Head-to-head comparison of CAMPYAIR aerobic culture medium versus standard microaerophilic culture for Campylobacter isolation from clinical samples.

Campylobacter spp. are considered the most frequent cause of acute gastroenteritis worldwide. However, outside high-income countries, its burden is poorly understood. Limited published data suggest that Campylobacter prevalence in low- and middle-income countries is high, but their reservoirs and age distribution are different. Culturing Campylobacter is expensive due to laboratory equipment and supplies needed to grow the bacterium (e.g., selective culture media, microaerophilic atmosphere, and a 42°C incubator). These requirements limit the diagnostic capacity of clinical laboratories in many resource-poor regions, leading to significant underdiagnosis and underreporting of isolation of the pathogen. CAMPYAIR, a newly developed selective differential medium, permits Campylobacter isolation without the need for microaerophilic incubation. The medium is supplemented with antibiotics to allow Campylobacter isolation in complex matrices such as human feces. The present study aims to evaluate the ability of the medium to recover Campylobacter from routine clinical samples. A total of 191 human stool samples were used to compare the ability of CAMPYAIR (aerobic incubation) and a commercial Campylobacter medium (CASA, microaerophilic incubation) to recover Campylobacter. All Campylobacter isolates were then identified by MALDI-TOF MS. CAMPYAIR showed sensitivity and specificity values of 87.5% (95% CI 47.4%-99.7%) and 100% (95% CI 98%-100%), respectively. The positive predictive value of CAMPYAIR was 100% and its negative predictive value was 99.5% (95% CI 96.7%-99.9%); Kappa Cohen coefficient was 0.93 (95% CI 0.79-1.0). The high diagnostic performance and low technical requirements of the CAMPYAIR medium could permit Campylobacter culture in countries with limited resources.

Dysbiosis of Gut Microbiome Is Associated With Rupture of Cerebral Aneurysms.

BACKGROUND AND PURPOSE: Environmental factors are important with respect to the rupture of cerebral aneurysms. However, the relationship between the gut microbiome, an environmental factor, and aneurysm rupture is unclear. Therefore, we compared the gut microbiome in patients with unruptured intracranial aneurysms (UIAs) and ruptured aneurysms (RAs) to identify the specific bacteria causing the rupture of cerebral aneurysms. METHODS: A multicenter, prospective case-control study was conducted over one year from 2019 to 2020. The fecal samples of patients with stable UIAs and RAs immediately after onset were collected. Their gut microbiomes were analyzed using 16S rRNA sequencing. Subsequently, a phylogenetic tree was constructed, and polymerase chain reaction was performed to identify the specific species. RESULTS: A total of 28 RAs and 33 UIAs were included in this study. There was no difference in patient characteristics between RAs and UIAs: age, sex, hypertension, dyslipidemia, diabetes status, body mass index, and smoking. No difference was observed in alpha diversity; however, beta diversity was significantly different in the unweighted UniFrac distances. At the phylum level, the relative abundance of Campylobacter in the RA group was larger than that in the UIA group. Furthermore, the gut microbiome in the RA and UIA groups exhibited significantly different taxonomies. However, Campylobacter was focused on because it is widely known as pathogenic among these bacteria. Then, a phylogenetic tree of operational taxonomic units related to Campylobacter was constructed and 4 species were identified. Polymerase chain reaction for these species identified that the abundance of the genus Campylobacter and Campylobacter ureolyticus was significantly higher in the RA group. CONCLUSIONS: The gut microbiome profile of patients with stable UIAs and RAs were significantly different. The genus Campylobacter and Campylobacter ureolyticus may be associated with the rupture of cerebral aneurysms.

Bacteriophage-Mediated Modulation of Bacterial Competition during Selective Enrichment of Campylobacter.

