PBP2a Production Research Articles

Detection of mecA-mediated methicillin resistance and evaluation of disk-diffusion antimicrobial susceptibility characteristics of Staphylococcus saprophyticus isolates from geographically diverse locations: Staphylococcus saprophyticus antibiotic susceptibility testing

Staphylococcus saprophyticus, a common uropathogen, is usually susceptible to urine-concentrating antimicrobials, so routine AST is not recommended by CLSI. Our study evaluated the antimicrobial resistance profiles of 277 S. saprophyticus isolates from North America and a globally diverse cohort. Notably, 24% (67/277) of our isolates come from non-urinary sources. AST was performed against 12 antimicrobials using standard disk diffusion, PCR for mecA and mecC, PBP2a production assays, and cefinase. 5% (13/277) of isolates were mecA positive and cefinase positive, 63% (176/277) were mecA negative but cefinase positive, 4% (11/277) were mecA positive but cefinase negative, and 28% (77/277) were mecA and cefinase negative. All (277/277) isolates were susceptible to delafloxacin, ciprofloxacin, rifampin, linezolid, and nitrofurantoin and 95% (262/277) were susceptible to trimethoprim-sulfamethoxazole. Our results showed that regardless of using CLSI or EUCAST breakpoints oxacillin had low categorical agreement for mecA presence, making it unsuitable for surrogate testing, while cefoxitin disk diffusion had high very major error rate. If possible, PBP2a or mecA testing is recommended for guiding therapy for non-urinary infections. Our work supports CLSI guidelines on routine susceptibility to urinary tract antibiotics.

Infecciones por estafilococos

Staphylococci encompass a multitude of species with different pathological capacities in human beings, with Staphylococcus aureus (SA) standing out as the most virulent species. SA is capable of producing a multitude of clinical syndromes, both mediated by the toxin and by tissue invasion and destruction. Of note for their frequency and severity are skin and soft tissue infections, bone and joint infections, bacteremia, endocarditis, prosthetic device infections, and pneumonia (including hemorrhagic pneumonia in young patients). It can have various mechanisms of resistance, of which resistance to beta-lactams (methicillin-resistant SA, MRSA) mediated by the production of PBP2a (mecA gene) is of note for its therapeutic importance. Treatment of methicillin-sensitive SA is based on cloxacillin or cefazolin whereas treatment options for MRSA are vancomycin, daptomycin, or linezolid. Coagulase-negative staphylococci are less virulent, although they tend to be resistant to methicillin. They only cause infections in patients with predisposing conditions (immunosuppressed, prosthetic material, venous access, etc.), with the exception of Staphylococcus lugdunensis, which can cause infections similar to SA and which also tend to be sensitive to penicillin.

Staphylococcal cassette chromosome mec elements from Staphylococcus intermedius group (SIG) isolates from dogs in a center for veterinary diagnostics in Brazil

ABSTRACT: Methicillin resistance in the Staphylococcus intermedius group (SIG) has emerged in small animal practice. Methicillin-resistant SIG (MRSIG) members have been implicated as causes of infections in both companion animals and humans. Staphylococcal cassette chromosome mec (SCCmec) elements carry the mecA/C genes, which encode for the transpeptidase PBP2a (PBP2’) responsible for β-lactam antibiotic resistance in staphylococci. This study examined the SCCmec types of MRSIG isolates from different clinical specimens of dogs that exhibited methicillin MIC ≥ 0.5 μg/mL by an automated identification and susceptibility system in a Center for Veterinary Diagnostics in São Paulo, Brazil. Susceptibility to methicillin was determined by broth microdilution testing, and Oxoid® M.I.C.Evaluator® strips. PBP2a production was detected using a latex agglutination assay. SCCmec typing was performed according to the International Working Group on the Classification of Staphylococcal Cassette Chromosome Elements (IWG-SCC) guidelines. SCCmec type II (2A), SCCmec type III (3A), composite SCC structures consisting of a class A mec gene complex in addition to multiple ccr gene complexes, and non-typable SCCmec elements were reported in these MRSIG isolates. SCCmec type variants differing from those so far acknowledged by IWG-SCC were found, indicating new rearrangements in the genetic context of mecA in these canine MRSIG isolates.

