RESUMEN
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.
Asunto(s)
Antibacterianos , Infecciones por Campylobacter , Campylobacter coli , Campylobacter jejuni , Pollos , Granjas , Variación Genética , Campylobacter coli/genética , Campylobacter coli/efectos de los fármacos , Campylobacter coli/aislamiento & purificación , Animales , Campylobacter jejuni/genética , Campylobacter jejuni/efectos de los fármacos , Campylobacter jejuni/aislamiento & purificación , Pollos/microbiología , Antibacterianos/farmacología , Infecciones por Campylobacter/microbiología , Infecciones por Campylobacter/epidemiología , Infecciones por Campylobacter/veterinaria , Farmacorresistencia Bacteriana/genética , Ecuador/epidemiología , Pruebas de Sensibilidad Microbiana , Humanos , Microbiología de Alimentos , Secuenciación Completa del Genoma , Tetraciclina/farmacologíaRESUMEN
Campylobacter is gram-negative bacteria considered the predominant genera isolated from poultry samples and associated with gastroenteritis. Due to the problems in conventional cultural methods of time-consuming and technically demanding requirements, a rapid and feasible method for their identification and discrimination of the closely related spp. Including Campylobacter coli, Campylobacter fetus, and Campylobacter jejuni is needed. This study analyzes the chicken and sheep meats samples (n = 125) using culture and pre-enrichment-based Quadraplex real-time PCR by targeting OrfA, CstA, HipO, and 16 S rRNA genes of C. coli, C. fetus, C. jejuni and Campylobacter spp. Respectively. The analysis of 125 chicken and sheep meat samples by culture and real-time PCR showed high concordance between the results of the two methods. The present study show high prevalence of Campylobacter species (35% and 32% from chicken and meat respectively) of which C. jejuni were the most abundant. Reaction efficiencies were between 90 and 110%, and detect as low as 8.9 fg in C. jejuni. The need for quick detection and discrimination methods in sheep and chicken meat can be met using the described Quadraplex real-time PCR methodology.
Asunto(s)
Campylobacter coli , Campylobacter jejuni , Pollos , Carne , Reacción en Cadena en Tiempo Real de la Polimerasa , Animales , Pollos/microbiología , Ovinos/microbiología , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Campylobacter coli/genética , Campylobacter coli/aislamiento & purificación , Campylobacter coli/clasificación , Campylobacter jejuni/genética , Campylobacter jejuni/aislamiento & purificación , Campylobacter jejuni/clasificación , Carne/microbiología , Campylobacter fetus/genética , Campylobacter fetus/aislamiento & purificación , Campylobacter fetus/clasificación , Campylobacter/genética , Campylobacter/aislamiento & purificación , Campylobacter/clasificación , Microbiología de Alimentos , ADN Bacteriano/genéticaRESUMEN
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.
Asunto(s)
Campylobacter jejuni , Pollos , Evolución Molecular , Genoma Bacteriano , Campylobacter jejuni/genética , Campylobacter jejuni/efectos de los fármacos , Campylobacter jejuni/aislamiento & purificación , Campylobacter jejuni/clasificación , Brasil , Animales , Pollos/microbiología , Humanos , Infecciones por Campylobacter/microbiología , Infecciones por Campylobacter/veterinaria , Adaptación al Huésped/genética , Antibacterianos/farmacología , Farmacorresistencia Bacteriana Múltiple/genética , FilogeniaRESUMEN
Escherichia coli O157:H7 (E. coli O157:H7) and Campylobacter jejuni (C. jejuni) are pathogenic microorganisms that can cause severe clinical symptoms in humans and are associated with bovine meat consumption. Specific monitoring for E. coli O157: H7 or C. jejuni in meat is not mandatory under Chilean regulations. In this study, we analyzed 544 samples for the detection of both microorganisms, obtained from 272 bovine carcasses (280 kg average) at two slaughterhouses in the Bio-Bío District, Chile. Sampling was carried out at post-shower of carcasses and after channel passage through the cold chamber. Eleven samples were found to be positive for E. coli O157:H7 (4.0%) using microbiological and biochemical detection techniques and were subjected to a multiplex PCR to detect fliC and rfbE genes. Six samples (2.2%) were also found to be positive for the pathogenicity genes stx1, stx2, and eaeA. Twenty-two carcasses (8.0%) were found to be positive for C. jejuni using microbiological and biochemical detection techniques, but no sample with amplified mapA gene was found.
