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1.
Microorganisms ; 7(12)2019 Dec 10.
Artigo em Inglês | MEDLINE | ID: mdl-31835624

RESUMO

We investigated the anti-Campylobacter activity of pinocembrin and its mechanism of action, as well as Campylobacter responses to pinocembrin treatment at the genetic and phenotypic levels, using C. jejuni NCTC 11168 and a multidrug efflux system repressor mutant (11168ΔcmeR). At its minimal inhibitory concentration, pinocembrin significantly increased cell membrane permeability of Campylobacter. Interestingly, at sub-inhibitory concentrations, pinocembrin did not significantly alter membrane functionality and it increased bacterial fitness. Treatment with pinocembrin evoked decreased expression of ribosomal proteins and down-regulation of several NADH dehydrogenase I chain subunits and proteins involved in iron uptake. This suggests altered protein production and redox cycle and iron metabolism. Interestingly, the chelation of Fe ions during the treatment with pinocembrin increased C. jejuni survival, although there was no increase in the formation of reactive oxygen species. Pre-treatment of C. jejuni with sub-inhibitory concentrations of pinocembrin for 2 h resulted in a 1 log decrease in C. jejuni colony forming units in mice liver at 8 days post-infection, compared to untreated C. jejuni. These findings suggest that pinocembrin modulates the metabolic activity of C. jejuni and that pre-treatment of C. jejuni with pinocembrin influences its virulence potential in mice. This anti-Campylobacter potential of pinocembrin warrants further investigation.

2.
Infect Immun ; 87(12)2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31570559

RESUMO

Campylobacter jejuni is a leading cause of foodborne illnesses worldwide. Its porA gene encodes the major outer membrane protein (MOMP) that is abundantly expressed and has important physiological functions, including a key role in systemic infection and abortion induction in pregnant animals. Despite the importance of porA in C. jejuni pathogenesis, mechanisms modulating its expression levels remain elusive. At the 3' end of the porA transcript, there is a Rho-independent transcription terminator (named T porA in this study). Whether T porA affects the expression and function of MOMP remains unknown and is investigated in this study. Green fluorescent protein (GFP) fusion constructs with the porA promoter at the 5' end and an intact T porA or no T porA at the 3' end of the gfp coding sequence revealed that both the transcript level of gfp and its fluorescence signals were more than 2-fold higher in the construct with T porA than in the one without T porA Real-time quantitative PCR (qRT-PCR) analysis of the porA mRNA and immunoblot detection of MOMP in C. jejuni showed that disruption of T porA significantly reduced the porA transcript level and the expression of MOMP. An mRNA decay assay demonstrated that disruption of T porA resulted in a shortened transcript half-life of the upstream gfp or porA gene, indicating that T porA enhances mRNA stability. In the guinea pig model, the C. jejuni construct with an interrupted T porA was significantly attenuated in abortion induction. Together, these results indicate that T porA enhances the expression level of MOMP by stabilizing its mRNA and influences the virulence of C. jejuni.

3.
J Vet Diagn Invest ; 31(6): 818-827, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31646949

RESUMO

Rapid identification of the infecting Salmonella serovar from porcine diagnostic samples is vital to allow implementation of appropriate on-farm treatment and management decisions. Although identification at the serogroup level can be rapidly achieved at most veterinary diagnostic laboratories, final Salmonella serovar identification often takes several weeks because of the limited number of reference laboratories performing the complex task of serotyping. Salmonella serogroup B, currently the dominant serogroup identified from swine clinical samples in the United States, contains serovars that vary from highly pathogenic to minimally pathogenic in swine. We determined the frequency of detection of individual group B serovars at the Iowa State Veterinary Diagnostic Laboratory from 2008 to 2017, and validated a multiplex real-time PCR (rtPCR) to distinguish pathogenic serogroup B serovars from those of lesser pathogenicity. Our results indicate that, since 2014, Salmonella enterica ssp. enterica serovar 4,[5],12:i:- has been the dominant serovar identified from swine clinical samples at the ISU-VDL, with S. Typhimurium now the second most common serovar identified. We developed a rtPCR to allow rapid differentiation of samples containing S. 4,[5],12:i:- and S. Typhimurium from samples containing serovars believed to be of less pathogenicity, such as S. Agona and S. Derby. When combined with enrichment culture, this rtPCR has the ability to significantly improve the time to final serovar identification of the 2 most commonly identified pathogenic Salmonella serovars in swine, and allows rapid implementation of serovar-specific intervention strategies.

