adeS Genes Research Articles

Inside-out, antimicrobial resistance mediated by efflux pumps in clinical strains of Acinetobacter baumannii isolated from burn wound infections.

Acinetobacter baumannii belongs to the ESKAPE group. It is classified as a critical priority group by the World Health Organization and a global concern on account of its capacity to acquire and develop resistance mechanisms to multiple antibiotics. Data from the United States indicates 500 deaths annually. Resistance mechanisms of this bacterium include enzymatic pathways such as ß-lactamases, carbapenemases, and aminoglycoside-modifying enzymes, decreased permeability, and overexpression of efflux pumps. A. baumannii has been demonstrated to possess efflux pumps, which are classified as members of the MATE family, RND and MFS superfamilies, and SMR transporters. The aim of our work was to assess the distribution of efflux pumps and their regulatory gene expression in clinical strains of A. baumannii isolated from burned patients. METHODS: From the Clinical Microbiology Laboratory at the Instituto Nacional de Rehabilitación Luis Guillermo Ibarra Ibarra collection in Mexico, 199 strains were selected. Antibiotics susceptibilities were performed by broth microdilutions to determine minimal inhibitory concentrations. Phenotypic assays with efflux pump inhibitors were conducted using carbonyl cyanide 3-chlorophenylhydrazone (CCCP) and phenylalanine-arginine ß-naphthylamide (PAßN) in conjunction with amikacin, ceftazidime, imipenem, meropenem and levofloxacin. A search was conducted for structural genes that are linked to efflux pumps, and the relative expression of the adeR, adeS, and adeL genes was analyzed. RESULTS: Among a total of 199 strains, 186 exhibited multidrug resistance (MDR). Fluoroquinolones demonstrated the highest resistance rates, while minocycline and amikacin displayed comparatively reduced resistance rates (1.5 and 28.1, respectively). The efflux activity of fluorquinolones exhibited the highest phenotypic detection (from 85 to 100%), while IMP demonstrated the lowest activity of 27% with PAßN and 43.3% with CCCP. Overexpression was observed in adeS and adeL, with adeR exhibiting overexpression. Concluding that clinical strains of A. baumannii fromour institution exhibited efflux pumps as one of the resistance mechanisms.

Efflux pumps encoding genes (adeA and adeS) in relation to antibiotic resistance pattern in Acinetobacter baumannii strains isolated from Benha university hospital

Background Acinetobacter baumannii become a growing problem in hospitals as a predominant multidrug-resistant (MDR). This resistance capacity is generated by various mechanisms including efflux pumps that can direct antibiotics outwards and prevent antibiotics from affecting the bacteria. Methods Clinical samples were cultured on blood agar and MacConkey agar medium, identified by Gram stain and biochemical reactions, and then identified to the species level by Vitek2 automated system. The adeA and adeS genes among isolated strains were detected by conventional PCR. This study included 50 A. baumannii strains to assess the antibiotic resistance pattern of A. baumannii. Results A. baumannii strains were fully resistant to Piperacillin (100%) and lowest resistance to Imipenem (54%) was observed. Out of 50 isolates, 41 (82%) of A. baumanii isolates had adeA gene and 32 (64%) had adeS gene. The clinical strains that had adeA gene and adeS gene showed significantly higher resistance to Ciprofloxacin (92.7%) and (100%), respectively. Additionally, borderline significant differences were reported regarding Gentamycin (P = 0.065), Ceftazidime (P = 0.08), and Meropenem (P = 0.08), with the resistance being higher in the adeA positive group. Conclusion Efflux pumps genes (adeA, adeS) played a key role in antibiotic resistance of A. baumanii.

