Bruna Carrer Gomes

Prevalence and characterization of Enterococcus spp. isolated from Brazilian foods

Enterococci can be used in the food industry as starter or probiotic cultures. However, enterococci are also implicated in severe multi-resistant nosocomial infections. In this study, the prevalence of enterococci in selected Brazilian foodstuffs (raw and pasteurized milk, meat products, cheeses and vegetables) was evaluated. Phenotypic and PCR protocols were used for species identification. Tests for production of gelatinase, haemolysin, bacteriocin and bile salt hydrolysis were done with all enterococci isolates, whereas molecular determination of virulence markers (genes esp, gel, ace, as, efaA, hyl and cylA) and antibiotic resistance was checked only for Enterococcus faecium and Enterococcus faecalis isolates. The antibiotic-resistant isolates were assayed for biofilm formation and adhesion to mammalian cells. From the 120 food samples analyzed, 52.5% were positive for enterococci, meat and cheese being the most contaminated. E. faecium was the predominant species, followed by E. faecalis, E. casseliflavus and Enterococcus gallinarum. Phenotypic tests indicated that 67.7% of isolates hydrolyzed bile salts, 15.2% produced bacteriocin, 12.0% were beta-hemolytic and 18.2% produced gelatinase. Antibiotic resistance (gentamicin, tetracycline and erythromycin) and genes encoding for virulence traits were more frequent in E. faecalis than in E. faecium. Three E. faecium isolates were resistant to vancomycin. Among antibiotic-resistant isolates, 72.4% of E. faecalis were able to form biofilm and 13.8% to adhere to Caco-2 cells. Antibiotic-resistant E. faecalis and E. faecium isolates were grouped by RAPD-PCR and a scattered distribution was noted, indicating that resistance was not related to a particular clone. The spread of virulence/resistance traits in isolates of the two species and different RAPD-types suggest the pathogenic potential of both species. By contrast, the recovery of bacteriocinogenic E. faecium isolates with no virulence traits suggests their potential for biotechnological applications. In conclusion, our results showed that enterococci from Brazilian foods present important dualist aspects for food safety.

Influence of peroxyacetic acid and nisin and coculture with Enterococcus faecium on Listeria monocytogenes biofilm formation.

Biofilm formation is a matter of concern in food industries because biofilms facilitate the survival of pathogenic bacteria such as Listeria monocytogenes, which may contaminate food-processing equipment and products. In this study, nisin and two Enterococcus faecium strains were evaluated for their effect on biofilm formation by L. monocytogenes cultured in brain heart infusion broth and on stainless steel coupons. Elimination of preformed L. monocytogenes biofilms by peroxyacetic acid also was tested. Adhesion control experiments were performed with pure cultures of L. monocytogenes after swab collection of adhered cells, which were then enumerated on PALCAM agar plates and visualized by scanning electron microscopy. Formation of a biofilm was recorded when the number of adhered cells was at least 10(3) CFU/cm2. When L. monocytogenes was cocultured with E. faecium bac-, the number of adhered L. monocytogenes cells was 2.5 log lower (P = 0.002) when initially compared with the control culture, but after 6 h of incubation a biofilm was again detected. However, in coculture on stainless steel coupons, E. faecium bac+ inhibited L. monocytogenes adherence and did not allow biofilm formation for up to 48 h (P < 0.001). In the presence of nisin or after treatment with peroxyacetic acid, bacterial growth was reduced (P < 0.001) up to 4.6 and 5.6 log CFU/cm2, respectively, when compared with L. monocytogenes cultures on untreated coupons. However, after these treatments, cells were still present, and after 24 h of incubation, a renewed biofilm was detected in L. monocytogenes cultures treated with nisin. Although all tested conditions reduced L. monocytogenes growth to some extent, only coculture with E. faecium bac+ efficiently reduced biofilm formation, suggesting a potential control strategy for this pathogen.