4.
BMC Vet Res ; 15(1): 130, 2019 May 06.
Artigo em Inglês | MEDLINE | ID: mdl-31060608

RESUMO

BACKGROUND: Antimicrobial resistance (AMR) of bacterial pathogens is an emerging public health threat. This threat extends to pets as it also compromises our ability to treat their infections. Surveillance programs in the United States have traditionally focused on collecting data from food animals, foods, and people. The Veterinary Laboratory Investigation and Response Network (Vet-LIRN), a national network of 45 veterinary diagnostic laboratories, tested the antimicrobial susceptibility of clinically relevant bacterial isolates from animals, with companion animal species represented for the first time in a monitoring program. During 2017, we systematically collected and tested 1968 isolates. To identify genetic determinants associated with AMR and the potential genetic relatedness of animal and human strains, whole genome sequencing (WGS) was performed on 192 isolates: 69 Salmonella enterica (all animal sources), 63 Escherichia coli (dogs), and 60 Staphylococcus pseudintermedius (dogs). RESULTS: We found that most Salmonella isolates (46/69, 67%) had no known resistance genes. Several isolates from both food and companion animals, however, showed genetic relatedness to isolates from humans. For pathogenic E. coli, no resistance genes were identified in 60% (38/63) of the isolates. Diverse resistance patterns were observed, and one of the isolates had predicted resistance to fluoroquinolones and cephalosporins, important antibiotics in human and veterinary medicine. For S. pseudintermedius, we observed a bimodal distribution of resistance genes, with some isolates having a diverse array of resistance mechanisms, including the mecA gene (19/60, 32%). CONCLUSION: The findings from this study highlight the critical importance of veterinary diagnostic laboratory data as part of any national antimicrobial resistance surveillance program. The finding of some highly resistant bacteria from companion animals, and the observation of isolates related to those isolated from humans demonstrates the public health significance of incorporating companion animal data into surveillance systems. Vet-LIRN will continue to build the infrastructure to collect the data necessary to perform surveillance of resistant bacteria as part of fulfilling its mission to advance human and animal health. A One Health approach to AMR surveillance programs is crucial and must include data from humans, animals, and environmental sources to be effective.


Assuntos
Bactérias/efeitos dos fármacos , Bactérias/genética , Laboratórios/normas , Saúde Única , Medicina Veterinária/organização & administração , Sequenciamento Completo do Genoma , Animais , Infecções Bacterianas/epidemiologia , Infecções Bacterianas/microbiologia , Infecções Bacterianas/veterinária , Canadá/epidemiologia , Estados Unidos/epidemiologia
5.
Front Microbiol ; 10: 698, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31024487

RESUMO

Campylobacter jejuni is a leading cause of foodborne illnesses worldwide. As a microaerobic pathogen, C. jejuni is subjected to DNA damages caused by various stresses such as reactive oxygen species (ROS) and UV radiations. The base excision repair (BER) system plays an important role in preventing mutations associated with oxidative DNA damage, but the system remains poorly characterized in Campylobacter. In this study, a BER homolog encoded by cj0595c (named nth) in C. jejuni was analyzed for endonuclease III activity and for its role in maintaining genomic stability. It was found that inactivation of nth resulted in elevated frequencies of spontaneous fluoroquinolone-resistant (FQR) and oxidative stress resistant (OXR) mutants, compared with the wild-type strain in C. jejuni. Sequencing analysis of the FQR and OXR mutants revealed that the elevated mutation rates were associated with C → T or G → A transition in gyrA (FQR mutants) or perR (for OXR mutants). In an in vitro assay, a purified recombinant C. jejuni Nth protein demonstrated endonuclease III activity that recognized and excised the thymine glycol (Tg) base from a double stranded DNA. These findings indicate that Nth functions as a BER repair enzyme in C. jejuni and is important for the repair of DNA damage, protecting the bacteria from stresses encountered within a host and in the environment.

6.
Vet Pathol ; 56(4): 555-564, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-30895909

RESUMO

The aim of this study was to assess whether pregnant mice represent a useful model to study the reproductive pathology of Campylobacter jejuni IA3902 using the end point of positive microbial culture of the organism from the fetoplacental unit. Pregnant BALB/c and CD-1 mice (14 days' gestation) were inoculated orally and intraperitoneally (IP) with 1 × 109 colony-forming units/ml of C. jejuni IA3902. The organism was recovered by microbial culture from the fetoplacental unit in 10 of 10 BALB/c and 10 of 10 CD-1 IP-inoculated pregnant mice and in 29% (2/7) BALB/c and 38% (3/8) CD-1 orally inoculated pregnant mice. Gross reproductive lesions included necrosuppurative placentitis, fetal resorption, intrauterine fetal death, stillborn pups (dead neonates), and multifocal hepatitis. Histological changes consisted of locally extensive neutrophilic and necrotizing placentitis with intralesional bacterial colonies of C. jejuni, ulcerative endometritis, random multifocal hepatitis, and rare cholecystitis. Immunohistochemistry for the major outer membrane protein of C. jejuni revealed moderate to large numbers of the organism at the periphery of the placental discs, within trophoblasts and extracellularly, with invasion into the placental disc largely via the vascular network. The organism is trophic for neutral mucin, iron, and L-fucose within the murine placenta. C. jejuni IA3902 has affinity for the murine reproductive tract, specifically the fetoplacental unit, where it results in a necrotizing placentitis with positive microbial recovery after both IP and oral challenge in BALB/c and CD-1 pregnant mice.


Assuntos
Infecções por Campylobacter/microbiologia , Campylobacter jejuni/fisiologia , Animais , Infecções por Campylobacter/patologia , Modelos Animais de Doenças , Feminino , Morte Fetal , Imuno-Histoquímica/veterinária , Camundongos , Camundongos Endogâmicos BALB C , Placenta/patologia , Gravidez , Trofoblastos/patologia
7.
Appl Environ Microbiol ; 85(11)2019 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-30926726

