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1.  The Population Structure of Acinetobacter baumannii: Expanding Multiresistant Clones from an Ancestral Susceptible Genetic Pool 
PLoS ONE  2010;5(4):e10034.
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.
doi:10.1371/journal.pone.0010034
PMCID: PMC2850921  PMID: 20383326
2.  Molecular Surveillance of European Quinolone-Resistant Clinical Isolates of Pseudomonas aeruginosa and Acinetobacter spp. Using Automated Ribotyping 
Journal of Clinical Microbiology  2000;38(10):3636-3645.
Nosocomial isolates of Pseudomonas aeruginosa and Acinetobacter spp. exhibit high rates of resistance to antibiotics and are often multidrug resistant. In a previous study (D. Milatovic, A. Fluit, S. Brisse, J. Verhoef, and F. J. Schmitz, Antimicrob. Agents Chemother. 44:1102–1107, 2000), isolates of these species that were resistant to sitafloxacin, a new advanced-generation fluoroquinolone with a high potency and a broad spectrum of antimicrobial activity, were found in high proportion in 23 European hospitals. Here, we investigate the clonal diversity of the 155 P. aeruginosa and 145 Acinetobacter spp. sitafloxacin-resistant isolates from that study by automated ribotyping. Numerous ribogroups (sets of isolates with indistinguishable ribotypes) were found among isolates of P. aeruginosa (n = 34) and Acinetobacter spp. (n = 16), but the majority of the isolates belonged to a limited number of major ribogroups. Sitafloxacin-resistant isolates (MICs > 2 mg/liter, used as a provisional breakpoint) showed increased concomitant resistance to piperacillin, piperacillin-tazobactam, ceftriaxone, ceftazidime, amikacin, gentamicin, and imipenem. The major ribogroups were repeatedly found in isolates from several European hospitals; these isolates showed higher levels of resistance to gentamicin and imipenem, and some of them appeared to correspond to previously described multidrug-resistant international clones of P. aeruginosa (serotype O:12) and Acinetobacter baumannii (clones I and II). Automated ribotyping, when used in combination with more discriminatory typing methods, may be a convenient library typing system for monitoring future epidemiological dynamics of geographically widespread multidrug-resistant bacterial clones.
PMCID: PMC87449  PMID: 11015376
3.  Identification and Characterization of Type II Toxin-Antitoxin Systems in the Opportunistic Pathogen Acinetobacter baumannii 
Journal of Bacteriology  2013;195(14):3165-3172.
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.
doi:10.1128/JB.00237-13
PMCID: PMC3697630  PMID: 23667234
4.  Characterization of a Novel Plasmid Type and Various Genetic Contexts of blaOXA-58 in Acinetobacter spp. from Multiple Cities in China 
PLoS ONE  2014;9(1):e84680.
Background/Objective
Several studies have described the epidemiological distribution of blaOXA-58-harboring Acinetobacter baumannii in China. However, there is limited data concerning the replicon types of blaOXA-58-carrying plasmids and the genetic context surrounding blaOXA-58 in Acinetobacter spp. in China.
Methodology/Principal Findings
Twelve non-duplicated blaOXA-58-harboring Acinetobacter spp. isolates were collected from six hospitals in five different cities between 2005 and 2010. The molecular epidemiology of the isolates was carried out using PFGE and multilocus sequence typing. Carbapenemase-encoding genes and plasmid replicase genes were identified by PCR. The genetic location of blaOXA-58 was analyzed using S1-nuclease method. Plasmid conjugation and electrotransformation were performed to evaluate the transferability of blaOXA-58-harboring plasmids. The genetic structure surrounding blaOXA-58 was determined by cloning experiments. The twelve isolates included two Acinetobacter pittii isolates (belong to one pulsotype), three Acinetobacter nosocomialis isolates (belong to two pulsotypes) and seven Acinetobacter baumannii isolates (belong to two pulsotypes/sequence types). A. baumannii ST91 was found to be a potential multidrug resistant risk clone carrying both blaOXA-58 and blaOXA-23. blaOXA-58 located on plasmids varied from ca. 52 kb to ca. 143 kb. All plasmids can be electrotransformed to A. baumannii recipient, but were untypeable by the current replicon typing scheme. A novel plasmid replicase named repAci10 was identified in blaOXA-58-harboring plasmids of two A. pittii isolates, three A. nosocomialis isolates and two A. baumannii isolates. Four kinds of genetic contexts of blaOXA-58 were identified. The transformants of plasmids with structure of IS6 family insertion sequence (ISOur1, IS1008 or IS15)-ΔISAba3-like element-blaOXA-58 displayed carbapenem nonsusceptible, while others with structure of intact ISAba3-like element-blaOXA-58 were carbapenem susceptible.
Conclusion
The study revealed the unique features of blaOXA-58-carrying plasmids in Acinetobacter spp. in China, which were different from that of Acinetobacter spp. found in European countries. The diversity of the genetic contexts of blaOXA-58 contributed to various antibiotics resistance profiles.
doi:10.1371/journal.pone.0084680
PMCID: PMC3882262  PMID: 24400107
5.  Molecular Analysis of the Acinetobacter baumannii Biofilm-Associated Protein 
Applied and Environmental Microbiology  2013;79(21):6535-6543.
