Background and Objectives:In most laboratories, diagnosis of invasive aspergillosis (IA) depends on culture and histopathological examination of the involved tissues. Galactomannan (GM) is a polysaccharide antigen detectable in body fluids and represents an ideal assay for diagnosing invasive pulmonary aspergillosis. The main objectives of this study were to estimate the proportion of patients with proven, probable, and possible aspergillosis and to compare different diagnostic methods. Materials and Methods:Samples for culture, microscopy, and GM assay were taken from a total of 60 patients. The enrolled patients were divided into proven, probable, and possible aspergillosis groups. Results:Among the 60 patients in the study, there were 9 (15%) cases of probable aspergillosis. galactomannan assay was positive in 19 patients. Galactomannan assay had a higher sensitivity (66.7%) when compared to other fungal assays for diagnosing probable aspergillosis. Conclusion:Diagnosing invasive aspergillosis is extremely challenging, and biomarkers such as galactomannan assay are important for early diagnosis. Raising the cutoff to >1 can eliminate false-positive results associated with the GM assay.
Background and Objectives:Pseudomonas aeruginosa is a major nosocomial pathogen implicated in a wide range of hospital-acquired infections. Prompt detection and effective control of outbreaks caused by this organism are essential to minimize associated morbidity and mortality. Multiple strains may circulate within healthcare settings due to cross-transmission and persistent environmental reservoirs. Various molecular typing techniques have proven valuable in tracing the sources and transmission dynamics of outbreaks. The present study aimed to investigate the antibiogram profiles and enterobacterial repetitive intergenic consensus-polymerase chain reaction (ERIC-PCR) patterns of P. aeruginosa isolates recovered from non-respiratory clinical samples at a tertiary care hospital. Materials and Methods:A total of 157 clinical isolates phenotypically identified as P. aeruginosa were subjected to 16S rDNA-PCR for species confirmation. Among these, 150 isolates were confirmed as P. aeruginosa and subsequently analysed for antimicrobial susceptibility and molecular typing. Antimicrobial susceptibility testing was performed according to Clinical and Laboratory Standards Institute (CLSI) guidelines. Molecular typing was carried out using ERIC-PCR. The resulting ERIC-PCR profiles were analysed using PAST version 4.03 software, and a dendrogram was constructed based on Dice similarity coefficients to assess genetic relatedness among isolates. Fisher's exact test was employed to determine associations between specific ERIC types and antibiotic resistance profiles. Results:Overall, 84% of the P. aeruginosa isolates were identified as multidrug-resistant (MDR), with widespread distribution among both inpatients and outpatients. The highest resistance rate was observed against ticarcillin/clavulanic acid, followed by ceftazidime, whereas no resistance was detected against colistin sulphate. A total of 54 distinct antibiotypes were identified, indicating substantial variability in antimicrobial resistance patterns. All 150 isolates were successfully typed using ERIC-PCR, which revealed 41 distinct clusters at an approximate similarity coefficient of 70%, demonstrating considerable genetic diversity among the isolates. Conclusion:This study enhances the understanding of the epidemiology of P. aeruginosa infections in healthcare settings by characterizing both the molecular diversity and antimicrobial resistance patterns of clinical isolates. The high prevalence of MDR strains and the circulation of multiple genetically diverse strains among hospital patients highlight the ongoing risk of transmission within the healthcare environment. Notably, the findings suggest a comparatively higher rate of cross-infection among burn patients. These observations underscore the urgent need for stringent infection prevention and control measures, particularly in burn units, to limit the spread of MDR P. aeruginosa.
Background and Objectives:Apart from spontaneous deamidation and isomerization of Asp/Asn residues, factors like ageing, oxidative stress, salinity and heat stress may also form abnormal isoaspartate (iso-Asp) residues compromising protein structure and function. PIMT helps in restoration of such iso-Asp residues. Pseudomonas aeruginosa is a gram-negative bacteria responsible for diseases with varied degree of pathogenesis in humans and animals. They can survive a wide range of temperature between 25°C to 42°C. The objective of the current study was to monitor the expression dynamics of PIMT in Pseudomonas aeruginosa upon exposure to different thermal stresses. Materials and Methods:The mid log phase culture of Pseudomonas aeruginosa was exposed to different temperature (37°C, 42°C, 56°C, 80°C) for an hour. The expression levels of PIMT were quantified by Western blot and ELISA. Results:A relatively higher level of expression of PIMT was observed in the group exposed to 42°C, 56°C, 80°C in comparison to 37°C. The expression of PIMT increased around 1.5 times in the group exposed to 42°C. Conclusion:A temperature dependent overexpression of PIMT was observed in Pseudomonas aeruginosa.