Selective media using antimicrobial supplements generate unique microbial ecology to facilitate bacterial isolation. However, antibiotic-resistant bacteria indigenous to samples can interfere with the isolation process using selective media. Recent studies showed that extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli is highly prevalent on retail raw chicken and compromises the efficacy of Campylobacter isolation because ESBL-producing E. coli are resistant to antimicrobial supplements in Campylobacter-selective media and outgrows Campylobacter. The objective of this study was to improve Campylobacter isolation by inhibiting the growth of ESBL-producing E. coli using bacteriophages (phages). The supplementation of Campylobacter-selective media with E. coli phages reduced the level of ESBL-producing E. coli during the enrichment step. When E. coli phages were combined with the antimicrobial supplements of Campylobacter-selective media, antimicrobial synergy was observed, particularly with rifampicin, an antibiotic used in Preston medium. Although the same materials (i.e., phages and selective media) were used, the sequence of combining the materials markedly influenced the inhibition of ESBL-producing E. coli and the isolation of Campylobacter. These findings indicated that the modulation of microbial competition at the enrichment step was critical to the successful isolation of fastidious bacteria and that phages can be utilized to facilitate the selective enrichment of target bacteria by inhibiting their competitive bacteria. IMPORTANCE Phages are promising antimicrobial alternatives. In this study, we first demonstrated that phages can be used to facilitate selective isolation of fastidious bacteria that are prone to be outgrown by bacterial competitors during isolation. The effectiveness of a phage-based isolation method was primarily dependent on the antimicrobial synergy between phages and antibiotics used in selective media. The same approach could be applied to the development of isolation methods for other fastidious bacteria.

Enhancement of Campylobacter hepaticus culturing to facilitate downstream applications.

Campylobacter hepaticus causes Spotty Liver Disease (SLD) in chickens. C. hepaticus is fastidious and slow-growing, presenting difficulties when growing this bacterium for the preparation of bacterin vaccines and experimental disease challenge trials. This study applied genomic analysis and in vitro experiments to develop an enhanced C. hepaticus liquid culture method. In silico analysis of the anabolic pathways encoded by C. hepaticus revealed that the bacterium is unable to biosynthesise L-cysteine, L-lysine and L-arginine. It was found that L-cysteine added to Brucella broth, significantly enhanced the growth of C. hepaticus, but L-lysine or L-arginine addition did not enhance growth. Brucella broth supplemented with L-cysteine (0.4 mM), L-glutamine (4 mM), and sodium pyruvate (10 mM) gave high-density growth of C. hepaticus and resulted in an almost tenfold increase in culture density compared to the growth in Brucella broth alone (log10 = 9.3 vs 8.4 CFU/mL). The type of culture flask used also significantly affected C. hepaticus culture density. An SLD challenge trial demonstrated that C. hepaticus grown in the enhanced culture conditions retained full virulence. The enhanced liquid culture method developed in this study enables the efficient production of bacterial biomass and therefore facilitates further studies of SLD biology and vaccine development.

Improvement of the detection efficiency of 3M™ molecular detection system for Campylobacter in poultry using nitrogen-doped carbon nanodots.

This study was performed to examine whether the use of nitrogen-doped carbon nanodots (N-CNDs) can improve the detection sensitivity of the 3 M™ molecular detection system (MDS) for Campylobacter. N-CNDs were added to a Campylobacter enrichment broth (CEB) at concentrations of 5 and 10 mg/mL (NCEB-5 and NCEB-10, respectively). Campylobacter coli, C. jejuni, and C. lari were inoculated into the broths. The broth cultures were then irradiated with light-emitting diode (LED) at 425 nm for 1 h and incubated at 42 °C for 6 h, and then grown on modified charcoal cefoperazone deoxycholate agar (mCCDA). The detection rates of the MDS and a conventional method (plating an enriched sample on mCCDA and analyzing a colony on mCCDA with PCR) for Campylobacter in chicken and duck carcasses were compared. The detection rates from the MDS were compared after enrichment in CEB and NCEB-5 at 3, 5, 6, 7, 9, 12, and 24 h. When 5 mg/mL of N-CNDs was added to the CEB followed by irradiation at 425 nm, growth of the Campylobacter was accelerated. In addition, the qualitative test was more sensitive in the MDS than in the conventional method, and the detection time was shortened in CEB enriched with N-CNDs. These results indicate that adding N-CNDs to CEB can improve the detection efficiency of MDS.

The effect of partial depopulation on Campylobacter introduction in broiler houses.