Identification and characterization of mutations responsible for the \u03b2-lactam resistance in oxacillin-susceptible mecA-positive Staphylococcus aureus

Staphylococcus aureus strains that are susceptible to the β-lactam antibiotic oxacillin despite carrying mecA (OS-MRSA) cause serious clinical problems globally because of their ability to easily acquire β-lactam resistance. Understanding the genetic mechanism(s) of acquisition of the resistance is therefore crucial for infection control management. For this purpose, a whole-genome sequencing-based analysis was performed using 43 clinical OS-MRSA strains and 100 mutants with reduced susceptibility to oxacillin (MICs 1.0–256 µg/mL) generated from 26 representative OS-MRSA strains. Genome comparison between the mutants and their respective parent strains identified a total of 141 mutations in 46 genes and 8 intergenic regions. Among them, the mutations are frequently found in genes related to RNA polymerase (rpoBC), purine biosynthesis (guaA, prs, hprT), (p)ppGpp synthesis (relSau), glycolysis (pykA, fbaA, fruB), protein quality control (clpXP, ftsH), and tRNA synthase (lysS, gltX), whereas no mutations existed in mec and bla operons. Whole-genome transcriptional profile of the resistant mutants demonstrated that expression of genes associated with purine biosynthesis, protein quality control, and tRNA synthesis were significantly inhibited similar to the massive transcription downregulation seen in S. aureus during the stringent response, while the levels of mecA expression and PBP2a production were varied. We conclude that a combination effect of mecA upregulation and stringent-like response may play an important role in acquisition of β-lactam resistance in OS-MRSA.

Heterogeneous oxacillin-resistant phenotypes and production of PBP2A by oxacillin-susceptible/mecA-positive MRSA strains from Africa

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. 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. 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. 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.

Synergistic Anti-bacterial Effects of Phellinus baumii Ethyl Acetate Extracts and β-Lactam Antimicrobial Agents Against Methicillin-Resistant Staphylococcus aureus.

BackgroundThe development of new drugs or alternative therapies effective against methicillin-resistant Staphylococcus aureus (MRSA) is of great importance, and various natural anti-MRSA products are good candidates for combination therapies. We evaluated the antibacterial activities of a Phellinus baumii ethyl acetate extract (PBEAE) and its synergistic effects with β-lactams against MRSA.MethodsThe broth microdilution method was used to determine the minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) of the PBEAE. The PBEAE synergistic effects were determined by evaluating the MICs of anti-staphylococcal antibiotic mixtures, with or without PBEAE. Anti-MRSA synergistic bactericidal effects of the PBEAE and β-lactams were assessed by time-killing assay. An ELISA was used to determine the effect of the PBEAE on penicillin binding protein (PBP)2a production.ResultsThe MICs and MBCs of PBEAE against MRSA were 256-512 and 1,024-2,048 µg/mL, respectively. The PBEAE significantly reduced MICs of all β-lactams tested, including oxacillin, cefazolin, cefepime, and penicillin. However, the PBEAE had little or no effect on the activity of non-β-lactams. Time-killing assays showed that the synergistic effects of two β-lactams (oxacillin and cefazolin) with the PBEAE were bactericidal in nature (Δlog10 colony forming unit/mL at 24 hr: 2.34-2.87 and 2.10-3.04, respectively). The PBEAE induced a dose-dependent decrease in PBP2a production by MRSA, suggesting that the inhibition of PBP2a production was a major synergistic mechanism between the β-lactams and the PBEAE.ConclusionsPBEAE can enhance the efficacy of β-lactams for combined therapy in patients infected with MRSA.

