Arcobacter population dynamics in pigs on farrow-to-finish farms

Healthy pigs are an important reservoir for the emerging human pathogen Arcobacter which can result in contamination of porcine carcasses and pork and the spread of arcobacters into the environment. Up to now, the excretion of arcobacters by pigs has been studied, but information about the transmission routes in fattening pigs is lacking. The present study aimed to elucidate the Arcobacter population dynamics in pigs during the fattening period on four farrow-to-finish farms. On each farm, 30 clinically healthy, 12-week-old piglets were selected. Fecal samples were collected on 10 sampling occasions until a slaughter age of 30 weeks was reached. Arcobacter spp. were isolated by a selective method and identified by multiplex PCR. The genetic diversity was examined by amplified fragment length polymorphism and enterobacterial repetitive intergenic consensus PCR. The Arcobacter presence in the fecal samples on the four farms ranged from 11.3 to 50.0%, with excretion levels of up to 10(4) CFU/g feces. The ratio in which Arcobacter species were isolated varied between the farms and over time. Characterization revealed a high degree of genotypic diversity among the isolates. Arcobacter strains persisted and spread within the finishing unit during the fattening period. The occurrence of both unique and shared genotypes in pigs in adjacent and nonadjacent pens demonstrates that transmission routes other than fecal-oral transmission occur.     

Genotypic characterization of Salmonella by multilocus sequence typing, pulsed-field gel electrophoresis and amplified fragment length polymorphism

Molecular typing is an important tool in surveillance and outbreak investigations of human Salmonella infections. In this study, three molecular typing methods were used to investigate the discriminatory ability, reproducibility and the genetic relationship between 110 Salmonella enterica subspecies enterica isolates. A total of 25 serotypes were investigated that had been isolated from humans or veterinary sources in Denmark between 1995 and 2001. All isolates were genotyped by multilocus sequence typing (MLST), pulsed-field gel electrophoresis (PFGE) and amplified fragment length polymorphism (AFLP). When making genetic trees, all three methods resulted in similar clustering that often corresponded with serotype, although some serotypes displayed more diversity than others. Of the three techniques, MLST was the easiest to interpret and compare between laboratories. Unfortunately the seven housekeeping genes used in this MLST scheme lacked diversity and the ability to discriminate between isolates were higher with both PFGE and AFLP. The discriminatory power of AFLP and PFGE were similar but PFGE fingerprints were both easier to reproduce, interpret and less time-consuming to analyze when compared to AFLP. PFGE is the therefore the preferred molecular typing method for surveillance and outbreak investigations, whereas AFLP is most useful for local outbreak investigations.     

Use of Hugh and Leifson's medium as a simple screening test to aid in the differentiation of Arcobacter spp. from background flora during their isolation from foodstuffs

Aims:  To investigate the suitability of Hugh and Leifson's medium (HLM) as the basis of a simple screening test to differentiate between contaminants and Arcobacter spp. during their isolation from foodstuffs.
Methods and Results:  Characterized Arcobacter spp. were obtained from recognized culture collections. Wild-type isolates of Arcobacter spp. and contaminants were obtained using published isolation protocols. Retail packs of red meats were used as the source of the isolates. Eighteen defined Arcobacter spp. gave no reaction on HLM, as did 10 local wild-type isolates. Overall 163 contaminants were studied for oxidative reactions on HLM and 86% of isolates demonstrated this property.
Conclusions:  HLM can usefully serve as a simple and effective screening test to differentiate between Arcobacter spp. and contaminants.
Significance and Impact of the Study:  Arcobacter isolation procedures are still being developed, and no effective diagnostic media currently exist. Rapidly excluding most contaminants can markedly increase the efficiency of isolation procedures by removing the need for extensive biotyping or the requirement to isolate DNA and conduct PCR tests.     

