Multidrug Efflux System-mediated resistance in Staphylococcus aureus under a One Health approach.

The aim of the study was to track the spread of antimicrobial resistance among the different sectors of One Health through the detection of Multidrug-Efflux-System in multidrug-resistant Staphylococcus aureus isolates. Multidrug-resistant (MDR) and methicillin-resistant (MRSA) S. aureus isolates were selected: 25 of human, one of animal and eight of food origin. The efflux system genes norA, norB, norC, LmrS, tet38 and msrA were screened by PCR. The activity of the efflux systems was determined by the minimum inhibitory concentration (MIC) of tetracycline and ciprofloxacin in the presence and absence of CCCP and in the quantification of ethidium bromide efflux. Furthermore, biofilm formation was determined in the presence and absence of the CCCP. The molecular epidemiology of the isolates was traced with the aid of PFGE. The gene norC was the most prevalent, detected in all isolates and msrA was the least prevalent, detected in only two isolates from humans. There was no difference in the MICs of tetracycline and ciprofloxacin in the presence of CCCP, but 55.9% of isolates showed ethidium bromide efflux. The presence of CCCP decreased the biofilm formation. Regarding the molecular epidemiology, in three clusters was a mixture of the isolates from different origins. Therefore, S. aureus MDR with active multidrug efflux systems are circulating between One Health domains and it is necessary to consider strategies to decrease this circulation in order to prevent the dissemination of resistance mediated by MES.

Differences in the coinfective process of Staphylococcus aureus and Streptococcus agalactiae in bovine mammary epithelial cells infected by Mycobacterium avium subsp. paratuberculosis.

The interactions between Mycobacterium avium subsp. paratuberculosis (MAP) and the causative agents of bovine mastitis are still relatively unknown. Still, it is suspected that they may contribute to the worsening and persistence of mastitis within the mammary epithelial cells. Considering the growing economic implications of paratuberculosis and subclinical mastitis in dairy herds, this study aimed to determine the coinfection interaction between MAP and S. aureus or S. agalactiae in bovine mammary epithelial cells (MAC-T) in an ex-vivo model. For this purpose, internalisation tests of MAP + S. aureus or MAP + S. agalactiae were performed in MAC-T cells for 10, 30 and 120 min. The qPCR was performed to quantify internalised MAP at the time of exposure. Colony-forming units were counted on BHI agar medium for internalised subclinical mastitis bacteria at each time of infection. Viability tests of MAC-T cells, using the lactate dehydrogenase assay, were performed. The results showed that in the MAC-T cells previously infected by MAP and subsequently by S. aureus, there was a rapid internalisation in the first 10 min, maintaining a higher number of internalised bacteria during all exposure times. Regarding MAP + S. agalactiae, there were no changes in the internalisation patterns. The amount of MAP remained constant at all times evaluated, and there was no compromise in the viability of MAC-T cells during the tests. Thus, the results demonstrate the existence of an interaction between MAP + S. aureus, favouring internalisation and being able to contribute to the persistence of subclinical mastitis in dairy herds.

Polymorphism analysis of the apxIA gene of Actinobacillus pleuropneumoniae serovar 5 isolated in swine herds from Brazil.

The bacterium Actinobacillus pleuropneumoniae is the etiological agent of Contagious Porcine Pleuropneumonia, a disease responsible for economic losses in the swine industry worldwide. A. pleuropneumoniae is capable of producing proteinaceous exotoxins responsible for inducing hemorrhagic lesions, one of which is ApxI. Few studies have conducted an in-depth evaluation of polymorphisms of the nucleotides that make up the ApxI toxin gene. Here we analyze the polymorphisms of the apxIA gene region of A. pleuropneumoniae serovar 5 isolated from swine in different regions in Brazil and report the results of molecular sequencing and phylogenetic analysis. Analysis of the apxIA gene in 60 isolates revealed the presence of genetic diversity and variability. The polymorphisms in the nucleotide sequences determined the grouping of the Brazilian sequences and five more sequences from the GenBank database into 14 different haplotypes, which formed three main groups and revealed the presence of mutations in the nucleotide sequences. The estimation of selection pressures suggests the occurrence of genetic variations by positive selective pressure on A. pleuropneumoniae in large groups of animals in relatively small spaces. These conditions presumably favor the horizontal dissemination of apxIA gene mutations within bacterial populations with host reservoirs. As a result, the same serovar can demonstrate different antigenic capacities due to mutations in the apxIA gene. These alterations in sequences of the apxIA gene could occur in other areas of countries with intense swine production, which could lead to differences in the pathogenicity and immunogenicity of each serovar and have implications for the clinical status or diagnosis of A. pleuropneumoniae.