Production Of Various Virulence Factors Research Articles

The Antimicrobial Potency of Mesoporous Silica Nanoparticles Loaded with Melissa officinalis Extract.

Melissa officinalis is an important medicinal plant that is used and studied intensively due to its numerous pharmacological effects. This plant has numerous active compounds with biomedical potential; some are volatile, while others are sensitive to heat or oxygen. Therefore, to increase stability and prolong biological activities, the natural extract can be loaded into various nanostructured systems. In this study, different loading systems were obtained from mesoporous silica, like Mobile Composition of Matter family (MCM) with a hexagonal (MCM-41) or cubic (MCM-48) pore structure, simple or functionalized with amino groups (using 3-aminopropyl) such as triethoxysilane (APTES). Thus, the four materials were characterized from morphological and structural points of view by scanning electron microscopy, a BET analysis with adsorption-desorption isotherms, Fourier-transform infrared spectroscopy (FTIR) and a thermogravimetric analysis coupled with differential scanning calorimetry. Natural extract from Melissa officinalis was concentrated and analyzed by High-Performance Liquid Chromatography to identify the polyphenolic compounds. The obtained materials were tested against Gram-negative bacteria and yeasts and against both reference strains and clinical strains belonging to Gram-positive bacteria that were previously isolated from intra-hospital infections. The highest antimicrobial efficiency was found against Gram-positive and fungal strains. Good activity was also recorded against methicillin-resistant S. aureus, the Melissa officinalis extract inhibiting the production of various virulence factors.

Comprehensive virulence profiling and evolutionary analysis of specificity determinants in Staphylococcus aureus two-component systems.

In the Staphylococcus aureus genome, a set of highly conserved two-component systems (TCSs) composed of histidine kinases (HKs) and their cognate response regulators (RRs) sense and respond to environmental stimuli, which drive the adaptation of the bacteria. This study investigates the complex interplay between TCSs in S. aureus USA300, a predominant methicillin-resistant S. aureus strain, revealing shared and unique virulence regulatory pathways and genetic variations mediating signal specificity within TCSs. Using TCS-related mutants from the Nebraska Transposon Mutant Library, we analyzed the effects of inactivated TCS HKs and RRs on the production of various virulence factors, in vitro infection abilities, and adhesion assays. We found that the TCSs' influence on virulence determinants was not associated with their phylogenetic relationship, indicating divergent functional evolution. Using the co-crystallized structure of the DesK-DesR from Bacillus subtilis and the modeled structures of the four NarL TCSs in S. aureus, we identified interacting residues, revealing specificity determinants and conservation within the same TCS, even from different strain backgrounds. The interacting residues were highly conserved within strains but varied between species due to selection pressures and the coevolution of cognate pairs. This study unveils the complex interplay and divergent functional evolution of TCSs, highlighting their potential for future experimental exploration of phosphotransfer between cognate and non-cognate recombinant HK and RRs.IMPORTANCEGiven the widespread conservation of two-component systems (TCSs) in bacteria and their pivotal role in regulating metabolic and virulence pathways, they present a compelling target for anti-microbial agents, especially in the face of rising multi-drug-resistant infections. Harnessing TCSs therapeutically necessitates a profound understanding of their evolutionary trajectory in signal transduction, as this underlies their unique or shared virulence regulatory pathways. Such insights are critical for effectively targeting TCS components, ensuring an optimized impact on bacterial virulence, and mitigating the risk of resistance emergence via the evolution of alternative pathways. Our research offers an in-depth exploration of virulence determinants controlled by TCSs in S. aureus, shedding light on the evolving specificity determinants that orchestrate interactions between their cognate pairs.

Tyramine, one quorum sensing inhibitor, reduces pathogenicity and restores tetracycline susceptibility in Burkholderia cenocepacia

