Analysis of a Clostridium difficile PCR ribotype 078 100 kilobase island reveals the presence of a novel transposon, Tn6164.

BACKGROUND: Clostridium difficile is the main cause of antibiotic associated diarrhea. In the past decade, the number of C. difficile patients has increased dramatically, coinciding with the emergence of two PCR ribotypes 027 and 078. PCR ribotype 078 is also frequently found during C. difficile outbreaks in pigfarms. Previously, the genome of the PCR ribotype 078 strain M120, a human isolate, was described to contain a unique insert of 100 kilobases. RESULTS: Analysis of this insert revealed over 90 open reading frames, encoding proteins originating from transposons, phages and plasmids. The insert was shown to be a transposon (Tn6164), as evidenced by the presence of an excised and circularised molecule, containing the ligated 5'and 3'ends of the insert. Transfer of the element could not be shown through filter-mating experiments. Whole genome sequencing of PCR ribotype 078 strain 31618, isolated from a diarrheic piglet, showed that Tn6164 was not present in this strain. To test the prevalence of Tn6164, a collection of 231 Clostridium difficile PCR ribotype 078 isolates from human (n = 173) and porcine (n = 58) origin was tested for the presence of this element by PCR. The transposon was present in 9 human, tetracycline resistant isolates, originating from various countries in Europe, and none of the pig strains. Nine other strains, also tetracycline resistant human isolates, contained half of the transposon, suggesting multiple insertion steps yielding the full Tn6164. Other PCR ribotypes (n = 66) were all negative for the presence of the transposon. Multi locus variable tandem repeat analysis revealed genetic relatedness among transposon containing isolates. Although the element contained several potential antibiotic resistance genes, it did not yield a readily distinguishable phenotype. CONCLUSIONS: Tn6164 is a newly described transposon, occurring sporadically in C. difficile PCR ribotype 078 strains. Although no transfer of the element could be shown, we hypothesize that the element could serve as a reservoir of antibiotic resistance genes for other bacteria. Further research is needed to investigate the transfer capabilities of the element and to substantiate the possible role of Tn6164 as a source of antibiotic resistance genes for other gut pathogens.

A One Health Perspective on the Human-Companion Animal Relationship with Emphasis on Zoonotic Aspects.

Over time the human-animal bond has been changed. For instance, the role of pets has changed from work animals (protecting houses, catching mice) to animals with a social function, giving companionship. Pets can be important for the physical and mental health of their owners but may also transmit zoonotic infections. The One Health initiative is a worldwide strategy for expanding collaborations in all aspects of health care for humans, animals, and the environment. However, in One Health communications the role of particularly dogs and cats is often underestimated. OBJECTIVE: Evaluation of positive and negative One Health issues of the human-companion animal relationship with a focus on zoonotic aspects of cats and dogs in industrialized countries. METHOD: Literature review. RESULTS: Pets undoubtedly have a positive effect on human health, while owners are increasing aware of pet's health and welfare. The changing attitude of humans with regard to pets and their environment can also lead to negative effects such as changes in feeding practices, extreme breeding, and behavioral problems, and anthropozoonoses. For the human, there may be a higher risk of the transmission of zoonotic infections due to trends such as sleeping with pets, allowing pets to lick the face or wounds, bite accidents, keeping exotic animals, the importation of rescue dogs, and soil contact. CONCLUSIONS: One Health issues need frequently re-evaluated as the close human-animal relationship with pet animals can totally differ compared to decennia ago. Because of the changed human-companion animal bond, recommendations regarding responsible pet-ownership, including normal hygienic practices, responsible breeding, feeding, housing, and mental and physical challenges conforming the biology of the animal are required. Education can be performed by vets and physicians as part of the One Health concept.

Rat bite fever.

Rat bite fever (RBF) is a bacterial zoonosis for which two causal bacterial species have been identified: Streptobacillis moniliformis and Spirillum minus. Haverhill fever (HF) is a form of S. moniliformis infection believed to develop after ingestion of contaminated food or water. Here the infectious agents, their host species, pathogenicity (virulence factors and host susceptibility), diagnostic methods, therapy, epidemiology, transmission and prevention are described. Special emphasis is given on information from the field of laboratory animal microbiology and suggestions for future research.

Arcobacter spp. possess two very short flagellins of which FlaA is essential for motility.

