Expression of toll‐like receptors in T lymphocytes stimulated with N‐(3‐oxododecanoyl)‐L‐homoserine lactone from Pseudomonas aeruginosa

The establishment of chronic Pseudomonas aeruginosa infections is correlated with the disturbance of the host immune system. The P. aeruginosa quorum‐sensing molecule N‐3‐(oxododecanoyl)‐L‐homoserine lactone (3‐O‐C12‐HSL) has the potential to modulate the host immune system. The immune system recognizes pathogens via toll‐like receptors (TLRs). We found that 3‐O‐C12‐HSL induced TLR changes in monocytes. However, the role of T cells in P. aeruginosa infection has not been delineated. In order to understand this activity, we examined whether 3‐O‐C12‐HSL has an effect on the immune function and the expression of TLRs in T lymphocytes. Human peripheral blood mononuclear cells (PBMCs) cells were cultured with 0, 1, 10, 50, or 100 μM 3‐O‐C12‐HSL for 12 h. TLR2/TLR4 expression and T‐lymphocyte proliferation were increased in a dose‐dependent manner, and 100 μM 3‐O‐C12‐HSL significantly increased TLR2 expression. Moreover, tumor necrosis factor‐α production of these PBMCs was inhibited. To conclude, 3‐O‐C12‐HSL can induce lymphocyte cell proliferation. These findings provide a new perspective on our understanding of the persistence of the chronic inflammation that accompanies P. aeruginosa infection.     

Acyl Homoserine Lactones Induce Early Response in the Airway

Acyl homoserine lactones (AHLs) are intercellular signaling molecules used in quorum sensing by Gram‐negative bacteria. We studied the early effects on the rat airway of in vivo intratracheal administration of AHLs (i.e., P. aeruginosa and B. cepacia) to test the hypothesis that AHLs also act on the airway cells, modifying secretory mechanisms which are important in mucosal defense. One hour after treatment, N‐butyryl‐homoserine lactone (C4‐HL) had caused dilated extracellular spaces, loss of cilia, reduction of secretory material, and the presence of prenecrotic elements in the epithelium, while N‐octanoyl‐homoserine lactone (C8‐HL) caused a mild lesion in the epithelium. After treatment with either C4‐ or C8‐HL, reduced immunoreactivity was found using CC10 antibody. At ultrastructural examination, dilatation of the mitochondria was evident in ciliate and secretory cells, while solitary chemosensory cells appeared better preserved, showing aspects of nucleocytoplasmic activation. Using microarray analysis, we found down‐regulation of early gene Fos and Egr1 in all AHL‐treated specimens. In vivo pharmacological magnetic resonance imaging after C4‐ or C8‐HL treatment showed a slight increase in tracheal secretion at a first evaluation 5 min after administration, with no increase in the following minutes. In conclusion, AHLs induce an early mucosal response, and the chondriomas of ciliate and secretory cells are the main cytological target of AHL action. Our results show that AHL action is not limited to activation of conspecific bacteria, but also modifies innate airway defense mechanisms. Anat Rec, 292:439–448, 2009. © 2009 Wiley‐Liss, Inc.     

Dissemination of metallo‐β‐lactamase in Pseudomonas aeruginosa isolates in Egypt: mutation in blaVIM‐4

This study was designed to investigate the prevalence of metallo‐β‐lactamase (MBL) in Pseudomonas aeruginosa isolates collected from Suez Canal University Hospital in Ismailia, Egypt. Antibiotic susceptibility testing and phenotypic and genotypic screening for MBLs were performed on 147 isolates of P. aeruginosa. MICs were determined by agar dilution method for carbapenem that was ≥2 μg/mL for meropenem. MBL genes were detected by multiplex and monoplex PCR for P. aeruginosa‐harbored plasmids. Mutation profile of sequenced MBL genes was screened using online software Clustal Omega. Out of 147 P. aeruginosa, 39 (26.5%) were carbapenem‐resistant isolates and 25 (64%) were confirmed to be positive for MBLs. The susceptibility rate of P. aeruginosa toward polymyxin B and norfloxacin was 99% and 88%, respectively. Identification of collected isolates by API analysis and constructed phylogenetic tree of 16S rRNA showed that the isolates were related to P. aeruginosa species. The frequency of blaGIM‐1, blaSIM‐1, and blaSPM‐1 was 52%, 48%, and 24%, respectively. BlaVIM and blaIMP‐like genes were 20% and 4% and the sequences confirm the isolate to be blaVIM‐1, blaVIM‐2, blaVIM‐4, and blaIMP‐1. Three mutations were identified in blaVIM‐4 gene. Our study emphasizes the high occurrence of multidrug‐resistant P. aeruginosa‐producing MBL enzymes.     

