Linda A. Miller

Molecular Epidemiology of Methicillin-Resistant and Methicillin-Susceptible Staphylococcus aureus Isolates from Global Clinical Trials

ABSTRACT Determining the genetic characteristics of Staphylococcus aureus is important for better understanding of the global and dynamic epidemiology of this organism as we witness the emergence and spread of virulent and antibiotic-resistant clones. We genotyped 292 S. aureus isolates (105 methicillin resistant and 187 methicillin susceptible) using a combination of pulsed-field gel electrophoresis, multilocus sequence typing, and SCCmec typing. In addition, S. aureus isolates were tested for the presence of the Panton-Valentine leukocidin (PVL) genes. Isolates were recovered from patients with uncomplicated skin infections in 10 different countries during five phase III global clinical trials of retapamulin, a new topical antibiotic agent. The most common methicillin-resistant clone had multilocus sequence type 8, pulsed-field type USA300, and SCCmec type IV and possessed the PVL genes. This clone was isolated exclusively in the United States. The most common PVL-positive, methicillin-susceptible clone had multilocus sequence type 121 and pulsed-field type USA1200. This clone was found primarily in South Africa and the Russian Federation. Other clones were found at lower frequencies and were limited in their geographic distribution. Overall, considerable genetic diversity was observed within multilocus sequence type clonal complexes and pulsed-field types.

Increasing Role of Staphylococcus aureus and Community-Acquired Methicillin-Resistant Staphylococcus aureus Infections in the United States: A 10-Year Trend of Replacement and Expansion

BACKGROUND There is the need to properly characterize the temporal trend of U.S. Staphylococcus aureus infections, including methicillin-resistant S. aureus (MRSA) and community-acquired (CA) MRSA in inpatient and outpatient settings. METHODS The study used the Surveillance Network(®) surveillance database (Eurofins Medinet) and the National Hospitalization Discharge Survey for the period 1998-2007. CA-MRSA phenotype was defined by a resistance profile that includes susceptibility to gentamicin and cotrimoxazole, and coresistance to ciprofloxacin/clindamycin. Adjusted rates, rate ratios, and 95% confidence intervals (CIs) were computed using multivariate logistic regression. RESULTS The study consisted of 1,761,991 S. aureus isolates. Annual MRSA prevalence continuously increased over the 10-year period from 32.7% in 1998 to 53.8% in 2007 (odds ratio 2.4, 95% CI 2.3-2.5). CA-MRSA replaced competing strains by increasing its share of MRSA from 22.3% in 1998 to 66.1% in 2007 (odds ratio 6.7, 95% CI 6.5-6.9). MRSA-related hospitalization rate per 1,000 discharges doubled from 3.5 ± 0.9 in 1998 to 7.6 ± 1.5 in 2007 (RR 2.2, 95% CI 1.8-3.1), whereas CA-MRSA increased from 0.4 ± 0.14 hospitalizations per 1,000 discharges in 1998 to 3.1 ± 0.5 in 2007 (RR 8.1, 95% CI 5.2-14.1), By 2007, 81.5% of all MRSA isolates were categorized as CA-MRSA among children, whereas CA-MRSA represented 48.9% of MRSA isolates from the elderly. CONCLUSION MRSA not only replaced methicillin susceptible S. aureus (MSSA) isolates as a percentage of all S. aureus isolates, but its hospitalization rates increased over and above the replacement process. This trend also applies to CA-MRSA over hospital-acquired (HA) MRSA.

β-Lactamase-inhibitor combinations in the 21st century: current agents and new developments

Combinations of beta-lactams and beta-lactamase inhibitors have become one of the most successful antibacterial strategies in our global battle against bacterial infections. The success of these agents is particularly emphasized by the continued efficacy of Augmenting (amoxicillin and clavulanate) after nearly 20 years of clinical use. The clinical situation now dictates that second-generation beta-lactamase inhibitors capable of encompassing both class A and class C beta-lactamases would combat emerging resistance and provide a vital addition to our armory of hospital antibiotics. This realization has generated a renewed interest in beta-lactamase inhibitors and improved the prospects for the delivery of such agents in the future.

