Mupirocin Resistance among Methicillin-Resistant Staphylococcus aureus-Colonized Patients at Admission to a Tertiary Care Medical Center

All patients admitted to our tertiary care hospital from 1 December 2007 to 10 June 2008 were screened for methicillin (meticillin)-resistant Staphylococcus aureus (MRSA) nasal colonization, and the isolates were tested for mupirocin susceptibility by using Etest. Mupirocin resistance (MR) was noted to occur in 3.4% of MRSA carriers, and high-level MR was noted to occur in 0.62% of carriers.Mupirocin is an antimicrobial that inhibits the synthesis of bacterial proteins by competitive inhibition of bacterial isoleucyl-tRNA synthetase (2). Mupirocin was approved for decolonization of the anterior nares as part of a comprehensive program to control the introduction and spread of methicillin (meticillin)-resistant Staphylococcus aureus (MRSA) in health care facilities (Bactroban Nasal package insert; GlaxoSmithKline, Research Triangle Park, NC).MRSA infections continue to increase in incidence and have become endemic in several U.S. hospitals (5, 11). Recently in Illinois, Public Act 095-0312 was passed, mandating MRSA screening for all patients admitted to an intensive care unit as well as for non-intensive care unit patients at high risk for MRSA carriage (9). Nasal colonization with MRSA has been shown to be a risk factor for nosocomial transmission and MRSA infection (8). With the increased use of mupirocin, two different groups of mupirocin-resistant MRSA clones have emerged: those with high-level resistance (≥512 μg/ml) and those with low-level resistance (8 to 256 μg/ml) (7, 11, 12). High-level resistance is believed to be the result of a mutated gene on a plasmid, while low-level resistance is thought to be caused by a chromosomal mutation for a gene coding for the tRNA synthetase (15). Currently, little is known about the prevalence of mupirocin-resistant strains among MRSA-colonized patients upon admission to hospitals throughout the United States. Our study sought to determine the rate of mupirocin resistance among patients testing positive for MRSA nasal colonization upon admission to our tertiary care center.All patients admitted to Loyola University Health Systems from 1 December 2007 to 10 June 2008, with consent, had dual nasal swab specimens collected upon admission. The first nasal swab was screened for MRSA nasal colonization via PCR using the Cepheid Xpert MRSA assay. If the first nasal swab yielded a positive or indeterminate screen result, the second swab was automatically cultured, and MRSA isolates were tested for mupirocin susceptibility by using Etest (AB Biodisk, Solna, Sweden). Quality control for mupirocin Etest strips was performed weekly throughout the course of the study by using Staphylococcus aureus ATCC 29213. All quality control results fell within the tentative quality control range of 0.064 to 0.25 g/ml provided in the Etest package insert. The Etest that was used has not been cleared by the FDA. Presence of resistance was defined as ≥8 μg/ml. High-level resistance was defined as a MIC of ≥512, and low-level resistance was defined as a MIC of 8 to 256 μg/ml.During the study period, 14,840 patients were screened and 948 (6.3%) patients tested positive for MRSA by the PCR screen. Five hundred ninety-one of the patients with positive screen results had positive MRSA cultures. Dual swabs were not available on all patients with positive screen results, and the culture positivity rates among patients with positive screen results ranged from 69 to 78% per month. Twenty of 591 (3.4%) isolates were resistant to mupirocin; 17 (2.9%) isolates had low-level mupirocin resistance (MIC, 8 to 256 μg/ml), and 3 (0.5%) had high-level mupirocin resistance (MIC, ≥512 μg/ml). Of the 20 patients with mupirocin-resistant MRSA, 9 were females and 11 were males (P = 0.821). These patients ranged from 2 years to 95 years of age. Twelve patients were older than 60 years of age. One of the 40 pediatric patients colonized with MRSA had a low-level-mupirocin-resistance MRSA isolate. One of the three patients with high-level resistance had a previous history of multiple MRSA abscesses. None of these three patients had a history of mupirocin therapy in the past.Despite the reported increased incidence of mupirocin-resistant S. aureus from various regions of the world, the literature on this topic is limited to relatively isolated areas (4, 16, 18, 20, 21). The first large study of universal surveillance for MRSA in the United States, published in 2008, reported the use of mupirocin for nasal decolonization; however, it did not address the incidence of mupirocin resistance among the MRSA isolates (17). In a subsequent letter, the authors of this study reported that mupirocin resistance occurred in 5.6% of isolates before the surveillance began and 4.1% at the end of program''s first year of universal surveillance (16). Studies addressing the true prevalence of mupirocin-resistant MRSA throughout hospitals in United States are lacking.Mupirocin-resistant MRSA is present in our Chicago area patient population at admission, but it comprised only 3.4% of MRSA isolates, and high-level resistance was distinctly uncommon. Although 60% of mupirocin-resistant MRSA isolates were found in patients >60 years of age, it does not appear that increased age is a risk factor for mupirocin-resistant MRSA. Our data, in conjunction with previous published studies, indicate that low-level mupirocin resistance was more prevalent than high-level mupirocin resistance among MRSA isolates (1, 10, 14). The occurrence of high-level-mupirocin-resistance MRSA in patients without past histories of mupirocin exposure is cause for concern.The clinical significance of low- or high-level mupirocin resistance upon the efficacy of mupirocin decolonization remains unclear. Our study was not designed to address this issue. The published studies offer disparate results. Topical mupirocin achieves remarkably high concentrations (20,000 μg/ml) which far exceed the MICs of even high-level-mupirocin-resistance strains (3, 6). Semret and Miller found that there was no significant difference in MRSA clearance of all infected sites, regardless of whether the patients were colonized with mupirocin-sensitive MRSA (68% clearance) or mupirocin-resistant MRSA (52% clearance) (P = 0.365) but that when patients were colonized with MRSA only in the nares, clearance rates were slightly better in patients colonized with mupirocin-sensitive strains (85.7%) than in those colonized with mupirocin-resistant strains (44.4%) (P = 0.145) (19).The induction of mupirocin resistance through sporadic nasal decolonization of MRSA-colonized patients in the preoperative setting or for hospital admissions is also not well defined. Studies have shown the emergence of mupirocin-resistant isolates in some settings where mupirocin-containing MRSA decolonization regimens were used (13). Mupirocin-resistant MRSA has also been associated with an increase in in-hospital mortality, compared to the level associated with mupirocin-susceptible MRSA (10). Large-scale studies and epidemiologic data on mupirocin use, trends in mupirocin resistance, and eventual correlation with clinical outcomes are lacking.Our data indicate that high-level mupirocin resistance is distinctly uncommon in our patient population. Periodic monitoring would be useful for detecting changing trends in mupirocin resistance, especially since recently enacted state legislations mandate active MRSA surveillance programs, in turn helping to drive increased use of mupirocin decolonization treatment to curb MRSA transmission.     

Vancomycin MIC plus Heteroresistance and Outcome of Methicillin-Resistant Staphylococcus aureus Bacteremia: Trends over 11 Years

Vancomycin MICs (V-MIC) and the frequency of heteroresistant vancomycin-intermediate Staphylococcus aureus (hVISA) isolates are increasing among methicillin (meticillin)-resistant Staphylococcus aureus (MRSA) isolates, but their relevance remains uncertain. We compared the V-MIC (Etest) and the frequency of hVISA (Etest macromethod) for all MRSA blood isolates saved over an 11-year span and correlated the results with the clinical outcome. We tested 489 isolates: 61, 55, 187, and 186 isolates recovered in 1996-1997, 2000, 2002-2003, and 2005-2006, respectively. The V-MICs were ≤1, 1.5, 2, and 3 μg/ml for 74 (15.1%), 355 (72.6%), 50 (10.2%), and 10 (2.1%) isolates, respectively. We detected hVISA in 0/74, 48/355 (13.5%), 15/50 (30.0%), and 8/10 (80.0%) isolates with V-MICs of ≤1, 1.5, 2, and 3 μg/ml, respectively (P < 0.001). The V-MIC distribution and the hVISA frequency were stable over the 11-year period. Most patients (89.0%) received vancomycin. The mortality rate (evaluated with 285 patients for whose isolates the trough V-MIC was ≥10 μg/ml) was comparable for patients whose isolates had V-MICs of ≤1 and 1.5 μg/ml (19.4% and 27.0%, respectively; P = 0.2) but higher for patients whose isolates had V-MICs of ≥2 μg/ml (47.6%; P = 0.03). However, the impact of V-MIC and hVISA status on mortality or persistent (≥7 days) bacteremia was not substantiated by multivariate analysis. Staphylococcal chromosome cassette mec (SCCmec) typing of 261 isolates (including all hVISA isolates) revealed that 93.0% of the hVISA isolates were SCCmec type II. These findings demonstrate that the V-MIC distribution and hVISA frequencies were stable over an 11-year span. A V-MIC of ≥2 μg/ml was associated with a higher rate of mortality by univariate analysis, but the relevance of the V-MIC and the presence of hVISA remain uncertain. A multicenter prospective randomized study by the use of standardized methods is needed to evaluate the relevance of hVISA and determine the optimal treatment of patients whose isolates have V-MICs of ≥2.0 μg/ml.The treatment of methicillin (meticillin)-resistant Staphylococcus aureus (MRSA) bacteremia with vancomycin is often associated with a poor clinical outcome (6, 15, 28, 40). Treatment failure was reported among patients infected with isolates whose vancomycin MICs were ≥4 μg/ml (6, 9, 12, 25, 28, 42). This prompted the Clinical and Laboratory Standards Institute to lower the cutoffs for S. aureus susceptibility to ≤2 μg/ml for susceptible, 4 to 8 μg/ml for intermediate (vancomycin-intermediate S. aureus [VISA]), and 16 μg/ml for resistance (39). Within the susceptibility range, the MIC is reported to increase over time (14, 25, 35-40). This is often referred to as MIC creep (38). Additionally, isolates with heteroresistance (heteroresistant vancomycin-intermediate S. aureus [hVISA]) are emerging, and this has uncertain implications for laboratory detection and clinical management (2, 5, 15, 24, 40-42). The first isolate of hVISA to be identified was reported from Japan in 1997 (11). Since then, it has been reported worldwide at frequencies of 0 to 50% (2, 4, 6, 9, 12, 19, 20, 21, 24, 26, 27, 31, 40, 42, 44). This disparity in frequency is probably a result of its variable incidence and the different testing methodologies used. Likewise, the frequency of isolates with MICs of 1.5 to <4 μg/ml varies according to the testing method used (3, 32). The relevance of an MIC on the higher side of the susceptibility range and the presence of hVISA isolates remains uncertain (8, 19, 21). Therapeutic failure was reported in patients infected with isolates with vancomycin MICs of 2 μg/ml (6, 12, 28) and 1.5 or 1 μg/ml (25, 34, 37). Most clinical microbiology laboratories use automated testing methods that are known to underestimate the vancomycin MIC (13, 24). Additionally, most previous studies addressing the relevance of such isolates were observational and usually involved only a few patients and poorly selected controls (1, 4, 7, 9, 12, 14, 25, 35, 38, 42). At our institution, we found the frequency of hVISA isolates among isolates from patients with persistent MRSA bacteremia to be 14%; however, heteroresistance did not correlate with the mortality rate (19). In the current study, we tested all blood MRSA isolates collected over 11 years to determine whether the vancomycin MIC and the prevalence of hVISA have changed over time and to evaluate the effects of increasing vancomycin MICs and the hVISA frequency on patient outcomes.     