Burkholderia cenocepacia is an opportunistic respiratory pathogen of particular relevance to patients with cystic fibrosis (CF), primarily regulating its biological functions and virulence factors through two quorum sensing (QS) systems (CepI/R and CciI/R). The highly persistent incidence of multidrug resistant Burkholderia cenocepacia poses a global threat to public health. In this study, we investigated the effects of tyramine, one biogenic amine, on the QS systems of Burkholderia cenocepacia. Genetic and biochemical analyses revealed that tyramine inhibited the production of N-hexanoyl-homoserine (AHL) signaling molecules (C8-HSL and C6-HSL) by blocking the CepI/R and CciI/R systems. As a result, the inhibition of QS systems leads to reduced production of various virulence factors, such as biofilm formation, extracellular polysaccharides, lipase, and swarming motility. Notably, as a potential quorum sensing inhibitor, tyramine exhibits low toxicity in vivo in Galleria mellonella larvae and is well characterized by Lipinski’s five rules. It also shows high gastrointestinal absorption and the ability to cross the blood–brain barrier according to SwissADME database and ProTox-II server. Additionally, tyramine was found to enhance the efficacy of tetracycline in reducing the infectivity of Burkholderia cenocepacia in Galleria mellonella larvae infection model. Therefore, tyramine could be a promising candidate for combination therapy with traditional antimicrobials to improve their effectiveness against Burkholderia cenocepacia.

3-(Bromoacetyl) coumarin is a potential therapeutic agent against neonatal sepsis-associated Pseudomonas extremorientalis.

Neonatal sepsis is a severe bacterial infection that can lead to life-threatening complications in newborns. Pseudomonas extremorientalis is a Gram-negative bacterium andthese Gram-negative organismshave been identified as a major cause of neonatal sepsis. The virulence factors produced by this bacterium play a crucial role in its pathogenicity. Therefore, targeting these virulence factors could be a potential strategy to treat neonatal sepsis caused by P. extremorientalis. In this study, we investigated the efficacy of 3-(bromoacetyl) coumarin (3-BC) in reducing the virulence factors of P. extremorientalis strains isolated from neonatal sepsis. Our results showed that 3-BC effectively reduced the production of various virulence factors, including protease, elastase, siderophore, and exopolysaccharide in these strains. Furthermore, at a concentration of 125µg/ml, 3-BC also inhibited the biofilm formation ability of these strains in combination with ciprofloxacin. It was discovered that 3-BC was functionally effective in protecting C. elegans against bacterial infection. Moreover, the in vitro and in vivo outcomes were strongly correlated with docking studies of various activator proteins. Overall, our findings suggest that 3-BC could be a potential therapeutic agent for the treatment of neonatal sepsis caused by P. extremorientalis. Further studies are needed to explore the mechanism of action of 3-BC and its potential use in clinical settings.

Phenotypic Detection of Virulence Factors of Uropathogenic Enterobacteriaceae

Members of the family Enterobacteriaceae are the major cause of nosocomial infections, including approximately 70% of gastrointestinal infections, 60% to 70% of urinary tract infections (UTIs), and septicemias. Since 1990, members of Enterobacteriaceae have emerged as predominant pathogens causing UTIs. In this study, we performed phenotypic testing of the virulence factors in uropathogens. A total of 118 uropathogens belonging to the family Enterobacteriaceae were isolated from suspected UTI cases in patients aged > 18 years. Uropathogens were tested for virulence factors, such as hemolysin, phospholipase, gelatinase, and lipase production, as well as for mannose resistance, mannose-sensitive hemagglutination (MRHA, MSHA), serum resistance, and cell surface hydrophobicity. Among 118 uropathogenic Enterobacteriaceae isolates, E. coli was the most prevalent (62,52.5%), followed by Klebsiella (28,23.7%), Citrobacter (23,19.5%), and, Proteus species (5, 4.2%). Serum resistance was the most common virulence factor and was seen in 54(87%) of E. coli, 24 (85.7%) of Klebsiella species, 22 (95.7%) of Citrobacter species, and 5(100%) of Proteus species isolates. Furthermore, 10 (16.1%) isolates of E. coli showed mannose resistance hemagglutination and 7(11.3%) showed mannose-sensitive hemagglutination. Two (8.7%) isolates of Citrobacter species showed mannose resistance and mannose-sensitive hemagglutination. Hemolysis was observed in 33(53.2%) isolates of E. coli, 11(39.3%) of Klebsiella species, 15(65.2%) Citrobacter species, and 1(20%) Proteus isolate. Gelatinase production was observed in 14(22.6%) of E. coli, 6(21.4%) of Klebsiella species, 7(30.4%) of Citrobacter species, and 5(100%) of Proteus isolates. All E. coli, Klebsiella species, and Citrobacter species isolates tested negative for phospholipase. This study showed that the Enterobacteriaceae family plays a role in UTIs by evading the host immune response through the production of various virulence factors.

Multidrug-Resistant Staphylococcus aureus Isolates in a Tertiary Care Hospital, Kingdom of Bahrain.

