Modifications of hydrophobicity, in vitro adherence and cellular aggregation of Streptococcus mutans by Helichrysum italicum extract

AIMS: The purpose of the present study was to examine whether sublethal concentrations of Helichrysum italicum extract could affect some of the cariogenic properties of Streptococcus mutans. METHODS AND RESULTS: We studied the antibacterial activity of H. italicum (ethanolic extract) against oral streptococci (Strep. mutans ATCC 35668, Strep. salivarius ATCC 13419 and Strep. sanguis ATCC 10556) and its influence on cell-surface hydrophobicity, in vitro sucrose-dependent adherence to glass surface and cellular aggregation of Strep. mutans. The results indicate that all streptococci were susceptible to ethanolic extract with minimum inhibitory concentration (MIC) values of 31.25-62.50 microg x ml(-1). Sub-MIC concentrations of H. italicum (7.81-31.25 microg x ml(-1)) reduced the hydrophobicity and the adherence (almost 90%) to glass surface of Strep. mutans. The aggregation in the presence of dextran T2000 was also affected. CONCLUSION: The inhibitory activity of H. italicum extract on Strep. mutans is worthy of further study. SIGNIFICANCE AND IMPACT OF THE STUDY: There is considerable interest in the use of natural compounds as alternative methods to control undesirable micro-organisms.     

Evaluation of antiherpesvirus-1 and genotoxic activities of Helichrysum italicum extract

The antiherpes virus-1 and genotoxic activities of diethyl ether extract from flowering tops of Helichrysum italicum (Compositae) were investigated. The extract showed significant antiviral activity at concentrations ranging from 400 to 100 microg/ml. This activity was not due to cytotoxic effect of the extract since Vero cells exhibited altered morphology or growth characteristics indicative of cytotoxic effects at higher concentration (800 microg/ml). Moreover H. italicum extract showed no DNA-damaging activity at concentrations up to 2000 microg/disk.     

Detection of enterotoxigenic Staphylococcus aureus isolates in raw milk cheese

AIM: To develop an easy, rapid and efficient DNA extraction procedure for Staphylococcus aureus detection with a low number of steps and removing completely the PCR inhibitors, applicable to raw milk cheese samples, and to compare phenotypical and genotypical method to detect Staph. aureus isolates and staphylococcal enterotoxins (SEs) production. METHODS AND RESULTS: A total of 33 bovine and caprine raw milk cheese samples were analysed by means of both classic microbiological and molecular techniques. All samples were positive for Staph. aureus contamination. The DNA extraction protocol optimized was found to achieve a detection limit of 100 CFU g(-1) for Staph. aureus. None of the samples tested with immunological assays contained SEs but in 14 of 33 samples a mixture of se positive (sea, sec, sed, seg, sel, sej) isolates were identified. CONCLUSIONS: Staphylococcus aureus is a food-borne pathogen mainly detected in finished dairy products. The rapid and efficient detection of Staph. aureus isolates from dairy products is essential for consumer safety. The direct detection of pathogens from food is possible with careful attention to sample preparation and nucleic acid amplification optimization. SIGNIFICANCE AND IMPACT OF THE STUDY: This study shows that raw milk cheese samples can be tested for Staph. aureus contamination with a rapid, simple and reproducible procedure.     

Cellular effects of monohydrochloride of L-arginine, N-lauroyl ethylester (LAE) on exposure to Salmonella typhimurium and Staphylococcus aureus

AIMS: Here we study the effect of monohydrochloride of L-arginine, N(alpha)-lauroyl ethylester (LAE), a cationic preservative derived from lauric acid and arginine, on the cell envelopes of Salmonella typhimurium and Staphylococcus aureus at sub-lethal concentration such as their respective minimal inhibitory concentrations, 32 and 8 microg ml(-1), respectively. METHODS AND RESULTS: Bacterial populations were studied by using transmission electron and fluorescence microscopy (TEM and FM), flow cytometry (FC) and ion-flux across the cellular membrane. Cell integrity was altered mainly in the outer membrane of S. typhimurium, but there was no significant change in the cytoplasm. However, in Staph. aureus, clear zones, abnormal septation and mesosome-like structures were observed in the cytoplasm. Bacterial populations were double-stained with propidium iodide (PI) and SYTO-13 for FC analysis. In S. typhimurium the proportion of damaged cells after 24 h was 97% and in Staph. aureus 56.3%. LAE induced transmembrane ion flux, the increase of potassium leakage after 30 min of contact was 7.7 and 3.34 microg ml(-1) for Staph. aureus and S. typhimurium, respectively. Membrane disruption was detected by measuring the proton flow across the membrane. CONCLUSIONS: Disturbance in membrane potential and structural changes was caused by LAE, although cells were not disrupted. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first time the cellular effects of LAE on bacterial cells were studied.     

