Purine Nucleosides Interfere with c-di-AMP Levels and Act as Adjuvants To Re-Sensitize MRSA To ß-Lactam Antibiotics.

The purine-derived signaling molecules c-di-AMP and (p)ppGpp control mecA/PBP2a-mediated ß-lactam resistance in methicillin-resistant Staphylococcus aureus (MRSA) raise the possibility that purine availability can control antibiotic susceptibility. Consistent with this, exogenous guanosine and xanthosine, which are fluxed through the GTP branch of purine biosynthesis, were shown to significantly reduce MRSA ß-lactam resistance. In contrast, adenosine (fluxed to ATP) significantly increased oxacillin resistance, whereas inosine (which can be fluxed to ATP and GTP via hypoxanthine) only marginally increased oxacillin susceptibility. Furthermore, mutations that interfere with de novo purine synthesis (pur operon), transport (NupG, PbuG, PbuX) and the salvage pathway (DeoD2, Hpt) increased ß-lactam resistance in MRSA strain JE2. Increased resistance of a nupG mutant was not significantly reversed by guanosine, indicating that NupG is required for guanosine transport, which is required to reduce ß-lactam resistance. Suppressor mutants resistant to oxacillin/guanosine combinations contained several purine salvage pathway mutations, including nupG and hpt. Guanosine significantly increased cell size and reduced levels of c-di-AMP, while inactivation of GdpP, the c-di-AMP phosphodiesterase negated the impact of guanosine on ß-lactam susceptibility. PBP2a expression was unaffected in nupG or deoD2 mutants, suggesting that guanosine-induced ß-lactam susceptibility may result from dysfunctional c-di-AMP-dependent osmoregulation. These data reveal the therapeutic potential of purine nucleosides, as ß-lactam adjuvants that interfere with the normal activation of c-di-AMP are required for high-level ß-lactam resistance in MRSA. IMPORTANCE The clinical burden of infections caused by antimicrobial resistant (AMR) pathogens is a leading threat to public health. Maintaining the effectiveness of existing antimicrobial drugs or finding ways to reintroduce drugs to which resistance is widespread is an important part of efforts to address the AMR crisis. Predominantly, the safest and most effective class of antibiotics are the ß-lactams, which are no longer effective against methicillin-resistant Staphylococcus aureus (MRSA). Here, we report that the purine nucleosides guanosine and xanthosine have potent activity as adjuvants that can resensitize MRSA to oxacillin and other ß-lactam antibiotics. Mechanistically, exposure of MRSA to these nucleosides significantly reduced the levels of the cyclic dinucleotide c-di-AMP, which is required for ß-lactam resistance. Drugs derived from nucleotides are widely used in the treatment of cancer and viral infections highlighting the clinical potential of using purine nucleosides to restore or enhance the therapeutic effectiveness of ß-lactams against MRSA and potentially other AMR pathogens.

Delivery of antisense oligonucleotides using multi-layer coated gold nanoparticles to methicillin-resistant S. aureus for combinatorial treatment.

The spread of multidrug-resistant (MDR) bacterial infections has become a serious global threat. We introduce multi-layer coated gold nanoparticles (MLGNPs) delivering antisense oligonucleotides (ASOs) targeting the resistance gene of methicillin-resistant Staphylococcus aureus (MRSA), as a selective antimicrobial by restoring susceptibility. MLGNPs were prepared by multi-step surface immobilization of gold nanoparticles (GNPs) with polyethylenimine (PEI) and loaded with ASO targeting the mecA gene. The MLGNPs were shown to be efficiently internalized into various types of Gram-positive bacteria, including MRSA, Staphylococcus epidermidis, and Bacillus subtilis, which was superior to single-layer coated GNPs and free PEI polymer. The delivery of MLGNPs into MRSA resulted in up to 74% silencing of the mecA gene with high selectivity, in a dose-dependent manner. The treatment of MLGNPs to MRSA in the presence of oxacillin, a beta-lactam antibiotic, showed major suppression (~71%) of bacterial growth, due to the recovery of antibacterial sensitivity. Furthermore, the treatment of MLGNPs in a complex system showed preferential uptake into bacteria over mammalian cells, demonstrating the suitable characteristics of MLGNPs for selective delivery into bacteria. The current approach can be potentially applied for targeting various types of MDR bacterial infections by specific silencing of a resistance gene, as a combinatorial therapeutic used with conventional antibiotics.

