Untargeted Metabolomics Approach for the Discovery of Environment-Related Pyran-2-ones Chemodiversity in a Marine-Sourced Penicillium restrictum.

Very little is known about chemical interactions between fungi and their mollusc host within marine environments. Here, we investigated the metabolome of a Penicillium restrictum MMS417 strain isolated from the blue mussel Mytilus edulis collected on the Loire estuary, France. Following the OSMAC approach with the use of 14 culture media, the effect of salinity and of a mussel-derived medium on the metabolic expression were analysed using HPLC-UV/DAD-HRMS/MS. An untargeted metabolomics study was performed using principal component analysis (PCA), orthogonal projection to latent structure discriminant analysis (O-PLSDA) and molecular networking (MN). It highlighted some compounds belonging to sterols, macrolides and pyran-2-ones, which were specifically induced in marine conditions. In particular, a high chemical diversity of pyran-2-ones was found to be related to the presence of mussel extract in the culture medium. Mass spectrometry (MS)- and UV-guided purification resulted in the isolation of five new natural fungal pyran-2-one derivatives-5,6-dihydro-6S-hydroxymethyl-4-methoxy-2H-pyran-2-one (1), (6S, 1'R, 2'S)-LL-P880ß (3), 5,6-dihydro-4-methoxy-6S-(1'S, 2'S-dihydroxy pent-3'(E)-enyl)-2H-pyran-2-one (4), 4-methoxy-6-(1'R, 2'S-dihydroxy pent-3'(E)-enyl)-2H-pyran-2-one (6) and 4-methoxy-2H-pyran-2-one (7)-together with the known (6S, 1'S, 2'S)-LL-P880ß (2), (1'R, 2'S)-LL-P880γ (5), 5,6-dihydro-4-methoxy-2H-pyran-2-one (8), (6S, 1'S, 2'R)-LL-P880ß (9), (6S, 1'S)-pestalotin (10), 1'R-dehydropestalotin (11) and 6-pentyl-4-methoxy-2H-pyran-2-one (12) from the mussel-derived culture medium extract. The structures of 1-12 were determined by 1D- and 2D-MMR experiments as well as high-resolution tandem MS, ECD and DP4 calculations. Some of these compounds were evaluated for their cytotoxic, antibacterial, antileishmanial and in-silico PTP1B inhibitory activities. These results illustrate the utility in using host-derived media for the discovery of new natural products.

Whole-bacterium ribosome display selection for isolation of highly specific anti-Staphyloccocus aureus Affitins for detection- and capture-based biomedical applications.

Detection and capture methods using antibodies have been developed to ensure identification of pathogens in biological samples. Though antibodies have many attractive properties, they also have limitations and there are needs to expand the panel of available affinity proteins with different properties. Affitins, that we developed from the Sul7d proteins, are a solid class of affinity proteins, which can be used as substitutes to antibodies or to complement them. We report the generation and characterization of antibacterial Affitins with high specificity for Staphylococcus aureus. For the first time, ribosome display selections were carried out using whole-living-cell and naïve combinatorial libraries, which avoid production of protein targets and immunization of animals. We showed that Affitin C5 exclusively recognizes S. aureus among dozens of strains, including clinical ones. C5 binds staphylococcal Protein A (SpA) with a K D of 108 ± 2 nM and has a high thermostability (T m = 77.0°C). Anti-S. aureus C5 binds SpA or bacteria in various detection and capture applications, including ELISA, western blot analysis, bead-fishing, and fluorescence imaging. Thus, novel anti-bacteria Affitins which are cost-effective, stable, and small can be rapidly and fully designed in vitro with high affinity and specificity for a surface-exposed marker. This class of reagents can be useful in diagnostic and biomedical applications.

Identification and Biological Activities of Long-Chain Peptaibols Produced by a Marine-Derived Strain of Trichoderma longibrachiatum.

