Zohreh Tamanai-Shacoori

Direct enumeration of injured Escherichia coli cells harvested onto membrane filters

Abstract Four methods testing either biosynthesis capacity (Direct Viable Counts), respiratory activity (CTC and XTT reduction assays), or membrane integrity (LIVE/DEAD® BacLight™ VIABILITY kit) were performed to enumerate E. coli injured cells previously harvested onto 0.2-μm black membranes. Some of them remained viable but were no longer culturable when exposed to hyperosmotic or oxidative stresses. The damaged cellular functions were dependent on the stress applied, and the results provided by these methods (except the XTT reduction method which is not sensitive enough), gave information on cell behaviour to one stress, and on the different cellular targets of this stress. The osmotic shock in artificial seawater led to moderate plasma membrane damage (0.8 log reduction in counts determined with the LIVE/DEAD® BacLight™ VIABILITY kit after 5 days in seawater), and to similar reductions of respiration and elongation capacities (2.7 log reduction of CTC counts, and 2.6 log reduction of DVC counts, respectively). After a 30-min oxidative stress induced by peracetic acid (8 mg l−1), the plasma membranes remained intact (no reduction of LIVE/DEAD® BacLight™ VIABILITY counts), and respiration was less impaired than elongation capacity (2.1 log reduction of CTC counts and 3.1 log reduction of DVC counts, respectively).

Roseburia spp.: a marker of health?

The genus Roseburia consists of obligate Gram-positive anaerobic bacteria that are slightly curved, rod-shaped and motile by means of multiple subterminal flagella. It includes five species: Roseburia intestinalis, R. hominis, R. inulinivorans, R. faecis and R. cecicola. Gut Roseburia spp. metabolize dietary components that stimulate their proliferation and metabolic activities. They are part of commensal bacteria producing short-chain fatty acids, especially butyrate, affecting colonic motility, immunity maintenance and anti-inflammatory properties. Modification in Roseburia spp. representation may affect various metabolic pathways and is associated with several diseases (including irritable bowel syndrome, obesity, Type 2 diabetes, nervous system conditions and allergies). Roseburia spp. could also serve as biomarkers for symptomatic pathologies (e.g., gallstone formation) or as probiotics for restoration of beneficial flora.

Genetic Analysis of an Ambler Class A Extended-Spectrum Beta-Lactamase from Capnocytophaga ochracea

ABSTRACT A beta-lactamase gene (cfxA3, 966 bp) was isolated from a beta-lactam-resistant Capnocytophaga ochracea clinical isolate and amplified using primers from the cfxA gene of Bacteroides vulgatus. The MICs of third-generation cephalosporins were much higher than those of the transconjugant Escherichia coli strain. The deduced protein sequence, by comparison with CfxA2 of Prevotella intermedia, had a Y239D substitution and possessed the characteristics of a class A, group 2e beta-lactamase.

High prevalence of β-lactam and macrolide resistance genes in human oral Capnocytophaga species

