Transmission of dominant strains of Campylobacter jejuni and Campylobacter coli between farms and retail stores in Ecuador: Genetic diversity and antimicrobial resistance.

Thermotolerant Campylobacter is an important zoonotic pathogen known for causing gastroenteritis in humans, with poultry as its primary reservoir. A total of 468 samples were collected, of which 335 were chicken carcass samples (representing the food component), and 133 were chicken caeca samples (representing the animal component). These samples underwent culture, with colonies examined under a microscope. Species identification was achieved through multiplex PCR. Additionally, antimicrobial susceptibility profiles were determined using the Kirby-Bauer method, testing for sensitivity to gentamicin, ciprofloxacin, tetracycline, and erythromycin. Additionally, 55 C. jejuni (62.5%) and 33 C. coli (37.5%) isolates were selected for whole genome sequencing (WGS). A High prevalence of Campylobacter was observed, with rates of 95.5% (n = 127, CI95%: 92.5% - 98.5%) in the animal component and 72.5% (n = 243, CI95%: 69.9% - 75.1%) in the food component. Specifically, C. jejuni was detected in 33.1% (n = 42) of poultry farms and 38.3% (n = 93) of chicken carcasses, while C. coli was found in 64.6% (n = 82) of poultry farms and 60.5% (n = 147) of chicken carcasses. Antimicrobials with the highest rates of resistance (67%-100%) were ciprofloxacin and tetracycline, in both animal and food component isolates. Erythromycin resistance was notable, ranging from 22% to 33%, with only two C. jejuni isolates from retail were resistant to gentamicin. Furthermore, multidrug resistance was identified in 23% (20 isolates) of the Campylobacter isolates. Genetic analysis revealed the presence of fourteen resistance genes in both C. jejuni and C. coli isolates, including tet(O), blaOXA-460, blaOXA-184, blaOXA-489, blaOXA-193, blaOXA-784, blaOXA-603, aph(3')-IIIa, aad9, aph(2'')-If, aadE-Cc, sat4, and ant(6)-Ia. Additionally, twenty-five plasmids were detected in the 88 Campylobacter isolates examined. Interestingly, most isolates also harbored genes encoding putative virulence factors associated with pathogenicity, invasion, adherence, and production of cytolethal distending toxin (cdt): cheV, cheA, cheW, cheY, flaA, flgR, flaC, flaD, flgB, flgC, ciaB, ciaC. The WGS analysis showed the presence of several cgSTs in both animal and food components, with nine of them widely disseminated between components. Moreover, C. coli and C. jejuni isolates from different sources presented less than 11 single nucleotide polymorphisms (SNPs), suggesting clonality (16 isolates). Further analysis using SNP tree demonstrated widespread distribution of certain C. jejuni and C. coli clones across multiple farms and retail stores. This study presents, for the first-time, insights into the clonality, plasmid diversity, virulence, and antimicrobial resistance (AMR) of thermotolerant Campylobacter strains originating from the Ecuadorian poultry industry. The identification of AMR genes associated with the main antibiotics used in the treatment of campylobacteriosis in humans, highlights the importance of the prudent use of antimicrobials in the poultry industry. Additionally, this research remarks the need for regional studies to understand the epidemiology of this pathogen.

Campylobacter infection of young children in Colombia and its impact on the gastrointestinal environment.

Campylobacter infections are a leading cause of bacterial-derived gastroenteritis worldwide with particularly profound impacts on pediatric patients in low- and middle-income countries. It remains unclear how Campylobacter impacts these hosts, though it is becoming increasingly evident that it is a multifactorial process that depends on the host immune response, the gastrointestinal microbiota, various bacterial factors, and host nutritional status. Since these factors likely vary between adult and pediatric patients in different regions of the world, it is important that studies define these attributes in well-characterized clinical cohorts in diverse settings. In this study, we analyzed the fecal microbiota and the metabolomic and micronutrient profiles of asymptomatic and symptomatic pediatric patients in Colombia who were either infected or uninfected with Campylobacter during a case-controlled study on acute diarrheal disease. Here, we report that the microbiome of Campylobacter-infected children only changed in their abundance of Campylobacter spp. despite the inclusion of children with or without diarrhea. In addition to increased Campylobacter, computational models were used to identify fecal metabolites that were associated with Campylobacter infection and found that glucose-6-phosphate and homovanillic acid were the strongest predictors of infection in these pediatric patients, which suggests that colonocyte metabolism is impacted during infection. Despite changes to the fecal metabolome, the concentrations of intestinal minerals and trace elements were not significantly impacted by Campylobacter infection but were elevated in uninfected children with diarrhea.IMPORTANCEGastrointestinal infection with pathogenic Campylobacter species has long been recognized as a significant cause of human morbidity. Recently, it has been observed that pediatric populations in low- and middle-income countries are uniquely impacted by these organisms in that infected children can be persistently colonized, develop enteric dysfunction, and exhibit reduced development and growth. While the association of Campylobacter species with these long-term effects continues to emerge, the impact of infection on the gastrointestinal environment of these children remains uncharacterized. To address this knowledge gap, our group leveraged clinical samples collected during a previous study on gastrointestinal infections in pediatric patients to examine the fecal microbiota, metabolome, and micronutrient profiles of those infected with Campylobacter species and found that the metabolome was impacted in a way that suggests gastrointestinal cell metabolism is affected during infection, which is some of the first data indicating how gastrointestinal health in these patients may be affected.

