Ilana Lopes Baratella da Cunha Camargo

An RpoB Mutation Confers Dual Heteroresistance to Daptomycin and Vancomycin in Staphylococcus aureus

ABSTRACT We have previously reported the establishment of a Staphylococcus aureus laboratory strain, 10*3d1, having reduced susceptibility to daptomycin and heterogeneous vancomycin-intermediate S. aureus (VISA) phenotype. The strain was generated in vitro by serial daptomycin selection (Camargo, I. L., H. M. Neoh, L. Cui, and K. Hiramatsu, Antimicrob. Agents Chemother. 52:4289-4299, 2008). Here we explored the genetic mechanism of resistance in the strain by whole-genome sequencing and by producing gene-replaced strains. By genome comparison between 10*3d1 and its parent methicillin-resistant Staphylococcus aureus (MRSA) strain N315ΔIP, we identified five nonsynonymous single nucleotide polymorphisms (SNPs). One of the five mutations was found in the rpoB gene encoding the RNA polymerase β subunit. The mutation at nucleotide position 1862 substituted the 621st alanine by glutamic acid. The replacement of the intact rpoB with the mutated rpoB, designated rpoB(A621E), conferred N315ΔIP with the phenotypes of reduced susceptibility to daptomycin and hetero-VISA. The rpoB(A621E)-mediated resistance conversion was accompanied by a thickened cell wall and reduction of the cell surface negative charge. Being consistent with these phenotypic changes, microarray data showed that the expression of the dlt operon, which increases the cell surface positive charge, was enhanced in the rpoB(A621E) mutant. Other remarkable findings of microarray analysis of the rpoB(A621E) mutant included repression of metabolic pathways of purine, pyrimidine, arginine, the urea cycle, and the lac operon, enhancement of the biosynthetic pathway of vitamin B2, K1, and K2, and cell wall metabolism. Finally, mutations identified in rplV and rplC, encoding 50S ribosomal proteins L22 and L3, respectively, were found to be associated with the slow growth, but not with the phenotype of decreased susceptibility to vancomycin and daptomycin, of 10*3d1.

Serial Daptomycin Selection Generates Daptomycin-Nonsusceptible Staphylococcus aureus Strains with a Heterogeneous Vancomycin-Intermediate Phenotype

ABSTRACT In order to better understand the mechanism of daptomycin resistance, we generated a daptomycin-nonsusceptible derivative strain, strain 10*3d1 (MIC = 3.0 μg/ml), by in vitro exposure of methicillin-resistant Staphylococcus aureus strain N315ΔIP (MIC = 0.5 μg/ml) to daptomycin. We also obtained a daptomycin-susceptible phenotypic revertant strain, strain 10*3d1-10 (MIC = 1.0 μg/ml), by passaging 10*3d1 in drug-free medium for 10 days. The resultant triple-isogenic strains were analyzed for their phenotypes and gene expression by microarray analysis. No significant differences in the membrane fluidities of 10*3d1 and 10*3d1-10 compared to the membrane fluidity of N315ΔIP were observed. Resistant strain 10*3d1 had the highest membrane potential, followed by strains 10*3d1-10 and N315ΔIP. The vancomycin and teicoplanin MICs also increased. Teichoic acid genes (tagA, tagG), mprF encoding lysyl-phosphatidylglycerol, and cls encoding cardiolipin synthase were downregulated in 10*3d1 and 10*3d1-10. The vraF and vraG genes, which encode ATP binding cassette transporter proteins, were upregulated in 10*3d1. The vraSR two-component regulatory system was upregulated, and electron microscopy revealed that the cell wall of 10*3d1 was significantly thicker than that of the parental strain. Taken together, daptomycin exposure selected a daptomycin-nonsusceptible strain with a phenotype similar to that of heterogeneous vancomycin-intermediate S. aureus and a transcription profile that partially overlapped that of heterogeneous vancomycin-intermediate S. aureus.

