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1.  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
2.  Changing carbapenemase gene pattern in an epidemic multidrug-resistant Acinetobacter baumannii lineage causing multiple outbreaks in central Italy 
Objectives
Infections caused by multidrug-resistant (MDR) Acinetobacter baumannii are a challenging problem worldwide. Here, the molecular epidemiology and the genetic basis of antibiotic resistance in 111 MDR A. baumannii strains isolated from June 2005 to March 2009 from infected patients in 10 intensive care units (ICUs) in central Italy were investigated.
Methods
Epidemiological typing was performed by random amplification of polymorphic DNA, PCR-based sequence grouping and macrorestriction analysis. MICs of antibiotics were determined by the broth microdilution method. Genes for OXA carbapenemases, metallo-β-lactamases and the CarO porin were searched for by PCR.
Results
Molecular genotyping identified one predominant A. baumannii lineage, related to the international clonal lineage II, accounting for 95.6% of isolates. Isolates referable to this lineage were recovered from all ICUs surveyed and were resistant to nearly all classes of antimicrobials, with the exception of tigecycline and colistin. A high percentage (60.5%) of A. baumannii isolates showed elevated resistance to imipenem (MICs ≥ 128 mg/L), concomitant with resistance to meropenem. Carbapenem resistance was associated with the presence of either blaOXA-58-like (22.8%) or blaOXA-23-like (71.1%) carbapenemase genes. Molecular typing showed that the epidemic lineage encoding OXA-23 emerged in 2007 and displaced a genetically related clone encoding OXA-58 that had been responsible for previous ICU outbreaks in the same region.
Conclusions
Emergence of the OXA-23 epidemic lineage could result from selective advantage conferred by the blaOXA-23-like determinant, which provides increased resistance to carbapenems.
doi:10.1093/jac/dkq407
PMCID: PMC3031335  PMID: 21088019
genotyping; intensive care units; OXA-23
3.  Epidemic Diffusion of OXA-23-Producing Acinetobacter baumannii Isolates in Italy: Results of the First Cross-Sectional Countrywide Survey 
Journal of Clinical Microbiology  2014;52(8):3004-3010.
Carbapenem-resistant Acinetobacter baumannii (CRAb) is emerging worldwide as a public health problem in various settings. The aim of this study was to investigate the prevalence of CRAb isolates in Italy and to characterize their resistance mechanisms and genetic relatedness. A countrywide cross-sectional survey was carried out at 25 centers in mid-2011. CRAb isolates were reported from all participating centers, with overall proportions of 45.7% and 22.2% among consecutive nonreplicate clinical isolates of A. baumannii from inpatients (n = 508) and outpatients (n = 63), respectively. Most of them were resistant to multiple antibiotics, whereas all remained susceptible to colistin, with MIC50 and MIC90 values of ≤0.5 mg/liter. The genes coding for carbapenemase production were identified by PCR and sequencing. OXA-23 enzymes (found in all centers) were by far the most common carbapenemases (81.7%), followed by OXA-58 oxacillinases (4.5%), which were found in 7 of the 25 centers. In 6 cases, CRAb isolates carried both blaOXA-23-like and blaOXA-58-like genes. A repetitive extragenic palindromic (REP)-PCR technique, multiplex PCRs for group identification, and multilocus sequence typing (MLST) were used to determine the genetic relationships among representative isolates (n = 55). Two different clonal lineages were identified, including a dominant clone of sequence type 2 (ST2) related to the international clone II (sequence group 1 [SG1], SG4, and SG5) and a clone of ST78 (SG6) previously described in Italy. Overall, our results demonstrate that OXA-23 enzymes have become the most prevalent carbapenemases and are now endemic in Italy. In addition, molecular typing profiles showed the presence of international and national clonal lineages in Italy.
doi:10.1128/JCM.00291-14
PMCID: PMC4136168  PMID: 24920776
4.  Evidence of Diversity among Epidemiologically Related Carbapenemase-Producing Acinetobacter baumannii Strains Belonging to International Clonal Lineage II 
Journal of Clinical Microbiology  2012;50(3):590-597.
