- Volume 69, Issue 5, 2020
Volume 69, Issue 5, 2020
- Editorial
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Logic in the time of coronavirus
More LessMuch has happened here since the local news media trumpeted the first Australian COVID-19 fatality, and stirred up a medieval fear of contagion. We now need to take a step back to examine the logic underlying the use of our limited COVID-19 countermeasures. Emerging infectious diseases by their nature, pose new challenges to the diagnostic-treatment-control nexus, and push our concepts of causality beyond the limits of the conventional Koch-Henle approach to aetiology. We need to use contemporary methods of assessing causality to ensure that clinical, laboratory and public health measures draw on a rational, evidence-based approach to argumentation. The purpose of any aetiological hypothesis is to derive actionable insights into this latest emerging infectious disease. This review is an introduction to a conversation with medical microbiologists, which will be supported by a moderated blog.
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- Antimicrobial Resistance
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Same-day antimicrobial susceptibility test using acoustic-enhanced flow cytometry visualized with supervised machine learning
Purpose. Antimicrobial susceptibility is slow to determine, taking several days to fully impact treatment. This proof-of-concept study assessed the feasibility of using machine-learning techniques for analysis of data produced by the flow cytometer-assisted antimicrobial susceptibility test (FAST) method we developed.
Methods. We used machine learning to assess the effect of antimicrobial agents on bacteria, comparing FAST results with broth microdilution (BMD) antimicrobial susceptibility tests (ASTs). We used Escherichia coli (1), Klebsiella pneumoniae (1) and Staphylococcus aureus (2) strains to develop the machine-learning algorithm, an expanded panel including these plus E. coli (2), K. pneumoniae (3), Proteus mirabilis (1), Pseudomonas aeruginosa (1), S. aureus (2) and Enterococcus faecalis (1), tested against FAST and BMD (Sensititre, Oxoid), then two representative isolates directly from blood cultures.
Results. Our data machines defined an antibiotic-unexposed population (AUP) of bacteria, classified the FAST result by antimicrobial concentration range, and determined a concentration-dependent antimicrobial effect (CDE) to establish a predicted inhibitory concentration (PIC). Reference strains of E. coli, K. pneumoniae and S. aureus tested with different antimicrobial agents demonstrated concordance between BMD results and machine-learning analysis (CA, categoric agreement of 91 %; EA, essential agreement of 100 %). CA was achieved in 35 (83 %) and EA in 28 (67 %) by machine learning on first pass in a challenge panel of 27 Gram-negative and 15 Gram-positive ASTs. Same-day AST results were obtained from clinical E. coli (1) and S. aureus (1) isolates.
Conclusions. The combination of machine learning with the FAST method generated same-day AST results and has the potential to aid early antimicrobial treatment decisions, stewardship and detection of resistance.
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Hydroxyethoxy phenyl butanone, a new cosmetic preservative, does not cause bacterial cross-resistance to antimicrobials
More LessIntroduction. Biocide-induced cross-resistance to antimicrobials in bacteria has been described and is a concern for regulators. We have recently reported on a new protocol to predict the propensity of biocide to induce phenotypic resistance in bacteria.
Aim. To measure bacterial propensity to develop antimicrobial resistance following exposure to a new cosmetic preservative developed by L’Oréal R and I.
Methodology. Well-established antimicrobials including triclosan (TRI) and benzalkonium chloride (BZC) and a new molecule hydroxyethoxy phenyl butanone (HEPB) were investigated for their antimicrobial efficacy, effect on bacterial growth, and their potential to induce resistance to chemotherapeutic antibiotics using a new predictive protocol.
Results. The use of this predictive protocol with Staphylococcus aureus , Escherichia coli and Pseudomonas aeruginosa showed that TRI and BZC significantly affected bacterial growth, MICs and minimum bactericidal concentrations (MBCs). There was no change in antibiotic susceptibility profile following exposure to BZC, but E. coli became intermediate resistant to tobramycin following treatment with TRI (0.00002 % w/v). HEPB did not change the antimicrobial susceptibility profile in P. aeruginosa and S. aureus but E. coli became susceptible to gentamicin. TRI exposure resulted in bacterial susceptibility profile alteration consistent with the literature and confirmed the use of TRI as a positive control in such a test.