Asunto(s)
Mataderos , Campylobacter jejuni , Escherichia coli O157 , Proteínas de Escherichia coli , Microbiología de Alimentos , Animales , Bovinos , Campylobacter jejuni/aislamiento & purificación , Campylobacter jejuni/genética , Escherichia coli O157/aislamiento & purificación , Escherichia coli O157/genética , Chile , Proteínas de Escherichia coli/genética , Flagelina/genética , Carne/microbiología , Contaminación de Alimentos/análisis , Adhesinas Bacterianas/genética , Toxina Shiga I/genética , Toxina Shiga II/genética , Reacción en Cadena de la Polimerasa Multiplex , Proteínas Bacterianas/genética , Transaminasas , Carbohidrato EpimerasasRESUMEN
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.
Asunto(s)
Infecciones por Campylobacter , Campylobacter coli , Campylobacter jejuni , Campylobacter , Niño , Humanos , Campylobacter coli/genética , Reacción en Cadena de la Polimerasa , Infecciones por Campylobacter/diagnóstico , Infecciones por Campylobacter/microbiología , Heces/microbiologíaRESUMEN
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.
Asunto(s)
Antibacterianos , Infecciones por Campylobacter , Campylobacter jejuni , Factores de Virulencia , Humanos , Brasil , Infecciones por Campylobacter/microbiología , Antibacterianos/farmacología , Virulencia/genética , Campylobacter jejuni/genética , Campylobacter jejuni/patogenicidad , Campylobacter jejuni/efectos de los fármacos , Campylobacter jejuni/aislamiento & purificación , Células CACO-2 , Factores de Virulencia/genética , Genoma Bacteriano , Farmacorresistencia Bacteriana , Variación Genética , Pruebas de Sensibilidad Microbiana , Tipificación de Secuencias Multilocus , Secuenciación Completa del GenomaRESUMEN
AIMS: To evaluate the biofilm-forming capacity of thermotolerant Campylobacter (TC) strains from poultry production and to analyse the inhibitory capacity of Lactiplantibacillus plantarum LP5 against TC on different materials. METHODS AND RESULTS: Biofilm-forming capacity by Campylobacter jejuni and Campylobacter coli was analysed by cell adhesion in polystyrene plates. TC were classified as non-biofilm-forming (NBF, 1.3%), weak biofilm-forming (WBF, 68.4%), moderate biofilm-forming (MBF, 27.6%), and strong biofilm-forming (SBF, 2.7%). The inhibitory capacity of L. plantarum LP5 against TC was tested on stainless-steel, nylon, aluminium, and glass disks (treated group) and compared with biofilm-forming TC (control group). Lactiplantibacillus plantarum LP5 was inoculated, and then TC. Biofilm was removed in both experimental groups and TC and LP5 bacterial counts were performed. The L. plantarum LP5 presence reduced the formation of TC biofilm (P < 0.001). The material type and strain category influenced biofilm formation, with stainless-steel and the SBF strain being the material and TC having the highest adhesion (P < 0.001). Lactiplantibacillus plantarum LP5 formed a similar biofilm on all materials (P = 0.823). CONCLUSIONS: This trial showed very promising results; L. plantarum LP5 could be incorporated as a bio-protector of TC on different surfaces.
