Molecular genetic study of potentially bacteriocinogenic clinical and dairy Enterococcus spp. isolates from Bulgaria

Abstract

A total of 424 Enterococcus spp. isolates from clinical samples (n = 398) and traditional Bulgarian artisanal cheeses (n = 26) were studied in order to evaluate the incidence and expression of bacteriocin genetic determinants. Structural genes coding for enterocin A (entA), enterocin P (entP), enterocin B (entB), enterocin AS-48 (as-48) and enterolysin A (entlA) were detected by polymerase chain reaction (PCR), while reverse transcription PCR was performed to establish the gene expression. Of the total isolates, 163 (38.4 %) were potentially bacteriocinogenic. The genes typical of Enterococcus faecium (entA, entB and entP) were found in 84.0, 8.4 and 15.1 % of the E. faecium isolates, respectively. Focusing on E. faecalis isolates, the frequencies of entlA and as-48 were 19.3 and 0.7 %, respectively. No significant differences in the gene presence between clinical and dairy isolates were ascertained. Bacteriocin genes were found to be expressed at transcriptional level in 83.4 % of the bacteriocinogenic Enterococcus spp. isolates: a single gene in 113 samples (69.3 %), two genes in 21 (12.9 %) and three genes in 2 isolates (1.2 %). The present investigation may serve as a basis for further studies for elucidating the spread of the bacteriocin genetic determinants in the two enterococcal populations (clinical and dairy).

Introduction

Enterococci are lactic acid bacteria (LAB) of significance in medical microbiology, industrial microbiology and public health. They are part of the normal microflora of the gut, urogenital tract and mouth in humans and animals, and are widespread in soil, water, plants and foods (Müller et al. 2001; Giraffa 2002).

Enterococci have traditionally been regarded as low-grade pathogens. Therefore, enterococcal infections could arise as a complication after surgery, invasive procedures, prolonged in-patient stay and precedent antimicrobial therapy or in some hidden rare diseases of the patient (Batistão et al. 2012). During the last two decades, Enterococcus spp. (mostly E. faecalis and E. faecium) have emerged as an increasingly important cause of nosocomial and community-acquired infections, such as urinary tract infections, surgical site and burn wound infections, bacteremia and sepsis, endocarditis, cholecystitis, peritonitis, neonatal meningitis and others (Sood et al. 2008).

Also, enterococci have important implications in the dairy industry. They occur as nonstarter LAB in a variety of cheeses, especially traditional cheeses produced in southern Europe and Asia from raw or pasteurised milk, and in natural milk or whey starter cultures (Franz et al. 2003; Giraffa 2003). Enterococcal strains are involved in ripening of many artisanal cheeses and have also been used as components of cheese starter cultures (Foulquié Moreno et al. 2006). The positive influence of enterococci on cheese seems due to specific biochemical traits such as lipolytic and proteolytic activities, their capacity to use citrate and pyruvate as carbon sources, and production of aromatic volatile compounds. A few strains are even being promoted as probiotics, contributing to intestinal health by improving the microbial balance of the gut (Domann et al. 2007).

The difference between an enterococcal pathogen and an apparently safe dairy use strain is unclear. It is established that the molecular taxonomy of enterococci does not distinguish these two types of strains (Eaton and Gasson 2001). Many Enterococcus spp. strains of clinical and food origin produce antibacterial peptides (bacteriocins), generally called enterocins (Franz et al. 2007). Usually, they are small cationic peptide toxins with bactericidal activity, in most cases against closely related species, which enables microorganisms to occupy their ecological niches. Interest in enterocins has been stimulated by the fact that they are active against Gram-positive dairy-borne pathogens such as Listeria monocytogenes, Staphylococcus aureus, Bacillus cereus, and vegetative cells and spores of Clostridium botulinum (Franz et al. 2007).

Bacteriocins produced by Enterococcus spp. include enterocins A, B and P typical of E. faecium, as well as enterocin AS-48 and enterolysin A typical of E. faecalis.

The aim of this study was to evaluate the incidence and expression of bacteriocin structural genes among Enterococcus spp. strains isolated from clinical specimens and traditional Bulgarian artisanal cheeses.

Materials and methods

Clinical isolates

A collection of 398 non-duplicate clinically significant strains of Enterococcus spp. (294 E. faecalis and 104 E. faecium) was investigated. The isolates were recovered between June 2012 and March 2015 from 301 in-patients and 97 outpatients in seven large Bulgarian hospitals. They were obtained from urine (n = 152), surgical wound or abscesses (n = 117), genital tract samples (n = 78), blood (n = 21), lower respiratory tract samples (n = 15) and bile (n = 15).

