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Pseudomonas aeruginosa, Staphylococcus aureus, and Burkholderia cepacia are notorious pathogens known for their ability to form resilient biofilms, particularly within the lung environment of cystic fibrosis (CF) patients. The heightened concentration of NaCl, prevalent in the airway liquid of CF patients' lungs, has been identified as a factor that promotes the growth of osmotolerant bacteria like S. aureus and dampens host antibacterial defenses, thereby fostering favorable conditions for infections.In this study, we aimed to investigate how increased NaCl concentrations impact the development of multi-species biofilms in vitro, using both laboratory strains and clinical isolates of P. aeruginosa, S. aureus, and B. cepacia co-cultures. Employing a low-nutrient culture medium that fosters biofilm growth of the selected species, we quantified biofilm formation through a combination of adherent CFU counts, qPCR analysis, and confocal microscopy observations.Our findings reaffirmed the challenges faced by S. aureus in establishing growth within 1:1 mixed biofilms with P. aeruginosa when cultivated in a minimal medium. Intriguingly, at an elevated NaCl concentration of 145 mM, a symbiotic relationship emerged between S. aureus and P. aeruginosa, enabling their co-existence. Notably, this hyperosmotic environment also exerted an influence on the interplay of these two bacteria with B. cepacia. We demonstrated that elevated NaCl concentrations play a pivotal role in orchestrating the distribution of these three species within the biofilm matrix.Furthermore, our study unveiled the beneficial impact of NaCl on the biofilm growth of clinically relevant mucoid P. aeruginosa strains, as well as two strains of methicillin-sensitive and methicillin-resistant S. aureus. This underscores the crucial role of the microenvironment during the colonization and infection processes. The results suggest that hyperosmotic conditions could hold the key to unlocking a deeper understanding of the genesis and behavior of CF multi-species biofilms.

Background: Meningitis due to Cryptococcus neoformans/gattii is a fatal infection affecting immunocompromised population worldwide. Amphotericin B (AmB), fluconazole (FLC) and 5-flucytosine are the drugs of choice to treat the infection. We studied antifungal susceptibility pattern of clinical and environmental cryptococcal species using newer approach and analyze their resistant characteristics. Methods: Eighty clinical (54 C. neoformans and 26 C. gattii) and 18 environmental (14 C. neoformans and 4 C. gattii) isolates were subjected to antifungal susceptibility testing by automated (VITEK2C) method. Minimum inhibitory concentrations (MIC) were analyzed statistically. Genomic DNA of FLC resistant isolates was extracted and amplified to detect presence of CnAFR1 gene. Results: C. neoformans showed 1.85% and 21.4% AmB resistance, and 1.85% and 28.5% FLC- resistance, whereas C. gattii showed 25% and 50% FLC-resistance among clinical and environmental isolates respectively. MIC values were significantly (p < 0.05) different for the isolates from 2 sources. CnAFR1 gene sequence analysis revealed phylogenetic relationship among the resistant isolates. Conclusions: This pioneering study provides an insight into the sensitivity patterns of clinical and environmental cryptococcal isolates from south India. The recent emergence of AmB-resistance may transpire as a challenge for the clinicians. As the clinical and environmental isolates are phylogenetically evolved from CnAFR1 gene of Filobasidiella neoformans, the resistance is most probably an inherent attribute. This study emphasizes the need for speciation and antifungal susceptibility testing of cryptococcal isolates from clinical sources to institute appropriate antifungal therapy and to reduce the mortality and morbidity.

Objectives: Pseudomonas aeruginosa is important pathogens commonly cause nosocomial infections. The occurrence of multi-resistant organisms (MROs) of Pseudomonas aeruginosa strains have been increased worldwide and limiting the therapeutic options. The MRO of Pseudomonas aeruginosa phenotype can be mediated by a variety of resistance mechanisms and highly versatile property to mutate. Therefore the our study aimed to evaluate the resistance pattern of Pseudomonas aeruginosa collected from Riyadh tertiary care hospital, Kingdom of Saudi Arabia. Methods: During the period from 2019 to 2021 clinical samples were collected from microbiology lab at King Khalid University Hospital and analysed for the antibiotic susceptibility pattern. Results: Suggested that the rates of resistance for the three years were higher for isolates collected from patients older than 50 years if its compared with the strains collected from young age. A total of 1024 Pseudomonas aeruginosa isolates were collected during the last three years, the prevalence rate were 44.6%, 32.6% and 22.7% during the period of 2019, 2020, and 2021 respectively. Meanwhile, the highest percentages of multi drug resistance Pseudomonas aeruginosa strains were recovered from body fluids; about 38 (47.5%) out of 80 Pseudomonas aeruginosa isolates were MRO Pseudomonas aeruginosa. The rate of resistances showed that Imipenem was significantly higher in resistant among the clinical isolates (77.8%), then Meropenem (61%), Aztreonam (42%) and Ceftazidime (36%) than other antibiotics. Most of isolates were sensitive to colistin except (2.7%) were resistance. Moreover, antibiotic resistant bacteria have been observed with increasing frequency over the past three years. Conclusions: The current study reports that the susceptibility among P. aeruginosa isolates have been decreased in KSA, perhaps due to the massive use of antibiotics, the lack of adherence to approved infection control practices by hospitals, or due to the changes to the public health infrastructure.

