RESUMO
BACKGROUND: Healthcare-associated infections pose a potentially fatal threat to patients worldwide and Staphylococcus aureus is one of the most common causes of healthcare-associated infections. S. aureus is a common commensal pathogen and a frequent cause of bacteremia, with studies demonstrating that nasal and blood isolates from single patients match more than 80% of the time. Here we report on a contemporary collection of colonizing isolates from those with methicillin-resistant S. aureus (MRSA) bloodstream infections to evaluate the diversity within hosts, and detail the clinical features associated with concomitant nasal colonization. METHODS: Swabs of the bilateral anterior nares were obtained from patients diagnosed with MRSA bacteremia. A single colony culture from the blood and an average of 6 colonies from the nares were evaluated for MRSA growth. For the nares cultures, we typed multiple isolates for staphylococcal protein A (spa) and derived the clonal complexes. Demographic and clinical data were obtained retrospectively from the electronic medical record system and analysed using univariate and multivariable regression models. RESULTS: Over an 11-month period, 68 patients were diagnosed with MRSA bloodstream infection, 53 were swabbed, and 37 (70%) were colonized with MRSA in the anterior nares. We performed molecular typing on 213 nasal colonies. Spa types and clonal complexes found in the blood were also detected in the nares in 95% of the cases. We also found that 11% of patients carried more than one clone of MRSA in the nares. Male sex and history of prior hospitalization within the past 90 days increased odds for MRSA colonization. CONCLUSION: The molecular epidemiological landscape of colonization in the setting of invasive disease is diverse and defining the interplay between colonization and invasive disease is critical to combating invasive MRSA disease.
Assuntos
Bacteriemia , Infecção Hospitalar , Staphylococcus aureus Resistente à Meticilina , Infecções Estafilocócicas , Bacteriemia/epidemiologia , Portador Sadio , Infecção Hospitalar/epidemiologia , Humanos , Masculino , Staphylococcus aureus Resistente à Meticilina/genética , Nariz , Estudos Retrospectivos , Infecções Estafilocócicas/epidemiologia , Staphylococcus aureusRESUMO
OBJECTIVES: As part of an active MRSA surveillance programme in our neonatal ICU, we identified nares surveillance cultures from two infants that displayed heterogeneity in methicillin resistance between isolated subclones that lacked mecA and mecC. METHODS: The underlying mechanism for the modified Staphylococcus aureus (MODSA) methicillin-resistance phenotype was investigated by WGS. RESULTS: Comparison of finished-quality genomes of four MODSA and four MSSA subclones demonstrated that the resistance changes were associated with unique truncating mutations in the gene encoding the cyclic diadenosine monophosphate phosphodiesterase enzyme GdpP or a non-synonymous substitution in the gene encoding PBP2. CONCLUSIONS: These two cases highlight the difficulty in identifying non-mecA, non-mecC-mediated MRSA isolates in the clinical microbiology laboratory, which leads to difficulties in implementing appropriate therapy and infection control measures.
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Staphylococcus aureus Resistente à Meticilina , Infecções Estafilocócicas , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , Proteínas de Bactérias , Humanos , Recém-Nascido , Terapia Intensiva Neonatal , Staphylococcus aureus Resistente à Meticilina/genética , Testes de Sensibilidade Microbiana , Infecções Estafilocócicas/tratamento farmacológico , Infecções Estafilocócicas/epidemiologia , Staphylococcus aureusRESUMO
Methicillin-resistant Staphylococcus aureus (MRSA) strains carry either a mecA- or a mecC-mediated mechanism of resistance to beta-lactam antibiotics, and the phenotypic expression of resistance shows extensive strain-to-strain variation. In recent communications, we identified the genetic determinants associated with the stringent stress response that play a major role in the antibiotic resistant phenotype of the historically earliest "archaic" clone of MRSA and in the mecC-carrying MRSA strain LGA251. Here, we sought to test whether or not the same genetic determinants also contribute to the resistant phenotype of highly and homogeneously resistant (H*R) derivatives of a major contemporary MRSA clone, USA300. We found that the resistance phenotype was linked to six genes (fruB, gmk, hpt, purB, prsA, and relA), which were most frequently targeted among the analyzed 20 H*R strains (one mutation per clone in 19 of the 20 H*R strains). Besides the strong parallels with our previous findings (five of the six genes matched), all but one of the repeatedly targeted genes were found to be linked to guanine metabolism, pointing to the key role that this pathway plays in defining the level of antibiotic resistance independent of the clonal type of MRSA.