Determining spa-type of methicillin-resistant Staphylococcus aureus (MRSA) via high-resolution melting (HRM) analysis, Shiraz, Iran.

OBJECTIVES: Molecular typing methods are useful for rapid detection and control of a disease. Recently, the use of high-resolution melting (HRM) for spa typing of MRSA isolates were reported. This technique is rapid, inexpensive and simple for genotyping and mutation screening in DNA sequence. The aim of this study was to evaluate the ability of HRM-PCR to analysis spa genes amongst MRSA isolates. RESULTS: A total of 50 MRSA isolates were collected from two teaching hospitals in Shiraz, Iran. The isolates were confirmed as MRSA by susceptibility to cefoxitin and detection of mecA gene using PCR. We used HRM analysis and PCR-sequencing method for spa typing of MRSA isolates. In total, 15 different spa types were discriminate by HRM and sequencing method. The melting temperature of the 15 spa types, using HRM genotyping were between 82.16 and 85.66 °C. The rate of GC % content was 39.4-46.3. According to the results, spa typing of 50 clinical isolates via PCR-sequencing and HRM methods were 100% similar. Consequently, HRM method can easily identify and rapidly differentiate alleles of spa genes. This method is faster, less laborious and more suitable for high sample at lower cost and risk of contamination.

Epidemiological and molecular characterization of invasive Streptococcus pneumoniae isolated following introduction of 7-valent conjugate vaccine in Kinki region, Japan, 2008-2013.

Streptococcus pneumoniae is one of the most common bacteria causing community-acquired pneumonia and meningitis. The use of 7-valent pneumococcal conjugate vaccine (PCV7) has reduced the incidence of pneumococcal disease while changing pneumococcal population through herd immunity and non-vaccine pneumococci replacement. This study investigated molecular epidemiologic characteristics of pneumococcal strains in the Kinki region of Japan from 2008 to 2013. A total of 159 invasive pneumococcal isolates were characterized by serotyping, antibiotic susceptibility testing, PCR analysis of penicillin-binding protein genes, multilocus sequence typing (MLST), and pulsed-field gel electrophoresis (PFGE). In adult populations, pediatric PCV7 introduction decreased isolates expressing PCV7 serotypes via herd immunity and increased isolates expressing non-PCV7 serotypes. The rate of penicillin resistance and isolates with alterations in all three pbp genes was higher in PCV7 type isolates than in non-PCV7 type isolates. In MLST analysis, all of serotype 19F isolates were of the same sequence type, ST236, which is the antimicrobial-resistant clone Taiwan19F-14, and the majority of serotypes 23F and 19A isolates were of ST1437 and ST3111 respectively, which are the predominant clones of antimicrobial-resistant pneumococci in Japan. In PFGE profiles, serotype 6B-ST2224, serotype 19F-ST236, serotype 19A-ST3111, and serotype 23F-ST1437 formed six separate clusters composed of genetically identical strains, and genetically identical serotype 22F-ST433 formed two different clusters between the pre- and post-PCV7 period. The results of molecular analysis suggest the spread and persistence of these identical antimicrobial resistant clones in the Kinki region and genetic changes of epidemic clone serotype 22F-ST433 before and after pediatric PCV7 introduction.

Emergence of Ceftriaxone Resistance during a Case of Pneumococcal Meningitis with Fatal Evolution.

