A global view on carbapenem-resistant Acinetobacter baumannii.

To give an update on the molecular epidemiology and global distribution of carbapenemase encoding genes, we subjected 313 carbapenem-resistant Acinetobacter baumannii isolated from 114 study centers in 47 countries in five world regions, Africa, Asia, Europe, Latin America, and North America, to whole genome sequencing. Numbers of isolates investigated were proportional to the population size of the contributing countries. Molecular epidemiology was investigated using seven-loci and core genome multilocus sequence typing, whole-genome single nucleotide polymorphism phylogenies, and the intrinsic blaOXA-51-like variant. Carbapenemase encoding genes were identified by multiplex PCR and ResFinder. Among the total of 313 isolates, 289 (92.3%) were assigned to A. baumannii international clones (IC) IC1-IC8. IC2 predominated with 196 isolates (62.6%) and was spread worldwide, followed by IC5 with 44 isolates (14.1%) mainly confined to Latin America. Six isolates (1.9%) originating from Belgium, Egypt, Italy, and Pakistan represent the novel IC9. Acquired OXA-type carbapenemase genes were found in 300 (96%) isolates with blaOXA-23-like and blaOXA-40-like predominating, which constitutes a significant increase compared to our findings from 2010. Metallo-beta-lactamases were rare with seven isolates (2.2%). The distribution of ICs and carbapenemase determinants can vary widely among different geographical regions. IMPORTANCE Carbapenem-resistant Acinetobacter baumannii are of increasing public health importance, as they are resistant to last-line antibiotics. International clones with well-characterized resistance genes dominate globally; however, locally, other lineages with different properties may be of importance to consider. This study investigated isolates from a broad geographic origin from 114 hospitals in 47 countries and from five world regions ensuring the greatest possible diversity in an organism known for its propensity for clonal epidemic spread and reflecting the current global epidemiology of carbapenem-resistant A. baumannii. In Latin America, a lineage different from other geographic regions circulates, with a different resistance gene profile. This knowledge is important to adjust local infection prevention measures. In a global world with migration and increasing use of antimicrobials, multidrug-resistant bacteria will continue to adapt and challenge our healthcare systems worldwide.

In Vitro Activity of Cefiderocol against a Global Collection of Carbapenem-Resistant Acinetobacter baumannii Isolates.

Background: Cefiderocol is a novel siderophore cephalosporin with potent activity against multi-drug-resistant Gram-negative pathogens including carbapenem-resistant Acinetobacter baumannii (CRAB). Methods: The susceptibility of 313 non-duplicate CRAB isolates with defined carbapenem resistance mechanisms from a global collection to cefiderocol, ceftazidime, ceftazidime/avibactam, ceftolozane/tazobactam, ciprofloxacin, colistin, imipenem/relebactam, meropenem, meropenem/vaborbactam, minocycline, and piperacillin/tazobactam was determined using the broth microdilution method. Isolates were obtained from various body sites from patients in 47 countries in five world regions between 2012 and 2016. The identification of carbapenem resistance mechanisms and assignment to A. baumannii international clonal lineages were based on whole genome sequencing. Results: Cefiderocol showed greater activity than comparator antimicrobials of the ß-lactam class, including novel ß-lactams combined with ß-lactamase inhibitors, ciprofloxacin, and minocycline. Cefiderocol MIC50 and MIC90 values were 0.5 mg/L and 4 mg/L, respectively, while colistin had comparable activity with a higher MIC50 at 1 mg/L and a lower MIC90 value of 2 mg/L. Many isolates with elevated cefiderocol MICs ≥ 4 mg/L represented A. baumannii international clone (IC) 1 and harbored a metallo-ß-lactamase. Conclusions: While cefiderocol is a useful addition to the limited armamentarium of drugs targeting this problematic pathogen, a considerable part of CRAB isolates had elevated MIC values in a range of 4 -> 32 mg/L, including all isolates with a metallo-ß-lactamase.

The evolution of carbapenem resistance determinants and major epidemiological lineages among carbapenem-resistant Acinetobacter baumannii isolates in Germany, 2010-2019.