Assuntos
Antibacterianos/farmacologia , Resistência a Meticilina/genética , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Staphylococcus aureus Resistente à Meticilina/genética , Oxacilina/farmacologia , Proteínas de Bactérias/genética , Dinamarca , Guanina/metabolismo , Humanos , Staphylococcus aureus Resistente à Meticilina/isolamento & purificação , Testes de Sensibilidade Microbiana , Proteínas de Ligação às Penicilinas/genética , Reino Unido , Sequenciamento Completo do GenomaRESUMO
Penicillin-binding protein 4 (PBP4), a nonessential, low-molecular-weight penicillin-binding protein of Staphylococcus aureus, has been implicated in low-level resistance to ß-lactam antibiotics, although the mechanism is unknown. Mutations in PBP4 and its promoter were identified in a laboratory-generated mutant strain, CRB, which expresses high-level resistance to ß-lactams, including resistance to the new-generation cephalosporins active against methicillin-resistant strains of S. aureus These mutations did not appreciably alter the ß-lactam antibiotic binding affinity of purified recombinant mutant PBP4 compared to that of wild-type PBP4. Compared to the susceptible parent strain, COLnex, the CRB strain produces a highly cross-linked cell wall peptidoglycan, indicative of increased transpeptidase activity. The pbp4 promoter mutation of CRB was associated with greatly increased amounts of PBP4 in membranes compared to those in the COLnex parent. Replacement of the native promoter of COLnex with the mutant promoter of CRB resulted in increased amounts of PBP4 in membranes and a highly cross-linked cell wall. PBP4 can be repurposed to provide essential transpeptidase activity in vivo and confer high-level resistance to ß-lactam antibiotics, such as ceftobiprole and ceftaroline.
Assuntos
Staphylococcus aureus/efeitos dos fármacos , beta-Lactamas/farmacologia , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , Cefalosporinas/farmacologia , Cefalosporinas/uso terapêutico , Resistência a Meticilina/genética , Proteínas de Ligação às Penicilinas/genética , Proteínas de Ligação às Penicilinas/metabolismo , Infecções Estafilocócicas/tratamento farmacológico , Infecções Estafilocócicas/microbiologia , beta-Lactamas/uso terapêuticoRESUMO
OBJECTIVES: Recent surveillance of MRSA colonizing patients and healthcare workers in two African countries (Angola and São Tomé and Príncipe) reported the frequent recovery of oxacillin-susceptible MRSA (OS-MRSA): Staphylococcus aureus strains that gave positive results with the mecA DNA probe, but had low oxacillin MIC values characteristic of susceptible S. aureus. This apparent dissociation of the drug-resistant phenotype from mecA-the primary genetic determinant of resistance-prompted us to perform a more detailed analysis on nine of the African OS-MRSA strains. METHODS: Oxacillin MIC values were determined by Etest and population analysis profiles with and without induction of the stringent stress response by mupirocin. Biochemical profiling using SDS-PAGE followed by western blotting was used for the detection of PBP2A protein produced. RESULTS: Cultures of the African MRSA strains (ST88-IVa and ST8-V) showed heterogeneous oxacillin resistance in which the majority of cells exhibited low oxacillin MICs (≤0.75 mg/L), but highly resistant subpopulations were also present with oxacillin MIC values up to several hundred mg/L and with frequencies of 10(-4) to 10(-6). The same strains after induction of the stringent stress response by mupirocin 'converted' the heterogeneous phenotypes into a more homogeneous and higher level resistance. After induction by oxacillin and mupirocin, each of the nine African OS-MRSA strains produced PBP2A-the protein product of mecA. CONCLUSIONS: The resistant phenotype of OS-MRSA resembles the phenotypes of historically early MRSA clones. The nature of genetic determinants responsible for the heterogeneous phenotypes of OS-MRSA remains to be determined.