Acinetobacter baumannii is a multidrug-resistant pathogen associated with hospital outbreaks of infection across the globe, particularly in the intensive care unit. The ability of A. baumannii to survive in the hospital environment for long periods is linked to antibiotic resistance and its capacity to form biofilms. Here we studied the prevalence, expression, and function of the A. baumannii biofilm-associated protein (Bap) in 24 carbapenem-resistant A. baumannii ST92 strains isolated from a single institution over a 10-year period. The bap gene was highly prevalent, with 22/24 strains being positive for bap by PCR. Partial sequencing of bap was performed on the index case strain MS1968 and revealed it to be a large and highly repetitive gene approximately 16 kb in size. Phylogenetic analysis employing a 1,948-amino-acid region corresponding to the C terminus of Bap showed that BapMS1968 clusters with Bap sequences from clonal complex 2 (CC2) strains ACICU, TCDC-AB0715, and 1656-2 and is distinct from Bap in CC1 strains. By using overlapping PCR, the bapMS1968 gene was cloned, and its expression in a recombinant Escherichia coli strain resulted in increased biofilm formation. A Bap-specific antibody was generated, and Western blot analysis showed that the majority of A. baumannii strains expressed an ∼200-kDa Bap protein. Further analysis of three Bap-positive A. baumannii strains demonstrated that Bap is expressed at the cell surface and is associated with biofilm formation. Finally, biofilm formation by these Bap-positive strains could be inhibited by affinity-purified Bap antibodies, demonstrating the direct contribution of Bap to biofilm growth by A. baumannii clinical isolates.
doi:10.1128/AEM.01402-13
PMCID: PMC3811493  PMID: 23956398
6.  Endemic and Epidemic Acinetobacter Species in a University Hospital: an 8-Year Survey▿  
Journal of Clinical Microbiology  2009;47(11):3593-3599.
The prevalence of the currently known Acinetobacter species and related trends of antimicrobial resistance in a Dutch university hospital were studied. Between 1999 and 2006, Acinetobacter isolates from clinical samples were collected prospectively. Isolates were analyzed by amplified fragment length polymorphism fingerprinting. For species identification, a profile similarity cutoff level of 50% was used, and for strain identification, a cutoff level of 90% was used. Susceptibility for antimicrobial agents was tested by disk diffusion by following the CLSI guideline.
The incidences of Acinetobacter isolates ranged from 1.7 to 3.7 per 10,000 patients per year, without a trend of increase, during the study years. Twenty different species were distinguished. Acinetobacter baumannii (27%) and Acinetobacter genomic species (gen. sp.) 3 (26%) were the most prevalent. Other species seen relatively frequently were Acinetobacter lwoffii (11%), Acinetobacter ursingii (4%), Acinetobacter johnsonii (4%), and Acinetobacter junii (3%). One large cluster of A. baumannii, involving 31 patients, and 16 smaller clusters of various species, involving in total 39 patients, with at most 5 patients in 1 cluster, occurred. Overall, 37% of the A. baumannii isolates were fully susceptible to the tested antibiotics. There was a borderline significant (P = 0.059) trend of decreasing susceptibility. A. baumannii was the Acinetobacter species causing the largest burden of multiple-antibiotic resistance and transmissions in the hospital.
doi:10.1128/JCM.00967-09
PMCID: PMC2772585  PMID: 19794057
7.  Dissemination of multidrug resistant Acinetobacter baumannii in various hospitals of Antananarivo Madagascar 
This study reports the dissemination of multidrug-resistant (MDR) OXA-23-producing Acinetobacter baumannii clones in hospitals in Antananarivo, Madagascar. A total of 53 carbapenem-resistant A. baumannii isolates were obtained from September 2006 to March 2009 in five hospitals. These resistant strains represent 44% of all A. baumannii isolates. The double disk synergy test was performed to screen for production of metallo-beta-lactamases. Polymerase chain reaction (PCR) and DNA sequencing were performed for the detection of bla(AmpC), bla(OXA-51),bla(OXA-23), bla(OXA-24), bla(IMP), bla(VIM). The presence of the insertion sequence ISAba1 relative to blaOXA-23 and blaOXA-51 was assessed by PCR. Isolates were typed by Rep-PCR. All the isolates were MDR and produced the OXA-23 carbapenemase, which was confirmed by sequencing. PCR analysis for AmpC and OXA-51 gave positive results for all strains studied. No isolates produced metallo-beta-lactamases. In all isolates ISAba1 laid upstream of blaOXA-23. The A. baumannii isolates were separated into two genotypes; genotype A had a higher prevalence (41 strains) than genotype B (12 strains). Genotype A was present in four hospitals, whilst genotype B had spread in two hospitals. The high frequency of MDR OXA-23-producing A. baumannii in various hospitals in Antananarivo is curious since carbapenems are not available in Madagascar, but it emphasises the need for infection control procedures and strict adherence to them to prevent the spread of these resistant organisms in Antananarivo and also the need to control the use of carbapenems in the future.
doi:10.1186/1476-0711-9-17
PMCID: PMC2910008  PMID: 20591154
8.  Molecular Epidemiology of Multidrug-Resistant Acinetobacter baumannii in a Tertiary Care Hospital in Naples, Italy, Shows the Emergence of a Novel Epidemic Clone▿  
Journal of Clinical Microbiology  2010;48(4):1223-1230.