Variation in Susceptibility of Bloodstream Isolates of Candida glabrata to Fluconazole According to Patient Age and Geographic Location in the United States in 2001 to 2007

We examined the susceptibilities to fluconazole of 642 bloodstream infection (BSI) isolates of Candida glabrata and grouped the isolates by patient age and geographic location within the United States. Susceptibility of C. glabrata to fluconazole was lowest in the northeast region (46%) and was highest in the west (76%). The frequencies of isolation and of fluconazole resistance among C. glabrata BSI isolates were higher in the present study (years 2001 to 2007) than in a previous study conducted from 1992 to 2001. Whereas the frequency of C. glabrata increased with patient age, the rate of fluconazole resistance declined. The oldest age group (≥80 years) had the highest proportion of BSI isolates that were C. glabrata (32%) and the lowest rate of fluconazole resistance (5%).Candidemia is without question the most important of the invasive mycoses (6, 33, 35, 61, 65, 68, 78, 86, 88). Treatment of candidemia over the past 20 years has been enhanced considerably by the introduction of fluconazole in 1990 (7, 10, 15, 28, 29, 31, 40, 56-58, 61, 86, 90). Because of its widespread usage, concern about the development of fluconazole resistance among Candida spp. abounds (2, 6, 14, 32, 47, 53, 55, 56, 59, 60, 62, 80, 86). Despite these concerns, fluconazole resistance is relatively uncommon among most species of Candida causing bloodstream infections (BSI) (5, 6, 22, 24, 33, 42, 54, 56, 65, 68, 71, 86). The exception to this statement is Candida glabrata, of which more than 10% of BSI isolates may be highly resistant (MIC ≥ 64 μg/ml) to fluconazole (6, 9, 15, 23, 30, 32, 36, 63-65, 71, 87, 91). Suboptimal fluconazole dosing practices (low dose [<400 mg/day] and poor indications) may lead to an increased frequency of isolation of C. glabrata as an etiological agent of candidemia in hospitalized patients (6, 17, 29, 32, 35, 41, 47, 55, 60, 68, 85) and to increased fluconazole (and other azole) resistance secondary to induction of CDR efflux pumps (2, 11, 13, 16, 43, 47, 50, 55, 69, 77, 83, 84) and may adversely affect the survival of treated patients (7, 10, 29, 40, 59, 90). Among the various Candida species, C. glabrata alone has increased as a cause of BSI in U.S. intensive care units since 1993 (89). Within the United States, the proportion of fungemias due to C. glabrata has been shown to vary from 11% to 37% across the different regions (west, midwest, northeast, and south) of the country (63, 65) and from <10% to >30% within single institutions over the course of several years (9, 48). It has been shown that the prevalence of C. glabrata as a cause of BSI is potentially related to many disparate factors in addition to fluconazole exposure, including geographic characteristics (3, 6, 63-65, 71, 88), patient age (5, 6, 25, 35, 41, 42, 48, 63, 82, 92), and other characteristics of the patient population studied (1, 32, 35, 51). Because C. glabrata is relatively resistant to fluconazole, the frequency with which it causes BSI has important implications for therapy (21, 29, 32, 40, 41, 45, 56, 57, 59, 80, 81, 86, 90).Previously, we examined the susceptibilities to fluconazole of 559 BSI isolates of C. glabrata and grouped the isolates by patient age and geographic location within the United States over the time period from 1992 to 2001 (63). In the present study we build upon this experience and report the fluconazole susceptibilities of 642 BSI isolates of C. glabrata collected from sentinel surveillance sites throughout the United States for the time period from 2001 through 2007 and stratify the results by geographic region and patient age. The activities of voriconazole and the echinocandins against this contemporary collection of C. glabrata isolates are also reported.     

Development of a Bead Immunoassay To Measure Vi Polysaccharide-Specific Serum IgG after Vaccination with the Salmonella enterica Serovar Typhi Vi Polysaccharide

Vi polysaccharide from Salmonella enterica serotype Typhi is used as one of the available vaccines to prevent typhoid fever. Measurement of Vi-specific serum antibodies after vaccination with Vi polysaccharide by enzyme-linked immunosorbent assay (ELISA) may be complicated due to poor binding of the Vi polysaccharide to ELISA plates resulting in poor reproducibility of measured antibody responses. We chemically conjugated Vi polysaccharide to fluorescent beads and performed studies to determine if a bead-based immunoassay provided a reproducible method to measure vaccine-induced anti-Vi serum IgG antibodies. Compared to ELISA, the Vi bead immunoassay had a lower background and therefore a greater signal-to-noise ratio. The Vi bead immunoassay was used to evaluate serum anti-Vi IgG in 996 subjects from the city of Kolkata, India, before and after vaccination. Due to the location being one where Salmonella serotype Typhi is endemic, approximately 45% of the subjects had protective levels of anti-Vi serum IgG (i.e., 1 μg/ml anti-Vi IgG) before vaccination, and nearly 98% of the subjects had protective levels of anti-Vi serum IgG after vaccination. Our results demonstrate that a bead-based immunoassay provides an effective, reproducible method to measure serum anti-Vi IgG responses before and after vaccination with the Vi polysaccharide vaccine.Typhoid fever is caused by Salmonella enterica serotype Typhi (32). Humans are the only natural host and reservoir of S. enterica serotype Typhi (32, 41). Typhoid fever represents a spectrum of diseases ranging from an acute uncomplicated disease—including fever, headache, malaise, and disturbances of bowel function (constipation in adults and diarrhea in children)—to a more severe, complicated form of disease in 10 to 20% of infected patients that includes bleeding in the gastrointestinal tract, intestinal perforation (in 1 to 3% of hospital typhoid fever cases) and an altered mental state (32, 41). The case fatality rate is highly variable, depending on the medical treatment available and geographic location. For example, the average fatality rate is less than 1% overall but may range between 2% fatality in hospitalized patients in Pakistan and Vietnam and 50% fatality in hospitalized patients in some parts of Indonesia and Papua New Guinea (32, 41). Worldwide, typhoid fever remains a significant public health problem, with an estimated 17,000,000 cases of typhoid fever each year and up to 600,000 deaths (2, 10, 32, 41).Typhoid vaccines currently available are composed of purified Vi polysaccharide or live attenuated S. enterica serotype Typhi (Ty21a) organisms (10, 39). The Vi polysaccharide vaccine induces protective serum antibody responses that reach a maximum at 28 days after a single intramuscular vaccination with 25 μg purified Vi polysaccharide (39), a capsular polysaccharide (Vi for virulence) that increases the virulence of S. enterica serotype Typhi (32). Protective antibody levels have been estimated to be 1 μg/ml anti-Vi IgG antibody in the serum (20). Protective efficacy of the Vi polysaccharide vaccine as determined by protection against disease is modest, with only 55 to 72% of subjects protected against disease through 3 years postvaccination (1, 20, 39). The live attenuated Ty21a vaccine is administered orally as three or four doses of enteric capsules (39). Due to its use as an oral, mucosally administered vaccine, the Ty21a vaccine induces protection against typhoid fever by induction of mucosal IgA and serum IgG antibodies specific for lipopolysaccharide antigens (39). The protective efficacy of the Ty21a vaccine at 3 years postvaccination was reported to range from 42 to 67% when using three doses of Ty21a enteric capsules (11, 39). Next-generation vaccines that utilize Vi conjugated to protein carriers that provide superior induction of anti-Vi antibodies are currently in development (14, 21, 25, 36).Despite its ability to induce protective immune responses when used alone or conjugated to protein carriers, the use of Vi polysaccharide as a coating antigen in enzyme-linked immunosorbent assay (ELISA) to measure vaccine-induced anti-Vi antibody responses has been reported to be problematic. The use of polysaccharides (lipopolysaccharide [LPS], Haemophilus influenzae type b capsular polysaccharide, Vi polysaccharide) as coating antigens for immunoassays is plagued by problems such as a poor binding of polysaccharides to ELISA plates and inconsistent results (3, 15, 16, 26, 33). To increase binding of Vi antigen to ELISA plates and produce more-robust assays, others have biotinylated Vi and then added it to streptavidin-coated plates (12) or conjugated Vi to tyramine (22, 26). However, some reports indicate that Vi was used without any additional treatment as an ELISA coating antigen (7, 19, 21) although a Vi ELISA performed on plates was less sensitive than a radioimmunoassay procedure (19).Immunoassays based on the use of fluorescent beads as the solid surface have recently been developed and compared to ELISA for the measurement of antigen-specific antibodies for polysaccharides from Streptococcus pneumoniae, Neisseria meningitidis, or Haemophilus influenzae type b (HiB) (5, 8, 23, 27, 34, 35). The fluorescent bead assays were comparable to ELISA and sometimes were noted as having enhanced dynamic ranges or increased sensitivity (5, 8, 27, 35). An additional benefit of fluorescent bead immunoassays is their ability to be multiplexed to permit the simultaneous measurement of antibodies specific for different antigens (8, 23, 27, 34, 35). This study was performed to evaluate a fluorescent bead immunoassay for its ability to measure vaccine-induced antibodies specific for Salmonella serotype Typhi Vi polysaccharide. The performance of the fluorescent bead assay was compared to that of ELISA.     

