Common Clinical Isolates Research Articles

A ubiquitin-mediated post-translational degradation of Cyp51A contributes to a novel azole resistance mode in Aspergillus fumigatus

The airborne fungus Aspergillus fumigatus is a major pathogen that poses a serious health threat to humans by causing aspergillosis. Azole antifungals inhibit sterol 14-demethylase (encoded by cyp51A), an enzyme crucial for fungal cell survival. However, the most common mechanism of azole resistance in A. fumigatus is associated with the mutations in cyp51A and tandem repeats in its promoter, leading to reduced drug-enzyme interaction and overexpression of cyp51A. It remains unknown whether post-translational modifications of Cyp51A contribute to azole resistance. In this study, we report that the Cyp51A expression is highly induced upon exposure to itraconazole, while its ubiquitination level is significantly reduced by itraconazole. Loss of the ubiquitin-conjugating enzyme Ubc7 confers resistance to multiple azole antifungals but hinders hyphal growth, conidiation, and virulence. Western blot and immunoprecipitation assays show that deletion of ubc7 reduces Cyp51A degradation by impairing its ubiquitination, thereby leading to drug resistance. Most importantly, the overexpression of ubc7 in common environmental and clinical azole-resistant cyp51A isolates partially restores azole sensitivity. Our findings demonstrate a non-cyp51A mutation-based resistance mechanism and uncover a novel role of post-translational modification in contributing to azole resistance in A. fumigatus.

Detection and characterization of eravacycline heteroresistance in clinical bacterial isolates.

Eravacycline (ERV) has emerged as a therapeutic option for the treatment of carbapenem-resistant pathogens. However, the advent of heteroresistance (HR) to ERV poses a challenge to these therapeutic strategies. This study aimed to investigate ERV HR prevalence among common clinical isolates and further characterize ERV HR in carbapenem-resistant Klebsiella pneumoniae (CRKP). A total of 280 clinical pathogens from two centers were selected for HR and analyzed using population analysis profiling (PAP) and modified E-tests. The PAP assay revealed an overall ERV HR prevalence of 0.7% (2/280), with intermediate heterogeneity observed in 24.3% (68/280) of strains. The proportion of heteroresistant strains was 18.3% according to modified E-test results. A time-killing assay demonstrated that CRKP CFU increased significantly after 10 h of ERV treatment, contributing to the reduced bactericidal effect of ERV in vitro. Interestingly, dual treatment with ERV and polymyxin B effectively inhibited the total CFU, simultaneously reducing the required polymyxin B concentration. Furthermore, fitness cost measurements revealed a growth trade-off in CRKP upon acquiring drug resistance, highlighting fitness costs as crucial factors in the emergence of ERV HR in CRKP. Overall, the findings of the current study suggest that ERV HR in clinical strains presents a potential obstacle in its clinical application.

A mechanism study on the synergistic effects of rifapentine and fluconazole against fluconazole-resistant Candida albicans in vitro

Candida albicans (C. albicans) is one of the most common clinical isolates of systemic fungal infection. Long-term and inappropriate use of antifungal drugs can cause fungal resistance, which poses a great challenge to the clinical treatment of fungal infections. The combination of antifungal drugs and non-antifungal drugs to overcome the problem of fungal resistance has become a research hotspot in recent years. Our previous study found that the combination of rifapentine (RFT) and fluconazole (FLC) has a significant synergistic against FLC-resistant C. albicans. The present study aimed to further verify the synergistic effect between FLC and RFT against the FLC-resistant C. albicans 100, and explore the underlying mechanism. The growth curve and spot assay test not only showed the synergistic effect of FLC and RFT on FLC-resistant C. albicans in vitro but exhibited a dose-dependent effect on RFT, indicating that RFT may play a principal role in the synergic effect of the two drugs. Flow cytometry showed that the combined use of RFT and FLC arrested cells in the G2/M phase, inhibiting the normal division and proliferation of FLC-resistant C. albicans. Transmission electron microscopy (TEM) demonstrated that FLC at a low concentration could still cause a certain degree of damage to the cell membrane in the FLC-resistant C. albicans, as represented by irregular morphologic changes and some defects observed in the cell membrane. When FLC was used in combination with RFT, the nuclear membrane was dissolved and the nucleus was condensed into a mass. Detection of the intracellular drug concentration of fungi revealed that the intracellular concentration of RFT was 31–195 fold that of RFT alone when it was concomitantly used with FLC. This indicated that FLC could significantly increase the concentration of RFT in cells, which may be due to the damage caused to the fungal cell membrane by FLC. In short, the present study revealed a synergistic mechanism in the combined use of RFT and FLC, which may provide a novel strategy for the clinical treatment of FLC-resistant C. albicans.

