers specific for the flagellin A gene (present in C. jejuni and C. coli). The developed surface adhesion PCR (SA-PCR) technique had a detection limit of log₁₀4–5 and could be completed within 29 h. Results from SA-PCR analysis of a number of retail samples (n=50) correlated favourably with traditional plate culture results, i.e. 34 samples were found to contain Campylobacter by both methods.     

Model of inactivation of Campylobacter jejuni in poultry scalding

The purpose of this study was to develop a model of inactivation of Campylobacter jejuni in industrial scalding of chickens. Models can be used as a guide for broiler slaughterhouse operations for reducing levels of C. jejuni contamination on broiler carcasses. Mean concentrations of C. jejuni in terms of colony forming units (CFU) in scald tank water and in carcass rinse solution after scalding were 2.90 ± 0.07 and 3.86 ± 0.11 LogCFU/mL, respectively. Scald tank water temperature, flow rate, pH, and total solids in scalding process water were 54.15 ± 0.2 °C, 172.0 ± 8.4 L/min, 8.0 ± 0.01, and 2565 ± 114.3 mg/L, respectively. Inactivation models were developed by using mass balances and literature data for inactivation kinetics, of Campylobacter and the Arrhenius equation. Results of the inactivation models of scalding process indicate that high temperature and short time (less than 2 min) of scalding process were effective in reducing the number of viable cells. For this experimental data more than 50% of the Campylobacter are inactivated on surfaces of the chickens. The model fits the experimental data well and the values of the estimated parameters provide insight for this process. The model can be used for process design and potential process modifications.     

Prevalence,antimicrobial resistance,and molecular characterization of Campylobacter spp. in bulk tank milk and milk filters from US dairies

Campylobacter spp. are frequently isolated from dairy cows as commensal organisms. Sporadic Campylobacter infections in humans in the United States are generally attributed to poultry, but outbreaks are also commonly associated with dairy products, particularly unpasteurized or raw milk. Bulk tank milk samples and milk filters from US dairy operations were collected during the National Animal Health Monitoring System Dairy 2014 study and analyzed using real-time PCR and traditional culture techniques for the presence of thermophilic Campylobacter species. The weighted prevalence of operations from which we detected Campylobacter spp. in either bulk tank milk or milk filters was 24.9%. We detected Campylobacter spp. in a higher percentage of operations with 100–499 cows (42.8%) and 500 or more cows (47.5%) than in operations with 30–99 cows (6.5%). Campylobacter spp. were also more frequently detected in operations in the west than the east (45.9 and 22.6%, respectively). We isolated Campylobacter spp. from approximately half of PCR-positive samples, representing 12.5% (weighted prevalence) of operations. The majority (91.8%) of isolates were C. jejuni, but C. lari and C. coli were also isolated. We detected resistance to tetracycline in 68.4% of C. jejuni isolates, and resistance to ciprofloxacin and nalidixic acid in 13.2% of C. jejuni isolates. Based on pulsed-field gel electrophoresis, we found that dairy-associated C. jejuni were genotypically diverse, although clonal strains were isolated from different geographic regions. These results suggest that bulk tank milk can be contaminated with pathogenic Campylobacter spp., and that the consumption of unpasteurized or raw milk presents a potential human health risk.     

Survival of poultry-derived Campylobacter jejuni of multilocus sequence type clonal complexes 21 and 45 under freeze,chill, oxidative,acid and heat stresses

The application of multilocus sequence typing (MLST) for studying Campylobacter jejuni diversity reveals that MLST clonal complex (CC) 21 and CC-45 occupies significant proportion in the diverse population of C. jejuni. These two complexes are ecologically abundant and represent an interesting subpopulation for studying C. jejuni survival under different stress conditions. In the present study we characterize and compare 19 C. jejuni strains assigned to CC-21 and CC-45, isolated from chicken meat, based on laboratory stress models maintained in Muller-Hinton broth. Model conditions were mimicking freeze, chill, oxidative, acid and heat stresses. Results show that survival patterns varied between the strains. C. jejuni strains of CC-21 survived significantly better than C. jejuni strains of CC-45 under heat (P value = 0.022) and chill (P value = 0.001) stress models. On the other hand, C. jejuni strains of CC-45 showed significantly better survival compared to C. jejuni strains of CC-21 in response to oxidative (P value = 0.003) and freeze (P value = 0.021) stress models. C. jejuni strains assigned to the founder ST-45 showed significantly better survival (P value = 0.017) under heat stress model compared to their ancestral sequence types. However, an association between survival fitness and the diversification of a clonal group cannot be demonstrated directly from the obtained results. In conclusion, findings of the present study show that genotypic variations of C. jejuni might play a role in enabling certain lineages to be selected when encountering adverse and stressful environments. In future stress response studies, it is recommended to consider the effect of genotypic diversity among C. jejuni strains as that might bias the experimental findings.     