Dairy isolates

A total of 26 non-pathogenic Enterococcus spp. strains (11 E. faecalis and 15 E. faecium) were isolated from artisanal cheeses in different mountain regions of Bulgaria. They play an acknowledged role during the ripening.

Culture media

HiCrome Enterococcus faecium Agar Base (Himedia Labs) was applied for isolation of the Enterococcus spp. strains. E. faecalis forms blue colonies, while E. faecium gives green colonies, surrounded by yellowish colouring of the ambient.

Biochemical identification with commercial kits and systems

Species identification was done using API Rapid ID 32 Strep (bioMérieux), BBL Crystal Gram-positive ID kit (Becton Dickinson) and the automated system VITEK 2 (bioMérieux).

DNA isolation

Total DNA from all used strains was isolated with GenElute™ Bacterial Genomic DNA Kit (Sigma-Aldrich), according to the manufacturer’s instructions, from 3-ml overnight cultures inoculated with a single colony.

Molecular genetic genus and species identification

Polymerase chain reaction (PCR) amplification of the 16S ribosomal ribonucleic acid (rRNA) gene of Enterococcus spp. was used for genus identification of all isolates included in this study. The sodA genes encoding the enzyme manganese-dependent superoxide dismutase for the most common enterococci were detected with multiplex PCR. E. faecalis identification was confirmed by PCR for the eda1 gene (encoding 2-keto-3-deoxy-6-phosphogluconate aldolase, which is an enzyme taking part into the Entner-Doudoroff pathway and is species-specific for E. faecalis). Oligonucleotides used as primers for amplification were synthesized from Alpha DNA (Canada) and are listed in Table 1.

Table 1 Oligonucleotides used as amplification primers for genus-specific and species-specific identification of Enterococcus spp

PCR screening for the presence of bacteriocin genetic determinants

PCR amplification was performed in order to confirm the presence of genes coding for different class bacteriocins according to the current classification system of Franz et al. (2007): class II.1. [enterocin A (entA) and enterocin P (entP)], class II.3. [enterocin B (entB)], class III [enterocin AS-48 (as-48)] and class IV [enterolysin A (entlA)] bacteriocins. Oligonucleotides used as amplification primers (Alpha DNA) are shown in Table 2.

Table 2 Oligonucleotides used as primers for amplification of bacteriocin structural genes in Enterococcus spp

PCR was carried out with 10 ng of template DNA, 0.25 μM of each primer, 0.2 mM deoxyribonucleoside triphosphates, 1× reaction buffer, 2 mM MgCl₂ and 0.5 U Prime Taq DNA polymerase (Genet Bio) in a total volume of 25 μl. The DNA was amplified using the following protocol: initial denaturation (94 °C for 5 min), followed by 28–30 cycles of denaturation (94 °C for 35–45 s), annealing (50–60 °C, from 45 s to 1 min) and extension (72 °C, from 45 s to 1 min), with a single final extension of 7 min at 72 °C. PCR products were separated in 1 % agarose gel for 40–60 min at 140 V, stained with ethidium bromide (0.5 μg/ml) and detected by UV transillumination (wavelength 312 nm). Amplified genes were identified on the basis of fragment length presented in Tables 1 and 2.

Reverse transcription PCR

Reverse transcription PCR (RT-PCR) was performed to ascertain the bacteriocin gene expression at transcriptional level. Total bacterial RNA was isolated with GeneJET RNA Purification Kit (Thermo Scientific) according to the manufacturer’s instructions. The purified total RNA was used immediately or frozen quickly in liquid nitrogen and conserved at −80 °C. Immediately before the reverse transcription reaction, the RNA was treated with DNAse I (Thermo Scientific) by adding 1 μl of DNAse buffer (with MgCl₂ and 1 μl of DNAse I) to 8 μl of the RNA samples, followed by incubation for 30 min at 37 °C. The reaction was stopped by inactivating the enzyme by adding 1 μl of the provided EDTA solution and heating to 65 °C for 10 min, followed by chilling on ice. RevertAid Reverse Transcriptase kit (Thermo Scientific) was used for synthesis of the first-strand cDNA. The reaction was carried out with 4 μl of the reaction buffer, 2 μl of the bacteriocin-specific primer (at 10 μM concentration), 2 μl of the provided dNTPs mix, 1 μl M-MuLV reverse transcriptase, 1 μl RiboLock RNAse inhibitor and 8 μl of diH₂O in a total volume of 20 μl for 1 h at 37 °C. The reverse transcription was stopped by incubating the reaction mixture for 5 min at 70 °C, followed by chilling on ice. The obtained single-strand cDNAs were further used as matrices for PCR reactions, which were carried out in a MJ Research PTC-200 Thermal Cycler under the same conditions as the standard PCR amplifications for bacteriocin genetic determinants.

