Phenotypic and genotypic characterization of Enterococcus cecorum strains associated with infections in poultry

Background From the beginning of the 21st century Enterococcus cecorum has emerged as a significant health problem for poultry raised under intensive production systems. To obtain new insights into this bacterial species, we investigated 82 clinical isolates originating from different poultry flocks in Poland between 2011 and 2014. Results Phenotypically, isolates from clinical cases showed ability to growth at low temperatures (4 °C, 10 °C), and differences in growth at 45 °C (74.4 %). Survival at high temperatures (60 °C, 70 °C) was observed for 15, 30 min. More than half of strains survived at 60 °C even after prolonged incubation (1 h), but none survived after 1 h at 70 °C. Total growth inhibition was observed on agar supplemented with tergitol or potassium tellurite. Relatively high number of isolates gave positive reactions for β-galactosidase (βGAL 80 %), Voges Proskauer test (60 %), less for β-mannosidase (17 %), glycogen and mannitol (12 %). The metabolic fingerprinting for E. cecorum obtained in Biolog system revealed ability to metabolise 22 carbon sources. Only 27/82 strains contained ≥ 1 virulence genes of tested 7, however 2.4 % isolates carried 6. Increased antimicrobial resistance was observed to enrofloxacin (87 %), teicoplanin (85 %), doxycycline (83 %), erythromycin (46 %). Most strains (75/82) showed multidrug resistance. The single isolate was resistant to vancomycin (VRE) and high level gentamicin (HLGR). Linezolid resistance among clinical isolates was not found. PFGE revealed diversity of E. cecorum from cases. It could be assumed that transmission of pathogenic strains between flocks regardless of type of production or geographical region may be possible. Conclusions Clinical infections in poultry caused by E. cecorum may indicated on new properties of this bacterial species, previously known as a commensal. Despite many common phenotypic features, differences were found among clinical isolates. Several, widely distributed pathogenic E. cecorum strains seemed to be responsible for infection cases found in different poultry types. Electronic supplementary material The online version of this article (doi:10.1186/s12917-016-0761-1) contains supplementary material, which is available to authorized users.


Background
First time Enterococcus cecorum was isolated from cecal flora of chickens and described as Streptococcus cecorum in 1983, thereafter well known as commensal in gastrointestinal tract of various mammals and birds [1]. On the other hand, Enterococcus cecorum belongs to opportunistic pathogens and may also play a role as etiological agent of diseases in humans (nosocomial infections) [2,3], chickens [4], and racing pigeons [5]. Recently, this bacteria appears to be a new threat ("emerging pathogen") to poultry industry worldwide [6][7][8][9][10][11][12][13][14][15]. E. cecorum has been increasingly recognized as a cause of enterococcal spondylitis (ES), previously called enterococcal vertebral osteoarthritis (EVOA) in chickens [12]. Disease outbreaks were diagnosed mostly in broiler chicken flocks raised under an intensive production system. Clinically affected birds suffered from locomotor problems due to compression of the spinal cord at the thoracic vertebrae resulting from E. cecorum induced osteomyelitis and due to femoral head necrosis (FHN) [6,7,9,12,13]. Disease outbreaks can lead to high morbidity, mortality, culling, carcass condemnations, and may result in severe economic losses within a short time [9].
Recently, poultry or domestic animals (cats, dogs) are thought to be a possible source for transmission leading to E. cecorum-associated septicaemia in humans [2,3].
Various methods using conventional biochemical tests and molecular techniques have been commonly used for identification and typing enterococci [16][17][18]. Pulsed field gel electrophoresis (PFGE) is considered to be the "gold standard" for subtyping enterococci and has been used extensively for molecular epidemiological characterization of enterococcal outbreaks [19,20]. The PCR assay based on specific amplification followed by sequencing and nucleotide sequence comparison of target genes (such as 16S ribosomal RNA, sodA, ddl, tuf, groESL) or tDNA-PCR have served for the genotypic identification of enterococci [21][22][23].
Despite of available literature biochemical and molecular analysis of E. cecorum strains with poultry origin isolated in Europe are still limited. Moreover, there is not enough data regarding the properties of isolates, usually referred as pathogenic for poultry [1,7,8,10]. The purpose of this study was phenotypic characterization of clinical E. cecorum isolates associated with infections in poultry and investigation their genetic relatedness.