Methicillin-resistant Staphylococcus aureus (MRSA) is a ubiquitous pathogen associated with a wide spectrum of human infections. In recent decades, MRSA infections have been increasingly reported in individuals without established risk factors, infecting immunocompetent members of the community. This emergence is attributed to the production of various virulence factors, notably Panton-Valentine leukocidin(PVL). The aim of this study was to better understand the prevalence, antibiotic resistance profiles, and molecular characteristics of S. aureus and MRSA in a tertiary care hospital in the Kingdom of Bahrain. This cross-sectional study was carried out in a tertiary hospital for a one-year period, from December 2020 to December 2021. A total of 161 consecutive S. aureus isolates were collected. Antibiotic susceptibility was tested using BD Phoenix™ automated identification and susceptibility testing system. Molecular analysis was conducted via conventional PCR and conventional multiplex PCR for SCCmec typing. In this study, 161 S. aureus isolates were investigated, 60% (n=97) were characterized as MRSA, of which, 12% (n=12) were healthcare-associated methicillin-resistant Staphylococcus aureus (HA-MRSA) while 88% (n=85) were community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA). No statistically significant difference (P>0.05) in antibiotic resistance trends between HA-MRSA and CA-MRSA was detected. Multidrug resistance (MDR) amounted to 19% (n=30) of all S. aureus isolates, 14% (n=9) of methicillin-susceptible Staphylococcus aureus (MSSA) isolates, and 22% (n=21) of MRSA isolates. SCCmec typing demonstrated a high prevalence of type IV (61%, n=59), followed by type V (32%, n=31), then type II (4%, n=4), and type III (3%, n=3). The PVL prevalence was 39% (n=25) in MSSA and 62% (n=60) in MRSA, 33% (n=4) in HA-MRSA, and 66% (n=56) in CA-MRSA. This study demonstrated the emergence of PVL-producing CA-MRSA in a tertiary care hospital, as well as the detection of PVL-producing MDR strains. This development prompts serious measures to be taken in order to sustain a healthy clinical environment.

Paecilomycone Inhibits Quorum Sensing in Gram-Negative Bacteria.

Pseudomonas aeruginosa is an opportunistic pathogen that causes major health care concerns due to its virulence and high intrinsic resistance to antimicrobial agents. Therefore, new treatments are greatly needed. An interesting approach is to target quorum sensing (QS). QS regulates the production of a wide variety of virulence factors and biofilm formation in P. aeruginosa. This study describes the identification of paecilomycone as an inhibitor of QS in both Chromobacterium violaceum and P. aeruginosa. Paecilomycone strongly inhibited the production of virulence factors in P. aeruginosa, including various phenazines, and biofilm formation. In search of the working mechanism, we found that paecilomycone inhibited the production of 4-hydroxy-2-heptylquinoline (HHQ) and 3,4-dihydroxy-2-heptylquinoline (PQS), but not 2'-aminoacetophenone (2-AA). Therefore, we suggest that paecilomycone affects parts of QS in P. aeruginosa by targeting the PqsBC complex and alternative targets or alters processes that influence the enzymatic activity of the PqsBC complex. The toxicity of paecilomycone toward eukaryotic cells and organisms was low, making it an interesting lead for further clinical research. IMPORTANCE Antibiotics are becoming less effective against bacterial infections due to the evolution of resistance among bacteria. Pseudomonas aeruginosa is a Gram-negative pathogen that causes major health care concerns and is difficult to treat due to its high intrinsic resistance to antimicrobial agents. Therefore, new targets are needed, and an interesting approach is to target quorum sensing (QS). QS is the communication system in bacteria that regulates multiple pathways, including the production of virulence factors and biofilm formation, which leads to high toxicity in the host and low sensitivity to antibiotics, respectively. We found a compound, named paecilomycone, that inhibited biofilm formation and the production of various virulence factors in P. aeruginosa. The toxicity of paecilomycone toward eukaryotic cells and organisms was low, making it an interesting lead for further clinical research.

Guaiacol augments quorum quenching potential of ciprofloxacin against Pseudomonas aeruginosa.