2,5‐Piperazinedione inhibits quorum sensing‐dependent factor production in Pseudomonas aeruginosa PAO1

The effects of 2,5‐piperazinedione in reducing the production of quorum sensing (QS)‐dependent factors in Pseudomonas aeruginosa PAO1 were assessed both in vitro and in vivo. 2,5‐Piperazinedione exhibited a 69% reduction in the azocasein‐degrading proteolytic activity and a 48% reduction in the elastolytic activity of PAO1. Further, it showed 85% and 96% reduction in the production of pyocyanin and extracellular polymeric substances (EPS) of PAO1, respectively. In the swimming inhibition assay, 2,5‐piperazinedione‐treated PAO1 cells exhibited poor swimming motility in swim agar medium. In the in vivo analysis, an enhanced survival of PAO1‐preinfected Caenorhabditis elegans was observed after treatment with 2,5‐piperazinedione. Regarding the mode of action, in the molecular docking analysis, 2,5‐piperazinedione interacts with the amino acid residue of the LasR receptor protein required for binding the natural ligand N ‐3‐oxododecanoyl‐l‐homoserine lactone (3‐oxo‐C12‐HSL). This demonstrates the probability of 2,5‐piperazinedione to interfere with the binding process of 3‐oxo‐C12‐HSL to its receptor protein. Thus, the findings of the present study reveal the potential of 2,5‐piperazinedione in reducing the QS‐dependent phenotypic features of PAO1. (© 2012 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Pleiotropic effects of acyltransferases on various virulence‐related phenotypes of Pseudomonas aeruginosa

Pseudomonas aeruginosa, an opportunistic pathogen causing various infections, expresses various virulence factors under the control of quorum sensing (QS), a cell density–sensing mechanism. Because the major signal molecules of QS are acyl homoserine lactones (acyl‐HSLs), acyltransferases, the enzymes that act upon acyl group transfer could affect the QS signaling and QS‐related virulence phenotypes. In this study, we overexpressed acyltransferases of P. aeruginosa and screened them for the activity influencing the QS and QS‐related virulence phenotypes. Among seven acyltransferases tested in this study, two acyltransferases, PA3984 (apolipoprotein N‐acyltransferase) and PA2537 (putative acyltransferase), significantly affected both growth of P. aeruginosa and the activity of LasR, a major QS regulator, when overexpressed. These acyltransferases also reduced virulence and swarming motility of P. aeruginosa. The other acyltransferase, PA3646 (UDP‐3‐O‐[3‐hydroxylauroyl] glucosamine N‐acyltransferase), reduced the LasR activity, swarming motility, protease production and virulence without any influence on growth. These effects by PA3646 over‐expression were caused by less production of QS signal. PA3644 (UDP‐N‐acetylglucosamine acyltransferase) enhanced biofilm formation and swarming motility with no effect on the growth and QS activity. These results suggest that acyltransferases may be an important factor regulating the cellular activity about virulence‐related phenotypes.     

Evaluation of phytochemicals from medicinal plants of Myrtaceae family on virulence factor production by Pseudomonas aeruginosa

Virulence factors regulated by quorum sensing (QS) play a critical role in the pathogenesis of an opportunistic human pathogen, Pseudomonas aeruginosa in causing infections to the host. Hence, in the present work, the anti‐virulence potential of the medicinal plant extracts and their derived phytochemicals from Myrtaceae family was evaluated against P. aeruginosa. In the preliminary screening of the tested medicinal plant extracts, Syzygium jambos and Syzygium antisepticum demonstrated a maximum inhibition in QS‐dependent violacein pigment production by Chromobacterium violaceum DMST 21761. These extracts demonstrated an inhibitory activity over a virulence factor, pyoverdin, production by P. aeruginosa ATCC 27853. Gas chromatography–mass spectrometric (GC‐MS) analysis revealed the presence of 23 and 12 phytochemicals from the extracts of S. jambos and S. antisepticum respectively. Three top‐ranking phytochemicals, including phytol, ethyl linoleate and methyl linolenate, selected on the basis of docking score in molecular docking studies lowered virulence factors such as pyoverdin production, protease and haemolytic activities of P. aeruginosa to a significant level. In addition, the phytochemicals reduced rhamnolipid production by the organism. The work demonstrated an importance of plant‐derived compounds as anti‐virulence drugs to conquer P. aeruginosa virulence towards the host.     

