Showing papers in "Antimicrobial Agents and Chemotherapy in 2008"

Clinical and Economic Impact of Common Multidrug-Resistant Gram-Negative Bacilli

Abstract: During the last decade, the efforts to combat multidrug-resistant (MDR) microorganisms mainly focused on gram-positive bacteria, namely, methicillin-resistant Staphylococcus aureus and vancomycin-resistant enterococci. While a large number of hospitals have implemented more rigorous infection

Relationship between Vancomycin MIC and Failure among Patients with Methicillin-Resistant Staphylococcus aureus Bacteremia Treated with Vancomycin

Abstract: There is growing concern that vancomycin has diminished activity for methicillin-resistant Staphylococcus aureus (MRSA) infections, with vancomycin MICs at the high end of the CLSI susceptibility range. Despite this growing concern, there are limited clinical data to support this notion. To better elucidate this, a retrospective cohort study was conducted among patients with MRSA bloodstream infections who were treated with vancomycin between January 2005 and May 2007. The inclusion criteria were as follows: at least 18 years old, nonneutropenic, with an MRSA culture that met the CDC criteria for bloodstream infection, had received vancomycin therapy within 48 h of the index blood culture, and survived >24 h after vancomycin administration. Failure was defined as 30-day mortality, bacteremia >or=10 days on vancomycin therapy, or a recurrence of MRSA bacteremia within 60 days of vancomycin discontinuation. Classification and regression tree (CART) analysis identified the vancomycin MIC breakpoint associated with an increased probability of failure. During the study period, 92 patients met the inclusion criteria. The vancomycin MIC breakpoint derived by CART analysis was >or=1.5 mg/liter. The 66 patients with vancomycin MICs of >or=1.5 mg/liter had a 2.4-fold increase in failure compared to patients with MICs of or=1.5 mg/liter was independently associated with failure (adjusted risk ratio, 2.6; 95% confidence interval, 1.3 to 5.4; P = 0.01). These data strongly suggest that patients with MRSA bloodstream infections with vancomycin MICs of >or=1.5 mg/liter respond poorly to vancomycin. Alternative anti-MRSA therapies should be considered for these patients.

Larger Vancomycin Doses (at Least Four Grams per Day) Are Associated with an Increased Incidence of Nephrotoxicity

Abstract: Recent guidelines recommend vancomycin trough concentrations between 15 and 20 mg/liter. In response, some clinicians increased vancomycin dosing to >or=4 g/day. Scant data are available regarding toxicities associated with higher vancomycin doses. The purpose of this study was to examine vancomycin-associated nephrotoxicity at >or=4 g/day. To accomplish the study objective, a cohort study among a random selection of patients receiving vancomycin or linezolid between 2005 and 2006 was performed. Patients were included if they (i) were >or=18 years of age, (ii) were nonneutropenic, (iii) were on therapy for >48 h, (iv) had baseline serum creatinine levels of or=4 g/day), and linezolid. Nephrotoxicity was defined as a serum creatinine increase of 0.5 mg/dl or 50%, whichever was greater, after therapy initiation. Stratified Kaplan-Meier analysis and Cox modeling were used to compare times to nephrotoxicity across groups. During the study, 246 patients on vancomycin (26 patients taking >or=4 g/day and 220 patients taking or=4 g vancomycin/day, those receiving or=4 g vancomycin/day, a total body weight of >or=101.4 kg, estimated creatinine clearance of

Surveillance for Neuraminidase Inhibitor Resistance among Human Influenza A and B Viruses Circulating Worldwide from 2004 to 2008

Abstract: The surveillance of seasonal influenza virus susceptibility to neuraminidase (NA) inhibitors was conducted using an NA inhibition assay. The 50% inhibitory concentration values (IC 50 s) of 4,570 viruses collected globally from October 2004 to March 2008 were determined. Based on mean IC 50 s, A(H3N2) viruses (0.44 nM) were more sensitive to oseltamivir than A(H1N1) viruses (0.91 nM). The opposite trend was observed with zanamivir: 1.06 nM for A(H1N1) and 2.54 nM for A(H3N2). Influenza B viruses exhibited the least susceptibility to oseltamivir (3.42 nM) and to zanamivir (3.87 nM). To identify potentially resistant viruses (outliers), a threshold of a mean IC 50 value + 3 standard deviations was defined for type/subtype and drug. Sequence analysis of outliers was performed to identify NA changes that might be associated with reduced susceptibility. Molecular markers of oseltamivir resistance were found in six A(H1N1) viruses (H274Y) and one A(H3N2) virus (E119V) collected between 2004 and 2007. Some outliers contained previously reported mutations (e.g., I222T in the B viruses), while other mutations [e.g., R371K and H274Y in B viruses and H274N in A(H3N2) viruses) were novel. The R371K B virus outlier exhibited high levels of resistance to both inhibitors (>100 nM). A substantial variance at residue D151 was observed among A(H3N2) zanamivir-resistant outliers. The clinical relevance of newly identified NA mutations is unknown. A rise in the incidence of oseltamivir resistance in A(H1N1) viruses carrying the H274Y mutation was detected in the United States and in other countries in the ongoing 2007 to 2008 season. As of March 2008, the frequency of resistance among A(H1N1) viruses in the United States was 8.6% (50/579 isolates). The recent increase in oseltamivir resistance among A(H1N1) viruses isolated from untreated patients raises public health concerns and necessitates close monitoring of resistance to NA inhibitors.