Serotype Replacement and Multiple Resistance in Streptococcus pneumoniae After the Introduction of the Conjugate Pneumococcal Vaccine

BACKGROUND The pneumococcal conjugate vaccine (PCV7), introduced in February 2000, covered 82% of the U.S. pediatric population in 2005. Changes over time in serogroup prevalence and multidrug-resistance (MR) to antimicrobials were evaluated using the U.S. SENTRY surveillance program. METHODS The study included 704 U.S. isolates, with equal numbers before (1998-1999) and after the introduction of the vaccine (2003-2004). Demographic data, serotype, and resistance profiles for five antimicrobial classes were analyzed. Strains displaying resistance to >or=2 classes were considered MR. Statistical analysis was performed using logistic regression. RESULTS Prevalence of PCV7 serotypes was 68.5% in the prevaccine years, dropping to 29.3% in the postvaccine period. Among PCV7 serotypes, only 19F persisted, with nonvaccine (NV) serotype 19nonF strains increasing from 3% to 20% of total p<0.001. NV serotypes were 1.9 times (95% confidence interval [CI] 1.1-3.1) more likely to acquire MR over time. Although PCV7 serotypes constituted 84% of all MR isolates in the prevaccine era, MR was unchanged in the postvaccine period due to increased prevalence and acquisition of resistance by NV serotypes. MR among invasive isolates did not change over time, but increased among noninvasive NV isolates by 17% (95% CI 12-22%). CONCLUSIONS The complete switch in prevalence of PCV7 by NV serotypes has been aided by a herd effect in adults and older children. NV serotypes have acquired MR at a rate that is proportional to the replacement process.

A Geographic Variant of the Staphylococcus aureus Panton-Valentine Leukocidin Toxin and the Origin of Community-Associated Methicillin-Resistant S. aureus USA300

BACKGROUND The majority of recent community-associated methicillin-resistant Staphylococcus aureus (MRSA) infections in the United States have been caused by a single clone, USA300. USA300 secretes Panton-Valentine leukocidin (PVL) toxin, which is associated with highly virulent infections. METHODS We sequenced the PVL genes of 174 S. aureus isolates from a global clinical sample. We combined phylogenetic reconstruction and protein modeling methods to analyze genetic variation in PVL. RESULTS Nucleotide variation was detected at 12 of 1726 sites. Two PVL sequence variants, the R variant and the H variant, were identified on the basis of a substitution at nt 527. Of sequences obtained in the United States, 96.7% harbor the R variant, whereas 95.6% of sequences obtained outside the United States harbor the H variant; 91.3% of MRSA isolates harbor the R variant, and 91.3% of methicillin-susceptible strains harbor the H variant. A molecular model of PVL shows 3 mechanisms by which the amino acid substitution may affect PVL function. CONCLUSIONS All sampled PVL genes appear to share a recent common ancestor and spread via a combination of clonal expansion and horizontal transfer. US isolates harbor a variant of PVL that is strongly associated with MRSA infections. Protein modeling reveals that this variant may have functional significance. We propose a hypothesis for the origin of USA300.

Acinetobacter baumannii 2002-2008: increase of carbapenem-associated multiclass resistance in the United States.

