Prevalence and Characterization of Heterogeneous Vancomycin-Intermediate Staphylococcus aureus Isolates from 14 Cities in China

The prevalence of heterogeneous vancomycin-intermediate Staphylococcus aureus (hVISA) among 1,012 vancomycin-susceptible methicillin (meticillin)-resistant S. aureus isolates collected from 14 cities in China from 2005 to 2007 was 13 to 16%, as determined by a combination of (i) measurement by the modified population analysis profile-area under the curve method (PAP-AUC) and (ii) estimation from the measured sensitivity and specificity of a screening method. Two hundred isolates from blood were chosen as a subset for measurement of the sensitivities and the specificities of several previously described screening methods by using the results of PAP-AUC as the reference. During this testing, one isolate was found to be a vancomycin-intermediate S. aureus (VISA) strain so was not used in the evaluation of the screening tests. Of the other 199 isolates, 26 (13.1%) were hVISA, as assessed by PAP-AUC. A screening cascade of culturing the isolates on brain heart infusion agar containing teicoplanin (5 mg/liter) and then subjecting the positive isolates to a macro-Etest method was applied to the 812 non-blood isolates, yielding 149 positive results. From these results and by adjusting for sensitivity (0.423) and specificity (0.861), the prevalence was estimated to be 15.7%. The precision of that estimate was assessed by reapplying the screening cascade to 120 randomly selected isolates from the 812 non-blood isolates and simultaneously determining their heterogeneous vancomycin-intermediate susceptibility status by PAP-AUC. Because PAP-AUC is impractical for use with large numbers of isolates, the screening-based estimation method is useful as a first approximation of the prevalence of hVISA. Of the 27 VISA or hVISA isolates from blood, 22.2% and 74.1% were staphylococcal chromosome cassette mec types II and III, respectively, while 77.8% and 22.2% were agr type 1 and agr type 2, respectively; the MIC ranges were 0.5 to 4 mg/liter for vancomycin and 0.25 to 1 mg/liter for daptomycin.Since their first report in Japan in 1997 (11), heterogeneous vancomycin-intermediate (heteroresistant) Staphylococcus aureus (hVISA) strains have been a new concern with respect to methicillin (meticillin)-resistant S. aureus (MRSA) strains that display reduced susceptibility to glycopeptides (12, 13), because such strains are potentially associated with the clinical failure of vancomycin treatment (4). Vancomycin heteroresistance among S. aureus isolates was one of the reasons that Tenover and Moellering cited for the Clinical and Laboratory Standards Institute decision in 2006 to lower the vancomycin MIC breakpoints for S. aureus from ≤4 mg/liter to ≤2 mg/liter for susceptible, from 8 to 16 mg/liter to 4 to 8 mg/liter for intermediate, and from ≥32 mg/liter to ≥16 mg/liter for resistant (24).These new MIC breakpoints were not sensitive enough to distinguish hVISA strains from vancomycin-susceptible S. aureus strains (31). Currently, a particular isolate of S. aureus is defined as hVISA if the large majority of cells of a population cannot grow in the presence of vancomycin concentrations in excess of 2 mg/liter but if some proportions of cells can form colonies, e.g., at frequencies as low as 10⁻⁶, in the presence of higher concentrations (13, 30). Many traditional methods have proved unreliable at detecting hVISA (30). Agar screening methods with different media, inocula, and glycopeptide concentrations have been investigated; but their sensitivities and specificities have varied considerably, making them also unreliable for hVISA detection (8, 30). The macro-Etest method (MET) has shown high degrees of sensitivity and specificity (27), but its use for routine clinical work has been hampered by its high cost. In 2001, a modified population analysis profile (PAP)-area under the curve (AUC) method (PAP-AUC) was proposed as a reliable, albeit labor-intensive, test for the identification of hVISA (29).With the development of the various detection methods, the prevalence of hVISA has been reported worldwide, e.g., in France (0.7%); Australia (9.4%); the United States (0.3 to 2.3%); and several Asian countries, including Japan (1.3% to 20%), India (6.3%), South Korea (6.1%), and Singapore (2.3%) (3, 4, 12, 21a, 23). However, little information from China has been available. In a study conducted in 2004, a total of 84 MRSA isolates obtained from 1997 to 2000 from China were investigated, but none of them was identified as hVISA (23). Given the high prevalence of MRSA strains (32) and the increasing use of glycopeptides in China, the emergence of hVISA, vancomycin-intermediate S. aureus (VISA), and vancomycin-resistant S. aureus strains in this region might be anticipated. Therefore, we have conducted a survey using the PAP-AUC method with more MRSA isolates and isolates from more study centers to investigate the epidemiology of hVISA in mainland China. As part of the study, we evaluated several agar screening methods and MET for their potential wider use in more clinical microbiology laboratories in China.The 26 hVISA isolates and the 1 VISA isolate identified were further characterized by antibiotic susceptibility testing and molecular typing. These data provide baseline information on the epidemiology of hVISA isolated from patients in mainland China.     

Increased Temperature Enhances the Antimicrobial Effects of Daptomycin,Vancomycin, Tigecycline,Fosfomycin, and Cefamandole on Staphylococcal Biofilms

Implant-related infections are serious complications of trauma and orthopedic surgery and are most difficult to treat. The bacterial biofilms of 34 clinical Staphylococcus sp. isolates (Staphylococcus aureus, n = 14; coagulase-negative staphylococci, n = 19) were incubated with daptomycin (DAP; 5, 25, or 100 mg/liter), vancomycin (VAN; 5, 25, or 100 mg/liter), tigecycline (TGC; 1, 5, or 25 mg/liter), fosfomycin (FOM; 100, 250, or 1,000 mg/liter), and cefamandole (FAM; 50, 100, or 500 mg/liter) for 24 h at three different ambient temperatures: 35°C, 40°C, and 45°C. To quantify the reduction of the biomass, the optical density ratio (ODr) of stained biofilms and the number of growing bacteria were determined. Increasing the temperature to 45°C or to 40°C during incubation with FAM, FOM, TGC, VAN, or DAP led to a significant but differential reduction of the thickness of the staphylococcal biofilms compared to that at 35°C (P < 0.05). Growth reduction was enhanced for DAP at 100 mg/liter at 35°C, 40°C, and 45°C (log count reductions, 4, 3.6, and 3.3, respectively; P < 0.05). A growth reduction by 2 log counts was detected for FAM at a concentration of 500 mg/liter at 40°C and 45°C (P = 0.01). FOM at 1,000 mg/liter reduced the bacterial growth by 1.2 log counts (not significant). The antibacterial activity of antimicrobial agents is significantly but differentially enhanced by increasing the ambient temperature and using high concentrations. Adjuvant hyperthermia may be of value in the treatment of biofilm-associated implant-related infections.Implant-related infections are severe complications of trauma and orthopedic surgery that frequently require long-term antimicrobial treatment, supportive management, and multiple additional surgical procedures (53). Staphylococcus aureus and coagulase-negative staphylococci (CoNS), primarily Staphylococcus epidermidis, are the most common organisms associated with implant-related infections after trauma and orthopedic surgery (13).Bacterial biofilms develop on the surfaces of the implants (12, 13, 30, 41). The biofilm consists of a structured community of bacterial cells enclosed in a self-produced polymeric matrix that adheres to an inert or living surface. The bacteria embedded in the biofilm are quasiprotected by this self-made polymeric matrix; thus, resistance to antimicrobials is increased such that the concentrations needed to kill the biofilm bacteria are 500 to 5,000 times higher than the levels needed to kill planktonic bacteria (8, 12, 13). Although removal of the colonized foreign material may be the most effective means to treat biofilm-associated infections (14), the implant must sometimes be retained because of technical or physiological complications. In this case, any hope for cure or at least the stability of the patient lies in antimicrobial treatment alone.Antimicrobial agents for the treatment of staphylococcal infections include beta-lactam antibiotics and, in the case of methicillin resistance, vancomycin (VAN), fosfomycin (FOM), tigecycline (TGC), or daptomycin (DAP) (32, 38). FOM is a small-molecule antibiotic with a wide antibacterial spectrum and excellent tissue penetration, representing an excellent alternative antimicrobial agent for the treatment of deep-seated infections (18, 27). Both TGC and DAP are highly active against Gram-positive cocci resistant to commonly used antibiotics, including methicillin-resistant staphylococci (18, 43). Antimicrobial agents may reduce biofilm and bacterial growth (35, 39). In a previous study, we demonstrated that the antibiotic concentrations achieved under normal physiological conditions did not decrease the growth of established staphylococcal biofilms (21). Bacterial biofilm growth was significantly reduced by the use of antimicrobials at excessive concentrations or when antimicrobials were used in combination with azithromycin (21, 37).Because the effects of antibiotics on established biofilms are unsatisfactory, other measures to reduce biofilm thickness and to kill biofilm bacteria may be helpful (37). Adjunctive therapy to enhance the activity of an antimicrobial to save an infected but unremovable implant or to improve the engraftment of a new implant after reimplantation may be a benefit for the patient (13, 16). Experimental measures taken to reduce bacterial biofilms included antibiotic combination therapy, particularly with rifampin; therapy with a combination of antibiotics and chemicals like EDTA or N-acytylcysteine; or therapy with a combination of antibiotics and physical measures, such as ultrasound or an electric current (7, 15, 39, 51). Low-frequency ultrasound was used to prevent uncontrolled heating and associated tissue damage (5, 6). For physical therapy, however, ultrasound of 1 MHz (unpulsed) had been described to have a beneficial effect on osteoarthritis (45). Further, application of heat has traditionally been used in physical therapy with various rates of success and minimal harm (9).Increasing the temperature induced the formation of thicker biofilms (40). Although the amount of heating was never measured in the ultrasound or electric current experiments, heating may have an influence on the effects of antibiotics on biofilms. Thus, we investigated the effects of stepwise increases in the ambient temperature together with antibiotic treatment on biofilm thickness and bacterial growth. Static biofilms of clinical isolates from orthopedic implant infections or preoperative skin isolates, including S. aureus and CoNS (S. epidermidis, S. lugdunensis, S. hominis, and S. capitis), were incubated at an ambient temperature of 35, 40, or 45°C for 24 h with DAP, VAN, FOM, TGC, or FAM. Three concentrations of each of the antimicrobial agents were investigated.(Some of the data presented here were presented as poster K2069 at the 48th Annual Interscience Conference on Antimicrobial Agents and Chemotherapy-Infectious Diseases Society of America 46th Annual Meeting, San Francisco, CA, 2009.)     