Poultry is seen as the main reservoir for Campylobacter. Control of this zoonotic pathogen in primary production could potentially reduce the colonization in broiler flocks and consequently reduce the number of human infections. In the present study, 20 broiler flocks from 10 farms, were sampled immediately before and 5 to 7 d after partial depopulation (thinning) for the presence of Campylobacter using cecal droppings and overshoes. At the time of thinning, the catching crew, transportation vehicles, forklift, and transport containers were sampled for the presence of Campylobacter. Samples were cultivated; presumed positive isolates were confirmed by PCR. The isolates were molecularly typed by flaA restriction analysis and pulsed field gel electrophoresis. Results show that all flocks were thinned using Campylobacter-contaminated equipment and materials. One-third of the broiler flocks became colonized after thinning. In 67% of the colonization cases, identical strains were found matching those of container systems, transport trucks, and/or forklifts. This identifies thinning as an important risk factor for Campylobacter introduction into broiler houses. Setup and compliance with biosecurity practices during thinning is essential to prevent Campylobacter colonization of broiler flocks.

Bacterial shifts on broiler carcasses at retail upon frozen storage.

Freezing broiler carcasses, industrially or at home, not only delays spoilage, but also is expected to increase food safety by hampering growth of food pathogens. However, detailed knowledge on microbial changes after a short or longer freezing period of fresh broiler meat in home freezing setting is lacking and no comparison between different freezing periods has been published yet. The present study combined classical isolation techniques and identification by MALDI-TOF MS with 16S rRNA amplicon sequencing to assess bacterial contamination on broiler carcasses that were either bought fresh and then frozen for short periods (total n = 20) in home freezing, or industrial frozen one (total n = 4) at retail. Changes in total aerobic bacteria (TAB) were also studied on 78 freshly bought broiler carcasses that were then stored frozen for up to 6 months in domestic freezers. Salmonella and Campylobacter were examined to assess the effect of freezing on controlling common foodborne pathogens. The contamination level of mesophilic and psychrotrophic TAB was numerically equal on carcasses at retail, either fresh or frozen at different time points. After short and long freezing period, a decrease in counts of mesophilic TAB was observed, while changes in counts of psychrotrophic TAB were rarely observed. No correlation between home freezing period and TAB load, either mesophilic (R = -0.006, p = 0.949) or psychrotrophic (R = 0.080, p = 0.389), was observed. No Salmonella and Campylobacter was detected on industrial frozen carcasses but on fresh carcasses at retail, either pre-freezing or after freezing. The bacterial communities were influenced by freezing, in which some genera showed significantly changes in relative abundance after freezing. In conclusion, from a food safety point of view, freezing of meat products does not serve as safety hurdle, and freezing should only be considered as a method for extending shelf life compared with fresh chicken meat. Applying hygienic slaughter procedures to keep the initial contamination as low as possible, and the maintenance of the cold chain during further processing are the key factors in food safety.

The impact of key processing stages and flock variables on the prevalence and levels of Campylobacter on broiler carcasses.

This study examined the impact of key processing stages and flock variables on the prevalence of Campylobacter on broiler carcasses. Overall, the prevalence of Campylobacter was 62% in caeca, and 68%, 65% and 62% in neck skin samples collected after evisceration, final wash and carcass chilling, respectively. Campylobacter were found in 32% of caeca, and 52%, 40% and 32% of neck skin samples collected after evisceration, final wash and carcass chilling, respectively from first thin broiler batches. Final thin broiler batches were more frequently contaminated with prevalences of 83% found in caeca, 80% in neck skin samples collected after evisceration and 83% found in neck skin samples collected after both final wash and carcass chilling stages (p < 0.05). Thinning status had a significant effect on Campylobacter counts with significantly higher counts observed in samples from final thin batches (p < 0.05). Highest Campylobacter concentrations in neck skin samples were observed at the evisceration stage in both first and final thin samples, with counts ranging from 2.0 to 3.8 log10 CFU/g and 2.3 to 4.8 log10 CFU/g in first and final thin batches, respectively. All first thin samples had counts below the European Union (EU) Process Hygiene Criterion threshold level of 3 log10 CFU/g after chilling while 52% of final thin batches had counts above this limit.