Carbapenem-resistant Acinetobacter baumannii strains belonging to international clonal lineage II (ICL-II) have become predominant in intensive care units (ICUs) throughout Italy. Between 2005 and 2009, the carbapenem-hydrolyzing class D β-lactamase (CHDL) blaOXA-23 gene became more prevalent than blaOXA-58 among epidemic ICL-II strains showing extensive genetic similarity. These findings posed the question of whether CHDL gene replacement occurred in the homogeneous ICL-II population or a new OXA-23 clone(s) emerged and spread in ICUs. In this study, the changes in the ICL-II A. baumannii population and CHDL gene carriage were investigated in 30 genetically related isolates collected during the blaOXA-58-to-blaOXA-23 transition period. Pulsotyping, randomly amplified polymorphic DNA (RAPD) analysis, and multilocus sequence typing (MLST) results were combined with multilocus variable-number tandem-repeat (VNTR) analysis (MLVA-8), siderotyping, and plasmid profiling to improve genotype-based discrimination between isolates. Pulsotyping, RAPD analysis, and MLST clustered isolates into a single type. MLVA-8 identified 19 types that clustered into three complexes. All OXA-23-producing isolates formed a single complex, while OXA-58 producers were split into two complexes. Southern blot analysis of the physical localization and genetic context of the CHDL genes showed that blaOXA-58 was invariably located on plasmids, while blaOXA-23 was present within Tn2006 on the chromosome or both the chromosome and plasmids. These data indicate that the apparently homogeneous population of CHDL-producing ICL-II strains was composed of several independent strains and that, between 2005 and 2009, distinct OXA-23 producers displaced the preexisting OXA-58 producers. Thus, MLVA-8 appears to be a suitable tool not only for investigating A. baumannii population structure but also for high-resolution epidemiological typing.
doi:10.1128/JCM.05555-11
PMCID: PMC3295171  PMID: 22205821
5.  Molecular Analysis of Acinetobacter baumannii Strains Isolated in Lebanon Using Four Different Typing Methods 
PLoS ONE  2014;9(12):e115969.
This study analyzed 42 Acinetobacter baumannii strains collected between 2009–2012 from different hospitals in Beyrouth and North Lebanon to better understand the epidemiology and carbapenem resistance mechanisms in our collection and to compare the robustness of pulsed field gel electrophoresis (PFGE), multilocus sequence typing (MLST), repetitive sequence-based PCR (rep-PCR) and blaOXA-51 sequence-based typing (SBT). Among 31 carbapenem resistant strains, we have detected three carbapenem resistance genes: 28 carried the blaOXA-23 gene, 1 the blaOXA-24 gene and 2 strains the blaOXA-58 gene. This is the first detection of blaOXA-23 and blaOXA-24 in Lebanon. PFGE identified 11 types and was the most discriminating technique followed by rep-PCR (9 types), blaOXA-51 SBT (8 types) and MLST (7 types). The PFGE type A'/ST2 was the dominant genotype in our collection present in Beyrouth and North Lebanon. The clustering agreement between all techniques was measured by adjust Wallace coefficient. An overall agreement has been demonstrated. High values of adjust Wallace coefficient were found with followed combinations: PFGE to predict MLST types  = 100%, PFGE to predict blaOXA-51 SBT = 100%, blaOXA-51 SBT to predict MLST = 100%, MLST to predict blaOXA-51 SBT = 84.7%, rep-PCR to predict MLST = 81.5%, PFGE to predict rep-PCR = 69% and rep-PCR to predict blaOXA-51 SBT = 67.2%. PFGE and MLST are gold standard methods for outbreaks investigation and population structure studies respectively. Otherwise, these two techniques are technically, time and cost demanding. We recommend the use of blaOXA-51 SBT as first typing method to screen isolates and assign them to their corresponding clonal lineages. Repetitive sequence-based PCR is a rapid tool to access outbreaks but careful interpretation of results must be always performed.
doi:10.1371/journal.pone.0115969
PMCID: PMC4277430  PMID: 25541711
6.  Co-occurrence of carbapenem and aminoglycoside resistance genes among multidrug-resistant clinical isolates of Acinetobacter baumannii from Cracow, Poland 
Background
Acinetobacter baumannii is a significant hospital pathogen, possessing a considerable degree of antimicrobial resistance. A. baumannii resistance to carbapenems and aminoglycosides is mostly conferred by class D OXA carbapenemases and aminoglycoside-modifying enzymes, respectively. The aim of this study was to determine the prevalence of selected genes encoding OXA carbapenemases and aminoglycoside-modifying enzymes in multidrug-resistant strains of A. baumannii.
Material/Methods
The study included 61 carbapenem-resistant and aminoglycoside-nonsusceptible A. baumannii isolates, collected between 2009 and 2011 in Cracow, Poland. Selected resistance genes, including: blaOXA-51-like, blaOXA-23-like, blaOXA-40-like, blaOXA-58-like, aac(6′)-Ih, aac(3)-Ia, aac(3)-IIa, aac(6′)-Ib, aph(3′)-Ia and aph(3′)-VI, were detected by PCR method.
Results
The blaOXA-51-like genes were detected in all isolates, while acquired carbapenemase encoding genes were found in 96.7% of tested strains. Presence of blaOXA-40-like and blaOXA-23-like genes was observed among 65.6% and 27.9% of isolates, respectively. Assayed aminoglycoside resistance genes were found to harbor 98.4% of isolates. Among tested strains, we observed the following percentages of resistance determinants: aac(3)-Ia – 78.7%, aph(3′)-VI – 78.7% and aph(3′)-Ia – 27.9%. Analysis of co-occurrence of carbapenem and aminoglycoside resistance genes revealed the highest percentage of strains possessing blaOXA-40-like, aac(3)-Ia, and aph(3′)-VI genes (44.3%).
