Clinical Isolates Research Articles

Antimicrobial resistance and population genomics of emerging multidrug-resistant Salmonella 4,[5],12:i:- in Guangdong, China.

Salmonella 4,[5],12:i:-, a monophasic variant of Salmonella Typhimurium, has emerged as a global cause of multidrug-resistant salmonellosis and has become endemic in many developing and developed countries, especially in China. Here, we have sequenced 352 clinical isolates in Guangdong, China, during 2009-2019 and performed a large-scale collection of Salmonella 4,[5],12:i:- with whole genome sequencing (WGS) data across the globe, to better understand the population structure, antimicrobial resistance (AMR) genomic characterization, and transmission routes of Salmonella 4,[5],12:i:- across Guangdong. Salmonella 4,[5],12:i:- strains showed broad genetic diversity; Guangdong isolates were found to be widely distributed among the global lineages. Of note, we identified the formation of a novel Guangdong clade (Bayesian analysis of population structure lineage 1 [BAPS1]) genetically diversified from the global isolates and likely emerged around 1990s. BAPS1 exhibits unique genomic features, including large pan-genome, decreased ciprofloxacin susceptibility due to mutation in gyrA and carriage of plasmid-mediated quinolone resistance (PMQR) genes, and the multidrug-resistant IncHI2 plasmid. Furthermore, high genetic similarity was found between strains collected from Guangdong, Europe, and North America, indicating the association with multiple introductions from overseas. These results suggested that global dissemination and local clonal expansion simultaneously occurred in Guangdong, China, and horizontally acquired resistance to first-line and last-line antimicrobials at local level, underlying emergences of extensive drug and pan-drug resistance. Our findings have increased the knowledge of global and local epidemics of Salmonella 4,[5],12:i:- in Guangdong, China, and provided a comprehensive baseline data set essential for future molecular surveillance.IMPORTANCESalmonella 4,[5],12:i:- has been regarded as the predominant pandemic serotype causing diarrheal diseases globally, while multidrug resistance (MDR) constitutes great public health concerns. This study provided a detailed and comprehensive genome-scale analysis of this important Salmonella serovar in the past decade in Guangdong, China. Our results revealed the complexity of two distinct transmission modes, namely global transmission and local expansion, circulating in Guangdong over a decade. Using phylogeography models, the origin of Salmonella 4,[5],12:i:- was predicted from two aspects, year and country, that is, Salmonella 4,[5],12:i:- emerged in 1983, and was introduced from the UK, and subsequently differentiated into the local endemic lineage circa 1991. Additionally, based on the pan-genome analysis, it was found that the gene accumulation rate in local endemic BAPS 1 lineage was higher than in other lineages, and the horizontal transmission of MDR IncHI2 plasmid associated with high resistance played a major role, which showed the potential threat to public health.

Species distribution and antimicrobial susceptibility of Burkholderia cepacia complex isolates in clinical infections: Experience from a tertiary care hospital, Southern India

PurposeBurkholderia cepacia complex (Bcc) is a diverse group of environmental bacteria associated with opportunistic infections. The identification of Bcc using conventional methods poses challenges. Bcc infections are difficult to treat due to intrinsic antibiotic resistance. The study aimed to investigate the species distribution and antimicrobial susceptibility of clinical Bcc isolates. MethodsA total of 153 Bcc isolates obtained from clinical samples were analysed. Species identification was carried out using automated methods, including MALDI-TOF MS and VITEK2. Antimicrobial susceptibility testing was performed using the disc diffusion method. ResultsBurkholderia cenocepacia (70.5%) emerged as the most prevalent species, followed by Burkholderia contaminans (9.8%) and Burkholderia cepacia (7.2%). Ventilator-associated pneumonia (38.6%) was the most common infection, followed by sepsis (28.1%). Co-existence of Bcc with other pathogens in many cases suggested potential co-infection scenarios. Antimicrobial susceptibility revealed that ceftazidime, co-trimoxazole and meropenem were the most effective drugs, while levofloxacin proved to be the least effective. Moderate susceptibility was noted to minocycline, with 4.6% of isolates exhibiting multi-drug resistance. ConclusionThis study provides valuable insights into the prevalence, clinical associations, and antibiotic susceptibility of Bcc in India. It highlights the importance of Bcc as a nosocomial pathogen, especially in vulnerable patient populations. The findings contribute to understanding Bcc infections, their distribution, and emphasize the necessity for accurate identification methods in clinical settings.

