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Abstract Tuberculosis (TB) remains one of the leading infectious disease killers in the world. The ongoing development of novel anti-TB medications has yielded potent compounds that often target single sites with well-defined mechanisms of action. However, despite the identification of resistance-associated mutations through target deconvolution studies, comparing these findings with the diverse Mycobacterium tuberculosis populations observed in clinical settings is often challenging. To address this gap, we constructed an open-access database encompassing genetic variations from > 50,000 clinical isolates, spanning the entirety of the M. tuberculosis protein-encoding genome. This resource offers a valuable tool for investigating the prevalence of target-based resistance mutations in any drug target within clinical contexts. To demonstrate the practical application of this dataset in drug discovery, we focused on drug targets currently undergoing phase II clinical trials. By juxtaposing genetic variations of these targets with resistance mutations derived from laboratory-adapted strains, we identified multiple positions across three targets harbouring resistance-associated mutations already present in clinical isolates. Furthermore, our analysis revealed a discernible correlation between genetic diversity within each protein and their predicted essentiality. This meta-analysis, openly accessible via a dedicated dashboard, enables comprehensive exploration of genetic diversity pertaining to any drug target or resistance determinant in M. tuberculosis.

Pseudomonas aeruginosa is one of the biofilm-forming bacteria implicated in many infections resulting in prolonged hospital stay, morbidity and high mortality, with ultimate economic burden and retardation of antibiotics effectiveness. This study determined the biofilm production potential and antimicrobial resistance pattern in clinical isolates of Pseudomonas aeruginosa from Benue State University Teaching Hospital, Makurdi. All suspected Pseudomonas aeruginosa isolates from samples submitted to the Medical Microbiology Laboratory Unit of the Hospital from August, 2023 to January 2024, were collected, purified and identified using standard microbiological techniques. Antimicrobial susceptibility testing was carried out using the disk diffusion method and EUCAST interpretative criteria. A total of 200 samples were randomly collected from different sources, 50 samples per source (Ear, wound, urine and blood). The occurrence of Pseudomonas aeruginosa from all the sources was 40.5% (81/200). Of the 81 Pseudomonas aeruginosa isolates, 67% (54) were biofilm producers (How did you determine this? Not mentioned above). The distribution of biofilm producing Pseudomonas aeruginosa isolates in the different samples evaluated were 44.4% (36) from ear swab, 27.2% (22) from wound swab, 18.5% (15) from urine and 9.9% (8) from blood. The isolates were resistant to Ticarcillin-Clavulanic acid (73%), Ceftazidine (32%), Ciprofloxacin (28%), Amikacin (26%) and Imipenem (1 %). Statistical analysis showed that there was no significant difference in antibiotics resistance pattern of biofilm producers and nonbiofilm producing Pseudomonas aeruginosa isolates. In this study, 25(31%) of the isolates were multidrug resistant (MDR), being resistant to one or more agents in three or more antibiotic classes. This study reported biofilm producing P. aeruginosa with high resistance profile from Makurdi and calls for immediate surveillance of biofilm production in all clinical isolates to reduce the spread of resistance among patients. [ABSTRACT FROM AUTHOR]

A new Mycoplasma hominis phenotype forming mini-colonies (MC) on agar and distinct from the phenotype forming typical colonies (TC) not only in size, but also in morphology, growth rate, and resistance to adverse factors, has been previously identified. In this study, the phenotype of colonies was determined and a comparative analysis of the amino acid sequence of the main variable antigen Vaa of the laboratory strain N-34 and seven clinical isolates of M. hominis was performed. It is demonstrated that the amino acid sequence of Vaa in clinical isolates forming TC (similar to the laboratory strain N-34) is entirely analogous to that of laboratory strain. Clinical isolates forming MC carry amino acid substitutions in the variable C-terminal region of Vaa, which can contribute to adhesion to eukaryotic cells and immune evasion. The connection between colony phenotype and amino acid sequence of Vaa is established. [ABSTRACT FROM AUTHOR]

Integrons are genetic elements that store, express and exchange gene cassettes. These elements are characterized by containing a gene that codes for an integrase (intI), a cassette integration site (attI) and a variable region holding the cassettes. Using bioinformatics and molecular biology methods, a functional integron found in Aeromonas sp. 3925, a strain isolated from diarrheal stools, is described. To confirm the integron class, a phylogenetic analysis with amino acid sequences was conducted. The integrase was associated to class 4 integrases; however, it is clearly different from them. Thus, we classified the associated element as a class 4-like integron. We found that the integrase activity is not under the control of the SOS or catabolic repression, since the expression was not increased in the presence of mitomycin or arabinose. The class-4-like integron is located on the chromosome and contains two well-defined gene cassettes: aadA1 that confers resistance to streptomycin and lpt coding for a lipoprotein. It also includes eight Open Reading frames (ORFs) with unknown functions. The strain was characterized through a Multilocus Phylogenetic Analyses (MLPA) of the gyrB, gyrA, rpoD, recA, dnaJ and dnaX genes. The phylogenetic results grouped it into a different clade from the species already reported, making it impossible to assign a species. We resorted to undertaking complete genome sequencing and a phylogenomic analysis. Aeromonas sp. 3925 is related to A. media and A. rivipollensis clusters, but it is clearly different from these species. In silico DNA-DNA hybridization (isDDH) and Average Nucleotide Identity (ANI) analyses suggested that this isolate belongs to the genomospecies paramedia. This paper describes the first class 4-like integron in Aeromonas and contributes to the establishment of genomospecies paramedia. [ABSTRACT FROM AUTHOR]

ObjectiveTo compare genetic characteristics of Bordetella pertussis isolates from child patients in Xishuangbanna, Yunnan province with those of vaccine isolate and Bordetella pertussis isolates from patients of other regions for providing evidence to pertussis prevention and control. MethodsFour Bordetella pertussis strains (named BP-L1, BP-L2, BP-L3, BP-L4) were isolated from nasopharyngeal swab specimens of child pertussis patients in the People′s Hospital of Xishuangbanna Prefecture, Yunnan province during October – December 2019 and the whole genome of the isolated strains were sequenced; the ptxP, ptxA, prn, fim3 genotype of the isolated strains were determined with MegAlign 7.1.0. According to the gene sequences published in National Center for Biotechnology Information (NCBI) database, the genotypes of the isolated strains were compared with those of CS strain of acellular pertussis vaccine innoculated in China and of the Bordetella pertussis strains prevalent in other countries and regions. Phylogenetic trees were constructed using RAxML software based on single nucleotide polymorphism (SNP) of 28 Bordetella pertussis strains. ResultsGene rearrangement due to 33 – 36 insertion sequences of the four isolated strains were identified in comparison with those of the CS strain. The similarity between ptxP, ptxA, prn, fim3 gene sequence of the isolated strains and CS strain was 99.2% – 100%. The results of phylogenetic tree showed that CS strain and reference strain TohamaⅠ clustered into a branch and formed an independent evolutionary branch. In recent years, Bordetella pertussis, which is prevalent in the world, can be divided into two clusters; the isolated BP-L1 and BP-L4 were clustered with the Bordetella pertussis prevalent in most European and American countries, which mainly included ptxP3/ptxA1/prn2 strains; while, the isolated BP-L2 and BP-L3 were clustered with Bordetella pertussis inducing endemic in Asia and mainly including ptxP1/ptxA1/prn1 strains. ConclusionCompared with the vaccine strains, the main virulence protein genes and evolutionary branches of the Bordetella pertussis strains clinically isolated in Xishuangbanna, Yunnan province changed significantly and the changes are consistent with the genetic evolution trend of the Bordetella pertussis prevalent globally in recent years.

