The Population Structure of Acinetobacter baumannii: Expanding Multiresistant Clones from an Ancestral Susceptible Genetic Pool

Outbreaks of hospital infections caused by multidrug resistant Acinetobacter baumannii strains are of increasing concern worldwide. Although it has been reported that particular outbreak strains are geographically widespread, little is known about the diversity and phylogenetic relatedness of A. baumannii clonal groups. Sequencing of internal portions of seven housekeeping genes (total 2,976 nt) was performed in 154 A. baumannii strains covering the breadth of known diversity and including representatives of previously recognized international clones, and in 19 representatives of other Acinetobacter species. Restricted amounts of diversity and a star-like phylogeny reveal that A. baumannii is a genetically compact species that suffered a severe bottleneck in the recent past, possibly linked to a restricted ecological niche. A. baumannii is neatly demarcated from its closest relative (genomic species 13TU) and other Acinetobacter species. Multilocus sequence typing analysis demonstrated that the previously recognized international clones I to III correspond to three clonal complexes, each made of a central, predominant genotype and few single locus variants, a hallmark of recent clonal expansion. Whereas antimicrobial resistance was almost universal among isolates of these and a novel international clone (ST15), isolates of the other genotypes were mostly susceptible. This dichotomy indicates that antimicrobial resistance is a major selective advantage that drives the ongoing rapid clonal expansion of these highly problematic agents of nosocomial infections.     

Synthetic arylquinuclidine derivatives exhibit antifungal activity against Candida albicans, Candida tropicalis and Candida parapsilopsis

Background

Severely burned patients may develop life-threatening nosocomial infections due to Pseudomonas aeruginosa, which can exhibit a high-level of resistance to antimicrobial drugs and has a propensity to cause nosocomial outbreaks. Antiseptic and topical antimicrobial compounds constitute major resources for burns care but in vitro testing of their activity is not performed in practice.

Results

In our burn unit, a P. aeruginosa clone multiresistant to antibiotics colonized or infected 26 patients over a 2-year period. This resident clone was characterized by PCR based on ERIC sequences. We investigated the susceptibility of the resident clone to silver sulphadiazine and to the main topical antimicrobial agents currently used in the burn unit. We proposed an optimized diffusion assay used for comparative analysis of P. aeruginosa strains. The resident clone displayed lower susceptibility to silver sulphadiazine and cerium silver sulphadiazine than strains unrelated to the resident clone in the unit or unrelated to the burn unit.

Conclusions

The diffusion assay developed herein detects differences in behaviour against antimicrobials between tested strains and a reference population. The method could be proposed for use in semi-routine practice of medical microbiology.     

Comparative genomic analysis of Acinetobacter baumannii clinical isolates reveals extensive genomic variation and diverse antibiotic resistance determinants

Background

Acinetobacter baumannii is an important nosocomial pathogen that poses a serious health threat to immune-compromised patients. Due to its rapid ability to develop multidrug resistance (MDR), A. baumannii has increasingly become a focus of attention worldwide. To better understand the genetic variation and antibiotic resistance mechanisms of this bacterium at the genomic level, we reported high-quality draft genome sequences of 8 clinical isolates with various sequence types and drug susceptibility profiles.

Results

We sequenced 7 MDR and 1 drug-sensitive clinical A. baumannii isolates and performed comparative genomic analysis of these draft genomes with 16 A. baumannii complete genomes from GenBank. We found a high degree of variation in A. baumannii, including single nucleotide polymorphisms (SNPs) and large DNA fragment variations in the AbaR-like resistance island (RI) regions, the prophage and the type VI secretion system (T6SS). In addition, we found several new AbaR-like RI regions with highly variable structures in our MDR strains. Interestingly, we found a novel genomic island (designated as GI_BJ4) in the drug-sensitive strain BJ4 carrying metal resistance genes instead of antibiotic resistance genes inserted into the position where AbaR-like RIs commonly reside in other A. baumannii strains. Furthermore, we showed that diverse antibiotic resistance determinants are present outside the RIs in A. baumannii, including antibiotic resistance-gene bearing integrons, the bla_OXA-23-containing transposon Tn2009, and chromosomal intrinsic antibiotic resistance genes.