High-Level Rifampin Resistance Correlates with Multiple Mutations in the rpoB Gene of Pulmonary Tuberculosis Isolates from the Afghanistan Border of Iran

The aim of this study was to investigate the significance of multiple mutations in the rpoB gene as well as predominant nucleotide changes and their correlation with high levels of resistance to rifampin (rifampicin) in Mycobacterium tuberculosis isolates that were randomly collected from the sputa of 46 patients with primary and secondary cases of active pulmonary tuberculosis from the southern region (Afghanistan border) of Iran where tuberculosis is endemic. Drug susceptibility testing was performed using the CDC standard conventional proportional method. DNA extraction, rpoB gene amplification, and DNA sequencing analysis were performed. Thirty-five (76.09%) isolates were found to have multiple mutations (two to four) in the rpoB (β-subunit) gene. Furthermore, we demonstrate that the combination of mutations with more prevalent nucleotide changes were observed in codons 523, 526, and 531, indicating higher frequencies of mutations among patients with secondary infection. In this study, 76.08% (n = 35) of all isolates found to have mutation combinations involving nucleotide changes in codons 523 (GGG→GCG), 531 (TCG→TTG or TTC), and 526 (CAC→CGC, TTC, AAC, or CAA) demonstrated an association with higher levels of resistance to rifampin (MIC, ≥100 μg/ml).In bacterial populations, the generation of antibiotic resistance depends on the rate of emergence of resistant mutants (1, 19, 23). Correlations between high mutation rates, the geographic distribution of mutations, antibiotic resistance, and virulence in bacteria have been reported in several studies (9, 20, 33, 37). Knowledge of geographic variations is important for monitoring rifampin (rifampicin) resistance within a defined population of patients infected with Mycobacterium tuberculosis, as the prevalence of the mutations studied so far varies for M. tuberculosis strains isolated from different countries (24, 26, 29, 33, 36). In 2004, the prevalence of tuberculosis in Iran was reported to be 17 per 100,000, and at the southern border of Iran (Zabol province) where tuberculosis is endemic, the prevalence was 141 per 100,000 (20). Rifampin resistance is of particular epidemiologic importance, since it represents a valuable surrogate marker for multidrug-resistant (MDR) tuberculosis strains, and the prevalence of MDR strains is a significant obstacle to tuberculosis therapy (4, 21, 26). DNA sequencing studies indicate that more than 95% of rifampin-resistant M. tuberculosis strains have mutations within the 81-bp hot-spot region (codons 507 to 533) of the RNA polymerase β-subunit (rpoB) gene (4, 19, 32). Over the last 15 years, Kapur et al. and Telenti et al. have identified the molecular basis of rifampin resistance in M. tuberculosis (9, 29). Thus, it is important to determine the molecular bases of mutations and their distribution at the level of each country prior to molecular testing introduction for routine diagnostics (9, 11, 13, 15, 16, 23).In this study, we investigated the significance of multiple mutations in the rpoB gene and their correlation with highly prevalent nucleotide changes in codons 523, 531, and 526 and also demonstrated the highly prevalent nucleotide changes observed in the last nine codons of the β-subunit (523 to 531) that are associated with higher levels of resistance to rifampin (MIC, ≥100 μg/ml) in patients bearing secondary M. tuberculosis infection.     

Characteristics of Meropenem Heteroresistance in Klebsiella pneumoniae Carbapenemase (KPC)-Producing Clinical Isolates of K. pneumoniae

Meropenem heteroresistance was investigated in six apparently meropenem-susceptible, Klebsiella pneumoniae carbapenemase (KPC)-producing K. pneumoniae (KPC-KP) clinical isolates, compared with that in carbapenemase-negative, meropenem-susceptible controls. In population analyses, the KPC-KP isolates grew at meropenem concentrations of 64 to 256 μg/ml. Heteroresistant colonies had significantly elevated expression of the bla_KPC gene compared with the native populations but did not retain heteroresistance when subcultured in drug-free media. Time-kill assays indicated that meropenem alone was not bactericidal against KPC-KP but efficiently killed the control strains.Since the beginning of the last decade, Klebsiella pneumoniae carbapenemase (KPC)-producing K. pneumoniae (KPC-KP) isolates have been increasingly detected in the United States and subsequently in several regions worldwide (3, 4, 13, 17, 21). KPC enzymes efficiently hydrolyze all β-lactam molecules (1, 22), conferring various levels of resistance to all β-lactam compounds, including carbapenems (13). However, KPC-producing K. pneumoniae may appear susceptible to carbapenems, mainly meropenem (2, 13), by reference CLSI agar dilution or broth microdilution methods as well as by automated systems (6, 15, 17). Characteristically, it has been reported that automated systems may identify as many as 87% of KPC-KP isolates to be susceptible to meropenem (13). The detection of the susceptibility level of KPC-KP isolates to carbapenems has been shown to be difficult due to the phenotypic heterogeneity that they commonly exhibit (3, 10, 13). For instance, in agar diffusion methods such as disk diffusion or Etest, the heterogeneous growth to carbapenems of KPC-KP results in the appearance of scattered colonies within the inhibition zones (9, 13).These issues raise the need for cautious evaluation of susceptibility testing in KPC-KP isolates that are recovered in clinical laboratories. In our clinical laboratories, several KPC-KP isolates that appear susceptible by automated susceptibility assays or reference dilution assays contain heterogeneous subpopulations (D. Sofianou and K. Themeli-Digalaki, personal communications). It has been also shown that among Greek KPC-KP isolates, meropenem tends to exhibit lower MICs than imipenem or ertapenem (17, 20). In that respect, the aim of the present study was to characterize the heterogeneous mode of growth of apparently meropenem-susceptible KPC-KP clinical isolates by population analyses and bactericidal assays.     

Staphylococcus aureus Induces Microglial Inflammation via a Glycogen Synthase Kinase 3β-Regulated Pathway