Statistical analysis

The distribution of bacteriocin structural genes with respect to isolate origin was compared using the Student’s t test. A P value below 0.05 was considered to be statistically significant.

Results

Incidence of bacteriocin structural genes among all studied Enterococcus spp. isolates

A total of 163 of the 424 studied enterococcal isolates (38.4 %) were potentially bacteriocinogenic, according to the PCR screening. The genes typical of E. faecium (entA, entB and entP) were found in 84.0, 8.4 and 15.1 % of the E. faecium isolates, respectively. Focusing on E. faecalis isolates, the frequencies of entlA and as-48 were 19.3 and 0.7 %, respectively. The structural gene for enterocin A was widely distributed among E. faecium isolates included in this study, whereas that of enterocin B generally (70 %) occurred in the presence of entA.

Figures 1 and 2 show the electrophoretograms for four bacteriocin-encoding genes for which amplification products were obtained (entlA, as-48, entA and entB).

Fig. 1

Amplification results for genes encoding entrolysin A (a) and enterocin AS-48 (b) in E. faecalis. a Lines 1–5, specific amplification products corresponded to the entlA gene (1007 bp); line 6, (−) negative control; line 7, 100-bp Ladder. b Line 1, 100-bp Ladder; lines 2, 3, specific amplification products corresponded to the as-48 gene (377 bp); line 4, (−) negative control

Fig. 2

Amplification results for genes encoding enterocin A (a) and enterocin B (b) in E. faecium. a Line 1, 100-bp Ladder; lines 2–12, specific amplification products corresponded to the entA gene (125 bp); line 13, (−) negative control. b Line 1, 100-bp Ladder; lines 2, 3, 7, specific amplification products corresponded to the entB gene (160 bp); lines 4–6, 8–9, (−) negative PCR products; line 10, (−) negative control

Comparative frequency of bacteriocin structural genes among Enterococcus spp. isolates from clinical samples and artisanal cheeses

The bacteriocin gene incidence in the two different collections (clinical, pathogenic and dairy, non-pathogenic Enterococcus spp. isolates) is presented in Table 3, in which, noticeably, is the more widespread dissemination of genes typical of E. faecium (entA, entB and entP) among cheese isolates than that in clinical isolates, whereas the genes of E. faecalis (entlA and as-48) were found more frequently in the pathogenic enterococci. No significant differences in the gene presence between clinical and dairy isolates were ascertained. Four of our cheese E. faecium isolates carried the enterocin A and B structural genes (entA and entB) simultaneously, while another four isolates carried the combination of genes entA + entP.

Table 3 PCR detection of bacteriocin structural genes among 424 Enterococcus spp. isolates from different sources

Expression of bacteriocin structural genes among the bacteriocinogenic Enterococcus spp. isolates

Bacteriocin genes were found to be expressed at transcriptional level in 83.4 % (n = 136) of the 163 potentially bacteriocinogenic isolates Enterococcus spp. isolates: a single gene in 113 samples (69.3 %), two genes in 21 (12.9 %) and three genes in 2 isolates (1.2 %). No gene expression appeared in 16.5 % (n = 27) of the Enterococcus spp. strains presenting bacteriocin genes (Fig. 3). The most frequent co-expressed genes were entA and entP (8 % of the potentially bacteriocinogenic strains), followed by entA + entB (3.7 %). Intriguingly, two isolates (1.2 %) expressed three genes simultaneously, entA + entB + entP.

Fig. 3

Expression of bacteriocin structural genes (as percentages) among the potentially bacteriocinogenic Enterococcus spp. isolates (n = 163)

Discussion

Enterococci are usually isolated from fermented foods, in which they contribute to the ripening/organoleptic characteristics (Giraffa 2003), but nowadays are considered emerging pathogens, due to an increase of antibiotic resistances and production of virulence traits Morrison et al. 1997). Both groups, GRAS (generally recognized as safe) Enterococcus spp. strains and clinically significant Enterococcus spp. strains, have a potential to produce different bacteriocins, some of which have been well characterized, e.g., enterocins A, B, P, AS-48, enterolysin A, etc.

