Genetic characteristics and potential pathogenic agents in Campylobacter upsaliensis based on genomic analysis.

Campylobacter upsaliensis was the most common Campylobacter species in pets' gastrointestinal tracts and has been isolated from patients with bacteremia, hemolytic-uremic syndrome, spontaneous abortion, and Guillain-Barré syndrome. However, the genetic characteristics and the full extent of its significance as a human pathogen remain to be fully understood. This study involved an investigation for genomic analysis of 154 strains from different sources and additional antimicrobial resistance profiles of 26 strains for this species. The genomes contained 1,558-1,971 CDS and the genome sizes were estimated to vary from 1.53 Mb to 1.86 Mb, with an average GC content of 34.71%. The entire analyzed genomes could be divided into three clades (A, B, and C) based on ANI and phylogenomic analysis. Significantly, nearly all strains in Clade B were isolated from patient samples, and the virulence-related sequences FlgD, GmhA, and CdtC might serve as determining factors for the classification of Clade B. Half of the tested isolates had MIC values over 64 µg mL-1 for nalidixic acid, gentamicin, and streptomycin. Isolates from pets in China carried more resistant elements in the genomes. This study both provided a comprehensive profile of C. upsaliensis for its genomic features and suggested some pathogenic agents for human infection with this species.

Multidrug resistance and high genotypic diversity in Campylobacter upsaliensis from household dogs in Metro Manila, Philippines.

Campylobacter upsaliensis is an emerging pathogen implicated in human gastroenteritis. Contact with pets, especially dogs, has been identified as a risk factor. Fourteen (14) C. upsaliensis isolates were obtained from household dogs in Metro Manila, Philippines. Antimicrobial susceptibility testing (AST) determined high resistance to ciprofloxacin (92.86%), erythromycin (100%), and tetracycline (100%). Multilocus sequence typing (MLST) identified 14 novel sequence types and 1 novel clonal complex, which is suggestive of a high genotypic diversity within C. upsaliensis. AST results indicate that antimicrobial stewardship and education of pet owners should be emphasised in veterinary practice.

Survival capability of Campylobacter upsaliensis under environmental stresses.

OBJECTIVE: Campylobacter upsaliensis has been recognized as an emerging pathogen. However, little is known about its survival in the environment. To evaluate its survival capability, we estimated the reduction in viable counts of C. upsaliensis after aerobic exposure to starvation in phosphate-buffered saline (PBS), acidity (pH = 4.3), high osmolarity (4% NaCl), and dryness in wet pulp disks at different temperatures. Also, survival in dog feces and dog food at variable temperate was assessed. RESULTS: Campylobacter upsaliensis remained culturable under starvation for 4 days at 25 °C and for 10 weeks at 4 °C. C. upsaliensis was also recoverable after exposure to high osmolality for 9 days, dryness for 5 days, and acidity for 2 days, respectively. Similarly, C. upsaliensis survived in dog feces and dog food for several days at 25 °C and weeks at 4 °C. The survival capability of the organism was dependent on the water content, and also temperature. Notably, the tested C. upsaliensis strain was less resilient under all tested conditions than a C. jejuni strain used as a control. The findings showed that C. upsaliensis is able to survive under various environmental stresses, suggesting that it could pose a potential threat to public health.

Campylobacter upsaliensis isolated from a giant hepatic cyst.

Campylobacter upsaliensis is an enteropathogenic bacterium in animals, and is also rarely isolated from humans, where it can cause enteritis and bacteremia. This report describes the first case of isolation of C. upsaliensis from an infected giant hepatic cyst. This bacterium could not be cultured from abscess punctuate in a usual Campylobacter-selection medium (charcoal cefoperazone deoxycholate agar medium), because of high concentration of cefoperazone as a selection agent. It could not identified by matrix-assisted laser desorption ionization-time of flight mass spectrum. Rather, it was identified as C. upsaliensis by whole genome sequencing, including by multilocus sequence typing.

Occurrence and Antimicrobial Resistance Profiles of Campylobacter jejuni, Campylobacter coli, and Campylobacter upsaliensis in Beef Cattle on Cow-Calf Operations in South Africa.