A proinflammatory role for glycogen synthase kinase 3β (GSK-3β) has been demonstrated. Here, we addressed its roles on heat-inactivated Staphylococcus aureus-induced microglial inflammation. Heat-inactivated S. aureus induced tumor necrosis factor alpha (TNF-α) and nitric oxide (NO) production, at least in part, via a Toll-like receptor 2-regulated pathway. Neutralization of TNF-α largely blocked heat-inactivated S. aureus-induced NO. Heat-inactivated S. aureus activated GSK-3β, and inhibiting GSK-3β reduced TNF-α production as well as inducible NO synthase (iNOS)/NO biosynthesis. While activation of NF-κB was essential for heat-inactivated S. aureus-induced TNF-α and NO, inhibiting GSK-3β blocked heat-inactivated S. aureus-induced NF-κB p65 nuclear translocation. Additionally, inhibiting GSK-3β enhanced heat-inactivated S. aureus-induced interleukin-10 (IL-10) production (IL-10 is an anti-inflammatory cytokine which inhibits TNF-α production). Neutralization of IL-10 reduced TNF-α downregulation caused by GSK-3β inhibition. These results suggest that GSK-3β regulates heat-inactivated S. aureus-induced TNF-α and NO production in microglia mainly by activating NF-κB and probably by inhibiting IL-10.Staphylococcus aureus, a gram-positive bacterium, causes a variety of diseases, such as bacteremia, peritonitis, subcutaneous and brain abscess, and life-threatening staphylococcal septic shock (15). The mechanisms that lead to staphylococcal septic shock are multifactorial but involve especially immunogenic and toxic injuries (10, 40). Cell wall components and secreted virulence factors, including enterotoxins and exotoxins, have been shown to be inflammatory and cytotoxic to the host. Pathogen-associated molecular patterns are recognized by the innate immune system through a family of pattern recognition receptors, such as Toll-like receptors (TLRs) (2, 6, 26). Microglia, the resident macrophages in the brain, express TLR2 to recognize S. aureus peptidoglycan and play a critical role in neuroinflammation (7, 35, 37). Induction of neuroinflammation by S. aureus is partially mediated by TLR2- and nuclear factor-κB (NF-κB)-regulated pathways (23, 26, 36, 51).Infection of S. aureus causes the deregulated production of inflammatory cytokines, including tumor necrosis factor alpha (TNF-α), interleukin-6 (IL-6), and IL-10, and chemokines, including monocyte chemoattractant protein 1 (MCP-1) and RANTES (regulated on activation, normal T cell expressed and secreted protein) (24, 25, 32, 45). TNF-α, a potent proinflammatory cytokine, causes severe inflammatory responses, including cytokine and chemokine production and inducible nitric oxide (NO) synthase (iNOS)/NO biosynthesis in S. aureus infection (49). The deregulated generation of NO contributes to S. aureus-induced circulatory failure and liver injury (34). IL-10, a potent anti-inflammatory cytokine, inhibits the synthesis of the proinflammatory cytokines (TNF-α, IL-1, IL-6, IL-12, IL-18, and IL-10 itself), chemokines (IL-8, MCP-1, and RANTES), and iNOS/NO (4, 30, 43). IL-10 knockout mice display high mortality and are more susceptible to S. aureus-induced brain abscess (48). Exogenous IL-10 inhibits lethal sepsis, hepatic injury, and TNF-α production induced by staphylococcal enterotoxin B in mice (46, 48).Inhibiting glycogen synthase kinase 3β (GSK-3β) downregulates TLR-mediated inflammatory responses but increases IL-10 production (41, 53). Since NF-κB is important for inflammatory activation, GSK-3β is also involved in activating NF-κB in response to inflammatory stimuli (17-21, 29, 44, 50, 52). Therefore, GSK-3β inhibitors have been used to confer anti-inflammation against TNF-α administration, endotoxemia, experimental colitis, type II collagen-induced arthritis, ovalbumin-induced asthma, and experimental autoimmune encephalomyelitis (5, 12-14, 18, 20, 31, 41, 50, 52). Notably, current studies also show the effects of GSK-3β inhibition in reducing gram-negative coccobacillus Francisella-induced inflammation (55). GSK-3β inhibitors have also been widely used to reduce microglial inflammation and neurotoxicity (31, 54). In search of strategies against S. aureus-induced microglial inflammation, we investigated the possible effects of GSK-3β inhibition. In the present study, we report that inhibiting GSK-3β blocks NF-κB activation, TNF-α production, and iNOS/NO biosynthesis, but increases IL-10 production in heat-inactivated S. aureus-stimulated microglia.     

γδ T Cells but Not NK Cells Are Essential for Cell-Mediated Immunity against Plasmodium chabaudi Malaria

Blood-stage Plasmodium chabaudi infections are suppressed by antibody-mediated immunity and/or cell-mediated immunity (CMI). To determine the contributions of NK cells and γδ T cells to protective immunity, C57BL/6 (wild-type [WT]) mice and B-cell-deficient (J_H^−/−) mice were infected with P. chabaudi and depleted of NK cells or γδ T cells with monoclonal antibody. The time courses of parasitemia in NK-cell-depleted WT mice and J_H^−/− mice were similar to those of control mice, indicating that deficiencies in NK cells, NKT cells, or CD8⁺ T cells had little effect on parasitemia. In contrast, high levels of noncuring parasitemia occurred in J_H^−/− mice depleted of γδ T cells. Depletion of γδ T cells during chronic parasitemia in B-cell-deficient J_H^−/− mice resulted in an immediate and marked exacerbation of parasitemia, suggesting that γδ T cells have a direct killing effect in vivo on blood-stage parasites. Cytokine analyses revealed that levels of interleukin-10, gamma interferon (IFN-γ), and macrophage chemoattractant protein 1 (MCP-1) in the sera of γδ T-cell-depleted mice were significantly (P < 0.05) decreased compared to hamster immunoglobulin-injected controls, but these cytokine levels were similar in NK-cell-depleted mice and their controls. The time courses of parasitemia in CCR2^−/− and J_H^−/− × CCR2^−/− mice and in their controls were nearly identical, indicating that MCP-1 is not required for the control of parasitemia. Collectively, these data indicate that the suppression of acute P. chabaudi infection by CMI is γδ T cell dependent, is independent of NK cells, and may be attributed to the deficient IFN-γ response seen early in γδ T-cell-depleted mice.Malaria remains a leading cause of morbidity and mortality, annually killing about 2 million people worldwide (32, 33). Despite decades of research, malaria is a reemerging disease because of increasing drug resistance by malarial parasites and insecticide resistance by the mosquito vector. Most infected individuals do not succumb to malaria but develop clinical immunity where parasite replication is controlled to some degree by the immune system without eliciting clinical disease or sterile immunity (14, 38).Understanding the immunologic pathways leading to the control of blood-stage parasite replication is important for defining the mechanisms of disease pathogenesis and improving vaccines currently in development. The early events of the immune response depend upon activation of the innate immune system, which regulates the downstream adaptive immune response needed to control or cure (44). Natural killer (NK) and γδ T cells function early in the immune response to pathogens as components of the innate immune system. Both cell types have been proposed to play significant roles in the subsequent clearance of blood-stage malarial parasites by activating the adaptive immune system (35, 43, 44). The mechanism by which they accomplish this appears to be mediated via their secretion of gamma interferon (IFN-γ) induced by cytokines such as interleukin-12 (IL-12), tumor necrosis factor alpha (TNF-α), and IL-6 produced by other components of the innate immune system, including macrophages and dendritic cells (17, 25, 26, 37, 49).Blood-stage malaria parasites are cleared by mature isotypes of antibodies and/or by antibody-independent but T-cell-dependent mechanisms of immunity (2, 15, 22). Both responses require CD4⁺ αβ T cells; in addition, the expression of cell-mediated immunity (CMI) during both acute and chronic malaria is dependent on γδ T cells activated by CD4⁺ αβ T cells (29, 47, 49, 50). Wild-type (WT) mice depleted of γδ T cells by antibody treatment or gene knockout suppress P. chabaudi parasitemia by antibody-mediated immunity (AMI) (21, 52). Mice depleted of B cells by the same procedures also cure their acute infections in the same timeframe as intact control mice but then develop chronic low-grade parasitemia of long-lasting duration, indicating that B cells and their antibodies are needed to sterilize the infection as we originally reported (15, 48) and has since been confirmed by others (51). B-cell-deficient mice depleted of γδ T cells cannot suppress P. chabaudi parasitemia (49, 50, 52).The prominent role played by IFN-γ in immunity to malaria is generally accepted by most researchers. P. chabaudi malaria is more severe in WT mice treated with neutralizing antibody and in IFN-γ^−/− mice, as indicated by the increased magnitude and duration of parasitemia and mortality in mice deficient in IFN-γ versus intact controls (24, 39, 46). In B-cell-deficient animals, the similar neutralization of IFN-γ by treatment with anti-IFN-γ monoclonal antibody (MAb) or gene knockout of IFN-γ has an even greater effect on the time course of parasitemia, which remains at high levels and fails to cure (1, 46), indicating that IFN-γ is essential for the expression of anti-parasite CMI and contributes to AMI in this model system.The early source of IFN-γ remains controversial, with both NK cells and γδ T cells being proposed to produce this critical cytokine necessary for the activation of the adaptive immune response and the development of protective immunity (9). The results of earlier genetic studies failed to correlate susceptibility to P. chabaudi infection with NK activity (31, 44). Subsequently, Mohan et al. (25) reported that NK cell activity against tumor cell targets correlates with protection against P. chabaudi; anti-asialo GM1 polyclonal antibody depletion of NK cells results in significantly increased levels of peak parasitemia and a prolonged duration of infection compared to controls. The mode of action by which NK cells function appears to be via the secretion of cytokines (25) rather than direct cytotoxicity against the blood-stage parasites. The surface expression of lysosome-associated membrane protein 1 (LAMP-1) by subsets of human NK cells exposed to Plasmodium falciparum-infected erythrocytes may suggest otherwise (20). NK cells in collaboration with dendritic cells are responsible for optimal IFN-γ production dependent upon IL-12 (17, 36, 39, 40). In contrast to the findings of Mohan et al., other studies indicate similar P. chabaudi parasitemia in depleted mice and intact controls after NK1.1 MAb depletion of NK cells (19, 41, 53). Using microarray analysis of blood cells from P. chabaudi-infected mice, Kim et al. (18) reported a rapid production of IFN-γ and activation of IFN-γ-mediated signaling pathways as early as 8 h after infection; however, NK cells did not express IFN-γ or exhibit IFN-γ-mediated pathways in their analysis. At this time, NK cells are replicating and migrating from the spleen to the blood. In humans with P. falciparum malaria, increased production of IFN-γ by PBMC in response to parasitized RBCs correlates with protection from high-density parasitemia and clinical malaria (10, 11); early IFN-γ production by PBMC obtained from malaria naive donors is primarily by γδ T cells and not by NK cells (26). Animal models by definition do not exactly mimic the human condition, and the experimental malaria in mice uses distinct species from those that infect humans. Nevertheless, analysis of protective immunity provides important information on how a protective immune response to Plasmodium may be elicited.Whether both NK cells and γδ T cells have essential roles during the early stages of the immune response to blood-stage malaria remains to be determined. Likewise, whether these cells function early in CMI to malaria parasites is unknown. To address these issues, we infected NK-cell- or γδ-T-cell-depleted J_H^−/− mice with blood-stage P. chabaudi. The resulting time course of parasitemia was monitored and compared to control mice. In addition, spleen cells from depleted and control mice were profiled by cytofluorimetry, and the serum levels of inflammatory cytokines were measured.     

