flaA Restriction Fragment Length Polymorphism Research Articles

Assessment of biofilm formation by Campylobacter spp. isolates mimicking poultry slaughterhouse conditions

This research aimed to assess the biofilm formation ability of Campylobacter strains under temperature and oxygen stress conditions, similar to those found in the industrial environment, to explain the persistence of this pathogen on the poultry slaughter line. A collection of C. jejuni and C. coli isolates (n = 143) obtained from poultry samples (cecal content and neck skin), collected at slaughterhouse level, from diverse flocks, on different working days, was genotyped by flaA-restriction fragment length polymorphism (RFLP) typing method. A clustering analysis resulted in the assignment of 10 main clusters, from which 15 strains with different flaA-RFLP genotypes were selected for the assessment of biofilm formation ability and antimicrobial susceptibility. Biofilm assays, performed by crystal violet staining method, were conducted with the goal of mimicking some conditions present at the slaughterhouse environment, based on temperature, atmosphere, and contamination levels. Results indicated that many C. jejuni strains with similar flaA-RFLP profiles were present at the slaughterhouse on different processing days. All the strains tested (n = 15) were multidrug-resistant except for one. Biofilm formation ability was strain-dependent, and it appeared to have been affected by inoculum concentration, temperature, and tolerance to oxygen levels. At 10°C, adherence levels were significantly lower than at 42°C. Under microaerobic and aerobic atmospheres, at 42°C, 3 strains (C. jejuni 46E, C. jejuni 61C, and C. coli 65B) stood out, exhibiting significant levels of biofilm formation. C. jejuni strains 46E and 61C were inserted in clusters with evidence of persistence at the slaughterhouse for a long period of time. This study demonstrated that Campylobacter strains from broilers are capable of forming biofilms under conditions resembling the slaughterhouse environment. These results should be seen as a cue to improve the programs of hygiene implemented, particularly in those zones that can promote biofilm formation.

Antimicrobial Resistance Profiles, Virulence Genes, and Genetic Diversity of Thermophilic Campylobacter Species Isolated From a Layer Poultry Farm in Korea.

Thermophilic Campylobacter species are among the major etiologies of bacterial enteritis globally. This study aimed at assessing the antimicrobial resistance (AMR) profiles, virulence genes, and genetic diversity of thermophilic Campylobacter species isolated from a layer poultry farm in South Korea. One hundred fifty-three chicken feces were collected from two layer poultry farms in Gangneung, South Korea. The Campylobacter species were isolated by cultural techniques, while PCR and sequencing were used for species confirmation. Antimicrobial susceptibility testing for six antimicrobials [ciprofloxacin (CIP), nalidixic acid (NAL), sitafloxacin (SIT), erythromycin (ERY), tetracycline (TET), and gentamicin (GEN)] was carried out by broth microdilution. Three AMR and nine virulence genes were screened by PCR. Genotyping was performed by flaA-restriction fragment length polymorphism (RFLP) and multilocus sequence typing (MLST). Of the 153 samples, Campylobacter spp. were detected in 55 (35.9%), with Campylobacter jejuni and Campylobacter coli being 49 (89.1%) and six (10.9%), respectively. High-level resistance was observed for CIP (100%), NAL (100%), and TET (C. jejuni, 93.9%; C. coli: 83.3%). No resistance was observed for SIT. The missense mutation (C257T) in gyrA gene was confirmed by sequencing, while the tet(O) gene was similar to known sequences in GenBank. The rate of multidrug-resistant (MDR) strains was 8.2%, and they all belonged to C. jejuni. All Campylobacter isolates possessed five virulence genes (cdtB, cstII, flaA, cadF, and dnaJ), but none possessed ggt, while the rates for other genes (csrA, ciaB, and pldA) ranged between 33.3 and 95.9%. The flaA-RFLP yielded 26 flaA types (C. jejuni: 21 and C. coli: five), while the MLST showed 10 sequence types (STs) for C. jejuni and three STs for C. coli, with CC-607 (STs 3611) and CC-460 (ST-460) being predominant. Among the 10 STs of C. jejuni, three were newly assigned. The findings of this study highlight the increased resistance to quinolones and TET, the virulence potential, and the diverse genotypes among Campylobacter strains isolated from the layer poultry farm.

