Campylobacter Upsaliensis Research Articles

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

ABSTRACT 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.

Campylobacteriosis in Poultry: A Review

Campylobacter is common in poultry, including layer and broiler chickens, geese, ducks, and turkeys. This review aimed to emphasize the prevalence of campylobacteriosis, recent poultry diagnoses, and strict prevention measures. Campylobacter species colonize the intestines of poultry and waterfowl but are generally nonpathogenic in poultry. However, they are the most common bacterial cause of sporadic human enteritis in both developed and developing countries. The main species responsible for campylobacteriosis is Campylobacter jejuni, followed by Campylobacter coli. A number of other Campylobacter species, such as Campylobacter lari, fetus, upsaliensis, and hyointestinalis are rarely associated with campylobacteriosis. Campylobacter hepaticus is the species linked to spotty liver disease in layers and breeder chickens, and it may be the etiological agent of the disease previously known as avian vibrionic hepatitis. The most prevalent infection source for Campylobacter is environmental contamination from poultry droppings. However, some Campylobacter species can be transmitted vertically, either on the surface of eggs or via trans-ovarian transmission in addition to consumption of contaminated feed or water. Due to the non-specific clinical signs such as diarrhea and weight loss, diagnosing campylobacteriosis in poultry requires culture or polymerase chain reaction tests. Little is known about the available vaccine or effective antibiotic treatment due to the rapid development of antibiotic resistance. Therefore, strict biosecurity measures play a crucial role in preventing Campylobacter infection in commercial poultry. These measures include decontaminating housing between flocks, preventing the entry of rodents, wild birds, and animals, and eradicating insects. To control campylobacteriosis and reduce infection risks, it is important to implement efficient on-farm biosecurity measures, conduct regular inspections of workers at meat processing plants and poultry farms, and ensure thorough preparation of chicken meat and eggs before consumption. These measures are vital in minimizing the Campylobacter transmission from both broiler and laying chickens, thereby reducing the risk of foodborne diseases caused by contaminated food.

#67 Treatment of Campylobacter upsaliensis and Helicobacter canis in twin brothers with XLA

Abstract Background X-linked agammaglobulinemia (XLA) is a primary immunodeficiency associated with mutations in the B cell tyrosine kinase gene leading to failure in B cell maturation, resulting in antibody deficiency and increased susceptibility to infections. Patients with XLA are at risk of severe disease with infections with Campylobacter and closely related Helicobacter species and warrant treatment with close follow up given risk of recurrence. Method We present follow up of two 5-year-old fraternal twins with XLA who were diagnosed with C. upsaliensis and H. canis respectively via microbial cell-free DNA (mcfDNA) and underwent successful treatment with multi-drug antimicrobial therapy with 12 months of follow-up. Results Patient #1 was diagnosed with XLA at 11 months of age. At 3 years of age, he presented with fever and diarrhea, with stool cultures that grew C. jejuni. Despite treatment with azithromycin, his symptoms persisted and repeat stool cultures grew C. upsaliensis. Testing via mcfDNA confirmed C. upsaliensis. Patient #2 was diagnosed with XLA at 12 months of age. He developed fever, erythema nodosum (EN) like lesions and arthritis at 3.5 years old. Given his twin’s diagnosis of Campylobacter sp. enteritis, he was given a presumptive diagnosis of Campylobacter reactive arthritis and EN. Despite treatment with Naprosyn and 14 days of azithromycin, his symptoms persisted and stool PCR was positive for Campylobacter sp. mcfDNA testing confirmed C. upsaliensis and H.canis. Given failure of initial antimicrobial therapy, both boys were treated with a 4-week course of IV ertapenem, and oral ciprofloxacin and amoxicillin, and both developed neutropenia and concern for drug toxicity. Therapy was transitioned to triple drug therapy with oral doxycycline, ciprofloxacin and amoxicillin. Doxycycline was discontinued after 3 weeks due to patients’ young age. Ciprofloxacin and amoxicillin were continued for 12 months. Both boys had complete resolution of symptoms after 12 months of therapy and remain without recurrence 10 weeks after discontinuation of antimicrobial therapy. Conclusion Physicians should be aware of the treatment challenges of chronic Campylobacter and Helicobacter infection in boys with XLA and other immunocompromised hosts. Successful treatment may require prolonged multi-drug antimicrobial therapy, with monitoring for drug toxicities.