Serologic Assay To Quantify Human Immunoglobulin G Antibodies to the Staphylococcus aureus Iron Surface Determinant B Antigen

A direct binding Luminex assay has been developed and validated for the detection of human immunoglobulin G (IgG) antibodies to the Staphylococcus aureus iron surface determinant B protein (IsdB) in serum following natural infection or immunization with investigational Saccharomyces cerevisiae-derived IsdB-based vaccines. To ensure that IsdB-specific IgG antibodies are measured following immunization with S. cerevisiae-derived IsdB, an Escherichia coli-produced IsdB antigen is used in the assay. The IsdB antigen is covalently conjugated to maleimide microspheres via an engineered carboxy-terminal cysteine residue. Antibody titers are determined in a direct binding format, where the phycoerythrin-labeled monoclonal antibody (HP6043) specific for IgG1 to IgG4 binds to human serum IgG antibodies. Fluorescent signal emitted from bound HP6043 is directly proportional to an individual''s antibody levels. A pooled human reference serum from vaccinees with high titers to IsdB is used to generate a 12-point standard curve. The correlation of mean fluorescent intensity (MFI) units to μg/ml of IsdB-specific IgG is made by interpolating the MFI data through a four-parameter curve-fitting algorithm. The assay is sensitive to 1.06 μg/ml with a dynamic range of 2.1 to 10,625 μg/ml. The overall specificity of the assay is >96% and the linearity (parallelism) of the assay is −4% per 10-fold dilution. The total precision of the assay was 16.6% relative standard deviation across three different IsdB antigen lots, three different microsphere lots, two secondary antibody lots, and three different operators. The assay has proven useful for evaluating the immune response following the administration of different dosages and formulations of investigational IsdB-based vaccines.Staphylococcus aureus is a gram-positive commensal bacterium of the nares and skin and is a common cause of both community-acquired and nosocomial disease (30). S. aureus infections can cause a spectrum of diseases, including impetigo, respiratory tract infections, toxic shock syndrome, food poisoning, endocarditis, septic arthritis, and scalded skin syndrome (6, 20). Up to 40% of children and adults can have transient, asymptomatic colonization with S. aureus (16). Due to the widespread use of antibiotics, numerous strains of S. aureus exhibit broad-spectrum antibiotic drug resistance. Multiple antibiotic-resistant strains are isolated in approximately 60% of community-acquired infections and upwards of 80% of nosocomial acquired infections (21).An estimated 2 million patients develop nosocomial infections in the United States annually (31). In 2003 the hospital expenditures associated with S. aureus infections were estimated at $14.5 billion (32). Current estimates indicate that hospital-acquired S. aureus infections have dramatically increased over the past two decades (3, 8, 22, 24, 47). The increase in nosocomial infections has a direct impact on the health care industry in terms of length of stay, total hospitalization costs, and in-hospital mortality. An analysis of an inpatient sample database for the years 2000 and 2001 revealed that approximately 300,000 hospital inpatients were diagnosed with S. aureus infections prior to discharge. Additionally, inpatients diagnosed with S. aureus infections had five times the risk of hospital death (11.2% versus 2.3%) than S. aureus infection-free inpatients. With a continued rise of antibiotic-resistant S. aureus, inpatient diagnosis of infection represents a significant burden to both patients and the health care system.S. aureus is adept at colonizing wounds and disabling the immune system by expressing factors such as protein A, which binds to the Fc region of antibodies (15). In addition, S. aureus has the ability to adhere to surgical implants, such as catheters and joint replacements, and can form biofilms that are difficult to eradicate. Iron is critical for the survival of S. aureus. The bacteria express several proteins, such as the IsdA to -E family, for the purpose of scavenging iron from heme from its host during the initial stages of infections (39). In addition to extracting iron from heme, the Isd protein cascade transports heme across the bacterium cell wall to the cytoplasm. Many of these iron-scavenging proteins are conserved across numerous S. aureus strains (11) and represent good targets for drug and vaccine development.Due to the increasing number of antibiotic-resistant strains and the high morbidity and mortality associated with infection, there is a need to develop new and innovative therapies. Immunological approaches, such as prophylactic antibody treatment or vaccination, have the potential to prevent infection and disease. Vaccines against S. aureus have targeted capsular polysaccharides serotypes 5 and 8 (12), adhesion factors such as clumping factor A, fibronectin, and collagen binding proteins (2, 4, 14, 41), toxins such as alpha-toxin, enterotoxins, and toxic shock syndrome toxin (5, 18, 19, 40), and the surface-associated polysaccharide poly-N-acetyl-β-(1-6)-glucosamine (27). We have developed an IsdB-based investigational vaccine for the prevention of S. aureus infections following surgery and for patients with indwelling catheters (25). A truncated form of the IsdB protein is expressed in Saccharomyces cerevisiae and serves as the immunogen for the investigational S. aureus vaccine. Low to high levels of antibodies are generated against IsdB following S. aureus infection, and IsdB-based vaccines have been shown effective in animal models as a single antigen (25) or as a component of a multivalent vaccine (43). An IsdB-based prophylactic vaccine at a dose level of 60 μg is currently being tested in a clinical trial for the prevention of S. aureus disease following cardiothoracic surgery.To evaluate the immunogenicity of several different formulations of IsdB-based vaccines, we developed and validated a serologic assay to measure serum levels of IsdB-specific IgG antibodies. Because several different assays to measure the immunogenicity of vaccines have been successfully developed using the open platform Luminex xMAP technology (7, 9, 26, 34, 35, 37, 38, 42, 44), we developed and validated this assay to detect antibodies against IsdB using this technology. The assay uses maleimide-modified microspheres conjugated to the IsdB protein via a carboxyl cysteine. The assay was shown to be rugged, with less than a 10% change in antibody concentrations to three different operators, three IsdB antigen lots, three IsdB-microsphere lots, and two secondary detection antibody lots. The assay was also shown to be acceptably specific and precise and is considered fit for its intended purpose of monitoring antibody levels following vaccination. The assay has proven valuable in monitoring immune responses elicited following natural infection and by an IsdB-based experimental vaccine against staphylococcal infections.     

Activity of MGCD290, a Hos2 Histone Deacetylase Inhibitor,in Combination with Azole Antifungals against Opportunistic Fungal Pathogens

