Molecular epidemiology and drug resistance of Acinetobacter baumannii isolated from a regional hospital in the Brazilian Amazon region

Abstract INTRODUCTION: In this study, we report a clonal dissemination of carbapenem resistant Acinetobacter baumannii isolates due to the acquisition of blaOXA-23 in a regional hospital located in Brazilian Amazon Region. METHODS: The isolates were identified by MALDI-TOF and the carbapenemase-encoding genes were detected by multiplex-PCR. The genetic similarity was investigated by pulsed-field gel electrophoresis (PFGE). RESULTS: Only 10 (55.6%) isolates harbored the gene bla OXA-23. PFGE analysis revealed that these isolates belong to a single clone. CONCLUSIONS: This dissemination strategy indicates the need for surveillance, adoption of control procedures defined in guidelines, and the careful administration of antimicrobials should be reinforced.

High rate of detection of OXA-23-producing Acinetobacter from two general hospitals in Brazil

Abstract INTRODUCTION In recent decades, the prevalence of carbapenem-resistant Acinetobacter isolates has increased, and the production of oxacillinase (OXA)-type carbapenemases is the main mechanism underlying resistance. We evaluated OXA production from 114 Acinetobacter isolates collected between March and December 2013 from different clinical specimens of patients in two hospitals (Hospital 1 [n = 61] and Hospital 2 [n = 53]) located in Niterói, Rio de Janeiro, Brazil. We also evaluated the genetic diversity of OXA-producing isolates. METHODS All the isolates were identified through the automated system Vitek II and matrix-assisted laser desorption ionization-time of flight mass spectrometry MALDI-TOF MS as belonging to the A. baumannii-A. calcoaceticuscomplex. Antimicrobial susceptibility profiles were verified through agar diffusion tests. The presence of OXA-encoding genes was confirmed by PCR. The genetic diversity of isolates positive for carbapenemase production was analyzed through pulsed-field gel electrophoresis. RESULTS There was a high rate of resistance to carbapenems in the isolates (imipenem: 96%; meropenem: 92%) from both hospitals. Moreover, a high percentage (95.6%) of OXA-23-positive isolates was observed for both hospitals, indicating that this was the main mechanism of carbapenem-resistance among the studied population. In addition, most isolates (96.5%) were positive for bla OXA-51. A high genetic diversity and a few major genotypes were found among the OXA-23-positive isolates analyzed. Only intra-hospital dissemination was observed. CONCLUSIONS The elevated dissemination of bla OXA-23-like observed among Acinetobacter isolates from both the studied hospitals highlights the need for continuous epidemiological surveillance in these institutions.

Application of carbapenem inactivation method in detection of carbapenemase-producing Acinetobacter baumannii / 中国感染与化疗杂志

Objective To evaluate the utility of carbapenem inactivation method (CIM) in detecting carbapenemase-producing Acinetobacter baumannii.Methods A total of 121 strains of A. baumannii were identified and subjected to antimicrobial susceptibility testing by VITEK compact. Carbapenem inactivation method (CIM) was applied to detect the carbapenemase in the A. baumannii strains. The OXA-23 type carbapenemase-encoding genes were analyzed by common PCR method.Results Six-eight of the 121 strains showed resistance to imipenem and meropenem. PCR showed that 65 of the 68 strains carried OXA-23 gene. CIM was positive in 66 of the 68 strains. And 52 of the 121A. baumannii strains were susceptible to imipenem and meropenem. PCR showed that OXA-23 gene was negative in 49 of the 52 strains. CIM was negative in the 52 strains of non-carbapenemase-producing A. baumannii. Only one strain was resistant to imipenem but susceptible to meropenem. CIM was negative but QXA-23 was positive for this strain. The sensitivity and the specificity of CIM was 94.2% and 98.1% respectively in detecting carbapenemase-producing A. baumannii.Conclusions The results of CIM were consistent with the results obtained by PCR to detect the encoding gene of OXA-23. CIM is inexpensive, easier to operate and interpret than PCR method. CIM is applicable to detect OXA-23 type carbapenemase rapidly inA. baumannii.