Objective: This study aims to investigate the distribution and drug resistance of bacteria in clinical blood culture specimens from children in Jiangxi province in recent years and to provide a foundation for preventing and treating bloodstream infection diseases in children. Methods: The study involved a statistical analysis of the isolation and drug resistance of bacterial strains obtained from blood culture specimens of children in Jiangxi province between 2017 and 2021. The analysis was performed using the WHONET 5.6 software. Results: A total of 7,977 bacterial strains were isolated from the blood samples of children between 2017 and 2021. Of these, 2,334 strains (29.3%) were identified as Gram-negative bacteria, and 5,643 strains (70.7%) were identified as Gram-positive bacteria. The most commonly isolated pathogens were coagulase-negative Staphylococcus, Escherichia coli, and Staphylococcus aureus. Among the Gram-negative bacteria, Escherichia coli (840 strains, 36.0%), Klebsiella pneumoniae (385 strains), Salmonella (283 strains), Acinetobacter baumannii (137 strains), and Pseudomonas aeruginosa (109 strains) were the most prevalent. Among the Gram-positive bacteria, coagulase-negative Staphylococcus (3,424 strains, 60.7%), Staphylococcus aureus (679 strains), Streptococcus pneumoniae (432 strains), Enterococcus sp. (292 strains), and Streptococcus agalactiae (192 strains) were the most common. Resistance to third-generation cephalosporins (cefotaxime/ceftriaxone) was observed in 45.9% and 56.0% of Escherichia coli and Klebsiella pneumoniae strains, respectively, while resistance to carbapenems was observed in 4.6% and 20.3% of these strains, respectively. Resistance to third-generation cephalosporins (cefotaxime/ceftriaxone) was observed in 15.5% of Salmonella strains, while resistance to imipenem was absent. Carbapenem resistance was observed in 17.1% (20/117) and 13% (14/108) of Acinetobacter baumannii and Pseudomonas aeruginosa strains, respectively. Methicillin-resistant Staphylococcus aureus (MRSA) was detected in 32.7% of the strains, while methicillin-resistant coagulase-negative Staphylococcus was detected in 64.3% of the coagulase-negative Staphylococcus strains. No Staphylococcus bacteria resistant to vancomycin were detected. Four strains of vancomycin-resistant Enterococcus faecium were detected over the 5-year period, and one strain of linezolid-resistant Enterococcus faecalis was detected. Conclusion: Gram-positive cocci were the most commonly isolated clinical pathogens in blood specimens from children in Jiangxi province. The composition of the pathogen species showed a slight change over the years. The detection ratios of pathogens varied with age group and season. Although the isolation rate of common carbapenem-resistant Enterobacter bacteria has decreased, it remains high. It is necessary to monitor the antimicrobial resistance of pathogens causing bloodstream infections in children more closely, and antimicrobial agents should be used with caution. [ABSTRACT FROM AUTHOR]

Antimicrobial resistance (AMR) is one of the top ten threats to public health, as reported by the World Health Organization (WHO). One of the causes of the growing AMR problem is the lack of new therapies and/or treatment agents; consequently, many infectious diseases could become uncontrollable. The need to discover new antimicrobial agents that are alternatives to the existing ones and that allow mitigating this problem has increased, due to the rapid and global expansion of AMR. Within this context, both antimicrobial peptides (AMPs) and cyclic macromolecules, such as resorcinarenes, have been proposed as alternatives to combat AMR. Resorcinarenes present multiple copies of antibacterial compounds in their structure. These conjugate molecules have exhibited antifungal and antibacterial properties and have also been used in anti-inflammatory, antineoplastic, and cardiovascular therapies, as well as being useful in drug and gene delivery systems. In this study, it was proposed to obtain conjugates that contain four copies of AMP sequences over a resorcinarene core. Specifically, obtaining (peptide)4-resorcinarene conjugates derived from LfcinB (20–25): RRWQWR and BF (32–34): RLLR was explored. First, the synthesis routes that allowed obtaining: (a) alkynyl-resorcinarenes and (b) peptides functionalized with the azide group were established. These precursors were used to generate (c) (peptide)4-resorcinarene conjugates by azide-alkyne cycloaddition CuAAC, a kind of click chemistry. Finally, the conjugates' biological activity was evaluated: antimicrobial activity against reference strains and clinical isolates of bacteria and fungi, and the cytotoxic activity over erythrocytes, fibroblast, MCF-7, and HeLa cell lines. Our results allowed establishing a new synthetic route, based on click chemistry, for obtaining macromolecules derived from resorcinarenes functionalized with peptides. Moreover, it was possible to identify promising antimicrobial chimeric molecules that may lead to advances in the development of new therapeutic agents. [ABSTRACT FROM AUTHOR]

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Abstract Background Leishmania infantum is the most important etiological agent of visceral leishmaniasis in the Americas and Mediterranean region, and the dog is the main host. Miltefosine was authorized to treat canine leishmaniasis (CanL) in Brazil in 2017, but there is a persistent fear of the emergence of parasites resistant not only to this drug but, through cross-resistance mechanisms, also to meglumine antimoniate and amphotericin B. Additionally, the literature shows that acquisition of resistance is followed by increased parasite fitness, with higher rates of proliferation, infectivity and metacyclogenesis, which are drivers of parasite virulence. In this context, the aim of this study was to analyze the impact of treating a dog with miltefosine and allopurinol on the generation of parasites resistant to miltefosine, amphotericin B and meglumine antimoniate. Methods In vitro susceptibility tests were conducted against miltefosine, amphotericin B and meglumine antimoniate with T0 (parasites isolated from a dog before treatment with miltefosine plus allopurinol), T1 (after 1 course of treatment) and T2 (after 2 courses of treatment) isolates. The rates of cell proliferation, infectivity and metacyclogenesis of the isolates were also evaluated. Results The results indicate a gradual increase in parasite resistance to miltefosine and amphotericin B with increasing the number of treatment courses. An increasing trend in the metacyclogenesis rate of the parasites was also observed as drug resistance increased. Conclusion The data indicates an increased L. infantum resistance to miltefosine and amphotericin B after the treatment of a dog with miltefosine plus allopurinol. Further studies with a larger number of L. infantum strains isolated from dogs with varied immune response profiles and undergoing different treatment regimes, are advocated. Graphical Abstract

Integrons are genetic elements that store, express and exchange gene cassettes. These elements are characterized by containing a gene that codes for an integrase (intI), a cassette integration site (attI) and a variable region holding the cassettes. Using bioinformatics and molecular biology methods, a functional integron found in Aeromonas sp. 3925, a strain isolated from diarrheal stools, is described. To confirm the integron class, a phylogenetic analysis with amino acid sequences was conducted. The integrase was associated to class 4 integrases; however, it is clearly different from them. Thus, we classified the associated element as a class 4-like integron. We found that the integrase activity is not under the control of the SOS or catabolic repression, since the expression was not increased in the presence of mitomycin or arabinose. The class-4-like integron is located on the chromosome and contains two well-defined gene cassettes: aadA1 that confers resistance to streptomycin and lpt coding for a lipoprotein. It also includes eight Open Reading frames (ORFs) with unknown functions. The strain was characterized through a Multilocus Phylogenetic Analyses (MLPA) of the gyrB, gyrA, rpoD, recA, dnaJ and dnaX genes. The phylogenetic results grouped it into a different clade from the species already reported, making it impossible to assign a species. We resorted to undertaking complete genome sequencing and a phylogenomic analysis. Aeromonas sp. 3925 is related to A. media and A. rivipollensis clusters, but it is clearly different from these species. In silico DNA-DNA hybridization (isDDH) and Average Nucleotide Identity (ANI) analyses suggested that this isolate belongs to the genomospecies paramedia. This paper describes the first class 4-like integron in Aeromonas and contributes to the establishment of genomospecies paramedia.