We report on the in vitro activity of the Hos2 fungal histone deacetylase (HDAC) inhibitor MGCD290 (MethylGene, Inc.) in combination with azoles against azole-resistant yeasts and molds. Susceptibility testing was performed by the CLSI M27-A3 and M38-A2 broth microdilution methods. Testing of the combinations (MGCD290 in combination with fluconazole, posaconazole, or voriconazole) was performed by the checkerboard method. The fractional inhibitory concentrations were determined and were defined as <0.5 for synergy, ≥0.5 but <4 for indifference, and ≥4 for antagonism. Ninety-one isolates were tested, as follows: 30 Candida isolates, 10 Aspergillus isolates, 15 isolates of the Zygomycetes order, 10 Cryptococcus neoformans isolates, 8 Rhodotorula isolates, 8 Fusarium isolates, 5 Trichosporon isolates, and 5 Scedosporium isolates. MGCD290 showed modest activity when it was used alone (MICs, 1 to 8 μg/ml) and was mostly active against azole-resistant yeasts, but the MICs against molds were high (16 to >32 μg/ml). MGCD290 was synergistic with fluconazole against 55 (60%) of the 91 isolates, with posaconazole against 46 (51%) of the 91 isolates, and with voriconazole against 48 (53%) of the 91 isolates. Synergy between fluconazole and MGCD290 was observed against 26/30 (87%) Candida isolates. All 23 of the 91 Candida isolates that were not fluconazole susceptible demonstrated a reduced fluconazole MIC that crossed an interpretive breakpoint (e.g., resistant [MIC, ≥64 μg/ml] to susceptible [MIC, ≤8 μg/ml]) when fluconazole was combined with MGCD290 at 0.12 to 4 μg/ml. The activity of fluconazole plus MGCD290 was also synergistic against 6/10 Aspergillus isolates. Posaconazole plus MGCD290 demonstrated synergy against 14/15 Zygomycetes (9 Rhizopus isolates and 5 Mucor isolates). Voriconazole plus MGCD290 demonstrated synergy against six of eight Fusarium isolates. Thus, MGCD290 demonstrated in vitro synergy with azoles against the majority of clinical isolates tested, including many azole-resistant isolates and genera inherently resistant to azoles (e.g., Mucor and Fusarium). Further evaluation of fungal HDAC inhibitor-azole combinations is indicated.At present, the azole class of antifungal agents constitutes one of the cornerstones of therapy for opportunistic mycoses due to many yeasts and molds (3, 16, 20, 24, 28, 30, 31, 33). Unfortunately, the clinical efficacy of this class of agents may be compromised by intrinsic or acquired resistance (11, 25, 27, 30). Resistance to azoles has been studied most extensively in Candida spp., in which the upregulation of genes encoding the lanosterol demethylase target enzyme (ERG11) and the Candida drug resistance (CDR) efflux pumps may occur upon exposure of the organism to azole antifungal agents. These alterations in gene regulation can result in increases in MICs and compromised clinical efficacy (25, 27, 30, 34). Given the proven safety, efficacy, and ease of use of these agents, the availability of strategies that may be used to avoid the emergence of resistance is important. Combination antifungal therapy with agents of different mechanistic classes could promote fungal killing and clinical efficacy and provide an alternative to monotherapy for patients with infections caused by multiresistant species and for patients who fail to respond to standard treatments.Histone deacetylases (HDACs) are a family of enzymes which deacetylate lysines on core histones and other cellular proteins (9, 10, 32). They play an important role in gene regulation and also in the control of other cellular functions, such as proliferation, cell death, and motility (9, 10, 22, 32). Inhibitors of HDACs belong to several chemical classes that act by binding to the catalytic site of the enzyme, causing cell cycle arrest, apoptosis, and terminal differentiation (9, 22). HDAC enzymes have been explored as potential targets in the treatment of cancer cells and infections caused by several eukaryotic microorganisms (1a, 7, 9, 22, 26, 29). Modulation of fungal gene expression through fungal HDAC inhibition may be an alternative approach to the treatment of fungal infections (17, 29). Smith and Edlind (29) have shown in Candida albicans and two other Candida spp. that trichostatin A, a nonselective HDAC inhibitor with cytoxic properties in mammalian cells, markedly decreased the upregulation of the ERG11 and CDR genes following exposure to sterol biosynthesis inhibitors, such as fluconazole and terbinafine.We previously examined the potential chemosensitizing interaction between a novel Hos2 inhibitor, MGCD290, developed by MethylGene, Inc. (Montreal, Quebec, Canada), and three triazole antifungal agents (fluconazole, itraconazole, and voriconazole) against a panel of 45 clinical isolates of Candida spp. (16 of which were fluconazole resistant) and 16 clinical isolates of Aspergillus spp. In the previous study, MGCD290 displayed synergy with fluconazole against 76% of the Candida isolates tested and with voriconazole and itraconazole against 69% of the Aspergillus isolates tested (8a). Our results suggest a potential clinical use for the combination of HDAC inhibitors and azoles in the treatment of fungal infections.In the present study, we expand upon our initial findings by examining the interaction between MGCD290 and three triazoles (fluconazole, voriconazole, and posaconazole) against a larger and more diverse collection of yeasts and molds, most of which were azole resistant.     

Importance of Conserved Residues of the Serine Protease Autotransporter β-Domain in Passenger Domain Processing and β-Barrel Assembly

Serine protease autotransporters of the family Enterobacteriaceae (SPATE) comprise a family of virulence proteins secreted by enteric Gram-negative bacteria via the autotransporter secretion pathway. A SPATE polypeptide contains a C-terminal translocator domain that inserts into the bacterial outer membrane as a β-barrel structure and mediates secretion of the passenger domain to the extracellular environment. In the present study, we examined the role of conserved residues located in the SPATE β-barrel-forming region in passenger domain secretion. Thirty-nine fully conserved residues in Tsh were mutated by single-residue substitution, and defects in their secretion phenotypes were assessed by cell fractionation and immunochemistry. A total of 22 single mutants exhibited abnormal phenotypes in different cellular compartments. Most mutants affecting secretion are charged residues with side chains pointing into the β-barrel interior. Seven mutants showed notable abnormalities in processing (constructs with the E1231A, E1249A, and R1374A mutations) and β-barrel assembly or insertion into the outer membrane (constructs with the G1158Y, F1360A, Y1375A, and F1377A mutations). The phenotypes of the β-barrel assembly/insertion mutants and the presence of a processed Tsh passenger domain in the periplasm support the possibility that the translocator domain must undergo extensive folding prior to insertion into the outer membrane. Results from double-mutation experiments further demonstrate that F1360 and F1377 affect β-barrel insertion/assembly at different times. In light of these new data, a more refined model for the mechanism of SPATE secretion is presented.In Gram-negative bacteria, the type V, or autotransporter (AT), pathway is among the most commonly utilized protein secretion pathways (14). A nascent AT polypeptide consists of an N-terminal signal sequence involved in inner membrane (IM) translocation, a passenger domain to be secreted to the extracellular environment, and a C-terminal translocator domain further comprised of an N-terminal α-helical linker and a C-terminal β-domain (14, 20). Following its IM translocation mediated by the Sec translocase and the removal of the signal peptide by the signal peptidase, an AT is released into the periplasm (14, 20). The extent of AT folding in the periplasm is still under investigation, although recent evidence suggests the presence of some tertiary structure in this compartment (2, 12, 16, 32) and the involvement of periplasmic chaperones in the secretion event (11, 25, 28). After an AT transits through the periplasm, the outer membrane (OM) assembly Bam (YaeT/Omp85) complex assists in an AT insertion into the OM (15, 37); there the AT translocator domain forms a pore-like β-barrel. In conventional ATs, only a single protein''s translocator domain is required to form a complete β-barrel (14). In the OM, the β-barrel tethers the folded passenger domain via the flexible α-helical linker (6, 14, 20). In many cases, the tethered passenger domain is eventually cleaved and released from the cell surface (6, 14, 20).Most ATs characterized to date possess virulence functions (6). Among the numerous conventional ATs is a subfamily named the serine protease ATs of the family Enterobacteriaceae (SPATE), which is a group of multifunctional ATs implicated in the pathogenicity of enteric bacterial pathogens (44). SPATE share many conserved elements: all of these ATs possess a requisite serine protease motif [GDSGS(P)] in the passenger domain and an α-helical linker motif (EVNNLNKRMGDLRD) in which the double asparagines serve as the cleavage site between the passenger and translocator (6, 18). Cleavage releases a SPATE into the extracellular environment, its final secretion destination (44). Considering the ubiquity of conventional ATs in pathogenic bacteria and their participation in disease development (6), a better understanding of their secretion mechanism might help improve current therapeutic means. To study the secretion mechanism of conventional ATs, we used the temperature-sensitive hemagglutinin (Tsh), a SPATE from avian-pathogenic Escherichia coli, as a model (24).This study focused on a conventional AT''s translocator domain, which has been shown to be essential for the secretion of the passenger domain (23). The crystal structure of the translocator domain of a SPATE protein, EspP, revealed a β-barrel formed by 12 antiparallel β-strands, with the α-helical linker plugging the interior of the β-barrel (1). While these features of the EspP translocator resemble those of the crystallized translocator domain of NalP (22), the processing sites between the passenger and translocator in these two ATs are remarkably different. In NalP the processing site is close to the opening of the β-barrel, and thus, the α-helical linker extends throughout the length of the channel (22). In contrast, cleavage in EspP is assisted by an aspartate residue located in the midlength of the β-barrel through autoproteolysis (3), hence leaving only half of the α-helical linker in the β-barrel (1). Autoproteolytic release of the passenger domain is unique, and the processing of other ATs requires OM proteases (5, 31), the GDSGS motif located in the passenger (8, 23), or other unknown factors.Considering the different roles of the translocator domain in an AT''s secretion process, it is possible that residues located in the translocator have different secretion functions. For instance, some residues might be the recognition sites for OM assembly factors or periplasmic chaperones; some might play a structural role stabilizing the β-barrel in the OM; and others might be responsible for promoting the processing of the passenger domain, as seen in EspP (3). Indeed, a number of residues within the conserved α-linker motif have been shown by this laboratory to be vital for passenger secretion (18). Here, we focus on the remaining region of the SPATE translocator, the β-domain. Mutations introduced into residues in this domain resulted in many defective phenotypes related to passenger secretion. Notably, several mutants impaired processing of the Tsh passenger domain, and some prevented proper assembly or insertion of the β-domain into the OM. Two key residues involved in the assembly/insertion of the β-barrel at different times were also identified. Based on our results, as interpreted in conjunction with recently published structural and biochemical data, we have proposed a more refined model for the SPATE secretion mechanism.     

