Impact of the Stringent Stress Response on the Expression of Methicillin Resistance in Staphylococcaceae Strains Carrying mecA, mecA1 and mecC

The acquisition of the resistance determinant mecA by Staphylococcus aureus is of major clinical importance, since it confers a resistant phenotype to virtually the entire large family of structurally diverse β-lactam antibiotics. While the common resistance determinant mecA is essential, the optimal expression of the resistance phenotype also requires additional factors. Previous studies showed that the great majority of clinical isolates of methicillin-resistant S. aureus (MRSA) have a heterogeneous resistant phenotype, and we observed that strains carrying methicillin genetic determinants other than mecA also produce similar heterogeneous phenotypes. All these strains were able to express high and homogeneous levels of oxacillin resistance when sub-inhibitory concentrations of mupirocin, an effector of the stringent stress response, were added to growth media. Our studies show that the gene gmk, involved in guanine metabolism, was one of the first genes to exhibit mutations in homoresistant (H*R) derivatives obtained through serial passages (with increasing concentrations of oxacillin) of the prototype mecC-carrying MRSA strain LGA251. All these observations led us to propose that a common molecular mechanism for the establishment of high and homogeneous oxacillin resistance must be present among isolates carrying different methicillin resistance determinants. In this work, we tested this hypothesis using whole-genome sequencing (WGS) to compare isogenic populations differing only in their degrees of oxacillin resistance and carrying various methicillin genetic determinant

Redefining a Structural Variant of Staphylococcal Cassette Chromosome mec, SCCmec Type VI

Previously we identified a SCCmec variant similar in size to type IV but with a new ccrAB allotype, allotype 4. We addressed the epidemiological relevance of this variant and found it among several strains belonging to the same clone. We propose to rename this structural variant SCCmec type VI

Update to the Multiplex PCR Strategy for Assignment of mec Element Types in Staphylococcus aureus▿

Staphylococcal cassette chromosome mec (SCCmec) typing is important for the identification and definition of methicillin-resistant Staphylococcus aureus clones, and for routine purposes, multiplex PCR assays are the most adequate for SCCmec typing. Here, we describe an update to the multiplex PCR strategy for SCCmec typing that we described in 2002 so that SCCmec types IV and V may be properly identified

The mechanism of heterogeneous beta-lactam resistance in MRSA: key role of the stringent stress response.

All methicillin resistant S. aureus (MRSA) strains carry an acquired genetic determinant--mecA or mecC--which encode for a low affinity penicillin binding protein -PBP2A or PBP2A'--that can continue the catalysis of peptidoglycan transpeptidation in the presence of high concentrations of beta-lactam antibiotics which would inhibit the native PBPs normally involved with the synthesis of staphylococcal cell wall peptidoglycan. In contrast to this common genetic and biochemical mechanism carried by all MRSA strains, the level of beta-lactam antibiotic resistance shows a very wide strain to strain variation, the mechanism of which has remained poorly understood. The overwhelming majority of MRSA strains produce a unique--heterogeneous--phenotype in which the great majority of the bacteria exhibit very poor resistance often close to the MIC value of susceptible S. aureus strains. However, cultures of such heterogeneously resistant MRSA strains also contain subpopulations of bacteria with extremely high beta-lactam MIC values and the resistance level and frequency of the highly resistant cells in such strain is a characteristic of the particular MRSA clone. In the study described in this communication, we used a variety of experimental models to understand the mechanism of heterogeneous beta-lactam resistance. Methicillin-susceptible S. aureus (MSSA) that received the mecA determinant in the laboratory either on a plasmid or in the form of a chromosomal SCCmec cassette, generated heterogeneously resistant cultures and the highly resistant subpopulations that emerged in these models had increased levels of PBP2A and were composed of bacteria in which the stringent stress response was induced. Each of the major heterogeneously resistant clones of MRSA clinical isolates could be converted to express high level and homogeneous resistance if the growth medium contained an inducer of the stringent stress response

Evidence for a purifying selection acting on the β-lactamase locus in epidemic clones of methicillin-resistant <it>Staphylococcus aureus</it>