Multifunctional in vitro, in silico and DFT analyses on antimicrobial BagremycinA biosynthesized by Micromonospora chokoriensis CR3 from Hieracium canadense

Among the actinomycetes in the rare genera, Micromonospora is of great interest since it has been shown to produce novel therapeutic compounds. Particular emphasis is now on its isolation from plants since its population from soil has been extensively explored. The strain CR3 was isolated as an endophyte from the roots of Hieracium canadense, and it was identified as Micromonospora chokoriensis through 16S gene sequencing and phylogenetic analysis. The in-vitro analysis of its extract revealed it to be active against the clinical isolates of methicillin-resistant Staphylococcus aureus (MRSA) and Candida tropicalis (15 mm). No bioactivity was observed against Gram-negative bacteria, Escherichia coli ATCC 25922, and Klebsiella pneumoniae ATCC 706003. The Micromonospora chokoriensis CR3 extract was also analyzed through the HPLC-DAD-UV–VIS resident database, and it gave a maximum match factor of 997.334 with the specialized metabolite BagremycinA (BagA). The in-silico analysis indicated that BagA strongly interacted with the active site residues of the sterol 14-α demethylase and thymidylate kinase enzymes, with the lowest binding energies of − 9.7 and − 8.3 kcal/mol, respectively. Furthermore, the normal mode analysis indicated that the interaction between these proteins and BagA was stable. The DFT quantum chemical properties depicted BagA to be reasonably reactive with a HOMO-LUMO gap of (ΔE) of 4.390 eV. BagA also passed the drug-likeness test with a synthetic accessibility score of 2.06, whereas Protox-II classified it as a class V toxicity compound with high LD50 of 2644 mg/kg. The current study reports an endophytic actinomycete, M. chokoriensis, associated with H. canadense producing the bioactive metabolite BagA with promising antimicrobial activity, which can be further modified and developed into a safe antimicrobial drug.

Immortalized hepatocyte-like cells: A competent hepatocyte model for studying clinical HCV isolate infection.

More than 58 million individuals worldwide are inflicted with chronic HCV. The disease carries a high risk of end stage liver disease, i.e., cirrhosis and hepatocellular carcinoma. Although direct-acting antiviral agents (DAAs) have revolutionized therapy, the emergence of drug-resistant strains has become a growing concern. Conventional cellular models, Huh7 and its derivatives were very permissive to only HCVcc (JFH-1), but not HCV clinical isolates. The lack of suitable host cells had hindered comprehensive research on patient-derived HCV. Here, we established a novel hepatocyte model for HCV culture to host clinically pan-genotype HCV strains. The immortalized hepatocyte-like cell line (imHC) derived from human mesenchymal stem cell carries HCV receptors and essential host factors. The imHC outperformed Huh7 as a host for HCV (JFH-1) and sustained the entire HCV life cycle of pan-genotypic clinical isolates. We analyzed the alteration of host markers (i.e., hepatic markers, cellular innate immune response, and cell apoptosis) in response to HCV infection. The imHC model uncovered the underlying mechanisms governing the action of IFN-α and the activation of sofosbuvir. The insights from HCV-cell culture model hold promise for understanding disease pathogenesis and novel anti-HCV development.

Molecular Diagnostics of Cryptococcus spp. and Immunomics of Cryptococcosis-Associated Immune Reconstitution Inflammatory Syndrome

Cryptococcal infection poses a significant global public health challenge, particularly in regions near the equator. In this review, we offer a succinct exploration of the Cryptococcus spp. genome and various molecular typing methods to assess the burden and genetic diversity of cryptococcal pathogens in the environment and clinical isolates. We delve into a detailed discussion on the molecular pathogenesis and diagnosis of immune reconstitution inflammatory syndrome (IRIS) associated with cryptococcosis, with a specific emphasis on cryptococcal meningitis IRIS (CM-IRIS). Our examination includes the recent literature on CM-IRIS, covering host cellulomics, proteomics, transcriptomics, and genomics.

