Partial gene sequences of phenylalanyl-tRNA synthase alpha subunit (pheS) and RNA polymerase alpha subunit (rpoA) were evaluated for species delineation and detection of recombination among enterococci populations recovered from a bathing beach impacted by low tide river flow. At inter-species level, a maximum similarity of 86.5% and 94.8% was observed among the enterococci pheS and rpoA sequence, respectively. A superimposed plot of delimited pairwise similarity values obtained for 266 pair-wise observations revealed that while there was a harmony between species identity obtained from both genes, pheS was more discriminatory than rpoA. The difference was more pronounced for inter-species comparison. A number of putative recombination events between indigenous and non-indigenous strains was detected based on a library of aligned sequences. Virulence genes cyl, esp, gelE and asa were detected in 7, 22, 100 and 63%, respectively among river isolates but at lower proportion of 0, 20, 67 and 42%, respectively among beach water isolates. Random amplified polymorphic DNA profiling presented evidence suggesting low tide river as a source of fecal enterococci entering the recreation beach water. Multilocus sequence typing analysis of a number of Enterococcus faecalis isolates presented four sequence types, ST59, 117, 181 and 474. The presence of genetically diverse fecal enterococci with associated virulence traits and a background of recombination events in surface recreational water could present a potential public health risk.
Median enterococci counts of beach water samples gradually increased at statistically significant levels (χ2: 26.53, df: 4; p<0.0001) with increasing proximity to river influx. The difference in proportion of antibiotic resistant enterococci in beach water and river water samples was statistically significant (p<0.05) for the tested antibiotics with river isolates generally presenting higher resistance frequencies. Virulence genes cyl, esp, gelE and asa were detected at varying frequencies (7.32%, 21.95%, 100% and 63.41% respectively) among river isolates. On the other hand, the prevalence of these genes was lower (0%, 20%, 67.27% and 41.82% respectively) among beach water isolates. Multi-Locus-Sequence-Typing analysis of Enterococcus faecalis presented four sequence types (ST) one of which shared six out of seven tested loci with ST6, a member of the clonal complex of multi-drug resistant strains associated with hospital outbreaks.
Bacteriocin-producing Pseudomonas putida strain FStm2 isolated from shark showed broad range of antibacterial activity against all pathogens tested except Bacillus subtilis ATCC11774, MRSA N32064, Proteus mirabilis ATCC12453, Enterococcus faecalis ATCC14506, Salmonella typhimurium ATCC51312, Salmonella mutan ATCC25175, and Aeromonas hydrophila Wbf314. Of the three growth media tested in this study, TSB was observed to support the bacteriocin activity the most. While the highest bacteriocin activity was observed for media supplemented with 1 % NaCl, there was an observed reduction in bacteriocin activity with increasing salt concentration. Although the least bacteriocin activity was observed for marine broth, addition of increasing amounts of tryptone, glucose, or yeast extract increased bacteriocin activity. This was, however, contrary to the effect observed when MgSO4 and MnSO4 were added as supplements. In the presence of α-amylase, lipase, DNase, and RNase, a positive effect on bacteriocin production was observed. Proteinase K strongly inhibited bacteriocin production. Furthermore, the bacteriocins produced were heat stable within the temperature range of 30-70 °C. Bacteriocin activity also was not affected within a wide pH range of 3-9. Exposure to detergents did not inhibit the activity of the bacteriocin at the concentrations tested. Instead, a positive effect on the relative activity of produced bacteriocin was observed as sodium dodecyl sulfate (SDS), EDTA, and Tween 20 at 1 % concentration all improved bacteriocin activity when the cell-free supernatant was tested against Serratia marcescens ATCC 13880. The bacteriocin was purified by ammonium sulfate precipitation and gel filtration on a Superdex-200 column. SDS-PAGE analysis of the partially purified bacteriocin revealed an apparent molecular weight of ~32 kDa.
We report the first study on the occurrence of antibiotic-resistant enterococci in coastal bathing waters in Malaysia. One hundred and sixty-five enterococci isolates recovered from two popular recreational beaches in Malaysia were speciated and screened for antibiotic resistance to a total of eight antibiotics. Prevalence of Enterococcus faecalis and Enterococcus faecium was highest in both beaches. E. faecalis/E. faecium ratio was 0.384:1 and 0.375:1, respectively, for isolates from Port Dickson (PD) and Bagan Lalang (BL). Analysis of Fisher's exact test showed that association of prevalence of E. faecalis and E. faecium with considered locations was not statistically significant (p < 0.05). Chi-square test revealed significant differences (χ(2) = 82.630, df = 20, p < 0.001) in the frequency of occurrence of enterococci isolates from the considered sites. Resistance was highest to nalidixic acid (94.84 %) and least for chloramphenicol (8.38 %). One-way ANOVA using Tukey-Kramer multiple comparison test showed that resistance to ampicillin was higher in PD beach isolates than BL isolates and the difference was extremely statistically significant (p < 0.0001). Frequency of occurrence of multiple antibiotic resistance (MAR) isolates were higher for PD beach water (64.29 %) as compared to BL beach water (13.51 %), while MAR indices ranged between 0.198 and 0.48. The results suggest that samples from Port Dickson may contain MAR bacteria and that this could be due to high-risk faecal contamination from sewage discharge pipes that drain into the sea water.