Six long-chain peptaibols, 1 - 6, were identified from agar cultures of a marine-derived Trichoderma longibrachiatum Rifai strain (MMS151) isolated from blue mussels. The structure elucidation was carried out using electrospray ionization ion trap mass spectrometry (ESI-IT-MS) and GC/EI-MS. The long-chain peptaibols exhibited the general building scheme Ac-Aib-Ala-Aib-Ala-Aib-XXX-Gln-Aib-Vxx-Aib-Gly-XXX-Aib-Pro-Vxx-Aib-XXX-Gln-Gln-Pheol and were similar or identical to recurrent 20-residue peptaibols produced by Trichoderma spp. Three new sequences were identified and were called longibrachins A-0, A-II-a, and A-IV-b. The isolated peptaibols were assayed for cytotoxic, antibacterial, and antifungal activities, and acute toxicity on Dipteran larvae.

16S rRNA gene pyrosequencing reveals shift in patient faecal microbiota during high-dose chemotherapy as conditioning regimen for bone marrow transplantation.

Gastrointestinal disturbances are a side-effect frequently associated with haematological malignancies due to the intensive cytotoxic treatment given in connection with bone marrow transplantation (BMT). However, intestinal microbiota changes during chemotherapy remain poorly described, probably due to the use of culture-based and low-resolution molecular methods in previous studies. The objective of our study was to apply a next generation DNA sequencing technology to analyse chemotherapy-induced changes in faecal microbiota. We included eight patients with non-Hodgkin's lymphoma undergoing one course of BMT conditioning chemotherapy. We collected a prechemotherapy faecal sample, the day before chemotherapy was initiated, and a postchemotherapy sample, collected 1 week after the initiation of chemotherapy. Total DNA was extracted from faecal samples, denaturing high-performance liquid chromatography based on amplification of the V6 to V8 region of the 16S ribosomal RNA (rRNA) gene, and 454-pyrosequencing of the 16 S rRNA gene, using PCR primers targeting the V5 and V6 hypervariable 16S rRNA gene regions were performed. Raw sequence data were screened, trimmed, and filtered using the QIIME pipeline. We observed a steep reduction in alpha diversity and significant differences in the composition of the intestinal microbiota in response to chemotherapy. Chemotherapy was associated with a drastic drop in Faecalibacterium and accompanied by an increase of Escherichia. The chemotherapy-induced shift in the intestinal microbiota could induce severe side effects in immunocompromised cancer patients. Our study is a first step in identifying patients at risk for gastrointestinal disturbances and to promote strategies to prevent this drastic shift in intestinal microbiota.

Detection of clonally related Escherichia coli isolates producing different CMY ß-lactamases from a cystic fibrosis patient.

OBJECTIVES: This study reports details on Escherichia coli isolates recovered from a cystic fibrosis (CF) patient in order to understand how this pathogen adapts to and resists broad-spectrum antipseudomonal therapy in this context. METHODS: Five E. coli isolates were obtained from various clinical samples (airways, urine or dialysis catheter) over a 7 month period covering a double-lung transplantation. All isolates were analysed in terms of clonality [enterobacterial repetitive intergenic consensus (ERIC)-PCR and multilocus sequence typing], virulence profiles (phylogroup and search for 15 virulence genes), growth patterns (morphotype, biofilm-forming ability and growth rate), hypermutability and antimicrobial susceptibility, with molecular characterization of ß-lactamases and porins. RESULTS: The five isolates shared similar ERIC-PCR profiles and sequence types (ST1193) and exhibited the same virulence profile. The respiratory isolates were strong mutators, exhibited mucoid or small-colony morphotypes, exhibited strong biofilm-forming ability and grew slowly compared with the others. All isolates were highly resistant to ceftazidime. The respiratory isolates showed reduced susceptibility to cefepime and high resistance to aztreonam. The patient had received a 31 day course of ceftazidime/aztreonam until transplantation. All isolates harboured the same wild-type chromosomal AmpC. A CMY-2 enzyme was detected in the non-respiratory isolates. The respiratory isolates harboured L293S and V211A/L293S new CMY-2 variants, which were designated CMY-94 and CMY-95, respectively. OmpF porin loss was observed in the non-respiratory isolates. CONCLUSIONS: Our study shows that, similarly to Pseudomonas aeruginosa, E. coli can undergo phenotypic and genomic changes in the CF context. For the first time, we identified an in vivo expanded-spectrum evolution of the CMY-2 ß-lactamase, during bacterial persistence in the CF lung.