Antiseptic management of critical wounds: differential bacterial response upon exposure to antiseptics and first insights into antiseptic/phage interactions - experimental studies.

With the antibiotic crisis, the topical antibacterial control including chronic wounds gains increasing importance. However, little is known regarding tolerance development when bacteria face repetitive exposure to the identical antiseptics as commonly found in clinical practice. We exposed clinical isolates foremost of chronic wounds in vitro to dilutions of two antisepctics used for wound therapy: polyhexanide or octenidine. Adaptive response was determined by growth/kill curves, minimal inhibitory concentration (MIC), and whole genome sequencing. Antiseptic/bacteriophage combinations were studied by liquid-infection assays and bacterial plating. Polyhexanide acted stronger against Escherichia coli and Proteus mirabilis while octenidine was more potent against Staphylococcus aureus. Otherwise, the antiseptic efficacy varied across isolates of Klebsiella pneumoniae, Pseudomonas aeruginosa, and Acinetobacter baumannii. Upon repetitive exposure with constant antiseptic concentrations P. aeruginosa and P. mirabilis adaptation was evident by a reduced lag-phase and a two-fold increased MIC. Under increasing octenidine concentrations, P. aeruginosa adapted to an eightfold higher dosage with mutations in smvA, opgH and kinB affecting an efflux pump, alginate and biofilm formation, respectively. S. aureus adapted to a fourfold increase of polyhexanide with a mutation in the multiple peptide resistance factor MprF, also conferring cross-resistance to daptomycin. Antiseptic/bacteriophage combinations enhanced bacterial inhibition and delayed adaptation. Different bacterial species/strains respond unequally to low-level antiseptic concentrations. Bacterial adaptation potential at phenotypic and genotypic levels may indicate the necessity for a more nuanced selection of antiseptics. Bacteriophages represent a promising yet underexplored strategy for supporting antiseptic treatment which may be particularly beneficial for the management of critical wounds.

Molecular genetic determinants of virulence of Streptococcus agalactiae isolated from pregnant women and newborns in St. Petersburg and the Leningrad region in 2010–2023

Introduction. Colonization of the reproductive organs of pregnant women with group B streptococci (GBS; Streptococcus agalactiae) can lead to severe perinatal and neonatal pathology. In modern conditions, aside from the prevention of antenatal infection of the fetus during childbirth using antibacterial drugs, vaccination is also necessary. In this regard, surveillance of GBS genotypes is an important task. Objective. To determine the molecular genetic determinants of virulence of Streptococcus agalactiae isolated from pregnant women and newborns, and to monitor the distribution of capsular polysaccharides types and pili profiles in GBS clinical isolates. Materials and methods. The study used clinical isolates of GBS (n = 420) isolated from pregnant women and newborns in 2010–2023.The bacteriological method was used for isolation of S. agalactiae. PCR method was used to determine the types of capsular polysaccharides, pili, and whether the strains belonged to the hypervirulent sequence type ST-17. Results. During 13 years of observation, the predominance of Ia, III and V genotypes of GBS capsular polysaccharides was noted both in pregnant women and in newborns. The frequency of occurrence of genotype Ib increased from 0.7 to 6.7%, genotype V from 12.1 to 24.4%, and the prevalence of genotype III decreased significantly from 41.1 to 21.1%. Hypervirulent sequence type ST-17 was detected in 6 pregnant women and 2 newborns. However, there were no signs of neonatal infection in these children. More than half of all clinical isolates of S. agalactiae had pili genotypes PI-1 + PI-2a, as well as pili genotypes PI-2a and PI-1 + PI-2b. The distribution of pili types did not change over 13 years of the surveillance period. Conclusion. Surveillance of the GBS capsular polysaccharides and pili genotypes is necessary for the development of effective preventive vaccines.

Machine Learning Models Identify Inhibitors of New Delhi Metallo-β-lactamase.