Conclusions
The blaOXA-40-like and aac(3)-Ia/aph(3′)-VI were the most prevalent genes encoding acquired OXA carbapenemases and aminoglycoside-modifying enzymes, respectively, among A. baumannii strains in Cracow, Poland. Genes conferring resistance to carbapenems and aminoglycosides coexisted in the clinical strains of A. baumannii. The phenomenon of A. baumannii resistance indicates the necessity of monitoring for the presence of the resistance genes.
doi:10.12659/MSMBR.889811
PMCID: PMC3936949  PMID: 24463791
Acinetobacter baumannii; carbapenem resistance detection; aminoglycoside resistance detection
7.  Contribution of a Plasmid-Borne blaOXA-58 Gene with Its Hybrid Promoter Provided by IS1006 and an ISAba3-Like Element to β-Lactam Resistance in Acinetobacter Genomic Species 13TU ▿  
The contribution of the blaOXA-58 gene and its promoter to β-lactam resistance has not been validated in Acinetobacter spp. other than Acinetobacter baumannii. We identified a multidrug-resistant (including carbapenem resistance) Acinetobacter genomic species 13TU in which blaOXA-58 was the only detected carbapenemase gene. The blaOXA-58 gene was plasmid located, flanked by ISAba3 (downstream) and an ISAba3-like element (upstream). An IS1006 element was inserted into ISAba3-like (IS1006-ΔISAba3-like) to generate a hybrid promoter for blaOXA-58, with a −35 promoter located in IS1006 and a −10 promoter in ISAba3-like. The reference strain of Acinetobacter genomic species 13TU, ATCC 17903, revealed higher MICs of amoxicillin, ticarcillin, and piperacillin and heteroresistance to imipenem and meropenem when it was transformed with a shuttle vector containing a fragment encompassing ΔISAba3-like-blaOXA-58, compared to the same host containing only blaOXA-58. When the fragment was changed from ΔISAba3-like-blaOXA-58 to IS1006-ΔISAba3-like-blaOXA-58, the ATCC 17903 transformant revealed a markedly higher level of blaOXA-58 transcription (12-fold), increased cefuroxime and piperacillin-tazobactam MICs, and homoresistance to imipenem and meropenem. Different roles of the insertion elements preceding the blaOXA-58 gene in Acinetobacter genomic species 13TU are demonstrated. The ISAba3-like--blaOXA-58 construct can mediate resistance to penicillin derivatives but only heteroresistance to carbapenems. The insertion of IS1006 into ISAba3-like, generating a hybrid promoter, could further enhance the transcription of blaOXA-58 and mediate homoresistance to carbapenems and also enhanced resistance to piperacillin-tazobactam.
doi:10.1128/AAC.00128-10
PMCID: PMC2916316  PMID: 20516281
8.  Molecular Epidemiology of Clinical Isolates of Carbapenem-Resistant Acinetobacter spp. from Chinese Hospitals▿  
Antimicrobial Agents and Chemotherapy  2007;51(11):4022-4028.
Carbapenem resistance in Acinetobacter spp. is an emerging problem in China. We investigated the molecular epidemiology and carbapenemase genes of 221 nonrepetitive imipenem-resistant clinical isolates of Acinetobacter spp. collected from 1999 to 2005 at 11 teaching hospitals in China. Genotyping by pulsed-field gel electrophoresis (PFGE) found 15 PFGE patterns. Of these, one (clone P) was identified at four hospitals in Beijing and another (clone A) at four geographically disparate cities. Most imipenem-resistant isolates exhibited high-level resistance to all β-lactams and were only susceptible to colistin. blaOXA-23-like genes were found in 97.7% of isolates. Sequencing performed on 60 representative isolates confirmed the presence of the blaOXA-23 carbapenemase gene. Analysis of the genetic context of blaOXA-23 showed the presence of ISAba1 upstream of blaOXA-23. All of the 187 A. baumannii isolates identified by amplified RNA gene restriction analysis carried a blaOXA-51-like oxacillinase gene, while this gene was absent from isolates of other species. Sequencing indicated the presence of blaOXA-66 for 18 representative isolates. Seven isolates of one clone (clone T) carried the plasmid-mediated blaOXA-58 carbapenemase gene, while one isolate of another clone (clone L) carried the blaOXA-72 carbapenemase gene. Only 1 isolate of clone Q carried the blaIMP-8 metallo-β-lactamase gene, located in a class 1 integron. Of 221 isolates, 77.8% carried blaPER-1-like genes. Eleven different structures of class 1 integrons were detected, and most integrons carried genes mediating resistance to aminoglycosides, rifampin, and chloramphenicol. These findings indicated clonal spread of imipenem-resistant Acinetobacter spp. and wide dissemination of the OXA-23 carbapenemase in China.
doi:10.1128/AAC.01259-06
PMCID: PMC2151426  PMID: 17846127
9.  Emergence and Distribution of Plasmids Bearing the blaOXA-51-Like Gene with an Upstream ISAba1 in Carbapenem-Resistant Acinetobacter baumannii Isolates in Taiwan ▿  
Antimicrobial Agents and Chemotherapy  2010;54(11):4575-4581.