Effect of plant phenolic compounds on biofilm formation by Pseudomonas aeruginosa

In the natural environment, bacteria predominantly exist in matrix‐enclosed multicellular communities associated with various surfaces, referred to as biofilms. Bacteria in biofilms are extremely resistant to antibacterial agents thus causing serious problems for antimicrobial therapy. In this study, we showed that different plant phenolic compounds, at concentrations that did not or weakly suppressed bacterial growth, increased the capacity of Pseudomonas aeruginosa PAO1 to form biofilms. Biofilm formation of P. aeruginosa PAO1 was enhanced 3‐ to 7‐fold under the action of vanillin and epicatechin, and 2‐ to 2.5‐fold in the presence of 4‐hydroxybenzoic, gallic, cinnamic, sinapic, ferulic, and chlorogenic acids. At higher concentrations, these compounds displayed an inhibiting effect. Similar experiments carried out for comparison with Agrobacterium tumefaciens C58 showed the same pattern. Vanillin, 4‐hydroxybenzoic, and gallic acids at concentrations within the range of 40 to 400 μg/mL increased the production of N–3‐oxo‐dodecanoyl‐homoserine lactone in P. aeruginosa PAO1 which suggests a possible relationship between stimulation of biofilm formation and Las Quorum Sensing system of this bacterium. Using biosensors to detect N‐acyl‐homoserine lactones (AHL), we demonstrated that the plant phenolics studied did not mimic AHLs.     

Occurrence of diversified N‐acyl homoserine lactone mediated biofilm‐forming bacteria in rice rhizoplane

Quorum sensing (QS)‐mediated biofilm‐forming rhizobacteria are indispensable due to their competitiveness in the crop rhizosphere. In the present work, we have reported on the occurrence of diversified bacterial species capable of producing N‐acyl homoserine lactone (AHL) as the QS signal in the roots of a rice plant grown under field conditions. The AHL‐producing bacteria were directly isolated from the rice root by the biosensor reporter (Chromobacterium violaceum CV026) overlay method and characterized for biofilm production by the microtiter plate method. A total of 48 QS‐positive bacterial isolates were purified from different aged (7, 20, 24, 26, and 36 days) rice seedlings. The in vitro biofilm production and genetic diversity as revealed by BOX‐PCR fingerprinting showed high variability among the isolates. Most of the best biofilm‐forming isolates produced a N‐butyryl dl ‐homoserine lactone (a C4‐AHL type) signal in the medium. The 16S ribosomal RNA (rRNA) gene sequence of these putative elite isolates identified that they were close to Aeromonas hydrophila (QS7‐4; QS36‐2), A. enteropelongenes (QS20‐8), A. veronii (QS36‐3), Enterobacter sp. (QS20‐11), Klebsiella pneumoniae (QS24‐6), Kosakonia cowanii (QS24‐21), Providentia rettigeri (QS24‐2), Sphingomonas aquatilis (QS24‐17), and Pseudomonas sihuiensis (QS24‐20). These strains profusely colonized the rice root upon inoculation and formed biofilms on the surface of the root under gnotobiotic conditions. Developing inoculants from these strains would ensure competitive colonization on the rhizoplane of the crop through their biofilm and thereby improve plant growth and health.     