The molecular epidemiology of multidrug-resistant Acinetobacter baumannii was investigated in two intensive care units of the V. Monaldi university hospital in Naples, Italy, from May 2006 to December 2007. Genotype analysis by pulsed-field gel electrophoresis (PFGE), trilocus sequence-based typing (3LST), and multilocus sequence typing (MLST) of A. baumannii isolates from 71 patients identified two distinct genotypes, one assigned to PFGE group A, 3LST group 1, and ST2 in 14 patients and the other to PFGE group B, 3LST group 6, and ST78 in 71 patients, that we named ST2/A and ST78/B, respectively. Of these, ST2/A corresponded to European clone II identified in the same hospital during 2003 and 2004; ST78/B was a novel genotype that was isolated for the first time in May 2006 but became prevalent during 2007. The ST78/B profile was also identified in five patients from two additional hospitals in Naples during 2007. The ST2/A and ST78/B isolates were resistant to all antimicrobials tested, including carbapenems, but were susceptible to colistin. Both ST2/A and ST78/B isolates possessed a plasmid-borne carbapenem-hydrolyzing oxacillinase gene, blaOXA-58, flanked by ISAba2 and ISAba3 elements at the 5′ and 3′ ends, respectively. The selection of the novel ST78/B A. baumannii clone might have been favored by the acquisition of the blaOXA-58 gene.
doi:10.1128/JCM.02263-09
PMCID: PMC2849555  PMID: 20181918
9.  Diversity and Clinical Impact of Acinetobacter baumannii Colonization and Infection at a Military Medical Center ▿  
Journal of Clinical Microbiology  2010;49(1):159-166.
The epidemiology of Acinetobacter baumannii emerging in combat casualties is poorly understood. We analyzed 65 (54 nonreplicate) Acinetobacter isolates from 48 patients (46 hospitalized and 2 outpatient trainees entering the military) from October 2004 to October 2005 for genotypic similarities, time-space relatedness, and antibiotic susceptibility. Clinical and surveillance cultures were compared by amplified fragment length polymorphism (AFLP) genomic fingerprinting to each other and to strains of a reference database. Antibiotic susceptibility was determined, and multiplex PCR was performed for OXA-23-like, -24-like, -51-like, and -58-like carbapenemases. Records were reviewed for overlapping hospital stays of the most frequent genotypes, and risk ratios were calculated for any association of genotype with severity of Acute Physiology and Chronic Health Evaluation II (APACHE II) score or injury severity score (ISS) and previous antibiotic use. Nineteen genotypes were identified; two predominated, one consistent with an emerging novel international clone and the other unique to our database. Both predominant genotypes were carbapenem resistant, were present at another hospital before patients' admission to our facility, and were associated with higher APACHE II scores, higher ISSs, and previous carbapenem antibiotics in comparison with other genotypes. One predominated in wound and respiratory isolates, and the other predominated in wound and skin surveillance samples. Several other genotypes were identified as European clones I to III. Acinetobacter genotypes from recruits upon entry to the military, unlike those in hospitalized patients, did not include carbapenem-resistant genotypes. Acinetobacter species isolated from battlefield casualties are diverse, including genotypes belonging to European clones I to III. Two carbapenem-resistant genotypes were epidemic, one of which appeared to belong to a novel international clone.
doi:10.1128/JCM.00766-10
PMCID: PMC3020478  PMID: 21084513
10.  Carbapenem-resistant Acinetobacter baumannii and Klebsiella pneumoniae across a hospital system: impact of post-acute care facilities on dissemination 
Background
Resistance to carbapenems among Acinetobacter baumannii and Klebsiella pneumoniae presents a serious therapeutic and infection control challenge. We describe the epidemiology and genetic basis of carbapenem resistance in A. baumannii and K. pneumoniae in a six-hospital healthcare system in Northeast Ohio.
Methods
Clinical isolates of A. baumannii and K. pneumoniae distributed across the healthcare system were collected from April 2007 to April 2008. Antimicrobial susceptibility testing was performed followed by molecular analysis of carbapenemase genes. Genetic relatedness of isolates was established with repetitive sequence-based PCR (rep-PCR), multilocus PCR followed by electrospray ionization mass spectrometry (PCR/ESI-MS) and PFGE. Clinical characteristics and outcomes of patients were reviewed.
Results
Among 39 isolates of A. baumannii, two predominant genotypes related to European clone II were found. Eighteen isolates contained blaOXA-23, and four isolates possessed blaOXA-24/40. Among 29 K. pneumoniae isolates with decreased susceptibility to carbapenems, two distinct genotypes containing blaKPC-2 or blaKPC-3 were found. Patients with carbapenem-resistant A. baumannii and K. pneumoniae were elderly, possessed multiple co-morbidities, were frequently admitted from and discharged to post-acute care facilities, and experienced prolonged hospital stays (up to 25 days) with a high mortality rate (up to 35%).
Conclusion
In this outbreak of carbapenem-resistant A. baumannii and K. pneumoniae across a healthcare system, we illustrate the important role post-acute care facilities play in the dissemination of multidrug-resistant phenotypes.
doi:10.1093/jac/dkq191
PMCID: PMC2904665  PMID: 20513702
LTCF; LTACH; molecular epidemiology; MLST; PFGE; rep-PCR; KPC
11.  Occupational Transmission of Acinetobacter baumannii from a United States Serviceman Wounded in Iraq to a Health Care Worker 
Background
Acinetobacter baumannii is increasingly recognized as being a significant pathogen associated with nosocomial outbreaks in both civilian and military treatment facilities. Current analyses of these outbreaks frequently describe patient-to-patient transmission. To date, occupational transmission of A. baumannii from a patient to a health care worker (HCW) has not been reported. We initiated an investigation of an HCW with a complicated case of A. baumannii pneumonia to determine whether a link existed between her illness and A. baumannii–infected patients in a military treatment facility who had been entrusted to her care.
Methods
Pulsed-field gel electrophoresis and polymerase chain reaction/electrospray ionization mass spectrometry, a form of multilocus sequencing typing, were done to determine clonality. To further characterize the isolates, we performed a genetic analysis of resistance determinants.