The present study aims to investigate the antimicrobial as well as antivirulence potential and the principle mechanism of action of guaiacol against Pseudomonas aeruginosa. Quorum sensing inhibition and membrane disruption studies were performed to check the effect of guaiacol on the virulence of P. aeruginosa. Production of various virulence factors and biofilm formation was studied at a sub-MIC concentration of guaiacol alone (1/8 MIC) and in combination with ciprofloxacin (1/2 FIC). Guaiacol exhibited synergistic interactions with ciprofloxacin and further reduced the production of all virulence factors and biofilm formation. Using crystal violet (CV) assay and quantification of exopolysaccharide, we observed weak biofilm formation, together with reduced motilities at sub-MIC, which was further visualized by confocal laser microscopy and Field Emission Scanning Electron Microscopy. The antibacterial activity of guaiacol against P. aeruginosa upon 2 × MIC exposure coincided with enhanced membrane permeability leading to disruption and release of cellular material as quantified by CV uptake assay and sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). The results demonstrated that sub-MICs of guaiacol in combination with ciprofloxacin can act as a potent alternate compound for attenuation of quorum sensing in P. aeruginosa. The study reports that guaiacol in combination with ciprofloxacin at 1/2 FIC significantly compromised the bacterial growth and motilities alongside inducing quorum quenching potential. This was accompanied by inhibition of biofilm which subsequently decreased EPS production at sub-MIC concentration. Furthermore, guaiacol in combination displayed a severe detrimental effect on bacterial membrane disruption, thereby enhancing cellular material release. For the first time, the potential of guaiacol in combination with ciprofloxacin in attenuation of virulence factors, and biofilm formation in Pseudomonas aeruginosa was described. Results corroborate how plant bioactive in synergism with antibiotics can act as an alternate treatment regime to tackle the menace of drug resistance.

Biofilm neutrophils interactions under hypoxia: A mathematical modeling study

Neutrophils are important to the defense of the host against bacterial infection. Pathogens and the immune system cells create via respiration, a hypoxic environment in infected regions. Hypoxic conditions affect both the neutrophil’s ability to eradicate the infection and also change the behavior of the bacterial-pathogens by eliciting the production of various virulence factors, the creation of bacterial biofilm and the initialization of anaerobic metabolism. In this work interactions of bacterial biofilm and neutrophils are studied in a domain where oxygen is diffusing into the environment and is being consumed by biofilm. Within a hypoxic environment, bacteria grow anaerobically and secrete higher levels of toxin that diffuses and lyses neutrophils. A mathematical model explicitly representing the biofilm volume fraction, oxygen, and diffusive virulence factors (toxin) as well as killing of bacteria by neutrophils is developed and studied first in 1D and then in 2D. Stability analysis and numerical simulations showing the effects of oxygen and toxin concentration on neutrophil–bacteria interactions are presented to identify different possible scenarios that can lead to elimination of the infection or its persistence as a chronic infection. Specifically, when bacteria are allowed to utilize anaerobic breathing and or to produce toxin, their fitness is enhanced against neutrophils attacks. A possible insight on how virulent bacterial colonies can synergistically resist neutrophils and survive is presented.

Sphingolipids: Regulators of azole drug resistance and fungal pathogenicity.

In recent years, the role of sphingolipids in pathogenic fungi, in terms of pathogenicity and resistance to azole drugs, has been a rapidly growing field. This review describes evidence about the roles of sphingolipids in azole resistance and fungal virulence. Sphingolipids can serve as signaling molecules that contribute to azole resistance through modulation of the expression of drug efflux pumps. They also contribute to azole resistance by participating in various microbial pathways such as the unfolded protein response (UPR), pH-responsive Rim pathway, and pleiotropic drug resistance (PDR) pathway. In addition, sphingolipid signaling and eisosomes also coordinately regulate sphingolipid biosynthesis in response to azole-induced membrane stress. Sphingolipids are important for fungal virulence, playing roles during growth in hosts under stressful conditions, maintenance of cell wall integrity, biofilm formation, and production of various virulence factors. Finally, we discuss the possibility of exploiting fungal sphingolipids for the development of new therapeutic strategies to treat infections caused by pathogenic fungi.

