Genus Campylobacter Research Articles

RAPID METHODS OF THE DETECTION OF BACTERIA OF THE GENUS CAMPYLOBACTER IN FOOD PRODUCTS

Introduction. Campylobacteriosis is an acute intestinal disease caused by Campylobacter spp., which manifests with symptoms of enterocolitis and gastroenteritis. The causative agents of campylobacteriosis are C. jejuni, C. coli, C. lari, C. upsaliensis, and C. helveticus. The incidence of campylobacteriosis is recorded worldwide as sporadic cases and foodborne or waterborne outbreaks. The industrialization of poultry production has led both to the acceleration of the evolution of commensal pathogens from the genus of Campylobacter, increased contamination of raw products and importance Campylobacter as foodborne pathogens. Material and methods. The main objects were raw poultry products and swabs from the environment of poultry processing enterprises. Cultural, biochemical, immunological methods, methods for the detection of the sensitivity of microorganisms to antibiotics, PCR analysis were used. Results and discussion. The methods of rapid detection of thermophilic campylobacters using combined schemes of bacteriological and molecular genetic analysis are developed. Because C. jejuni makes 85-90% of food isolates of campylobacters, for the purposes of production control the detection of thermophilic Сampylobacter with a minimum set of cultural and biochemical tests of identification is allowed. This will reduce the duration of analyses up to 3-4 days and decrease their labor-cost. The control critical points of production of poultry products in which it is necessary to control the presence of thermophilic campylobacters are indicated. These are the stages of slaughter, scalding, washing, and processing on the conveyor of carcasses, contact cooling baths, the areas of semi-finished products manufacturing and the packaging. Conclusion. The obtained results were used to develop Guidelines “Methods of rapid determination of bacteria of the genus Campylobacter in food products and evaluation of their antibiotic resistance”.

Trends in fluoroquinolone resistance in Campylobacter.

Members of the genus Campylobacter remain a leading cause of bacterial gastroenteritis worldwide. Infection is usually self-limiting but in severe cases may require antibiotic treatment. In a recent statement by the World Health Organization (WHO) Campylobacter was named as one of the 12 bacteria that pose the greatest threat to human health because they are resistant to antibiotics. In this mini review we describe recent trends in fluoroquinolone (FQ) (particularly ciprofloxacin) resistance in strains of members of the genus Campylobacter isolated from livestock and clinical samples from several countries. Using evidence from phenotyping surveys and putative resistance prediction from DNA sequence data, we discuss the acquisition and spread of FQ resistance and the role of horizontal gene transfer and describe trends in FQ-resistance in samples from livestock and clinical cases. This review emphasises that FQ resistance remains common among isolates of members of the genus Campylobacter from various sources.

Comparative genomics of Campylobacter concisus: Analysis of clinical strains reveals genome diversity and pathogenic potential

In recent years, an increasing number of Campylobacter species have been associated with human gastrointestinal (GI) diseases including gastroenteritis, inflammatory bowel disease, and colorectal cancer. Campylobacter concisus, an oral commensal historically linked to gingivitis and periodontitis, has been increasingly detected in the lower GI tract. In the present study, we generated robust genome sequence data from C. concisus strains and undertook a comprehensive pangenome assessment to identify C. concisus virulence properties and to explain potential adaptations acquired while residing in specific ecological niche(s) of the GI tract. Genomes of 53 new C. concisus strains were sequenced, assembled, and annotated including 36 strains from gastroenteritis patients, 13 strains from Crohn’s disease patients and four strains from colitis patients (three collagenous colitis and one lymphocytic colitis). When compared with previous published sequences, strains clustered into two main groups/genomospecies (GS) with phylogenetic clustering explained neither by disease phenotype nor sample location. Paired oral/faecal isolates, from the same patient, indicated that there are few genetic differences between oral and gut isolates which suggests that gut isolates most likely reflect oral strain relocation. Type IV and VI secretion systems genes, genes known to be important for pathogenicity in the Campylobacter genus, were present in the genomes assemblies, with 82% containing Type VI secretion system genes. Our findings indicate that C. concisus strains are genetically diverse, and the variability in bacterial secretion system content may play an important role in their virulence potential.

