Influence of the Type of Diet on the Incidence of Pathogenic Factors and Antibiotic Resistance in Enterococci Isolated from Faeces in Mice

International Journal of Molecular Sciences Article Influence of the Type of Diet on the Incidence of Pathogenic Factors and Antibiotic Resistance in Enterococci Isolated from Faeces in Mice Beatriz Sánchez 1, Antonio Cobo 1,†, Marina Hidalgo 1, Ana M. Martínez-Rodríguez 2, Isabel Prieto 3, Antonio Gálvez 1and Magdalena Martínez-Cañamero 1,* 1Área de Microbiología, Departamento de Ciencias de la Salud, Universidad de Jaén, Paraje de Las Lagunillas s/n, 23071 Jaen, Spain 2 Departamento de Estadística e Investigación Operativa, Universidad de Jaén, Paraje de Las Lagunillas s/n, 23071 Jaen, Spain 3Área de Fisiología, Departamento de Ciencias de la Salud, Universidad de Jaén, Paraje de Las Lagunillas s/n, 23071 Jaen, Spain *Correspondence: canamer[email protected] † Current address: Departamento de Microbiología, Universidad de Granada, Campus de la Cartuja s/n, 18071 Granada, Spain. Received: 30 July 2019; Accepted: 28 August 2019; Published: 2 September 2019   Abstract: A comparative study on potential risks was carried out in a collection of 50 enterococci isolated from faeces of mice fed a standard or a high-fat diet enriched with extra virgin olive oil, refined olive oil or butter, at the beginning, after six weeks and after twelve weeks of experiments. Strains were biochemically assessed and genetically characterized. E. faecalis and E. casseliflavus were the most frequently isolated species in any diet and time points. Apart from the fact of not having isolated any strain from the virgin olive oil group during the last balance, we found statistically significant differences (p<0.05) among the diets in the percentage of antibiotic resistance and in the presence of the enterococcal surface protein gene (esp), as well as a tendency (p<0.1) for the presence of the tyrosine decarboxylase gene (tdc) to increase over time in the group of isolates from the standard diet. When the resistance of the strains to virgin or refined olive oil was studied, only the group of enterococci from high fat diets showed a significantly higher percentage of resistance to refined olive oil (p<0.05) , while both types of oil equally inhibited those isolated from the standard diet (p>0.05). Keywords: enterococci; virulence; antibiotic resistance; olive oil; high fat diets 1. Introduction The intestinal microbial diversity will be determined by the interactions among the organisms that compose it, the genetics of the host and the diets applied [ 1 ]. Diet, in fact, has a marked influence on the intestinal microbiota of the host [ 2 ] and much attention has been drawn specifically on high fat diets (HFD) because of their influence on health [ 3 ]. While studying the dissimilar effect of fats with different degrees of saturation, we have previously reported several studies comparing the influence of virgin olive oil (EVOO) and butter (BT) on the intestinal microbiota of mice, both using genotyping methods [ 4 ] and massive sequencing [ 5 ]. In these works, we presented evidence supporting a link between specific diets, physiological parameters and some bacterial taxa. Moreover, by comparing EVOO and refined olive oil (ROO) diets, the possible effect of virgin olive oil polyphenols was uncovered [ 6 ]. These new culture-independent technologies can, however, address only global taxa, giving no discriminative data on how the different strains of a certain bacterial group are evolving Int. J. Mol. Sci. 2019,20, 4290; doi:10.3390/ijms20174290 www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2019,20, 4290 2 of 16 in response to the diet, even though this strain-level change is most probably the first one to occur in the bowels. Intestinal microorganisms will deal with the capacity of self-regulation of the system, the available food and