Background and Objectives: Malassezia spp. are lipid-dependent yeasts that colonize the skin of humans and animals and may act as opportunistic pathogens. This study aimed to characterize virulence-associated enzymes and their encoding genes in Malassezia spp. isolated from companion animals in northern Iran. Materials and Methods: A total of 300 clinical samples were collected from dogs and cats with dermatological or otic lesions. Species identification was performed using ITS-based PCR. Multiplex PCR was used to detect LIP, PLB, and KER genes. Enzymatic activity was evaluated using the Pz index. Statistical analyses included Chi-square and Spearman’s cor- relation tests (p < 0.05). Results: Eighty isolates were identified as Malassezia pachydermatis (87.5%) and Malassezia nana (12.5%). LIP, PLB, and KER genes were detected in 90%, 62.5%, and 50% of isolates, respectively. Strong enzymatic activity (Pz ≤ 0.69) was observed in 41.2% of isolates. A significant association was found between the presence of the PLB gene and phospholipase activity. Conclusion: Malassezia pachydermatis was the predominant species. Virulence-associated genes were associated with high- er enzymatic activity, supporting their role in pathogenic potential.
Background and Objectives:Flavoprotein monooxygenases (FPMOs) participate in various biological processes, including lignin degradation, natural product biosynthesis, and xenobiotic detoxification. This study aimed to investigate the heterologous expression of FPMO and assess its functional activity by examining its capacity to inactivate ampicillin. Materials and Methods:In silico analyses were performed to predict the enzyme's secondary and tertiary structures. The target gene was isolated from Pseudomonas aeruginosa, cloned into the pET-22b vector, and expressed in Escherichia coli BL21 (DE3). Heterologous protein expression was examined using SDS-PAGE, and the protein was purified through nickel-affinity chromatography. Enzymatic activity was measured by spectrophotometric monitoring of NADPH oxidation at 340 nm. Antibacterial activity was tested using an agar well-diffusion assay, measuring the inhibition zone diameter in E. coli treated with ampicillin. Results:Homology modeling indicated that the 3D structure of FPMO is very similar to cyclohexanone monooxygenase, supporting its classification as a Baeyer-Villiger monooxygenase (BVMO). Molecular docking proposed that ampicillin might be a substrate for FPMO, with predicted interactions at Ile141 and Val159. Enzymatic assays confirmed that FPMO catalyzes the oxidation of ampicillin, using FAD and NADPH as cofactors. Additionally, well-diffusion tests showed decreased ampicillin antibacterial activity after treatment. The enzyme's activity was further confirmed using an ampicillin inactivation assay. Conclusion:The heterologously expressed protein was functionally active. These findings suggest that the studied FPMO may play a role in antibiotic resistance in P. aeruginosa by oxidatively inactivating ampicillin.
Background and Objectives: The ability of Aspergillus species to produce various extracellular enzymes has a pathogenic role in humans. These enzymes not only play a role in metabolism but also act as virulence factors. The secretion of hydro- lytic enzymes facilitates tissue invasion and is considered a virulence factor for several fungi. The current study aimed to compare the enzymatic activity of A. flavus section Flavi isolated from otomycosis and environmental samples. Materials and Methods: In the present study, eighty-one isolates of A. flavus including 41 clinical strains from otomycosis and 40 environmental isolates were identified using the calmodulin gene. The enzymatic activity of the isolates was assessed using standard culture media. Results: All isolates exhibited phospholipase and urease activity. Esterase and hemolysin activity in otomycosis isolates were higher than in environmental isolates (88% > 62%), (98% > 72%). High levels of both extracellular and intracellular catalase activity were detected among otomycosis isolates. Conclusion: All isolates were phenotypically identified as Aspergillus section Flavi and then characterized using the calm- odulin gene. The mean activity of intracellular catalase enzymes (1.29) in both groups was higher than the mean activity of extracellular catalase enzymes (0.85). Additionally, the esterase (88%) and hemolysin (98%) activity of the clinical isolates was higher than that of the environmental isolates (esterase 62%, hemolysin 72%). All isolates exhibited phospholipase and urease activity.
