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Staphylococcus aureusgamma-hemolysin CB (HlgCB) is a core-genome encoded pore-forming toxin that targets the C5a receptor, similarly as the phage-encoded Panton-Valentine Leucocidin. Absolute quantification by mass spectrometry of HlgCB in 39 community-acquired pneumonia (CAP) isolates showed considerable variations in HlgC and HlgB yields between isolates. Interestingly, when testing the hypothesis that HlgCB might be associated with severeS. aureusCAP, we found that a high level of HlgCB synthesis was associated with mortality in a rabbit model of pneumonia. To decipher the molecular basis for the variation inhlgCB andhlgB expression and protein production among strains, different regulation levels were analyzed in representative clinical isolates and reference strains. Although HlgC and HlgB are encoded on a single operon, their levels were dissociated in 10% of the clinical strains studied. HlgCB amount and HlgC/HlgB ratio were found to both depend on promotor activity, mRNA stability and translatability, and on the presence of an individualhlgB mRNA processed from thehlgCB transcript. Strikingly, toe-printing andin vitrotranslation assays revealed that a single SNP in the 5’-UTR ofhlgCB mRNA strongly impairedhlgC translation in the USA300 strain, leading to a strong decrease in HlgC but not in HlgB; the level of HlgB is likely to have been maintained by the presence of the processedhlgB mRNA. This work illustrates the complexity of virulence factor expression in clinical strains and demonstrates a butterfly effect, where subtle genomic variations have a major impact on phenotype and virulence.Author SummaryThe Gram-positive bacteriumStaphylococcus aureuscan provoke a wide range of infections due to its ability to produce a large diversity of virulence factors, including immune evasion molecules, adhesins, and toxins. Some of these toxin-encoding genes are localized in mobile genetic elements, and are thus not present in all strains, whilst others are encoded in the core-genome and present in all strains. Gamma-hemolysin CB is a core-genome encoded toxin but its amount varies between community-acquired pneumonia isolates. The regulation mechanisms underlying this variation however, are not well characterized. Here, we show that gamma-hemolysin expression levels vary largely among clinical strains and that, when highly produced, it induces high mortality in a rabbit model of pneumonia. The molecular basis for the variation in gamma-hemolysin expression depends on multiple mechanisms including promoter strength, transcript stability and processing, and translatability (i.e. the amount of protein that is synthetized by the ribosome for a given transcript). Incredibly, all these factors rely on a subtle genetic modification. This work emphasizes the importance of the disparity in virulence factor expression among clinical isolates and points the extreme complexity of the molecular mechanisms underlying their regulation, rendering the prediction of virulence for a clinical isolate difficult.

Objectives: As daptomycin adjunction is currently under clinical evaluation in the multicentre phase II AddaMAP study to improve the prognosis of pneumococcal meningitis, the present work aimed at evaluating the in vitro antimicrobial activity of daptomycin-based combinations against some of the most frequent species responsible for bacterial meningitis. Methods: Clinically relevant strains of Streptococcus pneumoniae, Listeria monocytogenes, Haemophilus influenzae and Neisseria meningitidis were obtained from National Reference Centers. The antimicrobial activity of amoxicillin, cefotaxime and rifampicin, either alone or in association with daptomycin, was explored through the determination of minimum inhibitory concentration (MIC) and fractional inhibitory concentration index (FICI) as well as time–kill assay (TKA) using the broth microdilution method. Results: All species taken together, the adjunction of daptomycin had no deleterious impact on the antimicrobial activity of amoxicillin, cefotaxime or rifampicin in vitro. Regarding Gram-positive bacteria, FICI and TKA analysis confirmed a global improvement of growth inhibition and bactericidal activity due to the adjunction of daptomycin. The synergistic effect prevailed for L. monocytogenes as demonstrated by FICI mainly 50%. In addition, daptomycin-based associations did not modify the activity of β-lactam antibiotics or rifampicin against Gram-negative bacteria, notably N. meningitidis. Conclusion: These results bring comforting evidence towards the clinical potential of daptomycin adjunction in the treatment of bacterial meningitis, which supports the ongoing AddaMAP clinical trial.

Background Mechanical ventilation for pneumonia may contribute to lung injury due to factors that include mitochondrial dysfunction, and mesenchymal stem cells may attenuate injury. This study hypothesized that mechanical ventilation induces immune and mitochondrial dysfunction, with or without pneumococcal pneumonia, that could be mitigated by mesenchymal stem cells alone or combined with antibiotics. Methods Male rabbits underwent protective mechanical ventilation (8 ml/kg tidal volume, 5 cm H2O end-expiratory pressure) or adverse mechanical ventilation (20 ml/kg tidal-volume, zero end-expiratory pressure) or were allowed to breathe spontaneously. The same settings were then repeated during pneumococcal pneumonia. Finally, infected animals during adverse mechanical ventilation received human umbilical cord–derived mesenchymal stem cells (3 × 106/kg, intravenous) and/or ceftaroline (20 mg/kg, intramuscular) or sodium chloride, 4 h after pneumococcal challenge. Twenty-four-hour survival (primary outcome), lung injury, bacterial burden, immune and mitochondrial dysfunction, and lung transcriptomes (secondary outcomes) were assessed. Results High-pressure adverse mechanical ventilation reduced the survival of infected animals (0%; 0 of 7) compared with spontaneous breathing (100%; 7 of 7) and protective mechanical ventilation (86%; 6 of 7; both P < 0.001), with higher lung pathology scores (median [interquartile ranges], 5.5 [4.5 to 7.0] vs. 12.6 [12.0 to 14.0]; P = 0.046), interleukin-8 lung concentrations (106 [54 to 316] vs. 804 [753 to 868] pg/g of lung; P = 0.012), and alveolar mitochondrial DNA release (0.33 [0.28 to 0.36] vs. 0.98 [0.76 to 1.21] ng/μl; P < 0.001) compared with infected spontaneously breathing animals. Survival (0%; 0 of 7; control group) was improved by mesenchymal stem cells (57%; 4 of 7; P = 0.001) or ceftaroline alone (57%; 4 of 7; P < 0.001) and improved even more with a combination treatment (86%; 6 of 7; P < 0.001). Mesenchymal stem cells reduced lung pathology score (8.5 [7.0 to 10.5] vs. 12.6 [12.0 to 14.0]; P = 0.043) and alveolar mitochondrial DNA release (0.39 (0.34 to 0.65) vs. 0.98 (0.76 to 1.21) ng/μl; P = 0.025). Mesenchymal stem cells combined with ceftaroline reduced interleukin-8 lung concentrations (665 [595 to 795] vs. 804 [753 to 868] pg/g of lung; P = 0.007) compared to ceftaroline alone. Conclusions In this preclinical study, mesenchymal stem cells improved the outcome of rabbits with pneumonia and high-pressure mechanical ventilation by correcting immune and mitochondrial dysfunction and when combined with the antibiotic ceftaroline was synergistic in mitigating lung inflammation. Editor’s Perspective What We Already Know about This Topic What This Article Tells Us That Is New