Assuntos
Antibacterianos/farmacologia , Proteínas de Bactérias/biossíntese , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Staphylococcus aureus Resistente à Meticilina/isolamento & purificação , Oxacilina/farmacologia , Proteínas de Ligação às Penicilinas/biossíntese , África/epidemiologia , Angola/epidemiologia , Proteínas de Bactérias/genética , Farmacorresistência Bacteriana Múltipla/genética , Eletroforese em Gel de Poliacrilamida , Monitoramento Epidemiológico , Humanos , Staphylococcus aureus Resistente à Meticilina/metabolismo , Testes de Sensibilidade Microbiana , Proteínas de Ligação às Penicilinas/genética , Fenótipo , Infecções Estafilocócicas/epidemiologia , Infecções Estafilocócicas/microbiologia , Resistência beta-Lactâmica/genéticaRESUMO
In November 2011, The Rockefeller University Center for Clinical and Translational Science (CCTS), the Laboratory of Microbiology and Infectious Diseases, and Clinical Directors Network (CDN) launched a research and learning collaborative project with six community health centers in the New York City metropolitan area to determine the nature (clonal type) of community-acquired Staphylococcus aureus strains causing skin and soft tissue infections (SSTIs). Between November 2011 and March 2013, wound and nasal samples from 129 patients with active SSTIs suspicious for S. aureus were collected and characterized by molecular typing techniques. In 63 of 129 patients, the skin wounds were infected by S. aureus: methicillin-resistant S. aureus (MRSA) was recovered from 39 wounds and methicillin-sensitive S. aureus (MSSA) was recovered from 24. Most-46 of the 63-wound isolates belonged to the CC8/Panton-Valentine leukocidin-positive (PVL(+)) group of S. aureus clone USA300: 34 of these strains were MRSA and 12 were MSSA. Of the 63 patients with S. aureus infections, 30 were also colonized by S. aureus in the nares: 16 of the colonizing isolates were MRSA, and 14 were MSSA, and the majority of the colonizing isolates belonged to the USA300 clonal group. In most cases (70%), the colonizing isolate belonged to the same clonal type as the strain involved with the infection. In three of the patients, the identity of invasive and colonizing MRSA isolates was further documented by whole-genome sequencing.
Assuntos
Portador Sadio/microbiologia , Genótipo , Tipagem Molecular , Infecções dos Tecidos Moles/microbiologia , Infecções Estafilocócicas/microbiologia , Infecções Cutâneas Estafilocócicas/microbiologia , Staphylococcus aureus/classificação , Portador Sadio/epidemiologia , Centros Comunitários de Saúde , Infecções Comunitárias Adquiridas/epidemiologia , Infecções Comunitárias Adquiridas/microbiologia , Variação Genética , Humanos , Resistência a Meticilina , Epidemiologia Molecular , Cidade de Nova Iorque/epidemiologia , Nariz/microbiologia , Infecções dos Tecidos Moles/epidemiologia , Infecções Estafilocócicas/epidemiologia , Infecções Cutâneas Estafilocócicas/epidemiologia , Staphylococcus aureus/genética , Staphylococcus aureus/isolamento & purificação , Ferimentos e Lesões/microbiologiaRESUMO
Objective: Dual carbapenemase-producing organisms (DCPOs) are an emerging threat that expands the spectrum of antimicrobial resistance. There is limited literature on the clinical and genetic epidemiology of DCPOs. Methods: DCPO isolates were identified by Xpert® Carba-R PCR testing of routine diagnostic cultures performed from 2018 to 2021 at a New York City health system. WGS was performed by Illumina and/or PacBio. Medical records of patients were reviewed for clinical and epidemiological data. Results: Twenty-six DCPO isolates were obtained from 13 patients. Klebsiella pneumoniae (nâ=â22) was most frequent, followed by Pseudomonas aeruginosa (nâ=â2), Escherichia coli (nâ=â1) and Enterobacter cloacae (nâ=â1). The most common DCPO combination was blaNDM/blaOXA-48-like (nâ=â16). Notably, 1.05% (24/2290) of carbapenem-resistant Enterobacterales isolates were identified as DCPOs. The susceptibility profiles matched the identified resistance genes, except for a K. pneumoniae (blaKPC/blaOXA-48-like) isolate that was phenotypically susceptible to meropenem. Eleven patients were hospitalized within the year prior to admission, and received antibiotic(s) 1â month prior. Seven patients were originally from outside the USA. Hypertension, kidney disease and diabetes were frequent comorbidities. Death in two cases was attributed to DCPO infection. WGS of eight isolates showed that carbapenemases were located on distinct plasmids, except for one K. pneumoniae isolate where NDM and KPC carbapenemases were located on a single IncC-type plasmid backbone. Conclusions: Here we characterized a series of DCPOs from New York City. Foreign travel, prior hospitalization, antibiotic usage and comorbidities were common among DCPO cases. All carbapenemases were encoded on plasmids, which may facilitate horizontal transfer.
