Leyre Lavilla Lerma

New insights in antibiotic resistance of Lactobacillus species from fermented foods

Abstract Bacteria belonging to the genus Lactobacillus are used as starter cultures or that develop naturally as fermenting microbiota in the production of various foods. On the detrimental side, lactobacilli may act as reservoir of antibiotic resistance genes, which can spread to commensal bacteria in humans or animals, or to food-associated pathogens. In the last decade, advances in molecular biology and in genome sequencing have provided more information on antibiotic resistances in foodborne bacteria. The aim of this review was to consider and provide an up-to-date status on phenotypic and genotypic antibiotic resistance profiles in Lactobacillus species from fermented foods and also to highlight new information on the distribution of glycopeptide and chloramphenicol resistance genes in Lactobacillus genomes. In silico screening of vanZ (glycopeptide resistance) and cat (chloramphenicol resistance)-like sequences in Lactobacillus species isolated from fermented foods revealed for the first time the occurrence of vanZ and cat genes in Lactobacillus species being highly conserved genes in the chromosome of each species, presumably non-transferable. Further studies involving genome sequences of Lactobacillus isolated from fermented foods, especially those relying on spontaneous fermentation, is crucial to increase knowledge on the potential presence and spread of antibiotic resistance genes via the food route.

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 controversial nature of the Weissella genus: technological and functional aspects versus whole genome analysis-based pathogenic potential for their application in food and health.

Despite the use of several Weissella (W.) strains for biotechnological and probiotic purposes, certain species of this genus were found to act as opportunistic pathogens, while strains of W. ceti were recognized to be pathogenic for farmed rainbow trout. Herein, we investigated the pathogenic potential of weissellas based on in silico analyses of the 13 whole genome sequences available to date in the NCBI database. Our screening allowed us to find several virulence determinants such as collagen adhesins, aggregation substances, mucus-binding proteins, and hemolysins in some species. Moreover, we detected several antibiotic resistance-encoding genes, whose presence could increase the potential pathogenicity of some strains, but should not be regarded as an excluding trait for beneficial weissellas, as long as these genes are not present on mobile genetic elements. Thus, selection of weissellas intended to be used as starters or for biotechnological or probiotic purposes should be investigated regarding their safety aspects on a strain to strain basis, preferably also by genome sequencing, since nucleotide sequence heterogeneity in virulence and antibiotic resistance genes makes PCR-based screening unreliable for safety assessments. In this sense, the application of W. confusa and W. cibaria strains as starter cultures or as probiotics should be approached with caution, by carefully selecting strains that lack pathogenic potential.

Antibiotic Multiresistance Analysis of Mesophilic and Psychrotrophic Pseudomonas spp. Isolated from Goat and Lamb Slaughterhouse Surfaces throughout the Meat Production Process

ABSTRACT The aim of this study was to investigate the phenotypic and genotypic antibiotic resistance profiles of pseudomonads isolated from surfaces of a goat and lamb slaughterhouse, which were representative of areas that are possible sources of meat contamination. Mesophilic (85 isolates) and psychrotrophic (37 isolates) pseudomonads identified at the species level generally were resistant to sulfamethoxazole, erythromycin, amoxicillin, ampicillin, chloramphenicol, trimethoprim, rifampin, and ceftazidime (especially mesophiles), as well as colistin and tetracycline (especially psychrotrophes). However, they generally were sensitive to ciprofloxacin, gentamicin, imipenem, and kanamycin regardless of species identity. Worryingly, in the present study, we found multidrug resistance (MDR) to up to 13 antibiotics, which was related to intrinsic and acquired resistance mechanisms. Furthermore, a link between various antimicrobial resistance genes was shown for beta-lactams and tetracycline, trimethoprim, and sulfonamides. The distribution and resistome-based analysis of MDR pseudomonads in different slaughterhouse zones indicated that the main sources of the identical or related pseudomonad strains were the animals (feet and wool) and the slaughterhouse environment, being disseminated from the beginning, or entrance environment, to the environment of the finished meat products. Those facts must be taken into consideration to avoid cross-contamination with the subsequent flow of mobile resistance determinants throughout all slaughterhouse zones and then to humans and the environment by the application of adequate practices of hygiene and disinfection measures, including those for animal wool and feet and also the entrance environment.