We developed a rat model of methicillin-resistant Staphylococcus epidermidis (MRSE) osteitis without implant to compare the efficacy of vancomycin, linezolid, daptomycin, ceftaroline, and rifampin either alone or in association with rifampin. A clinical strain of MRSE was inoculated into the proximal tibia. Following a 1-week infection period, rats received either no treatment or 3, 7, or 14 days of human-equivalent antibiotic regimen. Quantitative bone cultures were performed throughout the 14-day period. The mean ± SD quantity of staphylococci in the bone after a 1-week infection period was 4.5 ± 1.0 log(10) CFU/g bone, with this bacterial load remaining stable after 3 weeks of infection (4.9 ± 1.4 log(10) CFU/g bone). Vancomycin monotherapy was the most slowly bactericidal treatment, whereas ceftaroline monotherapy was the most rapidly bactericidal treatment. The addition of rifampin significantly increased the bacterial reduction for vancomycin, linezolid, and daptomycin. All tibias were sterilized after 2 weeks of treatment except for animals receiving vancomycin or daptomycin alone (66.6% and 50% of sterilization, respectively). These results show that ceftaroline and linezolid alone remain good options in the treatment of MRSE osteitis without implant. The combination with rifampin increases the antibiotic effect of vancomycin and daptomycin lines.

Introduction La ventilation mecanique (VM) est associee a une surmortalite au cours de la pneumopathie grave. Les dysfonctions mitochondriales pourraient contribuer aux alterations immunitaires et aux defaillances d’organe au cours du sepsis. L’objectif etait d’etudier l’impact de la VM sur les dysfonctions immunitaires et mitochondriales au cours de la pneumopathie a Streptococcus pneumoniae (S.p.), et d’evaluer l’effet des cellules souches/stromales mesenchymateuses (CSMs). Materiels et methodes Des lapins etaient soumis a une VM protectrice ou agressive pendant 24 heures ou gardes en ventilation spontanee (VS). Une pneumopathie etait obtenue par instillation endo-bronchique de S.p. Les lapins non infectes etaient utilises comme controle (n = 7/groupe). La survie, la charge bacterienne, les dommages pulmonaires, la reponse immunitaire, les alterations mitochondriales (ADN mitochondrial [ADNmt], pool mitochondrial, potentiel de membrane mitochondrial [ΔΨm]) etaient mesures dans le poumon (ou LBA) et le sang. Au cours de la pneumopathie a S.p. ventilee, les animaux etaient randomises et recevaient a 4 hrs : NaCl, CSMs humaines derivees de la gelee de Wharton du cordon ombilical (3 × 106/kg, i.v.), Ceftaroline (20 mg/kg, i.m.) ou les 2 traitements (CSMs et Ceftaroline). Resultats La VM agressive seule s’associait a des dommages et a une inflammation pulmonaires (afflux de neutrophiles, liberation d’IL-8), ainsi qu’a des alterations mitochondriales (augmentation des niveaux d’ADNmt et du ΔΨm des neutrophiles alveolaires, diminution du pool mitochondrial pulmonaire). Compares a ceux sous VM protectrice, les animaux sous VM agressive developpaient des pneumopathies multifocales, avec une surmortalite (100 % vs 14 % ; p Conclusion Les CSMs ameliorent le pronostic de la pneumopathie a pneumocoque ventilee, via la correction de dysfonctions immunitaires et mitochondriales, representant ainsi une strategie adjuvante a l’antibiotherapie.

This study investigated the in vivo efficacy of three bacteriophages combined compared with linezolid in two mouse models (nondiabetic and diabetic) of Staphylococcus aureus foot infection. In both models, a single injection of bacteriophages in the hindpaw showed significant antibacterial efficacy. Linezolid was as effective as bacteriophages in nondiabetic animals but ineffective in diabetic animals. These findings further support preclinical and clinical studies for the development of phage therapy.

Staphylococcus aureus pneumonia is associated with high mortality irrespective of antibiotic susceptibility. Both MRSA and MSSA strains produce powerful cytotoxins: alpha-hemolysin(Hla) and up to five leukocidins – LukSF-PV, HlgAB, HlgCB, LukED and LukGH (LukAB) – to evade host innate defense mechanisms. Neutralizing cytotoxins has been shown to provide survival benefit in rabbit S. aureus pneumonia models. We studied the mechanisms of protection of ASN100, a combination of two human monoclonal antibodies (mAbs), ASN-1 and ASN-2, that together neutralize Hla and the five leukocidins, in rabbit MRSA and MSSA pneumonia models. Upon prophylactic passive immunization, ASN100 displayed dose-dependent increase in survival and was fully protective against all S. aureus strains tested at 5 or 20 mg/kg doses. Macroscopic and microscopic lung pathology, edema rate, and bacterial burden were evaluated 12 hours post infection and reduced by ASN100. Pharmacokinetic analysis of ASN100 in bronchoalveolar-lavage fluid from uninfected animals detected efficient penetration to lung epithelial lining fluid reaching peak levels between 24 and 48 hours post dosing that were comparable to the mAb concentration measured in serum. These data confirm that the ASN100 mAbs neutralize the powerful cytotoxins of S. aureus in the lung and prevent damage to the mucosal barrier and innate immune cells.