Study the Role of the Efflux Pump in Multidrug-Resistant Acinetobacter baumannii

Thirty-one A. baumannii isolates were obtained from Shar and Burn hospitals in Sulaimani City, KRG-Iraq. Bacterial identification was confirmed by detecting both 16S rRNA and blaOXA-51 genes. The detection of carbapenem resistant isolates was carried out using Kirby- Bauer, RAPDEC Carba NP and combined disc assays. A high rate of drug resistance was observed, as 74.2% of the isolates were multidrug-resistant (MDR) and 38.7% were extensively drug-resistant (XDR), while carbapenem-resistant A. baumannii (CRAB) comprised approximately 58% of the isolates. Among CRAB isolates, carbapenemase production was detected by using the RAPDEC Carba NP and the combined disc assays in 88.8% (16/18) and 55.5% (10/18) of the isolates respectively. Phenotypic activity of efflux pump was detected among 22.5% of all isolates and 38.8% of CRAB isolates. PCR successfully identified the adeA, adeB, adeC, adeR, and adeS genes in 83.9%, 90.3%, 58%, 80.6%, and 90.3% of the isolates, respectively.
 Eight mutations in adeR and 15 mutations in adeS regulatory genes were recorded. We conclude that carbapenem resistance mechanisms in A. bumanni isolates included mutations in adeRS regulatory genes and production of carbapenemase.

Evaluation of Antibiotic Resistance and adeABC, adeR, adeS Efflux Pump Genes among Foodborne and Clinical Acinetobacter spp. in Türkiye.

The adeABC efflux pump has a crucial role in the resistance of Acinetobacter baumannii strains to antimicrobial agents; it is encoded by adeABC, adeR, adeS genes. We evaluated antibiotic resistance, efflux pump genes, clonal relationships, and analyzed a probable correlation that can exist between antibiotic resistance and the aforementioned genes. We conducted this study on 27 food-originated and 50 human clinical Acinetobacter spp. in Southern Türkiye. MALDI-TOF system and disc diffusion/agar dilution (colistin) methods were used for the identification and antibiotic susceptibility. The efflux pump genes and genetic relatedness of the two groups were investigated by (PCR) and (PFGE) methods. Foodborne A. dijkshoorniae strain was multidrug- resistant (MDR), and none of them resistant to colistin. Most of the clinical isolates (92%) were Extensive-Drug Resistant (XDR); highest resistant to ceftazidime, piperacillin-tazobactam, and imipenem (47, 94%), and were lowest to colistin (7, 14%), respectively. adeABC, and adeR, adeS genes were (23, 85.2%), (9, 33.3%), (27, 100%) and (10, 37.3%), (18, 66.7%) in foodborne strains respectively. These rates were (43, 86%), (48, 96%), (50, 100%), and (34, 68%), (48, 96.7%) in clinical strains respectively. A positive correlation existed between adeA gene positivity and piperacillintazobactam, ceftazidime, gentamycin, imipenem (P=0.048), amikacin (P=0.007) and trimethoprimsulfamethoxazole (P=0.029) resistance in clinical strains. A positive correlation of trimethoprimsulfamethoxazole resistance and adeS gene positivity was seen in foodborne strains (P=0.018). Multiple-efflux pump genes rise in parallel to multidrug-resistance in clinical isolates, while susceptible to diverse antibiotics; food may be a potential provenance for the dissemination of adeABC, adeR and adeS genes.

Molecular Characterization of Tigecycline Non-Susceptibility among Extensively Drug-Resistant Acinetobacter baumannii Isolates of Clinical Origin

Introduction: Tigecycline (TGC) is one of the last-resort therapeutic agents for treating infections caused by extensively drug resistant Acinetobacter baumannii isolates. Although resistance to TGC is not common, non-susceptible A. baumannii (NSAB) isolates have been described. In the current study, we aimed to assess the molecular mechanisms mediating TGC non-susceptibility in 5 clinical isolates of A. baumannii with reduced susceptibility to TGC. Methods: Susceptibility of isolates to TGC as well as various classes of antibiotics was evaluated by broth dilution and disk diffusion methods, respectively. The presence of tetX and tetX1 genes was investigated by PCR. The nucleotide sequences of adeR and adeS genes were assessed by PCR amplicon sequencing. To evaluate the association between reduced susceptibility to TGC and upregulation of AdeABC efflux pump, transcriptional level of adeB gene was quantified by RT-qPCR analysis. Results: All 5 TGC-NSAB isolates had a TGC MIC of ≥4 mg/L and were resistant to all antimicrobials tested by disk diffusion method except for minocycline and doxycycline for which a susceptibility rate of 40% and 20% was observed, respectively. The tetX/X1 genes were not detected in any isolates. All TGC non-susceptible isolates harbored genetic alterations in the adeRS operon, including AdeS G186V, N268H, and D60N and AdeR A136V and V120I substitutions among, which G186V and D60N were predicted by PROVEAN tool analysis as inactivating alterations. Reduced TGC susceptibility was associated with upregulation of AdeABC efflux pump in all TGC non-susceptible isolates. Conclusion: It can be concluded from our results that reduced susceptibility to TGC in the studied isolates was mainly mediated by genetic alterations in the AdeRS system, which resulted in overexpression of AdeABC efflux pump. Emergence of TGC non-susceptibility among isolates that had not been previously exposed to TGC is an issue of great concern.

