Damien Roux

Parallel bacterial evolution within multiple patients identifies candidate pathogenicity genes

Bacterial pathogens evolve during the infection of their human host, but separating adaptive and neutral mutations remains challenging. Here we identify bacterial genes under adaptive evolution by tracking recurrent patterns of mutations in the same pathogenic strain during the infection of multiple individuals. We conducted a retrospective study of a Burkholderia dolosa outbreak among subjects with cystic fibrosis, sequencing the genomes of 112 isolates collected from 14 individuals over 16 years. We find that 17 bacterial genes acquired nonsynonymous mutations in multiple individuals, which indicates parallel adaptive evolution. Mutations in these genes affect important pathogenic phenotypes, including antibiotic resistance and bacterial membrane composition and implicate oxygen-dependent regulation as paramount in lung infections. Several genes have not previously been implicated in pathogenesis and may represent new therapeutic targets. The identification of parallel molecular evolution as a pathogen spreads among multiple individuals points to the key selection forces it experiences within human hosts.

A carbon monoxide-releasing molecule (CORM-3) exerts bactericidal activity against Pseudomonas aeruginosa and improves survival in an animal model of bacteraemia

The search for new molecules to fight Pseudomonas aeruginosa is of paramount importance. Carbon monoxide (CO) is known to act as an effective inhibitor of the respiratory chain in P. aeruginosa, but the practical use of this gas as an antibacterial molecule is hampered by its toxicity and difficulty to manipulate. Here, we show that a water‐soluble CO releaser (CORM‐3) possesses bactericidal properties against laboratory and antibiotic‐resistant P. aeruginosa. CORM‐3 reduced the bacterial count by 4 logs 180 min after in vitro treatment. CORM‐3‐treated bacteria had a lower O2 consumption than vehicle‐treated bacteria, and the decrease in O2 consumption temporally preceded the bactericidal action of CORM‐3. These results support the hypothesis that the antimicrobial effect of CORM‐3 is mediated by an interaction of CO liberated by the carrier with the bacterial respiratory chain. The antibacterial effect occurred at concentrations of CORM‐3 that are 50fold lower than toxic concentrations for eukaryotic cells. CORM‐3 treatment compared to vehicle treatment decreased bacterial counts in the spleen and increased survival in immunocompetent and immunosuppressed mice following P. aeruginosa bacteremia. Our results suggest that CORMs could form the basis for developing a new therapeutic strategy against P. aeruginosa‐induced infection.—Desmard, M., Davidge, K. S., Bouvet, O., Morin, D., Roux, D., Foresti, R., Ricard, J. D., Denamur, E., Poole, R. K., Montravers, P., Motterlini, R., Boczkowski, J. A carbon monoxide‐releasing molecule (CORM‐3) exerts bactericidal activity against Pseudomonas aeruginosa and improves survival in an animal model of bacteraemia. FASEB J. 23, 1023–1031 (2009)

Antibody to a conserved antigenic target is protective against diverse prokaryotic and eukaryotic pathogens

Significance Poly-N-acetylglucosamine (PNAG) has been identified as a conserved surface polysaccharide produced by major bacterial, fungal, and protozoal parasites, including malarial sporozoites and blood-stage forms, which can all be targeted for vaccination using this single antigen. Surface carbohydrates are among the most successful vaccines against human microbial pathogens but have tremendous variability that complicates vaccine development. The species of bacteria, fungi, and protozoa shown here to produce PNAG lack an identifiable genetic locus for this antigen’s biosynthetic proteins based on known loci, indicative of a possible evolutionary convergent acquisition of PNAG synthesis with potential important significance for microbial biology. Microbial capsular antigens are effective vaccines but are chemically and immunologically diverse, resulting in a major barrier to their use against multiple pathogens. A β-(1→6)–linked poly-N-acetyl-d-glucosamine (PNAG) surface capsule is synthesized by four proteins encoded in genetic loci designated intercellular adhesion in Staphylococcus aureus or polyglucosamine in selected Gram-negative bacterial pathogens. We report that many microbial pathogens lacking an identifiable intercellular adhesion or polyglucosamine locus produce PNAG, including Gram-positive, Gram-negative, and fungal pathogens, as well as protozoa, e.g., Trichomonas vaginalis, Plasmodium berghei, and sporozoites and blood-stage forms of Plasmodium falciparum. Natural antibody to PNAG is common in humans and animals and binds primarily to the highly acetylated glycoform of PNAG but is not protective against infection due to lack of deposition of complement opsonins. Polyclonal animal antibody raised to deacetylated glycoforms of PNAG and a fully human IgG1 monoclonal antibody that both bind to native and deacetylated glycoforms of PNAG mediated complement-dependent opsonic or bactericidal killing and protected mice against local and/or systemic infections by Streptococcus pyogenes, Streptococcus pneumoniae, Listeria monocytogenes, Neisseria meningitidis serogroup B, Candida albicans, and P. berghei ANKA, and against colonic pathology in a model of infectious colitis. PNAG is also a capsular polysaccharide for Neisseria gonorrhoeae and nontypable Hemophilus influenzae, and protects cells from environmental stress. Vaccination targeting PNAG could contribute to immunity against serious and diverse prokaryotic and eukaryotic pathogens, and the conserved production of PNAG suggests that it is a critical factor in microbial biology.

