Clinical Validation of Innovative Optical-Sensor-Based, Low-Cost, Rapid Diagnostic Test to Reduce Antimicrobial Resistance.
ABSTRACT: The antibiotic susceptibility test determines the most effective antibiotic treatment for bacterial infection. Antimicrobial stewardship is advocated for the rational use of antibiotics to preserve their efficacy in the long term and provide empirical therapy for disease management. Therefore, rapid diagnostic tests can play a pivotal role in efficient and timely treatment. Here, we developed a novel, rapid, affordable, and portable platform for detecting uropathogens and reporting antibiogram to clinicians in just 4 h. This technology replicates the basic tenets of clinical microbiology including bacterial growth in indigenously formulated medium, and measurement of inhibition of bacterial growth in presence of antibiotic/s. Detection is based on chromogenic endpoints using optical sensors and is analyzed by a lab-developed algorithm, which reports antibiotic sensitivity to the antibiotics panel tested. To assess its diagnostic accuracy, a prospective clinical validation study was conducted in two tertiary-care Indian hospitals. Urine samples from 1986 participants were processed by both novel/index test and conventional Kirby Bauer Disc Diffusion method. The sensitivity and specificity of this assay was 92.5% and 82%, respectively (p < 0.0005). This novel technology will promote evidence-based prescription of antibiotics and reduce the burden of increasing resistance by providing rapid and precise diagnosis in shortest possible time.
Project description:Periodic monitoring of antibiotic susceptibility patterns in clinical settings is vital to ascertain the potency as well as re-establishing empirical therapy. This retrospective study aimed to evaluate the antibiotic susceptibility patterns of pathogens isolated from routine laboratory specimens at Ndola Teaching Hospital. A retrospective study was conducted on routine specimens received between May 2016 and July 2018. Specimens were cultured on standard media and Kirby-Bauer disc diffusion method was used for susceptibility testing in accordance with the Clinical and Laboratory Standard Institute's recommendations. A total of 693 specimens were analyzed, of which 65.9% (457) specimens came from inpatient departments and 49.1% (340) came from female patients. The commonest specimens were urine (58.6%), blood (12.7%) and wound swabs (8.5%), and the most common microorganisms were coliform (29.3%), Staphylococcus aureus (15.4%), coagulase negative Staphylococci (CoNS, 13.4%), and Escherichia coli (13%). The highest percentage of resistance to any particular antibiotic was co-trimoxazole (91.7%, 33) followed by nalidixic acid (75.2%, 279), norfloxacin (69.0%, 100), ceftazidime (55.7%, 185), nitrofurantoin (46.6%, 191), chloramphenicol (43%, 111) and ciprofloxacin (8.6%, 271). Furthermore, patient location had resistance effect on coliform (p = 0.014), CoNS (p = 0.031), Streptococcus species (p = 0.024) and Klebsiella species (p = 0.004) to nitrofurantoin, ceftazidime, nitrofurantoin and chloramphenicol, respectively. Besides coliform, resistance of Enterobacter species to ceftazidime and Proteus species to nalidixic acid were more from female patients. Generally, the most effective antibiotics were chloramphenicol and nitrofurantoin with addition of ceftazidime on blood pathogens and ciprofloxacin on wound swab pathogens. The common isolates were coliform, S. aureus, coagulase negative Staphylococci and Escherichia coli. The resistance of most bacteria to ceftazidime and nitrofurantoin were influenced by both gender and location. Our study presents a broad overview of the resistance profiles of bacterial isolates. However, more nosocomial prevalence and antibiogram studies on individual routine specimens are required to provide a more detailed picture of resistance patterns.
Project description:Antibiotic resistance is a serious threat against humankind and the need for new therapeutics is crucial. Without working antibiotics, diseases that we thought were extinct will come back. In this paper two new mannitol bisphosphate analogs, 1,6-dideoxy-1,6-diphosphoramidate mannitol and 1,6-dideoxy-1,6-dimethansulfonamide mannitol, have been synthesized and evaluated as potential inhibitors of the enzyme GmhB in the biosynthesis of lipopolysaccharides. 1,6-Dideoxy-1,6-diphosphoramidate mannitol showed promising result in computational docking experiments, but neither phosphate analog showed activity in the Kirby-Bauer antibiotic susceptibility test.
