ofAcinetobacter Baumannii Research Articles

RND-Type Efflux Pumps in Multidrug-Resistant Clinical Isolates of Acinetobacter baumannii: Major Role for AdeABC Overexpression and AdeRS Mutations

Increased expression of chromosomal genes for resistance-nodulation-cell division (RND)-type efflux systems plays a major role in the multidrug resistance (MDR) of Acinetobacter baumannii. However, the relative contributions of the three most prevalent pumps, AdeABC, AdeFGH, and AdeIJK, have not been evaluated in clinical settings. We have screened 14 MDR clinical isolates shown to be distinct on the basis of multilocus sequence typing (MLST) and pulsed-field gel electrophoresis (PFGE) for the presence and overexpression of the three Ade efflux systems and analyzed the sequences of the regulators AdeRS, a two-component system, for AdeABC and AdeL, a LysR-type regulator, for AdeFGH. Gene adeB was detected in 13 of 14 isolates, and adeG and the intrinsic adeJ gene were detected in all strains. Significant overexpression of adeB was observed in 10 strains, whereas only 7 had moderately increased levels of expression of AdeFGH, and none overexpressed AdeIJK. Thirteen strains had reduced susceptibility to tigecycline, but there was no correlation between tigecycline MICs and the levels of AdeABC expression, suggesting the presence of other mechanisms for tigecycline resistance. No mutations were found in the highly conserved LysR regulator of the nine strains expressing AdeFGH. In contrast, functional mutations were found in conserved domains of AdeRS in all the strains that overexpressed AdeABC with two mutational hot spots, one in AdeS near histidine 149 suggesting convergent evolution and the other in the DNA binding domain of AdeR compatible with horizontal gene transfer. This report outlines the high incidence of AdeABC efflux pump overexpression in MDR A. baumannii as a result of a variety of single mutations in the corresponding two-component regulatory system.

Prevalence of Metallo-β-Lactamases ProducingAcinetobacter baumanniiin a Moroccan Hospital

Objective. To determine the prevalence of metallo beta-lactamases (MBL) among carbapenem resistant strains ofAcinetobacter baumanniiin our hospital.Methodology. During a period of 12 months (January–December 2010), 47 isolates ofAcinetobacter baumanniiwere collected from different clinical specimens of in-patients. Antimicrobial susceptibility was determined and interpreted using the disk diffusion method according to the Antibiogram Committee of the French Society for Microbiology guidelines. Imipenem nonsusceptible isolates were further screened for production of MBL.Results. AllAcinetobacter baumannii’ isolates were resistant to ticarcillin, ticarcilline/clavulanate, piperacillin, piperacillin/tazobactam, gentamicin, tobramycin, and ciprofloxacin, except an isolate that was sensitive to ceftazidime and cefepime. In addition to that, amikacin and trimethoprim/sulfamethoxazole were, respectively, sensitive by 59.5% and 53%. Among 57,4% (27/47) imipenem non-susceptible isolates ofAcinetobacter baumannii, 74% (20/27) were found to be MBL producers.Conclusion. Although the rate of imipenem non-susceptible isolates ofAcinetobacter baumanniseems to remain stable in 2005 (57%) and 2010 (57,5%), the prevalence of MBL producer strain is increasing (38% in 2005 versus 75% in 2010). The findings strongly suggest that there is a need to track the detection of MBL producers; moreover, a judicious use of carbapenems is necessary to prevent further spread of these organisms.

