Missense Mutations in PBP2A Affecting Ceftaroline Susceptibility Detected in Epidemic Hospital-Acquired Methicillin-Resistant Staphylococcus aureus Clonotypes ST228 and ST247 in Western Switzerland Archived since 1998

The development and maintenance of an arsenal of antibiotics is a major health care challenge. Ceftaroline is a new cephalosporin with activity against methicillin-resistant Staphylococcus aureus (MRSA); however, no reports concerning MRSA ceftaroline susceptibility have been reported in Switzerland. We tested the in vitro activity of ceftaroline against an archived set of 60 MRSA strains from the University Hospital of Geneva collected from 1994 to 2003. Our results surprisingly revealed ceftaroline-resistant strains (MIC, >1 μg/ml in 40/60 strains; EUCAST breakpoints, susceptible [S], ≤1 μg/ml; resistant [R], >1 μg/ml) were present from 1998 to 2003. The detected resistant strains predominantly belonged to sequence type 228 (ST228) (South German clonotype) but also to ST247 (Iberian clonotype). A sequence analysis of these strains revealed missense mutations in the penicillin-binding protein 2A (PBP2A) allosteric domain (N146K or E239K and N146K-E150K-G246E). The majority of our ST228 PBP2A mutations (N146K or E150K) were distinct from ST228 PBP2A allosteric domain mutations (primarily E239K) recently described for MRSA strains collected in Thailand and Spain during the 2010 Assessing Worldwide Antimicrobial Resistance Evaluation (AWARE) global surveillance program. We also found that similar allosteric domain PBP2A mutations (N146K) correlated with ceftaroline resistance in an independent external ST228 MRSA set obtained from the nearby University Hospital of Lausanne, Lausanne, Switzerland, collected from 2003 to 2008. Thus, ceftaroline resistance was observed in our archived strains (including two examples of an MIC of 4 µg/ml for the Iberian ST247 clonotype with the triple mutation N146K/E150K/G246E), at least as far back as 1998, considerably predating the commercial introduction of ceftaroline. Our results reinforce the notion that unknown parameters can potentially exert selective pressure on PBP2A that can subsequently modulate ceftaroline resistance.     

Contribution of Selected Gene Mutations to Resistance in Clinical Isolates of Vancomycin-Intermediate Staphylococcus aureus

Infections with vancomycin-intermediate Staphylococcus aureus (VISA) have been associated with vancomycin treatment failures and poor clinical outcomes. Routine identification of clinical isolates with increased vancomycin MICs remains challenging, and no molecular marker exists to aid in diagnosis of VISA strains. We tested vancomycin susceptibilities by using microscan, Etest, and population analyses in a collection of putative VISA, methicillin-resistant S. aureus, and methicillin-sensitive S. aureus (VSSA) infectious isolates from community- or hospital-associated S. aureus infections (n = 77) and identified 22 VISA and 9 heterogeneous VISA (hVISA) isolates. Sequencing of VISA candidate loci vraS, vraR, yvqF, graR, graS, walR, walK, and rpoB revealed a high diversity of nonsynonymous single-nucleotide polymorphisms (SNPs). For vraS, vraR, yvqF, walK, and rpoB, SNPs were more frequently present in VISA and hVISA than in VSSA isolates, whereas mutations in graR, graS, and walR were exclusively detected in VISA isolates. For each of the individual loci, SNPs were only detected in about half of the VISA isolates. All but one VISA isolate had at least one SNP in any of the genes sequenced, and isolates with an MIC of 6 or 8 μg/ml harbored at least 2 SNPs. Overall, increasing vancomycin MICs were paralleled by a higher proportion of isolates with SNPs. Depending on the clonal background, SNPs appeared to preferentially accumulate in vraS and vraR for sequence type 8 (ST8) and in walK and walR for ST5 isolates. Taken together, by comparing VISA, hVISA, and VSSA controls, we observed preferential clustering of SNPs in VISA candidate genes, with an unexpectedly high diversity across these loci. Our results support a polygenetic etiology of VISA.     

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.     

In Vitro Activity of Ceftaroline against Community-Associated Methicillin-Resistant,Vancomycin-Intermediate,Vancomycin-Resistant,and Daptomycin-Nonsusceptible Staphylococcus aureus Isolates

