Appraisal of wastewater final effluents and river water as reservoirs of cholera and non-cholera causing vibrio species : case study of the Amatole and OR Tambo District Municipalities
- Authors: Nontongana, Nolonwabo
- Date: 2018
- Subjects: Vibrio -- South Africa -- Eastern Cape Vibrio cholerae Disease Reservoirs
- Language: English
- Type: Thesis , Doctoral , Microbiology
- Identifier: http://hdl.handle.net/10353/9929 , vital:35146
- Description: Vibrio infections remain a serious threat to public health. In the last decade, Vibrio disease outbreaks have created a painful awareness of the personal, economic, societal, and public health costs associated with the impact of inadequately treated wastewater effluents. This study was therefore designed to assess the occurrence of cholera and non-cholera causing Vibrio species in the final effluents of wastewater treatment plants and river waters in the Amatole (BT WWTP) and OR Tambo District municipalities (MT WWTP and QN River) of the Eastern Cape Province. Samples were collected monthly from December 2016 to November 2017 from the final effluent, 500 meters upstream and downstream of the discharge points and analysed for physicochemical parameters, Vibrio pathogens prevalence and their antibiogram characteristics using both culture-based and molecular techniques. Samples were collected aseptically using sterile 1L glass bottles containing 0.5 ml of sterile sodium thiosulphate solution and transported on ice to the laboratory for analyses within 6hrs of collection. The membrane filtration method was used for enumeration of presumptive Vibrio densities on thiosulfate citrate bile salt (TCBS) agar plates. Polymerase chain reaction (PCR) was then used to confirm the identities of the presumptive Vibrio species using the species-specific primers. The confirmed isolates were further subjected to molecular characterization to confirm their respective pathotypes. Antimicrobial susceptibility testing was done by the standard disc diffusion method recommended by the Clinical and Laboratory Standards Institute. The recovered Vibrio species were tested against a panel of 17 antibiotics. Physicochemical parameters measured include pH, temperature, electrical conductivity, salinity, turbidity, total dissolved solid (TDS), dissolved oxygen (DO), biochemical oxygen demand (BOD) and chlorine (only for BT WWTP). Unacceptably high levels of the assayed parameters were observed in many cases for TDS (66 - 879 mg/l), turbidity (2.0 -722.33 NTU) and DO (2.5 – 9.7 mg/l) as well as chlorine (0.2 – 3.2 mg/L). Presumptive Vibrio densities varied from 2.91 to 3.91 log10 CFU/100 ml and 2.67 to 3.18 log CFU/100ml, for BT WWTP and MT WWTP respectively. The densities for the QN River ranged between 2.51 to 3.99 log10 CFU/100mL. Out of 720 presumptive isolates recovered, 619 (86 percent) were found to be positive for the Vibrio genus. Molecular confirmation of the presumptive Vibrio species revealed the presence of V. fluvialis (16), V. vulnificus (12), V aliginolyticus (9), V. parahaemolyticus (37) and V. cholerae (5) isolates were confirmed. The susceptibility against 17 different antibiotics by the recovered species were examined. V. cholerae was notably resistant against nalidixic acid (3) and Ampicilin (2), all the V. vulnificus isolates were resistant against ampicillin (16), V. fluvialis showed resistance against ciprofloxacin and nalidixic acid, all (9) isolates for V. alginolyticus were resistant against ampicillin. V. parahaemolyticus showed resistance against cefutoxime (16), cefuxime (8) and ampicillin (13). The recovery of Vibrio in the discharged effluents throughout the sampling period even in adequately disinfected effluents is not acceptable considering that Vibrio are pathogenic bacteria. The findings of this study underline the need for constant monitoring of the physicochemical and microbiological qualities of discharged effluents and might also be suggestive for a review of the disinfection methods used at the treatment works as this might pose adverse health risk to the communities which still rely heavily on these surface waters.
