Heterogeneous Expression Of Resistance Research Articles

Ceftaroline is active against heteroresistant methicillin-resistant Staphylococcus aureus clinical strains despite associated mutational mechanisms and intermediate levels of resistance.

Methicillin-resistant Staphylococcus aureus (MRSA) is an important infectious human pathogen responsible for diseases ranging from skin and soft tissue infections to life-threatening endocarditis. β-Lactam resistance in MRSA involves acquisition of penicillin-binding protein 2a (PBP2a), a protein with low affinity for β-lactams that mediates cell wall assembly when the normal staphylococcal PBPs (PBP1 to -4) are blocked by these agents. Many MRSA strains display heterogeneous expression of resistance (HeR) against β-lactam antibiotics. The β-lactam-mediated homoresistant (HoR) phenotype is associated with both expression of the mecA gene and activation of the LexA-RecA-mediated SOS response, a regulatory network induced in response to DNA damage. Ceftaroline (CPT) is the only FDA-approved cephalosporin targeting PBP2a. We investigated the mechanistic basis of CPT activity against HeR-MRSA strains, including a set of strains displaying an intermediate level of resistance to CPT. Mechanistically, we found that 1 exposure of HeR-MRSA to subinhibitory concentrations of CPT selected for the HoR derivative activated the SOS response and increased mutagenesis. Importantly, CPT-selected HoR cells remained susceptible to CPT while still being resistant to most β-lactams, and 2-CPT activity in HeR-MRSA resided in an attenuated induction of mecA expression in comparison to other β-lactams. In addition, 3-CPT intermediate-resistant strains displayed a significant increase in CPT-induced mecA expression accompanied by mutations in PBP2, which together may interfere with the complete repression by CPT of both PBP2a and PBP2a-PBP2 interactions and thus be a determining factor in the low level of CPT resistance in the absence of mecA gene mutations. The present study provides mechanistic evidence that CPT represents an alternative therapeutic option for the treatment of heteroresistant MRSA strains.

Fate of Mutation Rate Depends on agr Locus Expression during Oxacillin-Mediated Heterogeneous-Homogeneous Selection in Methicillin-Resistant Staphylococcus aureus Clinical Strains

Methicillin-resistant Staphylococcus aureus (MRSA) strains are characterized by a heterogeneous expression of resistance. We have previously shown in clinical oxacillin-susceptible, mecA-positive MRSA strains that selection from a very heterogeneous (HeR) to highly homogeneous (HoR) resistant phenotype was mediated by acquisition of mutations through an oxacillin-induced SOS response. In the present study, we used a spotted DNA microarray to evaluate differential gene expression during HeR-HoR selection and found increased expression of the agr two-component regulatory system. We hypothesized that increased expression of agr represents a mechanistically relevant component of this process. We demonstrated that inactivation of agr during the HeR-HoR selection process results in a significant increase in mutation rate; these effects were reversed by complementing the agr mutant. Furthermore, we found that extemporal ectopic expression of agr and, more specifically, RNAII in agr-null mutant HeR cells suppressed mutation frequency and the capacity of these cells to undergo the HeR-HoR selection. These findings sustain the concept that increased expression of agr during HeR-HoR selection plays a critical role in regulating the β-lactam-induced increased mutation rate in very heterogeneous MRSA strains. Moreover, they indicate that a temporally controlled increase in agr expression is required to tightly modulate SOS-mediated mutation rates, which then allows for full expression of oxacillin homogeneous resistance in very heterogeneous clinical MRSA strains.

Carbapenem-non-susceptible Enterobacteriaceae in Europe: conclusions from a meeting of national experts

The emergence and global spread of carbapenemase-producing Enterobacteriaceae is of great concern to health services worldwide. These bacteria are often resistant to all beta-lactam antibiotics and frequently co-resistant to most other antibiotics, leaving very few treatment options. The epidemiology is compounded by the diversity of carbapenem-hydrolysing enzymes and the ability of their genes to spread between different bacterial species. Difficulties are also encountered by laboratories when trying to detect carbapenemase production during routine diagnostic procedures due to an often heterogeneous expression of resistance. Some of the resistance genes are associated with successful clonal lineages which have a selective advantage in those hospitals where antimicrobial use is high and opportunities for transmission exist; others are more often associated with transmissible plasmids. A genetically distinct strain of Klebsiella pneumoniae sequence type (ST) 258 harbouring the K. pneumoniae carbapenemases (KPC) has been causing epidemics of national and international proportions. It follows the pathways of patient referrals, causing hospital outbreaks along the way. Simultaneously, diverse strains harbouring New Delhi metallo-beta-lactamase (NDM-1) are repeatedly being imported into Europe, commonly via patients with prior medical exposure in the Indian subcontinent. Since the nature and scale of carbapenem-non-susceptible Entrobacteriaceae as a threat to hospital patients in Europe remains unclear, a consultation of experts from 31 countries set out to identify the gaps in diagnostic and response capacity, to index the magnitude of carbapenem-non-susceptibility across Europe using a novel five-level staging system, and to provide elements of a strategy to combat this public health issue in a concerted manner

