First detection of Shiga toxin producing Escherichia coli O157:H7 (HlyA gene) and enumeration of most probable number (MPN) of fecal coliforms and Escherichia coli in cage cultured oysters (Crassostrea iredalei) and water from southern Malaysia

Aims@#Escherichia coli O157:H7 is known to be transmitted via fecal-oral route, where water plays a role in the transmission process. Oysters as bivalves, bio accumulate pathogens from the water through filter feeding and are suspected to play a role as disease transmission vector. In Malaysia, the data on oyster’s microbiological quality are limited. Hence, it was vital to conduct oyster related studies in Malaysia. The main objectives of this study include the enumeration of most probable number (MPN) of fecal coliforms and E. coli and isolation of E. coli from oyster (Crassostrea iredalei) and water sample for the detection of 16S rRNA and HlyA (Hemolysin A) genes of E. coli O157:H7. @*Methodology and results@#A total of 120 oysters and water samples (n=6) were collected from a fisherman village located in southern Malaysia. Total fecal coliforms and E. coli were determined using the MPN procedure. Colonies of E. coli were identified based on Gram staining, biochemical test, and PCR detection for the presence of 16S rRNA and HlyA gene of E. coli O157:H7. The enumeration results showed that the MPN of the fecal coliforms and E. coli found in the collected oyster samples do not meet the standard to be directed for human consumption (0.72 ± 0.19 × 104 MPN/100 g and 0.13 ± 0.03 × 10 4 MPN/100 g, respectively). The PCR assays showed that 16 out of the 104 (15.38%) of E. coli isolated from water and oysters showed the presence of HlyA gene. The phylogenetic tree analysis showed there were genetic relationships between the HlyA gene of the E. coli isolated in this study with the ones isolated from calf and human faeces.@*Conclusion, significance and impact of study@#The detection of Shiga toxin producing E. coli O157:H7 (HlyA gene) in cage cultured oysters (C. iredalei) and water from southern Malaysia was first time reported here. In the future, more study can be conducted to study the expression of the HlyA gene and confirm of its identity as E. coli O157:H7 using different target genes such as eaeA (encodes a 94 kD outer membrane protein called intimin) and Stx1 (Shiga toxin, Shigella dysenteriae type 1).

Molecular identification of antibiotic-resistant bacteria isolated from used contact lens cases

Aims@#The contact lens (CL) has become one of the most convenience refractive devices used in vision correction, occupational and in cosmetics purposes. Despite the convenience of CL usage, poor hygiene might cause eye infections due to microbial contamination. In this work, a random collection of used CL cases among Universiti Malaysia Terengganu (UMT) students had shown the emergence of antibiotic-resistant bacteria towards commonly used antibiotics to treat eye infections.@*Methodology and results@#The study was carried out from 28 CL cases samples with the duration of one to three months of use. Bacteria that were successfully isolated from the CL cases were then exposed to the commonly prescribed antibiotics followed by identification through the partial 16S rDNA sequencing. Our finding exhibited that the rate of contamination is over 50% where 32 bacteria were isolated, with 20 (62.5%) of the isolates were Gram-positive bacteria. Approximately 31% of the isolated bacteria are resistant and intermediate resistant to the commonly used antibiotics to treat eye infection, especially erythromycin and chloramphenicol. The isolated bacteria were genotypic identified as Bacillus cereus, B. anthracis, Acinetobacter variabilis, Klebsiella pneumoniae, and Serratia marcescens. These bacteria are known as a common cause for microbial keratitis, except for A. variabilis, where the association of this bacteria in causing microbial keratitis is relatively rare.@*Conclusion, significance and impact of study@#This study highlights the emergence of antibiotic-resistant bacteria that can cause severe eye infections among CL wearer. The high percentage of contamination (>50%) found from the isolates reflected on the lack of hygiene practice on the CL handling. Thus, it is crucial to perceive this study as microbial contamination will lead to more serious eye infection disease such as conjunctivitis and keratitis.

The implication of amino acid mutations at flavivirus NS1-2A cleavage site on NS1’protein production

Aims: The presence of a C-terminally extended form of NS1 (NS1’ protein) has been previously reported in encephalitic flaviviruses, due to the presence of -1 programmed ribosomal frameshift at the N-terminal of NS2A protein. This present study is aimed to further confirm that the NS1’ protein production is independent of the authentic cleavage at NS1-2A junction. Methodology and results: Six different constructs (P1-Leu, P2-Asp, P3-Phe, P3-Leu, P3-Gly and P5-8 Ala) containing various mutations at conserved and variable amino acids at C-terminal of NS1 protein were generated by site-directed mutagenesis and analysed with transient polyprotein expression assay. While analysis on the NS1-2A cleavage of the mutants exhibited extremely poor to efficient cleavage ranging from 6-89%, significant amount of NS1’ being expressed in all mutants irrespective of their NS1-2A cleavage outcome. Conclusion, significance and impact study: In this analysis, we showed for the first time that the abolishment of the authentic NS1-2A cleavage in Murray Valley encephalitis virus (MVEV) did not impact on NS1’ production. This observation extend on previous studies to show that NS1 and NS2A proteins are the product of NS1-2A cleavage which is catalysed by an unknown host protease while NS1’ protein is a product of ribosomal frameshift, independent of the authentic cleavage at NS1-2A junction.