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- Date Issued: 2018
Molecular epidemiology of vibrio pathogens in selected surface waters and treated final effluents of wastewater treatment plants in Chris Hani District Municipality in the Eastern Cape Province, South Africa
- Authors: Ayodeji, Onsula Charles
- Date: 2018
- Subjects: Sewage disposal plants Sewage -- Purification Water -- Purification -- South Africa -- Eastern Cape
- Language: English
- Type: Thesis , Doctoral , Microbiology
- Identifier: http://hdl.handle.net/10353/10464 , vital:35478
- Description: Access to clean and safe water is essential for the survival of human beings. Nevertheless pollution from wastewater treatment plants (WWTPs) effluents often impacts the microbiological qualities of surface waters. Availability of freshwater resources is indispensable for preventing waterborne diseases. The current study evaluated the physicochemical properties and abundance of Vibrio species in selected rivers and WWTPs in the Eastern Cape Province of South Africa, over a one year sampling regime (December, 2016 to November, 2017). Water samples were collected monthly at designated locations along the banks of two rivers, treated effluents from 500 m upstream and downstream discharge points of two WWTPs and examined for physicochemical qualities, prevalence of Vibrio pathogens and their antibiogram profiles using both cultural and molecular techniques. The findings reveal the qualities of Tsomo and Great Fish river qualities with respect to electrical conductivity (EC) (137.75 – 377.69 mg/l), total suspended solids (TSS) (52.00 – 725.19 mg/l) and temperature (12.7 – 23 oC) and the treated effluents of WW-AD and WW-CR WWTPs with respect to EC (525.73 – 1071.89 mg/l), free chlorine (0.00 mg/l – 0.68 mg/l), temperature (12.4 – 28.8 oC), TSS (14.67 – 276.44 mg/l) exceeds the permissible limits set for effluent discharged to freshwater by South Africa guidelines. Other physicochemical qualities such as turbidity (63.11 – 797.17 NTU), BOD (3.30 – 5.26 mg/l) and TDS (68.88 – 148.14 mg/l) for Tsomo and Great Fish river and turbidity (20.33 – 310 NTU), biological oxygen demand (BOD) (1.28 – 4.96 mg/l), and total dissolve solid (TDS) (262.89 – 534.89 mg/l) for WWTPs WW-AD and WW-CR did not comply with WHO and EU standards as no regulation is set for them in the South African guidelines for domestic water usage. Statistical analysis revealed that pH, electrical conductivities, dissolved oxygen and total dissolved solid were significantly different whereas temperature did not differ significantly with respect to the four seasons (P < 0.05). The Vibrio densities for Great Fish river ranged between 0 and 3.29 log10 CFU/ml with the highest obtained in the spring. The Tsomo River Vibrio densities varied between 0 to 3.56 log10 CFU/ml and the maximum densities recorded during summer. The presumptive Vibrio densities in WW-AD and WW-CR WWTPs ranged from 0 to 3.67 log10 CFU/ml and 0 to 4.42 log10 CFU/ml with autumn and spring having the highest loads respectively. Molecular identification of the presumptive Vibrio species revealed 424 positive for the Vibrio genus. Of these, 21.69 percent, 11.79 percent, 8.25 percent and 2.12 percent were confirmed as V. cholerae, V. mimicus, V. parahaemolyticus and V. fluvialis respectively. The presence of potentially Vibrio pathogens in the rivers and treated effluents suggests potential public health threat to the communities relying on receiving watersheds where the effluents are discharged. Results of antibiotic sensitivity testing revealed high sensitivities against Ofloxacin (85.54 percent), Cefuroxime (81.93 percent), and Ciprofloxacin (74.70 percent), whereas resistance against other antibiotics follow this order: Imipenem (42.17 percent), Amoxicillin-Clavulanic Acid (54.17 percent), Ampicillin (63.54 percent), Nitrofurantoin (71.79 percent) and Polymyxin B (97.44 percent). Surprisingly, the resistance of Vibrio species against the known effective Carbapenems was relatively high (Meropenem (38.54 percent) and Imipenem (58.88 percent). Multiple antibiotic resistance phenotypes (MARP) of the isolates were resistant to two or more antibiotics whereas the calculated multiple antibiotic resistance index (MARI) ranged from 0.2 to 0.7. The observed high multiple antibiotics index suggest the recovered Vibrio pathogens are of high antimicrobial usage origin. Increase in antimicrobial resistance profiles towards conventionally used antibiotics as indicated in this study calls for adequate sanitation facilities and proper surveillance programs towards monitoring of antimicrobial resistance determinants in wastewater treatment effluents and receiving watersheds. This will in turn enhance early detection of resistant strains of public health importance, and supports the prompt notification and investigation of outbreaks. It further advocates the need for constant monitoring programme by the relevant regulatory agencies to ensure total compliance of the wastewater treatment facilities to the stipulated standards.