Outbreak caused by Proteus mirabilis isolates producing weakly expressed TEM-derived extended-spectrum ß-lactamase in spinal cord injury patients with recurrent bacteriuria.

We performed a retrospective extended-spectrum ß-lactamase (ESBL) molecular characterization of Proteus mirabilis isolates recovered from urine of spinal cord injury patients. A incorrectly detected TEM-24-producing clone and a new weakly expressed TEM-derived ESBL were discovered. In such patients, ESBL detection in daily practice should be improved by systematic use of a synergy test in strains of P. mirabilis resistant to penicillins.

Could Azithromycin Be Part of Pseudomonas aeruginosa Acute Pneumonia Treatment?

Azithromycin (AZM) is a 15-membered-ring macrolide that presents a broad-spectrum antimicrobial activity against Gram-positive bacteria and atypical microorganisms but suffers from a poor diffusion across the outer-membrane of Gram-negative bacilli, including Pseudomonas aeruginosa (PA). However, AZM has demonstrated clinical benefits in patients suffering from chronic PA respiratory infections, especially cystic fibrosis patients. Since the rise of multidrug-resistant PA has led to a growing need for new therapeutic options, this macrolide has been proposed as an adjunctive therapy. Clinical trials assessing AZM in PA acute pneumonia are scarce. However, a careful examination of the available literature provides good rationales for its use in that context. In fact, 14- and 15-membered-ring macrolides have demonstrated immunomodulatory and immunosuppressive effects that could be of major interest in the management of acute illness. Furthermore, growing evidence supports a downregulation of PA virulence dependent on direct interaction with the ribosomes, and based on the modulation of several key regulators from the Quorum Sensing network. First highlighted in vitro, these interesting properties of AZM have subsequently been confirmed in the animal models. In this review, we systematically analyzed the literature regarding AZM immunomodulatory and anti-PA effects. In vitro and in vivo studies, as well as clinical trials were reviewed, looking for rationales for AZM use in PA acute pneumonia.

Antibiotic resistance heterogeneity and LasR diversity within Pseudomonas aeruginosa populations from pneumonia in intensive care unit patients.

This study investigated within-host heterogeneity of 66 Pseudomonas aeruginosa populations from pneumonia in 51 critically ill ventilated patients by examining 30 colonies per bronchoalveolar lavage (BAL). Differences in antibiotic susceptibility and quorum-sensing (QS) phenotypes were observed between the members of 14 (21.2%) and 10 (15.2%) populations, respectively. A significant association was found between QS deficiency and ceftazidime resistance. QS deficiency was associated with various lasR modifications, and was observed in 25 of 51 (49.0%) patients, including seven patients who received ≤48 h of ventilation. This study confirms the need to examine diverse colonies when analysing BAL cultures, particularly in ß-lactam-exposed patients, to avoid missing ceftazidime- or imipenem-resistant isolates.

Occurrence of ST23 complex phylogroup A Escherichia coli isolates producing extended-spectrum AmpC beta-lactamase in a French hospital.

Extended-spectrum AmpC beta-lactamase (ESAC) Escherichia coli producers were investigated over a 5-year period. Eleven isolates presenting a strong ampC promoter and different strategic AmpC mutations, including two newly described modifications (A292V and an L-A-A insertion at 295), were characterized. All the isolates belonged to phylogenetic group A and to the ST23 complex.

Rapid genetic and phenotypic changes in Pseudomonas aeruginosa clinical strains during ventilator-associated pneumonia.