International audience; Pneumonia may involve methicillin-resistant Staphylococcus aureus (MRSA), with elevated rates of antibiotics failure. The present study aimed to assess the effect of statins given prior to pneumonia development. Spontaneously breathing (SB) or mechanically ventilated (MV) rabbits with pneumonia received atorvastatin alone, linezolid (LNZ) alone, or a combination of both (n = 5 in each group). Spontaneously breathing and MV untreated infected animals (n = 11 in each group), as well as uninfected animals (n = 5 in each group) were used as controls. Microbiological features and inflammation were evaluated. Data are presented as medians (interquartile range). Linezolid alone tended to reduce pulmonary MRSA load in both SB and MV rabbits, but failed to prevent bacteremia (59%) in the latter. Linezolid alone dampened TNF-α lung production in both SB and MV rabbits (e.g., 2226 [789] vs. 11478 [10251] pg/g; p = 0.022). Statins alone did the same in both SB and MV animals (e.g., 2040 [133]; p = 0.016), and dampened systemic inflammation in the latter, possibly through TLR2 down-regulation within the lung. However, the combination of LNZ and statin led to an increased rate of bacteremia in MV animals up to 75%. Statins provide an anti-inflammatory effect in rabbits with MRSA pneumonia, especially in MV ones. However, dampening the systemic inflammatory response with statins could impede blood defenses against MRSA.

IF 4.307; International audience; BackgroundAntibiotic streamlining is pivotal to reduce the emergence of resistant bacteria. However, whether streamlining is frequently performed and safe in difficult situations, such as bacteremic pneumococcal pneumonia (BPP), has still to be assessed.MethodsAll adult patients admitted to Dijon Hospital (France) from 2005 to 2013 who had BPP without complications, and were alive on the third day were enrolled. Clinical, biological, radiological, microbiological and therapeutic data were recorded. A first analysis was conducted to assess factors associated with being on amoxicillin on the third day. A second analysis, adjusting for a propensity score, was performed to determine whether 30-day mortality was associated with streamlining to amoxicillin monotherapy.ResultsOf the 196 patients hospitalized for BPP, 161 were still alive on the third day and were included in the study. Treatment was streamlined to amoxicillin in 60 patients (37%). Factors associated with not streamlining were severe pneumonia (OR 3.11, 95%CI [1.23–7.87]) and a first-line antibiotic combination (OR 3.08, 95%CI [1.34–7.09]). By contrast, starting with amoxicillin monotherapy correlated inversely with the risk of subsequent treatment with antibiotics other than amoxicillin (OR 0.06, 95%CI [0.01–0.30]). The Cox model adjusted for the propensity-score analysis showed that streamlining to amoxicillin during BPP was not significantly associated with a higher risk of 30-day mortality (HR 0.38, 95%CI [0.08–1.87]).ConclusionsStreamlining to amoxicillin is insufficiently implemented during BPP. This strategy is safe and potentially associated with ecological and economic benefits; therefore, it should be further encouraged, particularly when antibiotic combinations are started for severe pneumonia.

Introduction Les donnees epidemiologiques actuelles sur les meningites bacteriennes de l’adulte font etat d’une large prevalence de Streptococcus pneumoniae [Clin Microbiol Infect. 2016 ;22 Suppl 3 :S37-62]. Du fait de facteurs de virulence intrinseques, cette espece conduit a la morbi-mortalite la plus elevee [N Engl J Med. 2004 ;351(18) :1826-8]. Bien que le traitement repose sur l’utilisation des beta-lactamines, plusieurs travaux de recherche suggerent un risque d’aggravation des lesions ischemiques cerebrales via l’effet pro-inflammatoire d’endotoxines liberees lors de la lyse bacterienne [Future Microbiol. 2015 ;10(7) :1195-213]. L’ajout de daptomycine, lipopeptide bactericide non bacteriolytique, est actuellement en cours d’evaluation clinique dans le but de reduire la mortalite et les sequelles des meningites a pneumocoques [ADDAMAP, www.infectiologie.com/fr/projets.html ]. Mais l’impact d’une telle adjonction sur l’efficacite du traitement de reference des autres meningites bacteriennes est encore inconnu. Ce projet prevoit d’evaluer in vitro l’efficacite d’associations a base de daptomycine sur les principales especes responsables de meningites bacteriennes chez l’adulte. Materiels et methodes Plusieurs souches d’interet epidemiologique ont ete selectionnees avec les centres nationaux de reference pour cinq especes bacteriennes : Streptococcus pneumoniae, Neisseria meningitidis, Listeria monocytogenes, Haemophilus influenzae et Streptococcus agalactiae. L’activite antimicrobienne de l’amoxicilline, du cefotaxime, de la rifampicine et de la daptomycine – seules ou en association – a ete evaluee par determination des concentrations minimales inhibitrices (CMI), des indices de concentration inhibitrice fractionnaire (FICI) et des vitesses de bactericidie. Resultats L’adjonction de daptomycine ne semble pas diminuer l’efficacite des beta-lactamines ou de la rifampicine sur les especes bacteriennes testees. Au contraire, les resultats de FICI et vitesses de bactericidie confirment une augmentation du potentiel bactericide lorsque la daptomycine est associee aux autres antibiotiques. Prenant l’exemple de Listeria monocytogenes, la valeur des FICI pour toutes les associations comprenant la daptomycine est comprise entre 0,9 et 0,2, temoignant d’un effet additif ou synergique–variant selon les souches–a l’origine d’une augmentation significative des vitesses de bactericidie, en moyenne quatre fois superieures a celles obtenues avec les antibiotiques seuls. Conclusion Ces resultats in vitro apportent des donnees rassurantes en faveur de l’adjonction de daptomycine au traitement probabiliste des meningites bacteriennes de l’adulte. Des etudes complementaires sont actuellement en cours afin de mieux comprendre cette potentialisation de l’effet bactericide des beta-lactamines et de la rifampicine, notamment l’impact de la daptomycine sur la membrane bacterienne.

