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. 2022 Feb 12;100(3):skac030. doi: 10.1093/jas/skac030

Whole genome sequence analyses-based assessment of virulence potential and antimicrobial susceptibilities and resistance of Enterococcus faecium strains isolated from commercial swine and cattle probiotic products

Pragathi B Shridhar 1, Raghavendra G Amachawadi 2,✉, Mike Tokach 3, Isha Patel 4, Jayanthi Gangiredla 4, Mark Mammel 4, T G Nagaraja 1
PMCID: PMC8908542  PMID: 35150575

Abstract

Enterococcus faecium is one of the more commonly used bacterial species as a probiotic in animals. The organism, a common inhabitant of the gut of animals and humans, is a major nosocomial pathogen responsible for a variety infections in humans and sporadic infections in animals. In swine and cattle, E. faecium-based probiotic products are used for growth promotion and gut functional and health benefits. The objective of this study was to utilize whole genome sequence-based analysis to assess virulence potential, detect antimicrobial resistance genes, and analyze phylogenetic relationships of E. faecium strains from commercial swine and cattle probiotics. Genomic DNA extracted from E. faecium strains, isolated from commercial probiotic products of swine (n = 9) and cattle (n = 13), were sequenced in an Illumina MiSeq platform and analyzed. Seven of the nine swine strains and seven of the 13 cattle strains were identified as Enterococcus lactis, and not as E. faecium. None of the 22 probiotic strains carried major virulence genes required to initiate infections, but many carried genes involved in adhesion to host cells, which may benefit the probiotic strains to colonize and persist in the gut. Strains also carried genes encoding resistance to a few medically important antibiotics, which included aminoglycosides [aac(6ʹ)-Ii, aph(3ʹ)-III, ant(6)-Ia], macrolide, lincosamide and streptogramin B (msrC), tetracyclines [tet(L) and tet(M)], and phenicols [cat-(pc194)]. The comparison of the genotypic to phentypic AMR data showed presence of both related and unrelated genes in the probiotic strains. Swine and cattle probiotic E. faecium strains belonged to diverse sequence types. Phylogenetic analysis of the probiotic strains, and strains of human (n = 29), swine (n = 4), and cattle (n = 4) origin, downloaded from GenBank, indicated close clustering of strains belonging to the same species and source, but a few swine and cattle probiotic strains clustered closely with other cattle and human fecal strains. In conclusion, the absence of major virulence genes characteristic of the clinical E. faecium strains suggests that these probiotic strains are unlikely to initiate opportunistic infection. However, the carriage of AMR genes to medically important antibiotics and close clustering of the probiotic strains with other human and cattle fecal strains suggests that probiotic strains may pose risk to serve as a source of transmitting AMR genes to other gut bacteria.

Keywords: antimicrobial resistance genes, cattle, Enterococcus faecium, probiotics, swine, virulence genes, whole genome sequencing

Lay Summary

Probiotics, also called direct-fed microbials, are widely used in swine and cattle production systems, as an alternative for antibiotics. The benefits of feeding probiotic products include growth promotion and gut functional benefits. One of the more common bacterial species used in swine and cattle commercial probiotic products is Enterococcus faecium. The species is also a member of the normal flora of hindgut of humans and animals. In recent years, the species has emerged as a major hospital-acquired infection in humans, mainly because of the propensity to become resistant to antibiotics. In the United States, the species is considered as generally recognized as safe. In this study, the virulence and antimicrobial resistance genes profiles of 9 and 13 E. faecium strains isolated from commercial swine and cattle probiotics, respectively, were assessed by sequencing the whole genome DNA. The analysis indicated that 14 of 22 strains were Enterococcus lactis, and not E. faecium. The absence of major virulence genes characteristic of the clinical E. faecium strains suggests that the strains are unlikely to initiate opportunistic infection. However, the carriage of genes that confer resistance to medically important antibiotics suggests that probiotic strains may pose risk as a source of antimicrobial resistance genes to other bacteria.

Whole genome sequencing of Enterococcus faecium strains isolated from commercial swine and cattle probiotics revealed carriage of genes that confer resistance to medically important antimicrobials.

Introduction

Enterococcus faecium, a lactic acid producer, is one of the more widely used bacterial species, next only to Lactobacillus acidophilus, in the commercial probiotic products of swine and cattle. The reported benefits of feeding E. faecium-based probiotic products include growth promotion and other specific gut functional and health benefits (Franz et al., 2011). In pigs, the gut functional benefits include increased intestinal transport and gut barrier functions and competitive exclusion of pathogens, resulting in reduced neonatal and post-weaning diarrhea in piglets (Pollmann et al., 2005; Scharek et al., 2005; Lodemann et al., 2006; Taras et al., 2006). Feeding of E. faecium-based probiotics has shown to increase milk production during early lactation in dairy cows (Nocek et al., 2003) and control of diarrhea in milk-fed calves (Masucci et al., 2011). The milk production response is attributed to favorable alterations in ruminal fermentation, such as stimulation of ruminal bacterial activities, specifically of lactate-utilizing bacteria resulting in reduced risk of ruminal subacute acidosis, and reduced methanogens and methane production (Ghorbani et al., 2002; Nocek et al., 2002; Masucci et al., 2011; Pang et al., 2014; Mamuad et al., 2019).