Five centuries of genome evolution and multi-host adaptation of Campylobacter jejuni in Brazil.

Consumption of raw, undercooked or contaminated animal food products is a frequent cause of Campylobacter jejuni infection. Brazil is the world's third largest producer and a major exporter of chicken meat, yet population-level genomic investigations of C. jejuni in the country remain scarce. Analysis of 221 C. jejuni genomes from Brazil shows that the overall core and accessory genomic features of C. jejuni are influenced by the identity of the human or animal source. Of the 60 sequence types detected, ST353 is the most prevalent and consists of samples from chicken and human sources. Notably, we identified the presence of diverse bla genes from the OXA-61 and OXA-184 families that confer beta-lactam resistance as well as the operon cmeABCR related to multidrug efflux pump, which contributes to resistance against tetracyclines, macrolides and quinolones. Based on limited data, we estimated the most recent common ancestor of ST353 to the late 1500s, coinciding with the time the Portuguese first arrived in Brazil and introduced domesticated chickens into the country. We identified at least two instances of ancestral chicken-to-human infections in ST353. The evolution of C. jejuni in Brazil was driven by the confluence of clinically relevant genetic elements, multi-host adaptation and clonal population growth that coincided with major socio-economic changes in poultry farming.

Novel rpsK / rpsD primer-probe assay improves detection of Campylobacter jejuni and Campylobacter coli in human stool.

Campylobacter causes bacterial enteritis, dysentery, and growth faltering in children in low- and middle-income countries (LMICs). Campylobacter spp. are fastidious organisms, and their detection often relies on culture independent diagnostic technologies, especially in LMICs. Campylobacter jejuni and Campylobacter coli are most often the infectious agents and in high income settings together account for 95% of Campylobacter infections. Several other Campylobacter species have been detected in LMIC children at an increased prevalence relative to high income settings. After doing extensive whole genome sequencing of isolates of C. jejuni and C. coli in Peru, we observed heterogeneity in the binding sites for the main species-specific PCR assay (cadF) and designed an alternative rpsKD-based qPCR assay to detect both C. jejuni and C. coli. The rpsKD-based qPCR assay identified 23% more C.jejuni/ C.coli samples than the cadF assay among 47 Campylobacter genus positive cadF negative samples verified to have C. jejuni and or C. coli with shotgun metagenomics. This assay can be expected to be useful in diagnostic studies of enteric infectious diseases and be useful in revising the attribution estimates of Campylobacter in LMICs.

Insights on the genomic diversity, virulence and resistance profile of a Campylobacter jejuni strain isolated from a hospitalized patient in Brazil.

Campylobacteriosis is currently recognized as one of the major causes of foodborne bacterial diseases worldwide. In Brazil, there is insufficient data to estimate the impact of Campylobacter in public health. The aim of this present study was to characterize a C. jejuni CJ-HBSJRP strain isolated from a hospitalized patient in Brazil by its ability to invade human Caco-2 epithelial cells, to survive in U937 human macrophages, and to assess its phenotypic antimicrobial resistance profile. In addition, prophages, virulence and antimicrobial resistance genes were search using whole-genome sequencing data. The genetic relatedness was evaluated by MLST and cgMLST analysis by comparison with 29 other C. jejuni genomes isolated from several countries. The CJ-HBSJRP strain showed an invasion percentage of 50% in Caco-2 polarized cells, 37.5% of survivability in U937 cells and was phenotypically resistant to ampicillin, ciprofloxacin and nalidixic acid. A total of 94 virulence genes related to adherence, biofilm, chemotaxis, immune modulation, invasion process, metabolism, motility and toxin were detected. The resistance genes blaOXA-605 (blaOXA-61), cmeB and mutations in the QRDR region of gyrA were also found and none prophages were detected. The MLST analysis showed 23 different STs among the strains studied. Regarding cgMLST analysis, the CJ-HBSJRP strain was genetically distinct and did not group closely to any other isolate. The results obtained reinforce the pathogenic potential of the CJHBSJRP strain and highlighted the need for more careful attention to Campylobacter spp. infections in Brazil since this pathogen has been the most commonly reported zoonosis in several countries worldwide.

Positive cases of bovine genital campylobacteriosis and bovine trichomonosis in the eradication program of La Pampa, Argentina: 2008-2021.

Bovine genital campylobacteriosis, caused by the gram-negative bacteria Campylobacter fetus venerealis, and bovine trichomonosis, caused by the parasite protozoan Tritrichomonas foetus, are venereal diseases that occur with long intercalving periods and abortion. The control of both diseases relies on microbiological testing and culling infected bulls. Vaccination and antibiotic treatment may help in controlling campylobacteriosis but are not recommended for trichomonosis control. Several regions of the world have active control programs for trichomonosis, not campylobacteriosis. In Argentina, the state of La Pampa aims to eradicate trichomonosis and campylobacteriosis by imposing annual diagnostic testing of every bull and slaughtering positive animals. Prior studies indicated a declining trend in the prevalence of campylobacteriosis and trichomonosis in La Pampa. It was also proposed that the prevalence of one disease could be estimated from the prevalence of the other. The purpose of this retrospective analysis of data gathered from 2008 to 2021 was to determine the La Pampa program's efficacy. Descriptive statistics were employed to determine the reason behind the correlation between tricomonosis and campylobacteriosis diagnostic results. The outcomes refute the notion that this program of venereal eradication was a success. Furthermore, an excess of false positives in both diagnoses may have contributed to the correlation between the prevalences of campylobactriosis and trichomonosis. The practice of killing animals without verifying positive results hinders the determination of disease prevalence and results in the death of numerous healthy animals.