Isolation and characterization of the emerging foodborn pathogen Arcobacter from human stool

At present, isolation of arcobacters from human specimens is performed by slightly of not modified Campylobacter, Yersinia or Leptospira isolation techniques, and knowledge if arcobacters are part of the human commensal flora is lacking. Therefore, an Arcobacter selective isolation procedure was validated for the examination of human fecal specimens, and the presence and characteristics of Arcobacter in feces of asymptomatic humans was examined in order to assess the clinical relevance of arcobacters in diarrheal stool. With this method, Arcobacter was isolated from seven of 500 (1.4%) stool samples of healthy people with Arcobacter cryaerophilus as the only species present. Seven A. cryaerophilus genotypes were detected and only one genotype was found per person. Neither A. butzleri nor A. skirrowii were isolated, therefore the presence of those latter species in clinical samples requires further attention. Though the pathogenic role and potential virulence factors of arcobacters have to be further examined, the current status of arcobacters as emerging pathogens remains justified.     

Mother-to-child transmission of and multiple-strain colonization by Bacteroides fragilis in a cohort of mothers and their children

Bacteroides fragilis represents an early infant colonizer with important host interactions. Our knowledge about the diversity, transmission, and persistence of this bacterium, however, is limited. Here, we addressed these questions using a combination of multilocus sequence typing (MLST) and variable-number tandem repeat (VNTR) sequence analyses. We used both culture-dependent and -independent typing. We genotyped B. fragilis in fecal samples from a cohort of 93 mothers and their children, with samples taken from the mothers and from the children at the ages 1 to 10 days, 4 months, 1 year, and 2 years. By MLST we found two main B. fragilis groups, which we denoted clades A and B. Direct typing of stool samples using the icd gene revealed seven sequence types, five within clade A and two within clade B. A single clade A sequence type, however, represented 79% of all the sequences. This sequence type was further subtyped using VNTR. VNTR subtyping revealed 16 different VNTR types. Based on the distribution patterns of these, we show mother-to-child transmission and multiple-strain colonization. We argue that negative host selection promotes the coexistence of multiple strains. The significance of our findings is that we have started unraveling the transmission and persistence patterns of one of the most important human gut colonizers.     

Isolation of Arcobacter spp. from a brackish environment

Arcobacter spp. were isolated from water and mussels of two brackish lakes near Messina (Italy). The isolates were phenotypically characterized on the basis of a large battery of cultural and biochemical tests. By comparison with the reference strains Arcobacter butzleri ATCC 49616 and A. cryaerophilus ATCC 43157 they may be considered Arcobacter butzleri-like bacteria. The current isolation suggests that the brackish environment may play an important role in the survival and transmission of Arcobacter spp. also by seafood cultured in the examined waters.     

Typing of Campylobacter jejuni and Campylobacter coli isolated from live broilers and retail broiler meat by flaA-RFLP, MLST, PFGE and REP-PCR

We analyzed 100 Campylobacter spp. isolates (C. jejuni and C. coli) from Grenada, Puerto Rico and Alabama, which were collected from live broilers or retail broiler meat. We analyzed these isolates with four molecular typing methods: restriction fragment length polymorphism of the flaA gene (flaA-RFLP), multilocus sequence typing (MLST), pulsed-field gel electrophoresis (PFGE), and automated repetitive extragenic palindromic polymerase chain reaction (REP-PCR) using the DiversiLab system. All methods performed similarly for the typing of C. jejuni and C. coli. The DNA extraction method appears to influence the results obtained with REP-PCR. This method was better for the typing of C. jejuni than C. coli, however both REP-PCR and flaA-RFLP generated types that were indistinguishable between C. jejuni and C. coli and appeared to be random, without any relationship to species, location, or source of isolates. PFGE and MLST generated typing results that had a better correlation with the geographic location of the isolates and showed higher concordance with the Wallace coefficient. The adjusted Rand coefficient did not show higher concordance among the methods, although the PFGE/MLST combination exhibited the highest concordance. PFGE and MLST revealed a better discriminatory power for C. coli isolates than REP-PCR or flaA-RFLP. The use of readily available online tools to calculate the confidence interval of the Simpson's index of diversity and the adjusted Rand and Wallace coefficients helped estimate the discriminatory power of typing methods. Further studies using different C. jejuni and C. coli strains may expand our understanding of the benefits and limitations of each of these typing methods for epidemiological studies of Campylobacter spp.     

