Kevin J. Towner

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.

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

ABSTRACT 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.

Endemic Carbapenem Resistance Associated with OXA-40 Carbapenemase among Acinetobacter baumannii Isolates from a Hospital in Northern Spain

ABSTRACT Eighty-two carbapenem-resistant isolates of Acinetobacter baumannii from a single hospital in Bilbao were typed into two major clusters and several subclusters. Disk synergy tests and PCR indicated the production of a zinc-independent OXA-class carbapenemase. Sequencing identified this enzyme, OXA-40, as a variant of the OXA-24-OXA-25-OXA-26 cluster.

OXA‐51‐like β‐lactamases and their association with particular epidemic lineages of Acinetobacter baumannii

Sixty diverse clinical Acinetobacter baumannii isolates of worldwide origin were assigned to sequence groups, based on a multiplex PCR for the ompA, csuE and bla(OXA-51-like) genes. The majority (77%) of isolates belonged to sequence groups 1 and 2 (SG1 and SG2), with sequence group 3 (SG3) and non-grouped isolates accounting for the remainder. The isolates were not closely related according to pulsed-field gel electrophoresis (PFGE), and the majority were sensitive to imipenem and meropenem. The construction of a linkage map of OXA-51-like beta-lactamase sequence relationships revealed two closely related clusters of enzymes, one focused around OXA-66 and the other around OXA-69. Isolates belonging to SG1 encoded an enzyme from the OXA-66 cluster, while those belonging to SG2 encoded an enzyme from the OXA-69 cluster. All SG3 isolates encoded OXA-71, which does not form part of a close enzyme grouping. Major multinational lineages accounted for a significant proportion of A. baumannii clinical isolates, and the evolution of the OXA-51-like enzymes appears to be an ongoing process.

Evaluation of an Isothermal Signal Amplification Method for Rapid Detection of Methicillin-Resistant Staphylococcus aureus from Patient-Screening Swabs

ABSTRACT 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 × 105 and 106 CFU/assay, equivalent to 4 × 106 to 2 × 107 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 102 to 105 CFU/assay (below the CytAMP detection limit of 2 × 105 CFU/assay), and the sixth contained 106 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.

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.

Outbreak in Croatia caused by a new carbapenem-resistant clone of Acinetobacter baumannii producing OXA-72 carbapenemase.