Interactions between metabolically active bacteria and host gene expression at the cecal mucosa in pigs of diverging feed efficiency.

Little is known about the role of the gut mucosal microbiota and microbe-host signaling in the variation of pig's feed efficiency (FE). This study therefore aimed to investigate the FE-related differences in the metabolically active mucosal bacterial microbiota and expression of genes for innate immune response, barrier function, nutrient uptake, and incretins in the cecum of finishing pigs. Pigs (n = 72) were ranked for their residual feed intake (RFI; metric for FE) between days 42 and 91 postweaning and were stratified within litter and sex into high (HRFI; n = 8) and low RFI (LRFI; n = 8). Cecal mucosa and digesta were collected on day 137-141 of life. After isolating total RNA from the mucosa, the RNA was transcribed into cDNA which was used for gene expression analysis, total bacterial quantification, and high-throughput sequencing (Illumina MiSeq) of the hypervariable V3-V4 region of the 16S rRNA gene. The RFI differed by 2.1 kg between low RFI (LRFI; good FE) and high RFI (HRFI; poor FE) pigs (P < 0.001). The cecal mucosa was mainly colonized by Helicobacteraceae, Campylobacteraceae, Veillonellaceae, Lachnospiraceae, and Prevotellaceae. Despite the lack of differences in microbial diversity and absolute abundance, RFI-associated compositional differences were found. The predominant genus Campylobacter tended (P < 0.10) to be 0.4-fold more abundant in LRFI pigs, whereas low abundant Escherichia/Shigella (P < 0.05), Ruminobacter (P < 0.05), and Veillonella (P < 0.10) were 3.4-, 6.6-, and 4.4-fold less abundant at the cecal mucosa of LRFI compared to HRFI pigs. Moreover, mucin 2 and zona occludens-1 were less expressed (P < 0.05) in the cecal mucosa of LRFI compared to HRFI pigs. Cecal mucosal expression of monocarboxylate transporter-1, glucagon-like peptide-1, and peptide YY further tended (P < 0.10) to be downregulated in LRFI compared to HRFI pigs, indicating an enhanced VFA uptake and signaling in HRFI pigs. Sparse partial least square regression and relevance networking support the hypothesis that certain mucosal bacteria and luminal microbial metabolites were more associated than others with differences in RFI and cecal gene expression. However, present results do not allow the determination of whether mucosal bacterial changes contributed to variation in FE or were rather a consequence of FE-related changes in the pig's physiology or feeding behavior.

Campylobacter blaseri sp. nov., isolated from common seals (Phoca vitulina).

During a study to assess the faecal microbiome of common seals (Phoca vitulina) in a Dutch seal rehabilitation centre, 16S rRNA gene sequences of an unknown Campylobacter taxon were identified. Campylobacter isolates, which differed from the established Campylobacter taxa, were cultured and their taxonomic position was determined by a polyphasic study based on ten isolates. The isolates were characterized by 16S rRNA and atpA gene sequence analyses and by conventional phenotypic testing. Based on the whole genome sequences, the average nucleotide identity and core genome phylogeny were determined. The isolates formed a separate phylogenetic clade, divergent from all other Campylobacter taxa and most closely related to Campylobacter corcagiensis, Campylobacter geochelonis and Campylobacter ureolyticus. The isolates can be distinguished phenotypically from all other Campylobacter taxa based on their lack of motility, growth at 25 °C and growth on MacConkey agar. This study shows that these isolates represent a novel species within the genus Campylobacter, for which the name Campylobacter blaseri sp. nov. is proposed. The type strain for this novel species is 17S00004-5T (=LMG 30333T=CCUG 71276T).

Functional analysis of the Helicobacter pullorum N-linked protein glycosylation system.

