Synergistic effect of tryptamine-urea derivatives to overcome the chromosomally-mediated colistin resistance in Klebsiella pneumoniae.

Colistin is considered as the last-resort antibiotics to treat multi-drug resistant Gram-negative bacterial infections in humans. However, the clinical use of colistin was limited because of the apparition of chromosomal mutations and mobile colistin resistance genes in bacterial isolates. One promising strategy is to combine existing antibiotics with promising non-antibiotics to overcome the widespread emergence of antibiotic-resistant pathogens. Moreover, colistin resistance would be regulated by two component systems PhoP/PhoQ which leads to permanent synthesis of cationic groups compensating for Mg2+ deficiency. In this study, the synthesis of a small library of tryptamine urea derivatives was carried out. In addition, antibiotic susceptibility, antibiotic adjuvant screening and checkerboard assays were used to investigate the antibacterial activity of these synthesized compounds and the potential synergistic activity of their combination with colistin. Conformational analysis of the docked binding modes of the active compound in the predicted binding pocket of bacterial response regulator PhoP were carried out, to see if the active compound inhibits PhoP which is involved in colistin resistance. Finally, hemolytic activity studies have been conducted on the most active compound.

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.

Antimicrobial activity of antibiotics on biofilm formed by Staphylococcus aureus and Pseudomonas aeruginosa in an open microfluidic model mimicking the diabetic foot environment.

BACKGROUND: Diabetic foot infections (DFIs) represent a public health problem because of their frequency and the severity of their consequences, i.e. amputation and mortality. Polymicrobial biofilms on the skin surface of these ulcers complicate wound healing. Few in vitro models exist to study the antibiotics activity in this context. OBJECTIVES: This study evaluated the in vitro activity of antibiotics against the two main bacteria isolated in DFI, Staphylococcus aureus and Pseudomonas aeruginosa, using a dynamic system (BioFlux™ 200) and a chronic wound-like medium (CWM) that mimic the foot ulcer environment. METHODS: Reference strains and two pairs of clinical S. aureus and P. aeruginosa isolated together from a DFI were cultivated in brain heart infusion and CWM media during 72 h at 37°C, alone and combined in the BioFlux™ 200 system. Antibiotic activity was evaluated after a mechanical debridement. The activities were measured by the reduction of biofilm percentage of bacteria in the microfluidic system using microscopy. RESULTS: Daptomycin for S. aureus and ceftazidime for P. aeruginosa showed excellent activity to reduce biofilm biomass, whereas linezolid action was more mitigated and dalbavancin was ineffective. Ceftazidime + daptomycin presented the most potent in vitro activity on a mixed biofilm. CONCLUSIONS: The combination of CWM and the BioFlux™ microfluidic system represents a valuable tool to screen the potential antimicrobial activity of antibiotics under conditions mimicking those encountered in DFI. It could help clinicians in their management of chronic wounds.

Contribution of efflux and mutations in fluoroquinolone susceptibility in MDR enterobacterial isolates: a quantitative and molecular study.

OBJECTIVES: The emergence of MDR strains is a public health problem in the management of associated infections. Several resistance mechanisms are present, and antibiotic efflux is often found at the same time as enzyme resistance and/or target mutations. However, in the laboratory routinely, only the latter two are identified and the prevalence of antibiotic expulsion is underestimated, causing a misinterpretation of the bacterial resistance phenotype. The development of a diagnostic system to quantify the efflux routinely would thus improve the management of patients. METHODS: A quantitative technique based on detection of clinically used fluoroquinolones was investigated in Enterobacteriaceae clinical strains with a high or basal efflux activity. The detail of efflux involvement was studied from MIC determination and antibiotic accumulation inside bacteria. WGS was carried out on selected strains to determine the genetic background associated with efflux expression. RESULTS: Only 1 Klebsiella pneumoniae isolate exhibited a lack of efflux whereas 13 isolates had a basal efflux and 8 presented efflux pump overexpression. The antibiotic accumulation evidenced the efficacy of the efflux mechanism in strains, and the contribution of dynamic expulsion versus target mutations in fluoroquinolone susceptibility. CONCLUSIONS: We confirmed that phenylalanine arginine ß-naphthylamide is not a reliable marker of efflux due to the affinity of the AcrB efflux pump for different substrates. We have developed an accumulation test that can be used efficiently on clinical isolates collected by the biological laboratory. The experimental conditions and protocols ensure a robust assay that with improvements in practice, expertise and equipment could be transferred to the hospital laboratory to diagnose the contribution of efflux in Gram-negative bacteria.