Patterns of Susceptibility of Aspergillus Isolates Recovered from Patients Enrolled in the Transplant-Associated Infection Surveillance Network

We analyzed antifungal susceptibilities of 274 clinical Aspergillus isolates from transplant recipients with proven or probable invasive aspergillosis collected as part of the Transplant-Associated Infection Surveillance Network (TRANSNET) and examined the relationship between MIC and mortality at 6 or 12 weeks. Antifungal susceptibility testing was performed by the Clinical and Laboratory Standards Institute (CLSI) M38-A2 broth dilution method for amphotericin B (AMB), itraconazole (ITR), voriconazole (VOR), posaconazole (POS), and ravuconazole (RAV). The isolate collection included 181 Aspergillus fumigatus, 28 Aspergillus niger, 27 Aspergillus flavus, 22 Aspergillus terreus, seven Aspergillus versicolor, five Aspergillus calidoustus, and two Aspergillus nidulans isolates and two isolates identified as Aspergillus spp. Triazole susceptibilities were ≤4 μg/ml for most isolates (POS, 97.6%; ITR, 96.3%; VOR, 95.9%; RAV, 93.5%). The triazoles were not active against the five A. calidoustus isolates, for which MICs were ≥4 μg/ml. AMB inhibited 93.3% of isolates at an MIC of ≤1 μg/ml. The exception was A. terreus, for which 15 (68%) of 22 isolates had MICs of >1 μg/ml. One of 181 isolates of A. fumigatus showed resistance (MIC ≥ 4 μg/ml) to two of three azoles tested. Although there appeared to be a correlation of higher VOR MICs with increased mortality at 6 weeks, the relationship was not statistically significant (R² = 0.61; P = 0.065). Significant relationships of in vitro MIC to all-cause mortality at 6 and 12 weeks for VOR or AMB were not found.Invasive aspergillosis (IA) is an important problem in immunocompromised patients, especially in persons who have received hematopoietic stem cell or solid organ transplantation. On the basis of recent treatment guidelines, voriconazole is recommended as the primary therapy for IA, with alternatives including lipid preparations of amphotericin B (AMB), caspofungin, micafungin, itraconazole (ITR), and posaconazole (POS) (28). In vitro resistance among Aspergillus species is uncommon and may be increasing (5, 9, 27, 30). Several studies report prevalence of triazole resistance of up to 4.2% among Aspergillus isolates, or as much as 2.1% among Aspergillus fumigatus isolates (13, 14, 23). Triazole cross-resistance has been reported in several studies (14, 18, 19). In contrast, other studies have described triazole resistance in <1% of isolates, even in the postvoriconazole era (8, 19). Because of the potential of increasing MICs to triazoles and widespread use of triazoles for IA treatment, surveillance of Aspergillus susceptibility, especially among isolates causing IA, is warranted.Examining the influence of antifungal resistance on clinical outcomes has been challenging because of the difficulty in establishing large cohorts of IA patients with available isolates and because of the low frequency of resistant isolates. Moreover, a myriad of factors besides isolate susceptibility may contribute to patient outcomes. Recent reports describe the challenges of in vitro-in vivo correlations of Aspergillus spp. with current susceptibility testing methodologies (2, 17, 21). This challenge is especially daunting when considering the number of host- and transplant-related variables that impact outcomes of this infection (16, 25). While the impact of resistant Aspergillus isolates on outcomes has been demonstrated in animal models for triazoles, echinocandins, and AMB, a paucity of human data is available (2, 7, 12, 13, 17). Herein, we describe in vitro susceptibility patterns of Aspergillus isolates from transplant recipients with proven or probable IA collected as part of the Transplant-Associated Infection Surveillance Network (TRANSNET). In addition, the in vitro MIC in relationship to all-cause mortality is examined.     

α-Galactosylceramide Promotes Killing of Listeria monocytogenes within the Macrophage Phagosome through Invariant NKT-Cell Activation

α-Galactosylceramide (α-GalCer) has been exploited for the treatment of microbial infections. Although amelioration of infection by α-GalCer involves invariant natural killer T (iNKT)-cell activation, it remains to be determined whether macrophages (Mφ) participate in the control of microbial pathogens. In the present study, we examined the participation of Mφ in immune intervention in infection by α-GalCer using a murine model of listeriosis. Phagocytic and bactericidal activities of peritoneal Mφ from C57BL/6 mice, but not iNKT cell-deficient mice, were enhanced after intraperitoneal injection of α-GalCer despite the absence of iNKT cells in the peritoneal cavity. High levels of gamma interferon (IFN-γ) and nitric oxide (NO) were detected in the peritoneal cavities of mice treated with α-GalCer and in culture supernatants of peritoneal Mφ from mice treated with α-GalCer, respectively. Although enhanced bactericidal activity of peritoneal Mφ by α-GalCer was abrogated by endogenous IFN-γ neutralization, this was only marginally affected by NO inhibition. Similar results were obtained by using a listeriolysin O-deficient strain of Listeria monocytogenes. Moreover, respiratory burst in Mφ was increased after α-GalCer treatment. Our results suggest that amelioration of listeriosis by α-GalCer is, in part, caused by enhanced killing of L. monocytogenes within phagosomes of Mφ activated by IFN-γ from iNKT cells residing in an organ(s) other than the peritoneal cavity.Listeria monocytogenes, a Gram-positive facultative intracellular bacterium, is the causative agent of listeriosis, with an overall mortality rate of 30% (76). A major virulence factor of L. monocytogenes is listeriolysin O (LLO), a 58-kDa protein encoded by the hly gene (26, 42, 65). LLO promotes intracellular survival of L. monocytogenes in professional phagocytes such as macrophages (Mφ) by promoting listerial escape from the phagosome into the cytosol (10, 22, 26, 42, 62, 65). Cells of the innate immune system play a pivotal role as a first line of defense against L. monocytogenes infection and among these, mononuclear phagocytes are critical (56, 61). Activation of Mφ by gamma interferon (IFN-γ) is mandatory for elimination of L. monocytogenes (31, 35). Nitric oxide (NO) synthesized by inducible NO synthase, which is localized in the cytosol of professional phagocytes, participates in killing of L. monocytogenes (48, 52, 69, 71). Similarly, reactive oxygen intermediates (ROI) play a role in killing of L. monocytogenes within the phagosome (52, 53, 59).Natural killer T (NKT) cells represent a unique T-lymphocyte population expressing NKR-P1B/C (NK1.1; CD161), which is a type 2 membrane glycoprotein of the C-type lectin superfamily (6). In the mouse, the majority of NKT cells express an invariant T-cell receptor (TCR) α chain encoded by Vα14/Jα18 gene segments and a TCRVβ highly biased toward Vβ8.2, Vβ7, and Vβ2 (invariant NKT [iNKT] cells) (6). In contrast to conventional T cells, which recognize antigenic peptides presented by polymorphic major histocompatibility complex class I or class II molecules, iNKT cells recognize glycolipid antigens, including α-galactosylceramide (α-GalCer), a synthetic glycolipid originally isolated from a marine sponge, presented by the nonpolymorphic antigen presentation molecule CD1d (6, 40). iNKT cells are highly versatile and promptly produce both type 1 and type 2 cytokines, such as IFN-γ and interleukin-4 (IL-4), respectively, upon activation through their TCRs (1, 15-17, 79). IL-15 is an essential growth factor of both iNKT cells and NK cells and, hence, both cell populations are absent in IL-15-deficient (IL-15^−/−) mice (58). The numbers of iNKT cells are also markedly reduced in SJL mice because of a large deletion in their TCRVβ genetic region (5, 78).In vivo administration of α-GalCer causes prompt release of various cytokines by iNKT cells, which are involved in the control of various diseases, e.g., tumor rejection and prevention of autoimmune diseases (33, 41, 67, 70). Although α-GalCer has been reported to enhance host resistance to some microbial pathogens (27-29, 37, 39, 44, 55, 64), its potential role in protection against intracellular bacterial infections remains enigmatic.We have recently described that α-GalCer ameliorates murine listeriosis, which is, in part, caused by accelerated infiltration of inflammatory cells into the liver (18), although iNKT cells themselves exacerbate disease (19). Because Mφ play a central role in the elimination of L. monocytogenes, we considered the possibility that Mφ participate in enhanced resistance to L. monocytogenes infection caused by α-GalCer treatment. In the present study, we examined the influence of α-GalCer on listericidal activities of Mφ using a virulent and an avirulent strain of L. monocytogenes.     

