Capturing the mutational landscape of the beta-lactamase TEM-1.

Adaptation proceeds through the selection of mutations. The distribution of mutant fitness effect and the forces shaping this distribution are therefore keys to predict the evolutionary fate of organisms and their constituents such as enzymes. Here, by producing and sequencing a comprehensive collection of 10,000 mutants, we explore the mutational landscape of one enzyme involved in the spread of antibiotic resistance, the beta-lactamase TEM-1. We measured mutation impact on the enzyme activity through the estimation of amoxicillin minimum inhibitory concentration on a subset of 990 mutants carrying a unique missense mutation, representing 64% of possible amino acid changes in that protein reachable by point mutation. We established that mutation type, solvent accessibility of residues, and the predicted effect of mutations on protein stability primarily determined alone or in combination changes in minimum inhibitory concentration of mutants. Moreover, we were able to capture the drastic modification of the mutational landscape induced by a single stabilizing point mutation (M182T) by a simple model of protein stability. This work thereby provides an integrated framework to study mutation effects and a tool to understand/define better the epistatic interactions.

Incidence and impact on clinical outcome of infections with piperacillin/tazobactam resistant Escherichia coli in ICU: a retrospective study.

BACKGROUND: Escherichia coli infections are frequent in ICU patients. The increased resistance to fluoroquinolones and amoxicillin/clavulanate of this pathogen mandates the prescription of broad-spectrum antibiotics such as piperacillin/tazobactam (PIP-TAZ) or third generation cephalosporins (3GC). METHODS: To assess incidence and impact on clinical outcome of infections with PIP-TAZ resistant E. coli in ICU patients, we conducted a retrospective cohort study with infections due to PIP-TAZ resistant (PIP-TAZ R) or to PIP-TAZ susceptible strains (PIP-TAZ S) between 1 January 2002 and 30 June 2004. RESULTS: Of 83 strains, 13 were PIP-TAZ R: 2 strains produced an extended-spectrum beta-lactamase (2%), 11 produced a high level penicillinase (13%). Prior amoxicillin or amoxicillin/clavulanate prescription was reported in 7 cases (54%) of infections with PIP-TAZ R isolates and in 15 cases (21%) of infections with PIP-TAZ S isolates (p = 0.03). Time of onset of the infection from hospital admission was longer in case of infections with PIP-TAZ R than with PIP-TAZ S isolates (22 +/- 32 vs 10 +/- 21 days, p = 0.01). The overall ICU mortality rate was 38%. Mortality and length of stay in ICU were similar in case of infections with PIP-TAZ R isolates and with PIP-TAZ S isolates. CONCLUSION: Infections with PIP-TAZ R E. coli are frequent in ICU patients. No prognostic impact of this pattern of resistance was found. Prescription of PIP-TAZ for empirical treatment of E. coli infections in ICU however exposes to inappropriate therapy.

Hypervirulent Klebsiella pneumoniae, a 5-year study in a French ICU.

PURPOSE: Hypervirulent Klebsiella pneumoniae (hvKp) has emerged as a leading cause of severe community-acquired pneumonia, liver abscess and disseminated infection in the Far East. Data regarding the incidence, clinical features and microbiological characteristics related to hvKp infections in the Western world are scarce. METHODOLOGY: The incidence, clinical features and microbiological characteristics of hvKp infections were investigated through a 5-year survey conducted in a single French intensive care unit. K. pneumoniae strains were screened for hypermucoviscosity based on a string test. Multilocus sequence typing and multiplex PCR analysis targeting virulence genes were performed on string test-positive strains. RESULTS: Over a 53-month period, a total of 59 infections due to K. pneumoniae were identified including 26 community-onset infections. Twelve hvKp infections were documented, accounting for 46.1 % of community-acquired K. pneumoniae. Community-acquired pneumonia (n=6), aspiration pneumonia (n=4) and liver abscess (n=2) represented initial sites and mode of infection. Compared to non-hvKp infections, patients with hvKp infections displayed higher rates of multi-organ failure (83.3 % vs 35.7 %; P=0.04), but mortality rates were not different (50 % vs 35 %; P=0.71). Strains K1/ST23 (n=5) and K2/ST86 (n=5) predominated. All hvKp strains displayed wild-type susceptibility. CONCLUSION: hvKp represent a potentially underestimated cause of fatal infections in the Western world.

