Detection of Mycoplasma spp. in free-living seabirds.

This study aimed to investigate the presence of Mycoplasma spp. and identify the species of mycoplasma isolates obtained from seabirds found on Brazilian coastal beaches. Tracheal and cloacal swab samples were collected from 50 seabirds rescued by three conservation and marine animal rehabilitation centers located in Brazil. The tracheal and cloacal samples were subjected to mycoplasma culture and the isolates were identified through PCR. A "Mollicutes-specific" 16S rRNA PCR reaction was employed for triage. Four species-specific PCR reactions were used to detect Mycoplasma gallisepticum, Mycoplasma synoviae, Mycoplasma meleagridis, or M. gallinarum. The Mollicutes positive and species negative samples were submitted do 16S rRNA sequencing. Eighteen (36%) of 50 seabirds tested positive for mycoplasma by culture. In the PCR for the genus, 28 (56%) of 50 seabirds were positive for Mycoplasma spp., with 13 (26%) detected in the trachea, one (2%) in the cloaca, and 14 (28%) in both sites. In the species-specific PCR, M. gallisepticum was detected in 17.8%, and M. meleagridis in 17.8%. Both species were detected in 14.3%. Of the isolates not characterized at species level, we obtained ten sequences and they were divided into three clusters. The first cluster was closely related to M. meleagridis, the second to M. synoviae, and the third grouped M. tully, M. gallisepticum, and M. imitans. Four and five of nine species of seabirds studied had mycoplasma detected by culture or PCR, respectively. Mycoplasmas were found in the majority of the animals studied, with the highest prevalence proportionally found in Sula leucogaster, and the lowest in Fregata magnificens. The phylogenetic analysis identified Mycoplasma spp. adapted to aquatic birds.

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.

Detection of Mycoplasma species at various anatomical sites of dogs from different types of kennels.

This work aimed to detect Mycoplasma cynos, M. canis, M. edwardii, and M. molare in different types of kennels, in addition to evaluating their distribution in different colonization sites. The dogs belonged to different kennels from armed forces (n = 3), shelters (n = 3), and commercial purposes (n = 2). Samples of the oropharynx, genital mucosa, and ear canal were collected from each dog (n = 98), totaling 294 samples. Aliquots were submitted to isolation and the samples confirmed as Mycoplasma spp. were subjected to conventional PCR for M. canis and multiplex PCR for M. edwardii, M. molare, and M. cynos detection. Of the 98 dogs studied, 63.3% (62) were positive in at least one anatomical site evaluated for Mycoplasma spp. Among the 111 anatomical sites positive for Mycoplasma spp., M. canis, M. edwardii, and M. molare were detected in 29.7% (33/111), 40.5% (45/111), and 2.70% (3/111), respectively. No animal was positive for M. cynos.

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.

First molecular detection of Spiroplasma spp. in ticks from horses in Brazil.

The class Mollicutes comprises microorganisms that lack a cell wall, highly dependent on their host to survive. Within Mollicutes, the genus Spiroplasma comprises motile helical microorganisms associated with various insects and other arthropods. This study aimed to detect and characterize Mollicutes microorganisms in ticks of different species of veterinary importance, using molecular techniques. These ticks were collected from dogs, cats, cattle, and horses from Rio de Janeiro's metropolitan regions. They were morphologically classified and pooled according to their species for subsequent DNA extraction. These samples were tested by PCR using class Mollicutes-specific primers (16S rRNA) and positive amplicons were sequenced. The obtained DNA sequences were compared with other Mollicutes sequences deposited in GenBank. We found that four out of 745 (0.54%) of the tick pools were positive for members of the class Mollicutes, identified as Spiroplasma spp.; of the positive pools, one comprised Amblyomma sculptum adults and three comprised Dermacentor nitens nymphs. The present study describes Spiroplasma spp. in ticks in Brazil for the first time. Nevertheless, due to few reports on these microorganisms, further studies on epidemiology, virulence, and pathogenicity are needed.

Mycoplasma spp. in captive snakes (Boa constrictor and Bothrops atrox) from Brazil / Mycoplasma spp. em cobras de cativeiro (Boa constrictor e Bothrops atrox) do Brasil

ABSTRACT: Although rare, mycoplasmas are included among the causes of respiratory diseases in reptiles and, in the order Squamata, three reports of these microorganisms causing diseases in pythons have already been reported. This study aimed to evaluate the occurrence of Mycoplasma species in captive snakes. A total of 26 snakes of the families Pythonidae (13), Boidae (7), Viperidae (5) and Colubridae (1) from RioZoo, Brazil, were evaluated. Animals were examined to determine clinical signs consistent with any infectious disease. Tracheal swab samples from snakes were collected in Frey medium and analyzed for the presence of Mycoplasma spp.by isolation and a genus-specific PCR. DNA sequencing analyses of six positive samples by PCR were carried out to identify the species. Using isolation 19.23% (5/26) was positive, while 65.38% (17/26) of the animals were positive by PCR. Based on the analyses of the six sequences obtained, there was similarity with a Mycoplasma spp. previously described in a phyton and, M. agassizii and M. testudineum reported in chelonians. This is the first report of Mycoplasma spp. in animals of the families Boidae and Viperidae. Mycoplasma spp. were detected in snakes with and without clinical signs. The mycoplasmas reported resented identity (range, 95% to 100%) to others already described in reptiles. There was no relationship between the presence of Mycoplasma spp. and clinical signs.

RESUMO: Embora raros, os micoplasmas estão incluídos entre as causas de doenças respiratórias em répteis e, na ordem Squamata, já foram realizados três relatos destes microrganismos causando doença em pítons. Este estudo teve como objetivo avaliar a ocorrência de espécies de Mycoplasma em serpentes em cativeiro. Foram avaliadas 26 serpentes das famílias Pythonidae (13), Boidae (7), Viperidae (5) e Colubridae (1) do RioZoo, Brasil. Os animais foram examinados para determinar sinais clínicos consistentes com qualquer doença infecciosa. Amostras de swab traqueal de cobras foram coletadas em meio Frey e analisadas por isolamento microbiológico e pela técnica da PCR para identificar Mycoplasma spp. As amostras positivas para o gênero Mycoplasma spp. foram submetidas ao sequenciamento genético para identificação das espécies. No isolamento, 19,23% (5/26) foram positivos, enquanto 65,38% (17/26) dos animais foram positivos por PCR. Com base nas análises das seis sequências obtidas, houve similaridade com o Mycoplasma spp. descrito anteriormente em um píton e M. agassizii e M. testudineum encontrados em quelônios. Este é o primeiro relato de Mycoplasma spp. em animais das famílias Boidae e Viperidae. Mycoplasma spp. foi detectado em serpentes com e sem sinais clínicos. Os micoplasmas encontrados apresentaram semelhança genética com outros já descritos em répteis. Não houve relação entre a presença de Mycoplasma spp. e sinais clínicos.