Breakthrough Invasive Candidiasis in Patients on Micafungin

For Candida species, a bimodal wild-type MIC distribution for echinocandins exists, but resistance to echinocandins is rare. We characterized isolates from patients with invasive candidiasis (IC) breaking through ≥3 doses of micafungin therapy during the first 28 months of its use at our center: MICs were determined and hot-spot regions within FKS genes were sequenced. Eleven of 12 breakthrough IC cases identified were in transplant recipients. The median duration of micafungin exposure prior to breakthrough was 33 days (range, 5 to 165). Seventeen breakthrough isolates were recovered: FKS hot-spot mutations were found in 5 C. glabrata and 2 C. tropicalis isolates; of these, 5 (including all C. glabrata isolates) had micafungin MICs of >2 μg/ml, but all demonstrated caspofungin MICs of >2 μg/ml. Five C. parapsilosis isolates had wild-type FKS sequences and caspofungin MICs of 0.5 to 1 μg/ml, but 4/5 had micafungin MICs of >2 μg/ml. The remaining isolates retained echinocandin MICs of ≤2 μg/ml and wild-type FKS gene sequences. Breakthrough IC on micafungin treatment occurred predominantly in severely immunosuppressed patients with heavy prior micafungin exposure. The majority of cases were due to C. glabrata with an FKS mutation or wild-type C. parapsilosis with elevated micafungin MICs. MIC testing with caspofungin identified all mutant strains. Whether the naturally occurring polymorphism within the C. parapsilosis FKS1 gene responsible for the bimodal wild-type MIC distribution is also responsible for micafungin MICs of >2 μg/ml and clinical breakthrough or an alternative mechanism contributes to the nonsusceptible echinocandin MICs in C. parapsilosis requires further study.Invasive candidiasis (IC) is an important, life-threatening infection in hospitalized patients. The echinocandins (micafungin, caspofungin, and anidulafungin) are the newest class of medications approved for the prophylaxis and treatment of IC. They act via noncompetitive inhibition of β-1,3-glucan synthase, the enzyme responsible for producing β-1,3-d-glucan in the fungal cell wall (41). These drugs have low toxicity and few drug-drug interactions and possess a broad spectrum of antifungal activity against Candida species, including those resistant to fluconazole. In clinical trials, the echinocandins have demonstrated noninferiority for the treatment of IC versus amphotericin B deoxycholate, liposomal amphotericin B, and fluconazole (25, 32, 44). The echinocandins are considered interchangeable for clinical use, and a recent study comparing micafungin to caspofungin for IC supports this notion (38). Based on the accumulated experience, echinocandins are now considered a first-line therapeutic choice for IC (37).The echinocandins exhibit a bimodal MIC distribution among Candida species. MICs of C. parapsilosis, C. guilliermondii, and C. famata MICs (MIC₉₀, 0.25 to 2 μg/ml) are up to 133 times higher than those of C. albicans, C. glabrata, C. tropicalis, C. krusei, and C. kefyr (MIC₉₀, 0.015 to 0.25 μg/ml) (42). However, this difference has not translated into consistent clinical failure (25, 38, 44), and the MIC breakpoint for echinocandin susceptibility was set at ≤2 μg/ml, which is inclusive of 99% of the wild-type distribution of all Candida species (9). Organisms with MICs of >2 μg/ml are considered “nonsusceptible,” but the breakpoint for resistance has yet to be determined owing to the paucity of clinical isolates available from patients failing echinocandin therapy and with MICs of >2 μg/ml.As echinocandin use has escalated, cases of echinocandin breakthrough IC have been described (6, 7, 13, 25, 39, 50), and nonsusceptible isolates (MIC > 2 μg/ml) have been recovered from patients who demonstrated treatment failure (9). Moreover, several of these nonsusceptible isolates possess nonsynonymous point mutations in genes encoding the β-1,3-glucan synthase enzyme complex (Fksp) (4, 13, 39, 47). These specific FKS “hot-spot” mutations reduce the susceptibility of the β-1,3-glucan synthase enzyme complex to echinocandin drugs, supporting a biological mechanism of resistance (14).In February 2006, micafungin became the formulary echinocandin at our hospital, a tertiary care center with multiple intensive care units, two dedicated hematopoietic stem cell transplant (HSCT) units, and an active solid organ transplant (SOT) service. Multiple patients with breakthrough IC while receiving micafungin therapy were noted. These cases were reviewed, and the Candida isolates recovered from these patients were screened for FKS gene mutations; results were correlated with MIC values.(This work was presented in part at the 49th Interscience Conference on Antimicrobial Agents and Chemotherapy, San Francisco, CA, 12 to 14 September 2009 [slide presentation M-1243]).     

Wild-Type MIC Distribution and Epidemiological Cutoff Values for Aspergillus fumigatus and Three Triazoles as Determined by the Clinical and Laboratory Standards Institute Broth Microdilution Methods

Antifungal susceptibility testing of Aspergillus species has been standardized by both the Clinical and Laboratory Standards Institute (CLSI) and the European Committee on Antimicrobial Susceptibility Testing (EUCAST). Recent studies suggest the emergence of strains of Aspergillus fumigatus with acquired resistance to azoles. The mechanisms of resistance involve mutations in the cyp51A (sterol demethylase) gene, and patterns of azole cross-resistance have been linked to specific mutations. Studies using the EUCAST broth microdilution (BMD) method have defined wild-type (WT) MIC distributions, epidemiological cutoff values (ECVs), and cross-resistance among the azoles. We tested a collection of 637 clinical isolates of A. fumigatus for which itraconazole MICs were ≤2 μg/ml against posaconazole and voriconazole using the CLSI BMD method. An ECV of ≤1 μg/ml encompassed the WT population of A. fumigatus for itraconazole and voriconazole, whereas an ECV of ≤0.25 μg/ml was established for posaconazole. Our results demonstrate that the WT distribution and ECVs for A. fumigatus and the mold-active triazoles were the same when determined by the CLSI or the EUCAST BMD method. A collection of 43 isolates for which itraconazole MICs fell outside of the ECV were used to assess cross-resistance. Cross-resistance between itraconazole and posaconazole was seen for 53.5% of the isolates, whereas cross-resistance between itraconazole and voriconazole was apparent in only 7% of the isolates. The establishment of the WT MIC distribution and ECVs for the azoles and A. fumigatus will be useful in resistance surveillance and is an important step toward the development of clinical breakpoints.Invasive aspergillosis (IA) is second only to candidiasis as the most important invasive mycosis affecting humans (5, 10, 18, 37, 40). Although several hundred species of Aspergillus have been described, relatively few are known to cause disease in humans. Aspergillus fumigatus remains the most common cause of IA, although the proportion of IA cases caused by this species has fallen from ∼90% of cases in the 1980s to between 50 and 60% of cases in the 1990s and into the 2000s (30, 36).Antifungal susceptibility testing of Candida as an aid in guiding clinical treatment has gained wide acceptance (3, 8, 16, 20, 22, 24, 39). The development and application of in vitro susceptibility testing of Aspergillus spp. have lagged behind that for Candida due to the difficulty in determining a clinical role for this testing. Confounding the issue are numerous factors that play either a positive or negative role in determining the outcome of therapy (14, 26). Given the increasing number of antifungal agents with systemic activity against Aspergillus spp. (13, 15, 21, 38, 41, 42, 44, 48, 49), it is recognized that antifungal susceptibility testing of these opportunistic pathogens may be useful in guiding the selection of antifungal agents for the treatment of IA (1, 34, 42, 44, 47). This is especially true for itraconazole (11, 34) and the newer extended-spectrum triazoles posaconazole and voriconazole (42, 44).In vitro antifungal susceptibility testing of azoles versus Aspergillus spp. has been standardized by both the CLSI (7) and European Committee on Antimicrobial Susceptibility Testing (EUCAST) (27). Both methods use a 96-well broth microdilution (BMD) format, RPMI 1640 broth medium, 48 h of incubation, and a MIC endpoint of complete inhibition of growth as determined by visual inspection. The EUCAST method employs a higher inoculum concentration (2 × 10⁵ to 5 × 10⁵ CFU/ml versus 0.4 × 10⁴ to 5 × 10⁴ CFU/ml) and additional glucose (2% versus 0.2% final concentration) in an effort to improve fungal growth. Using these methods, A. fumigatus isolates exhibiting high MICs to azole drugs have been described (2, 11, 19, 28, 29) and both methods have been shown to reliably detect those strains with defined azole resistance mechanisms (1, 4, 6, 17, 31-33, 42, 44).Mechanisms of elevated azole MICs in A. fumigatus discovered thus far involve the cyp51A (lanosterol demethylase) gene and include mutations resulting in amino acid substitutions at glycine 54 (G54) (12, 29, 35, 44) at G448 (50), and at methionine 220 (M220) (31, 44) and an amino acid substitution of leucine for histidine at position 98 (L98H) together with a tandem repeat (TR) of a 34-bp sequence in the promoter of the cyp51A gene (33). Cross-resistance to itraconazole and posaconazole has been associated with the G54 substitution, whereas cross-resistance to voriconazole and ravuconazole has been associated with the G448 substitution (44, 50). Both the M220 substitution and the L98H TR produce a cross-resistance pattern to all four azoles (44).Interpretive breakpoints based upon the correlation of in vitro data with clinical outcome have not been established for any Aspergillus-drug combination (14, 42, 44). In the absence of the necessary clinical data, one practical approach to the use of susceptibility testing data in detecting resistance or decreased susceptibility has been to define the wild-type (WT) distribution of MICs for the relevant drug-organism combinations (e.g., populations of organisms with no acquired resistance mechanisms) and set epidemiological cutoff values (ECVs) that would discriminate WT strains from those with acquired resistance mechanisms (25, 44-46). The clinical relevance of ECVs would still be uncertain, but ECVs could nonetheless serve as the foundation for the laboratory detection of acquired resistance (decreased susceptibility) and be used to monitor resistance development (45).Rodriguez-Tudela et al. (44) employed the EUCAST BMD method to define the WT MIC distribution of four triazole antifungal agents (itraconazole, posaconazole, ravuconazole, and voriconazole) for A. fumigatus. They also used studies of resistance mechanisms in A. fumigatus to demonstrate that ECVs of ≤1 μg/ml for itraconazole, ravuconazole, and voriconazole and ≤0.25 μg/ml for posaconazole identified the WT strains and provided separation of the WT population from those strains with resistance mutations in the cyp51A gene (44). Definition of the WT distribution and ECVs for azoles and A. fumigatus is important in order to have a clear understanding of which MICs can be considered microbiologically susceptible (25, 44, 46). Furthermore, these studies showed that the MIC phenotype obtained by means of a standardized methodology was sufficient to identify nonsusceptible strains of A. fumigatus and their pattern of cross-resistance without the need for sequence analysis of cyp51A to detect the underlying mutation (44).Similar studies using CLSI BMD to test itraconazole, posaconazole, ravuconazole, and voriconazole against 553 isolates of A. fumigatus confirmed the cross-resistance profiles reported by Rodriguez-Tudela et al. (44), indicating that both CLSI and EUCAST BMD methods may be used to sort out these different patterns (42). In the present study, we utilized a large, geographically diverse collection of A. fumigatus isolates tested against itraconazole, posaconazole, and voriconazole by the CLSI method to provide further documentation of the WT MIC distribution and ECVs for the azoles and A. fumigatus.