N-linked protein glycosylation systems operate in species from all three domains of life. The model bacterial N-linked glycosylation system from Campylobacter jejuni is encoded by pgl genes present at a single chromosomal locus. This gene cluster includes the pglB oligosaccharyltransferase responsible for transfer of glycan from lipid carrier to protein. Although all genomes from species of the Campylobacter genus contain a pgl locus, among the related Helicobacter genus only three evolutionarily related species (H. pullorum, H. canadensis and H. winghamensis) potentially encode N-linked protein glycosylation systems. Helicobacter putative pgl genes are scattered in five chromosomal loci and include two putative oligosaccharyltransferase-encoding pglB genes per genome. We have previously demonstrated the in vitro N-linked glycosylation activity of H. pullorum resulting in transfer of a pentasaccharide to a peptide at asparagine within the sequon (D/E)XNXS/T. In this study, we identified the first H. pullorum N-linked glycoprotein, termed HgpA. Production of histidine-tagged HgpA in the background of insertional knockout mutants of H. pullorum pgl/wbp genes followed by analysis of HgpA glycan structures demonstrated the role of individual gene products in the PglB1-dependent N-linked protein glycosylation pathway. Glycopeptide purification by zwitterionic-hydrophilic interaction liquid chromatography coupled with tandem mass spectrometry identified six glycosites from five H. pullorum proteins, which was consistent with proteins reactive with a polyclonal antiserum generated against glycosylated HgpA. This study demonstrates functioning of a H. pullorum N-linked general protein glycosylation system.

CONCEPTUAL APPROACHES TO THE RAPID DETECTION OF CAMPYLOBACTER SPP. IN MEAT OF SLAUGHTER ANIMALS

The modern approach to quality assurance of food products based on the ISO 9000 standards indicates the need for the implementation of quality management systems in processing plants. According to the analysis of scientific publication databases (Science Direct and Web of Science), it is established that only 0.5–1.7% of publications are related to studying meat of slaughter animals (except for birds) concerning the presence of Campylobacter. The priority method of investigation is PCR. Ready-to-use PCR test system was developed for the detection of Campylobacter spp. on the basis of selected gene-specific primers to bacteria of Campylobacter genus. Specificity of the test system is established for Gram-negative bacteria of Salmonella, Escherichia, and Proteus genera, and for oxidase-positive Aeromonas. Gene-specific primers for Campylobacter were selected and ready-to-use PCR test system was developed on their basis. It was found that the selected primers have 100% convergence to the genome of Campylobacter genus bacteria, the PCR efficiency is not less than 95%, and the detection limit is not more than 1× 10 4 CFU/g. When estimating the specificity of the primers, it was taken into account that the bacteria of Campylobacter genus may be incorporated in a consortium with intestine microbiome, mainly with Enterobacteriaceae and lactic acid bacteria. However, Bolton’s enrichment medium is selective and, during the cultivation process, suppresses the growth of Gram-positive lactic acid bacteria. It was found that the selected primers were 100% specific and did not give false positive reactions with this group of microorganisms. The developed test system was successfully validated in a cycle of qualitative tests in the FEPAS system and implemented into laboratory practice. It was proved that the developed test system may be used both in screening at the stages of Campylobacter enrichment and in identification of pure culture of the microorganism.

Antimicrobial Resistance in Campylobacter Species: Mechanisms and Genomic Epidemiology.

The Campylobacter genus is a large and diverse group of Gram-negative bacteria that are known to colonize humans and other mammals, birds, reptiles, and shellfish. While it is now recognized that several emerging Campylobacter species can be associated with human disease, two species, C. jejuni and C. coli, are responsible for the vast majority of bacterial gastroenteritis in humans worldwide. Infection with C. jejuni, in particular, has also been associated with a number of extragastrointestinal manifestations and autoimmune conditions, most notably Guillain-Barré syndrome. The antimicrobial drugs of choice for the treatment of severe Campylobacter infection include macrolides, such as erythromycin, clarithromycin, or azithromycin. Fluoroquinolones, such as ciprofloxacin, are also commonly used for empirical treatment of undiagnosed diarrheal disease. However, resistance to these and other classes of antimicrobial drugs is increasing and is a major public health problem. The US Centers for Disease Control and Prevention estimates that over 300,000 infections per year are caused by drug-resistant Campylobacter. In this chapter, we discuss the taxonomy of the Campylobacter genus, the clinical and global epidemiological aspects of Campylobacter infection, with an emphasis on C. jejuni and C. coli, and issues related to the treatment of infection and antimicrobial resistance mechanisms. We further discuss the use of next-generation sequencing for the detection and surveillance of antimicrobial resistance genes.