Background: The prone position (PP) has proven beneficial in patients with severe lung injury subjected to mechanical ventilation (MV), especially in those with lobar involvement. We assessed the impact of PP on unilateral pneumonia in rabbits subjected to MV. Methods: After endobronchial challenge with Enterobacter aerogenes, adult rabbits were subjected to either “adverse” (peak inspiratory pressure = 30 cm H2O, zero end-expiratory pressure; n = 10) or “protective” (tidal volume = 8 ml/kg, 5 cm H2O positive end-expiratory pressure; n = 10) MV and then randomly kept supine or turned to the PP. Pneumonia was assessed 8 h later. Data are presented as median (interquartile range). Results: Compared with the supine position, PP was associated with significantly lower bacterial concentrations within the infected lung, even if a “protective” MV was applied (5.93 [0.34] vs. 6.66 [0.86] log10 cfu/g, respectively; P = 0.008). Bacterial concentrations in the spleen were also decreased by the PP if the “adverse” MV was used (3.62 [1.74] vs. 6.55 [3.67] log10 cfu/g, respectively; P = 0.038). In addition, the noninfected lung was less severely injured in the PP group. Finally, lung and systemic inflammation as assessed through interleukin-8 and tumor necrosis factor-α measurement was attenuated by the PP. Conclusions: The PP could be protective if the host is subjected to MV and unilateral bacterial pneumonia. It improves lung injury even if it is utilized after lung injury has occurred and nonprotective ventilation has been administered.

International audience; Biofilms are complex communities of microorganisms embedded in an extracellular matrix and adherent to a surface. The development was described as a four-stage process leading to the formation of a mature biofilm which was resistant to immune system and antibiotic actions. In bone and joint infections (BJIs), the formation of biofilms is a leading cause of treatment failure. Here we study the capacity of 11 antibiotics commonly used in the treatment of BJIs to inhibit the biofilm formation on 29 clinical Staphylococcus aureus isolates by a new test called Antibiofilmogram(\textregistered) The minimal inhibitory concentration (MIC) and biofilm MIC (bMIC) were determined in vitro and showed similar values for clindamycin, fusidic acid, linezolid and rifampin. Reversely, daptomycin, fosfomycin, gentamicin and ofloxacin showed a bMIC distribution different from MIC with bMIC above breakpoint. Finally, cloxacillin, teicoplanin and vancomycin revealed an intermediate bMIC distribution with a strain-dependent pattern. A murine in vivo model of catheter-associated S. aureus infection was made and showed a significant reduction, but not total prevention, of catheter colonization with cloxacillin at bMIC, and no or limited reduction with cloxacillin at MIC. Antibiofilmogram(\textregistered) could be of great interest after surgical operations on contaminated prostheses and after bacteremia in order to prevent the colonization of the device.

Ventilator-associated pneumonia (VAP) is common during mechanical ventilation (MV). Beside obvious deleterious effects on muco-ciliary clearance, MV could adversely shift the host immune response towards a pro-inflammatory pattern through toll-like receptor (TLRs) up-regulation. We tested this hypothesis in a rabbit model of Staphylococcus aureus VAP. Pneumonia was caused by airway challenge with S. aureus, in either spontaneously breathing (SB) or MV rabbits (n = 13 and 17, respectively). Pneumonia assessment regarding pulmonary and systemic bacterial burden, as well as inflammatory response was done 8 and 24 hours after S. aureus challenge. In addition, ex vivo stimulations of whole blood taken from SB or MV rabbits (n = 7 and 5, respectively) with TLR2 agonist or heat-killed S. aureus were performed. Data were expressed as mean +/- standard deviation. After 8 hours of infection, lung injury was more severe in MV animals (1.40 +/- 0.33 versus [vs] 2.40 +/- 0.55, p = 0.007), along with greater bacterial concentrations (6.13 +/- 0.63 vs. 4.96 +/- 1.31 colony forming units/gram, p = 0.002). Interleukin (IL)-8 and tumor necrosis factor (TNF)-alpha serum concentrations reached higher levels in MV animals (p = 0.010). Whole blood obtained from MV animals released larger amounts of cytokines if stimulated with TLR2 agonist or heat-killed S. aureus (e.g., TNF-alpha: 1656 +/- 166 vs. 1005 +/- 89; p = 0.014). Moreover, MV induced TLR2 overexpression in both lung and spleen tissue. MV hastened tissue injury, impaired lung bacterial clearance, and promoted a systemic inflammatory response, maybe through TLR2 overexpression.

Ceftaroline (CPT) is a new cephalosporin exhibiting bactericidal activity against Gram-positive organisms, including methicillin-resistant Staphylococcus aureus (MRSA) and multidrug-resistant Streptococcus pneumoniae (MDRSP), as well as common Gram-negative pathogens. This study investigated the in vivo efficacy of a 48-hour simulated human dose regimen of CPT compared with ceftriaxone (CRO) against isolates of S. pneumoniae with different susceptibilities to penicillin in a rabbit pneumonia model. Three S. pneumoniae strains were used: CRO-susceptible penicillin-susceptible S. pneumoniae (CRO-S PSSP), CRO-susceptible penicillin-intermediate S. pneumoniae (CRO-S PISP), and CRO-resistant penicillin-resistant S. pneumoniae (CRO-R PRSP). Animals were randomized to the control group (no treatment) ( n = 22) or to a group given intravenous (IV) CPT human equivalent (HE) dosage (600 mg/12 h; n = 19) or IV CRO HE dosage (1 g/24 h; n = 19). The total doses needed to achieve the HE dosage were 71 and 82 mg/kg of body weight/24 h for CRO and CPT, respectively. One group of rabbits infected with the CRO-R PRSP strain received intramuscular (IM) administration of CPT (5 or 20 mg/kg twice daily; n = 5 for each). Evaluation of efficacy was based on bacterial counts in the lungs and spleen. For IV CPT and IV CRO, the mean areas under the concentration-time curves from 0 to 24 h (AUC 0–24 s) were 155 and 938 mg · h/liter, respectively, the maximum concentrations in serum ( C max s) were 20 and 158 mg/liter, respectively, and the minimum concentrations in serum ( C min s) were 1.3 and 6 mg/liter, respectively. Both agents effectively treated pulmonary infections caused by CRO-S PSSP or CRO-S PISP with complete bacterial eradication in the lungs and spleen after 2 days of treatment. Against PRSP, CPT demonstrated excellent bactericidal activity, reducing bacterial counts in the lungs and spleen by approximately 8 and 4 log units, respectively ( P < 0.001); CRO treatment resulted in a 2-log-unit reduction in the bacterial counts in lungs that did not reach statistical significance. Twice-daily IM CPT (5 mg/kg) reduced the bacterial burden by approximately 6 log units in the lungs and 3 log units in the spleen, and the 20-mg/kg dosage effectively eradicated PRSP infection. These findings further validate the in vivo bactericidal activity of CPT against pneumococci.