Rapid detection of enterotoxigenic Staphylococcus aureus harbouring genes for four classical enterotoxins, SEA, SEB, SEC and SED, by loop-mediated isothermal amplification assay

AIM: The aim of this study was to develop a loop-mediated isothermal amplification (LAMP) assay targeting the genes for the four classical enterotoxins, SEA, SEB, SEC and SED, in Staphylococcus aureus. METHODS AND RESULTS: Specific primers were designed which target each specific sequence of the enterotoxin genes. With 30 strains of Staph. aureus, the results of the LAMP assay to each enterotoxin, SEA, SEB, SEC and SED, completely accorded with the results of polymerase chain reaction (PCR) assay. Enterotoxin production, determined by a reverse passive latex agglutination assay, strongly correlated with the presence of the corresponding genes. Amplification was not observed when 14 strains of nonenterotoxigenic Staph. aureus and 20 strains consisting of 19 bacterial species other than Staph. aureus were tested. In addition, the sensitivity of the LAMP assay was generally higher than that of conventional PCR assay and it rapidly detected enterotoxigenic Staph. aureus strains within 60 min. CONCLUSIONS: The LAMP assay developed in this study is rapid, specific and sensitive for the detection of enterotoxigenic Staph. aureus. SIGNIFICANCE AND IMPACT OF THE STUDY: The method is suitable for clinical diagnosis and food safety applications.     

Screening of some plants from Northern Argentina for their antimicrobial activity

AIMS: Screening of antimicrobial activity in 25 plant species from Northern Argentina. METHODS AND RESULTS: Inhibition of microbial growth was measured by a microplate assay with an oxidation-reduction indicator (Alamar Blue). Test organisms were: Escherichia coli, Salmonella typhimurium, Pseudomonas aeruginosa, Klebsiella pneumoniae, Staphylococcus aureus and Enterococcus faecium. Weak inhibitory activities (MIC=0.5 mg dry matter ml(-1)) were found in methanolic extracts of Rivina humilis, Crateva tapia, Funastrum claucum and Schinopsis balansae. Stronger bacteriostatic power was detected in Vassobia breviflora (MIC=0.25 mg ml(-1) against Staphylococcus aureus, and 0.5 mg ml(-1) against Enterococcus faecium). This activity was purified five-fold by extraction with dichloromethane, and it was found equally effective against susceptible or antibiotic-resistant strains of Staph. aureus. In addition, the purified extract was synergistic with gentamicin, and it was bactericidal at 24 h, with a concentration of 0.25 mg ml(-1). CONCLUSION: There is a significant antimicrobial activity in Vassobia breviflora. SIGNIFICANCE AND IMPACT OF THE STUDY: Further studies will be required to disclose the potential importance of these findings.     

Behaviour of Staphylococcus aureus strains, producers of enterotoxins C1 or C2, during the manufacture and storage of Burgos cheese

Burgos cheese was manufactured from pasteurized ewes' milk inoculated with Staphylococcus aureus strains FRI 137 and FRI 361, at levels of ca 10(3) and 10(5) cfu/ml and stored at 4 degrees, 10 degrees and 15 degrees C and at room temperature (10 degrees-15 degrees C). Populations of Staph. aureus and mesophilic aerobes, pH, and production of thermonuclease and enterotoxins C1 and C2 were investigated. Aerobic counts increased during cheese-making and storage. With both test strains, important growth was observed only during the storage period, the larger levels corresponding to the higher temperatures. Although Staph. aureus strains attained populations of over 10(8) cfu/g, no enterotoxin was detected. Strain FRI 361 reached 10(7) cfu/g without production of a detectable amount of thermonuclease. With strain FRI 137, the minimal population associated with enzyme activity was influenced by the inoculum size. Staphylococcus aureus counts are better indicators of staphylococcal growth in Burgos cheese than the thermonuclease test.     