Molecular characterization of methicillin-resistant Staphylococcus aureus resistant to tigecycline and daptomycin isolated in a hospital in Brazil

We report the molecular characterization of methicillin-resistant Staphylococcus aureus (MRSA) with resistance to tigecycline and to daptomycin isolated from intensive-care-unit patients in Brazil over a 6-month period. Thirty-six isolates (25 from infection sites, 11 from nasal sites) recovered from 23 patients who presented with MRSA infection during this period were characterized by pulsed-field gel electrophoresis, multilocus sequence typing, staphylococcal cassette chromosome mec (SCCmec) typing, and antimicrobial susceptibility profiling. Ten isolates from six patients and two isolates from different patients were resistant to tigecycline and daptomycin, respectively. Eight pulsotypes were identified and one, type A, accounted for 21 isolates from 12 patients; type A isolates were SCCmecII as were a further nine isolates of other pulsotypes. All but four of the total isolates were sequence type (ST) 5 or ST105 and classified as clonal complex (CC) 5; the historically prevalent lineage in Brazil, ST239-SCCmecIII, was identified in only three patients. Tigecycline-resistant strains were all ST105-SCCmecII and two patients were nasally colonized by strains of the same pulsotype found in infection sites. Two ST5-SCCmecII were daptomycin resistant after 48 h incubation. The origin and mechanism of these resistant strains remains unknown and further studies are warranted to determine whether such clones are becoming endemic in Brazilian hospitals and to assess their impact on infection control practice.

Multilocus sequence typing of uropathogenic ESBL-producing Escherichia coli isolated in a Brazilian community.

In the present study, multilocus sequence typing (MLST), pulsed-field gel electrophoresis (PFGE) and polymerase chain reaction detection of three resistance genes were combined to characterize seven uropathogenic E. coli isolated from outpatients. Selected portions of seven housekeeping and three antibiotic-resistance genes of the isolates were sequenced. The seven isolates were classified into four different sequence types (STs) by MLST and five PGFE types. Three isolates had a novel allelic profile representing a new ST designated as ST528 and showed the same PFGE and resistance genes. Two isolates, both characterized as ST359, were differentiated by PFGE and shared only one of the antibiotic-resistance genes studied. Comparison of MLST results with those of PFGE and resistance genes demonstrated that Escherichia coli had acquired different antibiotic-resistance genes and DNA rearrangements, causing alterations in PFGE patterns but maintaining the same ST. Furthermore, this article also reports the first detection of a CTX-M-2 ESBL E. coli and SHV-5 in a Brazilian community.

Inactivation of formyltransferase (wbkC) gene generates a Brucella abortus rough strain that is attenuated in macrophages and in mice.

Rough mutants of Brucella abortus were generated by disruption of wbkC gene which encodes the formyltransferase enzyme involved in LPS biosynthesis. In bone marrow-derived macrophages the B. abortusDeltawbkC mutants were attenuated, could not reach a replicative niche and induced higher levels of IL-12 and TNF-alpha when compared to parental smooth strains. Additionally, mutants exhibited attenuation in vivo in C57BL/6 and interferon regulatory factor-1 knockout mice. DeltawbkC mutant strains induced lower protective immunity in C56BL/6 than smooth vaccine S19 but similar to rough vaccine RB51. Finally, we demonstrated that Brucella wbkC is critical for LPS biosynthesis and full bacterial virulence.

Different VanA Elements in E. faecalis and in E. faecium Suggest at Least Two Origins of Tn1546 Among VRE in a Brazilian Hospital

In 2009 during surveillance in a Brazilian hospital, many patients were confirmed to be colonized by vancomycin-resistant enterococci (VRE) and a few infection cases occurred. Among 14 isolates of Enterococcus faecalis, most had the same pulsotype, virulence profile (agg(+)elrA(+)gelE(+)), and were sequence type (ST)103, indicating dissemination of a clone. The 47 Enterococcus faecium were separated into four pulsotypes, the predominant virulence profile being esp(+)acm(+). All of them harbored the hospital marker IS16, and three randomly chosen isolates were ST412, belonging to the Clonal Complex 17. E. faecalis were all susceptible to penicillin and ampicillin, while all E. faecium were resistant to them. All isolates were susceptible to daptomycin and tigecycline. There were no rep-family genes common to all VRE. The VanA element of all E. faecium lost its left-side inverted repeat (IRL) region and had a specific IS insertion. On the other hand, all E. faecalis presented intact Tn1546. The size of plasmids containing the vanA gene as well as its rep-families varied between and within species. The lack of a vanA plasmid common to all VRE, together with the differences among VanA elements, despite the fact that some patients were colonized by both species during their hospitalization, leads us to suggest at least two different Tn1546 origins.