The worldwide spread of the metallo-β-lactamases (MBL), especially New Delhi metallo-β-lactamase-1 (NDM-1), is threatening the efficacy of β-lactams, which are the most potent and prescribed class of antibiotics in the clinic. Currently, FDA-approved MBL inhibitors are lacking in the clinic even though many strategies have been used in inhibitor development, including quantitative high-throughput screening (qHTS), fragment-based drug discovery (FBDD), and molecular docking. Herein, a machine learning-based prediction tool is described, which was generated using results from HTS of a large chemical library and previously published inhibition data. The prediction tool was then used for virtual screening of the NIH Genesis library, which was subsequently screened using qHTS. A novel MBL inhibitor was identified and shown to lower minimum inhibitory concentrations (MICs) of Meropenem for a panel of E. coli and K. pneumoniae clinical isolates expressing NDM-1. The mechanism of inhibition of this novel scaffold was probed utilizing equilibrium dialyses with metal analyses, native state electrospray ionization mass spectrometry, UV-vis spectrophotometry, and molecular docking. The uncovered inhibitor, compound 72922413, was shown to be 9-hydroxy-3-[(5-hydroxy-1-oxa-9-azaspiro[5.5]undec-9-yl)carbonyl]-4H-pyrido[1,2-a]pyrimidin-4-one.

Metabolomics unveil key pathways underlying the synergistic activities of aztreonam and avibactam against multidrug-resistant Escherichia coli.

Aztreonam/avibactam is effective against serious infections caused by Gram-negative bacteria including Enterobacterales harboring metallo-β-lactamases. While the utility of this combination has been established in vitro and in clinical trials, the purpose of this study is to enhance our understanding of the underlying mechanism responsible for their activities through metabolomic profiling of a multidrug-resistant Escherichia coli clinical isolate. Metabolomic analyses of time-dependent changes in endogenous bacterial metabolites in a clinical isolate of a multidrug-resistant E. coli treated with aztreonam and avibactam were performed. E. coli metabolomes were compared at 15min, 1h and 24h following treatments with either avibactam (4mg/L), aztreonam (4mg/L), or aztreonam (4mg/L) + avibactam (4mg/L). Drug treatment affected 326 metabolites with magnitude changes of at least 2-fold, most of which are involved primarily in peptidoglycan biosynthesis, nucleotide metabolism, and lipid metabolism.The feedstocks for peptidoglycan synthesis were depleted by aztreonam/avibactam combination;a significant downstream increase in nucleotide metabolites and a release of lipids were observed at the three timepoints. The findings indicate that the aztreonam/avibactam combination accelerates structural damage to the bacterial membrane structure and their actions were immediate and sustained compared to aztreonam or avibactam alone. By inhibiting the production of crucial cell wall precursors, the combination may have inflicted damages on bacterial DNA.

Changing patterns of bacterial profile and antimicrobial resistance in high-risk patients during the COVID-19 pandemic at a tertiary oncology hospital

Changing patterns of bacterial profile and antimicrobial resistance in high-risk patients during the COVID-19 pandemic at a tertiary oncology hospital

Safety and efficacy evaluation of halicin as an effective drug for inhibiting intestinal infections

Halicin, the first antibacterial agent discovered by artificial intelligence, exerts broad-spectrum antibacterial effects and has a unique structure. Our study found that halicin had a good inhibitory effect on clinical isolates of drug-resistant strains and Clostridium perfringens (C. perfringens). The safety of halicin was evaluated by acute oral toxicity, genotoxicity and subchronic toxicity studies. The results of acute toxicity test indicated that halicin, as a low-toxicity compound, had an LD50 of 2018.3 mg/kg. The results of sperm malformation, bone marrow chromosome aberration and cell micronucleus tests showed that halicin had no obvious genotoxicity. However, the results of the 90-day subchronic toxicity test indicated that the test rats exhibited weight loss and slight renal inflammation at a high dose of 201.8 mg/kg. Teratogenicity of zebrafish embryos showed that halicin had no significant teratogenicity. Analysis of intestinal microbiota showed that halicin had a significant effect on the intestinal microbial composition, but caused a faster recovery. Furthermore, drug metabolism experiments showed that halicin was poorly absorbed and quickly eliminated in vivo. Our study found that halicin had a good therapeutic effect on intestinal infection model of C. perfringens. These results show the feasibility of developing oral halicin as a clinical candidate drug for treating intestinal infections.