Prevention and Treatment of Virulent Bacterial Biofilms with an Enzymatic Nitric Oxide-Releasing Dressing

The use of percutaneous medical devices often results in nosocomial infections. Attachment of microorganisms to the surfaces of these medical devices triggers biofilm formation, which presents significant complications to the health of a patient and may lead to septicemia, thromboembolism, or endocarditis if not correctly treated. Although several antimicrobials are commonly used for prevention of biofilm formation, they have limited efficacy against formed biofilms. In this study, we report the use of an enzymatic, gaseous nitric oxide (gNO)-releasing dressing for the prevention and treatment of Acinetobacter baumannii, methicillin-resistant Staphylococcus aureus, and Pseudomonas aeruginosa biofilms. Results show that the bactericidal activity against biofilms of the test strains was dependent on time and rate of gNO release from the dressing. Following 6 h of treatment, gNO-releasing dressings significantly inhibited the growth of test strains relative to vehicle control dressings, demonstrating eradication of bacterial concentrations of up to 10(5) CFU/cm(2). Complete cell death was observed for both prevention of biofilm formation and treatment of 24-h-grown biofilms after 6 h of treatment with the gNO-releasing dressings. Further, gNO-releasing dressings were more efficient against formed biofilms than other antimicrobial agents currently used. These results demonstrate that the gNO-releasing dressing can produce sufficient levels of gNO over a therapeutically relevant duration for maximal bactericidal effects against virulent bacterial strains known to cause nosocomial infections.

Assessing the Burden of Acinetobacter baumannii in Maryland: A Statewide Cross-Sectional Period Prevalence Survey

Objective.To determine the prevalence ofAcinetobacter baumannii, an important healthcare-associated pathogen, among mechanically ventilated patients in Maryland.Design.The Maryland MDRO Prevention Collaborative performed a statewide cross-sectional active surveillance survey of mechanically ventilated patients residing in acute care and long-term care (LTC) facilities. Surveillance cultures (sputum and perianal) were obtained from all mechanically ventilated inpatients at participating facilities during a 2-week period.Setting.All healthcare facilities in Maryland that provide care for mechanically ventilated patients were invited to participate.Patients.Mechanically ventilated patients, known to be at high risk for colonization and infection withA. baumannii, were included.Results.Seventy percent (40/57) of all eligible healthcare facilities participated in the survey, representing both acute care (n= 30) and LTC (n= 10) facilities in all geographic regions of Maryland. Surveillance cultures were obtained from 92% (358/390) of eligible Patients.A. baumanniiwas identified in 34% of all mechanically ventilated patients in Maryland; multidrug-resistantA. baumanniiwas found in 27% of all Patients.A. baumanniiwas detected in at least 1 patient in 49% of participating facilities; 100% of LTC facilities had at least 1 patient with A.baumannii, compared with 31% of acute care facilities.A. baumanniiwas identified from all facilities in which 10 or more patients were sampled.Conclusions.A.baumanniiis common among mechanically ventilated patients in both acute care and LTC facilities throughout Maryland, with a high proportion of isolates demonstrating multidrug resistance.

Assessing the Burden ofAcinetobacter baumanniiin Maryland: A Statewide Cross-Sectional Period Prevalence Survey

To determine the prevalence of Acinetobacter baumannii, an important healthcare-associated pathogen, among mechanically ventilated patients in Maryland. The Maryland MDRO Prevention Collaborative performed a statewide cross-sectional active surveillance survey of mechanically ventilated patients residing in acute care and long-term care (LTC) facilities. Surveillance cultures (sputum and perianal) were obtained from all mechanically ventilated inpatients at participating facilities during a 2-week period. All healthcare facilities in Maryland that provide care for mechanically ventilated patients were invited to participate. Mechanically ventilated patients, known to be at high risk for colonization and infection with A. baumannii, were included. Seventy percent (40/57) of all eligible healthcare facilities participated in the survey, representing both acute care ([Formula: see text]) and LTC ([Formula: see text]) facilities in all geographic regions of Maryland. Surveillance cultures were obtained from 92% (358/390) of eligible patients. A. baumannii was identified in 34% of all mechanically ventilated patients in Maryland; multidrug-resistant A. baumannii was found in 27% of all patients. A. baumannii was detected in at least 1 patient in 49% of participating facilities; 100% of LTC facilities had at least 1 patient with A. baumannii, compared with 31% of acute care facilities. A. baumannii was identified from all facilities in which 10 or more patients were sampled. A. baumannii is common among mechanically ventilated patients in both acute care and LTC facilities throughout Maryland, with a high proportion of isolates demonstrating multidrug resistance.