Continuous infusion of meropenem-vaborbactam for a KPC-3-producing Klebsiella pneumoniae bloodstream infection in a critically ill patient with augmented renal clearance.

PURPOSE: To demonstrate the feasibility of continuous infusion of meropenem-vaborbactam to optimize the treatment of carbapenem-resistant Enterobacterales. METHODS: Report of a case of a Klebsiella pneumoniae carbapenemase (KPC)-producing K. pneumoniae bloodstream infection comfirmed by whole genome sequencing and therapeutic drug monitoring (TDM) of meropenem. RESULTS: A patient with augmented renal clearance (ARC) went into septic shock caused by an ST11 KPC-3-producing K. pneumoniae bloodstream infection that was successfully treated with a continuous infusion of meropenem-vaborbactam at a dosage of 1 g/1 g q4h as a 4-h infusion. TDM confirmed sustained concentrations of meropenem ranging from 8 to 16 mg/L throughout the dosing interval. CONCLUSION: Continuous infusion of meropenem-vaborbactam was feasible. It could be appropriate for optimizing the management of critically ill patients with ARC, as it resulted in antibiotic concentrations above the minimum inhibitory concentration for susceptible carbapenem-resistant Enterobacterales (up to 8 mg/L) throughout the dosing interval.

Effect of Antibiotic Exposure on Staphylococcus epidermidis Responsible for Catheter-Related Bacteremia.

Coagulase-negative staphylococci (CoNS) and especially Staphylococcus epidermidis are responsible for health care infections, notably in the presence of foreign material (e.g., venous or central-line catheters). Catheter-related bacteremia (CRB) increases health care costs and mortality. The aim of our study was to evaluate the impact of 15 days of antibiotic exposure (ceftobiprole, daptomycin, linezolid and vancomycin) at sub-inhibitory concentration on the resistance, fitness and genome evolution of 36 clinical strains of S. epidermidis responsible for CRB. Resistance was evaluated by antibiogram, the ability to adapt metabolism by the Biofilm Ring test® and the in vivo nematode virulence model. The impact of antibiotic exposure was determined by whole-genome sequencing (WGS) and biofilm formation experiments. We observed that S. epidermidis strains presented a wide variety of virulence potential and biofilm formation. After antibiotic exposure, S. epidermidis strains adapted their fitness with an increase in biofilm formation. Antibiotic exposure also affected genes involved in resistance and was responsible for cross-resistance between vancomycin, daptomycin and ceftobiprole. Our data confirmed that antibiotic exposure modified bacterial pathogenicity and the emergence of resistant bacteria.

Polymicrobial Biofilm Organization of Staphylococcus aureus and Pseudomonas aeruginosa in a Chronic Wound Environment.

Biofilm on the skin surface of chronic wounds is an important step that involves difficulties in wound healing. The polymicrobial nature inside this pathogenic biofilm is key to understanding the chronicity of the lesion. Few in vitro models have been developed to study bacterial interactions inside this chronic wound. We evaluated the biofilm formation and the evolution of bacteria released from this biofilm on the two main bacteria isolated in this condition, Staphylococcus aureus and Pseudomonas aeruginosa, using a dynamic system (BioFlux™ 200) and a chronic wound-like medium (CWM) that mimics the chronic wound environment. We observed that all species constituted a faster biofilm in the CWM compared to a traditional culture medium (p < 0.01). The percentages of biofilm formation were significantly higher in the mixed biofilm compared to those determined for the bacterial species alone (p < 0.01). Biofilm organization was a non-random structure where S. aureus aggregates were located close to the wound surface, whereas P. aeruginosa was located deeper in the wound bed. Planktonic biofilm-detached bacteria showed decreased growth, overexpression of genes encoding biofilm formation, and an increase in the mature biofilm biomass formed. Our data confirmed the impact of the chronic wound environment on biofilm formation and on bacterial lifecycle inside the biofilm.

Phenotypic and Genotypic Virulence Characterisation of Staphylococcus pettenkoferi Strains Isolated from Human Bloodstream and Diabetic Foot Infections.