Increased antibiotic resistance of Pseudomonas aeruginosa isolates from chronic rhinosinusitis patients grown in anaerobic conditions.

In chronic rhinosinusitis (CRS), the congestion and blockage of the nose can cause anaerobic conditions within the sinus cavities which may promote the expression of virulence and antibiotic resistance genes in invading pathogens. Pseudomonas aeruginosa is a facultative anaerobic bacteria and causes severe recalcitrant CRS. In this study, we aimed to evaluate the antimicrobial resistance of P. aeruginosa isolates of CRS patients in planktonic and biofilm form grown in aerobic and anaerobic conditions. P. aeruginosa clinical isolates of CRS patients (n = 25) were grown in planktonic and biofilm form in aerobic and anaerobic conditions. Minimum inhibitory concentrations (MIC) of planktonic forms and minimum biofilm eradication concentrations (MBEC) were determined. Additionally, metabolic activity by fluorescein diacetate assay, biofilm biomass by crystal violet assay and eDNA concentration were assessed in both conditions. P. aeruginosa planktonic cells grown in anaerobic condition exhibited increased gentamicin resistance (p < .01), whereas P. aeruginosa biofilms grown in anaerobic condition displayed significantly increased MBEC values for gentamicin (p < .0001) and levofloxacin (p < .001). The metabolic activity of anaerobic biofilms was significantly higher compared with aerobic biofilms (p < .0001). However, the biofilm biomass of isolates grown in aerobic conditions was higher than anaerobic conditions (p < .5). P. aeruginosa isolates from CRS patients grown in anaerobic conditions showed significantly increased resistance to antibiotics with an increased metabolic activity but decreased biofilm biomass. NA.

Proteome analysis, genetic characterization, and antibiotic resistance patterns of Klebsiella pneumoniae clinical isolates

Klebsiella pneumoniae (K. pneumoniae) is a member of the ESKAPE group and is responsible for severe community and healthcare-associated infections. Certain Klebsiella species have very similar phenotypes, which presents a challenge in identifying K. pneumoniae. Multidrug-resistant K. pneumoniae is also a serious global problem that needs to be addressed. A total of 190 isolates were isolated from urine (n = 69), respiratory (n = 52), wound (n = 48) and blood (n = 21) samples collected from various hospitals in the Al-Qassim, Saudi Arabia, between March 2021 and October 2022. Our study aimed to rapidly and accurately detect K. pneumoniae using the Peptide Mass Fingerprinting (PMF) technique, confirmed by real-time PCR. Additionally, screening for antibiotic susceptibility and resistance was conducted. The primary methods for identifying K. pneumoniae isolates were culture, Gram staining, and the Vitek® 2 ID Compact system. An automated MALDI Biotyper (MBT) instrument was used for proteome identification, which was subsequently confirmed using SYBR green real-time polymerase chain reaction (real-time PCR) and microfluidic electrophoresis assays. Vitek® 2 AST-GN66 cards were utilized to evaluate the antimicrobial sensitivity of K. pneumoniae isolates. According to our results, Vitek® 2 Compact accurately identified 178 out of 190 (93.68%) K. pneumoniae isolates, while the PMF technique correctly detected 188 out of 190 (98.95%) isolates with a score value of 2.00 or higher. Principal component analysis was conducted using MBT Compass software to classify K. pneumoniae isolates based on their structure. Based on the analysis of the single peak intensities generated by MBT, the highest peak values were found at 3444, 5022, 5525, 6847, and 7537 m/z. K. pneumoniae gene testing confirmed the PMF results, with 90.53% detecting entrobactin, 70% detecting 16 S rRNA, and 32.63% detecting ferric iron uptake. The resistance of the K. pneumoniae isolates to antibiotics was as follows: 64.75% for cefazolin, 62.63% for trimethoprim/sulfamethoxazole, 59.45% for ampicillin, 58.42% for cefoxitin, 57.37% for ceftriaxone, 53.68% for cefepime, 52.11% for ampicillin-sulbactam, 50.53% for ceftazidime, 52.11% for ertapenem, and 49.47% for imipenem. Based on the results of the double-disk synergy test, 93 out of 190 (48.95%) K. pneumoniae isolates were extended-spectrum beta-lactamase. In conclusion, PMF is a powerful analytical technique used to identify K. pneumoniae isolates from clinical samples based on their proteomic characteristics. K. pneumoniae isolates have shown increasing resistance to antibiotics from different classes, including carbapenem, which poses a significant threat to human health as these infections may become difficult to treat.

