Acinetobacter baumannii: evolution of a global pathogen.

Acinetobacter baumannii is an opportunistic nosocomial pathogen and one of the six most important multidrug-resistant microorganisms in hospitals worldwide. This human pathogen is responsible for a vast array of infections, of which ventilator-associated pneumonia and bloodstream infections are the most common, and mortality rates can reach 35%. Community-acquired infections have also been reported, but few strains have been recovered from environmental sources and infection reservoirs external to the hospital have not been identified. The majority of A. baumannii infections are caused by two main population clones with worldwide distribution. Infection outbreaks are often associated with multidrug resistance, including the recent emergence of strains resistant to all available antibiotics. Nevertheless, A. baumannii virulence traits and pathogenic potential have mostly remained elusive. The recent expansion of A. baumannii sequenced genomes has permitted the development of large-array phylogenomic and phenotypic analyses, which can offer valuable insights into the evolution and adaptation of A. baumannii as a human pathogen. This review summarises these recent advances, with particular focus on A. baumannii evolutionary and genomic aspects, and proposes new avenues of research.

Acinetobacter infection--an emerging threat to human health.

The genus Acinetobacter comprises a complex and heterogeneous group of bacteria, many of which are capable of causing a range of opportunistic, often catheter-related, infections in humans. However, Acinetobacter baumannii, as well as its close relatives belonging to genomic species 3 ("Acinetobacter pittii") and 13TU ("Acinetobacter nosocomialis"), are important nosocomial pathogens, often associated with epidemic outbreaks of infection, that are only rarely found outside of a clinical setting. These organisms are frequently pandrug-resistant and are capable of causing substantial morbidity and mortality in patients with severe underlying disease, both in the hospital and in the community. Several epidemic clonal lineages of A. baumannii have disseminated worldwide and seem to have a selective advantage over non-epidemic strains. The reasons for the success of these epidemic lineages remain to be elucidated, but could be related to the potential of these organisms to achieve very dynamic reorganization and rapid evolution of their genome, including the acquisition and expression of additional antibiotic resistance determinants, under fluctuating environmental and selective conditions.

Distribution of intrinsic plasmid replicase genes and their association with carbapenem-hydrolyzing class D beta-lactamase genes in European clinical isolates of Acinetobacter baumannii.

Ninety-six genetically diverse multidrug-resistant clinical isolates of Acinetobacter baumannii from 25 hospitals in 17 European countries were screened by PCR for specific carbapenemase-hydrolyzing class D ß-lactamase (CHDL) genes and by PCR-based replicon typing for the presence of 19 different plasmid replicase (rep) gene homology groups (GRs). Results were confirmed by DNA sequencing where necessary. All 96 isolates contained at least 1 (with a maximum of 4) of the 19 groups of rep genes. Groups detected were GR6 (repAci6; 93 isolates), GR2 (including repAci1 [67 isolates] and repAci2 [3 isolates]), GR16 (repApAB49; 12 isolates), GR12 (p2ABSDF0001; 10 isolates), GR3 (repAci3; 4 isolates), GR4 (repAci4; 3 isolates), GR10 (repAciX; 1 isolate), and GR14 (repp4AYE; 1 isolate). Variations in rep gene content were observed even among epidemiologically related isolates. Genes encoding OXA-58-like CHDLs (22 isolates) were associated with carriage of the repAci1, repAci3, repAci4, and repAciX genes, genes encoding OXA-40-like CHDLs (6 isolates) were associated with repAci2 and p2ABSDF0001, and genes encoding OXA-23-like CHDLs (8 isolates) were associated with repAci1. Most intrinsic Acinetobacter plasmids are non-self-transferable, but the almost ubiquitous repAci6 gene was strongly associated with a potential tra locus that could serve as a general system for plasmid mobilization and consequent horizontal transmission of plasmids and their associated antibiotic resistance genes among strains of A. baumannii.

Genome-assisted identification of putative iron-utilization genes in Acinetobacter baumannii and their distribution among a genotypically diverse collection of clinical isolates.