Comparative antibiogram of annual antimicrobial susceptibility trend in a tertiary care institution in Telangana with ICMR annual AMR report for the year 2021

Introduction: Antibiogram data can improve hospital antibiotic formulary decisions and local protocols such as surgical prophylaxis or empirical treatment guidelines. This study aims to compare the antimicrobial resistance (AMR) trends observed in our institution with the national data published by the Indian Council of Medical Research (ICMR). Methods: The annual antibiogram of all common clinical isolates received in the hospital in 2021 was constructed as per the consensus guidelines developed by the Clinical and Laboratory Standards Institute, and the antimicrobial susceptibility trends were compared with the Indian Council of Medical Research annual Antimicrobial Resistance Report for 2021. Results: A total of 719 first isolates (excluding duplicate isolates) of common organisms viz., Escherichia coli (n = 198), Klebsiella pneumoniae (n = 164), Pseudomonas aeruginosa (n = 70), methicillin-sensitive Staphylococcus aureus (MSSA) (n = 174), methicillin-resistant Staphylococcus aureus (MRSA) (n = 39), Enterococcus species (n = 74) were analyzed and described as in charts. Discussion: The percentage susceptibility of gram-positive isolates to vancomycin, linezolid, tetracycline, nitrofurantoin, and fosfomycin was comparable to the national ICMR AMR report, whereas there were differences in the susceptibility rates to other antimicrobial agents such as penicillin, ampicillin, erythromycin, clindamycin, and co-trimoxazole. The percentage susceptibility of P. aeruginosa isolates was comparable to the national ICMR AMR report for all anti-pseudomonal agents tested and analyzed. The gram-negative isolates showed better susceptibility rates to the beta-lactam antibiotics, fluoroquinolones, and carbapenems in comparison to the national ICMR AMR report. Conclusion: Analysis of antibiograms from different regions of our country would show us the trends and patterns of antimicrobial resistance.

Antimicrobial resistance among common clinical isolates from Wayanad district

: The emerging multi-drug resistant variants in different clinical isolates is leading to increased morbidity and mortality, due failure in treatment. The paucity of an accurate data of antimicrobial resistance from different geographical areas is a major setback to its control and management. The aim of this study was to analyse the occurrence of drug resistant organisms from different clinical samples in the district of Wayanad, and also to determine the most prevalent and emerging bacterial pathogens among them. : A seven-month retrospective study of different bacterial isolates from various clinical samples was conducted in a tertiary care hospital in Wayanad district. Clinical samples taken for the study included urine, pus, sputum and blood. : Data from 2125 clinical samples were studied, in which 661 were urine samples, 910 were pus samples, 225 were blood samples and 339 were sputum samples. The predominant bacteria identified from urine sample was among which the prevalence of extended spectrum beta lactamase (ESBL) producing was 40.61%, and the metalobetalactamase (MBL) producers 3.45%. was the predominant bacteria in the pus samples, in which Methicillin resistant (MRSA) was found to be 3.92%. was the most predominant bacteria in the blood samples, in which ESBL producing was noted as 3.57%. Klebsiella species were the predominant bacteria in the sputum samples, in which ESBL producing was 16.79% and MBL producers were 3.82%.: The study helped to identify the most predominant antibiotic resistant strains from each of the clinical samples in a resource limited setting like Wayanad. Similar studies would help in successfully formulating treatment strategies against bacterial infections, thereby reducing morbidity and mortality in patients.

Comparison of Autof Ms1000 and EXS3000 MALDI-TOF MS Platforms for Routine Identification of Microorganisms.