Herpes simplex virus type 1 (HSV-1) is the only FDA- and EMA- approved oncolytic virus, and accordingly, many potential oncolytic HSVs (oHSV) are in clinical development. The utilized oHSV parental strains are, however, mostly based on laboratory reference strains, which may possess a compromised cytolytic capacity in contrast to circulating strains of HSV-1. Here, we assess the phenotype of thirty-six circulating HSV-1 strains from Finland to uncover their potential as oHSV backbones. First, we determined their capacity for cell-to-cell versus extracellular spread, to find strains with replication profiles favorable for each application. Second, to unfold the differences, we studied the genetic diversity of two relevant viral glycoproteins (gB/UL27, gI/US7). Third, we examined the oncolytic potential of the strains in cells representing glioma, lymphoma, and colorectal adenocarcinoma. Our results suggest that the phenotype of a circulating isolate, including the oncolytic potential, is highly related to the host cell type. Nevertheless, we identified isolates with increased oncolytic potential in comparison with the reference viruses across many or all of the studied cancer cell types. Our research emphasizes the need for careful selection of the backbone virus in early vector design, and it highlights the potential of clinical isolates as backbones in oHSV development. [ABSTRACT FROM AUTHOR]

Introduction Staphylococcus aureus (S. aureus) has been recognized as a powerful pathogen that causes numerous infections. Today, S.aureus has acquired multiple resistance to a wide range of antibiotics, including beta-lactams. Beta-lactamases hydrolyze beta-lactam antibiotics and are expressed through activation of the blaZ gene. The aim of this study was to evaluate the frequency of blaZ gene in clinical isolates of S.aureus isolated from Hamadan hospitals. Materials and Methods In this descriptive cross-sectional study, all S.aureus strains were randomly selected and transferred to the laboratory. In clinical isolates confirmed by biochemical tests of genomic DNA extraction procedures using CinnaGen extraction kit) was performed according to the manufacturerchr('39')s protocol and 1% agarose gel was used to view the PCR product. Data were analyzed by SPSS software. Results From 50 hospitalized patients in Fatemieh and Sina hospitals, 32 men (64%), and 18 women (36%), isolates of S.aureus were isolated. The pattern of antibiotic resistance obtained was that penicillin (10 µg) with 100% resistance and cefazolin (30 µg) with 5% resistance had the highest and lowest resistance, respectively. Also, 94% of isolates were resistant to cefoxitin (30 µg) and 90% of isolates were resistant to oxacillin (1 µg). Of 50 S.aureus isolated from clinical isolates, 44 (88 %) contained the blaZ beta-lactamase gene. Conclusion Based on the findings of this study, isolates isolated from clinical specimens showed the same pattern of abundance. Therefore, it is likely that more or more drug resistance will be transmitted to hospitals.

Introduction Nosocomial infections are a major global problem. Using metallic oxide nanoparticles to counter bacterial infections as an alternative to antibiotics can be effective. The aim of this study was to evaluate the effect of iron and silver nanoparticles on biofilm formation in bacteria isolated from clinical specimens and the possibility of using these nanoparticles on laboratory surfaces. Materials and Methods This study was performed on three bacterial strains isolated from different wards of Imam Reza, Imam Hossein, and Taleghani hospitals in Kermanshah. The biochemical tests of the bacteria were identified and their antibiotic resistance was determined by the disk method. The microtiter plate method was used to investigate the disinfecting effect of silver and iron nanoparticles on biofilm formation. Data were analyzed by SPSS software. Results Results showed 9 blood samples (30%), 5 skin samples (16.67%), and 16 urine samples (53.33%). The highest rate of inhibition of Escherichia coli biofilm formation by silver and iron nanoparticles was 93% and 64%, respectively. The inhibition of biofilm formation in S. epidermidis by silver and iron nanoparticles was 86 and 64%, respectively, and the highest inhibition of biofilm formation by Pseudomonas aeruginosa by silver and iron nanoparticles was 96 and 88%, respectively. Conclusion The use of silver and iron nanoparticles has a high potential to kill the bacteria in the biofilm of P.aeruginosa.

Abstract Background The only licensed live Bacille Calmette-Guérin (BCG) vaccine used to prevent severe childhood tuberculosis comprises genetically divergent strains with variable protective efficacy and rates of BCG-induced adverse events. The whole-genome sequencing (WGS) allowed evaluating the genome stability of BCG strains and the impact of spontaneous heterogeneity in seed and commercial lots on the efficacy of BCG-vaccines in different countries. Our study aimed to assess sequence variations and their putative effects on genes and protein functions in the BCG-1 (Russia) seed lots compared to their progeny isolates available from immunocompetent children with BCG-induced disease (mainly, osteitis). Results Based on the WGS data, we analyzed the links between seed lots 361, 367, and 368 used for vaccine manufacture in Russia in different periods, and their nine progeny isolates recovered from immunocompetent children with BCG-induced disease. The complete catalog of variants in genes relative to the reference genome (GenBank: CP013741) included 4 synonymous and 8 nonsynonymous single nucleotide polymorphisms, and 3 frameshift deletions. Seed lot 361 shared variants with 2 of 6 descendant isolates that had higher proportions of such polymorphisms in several genes, including ppsC, eccD5, and eccA5 involved in metabolism and cell wall processes and reportedly associated with virulence in mycobacteria. One isolate preserved variants of its parent seed lot 361 without gain of further changes in the sequence profile within 14 years. Conclusions The background genomic information allowed us for the first time to follow the BCG diversity starting from the freeze-dried seed lots to descendant clinical isolates. Sequence variations in several genes of seed lot 361 did not alter the genomic stability and viability of the vaccine and appeared accumulated in isolates during the survival in the human organism. The impact of the observed variations in the context of association with the development of BCG-induced disease should be evaluated in parallel with the immune status and host genetics. Comparative genomic studies of BCG seed lots and their descendant clinical isolates represent a beneficial approach to better understand the molecular bases of efficacy and adverse events during the long-term survival of BCG in the host organism.

Abstract Background Genome sequencing and genetic polymorphism analysis of clinical isolates of M. tuberculosis is carried out to gain further insight into molecular pathogenesis and host-pathogen interaction. Therefore the functional evaluation of the effect of single nucleotide variation (SNV) is essential. At the same time, the identification of invariant sequences unique to M. tuberculosis contributes to infection detection by sensitive methods. In the present study, genome analysis is accompanied by evaluation of the functional implication of the SNVs in a MDR clinical isolate VPCI591. Result By sequencing and comparative analysis of VPCI591 genome with 1553 global clinical isolates of M. tuberculosis (GMTV and tbVar databases), we identified 141 unique strain specific SNVs. A novel intergenic variation in VPCI591 in the putative promoter/regulatory region mapping between embC (Rv3793) and embA (Rv3794) genes was found to enhance the expression of embAB, which correlates with the high resistance of the VPCI591 to ethambutol. Similarly, the unique combination of three genic SNVs in RNA polymerase β gene (rpoB) in VPCI591 was evaluated for its effect on rifampicin resistance through molecular docking analysis. The comparative genomics also showed that along with variations, there are genes that remain invariant. 173 such genes were identified in our analysis. Conclusion The genetic variation in M. tuberculosis clinical isolate VPCI591 is found in almost all functional classes of genes. We have shown that SNV in rpoB gene mapping outside the drug binding site along with two SNVs in the binding site can contribute to quantitative change in MIC for rifampicin. Our results show the collective effect of SNVs on the structure of the protein, impacting the interaction between the target protein and the drug molecule in rpoB as an example. The study shows that intergenic variations bring about quantitative changes in transcription in embAB and in turn can lead to drug resistance.

