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

Harikrishnan H, Ismail A, Banga Singh KK

Gut Pathog, 2013;5(1):38.
PMID: 24330657 DOI: 10.1186/1757-4749-5-38

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

Harikrishnan H, Banga Singh KK, Ismail A

PLoS One, 2017;12(8):e0182878.
PMID: 28846684 DOI: 10.1371/journal.pone.0182878

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

Obande GA, Banga Singh KK

Infect Drug Resist, 2020;13:455-483.
PMID: 32104017 DOI: 10.2147/IDR.S217571

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.
Fulltext A pentaplex PCR assay for the detection and differentiation of Shigella species

Ojha SC, Yean Yean C, Ismail A, Singh KK

Biomed Res Int, 2013;2013:412370.
PMID: 23509722 DOI: 10.1155/2013/412370

The magnitude of shigellosis in developing countries is largely unknown because an affordable detection method is not available. Current laboratory diagnosis of Shigella spp. is laborious and time consuming and has low sensitivity. Hence, in the present study, a molecular-based diagnostic assay which amplifies simultaneously four specific genes to identify invC for Shigella genus, rfc for S. flexneri, wbgZ for S. sonnei, and rfpB for S. dysenteriae, as well as one internal control (ompA) gene, was developed in a single reaction to detect and differentiate Shigella spp. Validation with 120 Shigella strains and 37 non-Shigella strains yielded 100% specificity. The sensitivity of the PCR was 100 pg of genomic DNA, 5.4 × 10(4) CFU/ml, or approximately 120 CFU per reaction mixture of bacteria. The sensitivity of the pentaplex PCR assay was further improved following preincubation of the stool samples in gram-negative broth. A preliminary study with 30 diarrhoeal specimens resulted in no cross-reaction with other non-Shigella strains tested. We conclude that the developed pentaplex PCR assay is robust and can provide information about the four target genes that are essential for the identification of the Shigella genus and the three Shigella species responsible for the majority of shigellosis cases.
A 9-year study of shigellosis in Northeast Malaysia: Antimicrobial susceptibility and shifting species dominance

Banga Singh KK, Ojha SC, Deris ZZ, Rahman RA

Z Gesundh Wiss, 2011 Jun;19(3):231-236.
PMID: 21654922

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.
Demonstration of an outer membrane protein that is antigenically specific for Acinetobacter baumannii

Islam AH, Singh KK, Ismail A

Diagn Microbiol Infect Dis, 2011 Jan;69(1):38-44.
PMID: 21146712 DOI: 10.1016/j.diagmicrobio.2010.09.008

Acinetobacter baumannii is an emerging nosocomial pathogen that is resistant to many types of antibiotics, and hence, a fast, sensitive, specific, and economical test for its rapid diagnosis is needed. Development of such a test requires a specific antigen, and outer membrane proteins (OMPs) are the prime candidates. The goal of this study was to find a specific OMP of A. baumannii and demonstrate the presence of specific IgM, IgA, and IgG against the candidate protein in human serum. OMPs of A. baumannii ATCC 19606 and 16 other clinical isolates of A. baumannii were extracted from an overnight culture grown at 37 °C. Protein profiles were obtained using sodium dodecyl sulfate polyacrylamide gel electrophoresis, and Western blot analysis was performed to detect the presence of IgM, IgA, and IgG against the OMP in host serum. An antigenic 34.4-kDa OMP was uniquely recognized by IgM, IgA, and IgG from patients with A. baumannii infection, and it did not cross-react with sera from patients with other types of infection. The band was also found in the other 16 A. baumannii isolates. This 34.4-kDa OMP is a prime candidate for development of a diagnostic test for the presence of A. baumannii.
Fulltext Wound contraction effects and antibacterial properties of Tualang honey on full-thickness burn wounds in rats in comparison to hydrofibre

Khoo YT, Halim AS, Singh KK, Mohamad NA

BMC Complement Altern Med, 2010;10:48.
PMID: 20815896 DOI: 10.1186/1472-6882-10-48

Full-thickness burn wounds require excision and skin grafting. Multiple surgical procedures are inevitable in managing moderate to severe full-thickness burns. Wound bed preparations prior to surgery are necessary in order to prevent wound infection and promote wound healing. Honey can be used to treat burn wounds. However, not all the honey is the same. This study aims to evaluate the wound contraction and antibacterial properties of locally-produced Tualang honey on managing full-thickness burn wounds in vivo.
Fulltext Long-term trends in the epidemiology of human enteropathogens in Malaysia