The aim of this study was to investigate and compare the molecular epidemiology and carbapenem resistance determinants in clinical Acinetobacter baumannii isolates collected during four multicentre surveillance studies conducted by the Paul-Ehrlich-Society for Infection Therapy. Isolates were collected prospectively from hospital in-patients at 17 medical centres in Germany over four periods of three- to six-months starting in October of each of 2010, 2013, 2016 and 2019. Species identification was performed by MALDI-TOF, gyrB multiplex polymerase chain reaction (PCR), and detection of the intrinsic blaOXA-51-like gene. Minimum inhibitory concentrations were determined by broth microdilution. The prevalence of carbapenemase-encoding genes was investigated by OXA-multiplex PCR and whole-genome sequencing. Molecular epidemiology was examined by rep-PCR and core-genome multi-locus sequence typing. A total of 302 A. baumannii isolates were collected. Resistance to imipenem and/or meropenem was detected in 58 isolates (19.2%) from 14 centres. The proportion of carbapenem-resistant isolates increased from 21.3% in 2010 to 33.3% in 2013, and then decreased to 13.8% in 2016 and 12.3% in 2019. Forty-six of these isolates were associated with the international clonal lineage IC2 and five with IC1. The most prevalent carbapenemase gene detected was blaOXA-23-like (n=51). Further carbapenem-resistance determinants were blaOXA-40-like (n=1), blaOXA-58-like (n=3) and blaNDM-1 (n=2). In one isolate, ISAba1 was detected upstream of blaOXA-51-like. In conclusion, IC2 was the most prevalent clonal lineage detected in this study. Interestingly, in Germany, carbapenem resistance seems to have decreased in A. baumannii between 2013 and 2019.

In vitro activity of sulbactam/durlobactam against global isolates of carbapenem-resistant Acinetobacter baumannii.

OBJECTIVES: To evaluate the activity of the novel broad-spectrum serine ß-lactamase inhibitor durlobactam (ETX2514) combined with sulbactam against global isolates of carbapenem-resistant Acinetobacter baumannii with defined carbapenem resistance mechanisms compared with reference antimicrobials with known activity against Acinetobacter spp. METHODS: The susceptibility of 246 carbapenem-resistant non-duplicate A. baumannii isolates to sulbactam/durlobactam, amikacin, colistin, imipenem/sulbactam/durlobactam, imipenem, meropenem, minocycline and sulbactam was tested using broth microdilution. Isolates were obtained from various body sites from patients in 37 countries and from six world regions between 2012 and 2016. Identification of carbapenem resistance mechanisms and assignment to A. baumannii clonal lineages was based on WGS. RESULTS: Sulbactam/durlobactam showed excellent activity comparable to colistin but superior to amikacin, minocycline and sulbactam. The sulbactam/durlobactam MIC50/90 values were 1/4 and 2/4 mg/L and the colistin MIC50/90 values were 0.5 and 1 mg/L, respectively. Comparatively, amikacin, minocycline and sulbactam MIC50/90 values were 256/≥512, 2/16 and 16/64 mg/L, respectively. CONCLUSIONS: Sulbactam/durlobactam had excellent in vitro potency against A. baumannii isolates, including those that were resistant to imipenem/meropenem, amikacin, minocycline and colistin, compared with other compounds. Sulbactam/durlobactam has the potential to become a useful addition to the limited armamentarium of drugs that can be used to treat this problem pathogen.

In vitro activity of the novel fluorocycline TP-6076 against carbapenem-resistant Acinetobacter baumannii.

The activity of the novel, fully synthetic fluorocycline antibiotic TP-6076 against carbapenem-resistant Acinetobacter baumannii (CRAB) isolates with defined carbapenem resistance mechanisms was compared against reference antimicrobials with known activity against Acinetobacter spp. The susceptibility of 323 non-duplicate CRAB isolates to TP-6076, amikacin, ampicillin/sulbactam (SAM), cefepime, colistin, doxycycline, eravacycline, imipenem, levofloxacin, meropenem, minocycline, rifampicin, sulbactam, tigecycline, tobramycin and trimethoprim/sulfamethoxazole (SXT) was determined by the broth microdilution method. TP-6076 showed greater activity than comparator antimicrobials of the tetracycline class, SAM, levofloxacin, amikacin, tobramycin, SXT and colistin. MIC50 and MIC90 values for TP-6076 were 0.06 mg/L and 0.25 mg/L, respectively. In comparison, doxycycline, eravacycline, minocycline and tigecycline MIC50/90 values were 32/≥64, 0.5/1, 4/8 and 1/2 mg/L, respectively. Compared with other compounds, TP-6076 was the most active antimicrobial against CRAB, including isolates that were resistant to other anti-Acinetobacter reference drugs including SAM, colistin, the aminoglycosides amikacin and tobramycin, and levofloxacin. TP-6076 is a promising new agent that may be a useful addition to the limited armamentarium of drugs targeting this problematic pathogen.

