Quorum sensing inhibitory effect of bergamot oil and aspidosperma extract against Chromobacterium violaceum and Pseudomonas aeruginosa.

Quorum sensing (QS) represents a major target for reducing bacterial pathogenicity and antibiotic resistance. This study identifies bergamot and aspidosperma as new potential sources of anti-QS agents. We investigated the anti-QS activity of plant materials on both Chromobacterium violaceum and Pseudomonas aeruginosa. Initially, we determined the minimum inhibitory concentrations (MICs) of plant materials using a broth microdilution method. Subsequently, we tested the effect of sub-MIC concentrations on QS-regulated traits and virulence factors production in test bacteria. Results revealed that bergamot and aspidosperma inhibited the ability of C. violaceum to produce violacein. Other QS-controlled phenotypes of C. violaceum, namely chitinolytic activity, motility, and biofilm formation, were also reduced by both plant materials. Moreover, QS-linked traits of P. aeruginosa were also reduced. Bergamot inhibited swarming but not swimming motility, while aspidosperma diminished both motility types in P. aeruginosa. Both plant materials also demonstrated antibiofilm activity and inhibited the production of protease and pyocyanin in P. aeruginosa. Furthermore, we tested the anti-QS effect of plant materials on the transcriptional level using RT-qPCR. Bergamot dramatically downregulated the C. violaceum autoinducer synthase gene cviI and the vioB gene involved in violacein biosynthesis, confirming the phenotypic observation on its anti-QS activity. Aspidosperma also reduced the expression of cviI and vioB but less drastically than bergamot. In P. aeruginosa, downregulation in the transcripts of the QS genes lasI, lasR, rhlI, and rhlR was also achieved by bergamot and aspidosperma. Therefore, data in the present study suggest the usefulness of bergamot and aspidosperma as sources of antivirulence agents.

Bactericidal activity of a substituted thiazole against multidrug-resistant Eggerthia catenaformis isolated from patients with dental abscess.

Human infections caused by the anaerobic bacterium Eggerthia catenaformis are rare. However, a growing number of case reports have presented the bacterium as the causative agent in many serious complications. This study provides data on the isolation and antibiotic susceptibility profiles of E. catenaformis from dental abscess. Identification of isolates was performed using matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). We also investigated the antibacterial activity of 5-acetyl-4-methyl-2-(3-pyridyl) thiazole (AMPT) on E. catenaformis isolates. Minimum inhibitory concentrations (MICs) were determined by an agar dilution method and bactericidal activity was evaluated by a time-kill assay. Moreover, the mechanism of action of AMPT was also explored by cell membrane disruption assay and scanning electron microscopy (SEM). MALDI-TOF MS results revealed unambiguous identification of all isolates with score values between 2.120 and 2.501. Isolates NY4 and NY9 (20% of isolates) were found resistant to multiple antibiotics judged by MIC values. As multidrug-resistant strains of E. catenaformis were not reported to date, we then confirmed the identity of NY4 and NY9 based on 16S rRNA gene sequence. Favorably, all isolates were susceptible to AMPT with an MIC range of 0.25-1 mg/L. Time-kill kinetics of AMPT indicated that it exhibited potent bactericidal activity against the multidrug-resistant isolates NY4 and NY9. Furthermore, this study also hypothesizes that AMPT exerts its antibacterial effect through damaging the cell membrane and thereby induce the release of intracellular components. AMPT could therefore be considered as a therapeutic option for infections caused by multidrug-resistant bacteria.

Effect of kitasamycin and nitrofurantoin at subinhibitory concentrations on quorum sensing regulated traits of Chromobacterium violaceum.

