In vitro and in vivo evaluation of two combined ß-lactamase inhibitors against carbapenem-resistant Acinetobacter baumannii.

The objective of this study was to evaluate the in vitro and in vivo efficacy of clavulanic acid (C/A) in combination with tazobactam against clinical strains of carbapenem-resistant Acinetobacter baumannii. The MIC of 24 clinical strains of A. baumannii was determined, and a checkerboard assay and time-kill curve analysis were performed in selected strains to determine the synergy between C/A and tazobactam. The efficacy of C/A in monotherapy and in combination with tazobactam was evaluated in vitro in cell culture experiments and in a murine peritoneal sepsis model. The C/A and C/A plus tazobactam MIC50 were 128 and <1 mg/L, respectively. The checkerboard assay showed that tazobactam (4 and 8 mg/L) demonstrated synergy with C/A against A. baumannii Ab40, an OXA-24 producer strain, and Ab293, a lacking OXA ß-lactamase strain. The time-kill curve assay showed both bactericidal and synergistic effects against Ab40 and Ab293, with C/A 1xMIC and tazobactam (4 and 8 mg/L) at 24 h. In the murine peritoneal sepsis model with Ab293 strain, the combination of C/A and tazobactam reduced bacterial loads in tissues and blood by 2 and 4 log10 CFU/g or mL compared with C/A alone. Combining C/A with tazobactam could be considered as a potential alternative strategy to treat A. baumannii in some cases, and future work with more strains is needed to confirm this possibility.

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.

Repositioning rafoxanide to treat Gram-negative bacilli infections.

OBJECTIVES: Repurposing drugs provides a new approach to the fight against MDR Gram-negative bacilli (MDR-GNB). Rafoxanide, a veterinary antihelminthic drug, has shown antibacterial activity in vitro against Gram-positive bacteria. We aimed to analyse the in vitro and in vivo efficacy of rafoxanide in combination with colistin against colistin-susceptible (Col-S) and colistin-resistant (Col-R) GNB. METHODS: A collection of Col-S and Col-R Acinetobacter baumannii, Pseudomonas aeruginosa and Klebsiella pneumoniae were used. Chequerboard and time-kill curve analyses were performed to determine the synergy between rafoxanide and colistin. Changes in membrane structure and permeability were analysed using transmission electron microscopy and fluorescence assays. A murine peritoneal sepsis model using Col-R strains of these pathogens was performed to study the efficacy of rafoxanide (10 mg/kg/24 h, IV), colistimethate sodium (CMS) (20 mg/kg/8 h, intraperitoneally) and rafoxanide (10 mg/kg/24 h, IV) plus CMS (20 mg/kg/8 h, intraperitoneally) for 72 h. RESULTS: Rafoxanide showed MICs ≥256 mg/L for all Col-S and Col-R strains. Chequerboard and time-kill curve analyses showed that rafoxanide (1 mg/L) is more synergistic with colistin against Col-R than Col-S strains. Col-R, but not Col-S, strains treated with rafoxanide demonstrated higher membrane permeabilization. Transmission electron microscopy visualization confirmed that Col-R strains suffer morphological changes. In the murine peritoneal sepsis model with Col-R strains, rafoxanide plus CMS, compared with CMS alone, increased mouse survival to 53.8% and 73.3%, and reduced bacterial loads in tissues and blood between 2.34 and 4.99 log10 cfu/g or mL, respectively. CONCLUSIONS: Rafoxanide repurposing, as monotherapy and in combination with CMS, may address the urgent need for new treatments for infections caused by MDR-GNB.

Extended-spectrum resistance to ß-lactams/ß-lactamase inhibitors (ESRI) evolved from low-level resistant Escherichia coli.