RESUMO
The epidemic community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) USA300 lineage has recently become a leading cause of hospital-associated bloodstream infections (BSIs). Here, we leveraged this recent introduction into hospitals and the limited genetic variation across USA300 isolates to identify mutations that contribute to its success in a new environment. We found that USA300 BSI isolates exhibit altered virulence regulation. Using comparative genomics to delineate the genes involved in this phenotype, we discovered repeated and independent mutations in the transcriptional regulator sarZ. Mutations in sarZ resulted in increased virulence of USA300 BSI isolates in a murine model of BSI. The sarZ mutations derepressed the expression and production of the surface protein ClfB, which was critical for the pathogenesis of USA300 BSI isolates. Altogether, these findings highlight ongoing evolution of a major MRSA lineage and suggest USA300 strains can optimize their fitness through altered regulation of virulence.
Assuntos
Infecção Hospitalar , Staphylococcus aureus Resistente à Meticilina , Sepse , Infecções Estafilocócicas , Animais , Camundongos , Staphylococcus aureus Resistente à Meticilina/genética , Virulência/genética , Infecção Hospitalar/epidemiologiaRESUMO
We describe a case of Lemierre's syndrome (LS) caused by a hypervirulent strain of Klebsiella pneumoniae in a 63-year-old female with hypertension, hyperlipidemia, and diabetes mellitus, who presented with right neck pain and fevers. Computerized tomography of the neck and chest revealed an occluded right internal jugular vein secondary to thrombosis and septic emboli in lungs. Blood cultures grew K. pneumoniae. The patient was treated with ampicillin-sulbactam and then transitioned to amoxicillin-clavulanate to complete a 6-week course of antibiotics, and a 3-month course of rivaroxaban. String test of the K. pneumoniae isolate was positive at 2 cm. Whole genome sequencing identified several genes associated with the hypervirulent strain, notably the genes encoding for aerobactin (iucA and iucB) and salmochelin (iroB) iron acquisition systems. LS can rarely be caused by K. pneumoniae. Clinicians should monitor for known complications, such as septic emboli in patients with LS.
RESUMO
BACKGROUND: Whole-genome sequencing (WGS) is increasingly used to map the spread of bacterial and viral pathogens in nosocomial settings. A limiting factor for more widespread adoption of WGS for hospital infection prevention practices is the availability of standardized tools for genomic epidemiology. METHODS: We developed the Pathogen Sequencing Phylogenomic Outbreak Toolkit (PathoSPOT) to automate integration of genomic and medical record data for rapid detection and tracing of nosocomial outbreaks. To demonstrate its capabilities, we applied PathoSPOT to complete genome surveillance data of 197 MRSA bacteremia cases from two hospitals during a 2-year period. RESULTS: PathoSPOT identified 8 clonal clusters encompassing 33 patients (16.8% of cases), none of which had been recognized by standard practices. The largest cluster corresponded to a prolonged outbreak of a hospital-associated MRSA clone among 16 adults, spanning 9 wards over a period of 21 months. Analysis of precise timeline and location data with our toolkit suggested that an initial exposure event in a single ward led to infection and long-term colonization of multiple patients, followed by transmissions to other patients during recurrent hospitalizations. CONCLUSIONS: We demonstrate that PathoSPOT genomic surveillance enables the detection of complex transmission chains that are not readily apparent from epidemiological data and that contribute significantly to morbidity and mortality, enabling more effective intervention strategies.