Conclusions

Our comparative genomic analysis revealed that extensive genomic variation exists in the A. baumannii genome. Transposons, genomic islands and point mutations are the main contributors to the plasticity of the A. baumannii genome and play critical roles in facilitating the development of antibiotic resistance in the clinical isolates.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-1163) contains supplementary material, which is available to authorized users.     

Acinetobacter spp. as nosocomial pathogens: Epidemiology and resistance features

The genus Acinetobacter is a major cause of nosocomial infections; it is increasingly being associated with various epidemics and has become a widespread concern in a variety of hospitals worldwide. Multi-antibiotic resistant Acinetobacter baumannii, is now recognized to be of great clinical significance. Numerous reports relay to the spread of A. baumannii in the hospital settings which leads to enhanced nosocomial outbreaks associated with high death rates. However, many other Acinetobacter spp. also can cause nosocomial infections. This review focused on the role of Acinetobacter spp. as nosocomial pathogens in addition to their persistence, antimicrobial resistance patterns and epidemiology.     

Genome Sequence of Acinetobacter baumannii TYTH-1

Acinetobacter baumannii has emerged recently as a major cause of health care-associated infections due to the extent of its antimicrobial resistance and its propensity to cause large nosocomial outbreaks. Here we report the genome sequence of Acinetobacter baumannii TYTH-1 isolated in Taiwan during 2008.     

The Success of Acinetobacter Species; Genetic,Metabolic and Virulence Attributes

An understanding of why certain Acinetobacter species are more successful in causing nosocomial infections, transmission and epidemic spread in healthcare institutions compared with other species is lacking. We used genomic, phenotypic and virulence studies to identify differences between Acinetobacter species. Fourteen strains representing nine species were examined. Genomic analysis of six strains showed that the A. baumannii core genome contains many genes important for diverse metabolism and survival in the host. Most of the A. baumannii core genes were also present in one or more of the less clinically successful species. In contrast, when the accessory genome of an individual A. baumannii strain was compared to a strain of a less successful species (A. calcoaceticus RUH2202), many operons with putative virulence function were found to be present only in the A. baumannii strain, including the csu operon, the acinetobactin chromosomal cluster, and bacterial defence mechanisms. Phenotype microarray analysis showed that compared to A. calcoaceticus (RUH2202), A. baumannii ATCC 19606^T was able to utilise nitrogen sources more effectively and was more tolerant to pH, osmotic and antimicrobial stress. Virulence differences were also observed, with A. baumannii ATCC 19606^T, A. pittii SH024, and A. nosocomialis RUH2624 persisting and forming larger biofilms on human skin than A. calcoaceticus. A. baumannii ATCC 19606^T and A. pittii SH024 were also able to survive in a murine thigh infection model, whereas the other two species were eradicated. The current study provides important insights into the elucidation of differences in clinical relevance among Acinetobacter species.     

Comparative analysis of surface-exposed virulence factors of Acinetobacter baumannii

Background

Acinetobacter baumannii is a significant hospital pathogen, particularly due to the dissemination of highly multidrug resistant isolates. Genome data have revealed that A. baumannii is highly genetically diverse, which correlates with major variations seen at the phenotypic level. Thus far, comparative genomic studies have been aimed at identifying resistance determinants in A. baumannii. In this study, we extend and expand on these analyses to gain greater insight into the virulence factors across eight A. baumannii strains which are clonally, temporally and geographically distinct, and includes an isolate considered non-pathogenic and a community-acquired A. baumannii.

Results

We have identified a large number of genes in the A. baumannii genomes that are known to play a role in virulence in other pathogens, such as the recently studied proline-alanine-alanine-arginine (PAAR)-repeat domains of the type VI secretion systems. Not surprising, many virulence candidates appear to be part of the A. baumannii core genome of virulent isolates but were often found to be insertionally disrupted in the avirulent A. baumannii strain SDF. Our study also reveals that many known or putative virulence determinants are restricted to specific clonal lineages, which suggests that these virulence determinants may be crucial for the success of these widespread common clones. It has previously been suggested that the high level of intrinsic and adaptive resistance has enabled the widespread presence of A. baumannii in the hospital environment. This appears to have facilitated the expansion of its repertoire of virulence traits, as in general, the nosocomial strains in this study possess more virulence genes compared to the community-acquired isolate.