The significance of Helicobacter pylori in water, food and environmental samples

Abstract Infection by Helicobacter pylori is recognized as the major cause of gastric disease and its prevalence is elevated worldwide. It is hypothesised that the transmission of H. pylori involves multiple pathways: iatrogenic, oral–oral and faecal–oral. Food and water are suspects of serving as vehicles in the faecal–oral route of H. pylori infection. However, the difficult cultivation of H. pylori from samples with high loads of accompanying microflora and its conversion to viable but nonculturable state (VNC) impair the elucidation of the real role of waterborne and foodborne infections by this pathogen. In this sense, it is crucial the development of methods for isolation of H. pylori from environmental samples. In this work, an overview of the present knowledge on epidemiology and transmission of H. pylori is presented, attempting to the possible role of water and foods in the spread of the infection.

Microbiological Food Safety Issues in Brazil: Bacterial Pathogens

The globalization of food supply impacts patterns of foodborne disease outbreaks worldwide, and consumers are having increased concern about microbiological food safety. In this sense, the assessment of epidemiological data of foodborne diseases in different countries has not only local impact, but it can also be of general interest, especially in the case of major global producers and exporters of several agricultural food products, such as Brazil. In this review, the most common agents of foodborne illnesses registered in Brazil will be presented, compiled mainly from official databases made available to the public. In addition, some representative examples of studies on foodborne bacterial pathogens commonly found in Brazilian foods are provided.

Vaginal lactobacilli as potential probiotics against Candida spp

Urogenital infections affect millions of people every year worldwide. The treatment of these diseases usually requires the use of antimicrobial agents, and more recently, the use of probiotic lactic acid bacteria (LAB) cultures for the management of vaginal infections has been extensively studied. In this work, 11 vaginal lactobacilli isolates, previously obtained from healthy patients, were studied to screen microorganisms with probiotic properties against Candida spp. The LAB were tested for their ability of auto-aggregation, co-aggregation with C. albicans, C. glabrata, C. krusei, and C. tropicalis, adhesion to Caco-2 epithelial cells and production of lactic acid and hydrogen peroxide (H2O2). All lactobacilli isolates tested were able to auto-aggregate (ranging from 25.3% to 75.4% assessed at 4 hours of incubation) and to co-aggregate with the four Candida species into different degrees; among them L. crispatus showed the highest scores of co-aggregation. The highest amount of lactic acid was produced by L. salivarius (13.9 g/l), followed by L. johnsonii (6.5 g/l), L. acidophilus (5.5 g/l), and L. jensenii (5.4 g/l). All isolates produced H2O2, but the highest levels (3 – 10 mg/l) were observed for L. acidophilus, L. crispatus, L. gasseri, L. johnsonii, and L. vaginalis. Only L. agilis, L. jensenii, L. johnsonii and L. ruminus were able to adhere to epithelial Caco-2 cells. Among the isolates evaluated, L agilis, L. jensenii, L. johnsonii, and L. ruminus exhibited simultaneously several desirable properties as potential probiotic strains justifying future studies to evaluate their technological properties in different pharmaceutical preparations for human use.

Fate of Helicobacter pylori artificially inoculated in lettuce and carrot samples

Helicobacter pylori is known, worldwide, as the causative agent of gastric diseases. However, its transmission route has not been completely understood. To evaluate the survival of H. pylori (a clinical and a reference strain) artificially inoculated on lettuce and carrot samples, portions of 25 g were inoculated with approximately 106 CFU/g of H. pylori and packed under normal and/or modified atmosphere (3% oxygen, 10% carbon dioxide, and 87% nitrogen). The inoculated food samples were stored at 8oC, with daily enumeration of H. pylori populations on Columbia blood agar (CBA) and/ or Helicobacter pylori Special Peptone Agar (HPSPA). When CBA with antibiotics was used, the clinical isolate H. pylori HP1 was detected for up to 72 h in sanitized lettuce and carrot. In sterilized carrot samples, H. pylori HP1 remained viable for up to 96 h. The CBA without antibiotics allowed the recovery of H. pylori ATCC 43629, from carrot samples stored in both atmospheres tested, for up to 120 hours. Our results reinforce that foodborne transmission of H. pylori cannot be disregarded yet.