Bovine campylobacteriosis in heifer: pathogenesis study and insights in the conventional and molecular diagnosis in an experimental bovine model and field cases.

Campylobacter fetus spp. is a bacterium associated to reproductive losses in cattle worldwide. It is a venereal infectious disease known as bovine campilobacteriosis, with high impact mainly in countries with extensive production systems. Here, we show pathogenesis and diagnostic methods for Campylobacter fetus detection in cervico-vaginal mucus (CVM) samples from heifers experimentally infected and field cases from herds with low reproductive performance by campylobacteriosis infection. Bacterial culture, direct immunofluorescence test and qPCR were used as diagnostic methods to evaluate detection of C. fetus. In the experimental model 30 Aberdeen Angus and crossbred heifers and 4 Aberdeen Angus bulls for natural mating were assigned to 3 groups experimentally challenged with C. fetus subsp. fetus (Cff), C. fetus subsps venerealis (Cfv) and C. fetus subsp venerealis biovar intermedius (Cfvi), respectively, and a negative control group, all followed for 9 months. Also, field samples of CVM and aborted fetuses were recollected from seven beef cattle farms. Bacteriological culture had the higher C. fetus detection rate in CVM being the most appropriate, followed by qPCR (with commercial extraction DNA kit), direct immunofluorescence test and qPCR (with in-house extraction DNA method), in both, experimental model and field cases. From experimental model after natural mating, 62.5% and 25% heifers got pregnant from Cff and Cfvi groups, respectively, while from Cfv no pregnancy was detected. The strain more frequently detected was Cfvi, followed by Cff and Cfv. Colonization of Cff in female genital tract with high number of carriers and presence in aborted fetuses was evidenced, suggesting a high risk to bovine reproductive health. Bacteriemia was not detected after genital infection. Given the low detection rate of either test, we suggest the use of both, PCR based methods and bacterial culture could result in higher detection rate in farms with endemic campylobacteriosis.

Hospitalizaciones por diarrea con sangre en un centro pediátrico de referencia en Uruguay / Hospitalizations due to bloody diarrhea in a pediatric reference center in Uruguay / Internações por diarreia sanguinolenta em um centro de referência pediátrica no Uruguai

Introducción: La diarrea con sangre es un motivo frecuente de admisión hospitalaria en niños, con gastroenteritis aguda; en la mayoría de los casos se tratan de infecciones leves y autolimitadas, pero pueden producirse complicaciones graves. Objetivos: Describir la etiología y características clínico- evolutivas de los niños menores de 15 años hospitalizados por diarrea con sangre en el Hospital Pediátrico, Centro Hospitalario Pereira Rossell entre los años 2012- 2023. Materiales y métodos: Estudio retrospectivo mediante revisión de historias y registros de laboratorio. Variables: demográficas, estado nutricional, hidratación, motivos de hospitalización, ingreso unidades de cuidados intensivos (UCI), enteropatógenos, tratamientos, evolución. Resultados: Se incluyeron 229 niños, mediana de edad de 8 meses; sexo masculino 61%; eutróficos 88%, bien hidratados 55%, con comorbilidades 11%, prematurez 6,5%. El motivo de hospitalización fue diarrea con sangre/disentería sin otro síntoma 45%. Se solicitó coprovirológico/coprocultivo en 98% y detección por técnicas de ácidos nucleicos en materia fecal 5,2%. Se identificó al menos un agente patógeno en 34,3%: Shigella sp. 38%; Salmonella sp. 19,5%; coinfecciones en 12%. Se indicaron antibióticos a 86%; ceftriaxona 62%, azitromicina 35%. Ingresaron a UCI 6,5% (15), presentaron complicaciones 10/14, fallo renal agudo 5 y alteraciones del medio interno 3. La mayoría presentó buena evolución. Conclusiones: La diarrea con sangre/disentería continúa siendo una causa importante de hospitalización afectando en su mayoría a niños sanos menores de 5 años. Los patógenos detectados con mayor frecuencia fueron bacterias principalmente Shigella sp., Salmonella sp. y E coli diarreogénicas. Se reportó alta prescripción de antibióticos, cumpliendo en la mayoría de los casos con las recomendaciones.