New putative iron-uptake genes were identified in published genomes of the opportunistic human pathogen Acinetobacter baumannii, and their occurrence was determined in a genotypically distinct collection of 50 clinical isolates by PCR and Southern blot assays. The results demonstrated that all A. baumannii isolates tested share the coding potential for two endogenous siderophores, a heme-acquisition and a ferrous iron-uptake system. A second heme-uptake cluster was detected in almost two thirds of isolates, without any apparent correlation with the clonal lineage of the strains. The wide distribution of multiple iron-acquisition systems among diverse A. baumannii clinical isolates argues for a contribution of iron uptake to the pathogenicity of this species.

Characterization of epidemiologically unrelated Acinetobacter baumannii isolates from four continents by use of multilocus sequence typing, pulsed-field gel electrophoresis, and sequence-based typing of bla(OXA-51-like) genes.

This study used a diverse collection of epidemiologically unrelated Acinetobacter baumannii isolates to compare the robustness of a multilocus sequence typing (MLST) scheme, based on conserved regions of seven housekeeping genes, gltA, gdhB, recA, cpn60, rpoD, gyrB, and gpi, with that of sequence-based typing of bla(OXA-51-like) genes (SBT-bla(OXA-51-like) genes). The data obtained by analysis of MLST and SBT-bla(OXA-51-like) genes were compared to the data generated by pulsed-field gel electrophoresis (PFGE). The topologies of the phylogenetic trees generated for the gyrB and gpi genes showed evidence of recombination and were inconsistent with those of the trees generated for the other five genes. MLST identified 24 sequence types (STs), of which 19 were novel, and 5 novel alleles. Clonality was demonstrated by eBURST analysis and standardized index of association values of >1 (P < 0.001). MLST data revealed that all isolates harboring the major bla(OXA-51-like) alleles OXA-66, OXA-69, and OXA-71 fell within the three major European clonal lineages. However, the MLST data were not always in concordance with the PFGE data, and some isolates containing the same bla(OXA-51-like) allele demonstrated <50% relatedness by PFGE. It was concluded that the gyrB and gpi genes are not good candidates for use in MLST analysis and that a SBT-bla(OXA-51-like) gene scheme produced results comparable to those produced by MLST for the identification of the major epidemic lineages, with the advantage of having a significantly reduced sequencing cost and time. It is proposed that studies of A. baumannii epidemiology could involve initial screening of bla(OXA-51-like) alleles to identify isolates belonging to major epidemic lineages, followed by MLST analysis to categorize isolates from common lineages, with PFGE being reserved for fine-scale typing.

Occurrence of OXA-107 and ISAba1 in carbapenem-resistant isolates of Acinetobacter baumannii from Croatia.

Carbapenem-resistant isolates of Acinetobacter baumannii from intensive care units at Split University Hospital, Split, Croatia, were studied. Most (100 of 106) had ISAba1 inserted upstream of a bla(OXA-107) gene, encoding an unusual OXA-51-type oxacillinase. Pulsed-field gel electrophoresis revealed that the isolates formed three clusters belonging to the sequence group 2 (European clone 1) lineage.

ATPase genes of diverse multidrug-resistant Acinetobacter baumannii isolates frequently harbour integrated DNA.

