High frequency of increased triclosan MIC among CC5 MRSA and risk of misclassification of the SCCmec into types.

BACKGROUND: Typing of staphylococcal cassette chromosome mec (SCCmec) elements is commonly used for studies on the molecular epidemiology of MRSA. OBJECTIVES: To perform an investigation centred on uncovering the reasons for misclassification of MRSA clonal complex 5 (CC5) SCCmec type II clinical isolates in our laboratory. METHODS: MRSA isolates from CC5 were subjected to WGS and SCCmec typing. RESULTS: This investigation led to the discovery that the classification failure was due to an insertion of IS1272 carrying the fabI gene on a transposable element (TnSha1) that confers increased MIC to the biocide triclosan. Genomic analysis revealed that fabI was present in 25% of the CC5 MRSA isolates sampled. The frequency of TnSha1 in our collection was much higher than that observed among publicly available genomes (0.8%; n = 24/3142 CC5 genomes). Phylogenetic analyses revealed that genomes in different CC5 clades carry TnSha1 inserted in different integration sites, suggesting that this transposon has entered CC5 MRSA genomes on multiple occasions. In at least two genotypes, ST5-SCCmecII-t539 and ST5-SCCmecII-t2666, TnSha1 seems to have entered prior to their divergence. CONCLUSIONS: Our work highlights an important misclassification problem of SCCmecII in isolates harbouring TnSha1 when Boye's method is used for typing, which could have important implications for molecular epidemiology of MRSA. The importance of increased-MIC phenotype is still a matter of controversy that deserves more study given the widespread use of triclosan in many countries. Our results suggest expanding prevalence that may indicate strong selection for this phenotype.

Biofilm production and distribution of pilus variants among Streptococcus agalactiae isolated from human and animal sources.

Streptococcus agalactiae (group B Streptococcus, GBS) is a major pathogen in humans and animals. Pili and biofilm may be important virulence factors in this bacterial species. Here, biofilm production and the distribution of pilus variants among 134 GBS isolates from human and animal sources were evaluated. Biofilm production was significantly enhanced in 1% glucose-supplemented medium (p < 0.05). Using this medium, most GBS strains were strong biofilm producers. Biomass was mainly composed of proteins, followed by extracellular DNA, while polysaccharides represented a minor portion. All GBS strains presented at least one pilus variant. PI-2a was the most common among human GBS while PI-2b was the most common among animal isolates. Human GBS harboring PI-2b and animal GBS harboring PI-2a presented significantly reduced biofilm production (p = 0.0033). In conclusion, strong biofilm production seems to be a common characteristic in GBS, and association of the clinical source with the pilus variant may be crucial for this.

A unique SaeS allele overrides cell-density dependent expression of saeR and lukSF-PV in the ST30-SCCmecIV lineage of CA-MRSA.

ST30 (CC30)-SCCmec IV (USA1100) is one of the most common community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA) lineages. ST30 isolates typically carry lukSF-PV genes encoding the Panton-Valentine leukocidin (PVL) and are responsible for outbreaks of invasive infections worldwide. In this study, twenty CC30 isolates were analyzed. All were very susceptible to non-ß-lactam antimicrobials, 18/20 harbored the lukSF-PV genes, only 1/20 exhibited agr-rnaIII dysfunction, and the majority was not able to form biofilm on inert surfaces. Analysis of lukSF-PV temporal regulation revealed that opposite to other CA-MRSA isolates, these genes were more highly expressed in early log phase than in stationary phase. This inverted lukSF-PV temporal expression was associated with a similar pattern of saeRS expression in the ST30 isolates, namely high level expression in log phase and reduced expression in stationary phase. Reduced saeRS expression in stationary phase was associated with low expression levels of the sae regulators, agr and agr-upregulator sarA, which activate the stationary phase sae-P1 promoter and overexpression of agr-RNAIII restored the levels of saeR and lukSF-PV trancripts in stationary phase. Altered SaeRS activity in the ST30 isolates was attributed to amino acid substitutions (N227S, E268K and S351T) in the HTPase_c domain of SaeS (termed SaeS(SKT)). Complementation of a USA300 saeS mutant with the saeS(SKT) and saeS alleles under the direction of the log phase sae-P3 promoter revealed that saeR and lukSF-PV transcription levels were more significantly activated by saeS(SKT) than saeS. In summary our data identify a unique saeS allele (saeS(SKT)) which appears to override cell-density dependent SaeR and PVL expression in ST30 CA-MRSA isolates. Further studies to determine the contribution of saeS(SKT) allele to the pathogenesis of infections caused by ST30 isolates are merited.