Background and Objectives: A selenium (Se) enriched Lactobacillus brevis was applied as mouthwash to relieve mouth ulcers and regulate oral pathogens. The persistence of four frequent oral pathogens (Pseudomonas, Klebsiella, Proteus, and Enterobacter) and the quantity of mouth lesions were to be measured after exposure to the above probiotic. Susceptibility of the pathogens was also examined. Materials and Methods: In this randomized double-blind controlled clinical trial, 60 ICU patients were allocated into three groups equally (to receive normal saline, chlorhexidine, and Se-probiotic) every 6 hours for 1 week. The utilized Lactobacil- lus brevis was previously separated from a traditional dairy product and its enrichment was accomplished through streaking on MRS plates supplemented with sodium selenite. Results: Klebsiella and Pseudomonas were the most frequent initial pathogens, from which Pseudomonas were totally omitted from the probiotic group. The Se-probiotic existed in the mouth cavity of the test group six hours after the last dose. In the probiotic group, the number of oral lesions were significantly reduced. Conclusion: Clearance of Pseudomonas from mouth cavity and significant decrease in the quantity of mouth ulcers are con- siderable achievements. Hence, the applied Se-supplemented mouthwash exhibited promising capability to be considered as a potential oral hygiene formulation.
Background and Objectives:This study aimed to investigate the effects of biogenic silver nanoparticles (AgNPs) on Herpes simplex virus type 1 (HSV-1), focusing on the influence of plant-derived capping agents and their metal cores on antiviral efficacy and mechanism of action. Materials and Methods:Biosynthesized AgNPs using aqueous extracts of Juglans regia (J. regia) and Malva sylvestris (M. sylvestris) were characterized, and their zeta potential was assessed. Molecular docking studies were performed to illuminate the interaction between AgNPs and viral particles. The antiviral activity was evaluated using viral titer quantification and a cytotoxicity assay, both performed on Vero cells. Results:AgNPs exhibited zeta potentials of -8.87 mV and -5.49 mV for J. regia and M. sylvestris, respectively, indicating their stability and potential for interaction with viral particles. Strong binding affinities between AgNPs and viral glycoprotein D (gD) were revealed by molecular docking, which obtained negative MolDock scores. The antiviral assays demonstrated a ten-thousand-fold reduction in viral titers. Conclusion:This research underscores the critical roles of capping agents and the metallic core in modulating the antiviral efficacy of plant-derived AgNPs against HSV-1, establishing them as a promising platform for novel antiviral therapeutics.
Background and Objectives: Second-generation bioethanol, a sustainable and environmentally friendly alternative to fossil fuels, can significantly contribute to the economy. However, saccharification accounts for approximately 20-25% of total bioethanol production costs, which can be reduced by using the improved enzyme cocktails. Therefore, the present study aimed to isolate and characterize potent cellulolytic Streptomyces strains capable of enhancing the hydrolytic conversion of lignocellulosic biomass. Materials and Methods: Degraded wood and soil samples were collected and enriched with paddy straw. The isolates were obtained using standard methods and characterized by 16S rRNA sequencing. The best isolate was subjected to whole-ge- nome sequencing to identify hydrolytic genes involved in agro-waste degradation. Saccharification assays were performed using crude culture filtrate in combination with the commercial cellulase cocktail Celluclast® to evaluate sugar release from alkali-pretreated paddy straw. Results: Among the 38 isolates obtained, five were identified as potential cellulolytic Streptomyces strains based on 16S rRNA gene sequencing: Streptomyces tunisiensis SSPJ1, Streptomyces griseoincarnatus SSPJ4, Streptomyces werraensis SSPJ14, Streptomyces ardesiacus SSPJ32, and Streptomyces tendae SSPJ48. S. griseoincarnatus SSPJ4 emerged as the most promising isolate, demonstrating significantly higher enzyme activities. S. griseoincarnatus SSPJ4 was subjected to whole-genome sequencing using the GALAXY tool for assembly and annotation, with an assembled genome of 7.3 Mb, 72.38% G+C content, and 6678 genes. Biosynthetic gene clusters (23) were identified using antiSMASH, and 226 proteins were annotated with CAZy domains, of which 121 belonged to the glycoside hydrolase (GH) family. Supplementation of Celluclast® with the crude enzyme preparation from S. griseoincarnatus SSPJ4 enhanced the saccharification of alkali-pre- treated paddy straw, resulting in a 1.55-fold higher sugar yield compared with Celluclast® alone. Conclusion: The findings demonstrate the considerable potential of S. griseoincarnatus SSPJ4 as a source of accessory lig- nocellulolytic enzymes for improving biomass saccharification. Its enzyme repertoire could be exploited to develop cost-ef- fective and efficient enzymatic formulations for second-generation bioethanol production.