Conclusions

Major genetic variation in known or putative virulence factors was seen across the eight strains included in this study, suggesting that virulence mechanisms are complex and multifaceted in A. baumannii. Overall, these analyses increase our understanding of A. baumannii pathogenicity and will assist in future studies determining the significance of virulence factors within clonal lineages and/or across the species.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-1020) contains supplementary material, which is available to authorized users.     

Genome Sequence of Acinetobacter baumannii AC12, a Polymyxin-Resistant Strain Isolated from Terengganu,Malaysia

Acinetobacter baumannii is a major cause of nosocomial infection worldwide. We report the draft genome sequence of A. baumannii AC12, a multidrug-resistant nosocomial strain with additional resistance to carbapenems and polymyxin. The genome data will provide insights into the genetic basis of antimicrobial resistance and its adaptive mechanism.     

Identification of an Acinetobacter baumannii Zinc Acquisition System that Facilitates Resistance to Calprotectin-mediated Zinc Sequestration

Acinetobacter baumannii is an important nosocomial pathogen that accounts for up to 20 percent of infections in intensive care units worldwide. Furthermore, A. baumannii strains have emerged that are resistant to all available antimicrobials. These facts highlight the dire need for new therapeutic strategies to combat this growing public health threat. Given the critical role for transition metals at the pathogen-host interface, interrogating the role for these metals in A. baumannii physiology and pathogenesis could elucidate novel therapeutic strategies. Toward this end, the role for calprotectin- (CP)-mediated chelation of manganese (Mn) and zinc (Zn) in defense against A. baumannii was investigated. These experiments revealed that CP inhibits A. baumannii growth in vitro through chelation of Mn and Zn. Consistent with these in vitro data, Imaging Mass Spectrometry revealed that CP accompanies neutrophil recruitment to the lung and accumulates at foci of infection in a murine model of A. baumannii pneumonia. CP contributes to host survival and control of bacterial replication in the lung and limits dissemination to secondary sites. Using CP as a probe identified an A. baumannii Zn acquisition system that contributes to Zn uptake, enabling this organism to resist CP-mediated metal chelation, which enhances pathogenesis. Moreover, evidence is provided that Zn uptake across the outer membrane is an energy-dependent process in A. baumannii. Finally, it is shown that Zn limitation reverses carbapenem resistance in multidrug resistant A. baumannii underscoring the clinical relevance of these findings. Taken together, these data establish Zn acquisition systems as viable therapeutic targets to combat multidrug resistant A. baumannii infections.     

Isolation and Characterization of Antimicrobial Compounds in Plant Extracts against Multidrug-Resistant Acinetobacter baumannii

The number of fully active antibiotic options that treat nosocomial infections due to multidrug-resistant Acinetobacter baumannii (A. baumannii) is extremely limited. Magnolia officinalis, Mahonia bealei, Rabdosia rubescens, Rosa rugosa, Rubus chingii, Scutellaria baicalensis, and Terminalia chebula plant extracts were previously shown to have growth inhibitory activity against a multidrug-resistant clinical strain of A. baumannii. In this study, the compounds responsible for their antimicrobial activity were identified by fractionating each plant extract using high performance liquid chromatography, and determining the antimicrobial activity of each fraction against A. baumannii. The chemical structures of the fractions inhibiting >40% of the bacterial growth were elucidated by liquid chromatography/mass spectrometry analysis and nuclear magnetic resonance spectroscopy. The six most active compounds were identified as: ellagic acid in Rosa rugosa; norwogonin in Scutellaria baicalensis; and chebulagic acid, chebulinic acid, corilagin, and terchebulin in Terminalia chebula. The most potent compound was identified as norwogonin with a minimum inhibitory concentration of 128 µg/mL, and minimum bactericidal concentration of 256 µg/mL against clinically relevant strains of A. baumannii. Combination studies of norwogonin with ten anti-Gram negative bacterial agents demonstrated that norwogonin did not enhance the antimicrobial activity of the synthetic antibiotics chosen for this study. In conclusion, of all identified antimicrobial compounds, norwogonin was the most potent against multidrug-resistant A. baumannii strains. Further studies are warranted to ascertain the prophylactic and therapeutic potential of norwogonin for infections due to multidrug-resistant A. baumannii.     

