Genomic characterisation of Campylobacter jejuni Cj26: A high-level ciprofloxacin/erythromycin-resistant strain isolated from a poultry carcass in southern Brazil.

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.

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.

Molecular markers associated with antimicrobial resistance and genotypes of Campylobacter jejuni and Campylobacter coli isolated from broiler and swine flocks in southeast Brazil.

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.

Virulence-Associated Genes and Antimicrobial Resistance of Aeromonas hydrophila Isolates from Animal, Food, and Human Sources in Brazil.

Aeromonads are natural inhabitants of aquatic environments and may be associated with various human or animal diseases. Its pathogenicity is complex and multifactorial and is associated with many virulence factors. In this study, 110 selected Aeromonas hydrophila isolates isolated from food, animals, and human clinical material from 2010 to 2015 were analyzed. Antimicrobial susceptibility testing was performed by the disk diffusion method, and polymerase chain reaction was conducted to investigate the virulence genes hemolysin (hlyA), cytotoxic enterotoxin (act), heat-labile cytotonic enterotoxin (alt), aerolysin (aerA), and DNase-nuclease (exu). At least 92.7% of the isolates had one of the investigated virulence genes. Twenty different virulence profiles among the isolates were recognized, and the five investigated virulence genes were observed in four isolates. Human source isolates showed greater diversity than food and animal sources. Antimicrobial resistance was observed in 46.4% of the isolates, and multidrug resistance was detected in 3.6% of the isolates. Among the 120 isolates, 45% were resistant to cefoxitin; 23.5% to nalidixic acid; 16.6% to tetracycline; 13.7% to cefotaxime and imipenem; 11.8% to ceftazidime; 5.9% to amikacin, gentamicin, and sulfamethoxazole-trimethoprim; and 3.9% to ciprofloxacin and nitrofurantoin. Overall, the findings of our study indicated the presence of virulence genes and that antimicrobial resistance in A. hydrophila isolates in this study is compatible with potentially pathogenic bacteria. This information will allow us to recognize the potential risk through circulating isolates in animal health and public health and the spread through the food chain offering subsidies for appropriate sanitary actions.

Detection of fluoroquinolone resistance by mutation ingyrA gene of Campylobacterspp. isolates from broiler and laying (Gallus gallus domesticus) hens,from Rio de Janeiro State, Brazil / Detecção de resistência às fluoroquinolonas através da mutação no gene gyrA em Campylobacter spp. isolados de frangos de corte e galinhas (Gallus gallus domesticus) poedeiras, no Estado do Rio de Janeiro, Brasil

Poultry are considered to be the main reservoir of Campylobacterspp. bacteria, an important pathogen for humans. Many studies have reported a rapid selection of fluoroquinolone-resistant strains following the widespread use of these antimicrobials in poultry production and human medicine. The main mechanism of fluoroquinolone resistance in Campylobacteris a mutation in the Quinolone Resistance Determinant Region (QRDR) in the gyrA gene, which codes for the subunit of the enzyme DNA gyrase, the target for fluoroquinolone. The aim of this study was to investigate the mutation in QRDR in the gyrA gene of Campylobacterstrains previously isolated from broiler carcasses and feces of laying hens. Thirty-eight strains of C. jejuniand 19 C. colistrains (n=57), previously characterized as resistant to ciprofloxacin and enrofloxacin by the disk diffusion method and minimum inhibitory concentration (MIC), were selected. For detection of the mutation, a fragment of 454pb QRDR in the gyrA gene was used for direct sequencing. All strains presented the QRDR mutation in the gyrA gene at codon 86 (Thr-86-Ile), which confers resistance to fluoroquinolones. Other known silent mutations were observed. This genotypic characterization of fluoroquinolone resistance inCampylobacterstrains has confirmed the prior phenotypic detection of the resistance. The Thr-86-Ile mutation was observed in all samples confirming that this is the predominant mutation in enrofloxacin and ciprofloxacin resistant strains of C. jejuniand C. coli.

As aves são consideradas o principal reservatório deCampylobacterspp., um importante patógeno para humanos e muitos estudos têm relatado uma rápida seleção de cepas resistentes às fluoroquinolonas após o uso destes antimicrobianos na produção avícola e na medicina humana. O principal mecanismo de resistência às fluoroquinolonas em Campylobacterconsiste na mutação na Região Determinantes de Resistência às Quinolonas (RDRQ) do gene gyrA, que codifica para a subunidade A da enzima DNA girase, alvo das fluoroquinolonas. O objetivo deste estudo foi investigar a mutação na RDRQ do gene gyrA em cepas de Campylobacterpreviamente isolados de carcaças de frangos de corte e fezes de galinhas poedeiras. Foram selecionadas 38 cepas de C. jejunie 19 cepas de C. coli(n=57), previamente caracterizadas como resistentes à ciprofloxacina e enrofloxacina, pelo método da difusão em disco e pela determinação da concentração inibitória mínima. Para detecção da mutação, foi utilizado sequenciamento direto de um fragmento de 454pb da RDRQ do gene gyrA gerado por PCR. Todas as cepas apresentaram a mutação na RDRQ do gene gyrA no códon 86 (Tre-86-Ile), que confere resistência às fluoroquinolonas e outras mutações silenciosas foram observadas. A caracterização genotípica da resistência às fluoroquinolonas em Campylobacterconfirmou a prévia detecção fenotípica dessa resistência e a mutação Tre-86-Ile foi observada na totalidade das amostras, comprovando ser esta a mutação predominante em cepas de C. jejunie C. coliresistentes à enrofloxacina e ciprofloxacina.