The physiologic and phenotypic alterations due to macrolide exposure in Campylobacter jejuni

Physiologic and phenotypic alterations in the context of antibiotic resistance have been extensively studied in some bacteria. However there are not enough data addressing these alterations due to macrolide resistance in Campylobacter jejuni. The present study examined the fitness cost imposed by different macrolide resistance mutations and the phenotypic alterations due to exposure to macrolides in C. jejuni. C. jejuni was induced with different macrolide agents to obtain different macrolide resistance mutations. The results revealed that the mutations significantly imposed defect variations on the doubling time and the relative fitness in the resistant strains when competed against the susceptible strain. Furthermore macrolides through induction or exposure to sub-MIC concentrations impaired the motility of C. jejuni in 0.4% MH agar plates. Electron microscope analysis revealed the absence of flagellar filaments from strains exposed to macrolides. SDS-PAGE demonstrated that macrolides have no effect on protein synthesis and immunoblotting analysis further confirmed that flagellin was fully synthesized within C. jejuni strains exposed to macrolides. Nevertheless C. jejuni strains exposed to macrolides demonstrated defect in their excreted flagellin into the supernatant compared to strains not exposed to macrolides. Accordingly we speculated that macrolides inhibited flagellar filament formation in the strains exposed to macrolides via affecting the secretion of flagellin without affecting the amount synthesized within the bacteria. Taken together these findings demonstrated that different macrolide resistance mutations imposed different fitness costs and exposure to macrolides resulted in phenotypic alterations such as inhibition of flagellar filament formation and loss of motility in C. jejuni.     

Mixed species biofilms of Listeria monocytogenes and Lactobacillus plantarum show enhanced resistance to benzalkonium chloride and peracetic acid

We investigated the formation of single and mixed species biofilms of Listeria monocytogenes strains EGD-e and LR-991, with Lactobacillus plantarum WCFS1 as secondary species, and their resistance to the disinfectants benzalkonium chloride and peracetic acid. Modulation of growth, biofilm formation, and biofilm composition was achieved by addition of manganese sulfate and/or glucose to the BHI medium. Composition analyses of the mixed species biofilms using plate counts and fluorescence microscopy with dual fluorophores showed that mixed species biofilms were formed in BHI (total count, 8-9 log₁₀ cfu/well) and that they contained 1-2 log₁₀ cfu/well more L. monocytogenes than L. plantarum cells. Addition of manganese sulfate resulted in equal numbers of both species (total count, 8 log₁₀ cfu/well) in the mixed species biofilm, while manganese sulfate in combination with glucose, resulted in 1-2 log₁₀ more L. plantarum than L. monocytogenes cells (total count, 9 log₁₀ cfu/well). Corresponding single species biofilms of L. monocytogenes and L. plantarum contained up to 9 log₁₀ cfu/well. Subsequent disinfection treatments showed mixed species biofilms to be more resistant to treatments with the selected disinfectants. In BHI with additional manganese sulfate, both L. monocytogenes strains and L. plantarum grown in the mixed species biofilm showed less than 2 log₁₀ cfu/well inactivation after exposure for 15 min to 100 μg/ml benzalkonium chloride, while single species biofilms of both L. monocytogenes strains showed 4.5 log₁₀ cfu/well inactivation and single species biofilms of L. plantarum showed 3.3 log₁₀ cfu/well inactivation. Our results indicate that L. monocytogenes and L. plantarum mixed species biofilms can be more resistant to disinfection treatments than single species biofilms.     

Prevalence and survival of Campylobacter in Egyptian dairy products

Campylobacter is a leading foodborne pathogen that poses an interesting paradox of being frequently transmitted by foodstuffs, yet showing sensitivity to food preservation procedures. In the present study, 227 samples of milk and dairy products were randomly collected and examined for the presence of Campylobacter spp. Samples were collected from the Egyptian area Abou-Homos, where the pathogen had been previously shown to be a major causative agent of intestinal disease. Potential Campylobacter isolates were speciated using a multiplex PCR assay targeting the housekeeping gene lpxA. Only raw milk and fresh Domiati cheese samples were found to contain Campylobacter jejuni at low incidence rates. Using a selected C. jejuni isolate recovered in this study, it was shown that the pathogen maintained its viability in Domiati cheese more than in yoghurt, yet it survived in both products more than expected. This suggests that this foodborne strain of C. jejuni may develop adaptive strategies that aid survival under food preservation conditions, which contradicts with what is known about this pathogen as a stress-sensitive organism.