Fermented Aloreña Table Olives as a Source of Potential Probiotic Lactobacillus pentosus Strains

A collection of 31 Lactobacillus pentosus strains isolated from naturally fermented Aloreña green table olives were screened in depth in the present study for their probiotic potential. Several strains could be considered promising probiotic candidates since they showed good growth capacity and survival under simulated gastro-intestinal conditions (acidic pH of 1.5, up to 4% of bile salts and 5 mM of nitrate), good ability to auto-aggregate which may facilitate their adhesion to host cells as multiple aggregates and the subsequent displacement of pathogens. Moreover, co-aggregation of lactobacilli with pathogenic bacteria was shown with Listeria innocua, Staphylococcus aureus, Escherichia coli, and Salmonella Enteritidis as good defense strategy against gut and food pathogens. Furthermore, they exhibited adherence to intestinal and vaginal cell lines, such property could be reinforced by their capacity of biofilm formation which is also important in food matrices such as the olive surface. Their antagonistic activity against pathogenic bacteria by means of acids and plantaricins, and also their different functional properties may determine their efficacy not only in the gastro-intestinal tract but also in food matrices. Besides their ability to ferment several prebiotics, the new evidence in the present study was their capacity to ferment lactose which reinforces their use in different food matrices including dairy as a dietary adjunct to improve lactose digestibility. Lactobacillus pentosus CF2-10N was selected to have the best probiotic profile being of great interest in further studies. In conclusion, spontaneous fermented Aloreña table olives are considered a natural source of potential probiotic L. pentosus to be included as adjunct functional cultures in different fermented foods.

Comparative proteomic analysis of a potentially probiotic Lactobacillus pentosus MP-10 for the identification of key proteins involved in antibiotic resistance and biocide tolerance

Probiotic bacterial cultures require resistance mechanisms to avoid stress-related responses under challenging environmental conditions; however, understanding these traits is required to discern their utility in fermentative food preparations, versus clinical and agricultural risk. Here, we compared the proteomic responses of Lactobacillus pentosus MP-10, a potentially probiotic lactic acid bacteria isolated from brines of naturally fermented Aloreña green table olives, exposed to sub-lethal concentrations of antibiotics (amoxicillin, chloramphenicol and tetracycline) and biocides (benzalkonium chloride and triclosan). Several genes became differentially expressed depending on antimicrobial exposure, such as the up-regulation of protein synthesis, and the down-regulation of carbohydrate metabolism and energy production. The antimicrobials appeared to have altered Lb. pentosus MP-10 physiology to achieve a gain of cellular energy for survival. For example, biocide-adapted Lb. pentosus MP-10 exhibited a down-regulated phosphocarrier protein HPr and an unexpressed oxidoreductase. However, protein synthesis was over-expressed in antibiotic- and biocide-adapted cells (ribosomal proteins and glutamyl-tRNA synthetase), possibly to compensate for damaged proteins targeted by antimicrobials. Furthermore, stress proteins, such as NADH peroxidase (Npx) and a small heat shock protein, were only over-expressed in antibiotic-adapted Lb. pentosus MP-10. Results showed that adaptation to sub-lethal concentrations of antimicrobials could be a good way to achieve desirable robustness of the probiotic Lb. pentosus MP-10 to various environmental and gastrointestinal conditions (e.g., acid and bile stresses).

Correlation between antibiotic and biocide resistance in mesophilic and psychrotrophic Pseudomonas spp. isolated from slaughterhouse surfaces throughout meat chain production

The aim of this study was to evaluate biocide susceptibility in mesophilic and psychrotrophic pseudomonads isolated from surfaces of a goat and lamb slaughterhouse, which was representative of the region. To determine biocide resistance in pseudomonads, we determined for the first time the epidemiological cut-off values (ECOFFs) of benzalkonium, cetrimide, chlorhexidine, hexachlorophene, P3 oxonia, polyhexamethylene guanidine hydrochloride (PHMG), topax 66 and triclosan being generally very similar in different Pseudomonas spp. with some exceptions. Thus, resistance of pseudomonads was mainly shown to triclosan, and in lesser extent to cetrimide and benzalkonium chloride depending on the species, however they were highly susceptible to industrial formulations of biocides. By means of statistical analysis, positive correlations between antibiotics, biocides and both antimicrobials in pseudomonads were detected suggesting a co- or cross resistance between different antimicrobials in goat and lamb slaughterhouse environment. Cross-resistance between biocides and antibiotics in pseudomonads were especially detected between PHMG or triclosan and different antibiotics depending on the biocide and the population type. Thus, the use of those biocides as disinfectant in slaughterhouse zones must be carefully evaluated because of the selection pressure effect of antimicrobials on the emergence of resistant bacteria which could be spread to the consumer. It is noteworthy that specific industrial formulations such as topax 66 and oxonia P3 showed few correlations with antibiotics (none or 1-2 antibiotics) which should be taken into consideration for disinfection practices in goat and lamb slaughterhouse.