The blaOXA-51-like gene with an upstream ISAba1 (ISAba1-blaOXA-51-like gene) was originally found on the chromosomes of carbapenem-resistant or -susceptible Acinetobacter baumannii isolates. However, a plasmid-borne ISAba1-blaOXA-51-like gene has recently been identified in Acinetobacter genomic species 13TU and several A. baumannii isolates in Taiwan, and all of the isolates are carbapenem resistant. This study aimed to characterize the plasmids bearing the ISAba1-blaOXA-51-like gene and their significance in A. baumannii. Among the 117 ISAba1-blaOXA-51-like-harboring isolates collected from 10 hospitals in Taiwan, 58 isolates (49.6%) from 24 clones had the genes located on plasmids that likely originated from a common progenitor. Among the 58 isolates, four had additional copy of the ISAba1-blaOXA-51-like gene on their chromosomes. Based on the analysis of these four isolates, the plasmid-located ISAba1-blaOXA-51-like gene appeared to be acquired via one-ended transposition (Tn6080). The isolates with a plasmid bearing the ISAba1-blaOXA-51-like gene had higher rates of resistance to imipenem (98% versus 46.6%; P < 0.001) and meropenem (98% versus 69%; P = 0.019) than those with the genes chromosomally encoded, which is most likely due to increased gene dosage provided by the higher copy number of associated plasmids. Transformation with a recombinant plasmid harboring only the ISAba1-blaOXA-51-like gene was enough to confer a high level of carbapenem resistance to A. baumannii, eliminating the possible contribution of other factors on the original plasmids. This study demonstrated that the carbapenem resistance-associated plasmids carrying the ISAba1-blaOXA-51-like gene are widespread in A. baumannii strains in Taiwan.
doi:10.1128/AAC.00764-10
PMCID: PMC2976157  PMID: 20713680
10.  Carbapenem Susceptibility Testing Errors Using Three Automated Systems, Disk Diffusion, Etest, and Broth Microdilution and Carbapenem Resistance Genes in Isolates of Acinetobacter baumannii-calcoaceticus Complex▿ 
Antimicrobial Agents and Chemotherapy  2011;55(10):4707-4711.
The Acinetobacter baumannii-calcoaceticus complex (ABC) is associated with increasing carbapenem resistance, necessitating accurate resistance testing to maximize therapeutic options. We determined the accuracy of carbapenem antimicrobial susceptibility tests for ABC isolates and surveyed them for genetic determinants of carbapenem resistance. A total of 107 single-patient ABC isolates from blood and wound infections from 2006 to 2008 were evaluated. MICs of imipenem, meropenem, and doripenem determined by broth microdilution (BMD) were compared to results obtained by disk diffusion, Etest, and automated methods (the MicroScan, Phoenix, and Vitek 2 systems). Discordant results were categorized as very major errors (VME), major errors (ME), and minor errors (mE). DNA sequences encoding OXA beta-lactamase enzymes (blaOXA-23-like, blaOXA-24-like, blaOXA-58-like, and blaOXA-51-like) and metallo-β-lactamases (MBLs) (IMP, VIM, and SIM1) were identified by PCR, as was the KPC2 carbapenemase gene. Imipenem was more active than meropenem and doripenem. The percentage of susceptibility was 37.4% for imipenem, 35.5% for meropenem, and 3.7% for doripenem. Manual methods were more accurate than automated methods. blaOXA-23-like and blaOXA-24-like were the primary resistance genes found. blaOXA-58-like, MBLs, and KPC2 were not present. Both automated testing and manual testing for susceptibility to doripenem were very inaccurate, with VME rates ranging between 2.8 and 30.8%. International variability in carbapenem breakpoints and the absence of CLSI breakpoints for doripenem present a challenge in susceptibility testing.
doi:10.1128/AAC.00112-11
PMCID: PMC3187004  PMID: 21807971
11.  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
12.  Rapid Identification of Carbapenemase Genes in Gram-Negative Bacteria with an Oligonucleotide Microarray-Based Assay 
PLoS ONE  2014;9(7):e102232.