Analysis on drug-resistant genes and homology in nosocomial infection of carba penem-resistant Acinetobacter baumannii / 国际检验医学杂志

Objective To analyze the drug resistance genes and homology in nosocomial infection caused by carbapenem‐resistant Acinetobacter baumannii(CRAB) .Methods Forty strains of CRAB were collected and the minimal inhibitory concentration (MIC) was detected by the agar dilution method .The main carbapenemase genes were detected by PCR .The PCR products were performed the DNA sequencing .The enterobacterial repetitive intergenic consensus (ERIC)‐polymerase chain reaction(PCR) was performed to analyze the genotypes and homology of these strains .The plasmid conjugation experiment was carried out to investigate the trans‐mission of resistant genes .Results Forty strains of CRAB maintained the good sensitivity only to polymyxin B (100% ) and cef‐operazone/sulbactam(57 .5% ) .All of them had different degrees of resistance to other drugs .The OXA‐23 and OXA‐51genes were detected by PCR amplified in all of the CRAB strains .The homology analysis showed that the strains could be divided into 4 types according to 80% of cluster similarity coefficient ,the type A1 (19 strains) was the major epidemic strain ,moreover the various de‐partments had cross infection ;the plasmid conjugation experiment failed .Conclusion There is an endemic spread of CRAB in our hospital and its resistant mechanism is mainly related with OXA‐23 gene expression ;in the rational antibiotics use at the same time , the isolation and prevention measures should be done well for avoiding the hospital cross infection .

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

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

The LAMP method applied in the detection of carbapenem-resistance acinetobacter baumannii OXA-23 genes / 国际检验医学杂志

Objective To establish a simple ,rapid,highly specific and sensitive molecular detection of carbapenem-resistance acinetobacter baumannii(CRAB)OXA-23 genes,and this method is used to detect the multiple drug-resistant acinetobacter bau-mannii in our hospital,and the purpose is to know the antibiotic resistance of CRAB OXA-23 genes .Methods The loop-mediated isothermal amplification(LAMP)was established for detection of the CRAB OXA-23 genes,and a set of specific primers were de-signed by special software,PrimerExplorer version 4.The LAMP assay was developed on using SYBR Green Ⅰ for fluorescent chromogenic reaction substances,improved through a series of optimization tests,and through macroscopic observation and electro-phoresis test comparison results.At the same time,the application of LAMP was used to test 41 multiple drug-resistant acineto-bacter baumanniis which were collected from December 2013 to March 2014 in our hospitalized patients.Results The ladder ban-ding was produced in CRAB OXA-23 genes strains by the LAMP detection through electrophoresis test,however,no ladder ban-ding was observed in the others .The color of the amplification product in genes strain CRAB OXA-23 changed from orange to green by adding 1 μL SYBR Green Ⅰ,however it was still orange in others.The sensitivity of the LAMP detection in pure cultrue was 5 cfu/μL of the CRAB OXA-23 genes cells.Application of LAMP was used to separate multiple drug-resistant acinetobacter baumanniis from hospitalized patients ,32 strains were tested in 41 strains,the positive rate was 78.04%.Conclusion Separation of the CRAB OXA-23 genes carry rate is higher in our hospital ,and they have very high resistance of commonly used antibacterial drugs.The LAMP method to test OXA-23 gene of CRAB was established in this research was simple ,fast,sensitive and specific. Therefore,it is especially suitable wider use at the grass-roots unit,and it is of great significance for selecting reasonable choice of antibiotics by clinical doctor.

Characterization of the Multidrug-Resistant Acinetobacter species Causing a Nosocomial Outbreak at Intensive Care Units in a Korean Teaching Hospital: Suggesting the Correlations with the Clinical and Environmental Samples, Including Respiratory Tract-rel / 대한임상미생물학회지

BACKGROUND: Acinetobacter spp. is an important nosocomial pathogen for which increasing resistance to multiple antimicrobial agents has been observed. Prevalence of multidrug-resistant (MDR) Acinetobacter spp. in the intensive care unit (ICU) at a teaching hospital in Korea started to increase in 2008. The aim of this study was to determine the source of pathogen spread and to characterize the emerging strains at an early stage of outbreak. METHODS: Samples from respiratory instruments and fomites in the ICUs, as well as from the healthcare workers, were cultured to identify the sources of MDR Acinetobacter spp. Antimicrobial susceptibility was determined by the CLSI disk diffusion method. Pulsed field gel electrophoresis (PFGE) was performed for clinical and environmental isolates in order to determine clonality. Carbapenemase genes were detected by multiplex PCR. Infection control measures including peer-monitoring of hand washing, environmental cleaning and standard precautions were enforced. RESULTS: Among the samples from the ICU tools (105) and healthcare worker's hands (44), 31 (30%) and 2 (5%) respective samples yielded MDR Acinetobacter spp. Among the environmental samples, 90% were from respiratory-related equipment. The majority of clinical and environmental MDR Acinetobacter spp. (44/55) belonged to the pulsotype A. baumannii and carried both bla(OXA-51)-like and bla(OXA-23)-like genes. Even though infection-control measures were enforced, prevalence of MDR Acinetobacter spp. continues to increase. CONCLUSION: An outbreak of MDR Acinetobacter spp. in a Korean hospital was caused by A. baumannii carrying the bla(OXA-23)-gene and was correlated with contaminated respiratory-related instruments in the ICUs. More intensive measures for nosocomial infection control are needed for successful prevention of Acinetobacter spread in hospitals.

