Frequency of Clustered Regularly Interspaced Short Palindromic Repeats ( CRISPRs ) in non-clinical Enterococcus faecalis and Enterococcus faecium strains

The fidelity of the genomes is defended by mechanism known as Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) systems. Three Type II CRISPR systems (CRISPR1-cas, CRISPR2 and CRISPR3-cas) have been identified in enterococci isolates from clinical and environmental samples. The aim of this study was to observe the distribution of CRISPR1-cas, CRISPR2 and CRISPR3-cas in non-clinical strains of Enterococcus faecalis and Enterococcus faecium isolates from food and fecal samples, including wild marine animals. The presence of CRISPRs was evaluated by PCR in 120 enterococci strains, 67 E. faecalis and 53 E. faecium. It is the first report of the presence of the CRISPRs system in E. faecalis and E. faecium strains isolated from wild marine animal fecal samples. The results showed that in non-clinical strains, the CRISPRs were more frequently detected in E. faecalis than in E. faecium. And the frequencies of CRISPR1-cas and CRISPR2 were higher (60%) in E. faecalis strains isolated from animal feces, compared to food samples. Both strains showed low frequencies of CRISPR3-cas (8.95% and 1.88%). In conclusion, the differences in the habitats of enterococcal species may be related with the results observe in distribution of CRISPRs systems.


Introduction
Enterococci are important Gram-positive bacteria recognized as part of gut microbiota in humans and animals, and they are widely distributed in soil, water, plants and foods (Cassenego et al., 2013;Lebreton et al., 2014;Pieniz et al., 2015;Santestevan et al., 2015;Prichula et al., 2016;Medeiros et al., 2017).Their ubiquity reflects its ability to survive in a variety of environmental stressors (Byappanahalli et al., 2012).Another important characteristic is their intrinsic resistance to some antimicrobials agents commonly prescribed to treat Gram-positive cocci such as cephalosporin, lincomycin, and low levels of penicillin and aminoglycosides.Furthermore, they are also able to acquire a variety of resistance genes via transposons or plasmids.Epidemiological data suggest that enterococci are important reservoirs for the spread of antibiotic resistance to different species of bacteria (Lebreton et al., 2014).
The Enterococcus genus comprises more than 50 species, and Enterococcus faecalis and Enterococcus faecium are the most frequent species isolated from humans, animals and food samples (Lebreton et al., 2014).Moreover, they are the most prevalent species cultured from humans' infected sites, including bacteremia, surgical site infections, and urinary tract infections.
The genomes of E. faecalis and E. faecium have suffered rearrangements over hundreds of millions of years in evolution, given them selective advantages to survive and disperse in the environment.As a result, the presence of the Enterococcus spp.has been investigated and monitored in a variety of habitats (Van Tyne and Gilmore, 2014).
To defend the genome against parasitic DNA invasion, and to maintain the fidelity of the genomes in stable ecosystems, there is a mechanism known as Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) systems (Sorek et al. 2008;Palmer and Gilmore, 2010;Magadán et al., 2012).The CRISPR systems provides a type of defense in prokaryotes, leading to a resistance to plasmid uptake and phage infections, and a barrier to horizontal gene transfection (Thomas and Nielsen, 2005;Wiedenheft et al., 2012).This immunity depends on the presence of specific target-derived spacer sequences, the intervening repeat palindromes short and highly conserved, and nuclease activity encoded by the cas genes (Haft et al., 2005;Makarova et al., 2006;Sorek et al., 2008).
Type II CRISPR-Cas loci consists of a CRISPR array, the type-specific cas9 gene, and cas1 and cas2 genes.In enterococci strains, three Type II CRISPR-Cas loci have been identified, the CRISPR locus lacking cas genes, thus consisting solely of repeat-spacer arrays (orphan CRISPR2), and those carrying these cas genes (CRISPR1-Cas and CRISPR3-Cas) (Makarova et al., 2011).Palmer and Gilmore (2010) using comparative genomics found that pathogenic enterococci have multiple rearrangements that occurred during evolution and is followed by the loss of CRISPRs.In contrast, commensal strains have smaller genomes with fewer rearrangements, as demonstrated by whole-genome alignments.The same authors found an inverse relationship between the presence of a CRISPR-cas locus and acquired antibiotic resistance in E. faecalis, suggesting that antibiotic use inadvertently selects for enterococcal strains with compromised genome defense (Palmer and Gilmore, 2010).
CRISPR1-Cas and CRISPR3-Cas loci occur with variable distribution in E. faecalis and they are absent in multi-resistant strains isolated from clinical samples (Palmer and Gilmore, 2010).In addition, CRISPR2 locus is conserved across the species.Since the CRISPR system is reactive to the environment, it might play a critical role in the adaptation of the host to its surroundings and explains the persistence of particular bacterial strains in ecosystems where phages are present (Horvath et al., 2008).
The distribution of CRISPRs in clinical enterococci strains have been studied (Palmer and Gilmore, 2010;Lindenstrauß et al., 2011;Burley and Sedgley, 2012;Hullahalli et al., 2015), however, in non-clinical strains are still poorly studied (Katyal et al., 2013;Lyons et al., 2015).Therefore, the goal of this study was to determine the distribution and frequency of CRISPR1-cas, orphan CRISPR2 and CRISPR3-cas in E. faecalis and E. faecium strains isolated from food and fecal samples, including wild marine animals.