Rapid molecular identification of carbapenemase genes in Gram-negative bacteria is crucial for infection control and prevention, surveillance and for epidemiological purposes. Furthermore, it may have a significant impact upon determining the appropriate initial treatment and greatly benefit for critically ill patients. A novel oligonucleotide microarray-based assay was developed to simultaneously detect genes encoding clinically important carbapenemases as well as selected extended (ESBL) and narrow spectrum (NSBL) beta-lactamases directly from clonal culture material within few hours. Additionally, a panel of species specific markers was included to identify Escherichia coli, Pseudomonas aeruginosa, Citrobacter freundii/braakii, Klebsiella pneumoniae and Acinetobacter baumannii. The assay was tested using a panel of 117 isolates collected from urinary, blood and stool samples. For these isolates, phenotypic identifications and susceptibility tests were available. An independent detection of carbapenemase, ESBL and NSBL genes was carried out by various external reference laboratories using PCR methods. In direct comparison, the microarray correctly identified 98.2% of the covered carbapenemase genes. This included blaVIM (13 out of 13), blaGIM (2/2), blaKPC (27/27), blaNDM (5/5), blaIMP-2/4/7/8/13/14/15/16/31 (10/10), blaOXA-23 (12/13), blaOXA-40-group (7/7), blaOXA-48-group (32/33), blaOXA-51 (1/1) and blaOXA-58 (1/1). Furthermore, the test correctly identified additional beta-lactamases [blaOXA-1 (16/16), blaOXA-2 (4/4), blaOXA-9 (33/33), OXA-10 (3/3), blaOXA-51 (25/25), blaOXA-58 (2/2), CTX-M1/M15 (17/17) and blaVIM (1/1)]. In direct comparison to phenotypical identification obtained by VITEK or MALDI-TOF systems, 114 of 117 (97.4%) isolates, including Acinetobacter baumannii (28/28), Enterobacter spec. (5/5), Escherichia coli (4/4), Klebsiella pneumoniae (62/63), Klebsiella oxytoca (0/2), Pseudomonas aeruginosa (12/12), Citrobacter freundii (1/1) and Citrobacter braakii (2/2), were correctly identified by a panel of species specific probes. This assay might be easily extended, adapted and transferred to point of care platforms enabling fast surveillance, rapid detection and appropriate early treatment of infections caused by multiresistant Gram-negative bacteria.
doi:10.1371/journal.pone.0102232
PMCID: PMC4113224  PMID: 25068267
13.  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
14.  Genetic Basis of Multidrug Resistance in Acinetobacter Clinical Isolates in Taiwan▿  
Multidrug-resistant (MDR) Acinetobacter spp. have emerged as a threat to public health. We investigated the various genes involved in resistance to fluoroquinolones, aminoglycosides, cephalosporins, and carbapenems in 75 clinical Acinetobacter isolates from a Taiwanese hospital. All isolates were tested for the gyrA mutations, the presence of integrons, blaAmpC, and carbapenem resistance genes. The Ser83Leu mutation in GyrA accounted for fluoroquinolone resistance. The presence of integrons containing aminoglycoside-modifying enzymes was associated with resistance to gentamicin and tobramycin but not with resistance to amikacin. The presence of an ISAba1 element upstream of blaAmpC was correlated with cephalosporin resistance. Although most Acinetobacter baumannii isolates with ISAba1-blaOXA-51-like were resistant to carbapenems, several isolates remained susceptible to carbapenems. Transformation by the introduction of ISAba1-blaOXA-23 or ISAba1-blaOXA-66 into A. baumannii ATCC 15151 (CIP 70.10), resulting in the overexpression of OXA-23 or OXA-66, respectively, suggested the role of the ISAba1 element as a strong promoter. The two transformants showed significantly increased resistance to piperacillin-tazobactam, imipenem, and meropenem. The cefepime resistance conferred by ISAba1-blaOXA-23 and the impact of ISAba1-blaOXA-66 on carbapenem resistance in A. baumannii are reported here for the first time. Continuous surveillance of antibiotic resistance genes in MDR Acinetobacter spp. and elucidation of their antibiotic resistance mechanisms are crucial for the development of therapy regimens and for the prevention of further dissemination of these antibiotic resistance genes.
doi:10.1128/AAC.01398-09
PMCID: PMC2863617  PMID: 20194701
15.  Molecular Characterization of Carbapenem Resistant Isolates of Acinetobacter baumannii in An Intensive Care Unit of A Tertiary Care Centre at Central India 
Objective: To detect genes encoding carbapenem resistance in Acinetobacter baumannii in an intensive care unit.
Methods: A. baumannii isolates were recovered from various clinical specimens of hospitalized patients admitted to the Medical and Surgical intensive care units of a tertiary care centre in Pune. Bacterial identification was performed by routine conventional microbial culture and biochemical tests using standard recommended techniques. Antibiotic sensitivity test was performed by standard Kirby Bauer disc diffusion technique. PCR amplification and automated sequencing was carried out.
Results: A total of 155 /368 (42.11%) isolates A. baumannii were found to have reduced susceptibility to imipenem (diameter of zones of inhibition ≤13mm) by disc diffusion method. Among these 155 isolates tested 130 (83.87%) isolates showed MIC values for imipenem and meropenem ranging from16-64 mg/L as per CLSI breakpoints. Among these 155 isolates, Carbapenemase production was confirmed by Modified Hodge test for 93 (60%) isolates. Out of 155 isolates, DDST was positive for 89 (57.41%), CDST was positive for 73(47.09%) and MBL (IP/IPI) E-test was positive for 105 (67.74%). blaOXA-51 gene was detected in 47/105 (44.76%), blaOXA-23 gene in 55/105 (52.38%) and blaOXA-58 like gene in 15/105 (14.28%).