OBJECTIVES: Escherichia coli is characterized by three resistance patterns to ß-lactams/ß-lactamase inhibitors (BLs/BLIs): (i) resistance to ampicillin/sulbactam and susceptibility to amoxicillin/clavulanic acid and piperacillin/tazobactam (RSS); (ii) resistance to ampicillin/sulbactam and amoxicillin/clavulanic acid, and susceptibility to piperacillin/tazobactam (RRS); and (iii) resistance to ampicillin/sulbactam, amoxicillin/clavulanic acid and piperacillin/tazobactam (RRR). These resistance patterns are acquired consecutively, indicating a potential risk of developing resistance to piperacillin/tazobactam, but the precise mechanism of this process is not completely understood. METHODS: Clinical isolates incrementally pressured by piperacillin/tazobactam selection in vitro and in vivo were used. We determined the MIC of piperacillin/tazobactam in the presence and absence of piperacillin/tazobactam pressure. We deciphered the role of the blaTEM genes in the new concept of extended-spectrum resistance to BLs/BLIs (ESRI) using genomic analysis. The activity of ß-lactamase was quantified in these isolates. RESULTS: We show that piperacillin/tazobactam resistance is induced in E. coli carrying blaTEM genes. This resistance is due to the increase in copy numbers and transcription levels of the blaTEM gene, thus increasing ß-lactamase activity and consequently increasing piperacillin/tazobactam MICs. Genome sequencing of two blaTEM-carrying representative isolates showed that piperacillin/tazobactam treatment produced two types of duplications of blaTEM (8 and 60 copies, respectively). In the clinical setting, piperacillin/tazobactam treatment of patients infected by E. coli carrying blaTEM is associated with a risk of therapeutic failure. CONCLUSIONS: This study describes for the first time the ESRI in E. coli. This new concept is very important in the understanding of the mechanism involved in the acquisition of resistance to BLs/BLIs.

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.

Effect of Hypoxia on the Pathogenesis of Acinetobacter baumannii and Pseudomonas aeruginosa In Vitro and in Murine Experimental Models of Infection.

Hypoxia modulates bacterial virulence and the inflammation response through hypoxia-inducible factor 1α (HIF-1α). Here we study the influence of hypoxia on Acinetobacter baumannii and Pseudomonas aeruginosa infections. In vitro, hypoxia increases the bactericidal activities of epithelial cells against A. baumannii and P. aeruginosa, reducing extracellular bacterial concentrations to 50.5% ± 7.5% and 90.8% ± 13.9%, respectively, at 2 h postinfection. The same phenomenon occurs in macrophages (67.6% ± 18.2% for A. baumannii at 2 h and 50.3% ± 10.9% for P. aeruginosa at 24 h). Hypoxia decreases the adherence of A. baumannii to epithelial cells (42.87% ± 8.16% at 2 h) and macrophages (52.0% ± 18.7% at 24 h), as well as that of P. aeruginosa (24.9% ± 4.5% in epithelial cells and 65.7% ± 5.5% in macrophages at 2 h). Moreover, hypoxia decreases the invasion of epithelial cells (48.6% ± 3.8%) and macrophages (8.7% ± 6.9%) by A. baumannii at 24 h postinfection and by P. aeruginosa at 2 h postinfection (75.0% ± 16.3% and 63.4% ± 5.4%, respectively). In vivo, hypoxia diminishes bacterial loads in fluids and tissues in animal models of infection by both pathogens. In contrast, mouse survival time was shorter under hypoxia (23.92 versus 36.42 h) with A. baumannii infection. No differences in the production of cytokines or HIF-1α were found between hypoxia and normoxia in vitro or in vivo We conclude that hypoxia increases the bactericidal activities of host cells against both pathogens and reduces the interaction of pathogens with host cells. Moreover, hypoxia accelerates the rate at which animals die despite the lower bacterial concentrations in vivo.

Synergistic activity of an OmpA inhibitor and colistin against colistin-resistant Acinetobacter baumannii: mechanistic analysis and in vivo efficacy.