Quorum sensing (QS) is a mechanism of intercellular communication in bacteria that received substantial attention as alternate strategy for combating bacterial resistance and the development of new anti-infective agents. The present investigation reports on the assessment of using subinhibitory concentrations of antibiotics for the inhibition of QS-regulated phenotypes in Chromobacterium violaceum. Primarily, the minimum inhibitory concentrations of a series of antibiotics were determined by a microdilution method. Subsequently, the inhibitory effects of selected antibiotics on QS-regulated traits, namely violacein and chitinase production, biofilm formation and motility were evaluated using C. violaceum CV026 and C. violaceum ATCC 12472. Results revealed that kitasamycin and nitrofurantoin exhibited the highest quorum sensing inhibitory (QSI) activity. The amount of violacein produced by C. violaceum was significantly reduced in the presence of either kitasamycin or nitrofurantoin. Moreover, the chitinolytic activity, biofilm formation, and motility were also impaired in kitasamycin or nitrofurantoin-treated cultures. We further confirmed QSI effects at the molecular level using molecular docking and real-time quantitative polymerase chain reaction (RT-qPCR). Results of molecular docking suggested that both antibiotics can interact with CviR transcriptional regulator of C. violaceum. Furthermore, RT-qPCR revealed the suppressive effect of kitasamycin and nitrofurantoin on five genes under the control of the CviI/CviR system: cviI, cviR, vioB, vioC, and vioD. Giving that kitasamycin and nitrofurantoin are being safely used for decades, this study emphasizes their potential application as antivirulence agents to disarm resistant bacterial strains, making their removal an easier task for the immune system or for another antibacterial agent.

Antibacterial and anti-quorum sensing activities of a substituted thiazole derivative against methicillin-resistant Staphylococcus aureus and other multidrug-resistant bacteria.

Management of infections caused by methicillin-resistant Staphylococcus aureus (MRSA) is still challenging. We herein report the antibacterial and anti-quorum sensing (anti-QS) activities of 5-acetyl-4-methyl-2-(3-pyridyl) thiazole (AMPT) against MRSA and other multidrug-resistant bacteria. Minimum inhibitory concentrations (MICs) were determined by agar dilution method and bactericidal activity was assessed by a time-kill assay. The anti-QS activity was evaluated using Chromobacterium violaceum. The effect of AMPT on virulence factors production by MRSA and biofilm formation by MRSA, C. violaceum and Pseudomonas aeruginosa was also assessed. AMPT was superior to vancomycin and teichoplanin against MRSA isolates. MIC50/90 values of AMPT (2/4 mg/L) were 2-4 folds lower than the values for vancomycin (4/16 mg/L) and 2-fold lower than the values for teichoplanin (4/8 mg/L). Results of time-kill assay against two multidrug-resistant MRSA isolates revealed bactericidal effect of AMPT after 4 h of treatment, with no bacterial cells detected after 24 h. Remarkably, AMPT exhibited anti-QS activity against both C. violaceum and MRSA at subinhibitory concentrations. Moreover, AMPT reduced haemolysin and protease production by MRSA and inhibited biofilm formation by MRSA, C. violaceum and P. aeruginosa but had no dispersion effect on preformed ones. Furthermore, molecular docking analysis revealed promising interactions between AMPT and AgrA as well as SarA in S. aureus confirming the antivirulence and antibiofilm activities. Favourably, no significant cytotoxicity of AMPT was observed on murine macrophage cell line. Taken altogether, these results suggest that AMPT could be considered an interesting lead compound in the search for treatment of MRSA infections.

Evaluation of fosfomycin combined with vancomycin against vancomycin-resistant coagulase negative staphylococci.

The pathogenic potential of coagulase-negative staphylococci (CoNS) is increasingly recognized worldwide. The aim of the present study was to evaluate different combinations of fosfomycin (FOS) with vancomycin (VAN) or oxacillin (OXA) against vancomycin-resistant clinical isolates of CoNS. Characterization of VAN resistance in selected isolates was also sought. Antibiotic susceptibility of isolates was tested by disc diffusion method, MICs of antibiotics were determined by the agar dilution method, and fosfomycin combinations were evaluated by a time-kill assay according to the guidelines of the CLSI. Moreover, oxacillin and glycopeptides (vancomycin and teichoplanin) resistances were also characterized phenotypically and genotypically in this study. Out of 258 staphylococci, 52 were CoNS (20.2%). All isolates were multidrug resistant with 75% (n = 39) oxacillin-resistant, most of them with oxacillin MIC levels of >32 mg/L. Moreover, vancomycin non-susceptibility was observed in 46.2% (n = 24) of the tested isolates with MIC range of 4-32 mg/L. Identification of selected isolates revealed that S. epidermidis was the most abundant among tested CoNS, followed by S. hominis, S. saprophyticus, and interestingly S. caseolyticus. Furthermore, Synergistic and bactericidal effects of FOS + VAN combination were observed in 3 of 9 isolates after 6 h of treatment and in all studied isolates at 24 h. On the other hand, FOS + OXA combinations were ineffective. This study provides evidence that fosfomycin combination with vancomycin could be considered as a therapeutic alternative for CoNS infections. It also sheds some light on the possible emergence of the otherwise harmless bacterium S. caseolyticus as a human pathogen.