Microbial community diversity of Jinghong laying hens at peak production based on 16S rRNA sequencing

ABSTRACTIn this study, the diversity within the duodenum (S), jejunum (K) and caecum (M) contents of the three different intestinal gut sections microbiota of 30-week-old (peak production stage) Jinghong laying hens were evaluated. The bacterial DNA was sequentially isolated and the V3 to V4 regions of 16S rRNA genes were amplified. Results showed that the average bacterial sequences from the duodenum, jejunum and caecum content were identified to be 175.33 ± 26.63, 64.00 ± 20.95 and 305.33 ± 4.16 OTUs, respectively. The inherent OTUs were found among duodenum (75), jejunum (2) and caecum (172). The caecum had the highest diversity (Shannon = 5.57 ± 0.06) among the three communities. Firmicutes (65.54%) and Proteobacteria (32.68%) were the predominant bacterial phyla in the duodenum content. Firmicutes (97.27%) was the most commonly detected phyla in the jejunum content. As to the caecum, the relatively prominent phyla were Bacteroidetes and Firmicutes and Fusobacteria, accounting for 48.70%, 28.91% and 15.93%, respectively. At the genus level, Lactobacillus, Helicobacter, Bacillus, Peptoclostridium and Campylobacter were the relatively abundant genera in the duodenum content, accounting for 51.76%, 28.07%, 5.89%, 5.04% and 1.70%, respectively. Within the jejunum content, Lactobacillus was the most commonly detected genera, which represent 96.26% of the total genera.

Ocurrence of Campylobacter spp. in chilled chicken carcasses slaughtered in the west region of Santa Catarina, Brazil

Background: The thermophilic bacteria of the genus Campylobacter are important agents of alimentary gastroenteritis, called campylobacteriosis. These microorganisms multiply in temperatures ranging from 25ºC to 46ºC, however, low temperatures are incompatible with their multiplication. For this reason, the seasons of the year may interfere with the level of contamination by Campylobacter sp. The main sources of transmission are contaminated meat and giblets from poultry during poorly conducted slaughter operations. The disease may present itself with a different range of forms of disease: from mild signs of gastrointestinal infection to more severe cases, such as the Guillain-Barré syndrome.Material, Methods &amp; Results: Due to the great importance of western Santa Catarina to the poultry industry, it was necessary to verify the occurrence of the pathogen in cold carcasses of broilers slaughtered in this region, and its variation through the seasons of the year. From January 2013 to February 2015 broiler carcasses were collected weekly, after the water cooling process, in slaughterhouses under Federal Inspection of the three largest microregions of western Santa Catarina in terms of number of broilers slaughtered, totaling 808 samples. The assessment of thermophilic Campylobacter was performed according to the methodology recommended by ISO 10272-1: 2006. Of the 808 samples analyzed, the frequency of isolation of thermophilic Campylobacter was 1.82% (8/440) in microregion 1, 4.95% (10/202) in microregion 2 and 13.86% (23/166) in the microregion 3, totaling 5.07% of positive samples (41/808). Comparing the microregions, it was verified that there was no significant difference (P &gt; 0.05) between the isolation frequencies of microregions 1 and 2. However, there was a significant difference (P &lt; 0.05) between the isolation rates of Microregion 3 in relation to microregions 1 and 2. Microregion 1 presented the lowest percentage of Campylobacter thermophilic isolates.Discussion: Although the rates found are lower than expected when compared to those already published, consumption of these products may provide risks to consumers, mainly through cross-contamination of foods that will be consumed raw, requiring greater controls in the entire poultry industry in order to ensure a safe product for the consumer. Analyzing each season of the year individually (winter and summer), 5.88% (14/238) of the samples processed during summer (October to March) were positive for the assessed microorganism and in 4,74% (27/570) of the samples analyzed in the winter (April to September), the presence of thermophilic Campylobacter was detected. Nevertheless, there was no significant difference (P &gt; 0.05) between the seasons. However, it is evident the need for a longer study to evaluate the actual influence of the seasons (winter and summer) on the isolation rate of thermophilic Campylobacter in broiler carcasses shortly after the cooling process. Even though the study demonstrated low levels of thermophilic Campylobacter isolation rates in the evaluated regions, just the fact of isolating the agent already shows that there is an imminent risk to consumers, mainly due to cross-contamination of foods that will be consumed raw; therefore, higher standards of microbiological controls should be practiced in the production chain and slaughter of poultry, respecting good manufacturing practices, hygiene and handling practices within the industry.

