Faculty of Applied Sciences

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Carbapenem resistance profiling of selected bacterial species belonging to the Enterobacteriaceae family in various water matrices
(2021) Dludla, Winile Nokwazi; Stenström, T. A.; Pillai, S. K. K.; Adegoke, Anthony Ayodeji
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.
Occurrence of vancomycin resistant enterococci (VRE) in two Durban wastewater treatment plants for effluent reuse
(2019) Madu, Chibuzor Ezinne; Stenström, Thor-Axel; Reddy, Poovendhree; Adegoke, Anthony Ayodeji
The presence of enterococci in improperly treated wastewater leads to pollution of the recipient water bodies which directly or indirectly affects the humans especially when antibiotic resistant strains are involved. In 2017 the World Health Organization listed vancomycin resistant enterococci (VRE) among those with highest priority for further surveillance and research, both among humans and in the receiving aquatic environment. The purpose of this study is to determine how efficient the WWTPs are in removing both vancomycin-resistant enterococci (VRE) and vancomycin-sensitive enterococci (VSE) from wastewater. One hundred (60 wastewater and 40 river) samples were collected from July 2016 to June 2017 which covered the warm and cold seasons of South Africa. Primary isolation and enumeration were carried out on Slanetz and Bartley agar supplemented with and without vancomycin (6 µg/mL) for vancomycin resistant enterococci (VRE) and total enterococci (TE) respectively. Presumptive enterococci were selected using Gram staining, growth on bile aesculin agar, catalase and pyrase tests. The presumptive enterococci isolates (202 VRE and 67 VSE) were confirmed and speciated using polymerase chain reaction (PCR). The identified Enterococcus isolates were subjected to antibiotic susceptibility testing (AST) to examine their resistance profile against fifteen antibiotics including vancomycin. Antibiotic resistance genes (van, tet and emeA) were detected by PCR. The TE and VRE counts of the two WWTPs influents ranged from 6.1 to 7.2 log10 CFU/100 mL (for TE) and 4.3 to 6.7 log10 CFU/100 mL (for VRE) while the effluent concentration of Plant II contained 1.5 to 4.4 log10 CFU/100 mL and 0.9 to 3.4 log10 CFU/100 mL for TE and VRE respectively. Neither TE nor VRE was detected in Plant I effluent. The TE and VRE counts of the recipient river samples were higher than the effluents. There were no visible seasonal effects based on the counts. The removal efficiencies in the two plants ranged from 95 to 100%, where chlorination played a major role. Two hundred and sixty- nine (202 VRE and 67 VSE) isolates were identified by PCR as Enterococcus. The most abundant species was E. faecium followed by E. faecalis while other species include E. hirae, E. gallinarum, E. durans, E. casseliflavus and E. cecorium. MALDI-TOF and PCR were used in parallel for the identification of the isolates, which resulted in 80.1% agreement for genus identification. The AST results showed that a large percentage (39 to 98%) were resistantto all other antibiotics except amoxicillin/clavulanic acid and imipenem to which the isolates showed high sensitivity. Four van genes (vanA, vanB, vanC1, vanC2/3) and 4 tet genes (tetK, tetL, tetM, tetO), and also the multidrug efflux pump gene, emeA were detected among the 269 enterococci isolates with vanA and tetL being the most prevalent. At least one virulence gene (ace, asa1, cylA, efaA, esp, gelE and hyl) occurred in 74% (67/88) of the isolates. The result showed that the two WWTPs are efficient in removing both enterococci and VRE from their influents. Thus these effluents had little or no effect to enterococci count of their interlinked recipients. Also, a majority of the isolates are not only antibiotic resistant strains but are also virulent. They therefore pose risk to public health.