Performance of four commercial real-time PCR assays for the detection of bacterial enteric pathogens in clinical samples

ObjectivesMany laboratories use culture-independent diagnostic tests for bacterial gastroenteritis (i.e. real-time polymerase chain reaction, RT-PCR) instead of culture because of better sensitivity, automation, and faster turnaround times. To address some gaps in initial evaluations and lack of intraassay comparisons for many commercial RT-PCRs, this study compared the ability of four commercially available RT-PCR tests (Ridagene, Fast Track Diagnostics, BD Max, and Prodesse Progastro) to detect five major bacterial enteric pathogens: Campylobacter, Salmonella, Shiga-toxin producing Escherichia coli (STEC), Shigella, and Yersinia. MethodsClinical stool specimens and contrived samples comprising commonly circulating species, serotypes, biovars, and/or toxin subtypes were used for the comparison. ResultsConcordance rates for RT-PCR and culture using culture-positive and culture-negative clinical stools were >90% for Campylobacter (97.5-100%), Salmonella (97.5-100%), Shigella (100%), and STEC (90-100%). However, the agreement between RT-PCR and culture for Y. enteroccolitica ranged from 70-90%. For the contrived sample set, stx2f was detected by one of four assays. Of note, no assay could detect Yersinia non-enterocolitica and Campylobacter upsaliensis. ConclusionsDepending on the prevalence of certain stx sub-types, Yersinia species, and Campylobacter species in a laboratory's jurisdiction, without further improvement in culture-independent tests, culture methods remain critical for the detection of these pathogens.

S1797 Curved Rod Threw a Curveball: Rare Case of Campylobacter upsaliensis Bacteremia, Chronic Diarrhea, and a Negative Stool PCR

Introduction: The genus Campylobacter includes 32 species, with C. jejuni and C. coli most commonly responsible for enteritis and extragastrointestinal manifestations such as bacteremia.[1,2] However, other species such as C. upsaliensis are increasingly found to be associated with disease.[3] We present a unique case of Campylobacter upsaliensis bacteremia in a patient with chronic diarrhea, a negative common, commercially available stool PCR test for Campylobacter, and otherwise negative workup. Case Description/Methods: A 64-year-old male with NASH cirrhosis, diabetes mellitus, and severe aortic stenosis s/p TAVR presented with non-bloody, watery bowel movements (up to 10/day) for more than 6 weeks. He denied fevers, chills, emesis, abdominal pain, recent antibiotic use, or travel. He lived alone with two dogs and a cat. Initial work-up was negative including fecal calprotectin, stool O&P, stool PCR (BD MAX Enteric Bacterial Panel), [4] and C. difficile. He underwent colonoscopy and biopsies were negative for microscopic colitis, showing only minimal architecture distortion which could be consistent with chronic infection. Subsequently, The MALDI-TOF identified Campylobacter upsaliensis from blood cultures. IV azithromycin was initiated, and repeat cultures remained negative. His symptoms improved and he was discharged home with a 14-day course of therapy. Discussion: Stool PCR has replaced stool culture in many acute clinical settings due to rapidity of results and wider availability. Although the stool PCR used was noted to target Campylobacter spp., only Campylobacter spp. jejuni and coli are included, impeding our ability to detect C. upsaliensis with this common testing method. In an otherwise negative work up it may be prudent to consider stool testing for other less common but emerging subspecies in patients with diarrhea.Figure 1.: Top: transverse colon; grossly normal. Bottom: ascending colon with erythematous friable mucosa.