A Comprehensive Analysis of In Vitro and In Vivo Genetic Fitness of Pseudomonas aeruginosa Using High-Throughput Sequencing of Transposon Libraries

High-throughput sequencing of transposon (Tn) libraries created within entire genomes identifies and quantifies the contribution of individual genes and operons to the fitness of organisms in different environments. We used insertion-sequencing (INSeq) to analyze the contribution to fitness of all non-essential genes in the chromosome of Pseudomonas aeruginosa strain PA14 based on a library of ∼300,000 individual Tn insertions. In vitro growth in LB provided a baseline for comparison with the survival of the Tn insertion strains following 6 days of colonization of the murine gastrointestinal tract as well as a comparison with Tn-inserts subsequently able to systemically disseminate to the spleen following induction of neutropenia. Sequencing was performed following DNA extraction from the recovered bacteria, digestion with the MmeI restriction enzyme that hydrolyzes DNA 16 bp away from the end of the Tn insert, and fractionation into oligonucleotides of 1,200–1,500 bp that were prepared for high-throughput sequencing. Changes in frequency of Tn inserts into the P. aeruginosa genome were used to quantify in vivo fitness resulting from loss of a gene. 636 genes had <10 sequencing reads in LB, thus defined as unable to grow in this medium. During in vivo infection there were major losses of strains with Tn inserts in almost all known virulence factors, as well as respiration, energy utilization, ion pumps, nutritional genes and prophages. Many new candidates for virulence factors were also identified. There were consistent changes in the recovery of Tn inserts in genes within most operons and Tn insertions into some genes enhanced in vivo fitness. Strikingly, 90% of the non-essential genes were required for in vivo survival following systemic dissemination during neutropenia. These experiments resulted in the identification of the P. aeruginosa strain PA14 genes necessary for optimal survival in the mucosal and systemic environments of a mammalian host.

Enhanced in vivo fitness of carbapenem-resistant oprD mutants of Pseudomonas aeruginosa revealed through high-throughput sequencing

Significance It is thought antibiotic resistance carries a fitness cost and reduces microbial virulence. Using high-throughput sequencing analysis of a transposon insertion bank in Pseudomonas aeruginosa, we found enhanced fitness for in vivo mucosal colonization and systemic spread of strains with transposon insertions in the oprD gene. This conferred resistance to carbapenem antibiotics as well as enhanced resistance to killing at acidic pH and by normal human serum along with increased cytotoxicity against murine macrophages. RNA-sequencing analysis revealed that oprD deficiency led to transcriptional changes in numerous genes that may contribute to the enhanced in vivo fitness observed. Thus, if carbapenem resistance develops during antibiotic therapy of P. aeruginosa infections, it may lead to enhanced fitness and virulence in infected hosts. An important question regarding the biologic implications of antibiotic-resistant microbes is how resistance impacts the organism’s overall fitness and virulence. Currently it is generally thought that antibiotic resistance carries a fitness cost and reduces virulence. For the human pathogen Pseudomonas aeruginosa, treatment with carbapenem antibiotics is a mainstay of therapy that can lead to the emergence of resistance, often through the loss of the carbapenem entry channel OprD. Transposon insertion-site sequencing was used to analyze the fitness of 300,000 mutants of P. aeruginosa strain PA14 in a mouse model for gut colonization and systemic dissemination after induction of neutropenia. Transposon insertions in the oprD gene led not only to carbapenem resistance but also to a dramatic increase in mucosal colonization and dissemination to the spleen. These findings were confirmed in vivo with different oprD mutants of PA14 as well as with related pairs of carbapenem-susceptible and -resistant clinical isolates. Compared with OprD+ strains, those lacking OprD were more resistant to killing by acidic pH or normal human serum and had increased cytotoxicity against murine macrophages. RNA-sequencing analysis revealed that an oprD mutant showed dramatic changes in the transcription of genes that may contribute to the various phenotypic changes observed. The association between carbapenem resistance and enhanced survival of P. aeruginosa in infected murine hosts suggests that either drug resistance or host colonization can cause the emergence of more pathogenic, drug-resistant P. aeruginosa clones in a single genetic event.