Background: Bacillus cereus is a well-known pathogen for self-limited foodborne illness, and rarely an opportunistic pathogen associated with invasive infections among immunocompromised patients. Nosocomial outbreaks have been rarely reported. Methods: Between August and November 2019, four preterm neonates in neonatal care units of a medical center developed late-onset B. cereus bacteremia. An investigation was carried out. Forty-eight environmental specimens were obtained from these neonatal units, skin surface and environmental objects of Patient 4 for the detection of this organism 19 days after the onset of illness of Patient 4. B. cereus isolates from Patient 4, five unrelated patients and environmental objects if identified were further characterized by pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). Results: All four infants survived after vancomycin-containing treatment. Patient 4 developed diffuse cerebritis, brain abscess with severe neurologic sequelae. Of the 48 environmental samplings, 26 specimens showed positive for B. cereus, with one major clone (sequence type 365) accounting for 73%. The isolate from Patient 4 (ST427) was identical to one isolate collected from environmental objects in the same unit. After extensive cleaning of the environment and re-institution of the sterilization procedure of hospital linens, which was ceased since two months before the outbreak, no more cases was identified in these units for at least one year. Conclusions: We documented a cluster of B. cereus bacteremia involving four preterm infants, which might be associated with cessation of the procedure for linen sterilization and was successfully controlled by re-institution of this procedure.

Fluorine-18-fluorodeoxyglucose ([18F]FDG) positron emission tomography (18F-FDG-PET) is widely used for the detection of inflammatory and infectious diseases. Although this modality has proven to be a useful diagnostic tool, reliable distinction of bacterial infection from sterile inflammation or even from a malignancy remains challenging. Therefore, there is a need for bacteria-specific tracers for PET imaging that facilitate a reliable distinction of bacterial infection from other pathology. The present study was aimed at exploring the potential of 2-[18F]-fluorodeoxysorbitol ([18F]FDS) as a tracer for detection of Enterobacterales infections. Sorbitol is a sugar alcohol that is commonly metabolized by bacteria of the Enterobacterales order, but not by mammalian cells, which makes it an attractive candidate for targeted bacterial imaging. The latter is important in view of the serious clinical implications of infections caused by Enterobacterales. Here we demonstrate that sorbitol-based PET can be applied to detect a broad range of clinical bacterial isolates not only in vitro, but also in blood and ascites samples from patients suffering from Enterobacterales infections. Notably, the possible application of [18F]FDS is not limited to Enterobacterales since Pseudomonas aeruginosa and Corynebacterium jeikeium also showed substantial uptake of this tracer. We conclude that [18F]FDS is a promising tracer for PET-imaging of infections caused by a group of bacteria that can cause serious invasive disease.

Background: The environment may facilitate transmission of health care-associated methicillin-resistant Staphylococcus aureus (MRSA) and the pathogen is frequently shed by patients. However, the molecular characteristics and genetic relatedness between clinical and environmental MRSA isolates remain largely unclear in the clinical setting. Methods: A total of 100 hospitalized patients with MRSA infection and 25 hospitalized patients without MRSA infection were enrolled in a medical center, Taiwan in 2019. Environmental and clinical MRSA isolates were characterized by antibiotic susceptibility testing and molecular methods. Results: In the study, we detected 17 MRSA isolates in the environment that surrounded 15 MRSA-infected patients and one environmental MRSA isolate from one patient without MRSA infection. The molecular analysis revealed a high genetic diversity within either environmental or clinical MRSA isolates, while the USA300 clone (pulsotype AI, SCCmec IV, ST8, PVL-positive) accounts for 39% (7/18) of environmental and 33% (7/21) of clinical MRSA isolates. Moreover, 13 of the 15 MRSA-infected patients had identical paired clinical-environmental MRSA isolates, which exhibited indistinguishable genetic relatedness and highly similar antibiotic susceptibility phenotype, suggesting a possible transmission cycle of MRSA in the hospital. Conclusions: The environmental MRSA was closely linked to MRSA isolated from patients, suggesting that the environment may act as a reservoir of MRSA. Besides, the USA300 MRSA has become a major clone in the hospital setting. An effective and rigorous approach to environmental cleaning and decontamination is suggested to eradicate MRSA in the hospital.

Summary: Background: Gut microbiota-derived short-chain fatty-acid (SFCA) acetate protects mice against RSV A2 strain infection by increasing interferon-β production and expression of interferon-stimulated genes (ISGs). However, the role of SFCA in RSV infection using strains isolated from patients is unknown. Methods: We first used RSV clinical strains isolated from infants hospitalized with RSV bronchiolitis to investigate the effects of in vitro SCFA-acetate treatment of human pulmonary epithelial cells. We next examined whether SCFA-acetate treatment is beneficial in a mouse model of RSV infection using clinical isolates. We sought to investigate the relationship of gut microbiota and fecal acetate with disease severity among infants hospitalized with RSV bronchiolitis, and whether treating their respiratory epithelial cells with SCFA-acetate ex-vivo impacts viral load and ISG expression. We further treated epithelial cells from SARS-CoV-2 infected patients with SCFA-acetate. Findings: In vitro pre-treatment of A549 cells with SCFA-acetate reduced RSV infection with clinical isolates and increased the expression of RIG-I and ISG15. Animals treated with SCFA-acetate intranasally recovered significantly faster, with reduction in the RSV clinical isolates viral load, and increased lung expression of IFNB1 and the RIG-I. Experiments in RIG-I knockout A549 cells demonstrated that the protection relies on RIG-I presence. Gut microbial profile was associated with bronchiolitis severity and with acetate in stool. Increased SCFA-acetate levels were associated with increasing oxygen saturation at admission, and shorter duration of fever. Ex-vivo treatment of patients’ respiratory cells with SCFA-acetate reduced RSV load and increased expression of ISGs OAS1 and ISG15, and virus recognition receptors MAVS and RIG-I, but not IFNB1. These SCFA-acetate effects were not found on cells from SARS-CoV-2 infected patients. Interpretation: SCFA-acetate reduces the severity of RSV infection and RSV viral load through modulation of RIG-I expression. Funding: FAPERGS (FAPERGS/MS/CNPq/SESRS no. 03/2017 - PPSUS 17/2551-0001380-8 and COVID-19 20/2551-0000258-6); CNPq 312504/2017-9; CAPES) - Finance Code 001.