Diversity of amino acid substitutions in PmrCAB associated with colistin resistance in clinical isolates of Acinetobacter baumannii.

This study aimed to investigate the mechanisms of colistin resistance in 64 Acinetobacter baumannii isolates obtained from patients with ventilator-associated pneumonia hospitalised in Greece, Italy and Spain. In total, 31 A. baumannii isolates were colistin-resistant. Several novel amino acid substitutions in PmrCAB were found in 27 colistin-resistant A. baumannii. Most substitutions were detected in PmrB, indicating the importance of the histidine kinase for colistin resistance. In two colistin-resistant isolates, 93 amino acid changes were observed in PmrCAB compared with A. baumannii ACICU, and homologous recombination across different clonal lineages was suggested. Analysis of gene expression revealed increased pmrC expression in isolates harbouring pmrCAB mutations. Complementation of A. baumannii ATCC 19606 and ATCC 17978 with a pmrAB variant revealed increased pmrC expression but unchanged colistin MICs, indicating additional unknown factors associated with colistin resistance. Moreover, a combination of PmrB and PmrC alterations was associated with very high colistin MICs, suggesting accumulation of mutations as the mechanism for high-level resistance. The pmrC homologue eptA was detected in 29 colistin-susceptible and 26 colistin-resistant isolates. ISAba1 was found upstream of eptA in eight colistin-susceptible and one colistin-resistant isolate and eptA was disrupted by ISAba125 in two colistin-resistant isolates. Whilst in most isolates an association of eptA with colistin resistance was excluded, in one isolate an amino acid substitution in EptA (R127L) combined with a point mutation in ISAba1 upstream of eptA contributed to elevated colistin MICs. This study helps to gain an insight into the diversity and complexity of colistin resistance in A. baumannii.

Investigation of Novel pmrB and eptA Mutations in Isogenic Acinetobacter baumannii Isolates Associated with Colistin Resistance and Increased Virulence In Vivo.

Colistin resistance in Acinetobacter baumannii is of great concern and is a threat to human health. In this study, we investigate the mechanisms of colistin resistance in four isogenic pairs of A. baumannii isolates displaying an increase in colistin MICs. A mutation in pmrB was detected in each colistin-resistant isolate, three of which were novel (A28V, I232T, and ΔL9-G12). Increased expression of pmrC was shown by semi-quantitative reverse transcription-PCR (qRT-PCR) for three colistin-resistant isolates, and the addition of phosphoethanolamine (PEtN) to lipid A by PmrC was revealed by mass spectrometry. Interestingly, PEtN addition was also observed in some colistin-susceptible isolates, indicating that this resistance mechanism might be strain specific and that other factors could contribute to colistin resistance. Furthermore, the introduction of pmrAB carrying the short amino acid deletion ΔL9-G12 into a pmrAB knockout strain resulted in increased pmrC expression and lipid A modification, but colistin MICs remained unchanged, further supporting the strain specificity of this colistin resistance mechanism. Of note, a mutation in the pmrC homologue eptA and a point mutation in ISAba1 upstream of eptA were associated with colistin resistance and increased eptA expression, which is a hitherto undescribed resistance mechanism. Moreover, no cost of fitness was observed for colistin-resistant isolates, while the virulence of these isolates was increased in a Galleria mellonella infection model. Although the mutations in pmrB were associated with colistin resistance, PEtN addition appears not to be the sole factor leading to colistin resistance, indicating that the mechanism of colistin resistance is far more complex than previously suspected and is potentially strain specific.