Staphylococcus pettenkoferi is a recently described coagulase-negative Staphylococcus identified in human diseases, especially in infections of foot ulcers in patients living with diabetes mellitus. To date, its pathogenicity remains underexplored. In this study, whole-genome analysis was performed on a collection of 29 S. pettenkoferi clinical strains isolated from bloodstream and diabetic foot infections with regard to their phylogenetic relationships and comprehensive analysis of their resistome and virulome. Their virulence was explored by their ability to form biofilm, their growth kinetics and in an in vivo zebrafish embryo infection model. Our results identified two distinct clades (I and II) and two subclades (I-a and I-b) with notable genomic differences. All strains had a slow bacterial growth. Three profiles of biofilm formation were noted, with 89.7% of isolates able to produce biofilm and harbouring a high content of biofilm-encoding genes. Two virulence profiles were also observed in the zebrafish model irrespective of the strains' origin or biofilm profile. Therefore, this study brings new insights in S. pettenkoferi pathogenicity.

In vitro activity of dalbavancin against Gram-positive bacteria isolated from diabetic foot osteomyelitis.

BACKGROUND: Diabetic foot infections (DFIs) represent a serious threat to public health because of their frequency and the severity of their consequences, i.e. osteomyelitis and amputation. The management of diabetic foot osteomyelitis (DFOM) requires prolonged antibiotic therapy. In Western countries, Gram-positive bacteria are the most commonly encountered pathogens. OBJECTIVES: This study evaluated the in vitro activity of dalbavancin, a novel lipoglycopeptide with extended half-life, recently marketed in Europe for acute bacterial skin and skin structure infections, on a panel of Gram-positive bacteria responsible for DFOM. METHODS: Dalbavancin activity was evaluated against a panel of Gram-positive bacterial strains isolated from bone biopsies performed by a trained surgeon among patients with suspected DFOM. MICs were determined using MIC Test Strips (Liofilchem) and confirmed with the EUCAST broth microdilution method. Three other antimicrobial agents (vancomycin, teicoplanin and ceftobiprole) were used as comparators. RESULTS: Dalbavancin showed excellent activity against all Gram-positive bacterial strains tested, including one teicoplanin-resistant Staphylococcus epidermidis isolate. With MIC50 and MIC90 values of 0.047 and 0.094 mg/L, respectively, dalbavancin showed the most potent in vitro activity among antimicrobial agents tested. CONCLUSIONS: With its efficacy, good tolerability and unique pharmacokinetic properties, dalbavancin appears to be a promising treatment for DFOM involving Gram-positive bacteria.

Investigating Pathogenicity and Virulence of Staphylococcus pettenkoferi: An Emerging Pathogen.

Staphylococcus pettenkoferi is a coagulase-negative Staphylococcus identified in 2002 that has been implicated in human diseases as an opportunistic pathogenic bacterium. Its multiresistant character is becoming a major health problem, yet the pathogenicity of S. pettenkoferi is poorly characterized. In this study, the pathogenicity of a S. pettenkoferi clinical isolate from diabetic foot osteomyelitis was compared with a Staphylococcus aureus strain in various in vitro and in vivo experiments. Growth kinetics were compared against S. aureus, and bacteria survival was assessed in the RAW 264.7 murine macrophage cell line, the THP-1 human leukemia monocytic cell line, and the HaCaT human keratinocyte cell line. Ex vivo analysis was performed in whole blood survival assays and in vivo assays via the infection model of zebrafish embryos. Moreover, whole-genome analysis was performed. Our results show that S. pettenkoferi was able to survive in human blood, human keratinocytes, murine macrophages, and human macrophages. S. pettenkoferi demonstrated its virulence by causing substantial embryo mortality in the zebrafish model. Genomic analysis revealed virulence factors such as biofilm-encoding genes (e.g., icaABCD; rsbUVW) and regulator-encoding genes (e.g., agr, mgrA, sarA, saeS) well characterized in S. aureus. This study thus advances the knowledge of this under-investigated pathogen and validates the zebrafish infection model for this bacterium.

Surgical site infection after hip replacement due to a novel Peptoniphilus species, provisionally named 'Peptoniphilus nemausus' sp. nov.

We report a case of surgical site infection after total hip prosthesis replacement due to an ofloxacin-resistant Peptoniphilus isolate belonging to an unknown species for which the name 'Peptoniphilus nemausus' sp. nov. is proposed. Follow-up was favourable under clindamycin and rifampin for 3 months in this patient whom had a Proteus mirabilis infection treated by fluoroquinolone.

OXA-48-producing Enterobacterales in different ecological niches in Algeria: clonal expansion, plasmid characteristics and virulence traits.

