Vincent Le Moigne
email: vincent.le-moigne@uvsq.fr

Jean-Louis Gaillard

Jean-Louis Herrmann

Vaccine strategies against cystic fibrosis pathogens

Keywords: cystic fibrosis, vaccine, Mycobacterium abscessus, Pseudomonas aeruginosa, Burkholderia spp

A great number of cystic fibrosis (CF) pathogens such as Pseudomonas aeruginosa, the Burkholderia cepacia and the Mycobacterium abscessus complex raised difficult therapeutic problems due to their intrinsic multi-resistance to numerous antibiotics. Vaccine strategies represent one of the key weapons against these multi-resistant bacteria in a number of clinical settings like CF. Different strategies are considered in order to develop such vaccines, linked either to priming the host response, or by exploiting genomic data derived from the bacterium.

Interestingly, virulence factors synthesized by various pathogens might serve as targets for vaccine development and have been, for example, evaluated in the context of CF.

Cystic fibrosis and microbial lung infections.

Cystic fibrosis (CF) is a disease which arises from a Mendelian defect due to a series of mutations in the cftr gene encoding the Cl -channel [START_REF] Riordan | Identification of the cystic fibrosis gene: cloning and characterization of complementary DNA[END_REF] . The resulting flaw in this protein is responsible for increasing the viscosity of the mucus, which promotes the accumulation and the attachment of bacteria to mucins. Chronic inflammation [START_REF] Ratjen | Cystic fibrosis: pathogenesis and future treatment strategies[END_REF] and early bacterial infection maintain a vicious circle and are each responsible for the lung damage which ensues. Lung infections in CF patients represent the most frequent but also the more serious manifestations since they are responsible for more than 90% of CF patient deaths [START_REF] Rowe | Cystic fibrosis[END_REF] . The microorganisms that may infect the respiratory system are bacteria, fungi and viruses. Bacterial colonization occurs very early in the natural history of the disease [START_REF] May | Bacterial Infection in cystic fibrosis[END_REF] . The first causative organisms are Haemophilus influenzae and Staphylococcus aureus. S. aureus is usually the first detected [START_REF] Marks | Clinical significance of Staphylococcus aureus in cystic fibrosis[END_REF] and its prevalence is rising [START_REF] Lipuma | The changing microbial epidemiology in cystic fibrosis[END_REF] . Affinity of S. aureus for CF mucus contributes to persistent colonization and progressive pulmonary damage increasing the potential for further infections to set in, for example Pseudomonas spp. [START_REF] Marks | Clinical significance of Staphylococcus aureus in cystic fibrosis[END_REF] . Pseudomonas aeruginosa colonization arises several months to several years after. Finally, several bacterial complexes are found responsible for severe infections in CF, in addition to be the most difficult to treat: the Burkholderia cepacia complex (Bcc) and the Mycobacterium abscessus complex, which has emerged recently as a threat in CF patients, and may present with Mycobacterium avium, the major non-tuberculous mycobacterium (NTM) present in CF lungs with a significant prevalence [START_REF] Roux | Multicenter study of prevalence of nontuberculous mycobacteria in patients withcystic fibrosis in France[END_REF][START_REF] Olivier | Nontuberculous mycobacteria[END_REF] .

Opportunistic pathogens becoming untreatable weapons in CF patients

P. aeruginosa is the environmental opportunistic pathogen in CF patients. It is the most commonly isolated bacterium that infects individuals with CF, with colonization and chronic infections that may affect up to 80% of adult CF patients [START_REF] Gibson | Pathophysiology and management of pulmonary infections in cystic fibrosis[END_REF] . P. aeruginosa establishes a chronic endobronchial infection which impacts on morbidity and mortality of CF patients. P. aeruginosa is also notable for its resistance to antibiotics, making it therefore a difficult to treat pathogen, which, once acquired, is rarely, if ever, eradicated. In addition, P. aeruginosa frequently colonized CF lungs as a biofilm, which reduces the patient's immune response and access by antibiotics [START_REF] Drenkard | Antimicrobial resistance of Pseudomonas aeruginosa biofilms[END_REF] . A second opportunistic pathogen, represented as a complex is the Burkholderia cepacia complex (Bcc). It is composed of 18 species that are able to cause opportunistic and lethal infections CF patients [START_REF] Lipuma | Update on the Burkholderia cepacia complex[END_REF] . The two most clinically relevant species are Burkholderia cenocepacia and Burkholderia multivorans [START_REF] Caraher | Invasion and biofilm formation of Burkholderia dolosa is comparable with Burkholderia cenocepacia and Burkholderia multivorans[END_REF] . These environmental, intracellular and biofilm-forming bacteria are extremely antibiotic resistant organism [START_REF] Caraher | Invasion and biofilm formation of Burkholderia dolosa is comparable with Burkholderia cenocepacia and Burkholderia multivorans[END_REF] . Bcc infections are rarely cleared from CF patients once they are colonized, as observed in P. aeruginosa infections. The third antibiotic-resistant bacterium found in CF patients with frequency between 3 to 7% [START_REF] Roux | Multicenter study of prevalence of nontuberculous mycobacteria in patients withcystic fibrosis in France[END_REF][START_REF] Olivier | Nontubercu-lous mycobacteria I: multicenter prevalence study in cystic fibrosis[END_REF] is Mycobacterium abscessus. It is a rapidly growing mycobacterium also existing as a complex: the Mycobacterium abscessus complex [START_REF] Leao | Proposal that Mycobacterium massiliense and Mycobacterium bolletii be united and reclassified as Mycobacterium abscessus subsp. bolletii comb. nov., designation of Mycobacterium abscessus subsp.abscessus subsp. nov. and emended description of Mycobacterium abscessus[END_REF] , with two subspecies M. abscessus abscessus and M. abscessus bolletii respectively. M. abscessus is, within the group of rapid-growers, responsible for a broad spectrum of diseases in humans. Lung infections are frequent, with CF patients particularly susceptible [START_REF] Roux | Multicenter study of prevalence of nontuberculous mycobacteria in patients withcystic fibrosis in France[END_REF][START_REF] Olivier | Nontubercu-lous mycobacteria I: multicenter prevalence study in cystic fibrosis[END_REF][START_REF] Pierre-Audigier | Age-related prevalence and distribution of nontuberculousmycobacterial species among patients with cystic fibrosis[END_REF] , in addition to muco-cutaneous infections often of nosocomial origin [START_REF] Wallace Rj | Nosocomial outbreaks/pseudo-outbreaks caused by nontuberculous mycobacteria[END_REF] . Recent reports of human-to-human transmission in the context of CF care have been described [START_REF] Aitken | Respiratory outbreak of Mycobacterium abscessus subspecies massiliense in a lung transplant and cystic fibrosis center[END_REF][START_REF] Bryant | Whole-genome sequencing to identify transmission of Mycobacterium absces-sus between patients with cystic fibrosis: a retrospective cohort study[END_REF] . M. abscessus raises very challenging therapeutic issues because of its natural resistance to most available antibiotics [START_REF] Ripoll | Nonmycobacterial virulence genes in the genome of the emerging pathogen Mycobacterium abscessus[END_REF][START_REF] Nash | A novel gene, erm(41), confers inducible macrolide resistance to clinical isolates of Mycobacterium abscessus but is absent from Mycobacterium chelonae[END_REF] . Severe, even fatal, infections in CF patients have been described due to therapeutic deadlock [START_REF] Sanguinetti | Fatal pulmonary infection due to multidrug-resistant Mycobacterium abscessus in a patient with cystic fibrosis[END_REF] . M. abscessus infection might represent a contraindication for lung transplantation in several countries [START_REF] Taylor | Mycobacterium abscessus chest wall and pulmonary infection in a cystic fibrosis lung transplant recipient[END_REF] , leaving CF patients without therapeutic options.