PCR-based detection of enterotoxigenic Staphylococcus aureus in the early stages of raw milk cheese making

AIMS: To define PCR-based detectability of Staphylococcus aureus in raw milk and intermediate products of raw milk cheese making in the presence of a complex background microflora by targetting different specific genes harboured by a single strain. METHODS AND RESULTS: The strain Staph. aureus FRI 137 harbouring nuc, sec, seg, seh and sei genes was used in this study. Raw milk artificially contaminated by different concentrations of Staph. aureus FRI 137 was employed in dairy processing resembling traditional raw milk cheese making. Samples of milk and curds were PCR-analysed after DNA extraction by targetting all the above genes. The pathogen was detected when the initial contamination was 10(4) CFU ml(-1) by amplification of nuc and seh genes. 10(5) and 10(7) CFU ml(-1) were needed when seg or sei and sec genes were targetted, respectively. Enrichment cultures from raw milk and curd samples proved to increase the detection limit of 1 log on average. CONCLUSIONS: The direct detection of the pathogen in the raw material and dairy intermediates of production can provide rapid results and highlight the presence of loads of Staph. aureus potentially representing the risk of intoxication. However, every target gene to be used in the analysis has to be studied in advance in a system similar to the real case in order to determine the level of contamination potentially predictable. SIGNIFICANCE AND IMPACT OF THE STUDY: The detection in real dairy systems of significant loads of Staph. aureus by multiple targets PCR can be more accurate.     

Behaviour of Staphylococcus aureus strains, producers of enterotoxins C1 or C2, during the manufacture and storage of Burgos cheese

Burgos cheese was manufactured from pasteurized ewes milk inoculated with Staphylococcus aureus strains FRI 137 and FRI 361, at levels of ca 10³ and 10⁵ cfu/ml and stored at 4°, 10° and 15°C and at room temperature (10°-15°C). Populations of Staph. aureus and mesophilic aerobes, pH, and production of thermonuclease and enterotoxins C₁ and C₂ were investigated. Aerobic counts increased during cheese-making and storage. With both test strains, important growth was observed only during the storage period, the larger levels corresponding to the higher temperatures. Although Staph. aureus strains attained populations of over 10⁸ cfu/g, no enterotoxin was detected. Strain FRI 361 reached 10⁷ cfu/g without production of a detectable amount of thermonuclease. With strain FRI 137, the minimal population associated with enzyme activity was influenced by the inoculum size. Staphylococcus aureus counts are better indicators of staphylococcal growth in Burgos cheese than the thermonuclease test.     

Effect of combined physico-chemical preservatives on enterocin AS-48 activity against the enterotoxigenic Staphylococcus aureus CECT 976 strain

AIMS: Control of the enterotoxigenic Staphylococcus aureus CECT 976 strain by enterocin AS-48 in laboratory cultures, and behaviour of the AS-48 activity in the presence of food preservatives. METHODS AND RESULTS: Enterocin AS-48 shows inhibitory activity on the majority of the Staphylococcus species tested. This enterocin has a bactericidal and bacteriolytic mode of action on S. aureus CECT 976, a strain selected for this study by its enterotoxigenic character (SEA production). The inhibitory effect of AS-48 was pH and temperature dependent, and enterocin activity was higher at pH 5. The minimum bactericidal concentration (MBC) of AS-48, decreased from 15 microg ml(-1) at 37 degrees C to 10 microg ml(-1) at 15 degrees C. Sublethally injured cells showed an increased sensitivity with a MBC of 5 microg ml(-1). In this way, the highest effectiveness of Ent AS-48 against S. aureus CECT 976 was obtained at 4 degrees C in combination with high concentrations of NaCl (6 and 7%). Interestingly, enterotoxin SEA production by strain CECT 976 was markedly inhibited by subinhibitory concentrations of Ent AS-48. These low concentrations also provoked a delay of bacterial growth. CONCLUSION: The results presented indicated that Ent AS-48 has a potential for application as a protective agent against S. aureus in foods. SIGNIFICANCE AND IMPACT OF THE STUDY: In this study, we have established the conditions for an efficient inhibition of growth and enterotoxin production by S. aureus CECT 976 in culture media by a combination of environmental factors and Ent AS-48.     

