Influence of salinity upon the phenotypic expression of antibiotic resistance in nonhalophilic and halophilic vibrios.

The purpose of the present work is to demonstrate the influence of different NaCI concentrations included in the Mueller Hinton medium, upon the antibiotic susceptibility of 10 non-halophilic and 28 halophilic Vibrio strains. The highest number of resistance aspects were recorded at 1% NaCl concentration for V. cholerae O1/non O1 strains and at 3% for V. parahaemolyticus, V. algynolyticus, V. vulnificus, V. fisheri, V. anguillarum and V. metschnikovii.

Antimicrobial activity of some new of 2-(4-ethyl-phenoximethyl) benzoic acid thioureides against planktonic cells.

The aim of the present study was to evaluate the antimicrobial activity of new 2-(4-ethyl-phenoxymethyl) benzoic acid thioureides. The synthesis of the new compounds was done in three steps starting with the synthesis of 2-(4-ethyl-phenoxymethyl) benzoic acid. In the second stage, the 2-(4-ethyl-phenoxymethyl)-benzoyl chloride was prepared and the new thioureides were synthesized in the third step by the reaction of 2-(4-ethyl-phenoxymethyl) benzoyl isothiocyanate with various primary aromatic amines. The original compounds were screened for their in vitro antimicrobial activity against Gram-positive (Staphylococcus aureus, Bacillus subtilis) and Gram-negative bacteria (Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa) as well as to fungal strains (Candida albicans, Aspergillus niger), using both reference and clinical, multidrug resistant strains. The qualitative screening of the susceptibility spectra of various microbial strains versus these compounds was performed by three adaptated diffusion methods: (1) impregnation of the filter paper disks with the respective substance solutions, (2) distribution of the tested solutions into agar wells and (3) spotting of the respective solutions on the solid medium previously inoculated with the microbial suspensions. The quantitative assay of the antimicrobial activity was performed by broth microdilution method in 96-well microplates in order to establish the minimal inhibitory concentration (MIC). The tested compounds exhibited specific antimicrobial activity with MICs ranging from 3.9 microg/mL to 250 microg/mL.

Cytokine profiles of HeLa and human diploid cells induced by different fractions of Vibrio parahaemolyticus cultures exposed to stress conditions.

Vibrio (V.) parahaemolyticus is an aquatic halophilic bacteria which produces gastroenteritis and in rare cases septicaemia after the consumption of raw or under-cooked contaminated seafood.The severity of diarrheal illness caused by this bacterium is closely related to the presence of two types of hemolysins (the thermostable direct hemolysin-TDH and TDH related hemolysin-TRH) and also of type III secretion system (TTSS) proteins. The TTSS type 1 induces a wide array of effects on infected HeLa cells such as autophagy, oncosis, cell rounding and lysis. Previous studies have shown that heat shock proteins have the ability to stimulate the production of interleukins in different cellular cultures. In our studies we have stimulated two cellular lines (HeLa and human diploid cells) with different V. parahaemolyticus culture fractions in order to observe the effect on cytokines production. Thus, the purpose of this study was to analyze the expression of IL-1, IL-2, IL-4, IL-6, IL-10 and TNF-alpha induced by the cell treatment with total cellular lysate, periplasmic fractions and culture supernatants extracted from V. parahaemolyticus exposed to normal and also to stress conditions. The ELISA assay of the cytokine profile of the HeLa and HDC cell lines stimulated with different bacterial fractions revealed that in the V. parahemolyticus cultures submitted to osmotic and heat shock stress are accumulating factors (probably heat shock proteins) which are exhibiting immunomodulatory activity, responsible for the induction of a pro-inflammatory response associated with increased levels of IL-6 and TNF-alpha expression, however balanced by the stimulation of the anti-inflammatory cytokine IL-4 synthesis.