OBJECTIVES: To investigate the prevalence and molecular characteristics of OXA-48-carbapenemase-producing Enterobacterales strains recovered from various ecological niches in Algeria. METHODS: In total, 3309 samples were collected from different ecological niches (human carriage, animal farms, wild animals, pets, food products, aquatic environment and wastewater treatment plants) distributed among six provinces in Algeria between December 2015 and April 2017. The potential presence of OXA-48-producing Enterobacterales isolates was screened on selective medium. Resistance and virulence profiles were characterized by PCR and sequencing. The clonal relatedness of the different isolates was studied using Rep-PCR and MLST. Conjugation was performed for all OXA-48-producing isolates. The plasmids were analysed by PCR-based replicon typing and WGS. RESULTS: A total of 78 OXA-48-producing Enterobacterales isolates were detected from 3309 samples (2.4%). OXA producers were observed in all the screened sources. The blaCTX-M-15 gene was only observed in two isolates. Clonality analysis revealed distinct lineages of the isolates and a clonal expansion of Klebsiella pneumoniae ST13. K. pneumoniae and Escherichia coli had few virulence factors. Plasmid analysis confirmed that all the isolates harboured a very similar transferable plasmid (belonging to IncL) with a similar structure to the pOXA-48a plasmid carried by K. pneumoniae strain Kp11978. CONCLUSIONS: This study suggests a global dissemination of OXA-48-producing Enterobacterales in different niches due mainly to the spread of an epidemic plasmid. Furthermore, it clearly shows that K. pneumoniae and commensal E. coli can be reservoirs of the blaOXA-48 gene, contributing to the dissemination and transfer of this gene to diverse bacteria among different sources.

Performance of the Accelerate Pheno™ system for identification and antimicrobial susceptibility testing of a panel of multidrug-resistant Gram-negative bacilli directly from positive blood cultures.

Objectives: To evaluate the performance of the Accelerate Pheno™ system for the identification and antimicrobial susceptibility testing (AST) of a panel of Gram-negative bacilli (GNB) with different resistance profiles (e.g. penicillinases, ESBLs, cephalosporinase overproduction, carbapenemases, impermeability) directly from positive blood cultures in <7 h. Methods: A panel of 105 clinical strains previously characterized for the presence of ß-lactamase-encoding genes was tested. Approximately 100 cfu of each isolate was inoculated into sterile blood culture bottles and incubated in a BD BACTEC™ FX automated system (Becton Dickinson, USA). Positive blood cultures were subjected to parallel testing using the Accelerate Pheno™ system and conventional culture methods [identification of isolated colonies by MALDI-TOF and VITEK® 2 system (bioMérieux, France), and AST by disc diffusion and Etest following EUCAST recommendations]. Results: The overall identification agreement between the Accelerate Pheno™ system and conventional culture methods was 100% (105/105). The overall categorical agreement between the system and culture-based AST was 94.9% (1169/1232), with rates for minor errors of 4.1% (51/1232), major errors 0.3% (4/1232) and very major errors 0.7% (8/1232). The Accelerate Pheno™ system produced AST results indicative of third-generation cephalosporinases (26/26) and carbapenem-resistant strains (52/55). Conclusions: The Accelerate Pheno™ system is an accurate, sensitive and easy-to-use test for the rapid identification and AST of MDR GNB in bloodstream infections. Given the burden of multidrug resistance, its implementation in the microbiology laboratory could be a useful tool for prompt management of sepsis.

OXA-48-like carbapenemases producing Enterobacteriaceae in different niches.

The emergence of carbapenem-resistant enterobacterial species poses a serious threat to public health worldwide. OXA-48-type carbapenem-hydrolyzing class D ß-lactamases are widely distributed among Enterobacteriaceae, with significant geographical differences. To date, 11 OXA-48-like variants have been identified, with classical OXA-48 being the most widespread. These enzymes show high-level hydrolytic activity against penicillins and low-level hydrolysis towards carbapenems. Since the first description of the OXA-48 carbapenemase in Turkey, bacterial strains producing the enzyme have been extensively reported in nosocomial and community outbreaks in many parts of the word, particularly in the Mediterranean area and European countries. The rapid spread of Enterobacteriaceae producing OXA-48-like enzymes in different ecosystems has become a serious issue recently. The number of reservoirs for such organisms is increasing, not only in hospitals, but also in the community, among animals (e.g., livestock, companion animals, and wildlife) and in the environment. This review aims to summarize the main characteristics of the OXA-48-type carbapenemases, covering genetic and enzymatic traits, their epidemiology, clonality and associated genes, correlation with extended-spectrum ß-lactamases (ESBLs) or plasmidic AmpC (pAmpC) in different bacterial species worldwide.