Treatment with antibiotics leads to the selection of isolates with increased resistance. We investigated if evolution towards resistance was associated with virulence changes, in the context of P. aeruginosa ventilator-associated pneumonia (VAP). Four patients were selected because they had multiple VAP episodes during short periods (12 days to 5 weeks), with emergence of resistance. We performed whole-genome sequencing of 12 P. aeruginosa from bronchoalveolar lavages or blood culture (3 isolates per patient). Production of quorum sensing-dependent virulence factors, serum resistance, cytotoxicity against A549 cells, biofilm production, and twitching motility were studied. Each patient was infected with a unique strain. For all patients, resistance development was explained by genetic events in ampD, mexR or oprD. Additional variations were detected in virulence- and/or fitness-associated genes (algB, gacA, groEL, lasR, mpl, pilE, pilM, rhlR) depending on the strain. We noticed a convergence towards quorum sensing deficiency, correlated with a decrease of pyocyanin and protease production, survival in serum, twitching motility and cytotoxicity. In one patient, changes in pilM and pilE were related to enhanced twitching. We show that the emergence of resistance in P. aeruginosa is associated with virulence modification, even in acute infections. The consequences of this short-term pathoadaptation need to be explored.

Real-time PCR assay for the detection and quantification of Legionella pneumophila in environmental water samples: utility for daily practice.

We developed a quantitative real-time PCR assay targeting the mip gene of Legionella pneumophila for a prospective study from September 2004 to April 2005. It was compared with a standard culture method (French guideline AFNOR T90-431), analysing 120 water samples collected to monitor the risk related to Legionellae at Nantes hospital and to investigate a case of legionellosis acquired from hospital environment. Samples from six distinct water distribution systems were analysed by DNA extraction, amplification and detection with specific primers and FRET probes. The detection limit was 100 genomic units of L. pneumophila per liter (GU/l), the positivity threshold about 600 GU/l and the quantification limit 800 GU/l. PCR results were divided into three groups: negative (n=63), positive but non-quantifiable (n=22) or positive (n=35). PCR showed higher sensitivity than culture, whereas four culture-positive samples appeared negative by PCR (PCR inhibitor detected for two of them). Although no correlation was observed between both methods and real-time PCR cannot substitute for the reference method, it represents an interesting complement. Its sensitivity, reproducibility and rapidity appear particularly interesting in epidemic contexts in order to identify the source of contamination or to evaluate critical points of contamination in water distribution systems.

Increased resistance to beta-lactams in a Klebsiella pneumoniae strain: role of a deletion downstream of the Pribnow box in the blaSHV-1 promoter.

Two hundred and five isolates of Klebsiella pneumoniae were collected from Nantes University Hospital in 2002. A new 30bp deletion was detected downstream of the -10 box of the SHV-1 promoter in a clinical K. pneumoniae isolate with a high amoxicillin/clavulanic acid minimum inhibitory concentration. Reverse transcription polymerase chain reaction revealed increased transcription of bla(SHV-1) mRNA. All conjugation mating assays failed. This new promoter was cloned upstream of the cat gene of the reporter plasmid pKK232-8 and compared with previously described bla(SHV-1) promoters. The deletion induced a 15-fold increase in promoter strength compared with the usual weak promoter. This study reports a new genetic event that increases bla(SHV-1) chromosomal gene expression, which may be of clinical relevance when associated with porin deficiency.

Pathogenic potential of Escherichia coli clinical strains from orthopedic implant infections towards human osteoblastic cells.

Escherichia coli is one of the first causes of Gram-negative orthopedic implant infections (OII), but little is known about the pathogenicity of this species in such infections that are increasing due to the ageing of the population. We report how this pathogen interacts with human osteoblastic MG-63 cells in vitro, by comparing 20 OII E. coli strains to two Staphylococcus aureus and two Pseudomonas aeruginosa strains. LDH release assay revealed that 6/20 (30%) OII E. coli induced MG-63 cell lysis whereas none of the four control strains was cytotoxic after 4 h of coculture. This high cytotoxicity was associated with hemolytic properties and linked to hlyA gene expression. We further showed by gentamicin protection assay and confocal microscopy that the non-cytotoxic E. coli were not able to invade MG-63 cells unlike S. aureus strains (internalization rate <0.01% for the non-cytotoxic E. coli versus 8.88 ± 2.31% and 4.60 ± 0.42% for both S. aureus). The non-cytotoxic E. coli also demonstrated low adherence rates (<7%), the most adherent E. coli eliciting higher IL-6 and TNF-α mRNA expression in the osteoblastic cells. Either highly cytotoxic or slightly invasive OII E. coli do not show the same infection strategies as S. aureus towards osteoblasts.