Results and Conclusions
A “look-back” analysis revealed that the multidrug resistant A. baumannii recovered from the HCW and from a patient in her care were indistinguishable by pulsed-field gel electrophoresis. In addition, polymerase chain reaction/electrospray ionization mass spectrometry indicated that the isolates were similar to strains of A. baumannii derived from European clone type II (Walter Reed Army Medical Center strain type 11). The exposure of the HCW to the index patient lasted for only 30 min and involved endotracheal suctioning without use of an HCW mask. An examination of 90 A. baumannii isolates collected during this investigation showed that 2 major and multiple minor clone types were present and that the isolates from the HCW and from the index patient were the most prevalent clone type. Occupational transmission likely occurred in the hospital; HCWs caring for patients infected with A. baumannii should be aware of this potential mode of infection spread.
doi:10.1086/589247
PMCID: PMC4074882  PMID: 18611162
12.  Complete Genome Analysis of Three Acinetobacter baumannii Clinical Isolates in China for Insight into the Diversification of Drug Resistance Elements 
PLoS ONE  2013;8(6):e66584.
Background
The emergence and rapid spreading of multidrug-resistant Acinetobacter baumannii strains has become a major health threat worldwide. To better understand the genetic recombination related with the acquisition of drug-resistant elements during bacterial infection, we performed complete genome analysis on three newly isolated multidrug-resistant A. baumannii strains from Beijing using next-generation sequencing technology.
Methodologies/Principal Findings
Whole genome comparison revealed that all 3 strains share some common drug resistant elements including carbapenem-resistant blaOXA-23 and tetracycline (tet) resistance islands, but the genome structures are diversified among strains. Various genomic islands intersperse on the genome with transposons and insertions, reflecting the recombination flexibility during the acquisition of the resistant elements. The blood-isolated BJAB07104 and ascites-isolated BJAB0868 exhibit high similarity on their genome structure with most of the global clone II strains, suggesting these two strains belong to the dominant outbreak strains prevalent worldwide. A large resistance island (RI) of about 121-kb, carrying a cluster of resistance-related genes, was inserted into the ATPase gene on BJAB07104 and BJAB0868 genomes. A 78-kb insertion element carrying tra-locus and blaOXA-23 island, can be either inserted into one of the tniB gene in the 121-kb RI on the chromosome, or transformed to conjugative plasmid in the two BJAB strains. The third strains of this study, BJAB0715, which was isolated from spinal fluid, exhibit much more divergence compared with above two strains. It harbors multiple drug-resistance elements including a truncated AbaR-22-like RI on its genome. One of the unique features of this strain is that it carries both blaOXA-23 and blaOXA-58 genes on its genome. Besides, an Acinetobacter lwoffii adeABC efflux element was found inserted into the ATPase position in BJAB0715.
Conclusions
Our comparative analysis on currently completed Acinetobacter baumannii genomes revealed extensive and dynamic genome organizations, which may facilitate the bacteria to acquire drug-resistance elements into their genomes.
doi:10.1371/journal.pone.0066584
PMCID: PMC3691203  PMID: 23826102
13.  Influence of relative humidity and suspending menstrua on survival of Acinetobacter spp. on dry surfaces. 
Journal of Clinical Microbiology  1996;34(12):2881-2887.
Acinetobacter spp. are being reported with increasing frequency as a cause of nosocomial infection and have been isolated from the skin of healthy individuals, patients, hospital staff, dry nonbiotic objects, and different pieces of medical equipment. Factors affecting the survival of Acinetobacter spp. under conditions closely similar to those found in the hospital environment were investigated in the present study to help us understand the epidemiology of nosocomial Acinetobacter infection. Bacterial cells were suspended in distilled water or bovine serum albumin and were dried onto glass coverslips and kept at different relative humidities. Cells washed from coverslips were used to determined viable counts. Freshly isolated strains of Acinetobacter spp. belonging to the clinically important Acinetobacter calcoaceticus-Acinetobacter baumannii complex were found to be more resistant to drying conditions (e.g., 30 days for A. baumannii 16/49) than American Type Culture Collection strains (e.g., 2 days for A. baumannii ATCC 9955). The majority of strains belonging to the Acb complex had survival times similar to those observed for the gram-positive organism Staphylococcus aureus tested in the experiment. Survival times were prolonged for almost all the strains tested when they were suspended in bovine serum albumin (e.g., 60 days for A. baumannii R 447) compared with those for strains suspended in distilled water (11 days for R 447). The survival times for strains at higher relative humidity (31 or 93%) were longer than those for strains of Acinetobacter kept at a relative humidity of 10% (11 days at 31% relative humidity and 4 days at 10% relative humidity for R447). These findings are consistent with the observed tendency of Acinetobacter spp. to survive on dry surfaces, and they can be transferred not only by moist vectors but also under dry conditions in a hospital environment during nosocomial infection outbreaks. The results obtained in the experiment support the previously suggested airborne spread of Acinetobacter spp. in hospital wards and repeated outbreaks after incomplete disinfection of contaminated dry surfaces.
PMCID: PMC229427  PMID: 8940416
14.  Diversity of Acinetobacter baumannii in Four French Military Hospitals, as Assessed by Multiple Locus Variable Number of Tandem Repeats Analysis 
PLoS ONE  2012;7(9):e44597.
Background
Infections by A. calcoaceticus-A. baumannii (ACB) complex isolates represent a serious threat for wounded and burn patients. Three international multidrug-resistant (MDR) clones (EU clone I-III) are responsible for a large proportion of nosocomial infections with A. baumannii but other emerging strains with high epidemic potential also occur.