This study investigated occurrence and antimicrobial resistance profiles of Campylobacter spp. isolates in beef cattle on five cow-calf operations in South Africa. A total of 537 fecal samples from adult beef cattle (n = 435) and rectal swabs from calves (n = 102) were screened for Campylobacter jejuni, Campylobacter coli, and Campylobacter upsaliensis by culture and polymerase chain reaction. Furthermore, 86 Campylobacter spp. isolates including 46 C. jejuni, 24 C. coli, and 16 C. upsaliensis were tested for antimicrobial resistance against a panel of 9 antimicrobials. Overall, Campylobacter spp. was detected in 29.7% of cattle. Among the 158 Campylobacter spp.-positive cattle, 61.8% carried C. jejuni, 25% carried C. coli, and 10% carried C. upsaliensis. Five animals (3.1%) had mixed infections: three cows carried C. jejuni and C. coli concurrently, one cow had both C. jejuni and C. upsaliensis, and one cow harbored C. coli and C. upsaliensis. Antimicrobial resistance profiling among 86 Campylobacter spp. isolates revealed that 52.3% of the isolates were resistant to one or more antimicrobials. Antimicrobial resistance was observed in 46.7% of C. jejuni isolates, 35.6% of C. coli, and 17.8% of C. upsaliensis. Thirty-six percent of isolates were resistant to clindamycin, 19.7% to nalidixic acid, 18.6% to tetracycline, and 17.4% to erythromycin. Lower resistance rates were recorded for azithromycin (8.1%), florfenicol (3.4%), gentamicin (4.8%), and telithromycin and ciprofloxacin (5.8%). Multidrug resistance (MDR) was observed in 32.5% of isolates. Significantly higher levels of MDR were detected among C. jejuni (36.9%) and C. coli (33.3%) isolates in comparison to C. upsaliensis (18.7%). Two main multiresistance patterns were detected: nalidixic acid/clindamycin (17.8%) and tetracycline/clindamycin (14.2%). To the best of our knowledge, this is the first study which has shown that beef cattle on cow-calf operations in South Africa constitute an important reservoir and a potential source of clinically relevant and antimicrobial resistant Campylobacter spp. strains.

Prevalence and risk factors associated with Campylobacter spp. occurrence in healthy dogs visiting four rural community veterinary clinics in South Africa.

Reports on the occurrence of Campylobacter spp. in dogs in South Africa are non-existent. This study investigated the prevalence of Campylobacter spp. in 481 dogs visiting four rural community veterinary clinics in South Africa. Dogs were screened for Campylobacter spp. by culture and polymerase chain reaction (PCR), and logistic regression analysis was performed to assess the association between sex, clinic, breed and age and the occurrence of Campylobacter spp. in dogs. The prevalence of Campylobacter spp. was 41.50% (95% confidence interval [CI], 37.39% - 46.04%). Campylobacter jejuni, C. upsaliensis and C. coli were detected in 29.31% (95% CI, 25.42% - 33.54%), 13.10% (95% CI, 10.37% - 16.42%) and 5.41% (95% CI, 3.71% - 7.82%) of dogs, respectively. Dogs carrying more than one species of Campylobacter spp. accounted for 6.23% (95% CI, 4.40% - 8.78%). Campylobacter upsaliensis and C. jejuni were detected in 3.74% (95% CI, 2.37% - 5.86%), whereas C. coli and C. jejuni were found in 2.49% (95% CI, 1.42% - 4.34%) of dogs. Age and clinic were the risk factors significantly associated with Campylobacter spp. occurrence, while age, breed and clinic were predictors of C. jejuni carriage. Furthermore, age was the only risk factor associated with a higher likelihood of carrying C. upsaliensis. The prevalence of Campylobacter spp. C. jejuni and C. upsaliensis increased significantly as dogs grew older. In addition, the odds of carrying Campylobacter spp. were higher in the Staffordshire bull terrier breed compared to crossbreed dogs. In conclusion, this study shows that dogs visiting rural community veterinary clinics in South Africa are reservoirs of Campylobacter spp. and may be potential sources of Campylobacter spp. for humans living in close proximity of the dog populations under study.