Genetic diversity of Campylobacter jejuni isolates from farm animals and the farm environment

The genetic diversity of Campylobacter jejuni isolates from farm animals and their environment was investigated by multilocus sequence typing (MLST). A total of 30 genotypes, defined by allelic profiles (assigned to sequence types [STs]), were found in 112 C. jejuni isolates originating in poultry, cattle, sheep, starlings, and slurry. All but two of these genotypes belonged to one of nine C. jejuni clonal complexes previously identified in isolates from human disease and retail food samples and one clonal complex previously associated with an environmental source. There was some evidence for the association of certain clonal complexes with particular farm animals: isolates belonging to the ST-45 complex predominated among poultry isolates but were absent among sheep isolates, while isolates belonging to the ST-61 and ST-42 complexes were predominant among sheep isolates but were absent from the poultry isolates. In contrast, ST-21 complex isolates were distributed among the different isolation sources. Comparison with MLST data from 91 human disease isolates showed small but significant genetic differentiation between the farm and human isolates; however, representatives of six clonal complexes were found in both samples. These data demonstrate that MLST and the clonal complex model can be used to identify and compare the genotypes of C. jejuni isolates from farm animals and the environment with those from retail food and human disease.     

Molecular Epidemiology of Campylobacter jejuni Isolates from Wild-Bird Fecal Material in Children's Playgrounds

In many countries relatively high notification rates of campylobacteriosis are observed in children under 5 years of age. Few studies have considered the role that environmental exposure plays in the epidemiology of these cases. Wild birds inhabit parks and playgrounds and are recognized carriers of Campylobacter, and young children are at greater risk of ingesting infective material due to their frequent hand-mouth contact. We investigated wild-bird fecal contamination in playgrounds in parks in a New Zealand city. A total of 192 samples of fresh and dried fecal material were cultured to determine the presence of Campylobacter spp. Campylobacter jejuni isolates were also characterized by pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST), and the profiles obtained were compared with those of human isolates. C. jejuni was isolated from 12.5% of the samples. MLST identified members of clonal complexes ST-45, ST-682, and ST-177; all of these complexes have been recovered from wild birds in Europe. PFGE of ST-45 isolates resulted in profiles indistinguishable from those of isolated obtained from human cases in New Zealand. Members of the ST-177 and ST-682 complexes have been found in starlings (Sturnus vulgaris) in the United Kingdom, and these birds were common in playgrounds investigated in New Zealand in this study. We suggest that feces from wild birds in playgrounds could contribute to the occurrence of campylobacteriosis in preschool children. Further, the C. jejuni isolates obtained in this study belonged to clonal complexes associated with wild-bird populations in the northern hemisphere and could have been introduced into New Zealand in imported wild garden birds in the 19th century.     

Multilocus sequence typing reveals high genetic diversity and epidemic population structure for the fish pathogen Yersinia ruckeri

Yersinia ruckeri is the causative agent of enteric redmouth in fish and one of the major bacterial pathogens causing losses in salmonid aquaculture. Previously typing methods, including restriction enzyme analysis, pulsed-field gel electrophoresis and multilocus enzyme electrophoresis (MLEE) have indicated a clonal population structure. In this work, we describe a multilocus sequence typing (MLST) scheme for Y.ruckeri based on the internal fragment sequence of six housekeeping genes. This MLST scheme was applied to 103 Y.ruckeri strains from diverse geographic areas and hosts as well as environmental sources. Sequences obtained from this work were deposited and are available in a public database (http://publmst.org/yruckeri/). Thirty different sequence types (ST) were identified, 21 of which were represented by a single isolate, evidencing high genetic diversity. ST2 comprised more than one-third of the isolates and was most frequently observed among isolates from trout. Two major clonal complexes (CC) were identified by eBURST analysis showing a common evolutionary origin for 94 isolates forming 21 STs into CC1 and for 6 isolates of 6 STs in the CC2. It was also possible to associate some unique ST with isolates from recent outbreaks in vaccinated salmonid fish.     