Staphylococcus aureus y resistencia antimicrobiana en dos hospitales de Tegucigalpa, 2011-2012

El objetivo de esta investigación es determinar la resistencia antimicrobiana del Staphylococcus aureus en dos hospitales de Tegucigalpa. Para lograrlo se siguió una metodología de estudio descriptivo, transversal. Se recolectaron todas las muestras de Staphylococcus aureus aisladas de pacientes, de los laboratorios de dos hospitales, durante un periodo de 5 meses, en cada institución; las que se trasladaron al Laboratorio de Bacteriología de la Escuela de Microbiología de la UNAH; para el estudio de sensibilidad antibiótica y la identificación de los SARM, mediante el método de Kirby-Bauer y según normas establecidas por el Clinical and Laboratory Standards Institute (CLSI); para los que se usaron diferentes antibióticos. También se realizó la determinación de la concentración inhibitoria mínima (CIM) a OX usando E- test y la determinación de la producción de PBP2a, por el método de aglutinación en látex. Los datos fueron analizados con estadística descriptiva con el paquete estadístico EPI-INFO, versión 3.5.1. para Windows. Se obtuvieron 90 aislamientos de S. aureus del laboratorio del Hospital Escuela y 106 de S. aureus en el laboratorio del IHSS. La mayoría de las bacterias aisladas provenían de exudados, los cuales presentaron resistencia a diferentes antibióticos y ninguna bacteria aislada presentó resistencia a Vancomicina. La resistencia a Oxacilina o SARM fue de un 14.3 % (IC95 % = 9.7 a 20). Se confirmó la resistencia antimicrobiana de S. aureus a Oxacilina o SARM por el método de Kirby-Bauer, Etest, PBP2.En conclusión, la prevalencia de SARM no es tan alta, basada en los parámetros para los países en vías de desarrollo y no se encontró resistencia a Vancomicina. DOI: http://dx.doi.org/10.5377/rct.v0i13.1712 Revista Ciencia y Tecnología, No. 13, diciembre 2013: 30-40

Frequency of <i>Staphylococcal</i> Cassette Chromosome <i>mec</i> Type IV and Type V in Clinical Isolates of Methicillin Resistant <i>Staphylococcus aureus</i>

MRSA is able to generate a modified variety of penicillin binding protein named as PBP2a instead of PBP which makes it resistant against penicillin and methicillin. Production of PBP2a is due to the presence of a gene on Staphylococcal cassette chromosome or SCC termed as “mec-A gene”. SCC is a mobile genetic element carrying many resistance genes. It is because of current antibiotic selection pressure that by now there are eight types (I - VIII) of SCCmec. This research has been designed to determine frequency of SCCmec type IV and V in clinical isolates of MRSA. A total of 70 presumptive MRSA isolates collected from a tertiary care hospital of Lahore were cultured on blood agar, incubated overnight at 37&degC aerobically. Next day, they were examined for cultural characteristics, colonial morphology, gram stain, and biochemical profile. Confirmation of MRSA was done by phenotypic disk diffusion method according to (CLSI) 2013 guidelines. mecA gene was also detected at molecular level. Molecular identification of SCCmec type IV and V was done by Nested PCR strategy. A total of 50 isolates were confirmed to be MRSA Molecular detection of SCCmec type IV and V revealed that 11 isolates (22%) possess SCCmec type IV and only 2 isolate (4%) carries SCCmec type V. It is obvious from results that SCCmec type IV and V are present in our population too. Larger study (with larger sample size) might be undertaken to find out actual emergence of SCCmec type IV and V in our population.