the competition for resources, and will have to defend themselves against chemical products or aggressive proteins, which will lead to many of these bacterial groups to develop strategies and defence mechanisms. Among these mechanisms, we can find the expression of virulence factors or antibiotic resistance. These defensive weapons will inescapably affect the host, potentially causing serious clinical consequences. One of the best-studied genera in this sense is the genus Enterococcus, in which several of these factors are known. Enterococci are intestinal bacteria widely known for their presence both in food and the oral-faecal route, and their utilization and safety remain a source of controversy [ 7 ]. As lactic acid bacteria, they have been important in food fermentations [ 8 ] and are also used as probiotics [ 7 ]. However, they are important nosocomial pathogens too, which prevents them from getting their GRAS (Generally Recognized as Safe) qualification [ 9 ]. This is worsened by their ability to survive adverse environmental conditions and heat treatments [ 10 ], which makes enterococci a widely distributed microbial group [ 11 ]. Enterococcal strains from food [ 12 ], environmental or clinical origins [ 13 ] are then very carefully studied to evaluate their safety. Enterococcus is therefore a good model of how certain diets can protect the host, promoting, or not, the growth of strains with different levels of safety once they have reached the intestine. Considering the antimicrobial effect of olive oil on bacterial taxa in vitro [ 14 ] and, in a murine model [ 6 ], it is also interesting to evaluate its potential role, when included in the diet, in selecting the strains that are going to thrive in the intestines of the host. Consequently, our objective has been to carry out a study on the safety level of enterococcal strains, isolated from faeces of mice fed with standard chow or enriched with EVOO, ROO or BT, by evaluating their antibiotic resistance, virulence factors, and the production of biogenic amines, as well as to ultimately determine if there are statistically significant differences among these enterococci isolated from the four different diets. 2. Results 2.1. Isolation and Identification of Bacterial Strains A previous collection of bacterial strains obtained from faeces of mice fed four different diets (SD, BT, EVOO and ROO) over a twelve-week period was screened for enterococcal phenotypic characteristics. All Gram positive cocci, facultative anaerobic, catalase negative, able to hydrolyse esculin in 40% bile salts and to grow from 10 to 45 ◦ C, in a media containing 6.5% NaCl or buffered at pH 9.6, were selected and their r16S gene was sequenced. In total, a collection of 50 enterococcal strains was obtained. As shown in Table 1, most of them belonged to the species E. faecalis [ 15 ] and E. casseliflavus [ 16 ], followed by E. gallinarum [ 5 ], E. hirae [ 2 ] and one strain of E. avium and E. durans each. Twelve strains were obtained at the beginning of the experiment, eighteen after six weeks and twenty strains after twelve weeks. Most strains were obtained in the BT group [ 17 ] and the lowest number of strains was found in the EVOO group [ 6 ], mostly because no strains were isolated from faeces on this diet at the end of the experiment (Figure 1a,b). To confirm this result statistically, a Poisson’s regression model was applied to the number of strains obtained based on two factors: diet and time (6 and 12 weeks). According to this model, the diet enriched with EVOO produced a significantly lower number of strains (23% lower; p= 0.0221). However, in spite of these dissimilarities, no significant difference was found in the species distribution of the strains with respect to diet or time when applied a Fisher’s exact test for count data. Int. J. Mol. Sci. 2019,20, 4290 3 of 16 (a) (b) Figure 1. Bar plot of number of