Conclusion: MBL production along with co- production of OXA enzymes are considered to be the important reason for resistance to imipenem in Acinetobacter in our health care settings. Hence, early detection of these drug resistant genes by molecular methods is essential in limiting the spread of infection due to these organisms.
doi:10.7860/JCDR/2014/7749.4398
PMCID: PMC4080003  PMID: 24995182
A. baumannii; Carbapenem; Multidrug resistant; Metallo beta-lactamase
16.  Imipenem-resistant Acinetobacter baumannii carrying the ISAba1-blaOXA-23,51 and ISAba1-blaADC-7 genes in Monteria, Colombia 
Brazilian Journal of Microbiology  2012;43(4):1274-1280.
The purpose of this study was to identify the genes coding for resistance to ceftazidime and imipenem and describe the molecular epidemiology of A. baumannii strains isolated from a clinical center in Colombia. Twenty isolates of imipenem-resistant A. baumannii from an equal number of patients with nosocomial infections were obtained. Primers were used to amplify genes blaIMP, blaVIM, blaOXA-23, blaOXA-24, blaOXA-58, blaOXA-51 and blaADC-7. To detect insertion sequences ISAba1/blaOXA-23, ISAba1/blaOXA-51 and ISAba1/blaADC-7, mapping by PCR using combinations of reverse primers ISAba1 and reverse primers of blaOXA-23, blaOXA-51 and blaADC-7 were used. The amplification products were purified and cloned into PCR 2.1-TOPO vector and transformed into chemically competent Escherichia coli TOP10. These amplicons were then sequenced. PFGE was performed on DNA of A. baumannii isolates digested with ApaI. Results. The DNA profiles obtained included 9 clusters with, four 2–7 isolates per profile, and 5 single-isolate profiles. Of the 20 isolates resistant to imipenem, 15 carried blaOXA-23 gene, 4 contained ISAba1 upstream of blaOXA-51 gene, and 6 contained ISAba1 upstream of blaOXA-23 gene. Eighteen of these isolates carried the blaADC-7 gene, with 9 of the isolates having ISAba1 located upstream of this gene. This is the first report of the ISAba1/ADC-7 associated with OXAs genes in A. baumannii isolates from Colombia.
doi:10.1590/S1517-83822012000400006
PMCID: PMC3769044  PMID: 24031953
Nosocomial pathogens; Antimicrobial resistance; PFGE
17.  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
18.  Dissemination of imipenem-resistant Acinetobacter baumannii with new plasmid-borne blaOXA-72 in Taiwan 
BMC Infectious Diseases  2013;13:319.
Background
The systemic surveillance of imipenem-resistant Acinetobacter baumannii (IRAB) from multicenters in Taiwan revealed the emergence of isolates with blaOXA-72. This study described their genetic makeup, mechanism of spread, and contribution to carbapenem resistance.
Methods
Two hundred and ninety-one non-repetitive isolates of A. baumannii were collected from 10 teaching hospitals from different geographical regions in Taiwan from June 2007 to September 2007. Minimal inhibitory concentrations (MICs) were determined by agar dilution. Clonality was determined by pulsed-field gel electrophoresis. Plasmid was extracted and digested by restriction enzymes, and subsequently analyzed by electrophoresis and Southern blot for blaOXA-72. The flanking regions of blaOXA-72 were determined by inverse PCR. The contribution of blaOXA-72 to imipenem MIC was determined by transforming plasmids carrying blaOXA-72 into imipenem-susceptible A. baumannii.
Results
Among 142 IRAB in Taiwan, 27 harbored blaOXA-72; 22 originated from Southern Taiwan, 5 from Central Taiwan, and none from Northern Taiwan. There were two major clones. The blaOXA-72 was identified in the plasmids of all isolates. Two genetic structures flanking plasmid-borne blaOXA-72 were identified and shared identical sequences in certain regions; the one described in previous literature was present in only one isolate, and the new one was present in the remaining isolates. Introduction of blaOXA-72 resulted in an increase of imipenem MIC in the transformants. The overexpression of blaOXA-72 mRNA in response to imipenem further supported the contribution of blaOXA-72.
Conclusions
In conclusion, isolates with new plasmid-borne blaOXA-72 were found to be disseminated successfully in Southern Taiwan. The spread of the resistance gene depended on clonal spread and dissemination of a new plasmid. BlaOXA-72 in these isolates directly led to their imipenem-resistance.
doi:10.1186/1471-2334-13-319
PMCID: PMC3728158  PMID: 23849336
Imipenem-resistant; Acinetobacter baumannii; Carbapenemase; BlaOXA-72
19.  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
20.  Characterization and PCR-Based Replicon Typing of Resistance Plasmids in Acinetobacter baumannii▿  
Antimicrobial Agents and Chemotherapy  2010;54(10):4168-4177.