Assuntos
Infecção Hospitalar/epidemiologia , Surtos de Doenças , Genômica , Epidemiologia Molecular , Adolescente , Adulto , Idoso , Bacteriemia/microbiologia , Infecção Hospitalar/microbiologia , Infecção Hospitalar/prevenção & controle , Infecção Hospitalar/transmissão , Surtos de Doenças/prevenção & controle , Feminino , Genoma Bacteriano , Hospitais , Humanos , Masculino , Staphylococcus aureus Resistente à Meticilina/genética , Staphylococcus aureus Resistente à Meticilina/isolamento & purificação , Pessoa de Meia-Idade , Filogenia , Infecções Estafilocócicas/epidemiologia , Infecções Estafilocócicas/transmissão , Sequenciamento Completo do Genoma , Adulto JovemRESUMO
An outbreak of methicillin-resistant Staphylococcus aureus (MRSA) infections on the skin and soft tissues of experimental macaques in the vivarium of The Rockefeller University, New York, triggered this observational and interventional study. We screened 14 macaques in the colony (samples from head, nares, and rectum) and their housing (40 environmental surfaces) four times in 1 year, for S. aureus colonization or contamination, while implementing enhanced decolonization and decontamination procedures. A total of 114 isolates of S. aureus were recovered and characterized (antibiograms, spa typing, multilocus sequence typing, pulsed-field gel electrophoresis [PFGE], mecA, Panton-Valentine Leukocidin, and arginine catabolic mobile element). Based on these results, six strains of S. aureus were identified: two MRSA strains (t16708/ST3862/PFGE-A, t16709/ST3862/PFGE-C) and one methicillin-sensitive S. aureus (t8397/ST3884/PFGE-D) were characterized for the first time in this study; strains belonging to spa types t189 and t4167 have been identified in primates in previous studies. None of these strains was common to the neighboring New York City human community. Thus, it seems probable that the animals were already colonized upon arrival to the University. We suggest screening primates for S. aureus carriage upon arrival to University vivaria and possible implementation of extensive decolonization procedures before any surgical interventions.
Assuntos
Macaca/microbiologia , Staphylococcus aureus Resistente à Meticilina/isolamento & purificação , Infecções Estafilocócicas/microbiologia , Animais , Antibacterianos/farmacologia , Arginina/genética , Proteínas de Bactérias/genética , Toxinas Bacterianas/genética , Eletroforese em Gel de Campo Pulsado/métodos , Exotoxinas/genética , Genótipo , Humanos , Leucocidinas/genética , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Testes de Sensibilidade Microbiana/métodos , Tipagem de Sequências Multilocus/métodos , Cidade de Nova Iorque , Proteínas de Ligação às Penicilinas/genética , Fatores de Virulência/genéticaRESUMO
Multidrug-resistant strains of Staphylococcus aureus continue to increase in frequency worldwide, both in hospitals and in the community, raising serious problems for the chemotherapy of staphylococcal disease. Ceftobiprole (BPR; BAL9141), the active constituent of the prodrug ceftobiprole medocaril (BAL5788), is a new cephalosporin which was already shown to have powerful activity against a number of bacterial pathogens, including S. aureus. In an effort to test possible limits to the antibacterial spectrum and efficacy of BPR, we examined the susceptibilities of the relatively few pandemic methicillin-resistant S. aureus (MRSA) clones that are responsible for the great majority of cases of staphylococcal disease worldwide. We also included in the tests the highly oxacillin-resistant subpopulations that are present with low frequencies in the cultures of these clones. Such subpopulations may represent a natural reservoir from which MRSA strains with decreased susceptibility to BPR may emerge in the future. We also tested the efficacy of BPR against MRSA strains with reduced susceptibility to vancomycin and against MRSA strains carrying the enterococcal vancomycin resistance gene complex. BPR was shown to be uniformly effective against all these resistant MRSA strains, and the mechanism of superb antimicrobial activity correlated with the strikingly increased affinity of the cephalosporin against penicillin-binding protein 2A, the protein product of the antibiotic resistance determinant mecA.