Role of the mecA Gene in Oxacillin Resistance in a Staphylococcus aureus Clinical Strain with a pvl -Positive ST59 Genetic Background

The most prevalent community-associated methicillin-resistant Staphylococcus aureus (C-MRSA) strains in Taiwan, sequence type 59 (ST59) clones, carry staphylococcal cassette chromosome mec (SCCmec) type V and, to a lesser extent, type IV. These strains show wide variation in sensitivity to oxacillin, but the reasons for this variation are unknown. Here we compared the sequences of the mecA genes from clinical strains of different SCCmec types and found that they contain different mecA promoter mutations. Analysis of mecA promoter activity by reporter gene fusions showed that single base substitutions in the promoter have a strong influence on mecA transcription. The different mecA variants, including promoter sequences, were expressed in the methicillin-sensitive Staphylococcus aureus (MSSA) strain C195 (ST59 background). PBP 2a production among the parental strains and strains with promoter mutant mecA genes showed a close correlation with mecA transcription levels. Furthermore, the quantity of PBP 2a also closely correlated with the level of oxacillin resistance in the C195 background. Our data suggest that mecA promoter mutations play an important role in determining the level of oxacillin resistance. The mecA promoter mutation G-25A (25 bases upstream of the mecA translation start site) was found to be associated with a high oxacillin MIC (256 μg/ml), G-7T conferred a moderate oxacillin MIC (32 to 64 μg/ml), strains with C-33T showed a low oxacillin MIC (4 to 8 μg/ml), and A-38G reversed the effect of the C-33T mutation, restoring the oxacillin resistance level in the A-38G C-33T double mutant. These observations may explain why C-MRSA strains in Taiwan carrying SCCmec type IV or V have such enormous variations in oxacillin MICs.

Comparison of Ferment Sugars, Produce Hemolysis and Measuring Growth in Methicillin-Resistant and Methicillin-Sensitive Staphylococcus aureus Isolates from Inpatients and Healthcare Workers in Gorgan Hospitals, North of Iran

The mec A gene in Staphylococcus aureus leads to production of new penicillin-binding protein called PBP2a.This change may follow some changes in other phenotypes. The aim of this study was the comparison of Ferment Sugars, Produce Hemolysis and Measuring Growth in MRSA and MSSA isolates. 188 Staphylococcus aureus isolates separated from inpatients and healthcare workers (healthy carriers)were studied.Bacterialcultures in blood agar environment at 37°C during 24h and at 4°C during other 24h were applied for studying hemolysis. Sugar fermentation carried out in phenol red Broth medium, containing glucose, galactose, arabinose, fructose, xylose, ramnose, mannose, sucrose, trehalose, raffinose or maltose. For determining bacterial growth,bacterial concentration of 103was taken each hour during 12 cultured in MHAand colonies were counted after 24h.The mean amount of hemolysis diameter in MRSA isolates was rather more than that of MSSA isolates. The difference between MRSA and MSSA isolates were significant as to fermenting ramnose, trehalose, galactose and xylose. The mean rate of growth in MRSAwere significantly different from that of MSSAisolates (p<0.05).Resistance to methicillin in Staphylococcus aureus isolates accompanies the increase of ability to ferment sugars. This phenomenon may be one of reasons for increased pathogenicity of MRSA isolates; So results shows the logarithmic phase is longer in MRSA isolates, This may implicate that PBP2a production in methicillin-resistant isolates follows slowing down nutrients entrance into the bacterium that in turn may causes slow growth.

Molecular Identification of Methicillin-Resistant &lt;i&gt;Staphylococcus aureus&lt;/i&gt; in Benin-City Nigeria

We use the molecular techniques of PCR and PFGE to identify MRSA from clinical isolates of Staphylococcus aureus causing infections among hospitalized patients in Benin-City, Nigeria. A total of 36 isolates were obtained from the University of Benin Teaching Hospital between July-September, 2007. The MRSA strains were selected according to their phenotypic characteristics (antibiotic resistant profiles), susceptibility to oxacillin by E-test, and detection of β-lactamase. This was verified by a latex agglutination test for PBP2a production combined with PCR for mecA gene carriage. Four isolates representing 11% were confirmed as MRSA according to the molecular techniques used with two PFGE types (H and L) and one agr type (1). Multi resistance to the various antibiotics used was observed in one of the clones. The isolation of MRSA in health institution indicates that adequate steps in limiting spread are urgently needed. Also, for the first time two MRSA clones according to the PFGE classifications have been identified in Nigeria.Keywords: methicillin-resistant Staphylococcus aureus, MRSA, PFGE, PCR, molecular techniques.