Virulence factors in vancomycin-resistant and vancomycin - susceptible Enterococcus faecalis from Brazil

Enterococci are members of commensal flora of animals and insects, but are also important opportunistic pathogens. Our objective was to observe if there was any difference of virulence in several groups of E. faecalis, mainly between vancomycin-resistant E. faecalis (VREFS) of colonization and infection. VREFS and vancomycin-sensitive E. faecalis from Brazil were screened for the presence of virulence factor genes. Phenotypic assays were used to assess in vitro expression, to understand the pathogenic potential of these isolates and to determine whether a correlation exists between virulence and antibiotic resistance. Different virulence profiles were found suggesting that the disseminating clone may have generated several variations. However, our study showed that one constellation of traits appeared most commonly: gelatinase, aggregation substance and esp (GEA). These factors are important because they have been implicated in cell aggregation and biofilm formation. Biofilm formation may promote the conjugation of plasmids harboring resistance and virulence genes, enhancing the probability of entry of new resistance genes into species. Curiously, the profile GEA was not exclusive to VREFS, it was the second most observed in VSEFS isolates from colonization and infection in hospitalized patients and also from rectal swabs of healthy volunteers. Such strains appear to represent the entry gateway to new resistance genes into E. faecalis and may contribute to the spreading of E. faecalis mainly in hospitals.

Draft Genome Sequence of Methicillin-Resistant Staphylococcus aureus Strain SA16, Representative of an Endemic Clone from a Brazilian Hospital

ABSTRACT Here we report the draft genome sequence of a bloodstream isolate of methicillin-resistant Staphylococcus aureus strain SA16. Strain SA16 is a sequence type 5 (ST5)-staphylococcal cassette chromosome mec type II (SCCmec II) clone and was the most prevalent isolate at a Brazilian hospital during the second half of 2009.

Molecular analysis of methicillin-resistant Staphylococcus aureus dissemination among healthcare professionals and/or HIV patients from a tertiary hospital

INTRODUCTION Methicillin-resistant Staphylococcus aureus (MRSA) is a nosocomial pathogen in community settings. MRSA colonized individuals may contribute to its dissemination; the risk of MRSA infection is increased in human immunodeficiency virus/acquired immune deficiency syndrome (HIV/AIDS) patients, although the prevalence of colonization in this group is not well established. The present study addressed this issue by characterizing MRSA isolates from HIV/AIDS patients and their healthcare providers (HCPs) to determine whether transmission occurred between these two populations. METHODS A total of 24 MRSA isolates from HIV-infected patients and five from HCPs were collected between August 2011 and May 2013. Susceptibility to currently available antimicrobials was determined. Epidemiological typing was carried out by pulsed-field gel electrophoresis, multilocus sequence typing, and Staphylococcus cassette chromosome (SCCmec) typing. The presence of heterogeneous vancomycin-intermediate Staphylococcus aureus (hVISA) and heterogeneous daptomycin-resistant Staphylococcus aureus (hDRSA) was confirmed by population analysis profile. Isolates characterized in this study were also compared to isolates from 2009 obtained from patients at the same hospital. RESULTS A variety of lineages were found among patients, including ST5-SCCmecII and ST30-SCCmecIV. Two isolates were Panton-Valentine leukocidin-positive, and hVISA and hDRSA were detected. MRSA isolates from two HCPs were not related to those from HIV/AIDS patients, but clustered with archived MRSA from 2009 with no known relationship to the current study population. CONCLUSIONS ST105-SCCmecII clones that colonized professionals in 2011 and 2012 were already circulating among patients in 2009, but there is no evidence that these clones spread to or between HIV/AIDS patients up to the 7th day of their hospitalization.

Sequence Analysis of Enterococcus faecium Strain 10/96A (VanD4), the Original Vancomycin-Resistant E. faecium Strain in Brazil

ABSTRACT Enterococcus faecium strain 10/96A (VanD4) was the first vancomycin-resistant enterococcus (VRE) isolated in Brazil. Subsequent Brazilian VRE strains have all had the VanA phenotype. Multilocus sequence typing showed that strain 10/96A was isolated sporadically, has a unique sequence type (ST 281), and was not the progenitor of the VRE strains isolated from hospital outbreaks in Brazil.