Background and Objectives:Diabetes mellitus (DM) increases the risk of oral candidiasis by impairing immunity and enhancing Candida virulence. Although Lacticaseibacillus rhamnosus GG (LGG) has antifungal potential, its effect in diabetes remains unclear. This study evaluated the efficacy of LGG against C. albicans and C. tropicalis in diabetic rats. Materials and Methods:A fungal suspension (1×107 CFU/mL) was applied to the tongue for three days in the controls and experimental groups of male alloxan-induced diabetic Sprague-Dawley rats. Then, LGG (0.3 mL of 1×108 CFU/mL) was administered for 28 days in the experimental groups. Weekly oral swabs were analyzed, and blood and tongue tissues were evaluated using biochemical, histopathological, and molecular methods (TLR2, TLR4, and Msp1/p75). Results:Diabetic rats showed weight loss and increased oral Candida growth. LGG reduced C. albicans and C. tropicalis counts and stabilized body weight. Haematological and biochemical parameters improved with probiotic treatment. Histopathological examination showed reduced inflammation, and molecular analyses indicated modulation of Msp1/p75 and TLR2/TLR4. Overall, LGG provided antifungal and immunomodulatory benefits in diabetic rats with oral candidiasis. Conclusion:This study assessed the efficacy of LGG against C. albicans and C. tropicalis in diabetic rats. Administration of LGG reduced Candida colonization and maintained normal physiological parameters. These results suggest that LGG has potential as a safe complementary antifungal therapy.
Background and Objectives: This systematic review aimed to determine whether phage cocktails and co-evolutionarily trained phages prevent or delay the emergence of resistance in multidrug-resistant (MDR) bacteria more effectively than single-phage therapy in sewage and wastewater-derived systems, and to characterize the underlying mechanisms and the maturity of the current evidence base. Materials and Methods: Following PRISMA guidelines, a systematic review was performed using the PubMed and Scopus databases, with screening conducted via Rayyan Premium. Results: Among 4,795 records, 20 studies (≈0.4%) fulfilled the inclusion criteria; this low fraction reflects a deliberately broad search combined with strict eligibility criteria targeting original experimental studies of phage activity against MDR pathogens specifically isolated from sewage or wastewater. Phage cocktails showed a wider range of host coverage, a delay in resistance development, and improved penetration of biofilms, particularly when paired with depolymerases or antibiotics. Phage co-evolution training resulted in a more prolonged suppression of multidrug-resistant bacteria. Conclusion: Phage cocktails, especially those that are co-evolved and enhanced with enzymes, show potential for applications in wastewater; however, additional long-term and large-scale research is necessary.
Background and Objectives: Salmonella gallinarum, causes fowl typhoid, continues to be a significant limitation to poultry production in developing nations, with continuously rising antimicrobial resistance. The current research was design and test an indigenous S. gallinarum ghost vaccine and to measure its immunogenicity and protective effect on layer birds. Materials and Methods: Bacteriological culture of suspected clinical cases of fowl typhoid was performed, and isolates were verified by PCR against the 16S rRNA gene, after which they were sequenced, and phylogenetic analysis was per- formed. To obtain bacterial ghost cells, a representative native isolate was subjected to a detergent-based chemical lysis pro- cedure, followed up by scanning electron microscopy. 120-day-old layer chicks were divided into six groups randomly and immunized at the age of seven days using oral, intramuscular, air-spray, intramuscular with oil adjuvant or eye-drop routes. There was a 1st dose at 26 days of age in all vaccinated groups and the challenge of virulent S. gallinarum at 42 and 93 days of age. ELISA was used to test humoral immune response at different time points. Results: The highest antibody titers were found in vaccinated groups compared to controls (p < 0.05), and the intramuscular route generated the best and most enduring response in systemic immunity. Mucosal pathways elicited not high but rather significant antibody responses and provided protection against clinical disease. Conclusion: The research contributes to the feasibility of ghost-based vaccination in the role of a sustainable alternative to the disease control measures that are highly reliant on antibiotics in endemic poultry production systems.