[Trends of antimicrobial resistance in microbial strains isolated from invasive infections in Romania. Experience of participation in EARSS (European Antimicrobial Resistance Surveillance System), 2002-2008]. / Tendintele rezistentei la antibiotice a izolatelor din infectii invazive în România. Experienta participarii în Sistemul European de Supraveghere a Rezistentei la Antibiotice (EARSS) în perioada 2002-2008.

EARSS (European Antimicrobial Resistance Surveillance System) is the biggest antimicrobial resistance surveillance project in the world financed from public finds, aiming to provide validated and comparable official data on antimicrobial resistance of invasive microbial strains (isolated from blood and CSF), belonging to 6 indicator bacterial species, i.e.: S. aureus, E. coli, E. faecium/faecalis, Str. pneumoniae, Ps. aeruginosa, K. pneumoniae. Romania reported data to EARSS since 2002 so far. Though the number of participating laboratories increased progressively from 12 to 35, the number of hospitals which reported for EARSS. as the number of strains included in the data base remained steady and relatively low. This issue is related to the particular position of Romania in the European context, in respect of the very low number of blood cultures performed in hospitals. Our paper is presenting the trends of antimicrobial resistance in the indicator strains in the 2002-2008 interval. During the 2002-2008 interval, Romania reported to EARSS a total number of 1276 bacterial strains, distributed by species as follows: 513 S aureus, 369 E. coli, 128 Streptococcus pneumoniae, 127 Enterococcus spp.. 71 Klebsiella pneumoniae, 68 Pseudomonas aeruginosa. Klebsiella pneumoniae and Pseudomonas aeruginosa were reported, according to the EARSS protocol, only for the 2005-2008 interval. It is difficult to describe trends, specially in Enterococcus, Streptococcus pneumonaie and the 2 species collected only since 2005, because of the low number of isolates, but there are several results that are supporting us to claim that antimicrobial resistance in invasive isolates is a real problem in Romanian hospitals, like in other Central, Southern and South Eastern European countries: more than 25% of S. aureus strains resistant to methicilline, with more than 50% in some years, high aminoglycozides resistance in more than 70-80% of Enterococcus faecium invasive strains, more than 80% of strains resistant to 3rd generation cephalosporines etc.

Subinhibitory concentrations of phenyl lactic acid interfere with the expression of virulence factors in Staphylococcus aureus and Pseudomonas aeruginosa clinical strains.

The discovery of intra- and intercellular communication systems (quorum sensing systems) regulating bacterial virulence has afforded a novel opportunity to control infectious bacteria, without interfering with their growth. In this study, we investigated the ability of subinhibitory concentrations (sIC) of phenyl lactic acid (PLA), known to be produced by Lactobacillus probiotic strains, to attenuate the virulence and pathogenicity of Pseudomonas aeruginosa (as experimental model of intercellular bacterial communication in Gram-negative bacteria) and Staphylococcus aureus (as experimental model of intercellular bacterial communication in Gram-positive bacteria) by interfering with the coordinated expression of different virulence factors implicated in the pathogenicity of these opportunistic strains. Our results showed that sIC of PLA decreased the ability of the tested strains to adhere both to the cellular and inert substrata and induced changes in the adherence patterns as well as in the cell morphology. The sIC of PLA induced a significant decrease of sheep red blood cells haemolysins, lecithinase and caseinase and stimulated lipase and gelatinase production by Pseudomonas strains. The sIC of PLA induced an important and constant increase of the Pseudomonas growth inhibition zones diameters for all tested antibiotics, demonstrating the potential use of PLA in the design of new synergic antimicrobial associations active on multiresistant and biofilm-growing P, aeruginosa strains. The present study has proved the role of sIC of PLA released by Lactobacillus probiotic strains in the attenuation of P. aeruginosa and S. aureus virulence and pathogenicity, by interfering with different processes depending on cell density and regulated by quorum sensing (i.e. growth rate, expression of adhesion molecules and secretion of soluble, enzymatic factors) and altering the success of these pathogens in the colonization of a sensitive host and the development of an infectious process. Our results demonstrate that this probiotic soluble products could be used as a new, ecological anti-infective strategy with great therapeutic and preventive value in the biomedical field (especially in the treatment of chronic infections produced by multiresistant and biofilm forming microorganisms), but also in the management of the environmental quality, agriculture and industrial field by reducing the chemical burden delivered in the external medium and by preventing the surfaces colonization with microorganisms and the development of natural biofilms.