Asunto(s)
Campylobacter coli , Campylobacter jejuni , Campylobacter , Lactobacillus plantarum , Biopelículas , AceroRESUMEN
OBJECTIVES: The draft genome sequence of Campylobacter jejuni (Cj26) was analysed to investigate genetic mechanisms of antimicrobial resistance, virulence-associated genes, and phylogenetic context. METHODS: Antimicrobial resistance was assessed by agar dilution and disk diffusion. Cj26 was sequenced using NovaSeq 6000 technology. The genome was assembled and annotated. Resistance genes and chromosomal mutations were analysed using the Center for Genomic Epidemiology services, and the multilocus sequence type, SVR-flaA, and porA were determined. The virulome was determined using the Virulence Factor Database. Plasmid detection and assembly were performed using Unicycler v0.5.0 software. To infer the core genome phylogeny, prokka v1.14.5 was employed in conjunction with IQtree v2.0.3. RESULTS: The Cj26 strain showed a high level of resistance to ciprofloxacin (32 µg/mL) and erythromycin (>128 µg/mL) and resistance to tetracycline and ampicillin. Multilocus sequence typing revealed that the strain belonged to sequence type 353. The substitutions Tre-86-Ile in gyrA and A2075G in 23s RNA were detected, along with the genes tetO, aph(3')-III, ant(6)-Ia, and blaOXA 460. A consistent relationship among accessory and core genes was identified. When compared to other sequence type 353 genomes from Brazil, Cj26 clustered with strains that had more antimicrobial resistance genes than the other clusters. CONCLUSION: This report provides insight into the antimicrobial resistance determinants found in a C. jejuni strain and offers a valuable resource for further studies on Campylobacter genomics and antimicrobial resistance.
Asunto(s)
Antiinfecciosos , Campylobacter jejuni , Animales , Ciprofloxacina/farmacología , Eritromicina , Aves de Corral , Filogenia , Brasil , Farmacorresistencia Bacteriana/genética , Antiinfecciosos/farmacología , GenómicaRESUMEN
Poultry products are recognized as the main source of Salmonella and Campylobacter jejuni infections in humans, while avian pathogenic Escherichia coli may have zoonotic potential and can be transmitted from chicken meat to humans. Biofilm formation contributes to their spread through the food chain. This study aimed to compare the adhesion of Salmonella Enteritidis, E. coli, and C. jejuni strains isolated from poultry, food implicated in outbreaks, and poultry slaughterhouses on three surfaces widely used in poultry production (polystyrene, stainless steel, and polyethylene). S. Enteritidis and E. coli adhesion on the three surfaces tested were not significantly different (p > 0.05). Interestingly, the number of C. jejuni cells on stainless steel (4.51-4.67 log10 CFU/cm.-2) was significantly higher (p = 0.0004) than that on polystyrene (3.80-4.25 log10 CFU/cm.-2), but similar (p > 0.05) to that on polyethylene (4.03-4.36 log10 CFU/cm.-2). However, C. jejuni adhesion was significantly lower (p < 0.05) than S. Enteritidis and E. coli adhesion, regardless of the surface evaluated. In addition, scanning electron microscopy analyses have shown an increased irregularity of the stainless steel surface when compared to polyethylene and polystyrene. These irregularities form small spaces ideal for microbial adhesion.
Asunto(s)
Campylobacter jejuni , Salmonella enteritidis , Humanos , Escherichia coli , Adhesión Bacteriana , Biopelículas , Poliestirenos , Acero Inoxidable , Microbiología de Alimentos , PolietilenoRESUMEN
This work describes the preparation, characterization and antimicrobial activity of four palladium(II) complexes, namely, [Pd(meg)(1,10-phen)] 1, [Pd(meg)(PPh3)2] 2, [Pd(og)(1,10-phen)] 3 and [Pd(og)(PPh3)2] 4, where meg = methyl gallate, og = octyl gallate, 1,10-phen = 1,10-phenanthroline and PPh3 = triphenylphosphine. As to the chemical structures, spectral and physicochemical studies of 1-4 indicated that methyl or octyl gallate coordinates a palladium(II) ion through two oxygen atoms upon deprotonation. A chelating bidentate phenanthroline or two triphenylphosphine molecules complete the coordination sphere of palladium(II) ion, depending on the complex. The metal complexes were tested against the Mycobacterium tuberculosis H37Rv strain and 2 exhibited high activity (MIC = 3.28 µg/mL). As to the tests with Campylobacter jejuni, complex 1 showed a significant effect in reducing bacterial population (greater than 7 log CFU) in planktonic forms, as well as in the biomass intensity (IBF: 0.87) when compared to peracetic acid (IBF: 1.11) at a concentration of 400 µg/mL. The effect provided by these complexes has specificity according to the target microorganism and represent a promising alternative for the control of microorganisms of public health importance.
