Comparative genomic analysis of ten clinical Streptococcus pneumoniae collected from a Malaysian hospital reveal 31 new unique drug-resistant SNPs using whole genome sequencing.

BACKGROUND: Streptococcus pneumoniae or pneumococcus is a leading cause of morbidity and mortality worldwide, specifically in relation to community-acquired pneumonia. Due to the overuse of antibiotics, S. pneumoniae has developed a high degree of resistance to a wide range of antibacterial drugs. METHODS: In this study, whole genome sequencing (WGS) was performed for 10 clinical strains of S. pneumoniae with different levels of sensitivity to standard antibiotics. The main objective was to investigate genetic changes associated with antibiotic resistance in S. pneumoniae. RESULTS: Our results showed that resistant isolates contain a higher number of non-synonymous single nucleotide polymorphisms (SNPs) as compared to susceptible isolates. We were able to identify SNPs that alter a single amino acid in many genes involved in virulence and capsular polysaccharide synthesis. In addition, 90 SNPs were only presented in the resistant isolates, and 31 SNPs were unique and had not been previously reported, suggesting that these unique SNPs could play a key role in altering the level of resistance to different antibiotics. CONCLUSION: Whole genome sequencing is a powerful tool for comparing the full genome of multiple isolates, especially those closely related, and for analysing the variations found within antibiotic resistance genes that lead to differences in antibiotic sensitivity. We were able to identify specific mutations within virulence genes related to resistant isolates. These findings could provide insights into understanding the role of single nucleotide mutants in conferring drug resistance.

Anti-infective activities of 11 plants species used in traditional medicine in Malaysia.

Treatment of drug resistant protozoa, bacteria, and viruses requires new drugs with alternative chemotypes. Such compounds could be found from Southeast Asian medicinal plants. The present study examines the cytotoxic, antileishmanial, and antiplasmodial effects of 11 ethnopharmacologically important plant species in Malaysia. Chloroform extracts were tested for their toxicity against MRC-5 cells and Leishmania donovani by MTT, and chloroquine-resistant Plasmodium falciparum K1 strain by Histidine-Rich Protein II ELISA assays. None of the extract tested was cytotoxic to MRC-5 cells. Extracts of Uvaria grandiflora, Chilocarpus costatus, Tabernaemontana peduncularis, and Leuconotis eugenifolius had good activities against L. donovani with IC50 < 50 µg/mL. Extracts of U. grandiflora, C. costatus, T. peduncularis, L. eugenifolius, A. subulatum, and C. aeruginosa had good activities against P. falciparum K1 with IC50 < 10 µg/mL. Pinoresinol isolated from C. costatus was inactive against L. donovani and P. falciparum. C. costatus extract and pinoresinol increased the sensitivity of Staphylococcus epidermidis to cefotaxime. Pinoresinol demonstrated moderate activity against influenza virus (IC50 = 30.4 ±â€¯11 µg/mL) and was active against Coxsackie virus B3 (IC50 = 7.1 ±â€¯3.0 µg/mL). ß-Amyrin from L. eugenifolius inhibited L. donovani with IC50 value of 15.4 ±â€¯0.01 µM. Furanodienone from C. aeruginosa inhibited L. donovani and P. falciparum K1 with IC50 value of 39.5 ±â€¯0.2 and 17.0 ±â€¯0.05 µM, respectively. Furanodienone also inhibited the replication of influenza and Coxsackie virus B3 with IC50 value of 4.0 ±â€¯0.5 and 7.2 ±â€¯1.4 µg/mL (Ribavirin: IC50: 15.6 ±â€¯2.0 µg/mL), respectively. Our study provides evidence that medicinal plants in Malaysia have potentials as a source of chemotypes for the development of anti-infective leads.

Demonstrating the utility of Escherichia coli asymptomatic bacteriuria isolates' virulence profile towards diagnosis and management-A preliminary analysis.