Predictors of Carbapenem-Resistant Klebsiella pneumoniae Acquisition among Hospitalized Adults and Effect of Acquisition on Mortality

Abstract: Carbapenem-resistant Klebsiella pneumoniae (CRKP) is an emerging nosocomial pathogen. Little is known about its risk factors or mortality. We performed a case-case-control study to assess the risks for CRKP isolation and a retrospective cohort study to assess mortality in three groups of hospitalized adults: (i) patients from whom CRKP was isolated, (ii) patients from whom carbapenem-susceptible Klebsiella spp. (CSKS) were isolated, and (iii) controls from whom no Klebsiella spp. were isolated. After adjustment for length of stay (LOS), the demographics, comorbidities, and exposures of each case group were compared with those of the controls. Significant covariates were incorporated into LOS-adjusted multivariable models. In the mortality study, we evaluated the effect of CRKP on in-hospital death. There were 48 patients with CRKP isolation (21 died [44%]), 56 patients with CSKS isolation (7 died [12.5%]), and 59 controls (1 died [2%]). Independent risk factors for CRKP isolation were poor functional status (odds ratio [OR], 15.4; 95% confidence interval [CI], 4.0 to 58.6; P < 0.001); intensive care unit (ICU) stay (OR, 17.4; 95% CI, 1.5 to 201.9; P = 0.02); and receipt of antibiotics (OR, 4.4; 95% CI, 1.0 to 19.2; P = 0.05), particularly fluoroquinolones (OR, 7.2; 95% CI, 1.1 to 49.4; P = 0.04). CRKP was independently associated with death when patients with CRKP were compared with patients with CSKS (OR, 5.4; 95% CI, 1.7 to 17.1; P = 0.005) and with controls (OR, 6.7; 95% CI, 2.4 to 18.8; P < 0.001). After adjustment for the severity of illness, CRKP isolation remained predictive of death, albeit with a lower OR (for the CRKP group versus the CSKS group, OR, 3.9; 95% CI, 1.1 to 13.6; and P = 0.03; for the CRKP group versus the controls, OR, 5.0; 95% CI, 1.7 to 14.8; and P = 0.004). CRKP affects patients with poor functional status, an ICU stay, and antibiotic exposure and is an independent predictor of death.

Quinolone-mediated bacterial death.

Abstract: The fluoroquinolones are broad-spectrum antibacterial agents that are becoming increasingly popular as bacterial resistance erodes the effectiveness of other agents (fluoroquinolone sales accounted for 18% of the antibacterial market in 2006) (41). One of the attractive features of the quinolones is their ability to kill bacteria rapidly, an ability that differs widely among the various derivatives. For example, quinolones differ in rate and extent of killing, in the need for aerobic metabolism to kill cells, and in the effect of protein synthesis inhibitors on quinolone lethality. Understanding the mechanisms underlying these differences could lead to new ways for identifying the most bactericidal quinolone derivatives. Before describing the types of damage caused by the quinolones, it is useful to define lethal activity. Operationally, it is the ability of drug treatment to reduce the number of viable cells, usually measured as CFU on drug-free agar after treatment. This assay is distinct from measurements that detect inhibition of growth (e.g., MIC), since with the latter bacteria are exposed to drug throughout the measurement. The distinction between killing and blocking growth is important because it allows susceptibility determinations to be related to particular biological processes. For example, inhibition of growth is typically reversed by the removal of drug, while cell death is not. Thus, biochemical events associated with blocking growth should be readily reversible, while those responsible for cell death should be difficult to reverse. Reversibility can be used to distinguish among quinolone derivatives and assign functions to particular aspects of drug structure. Moreover, protective functions, such as repair and stress responses, can be distinguished by whether their absence affects inhibition of growth, killing, or both. The intracellular targets of the quinolones are two DNA topoisomerases: gyrase and topoisomerase IV. Gyrase tends to be the primary target in gram-negative bacteria, while topoisomerase IV is preferentially inhibited by most quinolones in gram-positive organisms (28). Both enzymes use a double-strand DNA passage mechanism, and it is likely that quinolone biochemistry is similar for both. However, physiological differences between the enzymes exist, some of which may bear on quinolone lethality. In the present minireview we consider cell death through a two-part “poison” hypothesis in which the quinolones form reversible drug-topoisomerase-DNA complexes that subsequently lead to several types of irreversible (lethal) damage. Other consequences of quinolone treatment, such as depletion of gyrase and topoisomerase IV activity, are probably less immediate (42). To provide a framework for considering quinolone lethality, we begin by briefly describing the drug-topoisomerase-DNA complexes. Readers interested in a more comprehensive discussion of quinolones are referred to a previously published work (28).

Genetic Structures at the Origin of Acquisition of the β-Lactamase blaKPC Gene

Abstract: Genetic structures surrounding the carbapenem-hydrolyzing Ambler class A bla KPC gene were characterized in several KPC-positive Klebsiella pneumoniae and Pseudomonas aeruginosa strains isolated from the United States, Colombia, and Greece. The bla KPC genes were associated in all cases with transposon-related structures. In the K. pneumoniae YC isolate from the United States, the β-lactamase bla KPC-2 gene was located on a novel Tn 3 -based transposon, Tn 4401 . Tn 4401 was 10 kb in size, was delimited by two 39-bp imperfect inverted repeat sequences, and harbored, in addition to the β-lactamase bla KPC-2 gene, a transposase gene, a resolvase gene, and two novel insertion sequences, IS Kpn6 and IS Kpn7 . Tn 4401 has been identified in all isolates. However, two isoforms of this transposon were found: Tn 4401 a was found in K. pneumoniae YC and K. pneumoniae GR from the United States and Greece, respectively, and differed by a 100-bp deletion, located just upstream of the bla KPC-2 gene, compared to the sequence of Tn 4401 b, which was found in the Colombian isolates. In all isolates tested, Tn 4401 was flanked by a 5-bp target site duplication, the signature of a recent transposition event, and was inserted in different open reading frames located on plasmids that varied in size and nature. Tn 4401 is likely at the origin of carbapenem-hydrolyzing β-lactamase KPC mobilization to plasmids and its further insertion into various-sized plasmids identified in nonclonally related K. pneumoniae and P. aeruginosa isolates.

Novel Carbapenem-Hydrolyzing β-Lactamase, KPC-1, from a Carbapenem-Resistant Strain of.