Evaluation of culture-based and molecular detection methods for Campylobacter in New Zealand raw cows' milk.

AIMS: This study evaluated the performance of a commercial molecular detection method (mericon Campylobacter triple kit real-time/quantitative (q)PCR) and a selective plating medium (R&F Campylobacter jejuni/Campylobacter coli Chromogenic Plating Medium (CCPM)) against a culture-based reference method (ISO 10272-1:2017 detection procedure B) for the detection of Campylobacter from raw milk enrichment broths. METHODS AND RESULTS: New Zealand raw cows' milk and Ultra-High Temperature-processed milk samples were inoculated with 50, 125 and 500 colony forming units of C. jejuni and C. coli cocktail per analytical unit. Samples were tested for Campylobacter after 0, 24- and 48 h refrigeration. ISO 10272-1:2017 proved to be a sensitive detection method (77/80 positive samples); detection only failed for some milk samples tested 48 h postinoculation. CCPM was as effective as Cefoperazone Charcoal Deoxycholate Agar for selective plating of Campylobacter raw milk enrichments (78/80 positive samples). However, the qPCR detected Campylobacter in only 42/80 samples and qPCR reaction inhibition was observed. CONCLUSIONS: The ISO 10272-1:2017 method was a more sensitive method for Campylobacter detection from raw milk than the mericon Campylobacter triple kit qPCR, and CCPM was a useful complementary medium to mCCDA where one of these media is required by the standard. SIGNIFICANCE AND IMPACT OF THE STUDY: In regions where testing is required or recommended, optimized methods for Campylobacter detection from raw milk will reduce risk to the raw milk consumer. Although molecular methods are generally touted as a rapid alternative to culture, issues with inhibition due to matrix components mean that culture-based methods might provide the most sensitive option for Campylobacter detection in raw milk. Findings also emphasize the importance of minimizing the time between milk collection and testing for Campylobacter.

Campylobacter risk for the consumers of wastewater-irrigated vegetables based on field experiments.

Water scarcity is emerging as a major problem in water stressed regions such as Middle East countries which highlights the importance of agricultural reuse of wastewater as a valid alternative source. However, consumption of wastewater-irrigated crops has been implicated as a vehicle for transmission of bacterial infections such as campylobacteriosis. Understanding and minimizing public health threats associated with agricultural reuse of treated wastewater (TWW) are crucial elements in sustainable water resource management. To address this need, the present study was carried out to determine Campylobacter risk for the consumers of TWW-irrigated vegetables by field experiments as well as quantitative microbial risk assessment (QMRA) model. Campylobacter was monitored in secondary treated wastewater, TWW-irrigated soil and harvested vegetables by nested real-time PCR assay. Campylobacter was detected in 64% (16/25) of TWW samples, whereas analysis of TWW-irrigated soil and vegetable samples yielded no positive result for Campylobacter. The estimated mean annual Campylobacter disease burden ranged from 2.37 × 10-5 to 6.6 × 10-5 disability-adjusted life years (DALYs) per person per year (pppy) for vegetable consumers which was lower than the less stringent reference level of 10-4 DALYs pppy has been recommended by world health organization (WHO). Our results in regard to the QMRA estimates and field experiments suggest that the reuse of TWW for irrigation of vegetables doesn't pose a considerable risk to human health from the viewpoint of Campylobacter infections in a semi-arid area.

Antimicrobial Susceptibility of Campylobacter isolates in the Capital of North Macedonia.