OBJECTIVES To determine macrolide-lincosamide-streptogramin (MLS) resistance determinants in the Capnocytophaga genus and to describe the prevalence of β-lactam resistance genes in human oral Capnocytophaga species. METHODS Forty-eight Capnocytophaga isolates identified by analysis of 16S rRNA sequences were isolated from subgingival samples from 14 haematology patients (HPs), 11 periodontitis patients (PPs) and 17 healthy volunteers (HVs). MICs of β-lactam and MLS antibiotics were obtained for all isolates. blaCfxA, blaCSP-1 (encoding a new class A β-lactamase) and MLS resistance genes [erm(F), erm(B), erm(Q), erm(D), erm(C) and erm(A)] were evaluated using specific PCR and sequencing. RESULTS In HVs, which had the lowest prevalence of β-lactamase-producing isolates in comparison with the other groups (16%; P < 0.001), Capnocytophaga ochracea was the prominent species (68%; P < 0.03). In PPs, which had a high prevalence of β-lactamase-positive isolates (82%; P < 0.001), Capnocytophaga sputigena was more frequently identified (64%; P < 0.03). In HPs, 50% of isolates were β-lactamase-positive. The more rarely identified species (15%) Capnocytophaga gingivalis, Capnocytophaga granulosa and Capnocytophaga leadbetteri were isolated only from PPs and/or HPs. All β-lactam-resistant isolates (44%) were PCR-positive for blaCfxA (31%) or blaCSP-1 (12.5%). Interestingly, blaCSP-1 was identified only in a subgroup of the C. sputigena species. Twenty-nine percent of isolates were MLS resistant independently of species identification, β-lactamase production or patient group. The MLS-resistant isolates carried the erm(F) or erm(C) gene (93% and 7%, respectively), previously unknown in the Capnocytophaga genus. CONCLUSIONS Our findings illustrate that Capnocytophaga species are important contributors to the β-lactam and MLS resistance gene reservoir in the oral microbiome.

Development of SNAP-tag-mediated live cell labeling as an alternative to GFP in Porphyromonas gingivalis

Porphyromonas gingivalis is an anaerobic periodontal pathogen that resides in the complex multispecies microbial biofilm known as dental plaque. Effective reporter tools are increasingly needed to facilitate physiological and pathogenetic studies of dental biofilm. Fluorescent proteins are ideal reporters for conveniently monitoring biofilm growth, but are restricted by several environmental factors, such as a requirement of oxygen to emit fluorescence. We developed a fluorescent reporter plasmid, known as the SNAP-tag, for labeling P. gingivalis cells, which encode an engineered version of the human DNA repair enzyme O(6)-alkylguanine-DNA alkyltransferase. Fluorescent substrates containing O(6)-benzylguanine covalently and specifically bind to the enzyme via stable thioether bonds. For the present study, we constructed a replicative plasmid carrying SNAP26b under the control of the P. gingivalis endogenous trxB promoter. The P. gingivalis-expressing SNAP26 protein was successfully labeled with specific fluorophores under anaerobic conditions. Porphyromonas gingivalis biofilm formation was investigated using flow cells and confocal laser scanning microscopy. A specific distribution of a strong fluorescence signal was demonstrated in P. gingivalis-SNAP26 monospecies and bispecies biofilms with Streptococcus gordonii-GFPmut3(*). These findings show that the SNAP-tag can be applied to studies of anaerobic bacteria in biofilm models and is a useful and advantageous alternative to existing labeling strategies.