Abstract: Hospital Infections Program, National Center for Infectious Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia 30333; The R. W. Johnson Pharmaceutical Research Institute, Raritan, New Jersey 08869; and Unidad de Investigacion, Hospital Son Dureta, Andrea Doria, Palma de Mallorca, 07014, and Área de Microbiologia, Universidad de las Islas Baleares, Crtra. Valldemosa, Palma de Mallorca, 07071, Spain

Inhibition of quorum sensing-controlled virulence factor production in Pseudomonas aeruginosa by South Florida plant extracts.

Abstract: Quorum sensing (QS) is a key regulator of virulence and biofilm formation in Pseudomonas aeruginosa and other medically relevant bacteria. Aqueous extracts of six plants, Conocarpus erectus, Chamaesyce hypericifolia, Callistemon viminalis, Bucida buceras, Tetrazygia bicolor, and Quercus virginiana, were examined in this study for their effects on P. aeruginosa virulence factors and the QS system. C. erectus, B. buceras, and C. viminalis caused a significant inhibition of LasA protease, LasB elastase, pyoverdin production, and biofilm formation. Additionally, each plant presented a distinct effect profile on the las and rhl QS genes and their respective signaling molecules, suggesting that different mechanisms are responsible for efficacy. Extracts of all plants caused the inhibition of QS genes and QS-controlled factors, with marginal effects on bacterial growth, suggesting that the quorum-quenching mechanisms are unrelated to static or cidal effects.

Effects of Antibiotics on Quorum Sensing in Pseudomonas aeruginosa

Abstract: During infection, Pseudomonas aeruginosa employs bacterial communication (quorum sensing [QS]) to coordinate the expression of tissue-damaging factors. QS-controlled gene expression plays a pivotal role in the virulence of P. aeruginosa, and QS-deficient mutants cause less severe infections in animal infection models. Treatment of cystic fibrosis (CF) patients chronically infected with P. aeruginosa with the macrolide antibiotic azithromycin (AZM) has been demonstrated to improve the clinical outcome. Several studies indicate that AZM may accomplish its beneficial action in CF patients by impeding QS, thereby reducing the pathogenicity of P. aeruginosa. This led us to investigate whether QS inhibition is a common feature of antibiotics. We present the results of a screening of 12 antibiotics for their QS-inhibitory activities using a previously described QS inhibitor selector 1 strain. Three of the antibiotics tested, AZM, ceftazidime (CFT), and ciprofloxacin (CPR), were very active in the assay and were further examined for their effects on QS-regulated virulence factor production in P. aeruginosa. The effects of the three antibiotics administered at subinhibitory concentrations were investigated by use of DNA microarrays. Consistent results from the virulence factor assays, reverse transcription-PCR, and the DNA microarrays support the finding that AZM, CFT, and CPR decrease the expression of a range of QS-regulated virulence factors. The data suggest that the underlying mechanism may be mediated by changes in membrane permeability, thereby influencing the flux of N-3-oxo-dodecanoyl-L-homoserine lactone.

AdeIJK, a Resistance-Nodulation-Cell Division Pump Effluxing Multiple Antibiotics in Acinetobacter baumannii

Abstract: We have identified a second resistance-nodulation-cell division (RND)-type efflux pump, AdeIJK, in clinical isolate Acinetobacter baumannii BM4454. The adeI, adeJ , and adeK genes encode, respectively, the membrane fusion, RND, and outer membrane components of the pump. AdeJ belongs to the AcrB protein family (57% identity with AcrB from Escherichia coli ). mRNA analysis by Northern blotting and reverse transcription-PCR indicated that the genes were cotranscribed. Overexpression of the cloned adeIJK operon was toxic in both E. coli and Acinetobacter . The adeIJK genes were detected in all of the 60 strains of A. baumannii tested. The two latter observations suggest that the AdeIJK complex might contribute to intrinsic but not to acquired antibiotic resistance in Acinetobacter . To characterize the substrate specificity of the pump, we have constructed derivatives of BM4454 in which adeIJK (strain BM4579), adeABC (strain BM4561), or both groups of genes (strain BM4652) were inactivated by deletion-insertion. Determination of the antibiotic susceptibility of these strains and of BM4652 and BM4579, in which the adeIJK operon was provided in trans , indicated that the AdeIJK pump contributes to resistance to β-lactams, chloramphenicol, tetracycline, erythromycin, lincosamides, fluoroquinolones, fusidic acid, novobiocin, rifampin, trimethoprim, acridine, safranin, pyronine, and sodium dodecyl sulfate. The chemical structure of these molecules suggests that amphiphilic compounds are the preferred substrates. The AdeABC and AdeIJK efflux systems contributed in a more than additive fashion to tigecycline resistance.

Failures in Clinical Treatment of Staphylococcus aureus Infection with Daptomycin Are Associated with Alterations in Surface Charge, Membrane Phospholipid Asymmetry, and Drug Binding

Abstract: Increasingly frequent reports have described the in vivo loss of daptomycin susceptibility in association with clinical treatment failures. The mechanism(s) of daptomycin resistance is not well understood. We studied an isogenic set of Staphylococcus aureus isolates from the bloodstream of a daptomycin-treated patient with recalcitrant endocarditis in which serial strains exhibited decreasing susceptibility to daptomycin. Since daptomycin is a membrane-targeting lipopeptide, we compared a number of membrane parameters in the initial blood isolate (parental) with those in subsequent daptomycin-resistant strains obtained during treatment. In comparison to the parental strain, resistant isolates demonstrated (i) enhanced membrane fluidity, (ii) increased translocation of the positively charged phospholipid lysyl-phosphotidylglycerol to the outer membrane leaflet, (iii) increased net positive surface charge (P < 0.05 versus the parental strain), (iv) reduced susceptibility to daptomycin-induced depolarization, permeabilization, and autolysis (P < 0.05 versus the parental strain), (v) significantly lower surface binding of daptomycin (P < 0.05 versus the parental strain), and (vi) increased cross-resistance to the cationic antimicrobial host defense peptides human neutrophil peptide 1 (hNP-1) and thrombin-induced platelet microbicidal protein 1 (tPMP-1). These data link distinct changes in membrane structure and function with in vivo development of daptomycin resistance in S. aureus. Moreover, the cross-resistance to hNP-1 and tPMP-1 may also impact the capacity of these daptomycin-resistant organisms to be cleared from sites of infection, particularly endovascular foci.