Fitness cost of antibiotic susceptibility during bacterial infection

The quest to stem antibiotic resistance might be exacerbated by enhanced fitness and virulence displayed by the drug-resistant microbes. Fit foes Myriad publications from Consumer Reports to Mother Jones have warned us about the pending peril that is antibiotic resistance. Scary survival statistics and gloomy graphs that depict decreases in new antimicrobial drugs over time make clear the damper resistance puts on our ability to keep infectious diseases under control. Now, Roux et al. add another unwelcome wrinkle to the scenario—in addition to limiting our repertoire of curative drugs, antibiotic resistance also appears to enhance microbial fitness and virulence. Most studies have investigated the selective advantage sported by drug-resistant strains during antibiotic treatment. But outside of the therapeutic arena, the phenotypic changes that confer drug resistance are thought—without experimental validation—to be accompanied by the dampening of in vivo fitness, virulence, and transmission. In the new work, the authors used a saturated transposon library of Pseudomonas aeruginosa to pinpoint genes that contributed to in vivo fitness during lung infections in animal models and found that genes that conferred both intrinsic and acquired antibiotic resistance also imparted an in vivo fitness advantage to Pseudomonas during infection. The authors confirmed their findings in two additional pathogenic bacteria—Acinetobacter baumannii and Vibrio cholerae—in mouse and rabbit infection models. Together, these findings warn that the fight against antibiotic resistance might be harder than we thought, given the enhanced fitness and virulence of our drug-resistant adversaries. Advances in high-throughput DNA sequencing allow for a comprehensive analysis of bacterial genes that contribute to virulence in a specific infectious setting. Such information can yield new insights that affect decisions on how to best manage major public health issues such as the threat posed by increasing antimicrobial drug resistance. Much of the focus has been on the consequences of the selective advantage conferred on drug-resistant strains during antibiotic therapy. It is thought that the genetic and phenotypic changes that confer resistance also result in concomitant reductions in in vivo fitness, virulence, and transmission. However, experimental validation of this accepted paradigm is modest. Using a saturated transposon library of Pseudomonas aeruginosa, we identified genes across many functional categories and operons that contributed to maximal in vivo fitness during lung infections in animal models. Genes that bestowed both intrinsic and acquired antibiotic resistance provided a positive in vivo fitness advantage to P. aeruginosa during infection. We confirmed these findings in the pathogenic bacteria Acinetobacter baumannii and Vibrio cholerae using murine and rabbit infection models, respectively. Our results show that efforts to confront the worldwide increase in antibiotic resistance might be exacerbated by fitness advantages that enhance virulence in drug-resistant microbes.

Targeting Pan-Resistant Bacteria With Antibodies to a Broadly Conserved Surface Polysaccharide Expressed During Infection

BACKGROUND New therapeutic targets for antibiotic-resistant bacterial pathogens are desperately needed. The bacterial surface polysaccharide poly-β-(1-6)-N-acetyl-glucosamine (PNAG) mediates biofilm formation by some bacterial species, and antibodies to PNAG can confer protective immunity. By analyzing sequenced genomes, we found that potentially multidrug-resistant bacterial species such as Klebsiella pneumoniae, Enterobacter cloacae, Stenotrophomonas maltophilia, and the Burkholderia cepacia complex (BCC) may be able to produce PNAG. Among patients with cystic fibrosis patients, highly antibiotic-resistant bacteria in the BCC have emerged as problematic pathogens, providing an impetus to study the potential of PNAG to be targeted for immunotherapy against pan-resistant bacterial pathogens. METHODS The presence of PNAG on BCC was assessed using a combination of bacterial genetics, microscopy, and immunochemical approaches. Antibodies to PNAG were tested using opsonophagocytic assays and for protective efficacy against lethal peritonitis in mice. RESULTS PNAG is expressed in vitro and in vivo by the BCC, and cystic fibrosis patients infected by the BCC species B. dolosa mounted a PNAG-specific opsonophagocytic antibody response. Antisera to PNAG mediated opsonophagocytic killing of BCC and were protective against lethal BCC peritonitis even during coinfection with methicillin-resistant Staphylococcus aureus. CONCLUSIONS Our findings raise potential new therapeutic options against PNAG-producing bacteria, including even pan-resistant pathogens.