Like Campylobacter and Helicobacter spp., Arcobacter spp. possess two flagellin genes (flaA and flaB) located adjacent to each other. The aim of this study was to characterize the flagellin proteins of Arcobacter spp., because these proteins are known virulence factors in the Epsilonproteobacteria, to which these three species belong. With the exception of Arcobacter nitrofigilis, Arcobacter flagellins are almost half the size of those in other Epsilonproteobacteria. Arcobacter flagellin proteins lack a large part of the variable central region. The low homology observed among flagellins of different Arcobacter species indicates genetic heterology between the members of this genus. Unlike in other Epsilonproteobacteria, the transcription of flagellin genes is not regulated by sigma 28- or sigma 54-dependent promoters, which suggests that transcription must be regulated in a different way in Arcobacter spp. Mutational studies revealed that only FlaA is needed for the motility of Arcobacter spp. Quantitative PCR analysis showed that transcription of flaB is higher at 30 degrees C than at 37 degrees C. Mutation of flaB had no effect on motility or on flaA transcription while mutation of flaA abolished motility and increased the transcription of flaB. These results underline that the genus Arcobacter is an unusual taxon in the epsilon subdivision of the Proteobacteria.

The introduction of Arcobacter spp. in poultry slaughterhouses.

Despite the presence high levels of Arcobacter spp. on chicken carcasses, the source of arcobacter contamination in slaughterhouses still remains unclear. It has been hypothesised in the literature that Arcobacter species that contaminate carcasses originate in in-plant slaughterhouses and/or supply water. The present study aimed to determine the source of Arcobacter contamination in two poultry slaughterhouses in The Netherlands. Carcasses and intestinal tracts from 3 hen flocks and 2 broiler flocks were collected. Water draining off carcasses during processing in 2 slaughterhouses and supply water in one slaughterhouse were also taken. For one flock, cloacal swabs and faecal samples were taken on the farm before slaughtering. ERIC-PCR was applied to study the genetic diversity and relationship among the isolates. No Arcobacter spp. were found in the supply water but on almost all of the sampled carcasses and in carcass-draining-off water arcobacters were identified. Arcobacter spp. were detected in the gut systems of chickens, ranging from 20% to 85% in hens and 3.3% and 51% in broilers. Similar ERIC-PCR genotypes were detected in gut contents as well as on carcasses from the same flock. The present study demonstrated that Arcobacter spp. can be detected in chicken intestines at slaughter and could be brought in this way into slaughterhouses where the bacteria contaminate carcasses during processing.

Dogs as vectors of Streptobacillus moniliformis infection?

Rat bite fever is a bacterial zoonosis transmitted through the bite of rats. One of the two etiological agents that cause rat bite fever is Streptobacillus moniliformis. Rat bite fever is rare and very likely under diagnosed but occurs worldwide. Other animals, like dogs and cats that have mouthed a rat are often mentioned in the literature as potential risks for the attraction of rat bite fever. However, rat bite fever caused by the bite of a dog or cat has very seldom been documented. Therefore, to identify the possible risk for humans to become infected with S. moniliformis after having been bitten by a dog that has been in contact with rats, the presence of S. moniliformis in the mouth of these dogs was tested with molecular methods. Swabs taken from the mouth of 18 dogs with proven contacts with rats were tested for the presence of S. moniliformis DNA by PCR. An amplicon of the right size was obtained in 10 of the 18 dogs. Nucleotide sequencing of five amplicons of PCR positive samples demonstrated the presence of S. moniliformis DNA in the mouth of three dogs. A bite by these dogs therefore might infect humans with S. moniliformis and cause rat bite disease.

Zoonotic bacteria and parasites found in raw meat-based diets for cats and dogs.

Feeding raw meat-based diets (RMBDs) to companion animals has become increasingly popular. Since these diets may be contaminated with bacteria and parasites, they may pose a risk to both animal and human health. The purpose of this study was to test for the presence of zoonotic bacterial and parasitic pathogens in Dutch commercial RMBDs. We analysed 35 commercial frozen RMBDs from eight different brands. Escherichia coli serotype O157:H7 was isolated from eight products (23 per cent) and extended-spectrum beta-lactamases-producing E coli was found in 28 products (80 per cent). Listeria monocytogenes was present in 19 products (54 per cent), other Listeria species in 15 products (43 per cent) and Salmonella species in seven products (20 per cent). Concerning parasites, four products (11 per cent) contained Sarcocystis cruzi and another four (11 per cent) S tenella In two products (6 per cent) Toxoplasma gondii was found. The results of this study demonstrate the presence of potential zoonotic pathogens in frozen RMBDs that may be a possible source of bacterial infections in pet animals and if transmitted pose a risk for human beings. If non-frozen meat is fed, parasitic infections are also possible. Pet owners should therefore be informed about the risks associated with feeding their animals RMBDs.

Interaction of Arcobacter spp. with human and porcine intestinal epithelial cells.