We report a case of a 62-year-old man treated for Streptococcus pneumoniae meningitis by ceftriaxone and dexamethasone. After neurological improvement, neurological degradation by vasculitis occurred, despite effective concentrations of ceftriaxone in the serum and cerebrospinal fluid (CSF). S. pneumoniae with increased MICs to third-generation-cephalosporins (3GC) was isolated from the ventricular fluid 10 days after the isolation of the first strain. Isolate analysis showed that a mutation in the penicillin-binding protein 2X (PBP2X) has occurred under treatment.

Genomic insights on heterogeneous resistance to vancomycin and teicoplanin in Methicillin-resistant Staphylococcus aureus: A first report from South India.

Methicillin-resistant Staphylococcus aureus (MRSA) infection is an important clinical concern in patients, and is often associated with significant disease burden and metastatic infections. There is an increasing evidence of heterogeneous vancomycin-intermediate S. aureus (hVISA) associated treatment failure. In this study, we aim to understand the molecular mechanism of teicoplanin resistant MRSA (TR-MRSA) and hVISA. A total of 482 MRSA isolates were investigated for these phenotypes. Of the tested isolates, 1% were identified as TR-MRSA, and 12% identified as hVISA. A highly diverse amino acid substitution was observed in tcaRAB, vraSR, and graSR genes in TR-MRSA and hVISA strains. Interestingly, 65% of hVISA strains had a D148Q mutation in the graR gene. However, none of the markers were reliable in differentiating hVISA from TR-MRSA. Significant pbp2 upregulation was noted in three TR-MRSA strains, which had teicoplanin MICs of 16 or 32 µg/ml, whilst significant pbp4 downregulation was not noted in these strains. In our study, multiple mutations were identified in the candidate genes, suggesting a complex evolutionary pathway involved in the development of TR-MRSA and hVISA strains.

Detection of heterogeneous vancomycin-intermediate Staphylococcus aureus: A preliminary report from south India.

Background & objectives: Although there are reports of heterogeneous vancomycin-intermediate Staphylococcus aureus (hVISA) across the globe, there is a lack of reliable data on hVISA in India. The present study was undertaken to determine the rate of hVISA among the methicillin-resistant Staphylococcus aureus (MRSA) isolates, and to compare the brain heart infusion agar with vancomycin 4 µg/ml (BHIV4) method with population analysis profile-area under the curve (PAP-AUC) method for the detection of hVISA and to study the distribution of mobile genetic element that carries methicillin-resistance gene SCCmec (Staphylococcal cassette chromosome mec) types among these isolates. Methods: BHIV4 and PAP-AUC methods were employed to detect hVISA among 500 clinical isolates of MRSA. SCCmec typing of these isolates was performed by multiplex polymerase chain reaction. The clinical presentation, treatment with vancomycin and outcome was documented for patients with hVISA. Results: The rate of hVISA was 12.4 per cent by PAP-AUC method. Sensitivity, specificity, PPV, NPV and kappa agreement of BHIV4 with PAP-AUC was 58.06, 93.15, 54.55, 94.01 per cent and 0.498, respectively. The isolation of hVISA was significantly (P<0.01) higher in patients admitted to intensive care units and wards than in patients attending the outpatient departments. Only 38 per cent of the patients received vancomycin as therapy. Majority of the hVISA isolates carried SCCmec type V or IV. Interpretation & conclusions: The rate of hVISA isolation in our study was 12.4 per cent. The sensitivity of the BHIV4 screening test was low, and was in moderate agreement with PAP-AUC test. SCCmec type V was the predominant type seen in half of the isolates. More studies need to be done in different parts of the country on a large number of isolates to confirm our findings.

Molecular typing, antibiotic susceptibility, and biofilm production in nonencapsulated Streptococcus pneumoniae isolated from children in Japan.