Conclusion. Data produced on the propensity of a molecule to induce bacterial resistance is useful and appropriate when launching a new preservative.
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Bacterial quantification in tissue homogenates from in vivo pharmacodynamic studies using growth curves
More LessIntroduction. Quantification of bacterial load in tissue homogenates in in vivo pharmacodynamic studies is cumbersome and time-consuming.
Aim. We therefore developed a new method for quantifying bacterial load in tissue homogenates of animals treated with a β-lactam and β-lactamase inhibitor using growth curves.
Methods. The log10 colony-forming units (c.f.u.) ml−1 of 184 thigh and lung homogenates from female CD-1 mice infected intranasally and intramuscularly with 4 Pseudomonas aeruginosa , 4 Klebsiella pneumoniae , 3 Enterobacter cloacae and 2 Escherichia coli strains treated with a β-lactam drug and tazobactam were calculated using the standard approach of serial quantitative cultures and analysis of growth curves. Growth curves were obtained with continuous (every 10 min) monitoring of optical density at 630 nm (OD630) after 20 µl tissue homogenates were inoculated in total volume of 200 µl Mueller–Hinton broth in 96-well microtitration plates and incubated at 37 °C for 18 h.
Results. The best correlation between log10 c.f.u. ml−1 determined with the serial quantitative cultures and growth curves was found at the time point corresponding to an OD630 of 0.25 increase above the baseline OD (average of first five timepoints) (R 2=0.918–0.999). The median (range) differences between the two methods was −0.19 (−1.79–1.69) with 86–97 % of all isolates and species being within 1 log10 c.f.u. ml−1 with 1 h hands-on-time and <13 h of incubation for 96 samples. Pharmacodynamic analysis showed similar dose–response relationships and 1 log kill dose estimations (paired t-test, P=0.112).
Conclusion. The new technique resulted in comparable c.f.u. counts to those for the standard serial dilution/culture technique with minimal hands-on and turnaround times.
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First description of IncX3 NDM-5-producing plasmid within Escherichia coli ST448 in Mali
Carbapenem resistance in Enterobacteriaceae has become an increasingly worrying threat. So far, no epidemiological data regarding NDM-producing enterobacterial isolates has been available on these strains in West Africa. The aim of this study was to seek for carbapenemase-producing Enterobacteriaceae clinical strains isolated in Bamako Teaching Hospital in Mali. Of 50 strains isolated between May 2016 and September 2016, we found a ST448 E. coli harbouring an IncX3 plasmid with bla NDM-5 embedded in the ΔISAba125-ble MBL structure. This study reports the first description of NDM-5 in Mali isolated in an undescribed ST E. coli in West Africa.
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Rhein inhibits the growth of Propionibacterium acnes by blocking NADH dehydrogenase-2 activity
More LessIntroduction. Rhein (4, 5-dihydroxyanthraquinone-2-carboxylic acid) has various properties, including anti-inflammatory, antioxidant and anticancer activities. However, the mechanism underlying the role of rhein in antimicrobial activity remains largely unknown.
Aim. This study aims to identify potential natural compounds of rhein that are capable of inhibiting Cutibacterium acnes and elucidate the effects of rhein on NADH dehydrogenase-2 activity in C. acnes .
Methodology. The anti-C. acnes activity of compounds was analysed using minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC), the paper disc diffusion test and the checkerboard dilution test. To check whether rhein was inhibitory, putative type II NADH dehydrogenase (NDH-2) of C. acnes was analysed, cloned and expressed in Escherichia coli, and then NDH-2 purification was assessed with Ni-NTA before rhein inhibition of NADH dehydrogenase-2 activity was checked with ferricyanide [K3Fe(CN)6] as a substrate.
Results. The results showed that the MIC of rhein against C. acnes was 6.25 µg ml−1, while the MBC was 12.5 µg ml−1, and there was a 38 mm inhibition zone in the paper disc diffusion test. Rhein showed an additive two- to fourfold reduction of the MIC value with four antibiotics on the checkerboard dilution test. The purified NADH dehydrogenase gene product showed a size of approximately 51 kDa and had a V max of 23 µmol and a K m of 280 µm. The inhibitory effect of rhein against NADH dehydrogenase-2 activity was non-competitive with ferricyanide [K3Fe(CN)6] with a K i value of 3.5–4.5 µm.