Intracellular Activity of Antibiotics against Staphylococcus aureus in a Mouse Peritonitis Model

Antibiotic treatment of Staphylococcus aureus infections is often problematic due to the slow response to therapy and the high frequency of infection recurrence. The intracellular persistence of staphylococci has been recognized and could offer a good explanation for these treatment difficulties. Knowledge of the interplay between intracellular antibiotic activity and the overall outcome of infection is therefore important. Several intracellular in vitro models have been developed, but few experimental animal models have been published. The mouse peritonitis/sepsis model was used as the basic in vivo model exploring a quantitative ex vivo extra- and intracellular differentiation assay. The intracellular presence of S. aureus was documented by electron microscopy. Five antibiotics, dicloxacillin, cefuroxime, gentamicin, azithromycin, and rifampin (rifampicin), were tested in the new in vivo model; and the model was able to distinguish between their extra- and intracellular effects. The intracellular effects of the five antibiotics could be ranked as follows as the mean change in the log₁₀ number of CFU/ml (Δlog₁₀ CFU/ml) between treated and untreated mice after 4 h of treatment: dicloxacillin (3.70 Δlog₁₀ CFU/ml) > cefuroxime (3.56 Δlog₁₀ CFU/ml) > rifampin (1.86 Δlog₁₀ CFU/ml) > gentamicin (0.61 Δlog₁₀ CFU/ml) > azithromycin (0.21 Δlog₁₀ CFU/ml). We could also show that the important factors during testing of intracellular activity in vivo are the size, number, and frequency of doses; the time of exposure; and the timing between the start of infection and treatment. A poor correlation between the intracellular accumulation of the antibiotics and the actual intracellular effect was found. This stresses the importance of performing experimental studies, like those with the new in vivo model described here, to measure actual intracellular activity instead of making predictions based on cellular pharmacokinetic and MICs.Staphylococcus aureus is a major human pathogen that causes both community- and hospital-acquired infections (35). It causes a diverse array of infections ranging from relatively minor skin and wound infections to more serious and life-threatening diseases such as pneumonia (20, 46), endocarditis (48), osteomyelitis (17, 29), arthritis (1), and meningitis (40). Some of these types of S. aureus infections, e.g., endocarditis, are associated with high rates of mortality (25 to 50%), despite antimicrobial treatment (48, 49, 57). Furthermore, S. aureus infections are often persistent and are associated with treatment difficulties, such as a slow response to antibiotic treatment and recurrences, that lead to an extended duration of antimicrobial therapy (11, 13, 31). The antimicrobial treatment of S. aureus infections has also become more difficult due to the emergence of multidrug-resistant strains (3, 4).Several factors may help explain the capacity of staphylococci to avoid the actions of antibiotics. Biofilm formation might be the main reason for a deficient antibiotic effect when foreign bodies are involved in the staphylococci infections (12, 15, 53). Otherwise, the intracellular presence of the bacteria could offer a good explanation for the slow response to antibiotics, since bacteria located intracellularly might be protected from the effects of antibiotics (55).S. aureus has classically been classified as an extracellular pathogen (21). Conversely, several reports have established that S. aureus internalizes and survives within professional and even nonprofessional mammalian phagocytes (7, 19, 24, 25, 26, 27). The attitude is therefore changing toward classifying S. aureus as a facultative/opportunistic intracellular pathogen (13, 36, 41, 42, 55).Having an intracellular target for antimicrobial therapy is more complex than having an extracellular target, because intracellular antimicrobial activity further depends on the penetration into and accumulation in the cell, cellular metabolism, the subcellular disposition, and the bioavailability of the drug. The bacterial responsiveness to antibiotics can also change intracellularly (54, 55). Antimicrobial activity is therefore often impaired intracellularly (6, 56).To date, this knowledge of the intracellular presence of S. aureus has not influenced the choice of antibiotic to be used for the treatment for S. aureus infections. Penicillinase-stable penicillins, for instance, are considered the mainstay of treatment for methicillin-susceptible S. aureus infections (5, 23, 35), even though penicillins are usually considered not to penetrate cells (8, 30, 50).Recurrent S. aureus infections may also, at least partly, be explained by the intracellular presence of the bacteria. Gresham et al. demonstrated that polymorphonuclear neutrophils with intracellular S. aureus isolated from the peritoneums of infected mice could cause a new infection by intraperitoneal injection of these cells into healthy mice (24). They also demonstrated that intracellular survival was linked to the global regulator sar, which regulates multiple virulence factors in S. aureus. These two observations could together indicate that intracellular survival is a part of the pathogenesis of S. aureus.Appropriate models for the testing of the intracellular activities of antimicrobials against S. aureus are needed. Several in vitro models that use various cells and cell lines are available for the study of intracellular S. aureus (6, 10, 18, 24, 44, 51), but only a few in vivo models have been developed.Here we present an in vivo model that can be used to study the intracellular activities of antimicrobials against S. aureus.(Part of this study was presented at the 16th European Congress of Clinical Microbiology and Infectious Diseases, Nice, France.)     

The BpeAB-OprB Efflux Pump of Burkholderia pseudomallei 1026b Does Not Play a Role in Quorum Sensing,Virulence Factor Production,or Extrusion of Aminoglycosides but Is a Broad-Spectrum Drug Efflux System