Mutations Associated with Failure of Raltegravir Treatment Affect Integrase Sensitivity to the Inhibitor In Vitro

Abstract: Raltegravir (MK-0518) is a potent inhibitor of human immunodeficiency virus (HIV) integrase and is clinically effective against viruses resistant to other classes of antiretroviral agents. However, it can select mutations in the HIV integrase gene. Nine heavily pretreated patients who received salvage therapy including raltegravir and who subsequently developed virological failure under raltegravir therapy were studied. For each patient, the sequences of the integrase-coding region were determined and compared to that at the beginning of the treatment. Four different mutation profiles were identified in these nine patients: E92Q, G140S Q148H, N155H, and E157Q mutations. For four patients, each harboring a different profile, the wild-type and mutated integrases were produced, purified, and assayed in vitro. All the mutations identified altered the activities of integrase protein: both 3′ processing and strand transfer activities were moderately affected in the E92Q mutant; strand transfer was markedly impaired in the N155H mutant; both activities were strongly impaired in the G140S Q148H mutant; and the E157Q mutant was almost completely inactive. The sensitivities of wild-type and mutant integrases to raltegravir were compared. The E92Q and G140S Q148H profiles were each associated with a 7- to 8-fold decrease in sensitivity, and the N155H mutant was more than 14-fold less sensitive to raltegravir. At least four genetic profiles (E92Q, G140S Q148H, N155H, and E157Q) can be associated with in vivo treatment failure and resistance to raltegravir. These mutations led to strong impairment of enzymes in vitro in the absence of raltegravir: strand transfer activity was affected, and in some cases 3′ processing was also impaired.

Role of Global Regulators and Nucleotide Metabolism in Antibiotic Tolerance in Escherichia coli

Abstract: Bacterial populations produce a small number of persister cells that exhibit multidrug tolerance. Persister cells are largely responsible for the antibiotic recalcitrance of biofilm infections. The mechanism of persister cell formation largely remains unknown due to the challenges in identifying persister genes. We screened an ordered comprehensive library of 3,985 Escherichia coli knockout strains to identify mutants with altered antibiotic tolerance. Stationary-state cultures in 96-well plates were exposed to ofloxacin at a concentration which allows only tolerant persister cells to survive. The persister cell level of each culture was determined. A total of 150 mutants with decreased persistence were identified in the initial screen, and subsequent validation confirmed that neither the growth rate nor the ofloxacin MIC was affected for 10 of them. The genes affected in these strains were dnaJ and dnaK (chaperones), apaH (diadenosine tetraphosphatase), surA (peptidyl-prolyl cis-trans isomerase), fis and hns (global regulators), hnr (response regulator of RpoS), dksA (transcriptional regulator of rRNA transcription), ygfA (5-formyl-tetrahydrofolate cyclo-ligase), and yigB (flavin mononucleotide [FMN] phosphatase). The prominent presence of global regulators among these strains pointed to the likely redundancy of persister cell formation mechanisms: the elimination of a regulator controlling several redundant persister genes would be expected to produce a phenotype. This observation is consistent with previous findings for a possible role of redundant genes such as toxin/antitoxin modules in persister cell formation. ygfA and yigB were of special interest. The mammalian homolog of YgfA (methenyltetrahydrofolate synthetase) catalyzes the conversion of 5-formyl-tetrahydrofolate (THF) into the rapidly degraded 5,10-methenyl-THF, depleting the folate pool. The YigB protein is a phosphatase of FMN which would deplete the pool of this cofactor. Stochastic overexpression of these genes could lead to dormancy and, hence, tolerance by depleting the folate and FMN pools, respectively. Consistent with this scenario, the overexpression of both genes produced increased tolerance to ofloxacin.

Outcome of Vancomycin Treatment in Patients with Methicillin-Susceptible Staphylococcus aureus Bacteremia

Abstract: Limited data on the clinical outcome of vancomycin treatment compared with that of beta-lactam treatment in patients with methicillin-susceptible Staphylococcus aureus bacteremia (MSSA-B) are available. We used different and complementary approaches: (i) a retrospective cohort study using a propensity score to adjust for confounding by treatment assignment and (ii) a matched case-control study. Of all patients with S. aureus bacteremia (SAB) in two university-affiliated hospitals over a 7-year period, 294 patients with MSSA-B were enrolled in the cohort study. The cases for the case-control study were defined as patients who received vancomycin treatment for MSSA-B; the controls, who were patients that received beta-lactam treatment for MSSA-B, were selected at a 1:2 (case:control) ratio according to the objective matching scoring system and the propensity score system. In the cohort study, SAB-related mortality in patients with vancomycin treatment (37%, 10/27) was significantly higher than that in those with beta-lactam treatment (18%, 47/267) (P = 0.02). In addition, multivariate analysis revealed that vancomycin treatment was associated with SAB-related mortality when independent predictors for SAB-related mortality and propensity score were considered (adjusted odds ratio of 3.3, 95% confidence interval of 1.2 to 9.5). In the case-control study using the objective matching scoring system and the propensity score system, SAB-related mortality in case patients was 37% (10/27) and in control patients 11% (6/54) (P < 0.01). Our data suggest that vancomycin is inferior to beta-lactam in the treatment of MSSA-B.

Antimicrobial-resistant pathogens in intensive care units in Canada: results of the Canadian National Intensive Care Unit (CAN-ICU) study, 2005-2006.