Campylobacter culture fails to correctly detect Campylobacter in 30% of positive patient stool specimens compared to non-cultural methods.

Campylobacter diagnosis is hampered because many laboratories continue to use traditional stool culture, which is slow and suffers false-negative results. This large multi-site study used a composite reference method consisting of a new FDA-cleared immunoassay and four molecular techniques to compare to culture. Prospectively collected patient fecal specimens (1552) were first preliminarily categorized as positive or negative by traditional culture. All specimens were also tested by EIA, and any EIA-positive or culture-discrepant results were further characterized by 16S rRNA qPCR, eight species-specific PCR assays, bidirectional sequencing, and an FDA-cleared multiplex PCR panel. The five non-culture methods showed complete agreement on all positive and discrepant specimens which were then assigned as true-positive or true-negative specimens. Among 47 true-positive specimens, culture incorrectly identified 13 (28%) as negative, and 1 true-negative specimen as positive, for a sensitivity of 72.3%. Unexpectedly, among the true-positive specimens, 4 (8%) were the pathogenic species C. upsaliensis. Culture had a 30% false result rate compared to immunoassay and molecular methods. More accurate results lead to better diagnosis and treatment of suspected campylobacteriosis.

Characterization of Campylobacter jejuni, Campylobacter upsaliensis, and a novel Campylobacter sp. in a captive non-human primate zoological collection.

BACKGROUND: The aim of this study was to longitudinally investigate the prevalence and characterization of Campylobacter spp. from non-human primates primate (NHP) with a history of endemic diarrhea housed at Como Park Zoo. METHODS: Fecal samples from 33 symptom-free NHP belonging to eight different species were collected weekly for 9 weeks. Species-level characterization and phylogenetic analysis of isolates included biochemical testing and 16S rRNA sequencing. RESULTS: Campylobacter spp. were isolated from the feces of 42% (14/33) of the primates. Three Campylobacter spp. (C upsaliensis, C jejuni, and novel Campylobacter sp.) were identified from three NHP species. A possible positive host Campylobacter species-specificity was observed. However, no statistical association was observed between the isolation of Campylobacter spp. and age and sex of the animal. CONCLUSIONS: The study revealed the value of conducting repeated fecal sampling to establish the overall prevalence of Campylobacter in zoo-maintained NHP; it also importantly identifies a novel Campylobacter sp. isolated from white-faced saki monkeys.

Investigation of the Role of Campylobacter Infection in Suspected Acute Polyradiculoneuritis in Dogs.

BACKGROUND: Acute polyradiculoneuritis (APN) is an immune-mediated peripheral nerve disorder in dogs that shares many similarities with Guillain-Barré syndrome (GBS) in humans, in which the bacterial pathogen Campylobacter spp. now is considered to be a major triggering agent. Little information is available concerning the relationship between APN and Campylobacter spp. in dogs. HYPOTHESIS/OBJECTIVES: To estimate the association between Campylobacter spp. infection and APN. Associations with additional potential risk factors also were investigated, particularly consumption of raw chicken. ANIMALS: Twenty-seven client-owned dogs suffering from suspected APN and 47 healthy dogs, client-owned or owned by staff members. METHODS: Case-control study with incidence density-based sampling. Fecal samples were collected from each enrolled animal to perform direct culture, DNA extraction, and polymerase chain reaction (PCR) for detection of Campylobacter spp. In some cases, species identification was performed by sequence analysis of the amplicon. Data were obtained from the medical records and owner questionnaires in both groups. RESULTS: In cases in which the fecal sample was collected within 7 days from onset of clinical signs, APN cases were 9.4 times more likely to be positive for Campylobacter spp compared to control dogs (P < 0.001). In addition, a significant association was detected between dogs affected by APN and the consumption of raw chicken (96% of APN cases; 26% of control dogs). The most common Campylobacter spp. identified was Campylobacter upsaliensis. CONCLUSIONS AND CLINICAL IMPORTANCE: Raw chicken consumption is a risk factor in dogs for the development of APN, which potentially is mediated by infection with Campylobacter spp.