Shunmugam P, Kanapathy S, Chan SE, Singh KK

Braz J Infect Dis, 2012 Nov-Dec;16(6):603-4.
PMID: 23158263 DOI: 10.1016/j.bjid.2012.07.005
Fulltext Serum neopterin is elevated in patients infected with Shigella

Singh KK, Wan-Nurfahizul-Izzati W, Ismail A

Gut Pathog, 2010;2(1):9.
PMID: 20727206 DOI: 10.1186/1757-4749-2-9

Neopterin is produced by human macrophages/monocytes when stimulated with interferon-gamma. Production of neopterin is found in serum, cerebrospinal fluid (CSF) and urine of patients with infections by viruses, intracellular bacteria and parasites, autoimmune diseases, malignant tumors and patients in allograft rejection episodes.
Fulltext Evaluations of bacterial contaminated full thickness burn wound healing in Sprague Dawley rats Treated with Tualang honey

Sukur SM, Halim AS, Singh KK

Indian J Plast Surg, 2011 Jan;44(1):112-7.
PMID: 21713196 DOI: 10.4103/0970-0358.81459

AIM: The effect of Tualang honey on wound healing in bacterial contaminated full-thickness burn wounds was evaluated in 36 male Sprague Dawley rats.
MATERIALS AND METHODS: The rats were randomly divided into three groups (n = 12/group). Three full-thickness burn wounds were created on each rat. Each group of rats was inoculated with a different organism in the burn wounds: Group A was inoculated with Pseudomonas aeruginosa, Group B was inoculated with Klebsiella pneumoniae and Group C was inoculated with Acinetobacter baumannii. One wound on each rat was dressed with either Tualang honey, Chitosan gel or Hydrofibre silver. Each wound size was measured on day 3, 6, 9, 12, 15, 18 and 21 of the study.
RESULTS: The mean wound size of the Tualang honey-treated wounds was not statistically different than that of the Chitosan gel or Hydrofibre silver-treated wounds when the wounds were compared throughout the entire experiment (P > 0.05). However, comparing the mean wound size on day 21 alone revealed that the Tualang honey-treated wounds were smaller in comparison to that of the Chitosan gel and Hydrofibre silver-treated groups.
CONCLUSIONS: This study shows that topical application of Tualang honey on burn wounds contaminated with P. aeruginosa and A. baumannii gave the fastest rate of healing compared with other treatments.
Fulltext Draft genome sequence of Acinetobacter baumannii Ab10, a clinical isolate from Hospital University Sains Malaysia (HUSM), Malaysia

Nik Mohd Nazri NZH, Banga Singh KK, Ismail MI

Microbiol Resour Announc, 2024 Apr 11;13(4):e0108423.
PMID: 38501781 DOI: 10.1128/mra.01084-23

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).
Fulltext Antibacterial properties of tualang honey and its effect in burn wound management: a comparative study

Nasir NA, Halim AS, Singh KK, Dorai AA, Haneef MN

BMC Complement Altern Med, 2010;10:31.
PMID: 20576085 DOI: 10.1186/1472-6882-10-31

The use of honey as a natural product of Apis spp. for burn treatment has been widely applied for centuries. Tualang honey has been reported to have antibacterial properties against various microorganisms, including those from burn-related diagnoses, and is cheaper and easier to be absorbed by Aquacel dressing. The aim of this study is to evaluate the potential antibacterial properties of tualang honey dressing and to determine its effectiveness as a partial thickness burn wound dressing.
Comparative evaluation of five culture media with triplex PCR assay for detection of methicillin-resistant Staphylococcus aureus

Al-Talib H, Yean CY, Al-khateeb A, Singh KK, Hasan H, Al-Jashamy K, et al.