Sufentanil modifies the antibacterial activity of bupivacaine and ropivacaine

PurposeThe purpose of our study was to investigate the effect on the growth ofEscherichia coli (E. coli), Staphylococcus aureus (S. aureus), andEnterococcus faecalis (E. faecalis) of bupivacaine at a final concentration of 0.77 mg·mL−1, ropivacaine at 1.2 mg·mL−1, and sufentanil at 0.38 and 0.5 μg·mL−1 (alone or in combination with bupivacaine and ropivacaine).MethodsThe strains were diluted to approximately 3 × 104 cfu·mL−1 in Mueller-Hinton broth. The anesthetics (0.5 mL) were incubated with the bacterial suspensions (0.5 mL) for 24 hr at 37°C.ResultsBupivacaine inhibited the growth ofE. coli (59 ± 0.8%;P < 0.05) andS. aureus (22 ± 3.6%;P < 0.05). Ropivacaine also inhibited the growth ofE. coli (41 ± 1.2%;P < 0.05) andS. aureus (25.5 ± 4.1%;P < 0.05). Both anesthetics were ineffective againstE. faecalis. Sufentanil only inhibitedS. aureus (13.8 ± 3.1%;P < 0.05) at a concentration of 0.5 μg·mL−1. Sufentanil modified the antibacterial activity of bupivacaine and ropivacaine. It increased the inhibitory effect of bupivacaine onE. faecalis andS. aureus by 10 ±2.1% (P < 0.05) and onE. coli by 7% (P < 0.05). Sufentanil did not increase the inhibitory effect of ropivacaine on the growth ofS. aureus. On the other hand, sufentanil reduced the inhibitory effect of ropivacaine onE. coli by 11% (P < 0.05).ConclusionBoth bupivacaine and ropivacaine alone or combined with sufentanil inhibited the growth ofE. coli andS. aureus. E. faecalis was partially sensitive to a bupivacaine + sufentanil mixture. Sufentanil had a partial synergistic effect on bupivacaine and a partial antagonistic effect on ropivacaine’s antibacterial activity.RésuméObjectifL’activité antibactérienne de la bupivacaïne (0,77 mg·mL− 1), la ropivacaïne (1,2 mg·mL− 1), le sufentanil) 0,38 et 0,5 μg·mL− 1, seul ou combiné avec chaque anesthésique local a été évalué sur Escherichia coli (E. coli), Staphylococcus aureus (S. aureus) et Enterococcus faecalis (E. faecalis).MéthodeNous avons ajouté 0,5 mL du produit à évaluer à 0,5 mL d’une suspension bactérienne (≈ 3 × 104 ufc·mL− 1), préparée dans un bouillon de Mueller-Hinton. Le mélange a été incubé à 37°C pendant 24 h et l’évolution de la densité de culture a été étudiée.RésultatsLa bupivacaïne et la ropivacaïne inhibent respectivement la croissance de 59 ± 0,8 % et 41 ± 1,2 % (P < 0,05) d’E. coli, et de 22 ± 3,6% et 25,5 ± 4,1% (P < 0,05) du S. aureus par rapport au témoin. Ces produits sont inefficaces sur la souche d’E. faecalis. Le sufentanil agit seulement sur l’E. faecalis à une concentration de 0,5μg·mL− 1 (13,8 ± 3,1%; P < 0,05 d’inhibition). Le sufentanil modifie les activités de la bupivacaïne et de la ropivacaïne lorsqu’il est administré avec chaque drogue. Il augmente de 10 ± 2,1% (P < 0,05) le potentiel inhibiteur de la bupivacaïne sur l’E. faecalis et le S. aureus, et de 7% (P < 0,05) sur l’E. coli. Une action similaire mais non significative a été observée avec la ropivacaïne combinée au sufentanil sur le S. aureus. À l’inverse, le sufentanil réduit de 11% (P < 0,05) le pouvoir inhibiteur de la ropivacaïne sur l’E. coli.ConclusionLa bupivacaïne et la ropivacaïne, seules ou combinée avec le sufentanil ont une activité antibactérienne contre l’E. coli et le S. aureus. L’E. faecalis est partiellement sensible à un mélange de bupivacaïne et de sufentanil. Le sufentanil a un effet synergique partiel sur l’activité antibactérienne de la bupivacaïne et un effet antagoniste partiel sur la ropivacaïne.

Osmotic Stress-Induced Genetic Rearrangements in Escherichia coli H10407 Detected by Randomly Amplified Polymorphic DNA Analysis

ABSTRACT Randomly amplified polymorphic DNA (RAPD) analysis is a DNA polymorphism assay commonly used for fingerprinting genomes. After optimizing the reaction conditions, samples of Escherichia coli H10407 DNA were assayed to determine the influence of osmotic and/or oligotrophic stress on variations in RAPD banding patterns. Genetic rearrangements or DNA topology variations could be detected as changes in agarose gel electrophoresis banding profiles. A new amplicon generated using DNA extracted from bacteria prestarved by an osmotic stress and resuscitated in rich medium was observed. Enrichment improved recovery of mutator cells and allowed them to be detected in samples, suggesting that DNA modifications, such as stress-induced alterations and supercoiling phenomena, should be taken into consideration before beginning RAPD analyses.