Asunto(s)
Campylobacter jejuni , Complejos de Coordinación , Mycobacterium tuberculosis , Paladio/farmacología , Paladio/química , Cristalografía por Rayos X , Complejos de Coordinación/farmacología , Complejos de Coordinación/químicaRESUMEN
In this study a data dependent acquisition label-free based proteomics approach was used to identify pH-dependent proteins that respond in a growth phase independent manner in Campylobacter jejuni reference strain NCTC 11168. NCTC 11168 was grown within its pH physiological normal growth range (pH 5.8, 7.0 and 8.0, µ = â¼0.5 h-1) and exposed to pH 4.0 shock for 2 h. It was discovered that gluconate 2-dehydrogenase GdhAB, NssR-regulated globins Cgb and Ctb, cupin domain protein Cj0761, cytochrome c protein CccC (Cj0037c), and phosphate-binding transporter protein PstB all show acidic pH dependent abundance increases but are not activated by sub-lethal acid shock. Glutamate synthase (GLtBD) and the MfrABC and NapAGL respiratory complexes were induced in cells grown at pH 8.0. The response to pH stress by C. jejuni is to bolster microaerobic respiration and at pH 8.0 this is assisted by accumulation of glutamate the conversion of which could bolster fumarate respiration. The pH dependent proteins linked to growth in C. jejuni NCTC 11168 aids cellular energy conservation maximising growth rate and thus competitiveness and fitness.
Asunto(s)
Campylobacter jejuni , Campylobacter jejuni/genética , Campylobacter jejuni/química , Proteínas Bacterianas/metabolismo , Regulación Bacteriana de la Expresión Génica , Proteómica , Concentración de Iones de HidrógenoRESUMEN
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.
Asunto(s)
Infecciones por Campylobacter , Campylobacter jejuni , Animales , Ciprofloxacina/farmacología , Eritromicina/farmacología , Antibacterianos/farmacología , Campylobacter jejuni/genética , Aves de Corral , Mataderos , Brasil , ARN Ribosómico 23S/genética , Infecciones por Campylobacter/microbiología , Farmacorresistencia Bacteriana/genética , Macrólidos/farmacología , Pollos/microbiología , Pruebas de Sensibilidad MicrobianaRESUMEN
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.
