IgM-enriched immunoglobulin improves colistin efficacy in a pneumonia model by Pseudomonas aeruginosa.

We evaluated the efficacy of ceftazidime or colistin in combination with polyclonal IgM-enriched immunoglobulin (IgM-IG), in an experimental pneumonia model (C57BL/6J male mice) using two multidrug-resistant Pseudomonas aeruginosa strains, both ceftazidime-susceptible and one colistin-resistant. Pharmacodynamically optimised antimicrobials were administered for 72 h, and intravenous IgM-IG was given as a single dose. Bacterial tissues count and the mortality were analysed. Ceftazidime was more effective than colistin for both strains. In mice infected with the colistin-susceptible strain, ceftazidime reduced the bacterial concentration in the lungs and blood (-2.42 and -3.87 log10 CFU/ml) compared with colistin (-0.55 and -1.23 log10 CFU/ml, respectively) and with the controls. Colistin plus IgM-IG reduced the bacterial lung concentrations of both colistin-susceptible and resistant strains (-2.91 and -1.73 log10 CFU/g, respectively) and the bacteraemia rate of the colistin-resistant strain (-44%). These results suggest that IgM-IG might be useful as an adjuvant to colistin in the treatment of pneumonia caused by multidrug-resistant P. aeruginosa.

Efficacy of Fosfomycin and Its Combination With Aminoglycosides in an Experimental Sepsis Model by Carbapenemase-Producing Klebsiella pneumoniae Clinical Strains.

Carbapenemase-producing Klebsiella pneumoniae infections are an increasing global threat with scarce and uncertain treatment options. In this context, combination therapies are often used for these infections. The bactericidal and synergistic activity of fosfomycin plus amikacin and gentamicin was studied trough time-kill assays against four clonally unrelated clinical isolates of carbapenemase-producing K. pneumoniae, VIM-1, VIM-1 plus DHA-1, OXA-48 plus CTXM-15, and KPC-3, respectively. The efficacy of antimicrobials that showed synergistic activity in vitro against all the carbapenemase-producing K. pneumoniae were tested in monotherapy and in combination, in a murine peritoneal sepsis model. In vitro, fosfomycin plus amikacin showed synergistic and bactericidal effect against strains producing VIM-1, VIM-1 plus DHA-1, and OXA-48 plus CTX-M-15. Fosfomycin plus gentamicin had in vitro synergistic activity against the strain producing KPC-3. In vivo, fosfomycin and amikacin and its combination reduced the spleen bacterial concentration compared with controls groups in animals infected by K. pneumoniae producing VIM-1 and OXA-48 plus CTX-M-15. Moreover, amikacin alone and its combination with fosfomycin reduced the bacteremia rate against the VIM-1 producer strain. Contrary to the in vitro results, no in vivo efficacy was found with fosfomycin plus amikacin against the VIM-1 plus DHA-1 producer strain. Finally, fosfomycin plus gentamicin reduced the bacterial concentration in spleen against the KPC-3 producer strain. In conclusion, our results suggest that fosfomycin plus aminoglycosides has a dissimilar efficacy in the treatment of this severe experimental infection, when caused by different carbapenemase-producing K. pneumoniae strains. Fosfomycin plus amikacin or plus gentamycin may be useful to treat infections by OXA-48 plus CTX-M-15 or KPC-3 producer strains, respectively.

In Vitro Activity of Pentamidine Alone and in Combination with Antibiotics against Multidrug-Resistant Clinical Pseudomonas aeruginosa Strains.