Systematic Review of the Burden of Campylobacter-associated Disease

Bacteria of the genus Campylobacter spp. are one of the most common causes of gastroenteritis and can lead to serious sequelae. Several studies have estimated the disease burden of Campylobacter spp. with the quantitative metric of disability-adjusted life years (DALY). The aim of this systematic review is to give an overview of the information available about different countries and periods for which DALYs were calculated and how the different results are comparable. One of the most important transmission pathways for Campylobacter spp. is food. Therefore, special attention was given to studies that only estimated the foodborne disease burden of Campylobacter bacteria. With a systematic search for the period 1/1996-6/2016, one worldwide and 21 country-specific publications of the WHO were identified. Because of the different methods and the quality of the different data sets, the estimated results of all Campylobacter health outcomes of the country-specific studies vary from 0.4 DALYs per 100000 people in France to 109 DALY per population in Poland. The calculation of the attributable foodborne disease burden was based on the estimations of the incidences of all Campylobacter health outcomes with the associated uncertainty for each result. So the estimations of the foodborne disease burden show a large range from 0.5 DALYs per 100000 people in Greek to 21.2 DALYs per 100000 people in New Zealand. This span can only be partially explained by the country-specific variability in the food production, the consumption behavior and the incidence of Campylobacter bacteria.

Genome Comparison of Erythromycin Resistant Campylobacter from Turkeys Identifies Hosts and Pathways for Horizontal Spread of erm(B) Genes.

Pathogens in the genus Campylobacter are the most common cause of food-borne bacterial gastro-enteritis. Campylobacteriosis, caused principally by Campylobacter jejuni and Campylobacter coli, is transmitted to humans by food of animal origin, especially poultry. As for many pathogens, antimicrobial resistance in Campylobacter is increasing at an alarming rate. Erythromycin prescription is the treatment of choice for clinical cases requiring antimicrobial therapy but this is compromised by mobility of the erythromycin resistance gene erm(B) between strains. Here, we evaluate resistance to six antimicrobials in 170 Campylobacter isolates (133 C. coli and 37 C. jejuni) from turkeys. Erythromycin resistant isolates (n = 85; 81 C. coli and 4 C. jejuni) were screened for the presence of the erm(B) gene, that has not previously been identified in isolates from turkeys. The genomes of two positive C. coli isolates were sequenced and in both isolates the erm(B) gene clustered with resistance determinants against aminoglycosides plus tetracycline, including aad9, aadE, aph(2″)-IIIa, aph(3′)-IIIa, and tet(O) genes. Comparative genomic analysis identified identical erm(B) sequences among Campylobacter from turkeys, Streptococcus suis from pigs and Enterococcus faecium and Clostridium difficile from humans. This is consistent with multiple horizontal transfer events among different bacterial species colonizing turkeys. This example highlights the potential for dissemination of antimicrobial resistance across bacterial species boundaries which may compromise their effectiveness in antimicrobial therapy.

In Vitro Culturing and Storage of Campylobacter Genus Bacteria.

Experimental model for in vitro evaluation of Campylobacter genus bacteria growth kinetics, inhibition, or inactivation is proposed. The model allows quantitative evaluation of the sensitivity to various types of stress exposure and promotes detection of the regularities of their transformation into uncultivable forms. The model implies the use of 96-well plates for parallel culturing of several subpopulations of the test strain in media with various parameters. The proposed algorithm includes evaluation of the proportion of viable CFU to total level of planktonic and uncultivable cells in the population, which is estimated by the content of genomic DNA in the samples by quantitative PCR (or real-time PCR) with ciaB, cdtB, or 16S rRNA primers. The presence of biofilm matrix is detected by the intensity of staining of polystyrene plates. This model can be used for evaluation of the most significant types of exposure, including low-dose antibacterial treatment, promoting the formation of stable microorganism variants. The model has been used to study the effects of culturing conditions on the characteristics of C. jejuni populations. The most characteristic feature of C. jejuni is reduction of the count of viable cells up to complete disappearance of cultivable forms under favorable conditions of growth. The level of viable cells in the populations decreased 10-fold and more, on average, after 48-h incubation. Not all strains exhibit this property, some strains retain their viability, which is detected by the culturing method, and contributes to biofilm formation.