This study assessed the in vitro activities of ceftaroline and five comparator agents against a collection of Staphylococcus aureus isolates. Ceftaroline demonstrated potent activity against community-associated methicillin-resistant S. aureus (CA-MRSA) isolates and showed bactericidal activity against vancomycin-intermediate S. aureus (VISA), vancomycin-resistant S. aureus (VRSA), heteroresistant VISA (hVISA), and daptomycin-nonsusceptible S. aureus (DNSSA) isolates. Ceftaroline may represent a bactericidal treatment option for infections caused by these pathogens.The increasing prevalence of resistant Staphylococcus aureus strains, including methicillin-resistant S. aureus (MRSA), community-associated MRSA (CA-MRSA), and S. aureus strains with reduced susceptibility to vancomycin, emphasizes the need for innovative antimicrobials with activity against these pathogens (19, 24). Although the prevalence of S. aureus strains with reduced vancomycin susceptibility remains low, such strains have been associated with vancomycin treatment failure, limiting the treatment options for patients with such infections (3, 8). Ceftaroline is a novel, parenteral, broad-spectrum cephalosporin exhibiting bactericidal activity against Gram-positive organisms, including MRSA and multidrug-resistant Streptococcus pneumoniae (MDRSP), as well as common Gram-negative pathogens (6, 9, 21). Ceftaroline is currently in phase 3 development for the treatment of complicated skin and skin structure infections and community-acquired bacterial pneumonia. The study described here evaluated the in vitro activities of ceftaroline and five comparator agents against CA-MRSA, vancomycin-intermediate S. aureus (VISA), vancomycin-resistant S. aureus (VRSA), heteroresistant VISA (hVISA), and daptomycin-nonsusceptible S. aureus (DNSSA) isolates.(A preliminary report of these results was presented at the 48th Interscience Conference on Antimicrobial Agents and Chemotherapy-Infectious Diseases Society of America Annual Meeting, Washington, DC, 25 to 28 October 2008.)A total of 132 MRSA strains were selected for evaluation. CA-MRSA strains (n = 92) were isolated from patients admitted to St. John Hospital and Medical Center in Detroit, MI. These patients had positive MRSA cultures within 48 h of admission, in accordance with the definition of CA-MRSA described by the Centers for Disease Control and Prevention (2). DNSSA strains (n = 7) and hVISA strains (n = 3) were obtained from blood collected from patients at the same hospital. The hVISA isolates were identified by a modified population analysis profile method (12). VISA isolates (n = 20) and VRSA isolates (n = 10) were obtained via the Network on Antimicrobial Resistance in Staphylococcus aureus (NARSA) program, supported under NIAID/NIH contract HHSN272200700055C.All CA-MRSA isolates were typed by pulsed-field gel electrophoresis (PFGE) with the restriction enzyme SmaI, followed by visual interpretation and categorization. PCR methods were used to determine the staphylococcal cassette chromosome mec (SCCmec) type, the presence of Panton-Valentine leukocidin (PVL) genes lukS-PV and lukF-PV, and the presence of the arginine catabolic mobile element (ACME) via detection of the arcA locus.In vitro susceptibility tests were performed with the antimicrobials ceftaroline (lot CI 170-07; Forest Laboratories, Inc., New York, NY), vancomycin-HCl (Sigma), daptomycin (Cubist), clindamycin-HCl (Sigma), linezolid (Pfizer), and trimethoprim-sulfamethoxazole (Sigma), which were obtained individually and reconstituted to a 1:19 ratio. All antibiotics were received in powder form and were reconstituted according to guidelines of the Clinical and Laboratory Standards Institute (CLSI). MICs and minimum bactericidal concentrations (MBCs) were determined according to the guidelines of the CLSI (4, 5). Microdilution tests with cation-adjusted Mueller-Hinton broth were used to identify the MICs of all antimicrobial agents tested. The percentages of susceptible isolates were determined by using the CLSI breakpoints. For the testing of daptomycin, additional calcium was added to the broth for a final concentration of 50 mg/liter. The MICs were read visually and corresponded to the concentration in the well with the lowest drug concentration with no visible bacterial growth. The MBC was defined as the antibiotic concentration that reduced the number of viable cells by ≥99.9%. This was based on colony counts from the growth control well and rejection values determined by tables provided by Pearson (18).S. aureus ATCC 29213 and Enterococcus faecalis ATCC 29212 were used as the control strains for the MIC determinations, and S. aureus ATCC 25923 was used as the control strain for the MBC determinations.Approximately 58% of the CA-MRSA isolates were SCCmec type IV/IVa, whereas the majority of the other isolates were SCCmec type II (Table ​(Table1).1). All SCCmec type IVa isolates were positive for the PVL and ACME genes, whereas isolates of all other SCCmec types were negative for these genes. Of all the other S. aureus strains, only two DNSSA isolates were SCCmec type IVa and positive for the PVL and ACME genes.

TABLE 1.