BACKGROUND: Campylobacter infections are typically self-limited, but in cases with severe enteritis, immuno-compromised system and bacteremia, an appropriate antimicrobial treatment is demanding. Our study aim was to determine the isolation rate of Campylobacter among patients with acute enteritis in the capital of North Macedonia and its antimicrobial susceptibility. MATERIAL AND METHODS: A total number of 3820 patients clinically diagnosed as acute enteritis, were included in the study. Stool samples were collected and Campylobacter was isolated and identified by classical microbiological methods. Antimicrobial susceptibility of all isolates to Ceftriaxone, Amoxicillin-clavulonic acid, Erythromycin, Ciprofloxacin, Tetracycline and Gentamicin was determined by disc-diffusion technique. Additionally, minimal inhibitory concentrations of all Campylobacter isolates against erythromycin, ciprofloxacin and tetracycline were determined by Epsilon gradient tests. RESULTS: Campylobacter species was isolated in 97 patients. Although the mean isolation rate of Campylobacter spp. during the whole study period was 2.53%, a statistically significant increase was detected in 2016 and 2017, in comparison with the data from previous four years of the study. The isolation rate of Campylobacter spp. didn't reveal statistically significant difference between males and females (p > 0.05). 46.4 % of patients with Campylobacter enteritis were children at the age under 15 years. Forty-three C. jejuni isolates were susceptible to all six antibiotics, but the remaining 44 isolates revealed resistance to at least one antibiotic. C. coli isolates were resistant to 3 antibiotics simultaneously. Two C. coli isolates only, were susceptible to all 6 antibiotics. 40.90% of C. jejuni and 50% of C. coli isolates were resistant to beta-lactams, fluoroquinolones and tetracyclines, simultaneously. CONCLUSION: The increase of the isolation rate of Campylobacter from patients with acute enteritis indicates the need for permanent isolation and identification of Campylobacter from every clinically diagnosed patient, as acute enteritis. Erythromicin is the most effective antibiotic for treatment of Campylobacter enteritis in our patients. The high level of Campylobacter resistance to beta-lactams, fluoroquinolones and tetracyclines requires more rational approach in the treatment of Campylobacter enteritis.

Source attribution at the food sub-product level for the development of the Canadian Food Inspection Agency risk assessment model.

Decreasing the health burden caused by foodborne pathogens is challenging and it depends on the identification of the most significant hazards and food sources causing illnesses, so adequate mitigation strategies can be implemented. In this regard, the Canadian Food Inspection Agency (CFIA) has developed the Establishment-based Risk Assessment (ERA) model, so that a more effective and efficient allocation of resources can be assigned to the highest food safety risk areas. To assess risk, the model considers the type of food sub-products being manufactured by establishments and its scope is limited to the 17 most important foodborne pathogens representing the highest level of food safety risk. However, the information on source attribution at the sub-product level based on a structured approach is limited. To overcome this challenge, an expert elicitation was conducted in 2016 to estimate the relative contribution and associated certainty of each sub-product for 31 pathogen-commodity combinations to the total Canadian health burden associated with foodborne illnesses (expressed in DALYs). These DALYs represent 78% of the total Canadian health burden associated with federally-regulated food commodities considered within the model. A total of 49 Canadian experts recruited using a "snow ball" sampling strategy participated in the study by completing an electronic survey. Results of the elicitation displayed variable levels of health burden allocation between the pathogens and the different commodity sub-products. Assessment of the certainty levels showed some combinations being evaluated with more confidence (e.g., Campylobacter and eggs/poultry sub-products) than others, where a bimodal distribution of certainty was observed (e.g., Toxoplasma in pork sub-products). Furthermore, no participant raised concerns on the food classification scheme, suggesting their agreement with the proposed sub-products categorization of the elicitation. Relative contribution estimates will be included in the CFIA ERA model and used to enhance its applicability for risk prioritization and effective resource allocation during food establishment inspections. While substantial uncertainty around the central tendency estimates was found, these estimates provide a good basis for regulatory oversight and public health policy.

Peracetic acid reduces Campylobacter spp. on turkey skin: Effects of a spray treatment on microbial load, sensory and meat quality during storage.