In-vitro activity of the novel fluorocycline eravacycline against carbapenem non-susceptible Acinetobacter baumannii.

The activity of eravacycline was compared with that of anti-Acinetobacter reference antimicrobials against carbapenem non-susceptible Acinetobacter baumannii isolates associated with an acquired OXA or up-regulation of the intrinsic OXA-51-like enzyme. Antimicrobial susceptibility testing was performed by broth microdilution of 286 non-duplicate, carbapenem non-susceptible A. baumannii isolates to eravacycline, amikacin colistin, doxycycline, imipenem, levofloxacin, meropenem, minocycline, sulbactam, tigecycline and tobramycin. Eravacycline showed greater activity than the comparators of the tetracycline class, levofloxacin, amikacin, tobramycin and colistin. The eravacycline MIC50/90 values were 0.5/1 mg/L and those for tigecycline, minocycline and doxycycline were 1/2, 4/8 and 32/ ≥ 64 mg/L, respectively. In conclusion, eravacycline was the most potent antibiotic of those tested against A. baumannii, including isolates that were resistant to sulbactam, imipenem/meropenem, levofloxacin and amikacin/tobramycin. Eravacycline has the potential to become a useful addition to the limited armamentarium of drugs that can be used to treat this problem pathogen.

Determining vancomycin Etest MICs in patients with MRSA bloodstream infection does not support switching antimicrobials.

OBJECTIVES: Elevated vancomycin minimum inhibitory concentrations (MIC) have been reported to adversely affect clinical outcome in methicillin-resistant Staphylococcus aureus (MRSA) bloodstream infection (BSI). We therefore examined the association between vancomycin MIC and outcome considering various potential confounders. METHODS: Clinical data and bacterial isolates were prospectively collected from patients with MRSA BSI from 2006 to 2012 as part of the Invasive Staphylococcus aureus Infection Cohort (INSTINCT) study. Antimicrobial susceptibility was assessed by Etest, broth microdilution (BMD) and VITEK 2. Bacterial genotypes were determined by spa typing. Using univariate and Cox regression analyses, we investigated the impact of low (≤1.0 mg/L) and high (≥1.5 mg/L) vancomycin Etest MIC on clinical outcomes. RESULTS: Ninety-one MRSA BSI episodes were included, of which 79 (86.8%) were caused by spa types t003, t032 and t045. High vancomycin MICs were seen only if using Etest but not confirmed using standard reference BMD. When episodes were stratified into low and high vancomycin Etest MIC groups, 30-day overall mortality was 34.5% and 27.3%, respectively (P = 0.64, OR 0.71; 95% confidence interval [CI] 0.27-1.79). Variables significantly associated with all-cause mortality in the Cox model were age (P = 0.003), acute physiology score (P = 0.0006), and Charlson comorbidity index (P = 0.018). CONCLUSIONS: Vancomycin MICs may vary dependent on testing methodologies and local MRSA epidemiology. The patients' underlying disease and individual comorbidities rather than elevated vancomycin MICs determine adverse clinical outcomes in MRSA BSI. Routine Etest MIC testing of MRSA isolates is of limited value for treatment decisions.

Head-to-Head Comparison of Two Multi-Locus Sequence Typing (MLST) Schemes for Characterization of Acinetobacter baumannii Outbreak and Sporadic Isolates.

To compare the two Acinetobacter baumannii multi-locus sequence typing (MLST) schemes and to assess their suitability to aid in outbreak analysis we investigated the molecular epidemiology of 99 Acinetobacter baumannii isolates representing outbreak-related and sporadic isolates from 24 hospitals in four different countries (Germany, Poland, Sweden, and Turkey). Pulsed-field gel electrophoresis (PFGE) was used as the reference method to determine the epidemiologic relatedness of isolates and compared to MLST using both the Oxford and Pasteur scheme. Rep-PCR was used to define international clonal lineages (IC). We identified 26 unique outbreak strains and 21 sporadic strains. The majority of outbreaks were associated with carbapenem-resistant A. baumannii harbouring oxacillinase OXA-23-like and corresponding to IC 2. Sequence types (STs) obtained from the Oxford scheme correlate well with PFGE patterns, while the STs of the Pasteur scheme are more in accordance with rep-PCR grouping, but neither one is mirroring completely the results of the comparator. On two occasions the Oxford scheme identified two different STs within a single outbreak where PFGE patterns had only one band difference. The CCs of both MLST schemes were able to define clonal clusters that were concordant with the ICs determined by rep-PCR. IC4 corresponds to the previously described CC15 Pasteur (= CC103 Oxford). It can be concluded that both MLST schemes are valuable tools for population-based studies. In addition, the higher discriminatory power of the Oxford scheme that compares with the resolution obtained with PFGE can often aid in outbreak analysis.