In the present study, the most commonly occurring bacteriocin-encoding gene was the entA. Similar to our results, the entA was the most widespread gene among E. faecium isolates of different sources, whereas the entB was always associated with the presence of entA, as referred to in other investigations (De Vuyst et al. 2003; Poeta et al. 2007; Sabia et al. 2008). Enterocin B is a broad-spectrum bacteriocin of the class II.2. (Franz et al. 2007), that acts synergistically with enterocin A, a member of the pediocin family (Casaus et al. 1997). We established significantly higher prevalence of spread of entA among E. faecium isolates compared to that in vancomycin-resistant enterococci collected from clinical, dairy and animal samples in Italy (84.0 vs. 46.1 %, p < 0.01) (Sabia et al. 2008). In the Italian investigation mentioned above, entP and as-48 were not detected in vancomycin-resistant E. faecium and E. faecalis isolates, respectively.

Recently, Padilla et al. (2012) studied the presence of some bacteriocin determinants in clinical E. faecalis strains isolated in Chile during the period 2008–2009. Contrary to our results, they found significantly higher frequency for entlA (31.3 vs 19.7 %, p < 0.01) and as-48 (9.3 % / 0.7 %, p < 0.02). Moreover, they proved higher rates of incidence of these genes among isolates causing bacteremia and urinary tract infections (UTIs) compared to our analogous isolates: entlA for blood isolates – 42 % in Chile versus 28.6 % in our study; entlA, UTIs isolates – 30 %/19 %; as-48, blood isolates – 42 %/0 %, p < 0.001; as-48, UTIs isolates - 30 %/1 %, p < 0.001.

Previous studies showed that PCR detection of more than one bacteriocin-encoding gene in the same Enterococcus spp. isolate from artisanal cheeses is not unusual (Edalatian et al. 2012; Özden Tuncer et al. 2013). In the present study, there was a higher dissemination rate of entA among E. faecium cheese isolates (93.3 %) compared to that among E. faecium isolated from traditional Iranian raw milk cheeses (72.7 %), whereas the entB and entP revealed lower frequency rates in our isolates than among Iranian ones (26.7 vs. 54.5 %) (Edalatian et al. 2012). Recently, Özden Tuncer et al. (2013) isolated three bacteriocin-producer strains of E. faecium from artisanal Turkish tulum cheese and established the occurrence of eneterocin genes. All these strains bore the entA and entB genes, while two strains also contained the entP gene.

In our study, we chose to perform simple RT-PCR reactions to monitor solely whether or not the bacteriocins were expressed at RNA level. Despite the fact that, nowadays, multiplex quantitative PCR (qPCR) has been applied as a technique that allows the levels of individual enterocin gene expression to be monitored and determination of how expression is altered under different growth conditions (Williams and Chanos 2012), for the purposes of the present investigation this was not necessary due to the fact that the levels of bacteriocin production is often strongly influenced by the environmental conditions, such as temperature, pH, salts concentrations, presence of diverse sugars, etc. (Dimov et al. 2008; Raykova et al. 2008; Peykov et al. 2008). This means the exact quantitation of the levels of bacteriocin expression in controlled cultivation conditions would surely differ from the growth conditions of the strains’ natural environments, clinical or dairy. On the other hand, simple RT-PCR is sensitive enough to detect even minimal expression levels, thus avoiding to perform relatively more expensive and time-consuming RT-qPCR, which, even if it was carried out, would give us erroneous results.

In conclusion, more than one-third of the isolates included in the present study were revealed to be bacteriocinogenic. To our knowledge, this is the first investigation in Bulgaria for the incidence of bacteriocin-encoding genes among clinically relevant and non-pathogenic enterococci, which may serve as a basis for additional studies for elucidating the spread of the bacteriocin genetic determinants among the two different types of enterococcal populations.

Enterococcus spp. strains that produce bacteriocins are of increasing interest for science and industry because they seem to have an ecological advantage when compared with other nonproducing bacteria which inhabit the same ecosystem or which concur for colonization and for invasion in a particular ecological niche (Poeta et al. 2007). In addition, enterococci are good acidifiers in the presence of available sugars, and the inhibitory effect due to acid production could be considered an advantage in addition to bacteriocin production, and is equally responsible for survival and colonization (Sabia et al. 2008).