Matrix-assisted laser desorption-ionization-time of flight mass spectrometry (MALDI-TOF) has recently been widely used in clinical microbiology laboratories, with the advantages of being reliable, rapid, and cost-effective. Here, we reported the performance of two MALDI-TOF MS instruments, EXS3000 (Zybio, China) and Autof ms1000 (Autobio, China), which are commonly used in clinical microbiology field. A total of 209 common clinical common isolates, including 70 gram-negative bacteria strains, 58 gram-positive bacteria strains, 33 yeast strains, 15 anaerobic bacteria strains, and 33 mold strains, and 19 mycobacterial strains were tested. All strains were identified by EXS3000 (Zybio, China) and Autof ms1000 (Autobio, China). Sequence analysis of 16S rRNA or ITS regions was used to verify all strains. Current study found that species-level discrimination was found to be 191 (91.39%) and 190 (90.91%) by EXS3000 and Autof ms1000, respectively. Genus-level discrimination was 205 (98.09%) by the EXS3000 and 205 (98.09%) by the Autof ms1000, respectively. The correct results at species level of the EXS3000 were 91.43% (64/70) for gram-negative bacteria, 93.1% (54/58) for gram-positive cocci, 93.94% (31/33) for yeast, 100% (15/15) for anaerobes and 81.82% (27/33) for filamentous fungi. The correct results at species level of the Autof ms1000 were 92.86% (65/70) for gram-negative bacteria, 91.38% (53/58) for gram-positive cocci, 93.94% (31/33) for yeast, 100% (15/15) for anaerobes and 78.79% (26/33) for filamentous fungi. Although the results show that the EXS3000 and Autof ms1000 systems are equally good choices in terms of analytical efficiency for routine procedures, the test result of EXS3000 is slightly better than Autof ms1000. It's worth mentioning that the target plate of the EXS 3000 instrument is reusable, but the target plate of the Autof ms1000 is disposable, making the EXS3000 more effective in reducing costs.

S5.3a Unraveling the genetic determinants of virulence in Cryptococcus neoformans

S5.3 Cellular pleomorphism and fungal virulence, September 22, 2022, 3:00 PM - 4:30 PM Cryptococcus neoformans is a human pathogenic basidiomycete yeast that can cause cryptococcal meningitis (CM), predominantly in immunocompromised individuals. The patient outcome depends on both host and pathogen-specific factors, including C. neoformans genetics. A groundbreaking 2012 study was the first to show that patient outcome is associated with genetic differences between C. neoformans isolates. Subsequent population-wide sequencing studies have revealed over 100 sequence types (ST) of C. neoformans that are associated with both geographic location and clinical outcome. All these studies have been broad, examining the severity of disease cryptococcal phenotypes in a collection of highly diverse strains. We chose a narrow focus and collected various genotypic and phenotypic data from a single ST: ST93. ST93 is a common sequence type isolated from patients globally and is the most common clinical isolate found in the sub-Saharan African country of Uganda. Previously, we performed whole genome sequencing on 38 ST93 Ugandan clinical isolates. We identified 652 unique SNPs in this ST93 population compared to the H99 reference genome. We also showed that ST93 contained two subpopulations: ST9A and ST93B. In the current study, we further characterized the genotypic, phenotypic, and virulence differences between these 38 clinical isolates. Using Illumina sequence data, we identified a pattern of linkage disequilibrium that suggested that ST93A and ST93B are evolving separately. We performed long-read sequencing on each isolate to investigate chromosomal changes and large structural variations, allowing us to identify a chromosomal translocation event wherein parts of chromosome 11 had recombined with chromosome 3. Additionally, we characterized several in vitro phenotypes for each isolate and identified three distinct phenotypic clusters based on cell wall challenge and growth experiments. Next, we infected mice with 35 isolates and observed eight different disease manifestations, including isolates that caused non-CNS infections. Overall, by working within a single sequence type, we can gain a deeper understanding of how some small genetic changes can impact strain-specific phenotypes while others have no discernable effect. Eventually, these data can be used to provide valuable information about how each clinical isolate impacts patient outcomes.

Antimicrobial susceptibility and minimum inhibitory concentration distribution of common clinically relevant non-tuberculous mycobacterial isolates from the respiratory tract