Abstract: Between 1 September 2005 and 30 June 2006, 19 medical centers collected 4,180 isolates recovered from clinical specimens from patients in intensive care units (ICUs) in Canada. The 4,180 isolates were collected from 2,292 respiratory specimens (54.8%), 738 blood specimens (17.7%), 581 wound/tissue specimens (13.9%), and 569 urinary specimens (13.6%). The 10 most common organisms isolated from 79.5% of all clinical specimens were methicillin-susceptible Staphylococcus aureus (MSSA) (16.4%), Escherichia coli (12.8%), Pseudomonas aeruginosa (10.0%), Haemophilus influenzae (7.9%), coagulase-negative staphylococci/Staphylococcus epidermidis (6.5%), Enterococcus spp. (6.1%), Streptococcus pneumoniae (5.8%), Klebsiella pneumoniae (5.8%), methicillin-resistant Staphylococcus aureus (MRSA) (4.7%), and Enterobacter cloacae (3.9%). MRSA made up 22.3% (197/884) of all S. aureus isolates (90.9% of MRSA were health care-associated MRSA, and 9.1% were community-associated MRSA), while vancomycin-resistant enterococci (VRE) made up 6.7% (11/255) of all enterococcal isolates (88.2% of VRE had the vanA genotype). Extended-spectrum beta-lactamase (ESBL)-producing E. coli and K. pneumoniae occurred in 3.5% (19/536) and 1.8% (4/224) of isolates, respectively. All 19 ESBL-producing E. coli isolates were PCR positive for CTX-M, with bla CTX-M-15 occurring in 74% (14/19) of isolates. For MRSA, no resistance against daptomycin, linezolid, tigecycline, and vancomycin was observed, while the resistance rates to other agents were as follows: clarithromycin, 89.9%; clindamycin, 76.1%; fluoroquinolones, 90.1 to 91.8%; and trimethoprim-sulfamethoxazole, 11.7%. For E. coli, no resistance to amikacin, meropenem, and tigecycline was observed, while resistance rates to other agents were as follows: cefazolin, 20.1%; cefepime, 0.7%; ceftriaxone, 3.7%; gentamicin, 3.0%; fluoroquinolones, 21.1%; piperacillin-tazobactam, 1.9%; and trimethoprim-sulfamethoxazole, 24.8%. Resistance rates for P. aeruginosa were as follows: amikacin, 2.6%; cefepime, 10.2%; gentamicin, 15.2%; fluoroquinolones, 23.8 to 25.5%; meropenem, 13.6%; and piperacillin-tazobactam, 9.3%. A multidrug-resistant (MDR) phenotype (resistance to three or more of the following drugs: cefepime, piperacillin-tazobactam, meropenem, amikacin or gentamicin, and ciprofloxacin) occurred frequently in P. aeruginosa (12.6%) but uncommonly in E. coli (0.2%), E. cloacae (0.6%), or K. pneumoniae (0%). In conclusion, S. aureus (MSSA and MRSA), E. coli, P. aeruginosa, H. influenzae, Enterococcus spp., S. pneumoniae, and K. pneumoniae are the most common isolates recovered from clinical specimens in Canadian ICUs. A MDR phenotype is common for P. aeruginosa isolates in Canadian ICUs.

A naturally occurring proline-to-alanine amino acid change in Fks1p in Candida parapsilosis, Candida orthopsilosis, and Candida metapsilosis accounts for reduced echinocandin susceptibility.

Abstract: Candida parapsilosis has emerged as a common cause of invasive fungal infection, especially in Latin America and in the neonatal setting. C. parapsilosis is part of a closely related group of organisms that includes the species Candida orthopsilosis and Candida metapsilosis. All three species show elevated MICs for the new echinocandin class drugs caspofungin, micafungin, and anidulafungin relative to other Candida species. Despite potential impacts on therapy, the mechanism behind this reduced echinocandin susceptibility has not been determined. In this report, we investigated the role of a naturally occurring Pro-to-Ala substitution at amino acid position 660 (P660A), immediately distal to the highly conserved hot spot 1 region of Fks1p, in the reduced-echinocandin-susceptibility phenotype. Kinetic inhibition studies demonstrated that glucan synthase from the C. parapsilosis group was 1 to 2 logs less sensitive to echinocandin drugs than the reference enzyme from C. albicans. Furthermore, clinical isolates of C. albicans and C. glabrata which harbor mutations at this equivalent position also showed comparable 2-log decreases in target enzyme sensitivity, which correlated with increased MICs. These mutations also resulted in 2.4- to 18.8-fold-reduced Vmax values relative to those for the wild-type enzyme, consistent with kinetic parameters obtained for C. parapsilosis group enzymes. Finally, the importance of the P660A substitution for intrinsic resistance was confirmed by engineering an equivalent P647A mutation into Fks1p of Saccharomyces cerevisiae. The mutant glucan synthase displayed characteristic 2-log decreases in sensitivity to the echinocandin drugs. Overall, these data firmly indicate that a naturally occurring P660A substitution in Fks1p from the C. parapsilosis group accounts for the reduced susceptibility phenotype.