The prevalence of nonencapsulated Streptococcus pneumoniae (NESp) has increased with the introduction of pneumococcal conjugate vaccines in children; however, the bacteriological characteristics of NESp have not been sufficiently clarified. In this study, NESp strains isolated from the nasopharyngeal carriage of children from four nursery schools in Japan were analyzed for molecular type, antibiotic susceptibility, and biofilm productivity. A total of 152 putative S. pneumoniae strains were identified by optochin-susceptibility analysis, of which 21 were not serotypeable by slide agglutination, quellung reaction, or multiplex PCR. Among these 21 strains, three were lytA-negative and, therefore, not S. pneumoniae. The remaining 18 strains were positive for lytA, ply, pspK, and bile solubility and were confirmed as NESp. Therefore, the isolation rate of NESp in the S. pneumoniae strains in this study was 12.0% (18/149). Molecular-typing analyses classified five strains as two existing sequence types (STs; ST7502 and ST7786), and 13 strains formed four novel STs. Horizontal spread was suspected, because strains with the same ST were often isolated from the same nursery school. The NESp isolates were generally susceptible to most antimicrobials, with the exception of macrolides; however, all isolates possessed more than one abnormal penicillin-binding protein gene. Furthermore, NESp strains were more effective than encapsulated counterparts at forming biofilms, which showed obvious differences in morphology. These data indicated that NESp strains should be continuously monitored as emerging respiratory pathogens.

Penicillin-binding protein 3 is a common adaptive target among Pseudomonas aeruginosa isolates from adult cystic fibrosis patients treated with ß-lactams.

OBJECTIVE: Determining the mechanisms that modulate ß-lactam resistance in clinical Pseudomonas aeruginosa (P. aeruginosa) isolates can be challenging, as the molecular profiles identified in mutation-based or expression-based resistance determinant screens may not correlate with in vitro phenotypes. One of the lesser studied resistance mechanisms in P. aeruginosa is the modification of penicillin-binding protein 3 (pbpB/ftsI). This study reported that nonsynonymous polymorphisms within pbpB frequently occur among ß-lactam resistant sputum isolates, and are associated with unique antibiotic susceptibility patterns. METHODS: Longitudinally collected isolates (n = 126) from cystic fibrosis (CF) patients with or without recent ß-lactam therapy or of non-clinical origin were tested for susceptibility to six ß-lactams (aztreonam, ceftazidime, cefsulodin, cefepime, meropenem, and piperacillin). Known ß-lactam resistance mechanisms were characterised by polymerase chain reaction (PCR)-based methods, and polymorphisms in the transpeptidase-encoding domain of pbpB identified by sequencing. RESULTS: Twelve nonsynonymous polymorphisms were detected among 86 isolates (67%) from five CF patients with a history of ß-lactam therapy, compared with one polymorphism in 30 (3.3%) from three patients who had not received ß-lactam treatments. No nonsynonymous polymorphisms were found in ten environmental isolates. Multiple pbpB alleles, often with different combinations of polymorphisms, were detected within the population of strains from each CF patient for up to 2.6 years. Traditional patterns of ampC or mexA de-repression reduced expression of oprD or the presence of extended-spectrum ß-lactamases were not observed in resistant isolates with nonsynonymous polymorphisms in pbpB. CONCLUSION: This study's findings suggest that pbpB is a common adaptive target, and may contribute to the development of ß-lactam resistance in P. aeruginosa.

Spermine and oxacillin stress response on the cell wall synthesis and the global gene expression analysis in Methicillin-resistance Staphylococcus aureus.