Caracterização fenogenotípica da resistência antimicrobiana em Staphylococcus spp. isolados de mastite bovina

A utilização de antibióticos no controle das infecções intramamárias e na eliminação de prováveis fontes de infecção nas fazendas leiteiras se constitui em importante medida de controle. No entanto, o uso inadequado de antibióticos no tratamento da doença pode selecionar cepas resistentes e comprometer a eficiência do tratamento. Bactérias do gênero Staphylococcus spp. estão entre os principais agentes etiológicos da mastite bovina e são freqüentemente resistentes aos antimicrobianos, em especial aos beta-lactâmicos, principalmente por dois mecanismos distintos: a produção da enzima extracelular beta-lactamase, codificada pelo gene blaZ, e a produção de PBP2a ou PBP2´, uma proteína ligante de penicilina de baixa afinidade, codificada pelo gene mecA. A expressão do gene mecA é constitutiva ou induzida por antibióticos betalactâmicos, como a oxacilina e cefoxitina. O gene mecA está inserido no cromossomo através de um elemento genético móvel, denominado cassete estafilocócico cromossômico mec (SCCmec). O presente estudo avaliou o perfil fenogenotípico de resistência aos beta-lactâmicos em 250 isolados de Staphylococcus spp., utilizando os marcadores oxacilina e cefoxitina, de modo a produzir dados que possam contribuir para o conhecimento da resistência antimicrobiana em algumas propriedades leiteiras das regiões Sul-Fluminense e Metropolitana do Estado do Rio de Janeiro com o objetivo de subsidiar a implementação de medidas de controle dessa enfermidade. A avaliação da resistência foi feita a partir de 8 diferentes testes fenotípicos, sendo obtidos 54 perfis. Os testes de difusão em disco simples e ágar screen com oxacilina foram utilizados como "padrão ouro" para os cálculos dos valores de sensibilidade, especificidade e predição por serem preconizados pelo CLSI veterinário. O teste de difusão em disco simples com cefoxitina foi o de melhor desempenho na predição da resistência a oxacilina. Na avaliação genotípica, não foi detectado qualquer isolado positivo para o gene mecA, já os genes mecI e mecRI foram detectados igualmente em 11,6% (29/250) dos Staphylococcus spp. avaliados. Foram detectados os quatro tipos de cassete mec analisados (I, II, III e IV), sendo o tipo I o que teve mais ampla distribuição entre as regiões estudadas. Gene blaZ foi detectado em 5,2% (13/250) dos isolados, sendo que nestes, todo o sistema blaZ-blaI- blaR1 foi detectado em 23,1% (3/13) dos isolados.

Reduced expression of PBP-2A by neonatal mecA-positive coagulase-negative staphylococci (CoNS) blood isolates: -lactams are useful first-line agents for the treatment of neonatal CoNS sepsis, restricting the use of vancomycin

Vancomycin use for neonatal coagulase-negative staphylococci (CoNS) sepsis is based on a high CoNS carriage rate of mecA, encoding penicillin-binding protein (PBP)-2a, with low affinity for, and associated with resistance to, β-lactam antibiotics. The relationship between mecA gene carriage, phenotypic expression of the gene by PBP-2a production and in vitro resistance to the β-lactam antibiotics oxacillin, cefazolin and amoxicillin/clavulanate was determined for 85 CoNS blood isolates randomly obtained from our collection of isolates from neonates with CoNS sepsis. The relationship between mecA gene carriage, phenotypic expression of the gene by PBP-2a production and in vitro resistance to the β-lactam antibiotics oxacillin, cefazolin and amoxicillin/clavulanate was determined for randomly obtained CoNS blood isolates from our collection of isolates from neonates with CoNS sepsis. The mecA gene was detected using multiplex PCR, and PBP-2a expression was determined using a latex agglutination (LA) test (Oxoid). β-Lactam susceptibility was determined using the Phoenix automated system and, in addition, by Etest with interpretation of MIC values according to the reference MIC breakpoints adopted from the CLSI guidelines M100-S20, Infobase™. Among 85 CoNS blood isolates, 73 (86%) were mecA positive and 12 (14%) were mecA negative. None of the mecA-negative isolates expressed PBP-2a and all were β-lactam susceptible. All mecA-positive CoNS isolates were oxacillin resistant, although most oxacillin MICs were not very high, ranging from 2 to 8 mg/L for the majority of isolates. Only 8/73 (11%) mecA-positive CoNS isolates had oxacillin MICs ≥32 mg/L (range 32 to >256 mg/L). mecA-positive CoNS blood isolates, although fully resistant to oxacillin, were almost universally susceptible to cefazolin and amoxicillin/clavulanate, which was associated with a low expression rate of PBP-2a. β-Lactam antibiotics are useful for the treatment of neonatal CoNS sepsis, reserving vancomycin for selected cases.