Campylobacter upsaliensis isolated from dogs produces high titer of cytolethal distending toxin.

Cytolethal distending toxin (CDT) consisting of CdtA, CdtB and CdtC has been reported to be a possible virulence factor of campylobacters including Campylobacter upsaliensis. In our previous study, the cdtB gene-based PCR-restriction fragment length polymorphism (RFLP) assay for detection and differentiation of 7 Campylobacter species yielded 3 different RFLP patterns (Cu-I to Cu-III). In this study, entire cdt (Cucdt) genes of each pattern were sequenced to see whether there are any differences in cdt genes, its amino acid sequences and biological activity of CuCDT. We found that all 3 representative strains harbor the entire Cucdt genes and homology between prototype and newly determined Cucdt genes was 94 to 98% with cdtA, 93 to 94% with cdtB and 92 to 93% with cdtC, while that between amino acids of CuCDT was 95 to 99% with CdtA, 97 to 98% with CdtB and 92 to 93% with CdtC. Furthermore, CDT activity produced by C. upsaliensis strains was examined by cytotoxicity assay with HeLa cells. Interestingly, C. upsaliensis produced 64 to 2,340 times higher CDT titer in comparison to other campylobacters did. In addition, Cu-III showed 64 times higher CDT titer than Cu-II, although CDT production level was almost the same by western blotting. These data suggest that CDT produced by C. upsaliensis might contribute more to human diseases in comparison to that produced by other campylobacters and Cu-III CDT seems to be more toxic to HeLa cells in comparison to Cu-I and Cu-II CDTs.

A Cytolethal Distending Toxin Gene-Based Multiplex PCR Assay for Campylobacter jejuni, C. fetus, C. coli, C. upsaliensis, C. hyointestinalis, and C. lari.

In this study, we devised a multiplex PCR assay based on the gene of cytolethal distending toxin (cdt) B subunit to simultaneously detect and discriminate Campylobacter jejuni, C. fetus, C. coli, C. upsaliensis, C. hyointestinalis, and C. lari. Species-specific PCR products were successfully obtained from all 38 C. jejuni, 12 C. fetus, 39 C. coli, 22 C. upsaliensis, 24 C. hyointestinalis, and 7 C. lari strains tested. On the other hand, no specific PCR products were obtained from other campylobacters and bacterial species tested (41 strains in total). The proposed multiplex PCR assay is a valuable tool for detection and descrimination of 6 major Campylobacter species, that are associated with gastrointestinal diseases in humans.

Population Genetics and Antimicrobial Susceptibility of Canine Campylobacter Isolates Collected before and after a Raw Feeding Experiment.

In recent years, increasing numbers of consumers have become interested in feeding raw food for their pet dogs as opposed to commercial dry food, in the belief of health advantages. However, raw meat and internal organs, possibly contaminated by pathogens such as Campylobacter spp., may pose a risk of transmission of zoonoses to the pet owners. Campylobacter jejuni is the leading cause of bacterial gastroenteritis in humans but C. upsaliensis has also been associated with human disease. In this study we investigated the effect of different feeding strategies on the prevalence of Campylobacter spp. in Finnish dogs. We further characterized the isolates using multilocus sequence typing (MLST), whole-genome (wg) MLST and antimicrobial susceptibility testing. Dogs were sampled before and after a feeding period consisting of commercial raw feed or dry pellet feed. Altogether 56% (20/36) of the dogs yielded at least one Campylobacter-positive fecal sample. C. upsaliensis was the major species detected from 39% of the dogs before and 30% after the feeding period. Two C. jejuni isolates were recovered, both from raw-fed dogs after the dietary regimen. The isolates represented the same genotype (ST-1326), suggesting a common infection source. However, no statistically significant correlation was found between the feeding strategies and Campylobacter spp. carriage. The global genealogy of MLST types of dog and human C. upsaliensis isolates revealed weakly clonal population structure as most STs were widely dispersed. Major antimicrobial resistance among C. upsaliensis isolates was against streptomycin (STR MIC > 4 mg/l). Apart from that, all isolates were highly susceptible against the antimicrobials tested. Mutations were found in the genes rpsL or rpsL and rsmG in streptomycin resistant isolates. In conclusion, increasing trend to feed dogs with raw meat warrants more studies to evaluate the risk associated with raw feeding of pets in transmission of zoonoses to humans.

