Molecular Characterization of Antimicrobial Resistance and Virulence Genes of Bacterial Pathogens from Bovine and Caprine Mastitis in Northern Lebanon

Mastitis is an infectious disease encountered in dairy animals worldwide that is currently a growing concern in Lebanon. This study aimed at investigating the etiology of the main mastitiscausing pathogens in Northern Lebanon, determining their antimicrobial susceptibility profiles, and identifying their antimicrobial resistance (AMR) genes. A total of 101 quarter milk samples were collected from 77 cows and 11 goats presenting symptoms of mastitis on 45 dairy farms. Bacterial identification was carried out through matrix-assisted laser desorption/ionization-time of flight mass spectrometry. Antimicrobial susceptibility was tested by disc diffusion and broth microdilution methods. Molecular characterization included polymerase chain reaction (PCR) screening for genes encoding extended-spectrum beta-lactamases (ESBLs) and plasmid-mediated AmpC among Enterobacterales isolates, and virulence factors among Staphylococcus isolates. Escherichia coli isolates were subjected to phylogenetic typing by a quadruplex PCR method. The most frequently identified species were Streptococcus uberis (19.2%), Streptococcus agalactiae (15.1%), E. coli (12.3%), and Staphylococcus aureus (10.96%). Gram-positive bacteria were resistant to macrolides and etracycline, whereas gram-negative bacteria displayed resistance to ampicillin and tetracycline. Two ESBL genes, blaTEM (83.3%) and blaOXA (16.7%), and one AmpC beta-lactamase gene, blaCMY-II (16.7%), were detected among six E. coli isolates, which mainly belonged to phylogenetic group B1. Among Staphylococcus spp., the mecA gene was present in three isolates. Furthermore, four isolates contained at least one toxin gene, and all S. aureus isolates carried the ica operon. These findings revealed the alarming risk of AMR in the Lebanese dairy chain and the importance of monitoring antimicrobial usage

Investigação epidemiológica de patógenos bacterianos associados a manifestações clinicas no trato gastrintestinal

-As doenças do trato gastrintestinal têm uma distribuição global, afetando principalmente crianças em todo mundo. Especialmente nos países em desenvolvimento, considerando-se a etiologia bacteriana, estas doenças estão associadas com altas taxas de morbidade e mortalidade. Neste sentido, patógenos entéricos tais como Escherichia coli (EPEC, ETEC, EIEC, EAEC e EHEC) são reconhecidos como importantes agentes etiológicos, embora outros grupos microbianos possam estar envolvidos. Dentro de um projeto amplo, neste estudo nosso objetivo foi a detecção genética e prevalência de Escherichia coli enteropatogênica (EPEC, ETEC, EIEC, EAEC e EHEC) em amostras fecais de crianças (entre 0 a 5 anos de idade) apresentando diarréia em Juiz de Fora, Minas Gerais. A coprocultura foi realizada em meio seletivo EMB (Eosin Methylene Blue) a partir de 52 amostras fecais, e E. coli foi identificada presuntivamente por técnicas bioquímicas convencionais. A identidade bacteriana foi confirmada por amplificação específica da região de DNA codificadora do RNA ribossomal 16S, por PCR. As estirpes enteropatogênicas foram caracterizadas por técnica de PCR-multiplex, utilizando como alvo genes de virulência específicos. Das 52 amostras fecais analisadas, 109 E. coli foram identificadas a partir dos testes bioquímicos convencionais e 108 foram confirmadas por PCR específico, indicando uma correlação de 99,08% entre as duas metodologias. As estirpes patogênicas de E. coli foram detectadas em 36,51% das amostras fecais, e foram distribuídas da seguinte forma: EAEC (17,30%), EPEC (11,53%), EHEC (5,76%), ETEC (1,92%), e não foram detectadas estirpes de EIEC. De acordo com os nossos resultados, E. coli patogênica pode ser considerada altamente prevalente entre a população avaliada, o que representa uma grande importância na etiologia das infecções gastrintestinais na infância, em nossa região. A técnica de PCR-multiplex mostrou ser rápida, útil e sensível, e que permitiu a detecção destes patógenos entéricos, uma vez que métodos sorológicos convencionais não são eficientes, de acordo com a literatura

Pé diabético: análise microbiológica e atenção farmacêutica ao paciente morador de Juiz de Fora