OBJECTIVES: The aim of the study was to determine whether ATPase genes of genetically diverse Acinetobacter baumannii isolates are disrupted by potential genomic islands. METHODS: Random amplified polymorphic DNA (RAPD)-PCR, sequence grouping and PFGE were used to investigate the genetic diversity of 50 A. baumannii isolated from various clinical specimens. PCR analysis was then used to identify isolates with a potentially disrupted ATPase gene. Representative genetically distinct isolates were further characterized by PCR mapping and chromosome walking to analyse the flanking regions of the disrupted ATPase genes. RESULTS: Forty-one of the 50 isolates tested appeared to contain a disrupted ATPase gene. Sequence group and PFGE data for 10 ATPase PCR-negative representative isolates confirmed substantial genetic diversity. Seven isolates contained elements with ends showing high levels of sequence similarity to one or both extremities of AbaR1, the first resistance island described in A. baumannii. A further isolate, A25, possessed a highly conserved AbaR1-like 3'-end, but a divergent, though related, 5'-terminus that exhibited near identity with a distinct locus in A. baumannii ATCC 17978. A ninth isolate (A92) possessed a completely novel sequence abutting on its 5'-ATPase remnant. Three isolates appeared to lack 3'-ATPase gene segments, as was the case with the recently sequenced strain ACICU. Thus, 8 of the 10 ATPase-negative isolates investigated in detail had ATPase genes disrupted with AbaR1-like flanking regions. CONCLUSIONS: ATPase genes of diverse A. baumannii isolates are frequently disrupted by insertions matching AbaR1-related flanking sequences. However, the ATPase gene of isolate A92 was disrupted by a DNA sequence distinct from those found in AbaR1.

Guidelines for the laboratory diagnosis and susceptibility testing of methicillin-resistant Staphylococcus aureus (MRSA).

These evidence-based guidelines have been produced after a literature review of the laboratory diagnosis and susceptibility testing of methicillin-resistant Staphylococcus aureus (MRSA). We have considered the detection of MRSA in screening samples and the detection of reduced susceptibility to glycopeptides in S. aureus. Recommendations are given for the identification of S. aureus and for suitable methods of susceptibility testing and screening for MRSA and for S. aureus with reduced susceptibility to glycopeptides. These guidelines indicate what tests should be used but not when the tests are applicable, as aspects of this are dealt with in guidelines on control of MRSA. There are currently several developments in screening media and molecular methods. It is likely that some of our recommendations will require modification as the new methods become available.

Comparison of Acinetobacter baumannii isolates from United Kingdom hospitals with predominant Northern European genotypes by amplified-fragment length polymorphism analysis.

Acinetobacter baumannii isolates collected between 1999 and 2001 from 46 United Kingdom hospitals were compared with previously identified northern European genotypes by amplified-fragment length polymorphism (AFLP) analysis. Two predominant northern European genotypes associated with outbreaks in the mid-1980s had been superseded by new outbreak-associated genotypes.

Frequencies and mechanisms of resistance to moxifloxacin in nosocomial isolates of Acinetobacter baumannii.

OBJECTIVES: To compare the in vitro activity of moxifloxacin and ciprofloxacin against 226 nosocomial isolates of Acinetobacter baumannii from 44 hospitals in the UK. METHODS: MICs of ciprofloxacin and moxifloxacin were determined by Etest. PCR analysis was used to detect chromosomal mutations in the gyrA and parC genes. Isolates resistant to ciprofloxacin and susceptible to moxifloxacin were examined for the ability to generate spontaneous moxifloxacin-resistant isolates. RESULTS: Of 226 isolates, 49.1% were resistant to ciprofloxacin and 39.4% were moxifloxacin-resistant according to BSAC criteria. Approximately 20% of isolates resistant to ciprofloxacin remained susceptible to moxifloxacin. A GyrA mutation at Ser-83 was found in all ciprofloxacin-resistant isolates. Single mutations in both the gyrA and parC genes at codons Ser-83 and Ser-80, respectively, were found in ciprofloxacin- and moxifloxacin-resistant isolates. Isolates that were ciprofloxacin-resistant but moxifloxacin-susceptible generated spontaneous moxifloxacin-resistant mutants when grown on medium containing up to 8x their initial MIC. However, these mutants were not stable and none displayed high-level moxifloxacin resistance. CONCLUSIONS: Moxifloxacin retained in vitro activity against some ciprofloxacin-resistant clinical A. baumannii isolates. Mutations in both gyrA and parC were necessary for resistance to moxifloxacin in most isolates of A. baumannii.

Detection of methicillin-resistant Staphylococcus aureus (MRSA) in blood with the EVIGENE MRSA detection kit.