Assuntos
Cefalosporinas/farmacologia , Resistência a Meticilina/efeitos dos fármacos , Oxacilina/farmacologia , Staphylococcus aureus/efeitos dos fármacos , Antibacterianos/farmacologia , Proteínas de Bactérias/genética , Cromatografia Líquida de Alta Pressão , Humanos , Testes de Sensibilidade Microbiana , Infecções Estafilocócicas/microbiologia , Staphylococcus aureus/genética , Vancomicina/farmacologiaRESUMO
Addition of ß-lactam antibiotics to growing cultures of bacteria inhibit synthesis of the bacterial cell wall peptidoglycan accompanied by killing (loss of viable titer) and lysis (physical disintegration) of the cells. However, it has also been well established that these antibiotics are not effective in killing non-growing or slow-growing bacteria and the mechanism of this "antibiotic tolerance" is not well understood. In this study, we report on the genetic basis and phenotypic properties of an antibiotic tolerant derivative of the methicillin susceptible S. aureus strain 27s. Cultures were exposed to "pulses" of high concentrations of oxacillin followed by outgrowth of the surviving bacteria. This procedure quickly selected for antibiotic tolerant mutants with an increased ability to survive antibiotic treatment without increase in the MIC value for the antibiotic. Such mutants also exhibited longer lag phase, decreased lysis, virtually no change in antibiotic susceptibilities, cross tolerance to D-cycloserine and vancomycin, and increase in biofilm formation in the presence of high concentrations of oxacillin. Whole genome sequencing showed that these altered properties were linked to mutations in the atl and gdpP genes.
Assuntos
Biofilmes/crescimento & desenvolvimento , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Mutação , Oxacilina/farmacologia , Fenótipo , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/genética , Antibacterianos/farmacologia , Biofilmes/efeitos dos fármacos , Farmacorresistência Bacteriana , Genoma Bacteriano , Genótipo , Testes de Sensibilidade Microbiana , Infecções Estafilocócicas/tratamento farmacológico , Infecções Estafilocócicas/genética , Infecções Estafilocócicas/microbiologia , Staphylococcus aureus/crescimento & desenvolvimento , Sequenciamento Completo do GenomaRESUMO
BACKGROUND: Persons with acquired immune deficiency syndrome (AIDS) who use drugs appear to be at increased risk for colonization and infection with Staphylococcus aureus. Little is known about the nature of and risk factors responsible for this association. This study is among the first to prospectively follow carriage and infection in this uniquely high-risk population. METHODS: We prospectively followed the cases of 75 patients with AIDS in a residential drug treatment facility and screened for S. aureus nasal colonization and infection. RESULTS: Thirty-seven baseline cultures (49%) were positive for S. aureus, and 81% of subjects were colonized at least once during the study. Thirteen subjects experienced 17 infections. Pulsed-field gel electrophoresis and sequence-based typing methods revealed that 244 (92%) of the isolates belonged to either clonal type A or B. Clonal type A was methicillin-susceptible. Clonal type B consisted of 3 main subtypes (B1, B2, and B3), all with the same allelic profile (ST8) and staphylococcal protein A gene (spa) type (7). Of note, subtype B1 was methicillin-susceptible (ST8 and spa type 7), lacking mecA, whereas the other B clones were methicillin-resistant. Both clones were resistant to trimethoprim-sulfamethoxazole. Clonal type B isolates were relatively resistant, suggesting prior exposure to the health care setting. CONCLUSIONS: This study demonstrates a sustained high rate of S. aureus carriage and infection. It demonstrates the capacity of unique methicillin-resistant S. aureus clones with an established linkage to earlier outbreaks of methicillin-resistant S. aureus, as well as to human immunodeficiency virus--infected subjects, to persist in this residential setting. It also illustrates the apparent genetic instability or transmissibility of the staphylococcal chromosomal cassette mec type IV element.