Genetic expression of AdeR and AdeS genes in multidrug‑resistant Acinetobacter spp., isolated from patients in Menoufia University Hospitals

Background: Acinetobacter is a serious nosocomial pathogen causing critical morbidity and mortality. The drug resistance of this organism is alarmingly high leaving few options for treatment. Numerous mechanisms are involved in its resistance to drug therapy. The active efflux mechanisms are an important factor for the development of multidrug-resistant Acinetobacter which is regulated by the AdeRS operon. The aim of this study end to determine the expression of AdeR and AdeS genes in multidrug‑resistant strains of Acinetobacter isolated from patients in Menoufia University Hospitals. Methods: This study included 100 strains of drug-resistant Acinetobacter spp., isolated from patients in Menoufia University Hospitals. They were collected from different clinical samples. Acinetobacter spp., strains were identified and their antibiotic susceptibilities were determined. Real-time PCR was performed to detect the expression of AdeR and AdeS genes. Results: The resistance of Acinetobacter spp., isolates to tested antibiotics were (94%) to piperacillin-tazobactam, (90%) to ampicillin-sulbactam, (80%) to ceftazidime, (55%) to levofloxacin, (50%) to amikacin, and (~52%) to carbapenems. The AdeR and AdeS genes were expressed in (84%) and (88%) of isolates, respectively. The AdeR and AdeS genes were expressed in (88.4%) and (90.7%) of multidrug-resistant strains, respectively. Conclusion: The majority of Acinetobacter spp., isolates are highly resistant to the most commonly used antibiotics. Also, high expression of adeRS genes may be responsible for the observed resistance among Acinetobacter isolates that demonstrate the possible role of efflux pump regulator genes in multidrug-resistant Acinetobacter.

Efflux pump inhibitory activity of biologically synthesized silver nanoparticles against multidrug-resistant Acinetobacter baumannii clinical isolates.

This study was carried out to investigate the possible efflux pump inhibitory activity of biologically synthesized silver nanoparticles (AgNPs) against multidrug-resistant (MDR) Acinetobacter baumannii isolates. In this study, the physicochemical characteristics of synthesized AgNPs were investigated using scanning electron microscopy (SEM), transmission electron microscopy (TEM), X-ray diffraction (XRD), and Fourier transform infrared spectrophotometer (FTIR) methods. Subsequently, MDR A. baumannii isolates were recovered from clinical samples and the phenotypic cartwheel efflux assay and polymerase chain reaction (PCR) were used to elucidate the possible presence of efflux pump in MDR strains. After treatment of MDR strains with sub-minimum inhibitory concentration (MIC) concentration of AgNPs, the expression level of efflux pump genes was evaluated using a quantitative real-time PCR technique. The synthesized AgNPs had a spherical nanostructure, with mean size 38.89 nm, according to SEM and TEM data. XRD and FTIR results confirmed the synthesis of AgNPs. The results of PCR revealed that among 50 strains, 12 A. baumannii strains had efflux pump genes and the expression level of AdeA, AdeC, AdeS, AdeR, AdeI, AdeJ, and AdeK efflux pump genes was downregulated significantly after the treatment with AgNPs. In addition, the inhibitory effect of AgNPs on efflux pumps can be detected when the MIC of ethidium bromide (EtBr) with AgNPs is lower than that of EtBr alone. According to the results, the biologically synthesized AgNPs exhibit efflux pumps inhibitory activity, which may be one of the possible mechanisms of their antibacterial activity against MDR A. baumannii strains.