Multidrug-resistant (MDR) Pseudomonas aeruginosa is a public health problem causing both community and hospital-acquired infections, and thus the development of new therapies for these infections is critical. The objective of this study was to analyze in vitro the activity of pentamidine as adjuvant in combinations to antibiotics against seven clinical P. aeruginosa strains. The Minimum Inhibitory Concentration (MIC) was determined following standard protocols, and the results were interpreted according to the European Committee on Antimicrobial Susceptibility Testing (EUCAST) breakpoints; however, the gentamicin activity was interpreted according to the Clinical and Laboratory Standards Institute (CLSI) recommendations. The bactericidal in vitro activity was studied at 1×MIC concentrations by time-kill curves, and also performed in three selected strains at 1/2×MIC of pentamidine. All studies were performed in triplicate. The pentamidine MIC range was 400-1600 µg/mL. Four of the strains were MDR, and the other three were resistant to two antibiotic families. The combinations of pentamidine at 1×MIC showed synergistic activity against all the tested strains, except for pentamidine plus colistin. Pentamidine plus imipenem and meropenem were the combinations that showed synergistic activity against the most strains. At 1/2×MIC, pentamidine plus antibiotics were synergistic with all three analyzed strains. In summary, pentamidine in combination with antibiotics showed in vitro synergy against multidrug-resistant P. aeruginosa clinical strains, which suggests its possible use as adjuvant to antibiotics for the therapy of infections from MDR P. aeruginosa.

Virulence role of the outer membrane protein CarO in carbapenem-resistant Acinetobacter baumannii.

Novel approaches to treat carbapenem-resistant Acinetobacter baumannii (CRAB) infections are urgently needed and anti-virulence drugs represent promising alternatives, but our knowledge on potential targets is scarce. We searched for potential A. baumannii virulence factors by whole-genome sequencing-based comparisons of CRAB clinical isolates causing bloodstream infections secondary to ventilator-associated pneumonia from demographics and clinically homogeneous patients, who received optimal treatment but with different clinical outcomes. Thus, the carO gene was interrupted in CRAB isolates from surviving patients, while it was intact in isolates from non-surviving patients, and proteomic/immunoblot techniques corroborated it. When the virulence role of A. baumannii CarO was analyzed in model systems, isogenic ΔcarO mutants and a CRAB clinical isolate with truncated CarO, showed lower ability to adhere and invade A549 cells and in vivo virulence. This unnoticed virulence role for CarO postulate this A. baumannii outer membrane protein as a potential target for new therapies against CRAB infections.

Assessing the impact on intestinal microbiome and clinical outcomes of antibiotherapy optimisation strategies in haematopoietic stem cell transplant recipients: study protocol for the prospective multicentre OptimBioma study.

INTRODUCTION: Haematopoietic stem cell transplantation (HSCT) is a life-saving treatment for a number of haematological diseases. Graft versus host disease (GVHD) is its main complication and hampers survival. There is strong evidence that intestinal microbiota diversity of the recipient may increase the risk of GVHD worsening survival. Antibiotic regimens used during the early phase of the transplant may influence clinical outcomes by reducing intestinal microbiota diversity. Present guidelines of European Conference on Infections in Leukaemia exhort to optimising antibiotic use in haematological patients including HSCT recipients. The present study aims to investigate if, in HSCT recipients, the optimisation of antibacterial use may preserve intestinal microbiota composition reducing the incidence and severity of acute GVHD and improving relevant clinical outcomes. METHODS AND ANALYSIS: This is a prospective longitudinal observational study of two cohorts of HSCT recipients: (1) the intervention cohort includes patients treated in centres in which a predefined strategy of antibiotherapy optimisation is implemented, with the objective of optimising and reducing antibiotic administration according to clinical criteria and (2) the control cohort includes patients treated in centres in which a classic permissive strategy of antibiotic prophylaxis and treatment is used. Adult patient receiving a first HSCT as a treatment for any haematological condition are included. Clinical variables are prospectively recorded and up to five faecal samples are collected for microbiota characterisation at prestablished peritransplant time points. Patients are followed since the preconditioning phase throughout 1-year post-transplant and four follow-up visits are scheduled. Faecal microbiota composition and diversity will be compared between both cohorts along with acute GVHD incidence and severity, severe infections rate, mortality and overall and disease-free survival. ETHICS AND DISSEMINATION: The study was approved between 2017 and 2018 by the Ethical Committees of participant centres. Study results will be disseminated through peer-reviewed journals and national and international scientific conferences. TRIAL REGISTRATION NUMBER: NCT03727113.

Role of PstS in the Pathogenesis of Acinetobacter baumannii Under Microaerobiosis and Normoxia.

