Semi-mechanistic PK/PD modelling of meropenem and sulbactam combination against carbapenem-resistant strains of Acinetobacter baumannii.

Due to limited treatment options for carbapenem-resistant Acinetobacter baumannii (CR-AB) infections, antibiotic combinations are commonly used. In this study, we explored the potential efficacy of meropenem-sulbactam combination (MEM/SUL) against CR-AB. The checkerboard method was used to screen for synergistic activity of MEM/SUL against 50 clinical CR-AB isolates. Subsequently, time-kill studies against two CR-AB isolates were performed. Time-kill data were described using a semi-mechanistic pharmacokinetic/pharmacodynamic (PK/PD) model. Subsequently, Monte Carlo simulations were performed to estimate the probability of 2-log kill, 1-log kill or stasis at 24-h following combination therapy. The MEM/SUL demonstrated synergy against 28/50 isolates. No antagonism was observed. The MIC50 and MIC90 of MEM/SUL were decreased fourfold, compared to the monotherapy MIC. In the time-kill studies, the combination displayed synergistic killing against both isolates at the highest clinically achievable concentrations. At concentrations equal to the fractional inhibitory concentration, synergism was observed against one isolate. The PK/PD model adequately delineated the data and the interaction between meropenem and sulbactam. The effect of the combination was driven by sulbactam, with meropenem acting as a potentiator. The simulations of various dosing regimens revealed no activity for the monotherapies. At best, the MEM/SUL regimen of 2 g/4 g every 8 h demonstrated a probability of target attainment of 2-log10 kill at 24 h of 34%. The reduction in the MIC values and the achievement of a moderate PTA of a 2-log10 reduction in bacterial burden demonstrated that MEM/SUL may potentially be effective against some CR-AB infections.

Reduced susceptibility to biocides in Acinetobacter baumannii: association with resistance to antimicrobials, epidemiological behaviour, biological cost and effect on the expression of genes encoding porins and efflux pumps.

OBJECTIVES: The objective of this study was to analyse whether there is an association between reduced susceptibility to biocides in Acinetobacter baumannii and (i) antimicrobial resistance (co-resistance), (ii) prevalent (epidemic) clones, (iii) changes in the fitness or (iv) expression of genes related to efflux pumps and porins. METHODS: Susceptibility to biocides and antimicrobials was determined in 49 clonally unrelated isolates of A. baumannii. Biological cost, in terms of mean generation time, was determined by spectrophotometry. Quantitative real-time RT-PCR was used to determine the relative expression of genes encoding several efflux pumps and porins. RESULTS: Reduced susceptibility to chlorhexidine digluconate, benzalkonium chloride and Irgasan(®) was associated with resistance to aminoglycosides, tetracycline and ciprofloxacin (P < 0.05). The MICs of carbapenems, aminoglycosides, doxycycline and ciprofloxacin for isolate Ab70 (epidemic clone) exposed to these biocides increased by ≥2 dilutions. Reduced susceptibility to Orsan(®) was more frequent among prevalent clones than non-prevalent clones (P < 0.05). Mean generation times for Ab70 before and after exposure to benzalkonium chloride were 57.8 and 78.1 min, respectively (P = 0.02). Relative expression of abeS and adeB was increased in Ab46 and Ab70 after exposure to chlorhexidine digluconate, but was decreased for ompA and carO after exposure to Irgasan(®). CONCLUSIONS: Reduced susceptibility to biocides is associated with co-resistance to carbapenems, aminoglycosides, tetracycline and ciprofloxacin. Reduced susceptibility to Orsan(®) may be a marker of prevalent clones. Acquisition of reduced susceptibility to benzalkonium chloride has a biological cost. Exposure to biocides affects the relative expression of genes related to some efflux pump genes (increased expression) or porins (reduced expression).

Carbapenem-resistant Acinetobacter baumannii: epidemiology, surveillance and management.

Carbapenem-resistant Acinetobacter baumannii pose a significant threat to hospitalized patients, as therapeutic options are scarse. Alarmingly, rates of carbapenem-resistance in A. baumannii are on the rise and are slowly becoming a routine phenotype for this organism. This review focuses on infection control strategies for identification and control of A. baumannii, as well the available therapeutic options.

