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Efflux Pump Activity and Mutations Driving Multidrug Resistance in Acinetobacter baumannii at a Tertiary Hospital in Pretoria, South Africa

ABSTRACT: Acinetobacter baumannii (A. baumannii) has developed several resistance mechanisms. The bacteria have been reported as origin of multiple outbreaks. This study aims to investigate the use of efflux pumps and quinolone resistance-associated genotypic mutations as mechanisms of resistance in A. baumannii isolates at a tertiary hospital. A total number of 103 A. baumannii isolates were investigated after identification and antimicrobial susceptibility testing by VITEK2 followed by PCR amplification of blaOXA-51. Conventional PCR amplification of the AdeABC efflux pump (adeB, adeS, and adeR) and quinolone (parC and gyrA) resistance genes were performed, followed by quantitative real-time PCR of AdeABC efflux pump genes. Phenotypic evaluation of efflux pump expression was performed by determining the difference between the MIC of tigecycline before and after exposure to an efflux pump inhibitor. The Sanger sequencing method was used to sequence the parC and gyrA amplicons. A phylogenetic tree was drawn using MEGA 4.0 to evaluate evolutionary relatedness of the strains. All the collected isolates were blaOXA-51-positive. High resistance to almost all the tested antibiotics was observed. Efflux pump was found in 75% of isolates as a mechanism of resistance. The study detected parC gene mutation in 60% and gyrA gene mutation in 85%, while 37% of isolates had mutations on both genes. A minimal evolutionary distance between the isolates was reported. The use of the AdeABC efflux pump system as an active mechanism of resistance combined with point mutation mainly in gyrA was shown to contribute to broaden the resistance spectrum of A. baumannii isolates.

SUBMITTER: Nogbou N

PROVIDER: S-EPMC8516574 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Antibiotic-resistant strains of the Gram-negative pathogen Acinetobacter baumannii have emerged as a significant global health threat. One successful therapeutic option to treat bacterial infections has been to target the bacterial ribosome. However, in many cases, multidrug efflux pumps within the bacterium recognize and extrude these clinically important antibiotics designed to inhibit the protein synthesis function of the bacterial ribosome. Thus, multidrug efflux within A. baumannii and other highly drug-resistant strains is a major cause of failure of drug-based treatments of infectious diseases. We here report the first structures of the Acinetobacter drug efflux (Ade)J pump in the presence of the antibiotic eravacycline, using single-particle cryo-electron microscopy (cryo-EM). We also describe cryo-EM structures of the eravacycline-bound forms of the A. baumannii ribosome, including the 70S, 50S, and 30S forms. Our data indicate that the AdeJ pump primarily uses hydrophobic interactions to bind eravacycline, while the 70S ribosome utilizes electrostatic interactions to bind this drug. Our work here highlights how an antibiotic can bind multiple bacterial targets through different mechanisms and potentially enables drug optimization by taking advantage of these different modes of ligand binding. IMPORTANCE Acinetobacter baumannii has developed into a highly antibiotic-resistant Gram-negative pathogen. The prevalent AdeJ multidrug efflux pump mediates resistance to different classes of antibiotics known to inhibit the function of the 70S ribosome. Here, we report the first structures of the A. baumannii AdeJ pump, both in the absence and presence of eravacycline. We also describe structures of the A. baumannii ribosome bound by this antibiotic. Our results indicate that AdeJ and the ribosome use very distinct binding modes for drug recognition. Our work will ultimately enable structure-based drug discovery to combat antibiotic-resistant A. baumannii infection.

Project description:Acinetobacter baumannii is an opportunistic pathogen that is increasingly responsible for hospital-acquired infections. The increasing prevalence of carbapenem resistant A. baumannii has left clinicians with limited treatment options. Last line antimicrobials (i.e., polymyxins and glycylcyclines) are often used as treatment options. The aim of this study was to determine the prevalence of selected ?-lactamase genes from A. baumannii isolates obtained from patients with hospital-acquired infections and to determine the genetic relationship and epidemiological profiles among clinical A. baumannii isolates collected from two tertiary academic hospitals in the Tshwane region, South Africa (SA). Multiplex-PCR (M-PCR) assays were performed to detect selected resistance genes. The collected isolates' genetic relatedness was determined by using pulsed field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). The acquired oxacillinase (OXA) genes, notably blaOXA-23-like were prevalent in the A. baumannii isolates. The M-PCR assays showed that the isolates collected from hospital A contained the OXA-23-like (96%; n = 69/72) genes and the isolates collected from hospital B contained the OXA-23-like (91%; n = 63/69) and OXA-58-like (4%; n = 3/69) genes. Colistin resistance was found in 1% of the isolates (n = 2/141) and tigecycline intermediate resistance was found in 6% of the isolates (n = 8/141). The A. baumannii isolates were genetically diverse. Molecular epidemiological data showed that specific sequence types (STs) (ST106, ST229, ST258 and ST208) were established in both hospitals, while ST848 was established in hospital A and ST502, ST339 and the novel ST1552 were established in hospital B. ST848 (established in hospital A) was predominately detected in ICU wards whereas ST208, ST339 and the novel ST1552 (established in hospital B) were detected in ICUs and the general wards. The origin of the A. baumannii isolates in the hospitals may be due to the dissemination and adaptation of a diverse group of successful clones. Poor infection control and prevention strategies and possibly the overuse of antimicrobials contributed to the establishment of these A. baumannii clones in the studied hospitals.

