Synergistic combinations of antimicrobial peptides against biofilms of methicillin-resistant Staphylococcus aureus (MRSA) on polystyrene and medical devices.

OBJECTIVES: Antimicrobial research is being focused to look for more effective therapeutics against antibiotic-resistant infections such as those caused by methicillin-resistant Staphylococcus aureus (MRSA). In this regard, antimicrobial peptides (AMPs) appear to be a promising solution. The aim of the present study was to investigate the potential activity of temporin A, citropin 1.1, CA(1-7)M(2-9)NH2 and Pal-KGK-NH2 in synergistic activity against MRSA biofilms developed on polystyrene surface (PSS) and central venous catheter (CVC). METHODS: The study was subdivided into distinct phases to assess the ability of AMPs to inhibit biofilm formation, to identify possible synergy between AMPs, and to eradicate preformed biofilms on PSS and CVC using AMPs alone or in combination. RESULTS: Activity of the AMPs was particularly evident in the inhibition of biofilm formation on PSS and CVC, whilst the eradication of preformed biofilms was more difficult and was reached only after 24h of contact. The synergistic activity of AMP combinations, selected by their fractional inhibitory concentration index (FICI), led to an improvement in the performance of all of the molecules in the removal of different biofilms. CONCLUSION: Overall, AMPs could represent the next generation of antimicrobial agents for a prophylactic or therapeutic tool to control biofilms of antibiotic-resistant bacteria and/or biofilm-associated infections on different medical devices.

Lactobacillus spp. inhibit the growth of Cronobacter sakazakii ATCC 29544 by altering its membrane integrity.

The effect of the cell-free culture supernatants (CFCSs) from different Lacobacillus spp. on growth ability of Cronobacter sakazakii ATCC 29544 was investigated by time-killing studies. The antimicrobial effect was evaluated using crude and 2.5 × concentrated CFCSs. Most of the CFCSs showed a dose-dependent antimicrobial activity, with the greatest C. sakazakii growth inhibition exerted by the CFCS 2.5 × of Lactobacillus casei rhamnosus ATCC 7469. Indeed, C. sakazakii growth was completely inhibited after 4 h of incubation with the crude CFCSs of L. casei rhamnosus and Lactobacillus acidophilus and after only 2 h using the related 2.5 × CFCSs. The flow cytometric analysis revealed that CFCSs altered the permeability of C. sakazakii cell membrane, showing 55% of live cells after 30 min of treatment with 2.5 × CFCSs of L. casei rhamnosus and L. acidophilus, reaching 1% of live cells after 2 h of exposure. The CFCSs of L. casei rhamnosus and L. acidophilus have showed anti-Cronobacter activity, determining a progressively inhibition of C. sakazakii growth as result of alterations in its membrane permeability.

Antimicrobial Activity of Different Antimicrobial Peptides (AMPs) Against Clinical Methicillin-resistant Staphylococcus aureus (MRSA).

BACKGROUND: Antimicrobial research is being focused to look for more effective therapeutics against antibiotic-resistant infections caused by methicillin-resistant Staphylococcus aureus (MRSA). In this direction, antimicrobial peptides (AMP) appear as promising tool. OBJECTIVES: This study evaluated the antimicrobial activity of different AMPs (Citropin 1.1, Temporin A, Pexiganan, CA(1-7)M(2-9)NH2, Pal-KGK-NH2, Pal-KKKK-NH2, LL-37) against human MRSA clinical isolates. METHODS: The Minimum Inhibitory Concentration (MIC) was assessed for each AMP; then, the most active ones (Citropin 1.1, Temporin A, CA(1-7)M(2-9)NH2 and Pal-KGK-NH2) were tested against selected MRSA strains by time-kill studies. RESULTS: The lowest MIC value was observed for Pal-KGK-NH2 (1 µg/ml), followed by Temporin A (4- 16 µg/ml), CA(1-7)M(2-9)NH2 (8-16 µg/ml) and Citropin 1.1 (16-64 µg/ml), while higher MICs were evidenced for LL-37, Pexiganan and Pal-KKKK-NH2 (> 128 µg/ml). In time-kill experiments, Citropin 1.1 and CA(1-7)M(2-9)NH2 showed a relatively high percentage of growth inhibition (>30 %) for all the tested MRSA clinical isolates, with a dose-dependent activity resulting in the highest percentage of bacterial growth inhibition (89.39%) at 2MIC concentration. CONCLUSION: Overall, our data demonstrated the potential of some AMPs against MRSA isolates, such as Citropin 1.1 and CA(1-7)M(2-9)NH2, that represents a promising area of development for different clinical applications.

Experimental approach for a possible integrated protocol to determine sanitizer activity against both planktonic bacteria and related biofilms.