Role of Sertaconazole in the Treatment of Dermatophytosis

Dermatophytosis is a major health burden worldwide and is now increasing day by day. Dermatophytosis is also becoming increasingly unresponsive to topical conventional antifungals now a day. Newer topical antifungals may be more effective in these patients. Sertaconazole is a new, broad spectrum, fungicidal and fungistatic imidazole with added antipruritic and anti-inflammatory activity that may be effective and beneficial in improving the quality of life for the patient with dermatophytoses. It is indicated in the European Union(EU) for the treatment of superficial skin mycoses such as dermatophytosis (including tinea corporis, tinea cruris, tinea manus, tinea barbae and tinea pedis), cutaneous candidiasis, pityriasis versicolor and seborrhoeic dermatitis of the scalp, and in the US for tinea pedis only. Sertaconazole has broad-spectrum antifungal activity against dermatophytes of the Trichophyton, Epidermophyton and Microsporum genera, and yeasts of the genera Candida and Cryptococcus; additionally, it is effective against opportunistic filamentous fungi and Gram-positive bacteria. Moreover, the antifungal activity of sertaconazole is maintained in clinical isolates of dermatophytes that show reduced susceptibility to other azoles. While the drug has good dermal penetration, this is not associated with systemic absorption. In clinical trials in patients with superficial mycoses, 2% sertaconazole cream applied twice daily was effective in the eradication of a range of dermatophytoses, and a significantly greater proportion of patients were cured compared with those receiving 2% miconazole cream twice-daily treatment. Both as a topical cream and suppository preparation, sertaconazole was generally well tolerated. Sertaconazole is a well-established antifungal agent, which is now available in a variety of formulations, and remains a useful treatment option particularly in patients with fungal infections resistant to other azoles. Medicine Today 2023 Vol.36 (1): 62-66

MOLECULAR IDENTIFICATION AND GENOTYPING OF METHICILLIN-RESISTANT STAPHYLOCOCCUS AUREUS (MRSA) IN DIFFERENT CLINICAL SAMPLES

Methicillin-resistant Staphylococcus aureus (MRSA) is a serious human pathogen that can spread in healthcare facilities and among the general public. This study was aimed to evaluate the prevalence and diversity of SCCmec types of this superbug among hospitalized patients. This study involved phenotypic identification and molecular confirmation of S. aureus based on the nuc gene, molecular detection of MRSA, SCCmec typing, and virulence factor profiling of MRSA clinical isolates obtained from hospitalized patients in Duhok province. Out of the 310 enrolled patients, 33 isolates (10.64%) were identified and confirmed as Staphylococcus aureus, of which 51.5% were identified as MRSA based on phenotypic and molecular targeting of the mecA gene. There were no discernible variations between the prevalence rates of this pathogen in different clinical sources, sexes, or age groups (p-values: 0.71, 0.39, and 0.15 respectively). The isolates had elevated rates of resistance to most antibiotic classes. They were classified as extensive drug-resistant (30.3%), multidrug-resistant (57.5%), and non-multidrug-resistant (12.1%). Additionally, SCCmec typing of MRSA by multiplex PCR identified three different SCCmec types and subtypes, including SCCmec type II (35.5%), followed by 17.64% of SCCmec type IV subtype d (IVd), and SCCmec type III (11.76%). However, 35.3% of the MRSA isolates were found to be non-typeable. Molecular profiling of major virulence factors and toxin genes revealed that 57.5% of the isolates were positive for the exfolitative toxin (ETA), 45.4% of the isolates carried TSST-1 (Toxic Shock Syndrome Toxin-1), the PVL (Panton-Valentine Leukocidin) cytotoxin was identified in 15% of the isolates, and 18.1% of the identified S. aureus isolates were positive for the ACME (arginine catabolic mobile element). The findings of the current investigation pointed out the circulating of highly virulent and extensively resistant MRSA strains among hospitalized patients, which may require active surveillance and better control policies