SCC mec elements, PVL and ACME genes, and ceftaroline MIC₉₀s and MBC₉₀s of CA-MRSA isolates^a

Fusidic Acid Resistance Determinants in Staphylococcus aureus Clinical Isolates

A total of 71 fusidic acid-resistant Staphylococcus aureus (45 methicillin-resistant and 26 methicillin-susceptible) isolates were examined for the presence of resistance determinants. Among 45 fusidic acid-resistant methicillin-resistant S. aureus (MRSA), isolates, 38 (84%) had fusA mutations conferring high-level resistance to fusidic acid (the MIC was ≥128 μg/ml for 22/38), none had fusB, and 7 (16%) had fusC. For 26 fusidic acid-resistant methicillin-susceptible S. aureus (MSSA), only 3 possessed fusA mutations, but 15 (58%) had fusB and 8 (31%) had fusC. Low-level resistance to fusidic acid (MICs ≤ 32 μg/ml) was found in most fusB- or fusC-positive isolates. For 41 isolates (38 MRSA and 3 MSSA), with fusA mutations, a total of 21 amino acid substitutions in EF-G (fusA gene) were detected, of which R76C, E444K, E444V, C473S, P478S, and M651I were identified for the first time. The nucleotide sequencing of fusB and flanking regions in an MSSA isolate revealed the structure of partial IS257-aj1-LP-fusB-aj2-aj3-IS257-partial blaZ, which is identical to the corresponding region in pUB101, and the rest of fusB-carrying MSSA isolates also show similar structures. On the basis of spa and staphylococcal cassette chromosome mec element (SCCmec) typing, two major genotypes, spa type t037-SCCmec type III (t037-III; 28/45; 62%) and t002-II (13/45; 29%), were predominant among 45 MRSA isolates. By pulsed-field gel electrophoresis analysis, 45 MRSA isolates were divided into 12 clusters, while 26 MSSA isolates were divided into 15 clusters. Taken together, the distribution of fusidic acid resistance determinants (fusA mutations, fusB, and fusC) was quite different between MRSA and MSSA groups.Fusidic acid has been used as a topical agent for skin infection and for some systemic infections caused by Staphylococcus aureus (12). Fusidic acid-resistant S. aureus has been reported in many countries, with the prevalence ranging from 0.3 to 52.5%, and the occurrences of resistance determinants were remarkably different among different countries (5, 6). The rate of fusidic acid resistance in S. aureus in our hospital each year is about 3 to 6%. Although this frequency is not very high, the understanding of fusidic acid resistance mechanisms still is very important.Two major fusidic acid resistance mechanisms have been reported in S. aureus: the alteration of the drug target site (4, 24, 25), which is due to mutations in fusA (encoding elongation factor G [EF-G]) (24, 28) or rplF (or FusE, encoding ribosome protein L6) (19, 25), and the protection of the drug target site by FusB family proteins, including fusB, fusC, and fusD (27, 30). Point mutations in fusA occur mainly in domain III of EF-G and usually permit normal colony size and growth rate, conferring the FusA class resistance (20, 25). Fusidic acid-resistant small-colony variant (SCV) isolates, referred to as the FusA-SCV class, were due mostly to mutations in domain V of EF-G (19, 25). Some fusA point mutations may compromise fitness during growth in vivo and in vitro, but these costs may be partly or fully compensated for by acquiring additional amino acid substitutions (24). Another subclass of mutations, located in rplF (32), is referred to as the FusE class, which confers fusidic acid resistance to SCV isolates.Acquired fusidic acid resistance genes found in Staphylococcus spp. include fusB, fusC, and fusD. The genes fusB and fusC were found in S. aureus and coagulase-negative staphylococci (22, 27, 30, 34), and fusD was an intrinsic factor causing fusidic acid resistance in Staphylococcus saprophyticus (30). The fusB determinant originally was found on the plasmid pUB101 in S. aureus (26). Later, the fusB determinant also was found on a transposon-like element (27) or in a staphylococcal pathogenicity island (29).In this study, we analyzed fusidic acid resistance determinants among methicillin-resistant S. aureus (MRSA) and methicillin-susceptible S. aureus (MSSA) isolates for which fusidic acid had a MIC of ≥2 μg/ml. The distribution of fusidic acid resistance determinants was found different in MRSA and MSSA groups. Furthermore, to understand the phylogenetic relationship of resistance determinant-containing isolates, genotyping also was performed.     

Anaerobic Resistance of Clinical Isolates of Staphylococcus aureus to Aminoglycosides

The anaerobic minimum inhibitory concentration of six aminoglycosides (amikacin, gentamicin, kanamycin, netilmicin, sisomicin, and tobramycin) averaged over 10-fold greater than the aerobic minimum inhibitory concentration for 50 clinical isolates of Staphylococcus aureus. Cultures from osteomyelitis and blood generally showed a somewhat greater increase in minimum inhibitory concentration due to anaerobiosis than did cultures from abscesses and wounds, and amikacin activity was most affected by anaerobiosis.     