Objective: Boric acid is an element that is found as compounds in the nature and has been shown to be effective in many areas. Being non-toxic for human and the environment and economical due to obtained naturally are the main remarkable properties. This study was conducted to investigate the disinfectant efficiency of boric acid. Methods: A suspension of lyophilized boric acid in different concentrations (1, 2, 4, 6%) was prepared then disinfectant activity against a total of 25 isolates including Staphylococcus aureus ATCC 29213, Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853, Enterococcus faecalis ATCC 29212, Streptococcus mutans ATCC 25175 and 20 different microorganisms with different resistance pattern (Acinetobacter baumannii, Klebsiella pneumoniae, Staphylococcus epidermidis, Candida albicans, Candida parapsilosis, Streptococcus mitis/oralis) isolated from blood samples was investigated by qualitative suspension method. In the qualitative suspension method; boric acid at different concentrations and selected bacteria were contacted for 1, 2, 5, 10, 30 minutes. At the end of the contact periods, neutralizing solution was added to stop the effects of the substances before culturing, and then cultured on tryptone soy agar. At the end of 48 hours incubation, After incubation, the bacteria were assessed for the presence or absence of proliferation. Results: The efficacy of boric acid at a concentration of 1% and the tested durations was not determined. It was found that boric acid at a concentration of 2% with 5 minutes contact time was efficient against almost all bacteria and to all at 10th minute, to all at 4% concentration since 5th minute and to all at 6% concentration since 1st minute. While efficacy was not determined at 1% concentration in Candida spp. species, it found to be effective in all at the 2% concentration from the 1st minute. Conclusion: It has been found that boric acid is an antimicrobial agent effective against bacteria and Candida spp. species even at low concentrations. It will be beneficial to carry out studies that examine variables such as safety and stability related to this substance, which is used for disinfection purposes. [ABSTRACT FROM AUTHOR]

Background: Leishmania infantum is the most important etiological agent of visceral leishmaniasis in the Americas and Mediterranean region, and the dog is the main host. Miltefosine was authorized to treat canine leishmaniasis (CanL) in Brazil in 2017, but there is a persistent fear of the emergence of parasites resistant not only to this drug but, through cross-resistance mechanisms, also to meglumine antimoniate and amphotericin B. Additionally, the literature shows that acquisition of resistance is followed by increased parasite fitness, with higher rates of proliferation, infectivity and metacyclogenesis, which are drivers of parasite virulence. In this context, the aim of this study was to analyze the impact of treating a dog with miltefosine and allopurinol on the generation of parasites resistant to miltefosine, amphotericin B and meglumine antimoniate. Methods: In vitro susceptibility tests were conducted against miltefosine, amphotericin B and meglumine antimoniate with T0 (parasites isolated from a dog before treatment with miltefosine plus allopurinol), T1 (after 1 course of treatment) and T2 (after 2 courses of treatment) isolates. The rates of cell proliferation, infectivity and metacyclogenesis of the isolates were also evaluated. Results: The results indicate a gradual increase in parasite resistance to miltefosine and amphotericin B with increasing the number of treatment courses. An increasing trend in the metacyclogenesis rate of the parasites was also observed as drug resistance increased. Conclusion: The data indicates an increased L. infantum resistance to miltefosine and amphotericin B after the treatment of a dog with miltefosine plus allopurinol. Further studies with a larger number of L. infantum strains isolated from dogs with varied immune response profiles and undergoing different treatment regimes, are advocated. [ABSTRACT FROM AUTHOR]

Background: Antibiotics have been widely used for the treatment of bacterial infections for decades. However, the rapid emergence of antibiotic-resistant bacteria has created many problems with a heavy burden for the medical community. Therefore, the use of nanoparticles as an alternative for antibacterial activity has been explored. In this context, metal nanoparticles have demonstrated broad-spectrum antimicrobial activity. This study investigated the antimicrobial activity of naked cerium oxide nanoparticles dispersed in aqueous solution (CNPs) and surface-stabilized using Pseudomonas aeruginosa as a bacterial model. Methods: Gelatin-polycaprolactone nanofibers containing CNPs (Scaffold@CNPs) were synthesized, and their effect on P. aeruginosa was investigated. The minimum inhibitory and bactericidal concentrations of the nanoparticls were determined in an ATCC reference strain and a clinical isolate strain. To determine whether the exposure to the nanocomposites might change the expression of antibiotic resistance, the expression of the genes shv, kpc, and imp was also investigated. Moreover, the cytotoxicity of the CNPs was assessed on fibroblast using flow cytometry. Results: Minimum bactericidal concentrations for the ATCC and the clinical isolate of 50 µg/mL and 200 µg/mL were measured, respectively, when the CNPs were used. In the case of the Scaffold@CNPs, the bactericidal effect was 50 µg/mL and 100 µg/mL for the ATCC and clinical isolate, respectively. Interestingly, the exposure to the Scaffold@CNPs significantly decreased the expression of the genes shv, kpc, and imp. Conclusions: A concentration of CNPs and scaffold@CNPs higher than 50 μg/mL can be used to inhibit the growth of P. aeruginosa. The fact that the scaffold@CNPs significantly reduced the expression of resistance genes, it has the potential to be used for medical applications such as wound dressings. [ABSTRACT FROM AUTHOR]

Colistin is frequently a last resort treatment for Pseudomonas aeruginosa infections caused by multidrug-resistant (MDR) and extensively drug resistant (XDR) strains, and detection of colistin resistance is essential for the management of infected patients. Therefore, we evaluated the recently developed MALDIxin test for the detection of colistin resistance in P. aeruginosa clinical strains using the routine matrix-assisted laser desorption ionization (MALDI) Biotyper Sirius system. The test is based on the detection by mass spectrometry of modified lipid A by the addition of 4-amino-l-arabinose (l-ara4N) molecules on one or two phosphate groups, in strains resistant to colistin. Overproduction of l-Ara4N molecules is mainly due to the constitutive activation of the histidine kinase (PmrB) or the response regulator (PmrA) following an amino-acid substitution in clinical strains. The performance of the test was determined on a panel of 14 colistin-susceptible and 14 colistin-resistant P. aeruginosa clinical strains, the reference strain PAO1 and positive control mutants PmrB (V28G), PmrB (D172), PhoQ (D240–247), and ParR (M59I). In comparison with the broth microdilution (BMD) method, all the susceptible strains (n=14) and 8/14 colistin-resistant strains were detected in less than 1h, directly on whole bacteria. The remaining resistant strains (n=6) were all detected after a short pre-exposure (4h) to colistin before sample preparation. Validation of the method on a larger panel of strains will be the next step before its use in diagnostics laboratories. Our data showed that the MALDIxin test offers rapid and efficient detection of colistin resistant P. aeruginosa and is thus a valuable diagnostics tool to control the spread of these emerging resistant strains.

Background: The emergence of multidrug-resistant bacteria has become a serious healthcare problem worldwide. Methods: In this study, 192 clinical specimens collected from a tertiary hospital in Al Jouf, Saudi Arabia were processed in order to isolate gram-negative bacteria. Bacterial isolation was performed using standard microbiological techniques and the MicroScan WalkAway Plus automated analyzer. Phenotypic methods and PCR were done to characterize bacterial strains, and DNA sequencing was used for the molecular characterization of new Delhi metallo-β-lactamases NDM genes. Results: This study reports the presence of the blaNDM gene in 95 gram-negative bacterial isolates. Among the isolates, 71 (74%) carried blaNDM-1 and 24 (25%) carried blaNDM gene variants. Different specimen analyses revealed that the samples from the intensive care unit (ICU) showed the highest percentage (38%) of NDM-1 positive isolates. Molecular identification revealed 87% belonged to the Enterobacteriaceae family. The species profiling showed that 41% of the clinical isolates were Klebsiella pneumoniae, 25.3% of isolates were E. coli, and 9.5% were Enterobacter cloacae. Thirteen percent of the gram-negative isolates, Pseudomonas aeruginosa and Acinetobacter baumannii, belonged to the Pseudomonadaceae family and Gammaproteobacteria family, respectively. The antibiotic sensitivity profile of the 95 isolates revealed that all the isolates harboring the NDM gene showed resistance against ampicillin. Among the NDM-harboring isolates, the highest resistance was found against cefuroxime and co-amoxiclav antibiotics. The isolates were the most sensitive to tigecycline and amikacin. However, the ability of some NDM-harboring gram-negative isolates to grow in the presence of amikacin and tigecycline suggests that these NDM-producing strains carry an additional drug-resistance mechanism. Conclusions: The prevalence of NDM-harboring gram-negative bacterial isolates in hospital settings is an alarming situation. The Enterobacteriaceae family was found to be the most common carrier of the NDM gene, which indicates a need for more stringent use of antibiotics and better control in hospitals to minimize the risk of cross contamination and spread of NDM-positive strains. [ABSTRACT FROM AUTHOR]

