Mutations in genes lpxL1, bamA, and pmrB impair the susceptibility of cystic fibrosis strains of Pseudomonas aeruginosa to murepavadin.

Murepavadin is a peptidomimetic exhibiting specific inhibitory activity against Pseudomonas species. In the present study, its in vitro activity was assessed on 230 cystic fibrosis (CF) strains of Pseudomonas aeruginosa isolated from 12 French hospitals, in comparison with 12 other antipseudomonal antibiotics. Although murepavadin is still in preclinical stage of development, 9.1% (n = 21) of strains had a minimum inhibitory concentration (MIC) >4 mg/L, a level at least 128-fold higher than the modal MIC value of the whole collection (≤0.06 mg/L). Whole-genome sequencing of these 21 strains along with more susceptible isogenic counterparts coexisting in the same patients revealed diverse mutations in genes involved in the synthesis (lpxL1 and lpxL2) or transport of lipopolysaccharides (bamA, lptD, and msbA), or encoding histidine kinases of two-component systems (pmrB and cbrA). Allelic replacement experiments with wild-type reference strain PAO1 confirmed that alteration of genes lpxL1, bamA, and/or pmrB can decrease the murepavadin susceptibility from 8- to 32-fold. Furthermore, we found that specific amino acid substitutions in histidine kinase PmrB (G188D, Q105P, and D45E) reduce the susceptibility of P. aeruginosa to murepavadin, colistin, and tobramycin, three antibiotics used or intended to be used (murepavadin) in aerosols to treat colonized CF patients. Whether colistin or tobramycin may select mutants resistant to murepavadin or the opposite needs to be addressed by clinical studies.

Cefiderocol susceptibility of Achromobacter spp.: study of an accurately identified collection of 230 strains.

BACKGROUND: Achromobacter spp. are opportunistic pathogens, mostly infecting immunocompromised patients and patients with cystic fibrosis (CF) and considered as difficult-to-treat pathogens due to both intrinsic resistance and the possibility of acquired antimicrobial resistance. Species identification remains challenging leading to imprecise descriptions of resistance in each taxon. Cefiderocol is a broad-spectrum siderophore cephalosporin increasingly used in the management of Achromobacter infections for which susceptibility data remain scarce. We aimed to describe the susceptibility to cefiderocol of a collection of Achromobacter strains encompassing different species and isolation sources from CF or non-CF (NCF) patients. METHODS: We studied 230 Achromobacter strains (67 from CF, 163 from NCF patients) identified by nrdA gene-based analysis, with available susceptibility data for piperacillin-tazobactam, meropenem and trimethoprim-sulfamethoxazole. Minimal inhibitory concentrations (MICs) of cefiderocol were determined using the broth microdilution reference method according to EUCAST guidelines. RESULTS: Strains belonged to 15 species. A. xylosoxidans represented the main species (71.3%). MICs ranged from ≤ 0.015 to 16 mg/L with MIC50/90 of ≤ 0.015/0.5 mg/L overall and 0.125/2 mg/L against 27 (11.7%) meropenem-non-susceptible strains. Cefiderocol MICs were not related to CF/NCF origin or species although A. xylosoxidans MICs were statistically lower than those of other species considered as a whole. Considering the EUCAST non-species related breakpoint (2 mg/L), 228 strains (99.1%) were susceptible to cefiderocol. The two cefiderocol-resistant strains (A. xylosoxidans from CF patients) represented 3.7% of meropenem-non-susceptible strains and 12.5% of MDR strains. CONCLUSIONS: Cefiderocol exhibited excellent in vitro activity against a large collection of accurately identified Achromobacter strains, irrespective of species and origin.

The unusual structure of Ruminococcin C1 antimicrobial peptide confers clinical properties.