Arcobacter in Lake Erie beach waters: an emerging gastrointestinal pathogen linked with human-associated fecal contamination

The genus Arcobacter has been associated with human illness and fecal contamination by humans and animals. To better characterize the health risk posed by this emerging waterborne pathogen, we investigated the occurrence of Arcobacter spp. in Lake Erie beach waters. During the summer of 2010, water samples were collected 35 times from the Euclid, Villa Angela, and Headlands (East and West) beaches, located along Ohio's Lake Erie coast. After sample concentration, Arcobacter was quantified by real-time PCR targeting the Arcobacter 23S rRNA gene. Other fecal genetic markers (Bacteroides 16S rRNA gene [HuBac], Escherichia coli uidA gene, Enterococcus 23S rRNA gene, and tetracycline resistance genes) were also assessed. Arcobacter was detected frequently at all beaches, and both the occurrence and densities of Arcobacter spp. were higher at the Euclid and Villa Angela beaches (with higher levels of fecal contamination) than at the East and West Headlands beaches. The Arcobacter density in Lake Erie beach water was significantly correlated with the human-specific fecal marker HuBac according to Spearman's correlation analysis (r = 0.592; P < 0.001). Phylogenetic analysis demonstrated that most of the identified Arcobacter sequences were closely related to Arcobacter cryaerophilus, which is known to cause gastrointestinal diseases in humans. Since human-pathogenic Arcobacter spp. are linked to human-associated fecal sources, it is important to identify and manage the human-associated contamination sources for the prevention of Arcobacter-associated public health risks at Lake Erie beaches.     

Genetic Diversity and Ecological Success of Staphylococcus aureus Strains Colonizing Humans

The genetic determinants and phenotypic traits which make a Staphylococcus aureus strain a successful colonizer are largely unknown. The genetic diversity and population structure of 133 S. aureus isolates from healthy, generally risk-free adult carriers were investigated using four different typing methods: multilocus sequence typing (MLST), amplified fragment length polymorphism analysis (AFLP), double-locus sequence typing (DLST), and spa typing were compared. Carriage isolates displayed great genetic diversity which could only be revealed fully by DLST. Results of AFLP and MLST were highly concordant in the delineation of genotypic clusters of closely related isolates, roughly equivalent to clonal complexes. spa typing and DLST provided considerably less phylogenetic information. The resolution of spa typing was similar to that of AFLP and inferior to that of DLST. AFLP proved to be the most universal method, combining a phylogeny-building capacity similar to that of MLST with a much higher resolution. However, it had a lower reproducibility than sequencing-based MLST, DLST, and spa typing. We found two cases of methicillin-resistant S. aureus colonization, both of which were most likely associated with employment at a health service. Of 21 genotypic clusters detected, 2 were most prevalent: cluster 45 and cluster 30 each colonized 24% of the carrier population. The number of bacteria found in nasal samples varied significantly among the clusters, but the most prevalent clusters were not particularly numerous in the nasal samples. We did not find much evidence that genotypic clusters were associated with different carrier characteristics, such as age, sex, medical conditions, or antibiotic use. This may provide empirical support for the idea that genetic clusters in bacteria are maintained in the absence of adaptation to different niches. Alternatively, carrier characteristics other than those evaluated here or factors other than human hosts may exert selective pressure maintaining genotypic clusters.     

DNA identification and characterization of Campylobacter jejuni and Campylobacter coli isolated from caecal samples of chickens in Grenada

Aims: To speciate Campylobacter strains from the caeca of chickens in Grenada using PCR and to evaluate DNA‐based typing methods for the characterization of these isolates. Methods and Results: Isolates were speciated with two multiplex PCR assays and were typed with flaA‐RFLP, pulsed‐field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). Results confirmed that Campylobacter coli strains were more predominant than Campylobacter jejuni strains. From 56 isolates, 18 were misidentified using biochemical tests. PFGE typing gave the highest discriminatory power among the methods used (Simpson’s index of diversity, D = 0·9061). However, the combination of flaA‐RFLP, PFGE and MLST results gave the highest discrimination for subtyping of these isolates (D = 0·9857). A band position tolerance of 4% in Bio Numerics was the most appropriate for the analysis of this database. MLST profiles were generally concordant with PFGE and/or flaA‐RFLP types. Several isolates exhibited new MLST sequence types (STs), and 43 of the 49 Camp. coli strains belonged to the ST‐828 clonal complex. Conclusions: Campylobacter coli was the most prevalent species isolated from broilers and layers in Grenada, and a combination of restriction and sequence methods was most appropriate for the typing of Camp. coli isolates. Campylobacter coli STs clustered with described poultry‐associated Camp. coli STs by phylogenetic analysis. Significance and Impact of the Study: Further studies to understand the predominance of Camp. coli within Campylobacter spp. from chickens in Grenada may help elucidate the epidemiology of these pathogens in chickens.     