The study consisted of data for 55,330 U.S. Acinetobacter baumannii isolates from The Surveillance Network(®) database for the period 2002-2008. Risk factors were time, age, sex, census region, location (Ward or ICU), and isolate source. Antimicrobial susceptibility data were available for carbapenems, cephalosporins, aminoglycosides, fluoroquinolones, and β-lactam/β-lactamase inhibitor combinations. Multiclass resistance was defined as nonsusceptibility to carbapenems and two or more additional classes. Odds of resistance were obtained using a logistic regression model with cubic splines. Carbapenem-associated multiclass resistance has had a 3.7-fold (95% confidence interval [CI] 3.4-4.3) increase from 20.6% in 2002 to 49.2% in 2008. Among blood isolates the increase was by 2.2 times (95% CI 1.7-2.9). Subjects <18 years old had significantly (p < 0.001) lower rates in 2002 (6.9%) than those 65 years or older (21.5%), but by 2008 this difference diminished as rates increased to 44.2% and 54.2%, respectively. A similar divergence was also observed between ICU and Ward, with no differences in 2002, whereas in 2008 ICU isolates had significantly higher rates (55.2%, 95% CI 53.6%-56.9%) than Ward isolates (45.6%, 95% CI 44.2%-47.0%). Over half of all A. baumannii-resistant isolates were carbapenem and multiclass resistant in 2008. Rates among subjects <18 years old have increased faster than those of the elderly, and in the ICU as compared to Ward.

History of Biological Warfare: Catapults to Capsomeres

Claims that biologcal agents have been used as weapons of war can be found in both the written records and the art work of many early civilizations. In fact, warring factions have used, threatened to use, or accused their enemies of using various forms of biological warfare (BW) throughout recorded hstory. Nevertheless hstorical documentation proving the use of biological warfare has always been sparse, and such information has been clouded in h s century by governments that are unwilling to admit to using biological warfare or to lift the cloak of secrecy surroundmg these activities. The murkiness of the historical record may discourage academic pursuit of the subject but does add a certain mystique to attempts to chronicle the history of biological warfare. Another factor that has inhibited careful historical analysis of biological warfare is the value-laden nature of the subject itself: it is difficult to discuss h s means of warfare without eliciting emotional responses, and as the other papers in this section demonstrate, these responses often color reports of the use of biologcal warfare.’P2 The fact that moral or ethical judgments almost always accompany consideration of biological warfare issues has discouraged discussion withm the one group that should be most interested in the subject-microbiologists. T h s review chronicles the hstory of biologcal warfare from 300 B.C. to the present, using examples from many published sources. We have divided the chronology into s k historic periods on the basis of techca l innovations that changed the nature of biological weapons. One sgdicant feature of this survey is that it relates exclusively to the use of viable organisms in biological warfare.3We have omitted references to use of bacterial toxins or related chemical derivatives of microorganisms because we believe they can better be dealt with as examples of chemical warfare.

A Comparative In Vitro Surveillance Study of Gemifloxacin Activities against 2,632 Recent Streptococcus pneumoniae Isolates from across Europe, North America, and South America

ABSTRACT From 1997 to 1999, 94 study centers in 15 European, 3 North American, and 2 South American countries contributed 2,632 isolates ofStreptococcus pneumoniae to an international antimicrobial susceptibility testing study. Only 62.0% of isolates were susceptible to penicillin, while 22.3% were penicillin intermediate and 15.6% were penicillin resistant. Resistance to trimethoprim-sulfamethoxazole (24.4%), azithromycin (26.0%), and clarithromycin (27.1%) was also highly prevalent. For the penicillin-resistant isolates (n = 411), the MICs at which 90% of isolates are inhibited (MIC90s) for gemifloxacin, levofloxacin, ofloxacin, clarithromycin, and azithromycin were 0.03, 1, 2, >16, and >64 μg/ml, respectively. Similarly, for isolates resistant to both azithromycin and clarithromycin (n = 649), gemifloxacin, levofloxacin, ofloxacin, and penicillin MIC90s were 0.03, 1, 2, and 4 μg/ml, respectively. Overall rates of resistance to trovafloxacin (0.3%), levofloxacin (0.3%), grepafloxacin (0.6%), and ofloxacin (0.7%) were low. For ofloxacin-intermediate and -resistant isolates (n = 142), gemifloxacin had the lowest MIC90 (0.12 μg/ml) compared to the MIC90s of trovafloxacin (0.5 μg/ml), grepafloxacin (1 μg/ml), and levofloxacin (2 μg/ml). For allS. pneumoniae isolates tested, gemifloxacin MICs were ≤0.5 μg/ml, suggesting that gemifloxacin has the potential to be used as a treatment for pneumococcal infections, including those arising from isolates resistant to β-lactams and macrolides.