Objective: To determine the minimum inhibitory concentration (MIC) distribution of antibacterial drugs and the susceptibility of non-tuberculous mycobacterial (NTM) isolates to provide a reference basis for the clinical selection of an effective starting regimen. Methods: The common clinical isolates of NTM in the respiratory tract, which met the standards of the American Thoracic Society for NTM lung disease, were collected. The MICs of 81 isolates were determined using the microbroth dilution method (Thermo Fisher Scientific, USA), as recommended by the Clinical and Laboratory Standards Institute, USA. Results: Included were 43 Mycobacterium avium complex (MAC) strains, 24 M. abscessus complex (MAB) strains, and 14 M. kansasii strains. The sensitivity rates of MAC to clarithromycin and amikacin were 81.4% and 79.1%, respectively, while the sensitivity rates to linezolid and moxifloxacin were only 20.9% and 9.3%; the MIC of rifabutin was the lowest (MIC50% was just 2 μg/mL). After incubation for 3–5 days, the sensitivity rate of MAB to clarithromycin was 87.5%; this decreased to 50% after 14 days’ incubation. Most of them were susceptible to amikacin (91.6%), and most were resistant to moxifloxacin (95.8%), ciprofloxacin (95.8%), imipenem (95.8%), amoxicillin/clavulanate (95.8%), tobramycin (79.1%), doxycycline (95.8%) and trimethoprim/sulfamethoxazole (95.8%). intermediate (83.3%) and resistant (16.7%) to cefoxitin. The susceptibility to linezolid was only 33.3%. The sensitivity and resistance breakpoints of tigecycline were set to ≤0.5 and ≥8 μg/mL, respectively, and the sensitivity and resistance rates were 50% and 0%, respectively. M. kansasii was susceptible to clarithromycin, amikacin, linezolid, moxifloxacin, rifampicin and rifabutin (100%). Discussion: In Wenzhou, clarithromycin, amikacin and rifabutin have good antibacterial activity against MAC, while linezolid and moxifloxacin have high resistance. Amikacin and tigecycline have strong antibacterial activity against MAB, while most other antibacterial drugs are resistant to varying degrees. Most antibacterial drugs are susceptible to M. kansasii and have good antibacterial activity. Conclusion: The identification of NTM species and the detection of their MICs have certain guiding values for the treatment of NTM lung disease. Key Message The three most common respiratory non-tuberculous mycobacterial (NTM) isolates with clinical significance in the Wenzhou area were tested for drug susceptibility. The broth microdilution method was used to determine the minimum inhibitory concentration distribution of antibacterial drugs and the susceptibility of NTM isolates to provide a reference basis for the clinical selection of an effective starting regimen.

Combination Therapy with TCM Preparation Kumu Injection and Azithromycin against Bacterial Infection and Inflammation: In Vitro and In Vivo

Background Azithromycin (AZM) is one of the most common broad-spectrum antibiotics. However, drug resistance is increasing and combination therapy has attracted great attention. AZM is usually combined with traditional Chinese medicine (TCM) preparations with heat-clearing and detoxifying effects, including Kumu injection (KM) made from Picrasma quassioides (D. Don) Benn. Purpose The present study aimed to investigate synergistic antimicrobial and anti-inflammatory activities of KM plus AZM with the aim of understanding the mechanism of clinical efficacy of combination regimens. Methods Seven common bacterial clinical isolates and LPS-induced RAW 264.7 cells were used for assay of in vitro potency. The minimum inhibitory concentration (MIC) was determined for each drug, followed by synergy testing through the checkerboard method and fractional inhibitory concentration index (FICI) for quantifying combined antibacterial effects. The rat model of Klebsiella pneumoniae-induced pneumonia was developed and subjected to various drug treatments, namely, AZM, KM, or AZM plus KM, intravenously administered at 75 mg/kg once a day for one week. The combination effects then were evaluated according to pharmacodynamics and pharmacokinetic assessments. Results KM-AZM combination synergistically inhibits in vitro growth of all the test standard strains except Pseudomonas aeruginosa and also the drug-resistant strains of Staphylococcus aureus, Streptococcus pneumoniae, Shigella dysenteriae, Klebsiella pneumoniae, and Escherichia coli. Despite an additive effect against NO, KM plus AZM at an equal dose could synergistically suppress overrelease of the inflammatory cytokines TNF-α and IL-6 by LPS-induced RAW 264.7 cells. The combination significantly inhibited the proliferation of K. pneumoniae in the rat lungs, mainly by inactivating MAPKs and NF-κB signaling pathways. KM-AZM combination caused a onefold increase in apparent distribution volume of AZM, along with a significant decrease of AZM level in the livers and heart for pharmacokinetics. Conclusion KM-AZM combination displayed synergistic antibacterial and anti-inflammatory effects beneficial to the therapeutic potential against bacterial infection.