Investigation of antimicrobial susceptibility and genetic diversity among Staphylococcus pseudintermedius isolated from dogs in Rio de Janeiro.

Staphylococcus pseudintermedius is an opportunistic pathogen causing a variety of infections that are difficult to treat, especially because of the development of antimicrobial resistance. It has a clonal distribution around the world. To have a better understanding of the MRSP population, we search the presence of MRSP in colonized or infected dogs. Samples from 99 dogs with infections and 35 from asymptomatic dogs were collected. Isolates were identified by mass spectrometry and Multiplex-PCR. The mecA gene was confirmed by conventional PCR. MRSP strains were analyzed by whole-genome sequencing. 75 S. pseudintermedius were identified, most from infection cases. The species were isolated from 70 out of the 135 dogs. Penicillin and Trimethoprim/Sulfamethoxazole presented higher resistance rates. Forty-seven strains were classified as multi-drug resistant (MDR), and were more isolated from dogs with infection (P < 0.05). Eighteen samples were classified as MRSP, representing 24.0% of the population. Six of 16 MRSP sequenced samples belonged to the world spread clone ST71; others belonged to unknown clones. Most samples carried the SCCmec type IIIA. Twenty-one different genetic resistance determinants were found among MRPS strains. MRSP is circulating among infected and colonized dogs in Rio de Janeiro, Brazil.

RdJ detection tests to identify a unique MRSA clone of ST105-SCCmecII lineage and its variants disseminated in the metropolitan region of Rio de Janeiro.

Hospital bloodstream infection (BSI) caused by methicillin-resistant Staphylococcus aureus (MRSA) is a major cause of morbidity and mortality and is frequently related to invasive procedures and medically complex patients. An important feature of MRSA is the clonal structure of its population. Specific MRSA clones may differ in their pathogenic, epidemiological, and antimicrobial resistance profiles. Whole-genome sequencing is currently the most robust and discriminatory technique for tracking hypervirulent/well-adapted MRSA clones. However, it remains an expensive and time-consuming technique that requires specialized personnel. In this work, we describe a pangenome protocol, based on binary matrix (1,0) of open reading frames (ORFs), that can be used to quickly find diagnostic, apomorphic sequence mutations that can serve as biomarkers. We use this technique to create a diagnostic screen for MRSA isolates circulating in the Rio de Janeiro metropolitan area, the RdJ clone, which is prevalent in BSI. The method described here has 100% specificity and sensitivity, eliminating the need to use genomic sequencing for clonal identification. The protocol used is relatively simple and all the steps, formulas and commands used are described in this work, such that this strategy can also be used to identify other MRSA clones and even clones from other bacterial species.

Assessing in vivo and in vitro biofilm development by Streptococcus dysgalactiae subsp. dysgalactiae using a murine model of catheter-associated biofilm and human keratinocyte cell.