Characterization and frequency of antibiotic resistance related to membrane porin and efflux pump genes among Acinetobacter baumannii strains obtained from burn patients in Tehran, Iran

Objective: To explore the characterization and frequency of antibiotic resistance related to membrane porin and efflux pump genes among Acinetobacter baumannii (A. baumannii) strains obtained from burn patients in Tehran, Iran. Methods: In this cross-sectional descriptive study, 100 strains of A. baumannii isolated from burn patients visiting teaching hospitals of Tehran were collected from January 2016 to November 2017. After A. baumannii strains were confirmed, antimicrobial susceptibility testing was done via Kirby-Bauer disc diffusion method according to the Clinical and Laboratory Standards Institute guidelines. PCR amplification was performed for detection of β -lactamase adeR, OprD, adeS genes among A. baumannii strains. Results: All isolates (100%) were resistant to ceftazidime, cefotaxime, cefepime, ciprofloxacin, and piperacillin, and most isolates indicated high resistance (95%-97%) to meropenem, imipenem, gentamicin, ceftriaxone, trimethoprim-sulfamethoxazole, piperacillin-tazobactam, amikacin, and tetracycline. The most effective antibiotic against A. baumannii isolates was colistin (97% sensitivity), followed by tigecycline. The frequency of OprD, adeS, and adeR genes were 98%, 91%, and 77%, respectively. Conclusions: This study shows that the majority of A. baumannii isolates are highly resistant to the antibiotics most commonly used in burn patients. Also, high distribution of OprD and adeRS genes may be responsible for the observed resistances among A. baumannii isolates that demonstrate the possible role of both efflux pumps in simultaneous of carbapenemase production during antibiotic resistance.

Molecular characterization of multidrug resistant strains of Acinetobacter baumannii isolated from pediatric intensive care unit in a Chinese tertiary hospital

BackgroundAcinetobacter baumannii is a nosocomial pathogen which is reported as a major cause of morbidity and mortality in intensive care units (ICUs). However, there is a lack of analysis focused on multidrug-resistant Acinetobacter baumannii (MDRAB) infection among patients from pediatric intensive care unit (PICU) in China. The aim of this study was to investigate the molecular characterization of MDRAB isolated from PICU.MethodsIn this study, 86 isolates of MDRAB were collected from PICU patients, from the First Affiliated Hospital of Sun Yat-sen University. The minimal inhibitory concentrations (MICs) of the isolates against common antibiotics were determined. The carbapenemase-encoding resistance genes and AdeABC-AdeRS efflux system genes of these isolates were detected by PCR. Real-time PCR was performed to determine the relative expression of the relevant efflux pumps.ResultsAmong 86 strains of MDRAB, 76.7% (66/86) were carbapenem-resistant A. baumannii (CRAB). All 86 clinical isolates possessed the blaOXA-51 gene. BlaOXA-23 was detected as the second most frequent (90.7%) carbapenemase. Harboring AdeABC efflux pump genes was prevalent among the majority of the MDR isolates. Specially, the distributions of AdeABC-AdeRS efflux system genes in CRAB strains reached up to 90.0%. Compared with those of the CSAB strains, there was a statistically significant increasing distribution of the regulator AdeR and AdeS genes(p < 0.05). Moreover, CRAB strains showed significantly increased expression of AdeB(12.3- fold), but decreased expression of AdeR (3.3- fold)(p < 0.05).ConclusionThe present study showed a high distribution of multiple genes, mainly the genes of blaOXA-23/blaOXA-51 carbapenemase and AdeABC efflux pump, is responsible to distinct drug-resistance in PICU. It is urgent to strengthen the molecular epidemiological surveillance of pediatric MDRAB isolates to prevent further outbreaks. This study is of significant help for the clinicians to make therapeutic decisions and manage infection control in PICU.

Role of insertion sequence Aba-1 and AdeS in reduced tigecycline susceptibility in MDR-Acinetobacter baumannii clinical isolates from Cairo, Egypt