As such, antibiotic treatment exemplifies a clear challenge now faced with these opportunistic pathogens. We demonstrated for example a significant link between previous intravenous antibiotic courses and the isolation of M. abscessus in CF patient lungs, underlining the role of broad-spectrum antimicrobial therapy in the emergence of M. abscessus disease [START_REF] Catherinot | Mycobacterium avium and Mycobacterium abscessus complex target distinct cysticfibrosis patient subpopulations[END_REF] . And this is true for the continuous emergence of resistant P. aeruginosa or Bcc due to the repeated antibiotic therapeutic regimens given to CF patients [START_REF] Lopez | The global burden of disease, 1990-2020[END_REF] . Emergence of multi-resistant bacteria leads to therapeutic impasses with severe and fatal infections [START_REF] Lopez | The global burden of disease, 1990-2020[END_REF] .

Vaccine approaches

Pathogens can be divided into two groups according to whether vaccines exist against them or not. However, no human vaccine has been developed so far against the antibiotic-resistant pathogens described above. As such, the development of prophylactic or therapeutic vaccines is of extreme importance when confronted with bacteria of this high resistance.. When defining an appropriate target, making the correct choice and how such tools can be developed is a long and tedious process. Development of vaccine targets can profit from genome sequence comparisons [START_REF] Hoft | Optimizing vaccine development[END_REF] , and, using a process known as reverse vaccinology, might introduce a novel strategy to identify target antigens that might serve as potential vaccines [START_REF] Hoft | Optimizing vaccine development[END_REF] .

We chose this strategy for the development of a first vaccine against M. abscessus 26 (see below). Indeed, the presence of specific genes in opportunistic pathogen genomes that are absent from saprophytic bacterial genomes belonging to the same genus represents the key to unravel virulence factors that can then be targeted using a vaccine approach. A better understanding of the "virulence" genes contributes greatly to the development of new control strategies against these microorganisms. In addition, the choice of a virulence factor as a vaccine target has been shown to be relevant in the scientific literature [START_REF] Hoft | Optimizing vaccine development[END_REF] .

Vaccination can then be performed using recombinant proteins when expression and purification are possible; or using the expression of plasmid DNA with modified eukaryotic gene sequences, as has been performed with M. abscessus [START_REF] Moigne | Bacterial phospholipases C as vaccine candidate antigens against cystic fibrosis respiratory pathogens: the Mycobacterium abscessus model[END_REF] . In fact, in the different vaccine strategies, DNA vaccines exemplify one of these new strategies and has been used successfully in the context of infectious diseases [START_REF] Tighe | Gene vaccination: plasmid DNA is more than just a blueprint[END_REF] . Over the last 15 years, DNA vaccines have proved effective in animal models including candidates against HIV, malaria and influenza [START_REF] Klinman | FDA guidance on prophylactic DNA vaccines: analysis and recommendations[END_REF] .

DNA vaccines have been extensively evaluated in humans with a recent review identifying 72

Phase I, 20 Phase II and two Phase III human trials [START_REF] Kutzler | DNA vaccines: ready for prime time?[END_REF] . Mycobacteria, such as Mycobacterium tuberculosis, have received particular attention in this respect [START_REF] Tascon | Vaccination against tuberculosis by DNA injection[END_REF][START_REF] Huygen | Immunogenicity and protective efficacy of a tuberculosis DNA vaccine[END_REF] . Finally, some common antigens might be present in a variety of different CF pathogens, and the development of a vaccine might have the potential for conferring cross-protection against several CF pathogens (see below).

Vaccines against opportunistic CF pathogens

Some infections [START_REF] Pillarisetti | Infection, inflammation, and lung function decline in infants with cystic fibrosis[END_REF][START_REF] Sagel | Impact of pseudomonas and staphylococcal infection on inflammation and clinical status in young children with cystic fibrosis[END_REF] frequently associated with impaired respiratory function in patients with cystic fibrosis are the subject of vaccine development: for example infections with P.

aeruginosa.

The development of a vaccine against P. aeruginosa has so far mobilized many research teams, even though no human trials have been conducted yet. Numerous vaccine attempts have been made against this pathogen that is generally considered to be the most targeted among pathogens infecting cystic fibrosis patients, and in order to obtain a state of the art overview in this domain, you would do well to explore the following recent reviews [START_REF] Grimwood | Vaccination against respiratory Pseudomonas aeruginosa infection[END_REF][START_REF] Savoia | New perspectives in the management of Pseudomonas aeruginosa infections[END_REF][START_REF] Johansen | Vaccines for preventing infection with Pseudomonas aeruginosa in cystic fibrosis[END_REF][START_REF] Sharma | Recent developments for Pseudomonas vaccines[END_REF] .

Major target antigens include the O-glycosylated lipopolysaccharide, cell-surface alginate, flagella, components of the Type III secretion system and outer membrane proteins [START_REF] Pier | Application of vaccine technology to prevention of Pseudomonas aeruginosa infections[END_REF] . The FliC flagellin protein widely considered as a virulence factor, has pro-inflammatory activity on respiratory epithelial cells. Flagellin has been one of the major targets for vaccines and therapeutic development expecially for CF patients. For example, as early as 1995, P. aeruginosa flagella were developed as a vaccine against P. aeruginosa and a Phase I study demonstrated that intramuscular immunization in healthy human adults results in high and long-lasting serum IgG flagella antibody titers and IgG, IgA and secretory IgA isotypes in the secretory immune system [START_REF] Döring | Parenteral application of a Pseudomonas aeruginosa flagella vaccine elicits specific anti-flagella antibodies in the airways of healthy individuals[END_REF] . Then, in a phase III study, an immunization with a bivalent vaccine for flagella subtypes significantly lowered the risk for initial P. aeruginosa infection in CF patients [START_REF] Döring | Flagella vaccine trial study, a double-blind randomized placebo-controlled phase III study of a Pseudomonas aeruginosa flagella vaccine in cystic fibrosis patients[END_REF] . Other proteins have been used in human trials: OprF and OprI that are outer membrane proteins are able to induce a specific antibody response in the lung after nasal and oral vaccinations, and are as such promising candidates for the development of antipseudomonas immunization [START_REF] Bumann | Systemic, nasal and oral live vaccines against Pseudomonas aeruginosa: a clinical trial of immunogenicity in lower airways of human volunteers[END_REF] . Furthermore, a fusion protein of the P. aeruginosa OprF fragment, OprI, and FliC promoted the clearance of P. aeruginosa in a pulmonary challenge model [START_REF] Weimer | A fusion protein vaccine containing OprF epitope 8, OprI, and type A and B flagellins promotes enhanced clearance of nonmucoid Pseudomonas aeruginosa[END_REF] . Another study which also tested these two outer membrane proteins as mucosal vaccines, lead to the development of airway immunogenicity against the pathogen with superior efficacy compared to systemic vaccination [START_REF] Sorichter | Immune responses in the airways by nasal vaccination with systemic boosting against Pseudomonas aeruginosa in chronic lung disease[END_REF] . We can add to this, as more recently, among the latest antigens and strategies tested, an assay using the conserved surface exopolysaccharide alginate, a virulence factor produced by mucoid strains, has been tried in mice [START_REF] Farjah | Immunological evaluation of an alginate-based conjugate as a vaccine candidate against Pseudomonas aeruginosa[END_REF] and conferred protection after intranasal challenge. It was also efficacious as a therapeutic vaccine. Previously, an assay was performed in humans with O-polysaccharide conjugated to toxin A and vaccinated children developed less chronic Pseudomonas lung infections than non-vaccinated children [START_REF] Zuercher | Antibody responses induced by long-term vaccination with an octovalent conjugate Pseudomonas aeruginosa vaccine in children with cystic fibrosis[END_REF][START_REF] Zuercher | Cellular immunity in healthy volunteers treated with an octavalent conjugate Pseudomonas aeruginosa vaccine[END_REF] . An element of alginate (polymannuronic acid) has also been conjugated to flagellin leading to protective efficacy in a mouse lung infection model [START_REF] Campodónico | Efficacy of a conjugate vaccine containing polymannuronic acid and flagellin against experimental Pseudomonas aeruginosa lung infection in mice[END_REF] . Some P. aeruginosa antigens conjugated to bovine serum albumin have also been tested in mice [START_REF] Rodrigues | Bovine serum albumin nanoparticle vaccine reduces lung pathology induced by live Pseudomonas aeruginosa infection in mice[END_REF] . However, despite more than 50 years of research efforts, a licensed vaccine against P. aeruginosa is still a long way off from being available for CF patients.