New thioureides of 2-(4-methylphenoxymethyl) benzoic acid with antimicrobial activity.

The purpose of this study was to evaluate in vitro, by means of qualitative and quantative methods, the antimicrobial activity of some new synthesized chemical compounds previously solubilized in DMF (Dimethyl formamide). The qualitative screening of the susceptibility spectra of different microbial strains versus these compounds was performed by three adaptated diffusion methods: paper filter disk impregnation with the tested substances solutions, the disposal of tested solutions in agar wells and the spotting of tested solutions on solid medium previously inoculated with microbial suspension. The quantitative assay of the antimicrobial activity was performed by broth microdilution method in 96-well microplates in order to establish the minimal inhibitory concentration (MIC).The antimicrobial activity was tested against Gram-positive strains (Staphylococcus) aureus sp., Bacillus subtilis sp.), Gram-negative (Escherichia coli sp, Pseudomonas aeruginosa sp., Klebsiella pneumonia sp.) and fungal strains (Candida albicans sp, Aspergillus nige rsp.). All tests were performed by comparison with the reference strains: K. pneumoniae IC 13420, E. coli IC 13529, S. aureus IC 13204, P. aeruginosa IC 13202, B. subtilis IC 12488, C. albicans IC 249, A. niger IC 13534. Our results indicated that the tested compounds exhibited specific antimicrobial activity, the highest activity being noticed against planktonic fungal cells (MIC ranging from 62.5 microg to 15.6 microg), followed by P. aeruginosa (MICs from 250 microg to 31.5 microg). Only few compounds exhibited antimicrobial activity against E. coli and K. pneumoniae. Antimicrobial activity against Gram positive bacteria was noticed for the most of the tested compounds, five of them exhibiting very low MIC values (MICs from 1000 microg to 62.5 microg).

The role of proteins expressed under the stress condition in virulence of some Vibrio strains.

In order to survive in changing environments, bacteria possess enormous adaptive capabilities that allow them to modulate their behavior and reprogram gene expression in response to environmental cues. Vibrios are inhabitants of estuarine and fresh waters and some species are pathogenic to humans, and marine vertebrates and invertebrates. Surface attachment is believed to be essential for colonization of all of these natural environments. Vibrio (V.) parahaemolyticus, an ubiquitous marine bacterium and human pathogen, seems to be particularly adapted to growth on surfaces or in biofilms. In response to its physical environment, V. parahaemolyticus induces the expression of a large number of genes. The purpose of the present study was to investigate the influence of different physicochemical parameters (temperature and osmolarity) on the virulence factors expression in Vibrio strains using different conditions simulating environmental stress factors. Some of the tested strains displayed a decreased adherence capacity to the inert substrate under stressful conditions, and the adherence capacity on HeLa cell was generally reduced, while the soluble enzymatic factors showed only slight changes. However, it is to be noticed that the haemolysins and Kanagawa enterotoxin were better expressed at higher temperature and osmolarity, these factors probably contributing to the bacterial adaptation and survival in the extern medium of certain Vibrio species.

Virulence and resistance markers in clinical and environmental Aeromonas strains isolated in Romania.