Bacterial isolates
Eighty two E. cecorum isolates of poultry-origin used in this study were obtained from archival bacterial collection deposited at Department of Pathology and Veterinary Diagnostics, or were obtained from clinical specimens submitted by veterinarians for routine diagnostic work to the Diagnostic Laboratory in Division of Avian Diseases, Faculty of Veterinary Medicine at the Warsaw University of Life Sciences-SGGW (Poland). Authors ensure that the ARRIVE guidelines were followed. Among 82 clinical strains collected between 2011 and 2014, 49 came from broiler chicken flocks (CB), 20 from broiler breeder flocks (BB), 10 from commercial layer flocks (CL), 2 from geese flocks (G) and 1 from turkey flock (T). According to adopted criteria in this study, one E. cecorum isolate represented one different flock in which clinical problems due to E. cecorum infection were reported by veterinarians on farms. Affected birds displayed a variety of clinical signs, however in all types of flocks the lameness, paralysis, hock sitting, weakness, pododermatitis, decreased water and food intake were usually noted. Subsequently, disease caused lower results of production, increased losses due to mortality and culling. Necropsies and pathological examinations revealed usually femoral head necrosis, (purulent) arthritis, fibrinous pericarditis, endocarditis, hepatitis and congested lungs. Characteristic osteomyelitis lesions at caudal thoracic vertebrae we found only in chicken flocks (mainly in CB). Isolates were recovered from tissue samples such as vertebral column, femoral heads, heart, liver, lungs or yolk sac, which were collected during necropsy.

Bacterial analysis
The tissue samples were inoculated onto Columbia agar with 5 % sheep blood (CA) (Graso, Poland) and agar plates with esculin (KAA, Biocorp, Poland; Enterococcosel Agar, Graso, Poland), then incubated at 37°C for 24 h in a CO 2 -enriched atmosphere. Bacteria were identified as Enterococcus based on their phenotypic properties such as colonial morphology, hemolysis (on CA), Gram-staining, catalase production (using a 3 % H 2 O 2 ), cytochrome oxidase production (OXItest, Erba Lachema s.r.o., Czech Republik), and esculin hydrolysis (Enterococcosel Agar, KAA). Pigment production was visually assayed by growing the bacteria on CA for 24 h and scraping off the growth with a white cotton swab. Motility was examined using Motility Test Agar (Graso, Poland). The ability to growth was estimated in 6.5 % NaCl (salt tolerance test) after 48 h at 37°C, and on different media (Graso, Poland) ( Table 2). Serological identification of Lancefield group was conducted by rapid latex agglutination method using Slidex Strepto Plus D (bioMérieux, France). Tests for E. cecorum growth were performed in BHI broth (Brain-heart infusion; bio-Mérieux, France) tubes preincubated at 4°C, 10°C, 45°C for 24 h. Then cultures in BHI broth were spread onto CA and incubated at 37°C. The growth response was assessed after 24 h and 48 h. The ability to survive at 60°C, 70°C was estimated for 15 min, 30 min, 1 h in BHI broth tubes, followed by incubation of inoculated CA plates. The results were recorded after 24 h and 48 h.