Streptococcus dysgalactiae subsp. dysgalactiae (SDSD) is an important agent of bovine mastitis. This infection causes an inflammatory reaction in udder tissue, being the most important disease-causing significant impact on the dairy industry. Therefore, it leads to an increase in dairy farming to meet commercial demands. As a result, there is a major impact on both the dairy industry and the environment including global warming. Recurrent mastitis is often attributed to the development of bacterial biofilms, which promote survival of sessile cells in hostile environments, and resistance to the immune system defense and antimicrobial therapy. Recently, we described the in vitro biofilm development on abiotic surfaces by bovine SDSD. In that work we integrated microbiology, imaging, and computational methods to evaluate the biofilm production capability of SDSD isolates on abiotic surfaces. Additionally, we reported that bovine SDSD can adhere and internalize human cells, including human epidermal keratinocyte (HEK) cells. We showed that the adherence and internalization rates of bovine SDSD isolates in HEK cells are higher than those of a SDSD DB49998-05 isolated from humans. In vivo, bovine SDSD can cause invasive infections leading to zebrafish morbidity and mortality. In the present work, we investigated for the first time the capability of bovine SDSD to develop biofilm in vivo using a murine animal model and ex-vivo on human HEK cells. Bovine SDSD isolates were selected based on their ability to form weak, moderate, or strong biofilms on glass surfaces. Our results showed that SDSD isolates displayed an increased ability to form biofilms on the surface of catheters implanted in mice when compared to in vitro biofilm formation on abiotic surface. A greater ability to form biofilm in vitro after animal passage was observed for the VSD45 isolate, but not for the other isolates tested. Besides that, in vitro scanning electron microscopy demonstrated that SDSD biofilm development was visible after 4 hours of SDSD adhesion to HEK cells. Cell viability tests showed an important reduction in the number of HEK cells after the formation of SDSD biofilms. In this study, the expression of genes encoding BrpA-like (biofilm regulatory protein), FbpA (fibronectin-binding protein A), HtrA (serine protease), and SagA (streptolysin S precursor) was higher for biofilm grown in vivo than in vitro, suggesting a potential role for these virulence determinants in the biofilm-development, host colonization, and SDSD infections. Taken together, these results demonstrate that SDSD can develop biofilms in vivo and on the surface of HEK cells causing important cellular damages. As SDSD infections are considered zoonotic diseases, our data contribute to a better understanding of the role of biofilm accumulation during SDSD colonization and pathogenesis not only in bovine mastitis, but they also shed some lights on the mechanisms of prosthesis-associated infection and cellulitis caused by SDSD in humans, as well.

Anti-Restriction Gene Homologs Are Highly Represented in Methicillin-Resistant and Multidrug-Resistant Staphylococcus aureus ST239 and ST398: Implications for Resistance Gene Acquisitions.

Multidrug resistance is commonly acquired by transferring DNA from one bacterium to another. However, the mechanisms that enhance the acquisitions of foreign genes are poorly understood, as well as the dynamics of their transmission between hosts in different environments. Here, genomic approaches were applied to evaluate the enrichment of the S. aureus chromosome with resistance traits in groups of genomes with or without anti-restriction genes and to analyze some evolutionary aspects of these acquisitions. Furthermore, the role played by an anti-restriction gene in improving multiresistance in MRSA was investigated by molecular cloning. A strong association was observed between the presence of anti-restriction gene homologs and patterns of multidrug resistance. Human isolates, mainly ST239-SCCmecIII, carry ardA-H1, and from animal sources, mainly CC398, carry ardA-H2. Increased DNA transfer was observed for clones that express the ardA-H1 allele, corroborating its role in promoting gene transfer. In addition, ardA-H1 was expressed in the dsDNA format in the BMB9393 strain. The evolution of successful multidrug-resistant MRSA lineages of the ST239 and ST398 was initiated not only by the entry of the mec cassette but also by the acquisition of anti-restriction gene homologs. Understanding the mechanisms that affect DNA transfer may provide new tools to control the spread of drug resistance.

Cellular Growth Arrest and Efflux Pumps Are Associated With Antibiotic Persisters in Streptococcus pyogenes Induced in Biofilm-Like Environments.