Little is known about the pathogenic mechanisms or potential virulence factors of Arcobacter spp. The aim of the study described here was to obtain more insights in the pathogenicity mechanisms of Arcobacter spp. by testing their ability to adhere to, invade and induce interleukin-8 expression in human Caco-2 and porcine IPI-2I cell lines. Eight Arcobacter strains were tested. Four strains were obtained from a culture collection, and represent the four Arcobacter spp. known to be associated with animals and humans. The other four strains were field isolates from the amniotic fluid of sows and from newborn piglets. All eight Arcobacter strains were able to adhere to both cell lines, and induced interleukin-8 production as early as 2 h after a 1h incubation period. This production was still increased 6 h postinfection. Differences in the cell association of the eight strains were obvious, with A. cibarius showing the highest adhesion ability. Invasion of intestinal epithelial cells was only observed for A. cryaerophilus strains. No correlation between invasiveness or strong adhesion of the tested strains and the level of interleukin-8 induction was observed.

Farmers' knowledge and expectations of antimicrobial use and resistance are strongly related to usage in Dutch livestock sectors.

Comprehensive strategies to improve on-farm antimicrobial use (AMU) are needed to contain antimicrobial resistance (AMR). Little is known about farmers' motivating and enabling factors, and about their influence on AMU. In a cross-sectional online survey, Dutch dairy, veal and pig farmers (n=457) reported their on-farm AMU as "Defined Daily Dose Animal" per year (DDDAF) and completed a detailed questionnaire on their view, knowledge and behavior towards AMU and AMR. Exploratory factor analysis (EFA) on the questionnaire items identified four psychological factors labeled as 'referent beliefs', 'perceived risk', 'knowledge', and 'undesired attitude to regulations'. Linear regression was done to explore the relationship between the obtained factors and on-farm AMU across the three animal sectors. Dairy farmers showed the highest factor scores for 'knowledge' and the lowest for 'perceived risk'. 'Knowledge' scores were significantly and inversely related to AMU (P=0.0004). Borderline significant associations with AMU were found for 'perceived risk' and 'undesired attitude to regulations' (negative and positive relationships respectively). There were no apparent differences for these relationships between the three livestock sectors. Behavioral interventions in farmers such as educational campaigns or increased support by veterinarians could empower farmers with more prudent and rational practices, eventually reducing AMU in food animals.

Arcobacter, what is known and unknown about a potential foodborne zoonotic agent!

Since the introduction of the genus Arcobacter in 1991, the association of Arcobacter butzleri, Arcobacter cryaerophilus and Arcobacter skirrowii with humans and animals has been clearly established. These bacteria have been detected world wide in products of animal origin and in healthy animals as well as in surface water. A fourth species Arcobacter cibarius was recently discovered on chicken carcasses. Although evidence was found for the connection of Arcobacter spp. with human and animal illness, Arcobacter spp. can be pathogens, opportunistic pathogens and commensals. Their potential as zoonotic foodborne and waterborne agents, the routes of transmission and the pathogenic mechanisms of these bacteria are largely unknown. Production of toxins or other virulence factors has not been demonstrated but adhesive and/or invasive properties were apparent. Antibiotic resistance is present in Arcobacter strains to significant levels. The tools to genetically access Arcobacter-like transformation of strains, construction of mutants are not yet available. Nor have genes (i.e. potential virulence factors) been cloned, expressed and characterized in other host organisms. Therefore those interested in the microbiology of these organisms eagerly await publication of the complete nucleotide sequence of the Arcobacter genome. The abundant presence of four Arcobacter species in foods of animal origin and the recovery of these bacteria from surface and drinking water suggest an important role of these bacteria as foodborne or waterborne agent and possibly as zoonotic agent.

Explanatory Variables Associated with Campylobacter and Escherichia coli Concentrations on Broiler Chicken Carcasses during Processing in Two Slaughterhouses.