Enterococcus faecium and other enterococcal species are hindgut commensals of humans and animals; however, E. faecium has emerged as one of the major nosocomial pathogens in humans (Lee et al., 2019). It is one among the group referred to as “ESKAPE” pathogens (E. faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumanii, Pseudomonas aeruginosa, and Enterobacter spp.), which have the propensity to become resistant to antimicrobial drugs and are responsible for the majority of the nosocomial human infections (Rice, 2008). The E. faecium is responsible for the majority of the Enterococcus health care-associated infections resistant to vancomycin, a critically important antibiotic used to treat a variety of severe Gram-positive bacterial infections in humans (Lee et al., 2019).

Testing for antimicrobial resistance and virulence characteristics of the bacterial probiotic is one of the guidelines implemented by Food and Agriculture Organization-World Health Organization in order to claim a product as probiotic (FAO/WHO, 2002). In Europe, the Food Safety Authority (EFSA) has established that the nature of any AMR of a microorganism should be determined prior to approval of the product as a probiotic for use in animals (EFSA, 2012). Enterococcus faecium contained in human probiotic products has been evaluated for the presence of virulence and antimicrobial resistance genes (Natarajan and Parani, 2015; Ghattargi et al., 2018). In a previous study, we determined the phenotypic susceptibilities and resistance to antimicrobials, detected virulence genes, and assessed genetic diversity based on pulsed-field gel electrophoresis (PFGE) of E. faecium strains isolated from commercial probiotic products used in swine and cattle (Amachawadi et al., 2018). Enterococcus faecium strains from 15 probiotic products (6 swine and 9 cattle) exhibited phenotypic resistance to at least one antimicrobial and a high proportion of strains was resistant to lincomycin, followed by tetracycline, daptomycin, ciprofloxacin, kanamycin, and penicillin.

Whole genome sequencing has become a routine procedure to characterize and predict AMR in bacteria (Zankari et al., 2012; Tyson et al., 2018) and is currently used by the National Antimicrobial Resistance Monitoring System to characterize pathogens that move through the food supply to cause human illnesses (FDA, 2017). The objective of this study was to utilize whole genome sequence-based analyses to characterize virulence and AMR gene profiles and phylogenetic relationships of E. faecium strains isolated from commercial swine and cattle probiotics.

Materials and Methods

Enterococcus faecium strains

Twenty-two E. faecium strains, isolated from commercial probiotic products of swine (n = 9; identified as A to I) and cattle (n = 13: identified as J to V), were used. The isolation, species confirmation, and phenotypic AMR profiles have been reported previously (Amachawadi et al., 2018).

DNA extraction and whole genome sequencing

A single colony of each E. faecium strain grown on the blood agar (Remel Inc., Lenexa, KS) was inoculated into Luria Bertani (LB) broth (Becton and Dickinson, Franklin Lakes, NJ) and incubated on a shaker at 37 °C. Genomic DNA was extracted from overnight cultures using the Qiagen DNeasy blood and tissue kit (Qiagen, Inc., Valencia, CA). The purity of the DNA was determined spectrophotometrically using the Nanodrop (Thermo Scientific, Waltham, MA). Genomic libraries of the strains were constructed using Nextera XT DNA Library Preparation kit (Illumina, Inc., San Diego, CA) and whole genome sequencing was performed on an Illumina MiSeq platform (Illumina, Inc., San Diego, CA) using the MiSeq version 2 reagent kit with 2 × 250 cycles. De novo assembly of the quality-controlled trimmed sequenced reads was performed using the SPAdes genome assembler version 3.8.2 (Bankevich et al., 2012).

Sequence analyses

The draft genome sequences were annotated using RAST (Rapid Annotation using Subsystem Technology; https://rast.nmpdr.org/). The number of genes categorized as those associated with virulence, disease and defense, mobile elements (plasmids, phages, prophages, and transposable elements), membrane transport, iron acquisition and metabolism, and stress response in each strain were determined using RAST (Overbeek et al., 2014). Virulence genes and AMR genes of the draft genomes of E. faecium strains were determined using Virulence Factor Database (VFDB; http://www.mgc.ac.cn/VFs/main.htm; Chen et al., 2005) and ResFinder 3.2 (https://cge.cbs.dtu.dk/services/ResFinder/) (Zankari et al., 2012), respectively. Plasmid sequences were identified using PlasmidFinder 2.1 (https://cge.cbs.dtu.dk/services/PlasmidFinder/; Joensen et al., 2014). The sequence types (ST) of the strains were determined in silico using MLST 2.0 (https://cge.cbs.dtu.dk/services/MLST/; Wirth et al., 2006; Jaureguy et al., 2008).

