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Vegetable-Derived Carbapenemase-Producing High-Risk Klebsiella pneumoniae ST15 and Acinetobacter baumannii ST2 Clones in Japan: Coexistence of bla _NDM-1, bla _OXA-66, bla _OXA-72, and an AbaR4-Like Resistance Island in the Same Sample.

ABSTRACT: This study was conducted to characterize carbapenemase-producing Klebsiella pneumoniae and Acinetobacter baumannii isolated from fresh vegetables in Japan. Two K. pneumoniae isolates (AO15 and AO22) and one A. baumannii isolate (AO22) were collected from vegetables in the city of Higashihiroshima, Japan, and subjected to antimicrobial susceptibility testing, conjugation experiments, and complete genome sequencing using Illumina MiniSeq and Oxford Nanopore MinION sequencing platforms. The two K. pneumoniae isolates were clonal, belonging to sequence type 15 (ST15), and were determined to carry 19 different antimicrobial resistance genes, including bla _NDM-1 Both the isolates carried bla _NDM-1 on a self-transmissible IncFII(K):IncR plasmid of 122,804 bp with other genes conferring resistance to aminoglycosides [aac(6')-Ib, aadA1, and aph(3')-VI], β-lactams (bla _CTX-M-15, bla _OXA-9, and bla _TEM-1A), fluoroquinolones [aac(6')-Ib-cr], and quinolones (qnrS1). A. baumannii AO22 carried bla _OXA-66 on the chromosome, while bla _OXA-72 was found as two copies on a GR2-type plasmid of 10,880 bp. Interestingly, A. baumannii AO22 harbored an AbaR4-like genomic resistance island (GI) of 41,665 bp carrying genes conferring resistance to tetracycline [tet(B)], sulfonamides (sul2), and streptomycin (strAB). Here, we identified Japanese carbapenemase-producing Gram-negative bacteria isolated from vegetables, posing a food safety issue and a public health concern. Additionally, we reported a GR2-type plasmid carrying two copies of bla _OXA-72 and an AbaR4-like resistance island from a foodborne A. baumannii isolate.IMPORTANCE Carbapenemase-producing Gram-negative bacteria (CPGNB) cause severe health care-associated infections and constitute a major public health threat. Here, we investigated the genetic features of CPGNB isolated from fresh vegetable samples in Japan and found CPGNB, including Klebsiella pneumoniae and Acinetobacter baumannii, with dissimilar carbapenemases. The NDM carbapenemase, rarely described in Japan, was detected in two K. pneumoniae isolates. The A. baumannii isolate identified in this study carried bla _OXA-66 on the chromosome, while bla _OXA-72 was found as two copies on a GR2-type plasmid. This study indicates that even one fresh ready-to-eat vegetable sample might serve as a significant source of genes (bla _NDM-1, bla _OXA-72, bla _CTX-M-14b, and bla _CTX-M-15) encoding resistance to frontline and clinically important antibiotics (carbapenems and cephalosporins). Furthermore, the detection of these organisms in fresh vegetables in Japan is alarming and poses a food safety issue and a public health concern.

SUBMITTER: Soliman AM

PROVIDER: S-EPMC8091017 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundCarbapenem-Resistant Enterobacterales (CRE) has been categorized as pathogens of critical priority by World Health organization (WHO) as they pose significant threat to global public health. Carbapenemase production considered as the principal resistance mechanism against carbapenems and with the recent surge and expansion of carbapenemases and its variants among clinically significant bacteria in India, the present study reports expansion blaOXA-78 and blaOXA-58 of in CRE of clinical origin.MethodsBacterial isolates were collected from a tertiary referral hospital and identified through VITEK® 2 Compact automated System (Biomerieux, France). Rapidec® Carba NP (Biomerieux, France) was used to investigate carbapenemase production followed by antibiotic susceptibility testing through Kirby-Bauer Disc Diffusion method and agar dilution method. Class D carbapenemase genes were targeted through PCR assay followed by investigation of horizontal transmission of blaOXA-58 and blaOXA-78. Whole genome sequencing was carried out using Illumina platform to investigate the genetic context of blaOXA-58 and blaOXA-78 genes and further characterization of the CRE isolates.ResultsThe carbapenem-resistant Escherichia coli (BJD_EC456) and Serratia marcescens (BJD_SM81) received during the study from the tertiary referral hospital were isolated from sputum and blood samples respectively. PCR assay followed by whole genome sequencing revealed that the isolates co-harbor blaOXA-58 and blaOXA-78, a variant of blaOXA-51. Horizontal transfer of blaOXA-58 and blaOXA-78 genes were unsuccessful as these genes were located on the chromosome of the study isolates. Transposon Tn6080 was linked to blaOXA-78 in the upstream region while the insertion sequences ISAba26 and ISCfr1 were identified in the upstream and downstream region of blaOXA-58 gene respectively. In addition, both the isolates were co-harboring multiple antibiotic resistance genes conferring clinical resistance towards beta-lactams, aminoglycosides, fluroquinolones, sulphonamides, tetracyclines. BJD_EC180 belonged to ST2437 while BJD_SM81 was of an unknown sequence type. The nucleotide sequences of blaOXA-78 (OQ533021) and blaOXA-58 (OQ533022) have been deposited in GenBank.ConclusionsThe study provides a local epidemiological information regarding carbapenem resistance aided by transposon and insertion sequences associated blaOXA-78 and blaOXA-58 genes associated and warrants continuous monitoring to prevent their further dissemination into carbapenem non-susceptible strains thereby contributing to carbapenem resistance burden which is currently a global concern.