Background and Objectives: Antimicrobial resistance (AMR) poses a critical global health threat, with multidrug-resistant (MDR), extensively drug-resistant (XDR), and pandrug-resistant (PDR) pathogens increasingly prevalent in healthcare set- tings. The study aimed to provide a comprehensive analysis of AMR among clinical bacterial isolates, including detailed antibiogram profiling and prevalence of MDR, XDR, and PDR phenotypes, to inform local treatment guidelines and antimi- crobial stewardship strategies. Materials and Methods: A retrospective cross-sectional analysis was conducted on 2,945 clinical specimens (blood, wound, sputum, urine, vaginal swabs, and others). Bacterial identification was performed using VITEK 2 automated system. Anti- microbial susceptibility testing was conducted according to Clinical and Laboratory Standards Institute (CLSI) guidelines. MDR, XDR, and PDR classifications were applied. Results: Out of 2,851 major clinical specimens, Gram-negative bacteria predominated (53%), with highest isolation rates from wound, sputum, and urine samples. Gram-positive organisms accounted for 30%, while Candida species represented 10%. Among Gram-negative isolates, 55% were classified as MDR, 15% as XDR, and 2% as PDR. Gram-positive bacteria showed lower resistance rates with 30% MDR, 4% XDR, and no PDR detected. Species-specific analysis revealed that Klebsiella pneumoniae and Acinetobacter baumannii exhibited the highest XDR and PDR rates, with A. baumannii showing 45% XDR and 10% PDR prevalence. Among Gram-positive organisms, MRSA demonstrated 100% MDR prevalence. The most effective antimicrobials against resistant isolates were carbapenems, amikacin, and vancomycin (for Gram-positives). Conclusion: This study demonstrates alarmingly high rates of MDR, XDR, and PDR among clinical bacterial isolates, with Gram-negative bacteria showing significantly greater resistance burden than Gram-positive organisms.
Background and Objectives: Catheter-associated urinary tract infections (CAUTIs) are common healthcare-associated infections mainly caused by biofilm formation on urinary catheters. This study aimed to provide evidence-based catheter management strategies to reduce CAUTI risk through biofilm prevention. Materials and Methods: This prospective observational study included 109 adult patients undergoing urinary catheteriza- tion between September 2017 and January 2018. Urine samples were collected after catheter insertion and before catheter removal. Bacteriuria was screened using flow cytometry and confirmed by urine culture. Removed catheters were cultured to assess biofilm formation, while isolates were tested for biofilm-forming ability using Congo Red Agar (CRA). Results: The mean catheterization duration was 5.6 ± 2.1 days, and most patients were catheterized for ≥5 days. Bacteriuria was significantly associated with catheterization (p = 0.029). Antibiotic use reduced bacteriuria incidence (p < 0.001) but did not significantly reduce biofilm formation (73.4%, p > 0.05). CRA-positive isolates showed 1.5-fold greater biofilm-forming potential. Female sex, early bacteriuria, and catheterization >5 days were associated with biofilm formation. Conclusion: Urinary catheterization promotes biofilm and increases CAUTI risk. Although antibiotics reduced bacteriuria, they showed limited efficacy against biofilms. Early bacteriuria screening, routine monitoring, biofilm prevention, and limit- ing catheter duration may help reduce CAUTI incidence.