Comparative genomics of multidrug resistance in Acinetobacter baumannii

Acinetobacter baumannii is a species of nonfermentative gram-negative bacteria commonly found in water and soil. This organism was susceptible to most antibiotics in the 1970s. It has now become a major cause of hospital-acquired infections worldwide due to its remarkable propensity to rapidly acquire resistance determinants to a wide range of antibacterial agents. Here we use a comparative genomic approach to identify the complete repertoire of resistance genes exhibited by the multidrug-resistant A. baumannii strain AYE, which is epidemic in France, as well as to investigate the mechanisms of their acquisition by comparison with the fully susceptible A. baumannii strain SDF, which is associated with human body lice. The assembly of the whole shotgun genome sequences of the strains AYE and SDF gave an estimated size of 3.9 and 3.2 Mb, respectively. A. baumannii strain AYE exhibits an 86-kb genomic region termed a resistance island—the largest identified to date—in which 45 resistance genes are clustered. At the homologous location, the SDF strain exhibits a 20 kb-genomic island flanked by transposases but devoid of resistance markers. Such a switching genomic structure might be a hotspot that could explain the rapid acquisition of resistance markers under antimicrobial pressure. Sequence similarity and phylogenetic analyses confirm that most of the resistance genes found in the A. baumannii strain AYE have been recently acquired from bacteria of the genera Pseudomonas, Salmonella, or Escherichia. This study also resulted in the discovery of 19 new putative resistance genes. Whole-genome sequencing appears to be a fast and efficient approach to the exhaustive identification of resistance genes in epidemic infectious agents of clinical significance.     

A multi-center blinded study on the efficiency of phenotypic screening methods to detect glycopeptide intermediately susceptible Staphylococcus aureus (GISA) and heterogeneous GISA (h-GISA)

Background

Staphylococcus aureus, one of the most frequently isolated pathogens in both hospitals and the community, has been particularly efficient at developing resistance to antimicrobial agents. In developed countries, as methicillin-resistant S. aureus (MRSA) has prevailed and, furthermore, as S. aureus with reduced susceptibility to vancomycin has emerged, the therapeutic options for the treatment of S. aureus infections have become limited. In developing countries and especially African countries very little is known concerning the resistance of S. aureus to antibiotics. In Madagascar no data exist concerning this resistance.

Objective

To update the current status of antibiotic resistance of S. aureus in Antananarivo, Madagascar.

Methods

Clinical S. aureus isolates were collected from patients at the Institut Pasteur of Madagascar from January 2001 to December 2005. Susceptibility tests with 18 antibiotics were performed by the disk diffusion method.

Results

Among a total of 574 isolates, 506 were from community-acquired infections and 68 from nosocomial infections. There was no significant difference in the methicillin resistance rate between community-acquired strains (33 of 506; 6.5%) and nosocomial strains (3 of 68, 4.4%). Many MRSA isolates were resistant to multiple classes of antibiotics. Resistance to tetracyclin, trimethoprim-sulfamethoxazole and erythromycin was more common. Among MRSA isolates resistance rates to rifampicin, fusidic acid, gentamicin and ciprofloxacin were lower than that observed with other drugs easily available in Madagascar. No isolates were resistant to glycopeptides.

Conclusion

The rate of methicillin-resistant S. aureus is not different between community-acquired and nosocomial infections and is still rather low in Madagascar.     