Isoniazid: An Exploratory Review

Isoniazid (INH) is one of the most successful tuberculosis medications in the market today. In particular, isoniazid is used as a prophylaxis medication to avoid resurgence of illness in those who have underlying Mycobacterium tuberculosis (MTB) infection. The mode of action of isoniazid is complicated and incorporates a number of distinct aspects in which various biomolecular routes are impacted, including mycolic acid production. Catalase-peroxidase (KatG) activates the prodrug isoniazid and enzymes such as β-ketoacyl ACP synthase (KasA) and enoyl acyl carrier protein (ACP) reductase target the active isoniazid products. Various genes in diverse biochemical networks and pathways are involved in the physiological mechanisms of isoniazid resistance. Isoniazid resistance is the most common of all clinical drug-resistant isolates, with incidence in some areas of up to 20 to 30%. In this review article, several existing components that may influence to the complexities of isoniazid function including mechanism of action, resistance mechanisms in MTB, along with their history, different synthetic procedures, uses, dosage forms, side effects, adverse drug reactions, physico-chemical characteristics, ADME properties, contraindications as well as future perspectives are discussed. Studies of pharmacokinetics have found that the cause of the drug mediated hepatotoxicity is possible by metabolism of isoniazid. Because of inter-individual heterogeneity of polymorphism that affect isoniazid metabolism rates, customized medicines may be required in various populations to prevent hepatotoxicity. The isoniazid multidrug combination treatment which would proved to be effective tuberculosis treatment in future. Further exploration is needed for better comprehension of pathogenesis mechanism of Mycobacterium tuberculosis (MTB) and drug resistance studies are required for building up better therapeutics and diagnostic against tuberculosis.

Serratia, No Longer an Uncommon Opportunistic Pathogen - Case Series & Review of Literature.

Serratia spp. is a common enteric bacterium generally thought not to be pathogenic in the gastrointestinal tract. Serratia marcescens is a member of the genus Serratia, which is a part of the family Enterobacteriales. Of all Serratia species, S. marcescens is the most common clinical isolate and the most important human pathogen. We discuss here four cases of Serratia marcescens which were reported in our laboratory at the Department of Microbiology Government Medical College and Hospital Chandigarh within six months of duration. All the samples were processed and identified using standard microbiological techniques. The isolates of Serratia marcescens were identified, depending upon their biochemical and morphological characteristics, and further confirmed by MALDI-TOF-MS, PGIMER Chandigarh. In one of the four cases, polymicrobial infection was observed, and among the cases, one patient was diabetic and the rest three patients were immunocompetent. The importance of detection of Serratia marcescens is related to the concern regarding its increased spread in hospital settings as nosocomial infection. We need to identify and isolate this pathogen not thinking of it only as a contaminant and opportunistic pathogen but as a pathogen which can lead to serious infections in hospital settings.

Phytochemical analysis and in-vitro antimicrobial screening of the leaf extract of Senna occidentalis (Fabaceae)

Abstract. Tamasi AA, Shoge MO, Adegboyega TT, Chukwuma EC. 2021. Phytochemical analysis and in-vitro antimicrobial screening of the leaf extract of Senna occidentalis (Fabaceae). Biofarmasi J Nat Prod Biochem 19: 57-64. Due to the use of Senna occidentalis as an antimalarial, anti-inflammatory, antioxidant, hepatoprotective, and antibacterial agent traditionally, this study examined the phytochemical composition and antimicrobial activity of the isolated methanol, ethyl acetate, and hexane fractions of the leaves of Senna occidentalis. Qualitative and quantitative phytochemical analyses were carried out using standard procedures. Antimicrobial activity was performed by using standard procedures against known common clinical isolates. Qualitative phytochemical analysis showed the abundant presence of saponins, tannins, flavonoids, and terpenoids in the ethyl acetate fraction. Cardiac glycosides were not detected in all the tested isolated fractions while steroids were found to be present and abundant in ethyl acetate and hexane fractions respectively. Quantitative phytochemical analysis showed that the methanol fraction contained 4%w/w alkaloids, 0.51%w/w flavonoids, 9.5%w/w saponins, 1.3%w/w tannins 5.5%w/w terpenoids, and 0.6%w/w total phenol content. The antimicrobial susceptibility test shows a range of inhibitory zone of 10-16 mm. Hexane fraction has the highest zone of inhibition against Candida albicans. The lowest MIC values of 0.6mg/mL and 0.2mg/mL were observed in ethyl acetate fraction against Candida albicans and Escherichia coli respectively. The highest MIC was 20mg/mL was observed in the ethyl acetate fraction against Staphylococcus aureus. The MMC values were varied widely. The MMC value against Bacillus subtilis, Candida albicans, and Trichophyton rubrum was 5mg/mL while the MMC value against Staphylococcus aureus, Pseudomonas aeruginosa, and Escherichia coli was 20mg/mL. The results from this study show that the leaves of Senna occidentalis can serve as a potential source of some phytochemicals and also have the potential to be developed as a source of antibiotics.