Detection of blaOXA-23 in Acinetobacter spp. isolated from patients of a university hospital

INTRODUCTION: Acinetobacter spp. have emerged as notorious pathogens involved in healthcareassociated infections. Carbapenems are important antimicrobial agents for treating infections due to multidrug resistant Acinetobacter spp. Different mechanisms may confer resistance to these drugs in the genus, particularly production of class D carbapenemases. OXA-23-like family has been pointed out as one of the predominant carbapenamases among Acinetobacter. The present work aimed to investigate the occurrence of OXA-23-like carbapenemases among Acinetobacter isolates recovered from patients of a university hospital in Niterói, RJ, Brazil. METHODS: Antimicrobial susceptibility profiles were determined by disk-diffusion. Imipenem resistant isolates were submitted to Modified Hodge Test in order to screen for carbapenemase production, and later to polymerase chain reaction (PCR) to investigate the presence of blaOXA-23. RESULTS: Imipenem and meropenem resistance rates were 71.4% and 69.7%, respectively. The Modified Hodge Test revealed carbapenemase production among 76 (89.4%) of the 85 imipenem resistant isolates analyzed; according to PCR results, 81 isolates (95.4%) carried the blaOXA-23 gene. CONCLUSIONS: OXA-23-like enzymes may be an important mechanism of carbapenem resistance among isolates present in the hospital studied.

Preparation, crystallization and preliminary X-ray crystallographic analysis of OXA-23, a carbapenemase conferring widespread antibiotic resistance.

OXA-23, a class D carbapenemase that confers widespread antibiotic resistance hydrolyzes the β-lactam rings of β-lactam antibiotics, presenting an enormous challenge to infection control, particularly in the eradication of pathogenic bacteria such as Acinetobacter baumannii, one of six top-priority dangerous pathogens. Thus, the enzyme is a potential target for developing antimicrobial agents against pathogens producing carbapenemases. In this study, OXA-23 was purified and crystallized at 298 K and X-ray diffraction data from OXA-23 crystal were collected at 2.03 Å resolution using synchrotron radiation. The crystal of OXA-23 belonged to space group P41 with unit cell parameters a = 82.47, b = 82.47 and c = 172.01 Å. Analysis of the packing density showed that the asymmetric unit probably contained two molecules with a solvent content of 73.64%.

Characterization of Acinetobacter baumannii Co-producing Carbapenemases OXA-23 and OXA-66, and armA 16S Ribosomal RNA Methylase at a University Hospital in South Korea / 대한임상미생물학회지

BACKGROUND: In the present study, the resistance mechanisms against carbapenems and aminoglycosides for 23 strains of multi-drug-resistant Acinetobacter baumannii isolated at a university hospital were investigated. METHODS: The minimal inhibitory concentrations (MICs) were determined via broth microdilution or Etest. The genes encoding OXA-type carbapenemases and 16S rRNA methylase were identified using multiplex PCR, and the amplified products were sequenced. Conjugation experiments were conducted, and an epidemiologic study was performed using enterobacterial repetitive intergenic consensus (ERIC)-PCR. RESULTS: In the isolates, the MICs of the tested aminoglycosides, including arbekacin, were >1024 microg/mL; the MICs of aztreonam, cefepime, ceftazidime, and ciprofloxacin ranged from 64 to 128 microg/mL; and the MICs of carbapenem ranged from 32 to 64 microg/mL, as determined through the broth microdilution test. According to the E-test, the MICs of ampicillin/sulbactam and colistin were 8 and 0.25 to 0.38 microg/mL, respectively. Sequence analysis confirmed that all of the isolates expressed carbapenemases OXA-23 and OXA-66, as well as armA 16S rRNA methylase. In addition, ISAba1 was identified upstream of the gene encoding OXA-23. OXA-23 and armA were not transferred to Escherichia coli J53 cells in the transconjugation experiments. ERIC-PCR molecular fingerprinting produced a single pattern in all cases. CONCLUSION: The co-production of OXA-23 and armA 16S rRNA methylase may be attributed to the multidrug resistance of the A. baumannii isolates in the present study. Stricter surveillance and more rapid detection are necessary to prevent the spread of this type of resistance in the future.