RESUMO

Campylobacter is a major foodborne pathogen in humans and a significant cause of abortion in sheep. Although ruminants are increasingly recognized as important reservoirs for Campylobacter species, limited information is available about the molecular epidemiology and antimicrobial resistance (AMR) profiles of sheep Campylobacter Here, we describe a two-trial study that examined Campylobacter profiles in sheep and determined whether in-feed tetracycline (TET) influenced the distribution and AMR profiles of Campylobacter Each trial involved 80 commercial sheep naturally infected with Campylobacter: 40 of these sheep were medicated with tetracycline in feed, while the other 40 received feed without antibiotics. Fecal and bile samples were collected for the isolation of Campylobacter The bacterial isolates were analyzed for antimicrobial susceptibility and genotypes. The results revealed that 87.0% and 61.3% of the fecal and bile samples were positive for Campylobacter (Campylobacter jejuni and Campylobacter coli), with no significant differences between the medicated and nonmedicated groups. All but one of the tested Campylobacter isolates were resistant to tetracycline. Although fluoroquinolone (FQ) resistance remained low in C. jejuni (1.7%), 95.0% of the C. coli isolates were resistant to FQ. Genotyping revealed that C. jejuni sequence type 2862 (ST2862) and C. coli ST902 were the predominant genotypes in the sheep. Feed medication with tetracycline did not affect the overall prevalence, species distribution, and AMR profiles of Campylobacter, but it did increase the total Campylobacter counts in bile and gallbladder. These findings identify predominant Campylobacter clones, reveal the high prevalence of FQ-resistant C. coli, and provide new insights into the epidemiology of Campylobacter in sheep.IMPORTANCE Campylobacter is a major cause of foodborne illness in humans, and antibiotic-resistant Campylobacter is considered a serious threat to public health in the United States and worldwide. As a foodborne pathogen, Campylobacter commonly exists in the intestinal tract of ruminant animals, such as sheep and cattle. Results from this study reveal the predominant genotypes and high prevalence of tetracycline (TET) and fluoroquinolone (FQ) resistance in sheep Campylobacter The finding on fluoroquinolone resistance in sheep Campylobacter is unexpected, as this class of antibiotics is not used for sheep in the United States, and it may suggest the transmission of fluoroquinolone-resistant Campylobacter from cattle to sheep. Additionally, the results demonstrate that in-feed medication with tetracycline increases Campylobacter counts in gallbladders, suggesting that the antibiotic promotes Campylobacter colonization of the gallbladder. These findings provide new information on Campylobacter epidemiology in sheep, which may be useful for curbing the spread of antibiotic-resistant Campylobacter in animal reservoirs.

8.
J Anim Sci ; 97(5): 2139-2153, 2019 Apr 29.
Artigo em Inglês | MEDLINE | ID: mdl-30888017

RESUMO

This study aimed to evaluate the effects of a source of dietary soluble (SF) and insoluble fiber (IF) without or with exogenous carbohydrases (xylanase, ß-glucanase, and pectinase) on diarrhea incidence, selected immune responses, and growth performance in enterotoxigenic Escherichia coli (ETEC)-challenged pigs. Sixty weaned pigs (6.9 ± 0.1 kg BW, ~23 d of age) were blocked by initial BW and placed in individual pens. Pens were randomly assigned to one of six treatments (n = 10 per treatment), including a nonchallenged control (NC), a positive challenge control (PC), the PC + a soluble fiber diet (10% sugar beet pulp) without (SF-) or with carbohydrases (SF+), and PC + an IF diet (15% corn distillers dried grains with solubles) without (IF-) or with carbohydrases (IF+). The control diet was primarily based on corn and soybean meal with 13.5% whey powder. The two sources of fiber were added at the expense of cornstarch in the control diet. Pigs were orally inoculated with 6 mL hemolytic F18 ETEC (~3.5 × 109 cfu/mL) or sham infected with 6 mL phosphate-buffered saline on day 7 (0 d postinoculation, dpi) postweaning. All ETEC challenged pigs were confirmed to be genetically susceptible to F18 ETEC. Pigs had free access to feed and water throughout the 14-d trial. Pig BW and feed intake were recorded on dpi -7, 0, and 7 or 8. Fecal swabs were collected on dpi -7, 0, 1, 2, 3, 5, and 7 or 8 to evaluate hemolytic E. coli shedding. Fecal score was visually ranked daily postchallenge to evaluate diarrhea incidence. Blood samples were collected on dpi -1, 3, and 7 or 8 at necropsy and intestinal tissues were collected at necropsy. Pigs on PC had lower dpi 1 to 7 ADG and ADFI than those on NC (P < 0.05). Compared with PC pigs, SF+ pigs had greater ADG during both pre- and postchallenge period (P < 0.05). The IF- increased postchallenge diarrhea incidence compared with PC (P < 0.05). Pigs on SF- had lower ileal E. coli attachment than PC (P < 0.05). The SF+ reduced haptoglobin and IF+ reduced C-reactive protein on dpi 3 compared with PC (P < 0.05). Compared with PC pigs, SF+ pigs tended to have lower ileal tumor necrosis factor alpha and greater ileal occludin (OCLN) mRNA (P < 0.10) and had greater (P < 0.05) colonic OCLN mRNA levels. Collectively, IF- increased incidence of diarrhea and fecal E. coli shedding compared with PC. The SF+ pigs had improved growth compared with PC pigs, likely due in part to a reduction in inflammatory intermediates.