strains isolated within each species distributed according to time ( a ) and diet (b). Table 1. Detection of virulence factors and biogenic amines gene products. Virulence Factors Biogenic Amines Species agg gelE cylA cylM cylB esp efaAfs efaAfm cob cpd ccf tdc 0S1-1 E. faecalis + 0S1-2 E. casseliflavus + 0S2-1 E. faecalis + + 0B1-2 E. casseliflavus + 0B2-4 E. faecalis + 0B3-1 E. casseliflavus + 0O5-2 E. faecalis + + + 0O5-4 E. faecalis + + 0O7-3 E. faecalis + 0V3-1 E. casseliflavus + 0V5-1 E. faecalis + + 0V5-2 E. faecalis + + + 6S3-3 E. casseliflavus + + + 6S3-4 E. gallinarum + 6S4-1 E. casseliflavus 6S4-2 E. faecalis + + 6S5-1 E. faecalis + 6S5-3 E. faecalis + + + 6B1-1 E. casseliflavus + 6B1-2 E. gallinarum + 6B1-3 E. hirae + + 6B2-1 E. faecalis + + 6B3-1 E. avium + + 6B3-2 E. casseliflavus + 6O5-2 E. faecalis + + 6O5-3 E. faecalis + 6O5-4 E. faecalis + + 6O6-1 E. faecalis + + 6O7-1 E. casseliflavus + 6V3-1 E. casseliflavus + 6V5-1 E. faecalis + + 6V6-1 E. casseliflavus + + 12S6-2 E. faecalis + 12S6-3 E. casseliflavus + + 12S7-2 E. casseliflavus + 12S7-3 E. gallinarum + + 12S7-4 E. casseliflavus + + 12B1-3 E. hirae + 12B1-4 E. faecalis + + + 12B2-4 E. casseliflavus + + 12B2-5 E. durans 12B3-2 E. casseliflavus + 12B3-4 E. faecalis + 12B3-5 E. faecalis + 12O5-1 E. gallinarum + + 12O5-2 E. casseliflavus + 12O5-3 E. gallinarum + 12O6-1 E. casseliflavus + + 12O6-3 E. casseliflavus + 12O7-1 E. casseliflavus + Int. J. Mol. Sci. 2019,20, 4290 4 of 16 2.2. RAPD Classification When subjected to RAPD-PCR genotyping, the fifty strains clustered in three different groups (Figure 2). Group 1 was deeply branched and included only five strains, all of which were isolated from standard diet fed mice. Group 2 contained 40% of the isolates, all of them from the SD group or from the other groups at t= 0. Finally, the third big cluster grouped the rest of the strains, 52%, most of them (21 out of 26) isolated from mice fed one of the three high fat diets (BT, EVOO or ROO). Pearson correlation [0.0%-100.0%] RAPD-M13 100 95 90 85 80 75 70 65 60 55 50 45 40 35 12O5-2 12O6-3 12B2-5 12S6-2 12S6-3 12O5-3 12B3-4 12O5-1 6B1-3 6B2-1 6B1-1 6B3-1 6B3-2 0B1-2 6O5-3 6O5-4 6B1-2 0B2-4 6V6-1 12B1-4 12B2-4 12S7-4 12O6-1 12O7-1 12B3-5 12B1-3 6S5-3 0V5-2 0O5-2 6S5-1 0S1-1 0S2-1 0S1-2 6V3-1 0O7-3 0V3-1 6V5-1 0B3-1 0V5-1 6O5-2 6O6-1 6S3-2 6S3-3 6S3-1 6S4-2 12S7-3 G3-3 G3-2 G3-1 G3 G2 G1 Figure 2. Pearson coefficient-based analysis of the RAPD profiles of the strains isolated. The initial number indicates the week of isolation. Int. J. Mol. Sci. 2019,20, 4290 5 of 16 2.3. PCR Amplification of Virulence Factors Specific PCR reactions were performed to detect the presence of genes related to virulence factors (Table 1). Thirty-six isolates presented at least one virulence-related gene, out of which, six strains showed positive for two virulence factors and only one isolate presented three. Five strains showed positive for the aggregation substance gene (agg). Gelatinase gene (gelE) was amplified in only one isolate. Sex pheromone genes (cpd and ccf) were present in one and four strains, respectively, while cob was not detected. Of the cytolysin genes, only cylB was present, in only one strain, and genes related to cell wall adhesions (efaAfs and efaAfm) were detected in two strains each. In contrast, the gene coding for the enterococcal surface protein (esp) was present in most of the strains (62%). Since the rest of virulence factors did not reach the number of positives needed for any statistical study, we centered our attention in the esp gene distribution. Contingent tables followed by Fisher’s exact test were performed to compare the percentage of presence of the gene in the strains isolated at the three different time-points and between each two different time-points, but no signification was detected. On the contrary, we found signification when comparing in the same way the percentage