Prevalence and Sequence Variation of Panton-Valentine Leukocidin in Methicillin-Resistant and Methicillin-Susceptible Staphylococcus aureus Strains in the United States

ABSTRACT Panton-Valentine leukocidin (PVL), encoded by the lukSF-PV genes, is a putative virulence factor and marker for community-associated methicillin-resistant Staphylococcus aureus. Here we report the prevalence of PVL among a representative sample of 1,055 S. aureus infection isolates from the United States and describe the sequence variation of the lukSF-PV genes. We performed multilocus sequence typing (MLST) on all isolates and sequenced fragments of the lukSF-PV genes from a sample of 86 isolates. We assigned isolates to a PVL R or H sequence type based on a polymorphism that results in an amino acid change from arginine (R) to histidine (H). Overall, we found that 36% of S. aureus isolates were positive for lukSF-PV. Among the 86 we typed, we identified 72 R variants and 14 H variants. Among the 47 methicillin-resistance S. aureus (MRSA) isolates, 43 harbored the R variant, and among the 39 methicillin-susceptible S. aureus (MSSA) isolates, 29 harbored the R variant. Almost all (97%) of the R variants were found in MLST clonal complex 8 (CC8), while the H variant was broadly distributed among 6 CCs. Within CC8, all 38 MRSA (USA300) and all 28 MSSA isolates harbored the R variant. Of the 20 isolates from blood and the lower respiratory tract, 19 (95%) harbored the R variant. While the R variant had been linked primarily to USA300 MRSA, we found that all CC8 MSSA isolates also contained the R variant, suggesting that some strains of USA300 may have lost methicillin resistance as an adaptation in the community.

Comparative in vitro activity of gemifloxacin, ciprofloxacin, levofloxacin and ofloxacin in a North American surveillance study

The in vitro activity of gemifloxacin, a new fluoroquinolone, was compared to three marketed fluoroquinolones; ciprofloxacin, levofloxacin and ofloxacin against over 4,000 recent clinical isolates covering 29 species isolated in the United States and Canada between 1997-1999. Based on MIC(90)s, gemifloxacin was the most potent fluoroquinolone tested against a majority of Gram-positive isolates: Streptococcus pneumoniae, penicillin resistant S. pneumoniae, macrolide resistant S. pneumoniae, ciprofloxacin non-susceptible (MIC > or = 4 microg/mL) S. pneumoniae, S. pyogenes, S. agalactiae, viridans streptococci, Enterococcus faecalis, methicillin susceptible Staphylococcus aureus, methicillin resistant S. aureus, S. epidermidis, S. hemolyticus, and S. saprophyticus. Against Enterobacteriaceae and aerobic non-Enterobacteriaceae Gram-negatives, gemifloxacin was usually comparable to ciprofloxacin and levofloxacin and more potent than ofloxacin for the following species: Citrobacter freundii, Enterobacter aerogenes, E. cloacae, Escherichia coli, Klebsiella oxytoca, Klebsiella pneumoniae, Morganella morganii, Proteus mirabilis, P. vulgaris, Providencia stuartii, Serratia marcescens, Acinetobacter lwoffii, A. baumannii, Burkholderia cepacia, Haemophilus influenzae, H. parainfluenzae, Moraxella catarrhalis, Pseudomonas aeruginosa, and Stenotrophomonas maltophilia. Gemifloxacin was generally 16-64 fold more potent than the other fluoroquinolones tested against Gram-positive organisms and retains excellent activity comparable with ciprofloxacin and levofloxacin against a majority of Gram-negative pathogens.