Development of homogeneous expression of resistance in methicillin-resistant Staphylococcus aureus clinical strains is functionally associated with a β-lactam-mediated SOS response

One of the main characteristics of methicillin-resistant Staphylococcus aureus (MRSA) from both hospitals and community is their heterogeneous expression of resistance. Recently, we reported new heterogeneous MRSA isolates phenotypically susceptible to oxacillin despite being mecA positive. These low-level mecA-mediated resistance MRSA strains are very heterogeneous in expression (HeR) and are likely to be clinically relevant since exposure of such isolates to beta-lactams can result in high-level homotypic resistance (HoR). We hypothesized that HeR to HoR selection in these clinically relevant strains may be determined by the pre-existence of a hypermutable population that favours its selection in the presence of oxacillin. Using established procedures, SA13011 HeR to HoR selection was performed by using subinhibitory concentrations of oxacillin and examined for mutability. Real-time RT-PCR and transcriptional profiling by DNA microarray were used to compare gene expression between both populations and related genetically modified SA13011 strain. We found that HeR/HoR selection by oxacillin was associated with increased mutation rate and oxacillin-mediated SOS response. We determined increased expression of both mecA and SOS response lexA/recA regulators. Mutational inactivation of lexA repressor resulted in a significant decrease in both mutation rate and oxacillin resistance in the HoR cells. Complementation of the lexA mutant strain restored oxacillin resistance to the high levels observed in the corresponding HoR wild-type strain. The present results support the notion that SOS response is mechanistically involved in generating mutations that, in addition to mecA induction, allow the selection of a highly oxacillin-resistant population.

Factors influencing methicillin resistance in staphylococci.

Methicillin resistance in staphylococci is due to an acquired penicillin-binding protein, PBP2' (PBP2a). This additional PBP, encoded by mecA, confers an intrinsic resistance to all beta-lactams and their derivatives. Resistance levels in methicillin-resistant Staphylococcus aureus (MRSA) depend on efficient PBP2' production and are modulated by chromosomal factors. Depending on the genetic background of the strain that acquired mecA, resistance levels range from phenotypically susceptible to highly resistant. Characteristic for most MRSA is the heterogeneous expression of resistance, which is due to the segregation of a more highly resistant subpopulation upon challenge with methicillin. Maximal expression of resistance by PBP2' requires the efficient and correct synthesis of the peptidoglycan precursor. Genes involved in cell-wall precursor formation and turnover, regulation, transport, and signal transduction may determine the level of resistance that is expressed. At this stage, however, there is no information available on the functionality or efficacy of such factors in clinical isolates in relation to methicillin resistance levels.

Methicillin resistant staphylococci: mechanisms of resistance

Methicillin-resistant Staphylococcus aureus (MRSA) is a major nosocomial pathogen causing severe morbidity and mortality in many hospitals in Europe, in the United States and worldwide. Once these organisms are introduced into an institution they are particularly difficult to eradicate. Colonization with MRSA is a major problem since 30%-60% of patients who become colonized eventually develop infection, and these patients serve as reservoir for further nosocomial spread. There are a number of difficulties associated with the isolation and identification of MRSA in clinical laboratories. As a consequence of the resistance of many of these organisms, a number of factors affect the expression of methicillin resistance, e.g. the expression of methicillin resistance is influenced both by environmental factors and cell genotype. Although heterogeneous expression of resistance is common, some MRSA strains have a much more homogeneous display of resistance and may even be more of a problem. Antibiotic resistance and phagotyping patterns vary between the strains. In addition to the laboratory problems, the hospital needs a system for the immediate recognition of new patients carrying new importation of MRSA. Moreover, the concept of borderline resistance (that is thought to be due to very high level β-lactamase production) is still poorly defined in many respects, with a number of biological, diagnostic and clinical questions still unanswered. There is some debate over whether to report such strains as susceptible or resistant. There is increasing evidence that the factors accounting for the reduced susceptibility of the borderline Staphylococcus aureus strains to penicillinase-resistant penicillins are probably more complex than originally believed. Biomedical Reviews 1994; 3: 65-71.