Most Burkholderia pseudomallei strains are intrinsically aminoglycoside resistant, mainly due to AmrAB-OprA-mediated efflux. Rare naturally occurring or genetically engineered mutants lacking this pump are aminoglycoside susceptible despite the fact that they also encode and express BpeAB-OprB, which was reported to mediate efflux of aminoglycosides in the Singapore strain KHW. To reassess the role of BpeAB-OprB in B. pseudomallei aminoglycoside resistance, we used mutants overexpressing or lacking this pump in either AmrAB-OprA-proficient or -deficient strain 1026b backgrounds. Our data show that BpeAB-OprB does not mediate efflux of aminoglycosides but is a multidrug efflux system which extrudes macrolides, fluoroquinolones, tetracyclines, acriflavine, and, to a lesser extent, chloramphenicol. Phylogenetically, BpeAB-OprB is closely related to Pseudomonas aeruginosa MexAB-OprM, which has a similar substrate spectrum. AmrAB-OprA is most closely related to MexXY, the only P. aeruginosa efflux pump known to extrude aminoglycosides. Since BpeAB-OprB in strain KHW was also implicated in playing a major role in export of acylated homoserine lactone (AHL) quorum-sensing molecules and in expression of diverse virulence factors, we explored whether this was also true in the strain 1026b background. The results showed that BpeAB-OprB was not required for AHL export, and mutants lacking this efflux system exhibited normal swimming motility and siderophore production, which were severely impaired in KHW bpeAB-oprB mutants. Biofilm formation was impaired in 1026b Δ(amrRAB-oprA) and Δ(amrRAB-oprA) Δ(bpeAB-oprB) mutants. At present, we do not know why our BpeAB-OprB susceptibility and virulence factor expression results with 1026b and its derivatives are different from those previously published for Singapore strain KHW.Efflux pumps of the resistance-nodulation-cell division (RND) family play a pivotal role in intrinsic and acquired drug resistance of many Gram-negative pathogens, including Pseudomonas aeruginosa (reviewed in references 19 and 30), Acinetobacter baumannii (11, 40), Stenotrophomonas maltophilia (46, 47), and others. Burkholderia pseudomallei is the etiologic agent of melioidosis (6, 42), a rare but serious disease endemic to Southeast Asia, Northern Australia, and other parts of the tropics around the world (10). Treatment of melioidosis is challenging (43) and is complicated by the bacterium''s intrinsic antibiotic resistance. Although genome analysis revealed numerous antibiotic resistance mechanisms (16), they have not yet been well studied. The genomes of strain K96243 and other sequenced strains encode at least 10 RND pumps (16, 18). Of these, only two have been characterized in detail. AmrAB-OprA of strain 1026b was shown to confer high-level resistance to aminoglycosides and macrolides (27). BpeAB-OprB of strain KHW was reported to mediate efflux of aminoglycosides and macrolides (5) as well as to play an important role in virulence and quorum sensing (3, 4). Specifically, BpeAB-OprB was shown to mediate efflux of the aminoglycosides gentamicin and streptomycin, the macrolide erythromycin, and the dye acriflavine (5). In strain KHW, this pump is also required for optimal production of biofilms, siderophores, and phospholipase C (4). Finally, excretion of acyl homoserine lactone (AHL) quorum-sensing molecules is dependent on BpeAB-OprB function (3, 4). Using a surrogate P. aeruginosa strain, we recently showed that a third pump, BpeEF-OprC, mediates efflux of chloramphenicol and trimethoprim (17), and its properties in B. pseudomallei are currently being studied in our laboratory. During these studies, we noticed that a 1026b derivative missing AmrAB-OprA was aminoglycoside susceptible, despite the fact that it expressed BpeAB-OprB. Since this finding is contrary to what was previously published about this pump in strain KHW, we decided to study the expression and antibiotic resistance profile of this pump in strain 1026b, using defined mutants. Our results indicate that in strain 1026b, BpeAB-OprB does not mediate efflux of aminoglycosides but extrudes other antibiotics, including fluoroquinolones, clindamycin, macrolides, and tetracyclines. Of these, doxycycline is of therapeutic importance for melioidosis. Strain 1026b BpeAB-OprB mutants were not impaired in extrusion of AHLs, swimming motility, or siderophore production, but AmrAB-OprA BpeAB-OprB mutants were impaired in biofilm formation.     

Activity of Telavancin against Staphylococci and Enterococci Determined by MIC and Resistance Selection Studies

This study used CLSI broth microdilution to test the activity of telavancin and comparator antimicrobial agents against 67 methicillin (meticillin)-resistant Staphylococcus aureus (MRSA) isolates. Twenty-six vancomycin-intermediate S. aureus (VISA) strains were among the isolates tested; all strains were susceptible to telavancin at ≤1 μg/ml, whereas 12/26 (46%) of these isolates were nonsusceptible to daptomycin at the same concentration. All strains were susceptible to quinupristin-dalfopristin, while resistance was found to all other drugs tested. Telavancin demonstrated potent activity against all vancomycin-susceptible isolates as well as against heterogeneously VISA and VISA resistance phenotypes. In multistep resistance selection studies, telavancin yielded one stable mutant after 43 days in one MRSA strain out of the 10 MRSA strains tested with the MIC rising eightfold from 0.25 μg/ml (parent) to 2 μg/ml. MICs for this clone did not increase further when passages were continued for the maximum 50 days. In contrast, daptomycin selected stable resistant clones (MIC increase of >4×) after 14 to 35 days in 4 of 10 MRSA strains with MICs increasing from 1 to 2 μg/ml (parents) to 4 to 8 μg/ml (resistant clones). Sequencing analysis of daptomycin resistance determinants revealed point mutations in the mprF genes of all four stable daptomycin-resistant clones. Teicoplanin gave rise to resistant clones after 14 to 21 days in 2 of 10 MRSA strains with MICs rising from 1 to 2 μg/ml (parents) to 4 to 16 μg/ml (stable resistant clones). Linezolid selected stable resistant clones after 22 to 48 days in 2 of 10 MRSA strains with MICs rising from 2 to 4 μg/ml (parents) to 32 μg/ml (resistant clones). Vancomycin yielded no resistant clones in 10 MRSA strains tested; however, MICs increased two- to fourfold from 1 to 8 μg/ml to 2 to 16 μg/ml after 50 days. No cross-resistance was found with any clone/antimicrobial combination. The two enterococci developed resistance to daptomycin, and one developed resistance to linezolid. Single-step mutation frequencies for telavancin (<4.0 × 10⁻¹¹ to <2.9 × 10⁻¹⁰ at 2× MIC) were lower than the spontaneous mutation frequencies obtained with the comparators.Staphylococcus aureus is becoming increasingly resistant to antibiotics. Methicillin (meticillin)-resistant S. aureus (MRSA) strains are increasingly encountered all over the world and cannot be treated with existing ß-lactams. Additionally, the majority of hospital-acquired methicillin-resistant (and also some methicillin-susceptible) strains are resistant to all currently available quinolones. The situation has become more complicated by the appearance of heterogeneously vancomycin-intermediate S. aureus (hVISA) strains, vancomycin-intermediate S. aureus (VISA) strains, and recently nine reported vancomycin-resistant S. aureus (VRSA) strains (1). Two recent papers emphasize the recent spread of VISA strains in Turkey (47) and France (15), and a recent alert from the New York City Department of Health (11) has documented six cases of VISA infections in New York City, NY, between February and October 2007, which led to four fatalities. It seems clear that VISA phenotypes occur everywhere but that they are not being routinely detected due to lack of standardized methodology (1, 20). Recently, Rybak et al. (43) have indicated, with Etest macromethod and population analysis testing, that the incidence of hVISA strains has increased over the past 22 years to an overall incidence of 2.2%. Yusof et al. (55) have recently described the utility of the Etest macromethod using a double-sided vancomycin-teicoplanin Etest strip which accurately differentiates between hVISA and VISA strains. Utilization of the latter method will surely increase reports of the incidence of hVISA and VISA strains. As of this time, the pathogenicity of VRSA strains awaits confirmation.The situation has become further complicated by the appearance and rapid spread, especially in the United States, of community-acquired MRSA strains that are especially virulent, possibly by virtue of production of Panton-Valentine leukocidin (4, 10, 16, 28, 34, 37). Although these strains are currently more susceptible to antimicrobial agents than hospital-acquired strains are, this situation will surely change. Additionally, treatment of the community-acquired MRSA strains with glycopeptides will increase the selective pressure leading to nonsusceptibility to vancomycin and teicoplanin. Recently, we and others have documented clinical development of daptomycin resistance after daptomycin therapy (25), and not all VISA strains are daptomycin susceptible (1, 19, 25, 26). There is an urgent need for new agents to treat MRSA infections.Telavancin is an investigational lipoglycopeptide active (MICs of ≤1 μg/ml) against gram-positive organisms including MRSA (17, 18, 22, 29, 31, 38, 48). Barcia-Macay and coworkers (3) have reported telavancin MICs of 0.5 μg/ml against two strains of VISA and of 2 to 4 μg/ml against two strains of VRSA. Leuthner and coworkers in a study of 50 glycopeptide-nonsusceptible staphylococci and 3 VRSA strains showed telavancin to be potent against all 37 hVISA and VISA strains (13 coagulase-negative strains; resistance phenotypes not differentiated from one another) with MICs of <1 μg/ml, with higher MICs of 2 to 4 μg/ml against the 3 VRSA strains (33). The antibacterial mode of action of telavancin results from inhibition of bacterial cell wall synthesis and interference with the barrier function of the bacterial cell membrane (22). The mode of action of daptomycin also involves targeting the bacterial cell membrane to initiate antimicrobial activity (2, 24, 26). This fact is supported by the involvement of staphylococcal genes regulating cell membrane surface charge (e.g., mprF) (39) and fatty acid synthesis (e.g., yycG) (36) in the development of daptomycin nonsusceptibility.In an effort to expand the comparative activity of telavancin against MRSA strains of various resistance phenotypes, we have investigated the activity of telavancin against MRSA strains by determining the activities of telavancin and comparator agents against 67 vancomycin-susceptible and -nonsusceptible MRSA strains using broth microdilution and also by testing the potential of telavancin and the comparator agents vancomycin, teicoplanin, daptomycin, and linezolid to select for resistance in 10 MRSA strains as well as two strains of enterococci by single-step and multistep selection methodology. Additionally, we conducted molecular genetic studies to characterize the mechanism(s) of daptomycin resistance in isolated clones.(Part of this study was presented at the 48th Interscience Conference on Antimicrobial Agents and Chemotherapy and the 46th Annual Meeting of the Infectious Disease Society of America, a joint meeting held in Washington, DC, in 2008 [12].)     