Suitable restriction enzyme for standardization of pulsed-field gel electrophoresis protocol and interlaboratory comparison of Acinetobacter baumannii.

BACKGROUND/PURPOSE: Interlaboratory comparison of pulsed-field gel electrophoresis (PFGE) patterns is difficult. A key reference of standardized PFGE protocol for Acinetobacter baumannii may address this issue. This study aimed to determine restriction enzymes with rare cutting sites on A baumannii genomes and evaluate their cost-effectiveness, discriminatory power, and interlaboratory consistence of band assignments. METHODS: There were 42 A baumannii isolates collected, including nine from three hospital outbreaks and 33 sporadic isolates. The numbers of cutting sites for the restriction enzymes were explored using the "Restriction Digest and PFGE" program. The cost-effectiveness for PFGE analysis was evaluated for the tested restriction enzymes, while its discriminatory ability was expressed through a discriminatory index and 95% confidence interval. The interlaboratory consistence of band assignments was evaluated for the 42 A baumannii isolates. RESULTS: ApaI was the most cost-effective restriction enzyme for a PFGE protocol for A baumannii. Both AscI and AsiSI were reasonable in terms of costs. ApaI, AscI, and AsiSI exhibited similar discriminatory indices. ApaI generated more than 40 fragments that were close and not easy to resolve, resulting in less consistence of band assignments. AscI and AsiSI generated 10-20 fragments that were clearly resolved, resulting in higher consistence of band assignments. AscI exhibited a close discriminatory power to that of AsiSI and at half of the cost of AsiSI for PFGE analysis. CONCLUSION: We recommend AscI as the primary enzyme and AsiSI as the secondary enzyme for standardizing the PFGE protocol and interlaboratory comparisons of A baumannii.

[Molecular characterization of the multidrug-resistant Acinetobacter baumannii strains and assessment of their sensitivity to the phage AP22].

The molecular analysis of 130 multidrug-resistant nosocomial Acinetobacter baumannii strains was performed. The strains were obtained from patients admitted to different Russian hospitals (Chelyabinsk, Moscow, Nizhni Novgorod, and St. Petersburg) in 2005-2010. Species identification was performed using the amplified 16S rRNA gene restriction analysis and by determining intrinsic for A. baumannii blaQXA-51-like genes using PCR. The genetic typing of the strains was performed by RAPD-PCR. All strains fell into two clusters: A and B with dominant RAPD-groups A1 and B1, respectively, including 82% (107 of 130) of all studied strains. The susceptibility to the bacteriophage AP22 of the strains was determined. The phage was found to infect specifically and to constitute 69% of 130 strains and 82% (88 of 107) of the A. baumannii strains from the dominant RAPD groups. The ability of the bacteriophage AP22 to constitute a broad range of the clinically relevant A. baumannii strains makes it an attractive candidate for designing the phage cocktails intended to control the A. baumannii-associated nosocomial infections. Moreover, the phage can be used for the identification of A. baumannii in bacteriological analysis of clinical materials.

Analysis of multilocus sequence typing schemes for 35 different bacteria revealed that gene loci of 10 bacteria could be replaced to improve cost-effectiveness.

Although multilocus sequence typing (MLST) has been widely used for bacterial typing, the contribution of the gene loci to the discriminatory power of each MLST scheme is unknown. We analyzed the discriminatory powers of 36 MLST schemes using all combinations of the 7 loci and contributions of each locus to the schemes. In 10 schemes, sequencing 6 loci can achieve the discriminatory powers of 7 loci. For the other 26 schemes, the median marginal increase in discriminatory power when 7 instead of 6 loci were used is 0.0004. Sequencing the 7 loci of 50 strains each of Pseudomonas aeruginosa and Acinetobacter baumannii revealed that the discriminatory power for P. aeruginosa was 0.9861 when either 6 (without trp) or 7 loci were used and that for A. baumannii was 0.9363 when 5, 6, or 7 loci were used. Genes that have no additional or minimal contribution to the overall discriminatory powers should be replaced.

The optimization of a rapid pulsed-field gel electrophoresis protocol for the typing of Acinetobacter baumannii, Escherichia coli and Klebsiella spp.