International audience; Background - Chronic kidney disease (CKD) is frequent in individuals infected with human immunodeficiency virus (HIV). Progression to end-stage renal disease can be slowed by appropriate medical management. Methods - To assess whether active promotion of guidelines improves CKD management, we conducted a cluster randomized controlled trial within the French Hospital Database on HIV (FHDH-ANRS CO4). We randomized 46 centers participating in the FHDH to either simple information on guideline availability or active promotion with a multifaceted and repeated intervention comprising reminders and audit feedback and targeting of local opinion leaders carried out between April 2009 and April 2010. Outcome measure was CKD management adequacy assessed before and 2 years after the beginning of the intervention in HIV-infected patients with moderate to severe CKD. CKD management was considered adequate in case of referral to a nephrologist or if proteinuria, blood pressure, low-density lipoprotein cholesterol level, and glycemia had been measured during the previous year and medications had been prescribed when necessary. Results - Three hundred six patients were enrolled, of whom 238 (78%) completed the 2 years of follow-up. During the study period, the percentage of patients receiving adequate CKD management improved from 64.1% to 70.4% (+6.3%) in the active arm and from 68.3% to 75.6% (+7.3%) in the control arm (adjusted mean difference, -0.7 percentage points [95% confidence interval: -9.2 to 7.9]; P = .95). The biggest impact of active promotion was on the management of proteinuria and blood pressure. Conclusions - Adequate compliance with CKD management guidelines improved slightly between 2009 and 2011, with no difference between the simple information and active promotion arms. Clinical trials registration - CCTIRS 10.150 and CNIL DR-2010-379.

International audience; Aminoglycosides are of major importance in treating Pseudomonas aeruginosa pneumonia (PAP). However, their efficacy may be compromised by low-level resistance caused by the inducible MexXY multidrug efflux pump. In the present study, the impact of the MexXY efflux pump was investigated in vivo in an experimental model of PAP in rabbits treated with intravenous tobramycin. Three strains were used to induce PAP in rabbits: PAO1 (wild-type strain; MIC 1 mg/L), mutant 11B (mexX::Tn501; no expression of MexXY; MIC 0.5 mg/L) and mutant MutGR1 (MexZ null; constitutive expression of MexXY; MIC 2 mg/L). Five hours after inoculation, treatment with tobramycin (10 mg/kg) was implemented (peak serum concentration 30 mg/L). The animals were killed humanely 48 h after inoculation, and the residual pulmonary bacterial concentration was determined. Selection of bacteria expressing MexXY was determined by plating lung homogenates on agar plates containing antibiotic. Mean bacterial counts (log(10) CFU/g) for treated vs. untreated rabbits were 6.26 and 8.13 (p < 0.0001), 6.00 and 8.38 (p < 0.001), and 7.25 and 8.79 (p 0.04) for PAO1, 11B and MutGR1, respectively, with an overall mortality rate of 0% vs. 8.9% (p < 0.01). MexXY-overexpressing bacteria were recovered from three (21%) treated rabbits. The C(max)/MIC ratio was the parameter that was best associated with tobramycin efficacy. The bacteria overexpressing MexXY, recovered from lung, occurred with a C(max)/MIC window of 19-26. It was concluded that the experimental PAP model highlights poor tobramycin bacteriological efficacy in vivo, contrasting with survival gain, and that the contribution of the MexXY system to this low level of tobramycin efficacy is modest. Finally, this model appears to be suitable for the investigation of new anti-pseudomonal therapeutic strategies.

Objective Ventilatory strategies combining low tidal volume (VT) with positive end-expiratory pressure (PEEP) are considered to be lung protective. The influence of the PEEP level was investigated on bacteriology and histology in a model of ventilator-associated pneumonia. Subjects Nineteen New Zealand rabbits. Interventions The animals were mechanically ventilated with a positive inspiratory pressure of 15 cmH2O and received either a zero end-expiratory pressure (ZEEP, n=6), a 5 cmH2O PEEP (n=5) or a 10 cmH2O PEEP (n=4). An inoculum of Enterobacter aerogenes was then instilled intrabronchially. The non-ventilated pneumonia group (n=4) was composed of spontaneously breathing animals which received the same inoculum. Pneumonia was assessed 24 h later. Main results The lung bacterial burden was higher in mechanically ventilated animals compared with spontaneously breathing animals. All animals from the latter group had negative spleen cultures. The spleen bacterial concentration was found to be lower in the 5 cmH2O PEEP group when compared to the ZEEP and 10 cmH2O PEEP groups (3.1±1.5 vs 4.9±1.1 and 5.0±1.3 log10 cfu/g, respectively; p