Infections caused by multidrug resistant (MDR) Acinetobacter baumannii (A. baumannii) especially in intensive care units have limited therapeutic options. Overexpression of the adeABC efflux pump may be caused either by the ISAba-1 insertion or by specific point mutations in adeR and adeS, therefore, plays a major role in conferring MDR-A. baumannii. We aimed in our study to monitor the tigecycline (TGC) susceptibility and to study the role of ISAba-1 and the adeS regulator within the AdeABC efflux pump among MDR-A. baumannii clinical isolates. MDR-A. baumannii (63) isolated from ICU patients were identified by detection of OXA-51-like gene. TGC MIC was determined by E-test and broth microdilution. PCR analysis of adeR, adeS, adeB and ISAba1 genes were done with further sequencing of adeS gene. Reduced susceptibility to TGC (MIC: 3–4 mg/L) was noticed in 6/63 (9.5%) MDR-A. baumannii isolates, ISAba-1 was detected in three isolates that two of which showed amino acid substitutions in the adeS operon. We concluded that the amino acids mutations in the adeS gene in presence of insertion ISAba-1 may play a role in conferring reduced TGC susceptibility of MDR-A. baumannii.

Investigation of mutations in adeR and adeS gene regions in gentamicine resistant Acinetobacter baumannii isolates

ABSTRACTThis study evaluated the relationship between aminoglycoside resistance and the active efflux pump and investigated the role of the active efflux pump in resistance mechanisms. In addition, the mutations related to aminoglycoside resistance were investigated in Acinetobacter baumannii isolates obtained from different clinical specimens. The study included 32 A. baumannii isolates. They were identified and their susceptibilities were determined using conventional techniques and an automated system. Total genomic RNA, DNA and cDNA were obtained using commercial extraction kits. Primers for adeR and adeS were designed using sequences in GenBank. All isolates were subjected to polymerase chain reaction (PCR) to determine the presence of adeR and adeS. The PCR products were electrophoresed for the optimization study. Subsequently, real-time PCR was performed to determine the expression levels of the adeR and adeS genes. Sequence analysis of the two adeRS operons in our isolates showed five mutations differing from those of other isolates. Isolate A21 had three mutations: (Tyr31Phe), (Val136Ala) and (Leu142Ile); isolate A24 had two mutations: (Asn115His) and (Leu142Ile). In our study, the examined gene regions that play a role in the resistance mechanisms of A. baumannii were considered important. The results indicated that adeR and adeS expression clearly affect aminoglycoside resistance. However, gene expression alone does not seem sufficient to explain that. These results could help to design improved active efflux pump inhibitors.

Efflux Pump AdeABC Assessment in Acinetobacter baumannii Strains Isolated in a Teaching Hospital

Over the past twenty years the worldwide clinically impact of Acinetobacter baumannii (A. baumannii) demonstrated its etiopathogenetic relevance. During a previously retrospective study in a teaching hospital, between January 2011 and February 2015, we observed increasingly infections caused by A. baumannii associated with antibiotic multi-resistance. Tigecycline, the first member of the glycylcycline class, is an effective option for the treatment of such infections even if, due to its increased clinical use, tigecycline resistant isolates have recently emerged. In A. baumannii several mechanisms are associated with a tigecycline decrease susceptibility, among these, expression efflux pump AdeABC and the presence of insertion sequence (IS) in the adeRS operon.About that, we decided to analyze adeB and adeS genes in 24 MDR A. baumannii clinical isolates, selected on the different tigecycline phenotype. The study of adeB and adeS genes was performed by an in-housepolymerase chain reaction (PCR) and by Sanger sequencing method. According to literature adeB and adeS genes were detected in all MDR A. baumannii isolates tested. Therefore our attention has focused on two resistant tigecycline clinical strains (ACI 2313 and ACI 1213), with a MIC value >8. In particular the ACI 2313 strains, showed the presence of an IS in the adeS gene. Then, adeS sequence analysis identified ISAba1 insertion. Moreover, adeB gene expression was evaluated by an in-house SYBR Green I-based real-time RT-PCR. We found an over expression of adeB gene in ACI 2313 strain, according to IS presence on adeS gene, while the lack of adeB overexpression in ACI 1213, still resistant to tigecycline, could be due to different resistance mechanisms.