Daptomycin-Oxacillin Combinations in Treatment of Experimental Endocarditis Caused by Daptomycin-Nonsusceptible Strains of Methicillin-Resistant Staphylococcus aureus with Evolving Oxacillin Susceptibility (the “Seesaw Effect”)

In vivo development of daptomycin resistance (DAP^r) among Staphylococcus aureus strains, especially methicillin-resistant S. aureus (MRSA) strains, in conjunction with clinical treatment failures, has emerged as a major problem. This has raised the question of DAP-based combination regimens to enhance efficacy against such strains. We studied five recent DAP-susceptible (DAP^s)/DAP^r clinical MRSA strain pairs obtained from patients who failed DAP monotherapy regimens, as well as one DAP^s/DAP^r MRSA strain pair in which the resistant strain was generated by in vitro passage in DAP. Of note, we identified a DAP-oxacillin (OX) “seesaw” phenomenon in vitro in which development of DAP^r was accompanied by a concomitant fall in OX resistance, as demonstrated by 3- to 4-fold decreases in the OX MIC, a susceptibility shift by population analyses, and enhanced early killing by OX in time-kill assays. In addition, the combination of DAP and OX exerted modest improvement in in vitro bactericidal effects. Using an experimental model of infective endocarditis and two DAP^s/DAP^r strain pairs, we demonstrated that (i) OX monotherapy was ineffective at clearing DAP^r strains from any target tissue in this model (heart valve, kidneys, or spleen) and (ii) DAP-OX combination therapy was highly effective in DAP^r strain clearances from these organs. The mechanism(s) of the seesaw effect remains to be defined but does not appear to involve excision of the staphylococcal cassette chromosome mec (SCCmec) that carries mecA.Daptomycin (DAP) is a cyclic lipopeptide antibiotic active against a wide range of Gram-positive organisms, including methicillin-resistant Staphylococcus aureus (MRSA), vancomycin (VAN)-intermediate S. aureus (VISA), and vancomycin-resistant S. aureus (VRSA) strains (39, 54). However, an increasing number of reports has described the in vivo loss of DAP susceptibility in association with DAP clinical treatment failures in S. aureus infections (2, 17, 47). Clearly, innovative approaches to this problem need to be expeditiously applied to enhance/retain DAP efficacy, including dose escalations and combination therapy strategies.It has been previously reported that in some clinical VISA and VRSA strains, as well as in vitro-selected VISA strains, as the VAN MIC was observed to rise, the concomitant MIC to semisynthetic antistaphylococcal penicillins (e.g., oxacillin [OX] or nafcillin) progressively fell. This phenomenon was termed the “seesaw effect” (42-44). This loss of methicillin resistance has most often been due to excision of the staphylococcal cassette chromosome mec element (SCCmec) that carries mecA, the gene encoding penicillin-binding protein 2a (PBP2a), which is responsible for the MRSA phenotype (11, 12, 37). Although uncommon in vivo (3), the seesaw effect was recently observed in a clinical VISA isolate with a retained mecA gene obtained during VAN therapy; of interest, after discontinuation of VAN therapy, the methicillin resistance phenotype was restored (32). Importantly, the combination of VAN and nafcillin has been shown to exert synergistic killing activities in vitro and in an experimental model of infective endocarditis (IE) (8) with both VISA and VRSA strains (14).We have found a similar seesaw relationship in S. aureus strains that progressively acquire DAP^r during DAP exposure, and their respective OX MICs declined in parallel (29). In the current study, we investigated (i) the extent of this seesaw effect in vitro by utilizing five clinical DAP^s/DAP^r strain pairs and (ii) whether combination regimens of DAP and OX would enhance both the in vitro and in vivo efficacies of these single agents against DAP^r strains.Note that although the currently accepted term for reduced in vitro susceptibility to daptomycin is “nonsusceptible,” we use the terms “daptomycin resistant” (DAP^r) and “daptomycin susceptible” (DAP^s) in this paper for a more facile presentation.     

Underestimation of Vancomycin and Teicoplanin MICs by Broth Microdilution Leads to Underdetection of Glycopeptide-Intermediate Isolates of Staphylococcus aureus

Broth microdilution was compared with tube macrodilution and a simplified population analysis agar method for evaluating vancomycin and teicoplanin MICs and detecting glycopeptide-intermediate isolates of Staphylococcus aureus. Modal vancomycin and teicoplanin MICs recorded by tube macrodilution and the agar plate assay, which both used inocula of 10⁶ CFU, were significantly higher (2 μg/ml) against a panel of borderline glycopeptide-susceptible and glycopeptide-intermediate methicillin-resistant S. aureus (MRSA) bloodstream isolates compared to broth microdilution (1 μg/ml). Vancomycin and teicoplanin MIC distributions by tube macrodilution and agar testing were also markedly different from those evaluated by broth microdilution. The 20-fold-lower inoculum size used for broth microdilution compared to macrodilution and agar MIC assays explained in part, but not entirely, the systematic trend toward lower vancomycin and teicoplanin MICs by microdilution compared to other methods. Broth microdilution assay led to underdetection of the vancomycin-intermediate S. aureus (VISA) phenotype, yielding only three VISA isolates, for which vancomycin MICs were 4 μg/ml compared to 8 and 19 VISA isolates detected by macrodilution and agar testing, respectively. While macrodilution and agar testing detected 7 and 22 isolates with elevated teicoplanin MICs (8 μg/ml), respectively, broth microdilution failed to detect such isolates. Detection rates of isolates with elevated vancomycin and teicoplanin MICs by macrodilution and agar testing assays were higher at 48 h than at 24 h. In conclusion, the sensitivity of broth microdilution MIC testing is questionable for reliable detection and epidemiological surveys of glycopeptide-intermediate resistance in S. aureus isolates.Since 1997, two major categories of vancomycin resistance in Staphylococcus aureus have been defined. The first category refers to vancomycin-resistant S. aureus (VRSA) clinical isolates with exogenously acquired, vanA-mediated high-level resistance (vancomycin MICs, ≥16 μg/ml) (7, 45); the second category includes vancomycin-intermediate S. aureus (VISA) isolates that developed low-level resistance (vancomycin MICs, ≥4 to <16 μg/ml) via complex, incompletely defined endogenous mechanisms (6, 10, 21, 51). Since VISA isolates are almost uniformly cross-resistant to teicoplanin (21, 30), they are frequently designated glycopeptide-intermediate S. aureus (GISA) (50). In contrast to vancomycin, widely different teicoplanin susceptibility breakpoints have been proposed by different national or international committees, varying from 2 (13) to 8 (10) μg/ml, which leads to a confusing situation.Soon after their initial discovery in Japan (23), it was realized that a large proportion of VISA isolates, referred to as hVISA, show heterogeneous expression of vancomycin-intermediate resistance, including a minority population (perhaps as few as 10⁻⁶ cells) for which the vancomycin MIC is ≥4 μg/ml, while the majority of bacteria are still vancomycin susceptible (vancomycin MICs, ≤2 μg/ml) (10, 21, 22, 24, 51). No mechanistic model explaining heterogeneous expression of glycopeptide resistance has been provided. hVISA/hGISA are assumed to be precursors of VISA/GISA strains, with glycopeptides providing the selective pressure for conversion (2, 14, 22, 24, 33, 39, 44, 55). On the other hand, serial passages on antibiotic-free media frequently lead to gradual dilution and eventual elimination of the resistant subpopulation (2, 21, 24). These data potentially challenge the previously established distinction between hGISA and GISA (21, 29, 51).Despite repeated efforts to create one, there is no standard molecular or phenotypic assay allowing reliable detection of GISA and hGISA clinical or laboratory isolates (5, 30). This situation can be explained by (i) the multifactorial molecular basis of hGISA/GISA phenotypes, which did not reveal any ubiquitous, single, specific molecular marker for their detection (24-27, 41), and (ii) the variable, phenotypic expression of low-level glycopeptide resistance, which is significantly influenced by several technical parameters, including the compositions of liquid or solid test media and varying time frames and inoculum sizes.Standard CLSI-recommended broth microdilution and agar MIC-testing methods (9) were reported to have suboptimal sensitivity for detecting some hGISA isolates (21, 51) because they use relatively small inocula (5 × 10⁴ CFU/well and 1 × 10⁴ CFU/spot, respectively). Accordingly, specifically designed agar screening or population analysis profiles, as well as modified Etest methods, were developed for improved detection of hGISA and GISA by integrating requirements for larger bacterial inocula and longer incubation periods (5, 17, 24, 48, 51, 54, 58, 60). Nevertheless, standardization of these elaborated, labor-intensive susceptibility test methods is difficult (17, 48, 58, 60), and their relationships with standard glycopeptide MIC breakpoints are not well defined. Finally, the recent revisions of vancomycin MIC breakpoints by CLSI (10) and of both teicoplanin and vancomycin MIC breakpoints by the European Committee on Antimicrobial Susceptibility Testing (EUCAST) (13), which were based on glycopeptide susceptibility surveys of S. aureus clinical isolates (15, 51, 59), hamper analysis of hGISA/GISA prevalence data reported before 2006.Despite the lack of standardized hGISA detection methods, a number of clinical reports have linked vancomycin therapeutic failure of methicillin-resistant S. aureus (MRSA) infections with the presence of VISA or hVISA isolates or with emergence of vancomycin-intermediate resistance during glycopeptide therapy (3, 8, 24, 25, 33, 36-38, 44, 51, 52). Even higher rates of vancomycin treatment failures were reported for bacteremic patients infected with MRSA isolates for which vancomycin MICs (2 μg/ml) were still in the susceptible range than for those with lower vancomycin MICs (<2 μg/ml) (3, 11, 18-20, 31, 32, 34, 35, 43, 46, 51). An emerging creep of vancomycin and teicoplanin MICs against MRSA in the last decade, which was suggested by large-scale epidemiological studies (19, 28, 47, 51, 56), has been challenged by more recent data (1, 42). Collectively, most of the discrepancies in the clinical and epidemiological results might have resulted from the lack of reliable, sensitive detection methods for hGISA and GISA.During a retrospective surveillance study that explored the prevalence of intermediate glycopeptide resistance in MRSA bloodstream isolates from our institution, we discovered that vancomycin MICs, assayed by the reference macrodilution (tube) method (9), were 2 μg/ml for a vast majority of our nosocomial isolates. Since these MIC estimates were significantly higher than those currently reported in clinical and epidemiological MRSA surveillance studies, in which the modal vancomycin MIC assayed by the broth microdilution (1, 18, 24, 42, 51) or agar dilution (40, 59) method was 1 μg/ml, we evaluated the impacts of three different susceptibility-testing methods, namely, broth microdilution, tube macrodilution, and a simplified population analysis assay, on glycopeptide MIC distributions for our panel of MRSA isolates. A detailed analysis of parameters that potentially contributed to assay-dependent differences in vancomycin and teicoplanin MIC estimates, such as the inoculum size, time of incubation, and medium composition, was performed. A novel approach, combining broth macrodilution and agar testing, is proposed for discriminating glycopeptide-susceptible from hGISA and GISA isolates.     

