The sponges Hymeniacidon perlevis and Halichondria panicea are reservoirs of antibiotic-producing bacteria against multi-drug resistant Staphylococcus aureus.

AIMS: Evaluation of the antibacterial activity of cultivable bacteria associated with the marine sponges Hymeniacidon perlevis and Halichondria panicea against multi-drug-resistant Staphylococcus aureus. METHODS AND RESULTS: One hundred and fourteen bacterial isolates were recovered from H. perlevis and H. panicea. Antibacterial action was demonstrated by 70% of the isolates against reference strain Staphylococcus aureus ATCC 29213 and by 31·6% against Pseudomonas aeruginosa ATCC 27853 in agar overlay assays. Antibacterial potential was further analysed against 36 multi-drug-resistant hospital Staphylococcus aureus strains with diverse resistance profiles. Among the 80 isolates positive against S. aureus ATCC 29213, 76·3% were active against at least one clinical S. aureus pathogen and 73·6% inhibited one or more methicillin-resistant (MRSA) and vancomycin non-susceptible S. aureus strains. In addition, 41·3% inhibited all vancomycin nonsusceptible MRSA strains. CONCLUSIONS: Culturable bacteria associated to H. perlevis and H. panicea are promising sources of antibacterial compounds of great pharmaceutical interest. SIGNIFICANCE AND IMPACT OF THE STUDY: This study was the first to explore the antibacterial potential of culturable bacteria associated with the marine sponges H. perlevis and H. panicea against MDR bacteria. This is the first report of antibacterial activity by Aquimarina, Denitrobaculum, Maribacter and Vagococcus isolates against MDR S. aureus strains, including vancomycin nonsusceptible and methicillin-resistant ones, against which new antibiotics are urgently needed.

Characterization of Putative Virulence Factors of Pseudomonas aeruginosa Strain RBS Isolated from a Saltern, Tunisia: Effect of Metal Ion Cofactors on the Structure and the Activity of LasB.

Pseudomonas aeruginosa is a ubiquitous Gram-negative bacterium able to survive in diverse environments such as soil, plants, freshwater, and seawater. P. aeruginosa can be an opportunistic pathogen to humans when their immune system is deficient. Its pathogenicity may be linked to the production of virulence factors. We isolated P. aeruginosa strain RBS from the saltern of Sfax in Tunisia. In this study, we characterized the halotolerance, antibiotic susceptibility, and some virulence factors of strain RBS. High NaCl concentrations inhibited growth and motility. However, biofilm formation was enhanced to protect bacteria against salt stress. Among the 18 antibiotics tested, quinolones and tetracycline showed a significant inhibitory effect on growth, motility, and biofilm formation of strain RBS. ß-Lactams, however, did not have any inhibitory effect on neither bacterial growth nor motility. In some cases, resistance was due, in part, to biofilm formation. We also showed that RBS produces two proteases, LasB and AprA, which have been shown to be implicated in host infection. LasB was further characterized to study the role of metal ions in enzyme stability. It possesses two distinct metal ion-binding sites coordinating a calcium and a zinc ion. The effect of metal ion chelation was evaluated as well as substitutions of residues involved in metal ion binding. Impairing metal ion binding of LasB led to a loss of activity and a sharp decrease of stability. Our findings suggest that the binding of both metal ions is interdependent as the two metal ions' binding sites are linked via a hydrogen bond network.

Assessment of methods of detection of water estrogenicity for their use as monitoring tools in a process of estrogenicity removal.