Terminalia chebula and Ficus racemosa principles mediated repression of novel drug target Las R – the transcriptional regulator and its controlled virulence factors produced by multiple drug resistant Pseudomonas aeruginosa - Biocompatible formulation against drug resistant bacteria

Pseudomonas aeruginosa– major group of an aerobic bacteria associated with nosocomial and other life threatening infections. Diverse virulence factors produced by P. aeruginosa is due to distinct molecular cell signaling mechanism termed as quorum sensing (QS). Interfering with normal QS mechanism by active biomolecules is an effective strategy for attenuating its virulence. With this objective, the present study is undertaken to evaluate the inhibition of quorum sensing of clinical isolate of P. aeruginosa by repression of Las R-a transcriptional regulator for QS by ethanol extract of Terminalia chebula and Ficus racemosa. Las R repression by the plant extracts was measured in inhibition of various virulence factors like biofilm, pyocyanin production, total proteolytic activity, swarming and twisting motility. Fabrication of the extracted metabolites on the wound dressing and its effect on anti bacterial activity was also investigated. Compatibility of plant extracts on zebra fish development and blood cells was further studied. P. aeruginosa was isolated from the post operative patient and the isolated pure culture was identified by cultural, biochemical, molecular characteristics. Active principles of both the plants were readily extracted in ethanol and effectively repressed the expression of Las R. Both the tested plant extracts effectively repressed Las R expression which in turn affect the production of various virulence factors like biofilm formation, pyocyanin production, swarming motility, twisting motility, total proteolytic activity, cell adhesion and signaling molecule acyl honoserine lactone (AHL) production. Plant extract treatment brought about drastic reduction of all the tested virulence factors and AHL production. Extracted metabolites were fabricated on the wound dressing material adopting simple dip or immersion method reveals uniform coating, effective embedding of phytochemicals with the fibers and retained the anti bacterial activity against P. aeruginosa. Biocompatibility studies with zebra fish model shows both the tested plant extracts treatment was not exhibited any sign of toxicity on the developmental stages of Zebra fish. Hemolysis and changes in anti oxidative enzymes were not recorded in the plant extracts treated blood which demonstrated the best biocompatibility of the tested plant extracts. These results shows that the presence of potential phytochemicals in the ethanolic extract of Terminalia chebula and Ficus racemosa effectively represses the Las R followed by inhibition of quorum sensing mediated virulence factors production may be useful in the lead of anti bacterial drugs.

Terpinen-4-ol attenuates quorum sensing regulated virulence factors and biofilm formation in Pseudomonas aeruginosa.

Aim: To investigate the effects of Terpinen-4-ol on quorum sensing (QS)-regulated biofilm formation and virulence factors production in Pseudomonas aeruginosa. Materials & methods: QS inhibition, molecular docking analysis and gene expression studies were performed to check attenuation effect of Terpinen-4-ol on virulence of P. aeruginosa. Production of various virulence factors and biofilm formation were studied at sub-MIC of Terpinen-4-ol alone and in combination with ciprofloxacin. Results: Terpinen-4-ol at sub-MIC exhibited QS inhibition and downregulated all key QS genes. Molecular docking analysis showed high binding affinities of Terpinen-4-ol with QS receptors. Terpinen-4-ol exhibited synergistic interaction with ciprofloxacin and further reduced production of all the virulence factors and biofilms formation. Conclusion: Terpinen-4-ol could be developed into antivirulence drug after its in vivo evaluation for treatment strategies.

An Alanine-Rich Peptide Attenuates Quorum Sensing-Regulated Virulence and Biofilm Formation in Staphylococcus aureus.

Background: Alanine-rich proteins/peptides (ARP), with bioactivity of up to 20 amino acid residues, can be observed by the body easily during gastrointestinal digestion. Objective: Populus trichocarpa extract's capability to attenuate quorum sensing-regulated virulence and biofilm formation in Staphylococcus aureus is described. Methods: PT13, an ARP obtained from P. trichocarpa, was tested for its activity against S. aureus using the broth microdilution test; a crystal-violet biofilm assay was performed under a scanning electron microscope. The production of various virulence factors was estimated with PT13 treatment. Microarray gene expression profiling of PT13-treated S. aureus was conducted and compared with an untreated control. Exopolysaccharides (EPS) was estimated to observe the PT13 inhibition activity. Results: PT13 was antimicrobial toward S. aureus at different concentrations and showed a similar growth rate in the presence and absence of PT13 at concentrations ≤8 μg/mL. Biofilm production was interrupted even at low concentrations, and biofilm-related genes were down-regulated when exposed to PT13. The genes encoding cell adhesion and bacterial attachment protein were the major genes suppressed by PT13. In addition, hemolysins, clumping activity, and EPS production of S. aureus decreased after treatment in a concentration-dependent manner. Conclusions: A long-chain PT13 with effective actions that, even at low concentration levels, not only regulated the gene expression in the producer organism but also blocked the virulence gene expression in this Gram-positive human pathogen is described. Highlights: We identified a PT13 as a potential antivirulence agent that regulated production of bacterial virulence determinants (e.g., toxins, enzymes and biofilm), downwards and it may be a promising anti-virulence agent to be further developed as an anti-infective agent.