The persistence of pathogenic bacteria in industrial settings is linked to biofilm embedded bacteria resistance to antimicrobial and disinfectant methods effective against planktonic cells. We proposed an experimental approach to evaluate sanitizers effectiveness against both planktonic microorganisms and related biofilms as possible integration of the official EN 1276 procedure. Firstly, the efficacy of three chemicals sanitizers was tested on planktonic cells of Escherichia coli O157:H7 ATCC 35150, Staphylococcus aureus ATCC 43387, Pseudomonas aeruginosa ATCC 9027, Enterococcus faecalis ATCC 29212 and Candida albicans ATCC 14053 using the suspension test indicated by EN 1276 in both dirty and clear simulated conditions (0.3% or 0.03% of bovine serum albumen). The sanitizers were tested against the related biofilms developed on stainless steel for 48 h at room temperature. The sanitizers (SANI 626, SUPERIG, IGIEN 155) reached 5-logarithmic reduction at the manufacture's recommended concentrations after 30 s and 5 min against planktonic microorganisms but, sometimes, the organic load interfered with their activity. The same concentrations tested with the proposed protocol weren't effective against biofilms and a log reduction >3 was reached using higher concentrations of the sanitizers and 15 min of contact, with the exception of IGIEN 155. The efficacy of a disinfectant/sanitizer is assessed against planktonic microorganisms and bacteria adhered to surface, while those embedded in biofilms are not taken into consideration. The proposed protocol could be used to evaluate the effectiveness of a sanitizer also against microorganisms organized in biofilms, in order to give to the users more detailed information on its activity.

Development of a rapid PCR protocol to detect Vibrio parahaemolyticus in clams.

Vibrio parahaemolyticus is part of the natural microflora of estuarine and coastal marine waters and can be also present in seafood, especially shellfish and bivalve molluscs. In this study we compared the reference cultural method ISO 6887-3 with two molecular methods, multiplex PCR and real-time PCR, for the detection of two distinct genetic markers (tlh species-specific gene and tdh virulence gene) of V. parahaemolyticus in bivalve mollusc. The analyses were performed on clams inoculated with V. parahaemolyticus ATCC 43996 at T0 and after a 3 and 6 h of pre-enrichment in alkaline saline peptone water. Counts on agar plates were largely inaccurate, probably due to other Vibrio species grown on the TCBS selective agar. Multiplex PCR assays, performed using primers pairs for tdh and tlh genes, showed a detection limit of 104 CFU/g of shell stock within 6 h of pre-enrichment, respecting however the action level indicated by the National Seafood Sanitation Program guideline. Detection by tdh gene in real-time PCR reached the definitely highest sensitivity in shorter times, 101 CFU/g after 3 h of pre-enrichment, while the sensitivity for the tlh gene was not promising, detecting between 105 and 106 CFU/g after 6 h of pre-enrichment. Our findings provide a rapid routine method of detection of V. parahaemolyticus based on tdh gene by real-time PCR for commercial seafood analysis to identify the risk of gastrointestinal diseases.

Chitosans inhibit the growth and the adhesion of Klebsiella pneumoniae and Escherichia coli clinical isolates on urinary catheters.

OBJECTIVE: The aim of this study was to determine the antimicrobial activity of different chitosans (CS) against typical colonizing pathogens of the urinary tract and to assess their efficacy against bacterial adhesion and the subsequent biofilm formation on urinary catheters. METHODS: The antimicrobial activity of high and low molecular weight CS (50 and 150 kDa) at pH 5.0 and 6.0 was tested against Klebsiella pneumoniae and Escherichia coli clinical isolates by time-kill studies. The anti-adhesion assays on Foley urinary catheters were performed in Artificial Urine Medium (AUM) with the addition of each CS (AUM-CS) at the same pH values. Finally, the efficacy over time of chitosan treatments on bacterial adhesion on urinary catheters was determined. RESULTS: A viability reduction of K. pneumoniae and E. coli isolates, regardless of pH value, was evidenced in time-kill studies, in particular in the presence of CS 50 kDa. As regards the anti-adhesion efficacy on urinary catheters, high and low molecular weight CS evidenced a higher efficacy to reduce bacterial adhesion at pH 5.0. A low number of viable K. pneumoniae and E. coli cells were recovered from catheters after CS treatments, highlighting a promising efficacy over time. CONCLUSION: Our data show the potential of chitosans to reduce or prevent not only the adhesion of well-known human uropathogens on urinary catheters but also the re-growth ability of the uropathogens.

A dual-species microbial model for studying the dynamics between oral streptococci and periodontal pathogens during biofilm development on titanium surfaces by flow cytometry.