Colistin is frequently a last resort treatment for Pseudomonas aeruginosa infections caused by multidrug-resistant (MDR) and extensively drug resistant (XDR) strains, and detection of colistin resistance is essential for the management of infected patients. Therefore, we evaluated the recently developed MALDIxin test for the detection of colistin resistance in P. aeruginosa clinical strains using the routine matrix-assisted laser desorption ionization (MALDI) Biotyper Sirius system. The test is based on the detection by mass spectrometry of modified lipid A by the addition of 4-amino-l-arabinose (l-ara4N) molecules on one or two phosphate groups, in strains resistant to colistin. Overproduction of l-Ara4N molecules is mainly due to the constitutive activation of the histidine kinase (PmrB) or the response regulator (PmrA) following an amino-acid substitution in clinical strains. The performance of the test was determined on a panel of 14 colistin-susceptible and 14 colistin-resistant P. aeruginosa clinical strains, the reference strain PAO1 and positive control mutants PmrB (V28G), PmrB (D172), PhoQ (D240–247), and ParR (M59I). In comparison with the broth microdilution (BMD) method, all the susceptible strains (n =14) and 8/14 colistin-resistant strains were detected in less than 1h, directly on whole bacteria. The remaining resistant strains (n =6) were all detected after a short pre-exposure (4h) to colistin before sample preparation. Validation of the method on a larger panel of strains will be the next step before its use in diagnostics laboratories. Our data showed that the MALDIxin test offers rapid and efficient detection of colistin resistant P. aeruginosa and is thus a valuable diagnostics tool to control the spread of these emerging resistant strains. [ABSTRACT FROM AUTHOR]

Histoplasma capsulatum is a dimorphic, thermal, and nutritional fungus. In the environment and at an average temperature of 28 °C, it develops as a mold that is composed of infecting particles. Once in the host or in cultures at 37 °C, it undergoes a transition into the parasitic form. In the present work, we performed chemical extraction and characterization using chromatography techniques of the associated lipid composition of the external surface of the cell wall of the mycelial phase of two isolates of the H. capsulatum: one clinical and one environmental. Several differences were evidenced in the fatty acids in the phospholipid composition. Surface pressure–area isotherms and compression module curves of the Amphotericin B and lipid extract monolayers, as well as (AmB)-lipid extract mixed monolayers were recorded. Results show a high affinity of AmB towards lipid extracts. The most stable monolayers were formed by AmB + environmental with a mass ratio of 1:3 and AmB + clinical with a mass ratio of 1:2. Knowledge of the AmB aggregation processes at a molecular level and the characterization of the lipid extracts allows the possibility to understand the interaction between the AmB and the lipid fractions of H. capsulatum.

The tuberculosis (TB) disease is still widely found even though BCG vaccine given to many people. Ineffectiveness of the BCG vaccine is one of causes that make the difficulties in preventing TB transmission. Objective of the research was to determine the immunogenicity of KatG protein of M. tuberculosis clinical isolate L19 in mice. The KatG protein as antigen was prepared by expression of the katG gene of M. tuberculosis clinical isolate L19 in Escherichia coli BL21 using pColdII-DNA vector. After purification by affinity chromatography, the KatG was vaccinated to mice to detect its immunogenicity. The expression of katG in E. coli BL21 could result in KatG protein with molecular weight 80 kDa in sodium dodecyl sulfate gel electrophoresis (SDS-PAGE). The pure KatG protein could significantly stimulate the immune response of mice by triggering the antibodies production of IgG1, IgG2a, IgG2b, IgG2c, IgG3, and IgM. The highest antibody level was obtained when the mice were vaccinated by KatG L19 with the dose of 45 μg/ml. Of the antibodies, the IgG2c isotype was dominantly produced in the blood serum. The KatG protein exhibited a high immunogenici­ty in mice, so it is possible to develop as a vaccine candidate for TB. A clinical test should be performed in a future to ensure its safety as a therapeutic protein.

Vibrio vulnificus, a pathogenic bacterium that causes serious infections in humans, requires iron for growth. Clinical isolate, V. vulnificus M2799, secretes a catecholate siderophore, namely, vulnibactin, to capture iron (III) from the environment. Growth experiments using a deletion mutant indicated that VuuB, a member of the FAD-containing siderophore-interacting protein family, plays a crucial role in Fe3+-vulnibactin reduction. IutB, a member of the ferric-siderophore reductase family, stands a substitute for VuuB in its absence. It remained unclear why V. vulnificus M2799 has two proteins with relevant functions. Here we biochemically characterized VuuB and IutB using purified recombinant proteins. Purified VuuB, a flavoprotein, catalyzed the reduction of Fe3+-nitrilotriacetic acid as its electron acceptor, in the presence of NADH as its electron donor and FAD as its cofactor. IutB catalyzed the reduction of Fe3+-nitrilotriacetic acid, in the presence of NADH, NADPH, or reduced glutathione as its electron donor. The optimal pH values and temperatures of VuuB and IutB were 7.0 and 37 °C, and 8.5 and 45 °C, respectively. On analyzing their ferric-chelate reductase activities, both VuuB and IutB were found to catalyze the reduction of Fe3+-aerobactin, Fe3+-vibriobactin, and Fe3+-vulnibactin. When the biologically relevant substrate, Fe3+-vulnibactin, was used, the levels of ferric-chelate reductase activities were similar between VuuB and IutB. Finally, the mRNA levels were quantified by qRT-PCR in M2799 cells cultivated under low-iron conditions. The number of vuuB mRNA was 8.5 times greater than that of iutB. The expression ratio correlated with the growth of their mutants in the presence of vulnibactin. [ABSTRACT FROM AUTHOR]

Background: Genome sequencing and genetic polymorphism analysis of clinical isolates of M. tuberculosis is carried out to gain further insight into molecular pathogenesis and host-pathogen interaction. Therefore the functional evaluation of the effect of single nucleotide variation (SNV) is essential. At the same time, the identification of invariant sequences unique to M. tuberculosis contributes to infection detection by sensitive methods. In the present study, genome analysis is accompanied by evaluation of the functional implication of the SNVs in a MDR clinical isolate VPCI591. Result: By sequencing and comparative analysis of VPCI591 genome with 1553 global clinical isolates of M. tuberculosis (GMTV and tbVar databases), we identified 141 unique strain specific SNVs. A novel intergenic variation in VPCI591 in the putative promoter/regulatory region mapping between embC (Rv3793) and embA (Rv3794) genes was found to enhance the expression of embAB, which correlates with the high resistance of the VPCI591 to ethambutol. Similarly, the unique combination of three genic SNVs in RNA polymerase β gene (rpoB) in VPCI591 was evaluated for its effect on rifampicin resistance through molecular docking analysis. The comparative genomics also showed that along with variations, there are genes that remain invariant. 173 such genes were identified in our analysis. Conclusion: The genetic variation in M. tuberculosis clinical isolate VPCI591 is found in almost all functional classes of genes. We have shown that SNV in rpoB gene mapping outside the drug binding site along with two SNVs in the binding site can contribute to quantitative change in MIC for rifampicin. Our results show the collective effect of SNVs on the structure of the protein, impacting the interaction between the target protein and the drug molecule in rpoB as an example. The study shows that intergenic variations bring about quantitative changes in transcription in embAB and in turn can lead to drug resistance. [ABSTRACT FROM AUTHOR]

Abstract Background The sexual stages (gametocytes) of Plasmodium falciparum do not directly contribute to the pathology of malaria but are essential for transmission of the parasite from the human host to the mosquito. Mature gametocytes circulate in infected human blood for several days and their circulation time has been modelled mathematically from data of previous in vivo studies. This is the first time that longevity of gametocytes is studied experimentally in vitro. Methods The in vitro longevity of P. falciparum gametocytes of 1 clinical isolate and 2 laboratory strains was assessed by three different methods: microscopy, flow cytometry and reverse transcription quantitative real-time PCR (RT-qPCR). Additionally, the rate of gametocytogenesis of the used P. falciparum strains was compared. Results The maximum in vitro lifespan of P. falciparum gametocytes reached almost 2 months (49 days by flow cytometry, 46 days by microscopy, and at least 52 days by RT-qPCR) from the starting day of gametocyte culture to death of last parasite in the tested strains with an average 50% survival rate of 6.5, 2.6 and 3.5 days, respectively. Peak gametocytaemia was observed on average 19 days after initiation of gametocyte culture followed by a steady decline due to natural decay of the parasites. The rate of gametocytogenesis was highest in the NF54 strain. Conclusions Plasmodium falciparum mature gametocytes can survive up to 16–32 days (at least 14 days for mature male gametocytes) in vitro in absence of the influence of host factors. This confirms experimentally a previous modelling estimate that used molecular tools for gametocyte detection in treated patients. The survival time might reflect the time the parasite can be transmitted to the mosquito after clearance of asexual parasites. These results underline the importance of efficient transmission blocking agents in the fight against malaria.