Phylogenetic analysis

The phylogenetic relationship between sequence types (ST), based on in silico MLST, of the E. faecium strains was determined using PHYLOViZ-2.0 (Nascimento et al., 2017), which implements goeBURST algorithm (Global Optimal eBURST; Feil et al., 2004). The algorithm assigns isolates into clonal complexes (CCs) based on the differences in their allelic profiles. Clonal complexes are defined in three levels (SLV, DLV, and TLV) based on the differences in one or more housekeeping genes. The relationship of the analyzed isolates is represented as an unrooted tree. Additionally, the WGS of E. faecium strains of human (n = 29), cattle (n = 4), and swine (n = 4) origin were downloaded from GenBank (Supplementary Table 1), and their STs were determined and included in the goeBURST analysis.

Parsnp v1.2 (http://harvest.readthedocs.io/en/latest/content/parsnp.html) was used to align the core genomes of E. faecium strains from the present study (n = 22; A to V). Whole genome sequences of E. faecium strains of human (n = 29), cattle (n = 4), and swine (n = 4) origin were also included in the analysis. The alignment of core genomes was followed by the construction of maximum likelihood tree. The phylogenetic tree was subsequently imported to FigTree 1.4.3 software (http://tree.bio.ed.ac.uk/software/figtree/; Rambaut, 2012) for better visualization.

Results

Species confirmation

The 22 probiotic strains from 9 swine and 13 cattle products were confirmed as E. faecium based on the 16S rRNA gene sequence in the draft genome sequences using SpeciesFinder 2.0 (Larsen et al., 2014). The draft genome sequences of all 9 swine probiotic strains and 9 of 13 cattle probiotic strains matched with that of the E. faecium T-110, the reference sequence in the database (a human probiotic strain; GenBank accession no. CP006030). The draft genome sequences of three cattle probiotic strains matched with that of a human E. faecium strain (blood of a hospitalized patient; GenBank accession no. CP011281). The draft genome sequence of one cattle strain (probiotic M) matched with that of an E. faecium strain (GenBank accession no. JX409651), but confidence of the result was reported as fail. In the National Center for Biotechnology Information (NCBI) database, seven of the nine swine probiotic strains (B, C, D, E, F, G, and H) and seven of the 12 cattle probiotic strains (M, N, P, Q, R, T, and U) were identified as Enterococcus lactis (Table 1).

Table 1.

Species identification comparison of Enterococcus faecium strains isolated from commercial swine (n = 9) and cattle (n = 13) probiotic products

Probiotic product code Species identity by the SpeciesFinder1 Species identity from the National Center for Biotechnology Information database
Swine probiotics
A Enterococcus faecium Enterococcus faecium
B Enterococcus faecium Enterococcus lactis
C Enterococcus faecium Enterococcus lactis
D Enterococcus faecium Enterococcus lactis
E Enterococcus faecium Enterococcus lactis
F Enterococcus faecium Enterococcus lactis
G Enterococcus faecium Enterococcus lactis
H Enterococcus faecium Enterococcus lactis
I Enterococcus faecium Enterococcus faecium
Cattle probiotics
J Enterococcus faecium Enterococcus faecium
K Enterococcus faecium Enterococcus faecium
L Enterococcus faecium Enterococcus faecium
M Enterococcus faecium Enterococcus lactis
N Enterococcus faecium Enterococcus lactis
O Enterococcus faecium Enterococcus faecium
P Enterococcus faecium Enterococcus lactis
Q Enterococcus faecium Enterococcus lactis
R Enterococcus faecium Enterococcus lactis
S Enterococcus faecium Enterococcus faecium
T Enterococcus faecium Enterococcus lactis
U Enterococcus faecium Enterococcus lactis
V Enterococcus faecium Enterococcus faecium
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SpeciesFinder at the Center for Genomic Epidemiology (https://www.genomicepidemiology.org/).

Rapid annotation using subsystem technology

Average genome size, no. of contigs, and N50 of swine probiotic strains were 2.72 Mb (2.54–2.92 Mb), 247 (176–423), and 75644 (51101–97268), respectively. For cattle probiotic strains, average genome size, number of contigs, and N50 of cattle probiotic strains are 2.91 Mb (2.52–3.86 Mb), 917 (155–4686), and 33548 (11508–50978), respectively. The functional categories of genes detected in the swine and cattle probiotic strains are shown in Table 2. Based on the RAST subsystem annotation, average number of genes associated with mobile genetic elements (plasmids, phages, prophages, and transposable elements) were 16 (Range = 5–30) and 14 (Range = 5–27) in swine and cattle probiotic strains, respectively (Table 2). Among the plasmid sequences detected, pAMbeta and pIP816 belonging to rep1 family of plasmids were the most common and were present in swine (n = 5) and cattle (n = 6) probiotic strains. Other plasmid sequences detected included pGL (rep29 family), p200B (rep18a family), pRE25 (rep2 family), pKL0018 (rep1 family), DOp2 (repUS1 family), and DOp1 (repUS43 family; data not shown).

Table 2.