Introduction: Bloody diarrhea is a common reason for hospital admission in children with acute gastroenteritis; In most cases these are mild and self-limiting infections, but serious complications can occur. Goals: To describe the etiology and clinical-evolutionary characteristics of children under 15 years of age hospitalized for bloody diarrhea at the Pediatric Hospital, Centro Hospitalario Pereira Rossell between the years 2012-2023. Materials and methods: Retrospective study through review of histories and laboratory records. Variables: demographics, nutritional status, hydration, reason for hospitalization, intensive care unit (ICU) admission, enteropathogens, treatments, evolution. Results: 229 children were included, median age 8 months; male sex 61%; eutrophic 88%, well hydrated 55%, with comorbidities 11%, prematurity 6.5%. The reason for hospitalization was bloody diarrhea/dysentery without other symptoms 45%. Coprovirological/coproculture was requested in 98% and detection by nucleic acid techniques in fecal matter was requested in 5,2%. At least one pathogenic agent was identified in 34,3%: Shigella sp. 38%; Salmonella sp 19,5%; coinfections in 12%. Antibiotics were indicated for 86%; ceftriaxone 62%, azithromycin 35%. Were admitted to the ICU 6,5% (15), 10/14 had complications, 5 had acute kidney failure and 3 had alterations in the internal environment. The majority had a good evolution. Conclusions: Bloody diarrhea/dysentery continues to be an important cause of hospitalization, affecting mostly healthy children under 5 years of age. The most frequently detected pathogens were bacteria, mainly Shigella sp., Salmonella sp. and diarrheagenic E coli. High prescription of antibiotics was reported, complying in most cases with the recommendations.

Introdução: A diarreia com sangue é um motivo comum de internação hospitalar em crianças com gastroenterite aguda; Na maioria dos casos, estas são infecções leves e autolimitadas, mas podem ocorrer complicações graves. Metas: Descrever a etiologia e as características clínico-evolutivas de crianças menores de 15 anos internadas por diarreia sanguinolenta no Hospital Pediátrico Centro Hospitalario Pereira Rossell entre os anos de 2012-2023. Materiais e métodos: Estudo retrospectivo por meio de revisão de histórias e registros laboratoriais. Variáveis: dados demográficos, estado nutricional, hidratação, motivo da internação, internação em unidade de terapia intensiva (UTI), enteropatógenos, tratamentos, evolução. Resultados: foram incluídas 229 crianças, mediana de idade 8 meses; sexo masculino 61%; eutrófico 88%, bem hidratado 55%, com comorbidades 11%, prematuridade 6,5%. O motivo da internação foi diarreia sanguinolenta/disenteria sem outros sintomas 45%. O estudo coprovirologico/coprocultivo foi solicitado em 98% e a detecção por técnicas de ácidos nucleicos em matéria fecal foi solicitada em 5,2%. Pelo menos um agente patogênico foi identificado em 34,3%: Shigella sp. 38%; Salmonella sp. 19,5%; coinfecções em 12%. Os antibióticos foram indicados para 86%; ceftriaxona 62%, azitromicina 35%. Foram internados em UTI 6,5% (15), 10/14 apresentaram complicações, 5 tiveram insuficiência renal aguda e 3 apresentaram alterações no meio interno, a maioria teve boa evolução. Conclusões: A diarreia/disenteria com sangue continua a ser uma causa importante de hospitalização, afetando sobretudo crianças saudáveis ​​com menos de 5 anos de idade. Os patógenos mais frequentemente detectados foram bactérias, principalmente Shigella sp., Salmonella sp. e E. coli diarreiogênica. Foi relatada elevada prescrição de antibióticos, cumprindo na maioria dos casos as recomendações.

A cross-sectional study on Campylobacter fetus subsp. venerealis prevalence and associated factors in Brazilian southern cattle farms.

Cattle farming is a major livestock activity with economic relevance in Rio Grande do Sul (RS), Brazil. However, this activity is still considered of intermediate to low technological level, and in this region, there are few epidemiologic reports of Campylobacter fetus subsp. venerealis (Cfv), the causative agent of bovine genital campylobacteriosis (BGC). Thus, we designed a cross-sectional study to assess the prevalence and Cfv-associated factors in cattle farms in RS, Brazil. In total, 99 farms were randomly selected to participate in the survey. Preputial mucus samples from selected bulls were collected twice (within a 15-day interval) and subjected to Cfv molecular detection. A farm was considered positive when at least one sample was positive for Cfv. Our findings indicate that the farm-level Cfv prevalence in RS is 67.67%. On average, the chance of a farm using natural service to be Cfv-positive increased approximately twice compared to farms that do not use natural service. We also determined that Cfv routine tests reduce the chance of a farm being positive by 92%. Therefore, both Cfv detection tests and the reduction of natural services decrease the chance of a farm being positive for Cfv. Finally, we conclude that Cfv is widely spread in Southern Brazil cattle farms and it is urgent the implementation of control measures to reduce Cfv prevalence in the target population.

Head-to-head comparison of CAMPYAIR aerobic culture medium versus standard microaerophilic culture for Campylobacter isolation from clinical samples.