The emergence of superbugs developing resistance to antibiotics and the resurgence of microbial infections have led scientists to start an antimicrobial arms race. In this context, we have previously identified an active RiPP, the Ruminococcin C1, naturally produced by Ruminococcus gnavus E1, a symbiont of the healthy human intestinal microbiota. This RiPP, subclassified as a sactipeptide, requires the host digestive system to become active against pathogenic Clostridia and multidrug-resistant strains. Here we report its unique compact structure on the basis of four intramolecular thioether bridges with reversed stereochemistry introduced posttranslationally by a specific radical-SAM sactisynthase. This structure confers to the Ruminococcin C1 important clinical properties including stability to digestive conditions and physicochemical treatments, a higher affinity for bacteria than simulated intestinal epithelium, a valuable activity at therapeutic doses on a range of clinical pathogens, mediated by energy resources disruption, and finally safety for human gut tissues.

Poly-L-Lysine to Fight Antibiotic Resistances of Pseudomonas aeruginosa.

Pseudomonas aeruginosa is a major hospital-associated pathogen that can cause severe infections, most notably in patients with cystic fibrosis (CF) or those hospitalized in intensive care units. Given its remarkable ability to resist antibiotics, P. aeruginosa eradication has grown more challenging. Therefore, there is an urgent need to discover and develop new strategies that can counteract P. aeruginosa-resistant strains. Here, we evaluated the efficacy of poly-L-lysine (pLK) in combination with commonly used antibiotics as an alternative treatment option against P. aeruginosa. First, we demonstrated by scanning electron microscopy that pLK alters the integrity of the surface membrane of P. aeruginosa. We also showed using a fluorometry test that this results in an enhanced permeability of the bacteria membrane. Based on these data, we further evaluated the effect of the combinations of pLK with imipenem, ceftazidime, or aztreonam using the broth microdilution method in vitro. We found synergies in terms of bactericidal effects against either sensitive or resistant P. aeruginosa strains, with a reduction in bacterial growth (up to 5-log10 compared to the control). Similarly, these synergistic and bactericidal effects were confirmed ex vivo using a 3D model of human primary bronchial epithelial cells maintained in an air-liquid interface. In conclusion, pLK could be an innovative antipseudomonal molecule, opening its application as an adjuvant antibiotherapy against drug-resistant P. aeruginosa strains.

6-Polyaminosteroid Squalamine Analogues Display Antibacterial Activity against Resistant Pathogens.

A series of 6-polyaminosteroid analogues of squalamine were synthesized with moderate to good yields and evaluated for their in vitro antimicrobial properties against both susceptible and resistant Gram-positive (vancomycin-resistant Enterococcus faecium and methicillin-resistant Staphylococcus aureus) and Gram-negative (carbapenem-resistant Acinetobacter baumannii and Pseudomonas aeruginosa) bacterial strains. Minimum inhibitory concentrations against Gram-positive bacteria ranged from 4 to 16 µg/mL for the most effective compounds, 4k and 4n, and showed an additive or synergistic effect with vancomycin or oxacillin. On the other hand, the derivative 4f, which carries a spermine moiety like that of the natural trodusquemine molecule, was found to be the most active derivative against all the resistant Gram-negative bacteria tested, with an MIC value of 16 µg/mL. Our results suggest that 6-polyaminosteroid analogues of squalamine are interesting candidates for Gram-positive bacterial infection treatments, as well as potent adjuvants to fight Gram-negative bacterial resistance.

Modified Fluoroquinolones as Antimicrobial Compounds Targeting Chlamydia trachomatis.

Chlamydia trachomatis causes the most common sexually transmitted bacterial infection and trachoma, an eye infection. Untreated infections can lead to sequelae, such as infertility and ectopic pregnancy in women and blindness. We previously enhanced the antichlamydial activity of the fluoroquinolone ciprofloxacin by grafting a metal chelating moiety onto it. In the present study, we pursued this pharmacomodulation and obtained nanomolar active molecules (EC50) against this pathogen. This gain in activity prompted us to evaluate the antibacterial activity of this family of molecules against other pathogenic bacteria, such as Neisseria gonorrhoeae and bacteria from the ESKAPE group. The results show that the novel molecules have selectively improved activity against C. trachomatis and demonstrate how the antichlamydial effect of fluoroquinolones can be enhanced.

Mechanisms of Resistance to Ceftolozane/Tazobactam in Pseudomonas aeruginosa: Results of the GERPA Multicenter Study.