Molecular Characterization and Epidemiology of Extended-Spectrum- β-Lactamase-Producing Escherichia coli and Klebsiella pneumoniae Isolates Causing Health Care-Associated Infection in Thailand, Where the CTX-M Family Is Endemic

Abstract: Extended-spectrum β-lactamase (ESBL)-producing Escherichia coli and Klebsiella pneumoniae have rapidly spread worldwide and pose a serious threat for health care-associated (HA) infection. We conducted molecular detection and characterization of ESBL-related bla genes, including bla TEM , bla SHV , bla CTX-M , bla VEB , bla OXA , bla PER , and bla GES , among 362 isolates of ESBL-producing E. coli ( n = 235) and ESBL-producing K. pneumoniae ( n = 127) collected from patients who met the definition of HA infection at two major university hospitals in Thailand from December 2004 to May 2005. The prevalence of ESBL-producing E. coli and ESBL-producing K. pneumoniae , patient demographics and the susceptibilities of these bacteria to various antimicrobial agents were described. A total of 87.3% of isolates carried several bla genes. The prevalence of bla CTX-M was strikingly high: 99.6% for ESBL-producing E. coli (CTX-M-14, -15, -27, -40, and -55) and 99.2% for ESBL-producing K. pneumoniae (CTX-M-3, -14, -15, -27, and -55). IS Ecp1 was found in the upstream region of bla CTX-M in most isolates. Up to 77.0% and 71.7% of ESBL-producing E. coli and ESBL-producing K. pneumoniae , respectively, carried bla TEM ; all of them encoded TEM-1. ESBL-producing K. pneumoniae carried bla SHV at 87.4% (SHV-1, -2a, -11, -12, -27, -71, and -75) but only at 3.8% for ESBL-producing E. coli (SHV-11 and -12). bla genes encoding VEB-1 and OXA-10 were found in both ESBL-producing E. coli (8.5% and 8.1%, respectively) and ESBL-producing K. pneumoniae (10.2% and 11.8%, respectively). None of the isolates were positive for bla PER and bla GES . Pulsed-field gel electrophoresis analysis demonstrated that there was no major clonal relationship among these ESBL producers. This is the first study to report CTX-M-3, CTX-M-27, CTX-M-40, SHV-27, SHV-71, and SHV-75 in Thailand and to show that CTX-M ESBL is highly endemic in the country.

Whole-Genome Pyrosequencing of an Epidemic Multidrug-Resistant Acinetobacter baumannii Strain Belonging to the European Clone II Group

Abstract: The whole-genome sequence of an epidemic, multidrug-resistant Acinetobacter baumannii strain (strain ACICU) belonging to the European clone II group and carrying the plasmid-mediated bla(OXA)(-)(58) carbapenem resistance gene was determined. The A. baumannii ACICU genome was compared with the genomes of A. baumannii ATCC 17978 and Acinetobacter baylyi ADP1, with the aim of identifying novel genes related to virulence and drug resistance. A. baumannii ACICU has a single chromosome of 3,904,116 bp (which is predicted to contain 3,758 genes) and two plasmids, pACICU1 and pACICU2, of 28,279 and 64,366 bp, respectively. Genome comparison showed 86.4% synteny with A. baumannii ATCC 17978 and 14.8% synteny with A. baylyi ADP1. A conspicuous number of transporters belonging to different superfamilies was predicted for A. baumannii ACICU. The relative number of transporters was much higher in ACICU than in ATCC 17978 and ADP1 (76.2, 57.2, and 62.5 transporters per Mb of genome, respectively). An antibiotic resistance island, AbaR2, was identified in ACICU and had plausibly evolved by reductive evolution from the AbaR1 island previously described in multiresistant strain A. baumannii AYE. Moreover, 36 putative alien islands (pAs) were detected in the ACICU genome; 24 of these had previously been described in the ATCC 17978 genome, 4 are proposed here for the first time and are present in both ATCC 17978 and ACICU, and 8 are unique to the ACICU genome. Fifteen of the pAs in the ACICU genome encode genes related to drug resistance, including membrane transporters and ex novo acquired resistance genes. These findings provide novel insight into the genetic basis of A. baumannii resistance.

Treatment of Scedosporiosis with Voriconazole: Clinical Experience with 107 Patients

Abstract: The efficacy of voriconazole in 107 patients with scedosporiosis was analyzed. Principal infection sites were the lungs/sinuses (24%), central nervous system (CNS) (20%), and bone (18%), while 21% of patients had disseminated infection. Solid organ transplantation (22%), hematological malignancy (21%), and surgery/trauma (15%) were the predominant underlying conditions. A successful therapeutic response was achieved in 57% of patients (median, 103 therapy days), with > 98% of those responding receiving > or = 28 days of therapy. Patients receiving primary therapy showed a 61% response versus 56% for the others. The best therapeutic responses were seen for skin/subcutaneous (91%) or bone (79%) infections, and the lowest for CNS infections (43%). Patients without major immune suppression (72%) or those with solid organ transplantation (63%) or various hematological conditions (60%) showed the best responses by underlying condition. Median known survival time was 133 days (therapy successes, 252 days; failures, 21 days). In all, 43 (40%) patients died, 73% due to scedosporiosis. Patients with Scedosporium prolificans infection had significantly reduced survival times (P = 0.0259) and were more likely to die from fungal infection (P = 0.002) than were Scedosporium apiospermum-infected patients. In a subset of 43 patients where voriconazole baseline MICs were available, response to voriconazole was higher for S. apiospermum-infected patients (54% response; MIC(50), 0.25 microg/ml) than for S. prolificans-infected patients (40% response; MIC(50), 4.0 microg/ml). Voriconazole demonstrated clinically useful activity in the treatment of both S. apiospermum and S. prolificans infections and was well tolerated.

First Molecular Characterization of Group B Streptococci with Reduced Penicillin Susceptibility

Abstract: Group B streptococci (GBS; Streptococcus agalactiae) are the leading cause of neonatal invasive diseases and are also important pathogens for adults. Penicillins are the drugs of first choice for the treatment of GBS infections, since GBS have been regarded to be uniformly susceptible to penicillins so far. Here we characterize the first strains of GBS with reduced penicillin susceptibility (PRGBS) identified in Japan. Fourteen PRGBS strains were clinically isolated from the sputa of elderly patients from 1995 to 2005; and the MICs of penicillin, oxacillin, and ceftizoxime ranged from 0.25 to 1 μg/ml, 2 to 8 μg/ml, and 4 to 128 μg/ml, respectively. Moreover, some strains were also insusceptible to ampicillin, cefazolin, cefepime, and cefotaxime. All the PRGBS isolates tested possessed a few amino acid substitutions adjacent to the conserved SSN and KSG motifs (amino acids 402 to 404 and 552 to 554, respectively) of PBP 2X, and the amino acid substitutions could be classified into two types, Q557E and V405A. Western blotting analysis of the 14 clinical isolates with anti-PBP 2X-specific serum suggested that the amount of PBP 2X among the 14 PRGBS isolates was reduced, although the 2 ATCC strains produced a significant amount of PBP 2X. The introduction of PRGBS-derived PBP 2X genes into penicillin-susceptible strains through allelic exchange elevated their penicillin insusceptibility, suggesting that these altered PBP 2X genes are responsible for the penicillin insusceptibility in PRGBS strains. In this study, we characterized for the first time PRGBS strains on a molecular basis, although several reports have so far mentioned the existence of β-lactam-insusceptible GBS from a phenotypic standpoint.