Campylobacter spp. are considered the most frequent cause of acute gastroenteritis worldwide. However, outside high-income countries, its burden is poorly understood. Limited published data suggest that Campylobacter prevalence in low- and middle-income countries is high, but their reservoirs and age distribution are different. Culturing Campylobacter is expensive due to laboratory equipment and supplies needed to grow the bacterium (e.g., selective culture media, microaerophilic atmosphere, and a 42°C incubator). These requirements limit the diagnostic capacity of clinical laboratories in many resource-poor regions, leading to significant underdiagnosis and underreporting of isolation of the pathogen. CAMPYAIR, a newly developed selective differential medium, permits Campylobacter isolation without the need for microaerophilic incubation. The medium is supplemented with antibiotics to allow Campylobacter isolation in complex matrices such as human feces. The present study aims to evaluate the ability of the medium to recover Campylobacter from routine clinical samples. A total of 191 human stool samples were used to compare the ability of CAMPYAIR (aerobic incubation) and a commercial Campylobacter medium (CASA, microaerophilic incubation) to recover Campylobacter. All Campylobacter isolates were then identified by MALDI-TOF MS. CAMPYAIR showed sensitivity and specificity values of 87.5% (95% CI 47.4%-99.7%) and 100% (95% CI 98%-100%), respectively. The positive predictive value of CAMPYAIR was 100% and its negative predictive value was 99.5% (95% CI 96.7%-99.9%); Kappa Cohen coefficient was 0.93 (95% CI 0.79-1.0). The high diagnostic performance and low technical requirements of the CAMPYAIR medium could permit Campylobacter culture in countries with limited resources.

First Report of Campylobacter hepaticus Isolation in Laying Hens and Broiler Breeders with Spotty Liver Disease in Costa Rica.

Poultry producers in Costa Rica have informally reported a spotty liver disease-like syndrome for more than 20 yr. However, despite many attempts, the infectious agent responsible for this syndrome had not been identified. Therefore, following current knowledge of spotty liver disease diagnosis, we invited veterinarians and poultry producers to submit samples to the diagnostic laboratories of the Veterinary Medicine School, Universidad Nacional, to identify the infectious agent of this syndrome. Veterinarians and poultry producers were instructed to collect gallbladders and livers aseptically and send them for pathology examinations and bacterial cultures in less than 24 hr after collection. Samples were processed for standard histopathologic studies and cultured under aerophilic, anaerobic, and microaerophilic conditions. Campylobacter-like colonies were isolated and identified by biochemical and PCR tests. Here we report for the first time the isolation, biochemical characterization, and molecular confirmation of Campylobacter hepaticus in laying hens and broiler breeders with spotty liver disease in Costa Rica.

Nota de investigación- Primer reporte de aislamiento de Campylobacter hepaticus en gallinas de postura y reproductoras pesadas con necrosis hepática focal en Costa Rica. Los productores avícolas en Costa Rica han reportado extraoficialmente un síndrome similar a la necrosis hepática focal durante más de 20 años. Sin embargo, a pesar de muchos intentos, el agente infeccioso responsable de este síndrome no había sido identificado. Por ello, siguiendo los conocimientos actuales relacionados con la necrosis hepática focal, se invitó a los veterinarios y a los productores avícolas a enviar muestras a los laboratorios de diagnóstico de la Facultad de Medicina Veterinaria de la Universidad Nacional, para identificar el agente infeccioso de este síndrome. Se instruyó a los veterinarios y productores avícolas para recolectar vesículas biliares e hígados asépticamente y enviarlos para exámenes patológicos y para cultivos bacterianos en menos de 24 horas después de la recolección. Las muestras se procesaron para estudios histopatológicos estándar y se cultivaron en condiciones aerófilas, anaeróbicas y microaerófilas. Las colonias sugestivas de Campylobacter se aislaron e identificaron mediante pruebas bioquímicas y por PCR. Aquí se reporta por primera vez el aislamiento, caracterización bioquímica y confirmación molecular de Campylobacter hepaticus en gallinas de postura y reproductoras pesadas con la necrosis hepática focal en Costa Rica.

SVR-flaA typing of erythromycin- and ciprofloxacin-resistant Campylobacter jejuni strains isolated from poultry slaughterhouses in southern Brazil.

The emergence of fluoroquinolone and macrolide resistance in C. jejuni, a recognized zoonotic pathogen, has increased worldwide. This study aimed to investigate phenotypic resistance to ciprofloxacin and erythromycin, the molecular mechanisms involved, and the strain of C. jejuni isolated from broiler carcasses. Eighty C. jejuni isolates from broiler carcasses in southern Brazil were investigated for their susceptibility to ciprofloxacin and erythromycin at minimal inhibitory concentrations. Mismatch amplification mutation assay-polymerase chain reaction (MAMA-PCR) was performed to detect substitutions of Thr-86-Ile, A2074C, and A2075G of domain V in the 23S rRNA. The presence of ermB gene and CmeABC operon were investigated by PCR. DNA sequencing was used to detect substitutions in the L4 and L22 proteins of the erythromycin-resistant strains. The Short Variable Region (SVR) of flaA was used to type all the strains resistant to both antimicrobials. Ciprofloxacin and erythromycin resistance were detected in 81.25% and 30.00% of the strains, respectively, and minimal inhibitory concentration values ranged from ≤ 0.125 to 64 µg/mL for ciprofloxacin and 0.5 to > 128 µg/mL for erythromycin. The Thr-86-Ile mutation in gyrA was observed in 100% of the ciprofloxacin-resistant strains. Mutations in both the A2074C and A2075G positions of 23S rRNA were observed in 62.5% of the erythromycin-resistant strains, while 37.5% had only the mutation A2075G. None of the strains harbored CmeABC operon, and ermB was not detected. Using DNA sequencing, the amino acid substitution T177S was detected in L4, and substitutions I65V, A103V, and S109A were detected in L22. Twelve flaA-SVR alleles were identified among the strains, with the most common SVR-flaA allele, type 287, covering 31.03% of ciprofloxacin- and erythromycin-resistant isolates. The present study revealed a high incidence and high levels of resistance to ciprofloxacin and erythromycin, as well as broad molecular diversity in C. jejuni isolates from broiler carcasses.