New synergistic benzoquinone scaffolds as inhibitors of mycobacterial cytochrome bc1 complex to treat multi-drug resistant tuberculosis

Through a comprehensive molecular docking study, a unique series of naphthoquinones clubbed azetidinone scaffolds was arrived with promising binding affinity to Mycobacterial Cytbc1 complex, a drug target chosen to kill multi-drug resistant Mycobacterium tuberculosis (MDR-Mtb). Five compounds from series-2, 2a, 2c, 2g, 2h, and 2j, showcased significant in vitro anti-tubercular activities against Mtb H37Rv and MDR clinical isolates. Further, synergistic studies of these compounds in combination with INH and RIF revealed a potent bactericidal effect of compound 2a at concentration of 0.39 μg/mL, and remaining (2c, 2g, 2h, and 2j) at 0.78 μg/mL. Exploration into the mechanism study through chemo-stress assay and proteome profiling uncovered the down-regulation of key proteins of electron-transport chain and Cytbc1 inhibition pathway. Metabolomics corroborated these proteome findings, and heightened further understanding of the underlying mechanism. Notably, in vitro and in vivo animal toxicity studies demonstrated minimal toxicity, thus underscoring the potential of these compounds as promising anti-TB agents in combination with RIF and INH. These active compounds adhered to Lipinski's Rule of Five, indicating the suitability of these compounds for drug development. Particular significance of molecules NQ02, 2a, and 2h, which have been patented (Published 202141033473).

Exploring the extrachromosomal plasmid rDNA of Naegleria fowleri AY27 genotype II: A human brain-eating amoeba via high-throughput sequencing

Naegleria fowleri, also known as brain-earing amoeba, causes severe and rapidly fatal CNS infection in humans called primary amebic meningoencephalitis (PAM). The DNA from the N. fowleri clinical isolate was sequenced for circular extrachromosomal ribosomal DNA (CERE - rDNA). The CERE contains 18 S, 5.8 S, and 28 S ribosomal subunits separated by internal transcribed spacers, 5 open reading frames (ORFs), and mostly repeat elements comprising 7268 bp out of 15,786 bp (46%). A wide variety of variations and recombination events were observed. Finally, the ORFs that comprised only 4 hypothetical proteins were modeled and screened against Zinc drug-like compounds. Two compounds [ZINC77564275 (ethyl 2-(((4-isopropyl-4 H-1,2,4-triazol-3-yl) methyl) (methyl)amino) oxazole-4-carboxylate) and ZINC15022129 (5-(2-methoxyphenoxy)-[2,2’-bipyrimidine]-4,6(1 H,5 H)-dione)] were finalized as potential druggable compounds based on ADME toxicity analysis. We propose that the compounds showing the least toxicity would be potential drug candidates after laboratory experimental validation is performed.

Antifungal activity of nisin against clinical isolates of azole-resistant Candida tropicalis