Average number of functional categories of genes, based on Rapid Annotation using Subsystem Technology analysis of whole genome sequences of Enterococcus faecium strains isolated from commercial swine (n = 9) and cattle (n = 13) probiotic products

Source Functional categories of genes, Mean (Range)
Virulence, disease, and defense Phages, prophages, transposable elements, and plasmids Membrane transport Iron acquisition and metabolism Stress response
Swine (n = 9) 56 (52–64) 16 (5–30) 56 (48–65) 22 (21–25) 72 (70–74)
Cattle (n = 13) 62 (52–78) 14 (5–27) 57 (49–67) 25 (21–40) 72 (68–78)
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Virulence genes

All 22 strains isolated from swine and cattle probiotic products carried bopD (biofilm on plastic surface), upps (encodes for undecaprenyl pyrophosphate synthase), and cdsA (encodes for phosphatidate cytidylyltransferase) genes. The strains also carried acm (collagen adhesion; seven swine and eight cattle probiotic strains), efa (Enterococcus faecalis antigen A; all swine and 12 cattle strains), ebp (endocarditis- and biofilm-associated pili; ebpA, B, C present only in a few swine and cattle strains), and srtC (sortase C; four swine and 12 cattle strains; Table 3). The prevalence of virulence genes in each of the 22 E. faecium strains isolated from the commercial probiotics is shown in Supplementary Table 2.

Table 3.

Virulence genes identified from whole genome sequencing of Enterococcus faecium strains isolated from commercial swine (n = 9) and cattle (n = 13) probiotic products

Virulence genes Product Function Swine (n = 9) Cattle (n = 13)
Adherence
 acm Collagen adhesin Adherence to collagen 7 8
 efaA Enterococcus faecalis antigen A Adherence to biotic and abiotic surfaces 9 12
 ebpA Endocarditis- and biofilm-associated pili Adherence to host extracellular matrix proteins 4 8
 ebpB 3 9
 ebpC 3 8
 srtC Sortase C Involved in biofilm formation by polymerizing ebp pili 4 12
Biofilm formation
 bopD Biofilm on plastic Sugar binding transcriptional regulator 9 13
Genes for evasion of immune system
 upps Undecaprenyl pyrophosphate synthase Evasion of host immune system 9 13
 cdsA Phosphatidate cytidylyltransferase Evasion of host immune system 9 13
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Antimicrobial resistance genes

The two most prevalent AMR genes were aac(6ʹ)-Ii (21/22 strains), which encodes for acetyl transferase that confers resistance by enzymatic modification of aminoglycoside, and msrC (22/22 strains), which encodes for a ribosomal protection protein and confers resistance to macrolide, lincosamide and streptogramin B (MLS) (Table 4). The other AMR genes detected were aph(3ʹ)-III and ant(6)-Ia) (2/22 strains) that encode for phosphotransferase and nucleotidyltransferase, respectively, and confer resistance by enzymatic modification of aminoglycosides, tet(L) and tet(M) that encode for efflux pump and ribosomal protection protein, respectively, to confer tetracycline resistance (3/22 strains), and cat-(pc194) (2/22 strains) that encode for acetyl transferase, which confers resistance by enzymatic modification of chloramphenicol.

Table 4.

Antimicrobial resistance genes identified from whole genome sequencing of Enterococcus faecium strains isolated from commercial swine (n = 9) and cattle (n = 13) probiotic products

Antimicrobial resistance gene Product/resistance mechanism Gene function Swine (n = 9) Cattle (n = 13)
aac(6ʹ)-Ii 6ʹ-N-aminoglycoside acetyltransferase/Enzymatic modification Aminoglycoside resistance 9 12
aph(3ʹ)-III 3ʹ-aminoglycoside O-phosphotransferase type IIIa/Enzymatic modification Aminoglycoside resistance 0 2
ant(6)-Ia aminoglycoside nucleotidyltransferases/Enzymatic modification Aminoglycoside resistance 0 2
tet(L) Efflux pump Tetracycline resistance 1 2
tet(M) Ribosomal protection protein/Ribosomal protection Tetracycline resistance 1 1
msrC ABC-F ATP-binding protein/Ribosomal protection Macrolide, lincosamide, and streptogramin B resistance 9 13
cat-(pc194) Chloramphenicol acetyl transferase/Enzymatic modification Phenicol resistance 0 2
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Genotypic to phenotypic resistance (Amachawadi et al., 2018) concordance is shown in Table 5. The three swine strains (C, E, and H) and four cattle strains (L, N, S, and U) that were pan susceptible to antimicrobials in NARMS Gram-positive panel (CMV3AGPF) carried an aminoglycoside resistance gene, aac(6ʹ)-Ii, and a MLS resistance gene, msrC. Strains that showed phenotypic resistance to kanamycin, an aminoglycoside (J, K, and V), and lincomycin (B, D, F, G, I, J, K, M, O, T, and V) carried genes related to the resistant antibiotic, aac(6ʹ)-Ii and msrC, respectively. The two strains (J and K) that were phenotypically resistant to erythromycin, a macrolide, carried the msrC gene. Of the three strains phenotypically resistant to tetracycline, only one strain (A) carried the tet(L) and tet(M)genes and the other two did not carry any tet genes. Strains phenotypically resistant to chloramphenicol, ciprofloxacin, daptomycin, and penicillin did not have any relevant AMR genes. The two cattle strains phenotypically resistant to ciprofloxacin were positive for all the AMR genes detected in the probiotic strains, including phenicol resistance gene, cat-(pc194). However, the swine strain A, phenotypically resistant to ciprofloxacin, contained only aac(6ʹ)-Ii) and msrC genes. Three E. faecium strains of cattle probiotics (J, K, and V) that were categorized as MDR because of phenotypic resistance to ≥ 3 classes (phenicols, macrolides, aminoglycosides, lincosamides, Beta-lactams, and tetracyclines) of antimicrobials (chloramphenicol, erythromycin, kanamycin, lincomycin, penicillin, and tetracycline) contained resistance genes for aminoglycoside [aac(6ʹ)-Ii] and MLS (msrC) resistance. The prevalence of AMR genes in all 22 E. faecium strains is shown in Supplementary Table 3.