Prevalence and phylogenetic relationship of Clostridioides difficile strains in fresh poultry meat samples processed in different cutting plants

Clostridioides difficile is one of the most frequent causes of nosocomial infections in humans leading to (antibiotic-associated) diarrhea and severe pseudomembranous colitis. With an increasing frequency, C. difficile infections (CDI) are also observed independently of hospitalization and the age of the patients in an ambulant setting. One potential source of so-called community-acquired CDI is a zoonotic transmission to humans based on direct contact with animals or the consumption of food.To estimate the exposure of humans with C. difficile via food, we screened 364 different retail fresh poultry meat products purchased in Berlin and Brandenburg, Germany and further characterized the isolates. None of the 42 turkey or chicken meat samples without skin was contaminated. However, 51 (15.8%) of 322 tested fresh chicken meat samples with skin were C. difficile-positive. The vast majority (84.3%) of all isolates exhibited toxin genes tcdA and tcdB, whereas the binary toxin cdtA/B was absent. Most of the isolates (50/51) were susceptible to all six investigated antimicrobials. However, one non-toxigenic strain was multidrug resistant to the antimicrobials clindamycin and erythromycin. The isolates were mainly represented by PCR-ribotypes (RT) 001, RT002, RT005, and RT014, which were already associated with human CDI cases in Germany and were partially detected in poultry. The relatively high contamination rate of fresh retail chicken meat with skin purchased in Germany indicates chicken meat as a potential source of human infections. Moreover, we identified cutting plants with a higher rate of a C. difficile-contamination (21.4–32.8%). To compare the phylogenetic relationship of the isolated strains from certain cutting plants over several months in 2018 and 2019, we analyzed them using NGS followed by core genome MLST. Interestingly, highly related strains (0–3 alleles distance) of common clinical RT001 and RT002 isolates, as well as of the non-toxigenic RT205 isolates were detectable in same cutting plants over a period of three and 16 months, respectively.The continuous contamination with the same strain could be explained by the longterm persistence of this strain within the cutting plant (e.g., within the scalder), or with a recurring entry e.g. from the same fattening farm.

Molecular and phenotypic characterization of Salmonella Typhimurium monophasic variant (1,4,[5],12:i:-) from Colombian clinical isolates

ResumenIntroducción. La variante monofásica (1,4,[5],12:i:-) de Salmonella Typhimurium ocupa los primeros lugares en los programas de vigilancia de Salmonella a nivel mundial. En Colombia, Salmonella enterica variante monofásica alcanza el cuarto lugar en cuanto a los aislamientos clínicos recuperados por medio de la vigilancia por laboratorio del Grupo de Microbiología del Instituto Nacional de Salud, pero se desconoce si dichos aislamientos están relacionados con la variante monofásica de Typhimurium que circula a nivel global, y con sus características genéticas y fenotípicas.Objetivo. Caracterizar los aislamientos de Salmonella monofásica recuperados en Colombia entre el 2015 y el 2018 por el Grupo de Microbiología del Instituto Nacional de Salud. Materiales y métodos. Se analizaron 286 aislamientos clínicos de Salmonella enterica variante monofásica mediante PCR o secuenciación del genoma completo (Whole Genome Sequencing, WGS) para confirmar si correspondían a Salmonella Typhimurium variante monofásica, en tanto que, en 54 aislamientos, se determinó la estructura genética del operón que codifica la segunda fase flagelar y, en 23, se evaluó la motilidad, el crecimiento y la expresión de las proteínas de membrana externa.Resultados. El 61 % (n=174) de los aislamientos de Salmonella monofásica correspondió a Salmonella Typhimurium serovar monofásico. El 64,8 % (n=35/54) se relacionó con el clon europeo-español y, el 13 % (n=7/54), con el estadounidense. En dos aislamientos de orina se encontró una diferencia significativa en la motilidad y el crecimiento, así como ausencia de la porina OmpD en medio mínimo M9.Conclusiones. En el periodo de estudio, circuló en Colombia la variante monofásica de Salmonella Typhimurium relacionada con el clon europeo-español, y se registró ausencia total del operón fljAB. Los resultados evidenciaron cambios fenotípicos en los aislamientos provenientes de muestras de orina que sugieren adaptación en procesos invasivos.

Comparative Genomic Analysis Reveals Genetic Mechanisms of the Variety of Pathogenicity, Antibiotic Resistance, and Environmental Adaptation of Providencia Genus.