Methicillin-resistant Staphylococcus aureus (MRSA) is a rapidly emerging bacteria causing infection, which has developed resistance to most of the beta-lactam antibiotics because of newly acquired low-affinity penicillin-binding protein (PBP2a), which can continue to build the cell wall when beta-lactams block other PBPs. Exogenous spermine exerts a dose-dependent inhibition effect on the growth of Escherichia coli, Salmonella enterica serovar, and S. aureus. Selection of an MRSA Mu50 derivative which harbors mutation on PBP2 gene (named as MuM) showing spermine resistance and which confers a complete abolishment of spermine-beta-lactam synergy was identified. To further investigate the gene expression changes, a transcriptome profiling of MuM against Mu50 (wild-type) without any treatment, MuM and Mu50 in response to high dose spermine and Mu50 in response to spermine-beta-lactam synergy at 15, 30 and 60 min time points was performed. Functional annotation was further performed to delineate the metabolic pathways associated with the significant genes. A significant down-regulation in the iron regulatory system, potassium channel uptake and polyamine transport system with an up-regulation in general stress response sigB dependent operon in MuM strain at 15, 30 and 60 min time points with spermine treatment compared to Mu50 strain was observed. Analysis of spermine-dependent synergy with beta-lactams on cell wall synthesis revealed that it significantly reduces the degree of cross-linkage on cell wall with no change in trypsin digestion pattern of purified PBPs and without affecting PBPs expression or PBPs acylation by Bocillin. A strong relation between PBP2 protein and general stress sigB response, iron, potassium and polyamine transport systems was observed. SigB regulon should be activated on stress, which was not seen in some of our previous studies where it was down-regulated in wild-type Mu50 strain with spermine stress. Here, an intriguing finding is made where there seems to be a correction of this abnormal response of no SigB induction to a significant induction by PBP2 mutation. In MuM strain, a significant down-regulation of KdpABC operon genes at 15, 30 and 60 min time points on spermine stress is seen, which seems to be absent without spermine treatment. Since KCL has been found to protect the cell against spermine stress in wild-type strain by induction of KdpABC operon, it fails to do so in MuM strain underlying the importance of PBP2 protein in spermine stress. Analysis of spermine-dependent synergy with beta-lactams on cell wall synthesis revealed that it significantly reduces the degree of cross-linkage on cell wall with no change in trypsin digestion patterns of purified PBPs and without affecting PBPs expression or PBPs acylation by Bocillin. Furthermore, spermine does not help in enhancing the binding of beta-lactams to PBPs and binding of spermine to PBPs does not cause conformational changes to PBPs, as tested with trypsin digestion patterns. Future studies on the molecular mechanism of spermine interactions with these systems hold great potential for the development of new therapeutics for MRSA infections.

Prevalence of Haemophilus influenzae with alteration of PBP 3 sequence over a 1-year period in a French hospital: focus on a clinical failure after ceftriaxone treatment.

Among 547 Haemophilus influenzae isolates recovered in our center, 45 displayed a phenotype of loss of PBP 3 affinity (8.2%). Two isolates with 6 substitutions in PBP 3 showed decreased susceptibility to third-generation cephalosporins. Clinical data revealed clinical failure after ceftriaxone treatment in a context of bronchitis in a patient with pulmonary sarcoidosis.

Methicillin-resistant Staphylococcus aureus intracranial abscess: An analytical series and review on molecular, surgical and medical aspects.

PURPOSE: Intracranial abscess caused by methicillin-resistant Staphylococcus aureus (MRSA) is rare and unexplored. The aim of the present study is to examine the prevalence, clinical and molecular characteristics, treatment options and outcome of MRSA intracranial abscess over a period of 6 years. PATIENTSAND METHODS: A total of 21 patients were included in this retrospective study. The demographic and clinical details of all the patients were collected. Molecular typing including staphylococcal cassette chromosome mec typing, spa typing and polymerase chain reaction of Panton-Valentine leucocidin toxin (PVL) gene for the latter 6 isolates was performed. RESULTS: The paediatric population was the most affected group (33.3%). The primary route of infection was post-operative/trauma in 7 (33.3%) cases. All the patients were treated surgically either by aspiration or excision. Fifteen (71%) patients received anti-MRSA treatment with vancomycin or linezolid, where linezolid-treated patients showed better prognosis. Of the 11 patients who were on follow-up, unfavourable outcome was observed in 3 (27.3%) cases and 8 (72.7%) cases improved. The molecular typing of six isolates revealed four community-associated (CA) MRSA, one each of livestock-associated (LA) and healthcare-associated MRSA with PVL gene noted in all. CONCLUSION: We propose that timely diagnosis, surgical intervention and appropriate anti-MRSA treatment would contribute to better outcome. The occurrence of CA-MRSA and LA-MRSA infection in the central nervous system signifies the threat from the community and livestock reservoir, thus drawing attention towards surveillance and tracking to understand the epidemiology and implement infection control measures.