Assuntos
Ração Animal/análise , Diarreia/veterinária , Fibras na Dieta/metabolismo , Escherichia coli Enterotoxigênica/fisiologia , Glicosídeo Hidrolases/metabolismo , Animais , Diarreia/metabolismo , Dieta/veterinária , Infecções por Escherichia coli/metabolismo , Fezes/microbiologia , Fermentação , Glicosídeo Hidrolases/genética , Íleo/metabolismo , Intestinos/microbiologia , Soja , Suínos , Doenças dos Suínos/imunologia , Doenças dos Suínos/metabolismo , Doenças dos Suínos/microbiologia , Desmame , Zea mays
9.
Foodborne Pathog Dis ; 16(2): 94-103, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30688527

RESUMO

Hemolytic Escherichia coli are important pathogens in neonatal and weaned pigs. In this study, we analyzed 95 hemolytic E. coli isolated from intestinal contents or fecal samples of diarrheic piglets in 15 states of the United States between November 2013 and December 2014. Phenotypic antimicrobial susceptibility was determined through Sensititre BOFO6F plates for all the strains. They were all resistant to clindamycin, penicillin, tiamulin, tilmicosin, and highly resistant to oxytetracycline (91.6%), chlortetracycline (78.9%), ampicillin (75.8%), and sulfadimethoxine (68.4%). 86.2% of them were multidrug resistant. Whole genome sequencing (WGS) showed that 55 strains were enterotoxigenic E. coli (ETEC) and 40 strains were non-ETEC, and the strains belonged to 22 known and 2 novel sequence types (STs). ST100 and ST10 were the main and predominant STs in ETEC strains, whereas the non-ETEC strains were diverse with ST23 and ST761 as the main STs. Antibiotic resistance gene/mutation profiling of the genomes confirmed the results of antimicrobial susceptibility test. Notably, significant differences were found in the susceptibility to enrofloxacin between ETEC and non-ETEC (58.2% vs. 5.0%) and gentamicin (32.7% vs. 7.5%). ampH, ampC2, and ampC1 were the most common beta-lactamase genes in all E. coli strains, and extended-spectrum beta-lactamase (ESBL) genes were rare in these isolates. This study provides new insights into antibiotic resistance and genotypes of intestinal pathogenic E. coli associated with swine disease in the United States, and support the utility of WGS in accurate prediction of resistance to most antibiotics.

10.
J Med Microbiol ; 68(2): 136-142, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30540246

RESUMO

PURPOSE: This study was aimed at investigating the occurrence and genetic mechanisms of resistance to ciprofloxacin, tetracycline and erythromycin in clinical isolates of Campylobacter jejuni recovered from human cases of acute gastroenteritis in Turkey. METHODOLOGY: MIC values of each antibiotic were determined with the epsilometer test (E-test). Resistance genes/mutations were first screened by PCR and analysed by subsequent DNA sequencing. RESULTS: From a total of 152 C. jejuni isolates tested, 113 (74.3%), 38 (25%) and 9 (5.9%) were found to be resistant to ciprofloxacin, tetracycline and erythromycin, respectively. Sequence analysis of ciprofloxacin-resistant isolates showed that all resistant strains (n=113) carried Thr-86-Ile substition in the gyrA gene, which is the most frequently observed mutation in fluoroquinolone-resistant Campylobacter. All of the tetracycline-resistant isolates (n=38) carried the tetO gene. All of the erythromycin-resistant isolates (n=9) harboured the point mutation A2075G in the 23S rRNA gene, which is the most common mutation conferring macrolide resistance in C. jejuni. CONCLUSION: The phenotypic susceptibility testing results were found to agree well with those obtained by genetic detection methods for the C. jejuni isolates tested. The findings of this study showed a very high level of resistance to ciprofloxacin and to a lesser extent to tetracycline while resistance to erythromycin remained at a low level. Thus, erythromycin may be considered as the first choice for treatment of Campylobacter infections in this geographical region when indicated.


Assuntos
Antibacterianos/farmacologia , Infecções por Campylobacter/microbiologia , Campylobacter jejuni/efeitos dos fármacos , Diarreia/microbiologia , Gastroenterite/microbiologia , Campylobacter jejuni/genética , Campylobacter jejuni/isolamento & purificação , Ciprofloxacino/farmacologia , DNA Bacteriano/química , DNA Bacteriano/isolamento & purificação , Testes de Sensibilidade a Antimicrobianos por Disco-Difusão , Farmacorresistência Bacteriana , Eritromicina/farmacologia , Fezes/microbiologia , Humanos , Testes de Sensibilidade Microbiana , RNA Bacteriano/genética , RNA Ribossômico 23S/genética , Tetraciclina/farmacologia , Turquia
11.
Microbiology ; 164(11): 1394-1404, 2018 11.
Artigo em Inglês | MEDLINE | ID: mdl-30383520

RESUMO

Mycoplasma hyopneumoniae is the causative agent of enzootic pneumonia in swine, an important disease worldwide. It has finite biosynthetic capabilities, including a deficit in de novo nucleotide synthesis. The source(s) for nucleotides in vivo are unknown, but mycoplasmas are known to carry membrane-bound nucleases thought to participate in the acquisition of nucleotides from host genomic DNA. Recent research has demonstrated that neutrophils can produce extracellular traps (NETs), chromatin NETs decorated with granular proteins to interact with and eliminate pathogens. We hypothesized that M. hyopneumoniae could utilize its membrane nuclease to obtain nucleotides from extracellular traps to construct its own DNA. Using the human monocytic cell line THP-1, we induced macrophage extracellular traps (METs), which are structurally similar to NETs. The thymidine analogue ethynyl deoxyuridine (EdU) was incorporated into THP-1 DNA and METs were induced. When incubated with M. hyopneumoniae, METs were degraded and the modified nucleotide label could be co-localized within M. hyopneumoniae DNA. When the nucleases were inhibited, MET degradation and nucleotide transfer were also inhibited. Controls confirmed that the EdU originated directly from the METs and not from free nucleotides arising from intracellular pools released during extrusion of the chromosomal DNA. M. hyopneumoniae incorporated labelled nucleotides more efficiently when 'fed' on METs than from free nucleotides in the medium, suggesting a tight linkage between nuclease degradation of DNA and nucleotide transport. These results strongly suggest that M. hyopneumoniae could degrade extracellular traps formed in vivo during infection and incorporate those host nucleotides into its own DNA.