For some pneumococci the fluoroquinolone MICs are low but the mutant prevention concentrations (MPCs) are high; this difference defines in vitro the mutant selection window (MSW). We investigated in vivo the bacterial reduction and the occurrence of resistant mutants with moxifloxacin (MFX; 400 mg once daily) or levofloxacin (LVX; 500 mg twice daily) in treatments similar to those in humans with experimental pneumonia due to pneumococci (expPP) exhibiting various MICs and MPCs. The MIC/MPC for MFX and LVX and genotypes were as follows: strain 16089, 0.125/0.125 and 0.5/0.5 (wild type); strain MS1A, 0.25/0.25 and 1/2 (efflux); strain MS2A, 0.25/4 and 1.75/28 ( parC79 ); strain MR3B4, 0.25/4 and 2/32 ( parC79 ); strain M16, 0.5/2 and 8/32 ( parC83 ); strain Gyr-1207, 1.5/3 and 8/16 ( gyrA ); and strain MQ3A, 4/4 and 16/64 ( parC and gyrA ). Both drugs were efficient with wild type-expPP, but only MFX was efficient with efflux-expPP. No bacterial reduction was observed for parC -expPPs due to mutants observed in 18 to 100% of animals, depending on the strain and the drug tested. These mutants showed unbound area under the concentration-time curve and MICs of from 50 to 164 for MFX. The in vivo pharmacodynamic boundaries of the MSW were different for MFX and LVX. We conclude that, after LVX or MFX treatment, mutants occur in vivo if there is a preexisting parC mutation, since the drug concentrations fall below the MPCs of these strains. Since the MPC determination cannot be routinely determined, these phenotypes or genotypes should be detected by simple tests to guide the therapeutic options.

Ceftaroline, the active metabolite of the prodrug ceftaroline fosamil, is a cephalosporin with broad-spectrum in vitro activity against Gram-positive organisms, including methicillin-resistant Staphylococcus aureus (MRSA) and multidrug-resistant Streptococcus pneumoniae (MDRSP), and common Gram-negative pathogens. This study investigated the in vivo activity of ceftaroline fosamil compared with clindamycin, linezolid, and vancomycin in a severe pneumonia model due to MRSA-producing Panton-Valentine leukocidin (PVL). A USA300 PVL-positive clone was used to induce pneumonia in rabbits. Infected rabbits were randomly assigned to no treatment or simulated human-equivalent dosing with ceftaroline fosamil, clindamycin, linezolid, or vancomycin. Residual bacterial concentrations in the lungs and spleen were assessed after 48 h of treatment. PVL expression was measured using a specific enzyme-linked immunosorbent assay (ELISA). Ceftaroline, clindamycin, and linezolid considerably reduced mortality rates compared with the control, whereas vancomycin did not. Pulmonary and splenic bacterial titers and PVL concentrations were greatly reduced by ceftaroline, clindamycin, and linezolid. Ceftaroline, clindamycin, and linezolid were associated with reduced pulmonary tissue damage based on significantly lower macroscopic scores. Ceftaroline fosamil, clindamycin, and, to a lesser extent, linezolid were efficient in reducing bacterial titers in both the lungs and spleen and decreasing macroscopic scores and PVL production compared with the control.

In multivariate analysis, a leukocyte score that adds 1 point each for neutropenia, lymphopenia and monocytopenia was associated with 30-day mortality in 192 patients with bacteremic pneumococcal pneumonia. By reflecting immunoparalysis, this score could improve clinical outcome predictions in BPP., Background Bacteremic pneumococcal pneumonia (BPP) is associated with high and early mortality. A simple procedure to predict mortality is crucial. Methods All adult patients with BPP admitted from 2005 through 2013 to the University Hospital of Dijon, France, were enrolled to study 30-day mortality and associated factors, particularly leukocyte counts. A simple leukocyte score was created by adding 1 point each for neutropenia (

ObjectivesCommunity-acquired Staphylococcus aureus necrotizing pneumonia is a life-threatening disease. Panton Valentine Leukocidin (PVL) has been associated with necrotizing pneumonia. PVL triggers inflammasome activation in human macrophages leading to IL-1β release. IL-1β activates lung epithelial cells to release IL-8. This study aimed to assess the relevance of this inflammatory cascade in vivo and to test the potential of an IL-1 receptor antagonist (IL-1Ra/Kineret) to decrease inflammation-mediated lung injury.MethodsWe used the sequential instillation of Heat-killed S. aureus and PVL or S. aureus infection to trigger necrotizing pneumonia in rabbits. In these models, we investigated inflammation in the presence or absence of IL-1Ra/Kineret.ResultsWe demonstrated that the presence of PVL was associated with IL-1β and IL-8 release in the lung. During PVL-mediated sterile pneumonia, Kineret/IL-1Ra reduced IL-8 production indicating the relevance of the PVL/IL-1/IL-8 cascade in vivo and the potential of Kineret/IL-1Ra to reduce lung inflammation. However, Kineret/IL-1Ra was ineffective in blocking IL-8 production during infection with S. aureus. Furthermore, treatment with Kineret increased the bacterial burden in the lung.ConclusionsOur data demonstrate PVL-dependent inflammasome activation during S.aureus pneumonia, indicate that IL-1 signaling controls bacterial burden in the lung and suggest that therapy aimed at targeting this pathway might be deleterious during pneumonia.

Summary Concerns have recently emerged about the quality of generic vancomycin products. Our aim is to analyze serum vancomycin concentrations measured 48 hours after the start of an empirical treatment regimen in patients with acute myeloid leukemia (AML) who received one of the two generic vancomycin products available in France. Patients and methods Seventy-nine AML patients treated with vancomycin during two study periods were included in the study. Our vancomycin dosing regimen was based on the patients’ total body weight adjusted for renal clearance. Results A total of 93 serum vancomycin concentrations were collected: 31 in period 1 and 62 in period 2. In bivariate analysis, the mean serum vancomycin concentrations were not significantly different (19.9 ± 11.2 mg/L in period 1 vs 18.9 ± 6.0 mg/L in period 2, P = 0.64). In the final generalized estimating equations model, serum vancomycin concentrations correlated statistically with a positive coefficient for age ( P P = 0.001) and hemoglobin level ( P = 0.021). Conclusion Serum vancomycin concentrations measured 48 hours after the start of an empirical treatment were not influenced by the nature of the generic product but correlated with age, sex and hemoglobin level in AML patients.