Prevalence and molecular identification of &lt;i&gt;Campylobacter&lt;/i&gt; species isolates from poultry and humans in Sokoto, Nigeria

Prevalence and molecular identification of Campylobacter species isolates from poultry and humans were conducted using culture, biochemical reaction and Polymerase Chain Reaction (PCR) techniques. A total of 798 (506 poultry and 292 human) samples were identified biochemically, out of which 312(39.1%) were positive for Campylobacter species. Campylobacter jejuni, C. coli and C. lari had 38 (23.8%) out of 160, 63(39.4%) out of 160, 59(36.9%) out of 160 prevalence rates, respectively in humans while 29(19.1%) out of 152, 79(52.0%) out of 152 and 44(28.9%) out of 152 were the rates for the species in the same order in poultry. Campylobacter isolates were kept at -20oC in 15% glycerol and 85% tryptone broth until used while some were identified 24hrs post isolation. Single and multiplex PCR were used to confirm the genus Campylobacter and three Campylobacter species, respectively. All the 130(100 stored and 30 fresh) isolates were members of the genus Campylobacter. The single PCR band view of stored isolates also revealed other bands in addition to 439 bp which is specific for the genus Campylobacter, while the fresh isolates had distinct bands at 439bp only. Multiplex PCR revealed 2(6.7%) out of 30 were positive for stored isolates out of 30, 1(50%) each for C. jejuni and C. Coli. However, 1 of the stored isolate was positive for both spp. On the other hand, 6(20.0%) of out 60 fresh isolates were positive, with 5(83.3%) and 1(16.7%) for C. jejuni and C. coli, respectively. The possibilities of improper identification using conventional method have been revealed in the study. PCR can identify Campylobacter species more accurately than biochemical method, though storage of isolates, integrity of extracted DNA and PCR conditions can affect result. However, the use of both methods should be encouraged in regular and effective surveillance of Campylobacter species in poultry and humans.Keywords: Campylobacter species, Humans, Molecular identification , Nigeria, Prevalence, Poultry, Sokoto

Optimization of microbiological methods for food control based on the differential-diagnostic media for the isolation and cultivation of bacteria of the genus Campylobacter

А screening study on the detection of campylobacteria in raw food products, semi-finished products and objects in the external environment in the poultry processing industry was conducted. The highest level of detection of campylobacteria is set for raw poultry products, including carcasses of broilers, turkeys and quail. A general accordance of getting Campylobacter in raw materials and food products with inadequate sanitary treatment of separate areas of production has been established: in most cases Campylobacter spp. was extracted from the samples, contaminated with coliform bacteria and Salmonella. It is shown that the frequency of contamination of raw poultry with pathogens is largely dependent on the cooling of the carcasses. When using the immersion method, the conditions for cross contamination with pathogens through water bath cooling are present (45% of samples infected with Campylobacter spp.). Under combined use of super-cooled water and aerosols Campylobacter were also detected quite often in 27% of samples. Contamination by pathogens was the lowest in evaporative cooling method with the use of antimicrobial hydrospray (less than 5% positive samples), allowing to recognize this method as the most promising for the production of microbiological safe products. The work on optimization of nutrient medium composition and adaptation recommended methodological scheme of analysis for detection and species identification of bacteria of the genus Campylobacter was carried out. Formulation of traditionally used growth media was modified, and balanced composition of growth and selective components was matched in accordance with the requirements of existing standards. Given the urgency of increasing the effectiveness of the methods of control of campylobacteria in foods and the lack of domestic analogues of the culture media in the Russian Federation, an optimized method for the production of dry nutrient media for the detection, identification and storage of campylobacteria isolated from food products and clinical material was developed. The conducted study allowed to develop Technical conditions 21.20.23-006-01897222-2016 «Dry culture medium for detection of bacteria of the genus Campylobacter» and «Instruction for use of culture media». Depending on the purpose of the medium produced in the following versions: the selective broth for enrichment of campylobacteria; differential selective agar for isolating and quantifying of Campylobacter spp.; semi-solid nutrient agar for cryostorage of Campylobacter strains. The list of criteria for assessing the quality of commercially available lots of dry media included: solubility, pH, gel strength of agar, the content of amine nitrogen, specificity, selectivity, growth and inhibitory properties. The practical application of these media in terms of the national laboratories will significantly simplify the use of existing standards developed based on international ISO standards, but not adapted to the main range of commercial media and reagents used in routine food control for the presence of campylobacteria.

