NLRP11 is a pattern recognition receptor for bacterial lipopolysaccharide in the cytosol of human macrophages.

Endotoxin-bacterial lipopolysaccharide (LPS)-is a driver of lethal infection sepsis through excessive activation of innate immune responses. When delivered to the cytosol of macrophages, cytosolic LPS (cLPS) induces the assembly of an inflammasome that contains caspases-4/5 in humans or caspase-11 in mice. Whereas activation of all other inflammasomes is triggered by sensing of pathogen products by a specific host cytosolic pattern recognition receptor protein, whether pattern recognition receptors for cLPS exist has remained unclear, because caspase-4, caspase-5, and caspase-11 bind and activate LPS directly in vitro. Here, we show that the primate-specific protein NLRP11 is a pattern recognition receptor for cLPS that is required for efficient activation of the caspase-4 inflammasome in human macrophages. In human macrophages, NLRP11 is required for efficient activation of caspase-4 during infection with intracellular Gram-negative bacteria or upon electroporation of LPS. NLRP11 could bind LPS and separately caspase-4, forming a high-molecular weight complex with caspase-4 in HEK293T cells. NLRP11 is present in humans and other primates but absent in mice, likely explaining why it has been overlooked in screens looking for innate immune signaling molecules, most of which have been carried out in mice. Our results demonstrate that NLRP11 is a component of the caspase-4 inflammasome activation pathway in human macrophages.

iTRAQ-Based Quantitative Proteomic Analysis of Acinetobacter baumannii under Hypoxia and Normoxia Reveals the Role of OmpW as a Virulence Factor.

Acinetobacter baumannii needs to adapt to hypoxia during infection. Understanding its proteome regulation during infection would allow us to determine new targets to develop novel treatments. iTRAQ proteomic analysis of A549 cell infection by the ATCC 17978 strain was performed. A total of 175 proteins were differentially expressed under hypoxia versus normoxia. We selected the hypoxia-downregulated protein OmpW to analyze its role as a virulence factor. The loss of OmpW decreased the adherence and invasion of A. baumannii in these host cells, without affecting its bacterial growth. Moreover, A549 cell viability with ΔOmpW infection was higher than that with the wild-type strain. ΔOmpW presented less biofilm formation. Finally, the minimum lethal dose required by the ΔOmpW mutant was higher than that of the wild-type strain in a murine peritoneal sepsis model, with lower bacterial loads in tissues and fluids. Therefore, OmpW seems to be a virulence factor necessary for A. baumannii pathogenesis. IMPORTANCE Acinetobacter baumannii causes infections that are very difficult to treat due to the high rate of resistance to most and sometimes all of the antimicrobials used in the clinical setting. There is an important need to develop new strategies to combat A. baumannii infections. One alternative could be blocking specific bacterial virulence factors that this pathogen needs to infect cells. Pathogens modulate their protein expression as a function of the environment, and several studies have reported that hypoxia occurs in a wide range of infections. Therefore, it would be interesting to determine the proteome of A. baumannii under hypoxia in order to find new virulence factors, such as the outer membrane protein OmpW, as potential targets for the design of novel therapies.

Analyzing Possible Native Functions of the Quinolone Resistance Gene qnr in Vibrio vulnificus.

The worldwide distribution of qnr genes found on plasmids and their presence on the chromosomes of aquatic bacteria, such as Vibrio vulnificus, one of the suspected sources, suggests an origin before the development of synthetic quinolones. However, their native function remains unknown. Previous work indicated that expression of qnrVv in V. vulnificus was induced by cold shock. To investigate its role further, we constructed single in-frame deletion mutants in qnrVv and cspA (the gene for cold shock protein) and a double mutant in qnrVv and cspA in V. vulnificus ATCC 17562 to evaluate the response to different environmental conditions and stresses and to exposure to various DNA-damaging agents. We found that qnrVv is involved in resistance to ciprofloxacin, levofloxacin, and mitomycin C and in the cold shock response in V. vulnificus Moreover, ΔqnrVv and ΔcspA mutants showed slower growth when they were treated with bile salts at 37°C and then shifted to 15°C (cold shock) without bile salts in the medium, with the effect being stronger in the double mutant. This transition may mimic what happens when V. vulnificus is ingested into the gastrointestinal tract and released in its natural environment. Cold shock and bile salts induced the expression of cspA and DNA gyrase and topoisomerase IV genes. However, no induction was found in the ΔqnrVv mutant, suggesting that the qnrVv gene is involved in the response to DNA damage and nucleic acid secondary structure.

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.

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.

The anthelmintic oxyclozanide restores the activity of colistin against colistin-resistant Gram-negative bacilli.