of presence of esp in the strains among all diets ( p= 0.0234; Figure 3a), with the lowest values obtained in the SD group. This difference turned out to be more significant when comparing the SD diet versus the three high fat diets (p=0.0037; Figure 3b). (a) (b) Figure 3. Bar plot of number of strains with presence (positives) or absence (negatives) of the gene esp, distributed among the four diets; ( a ) Fisher’s exact test p= 0.0234) or between SD and high fat diets; ( b ) Fisher’s exact test p= 0.0037). SD, standard diet; BT, butter enriched diet; EVOO, extra virgin olive oil enriched diet; ROO, refined olive oil enriched diet; F, all high fat diets. 2.4. Biogenic Amine Production Possible production of biogenic amines was evaluated through PCR amplification of the genes coding for the corresponding carboxylases. No amplification products of the genes hdc1,hdc2 or odc were obtained. However, 29 strains were positive in the case of the gene coding for tyrosine decarboxylase (tyr) (Table 1). Again, contingent tables followed by Fisher’s exact test were performed in order to uncover differences among diets and also among all time-points. No significant differences were found in any case. When studying diets, a p -value of 0.4192 was obtained. When comparing data distribution between initial and final time points, the p -value, though still not significant, was lower ( p= 0.1362) with five and thirteen positive strains, respectively (Figure 4a). Since there were no strains isolated in the EVOO group at 12 weeks, we lacked one time-point in the contingency table with respect to his diet. In fact, we have shown above that, on this diet, the number of strains is significantly smaller. Given that this could introduce an additional factor not relevant for the question asked and that this produces a sparse contingency table, we decided to repeat the statistical analysis Int. J. Mol. Sci. 2019,20, 4290 6 of 16 considering only those diets that present data along the three time points (SD, BT and ROO). In this case, the p-value (p=0.0968) is significant at 90% (Figure 4b). (a) (b) Figure 4. Bar plot of the number of strains with presence (positives) or absence (negatives) of the gene tdc, at the beginning or the end of the experiment considering strains from all diets; ( a ) Fisher’s exact test p= 0.1362) or from all but an extra virgin olive oil enriched diet; ( b ) Fisher’s exact test p=0.0968). 2.5. Antibiotic Resistance Resistance level was tested for fifteen different antibiotics and results were marked as R (resistance), I (intermediate) or S (susceptible) (Table 2). No antibiotic presented the same effect on all the strains; on the contrary, there was a high variability except in the case of kanamycin, norfloxacyn and levofloxacin, where most strains were sensitive, with only five resistant strains against the first antibiotic and six strains against each of the other two. Thirty-one strains were resistant to five or more antibiotics, and three isolates had no resistance at all. Then, the percentage of antibiotics to which each strain was resistant was calculated and these data were grouped by diet and by time-point and were compared using a Kruskal–Wallis test. No significant differences were found among the three time-points, but there was significance when the diets were compared ( p= 0.0225; Figure 5a). A pair comparison uncovered the fact that strains isolated from mice under SD diet presented less resistances than any of the three high fat diets, this difference being statistically significant when compared with the ROO diet ( p= 0.0140). If the initial time point was not considered, the significance of the difference with the ROO diet improved ( p= 0.0039), even reaching 90% significance also in the comparison between SD and BT (p=0.0804; Figure 5b). (a) (b) Figure 5. Boxplots of percentage of resistances in each strain grouped by diet considering