The virulence and resistance (R) features of 37 Aeromonas strains from diarrheal cases and 150 from the aquatic environment (isolated during cold and warm season) were tested at different incubation temperatures (4 degrees C, 28 degrees C and 37 degrees C). When incubated at 4 degrees C temperature, the Aeromonasstrains isolated during the cold season expressed the highest number of virulence factors by comparison with the strains isolated during warm season and from diarrhoeal cases, the virulence spectrum increasing simultaneously with the incubation temperature (i.e. 28 degrees C and 37 degrees C) for all strains. Mucinase was the unique virulence factor constantly present in all three categories of strains at all three incubation temperatures. The aquatic as well as clinical strains exhibited similar R levels to ampicillin and colistin, while for the other tested antibiotics, the aquatic strains generally proved higher R levels than clinical strains, excepting cephtazidime. Plasmids of molecular weights ranging between 1904-21226 bp, were isolated in 36.5% of Aeromonas strains, some of them being correlated with specific R patterns. The large virulence spectrum correlated with high R in Aeromonas strains isolated from the aquatic medium is pleading for the significant role of these bacteria in the pathogenic potential of the natural reservoir possibly implicated in human pathology.

[Effect of transferrin on the expression of virulence factors in Listeria monocytogenes strains]. / Studiul influentei transferinei asupra exprimarii factorilor de virulenta la tulpinile de L. monocytogenes.

Iron is an essential element for the great majority of microorganisms, which developed transport systems for iron acquisition, because during the colonization and invasion processes the microorganisms encounter iron-limiting conditions. The bacterial genes implicated in the iron transport and those codifying for other virulence factors are simultaneously depressed. The purpose of this study was to investigate the role of transferrin on the virulence factors expression in food isolated Listeria monocytogenes strains. Our results showed that transferrin stimulates the bacterial growth rates, as well as the adherence and invasion capacity (27 x 10(4) CFU/ml vs 13 x 10(4) CFU/ml) to cellular substrate and inert one (as shown by slime test). Transferrin also induced the secretion of haemolysins, amylases and lecithinases, demonstrating the role of iron ions in the virulence genes derepression and the simulation of bacterial growth rate.

[Discordancies between classical and API 20E microtest biochemical identification of Vibrio and Aeromonas strains]. / Discordante între testele biochimice clasice si galeriile microtest API 20E în diagnosticul diferential al tulpinilor din genurile Vibrio si Aeromonas.

The present study reveals some discordancies of diagnosis considering Vibrionaceae strains when tested by comparison with API 20E (BioMerieux) system and classical biochemical reactions, aspects already mentioned in the literature by other authors. If any misidentification has not an important impact (e.g. identification of Aeromonas schubertii as A. sobria), others as misidentification of Aeromonas as Vibrio strains or the failure to recognize Vibrio strains raise major epidemiological problems. The present study was performed on 25 aquatic strains, comparatively identified by means of API 20E system and classical biochemical tests (using media prepared in Vibrio Laboratory of Cantacuzino Institute). All 25 strains were identified as Vibrio fluvialis by API 20E system, while using the classical tests one strain proved to belong to Pseudomonadaceae (O/F test of oxidative type), one was identified as Aeromonas schubertii, 2 Aeromonas hydrophila and 4 Aeromonas caviae. Discordancies between API 20 E system and classical tests were registered for: gelatinase (20%), LDC 12% and ADH, ODH, O/F test, saccharose, manite, inosite (4% each). The misidentification of Aeromonas as Vibrio strains by API 20E hinders the recognition of Aeromonas infections implicated more and more frequently in human pathology in immunodeficient as well as in immunocompetent hosts. Our results demonstrate that the use of API 20E system has some limits in the diagnostic of some bacterial genera (Vibrio, Aeromonas), and the introduction of these systems in the current laboratory practice requires previous information and studies regarding their efficiency and availability for the diagnostic of particular bacterial genera and species.

[Comparative study of classical and commercial microtest API galleries in the diagnosis of cholera infection]. / Studiul comparativ al eficientei testelor conventionale si a kiturilor comerciale (microteste) API în diagnosticul infectiei holerice.