Genotyping of Pseudomonas aeruginosa isolates from lung transplant recipients and aquatic environment–detected in‐hospital transmission

Lung infection with Pseudomonas aeruginosa is common in lung transplant recipients and may lead to severe complications. Bacteriological surveillance aims to detect transmission of microbes between hospital environment and patients. We sought to determine whether genotyping of P. aeruginosa isolates could improve identifications of pathways of infection. From 2004 to 2009, we performed genotyping with multiple‐locus variable number of tandem repeats analysis (MLVA) and pulsed‐field gel electrophoresis (PFGE) of P. aeruginosa isolates cultured from lung transplant recipients at Sahlgrenska University Hospital, Gothenburg. During a small outbreak in 2008, cultivation and genotyping of isolates from sink and drains samples from the hospital ward were performed. Pseudomona aeruginosa from 11/18 patients were genotyped to unique strains. The remaining seven patients were carriers of a P. aeruginosa strain of cluster A genotype. Pseudomona aeruginosa was isolated in 4/8 water samples, typed by MLVA also as cluster A genotype and confirmed by PFGE to be similar or identical to the isolates from four transplanted patients. In conclusion, genotyping of isolates revealed a clonal relationship between patient and water isolates, indicating in‐hospital transmission of P. aeruginosa. We suggest genotyping with MLVA for rapid routine surveillance, with the PFGE method used for extended, confirmatory analyses.     

A multiresistant clone of Pseudomonas aeruginosa sequence type 773 spreading in a burn unit in Orumieh,Iran

Herein, we describe the phenotypic and genotypic characterization of a multiresistant clone of Pseudomonas aeruginosa disseminating in a burn unit in Orumieh, Iran. A total of 58 isolates of P. aeruginosa were collected during August 2007 and June 2008. Minimum inhibitory concentrations (MICs) of P. aeruginosa isolates were determined against 11 antimicrobial agents by E test. Serotyping, pulsed‐field gel electrophoresis (PFGE), and multilocus sequence typing (MLST) were used for studying the clonal relationship among the isolates. Antibiotic susceptibility testing revealed that most of the isolates were multidrug resistant and colistin was the antibiotic with the highest activity. Pseudomonas aeruginosa isolates fell into nine different serotypes, and O10 and O11 were the most common. PFGE analyses showed 12 different genotypes and 68.1% of isolates showed more than 80% similarity, indicating possible clonal relatedness. These isolates were found to belong to the same sequence type, ST773. This sequence type has earlier been reported from China, and a double locus variant of this ST has been found earlier in France in a PER‐1 extended‐spectrum β‐lactamase‐producing P. aeruginosa.     

Pathogenic potential and genetic diversity of environmental and clinical isolates of Pseudomonas aeruginosa

The aim of this study was to investigate the occurrence of virulence genes among clinical and environmental isolates of Pseudomonas aeruginosa and to establish their genetic relationships by Enterobacterial Repetitive Intergenic Consensus PCR (ERIC‐PCR). A total of 60 P. aeruginosa isolates from environmental and clinical sources were studied. Of these, 20 bacterial isolates were from soil, 20 from water, and 20 from patients with cystic fibrosis. Analysis of ERIC‐PCR demonstrated that the isolates of P. aeruginosa showed a considerable genetic variability, regardless of their habitat. Numerous virulence genes were detected in both clinical and environmental isolates, reinforcing the possible pathogenic potential of soil and water isolates. The results showed that the environmental P. aeruginosa has all the apparatus needed to cause disease in humans and animals.     

Protease inhibitors elicit anti‐inflammatory effects in CF mice with Pseudomonas aeruginosa acute lung infection

Pseudomonas aeruginosa is the major respiratory pathogen in patients with cystic fibrosis (CF). P. aeruginosa‐secreted proteases, in addition to host proteases, degrade lung tissue and interfere with immune processes. In this study, we aimed at evaluating the possible anti‐inflammatory effects of protease inhibitors Marimastat and Ilomastat in the treatment of P. aeruginosa infection. Lung infection with the P. aeruginosa PAO1 strain was established in wild‐type and cystic fibrosis transmembrane conductance regulator (CFTR) knock‐out C57BL/6 mice expressing a luciferase gene under control of bovine interleukin (IL)‐8 promoter. After intratracheal instillation with 150 µM Marimastat and Ilomastat, lung inflammation was monitored by in‐vivo bioluminescence imaging and bacterial load in the lungs was assessed. In vitro, the effects of protease inhibitors on PAO1 growth and viability were evaluated. Acute lung infection was established in both wild‐type and CFTR knock‐out mice. After 24 h, the infection induced IL‐8‐dependent bioluminescence emission, indicating lung inflammation. In infected mice with ongoing inflammation, intratracheal treatment with 150 µM Marimastat and Ilomastat reduced the bioluminescence signal in comparison to untreated, infected animals. Bacterial load in the lungs was not affected by the treatment, and in vitro the same dose of Marimastat and Ilomastat did not affect PAO1 growth and viability, confirming that these molecules have no additional anti‐bacterial activity. Our results show that inhibition of protease activity elicits anti‐inflammatory effects in cystic fibrosis (CF) mice with acute P. aeruginosa lung infection. Thus, Marimastat and Ilomastat represent candidate molecules for the treatment of CF patients, encouraging further studies on protease inhibitors and their application in inflammatory diseases.     