Conclusion. This study provided evidence of the inhibitory effects of rhein on the growth of C. acnes by blocking of NADH dehydrogenase-2 activity. This mechanism of inhibitory activity in the reduction of ROS formation and ATP productivity should be further tested in C. acnes and the question of whether rhein inhibits the natural growth of C. acnes should be investigated.
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- Disease, Diagnosis and Diagnostics
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Point of care testing of Influenza A/B and RSV in an adult respiratory assessment unit is associated with improvement in isolation practices and reduction in hospital length of stay
More LessIntroduction. Every winter seasonal influenza and other viral respiratory infections increase pressure on the health services and are associated with nosocomial infection and morbidity.
Aim. To compare provision of point-of-care (POC) testing with laboratory-based testing for influenza and RSV detection on an adult respiratory assessment unit to assess the impact on isolation practices and length of stay (LOS).
Methodology. Prospective interrupted ‘on-off’ study in adults admitted to the respiratory unit between December 2018 and April 2019 with a suspected respiratory tract infection. Nasopharyngeal samples were tested using either the GeneXpert rapid POC test for influenza and RSV (on-period), or were sent to the laboratory for multiplex PCR testing against a panel of 12 respiratory viruses (off-period). Outcome measures were time to patient isolation for infection control, LOS and turnaround time from admission to test results.
Results. Of 1145 patients evaluated, 755 were tested with POC and 390 with laboratory multiplex; a respiratory virus was identified in 164 (21.7 %) and 138 (35.4 %) patients respectively. A positive POC test was associated with a shorter time to isolation (mean difference 16.9 h, P<0.001), shorter LOS (mean difference 15.5 h, P=0.05,) and shorter turnaround time (mean difference 28.3 h, P<0.001), compared to laboratory testing.
Conclusion. Use of GeneXpert POC testing for Flu/RSV is associated with rapid reporting of results with significant improvements in isolation practices and reductions in LOS.
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Ability of quantitative PCR to discriminate Pneumocystis jirovecii pneumonia from colonization
Introduction. Pneumocystis jirovecii pneumonia (PCP) is a severe disease affecting immunocompromised patients. Diagnosis is difficult due to the low sensitivity of direct examination and inability to grow the pathogen in culture. Quantitative PCR in bronchoalveolar lavage fluid (BAL) has high sensitivity, but limited specificity for distinguishing PCP from colonization.
Aim. To assess the performance of an in-house quantitative PCR to discriminate between PCP and colonization.
Methodology. This was a single-centre retrospective study including all patients with a positive PCR result for P. jirovecii in BAL between 2009 and 2017. Irrespective of PCR results, PCP was defined as the presence of host factors and clinical/radiological criteria consistent with PCP and (i) the presence of asci at direct examination of respiratory sample or (ii) anti-PCP treatment initiated with clinical response and absence of alternative diagnosis. Colonization was considered for cases who did not receive anti-PCP therapy with a favourable outcome or an alternative diagnosis. Cases who did not meet the above mentioned criteria were classified as ‘undetermined’.
Results. Seventy-one patients with positive P. jirovecii PCR were included (90 % non-HIV patients). Cases were classified as follows: 37 PCP, 22 colonization and 12 undetermined. Quantitative PCR values in BAL were significantly higher in patients with PCP versus colonization or undetermined (P<0.0001). The cut-off of 5×103 copies/ml was able to discriminate PCP cases from colonization with 97 % sensitivity, 82 % specificity, 90 % positive predictive value and 95 % negative predictive value.
Conclusions. Our quantitative PCR for P. jirovecii in BAL was reliable to distinguish PCP cases from colonization in this predominantly non-HIV population.
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- Medical Mycology
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Epidemiology of yeast species causing bloodstream infection in Tehran, Iran (2015–2017); superiority of 21-plex PCR over the Vitek 2 system for yeast identification
Introduction. Given the limited number of candidaemia studies in Iran, the profile of yeast species causing bloodstream infections (BSIs), especially in adults, remains limited. Although biochemical assays are widely used in developing countries, they produce erroneous results, especially for rare yeast species.
Aim. We aimed to assess the profile of yeast species causing BSIs and to compare the accuracy of the Vitek 2 system and 21-plex PCR.