We report the first study on the occurrence of high-level aminoglycoside-resistant (HLAR) Enterococci in coastal bathing waters and beach sand in Malaysia. None of the encountered isolates were resistant to high levels of gentamicin (500 μg/mL). However, high-level resistance to kanamycin (2,000 μg/mL) was observed in 14.2 % of tested isolates, the highest proportions observed being among beach sand isolates. High-level resistance to kanamycin was higher among Enterococcus faecalis and Enterococcus faecium than Enterococcus spp. Chi-square analysis showed no significant association between responses to tested antibiotics and the species allocation or source of isolation of all tested Enterococci. The species classification of encountered Enterococci resistance to vancomycin was highest among Enterococcus spp. (5.89 %) followed by E. faecium (4.785) and least among E. faecalis. A total of 160 isolates were also tested for virulence characteristics. On the whole, caseinase production was profoundly highest (15.01 %) while the least prevalent virulence characteristic observed among tested beach Enterococci was haemolysis of rabbit blood (3.65 %). A strong association was observed between the source of isolation and responses for each of caseinase (C = 0.47, V = 0.53) and slime (C = 0.50, V = 0.58) assays. Analysis of obtained spearman's coefficient showed a strong correlation between caseinase and each of the slime production (p = 0.04), gelatinase (p = 0.0035) and haemolytic activity on horse blood (p = 0.004), respectively. Suggestively, these are the main virulent characteristics of the studied beach Enterococci. Our findings suggest that recreational beaches may contribute to the dissemination of Enterococci with HLAR and virulence characteristics.
There is currently no established bacteriological beach quality monitoring (BQM) program in place in Malaysia. To initiate cost-effective, sustainable bacteriological BQM schemes for the ultimate goal of protecting public health, policy decision makers need to be provided robust, indigenous empirical findings that validate appropriate water quality parameters for inclusion in such monitoring programs. This is the first study that assesses the validity of enterococci as an ideal indicator for bacteriological BQM in Malaysia using a multivariate approach. Beach water and sand samples from 7 beach locations were analyzed for a total of twenty-one microbial and non-microbial water quality parameters. A multivariate approach incorporating cluster analyses (CA), principal component analyses (PCA), and factor analysis (FA) was also adopted. Apart from the weak correlations of Staphylococcus aureus with concentrations of Vibro species (r = 0.302, p = 0.037) and total coliforms (r = 0.392, p = 0.006) in seawater, no correlation existed between S. aureus concentration and other parameters. Faecal coliforms failed to correlate with any of the tested parameters. Enterococci also correlated with more quality parameters than faecal coliforms or any other indicator. Multiple linear regressions highlighted a significant, best fit model that could predict enterococci concentrations in relation to other parameters with a maximum predictive success of 69.64%. PCA/FA clearly delineated enterococci and faecal coliforms as parameters that weighed strongly for BQM while Staphylococcus aureus, faecal coliforms and enterococci weighed strongly for beach sand quality monitoring. On the whole, higher correlations of enterococci levels with other parameters than was observed for faecal coliforms suggest that the former be considered a preferred parameter of choice for BQM in Malaysia. Our findings provide meaningful evidence particularly as it relates to the correlation of Enterococci with pathogens and other non-microbial parameters. It also provides empirical data to validate the applicability of the enterococci indicator paradigm for bacteriological beach quality monitoring in Malaysia. The current study thus provides policy decision makers evidenced based approach to parameter streamlining for optimized beach sampling and sustainable bacteriological quality monitoring.
Despite the growing demand of tourism in Malaysia, there are no resolute efforts to develop beaches as tourist destinations. With no incentives to monitor public beaches or to use them in a sustainable manner, they might eventually degenerate in quality as a result of influx of pollutants. This calls for concerted action plans with a view to promoting their sustainable use. The success of such plans is inevitably anchored on the availability of robust quality monitoring schemes. Although significant efforts have been channelled to collation and public disclosure of bacteriological quality data of rivers, beach water monitoring appears left out. This partly explains the dearth of published information related to beach water quality data. As part of an on-going nation-wide surveillance study on the bacteriological quality of recreational beaches, this paper draws on a situation analysis with a view to proffering recommendations that could be adapted for ensuring better beach water quality in Malaysia.