Methodology/Principal Findings
We automatized a Multiple locus variable number of tandem repeats (VNTR) analysis (MLVA) protocol and used it to investigate the genetic diversity of 136 ACB isolates from four military hospitals and one childrens hospital. Acinetobacter sp other than baumannii isolates represented 22.6% (31/137) with a majority being A. pittii. The genotyping protocol designed for A.baumannii was also efficient to cluster A. pittii isolates. Fifty-five percent of A. baumannii isolates belonged to the two international clones I and II, and we identified new clones which members were found in the different hospitals. Analysis of two CRISPR-cas systems helped define two clonal complexes and provided phylogenetic information to help trace back their emergence.
Conclusions/Significance
The increasing occurrence of A. baumannii infections in the hospital calls for measures to rapidly characterize the isolates and identify emerging clones. The automatized MLVA protocol can be the instrument for such surveys. In addition, the investigation of CRISPR/cas systems may give important keys to understand the evolution of some highly successful clonal complexes.
doi:10.1371/journal.pone.0044597
PMCID: PMC3440325  PMID: 22984530
15.  An investigation of drug-resistant Acinetobacter baumannii infections in a comprehensive hospital of East China 
Background
To investigate the drug resistant gene profiles and molecular typing of Acinetobacter baumannii isolates collected from clinical specimens in a comprehensive hospital, Jiangsu province.
Methods
This study included 120 patients in a comprehensive hospital with drug-resistant A. baumannii infections on clinical specimens from October 2011 to December 2013. Antibiotic susceptibility test was determined by Vitek 2 Compact system. OXA-51, OXA-23, OXA-24, OXA-58, VIM, IMP, SHV, GES, TEM, AmpC, qacEΔ1-sul1, intI l, CarO, aac(6′)-Ib, and aac(6′)-II were analyzed by PCR. The analysis of molecular typing for 50 multidrug resistant A. baumannii isolates was performed by PFGE.
Results
A total of 64(53%) isolates were multidrug-resistant A.baumannii. The antibiotic susceptibility tests showed that the resistant rates to common antibiotics of mutidrug-resistant A. baumannii were extremely high, most of which over 60%. One hundred and ten isolates harbored OXA-51 (91.7%), 100 for OXA-23(83.3%), 103 for VIM-1(85.8%), 90 for AmpC(75.00%), 50 for aac(6′)-Ib(41.7%), 77 for the loss of CarO (64.2%), 85 for intl1(70.8%), and 64 for qacEΔ1-sul1(53.33%), while OXA-24 was undetected. Fifty multidrug-resistant A. baumannii isolates belong to 14 clones according to the PFGE DNA patterns. Main clone A includes 24 isolates, while clone B and clone C includes 6 and 9 isolates, respectively and others with no common source identified.
Conclusion
There is high morbidity of A. baumannii infections in the hospital, especially in ICU and sputum is the most common sample type.The mainly drug-resistant genes of A. baumannii are OXA-51, OXA-23, and VIM-1 in the hospital. Clonal dissemination provides evidence for the prevalence of multidrug-resistant A. baumannii among clinical isolates. It is suggested that there is an urgent need for effective control and prevention measures.
doi:10.1186/s12941-015-0066-4
PMCID: PMC4328433  PMID: 25643932
Acinetobacter baumannii; Drug-resistant gene; PCR; PFGE
16.  Molecular Epidemiology of Sequential Outbreaks of Acinetobacter baumannii in an Intensive Care Unit Shows the Emergence of Carbapenem Resistance 
Journal of Clinical Microbiology  2004;42(3):946-953.
The molecular epidemiology of multidrug-resistant Acinetobacter baumannii was investigated in the medical-surgical intensive care unit (ICU) of a university hospital in Italy during two window periods in which two sequential A. baumannii epidemics occurred. Genotype analysis by pulsed-field gel electrophoresis (PFGE) of A. baumannii isolates from 131 patients identified nine distinct PFGE patterns. Of these, PFGE clones B and I predominated and occurred sequentially during the two epidemics. A. baumannii epidemic clones showed a multidrug-resistant antibiotype, being clone B resistant to all antimicrobials tested except the carbapenems and clone I resistant to all antimicrobials except ampicillin-sulbactam and gentamicin. Type 1 integrons of 2.5 and 2.2 kb were amplified from the chromosomal DNA of epidemic PFGE clones B and I, respectively, but not from the chromosomal DNA of the nonepidemic clones. Nucleotide analysis of clone B integron identified four gene cassettes: aacC1, which confers resistance to gentamicin; two open reading frames (ORFs) coding for unknown products; and aadA1a, which confers resistance to spectinomycin and streptomycin. The integron of clone I contained three gene cassettes: aacA4, which confers resistance to amikacin, netilmicin, and tobramycin; an unknown ORF; and blaOXA-20, which codes for a class D β-lactamase that confers resistance to amoxicillin, ticarcillin, oxacillin, and cloxacillin. Also, the blaIMP allele was amplified from chromosomal DNA of A. baumannii strains of PFGE type I. Class 1 integrons carrying antimicrobial resistance genes and blaIMP allele in A. baumannii epidemic strains correlated with the high use rates of broad-spectrum cephalosporins, carbapenems, and aminoglycosides in the ICU during the study period.
doi:10.1128/JCM.42.3.946-953.2004
PMCID: PMC356829  PMID: 15004037
17.  Co-production of ESBL and AmpC β-Lactamases in Clinical Isolates of A. baumannii and A. lwoffii in a Tertiary Care Hospital From Northern India 
Background: Acinetobacter baumannii is an important cause of health care associated infections which are difficult to control and treat, because of widespread antimicrobial resistance which is possessed by this organism.
