Azithromycin regulates bacterial virulence and immune response in a murine model of ceftazidime-treated Pseudomonas aeruginosa acute pneumonia.

Pseudomonas aeruginosa (PA) remains one of the leading causes of nosocomial acute pneumonia. The array of virulence factors expressed by PA and the intense immune response associated with PA pneumonia play a major role in the severity of these infections. New therapeutic approaches are needed to overcome the high resistance of PA to antibiotics and to reduce the direct damage to host tissues. Through its immunomodulatory and anti-virulence effects, azithromycin (AZM) has demonstrated clinical benefits in patients with chronic PA respiratory infections. However, there is relatively little evidence in PA acute pneumonia. We investigated the effects of AZM, as an adjunctive therapy combined with ceftazidime (CAZ), in a murine model of PA acute pneumonia. We observed that the combined therapy (i) reduces the weight loss of mice 24 h post-infection (hpi), (ii) decreases neutrophil influx into the lungs at 6 and 24 hpi, while this effect is absent in a LPS-induced pneumonia or when PA is pretreated with antibiotics and mice do not receive any antibiotics, and that (iii) AZM, alone or with CAZ, modulates the expression of PA quorum sensing regulators and virulence factors (LasI, LasA, PqsE, PhzM, ExoS). Our findings support beneficial effects of AZM with CAZ on PA acute pneumonia by both bacterial virulence and immune response modulations. Further investigations are needed to clarify the exact underlying mechanisms responsible for the reduction of the neutrophils influx and to better discriminate between direct immunomodulatory properties of AZM, and indirect effects on neutrophilia resulting from bacterial virulence modulation.

[How to improve vaccination coverage in France? Qualitative study among health care workers]. / Comment améliorer la couverture vaccinale en France ? Étude qualitative auprès de professionnels de santé.

BACKGROUND: The risk benefit ratio has been clearly proven for a long time to be in favor of vaccination. However, the vaccine distrust is still increasing in the French population and vaccination coverage could be improved. There is a real need to increase confidence in vaccination. In this study, we interviewed heath care workers to collect their opinion concerning vaccination hesitancy amongst their patients. METHODS: A qualitative study, using semi structured interviews, was performed. All the Pharmacists and doctors were interviewed by the same person using an interview guide prepared by a multidisciplinary team. Interviews were recorded and transcribed verbatim, then used as a basis for analysis and synthesis of the areas where improvement seems possible for the health care workers interviewed. RESULTS: Data saturation was obtained after 10 interviews. Analysis of the verbatim allowed the classification of the leads proposed by health care workers in three main themes: improvement of transparency and restoration of the trust for vaccine policy, improved initial and further training of health care workers, and a better communication towards the population. CONCLUSION: Results provide helpful insights into practical avenues to improve the vaccination confidence. If some of them need an in-depth reflection, others could be easily implemented in order to increase the adhesion of the population to vaccination, and consequently the vaccination coverage.

Vaccine distrust: Investigation of the views and attitudes of parents in regard to vaccination of their children.

BACKGROUND: In France, many parents have lost confidence in vaccinations, which has a direct impact on immunization coverage. Pharmacists, like other health professionals, often encounter parents exhibiting vaccine distrust. METHODS: Using a survey distributed in a school and in a number of volunteering pharmacies, the objective of this study was to gain a better understanding of the views and the attitude of parents in regard to vaccination of their children. RESULTS: Our results show that the main concerns were in regard to vaccine adjuvants, the risk of short- and long-term adverse effects, and the risk of developing a disease or a disability as a result of vaccination. The parents, although they tended to express a degree of reluctance and apprehension, in general, they were not opposed to vaccination, and they sought objective scientific information and full transparency regarding all aspects of vaccine composition, adverse effects, and effectiveness. Cooperation of all the parties involved in the health system on this subject is essential for a seamless chain of care and to improve vaccination coverage. CONCLUSION: The information collected, combined with a review of the international literature, allow avenues for dialogue adapted to parents' opinions to be established and thus assist health professionals to communicate effectively regarding vaccines, which is a bona fide public health issue.