Acinetobacter baumannii is an opportunistic pathogen, especially in intensive care units, and multidrug-resistant isolates have increasingly been reported during the last decade. Despite recent progress in knowledge of antibiotic resistance mechanisms in A. baumannii, little is known about the genetic factors driving isolates toward multidrug resistance. In the present study, the A. baumannii plasmids were investigated through the analysis and classification of plasmid replication systems and the identification of A. baumannii-specific mobilization and addiction systems. Twenty-two replicons were identified by in silico analysis, and five other replicons were identified and cloned from previously uncharacterized A. baumannii resistance plasmids carrying the OXA-58 carbapenem-hydrolyzing oxacillinase. Replicons were classified into homology groups on the basis of their nucleotide homology. A novel PCR-based replicon typing scheme (the A. baumannii PCR-based replicon typing [AB-PBRT] method) was devised to categorize the A. baumannii plasmids into homogeneous groups on the basis of the nucleotide homology of their respective replicase genes. The AB-PBRT technique was applied to a collection of multidrug-resistant A. baumannii clinical isolates carrying the blaOXA-58 or blaOXA-23 carbapenemase gene. A putative complete conjugative apparatus was identified on one plasmid whose self-conjugative ability was demonstrated in vitro. We showed that this conjugative plasmid type was widely diffused in our collection, likely representing the most important vehicle promoting the horizontal transmission of A. baumannii resistance plasmids.
doi:10.1128/AAC.00542-10
PMCID: PMC2944597  PMID: 20660691
21.  Extensively drug-resistant Acinetobacter baumannii in a Thai hospital: a molecular epidemiologic analysis and identification of bactericidal Polymyxin B-based combinations 
Background
Limited knowledge of the local molecular epidemiology and the paucity of new effective antibiotics has resulted in an immense challenge in the control and treatment of extensively drug-resistant (XDR) Acinetobacter baumannii infections in Thailand. Antimicrobial combination regimens may be the only feasible treatment option in such cases. We sought to characterize the local molecular epidemiology and assess the bactericidal activity of various antibiotics individually and in combination against XDR A. baumannii in a Thai hospital.
Methods
All XDR A. baumannii isolates from Thammasat University Hospital were collected between October 2010 and May 2011. Susceptibility testing was conducted according to reference broth dilution methods. Pulse-field gel electrophoresis was used to genotype the isolates. Carbapenemase genes were detected using polymerase chain reaction. In vitro testing of clinically-relevant concentrations of imipenem, meropenem, doripenem, rifampicin and tigecycline alone and in combination with polymyxin B was conducted using multiple combination bactericidal testing.
Results
Forty-nine polymyxin B-susceptible XDR A. baumannii isolates were identified. blaOXA-23 and blaOXA-51 genes were detected in all isolates. Eight clonally related clusters were identified, resulting in the initiation of several infection control measures. Imipenem, meropenem, doripenem, rifampicin, and tigecycline in combination with PB respectively, exhibited bactericidal killing in 100%, 100%, 98.0%, 100% and 87.8% isolates respectively at 24 hours.
Conclusion
Molecular epidemiologic analysis can aid the early detection of infection outbreak within the institution, resulting in the rapid containment of the outbreak. Imipenem/meropenem/rifampicin in combination with polymyxin B demonstrated consistent bactericidal effect against 49 blaOXA-23-harbouring XDR A. baumannii clinical isolates, suggesting a role of combination therapy in the treatment of these infections.
doi:10.1186/s13756-015-0043-x
PMCID: PMC4314787  PMID: 25648393
Combination therapy; Carbapenem resistance; Acinetobacter baumannii
22.  Molecular Epidemiology of Carbapenem Non-Susceptible Acinetobacter baumannii in France 
PLoS ONE  2014;9(12):e115452.
Carbapenem-resistant Acinetobacter baumannii have emerged globally. The objective of this study was to investigate the epidemiology, clonal diversity and resistance mechanisms of imipenem non-susceptible A. baumannii isolates in France. Between December 2010 and August 2011, 132 notifications were collected, including 37 outbreaks corresponding to 242 cases (2 to 55 per cluster). Multilocus sequence typing, pulsed-field gel electrophoresis (PFGE) and characterisation of carbapenemase-encoding genes were performed on 110 non-repetitive isolates. Gene blaOXA-23 was the most frequently detected (82%), followed by blaOXA-24 (11%) and blaOXA-58 (7%). Eleven sequence types (ST) were distinguished, among which sequence types ST1, ST2 (64%), ST20, ST25, ST85 and ST107. Isolates from epidemiological clusters had the same ST and resistance genes, indicating probable transmission within centres. In contrast, PFGE types of isolates differed among centres, arguing against transmission among centers. This study provides the first epidemiological snapshot of the population of A. baumannii with reduced susceptibility to carbapenems from France, and further underlines the predominance of international clones.
doi:10.1371/journal.pone.0115452
PMCID: PMC4269441  PMID: 25517732
23.  Trends in the molecular epidemiology of carbapenem resistant acinetobacter baumannii in a tertiary Greek hospital 
Hippokratia  2011;15(4):343-345.