Roles of viable but non-culturable state in the survival of Campylobacter jejuni.

Despite being considered fragile and fastidious, Campylobacter jejuni is the most prevalent cause of foodborne bacterial gastroenteritis, and chicken meat is considered the main vehicle of transmission to humans. This agent can survive adverse conditions in the form of biofilms, but extreme stress (nutritional, oxidative and thermal) promotes the acquisition of a state called viable but not culturable (VBNC). The emergence of this pathogen worldwide and the recent international requirements in its control instigated us to qualitatively and quantitatively estimate the time required for the acquisition of the VBNC form in 27 strains of C. jejuni, characterize morphological aspects, determine its adaptive and invasive potential and perform comparative metabolomic evaluation. Extreme stress promoted the complete acquisition of the VBNC form in a mean time of 26 days. Starting from an average initial count of 7.8 log CFU/mL, the first four days determined the greatest average reduction of the culturable form of 3.2 log CFU/mL. The scanning and transmission image analyses showed a transition from the typical viable form (VT) to the VBNC form, with initial acquisition of the straight rod shape, followed by loss of the flagella and subdivision into two to 11 imperfect cocci arranged in a chain and rich in cellular content, until their individual release. RT-PCR identified the presence of ciaB and p19 transcripts in the 27 cultivable C. jejuni strains, a character maintained in the VBNC form only for p19 and in 59.3% (16/27) of the VBNC strains for the ciaB gene. The average inoculation of 1.8 log CFU/mL of C. jejuni VBNC into primary chicken embryo hepatocyte cells promoted the occurrence of apoptosis processes significantly after 24 hours of contact by one of the strains tested. In C. jejuni VBNC, we detected higher expression of metabolites linked to protective and adaptation mechanisms and of volatile organic precursor compounds indicative of metabolism interruption. The oscillations in the time of acquisition of the VBNC form together with the presence of transcripts for ciaB and p19, the identification of cell lysis and metabolites that ensure the maintenance of the pathogen alert to the fact that C. jejuni VBNC remains virulent and adapted to stress, which makes evident the potential danger of this latent form, which is not detectable by official methodologies.

Antimicrobial resistance genotypes and phenotypes of Campylobacter coli isolated from different sources over a 16-year period in Brazil.

OBJECTIVES: This study aimed to identify antimicrobial resistance genotypes in 63 Campylobacter coli strains isolated from humans (12), animals (21), the environment (20), and food (10) in Brazil using whole genome sequencing (WGS) tools, comparing them with results obtained by antimicrobial susceptibility testing (AST) against some important antimicrobials in clinical use. METHODS: Phenotypic resistance profiles were determined by minimal inhibitory concentrations and the disk diffusion technique. The prediction of the resistance genes was performed using ABRicate v.0.8 and the Resistance Gene Identifier software of the CARD. RESULTS: The percentage of C. coli strains phenotypically resistant to antimicrobials were: ampicillin, 44.4%; doxycycline, 20.6%; tetracycline, 20.6%; ciprofloxacin, 12.7%; nalidixic acid, 12.7%; streptomycin, 6.3%; erythromycin, 4.8%; and gentamicin, 1.6%. The genes blaOXA-605 / blaOXA-61,tet(O), cmeB, aadE-Cc, aph (3 ') - IIIa, sat4 and aad9 were detected in 54%, 22.2%, 9.5%,6.3%, 1.6%, 1.6%, and 1.6% strains, respectively. Mutations T86I in the QRDR region of gyrA were detected in 8 (12.7%) strains. The agreement between AST and WGS was 100%, 92.9%, 82.4%, and 80% for quinolones, tetracycline, ß-lactam, and aminoglycoside classes, respectively. CONCLUSIONS: The rates of C. coli strains resistant to ß- lactams and quinolones may represent a public health concern. The partial agreement between AST and WGS shows that improvement in antibiotic resistance databases may be required to minimize this discrepancy observed in some antimicrobial classes and to become an acceptable tool to both clinical microbiologists and regulatory agencies.

Characterization of the cervicovaginal microbiota of female beef cattle harboring Campylobacter fetus subsp. venerealis using 16S rDNA gene sequencing.

Bovine genital campylobacteriosis (BGC) is a leading cause of return to estrus in cows. The etiologic agent, Campylobacter fetus subsp. venerealis (Cfv) is transmitted by venereal route. Hence, the surrounding reproductive tract microenvironment could play a role in return to estrus in cows. The presence of Cfv in cervicovaginal mucus of Angus breed females provide three experimental groups, which were subject to bacteriome analyses: 10 Cfv-positive cows (CVP), 10 Cfv-negative cows (CVN), and 10 nonsexually active heifers (NSA). Cows with return to estrus showed higher bacterial richness than NSA. Beta diversity analysis showed a significant difference (P = 0.006) in bacterial composition among the three groups analyzed (CVP, CVN, and NSA). However, no significant difference was found when comparing the CVP versus CVN groups. Ureaplasma and Pseudomonas were the genera most frequently observed in NSA, being Ureaplasma the predictor genus to that group, whereas Alistipes, Bacteroides, Rikenellaceae_RC9_gut_group, UCG-005, and UCG-10 were both significantly more abundant and predictors genera in cows with return to estrus. Our results provide an overview of the cervicovaginal bacterial microbiota in cows harboring Cfv and improve the knowledge of the pathogenesis of BGC.