Assuntos
DNA/genética , Armadilhas Extracelulares/genética , Macrófagos/metabolismo , Mycoplasma hyopneumoniae/genética , Mycoplasma hyopneumoniae/metabolismo , Linhagem Celular , Desoxiuridina/análogos & derivados , Desoxiuridina/metabolismo , Armadilhas Extracelulares/metabolismo , Humanos , Nucleotídeos/metabolismo , Coloração e Rotulagem , Células THP-1
12.
Avian Dis ; 62(1): 79-85, 2018 03.
Artigo em Inglês | MEDLINE | ID: mdl-29620465

RESUMO

Spotty liver disease (SLD) is characterized by multifocal liver lesions, mortality, and drop in egg production. The disease is emerging in Europe and Australia, particularly in free-range and floor-raised layer flocks. Campylobacter hepaticus has been recently identified as the causative agent of SLD. We report the isolation and characterization of C. hepaticus from livers of laying hens affected with SLD in the United States. Two isolates were characterized and found to be highly similar to those described from SLD cases in the United Kingdom and Australia. Initial isolation of C. hepaticus from liver samples required microaerophilic and thermophilic conditions and incubation for a particularly long duration (approximately 7 days). Morphologic and phenotypic properties of the isolates were typical for Campylobacter spp. Phylogenetic analysis based on 16S ribosomal RNA gene sequences and detection of a glycerol kinase gene confirmed the identity of the isolates as C. hepaticus. To the authors' knowledge, this is the first report of C. hepaticus isolation from layer chickens with SLD in the United States. With the increasing changes in the egg industry from conventional cages to cage-free housing systems, the incidence and economic impact of SLD could become significant.


Assuntos
Infecções por Campylobacter/veterinária , Campylobacter/isolamento & purificação , Galinhas , Hepatopatias/veterinária , Doenças das Aves Domésticas/epidemiologia , Animais , Campylobacter/classificação , Campylobacter/fisiologia , Infecções por Campylobacter/epidemiologia , Infecções por Campylobacter/microbiologia , Feminino , Incidência , Fígado/patologia , Hepatopatias/epidemiologia , Hepatopatias/microbiologia , Filogenia , Doenças das Aves Domésticas/microbiologia , Prevalência , RNA Bacteriano/análise , RNA Ribossômico 16S/análise , Análise de Sequência de RNA/veterinária , Estados Unidos/epidemiologia
13.
Foodborne Pathog Dis ; 15(6): 377-385, 2018 06.
Artigo em Inglês | MEDLINE | ID: mdl-29638171

RESUMO

Campylobacter spp. can be pathogenic to humans and often harbor antimicrobial resistance genes. Data on resistance in relation to fluoroquinolone use in beef cattle are scarce. This cross-sectional study of preharvest cattle evaluated Campylobacter prevalence and susceptibility to nalidixic acid and ciprofloxacin in feedlots that previously administered a fluoroquinolone as primary treatment for bovine respiratory disease. Twenty fresh fecal samples were collected from each of 10 pens, in each of five feedlots, 1-2 weeks before harvest. Feces were cultured for Campylobacter using selective enrichment and isolation methods. Genus and species were confirmed via PCR. Minimum inhibitory concentrations (MICs) of ciprofloxacin and nalidixic acid were determined using a micro-broth dilution method and human breakpoints. Antimicrobial use within each pen was recorded. Data were analyzed using generalized linear mixed-models (prevalence) and survival analysis (MICs). Overall, sample-level prevalence of Campylobacter was 27.2% (272/1000) and differed significantly among feedlots (p < 0.01). Campylobacter coli was the most common species (55.1%; 150/272), followed by Campylobacter hyointestinalis (42.6%; 116/272). Within-pen prevalence was not significantly associated with the number of fluoroquinolone treatments, sex, body weight, or metaphylaxis use, but was associated with the number of days cattle were in the feedlot (p = 0.03). The MICs for the majority of Campylobacter isolates were above the breakpoints for nalidixic acid (68.4%; 175/256) and for ciprofloxacin (65.6%; 168/256). Distributions of MICs for nalidixic acid (p ≤ 0.01) and ciprofloxacin (p ≤ 0.05) were significantly different among feedlots, and by Campylobacter species. However, fluoroquinolone treatments, sex, body weight, days on feed, and metaphylaxis were not significantly associated with MIC distributions within pens. We found no evidence that the number of fluoroquinolone treatments within feedlot pens significantly affected the within-pen fecal prevalence or quinolone susceptibilies of Campylobacter in feedlots that used a fluoroquinolone as primary treatment for bovine respiratory disease.


