Conjugative transfer of plasmid p_8N_qac(MN687830.1) carrying qacA gene from Staphylococcus aureus to Escherichia coli C600: potential mechanism for spreading chlorhexidine resistance.

The methicillin resistant Staphylococcus aureus (MRSA) is recognized by its ability to acquire and transferring resistance genes through interspecies conjugative plasmids. However, transference of plasmids from Gram-positive cocci to Gram-negative bacilli is not well characterized. In this report, we describe the transfer of a conjugative plasmid carrying qacA from MRSA to Escherichia coli C600. We performed a conjugation experiment using a chlorhexidine resistant MRSA isolate (ST-105/SCCmec type III) carrying the gene qacA and qacC as the donor and a chlorhexidine susceptible E. coli C600 isolate as the receptor. Transconjugants were selected using MacConkey agar plates containing chlorhexidine in concentrations ranging from 0.25 to 16 g.L-1. To genotypically confirm the transfer of the resistance gene, the transconjugants were screened by Polymerase Chain Reaction (PCR) and submitted to Sanger's sequencing. MRSA isolates successfully transferred the chlorhexidine resistance gene (qacA) to the recipient E. coli strain C600. The E. coli transconjugant exhibited an important reduction of chlorhexidine susceptibility, with MICs increasing from ≤ 0.25 to ≥ 16 g.L-1 after conjugation. The qacA gene was detected by PCR as well as in the Sanger's sequencing analysis of DNA from transconjugant plasmids. To the best of our knowledge, this is the first report of the plasmid p_8N_qac(MN687830.1) carrying qacA and its transfer by conjugation from a MRSA to an E. coli. These findings increase concerns on the emergence of resistance dissemination across the genus and emphasizes the importance of continuous antiseptic stewardship.

Susceptibility to chlorhexidine and mupirocin among methicillin-resistant Staphylococcus aureus clinical isolates from a teaching hospital.

Despite the widespread use of chlorhexidine (CHX) to prevent infection, data regarding the in vitro action of CHX against methicillin-resistant Staphylococcus aureus (MRSA) are limited. Clinical isolates from Hospital das Clinicas, Sao Paulo, Brazil, identified during 2002/2003 and 2012/2013 were studied to describe the susceptibility to CHX and mupirocin, molecular characteristics, and virulence profile of MRSA. Susceptibility test to Mupirocin was performed by the disk diffusion method and to CHX by the agar dilution technique. PCR for virulence genes, mecA gene and Staphylococcal Cassette Chromosome mec (SCCmec) types were investigated as well. Mupirocin- and CHX-resistant isolates were sequenced using the IlluminaTM plataform. Two hundred and sixteen MRSA clinical isolates were evaluated: 154 from infected and 62 from colonized patients. Resistance to mupirocin was observed in four isolates assigned as SCCmec type III and STs (ST05; ST239 and ST105) carrying mupA and blaZ, two of them co-harboring the ileS gene. Only one isolate assigned as SCCmec type III was resistant to CHX (MIC of 8.0 µg.mL-1) and harbored the qacA gene. Resistance to chlorhexidine and mupirocin were found in isolates carrying qacA and mupA in our hospital. Since these genes are plasmid-mediated, this finding draws attention to the potential spread of resistance to mupirocin in our hospital.

Prevalence of methicillin-resistant Staphylococcus aureus colonization in individuals from the community in the city of Sao Paulo, Brazil.