#9: Diagnosis of Campylobacter upsaliensis and Helicobacter canis by plasma-based Next-Generation Sequencing for Microbial Cell-free DNA in twin brothers with XLA

Abstract Background X-linked agammaglobulinemia is a primary immunodeficiency associated with mutations in the B cell tyrosine kinase (BTK) gene leading to failure in B cell maturation and defective antibody production. Campylobacter and closely related Helicobacter species can cause persistent bacteremia, enteritis and cellulitis in patients with XLA and are difficult to diagnose and eradicate. In this context, detection of circulating microbial cell-free DNA (mcfDNA) by next-generation sequencing (NGS) can be used to identify infectious agents in susceptible immunocompromised hosts. Methods We report 4-year-old fraternal twins with XLA presenting with disparate clinical manifestations due to C. upsaliensis and coinfection with C. upsaliensis and H. canis respectively. Results Patient #1 was diagnosed with XLA at 11 months-old and was started on weekly immunoglobulin replacement. At 2.5 yo he developed recurrent fever, abdominal pain and diarrhea. Endoscopy/colonoscopy revealed focal ileocolitis without ulceration or granulomas. Stool infectious workup, including bacterial stool cultures, was negative. At 3 yo he developed gram- rod bacteremia that was not further identified but successfully treated with ceftriaxone. He presented 2 months later with fever and diarrhea; blood cultures were negative, but stool culture grew C. jejuni. His symptoms recrudesced after two months and he was treated for presumed recurrent Campylobacter infection. Intermittent fevers and diarrhea recurred, and repeat stool culture grew C. upsaliensis identified by MALDI-TOF resulting in a 3-month course of azithromycin. Stool PCR remained positive for Campylobacter species after one month of therapy. Fever and diarrhea recurred after completion of therapy and stool culture again grew C. upsaliensis but sensitivities could not be obtained. McfDNA testing confirmed C. upsaliensis and therapy with ertapenem, ciprofloxacin, amoxicillin and doxycycline was initiated. He remained symptom free three months into therapy. Patient #2 was diagnosed with XLA at 1 yo and started weekly immunoglobulin replacement. At 3.5 yo he developed fever, erythema nodosum (EN) and arthritis. Given his twin’s diagnosis of Campylobacter enteritis with likely bacteremia, a presumptive diagnosis of Campylobacter related reactive arthritis and EN was made. Treatment with Naprosyn and 14 days of azithromycin failed to prevent the return of EN post therapy. He had multiple courses of azithromycin each followed by return of EN rash. Following completion of therapy, he presented with high fevers, worsening rash, leukocytosis and elevated ESR. Blood and stool cultures were obtained and returned negative. Despite completion of a 3 month course of azithromycin, stool PCR remained positive for Campylobacter species and his symptoms persisted. The recrudescence of fevers and worsening rash 2 months after completion of therapy prompted repeat blood and stool cultures that returned negative. McfDNA testing was obtained in that context and identified Helicobacter canis and Campylobacter upsaliensis. Treatment with ertapenem, ciprofloxacin, amoxicillin and doxycycline was initiated and he remained symptom free three months into therapy. Conclusions Physicians should be aware of the varied presentations of chronic Campylobacter and Helicobacter infection in XLA patients. Plasma NGS for circulating mcfDNA in immunocompromised patients offers a rapid, non-invasive means of detecting these fastidious organisms that can be difficult to diagnose using more conventional means.

Exploring the role of healthy dogs as hosts of enterohepatic Helicobacter species using cultivation-dependent and -independent approaches.