Staphylococcus aureus, thermostable nuclease and staphylococcal enterotoxins in raw ewes' milk Manchego cheese

Growth and survival of two enterotoxigenic strains of Staphylococcus aureus were studied during manufacture and ripening of eight batches of raw ewes' milk Manchego cheese. Only 2-3 generations of Staph. aureus occurred in the vat and during pressing. The death rate of Staph. aureus (mean decrease in log cfu/g/week of ripening) from day 1 to day 60 was 0.421 in cheese made with 1% Streptococcus lactis starter and 0.404 in cheese made without starter. Thermostable nuclease was produced in the vat by growing Staph. aureus cells; it was inactivated by rennet during the first 24 h and synthesized again by surviving cells of Staph. aureus from day 1 to day 60. Staphylococcal enterotoxins A, B, C and D were not detected in any batches of cheese, even though Staph. aureus counts exceeded 10(7) cfu/g.     

Isolation and characterization of staphylococci from goats milk produced in Northern Ireland

Goats milk was examined for total viable bacteria and staphylococci (Baird-Parker medium, Schleifer & Kramer's (SK) medium, SK medium with a reduced sodium azide content (SKR) and SK and SKR with 5% added sheep blood). Staphylococcus aureus, Staph. epidermidis and Staph. simulans were the predominant species isolated overall. SK medium proved inhibitory with respect to the isolation of Staph. caprae and Staph. chromogenes. This was reduced by plating on the modified SK media. Representative strains of each species isolated were examined for production of enterotoxins A-E. Enterotoxin C alone was produced by 35% of the Staph. aureus strains tested. None of the other staphylococcal species examined produced any of the known enterotoxins.     

Detection of enterotoxins and TSST-1 secreted by Staphylococcus aureus isolated from ruminant mastitis. Comparison of ELISA and immunoblot

J.A. ORDEN, J. GOYACHE, J. HERNÁNDEZ, A. DOMÉNECH, G. SUÁREZ AND E. GÓMEZ-LUCÍA. 1992. The production of staphylococcal enterotoxins (SE) and toxic shock syndrome toxin-1 (TSST-1) was studied in 81 strains of Staphylococcus aureus isolated from cases of mastitis in cattle, goats and sheep. SE and TSST-1 were detected by two techniques: ELISA double antibody sandwich, and an immunoblot technique combined with a semiautomated electrophoresis system. More Staph. aureus strains isolated from sheep produced enterotoxins than those from goats and cattle. SEC was the predominant type in all isolates from these animal species. The highest proportion of strains producing TSST-1 were obtained from sheep, twice as many as those from goats or cows. The two techniques gave similar results. as all the strains positive by immunoblot were also positive by ELISA, and only three were positive by ELISA but negative by immunoblot.     

Antimicrobial activity of Mitracarpus scaber extract and isolated constituents

The antimicrobial activity of a methanol extract and isolated constituents of Mitracarpus scaber, a species used in folk medicine by West African native people, was evaluated against Staphylococcus aureus and Candida albicans strains. The mitracarpus methanol extract possesses both antibacterial and antimycotic activities (minimum inhibitory concentration-MIC 31.25 and 62.50 microg ml-, respectively). This extract was subsequently fractioned and monitored by bioassays leading to the isolation of seven compounds screened for antibacterial and antimycotic activities. Among these compounds, gallic acid and 3,4,5-trimethoxybenzoic acid inhibited the growth of Staph. aureus (MIC 3.90 and 0.97 microg ml-). 4-Methoxyacetophenone and 3,4,5-trimethoxyacetophenone effectively inhibited C. albicans (MIC 1.95 microg ml-). The other compounds (kaempferol-3-O-rutinoside, rutin and psoralen) which were also isolated showed low antibacterial and antimycotic activities (125-500 microg ml-).     

Detection of enterotoxins and TSST-1 secreted by Staphylococcus aureus isolated from ruminant mastitis. Comparison of ELISA and immunoblot.

