Rubén Pérez Pulido

Biocide tolerance in bacteria.

Biocides have been employed for centuries, so today a wide range of compounds showing different levels of antimicrobial activity have become available. At the present time, understanding the mechanisms of action of biocides has also become an important issue with the emergence of bacterial tolerance to biocides and the suggestion that biocide and antibiotic resistance in bacteria might be linked. While most of the mechanisms providing antibiotic resistance are agent specific, providing resistance to a single antimicrobial or class of antimicrobial, there are currently numerous examples of efflux systems that accommodate and, thus, provide tolerance to a broad range of structurally unrelated antimicrobials, both antibiotics and biocides. If biocide tolerance becomes increasingly common and it is linked to antibiotic resistance, not only resistant (even multi-resistant) bacteria could be passed along the food chain, but also there are resistance determinants that can spread and lead to the emergence of new resistant microorganisms, which can only be detected and monitored when the building blocks of resistance traits are understood on the molecular level. This review summarizes the main advances reached in understanding the mechanism of action of biocides, the mechanisms of bacterial resistance to both biocides and antibiotics, and the incidence of biocide tolerance in bacteria of concern to human health and the food industry.

Microbiological Study of Lactic Acid Fermentation of Caper Berries by Molecular and Culture-Dependent Methods

ABSTRACT Fermentation of capers (the fruits of Capparis sp.) was studied by molecular and culture-independent methods. A lactic acid fermentation occurred following immersion of caper berries in water, resulting in fast acidification and development of the organoleptic properties typical of this fermented food. A collection of 133 isolates obtained at different times of fermentation was reduced to 75 after randomly amplified polymorphic DNA (RAPD)-PCR analysis. Isolates were identified by PCR or 16S rRNA gene sequencing as Lactobacillus plantarum (37 isolates), Lactobacillus paraplantarum (1 isolate), Lactobacillus pentosus (5 isolates), Lactobacillus brevis (9 isolates), Lactobacillus fermentum (6 isolates), Pediococcus pentosaceus (14 isolates), Pediococcus acidilactici (1 isolate), and Enterococcus faecium (2 isolates). Cluster analysis of RAPD-PCR patterns revealed a high degree of diversity among lactobacilli (with four major groups and five subgroups), while pediococci clustered in two closely related groups. A culture-independent analysis of fermentation samples by temporal temperature gradient electrophoresis (TTGE) also indicated that L. plantarum is the predominant species in this fermentation, in agreement with culture-dependent results. The distribution of L. brevis and L. fermentum in samples was also determined by TTGE, but identification of Pediococcus at the species level was not possible. TTGE also allowed a more precise estimation of the distribution of E. faecium, and the detection of Enterococcus casseliflavus (which was not revealed by the culture-dependent analysis). Results from this study indicate that complementary data from molecular and culture-dependent analysis provide a more accurate determination of the microbial community dynamics during caper fermentation.

Inhibition of Listeria monocytogenes by enterocin EJ97 produced by Enterococcus faecalis EJ97

Enterocin EJ97 from Enterococcus faecalis EJ97 showed a concentration-dependent antimicrobial activity against Listeria monocytogenes CECT 4032. Activity of enterocin EJ97 against L. monocytogenes CECT 4032 increased slightly at 4 degrees C, and cold-adapted cells did not show any increased resistance. Sensitivity of L. monocytogenes CECT 4032 to enterocin EJ97 was not modified by the addition of sodium benzoate, sodium acetate, NaCl or sodium tripolyphosphate. Anti-listeria activity was enhanced by potassium nitrate, and especially by sodium nitrite at concentrations of 50 microg/ml or above. E. faecalis EJ97 produced bacteriocin activity during cocultivation with L. monocytogenes CECT 4032 at 37 degrees C and also at 15 degrees C, but not at 4 degrees C. Growth of L. monocytogenes CECT 4032 was inhibited by bacteriocin produced during cocultivation at 37 and 15 degrees C, and the degree of inhibition was influenced by the incubation temperature and the initial concentrations of enterococci and listeria. E. faecalis EJ97 also produced bacteriocin during cocultivation in half-skimmed milk, although its capacity to control L. monocytogenes was limited to populations of 10(3) CFU/ml or lower.