Methods of monitoring of estrogenicity in water were gathered, compared, and tested within the context of their practical use as measurement and design tools, in the development of a process of degradation of estrogenic endocrine disruptors. In this work, the focus was put on in vitro assays, with the use of analytical techniques as additional analysis when possible. Practically, from a literature review, four methods that seemed most suitable to practical use required in a process development were tested: the Yeast Estrogen Screen assay, the Lyticase-assisted Yeast Estrogen Screen assay (LYES), the MMV-LUC assay and the HPLC-UV analytical method. Dose-response curves in response to estrogenic standard 17ß-estradiol were compared. Bisphenol A estrogenicity was measured by the methods as well. The model for the calculation of estradiol equivalents as measurements units was adapted. The methods were assessed in terms of ranges of detection, time of experiment, cost, ease of the experiment, reproducibility, etc. Based on that assessment, the LYES assay was selected and successfully applied to the monitoring of estrogenicity removal from 17ß-estradiol and bisphenol A. More precisely, the bioassay allowed the acquisition of kinetic curves for a laboratory-scaled process of estrogenicity removal by immobilized enzymes in a continuous packed-bed reactor. The LYES assay was found to have a real methodological potential for scale-up and design of a treatment process. The HPLC-UV method showed good complementarity with the LYES assay for the monitoring of bisphenol A concentrations in parallel with estrogenicity, reporting no significant estrogenicity from degradation byproducts, among others.

The stress proteome of Enterococcus faecalis.

Enterococcus faecalis is a resident bacterium of the intestinal tract of humans and animals. This bacterium can be responsible for serious diseases and is one of the largest causes of hospital-based infections. This hardy organism resists many kinds of stresses and is used as a major indicator of the hygienic quality of food, milk, and drinking water. On the other side, enterococci seem to have beneficial role in the development of cheese aroma and are added in certain starter cultures. Since ten years, our laboratory has used the two-dimensional electrophoresis (2-DE) technique to study the response of E. faecalis to physical or chemical stresses as well as to glucose and total starvation. Twenty-seven protein spots on 2-D gels have been identified by N-terminal sequencing or Western blotting which make up the first proteome database of this species. The proteins were classified in four different groups according to their function and their regulation. The first group comprises well-characterized proteins with known protective functions towards stresses. The second group contains enzymes of catabolic pathways. Their implication in stress resistance seems not obvious. A third group are proteins induced in glucose-starved cells belonging to the CcpA regulon. Induction of these enzymes under starvation may serve to increase the scavenging capacity of the cells for nutrients or may be important to mobilize endogenous energetic reserves. Lastly, nine N-terminal amino acid sequences or open reading frames (ORF) showed no homologies with sequences in databases. A comprehensive description of stress proteins of E. faecalis and analysis of their patterns of expression under different environmental conditions would greatly increase our understanding of the molecular mechanisms underlying the extraordinary capacity of this bacterium to survive under hostile conditions.

Identification and characterization of gsp65, an organic hydroperoxide resistance (ohr) gene encoding a general stress protein in Enterococcus faecalis.

The Enterococcus faecalis general stress protein Gsp65 has been purified from two-dimensional gel electrophoresis. Determination of its N-terminal sequence and characterization of the corresponding gene revealed that the gsp65 product is a 133-amino-acid protein sharing homologies with organic hydroperoxide resistance (Ohr) proteins. Transcriptional analysis of gsp65 gave evidence for a monocistronic mRNA initiated 52 nucleotides upstream of the ATG start codon and for an induction in response to hydrogen peroxide, heat shock, acid pH, detergents, ethanol, sodium chloride, and tert-butylhydroperoxide (tBOOH). A gsp65 mutant showed increased sensitivity to the organic hydroperoxide tBOOH and to ethanol.

Impact of thermal variations on biochemical and physiological traits in Pectinatus sp.

The influence of temperature on cellular fatty acid composition and on heat stress tolerance was studied in the two species of Pectinatus, an anaerobic gram-negative bacterium. Cellular fatty acid (FA) patterns were determined for Pectinatus species cultivated in MRS medium at various defined conditions of temperature and pH. Our study shows that fluctuations of growth temperature and pH induced important changes in the ratio of unsaturated FAs (UFAs) to saturated FAs (SFAs). The major differences in the FA composition as a function of growth temperature concerned C15:0 and C17:0 for the SFAs and C15:1 and C17:1 for the UFAs. The most significant adaptation of lipid composition to lower growth temperatures was the strong increase of UFAs, particularly for C15:1 and C17:1 concomitantly with a decrease of SFAs (C15:0 and C17:0). When the pH of the culture medium was lowered from 6.2 to 4.0, a notable drop in the synthesis of the UFAs C15:1 and C17:1 was observed together with an important increase of C18-cyclopropane (C18-cyc) and high carbon number SFAs. Thermal modifications also provoked changes in Pectinatus behaviour. We observed that P. cerevisiiphilus was more heat sensitive than P. frisingensis. Mild exponential phase cells were treated for 1 h, at 40 degrees C for P. cerevisiiphilus or at 41 degrees C for P. frisingensis. This thermal adaptation induced tolerance against heat challenge (49 and 50 degrees C for P. cerevisiiphilus and P. frisingensis, respectively). Survival of P. cerevisiiphilus and P. frisingensis adapted cells was, respectively, 3400- and 790-fold higher than control. Interestingly, adapted cells of P. cerevisiiphilus were more thermotolerant than P. frisingensis pretreated cells.