Handling and consumption of Campylobacter-contaminated poultry meat is the most common cause of human campylobacteriosis. While many studies deal with interventions to reduce Campylobacter spp. on chicken carcasses, studies on other poultry species are rare. In the present study, a spray treatment with peracetic acid (PAA) on turkey carcasses was evaluated. For this, parts of breast fillets with skin and Campylobacter (C.) jejuni DSM 4688 (108 cfu/ml) inoculated drumsticks were sprayed for 30 s with PAA (1200 ppm) or water as control solution. Samples were packaged under modified atmosphere and stored at 4°C until analysis on day 1, 6 and 12. The breast fillets were used for determination of the total viable count, sensory and meat quality examination as well as myoglobin content and biogenic amines. The drumsticks were used for C. jejuni counts. PAA had a significant effect in reducing total viable counts on all days by up to 1.2 log10 compared to the untreated control. Treatment with water alone showed no effect. C. jejuni counts were significantly reduced by PAA (0.9-1.3 log10), while water achieved a 0.5 log10 reduction on C. jejuni counts on day 1. No differences in sensory, pH, electrical conductivity and myoglobin content could be found. The skin of the PAA treated fillets had lower redness values than the water control on day 1, whereas on day 12 parts of the water treated muscles were lighter than the untreated control. A lower putrescine content of the water sprayed fillets in comparison to the control sample on day 12 was the only significant difference concerning the biogenic amines. Results from this study indicate that a spray treatment with 1200 ppm PAA would be a useful measure to lower the Campylobacter spp. counts on turkey carcasses without having a negative influence on product quality.

Population Dynamics of Thermotolerant Campylobacter in Broilers Reared on Reused Litter.

A study using sentinel broiler chickens was performed to address Campylobacter persistence in litter that was reused for successive flocks. Cloacal swabs, litter, drag swabs, darkling beetles, feed, and drinking water were weekly sampled and analyzed by standard microbiological procedures. Thermotolerant Campylobacter isolated strains were confirmed by polymerase chain reaction and subtyped by pulsed-field gel electrophoresis analysis. Campylobacter was not detected in samples collected immediately after downtime between broiler flocks. However, Campylobacter-positive samples were first detected at 21 d. After Campylobacter was initially isolated from the cloacal swabs, reused litter, drag swabs, or darkling beetles, these samples remained Campylobacter positive in the following weeks until the end of the rearing period. Campylobacter-positive cloacal swabs obtained from sentinel broilers ranged from 97.3% to 100% at 42 d. All isolated strains were identified as Campylobacter jejuni. Among the subtypes identified, an indistinguishable C. jejuni strain was predominant in sentinel broilers and was also detected in the other environmental samples analyzed, suggesting a common and persistent contamination source within the flocks. Sentinel broilers may have contributed to amplify the Campylobacter level, maintaining flock and broiler house contamination until the end of the production cycle.

Comparison of media and growth conditions for culturing enterohepatic Helicobacter species.

This research aims to compare the culturing conditions for enterohepatic Helicobacter, evaluating culture media, incubation atmosphere and susceptibility to antimicrobials used to generate selective conditions. Four common media for the closely related genus Campylobacter (Columbia, Bolton, Brucella and CCDA agar), as well as the need for hydrogen in the microaerobic incubation atmosphere, were evaluated. Serial dilutions of 13 strains belonging to six species (H. apodemus, H. bilis, H. canicola, H. canis, H. equorum and Helicobacter sp.) were inoculated in each media and incubated at 37°C for 48 to 96 h using CampyGen (OXOID) and gaseous exchange (including hydrogen) in parallel. Columbia or Brucella agars were the most appropriate for culturing EHH (P < 0·05). However, there was no significant difference between the atmospheres evaluated (P = 0·13). In addition, minimal inhibitory concentration for six antibiotics showed that all isolates were resistant to trimethoprim, whereas for the rest of the antibiotics (cephalothin, cefoperazone, cefsulodin, teicoplanin and vancomycin) the inhibition range was between 8 and 64 µg ml- 1 . Our findings suggest that Columbia or Brucella media, regardless of the use of hydrogen, can be used for the EHH isolation. In addition, the concentration of antibiotics included in commercial campylobacteria supplements is suitable for EHH species recovery. SIGNIFICANCE AND IMPACT OF THE STUDY: Enterohepatic Helicobacter (EHH) infections have been associated with several diseases in humans such as acute gastroenteritis, inflammatory bowel disease and hepatobiliary diseases. Although they are frequently detected in clinical samples by molecular methods, only occasionally they are isolated using culture conditions described for the taxonomic related pathogen Campylobacter sp. This is because the optimal conditions for the isolation of EHH have not yet been described, which results in an underestimation of the prevalence and clinical importance of these emerging pathogens. Therefore, this study provides insight for culturing EHH species.