Cholera still remains in the top of causes generating global mortality and morbidity, as revealed by the latest WHO reports. In 2000, on CDC/Atlanta website, the cholera agent was mentioned as potential biological weapon for bioterrorism actions in 9460 publications. Considering these aspects, a correct and rapid diagnosis is necessary in order to take appropriate epidemiological measures and to prevent the secondary transmission. Our study evaluated the efficiency of microtest systems API20E, API 20 NE and RapID 20E, by comparison with classical biochemical tests concerning the diagnosis of 150 Vibrio cholerae strains isolated during cholera epidemics of Romania (1990-1995). Our results demonstrated that classical biochemical identification still remains the most secure diagnostic method for Vibrio cholerae strains. The kits API 20NE gave the highest number of results in concordance with the classical tests. For these reasons, the confirmation of cholera infection must be performed in a National Reference Center. The limits of API galleries concerning the diagnosis of Vibrio cholerae strains must be taken into account and in case of suspect clinical symptoms, the isolated strains must be sent to the Cantacuzino Institute, even if the microtest galleries have not identified V. cholerae strains.

[Study of antibiotic influence on adherence capacity of gram positive and gram negative bacteria to the cellular substrate]. / Studiul influentei antibioticelor asupra capacitatii de aderenta a bacteriilor gram-pozitive si gram-negative la substratul celular.

Bacterial adherence to the cellular substrate (skin and mucosa) represents a precondition of infectious pathology. It was demonstrated that bacteria which adhere and form biofilms on catheters and other inert materials used in medicine are resistant to the therapeutic antibiotic concentrations being protected by the biofilm mathrix and generating severe and hard to treat infections. There are only few studies on the influence of antibiotics on the bacterial adhesins synthesis and bacterial adherence to the cellular substrate. The purpose of this study was to investigate the influence of subinhibitory concentrations of antibiotics on adherence capacity of Listeria monocytogenes, Vibrio cholerae and Aeromonas hydrophyla to the cellular substrate represented by HEp-2 cells. Suspensions (approximately 10(10) cells/ml) of bacterial cultures developed on solid media were incubated for 30 minutes in the presence of subinhibitory concentrations of penicillin, ampicillin, amoxicilin with clavulanic acid, ceftazidim, norfloxacin, kanamycin, chloramphenicole and vancomycin. Study of bacterial adherence to the cellular substrate was done by Cravioto's modified method. The quantitative evaluation of adherence/invasion capacity of bacterial suspensions pretreated with antibiotics was done by comparing the adherence/invasion index with controls without antibiotics. Penicillin, amoxicillin with clavulanic acid and vancomycin have significantly stimulated the adherence of Listeria monocytogenes strain and inhibited the adherence of Vibrio cholerae and Aeromonas hydrophyla strains. Ampicillin and chloramphenicole exhibited no significant effect on bacterial adherence capacity. The influence of kanamycin, ceftazidim and norfloxacin could not be interpreted due to the occurrence of a severe cytotoxic effect manifested by cell monolayer detaching, probably due to the action of antibiotic suspensions or to the increase of bacterial virulence under the selective pressure of the antibiotic.

[Factors influencing the capacity of cellular substrate adherence of vibrio cholerae O1 and non O1 strains]. / Studiul factorilor care influenteaza capacitatea de aderenta la substratul celular a tulpinilor de Vibrio cholerae O1 si non O1.