Biofilm inhibition, modulation of virulence and motility properties by FeOOH nanoparticle in Pseudomonas aeruginosa.

Biofilm formation is one of the resistance mechanisms of Pseudomonas aeruginosa against antimicrobial compounds. Biofilm formation also characterizes for the infection and pathogenesis of P. aeruginosa, along with production of various virulence factors. With recent development of nanotechnology, the present study aims to employ the synthetic iron nanoparticle (FeOOH-NP) as an active agent to inhibit the formation of P. aeruginosa biofilm. The FeOOH-NP was synthesized and characterized with rod shape and average size of 40nm. Inhibition of biofilm formation by the FeOOH-NP is in a concentration-dependent manner, with inhibition of biofilm formation increased as the FeOOH-NP concentration increased. Microscopic observations also confirmed the disruption ofthe biofilm architecturein the presence of the FeOOH-NP. In addition, the presence of the FeOOH-NP was also found to modulate bacterial motility as well as some other important virulence factors produced simultaneously with biofilm formation. These findings provide insights to anti-biofilm effect of a new iron NP, contributing to the search for an effective agent to combat P. aeruginosa infections resulted from biofilm formation.

Changing Virulence Factors among Vaginal Non-albicans Candida species

Background: Vulvovaginal candidiasis (VVC) is caused by overgrowth of Candida species in the female lower genital tract and most commonly caused by Candida albicans. The production of various virulence factors may attribute to their pathogenicity. Hence, this study was aimed to determine the production of various virulence factors of Candida spp. causing VVC. Materials and Methods: A total of 51 Candida spp. were isolated prospectively from 50 patients among 211 clinically suspected cases of VVC. The haemolytic activity, biofilm production, proteinase activity, phospholipase activity and esterase activity were detected by standard methods. Statistical analysis was performed using OpenEpi version 3.01. Results: Haemolytic activity was observed in 42 Candida isolates (82.4%), biofilm activity in 21 Candida isolates (41.2%), proteinase and esterase activity in 19 Candida isolates (37.3%) each and phospholipase activity in 15 Candida isolates (29.4%). Phospholipase activity was observed in all of the C. albicans strains, whereas all strains of Candida krusei were able to produce biofilm. All strains of Candida parapsilosis and 87% strains of Candida glabrata were haemolytic. Five of the eight C. glabrata strains were found to produce strong proteinase (Prz score ≤0.63). About 30.4% strains of C. glabrata and 20% strains of C. krusei were found to be positive for esterase activity. This is one of the few studies which revealed esterase activity among C. glabrata and C. krusei strains. Conclusions: This study highlighted that there is a change in the virulence factors among the non-albicans Candida species, especially C. glabrata strains which were haemolytic and produce strong proteinase activity and esterase activity. It may be one of the explanation of the most common causative agent of VVC in our study. Multicentric studies from this area might be required to get a more generalised conclusion.

Apoptosis induced by Pseudomonas aeruginosa: a lonely killer?