With reference to the Burkholderia cepacia complex (Bcc), several virulence factors associated with human infection were tested for their potential as vaccine candidates [START_REF] Casey | Exploiting molecular virulence determinants in Burkholderia to develop vaccine antigens[END_REF] . Such an approach was undertaken by a group that sought immunoreactive proteins expressed by both Burkholderia cenocepacia and Burkholderia multivorans [START_REF] Shinoy | Immunoproteomic analysis of proteins expressed by two related pathogens, Burkholderia multivorans and Burkholderia cenocepacia, during human infection[END_REF] . A recent review summarizes in detail all vaccination experiments that have been undertaken against Bcc 51 . As a recent example, the flagellar protein FliC from Burkholderia pseudomallei and considered as a virulence factor has been shown to confer protection in mice [START_REF] Musson | CD4+ T cell epitopes of FliC conserved between strains of Burkholderia: implications for vaccines against melioidosis and cepacia complex in cystic fibrosis[END_REF] . This study shows that the epitopes of interest in B. pseudomallei FliC cross-react with orthologous B. multivorans and B. cenocepacia FliC sequences suggesting protection can be conferred against members of the Bcc. Other proteins like OMPs from different Burkolderia species are also able to generate protection in mice [START_REF] Makidon | Induction of immune response to the 17 kDa OMPA Burkholderia cenocepacia polypeptide and protection against pulmonary infection in mice after nasal vaccination with an OMP nanoemulsion-based vaccine[END_REF][START_REF] Bertot | Nasal immunization with Burkholderia multivorans outer membrane proteins and the mucosal adjuvant adamantylamide dipeptide confers efficient protection against experimental lung infections with B. multivorans and B. cenocepacia[END_REF] .

Vaccination in the context of mycobacteria.

The most widely used global vaccine is the "Bacille Calmette et Guérin" (BCG) strain of Mycobacterium bovis used in the fight against tuberculosis, a disease caused by Mycobacterium tuberculosis, which presently kills 1.3 million individuals around the world every year [START_REF]Global tuberculosis report[END_REF] . This technique harnessed the historically defined strategy developed by Jenner [START_REF] Jenner | The Three Original Publications on Vaccination Against Smallpox[END_REF] , by using the antigenic repertoire of a non-pathogenic strain for human, in order to confer protection against the human pathogen. Despite its widespread use, BCG is still a controversial vaccine, and its deficiencies have lead to the development of new research axes, using either purified compounds or a DNA vaccines in a quest to improve anti-mycobacterial vaccines. Several mycobacterial proteins (Ag85A/B, 65-kDa heat shock protein, hsp65, 36-kDa proline-rich antigen, MPB83, MPB70, CFP-10 and ESAT-6) have been evaluated as DNA vaccines in experimental models [START_REF] Montgomery | Induction of humoral and cellular immune responses by vaccination with M. tuberculosis antigen 85 DNA[END_REF][START_REF] Chambers | Vaccination of mice and cattle with plasmid DNA encoding the Mycobacterium bovis antigen MPB83[END_REF][START_REF] Vordermeier | Use of synthetic peptides derived from the antigens ESAT-6 and CFP-10 for differential diagnosis of bovine tuberculosis in cattle[END_REF][START_REF] Huygen | On the use of DNA vaccines for the prophylaxis of mycobacterial diseases[END_REF][START_REF] Romano | DNA vaccines against mycobacterial diseases[END_REF][START_REF] Oksanen | An adult zebrafish model for preclinical tuberculosis vaccine development[END_REF][START_REF] Feng | Enhanced immune response and protective effects of nano-chitosan-based DNA vaccine encoding T cell epitopes of Esat-6 and FL against Mycobacterium tuberculosis infection[END_REF] . By virtue of its strong capacity to induce CD4+mediated Th1 and CD8+-mediated cytotoxic T-lymphocyte responses [START_REF] Hoft | Optimizing vaccine development[END_REF] , DNA vaccine approaches are particularly attractive for their preventive and therapeutic activity against intracellular pathogens such as pathogenic mycobacteria [START_REF] Romano | DNA vaccines against mycobacterial diseases[END_REF] . The majority of these studies were conducted in the fields of human [START_REF] Tascon | Vaccination against tuberculosis by DNA injection[END_REF][START_REF] Huygen | Immunogenicity and protective efficacy of a tuberculosis DNA vaccine[END_REF][START_REF] Montgomery | Induction of humoral and cellular immune responses by vaccination with M. tuberculosis antigen 85 DNA[END_REF][START_REF] Huygen | On the use of DNA vaccines for the prophylaxis of mycobacterial diseases[END_REF][START_REF] Romano | DNA vaccines against mycobacterial diseases[END_REF] and bovine tuberculosis [START_REF] Chambers | Vaccination of mice and cattle with plasmid DNA encoding the Mycobacterium bovis antigen MPB83[END_REF][START_REF] Vordermeier | Use of synthetic peptides derived from the antigens ESAT-6 and CFP-10 for differential diagnosis of bovine tuberculosis in cattle[END_REF] ; and even in leprosy (immuno-dominant 35-kDa protein [START_REF] Martin | DNA encoding a single mycobacterial antigen protects against leprosy infection[END_REF] ). Few DNA vaccines have been developed which are related to pathogenic or opportunistic NTM, for example M. avium (35-kDa protein [START_REF] Martin | Protection against virulent Mycobacterium avium infection following DNA vaccination with the 35-kilodalton antigen is accompanied by induction of gamma interferon-secreting CD4(+) T cells[END_REF] ; p85A-EGFP, p65K-EGFP 66 ); Mycobacterium ulcerans [START_REF] Tanghe | Protective efficacy of a DNA vaccine encoding antigen 85A from Mycobacterium bovis BCG against Buruli ulcer[END_REF] or Mycobacterium marinum [START_REF] Oksanen | An adult zebrafish model for preclinical tuberculosis vaccine development[END_REF][START_REF] Feng | Enhanced immune response and protective effects of nano-chitosan-based DNA vaccine encoding T cell epitopes of Esat-6 and FL against Mycobacterium tuberculosis infection[END_REF] . None were developed in the context of tackling infections in inherited diseases such as CF.

