Draft genome sequence of Acinetobacter baumannii Ab10, a clinical isolate from Hospital University Sains Malaysia (HUSM), Malaysia.

Acinetobacter baumannii strain Ab10 retrieved in Malaysia in 2017 represents a pathogen carrying multiple antibiotic-resistant genes (blaOXA-23, ant(3")-Ila, blaADC-32, and blaOXA-699). We introduce the 3.89 Mbp genome sequence from short-read sequencing (Illumina's NovaSeq6000).

Phenotypic Characterization and Comparative Genomic Analysis of Novel Salmonella Bacteriophages Isolated from a Tropical Rainforest.

Salmonella infections across the globe are becoming more challenging to control due to the emergence of multidrug-resistant (MDR) strains. Lytic phages may be suitable alternatives for treating these multidrug-resistant Salmonella infections. Most Salmonella phages to date were collected from human-impacted environments. To further explore the Salmonella phage space, and to potentially identify phages with novel characteristics, we characterized Salmonella-specific phages isolated from the Penang National Park, a conserved rainforest. Four phages with a broad lytic spectrum (kills >5 Salmonella serovars) were further characterized; they have isometric heads and cone-shaped tails, and genomes of ~39,900 bp, encoding 49 CDSs. As the genomes share a <95% sequence similarity to known genomes, the phages were classified as a new species within the genus Kayfunavirus. Interestingly, the phages displayed obvious differences in their lytic spectrum and pH stability, despite having a high sequence similarity (~99% ANI). Subsequent analysis revealed that the phages differed in the nucleotide sequence in the tail spike proteins, tail tubular proteins, and portal proteins, suggesting that the SNPs were responsible for their differing phenotypes. Our findings highlight the diversity of novel Salmonella bacteriophages from rainforest regions, which can be explored as an antimicrobial agent against MDR-Salmonella strains.

Prevalence of Multidrug-Resistant and Extended-Spectrum Beta-Lactamase-Producing Shigella Species in Asia: A Systematic Review and Meta-Analysis.

Shigellosis remains one of the leading causes of morbidity and mortality worldwide and is the second leading cause of diarrheal mortality among all age groups. However, the global emergence of antimicrobial-resistant Shigella strains, limiting the choice of effective drugs for shigellosis, has become the major challenge in the treatment of Shigella infections. The aim of this systematic review and meta-analysis was to provide an updated picture of the prevalence of antimicrobial-resistant Shigella species in Asia. A comprehensive and systematic search was performed on three electronic databases (PubMed, ScienceDirect and Scopus), in which 63 eligible studies published between 2010 and 2022 were identified. From our meta-analysis of proportions using a random-effects model, the overall prevalence of Shigella spp. in Asian patients was estimated to be 8.0% (95% CI: 5.5-10.5). The pooled prevalence rates of multidrug-resistant (MDR) and extended-spectrum beta-lactamase (ESBL)-producing Shigella strains were 68.7% (95% CI: 59.9-77.5) and 23.9% (95% CI: 12.9-34.8), respectively. Concerning recommended antimicrobial drugs for Shigella, the prevalence of resistance was highest for ciprofloxacin (29.8%) and azithromycin (29.2%), followed by ceftriaxone (23.8%), in spite of their importance as first- and second-line treatments for shigellosis. In contrast, resistance to carbapenems, such as ertapenem (0.0%), imipenem (0.1%) and meropenem (0.0%), was almost non-existent among the 49 tested antibiotics. The significantly high prevalence estimation suggests that the multidrug-resistant Shigella is a pressing threat to public health worthy of careful and justified interventions. Effective antibiotic treatment strategies, which may lead to better outcomes for the control and treatment of shigellosis in Asia, are essential.

Mass spectrometry-based immunopeptidomics and computational vaccinology strategies for the identification of universal Shigella immunogenic candidates.

Shigella is a Gram-negative bacteria that cause shigellosis. Treatment with antibiotics cannot be sustained to control the bacterial infection due to the risk of antibiotic resistance. Vaccine development against the highly prevalent Shigella serotypes could provide a generous benefit in reducing the occurrence of shigellosis. The present study is aimed to identify the peptides that could be the ideal candidates for the Shigella vaccine development. THP-1 human macrophage cell lines were infected with clinical strains of Shigella flexneri 2a. The bacterial peptides bound on HLA class II molecules of infected THP-1 were analyzed and identified using the immunopeptidomics approach. Following mass spectrometry identification, a total of 14 proteins were predicted by PSORTb, CELLO, and Gneg-mPLoc as outer membrane proteins (OMPs) of Shigella. Of which, 12 OMPs were found to be conserved among Shigella species and had no significance with human proteomes. Outer membrane receptor FepA and TonB-dependent receptor were among the OMPs predicted to possess the high number of immunogenic B- and T-cell epitopes. The epitopes with high antigenicity from FepA and TonB were identified as potential peptide candidates for Shigella vaccine development. The immunoreactivity of the constructed recombinant proteins were determined using the Shigella-infected human and rabbit sera, respectively. Their protective efficacy and immune responses in controlling the Shigella infection will further be investigated in experimental animal models.

