Camila Chioda de Almeida

Multidrug resistance is a significant threat to global public health. This study aimed to detect and characterize antimicrobial-resistant microorganisms in swine feces and carcasses obtained from slaughterhouses. A total of 214 samples were collected, comprising 111 swine feces and 103 from carcasses, across two different slaughterhouses. Polymerase chain reaction was used to detect antimicrobial resistance genes, identifying 22 resistant isolates that were subsequently confirmed to be Escherichia coli. Pulsed-field gel electrophoresis was performed on these 22 isolates to investigate their genetic similarity. The Kirby–Bauer disk diffusion method characterized phenotypic resistance to different antimicrobials. The blaCTX-M-1 gene was detected in all 22 E. coli isolates. Additionally, 90.9% of the strains were considered multidrug-resistant, exhibiting resistance to at least three different antimicrobial classes. The isolates exhibited high genetic diversity. The presence of these multidrug-resistant bacteria in swine and animal-derived foods emphasizes the importance of sanitation measures during production to protect public health.

O Brasil é um dos maiores exportadores de produtos derivados de frango para o mundo e a preocupação de consumidores em relação à contaminação de alimentos com bactérias multiresistentes e capazes de causar doenças vêm aumentanto com os anos, sendo um alerta à saúde púbica. O estudo teve como objetivo caracterizar cepas de Escherichia coli e Klebsiella pneumoniae resistentes a Beta Lactâmicos isoladas da cadeia produtiva de frango e amostras clínicas humanas (cloaca, carne de frango e fezes humanas). Foram utilizados 36 isolados, sendo 28 Escherichia coli e 8 Klebsiella pneumoniae portadores do gene blaCTX-M-15, obtidos de amostras de fezes e carne de aves de corte, provenientes de criadouros e abatedouros avícolas, respectivamente, e amostras de fezes humanas de laboratório de análises clínicas humano. Todos os estabelecimentos eram localizados no Estado de São Paulo, Brasil. A caracterização de genes de virulência foi realizada por meio de PCR (Reação em Cadeia da Polimerase), a resistência a antimicrobianos pelo método de difusão em disco, sorotipagem por teste de aglutinação e patogenicidade por meio de teste de mortalidade in vivo em pintainhos de um dia de vida. Os isolados demonstraram uma alta frequência de genes de virulência relacionados à Escherichia coli, como iutA, iss, hlyF, ompT e iroN. O gene mrkD também foi encontrado em grande parte dos isolados. Todos os isolados foram resistentes a pelo menos três diferentes classes de antimicrobianos e 21.4% (6) das cepas de Escherichia coli demonstraram alta patogenicidade no teste in vivo. Esses resultados evidenciaram o potencial aumento do gene blaCTX-M-15 associado a genes de virulência e multiresistência em Escherichia coli e Klebsiella pneumoniae, ambas enterobactérias podem ser encontradas em fezes de aves e possivelmente contaminar produtos como a carne, aumentando o risco de infecção em humanos. Isso destaca a necessidade de cuidados sanitários rigorosos na produção de animais e alimentos no Brasil.

Staphylococcus aureus is an important agent in bovine mastitis, and some specific virulence factors may be implicated in this disease. Therefore, this study aimed to investigate the importance of the presence of coagulase, superantigens, genotypic and phenotypic resistance, and pulsotypes in 65 S. aureus isolates from bovine clinical and subclinical mastitis in the Southeast of Brazil. A high correlation was observed between the genes coa and see, as well as between the sei and the see and seh. High resistance rates were observed for penicillin (95.4%), tetracycline (89.2%), cefoxitin (86.1%), oxacillin (84.6%), erythromycin (84.6%), clindamycin (84.6%), chloramphenicol (81.5%), ceftriaxone (80.0%), and ampicillin (80.0%). Analysis of antimicrobial resistance profiles showed that 89.2% of isolates were multi-drug-resistant. No mecA-positive S. aureus isolates were detected. It was observed that seven isolates were resistant to all the β-lactam tested while being susceptible to cefoxitin, which could be indicative of borderline methicillin resistance in S. aureus⁠. High genetic diversity with no specific virulence profile being predominant was observed. Thus, this study observed a high correlation between the coa and enterotoxins genes, and demonstrates that there is no predominant pulsotype causing intramammary infection and that there is a high rate of antibiotic resistance in S. aureus isolates from dairy farms in the southeast regions of Brazil.