Streptococcus pyogenes (group A Streptococcus-GAS) is an important pathogen for humans. GAS has been associated with severe and invasive diseases. Despite the fact that these bacteria remain universally susceptible to penicillin, therapeutic failures have been reported in some GAS infections. Many hypotheses have been proposed to explain these antibiotic-unresponsive infections; however, none of them have fully elucidated this phenomenon. In this study, we show that GAS strains have the ability to form antimicrobial persisters when inoculated on abiotic surfaces to form a film of bacterial agglomerates (biofilm-like environment). Our data suggest that efflux pumps were possibly involved in this phenomenon. In fact, gene expression assays by real-time qRT-PCR showed upregulation of some genes associated with efflux pumps in persisters arising in the presence of penicillin. Phenotypic reversion assay and whole-genome sequencing indicated that this event was due to non-inherited resistance mechanisms. The persister cells showed downregulation of genes associated with protein biosynthesis and cell growth, as demonstrated by gene expression assays. Moreover, the proteomic analysis revealed that susceptible cells express higher levels of ribosome proteins. It is remarkable that previous studies have reported the recovery of S. pyogenes viable cells from tissue biopsies of patients presented with GAS invasive infections and submitted to therapy with antibiotics. The persistence phenomenon described herein brings new insights into the origin of therapeutic failures in S. pyogenes infections. Multifactorial mechanisms involving protein synthesis inhibition, cell growth impairment and efflux pumps seem to play roles in the formation of antimicrobial persisters in S. pyogenes.

Association of DNA sequence-independent genetic regulatory mechanisms with apical periodontitis: A scoping review.

OBJECTIVE: Different studies in the last decade have proposed that gene expression alterations that are independent of the DNA sequence may also play an important role in periapical disease. The present study aimed to assess the available evidence supporting a relationship between these alterations and apical periodontitis through a scoping review. DESIGN: Specific strategies were developed for different databases (MEDLINE via PubMed, Cochrane Library, Scopus, Web of Science, and Virtual Health Library) and a search performed by March 1st, 2019. The evidence sources were selected according to the eligibility criteria and underwent a critical appraisal of methodological quality. RESULTS: The initial search retrieved 212 references, with eight eligible articles after the removal of replicates and application of exclusion criteria. Five studies identified altered DNA methylation on inflammatory response genes (FOXP3, CXCL3, FADD, MMP2, MMP9, IFNG, IL4, IL12) on AP patients. Three others identified the alterations on the expression of several microRNAs (miR-29b, 106b, 125b, 143, 146a, 155, 198) during AP. No evidence was identified regarding mechanisms of histone methylation, or of epigenetic heritability or stability. CONCLUSIONS: There is available evidence for the involvement of different genetic regulatory mechanisms independent of changes in DNA sequence in the development or severity of apical periodontitis. However, due to methodological limitations, further research must be performed before novel therapies and diagnostic tools for AP may arise from these data.

Local Diversification of Methicillin- Resistant Staphylococcus aureus ST239 in South America After Its Rapid Worldwide Dissemination.

The global spread of specific clones of methicillin-resistant Staphylococcus aureus (MRSA) has become a major public health problem, and understanding the dynamics of geographical spread requires worldwide surveillance. Over the past 20 years, the ST239 lineage of MRSA has been recognized as an emerging clone across the globe, with detailed studies focusing on isolates from Europe and Asia. Less is known about this lineage in South America, and, particularly, Brazil where it was the predominant lineage of MRSA in the early 1990s to 2000s. To gain a better understanding about the introduction and spread of ST239 MRSA in Brazil we undertook a comparative phylogenomic analysis of ST239 genomes, adding seven completed, closed Brazilian genomes. Brazilian ST239 isolates grouped in a subtree with those from South American, and Western, romance-language-speaking, European countries, here designated the South American clade. After an initial worldwide radiation in the 1960s and 1970s, we estimate that ST239 began to spread in South America and Brazil in approximately 1988. This clone demonstrates specific genomic changes that are suggestive of local divergence and adaptational change including agrC single-nucleotide polymorphisms variants, and a distinct pattern of virulence-associated genes (mainly the presence of the chp and the absence of sea and sasX). A survey of a geographically and chronologically diverse set of 100 Brazilian ST239 isolates identified this virulence genotype as the predominant pattern in Brazil, and uncovered an unexpectedly high prevalence of agr-dysfunction (30%). ST239 isolates from Brazil also appear to have undergone transposon (IS256) insertions in or near global regulatory genes (agr and mgr) that likely led to rapid reprogramming of bacterial traits. In general, the overall pattern observed in phylogenomic analyses of ST239 is of a rapid initial global radiation, with subsequent local spread and adaptation in multiple different geographic locations. Most ST239 isolates harbor the ardA gene, which we show here to have in vivo anti-restriction activity. We hypothesize that this gene may have improved the ability of this lineage to acquire multiple resistance genes and distinct virulence-associated genes in each local context. The allopatric divergence pattern of ST239 also may suggest strong selective pressures for specific traits in different geographical locations.