The rapid emergence of invasive infections caused by azole-resistant Candida tropicalis has become a public health concern, and there is an urgent need for alternative treatment strategies. Studies have demonstrated the antibacterial effects of nisin, a well-known peptide naturally produced by Lactococcus lactis subsp. lactis. However, there is scant information about the antifungal effect of nisin against C. tropicalis. The present study aims to investigate the in vitro antifungal activity of nisin against clinical isolates of azole-resistant C. tropicalis strains, as well as its inhibitory effect on biofilm formation. A total of 35 C. tropicalis strains isolated from patients with invasive fungal infections were divided into the azole-resistant group and the azole-sensitive group, containing 21 and 14 strains, respectively. The relative expression levels of the ERG11 and UPC2 genes in the azole-resistant group were higher than those in the azole-sensitive group (p &amp;lt; 0.0001), while no significant differences were observed in the expression levels of the MDR1 and CDR1 genes. The minimum inhibitory concentration of nisin against C. tropicalis ranged from 2 to 8 μg/mL. Nisin treatment inhibited the growth of azole-resistant C. tropicalis, with over a four-fold reduction in OD600 nm values observed at the 8-h time point, while it promoted the transition of C. tropicalis from the spore phase to the hyphal phase, as observed on cryo-scanning electron microscopy. The results of biofilm quantification using crystal violet staining indicated a significant decrease in OD570 nm values in the nisin-treated group compared to the controls (p &amp;lt; 0.0001). Among the 21 azole-resistant C. tropicalis strains, the biofilm formation was inhibited in 17 strains (17/21, 81%), and more than 85% inhibition of biofilm formation was observed in the representative strains. With regard to the molecular mechanisms, the expression of the BCR1 and UPC2 genes in the azole-resistant strains was down-regulated on nisin treatment (p &amp;lt; 0.05). In conclusion, we demonstrated, for the first time, that nisin has antifungal activity and significant anti-biofilm activity against clinical isolates of azole-resistant C. tropicalis strains. Based on the findings, nisin could be a promising alternative antifungal agent for combating azole-resistant C. tropicalis infections.

Detection of Biofilm Production and Antibiotic Susceptibility Pattern among Clinically Isolated Staphylococcus aureus.

The increasing antibiotic resistance and the ability to form biofilms in medical devices have become the leading cause of severe infections associated with Staphylococcus aureus (S. aureus). Since the bacteria living in biofilms can exhibit 10- to 1,000-fold increase in antibiotic resistance and implicate chronic infectious diseases, the detection of S. aureus ability to form biofilms is of great importance for managing, minimizing, and effectively treating infections caused by it. This study aimed to compare the tube and tissue culture methods to detect biofilm production and antibiotic susceptibility in MRSA and MSSA. The S. aureus isolates were identified by the examination of the colony morphology, Gram staining, and various biochemical tests. Antimicrobial susceptibility testing of all isolates was performed by the modified Kirby-Bauer disc diffusion method as recommended by CLSI guidelines. MRSA screening was performed phenotypically using a cefoxitin disc (30 µg). Isolates were tested for inducible resistance using the D-test, and two phenotypic methods detected biofilm formation. Among 982 nonrepeated clinical specimens, S. aureus was isolated from 103 (10.48%). Among 103 clinical isolates of S. aureus, 54 (52.42%) isolates were MRSA, and 49 (47.57%) were MSSA. Among 54 MRSA isolates, the inducible MLSB phenotype was observed in 23/54 (42.59%) with a positive D-test. By TCP method, 26 (48.1%) MRSA isolates were strong biofilm producers, whereas, among all MSSA isolates, only 6 (12.2%) were strong biofilm producers. MRSA showed strong biofilm production in comparison with MSSA. The TCP method is a recommended reliable method to detect the biofilm among S. aureus isolates, and the TM method could be useful for the screening of biofilm production in S. aureus in the routine clinical laboratory.

Synthetic phage-based approach for sensitive and specific detection of Escherichia coli O157

Escherichia coli O157 can cause foodborne outbreaks, with infection leading to severe disease such as hemolytic-uremic syndrome. Although phage-based detection methods for E. coli O157 are being explored, research on their specificity with clinical isolates is lacking. Here, we describe an in vitro assembly-based synthesis of vB_Eco4M-7, an O157 antigen-specific phage with a 68-kb genome, and its use as a proof of concept for E. coli O157 detection. Linking the detection tag to the C-terminus of the tail fiber protein, gp27 produces the greatest detection sensitivity of the 20 insertions sites tested. The constructed phage detects all 53 diverse clinical isolates of E. coli O157, clearly distinguishing them from 35 clinical isolates of non-O157 Shiga toxin-producing E. coli. Our efficient phage synthesis methods can be applied to other pathogenic bacteria for a variety of applications, including phage-based detection and phage therapy.