Assuntos
Antibacterianos/uso terapêutico , Infecções por Campylobacter/veterinária , Campylobacter/efeitos dos fármacos , Doenças dos Bovinos/epidemiologia , Enrofloxacina/uso terapêutico , Quinolonas/farmacologia , Animais , Antibacterianos/farmacologia , Campylobacter/isolamento & purificação , Infecções por Campylobacter/tratamento farmacológico , Infecções por Campylobacter/epidemiologia , Infecções por Campylobacter/microbiologia , Bovinos , Doenças dos Bovinos/tratamento farmacológico , Doenças dos Bovinos/microbiologia , Estudos Transversais , Farmacorresistência Bacteriana , Kansas/epidemiologia , Prevalência , Texas/epidemiologia
14.
Am J Vet Res ; 79(1): 107-114, 2018 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-29287162

RESUMO

OBJECTIVE To compare the pharmacokinetics of 2 commercial florfenicol formulations following IM and SC administration to sheep. ANIMALS 16 healthy adult mixed-breed sheep. PROCEDURES In a crossover study, sheep were randomly assigned to receive florfenicol formulation A or B at a single dose of 20 mg/kg, IM, or 40 mg/kg, SC. After a 2-week washout period, each sheep was administered the opposite formulation at the same dose and administration route as the initial formulation. Blood samples were collected immediately before and at predetermined times for 24 hours after each florfenicol administration. Plasma florfenicol concentrations were determined by high-performance liquid chromatography. Pharmacokinetic parameters were estimated by noncompartmental methods and compared between the 2 formulations at each dose and route of administration. RESULTS Median maximum plasma concentration, elimination half-life, and area under the concentration-time curve from time 0 to the last quantifiable measurement for florfenicol were 3.76 µg/mL, 13.44 hours, and 24.88 µg•h/mL, respectively, for formulation A and 7.72 µg/mL, 5.98 hours, and 41.53 µg•h/mL, respectively, for formulation B following administration of 20 mg of florfenicol/kg, IM, and 2.63 µg/mL, 12.48 hours, and 31.63 µg•h/mL, respectively, for formulation A and 4.70 µg/mL, 16.60 hours, and 48.32 µg•h/mL, respectively, for formulation B following administration of 40 mg of florfenicol/kg, SC. CONCLUSIONS AND CLINICAL RELEVANCE Results indicated that both formulations achieved plasma florfenicol concentrations expected to be therapeutic for respiratory tract disease caused by Mannheimia haemolytica or Pasteurella spp at both doses and administration routes evaluated.


Assuntos
Antibacterianos/farmacocinética , Ovinos/metabolismo , Tianfenicol/análogos & derivados , Animais , Área Sob a Curva , Cromatografia Líquida de Alta Pressão/veterinária , Estudos Cross-Over , Composição de Medicamentos/veterinária , Meia-Vida , Injeções Intramusculares/veterinária , Injeções Subcutâneas/veterinária , Masculino , Tianfenicol/administração & dosagem , Tianfenicol/farmacocinética
15.
Appl Environ Microbiol ; 83(24)2017 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-29030436

RESUMO

Campylobacter jejuni is a leading cause of foodborne illnesses worldwide. As a microaerophilic organism, C. jejuni must be able to defend against oxidative stress encountered both in the host and in the environment. How Campylobacter utilizes a mutation-based mechanism for adaptation to oxidative stress is still unknown. Here we present a previously undescribed phenotypic and genetic mechanism that promotes the emergence of oxidative stress-resistant mutants. Specifically, we showed that a naturally occurring mutator phenotype, resulting from a loss of function mutation in the DNA repair enzyme MutY, increased oxidative stress resistance (OXR) in C. jejuni We further demonstrated that MutY malfunction did not directly contribute to the OXR phenotype but increased the spontaneous mutation rate in the peroxide regulator gene perR, which functions as a repressor for multiple genes involved in oxidative stress resistance. Mutations in PerR resulted in loss of its DNA binding function and derepression of PerR-controlled oxidative stress defense genes, thereby conferring an OXR phenotype and facilitating Campylobacter survival under oxidative stress. These findings reveal a new mechanism that promotes the emergence of spontaneous OXR mutants in bacterial organisms.IMPORTANCE Although a mutator phenotype has been shown to promote antibiotic resistance in many bacterial species, little is known about its contribution to the emergence of OXR mutants. This work describes the link between a mutator phenotype and the enhanced emergence of OXR mutants as well as its underlying mechanism involving DNA repair and mutations in PerR. Since DNA repair systems and PerR are well conserved in many bacterial species, especially in Gram positives, the same mechanism may operate in multiple bacterial species. Additionally, we developed a novel method that allows for rapid quantification of spontaneous OXR mutants in a bacterial population. This method represents a technical innovation and may also be applied to other bacterial species. These findings significantly advance our understanding of bacterial mechanisms for survival under oxidative stress.


Assuntos
Proteínas de Bactérias/genética , Campylobacter jejuni/genética , Campylobacter jejuni/metabolismo , Mutação , Estresse Oxidativo , Proteínas de Bactérias/metabolismo , Fenótipo
16.
Appl Environ Microbiol ; 83(24)2017 12 15.
Artigo em Inglês | MEDLINE | ID: mdl-28970227