Geographical strain variations of Candida albicans causing different clinical conditions in susceptible individuals have been reported. In this study, the distribution of diploid sequence type of C. albicans was investigated in Mwanza, Tanzania. A total of 64 C. albicans were selected on the basis of their antifungal susceptibility patterns, followed by multilocus sequence typing (MLST) to establish the circulating sequence types (STs). Forty-eight MLST were obtained out of 64 isolates amounting to 75% population structure differences. Out of these STs, 27 (56.3%) were new diploid ST types. C. albicans isolates with new ST were more diverse than isolates with known STs (27/29, 93.1% vs. 21/35, 60%, p 0.002). In conclusion, C. albicans from clinical specimens were highly diverse, with more than half of the detected diploid ST not previously reported in the MLST database, thus confirming the genetic differences of C. albicans from different geographical regions.

Polymyxins are important therapeutic options for treating infections, mainly those caused by carbapenem-resistant Klebsiella pneumoniae. Specific chemical characteristics of polymyxins make it difficult to perform antimicrobial susceptibility testing, especially within the clinical laboratory. Here we aimed to evaluate the performance of three phenotypic methods: Rapid NP Polymyxin Test, ColiSpot test and the SuperPolymyxin medium. To accomplish this, 170 non-duplicate clinical K. pneumoniae isolates were analysed (123 colistin-resistant and 47 susceptible). The sensitivity and specificity obtained for Rapid Polymyxin NP Test, Colispot and SuperPolymyxin medium were, respectively, 90% and 94%, 74% and 100%, and 82% and 85%. Very major errors occurred more frequently in low-level colistin-resistant isolates (MICs 4 and 8 μg/mL). Rapid Polymyxin NP proved to be a method capable of identifying colistin-resistant strains in acceptable categorical agreement. However, major errors and very major errors of this method were considered unacceptable for colistin-resistance screening. Although the Colispot test is promising and easy to perform and interpret, the results did not reproduce well in the isolates tested. The colistin-containing selective medium (SuperPolymyxin) showed limitations, including quantification of mucoid colonies and poor stability. Nevertheless, Colispot and SuperPolymyxin medium methods did not present acceptable sensitivity, specificity and categorical agreement. It is essential to use analytical tools that faithfully reproduce bacterial resistance in vitro, especially in last-line drugs, such as polymyxins, when misinterpretation of a test can result in therapeutic ineffectiveness.

Background: The present study is one of the first to provide a picture of antimicrobial resistance for a range of bacteria and antimicrobial classes in Gabon, Central Africa. Methods: During the year 2010, 146 urine cytology, 143 blood cultures, 107 vaginal swabs, 23 urethral swabs, and 18 other culture examinations were positives. All isolates were tested for antibiotic sensitivity. Results: Four hundred thirty-seven microorganisms were isolated: 210 enterobacteria, 166 staphylococci, 38 streptococci, 14 Acinetobacter, and nine Stenotrophomonas. Of the Klebsiella isolates, 18% and 30% were found to be resistant to selected third-generation cephalosporins (3CG) and fourth-generation cephalosporins (4CG), respectively. Sixty-seven percent of Escherichia coli isolates were resistant to amoxicillin with clavulanic acid. Between 3% and 30% of E. coli isolates were resistant to selected 3CG. All Enterobacter cloacae isolates were sensitive to imipenem. Resistance to quinolones/fluoroquinolones was seen in 21–50% of E. coli isolates. Twenty-six percent of E. cloacae showed resistance to ceftazidime and 37% to cefotaxime. The resistance rate to quinolones ranged between 58% and 78%. Thirty-two percent of Staphylococcus isolates were resistant to gentamicin. Low resistance rates to teicoplanin (2–4%) were observed. Thirty-seven percent of isolated Staphylococcus aureus and 61% of isolated Staphylococcus saprophyticus were resistant to both penicillin G and oxacillin. Streptococcus isolates had low resistance rates to erythromycin, ceftriaxone, and ciprofloxacin (5%, 7%, and 14%, respectively) and were highly resistant to tetracycline, gentamicin, and sulfamethoxazole–trimethoprim (92%, 91%, and 62%, respectively). Conclusions: The antimicrobial resistance profiles seen here are of concern. To control the spread of drug-resistant bacteria, clinicians should be cognizant of their local antimicrobial resistance patterns.

The emergence of Klebsiella pneumoniae producing carbapenemase (KPC) has now become a global concern. As a part of a nationwide multicentre surveillance study in Cuba, three K. pneumoniae clinical isolates resistant to carbapenems were detected for a 1-month period (September to October 2011). PCR and sequence analysis revealed that the three strains harboured blaKPC-2. They showed resistance or intermediate susceptibility to expanded-spectrum cephalosporins, other ß-lactams, a ß-lactam/ß-lactamase inhibitor combination, and gentamicin. Two strains were susceptible only to colistin, whereas the other strain showing colistin resistance was susceptible to fluoroquinolones. These blaKPC-2-positive K. pneumoniae strains were classified into ST1271 (CC29), a novel clone harbouring blaKPC-2, and were revealed to be genetically identical by PCR-based DNA fingerprinting. The three patients infected with the KPC-producing K. pneumoniae had common risk factors, and had no overseas travel experience outside Cuba, suggesting local acquisition of the resistant pathogen. This is the first report of a KPC-producing K. pneumoniae in Cuba. Although detection of KPC in Enterobacteriaceae is still rare in Cuba, our finding indicated that KPC-producing bacteria are a global concern and highlighted the need to identify these microorganisms in clinical laboratories.