Biochemical tests
Identification to the species level based on biochemical characterization was performed by API rapid ID 32 STREP (bioMérieux, France) and on the basis of carbon source utilisation using Biolog system (Biolog Inc., Hayward, USA). Isolates (n = 13) were determined according to Biolog GP2 MicroPlates, which performed 95 discrete tests simultaneously and gave a characteristic reaction pattern (metabolic fingerprint). The MicroPlates were incubated at 37°C and read visually after 4 h and 24 h. The metabolic fingerprint patterns were compared and identified using the MicroLog™ 4.20.05 database software.
Production of gelatinase was additionally determined using Difco Nutrient Gelatin (BD, USA) according to the manufacturer's recommendations. The tubes inoculated with E. cecorum ATCC 43198, S. aureus ATCC 25923 (gelatinase positive), E. coli ATCC 25922 (gelatinase negative) and an uninoculated tube were used for quality control testing.

Molecular identification
Rapid extraction of bacterial genomic DNA was carried out by using boiling method. PCR assay targeting sodA gene was performed for identification and determination the diversity of 82 E. cecorum strains [22]. PCR products were visualized after electrophoresis on agarose gel (2 %) by staining with ethidium bromide, then purified using GeneMATRIX PCR/DNA Clean-Up Purification Kit (EURx, Poland) and submitted for sequencing to commercial services (IBB PAN, Genomed, Poland). The sodA gene sequences were analyzed with NCBI BLAST. The genetic distances based on the partial sequences of sodA was calculated by the two-parameter method of Kimura by using the MEGA6, and the phylogenetic tree was constructed using the Neighbor-Joining method (NJ) with 1000 bootstrap replicates.

PFGE
The standard PFGE procedure was adapted from previously published studies with minor modifications [18,26,27]. The 82 E. cecorum strains were cultured overnight on CA and then suspended in sterile saline to obtain the density of 3.5 on McFarland scale and centrifuged 10 min. at 4000 rpm/min. The bacterial pellets were mixed with 150 μl Tris-EDTA buffer solution (10 mM Tris-HCl, 1 mM disodium EDTA, pH 8.0) and 150 μl liquid 2 % agarose (InCert Agarose, Lonza, Rockland, USA) and small discs were formed (20 μl). The solidified discs were incubated at 37°C for 18 h in 1 ml of EC buffer (6 mM Tris-HCl pH 8.0, 1 M NaCl, 0.1 M EDTA, 0.2 % deoxycholate, 0.2 % sarkosyl) containing 10 mg lysozyme (A&A Biotechnology, Poland), and 0.02 mg RNase A (Thermo Fisher Scientific Inc., USA). DNA discs were washed 3 times in 5 ml EBS solution (0.5 M EDTA pH 9.0, 1 % sarkosyl) and incubated overnight at 50°C in 1 ml EBS solution containing 1 mg of proteinase K (ESP buffer) (A&A Biotechnology, Poland). Then the discs were washed 4 times (each time upside down for 30 times at room temperature) with 10 ml TE buffer (10 mM Tris, 1 mM EDTA, pH 8.0) and stored in 1 ml TE buffer at 4°C. Subsequently, each disc was preincubated in 100 μl restriction buffer for 30 min at room temperature. The agarose discs were digested with SmaI (20 U/μl; Fermentas, Lithuania) overnight (at 37°C). The restriction fragments were separated by clamped homogenous electric field (CHEF) electrophoresis with a CHEF-DR II System (Bio-Rad Laboratories, USA) in a 1.2 % (w/v) agarose gel using pulse time at 0.5 s followed by 35 s at 6 V/cm and temperature 14°C for 24 h [17]. Afterwards the gel was stained with ethidium bromide for 30 min, then washed in distilled water for 30 min, photographed under UV light and documented in the system VersaDoc (Bio-Rad Laboratories, USA). Lambda Ladder PFG marker (New England Biolabs Inc., USA) was used as molecular size marker. Gel images were analyzed by Gel Compar II version 6.6 (Applied Maths, Belgium) and cluster analysis was performed by UPGMA using dice similarity coefficient with optimization set at 1 % and position tolerance at 1 %. Isolates were clustered using an 80 % homology cut-off, above which strains were considered to be closely related and assigned to the same PFGE type [19]. Table 1 shows results of conventional tests and effects of different temperatures on the growth and survival of E. cecorum strains. Bacterial growth was characterized on 7 different microbiological media (Table 2).