Introduction Mechanical ventilation (MV) could prime the lung toward an inflammatory response if exposed to another insult such as bacterial invasion. The underlying mechanisms are not so far clear. Toll-like receptors (TLRs) allow the host to recognize selectively bacterial pathogens and in turn to trigger an immune response. We therefore hypothesized that MV modulates TLR2 expression and in turn modifies responsiveness to agonists such as bacterial lipopeptide (BLP). Method Both in vitro and in vivo experiments were conducted. First, TLR2 expression and protein were measured in the A549 pulmonary epithelial cell line submitted to 8-hour cyclic stretch (20% elongation; 20/minute rate). After a 24-hour period of cyclic stretch, the inflammatory response of the A549 cells to the synthetic BLP, Pam3CSK4, was tested after 8 hours of exposure. In a second set of experiments, healthy anesthetized and paralyzed rabbits were submitted to 8-hour MV (tidal volume = 12 ml/kg, zero end-expiratory pressure; FIO2 = 50%; respiratory rate = 20/minute) before being sacrificed for TLR2 lung expression assessment. The lung inflammatory response to BLP was then tested in animals submitted to 24-hour MV before being sacrificed 8 hours after the tracheal instillation of Pam3CSK4. Results Cyclic stretch of human pulmonary epithelial cell lines increased both TLR2 mRNA and protein expression. Cells submitted to cyclic stretch also increased IL-6 and IL-8 secretion in response to Pam3CSK4, a classical TLR2 ligand. A mild-stretch MV protocol induced a 60-fold increase of TLR2 mRNA expression in lung tissue when compared with spontaneously breathing controls. Moreover, the combination of MV and airway exposure to Pam3CSK4 acted synergistically in causing lung inflammation and injury. Conclusions Mild-stretch MV increases lung expression of TLR2 and sensitizes the lung to bacterial TLR2 ligands. This may account for the propensity of mechanically ventilated patients to develop acute lung injury in the context of airway bacterial colonization/infection.

We investigated the efficacy of 2 formulations of Augmentin on experimental pneumonia due to Haemophilus influenzae (HI) in rabbits. Two strains were used (H128 and 401285) with amoxicillin/clavulanic acid MICs of 1/0.5 mg/l and 4/2 mg/l. Pneumonia was induced in immunocompetent rabbits by inoculation of 10 log(10) CFU HI. The treatments were infused by using computer controlled pumps in order to mimic the human pharmacokinetic (PK) profile of either conventional Augmentin treatment (875/125 mg twice daily) or the sustained release formulation (SR: 2000/125 mg twice daily). After 2 d of treatment, the bacterial concentrations in the lungs were similar for both strains and both treatments: isolate H128, conventional Augmentin reduced bacterial numbers to 3.8+/-2.1 log(10) CFU/g and Augmentin SR to 3.1+/-2.4 log(10) CFU/g; isolate 401285, conventional Augmentin to 3.5+/-2. Thus, both treatments demonstrated similar efficacy against H. influenzae pneumonia in this model, even when induced by a strain with an amoxicillin/clavulanic acid MIC of 4/2 mg/l. These results support current breakpoints for conventional Augmentin against H. influenzae and suggest that Augmentin SR is at least as effective against these isolates.

Objective: Streptococcus pneumoniae is a leading cause of community-acquired pneumonia and is responsible for early-onset ventilator-associated pneumonia as well. In intensive care units, community-acquired pneumonia is still associated with a mortality rate of up to 30%, especially when mechanical ventilation is required. Our objective was to study to what extent MV could influence the efficacy of moxifloxacin in a rabbit model of pneumonia. Design: Prospective experimental study. Setting: University hospital laboratory. Subjects: Male New Zealand White rabbits (n = 75). Interventions: S. pneumoniae (16089 strain; minimal inhibitory concentration for moxifloxacin = 0.125 mg/L) was instilled intrabronchially. Four hours later, a human-like moxifloxacin treatment was initiated in spontaneously breathing (SB) and mechanically ventilated (MV) animals. Untreated rabbits were used as controls. Survivors were killed 48 hrs later. Pneumonia was assessed and moxifloxacin pharmacokinetics were analyzed. Measurements and Main Results: Moxifloxacin treatment was associated with an improvement in survival in the SB animals (13 of 13 [100%] vs. eight of 37 [21.6%] controls). The survival rate was less influenced by treatment in MV rabbits (seven of 15 [46.1%] vs. one of eight [12.5%] controls). The lung bacterial burden was greater in MV compared with SB rabbits (5.1 ± 2.4 vs. 1.6 ± 1.4 log 10 colony-forming units/g, respectively). Nearly all the untreated animals presented bacteremia as reflected by a positive spleen culture. No bacteremia was found in SB animals treated with moxifloxacin. In contrast, three of 13 (23.1%) moxifloxacin-treated and MV animals had positive spleen cultures. The apparent volume of distribution of moxifloxacin was lower in MV compared with SB rabbits. Conclusions: In our model of moxifloxacin-treated S. pneumoniae pneumonia, mechanical ventilation was associated with a higher mortality rate and seemed to promote bacterial growth as well as systemic spread of the infection. In addition, the volume of distribution of moxifloxacin was reduced in the presence of mechanical ventilation. Although the roles of factors such as anesthesia, paralysis, and endotracheal tube insertion could not be established, these results suggest that mechanical ventilation may impair host lung defense, rendering antibiotic therapy less effective.