Impact of mecA promoter mutations on mecA expression and β-lactam resistance levels

The reason for the extremely low-level oxacillin resistance in a so-called ‘drug clone’, a methicillin-resistant Staphylococcus aureus circulating among injection drug users in Zurich, Switzerland, could be traced back to the mecA promoter sequence and particularly to the strain's genetic background. Sequencing of its mec complex identified a point mutation (TATA CT to TATA TT), creating a perfect palindrome in the −10 region of the mecA promoter/operator region containing the binding sites for the mecA repressors MecI and BlaI. Two strains with vastly different β-lactam resistance phenotypes, the low-level resistant drug clone type strain CHE482 and the highly homogeneously resistant strain COLn, were cured of their SCC mec elements and subsequently transformed with plasmids containing mecA under the control of either the wild-type or mutant promoter. Expression studies showed that this mutation had significant effects on both mecA transcription and corresponding PBP2a production, but only small effects on β-lactam resistance levels within a given genetic background. A further mutation in the mecA ribosomal binding site (GGAG G to GGAG T), common to SCC mec type IV strains, was found to have no discernable effect on mecA transcription and PBP2a content, and only minimal effects on β-lactam resistance. Factors associated with the genetic backgrounds into which these differently controlled mecA genes were introduced had a much higher impact on β-lactam resistance levels than the rates of mecA transcription. The tight repression of mecA expression in this drug clone in the absence of β-lactams could contribute to the apparent fitness of this fast growing strain.

PBP 2a mutations producing very-high-level resistance to beta-lactams.

Resistance to the beta-lactam class of antibiotics in methicillin-resistant Staphylococcus aureus (MRSA) is mediated by PBP 2a, a synthetic bacterial cell wall penicillin-binding protein with a low affinity of binding to beta-lactams that is encoded by mecA. Beta-lactams that bind to PBP 2a with a high affinity and that are highly active against MRSA are under development. The potential for the emergence of resistance to such compounds was investigated by passage of homogeneous MRSA strain COL in L-695,256, an investigational carbapenem. A highly resistant mutant, COL52, expressed PBP 2a in which a two-amino-acid deletion mutation and three single-amino-acid substitution mutations were present. To examine the effects of these mutations on the resistance phenotype and PBP 2a production, plasmids carrying (i) PBP 2a with two or three of the four mutations, (ii) wild-type PBP 2a, or (iii) COL52 PBP 2a were introduced into methicillin-susceptible COL variants COLnex and COL52ex, from which the staphylococcus cassette chromosome mec (SCCmec) has been excised, as indicated by the "ex" suffix. Two amino acids substitutions, E-->K(237) within the non-penicillin-binding domain and V-->E(470) near the SDN(464) conserved penicillin-binding motif in the penicillin-binding domain in COL52, were important for high-level resistance. The highest level of resistance was observed when all four mutations were present. The emergence of PBP 2a-mediated resistance to beta-lactams that bind to PBP 2a with a high affinity is likely to require multiple mutations in mecA; chromosomal mutations appear to have a minor role.