Antibacterial Activities of Iron Chelators against Common Nosocomial Pathogens

The activities of iron chelators (deferoxamine, deferiprone, Apo6619, and VK28) were evaluated against type strains of Acinetobacter baumannii, Pseudomonas aeruginosa, Staphylococcus aureus, Klebsiella pneumoniae, and Escherichia coli. Deferiprone, Apo6619, and VK28 each inhibited growth in standard and RPMI tissue culture medium, while deferoxamine had no effect. Additionally, time-kill assays revealed that VK28 had a bacteriostatic effect against S. aureus. Therefore, these newly developed iron chelators might provide a nontraditional approach for treatment of bacterial infections.

In Vitro and In Vivo Biological Activities of Iron Chelators and Gallium Nitrate against Acinetobacter baumannii

We investigated the ability of compounds interfering with iron metabolism to inhibit the growth of Acinetobacter baumannii. Iron restriction with transferrin or 2,2-bipyridyl significantly inhibited A. baumannii growth in vitro. Gallium nitrate alone was moderately effective at reducing A. baumannii growth but became bacteriostatic in the presence of serum or transferrin. More importantly, gallium nitrate treatment reduced lung bacterial burdens in mice. The use of gallium-based therapies shows promise for the control of multidrug-resistant A. baumannii.

Molecular analysis of imipenem-resistantAcinetobacter baumanniiisolated from US service members wounded in Iraq, 2003–2008

Global dissemination of imipenem-resistant (IR) clones of Acinetobacter baumannii - A. calcoaceticus complex (ABC) have been frequently reported but the molecular epidemiological features of IR-ABC in military treatment facilities (MTFs) have not been described. We characterized 46 IR-ABC strains from a dataset of 298 ABC isolates collected from US service members hospitalized in different US MTFs domestically and overseas during 2003-2008. All IR strains carried the bla(OXA-51) gene and 40 also carried bla(OXA-23) on plasmids and/or chromosome; one carried bla(OXA-58) and four contained ISAbal located upstream of bla(OXA-51). Strains tended to cluster by pulsed-field gel electrophoresis profiles in time and location. Strains from two major clusters were identified as international clone I by multilocus sequence typing.

Characterization of eDNA from the Clinical StrainAcinetobacter baumanniiAIIMS 7 and Its Role in Biofilm Formation

Release of extracellular DNA (eDNA) was observed during in vitro growth of a clinical strain of Acinetobacter baumannii. Membrane vesicles (MV) of varying diameter (20–200 nm) containing DNA were found to be released by transmission electron microscopy (TEM) and atomic force microscopy (AFM). An assessment of the characteristics of the eDNA with respect to size, digestion pattern by DNase I/restriction enzymes, and PCR-sequencing, indicates a high similarity with genomic DNA. Role of eDNA in static biofilm formed on polystyrene surface was evaluated by biofilm augmentation assay using eDNA available in different preparations, for example, whole cell lysate, cell-free supernatant, MV suspension, and purified eDNA. Biofilm augmentation was seen up to 224.64%, whereas biofilm inhibition was 59.41% after DNase I treatment: confirming that eDNA facilitates biofilm formation in A. baumannii. This is the first paper elucidating the characteristics and role of eDNA in A. baumannii biofilm, which may provide new insights into its pathogenesis.