While the presence of NTM is demonstrated in 7 to 13% of CF patients [START_REF] Olivier | Nontuberculous mycobacteria[END_REF] , infections by M. tuberculosis are rare in CF patients [START_REF] Morand | Mediastinal tuberculosis in an adult patient with cystic fibrosis[END_REF][START_REF] Asherova | Cystic fibrosis complicated by multiresistant tuberculosis[END_REF] . BCG remains a currently recommended vaccine in children at risk of exposure to TB; and as a consequence of partial efficacy against NTM, continues to be recommended in CF children. The two mycobacteria that together are responsible for the majority of infections in cystic fibrosis patients are M. avium and M. abscessus, a slow and a rapid growing mycobacteria (SGM and RGM), respectively, and to our knowledge, no vaccine approach against these bacteria has been considered in the context of CF. As mentioned above, development of vaccine targets can benefit from knowledge derived from genome sequence comparison [START_REF] Hoft | Optimizing vaccine development[END_REF] . Genome sequence comparison between the M. abscessus genome and Mycobacterium chelonae, Mycobacterium smegmatis genomes (two rapid growing NTM, which are less or non-pathogenic respectively), allowed the unraveling of several key virulence factors [START_REF] Ripoll | Nonmycobacterial virulence genes in the genome of the emerging pathogen Mycobacterium abscessus[END_REF] . As described in each genome sequences, several of these virulence factors were acquired by horizontal gene transfer, from non-fermenting Gram negative bacteria such as those found in CF patient lungs: P. aeruginosa and Bcc [START_REF] Ripoll | Nonmycobacterial virulence genes in the genome of the emerging pathogen Mycobacterium abscessus[END_REF] . Among others we characterized MAB_0555, a phospholipase C (PLC) with the highest homology with PLC-N from P. aeruginosa [START_REF] Bakala | Mycobacterium abscessus Phospholipase C expression is induced during coculture within amoebae and enhances M. abscessus virulence in mice[END_REF] . We have demonstrated the impact of MAB_0555 PLC in the virulence of M. abscessus in mice [START_REF] Bakala | Mycobacterium abscessus Phospholipase C expression is induced during coculture within amoebae and enhances M. abscessus virulence in mice[END_REF] , when pre-cultivated on amoeba. We have also shown that the recombinant protein or the plasmid encoding PLC conferred protection, after an aerosol or IV challenge (Figure 1), in Δ508 or CF mice 71 only [START_REF] Moigne | Bacterial phospholipases C as vaccine candidate antigens against cystic fibrosis respiratory pathogens: the Mycobacterium abscessus model[END_REF] . The two formulations (Figure 1) gave quite similar results, namely a diminution of bacterial load in lungs three weeks after a M. abscessus aerosol challenge [START_REF] Moigne | Bacterial phospholipases C as vaccine candidate antigens against cystic fibrosis respiratory pathogens: the Mycobacterium abscessus model[END_REF] . PLC are also present in other CF pathogens like P. aeruginosa and the immune cross reactivity between M. abscessus PLC and P. aeruginosa PLC could lead to a vaccine protective against both mycobacterial and Gram negative infection in CF patients, as we were able to show the recognition of the MAB_0555 PLC by sera from CF patients only infected by P. aeruginosa. This approach is currently underway in our laboratory with other target designs recently unraveled [START_REF] Roux | Overexpression of proinflammatory TLR-2-signalling lipoproteins in hypervirulent mycobacterial variants[END_REF] .

Conclusion

Vaccination is effective in preventing infections and we advocate their use in patients with CF, specifically for the prevention of respiratory infections. In addition to the traditional vaccination schedules, [START_REF] Malfroot | Immunisation in the current management of cystic fibrosis patients[END_REF] evaluation studies to demonstrate the immunological and clinical efficacy of novel vaccines against multi-resistant bacteria remain necessary in this particular patient population.


Anne-Laure Lefebvre

Vincent Le Moigne

Audrey Bernut

Carole Veckerlé

Fabrice Compain

Jean-Louis Herrmann

Laurent Kremer

Michel Arthur
email: michel.arthur@crc.jussieu.fr

Jean-Luc Mainardi
email: jean-luc.mainardi@crc.jussieu.fr

Inhibition of the ␤-Lactamase Bla Mab by Avibactam Improves the In Vitro and In Vivo Efficacy of Imipenem against Mycobacterium abscessus

Keywords: ␤-lactamase inhibitor, avibactam, Mycobacterium abscessus, cystic fibrosis, imipenem

Mycobacterium abscessus pulmonary infections are treated with a macrolide (clarithromycin or azithromycin), an aminoglycoside (amikacin), and a ␤-lactam (cefoxitin or imipenem). The triple combination is used without any ␤-lactamase inhibitor, even though M. abscessus produces the broad-spectrum ␤-lactamase Bla Mab . We determine whether inhibition of Bla Mab by avibactam improves the activity of imipenem against M. abscessus. The bactericidal activity of drug combinations was assayed in broth and in human macrophages. The in vivo efficacy of the drugs was tested by monitoring the survival of infected zebrafish embryos. The level of Bla Mab production in broth and in macrophages was compared by quantitative reverse transcription-PCR and Western blotting. The triple combination of imipenem (8 or 32 g/ml), amikacin (32 g/ml), and avibactam (4 g/ml) was bactericidal in broth (Ͻ0.1% survival), with 3.2-and 4.3-log 10 reductions in the number of CFU being achieved at 72 h when imipenem was used at 8 and 32 g/ml, respectively. The triple combination achieved significant intracellular killing, with the bacterial survival rates being 54% and 7% with the low (8 g/ml) and high (32 g/ml) dosages of imipenem, respectively. In vivo inhibition of Bla Mab by avibactam improved the survival of zebrafish embryos treated with imipenem. Expression of the gene encoding Bla Mab was induced (20-fold) in the infected macrophages. Inhibition of Bla Mab by avibactam improved the efficacy of imipenem against M. abscessus in vitro, in macrophages, and in zebrafish embryos, indicating that this ␤-lactamase inhibitor should be clinically evaluated. The in vitro evaluation of imipenem may underestimate the impact of Bla Mab , since the production of the ␤-lactamase is inducible in macrophages.