To investigate the impact of low levels of fluoroquinolone resistance on the emergence of resistant mutants, we examined the mutant selection window (MSW) hypothesis in experimental pneumonia in rabbits infected with pneumococci with various susceptibility levels to fluoroquinolones and treated with gatifloxacin using a human-like regimen (equivalent to 400 mg once daily). The MSW corresponds to the range of concentrations between the minimal inhibitory concentration (MIC) and the mutant prevention concentration (MPC), which is the antibiotic concentration that prevents selection of resistant mutants.Five pneumococcal strains were tested and were defined as follows [MIC of ciprofloxacin (mg/L)/MIC of gatifloxacin (mg/L)/MPC of gatifloxacin (mg/L)/involved quinolone resistance mechanisms]: strain 16089=0.5/0.25/0.25/wild-type; strain MS1A=2/0.5/1/efflux; strain MS2A=8/1/8/parC S79F; strain MR3B4=10/1/8/parC S79T; strain Gyr-1207=6/4/4/gyrA S81F.A 48 h human-like treatment with gatifloxacin was significantly bactericidal on pneumonia induced by strain 16089 (6 log(10) killing) as well as the efflux derivative strain MS1A (5 log(10) killing). However, a small number of parC-gyrA mutants were recovered in 26% of the animals infected with this efflux strain. As expected, no decrease in viable bacteria counts was observed when pneumonia was induced by the gyrA resistant strain. In contrast, because of the enrichment of highly resistant mutants in 100% of the animals, no significant bacterial reduction was observed after treatment of pneumonia induced by the two susceptible parC mutated strains. A classification and regression tree (CART) analysis identified T(MSW) (percentage of the time during which gatifloxacin serum concentrations are inside the MSW) and AUC(MSW) (area under curve between MIC and MPC values) as the best parameters associated with the enrichment of resistant pneumococci.This study shows that the acquisition of a low level of fluoroquinolone resistance (especially a parC mutation and to a lesser extent an efflux mechanism) is associated with a clearly lower potential for preventing resistance development. These data support the concept that resistant mutants are selectively enriched when antibiotic concentrations fall inside the mutant selection window and suggest that in vivo dynamic models have to be used to predict the relative abilities of quinolones to prevent mutant selection.

Background. We measured the effect of low-level fluoroquinolone resistance in Streptococcus pneumoniae on the development of high-level resistance within the context of the mutant selection window. Methods. Rabbits infected with S. pneumoniae were treated with ciprofloxacin or moxifloxacin concentrations that simulated pharmacokinetics in treated humans; bacteria obtained from lungs were examined for fluoroquinolone susceptibility. Results. Ciprofloxacin enriched resistant mutants from a wild-type strain; moxifloxacin did not. However, moxifloxacin enriched resistant mutants from a parC mutant; the drug concentration at the top of the selection window was determined. Conclusions. A parC resistance mutation facilitates the enrichment of high-level resistance, as was predicted by in vitro measurements.

Newer fluoroquinolones, such as levofloxacin, have shown an enhanced in vitro and in vivo activity against penicillin-resistant Streptococcus pneumoniae infections. The frequency of S. pneumoniae with reduced susceptibility to quinolones, although currently low, raises the question of the therapeutic efficacy of levofloxacin on infection due to such strains. We used an animal model of penicillin-resistant pneumococcal pneumonia using six strains with various levels of susceptibility to ciprofloxacin and levofloxacin in rabbits to induce pneumonia, and simulated a human-like treatment of 500 mg twice a day for 48 h. Strains' susceptibility profiles for ciprofloxacin and levofloxaxin were (ciprofloxacin/levofloxacin MIC, mg/L; genotype): 0.5/0.5 (Cip0.5), 2/1 (Cip2), 4/1.75 (Cip4), 8/1.75 (parC mutation) (Cip8), 10/2 (parC mutation) (Cip10), 64/16 (parC and gyrA mutations) (Cip64), respectively. All the strains induced a crude pneumonia in all rabbits. Significant bacterial reductions at the end of treatment in lung and spleen were observed for the four former strains (P0.05) but not for the latter two. An AUC/MIC ratio of at least 32 identified 95% of an at least bacteriostatic effect (P = 0.038) and 76% of a bactericidal effect (P = 0.09). Mutants were detected in treated animals infected with strains harbouring parC mutations (Cip8 and Cip10) and when the AUC/MIC ratio was between 13 and 31. We conclude that levofloxacin is effective against experimental pneumonia due to pneumococci with MIC1.5 mg/L, ineffective on experimental pneumonia due to pneumococci with MICor = 2 mg/L, and could be associated with the appearance of mutants when a parC mutation is pre-existing.

Newer fluoroquinolones, such as levofloxacin, have shown an enhanced in vitro and in vivo activity against penicillin-resistant Streptococcus pneumoniae infections. The frequency of S. pneumoniae with reduced susceptibility to quinolones, although currently low, raises the question of the therapeutic efficacy of levofloxacin on infection due to such strains. We used an animal model of penicillin-resistant pneumococcal pneumonia using six strains with various levels of susceptibility to ciprofloxacin and levofloxacin in rabbits to induce pneumonia, and simulated a human-like treatment of 500 mg twice a day for 48 h. Strains' susceptibility profiles for ciprofloxacin and levofloxaxin were (ciprofloxacin/levofloxacin MIC, mg/L; genotype): 0.5/0.5 (Cip0.5), 2/1 (Cip2), 4/1.75 (Cip4), 8/1.75 (parC mutation) (Cip8), 10/2 (parC mutation) (Cip10), 64/16 (parC and gyrA mutations) (Cip64), respectively. All the strains induced a crude pneumonia in all rabbits. Significant bacterial reductions at the end of treatment in lung and spleen were observed for the four former strains (P or = 2 mg/L, and could be associated with the appearance of mutants when a parC mutation is pre-existing.

Although the mucoid form of Pseudomonas aeruginosa (Pa) is largely responsible for the progression of lung disease in cystic fibrosis (CF), the relationship between factors relating daily-care regimes to mucoidy acquisition are as yet poorly investigated. Fifty-two CF patients registered at the CF centre of Dijon, France, were retrospectively evaluated from the date of Pa colonization either to the first -positive sputum culture for mucoid Pa (n = 26) or to the last culture in which the Pa remained non-mucoid (n = 26). All clinical, pathological and therapeutic events were recorded. The association between the parameters collected and mucoid transition of Pa was assessed in a Cox model with time-dependant covariables. The mean follow-up was 4.7 ± 4.3 years. Three independent parameters were associated with the higher risk of mucoid transition of Pa: persistence of Pa in sputum (OR 7.89; p