In Vivo Efficacy of Ceftaroline Fosamil in a Methicillin-Resistant Panton-Valentine Leukocidin-Producing Staphylococcus aureus Rabbit Pneumonia Model

Ceftaroline, the active metabolite of the prodrug ceftaroline fosamil, is a cephalosporin with broad-spectrum in vitro activity against Gram-positive organisms, including methicillin-resistant Staphylococcus aureus (MRSA) and multidrug-resistant Streptococcus pneumoniae (MDRSP), and common Gram-negative pathogens. This study investigated the in vivo activity of ceftaroline fosamil compared with clindamycin, linezolid, and vancomycin in a severe pneumonia model due to MRSA-producing Panton-Valentine leukocidin (PVL). A USA300 PVL-positive clone was used to induce pneumonia in rabbits. Infected rabbits were randomly assigned to no treatment or simulated human-equivalent dosing with ceftaroline fosamil, clindamycin, linezolid, or vancomycin. Residual bacterial concentrations in the lungs and spleen were assessed after 48 h of treatment. PVL expression was measured using a specific enzyme-linked immunosorbent assay (ELISA). Ceftaroline, clindamycin, and linezolid considerably reduced mortality rates compared with the control, whereas vancomycin did not. Pulmonary and splenic bacterial titers and PVL concentrations were greatly reduced by ceftaroline, clindamycin, and linezolid. Ceftaroline, clindamycin, and linezolid were associated with reduced pulmonary tissue damage based on significantly lower macroscopic scores. Ceftaroline fosamil, clindamycin, and, to a lesser extent, linezolid were efficient in reducing bacterial titers in both the lungs and spleen and decreasing macroscopic scores and PVL production compared with the control.     

Topoisomerase Mutations in Fluoroquinolone-Resistant and Methicillin-Susceptible and -Resistant Clinical Isolates of Staphylococcus aureus

The incidence of the various mutations in the genes encoding topoisomerase IV and DNA gyrase in fluoroquinolone-resistant clinical isolates of Staphylococcus aureus is not known. Using restriction fragment length polymorphism analysis and DNA sequencing, we found that in fluoroquinolone- and methicillin-resistant strains, mutations in grlA and gyrA are quite likely to be present together. For fluoroquinolone-resistant but methicillin-susceptible strains, mutations in grlA alone are more common.     

In Vitro Activity of Ceftaroline against Methicillin-Resistant Staphylococcus aureus and Heterogeneous Vancomycin-Intermediate S. aureus in a Hollow Fiber Model

Ceftaroline is a broad-spectrum injectable cephalosporin exhibiting bactericidal activity against a variety of bacteria, including methicillin-resistant Staphylococcus aureus (MRSA). Using a two-compartment in vitro pharmacokinetic/pharmacodynamic (PK/PD) model, we evaluated the activity of ceftaroline at 600 mg every 8 h (q8h) and q12h in comparison with that of vancomycin at 1,000 mg q12h over a 72-h time period against six clinical MRSA isolates, including two heterogeneous vancomycin-intermediate S. aureus (hVISA) isolates. The MIC and minimum bactericidal concentration ranged between 0.125 to 2 and 0.5 to 2 μg/ml for ceftaroline and vancomycin, respectively. In the PK/PD model, ceftaroline was superior to vancomycin against all isolates (P < 0.05), except one to which it was equivalent. No difference in activity was observed between both q8 and q12h dosing regimens of ceftaroline. Bacterial regrowth was observed after 32 h for two isolates treated with ceftaroline. This regrowth was uncorrelated to resistance, instability of the drug, or tolerance. However, subpopulations with higher MICs to ceftaroline were found by population analysis for these two isolates. Finally, and in contrast to ceftaroline, MIC elevations up to 8 to 12 μg/ml were observed with vancomycin for the hVISA isolates. In conclusion, in addition to a lower potential to select resistant mutants, ceftaroline demonstrated activity equal to or greater than vancomycin against MRSA isolates. Although further in vitro and in vivo investigations are warranted, ceftaroline appears to be a promising alternative for the treatment of MRSA infections.Bacteria exhibit an amazing ability to adapt to changes in their environment, and perhaps nowhere is this more visible than in the development of resistance to antibacterial agents. Antibiotic use and misuse have largely contributed to the emergence of multidrug-resistant (MDR) pathogens in the health care setting, leading to a critical need for the development of new antibacterial agents (23). Among the various MDR gram-positive pathogens, methicillin-resistant Staphylococcus aureus (MRSA) strains, particularly those that are heteroresistant to vancomycin, heterogeneous vancomycin-intermediate S. aureus (hVISA), represent a serious clinical concern because they have been associated with poor treatment outcomes, excess health care costs, and prolonged hospital stay (10, 11, 15). In MRSA, resistance to β-lactam antibiotics results from the production of a low-affinity penicillin-binding protein (PBP) called PBP2′ or PBP2a, that is not inhibited by most β-lactams owing to inefficient formation of a covalent enzyme-inhibitor complex (25). New compounds with higher affinity for PBP2a offer the potential to overcome this resistance and represent a novel option for treatment of infections caused by MRSA, including isolates with reduced susceptibility to glycopeptides (20, 21).Ceftaroline is an investigational parenteral broad-spectrum cephalosporin that exhibits bactericidal activity against gram-positive and -negative pathogens, including MRSA, hVISA, and MDR Streptococcus pneumoniae (5, 18, 28). Administered as a water-soluble semisynthetic N-phosphono-type prodrug (ceftaroline fosamil), ceftaroline is currently in phase III development for treatment of complicated skin and skin structure infections (cSSSI) and community-acquired pneumonia (3). Several in vivo models have been used to compare the activity of ceftaroline versus those of vancomycin, linezolid, arbekacin, and teicoplanin against MRSA isolates (8, 9). In thigh muscle infection and endocarditis models, ceftaroline demonstrated a highly bactericidal effect and was significantly more active than linezolid against MRSA and hVISA isolates. Additionally, statistically significant differences between ceftaroline and vancomycin therapy were also observed against hVISA isolates, but not against MRSA, despite the fact that both agents achieved a bactericidal effect (9). Several factors, such as MIC values and the inoculum size, may affect the killing activity of β-lactam antimicrobials (24, 27). Until now, no in vitro studies of ceftaroline have been carried out using conditions that take into consideration the pharmacokinetic and pharmacodynamic (PK/PD) parameters of this agent. Thus, as the primary objective of this study, we evaluated the in vitro activity of ceftaroline in comparison with that of vancomycin against clinical MRSA and hVISA isolates using an experimental PK/PD model simulating human PK (16, 22, 26).(A portion of this work was presented at the 48th Interscience Conference on Antimicrobial Agents and Chemotherapy-46th Annual Meeting of the Infectious Diseases Society of America, Washington, DC, 25 to 28 October 2008 [29].)     