Asunto(s)
Infecciones por Campylobacter , Campylobacter jejuni , Embrión de Pollo , Animales , Humanos , Campylobacter jejuni/genética , Infecciones por Campylobacter/microbiología , Biopelículas , Adaptación Fisiológica , MetabolómicaRESUMEN
Bacillus subtilis (BS) has been used as an excellent probiotic; however, some BS strains seem to be opportunist pathogens or do not present inhibitory effects in the pathogenic bacteria, so the characterization of BS strains for use in animals is mandatory. This study aimed to select nonpathogenic strains of BS, which can inhibit Salmonella spp., avian pathogenic Escherichia coli (APEC), and Campylobacter jejuni (CJ) using a chicken embryo as a model. We tested nine (9) strains of BS isolated from several sources (named A to I) in in vitro by tests of mucin degradation activity, haemolytic activity, apoptosis, and necrosis in fibroblasts from chickens. After the in vitro test, we tested the remaining seven (7) strains (strains A to G) in a chicken embryo (CE) as an in vivo model and target animal. We inoculated 3 log CFU/CE of each strain via allantoic fluid at the 10th day postincubation (DPI). Each treatment group consisted of eight CEs. At the 17th DPI we checked CE mortality, gross lesions, CE weight, and whether BS strains were still viable. To perform the cytokine, total protein, albumin, and reactive C protein analysis, we collected the CE blood from the allantoic vessel and intestine fragments in the duodenum portion for histomorphometric analysis. After the results in CEs, we tested the inhibition capacity of the selected BS strains for diverse strains of Salmonella Heidelberg (SH), S. Typhimurium (ST), S. Enteritidis (SE), S. Minnesota (SM), S. Infantis (SI), Salmonella var. monophasic (SVM), APEC and C. jejuni. After the in vitro trial (mucin degradation activity, haemolytic activity, apoptosis, and necrosis), we removed two (2) strains (H and I) that showed ß-haemolysis, mucin degradation, and/or high apoptosis and necrosis effects. Although all strains of BS were viable in CEs at the 17th DPI, we removed four (4) strains (A, B, D, F) once they led to the highest mortality in CEs or a high albumin/protein ratio. C. jejuni inoculated with strain G had greater weight than the commercial strain, which could be further used for egg inoculation with benefits to the CE. From the tests in CEs, we selected the strains C, E, and G for their ability to inhibit pathogenic strains of relevant foodborne pathogens. We found that the inhibition effect was strain dependent. In general, strains E and/or G presented better or similar results than commercial control strains in the inhibition of SH, ST, SI, APEC, and two (2) strains of CJ. In this study, we selected BS strains C, E and G due to their in vitro and in vivo safety and beneficial effects. In addition, we emphasize the value of CE as an in vivo experimental model for assessing BS's safety and possible benefits for poultry and other animals.
Asunto(s)
Campylobacter jejuni , Infecciones por Escherichia coli , Probióticos , Embrión de Pollo , Animales , Pollos/microbiología , Bacillus subtilis , Escherichia coli , Mucinas , NecrosisRESUMEN
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.
Asunto(s)
Infecciones por Campylobacter , Campylobacter jejuni , Campylobacter , Gastroenteritis , Animales , Infecciones por Campylobacter/epidemiología , Infecciones por Campylobacter/microbiología , Infecciones por Campylobacter/veterinaria , Campylobacter jejuni/genética , Ácido Edético/análogos & derivados , Humanos , Aves de Corral/microbiología , Prevalencia , Factores de Virulencia/genéticaRESUMEN
Bovine by-products, such as liver, could be an underestimated source of Campylobacter jejuni. Therefore, our aims were to evaluate the occurrence of C. jejuni and other Campylobacteraceae in retail beef liver and characterize their antibiotic resistance (ciprofloxacin, tetracycline, erythromycin and gentamicin) and potential genetic relationship by flagellin gene restriction fragment length polymorphism (flaA-RFLP) and multilocus sequence typing with clinical strains. Seventy-six out of 206 samples (36·9%) were positive for Campylobacter and related organisms. Arcobacter butzleri was the most frequently isolated species (21·8%), followed by C. jejuni (9·7%), C. fetus (7·8%) and C. coli (1%). The C. jejuni strains showed resistance to tetracycline (17·2%) or ciprofloxacin (6·9%), with only one strain resistant to both antibiotics. Meanwhile, 8·3% of ciprofloxacin resistance was observed in C. fetus. The other species showed no resistance. Most of the clonal complexes (CC) in which the C. jejuni genotypes were grouped (CC-21, 42, 48 and 52), coincided with genotypes of clinical strains previously reported in Chile. As such, this study provides evidence that beef liver could be an underestimated route for resistant C. jejuni to humans. Further studies should assess whether this food could play a role in the transmission of other emerging Campylobacteraceae such as those reported here.