Methodology. Yeast blood isolates were retrospectively collected from patients recruited from two tertiary care training hospitals in Tehran from 2015 to 2017. Relevant clinical data were mined. Identification was performed by automated Vitek 2, 21-plex PCR and sequencing of the internal transcribed spacer region (ITS1-5.8S-ITS2).
Results. In total, 137 yeast isolates were recovered from 107 patients. The overall all-cause 30-day mortality rate was 47.7 %. Fluconazole was the most widely used systemic antifungal. Candida albicans (58/137, 42.3 %), Candida glabrata (30/137, 21.9 %), Candida parapsilosis sensu stricto (23/137, 16.8 %), Candida tropicalis (10/137, 7.3 %) and Pichia kudriavzevii (Candida krusei) (4/137, 2.9 %) constituted almost 90 % of the isolates and 10 % of the species detected were rare yeast species (12/137; 8.7 %). The 21-plex PCR method correctly identified 97.1 % of the isolates, a higher percentage than the Vitek 2 showed (87.6 %).
Conclusion. C. albicans was the main cause of yeast-derived fungaemia in this study. Future prospective studies are warranted to closely monitor the epidemiological landscape of yeast species causing BSIs in Iran. The superiority of 21-plex PCR over automated Vitek 2 indicates its potential clinical utility as an alternative identification tool use in developing countries.
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- Microbiome and Microbial Ecology in Health
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Diversity of navel microbiome in young adults
More LessIntroduction. Human skin microbial communities represent a tremendous source of genetic diversity that evolves as a function of human age. Microbiota differs between regions of oily and moist skin, and appears to stabilize with age.
Aim. We have a minimal understanding of the time frame required for the stabilization of skin microbiota, and the role played by gender. In the current study, we examined the microbiota present in the navel region of college-attending young adults in the age group of 18–25 years and investigated if diversity is associated with gender (male and female).
Method. The study involved 16 female and six male subjects. Isolated DNA samples from navel swabs were processed using the Nextera XT library preparation kit and sequenced using the MiSeq platform. Data were analysed using QIIME and statistical analysis performed in R.
Results. Microbiota of navel skin is dominated by Corynebacterium and Staphylococcus and includes opportunistic pathogens like Clostridium and Pseudomonas . Also present as the major component of the flora were the organisms normally associated with the gastrointestinal tract such as Acinetobacter , Campylobacter , Klebsiella and organisms from the Enterobacteriaceae and Moraxellaceae families. Comparison of alpha and beta diversity of the microbiota in the male and female navel regions suggests that the flora is not statistically different (P>0.05). However, pairwise comparison suggests that the abundance of 12 specific genera varied with gender, including higher abundance of Klebsiella and Enterobacter in females.
Conclusion. Our findings indicate that the navel skin microbiota of young adults has a core microbiota of Corynebacterium and Staphylococcus . We also noted the presence of a significant number of opportunistic pathogens. A minor gender difference in the abundance of individual organisms was also observed.
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- Molecular and Microbial Epidemiology
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Molecular identification of Nocardia species causing endophthalmitis using multilocus sequence analysis (MLSA): a 10-year perspective
Introduction. Nocardia spp. can cause several ocular infections, such as keratitis, endophthalmitis and scleral abscesses. Molecular identification of Nocardia spp. by 16S rDNA sequencing is the gold standard method at present for species identification, but closely related species can only be identified by multilocus sequence analysis (MLSA) of housekeeping genes.
Aim. The major objective was to profile Nocardia species, antibiotic susceptibility patterns and clinical outcomes in endophthalmitis patients.
Methodology. Between January 2009 and December 2018, endophthalmitis patients who were diagnosed with Nocardia infection based on microscopic and culture characteristics were selected. Antibacterial susceptibility tests were performed and Nocardia speciation was performed using MLSA and phylogenetic tree analysis of the 16 s rRNA gene and the gyrB, hsp65 and secA1 genes.