Elevated Linezolid Resistance in Clinical cfr-Positive Staphylococcus aureus Isolates Is Associated with Co-Occurring Mutations in Ribosomal Protein L3

Resistance to linezolid (LZD) occurs through mutations in 23S rRNA and ribosomal proteins L3 and L4 or through methylation of 23S rRNA by Cfr. Here we report novel L3 mutations, ΔSer145/His146Tyr and ΔMet169-Gly174, co-occurring with cfr in LZD-resistant Staphylococcus aureus isolates recovered from a hospital outbreak in Madrid, Spain. LZD MIC values (16, 32, or 64 μg/ml) correlated with the presence and severity of the L3 mutation. All isolates had TR-700 (torezolid) MIC values of ≤2 μg/ml.Linezolid (LZD) resistance was first associated with mutations in the domain V region of 23S rRNA genes (G2576T) (7, 30). Over time, a variety of 23S rRNA mutations have been identified, and these remain the most commonly reported class of mutation leading to LZD resistance (5, 9, 10). In rare cases, mutations in ribosomal protein L4 have also been associated with LZD resistance (8, 17, 32). More recently, a variety of mutations in ribosomal protein L3 have also been identified in both laboratory and clinically derived staphylococci associated with reduced susceptibility to oxazolidinones (15-17). Cfr-based LZD resistance, however, is potentially more worrisome than mutation-based chromosomally encoded resistance mechanisms (25, 29). The cfr gene encodes a methyltransferase which confers LZD resistance via methylation of carbon-8 on 23S rRNA base A2503 (6). Cfr is generally plasmid borne and transposon associated and therefore likely to be horizontally transmitted (11). Strains carrying cfr are resistant not only to LZD but also to phenicols, lincosamides, pleuromutilins, and streptogramin A class antibiotics (19), as well as 16-membered ring macrolides (28). Thus, selective pressure due to the use of any of these drug classes may lead to the spread of this resistance determinant.The emergence of cfr and identification of additional LZD resistance mechanisms, including L3 mutations, raise the potential for multiple mechanisms to occur within a single strain. Our previous work identified coupled 23S rRNA and L3 mutations in both a laboratory LZD serially passaged Staphylococcus aureus strain (17) and a clinical Staphylococcus epidermidis isolate (15). Another recent report documented a Spanish outbreak of LZD^r strains (S. aureus, S. epidermidis, Enterococcus faecium, and Enterococcus faecalis) which included strains possessing cfr alone, 23S rRNA mutations alone, or 23S rRNA G2576T mutations in conjunction with cfr (2). Finally, an outbreak of LZD^r S. epidermidis in Ohio was comprised of isolates possessing an L4 mutation in conjunction with either the cfr gene or mutations in 23S rRNA (1). These reports are the first to demonstrate the co-occurrence of cfr with any other LZD resistance mechanism; however, given the low fitness cost of Cfr methylation (12), strains with resistance due to multiple mechanisms may not be unexpected. Characterization of such isolates and the evaluation of antibacterial activities of clinically relevant oxazolidinones are thus of high interest.Previous reports of S. aureus cfr-positive clinical isolates and laboratory-generated cfr-transformed S. aureus strains typically cite LZD MIC values in the range of 8 to 16 μg/ml (13, 14, 21, 29). Analysis of cfr-positive LZD^r MRSA from a 2008 hospital outbreak in Madrid, Spain, identified 18 isolates: 1 environmental and 12 patient intensive care unit (ICU) isolates, 3 patient isolates from other wards, and 2 additional patient isolates predating the outbreak that were identified in a retrospective study (22). LZD MIC values for these strains were 8 (n = 4), 16 (n = 13), or 32 (n = 1) μg/ml (22), all of which are above the LZD breakpoint of 4 μg/ml. This study investigated whether additional oxazolidinone resistance mechanisms could account for the variability in LZD resistance levels among these clinical cfr isolates.(Portions of this work were presented at the 50th Interscience Conference on Antimicrobial Agents and Chemotherapy [J. B. Locke, G. Morales, M. Hilgers, Kedar G. C., S. Rahawi, J. J. Picazo, K. J. Shaw and J. L. Stein, abstr. C1-1431], Boston, MA, 12 to 15 September 2010.)MICs of clinically relevant oxazolidinones, LZD (ChemPacific, Inc., Baltimore, MD), TR-700 (torezolid, formerly known as DA-7157; Trius Therapeutics, Inc., San Diego, CA), and radezolid (RZD) (RX-1741; Medicilon, Chicago, IL), as well as tiamulin (TIA) (Wako Pure Chemical Industries, Ltd., Richmond, VA), chloramphenicol (CHL) (Sigma-Aldrich Corp., St. Louis, MO), and vancomycin (VAN) (Sigma), were determined via broth microdilution in accordance with CLSI guidelines as previously described (3, 17). Quality control of oxazolidinones was performed via nuclear magnetic resonance (NMR), liquid chromatography-mass spectrometry (LC-MS), and biological activity assays. MIC values reported for each strain/drug combination were determined in at least three independent experiments, all yielding identical results. LZD MIC values for a panel of the 6 representative strains included in this study fell into three groups: group 1 (16 μg/ml; n = 1), group 2 (32 μg/ml; n = 4), or group 3 (64 μg/ml; n = 1), which corresponded to TR-700 MIC values of 0.5, 1, or 2 μg/ml and radezolid MIC values of 4, 4, or 8 μg/ml, respectively (Table ​(Table1).1). Both TR-700 and LZD MIC values in this study were 2-fold higher than those originally reported for these strains (22). As expected, the cfr isolates were resistant to TIA and CHL (Table ​(Table11).