Pseudomonas aeruginosa is an opportunistic human pathogen that has developed antibiotic resistance (AR) and causes a range of illnesses, including respiratory pneumonia, gastrointestinal infections, keratitis, otitis media and bacteremia in patients with compromised immune system. The production of metallo-β-lactamases (MBLs) is one of the major mechanisms of AR in this bacterium with ensuing infections difficult to treat. The main goal of this study was to provide a quantitative estimate of MBLs producing clinical P. aeruginosa isolates among the Nepalese patients and determine if MBL correlates with multi-drug resistance (MDR). Preferred Reporting Items for Systematic Reviews and Meta-Analysis (PRISMA) guideline was followed for meta-analysis of relevant literature using PubMed, Research4Life, and Google Scholar. The prevalence of MBLs in P. aeruginosa from clinical samples was determined using R 4.1.2 for data pooled from studies published until 2021. The meta-analysis of a total of 19 studies selected (of 6038 studies for which titles and abstracts were reviewed) revealed the prevalence of MBLs producing P. aeruginosa (MBL-PA) was 14 % (95 % CI: 0.10-0.19) while MDR isolates among P. aeruginosa was 42 % (95 % CI: 0.30-0.55) in Nepal. Combined Disc Test was predominantly used phenotypic method for confirming MBLs phenotypes among the studies. Sputum was the most common specimen from which MBL-PA was recovered. A significant positive correlation was observed between MDR and MBL production in P. aeruginosa. We conclude that MBL producing strains are widespread among the clinical isolates of P. aeruginosa in Nepal and responsible for emerging MDR strains. It is paramount that antibiotics prescription against the bacterium should be monitored closely and alternative therapeutic modalities against MBL-PA explored., Competing Interests: Declaration of competing interest Lok R. Pokhrel reports financial support was provided by East Carolina University., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Geographical strain variations of Candida albicans causing different clinical conditions in susceptible individuals have been reported. In this study, the distribution of diploid sequence type of C. albicans was investigated in Mwanza, Tanzania. A total of 64 C. albicans were selected on the basis of their antifungal susceptibility patterns, followed by multilocus sequence typing (MLST) to establish the circulating sequence types (STs). Forty-eight MLST were obtained out of 64 isolates amounting to 75% population structure differences. Out of these STs, 27 (56.3%) were new diploid ST types. C. albicans isolates with new ST were more diverse than isolates with known STs (27/29, 93.1% vs. 21/35, 60%, p 0.002). In conclusion, C. albicans from clinical specimens were highly diverse, with more than half of the detected diploid ST not previously reported in the MLST database, thus confirming the genetic differences of C. albicans from different geographical regions., (© 2020 The Author(s).)

Polymyxins are important therapeutic options for treating infections, mainly those caused by carbapenem-resistant Klebsiella pneumoniae . Specific chemical characteristics of polymyxins make it difficult to perform antimicrobial susceptibility testing, especially within the clinical laboratory. Here we aimed to evaluate the performance of three phenotypic methods: Rapid NP Polymyxin Test, ColiSpot test and the SuperPolymyxin medium. To accomplish this, 170 non-duplicate clinical K. pneumoniae isolates were analysed (123 colistin-resistant and 47 susceptible). The sensitivity and specificity obtained for Rapid Polymyxin NP Test, Colispot and SuperPolymyxin medium were, respectively, 90% and 94%, 74% and 100%, and 82% and 85%. Very major errors occurred more frequently in low-level colistin-resistant isolates (MICs 4 and 8 μg/mL). Rapid Polymyxin NP proved to be a method capable of identifying colistin-resistant strains in acceptable categorical agreement. However, major errors and very major errors of this method were considered unacceptable for colistin-resistance screening. Although the Colispot test is promising and easy to perform and interpret, the results did not reproduce well in the isolates tested. The colistin-containing selective medium (SuperPolymyxin) showed limitations, including quantification of mucoid colonies and poor stability. Nevertheless, Colispot and SuperPolymyxin medium methods did not present acceptable sensitivity, specificity and categorical agreement. It is essential to use analytical tools that faithfully reproduce bacterial resistance in vitro , especially in last-line drugs, such as polymyxins, when misinterpretation of a test can result in therapeutic ineffectiveness., (© 2020 The Authors.)

Introduction: The clinical significance of enterococci is mostly related to its antibiotic resistance which contributes to colonization and infection, in particular amongst the hospitalized patients. The present review has examined the literature to provide a comprehensive data on enterococci antibiotic resistance during the last 20 years in Iran., Methods: Search engines such as Google Scholar and PubMed were used to identify all Persian and English-language articles investigating enterococci in Iran from 1996 to 2017. The search terms were "enterococci", "enterococcal", "enterococcus", "Iran", "bacterial resistance", "antibiotic resistance" and "resistance"., Results: Decrease in the resistance trend against ampicillin, gentamycin and ciprofloxacin was observed over a period of 15 years (2001 to 2016) in Iran. During a 10 years period from 2001 to 2015, the rate of resistance among Enterococcus faecalis species was less than Enterococcus faecium . The resistancerate, however, was considerably increased for both species during this period. The mean resistance rates for vancomycin, gentamicin, ciprofloxacin, erythromycin, nitrofurantoin, chloramphenicol, trimethoprim-sulfametoxazol, imipenem and teicoplanin were higher among complicated cases (patients with underlying debilitating disorders) compared to general cases (hospitalized or outpatients with no specific underlying disorder)., Conclusions: E. faecalis and E. faecium showed a rise in the mean resistance against all the antibiotics during a 10-year period from 2010 to 2015. With the exception of penicillin and ampicillin, resistance to all antibiotics was higher amongst complicated cases compared to general patients.