I n the context of cystic fibrosis, Mycobacterium abscessus has emerged in recent years as an important opportunistic lung pathogen increasingly responsible for mortality [START_REF] Sermet-Gaudelus | Mycobacterium abscessus and children with cystic fibrosis[END_REF][START_REF] Esther | Chronic Mycobacterium abscessus infection and lung function decline in cystic fibrosis[END_REF][START_REF] Olivier | Nontuberculous mycobacteria. I. Multicenter prevalence study in cystic fibrosis[END_REF][START_REF] Levy | Multicenter cross-sectional study of nontuberculous mycobacterial infections among cystic fibrosis patients, Israel[END_REF][START_REF] Roux | Multicenter study of prevalence of nontuberculous mycobacteria in patients with cystic fibrosis in France[END_REF]. These infections are extremely difficult to treat and to eradicate, as M. abscessus is naturally resistant to most antibiotics, including antituberculous agents [START_REF] Brown-Elliott | Antimicrobial susceptibility testing, drug resistance mechanisms, and therapy of infections with nontuberculous mycobacteria[END_REF][START_REF] Novosad | Treatment of Mycobacterium abscessus infection[END_REF]. The recommended treatment for pulmonary infections relies on the combination of a macrolide (clarithromycin or azithromycin), an aminoglycoside (amikacin), and an intravenous ␤-lactam (cefoxitin or imipenem) for at least 12 to 16 months [START_REF] Brown-Elliott | Antimicrobial susceptibility testing, drug resistance mechanisms, and therapy of infections with nontuberculous mycobacteria[END_REF][START_REF] Floto | US Cystic Fibrosis Foundation and European Cystic Fibrosis Society consensus recommendations for the management of non-tuberculous mycobacteria in individuals with cystic fibrosis[END_REF]. Resistance to macrolide, present in 40 to 60% of the isolates [START_REF] Bastian | Assessment of clarithromycin susceptibility in strains belonging to the Mycobacterium abscessus group by erm(41) and rrl sequencing[END_REF], leads to a cure rate of only 25 to 40% (whereas the cure rate is 88 to 95% for patients infected with macrolide-susceptible isolates). Cefoxitin and imipenem are the most active ␤-lactams, in spite of moderate in vitro activity with MICs (MIC 50 s) of 32 and 16 g/ml, respectively [START_REF] Lavollay | In vitro activity of cefoxitin and imipenem against Mycobacterium abscessus complex[END_REF].

We have previously shown that the production of a broad-spectrum ␤-lactamase, the M. abscessus ␤-lactamase (Bla Mab ), is a major determinant of ␤-lactam resistance in M. abscessus [START_REF] Soroka | Characterization of broad-spectrum Mycobacterium abscessus class A beta-lactamase[END_REF][START_REF] Dubee | ␤-Lactamase inhibition by avibactam in Mycobacterium abscessus[END_REF]. In contrast to the ␤-lactamase BlaC from M. tuberculosis, Bla Mab is not inactivated by clavulanate, sulbactam, and tazobactam, since these inhibitors are hydrolyzed by the ␤-lactamase. However, Bla Mab is efficiently inhibited by avibactam [START_REF] Dubee | ␤-Lactamase inhibition by avibactam in Mycobacterium abscessus[END_REF], a ␤-lactamase inhibitor approved by the FDA in 2014 [START_REF] Falcone | Spotlight on ceftazidime/avibactam: a new option for MDR Gram-negative infections[END_REF][START_REF] Bush | A resurgence of beta-lactamase inhibitor combinations effective against multidrug-resistant Gram-negative pathogens[END_REF]. Deletion of the gene encoding Bla Mab or chemical inhibition of the ␤-lactamase activity by avibactam extends the spectrum of ␤-lactams active against M. abscessus, as previously shown for an extensive evaluation of the amoxicillin-avibactam combination [START_REF] Dubee | ␤-Lactamase inhibition by avibactam in Mycobacterium abscessus[END_REF].

Recently, we have reported that the rate of killing of an M. abscessus Bla Mab -deficient mutant by the combination of imipenem and amikacin is significantly higher than that of the parental strain [START_REF] Lefebvre | Bactericidal and intracellular activity of ␤-lactams against Mycobacterium abscessus[END_REF]. In macrophages, the difference was even more pronounced, since a 100-fold reduction in the number of intracellular bacteria was observed for the Bla Mab -deficient mutant, whereas the combination was only bacteriostatic for the wild-type strain [START_REF] Lefebvre | Bactericidal and intracellular activity of ␤-lactams against Mycobacterium abscessus[END_REF]. Together, the results obtained with the Bla Mab -deficient mutant indicated that the production of the ␤-lactamase may limit the efficacy of imipenem, despite the fact that this drug is used in the absence of a ␤-lactamase inhibitor in the recommended treatment of pulmonary infections due to M. abscessus. In this study, we evaluated whether Bla Mab inhibition by avibactam could potentiate the effects of imipenem in vitro, in macrophages, and in zebrafish embryos. The zebrafish embryo model has been developed to assess the in vivo activity of antibiotics against M. abscessus [START_REF] Bernut | In vivo assessment of drug efficacy against Mycobacterium abscessus using the embryonic zebrafish test system[END_REF]. Unexpectedly, we found that the production of Bla Mab has a greater impact on the activity of imipenem in macrophages than in in vitro cultures, prompting us to determine and compare the level of Bla Mab production in planktonically and intracellularly growing M. abscessus bacteria.

RESULTS

In vitro killing of M. abscessus CIP104536 S by imipenem alone or in combination with amikacin and avibactam. Imipenem was tested at 8 g/ml and 32 g/ml, doses that correspond to concentrations equal to 4-and 16-fold the MICs of the drug against M. abscessus CIP104536 with a smooth morphotype (CIP104536 S), respectively (15). Amikacin was tested at 4-fold the MIC (32 g/ml). Avibactam was tested at 4 g/ml, which is the concentration used for susceptibility testing in Enterobacteriaceae [START_REF] Shields | Effects of Klebsiella pneumoniae carbapenemase subtypes, extended-spectrum ␤-lactamases, and porin mutations on the in vitro activity of ceftazidime-avibactam against carbapenem-resistant K. pneumoniae[END_REF][START_REF] Hackel | Assessment of the in vitro activity of ceftazidime-avibactam against multidrug-resistant Klebsiella spp. collected in the INFORM global surveillance study, 2012 to 2014[END_REF]. Reductions in the log 10 number of CFU of M. abscessus CIP104536 S were observed for imipenem at 8 g/ml when it was tested alone (1.7 log 10 ) or in combination with amikacin (2.3 log 10 ) or avibactam (1.9 log 10 ) (Fig. 1a; see Table S2 in the FIG 1 Time-kill curves of the activity of imipenem (IPM) alone or in combination with the ␤-lactamase inhibitor avibactam (AVI) and amikacin (AMK) against M. abscessus CIP104536 S. (a) Time-kill curves of imipenem at 8 g/ml alone or in combination with avibactam (4 g/ml) and amikacin (32 g/ml). The number of CFU was determined after 0, 48, and 72 h of exposure to the antibiotics. (b) Time-kill curves of imipenem at 32 g/ml alone or in combination with avibactam (4 g/ml) and amikacin (32 g/ml).

supplemental material for the results of statistical analysis). The triple combination of imipenem-amikacin-avibactam was bactericidal (less than 0.1% survival), achieving a 3.2-log 10 reduction in the number of CFU. The triple combination was more active than imipenem plus avibactam (3.2-versus 1.9-log 10 reductions; P Ͻ 0.05), but the difference from the results obtained with imipenem plus amikacin was not significant (3.2-versus 2.3-log 10 reductions; P ϭ 0.12). Increasing the imipenem concentration from 8 g/ml (Fig. 1a) to 32 g/ml (Fig. 1b) moderately increased the activity of imipenem alone (1.7versus 2.2-log 10 reductions), of imipenem combined with amikacin (2.3-versus 2.7log 10 reductions), and of imipenem combined with avibactam (1.9-versus 2.3-log 10 reductions), but none of these differences were statistically significant. Imipenem at 32 g/ml combined with amikacin and avibactam was the most active drug combination, achieving a 4.3-log 10 reduction.