Bovine campylobacteriosis in bulls: insights in the conventional and molecular diagnosis.

Campylobacter fetus is a gram-negative motile bacterium, with two subspecies relevant to cattle health: C. fetus subsp. venerealis (Cfv) and C. fetus subsp. fetus (Cff). Both subspecies are associated with reproductive losses in cattle. In this study, we evaluated the identification of C. fetus for the diagnosis of bovine campylobacteriosis through bacteriological culture, direct immunofluorescence (DIF) and molecular tests in preputial smegma (PS) samples of three Angus bulls challenged with Cfv, Cfv biovar intermedius (Cfvi) or Cff, respectively, in an experiment imitating the natural infection. Two DNA extraction protocols were tested (in-house thermal extraction and commercial kit). Aspiration and scraping collection for PS were compared by conventional tests. Additionally, bacteremia was also evaluated in blood samples. Bulls were challenged by natural mating with heifers that had been experimentally infected with C. fetus subspecies; which led to infection. The Cfv- and Cfvi-bulls were positive for at least 9 months. Although Cff is not considered a venereal strain, in this study it was transmissible to bull from heifers experimentally infected, as evidenced by its colonization and persistence in the preputial cavity for 5 to 6 months. This finding suggests a potential risk of dissemination within herds. The results obtained by bacteriological culture or direct immunofluorescence (DIF) showed no significant differences, regardless the sampling device used (aspiration with Cassou pipette, metal and plastic scraper). C. fetus qPCR, on the other hand, yielded better results with an in-house DNA extraction method than with a commercial kit (75% vs 66.6%). Furthermore, qPCR diagnosis was more efficient than culture (66.6%) or DIF (56%). Bacteremia in whole blood samples was negative by qPCR and bacteriological culture in all samples. Altogether, this study demonstrated the transmission of Cff from heifers to bull and also showed that PCR-based methods are promising for the diagnosis of Bovine Genital Campylobacteriosis from clinical samples of PS.

Investigation of campylobacteriosis and genital trichomoniasis in bulls from rural properties in the Pantanal of Mato Grosso state, Brazil / Investigação de Campilobacteriose e Tricomonose genital em touros de propriedades rurais do Pantanal Mato-Grossense, Brasil

Bovine genital campylobacteriosis (BGC) and bovine trichomoniasis (BT) are diseases of cattle that are primarily transmitted through sexual contact. Although bulls may be asymptomatic, these infectious diseases contribute to reproductive failure, embryonic death, and abortion in cows. Infection in cattle causes significant economic losses. BGC is caused by two bacterial subspecies, Campylobacter fetus subsp. venerealis and Campylobacter fetus subsp. fetus, whereas the protozoan Tritrichomonas foetus causes BT. Mato Grosso state has the largest bovine herd in Brazil, particularly in the Pantanal region. This area encompasses vast expanses of land characterized by annual floods and a predominant reliance on natural breeding for animal reproduction. These conditions create a favorable environment for the occurrence of BGC and BT within the herd. Given the lack of up-to-date data regarding the prevalence of these diseases, this study aimed to examine the presence of Campylobacter spp. and Tritrichomonas foetus in samples from 100 bulls in the municipalities of Poconé, Santo Antônio de Leverger, and Nossa Senhora do Livramento located in the Pantanal region of Mato Grosso state. Preputial smegma samples were retrieved using preputial swabs and stored in a saline solution at -80ºC for subsequent analysis. Polymerase chain reaction was used to identify the presence of Campylobacter fetus subsp. venerealis, Campylobacter fetus subsp. fetus, and Tritrichomonas foetus. Despite a questionnaire revealing epidemiological conditions conducive to the persistence and spread of these pathogens, they were not detected in the bulls evaluated on rural properties in the Pantanal of Mato Grosso region.(AU)

Campilobacteriose genital bovina (CGB) e Tricomonose bovina (TB) são doenças infectocontagiosas de transmissão venérea, assintomáticas nos touros, sendo consideradas como importantes enfermidades causadoras de falha reprodutiva, morte embrionária ou abortamento, ocasionando perdas econômicas significativas em rebanhos bovinos infectados. CGB é causada pela bactéria Campylobacter fetus subsp. venerealis e Campylobacter fetus subsp. fetus, e TB pelo protozoário Tritrichomonas foetus. O estado de Mato Grosso é detentor do maior rebanho bovino do Brasil, envolve a região do Pantanal Mato-Grossense que possui grandes extensões de terra, com ciclo anual de enchentes e a reprodução dos animais realizada predominantemente por monta natural, condições estas, favoráveis a presença de CGB e TB no rebanho. Considerando a carência de informações recentes sobre a ocorrência dessas enfermidades no estado de Mato Grosso, o objetivo deste estudo foi investigar a presença de Campylobacter spp. e Tritrichomonas foetus em 100 touros provenientes dos municípios de Poconé, Santo Antônio de Leverger e Nossa Senhora do Livramento, localizados na região pantaneira do estado de Mato Grosso. Amostras de esmegma prepucial foram coletadas por meio de escarificação via swab prepucial e armazenadas em solução salina a -80ºC. Para a detecção de Campylobacter fetus subsp. venerealis, Campylobacter fetus subsp. fetus, e Tritrichomonas foetus, foi realizada a reação em cadeia pela polimerase (PCR). Apesar do questionário aplicado nas propriedades revelar condições epidemiológicas que favorecem a manutenção e disseminação desses patógenos, este estudo não identificou a presença dos referidos agentes em touros avaliados nas propriedades rurais do pantanal Mato-Grossense.(AU)