Structural Insights into Substrate Binding and Antibiotic Inhibition of Enterobacterial Penicillin-Binding Protein 6.

Shigella sonnei remains the second most common cause of shigellosis in young children and is now increasingly dominant across developing countries. The global emergence of drug resistance has become a main burden in the treatment of S. sonnei infections and ß-lactam antibiotics, such as pivmecillinam and ceftriaxone, are recommended to be used as second-line treatment. They work by inhibiting the biosynthesis of the peptidoglycan layer of bacterial cell walls, in which the final transpeptidation step is facilitated by penicillin-binding proteins (PBPs). In this study, using protein homology modelling, we modelled the structure of PBP6 from S. sonnei and comprehensively examined the molecular interactions between PBP6 and its pentapeptide substrate and two antibiotic inhibitors. The docked complex of S. sonnei PBP6 with pentapeptides showed that the substrate bound to the active site groove of the DD-carboxypeptidase domain, via hydrogen bonding interactions with the residues S79, V80, Q101, G144, D146 and R240, in close proximity to the catalytic nucleophile S36 for the nucleophilic attack. Two residues, R240 and T208, were found to be important in ligand recognition and binding, where they formed strong hydrogen bonds with the substrate and ß-lactams, respectively. Our results provide valuable information on the molecular interactions essential for ligand recognition and catalysis by PBP6. Understanding these interactions will be helpful in the development of effective drugs to treat S. sonnei infections.

Sequence-Specific Electrochemical Genosensor for Rapid Detection of blaOXA-51-like Gene in Acinetobacter baumannii.

Acinetobacter baumannii (A. baumannii) are phenotypically indistinguishable from the Acinetobacter calcoaceticus−A. baumannii (ACB) complex members using routine laboratory methods. Early diagnosis plays an important role in controlling A. baumannii infections and this could be assisted by the development of a rapid, yet sensitive diagnostic test. In this study, we developed an enzyme-based electrochemical genosensor for asymmetric PCR (aPCR) amplicon detection of the blaOXA-51-like gene in A. baumannii. A. baumanniiblaOXA-51-like gene PCR primers were designed, having the reverse primer modified at the 5' end with FAM. A blaOXA-51-like gene sequence-specific biotin labelled capture probe was designed and immobilized using a synthetic oligomer (FAM-labelled) deposited on the working electrode of a streptavidin-modified, screen-printed carbon electrode (SPCE). The zot gene was used as an internal control with biotin and FAM labelled as forward and reverse primers, respectively. The blaOXA-51-like gene was amplified using asymmetric PCR (aPCR) to generate single-stranded amplicons that were detected using the designed SPCE. The amperometric current response was detected with a peroxidase-conjugated, anti-fluorescein antibody. The assay was tested using reference and clinical A. baumannii strains and other nosocomial bacteria. The analytical sensitivity of the assay at the genomic level and bacterial cell level was 0.5 pg/mL (1.443 µA) and 103 CFU/mL, respectively. The assay was 100% specific and sensitive for A. baumannii. Based on accelerated stability performance, the developed genosensor was stable for 1.6 years when stored at 4 °C and up to 28 days at >25 °C. The developed electrochemical genosensor is specific and sensitive and could be useful for rapid, accurate diagnosis of A. baumannii infections even in temperate regions.

Current State of COVID-19 Pandemic in Africa: Lessons for Today and the Future.

This study is a cross-sectional, observational analysis of the COVID-19 pandemic in Africa, to understand the progression of the disease across the continent. Published data on COVID-19 from 20 January 2020 to 21 June 2021 were obtained and analyzed. Case fatality ratios, as well as case growth rates and other indices were computed. On 21 June 2021, a total of 178,210,532 confirmed cases and 3,865,978 deaths had been recorded worldwide. While the Americas recorded the highest number of cases, Southern Africa recorded the majority of African cases. Fatality rate since from 20 February 2020 to 21 June 2021 was highest in the Americas (2.63%) and low in the South Eastern Asia region (1.39%), globally increasing from 2.17% at the end of January to 6.36% in May 2020 and decreasing to a range between 2.14% to 2.30% since January 2021. In Africa, the infection rate per 100,000 persons was up to 3090.18, while deaths per 100,000 and case fatality ratio were as high as 119.64 and 5.72%, respectively, among the 20 most-affected countries. The testing rate per million population was highest in Botswana (512,547.08). Fatality appears to be increasing in some regions of Africa. The rate of infection and fatality in Africa could still likely take an upward turn. Strict control measures are required, considering the continent's weak healthcare systems.