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- Date Issued: 2018
Characterisation of human pathogenic vibrios and methicillin-resistant staphylococcus aureus (MRSA) from wild and tank cultured dusky kob (agyrosomus japonicus) in the Eastern Cape Province, South Africa
- Authors: Justine, Fri
- Date: 2017
- Subjects: Marine microbiology Marine microbiology--South Africa--Eastern Cape Staphylococcus aureus infections--South Africa--Eastern Cape
- Language: English
- Type: Thesis , Doctoral , Microbiology
- Identifier: http://hdl.handle.net/10353/16047 , vital:40607
- Description: Introduction: Marine finfish including dusky kob (Agyrosomus japonicus), in addition to its high protein value, is bestowed with numerous health benefits including being heart friendly due to high proportion of omega 3 fatty acids. They are therefore used as powerful preventive measures against cardiovaslcular diseases. They are also important in neurological development during gestation and infancy, thus a regular recommendation by nutritionists. However, fish and its environment may be contaminated by bacteria pathogens. Human infections caused by members of the genus Vibrio, and Staphylococcus especially Methicillin-resistant S. aureus (MRSA) are of increased concern worldwide especially with increased Vibrio disease outbreaks and rapid spread of MRSA in the community. Methicillin-resistant Staphylococcus aureus is regarded as an emerging zoonotic agent, therefore marine fish may be considered as a reservoir for infection. The emergence of multi-drug resistant organisms in both farm animals and humans has also been a challenge that hurdles chemotherapy worldwide. Methods: We enumerated the bacteria flora of skin, gill and gut of marine dusky kob (Agyrosomus japonicus) raised in a recirculatory aquaculture system using metagenomic sequencing of the 16S rRNA. We further screened 120 dusky kob fishes (skin, gill and gut) and 80 water samples from two fish farms and Kariega estuary collected over a 10 month period for human pathogenic vibrios, and MRSA using standard procedures. Vibrio species were further subjected to species specific PCRs delineating them into V. cholerae, V. parahaemolyticus, V. vulnificus, and V. fluvialis. This was followed by elucidation of the genotypic virulence determinants. The Reverse Fragment Length Polymorphism (RFLP) of the cytotoxin hemolysin (vvhA) gene of V. vulnificus strains was carried out to determine the associated biotypes. A total of 277 Vibrio strains consisting of 126 V. fluvialis, 45 Vibrio vulnificus, 30 V. parahaemolyticus and 76 vibrios belonging to other species were subjected to susceptibility testing to 15 antibiotics by the disc diffusion method and resistant strains were further evaluated for their genotypic antimicrobial resistant determinants by polymerase chain reaction followed by plasmid profiling. For MRSA screening, we evaluated the reliability of cefoxitin disc diffusion, oxacillin screen agar and BrillianceTM MRSA 2 agar tests, in detecting methicillin resistance from fish (environmental) samples using PCR detection of the mecA as the gold standard. A total of 33 mecA positive S. aureus strains (MRSA) were subjected to PCR detection of enterotoxins (Staphylococcal enterotoxin A-E) and Panton Valentine Leucocidin (PVL) encoding genes. The SCCmec types were also investigated by multiplex PCR targeting genes encoding type I-V. The antibiogram profiles of MRSA strains to 12 antibiotics by the disc diffusion method and to vancomycin by broth micro dilution method were elucidated. Polymerase chain reaction was employed to evaluate the presence of antibiotic resistant genes. The nucleotide sequences of the rpoB gene were analysed to determine mutations that could lead to rifampicin resistance. Results: A broad diversity of pathogenic and non-pathogenic bacterial species of different taxonomic groups was observed with Proteobacteria revealed as the most common bacteria phyla. Fifty one families were identified with Enterobacteriaceae the most frequently detected in all the anatomical sites and the gut showing the highest species diversity. A total of 606 (60.23 percent ) presumptive Vibrio isolates were confirmed by PCR of which V. fluvialis