The bacterial genus Providencia is Gram-negative opportunistic pathogens, which have been isolated from a variety of environments and organisms, ranging from humans to animals. Providencia alcalifaciens, Providencia rettgeri, and Providencia stuartii are the most common clinical isolates, however, these three species differ in their pathogenicity, antibiotic resistance and environmental adaptation. Genomes of 91 isolates of the genus Providencia were investigated to clarify their genetic diversity, focusing on virulence factors, antibiotic resistance genes, and environmental adaptation genes. Our study revealed an open pan-genome for the genus Providencia containing 14,720 gene families. Species of the genus Providencia exhibited different functional constraints, with the core genes, accessory genes, and unique genes. A maximum-likelihood phylogeny reconstructed with concatenated single-copy core genes classified all Providencia isolates into 11 distant groups. Comprehensive and systematic comparative genomic analyses revealed that specific distributions of virulence genes, which were highly homologous to virulence genes of the genus Proteus, contributed to diversity in pathogenicity of Providencia alcalifaciens, Providencia rettgeri, and Providencia stuartii. Furthermore, multidrug resistance (MDR) phenotypes of isolates of Providencia rettgeri and Providencia stuartii were predominantly due to resistance genes from class 1 and 2 integrons. In addition, Providencia rettgeri and Providencia stuartii harbored more genes related to material transport and energy metabolism, which conferred a stronger ability to adapt to diverse environments. Overall, our study provided valuable insights into the genetic diversity and functional features of the genus Providencia, and revealed genetic mechanisms underlying diversity in pathogenicity, antibiotic resistance and environmental adaptation of members of this genus.

The stuA gene controls development, adaptation, stress tolerance, and virulence of the dermatophyte Trichophyton rubrum

The APSES family, comprising of the transcriptional regulators Asm1p, Phd1p, Sok2p, Efg1p, and StuA, is found exclusively in fungi and has been reported to control several cellular processes in these organisms. However, its function in dermatophytes has not yet been completely understood. Here, we generated two null mutant strains by deleting the stuA gene in the dermatophyte Trichophyton rubrum, the most common clinical isolate obtained from human skin and nail mycoses. The functional characterization of the knocked-out strains revealed the involvement of stuA in germination, morphogenesis of conidia and hyphae, pigmentation, stress responses, and virulence. Although the mutant strains could grow under several nutritional conditions, growth on the keratin medium, human nails, and skin was impaired. The co-culture of stuA mutants with human keratinocytes revealed enhanced development. Moreover, a stuA mutant grown on the keratin substrate showed a marked decrease in the transcript numbers of the hydrophobin encoding gene (hypA), suggesting the involvement of stuA in the molecular mechanisms underlying mechanosensing during the fungi-host interaction. In addition, bioinformatics analyses revealed the potential involvement of StuA in different biological processes such as oxidation-reduction, phosphorylation, proteolysis, transcription/translation regulation, and carbohydrate metabolism. Cumulatively, the present study suggested that StuA is a crosstalk mediator of many pathways and is an integral component of the infection process, implying that it could be a potential target for antifungal therapy.

Microbiological analysis of primary infected root canals with symptomatic and asymptomatic apical periodontitis of young permanent teeth

Background/Aim: Understanding the composition of bacteria in infected root canals is important for ameliorating the treatment strategies that lead to the elimination of pathogens and infection control, but also prevent reinfection. Aim of this study was to investigate microbial composition of primary infected root canals with apical periodontitis of young permanent teeth, originating form school children in Serbia, and its association with clinical symptoms. Material and Methods: To determine the bacterial composition of infected root canals in children, 35 endodontic samples were obtained. The identification of cultured bacteria was performed by MALDI-TOF MS analysis. The presence or absence of clinical symptoms were recorded. Results: Facultative anaerobes were 2,2 times more frequent than obligate anaerobes. The most common facultative anaerobes belonged to following genera, Streptococcus (58 isolates), Actinomyces (10) and Enterococcus (8), while predominant obligate anaerobes, belonged to genera Veillonella (15), Prevotella (9) and Fusobacterium (8). The most common clinical isolates recovered from infected root canals with symptomatic apical periodontitis were Veillonella parvula (10) and Fusobacterium nucleatum (7), while from the asymptomatic ones, they were Streptococcus mitis/Streptococcus oralis (5). Prevalence of Parvimonas micra, Prevotella buccae and Streptococcus constellatus within the root canals might be associated to clinical symptoms. Conclusions: Species of genera Streptococcus and Veillonella were the most common isolates from primary infected root canals with apical periodontitis in Serbian school children. Facultative anaerobes were predominant over obligate anaerobes. The prevalence of obligate anaerobes was much higher in symptomatic compared to asymptomatic root canal infections. No specific bacterial strain might be associated to a single examined clinical symptom (pain, tenderness to percussion or swelling), but majority of the strains are associated to all of the examined three symptoms.