all time-points; (a)pSD vs. ROO = 0.0225) or without initial time data; (b)pSD vs. ROO = 0.0039). Int. J. Mol. Sci. 2019,20, 4290 7 of 16 Table 2. Antibiotic resistance. R: Resistant I: Intermediate S: Susceptible. Antibiotics. PENP: penicillin pneumo; PENS: penicillin strepto; AMPE: ampicillin; CTXP: cetofaxim pneumo; CTXS: cetoaxim strepto; IMIE: imipenem; KAHES: kanamycin; GEHES: gentamycin; FQPR: norfloxacin; MXFPS: moxifloxacin; LVXS: levofloxacin strepto; LVXP: levofloxacin pneumo; ERYPS: erythromycin; TELPS: telithromycin; QDAE: quinupristin-dalfopristin; TEPS: tetracyclin; RFAPS: rifampicin; TSU: cotrimoxazol; LNZEP: linezolid pneumo; LNZS: linezolid strepto; FOSP: fosfomycin; FURES: nitrofurantoin; VAN: vancomycin; TEC: teicoplanin. Antibiotic Resistance PEN PENS AMPE CTXP CTXS/IMIE KAHES/GEHES FQPR/MXFPS LVXS/LVXP ERYPS/TELPS QDAE TETPS/RFAPS TSU/LNZEP LNZS FOSP7FURES VAN/TEC 0S1-1 R R I R I S S S R R R R R I R 0S1-2 R R I R I S S S R R I I R S R 0S2-1 R R R R I S S S R R R R R R R 0B1-2 I S S R I S S S S S S S S S S 0B2-4 R R R R I S S S R R R R R R R 0B3-1 R R R I I S I S R R R R R R R 0O5-2 R R R R R S S S R R R I R R R 0O5-4 R I S R I S S I S I R S I I S 0O7-3 R R I R I S S S R R R I R I R 0V3-1 R R R I I S S S R R R I R R R 0V5-1 R R R R I S S S R R R I R R R 0V5-2 I I S R I S S S I R R S S S S 6S3-3 R R I I I S S S I S S S S S S 6S3-4 I S S I I S S S S S S S S S S 6S4-1 R R I R I S S S R R R I R S R 6S4-2 R R S R I S S S I S S S S S S 6S5-1 I I S I I S S S I I I S S I S 6S5-3 I I S S S S S S I I R S S I S 6B1-1 R I S R I I S S I R R S S I S 6B1-2 R R R R I S I S R R R I R R R 6B1-3 I S S R I S S S I I I S I S S 6B2-1 R R R R R R R R R S I I S S S 6B3-1 R R R R I S R S R R R I R R R 6B3-2 R I S R I S S S R R R S S I S 6O5-2 R R R I S S S R R R R I R R R 6O5-3 R R R R I S S S R R R I R R R 6O5-4 R R R R R S S S R R R I R R R 6O6-1 R R R R I S S S R R R I R R R 6O7-1 R I S R I S S S I R R S S S S 6V3-1 R R R R I S I S R R R I R R R 6V5-1 I I S R I S S S R R R S S I S 6V6-1 I I S I I S S S I R R S S S S 12S6-2 R R I S S S S S R R R R R I R 12S6-3 I I S R I S S S I I I S S I S 12S7-2 I I S R I S S S I I I S S I S 12S7-3 R R S I S S S S I S S I S S S 12S7-4 R R I R I S S S R R R R R S R 12B1-3 R R R R R R R R R S R I S S S 12B1-4 R R R R R R R R R S R I S S S 12B2-4 R I S R I S S S I R R S S R S 12B2-5 R I S R I S S S I R R S S R S 12B3-2 R R R R R R R R R S R I S S S 12B3-4 R R R R R R R R R I R S S I I 12B3-5 I I S I I S S S I S R S S I S 12O5-1 R R S R I S S S R R R I R I S 12O5-2 R R R R R S R I R R R I R R R 12O5-3 I S S R I S S S S S R S S I S 12O6-1 R R R R R S S S R R R I R R R 12O6-3 R R R R I S S S R R R I R S R 12O7-1 R R R R R S S S R R R I R R R Int. J. Mol. Sci. 2019,20, 4290 8 of 16 Results on the genetic determinants of resistance for the isolates are shown in Table 3. All strains but one tested positive to at least one genetic determinant studied. Among the β -lactamase tested, bla PSE and bla TEM were the most frequent with presence in 23 and 24 strains, respectively, while tetE was the most prominent tetracycline resistance gene with 26 positives. Additionally, 34 strains presented genes for efflux pumps. Table 3. Genetic determinants of resistance. β-Lactamases Tetracyclines Efflux Pumps Group I Group II blaCTX-M blaCTX-M2 blaPSE blaTEM tetB tetC tetD tetA tetE tetG acrB aadA1 mdfA 0S1-1 + + + + + 0S1-2 + + + + + + + 0S2-1 + + + + + 0B1-2 + 0B2-4 + + 0B3-1 + + 0O5-2 + + + 0O5-4 + 0O7-3 + + + + + 0V3-1 + + + + + + 0V5-1 + + + 0V5-2 + + 6S3-3 + + + + + 6S3-4 + + + + + + + 6S4-1 + + + 6S4-2 + + + + 6S5-1 + + + + 6S5-3 + + + + + 6B1-1 + + + 6B1-2 + + + 6B1-3 + + + 6B2-1 + + + + 6B3-1 + + + + 6B3-2 + + + + 6O5-2 + + + 6O5-3 + + + + 6O5-4 + + + + 6O6-1 + + + + 6O7-1 + + + + 6V3-1 + + 6V5-1 + + + + + + 6V6-1 + + 12S6-2 + + 12S6-3 + + + + + + + 12S7-2 + + 12S7-3 + + + + 12S7-4 + + + 12B1-3 + + + + + 12B1-4 + + 12B2-4 + + + 