Antibiotic susceptibility
One (0.82 %) out of the 82 clinical E. cecorum was susceptible to 13 antibiotics tested, the rest were resistant to one or more antibiotics (Table 4). All isolates were susceptible to amoxicillin/clavulanic acid (AUG) and

Molecular identification
The obtained sequences sodA gene fragment showed similarity to E. cecorum (BLAST database) and allowed for identification strains. Dendrogram showed the genetic similarity between reference strain of E. cecorum and clinical isolates based on the sodA gene sequences (Fig. 2). Phylogenetic analysis supported the separation of clinical isolates into three main groups (A, B, C). Genetic distances between groups ranged from 0.00 to 0.04 ( Table 5). The group A comprised 69 strains (CB n = 43, BB n = 15, CL n = 10, G n = 1) and had one subgroup (A') with 5 strains (BB n = 4, CB n = 1). Five CB isolates were clustered together in the group B, and three isolates (BB, G, T) in the group C (all isolates from 2014). Among all groups, the group C revealed the highest values of genetic distance with B group (0.04) and with reference strain (0.03).

Discussion
In order to characterize clinical E. cecorum, we investigated 82 strains isolated from clinical samples originated from different poultry flocks (1 isolate per flock). Our observations were consistent with reports on a succession of disease outbreaks in broiler flocks raised in the intensive farming systems [9]. Previously, clinical E. cecorum was not described in commercial chicken layers or geese flocks. We found that the problem may affect hens or other bird species than chicken. Our results were consistent with the literature in regarding on certain characteristics traditionally considered to be typical for the genus Enterococcus or E. cecorum including intestinal isolates of poultry origin [28,29]. According to the literature, E. cecorum are often NaCl sensitive [17,30], and intestinal E. cecorum of poultry may be also NaCl-resistant [28]. In our study, clinical isolates appeared to be less salt-tolerant, however no complete inhibition of growth was observed. Authors suggested possible higher ability to survive clinical E. cecorum in saline environment or even higher resistance to chlorine-based disinfectants. Previous research demonstrated no growth of poultry cecal E. cecorum on Slanetz medium, and on KAA agar [1], while clinical strains showed variable growth on these media. The growth was clearly more abundant on bile esculine azide agar than on esculin azide agar with kanamycin. Based on results, we suggested that complete growth inhibition on a solid medium supplemented with tergitol or with potassium tellurite may be used in identification of this enterococcal species. According to the literature, Enterococcus species are able to survive a range of stresses and hostile environments [31], but E. cecorum was described as unable to grow at 10°C or survive 30 min at 60°C [5,29]. In contrast to above authors, clinical isolates were able to grow at low temperatures (4°C, 10°C) and some of them might survive even longer heating at 60°C for 1 h and even 70°C for  We confirmed the efficacy of two biochemical systems for identification poultry-origin E. cecorum strains. Instead of doubtful or unacceptable profile in API, all strains were properly recognized by sodA gene sequencing. We found, that almost all clinical strains gave positive reactions in 10 biochemical tests, and negative in 5 tests (API). Similar results were reported for other E. cecorum including commensal or reference strains with some exceptions [1,17,28,32]. We observed that all of strains were able to metabolise α-cyclodextrin. Makrai et al. [10] observed differences among clinical isolates in metabolism of both αand β-cyclodextrin. We noted relatively high positive reactions for βGAL, βGAR, VP, opposed to reference E. cecorum strain and despite the discrepancies in the literature [17]. In contrast to other studies [2,17], some clinical E. cecorum showed ability to produce urease, β-mannosidase, and metabolize glycogen. On the other hand, results for βmannosidase, glycogen, mannitol were lower for clinical isolates than reported for standard strains. Our results were consistent with Borst et al. [33] who noted that pathogenic E. cecorum isolates are more deficient in mannitol metabolism. Recently molecular aspects for the defect mannitol metabolism in pathogenic strains were investigated [34]. Based on comparative analysis of our results with study of Makrai et al. [10], it could be assumed, that all clinical E. cecorum may metabolise 18 carbon sources (adenosine, arbutin, D-cellobiose, dextrin, Dfructose, D-mannose, D-psicose, D-raffinose, D-trehalose, gentiobiose, maltose, maltotriose, N-acetyl-D-glucosamine, N-acetyl-β-D-mannosamine, pyruvic acid methyl ester, salicin, sucrose, α-D-glucose). Similarly to above mentioned authors, clinical isolates may give differences in 3 tests: α-D-lactose, D-ribose, 2deoxy adenosine.
Recently several potential mediators of virulence were found in pathogenic E. cecorum isolated from chickens in the southeast US. These virulence determinants conserved in pathogenic EC were found to be similar to those utilized by other medically important enterococci [33]. In the present study, only 32.9 % clinical E. cecorum strains contained one or more virulence genes. E. cecorum from chicken flocks contained mainly asa1/ gelE/ace genes. The pathogenicity of E. cecorum may be associated with other species-specific virulence factors. Similar observations were presented by Jackson et al. [35] who detected only few virulence genes among US E. cecorum isolates, and the incidences of virulence determinants tested were lower than ours. In our study the most of positive isolates contained two asa1/gelE or four asa1/gelE/efaA/ace virulence genes. We speculated about possible linkage between asa1/gelE (74 % of all virulence positive isolates) or efaA/ace (33.3 % of all virulence positive isolates) in clinical E. cecorum. It may have impact on pathogenesis and clinical course of infection.
