Insertional inactivation of oprD in carbapenem-resistant Pseudomonas aeruginosa strains isolated from burn patients in Tehran, Iran

Abstract

In this study, we report the insertion sequence ISPpu21 in the oprD porin gene of carbapenem-resistant Pseudomonas aeruginosa isolates from burn patients in Tehran, Iran. Antibiotic susceptibility tests for P. aeruginosa isolates were determined. Production of metallo-β-lactamases (MBLs) and carbapenemase was evaluated and the β-lactamase-encoding and aminoglycoside-modifying enzyme genes were investigated by PCR and sequencing methods. The mRNA transcription level of oprD and mex efflux pump genes were evaluated by real-time PCR. The outer membrane protein profile was determined by SDS–PAGE. The genetic relationship between the P. aeruginosa isolates was assessed by random amplified polymorphic DNA PCR. In all, 10.52% (10/95) of clinical isolates of P. aeruginosa harboured the ISPpu21 insertion element in the oprD gene. The extended-spectrum β-lactamase-encoding gene in ISPpu21-carrying isolates was blaTEM. PCR assays targeting MBL and carbapenemase-encoding genes were also negative in all ten isolates. The rmtA, aadA, aadB and armA genes were positive in all ISPpu21 harbouring isolates. The relative expression levels of the mexX, mexB, mexT and mexD genes in ten isolates ranged from 0.1- to 1.4-fold, 1.1- to 3.68-fold, 0.3- to 8.22-fold and 1.7- to 35.17-fold, respectively. The relative expression levels of the oprD in ten isolates ranged from 0.57- to 35.01-fold, which was much higher than those in the control strain P. aeruginosa PAO1. Evaluation of the outer membrane protein by SDS–PAGE suggested that oprD was produced at very low levels by all isolates. Using random amplified polymorphic DNA PCR genotyping, eight of the ten isolates containing ISPpu21 were shown to be clonally related. The present study describes a novel molecular mechanism, ISPpu21 insertion of the oprD gene, associated with carbapenem resistance in clinical P. aeruginosa isolates.

Quercetin potentiates meropenem activity among pathogenic carbapenem‐resistantPseudomonas aeruginosaandAcinetobacter baumannii

“…Although, PCR screening of clinically important carbapenemases indicated diverse distribution of several carbapenemases, bla NDM and bla OXA, particularly, bla OXA‐23 ‐like gene was found to be prevalent. Furthermore, most of the earlier studies have demonstrated efflux pump ( mexB and adeB ) overexpression and porin (OPrD, CarO and 33‐36 KD porin) loss to be secondary mechanisms of carbapenem resistance development among P. aeruginosa and A. baumannii (Poirel and Nordmann ; Shariati et al ; Chen et al ; Novović et al ). While, Shariati et al (), earlier reported 1·1–3·68 fold overexpression of mexB and approximately twofold reduced expression of OprD among carbapenem‐resistant P. aeruginosa, this study demonstrated approximately 16‐fold overexpression of mexB and complete loss of OprD among most of the CRPsA.…”

“…Furthermore, most of the earlier studies have demonstrated efflux pump ( mexB and adeB ) overexpression and porin (OPrD, CarO and 33‐36 KD porin) loss to be secondary mechanisms of carbapenem resistance development among P. aeruginosa and A. baumannii (Poirel and Nordmann ; Shariati et al ; Chen et al ; Novović et al ). While, Shariati et al (), earlier reported 1·1–3·68 fold overexpression of mexB and approximately twofold reduced expression of OprD among carbapenem‐resistant P. aeruginosa, this study demonstrated approximately 16‐fold overexpression of mexB and complete loss of OprD among most of the CRPsA. Similarly, the present study exhibited approximately 17·5‐fold overexpression of adeB among CRAB which was consistent with that of earlier findings of approximately 12·3‐fold adeB overexpression demonstrated by Chen et al ().…”

“…In general, CRPsA and CRAB mostly acquired carbapenem resistance through expression of VIM and OXA type of carbapenemases respectively (Poirel and Nordmann 2006;Chen et al 2018;Subramaniyan and Sundaram 2018). Further, overexpression of major efflux pump genes (mexB and adeB) and loss of outer membrane proteins (OMPs: OprD, CarO and 33-36 kDa porins) also conferred carbapenem resistance to CRPsA and CRAB (Poirel and Nordmann 2006;Shariati et al 2017;Chen et al 2018;Novovi c et al 2018). Although, both expression of carbapenemases and alteration in membrane permeability contributed in carbapenem resistance development among CRPsA and CRAB, loss of OprD and efflux pump overexpresion have been considered to be crucial for carbapenem resistance development among P. aeruginosa (Karampatakis et al 2018).…”

The possible role of bacteria, viruses, and parasites in initiation and exacerbation of irritable bowel syndrome

“…People infected with high doses of bacteria, or those exhibiting different immune responses, including female sex, are more likely to suffer from IBS after being infected with salmonella (McKendrick & Read, 1994;Mearin et al, 2005). 10 | P. AERUGINOSA AND IBS P. aeruginosa is a Gram-negative aerobic bacillus with a high antibiotic resistance that induces a variety of important infections such as cystic fibrosis and burn infections (Oliver, Cantón, Campo, Baquero, & Blázquez, 2000;Shariati, Azimi et al, 2018). Results of a recent study revealed a higher prevalence and level of the Pseudomonas genus in fecal and small intestinal samples from IBS patients than control subjects and considered P. aeruginosa as the predominant isolated species (Kerckhoffs et al, 2011).…”

<p>How Phages Overcome the Challenges of Drug Resistant Bacteria in Clinical Infections</p>

“…84,85 Different studies in recent years have shown that Gram-positive and Gram-negative bacteria, such as vancomycin-resistant enterococci, methicillinresistant S. aureus (MRSA), Enterobacteriaceae with extended-spectrum b-lactamase and XDR A. baumannii and P. aeruginosa have caused the highest mortality among infected patients. 86,87 On the other hand, it should be noted that of extensively drug-resistant tuberculosis (XDR-TB), MDR Clostridium difficile and newly identified transmissible carbapenamase, New Delhi metallo-betalactamases (NDM) in Enterobacteriaceae are expanding all over the world, especially in developing countries, and may become a big problem in the coming years. 3,88,89 Intrinsic properties, such as external barriers, which prevent the entry of drugs into bacteria, natural mutations in antibiotic targets and acquired features, such as sequestration of the drug, efflux pumps and enzyme-dependent drug alterations cause such high levels of resistance to existing antibiotics.…”