Enterohepatic Helicobacter (EHH) species have been increasingly associated with acute gastroenteritis, inflammatory bowel disease and hepatobiliary diseases in humans. However, their host range and transmission routes are poorly understood. Therefore, the aim of this study was to determine the presence of EHH in healthy dogs using both cultivation-dependent and -independent methods. Three hundred and ninety faecal samples from domestic dogs without gastrointestinal symptoms were analysed between June 2018 and July 2019 in Valdivia (South of Chile). Samples were inoculated on selective medium and in parallel were filtrated over an antibiotic-free blood agar. Both media were incubated in a microaerobic atmosphere at 37°C for 7days. Colonies were identified by PCR and phylogenetic analysis. A subset of 50 samples (half of them positive for EHH by cultivation and the remaining half negative) was analysed by PCR-Denaturing Gradient Gel Electrophoresis (PCR-DGGE) for direct detection. Cultivation method detected EHH in 15.4% (60/390) of the samples, being the most prevalent species H. canis (5.8%, 23/390) and H. canicola (5.1%, 20/390), followed by H. bilis (3.6%, 14/390) and 'H. winghamensis' (1.3%, 5/390). In contrast, PCR-DGGE method detected Helicobacter DNA in almost all (96%, 48/50) tested samples. On the other hand, the method used also allowed to isolate other Campylobacterales, in fact 44.3% (173/390) of the samples were positive for Campylobacter upsaliensis (43.3%, 169/390) followed by C. jejuni (2.0%, 8/390). Moreover, two strains that presented Campylobacter-like morphology were finally identified as Anaerobiospirillum succiniciproducens. Our results indicate that healthy domestic dogs commonly carry EHH and other Campylobacter species. However, further studies are needed to determine whether and how these Helicobacter and Campylobacter species can be transmitted to humans.

Campylobacter Colonization, Environmental Enteric Dysfunction, Stunting, and Associated Risk Factors Among Young Children in Rural Ethiopia: A Cross-Sectional Study From the Campylobacter Genomics and Environmental Enteric Dysfunction (CAGED) Project.

Livestock farming provides a possible mechanism by which smallholder farmers can meet their household need for animal source foods (ASF), which may reduce the risk of stunting. However, direct/indirect contacts with domestic animals may increase colonization by Campylobacter spp., which has been associated with Environmental Enteric Dysfunction (EED) and stunting. A cross-sectional study involving 102 randomly selected children between 12 and 16 months of age was conducted in rural eastern Ethiopia to establish prevalence rates of Campylobacter colonization, EED, and stunting, and evaluate potential risk factors. Data were collected between September and December 2018. The prevalence of EED and stunting was 50% (95% CI: 40–60%) and 41% (95% CI: 32–51%), respectively. Among enrolled children, 56% had consumed some ASF in the previous 24 h; 47% had diarrhea and 50% had fever in the past 15 days. 54, 63, 71 or 43% of households owned at least one chicken, cow/bull, goat, or sheep; 54 (53%) households kept chickens indoors overnight and only half of these confined the animals. Sanitation was poor, with high levels of unimproved latrines and open defecation. Most households had access to an improved source of drinking water. The prevalence of Campylobacter colonization was 50% (95% CI: 41–60%) by PCR. In addition to the thermotolerant species Campylobacter jejuni, Campylobacter coli and Campylobacter upsaliensis, non-thermotolerant species related to Campylobacter hyointestinalis and Campylobacter fetus were frequently detected by Meta-total RNA sequencing (MeTRS). Current breastfeeding and ASF consumption increased the odds of Campylobacter detection by PCR, while improved drinking water supply decreased the odds of EED. No risk factors were significantly associated with stunting. Further studies are necessary to better understand reservoirs and transmission pathways of Campylobacter spp. and their potential impact on child health.