Whole genome sequencing (WGS) can provide a comprehensive analysis of Mycobacterium tuberculosis mutations that cause resistance to anti-tuberculosis drugs. With the deployment of bench-top sequencers and rapid analytical software, WGS is poised to become a useful tool to guide treatment. However, direct sequencing from clinical specimens to provide a full drug resistance profile remains a serious challenge. This article reviews current practices for extracting M. tuberculosis DNA and possible solutions for sampling sputum. Techniques under consideration include enzymatic digestion, physical disruption, chemical degradation, detergent solubilization, solvent extraction, ligand-coated magnetic beads, silica columns, and oligonucleotide pull-down baits. Selective amplification of genomic bacterial DNA in sputum prior to WGS may provide a solution, and differential lysis to reduce the levels of contaminating human DNA is also being explored. To remove this bottleneck and accelerate access to WGS for patients with suspected drug-resistant tuberculosis, it is suggested that a coordinated and collaborative approach be taken to more rapidly optimize, compare, and validate methodologies for sequencing from patient samples.

Water is considered to play a role in the dissemination of antibiotic-resistant Gram-negative bacteria including those encoding Extended-spectrum beta-lactamases (ESBL) and carbapenemases. To investigate the role of water for their spread in more detail, we characterized ESBL/Carbapenemase-producing bacteria from surface water and sediment samples using phenotypic and genotypic approaches. ESBL/Carbapenemase-producing isolates were obtained from water/sediment samples. Species and antibiotic resistance were determined. A subset of these isolates (n = 33) was whole-genome-sequenced and analyzed for the presence of antibiotic resistance genes and virulence determinants. Their relatedness to isolates associated with human infections was investigated using multilocus sequence type and cgMLST-based analysis. Eighty-nine percent of the isolates comprised of clinically relevant species. Fifty-eight percent exhibited a multidrug-resistance phenotype. Two isolates harbored the mobile colistin resistance gene mcr-1. One carbapenemase-producing isolate identified as Enterobacter kobei harbored blaVIM–1. Two Escherichia coli isolates had sequence types (ST) associated with human infections (ST131 and ST1485) and a Klebsiella pneumoniae isolate was classified as hypervirulent. A multidrug-resistant (MDR) Pseudomonas aeruginosa isolate encoding known virulence genes associated with severe lung infections in cystic fibrosis patients was also detected. The presence of MDR and clinically relevant isolates in recreational and surface water underlines the role of aquatic environments as both reservoirs and hot spots for MDR bacteria. Future assessment of water quality should include the examination of the multidrug resistance of clinically relevant bacterial species and thus provide an important link regarding the spread of MDR bacteria in a One Health context.

ABSTRACT Lassa virus (LASV), a member of the family Arenaviridae, is the causative agent of Lassa fever. Lassa virus is endemic in West African countries, such as Nigeria, Guinea, Liberia, and Sierra Leone, and causes outbreaks annually. Lassa fever onset begins with “flu-like” symptoms and may develop into lethal hemorrhagic disease in severe cases. Although Lassa virus is one of the most alarming pathogens from a public health perspective, there are few licensed vaccines or therapeutics against Lassa fever. The fact that animal models are limited and the fact that mostly laboratory-derived viruses are used for studies limit the successful development of countermeasures. In this study, we demonstrated that the LASV isolate LF2384-NS-DIA-1 (LF2384), which was directly isolated from a serum sample from a fatal human Lassa fever case in the 2012 Sierra Leone outbreak, causes uniformly lethal infection in outbred Hartley guinea pigs without virus-host adaptation. This is the first report of a clinically isolated strain of LASV causing lethal infection in outbred guinea pigs. This novel guinea pig model of Lassa fever may contribute to Lassa fever research and the development of vaccines and therapeutics. IMPORTANCE Lassa virus, the causative agent of Lassa fever, is a zoonotic pathogen causing annual outbreaks in West African countries. Human patients can develop lethal hemorrhagic fever in severe cases. Although Lassa virus is one of the most alarming pathogens from a public health perspective, there are few available countermeasures, such as antiviral drugs or vaccines. Moreover, the fact that animal models are not readily accessible and the fact that mostly laboratory viruses, which have been passaged many times after isolation, are used for studies further limits the successful development of countermeasures. In this study, we demonstrate that a human isolate of Lassa virus causes lethal infection uniformly in Hartley guinea pigs. This novel animal model of Lassa fever may contribute to Lassa fever research and the development of vaccines and therapeutics.

Water is considered to play a role in the dissemination of antibiotic-resistant Gram-negative bacteria including those encoding Extended-spectrum beta-lactamases (ESBL) and carbapenemases. To investigate the role of water for their spread in more detail, we characterized ESBL/Carbapenemase-producing bacteria from surface water and sediment samples using phenotypic and genotypic approaches. ESBL/Carbapenemase-producing isolates were obtained from water/sediment samples. Species and antibiotic resistance were determined. A subset of these isolates (n = 33) was whole-genome-sequenced and analyzed for the presence of antibiotic resistance genes and virulence determinants. Their relatedness to isolates associated with human infections was investigated using multilocus sequence type and cgMLST-based analysis. Eighty-nine percent of the isolates comprised of clinically relevant species. Fifty-eight percent exhibited a multidrug-resistance phenotype. Two isolates harbored the mobile colistin resistance gene mcr-1. One carbapenemase-producing isolate identified as Enterobacter kobei harbored bla VIM–1. Two Escherichia coli isolates had sequence types (ST) associated with human infections (ST131 and ST1485) and a Klebsiella pneumoniae isolate was classified as hypervirulent. A multidrug-resistant (MDR) Pseudomonas aeruginosa isolate encoding known virulence genes associated with severe lung infections in cystic fibrosis patients was also detected. The presence of MDR and clinically relevant isolates in recreational and surface water underlines the role of aquatic environments as both reservoirs and hot spots for MDR bacteria. Future assessment of water quality should include the examination of the multidrug resistance of clinically relevant bacterial species and thus provide an important link regarding the spread of MDR bacteria in a One Health context. [ABSTRACT FROM AUTHOR]

Candida albicans is the most frequently isolated opportunistic fungal pathogen in humans. However, patients with cervical lymphadenitis caused by Candida infection are rarely reported, and few studies have focused on the mechanisms underlying chronic Candida infection. In this study, we isolated a C. albicans strain (JL01) from a recurrent cervical lymphadenitis patient. The clinical isolate was identified by morphological observation and confirmed by DNA sequencing of the internal transcribed spacer (ITS) regions. Strain JL01 is resistant to azole antifungal drugs, but sensitive to amphotericin B. The strain is able to adapt to oxidative and osmotic stresses but is defective in filamentous and invasive growth. The strain displays attenuated virulence in a murine systemic infection model. RNA-sequencing analysis revealed that JL01 has a distinct gene expression profile compared with C. albicans reference strain SC5314; hundreds of transcripts were significantly dysregulated, including those related to morphogenesis and pathogenesis. Taken together, our clinical, virulence, morphological, and biological analyses suggest that the azole resistance, oxidative and osmotic stress tolerance, invasive defect, hypovirulence, and impaired interaction with the host immune system of strain JL01 may correlate with its ability to cause cervical lymphadenitis in the patient. Our research may contribute to elucidating the mechanism(s) underlying the drug resistance and immune escape of C. albicans in chronic fungal infection. [ABSTRACT FROM AUTHOR]