Aims: The aim of the present study was to know the prevalence of ESBLs and AmpC β-lactamases in clinical isolates of Acinetobacter spp. which were cultured from various clinical specimens by using different phenotypic methods.
Settings and Design: Study was conducted over a period of one year at the Microbiology Department of a tertiary care teaching hospital. A total of 100 consecutive, non-duplicate strains of Acinetobacter species which were isolated from various clinical samples were included.
Materials and Methods: All the isolates were identified by standard microbiological procedures and antimicrobial susceptibility testing was done by Kirby-Bauer disc diffusion technique. Isolates which showed reduced susceptibilities to third generation cephalosporins were tested for ESBL production by CLSI double disc synergy method and also by using sulbactam as an inhibitory agent. Isolates which showed reduced susceptibilities to cefoxitin were tested for AmpC detection by doing AmpC disc test.
Statistical Analysis: SPSS, version 17 was used to calculate p-value. If the p-value was <0.05, it was considered to be significant.
Results: Out of 100 isolates, 82 were Acinetobacter baumannii and 18 were Acinetobacter lwoffii. ESBL were mentioned in 4% of the Acinetobacter isolates and in 77% of the isolates by using clavulanic acid and sulbactam as inhibitory agents respectively. AmpC β-lactamase production was detected in 60% isolates of Acinetobacter spp. Co-production of both ESBL and AmpC enzymes were seen in 29% of the Acinetobacter strains.
Conclusion: Failure in detecting β-lactamases contributes to their uncontrolled spread and therapeutic failures. Hence, these β-lactamases should be detected routinely and they should be reported to clinicians in time, so that inappropriate use of antibiotics can be stopped in time.
doi:10.7860/JCDR/2014/8008.4289
PMCID: PMC4064843  PMID: 24959443
Acinetobacter; Extended spectrum β-lactamases; AmpC β-lactamases; Multi-drug resistance; Cephalosporins
18.  Comparative Genomics of Multidrug Resistance in Acinetobacter baumannii 
PLoS Genetics  2006;2(1):e7.
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.
Synopsis
The bacterial species Acinetobacter baumannii is a major cause of hospital-acquired infection throughout the world, and it is an increasing public health concern due to its increasing resistance to antibiotic treatment. Coincidently, a high incidence of multidrug-resistant A. baumannii bloodstream infections was recently reported in US Army service members injured during Afghanistan and Iraq/Kuwait military operations. A. baumannii exhibits a remarkable ability to rapidly develop antibiotic resistance, which led from fully susceptible to multidrug-resistant strains within three decades. The authors used whole-genome sequencing and bioinformatic analyses to identify the complete repertoire of resistance genes exhibited by the multidrug-resistant A. baumannii strain AYE, which is epidemic in France, and to investigate the mechanisms of their acquisition by comparison with the fully susceptible A. baumannii strain SDF, which is associated with human body lice. This study led to the discovery in the AYE genome of an 86-kb region called a resistance “island”—the largest identified to date—that contains a cluster of 45 resistance genes. The homologous location in the susceptible strain, curiously, exhibited a 20-kb genomic island that is devoid of resistance markers. This ability to “switch” its genomic structure probably explains the unmatched speed at which A. baumannii captures resistance markers when under antibacterial pressure, such as is found in hospital intensive care units.
doi:10.1371/journal.pgen.0020007
PMCID: PMC1326220  PMID: 16415984
19.  Antimicrobial susceptibility profiling and genomic diversity of Acinetobacter baumannii isolates: A study in western Iran 
Iranian Journal of Microbiology  2013;5(3):195-202.
Background and Objective
Acinetobacter baumannii is an aerobic non-motile Gram-negative bacterial pathogen that is resistant to most antibiotics. Carbapenems are the most common antibiotics for the treatment of infections caused by this pathogen. Mechanisms of antibiotic-resistance in A. baumannii are mainly mediated by efflux pumps-lactamases. The aim of this study was to determine antibiotic susceptibility, the possibility of existence of OXAs genes and fingerprinting by Pulsed-Field Gel Electrophoresis (PFGE) among clinical isolates of Acinetobacter collected from Kermanshah hospitals.
Materials and Methods
One hundred and four isolates were collected from patients attending Imam Reza, Taleghani and Imam Khomeini hospitals of Kermanshah (Iran). Isolates were identified by biochemical tests and API 20NE kit. The susceptibility to different antibiotics was assessed with Kirby-Bauer disk diffusion method. PCR was performed for detection of bla OXA-23, bla OXA-24, bla OXA-51 and bla OXA-58 beta-lactamase genes. Clonal relatedness was estimated by PFGE (with the restriction enzyme Apa I) and DNA patterns were analyzed by Gel compare II 6.5 software.
Results
All isolates showed high-level of resistance to imipenem, meropenem as well as to other antimicrobial agents, while no resistance to polymyxin B, colistin, tigecylcine and minocycline was observed. The bla OXA-23like and bla OXA-24 like were found among 77.9% and 19.2% of the isolates, respectively. All isolates were positive for bla OXA-51, but none produced any amplicon for bla OXA-58. PFGE genotype analysis suggested the existence of eight clones among the 104 strains [A (n = 35), B (n = 29), C (n = 19), D (n = 10), E (n = 4), F (n = 3), G (n = 3), H (n = 1)]. Clone A was the dominant clone in hospital settings particularly infection wards so that the isolates in this group, compared to the other clones, showed higher levels of resistance to antibiotics.