-Diabetes Mellitus é uma enfermidade crônica que predispõe a uma série de complicações, sendo o pé diabético a mais relevante diante do impacto socioeconômico nos sistemas de saúde, em todo mundo. O estudo objetivou avaliar a ocorrência de bactérias e fungos em lesões de pé, o perfil de susceptibilidade a drogas antimicrobianas, a terapia medicamentosa bem como a inter-relação de variáveis que levam à ocorrência do pé diabético. Trata-se de um estudo descritivo e observacional de 10 pacientes diabéticos portadores de lesão dos pés do Programa de Atendimento Interdisciplinar ao Diabético do Núcleo Interdisciplinar de Estudo e Pesquisa em Nefrologia da UFJF em Juiz de Fora - MG, entre junho a outubro de 2007. Para a análise de fatores de risco do pé diabético foi inserido um grupo controle com 20 pacientes diabéticos sem lesão de pé. Dentre os pacientes avaliados 70% eram do sexo masculino, com idade média de 65 anos, 60% aposentados, 80% concluíram o ensino fundamental, renda entre 1 e 3 salários mínimos. Como fatores desencadeantes das lesões destacam-se arteriopatia (100%) e neuropatia (60%). A HAS foi a patologia mais associada ao DM. Na avaliação microbiológica foram isolados 45% BGN, 38% CGP, 17% leveduras do gênero Candida, sendo detectada alguma resistência antimicrobiana. Na avaliação terapêutica foi observada extensa utilização de medicamentos pelos pacientes. A prescrição estava adequada à maioria das indicações, no entanto foram identificadas muitas interações medicamentosas, sugerindo um acompanhamento de equipe multiprofissional, incluindo o Farmacêutico, para adoção estratégias racionais na terapia medicamentosa

Microbiological and chemical analysis of land snails commercialised in Sicily

In this study 160 samples of snails belonging to the species Helix aspersa maxima and Helix aspersa muller were examined for chemical and microbiological analysis. Samples came from Greece and Poland. Results showed mean concentration of cadmium (0.35±0.036 mg/kg) and lead (0.05±0.013 mg/kg) much higher than the limit of detection. Mercury levels in both species were not detected. Microbiological analysis revealed the absence of Salmonella spp. and Clostridium spp. in both examined species. E. coli and K. oxytoca were observed in Helix aspersa maxima and Helix aspersa muller. Furthermore, one case of fungi positivity in samples of Helix aspersa muller was found. The reported investigations highlight the need to create and adopt a reference legislation to protect the health of consumers

Preliminary Results on the Prevalence of Salmonella spp. in Marine Animals Stranded in Sicilian Coasts: Antibiotic Susceptibility Profile and ARGs Detection in the Isolated Strains

The presence of Salmonella spp. in marine animals is a consequence of contamination from terrestrial sources (human activities and animals). Bacteria present in marine environments, including Salmonella spp., can be antibiotic resistant or harbor resistance genes. In this study, Salmonella spp. detection was performed on 176 marine animals stranded in the Sicilian coasts (south Italy). Antibiotic susceptibility, by disk diffusion method and MIC determination, and antibiotic resistance genes, by molecular methods (PCR) of the Salmonella spp. strains, were evaluated. We isolated Salmonella spp. in three animals, though no pathological signs were detected. Our results showed a low prevalence of Salmonella spp. (1.7%) and a low incidence of phenotypic resistance in three Salmonella spp. strains isolated. Indeed, of the three strains, only Salmonella subsp. enterica serovar Typhimurium from S. coeruleoalba and M. mobular showed phenotypic resistance: the first to ampicillin, tetracycline, and sulphamethoxazole, while the latter only to sulphamethoxazole. However, all strains harbored resistance genes (blaTEM, blaOXA, tet(A), tet(D), tet(E), sulI, and sulII). Although the low prevalence of Salmonella spp. found in this study does not represent a relevant health issue, our data contribute to the collection of information on the spread of ARGs, elements involved in antibiotic resistance, now considered a zoonosis in a One Health approach

First Molecular Survey to Detect <i>Mycoplasma gallisepticum</i> and <i>Mycoplasma synoviae</i> in Poultry Farms in a Strategic Production District of Sicily (South-Italy)

Mycoplasmas are recognized as avian pathogens, which may cause both respiratory disease and synovial infections in poultry, resulting in severe economic losses. Our study aims to determine the occurrence of Mycoplasma gallisepticum (MG) and Mycoplasma synoviae (MS) among commercial and rural laying hens located in Ragusa province (South Italy), using a duplex real time PCR. Four hundred tracheal swabs were collected from seven commercial (200 swabs) and 25 rural (200 swabs) farms without any clinical disease history. Out of 400 swabs collected, 50 (12.5%) and 93 (23.25%) were positive for MG and MS, respectively. In particular, 9 (18%) and 22 (23.65%) positive swabs for MG and MS, respectively, originated from commercial farms, compared to 41 (82%) and 71 (76.34%) obtained from rural farms. Data obtained show a lower prevalence of MG than MS in the studied farms. Moreover, both pathogens were spread in rural and commercial farms. PCR could be concluded as a rapid and sensitive method for the identification of MG and MS in areas where commercial farms that are declared Mycoplasma-free and rural flocks coexist. These data highlight the importance of surveillance also in rural poultry to monitoring the occurrence of mycoplasmas strains in strategic productive districts