Project description:The objective of this study was to determine the effect of chlorine induced sublethal oxidative stress against homologous and heterologous stress adaptations in five Listeria monocytogenes (Lm) strains. Lm cells were exposed to gradually increasing sublethal concentrations of total chlorine/day: 250 ppm (day 1), 270 ppm (day 2), 290 ppm (day 3), 310 ppm (day 4), 330 ppm (day 5), 350 ppm (day 6), and 375 ppm (day 7) in tryptic soy broth (TSB). Changes in minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of Lm cells exposed to chlorine and control (non-adapted cells) were determined by the macro-dilution method. Chlorine-adapted Lm cells were also evaluated for changes in antibiotic resistance using the Kirby-Bauer disk diffusion and MIC double dilution assay as per the Clinical and Laboratory Standards Institute (CLSI, 2016) guidelines. In four Lm strains (Scott A, V7, FSL-N1-227 and FSL-F6-154) after adapted to sublethal chlorine, the MIC (600 ppm) and MBC (700 ppm) values of chlorine were slightly higher as compared to control (500 ppm MIC, and 600 ppm MBC). The Kirby-Bauer and MIC double dilution assays showed some significant changes in antibiotic susceptibility patterns for antibiotics such as streptomycin, gentamicin and ceftriaxone (p < 0.05). However, the changes in zones of inhibition and MIC values to all antibiotics tested for the chlorine-adapted and non-adapted (control) Lm cells were still within the susceptible range. Transmission electron microscopy studies showed that changes in cell wall and membrane integrity resulting, from the elongation of cells, may contribute to the possible routes of its increase in tolerance to chlorine and selective antibiotics. These findings indicate that the continuous exposure of Lm cells to chlorine may lead to significant changes in homologs and heterologous stress adaptation.
Project description:Rapid antimicrobial susceptibility testing is important for efficient and timely therapeutic decision making. Due to globally spread bacterial resistance, the efficacy of antibiotics is increasingly being impeded. Conventional antibiotic tests rely on bacterial culture, which is time-consuming and can lead to potentially inappropriate antibiotic prescription and up-front broad range of antibiotic use. There is an urgent need to develop point-of-care platform technologies to rapidly detect pathogens, identify the right antibiotics, and monitor mutations to help adjust therapy. Here, we report a biosensor for rapid (<90 min), real time, and label-free bacteria isolation from whole blood and antibiotic susceptibility testing. Target bacteria are captured on flexible plastic-based microchips with printed electrodes using antibodies (30 min), and its electrical response is monitored in the presence and absence of antibiotics over an hour of incubation time. We evaluated the microchip with Escherichia coli and methicillin-resistant Staphylococcus aureus (MRSA) as clinical models with ampicillin, ciprofloxacin, erythromycin, daptomycin, gentamicin, and methicillin antibiotics. The results are compared with the current standard methods, i.e. bacteria viability and conventional antibiogram assays. The technology presented here has the potential to provide precise and rapid bacteria screening and guidance in clinical therapies by identifying the correct antibiotics for pathogens.
Project description:Bacteraemia is a common cause of fever among patients presenting to hospitals in sub-Saharan Africa. The worldwide rise of antibiotic resistance makes empirical therapy increasingly difficult, especially in resource-limited settings.To describe the incidence of bacteraemia in febrile adults presenting to Maputo Central Hospital (MCH), an urban referral hospital in the capital of Mozambique, and characterise the causative organisms and antibiotic susceptibilities. We aimed to describe the antibiotic prescribing habits of local doctors, to identify areas for quality improvement.Inclusion criteria were: (i) ≥18 years of age; (ii) axillary temperature ≥38°C or ≤35°C; (iii) admission to MCH medical wards in the past 24 hours; and (iv) no receipt of antibiotics as an inpatient. Blood cultures were drawn from enrolled patients and incubated using the BacT/Alert automated system (bioMérieux, France). Antibiotic susceptibilities were tested using the Kirby-Bauer disc diffusion method.Of the 841 patients enrolled, 63 (7.5%) had a bloodstream infection. The most common isolates were Staphylococcus aureus, Escherichia coli, and non-typhoidal Salmonella. Antibiotic resistance was common, with 20/59 (33.9%) of all bacterial isolates showing resistance to ceftriaxone, the broadest-spectrum antibiotic commonly available at MCH. Receipt of insufficiently broad empirical antibiotics was associated with poor in-hospital outcomes (odds ratio 8.05; 95% confidence interval 1.62 - 39.91; p=0.04).This study highlights several opportunities for quality improvement, including educating doctors to have a higher index of suspicion for bacteraemia, improving local antibiotic guidelines, improving communication between laboratory and doctors, and increasing the supply of some key antibiotics.