Project description:The emergence of carbapenemase-producing Enterobacterales (CPE) infections is a major global public health threat. Rapid and accurate detection of pathogenic bacteria is essential to optimize treatment and timely avoid further transmission of these bacteria. Here, we aimed to develop a rapid on site visualization detection method for CPE using improved recombinase polymerase amplification (RPA) combined with lateral flow strip (LFS) method, based on four most popular carbapenemase genes: bla KPC, bla NDM, bla OXA-48-like, and bla IMP. All available allelic variants of the above carbapenemases were downloaded from the β-lactamase database, and the conserved regions were used as targets for RPA assay. Five primer sets were designed targeting to each carbapenemase gene and the RPA amplification products were analyzed by agarose gel electrophoresis. FITC-labeled specific probes were selected, combined with the best performance primer set (Biotin-labeled on the reverse primer), and detected by RPA-LFS. Mismatches were made to exclude the false positive signals interference. This assay was evaluated in 207 clinically validated carbapenem-resistant Enterobacterales (CRE) isolates and made a comparison with conventional PCR. Results showed that the established RPA-LFS assay for CPE could be realized within 30 min at a constant temperature of 37°C and visually detected amplification products without the need for special equipment. This assay could specifically differentiate the four classes of carbapenemases without cross-reactivity and shared a minimum detection limit of 100 fg/reaction (for bla KPC, bla NDM, and bla OXA-48-like) or 1000 fg/reaction (for bla IMP), which is ten times more sensitive than PCR. Furthermore, the detection of 207 pre-validated clinically CRE strains using the RPA-LFS method resulted in 134 bla KPC, 69 bla NDM, 3 bla OXA-48-like, and 1 bla IMP. The results of the RPA-LFS assay were in consistent with PCR, indicating that this method shared high sensitivity and specificity. Therefore, the RPA-LFS method for CPE may be a simple, specific, and sensitive method for the rapid diagnosis of carbapenemase Enterobacterales.

Project description:Aim of this study was to genetically characterize two carbapenemase-producing Escherichia coli strains obtained from a pediatric patient affected by diarrhea, expressing OXA-181 and/or NDM-5 type enzymes. The above microorganisms were collected in the same Desenzano hospital (Northern Italy) where the bla NDM- 5 gene was detected for the first time in Italy 3 years ago. One strain (5P), belonged to sequence type ST405/ST477 (according to Pasture/Oxford schemes) and serotype O102:H6. It was characterized by a 130562 bp multi-replicon plasmid IncFII/IncFIA/IncFIB (pVSI_NDM-5) enclosing two main antibiotic resistance islands: (i) ARI-I, 10030 bp in size, carried genes coding for β-lactam- (bla OXA- 1, bla CTX-M- 15), fluoroquinolone/aminoglycoside- (aac(6')-lb-cr) and phenicol- resistance (catB3), (ii) ARI-II, 15326 bp in size, carried genes coding for sulfonamide- (sul1), β-lactam- (bla NDM- 5, bla TEM- 1 B), phenicol- (catB3), trimethoprim- (dfrA17), antiseptic- (qacEΔ1), and aminoglycoside- (aadA5, rmtB) resistance. The other isolate (5M), belonged to sequence type ST2659/ST759 and serotype O50/02:H18, and carried four plasmids: a 153866 bp multi-replicon IncFII/IncFIA/IncFIB (pISV_IncFII_NDM-5), an 89866 bp IncI1 plasmid, a 51480 bp IncX3 plasmid (pISV_IncX3_OXA181), and a 41143 bp IncI plasmid (pISV_IncI_CMY-42). pISV_IncFII_NDM-5 carried two main antibiotic resistance islands: (i) ARI-III, 12220 bp in size, carried genes coding for β-lactam- (bla OXA- 1), fluoroquinolone/aminoglycoside- (aac(6')-lb-cr), tetracycline- (tet(B)) and phenicol- resistance (catB3, catA1), and ii) ARI-IV, 26527 bp in size, carried determinants coding for macrolide- (erm(B), mph(A)), sulfonamide- (sul1), beta-lactam- (bla NDM- 5, bla TEM- 1 B), trimethoprim- (dfrA14, dfrA12), antiseptic- (qacEΔ1), and aminoglycoside- resistance (aadA5). pISV_IncI_CMY-42 harbored the bla CMY- 42 gene coding for beta-lactam resistance, pISV_IncX3_OXA181 harbored genes encoding fluoroquinolone- (qnrS1) and beta-lactams- resistance (bla OXA- 181). In conclusion, the detection of two different NDM-5 E. coli strains from a pediatric patient with a history of travel to the Far East countries strongly highlight an increasing trend and risk of importation from such areas.

