- Title
- 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
- Creator
- Justine, Fri
- Subject
- Marine microbiology Marine microbiology--South Africa--Eastern Cape Staphylococcus aureus infections--South Africa--Eastern Cape
- Date Issued
- 2017
- Date
- 2017
- Type
- Thesis
- Type
- Doctoral
- Type
- Microbiology
- Identifier
- http://hdl.handle.net/10353/16047
- Identifier
- 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.
- Format
- 194 leaves
- Format
- Publisher
- University of Fort Hare
- Publisher
- Faculty of Science and Agriculture
- Language
- English
- Rights
- University of Fort Hare
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UFH Department of Biochemistry & Microbiology
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