Detection of an IS21 insertion sequence in the mexR gene of Pseudomonas aeruginosa increasing beta-lactam resistance.

To understand the regulation of the MexAB OprM efflux system in a clinical strain of Pseudomonas aeruginosa presenting a decreased susceptibility to ticarcillin and aztreonam, the mexR repressor gene was amplified by polymerase chain reaction (PCR) and was shown to be disrupted by an insertion sequence of more than 2 kb, with characteristic direct and inverted repeat sequences. Sequencing revealed a 2131-bp IS21 insertion sequence. A reverse transcription PCR method was used to quantify mexA transcripts and showed an increased transcription rate of mexA in this strain, compared with a PAO1 control strain. The nalB phenotype in P. aeruginosa may be due to point mutations, but also to the presence of an insertion sequence in the mexR regulator gene.

Comparison of two RT-PCR methods for quantifying ampC specific transcripts in Escherichia coli strains.

In Escherichia coli, beta-lactam resistance usually depends on beta-lactamase production. AmpC chromosomal cephalosporinase hyperproduction is generally due to mutations in the ampC gene promoter. In order to study ampC expression in E. coli clinical strains, we have compared two methods: conventional and real-time reverse transcription-polymerase chain reaction (RT-PCR). With both methods, ampC mRNA was found to be greatly increased in strains presenting -42 or -32 mutations in the ampC promoter, and moderately increased when a -11 mutation was present in the Pribnow box. Real-time RT-PCR represents a powerful tool combining amplification, fluorescent detection and analysis.

Comparison of three methods to study biofilm formation by clinical strains of Escherichia coli.

Biofilm formation seems to be a key factor in many bacterial infections, particularly those involving prosthetic implants or urinary catheters, where Escherichia coli is frequently involved. We have determined the ability to form biofilm in vitro of 34 E. coli isolates by 3 different methods (crystal violet staining, BioFilm Ring Test®, and resazurin assay) and tried to correlate biofilm production with phylogenetic background and with the presence of different genes involved in biofilm synthesis. Only 3 isolates (including positive control E. coli ATCC 25922) were classified as strong biofilm producers (1B1, 1D, and 1B2 = control) by the 3 methods, 2 isolates by 2 different methods, and 5 additional isolates by only 1 method. All isolates possessed the csgA gene belonging to the csgABC operon encoding curli, and its regulator csgD. By contrast, only 76% possessed pgaA gene which is part of the pgaABCD operon encoding a polysaccharide adhesin. Interestingly, one of the strong biofilm producers did not harbor pgaA. In the second part, we have selected 5 specific isolates to study the impact of various experimental conditions on biofilm formation. For all these isolates, biofilm production was decreased in anaerobiosis and increased in LB medium compared with brain heart infusion medium, but at various degrees for the different isolates. These results underline the problems encountered in comparing the different published studies using various methods to study biofilm formation in vitro and the great need of standardization.

Orthopaedic-implant infections by Escherichia coli: molecular and phenotypic analysis of the causative strains.

OBJECTIVES: Little is known about Escherichia coli Orthopaedic Implant Infections (OII) pathogenesis. Thus, we compared 30 clinical strains isolated in this context with 30 clinical strains of faecal origin, in order to identify phenotypic and genetic features related to E. coli OII. METHODS: Phylogenetic analysis and detection of 19 virulence genes were performed by PCR. Ability to form biofilm was studied using the crystal violet reference method and the innovative BioFilm Ring Test(®). RESULTS: Most of the OII isolates (56.7%) belonged to the virulence-associated phylogenetic group B2, but did not present a specific set of virulence factors. S fimbriae was the only adhesin significantly associated with OII isolates. Isolates varied greatly in their ability to form biofilm but OII isolates did not produce significantly more biofilm in vitro than isolates of faecal origin, whatever the method used. CONCLUSIONS: Neither a specific pathogenic signature nor an increased ability to form biofilm in vitro was detected in E. coli strains isolated from OII. Nevertheless, genetic properties of these isolates could provide a clue to their origin. Hence, we found that virulence factors of uropathogenic strains and urological disorders were frequently detected among our OII cohort.