Population Pharmacokinetic Analysis of Vancomycin Using Serum Cystatin C as a Marker of Renal Function

We determined the population pharmacokinetics of vancomycin (VAN) using the glomerular filtration rate (GFR) estimated from the serum cystatin C concentration. We examined the predictive performance of the trough serum VAN concentration for determination of the initial dose by using a new model for the analysis of the population pharmacokinetic parameters. Data for 86 patients were used to estimate the values of the population pharmacokinetic parameters. Analysis with a nonlinear mixed-effects modeling program was done by using a one-compartment model. Data for 78 patients were used to evaluate the predictive performance of the new model for the analysis of population pharmacokinetic parameters. The estimated GFR values determined by using Hoek''s formula correlated linearly with VAN clearance (VAN clearance [ml/min] = 0.825 × GFR). The mean volume of distribution was 0.864 (liters/kg). The interindividual variability of VAN clearance was 19.8%. The accuracy of the prediction determined by use of the new model was statistically better than that determined by use of the Japanese nomogram-based model because the 95% confidence interval (−3.45 to −1.38) of the difference in each value of the mean absolute error (−2.41) did not include 0. Use of the serum cystatin C concentration as a marker of renal function for prediction of serum VAN concentrations may be useful.Vancomycin (VAN) has been widely used for the treatment of methicillin-resistant Staphylococcus aureus (MRSA) infections. It is mainly eliminated via the kidneys and has a narrow therapeutic range; high doses cause nephrotoxicity, particularly if it is used in combination with an aminoglycoside antibiotic (6, 28). The area under the blood concentration-time curve (AUC)/MIC is the pharmacodynamic parameter that best correlates with a successful outcome after the use of VAN (20, 27). Therefore, it is believed that therapeutic drug monitoring (TDM) is appropriate for VAN therapy (3, 16, 18, 29).The initial VAN dosage regimen is usually selected by use of a nomogram that uses the serum creatinine concentration as a marker of renal function. Our previous studies indicated that the nomogram that uses the serum creatinine concentration does not accurately predict the serum VAN trough concentration, particularly in elderly individuals (33). This is probably caused by using the serum creatinine concentration as a marker of renal function because this leads to an overestimation of the glomerular filtration rate (GFR) (17). A more accurate marker of GFR is needed for the appropriate use of VAN because renal function is one of the most important factors affecting the clearance of VAN.It has been reported that the serum cystatin C concentration is a better marker of renal function than the serum creatinine concentration (5). A recent meta-analysis demonstrated that the serum cystatin C concentration is superior to the serum creatinine concentration for use for the detection of an impaired GFR (7). Some studies reported that the serum cystatin C concentration is a better marker of drug clearance than the serum creatinine concentration (15, 23). Recently, we have shown that the serum cystatin C concentration is a better marker for determination of the initial dose in VAN therapy. In a previous study, GFR was estimated on the basis of the serum cystatin C concentration in place of the creatinine clearance, which is the parameter usually used to determine the population pharmacokinetics of VAN (32). However, a means of population pharmacokinetic analysis that uses the serum cystatin C concentration as a marker of renal function is lacking.We approached the population pharmacokinetic analysis of VAN using the serum cystatin C concentration and a one-compartment model for adult patients infected with MRSA. Covariate selection revealed that total body weight (TBW) affected the volume of distribution, whereas renal function (estimated from GFR by use of the serum cystatin C concentration) affected VAN clearance. We also compared the predictive performance of the trough serum VAN concentration for determination of the initial dose with that of the use of the nomogram and the serum creatinine concentration.     

Comparative Efficacies of Human Simulated Exposures of Telavancin and Vancomycin against Methicillin-Resistant Staphylococcus aureus with a Range of Vancomycin MICs in a Murine Pneumonia Model

Telavancin displays potent in vitro and in vivo activity against methicillin-resistant Staphylococcus aureus (MRSA), including strains with reduced susceptibility to vancomycin. We compared the efficacies of telavancin and vancomycin against MRSA strains with vancomycin MICs of ≥1 μg/ml in a neutropenic murine lung infection model. Thirteen clinical MRSA isolates (7 vancomycin-susceptible, 2 vancomycin-heteroresistant [hVISA], and 4 vancomycin-intermediate [VISA] isolates) were tested after 24 h, and 7 isolates (1 hVISA and 4 VISA isolates) were tested after 48 h of exposure. Mice were administered subcutaneous doses of telavancin at 40 mg/kg of body weight every 12 h (q12h) or of vancomycin at 110 mg/kg q12h; doses were designed to simulate the area under the concentration-time curve for the free, unbound fraction of drug (fAUC) observed for humans given telavancin at 10 mg/kg q24h or vancomycin at 1 g q12h. Efficacy was expressed as the 24- or 48-h change in lung bacterial density from pretreatment counts. At dose initiation, the mean bacterial load was 6.16 ± 0.26 log₁₀ CFU/ml, which increased by averages of 1.26 ± 0.55 and 1.74 ± 0.68 log in untreated mice after 24 and 48 h, respectively. At both time points, similar CFU reductions were noted for telavancin and vancomycin against MRSA, with vancomycin MICs of ≤2 μg/ml. Both drugs were similarly efficacious after 24 and 48 h of treatment against the hVISA strains tested. Against VISA isolates, telavancin reduced bacterial burdens significantly more than vancomycin for 1 of 4 isolates after 24 h and for 3 of 4 isolates after 48 h. These data support the potential utility of telavancin for the treatment of MRSA pneumonia caused by pathogens with reduced susceptibility to vancomycin.Over the course of the last 15 years, methicillin resistance among Staphylococcus aureus strains has increased steadily. Recent surveys report methicillin-resistant S. aureus (MRSA) rates of upwards of 50% for hospitalized patients with staphylococcal infections and upwards of 60% for patients in intensive care units (27). Considering that S. aureus accounts for 20 to 30% of hospital-acquired pneumonia cases, MRSA is a clinically important pathogen to consider in empirically choosing a regimen to treat pneumonia (22, 33).Vancomycin has long been regarded as the drug of choice for the treatment of MRSA infections. Current practice guidelines for the treatment of health care-associated pneumonia recommend vancomycin as a first-line therapy (2). Despite its being a first-line recommendation, studies evaluating the clinical success of vancomycin treatment in patients with MRSA pneumonia have observed failure in 45% to 77% of patients (15, 24). One possible explanation could be the recently reported vancomycin MIC creep detected among S. aureus strains (32). Over the last decade, an increase in vancomycin MICs has been noted by some centers, despite values staying within the Clinical and Laboratory Standards Institute (CLSI)-defined susceptibility range of ≤2 μg/ml (6). Among selected patients with MRSA bacteremia treated with vancomycin, Sakoulas and colleagues found significantly more treatment failures for patients infected with isolates possessing vancomycin MICs of 1 to 2 μg/ml (90.5%) than for those infected with isolates with vancomycin MICs of ≤0.5 μg/ml (44.4%) (31).In addition to MIC creep, another explanation for vancomycin failures is the increasing appearance of vancomycin-intermediate S. aureus (VISA) and heterogeneous vancomycin-intermediate S. aureus (hVISA) strains (3). The simultaneous increases in frequency of these isolates are expected, as hVISA isolates are thought to be the strains immediately preceding VISA in its evolution (12). The parent strains of hVISA have vancomycin MICs ranging from 1 to 4 μg/ml. However, when these strains are subjected to increasing concentrations of vancomycin, a subpopulation of resistant clones develops, with MICs of at least 8 μg/ml (21).Clinically, poor outcomes have been reported for both VISA and hVISA infections treated with vancomycin (21). In one study, hVISA-infected bacteremia patients were shown to have a significantly longer length of hospital stay, to have a greater proportion of high-bacterial-load infections, and to more commonly fail vancomycin therapy than those with vancomycin-susceptible MRSA infections (5). For this reason, it is necessary to characterize other treatment options not only for MRSA but also for the increasingly prevalent hVISA and VISA isolates.The recently approved lipoglycopeptide telavancin is the newest option for the treatment of resistant S. aureus. In vitro studies of telavancin demonstrated potent activity against a number of Gram-positive organisms, including MRSA, hVISA, and VISA (7, 8, 18). A murine lung infection model of human simulated exposures of telavancin and vancomycin showed an increased efficacy of telavancin against a single MRSA isolate (28). Similar results were noted against an hVISA and 2 VISA isolates in a murine bacteremia model (10). The present study was designed to merge these two findings by comparing the efficacies of human simulated exposures to telavancin and vancomycin against a number of MRSA isolates, including hVISA and VISA isolates, in a murine lung infection model.(These data were presented in part at the 49th Interscience Conference on Antimicrobial Agents and Chemotherapy.)     