Apoptosis is a fundamental biological process allowing tissue homeostasis through the regulation of cell populations by eliminating unnecessary elements. During infection, pathogens have evolved to take advantage of this process for their own and are able to induce the apoptosis of cells, i.e. immune cells by the host itself. Pseudomonas aeruginosa is one of the most studied opportunistic bacteria due to its significant involvement worldwide in pneumonia, corneal infections and wound burns. Several research groups have pointed out the ability of these bacteria to interfere and/or evade host immune system by inducing apoptosis of the targeted cells. In May 2014, looking up ‘Pseudomonas aeruginosa’ and ‘apoptosis’ keywords in PubMed search engine retrieve more than 300 hits. Pseudomonas aeruginosa seems to induce apoptosis through direct interaction with the host cells (the most studied system being the type-III secretion system: T3SS) or through secreting factors such as pyocyanin. T3SS, the most well-studied virulence apparatus of P. aeruginosa is composed of a needle complex through which exoenzymes are injected into the host cells (Galle et al., 2012,2012). Recently, Beyaert's laboratory described an exotoxin-independent function of the T3SS in the killing of macrophages in an acute lung infection model (Galle et al., 2012,2012). Although, T3SS is a major virulence system, it is not fully required for the bacteria to display virulence as T3SS negative strains are shown to exihibit signficant virulence (example of Elsen's paper). In short, a wide variety of P. aeruginosa virulence factors are involved in inducing apoptosis by several distinct mechanisms, from the activation of the mitochondrial pathway, the generation of reactive oxygen species to the activation of the caspase pathways (Table 1). The ability of P. aeruginosa to induce apoptosis in various in vitro model of infection (macrophages, neutrophils, epithelial cells …) or in vivo models such as lungs, cornea and burn wounds infections is not mediated by a single bacterial cell but rather by a multicellular population of P. aeruginosa. Members of such population interact with each other through a number of chemical signals known globally on for quorum sensing (QS). Quoting Rutherford and Bassler (2012), ‘Quorum sensing is a bacterial cell-cell communication process that involves the production, detection, and response to extracellular signaling molecules called autoinducers’. QS molecules were shown to regulate virulence factors such as toxins, exotoxin A, pyocyanin, … and so in fine apoptosis (Rutherford and Bassler, 2012). The best described QS signalling systems in P. aeruginosa are the N-acyl homoserine lactones systems Las and Rhl. The Las system produces and responds to N-oxododecanoyl homoserine lactone and the Rhl system to N-butanoyl homoserine lactone respectively. Las system is known to control the production of various virulence factors involved in host cell damages such as exotoxin A (Jones et al., 1993). On the other hand, Rhl system was described to repress the expression of genes responsible for the assembly and function of the T3SS (Bleves et al., 2005). Last but not least, QS molecule such as 3-oxododecanoyl-L-homoserine lactone (3-oxo-C12-HSL) itself has been shown to induce apoptosis. Several studies have demonstrated that incubation of different cell lines with 3-oxo-C12-HSL molecule resulted in the induction of apoptosis involving calcium signalling, the mitochondrial pathway and caspase activations (Table 2). Interestingly, N-butanoyl-L-homoserine lactone (known as C4-HSL, the second major QS molecule in P. aeruginosa) harbouring a shorter fatty acid chain has not been shown to induce apoptosis compared with 3-oxo-C12-HSL (Tateda et al., 2003; Holban et al., 2014). Knowing that P. aeruginosa is able to induce apoptosis through its QS systems molecule, studies focusing on apoptosis induction should be considered with the context of QS signalling. Particularly, QS considerations should be taken into account when comparing studies using different multiplicity of infection or bacteria preparation protocols, processes that influence QS molecules concentration and/or bacteria population numbers. None declared.

Prevalence of exfoliative toxin A and B genes in Staphylococcus aureus isolated from clinical specimens

The pathogenicity of Staphylococcus aureus is based on the production of various virulence factors. The frequency of these factors can markedly differ according to the geographical region. In this study, we investigated the prevalence of two frequent isoforms of exfoliative toxins and mecA genes using PCR in 197 clinical isolates obtained from clinical samples during the years 2011 and 2012. The samples were obtained from an educational hospital in northern Tehran, Iran. In addition, the antibiotic susceptibility pattern was studied for each isolate using the disc diffusion method. In this study, 186 (94.4%), 15 (7.6%) and 172 (86.3%) of the 197 isolates expressed the eta, etb and mecA genes, respectively. In addition, 164 (88.2%) and 12 (80%) strains, which harbored the eta and etb genes, respectively, were MRSA (methicillin resistant S. aureus). Furthermore, the prevalence of the mecA, eta and etb genes was higher among the wound samples (61.2%, 55.8% and 6.09%, respectively). We observed high rates of MDR (multi drug resistance) among our isolates. A significant correlation was detected between the presence of the mecA gene and the resistance to oxacillin, gentamicin, kanamycin, erythromycin, tetracycline, cotrimoxazole, clindamycin, and cephazolin as well as the multi-drug resistant property (P<0.05). In addition to penicillin, the MDR properties and resistances to the tested antibiotics in the etb-positive strains were significantly lower compared to the etb-negative strains (P<0.05). The prevalence of the eta, etb and mecA genes might be due to the specific geographic region.