The production of staphylococcal enterotoxins (SE) and toxic shock syndrome toxin-1 (TSST-1) was studied in 81 strains of Staphylococcus aureus isolated from cases of mastitis in cattle, goats and sheep. SE and TSST-1 were detected by two techniques: ELISA double antibody sandwich, and an immunoblot technique combined with a semiautomated electrophoresis system. More Staph. aureus strains isolated from sheep produced enterotoxins than those from goats and cattle. SEC was the predominant type in all isolates from these animal species. The highest proportion of strains producing TSST-1 were obtained from sheep, twice as many as those from goats or cows. The two techniques gave similar results, as all the strains positive by immunoblot were also positive by ELISA, and only three were positive by ELISA but negative by immunoblot.     

Staphylococcus aureus, thermostable nuclease and staphylococcal enterotoxins in raw ewes' milk Manchego cheese

Growth and survival of two enterotoxigenic strains of Staphylococcus aureus were studied during manufacture and ripening of eight batches of raw ewes' milk Manchego cheese. Only 2–3 generations of Staph. aureus occurred in the vat and during pressing. The death rate of Staph. aureus (mean decrease in log cfu/g/week of ripening) from day 1 to day 60 was 0.421 in cheese made with 1% Streptococcus lactis starter and 0.404 in cheese made without starter. Thermostable nuclease was produced in the vat by growing Staph. aureus cells; it was inactivated by rennet during the first 24 h and synthesized again by surviving cells of Staph. aureus from day 1 to day 60. Staphylococcal enterotoxins A, B, C and D were not detected in any batches of cheese, even though Staph. aureus counts exceeded 10⁷ cfu/g.     

Effect of subtherapeutic concentrations of tylosin on the inhibitory stringency of a mixed anaerobe continuous-flow culture of chicken microflora against Escherichia coli O157:H7

AIMS: The aim of this study was twofold: first to determine the effect of subtherapeutic concentrations of tylosin, a macrolide antibiotic used for growth promotion, on a mixed anaerobic continuous-flow fermentation culture of chicken gastrointestinal microorganisms (CCF) and secondly, to determine if these concentrations would allow persistence of Escherichia coli O157:H7 in CCF. METHODS AND RESULTS: CCF was treated with tylosin at 10.0, 20.0 and 40.0 microg ml(-1). Tylosin treatment resulted in a significant (P < 0.0001) decrease in total volatile fatty acids (VFAs) from a mean concentration of 101 +/- 10.8 micromol ml(-1) in control cultures to 32.0 +/- 6.3 and 40.2 +/- 9.6 micromol ml(-1) in 10 and 40 microg ml(-1) treated cultures, respectively. Untreated CCF challenged with E. coli O157:H7 cleared the challenge microorganism in 7 days at a rate of 0.96 log10 CFU ml(-1) day(-1). In contrast, E. coli O157:H7 persisted in all tylosin treated cultures. CONCLUSIONS: In the presence of tylosin, E. coli O157:H7 was able to persist in the CCF culture. The significant decrease in the production of VFAs may have been a contributing factor. SIGNIFICANCE AND IMPACT OF THE STUDY: The use of low-level, growth-promoting antimicrobials may compromise the ability of normal microflora that serve as a natural host defence against infection.     

Different antibacterial actions of isoflavones isolated from Erythrina poeppigiana against methicillin-resistant Staphylococcus aureus