In vitro evaluation of the probiotic potential of bacteriocin producer Lactobacillus sakei 1.

Lactobacillus sakei 1 is a food isolate that produces a heat-stable antimicrobial peptide (sakacin 1, a class IIa bacteriocin) inhibitory to the opportunistic pathogen Listeria monocytogenes. Bacterial isolates with antimicrobial activity may be useful for food biopreservation and also for developing probiotics. To evaluate the probiotic potential of L. sakei 1, it was tested for (i) in vitro gastric resistance (with synthetic gastric juice adjusted to pH 2.0, 2.5, or 3.0); (ii) survival and bacteriocin production in the presence of bile salts and commercial prebiotics (inulin and oligofructose); (iii) adhesion to Caco-2 cells; and (iv) effect on the adhesion of L. monocytogenes to Caco-2 cells and invasion of these cells by the organism. The results showed that L. sakei 1 survival in gastric environment varied according to pH, with the maximum survival achieved at pH 3.0, despite a 4-log reduction of the population after 3 h. Regarding the bile salt tolerance and influence of prebiotics, it was observed that L. sakei 1 survival rates were similar (P > 0.05) for all de Man Rogosa Sharpe (MRS) broth formulations when tests were done after 4 h of incubation. However, after incubation for 24 h, the survival of L. sakei 1 in MRS broth was reduced by 1.8 log (P < 0.001), when glucose was replaced by either inulin or oligofructose (without Oxgall). L. sakei 1 was unable to deconjugate bile salts, and there was a significant decrease (1.4 log) of the L. sakei 1 population in regular MRS broth plus Oxgall (P < 0.05). In spite of this, tolerance levels of L. sakei 1 to bile salts were similar in regular MRS broth and in MRS broth with oligofructose. Lower bacteriocin production was observed in MRS broth when inulin (3,200 AU/ml) or oligofructose (2,400 AU/ml) was used instead of glucose (6,400 AU/ml). L. sakei 1 adhered to Caco-2 cells, and its cell-free pH-neutralized supernatant containing sakacin 1 led to a significant reduction of in vitro listerial invasion of human intestinal Caco-2 cells.

Elongated cells of Listeria monocytogenes in biofilms in the presence of sucrose and bacteriocin-producing Leuconostoc mesenteroides A11

Listeria monocytogenes is a foodborne pathogen which may survive in biofilms and persist in food processing plants. In this study, the ability of Leuconostoc mesenteroides (bac+ and bac-) to inhibit biofilm formation by L. monocytogenes ATCC 19115 was studied with stainless steel coupons immersed in BHI broth and BHI broth plus sucrose in combination with the Lactic Acid Bacteria (LAB). Adhered cells were collected with swabs and enumerated on selective agars (Oxford for listeria and MRS for leuconostoc). Leuconostoc mesenteroides bac+ in co-culture with L. monocytogenes was effective to inhibit biofilm formation by listeria for up to 3 hours of incubation, but at 24 hours, biofilm was present in all conditions tested, as confirmed by observations of stainless steel coupons under Scanning Electron Microscopy (SEM). It was also observed that in the presence of L. mesenteroides bac+ in BHI plus sucrose, a high number of elongated cells of L. monocytogenes was present, which may indicate an adaptation response of the pathogen to stress conditions with important implications for food safety.

Correlation between API 20 STREP and multiplex PCR for identification of Enterococcus spp. isolated from Brazilian foods

We evaluated the suitability of API 20 STREP and multiplex PCR to speciate 52 Enterococcus spp. obtained from Brazilian foods. A high percentage of isolates (78.9%) presented discrepant results between evaluated tests. Similar results were obtained for six E. faecalis and five E. faecium. The PCR multiplex was more effective than API 20 STREP for complete identification of the isolates.