There is a great interest in developing novel anti-biofilm materials in order to decrease medical device-associated bacterial infections causing morbidity and high healthcare costs. However, the testing of novel materials is often done using bacterial lab strains that may not exhibit the same phenotype as clinically relevant strains infecting medical devices. Furthermore, no consensus of strain selection exists in the field, making results very difficult to compare between studies. In this work, 19 clinical isolates of Pseudomonas aeruginosa originating from intubated patients in an intensive care unit have been characterized and compared to the lab reference strain PAO1 and a rmlC lipopolysaccharide mutant of PAO1. The adhesion and biofilm formation was monitored, as well as cell properties such as hydrophobicity, zeta potential and motility. Two groups of isolates were observed: one with high adhesion to polymer surfaces and one with low adhesion (the latter including PAO1). Furthermore, detailed biofilm assays in a flow system were performed using five characteristic isolates from the two groups. Confocal microscopy showed that the adhesion and biofilm formation of four of these five strains could be reduced dramatically on zwitterionic surface coatings. However, one isolate with pronounced swarming colonized and formed biofilm also on the antifouling surface. We demonstrate that the biofilm properties of clinical isolates can differ greatly from that of a standard lab strain and propose two clinical model strains for testing of materials designed for prevention of biofilm formation in the respiratory tract. The methodology used could beneficially be applied for screening of other collections of pathogens to identify suitable model strains for in vitro biofilm testing., Statement of Significance: Medical-device associated infections present a great challenge in health care. Therefore, much research is undertaken to prevent bacterial colonization of new types of biomaterials. The work described here characterizes, tests and presents a number of clinically relevant bacterial model strains for assessing biofilm formation by Pseudomonas aeruginosa. Such model strains are of importance as they may provide better predictability of lab testing protocols with respect to how well materials would perform in an infection situation in a patient. Furthermore, this study uses the strains to test the performance of polymer surfaces designed to repel bacterial adhesion and it is shown that the biofilm formation for four out of the five tested bacterial strains was reduced., (Copyright © 2018 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

Malaria remains a major cause of childhood deaths in resource-limited settings. In the absence of an effective vaccine, drugs and other interventions have played very significant roles in combating the scourge of malaria. The recent reports of resistance to artemisinin necessitate the need for new antimalarial drugs with novel mechanisms of action. Towards the development of new, affordable and easily accessible antimalarial drugs for endemic regions, the Medicines for Malaria Venture (MMV) assembled a total of 400 active antimalarial compounds called the Malaria Box. The potency and the efficacy of the Malaria Box Compounds have been determined mainly using laboratory strains of P. falciparum. This study investigated the potency of twenty compounds from the Malaria Box against four clinical isolates from Ghana, using optimized in vitro growth inhibitory assays. Seven out of the 20 compounds screened had 50% inhibitory concentration (IC 50 ) below 500 nM. The most active among the selected compounds was MMV006087 (average IC 50 of 30.79 nM). Variations in the potency of the Malaria Box Compounds were observed between P. falciparum clinical isolates and Dd2 strain. We also investigated the sensitivity of the clinical isolates to chloroquine and artesunate. The N093 clinical isolate was found to be resistant to chloroquine but showed high sensitivity to artesunate. The results underscore the importance of including clinical isolates with different drug-resistant backgrounds, in addition to laboratory strains, in validating potential compounds during antimalarial compound screening programs., (Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.)

The ability of Plasmodium falciparum infected erythrocytes (Pf-IEs) to activate endothelial cells has been described; however, the interaction of the endothelium with Pf-IEs field isolates from patients has been less characterized. Previous reports have shown that isolates alter the endothelial permeability and apoptosis. In this study, the adhesion of 19 uncomplicated malaria isolates to Human Dermal Microvascular Endothelial Cells (HDMEC), and their effect on the expression of ICAM-1 and proinflammatory molecules (sICAM-1, IL-6, IL-8, and MCP-1) was evaluated. P. falciparum isolates adhered to resting and TNFα-activated HDEMC cells at different levels. All isolates increased the ICAM-1 expression on the membrane (mICAM-1) of HDMEC and increased the release of its soluble form (sICAM-1), as well the production of IL-6, IL-8 and MCP-1 by HDMEC with no signs of cell apoptosis. No correlation between parasite adhesion and production of cytokines was observed. In conclusion, isolates from uncomplicated malaria can induce a proinflammatory response in endothelial cells that may play a role during the initial inflammatory response to parasite infection; however, a continuous activation of the endothelium can contribute to pathogenesis., (Copyright © 2016 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.)