Asunto(s)
Infecciones por Campylobacter , Campylobacter jejuni , Campylobacteraceae , Animales , Bovinos , Antibacterianos/farmacología , Infecciones por Campylobacter/epidemiología , Infecciones por Campylobacter/veterinaria , Campylobacter jejuni/genética , Ciprofloxacina/farmacología , Hígado , Pruebas de Sensibilidad Microbiana , TetraciclinaRESUMEN
Campylobacter jejuni infection is considered the most frequent factor associated with Guillain-Barré syndrome (GBS). In 2019, a large outbreak of GBS was detected in Peru, being associated with C. jejuni detected in stool samples from these patients. The aim of this study was to determine the molecular epidemiology of C. jejuni strains (ST-2993) associated with a large GBS outbreak in Peru. In this study, 26 C. jejuni strains belonging to the ST-2293, obtained from 2019 to 2020, were sequenced using Illumina technology. Five low-quality sequences were removed using bioinformatics, and 21 genomes (17 clinical strains and 4 chicken strains) were considered in the phylogenetic analysis and comparative genomics. Phylogenetic reconstruction, including genomes from international databases, showed a connection between Peruvian and Chinese GBS strains, both of them having lipooligosaccharides (LOS) locus genes related to molecular mimicry with gangliosides in peripheral nerves. Also, ST-2993 was detected in Amazon strains recovered many years before the 2019 outbreak, but with no epidemiological connection with GBS. Besides, a close relationship between human and chicken C. jejuni strains indicated chicken as one of the probable reservoirs. Finally, comparative genomics revealed differences between Chinese and Peruvian strains, including the presence of a prophage inserted into the genome. In conclusion, C. jejuni ST-2993 strains recovered from the GBS outbreak are closely related to Peruvian Amazon strains. Moreover, ST-2993 has been circulated in Peru since 2003 in the Peruvian Amazonia, showing the necessity to reinforce the epidemiological surveillance of C. jejuni to improve the prevention and control of future GBS outbreaks. IMPORTANCE This article describes the molecular epidemiology of C. jejuni strains (ST-2993) associated with a large Guillain-Barré Syndrome (GBS) outbreak in Peru, sequencing several strains recovered from GBS patients and chickens from 2019 to 2020. Phylogenetic analysis showed a connection between Peruvian and Chinese GBS strains, both of them having lipooligosaccharides (LOS) locus genes related to molecular mimicry with gangliosides in peripheral nerves. Also, ST-2993 strains were detected in isolates recovered many years before the 2019 outbreak, but with no epidemiological connection with GBS. Besides, a close relationship between human and chicken strains indicated those animals as a probable reservoir. This information will help to understand the real situation of GBS in Peru and its causal agent, C. jejuni ST-2993, showing the necessity to increase epidemiological tracking of these kinds of pathogens to detect them and avoid GBS outbreaks in the future.
Asunto(s)
Infecciones por Campylobacter , Campylobacter jejuni , Síndrome de Guillain-Barré , Humanos , Animales , Campylobacter jejuni/genética , Perú/epidemiología , Epidemiología Molecular , Filogenia , Pollos , Síndrome de Guillain-Barré/epidemiología , Infecciones por Campylobacter/epidemiología , Gangliósidos , Brotes de EnfermedadesRESUMEN
This study aimed to identify molecular markers associated with antimicrobial resistance and genotype isolates of Campylobacter spp. from broiler and swine flocks due to its importance to one-health. C. jejuni (n=27) and C. coli (n = 35) strains were screened for the antimicrobial genetic markers C257T in gyrA, A2074C and A2075G in 23S rRNA, CmeABC, ermB, tetO and blaOXA61 by PCR. Fifteen strains had SVR-flaA and porA genes sequenced to evaluate their genetic diversity. Among C. jejuni strains 62.96% had C257T mutation and only one strain had A2075G mutation. CmeA, cmeB, cmeC, tetO and blaOXA61 were detected respectively in 92.59%, 100%, 100%, 85.19%, 85.19% of the strains. All C. coli had C257T mutation; 48.75% had A2075G and cmeA, cmeB, cmeC, tetO, blaOXA61 were detected in 8.57%, 94.29%, 91.43%, 91.43%, 80%, respectively. Twelve porA and SVR-flaA alleles were detected, with a Simpson index of diversity value of 0.962.