First report of cavitary pneumonia due to community-acquired Acinetobacter pittii, study of virulence and overview of pathogenesis and treatment.

BACKGROUND: Acinetobacter pittii is a nosocomial pathogen rarely involved in community-acquired infections. We report for the first time that A. pittii can be responsible for cavitary community-acquired pneumonia and study its virulence, and discuss its pathogenesis and treatment options. CASE PRESENTATION: A 45-year-old woman with a history of smoking and systemic lupus was admitted to Nimes University Hospital (France) with coughing and sputum lasting for three weeks. Thoracic CT scanner showed cavitary pneumonia. Broncho-alveolar lavage cultures found community-acquired Acinetobacter calcoaceticus-baumannii complex. The clinical outcome was favourable after twenty-one days of antimicrobial treatment by piperacillin/tazobactam and amikacin then cefepime. Multilocus sequence typing (MLST) analyses identified an A. pittii ST249. Despite the atypical clinical presentation with an unexpected partial destruction of lung parenchyma, we found very low virulence potential of the A. pittii strain with nematode killing assays and biofilm formation test. The median time required to kill 50% of the nematodes was 7 ± 0.3 days for A. pittii ST249, 7 ± 0.2 days for A. baumanii NAB ST2 and 8 ± 0.2 days for E. coli OP50, (p > 0,05). A. pittii ST249 showed significantly slower biofilm formation than A. baumanii NAB ST2: BFI = 8.83 ± 0.59 vs 3.93 ± 0.27 at 2 h (p < 0.0001), BFI = 6.3 ± 0.17 vs 1.87 ± 0.12 at 3 h (p < 0.0001) and BFI = 3.67 ± 0.41 vs 1.7 ± 0.06 after 4 h of incubation (p < 0.01). CONCLUSIONS: Community-acquired A. pittii should be considered as possible cause of sub-acute cavitary pneumonia particularly in a smoking and/or immunocompromised patient despite its low virulence potential.

Development of an algorithm for phenotypic screening of carbapenemase-producing Enterobacteriaceae in the routine laboratory.

BACKGROUND: Carbapenemase-producing Enterobacteriaceae (CPE) are difficult to identify among carbapenem non-susceptible Enterobacteriaceae (NSE). We designed phenotypic strategies giving priority to high sensitivity for screening putative CPE before further testing. METHODS: Presence of carbapenemase-encoding genes in ertapenem NSE (MIC > 0.5 mg/l) consecutively isolated in 80 French laboratories between November 2011 and April 2012 was determined by the Check-MDR-CT103 array method. Using the Mueller-Hinton (MH) disk diffusion method, clinical diameter breakpoints of carbapenems other than ertapenem, piperazicillin+tazobactam, ticarcillin+clavulanate and cefepime as well as diameter cut-offs for these antibiotics and temocillin were evaluated alone or combined to determine their performances (sensitivity, specificity, positive and negative likelihood ratios) for identifying putative CPE among these ertapenem-NSE isolates. To increase the screening specificity, these antibiotics were also tested on cloxacillin-containing MH when carbapenem NSE isolates belonged to species producing chromosomal cephalosporinase (AmpC) but Escherichia coli. RESULTS: Out of the 349 ertapenem NSE, 52 (14.9%) were CPE, including 39 producing OXA-48 group carbapenemase, eight KPC and five MBL. A screening strategy based on the following diameter cut offs, ticarcillin+clavulanate <15 mm, temocillin <15 mm, meropenem or imipenem <22 mm, and cefepime <26 mm, showed 100% sensitivity and 68.1% specificity with the better likelihood ratios combination. The specificity increased when a diameter cut-off <32 mm for imipenem (76.1%) or meropenem (78.8%) further tested on cloxacillin-containing MH was added to the previous strategy for AmpC-producing isolates. CONCLUSION: The proposed strategies that allowed for increasing the likelihood of CPE among ertapenem-NSE isolates should be considered as a surrogate for carbapenemase production before further CPE confirmatory testing.

First Description of Two Sequence Type 2 Acinetobacter baumannii Isolates Carrying OXA-23 Carbapenemase in Pagellus acarne Fished from the Mediterranean Sea near Bejaia, Algeria.