Acinetobacter baumannii is a successful pathogen responsible for infections with high mortality rate. During the course of infection it can be found in microaerobic environments, which influences virulence factor expression. From a previous transcriptomic analysis of A. baumannii ATCC 17978 under microaerobiosis, we know the gene pstS is overexpressed under microaerobiosis. Here, we studied its role in A. baumannii virulence. pstS loss significantly decreased bacterial adherence and invasion into A549 cells and increased A549 cell viability. pstS loss also reduced motility and biofilm-forming ability of A. baumannii. In a peritoneal sepsis murine model, the minimum lethal dose required by A. baumannii ATCC 17978 ΔpstS was lower compared to the wild type (4.3 vs 3.2 log colony forming units/mL, respectively), and the bacterial burden in tissues and fluids was lower. Thus, the loss of the phosphate sensor PstS produced a decrease in A. baumannii pathogenesis, supporting its role as a virulence factor.

Pangenome of Acinetobacter baumannii uncovers two groups of genomes, one of them with genes involved in CRISPR/Cas defence systems associated with the absence of plasmids and exclusive genes for biofilm formation.

Acinetobacter baumannii is an opportunistic bacterium that causes hospital-acquired infections with a high mortality and morbidity, since there are strains resistant to virtually any kind of antibiotic. The chase to find novel strategies to fight against this microbe can be favoured by knowledge of the complete catalogue of genes of the species, and their relationship with the specific characteristics of different isolates. In this work, we performed a genomics analysis of almost 2500 strains. Two different groups of genomes were found based on the number of shared genes. One of these groups rarely has plasmids, and bears clustered regularly interspaced short palindromic repeat (CRISPR) sequences, in addition to CRISPR-associated genes (cas genes) or restriction-modification system genes. This fact strongly supports the lack of plasmids. Furthermore, the scarce plasmids in this group also bear CRISPR sequences, and specifically contain genes involved in prokaryotic toxin-antitoxin systems that could either act as the still little known CRISPR type IV system or be the precursors of other novel CRISPR/Cas systems. In addition, a limited set of strains present a new cas9-like gene, which may complement the other cas genes in inhibiting the entrance of new plasmids into the bacteria. Finally, this group has exclusive genes involved in biofilm formation, which would connect CRISPR systems to the biogenesis of these bacterial resistance structures.

Prevalence and clinical impact of Streptococcus pneumoniae nasopharyngeal carriage in solid organ transplant recipients.

BACKGROUND: S. pneumoniae is the leading cause of community-acquired pneumonia in the solid organ transplant recipient (SOTR); nevertheless, the prevalence of colonization and of the colonizing/infecting serotypes has not been studied in this population. In this context, the aim of the present study was to describe the rate, characteristics, and clinical impact of S. pneumoniae nasopharyngeal carriage. METHODS: A prospective observational cohort of Solid Organ Transplant recipients (SOTR) was held at the University Hospital Virgen del Rocío, Seville, Spain with the aim to evaluate the S. pneumoniae colonization and the serotype prevalence in SOTR. Two different pharyngeal swabs samples from 500 patients were included in two different seasonal periods winter and spring/summer. Optochin and bile solubility tests were performed for the isolation of thew strains. Antimicrobial susceptibility studies (MICs, mg/l) of levofloxacin, trimethoprim-sulfamethoxazole, penicillin, amoxicillin, cefotaxime, ceftriaxone, erythromycin, azithromycin and vancomycin for each isolate were determined by E-test strips. Capsular typing was done by sequential multiplex PCR reactions. A multivariate logistic regression analysis of factors potentially associated with pneumococcal nasopharyngeal carriage and disease was performed. RESULTS: Twenty-six (5.6%) and fifteen (3.2%) patients were colonized in winter and spring/summer periods, respectively. Colonized SOT recipients compared to non-colonized patients were more frequently men (79.5% vs. 63.1%, P < 0.05) and cohabitated regularly with children (59% vs. 32.2%, P < 0.001). The most prevalent serotype in both studied periods was 35B. Forty-five percent of total isolates were included in the pneumococcal vaccine PPV23. Trimethoprim-sulfamethoxazole and macrolides were the less active antibiotics. Three patients had non-bacteremic pneumococcal pneumonia, and two of them died. CONCLUSIONS: Pneumococcal colonization in SOTR is low with the most colonizing serotypes not included in the pneumococcal vaccines.