Innate immune evasion of Escherichia coli clinical strains from orthopedic implant infections.

Escherichia coli is one of the first causes of Gram-negative orthopedic implant infections (OII). Those infections, usually hematogenous, mostly originate from the urinary tract. We investigated the strategies developed by E. coli in this context to evade host innate immune responses, i.e. complement and polymorphonuclear neutrophils (PMN). Twenty strains from OII were compared with 20 strains from bacteremia in patients with non-infected orthopedic implant. In both groups, 6/20 (30 %) strains lysed PMNs, due to the production of the pore-forming toxin α-hemolysin (HlyA). For the others, resistance to phagocytic killing by PMN was not significantly different between both groups. In contrast, resistance to complement-mediated serum killing was significantly higher in OII strains than in the others (65 % vs 10 %; P <0.001). In E. coli, different mechanisms have been involved in complement resistance. Here, serum resistance was not linked to a group 2 capsule, or a loss of outer membrane permeability, or the recruitment of the complement inhibitor C4bp, but was significantly associated with the synthesis of long-chain LPS, regardless of the O-antigen. Thus, serum resistance could promote seeding of peri-implant tissues by helping E. coli to either persist in blood and reach the site of infection or overcome localized complement activation.

Prevalence of plasmid-mediated quinolone resistance determinants in ESBL Enterobacteriaceae clinical isolates over a 1-year period in a French hospital.

The prevalence of plasmid-mediated quinolone resistance (PMQR) determinants (qnrA, qnrB, qnrS, aac(6')-Ib-cr, and qepA) was investigated in a collection of 47 extended-spectrum ß-lactamase (ESBL) producing enterobacterial isolates with reduced susceptibility to fluoroquinolones, recovered at Nantes University hospital, in 2006. qnr, aac(6')-Ib-cr, and qepA genes were screened by PCR, and positive results were subsequently confirmed by sequencing. The epidemiological relationship between positive isolates was studied by pulsed-field gel electrophoresis (PFGE). qnr-positive isolates were analyzed for antimicrobial susceptibility and presence of mutations in the quinolone resistance-determining region (QRDR) of gyrA and parC genes. ESBL genes were characterized by PCR and sequencing. Conjugation experiments were performed to determine whether the qnr-carrying plasmids were self-transferable. Two Klebsiella pneumoniae isolates (4.3%), not clonally related, harboured a qnrS1 gene, whereas no qnrA- or qnrB-positive isolate was detected. The aac(6')-Ib-cr gene was detected in 11 Escherichia coli and one K. pneumoniae isolates. None of the 47 isolates carried the qepA gene. ESBLs associated with QnrS1 were CTX-M-14 and CTX-M-15. The CTX-M-15 producing isolate was highly resistant to fluoroquinolones and harboured three mutations in the QRDR and two PMQR determinants (qnrS1 and aac(6')-Ib-cr). The CTX-M-14-producing isolate exhibited reduced susceptibility or resistance to fluoroquinolones without resistance to nalidixic acid. This strain harboured only a qnr gene on a single 170 kb transferable plasmid, without any mutation in the QRDR. In conclusion, our study showed that aac(6')-Ib-cr gene had occurred in multiclonal ESBL-producing enterobacterial isolates collected at Nantes University hospital in 2006, with a higher prevalence than qnr genes.

Assessment of ica operon carriage and biofilm production in Staphylococcus epidermidis isolates causing bacteraemia in bone marrow transplant recipients.