Molecular typing of Clostridioides difficile isolates from clinical and non‐clinical samples in Iran

Clostridioides difficile is a major cause of nosocomial infectious diarrhea in hospitalized patients throughout the world. We aimed to characterize C. difficile isolates among hospitalized patients, hospital staffs, and hospital environment samples obtained in three tertiary care hospitals of Iran with regard to their molecular types between June 2016 and November 2017. The toxigenicity of C. difficile isolates was determined by toxigenic culture and multiplex‐PCR. Toxigenic C. difficile isolates collected were ribotyped using capillary gel electrophoresis‐based PCR and the database of WEBRIBO ( http://webribo.ages.at ). Of 500 clinical and non‐clinical samples, toxigenic C. difficile were identified in 35 of 250 stool samples (14%) and in 3 of 250 swabs (1.2%). The most frequently found ribotypes (RTs) were 039, AI‐12, and AI‐21 (15.8, 10.52, and 10.52% of all isolates, respectively). Further RTs were: 017, 001, AI‐3, AI‐15, AI‐18, AI‐10, AI‐4, and PR21195 (as new ribotype). The epidemic RTs (027 and 078) seen in the Europe, North America, and Asia were completely absent in this study.     

Response of leaf-associated bacterial communities to primary acyl-homoserine lactone in the tobacco phyllosphere

The phyllosphere is inhabited by large populations of epiphytic bacteria that are able to modulate their phenotypes and behavior by quorum sensing (QS). However, the impact of acyl-homoserine lactones (AHLs) involved in QS on the ecology of bacteria in their natural habitat remains unclear. Therefore, we used a bioassay and liquid chromatography-mass spectrometry to detect AHLs in the tobacco phyllosphere. Our results identified several AHLs in the tobacco phyllosphere, the majority of which were short-chain AHLs. Furthermore, the addition of an exogenous N-(3-oxohexanoyl) homoserine lactone (3OC6HSL), which is seen in the naturally occurring tobacco phyllosphere, generated variability in the composition of the bacterial community as determined by denaturing gradient gel electrophoresis (DGGE) analysis and phospholipid fatty acid (PLFA) analysis. Notably, the ratio of Gram-positive (GP) bacteria increased in response to treatment with 1 μM AHL, but decreased incipiently when treated with 10 μM AHL. These observations provide insight into the composition of the leaf-colonizing epiphyte community responsible for AHLs, particularly GP bacteria as they do not use AHLs as signaling molecules for QS.     

Biofilm formation of ica operon‐positive Staphylococcus epidermidis from different sources

Information on the prevalence of biofilm‐related factors (PIA, Bhp, Aap, Embp) in Staphylococcus epidermidis of animal origin is scarce. In this study, 263 S. epidermidis isolates of diverse origin (animal, farmers, patients, and laboratory staff) were investigated for the presence of the ica operon (icaRADBC). The icaRADBC‐positive isolates were further characterized by means of biofilm formation, presence of other biofilm‐related genes, antimicrobial resistance, and population structure. Of all isolates, 28.5% (n = 75) were icaRADBC‐positive, including 16.5% of animal origin, 29.1% farmer isolates, and 44.6% hospital‐associated isolates (including patients and laboratory staff isolates). Most icaRADBC‐positive isolates carried embp (n = 73), aap (n = 57), bhp (n = 22), and IS256 (n = 29). Statistical differences were found between animal and patient isolates for the presence of icaRADBC, bhp, and aap. No statistically significant relation was found between the presence of one or more genes and the level of biofilm formation. Most icaRADBC‐positive isolates belonged to the clonal complex 5 (formerly 2) and most sequence types corresponded to types previously observed in community and nosocomial S. epidermidis populations. Although the prevalence of S. epidermidis in the nasal cavity of bovines and poultry is low, some isolates belong to STs related to ica‐positive clinical strains.     