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Antiinfecciosos , Infecciones por Campylobacter , Campylobacter coli , Campylobacter jejuni , Enfermedades de los Porcinos , Animales , Antibacterianos/farmacología , Antiinfecciosos/farmacología , Brasil/epidemiología , Campylobacter coli/genética , Infecciones por Campylobacter/epidemiología , Infecciones por Campylobacter/veterinaria , Pollos , Farmacorresistencia Bacteriana/genética , Genotipo , PorcinosRESUMEN
Campylobacteriosis is one of the most common types of bacterial gastroenteritis affecting humans, and poultry is considered a major source of the causative organism, Campylobacter spp. Broilers may arrive contaminated at slaughterhouses, and transport crates could be considered a potential source of contamination. Thus, cleaning and disinfection procedures are crucial to avoid cross-contamination among flocks. Despite its public health importance in Latin American countries, virulence factors of Campylobacter jejuni remain poorly studied in this region. Thus, this study aimed to: 1) determine the occurrence of contaminated crates at a poultry slaughterhouse, 2) compare the contamination before and after the cleaning and disinfection procedures, and 3) detect virulence-associated genes in C. jejuni strains by PCR. Campylobacter spp. were recovered from 8 of the 10 flocks evaluated, and C. jejuni was detected as the main species. There was no significant difference in the Campylobacter detection or quantification between crates at the reception platform and crates after the cleaning/disinfection processes. However, crates after 24 h of natural drying, presented a significant (P < 0.05) lower amount of Campylobacter cells than before the cleaning and disinfection processes. A negative relationship (R2 = 0.210, P = 0.045) between environmental conditions and Campylobacter quantification was found for transport crates after 24 h of natural drying. There was no significant difference (P > 0.05) in the detection of two C. jejuni virulence genes, flaA (encode a major flagellin protein) and cadF (encode an adhesion and fibronectin-binding protein), among various stages of the cleaning and disinfection processes. Our results demonstrate the high contamination levels of Campylobacter strains in broiler flocks and the potential involvement of poultry transport crates in transmitting these bacteria. This study also suggests that ineffective cleaning and disinfection procedures can increase Campylobacter contamination and facilitate the spread of bacteria in poultry establishments.
Asunto(s)
Infecciones por Campylobacter , Campylobacter jejuni , Campylobacter , Enfermedades de las Aves de Corral , Mataderos , Animales , Campylobacter/genética , Infecciones por Campylobacter/epidemiología , Infecciones por Campylobacter/prevención & control , Infecciones por Campylobacter/veterinaria , Campylobacter jejuni/genética , Pollos/microbiología , Desinfección/métodos , Microbiología de Alimentos , Aves de Corral/microbiología , Enfermedades de las Aves de Corral/microbiologíaRESUMEN
A wide variety of microorganism species have been used as probiotics to improve the intestinal microbial balance, control and prevent the colonization of pathogenic bacteria and promote growth in animal production. Based on the various beneficial factors of probiotic agents, this study aimed to evaluate the effectiveness of three different formulations of probiotics strains through the analysis of antagonistic capacity against common pathogens from chickens gastrointestinal tract. Three formulations composed by Bacillus, Lactobacillus, Saccharomyces, Enterococcus, Bifidobacterium and Pediococcus were tested in vitro by the method of probiotic culture spot in plates seeded with inocula of Escherichia coli, Salmonella Heidelberg and Campylobacter jejuni. The inhibition halos were measured and classified according to the degree of pathogenic bacteria inhibition. The formulation containing an association among Bacillus, Lactobacillus and Saccharomyces presented better results when compared to the other formulations.