Identification of general stress genes in Enterococcus faecalis.

The susceptibility and the acquisition of tolerance of E. faecalis ATCC 19433 to heat, ethanol, bile salts, NaCl, H2O2 and pH shifts were determined. During exposure to these environmental stresses, protein synthesis analysed by 2-D electrophoresis revealed 167 stress proteins. Six stress protein were found to be induced by at least six of the eight treatments and considered to be general stress proteins (Gsp). Western blotting identified two of these Gsp as DnaK and GroEL. Analysis of the four other Gsp revealed that at least two of them were not previously described.

The oxidative stress response in Enterococcus faecalis: relationship between H2O2 tolerance and H2O2 stress proteins.

The hydrogen peroxide (H2O2) stress response in Enterococcus faecalis ATCC19433 was investigated. A 2.4 mmol l-1 H2O2 pretreatment conferred protection against a lethal concentration (45 mmol l-1) of this agent. The relatively high concentrations of H2O2 used for adaptation and challenge treatments in Ent. faecalis emphasised the strong resistance towards oxidative stress in this species. Various stresses (NaCl, heat, ethanol, acidity and alkalinity) induced weak or strong H2O2 cross-protection. This paper describes the involvement of protein synthesis in the active response to lethal dose of H2O2, in addition to the impressive enhancement of synthesis of five H2O2 stress proteins. Combined results suggest that these proteins might play an important role in the H2O2 tolerance response.

A new theta-type thermosensitive replicon from Lactococcus lactis as an integration vector for Enterococcus faecalis.

We isolated a replication thermosensitive mutant of the theta-type lactococcal pUCL22 replicon. An improved version of this thermosensitive replicon was obtained by fusioning the replication repA gene with the downstream repB gene. The resulting plasmid was named pUCB3522Ts. It is highly instable at 42 degrees C in Enterococcus faecalis. Integration into the chromosome via homologous recombination was monitored using the npr gene of E. faecalis JH2-2 as a target. A 513 bp PCR amplification product from an internal region of this npr gene was cloned into pUCB3522Ts. Integration of this construction into the JH2-2 npr gene was selected by shift temperature, from 30 degrees C to 42 degrees C. 85% of the analysed clones showed integration into the npr gene, demonstrating the practicality of this thermosensitive replicon as a genetic integrative tool for E. faecalis.

Molecular analysis of the replication region of the theta-replicating plasmid pUCL287 from Tetragenococcus (Pediococcus) halophilus ATCC33315.

The complete nucleotide sequence of the 8.7-kb theta-replicating plasmid pUCL287 from Tetragenococcus halophilus (formerly Pediococcus halophilus) ATCC33315 has been determined. The replication region was identified and analyzed. Its nucleotide sequence contains an untranslated region, the replication origin, followed by two open reading frames (ORFs) encoding two proteins of 311 (RepA287) and 168 (RepB287) amino acids, respectively. Evidence is presented to show that RepA287 represents the plasmid replication protein. RepB287, which is non-essential for replication, is involved in the plasmid copy-number control and segregational stability. The roles of lactococcal proteins homologous to RepB287 have not been defined so far. Nevertheless, the structural organization of the pUCL287 replication region is remarkably similar to those of well known theta-replicating lactococcal plasmids despite the absence of homology of the replication origin and of the replication protein, and this suggests that pUCL287 uses the same mechanism of replication. Nucleotide sequence comparisons show that pSMB74, a pediococcal plasmid encoding bacteriocin production, is a member of the pUCL287 replicon family.