Mercury (Hg) is one of the most toxic metals and is not essential for any organism. In this study, the potential of maize plants in association with bacteria to treat oxisol contaminated with Hg (II) was evaluated. The experiment was conducted in a controlled environment, and pots with 2 kg of oxisol were contaminated with HgCl2 solution at a dose of 36 mg kg-1 of Hg in a 7x4 factorial scheme: control (soil without Hg(II) and microorganisms), T2= (soil with Hg(II) and without microorganisms), and T3= soil with Hg(II) + Enterobacter cloacae, T4= Hg(II) + Bacillus subtilis, T5= Hg(II) + Enterobacter sp., T6= Hg(II) + Staphylococcus epidermidis, and T7= Hg(II) + Bacillus sp. Total Hg quantification was performed by atomic absorption spectrophotometry. At the end of the experiment, the soil pH was significantly lower (0.3 to 0.4 pH unit) in the T2 (no inoculation), Enterobacter cloacae, Enterobacter sp. and Bacillus sp. treatments. Neither contamination of soil with Hg nor plant associations with bacteria led to differences in the root dry mass of maize plants. Maize plants associated with Staphylococcus epidermidis and Bacillus sp. bacteria had lower shoot biomass (71 and 50%) compared to the treatment 2. The best remedial effect was observed with the association of maize plants with Bacillus sp., which recovered 19.67% of Hg(II) in the soil when compared to control and treatment 2 and treatment with B. subtilis. The recommendation is the use of B. subtilis to decrease the toxicity caused by Hg(II).

Staphylococcus aureus is a commonly reported cause of buffalo mastitis. However, its prevalence may be overestimated. The aim of this study was to compare S. aureus identification by conventional phenotypic and genotypic assays versus Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry (MALDI-TOF MS) and novel real-time quantitative PCR tests for the cytochrome oxidase subunit D II (cydB) and staphylocoagulase (coa) genes. From 408 samples obtained from buffalo milk/milking environment, 32 putative S. aureus strains were identified based on characteristic growth on Baird Parker agar, positive catalase reaction, ability to clot rabbit plasma, and positive Sa442 PCR assay. However, in further testing, only 10 of these strains were positive in latex agglutination tests and by MALDI-TOF MS, only eight of the 32 strains were S. aureus while the rest were S. chromogenes (19), S. agnetis (3), S. cohnii (1), or S. xylosus (1). All eight strains identified as S. aureus by MALDI-TOF analysis and confirmed by 16S RNA gene sequencing were positive in a S. aureus-specific cydB PCR test. As well, 7/8 S. aureus strains were PCR positive in a real-time coa PCR test as were 2/69 S. chromogenes and the lone S. xylosus strain tested.

The interest in production of buffalo milk is increasing in Brazil due to its physico-chemical and nutritional characteristics. However, just as sheep, goats and cows are susceptible to mastitis, so are buffaloes, which is mainly caused by Staphylococcus aureus. In this regard, biofilm formation, the ability to escape host immune defense and virulence factor, is presumably a key factor for acute and chronic intra-mammary infection in buffaloes. In this study, biofilm forming capabilities and virulence genes were evaluated in S. aureus isolated from buffalo milk and milking environments, using phenotypic and genotypic assays. Thirty two S. aureus strains isolated from buffalo milk, milking machines and milkers’ hands were obtained from a farm in Analândia, São Paulo State, Brazil. Samples were collected in April, June, October and November 2013. These strains were tested for the presence of sa442, icaA, icaD, clfA, clfB, sarA and hla genes, slime production using Congo Red Agar (CRA), and biofilm formation using microtiter assay. All samples of S. aureus were positive for CRA and microtiter assay. Although, icaA and icaD genes were simultaneously detected in nine of the 32 samples, none of the samples were positive for icaA; only seven were positive for icaD gene. This suggests that other factors may be involved in biofilm formation. Seventeen strains of S. aureus were positive for sarA gene, nine for clfA gene, 16 for clfB and 16 for hla. A great variability in SmaI restriction profiles of S. aureus strains was observed. Thirty isolates were typified and two strains were not by SmaI restriction. In addition, 18 pulse types were detected. It is hypothesized that biofilm can be produced by the expression of icaD gene only. Our findings suggest that S. aureus strains from buffalo milk and milking environment are similar, which contradict the findings obtained from bovine strains. This behavior may contribute to the persistence of mastitis in buffalo caused by S. aureus, which results in a potential zoonotic problem. Our results may bring new insights into the development of novel strategies for the prevention and treatment of bubaline mastitis. Key words: Staphylococcus aureus, buffalo milk, biofilm, milking machine, milkers’ hands, utensils, mastitis.