Mechanisms of tigecycline resistance in carbapenems-resistant Acinetobacter baumannii strains

Objective To investigate the mechanisms of tigecycline nonsusceptibility in carbapenems-resistant Acinetobacter baumannii (CRAB) strains in order to provide a theoretical basis for a reasonable use of antibiotics and the control of nosocomial infection. Methods Susceptibility testing of 120 non-duplicate CRAB strains to tigecycline was performed by using the broth microdilution method. Minimal inhibitory concentrations (MIC) of tigecycline against the A. baumannii strains were determined by using the broth microdilution method before and after exposing the strains to Carbonylcyanide-m-chlorophenylhydrazone (CCCP), which was the efflux pump inhibitor. Polymerase chain reaction (PCR) was used to amply the efflux pumps genes including adeB, adeJ, adeG, abeM, adeE, adeRS, tetX and tetX1. The real-time PCR was performed to measure the expression of efflux pumps genes including adeB, adeJ, adeG, abeM and adeE. Results A total of 120 CRAB strains were collected including 13 (10.8%) tigecycline non-susceptible A. baumannii (TNAB) strains and 107 (89.2%) tigecycline susceptible A. baumannii (TSAB) strains. The MIC values of tigecline to the 120 CRAB strains were in a range of 0.25 μg/ml to 8 μg/ml. The adeR and adeJ genes were detected in 90.0% and 92.5% of the 120 CRAB strains, respectively. The positive rates of adeB, adeS, adeG and abeM genes among the 120 CRAB strains were all 94.2%. None of the three genes including adeE, tetX and tetX1 were detected. The mean expression levels of adeB and adeJ in TNAB strains were respectively increased by 18.69 folds and 5.46 folds as compared with those in sensitive strains. No significant increase in the expression of adeG and abeM genes was observed in TNAB strains. A 4-fold decrease in the MIC was observed in 8 out of 13 TNAB isolates treated with 10 μg/ml of CCCP. The CCCP could partially reverse the resistance pattern of tigecycline. Conclusion The efflux pump systems of adeABC and adeIJK rather than the abeFGH and abeM systems might play an important role in reducing the tigecycline susceptibility in carbapenems-resistant A. baumannii strains. Key words: Acinetobacter baumannii; Tigecycline; Efflux pumps; Resistance mechanisms

Role of efflux pumps in resistance to tigecycline in Acinetobacter baumannii

To investigate the role of active efflux pumps in mediating the resistance of Acinetobacter baumannii to tigecycline. The in vitro susceptibility of Acinetobacter baumannii to tigecycline was determined by broth microdilution method. Minimal inhibitory concentrations (MICs) of tigecycline against Acinetobacter baumannii strains were determined by broth microdilution method with or without efflux pump inhibitors of Carbonylcyanide-m-chlorophenylhydrazone (CCCP). Efflux pumps genes including adeB, adeJ and abeM were amplified by polymerase chain reaction (PCR) and sequenced. The expression levels of these genes were measured using quantitative real-time transcription- polymerase chain reaction (RT-PCR). The modulating gene adeR and adeS were detected by PCR amplification, deoxyribonucleic acid (DNA) sequencing were performed with specific primers and were compared with the published sequence online. The sensitive rate of Acinetobacter baumannii to tigecycline was 22.39% (15/67), active efflux of drugs existed. The adeB gene was positive in 88.06% (59/67) out of 67 Acinetobacter baumannii strains, and adeJ and abeM were 100% positive. adeB and adeJ genes expression in clinical isolates of resistant Acinetobacter baumannii increased 17.88-fold and 6.07-fold than sensitive strains, respectively. AbeM gene expression in resistant strains had not significant increase. The sequence of adeR and adeS genes in clinical isolates of sensitive and resistant strains were the same except several point mutations. Efflux pump systems of AdeABC and AdeIJK may play a role in Acinetobacter baumannii resistant to tigecycline in western China, AbeM may be irrelevant to it. The point mutations of adeR and adeS may not up-regulate the transcription activities of adeB in this study.

Relationship of drug resistance in acinetobacter baumannii isolated from the burn patients with the expression alteration of efflux pump adeAB