Therapeutic options for coronaviruses remain limited. To address this unmet medical need, we screened 5406 compounds, including United States Food and Drug Administration (FDA)-approved drugs and bioactives, for activity against a South Korean Middle East respiratory syndrome coronavirus (MERS-CoV) clinical isolate. Among 221 identified hits, 54 had therapeutic indexes (TI) greater than 6, representing effective drugs. The time-of-addition studies with selected drugs demonstrated eight and four FDA-approved drugs which acted on the early and late stages of the viral life cycle, respectively. Confirmed hits included several cardiotonic agents (TI > 100), atovaquone, an anti-malarial (TI > 34), and ciclesonide, an inhalable corticosteroid (TI > 6). Furthermore, utilizing the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), we tested combinations of remdesivir with selected drugs in Vero-E6 and Calu-3 cells, in lung organoids, and identified ciclesonide, nelfinavir, and camostat to be at least additive in vitro. Our results identify potential therapeutic options for MERS-CoV infections, and provide a basis to treat coronavirus disease 2019 (COVID-19) and other coronavirus-related illnesses.

Aim. Study the prevalence and intensity of anti-hemoglobin and anti-lactoferrin activity in enterococci as representatives of symbiotic intestine microflora and causative agents of infection-inflammatory diseases in children. Materials and methods. 95 strains of enterococci were used in the study, that were isolated from intestines of children during examination for dysbiosis, and 34 clinical isolates. Strain identification was carried out by using multiplex PCR. Anti-lactoferrin and anti-hemoglobin activity were determined by O.V.Bukharin et al. (2005). Results. A higher level of prevalence and intensity of anti-lactoferrin, anti-hemoglobin activity, as well as a combination of those properties was established in clinical isolates of Enterococcus faecalis and E. faecium compared with strains, isolated from intestines of healthy individuals. Conclusion. The studied properties could be used for differentiation of pathogenic and non-pathogenic strains of enterococci and during selection of inhibitory preparations, suitable for therapy of enterococci infections.

Background: Enterococci are part of the normal flora of both the human and animal gastrointestinal tract. The aim of the present study was to compare vancomycin resistance and vanA gene in clinical and stool isolates in children specimens. Methods: One hundred enterococcal isolates were identified by laboratory tests and then they were examined for their antibiotic resistance. Resistant isolates were also studied by PCR for vanA gene. Results: Of 50 stool isolates, 48 were Enterococcus faecium and 2 isolates were identified as Enterococcus faecalis. In our 50 clinical isolates, 23 were belonged to Enterococcus faecium while 27 isolates were Enterococcus faecalis. The stool isolates showed higher resistance to vancomycin than those from clinical isolates. In our stool isolates the rate of vancomycin resistance was 52%, while the vancomycin resistance rate in the clinical isolates was 32%. According to PCR results, in the 26 vancomycin resistant isolates from stool specimens, vanA gene was found in 1 isolate, though in our 16 vancomycin resistant isolates from clinical specimens, vanA gene was found in 10 isolates. Conclusion: Vancomycin resistant enterococci are of public health concern, especially in hospitals. Our results showed that stool isolates had higher resistance to antibiotics than those from clinical isolates. In the stool isolates the rate of vancomycin resistance was 52%, while the vancomycin resistance rate in our clinical isolates was 32%, but vanA gene was more prevalent among clinical isolates than stool isolates. [ABSTRACT FROM AUTHOR]

Background and Objectives: Enterococci as a part of the normal flora of human gastrointestinal tracts can cause infections in children and adults who have a weaker immune system. The aim of this study was to determine and compare vancomycin, gentamicin, erythromycin and tetracycline resistance in enterococci isolated from Tabriz Children's Hospital. Materials and Methods: In this laboratory study, between May 2016 and May 2017, one hundred enterococci isolates (fifty clinical isolates from variant specimens and fifty stool isolates from different parts of Tabriz Children's Hospital) were collected. After determinig the type, they were tested for their resistance against antibiotics (vancomycin, gentamicin, erythromycin, tetracycline) by disk diffusion. Data analysis was performed using T-test. Results: Out of 50 clinical isolates, 23 (46%) isolates belonged to Enterococcus faecium while 27 (54%) isolates were Enterococcus faecalis. Among 50 stool isolates, 48 (96%) isolates were Enterococcus faecium and 2 (4%) isolates were Enterococcus faecalis. The results also indicated that there was a significant difference between the clinical and stool isolates in regard to vancomycin (p=0.004), but no significant difference was observed between the clinical and stool isolates in regard to the other antibiotics. Conclusion: Considering the results, stool isolates have higher resistance to antibiotics than those of clinical isolates. Increasing antibiotic resistant strains, especially vancomycin among enterococci, is a serious threat to the general public, especially hospitalized patients, and limits the treatment options for patients with nosocomial infections. [ABSTRACT FROM AUTHOR]

Vibrio vulnificus , the causative agent of serious, often fatal, infections in humans, requires iron for its pathogenesis. As such, it obtains iron via both vulnibactin and heme-mediated iron-uptake systems. In this study, we identified the heme acquisition system in V . vulnificus M2799. The nucleotide sequences of the genes encoding heme receptors HupA and HvtA and the ATP-binding cassette (ABC) transport system proteins HupB, HupC, and HupD were determined, and then used in the construction of deletion mutants developed from a Δ ics strain, which could not synthesize vulnibactin. Growth experiments using these mutants indicated that HupA and HvtA are major and minor heme receptors, respectively. The expressions of two proteins were analyzed by the quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR). Furthermore, complementation analyses confirmed that the HupBCD proteins are the only ABC transport system shared by both the HupA and HvtA receptors. This is the first genetic evidence that the HupBCD proteins are essential for heme acquisition by V . vulnificus . Further investigation showed that hupA , hvtA , and hupBCD are regulated by Fur. The qRT-PCR analysis of the heme receptor genes revealed that HupR, a LysR-family positive transcriptional activator, upregulates the expression of hupA , but not hvtA . In addition, ptrB was co-transcribed with hvtA , and PtrB had no influence on growth in low-iron CM9 medium supplemented with hemin, hemoglobin, or cytochrome C. [ABSTRACT FROM AUTHOR]

El artículo discurre sobre la expresión del virus del papiloma humano tipo16 en Escherichia coli. Los autores comentan sobre un estudio de partículas similares al virus de la proteína L1 y examinan el rol de E. coli como un hospedero de proteínas recombinantes. También se considera la construcción del plasmidio pETHPV16mye-His en un entorno laboratorio.