Results. A total of 43 culture-proven patients were identified during the study period. All isolates were 100 % sensitive to amikacin and 98 % resistant to ceftazidime. Fluoroquinolone sensitivity was observed in the range of 58 to 72 %. Year-wise analysis of antibiotic resistance patterns revealed there was a significant increase in resistance to fluoroquinolones. Twenty-two isolates were stored and six different species were identified. Nocardia farcinica (n=10) was found to be the most predominant, followed by Nocardia cyriacigeorgica (n=4), Nocardia otitidiscaviarum (n=3), Nocardia amikacinitolerans (n=2), Nocardia puris (n=2) and Nocardia higoensis (n=1).
Conclusions. N. farcinica is the major pathogen, and this is the first report to identify N. otitidiscaviarum , N. amikacinitolerans and N. higoensis as causing endophthalmitis. Overall, visual outcomes were mostly poor even after aggressive management.
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IncN3 and IncHI2 plasmids with an In1763 integron carrying bla IMP-1 in carbapenem-resistant Enterobacterales clinical isolates from the UK
Introduction. Imipenemase (IMP) carbapenemase genes are relatively rare among Enterobacterales in the UK. Emergence in multiple hospitals, in different strains and species, prompted an investigation into their genetic context.
Aim. Our goal was to identify and describe the elements carrying bla IMP genes in a variety of Enterobacterales from five hospitals in the UK.
Methodology. Long-read nanopore sequencing was carried out on 18 IMP-positive isolates belonging to 6 species. The locations of the bla IMP genes and other associated genetic elements were identified.
Results. Ten out of 18 isolates carried bla IMP-1 on an IncN3 plasmid (52–57 kb) in an In1763 class 1 integron. These plasmids also contained genes encoding type IV secretion and conjugal transfer proteins. Five out of 18 isolates carried bla IMP-1 in the same In1763 integron in much larger IncHI2 plasmids. A further isolate carried the In1763 integron in a chromosomally located plasmid fragment. Two isolates carried bla IMP-4 in IncHI2 plasmids. The isolates included three representatives of sequence type 20 of Klebsiella pneumoniae , with one carrying a distinct plasmid from the other two.
Conclusion. Highly similar IncN3 plasmids were found in a range of Enterobacterales , mostly K. pneumoniae and the Enterobacter cloacae complex, from three of four London hospitals, with the same In1763 integron carrying bla IMP-1 also being found in IncHI2 plasmids and chromosomally. These plasmids carried multiple elements facilitating self-transmission. Strain typing alone was not sufficient to investigate cross-infection among this set of isolates, many of which appeared to be unrelated until plasmid analysis was undertaken, and vice versa.
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- Pathogenesis, Virulence and Host Response
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Cronobacter sakazakii induces necrotizing enterocolitis by regulating NLRP3 inflammasome expression via TLR4
More LessIntroduction. Neonatal infection with Cronobacter sakazakii can cause severe intestinal damage and necrotizing enterocolitis (NEC). The inflammasome and Toll-like receptors mediate intestinal damage caused by other intestinal pathogens causing NEC, but the exact mechanism is unclear.
Aim. We evaluated the molecular mechanisms underlying C. sakazakii -induced NEC.
Methodology. The effects of C. sakazakii treatment on two cell lines and a Sprague–Dawley rat model of NEC were evaluated by a cell death assay, western blot and real-time PCR analyses of the NLRP3 inflammasome and downstream factors, and observation of cell and intestinal damage.
Results. C. sakazakii caused cellular damage in vitro, as well as intestinal damage in an animal model. NLRP3, caspase-1, TLR4 and MyD88, as well as the downstream factor IL-1β, were upregulated in C. sakazakii -infected J774A.1 and HT-29 cells. Western blotting showed that C. sakazakii-infected J774A.1 and HT-29 cells and the NEC rat model had higher expression levels of N-terminal gasdermin D (GSDMD) compared with those in the control groups. C. sakazakii and its components promote NF-κB expression via the TLR4/MyD88 signalling pathway, thereby regulating the NLRP3 inflammasome and mediating GSDMD cleavage, resulting in pyroptosis-induced intestinal damage.
Conclusion. We found that C. sakazakii upregulates NF-κB via TLR4/MyD88 to promote activation of the NLRP3 inflammasome, leading to the up-regulation of downstream caspase-1, release of IL-1β, GSDMD-mediated pyroptosis and development of NEC. These findings clarify the mechanisms by which C. sakazakii contributes to NEC.
