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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.  bla  SHV ,  bla  OXA ,  bla  TEM  and  bla  CTX M . The most prevalent carbapenemase genes were  bla  NDM-1 ,  bla  OXA-48  and  bla  KPC-2 . They were found in all types of isolates, while  bla  OXA-162 , a rare  bla  OXA-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 ( bla  SHV-145 ,  qacEdelta1 ,  sul1 ,  aadA1 ,  aadA2 ) and clinical ( bla  OXA-1 ,  bla  SHV-106 , bla  TEM-150 ,  aac(3)Iia ,  dfrA14 ,  oqxA10 ;  oqxB17 , catB3 ,  tetD ) reservoirs of AR. Moreover, some ARGs ( oqxA10 ;  bla  SHV-145 ;  bla  SHV-100 ,  aac(6')Il ,  aph(3')VI ,  armA ,  arr2 ,  cmlA5 ,  bla  CMY-4 ,  mphE ,  msrE ,  oqxB13 ,  bla  OXA-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  bla  OXA-162 , which was co-expressed in our strains with  bla  CTX-M-15  and  bla  OXA-1 .

plos one

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