TABLE 1.

Characteristics of clinical and laboratory-derived S. aureus strains possessing L3 mutations and/or the cfr methyltransferase gene

Transduction and Elimination of Resistance Determinants in Methicillin-Resistant Staphylococcus aureus

Elimination and transduction of drug resistance was examined in methicillin-resistant strains of Staphylococcus aureus. Irreversible spontaneous loss and "curing" by aging of cultures and by treatment with ethidium bromide indicated that the determinants for penicillinase production and chloramphenicol resistance, and probably also for neomycin resistance, were located extrachromosomally. On the other hand, the determinants of resistance to erythromycin, streptomycin, tetracycline, and methicillin could not be eliminated by acridines, ethidium bromide, rifampin, sodium dodecyl sulfate, ultraviolet (UV) irradiation, growth at 43.5 C, aging of cultures, or combinations of these treatments. The stimulation of transduction frequency by UV irradiation of phage in the case of the stable markers, but not in the case of the unstable ones, supported further the hypothesis of chromosomal location of the markers of methicillin, erythromycin, tetracycline, and streptomycin resistance and extrachromosomal location of the determinants for penicillinase production and chloramphenicol resistance. Neomycin resistance could not be transduced. Joint elimination and co-transduction of the determinants for penicillinase production and resistance to chloramphenicol and neomycin were not observed, indicating the location of these markers on separate, mutually compatible plasmids. Co-transduction of chromosomal resistance determinants was usually less than 1%, which makes the location of these genes in a circumscribed area of the chromosome improbable.     

Fitness Cost of VanA-Type Vancomycin Resistance in Methicillin-Resistant Staphylococcus aureus