Project description:Background:Antibiotic resistance poses burden to the community and health-care services. Efforts are being made at local, national and global level to combat the rise of antibiotic resistance including antibiotic stewardship. Surveillance to antibiotic resistance is of importance to aid in planning and implementing infection prevention and control measures. The study was conducted to assess the resistance pattern to cefepime, clindamycin and meropenem, which are reserved antibiotics for use at tertiary hospitals in Tanzania. Methods:A hospital-based antibiotic resistance surveillance was conducted between July and November 2019 at Muhimbili National Hospital and Bugando Medical Center, Tanzania. All organisms isolated were identified based on colony morphology, Gram staining and relevant biochemical tests. Antibiotic susceptibility testing was performed on Muller-Hinton agar using Kirby-Bauer disc diffusion method. Antibiotic susceptibility was performed according to the protocol by National Committee for Clinical Laboratory Standards. Results:A total of 201 clinical samples were tested in this study. Urine (39.8%, n=80) and blood (35.3%, n=71) accounted for most of the collected samples followed by pus (16.9%, n=34). The bacterial resistance to clindamycin, cefepime and meropenem was 68.9%, 73.2% and 8.5%, respectively. About 68.4% Staphylococcus aureus isolates were resistant to clindamycin whereby 56.3%, 75.6%, 93.8% and 100% of the tested Escherichia coli, Klebsiella spp, Pseudomonas aeruginosa and Enterobacter cloacae, respectively, were cefepime resistant. About 8.5% of isolated Klebsiella spp were resistant and 6.4% had intermediate susceptibility to meropenem. Also, Pseudomonas aeruginosa was resistant by 31.2% and 25% had intermediate susceptibility to meropenem. Conclusion:The bacterial resistance to clindamycin and cefepime is high and low in meropenem. Henceforth, culture and susceptibility results should be used to guide the use of these antibiotics. Antibiotics with low resistance rate should be introduced to the reserve category and continuous antibiotic surveillance is warranted.
Project description:Antimicrobial resistance is a global issue currently resulting in the deaths of hundreds of thousands of people a year worldwide. Data present in the literature illustrate the emergence of many bacterial species that display resistance to known antibiotics; Acinetobacter spp. are a good example of this. We report here that Acinetobacter radioresistens has a Baeyer-Villiger monooxygenase (Ar-BVMO) with 100% amino acid sequence identity to the ethionamide monooxygenase of multidrug-resistant (MDR) Acinetobacter baumannii. Both enzymes are only distantly phylogenetically related to other canonical bacterial BVMO proteins. Ar-BVMO not only is capable of oxidizing two anticancer drugs metabolized by human FMO3, danusertib and tozasertib, but also can oxidize other synthetic drugs, such as imipenem. The latter is a member of the carbapenems, a clinically important antibiotic family used in the treatment of MDR bacterial infections. Susceptibility tests performed by the Kirby-Bauer disk diffusion method demonstrate that imipenem-sensitive Escherichia coli BL21 cells overexpressing Ar-BVMO become resistant to this antibiotic. An agar disk diffusion assay proved that when imipenem reacts with Ar-BVMO, it loses its antibiotic property. Moreover, an NADPH consumption assay with the purified Ar-BVMO demonstrates that this antibiotic is indeed a substrate, and its product is identified by liquid chromatography-mass spectrometry to be a Baeyer-Villiger (BV) oxidation product of the carbonyl moiety of the ?-lactam ring. This is the first report of an antibiotic-inactivating BVMO enzyme that, while mediating its usual BV oxidation, also operates by an unprecedented mechanism of carbapenem resistance.