Table 5.

Phenotypic and genotypic resistance concordance in Enterococcus faecium strains isolated from commercial swine (n = 9) and cattle (n = 13) probiotic products

Phenotypic resistance Product code Resistance breakpoint (μg/mL)1,2 Minimum inhibitory concentration (μg/mL)2 Genotypic resistance
Related Unrelated
Swine probiotics
 None C, E, H aac(6ʹ)-Ii, msrC
 Ciprofloxacin A ≥ 4 4 aac(6ʹ)-Ii, msrC
 Daptomycin A, B, I NA3 16, 16, 8 aac(6ʹ)-Ii, msrC
 Lincomycin B, D, F, G, I ≥ 8 8, 8, 8, 8, 8, msrC aac(6ʹ)-Ii
 Tetracycline A ≥16 32 tet(L), tet(M) aac(6ʹ)-Ii, msrC
Cattle probiotics
 None L, N, S, U aac(6ʹ)-Ii, msrC
 Chloramphenicol J, K, V ≥ 32 32, 32, 32 aac(6ʹ)-Ii, msrC
 Ciprofloxacin P ≥ 4 4 aac(6ʹ)-Ii, aph(3ʹ)-III, ant(6)-Ia,cat-(pc194, msrC, tet(L), tet(M)
 Ciprofloxacin Q ≥ 4 4 aac(6ʹ)-Ii, aph(3ʹ)-III, ant(6)-Ia,cat-(pc194),msrC, tet(L)
 Ciprofloxacin R ≥ 4 4 aac(6ʹ)-Ii, msrC
 Daptomycin O NA2 8 aac(6ʹ)-Ii, msrC
 Erythromycin J, K ≥ 8 8, 8 msrC aac(6ʹ)-Ii, msrC
 Kanamycin J, K, V ≥ 1,024 1,024, 1,024, 1,024 aac(6ʹ)-Ii msrC
 Lincomycin J, K, M, O, T, V ≥ 8 8, 8, 8, 8, 8, 8 msrC aac(6ʹ)-Ii, msrC
 Penicillin J ≥ 16 16 aac(6ʹ)-Ii, msrC
 Tetracycline J, K, V ≥ 16 32 aac(6ʹ)-Ii, msrC
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Breakpoints established by the Clinical Laboratory Standards Institute.

NA, Not applicable. A susceptibility breakpoint of ≥ 4 μg/mL for daptomycin exists but no resistant breakpoint has been established. In this study, isolates with minimum inhibitory concentration of ≥ 8 μg/mL were considered as resistant.

Sequence types

Enterococcus faecium strains isolated from swine probiotics belonged to six sequence types (ST1, 94, 160, 178, 296, 1513) and those isolated from cattle probiotics belonged to seven STs (94, 160, 178, 296, 611, 696, 1433; Table 6). The genomes of six strains carried novel alleles; hence, the nearest ST assigned by the MLST database (MLST 2.0) is reported (Table 6).

Table 6.

In silico multi-locus sequence typing of Enterococcus faecium strains isolated from commercial swine (n = 9) and cattle (n = 13) probiotic products

Sequence type (ST) Swine (n = 9) Cattle (n = 13)
1 11 0
94 1 2
160 1 3
178 21 11
296 2 2
611 0 21
696 0 2
1433 0 11
1513 2 0
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Contains novel alleles, hence the nearest STs were reported.

Phylogenetic relationship

The goeBURST analysis of the 59 E. faecium strains representing 36 STs revealed 18 clonal complexes (CC). Three major CCs were identified, which included the majority of the STs (19/36), with CC0 being the most common CC with 11 ST. Enterococcus faecium strains isolated from cattle probiotics with STs 160 and 611 belonged to CC1, which also clustered with strains of human (ST21, ST1054) and cattle origin (ST32). Enterococcus faecium strains isolated from cattle and swine probiotics with STs 94 and 178 belonged to CC3, which also clustered with a human strain (ST361). The remaining E. faecium strains of swine and cattle probiotics (ST1, ST296, ST696, ST1433, and ST1513) existed as singletons; they were clonally different and did not share their allelic profiles with other strains. Similarly, other strains of human, cattle, and swine origin belonging to different STs (ST19, ST54, ST156, ST214, ST218, ST430, ST736, ST904, and ST955) also existed as singletons (Figure 1).