Inquiring into the Gaps of Campylobacter Surveillance Methods.

Campylobacter is one of the most common pathogen-related causes of diarrheal illnesses globally and has been recognized as a significant factor of human disease for more than three decades. Molecular typing techniques and their combinations have allowed for species identification among members of the Campylobacter genus with good resolution, but the same tools usually fail to proceed to subtyping of closely related species due to high sequence similarity. This problem is exacerbated by the demanding conditions for isolation and detection from the human, animal or water samples as well as due to the difficulties during laboratory maintenance and long-term storage of the isolates. In an effort to define the ideal typing tool, we underline the strengths and limitations of the typing methodologies currently used to map the broad epidemiologic profile of campylobacteriosis in public health and outbreak investigations. The application of both the old and the new molecular typing tools is discussed and an indirect comparison is presented among the preferred techniques used in current research methodology.

Campylobacter ornithocola sp. nov., a novel member of the Campylobacter lari group isolated from wild bird faecal samples

During a study on the prevalence and diversity of campylobacteria in wild birds faecal samples from the city of Valdivia (southern Chile) 17 Gram-stain-negative, curved-rod-shaped isolates, were initially identified as Campylobacter lari by PCR-RFLP. Further identification by 16S rRNA sequence analysis revealed that they formed a distinct group in the genus Campylobacter. This unique position was confirmed by the results of analysis of rpoB, atpA and cpn60 gene sequences. The average nucleotide identity between the representative strain WBE38T and the type strain of the most closely related taxon C. larisubsp.concheus (LMG 11760) was 90.8 %. The oxidase and urease activity of the novel isolates enabled them to be phenotypically differentiated from species of the genus Campylobacter with validly published names. Therefore, on the basis of phenotypic, genetic and genomic characterizations, the results of this study clearly indicate that these strains represent a novel species within the genus Campylobacter, for which the name Campylobacter ornithocola sp. nov. is proposed, with the type strain WBE38T (=CECT 9147T=LMG 29815T).

Campylobacter pinnipediorum sp. nov., isolated from pinnipeds, comprising Campylobacter pinnipediorum subsp. pinnipediorum subsp. nov. and Campylobacter pinnipediorum subsp. caledonicus subsp. nov.

During independent diagnostic screenings of otariid seals in California (USA) and phocid seals in Scotland (UK), Campylobacter-like isolates, which differed from the established taxa of the genus Campylobacter, were cultured from abscesses and internal organs of different seal species. A polyphasic study was undertaken to determine the taxonomic position of these six isolates. The isolates were characterized by 16S rRNA gene and AtpA sequence analysis and by conventional phenotypic testing. The whole-genome sequences were determined for all isolates, and the average nucleotide identity (ANI) was determined. The isolates formed a separate phylogenetic clade, divergent from all other taxa of the genus Campylobacter and most closely related to Campylobactermucosalis. Although all isolates showed 100 % 16S rRNA gene sequence homology, AtpA and ANI analyses indicated divergence between the otariid isolates from California and the phocid isolates from Scotland, which warrants subspecies status for each clade. The two subspecies could also be distinguished phenotypically on the basis of catalase activity. This study shows clearly that the isolates obtained from pinnipeds represent a novel species within the genus Campylobacter, for which the name Campylobacter pinnipediorum sp. nov. is proposed. Within this novel species, the Californian isolates represent a separate subspecies, for which the name C. pinnipediorum subsp. pinnipediorum subsp. nov. is proposed. The type strain for both this novel species and subspecies is RM17260T (=LMG 29472T=CCUG 69570T). The Scottish isolates represent another subspecies, for which the name C. pinnipediorum subsp. caledonicus subsp. nov. is proposed. The type strain of this subspecies is M302/10/6T (=LMG 29473T=CCUG 68650T).