Asymptomatic bacteriuria (ASB) caused by Escherichia coli (E. coli) is a significant condition associated with pregnancy and is considered as prognostic for the development of symptomatic urinary tract infection (UTI). However, treating all ASB increases the use of antibiotics and leads to the development of multidrug resistance (MDR). Therefore, this study aimed to identify the distribution of UPEC associated virulence genes and antibiotic susceptibility among phylogroups of E. coli isolated from ASB in pregnancy. Moreover, the gene expression of selected virulence genes was also compared among two E. coli isolates (with different pathogenic potential) to determine its pathogenicity. One hundred and sixty E. coli isolates from midstream urine samples of pregnant women with ASB were subjected to PCR-based detection for its phylogroups and virulence genes. The antibiotic susceptibility of isolated strains was determined by the disc diffusion method. Expression of the virulence genes were determined through microarray analysis and quantitative Real-Time PCR. The prevalence of ASB in this study was 16.1%. Within ASB isolates, the occurrence of phylogroup B2 was the highest, and isolates from this group harboured most of the virulence genes studied. Overall, the most identified virulence genes among all phylogroups in descending order were fimH, chuA, kpsMTII, usp, fyuA, hlyA, iroN, cnf, papC, sfa, ompT, and sat. In this study, higher resistance to antibiotics was observed for ampicillin (77.5%), amoxicillin-clavulanate (54.4%), trimethoprim-sulfamethoxazole (46.9%) and amikacin (43.8%) compared to the other tested antibiotics and 51.9% of the tested isolates were MDR. Furthermore, hierarchical clustering and gene expression analysis demonstrated extreme polarization of pathogenic potential of E. coli causing ASB in pregnancy necessitating the need for bacterial isolate focused approach towards treatment of ASB.

Elastomeric biocomposite of silver-containing mesoporous bioactive glass and poly(1,8-octanediol citrate): Physiochemistry and in vitro antibacterial capacity in tissue engineering applications.

A novel series of silver-doped mesoporous bioactive glass/poly(1,8-octanediol citrate) (AgMBG/POC) elastomeric biocomposite scaffolds were successfully constructed by a salt-leaching technique for the first time and the effect of inclusion of different AgMBG contents (5, 10, and 20 wt%) on physicochemical and biological properties of pure POC elastomer was evaluated. Results indicated that AgMBG particles were uniformly dispersed in the POC matrix and increasing the AgMBG concentration into POC matrix up to 20 wt% enhanced thermal behaviour, mechanical properties and water uptake ability of the composite scaffolds compared to those from POC. The 20%AgMBG/POC additionally showed higher degradation rate in Tris(hydroxymethyl)-aminomethane-HCl (Tris-HCl) compared with pure POC and lost about 26% of its initial weight after soaking for 28 days. The AgMBG phase incorporation also significantly endowed the resulting composite scaffolds with efficient antibacterial properties against Escherichia coli and Staphylococcus aureus bacteria while preserving their favorable biocompatibility with soft tissue cells (i.e., human dermal fibroblast cells). Taken together, our results suggest that the synergistic effect of both AgMBG and POC make these newly designed AgMBG/POC composite scaffold an attractive candidate for soft tissue engineering applications.

Potency and Cytotoxicity of a Novel Gallium-Containing Mesoporous Bioactive Glass/Chitosan Composite Scaffold as Hemostatic Agents.

Chitosan-based hemostats are promising candidates for immediate hemorrhage control. However, they have some disadvantages and require further improvement to achieve the desired hemostatic efficiency. Here, a series of 1% Ga2O3-containing mesoporous bioactive glass-chitosan composite scaffolds (Ga-MBG/CHT) were constructed by the lyophilization process and the effect of various concentrations of Ga-MBG (10, 30, and 50 wt %) on the hemostatic function of the CHT scaffold was assessed as compared to that of Celox Rapid gauze (CXR), a current commercially available chitosan-coated hemostatic gauze. The prepared scaffolds exhibited >79% porosity and showed increased water uptake compared to that in CXR. The results of coagulation studies showed that pure CHT and composite scaffolds exhibited increased hemostatic performance with respect to CXR. Furthermore, the composite scaffold with the highest Ga-MBG content (50 wt %) had increased capability to enhancing thrombus generation, blood clotting, and platelet adhesion and aggregation than that of the scaffold made of pure CHT. The antibacterial efficacy and biocompatibility of the prepared scaffolds were also assessed by a time-killing assay and an Alamar Blue assay, respectively. Our results show that the antibacterial effect of 50% Ga-MBG/CHT was more pronounced than that of CHT and CXR. The cell viability results also demonstrated that Ga-MBG/CHT composite scaffolds had good biocompatibility, which facilitates the spreading and proliferation of human dermal fibroblast cells even with 50 wt % Ga-MBG loading. These results suggest that Ga-MBG/CHT scaffolds could be a promising hemostatic candidate for improving hemostasis in critical situations.

Next generation sequencing reveals the antibiotic resistant variants in the genome of Pseudomonas aeruginosa.