Resistance mechanisms of Pseudomonas aeruginosa to ceftolozane/tazobactam (C/T) were assessed on a collection of 420 nonredundant strains nonsusceptible to ceftazidime (MIC > 8 µg/ml) and/or imipenem (>4 µg/ml), collected by 36 French hospital laboratories over a one-month period (the GERPA study). Rates of C/T resistance (MIC > 4/4 µg/ml) were equal to 10% in this population (42/420 strains), and 23.2% (26/112) among the isolates resistant to both ceftazidime and imipenem. A first group of 21 strains (50%) was found to harbor various extended-spectrum ß-lactamases (1 OXA-14; 2 OXA-19; 1 OXA-35; 1 GES-9; and 3 PER-1), carbapenemases (2 GES-5; 1 IMP-8; and 8 VIM-2), or both (1 VIM-2/OXA-35 and 1 VIM-4/SHV-2a). All the strains of this group belonged to widely distributed epidemic clones (ST111, ST175, CC235, ST244, ST348, and ST654), and were highly resistant to almost all the antibiotics tested except colistin. A second group was composed of 16 (38%) isolates moderately resistant to C/T (MICs from 8/4 to 16/4 µg/ml), of which 7 were related to international clones (ST111, ST253, CC274, ST352, and ST386). As demonstrated by targeted mass spectrometry, cloxacillin-based inhibition tests, and gene blaPDC deletion experiments, this resistance phenotype was correlated with an extremely high production of cephalosporinase PDC. In part accounting for this strong PDC upregulation, genomic analyses revealed the presence of mutations in the regulator AmpR (D135N/G in 6 strains) and enzymes of the peptidoglycan recycling pathway, such as AmpD, PBP4, and Mpl (9 strains). Finally, all of the 5 (12%) remaining C/T-resistant strains (group 3) appeared to encode PDC variants with mutations known to improve the hydrolytic activity of the ß-lactamase toward ceftazidime and C/T (F147L, ΔL223-Y226, E247K, and N373I). Collectively, our results highlight the importance of both intrinsic and transferable mechanisms in C/T-resistant P. aeruginosa Which mutational events lead some clinical strains to massively produce the natural cephalosporinase PDC remains incompletely understood.

Performance of disc diffusion, MIC gradient tests and Vitek 2 for ceftolozane/tazobactam and ceftazidime/avibactam susceptibility testing of Pseudomonas aeruginosa.

OBJECTIVES: To assess performance of disc diffusion, gradient tests and Vitek 2 system to determine the susceptibility of clinical Pseudomonas aeruginosa strains to ceftolozane/tazobactam (C/T) and ceftazidime/avibactam (CZA). METHODS: Two-hundred non-duplicate P. aeruginosa strains isolated by 47 French medical laboratories were selected to cover a wide range of C/T and CZA MICs. Performance of C/T disc (30/10 µg, Bio-Rad), CZA discs (10/4 µg) (Thermo Fisher and Bio-Rad), C/T and CZA gradient tests (Etest, BioMérieux; MIC Test Strip, Liofilchem), and AST-XN12 card of Vitek 2 system (BioMérieux) were compared with a broth microdilution (BMD) method (Thermo Fisher). MIC and disc results were interpreted using current EUCAST breakpoints. RESULTS: Twenty percent and 17% of strains were resistant to C/T and CZA, respectively. All the methods tested satisfactorily determined the susceptibility of P. aeruginosa to C/T [Category Agreement (CA) ≥95%] except the disc diffusion method. Because of the high rates of Major Errors (MEs) (12.5%), this latter method tends to overestimate the resistance. For CZA, only the gradient tests yielded more than 90% of CA. The Vitek 2 system and disc diffusion misclassified 18.1%, 10.1% (disc Bio-Rad) and 11.9% (disc Thermo Fisher) of susceptible strains, respectively. CONCLUSIONS: The gradient tests (MIC Test Strip and Etest) and Vitek 2 card XN12 performed the best to determine the susceptibility of P. aeruginosa to C/T, whereas gradient tests were an acceptable alternative to BMD to assess CZA susceptibility.

The Efflux Pump MexXY/OprM Contributes to the Tolerance and Acquired Resistance of Pseudomonas aeruginosa to Colistin.