Figure 1.

Figure 1.

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geoBURST analysis of 67 Enterococcus faecium strains representing 36 Sequence Types (ST). Numbers within the circles represent STs, size of each circle is proportional to the number of isolates within each ST. Different colors represent the source of E. faecium strains; Red: Human strains; Blue: Cattle probiotic strains, Green: Swine probiotic strains; Purple: Cattle strains, Brown: Swine strains. Black link—drawn without recourse to tiebreak rules; Blue link—drawn using tiebreak rule 1 (number of single locus variants (SLV); Yellow link—drawn using tiebreak rule 4 or 5 (frequency found on the data set and ST number, respectively).

Phylogenetic analysis of E. faecium strains, based on core genome, revealed close clustering of the strains of the same source, with a few exceptions. One of the strains isolated from cattle probiotics (Probiotic S) clustered with a strain isolated from human feces (Accession no. LN999844). The seven swine probiotic strains and seven cattle probiotic strains identified as E. lactis, based on NCBI database, two human fecal strains (Accession nos. CP025685 and CP040878) and a cattle strain (MJDY01) clustered separately (Figure 2).

Figure 2.

Figure 2.

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Phylogenetic tree of E. faecium strains isolated from cattle and swine probiotic products (strains from the present study—A to V), and whole genome sequences downloaded from GenBank, using Parsnip v1.2 and visualized using FigTree 1.4.3. The colors of the branch tip labels indicate the source of the strains (Red: Human strains; Blue: Cattle probiotic strains; Green: Swine probiotic strains; Purple: Cattle strains; Brown: Swine strains).

Data availability

The whole genome sequences of the 8 strains of E. faecium and 14 strains of E. lactis have been deposited in NCBI GenBank under the BioProject number PRJNA746973 (https://www.ncbi.nlm.nih.gov/bioproject/?term=PRJNA746973).

Discussion

In this study, we determined the virulence and AMR genes profiles and phylogenetic relationships of 22 strains isolated from E. faecium-based commercial swine and cattle probiotics. Although Species-Finder database, based on 16S rRNA gene sequence, confirmed all 22 as E. faecium, the NCBI, which is a more updated database, identified seven swine (B, C, D, E, F, G, and H strains) and seven cattle strains (M, N, P, Q, R, T, and U) as E. lactis, and not as E. faecium. Enterococcus lactis was first isolated from Italian raw milk cheeses and was identified as a novel species based on 16S rRNA gene sequence analysis (Morandi et al., 2012). The isolates were closely related to E. hirae, E. durans, and E. faecium with 98.8%, 98.9%, and 99.4% similarity in 16S rRNA sequence, respectively. In addition to 16S rRNA sequence differences, the two species, E. faecium and E. lactis, can be differentiated by 16S-23S internal transcriber speacer analysis and phenotypically by sugar fermentations (Morandi et al., 2012). Historically, the known strains of E. faecium are divided into two clades, clade A, containing the hospital-associated strains, and clade B, containing the community-associated strains. The clade A is further divided into two sub-clades A1 and A2 to include clinical isolates in A1 and animal-associated strains in A2 (Belloso Daza et al., 2021). It is suggested that the split into two sub-clades likely happened after the introduction of antibiotics in human and animal settings, approximately 75 yr ago (Lebreton et al., 2013). Although 16S rRNA gene sequence is considered generally as a standard for bacterial taxonomy, variability in the number of copies and variability within the many copies of the 16S rRNA gene sequences, which may preclude accurate species identification, are not uncommon phenomenon in bacteria (Ibal et al., 2019; Belloso Daza et al., 2021). However, WGS analysis, which is becoming a more widely used tool, pangenome analysis, provides a more definitive method for microbial taxonomy. Belloso Daza et al. (2021) investigated the taxonomic relationship among strains of E. faecium of different origins and E. lactis using WGS and concluded that clade B strains differed from E. faecium clades A1 and A2 and should be reamed as E. lactis (Belloso Daza et al., 2021).

In the United States, the list of microbial species used as probiotics in animal feeds, more commonly called direct-fed microbials, is published in the Association of American Feed Control Official Manual and the list includes six species of Enterococcus (Feedstuffs.com., 2021). The six species, including E. faecium, have a “Generally Recognized as Safe” (GRAS) status. However, in Europe, according to the Qualified Presumption of Safety list from the European Food safety Authority (EFSA), the genus Enterococcus does not have the “GRAS” status (Ogier and Serror, 2008; Hanchi et al., 2018) because of the safety concerns of potential virulence and the propensity to acquire and transfer AMR.