Early Bactericidal Activity and Pharmacokinetics of the Diarylquinoline TMC207 in Treatment of Pulmonary Tuberculosis

Abstract: Tibotec Medicinal Compound 207 (TMC207) is a novel diarylquinoline with a unique mode of action that targets mycobacterial ATP synthase. TMC207 exhibits high in vitro activity against mycobacterial strains either susceptible or resistant to all first-line and many second-line drugs, including fluoroquinolones, and has shown exceptional in vivo activity against several mycobacterial species in different animal models. In this early bactericidal activity study, 75 treatment-naive patients with smear-positive pulmonary tuberculosis were randomized to once-daily oral TMC207 (25 mg, 100 mg, or 400 mg), 600 mg rifampin (RIF), or 300 mg isoniazid (INH) for 7 days. Sixteen-hour overnight sputum collected at baseline and on each treatment day was plated in serial dilutions on selective agar plates. The bactericidal activity was expressed as the log(10) decrease in CFU/ml sputum/day. Pharmacokinetic sampling was performed on day 7 of TMC207 administration up to 24 h postdose. The decreases in log(10) CFU counts (+/- standard deviation) from baseline to day 7 were 0.04 +/- 0.46 for 25 mg TMC207 (n = 14), 0.26 +/- 0.64 for 100 mg TMC207 (n = 14), 0.77 +/- 0.58 for 400 mg TMC207 (n = 14), 1.88 +/- 0.74 for INH (n = 11), and 1.70 +/- 0.71 for RIF (n = 14). Significant bactericidal activity of 400 mg TMC207 was observed from day 4 onward and was similar in magnitude to those of INH and RIF over the same period. The pharmacokinetics of TMC207 were linear across the dose range. In summary, TMC207 demonstrated bactericidal activity with a delayed onset and was well tolerated, and no study drug-related serious adverse events occurred.

Interpretation of antibiotic concentration ratios measured in epithelial lining fluid

Abstract: In spite of the development and wide use of antibiotics, pneumonia is still the leading cause of infection-related mortality worldwide (101), and antibiotic resistance in the major pathogens of pneumonia has become more frequent during the past several decades. In order to defeat and prevent antibiotic resistance, antibiotics need to be used based on pharmacokinetics (PK) and pharmacodynamics (PD) (6, 10, 35). With regard to the PK/PD of antibiotics, considerable effort has been devoted to directly measure the concentrations of antibiotics at infection sites, because the distributions of antibiotics may be different among a variety of tissues. However, even beyond the fact that these measurements are carried out in normal tissues, the techniques used for the measurement are variable in accuracy and reproducibility and the interpretation of their results is hindered by many confounding factors (74, 75). For pulmonary infections, concentrations of antibiotics in epithelial lining fluid (ELF) for extracellular pathogens and in alveolar macrophage (AM) cells for intracellular pathogens are thought to reflect antibiotic activity in pneumonia. Antibiotics whose concentrations are high at these extravascular sites, such as macrolides and fluoroquinolones, tend to be promoted for treatment of pulmonary infection over antibiotics like betalactams and aminoglycosides, even though clinical trials do not show differences in clinical outcome or even bacteriological response. In fact, it is less clear why the ratios of ELF to plasma concentrations are diverse between antibiotics and even between members of the same antibiotic class. The measured ELF-to-plasma concentration ratios may differ based on physicochemical characteristics intrinsic to the molecules. And also, as the ELF concentration of antibiotics is commonly measured by bronchoalveolar lavage (BAL), technical factors or errors in the method of measurement may create these differences. It is believed that these factors need to be clarified before the concept of ELF concentration should be connected to antibiotic outcomes such as bacterial eradication or clinical response. In the current review, data from published human studies were extracted and analyzed to interpret ELF concentrations of antibiotics measured by BAL, considering possible confounding factors.

Both Oral Metronidazole and Oral Vancomycin Promote Persistent Overgrowth of Vancomycin-Resistant Enterococci during Treatment of Clostridium difficile-Associated Disease

Abstract: For treatment of mild to moderate Clostridium difficile-associated disease (CDAD), oral metronidazole has been recommended as the preferred agent, in part due to concern that vancomycin may be more likely to promote colonization by vancomycin-resistant enterococci (VRE). We performed a prospective observational study to examine the effects of oral metronidazole or vancomycin treatment of CDAD on acquisition and concentration of VRE stool colonization. Before, during, and after 90 courses of CDAD therapy, stool samples were cultured for VRE, and the concentrations were quantified. Eighty-seven subjects (97%) had received antibiotics within the past month. For 56 treatment courses in which preexisting VRE colonization was present, metronidazole (n = 37 courses) and vancomycin (n = 19 courses), each promoted persistent VRE overgrowth during therapy, and the concentration decreased significantly in both groups by ∼2 weeks after completion of treatment (P <0.049). For 34 treatment courses in which baseline cultures were negative for VRE, new detection of VRE stool colonization occurred during 3 (14%) of the 22 courses of metronidazole and 1 (8%) of the 12 courses of vancomycin (P = 1.0). These results demonstrate that both oral metronidazole and oral vancomycin promote the overgrowth of VRE during treatment of CDAD. New CDAD treatments are needed that are less likely to disrupt the intestinal microflora and promote overgrowth of healthcare-associated pathogens.