The intrinsic resistance of Pseudomonas aeruginosa to polymyxins in part relies on the addition of 4-amino-4-deoxy-l-arabinose (Ara4N) molecules to the lipid A of lipopolysaccharide (LPS), through induction of operon arnBCADTEF-ugd (arn) expression. As demonstrated previously, at least three two-component regulatory systems (PmrAB, ParRS, and CprRS) are able to upregulate this operon when bacteria are exposed to colistin. In the present study, gene deletion experiments with the bioluminescent strain PAO1::lux showed that ParRS is a key element in the tolerance of P. aeruginosa to this last-resort antibiotic (i.e., resistance to early drug killing). Other loci of the ParR regulon, such as those encoding the efflux proteins MexXY (mexXY), the polyamine biosynthetic pathway PA4773-PA4774-PA4775, and Ara4N LPS modification process (arnBCADTEF-ugd), also contribute to the bacterial tolerance in an intricate way with ParRS. Furthermore, we found that both stable upregulation of the arn operon and drug-induced ParRS-dependent overexpression of the mexXY genes accounted for the elevated resistance of pmrB mutants to colistin. Deletion of the mexXY genes in a constitutively activated ParR mutant of PAO1 was associated with significantly increased expression of the genes arnA, PA4773, and pmrA in the absence of colistin exposure, thereby highlighting a functional link between the MexXY/OprM pump, the PA4773-PA4774-PA4775 pathway, and Ara4N-based modification of LPS. The role played by MexXY/OprM in the adaptation of P. aeruginosa to polymyxins opens new perspectives for restoring the susceptibility of resistant mutants through the use of efflux inhibitors.

Coordinate overexpression of two RND efflux systems, ParXY and TtgABC, is responsible for multidrug resistance in Pseudomonas putida.

Resistance Nodulation cell Division (RND) efflux pumps are known to contribute to the tolerance of Pseudomonas putida to aromatic hydrocarbons, but their role in antibiotic resistance has not been fully elucidated. In this study, two types of single-step multidrug-resistant (MDR) mutants were selected in vitro from reference strain KT2440. Mutants of the first type were more resistant to fluoroquinolones and ß-lactams except imipenem, and overproduced the efflux system TtgABC as a result of mutations occurring in regulator TtgR. In addition to TtgABC, mutants of the second type such as HPG-5 were found to upregulate a novel RND pump, dubbed ParXY/TtgC, which accommodates cefepim, fluoroquinolones and aminoglycosides. As demonstrated by gene deletion experiments, TtgABC and ParXY/TtgC are both under the positive control of a two-component system, PpeRS. Whole-genome sequence analyses revealed that mutant HPG-5 harbours a mutation inactivating the gene (sucD) of succinyl-CoA synthetase, an enzyme of the tricarboxylic cycle. Disruption of sucD in strain KT2440 reproduced the resistance phenotype of HPG-5, and activated the glyoxylate shunt. Finally, identification of two MDR clinical strains of P. putida that jointly overexpress TtgABC and ParXY/TtgC, of which one is a sucD mutant, highlights the role of these efflux systems as determinants of antibiotic resistance.

Production of Norspermidine Contributes to Aminoglycoside Resistance in pmrAB Mutants of Pseudomonas aeruginosa.

Emergence of resistance to polymyxins in Pseudomonas aeruginosa is mainly due to mutations in two-component systems that promote the addition of 4-amino-4-deoxy-l-arabinose to the lipopolysaccharide (LPS) through upregulation of operon arnBCADTEF-ugd (arn) expression. Here, we demonstrate that mutations occurring in different domains of histidine kinase PmrB or in response regulator PmrA result in coresistance to aminoglycosides and colistin. All seventeen clinical strains tested exhibiting such a cross-resistance phenotype were found to be pmrAB mutants. As shown by gene deletion experiments, the decreased susceptibility of the mutants to aminoglycosides was independent from operon arn but required the efflux system MexXY-OprM and the products of three genes, PA4773-PA4774-PA4775, that are cotranscribed and activated with genes pmrAB Gene PA4773 (annotated as speD2 in the PAO1 genome) and PA4774 (speE2) are predicted to encode enzymes involved in biosynthesis of polyamines. Comparative analysis of cell surface extracts of an in vitro selected pmrAB mutant, called AB16.2, and derivatives lacking PA4773, PA4774, and PA4775 revealed that these genes were needed for norspermidine production via a pathway that likely uses 1,3-diaminopropane, a precursor of polyamines. Altogether, our results suggest that norspermidine decreases the self-promoted uptake pathway of aminoglycosides across the outer membrane and, thereby, potentiates the activity of efflux pump MexXY-OprM.