Species of Enterococcus are ubiquitously distributed in the environment, primarily inhabiting human and animal gastrointestinal tracts, but also occur in soil, water, variety of foods, and on plants and insects (Franz et al., 2011). Enterococcus faecium is also a widely used bacterial species in probiotic products of humans to promote health, and in animals to promote performance (growth in swine and milk production in dairy cows) and health. The documented health benefits in animals include reduced risk of ruminal subacute acidosis in cattle and reduced incidence of diarrhea in piglets and baby calves (Franz et al., 2011). In children, clinical studies have shown that E. faecium-based probiotic products reduced the severity of diarrhea and length of the hospital stay (Chen et al., 2010).

Enterococcus species, particularly faecium and faecalis, are nosocomial pathogens that cause bacteremia, endocarditis, urinary tract infections, intra-abdominal and pelvic infections, and even death, more often in persons with serious underlying diseases and or are immunocompromised (Murray, 1990; Hoge et al., 1991). The emergence of E. faecium as a nosocomial pathogen was initially thought to be due to acquisition of AMR; however, AMR alone does not explain virulence. The major virulence factors of E. faecium, which contribute to the pathogenicity, include adhesins to mediate adherence and colonization, promote cell clumping and biofilm formation, proteolytic enzymes to degarde host proteins, and a cytolysin, which enhances virulence of pathogenic strains (Franz et al., 2011; Arias and Murray, 2012).

Enterococcus faecium contained in human probiotic products has been evaluated for their safety and efficacy with regard to health (Natarajan and Parani, 2015; Ghattargi et al., 2018). A couple of strains of E. faecium, T-110 and LBB.B1, which are contained in several commercial products, have been whole genome sequenced to assess safety. The analysis of T-110 has revealed the absence of most of the genes related to virulence and AMR and presence of a few adhesion genes (Natarajan and Parani, 2015). Noguchi et al. (2011) compared virulence genes and antimicrobial susceptibilities of clinical E. faecium strains with strains isolated from six human probiotic products and concluded that none of the probiotic strains contained any of 13 virulence genes assayed and clinical strains were resistant to levofloxacin, a fluorquinolone, whereas all probiotic strains were phenotypically susceptible (Noguchi et al., 2011).

None of the 22 probiotic strains carried major virulence genes, asa1, gelE, cylA, esp, and hyl, generally carried by pathogenic E. faecium strains (Fisher and Phillips, 2009; Lee et al., 2019). In our previous study, we used a multiplex PCR assay to report the absence of these genes in the probiotic strains (Amachawadi et al., 2018). Although 22 probiotic strains carried bopD gene, none carried the fsrABC operon, which controls the expression of the bopD gene (Bourgogne et al., 2006). The non-functional bopD gene due to lack of the fsrABC operon has been previously reported in human probiotic strains of E. faecium (Natarajan and Parani, 2015; Ghattargi et al., 2018). Similarly, the acm gene, which encodes for an adhesin that binds to the host collagen in seven of the 15 acm-positive strains, is likely to be non-functional due to the presence of non-sense mutation. Probiotic strains of E. faecium have been reported to carry non-functional acm genes (Natarajan and Parani, 2015; Ghattargi et al., 2018; Urshev and Yungareva, 2021). All 22 strains carried upps (encodes for undecaprenyl pyrophosphate synthase) and cdsA (encodes for phosphatidate cytidylyltransferase) genes, which play a role in the evasion of immune system. A number of strains carried several genes (acm, efaA, ebpA, ebpB, ebpC, srtC, and bopD) involved in adhesion to host proteins and other biotic and abiotic surfaces and in biofilm formation (Franz et al., 2011). Although adhesion is a critical process in an infectious process, adherence is also one of the main criteria for the selection of potential probiotics. Adherence to the gut epithelial cells and subsequent colonization extend the persistence of probiotic strains in the intestinal tract (Ouwehand et al., 1999; Ferreira et al., 2011). Investigation on the presence of virulence genes and their contributions to virulence in enterococci from several sources has shown that the occurrence of individual virulence factors is strain specific (Abriouel et al., 2008; Franz et al., 2011). More often, the strains become virulent by acquiring specific virulence-associated genes via mobile genetic elements (Leavis et al., 2007; van Schaik et al., 2010), possibly increasing their fitness to adapt to the animal or human host. For example, a E. faecium genetic lineage that developed and spread globally is hospital-adapted (CC 17) because of acquisition of a large pathogenicity island of > 60 kbp, esp gene, vancomycin-resistance gene, and acm gene (van Schaik et al., 2010). Studies have shown separate clustering of and distinct genomic differences between clinical and nonclinical E. faecium strains (Kim and Marco, 2014; Beukers et al., 2017). Genomes of clinical strains are significantly larger than non-clinical strains because of acquisition of mobile genetic elements, virulence, and AMR genes (Kim and Marco, 2014).