Characterisation by multilocus sequence and porA and flaA typing of Campylobacter jejuni isolated from samples of dog faeces collected in one city in New Zealand

AIMS: To investigate the prevalence of Campylobacter spp. and C. jejuni in dog faecal material collected from dog walkways in the city of Palmerston North, New Zealand, and to characterise the C. jejuni isolates by multilocus sequence typing (MLST) and porA and flaA antigen gene typing.METHODS: A total of 355 fresh samples of dogs faeces were collected from bins provided for the disposal of dog faeces in 10 walkways in Palmerston North, New Zealand, between August 2008–July 2009. Presumptive Campylobacter colonies, cultured on modified charcoal cefoperazone deoxycholate plates, were screened for genus Campylobacter and C. jejuni by PCR. The C. jejuni isolates were subsequently characterised by MLST and porA and flaA typing, and C. jejuni sequence types (ST) were assigned.RESULTS: Of the 355 samples collected, 72 (20 (95% CI=16–25)%) were positive for Campylobacter spp. and 22 (6 (95% CI=4–9)%) were positive for C. jejuni. Of the 22 C. jejuni isolates, 19 were fully typed by MLST. Ten isolates were assigned to the clonal complex ST-45 and three to ST-52. The allelic combinations of ST-45/flaA 21/porA 44 (n=3), ST-45/flaA 22/porA 53 (n=3) and ST-52/ flaA 57/porA 905 (n=3) were most frequent.CONCLUSIONS: The successful isolation of C. jejuni from canine faecal samples collected from faecal bins provides evidence that Campylobacter spp. may survive outside the host for at least several hours despite requiring fastidious growth conditions in culture. The results show that dogs carry C. jejuni genotypes (ST-45, ST-50, ST-52 and ST-696) that have been reported in human clinical cases.CLINICAL RELEVANCE: Although these results do not provide any evidence either for the direction of infection or for dogs being a potential risk factor for human campylobacteriosis, dog owners are advised to practice good hygiene with respect to their pets to reduce potential exposure to infection.

Formation of Biofilms by Foodborne Pathogens and Development of Laboratory In Vitro Model for the Study of Campylobacter Genus Bacteria Based on These Biofilms

We analyzed the formation of biofilms by 7 strains of Campylobacter genus bacteria and 18 strains of Enterobacteriaceae genus bacteria that were isolated from plant and animal raw materials, from finished products, and swabs from the equipment of the food industry. Biofilm formation on glass plates, slides and coverslips, microtubes made of polymeric materials and Petri dishes, and polystyrene plates of different profiles were analyzed. When studying the process of films formation, different effects on bacterial populations were simulated, including variation of growth factor composition of culture media, technique of creating of anaerobiosis, and biocide treatment (active chlorine solutions in a concentration of 100 mg/dm3). The formation of biofilms by the studied cultures was assessed by the formation of extracellular matrix stained with aniline dyes on glass and polystyrene surfaces after incubation; 0.1% crystal violet solution was used as the dye. The presence and density of biomatrix were assessed by staining intensity of the surfaces of contact with broth cultures or by optical density of the stained inoculum on a spectrophotometer. Biofilms were formed by 57% Campylobacter strains and 44% Enterobacteriaceae strains. The intensity of the film formation depended on culturing conditions and protocols, species and genus of studied isolates, and largely on adhesion properties of abiotic surfaces. In 30% of Enterobacteriaceae strains, the biofilm formation capacity tended to increase under the influence of chlorine-containing biocide solutions. Thus, we developed and tested under laboratory conditions a plate version of in vitro chromogenic model for evaluation of biofilm formation capacity of C. jejuni strains and studied stress responses to negative environmental factors.