was the most predominant, 193 (31.85 percent ). This was followed by Vibrio vulnificus 74 (12.21 percent ), and V. parahaemolyticus 33 (5.45 percent ). No V. cholerae strain was detected. Only one of the V. parahaemolyticus strains possessed the trh virulent gene with no tdh detected. Virulence factors of V. fluvialis detected were stn (13.5 percent ), hupO (10.4 percent ), and vfpA (1.0 percent ). V. vulnificus biotype 3, formerly restricted to Isreal was identified. Generally, resistance among Vibrio isolates varied considerably with highest resistances recorded to amoxicillin (76.2 percent ), ampicillin (67.5 percent ), erythromycin (38.3 percent ), and doxycycline (35.0 percent ) while higher susceptibilities were revealed by gentamicin (100 percent ), norfloxacin (97.8 percent ), florfenicol (90.3 percent ), tetracycline (87.7 percent ), and chloramphenicol (87.4 percent ). Multiple antibiotic resistance (MAR) (resistance to ≥ 2 classes of antibiotics) was 58.5 percent . The MAR index did not vary significantly between sites (P>0.05) and values of >0.20 were recorded in 40 percent (108/277) of all the isolates. The resistant genes ampC, blaOXA, tetA, tetM, dfr1, sul1, sul2, ermB, nptII and strA were detected in two or more Vibrio isolates while the SXT intergrase was detected only in one isolate. Seven of the 18 extreme drug resistant isolates possessed 1–3 plasmids of 1.5–10 kb range. A total of 321 presumptive Staphylococcus aureus isolates were recovered by culture out of which 202 (62.9 percent ) were identified as S. aureus by PCR. Of these, 33 (16.3 percent ) strains were mecA positive while the rest, 169 (83.7 percent ) were mecA negative. The sensitivity and specificity of MRSA detection by use of cefoxitin disc (30 μg) test was 93.9 percent and 91.7 percent , that for oxacillin screen agar test (6 μg/mL) was 81.8 percent and 92.3 percent while that of BrillianceTM MRSATM 2 agar was 87.9 percent and 94.1 percent respectively. Staphylococcal enterotoxin SEE was detected in 12.1 percent (4/33) of total strains followed by SEA 3.0 percent (1/33), and SEC 3.0 percent (1/33). SEB and SED were absent. Six of the isolates had the PVL gene, giving a prevalence of 18.2 percent . The most frequently detected SCCmec type was SCCmec type III (17/33; 51.5 percent ), closely followed by SCCmec subtype IVc (10/33; 30.3 percent ) and the least were SCCmec types I and II with only 3.0 percent (1/33) each. The rest four (4/33; 12.1 percent ) did not belong to any of the above types and were therefore considered to belong to other subtypes of SCCmec type IV not tested in the study. For antibiogram patterns, 12 (36.4 percent ) strains were resistant to doxycyline, 10 (30.3 percent ) to trimethroprim-sulfamethoxazole, 9 (27.3 percent ) to tetracycline and only 1 (3.0 percent ) strain was resistant to ciprofloxacin. Nineteen (57.6 percent ) of the MRSA strains had vancomycin MIC of ≤ 2 μg/mL, 4 (12.1 percent ) with MIC range of 4-8 μg/mL, and 10 (30.3 percent ) with values ≥ 16 μg/mL. Overall, 27 (91.percent) isolates showed simultaneous resistance to 3 or more antibiotics. The resistant genes tetM, tetA, ermB, BlaZ femA were detected in two or more strains resistant to various antimicrobials. Resistance to rifampicin was not caused by mutations in the rpoB gene. Conclusions: To the best of our knowledge, this study is the first study reporting a broad, and diverse bacteria flora of skin, gill, and gut of the South African dusky kob as well as the first study on human pathogenic vibrios, and MRSA from marine finfish especially those from recirculatory aquaculture systems. Also, our study revealed that Vibrio species with pathogenic potential are present in marine dusky kob and its environs. Dusky kob is contaminated with CA- MRSA and HA- MRSA that may cause difficult to treat infections with regular antibiotics. Some of the MRSA strains contained toxins that can cause food-borne gastroeneteritis. The present study revealed that MRSA and Vibrio species are potential reservoirs for antibiotic resistant genes, which could be disseminated to other bacterial species probably through plasmids, a public health risk.
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- Date Issued: 2017