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LPS stimulation during HCV infection induces MMP/TIMP1 imbalance in macrophages
Introduction. During chronic hepatitis C virus (HCV) infections, HCV antigens establish cross-tolerance of endotoxins, but additional lipopolysaccharide (LPS) stimulation effects in this condition are poorly understood.
Aim. This study aims to investigate the effects of the upregulated LPS on MMP and TIMP expression during chronic hepatitis C infection.
Methodology. In the present study, we analysed the effect of HCV antigens and LPS stimulation on peripheral blood mononuclear cells (PBMCs) both in vivo and in vitro. Macrophages from HCV patients were isolated and their association with endotoxin tolerance was examined. MMP/TIMP1 expression and the related signalling pathways in macrophages were analysed. The macrophage and Huh7.5 cell co-culture model was used to analyse the effects of the cross-tolerance on collagen I deposition.
Results. LPS levels were found to be significantly higher in HCV patients, particularly in those with HCV-induced liver fibrosis. In addition, although LPS serum level was occasionally upregulated in the patients, it did not induce intense immune response in PBMCs due to endotoxin cross-tolerance, and this was measured according to the changes in IL-6 and TNF-α levels. However, TIMP1 expression increased significantly during stimulation, exhibiting a tolerance/resistance phenotype, which was associated with TGF-β/Erk activation in macrophages. However, MMP levels did not increase due to endotoxin tolerance, which ultimately led to MMP/TIMP imbalance and influenced the deposition of collagen I.
Conclusion. Increased LPS stimulation of macrophage during HCV antigen-induced endotoxin cross-tolerance contributes to MMP/TIMP1 imbalance and collagen I deposition.
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- Prevention, Therapy and Therapeutics
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Sodium salicylate interferes with quorum-sensing-regulated virulence in chronic wound isolates of Pseudomonas aeruginosa in simulated wound fluid
More LessIntroduction. An important factor for delayed healing of chronic wounds is the presence of bacteria. Quorum sensing (QS), a cell density-dependent signalling system, controls the production of many virulence factors and biofilm formation in Pseudomonas aeruginosa .
Aim. Inhibition by sodium salicylate (NaSa) of QS-regulated virulence expression was evaluated in QS-characterized clinical wound isolates of P. aeruginosa, cultured in serum-containing medium.
Methodology. Fourteen clinical P. aeruginosa strains from chronic wounds were evaluated for the production of QS signals and virulence factors. Inhibition of QS by NaSa in P. aeruginosa clinical strains, wild-type PAO1 and QS reporter strains was evaluated using in vitro assays for the production of biofilm, pyocyanin, siderophores, alkaline protease, elastase and stapholytic protease.
Results. Six clinical strains secreted several QS-associated virulence factors and signal molecules and two were negative for all factors. Sub-inhibitory concentrations of NaSa downregulated the expression of the QS-related genes lasB, rhlA and pqsA and reduced the secretion of several virulence factors in PAO1 and clinical strains cultured in serum. Compared to serum-free media, the presence of serum increased the expression of QS genes and production of siderophores and pyocyanin but decreased biofilm formation.
Conclusions. Pseudomonas aeruginosa from chronic wound infections showed different virulence properties. While very few strains showed no QS activity, approximately half were highly virulent and produced QS signals, suggesting that the targeting of QS is a viable and relevant strategy for infection control. NaSa showed activity as a QS-inhibitor by lowering the virulence phenotypes and QS signals at both transcriptional and extracellular levels.
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Volumes and issues
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Volume 73 (2024)
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Volume 72 (2023 - 2024)
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Volume 71 (2022)
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Volume 70 (2021)
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Volume 69 (2020)
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Volume 68 (2019)
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Volume 67 (2018)
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Volume 66 (2017)
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Volume 65 (2016)
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Volume 64 (2015)
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Volume 63 (2014)
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Volume 62 (2013)
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Volume 61 (2012)
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Volume 60 (2011)
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Volume 59 (2010)
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Volume 58 (2009)
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Volume 56 (2007)
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Volume 55 (2006)
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Volume 54 (2005)
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Volume 53 (2004)
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Volume 47 (1998)
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Volume 35 (1991)
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Volume 6 (1973)
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Volume 5 (1972)
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Volume 4 (1971)
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Volume 3 (1970)
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Volume 2 (1969)
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Volume 1 (1968)