Whole genome sequencing snapshot of multi-drug resistant Klebsiella pneumoniae strains from hospitals and receiving wastewater treatment plants in Southern Romania

PLoS One. 2020 Jan 30;15(1):e0228079. doi: 10.1371/journal.pone.0228079. eCollection 2020.


We report on the genomic characterization of 47 multi-drug resistant, carbapenem resistant and ESBL-producing K. pneumoniae isolates from the influent (I) and effluent (E) of three wastewater treatment plants (WWTPs) and from Romanian hospital units which are discharging the wastewater in the sampled WWTPs. The K. pneumoniae whole genome sequences were analyzed for antibiotic resistance genes (ARGs), virulence genes and sequence types (STs) in order to compare their distribution in C, I and E samples. Both clinical and environmental samples harbored prevalent and widely distributed ESBL genes, i.e. blaSHV, blaOXA, blaTEM and blaCTX M. The most prevalent carbapenemase genes were blaNDM-1, blaOXA-48 and blaKPC-2. They were found in all types of isolates, while blaOXA-162, a rare blaOXA-48 variant, was found exclusively in water samples. A higher diversity of carbapenemases genes was seen in wastewater isolates. The aminoglycoside modifying enzymes (AME) genes found in all types of samples were aac(6'), ant(2'')Ia, aph(3'), aaD, aac(3) and aph(6). Quinolone resistance gene qnrS1 and the multi-drug resistance oqxA/B pump gene were found in all samples, while qnrD and qnrB were associated to aquatic isolates. The antiseptics resistance gene qacEdelta1 was found in all samples, while qacE was detected exclusively in the clinical ones. Trimethroprim-sulfamethoxazole (dfrA, sul1 and sul2), tetracyclines (tetA and tetD) and fosfomycin (fosA6, known to be located on a transpozon) resistance genes were found in all samples, while for choramphenicol and macrolides some ARGs were detected in all samples (catA1 and catB3 / mphA), while other (catA2, cmIA5 and aac(6')Ib / mphE and msrE) only in wastewater samples. The rifampin resistance genes arr2 and 3 (both carried by class I integrons) were detected only in water samples. The highly prevalent ARGs preferentially associating with aquatic versus clinical samples could ascribe potential markers for the aquatic (blaSHV-145, qacEdelta1, sul1, aadA1, aadA2) and clinical (blaOXA-1, blaSHV-106,blaTEM-150, aac(3)Iia, dfrA14, oqxA10; oqxB17,catB3, tetD) reservoirs of AR. Moreover, some ARGs (oqxA10; blaSHV-145; blaSHV-100, aac(6')Il, aph(3')VI, armA, arr2, cmlA5, blaCMY-4, mphE, msrE, oqxB13, blaOXA-10) showing decreased prevalence in influent versus effluent wastewater samples could be used as markers for the efficiency of the WWTPs in eliminating AR bacteria and ARGs. The highest number of virulence genes (75) was recorded for the I samples, while for E and C samples it was reduced to half. The most prevalent belong to three functional groups: adherence (fim genes), iron acquisition (ent, fep, fyu, irp and ybt genes) and the secretion system (omp genes). However, none of the genes associated with hypervirulent K. pneumoniae have been found. A total of 14 STs were identified. The most prevalent clones were ST101, ST219 in clinical samples and ST258, ST395 in aquatic isolates. These STs were also the most frequently associated with integrons. ST45 and ST485 were exclusively associated with I samples, ST11, ST35, ST364 with E and ST1564 with C samples. The less frequent ST17 and ST307 aquatic isolates harbored blaOXA-162, which was co-expressed in our strains with blaCTX-M-15 and blaOXA-1.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Anti-Bacterial Agents / pharmacology
  • Drug Resistance, Multiple, Bacterial / drug effects
  • Drug Resistance, Multiple, Bacterial / genetics*
  • Genes, Bacterial
  • Hospitals*
  • Humans
  • Integrons / genetics
  • Klebsiella pneumoniae / classification
  • Klebsiella pneumoniae / drug effects
  • Klebsiella pneumoniae / genetics*
  • Klebsiella pneumoniae / pathogenicity
  • Microbial Sensitivity Tests
  • Multilocus Sequence Typing
  • Quinolones / pharmacology
  • Romania
  • Virulence / genetics
  • Wastewater / microbiology*
  • Water Purification*
  • Whole Genome Sequencing*
  • beta-Lactams / metabolism


  • Anti-Bacterial Agents
  • Quinolones
  • Waste Water
  • beta-Lactams

Grant support

This study was supported by the Romanian Executive Agency for Higher Education, Research, Development and Inovation (https://uefiscdi.gov.ro/) research project PN-III-P4-ID-PCCF-2016-0114 POSCCE (RADAR). It was also partially supported by program CRCBABI project (642/2014) implemented in the National Institute for Infectious diseases “Matei Bals” and by ERANET-JPI-EC-AMR-AWARE-WWTP, PN-III-P1-1.1-PD-2016-2137 (PD 113/2018) and PN-III-P1-1.1-PD-2016-1798 (PD 148/2018). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.