RESUMO

Campylobacter jejuni clone SA is the major cause of sheep abortion and contributes significantly to foodborne illnesses in the United States. Clone SA is hypervirulent because of its distinct ability to produce systemic infection and its predominant role in clinical sheep abortion. Despite the importance of clone SA, little is known about its distribution and epidemiological features in cattle. Here we describe a prospective study on C. jejuni clone SA prevalence in 35 feedlots in 5 different states in the United States and a retrospective analysis of clone SA in C. jejuni isolates collected by National Animal Health Monitoring System (NAHMS) dairy studies in 2002, 2007, and 2014. In feedlot cattle feces, the overall prevalence of Campylobacter organisms was 72.2%, 82.1% of which were C. jejuni Clone SA accounted for 5.8% of the total C. jejuni isolates, but its prevalence varied by feedlot and state. Interestingly, starlings on the feedlots harbored C. jejuni in feces, including clone SA, suggesting that these birds may play a role in the transmission of Campylobacter In dairy cattle, the overall prevalence of clone SA was 7.2%, but a significant decrease in the prevalence was observed from 2002 to 2014. Whole-genome sequence analysis of the dairy clone SA isolates revealed that it was genetically stable over the years and most of the isolates carried the tetracycline resistance gene tet(O) in the chromosome. These findings indicate that clone SA is widely distributed in both beef and dairy cattle and provide new insights into the molecular epidemiology of clone SA in ruminants.IMPORTANCE C. jejuni clone SA is a major cause of small-ruminant abortion and an emerging threat to food safety because of its association with foodborne outbreaks. Cattle appear to serve as a major reservoir for this pathogenic organism, but there is a major gap in our knowledge about the epidemiology of clone SA in beef and dairy cattle. By taking advantage of surveillance studies conducted on a national scale, we found a wide but variable distribution of clone SA in feedlot cattle and dairy cows in the United States. Additionally, the work revealed important genomic features of clone SA isolates from cattle. These findings provide critically needed information for the development of preharvest interventions to control the transmission of this zoonotic pathogen. Control of C. jejuni clone SA will benefit both animal health and public health, as it is a zoonotic pathogen causing disease in both ruminants and humans.


Assuntos
Infecções por Campylobacter/veterinária , Campylobacter jejuni/isolamento & purificação , Doenças dos Bovinos/epidemiologia , Controle de Pragas , Estorninhos , Animais , Infecções por Campylobacter/epidemiologia , Infecções por Campylobacter/microbiologia , Campylobacter jejuni/genética , Bovinos , Doenças dos Bovinos/microbiologia , Colorado/epidemiologia , Iowa/epidemiologia , Kansas/epidemiologia , Missouri/epidemiologia , Prevalência , Estudos Prospectivos , Estudos Retrospectivos , Texas/epidemiologia , Estados Unidos/epidemiologia
17.
Foodborne Pathog Dis ; 14(8): 472-477, 2017 08.
Artigo em Inglês | MEDLINE | ID: mdl-28622473

RESUMO

We investigated the contribution of litter to the occurrence of Campylobacter on three broiler farms, which were known to have low (LO) and high (HI-A and HI-B) Campylobacter prevalence. For this purpose, we collected litter samples (n = 288) during and after two rearing cycles from each farm. We evaluated the occurrence of Campylobacter (using selective enrichment and quantitative real-time polymerase chain reaction [q-PCR] analysis) in the litter samples as well as the litter's pH and moisture content. Ceca from each flock (n = 144) were harvested at slaughter age and used to quantify Campylobacter colony-forming units (CFUs). Campylobacter was only retrieved from 7 litter samples that were collected from HI-A and HI-B during the growing period, but no Campylobacter was isolated from LO farms. The q-PCR analysis detected Campylobacter in pooled litter samples from all three farms. However, in litter collected during the same rotation, Campylobacter levels were significantly higher (p < 0.05) in HI-A and HI-B litter samples in comparison to those in LO. Cecal samples from HI-A and HI-B yielded relatively high numbers of Campylobacter CFUs, which were undetectable in LO samples. Litter's pH and moisture did not affect the overall occurrence of Campylobacter in litter and ceca on any of the farms. Our data suggest that Campylobacter was generally more abundant in litter that was collected from farms with highly colonized flocks. Therefore, better approaches for assessing the occurrence of Campylobacter in litter might be warranted in order to reduce the dissemination of these pathogens on and off poultry farms.


Assuntos
Infecções por Campylobacter/microbiologia , Campylobacter/isolamento & purificação , Galinhas/microbiologia , Doenças Transmitidas por Alimentos/microbiologia , Doenças das Aves Domésticas/microbiologia , Animais , Campylobacter/genética , Infecções por Campylobacter/epidemiologia , Inocuidade dos Alimentos , Doenças Transmitidas por Alimentos/epidemiologia , Humanos , Concentração de Íons de Hidrogênio , Aves Domésticas , Doenças das Aves Domésticas/epidemiologia , Prevalência
18.
Infect Immun ; 85(6)2017 06.
Artigo em Inglês | MEDLINE | ID: mdl-28373351

RESUMO

Campylobacter jejuni is a zoonotic pathogen, and a hypervirulent clone, named clone SA, has recently emerged as the predominant cause of ovine abortion in the United States. To induce abortion, orally ingested Campylobacter must translocate across the intestinal epithelium, spread systemically in the circulation, and reach the fetoplacental tissue. Bacterial factors involved in these steps are not well understood. C. jejuni is known to produce capsular polysaccharide (CPS), but the specific role that CPS plays in systemic infection and particularly abortion in animals remains to be determined. In this study, we evaluated the role of CPS in bacteremia using a mouse model and in abortion using a pregnant guinea pig model following oral challenge. Compared with C. jejuni NCTC 11168 and 81-176, a clone SA isolate (IA3902) resulted in significantly higher bacterial counts and a significantly longer duration of bacteremia in mice. The loss of capsule production via gene-specific mutagenesis in IA3902 led to the complete abolishment of bacteremia in mice and abortion in pregnant guinea pigs, while complementation of capsule expression almost fully restored these phenotypes. The capsule mutant strain was also impaired for survival in guinea pig sera and sheep blood. Sequence-based analyses revealed that clone SA possesses a unique CPS locus with a mosaic structure, which has been stably maintained in all clone SA isolates derived from various hosts and times. These findings establish CPS as a key virulence factor for the induction of systemic infection and abortion in pregnant animals and provide a viable candidate for the development of vaccines against hypervirulent C. jejuni.