Prevalence and Genetic Diversity of C. Jejuni Isolated from Broilers and their Environment Using flaA-RFLP Typing and MLST Analysis

Abstract Campylobacter is highly diverse genetically and also undergoes frequent intraspecific recombination. A major source of campylobacteriosis, which is transmitted to humans is found in poultry. The assessment of the genetic diversity among Campylobacter population is critical to our understanding of the epidemiology. The genetic diversity of Campylobacter jejuni isolates in broilers and their environment were investigated by flaA-restriction fragment length polymorphism (RFLP) and multilocus sequence typing (MLST). The study revealed that 92.3% of the examined broiler flocks were contaminated with Campylobacter spp. A total number of 35 different flaA types defined by flaA-RFLP were found in 448 C. jejuni isolates originated from broilers, litter, puddles, zones, anteroom and wild birds. The most dominant flaA type was XXV. MLST defined 20 sequence types (STs) belonging to 10 clonal complexes (CCs). Among all the STs 9 isolates (15%) were consigned to 2 different STs (ST-7413 and ST-4800), which could not be assigned. The most common CCs were ST-21 and ST-179. The ST-21 CC was common in broilers and environment (puddle water and concentric zones) and the ST-179 CC was specific to wild birds, but also was found in puddle water and concentric zones.

Molecular epidemiology of Campylobacter isolates from broiler slaughterhouses in Tripoli, North of Lebanon

ABSTRACT 1. The real burden of Campylobacter spp. in Lebanon is still unknown. The aims of this study were to unravel the epidemiology of Campylobacter spp. in broilers at slaughterhouses in Tripoli, North of Lebanon and to characterise their antibiotic resistance profiles. 2. From May to November 2015, sampling was performed through five repeated surveys from 15 slaughterhouses that sold chicken directly to Lebanese customers. Isolates were subjected to pulsed field gel electrophoresis (PFGE) and flaA-restriction fragment length polymorphism (flaA-RFLP). 3. All investigated slaughterhouses were found to be positive for Campylobacter spp. Campylobacter coli was the predominant species (38 isolates) followed by C. jejuni (eight isolates). A noticeable level of resistance was detected among isolates against ciprofloxacin (97% of C. coli and 87.5% of C. jejuni), amoxicillin (89% of C. coli and 75% of C. jejuni), gentamicin (79% of C. coli and 50% of C. jejuni), and co-amoxiclav (24% of C. coli and 25% of C. jejuni). Erythromycin and ertapenem resistance were observed only in C. coli with the following percentages 74% and 13% respectively, but not in C. jejuni. PFGE and flaA-RFLP using DdeI as restriction enzyme divided the strains into 27 and 25 types respectively. 4. The high observed genetic diversity of Campylobacter spp. revealed the complexity of the spread of this genus in broilers. This study highlighted the pressing need to monitor antibiotic resistance and to ensure food safety from ‘farm to fork’ in Lebanon.

Dairy Cattle, a Potential Reservoir of Human Campylobacteriosis: Epidemiological and Molecular Characterization of Campylobacter jejuni From Cattle Farms.

Campylobacter jejuni is a major foodborne pathogen that is increasingly found worldwide and that is transmitted to humans through meat or dairy products. A detailed understanding of the prevalence and characteristics of C. jejuni in dairy cattle farms, which are likely to become sources of contamination, is imperative and is currently lacking. In this study, a total of 295 dairy cattle farm samples from 15 farms (24 visits) in Korea were collected. C. jejuni prevalence at the farm level was 60% (9/15) and at the animal level was 23.8% (68/266). Using the multivariable generalized estimating equation (GEE) model based on farm-environmental factors, we estimated that a high density of cattle and average environmental temperature (7 days prior to sampling) below 24°C affects the presence and survival of C. jejuni in the farm environment. Cattle isolates, together with C. jejuni from other sources (chicken and human), were genetically characterized based on analysis of 10 virulence and survival genes. A total of 19 virulence profile types were identified, with type 01 carrying eight genes (all except hcp and virB11) being the most prevalent. The prevalence of virB11 and hcp was significantly higher in isolates from cattle than in those from other sources (p < 0.05). Multilocus sequence typing (MLST) of C. jejuni isolates from three different sources mainly clustered in the CC-21 and CC-48. Within the CC-21 and CC-48 clusters, cattle isolates shared an indistinguishable pattern with human isolates according to pulsed-field gel electrophoresis (PFGE) and flaA-restriction fragment length polymorphism (RFLP) typing. This suggests that CC-21 and CC-48 C. jejuni from dairy cattle are genetically related to clinical campylobacteriosis isolates. In conclusion, the farm environment influences the presence and survival of C. jejuni, which may play an important role in cycles of cattle re-infection, and dairy cattle represent potential reservoirs of human campylobacteriosis. Thus, environmental management practices could be implemented on cattle farms to reduce the shedding of C. jejuni from cattle, subsequently reducing the potential risk of the spread of cattle-derived C. jejuni to humans through the food chain.