Enhanced microbiological surveillance reveals that temporal case clusters contribute to the high rates of campylobacteriosis in a model agroecosystem.

Infections by pathogenic Campylobacter species were determined in diarrheic (n = 2,217) and non-diarrheic control (n = 104) people in Southwestern Alberta (SWA), Canada over a 1-year period using specialized and conventional isolation, and direct PCR. Overall, 9.9% of diarrheic individuals were positive for C. jejuni (9.1%), C. upsaliensis (0.6%), and C. coli (0.5%). No C. lari was detected. Four diarrheic individuals were co-infected with C. jejuni and C. coli, and four different individuals were co-infected with C. jejuni and C. upsaliensis. Two control individuals were positive for C. jejuni. Approximately 50% of stools containing C. jejuni and/or C. coli were deemed negative by conventional isolation. Direct PCR for C. jejuni was less effective than culture-based detection. Most C. jejuni infections occurred in people living in the urban centers, but the prevalence of the bacterium was lower in females than males living in urban locations, and both males and females living in rural locations. Although C. jejuni was detected throughout the year, a trend for higher infection rates was observed in the late spring to early fall with a peak in August. Forty-six C. jejuni subtype clusters were identified, including 44 temporal case clusters attributed to 28 subtype groupings. The majority of infections (70.3%) were linked to subtypes associated with beef cattle. We conclude that many occurrences of pathogenic Campylobacter species were not detected by the conventional laboratory methodology, and temporal case clusters of C. jejuni subtypes associated with cattle contribute to the high rates of campylobacteriosis in SWA.

Multi-step optimization of the filtration method for the isolation of Campylobacter species from stool samples.

The filtration method (FM) is the most effective isolation technique for Epsilobacteriaceae from stool samples. FM's different adaptations make it difficult to compare data between studies. This study was performed in three phases to optimize FM from a routine laboratory perspective. In July-September 2014 (part I), FM was performed on Mueller-Hinton agar containing 5% sheep blood and Columbia agar containing 5% sheep blood. In July 2016 (part II), FM was performed using 0.60-µm pore size polycarbonate filters (0.6-PC filter) and 0.45-µm pore size cellulose acetate filters (0.45-AC filter); in January 2018 (part III), the addition of hydrogen to incubators was studied. On 1146 stools analyzed in part I, the positive samples that showed no growth on the Butzler medium (n = 22/72, 30.6%) had improved growth of Epsilobacteriaceae when using the Columbia instead of the Mueller-Hinton medium (21/22 strains vs. 11/22, p < 0.05). In part II, on 718 stools, 91 strains grew with FM (12.7%), more with 0.6-PC filter (90/91) than with 0.45-AC filter (44/91) (p < 0.05). In part III, 578 stools were cultured, 98 Epsilobacteriaceae strains grew with FM, and 7% hydrogen finding significantly more Epsilobacteriaceae than without hydrogen (90/98, 91.8%, vs. 72/98, 73.5%; p < 0.05). The use of a Columbia medium containing 5% sheep blood with 0.6-PC filters incubated at 37 °C in a 7% hydrogen-enriched atmosphere led to an almost fourfold increase in the isolation rate of Epsilobacteriaceae among the studied combinations. Reference centers for Campylobacter should use standardized protocols to enable the comparison of prevalence in space and time.

Development and evaluation of a novel Campylobacter spp. enrichment medium.

Although rapid detection kits continue to be developed and used, the classical culture method is still accepted as a gold standard. Therefore accelerating the classical culture methods safely is important for the detection of Campylobacter. The aim of this study is to design and compare a novel developed medium with other confirmed media in naturally and experimentally contaminated food matrices. Besides classical culture methods, it is subjected to qPCR and FISH methods. In this study, Campylobacter counts are investigated in spiked milk, chicken breast meat, cumin, minced beef meat, celery and tomato puree. Also to evaluate the enrichment medium in naturally contaminated samples, Campylobacter detection is performed in 20 chicken neck skin samples obtained from different sales points. The study showed that the novel broth provides a faster detectable number of Campylobacter. It was found to provide detectable Campylobacter counts after eight hours of inoculation. The results have shown that there is a significant increase on Campylobacter count in the detection performed using the spiked foods. Furthermore, the entire natural contaminated chicken neck skin samples are found to be positive the same as the other mediums. As a result of the study, in the classic culture methods, designed enrichment medium is faster than the currently used enrichment mediums. It is an important point of view to develop fast and reliable diagnostic methods for assuring adequate public health.