The antibacterial activity of tramadol against bacteria associated with infectious complications after local or regional anesthesia.

BACKGROUND:Tramadol is a synthetic analog of codeine with opioid and local anesthetic properties. It is used as a central-acting analgesic, and recently, in subcutaneous or intradermal injections, as a local anesthetic. We investigated in vitro the antibacterial activity of tramadol in the absence of any local anesthetics against Escherichia coli, Staphylococcus aureus, Staphylococcus epidermidis, and Pseudomonas aeruginosa pathogens that can cause infectious complications after local or regional anesthesia. METHODS:Bacterial cultures were grown for 18 h, diluted in sterile physiological saline, and incubated for 6 or 24 h at 37°C with 6.25, 12.5, or 25 mg/mL tramadol. The mixtures were then plated onto blood agar and colony counts were recorded after 24 h incubation at 37°C. RESULTS:Tramadol had bactericidal activity against E. coli and S. epidermidis compared with controls: at 25 mg/mL for 6 h or at 12.5 mg/mL for 24 h, tramadol decreased by approximately 7 log10 (P < 0.001) the colony counts of E. coli (100% kill). Similar results were obtained with S. epidermidis, with approximately 6 log10 reduction (100% kill) when tramadol was used at 25 mg/mL for 24 h (P < 0.001). The antibacterial effect of 25 mg/mL tramadol was lower against S. aureus and P. aeruginosa, reducing the growth of these strains by approximately 3log10 after 24 h (P < 0.001). CONCLUSIONS:Tramadol has dose- and time-dependent bactericidal activity against E. coli and S. epidermidis, as well as antibacterial activity against S. aureus and P. aeruginosa. The antibacterial properties of tramadol may be useful for reducing the risk of bacterial infection after local or regional anesthesia.

Prevalence of oropharyngeal beta-lactamase-producing Capnocytophaga spp. in pediatric oncology patients over a ten-year period

BackgroundThe aim of this study was to evaluate the prevalence of beta-lactamase-producing Capnocytophaga isolates in young children hospitalized in the Pediatric Oncology Department of Hôpital Sud (Rennes, France) over a ten-year period (1993–2002).MethodsIn neutropenic children, a periodic survey of the oral cavity allows a predictive evaluation of the risk of systemic infections by Capnocytophaga spp. In 449 children with cancer, 3,053 samples were collected by oral swabbing and plated on TBBP agar. The susceptibility of Capnocytophaga isolates to five beta-lactams was determined.ResultsA total of 440 strains of Capnocytophaga spp. were isolated, 309 (70%) of which were beta-lactamase producers. The beta-lactamase-producing strains were all resistant to cefazolin, 86% to amoxicillin, and 63% to ceftazidime. The proportion of strains resistant to third-generation cephalosporins remained high throughout the ten-year study, while susceptibility to imipenem and amoxicillin combined with clavulanic acid was always conserved.ConclusionThese results highlight the risk of antibiotic failure in Capnocytophaga infections and the importance of monitoring immunosuppressed patients and testing for antibiotic susceptibility and beta-lactamase production.

Comparison of direct PCR and PCR amplification after DNA extraction for the detection of viable enterotoxigenic Escherichia coli in laboratory microcosms

Abstract Studies of laboratory microcosms have shown that enterotoxigenic Escherichia coli (ETEC) lose their culturability but remain viable when exposed to starvation conditions. A polymerase chain reaction (PCR) procedure was used to detect viable cells incubated in artificial seawater microcosms, either directly after boiling the water or after extracting the DNA. After 4 days of incubation, culturable bacterial counts decreased to −1 but viable bacteria were detected for up to 6 days in all cases. After 15 days, only direct PCR remained positive and after 21 days, all results were negative. The non-culturable state of ETEC may pose health problems, and this study demonstrated the efficiency of direct PCR to detect total viable starved Escherichia coli strains.