Screening for Acinetobacter baumannii Colonization by Use of Sponges

There is currently no consensus method for the active screening of Acinetobacter baumannii. The use of swabs to culture nostrils, pharynx, and skin surface of various anatomical sites is known to yield less-than-optimal sensitivity. In the present study, we sought to determine whether the use of sterile sponges to sample large areas of the skin would improve the sensitivity of the detection of A. baumannii colonization. Forty-six patients known to be colonized with A. baumannii, defined by a positive clinical culture for this organism as defined by resistance to more than two classes of antimicrobials, participated in the study. The screening sites included the forehead, nostrils, buccal mucosa, axilla, antecubital fossa, groin, and toe webs with separate rayon swabs and the forehead, upper arm, and thigh with separate sponges. Modified Leeds Acinetobacter medium (mLAM) agar plates that contained vancomycin and either aztreonam or ceftazidime were used as the selective medium. An enrichment culture grown overnight substantially increased the sensitivity for most sites. The sensitivity ranged between 69.6 and 82.6% for individual sponge sites and 21.7 to 52.2% for individual swab sites when mLAM plates with ceftazidime were inoculated after a 24-h enrichment period. The sponge and swab sites with the best sensitivity were the leg and the buccal mucosa, respectively (82.6% and 52.2%; P = 0.003). The combined sensitivity for the upper arm and leg with a sponge was 89.1%. The novel screening method using sterile sponges was easy to perform and achieved excellent sensitivity for the detection of A. baumannii colonization.

Mutation ofgyrA andparC in clinical isolates ofAcinetobacter baumannii and its relationship with antimicrobial drugs resistance in Taiwan

Acinetobacter baumannii is a species of non fermentative 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. Herein we have determined the mutation frequency of two hot spot residue 83 in gyrA of gyrase and residue 80 in parC of topoisomerase IV and performed a comparative screen the drug resistance ability in 128 clinical isolates ofAcinetobacter baumannii in Taiwan region. Low frequency of mutation was found (11.7%, 11.7%, and 10.2% ingyrA, parC, or both, respectively). Mutation of these sites was not correlated with drug resistance. Our study suggested that mutation ofgyrA andparC may play a minor role in quinolone resistance and other mechanisms may contribute to the drug resistance ofA. Baumannii.

An OXA-66/OXA-51-Like Carbapenemase and Possibly an Efflux Pump Are Associated with Resistance to Imipenem in Acinetobacter baumannii

We investigated the mechanisms involved in imipenem resistance in 23 clinical strains of Acinetobacter baumannii. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis showed the presence of a 30-kDa protein in imipenem-intermediate A. baumannii (IIAB) and imipenem-resistant A. baumannii (IRAB) strains; this protein was almost undetectable in imipenem-susceptible A. baumannii (ISAB) strains. The 30-kDa protein was identified as an OXA-51-like carbapenemase using two-dimensional gel electrophoresis and mass spectrometry. Similar to other recent findings, bla(OXA-51-like) genes were found to exist in all 23 clinical strains; however, the transcript levels of bla(OXA-51-like) in the IIAB and IRAB were higher than in the ISAB strains using reverse transcriptase PCR (RT-PCR) and real-time RT-PCR assays. This change was due to the presence of an insertion sequence, ISAba1, upstream of bla(OXA-51-like) in the IIAB and IRAB strains that was not present in the ISAB strains. The introduction of bla(OXA-66) (a bla(OXA-51)(-like) gene), identified in ISAB ab1254 and IRAB ab1266, into Escherichia coli TOP10 cells resulted in 3.95-fold and 7.90-fold elevations in resistance to imipenem, respectively. Furthermore, when ISAB ab8 and ISAB ab1254 and their in vitro-selected imipenem-resistant mutants ISAB ab8(r) and ISAB ab1254(r) were compared, the results showed no change in the bla(OXA-66)/bla(OXA-51-like) gene sequences, in expression of the gene, and in the outer membrane protein profiles. However, there was a four- to eightfold reduction in imipenem resistance upon adding carbonyl cyanide m-chlorophenylhydrazone. Taken together, these results suggest that the OXA-66/OXA-51-like carbapenemase contributes to intrinsic resistance to imipenem; however, drug export by an efflux pump may be more important and/or occur more frequently in imipenem-resistant A. baumannii. Furthermore, this is the first report of a Taiwanese strain of an OXA-66/OXA-51-like carbapenemase that confers imipenem resistance in A. baumannii.