Intramacrophage activity of imipenem alone or in combination with amikacin and avibactam. THP-1-derived macrophages were infected with M. abscessus CIP104536 S and exposed to various drugs for 2 days, and the surviving bacteria were enumerated by plating serial dilutions of macrophage lysates (Fig. 2; see Table S3 in the supplemental material for the results of statistical analysis). In the absence of antibiotic, M. abscessus CIP104536 S grew in macrophages, leading to a 100-fold increase in the number of CFU at 2 days. Imipenem at 8 g/ml (Fig. 2) partially prevented the intramacrophage growth of M. abscessus CIP104536 S (4.5-versus 100-fold increases in the number of CFU; P Ͻ 0.05). Amikacin was also active (8-fold increase in the number of CFU; P Ͻ 0.05). The combination of imipenem and amikacin was not more active than imipenem alone (4.5-versus 5.0-fold increases in the number of CFU, respectively; P ϭ 0.51). In contrast, avibactam improved the activity of imipenem, preventing the intracellular proliferation of M. abscessus CIP104536 S (1.1-versus 4.5-fold increases in the number of CFU; P Ͻ 0.05). The combination of imipenem (8 g/ml), amikacin, and avibactam was the only combination that reduced the number of intracellular bacteria (fold change in the number of CFU, 0.54). This value was significantly different from that obtained with imipenem combined with avibactam (fold change in the number of CFU, 0.54 versus 1.1; P Ͻ 0.05). Increasing the concentration of imipenem from 8 g/ml to 32 g/ml improved the activity of the drug tested alone (4.5-versus 1.6-fold change in the number of CFU) and in combination with amikacin (5.0-versus 1.1-fold change), FIG 2 Intracellular activity of imipenem (IPM; 8 g/ml and 32 g/ml) alone or in combination with the ␤-lactamase inhibitor avibactam (AVI; 16 g/ml) and amikacin (AMK; 8 g/ml) against M. abscessus CIP104536 S. Intracellular bacteria were enumerated, and the fold change in the number of CFU was determined at between days 0 and 2 postinfection. Bars represent standard deviations.

avibactam (1.1-versus 0.1-fold change), and amikacin and avibactam (0.54-versus 0.07-fold change). The latter differences were significant (P Ͻ 0.05). In conclusion, avibactam significantly improved the activity of imipenem when it was tested both with and without amikacin. Significant intracellular killing was obtained with the double and triple combinations involving imipenem at 32 g/ml and avibactam with or without amikacin.

Bla Mab is produced at a high level within macrophages. The expression of bla Mab was investigated at both the transcriptional and translational levels in M. abscessus bacteria grown in vitro or proliferating in infected macrophages. Quantitative reverse transcription-PCR (qRT-PCR) analyses indicated that the growth of M. abscessus within macrophages led to a 20-fold increase in the relative abundance of the bla Mab transcript in comparison to that in planktonic cultures performed in vitro in 7H9sB medium (Fig. 3a). Western blot analysis confirmed that Bla Mab is produced at a higher level in macrophages (Fig. 3b).

Avibactam increases the efficacy of imipenem in the zebrafish model of M. abscessus infection. The zebrafish model of M. abscessus infection ( 16) was used to assess the in vivo efficacy of imipenem following inhibition of Bla Mab by avibactam (Fig. 4). Although imipenem alone was active, avibactam further increased larval survival (P Ͻ 0.05 for both concentrations of imipenem tested). These results indicate that the production of Bla Mab during infection impairs the efficacy of imipenem and that chemical inhibition by avibactam overcomes the deleterious effect of the ␤-lactamase.

DISCUSSION

Here, we assessed whether the inhibition of the ␤-lactamase Bla Mab by avibactam improves the efficacy of imipenem against M. abscessus. The impact of the ␤-lactamase inhibitor was assessed in vitro and in infected macrophages by determining the killing activity of various drug combinations. Avibactam was also investigated by monitoring the survival of infected zebrafish embryos treated with imipenem alone or in combination with the inhibitor. In macrophages, avibactam significantly improved the activity of imipenem in all drug combinations tested, which included two dosages of imipenem with or without amikacin (Fig. 2). Imipenem combined with avibactam and amikacin was highly active in macrophages, leading to 93% intracellular killing at the highest dose tested. In the zebrafish model, imipenem alone increased the survival of the embryos, as previously reported [START_REF] Bernut | In vivo assessment of drug efficacy against Mycobacterium abscessus using the embryonic zebrafish test system[END_REF], and the efficacy of the drug was significantly improved by avibactam (Fig. 4). Amikacin could not be tested in this model due to its toxicity during larval development (data not shown). Time-kill curves showed that the triple combinations of imipenem (8 and 32 g/ml), avibactam, and amikacin were bactericidal (less than 0.1% survival; Fig. 1a andb). These associations were the most active, although statistical analysis did not demonstrate a significant impact of avibactam. The bla Mab transcript was 20-fold less abundant in M. abscessus bacteria proliferating in planktonic cultures than in M. abscessus bacteria in infected macrophages (Fig. 3). Thus, the high level of production of Bla Mab in the infected macrophages, which was confirmed by Western blotting, may account for the fact that avibactam has a greater impact on the efficacy of imipenem intracellularly than in vitro. The induction of bla Mab in macrophages indicates that the in vitro evaluation of imipenem may underestimate the impact of the ␤-lactamase Bla Mab . Of note, there is only a 2-fold difference in the MIC of imipenem against the Bla Mab -deficient mutant of M. abscessus and that against the parental strain (19).

Two ␤-lactams, cefoxitin and imipenem, are recommended for the treatment of pulmonary infections due to M. abscessus [START_REF] Floto | US Cystic Fibrosis Foundation and European Cystic Fibrosis Society consensus recommendations for the management of non-tuberculous mycobacteria in individuals with cystic fibrosis[END_REF]. Currently, there is no recommendation for the preferential use of one these drugs. We have previously shown that Bla Mab hydrolyzes imipenem with a moderate but significant catalytic efficacy (3 ϫ 10 4 M Ϫ1 s Ϫ1 ) [START_REF] Soroka | Characterization of broad-spectrum Mycobacterium abscessus class A beta-lactamase[END_REF]. Bla Mab is 4,000-fold less active for the hydrolysis of cefoxitin. The low efficacy of hydrolysis of cefoxitin by Bla Mab is likely to be irrelevant since the killing curve assay did not reveal any difference in the activity of cefoxitin, alone or in combination with amikacin, against a Bla Mab -deficient mutant of M. abscessus and the parental strain [START_REF] Lefebvre | Bactericidal and intracellular activity of ␤-lactams against Mycobacterium abscessus[END_REF]. In this study, we have shown that imipenem is highly active in macrophages, but this requires combination with avibactam due to the induction of bla Mab . Together, these data indicate that imipenem is intrinsically more active than cefoxitin, although this difference is compensated for by the higher level of hydrolysis of imipenem by the ␤-lactamase Bla Mab .