12B2-5 + + + 12B3-2 + + + + + + 12B3-4 + + + 12B3-5 12O5-1 + + + 12O5-2 + + + 12O5-3 + + 12O6-1 + + + + + + 12O6-3 + + + + 12O7-1 + + + + + + 2.6. Growth with Virgin and Refined Olive Oil Refined or virgin olive oil spotted directly on the lawn prevented any bacterial growth. Therefore, in order to see differences in the antimicrobial effect between both oils, we tried to emulate physiological conditions and made emulsions of the oils with cholic acid, lecithin or a mixture of both. Under these conditions, there was no total inhibition in any case and bacterial growth or partial inhibition alternated in all diets and with both types of oil (Table 4). When contingency tables were performed with these two options (growth/partial inhibition) and the four diets, no significant differences were detected among them. Int. J. Mol. Sci. 2019,20, 4290 9 of 16 When the antimicrobial effect of EVOO and ROO was checked with the filter paper technique, we found partial inhibition in all strains when confronted with virgin olive oil but growth/partial inhibition alternated again with refined olive oil (Table 4). The highest percentage of unaffected strains/the lowest percentage of partial inhibition was found in the group of isolates from the ROO diet (Figure 6a,b). Again, a contingency table was performed to check whether distribution of data among the diets was or not homogeneous, and these time differences were found at 90% of signification (p=0.0692), confirming a strong tendency (Figure 6a). Finally, each diet data were studied independently by means of a paired data analysis, with the McNemar’s test and exact p -values, in order to check signification of the differences in growth/partial inhibition when facing refined or virgin olive oil with the different techniques. This analysis could not be performed in the strains isolated from the EVOO diet because of its low number. With respect to the other three diets, no significant differences were found in the case of lecithin, cholic acid or the mixture of both. However, when studying data using filter paper (Figure 6b), significant differences were found for the ROO diet isolates ( p= 0.0019) and for the BT diet isolates ( p= 0.004), indicating a significantly lower number of partially inhibited strains when growing with refined olive oil with respect to growing with virgin olive oil in both cases. However, no significant differences at 95% were found for the SD diet isolates (p=0.062), indicating a higher number of partially inhibited strains. (a) (b) Figure 6. Bar plots of the number of strains with full growth or partial inhibition under refined olive oil; ( a ) according to diet (Fisher’s exact test p= 0.0692); ( b ) paired data analysis of strains isolated from BT (McNemar’s test p= 0.0040), ROO (McNemar’s test p= 0.0019) and SD (McNemar’s test p= 0.062). SD, standard diet; BT, butter enriched diet; EVOO, extra virgin olive oil enriched diet; ROO, refined olive oil enriched diet. Table 4. Refined (ROO) and virgin olive oil (EVOO) antimicrobial activity. PI: Partial inhibition. Cholic Acid 1% Lecitine 3% Lecitine 3% + Cholic Acid 1% Filter Paper ROO EVOO ROO EVOO ROO EVOO ROO EVOO 0S1-1 PI PI PI PI 0S1-2 PI PI 0S2-1 PI 0B1-2 PI 0B2-4 PI PI PI PI PI 0B3-1 PI PI Int. J. Mol. Sci. 2019,20, 4290 16 of 16 29. Swick, M.C.; Morgan-Linnell, S.K.; Carlson, K.M.; Zechiedrich, L. Expression of multidrug efflux pump genes acrAB-tolC,mdfA, and norE in Escherichia coli clinical isolates as a function of fluoroquinolone and multidrug resistance. Antimicrob. Agents Chemother. 2011,55, 921–924. [CrossRef] [PubMed] 30. Guerra, B.; Soto, S.M.; Argüelles, J.M.; Mendoza, M.C. Multidrug resistance is mediated by large plasmids carrying a class 1 integron in the emergent Salmonella enterica serotype [4,5,12:i:-]. Antimicrob. Agents Chemother. 2001,45, 1305–1308. [CrossRef] c 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).