Prevalence and distribution of Arcobacter species in various sources in Turkey and molecular analysis of isolated strains by ERIC-PCR

AIMS: To determine the prevalence of Arcobacter in various food, animal and water sources in Turkey and to subtype the isolated strains using enterobacterial repetitive intergenic consensus (ERIC)-PCR. METHODS AND RESULTS: A total of 806 samples consisting of chicken (100) and turkey meat (100); minced beef (27); rectal swabs from cattle (173), sheep (68) and dogs (62); cloacal swabs of broilers (100) and layers (100); gall bladders of cattle (50) and drinking water samples (26) were examined. A previously described membrane filtration method was used for the isolation. Isolates were identified at species level using multiplex-PCR and discriminated by ERIC-PCR for subtyping. Ninety-eight (12.1%) of the samples examined were found positive for arcobacters. Arcobacter spp. were isolated from 68%, 4%, 6.9%, 8% and 37% of chicken and turkey meats, rectal swabs and gall bladders of cattle and minced beef, respectively. No arcobacters were obtained from the rectal swabs of sheep and dogs, cloacal swabs of broilers and layers, and water samples examined. In total, 99 Arcobacter isolates were obtained. Of these isolates, 92 were identified as Arcobacter butzleri, five were Arcobacter skirrowii and two were Arcobacter cryaerophilus. Thirteen distinct DNA profiles among A. butzleri isolates were obtained by the ERIC-PCR. Of these profiles, eight were from chicken carcass, three from cattle rectal swab and two from minced beef meat isolates. Some of the isolates originated from different sources gave the same DNA profiles. All isolates of A. skirrowii and A. cryaerophilus gave different DNA profiles. CONCLUSIONS: Poultry carcasses, minced beef meat, rectal swabs and gall bladders of cattle were found to be positive for Arcobacter spp. A. butzleri was the predominant species isolated. In addition, large heterogeneity among the Arcobacter isolates was determined. SIGNIFICANCE AND IMPACT OF THE STUDY: Contamination of the poultry carcasses and minced beef meat, rectal and gall bladder samples of cattle with arcobacters poses a risk for both human and animal infections. Detection of several different Arcobacter strains may suggest multiple sources for contamination and infection.     

Presence of Arcobacter spp. in environmental waters correlates with high levels of fecal pollution

We investigated the presence of Arcobacter spp. in 205 water samples of freshwater, seawater and sewage in Spain. These bacteria were present in 55.1% of the samples (113/205) and were significantly associated for the first time with bacterial indicators of fecal pollution. The dominant species in the positive samples was Arcobacter butzleri (94%) followed by Arcobacter cryaerophilus (30%) and Arcobacter skirrowii (1.8%).     

Genotypic Diversity and Virulence Characteristics of Clinical and Environmental Vibrio vulnificus Isolates from the Baltic Sea Region

The genetic diversity of Vibrio vulnificus isolates from clinical and environmental sources originating from the Baltic Sea region was evaluated by multilocus sequence typing (MLST), and possible relationships between MLST clusters, potential genotypic and phenotypic traits associated with pathogenicity, and source of isolation were investigated. The studied traits included genotyping of polymorphic loci (16S rRNA, vcg, and pilF), presence/absence of potential virulence genes, including nanA, nab, and genes of pathogenicity regions, metabolic features, hemolytic activity, resistance to human serum, and cytotoxicity to human intestinal cells. MLST generated 35 (27 new) sequence types and divided the 53 isolates (including four reference strains) into two main clusters, with cluster I containing biotype 1 and 2 isolates of mainly environmental origin and cluster II containing biotype 1 isolates of mainly clinical origin. Cluster II isolates were further subdivided into two branches. Branch IIB included isolates from recent cases of wound infections that were acquired at the German Baltic Sea coastline between 2010 and 2011 and isolates from seawater samples of the same regions isolated between 1994 and 2010. Comparing the MLST data with the results of genotyping and phenotyping showed that strains of MLST cluster II possess a number of additional pathogenicity-associated traits compared to cluster I strains. Rapid microbiological methods such as matrix-assisted laser desorption ionization–time of flight (MALDI-TOF) mass spectrometry combined with typing of selected virulence-associated traits (e.g., serum resistance, mannitol fermentation, nanA, and pathogenicity region XII) could be used for risk assessment purposes regarding V. vulnificus strains isolated from the Baltic Sea region.     