The Cyclic Antibacterial Peptide Enterocin AS-48: Isolation, Mode of Action, and Possible Food Applications

Enterocin AS-48 is a circular bacteriocin produced by Enterococcus. It contains a 70 amino acid-residue chain circularized by a head-to-tail peptide bond. The conformation of enterocin AS-48 is arranged into five alpha-helices with a compact globular structure. Enterocin AS-48 has a wide inhibitory spectrum on Gram-positive bacteria. Sensitivity of Gram-negative bacteria increases in combination with outer-membrane permeabilizing treatments. Eukaryotic cells are bacteriocin-resistant. This cationic peptide inserts into bacterial membranes and causes membrane permeabilization, leading ultimately to cell death. Microarray analysis revealed sets of up-regulated and down-regulated genes in Bacillus cereus cells treated with sublethal bacteriocin concentration. Enterocin AS-48 can be purified in two steps or prepared as lyophilized powder from cultures in whey-based substrates. The potential applications of enterocin AS-48 as a food biopreservative have been corroborated against foodborne pathogens and/or toxigenic bacteria (Listeria monocytogenes, Bacillus cereus, Staphylococcus aureus, Escherichia coli, Salmonella enterica) and spoilage bacteria (Alicyclobacillus acidoterrestris, Bacillus spp., Paenibacillus spp., Geobacillus stearothermophilus, Brochothrix thermosphacta, Staphylococcus carnosus, Lactobacillus sakei and other spoilage lactic acid bacteria). The efficacy of enterocin AS-48 in food systems increases greatly in combination with chemical preservatives, essential oils, phenolic compounds, and physico-chemical treatments such as sublethal heat, high-intensity pulsed-electric fields or high hydrostatic pressure.

Antimicrobial Resistance in Enterococci

Enterococci show intrinsic low resistance to a large number of antibiotics (β-lactams, lincosamines, aminoglycosides and trimetoprim-sulfametoxazol). In addition, Enterococci can acquire new resistance to antimicrobial agents. This can happen by mutation or acquisition of extrachromosomal DNA, as plasmids or transposons. Resistance to erythromycin, aminoglycosides and tetracycline are common. Resistance to glycopeptide antibiotics and to newer antimicrobial substances may turn opportunistic enterococcal infections into high-risk infections, specially for immunocompromised patients. Enterococci isolated at different steps in the food chain also show a remarkable incidence of antimicrobial resistance. Heavy metal resistance and biocide tolerance could be factors in the co-selection of antibiotic resistance in the absence of antibiotic selective pressure, such as at certain steps of the food chain.

Effect of enterocin AS-48 in combination with biocides on planktonic and sessile Listeria monocytogenes

Enterocin AS-48 was tested on a cocktail of Listeria monocytogenes strains in planktonic and sessile states, singly or in combination with biocides benzalkonium chloride, cetrimide, hexadecylpyridinium chloride, didecyldimethylammonium bromide, triclosan, poly-(hexamethylen guanidinium) hydrochloride, chlorhexidine, hexachlorophene, and the commercial sanitizers P3 oxonia and P3 topax 66. Combinations of sub-inhibitory bacteriocin concentrations and biocide concentrations 4 to 10-fold lower than their minimum inhibitory concentrations (MIC) completely inhibited growth of the planktonic listeriae. Inactivation of Listeria in biofilms formed on polystyrene microtiter plates required concentrations of enterocin AS-48 greater than 50 μg/ml, and biocide concentrations ten to 100-fold higher. In combination with enterocin AS-48 (25 or 50 μg/ml), microbial inactivation increased remarkably for all biocides except P3 oxonia and P3 topax 66 solutions. Polystyrene microtiter plates conditioned with enterocin solutions (0.5-25 μg/ml) decreased the adherence and biofilm formation of the L. monocytogenes cell cocktail, avoiding biofilm formation for at least 24 h at a bacteriocin concentration of 25 μg/ml.

Annotated Genome Sequence of Lactobacillus pentosusMP-10, Which Has Probiotic Potential, from Naturally Fermented Aloreña Green Table Olives

Lactobacillus pentosus MP-10 was isolated from brines of naturally fermented Aloreña green table olives. MP-10 has potential probiotic traits, including inhibition of human pathogenic bacteria, survival at low pH (1.5), and bile salt tolerance (3%). Here, we report for the first time the annotated genome sequence of L. pentosus.

Phenotypic and Molecular Antibiotic Resistance Profile of Enterococcus faecalis and Enterococcus faecium Isolated from Different Traditional Fermented Foods