Curr Microbiol, 2010 Jul;61(1):1-6.
PMID: 20033170 DOI: 10.1007/s00284-009-9567-8

The emergence of methicillin-resistant Staphylococcus aureus (MRSA) is responsible for nosocomial and community-acquired infections. Hence, rapid and accurate laboratory diagnosis of MRSA is a vital constituent of control measures. The present study evaluated five different methods for the identification of MRSA. A total of 207 S. aureus clinical isolates that consisted of 89 MRSA and 118 methicillin-susceptible S. aureus (MSSA) strains confirmed by PCR were tested. MRSA strains were evaluated by five different methods: chromogenic MRSA agar (CMRSA), oxacillin resistance screening agar base (ORSAB), mannitol salt oxacillin agar (MSO), mannitol salt cefoxitin agar with two different concentrations of cefoxitin [4 microg/ml (MSC-4) and 6 microg/ml (MSC-6)]. The results of the different methods were compared to mecA PCR as the gold standard. MSC-6 showed only six false-positive MRSA in comparison with PCR. The sensitivities and specificities of MSC-6, MSC-4, MSO-4, ORSAB, and CMRSA were as follows: 98.9/94.9%, 100/83.1%, 89.9/87.3%, 97.8/96.6%, and 95.5/94.9%, respectively. In comparison with PCR, it was found that both MSC-6 and ORSAB were relatively the least expensive screening tests ($0.70 and $1.00, respectively). In conclusion, all methods were comparable, but MSC-6 was the least expensive medium for MRSA screening.
Development of a dry-reagent-based nucleic acid-sensing platform by coupling thermostabilised LATE-PCR assay to an oligonucleotide-modified lateral flow biosensor

Ang GY, Yu CY, Chan KG, Singh KK, Chan Yean Y

J Microbiol Methods, 2015 Nov;118:99-105.
PMID: 26342435 DOI: 10.1016/j.mimet.2015.08.024

In this study, we report for the first time the development of a dry-reagent-based nucleic acid-sensing platform by combining a thermostabilised linear-after-the-exponential (LATE)-PCR assay with a one-step, hybridisation-based nucleic acid lateral flow biosensor. The nucleic acid-sensing platform was designed to overcome the need for stringent temperature control during transportation or storage of reagents and reduces the dependency on skilled personnel by decreasing the overall assay complexity and hands-on time. The platform was developed using toxigenic Vibrio cholerae as the model organism due to the bacterium's propensity to cause epidemic and pandemic cholera. The biosensor generates result which can be visualised with the naked eyes and the limit of detection was found to be 1pg of pure genomic DNA and 10CFU/ml of toxigenic V. cholerae. The dry-reagent-based nucleic acid-sensing platform was challenged with 95 toxigenic V. cholerae, 7 non-toxigenic V. cholerae and 66 other bacterial strains in spiked stool sample and complete agreement was observed when the results were compared to that of monosialoganglioside (GM1)-ELISA. Heat-stability of the thermostabilised LATE-PCR reaction mixes at different storage temperatures (4-56°C) was investigated for up to 90days. The dry-reagent-based genosensing platform with ready-to-use assay components provides an alternative method for sequence-specific detection of nucleic acid without any cold chain restriction that is associated with conventional molecular amplification techniques.
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

Yu CY, Ang GY, Chan KG, Banga Singh KK, Chan YY

Biosens Bioelectron, 2015 Aug 15;70:282-8.
PMID: 25835520 DOI: 10.1016/j.bios.2015.03.048

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.
Fulltext A pentaplex PCR assay for the rapid detection of methicillin-resistant Staphylococcus aureus and Panton-Valentine Leucocidin

Al-Talib H, Yean CY, Al-Khateeb A, Hassan H, Singh KK, Al-Jashamy K, et al.

BMC Microbiol, 2009;9:113.
PMID: 19476638 DOI: 10.1186/1471-2180-9-113

Staphylococcus aureus is a major human pathogen, especially methicillin-resistant S. aureus (MRSA), which causes a wide range of hospital and community-acquired infections worldwide. Conventional testing for detection of MRSA takes 2-5 days to yield complete information of the organism and its antibiotic sensitivity pattern.
Effect of empagliflozin in patients with type 2 diabetes during Ramadan on volume status, ketonaemia, and hypoglycaemia

Goh KG, Zakaria MH, Raja Azwan RN, Bhajan Singh KK, Badrul Hisham MH, Hussein Z

Diabetes Metab Syndr, 2023 Jan;17(1):102680.
PMID: 36473336 DOI: 10.1016/j.dsx.2022.102680