Assuntos
Síndrome da Imunodeficiência Adquirida/complicações , Epidemiologia Molecular , Infecções Estafilocócicas/epidemiologia , Infecções Estafilocócicas/microbiologia , Staphylococcus aureus/classificação , Staphylococcus aureus/isolamento & purificação , Adulto , Antibacterianos , Portador Sadio , Farmacorresistência Bacteriana , Feminino , Genótipo , Humanos , Masculino , Pessoa de Meia-Idade , Nariz/microbiologia , Fenótipo , Filogenia , Fatores de Risco , Infecções Estafilocócicas/etiologia , Staphylococcus aureus/genéticaRESUMO
We identified mutated genes in highly resistant subpopulations of methicillin-resistant Staphylococcus aureus (MRSA) that are most likely responsible for the historic failure of the ß-lactam family of antibiotics as therapeutic agents against these important pathogens. Such subpopulations are produced during growth of most clinical MRSA strains, including the four historically early MRSA isolates studied here. Chromosomal DNA was prepared from the highly resistant cells along with DNA from the majority of cells (poorly resistant cells) followed by full genome sequencing. In the highly resistant cells, mutations were identified in 3 intergenic sequences and 27 genes representing a wide range of functional categories. A common feature of these mutations appears to be their capacity to induce high-level ß-lactam resistance and increased amounts of the resistance protein PBP2A in the bacteria. The observations fit a recently described model in which the ultimate controlling factor of the phenotypic expression of ß-lactam resistance in MRSA is a RelA-mediated stringent response. IMPORTANCE It has been well established that the level of antibiotic resistance (i.e., minimum concentration of a ß-lactam antibiotic needed to inhibit growth) of a methicillin-resistant Staphylococcus aureus (MRSA) strain depends on the transcription and translation of the resistance protein PBP2A. Here we describe mutated loci in an additional novel set of genetic determinants that appear to be essential for the unusually high resistance levels typical of subpopulations of staphylococci that are produced with unique low frequency in most MRSA clinical isolates. We propose that mutations in these determinants can trigger induction of the stringent stress response which was recently shown to cause increased transcription/translation of the resistance protein PBP2A in parallel with the increased level of resistance.
Assuntos
Farmacorresistência Bacteriana , Genes Bacterianos , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Staphylococcus aureus Resistente à Meticilina/genética , DNA Bacteriano/química , DNA Bacteriano/genética , DNA Bacteriano/isolamento & purificação , Regulação Bacteriana da Expressão Gênica , Genoma Bacteriano , Humanos , Dados de Sequência Molecular , Mutação , Análise de Sequência de DNARESUMO
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.
Assuntos
Antibacterianos/farmacologia , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Resistência beta-Lactâmica , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Relação Dose-Resposta a Droga , Regulação Bacteriana da Expressão Gênica , Humanos , Staphylococcus aureus Resistente à Meticilina/fisiologia , Testes de Sensibilidade Microbiana , Modelos Biológicos , Mutação , Oxacilina/farmacologia , Proteínas de Ligação às Penicilinas , Fenótipo , Estresse Fisiológico , Resistência beta-Lactâmica/genéticaRESUMO
The overwhelming majority of methicillin-resistant Staphylococcus aureus (MRSA) clinical isolates exhibit a peculiar heterogeneous resistance to ß-lactam antibiotics: in cultures of such strains, the majority of cells display only a low level of methicillin resistance--often close to the MIC breakpoint of susceptible strains. Yet, in the same cultures, subpopulations of bacteria exhibiting very high levels of resistance are also present with variable frequencies, which are characteristic of the particular MRSA lineage. The mechanism of heterogeneous resistance is not understood. We describe here an experimental system for exploring the mechanism of heterogeneous resistance. Copies of the resistance gene mecA cloned into a temperature-sensitive plasmid were introduced into the fully sequenced methicillin-susceptible clinical isolate S. aureus strain 476. Transductants of strain 476 expressed methicillin resistance in a heterogeneous fashion: the great majority of cells showed only low MIC (0.75 µg/ml) for the antibiotic, but a minority population of highly resistant bacteria (MIC >300 µg/ml) was also present with a frequency of â¼10(-4). The genetic backgrounds of the majority and minority cells were compared by whole-genome sequencing: the only differences detectable were two point mutations in relA of the highly resistant minority population of bacteria. The relA gene codes for the synthesis of (p)ppGpp, an effector of the stringent stress response. Titration of (p)ppGpp showed increased amounts of this effector in the highly resistant cells. Involvement of (p)ppGpp synthesis genes may explain some of the perplexing aspects of ß-lactam resistance in MRSA, since many environmental and genetic changes can modulate cellular levels of (p)ppGpp.