Levels of vancomycin in cerebrospinal fluid of adult patients receiving adjunctive corticosteroids to treat pneumococcal meningitis: a prospective multicenter observational study.

BACKGROUND: Evidence from a recent randomized controlled trial suggests that dexamethasone as adjunct therapy in adult pneumococcal meningitis reduces mortality and neurological sequelae. However, adding dexamethasone has the potential to reduce penetration of vancomycin into the cerebrospinal fluid (CSF). We sought to determine concentrations of vancomycin in serum and CSF of patients with suspected or proven pneumococcal meningitis receiving dexamethasone to assess the penetration of vancomycin into the CSF during steroid therapy. METHODS: In an observational open multicenter study, adult patients admitted to the intensive care unit because of suspected pneumococcal meningitis received recommended treatment for pneumococcal meningitis, comprising intravenous cefotaxime (200 mg per kg of body weight per day), vancomycin (administered as continuous infusion of 60 mg per kg of body weight per day after a loading dose of 15 mg per kg of body weight), and adjunctive therapy with dexamethasone (10 mg every 6 h). Vancomycin levels in CSF were measured on day 2 or day 3 of therapy and were correlated with protein levels in CSF and vancomycin levels in serum (determined at the same time as levels in CSF). RESULTS: Fourteen patients were included. Thirteen had proven pneumococcal meningitis; 1 patient, initially suspected of having pneumococcal meningitis, was finally determined to have meningitis due to Neisseria meningitidis. Mean levels of vancomycin in serum and CSF were 25.2 and 7.2 mg/L, respectively, and were positively correlated (r=0.6; P=.025). A positive correlation was also found between the ratio of vancomycin in CSF to vancomycin in serum and the level of protein in CSF (r=0.66; P=.01). CONCLUSIONS: Appropriate concentrations of vancomycin in CSF may be obtained even when concomitant steroids are used. Dexamethasone can, therefore, be used without fear of impeding vancomycin penetration into the CSF of patients with pneumococcal meningitis, provided that vancomycin dosage is adequate. This study is registered at http://www.ClinicalTrials.gov/ (registration number NCT00162578).

Evaluation of a strategy of screening multiple anatomical sites for methicillin-resistant Staphylococcus aureus at admission to a teaching hospital.

We compared the sensitivity of screening with nasal culture alone with that of a multiple-site screening method for the identification of carriers of methicillin-resistant Staphylococcus aureus at hospital admission. If nasal cultures alone had been used during the 1-year study, 27.0% of carriers of methicillin-resistant S. aureus would have been missed, which corresponds to 560 theoretical isolation days. If rectal screening had not been used, 431 theoretical isolation days would have been missed, and, if axillary screening had not been used, 99 theoretical isolation days would have been missed.

Consideration of age at admission for selective screening to identify methicillin-resistant Staphylococcus aureus carriers to control dissemination in a medical ward.

BACKGROUND: Methicillin-resistant Staphylococcus aureus (MRSA) has become an increasingly important pathogen responsible for hospital-acquired infections. Our study was to evaluate the efficiency of our selective screening program for methicillin-resistant Staphylococcus aureus (MRSA) carriers at admission to nonintensive care units. METHODS: During 6 months, all patients were screened at admission to an internal medicine ward, at which time they were classified as patients at risk of carriage (PRC) and those with no known risk factor. The amplitude of cross transmission was estimated using various indicators during this universal screening period and during the same calendar period of the preceding year (selective screening). RESULTS: The prevalence of MRSA carriage at admission was 5.5%. Among the 22 carriers identified, only 10 were PRC. Age >80 years was significantly associated with MRSA carriage upon admission (OR, 3.5; P < .01). All estimation indicators of MRSA dissemination amplitude were significantly lower during universal screening (relative risks varied from 2.79 to 26.4 according to indicators), demonstrating the need to broaden our criteria defining PRC. CONCLUSION: Adding patients >80 years of age to our PRC definition would increase screening sensitivity (15 carriers identified for 128 patients sampled) and would enable early implementation of barrier precautions for the additional carriers identified.