Background: Acinetobacter baumannii is responsible for a variety of nosocomial infections, especially in intensive care unit patients. Nosocomial outbreaks due to carbapenem-resistant A. baumannii strains have been reported in many countries, including Greece. The aim of the present study was to determine the trends of molecular epidemiology of carbapenem-resistant A. baumannii isolates in a 750-bed hospital in Thessaloniki, Greece, during 2009.
Methods: The study included 39 carbapenem-resistant A. baumannii isolates collected from patients hospitalized in the General Hospital Papageorgiou during 2009. They were tested for the presence of Ambler class D carbapenemases and class 1 integrons, and they were typed by pulsed-field gel electrophoresis.
Results: The blaOXA-58 gene was detected in all A. baumannii isolates. Among the 39 isolates, 18 were carrying a 2.2 kb integron, 18 were carrying a 2.5 kb integron, and 3 isolates had no class 1 integrons. Two different clones, each divided further into two subclones, were observed. Comparing the clones detected in 2009 with those of former years (2001- 2008), a significant difference was observed: three clones have disappeared, two clones continued to circulate in the hospital, while a new subclone emerged in February 2009.
Conclusions: A change was seen in the molecular epidemiology of carbapenem-resistant A. baumannii isolates during 2009. Molecular epidemiology studies provide useful data for the distribution of resistant bacteria in order to design effective prevention and control measures.
PMCID: PMC3876851  PMID: 24391417
acinetobacter baumannii; carbapenem-resistance; molecular epidemiology
24.  Carbapenem Resistance and Acinetobacter baumannii in Senegal: The Paradigm of a Common Phenomenon in Natural Reservoirs 
PLoS ONE  2012;7(6):e39495.
Incidence of carbapenem-resistant Acinetobacter baumannii is rising in several parts of the world. In Africa, data concerning this species and its resistance to carbapenems are limited. The objective of the present study was to identify the presence of A. baumannii carbapenem-resistant encoding genes in natural reservoirs in Senegal, where antibiotic pressure is believed to be low. From October 2010 to January 2011, 354 human head lice, 717 human fecal samples and 118 animal fecal samples were screened for the presence of A. baumannii by real time PCR targeting blaOXA51-like gene. For all samples positive for A. baumannii, the carbapenemase-hydrolysing oxacillinases blaOXA23-like and blaOXA24-like were searched for and sequenced, and the isolates harbouring an oxacillinase were genotyped using PCR amplification and sequencing of recA gene. The presence of A. baumannii was detected in 4.0% of the head lice, in 5.4% of the human stool samples and in 5.1% of the animal stool samples tested. No blaOXA24 gene was detected but six fecal samples and three lice were positive for blaOXA23-like gene. The blaOXA23-like gene isolated in lice was likely a new oxacillinase sequence. Finally, the A. baumannii detected in stools were all of recA genotype 3 and those detected in lice, of recA genotype 4. This study shows for the first time a reservoir of blaOXA23-like-positive gene in human head lice and stool samples in Senegal.
doi:10.1371/journal.pone.0039495
PMCID: PMC3380006  PMID: 22745768
25.  Molecular and Epidemiological Characterization of Carbapenem-Resistant Acinetobacter baumannii in Non-Tertiary Korean Hospitals 
Yonsei Medical Journal  2012;54(1):177-182.
Purpose
The increasing prevalence and global spread of carbapenem-resistant Acinetobacter baumannii (A. baumannii) has become a serious problem. The aim of this study was to investigate molecular and epidemiological characteristics of carbapenem-resistant A. baumannii isolates collected from Korean non-tertiary hospitals.
Materials and Methods
Thirty six non-duplicated carbapenem-resistant A. baumannii isolates were collected from 17 non-tertiary hospitals in Korea between 2004 and 2006. Isolates were typed by multilocus sequence typing and repetitive-sequence-based PCR (rep-PCR). Detection of genes encoding OXA carbapenemase and their relationship with ISAba1 was performed by PCR.
Results
Two clones were prevalent among 36 isolates: ST69 (17 isolates, 47.2%) and ST92 (19 isolates, 52.8%). Rep-PCR patterns were diverse and revealed that all isolates were clustered into eight band patterns. The ISAba1-activated blaOXA-23-like and ISAba1-activated blaOXA-51-like genes were prevalent among the carbapenem-resistant A. baumannii isolates.
Conclusion
The class D β-lactamase genes of A. baumannii were distributed nationwide in non-tertiary Korean hospitals.
doi:10.3349/ymj.2013.54.1.177
PMCID: PMC3521248  PMID: 23225816
Acinetobacter baumannii; Carbapenem-resistant; OXA carbapenemase

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