[Enterococci in human environment]. / Les entérocoques dans l'environnement proche de l'homme.

Enterococci, formerly confounded with faecal streptococci, are recognized since the beginning of the century as being faecal in origin and are generally searched for in waste waters and food products; their detection may in fact indicate the presence of enteropathogenic organisms. Although nearly ubiquitous, their preferred ecological niche is the intestine sphere. Rejected in the environment by means of human faeces or animal dejecta, they are scattered afterwards in diverse niches. Once in the external environment, their survival is linked with their exceptional aptitude to resist or grow in hostile environments that are usually detrimental to the development of most mesophilic microorganisms. However, a certain ambiguity exists concerning their relationships with human beings. In fact, certain enterococcus strains or species are used in the elaboration of some milk products. Conversely, others are opportunists and may cause severe infections to people from infants to adults. Moreover, undergoing adaptation perpetually, they present a multiresistance pattern to antibiotics. Thus, the barrier that separates bacteria as nonoffensive contaminants from powerful pathogens appears most fragile, suggesting that people must systematically consider suspect the presence of enterococci in their near environment.

Alkaline stress response in Enterococcus faecalis: adaptation, cross-protection, and changes in protein synthesis.

The alkaline shock response in Enterococcus faecalis was studied in this work. Cells adapted to an optimum pH of 10.5 were tolerate to pH 11.9 conditions but acquired sensitivity to acid damage. An analysis of stress proteins revealed that 37 polypeptides were amplified. Two of these are DnaK and GroEL. The combined results show that bile salts and alkaline stress responses are closely related.

Glucose starvation response in Enterococcus faecalis JH2-2: survival and protein analysis.

We investigated the survival of Enterococcus faecalis following starvation provoked by energy source glucose exhaustion. Inhibition of protein synthesis by chloramphenicol before 3 h of starvation resulted in a dramatic decrease in viable bacteria. Antibiotic treatment of cells after 3 or 6 h of starvation had a progressively lesser influence on bacterial survival. During the first 24 h of deprivation, a total of 42 proteins were identified as glucose-starvation-inducible; 4 temporal classes of proteins (A, B, C and D) were defined in relation to their enhanced synthesis after glucose exhaustion. Our results show that proteins from the two early classes (A and B) seem to be the most important for long-term survival in E. faecalis. One protein of each of these classes was analysed at the molecular level. The N-terminal sequence of one of them, belonging to class A, showed strong homology with the N-terminal sequence of carbamate kinase from Streptococcus faecium. This enzyme could be implicated in the development of alternative metabolic pathways of energy production and could be compared to the Cst proteins of Escherichia coli.

Relationship between the thermotolerance and the increase of DnaK and GroEL synthesis in Enterococcus faecalis ATCC19433.

Relationship between intrinsic thermal resistance, thermotolerance and heat shock proteins (hsp) synthesis is studied in Enterococcus faecalis. We showed that an impressive phenotypic heat resistance was induced by mild heat and a slight thermotolerance was developed by various sublethal pretreatments such as NaCl, SDS and bile salts. Hydrogen peroxide, acid and alkaline shifts or "thermomimetic" agent such as ethanol, did not enhance the survival of adapted cells against the lethal thermal shock (62 degrees C). The inhibition of protein synthesis by chloramphenicol or rifampin abolished thermotolerance. The immunological identification of the DnaK and GroEL proteins in E. faecalis allowed to study induction of these molecular chaperones under various conditions. Heat was the most efficient inductor of DnaK and GroEL synthesis. However, it was surprising that ethanol did not strongly induce these proteins. We also show that amplification of these hsp is not correlated to acquired thermotolerance with a linear relationship. A weak thermotolerance is not coupled from increased synthesis of DnaK and GroEL. So, we postulate that the high synthesis of the major hsp is not obligatory in the thermal cross-protection but that de novo protein synthesis is an absolute necessity in E. faecalis. Activation of preformed hsp or other factors depending or not on protein synthesis may be also necessary to enhance thermal resistance.