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].)     

Role for Fks1 in the Intrinsic Echinocandin Resistance of Fusarium solani as Evidenced by Hybrid Expression in Saccharomyces cerevisiae

The opportunistic mold Fusarium solani is intrinsically resistant to cell wall synthesis-inhibiting echinocandins (ECs), including caspofungin and micafungin. Mutations that confer acquired EC resistance in Saccharomyces cerevisiae and other normally susceptible yeast species have been mapped to the Fks1 gene; among these is the mutation of residue 639 from Phe to Tyr (F639Y) within a region designated hot spot 1. Fks1 sequence analysis identified the equivalent of Y639 in F. solani as well as in Scedosporium prolificans, another intrinsically EC-resistant mold. To test its role in intrinsic EC resistance, we constructed Fks1 hybrids in S. cerevisiae that incorporate F. solani hot spot 1 and flanking residues. Hybrid construction was accomplished by a PCR-based method that was validated by studies with Fks1 sequences from EC-susceptible Aspergillus fumigatus and paired EC-susceptible and -resistant Candida glabrata isolates. In support of our hypothesis, hybrid Fks1 incorporating F. solani hot spot 1 conferred significantly reduced EC susceptibility, 4- to 8-fold less than that of wild-type S. cerevisiae and 8- to 32-fold less than that of the same hybrid with an F639 mutation. We propose that Fks1 sequences represent determinants of intrinsic EC resistance in Fusarium and Scedosporium species and, potentially, other fungi.Serious fungal infections have increased in recent years as a consequence of increased immunosuppression associated with human immunodeficiency virus infection, organ and tissue transplants, and aggressive treatments for neoplastic and autoimmune disease. These infections typically are treated with ergosterol biosynthesis-inhibiting azole antifungals such as fluconazole. However, azoles have limitations: their activity is fungistatic, acquired resistance in normally susceptible yeast is not uncommon, and intrinsic low- to high-level resistance is demonstrated by many molds (3, 21). The membrane-disrupting antifungal amphotericin B is generally fungicidal and has broad-spectrum activity, and resistance to it is rare, but its use remains limited due to toxicity. The echinocandins (ECs) caspofungin (CSP), micafungin (MCF), and anidulafungin represent the most recently introduced group of antifungals. Importantly, ECs have fungicidal activity against most Candida species (including azole-resistant strains), fungistatic activity against Aspergillus species, and negligible toxicity (5, 20, 24). ECs act by inhibiting the synthesis of the cell wall polysaccharide β-1,3-glucan (7). This can result in cell lysis or more subtle cell wall changes that enhance susceptibility to innate immunity (25, 41).Acquired EC resistance in susceptible fungi is associated with specific mutations in the integral membrane protein Fks1 (or its paralog Fks2) (7, 35). Fks1 is believed to represent the β-1,3-glucan synthase catalytic subunit, although this has not been formally proven since only crude membrane preparations retain catalytic activity. Most resistance-conferring mutations cluster within so-called hot spot 1, which corresponds, within the model yeast Saccharomyces cerevisiae, to Fks1 residues Phe639 to Pro647 (F639-P647) (1, 2, 6, 7, 13, 18, 19, 26, 33, 34, 40). Fortunately, acquired EC resistance is rare. On the other hand, the intrinsic EC resistance of ascomycetous molds such as Fusarium solani and Scedosporium prolificans, zygomycetous molds such as Rhizopus oryzae, and the basidiomycetous yeasts Cryptococcus neoformans and Trichosporon asahii represents a major limitation to EC clinical use. Many of these fungi have emerged in recent years as important opportunistic pathogens (3, 27, 32, 37), and a contributing factor may be their intrinsic resistance to antifungals, including ECs (10, 22, 30).The basis for intrinsic EC resistance has been investigated in several of these fungi, but it remains unclear (14, 15, 28, 39). Notably, Ha et al. (14) reported that F. solani Fks1, when heterologously expressed in A. fumigatus, conferred a modest but potentially significant fourfold decrease in CSP susceptibility, the basis for which was not explored. However, expression in their system was abnormally low, and A. fumigatus itself exhibits some degree of intrinsic EC resistance (5), which together complicate the interpretation of this result. On the other hand, recent studies examining the basis for the intrinsically low EC susceptibility of Candida parapsilosis strongly implicated its Fks1 sequence; specifically, a hot spot 1 substitution equivalent to P647A (12).FKS1 initially was identified as the S. cerevisiae gene whose null mutation confers susceptibility to the calcineurin inhibitor FK506 (7, 8). This susceptibility results from the requirement for the calcineurin-mediated expression of FKS2; relatedly, single fks1Δ and fks2Δ disruptants are viable, but double disruption is lethal (31). Here, we exploit this FK506 susceptibility to construct Fks1 hybrids, replacing hot spot 1 from S. cerevisiae with that from F. solani (and, for comparison, A. fumigatus and Candida glabrata) to further test the hypothesis that intrinsic EC resistance is mediated by Fks1 sequence.     

Echinocandin Susceptibility Testing of Candida Species: Comparison of EUCAST EDef 7.1, CLSI M27-A3, Etest,Disk Diffusion,and Agar Dilution Methods with RPMI and IsoSensitest Media