Enterococcus strains
A total of 120 enterococci isolated from food and animal fecal samples were used in this study (Table 1).Among these, 67 were identified as E. faecalis and 53 E. faecium.The enterococci were isolated from different foods samples (beetroot, potato, parsley, raw meat, buffalos milk and dairy products, such as mozzarella cheese), and animal feces (wild fur seals, chickens, wild Magellanic penguins and wild green turtles) in Southern Brazil, from 2009 to 2015.Strains were randomly selected from the culture collection of the Department of Microbiology, Universidade Federal do Rio Grande do Sul.

DNA isolation
For DNA isolation, an aliquot of frozen bacterial cells was recovered in Brain Heart Infusion agar (BHA, Himedia  , India), and incubated at 35 °C for 24 h.Genomic DNA was extracted using the method described by Donato (2007).All strains were confirmed to genus and species by polymerase chain reaction (PCR) assays according to the protocol established by Prichula et al. (2016).The primers sequences and their amplification products are listed in Table 2.

Detection of CRISPR-associated (cas) genes and orphan locus lacking cas genes
Primers for CRISPR amplification reported by Palmer and Gilmore (2010) were used in PCR reaction.The primers and annealing temperatures used are listed in Table 2.The PCR was performed in a total volume of 25 µL containing: 1x PCR buffer (10 mM Tris-HCl [pH 9.0], (Invitrogen, Carlsbad, CA, USA), 1.5 mM of MgCl 2 (Invitrogen, Carlsbad, CA, USA), 200 μM of dNTPs (Ludwig Biotecnologia), 0.4 µM of each primer (Invitrogen, Carlsbad, CA, USA), 1 U of Taq polymerase (Invitrogen, Carlsbad, CA, USA), and 100 ng of genomic DNA.The reaction mixture was subjected to 30 cycles of denaturation at 94 °C for 1 min.;primer annealing for 1 min.(at appropriate temperature), and extension at 72 °C for 1 min., followed by a final extension at 72 °C for 10 min.The PCR products were analyzed on 1.2% agarose gels stained with SYBR safe (Invitrogen, Carlsbad, CA, USA) solution and visualized under UV light.

Sequencing of samples
In order to confirm the CRISPRs amplifications, PCR products amplified of CRISPR1-cas, orphan CRISPR2 and CRISPR3-cas genes were submitted to nucleotide sequence analysis.The DNA fragments were purified using illustra GFX™ PCR DNA and gel band purification kit (GE Healthcare-Buckinghamshire, United Kingdom -UK).
Sequencing was carried out with the Big Dye Terminator Cycle Sequencing Ready Reaction (Applied Biosystems) in an ABI-PRISM 3100 Genetic Analyzer (ABI), according to the protocol of the manufacturer.The sequences obtained were compared with homologous nucleotide sequences deposited in GenBank).

Detection of CRISPRs in enterococci isolates from food and animals fecal samples
The distribution and the frequency of the Type II CRISPRs (CRISPR1-cas, CRISPR2, and CRISPR3-cas) in non-clinical strains of E. faecalis and E. faecium present in Figure 1.The CRISPRs were more frequently detected in E. faecalis than in E. faecium strains.In E. faecalis strains, the CRISPR1-cas and CRISPR2 were detected in 44.77% and 88.07%, respectively.On the other hand, in E. faecium the CRISPR1-cas were observed in 5.66% and CRISPR2 in 32.72% of the strains.Both strains showed low frequencies of CRISPR3-cas genes (8.95% and 1.88%).
The frequency of CRISPRs in E. faecalis and E. faecium strains and source of isolation were related.A higher frequency of CRISPR1-cas and CRISPR2 in non-clinical were observed in E. faecalis isolates from fecal samples, in contrast to food samples.In fecal strains, the CRISPR1-cas was observed in 60% of the E. faecalis strains, whereas in food strains in 13.66%.In relation to CRISPR2, it was positive in 97.11% and 68.18% of the E. faecalis isolated from fecal and food samples, respectively.Similar frequency of CRISPR3-cas gene was detected in both samples.
Identical proportions of CRISPR1-cas and CRISPR3-cas (3.22%) were observed in E. faecium isolated from food products, but a substantially high prevalence of CRISPR2 was observed in E. faecium (48.38%).In addition, equal frequency of CRISPR1-cas and CRISPR2 (9.095%) were observed in E. faecium isolated from animals fecal samples.The CRISPR3-cas amplification was not detected in E. faecium strains.