Prevalence and distribution of pathogenic genes in Campylobacter jejuni isolated from poultry and human sources.

INTRODUCTION: Campylobacter jejuni is one of the most common bacterial causes of human gastroenteritis. Despite its public health importance, the virulence factors and mechanisms underlying C. jejuni pathogenesis remain poorly understood and the relationships between these genes and the sources of the strains are not clear. We aimed to determine the virulence profiles of C. jejuni isolated from poultry and human cases of Campylobacteriosis. METHODOLOGY: A total of 50 strains of C. jejuni isolated from poultry and human cases of Campylobacteriosis were screened by polymerase chain reaction (PCR) for the presence of six pathogenic genes (flaA, iam, wlaN, cdtA, cdtB, cdtC). RESULTS: A total of 40% (10/25) of the human isolates presented only one virulence marker. In contrast, 64% (16/25) of the poultry-derived strains showed four or five virulence markers. cdtA and flaA occurred more frequently in poultry-derived strains than in human strains. Ten different virulence profiles were observed among the human isolates and 11 among the poultry strains. Only four profiles were common to both sources: profiles 3 (flaA, cdtA, cdtB, and cdtC), 5 (cdtA and cdtB), 7 (flaA and cdtB), and 10 (iam, flaA, cdtA, cdtB, and cdtC). The human-derived strains had a higher Shannon diversity index (1.9396) and Simpson index (0.8367), indicating a more diversified population than found in poultry (1.7742 and 0.7333, respectively). CONCLUSIONS: We found variations in the genetic profiles of the circulating strains based on the isolation source and genes that are potentially pathogenic to humans were detected in poultry-derived strains.

Shotgun metagenomics of fecal samples from children in Peru reveals frequent complex co-infections with multiple Campylobacter species.

Campylobacter spp. are a major cause of bacterial diarrhea worldwide and are associated with high rates of mortality and linear growth faltering in children living in low- to middle-income countries (LMICs). Campylobacter jejuni and Campylobacter coli are most often the causative agents of enteric disease among children in LMICs. However, previous work on a collection of stool samples from children under 2 years of age, living in a low resource community in Peru with either acute diarrheal disease or asymptomatic, were found to be qPCR positive for Campylobacter species but qPCR negative for C. jejuni and C. coli. The goal of this study was to determine if whole-genome shotgun metagenomic sequencing (WSMS) could identify the Campylobacter species within these samples. The Campylobacter species identified in these stool samples included C. jejuni, C. coli, C. upsaliensis, C. concisus, and the potential new species of Campylobacter, "Candidatus Campylobacter infans". Moreover, WSMS results demonstrate that over 65% of the samples represented co-infections with multiple Campylobacter species present in a single stool sample, a novel finding in human populations.

"Candidatus Campylobacter infans" detection is not associated with diarrhea in children under the age of 2 in Peru.

A working hypothesis is that less common species of Campylobacter (other than C. jejuni and C. coli) play a role in enteric disease among children in low resource settings and explain the gap between the detection of Campylobacter using culture and culture independent methods. "Candidatus Campylobacter infans" (C. infans), was recently detected in stool samples from children and hypothesized to play a role in Campylobacter epidemiology in low- and middle-income countries (LMIC). This study determined the prevalence of C. infans in symptomatic and asymptomatic stool samples from children living in Iquitos, Peru. Stool samples from 215 children with diarrhea and 50 stool samples from children without diarrhea under the age of two were evaluated using a multiplex qPCR assay to detect Campylobacter spp. (16S rRNA), Campylobacter jejuni / Campylobacter coli (cadF gene), C. infans (lpxA), and Shigella spp. (ipaH). C. infans was detected in 7.9% (17/215) symptomatic samples and 4.0% (2/50) asymptomatic samples. The association between diarrhea and the presence of these targets was evaluated using univariate logistic regressions. C. infans was not associated with diarrhea. Fifty-one percent (75/146) of Campylobacter positive fecal samples were negative for C. jejuni, C. coli, and C. infans via qPCR. Shotgun metagenomics confirmed the presence of C. infans among 13 out of 14 positive C. infans positive stool samples. C infans explained only 20.7% of the diagnostic gap in stools from children with diarrhea and 16.7% of the gap in children without diarrhea. We posit that poor cadF primer performance better explains the observed gap than the prevalence of atypical non-C. jejuni/coli species.