Reduced methicillin resistance in a new Staphylococcus aureus transposon mutant that incorporates muramyl dipeptides into the cell wall peptidoglycan.

Screening of a new Tn551 library constructed in the background of a highly methicillin-resistant Staphylococcus aureus strain identified a new insertion site located on the SmaI B-fragment of the chromosome that reduced the minimal inhibitory concentration of the parent (1600 micrograms/ml) to 25-50 micrograms/ml in the mutant and caused heterogeneous expression of resistance and abnormality in peptidoglycan composition (absence of the unsubstituted pentapeptide and incorporation of alanylglutamate- and alanylisoglutamate-containing muropeptides). There was an accumulation of large amounts of the UDP-linked muramyl dipeptide in the cytoplasmic wall precursor pool of the mutant. Reduced (heterogeneous) antibiotic resistance and all the biochemical abnormalities were reproduced in genetic backcrosses by transduction with phage 80 alpha. Mutant RUSA235 appears to be impaired in the biosynthesis of the staphylococcal cell wall precursor muropeptide before the lysine addition step. We propose to provisionally call the gene inactivated in this mutant femF.

DETECTION OF METHICILLIN-RESISTANT STAPHYLOCOCCI

Susceptibility tests commonly used in the clinical laboratory for detection of methicillin-resistant strains of staphylococci are discussed. The biochemical and genetic basis of methicillin resistance, including heterogeneous expression of resistance, is summarized. The conditions that can affect results of disk diffusion, broth dilution, and agar screen tests and sensitivity and specificity of these tests are reviewed. DNA detection methodologies, which, with their greater accuracy, may replace susceptibility testing, also are discussed.

Methicillin-resistant staphylococci.

Strains of staphylococci resistant to methicillin were identified immediately after introduction of this drug. Methicillin-resistant strains have unusual properties, the most notable of which is extreme variability in expression of the resistance trait. The conditions associated with this heterogeneous expression of resistance are described. Methicillin resistance is associated with production of a unique penicillin-binding protein (PBP), 2a, which is bound and inactivated only at high concentrations of beta-lactam antibiotics. PBP2a appears to be encoded by the mec determinant, which also is unique to methicillin-resistant strains. The relationships between PBP2a and expression of resistance and implications for the mechanism of resistance are discussed. The heterogeneous expression of methicillin resistance by staphylococci poses problems in the detection of resistant strains. Experience with several susceptibility test methods is reviewed and guidelines for performance of these tests are given. Treatment of infections caused by methicillin-resistant staphylococci is discussed. Vancomycin is the treatment of choice. Alternatives have been few because methicillin-resistant strains often are resistant to multiple antibiotics in addition to beta-lactam antibiotics. New agents which are active against methicillin-resistant staphylococci are becoming available, and their potential role in treatment is discussed.

Methicillin-resistant staphylococci.

Strains of staphylococci resistant to methicillin were identified immediately after introduction of this drug. Methicillin-resistant strains have unusual properties, the most notable of which is extreme variability in expression of the resistance trait. The conditions associated with this heterogeneous expression of resistance are described. Methicillin resistance is associated with production of a unique penicillin-binding protein (PBP), 2a, which is bound and inactivated only at high concentrations of beta-lactam antibiotics. PBP2a appears to be encoded by the mec determinant, which also is unique to methicillin-resistant strains. The relationships between PBP2a and expression of resistance and implications for the mechanism of resistance are discussed. The heterogeneous expression of methicillin resistance by staphylococci poses problems in the detection of resistant strains. Experience with several susceptibility test methods is reviewed and guidelines for performance of these tests are given. Treatment of infections caused by methicillin-resistant staphylococci is discussed. Vancomycin is the treatment of choice. Alternatives have been few because methicillin-resistant strains often are resistant to multiple antibiotics in addition to beta-lactam antibiotics. New agents which are active against methicillin-resistant staphylococci are becoming available, and their potential role in treatment is discussed.