Analysis of quorum sensing-dependent virulence factor production and its relationship with antimicrobial susceptibility in Pseudomonas aeruginosa respiratory isolates

Pseudomonas aeruginosa is an opportunistic pathogen causing severe respiratory infections. The pathogenesis of these infections is multifactorial and the production of many virulence factors is regulated by quorum sensing (QS), a cell-to-cell communication mechanism. The two well defined QS systems in P. aeruginosa, the las and rhl systems, rely on N-acyl homoserine lactone signal molecules, also termed autoinducers. We assessed the activity of QS-dependent virulence factors (including elastase, alkaline protease, pyocyanin and biofilm production) in respiratory isolates of P. aeruginosa and their relationship with antimicrobial susceptibility. We identified sixteen isolates displaying impaired phenotypic activity; among them, eleven isolates were also defective in autoinducer production, and therefore considered QS-deficient. Six of the QS-deficient isolates failed to amplify one or more of the four QS regulatory genes (lasI, lasR, rhlI, rhlR) with PCR: one isolate was negative for rhlR, two isolates were negative for rhlI and rhlR and three isolates were negative for all four genes. The isolates that were negative for virulence factor production were generally less susceptible to the antimicrobials and statistically significant correlations were observed between the lack of elastase production and resistance to piperacillin and ceftazidime; between failure in alkaline protease production and resistance to tobramycin, piperacillin, piperacillin-tazobactam, cefepime, imipenem and ciprofloxacin; and between failure in pyocyanin production and resistance to amikacin, tobramycin, ceftazidime, ciprofloxacin and ofloxacin. The results obtained indicate that, despite the pivotal role of QS in the pathogenesis of P. aeruginosa respiratory infections, QS-deficient strains are still capable of causing infections and tend to be less susceptible to antimicrobials.     

Bacillus pumilus of Palk Bay origin inhibits quorum-sensing-mediated virulence factors in Gram-negative bacteria

The aim of the current study was to inhibit quoring-sensing(QS)-mediated virulence factors of representative Gram-negative bacteria by marine bacterial isolates. Bacteria isolated from Palk Bay sediments were screened for anti-QS activity. Eleven strains inhibited QS signals in Chromobacterium violaceum (ATCC 12472) and C. violaceum CV026. The marine bacterial strain S8-07 reduced the accumulation of N-acyl homoserine lactone (AHLs) and showed significant inhibition of LasA protease(76%), LasB elastase(84%), caseinase(70%), pyocyanin (84%), pyoverdin and biofilm formation(87%) in Pseudomonas aeruginosa PAO1. Strain S8-07 also showed highly significant reduction (90%) in prodigiosin, secreted casienase (92%), hemolytic activity (73%) and biofilm formation (61%) in Serratia marcescens. Strain S8-07, identified as Bacillus pumilus (accession number FJ584416), showed distinct profiles of inhibition against the virulence factors of both P. aeruginosa PAO1 (las, rhl) and S. marcescens (shl). Polar extraction and proteinase K treatment of the culture supernatant confirmed that the anti-QS activity of S8-07 was indeed due to a protein molecule. Acidification assay and HPLC analysis revealed that the degradation of AHL was not due to lactonase activity, but rather, was due to acylase activity of S8-07. Thus, novel anti-QS acylase activity is reported for the first time from a B. pumilus strain of marine origin.     

N-acylhomoserine lactones undergo lactonolysis in a pH-, temperature-, and acyl chain length-dependent manner during growth of Yersinia pseudotuberculosis and Pseudomonas aeruginosa