The assessments of the efficacy of drug combinations in vitro (Fig. 1), in macrophages (Fig. 2), and in the zebrafish (Fig. 4) indicate that the triple combination of avibactam, imipenem, and amikacin should be clinically evaluated, particularly in infections due to clarithromycin-resistant M. abscessus, which are often not cured by the recommended treatments [START_REF] Novosad | Treatment of Mycobacterium abscessus infection[END_REF][START_REF] Koh | Clinical significance of differentiation of Mycobacterium massiliense from Mycobacterium abscessus[END_REF][START_REF] Jarand | Clinical and microbiologic outcomes in patients receiving treatment for Mycobacterium abscessus pulmonary disease[END_REF][START_REF] Harada | Clinical and microbiological differences between Mycobacterium abscessus and Mycobacterium massiliense lung diseases[END_REF]. Unfortunately, avibactam is currently manufactured in combination with ceftazidime, which has no activity against M. abscessus [START_REF] Dubee | ␤-Lactamase inhibition by avibactam in Mycobacterium abscessus[END_REF]. Formulation of avibactam independently from any ␤-lactam partner would be necessary to provide cystic fibrosis patients access to the avibactam-imipenem-amikacin combination.

MATERIALS AND METHODS

Bacterial strains and growth conditions. M. abscessus CIP104536 (ATCC 19977) with a smooth (S) or rough (R) morphotype and the respective ␤-lactamase-deficient derivatives (Δbla Mab ) [START_REF] Dubee | ␤-Lactamase inhibition by avibactam in Mycobacterium abscessus[END_REF] were grown in Middlebrook 7H9 broth (BD-Difco, Le Pont de Claix, France) supplemented with 10% (vol/vol) oleic acid, albumin, dextrose, and catalase (OADC; BD-Difco) and 0.05% (vol/vol) Tween 80 (Sigma-Aldrich) (7H9sB) at 30°C with shaking (150 rpm) [START_REF]M24-A2. Susceptibility testing of mycobacteria, nocardiae, and other aerobic actinomycetes[END_REF].

Antibiotics. Amikacin was provided by Bristol-Myers Squibb (Rueil-Malmaison, France), and imipenem was provided by Mylan (Saint-Priest, France). Avibactam was provided by AstraZeneca. Water was the solvent used to prepare stock solutions, which were freshly prepared for each experiment and filtered using a sterilized 0.22-m-pore-size polycarbonate syringe filter (Millipore, Saint-Quentin-en-Yvelines, France).

Time-kill assay. Bottles of 20 ml of 7H9sB containing imipenem, amikacin, and avibactam alone or in combination were inoculated with exponentially growing bacteria of M. abscessus CIP104536 S (7 ϫ 10 6 CFU/ml) and incubated with shaking (150 rpm) at 30°C for 72 h. Bacteria were enumerated at 0, 48, and 72 h by plating serial dilutions prepared in sterile saline solution on lysogeny broth (LB) plates. The plates were incubated for 4 days at 30°C. The detection limit was 2 log 10 CFU/ml. The experiments were performed in triplicate.

Activity of imipenem alone or in combination with amikacin and avibactam in THP-1 macrophages. The activity of antibiotics was studied as previously described [START_REF] Lefebvre | Bactericidal and intracellular activity of ␤-lactams against Mycobacterium abscessus[END_REF]. Briefly, THP-1 cells were seeded into 24-well plates (5 ϫ 10 5 cells per 1-ml well), differentiated for 24 h, and infected with M. abscessus CIP104536 S at a multiplicity of infection of 10 for 3 h. Imipenem (8 and 32 g/ml), amikacin (8 g/ml), and avibactam (16 g/ml) alone or in combination were added to each well. The plates were incubated with 5% CO 2 at 37°C for 2 days. Macrophages were lysed with deionized water. Dilutions were plated onto LB agar plates, and the CFU were enumerated after 4 days of incubation at 30°C. The experiments were performed in triplicate.

Determination of bla Mab mRNA by qRT-PCR. Extraction of bacterial RNA from M. abscessus-infected macrophages was performed as previously described [START_REF] N=goma | Mycobacterium abscessus phospholipase C expression is induced during coculture within amoebae and enhances M. abscessus virulence in mice[END_REF]. Briefly, bacterial cells were harvested after different times postinfection, washed once with Dulbecco's modified Eagle's medium alone, and resuspended in 4 M guanidine thiocyanate solution containing ␤-mercaptoethanol. RNA was extracted and cDNA was prepared as previously described [START_REF] N=goma | Mycobacterium abscessus phospholipase C expression is induced during coculture within amoebae and enhances M. abscessus virulence in mice[END_REF]. See Table S1 in the supplemental material for the sequences of the primers used for qRT-PCR. Controls without reverse transcriptase were used with each RNA sample to rule out DNA contamination [START_REF] N=goma | Mycobacterium abscessus phospholipase C expression is induced during coculture within amoebae and enhances M. abscessus virulence in mice[END_REF]. sigA gene RNA was included as an internal control.

Mouse anti-Bla Mab antibodies. Purified Bla Mab [START_REF] Dubee | ␤-Lactamase inhibition by avibactam in Mycobacterium abscessus[END_REF] was subcutaneously injected into five BALB/c mice (Janvier, France) (20 g per mouse) with incomplete Freund's adjuvant (1/1, vol/vol) on days 1, 28, and 57. At 1 week after days 28 and 57, blood samples were obtained from the retroorbital plexus, centrifuged, and stored at Ϫ20°C until use. All procedures were performed according to institutional and national ethical guidelines under agreement 92-033-01 (Préfecture des Hauts-de-Seine, Boulogne-Billancourt, France).

Immunodetection of Bla Mab in protein extracts. Bacterial pellets were obtained from M. abscessus cells grown in vitro and in human macrophages as previously described [START_REF] N=goma | Mycobacterium abscessus phospholipase C expression is induced during coculture within amoebae and enhances M. abscessus virulence in mice[END_REF]. Briefly, bacteria were resuspended in 5 ml of cooled phosphate-buffered saline (PBS) containing 1% Triton X-100 for 5 min at room temperature. Then, 2.5 ml of cold PBS was added and the suspension was centrifuged at 2,500 ϫ g for 15 min. Bacteria were resuspended in 0.5 ml Tris-buffered saline (TBS) and lysed by sonication 3 times for 30 s each time with 1-min cooling intervals on ice. After centrifugation at 14,000 ϫ g for 10 min, the protein concentration was determined using the Bradford assay with bovine serum albumin as the standard. Proteins in crude extracts (3 g) were separated by 15% SDS-PAGE and transferred to a nitrocellulose membrane (Bio-Rad, Hercules, CA, USA), and Bla Mab was detected with mouse anti-Bla Mab antiserum (1/6,000). A peroxidase-conjugated goat anti-mouse immunoglobulin antibody (IgG; 1/4,000) was used as the secondary antibody. Rat anti-KasA antibodies were used as a loading control [START_REF] Kremer | Inhibition of InhA activity, but not KasA activity, induces formation of a KasA-containing complex in mycobacteria[END_REF].