Aspergillus section Fumigati: antifungal susceptibility patterns and sequence-based identification.

Abstract: This study analyzed 28 Aspergillus strains belonging to the section Fumigati that were isolated from clinical samples in Spain. All isolates sporulated slowly and were unable to grow at 48°C. Phylogenetic analysis based on sequencing of partial sequences of the β-tubulin and rodlet A genes was used to classify the 28 strains into six different clades ( Neosartorya hiratsukae , Neosartorya pseudofischeri , Aspergillus viridinutans , Aspergillus lentulus , Aspergillus fumigatiaffinis , and Aspergillus fumisynnematus ). Antifungal susceptibility testing showed heterogeneous patterns and grouped the strains together by species. Most A. lentulus and A. fumigatiaffinis isolates showed high MICs of amphotericin B (geometric mean [GM] MICs, ≥4.5 μg/ml), itraconazole (GM MICs, ≥6 μg/ml), voriconazole (GM MICs, ≥3 μg/ml), and ravuconazole (GM MICs, ≥3 μg/ml); N pseudofischeri and A. viridinutans showed high MICs of itraconazole (GM MICs, ≥8 μg/ml), voriconazole (GM MICs, ≥3.33 μg/ml), and ravuconazole (GM MICs, ≥2 μg/ml); and N. hiratsukae and A. fumisynnematus were susceptible to all the antifungals tested. In conclusion, a number of different species whose morphological features resemble those of Aspergillus fumigatus could succeed in producing invasive infections in the susceptible host. In addition, some of them showed high MICs for most of the antifungals available for the treatment of patients infected with these organisms. The epidemiology and clinical relevance of these species should therefore be addressed.

Plasmid-Mediated qepA Gene among Escherichia coli Clinical Isolates from Japan

Abstract: Seven hundred fifty-one Escherichia coli clinical isolates collected from 140 Japanese hospitals between 2002 and 2006 were screened for the qepA and qnr genes. Two E. coli isolates (0.3%) harbored qepA, but no qnr was identified. The results suggested a low prevalence of E. coli harboring qepA or qnr in Japan.

Antimicrobial Resistance of Old and Recent Staphylococcus aureus Isolates from Poultry: First Detection of Livestock-Associated Methicillin-Resistant Strain ST398

Abstract: The susceptibilities of 12 antimicrobial agents for two collections of Staphylococcus aureus, isolated in the 1970s and in 2006 from poultry, were determined. For eight antibiotics, the percentage of resistance was significantly higher in the recent isolates. Ten recent isolates were methicillin resistant and had spa types t011 and t567, belonging to multilocus sequence type 398. This is the first report of "livestock-associated" methicillin resistant S. aureus from healthy poultry.

Spread of OXA-48-positive carbapenem-resistant Klebsiella pneumoniae isolates in Istanbul, Turkey.

Abstract: The first outbreak of carbapenem-resistant Klebsiella pneumoniae isolates producing the plasmid-encoded carbapenem-hydrolyzing oxacillinase OXA-48 is reported. The 39 isolates belonged to two different clones and were collected at the University Hospital of Istanbul, Turkey, from May 2006 to February 2007, and they coproduced various β-lactamases (SHV-12, OXA-9, and TEM-1 for clone A and CTX-M-15, TEM-1, and OXA-1 for clone B).

First Report of cfr-Mediated Resistance to Linezolid in Human Staphylococcal Clinical Isolates Recovered in the United States

Abstract: Linezolid resistance has dominantly been mediated by mutations in 23S rRNA or ribosomal protein L4 genes. Recently, cfr has demonstrated the ability to produce a phenotype of resistance to not only oxazolidinones, but also other antimicrobial classes (phenicols, lincosamides, pleuromutilins, and streptogramin A). We describe the first detection of cfr-mediated linezolid resistance in Staphylococcus aureus and Staphylococcus epidermidis recovered from human infection cases monitored during the 2007 LEADER Program.

Diversity of Extended-Spectrum β-Lactamases and Class C β-Lactamases among Cloacal Escherichia coli Isolates in Belgian Broiler Farms

Abstract: A total of 295 ceftiofur-resistant Escherichia coli isolates were obtained from 489 cloacal samples collected at five different Belgian broiler farms with the aim to evaluate the diversity of this resistance at the farm level. Strains were examined for resistance against β-lactam antibiotics and other antimicrobial agents by using disk diffusion tests. Three different β-lactam resistance phenotypes suggested the presence of an extended-spectrum β-lactamase (ESBL), a class C β-lactamase, or the combination of an ESBL with a class C β-lactamase. Seventy-six percent of these isolates also showed acquired resistance to other antimicrobial agents. After genotyping by repetitive extragenic palindromic-PCR, 51 unrelated E. coli strains were selected for further analyses. Isoelectric focusing and sequencing of the amplicons obtained in PCRs for the detection of genes encoding broad-spectrum β-lactamase enzymes revealed the following ESBLs: TEM-52 (13.2%), TEM-106 (2%), CTX-M-1 (27.4%), CTX-M-2 (7.8%), CTX-M-14 (5.9%), and CTX-M-15 (2%). The only plasmidic AmpC β-lactamase found in this study was the CMY-2 enzyme (49%). Mutations in the promoter and attenuator regions of the chromosomal ampC gene were found only in association with blaCMY-2 genes and ESBL genes. The combination of an ESBL (CTX-M-1) with a plasmidic AmpC β-lactamase (CMY-2) was found in 7.8% of the isolates. These data show that ceftiofur-resistant E. coli strains are often present in cloacal samples of broilers at the farm level in Belgium. The diversity of broad-spectrum β-lactamases among these isolates is high, and they may act as a reservoir of ESBL and ampC genes.