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 Infections in Children

1. Rebecca G. Same, MD* 2. Pranita D. Tamma, MD, MHS* 1. *Division of Pediatric Infectious Diseases, Department of Pediatrics, The Johns Hopkins University School of Medicine, Baltimore, MD * Abbreviations: CDC: : Centers for Disease Control and Prevention CIDT: : culture-independent diagnostic testing GBS: : Guillain-Barre syndrome HIV: : human immunodeficiency virus IBD: : inflammatory bowel disease Ig: : immunoglobulin Campylobacter is one of the 2 most common causes of foodborne illness in the United States. It most commonly occurs in children younger than 5 years of age. Campylobacter species can cause a wide range of syndromes, from asymptomatic infections to severe systemic infections. After completing this article, readers should be able to: 1. Recognize that Campylobacter is a common cause of foodborne illness in the United States and internationally. 2. Understand the indications for testing and the treatment of Campylobacter infection. Campylobacter species are an important cause of infection throughout the world, especially in young children. Campylobacter jejuni is the most common Campylobacter species in the United States, where it is 1 of the top 2 causes of foodborne illness. (1) Other important species include Campylobacter upsaliensis , Campylobacter lari , and Campylobacter fetus , which can cause serious systemic infections in all age groups, particularly in immunocompromised patients. Campylobacter species infections cause gastroenteritis and typically present with diarrhea that may or may not be bloody, emesis, and abdominal pain. Campylobacter infection in children can mimic intussusception, appendicitis, or inflammatory bowel disease (IBD). Immune-mediated complications of Campylobacter infection include reactive arthritis and Guillain-Barre syndrome (GBS). Campylobacter infection is a nationally notifiable disease, and confirmed cases should be reported through the Centers for Disease Control and Prevention (CDC) Foodborne Diseases Active Surveillance Network. Most Campylobacter infections are mild and self-limited and require only supportive care, although some may lead to severe dehydration. Serious infection or infections in immunocompromised hosts may benefit from treatment with macrolide antibiotics. Increasing …

Prevalence of most common human pathogenic Campylobacter spp. in dogs and cats in Styria, Austria.

The aim of this study was to determine the frequency of occurrence of most common human pathogenic Campylobacter species, Campylobacter jejuni (C. jejuni) and Campylobacter coli (C. coli), in dogs and cats in Styria, Austria. In the period from April 2010 to April 2012, 842 faecal samples from dogs and cats from Styria, Austria were examined for Campylobacter (C.) species (spp.). All samples were subjected to qualitative microbiological culture testing, and additionally, some of them have been studied using qualitative real‐time PCR. In microbiological culture, 5.9% of all samples investigated were C. spp. positive. With 3.1% out of positive samples, C. jejuni was the most common type. Campylobacter upsaliensis (C. upsaliensis) was detected only in 0.5% of the samples. The remaining positive samples (2.4%) were classified as C. species (sp.). C. coli could not be found in any of the samples. A higher prevalence of C. jejuni was found in kittens with 14.3% and in diarrhoeic dogs (7.4%) and cats (23.8%). The real‐time PCR revealed for dogs and cats together, 27% of C. jejuni‐positive faecal and 8% positive faecal swap samples. The obtained C. jejuni strains underwent antibiotic resistance testing using three different tests (agar diffusion, MIC testing and E‐test) with different numbers of antibiotics. From the antibiotics used in this study, several showed high test‐dependent resistance rates (cephalexin, cefovecin, kanamycin, sulfamethoxazole/trimethoprim, penicillin G, ciprofloxacin, enrofloxacin, marbofloxacin, nalidixic acid). Overall, the prevalence of C. spp. in this study was very low compared to others, with the exception of C. jejuni in kittens and diarrhoeic animals. The results of the real‐time PCR suggest that the rate of colonization of C. jejuni was actually higher than the results of the culture showed. As the resistance rates of C. jejuni isolates partly were very high, possible transmission of (multi‐) resistant C. jejuni strains to humans especially from kittens and diarrhoeic animals must be expected.