Conclusion
The bla OXA-51-like and bla OXA-23like were the predominant mechanisms of resistance to imipenem in A. baumannii. A high prevalence of clone A, B and C in different parts of the healthcare system showed that hospitalized patients should be safeguarded to prevent the spread of these clones. Early recognition of the presence of carbapenem-resistant A. baumannii clones is useful for preventing their spread within the hospital environment.
PMCID: PMC3895554  PMID: 24475323
Acinetobacter; beta-lactamase; carbapenemase; Pulsed-Field Gel Electrophoresis; Kermanshah
20.  Acinetobacter baumannii isolates from pets and horses in Switzerland: molecular characterization and clinical data 
Journal of Antimicrobial Chemotherapy  2011;66(10):2248-2254.
Objectives
We investigated whether Acinetobacter baumannii isolates of veterinary origin shared common molecular characteristics with those described in humans.
Methods
Nineteen A. baumannii isolates collected in pets and horses were analysed. Clonality was studied using repetitive extragenic palindromic PCR (rep-PCR) and multilocus sequence typing (MLST). PCR and DNA sequencing for various β-lactamase, aminoglycoside-modifying enzyme, gyrA and parC, ISAba1 and IS1133, adeR and adeS of the AdeABC efflux pump, carO porin and class 1/2/3 integron genes were performed.
Results
Two main clones [A (n = 8) and B (n = 9)] were observed by rep-PCR. MLST indicated that clone A contained isolates of sequence type (ST) ST12 (international clone II) and clone B contained isolates of ST15 (international clone I). Two isolates of ST10 and ST20 were also noted. Seventeen isolates were resistant to gentamicin, 12 to ciprofloxacin and 3 to carbapenems. Isolates of ST12 carried blaOXA-66, blaADC-25, blaTEM-1, aacC2 and IS1133. Strains of ST15 possessed blaOXA-69, blaADC-11, blaTEM-1 and a class 1 integron carrying aacC1 and aadA1. ISAba1 was found upstream of blaADC (one ST10 and one ST12) and/or blaOXA-66 (seven ST12). Twelve isolates of different STs contained the substitutions Ser83Leu in GyrA and Ser80Leu or Glu84Lys in ParC. Significant disruptions of CarO porin and overexpressed efflux pumps were not observed. The majority of infections were hospital acquired and in animals with predisposing conditions for infection.
Conclusions
STs and the molecular background of resistance observed in our collection have been frequently described in A. baumannii detected in human patients. Animals should be considered as a potential reservoir of multidrug-resistant A. baumannii.
doi:10.1093/jac/dkr289
PMCID: PMC3172040  PMID: 21733964
MLST; MDR; PCR/ESI-MS; rep-PCR; T5000
21.  Novel Acquired Metallo-β-Lactamase Gene, blaSIM-1, in a Class 1 Integron from Acinetobacter baumannii Clinical Isolates from Korea 
Antimicrobial Agents and Chemotherapy  2005;49(11):4485-4491.
Carbapenem resistance mediated by acquired carbapenemase genes has been increasingly reported, particularly for clinical isolates of Pseudomonas aeruginosa and Acinetobacter spp. Of 1,234 nonduplicate isolates of carbapenem-resistant Pseudomonas spp. and Acinetobacter spp. isolated at a tertiary-care hospital in Seoul, Korea, 211 (17%) were positive for metallo-β-lactamase (MBL). Of these, 204 (96%) had either the blaIMP-1 or blaVIM-2 allele. In addition, seven Acinetobacter baumannii isolates were found to have a novel MBL gene, which was designated blaSIM-1. The SIM-1 protein has a pI of 7.2, is a new member of subclass B1, and exhibits 64 to 69% identity with the IMP-type MBLs, which are its closest relatives. All SIM-1-producing isolates exhibited relatively low imipenem and meropenem MICs (8 to 16 μg/ml) and had a multidrug resistance phenotype. Expression of the cloned blaSIM-1 gene in Escherichia coli revealed that the encoded enzyme is capable of hydrolyzing a broad array of β-lactams, including penicillins, narrow- to expanded-spectrum cephalosporins, and carbapenems. The blaSIM-1 gene was carried on a gene cassette inserted into a class 1 integron, which included three additional cassettes (arr-3, catB3, and aadA1). The strains were isolated from sputum and urine specimens from patients with pneumonia and urinary tract infections, respectively. All patients had various underlying diseases. Pulsed-field gel electrophoresis of SmaI-digested genomic DNAs showed that the strains belonged to two different clonal lineages, indicating that horizontal transfer of this gene had occurred and suggesting the possibility of further spread of resistance in the future.
doi:10.1128/AAC.49.11.4485-4491.2005
PMCID: PMC1280121  PMID: 16251286
22.  Genomic epidemiology of a protracted hospital outbreak caused by multidrug-resistant Acinetobacter baumannii in Birmingham, England 
Genome Medicine  2014;6(11):70.
Background
Multidrug-resistant Acinetobacter baumannii commonly causes hospital outbreaks. However, within an outbreak, it can be difficult to identify the routes of cross-infection rapidly and accurately enough to inform infection control. Here, we describe a protracted hospital outbreak of multidrug-resistant A. baumannii, in which whole-genome sequencing (WGS) was used to obtain a high-resolution view of the relationships between isolates.
Methods
To delineate and investigate the outbreak, we attempted to genome-sequence 114 isolates that had been assigned to the A. baumannii complex by the Vitek2 system and obtained informative draft genome sequences from 102 of them. Genomes were mapped against an outbreak reference sequence to identify single nucleotide variants (SNVs).
