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.
A sacred ritual well with continuously discharging of methane gas through its water body was studied for physicochemical and microbiological quality in three seasons and during ritual mass bathing. Most of the physicochemical parameters showed significant seasonal variations (P<0.05) and a sharp fluctuation during mass bathing. Dissolved oxygen (DO) was found negatively correlated with temperature (r=-0.384, P<0.05), biochemical oxygen demand (BOD) (r=-0.58, P<0.001) and ammonia (r=-0.738, P<0.001), while BOD showed positive correlation with chemical oxygen demand (COD) (r=0.762, P<0.001) and ammonia (r=0.83, P<0.001). Simple regression analysis also yielded significant linear relationship in DO vs. temperature (r(2)=0.147, P<0.05), DO vs. ammonia (r(2)=0.544, P<0.001) and BOD vs. DO (r(2)=0.336, P<0.001). A total of eight microbial indicators were studied and found that all of them increased unusually during mass bathing comparing with their respective seasonal values. Total coliforms (TC) were found positively correlated with fecal coliforms (FC) (r=0.971), FC with Escherichia coli (EC) (r=0.952), EC with intestinal enterococci (IE) (r=0.921), fecal streptococci (FS) with IE (r=0.953) and Staphylococcus aureus (SA) with Pseudomonas aeruginosa (PA) (r=0.946), which were significant at P<0.001. Some regression models showed significant linear relationship at P<0.001 with r(2) value of 0.943 for FC vs. TC, 0.907 for EC vs. FC, 0.869 for FS vs. FC, 0.848 for IE vs. EC and 0.909 for IE vs. FS. The overall results found in this study revealed that well water is suitable for bathing purpose but the religious activity considerably worsen its quality.
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.