Abstract Background The β-lactamase (bla) locus, which confers resistance to penicillins only, may control the transcription of mecA, the central element of methicillin resistance, which is embedded in a polymorphic heterelogous chromosomal cassette (the SCCmec element). In order to assess the eventual correlation between bla allotypes and genetic lineages, SCCmec types and/or β-lactam resistance phenotypes, the allelic variation on the bla locus was evaluated in a representative collection of 54 international epidemic methicillin-resistant Staphylococcus aureus (MRSA) clinical strains and, for comparative purposes, also in 24 diverse methicillin-susceptible S. aureus (MSSA) strains. Results Internal fragments of blaZ (the β-lactamase structural gene) were sequenced for all strains. A subset of strains, representative of blaZ allotypes, was further characterized by sequencing of internal fragments of the blaZ transcriptional regulators, blaI and blaR1. Thirteen allotypes for blaZ, nine for blaI and 12 for blaR1 were found. In a total of 121 unique single-nucleotide polymorphisms (SNP) detected, no frameshift mutations were identified and only one nonsense mutation within blaZ was found in a MRSA strain. On average, blaZ alleles were more polymorphic among MSSA than in MRSA (14.7 vs 11.4 SNP/allele). Overall, blaR1 was the most polymorphic gene with an average of 24.8 SNP/allele. No correlation could be established between bla allotypes and genetic lineages, SCCmec types and/or β-lactam resistance phenotypes. In order to estimate the selection pressure acting on the bla locus, the average dN/dS values were computed. In the three genes and in both collections dN/dS ratios were significantly below 1. Conclusions The data strongly suggests the existence of a purifying selection to maintain the bla locus fully functional even on MRSA strains. Although, this is in agreement with the notion that in most clinical MRSA strains mecA gene is under the control of the bla regulatory genes, these findings also suggest that the apparently redundant function of blaZ gene for the MRSA resistant phenotype is still important for these strains. In addition, the data shows that the sensor-inducer blaR1 is the primary target for the accumulation of mutations in the bla locus, presumably to modulate the response to the presence of β-lactam antibiotic.</p

Correction: The Mechanism of Heterogeneous Beta-Lactam Resistance in MRSA: Key Role of the Stringent Stress Response

Os estudos sobre as desigualdades sociais no Brasil concentram-se, em geral, nas dimensões objetivas do fenômeno, como sexo, cor, renda, ocupação e escolaridade. A amplitude do fosso entre ricos e pobres, graças às pesquisas já realizadas, é razoavelmente conhecida, mas pouco se sabe sobre a maneira como os brasileiros vêem essa disparidade. Em 2000, como parte do International Social Survey Programme tentou-se preencher essa lacuna com um survey aplicado a representantes das elites nacionais, entendendo-se por "elites" indivíduos pertencentes ao grupo dos 10% mais ricos do país. A pesquisa, de abrangência nacional - 2 mil entrevistas em 195 municípios -, buscou revelar diferenças entre as percepções da "elite" e do "povo" acerca da desigualdade. As questões levantadas pelo survey diziam respeito à imagem que os dois grupos têm da sociedade brasileira e da forma como ela está estruturada; as remunerações que seriam adequadas para trabalhadores de diferentes níveis de qualificação; os valores que deveriam prevalecer na distribuição da riqueza do país; os maiores problemas do Brasil; e as estratégias preferenciais de cada grupo para a redução da pobreza, com destaque para o papel do Estado. As respostas revelaram convergências e divergências surpreendentes entre povo e elite. Os dois grupos percebem a extensão das desigualdades sociais no país, mas tendem a defender estratégias distintas para reduzi-las, transferindo a solução das desigualdades ao Estado. Essas aproximações e diferenças de pontos de vista podem oferecer elementos importantes para se compreender os mecanismos de legitimação das desigualdades

The <i>mecR2</i> function is not dependent of <i>mecR1</i> neither of the β-lactamase locus.

<p>(A) Prototype strain HT0350 is negative for <i>mecR1-mecI</i> and for the β-lactamase locus. Co-overexpression of <i>mecI</i> and <i>mecR2</i> in strain HT0350 (HT0350+<i>mecI-mecR2</i>), reverts the effect of <i>mecI</i> overexpression (HT0350+<i>mecI</i>). (B) The strategy used to delete <i>mecR2</i> in prototype strain N315 generated an intermediate mutant that has lost the β-lactamase plasmid. The chromosomal <i>mecR2</i> deletion was then transduced back to the parental strain generating a β-lactamase positive <i>mecR2</i> null mutant. In both variants, the deletion of <i>mecR2</i> caused a sharp decrease of the resistance level to oxacillin. (C) Prototype strain HU25 is positive for <i>mecR2</i> and the β-lactamase locus and has a truncated non-functional MecI and, as such, the <i>mecA</i> expression is under the exclusive control of <i>bla</i> regulatory genes. Deletion of <i>mecR2</i> in strain HU25 (HU25::Δ<i>mecR2</i>) has no effect on the phenotypic expression of oxacillin resistance.</p