Because E. faecium is Gram-positive, it is intrinsically resistant to low levels of aminoglycoside (Moellering and Weinberg, 1971). The presence of aac(6ʹ)-Ii, which encodes for 6ʹ-N-aminoglycoside acetyltransferase that cleaves the 6ʹ-amino group of aminoglycoside antibiotics, in all 22 strains of E. faecium suggests moderate level of aminoglycoside resistance (Chow, 2000). Additionally, two cattle strains (P and Q) possessed aph(3ʹ)-III and ant(6)-Ia genes that encode for aminoglycoside modifying enzymes, 3ʹ-aminoglycoside O-phosphotransferase type IIIa and aminoglycoside nucleotidyltransferases, respectively, which allow the organisms to resistant to high concentrations of aminoglycosides (Costa et al., 1993). However, both strains were phenotypically susceptible to kanamycin (MIC < 1.024 µg/mL), suggesting that the genes were likely non-functional. The comparison of the phenotypic susceptbility and resistance to genotypic data showed presence of both related and unrelated genes in the 22 probiotic strains. The strains that were phenotypically resistant to kanamycin and lincomycin carried AMR genes related to the antibiotic class. However, the seven strains pan-susceptbile to NARMS Gram positive panel contained two AMR genes, aac(6ʹ)-Ii and msrC, which likely means that they were nonfunctional. The strains that were phenotypically resistant to chloramphenicol, ciprofloxacin, daptomycin, and penicillin did not have related AMR genes. It is possible that the organisms had nonspecific efflux pumps. Tyson et al. (2018) have compared phenotypic resistance with genotypic data, derived from WGS of 100 E. faecium strains from animal and food sources, and have reported high degree of concordance for the 11 antibiotics tested (Tyson et al., 2018).

Over the years, the public health implication of vancomycin-resistant E. faecium in human clinical strains is well recognized (Treitman et al., 2005; Deshpande et al., 2007). None of the probiotic strains in our study carried genes for vancomycin resistance. However, they carried genes encoding resistance for other major clinically important antibiotics (aminoglycosides, tetracyclines, macrolides, lincosamide, streptogramin B, and phenicol). The WGS analysis of a probiotic strain E. faecium LBB.E81, another human probiotic strain, revealed the presence of aac(6ʹ)-Ii and msrC (Urshev and Yungareva, 2021).

Swine and cattle probiotic E. faecium strains belonged to diverse sequence types. Some of the STs have been previously reported in E. faecium strains from various sources (ST696 from human clinical case and waste water, ST94 from human clinical case and waste water, ST296 from waste water, ST178 from waste water, and ST160 in National collection of type culture; Gouliouris et al., 2018). Phylogenetic analysis revealed close clustering of E. faecium strains belonging to the same source with a few exceptions. Some of the 22 probiotic strains clustered closely with cattle and human fecal strains, which suggest movement of E. faecium strains across different species and the ability of the strains to adapt to various ecological niches. Swine and cattle probiotic strains identified as E. lactis clustered separately along with two human fecal (CP025685) and CP040878) and one cattle fecal (MJDY01) strains. The two human and one cattle fecal strains are now listed as E. lactis in the updated NCBI database.

In conclusion, 14 of the 22 probiotic strains were identified as E. lactis, not as E. faecium. None of the strains investigated carried any of the major virulence genes characteristic of the clinical E. faecium strains, suggesting that these probiotic strains are unlikely to initiate opportunistic infection. However, a number of strains carried several genes involved in adhesion to host proteins and other biotic and abiotic surfaces, which may be a beneficial feature for probiotic strains. Close clustering of the probiotic strains with other human and cattle fecal strains, and the presence of AMR genes suggest the potential of these strains to survive in different ecological niche and transfer AMR genes to medically important antimicrobials to other gut bacteria. The study also illustrates the utility of WGS in assessing the safety of probiotic strains.

Supplementary Material

skac030_suppl_Supplementary_Tables
Click here for additional data file. (34KB, docx)

Acknowledgments

This is contribution no. 22-021-J from the Kansas Agricultural Experiment Station, Manhattan, KS. The study was supported in part by a grant from the National Pork Board (Award # 15-024). The funders had no role in the study design, data collection and analyses, preparation of the manuscript, or decision to publish.

Glossary

Abbreviations

AMR

antimicrobial resistance

CC

clonal complex

DNA

deoxyribonucleic acid

EFSA

European Food Safety Authority

FAO-WHO

Food and Agriculture Organization-World Health Organization

FDA

Food and Drug Administration

GRAS

Generally Recognized as Safe

LB

Luria Bertani

MLST

multi-locus sequence typing

NCBI

National Center for Biotechnology Information

PFGE

pulsed-field gel electrophoresis

RAST

Rapid Annotation Using Subsystem Technology

rRNA

ribosomal ribonucleic acid

ST

sequence type

VFDB

virulence factor database

WGS

whole genome sequencing

Conflict of Interest Statement

None of the authors has any conflict of interest with the publication of the study.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

skac030_suppl_Supplementary_Tables
Click here for additional data file. (34KB, docx)

Data Availability Statement

The whole genome sequences of the 8 strains of E. faecium and 14 strains of E. lactis have been deposited in NCBI GenBank under the BioProject number PRJNA746973 (https://www.ncbi.nlm.nih.gov/bioproject/?term=PRJNA746973).

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