Spread in an Italian Hospital of a Clonal Acinetobacter baumannii Strain Producing the TEM-92 Extended-Spectrum β-Lactamase

Clinical isolates of Acinetobacter baumannii (n = 470) were collected during a 7-year period and investigated for the genetic determinants of resistance to expanded-spectrum beta-lactams. Thirty-one isolates produced the TEM-92 extended-spectrum beta-lactamase (ESBL) and were clonally related. This is the first report of A. baumannii producing a TEM-type ESBL.

Crystal structure of the carbapenemase OXA-24 reveals insights into the mechanism of carbapenem hydrolysis

Combating bacterial resistance to beta-lactams, the most widely used antibiotics, is an emergent and clinically important challenge. OXA-24 is a class D beta-lactamase isolated from a multiresistant epidemic clinical strain of Acinetobacter baumannii. We have investigated how OXA-24 specifically hydrolyzes the last resort carbapenem antibiotic, and we have determined the crystal structure of OXA-24 at a resolution of 2.5 A. The structure shows that the carbapenem's substrate specificity is determined by a hydrophobic barrier that is established through the specific arrangement of the Tyr-112 and Met-223 side chains, which define a tunnel-like entrance to the active site. The importance of these residues was further confirmed by mutagenesis studies. Biochemical and microbiological analyses of specific point mutants selected on the basis of structural criteria significantly reduced the catalytic efficiency (k(cat)/K(m)) against carbapenems, whereas the specificity for oxacillin was noticeably increased. This is the previously unrecognized crystal structure that has been obtained for a class D carbapenemase enzyme. Accordingly, this information may help to improve the development of effective new drugs to combat beta-lactam resistance. More specifically, it may help to overcome carbapenem resistance in A. baumannii, probably one of the most worrying infectious threats in hospitals worldwide.

Activity of Tigecycline (GAR-936) against Acinetobacter baumannii Strains, Including Those Resistant to Imipenem

We determined the in vitro activities of tigecycline and imipenem against 49 isolates of Acinetobacter baumannii, including those resistant to imipenem. The MIC at which 50% of the isolates were inhibited (MIC(50)) and the MIC(90) for tigecycline and imipenem were 2 and 2 mg/liter and 32 and 128 mg/liter, respectively, with 92 and 20%, respectively, of the strains being susceptible. Tigecycline did not show bactericidal activity in the time-kill studies (n = 9 strains). Imipenem showed bactericidal activity against seven out of nine strains. These in vitro results show that tigecycline has good in vitro bacteriostatic activity against A. baumannii, including strains resistant to imipenem.

Presence of the Tet M determinant in a clinical isolate of Acinetobacter baumannii.

The tet(M) gene encodes a protein which is related to tetracycline ribosomal protection, one of the mechanisms of tetracycline resistance. A tet(M) gene that is 100% homologous to the tet(M) gene of Staphylococcus aureus has been found in a clinical isolate of Acinetobacter baumannii, which also carries the tet(A) gene encoding a tetracycline efflux pump.

Chromosomal integration of a cephalosporinase gene from Acinetobacter baumannii into Oligella urethralis as a source of acquired resistance to beta-lactams.

Clinical Oligella urethralis isolate COH-1, which was uncommonly resistant to penicillins and narrow-spectrum cephalosporins, was recovered from a 55-year-old patient with a urinary tract infection. Shotgun cloning into Escherichia coli and expression experiments gave recombinant clones expressing either an AmpC beta-lactamase-type phenotype of resistance or a carbenicillin-hydrolyzing beta-lactamase-type phenotype of resistance. The AmpC beta-lactamase identified (ABA-1), which had a pI value of 8.2, had 98% amino acid identity with a chromosomally encoded cephalosporinase of Acinetobacter baumannii. A 820-bp insertion sequence element, ISOur1, belonging to the IS6 family of insertion sequence elements, was identified immediately upstream of bla(ABA-1), providing a -35 promoter sequence and likely giving rise to a hybrid promoter region. The carbenicillin-hydrolyzing beta-lactamase identified (CARB-8), which had a pI value of 6.4, differed from CARB-5 by two amino acid substitutions. Hybridization of CeuI fragment I-restricted DNA fragments of O. urethralis COH-1 with bla(ABA-1)-, bla(CARB-8)-, and 16S rRNA-specific probes indicated the chromosomal integration of the beta-lactamase genes. PCR and hybridization experiments failed to detect bla(CARB-8)- and bla(ABA-1)-like genes in three O. urethralis reference strains, indicating that the beta-lactamase genes identified were the source of acquired resistance in O. urethralis COH-1. This is one of the few examples of the interspecies transfer and the chromosomal integration of a gene encoding a naturally occurring beta-lactamase.

