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Role of plasmid carrying bla NDM in mediating antibiotic resistance among Acinetobacter baumannii clinical isolates from Egypt.

ABSTRACT: We investigated antibiotic resistance levels among bla NDM -positive (n?=?9) and -negative (n?=?65) A. baumannii clinical isolates collected in 2010 and 2015 from Alexandria Main University Hospital, Egypt using disc diffusion and minimum inhibitory concentration (MIC) determination. Plasmids from bla NDM -positive isolates were transformed into a carbapenem-susceptible A. baumannii (CS-AB) isolate to assess the role of plasmid transfer in mediating carbapenem resistance. Imipenem, meropenem, and ertapenem MIC90 values against bla NDM -positive isolates were 128,?>?256, and 256 µg/mL, respectively. Plasmid isolation and polymerase chain reaction revealed that bla NDM was plasmid mediated. The plasmids were electroporated into the cells of a CS-AB isolate at an efficiency of 1.3?×?10-8 to 2.6?×?10-7, transforming them to bla NDM -positive carbapenem-resistant cells with an imipenem MIC increase of 256-fold. In addition to carbapenem resistance, the bla NDM -positive isolates also exhibited higher levels of cephalosporins, tetracycline, aminoglycosides, fluoroquinolones, and colistin resistance than the bla NDM -negative isolates. Acquisition of bla NDM -carrying plasmids dramatically increased imipenem resistance among A. baumannii isolates. Intriguingly, bla NDM -positive isolates also showed a high degree of resistance to antibiotics of different classes. The potential co-existence of different resistance determinants on A. baumannii plasmids and their possible transfer owing to the natural competence of the pathogen are especially alarming. More effective infection control and antibiotic stewardship programs are needed to curb the spread and treat such infections in both hospital and community settings.

SUBMITTER: Abouelfetouh A

PROVIDER: S-EPMC7076099 | biostudies-literature | 2020 Apr

REPOSITORIES: biostudies-literature

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Role of plasmid carrying bla NDM in mediating antibiotic resistance among Acinetobacter baumannii clinical isolates from Egypt.

Abouelfetouh Alaa A Torky Aisha S AS Aboulmagd Elsayed E

3 Biotech 20200316 4

We investigated antibiotic resistance levels among bla NDM -positive (n = 9) and -negative (n = 65) A. baumannii clinical isolates collected in 2010 and 2015 from Alexandria Main University Hospital, Egypt using disc diffusion and minimum inhibitory concentration (MIC) determination. Plasmids from bla NDM -positive isolates were transformed into a carbapenem-susceptible A. baumannii (CS-AB) isolate to assess the role o ...[more]

PMID: 32206504

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Project description:NDM-5 carbapenemase was mainly identified in Escherichia coli, while the rapid transmission of bla NDM-5 among Enterobacteriaceae has raised serious public attention. This study identified 14 NDM-5-producing Klebsiella pneumoniae isolates from 107 carbapenem-resistant K. pneumoniae isolates, recovered from blood, urine, and normally sterile body fluids of pediatric patients from January 2016 to December 2018. All NDM-5-producing isolates were highly resistant to ?-lactams, while tigecycline and polymyxin B exhibited excellent antimicrobial activity. These 14 strains belonged to 9 different sequence types (STs) and displayed various pulsed-field gel electrophoresis (PFGE) patterns, suggesting that they were not clonally related. S1-PFGE followed by Southern blotting showed that the bla NDM-5 gene was located on an ?46-kb IncX3 plasmid in all strains. All bla NDM-5-carrying plasmids were successfully transferred into recipient E. coli J53. PCR-based sequencing demonstrated that all of the bla NDM-5-carrying plasmids shared highly similar backbones, with nucleotide sequence identity of >99%. Moreover, this plasmid displayed high sequence similarity to the previously reported epidemic IncX3 bla NDM-5-carrying plasmids, with dynamic changes observed only in bla NDM-5-surrounding elements. Interestingly, the IncX3 bla NDM-5-carrying plasmids showed strong stability in clinical isolates when cultured in antibiotic-free medium. However, after the conjugation inhibitor linoleic acid was added, a gradual increase in the level of IncX3 plasmid loss could be observed. Clinical isolates displayed 10% to 15% bla NDM-5-carrying plasmid loss after coculture with linoleic acid for 5?days. These results showed that the IncX3 plasmid facilitated the dissemination of bla NDM-5 among multiclonal K. pneumoniae strains in children and that conjugal transfer contributed significantly to IncX3 plasmid stability within K. pneumoniae IMPORTANCE The emergence and spread of New Delhi metallo-?-lactamase (NDM)-producing Enterobacteriaceae have been a serious challenge to public health, and NDM-5 shows increased resistance to carbapenems compared with other variants. NDM-5 has been identified mostly in E. coli but has rarely been described in K. pneumoniae and other Enterobacteriaceae isolates. Here, we present the dissemination of highly similar 46-kb IncX3 bla NDM-5-carrying plasmids among multiclonal K. pneumoniae strains in children, highlighting the horizontal gene transfer of bla NDM-5 among K. pneumoniae strains via the IncX3 plasmid. Moreover, the IncX3 bla NDM-5-carrying plasmids displayed strong stability in clinical strains when cultured in antibiotic-free medium, and the plasmid maintenance was attributed partly to conjugal transfer. Plasmid conjugation is mediated by the type IV secretion system (T4SS), and T4SS is conserved among all epidemic IncX3 bla NDM-5-carrying plasmids. Therefore, combining conjugation inhibition and promotion of plasmid loss would be an effective strategy to limit the conjugation-assisted persistence of IncX3 bla NDM-5-carrying plasmids.