Motility and biofilm formation of the emerging gastrointestinal pathogen Campylobacter concisus differs under microaerophilic and anaerobic environments.

Campylobacter concisus has been isolated from patients with gastroenteritis and inflammatory bowel disease (IBD), as well as healthy subjects. While strain differences may plausibly explain virulence differentials, an alternative hypothesis posits that the pathogenic potential of this species may depend on altered ecosystem conditions in the inflamed gut. One potential difference is oxygen availability, which is frequently increased under conditions of inflammation and is known to regulate bacterial virulence. Hence, we hypothesized that oxygen influences C. concisus physiology. We therefore characterized the effect of microaerophilic or anaerobic environments on C. concisus motility and biofilm formation, two important determinants of host colonization and dissemination. C. concisus isolates (n = 46) sourced from saliva, gut mucosal biopsies and feces of patients with IBD (n = 23), gastroenteritis (n = 8) and healthy subjects (n = 13), were used for this study. Capacity to form biofilms was determined using crystal violet assay, while assessment of dispersion through soft agar permitted motility to be assessed. No association existed between GI disease and either motility or biofilm forming capacity. Oral isolates exhibited significantly greater capacity for biofilm formation compared to fecal isolates (p<0.03), and showed a strong negative correlation between motility and biofilm formation (r = -0.7; p = 0.01). Motility significantly increased when strains were cultured under microaerophilic compared to anaerobic conditions (p<0.001). Increased biofilm formation under microaerophillic conditions was also observed for a subset of isolates. Hence, differences in oxygen availability appear to influence key physiological aspects of the opportunistic gastrointestinal pathogen C. concisus.

Evaluation of potassium clavulanate supplementation of Bolton broth for enrichment and detection of Campylobacter from chicken.

Culture-based detection of Campylobacter can be affected by competing flora, temperature, incubation time, and presence of blood. The presence of extended-spectrum ß-lactamase (ESBL)-producing Escherichia coli in poultry has become one of the most common factors interfering with the detection of Campylobacter. In the present study, we evaluated potassium clavulanate (ESBL inhibitor) as a supplement in Bolton broth (C-Bolton broth) for enrichment and detection of Campylobacter. First, we determined growth kinetics of Campylobacter in the presence of different concentrations of ESBL E. coli in C-Bolton broth during enrichment. The effects of other factors such as incubation time, incubation temperature, and presence of blood on Campylobacter detection in C-Bolton broth were also investigated. The growth of Campylobacter co-cultured at a low concentration (2 and 4 log10 CFU/mL) of ESBL E. coli was similar to that of Campylobacter alone in C-Bolton broth, and Campylobacter co-cultured at a high concentration (6 and 8 log10 CFU/mL) of ESBL E. coli showed slower growth than the pure Campylobacter culture. The Campylobacter detection limit was 1 log10 CFU/mL when mixed with 2, 4, or 6 log10 CFU/mL of E. coli and 3 log10 CFU/mL when mixed with 8 log10 CFU/mL of E. coli after 48 h enrichment in the broth. Campylobacter detection from chicken feces and litter samples was not affected by incubation time, or presence of blood in the broth. A modified procedure of enrichment in C-Bolton broth at 37°C for 24 h without blood showed a significantly (P ≤ 0.05) higher detection rate and a lower false-negative rate than the ISO 10272-1:2006 method for Campylobacter detection from chicken feces and litter samples. In summary, the present study demonstrates the efficacy of Bolton broth supplemented with potassium clavulanate in the detection of Campylobacter mixed with ESBL E. coli, and an improved procedure to detect Campylobacter from chicken feces and litter samples.