The clinical significance of coagulase-negative staphylococci isolated from blood culture is typically assessed on the basis of a combination of clinical and microbiological criteria. However, these criteria are difficult to apply to haematology patients who are highly immunosuppressed and from whom blood cultures are obtained most frequently through a central venous catheter. This study analysed 112 episodes of Staphylococcus epidermidis bacteraemia that occurred in 79 bone marrow transplant recipients. In 73 (65%) episodes, only one blood culture set was positive for S. epidermidis, while 39 (35%) episodes grew S. epidermidis from multiple blood cultures. Nine patients had two or more episodes of bacteraemia with the same strain, as determined by pulsed-field gel electrophoresis (PFGE). The PFGE method also showed that 34 (31%) isolates belonged to seven clusters, indicating the persistence of certain clones in the environment. Of the 109 isolates analysed, 59 (54%) produced biofilm and 91 (83.5%) carried the ica operon. Isolates that produced biofilm were observed to colonise central venous catheters faster than non-biofilm-producing isolates (18 vs. 37 days; p 0.03). No clinical features were associated with carriage of the ica operon, but the ica operon was carried more frequently by the isolates that formed clusters.

Risk-factors for gastrointestinal colonisation with resistant Enterobacteriaceae among hospitalised patients: a prospective study.

This study assessed the incidence of gastrointestinal colonisation by resistant Enterobacteriaceae among hospitalised patients, and identified risk-factors for ceftazidime and ofloxacin resistance. A prospective cohort study was performed in five wards in a French teaching hospital during a 2-year period. Patients hospitalised for > 48 h were enrolled between 17 April 2000 and 30 April 2002. A rectal swab was taken at admission, then once-weekly and/or on the day of discharge. In total, 933 patients were investigated and 585 amoxycillin-resistant isolates were obtained. Resistance rates for ceftazidime and ofloxacin were 9.4% and 4.8%, respectively. Multivariate analysis indicated that previous hospitalisation (p < 0.004) and exposure to amoxycillin-clavulanate (p < 0.003) and ceftriaxone (p < 0.002) were associated significantly with ceftazidime resistance. Hospitalisation in the urology ward (p < 0.02) and previous exposure to fluoroquinolones (p < 0.01) were the two independent risk-factors associated with ofloxacin resistance. The results of the study confirmed that antibiotic use selected resistant Enterobacteriaceae from the gut flora. Resistance was observed mostly in patients with previous antibiotic exposure and previous hospitalisation in wards with a high antibiotic selection pressure.

Subversion of human intestinal mucosa innate immunity by a Crohn's disease-associated E. coli.

Adherent-invasive Escherichia coli (AIEC), associated with Crohn's disease, are likely candidate contributory factors in the disease. However, signaling pathways involved in human intestinal mucosa innate host response to AIEC remain unknown. Here we use a 3D model of human intestinal mucosa explant culture to explore the effects of the AIEC strain LF82 on two innate immunity platforms, i.e., the inflammasome through evaluation of caspase-1 status, and NFκB signaling. We showed that LF82 bacteria enter and survive within a few intestinal epithelial cells and macrophages, without altering the mucosa overall architecture. Although 4-h infection with a Salmonella strain caused crypt disorganization, caspase-1 activation, and mature IL-18 production, LF82 bacteria were unable to activate caspase-1 and induce IL-18 production. In parallel, LF82 bacteria activated NFκB signaling in epithelial cells through IκBα phosphorylation, NFκBp65 nuclear translocation, and TNFα secretion. In addition, NFκB activation was crucial for the maintenance of epithelial homeostasis upon LF82 infection. In conclusion, here we decipher at the whole-mucosa level the mechanisms of the LF82-induced subversion of innate immunity that, by maintaining host cell integrity, ensure intracellular bacteria survival.

Mutations in the ampC promoter of Escherichia coli isolates resistant to oxyiminocephalosporins without extended spectrum beta-lactamase production.

Escherichia coli strains showing an increased resistance to oxyiminocephalosporins without an extended spectrum beta-lactamase production have been screened for mutations in their ampC beta-lactamase gene promoter. Mutations were found by direct sequencing of seven promoters at positions -42, -32 (box -35), -18, -1, +5, +24 (attenuator), +31 (attenuator) and +58. By using rapid and simple methods, three of these mutations (-42, -32 and +24), which could enhance transcription, were searched in 37 resistant and 25 sensitive isolates. The -42 mutation was present in 33 of the 37 promoters from the resistant isolates. The -32 and +24 mutations were present only in three and two promoters, respectively, and they were combined in the most resistant strain of the study. The +24 mutation was detected in another strain associated with a 1-bp insertion between the -35 and -10 conserved sequences. None of these mutations was detected in the ampC beta-lactamase from the sensitive isolates.