Five-year trends for ventilator-associated pneumonia: Correlation between microbiological findings and antimicrobial drug consumption.

The epidemiology of multidrug-resistant bacteria (MDRB) has changed significantly in European healthcare settings, with a decrease in frequency of meticillin-resistant Staphylococcus aureus and an increase in extended-spectrum ß-lactamase (ESBL)-producing Enterobacteriaceae. Little is known about the effects of these changes on ventilator-associated pneumonia (VAP). A retrospective 5-year trend analysis of ICU antibiotic consumption and resistance in bacteria causing VAP was undertaken. Poisson regression analysis between complete microbiological data and antibiotic consumption was performed. In total, 252 episodes of VAP in 184 patients were identified between 2007 and 2011, from which 364 causal bacteria were isolated. Enterobacteriaceae isolation rates increased significantly over this period [from 6.64 to 10.52 isolates/1000 patient-days; P=0.006], mostly due to an increase in AmpC-producing Enterobacteriaceae (APE) (2.85-4.51 isolates/1000 patient-days; P=0.013), whereas the number of episodes due to S. aureus and Pseudomonas aeruginosa remained stable. A positive association was found between the increase in APE infections and an increase in past-year antibiotic consumption: amoxicillin/clavulanic acid (P=0.003), ceftazidime and cefepime (P=0.007), carbapenems (P=0.002), fluoroquinolones (P=0.012), macrolides (P=0.002) and imidazoles (P=0.004). No such association was found for the emergence of resistance in P. aeruginosa. These results indicate a change in the epidemiology of VAP, with Enterobacteriaceae exceeding P. aeruginosa and S. aureus. Moreover, a positive correlation was observed between antibiotic consumption and the incidence of potentially MDRB such as APE. No such correlation was found for ESBL-producing Escherichia coli and antibiotic-resistant P. aeruginosa.

Risks of nonsteroidal antiinflammatory drugs in undiagnosed intensive care unit pneumococcal pneumonia: younger and more severely affected patients.

PURPOSE: The purpose of this study is to investigate whether exposure to nonsteroidal antiinflammatory drugs (NSAIDs) at the early stage of severe pneumococcal community-acquired pneumonia (CAP) requiring intensive care unit (ICU) admission may affect its presentation and outcome. MATERIAL AND METHODS: Medical records of ICU adult patients (12-year period) with a pneumococcal CAP diagnosis were retrospectively analyzed according to previous NSAID exposure. RESULTS: One hundred six confirmed pneumococcal CAP were identified, 20 received NSAIDs within 4 (2-6) days before admission. Nonsteroidal antiinflammatory drug-exposed patients were younger (43.3 vs 62.2 years; P < .0001), had less frequently at least one chronic comorbid condition (40% vs 75%; P = .003), had more often complicated pleural effusions (20% vs 2.3%; P = .01), and more frequent pleuropulmonary complications (odds ratio: 5.75 [1.97-16.76]). Nonsteroidal antiinflammatory drug patients required more often noninvasive ventilatory support (25% vs 4.6%; P = .003). Intensive care unit length of stay and mortality were similar. CONCLUSIONS: We report as severe pneumococcal pneumonia in young and healthy patients exposed to NSAIDs as in older, more comorbid, and nonexposed ones. Nonsteroidal antiinflammatory drug use may mask initial symptoms and delay antimicrobial therapy, thus predisposing to worse outcomes.

Influence of tracheal suctioning systems on health care workers' gloves and equipment contamination: a comparison of closed and open systems.

The impact of tracheal suctioning with an open or a closed system on equipment and health care workers contamination with multidrug-resistant pathogens was compared. Only the closed system reduced hand and equipment contamination during tracheal suctioning. This equipment could be systematically used to reduce risk of cross contamination in the intensive care unit.

Impact of the reinforcement of a methicillin-resistant Staphylococcus aureus control programme: a 3-year evaluation by several indicators in a French university hospital.