Characteristics of Staphylococcus aureus isolated from acute, sub-acute and sub-clinical staphylococcosis in rabbits

Staphylococcus aureus bacteria isolated from different clinical presentations of staphylococcosis in rabbits were examined for the production of various virulence factors using biochemical and immunological tests. In the total of 106 S. aureus isolates; toxic shock syndrome toxin-1, staphylococcal enterotoxin-C, DNase, α-haemolysin, β-haemolysin, δ-haemolysin, protein A and clumping factor were observed with a frequency of 33.2, 16.98, 83.96, 69.81, 36.79, 100, 78.30 and 54.72 percent, respectively. No SE-A, SE-B and SE-D producing isolates were recovered in this study. All the S. aureus isolates from acute staphylococcosis produced TSST-1, SE-C and protein A. While δ–haemolysin and clumping factor were not detected in any acute isolates, these factors were observed at a relatively higher frequency in isolates from sub-acute and sub-clinical staphylococcosis. Coagulase type III was observed more predominantly with a frequency of 45.28%, while coagulase types V and VII were not observed in any isolate. Most of the virulence factors belonged to coagulase type III followed by type VI. TSST-1 and SE-C along with coagulase types III and VI could be correlated with the acute and sub-acute staphylococcal infections in rabbits in this study.

In vivo effects of single or combined N-acyl homoserine lactone quorum sensing signals on the performance of Macrobrachium rosenbergii larvae

Three experiments, each of 8-days, were conducted to evaluate the effects of (5) N-acyl homoserine lactone (AHL) either alone or in combination on the development and survival of Macrobrachium rosenbergii larvae. Experiment 1 was carried out as preliminary trial to confirm whether AHL mixture added daily at 1 mg/L concentration could have any negative effect on the larval performance. In experiment 2, it was verified whether the negative effect on the larvae was due to individual AHL or their interactions and experiment 3 was carried out to determine the critical threshold dose of AHL mixture. Results revealed that AHL mixture added daily at 1 mg/L concentration significantly (P<0.01) reduced the development and survival of the larvae. Except for C4-HST, there was no significant effect (P>0.05) of any of the AHL molecules added individually at 1 mg/L concentration on the development and survival of the prawn larvae. The AHL molecule C4-HST significantly (P<0.01) reduced the larval stage index (LSI) and survival of prawn larvae by about 9.1 and 8.7%, respectively. In contrast, the AHL mixture added daily at 1 mg/L concentration significantly (P<0.01) reduced the LSI and survival of the larvae by 12 and 41%, respectively. Thus the poor development and low survival of the larvae is probably due to the interaction of different AHL molecules which could have instigated the production of various virulence factors in micro-organisms associated with the larvae. Using the AHL mixture, the dose response relationship with respect to the performance of the larvae showed that the development of the larvae was already negatively affected by adding the mixture to a final concentration of 0.125 mg/L while at that concentration, the survival was still unaffected. This indicate that the standing microbial communities associated with the prawn larvae can become virulent when a quorum or critical threshold level is reached through the daily addition of the AHL mixture at a concentration of 0.125 mg/L.

PecS Is a Global Regulator of the Symptomatic Phase in the Phytopathogenic Bacterium Erwinia chrysanthemi 3937

Pathogenicity of the enterobacterium Erwinia chrysanthemi (Dickeya dadantii), the causative agent of soft-rot disease in many plants, is a complex process involving several factors whose production is subject to temporal regulation during infection. PecS is a transcriptional regulator that controls production of various virulence factors. Here, we used microarray analysis to define the PecS regulon and demonstrated that PecS notably regulates a wide range of genes that could be linked to pathogenicity and to a group of genes concerned with evading host defenses. Among the targets are the genes encoding plant cell wall-degrading enzymes and secretion systems and the genes involved in flagellar biosynthesis, biosurfactant production, and the oxidative stress response, as well as genes encoding toxin-like factors such as NipE and hemolysin-coregulated proteins. In vitro experiments demonstrated that PecS interacts with the regulatory regions of five new targets: an oxidative stress response gene (ahpC), a biosurfactant synthesis gene (rhlA), and genes encoding exported proteins related to other plant-associated bacterial proteins (nipE, virK, and avrL). The pecS mutant provokes symptoms more rapidly and with more efficiency than the wild-type strain, indicating that PecS plays a critical role in the switch from the asymptomatic phase to the symptomatic phase. Based on this, we propose that the temporal regulation of the different groups of genes required for the asymptomatic phase and the symptomatic phase is, in part, the result of a gradual modulation of PecS activity triggered during infection in response to changes in environmental conditions emerging from the interaction between both partners.