Inverse PCR for subtyping of <Emphasis Type="Italic">Acinetobacter baumannii</Emphasis> carrying IS<Emphasis Type="Italic">Aba</Emphasis>1

Acinetobacter baumannii has been prevalent in nosocomial infections, often causing outbreaks in intensive care units. ISAba1 is an insertion sequence that has been identified only in A. baumannii and its copy number varies among strains. It has been reported that ISAba1 provides a promoter for bla_OXA-51-like, bla_OXA-23-like, and bla_ampC, which are associated with the resistance of A. baumannii to carbapenems and cephalosporins. The main purpose of this study was to develop a novel inverse PCR method capable of typing A. baumannii strains. The method involves three major steps: cutting of genomic DNA with a restriction enzyme, ligation, and PCR. In the first step, bacterial genomic DNA was digested with DpnI. In the second step, the digested genomic DNAs were ligated to form intramolecular circular DNAs. In the last step, the ligated circular DNAs were amplified by PCR with primers specific for ISAba1 and the amplified PCR products were electrophoresed. Twenty-two clinical isolates of A. baumannii were used for the evaluation of the inverse PCR (iPCR) typing method. Dendrogram analysis revealed two major clusters, similar to pulsed-field gel electrophoresis (PFGE) results. Three ISAba1-associated genes — bla_ampC, bla_OXA-66-like, and csuD — were amplified and detected in the clinical isolates. This novel iPCR typing method is comparable to PFGE in its ability to discriminate A. baumannii strains, and is a promising molecular epidemiological tool for investigating A. baumannii carrying ISAba1.     

Identification and Characterization of Type II Toxin-Antitoxin Systems in the Opportunistic Pathogen Acinetobacter baumannii

Acinetobacter baumannii is an opportunistic pathogen that causes nosocomial infections. Due to the ability to persist in the clinical environment and rapidly acquire antibiotic resistance, multidrug-resistant A. baumannii clones have spread in medical units in many countries in the last decade. The molecular basis of the emergence and spread of the successful multidrug-resistant A. baumannii clones is not understood. Bacterial toxin-antitoxin (TA) systems are abundant genetic loci harbored in low-copy-number plasmids and chromosomes and have been proposed to fulfill numerous functions, from plasmid stabilization to regulation of growth and death under stress conditions. In this study, we have performed a thorough bioinformatic search for type II TA systems in genomes of A. baumannii strains and estimated at least 15 possible TA gene pairs, 5 of which have been shown to be functional TA systems. Three of them were orthologs of bacterial and archaeal RelB/RelE, HicA/HicB, and HigB/HigA systems, and others were the unique SplT/SplA and CheT/CheA TA modules. The toxins of all five TA systems, when expressed in Escherichia coli, inhibited translation, causing RNA degradation. The HigB/HigA and SplT/SplA TA pairs of plasmid origin were highly prevalent in clinical multidrug-resistant A. baumannii isolates from Lithuanian hospitals belonging to the international clonal lineages known as European clone I (ECI) and ECII.     

Draft genome sequence of a multidrug-resistant Acinetobacter baumannii ST15 (CC15) isolated from Brazil

Acinetobacter baumannii is an important pathogen frequently associated with nosocomial outbreaks around the world. In Brazil, A. baumannii has become particularly problematic because of its prevalence and the carbapenems resistance. Here, we report the draft genome sequence of a multidrug-resistant A. baumannii (ST15/CC15) isolated in 2009 from the state of Espírito Santo (Southeast Brazil). We observed important resistance determinant genes in an estimated genome size of 4,102,788 bp with 3,862 predicted coding regions. A detailed report of the genomic data analysis might help to understand the specific features of highly successful strains belonged to a relevant complex clonal in different Brazilian geographical regions.     

A comparative study of class 1 integrons in Acinetobacter baumannii

Multidrug resistance (MDR) in Acinetobacter baumannii is increasingly reported and has become a significant public concern. The method responsible for the acquisition of resistance genes via integrons from the environment or intra-species in A. baumannii remains to be understood. This study was performed to investigate the transmission route of these integrons using a comparative analysis of published A. baumannii complete genomes. The phylogenetic analysis of A. baumannii type 1 integrases (IntI1) showed that the integrons could be transferred across the two evolutionary lineages, the international clone I (IC I) and clone II (IC II) strains. In addition, the integrons in A. baumannii strains were mainly responsible for the transfer of resistance genes for two types of long-term usage antibiotics and antiseptics, such as aminoglycosides, chloramphenicol and the quaternary-ammonium-compound family. The in silico comparative analysis of known integron integrases revealed that the intI genes were phylogenetically related among A. baumannii strains and some microorganisms living in a sediment community, implicating that the integrons of A. baumannii might have originated from those microorganisms belonging to the β-preoteobacterial class in the sediment environment. The data suggest that the gain of class 1 integrons in A. baumannii strains may have started before the antibiotic era. This report shows that the origins of A. baumannii class 1 integrons may be the soil environment and that the resistance genes included in integrons are horizontally transferred across all the A. baumannii genomes, including IC I and IC II.     