Vitamin D deficiency attenuates endothelial function by reducing antioxidant activity and vascular eNOS expression in the rat microcirculation.

This study examined the effects of vitamin D deficiency on vascular function and tissue oxidative status in the microcirculation; and whether or not these effects can be ameliorated with calcitriol, the active vitamin D metabolite. Three groups (n = 10 each) of male Sprague Dawley rats were fed for 10 weeks with control diet (CR), vitamin D-deficient diet without (DR), or with oral calcitriol supplementation (0.15 µg/kg) for the last four weeks (DSR). After 10 weeks, rats were sacrificed; mesenteric arterial rings were studied using wire myograph. Oxidative stress biomarkers malondialdehyde (MDA) levels and superoxide dismutase (SOD) activity were measured in the mesenteric arterial tissue. Vascular protein expression of endothelial nitric oxide synthase (eNOS) was determined by Western blotting. Acetylcholine-induced endothelium-dependent relaxation of DR was lower than CR. eNOS expression and SOD activity were lower in mesenteric arterial tissue of DR compared to CR. Calcitriol supplementation to DSR did not ameliorate the above parameters; in fact, augmented endothelium-dependent contraction was observed. Serum calcium was higher in DSR compared to CR and DR. In conclusion, vitamin D deficiency impaired microvascular vasodilation, associated with eNOS downregulation and reduced antioxidant activity. Calcitriol supplementation to vitamin D-deficient rats at the dosage used augmented endothelium-dependent contraction, possibly due to hypercalcaemia.

Current and Future Perspectives on Isothermal Nucleic Acid Amplification Technologies for Diagnosing Infections.

Nucleic acid amplification technology (NAAT) has assumed a critical position in disease diagnosis in recent times and contributed significantly to healthcare. Application of these methods has resulted in a more sensitive, accurate and rapid diagnosis of infectious diseases than older traditional methods like culture-based identification. NAAT such as the polymerase chain reaction (PCR) is widely applied but seldom available to resource-limited settings. Isothermal amplification (IA) methods provide a rapid, sensitive, specific, simpler and less expensive procedure for detecting nucleic acid from samples. However, not all of these IA techniques find regular applications in infectious diseases diagnosis. Disease diagnosis and treatment could be improved, and the rapidly increasing problem of antimicrobial resistance reduced, with improvement, adaptation, and application of isothermal amplification methods in clinical settings, especially in developing countries. This review centres on some isothermal techniques that have found documented applications in infectious diseases diagnosis, highlighting their principles, development, strengths, setbacks and imminent potentials for use at points of care.

Reverse vaccinology approach for the identification and characterization of outer membrane proteins of Shigella flexneri as potential cellular- and antibody-dependent vaccine candidates.

PURPOSE: In the developing world, bacillary dysentery is one of the most common communicable diarrheal infections. There are approximately 169 million cases of shigellosis reported worldwide. The disease is transmitted by a group of Gram-negative intracellular enterobacteria known as Shigella flexneri, S. sonnei, S. dysenteriae, and S. boydii. Conventional treatment regimens for Shigella have been less effective due to the development of resistant strains against antibiotics. Therefore, an effective vaccine for the long term control of Shigella transmission is urgently needed. MATERIALS AND METHODS: In this study, a reverse vaccinology approach was employed to identify most conserved and immunogenic outer membrane proteins (OMPs) of S. flexneri 2a. RESULTS: Five OMPs including fepA, ompC, nlpD_1, tolC, and nlpD_2 were identified as potential vaccine candidates. Protein-protein interactions analysis using STRING software (https://string-db.org/) revealed that five of these OMPs may potentially interact with other intracellular proteins which are involved in beta-lactam resistance pathway. B- and T-cell epitopes of the selected OMPs were predicted using BCPred as well as Propred I and Propred (http://crdd.osdd.net/raghava/propred/), respectively. Each of these OMPs contains regions which are capable to induce B- and T-cell immune responses. CONCLUSION: Analysis acquired from this study showed that five selected OMPs have great potential for vaccine development against S. flexneri infection. The predicted immunogenic epitopes can also be used for development of peptide vaccines or multi-epitope vaccines against human shigellosis. Reverse vaccinology is a promising strategy for the discovery of potential vaccine candidates which can be used for future vaccine development against global persistent infections.