This study compared nine susceptibility testing methods and 12 endpoints for anidulafungin, caspofungin, and micafungin with the same collection of blinded FKS hot spot mutant (n = 29) and wild-type isolates (n = 94). The susceptibility tests included EUCAST Edef 7.1, agar dilution, Etest, and disk diffusion with RPMI-1640 plus 2% glucose (2G) and IsoSensitest-2G media and CLSI M27A-3. Microdilution plates were read after 24 and 48 h. The following test parameters were evaluated: fks hot spot mutants overlapping the wild-type distribution, distance between the two populations, number of very major errors (VMEs; fks mutants misclassified as susceptible), and major errors (MEs; wild-type isolates classified as resistant) using a wild-type-upper-limit value (WT-UL) (two twofold-dilutions higher than the MIC₅₀) as the susceptibility breakpoint. The methods with the lowest number of errors (given as VMEs/MEs) across the three echinocandins were CLSI (12%/1%), agar dilution with RPMI-2G medium (14%/0%), and Etest with RPMI-2G medium (8%/3%). The fewest errors overall were observed for anidulafungin (4%/1% for EUCAST, 4%/3% for CLSI, and 3%/9% for Etest with RPMI-2G). For micafungin, VME rates of 10 to 71% were observed. For caspofungin, agar dilution with either medium was superior (VMEs/MEs of 0%/1%), while CLSI, EUCAST with IsoSensitest-2G medium, and Etest were less optimal (VMEs of 7%, 10%, and 10%, respectively). Applying the CLSI breakpoint (S ≤ 2 μg/ml) for CLSI results, 89.2% fks hot spot mutants were classified as anidulafungin susceptible, 60.7% as caspofungin susceptible, and 92.9% as micafungin susceptible. In conclusion, no test was perfect, but anidulafungin susceptibility testing using the WT-UL to define susceptibility reliably identified fks hot spot mutants.Three echinocandin class drugs, anidulafungin, caspofungin, and micafungin, are licensed for the treatment of invasive candidiasis. They are among the preferred agents for invasive candidiasis, as a number of recent fungemia surveys have reported a considerable proportion of cases involving species with reduced susceptibility to fluconazole (3, 4, 24, 28, 31, 37, 44). Additionally, anidulafungin has been associated with an improved success rate, even in cases involving fluconazole-susceptible species (39). Following increased use, sporadic cases of failures associated with elevated MICs have been reported. In the majority of cases, these failures have been associated with mutations in two hot spot regions of FKS genes, which encode the target and major subunit of the 1,3-ß-d-glucan synthase complex (5, 7, 22, 25, 26, 33, 34). Consequently, close monitoring and robust susceptibility testing methods have become increasingly important.EUCAST and CLSI have developed standard methods based on broth dilution for the susceptibility testing of yeasts (9, 41). Methodological differences include glucose concentration, inoculum size, shape of microtiter wells (flat or round), and end-point reading (visual or spectrophotometric), but the methods are more alike than different and in general generate similar results (11, 42). Recently, CLSI proposed an S value of ≤2 μg/ml as a tentative susceptibility breakpoint for caspofungin, micafungin, and anidulafungin for Candida spp., taking into account analysis of mechanisms of resistance, an epidemiological MIC population distribution, parameters associated with success in pharmacodynamic models, and results of clinical efficacy studies (9, 38). As no significant differences in clinical response were noted among the various species, results for all species were merged, and a susceptibility breakpoint of 2 μg/ml was found to encompass the vast majority of isolates, while not bisecting the population of Candida parapsilosis. The crucial issue is whether current susceptibility testing methods and breakpoints clearly and reliably identify isolates with resistance mechanisms associated with treatment failures (5, 7, 8, 13, 14, 16, 18, 22, 25, 26, 33, 40). Not only have cases involving isolates classified as susceptible using the reference methods been shown to contain resistance mutations (5, 7, 13, 14, 22, 25), but also recent studies suggest that a breakpoint of an S value of ≤2 μg/ml may be too high for anidulafungin and micafungin, considering the 1,3-ß-d-glucan synthase kinetic inhibition data of wild-type and mutant enzymes from resistant strains (17, 18). Finally, we recently reported a resistant Candida albicans isolate that failed to be identified as resistant when the reference methodologies were used, while Etest, agar dilution, and disk diffusion methods correctly identified it (5).We therefore undertook a comparative study of the two references methods, a modified EUCAST microdilution method using IsoSensitest medium, agar dilution, and disk and Etest diffusion using RPMI-1640 as well as IsoSensitest medium to evaluate their ability to reliably discriminate between a well-characterized panel of wild-type and fks hot spot mutant Candida isolates. The semisynthetic IsoSensitest medium was chosen as an alternative medium due to this medium having previously been shown to be appropriate for amphotericin B MIC testing (10).     

Complementation of a Saccharomyces cerevisiae ERG11/CYP51 (Sterol 14α-Demethylase) Doxycycline-Regulated Mutant and Screening of the Azole Sensitivity of Aspergillus fumigatus Isoenzymes CYP51A and CYP51B

Aspergillus fumigatus sterol 14α-demethylase isoenzymes CYP51A and CYP51B were heterologously expressed in a Saccharomyces cerevisiae mutant (YUG37-erg11), wherein native ERG11/CYP51 expression is controlled using a doxycycline-regulatable promoter. When cultured in the presence of doxycycline, recombinant YUG37-pcyp51A and YUG37-pcyp51B yeasts were able to synthesize ergosterol and grow; a control strain harboring reverse-oriented cyp51A could not. YUG37-pcyp51A and YUG37-pcyp51B constructs showed identical sensitivity to itraconazole, posaconazole, clotrimazole, and voriconazole. Conversely, YUG37-pcyp51A withstood 16-fold-higher concentrations of fluconazole than YUG37-pcyp51B (8 and 0.5 μg ml⁻¹, respectively).Azoles are used for treatment of Aspergillus infections (11, 13) and also in prophylactic drug regimens for immunocompromised patients (8). The emergence (4, 14, 30, 31, 32) and potential for spread (2) of azole-resistant Aspergillus (hereafter focusing on Aspergillus fumigatus) have highlighted the need to develop diagnostic tools (6, 9) and novel antifungal agents (15). These requirements demand better understanding of the mechanisms that mediate azole resistance in Aspergillus.Cytochrome P450 (CYP) was first investigated in A. fumigatus in 1990 (1); genome sequencing has revealed approximately 70 genes from this superfamily (29) that have not been fully annotated (manually verified) as for the 111 members of the Aspergillus nidulans cytochrome P450 complement (CYPome) (16). Given their importance in other pathogenic fungi (e.g., Candida albicans [18, 19, 21, 36]), the significance of mutations in A. fumigatus sterol 14α-demethylase, the CYP51 protein target of azoles, has attracted particular attention. Since the discovery that A. fumigatus possesses two genes (cyp51A and cyp51B) encoding sterol 14α-demethylase-like enzymes (26), it has been reasoned that the relative importance of each for ergosterol biosynthesis and/or resistance phenotypes observed in the clinic might differ. To date, the most prevalent mechanism of azole resistance in A. fumigatus appears to be the modification of CYP51A (5, 22, 25, 27, 28). Missense mutations in cyp51A are associated with cross-resistance, elevated MICs to azoles, and increased CYP51A expression (25, 27).Research has demonstrated the essentiality of the erg11 gene family (cyp51A and cyp51B) in A. fumigatus despite neither member being essential individually (15). It has also been postulated that CYP51A might provide the major 14α-demethylase activity required for growth in A. fumigatus and that CYP51B may serve a redundant or alternative function under certain growth conditions (28). However, despite the research interest surrounding A. fumigatus, it has not yet been shown that cyp51A and cyp51B both encode functional sterol 14α-demethylase. We investigated the use of a doxycycline-regulated Saccharomyces cerevisiae erg11/cyp51 (sterol 14α-demethylase) mutant to heterologously express A. fumigatus CYP51A and CYP51B in order to demonstrate complementation for ergosterol biosynthesis. The azole sensitivity of yeast transformants expressing A. fumigatus CYP51A and CYP51B was then screened.     

Evaluation of a DNA Microarray,the Check-Points ESBL/KPC Array,for Rapid Detection of TEM,SHV, and CTX-M Extended-Spectrum β-Lactamases and KPC Carbapenemases

Extended-spectrum ß-lactamases (ESBLs) and Klebsiella pneumoniae carbapenemases (KPC carbepenemases) have rapidly emerged worldwide and require rapid identification. The Check-Points ESBL/KPC array, a new commercial system based on genetic profiling for the direct identification of ESBL producers (SHV, TEM, and CTX-M) and of KPC producers, was evaluated. Well-characterized Gram-negative rods (Enterobacteriaceae, Pseudomonas aeruginosa, Acinetobacter baumannii) expressing various ß-lactamases (KPC-2, SHV, TEM, and CTX-M types) were used as well as wild-type reference strains and isolates harboring ß-lactamase genes not detected by the assay. In addition, phenotypically confirmed ESBL producers isolated in clinical samples over a 3-month period at the Bicetre hospital were analyzed using the Check-Points ESBL/KPC array and by standard PCR. The Check-Points ESBL/KPC array allowed fast detection of all TEM, SHV, and CTX-M ESBL genes and of the KPC-2 gene. The assay allowed easy differentiation between non-ESBL TEM and SHV and their ESBL derivatives. None of the other tested ß-lactamase genes were detected, underlining its high specificity. The technique is suited for Enterobacteriaceae but also for P. aeruginosa and A. baumannii. However, for nonfermenters, especially P. aeruginosa, a 1:10 dilution of the total DNA was necessary to detect KPC-2 and SHV-2a genes reliably. The Check-Points ESBL/KPC array is a powerful high-throughput tool for rapid identification of ESBLs and KPC producers in cultures. It provided definitive results within the same working day, allowing rapid implementation of isolation measures and appropriate antibiotic treatment. It showed an interesting potential for routine laboratory testing.Extended-spectrum ß-lactamases (ESBLs) and Klebsiella pneumoniae carbapenemase (KPC) are reported increasingly in Gram-negative bacilli (GNB) (5, 6, 17, 18, 25, 30). KPC producers, initially identified in the United States, are now reported worldwide, and illnesses caused by them have become endemic in some regions (25). Isolates expressing KPC enzymes may be reported as susceptible to carbapenems due to heterogeneous and variable levels of expression of β-lactam resistance.The vast majority of ESBLs belong to the TEM, SHV, and CTX-M types (5, 18, 28). These ß-lactamases are encoded by plasmid-located genes and therefore can very easily spread among Enterobacteriaceae (6, 14, 16). More than 160 TEM-type and 110 SHV-type ß-lactamases have been identified worldwide. Amino acid substitutions at many sites in TEM-1 ß-lactamases have been documented, but those at positions 104, 164, 238, and 240 most often lead to an ESBL phenotype (5, 28). As with TEM, SHV-type ESBLs have one or more amino acid substitutions located around the active site compared to SHV-1: substitutions at positions 238 and/or 240 are the most common and are associated with resistance to ceftazidime, cefotaxime, and aztreonam. Less commonly, an alteration at positions 146 or 179 provides ceftazidime resistance (28).Unlike TEM/SHV enzymes, all the CTX-M enzymes are ESBLs (6, 28). More than 80 CTX-M-variants, sharing 71 to 98% amino acid sequence identities, have now been described and are divided now into five groups (groups CTX-M-1, CTX-M-2, CTX-M-9, CTX-M-8, and CTX-M-25) based on amino acid sequence identity (5).Detection of ESBLs is primarily based on phenotypic testing, such as evidencing a synergy image using the double-disk synergy test performed with expanded-spectrum cephalosporins (ESC) and ticarcillin-clavulanic acid disks (3, 10, 23). This test is not always obvious and is usually time-consuming since it requires subculturing or the use of cloxacillin-containing plates to inhibit the naturally occurring and plasmid-mediated cephalosporinases. Unambiguous identification of KPCs by phenotypic methods is relatively difficult (25). Over the last 20 years, alternative strategies aimed at replacing or complementing traditional phenotypic methods have been proposed. Standard PCR and gene sequencing is still the most widely used technique. Other molecular detection techniques for ESBLs and KPC genes have been proposed, but none have been really suited for routine detection (1, 4, 8, 9, 11, 13, 15, 19, 20, 22, 24, 26, 27, 29, 31, 39), since usually only one ESBL/KPC gene is detected at a time. Finally, the presence of narrow-spectrum variants of TEM and SHV types may complicate significantly the molecular detection of TEM/SHV-type ESBLs (28).Microarray technology has recently been developed for the typing of Salmonella isolates (37, 38). This technology has the potential to detect an almost unlimited number of genes within one reaction mixture. Here, a new commercial DNA-based test, the Check-Points ESBL/KPC array, aimed at identifying TEM-, SHV-, and CTX-M-type ESBLs as well as KPC-type carbapenemases, was evaluated by comparing its performance with that of standard PCR on well-characterized reference strains and on 40 ESBL producers isolated at the Bicetre hospital from January to March 2009.     