Because none of the investigated strains harbored hyl gene, we suggest that this virulence determinant may be not widespread among clinical isolates. Our results were consistent with other authors who described the lack of hyl in E. cecorum from poultry carcass rinsates, diseased chickens [35] and pigeons [5]. According to the literature, hyaluronidase is a degradative enzyme that is associated with tissue damage. Among Enteroccocus species the hyl gene has been reported more often in ampicillinresistant VRE E. faecium isolates [36]. We suggest that   hyl is not specific for E. cecorum and could has minor role in pathogenicity of E. cecorum, however more studies are needed to elucidate this aspect. The present study showed lack of correlation between the presence of gelE gene and its expression. The literature provide no data in regard this aspect on E. cecorum, however similar observations are available for E. faecalis [37].
Generally, pathogenic isolates from poultry were found to be significantly more drug resistant than commensal strains [33]. In the present study almost all of clinical isolates showed high level of antibiotic resistance and 91.5 % of them showed multidrug resistance (resistance to ≥ 2 antimicrobials). Other authors identified lower multidrug resistance in E. cecorum from carcass rinsates and diseased poultry, however the panel of used antibicrobials were not completely the same [35]. Affected flocks were treated against E. cecorum usually with amoxicillin, doxycycline or enrofloxacin. All of the above antibiotics were tested in this study. Similarly to other authors, the overwhelming majority of the isolates were susceptible to penicillin, which appear to be drug of choice [4,5,7,10,14,15]. However, the majority of E. cecorum was resistant to enrofloxacin > teicoplanin > doxycycline > erythromycin. Our results were opposed to clinical E. cecorum from other countries, in which sensitivity to enrofloxacin (in Germany, Holland, Hungary, South Africa), doxycycline (in Germany, Hungary) and macrolides (in Belgium, Germany) were identified [4,5,7,10,14,15]. Similarly to the isolates from Canada, USA, Holland and Belgium, clinical E. cecorum from Poland showed the increased resistance to tetracycline or erythromycin (macrolides) [4,7,27,33,35].
This antimicrobial resistance pattern may be common and characteristic for pathogenic E. cecorum. The presented study indicated on the presence clinical E. cecorum (1.2 %) with the resistance to vancomycin (VRE) and to high level gentamicin (HLGR). Similarly to Jackson et al. [35], we found out that none of the isolates were resistant to linezolid. According to the literature, enterococci have both an intrinsic and acquired resistance to antibiotics which complicate treatment of infections. The acquired resistance includes resistance to i.a. chloramphenicol, tetracyclines, fluoroquinolones, aminoglycosides (high levels), and vancomycin. Enterococci have demonstrated a huge potential for acquiring and disseminating resistant genes. We found, that the high level of the resistance to enrofloxacin, doxycycline, tetracycline in E. cecorum isolates is probably related to the wide use of these antibiotics in poultry production. In previous years erythromycin was also commonly applied in the therapy of poultry. Other authors confirmed the presence of resistance genes (including van genes) among E. cecorum from broilers or retail chicken meat [38,39]. We suggest, that poultry may play an important role as reservoirs of antibiotic resistant E. cecorum in the environment. However, further studies are needed to investigate the resistance genes in clinical isolates.
In the present study sodA gene fragment was successfully used to confirm phenotypic identification of E. cecorum, however it was not sufficiently discriminative to differentiate them from each other. In the collection it was possible to distinguish for three phylogenetic groups and one subgroup. The strains from group B showed the same type of production (CB), year of No clear genetic differences were observed between clinical strains and reference strain. Recent data indicated that pathogenic E. cecorum from the southeast US were clonal, however comparative genomic analysis revealed fundamental differences in their genomes [34]. According to the previous report, isolates recovered from spinal abscesses were highly similar and could be detected by using PFGE [33]. In our study, PFGE results showed the genetic heterogeneity between clinical E. cecorum isolates, that is consistent with the other studies [18]. Therefore, the usage of PFGE in distinguishing pathogenic strains may be difficult and limited. This genetic diversity was seen between poultry flocks, however some clustering was visible in relation of type of production (CB, CL). Moreover, some temporal and geographical clustering was visible. Many CB isolates from the same year and geographical origin were clustered together (pulsotype A, D, L, P) indicating their close genetic relationship. Some CL isolates from the same location but different years were grouped into a single pulsotype (U) indicating on the possible horizontal transmission among CL flocks in this area. We found that CB and BB isolates from the same year which were clustered together into separate pulsotypes (I, M). Based on the relatively close relationship between isolates from geese and chicken flocks, it could be assumed that isolates from the single clonal lineage may cause outbreaks in different bird species. The results may suggest the transmission of potential diseasecausing E. cecorum between flocks.