Acquisition of class C ß-lactamase PAC-1 by ST664 strains of Pseudomonas aeruginosa.

Four ST664 (serotype O:5) strains of Pseudomonas aeruginosa highly resistant to antibiotics including ceftolozane/tazobactam and ceftazidime/avibactam but susceptible to colistin, were found to harbor the rare class C ß-lactamase PAC-1 encoding gene on a chromosomally-located Tn1721-like transposon. Gene bla PAC-1 was associated with the 16S rRNA methylase determinant rmtF2, that confers pan-aminoglycoside resistance. These genotypically-related strains were isolated in repatriated patients from Mauricius and Afghanistan and close to a lineage reported in Nepal, Pakistan and India.

Azetidine-Containing Alkaloids Produced by a Quorum-Sensing Regulated Nonribosomal Peptide Synthetase Pathway in Pseudomonas aeruginosa.

Pseudomonas aeruginosa displays an impressive metabolic versatility, which ensures its survival in diverse environments. Reported herein is the identification of rare azetidine-containing alkaloids from P. aeruginosa PAO1, termed azetidomonamides, which are derived from a conserved, quorum-sensing regulated nonribosomal peptide synthetase (NRPS) pathway. Biosynthesis of the azetidine motif has been elucidated by gene inactivation, feeding experiments, and biochemical characterization in vitro, which involves a new S-adenosylmethionine-dependent enzyme to produce azetidine 2-carboxylic acid as an unusual building block of NRPS. The mutants of P. aeruginosa unable to produce azetidomonamides had an advantage in growth at high cell density in vitro and displayed rapid virulence in Galleria mellonella model, inferring functional roles of azetidomonamides in the host adaptation. This work opens the avenue to study the biological functions of azetidomonamides and related compounds in pathogenic and environmental bacteria.

Mutations in Gene fusA1 as a Novel Mechanism of Aminoglycoside Resistance in Clinical Strains of Pseudomonas aeruginosa.

Resistance of clinical strains of Pseudomonas aeruginosa to aminoglycosides can result from production of transferable aminoglycoside-modifying enzymes, of 16S rRNA methylases, and/or mutational derepression of intrinsic multidrug efflux pump MexXY(OprM). We report here the characterization of a new type of mutant that is 4- to 8-fold more resistant to 2-deoxystreptamine derivatives (e.g., gentamicin, amikacin, and tobramycin) than the wild-type strain PAO1. The genetic alterations of three in vitro mutants were mapped on fusA1 and found to result in single amino acid substitutions in domains II, III, and V of elongation factor G (EF-G1A), a key component of translational machinery. Transfer of the mutated fusA1 alleles into PAO1 reproduced the resistance phenotype. Interestingly, fusA1 mutants with other amino acid changes in domains G, IV, and V of EF-G1A were identified among clinical strains with decreased susceptibility to aminoglycosides. Allelic-exchange experiments confirmed the relevance of these latter mutations and of three other previously reported alterations located in domains G and IV. Pump MexXY(OprM) partly contributed to the resistance conferred by the mutated EF-G1A variants and had additive effects on aminoglycoside MICs when mutationally upregulated. Altogether, our data demonstrate that cystic fibrosis (CF) and non-CF strains of P. aeruginosa can acquire a therapeutically significant resistance to important aminoglycosides via a new mechanism involving mutations in elongation factor EF-G1A.

Toxic Electrophiles Induce Expression of the Multidrug Efflux Pump MexEF-OprN in Pseudomonas aeruginosa through a Novel Transcriptional Regulator, CmrA.