The association of the pioneer organisms Streptococcus mutans ATCC 25175 or Streptococcus oralis ATCC 9811 with secondary colonizers Fusobacterium nucleatum ATCC 25586 or Porphyromonas gingivalis ATCC 33277 during biofilm development on titanium surfaces was evaluated by flow cytometry (FCM) using specific polyclonal antibodies. ELISA and FCM were employed, revealing high antibody sensitivity and specificity. Biofilm formation of four dual-species combinations was analyzed by crystal violet staining, while the association between streptococci and periodontal pathogens was assessed using FCM. Dual-species association between S. oralis and P. gingivalis or F. nucleatum showed a proportional decrease in S. oralis during biofilm development, with a concomitant increase in P. gingivalis or F. nucleatum. This trend was not observed in either of the dual-species associations of S. mutans with the periodontal pathogens. Our dual-species microbial model, which employed FCM, proved to be useful in the study of partnerships between bacteria in oral associations, showing that the presence of primary colonizers is required for the establishment of secondary colonizers in biofilms. The proposed experimental approach is technically simple to prepare and analyze, and also proved to be reproducible; hence, it is well-suited for investigating the development and dynamics of oral communities.

Characterization of biosurfactants produced by Lactobacillus spp. and their activity against oral streptococci biofilm.

Lactic acid bacteria (LAB) can interfere with pathogens through different mechanisms; one is the production of biosurfactants, a group of surface-active molecules, which inhibit the growth of potential pathogens. In the present study, biosurfactants produced by Lactobacillus reuteri DSM 17938, Lactobacillus acidophilus DDS-1, Lactobacillus rhamnosus ATCC 53103, and Lactobacillus paracasei B21060 were dialyzed (1 and 6 kDa) and characterized in term of reduction of surface tension and emulsifying activity. Then, aliquots of the different dialyzed biosurfactants were added to Streptococcus mutans ATCC 25175 and Streptococcus oralis ATCC 9811 in the culture medium during the formation of biofilm on titanium surface and the efficacy was determined by agar plate count, biomass analyses, and flow cytometry. Dialyzed biosurfactants showed abilities to reduce surface tension and to emulsifying paraffin oil. Moreover, they significantly inhibited the adhesion and biofilm formation on titanium surface of S. mutans and S. oralis in a dose-dependent way, as demonstrated by the remarkable decrease of cfu/ml values and biomass production. The antimicrobial properties observed for dialyzed biosurfactants produced by the tested lactobacilli opens future prospects for their use against microorganisms responsible of oral diseases.

Identification and functional traits of lactic acid bacteria isolated from Ciauscolo salami produced in Central Italy.

Lactic acid bacteria (LAB) from Ciauscolo salami produced in Marche Region of Central Italy, and LAB strains belonging to our laboratory collection were examined for their capability to survive at low pH and bile, to adhere to Caco-2 cells, and for antibiotic resistance. LAB from Ciauscolo were identified by ARDRA and RAPD-PCR. Our study showed that all LAB strains had good adaptation to gastric juice and moderate tolerance to bile. The adhesiveness was variable among strains but significantly lower in LAB from food. Antibiotic resistance was broadly spread among food strains, with level of resistance exceeding 15% for all the antibiotics tested. The resistance determinants erm(B) and tet(M) were found in nine strains of food origin (21.4%) while tet(L) in one strain of our collection (5%). Our work suggests that fermented foods are valuable sources of bacterial strains with functional traits of intestinal lactobacilli. These bacteria may be further studied for their use in probiotic applications.

In vitro activity of Carvacrol against titanium-adherent oral biofilms and planktonic cultures.

OBJECTIVE: The aim of this study was to test the effect of Carvacrol against oral pathogens and their preformed biofilms on titanium disc surface. METHODS: Minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC) and biofilm inhibitory concentration (BIC) were performed to evaluate Carvacrol antibacterial activity, while flow cytometry (FCM) was used to verify the Carvacrol effect on esterase activity and membrane permeability. Carvacrol was tested in vitro on single- and multi-species biofilms formed on titanium disc by Streptococcus mutans ATCC 25175, Porphyromonas gingivalis ATCC 33277 or Fusobacterium nucleatum ATCC 25586, in different combinations, comparing its effect to that of chlorhexidine. RESULTS: The pathogens were sensitive to Carvacrol with MICs and MBCs values of 0.25 % and 0.50 % and BICs of 0.5 % for S. mutans ATCC 25175 and 1 % for P. gingivalis ATCC 33277 and F. nucleatum ATCC 25586. FCM analysis showed that treatment of planktonic cultures with Carvacrol caused an increase of damaged cells and a decrement of bacteria with active esterase activity. Moreover, Carvacrol demonstrated greater biofilm formation preventive property compared to chlorhexidine against titanium-adherent single- and multi-specie biofilms, with statistically significant values. CONCLUSIONS: Carvacrol showed inhibitory activity against the tested oral pathogens and biofilm formation preventive property on their oral biofilm; then, it could be utilized to control and prevent the colonization of microorganisms with particular significance in human oral diseases. CLINICAL RELEVANCE: This natural compound may be proposed in daily hygiene formulations or as an alternative agent supporting traditional antimicrobial protocols to prevent periodontal diseases in implanted patients.

