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Emergence of blaTEM, blaCTX-M, blaSHV and blaOXA genes in multidrug-resistant Enterobacteriaceae and Acinetobacter baumannii in Saudi Arabia

ABSTRACT: Multidrug-resistant (MDR) patterns due to extended-spectrum β-lactamase (ESBL) production in pathogenic bacteria are now becoming prevalent in hospitals worldwide, posing a public health challenge. The aim of the present study was to determine the antibiotic susceptibility patterns and distribution of the blaTEM, blaCTX-M, blaSHV and blaOXA ESBL resistance genes in MDREnterobacteriaceae and Acinetobacter baumannii (A. baumannii). A cross-sectional study was conducted between September 2017 and August 2018 in the King Abdullah Hospital (Bisha, Saudi Arabia). Bacterial isolates were collected from the clinical samples of patients; these were identified and screened for ESBL production and their antibiotic susceptibility was examined using standard microbiology methods. Multiplex-PCR runs were performed to identify genes encoding ESBL producers. DNA sequencing analysis was used to identify the specific gene variants. Of the 274 isolates, 173 (63.1%) exhibited MDR patterns to different antibiotics. A. baumannii revealed the highest resistance rates for cefuroxime (100%), gentamicin (88%) and amikacin (86%). Klebsiella pneumoniae (K. pneumoniae) isolates had the highest resistance rates for cefuroxime (98%), aztreonam and trimethoprim/sulfamethoxazole (87% for each). Escherichia coli (E. coli) exhibited high resistance rates for trimethoprim/sulfamethoxazole (92%) and cefuroxime (87%). Of the 173 MDR isolates, 78 (45.1%) exhibited ESBL production. Of these, 88.9% (72/78) carried ESBL genes. The most prevalent gene-encoding isolates were blaTEM (84.7%), followed by blaCTX-M (33.3%), blaSHV (2.7%) and blaOXA-1 (1.4%). A single blaTEM gene was predominantly produced by K. pneumoniae (60.7%), A. baumannii (78.9%) and Proteus mirabilis (80%), whereas blaCTX-M was harbored by E. coli (33.3%). The co-existence of two different genes in a single bacterium was revealed in 22.2% of isolates, commonly between blaTEM and blaCTX-M (19.4%). Sequencing analysis revealed that blaCTX-M-15 and blaTEM-1 were predominant variants of the blaCTX-M and blaTEM genes, respectively. The present study revealed a diversity of ESBL genes in Gram-negative bacterial isolates, with blaTEM being the most prevalent type. The emergence of various ESBL genes with several co-existing genotypes is alarming, rendering extensive surveillance studies necessary to understand the transmission and epidemiology of such resistant gene-carrying isolates.

SUBMITTER: Ibrahim M

PROVIDER: S-EPMC8549092 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Nosocomial infections caused by Acinetobacter spp. resistant to carbapenems are increasingly reported worldwide. Carbapenem-resistant Acinetobacter (CRA) is becoming a serious concern with increasing patient morbidity, mortality, and lengths of hospital stay. Therefore, the rapid detection of CRA is essential for epidemiological surveillance. Polymerase chain reaction (PCR) has been extensively used for the rapid identification of most pathogens. In this study, we have developed two multiplex real-time PCR assays to detect and differentiate A. baumannii and non-A. baumannii Acinetobacter spp, and common carbapenemase genes, including blaNDM, blaOXA-23-like, blaOXA-40-like, blaOXA-51-like, and blaOXA-58-like. We demonstrate the potential utility of these assays for the direct detection of blaNDM-, blaOXA-23-like-, blaOXA-40-like-, blaOXA-51-like-, and blaOXA-58-like-positive CRA in clinical specimens. Primers were specifically designed, and two multiplex real-time PCR assays were developed: multiplex real-time PCR assay1 for the detection of Acinetobacter baumannii 16S-23S rRNA internal transcribed spacer sequence, the Acinetobacter recA gene, and class-B-metalloenzyme-encoding gene blaNDM; and multiplex real-time PCR assay2 to detect class-D-oxacillinase-encoding genes (blaOXA-23-like, blaOXA-40-like, blaOXA-51-like,and blaOXA-58-like). The assays were performed on an ABI Prism 7500 FAST Real-Time PCR System. CRA isolates were used to compare the assays with conventional PCR and sequencing. Known amounts of CRA cells were added to sputum and fecal specimens and used to test the multiplex real-time PCR assays. The results for target and nontarget amplification showed that the multiplex real-time PCR assays were specific, the limit of detection for each target was 10 copies per 20 μL reaction volume, the assays were linear over six log dilutions of the target genes (r2 > 0.99), and the Ct values of the coefficients of variation for intra- and interassay reproducibility were less than 5%. The multiplex real-time PCR assays showed 100% concordance with conventional PCR when tested against 400 CRA isolates and their sensitivity for the target DNA in sputum and fecal specimens was 102 CFU/mL. Therefore, these novel multiplex real-time PCR assays allow the sensitive and specific characterization and differentiation of blaNDM-, blaOXA-23-like-, blaOXA-40-like-, blaOXA-51-like-, and blaOXA-58-like-positive CRA, making them potential tools for the direct detection of CRA in clinical specimens and the surveillance of nosocomial infections.

Project description:The rate of recovery of carbapenem-resistant Acinetobacter baumannii (CRAB) isolates has increased significantly in recent decades in Taiwan. This study investigated the molecular epidemiology of CRAB with a focus on the mechanisms of resistance and spread in isolates with blaOXA-23-like or blaOXA-24-like All 555 CRAB isolates in our multicenter collection, which were recovered from 2002 to 2010, were tested for the presence of class A, B, and D carbapenemase genes. All isolates with blaOXA-23-like or blaOXA-24-like were subjected to pulsed-field gel electrophoresis, and 82 isolates (60 isolates with blaOXA-23-like and 22 isolates with blaOXA-24-like) were selected for multilocus sequence typing to determine the sequence type (ST) and clonal group (CG) and for detection of additional β-lactamase and aminoglycoside resistance genes. The flanking regions of carbapenem and aminoglycoside resistance genes were identified by PCR mapping and sequencing. The localization of blaOXA was determined by S1 nuclease and I-CeuI assays. The numbers of CRAB isolates carrying blaOXA-23-like or blaOXA-24-like, especially those carrying blaOXA-23-like, increased significantly from 2008 onward. The blaOXA-23-like gene was carried by antibiotic resistance genomic island 1 (AbGRI1)-type structures located on plasmids and/or the chromosome in isolates of different STs (CG92 and novel CG786), whereas blaOXA-24-like was carried on plasmids in CRAB isolates of limited STs (CG92). No class A or B carbapenemase genes were identified. Multiple aminoglycoside resistance genes coexisted in CRAB. Tn6180-borne armA was found in 74 (90.2%) CRAB isolates, and 58 (70.7%) isolates had Tn6179 upstream, constituting AbGRI3. blaTEM was present in 38 (46.3%) of the CRAB isolates tested, with 35 (92.1%) isolates containing blaTEM in AbGRI2-type structures, and 61% of ampC genes had ISAba1 upstream. We conclude that the dissemination and spread of a few dominant lineages of CRAB containing various resistance island structures occurred in Taiwan.

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Emergence of blaTEM, blaCTX-M, blaSHV and blaOXA genes in multidrug-resistant Enterobacteriaceae and Acinetobacter baumannii in Saudi Arabia

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