In vitro activity of ceftazidime, ceftaroline and aztreonam alone and in combination with avibactam against European Gram-negative and Gram-positive clinical isolates.

Recent clinical isolates of key Gram-negative and Gram-positive bacteria were collected in 2012 from hospitalised patients in medical centres in four European countries (France, Germany, Italy and Spain) and were tested using standard broth microdilution methodology to assess the impact of 4 mg/L avibactam on the in vitro activities of ceftazidime, ceftaroline and aztreonam. Against Enterobacteriaceae, addition of avibactam significantly enhanced the level of activity of these antimicrobials. MIC(90) values (minimum inhibitory concentration that inhibits 90% of the isolates) of ceftazidime, ceftaroline and aztreonam for Escherichia coli, Klebsiella pneumoniae, Enterobacter cloacae, Enterobacter aerogenes, Citrobacter freundii and Morganella morganii were reduced up to 128-fold or greater when combined with avibactam. A two-fold reduction in the MIC(90) of ceftazidime to 8 mg/L was noted in Pseudomonas aeruginosa isolates when combined with avibactam, whereas little effect of avibactam was noted on the MIC values of the test compounds when tested against Acinetobacter baumannii isolates. Avibactam had little effect on the excellent activity of ceftazidime, ceftaroline and aztreonam against Haemophilus influenzae. It had no impact on the in vitro activity of ceftazidime and ceftaroline against staphylococci and streptococci. This study demonstrates that addition of avibactam enhances the activities of ceftazidime, ceftaroline and aztreonam against Enterobacteriaceae and P. aeruginosa but not against A. baumannii.

Nosocomial bloodstream infections due to Candida spp. in the USA: species distribution, clinical features and antifungal susceptibilities.

Candida spp. are among the most frequent nosocomial pathogens, contributing significantly to morbidity and mortality. Longitudinal data on the epidemiology of Candida bloodstream infections (BSIs) are still limited. Isolates and clinical data from 1218 episodes of Candida BSI were prospectively collected from patients in 52 hospitals in the USA between 1998 and 2006. Susceptibilities to amphotericin B, flucytosine, fluconazole, posaconazole, voriconazole, anidulafungin, caspofungin and micafungin were determined for 1077 Candida isolates by the CLSI reference broth microdilution method using the recently published species-specific clinical breakpoints. Candida albicans was the most prevalent species (50.7%), followed by Candida parapsilosis (17.4%), Candida glabrata (16.7%) and Candida tropicalis (10.2%). The prevalence of non-albicans Candida spp. increased over time. Patients had a mean age of 51 years and a mean length of hospital stay prior to BSI of 22 days. The main underlying conditions were gastrointestinal (20.1%) and pulmonary (13.0%) diseases. Intravenous catheters (19.1%) and the urinary tract (8.0%) were the most frequently determined likely sources, whilst in the majority of patients (61.1%) no source could be identified. Overall mortality was 38.1%. Of the isolates studied, 0.8% of C. albicans, 100.0% of C. glabrata, 2.9% of C. parapsilosis and 4.9% of C. tropicalis were non-susceptible to fluconazole, and 0.6% of C. albicans, 5.0% of Candida krusei, 7.6% of C. parapsilosis and 9.8% of C. tropicalis were non-susceptible to voriconazole. All echinocandins showed good activity against most Candida spp., including the majority of C. parapsilosis isolates, but only 38.1% of C. glabrata tested susceptible to caspofungin.

In vitro activity of the siderophore monosulfactam BAL30072 against meropenem-non-susceptible Acinetobacter baumannii.