Effects of landfill leachate treatment on hepatopancreas of Armadillidium vulgare (Crustacea, Isopoda).

The major environmental impact of landfills is emission of pollutants via the leachate and gas pathways. The hepatopancreas of the terrestrial isopod Armadillidium vulgare (Isopoda, Crustacea, Latreille 1804) plays an important role in the bioaccumulation of contaminants, such as heavy metals. To evaluate the effects of landfill leachate treatment, 2 different approaches were applied: 1) the detection of accumulation of trace elements (As, Cd, Cr, Cu, Sb, Zn, Pb, Ni, V) in hepatopancreatic cells, and 2) the evaluation of biological effect of contaminants on fresh hepatopancreatic cells by flow-cytometric analyses. The presence of 2 different cell types (herein referred to as "small" [S] cells and "big" [B] cells, in agreement with the literature based on morphological examinations) was detected for the first time by flow cytometry, which also highlighted their different response to stress stimuli. In particular, B cells appeared more sensitive to landfill leachate treatment, being more damaged in the short term, while S cells seemed more adaptive. Furthermore, S cells could represent a pool from which they are able to differentiate into B cells. These findings were also confirmed by principal component analyses, underlining that S SYBR Green I bright cells correlate with specific chemicals (Ca, Cu, Co), confirming their resistance to stress stimuli, and suggesting that the decrease of specific cell types may prime other elements to replace them in a homeostasis-preservation framework.

Antagonistic activity of Lactobacillus acidophilus ATCC 4356 on the growth and adhesion/invasion characteristics of human Campylobacter jejuni.

The aim of this research was to determine the potential probiotic activity of Lactobacillus acidophilus ATCC 4356 against several human Campylobacter jejuni isolates. The ability to inhibit the pathogen's growth was evaluated by co-culture experiments as well as by antimicrobial assays with cell-free culture supernatant (CFCS), while interference with adhesion/invasion to intestinal Caco-2 cells was studied by exclusion, competition, and displacement tests. In the co-culture experiments L. acidophilus ATCC 4356 strain reduced the growth of C. jejuni with variable percentages of inhibition related to the contact time. The CFCS showed inhibitory activity against C. jejuni strains, stability to low pH, and thermal treatment and sensitivity to proteinase K and trypsin. L. acidophilus ATCC 4356 was able to reduce the adhesion and invasion to Caco-2 cells by most of the human C. jejuni strains. Displacement and exclusion mechanisms seem to be the preferred modalities, which caused a significant reduction of adhesion/invasion of pathogens to intestinal cells. The observed inhibitory properties of L. acidophilus ATCC 4356 on growth ability and on cells adhesion/invasion of C. jejuni may offer potential use of this strain for the management of Campylobacter infections.

Increased levels of erythropoietin in nipple aspirate fluid and in ductal cells from breast cancer patients.

BACKGROUND: Erythropoietin (Epo) is an important regulator of erythropoiesis, and controls proliferation and differentiation of both erythroid and non-erythroid tissues. Epo is actively synthesized by breast cells during lactation, and also plays a role in breast tissues promoting hypoxia-induced cancer initiation. Our aims are to perform an exploratory investigation on the Epo accumulation in breast secretions from healthy and cancer patients and its localization in breast cancer cells. METHODS: Epo was determined by ELISA, immunoprecipitation, western blot and immunocytochemical analyses in 130 Nipple Aspirate Fluids (NAF) from 102 NoCancer and 28 Breast Cancer (BC) patients, comparing results with those found in 10 milk, 45 serum samples and breast cancer cell lines. RESULTS: Epo levels in NAFs were significantly higher than those in milk and serum. No difference in Epo electrophoretic mobility was found among NAF, milk and serum samples, and conditioned cell culture medium. Immunolocalization of intracellular Epo in ductal cells floating in BC NAFs was similar to those of cancer cell lines. No significant correlation between TNM classification and Epo in NAFs from BC patients was found. Significantly higher Epo concentration was found in NAF from BC patients compared to NoCancer. CONCLUSION: We demonstrate that breast epithelial cells are a source of Epo in breast microenvironment, suggesting the presence of a paracrine/autocrine Epo function in NAFs, triggering off intracellular signaling cascade with subsequent BC initiation.