High-Throughput flaA Short Variable Region Sequencing to Assess Campylobacter Diversity in Fecal Samples From Birds.

Current approach to identify sources of human pathogens is largely dependent on the cultivation and isolation of target bacteria. For rapid pathogen source identification, culture-independent strain typing method is necessary. In this study, we designed new primer set that broadly covers flaA short variable region (SVR) of various Campylobacter species, and applied the flaA SVR sequencing method to examine the diversity of Campylobacter spp. in geese fecal samples (n = 16) with and without bacteria cultivation. Twenty-three Campylobacter strains isolated from the 16 geese fecal samples were grouped similarly by conventional flaA restriction fragment length polymorphism (RFLP) method and by the flaA SVR sequencing method, but higher discriminant power was observed in the flaA SVR sequencing approach. For culture-independent flaA SVR sequencing analysis, we developed and optimized the sequence data analysis pipeline to identify as many genotypes as possible, while minimizing the detection of genotypes generated by sequencing errors. By using this pipeline, 51,629 high-quality flaA sequence reads were clustered into 16 operational taxonomic units (=genotypes) by using 98% sequence similarity and >50 sequence duplicates. Almost all flaA genotypes obtained by culture-dependent method were also identified by culture-independent flaA SVR MiSeq sequencing method. In addition, more flaA genotypes were identified probably due to high throughput nature of the MiSeq sequencing. These results suggest that the flaA SVR sequencing could be used to analyze the diversity of Campylobacter spp. without bacteria isolation. This method is promising to rapidly identify potential sources of Campylobacter pathogens.

Prevalence, genetic diversity and antimicrobial susceptibility of Campylobacter jejuni isolated from retail food in China

The aim of this study was to determine the prevalence, antimicrobial resistance and molecular epidemiology of Campylobacter jejuni isolated from retail food samples in China and assess the potential risk to consumer' health. A total of 34 strains of C. jejuni isolated from 1798 retail food samples in China were identified using conventional methods and duplex PCR aimed 16S and hipo genes. There are 59 positive samples by conventional method, and the prevalence of C. jejuni was 3.30% (59/1798). Using flaA RFLP-PCR (flaA Restriction Fragment Length Polymorphism) typing with a relative similarity coefficient of 0.81, the 34 isolates and 1 reference strains were grouped into 4 clusters and 2 singletons. Antibiotic resistance profiles determined using the disk diffusion method showed that a high proportion of C. jejuni isolates were resistant to the tested antibiotics, particularly for ciprofloxacin (88.5%) and nalidixic acid (88.5%). Most isolates were multidrug resistant, with 20 strains showing resistance to 5–8 antibiotics and 2 strains showing resistance to 9 antibiotics. Moreover, analysis using MAMA-PCR to determine the gyrA mutation status (C257T) indicated that approximately 30 strains had mutations, and these findings were consistent with disk diffusion assay results. Examination for the presence of 11putative virulence genes showed that the majority of these genes were detected in all C. jejuni strains. There was no direct evidence to suggest a connection between antibiotic resistance and virulence genes. This study is the first to explore the potential sources of C. jejuni contamination in retail foods in China and provide comprehensive surveillance on its incidence in retail foods. These data will ensure more accurate treatment of human campylobacteriosis using effective antibiotics.