Assuntos
Aborto Séptico/microbiologia , Cápsulas Bacterianas/metabolismo , Infecções por Campylobacter/microbiologia , Campylobacter jejuni/patogenicidade , Polissacarídeos Bacterianos/metabolismo , Animais , Cápsulas Bacterianas/genética , Infecções por Campylobacter/metabolismo , Campylobacter jejuni/genética , Feminino , Regulação Bacteriana da Expressão Gênica , Cobaias , Camundongos , Mutação , Polissacarídeos Bacterianos/genética , Gravidez , Ovinos , Virulência , Fatores de Virulência/genética
19.
Sci Rep ; 7(1): 494, 2017 03 29.
Artigo em Inglês | MEDLINE | ID: mdl-28356558

RESUMO

Antibiotic resistance, particularly to fluoroquinolones and macrolides, in the major foodborne pathogen Campylobacter is considered a serious threat to public health. Although ruminant animals serve as a significant reservoir for Campylobacter, limited information is available on antibiotic-resistant Campylobacter of bovine origin. Here, we analyzed the antimicrobial susceptibilities of 320 C. jejuni and 115 C. coli isolates obtained from feedlot cattle farms in multiple states in the U.S. The results indicate that fluoroquinolone resistance reached to 35.4% in C. jejuni and 74.4% in C. coli, which are significantly higher than those previously reported in the U.S. While all fluoroquinolone resistant (FQR) C. coli isolates examined in this study harbored the single Thr-86-Ile mutation in GyrA, FQR C. jejuni isolates had other mutations in GyrA in addition to the Thr-86-Ile change. Notably, most of the analyzed FQR C. coli isolates had similar PFGE (pulsed field gel electrophoresis) patterns and the same MLST (multilocus sequence typing) sequence type (ST-1068) regardless of their geographic sources and time of isolation, while the analyzed C. jejuni isolates were genetically diverse, suggesting that clonal expansion is involved in dissemination of FQR C. coli but not C. jejuni. These findings reveal the rising prevalence of FQR Campylobacter in the U.S. and provide novel information on the epidemiology of antibiotic-resistant Campylobacter in the ruminant reservoir.


Assuntos
Antibacterianos/farmacologia , Infecções por Campylobacter/veterinária , Campylobacter/efeitos dos fármacos , Doenças dos Bovinos/epidemiologia , Doenças dos Bovinos/microbiologia , Farmacorresistência Bacteriana , Fluoroquinolonas/farmacologia , Animais , Campylobacter/genética , Campylobacter/isolamento & purificação , Bovinos , Análise por Conglomerados , Farmacorresistência Bacteriana Múltipla , Variação Genética , Testes de Sensibilidade Microbiana , Prevalência , Estados Unidos/epidemiologia
20.
J Antimicrob Chemother ; 72(6): 1581-1588, 2017 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-28186558

RESUMO

Objectives: To identify and characterize a novel cfr variant that recently emerged and confers multidrug resistance in Campylobacter , a major foodborne pathogen. Methods: WGS was initially used to identify the cfr (C) gene in Campylobacter isolates and its function was further verified by cloning into an antibiotic-susceptible Campylobacter jejuni strain. Distribution of cfr (C) in various Campylobacter isolates was determined by PCR analysis. Genotyping of cfr (C)-positive strains was done by PFGE and MLST. Results: The cfr (C) gene is predicted to encode a protein that shares 55.1% and 54.9% identity with Cfr and Cfr(B), respectively. cfr (C) was located on a conjugative plasmid of ∼48 kb. Cloning of cfr (C) into C. jejuni NCTC 11168 and conjugative transfer of the cfr (C)-containing plasmid confirmed its role in conferring resistance to phenicols, lincosamides, pleuromutilins and oxazolidinones, and resulted in an 8-256-fold increase in their MICs in both C. jejuni and Campylobacter coli . The cfr (C) gene was detected in multiple C. coli (34 of 344; 10%) isolates derived from different cattle farms in different states, and molecular typing of the cfr (C)-positive C. coli isolates revealed its spread mainly via clonal expansion. Conclusions: These results identify cfr (C) as a new multidrug resistance mechanism in Campylobacter and suggest the potential transmission of this mechanism via the foodborne route, warranting enhanced efforts to monitor its spread in Campylobacter and other foodborne pathogens.


Assuntos
Campylobacter/genética , Farmacorresistência Bacteriana Múltipla/genética , Microbiologia de Alimentos , Genes MDR , Plasmídeos , Animais , Antibacterianos/farmacologia , Campylobacter/efeitos dos fármacos , Campylobacter/isolamento & purificação , Campylobacter/patogenicidade , Campylobacter jejuni/genética , Bovinos/microbiologia , Clonagem Molecular , DNA Bacteriano/genética , Genoma Bacteriano , Genótipo , Humanos , Testes de Sensibilidade Microbiana , Tipagem de Sequências Multilocus , Reação em Cadeia da Polimerase , Análise de Sequência de DNA , Estados Unidos/epidemiologia
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