To determine the occurrence of carbapenem-resistantAcinetobacter baumanniiin fish fished from the Mediterranean Sea near the Bejaia coast (Algeria), we studied 300 gills and gut samples that had been randomly and prospectively collected during 1 year. After screening on selective agar media, using PCR arrays and whole-genome sequencing, we identified for the first time two OXA-23-producingA. baumanniistrains belonging to the widespread sequence type 2 (ST2)/international clone II and harboring aminoglycoside-modifying enzymes [aac(6')-Ib andaac(3')-I genes].

Modulation of Membrane Influx and Efflux in Escherichia coli Sequence Type 131 Has an Impact on Bacterial Motility, Biofilm Formation, and Virulence in a Caenorhabditis elegans Model.

Energy-dependent efflux overexpression and altered outer membrane permeability (influx) can promote multidrug resistance (MDR). The present study clarifies the regulatory pathways that control membrane permeability in the pandemic clone Escherichia coli sequence type 131 (ST131) and evaluates the impact of efflux and influx modulations on biofilm formation, motility, and virulence in the Caenorhabditis elegans model. Mutants of two uropathogenic E. coli (UPEC) strains, MECB5 (ST131; H30-Rx) and CFT073 (ST73), as well as a fecal strain, S250 (ST131; H22), were in vitro selected using continuous subculture in subinhibitory concentrations of ertapenem (ETP), chloramphenicol (CMP), and cefoxitin (FOX). Mutations in genes known to control permeability were shown for the two UPEC strains: MECB5-FOX (deletion of 127 bp in marR; deletion of 1 bp and insertion of an IS1 element in acrR) and CFT073-CMP (a 1-bp deletion causing a premature stop in marR). We also demonstrated that efflux phenotypes in the mutants selected with CMP and FOX were related to the AcrAB-TolC pump, but also to other efflux systems. Alteration of membrane permeability, caused by underexpression of the two major porins, OmpF and OmpC, was shown in MECB5-ETP and mutants selected with FOX. Lastly, our findings suggest that efflux pump-overproducing isolates (CMP mutants) pose a serious threat in terms of virulence (significant reduction in worm median survival) and host colonization. Lack of porins (ETP and FOX mutants) led to a high level of antibiotic resistance in an H30-Rx subclone. Nevertheless, this adaptation created a physiological disadvantage (decreased motility and ability to form biofilm) associated with a low potential for virulence.

High Prevalence of SXT/R391-Related Integrative and Conjugative Elements Carrying blaCMY-2 in Proteus mirabilis Isolates from Gulls in the South of France.

The genetic structures involved in the dissemination of blaCMY-2 carried by Proteus mirabilis isolates recovered from different gull species in the South of France were characterized and compared to clinical isolates. blaCMY-2 was identified in P. mirabilis isolates from 27/93 yellow-legged gulls and from 37/65 slender-billed gulls. It was carried by a conjugative SXT/R391-like integrative and conjugative element (ICE) in all avian strains and in 3/7 human strains. Two clinical isolates had the same genetic background as six avian isolates.

Pyomelanin-producing Pseudomonas aeruginosa selected during chronic infections have a large chromosomal deletion which confers resistance to pyocins.

When bacterial lineages make the transition from free-living to permanent association with hosts, they can undergo massive gene losses, for which the selective forces within host tissues are unknown. We identified here melanogenic clinical isolates of Pseudomonas aeruginosa with large chromosomal deletions (66 to 270 kbp) and characterized them to investigate how they were selected. When compared with their wild-type parents, melanogenic mutants (i) exhibited a lower fitness in growth conditions found in human tissues, such as hyperosmolarity and presence of aminoglycoside antibiotics, (ii) narrowed their metabolic spectrum with a growth disadvantage with particular carbon sources, including aromatic amino acids and acyclic terpenes, suggesting a reduction of metabolic flexibility. Despite an impaired fitness in rich media, melanogenic mutants can inhibit their wild-type parents and compete with them in coculture. Surprisingly, melanogenic mutants became highly resistant to two intraspecific toxins, the S-pyocins AP41 and S1. Our results suggest that pyocins produced within a population of infecting P. aeruginosa may have selected for bacterial mutants that underwent massive gene losses and that were adapted to the life in diverse bacterial communities in the human host. Intraspecific interactions may therefore be an important factor driving the continuing evolution of pathogens during host infections.