BACKGROUND AND AIMS: Patients with type 2 diabetes (T2D) carry higher risk of glycaemic variability during Ramadan. Glucose-lowering medications such as SGLT2 inhibitors are also associated with genitourinary infection, acute kidney injury, and euglycaemic diabetic ketoacidosis. Limited data is available on the effects of SGLT2 inhibitors on T2D patients during Ramadan. We investigated effects of empagliflozin use in fasting T2D patients.
METHODS: This was a prospective cohort study in a single diabetes centre in Malaysia. Empagliflozin group were on study drug for at least three months. For control group, subjects not receiving SGLT2 inhibitors were recruited. Follow-up were performed before and during Ramadan fasting. Anthropometric measurements, blood pressure, renal profile, and blood ketone were recorded during visits. Hypoglycaemia symptoms were assessed via hypoglycaemia symptom rating questionnaire (HypoSRQ).
RESULTS: We recruited a total of 98 subjects. Baseline anthropometry, blood pressure, and renal parameters were similar in two groups. No significant changes in blood pressure, weight, urea, creatinine, eGFR, or haemoglobin levels during Ramadan was found in either group. Likewise, no difference was detected in blood ketone levels (empagliflozin vs control, 0.17 ± 0.247 mmol/L vs 0.13 ± 0.082 mmol/L, p = 0.304) or hypoglycaemia indices (empagliflozin vs control, 19.1% vs 16%, p = 0.684).
CONCLUSIONS: Ramadan fasting resulted in weight loss and reduction in eGFR levels in patients with T2D. Empagliflozin use during Ramadan is safe and not associated with increased risk of dehydration, ketosis, or hypoglycaemia. Therefore, empagliflozin is a viable glucose-lowering drug for patients with T2D planning for Ramadan fasting.
Fulltext Prevalence of Multidrug-Resistant and Extended-Spectrum Beta-Lactamase-Producing Shigella Species in Asia: A Systematic Review and Meta-Analysis

Salleh MZ, Nik Zuraina NMN, Hajissa K, Ilias MI, Banga Singh KK, Deris ZZ

Antibiotics (Basel), 2022 Nov 18;11(11).
PMID: 36421297 DOI: 10.3390/antibiotics11111653

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.
Potential of Phytomolecules in Alliance with Nanotechnology to Surmount the Limitations of Current Treatment Options in the Management of Osteoarthritis

Mourya A, Shubhra, Bajwa N, Baldi A, Singh KK, Pandey M, et al.

Mini Rev Med Chem, 2023;23(9):992-1032.
PMID: 35546778 DOI: 10.2174/1389557522666220511140527

Osteoarthritis (OA), a chronic degenerative musculoskeletal disorder, progressively increases with age. It is characterized by progressive loss of hyaline cartilage followed by subchondral bone remodeling and inflammaging. To counteract the inflammation, synovium releases various inflammatory and immune mediators along with metabolic intermediates, which further worsens the condition. However, even after recognizing the key molecular and cellular factors involved in the progression of OA, only disease-modifying therapies are available such as oral and topical NSAIDs, opioids, SNRIs, etc., providing symptomatic treatment and functional improvement instead of suppressing OA progression. Long-term use of these therapies leads to various life-threatening complications. Interestingly, mother nature has numerous medicinal plants containing active phytochemicals that can act on various targets involved in the development and progression of OA. Phytochemicals have been used for millennia in traditional medicine and are promising alternatives to conventional drugs with a lower rate of adverse events and efficiency frequently comparable to synthetic molecules. Nevertheless, their mechanism of action in many cases is elusive and uncertain. Even though many in vitro and in vivo studies show promising results, clinical evidence is scarce. Studies suggest that the presence of carbonyl group in the 2nd position, chloro in the 6th and an electron- withdrawing group at the 7th position exhibit enhanced COX-2 inhibition activity in OA. On the other hand, the presence of a double bond at the C2-C3 position of C ring in flavonoids plays an important role in Nrf2 activation. Moreover, with the advancements in the understanding of OA progression, SARs (structure-activity relationships) of phytochemicals and integration with nanotechnology have provided great opportunities for developing phytopharmaceuticals. Therefore, in the present review, we have discussed various promising phytomolecules, SAR as well as their nano-based delivery systems for the treatment of OA to motivate the future investigation of phytochemical-based drug therapy.
Fulltext Sequence-Specific Electrochemical Genosensor for Rapid Detection of blaOXA-51-like Gene in Acinetobacter baumannii

Kanapathy S, Obande GA, Chuah C, Shueb RH, Yean CY, Banga Singh KK

Microorganisms, 2022 Jul 13;10(7).
PMID: 35889132 DOI: 10.3390/microorganisms10071413

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.

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