Over the last two decades worldwide levels of antibiotic resistance have risen leading to the appearance of multidrug resistant microorganisms. Acinetobacter baumannii is a known skin pathogen which has emerged as a major cause of nosocomial outbreaks due to its capacity to colonize indwelling medical devices and natural antibiotic resistance. With chitosan being an effective antimicrobial agent against antibiotic resistant microorganisms, the aim of this work was to access its potential as an alternative to traditional antimicrobials in the management of A. baumannii growth. What the results showed was that both chitosan MW’s tested were active upon A. baumannii ’s planktonic and sessile growth. For planktonic growth MICs and MBCs were obtained at relatively low concentrations (0.5–2 mg/mL) while for sessile growth chitosan proved to be an effective inhibitor of A. baumannii ’s adhesion and biofilm formation. Considering these results chitosan shows a high potential for control of A. baumannii infections. [ABSTRACT FROM AUTHOR]

Objective: Thiosemicarbazone (TSC) containing three new mononuclear ruthenium(II)-arene complexes were synthesized so as to contribute to the development of ruthenium complexes with pharmacologically attracted properties. Methods: Reactions of the ruthenium(II)-arene dimer [{RuCl(μ-Cl)(η6-p-cymene)}2] (1) with the respective TSC1-3 (1:2 molar ratio) in methanol resulted in p-cymene containing new conformationally rigid half-sandwich organometallic ruthenium(II)-arene complexes; [(η6-p-cymene)Ru(TSC1-κ3O,N,S)]Cl (I), [(η6-p-cymene)Ru(Cl)(TSC2-κ2N,S)]Cl (II), and [(η6-p-cymene)Ru(Cl)(TSC3- κ2N,S)]Cl (III). The molecular structures of complexes I, II and III were elucidated on the spectroscopic data obtained by the application of 1H NMR, Fourier transform infrared (FT-IR), UV-vis and elemental analysis techniques. In vitro antimicrobial activities of the synthesized three ruthenium(II)-arene complexes were evaluated using the disc diffusion method. Results: The spectroscopic data indicated that TSC1 was bounded to the metal as a tridentate ligand with its thione sulfur atom, phenolic oxygen atom and azomethine nitrogen atom in the complex I, while TSC2 and TSC3 were bounded to metal center as bidentate manner through their thione sulfur atom and imine nitrogen (C=N) atom in the complexes II and III, respectively. The obtained antimicrobial activity results showed that these complexes efficiently inhibit the growth of Gram-positive bacterial strains. Conclusion: The TSC1-3 containing ruthenium(II)-arene complexes were successfully synthesized and their molecular structures were also determined by the spectroscopic methods. All ruthenium(II)-arene complexes showed higher antibacterial activities against Gram-positive bacterial strains than the Gram-negative ones. [ABSTRACT FROM AUTHOR]

Objective In Streptococcus mutans , a Gram-positive pathogen of dental caries, several surface proteins are anchored by the activity of sortase enzyme. Although various reports have shown that constructed S. mutans mutants deficient of sortase as well as laboratory reference strains with a sortase gene mutation have low cariogenic potential, no known studies have investigated clinical isolates with sortase defects. Here, we examined the cariogenic properties of S. mutans clinical isolates with sortase defects as well as caries status in humans harboring such defective isolates. Design Sortase-defective clinical isolates were evaluated for biofilm formation, sucrose-dependent adhesion, stress-induced dextran-dependent aggregation, acid production, and acid tolerance. Additionally, caries indices of subjects possessing such defective isolates were determined. Results Our in vitro results indicated that biofilm with a lower quantity was formed by sortase-defective as compared to non-defective isolates. Moreover, impairments of sucrose-dependent adhesion and stress-induced dextran-dependent aggregation were found among the isolates with defects, whereas no alterations were seen in regard to acid production or tolerance. Furthermore, glucan-binding protein C, a surface protein anchored by sortase activity, was predominantly detected in culture supernatants of all sortase-defective S. mutans isolates. Although the sortase-defective isolates showed lower cariogenic potential because of a reduction in some cariogenic properties, deft/DMFT indices revealed that all subjects harboring those isolates had caries experience. Conclusions Our findings suggest the impairment of cariogenic properties in S. mutans clinical isolates with sortase defects, though the detection of these defective isolates seemed not to imply low caries risk in the subjects harboring them. [ABSTRACT FROM AUTHOR]

Background: The sexual stages (gametocytes) of Plasmodium falciparum do not directly contribute to the pathology of malaria but are essential for transmission of the parasite from the human host to the mosquito. Mature gametocytes circulate in infected human blood for several days and their circulation time has been modelled mathematically from data of previous in vivo studies. This is the first time that longevity of gametocytes is studied experimentally in vitro. Methods: The in vitro longevity of P. falciparum gametocytes of 1 clinical isolate and 2 laboratory strains was assessed by three different methods: microscopy, flow cytometry and reverse transcription quantitative real-time PCR (RTqPCR). Additionally, the rate of gametocytogenesis of the used P. falciparum strains was compared. Results: The maximum in vitro lifespan of P. falciparum gametocytes reached almost 2 months (49 days by flow cytometry, 46 days by microscopy, and at least 52 days by RT-qPCR) from the starting day of gametocyte culture to death of last parasite in the tested strains with an average 50% survival rate of 6.5, 2.6 and 3.5 days, respectively. Peak gametocytaemia was observed on average 19 days after initiation of gametocyte culture followed by a steady decline due to natural decay of the parasites. The rate of gametocytogenesis was highest in the NF54 strain. Conclusions: Plasmodium falciparum mature gametocytes can survive up to 16-32 days (at least 14 days for mature male gametocytes) in vitro in absence of the influence of host factors. This confirms experimentally a previous modelling estimate that used molecular tools for gametocyte detection in treated patients. The survival time might reflect the time the parasite can be transmitted to the mosquito after clearance of asexual parasites. These results underline the importance of efficient transmission blocking agents in the fight against malaria. [ABSTRACT FROM AUTHOR]

Objectives Borrelia miyamotoi has been shown to infect humans in Eurasia and North America causing hard tick-borne relapsing fever (HTBRF). In vitro cultivation of B. miyamotoi was described recently; but clinical isolation of relapsing fever Borrelia is cumbersome. Our aim was to develop a straightforward protocol enabling B. miyamotoi isolation directly from the blood of patients. Methods Modified Kelly-Pettenkorfer (MKP-F) medium, with or without anticoagulants, or blood from healthy human volunteers, was spiked with B. miyamotoi spirochaetes in vitro . Subsequently, either media or plasma was used for cultivation directly, or after an additional centrifugation step. This isolation protocol was tested in a clinical setting on patients suspected of HTBRF. Results Dipotassium-EDTA, trisodium citrate and lithium heparin inhibited growth of B. miyamotoi at concentrations ≥250 μg/mL, 2.5 mM and 1 IU/mL, respectively. However, when plasma originating from human blood containing B. miyamotoi spirochaetes was subjected to an additional centrifugation step at 8000 g , suspended and inoculated into fresh MKP-F media, positive cultures were observed within 2 weeks. Of importance, this straightforward protocol allowed for isolation of B. miyamotoi from six out of nine patients with confirmed HTBRF. Conclusions Direct culture from K 2 -EDTA, trisodium citrate and lithium heparin plasma containing B. miyamotoi is hampered due to anticoagulants. Using a simple centrifugation protocol we were able to circumvent this detrimental effect, allowing for the first clinical isolation of B. miyamotoi. This will be of value for future research on the pathogenesis, genetics, diagnosis, therapy and epidemiology of HTBRF and other tick-borne relapsing fevers. [ABSTRACT FROM AUTHOR]

Vibrio vulnificus, an opportunistic pathogen that causes a serious, often fatal, infection in humans, requires iron for its growth. This bacterium utilizes iron from the environment via the vulnibactin-mediated iron uptake system. The mechanisms of vulnibactin biosynthesis, vulnibactin export, and ferric-vulnibactin uptake systems have been reported, whereas the ferric-vulnibactin reduction mechanism in the cell remains unclear. The results of our previous study showed that VuuB, a member of the flavin adenine dinucleotide-containing siderophore-interacting protein family, is a ferric-vulnibactin reductase, but there are other reductases that can complement for the defective vuuB. The aim of this study was to identify these proteins that can complement the loss of function of VuuB. We constructed mutants of genes encoding putative reductases in V. vulnificus M2799, and analyzed their growth under low-iron conditions. Complementation analyses confirmed that IutB, which functions as a ferric-aerobactin reductase, participates in ferric-vulnibactin reduction in the absence of VuuB. This is the first genetic evidence that ferric-vulnibactin is reduced by a member of the ferric-siderophore reductase protein family. In the aerobactin-utilization system, IutB plays a major role in ferric-aerobactin reduction in V. vulnificus M2799, and VuuB and DesB can compensate for the defect of IutB. Furthermore, the expression of iutB and desB was found to be regulated by iron and a ferric uptake regulator. [ABSTRACT FROM AUTHOR]