This study aimed at identifying explanatory variables that were associated with Campylobacter and Escherichia coli concentrations throughout processing in two commercial broiler slaughterhouses. Quantative data on Campylobacter and E. coli along the processing line were collected. Moreover, information on batch characteristics, slaughterhouse practices, process performance, and environmental variables was collected through questionnaires, observations, and measurements, resulting in data on 19 potential explanatory variables. Analysis was conducted separately in each slaughterhouse to identify which variables were related to changes in concentrations of Campylobacter and E. coli during the processing steps: scalding, defeathering, evisceration, and chilling. Associations with explanatory variables were different in the slaughterhouses studied. In the first slaughterhouse, there was only one significant association: poorer uniformity of the weight of carcasses within a batch with less decrease in E. coli concentrations after defeathering. In the second slaughterhouse, significant statistical associations were found with variables, including age, uniformity, average weight of carcasses, Campylobacter concentrations in excreta and ceca, and E. coli concentrations in excreta. Bacterial concentrations in excreta and ceca were found to be the most prominent variables, because they were associated with concentration on carcasses at various processing points. Although the slaughterhouses produced specific products and had different batch characteristics and processing parameters, the effect of the significant variables was not always the same for each slaughterhouse. Therefore, each slaughterhouse needs to determine its particular relevant measures for hygiene control and process management. This identification could be supported by monitoring changes in bacterial concentrations during processing in individual slaughterhouses. In addition, the possibility that management and food handling practices in slaughterhouses contribute to the differences in bacterial contamination between slaughterhouses needs further investigation.

Pre-scald brushing for removal of solids and associated broiler carcass bacterial contamination.

The aim of the study was to investigate the effect of brushing prior to scalding on reducing the E. coli and Enterobacteriaceae concentrations on carcasses. Three visits were arranged to a commercial slaughterhouse in which carcasses were cleaned in a separate line. Ten batches were sampled to compare the E. coli and Enterobacteriaceae concentrations on carcasses before and after a stand-alone brushing unit. Per batch, 8 carcasses before and 8 after brushing were sampled by the whole-carcass rinse method. Furthermore, the dry matter content and the pH were determined in these samples, as these parameters indirectly (dry matter) or directly (pH) influence the scalding lethality. Results revealed a small but statistically significant reduction (P < 0.001) in E. coli and Enterobacteriaceae concentrations on the brushed carcasses. The concentrations on whole carcasses were reduced on average by 0.3 log for both E. coli and Enterobacteriaceae. Rinse samples from treated carcasses had significantly less dry matter on average by 2.5 g (P < 0.001) and significantly higher pH by 0.08 units (P < 0.001). Although these differences are statistically significant, they might have rather low biological relevance; thus, further optimization of brushes is needed for more relevant results. This study confirms that brushing reduces bacterial concentrations on carcasses, which may be increased potentially by enlarging the brushed surface of the carcass. Further in-line investigations are needed to observe the effect of brushing on bacterial concentrations in scalding water and on carcasses after scalding and at the end of processing.

A comparison of fluctuations of Campylobacter and Escherichia coli concentrations on broiler chicken carcasses during processing in two slaughterhouses.

The causes of differences in Campylobacter and Escherichia coli concentrations on broiler chicken carcasses after chilling between slaughterhouses are not fully identified. Therefore, it is a challenge for slaughterhouses to comply with Process Hygiene Criteria for broiler meat. The aim of the study was to identify which processing steps contribute to increases or decreases in Campylobacter and E. coli concentrations within and between two slaughterhouses. Identifying the processing steps with variable performance could explain the differences in bacterial concentrations after chilling between slaughterhouses. Thermotolerant Campylobacter and E. coli concentrations on carcasses during broiler processing were measured during the summer period in 21 trials after bleeding, scalding, defeathering, evisceration and chilling. In two slaughterhouses with comparable Campylobacter and E. coli concentrations in the incoming batches (after bleeding), the mean log10 concentrations are found to be significantly different after chilling. Campylobacter concentrations decreased by 1.40 log10 in Slaughterhouse 1 and by 1.86 log10 in Slaughterhouse 2, whereas E. coli decreased by 2.19 log10 in Slaughterhouse 1 and by 2.84 log10 in Slaughterhouse 2. Higher concentrations of Campylobacter and E. coli on carcasses after chilling were observed in Slaughterhouse 1 in which an increase in concentrations was observed after evisceration. The effect of processing on Campylobacter and E. coli concentrations in Slaughterhouse 1 did not differ between batches. In Slaughterhouse 2, the effect of processing on the concentrations of both bacteria varied over batches. Changes in E. coli concentration levels during processing were similar to Campylobacter except for defeathering. E. coli concentration significantly decreased after defeathering in both slaughterhouses, whereas Campylobacter increased in Slaughterhouse 2 and in Slaughterhouse 1 no significant changes were observed. The patterns of increases and decreases in bacterial concentrations during processing are specific for each slaughterhouse. Inhomogeneous patterns potentially explain the differences in concentrations after chilling between slaughterhouses. Critical processing steps should be validated in each slaughterhouse by longitudinal studies and potentially based on E. coli. E. coli has a potential to be used as an indicator of processing hygiene, because the impact of most of the studied processing steps was similar as for Campylobacter.

Effect of fermented feed on shedding of Enterobacteriaceae by fattening pigs.