Due to the significant increase in antimicrobial resistance in Gram-negative bacilli (GNB), development of non-antimicrobial therapeutic alternatives, which can be used together with the few and non-optimal available antimicrobial agents such as colistin, has become an urgent need. In this context, dysregulation of the bacterial cell wall could be a therapeutic adjuvant to the activity of colistin. The aim of this study was to analyse the activity of oxyclozanide, an anthelmintic drug, in combination with colistin against colistin-susceptible (Col-S) and colistin-resistant (Col-R) GNB. Three Col-S reference strains and 13 clinical isolates (1 Col-S, 12 Col-R) of Acinetobacter baumannii, Pseudomonas aeruginosa and Klebsiella pneumoniae were studied. Microdilution assays and time-kill curves were performed to examine the activity of oxyclozanide in combination with colistin. The outer membrane protein (OMP) profile, membrane permeability and cell wall structure of Col-S and Col-R A. baumannii, P. aeruginosa and K. pneumoniae in the presence of oxyclozanide were assessed by SDS-PAGE, fluorescence microscopy and transmission electron microscopy, respectively. Oxyclozanide in combination with colistin increased the activity of colistin against Col-S and Col-R A. baumannii, P. aeruginosa and K. pneumoniae. Time-kill curves showed synergistic activity between oxyclozanide and colistin against these bacterial isolates. Moreover, Col-R A. baumannii, P. aeruginosa and K. pneumoniae in the presence of oxyclozanide presented greater permeability and disruption of their cell wall than Col-S strains, without modification of their OMP profile. These data suggest that combination of oxyclozanide and colistin may be a new alternative for the treatment of Col-R GNB infections.

Synergistic Activity of Niclosamide in Combination With Colistin Against Colistin-Susceptible and Colistin-Resistant Acinetobacter baumannii and Klebsiella pneumoniae.

Colistin is among the few antibiotics effective against multidrug-resistant Acinetobacter baumannii and Klebsiella pneumoniae clinical isolates. However, in the last few years, colistin-resistant A. baumannii and K. pneumoniae strains have emerged. Therefore, combination therapies, between colistin and other old drugs, restoring the activity of colistin are required. The main objective of this study was to analyse the activity of niclosamide, an anthelmintic drug, in combination with colistin against colistin-susceptible (Col-S) and colistin-resistant (Col-R) A. baumannii and K. pneumoniae. The MIC were determined by microdilution assay and the time-kill curves were performed. The zeta potential of Col-S and Col-R of A. baumannii and K. pneumoniae in presence of niclosamide was assessed. Niclosamide in combination with colistin showed improved activity against Col-S and Col-R A. baumannii and K. pneumoniae. Time-killing curves showed synergic activity between niclosamide and colistin against Col-S and Col-R A. baumannii and K. pneumoniae, especially when niclosamide or colistin was added for second time at 4 h of the 24 h killing curve. Col-R A. baumannii and K. pneumoniae in presence of niclosamide exhibited a greater negative charge (-34.95 ± 0.35 mV and -38.85 ± 0.92 mV; P < 0.05) than Col-R A. baumannii and K. pneumoniae in absence of niclosamide (-26.85 ± 3.65 mV and -35.27 ± 0.72 mV). These data suggest that niclosamide might be combined with colistin, being a potential alternative for treatment of Col-R Gram-negative bacilli infections.

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.

Peptidoglycan recycling contributes to intrinsic resistance to fosfomycin in Acinetobacter baumannii.

Background: Acinetobacter baumannii is intrinsically resistant to fosfomycin; however, the mechanisms underlying this resistance are poorly understood. Objectives: To identify and characterize genes that contribute to intrinsic fosfomycin resistance in A. baumannii. Methods: More than 9000 individual transposon mutants of the A. baumannii ATCC 17978 strain (fosfomycin MIC ≥1024 mg/L) were screened to identify mutations conferring increased susceptibility to fosfomycin. In-frame deletion mutants were constructed for the identified genes and their susceptibility to fosfomycin was characterized by MIC determination and growth in the presence of fosfomycin. The effects of these mutations on membrane permeability and peptidoglycan integrity were characterized. Susceptibilities to 21 antibiotics were determined for the mutant strains. Results: Screening of the transposon library identified mutants in the ampD and anmK genes, both encoding enzymes of the peptidoglycan recycling pathway, that demonstrated increased susceptibility to fosfomycin. MIC values for in-frame deletion mutants were ≥42-fold (ampD) and ≥8-fold (anmK) lower than those for the parental strain, and growth of the mutant strains in the presence of 32 mg/L fosfomycin was significantly reduced. Neither mutation resulted in increased cell permeability; however, the ampD mutant demonstrated decreased peptidoglycan integrity. Susceptibility to 21 antibiotics was minimally affected by mutations in ampD and anmK. Conclusions: This study demonstrates that AmpD and AnmK of the peptidoglycan recycling pathway contribute to intrinsic fosfomycin resistance in A. baumannii, indicating that inhibitors of these enzymes could be used in combination with fosfomycin as a novel treatment approach for MDR A. baumannii.