Results
We found that the pulsotype 27 outbreak strain was distinct from all other genome-sequenced strains. Seventy-four isolates from 49 patients could be assigned to the pulsotype 27 outbreak on the basis of genomic similarity, while WGS allowed 18 isolates to be ruled out of the outbreak. Among the pulsotype 27 outbreak isolates, we identified 31 SNVs and seven major genotypic clusters. In two patients, we documented within-host diversity, including mixtures of unrelated strains and within-strain clouds of SNV diversity. By combining WGS and epidemiological data, we reconstructed potential transmission events that linked all but 10 of the patients and confirmed links between clinical and environmental isolates. Identification of a contaminated bed and a burns theatre as sources of transmission led to enhanced environmental decontamination procedures.
Conclusions
WGS is now poised to make an impact on hospital infection prevention and control, delivering cost-effective identification of routes of infection within a clinically relevant timeframe and allowing infection control teams to track, and even prevent, the spread of drug-resistant hospital pathogens.
doi:10.1186/s13073-014-0070-x
PMCID: PMC4237759  PMID: 25414729
23.  AbaR7, a Genomic Resistance Island Found in Multidrug-resistant Acinetobacter baumannii Isolates in Daejeon, Korea 
Annals of Laboratory Medicine  2012;32(5):324-330.
Background
Acinetobacter baumannii resistance islands (AbaRs) have been recently recognized as mobile genetic elements that harbor multiple resistance determinants and are associated with multidrug resistance (MDR). In the present study, we aimed to determine the AbaRs conferring multiple antimicrobial resistance and their clonal relatedness to MDR A. baumannii clinical isolates obtained from a university hospital in Daejeon, Korea.
Methods
This study included 29 MDR A. baumannii strains isolated in Daejeon, Korea. The minimal inhibitory concentrations (MICs) were determined by Etest. A. baumannii isolates were characterized using the 2 multiplex PCR assays and multilocus sequence typing (MLST) scheme. To detect and characterize AbaRs, PCR and PCR mapping experiments were performed.
Results
Twenty-seven of the 29 isolates belonged to the European (EU) clone II lineage and contained 5 sequence types (STs) (75, 92, 137, 138, and 357). In this study, ST357 was confirmed for the first time in Korea. Only 2 of the 29 isolates belonged to the EU clone I lineage, and were confirmed as ST109. These 2 isolates harbored the 22-kb AbaR7 aacC1-orfP-orfQ-aadA1 gene cassette array. In contrast, AbaR was not found in EU clone II isolates.
Conclusions
This is the first study that attempted to determine the AbaRs in MDR A. baumannii isolates in Korea. We found 2 EU clone I isolates (ST109) that harbored AbaR7.
doi:10.3343/alm.2012.32.5.324
PMCID: PMC3427819  PMID: 22950067
A. baumannii; Multidrug resistance; Multilocus sequence typing; PCR
24.  First Identification and Characterization of an AdeABC-Like Efflux Pump in Acinetobacter Genomospecies 13TU▿ †  
Non-Acinetobacter baumannii spp. are emerging among clinical Acinetobacter isolates causing nosocomial infections, and some (such as genomospecies 13TU) appear to be multidrug resistant. The prevalence of non-Acinetobacter baumannii spp. in the hospital setting is likely understated due to poor identification techniques. We report the first identification of an AdeABC-type efflux pump in an Acinetobacter genomospecies 13TU clinical isolate, its contribution to multidrug resistance, and the coexistence of three Ade-type efflux pumps in this strain.
doi:10.1128/AAC.01142-10
PMCID: PMC3067064  PMID: 21199925
25.  Identification, Genotypic Relation, and Clinical Features of Colistin-Resistant Isolates of Acinetobacter Genomic Species 13BJ/14TU from Bloodstreams of Patients in a University Hospital 
Journal of Clinical Microbiology  2014;52(3):931-939.
Colistin resistance remains rare among clinical isolates of Acinetobacter species. We noted the emergence of colistin-resistant bloodstream isolates of the Acinetobacter genomic species (GS) 13BJ/14TU from patients at a university hospital between 2003 and 2011. We report here, for the first time, the microbiological and molecular characteristics of these isolates, with clinical features of Acinetobacter GS 13BJ/14TU bacteremia. All 11 available patient isolates were correctly identified as Acinetobacter GS 13BJ/14TU using partial rpoB gene sequencing but were misidentified using the phenotypic methods Vitek 2 (mostly as Acinetobacter baumannii), MicroScan (mostly as A. baumannii/Acinetobacter haemolyticus), and the API 20 NE system (all as A. haemolyticus). Most isolates were susceptible to commonly used antibiotics, including carbapenems, but all were resistant to colistin, for which it is unknown whether the resistance is acquired or intrinsic. However, the fact that none of the patients had a history of colistin therapy strongly suggests that Acinetobacter GS 13BJ/14TU is innately resistant to colistin. The phylogenetic tree of multilocus sequence typing (MLST) showed that all 11 isolates formed a separate cluster from other Acinetobacter species and yielded five sequence types. However, pulsed-field gel electrophoresis (PFGE) revealed 11 distinct patterns, suggesting that the bacteremia had occurred sporadically. Four patients showed persistent bacteremia (6 to 17 days), and all 11 patients had excellent outcomes with cleared bacteremia, suggesting that patients with Acinetobacter GS 13BJ/14TU-associated bacteremia show a favorable outcome. These results emphasize the importance of precise species identification, especially regarding colistin resistance in Acinetobacter species. In addition, MLST offers another approach to the identification of Acinetobacter GS 13BJ/14TU, whereas PFGE is useful for genotyping for this species.
doi:10.1128/JCM.02868-13
PMCID: PMC3957785  PMID: 24403305

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