Transcription of the gene mediating methicillin resistance in Staphylococcus aureus (mecA) is corepressed but not coinduced by cognate mecA and beta-lactamase regulators.

Resistance to beta-lactam antibiotics in staphylococci is mediated by mecA and blaZ, genes encoding a penicillin-binding protein (PBP2a) with low beta-lactam affinity and beta-lactamase, respectively. The mec and bla regulators, mecR1-mecI and blaR1-blaI, respectively, encode inducer-repressors with sufficient amino acid homology to suggest that they could coregulate PBP2a production. In order to test this hypothesis, plasmids containing mec and bla regulatory sequences were introduced into Staphylococcus aureus containing a chromosomal mecA-lacZ transcriptional fusion. Corepression was confirmed by demonstrating a gene dosage-dependent reduction in beta-galactosidase activity by either MecI or BlaI and additive repression when both were present. Both MecI-MecI and BlaI-BlaI homodimer and MecI-BlaI heterodimer interactions were demonstrated in the yeast two-hybrid assay, and purified MecI and BlaI protected the same mec promoter-operator sequences. However, MecI was approximately threefold more effective at mecA-lacZ transcriptional repression than was BlaI. While MecI and BlaI displayed similar activity as repressors of mecA transcription, there was a marked difference between MecR1 and BlaR1 in the rate and specificity of induction. Induction through BlaR1 by a beta-lactam was 10-fold greater than through MecR1 at 60 min and was 81% of maximal by 2 h, while induction through MecR1 never exceeded 20% of maximal. Furthermore, complementation studies showed that MecI- or BlaI-mediated mecA transcriptional repression could be relieved by induction through homologous but not heterologous sensor-inducer proteins, demonstrating the repressor specificity of induction.

Methicillin-resistant Staphylococcus aureus. Mechanisms of resistance and implications for treatment.

The frequency of methicillin-resistant Staphylococcus aureus (MRSA) continues to increase steadily, with nosocomial isolates approaching 50% of the total tested. Primarily isolated in hospitals, strains of MRSA have now spread into the community, complicating the management of this sometimes-fatal pathogen. Methicillin resistance in S aureus is mediated by the mecA gene, which encodes for a novel penicillin-binding protein (PBP), PBP-2a. In MRSA, exposure to methicillin inactivates the 4 high-binding-affinity PBPs normally present. PBP-2a, which displays a low affinity for methicillin, takes over the functions of these PBPs, permitting the cell to grow. Regulation of the methicillin-resistant phenotype and production of PBP-2a are influenced by the action of other genes. Two genes located upstream from mecA--mecR1 and mecI--control expression of PBP-2a. Antibiotics with high affinity for PBP-2a have displayed efficacy against MRSA in vivo, but none of these agents has made it beyond the investigational stage. Vancomycin remains the drug of choice for treatment of infections caused by MRSA, although it is intrinsically less active than the antistaphylococcal penicillins. Combinations of vancomycin with ss-lactam antibiotics may be synergistic in vivo against MRSA strains, including those with intermediate susceptibility to vancomycin. Given the increasing prevalence of MRSA in hospitals and in community settings, alternative approaches are needed for treatment of infections caused by MRSA.

Regulation of β-Lactamase Synthesis as a Novel Site of Action for Suppression of Methicillin Resistance in Staphylococcus aureus