Objective To observe the expressions of efflux pump gene cluster adeAB in acinetobacter baumannii isolated from the burn patients and the expression changes of its upstream regulatory genes adeR and adeS and determine the influence of those genes on drug resistance of acinetobacter baumannii.Methods Nine drug-resistant strains and nine sensitive strains of acinetobacter baumannii isolated from the burn patients treated between June 2012 and March 2013 were used.After strain identification using 16SrDNA sequencing,acinetobacter baumannii standard strain ATCC19606 was employed as the control.mRNA expressions of efllux pump genes adeA and adeB and their upstream regulatory genes adeR and adeS were detected by real-time quantitative PCR.Results (1) adeA and adeB genes presented higher expressions in drug-resistant strains (3.71 ±0.95,76.16 ± 8.75) than in sensitive strains (0.92 ± 0.94,0.72 ± 0.78) (F =38.71,663.65 respectively,both P ＜ 0.05).(2) adeS and adeR genes showed higher expressions in drug resistant strains (18.02 ± 6.71,3.02 ± 2.69) than in sensitive strains (1.64±1.51,0.76±0.61) (F =51.04,5.57 respectively,bothP＜0.05).Conclusions The over-expression of efflux pump gene cluster adeAB induced by the expression alteration of its upstream regulatory genes adeR and adeS is closely associated with the drug resistance of acinetobacter baumannii in the burn patient.Besides,the regulatory genes may depend on adeS to sense the nosocomial environment condition,activate or inactivate adeR and hence regulate efflux pump expression. Key words: Burns; Multidrug resistance-associated proteins; Genes, regulator; Acinetobacter baumannii

Relationship between the AdeABC efflux system gene content, netilmicin susceptibility and multidrug resistance in a genotypically diverse collection of Acinetobacter baumannii strains

To assess the occurrence of the genes of the AdeABC efflux system and their association with antimicrobial resistance in Acinetobacter baumannii. A set of 116 strains selected for their diversity both in genotypic properties and geographic origin was investigated for the presence of the structural (adeA, adeB and adeC) and regulatory (adeR and adeS) genes of the AdeABC system by PCR, for resistance to 11 antimicrobials by disc diffusion, for MIC of netilmicin and for the presence of aacC2 and aacA4, encoding netilmicin-modifying enzymes. Ninety-five strains were positive for adeA, adeB, adeR and adeS, 10 were positive for 1 to 3 of these genes and 11 were negative for all genes. The adeC gene was found in 49 strains with one or more of the other genes. Forty-one strains were resistant to a maximum of one agent and 75 strains to two or more agents. Netilmicin MICs showed an almost bimodal distribution with respective peaks of 0.5-1 and 8 mg/L; aacC2 or aacA4 was found in six strains with netilmicin MIC of >or=64 mg/L. All 61 strains with netilmicin MICs >or= 4 mg/L were both adeABRS-positive and resistant to two or more agents, whereas netilmicin MICs <or= 2 mg/L (n = 51) were found for all strains resistant to a maximum of one agent and those negative for one or more of the adeABRS genes. The AdeABC genes are common in A. baumannii, but may be absent in some, mostly fully susceptible strains. Decreased susceptibility to netilmicin (MIC 4-32 mg/L) is associated with both the presence of these genes and multidrug resistance and may be indicative of AdeABC overexpression.

AdeABC multidrug efflux pump is associated with decreased susceptibility to tigecycline in Acinetobacter calcoaceticus–Acinetobacter baumannii complex

To investigate the role of the AdeABC multidrug efflux pump in the decreased susceptibility of clinical isolates of Acinetobacter calcoaceticus-Acinetobacter baumannii complex to tigecycline. Gene expression was analysed by Taqman RT-PCR. A single cross-over achieved insertional inactivation of the adeB gene with a suicide plasmid construct carrying an adeB fragment obtained by PCR. Analysis of the adeRS locus was performed by PCR and sequencing. Ribotyping was performed with the RiboPrinter system. MICs were determined by Etest. Expression analysis revealed constitutive overexpression of adeABC in less-susceptible clinical isolates G5139 and G5140 (tigecycline MIC=4 mg/L) when compared with the isogenic clinical isolates G4904 and G5141 (MIC=1.5 mg/L). Insertional mutants GC7945 (adeB knockout in G5139) and GC7951 (adeB knockout in G5140) were obtained, which resulted in tigecycline MICs of 0.5 mg/L. As reported previously, the expression of adeABC is regulated by the two-component signalling system encoded by the adeR and adeS genes. PCR and sequencing analyses revealed an insertion of an IS(ABA-1) element in the adeS gene of G5139 and G5140. The results of this study suggest that decreased susceptibility to tigecycline in the A. calcoaceticus-A. baumannii complex is associated with the overexpression of the AdeABC multidrug efflux pump.