Assuntos
Antibacterianos/farmacologia , Ligases/genética , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Oxacilina/farmacologia , Antibacterianos/administração & dosagem , Proteínas de Bactérias/genética , Genoma Bacteriano , Staphylococcus aureus Resistente à Meticilina/genética , Testes de Sensibilidade Microbiana , Oxacilina/administração & dosagem , Proteínas de Ligação às Penicilinas , Resistência beta-Lactâmica/genéticaRESUMO
Expression of high-level beta-lactam resistance is known to be thermosensitive in many methicillin-resistant Staphylococcus aureus (MRSA) strains, including strain COL, in which the high methicillin MIC for cultures grown at 37 degrees C (800 microg/ml) was reduced to 12 microg/ml at 42 degrees C. COL grew faster at 42 degrees C than at 37 degrees C and at the higher temperature produced cell walls of abnormal composition: there was an over-representation of the monomeric muropeptide without the oligoglycine chain and an increase in the representation of multimers that contained this wall component as the donor molecule. Screening of a Tn551 insertional library for mutants, in which the high and homogenous beta-lactam antibiotic resistance of strain COL is retained at 42 degrees C, identified mutant C245, which expressed high-level methicillin resistance and produced a cell wall of normal composition independent of the temperature. The Tn551 inactivated gene was found, by homology search, to encode for a sodium-dependent symporter, homologues of which are ubiquitous in both prokaryotic and eukaryotic genomes. Inactivation of this putative symporter in several heteroresistant clinical MRSA isolates caused striking increases in the level of their beta-lactam resistance.
Assuntos
Parede Celular/metabolismo , Temperatura Alta , Sódio/metabolismo , Staphylococcus aureus/efeitos dos fármacos , Simportadores/metabolismo , Resistência beta-Lactâmica , Antibacterianos/farmacologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Elementos de DNA Transponíveis , Humanos , Meticilina/farmacologia , Resistência a Meticilina , Testes de Sensibilidade Microbiana , Mutação , Proteínas de Ligação às Penicilinas , Staphylococcus aureus/genética , Staphylococcus aureus/crescimento & desenvolvimento , Staphylococcus aureus/metabolismo , Simportadores/químicaRESUMO
A total of 202 methicillin-resistant Staphylococcus aureus (MRSA) single-patient isolates recovered between January and June 1998 in two hospitals in Miami, Florida, were characterized by a combination of several molecular typing techniques: multilocus sequence typing, spaA typing, pulsed-field gel electrophoresis, and determination of the structure of the SCCmec element. The overwhelming majority of the isolates-187 of 202, or 93%-belonged to one of three internationally spread epidemic clones which were identified on the basis of their multilocus sequence type (ST) as E-MRSA-16 (ST36), the New York clone V (ST8), and the New York/Japan clone (ST5; SCCmec II) and its single- and double-locus variants. The rest of the isolates (15 of 202, or 7%) were more genetically diverse and were each recovered from a few patients only. Of the 23 MRSA strains isolated from confirmed human immunodeficiency virus-positive patients, as many as 17 (or 70%) belonged to a single ST8 clone carrying SCCmec type IV. The data provide further evidence for the conclusion of earlier studies that most MRSA disease in hospitals is caused by relatively few pandemic clones.