Isolation, identification and differentiation of Campylobacter spp. using multiplex PCR assay from goats in Khartoum State, Sudan

The aim of this study was to identify and characterize thermophilic Campylobacter species in faecal samples from goats in Khartoum State, Sudan, by application of multiplex polymerase chain reaction. Campylobacteriosis is a zoonotic disease of global concern, and the organisms can be transmitted to human via food, water and through contact with farm animals and pets. There are five clinically related Campylobacter species: Campylobacter jejuni (C. jejuni). Campylobacter coli, Campylobacter lari, Campylobacter upsaliensis and Campylobacter fetus. Conventional cultural methods to diagnose campylobacteriosis are tedious and time consuming. Wide ranges of genes have been reported to be used for PCR-based identification of Campylobacter spp. We used a multiplex PCR assay to simultaneously detect genes from the major five clinically significant Campylobacter spp. The genes selected were hipO (hippuricase) and 23S rRNA from glyA (serine hydroxymethyl transferase) from each of C. jejuni. C. coli, C. lari, and C. upsaliensis; and sapB2 (surface layer protein) from C. fetus subsp. fetus. The assay was used to identify Campylobacter isolates recovered from 336 cultured faecal samples from goats in three localities in Khartoum State. C. coli was the most predominant isolate (234; 69.6%), followed by C. jejuni (19; 5.7%), C. upsaliensis (13; 3.9%), C. fetus subsp. fetus (7; 2.1%) and C. lari (6; 1.8%). Twenty-nine goats showed mixed infection with Campylobacter spp., 21 of which harbored two Campylobacter spp., while eight animals were infected with three species. Ten out of twelve goats that displayed diarrhea harbored C. coli only. C. coli, C. jejuni and C. upsaliensis showed significant variation with localities. The prevalence of C. coli was significantly higher (87; 25.9%) in goats from Omdurman, whereas C. jejuni and C. upsaliensis were significantly higher (11; 3.3%, 9; 2.7%) in goats from Khartoum. The multiplex PCR assay was found to be rapid and easy to perform and had a high sensitivity and specificity for characterizing the isolates, even in mixed cultures. The study demonstrated the significance of goats as reservoirs in the dissemination of Campylobacter spp. which could be considered as potential agent of caprine enteritis and abortion as well as contamination of the wider environment posing serious public health concern in Khartoum State.

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.

Isolation of Campylobacter spp. from Client-Owned Dogs and Cats, and Retail Raw Meat Pet Food in the Manawatu, New Zealand.

Campylobacter causes acute gastroenteritis in people worldwide and is frequently isolated from food, animals and the environment. The disease is predominately food-borne but many routes of transmission and sources of infection have been described, including contact with pets. The prevalence of Campylobacter spp. in dogs and cats varies widely, and data on New Zealand pets are limited. This study aimed to investigate the prevalence of Campylobacter spp. in dogs, cats and retail raw meat pet food products in New Zealand and to characterize Campylobacter jejuni isolates using multilocus sequence typing (MLST). Ninety dogs and 110 cats examined at the Massey University Veterinary Teaching Hospital for elective procedures, and fifty locally purchased retail raw meat pet diets were sampled. Two culture protocols combining Bolton broth enrichment and mCCDA and CAT agars in a microaerobic atmosphere at 42°C and 37°C with species identification using PCR were performed. The prevalence of Campylobacter spp., C.jejuni, Campylobacter upsaliensis and Campylobacter helveticus was 36%, 13%, 23% and 1% in dogs and 16%, 5%, 5% and 7% in cats, respectively. One dog had Campylobacter lari confirmed, and three dogs and one cat had multiple Campylobacter spp. detected. Significantly more animals tested positive using CAT than mCCDA agar (P<0.001). Being neutered, vaccinated for Bordetella bronchiseptica, fed dry diets and brought in for neutering were protective factors for dogs, whereas attendance for dental treatment was a risk factor for cats. Campylobacter spp. were isolated from 28%, C.jejuni 22%, C.lari 6% and Campylobacter coli 6% of food samples. Six isolates positive by Campylobacter genus PCR were identified as Arcobacter butzleri. Poultry meat was more likely to be positive than non-poultry meat (P=0.006). Of the 13 C.jejuni pet isolates with full MLST profiles, eight were of different sequence types (ST) and all nine food isolates were of different STs.