A Retrospective Analysis of Treatment and Clinical Outcomes among Patients with Methicillin-Susceptible Staphylococcus aureus Bloodstream Isolates Possessing Detectable mecA by a Commercial PCR Assay Compared to Patients with Methicillin-Resistant Staphylococcus aureus Bloodstream Isolates.

mecA-positive Staphylococcus aureus isolates phenotypically susceptible to cefoxitin (mecA-methicillin-sensitive S. aureus [MSSA]) have been identified. We describe the treatment and outcomes among patients with mecA-MSSA bloodstream infections (BSI) and MRSA BSI matched 1:1 for age, BSI origin, and BSI type (n = 17 per group). Compared to MRSA BSI patients, mecA-MSSA BSI patients more often experienced clinical failure (58.8% and 11.8%, P = 0.010), driven largely by persistent bacteremia (35.3% and 11.8%). mecA-MSSA BSI patients may be at higher risk for poor clinical outcomes.

Characterization of SCCmec and spa types of methicillin-resistant Staphylococcus aureus isolates from health-care and community-acquired infections in Kerman, Iran.

Spread of methicillin-resistant Staphylococcus aureus (MRSA) isolates is a worldwide problem. Molecular typing is a useful tool to understand MRSA epidemiology. Herein, we determined vancomycin-resistant, SCCmec and spa types among MRSA isolates recovered from healthcare and community-acquired infections in Kerman, Iran. A total of 170 S. aureus isolates were collected from different patients who were admitted to affiliated hospitals of Kerman University of Medical science. MRSA and vancomycin-resistant S. aureus (VRSA) isolates were detected by phenotypic methods. Polymerase chain reaction (PCR) technique was used for detection of mecA, vanA and vanB genes. Staphylococcal cassette chromosomemec (SCCmec) and spa typing were used for molecular typing of among MRSA isolates. Overall, 53% of isolates were considered as MRSA. Two MRSA isolates were resistant to vancomycin and vanA was detected in only one of VRSA isolates. SCCmec type III belonged to spa types t030 and t459 which they were the dominant spa types among community-associated MRSA (CA-MRSA) and healthcare-acquired MRSA (HA-MRSA) isolates. Our findings showed that the SCCmec type I and III spread from hospital settings to community, although the SCCmec type IV spread from community to healthcare systems. We have also reported VRSA isolates from hospitalized patients, therefore, appropriate policies should be enforced in order to prevent the spread of antibiotic resistance isolates in hospitals settings.

Ceftaroline-Resistant, Daptomycin-Tolerant, and Heterogeneous Vancomycin-Intermediate Methicillin-Resistant Staphylococcus aureus Causing Infective Endocarditis.

We report a case of infective endocarditis (IE) caused by ceftaroline-resistant, daptomycin-tolerant, and heterogeneous vancomycin-intermediate methicillin-resistant S. aureus (MRSA). Resistance to ceftaroline emerged in the absence of drug exposure, and the E447K substitution in the active site of PBP2a previously associated with ceftaroline resistance was identified. Additionally, we present evidence of patient-to-patient transmission of the strain within the same unit. This case illustrates the difficulties in treating MRSA IE in the setting of a multidrug-resistant phenotype.

[Virtual screening and antibacterial activity of lead compounds targeting to penicillin-binding protein 3 (PBP3) of Pseudomonas aeruginosa].