Prevalence and fate of antibiotics and its derivatives in sewage treatment in Durban and the receiving environment
(2019) Faleye, Adekunle Christopher; Stenström, Thor-Axel; Ramluckan, Krishan; Adegoke, Anthony Ayodeji; Bux, Faizal
Antibiotics are released to the environment either directly in an unchanged form or partially metabolized. The discharge is usually through untreated waste or through wastewater treatment effluents. The stable antibiotics in reduced amounts persist through the wastewater treatment processes and end up in receiving waters, where they may impact crops through irrigation or affect drinking water intakes. Antibiotics in the waste and sludge fractions may similarly impact crops and arable land through their use as fertilizers. Conventional wastewater treatment plants are not designed for the removal of antibiotics but may to a varying extent reduce their concentrations. Their quantitative occurrence within the water matrices depends on the frequency and quantities of use for therapeutic purposes or as growth promoters in animal production. Additional inputs may emanate from individual waste discharges. Antibiotics present in sub-inhibitory concentrations may predicate for resistance among the resident bacteria in the water matrix, biofilms or in humans and animals. In South Africa antibiotics are extensively used both in human therapy and in animal husbandry without clearly followed regulations and are sometimes readily available. The available studies have focused on the presence of antibiotic resistant bacteria in wastewater influent and effluent but there is a paucity of information relating to these antibiotics as emerging contaminants in South Africa wastewater. In this thesis a rapid and sensitive analytical methodology was initially assessed and applied, based on the use of HPLC/diode array UV detector for six antibiotics (ethionamide (ETI), metronidazole (MET), trimethoprim (TRI), ciprofloxacin (CIP), sulfisoxazole (SUF) and albendazole (ALB). Validation of the method was performed by screening assessment in selected wastewater treatment plants (WWTPs) with the aim of determining the sensitivity of the equipment (Shimadzu 2020), assess the limit of detection, optimize the extraction procedure (solid phase extraction) and screen for the most prevalent antibiotics. The percentage recovery for the optimized method using wastewater sample was above 65 % for all antibiotics of interest. The limit of detection, which ranges from 0.03 to 0.48 mg L-1, enables the determination of a range of concentration of antibiotics in polluted sample such as the wastewater influent sample. Furthermore in this thesis, a more advanced, online solid phase extraction – high performance liquid chromatography mass spectrometry (SPE-HPLC-MS) method, was applied to measure the concentration of these and an additional seven antibiotics, norfloxacin (NOR), ofloxacin (OFL), clindamycin (CLI), sulfamethoxazole (SUL), erythromycin (ERY), clarithromycin (CLA), azithromycin (AZI) and roxithromycin (ROX) in ng L-1 concentrations. The quantity and occurrence of the selected antibiotics was assessed in untreated wastewater in four wastewater treatment plants in Durban, KwaZulu-Natal (KZN), at different treatment stages and in the effluent and recipient surface water environment. In the influent the additive concentration of the antibiotics associated to the separated sediment fraction through centrifugation and in the supernatant of samples collected were accounted for and analyzed. The limit of detection (LOD) and the limit of quantification (LOQ), ranged from 0.07 – 0.33 ng L-1 and 0.23 – 1.09 ng L-1, respectively for the 13 assessed antibiotics and the percentage recovery were in the range of 51 to 111 %. The percentage of antibiotics recovered from the sediment (centrifuged) samples, which would have been lost to filtration if not analyzed in parallel, were in the range of 2.6% – 97% (n = 32), while the frequency of detection in the influent samples for the sampling period ranges from 62.5 – 100 % (n = 32). All the studied antibiotics were detected in the influent of each WWTP and the concentration was in the rage of 1.3 ng L-1 (AZI) – 81748 ng L-1 (CIP). The antibiotics with the highest concentrations (median) detected in the receiving water (downstream) for each of the four WWTPs in KZN, were TRI (217 ng L-1), SUL (239 ng L-1), CIP (708 ng L-1) and ALB (325 ng L-1) respectively. The overall percentage removal efficiency for the four WWTPs ranged from 21 % - 100 %. The most effective treatment steps were assessed with the focus on activated sludge filter and trickling filter. Within these, it was actually the sedimentation treatment stages (secondary clarifier), after these steps that played the most vital role in the reduction of antibiotics where > 70 % of the antibiotics was removed. Finally, the impact of post chlorination was analyzed for the effluent of the WWTPs. The presence of transformation product as a result of post chlorination was examined in a parallel study using a controlled experiment and full scale analysis. The efficiency of chlorine in the reduction of antibiotics was more of transformation of antibiotics than degradation. The oxidative ability of chlorine enhances its reaction with antibiotics thereby transforming the antibiotics. The percentage reduction of antibiotics in relation to chlorination was >85 % (pilot experiment) and ranged between 14 % - 97 % in the field experiments. Likewise, UV was effective in the degradation of antibiotics, with longer exposure time producing higher degradation. Future research should focus on determining the toxicological impact of these transformation products. The concentration of the antibiotics in the downstream samples were generally low when compared to their influent concentrations.