Genetic Diversity of Campylobacter jejuni Isolates from Farm Animals and the Farm Environment

The genetic diversity of Campylobacter jejuni isolates from farm animals and their environment was investigated by multilocus sequence typing (MLST). A total of 30 genotypes, defined by allelic profiles (assigned to sequence types [STs]), were found in 112 C. jejuni isolates originating in poultry, cattle, sheep, starlings, and slurry. All but two of these genotypes belonged to one of nine C. jejuni clonal complexes previously identified in isolates from human disease and retail food samples and one clonal complex previously associated with an environmental source. There was some evidence for the association of certain clonal complexes with particular farm animals: isolates belonging to the ST-45 complex predominated among poultry isolates but were absent among sheep isolates, while isolates belonging to the ST-61 and ST-42 complexes were predominant among sheep isolates but were absent from the poultry isolates. In contrast, ST-21 complex isolates were distributed among the different isolation sources. Comparison with MLST data from 91 human disease isolates showed small but significant genetic differentiation between the farm and human isolates; however, representatives of six clonal complexes were found in both samples. These data demonstrate that MLST and the clonal complex model can be used to identify and compare the genotypes of C. jejuni isolates from farm animals and the environment with those from retail food and human disease.     

RecA gene sequence and Multilocus Sequence Typing for species-level resolution of Burkholderia cepacia complex isolates

Aim:  To identify, by means of recA sequencing and multilocus sequence typing (MLST), Burkholderia cepacia complex (BCC) isolates of environmental and clinical origin, which failed to be identified by recA RFLP and species-specific PCR.
Methods and Results:  By using recA sequence-based identification, 17 out of 26 BCC isolates were resolved at the level of species and lineage (ten Burkholderia cenocepacia IIIB, two Burkholderia arboris and five Burkholderia lata ). By using MLST method, 24 BCC isolates were identified. MLST confirmed recA sequence results, and, furthermore, enabled to identify isolates of the BCC5 group, and showed relatedness with Burkholderia contaminans for one of the two isolates not identified.
Conclusions:  recA sequence-based identification allowed to resolve, at the level of species and lineage, 65·4%, of the BCC isolates examined, whilst MLST increased this percentage to 88·5%.
Significance and Impact of the Study:  BCC isolates previously not resolved by recA RFLP and species-specific PCR were successfully identified by means of recA sequencing and MLST, which represent the most appropriate methods to identify difficult strains for epidemiological purposes and cystic fibrosis patients management.     

Detection of Arcobacter spp. in the coastal environment of the Mediterranean Sea

The occurrence of Arcobacter spp. was studied in seawater and plankton samples collected from the Straits of Messina, Italy, during an annual period of observation by using cultural and molecular techniques. A PCR assay with three pairs of primers targeting the 16S and 23S rRNA genes was used for detection and identification of Arcobacter butzleri, Arcobacter cryaerophilus, and Arcobacter skirrowii in cultures and environmental samples. Only one of the Arcobacter species, A. butzleri, was isolated from seawater and plankton samples. With some samples the A. butzleri PCR assay gave amplified products when cultures were negative. A. cryaerophilus and A. skirrowii were never detected by culture on selective agar plates; they were detected only by PCR performed directly with environmental samples. Collectively, our data suggest that culturable and nonculturable forms of Arcobacter are present in marine environments. The assay was useful for detecting Arcobacter spp. both as free forms and intimately associated with plankton. This is the first report showing both direct isolation of A. butzleri and the presence of nonculturable Arcobacter spp. in the coastal environment of the Mediterranean Sea.