First case report of fatal sepsis due to Campylobacter upsaliensis.

We encountered a rare case of severe fatal infection in a 70-year-old woman due to Campylobacter upsaliensis, identified by PCR amplification and sequencing analysis of the 16S rRNA gene using DNA extracted from the isolates. To our knowledge, fatal sepsis due to this organism has never been described to date.

Molecular characterisation of a type III restriction-modification system in Campylobacter upsaliensis.

Two examples of Campylobacter upsaliensis RM3195 and JV21 strains are shown to carry putative type III restriction (res)-modification (mod) enzyme gene clusters, following genome sequence analyses. It is suggested that the cluster is composed of at least three structural genes, res, internal methylase gene and mod, in the strains, based on the nucleotide sequence information. A ribosome binding site, a putative promoter consisting of a consensus sequence at the -10-like structure and a semiconserved T-rich region and a putative intrinsic p-independent transcriptional terminator were identified for the gene cluster in the two strains. Using two primer pairs, f-/r-res and f-/r-mod, 34 of 41 C. upsaliensis isolates generated two expected amplicons of the res and mod gene segments, and using another primer pair, the same number of isolates also generated an amplicon of the res and mod gene segments cluster, including the third internal methylase gene. Thus, C. upsaliensis isolates frequently carried putative type III R-M gene clusters, encoding the three enzymes. Interestingly, two possible overlaps were identified within the three tandem structural genes. In addition, the type III R-M gene cluster loci appear to be very similar among the C. upsaliensis isolates and very different from other thermophilic campylobacters.

Association of Campylobacter upsaliensis with persistent bloody diarrhea.

Campylobacter upsaliensis is a zoonotic, emerging pathogen that is not readily recovered in traditional stool culture. This case represents the first report of persistent bloody diarrhea with C. upsaliensis that was confirmed by filtration culture, PCR, and sequencing.

Campylobacter spp., C. jejuni and C. upsaliensis infection-associated factors in healthy and ill dogs from clinics in Cordoba, Spain. Screening tests for antimicrobial susceptibility.

A microbiological and epidemiological survey examining Campylobacter species-related infection in dogs from clinics in Cordoba (Southern Spain) was performed from May 2007 to March 2008. Three-hundred and six rectal swabs were taken from both ill and healthy dogs, and an epidemiological questionnaire was filled out. The isolated bacterial strains were identified by means of multiplex polymerase chain reaction (PCR). The prevalence factors for Campylobacter spp., C. jejuni and C. upsaliensis infection were determined by logistic regression. An antimicrobial susceptibility screening test was performed using the agar disk diffusion method. Campylobacter species were isolated from 102 samples. Forty strains (35.2%) were identified as Campylobacter jejuni, 60 strains (58.8%) as Campylobacter upsaliensis and two strains (2%) as Campylobacter spp. The sampling season was a prevalence factor for C. jejuni infection, while the age of the dog was a prevalence factor for Campylobacter spp. and C. upsaliensis infection. Enteric disease was a prevalence factor for Campylobacter spp., C. jejuni and C. upsaliensis infection. The antimicrobial susceptibility test indicated that gentamicin and erythromycin were the most effective antibiotics, with 97.8% and 88% strains sensitive to the antibiotics, respectively.

Multilocus sequence typing of human and canine C. upsaliensis isolates.