The multidrug efflux system MexEF-OprN is produced at low levels in wild-type strains of Pseudomonas aeruginosa However, in so-called nfxC mutants, mutational alteration of the gene mexS results in constitutive overexpression of the pump, along with increased resistance of the bacterium to chloramphenicol, fluoroquinolones, and trimethoprim. In this study, analysis of in vitro-selected chloramphenicol-resistant clones of strain PA14 led to the identification of a new class of MexEF-OprN-overproducing mutants (called nfxC2) exhibiting alterations in an as-yet-uncharacterized gene, PA14_38040 (homolog of PA2047 in strain PAO1). This gene is predicted to encode an AraC-like transcriptional regulator and was called cmrA (for chloramphenicol resistance activator). In nfxC2 mutants, the mutated CmrA increases its proper gene expression and upregulates the operon mexEF-oprN through MexS and MexT, resulting in a multidrug resistance phenotype without significant loss in bacterial virulence. Transcriptomic experiments demonstrated that CmrA positively regulates a small set of 11 genes, including PA14_38020 (homolog of PA2048), which is required for the MexS/T-dependent activation of mexEF-oprN PA2048 codes for a protein sharing conserved domains with the quinol monooxygenase YgiN from Escherichia coli Interestingly, exposure of strain PA14 to toxic electrophilic molecules (glyoxal, methylglyoxal, and cinnamaldehyde) strongly activates the CmrA pathway and upregulates MexEF-OprN and, thus, increases the resistance of P. aeruginosa to the pump substrates. A picture emerges in which MexEF-OprN is central in the response of the pathogen to stresses affecting intracellular redox homeostasis.

Amino Acid Substitutions Account for Most MexS Alterations in Clinical nfxC Mutants of Pseudomonas aeruginosa.

Multidrug-resistant mutants ofPseudomonas aeruginosathat overproduce the active efflux system MexEF-OprN (callednfxCmutants) have rarely been characterized in the hospital setting. Screening of 221 clinical strains exhibiting a reduced susceptibility to ciprofloxacin (a substrate of MexEF-OprN) and imipenem (a substrate of the negatively coregulated porin OprD) led to the identification of 43 (19.5%)nfxCmutants. Subsequent analysis of 22 nonredundant mutants showed that, in contrast to theirin vitro-selected counterparts, only 3 of them (13.6%) harbored a disruptedmexSgene, which codes for the oxidoreductase MexS, whose inactivation is known to activate themexEF-oprNoperon through a LysR-type regulator, MexT. Nine (40.9%) of the clinicalnfxCmutants contained single amino acid mutations in MexS, and these were associated with moderate effects on resistance and virulence factor production in 8/9 strains. Finally, the remaining 10 (45.5%)nfxCmutants did not display mutations in any of the regulators known to controlmexEF-oprNexpression (themexS,mexT,mvaT, andampRgenes), confirming that other loci are responsible for pump upregulation in patients. Collectively, these data demonstrate thatnfxCmutants are probably more frequent in the hospital than previously thought and have genetic and phenotypic features somewhat different from those ofin vitro-selected mutants.

Phenotype and toxicity of the recently discovered exlA-positive Pseudomonas aeruginosa strains collected worldwide.

We recently identified a hypervirulent strain of Pseudomonas aeruginosa, differing significantly from the classical strains in that it lacks the type 3 secretion system (T3SS), a major determinant of P. aeruginosa virulence. This new strain secretes a novel toxin, called ExlA, which induces plasma membrane rupture in host cells. For this study, we collected 18 other exlA-positive T3SS-negative strains, analyzed their main virulence factors and tested their toxicity in various models. Phylogenetic analysis revealed two groups. The strains were isolated on five continents from patients with various pathologies or in the environment. Their proteolytic activity and their motion abilities were highly different, as well as their capacity to infect epithelial, endothelial, fibroblastic and immune cells, which correlated directly with ExlA secretion levels. In contrast, their toxicity towards human erythrocytes was limited. Some strains were hypervirulent in a mouse pneumonia model and others on chicory leaves. We conclude that (i) exlA-positive strains can colonize different habitats and may induce various infection types, (ii) the strains secreting significant amounts of ExlA are cytotoxic for most cell types but are poorly hemolytic, (iii) toxicity in planta does not correlate with ExlA secretion.