Project description:BACKGROUND:Neisseria gonorrhoeae (commonly known as gonorrhea) has developed resistance to all first-line therapy in Southeast Asia. East Africa has historically had absent or rudimentary gonorrhea surveillance programs and, while the existence of antimicrobial-resistant gonorrhea is recognized, the extent of its resistance is largely unknown. In 2016, the World Health Organization's Enhanced Gonococcal Antimicrobial Surveillance Program (EGASP) was initiated in Uganda to monitor resistance trends. OBJECTIVE:This study characterizes gonorrhea and antibiotic resistance in a large surveillance program of men with urethral discharge syndrome from Kampala, Uganda. METHODS:Men attending sentinel clinics with urethritis provided demographic information, behavior data, and a urethral swab in line with the World Health Organization's EGASP protocols for culture, identification, and antibiotic-sensitivity testing using 2 methods-disk diffusion (Kirby-Bauer test) and Etest (BioMérieux Inc). A subset of samples underwent detailed antimicrobial resistance testing. RESULTS:Of 639 samples collected from September 2016 to February 2018, 400 (62.6%) were culture-positive though 414 (64.8%) had microscopic evidence of gonorrhea. The mean age of the men from whom the samples were collected was 26.9 (SD 9.6) years and 7.2% (46/639) reported having HIV. There was high-level resistance to ciprofloxacin, tetracycline, and penicillin (greater than 90%) by Kirby-Bauer disk diffusion and 2.1% (4/188) had reduced azithromycin sensitivity by Etest. Of the early isolates that underwent detailed characterization, 60.3% (70/116) were culture-positive, 94% (66/69) isolates were either ciprofloxacin-resistant or ciprofloxacin-intermediate by Etest, 96% (65/68) were azithromycin-sensitive, and 96% (66/69) were gentamicin-sensitive. Resistance profiles were comparable between methods except for ceftriaxone (disk diffusion: 68/69, 99%; Etest: 67/69, 97%) and for gentamicin (disk diffusion: 2/8, 25%; Etest: 66/69, 96%) sensitivity. CONCLUSIONS:This is the first report from a systematic gonorrhea surveillance program in Uganda. Findings demonstrated resistance or increased minimum inhibitory concentration to all key antigonococcal antibiotics. There was evidence of poor antibiotic stewardship, near-universal resistance to several antibiotics, and emerging resistance to others. Individuals in the population sampled were at exceptionally high risk of STI and HIV infection requiring intervention. Ongoing surveillance efforts to develop interventions to curtail antimicrobial-resistant gonorrhea are needed.
Project description:BACKGROUND:Paenibacillus sp. strain VT-400, a novel spore-forming bacterium, was isolated from patients with hematological malignancies. METHODS:Paenibacillus sp. strain VT-400 was isolated from the saliva of four children with acute lymphoblastic leukemia. The genome was annotated using RAST and the NCBI Prokaryotic Genome Annotation Pipeline to characterize features of antibiotic resistance and virulence factors. Susceptibility to antibiotics was determined by the Kirby-Bauer disc diffusion method. We used a mouse model of pneumonia to study virulence in vivo. Mice were challenged with 7.5 log10-9.5 log10 CFU, and survival was monitored over 7 days. Bacterial load was measured in the lungs and spleen of surviving mice 48 h post-infection to reveal bacterial invasion and dissemination. RESULTS:Whole-genome sequencing revealed a large number of virulence factors such as hemolysin D and CD4+ T cell-stimulating antigen. Furthermore, the strain harbors numerous antibiotic resistance genes, including small multidrug resistance proteins, which have never been previously found in the Paenibacillus genus. We then compared the presence of antibiotic resistance genes against results from antibiotic susceptibility testing. Paenibacillus sp. strain VT-400 was found to be resistant to macrolides such as erythromycin and azithromycin, as well as to chloramphenicol and trimethoprim-sulphamethoxazole. Finally, the isolate caused mortality in mice infected with ?8.5 log10 CFU. CONCLUSIONS:Based on our results and on the available literature, there is yet no strong evidence that shows Paenibacillus species as an opportunistic pathogen in immunocompromised patients. However, the presence of spore-forming bacteria with virulence and antibiotic resistance genes in such patients warrants special attention because infections caused by spore-forming bacteria are poorly treatable.
Project description:Silver nanoparticles (AgNPs) are promising alternatives to antibiotics. The aims of this study were to produce AgNPs using two biological methods and determine their antibacterial activity against Pseudomonas aeruginosa and Staphylococcus pseudintermedius. AgNPs were biosynthesized from an infusion of Curcuma longa (turmeric) and the culture supernatant of E. coli. Characterization was achieved by ultraviolet-visible spectroscopy and by Transmission Electron Microscopy (TEM). The antibacterial properties of NPs from C. longa (ClAgNPs) and E. coli (EcAgNPs), alone and in combination with carbenicillin and ampicillin, were investigated through the Kirby-Bauer disk diffusion assay and the minimum inhibitory concentration (MIC). Dimensions of NPs ranged from 11.107 ± 2.705 nm (ClAgNPs) to 27.282 ± 2.68 nm (EcAgNPs). Kirby-Bauer and MIC assays showed great antibacterial abilities for both NPs alone and in combination with antibiotics. EcAgNPs alone showed the most powerful antibacterial activities, resulting in MIC values ranging from 0.438 ± 0.18 µM (P. aeruginosa) to 3.75 ± 3.65 µM (S. pseudintermedius) compared to those of ClAgNPs: 71.8 ± 0 µM (P. aeruginosa) and 143.7 ± 0 µM (S. pseudintermedius). The antibiofilm abilities were strain-dependent, but no statistical differences were found between the two NPs. These results suggest the antibacterial potential of AgNPs for the treatment of infectious diseases.