Borrelia burgdorferi Resistance to a Major Skin Antimicrobial Peptide Is Independent of Outer Surface Lipoprotein Content

We hypothesize a potential role for Borrelia burgdorferi OspC in innate immune evasion at the initial stage of mammalian infection. We demonstrate that B. burgdorferi is resistant to high levels (>200 μg/ml) of cathelicidin and that this antimicrobial peptide exhibits limited binding to the spirochetal outer membrane, irrespective of OspC or other abundant surface lipoproteins. We conclude that the essential role of OspC is unrelated to resistance to this component of innate immunity.Borrelia burgdorferi, a spirochete and the causal organism of Lyme disease, is naturally transmitted to mammals through the bite of infected Ixodes ticks (5, 8). A significant change in B. burgdorferi gene expression accompanies transmission between these diverse environments. This was first described for the inverse relationship between two abundant outer surface proteins of B. burgdorferi, in which synthesis of OspA declines and that of OspC increases during tick feeding (41). We and others have demonstrated the essential nature of OspC for colonization of the murine host (23, 35, 42, 45, 47, 49). These findings suggest a critical role for OspC in evasion of host innate immunity immediately after transmission (47). However, the essential contribution of OspC to early mammalian infection by B. burgdorferi remains undefined.Microorganisms induce a variety of responses from the skin epithelial cells of their hosts, including the production of antimicrobial peptides, which are recognized as integral components of the innate immune system (20, 22). Defensins and cathelicidins comprise two major families of cationic antimicrobial peptides secreted by human and other mammalian skin neutrophils (20). Mouse neutrophils lack α defensins (14, 24), but about 30 cathelicidin members have been identified in various mammalian species, including mice (21, 50). These small, cationic, amphipathic molecules are primarily stored as inactive propeptides in the secretory granules of skin neutrophils. The mature bioactive peptides assume an α-helical structure in solution and preferentially interact with negatively charged cell surface components of a broad spectrum of bacteria and fungi, in which they disrupt cell membrane integrity (6, 9, 12, 20, 34). The importance of the sole murine cathelicidin, known as mCRAMP (mouse cathelin-related antimicrobial peptide) (19, 36), to innate host defense is well established, and mCRAMP has been shown to provide protection against bacterial skin infections in mice (33).     

Resistance to Colistin in Acinetobacter baumannii Associated with Mutations in the PmrAB Two-Component System

The mechanism of colistin resistance (Col^r) in Acinetobacter baumannii was studied by selecting in vitro Col^r derivatives of the multidrug-resistant A. baumannii isolate AB0057 and the drug-susceptible strain ATCC 17978, using escalating concentrations of colistin in liquid culture. DNA sequencing identified mutations in genes encoding the two-component system proteins PmrA and/or PmrB in each strain and in a Col^r clinical isolate. A colistin-susceptible revertant of one Col^r mutant strain, obtained following serial passage in the absence of colistin selection, carried a partial deletion of pmrB. Growth of AB0057 and ATCC 17978 at pH 5.5 increased the colistin MIC and conferred protection from killing by colistin in a 1-hour survival assay. Growth in ferric chloride [Fe(III)] conferred a small protective effect. Expression of pmrA was increased in Col^r mutants, but not at a low pH, suggesting that additional regulatory factors remain to be discovered.Among gram-negative pathogens that are reported as “multidrug resistant” (MDR), Acinetobacter baumannii is rapidly becoming a focus of significant attention (1, 7, 25, 32, 38, 39, 46, 51). In intensive care units, up to 30% of A. baumannii clinical isolates are resistant to at least three classes of antibiotics, often including fluoroquinolones and carbapenems (25).The emergence of MDR gram-negative pathogens, including A. baumannii, has prompted increased reliance on the cationic peptide antibiotic colistin (12). Regrettably, increasing colistin use has led to the discovery of resistant strains (10, 11, 22, 26). For example, in a recent study, 12% of carbapenemase-producing Enterobacteriaceae were found to be colistin resistant (Col^r) (6). Although still uncommon, A. baumannii isolates resistant to all available antimicrobial agents have been reported (26, 45) and are of enormous concern, given their potential to spread in the critical care environment.Colistin and other polymyxins are cyclic cationic peptides produced by the soil bacterium Bacillus polymyxa that act by disrupting the negatively charged outer membranes of gram-negative bacteria (37, 50). The following three distinct mechanisms that give rise to colistin resistance are known: (i) specific modification of the lipid A component of the outer membrane lipopolysaccharide, resulting in a reduction of the net negative charge of the outer membrane; (ii) proteolytic cleavage of the drug; and (iii) activation of a broad-spectrum efflux pump (13, 14, 49). The mechanism of colistin resistance in Acinetobacter spp. is not yet known. Heteroresistance to colistin in A. baumannii has been described (17, 24), but it is uncertain whether the basis for this resistance is the presence of a genetically distinct population of cells or whether variation in the regulatory program among genetically identical cells may be sufficient for the expression of resistance.In Salmonella enterica, the two-component signaling systems PmrAB and PhoPQ are involved in sensing environmental pH, Fe³⁺, and Mg²⁺ levels, leading to altered expression of a set of genes involved in lipid A modification (14, 43, 53). A small adapter protein, PmrD, serves as an interface between the two-component systems by stabilizing the activated form of PmrA in S. enterica (19), but other mechanisms of coordinated regulation are described for other species (52). Mutations causing constitutive activation of PmrA and PmrB are associated with colistin resistance (31, 33). Interestingly, the phoPQ and pmrD genes do not appear to be present in Acinetobacter spp., based on computational analysis of the genome sequences (2).PmrA-regulated resistance to colistin in S. enterica and P. aeruginosa results from modification of lipid A with 4-deoxy-aminoarabinose (Ara4N) or phosphoethanolamine via activation of ugd, the pmrF (or pbgP) operon, and pmrC, which encode UDP-glucose dehydrogenase (the first step in Ara4N biosynthesis), Ara4N biosynthetic enzymes, and lipid A phosphoethanolamine transferase, respectively (8, 15, 21, 41, 48). The Ara4N biosynthesis and attachment genes are not present in A. baumannii or Neisseria meningitidis (36, 47). N. meningitidis is intrinsically resistant to polymyxins, demonstrating that Ara4N modification of lipid A is not required for resistance. Mutations in the pmrC ortholog lptA, encoding the lipid A phosphoethanolamine transferase, reduce colistin resistance in N. meningitidis, suggesting that this modification alone may be sufficient for conferring colistin resistance (49). Here we show that the PmrAB system is involved in regulating colistin resistance in A. baumannii by identification of mutations in resistant isolates that exhibit constitutive expression of pmrA.