Background and Objectives: As an important opportunistic pathogen, Pseudomonas aeruginosa is associated with severe infections and increasing antimicrobial resistance. The growing prevalence of multidrug-resistant (MDR) strains has re- newed interest in bacteriophages as alternative antibacterial agents. The present study sought to isolate and characterize lytic bacteriophages from hospital wastewater active against antibiotic-resistant clinical P. aeruginosa isolates. Materials and Methods: Fifty clinical isolates of P. aeruginosa were obtained and tested for antimicrobial susceptibility. Bacteriophages were isolated from hospital wastewater by enrichment and spot assay screening, followed by plaque purifi- cation and titration using the double-layer agar method. The host range of purified phages was evaluated against the clinical isolates. Adsorption assays and one-step growth experiments were performed to determine adsorption efficiency, latent peri- od, and burst size. Transmission electron microscopy (TEM) was used to examine phage morphology. Results: A considerable proportion of the clinical isolates showed multidrug-resistant phenotypes, and a considerable num- ber were also classified as extensively drug-resistant. Four lytic bacteriophages, designated HSP1-HSP4, were successfully isolated and purified. All produced clear plaques and reached high titers. Host range analysis showed variable lytic spectra, with HSP1 exhibiting the broadest activity and showing lytic activity against 70% of the tested isolates, followed by HSP2 (58%), HSP3 (48%), and HSP4 (40%). Adsorption analysis indicated that over 80% of phage particles attached to their host cells within 10 minutes, while HSP1 exhibited the highest adsorption efficiency at 93.6%. A short latent period of approxi- mately 20 minutes was observed for all four phages, and burst sizes ranged from 56 to 65 PFU/cell. Transmission electron microscopy revealed isometric heads and contractile tails, morphologically consistent with tailed phages of the class Cau- doviricetes. Conclusion: Hospital wastewater can serve as a practical source for the isolation of lytic bacteriophages active against anti- biotic-resistant P. aeruginosa. The in vitro properties of the isolated phages support their further evaluation as candidate an- tibacterial agents against resistant P. aeruginosa. However, genomic characterization, phage-antibiotic interaction analyses, and in vivo investigations are required before their broader applicability can be determined.
Background and Objectives: Bladder cancer (BCa) is the most prevalent malignancy of the urinary tract and is often diagnosed at a late stage. Recurrence remains a major challenge for current treatment strategies. Over the past decade, the antitumor potential of oncolytic viruses (OVs) has been increasingly recognized, with several studies evaluating their safety and efficacy in BCa treatment. In this systematic review, we present clinical trials and animal studies to comprehensively evaluate the safety and therapeutic efficiency of various OVs in BCa. Materials and Methods: After receiving the PROSPERO registration code, four electronic databases PubMed, Web of Sci- ence, Scopus, and Embase, were systematically searched to identify studies evaluating the safety and treatment potential of OVs in BCa. Two reviewers independently conducted the screening process, and data from clinical trials and animal studies were systematically extracted into Excel for subsequent synthesis and reporting. All steps of this systematic review adhered to the guidelines outlined in the Cochrane Handbook for Systematic Reviews of Interventions, upholding high standards of methodological rigor and transparency. Results: The initial search strategy yielded 1,061 records. After removing duplicates, 809 articles were screened for rel- evance. Following the exclusion of 761 studies, 48 were deemed eligible for data extraction, comprising 17 clinical trials and 29 animal studies. Various oncolytic viruses were investigated, either as monotherapies or in combination with other agents, including Vaccinia virus, Adenovirus, Oncolytic adenovirus ONYX, Coxsackievirus A21 (CVA21), oncolytic CVA21 (V937), attenuated measles virus (MV-NIS virus), recombinant adenovirus (rAd), and a serotype 5 adenovirus engineered to express GM-CSF. All studies reported their most effective doses administered within safe parameters. Conclusion: The common virus was the non-replicative recombinant adenovirus serotype 5 (Ad5), which encodes human interferon alfa-2b (IFNα2b) a cytokine with anti-tumor properties. It is administered via intravesical instillation and has received FDA approval
Background and Objectives:Non-tuberculous mycobacteria (NTM), especially Mycobacterium abscessus, are emerging causes of pulmonary disease and are often misdiagnosed as tuberculosis in high TB burden countries. Conventional culture methods are time-consuming and may reduce viable organism recovery. Rapidly growing mycobacteria (RGM) medium enables direct inoculation of respiratory specimens and improved recovery of rapidly growing NTM. This study evaluated the utility of RGM medium for isolation of M. abscessus from presumptive pulmonary tuberculosis patients and characterized the isolates using molecular and phylogenetic analyses. Materials and Methods:A total of 223 sputum samples collected from presumptive pulmonary tuberculosis patients. Untreated sputum samples were inoculated onto RGM medium with selective antibiotics and incubated at 30°C for 10 days. Suspected colonies were confirmed by Ziehl-Neelsen staining. Identification was performed by PCR and sequencing of hsp65 and rpoB genes. Phylogenetic analysis was done using the Neighbour-Joining method in MEGA12. Mycobacterium abscessus isolates were further analysed using the GenoType NTM-DR assay. Results:Among the 223 sputum samples processed, 7.6% (17/223) demonstrated positive growth on RGM medium. Of the culture-positive isolates, 64.7% (11/17) were identified as Mycobacterium abscessus, while 35.3% (6/17) were identified as other non-tuberculous mycobacterial (NTM) species. All isolates were positive for the rpoB and hsp65 genes. Eleven M. abscessus isolates were further analysed using the GenoType NTM-DR assay, and no resistance-associated mutations were detected in the erm(41), rrl, or rrs genes. Additionally, all isolates were found to be susceptible to macrolides and aminoglycosides. Conclusion:RGM medium demonstrated good utility for recovery of clinically significant rapidly growing mycobacteria, particularly M. abscessus, directly from sputum samples. Molecular and phylogenetic analyses enabled accurate species identification and characterization. The absence of resistance-associated mutations suggests retained susceptibility to key therapeutic agents. These findings support the use of RGM medium for improved detection of pulmonary NTM infections in TB-endemic settings.