Project description:IntroductionMultidrug-resistant Acinetobacter baumannii (A. baumannii) has become one of the greatest threats worldwide to the therapeutic management of infections. Our previous research confirmed an in vitro synergistic effect of amlodipine and imipenem against A. baumannii, and this study is designed to understand its mechanism.MethodsSixty-four non-duplicate A. baumannii isolates were collected and tested for antimicrobial susceptibility by the disk diffusion method. PCR amplification and sequencing were used to identify the presence of the adeB, adeE, adeH, adeJ, abeM and abeS efflux pump genes. The minimal inhibitory concentrations of imipenem, imipenem+amlodipine and imipenem+carbonyl cyanide m-chlorophenyl-hydrazone against these isolates were also determined by the broth microdilution method before and after siRNA silencing of the expression of the adeABC efflux pump.ResultsIn this study, the combination of amlodipine with imipenem showed synergistic antimicrobial activity against sixty-four A. baumannii isolates when compared with the activity of imipenem alone (p<0.025). In the multidrug-resistant group, AML was more effective than carbonyl cyanide m-chlorophenyl-hydrazone (p<0.001). The efflux pump genes adeB, adeE, adeH, adeJ, abeM and abeS were detected in 100% (4/64), 75% (48/64), 0% (0/64), 100% (64/64), 96.9% (62/64) and 96.9% (62/64) of the sixty-four A. baumannii isolates, respectively. The expression of the adeABC efflux pump genes in the multidrug-resistant group (5.05±19.25) is clearly higher than in the non-multidrug-resistant group (0.17±0.20), (p = 0.01). A gene silencing test verified that the mRNA expression levels of adeABC were decreased at 12 h and increased at 24 h, while the reversal of imipenem resistance by amlodipine disappeared at 12 h and reappeared at 24 h.ConclusionsThe combination of amlodipine with imipenem exhibits an in vitro synergistic antimicrobial effect on multidrug-resistant A. baumannii, which may be due to the inhibition of the AdeABC efflux pump.

Project description:IntroductionCarbapenem-resistant Acinetobacter baumannii has been responsible for an increasing number of hospital-acquired infections globally. The study investigated the prevalence of carbapenemase-encoding genes in clinical multidrug-resistant A. baumannii strains.Materials and methodsA total of 100 nonduplicate multidrug-resistant A. baumannii strains were cultured from clinical samples obtained from healthcare facilities in the O. R. Tambo district. The strains were confirmed by detecting the intrinsic bla OXA-51-like gene. Antimicrobial susceptibility testing was performed by VITEK® 2 and autoSCAN-4 systems. The MIC of imipenem and meropenem was rechecked by E-test. Colistin MIC was confirmed by the broth microdilution method. Real-time PCR was performed to investigate the presence of carbapenemase-encoding genes.ResultsMost strains showed high resistance rates (>80%) to the antibiotics tested. Resistance to amikacin, tetracycline, and tigecycline were 50%, 64%, and 48%, respectively. All strains were fully susceptible to colistin. The bla OXA-51-like was detected in all strains whilst bla OXA-23-like, bla OXA-58-like, bla OXA-24-like, bla IMP-1, bla VIM, and bla NDM-1 were found in 70%, 8%, 5%, 4%, 3%, and 2% of strains, respectively. None of the tested strains harboured the genes bla SIM and bla AmpC. The coexistence of bla OXA-23-like, and bla IMP-1 or bla OXA-58-like was detected in 1% and 2% strains, respectively. A distinct feature of our findings was the coharbouring of the genes bla OXA-23-like, bla OXA-58-like, and bla IMP-1 in 2% strains, and this is the first report in the Eastern Cape Province, South Africa. The intI1 was carried in 80% of tested strains whilst ISAba1/bla OXA-51-like and ISAba1/bla OXA-23-like were detected in 15% and 40% of the strains, respectively. The detection of bla OXA-23-like, ISAba1/bla OXA-51-like, ISAba1/bla OXA-23-like, and bla OXA-23-like, bla OXA-58-like, and bla IMP-1 carbapenemases in strains had a significant effect on both imipenem and meropenem MICs.ConclusionsResults showed a high level of oxacillinases producing A. baumannii circulating in our study setting, highlighting the need for local molecular surveillance to inform appropriate management and prevention strategies.

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Efflux Pump Activity and Mutations Driving Multidrug Resistance in Acinetobacter baumannii at a Tertiary Hospital in Pretoria, South Africa

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