In vitro Activity of Pentamidine Alone and in Combination With Aminoglycosides, Tigecycline, Rifampicin, and Doripenem Against Clinical Strains of Carbapenemase-Producing and/or Colistin-Resistant Enterobacteriaceae.

Enterobacteriaceae cause different types of community- and hospital-acquired infections. Moreover, the spread of multidrug-resistant Enterobacteriaceae is a public health problem and the World Health Organization pointed them among the pathogens in which the search of new antibiotics is critical. The objective of this study was to analyze the in vitro activity of pentamidine alone and in combination with gentamicin, tobramycin, amikacin, tigecycline, rifampicin, or doripenem against eight clinical strains of carbapenemase-producing and/or colistin-resistant Enterobacteriaceae: five carbapenemase-producing Klebsiella pneumoniae, one carbapenemase-producing Escherichia coli, and two colistin-resistant Enterobacter cloacae. MIC and MBC were determined following standard protocols. MIC results were interpreted for all the antibiotics according to the EUCAST breakpoints but for rifampicin in which the French FSM breakpoint was used. Bactericidal and synergistic activity of pentamidine alone and in combination with antibiotics at concentrations of 1xMIC was measured by time-kill curves. For one selected strain, K. pneumoniae OXA-48/CTX-M-15 time-kill curves were performed also at 1/2xMIC of pentamidine. All studies were performed in triplicate. Pentamidine MIC range was 200-800 µg/mL. The 50, 12.5, 62.5, 87.5, and 62.5% of the strains were susceptible to gentamicin, tobramycin, amikacin, tigecycline, and doripenem, respectively. Only the two E. cloacae strains were susceptible to rifampicin. Pentamidine alone at 1xMIC showed bactericidal activity against all strains, except for the E. cloacae 32 strain. The bactericidal activity of pentamidine alone was also observed in combination. The combinations of pentamidine were synergistic against E. cloacae 32 with amikacin and tobramycin at 24 h and with tigecycline at 8 h. Pentamidine plus rifampicin was the combination that showed synergistic activity against more strains (five out of eight). Pentamidine plus doripenem did not show synergy against any strain. At 1/2xMIC, pentamidine was synergistic with all the studied combinations against the K. pneumoniae OXA-48/CTX-M-15 strain. In summary, pentamidine alone and in combination shows in vitro activity against carbapenemase-producing and/or colistin-resistant Enterobacteriaceae. Pentamidine appears to be a promising option to treat infections caused by these pathogens.

Efficacy of Colistin and Its Combination With Rifampin in Vitro and in Experimental Models of Infection Caused by Carbapenemase-Producing Clinical Isolates of Klebsiella pneumoniae.

Despite the relevance of carbapenemase-producing Klebsiella pneumoniae (CP-Kp) infections there are a scarce number of studies to evaluate in vivo the efficacy of combinations therapies. The bactericidal activity of colistin, rifampin, and its combination was studied (time-kill curves) against four clonally unrelated clinical isolates of CP-Kp, producing VIM-1, VIM-1 plus DHA-1(acquired AmpC ß-lactamase), OXA-48 plus CTX-M-15 (extended spectrum ß-lactamase) and KPC-3, respectively, with colistin MICs of 0.5, 64, 0.5, and 32 mg/L, respectively. The efficacies of antimicrobials in monotherapy and in combination were tested in a murine peritoneal sepsis model, against all the CP-Kp. Their efficacies were tested in the pneumonia model against the OXA-48 plus CTX-M-15 producers. The development of colistin-resistance was analyzed for the colistin-susceptible strains in vitro and in vivo. In vitro, colistin plus rifampin was synergistic against all the strains at 24 h. In vivo, compared to the controls, rifampin alone reduced tissue bacterial concentrations against VIM-1 and OXA-48 plus CTX-M-15 strains; CMS plus rifampin reduced tissue bacterial concentrations of these two CP-Kp and of the KPC-3 strain. Rifampin and the combination increased the survival against the KPC-3 strain; in the pneumonia model, the combination also improved the survival. No resistant mutants appeared with the combination. In conclusion, CMS plus rifampin had a low and heterogeneous efficacy in the treatment of severe peritoneal sepsis model due to CP-Kp producing different carbapenemases, increasing survival only against the KPC-3 strain. The combination showed efficacy in the less severe pneumonia model. The combination prevented in vitro and in vivo the development of colistin resistant mutants.