A total of 200 blood cultures containing putative staphylococci were analyzed by a commercial gene probe hybridization assay (EVIGENE; Statens Serum Institut, Copenhagen, Denmark), and 18 were identified as methicillin-resistant Staphylococcus aureus (MRSA) positive. Of these, 17 were positive by PCR and 16 were positive by culture. Detailed analysis of the discrepant results showed that the EVIGENE kit allowed specific identification of MRSA in blood cultures without any of the drawbacks associated with PCR.

Evaluation of an isothermal signal amplification method for rapid detection of methicillin-resistant Staphylococcus aureus from patient-screening swabs.

A new molecular assay (CytAMP) utilizing isothermal signal-mediated amplification of RNA was evaluated for rapid detection of methicillin (oxacillin)-resistant Staphylococcus aureus (MRSA). The assay targeted the coa (coagulase) and mecA genes, thereby simultaneously identifying S. aureus and methicillin (oxacillin) resistance. Results were obtained in approximately 3.5 h as a color signal in 96-well microtiter plates. The detection limit was between 2 x 10(5) and 10(6) CFU/assay, equivalent to 4 x 10(6) to 2 x 10(7) CFU/ml in an overnight broth. This level of growth was obtained with an initial inoculum of 10 to 50 CFU. The CytAMP assay and a mecA-femB PCR assay both detected 113 MRSA strains among 396 clinical isolates of bacteria (CytAMP sensitivity and specificity were both 100% relative to those of PCR). Conventional culture detected 109 MRSA strains, but with 19 false-positive and 23 false-negative results relative to both molecular methods. Discrepancies were also observed for 100 enrichment broths containing MRSA screening swabs, with 11 broths culture negative but PCR positive. CytAMP and PCR were more in agreement, but six broths were CytAMP negative and PCR positive. Five of these contained 10(2) to 10(5) CFU/assay (below the CytAMP detection limit of 2 x 10(5) CFU/assay), and the sixth contained 10(6) CFU/assay. Overall, culture and CytAMP had similar sensitivities and specificities relative to those of PCR, but the CytAMP assay enabled swabs to be analyzed as a batch following overnight incubation in enrichment broth, with results reported before 12 noon the next day.

Population structure and antibiotic resistance of Acinetobacter DNA group 2 and 13TU isolates from hospitals in the UK.

A total of 287 Acinetobacter isolates belonging to DNA groups 2 (A. baumannii) and 13TU was collected consecutively from 46 hospitals and typed by randomly amplified polymorphic DNA fingerprinting with primers DAF-4 and ERIC-2. With a similarity coefficient of >/=72% as a cut-off value, 37 clusters of genotypically similar isolates (genotypes) were recognised. Four major clusters, found in 15, 12, 12 and 8 hospitals respectively, accounted for 42% of isolates, but only three of these predominant clusters were associated with outbreaks of infection in individual hospitals. Many of the isolates were resistant to multiple antibiotics, including expanded-spectrum beta-lactam agents, aminoglycosides, tetracyclines and fluoroquinolones, but >98% remained susceptible to carbapenems and colistin. Overall, the study demonstrated that a heterogeneous population of Acinetobacter DNA group 2 and 13TU isolates, frequently showing multiple resistance to antibiotics, was causing infections in UK hospitals, and that four predominant genotypes appeared to have disseminated among geographically distinct locations.

Antibiotic resistance among clinical isolates of Acinetobacter in the UK, and in vitro evaluation of tigecycline (GAR-936).