Mapping the carriage of flaA-restriction fragment length polymorphism Campylobacter genotypes on poultry carcasses through the processing chain and comparison to clinical isolates

Poultry are considered a major source for campylobacteriosis in humans. A total of 1866 Campylobacter spp. isolates collected through the poultry processing chain were typed using flaA-restriction fragment length polymorphism to measure the impact of processing on the genotypes present. Temporally related human clinical isolates (n = 497) were also typed. Isolates were obtained from whole chicken carcass rinses of chickens collected before scalding, after scalding, before immersion chilling, after immersion chilling and after packaging as well as from individual caecal samples. A total of 32 genotypes comprising at least four isolates each were recognised. Simpson's Index of Diversity (D) was calculated for each sampling site within each flock, for each flock as a whole and for the clinical isolates. From caecal collection to after packaging samples the D value did not change in two flocks, decreased in one flock and increased in the fourth flock. Dominant genotypes occurred in each flock but their constitutive percentages changed through processing. There were 23 overlapping genotypes between clinical and chicken isolates. The diversity of Campylobacter is flock dependant and may alter through processing. This study confirms that poultry are a source of campylobacteriosis in the Australian population although other sources may contribute.

Absence of tetracycline resistance inCampylobacter coliisolates from Finnish finishing pigs treated with chlortetracycline

To determine whether therapeutic treatment of pigs with chlortetracycline affects the susceptibility of their Campylobacter isolates for tetracycline, ciprofloxacin and erythromycin. Minimum inhibitory concentrations (MICs) and presence of a tetracycline resistance gene tet(O) were studied in Campylobacter collected before, during and after chlortetracycline treatment. Tetracycline MICs and the presence of tet(O) for additional Campylobacter coli isolates collected previously from seven farrowing farms were also determined. Isolates with ciprofloxacin MICs above the epidemiological cut-off value (ECOFF) were subtyped by flaA restriction fragment length polymorphism (RFLP). Tetracycline MICs of 221 Camp. coli isolates remained under the ECOFF at all sampling stages as well as the MICs for 63 isolates from the other farms. The ciprofloxacin MIC was above the ECOFF for 22% of the isolates, and one Camp. coli isolate had an erythromycin MIC above the ECOFF. None of the studied 300 Campylobacter isolates from nine herds carried tet(O). flaA-RFLP typing revealed the heterogeneity of Camp. coli isolates with high ciprofloxacin MICs. Use of chlortetracycline did not increase the MIC values for the antimicrobials studied. This study demonstrated that susceptibility of Camp. coli isolates is not affected by chlortetracycline therapy if tet(O) is not present in Camp. coli population.

Epidemiology, Relative Invasive Ability, Molecular Characterization, and Competitive Performance of Campylobacter jejuni Strains in the Chicken Gut

One hundred forty-one Campylobacter jejuni isolates from humans with diarrhea and 100 isolates from retailed poultry meat were differentiated by flaA typing. The bacteria were isolated in a specific geographical area (Dunedin) in New Zealand over a common time period. Twenty nine flaA types were detected, one of which (flaA restriction fragment length polymorphism type 15 [flaA-15]) predominated among isolates from humans ( approximately 30% of isolates). This strain was of low prevalence (5% of isolates) among poultry isolates. flaA-15 strains were five to six times more invasive of HEp2 cells in an in vitro assay than a flaA type (flaA-3) that was commonly encountered on poultry meat (23% of isolates) but was seldom associated with human illness (5%). Competitive-exclusion experiments with chickens, utilizing real-time quantitative PCR to measure the population sizes of specific strains representing flaA-15 (T1016) and flaA-3 (Pstau) in digesta, were carried out. These experiments showed that T1016 always outcompeted Pstau in the chicken intestine. Genomic comparisons of T1016 and Pstau were made using DNA microarrays representing the genome of C. jejuni NCTC 11168. These comparisons revealed differences between the strains in the gene content of the Cj1417c-to-Cj1442c region of the genome, which is associated with the formation of capsular polysaccharide. The strains differed in Penner type (T1016, O42; Pstau, O53). It was concluded that poultry meat was at least one source of human infection with C. jejuni, that some Campylobacter strains detected in poultry meat are of higher virulence for humans than others, and that bacterial attributes affecting strain virulence and commensal colonization ability may be linked.