Variation in the limit-of-detection of the ProSpecT Campylobacter microplate enzyme immunoassay in stools spiked with emerging Campylobacter species

Campylobacter enteritis in humans is primarily associated with C. jejuni/coli infection. The impact of other Campylobacter spp. is likely to be underestimated due to the bias of culture methods towards Campylobacter jejuni/coli diagnosis. Stool antigen tests are becoming increasingly popular and appear generally less species-specific. A review of independent studies of the ProSpecT® Campylobacter Microplate enzyme immunoassay (EIA) developed for C. jejuni/coli showed comparable diagnostic results to culture methods but the examination of non-jejuni/coli Campylobacter spp. was limited and the limit-of-detection (LOD), where reported, varied between studies. This study investigated LOD of EIA for Campylobacter upsaliensis, Campylobacter hyointestinalis and Campylobacter helveticus spiked in human stools. Multiple stools and Campylobacter isolates were used in three different concentrations (104–109CFU/ml) to reflect sample heterogeneity.All Campylobacter species evaluated were detectable by EIA. Multivariate analysis showed LOD varied between Campylobacter spp. and faecal consistency as fixed effects and individual faecal samples as random effects. EIA showed excellent performance in replicate testing for both within and between batches of reagents, in agreement between visual and spectrophotometric reading of results, and returned no discordance between the bacterial concentrations within independent dilution test runs (positive results with lower but not higher concentrations). This study shows how limitations in experimental procedures lead to an overestimation of consistency and uniformity of LOD for EIA that may not hold under routine use in diagnostic laboratories. Benefits and limitations for clinical practice and the influence on estimates of performance characteristics from detection of multiple Campylobacter spp. by EIA are discussed.

Substitute sweeteners: diverse bacterial oligosaccharyltransferases with unique N-glycosylation site preferences.

The central enzyme in the Campylobacter jejuni asparagine-linked glycosylation pathway is the oligosaccharyltransferase (OST), PglB, which transfers preassembled glycans to specific asparagine residues in target proteins. While C. jejuni PglB (CjPglB) can transfer many diverse glycan structures, the acceptor sites that it recognizes are restricted predominantly to those having a negatively charged residue in the −2 position relative to the asparagine. Here, we investigated the acceptor-site preferences for 23 homologs with natural sequence variation compared to CjPglB. Using an ectopic trans-complementation assay for CjPglB function in glycosylation-competent Escherichia coli, we demonstrated in vivo activity for 16 of the candidate OSTs. Interestingly, the OSTs from Campylobacter coli, Campylobacter upsaliensis, Desulfovibrio desulfuricans, Desulfovibrio gigas, and Desulfovibrio vulgaris, exhibited significantly relaxed specificity towards the −2 position compared to CjPglB. These enzymes glycosylated minimal N-X-T motifs in multiple targets and each followed unique, as yet unknown, rules governing acceptor-site preferences. One notable example is D. gigas PglB, which was the only bacterial OST to glycosylate the Fc domain of human immunoglobulin G at its native ‘QYNST’ sequon. Overall, we find that a subset of bacterial OSTs follow their own rules for acceptor-site specificity, thereby expanding the glycoengineering toolbox with previously unavailable biocatalytic diversity.

A new variant of cytolethal distending toxin in a clinical isolate of Campylobacter hyointestinalis.