Species Distribution of Clinical Acinetobacter Isolates Revealed by Different Identification Techniques

A total of 2582 non-duplicate clinical Acinetobacter spp. isolates were collected to evaluate the performance of four identification methods because it is important to identify Acinetobacter spp. accurately and survey the species distribution to determine the appropriate antimicrobial treatment. Phenotyping (VITEK 2 and VITEK MS) and genotyping (16S rRNA and rpoB gene sequencing) methods were applied for species identification, and antimicrobial susceptibility test of imipenem and meropenem was performed with a disk diffusion assay. Generally, the phenotypic identification results were quite different from the genotyping results, and their discrimination ability was unsatisfactory, whereas 16S rRNA and rpoB gene sequencing showed consistent typing results, with different resolution. Additionally, A. pittii, A. calcoaceticus and A. nosocomialis, which were phylogenetically close to A. baumannii, accounted for 85.5% of the non-A. baumannii isolates. One group, which could not be clustered with any reference strains, consisted of 11 isolates and constituted a novel Acinetobacter species that was entitled genomic species 33YU. None of the non-A. baumannii isolates harbored a bla _OXA-51-like gene, and this gene was disrupted by ISAba19 in only one isolate; it continues to be appropriate as a genetic marker for A. baumannii identification. The resistance rate of non-A. baumannii isolates to imipenem and/or meropenem was only 2.6%, which was significantly lower than that of A. baumannii. Overall, rpoB gene sequencing was the most accurate identification method for Acinetobacter species. Except for A. baumannii, the most frequently isolated species from the nosocomial setting were A. pittii, A. calcoaceticus and A. nosocomialis.     

耐碳青霉烯鲍曼不动杆菌的临床分布及基因组流行病学分析

[背景]鲍曼不动杆菌是造成临床感染的重要病原菌之一,其对碳青霉烯类抗生素的耐药形势日益严重,利用基因组测序技术解析其临床分布特征和流行病学规律有助于临床感染的有效防治.[目的]研究沧州市中心医院2018年检出的200株耐碳青霉烯鲍曼不动杆菌(carbapenem-resistant Acinetobacter baum...     

Deciphering the multifactorial nature of Acinetobacter baumannii pathogenicity

Background

Acinetobacter baumannii is an emerging bacterial pathogen that causes a broad array of infections, particularly in hospitalized patients. Many studies have focused on the epidemiology and antibiotic resistance of A. baumannii, but little is currently known with respect to its virulence potential.

Methodology/Principal Findings

The aim of this work was to analyze a number of virulence-related traits of four A. baumannii strains of different origin and clinical impact for which complete genome sequences were available, in order to tentatively identify novel determinants of A. baumannii pathogenicity. Clinical strains showed comparable virulence in the Galleria mellonella model of infection, irrespective of their status as outbreak or sporadic strains, whereas a non-human isolate was avirulent. A combined approach of genomic and phenotypic analyses led to the identification of several virulence factors, including exoproducts with hemolytic, phospholipase, protease and iron-chelating activities, as well as a number of multifactorial phenotypes, such as biofilm formation, surface motility and stress resistance, which were differentially expressed and could play a role in A. baumannii pathogenicity.

Conclusion/Significance

This work provides evidence of the multifactorial nature of A. baumannii virulence. While A. baumannii clinical isolates could represent a selected population of strains adapted to infect the human host, subpopulations of highly genotypically and phenotypically diverse A. baumannii strains may exist outside the hospital environment, whose relevance and distribution deserve further investigation.