OBJECTIVE: This study was carried out to obtain lead compounds targeting penicillin-binding protein 3 (PBP3) of Pseudomonas aeruginosa by virtual screening. METHODS: UCSF dock 6.5 was used for the virtual screening from a database containing 1.04 million small molecules. Hit compounds with simple structures were synthesized and then evaluated for their antibacterial activities. RESULTS: Grid score was used for the first round of screening, and 60000 small molecules whose scores lower than -30 kcal/mol were screened out from the database. These molecules were subjected to the second round of screening using amber score. Approximately 200 hit compounds with scores lower than -20 kcal/mol were analyzed and 4 of them were selected as lead compounds and then synthesized. The minimal inhibition concentrations (MICs) of the lead compounds were between 175-275 µg/mL, which were lower than that of Sulfadiazine (500 µg/mL) significantly. Meanwhile, these compounds were effective for both Gram-negative and Gram-positive bacteria. CONCLUSION: The lead compounds had potential to become new antibacterial agents for conquering the drug resistance of P. aeruginosa.

Antibiotic combination therapy can select for broad-spectrum multidrug resistance in Pseudomonas aeruginosa.

Combination therapy with several antibiotics is one strategy that has been applied in order to limit the spread of antimicrobial resistance. We compared the de novo evolution of resistance during combination therapy with the ß-lactam ceftazidime and the fluoroquinolone ciprofloxacin with the resistance evolved after single-drug exposure. Combination therapy selected for mutants that displayed broad-spectrum resistance, and a major resistance mechanism was mutational inactivation of the repressor gene mexR that regulates the multidrug efflux operon mexAB-oprM. Deregulation of this operon led to a broad-spectrum resistance phenotype that decreased susceptibility to the combination of drugs applied during selection as well as to unrelated antibiotic classes. Mutants isolated after single-drug exposure displayed narrow-spectrum resistance and carried mutations in the MexCD-OprJ efflux pump regulator gene nfxB conferring ciprofloxacin resistance, or in the gene encoding the non-essential penicillin-binding protein DacB conferring ceftazidime resistance. Reconstruction of resistance mutations by allelic replacement and in vitro fitness assays revealed that in contrast to single antibiotic use, combination therapy consistently selected for mutants with enhanced fitness expressing broad-spectrum resistance mechanisms.

PBP2a mutations causing high-level Ceftaroline resistance in clinical methicillin-resistant Staphylococcus aureus isolates.

Ceftaroline is the first member of a novel class of cephalosporins approved for use in the United States. Although prior studies have identified eight ceftaroline-resistant methicillin-resistant Staphylococcus aureus (MRSA) isolates in Europe and Asia with MICs ranging from 4 to 8 mg/liter, high-level resistance to ceftaroline (>32 mg/liter) has not been described in MRSA strains isolated in the United States. We isolated a ceftaroline-resistant (MIC > 32 mg/liter) MRSA strain from the blood of a cystic fibrosis patient and five MRSA strains from the respiratory tract of this patient. Whole-genome sequencing identified two amino acid-altering mutations uniquely present in the ceftaroline-binding pocket of the transpeptidase region of penicillin-binding protein 2a (PBP2a) in ceftaroline-resistant isolates. Biochemical analyses and the study of isogenic mutant strains confirmed that these changes caused ceftaroline resistance. Thus, we identified the molecular mechanism of ceftaroline resistance in the first MRSA strain with high-level ceftaroline resistance isolated in the United States.

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

OBJECTIVES: 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, ß-lactam antibiotics. The relationship between mecA gene carriage, phenotypic expression of the gene by PBP-2a production and in vitro resistance to the ß-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. METHODS: The relationship between mecA gene carriage, phenotypic expression of the gene by PBP-2a production and in vitro resistance to the ß-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). ß-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™. RESULTS: 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 ß-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. CONCLUSIONS: ß-Lactam antibiotics are useful for the treatment of neonatal CoNS sepsis, reserving vancomycin for selected cases.