OBJECTIVES: In this study, the effect of the Piper betle L. extract towards S. mutans (with/without sucrose) using scanning electron microscopy (SEM) and on partially purified cell-associated glucosyltransferase activity were determined.
MATERIAL AND METHODS: S. mutans were allowed to adhere to glass beads suspended in 6 different Brain Heart Infusion broths [without sucrose; with sucrose; without sucrose containing the extract (2 mg mL(-1) and 4 mg mL(-1)); with sucrose containing the extract (2 mg mL(-1) and 4 mg mL(-1))]. Positive control was 0.12% chlorhexidine. The glass beads were later processed for SEM viewing. Cell surface area and appearance and, cell population of S. mutans adhering to the glass beads were determined upon viewing using the SEM. The glucosyltransferase activity (with/without extract) was also determined. One- and two-way ANOVA were used accordingly.
RESULTS: It was found that sucrose increased adherence and cell surface area of S. mutans (p<0.001). S. mutans adhering to 100 µm² glass surfaces (with/without sucrose) exhibited reduced cell surface area, fluffy extracellular appearance and cell population in the presence of the Piper betle L. leaves extract. It was also found that the extract inhibited glucosyltransferase activity and its inhibition at 2.5 mg mL(-1) corresponded to that of 0.12% chlorhexidine. At 4 mg mL(-1) of the extract, the glucosyltransferase activity was undetectable and despite that, bacterial cells still demonstrated adherence capacity.
CONCLUSION: The SEM analysis confirmed the inhibitory effects of the Piper betle L. leaves extract towards cell adherence, cell growth and extracellular polysaccharide formation of S. mutans visually. In bacterial cell adherence, other factors besides glucosyltransferase are involved.
MATERIALS AND METHODS: We studied the samples of the pharyngeal mucosa smears taken from children aged 1-15 years with X-ray confirmed pneumonia. The selection of DNA probes for specific detection of community-acquired pneumonia pathogens (S. pneumoniae, H. influenzae, M. pneumoniae, C. pneumonia, and L. pneumophila) and development of the microarray design were carried out using the disprose program. The nucleotide sequences of pathogens were obtained from NCBI Nucleotide database. In the research we used CustomArray microarrays (USA). For a pooled sample containing S. pneumoniae and H. influenzae DNA, we performed a sequential selection of the best combinations of hybridization parameters: DNA fragment size, DNA amount, hybridization temperature. The selection criteria were: the percentage of effective probes with a standardized hybridization signal (SHS) ≥3 Z, and the excess of SHS levels of effective specific probes compared to SHS of effective nonspecific probes. We selected the probes to detect of S. pneumoniae and H. influenzae characterized by an effective hybridization signal under optimal conditions. The developed microarray was tested under the selected conditions on clinical samples containing S. pneumoniae or H. influenzae DNA. Using ROC analysis there were established threshold values for the signals of specific probes at optimal sensitivity points and the test specificity, the excess of which was interpreted as the evidence of pathogen presence in a sample.
RESULTS: A microarray design included 142 DNA probes to detect S. pneumoniae, H. influenzae, M. pneumoniae, C. pneumoniae, and L. pneumophila, the probes being synthesized onto slides. Using the example of clinical samples containing S. pneumoniae and/or H. influenza DNA, we selected optimal parameters for DNA hybridization on microarrays, which enabled to identify bacterial pathogens of community-acquired pneumonia with sufficient efficiency, specificity and reproducibility: the amount of hybridized DNA was 2 μg, the DNA fragment size: 300 nt, hybridization temperature: 47°C. There was selected a list of probes for specific detection of S. pneumoniae and H. influenzae characterized by an effective hybridization signal under the identified conditions. We determined the threshold values of standardized probe signals for specific detection of S. pneumoniae (4.5 Z) and H. influenzae (4.9 Z) in clinical samples.
CONCLUSION: A DNA microarray was developed and synthesized for parallel indication of bacterial pathogens of community-acquired pneumonia. There were selected the optimal parameters for DNA hybridization on a microarray to identify bacterial pathogens - S. pneumoniae and H. influenzae, and determined the threshold values of significant probe signals for their specific detection. The interpretation of the microarray hybridization results corresponds to those obtained by PCR. The microarray can be used to improve laboratory diagnostics of community-acquired pneumonia pathogens.
METHODOLOGY/PRINCIPAL FINDINGS: We performed a literature review, whole genome sequencing on 145 GBS isolates collected from six Southeast Asian countries, and phylogenetic analysis on 7,468 GBS sequences including 227 variants of ST283 from humans and animals. Although almost absent outside Asia, ST283 was found in all invasive Asian collections analysed, from 1995 to 2017. It accounted for 29/38 (76%) human isolates in Lao PDR, 102/139 (73%) in Thailand, 4/13 (31%) in Vietnam, and 167/739 (23%) in Singapore. ST283 and its variants were found in 62/62 (100%) tilapia from 14 outbreak sites in Malaysia and Vietnam, in seven fish species in Singapore markets, and a diseased frog in China.
CONCLUSIONS: GBS ST283 is widespread in Southeast Asia, where it accounts for a large proportion of bacteraemic GBS, and causes disease and economic loss in aquaculture. If human ST283 is fishborne, as in the Singapore outbreak, then GBS sepsis in Thailand and Lao PDR is predominantly a foodborne disease. However, whether transmission is from aquaculture to humans, or vice versa, or involves an unidentified reservoir remains unknown. Creation of cross-border collaborations in human and animal health are needed to complete the epidemiological picture.