Pulsed-field gel electrophoresis (PFGE) is the most common genotyping method used for the typing of a number of bacterial species. Generally, investigators use their own custom-developed protocol, but a standardized PFGE protocol would allow the comparison of typing results between laboratories and the tracing of strains around the country. In the present study, we optimized a PFGE protocol for subtyping of Acinetobacter baumannii, Escherichia coli and Klebsiella spp., which are commonly isolated from nosocomial infections in many hospitals. Reproducibility of our PFGE procedure was studied three times at 2- to 3-week intervals. Epidemiological concordance of the optimized PFGE procedure was tested on seven isolates of A. baumannii from a previous outbreak and seven A. baumannii isolates randomly selected among the clinical isolates. The optimized PFGE procedure was evaluated on a total of 174 clinical isolates including 62 A. baumannii, 50 E. coli, and 62 Klebsiella spp. The inter-laboratory reproducibility of the optimized protocol was tested at four laboratories. The optimized procedure is completed in 28 h after culturing. It is likely to be cost-effective, due to the reduction in the time, reagent volume and enzyme concentration needed. The procedure showed high concordance with epidemiological data. There were no non-typeable isolates among the tested bacteria. It is reproducible and versatile. This protocol can be used to identify outbreaks and monitor the spreading rate of nosocomial infections caused by the tested bacterial isolates. Furthermore, due to its high intra- and inter-laboratory reproducibility, the protocol has the potential to be useful for comparing PFGE fingerprinting profiles of the isolates from different settings.

Epidemiologic, clinical, and economic evaluation of an outbreak of clonal multidrug-resistant Acinetobacter baumannii infection in a surgical intensive care unit.

OBJECTIVES: To determine risk factors for acquisition of multidrug-resistant (MDR) Acinetobacter baumannii infection during an outbreak, to describe the clinical manifestations of infection, and to ascertain the cost of infection. DESIGN: Case-control study. SETTING: Surgical intensive care unit in a 400-bed urban teaching hospital and level 1 trauma center. PATIENTS: Case patients received a diagnosis of infection due to A. baumannii isolates with a unique pattern of drug resistance (ie, susceptible to imipenem, variably susceptible to aminoglycosides, and resistant to all other antibiotics) between December 1, 2004, and August 31, 2005. Case patients were matched 1 : 1 with concurrently hospitalized control patients. Isolates' genetic relatedness was established by pulsed-field gel electrophoresis. RESULTS: Sixty-seven patients met the inclusion criteria. Case and control patients were similar with respect to age, duration of hospitalization, and Charlson comorbidity score. MDR A. baumannii infections included ventilator-associated pneumonia (in 56.7% of patients), bacteremia (in 25.4%), postoperative wound infections (in 25.4%), central venous catheter-associated infections (in 20.9%), and urinary tract infections (in 10.4%). Conditional multiple logistic regression was used to determine statistically significant risk factors on the basis of results from the bivariate analyses. The duration of hospitalization and healthcare charges were modeled by multiple linear regression. Significant risk factors included higher Acute Physiology and Chronic Health Evaluation II score (odds ratio [OR], 1.1 per point increase; P=.06), duration of intubation (OR, 1.4 per day intubated; P<.01), exposure to bronchoscopy (OR, 22.7; P=.03), presence of chronic pulmonary disease (OR, 77.7; P=.02), receipt of fluconazole (OR, 73.3; P<.01), and receipt of levofloxacin (OR, 11.5; P=.02). Case patients had a mean of $60,913 in attributable excess patient charges and a mean of 13 excess hospital days. INTERVENTIONS: Infection control measures included the following: limitations on the performance of pulsatile lavage wound debridement, the removal of items with upholstered surfaces, and the implementation of contact isolation for patients with suspected MDR A. baumannii infection. CONCLUSIONS: This large outbreak of infection due to clonal MDR A. baumannii caused significant morbidity and expense. Aerosolization of MDR A. baumannii during pulsatile lavage debridement of infected wounds and during the management of respiratory secretions from colonized and infected patients may promote widespread environmental contamination. Multifaceted infection control interventions were associated with a decrease in the number of MDR A. baumannii isolates recovered from patients.

Evaluation of the 10th External Quality Assessment Scheme results in clinical microbiology laboratories in Tehran and districts.