A collection of 55 enterococci (41 Enterococcus faecium and 14 E. faecalis strains) isolated from various traditional fermented foodstuffs of both animal and vegetable origins, and water was evaluated for resistance against 15 antibiotics. Lower incidence of resistance was observed with gentamicin, ampicillin, penicillin and teicoplanin. However, a high incidence of antibiotic resistance was detected for rifampicin (12 out of 14 of isolates), ciprofloxacin (9/14), and quinupristin/dalfopristin (8/14) in E. faecalis strains. Enterococcus faecium isolates were resistant to rifampicin (25/41), ciprofloxacin (23/41), erythromycin (18/41), levofloxacin (16/41), and nitrofurantoin (15/41). One Enterococcus faecalis and two E. faecium strains were resistant to vancomycin (MIC>16 μg/mL). Among 55 isolates, 27 (19 E. faecium and eight E. faecalis) were resistant to at least three antibiotics. High level of multidrug resistance to clinically important antibiotics was detected in E. faecalis strains (57% of E. faecalis versus 46% of E. faecium), which showed resistance to six to seven antibiotics, especially those isolated from foods of animal origin. So, it is necessary to re-evaluate the use of therapeutic antibiotics in stock farms at both regional and international levels due to the high number of multiple resistant (MR) bacteria. Fifty-six MR E. faecalis and E. faecium strains selected from this and previous studies (Valenzuela et al., 2008, 2010) were screened by polymerase chain reaction for antibiotic resistance genes, revealing the presence of tet(L), tet(M), ermB, cat, efrA, efrB, mphA, or msrA/B genes. The ABC Multidrug Efflux Pump EfrAB was detected in 96% of E. faecalis strains and also in 13% of E. faecium strains; this is the first report describing EfrAB in this enterococcal species. The efflux pump-associated msrA/B gene was detected in 66.66% of E. faecium strains, but not in E. faecalis strains.

The human gastrointestinal tract and oral microbiota in inflammatory bowel disease: a state of the science review.

Inflammatory bowel disease (IBD) includes a spectrum of diseases from ulcerative colitis (UC) to Crohns disease (CD). Many studies have addressed the changes in the microbiota of individuals affected by UC and CD. A decrease in biodiversity and depletion of the phyla Bacteroidetes and Firmicutes has been reported, among others. Changes in microbial composition also result in changes in the metabolites generated in the gut from microbial activity that may involve the amount of butyrate and other metabolites such as H2S being produced. Other factors such as diet, age, or medication need to be taken into consideration when studying dysbiosis associated with IBD. Diverse bacterial species have been associated specifically or non‐specifically to IBD, but none of them have been demonstrated to be its ethiological agent. Recent studies also suggest that micro‐eukaryotic populations may also be altered in IBD patients. Last, but not least, viruses, and specially bacteriophages, can play a role in controlling microbial populations in the gastrointestinal tract. This may affect both bacterial diversity and metabolism, but possible implications for IBD still remain to be solved. Dysbiosis in the oral microbiome associated with IBD remains an emerging field for future research.

Genetic determinants of antimicrobial resistance in Gram positive bacteria from organic foods.

Bacterial biocide resistance is becoming a matter of concern. In the present study, a collection of biocide-resistant, Gram-positive bacteria from organic foods (including 11 isolates from genus Bacillus, 25 from Enterococcus and 10 from Staphylococcus) were analyzed for genes associated to biocide resistance efflux pumps and antibiotic resistance. The only qac-genes detected were qacA/B (one Bacillus cereus isolate) and smr (one B. cereus and two Staphylococcus saprophyticus isolates). Efflux pump genes efrA and efrB genes were detected in Staphylococcus (60% of isolates), Bacillus (54.54%) and Enterococcus (24%); sugE was detected in Enterococcus (20%) and in one Bacillus licheniformis; mepA was detected in Staphylococcus (60%) and in one Enterococcus isolate (which also carried mdeA), and norE gene was detected only in one Enterococcus faecium and one S. saprophyticus isolate. An amplicon for acrB efflux pump was detected in all but one isolate. When minimal inhibitory concentrations (MICs) were determined, it was found that the addition of reserpine reduced the MICs by eight fold for most of the biocides and isolates, corroborating the role of efflux pumps in biocide resistance. Erythromycin resistance gene ermB was detected in 90% of Bacillus isolates, and in one Staphylococcus, while ereA was detected only in one Bacillus and one Staphyloccus, and ereB only in one Staphylococcus. The ATP-dependent msrA gene (which confers resistance to macrolides, lincosamides and type B streptogramins) was detected in 60% of Bacillus isolates and in all staphylococci, which in addition carried msrB. The lincosamide and streptogramin A resistance gene lsa was detected in Staphylococcus (40%), Bacillus (27.27%) and Enterococcus (8%) isolates. The aminoglycoside resistance determinant aph (3_)-IIIa was detected in Staphylococcus (40%) and Bacillus (one isolate), aph(2_)-1d in Bacillus (27.27%) and Enterococcus (8%), aph(2_)-Ib in Bacillus (one isolate), and the bifunctional aac(6_)1e-aph(2_)-Ia in Staphylococcus (20%), Enterococcus (8%) and Bacillus (one isolate). Chloramphenicol resistance cat gene was detected in Enterococcus (8%) and Staphylococcus (20%), and blaZ only in Staphylococcus (20%). All other antibiotic or biocide resistance genes investigated were not detected in any isolate. Isolates carrying multiple biocide and antibiotic determinants were frequent among Bacillus (36.36%) and Staphylococcus (50%), but not Enterococcus. These results suggest that biocide and antibiotic determinants may be co-selected.