Role of hospital stay and antibiotic use on Pseudomonas aeruginosa gastrointestinal colonization in hospitalized patients.

The prospective cohort study presented here assessed the risk factors associated with Pseudomonas aeruginosa gastrointestinal colonization (PAGIC) in 933 patients hospitalized in five different wards in a French university hospital. A total of 195 patients were colonized. By logistic regression, hospitalization in an intensive care unit and length of hospital stay were independent risk factors. A significant association was observed between fluoroquinolone use and PAGIC caused by an ofloxacin-resistant strain (p < 0.0001), imipenem use and PAGIC caused by an imipenem-resistant strain (p < 0.0002) and ceftazidime use and PAGIC caused by a ceftazidime-resistant strain (p < 0.02). The ecological impact of antibiotic use is of great clinical relevance and clinicians should consider antimicrobial resistance in order to limit the development and dissemination of resistant microorganisms.

Escherichia coli: epidemiology and analysis of risk factors for infections caused by resistant strains.

This study analyzes the epidemiology of hospital and community-acquired infections caused by Escherichia coli. The antimicrobial resistance pattern was used to characterize the isolates, and a prospective observational study was performed to assess the relationship between antimicrobial use and bacterial resistance. The study was conducted during a 3-month period in a 1,200-bed tertiary care hospital in Nantes, France. An E. coli infection was diagnosed in 3.8% of the patients (507 of 13,384) admitted to the hospital between 1 January and 31 March 1996. Of the 507 isolates, 205 (40.4%) were resistant to at least one antimicrobial; 40% were resistant to amoxicillin, 30% to amoxicillin/clavulanate, 38% to ticarcillin, and 16% to trimethoprim-sulfamethoxazole, while resistance to other antimicrobials was low. Prior receipt of antimicrobial and/or immunosuppressive therapy was significantly associated with infection caused by a resistant organism.

Regulation of human renin secretion and renin transcription by quantitative PCR in cultured chorionic cells: synergistic effect of cyclic AMP and protein kinase C.

A quantitative PCR was developed to measure human renin mRNA in chorionic cells under various stimuli. An internal standard consisting of a mutated renin mRNA with an insertion of 60 bp was designed to quantify the reaction. Quantitative PCR is a suitable tool for studying human renin gene transcription from a low number of cells. Forskolin alone had no effect on either renin mRNA levels or renin production whereas 10(-7)M PMA stimulated renin mRNA levels and renin production 5- and 2.6-fold, respectively, after 24 hours incubation. PMA and forskolin acted synergistically to increase both renin mRNA levels and renin production in a dose-dependent manner.

Analysis of the effects of -42 and -32 ampC promoter mutations in clinical isolates of Escherichia coli hyperproducing ampC.

Escherichia coli usually produces only very small amounts of a constitutive AmpC beta-lactamase, but clinical strains overproducing this enzyme have been isolated. Three different ampC promoters of E. coli clinical strains were cloned upstream of the chloramphenicol acetyltransferase (CAT) gene in the pKK232-8 reporter plasmid and their relative strengths were compared by two different methods. The strength of the promoters from AmpC hyperproducers was 70- to 120-fold higher than those from a low-level AmpC producer. One of the strong promoters, which differs from strain K12 at bases -88, -82, -42, -18, -1 and +58, was mutated to abolish the -42 mutation. This change resulted in a 43-fold decrease in CAT concentration. In another promoter, with eight different mutations at positions -88, -82, -32, -18, -1, +5, +24 and +58, the -32T-->A transversion, which created perfect homology with the -35 consensus sequence, was reverted; this led to a 13-fold decrease in CAT concentration. The -42 and -32 mutations play an important role in E. coli resistance to beta-lactams by increasing ampC transcription.

-11 Mutation in the ampC promoter increasing resistance to beta-lactams in a clinical Escherichia coli strain.