We evaluated the performance of microbiology laboratories in the 10th run of the external quality assessment scheme (EQAS) in Tehran and districts. Each laboratory was sent 2 species of bacteria for identification. Of the 487 laboratories that participated, 437 returned their findings. While 77.0% and 69.9% correctly identified Staphylococcus saprophyticus and Citrobacter freundii respectively, only 29.8% correctly identified Acinetobacter baumanii, 25.3% identified Enterococcus faecalis and 35.6% identified Enterobacter agglomerans. However 78.7% and 79.5% of the laboratories reported correct -results for susceptibility testing for S. saprophyticus and C. freundii respectively.

Standardization and interlaboratory reproducibility assessment of pulsed-field gel electrophoresis-generated fingerprints of Acinetobacter baumannii.

A standard procedure for pulsed-field gel electrophoresis (PFGE) of macrorestriction fragments of Acinetobacter baumannii was set up and validated for its interlaboratory reproducibility and its potential for use in the construction of an Internet-based database for international monitoring of epidemic strains. The PFGE fingerprints of strains were generated at three different laboratories with ApaI as the restriction enzyme and by a rigorously standardized procedure. The results were analyzed at the respective laboratories and also centrally at a national reference institute. In the first phase of the study, 20 A. baumannii strains, including 3 isolates each from three well-characterized hospital outbreaks and 11 sporadic strains, were distributed blindly to the participating laboratories. The local groupings of the isolates in each participating laboratory were identical and allowed the identification of the epidemiologically related isolates as belonging to three clusters and identified all unrelated strains as distinct. Central pattern analysis by using the band-based Dice coefficient and the unweighted pair group method with mathematical averaging as the clustering algorithm showed 95% matching of the outbreak strains processed at each local laboratory and 87% matching of the corresponding strains if they were processed at different laboratories. In the second phase of the study, 30 A. baumannii isolates representing 10 hospital outbreaks from different parts of Europe (3 isolates per outbreak) were blindly distributed to the three laboratories, so that each laboratory investigated 10 epidemiologically independent outbreak isolates. Central computer-assisted cluster analysis correctly identified the isolates according to their corresponding outbreak at an 87% clustering threshold. In conclusion, the standard procedure enabled us to generate PFGE fingerprints of epidemiologically related A. baumannii strains at different locations with sufficient interlaboratory reproducibility to set up an electronic database to monitor the geographic spread of epidemic strains.

Assessment of intra-species diversity among strains of Acinetobacter baumannii isolated from sites contaminated with petroleum hydrocarbons.

A total of 96 crude oil-degrading bacterial strains were isolated from 5 geographically diverse sites in India that were contaminated with different types of petroleum hydrocarbons. The strains were identified by sequencing the genes that encode for 16S rRNA. Out of the 96 isolates, 25 strains were identified as Acinetobacter baumannii and selected for the study. All of the selected strains could degrade the total petroleum hydrocarbon fractions of crude oil. These 25 strains were biochemically profiled and grouped into 8 phenovars on the basis of multivariate analysis of their substrate utilization profiles. PCR-based DNA fingerprinting was performed using intergenic repetitive DNA sequences, which divided the selected 25 strains into 7 specific genomic clusters. tRNA intergenic spacer length polymorphism was performed to determine the intra-species relatedness among these 25 strains. It delineated the strains into 8 genomic groups. The present study detected specific variants among the A. baumannii strains with differential degradation capacities for different fractions of crude oil. This could play a significant role in in situ bioremediation. The study also revealed the impact of environmental factors that cause intra-species diversity within the selected strains of A. baumannii.

Assessment of Acinetobacter baumannii susceptibility to antiseptics and disinfectants.

Disinfection and antisepsis are of primary importance in controlling outbreaks of Acinetobacter baumannii, a nosocomial pathogen that frequently shows multiple antibiotic resistance. In this study we assessed the susceptibility of nine A. baumannii strains isolated during a sustained intensive care unit outbreak, to several antiseptics and disinfectants based on European Standards. While the tested strains showed diverse antibiotic resistance patterns, they were equally sensitive to the biocides assessed in vitro. We observed neither evidence of development of resistance to biocides over time, nor a correlation between resistance to antibiotics and a decreased susceptibility to antiseptics or disinfectants.