Mechanisms of Acquiring Carbapenem-resistance in Acinetobacter Species / 대한임상미생물학회지

A rapid dissemination of carbapenem-resistant Acinetobacter spp. represents a significant clinical threat. Production of OXA carbapenemases and metallo-beta- lactamases (MBLs) is the most important mechanism in acquiring carbapenem resistance in Acinetobacter spp. Carbapenem resistance has also ascribed to non- enzymatic mechanisms, including changes in outer membrane proteins, alterations in the affinity or expression of penicillin-binding proteins, and overexpression of efflux pumps. The most important mechanism in A. baumannii isolates from Korea is the production of OXA-23, while that in other species of Acinetobacter is the production of metallo-beta-lactamases.

Outbreaks of Imipenem Resistant Acinetobacter Baumannii Producing OXA-23 beta-Lactamase in a Tertiary Care Hospital in Korea

PURPOSE: Since November 2006, imipenem-resistant Acinetobacter baumannii isolates have increased in Kyung Hee University Hospital in Seoul, Korea. The purpose of this study was to determine the genetic basis and molecular epidemiology of outbreak isolates. MATERIALS AND METHODS: Forty-nine non-repetitive isolates of the 734 IRAB strains were investigated in order to determine their characteristics. The modified Hodge and the ethylenediaminetetraacetic acid (EDTA)-disk synergy test were performed for the screening of carbapenemase and metallo-beta-lactamase production. Multiplex polymerase chain reaction (PCR) assays were performed for the detection of genes encoding for OXA-23-like, OXA-24-like, OXA-58-like and OXA-51-like carbapenemase. Pulsed-field gel electrophoresis (PFGE) was performed for strain identification. RESULTS: All isolates showed 100% resistance to ciprofloxacin and gentamicin, 97.9% resistance to cefepime, piperacillin/tazobactam, aztreonam, ceftazidime and piperacillin, 93.9% resistance to tobramycin and 57.1% resistance to amikacin. All of the 49 isolates (100%) showed positive results in the modified Hodge test and negative results in the EDTA-disk synergy test. They all (100%) possessed the encoding gene for an intrinsic OXA-51-like carbapenemase and an acquired OXA-23-like carbapenemase in the multiplex PCR assay. PFGE patterns revealed that all isolates were clonally related from A1 to A14. CONCLUSION: It is concluded that all of the 49 IRAB isolates acquired resistance to imipenem by producing OXA-23 carbapenemase and they might have originated from a common source.

Prevalence of Imipenem-Resistant Pseudomonas aeruginosa Isolates and Mechanisms of Resistance / 대한임상미생물학회지

BACKGROUND: Spread of imipenem-resistant Pseudomonas aeruginosa isolates is an important clinical threat. The aim of this study is to survey the prevalence of carbapenem-resistant P.aeruginosa isolates in a university hospital, Busan, Korea, and to determine the mechanisms of the resistance. METHODS: P.aeruginosa isolates from the patients in Kosin University Gospel Hospital were collected during the period of June through September, 2004. Antimicrobial susceptibilities were tested by the disk diffusion method, and production of carbapenemase and metallo-beta-lactamase was determined by the modified Hodge and EDTA-disk synergy tests, respectively. MICs were determined by the agar dilution method, and pIs of beta-lactamases were determined by the isoelectric focusing. Genotypes of carbapenemases were determined by direct sequencing of amplified products. RESULTS: A total of 77 clinical isolates of P.aeruginosa were collected. Twenty-two (55.0%) and 15 (37.5%) isolates showed positive results in the modified Hodge and EDTA-disk synergy tests, re-spectively. Searches for bla OXA-23 and bla IMP-1 genes showed positive results in 15 and 12 isolates, respectively. MIC ranges of imipenem and meropenem to OXA-23-producing isolates were 8-16 microgram/mL and 2-32 microgram/mL, respectively, and those to IMP-1-producing isolates were 2-> or =256 microgram/mL and 2-128 microgram/mL, respectively. CONCLUSION: Production of OXA-23 or IMP-1 is the most prevalent mechanism of imipenem-resistance in P.aeruginosa isolates in a university hospital, Busan, Korea. Periodical surveys are necessary to monitor the spreading of imipenem-resistant isolates and emerging new mechanisms of imipenem-resistance.