Discussion
Differences in the distribution and the frequency of the Type II CRISPRs (CRISPR1-cas, CRISPR2, and CRISPR3-cas) in non-clinical E. faecalis and E. faecium were observed.These differences observed in CRISPRS distributions among enterococci species were reported before by Palmer and Gilmore (2010), Lindenstrauß et al. (2011), Katyal et al. (2013), Hullahalli et al. (2015) and Lyons et al. (2015).The incidence of CRISPR1-cas gene in Enterococcus species is recognized; sometimes cas sequences are distinctly different from each other, showing species-level evolution.CRISPR2 loci were detected in high frequency in all isolates evaluated, manly in animal fecal samples.Similar results were identified in other reports that evaluated E. faecalis strains (Palmer and Gilmore, 2010;Hullahalli et al., 2015).Hullahalli et al. (2015) showed that CRISPR2 locus is ubiquitous in E. faecalis.Until today, there are only a few reports evaluating the distribution of CRISPRs in non-clinical enterococci strains, isolated from food and animals fecal samples (Katyal et al., 2013;Lindenstrauß et al., 2011;Lyons et al., 2015).It is the first report of the presence of CRISPRs in E. faecalis and E. faecium isolates from fecal samples of wild marine animals.The high frequency of CRISPRs in E. faecalis strains isolated from animal fecal samples, especially in fecal samples of wild animal could be related with low frequency of antibiotic resistance genes in these strains (Santestevan et al., 2015;Prichula et al., 2016).To fecal strains, the results are partial in agreement with Lyons et al. 2015 andKatyal et al. 2003   CRISPR3-cas.We observed for the first time the presence of this gene in animal fecal samples.
Our results demonstrated a low frequency of CRISPRs Type II in E. faecium strains isolated from animal and food samples.A similar result was observed to Palmer and Gilmore (2010) in clinical E. faecium strains.There is only one study which tested CRISPRs in environmental E. faecium from strains, and the authors did not detect any CRISPRs (Lindenstrauß et al., 2011).So, our data is the first report of the presence of CRISPR1-cas and CRISPR2 in E. faecium isolated from animal feces.
For some enterococcal lineages, CRISPR system has been recognized as a prokaryotic self-defense that provides a type of acquired immunity.Palmer and Gilmore (2010) observed an inverse correlation between CRISPRs presence and antibiotic resistance.In this study, we also observed that strains which contain less antibiotic resistant genes tended to have more cas genes (data not show).
In previous studies from our group evaluating the same strains, was observed in food strains a large number of antibiotic resistance genes (data not published), and in animals strains, mainly wild animals, a low frequency of antibiotic resistance genes (Santestevan et al., 2015;Prichula et al., 2016).
In the conclusion, we detected different proportions and distributions of CRISPRs in E. faecalis and E. faecium isolates from food and fecal samples.The differences in the habitats of enterococcal species influenced this result.The CRISPRs genes were obtained more frequently between E. faecalis strains isolated from fecal samples of animals, highlighting to wild marine animals fecal strains.These strains isolated from wild animals are theoretically under an extremely small selective pressure when compared to strains isolated from food and clinical samples.

Figure 1 .
Figure 1.Distribution of CRISPR systems in non-clinical Enterococcus faecalis and Enterococcus faecium isolated from food and animal fecal samples.

Table 2 .
Primers used in the PCRs carried out in this study.
Lyons et al. (2015)Rs in E. faecalis isolated from fecal specimens.AlthoughLyons et al. (2015)described a high frequency of CRISPR1-cas in Enterococcus spp.isolates from animal fecal samples; CRISPR1-cas was not detected in E. faecalis strains.Instead, Katyal et al. (2013) reported a similar high frequency of CRISPR1-cas (52%) in E. faecalis isolates from pig feces; they have not reported the presence of