Antimicrobial peptides (AMPs) are promising candidates for new antibiotic classes but often display an unacceptably high toxicity towards human cells. A naturally produced C-terminal fragment of PGLa, named PGLa-H, has been reported to have a very low haemolytic activity while maintaining a moderate antibacterial activity. A sequential tandem repeat of this fragment, diPGLa-H, was designed, as well as an analogue with a Val to Gly substitution at a key position. These peptides showed markedly improved in vitro bacteriostatic and bactericidal activity against both reference strains and multidrug resistant clinical isolates of Gram-negative and Gram-positive pathogens, with generally low toxicity for human cells as assessed by haemolysis, cell viability, and DNA damage assays. The glycine substitution analogue, kiadin, had a slightly better antibacterial activity and reduced haemolytic activity, which may correlate with an increased flexibility of its helical structure, as deduced using molecular dynamics simulations. These peptides may serve as useful lead compounds for developing anti-infective agents against resistant Gram-negative and Gram-positive species., (Copyright © 2016 Elsevier B.V. All rights reserved.)

The spread of multidrug resistant Mycobacterium tuberculosis is one of the major challenges in tuberculosis control. In Eurasia, the spread of multidrug resistant tuberculosis is driven by the M. tuberculosis Beijing genotype. In this study, we examined whether selective advantages are present in the proteome of Beijing isolates that contribute to the emergence of this genotype. To this end, we compared the proteome of M. tuberculosis Beijing to that of M. tuberculosis H37Rv, both in the presence and absence of the first-line antibiotic rifampicin. During rifampicin exposure, both M. tuberculosis genotypes express proteins belonging to the DosR dormancy regulon, which induces a metabolically hypoactive-, drug tolerant phenotype. However, these markers of rifampicin tolerance were already more abundant in the M. tuberculosis Beijing isolate prior to drug exposure. To determine whether the a priori high abundance of specific proteins contribute to the formation of antibiotic resistance in M. tuberculosis Beijing, we quantified the abundance of 33 selected proteins in 27 clinical isolates from the five most common M. tuberculosis lineages using parallel reaction monitoring. The observed pre-existing high abundance of dormancy proteins in Beijing strains provides an evolutionary advantage that allows these strains to persist for prolonged periods during rifampicin treatment., Significance: M. tuberculosis is the leading cause of death by a bacterial infection worldwide. Treatment-regimen to eradicate this pathogen make use of the first-line antibiotic rifampicin, which is considered to be the cornerstone of modern day anti-tuberculosis treatment. Despite the potency of rifampicin, there is an increasing occurrence of rifampicin resistant mutants in a specific cluster of M. tuberculosis, the Beijing genotype. Using both a data dependent acquisition and a targeted proteomic approach we identified markers of rifampicin tolerance to be high abundant in members of the M. tuberculosis Beijing genotype, already prior drug exposure. The identification of this M. tuberculosis Beijing specific trait will contribute to improved diagnostics and treatment of M. tuberculosis., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Coxsackie-B-viruses (CVB) cause a wide variety of diseases, ranging from mild syndromes to life-threatening conditions such as pancreatitis, myocarditis, meningitis and encephalitis. Especially newborns and young infants develop severe diseases and long-term sequelae may occur among survivors. Due to lack of specific antiviral therapy the current treatment of CVB infection is limited to symptomatic treatment. Here we analyzed the antiviral activity of a soluble receptor fusion protein, containing the extracellular part of the coxsackievirus and adenovirus receptor (CAR) fused to the constant domain of the human IgG - sCAR-Fc - against laboratory and clinical CVB strains. We found a high overall antiviral activity of sCAR-Fc against various prototypic laboratory strains of CVB, with an inhibition of viral replication up to 3 orders of magnitude (99.9%) at a concentration of 2.5 μg/ml. These include isolates that are not dependent on CAR for infection and isolates that are resistant against pleconaril, the currently most promising anti-CVB therapeutic. A complete inhibition was observed using higher concentration of sCAR-Fc. Further analysis of 23 clinical CVB isolates revealed overall high antiviral efficiency (up to 99.99%) of sCAR-Fc. In accordance with previous data, our results confirm the strong antiviral activity of sCAR-Fc against laboratory CVB strains and demonstrate for the first time that sCAR-Fc is also highly efficient at neutralizing clinical CVB isolates. Importantly, during the sCAR-Fc inhibition experiments, no naturally occurring resistant mutants were observed., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Clinical isolates from protozoan parasites such as Giardia lamblia are at present practically impossible to culture. By using simple cyst purification methods, we show that Giardia whole genome sequencing of clinical stool samples is possible. Immunomagnetic separation after sucrose gradient flotation gave superior results compared to sucrose gradient flotation alone. The method enables detailed analysis of a wide range of genes of interest for genotyping, virulence and drug resistance., (Copyright © 2014 European Society of Clinical Microbiology and Infectious Diseases. Published by Elsevier Ltd. All rights reserved.)

Background: The present study is one of the first to provide a picture of antimicrobial resistance for a range of bacteria and antimicrobial classes in Gabon, Central Africa., Methods: During the year 2010, 146 urine cytology, 143 blood cultures, 107 vaginal swabs, 23 urethral swabs, and 18 other culture examinations were positives. All isolates were tested for antibiotic sensitivity., Results: Four hundred thirty-seven microorganisms were isolated: 210 enterobacteria, 166 staphylococci, 38 streptococci, 14 Acinetobacter, and nine Stenotrophomonas. Of the Klebsiella isolates, 18% and 30% were found to be resistant to selected third-generation cephalosporins (3CG) and fourth-generation cephalosporins (4CG), respectively. Sixty-seven percent of Escherichia coli isolates were resistant to amoxicillin with clavulanic acid. Between 3% and 30% of E. coli isolates were resistant to selected 3CG. All Enterobacter cloacae isolates were sensitive to imipenem. Resistance to quinolones/fluoroquinolones was seen in 21-50% of E. coli isolates. Twenty-six percent of E. cloacae showed resistance to ceftazidime and 37% to cefotaxime. The resistance rate to quinolones ranged between 58% and 78%. Thirty-two percent of Staphylococcus isolates were resistant to gentamicin. Low resistance rates to teicoplanin (2-4%) were observed. Thirty-seven percent of isolated Staphylococcus aureus and 61% of isolated Staphylococcus saprophyticus were resistant to both penicillin G and oxacillin. Streptococcus isolates had low resistance rates to erythromycin, ceftriaxone, and ciprofloxacin (5%, 7%, and 14%, respectively) and were highly resistant to tetracycline, gentamicin, and sulfamethoxazole-trimethoprim (92%, 91%, and 62%, respectively)., Conclusions: The antimicrobial resistance profiles seen here are of concern. To control the spread of drug-resistant bacteria, clinicians should be cognizant of their local antimicrobial resistance patterns., (Copyright © 2014 The Authors. Published by Elsevier Ltd.. All rights reserved.)