Comparison of the bile salts and sodium dodecyl sulfate stress responses in Enterococcus faecalis.

The resistance to detergents and detergent-induced tolerance of a gastrointestinal organism, Enterococcus faecalis ATCC 19433, were examined. The most remarkable observation was the rapid response of cells in contact with bile salts and sodium dodecyl sulfate (SDS). The killing by high concentrations of detergents was nearly instantaneous. A 5-s adaptation with moderate sublethal concentrations of bile salts or SDS (0.08 or 0.01%, respectively) was sufficient to induce significant adaptation against homologous lethal conditions (0.3% bile salts or 0.017% SDS). However, resistance to a subsequent lethal challenge progressively increased further to a maximum reached after 30 min of adaptation. Furthermore, extremely strong cross-resistances were observed with bile salts- and SDS-adapted cells. However, no relationship seems to exist between levels of tolerance and de novo-synthesized proteins, since blockage of protein synthesis during adaptation had no effect on induction of resistance to bile salts and SDS. We conclude that this induced tolerance to detergent stress is independent of protein synthesis. Nevertheless, the stress-induced protein patterns of E. faecalis ATCC 19433 showed significant modifications. The rates of synthesis of 45 and 34 proteins were enhanced after treatments with bile salts and SDS, respectively. In spite of the overlap of 12 polypeptides, the protein profiles induced by the two detergents were different, suggesting that these detergents trigger different responses in E. faecalis. Therefore, bile salts cannot be substituted for SDS in biochemical detergent shock experiments with bacteria.

Starvation-induced multiresistance in Enterococcus faecalis JH2-2.

Compared with growing bacteria, carbohydrate-starved cells of Enterococcus faecalis show development of a multiresistance state against heat, H2O2, acid, and ethanol, but not against UV irradiation. The kinetics of acquisition of resistance is different according to the stress. Three hours of starvation provide maximal resistance against ethanol, while the tolerance to heat, H2O2, and acid increases progressively with the duration of starvation. Chloramphenicol treatment does not abolish the ethanol tolerance. Protein synthesis inhibition during the transitional growth phase and the first hours of starvation partially inhibit the acquisition of heat and oxidative resistances. Antibiotic treatment after 3 h of starvation does not affect the increase of these resistances. We suggest that synthesis of specific proteins revealed by 2-D gel analysis in the first 3 h of starvation, followed by a second mechanism related to protein degradation or alteration, is necessary for acquisition of maximal resistance towards heat and oxidative stresses.

Defense against lethal treatments and de novo protein synthesis induced by NaCl in Enterococcus faecalis ATCC 19433.

Enterococcus faecalis was strongly resistant to high osmotic pressure in complex medium; however, when it was subjected to a moderate osmotic stress [6.5% (w/v) NaCl or 52% (w/v) sucrose] for 2 h, it showed cross-protection against ethanol (22%), detergents stresses [bile sales (0.3%) and SDS (0.017%)], hydrogen peroxide challenge (45 mM), and to a minor extent against lethal temperature (62 degrees C). In response to salt stress [6.5% (w/v) NaCl], E. faecalis induced a large number of stress proteins. In addition, NaCl strongly induced the synthesis of many proteins more than tenfold. Although the acquired thermotolerance was inhibited markedly by chloramphenicol, the other NaCl-induced cross-tolerances seemed not to be correlated with de novo protein synthesis. The relationship between the stress protein synthesis and the induction of different types of cross-protection is discussed.

Relationship between stress response toward bile salts, acid and heat treatment in Enterococcus faecalis.

Stress tolerance and cross-protection in Enterococcus faecalis ATCC19433 were examined after exposure to bile salts, acid or heat shock. Bile salts and heat adapted cells demonstrated induced homologous tolerance and cross-resistance. No cross-protection of heat adapted cells against acid stress is observed and pretreatment with bile salts even sensitized the cells to this challenge. Whole-cell protein extract analysis revealed that each treatment induced a battery of stress proteins. Some of these polypeptides are induced by more than one treatment. The greatest overlap is observed between bile salts and heat treatments. Eighteen stress proteins, including DnaK and GroEL, are common between these stresses.