OBJECTIVES: The activity of BAL30072 was compared with that of anti-Acinetobacter reference drugs against meropenem-non-susceptible Acinetobacter baumannii isolates associated with up-regulation of the intrinsic OXA-51-like enzyme or an acquired OXA. METHODS: Antimicrobial susceptibility testing was investigated by broth microdilution of 310 non-duplicate, meropenem-non-susceptible A. baumannii isolates to BAL30072, amikacin ampicillin/sulbactam, aztreonam, cefepime, colistin, imipenem, levofloxacin, meropenem, rifampicin, tigecycline and tobramycin. RESULTS: BAL30072 showed greater activity than the ß-lactam comparators, levofloxacin, amikacin, tobramycin and rifampicin. The activity of BAL30072 was comparable to that of tigecycline, with an MIC(50) of 2 mg/L. Elevated BAL30072 MICs were found, but there was no correlation with elevated MICs of the other antimicrobials. CONCLUSIONS: BAL30072 is a promising new agent with good activity against carbapenem-non-susceptible A. baumannii.

Nosocomial bloodstream infections due to Acinetobacter baumannii, Acinetobacter pittii and Acinetobacter nosocomialis in the United States.

OBJECTIVES: To compare the clinical features and antimicrobial susceptibilities of the clinically most important Acinetobacter species Acinetobacter baumannii, Acinetobacter pittii (formerly Acinetobacter genomic species 3) and Acinetobacter nosocomialis (formerly Acinetobacter genomic species 13TU). METHODS: 295 Acinetobacter isolates collected prospectively from patients with bloodstream infections (BSI) in 52 US hospitals were identified to species level. Clinical and microbiological features were compared between species. RESULTS: A. baumannii (63%) was the most prevalent species, followed by A. nosocomialis (21%), and A. pittii (8%). Intravascular catheters (15.3%) and the respiratory tract (12.9%) were the most frequent sources of BSI. A higher overall mortality was observed in patients with A. baumannii BSI than in patients with BSI caused by A. nosocomialis and A. pittii (36.9% vs. 16.4% and 13.0%, resp., p < 0.001). The most active antimicrobial agents as determined by broth microdilution were tigecycline (99.6% of isolates susceptible), colistin (99.3%), amikacin (98.5%), and imipenem (95.2%). 27 isolates (10.0%) were multi-drug resistant, all but one of these were A. baumannii. CONCLUSIONS: About one third of Acinetobacter BSI in our study were caused by A. nosocomialis or A. pittii. Patients with A. baumannii BSI had a less favorable outcome.

Evaluation of quantitative antibiotic susceptibility testing by Vitek 2 as a routine method to predict strain relatedness of coagulase-negative staphylococci isolated from blood cultures.

To test the hypothesis that the strain relatedness of coagulase-negative staphylococci (CoNS) recovered from blood cultures can be inferred from automated antibiotic susceptibility testing (AST) results generated by Vitek 2, concordant or discordant AST results were compared with pulsed-field gel electrophoresis (PFGE) typing results for 119 CoNS blood culture isolate pairs. Concordant AST results were highly predictive of the strain relatedness of CoNS isolates.

Molecular evolution of methicillin-resistant Staphylococcus aureus in the metropolitan area of Cologne, Germany, from 1984 to 1998.