AIMS: To screen six isoflavones isolated from Erythrina poeppigiana (Leguminosae) for their antibacterial activity against methicillin-resistant Staphylococcus aureus (MRSA). METHODS AND RESULTS: Stem bark of E. poeppigiana was macerated with acetone and the methylene chloride-soluble fraction of the residue was applied to repeated silica gel column chromatography and eluted. Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) were determined by a broth dilution method. Inactive compounds that failed inhibiting bacterial growth at 25 microg ml(-1) were further investigated for their combination effects with methicillin and oxacillin. Of the isolated isoflavones, 5,7,4'-trihydroxy-8,3'-di(gamma,gamma-dimethylallyl)isoflavone (isolupalbigenin) exhibited the highest anti-MRSA activity (MICs: 1.56-3.13 microg ml(-1); MBCs: 6.25-12.5 microg ml(-1)), followed by 5,7,4'-trihydroxy-6-gamma,gamma-dimethylallylisoflavone (erythrinin B). Inactive compounds were combined with methicillin or oxacillin, 5,4'-dihydroxy-(3',4'-dihydro-3'-hydroxy)-2',2'-dimethylpyrano[5',6':6,7]isoflavone (M-Wi-2) intensifying the susceptibility of MRSA strains to these antibiotics. In all but one strain, the MIC values of methicillin were reduced from > or =100 to 6.25-12.5 microg ml(-1) in the presence of M-Wi-2 (25 microg ml(-1)). CONCLUSIONS: Isoflavones from E. poeppigiana showed two different antibacterial activities against MRSA: direct growth inhibition and intensification of methicillin sensitivity. SIGNIFICANCE AND IMPACT OF THE STUDY: Isolupalbigenin and M-Wi-2 could lead to the development of compounds for new approaches against MRSA infection.     

Accumulation of ppGpp and ppGp in Staphylococcus aureus 8325-4 following nutrient starvation

AIM: To investigate the accumulation of highly phosphorylated guanosine nucleotides in Staphylococcus aureus 8325-4 following nutrient deprivation. METHODS AND RESULTS: Nutrient shiftdown of Staph. aureus, HPLC of nucleotides and Western blotting of cell-free extracts. ppGpp rapidly accumulated when cells were deprived of isoleucine following addition of mupirocin, or after carbon deprivation. In contrast, total amino acid starvation led to delayed production of ppGp, which suggests that Staph. aureus exhibits a unique response to total amino acid deprivation compared with other eubacteria. Intracellular ppGp was observed at high levels under all starvation conditions, which suggests that this nucleotide is linked to nutrient limitation and may therefore be involved in regulating the stringent response in Staph. aureus. pppGpp was not observed under any nutrient-limiting condition. Western blot analysis of whole-cell extracts from Staph. aureus 8325-4, showed that antibodies to RelA and SpoT cross-reacted under conditions that detected these proteins in Escherichia coli. CONCLUSIONS: Staph. aureus produces ppGpp and ppGp following nutrient limitation. Immunological analysis indicates that Staph. aureus contains RelA and SpoT proteins, similar to those produced by E. coli. SIGNIFICANCE AND IMPACT OF THE STUDY: This study provides a new example of the diversity of metabolic regulations in bacteria.     

The synergistic effect of nisin and lactoferrin on the inhibition of Listeria monocytogenes and Escherichia coli O157:H7

AIMS: The goal of this study was to determine whether nisin and lactoferrin would act synergistically to inhibit the growth of Listeria monocytogenes and Escherichia coli O157:H7. METHODS AND RESULTS: Lactoferrin and nisin separately or in combination were suspended in peptone yeast glucose broth and following inoculation with L. monocytogenes or E. coli O157:H7 growth inhibition of each pathogen was determined. At 1000 microg ml(-1) lactoferrin L. monocytogenes was effectively inhibited. However, E. coli O157:H7 initially was inhibited and then grew to cell density similar to the control. A combination of 500 microg ml(-1) of lactoferrin and 250 IU ml(-1) of nisin effectively inhibited the growth of E. coli O157:H7, whereas, 250 microg ml(-1) of lactoferrin and 10 IU ml(-1) of nisin were inhibitory to L. monocytogenes. CONCLUSIONS: The results suggest that lactoferrin and nisin act synergistically to inhibit the growth of L. monocytogenes and E. coli O157:H7. SIGNIFICANCE AND IMPACT OF THE STUDY: Natural preservatives that are active against gram-positive and gram-negative pathogens are desirable to the food industry and consumers. This study demonstrates that lactoferrin and nisin work synergistically reducing the levels required independently inhibiting growth of two major foodborne pathogens. Previous reported results indicated a low level of antimicrobial activity; however, this work was not performed in low divalent cation concentration media. It has been suggested that nondivalent cation-limiting medium such as trypticase soy broth (TSB), can reduce or completely eliminate the inhibitory activity. Further knowledge of these interactions can increase the understanding of the antimicrobial activity of lactoferrin. This should make the use of these compounds by industry more attractive.