Prevalence, antibiotic resistance trends, virulence and effect of some medicinal plants on Staphylococcus froms school children in the Mariental district, Namibia

A dissertation submitted in fulfillment of the requirements for the Degree of Doctor of Philosophy (Microbiology)

The main aim of this study was to investigate potentially pathogenic community-associated staphylococci in school children. Objectives were: To determine the prevalence of nasal Staphylococcus aureus and coagulase-negative staphylococci (CoNS) in school children from the Mariental District; to characterize the bacteria in terms of their antibiograms and drug resistance patterns; to screen bacterial isolates for their ability to produce enterotoxins and produce biofilms as potential virulence factors; and to assess the antimicrobial and anti-biofilm activity of crude methanolic extracts of Aptosimum albomarginatum (Marloth and Engl.) roots, Albizia anthelmintica (A. Rich Brongn.) twigs and Dicoma schinzii (O. Hoffm.) against staphylococci (including multi-drug resistant strains) isolated from the learners. To our knowledge, this is the first study undertaken to report on the prevalence, antibiotic resistance trends and virulence characteristics of potentially pathogenic staphylococci among school children in the Mariental District. This was a cross-sectional study involving five schools. With informed consent from parents/guardians, nasal specimens (swabs) were obtained from 272 randomly selected learners aged 6-14 years. Specimens from swabs were enriched for Staphylococcus in brain heart infusion broth prior to isolation on Staphylococcus medium no. 110 and tryptone soy agar. Pure cultures were obtained from mixed cultures, Gram-stained and biochemically tested for identification of S. aureus and CoNS. Methicillin-resistant Staphylococcus aureus (MRSA) and methicillin-resistant coagulase-negative staphylococci (MRCoNS) were identified by their resistance towards cefoxitin using Kirby-Bauer disk diffusion assay. By disk diffusion assay, 352 S. aureus and 81 coagulase-negative staphylococcal isolates underwent susceptibility testing against the antibiotics ampicillin, cefoxitin, ciprofloxacin, erythromycin, gentamicin, rifampicin and tetracycline. The American Type Culture Collection reference strains S. aureus ATCC 25923 and S. aureus ATCC 33591 were used for quality control. Isolates were classified as multi-drug resistant when they displayed resistance towards three or more classes of antibiotics. Twenty-two multi-drug resistant MRSA nasal isolates and the multi-drug resistant MRSA reference strain S. aureus ATCC 33591 were screened for production of enterotoxins A-D, using a SET-RPLA toxin detection kit. The microtiter plate assay was employed to determine biofilm production in 10 nasal S. aureus isolates (including one MRSA isolate), as well as S. aureus ATCC 25923 and S. aureus ATCC 33591. Crude methanolic plant extracts were prepared by maceration, filtration, rotary evaporation and freeze-drying. Qualitative chemical assays and thin layer chromatography (TLC) were used to screen for flavonoids, saponins and anthraquinones in the plant material. To test for antimicrobial activity of the crude methanolic extracts, disk diffusion assays were used. The antibiotics gentamicin and chloramphenicol were used as positive controls, and discs flooded with dimethyl sulfoxide (DMSO) as negative control. A microtiter plate assay determined if extracts could inhibit and/or eradicate S. aureus and MRSA biofilms. Four hundred and thirty-three isolates from 272 swabs were morphologically and biochemically identified as Staphylococcus bacteria. Of these isolates, 352 (81.3%) were S. aureus, while 81 (18.7%) were CoNS. Furthermore, 51/433 (11.8%) isolates were MRSA and 7/433 (1.6%) MRCoNS. The overall prevalence of S. aureus in the study population of 272 learners was 80.5%, and that of CoNS was 25.0%. Methicillin-resistant S. aureus was isolated from 48/272 (17.6%) learners and MRCoNS in only 7/272 (2.6%) learners. From the study participants who reported getting nosebleeds, 75.4% were colonized with S. aureus, whereas 81.4% who were exposed to cigarette smoke from a household member carried this bacterium. Out of 433 staphylococcal isolates, 96.0% S. aureus and 66.7% CoNS were resistant to ampicillin, with this resistance being significantly higher in S. aureus (P < 0.0001). Ciprofloxacin and gentamicin were most effective against S. aureus isolates, with 99.7% and 93.2% of isolates that were susceptible to these drugs, respectively. Ciprofloxacin was also the most effective drug against isolates of