Diversity, Distribution and Quantification of Antibiotic Resistance Genes in Goat and Lamb Slaughterhouse Surfaces and Meat Products

The distribution and quantification of tetracycline, sulfonamide and beta-lactam resistance genes were assessed in slaughterhouse zones throughout meat chain production and the meat products; this study represents the first to report quantitatively monitor antibiotic resistance genes (ARG) in goat and lamb slaughterhouse using a culture independent approach, since most studies focused on individual bacterial species and their specific resistance types. Quantitative PCR (qPCR) revealed a high prevalence of tetracycline resistance genes tetA and tetB in almost all slaughterhouse zones. Sulfonamide resistance genes were largely distributed, while beta-lactam resistance genes were less predominant. Statistical analysis revealed that resistant bacteria, in most cases, were spread by the same route in almost all slaughterhouse zones, except for tetB, blaCTX and blaTEM genes, which occurred in few zones as isolated ‘hot spots.’ The sum of all analyzed ARG indicated that slaughterhouse surfaces and end products act as reservoirs of ARG, mainly tet genes, which were more prevalent in slaughtering room (SR), cutting room (CR) and commercial meat products (MP). Resistance gene patterns suggest they were disseminated throughout slaughterhouse zones being also detected in commercial meat products, with significant correlations between different sampling zones/end products and total resistance in SR, CR and white room (WR) zones, and also refrigerator 4 (F4) and MP were observed. Strategically controlling key zones in slaughterhouse (SR, CR and WR) by adequate disinfection methods could strategically reduce the risks of ARG transmission and minimize the issues of food safety and environment contamination.

Complete Genome Sequence of a Potential Probiotic, Lactobacillus pentosus MP-10, Isolated from Fermented Aloreña Table Olives

ABSTRACT We report here a 3,698,214-bp complete genome sequence of a potential probiotic Lactobacillus pentosus strain, MP-10, isolated from brines of naturally fermented Aloreña green table olives; it is considered the largest sequenced genome among lactobacilli to date. The annotated genome sequence revealed the presence of 3,558 open reading frames (ORFs) and 87 structural RNAs.

Efficacy of "HLE"—a multidrug efflux-pump inhibitor—as a disinfectant against surface bacteria

Abstract We evaluated the efficacy of a new disinfectant product, HLE, to inhibit multiple species of planktonic and biofilm bacterial cultures. The HLE disinfectant comprised of EDTA, lactic acid and hydrogen peroxide, and our data indicated that the disinfectant had effective antimicrobial and anti‐biofilm activity even at low concentrations (0.15% to 0.4% HLE, v/v). Furthermore, the HLE disinfectant destabilized biofilm structures eradicated them due to the synergistic effect of EDTA and both antimicrobials (lactic acid and hydrogen peroxide), as revealed by confocal laser scanning microscopy. Additionally, sub‐inhibitory concentrations of HLE disinfectant, with EDTA as an efflux pump inhibitor, inhibited the expression of multidrug EfrAB, NorE and MexCD efflux pumps in both planktonic and biofilm cultures. This could provide an alternative way to disinfect surfaces to avoid spreading multi‐drug resistant strains in the food chain and the environment by decreasing efflux pump expression and consequently reducing the antibiotic selective pressure caused by systemic antibiotics and disinfectant use. HighlightsHLE disinfectant had effective antimicrobial and anti‐biofilm activity.HLE disinfectant destabilized biofilm structures eradicating them.Sub‐inhibitory concentrations of HLE inhibited multidrug efflux pumps.Disinfection based on HLE may avoid spreading multi‐drug resistant bacteria.