In Vitro Activity of Fosfomycin in Double and Triple Combinations with Imipenem, Ciprofloxacin and Tobramycin Against Multidrug-Resistant Escherichia coli.

The rates of urinary tract infection with multidrug-resistant (MDR) Escherichia coli have dramatically increased and the treatment of these infections with single and double antibiotic combinations became limited or ineffective. The present study aimed at finding effective antibiotic combinations against MDR uropathogenic E. coli. Antibiotic susceptibility testing of uropathogenic E. coli isolates (n = 29) showed that all the examined isolates were found to be MDR. The interaction of double and triple combinations of fosfomycin (FOS) with imipenem (IPM), ciprofloxacin (CIP) and tobramycin (TOB) against selected isolates (n = 8) by checkerboard method showed that all the examined combinations exhibited synergistic effects (FIC index < 1) against tested isolate. However, 1/8, 5/8 and 6/8 of the isolates remained resistant to the constituent antibiotics in FOS/IPM, FOS/CIP and FOS/TOB combinations, respectively. Notably, the triple combinations (FOS/IPM/CIP, FOS/IPM/TOB and FOS/CIP/TOB) increased the synergism against all selected isolates at MIC levels lower than the susceptible breakpoints. Furthermore, time-kill analysis demonstrated that FOS/IPM combination exhibited synergistic and bactericidal effects with UTI-9. However, the combination had no effect on UTI-13. The highest synergistic and bactericidal effects against both representative isolates were achieved by FOS/IPM/CIP, FOS/IPM/TOB and FOS/CIP/TOB combinations after 2 h of post-treatment and lasted up to 24 h. Therefore, we report here that the combinations of FOS with IPM, CIP and TOB could be beneficial against MDR uropathogenic E. coli at least in vitro. The effectiveness of these antibiotics increased in combination with FOS compared to individual antibiotics acting alone.

Contribution of the ATP-dependent protease ClpCP to the autolysis and virulence of Streptococcus pneumoniae.

The ATP-dependent caseinolytic proteases (Clp) are fundamental for stress tolerance and virulence in many pathogenic bacteria. The role of ClpC in the autolysis and virulence of Streptococcus pneumoniae is controversial. In this study, we tested the role of ClpC in a number of S. pneumoniae strains and found that the contribution of ClpC to autolysis is strain dependent. ClpC is required for the release of autolysin A and pneumolysin in serotype 2 S. pneumoniae strain D39. In vivo, ClpC is required for the growth of the pneumococcus in the lungs and blood in a murine model of disease, but it does not affect the overall outcome of pneumococcal disease. We also report the requirement of ClpP for the growth at elevated temperature and virulence of serotype 4 strain TIGR4 and confirm its contribution to the thermotolerance, oxidative stress resistance, and virulence of D39.

Control of virulence by the two-component system CiaR/H is mediated via HtrA, a major virulence factor of Streptococcus pneumoniae.

The CiaR/H two-component system is involved in regulating virulence and competence in Streptococcus pneumoniae. The system is known to regulate many genes, including that for high-temperature requirement A (HtrA). This gene has been implicated in the ability of the pneumococcus to colonize the nasopharynx of infant rats. We reported previously that deletion of the gene for HtrA made the pneumococcal strains much less virulent in mouse models, less able to grow at higher temperatures, and more sensitive to oxidative stress. In this report, we show that the growth phenotype as well as sensitivity to oxidative stress of Delta ciaR mutant was very similar to that of a Delta htrA mutant and that the expression of the HtrA protein was reduced in a ciaR-null mutant. Both the in vitro phenotype and the reduced virulence of Delta ciaR mutant could be restored by increasing the expression of HtrA.

Role of HtrA in the virulence and competence of Streptococcus pneumoniae.

HtrA is a major virulence factor of Streptococcus pneumoniae (the pneumococcus). Deletion of the gene for HtrA from strain D39 of the pneumococcus completely abolished its virulence in mouse models of pneumonia and bacteremia, while the virulence of a second strain (TIGR4) was dramatically reduced. HtrA-negative mutants induced much less inflammation in the lungs during pneumonia than the wild type. HtrA is involved in the ability of the pneumococcus to grow at high temperatures, to resist oxidative stress, and to undergo genetic transformation. The expression and cellular location of several known virulence factors of the pneumococcus were not affected by the lack of HtrA.