A Randomized Trial of the Amikacin Fosfomycin Inhalation System for the Adjunctive Therapy of Gram-Negative Ventilator-Associated Pneumonia: IASIS Trial

BACKGROUND: Clinical failures in ventilator‐associated pneumonia (VAP) caused by gram‐negative bacteria are common and associated with substantial morbidity, mortality, and resource utilization. METHODS: We assessed the safety and efficacy of the amikacin fosfomycin inhalation system (AFIS) for the treatment of gram‐negative bacterial VAP in a randomized double‐blind, placebo‐controlled, parallel group, phase 2 study between May 2013 and March 2016. We compared standard of care in each arm plus 300 mg amikacin/120 mg fosfomycin or placebo (saline), delivered by aerosol twice daily for 10 days (or to extubation if < 10 days) via the investigational eFlow Inline System (PARI GmbH). The primary efficacy end point was change from baseline in the Clinical Pulmonary Infection Score (CPIS) during the randomized course of AFIS/placebo, using the subset of patients with microbiologically proven baseline infections with gram‐negative bacteria. RESULTS: There were 143 patients randomized: 71 to the AFIS group, and 72 to the placebo group. Comparison of CPIS change from baseline between treatment groups was not different (P = .70). The secondary hierarchical end point of no mortality and clinical cure at day 14 or earlier was also not significant (P = .68) nor was the hierarchical end point of no mortality and ventilator‐free days (P = .06). The number of deaths in the AFIS group was 17 (24%) and 12 (17%) in the placebo group (P = .32). The AFIS group had significantly fewer positive tracheal cultures on days 3 and 7 than placebo. CONCLUSIONS: In this trial of adjunctive aerosol therapy compared with standard of care IV antibiotics in patients with gram‐negative VAP, the AFIS was ineffective in improving clinical outcomes despite reducing bacterial burden. TRIAL REGISTRY: ClinicalTrials.gov; No.: NCT01969799; URL: www.clinicaltrials.gov

Natural antibodies in normal human serum inhibit Staphylococcus aureus capsular polysaccharide vaccine efficacy.

BACKGROUND Vaccines against Streptococcus pneumoniae, Neisseria meningitidis, and Hemophilus influenzae type b induce functional opsonic or bactericidal antibodies to surface capsular polysaccharides (CP). Targeting the comparable Staphylococcus aureus CP seems logical, but to date such efforts have failed in human trials. Studies using immunization-induced animal antibodies have documented interference in opsonic and protective activities of antibodies to CP by antibodies to another S. aureus cell surface polysaccharide, poly-N-acetyl glucosamine (PNAG). Here we evaluated whether natural antibody to PNAG in normal human serum (NHS) had a similar deleterious effect. METHODS Functional and/or protective activities of antibody to S. aureus CP and PNAG antigens in patients with bacteremia, in mice immunized with combinations of CP and PNAG conjugate vaccines, and in serum samples of healthy subjects with natural antibody to PNAG, to which immunization-induced animal antibodies to CP antigens were added, were evaluated. RESULTS Antibodies to PNAG and CP that mutually interfered with opsonic killing of S. aureus were detected in 9 of 15 bacteremic patients. Active immunization of mice with combinations of PNAG and CP conjugate antigens always induced antibodies that interfered with each others functional activity. Non-opsonic natural antibodies to PNAG found in NHS interfered with the functional and protective activities of immunization-induced antibody to CP antigens during experimental infection with S. aureus. CONCLUSIONS Both immunization-induced animal antibodies and natural antibodies to PNAG in NHS interfere with the protective activities of immunization-induced antibody to S. aureus CP5 and CP8 antigens, representing potential barriers to successful use of CP-specific vaccines.

Prognostic significance of new immunohistochemical markers in refractory classical Hodgkin lymphoma: a study of 59 cases.

Although most classical Hodgkin lymphoma patients are cured, a significant minority fail after primary therapy and may die as result of their disease. To date, there is no consensus on biological markers that add value to usual parameters (which comprise the International Prognostic Score) used at diagnosis to predict outcome. We evaluated 59 patients (18 with primary refractory or early relapse disease and 41 responders) for bcl2, Ki67, CD20, TiA1 and c-kit expression by semi-quantitative immunohistochemical study and correlated the results with the response to treatment. The results showed that expression of bcl2 and CD20 in Hodgkin and Reed Sternberg cells, and expression of TiA1 in micro-environmental lymphocytes, and c-kit positive mast cells in microenvironment, were independent prognostic markers. These novel cHL markers could be used in association with clinical parameters to identify newly diagnosed patients with favorable or unfavorable prognosis and to better tailor treatment for different risk groups.