First Molecular Survey to Detect Mycoplasma gallisepticum and Mycoplasma synoviae in Poultry Farms in a Strategic Production District of Sicily (South-Italy)

Mycoplasmas are recognized as avian pathogens, which may cause both respiratory disease and synovial infections in poultry, resulting in severe economic losses. Our study aims to determine the occurrence of Mycoplasma gallisepticum (MG) and Mycoplasma synoviae (MS) among commercial and rural laying hens located in Ragusa province (South Italy), using a duplex real time PCR. Four hundred tracheal swabs were collected from seven commercial (200 swabs) and 25 rural (200 swabs) farms without any clinical disease history. Out of 400 swabs collected, 50 (12.5%) and 93 (23.25%) were positive for MG and MS, respectively. In particular, 9 (18%) and 22 (23.65%) positive swabs for MG and MS, respectively, originated from commercial farms, compared to 41 (82%) and 71 (76.34%) obtained from rural farms. Data obtained show a lower prevalence of MG than MS in the studied farms. Moreover, both pathogens were spread in rural and commercial farms. PCR could be concluded as a rapid and sensitive method for the identification of MG and MS in areas where commercial farms that are declared Mycoplasma-free and rural flocks coexist. These data highlight the importance of surveillance also in rural poultry to monitoring the occurrence of mycoplasmas strains in strategic productive districts

Diagnostic Findings in a Confirmed Outbreak of Brucella ovis Infection in a Traditional Sheep Farm in Sicily (South-Italy)

Aim of this study is to report a laboratory investigation performed following the isolation of Brucella ovis, causing ovine epididymitis, in a traditional sheep farm in Sicily (South Italy). This disease represents a newly emerging risk for Italian livestock and is listed among diseases of EU priority (EU Reg 2016/429). Blood samples from 56 rams and 143 ewes were analyzed by both Enzyme-Linked Immunosorbent Assay (ELISA) and Complement Fixation Test (CFT). Genital swabs from all rams and 15 lactating ewes were collected to perform real-time PCR. Eighteen serologically positive rams were slaughtered and postmortem-inspected. Samples of testicle, epididymis, lymph nodes, and urine were also collected in order to perform microbiological, molecular, and histopathological analysis. Twelve slaughtered rams showed anatomo-pathological lesions. Real-time PCR for B. ovis BOV_A0504 gene was positive for 13 testicles and epididymis and 11 urine while B. ovis was isolated from epididymis and testicles of 7 slaughtered rams. This is the first exhaustive laboratory report of a microbiological, molecular, and serological pattern of the disease in sheep in Italy. Despite the impact on health and animal welfare, the epidemiology of B. ovis infection is still unknown, particularly in our country where the disease is considered endemic

Detection of <i>Mycoplasma agalactiae</i> in Ticks (<i>Rhipicephalus bursa</i>) Collected by Sheep and Goats in Sicily (South-Italy), Endemic Area for Contagious Agalactia

The aim of this preliminary study was to investigate the presence of Mycoplasma agalactiae (Ma) or other Contagious Agalactia (CA) causative organisms, in hard ticks infesting milking sheep and goats in endemic areas for CA in Sicily (South-Italy). Although there is accumulating evidence to support the role of ticks in the transmission of blood-borne haemoplasmas, information regarding their role in the transmission of CA, remains scarce. Ticks (n = 152) were collected from 25 lactating sheep and goats from three farms with previous outbreaks of CA. Microbiological and biomolecular, as well as serological analysis were performed on milk, tick, and serum samples, respectively. Rhipicephalus bursa species predominated, comprising 84.8% of the sampled ticks. Mycoplasma-like colonies were isolated from 5/56 (8.9%) tick pools and were identified as Ma by specific PCR and 16S rRNA gene sequencing. Unexpectedly, the organism was isolated from R. bursa ticks recovered only from animals whose milk tested negative for the pathogen. This preliminary demonstration suggests the potential role for ticks to act as a reservoir for the organisms, with potential involvement in the spread and maintenance of CA. Further work is required to determine the location of the organisms within the body of the ticks and to assess transmission potential