To investigate the molecular evolution of methicillin-resistant Staphylococcus aureus (MRSA) in a large metropolitan area in Germany, 398 nonrepetitive MRSA isolates recovered from patients from various teaching and nonteaching hospitals in Cologne between 1984 and 1998 were characterized by pulsed-field gel electrophoresis (PFGE). On this basis, 95 representative isolates were selected and further investigated by multilocus sequence typing (MLST), spa typing, and staphylococcal cassette chromosome mec (SCCmec) typing. Overall, there were 9 MLST types and 16 spa types. The most prevalent sequence types (STs) were ST239 (38% of isolates), ST247 (29%), and ST228 (18%); the most prevalent spa types were 37 (32%) and 51 (29%). ST239 comprised five major PFGE types and various unique PFGE patterns, and ST5 comprised two PFGE types. While the same PFGE pattern was not observed among strains with different STs, spa type 37 was observed among strains representing two different STs (ST239 and ST241), and these belonged to the same clonal complex as single-locus variants. ST239 was the earliest predominant ST, with the highest prevalence from 1984 to 1988 (96%), followed by ST247 from 1989 to 1993 (83%) and ST228 from 1994 to 1998 (40%). Spa type 37 was the most prevalent from 1984 to 1988 (96%), spa type 51 was the most prevalent from 1989 to 1993 (83%), and spa types 1 and 458 were the most prevalent from 1994 to 1998 (26% and 14%, respectively). The prevalence of SCCmec type III decreased from 96% from 1984 to 1988 to 8% from 1989 to 1993, the prevalence of SCCmec type I increased from 4% from 1984 to 1988 to 97% from 1989 to 1993 and decreased to 62% from 1994 to 1998. While the genetic diversity of MRSA increased from 1984 to 1998, one prevalent ST usually accounted for most of the isolates in a given time period.

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.

Selection of topoisomerase mutations and overexpression of adeB mRNA transcripts during an outbreak of Acinetobacter baumannii.

OBJECTIVES: To investigate the mechanism of ciprofloxacin resistance in isolates of Acinetobacter baumannii during two hospital outbreaks and to determine the expression level of the gene encoding the AdeB efflux pump. METHODS: Isolates were previously typed by PFGE and their MICs determined by broth microdilution. The gyrA and parC genes were sequenced and the adeB gene examined by real-time reverse transcription PCR (RT-PCR). RESULTS: Two clonal lineages were responsible for the two hospital outbreaks. In both outbreaks, ciprofloxacin susceptibility was reduced during the course of the outbreak when compared with the index isolates. Mutations in gyrA and parC were found to have occurred during the outbreak. The MICs of non-fluoroquinolone antibiotics were raised in one clonal lineage and this was associated with a >10-fold increase in mRNA transcripts for adeB. CONCLUSIONS: We have witnessed the appearance of gyrA and parC mutations during outbreaks of A. baumannii. In parallel with these mutations, we observed up-regulation of the adeB gene associated with a decrease in susceptibility to non-fluoroquinolone antibiotics. These data illustrate the propensity for A. baumannii to develop multi-drug resistance rapidly.

In vitro activities of the beta-lactamase inhibitors clavulanic acid, sulbactam, and tazobactam alone or in combination with beta-lactams against epidemiologically characterized multidrug-resistant Acinetobacter baumannii strains.

Acinetobacter baumannii is an important nosocomial pathogen usually in the context of serious underlying disease. Multidrug resistance in these organisms is frequent. The beta-lactamase inhibitors clavulanic acid, sulbactam, and tazobactam have intrinsic activity against Acinetobacter strains. To evaluate their potential therapeutic usefulness, we determined the in vitro activity of ampicillin, sulbactam, ampicillin-sulbactam, cefoperazone, cefoperazone-sulbactam, piperacillin, piperacillin-sulbactam, tazobactam, piperacillin-tazobactam, amoxicillin, clavulanic acid, amoxicillin-clavulanic acid, ticarcillin, and ticarcillin-clavulanic acid against multidrug-resistant A. baumannii. All isolates were epidemiologically characterized by RAPD [random(ly) amplified polymorphic DNA] analysis and/or pulsed-field gel electrophoresis and represented different strain types, including sporadic strains, as well as outbreak-related strains. The MICs were determined by agar dilution on Mueller-Hinton agar (using fixed concentrations, as well as fixed ratios for beta-lactamase inhibitors) and the E-test. The majority of E-test results were within two dilutions of those recorded by agar dilution, with the exception of piperacillin-tazobactam. Sulbactam was superior to clavulanic acid and tazobactam and may represent an alternative treatment option for infections due to multiresistant A. baumannii strains. beta-Lactamase inhibitors have intrinsic activity but do not enhance activity of beta-lactams against A. baumannii. Testing with the inhibitor added at a fixed concentration as recommended for piperacillin-tazobactam and ticarcillin-clavulanic acid by the National Committee for Clinical Laboratory Standards may falsely suggest high activity or gives uninterpretable results due to trailing. If combinations are used for testing, fixed ratios may give more useful results.