In gram-negative bacterial pathogens, such as Pseudomonas aeruginosa and Yersinia pseudotuberculosis, cell-to-cell communication via the N-acylhomoserine lactone (AHL) signal molecules is involved in the cell population density-dependent control of genes associated with virulence. This phenomenon, termed quorum sensing, relies upon the accumulation of AHLs to a threshold concentration at which target structural genes are activated. By using biosensors capable of detecting a range of AHLs we observed that, in cultures of Y. pseudotuberculosis and P. aeruginosa, AHLs accumulate during the exponential phase but largely disappear during the stationary phase. When added to late-stationary-phase, cell-free culture supernatants of the respective pathogen, the major P. aeruginosa [N-butanoylhomoserine lactone (C4-HSL) and N-(3-oxododecanoyl)homoserine lactone (3-oxo-C12-HSL)] and Y. pseudotuberculosis [N-(3-oxohexanoyl)homoserine lactone (3-oxo-C6-HSL) and N-hexanoylhomoserine lactone (C6-HSL)] AHLs were inactivated. Short-acyl-chain compounds (e.g., C4-HSL) were turned over more extensively than long-chain molecules (e.g., 3-oxo-C12-HSL). Little AHL inactivation occurred with cell extracts, and no evidence for inactivation by specific enzymes was apparent. This AHL turnover was discovered to be due to pH-dependent lactonolysis. By acidifying the growth media to pH 2.0, lactonolysis could be reversed. By using carbon-13 nuclear magnetic resonance spectroscopy, we found that the ring opening of homoserine lactone (HSL), N-propionyl HSL (C3-HSL), and C4-HSL increased as pH increased but diminished as the N-acyl chain was lengthened. At low pH levels, the lactone rings closed but not via a simple reversal of the ring opening reaction mechanism. Ring opening of C4-HSL, C6-HSL, 3-oxo-C6-HSL, and N-octanoylhomoserine lactone (C8-HSL), as determined by the reduction of pH in aqueous solutions with time, was also less rapid for AHLs with more electron-donating longer side chains. Raising the temperature from 22 to 37 degrees C increased the rate of ring opening. Taken together, these data show that (i) to be functional under physiological conditions in mammalian tissue fluids, AHLs require an N-acyl side chain of at least four carbons in length and (ii) that the longer the acyl side chain the more stable the AHL signal molecule.     

Inactivation of the rhlA gene in Pseudomonas aeruginosa prevents rhamnolipid production,disabling the protection against polymorphonuclear leukocytes

Many of the virulence factors produced by the opportunistic human pathogen Pseudomonas aeruginosa are quorum‐sensing (QS) regulated. Among these are rhamnolipids, which have been shown to cause lysis of several cellular components of the human immune system, e.g. monocyte‐derived macrophages and polymorphonuclear leukocytes (PMNs). We have previously shown that rhamnolipids produced by P. aeruginosa cause necrotic death of PMNs in vitro. This raises the possibility that rhamnolipids may function as a ‘biofilm shield’in vivo, which contributes significantly to the increased tolerance of P. aeruginosa biofilms to PMNs. In the present study, we demonstrate the importance of the production of rhamnolipids in the establishment and persistence of P. aeruginosa infections, using an in vitro biofilm system, an intraperitoneal foreign‐body model and a pulmonary model of P. aeruginosa infections in mice. Our experimental data showed that a P. aeruginosa strain, unable to produce any detectable rhamnolipids due to an inactivating mutation in the single QS‐controlled rhlA gene, did not induce necrosis of PMNs in vitro and exhibited increased clearance compared with its wild‐type counterpart in vivo. Conclusively, the results support our model that rhamnolipids are key protective agents of P. aeruginosa against PMNs.     

Molecular epidemiological characteristics of Salmonella enterica serovars Enteritidis,Typhimurium and Livingstone strains isolated in a Tunisian university hospital

Enteritidis, Typhimurium and Livingstone are the main Salmonella enterica serovars recovered in Tunisia. Here, we aimed to assess the genetic diversity of fifty‐seven Salmonella enterica strains from different sampling periods, origins and settings using pulsed‐field gel electrophoresis (PFGE), multi‐locus sequence typing (MLST) and multi‐locus variable‐number tandem repeat analysis (MLVA). Salmonella Enteritidis, isolated from human and food sources from two regions in Sfax in 2007, were grouped into one cluster using PFGE. However, using MLVA these strains were divided into two clusters. Salmonella Typhimurium strains, recovered in 2012 and represent sporadic cases of human clinical isolates, were included in one PFGE cluster. Nevertheless, the MLVA technique, divided Salmonella Typhimurium isolates into six clusters with diversity index reaching (DI = 0.757). For Salmonella Livingstone which was responsible of two nosocomial outbreaks during 2000–2003, the PFGE and MLVA methods showed that these strains were genetically closely related. Salmonella Enteritidis and Salmonella Livingstone populations showed a single ST lineage ST11 and ST543 respectively. For Salmonella Typhimurium, two MLST sequence types ST19 and ST328 were defined. Salmonella Enteritidis and Salmonella Typhimurium strains were clearly differentiated by MLVA which was not the case using PFGE.