Inactivation of the Autolysis-Related Genes lrgB and yycI in Staphylococcus aureus Increases Cell Lysis-Dependent eDNA Release and Enhances Biofilm Development In Vitro and In Vivo.

Staphylococcus aureus ica-independent biofilms are multifactorial in nature, and various bacterial proteins have been associated with biofilm development, including fibronectin-binding proteins A and B, protein A, surface protein SasG, proteases, and some autolysins. The role of extracellular DNA (eDNA) has also been demonstrated in some S. aureus biofilms. Here, we constructed a Tn551 library, and the screening identified two genes that affected biofilm formation, lrgB and yycI. The repressive effect of both genes on the development of biofilm was also confirmed in knockout strains constructed by allelic recombination. In contrast, the superexpression of either lrgB or yycI by a cadmium-inducible promoter led to a decrease in biofilm accumulation. Indeed, a significant increase in the cell-lysis dependent eDNA release was detected when lrgB or yycI were inactivated, explaining the enhanced biofilm formed by these mutants. In fact, lrgB and yycI genes belong to distinct operons that repress bacterial autolysis through very different mechanisms. LrgB is associated with the synthesis of phage holin/anti-holin analogues, while YycI participates in the activation/repression of the two-component system YycGF (WalKR). Our in vivo data suggest that autolysins activation lead to increased bacterial virulence in the foreign body animal model since a higher number of attached cells was recovered from the implanted catheters inoculated with lrgB or yycI knockout mutants.

Complete Genome Sequence of a Variant of the Methicillin-Resistant Staphylococcus aureus ST239 Lineage, Strain BMB9393, Displaying Superior Ability To Accumulate ica-Independent Biofilm.

Biofilm is considered an important virulence factor in nosocomial infections. Herein, we report the complete genome sequence of a variant of methicillin-resistant Staphylococcus aureus, strain BMB9393, which is highly disseminated in Brazil. This strain belongs to the lineage ST239 and displays increased ability to accumulate ica-independent biofilm and to invade human epithelial cells.

Characterization of coagulase-negative staphylococci isolated from hospital indoor air and a comparative analysis between airborne and inpatient isolates of Staphylococcus epidermidis.

A total of 108 coagulase-negative staphylococci (CoNS) were collected from hospital indoor air. The majority of the isolates were able to produce biofilms and displayed multiresistance profiles. The most frequent species identified were Staphylococcus epidermidis (n=27) and Staphylococcus haemolyticus (n=17). Potential virulence traits (icaAD, aap, hld, atlE and sesB) and genotypic profiles were compared for S. epidermidis isolates from indoor air (n=27) and from patients (n=26) who had been admitted to the hospital 8-34 months after air sampling. Overall, the virulence factors tested were more frequently found among S. epidermidis recovered from clinical origin than from air sources (P=0.003). Indeed, the group of patient isolates exhibited superior ability to accumulate biofilms (P<0.0001). Despite this, genotyping using PFGE revealed that identical clones of S. epidermidis could be recovered from both patient and indoor air samples. In addition, some airborne isolates displayed virulence profiles and levels of biofilm accumulation similar to those found in patient isolates. Therefore, further studies are necessary to clarify the importance of hospital indoor air as a route of transmission for CoNS isolates (mainly S. epidermidis).