Objectives: Preventing bacterial contact with host cells can provide an additional approach to tackling MDR Acinetobacter baumannii. Recently, we identified AOA-2 as a potential blocker of A. baumannii outer membrane protein A without presenting bactericidal activity. Here, we aimed to study whether AOA-2 can increase the activity of colistin against colistin-resistant A. baumannii in vitro and in vivo. Methods: Reference and clinical A. baumannii strains susceptible and resistant to colistin (CST-S and CST-R) were used. Microdilution and time-kill curve assays were performed to determine the synergy between AOA-2 and colistin. SDS-PAGE assays with CST-S and CST-R outer membrane proteins and MALDI-TOF-TOF (MS-MS/MS) analysis were performed to determine the AOA-2 and colistin synergy mechanism. In a murine peritoneal sepsis model, the therapeutic efficacy of AOA-2 (10 mg/kg/24 h) in combination with a sub-optimal dose of colistin (10 mg/kg/24 h) against CST-R was evaluated by determining the bacterial load in tissues and blood, and mouse survival. Results: We showed that AOA-2 increased the in vitro colistin susceptibility of reference and clinical CST-S and CST-R strains. This combination also enhanced their killing activity after 24 h of drug exposure. This synergy is mediated by the overexpression of Omp25. In vivo, the combination of AOA-2 with colistin significantly reduced the bacterial load in tissues and blood, and increased mouse survival, compared with colistin monotherapy. Conclusions: We identified a novel class of antimicrobial agents that has proven to be effective in combination with colistin in an experimental model of severe infection by CST-R A. baumannii.

Overproduction of Outer Membrane Protein A by Acinetobacter baumannii as a Risk Factor for Nosocomial Pneumonia, Bacteremia, and Mortality Rate Increase.

Background: Outer membrane protein A (OmpA) is a porin involved in Acinetobacter baumannii pathogenesis. However, OmpA clinical implication in hospital-acquired infections remains unknown. We aimed to determine whether OmpA overproduction was a risk factor associated with pneumonia, bacteremia, and mortality. Methods: We analyzed demographic, microbiological, and clinical data from 100 patients included in a unicenter cohort and 246 included in a unicenter cohort and a multicenter cohort. Representative isolates were classified into 2 groups: (1) isolates from patients colonized by A. baumannii (16 from the unicenter and 20 from the multicenter cohort) and (2) isolates from bacteremic or nonbacteremic patients with pneumonia (PP) caused by A. baumannii (13 from the unicenter and 23 from the multicenter cohort) Expression of ompA was determined with quantitative reverse-transcription polymerase chain reaction. Results: Isolates from PP overexpressed more ompA than those from colonized patients from the unicenter (ratio, 1.76 vs 0.36; P < .001) and the multicenter (1.36 vs 0.91; P = .03) cohorts. Among isolates from PP, those from bacteremic patients overexpressed nonsignificantly more ompA than those from nonbacteremic patients in the unicenter (ratio, 2.37 vs 1.43; P = .06) and the multicenter (2.03 vs 0.91; P = .14) cohorts. Multivariate analysis in both cohorts together showed ompA overexpression as independent risk factor for pneumonia (P < .001), bacteremia (P = .005), and death (P = .049). Conclusions: These data suggest that ompA overexpression is an associated factor for pneumonia, bacteremia, and death due to A. baumannii.

Therapeutic efficacy of lysophosphatidylcholine in severe infections caused by Acinetobacter baumannii.

Due to the significant increase in antimicrobial resistance of Acinetobacter baumannii, immune system stimulation to block infection progression may be a therapeutic adjuvant to antimicrobial treatment. Lysophosphatidylcholine (LPC), a major component of phospholipids in eukaryotic cells, is involved in immune cell recruitment and modulation. The aim of this study was to show if LPC could be useful for treating infections caused by A. baumannii. A. baumannii ATCC 17978 was used in this study. Levels of serum LPC and levels of the inflammatory cytokines tumor necrosis factor alpha (TNF-α), interleukin-6 (IL-6), IL-1ß, and IL-10 were determined by spectrophotometric assay and enzyme-linked immunosorbent assay (ELISA), respectively, using a murine peritoneal sepsis model in which mice were inoculated with 5.3 log CFU/ml of A. baumannii. The therapeutic efficacy of LPC against A. baumannii in murine peritoneal sepsis and pneumonia models was assessed for 48 h after bacterial infection. At early time points in the murine model of peritoneal sepsis caused by A. baumannii, LPC was depleted and was associated with an increase of inflammatory cytokine release. Preemptive therapy with LPC in murine peritoneal sepsis and pneumonia models markedly enhanced spleen and lung bacterial clearance and reduced the numbers of positive blood cultures and the mouse mortality rates. Moreover, treatment with LPC reduced proinflammatory cytokine production. These data demonstrate that LPC is efficacious as a preemptive treatment in experimental models of peritoneal sepsis and pneumonia caused by A. baumannii.