Bacterial adherence to eukariotic cells represents an important step of tissue colonization and is mediated by specific molecules called adhesins. Bacterial adherence to cellular substrate is a very complex process consisting in specific interactions between the surface of host cell and bacterial cell surface respectively. Adherence to cellular substrate confers selective advantages to bacterial cells, as: rapid growth rate by shorter lag period and protection against antibodies and lysozime. Adherence and colonization of small bowel represent the early steps of cholera infection (1, 2). The purposes of this study were to characterize the adherence ability of 46 Vibrio cholerae O1 and non O1 strains with different sources of isolation (acute diarrhea, water sources) to HEp-2 cell; to determine the influence of different factors (culture media, bacterial culture growth phase, proteolytic enzymes, carbohydrates and polyvalent agglutinant anti V. cholerae O1 serum) on the bacterial adherence capacity. Adherence capacity was assayed using the qualitative Cravioto's method. The adherence ability was appreciated by semi quantitative ("+", "++" and "+++") and quantitative assays. The adherence pattern of the tested strains was predominantly a diffuse one. The agar medium proved to be the most appropriate for the early and maximal expression of adhesion molecules, by comparison with nutritive broth and alkaline peptone water. Manose in different concentrations (1% and 3%) inhibited the adherence ability, demonstrating the role of manose-sensitive haemagglutinating fimbriae (MSHA) in mediating the adherence of V. cholerae strains to cellular substrate. Trypsine has no notable effect on the adherence ability, suggesting that the major V. cholerae adhesion molecules are not essentially of protein nature, so that the afimbrial adhesins could also play an important role in bacterial adhesion to eukariotic cells. The agglutinant polyvalent anti-V. cholerae O1 serum had the most significant inhibitory effect on the adherence ability, which was completely abolished in the presence of sub-agglutinant dilutions of serum titer (1/60-1/120) and partially reduced at titers ranging from 1/240 to 1/920. This inhibitory effect could be explained by bacterial agglutination, but also by the specific blocking of some surface structure implicated in the adherence process (i.e. lipopolysaccharides, as demonstrated by the inhibitory effect of sub-agglutinant serum titers). The inhibitory effect of polyvalent anti-V. cholerae O1 serum was limited to O1, but was not evident for the non O1 serogroups, demonstrating that the serum antibodies are acting on serogroup specific antigenic fractions.

[ Study of exo-enzymatic profile and cytotoxic effect of bacterial culture supernatants in different growth phase]. / Studiul profilului exo-enzimatic si al citotoxicitatii supernatantelor de culturi bacteriene în diferite faze de crestere.

Bacterial quorum-sensing represents an ubiquitary regulating system in which the pheromones (small molecules with different chemical structures, i.e. homoserin-lactones, octapeptides, aminoacids) act as extracellular mediators of signaling and intercellular communication. This chemical system is implicated in the regulation of different physiological processes dependent on the cell density (i.e. biolumniscence, virulence factors expression, sporulation, conjugation, antibiotic secretion etc). It is also mentioned in the literature the implication of bacterial pheromones in the modulation of eukariotic cells division rate. The objectives of this study were: a) to determine the exo-enzymatic profile of bacterial cultures in different growth phase in order to establish potential relationships between the phenotypic expression of some virulence factors on one side and the growth phase and bacterial culture density, on the other side; b) to determine de cytotoxic effect and the influence of bacterial culture supernatants on the HEp-2 cell division rate. Supernatants of bacterial cultures in nutrient broth of 2, 5 and 24 hrs of Staphylococcus aureus and Proteus sp. were tested directly and also, after thermic inactivation (at 100 degrees C, for 5 minutes) for the presence of different enzymatic activities known as virulence factors (spot and Kanagawa haemolysins, CAMP-like factor, caseinase, amilase, lipase, lecithinase, mucinase, DNA-ase). The exo-enzymatic profile of bacterial cultures of 2 and 5 hrs proved to be similar, the tested supernatants exhibiting haemolytic activity, and for Staphylococcus aureus, amilase and caseinase activities. Supernatants of and 5 hrs bacterial cultures exhibited also cytotoxic effect on HEp-2 cells. Supernatants of bacterial cultures of 24 hrs exhibited neither enzymatic activities, nor cytotoxic effect on HEp-2 cells, probably due to the inhibition of phenotypic expression of enzymatic activities at high bacterial densities through the activation of the quorum-sensing system. Bacterial supernatants did not significantly influence the HEp-2 cells division rate.