Project description:In clinical practice, carbapenems and tigecycline are considered significant options for treating infections caused by multidrug-resistant Klebsiella spp. The continual evolution of resistance mechanisms to carbapenems and tigecycline is shattering the present condition. Meanwhile, convergence of the two resistance mechanisms in a single strain has been reported repeatedly, posing a significant threat to public health and safety. In this study, two carbapenem- and tigecycline-resistant Klebsiella species were obtained from patients and investigated using antimicrobial susceptibility testing, conjugation assay, whole-genome sequencing, and bioinformatics analysis. In Klebsiella variicola FK2020ZBJ35, an untransferable multidrug IncFIB(Mar)/IncHI1B-like plasmid carrying tmexCD2-toprJ2, blaIMP-4, and blaNDM-1 was discovered, as was a similar plasmid carrying tmexCD1-toprJ1 and blaIMP-4 in Klebsiella quasipneumoniae 2019SCSN059. Genetic context analysis found that two distinct tmexCD-toprJ variants were detected in comparable mobile units with genetic array int-int-hp-hp-tnfxB-tmexCD-toprJ and integrated into separate genetic locations. blaIMP-4 and blaNDM-1 were carried by an integron In1377 and a truncated Tn3000, respectively. These findings revealed that the carbapenem and tigecycline resistance genes carried by the two strains were located on mobile elements and might potentially transmit horizontally to additional strains. Furthermore, our findings showed that IncFIB(Mar)/IncHI1B-like plasmids represent a significant reservoir of essential resistance genes that warrants continued monitoring. IMPORTANCE Tigecycline is an essential antibiotic that is used to treat infections caused by carbapenem-resistant Klebsiella pneumoniae (CRKP). The emergence of high-level tigecycline-resistant CRKP poses a serious hazard to human health. This work screened two tigecycline-resistant CRKP strains from clinical patients and found a type of plasmid that encoded carbapenemase and TmexCD-ToprJ in Klebsiella. Importantly, one plasmid cocarried tmexCD-toprJ, blaNDM-1, and blaIMP-4, hinting that this plasmid could be a critical vector for superbug development. Furthermore, we discovered that the carbapenem and tigecycline resistance genes are located in mobile units by genetic structure analysis. Our research tracks the formation of clinically super-resistant Gram-negative bacteria.

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Vegetable-Derived Carbapenemase-Producing High-Risk Klebsiella pneumoniae ST15 and Acinetobacter baumannii ST2 Clones in Japan: Coexistence of bla _NDM-1, bla _OXA-66, bla _OXA-72, and an AbaR4-Like Resistance Island in the Same Sample.

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Vegetable-Derived Carbapenemase-Producing High-Risk Klebsiella pneumoniae ST15 and Acinetobacter baumannii ST2 Clones in Japan: Coexistence of bla NDM-1, bla OXA-66, bla OXA-72, and an AbaR4-Like Resistance Island in the Same Sample.

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OmicsDI is part of the ELIXIR infrastructure

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Vegetable-Derived Carbapenemase-Producing High-Risk Klebsiella pneumoniae ST15 and Acinetobacter baumannii ST2 Clones in Japan: Coexistence of bla _NDM-1, bla _OXA-66, bla _OXA-72, and an AbaR4-Like Resistance Island in the Same Sample.