Epidemiological studies showed that the use of fermented feed could significantly reduce Salmonella prevalence in pigs compared to the use of normal feed. Experimental challenge experiments with Salmonella have however never been conducted to reveal the efficacy of fermented feed in reducing Salmonella shedding and/or reducing the number of Salmonella-positive pigs. A longitudinal study was conducted to measure the effect of fermented feed, in particular of its components lactic acid and Lactobacillus plantarum, on gastrointestinal bacterial ecology (Salmonella, Enterobacteriaceae, lactobacilli, volatile fatty acids (VFAs), pH). Seeder pigs were used as a mode for Salmonella transmission within a pig herd. Bacteriological measurements were performed in faeces of the pigs. The results showed that fermented feed affected/reduced the Enterobacteriaceae population in faeces of the pigs. No differences were found in the number of positive pigs infected or in the number of shedding with Salmonella serovar Typhimurium fed fermented feed and between the normal feed group. S. serovar Goldcoast could not establish an infection in the seeder pigs in the fermented feed group as well as in the normal feed group. The pH of the faeces in the fermented feed groups was significantly higher than the pH of the faeces of the normal feed groups. The role of the undissociated form of the faecal VFAs on the significantly lower Enterobacteriaceae number in faeces of the pigs of the fermented feed groups could not be demonstrated because of the significant higher pH in the faeces of the pigs fed fermented feed.

Propidium monoazide does not fully inhibit the detection of dead Campylobacter on broiler chicken carcasses by qPCR.

A real time quantitative PCR combined with propidium monoazide (PMA) treatment of samples was implemented to quantify live C. jejuni, C. coli and C. lari on broiler chicken carcasses at selected processing steps in the slaughterhouse. The samples were enumerated by culture for comparison. The Campylobacter counts determined with the PMA-qPCR and the culture method were not concordant. We conclude that the qPCR combined with PMA treatment of the samples did not fully reduce the signal from dead cells.

Capnocytophaga canimorsus.

Capnocytophaga canimorsus is a commensal bacterium in the oral flora of dogs and cats. The bacterium is a zoonotic agent and has been isolated from humans, infected by dog or cat bites, scratches, licks or simply exposure to dogs or cats. Here the infectious agent, its pathogenicity and potential virulence factors, infection in animals and humans, diagnostic methods, prevalence, therapy and prevention are described. Suggestions for future research are given.

Pythium insidiosum: an overview.

Pythium insidiosum is an oomycete pathogenic in mammals. The infection occurs mainly in tropical and subtropical areas, particularly in horses, dogs and humans. Infection is acquired through small wounds via contact with water that contains motile zoospores or other propagules (zoospores or hyphae). The disease, though described as emerging has in fact already been described since 1884. Depending on the site of entry, infection can lead to different forms of pythiosis i.e. a cutaneous, vascular, ocular, gastrointestinal and a systemic form, which is rarely seen. The infection is not contagious; no animal-animal or animal-human transmission has been reported so far. Therapy includes radical surgery, antifungal drugs, immunotherapy or a combination of these therapies. The prevention to contract the disease in endemic areas is difficult. Avoiding stagnant waters could be of help, although the presence of P. insidiosum on grass and soil in enzootic areas renders this practice useless.

Competitive exclusion of Salmonella enterica serovar Enteritidis by Lactobacillus crispatus and Clostridium lactatifermentans in a sequencing fed-batch culture.

Competitive exclusion of Salmonella enterica serovar Enteritidis by a mixed culture of Lactobacillus crispatus and Clostridium lactatifermentans was studied in a sequencing fed-batch reactor mimicking the cecal ecophysiology of broiler chickens. Growth of serovar Enteritidis was inhibited by a mixed culture of L. crispatus and C. lactatifermentans at pH 5.8 but not by a monoculture of L. crispatus at the same pH. Moreover, experiments performed at pH 7.0 did not show growth inhibition of serovar Enteritidis. L. crispatus fermented lactose to lactate, and C. lactatifermentans fermented the lactate to acetate and propionate in a mixed culture of L. crispatus and C. lactatifermentans growing on lactose. In contrast, only lactate was produced from lactose by a monoculture of L. crispatus. At pH 5.8 considerable concentrations of acetate and propionate were present as undissociated acids, whereas only trace levels of undissociated lactate were present at pH 5.8 due to the low pK(a) of lactate. At pH 7.0 all three acids were present in their dissociated forms. We conclude that a mixed culture of L. crispatus and C. lactatifermentans inhibits growth of serovar Enteritidis under cecal growth conditions. The undissociated forms of acetate and propionate produced in the mixed culture inhibited the growth of serovar Enteritidis.