Project description:The emergence of New Delhi metallo-?-lactamase (NDM) in common enterobacterial species is a major concern for healthcare. Early reports have revealed that the spread of NDM involved diverse and heterogeneous plasmids. Recently, the involvement of a rare, IncX3 subtype plasmid has been increasingly recognized. Here, we studied the prevalence of IncX plasmid subtypes in 198 carbapenem-resistant Enterobacteriaceae, originating from a territory-wide active surveillance in Hong Kong in 2016. The complete sequences and biological features of the bla NDM-carrying plasmids were investigated. A total of 62 NDM-type, 21 OXA-48 type, 14 IMP-type, 8 KPC-type, 4 IMI-type producers, and 89 non-carbapenemase-producers were tested for presence of IncX subtypes. IncX3 (n = 60) was the most common subtype, followed by IncX4 (n = 6) and IncX1 (n = 2). The prevalence of IncX3 subtype in isolates producing NDM, other carbapenemase types and non-carbapenemase producers were 75.8, 21.3, and 3.4%, respectively (P < 0.001). An IncX3 plasmid (size ?50 kb) was confirmed to carry bla NDM in 47 isolates of different enterobacterial species. Thirteen IncX3 plasmids originating from six healthcare regions in Hong Kong were completely sequenced. The results showed that the IncX3 plasmids carrying bla NDM share a high degree of sequence identity with a previously reported plasmid, pNDM-HN380 (GenBank accession JX104760), over the backbone and genetic load regions. A blast search further revealed the occurrence of identical or nearly identical IncX3 plasmids carrying bla NDM in other part of China, Korea, Myanmar, India, Oman, Kuwait, Italy, and Canada. Two IncX3 carrying bla NDM were investigated further. Conjugation experiments demonstrated that the IncX3 plasmids could be efficiently transferred to multiple enterobacterial species at frequencies that are comparable or higher than the epidemic IncFII plasmid carrying bla CTX-M (pHK01). In addition, efficient transfer of the NDM plasmids occurred over a range of temperatures. In conclusion, this study demonstrated the important role played by IncX3 in the dissemination of NDM and the occurrence of pNDM-HN380-like plasmids in geographically widespread areas. The high mobility of IncX3 plasmid across different enterobacterial species highlights the ability of this plasmid replicon to be an important vehicle in worldwide dissemination of NDM.

Project description:Background:Antibiotic use is largely under-regulated in Egypt leading to the emergence of resistant isolates. Carbapenems are last resort agents to treat Acinetobacter baumannii infections resistant to other classes of antibiotics. However, carbapenem-resistant isolates are emerging at an alarming rate. This study aimed at phenotypically and molecularly characterizing seventy four carbapenem-unsusceptible A. baumannii isolates from Egypt to detect the different enzymes responsible for carbapenem resistance. Methods:Carbapenemase production was assessed by a number of phenotypic methods: modified Hodge test (MHT), carbapenem inactivation method (CIM), combined disc test (CDT), CarbAcineto NP test and boronic acid disc test. Polymerase chain reaction (PCR) was used to screen the isolates for the presence of some genes responsible for resistance to carbapenems, as well as some insertion sequences. Results:PCR amplification of class D carbapenemases revealed the prevalence of bla OXA-51 and bla OXA-23 in 100% of the isolates and of bla OXA-58 in only one isolate (1.4%). bla VIM and bla NDM-1 belonging to class B metallo-?-lactamases were present in 100 and 12.1% of the isolates, respectively. The prevalence of ISAba1, ISAba2 and ISAba3 was 100, 2.7 and 4.1%, respectively. None of the tested isolates carried bla OXA-40 , bla IMP , bla SIM , bla SPM , bla GIM or the class A bla KPC . Taking PCR as the gold standard method for the detection of different carbapenemases, the sensitivities of the MHT, CIM, CDT, CarbAcineto NP test and boronic acid disc/imipenem or meropenem test for this particular collection of isolates were 78.4, 68.9, 79.7, 95.9, and 56.8% or 70.3%, respectively. Conclusions:The widespread detection of carbapenem-resistant A. baumannii (CR-AB) has become a real threat to the efficacy of treatment regimens. Among the studied cohort of CR-AB clinical isolates, bla OXA-51 , bla OXA-23 and bla VIM were the most prevalent, followed by bla NDM-1 and bla OXA-58 . The genotypic detection of carbapenemases among CR-AB clinical isolates using PCR was most conclusive, followed closely by the phenotypic testing using CarbAcineto NP test.

Dataset Information

Role of plasmid carrying bla NDM in mediating antibiotic resistance among Acinetobacter baumannii clinical isolates from Egypt.

Publications

Role of plasmid carrying <i>bla</i> <sub><i>NDM</i></sub> in mediating antibiotic resistance among <i>Acinetobacter baumannii</i> clinical isolates from Egypt.

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