Loss of a 29-kilodalton outer membrane protein in Acinetobacter baumannii is associated with imipenem resistance.

We analyzed the possible causes of imipenem (IPM) resistance in multidrug-resistant isolates of Acinetobacter baumannii. Comparison of the outer membrane protein (OMP) profiles of two genomically related strains (Ab288 [IPM sensitive] and Ab242 [IPM resistant]) indicated the conspicuous loss of a 29-kDa polypeptide in the Ab242 strain. No carbapenemase activity was detected in any of these strains. The treatment of Ab288 with sodium salicylate resulted in IPM resistance and the loss of the 29-kDa OMP. In addition, IPM-resistant clones of Ab288 which were selected by repetitive culturing in increasing concentrations of this antibiotic also showed the absence of this 29-kDa OMP.

Comparison of Pulsed-Field Gel Electrophoresis and Amplified Fragment-Length Polymorphism for Epidemiological Investigations of Common Nosocomial Pathogens

To compare molecular typing by amplified fragment-length polymorphism (AFLP) analysis with pulsed-field gel electrophoresis (PFGE) with respect to the ability to differentiate between epidemiologically related and unrelated isolates of common nosocomial pathogens recovered during a period of endemicity. Retrospective laboratory analysis. Tertiary-care institution. 17 isolates of Acinetobacter baumannii, 22 isolates of Pseudomonas aeruginosa, and 22 vancomycin-resistant Enterococcusfaecium (VRE) were typed by both methods. AFLP generated comparable results to PFGE for A baumannii and P aeruginosa isolates; both methods identified epidemiologically related and unrelated isolates. However, strain typing of VRE isolates produced discordant results between the two methods. PFGE identified 10 different strain types and differentiated between all epidemiologically related and unrelated isolates. In contrast, AFLP generated only five different strain types, three of which contained both epidemiologically related and unrelated isolates. Molecular typing by AFLP is comparable to PFGE for A baumannii and P aeruginosa isolates. For VRE isolates, however, PFGE remains the method of choice.

Surveillance of an adult intensive care unit for long-term persistence of a multi-resistant strain of Acinetobacter baumannii.

Sporadic infections with Acinetobacter spp., punctuated with prolonged outbreaks of infection involving larger numbers of patients and a particular epidemic strain of Acinetobacter baumannii, have occurred in the adult intensive care unit (ICU) of Nottingham University Hospital since 1985. The aim of this study was to screen patients admitted to the ICU for three or more days during a non-outbreak period in 1994-1995 and to use DNA fingerprinting techniques to compare any isolates of Acinetobacter spp. with isolates obtained from the same ICU during the previous ten years. In the present study, almost 20% of the ICU patients screened during 1994-1995 became colonized with Acinetobacter spp. The commonest species isolated from patients was Acinetobacter baumannii; five different strains were identified by random amplified polymorphic DNA fingerprinting, including the epidemic strain responsible for outbreaks of infection in 1985-1986 and 1992-1993. Environmental sampling yielded Acinetobacter spp. from one or more samples on four occasions; Acinetobacter radioresistens was the commonest species isolated, and Acinetobacter baumannii (not the epidemic strain) was isolated on only one occasion from the environment. The long-term persistence of a potentially epidemic strain in the ICU, even during a non-outbreak period, indicates a need for continued vigilance. Consequently, periodic patient and environmental surveillance, combined with typing of isolates, is recommended for ICUs where significant outbreaks of Acinetobacter infection have occurred in the past.