Genetic diversity in fluoroquinolone and macrolide-resistant Campylobacter coli from pigs

The genetic diversity of 115 Campylobacter coli strains, isolated from pigs of 59 geographical distant farms in Switzerland, were characterized on the basis of their DNA fingerprints and resistance to macrolides and fluoroquinolones. Sequence analysis showed that the macrolide-resistant isolates had a point mutation in the 23S ribosomal RNA (rRNA) genes (A2075G) and that the fluoroquinolone-resistant isolates had a point mutation in the gyrase gene gyrA (C257T). One fluoroquinolone-resistant strain had an additional transition mutation in the gyrB gene (A1471C). The flaA restriction fragment length polymorphism (RFLP) genotyping revealed that 57% of the isolates were genetically different. Point mutations in the 23S rRNA and gyrA genes could be found in both genetically distant and genetically related isolates. Additionally, isolates with and without point mutations were found within individual farms and on different farms. This study showed that the ciprofloxacin and erythromycin-resistant C. coli population present on the pig farms is not issued from a common ancestral clone, but individual Campylobacter strains have most likely mutated independently to acquire resistances under the selective pressure of an antibiotic.

Tetracycline resistance of Australian Campylobacter jejuni and Campylobacter coli isolates

Tetracycline resistance in Campylobacter is encoded by the tet(O) gene and is usually associated with conjugative plasmids. Little was known about tetracycline resistance in Australian Campylobacter species, therefore we investigated this resistance in 41 Campylobacter jejuni and five Campylobacter coli strains from humans and healthy chickens. Tetracycline MICs were determined for each isolate using an agar dilution method. The distribution and localization of tet(O) on plasmid and chromosomal DNA was determined by Southern-blot experiments. The ability to transfer resistance to recipient strains was examined through conjugation studies. Identity of transconjugants was confirmed by PCR and flaA-restriction fragment length polymorphism analysis. High-level tetracycline resistance was observed, ranging from 32 to >256 mg/L. Plasmids were detected in 74% of isolates with plasmids between 30 and 40 kb in size most frequently isolated. tet(O) was present in all tetracycline-resistant isolates. In the majority of strains under study the tet(O) gene was chromosomally encoded. Tetracycline resistance of six C. jejuni strains in which tet(O) was plasmid mediated was transferred by conjugation to a C. jejuni recipient strain. Transfer did not occur between tetracycline-resistant C. jejuni strains and a C. coli recipient. No difference in MICs, plasmid carriage and tet(O) localization was detected between human and chicken isolates. These data indicate that the tet(O) gene, previously reported in Campylobacter strains throughout the world, is present in Australian Campylobacter. This study will lead to a greater understanding of antibiotic resistance distribution in Campylobacter spp. in Australia.

Restriction fragment length polymorphism analysis and random amplified polymorphic DNA analysis of Campylobacter jejuni strains isolated from patients with Guillain-Barré syndrome.

Campylobacter jejuni serotype O19 strains associated with the Guillain-Barré syndrome (GBS) and other strains were examined by restriction fragment length polymorphism (RFLP) analysis of polymerase chain reaction products of the flaA genes and by random amplified polymorphic DNA (RAPD) analysis. RFLP analysis showed that regardless of LIO serotype, geographic origins, or association with GBS, the O19 isolates shared an identical digestion pattern by each of four restriction endonucleases, DdeI, MboI, MseI, and AluI. In contrast, among C. jejuni O1 or O2 strains, RFLP patterns were different even among strains of the same LIO serotype. The results of the RAPD analysis were consistent with the flaA RFLP data. These data indicate that all of the O19 strains that were tested were closely related to one another whether they were or were not associated with GBS.

Cytotoxicity and motility of Helicobacter pylori.

To clarify the relationship between interleukin-8 (IL-8) production and virulent factors, we examined the motility and cytotoxicity of H. pylori, suggested to be a major cause of chronic gastritis and peptic ulcers. Our results demonstrated that among cytotoxic strains of H. pylori, high-motility strains induced more IL-8 than low-motility strains. There was no correlation between cytotoxicity and motility of H. pylori. Four restriction fragment length polymorphism (RFLP) patterns were observed in the flaA PCR products. There was no correlation between flaA RFLP and motility. In conclusion, our findings suggest that both cytotoxicity and motility are virulent factors in the pathogenesis of gastric mucosal injury.