CoNS, with 100.0% susceptibility. Of 352 S. aureus isolates, 51 (14.5%) were cefoxitin/methicillin-resistant. Only seven (8.6%) of 81 CoNS isolates were cefoxitin/methicillin-resistant. In total 31 antibiotic resistance patterns were observed, 27 for S. aureus and 14 for CoNS. Overall, the three most frequently observed patterns were AP, AP-E, and AP-T. Of all isolates, 12.5% were multi-drug resistant. These include 50 isolates of S. aureus and four CoNS. From the 51 MRSA isolates, 43.1% were multi-drug resistant. One of these MRSA isolates showed resistance towards 6/7 antibiotics tested with only ciprofloxacin that was effective against it. Methicillin-resistant CoNS was not multi-drug resistant, with the most common resistance pattern being AP-RP-FOX. Twenty-three multi-drug resistant MRSA isolates were screened for enterotoxins A-D. Of these, seven were enterotoxigenic. Enterotoxin A was the most prevalent, produced by five isolates. Enterotoxin B was found in one isolate, while enterotoxin C was produced by two isolates. One isolate tested positive for both enterotoxins B and C. Enterotoxin D was not detected in the isolates screened. All 12 S. aureus isolates, including two MRSA strains, which were evaluated for biofilm formation were strong biofilm formers in microtiter plates. Aptosimum albomarginatum root extract was a moderately active antimicrobial agent against 7/12 S. aureus isolates, including two MRSA isolates. Moderate antimicrobial activity was also observed with this extract in 9/54 multi-drug resistant isolates, of which two were MRSA with the same antibiotic resistance pattern of AP-T-RP-FOX. These two isolates may therefore be the same S. aureus strain. The root extract was the best biofilm inhibition agent, with highly active inhibition (86.0%) observed in S. aureus ATCC 33591 (MRSA), and moderate activity in four other isolates. This extract eradicated the biofilm of S. aureus isolate S110 S73 Pure by 40.0% (moderate activity). Flavonoids and saponins/triterpenes may contribute to the root’s antimicrobial and anti-biofilm properties. Root extract from D. schinzii displayed moderate antimicrobial activity against 8/12 S. aureus isolates, while the plant’s leaf extract was moderately active against 2/12 S. aureus isolates. The leaf extract also moderately inhibited biofilms in three isolates. Flavonoids, coumarins, saponins or triterpenes in the leaves may contribute to its antimicrobial and anti-biofilm properties. In conclusion, the high prevalence of S. aureus and presence of MRSA (including multi-drug resistant bacteria) among study participants calls for improvement in current hygiene practices at schools in the Mariental District to prevent staphylococcal disease. Nosebleeds and exposure to cigarette smoke were identified as possible risk factors for colonization with S. aureus in the children. Overall, multi-drug resistance in our study was relatively low. However, almost half of the MRSA isolates were multi-drug resistant, which is of concern. Results from toxin screening indicated that multi-drug resistant MRSA may produce enterotoxins, whereas S. aureus and MRSA can produce strong biofilms. Self-infection by these bacteria poses various health risks for the children. Nevertheless, learners should be encouraged to frequently wash their hands to prevent spread of antibiotic-resistant bacteria within the community. Ciprofloxacin and gentamicin may effectively be used to treat staphylococcal infections in this study population. Antimicrobial activity was observed with A. albomarginatum roots and D. schinzii roots and leaves, indicating potential use against staphylococcal infections. Noteworthy is this activity of A. albomarginatum root extract against multi-drug resistant strains, including MRSA. The root extract was the best anti-biofilm agent against S. aureus. It was highly active in inhibiting biofilm formation in one MRSA isolate, and moderately active in inhibiting formation in four isolates. The extract moderately eradicated the biofilm in one isolate. This activity may to some extent be attributed to the presence of the secondary metabolites flavonoids, saponins or triterpenes in the roots. The D. schinzii leaf extract moderately inhibited biofilms in three isolates. Flavonoids, coumarins, saponins or triterpenes may play a role its anti-biofilm activity. The present work thus supports the traditional medicinal use of A. albomarginatum roots and D. schinzii roots and leaves as natural antimicrobial agents, and A. albomarginatum roots and D. schinzii leaves as anti-biofilm agents, in infections involving the bacteria under study.