Conclusions
These data indicate that several, widely distributed pathogenic E. cecorum clones seemed to be responsible for infection cases found in different poultry types. The isolates causing infection in different CB flock in the same year and region may be somewhat genetically distinct from each other and from those that cause disease in CL or BB flocks in the same year and region. Phenotypically, clinical isolates were generally found to be very similar, however some properties or characteristics described in some isolates were not found in others. The study presented here is the first in Poland as well as one of the few in Europe which provides phenotypic and genotypic characterization of E. cecorum isolates associated with disease outbreaks in poultry flocks. Further research needs to focus on finding new virulence determinants of E. cecorum and recognition of transmission routes.

Additional files
Additional file 1: Alignment of partial sodA gene sequences from E. cecorum isolates and reference strain (ATCC 43198). Nucleotide differences are specified by the nucleotide, while dot represented no nucleotide changing. (TIF 12403 kb) Additional file 2: Alignment of partial 16S rRNA gene sequences from E. cecorum isolates and reference strain (ATCC 43198). Nucleotide differences are specified by the nucleotide, while dot represented no nucleotide changing. (TIF 60654 kb) Abbreviations BB, broiler breeder flocks; CA, Columbia agar with 5 % sheep blood; CB, chicken broiler flocks (commercial broilers); CL, commercial layer flocks; E. cecorum, Enterococcus cecorum; ES, enterococcal spondylitis; G, geese flocks; HLGR, high level gentamicin resistance; PFGE, Pulsed Field Gel Electrophoresis; T, turkey flock; VRE Vancomycin-Resistant Enterococcus