We have quantified the biological cost of VanA-type glycopeptide resistance due to the acquisition of the resistance operon by methicillin-resistant Staphylococcus aureus (MRSA) from Enterococcus sp. Exponential growths of recipient strain HIP11713, its transconjugant VRSA-1, VRSA-5, and VRSA-6 were compared in the absence or, except for HIP11713, in the presence of vancomycin. Induction of resistance was performed by adding vancomycin in both the preculture and the culture or the culture at only 1/50 the MIC. In the absence of vancomycin, the growth rates of the vancomycin-resistant S. aureus (VRSA) strains were similar to that of susceptible MRSA strain HIP11713. When resistance was induced, and under both conditions, there was a significant reduction of the growth rate of the VRSA strains relative to that of HIP11713 and to those of their noninduced counterparts, corresponding to a ca. 20% to 38% reduction in fitness. Competition experiments between isogenic VRSA-1 and HIP11713 mixed at a 1:1, 1:100, or 100:1 ratio revealed a competitive disadvantage of 0.4% to 3% per 10 generations of the transconjugant versus the recipient. This slight fitness burden can be attributed to the basal level of expression of the van genes in the absence of induction combined with a gene dosage effect due to the presence of the van operon on multicopy plasmids. These data indicate that VanA-type resistance, when induced, is highly costly for the MRSA host, whereas in the absence of induction, its biological cost is minimal. Thus, the potential for the dissemination of VRSA clinical isolates should not be underestimated.Staphylococcus aureus is one of the most common causes of hospital- and community-acquired infections, and treatment of staphylococcal diseases is complicated by the organism''s innate ability to become resistant to chemotherapy (15). Vancomycin is the drug of choice to treat infections due to methicillin-resistant S. aureus (MRSA), but an increase in vancomycin use has led to the emergence of two types of glycopeptide-resistant S. aureus strains. The first one, designated glycopeptide-intermediate-resistant S. aureus (GISA), is associated with a thickened and poorly cross-linked cell wall, resulting in an accumulation of d-alanyl-d-alanine (d-Ala-d-Ala) targets in the periphery that sequester glycopeptides (9). The second type, designated vancomycin-resistant S. aureus (VRSA), is due to the acquisition of the vanA operon carried by transposon Tn1546 from Enterococcus sp., resulting in high-level resistance (4, 5). VanA-type resistance results in the synthesis of a new cell wall utilizing precursors ending in d-alanyl-d-lactate (d-Ala-d-Lac) that have 1,000-fold-less affinity for glycopeptides associated with the elimination of the susceptible d-Ala-d-Ala-containing precursors to which vancomycin binds (8). The expression of resistance is regulated by a two-component system (VanS-VanR) that allows the inducible expression of the vanA operon in response to the presence of glycopeptides, vancomycin, or teicoplanin in the culture medium (3, 10). Since 2002, nine MRSA strains that were highly resistant to glycopeptides that harbor the vanA gene cluster on two types of plasmids have been reported in the United States. The first group is exemplified by transconjugant strain VRSA-1, which was isolated together with susceptible recipient strain HIP11713 and vancomycin-resistant Enterococcus faecalis donor strain DMC83006B from the foot ulcer of a diabetic patient (23, 24). The vanA operon was acquired in two steps: first, plasmid pAM830, carrying Tn1546, was transferred by conjugation from E. faecalis DMC83006B (12) to MRSA strain HIP11713, and Tn1546 was then transposed on resident plasmid pAM829, generating pLW1043 (pAM829::Tn1546) (20, 24). Strains VRSA-5 and VRSA-6, which belong to the second group, acquired and subsequently stably maintained an Inc18-like enterococcal plasmid carrying Tn1546 (26). We have studied these three clinical isolates that are representative of the two VRSA classes.Antibiotic resistance, by acquisition of a mobile genetic element or by mutation, is often associated with a reduced fitness of the bacterial host (1). More-fit variants can be selected during further evolution after either a loss of resistance or the occurrence of a compensatory mutation that restores bacterial fitness (16). The biological cost is one of the major indirect factors that determines the stability and dissemination of antibiotic resistance. Study of the fitnesses of MRSA and GISA strains revealed a decrease in the growth rates of the two types of strains (17). Worldwide dissemination of MRSA clones has been associated with their ability to compensate for the cost of harboring the staphylococcal chromosomal cassette mec element (11). In certain GISA isolates, the deletion of the mecA gene can partially compensate for the fitness cost imposed by vancomycin resistance, suggesting that the simultaneous resistance to β-lactams and glycopeptides is highly costly for S. aureus (17). High-level vancomycin resistance is associated with a sophisticated dual biochemical mechanism mediated by seven genes, vanRSHAXYZ, which combines the synthesis of modified late peptidoglycan precursors with the elimination of the chromosomal pathway for the synthesis of the susceptible cell wall. The biological cost resulting from this combinatorial mechanism of resistance to glycopeptides on the host is predicted to be high, which is consistent with the fact that only a few strains of VRSA have been isolated. Considering the major public health problem that would result from VRSA dissemination, we have evaluated the fitnesses of clinical isolates VRSA-1, VRSA-5, and VRSA-6.     

Evaluation of Vancomycin in Combination with Piperacillin-Tazobactam or Oxacillin against Clinical Methicillin-Resistant Staphylococcus aureus Isolates and Vancomycin-Intermediate S. aureus Isolates In Vitro