Molecular Epidemiology of Infection Caused by OXA-23 or IMP-1 beta-Lactamase-Producing Acinetobacter baumannii / 대한임상미생물학회지

BACKGROUND: The purposes of this study were to investigate the prevalence of imipenem-resistant clinical Acinetobacter baumannii isolates and to determine the mechanism of the resistance. METHODS: During the period of June to September 2004, susceptibility to imipenem of A. baumannii isolates from a hospital in Busan, Korea were investigated. The isolates were screened for the production of carbapenemase and metallo-beta-lactamase by Modified-Hodge and EDTA-disk synergy tests, respectively; minimum inhibitory concentrations (MICs) were determined by agar dilution method. Genes coding for GES, IMP, VIM, SMP-1, GIM-1 and OXA type beta-lactamases were searched by PCR amplification, and the PCR products were subjected to direct sequencing. Isoelectric points of beta-lactamases were estimated by isoelectric focusing and the epidemiological relationships of isolates were investigated by enterobacterial repetitive intergenic consensus (ERIC) PCR. RESULTS: Fifty eight strains of A. baumannii were isolated from clinical specimens during the surveillance period, and 14 isolates (24.1%) were resistant to imipenem. Of the 14 isolates, 9 were tested positive in Modified-Hodge test and 2 were also positive in EDTA-disk synergy test. Genes encoding OXA-23 and IMP-1 were detected in 7 and 2 isolates, respectively. In IEF studies, OXA-23 and IMP-1 enzymes had corresponding pIs at 6.7 and 9.0, respectively. Seven OXA-23-producing and 2 IMP-1-producing isolates showed the same ERIC PCR patterns. CONCLUSION: It is concluded that 7 and 2 A. baumannii isolates from the patients in a hospital in Busan acquired resistance to imipenem by producing OXA-23 and IMP-1 beta-lactamases, respectively. The isolates producing these beta-lactamases might be originated from a common source.

Prevalence of OXA-23 Extended-Spectrum beta-Lactamase-Producing Clinical Isolates of Acinetobacter baumannii in a University Hospital, Busan, Korea / 대한임상미생물학회지

BACKGROUND: Acinetobacter baumannii is a glucose-nonfermenting gram-negative rod and is a well-recognized nosocomial pathogen. In recent years, A. baumannii strains showing resistance to carbapenems by producing metallo-beta-lactamases or OXA-type beta-lactamases have increased, and it is considered to be a serious clinical problem. But genotypes of carbapenemases produced by A. baumannii isolates in Korea have been rarely reported. The purpose of this study was to investigate the prevalence of imipenem-resistant A. baumannii and to determine the mechanism of resistance. METHODS: During the period of January through September, 2003, susceptibilities to imipenem of A. baumannii isolates from patients admitted in Kosin University Gospel Hospital in Busan, Korea were investigated. The modified Hodge and EDTA-disk synergy tests were performed for screening of carbapenemase and metallo-beta-lactamase-production. Minimal inhibitory concentrations (MICs) were determined by the agar dilution method. For detection of IMP, VIM and OXA-type beta-lactamases genes, polymerase chain reactions (PCR) were performed, and the DNA sequences of OXA-type beta-lactamases genes were determined by using the dideoxy-chain termination method. The isoelectric points of beta-lactamases were determined by isoelectric focusing. Pulsed-field gel electrophresis (PFGE) of the SmaI-digested genomic DNA was performed. RESULTS: A total of 193 strains of A. baumannii were collected from patients during the surveillance period. Twenty-seven percents (52/193) of A. baumannii isolates were resistant to imipenem. Among the 52 imipenem-resistant isolates, 41 isolates (78.8%) showed positive results in the modified Hodge test, but none of the isolates showed positive results in the EDTA-disk synergy test. Thirty-eight modified Hodge test-positive isolates harbored blaOXA-23 gene, but none of the isolates harbored IMP- or VIM-type metallo-beta-lactamases genes. Analytical isoelectric focusing revealed that all the 38 isolates had a nitrocefin-positive band at pI of 6.65. Thirty-five OXA-23-producing isolatesshowed a similar PFGE pattern when digested by SmaI endonuclease. CONCLUSION: Thirty-eight clinical isolates of A. baumannii acquired resistance to imipenem by producing OXA-23 beta-lactamase. Among them were 35 isolates thought to be originated from the same source, because they contained a similar chromosomal type. To the best of our knowledge, this is the first time that OXA-23 beta-lactamase has been detected in Korea.