In this study, we investigated the prevalence of genital Chlamydia trachomatis isolated in Japan using a high-resolution genotyping method, the multilocus VNTR analysis (MLVA)-ompA typing method. Seventeen serotypes of C. trachomatis standard strain (A-L3) and 44 clinical isolates were obtained from clinical settings. Genotyping of the ompA gene allowed clinical isolates to be divided into nine serotypes: B (6.8%), D (15.9%), E (25%), F (20.5%), G (18.1%), H (6.8%), Ia (2.3%), J (2.3%), and K (2.3%). These isolates were further divided into 28 types after combining ompA genotyping data with MLVA data (Hunter-Gaston discriminatory index D, 0.949). Thus, our results demonstrated that MLVA could identify clinical isolates that could not be distinguished by ompA typing., (Copyright © 2014 Japanese Society of Chemotherapy and The Japanese Association for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.)

The present study reports a simple and robust method for synthesis of silver and gold nanoparticles using Coleus forskohlii root extract as reducing and stabilizing agent. Stable silver nanoparticles (AgNPs) and gold nanopoarticles (AuNPs) were formed on treatment of an aqueous silver nitrate (AgNO3) and chloroauric acid (HAuCl4) solutions with the root extract. The nanoparticles obtained were characterized by UV-Visible spectroscopy, Transmission electron microscopy (TEM), X-ray diffraction (XRD) and Fourier-transform infrared spectroscopy (FT-IR). UV-Vis and TEM analysis indicate that with higher quantities of root extract, the interaction is enhanced leading to size reduction of spherical metal nanoparticles. XRD confirms face-centered cubic phase and the diffraction peaks can be attributed to (111), (200), (222) and (311) planes for these nanoparticles. These synthesized Ag and Au nanoparticles were found to exhibit excellent bactericidal activity against clinically isolated selected pathogens such as Escherichia coli (E. coli), Pseudomonas aeruginosa (P. aeruginosa) and Staphylococcus aureus (S. aureus). The synthesized AgNPs were also found to function as an efficient green catalyst in the reduction of anthropogenic pollutant 4-nitrophenol (4-NP) to 4-aminophenol (4-AP) by sodium borohydride, which was apparent from the periodical color change from bright yellow to colorless, after the addition of AgNPs., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Staphylococcus aureus is a major human pathogen causing community- and hospital-acquired infections. Capsule production of S. aureus confers protection against host defence. There is a lack of information concerning the association of capsular polysaccharide (CP) expression and activity of the accessory gene regulator (agr) in clinical S. aureus isolates. Production of CP and agr expression were assessed in 195 S. aureus isolates from infected patients at a German University Hospital. Northern blot analysis revealed that S. aureus strains with a non-functional agr locus were more likely to be CP-negative than strains with a functional agr locus., (© 2013 The Authors Clinical Microbiology and Infection © 2013 European Society of Clinical Microbiology and Infectious Diseases.)

The emergence of Klebsiella pneumoniae producing carbapenemase (KPC) has now become a global concern. As a part of a nationwide multicentre surveillance study in Cuba, three K. pneumoniae clinical isolates resistant to carbapenems were detected for a 1-month period (September to October 2011). PCR and sequence analysis revealed that the three strains harboured bla KPC-2. They showed resistance or intermediate susceptibility to expanded-spectrum cephalosporins, other β-lactams, a β-lactam/β-lactamase inhibitor combination, and gentamicin. Two strains were susceptible only to colistin, whereas the other strain showing colistin resistance was susceptible to fluoroquinolones. These bla KPC -2-positive K. pneumoniae strains were classified into ST1271 (CC29), a novel clone harbouring bla KPC -2, and were revealed to be genetically identical by PCR-based DNA fingerprinting. The three patients infected with the KPC-producing K. pneumoniae had common risk factors, and had no overseas travel experience outside Cuba, suggesting local acquisition of the resistant pathogen. This is the first report of a KPC-producing K. pneumoniae in Cuba. Although detection of KPC in Enterobacteriaceae is still rare in Cuba, our finding indicated that KPC-producing bacteria are a global concern and highlighted the need to identify these microorganisms in clinical laboratories.