Increasing numbers of Campylobacter hyointestinalis have been isolated from humans and animals with gastroenteritis, although the virulence mechanism of this species remains largely unknown. Here, we show that C. hyointestinalis isolated from a patient with diarrhoea in Thailand produced a novel variant of cytolethal distending toxin (CDT). Sequencing of a 13 965 bp genomic region of C. hyointestinalis carrying the genes coding for Ch-CDT revealed three ORFs of 798, 804 and 537 bp, which code for the Ch-CdtA, Ch-CdtB and Ch-CdtC subunits, respectively. The deduced amino acid sequence of Ch-CdtA showed ∼38.9 % homology with the CdtA of Campylobacter coli, but sequences of Ch-CdtB and Ch-CdtC were homologous to CdtB (65.7 %) and CdtC (33.1 %) of Campylobacter upsaliensis, respectively. Filter-sterilized sonic lysate of C. hyointestinalis demonstrated distension and death of HeLa cells by arresting the cell cycle at the G(2)/M phase and phosphorylation of host histone H2AX, a sensitive marker of DNA double-strand breaks. Rabbit antiserum raised against recombinant Ch-CdtB was not reactive against the recombinant CdtB protein of Campylobacter jejuni. A reconstituted Ch-CDT holotoxin prepared using each of the recombinant subunit proteins demonstrated distension and death of HeLa cells, suggesting that the C. hyointestinalis isolate indeed produced functionally active Ch-CDT. Furthermore, the immunological distinctiveness of the Ch-CDT produced by C. hyointestinalis and the increasing prevalence of the species in patients and animals with gastroenteritis suggest that this species may be an important emerging zoonotic pathogen.

Low Prevalence of Campylobacteriosis in the Northern Region of India

Campylobacter is one of the most common bacterial enteropathogens of food borne origin in industrialized countries with C. jejuni being the most common species followed by C. coli. The prevalence of Campylobacters in and around Chandigarh, India was studied by phenotypic and genotypic methods. Fecal samples from 1145 diarrheal patients and 102 healthy subjects from hospital and community were cultured on Campylobacter media and identified by Gram stain, biochemical investigations and serotyping. Molecular identification of Campylobacter isolates was done using specific primers to unique regions of 16S rRNA, Campylobacter jejuni (hipO), Campylobacter coli (aspK), Campylobacter lari (glyA) and Campylobacter upsaliensis (lpxA) genes. Identification of specific genes to look for resistance to nalidixic acid, ciprofloxacin, tetracyclin and streptomycin was also done. Campylobacters were isolated from 2.6% of patients with diarrhea. Campylobacteriosis was more prevalent in children ≤5 years old and during summer season. The most frequent serotypes were S:27, B:2, Z5:52 and V:32. All the Campylobacters isolated by culture were confirmed genotypically by identification of 16S rRNA, hipO and aspK genes. Of the 30 isolates, 27 were C. jejuni and 3 were C. coli. No C. lari or C. upsaliensis were detected. Antibiotic resistance was 40% for nalidixic acid, 23.3% for ciprofloxacin, 50% for tetracyclin and 20% for streptomycin. Campylobacter prevalence is low in the region with C. jejuni being the most common species. A high degree of resistance was found for nalidixic acid and tetracyclin but moderate for ciprofloxacin and streptomycin.

PorA specific primers for the identification of Campylobacter species in food and clinical samples

Campylobacteriosis is a public health problem with considerable socio-economic impact. As the European Food Safety Authority has emphasized the importance of a surveillance programme for campylobacteriosis, the aim of the present study was the optimization of a specific and sensitive PCR protocol able to detect Campylobacter species responsible for gastrointestinal infections. Raw poultry meat samples were analysed for the presence of Campylobacter sp., by plating onto mCCD (Modified Charcoal-Cefoperazone-Deoxycholate) Agar and Campylobacter Selective Preston Agar and using four sets of species-specific primers for Campylobacter jejuni, Campylobacter coli, Campylobacter upsaliensis and Campylobacter lari designed to bridge the porA gene. The resulting primers demonstrated a sensitivity of 0.01 ng/μl for the C. coli-specific, C. lari-specific, and C. upsaliensis-specific primer sets and 0.5 ng/μl for the C. jejuni-specific primer sets using DNA from pure cultures. Non-specific amplification of non-target DNA was not observed indicating excellent specificity. The primers were useful for the analyses of poultry meat samples both for direct plating onto mCCDA, and for DNA extracted directly from the cells grown for 48 h in Preston enrichment broth. The sets of primers were also useful when used for species identification of human isolates.