Rapid progress in next generation sequencing and allied computational tools have aided in identification of single nucleotide variants in genomes of several organisms. In the present study, we have investigated single nucleotide polymorphism (SNP) in ten multi-antibiotic resistant Pseudomonas aeruginosa clinical isolates. All the draft genomes were submitted to Rapid Annotations using Subsystems Technology (RAST) web server and the predicted protein sequences were used for comparison. Non-synonymous single nucleotide polymorphism (nsSNP) found in the clinical isolates compared to the reference genome (PAO1), and the comparison of nsSNPs between antibiotic resistant and susceptible clinical isolates revealed insights into the genome variation. These nsSNPs identified in the multi-drug resistant clinical isolates were found to be altering a single amino acid in several antibiotic resistant genes. We found mutations in genes encoding efflux pump systems, cell wall, DNA replication and genes involved in repair mechanism. In addition, nucleotide deletions in the genome and mutations leading to generation of stop codons were also observed in the antibiotic resistant clinical isolates. Next generation sequencing is a powerful tool to compare the whole genomes and analyse the single base pair variations found within the antibiotic resistant genes. We identified specific mutations within antibiotic resistant genes compared to the susceptible strain of the same bacterial species and these findings may provide insights to understand the role of single nucleotide variants in antibiotic resistance.

Mechanisms of action and in vivo antibacterial efficacy assessment of five novel hybrid peptides derived from Indolicidin and Ranalexin against Streptococcus pneumoniae.

BACKGROUND: Antimicrobial peptides (AMPs) are of great potential as novel antibiotics for the treatment of broad spectrum of pathogenic microorganisms including resistant bacteria. In this study, the mechanisms of action and the therapeutic efficacy of the hybrid peptides were examined. METHODS: TEM, SEM and ATP efflux assay were used to evaluate the effect of hybrid peptides on the integrity of the pneumococcal cell wall/membrane. DNA retardation assay was assessed to measure the impact of hybrid peptides on the migration of genomic DNA through the agarose gel. In vitro synergistic effect was checked using the chequerboard assay. ICR male mice were used to evaluate the in vivo toxicity and antibacterial activity of the hybrid peptides in a standalone form and in combination with ceftriaxone. RESULTS: The results obtained from TEM and SEM indicated that the hybrid peptides caused significant morphological alterations in Streptococcus pneumoniae and disrupting the integrity of the cell wall/membrane. The rapid release of ATP from pneumococcal cells after one hour of incubation proposing that the antibacterial action for the hybrid peptides is based on membrane permeabilization and damage. The DNA retardation assay revealed that at 62.5 µg/ml all the hybrid peptides were capable of binding and preventing the pneumococcal genomic DNA from migrating through the agarose gel. In vitro synergy was observed when pneumococcal cells treated with combinations of hybrid peptides with each other and with conventional drugs erythromycin and ceftriaxone. The in vivo therapeutic efficacy results revealed that the hybrid peptide RN7-IN8 at 20 mg/kg could improve the survival rate of pneumococcal bacteremia infected mice, as 50% of the infected mice survived up to seven days post-infection. In vivo antibacterial efficacy of the hybrid peptide RN7-IN8 was signficantly improved when combined with the standard antibiotic ceftriaxone at (20 mg/kg + 20 mg/kg) as 100% of the infected mice survived up to seven days post-infection. DISCUSSION: Our results suggest that attacking and breaching the cell wall/membrane is most probably the principal mechanism for the hybrid peptides. In addition, the hybrid peptides could possess another mechanism of action by inhibiting intracellular functions such as DNA synthesis. AMPs could play a great role in combating antibiotic resistance as they can reduce the therapeutic concentrations of standard drugs.

Antimicrobial Activity of Novel Synthetic Peptides Derived from Indolicidin and Ranalexin against Streptococcus pneumoniae.

Antimicrobial peptides (AMPs) represent promising alternatives to conventional antibiotics in order to defeat multidrug-resistant bacteria such as Streptococcus pneumoniae. In this study, thirteen antimicrobial peptides were designed based on two natural peptides indolicidin and ranalexin. Our results revealed that four hybrid peptides RN7-IN10, RN7-IN9, RN7-IN8, and RN7-IN6 possess potent antibacterial activity against 30 pneumococcal clinical isolates (MIC 7.81-15.62µg/ml). These four hybrid peptides also showed broad spectrum antibacterial activity (7.81µg/ml) against S. aureus, methicillin resistant S. aureus (MRSA), and E. coli. Furthermore, the time killing assay results showed that the hybrid peptides were able to eliminate S. pneumoniae within less than one hour which is faster than the standard drugs erythromycin and ceftriaxone. The cytotoxic effects of peptides were tested against human erythrocytes, WRL-68 normal liver cell line, and NL-20 normal lung cell line. The results revealed that none of the thirteen peptides have cytotoxic or hemolytic effects at their MIC values. The in silico molecular docking study was carried out to investigate the binding properties of peptides with three pneumococcal virulent targets by Autodock Vina. RN7IN6 showed a strong affinity to target proteins; autolysin, pneumolysin, and pneumococcal surface protein A (PspA) based on rigid docking studies. Our results suggest that the hybrid peptides could be suitable candidates for antibacterial drug development.