Evaluation of imipenem in a zebrafish model of M. abscessus infection. The zebrafish model of M. abscessus infection was used to assess the in vivo activity of imipenem alone or in combination with avibactam. The TdTomato-expressing M. abscessus CIP104536 R derivative was injected in zebrafish embryos according to procedures described earlier [START_REF] Bernut | Deciphering and imaging pathogenesis and cording of Mycobacterium abscessus in zebrafish embryos[END_REF]. Briefly, systemic infections were carried by the injection of 150 CFU into the caudal vein of embryos at 30 h postfertilization. Infected larvae were exposed to various imipenem concentrations (180 and 360 g/ml) alone or in combination with avibactam at 50 g/ml. Drug-containing water was renewed daily for 5 days from day 1 to day 6 postinfection. The viability of the infected embryos was evaluated daily by assessment of cardiac activity. The zebrafish experiments were conducted in accordance with the guidelines from the European Union for the handling of laboratory animals (http://ec.europa.eu/environment/chemicals/lab_animals/home_en.htm) and approved by the Direction Sanitaire et Vétérinaire de l'Hérault et Comité d'Ethique pour l'Expérimentation Animale de la Région Languedoc Roussillon under reference number CEEA-LR-1145.

Statistical analysis. The Mann-Whitney U test and the Kruskal-Wallis test were used to compare the intracellular activities of the antibiotics. For the zebrafish infection model, experiments were performed at least in triplicate. Data from the replicates were pooled for the construction and comparison of survival curves. The efficacy of imipenem alone or in combination with avibactam was compared using the log-rank test. All statistical analyses were performed with EPI Info software (version 7.1.3; Centers for Disease Control and Prevention, Atlanta, GA).

SUPPLEMENTAL MATERIAL

Supplemental material for this article may be found at https://doi.org/10.1128/ AAC.02440-16.

TEXT S1, PDF file, 0.05 MB.

SUPPLEMENTARY TABLES

TABLE S1 Sequence of the primers

Primer Sequence F-MAB2875-Q-PCR 5'-TCCATTCGGGCAGCAGTTC -3' R-MAB2875-Q-PCR 5'-GTCGGCTTACCTTTGGTGG -3' F-SigA 5'-TCCGAGAAAGACAAGGCTTC -3' R-SigA 5'-CCAGCTCGACTTCCTCTTCG -3'

FIG 3 FIG 4

 34 FIG 3 Production of Bla Mab in vitro and in macrophages. (a) Quantification of bla Mab by qRT-PCR. RNA was isolated from wild-type strain CIP CIP104536 S grown in vitro in 7H9 medium for 48 h or in human J774 macrophages for 24 h, 48 h, 72 h, and 96 h. sigA rRNA was used as an internal standard. The values are the ratio of intramacrophage growth to in vitro growth. Results are expressed as the means Ϯ standard deviations from three experiments performed in triplicate. Bars represent standard deviations. (b) Immunodetection of Bla Mab . Protein extracts were prepared from wild-type strain CIP104536 S and its Δbla Mab derivative grown in vitro in 7H9 medium for 48 h or in human J774 macrophages for 1 h, 24 h, and 48 h. Proteins (3 g) were separated by 15% SDS-PAGE. Immunodetection was performed with a mouse immune serum specific for Bla Mab and a peroxidase-conjugated goat anti-mouse immunoglobulin antibody. Immunodetection of KasA was used as a loading control.

TABLE S2

 S2

Statistical analysis of the killing activity of imipenem (IPM) alone or in combination with avibactam (AVI) and amikacin (AMK) against M. abscessus CIP104536 S.

TABLE S3

 S3

							P value for the indicated comparison (Mann-Whitney U test)
			control	AMK32	IPM8	IPM32	AMK32+AVI4 IPM8+AMK32 IPM8+AVI4 IPM32+AMK32 IPM32+AVI4 IPM8+AMK32+AVI4 IPM32+AMK32+AVI4
			(9.44)	(6.33)	(5.09)	(4.58)		(6.58)	(4.50)	(4.92)	(4.12)	(4.48)	(3.57)	(2.48)
	control	(9.44)	NA	0.0495	0.0495	0.0495		0.0495	0.0495	0.0495	0.0495	0.0495	0.0495	0.0495
	AMK32	(6.33)		NA	0.2752	0.0495		0.5127	0.0495	0.1266	0.0495	0.0495	0.0495	0.0495
	IPM8	(5.09)			NA	0.1266		0.0495	0.1266	0.5127	0.1266	0.1266	0.0495	0.0495
	IPM32	(4.58)				NA		0.0495	0.8273	0.2752	0.2752	0.8273	0.1840	0.0495
	AMK32+AVI4	(6.58)						NA	0.0495	0.0495	0.0495	0.0495	0.0495	0.0495
	IPM8+AMK32	(4.50)							NA	0.5127	0.5127	0.8273	0.1266	0.0495
	IPM8+AVI4	(4.92)								NA	0.1266	0.3758	0.0495	0.0495
	IPM32+AMK32	(4.12)									NA	0.5127	0.8273	0.3758
	IPM32+AVI4	(4.48)										NA	0.1266	0.0495
	IPM8+AMK32+AVI4	(3.57)											NA	0.2752
	IPM32+AMK32+AVI4	(2.48)												NA
		Statistical analysis of the intracellular activity of imipenem (IPM) alone or in
	combination with avibactam (AVI) and amikacin (AMK) against M. abscessus CIP104536 S.
			control	AVI16	AMK8	IPM8	IPM32	AMK8+AVI16 IPM8+AMK8 IPM8+AVI16 IPM32+AMK8 IPM32+AVI16 IPM8+AMK8+AVI16 IPM32+AMK8+AVI16
			(102)	(151)	(8.00)	(4.54)	(1.57)	(104)	(4.96)	(1.13)	(1.14)	(0.10)	(0.54)	(0.07)
	control	(102)	NA	0.1266	0.0495	0.0495	0.0495	0.8273	0.0495	0.0495	0.0495	0.0495	0.0495	0.0495
	AVI16	(151)		NA	0.0495	0.0495	0.0495	0.0495	0.0495	0.0495	0.0495	0.0495	0.0495	0.0495
	AMK8	(8.00)			NA	0.0495	0.0495	0.0495	0.0495	0.0495	0.0495	0.0495	0.0495	0.0495
	IPM8	(4.54)				NA	0.0495	0.0495	0.5127	0.0495	0.0495	0.0495	0.0495	0.0495
	IPM32	(1.57)					NA	0.0495	0.0495	0.1266	0.0495	0.0495	0.0495	0.0495
	AMK8+AVI16	(104)						NA	0.0495	0.0495	0.0495	0.0495	0.0495	0.0495
	IPM8+AMK8	(4.96)							NA	0.0495	0.0495	0.0495	0.0495	0.0495
	IPM8+AVI16	(1.13)								NA	0.8273	0.0495	0.0495	0.0495
	IPM32+AMK8	(1.14)									NA	0.0495	0.0495	0.0495
	IPM32+AVI16	(0.10)										NA	0.0495	0.2752
	IPM8+AMK32+AVI16	(0.54)											NA	0.0495
	IPM32+AMK32+AVI16	(0.07)												NA

P value for the indicated comparison (Mann-Whitney U test)

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ACKNOWLEDGMENTS

We thank AstraZeneca for providing the avibactam. This work was supported by the Vaincre la Mucoviscidose and l'Association Grégory Lemarchal (Ph.D. fellowship to A.-L.L. and grant RF20150501376 to J.-L.M., J.-L.H., and L.K.) and the ANR DIMYVIR (to J.-L.H. and L.K.). J.-L.M. has received consulting fees (as a scientific advisor for ceftaroline) and reimbursement of travel expenses (attendance at the 54th Interscience Conference on Antimicrobial Agents and Chemotherapy, 2014) from AstraZeneca. All other authors have no conflicts to declare.