A survey was conducted of the antimicrobial susceptibilities of 595 Acinetobacter spp. isolated from routine clinical specimens in 54 sentinel laboratories throughout the UK during 2000. Isolates of the Acinetobacter baumannii complex (genomic groups 2, 3 and 13TU; n = 443) were distinguished from other genomic groups (n = 152) by PCR fingerprinting of tDNA spacer regions. MICs of amikacin, cefotaxime, ceftazidime, ciprofloxacin, colistin, gentamicin, imipenem, meropenem, minocycline, piperacillin, piperacillin/tazobactam, rifampicin, sulbactam and tetracycline were determined on IsoSensitest agar and interpreted, wherever possible, using BSAC breakpoints. Tigecycline (GAR-936), a new glycylcycline, was also tested. Resistance to cephalosporins, aminoglycosides and ciprofloxacin was widespread, but carbapenems, colistin, sulbactam, minocycline and tigecycline were each active against >80% of the isolates. Isolates of A. baumannii were more often resistant to cefotaxime, ceftazidime, piperacillin, piperacillin/tazobactam, ciprofloxacin, gentamicin and tetracyclines than those belonging to other genomic groups, but were less often resistant to colistin; no significant differences between genomic groups were noted in the susceptibilities to amikacin, carbapenems, rifampicin or sulbactam. The relative activities of the tetracyclines were minocycline > tigecycline > tetracycline. Thirteen carbapenem-resistant isolates (MICs > or =8 mg/L; 2.2%) were received from six centres; four centres sent single isolates; one sent three and one sent six. An allele of bla(IMP) was detected in one of these isolates, but the other 12 isolates either had carbapenemase-independent resistance, or undetectable carbapenemase activity combined with other resistance mechanisms. In conclusion, carbapenems, colistin and minocycline retained greatest activity against the Acinetobacter isolates collected. Tigecycline was less active than minocycline, but both agents overcame most tetracycline resistance.

Carriage of class 1 integrons and antibiotic resistance in clinical isolates of Acinetobacter baumannii from northern Spain.

A collection of 70 clinical isolates of Acinetobacter baumannii from Bilbao in northern Spain was examined by PCR for the presence of class 1 integron structures. The organisms comprised 21 distinct RAPD genotypes, with 10 distinct antibiogram patterns. Four different integron structures were detected in a total of 59 (84%) of the 70 isolates, with two predominant integron structures found in 20 and 30 isolates each. No clear antibiogram differences could be correlated with the presence or absence of integron structures, but sequence analysis of two of the internal integron regions indicated homology with genes encoding ANT(2'') adenyltransferase activity and AAC(6')-Ib acetyltransferase activity. Phenotypic analysis of aminoglycoside resistance profiles indicated that many isolates produced a combination of aminoglycoside-modifying enzymes, with most of the observed resistance to amikacin being associated with a gene encoding APH(3')-VI phosphotransferase, as detected by PCR. RAPD analysis indicated that all the Bilbao isolates producing APH(3')-VI were distinct from an epidemic integron-carrying and APH(3')-VI-producing Acinetobacter strain found in other regions of Spain. It is concluded that, although class 1 integrons are widely disseminated amongst clinical isolates of A. baumannii from the Bilbao region of Spain, at present they are not playing a major role in the dissemination of antibiotic resistance genes in this region.

Molecular epidemiology of quinolone resistance in Acinetobacter spp.

OBJECTIVE: To determine whether similar mutations to quinolone resistance in the gyrA subunit of DNA gyrase and the parC subunit of topoisomerase IV are occurring independently in genotypically unrelated clinical isolates of Acinetobacter spp., or whether worldwide clonal spread of particular resistant strains is occurring. METHODS: The genotypic relationships of 25 nosocomial isolates of Acinetobacter spp. from 15 locations in 11 different countries worldwide were examined by randomly amplified polymorphic DNA analysis. Quinolone resistance-determining regions of gyrA and parC were amplified by PCR and mutations were analyzed by restriction digestion with Hinfl and DNA sequencing. RESULTS: Twenty-four of the 25 Acinetobacter isolates were genotypically heterogeneous and 12 were resistant to both nalidixic acid and ciprofloxacin. Analysis of conserved gyrA and parC regions showed that all isolates with a ciprofloxacin MIC of 4 mg/L had a substitution of Ser83 with either Leu or Phe in the GyrA protein. Five of six isolates with ciprofloxacin MICs of 64 mg/L had additional substitutions of Ser80 with Leu in the ParC protein. CONCLUSIONS: Similar mutations to quinolone resistance, predominantly at codons 82--83 of gyrA, are occurring independently in genotypically distinct isolates of Acinetobacter spp. from different worldwide locations. Most isolates with high ciprofloxacin MICs also exhibited secondary mutations in parC at codons 79--80.