Emergence of KPC-2-Producing Raoultella ornithinolytica Isolated from a Hospital Wastewater Treatment Plant

Abstract

B acterial carbapenem resistance is a threat to the public health worldwide (1). Klebsiella pneumoniae carbapenemase (KPC) is the most common carbapenemase and can be obtained from carbapenem-resistant Enterobacteriaceae (CRE) that are distributed globally (2, 3). Although bla KPC genes are usually recovered from samples from patients and healthy humans, transmission of bla KPC genes between environmental isolates remains largely unknown (4). In this study, we isolated Raoultella ornithinolytica SRo1810 carrying bla KPC-2 from a wastewater treatment plant (WWTP) located at the First Affiliated Hospital of Zhejiang University (FAHZU). Comprehensive analysis of R. ornithinolytica SRo1810 was performed to understand the resistant profiles and molecular characteristics of this isolate. R. ornithinolytica SRo1810 was isolated from the WWTP in July 2017 by using selective medium as described previously (5). The carbapenemase gene was identified using PCR and DNA sequencing. The plasmid was characterized using S1-pulsed-field gel electrophoresis (S1-PFGE), and the location of bla KPC-2 was identified with Southern hybridization. A plasmid conjugation experiment was carried out using Escherichia coli J53 as the recipient strain (5). MICs were established using a broth dilution method and were interpreted according to the EUCAST guidelines (http://www.eucast.org/). Genomic DNA was extracted using a DNA kit (Omega Bio-tek, Norcross, GA, USA). The DNA was subsequently sequenced using Illumina-HiSeq 4000-PE150 (Illumina, San Diego, CA, USA) and PacBio RS II platforms (Pacific Biosciences, Menlo Park, CA, USA). After sequencing, the complete genomic sequence of R. ornithinolytica SRo1810 was generated using Unicycler (6) by combining the sequencing results. Bioinformatic analysis was conducted as described previously (7-9). As shown in Table S1 in the supplemental material, SRo1810 was resistant to all ␤-lactams tested but was susceptible to aminoglycosides, fluoroquinolones, tigecycline, and colistin (Table S1). Another ␤-lactamase gene, bla TEM-1B , was further identified using the ResFinder database (https://cge.cbs.dtu.dk/services/ResFinder/). Interestingly, a variant of fosA7, which encodes fosfomycin resistance via a glutathione transferase, was detected in the genome via BLAST search analysis (Fig. S1A and B). Antimicrobial susceptibility tests confirmed that SRo1810 was resistant to fosfomycin. bla TEM-1B and bla KPC-2 were located in the plasmid, while fosA7 was located on the chromosome. In addition, we investigated the genetic environment surrounding fosA7, and the results suggest that mobile genetic elements may promote the transmission of the fosA7 gene. The insertion sequences ISL3 and IS3 are located upstream of the fosA7 gene, and IS110 and IS6 are located downstream of the fosA7 gene (Fig. S1C). Southern blot analysis demonstrated that SRo1810 carried at least five plasmids with sizes of approximately 150 kb, 120 kb, 80 kb, 40 kb, and 30 kb. KPC-2 was located on an ϳ42-kb plasmid (designated pSRo1810-KPC; Fig...

The Current Burden of Carbapenemases: Review of Significant Properties and Dissemination among Gram-Negative Bacteria

“…This explains why carbapenemase studies on aquatic sources (rivers, lakes, and wastewater treatment plants), livestock, and wildlife are accumulating. For example, hospital wastewater treatment plants in China were shown to contain KPC-2-producing Raoultella ornithinolytica [270], which is an aquatic Gram-negative organism with emerging importance in hospital infections associated with invasive procedures [271]. According to a Swiss study in 2019, the environmental Gram-negative organism Zhongshania aliphaticivorans, which lives in marine sediments, was shown to produce ZHO-1.…”

“…Xin et al reported pollution of estuarine water with bacterial genera including Rheinheimera, Stenotrophomonas, Shewanella, Raoultella, Vibrio, Pseudoalteromonas, Algoriphagus, Bowmanella, and Thalassospira, which all harbor bla OXA-58 [155] conferring resistance to carbapenems and penicillin and an ability to hydrolyze cefpirome and cephalothin but not ceftazidime, cefotaxime, or cefepime. Citrobacter freundii OXA-204 [171] Hospital wastewater E. coli KPC-2 [64] Raoltella ornitholitica KPC-2 [270] Acinetobacter towneri OXA-58 [154] Coastal water A. towneri OXA-58 [155] Municipal water Acinetobacter lwoffii OXA-235 and OXA-278 [163] River water Klebsiella pneumoniae KPC-2 [216] Estuarine water…”

Genetic characterization and virulence determinants of multidrug-resistant NDM-1-producing Aeromonas caviae

“…Genes encoding a small multidrug resistance (SMR) efflux transporter were also found in the MRR, which were deduced to be associated with the efflux system ( Kazama et al, 1998 ; Shen et al, 2021 ). Furthermore, both pK433-NDM and pFAHZZU2447_NDM lacked a tra module, which encodes a primary pilus for conjugation ( Chi et al, 2020 ). In addition, the OriTFinder results indicated that pFAHZZU2447_NDM had incomplete conjugative modules with the absence of a transfer site ( oriT ) and type IV coupling protein (T4CP) ( Supplementary Table 1 ).…”

First report of Klebsiella pneumoniae co-producing OXA-181, CTX-M-55, and MCR-8 isolated from the patient with bacteremia

“…The number and size of plasmids in K. pneumoniae 5589 were detected by S1-PFGE of total DNA (Chi et al, 2020). The locations of plasmids harboring the bla OXA-181 and mcr-8 were determined by Southern blotting and hybridization with digoxigenin-labeled specific probes.…”