Impaired growth under iron-limiting conditions associated with the acquisition of colistin resistance in Acinetobacter baumannii.

Acquisition of colistin resistance in Acinetobacter baumannii has been associated with reduced bacterial fitness and virulence, although the mechanisms underlying this fitness loss have not been well characterised. In this study, the role played by environmental iron levels on the growth and survival of colistin-resistant strains of A. baumannii was assessed. Growth assays with the colistin-susceptible ATCC 19606 strain and its colistin-resistant derivative RC64 [colistin minimum inhibitory concentration (MIC) of 64 mg/L] demonstrated that the strains grew similarly in rich laboratory medium (Mueller-Hinton broth), whereas RC64 demonstrated impaired growth compared with ATCC 19606 in human serum (>100-fold at 24 h). Compared with RC64, ATCC 19606 grew in the presence of higher concentrations of the iron-specific chelator 2,2'-bipyridine and grew more readily under iron-limiting conditions in solid and liquid media. In addition, iron supplementation of human serum increased the growth of RC64 compared with unsupplemented human serum to a greater extent than ATCC 19606. The ability of 11 colistin-resistant clinical isolates with mutations in the pmrB gene to grow in iron-replete and iron-limiting conditions was assessed, demonstrating that eight of the strains showed reduced growth under iron limitation. Individual mutations in the pmrB gene did not directly correlate with a decreased capacity for growth under iron limitation, suggesting that mutations in pmrB may not directly produce this phenotype. Together these results indicate that acquisition of colistin resistance in A. baumannii can be associated with a decreased ability to grow in low-iron environments.

Draft Genome Sequences of Seven Multidrug-Resistant Acinetobacter baumannii Strains, Isolated from Respiratory Samples in Spain.

The draft genome sequences of seven multidrug-resistantAcinetobacter baumanniiclinical strains belonging to sequence types ST-208 and ST-218 are reported in this study. They were isolated from tracheobronchial aspirate of mechanically ventilated adult patients admitted to the intensive care unit of a Spanish tertiary hospital during 2010 to 2011.

Efficacy of ceftaroline versus vancomycin in an experimental foreign-body and systemic infection model caused by biofilm-producing methicillin-resistant Staphylococcus epidermidis.

In this study, the efficacy of ceftaroline versus vancomycin against biofilm-producing methicillin-resistant Staphylococcus epidermidis (MRSE) in a murine model of foreign-body and systemic infection was compared. Two bacteraemic biofilm-producing MRSE strains were used (SE284 and SE385). The minimum inhibitory concentrations (MICs) for strains SE284 and SE385, were, respectively, 0.25 mg/L and 0.5 mg/L for ceftaroline and 4 mg/L and 2 mg/L for vancomycin. The in vitro bactericidal activities of ceftaroline and vancomycin were evaluated using time-kill curves. A foreign-body and systemic infection model in neutropenic female C57BL/6 mice was used to ascertain in vivo efficacy. Animals were randomly allocated into three groups (n = 15) without treatment (controls) or treated with ceftaroline 50 mg/kg every 8 h or vancomycin 110 mg/kg every 6 h. In vitro, ceftaroline showed concentration-dependent bactericidal activity, whilst vancomycin presented time-dependent activity. In the experimental in vivo model, ceftaroline and vancomycin decreased the liver and catheter bacterial concentrations (P <0.05) and increased survival (P <0.05) for both strains. In conclusion, ceftaroline is as effective as vancomycin in the treatment of experimental foreign-body and systemic infection caused by biofilm-producing MRSE.