Designing and evaluation of an antibody-targeted chimeric recombinant vaccine encoding Shigella flexneri outer membrane antigens.

Shigellosis is one of the most common diseases found in the developing countries, especially those countries that are prone flood. The causative agent for this disease is the Shigella species. This organism is one of the third most common enteropathogens responsible for childhood diarrhea. Since Shigella can survive gastric acidity and is an intracellular pathogen, it becomes difficult to treat. Also, uncontrolled use of antibiotics has led to development of resistant strains which poses a threat to public health. Therefore, there is a need for long term control of Shigella infection which can be achieved by designing a proper and effective vaccine. In this study, emphasis was made on designing a candidate that could elicit both B-cell and T-cell immune response. Hence B- and T-cell epitopes of outer membrane channel protein (OM) and putative lipoprotein (PL) from S. flexneri 2a were computationally predicted using immunoinformatics approach and a chimeric construct (chimeric-OP) containing the immunogenic epitopes selected from OM and PL was designed, cloned and expressed in E. coli system. The immunogenicity of the recombinant chimeric-OP was assessed using Shigella antigen infected rabbit antibody. The result showed that the chimeric-OP was a synthetic peptide candidate suitable for the development of vaccine and immunodiagnostics against Shigella infection.

An impedimetric micro-immunosensing assay to detect Alzheimer's disease biomarker: Aß40.

A miniaturized biosensing platform, based on monoclonal amyloid-beta antibodies (mAßab) that were immobilized on a disc-shaped platinum/iridium (Pt/Ir) microelectrode surface coupled with an impedimetric signal transducer, was developed for the label-free and sensitive detection of amyloid-beta peptide fragment 1-40 (Aß40); a reliable biomarker for early diagnosis of Alzheimer's disease (AD). A Pt/Ir microelectrode was electropolymerized with poly (ortho-phenylenediamine), a conducting free amine-containing aromatic polymer; followed by crosslinking with glutaraldehyde for subsequent coupling of mAßab on the microelectrode surface. This modification strategy efficiently improved the impedimetric detection performance of Aß40 in terms of charge transfer resistance (∼400-fold difference) and normalized impedance magnitude percentage change (∼40% increase) compared with a passive adsorption-based immobilization method. The sensitivity of the micro-immunosensing assay was found to be 1056 kΩ/(pg/mL)/cm2 and the limit of detection was found to be 4.81 pg/mL with a dynamic range of 1-104 pg/mL (R2 = 0.9932). The overall precision of the assay, as measured by relative standard deviation, ranged from 0.84 to 5.15%, demonstrating its reliability and accuracy; while in respect to assay durability and stability, the immobilized mAßab were able to maintain 80% of their binding activity to Aß40 after incubation for 48 h at ambient temperature (25 °C). To validate the practical applicability, the assay was tested using brain tissue lysates prepared from AD-induced rats. Results indicate that the proposed impedimetric micro-immunosensing platform is highly versatile and adaptable for the quantitative detection of other disease-related biomarkers.

Outer membrane proteins analysis of Shigella sonnei and evaluation of their antigenicity in Shigella infected individuals.

Bacillary dysentery caused by infection with Shigella spp. remains as serious and common health problem throughout the world. It is a highly multi drug resistant organism and rarely identified from the patient at the early stage of infection. S. sonnei is the most frequently isolated species causing shigellosis in industrialized countries. The antigenicity of outer membrane protein of this pathogen expressed during human infection has not been identified to date. We have studied the antigenic outer membrane proteins expressed by S. sonnei, with the aim of identifying presence of specific IgA and IgG in human serum against the candidate protein biomarkers. Three antigenic OMPs sized 33.3, 43.8 and 100.3 kDa were uniquely recognized by IgA and IgG from patients with S. sonnei infection, and did not cross-react with sera from patients with other types of infection. The antigenic proteome data generated in this study are a first for OMPs of S. sonnei, and they provide important insights of human immune responses. Furthermore, numerous prime candidate proteins were identified which will aid the development of new diagnostic tools for the detection of S. sonnei.

Enzymatic electrochemical detection of epidemic-causing Vibrio cholerae with a disposable oligonucleotide-modified screen-printed bisensor coupled to a dry-reagent-based nucleic acid amplification assay.