Nearly all clinical isolates of methicillin resistant Staphylococcus aureus (MRSA) produce beta-lactamase as well as an additional low-affinity penicillin-binding protein called PBP2a or PBP2', the main factor for mediating methicillin resistance. Polidocanol (PDO), a dodecyl polyethyleneoxide ether, resensitizes clinical isolates of MRSA to methicillin; in addition, their resistance to benzylpenicillin (BP) is reduced. The action of PDO is based on the inhibition of the induced syntheses of PBP2a and beta-lactamase. Induction in our study was performed with 2-(2'-carboxyphenyl)benzoyl-6-aminopenicillanic acid (CBAP). Inducible PBP2a production in MRSA strains is under the control of the same regulatory system which is responsible for the induction of beta-lactamase synthesis. BlaR1, a membrane-spanning protein with a penicillin sensor and a signal transducer domain represents the starting point of this induction cascade. Based on its amphiphilic properties, it is likely that the action of PDO is located in the bacterial membrane. Therefore we investigated the possibility that BlaR1 might be the main target for PDO action. We were able to detect the BlaR1 sensor domain in resistant staphylococcal cells even in the noninduced state by fluorography. In a competition assay, CBAP was bound specifically, with a high affinity to the penicillin sensor. Moreover, the binding of CBAP was very stable. As concerns PDO, no significant interaction with the penicillin binding site of BlaR1 was detectable. This is why the BlaR1 transducer domain is thought to be the actual target area of PDO. In this case, PDO would interfere with the transmission of the signal, generated by the receptor binding of CBAP, through the membrane via BlaR1 into the staphylococcal cell. This assumption could be confirmed by the analysis of the concentration-effect relationship, whereafter PDO does not work as a competitive, but as a noncompetitive antagonist of CBAP. Our results demonstrate that BlaR1 could be an attractive new target for the development of new drugs to overcome methicillin resistance.

Genomic diversity ofmecregulator genes in methicillin-resistantStaphylococcus aureusandStaphylococcus epidermidis

Low-affinity penicillin-binding protein PBP-2a encoded by mecA is closely related to methicillin resistance in staphylococci, and expression of PBP-2a is controlled by regulator elements encoded by mecR1 and mecI which are located adjacent to mecA on the chromosome. Deletion or mutation which occurred in mec regulator gene is considered to be associated with constitutive production of PBP-2a. The distribution of the mec regulator genes in 176 strains of Staphylococcus aureus and 33 strains of S. epidermidis isolated from a single hospital was studied by polymerase chain reaction amplification. Most clinical isolates of methicillin-resistant S. aureus (MRSA) (94.3%) and S. epidermidis (MRSE) (83.9%) possessed both mecI and mecR1 genes (type I), whereas no mec regulator genes were detected in mecA-negative isolates. In contrast, 7 MRSA and 5 MRSE isolates were found to have incomplete regulator genes, and they were classified into three groups; strains which lacked only mecI gene (type II), strains which lacked mecI and 3'-end of mecR1 gene (type III), and strains which lacked both regulator genes (type IV). Analysis of mecI gene from all the strains having mecI by restriction fragment length polymorphism after Mse I digestion indicated that three MRSA strains possessed one of the known point mutations identified previously. These findings indicated the predominance of a single type of MRSA possessing both mecI and mecR1 in the study period and also suggested a high genomic diversity in mec regulator region of staphylococci.

BlaI and blaR1 regulate beta-lactamase and PBP 2a production in methicillin-resistant Staphylococcus aureus

For Staphylococcus aureus, it is hypothesized that two genes located upstream of the beta-lactamase gene, blaZ, are required for the inducible expression of beta-lactamase. blaR1 is predicted to encode a signal-transducing membrane protein, and blaI is predicted to encode a repressor protein. These same two genes may also regulate the production of penicillin-binding protein 2a (PBP 2a), a protein essential for expression of methicillin resistance. To confirm that these two genes encode products that can control both beta-lactamase and PBP 2a production, blaI, blaR1, and blaZ with a 150-nucleotide deletion at the 3' end were subcloned from a 30-kb staphylococcal beta-lactamase plasmid and three beta-lactamase-negative strains of methicillin-resistant S. aureus were transformed with the recombinant plasmid containing that insert. The production of PBP 2a and a nonfunctional beta-lactamase was detected by fluorography and by immunoblots with polyclonal antisera directed against each of the proteins. Whereas the parent strains did not produce beta-lactamase and constitutively produced PBP 2a, PBP 2a and a truncated beta-lactamase were now inducible in the transformants. Therefore, two plasmid-derived genes regulate the production of both PBP 2a and beta-lactamase.