The hepatitis C virus (HCV) NS4B inhibitors have shown potent inhibition of HCV replication in vitro. To assess the effect of viral diversity on the susceptibility to NS4B inhibitors, genotype (GT)-specific GT1a and GT1b replicon shuttle vectors were designed and created for cloning HCV NS4B genes from clinical isolates. For the GT1b NS4B shuttle vector, the S2204I adaptive mutation was introduced in NS5A to improve replication due to the replacement of the K1846T adaptive mutation in NS4B with NS4B from the clinical isolates. In addition to the adaptive mutations, a newly identified Huh-7 cell line, Huh-7-1C, which is highly permissive for both GT1a and GT1b replication, was used to further enhance the replication levels. HCV NS4B gene from clinical isolates was amplified and inserted into the corresponding GT1a and GT1b modified lab strain chimeric replicons. GT1a and GT1b chimeric replicons expressing diverse NS4B genes from corresponding subtypes of clinical isolates replicated at highly efficient levels for phenotypic analysis. Due to natural variation in their amino acid residues in NS4B, these isolates displayed varying drug susceptibilities to an NS4B inhibitor. In mixed populations with wild-type, the sensitivity of resistance detection of NS4B resistant mutants H94R and V105M was between 20% and 80%. The chimeric shuttle vectors can be used to characterize the activity of antiviral drugs targeting NS4B from diverse natural clinical isolates and aid in the development of novel compounds against HCV NS4B., (Copyright © 2013. Published by Elsevier B.V.)

Aim of the Study: To detect virulence factors in 54 Klebsiella pneumoniae isolates from different clinical specimens: urine (26), blood (11), pus (11), lung (four), cerebrospinal fluid (one) and ascitic fluid (one)., Material and Methods: PCR was used to investigate virulence genes encoding adhesins (fimH-1, mrkD, kpn, ycfM), siderophores (entB: enterobactin, iutA: aerobactin, irp-1, irp-2, ybtS, fyuA: yersiniabactin, iroN: catechols receptor), protectines or invasins (rmpA, magA, traT) and toxins (hlyA, cnf-1). The serum resistance, capsule and hypermucoviscosity, and ability to form biofilm and produce siderophores were sought by phenotypic assays. The in vivo virulence was assessed in mice infected by intraperitoneal way. Antimicrobial susceptibility was tested by diffusion method., Results: The most common virulence genes were fimH-1 (100%), mrkD (96.3%), ycfM (96.3%), and entB (100%). kpn and yersiniabactin genes were found at medium rates of 63% and 46.3% and at lower prevalence, were genes traT (1.8%), iroN (3.7%), iutA (5.5%) and rmpA (3.7%). magA, hlyA and cnf-1 genes were not detected. The capsule, serum resistance, biofilm formation, mannose-sensitive or -resistant haemagglutination and hypermucoviscosity were observed in 100%, 92.6%, 88.8%, 94.4%, 68.5% and 9.2% of isolates, respectively. The prevalence of siderophores was consistent with that of genotypic detection. The LD50 in mice was very low (<10(2) CFUs) for isolates with the most virulence factors. A rate of 74.1% of isolates showed a multidrug resistance (MDR) pattern., Conclusions: The distribution of virulence profiles according to the clinical origin suggests a role of enterobactin in urinary infections and yersiniabactin in the invasiveness. The fimbriae F1 and F3, capsule, enterobactin, serum resistance and biofilm formation, were commonly found in isolates, they seem to be at the basis of classic pathogenicity of K. pneumoniae. The invasiveness enhancers, aerobactin, yersiniabactin, catechols receptor, mucoid factor and hypermucoviscosity, detected concomitantly in some isolates, constitute a threat for vulnerable populations, even more if they are in combination with antibiotic resistance., (Copyright © 2012 Elsevier Masson SAS. All rights reserved.)

Endotoxins are lipopolysaccharides (LPS), and major constituents of the outer membrane of Gram-negative bacteria. Bordetella pertussis LPS were the only major antigens, of this agent of whooping-cough, that were not yet analyzed on isolates from the pre- and post-vaccination era. We compared here the LPS structures of four clinical isolates with that of the vaccine strain BP 1414. All physico-chemical analyses, including SDS-PAGE, TLC, and different MALDI mass spectrometry approaches were convergent. They helped demonstrating that, on the contrary to some other B. pertussis major antigens, no modification occurred in the dodecasaccharide core structure, as well as in the whole LPS molecules. These results are rendering these major antigens good potential vaccine components. Molecular modeling of this conserved LPS structure also confirmed the conclusions of previous experiments leading to the production of anti-LPS monoclonal antibodies and defining the main epitopes of these major antigens., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Background: Staphylococcus aureus produces various toxins and enzymes, and its presence can exacerbate skin conditions. Previous studies have shown that S. aureus is involved in skin deterioration, even in normal tissue. Biofilm strains show much greater resistance to antimicrobial agents and therefore require a much higher concentration of biocide than planktonic counterparts., Objective: As such, alternative strategies and more effective therapeutic agents against biofilm-producing S. aureus in skin are of great interest. Therefore, we turned our attention to differences in 50 clinical biofilm strains isolated from human facial skin., Methods: Based on S. aureus density on facial skin, we divided donors into two groups: relatively low density (LSG) and high density (HSG). In general, strong biofilm-forming strains were detected in the HSG donors. Two strains from each of the groups were submitted to gene microarray analysis to investigate expression differences and confirmed by RT-PCR., Results: In total, 111 of 7775 genes were differentially expressed between low (SA2 and SA7) vs. high (SA10 and SA33) biofilm-forming clinical strains. These genes include already well-known as biofilm formation related genes like icaABCD and lrgAB, and newly identified genes (sdrC, sspBCP) by RT-PCR. Comparison of gene expression differences between the two groups available at NCBI Gene Expression Omnibus accession number GSE44268., Conclusion: Our results suggest that S. aureus density in the skin is closely related to biofilm-forming ability, and we have identified several potential target genes that may be involved in regulating biofilm formation in situ., (Copyright © 2013 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights reserved.)