In this study, we developed a nucleic acid-sensing platform in which a simple, dry-reagent-based nucleic acid amplification assay is combined with a portable multiplex electrochemical genosensor. Preparation of an amplification reaction mix targeting multiple DNA regions of interest is greatly simplified because the lyophilized reagents need only be reconstituted with ultrapure water before the DNA sample is added. The presence of single or multiple target DNAs causes the corresponding single-stranded DNA (ssDNA) amplicons to be generated and tagged with a fluorescein label. The fluorescein-labeled ssDNA amplicons are then analyzed using capture probe-modified screen-printed gold electrode bisensors. Enzymatic amplification of the hybridization event is achieved through the catalytic production of electroactive α-naphthol by anti-fluorescein-conjugated alkaline phosphatase. The applicability of this platform as a diagnostic tool is demonstrated with the detection of toxigenic Vibrio cholerae serogroups O1 and O139, which are associated with cholera epidemics and pandemics. The platform showed excellent diagnostic sensitivity and specificity (100%) when challenged with 168 spiked stool samples. The limit of detection was low (10 colony-forming units/ml) for both toxigenic V. cholerae serogroups. A heat stability assay revealed that the dry-reagent amplification reaction mix was stable at temperatures of 4-56 °C, with an estimated shelf life of seven months. The findings of this study highlight the potential of combining a dry-reagent-based nucleic acid amplification assay with an electrochemical genosensor in a more convenient, sensitive, and sequence-specific detection strategy for multiple target nucleic acids.

Temperature-regulated expression of outer membrane proteins in Shigella flexneri.

BACKGROUND: Bacteria exist widely in a diversity of natural environments. In order to survive adverse conditions such as nutrient depletion, biochemical and biological disturbances, and high temperature, bacteria have developed a wide variety of coping mechanisms. Temperature is one of the most important factors that can enhance the expression of microbial proteins. This study was conducted to investigate how outer membrane proteins (OMPs) of the bacterium Shigella flexneri respond to stress, especially during fever when the host's body temperature is elevated. METHODS: OMPs of S. flexneri ATCC 12022 and clinical isolate SH057 were extracted from an overnight culture grown at 37, 38.5, and 40°C. Comparisons of the expressed proteins under the different growth conditions were based on equal numbers of bacterial cells loaded in the SDS-PAGE gels. Separated proteins were stained with Coomassie brilliant blue. Selected proteins showing increased expression at 38.5 and 40°C were characterized by performing MALDI-ToF-ToF. RESULTS: Different degrees of expression were demonstrated for different proteins expressed at 37°C compared to 38.5 and 40°C. The proteins with molecular sizes of 18.4, 25.6, and 57.0 kDa showed increased expression level at increasing temperature and were identified as Dps, WrbA, and PepA, respectively. CONCLUSION: This study revealed that strains of S. flexneri respond at the proteomic level during stress caused by elevated temperature by decreasing the expression of proteins, maintaining the level of important proteins, or enhancing the levels of proteins presumably involved in survival and virulence.

A 9-year study of shigellosis in Northeast Malaysia: Antimicrobial susceptibility and shifting species dominance.

AIMS: In Malaysia, Shigella spp. is the third most common bacterial agent responsible for childhood diarrhoea. This study was conducted to determine the prevalence and antimicrobial susceptibility patterns of Shigella spp. isolated from patients admitted to the Hospital Universiti Sains Malaysia from January 2001 to December 2009. SUBJECTS AND METHODS: A hospital-based retrospective study was used. Stool samples from patients were cultured using a standard culture method. Shigella spp. isolates were identified by biochemical and serological methods, and the antimicrobial susceptibility pattern was evaluated using the Kirby-Bauer disc-diffusion method. RESULTS: A total of 138 Shigella spp. were isolated from a total of 14,830 routine stool specimens, yielding an isolation rate of 0.93% that corresponded to 9.99% of the 1,381 bacterial pathogens isolated. Of these isolates, S. sonnei was the predominant species, followed by S. flexneri and S. boydii. Seasonal variation was noticed, and no significant differences were detected in the demographic data for S. flexneri and S. sonnei. The susceptibility of all isolated Shigella strains was tested against seven antibiotics. Ceftriaxone (99.1%), ciprofloxacin (98.4%), and nalidixic acid (93.8%) were effective against the Shigella strains, whereas tetracycline and trimethoprim-sulfamethoxazole exhibited high frequencies of resistance (58.4% and 53.8%, respectively). CONCLUSION: This study is important for public health education aimed at reducing the morbidity and mortality associated with Shigella spp. infection. Our results also will be helpful for paediatricians and microbiologists in the selection of appropriate antibiotics for the management of diarrhoea.