Background and Objectives: MALDI-TOF MS offers a unique combination of flexibility, accuracy, automation and has found benefit in many fields of biological research. Here we investigated MALDI-TOF analysis for the identification of anaerobic and microaerophilic bacteria isolated from clinical specimen and also compared the results with conventional biochemical characterization. Materials and Methods: The present study was conducted for a duration of 2 years and had obtained Ethical clearance. Bacteria obtained from 1514 specimens were subjected to MALDI-TOF in comparison with conventional techniques. MAL- DI-TOF was done by Extended Direct Transfer technique after a preparatory protein extraction. Results: Of the 957 isolates of anaerobes and 214 microaerophilic organisms, 552 anaerobes and 154 microaerophilic iso- lates were identified up to species level giving concordant results by conventional techniques. Rest of the strains could be identified only up to genus level by biochemical characterization. MALDI-TOF MS was able to identify more isolates to the species level compared with conventional methods, including several new genera and species including Clostridia, Peptost- reptococcus and Lactobacilli. Conclusion: Newly recognized and taxonomically rearranged genera, frequently isolated anaerobic and microaerophilic species as well as closely related species could be identified accurately by employing MALDI-TOF MS. Implementation of this technique for identification will shorten the Turn Around Time, reduce the cost in the Microbiology laboratory and improve the patient care with better clinical outcome
Brucellosis, endemic in the Middle East and similar regions, can present with pancytopenia, complicating diagnosis and treatment. We report a 62-year-old female with fever, fatigue, and weight loss. Persistent pancytopenia required prolonged triple antibiotic therapy. Clinicians must consider brucellosis-related pancytopenia and tailor therapy with careful monitoring.
Background and Objectives:Escherichia coli is the leading cause of urinary tract infections (UTIs) and has shown increasing resistance to trimethoprim-sulfamethoxazole (TMP-SMX). Resistance to TMP-SMX is commonly associated with the acquisition of resistance genes such as dfr and sul, often mediated by horizontal gene transfer. This study aimed to characterize the presence of dfr (dfrA1, dfrA5, dfrA7/17) and sul (sul1 and sul2) genes and to describe sequence variations in selected E. coli isolates. Materials and Methods:Urine culture samples were obtained from 678 patients suspected of having UTIs, and 21 samples yielded positive culture results. This study further analyzed 11 E. coli isolates, consisting of eight TMP-SMX-resistant isolates, two susceptible isolates, and one E. coli ATCC 25922 strain as a control. The clinical isolates were provided by the Clinical Microbiology Laboratory, Faculty of Medicine, Universitas Indonesia. PCR amplification and agarose gel electrophoresis were used to identify dfr and sul genes, followed by Sanger sequencing of selected amplicons. Sequence data were analyzed with BioEdit software and aligned against reference sequences from NCBI. Results:The distribution of resistance genes varies among resistant isolates. Some isolates carry the dfrA5 gene, while dfrA1 was detected in one isolate. The sul2 gene was present in a number of resistant isolates, while sul1 was identified but showed no differences from the reference sequence. Further sequence analysis revealed amino acid changes in the dfrA1, dfrA5, and sul2 genes, although these changes were not consistently found in all isolates. Conclusion:This study characterizes the distribution of dfr and sul genes and their sequence variations in a limited number of E. coli isolates. The results suggest that TMP-SMX-resistant isolates may contain diverse genetic determinants associated with resistance. Nevertheless, the limited isolate number restricts the broader interpretation of these findings. Larger-scale studies, supported by functional analyses, are required to determine the contribution of these genes and their sequence variations to antibiotic resistance.