Risk of Campylobacter infection in humans has been associated with many sources, including dogs. C. upsaliensis is the most common species found in canines, and has been occasionally isolated from symptomatic humans. This study aimed to investigate the genetic diversity of 41 C. upsaliensis isolates carried by dogs and from nine isolates carried by humans using Multilocus sequence typing (MLST). We identified considerable genetic diversity amongst the C. upsaliensis isolates from both dogs and humans, identifying 45 different sequence types (STs). All STs were new, apart from that of the reference strain. Only three STs were found in more than one isolate: ST-72 (2 isolates), ST-98 (2 isolates) and ST-104 (3 isolates). ST-104 was the only ST to be encountered in both dogs and humans. Thirty-one of the 45 STs were assigned to one of 13 clonal complexes (CCs). Four of these CCs contained STs originating from both humans and dogs. None of the CCs contained exclusively human isolates, and two isolates from dogs within the same kennel belonged to the same CC. The large amount of diversity found in both dog and human isolates of C. upsaliensis, combined with the relatively small database, made it difficult to assign strains to sources of infection. This emphasizes the need to increase the size of the database. Dog and human isolates occasionally grouped together, however there were insufficient human-derived isolates to determine whether or not dogs are a common source of infection. Although C. upsaliensis infection is rare in humans, dogs still remain a potential source, and are therefore a possible zoonotic risk. Further work is needed to investigate the epidemiology of C. upsaliensis infection in humans.

Preliminary study on the prevalence of Campylobacter in childhood diarrhoea in north Lebanon

Campylobacter species are a major cause of human diarrhoeal disease worldwide. In Lebanon, the true prevalence of Campylobacter infections and the species distribution in childhood diarrhoea are not known. This study in 2010 investigated the prevalence of Campylobacter species and its possible etiologic role in childhood diarrhoea in north Lebanon. A total of 90 stool samples from children [aged 1 month to 10 years] presenting with diarrhoea were collected from 5 hospitals. A polymerase chain reaction technique [PCR] was used for each sample for the amplification of all Campylobacter species followed by 5 PCR reactions for the amplification of C. jejuni, C. hyointestinalis, C. coli, C. fetusand C. upsaliensis. Of the 90 samples, 10 were positive for Campylobacter species [11.1%]: 1 for C .coli, 1 for C. jejuni, 2 for both C. jejuni and C. coli, and 6 could not be identified to the species level with the available primers. Campylobacter species is frequently associated with childhood diarrhoea in north Lebanon but Campylobacter infection may be significantly underdiagnosed because the search for Campylobacteria not part of the routine stool culture

Factors related to Campylobacter spp. carriage in client-owned dogs visiting veterinary clinics in a region of Ontario, Canada.

From July 2008 until May 2009, 240 client-owned pet dogs from seven veterinary clinics in the Region of Waterloo, Ontario, Canada participated in a study to determine pet-related management factors that may be associated with the presence of Campylobacter spp. in dogs. The prevalence of Campylobacter spp. carriage in our study population of pet dogs was 22%, with 19% of the dogs positive for C. upsaliensis, and 3% positive for C. jejuni. A significant risk factor from multivariable logistic regression models for both Campylobacter spp. and C. upsaliensis carriage was having homemade cooked food as the dog's diet or added to its diet, and a significant sparing factor for both models was treatment with antibiotics in the previous month. Increasing age of the dog decreased the odds of Campylobacter spp. and C. upsaliensis carriage. Based on the high prevalence of Campylobacter, and specifically C. upsaliensis, further research concerning pet dogs as a risk factor for campylobacteriosis in humans is warranted.

Prevalence of Campylobacter spp. in a cross-sectional study of dogs attending veterinary practices in the UK and risk indicators associated with shedding.

Campylobacteriosis is a major cause of gastroenteritis in humans and some studies have suggested that dog ownership is a risk factor for the condition. To determine the prevalence, species distribution, and risk indicators for Campylobacter spp. infecting dogs attending veterinary practices in UK, faecal samples were collected in a cross-sectional study from 249 dogs with and without clinical signs. The prevalence of Campylobacter spp. was 38% (95% CI 32, 44), with Campylobacter upsaliensis accounting for 94 (98%) of the isolates and Campylobacter jejuni for the remainder. Multivariable analysis indicated that younger dogs were more likely to carry C. upsaliensis and the high prevalence of this pathogen supports the hypothesis that dogs, particularly younger animals, may be an important source of C. upsaliensis infection for humans. However the prevalence of C. jejuni, the most common Campylobacter spp. associated with disease in humans, was low (1.2%, 95% CI 0.3, 3).