Vancomycin with piperacillin-tazobactam is used as empirical therapy for critically ill patients. Studies of this combination against methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-intermediate S. aureus (VISA) are limited, but β-lactams in combination with vancomycin have shown synergistic activity against MRSA and VISA. The goal of this study was to evaluate whether piperacillin-tazobactam and vancomycin were synergistic against MRSA and VISA in vitro. Bloodstream MRSA (n = 20) and VISA (n = 4) strains were selected. In vitro antimicrobial activities of piperacillin-tazobactam and oxacillin were evaluated by disk diffusion, and MICs were determined by Etest using Muller-Hinton agar with and without vancomycin at one-half the MIC. Time-kill studies evaluated 14 MRSA and all 4 VISA isolates using piperacillin-tazobactam at 300/35 mg/liter or oxacillin at 40 mg/liter alone and with vancomycin at one-half the MIC. Mean zones of inhibition for piperacillin-tazobactam and oxacillin increased with vancomycin against MRSA and VISA (P < 0.001 for all), and the MIC₉₀ decreased with vancomycin against MRSA and VISA to values meeting susceptibility criteria for S. aureus (P < 0.001 for both antibiotics against MRSA). In MRSA time-kill studies, the mean 24-h reductions in inoculum for piperacillin-tazobactam, piperacillin-tazobactam with vancomycin, and oxacillin with vancomycin were 3.53, 3.69, and 2.62 log₁₀ CFU/ml, respectively. The mean 24-h reductions in VISA inoculum for piperacillin-tazobactam, piperacillin-tazobactam with vancomycin, and oxacillin with vancomycin were 2.85, 2.93, and 3.45 log₁₀ CFU/ml, respectively. Vancomycin with piperacillin-tazobactam or oxacillin demonstrated synergistic activity against MRSA and VISA. The clinical implications of these combinations against MRSA and VISA should be investigated.     

Cloning and Occurrence of czrC,a Gene Conferring Cadmium and Zinc Resistance in Methicillin-Resistant Staphylococcus aureus CC398 Isolates

We recently reported a phenotypic association between reduced susceptibility to zinc and methicillin resistance in Staphylococcus aureus CC398 isolates from Danish swine (F. M. Aarestrup, L. M. Cavaco, and H. Hasman, Vet. Microbiol. 142:455-457, 2009). The aim of this study was to identify the genetic determinant causing zinc resistance in CC398 and examine its prevalence in isolates of animal and human origin. Based on the sequence of the staphylococcal cassette chromosome mec (SCCmec) element from methicillin-resistant S. aureus (MRSA) CC398 strain SO385, a putative metal resistance gene was identified in strain 171 and cloned in S. aureus RN4220. Furthermore, 81 MRSA and 48 methicillin-susceptible S. aureus (MSSA) strains, isolated from pigs (31 and 28) and from humans (50 and 20) in Denmark, were tested for susceptibility to zinc chloride and for the presence of a putative resistance determinant, czrC, by PCR. The cloning of czrC confirmed that the zinc chloride and cadmium acetate MICs for isogenic constructs carrying this gene were increased compared to those for S. aureus RN4220. No difference in susceptibility to sodium arsenate, copper sulfate, or silver nitrate was observed. Seventy-four percent (n = 23) of the animal isolates and 48% (n = 24) of the human MRSA isolates of CC398 were resistant to zinc chloride and positive for czrC. All 48 MSSA strains from both human and pig origins were found to be susceptible to zinc chloride and negative for czrC. Our findings showed that czrC is encoding zinc and cadmium resistance in CC398 MRSA isolates, and that it is widespread both in humans and animals. Thus, resistance to heavy metals such as zinc and cadmium may play a role in the coselection of methicillin resistance in S. aureus.Methicillin-resistant Staphylococcus aureus (MRSA) isolates mainly belonging to CC (clonal complex) 398 have emerged recently in livestock production around the world (7, 10, 20, 22). This clonal lineage is able to persist in the farm environment and therefore constitutes a large reservoir for transmission to humans working in such environments, which raises occupational health concerns (14, 22, 27). Resistance to antimicrobials, as well as other factors, may have contributed to the success of MRSA CC398 and its emergence in the animal reservoirs (1).Metal-containing compounds are widely used as a feed supplement or for the prevention of gastrointestinal diseases in food animals. Resistance to metals is widely disseminated among bacterial species, and we have shown previously that the use of copper in feed can coselect for resistance to macrolides and glycopeptides in Enterococcus faecium (5). Heavy-metal resistance, i.e., resistance to arsenic, mercury, and cadmium, has been described among S. aureus isolates, including MRSA (2, 17-19). We recently described a phenotypic association between reduced susceptibility to zinc and MRSA CC398 from pigs in Denmark (1). It was suspected that the genetic background for the metal resistance was genetically linked to the methicillin resistance, and it was hypothesized to be present in the staphylococcal cassette chromosome mec (SCCmec) element carried by these MRSA isolates, as metal resistance genes had been described previously in the SCCmec element type III of MRSA isolate ST239 (9).The purpose of this study was to identify the genetic determinant(s) involved in the observed zinc resistance phenotype among the CC398 MRSA isolates from animals in Denmark and to investigate its effects on susceptibility to several metallic compounds. Furthermore, the prevalence of resistance to zinc and the putative resistance gene were determined among a larger collection of swine and human S. aureus CC398 isolates from Denmark.