Carbapenem resistance profiling of selected bacterial species belonging to the Enterobacteriaceae family in various water matrices
Authors:   , Dludla Winile Nokwazi 
Published: June 2022

Carbapenems are broad-spectrum β-Lactams exhibiting bactericidal activity by binding to penicillin-binding proteins (PBPs). These antibiotics are the last resort drugs prescribed for severe infections when other administered ones have failed to produce enough responses in patients. Bacteria belonging to the Enterobacteriaceae family, including Escherichia coli and Klebsiella pneumoniae were identified as “priority pathogens” by the World Health Organization (WHO) in 2017 due to their resistance against carbapenems and have been ranked as “Priority 1: Critical” for research. Among the various types of carbapenem-resistant Enterobacteriaceae (CRE), carbapenemase-producing CRE (CP-CRE) have drawn the most attention since they can contribute to the overall challenge of antimicrobial resistance. Wastewater treatment plants (WWTPs) have been considered a potential hub for antibiotic resistance gene exchange and further release into the environment. Therefore, antimicrobial resistance surveillance using sewage isolates has been presented as a way to study the occurrence and spread of specific clonal groups or sequence types within a community or population. Thus, this study aimed to investigate the occurrence of carbapenem-resistant K. pneumoniae and E. coli and their virulence genes in selected WWTPs treating municipal wastewater in Durban, South Africa, and to assess the efficiency of these WWTPs in removing these microorganisms. The samples were taken from two WWTPS (WWTP I and WWTP II) monthly from six sampling points (influent, aeration/biofilter, pre-chlorination, post-chlorination, upstream and downstream from the WWTP’s discharge points) from January to September 2018. Primary isolation and enumeration were carried out on CHROMagarTM ECC and Klebsiella ChromoSelect Selective Agar Base, for E. coli and K. pneumoniae, respectively. A total of 120 carbapenem-resistant E. coli (CR E. coli) and 100 carbapenem-resistant K. pneumoniae (CR K. pneumoniae) were randomly selected, further identified using biochemical tests and confirmed using matrix-assisted laser desorption ionization-time of flight (MALDI-TOF), polymerase chain reaction (PCR) and 16S rRNA sequencing. The confirmed isolates were then subjected to antimicrobial susceptibility testing (AST) using eight antibiotics (third-generation cephalosporins and carbapenems) to determine their resistance profile. Extended-spectrum beta-lactamases (SHV, CTX-M, TEM, and OXA-1) and carbapenemase genes (NDM-1, OXA-48, IMP and VIM) and virulence genes such as stx 1 and 2, rfbE, hly, eae, and fliC were targeted using PCR. While carbapenem-resistant E. coli was detected in both WWTPs, the carbapenem-resistant K. pneumoniae was only detected in WWTP II. The total E. coli count in the influent ranged from 6.8 to 7.1±0.06 log10 CFU/100 mL (WWTP I) and 6.9 to 7.2±0.08 log10 CFU/100 mL (WWTP II) with a carbapenem resistance percentage of 0.01% and 0.07%, respectively. A complete reduction of CR E. coli was observed in both WWTPs surveyed and was not detected from the receiving water bodies. The total K. pneumoniae in the influent of WWTP II ranged between 7.1 to 7.3±0.1 log10 CFU/100 mL. For carbapenem-resistant K. pneumoniae, the highest percentage was detected in the aeration tank (3.03%), followed by the pre-chlorinated effluent (1.33%), with the influent having the least (0.34%). However, carbapenem-resistant K. pneumoniae was not detected from the post-chlorinated effluent and the receiving water bodies, indicating a complete removal. The third-generation cephalosporin resistance profile showed high resistance against cefixime in both E. coli and K. pneumoniae isolates. Among these, the influent of WWPT II showed the highest percentage of resistant E. coli against cefixime (92%) compared to WWTP I E. coli (62%) and WWTP II K. pneumoniae (81%). In addition, the resistance profile of carbapenems showed that most of the isolates were resistant to ertapenem in both WWTPs. Of the 120 E. coli isolates, 60 in WWTP I and II showed 100% and 95% resistance against ertapenem, respectively. Additionally, K. pneumoniae showed 100% resistance against ertapenem. The predominant ESBL genes detected in the E. coli isolates were TEM and SHV, showing a 100% carriage in both WWTPs. The least detected ESBL gene was OXA-1, at 98% and 52% carriage in WWTP I and II, respectively. The occurrence of the ESBL genes in K. pneumoniae isolates was slightly different between the sampling points. In the influent, the dominant genes were TEM and CTX-M at 75% and 62%, respectively. In the aeration and pre-chlorinated effluent, the dominant genes were TEM and SHV with 100% carriage. The least detected gene was the CTX-M at 13% in the influent, with no detection at the other sampling points. Both E. coli and K. pneumoniae isolates showed NDM-1 and OXA-48 to be the predominant genes identified of the carbapenemase-producing genes investigated. In the influent of WWTP I and II, the E. coli isolates that harboured NDM-1 were 100% and 82%, respectively. However, the modified Hodge test did not correlate with the detection of CRE since the test was positive for only 34 isolates (15%), while 168 isolates carried the carbapenemase genes based on PCR test. Out of the six virulence genes tested in CR E. coli, three (hly, rfbE, and eae) were detected. The predominant gene in both WWTPs was hly, with the highest percentage in WWTP II (88%) compared to WWTP I (33%). The least detected gene was eae, only detected in WWTP II at 8%. For K. pneumoniae, out of the three virulence genes (wabG, urea, and rmpA) tested, wabG was the only gene detected. This gene was detected at all the sampling points, with the highest percentage being in the pre-chlorinated effluent (50%), followed by the influent (11%) and the least in the aeration (10%). The investigated WWTPs showed carbapenem-resistant E. coli and K. pneumoniae isolates in their influent samples and the various stages of the treatment except in the post chlorinated effluent indicating their efficient removal during the disinfection process. Future research to determine the presence of viable but not culturable (VBNC) carbapenem-resistant Enterobacteriaceae in these treated effluent and recipient water bodies, and the use of advanced molecular methods capable of identifying these bacteria at lower concentrations is hereby recommended as these water bodies are routinely used for agricultural, industrial, and household purposes by the local communities.