Antibiofilm Synergy of β-Lactams and Branched Polyethylenimine against Methicillin-Resistant Staphylococcus epidermidis.

Microbial biofilms are ubiquitous in nature, and they pose a serious threat to public health. Staphylococcus epidermidis is the most common clinical isolate from healthcare- and medical device-related biofilm infections. No antibiotic currently on the market can eradicate pathogenic biofilms, which contain complex defense mechanisms composed of slimelike extracellular polymeric substances. Understanding the need to develop alternative approaches, we examine 600 Da branched polyethylenimine (BPEI) against methicillin-resistant Staphylococcus epidermidis (MRSE) biofilms. Here, a microtiter biofilm model is used to test the synergistic effects between the two components of our combination treatment: BPEI and β-lactam antibiotics. Electron microscopy was used to confirm the growth of MRSE biofilms from the model. Minimum biofilm eradication concentration assays, crystal violet assays, and biofilm kill curves suggest that BPEI exhibits antibiofilm activity and can potentiate β-lactams to eradicate MRSE biofilms.

BPEI-Induced Delocalization of PBP4 Potentiates β-Lactams against MRSA.

With its high morbidity rate and increasing resistance to treatment, methicillin-resistant Staphylococcus aureus (MRSA) is a grave concern in the medical field. In methicillin-susceptible strains, β-lactam antibiotics disable the penicillin binding proteins (PBPs) that cross-link the bacterial cell wall. However, methicillin-resistant strains have PBP2a and PBP4, which continue enzymatic activity in the presence of β-lactam antibiotics. The activity of PBP2a and PBP4 is linked to the presence of wall teichoic acid (WTA); thus, WTA has emerged as a target for antibiotic drug discovery. In this work, we disable WTA in situ using its anionic phosphodiester backbone to attract cationic branched polyethylenimine (BPEI). Data show that BPEI removes β-lactam resistance in common MRSA strains and clinical isolates. Fluorescence microscopy was used to investigate this mechanism of action. The results indicate that BPEI prevents the localization of PBP4 to the cell division septum, thereby changing the cellular morphology and inhibiting cell division. Although PBP4 is not required for septum formation, proper cell division and morphology require WTA; BPEI prevents this essential function. The combination of BPEI and β-lactams is bactericidal and synergistic. Because BPEI allows us to study the role of WTA in the cell wall without genetic mutation or altered translocation of biomolecules and/or their precursors, this approach can help revise existing paradigms regarding the role of WTA in prokaryotic biochemistry at every growth stage.

Aspergillus diversity in the environments of nosocomial infection cases at a university hospital.

Aspergillus species (sp.) that causes opportunistic infections have been increasingly found in human mainly immunosuppressive patients around the world every year. The main objective was to use a rapid and cheap molecular method for monitoring Aspergillus infections and epidemiological approaches. In order to identity Aspergilli species (spp.), a number of molecular methods including restriction fragment length polymorphism (RFLP) have been employed in accordance with ribosomal RNA amplification. The focus of this study — a group of hospitalized patients with clinical and subclinical signs of infection. All of the collected clinical specimens were transported to the medical mycology lab and examined for Aspergillus identification. The environmental specimens were collected from air and surfaces inspected for the Aspergillus within the hospital sources. At first, growth characteristics and microscopic features on mycological media for the identification of Aspergillus sp. were performed. For the confirmation of Aspergillus isolates which similarly found in clinical and environmental sources, molecular method polymerase chain reaction/restriction fragment length polymorphism was carried out. From the mentioned specimens, 102 fungal isolates included Candida spp., Aspergillus spp. and other fungi. Aspergillus flavus (47%), Aspergillus fumigatus (29.4%) and Aspergillus niger (23.5%) all were found as the most common clinical isolates. In addition, Aspergillus isolates from environmental were Aspergillus niger (43.7%), Aspergillus flavus (41.7%), Aspergillus fumigatus (14.6%). Therefore, polymerase chain reaction-restriction fragment length polymorphism with a single restriction enzyme can be very useful in the identification of Aspergillus spp., because of its facility in use, speed, robust, and high sensitivity of diagnosis.