A mutation was discovered in the Pribnow box of the ampC promoter in a clinical Escherichia coli strain. This -11 C-to-T transition created a perfect homology with the -10 consensus sequence. The new promoter was cloned upstream of the cat gene of pKK232-8 and induced a sixfold increase in promoter strength.

Two epidemiologically related cases of Rahnella aquatilis bacteremia.

Rahnella aquatilis was isolated from the blood cultures of two patients who were in different units of the same hospital. Both isolates were susceptible to aminoglycosides, fluoroquinolones, cotrimoxazole, piperacillin, third generation cephalosporins and amoxicillin-clavulanate, but resistant to amoxicillin, ticarcillin, and first generation cephalosporins. The synergistic activity of amoxicillin and clavulanic acid suggested the presence of a beta-lactamase, confirmed by a positive nitrocefin test and by analytical isoelectric focusing. Pulsed-field gel electrophoresis and ribotyping with the pKK3535 probe showed that the isolates shared the same banding pattern. The results of an epidemiological study suggested that an in-house total parenteral nutrition solution might be the source of this unusual gram-negative rod.

Phenotypic study of resistance of beta-lactamase-inhibitor-resistant TEM enzymes which differ by naturally occurring variations and by site-directed substitution at Asp276.

At this time an amino acid substitution at position 276 in the TEM-1 enzyme is associated with an additional substitution at position 69 in natural beta-lactamase-inhibitor-resistant (IRT) beta-lactamases. The effect of the Asn276-->Asp substitution on resistance was assessed with the Asn276Asp variant, generated by site-directed mutagenesis. The mutant was resistant to beta-lactamase inhibitors, but the MICs of amoxicillin combined with clavulanic acid or tazobactam were strikingly different for E. coli strains producing the Asn276Asp variant and those producing naturally occurring IRTs with single or double substitutions. The inhibitory effects of clavulanic acid and tazobactam were the same in IRTs with substitutions at position 69 (IRT-5 and IRT-6). The effect of clavulanic acid on the MICs of amoxicillin for the Asn276Asp variant was greater than that of tazobactam. In IRTs with double substitutions, at positions 69 plus 276 (IRT-4, IRT-7, and IRT-8) or 69 plus 275 (IRT-14), tazobactam was a more potent inhibitor than clavulanic acid. The effect of the Asn276-->Asp substitution on the values of the kinetic constants and the concentration required to inhibit by 50% the hydrolysis of benzylpenicillin confirms that this single mutation is responsible for resistance to beta-lactamase inhibitors. Molecular modeling of the Asn276Asp mutant shows that Asp276 can form two salt bonds with Arg244 close to the penicillin-binding cavity. The addition of the Asp276 mutation to that preexisting at position 69 confers a higher selective advantage to bacteria, as shown by the reduction in beta-lactamase inhibitor efficiencies of the double variants. Therefore, the emergence of multiple mutations in TEM beta-lactamases by virtue of the use of beta-lactamase inhibitors increases selection pressure resulting in the convergent evolution of resistant strains.

Detection of renin mRNA in aldosterone-producing adenomas by polymerase chain reaction.

1. Ribonucleic acid (RNA) was extracted from two normal human adrenal cortices and from five aldosterone-producing adenomas (APA). 2. The five APA could be categorized, on the basis of in vivo aldosterone responsiveness to angiotensin infusion and upright posture, into two APA responsive to both stimuli, two responsive only to angiotensin infusion, and one unresponsive to either stimulus. 3. RNA was reverse transcribed and coamplified by polymerase chain reaction (PCR) with an internal standard of renin complementary DNA (cDNA) containing a 60 base pair insertion. Renin mRNA in the APA was compared with normal adrenals. 4. Renin mRNA was greater than normal in the two APA responsive to both stimuli and less than, or similar to normal, in the two APA responsive only to angiotensin infusion. Renin mRNA was also less than, or similar to normal, in the APA unresponsive to either stimulus. 5. These findings support a possible role for adrenal renin in the development and biochemical behaviour of angiotensin-responsive APA.