Our objective was to evaluate the impact of the reinforcement of a methicillin-resistant Staphylococcus aureus (MRSA) control programme and to assess the impact of risk adjustment on the interpretation of data. A stepwise, retrospective analysis of 3-year prospectively collected data was performed in a 600-bed French teaching hospital in the Parisian area. A reinforcement of a pre-existing programme for limiting the spread of MRSA was implemented in 2002 and 2003 by increasing the frequency of the feedback of surveillance data, by using alcohol-based disinfectants, and by increasing patient screening. Different indicators were used to follow the change over time of MRSA transmission: the proportion of MRSA acquired in our hospital, the incidence of newly acquired MRSA/1,000 patient-days (PD) (incidence of newly acquired MRSA), the incidence of newly acquired MRSA isolated in at least one clinical specimen/1,000 PD (incidence of newly acquired clinical MRSA), and a risk-adjusted indicator, the incidence of newly acquired-MRSA isolated in at least one clinical specimen/1,000 PD of carriers identified at admission (incidence related to the risk of acquisition). The change over time of these indicators was studied with the chi-square test for trend. During the study, all indicators decreased significantly, with a mean drop of 0.07/1,000 PD for the incidence of newly acquired clinical MRSA, and a mean drop of 3.0/1,000 PD for the incidence related to the risk of acquisition. The proportion of MRSA acquired in our hospital decreased from 49.3% in 2002 to 24.1% in 2004. Concurrently, between 2002 and 2004, the number of patients screened on admission to hospital or at the time of intra-hospital transfer increased by 31% and the consumption of waterless alcohol-based hand disinfectants increased by 244%. The decreasing trend of all indicators emphasizes the effectiveness of the reinforcement of our MRSA control programme. From 2002 to 2004, the trend of the indicator related to the risk of acquisition over time is similar to those of other indicators. Further studies should be useful to assess if risk-adjustment is absolutely necessary when tracking rates within a single institution.

Resistance to ceftazidime is associated with a S220Y substitution in the omega loop of the AmpC beta-lactamase of a Serratia marcescens clinical isolate.

OBJECTIVES: The aim of this study was to characterize the ampC beta-lactamase gene of a clinical isolate of Serratia marcescens resistant to ceftazidime. METHODS: S. marcescens SMSA was isolated from an intra-abdominal wound of a patient previously treated with ceftazidime. A susceptible strain, SLS73, was used as a control. Susceptibility testing, PCR, DNA sequencing, molecular cloning, site-directed mutagenesis and determination of kinetic parameters were carried out to investigate the mechanism of resistance to ceftazidime. RESULTS: MICs of ceftazidime were 64 and 0.2 mg/L for SMSA and SLS73, respectively. Sequencing of the ampC gene of SMSA was carried out. When compared with the closest AmpC enzyme, the S. marcescens S3 beta-lactamase, the novel protein showed E57Q, Q129K and S220Y substitutions. The S220Y substitution is located in the omega loop. Introduced by mutagenesis in the ampC gene of SLS73, this substitution conferred the same level of resistance to ceftazidime. The catalytic efficiency (k(cat)/K(m)) of the mutated enzyme toward ceftazidime was increased by about 100-fold. CONCLUSIONS: We present another example of in vivo selection of broad-spectrum resistance by amino acid substitution in the omega loop of chromosomal AmpC beta-lactamase in S. marcescens.

Novel plasmid-encoded class C beta-lactamase (MOX-2) in Klebsiella pneumoniae from Greece.

Klebsiella pneumoniae KOL, a clinical strain resistant to various beta-lactams, was isolated from the stools of a patient from Greece. This strain harbored a new pI 9.1 plasmid-mediated AmpC beta-lactamase with unusually high levels of hydrolytic activity for cefoxitin and cefotetan that we named MOX-2. Sequencing of bla(MOX-2) revealed 93.2, 92.9, 92.7, and 73.1% identities with the deduced amino acid sequences of CMY-8, MOX-1, CMY-1, and the AmpC beta-lactamase of Aeromonas sobria, respectively.