Acinetobacter baumannii is a multidrug-resistant opportunistic pathogen that causes nosocomial infections and outbreaks, particularly in the intensive care unit (ICU) setting.1 Many outbreak strains belong to one of three worldwide lineages, known originally as European clones I, II and III. These correspond to sequence groups 2, 1 and 3, respectively, each of which includes a number of different genotypes defined by pulsed-field gel electrophoresis (PFGE).2 Only two previous reports have analysed carbapenem resistance inmultidrug-resistant isolates of A. baumannii from Croatia.3,4 In Split, the first carbapenem-resistant isolate of A. baumannii [meropenem minimum inhibitory concentration (MIC): 16 mg/mL] was isolated in 2002 at Split University Hospital. Between 2002 and 2008, >100 patient isolates were found to belong to the European clone 1 lineage, with most isolates carrying a blaOXA-107 gene associated with ISAba1.3 We now wish to report a new clone of multidrugresistant A. baumannii causing an outbreak in Split University Hospital following inward transfer of a patient. On 5 January 2009, a female aged 51 years was transferred to the intensive care unit (ICU) of Split University Hospital, Croatia, following brain surgery for glioblastoma at the General Hospital Mostar, Bosnia Herzegovina. According to the transfer letter, multidrug-resistant Acinetobacter sp. was isolated from a bronchial aspirate at Mostar General Hospital. No published data are available concerning the incidence, molecular basis and epidemiology of carbapenem-resistant isolates of Acinetobacter in Bosnia Herzegovina. On the day of transfer, blood cultures, cerebrospinal fluid and bronchial lavage were taken. Two days after transfer, carbapenemresistant A. baumannii was isolated from blood culture, and a day later from the bronchial lavage. Initial identification was made using the ATB 32GN and Vitek 2 systems (bioMérieux, Marcy l’Etoile, France), with A. baumannii confirmed by tRNA spacer fingerprinting and the presence of an OXA-51-type b-lactamase (specific to A. baumannii).2 Susceptibility to b-lactams (ceftazidime, cefepime, imipenem, meropenem), b-lactam/b-lactamase inhibitor combinations (ampicillin/sulbactam, piperacillin/tazobactam), aminoglycosides (amikacin, gentamicin) and fluoroquinolones (ciprofloxacin) was determined by disc-diffusion tests, and susceptibility to colistin by E-tests (AB Biodisk, Solna, Sweden). MICs were determined by broth microdilution according to Clinical and Laboratory Standards Institute recommendations. The A. baumannii isolates were resistant to all antimicrobials tested except colistin (MIC: 0.5 mg/mL) and ampicillin/sulbactam (MIC: 1 mg/mL). The patient was treated with intravenous ampicillin/sulbactam and colistin in contact isolation, but died two weeks following transfer. During the next 6 months (January to July 2009), 32 similar consecutive isolates were obtained from blood cultures, urine samples, catheter tip specimens, cerebrospinal fluid, throat and nasal swabs, and bronchial secretions collected from 23 different patients hospitalised in two ICUs and three different departments at Split University Hospital. PFGE following macrorestriction of genomic DNA with ApaI revealed that all isolates belonged to the European clone 2 lineage. All isolates also displayed the same multidrug resistance pattern (with no inhibition zone around imipenem or meropenem discs), but susceptibility to sulbactam and colistin (Table I). Bacterial DNA was extracted using a DNAze kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. The presence of genes encoding class D carbapenemases was detected by multiplex polymerase chain reaction using primers specific for the OXA-23, OXA-40, OXA-51 and OXA-58 gene families.5 Each isolate possessed a blaOXA-51-like and a blaOXA-40-like gene. Sequencing (both strands) of the blaOXA-51-like amplicons from a randomly selected subset of isolates by a commercial laboratory (Macrogen, Seoul, South Korea) revealed the presence of the OXA-90 gene (a variant of OXA-66), whereas sequencing of the blaOXA-40-like amplicons revealed the presence of a gene encoding OXA-72.2 OXA-40-like carbapenemases were originally described in A. baumannii isolates from the Iberian peninsula, but have now been reported worldwide, with sequence divergences between the members of this family varying from one to five amino acids.6,7 OXA-72was first described in an Acinetobacter isolate fromThailand in 2004, and has since been reported in a single A. baumannii isolate from mainland China and as a major mechanism of carbapenem resistance in a Taiwanese hospital.8 This is the first report of OXA-72 in Southeast Europe. PFGE analysis revealed that this new imported clone belonged to the European clone 2 lineage, in contrast to carbapenem-resistant isolates of A. baumannii from the same hospital during the preceding six years, which belonged to the European clone 1 lineage and encoded only the unusual OXA-51-type enzyme OXA-107, associated with ISAba1.3 These observations reinforce the continued importance of careful epidemiological surveillance and enhanced control measures following international patient transfers. Wherever possible, hospitals should routinely isolate and screen all patients admitted from hospitals in other countries. This is particularly important when the source country is known to have a high prevalence of multidrug-resistant bacteria or when no antimicrobial surveillance data are available. It is mandatory to rapidly identify the source and/or reservoir of colonisation/infection with multidrug-resistant A. baumannii in order to prevent further dissemination inside a hospital.

Direct comparison of two commercially available computer programs for analysing DNA fingerprinting gels.

Randomly amplified polymorphic DNA (RAPD) fingerprints were generated with M13 and DAF4 primers for 25 isolates of Acinetobacter spp. obtained from 16 different hospitals situated in 12 countries. The overall robustness of the algorithms and the reproducibility of the cluster analysis results generated by two commercially available computer programs (GelCompar and DENDRON) for analysing DNA fingerprinting gels were tested by examining the same set of fingerprinting data independently in two laboratories with the different software packages. Both programs were efficient at recognising and grouping strains with closely similar RAPD fingerprints, i.e., strains which might be expected to have a close epidemiological or evolutionary relationship. However, the relationships suggested for less closely related strains showed considerable variation in terms of the overall similarity or percentage correlation values suggested by the programs. It was concluded that both programs were useful tools for indicating close genotypic relationships between individual strains, but that epidemiological conclusions based on the similarity or correlation values (or the dendrograms derived from them) obtained for less closely related strains should be treated with considerable caution.

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

ABSTRACT 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 β-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.