Staphylococcus aureus (SA) is a commensal habitant of nasal cavities and skin. Colonization by community-acquired methicillin-resistant SA (CA-MRSA) is associated with infections in patients who have not been recently hospitalized. The aim of this study is to determine the prevalence of MRSA colonization in an outpatient population, currently unknown in Brazil. Three-hundred patients or caregivers from two teaching hospitals were included. A questionnaire was applied and nasal swabs were obtained from patients. Swabs were inoculated in brain heart infusion (BHI) with 2.5% NaCl and seeded in mannitol. Suspicious colonies were subjected to MALDI-TOF MS Microflex™ identification. Antimicrobial susceptibility test for oxacillin was performed for SA-positive samples by microdilution. Polymerase chain-reactions for detection of mecA and coA genes were performed for resistant samples. Data about MRSA carriers were compared with non-carriers. There were 127 S. aureus isolates, confirmed by MALDI-TOF. Only seven (2.3%) were MRSA and positive for mecA and coA genes. Factors associated with MRSA carriage were African ethnicity, skin diseases or antibiotic use. The majority of them were from Dermatology clinics. Prevalence of MRSA colonization in individuals from the community was low in our study (2.3%). This finding raises the hypothesis of inter-household transmission of SA, although we did not find any association between MRSA-colonization and the shared use of personal objects. Given the low prevalence of MRSA carriers observed, empirical antimicrobial coverage for MRSA in community-acquired infections should be not necessary.

Multidrug-resistant Stenotrophomonas maltophilia: Description of new MLST profiles and resistance and virulence genes using whole-genome sequencing.

OBJECTIVES: Stenotrophomonas maltophilia is an opportunistic pathogen that has high intrinsic and acquired antimicrobial resistance, with great genetic diversity. The aim of this study was to characterise four S. maltophilia clinical isolates displaying different susceptibility profiles using whole-genome sequencing. METHODS: The whole genomes of four clinical isolates of S. maltophilia from three patients were sequenced using Ion Torrent™ PGM technology. The isolates presented different susceptibilities to trimethoprim/sulfamethoxazole (SXT) and levofloxacin. RESULTS: Three new multilocus sequence typing (MLST) profiles were identified (ST144, ST172 and ST173), differing in virulence and resistance genes. The ST172 isolate had more genes related to toxins than related to motility or adhesion and had different types of efflux pumps than the other isolates. The SXT-resistant strains belonged to ST172 or ST144 and did not harbour the sul1, sul2 or dfrA resistance genes. Strains I and II, from the same patient and belonging to the same ST but differing in resistance to SXT, had all of the resistance genes searched for in common, except for the SmeABC efflux pump complex genes that were only found in the SXT-resistant strain. All strains, including the strain susceptible to levofloxacin, harboured the qnrB gene, which may question the importance of this gene in determining levofloxacin resistance in S. maltophilia. CONCLUSION: Here we describe three new MLST profiles. Resistance to SXT in these strains appears to be associated with efflux pumps.

High mortality of bloodstream infection outbreak caused by carbapenem-resistant P. aeruginosa producing SPM-1 in a bone marrow transplant unit.

PURPOSE: Carbapenem resistance in P. aeruginosa is increasing worldwide. In Brazil, SPM-1 is the main P. aeruginosa carbapenemase identified. Little is known about the virulence factor in SPM-1 clones.Methodolgy. We describe a carbapenem-resistant P. aeruginosa bloodstream infection (CRPa-BSI) outbreak in a bone marrow transplant Unit (BMT). Twenty-nine CRPa-BSI cases were compared to 58 controls. Microbiological characteristics of isolates, such as sensitivity, carbapenemase gene PCR for P. aeruginosa, and PFGE are described, as well as the whole-genome sequence (WGS) of three strains.Results/Key findings. The cultures from environmental and healthcare workers were negative. Some isolates harboured KPC and SPM. The WGS showed that the 03 strains belonged to ST277, presented the same mutations in outer membrane protein, efflux pump, and virulence genes such as those involved in adhesion, biofilm, quorum-sensing and the type III secretion system, but differ regarding the carbapenemase profile. A predominant clone-producing SPM harbouring Tn 4371 was identified and showed cross-transmission; no common source was found. Overall mortality rate among cases was 79 %. The first multivariate analysis model showed that neutropenia (P=0.018), GVHD prophylaxis (P=0.016) and prior use of carbapenems (P=0.0089) were associated with CRPa-BSI. However, when MASCC>21 points and platelets were added in the final multivariate analysis, only prior use of carbapenems remained as an independent risk factor for CRPa-BSI (P=0.043). CONCLUSIONS: The predominant clone belonging to ST277 showed high mortality. Carbapenem use was the only risk factor associated with CRPa-BSI. This finding is a wake-up call for the need to improve management in BMT units.