First isolation of Aerococcus viridans from clinical specimens collected on a pig farm in Poland

Originally isolated from a hospital environment and described in the early 1950s, Aerococcus viridans was the first subspecies in the Aerococcus genus of the Aerococcaceae family and Lactobacillales order (39). Seven distinct Gram-positive cocci bacteria of the genus have been validly published to date: A. christensenii, A. sanguinicola, A. suis, A. urinae, A. urinaeequi, A. urinaehominis and A. vaginalis (1, 7, 12, 19, 20, 30, 39). Once regarded to be contaminants commonly present in air and soil (17, 18, 39), they are now deemed to be associated with different infections in humans. Even though the pathogenicity of the members of the genus has not been fully elucidated thus far, the bacteria have been infrequently reported as causative agents of arthritis (35), endocarditis (15, 28), meningitis (24) and urinary tract infections (2, 7, 8, 19).

Aerococcus spp. have also been reported to be pathogenic for different animal species. Initially, A. viridans was recognised as the causative agent of gaffkaemia, a fatal disease of lobsters (3, 13, 32). According to some peer-reviewed studies analysing the problem of A. viridans infections in animals, the bacterium was subsequently associated with several cases of bovine mastitis (11, 21, 29, 31), septicaemia in immunodeficient laboratory mice (10), high mortality in a tilapia fishery (16), septicaemia in sea turtles (36) and genital infection in a captive elephant (9). Moreover, a growing body of evidence indicating the importance of A. viridans in a diverse spectrum of disorders in swine has been produced to date. The bacterium was successfully recovered from samples collected from pigs suffering from arthritis (22), meningitis (22, 27), pneumonia (22) and urinary tract infections (23), and most recently from aborted foetuses (25).

Peer-reviewed research works describing successful retrieval of A. viridans from clinical specimens collected from pigs are exceptionally rare and geographically limited to data only obtained from a few swineproducing countries. Similarly, reports delineating its antibiotic susceptibility and genetic diversity remain extremely scarce. Therefore, the aim of this study was to report the first isolation of A. viridans recovered from a specimen originating from a commercial pig farm located in Poland.

Of the seven samples examined, five were positive for bacteria by culture and five bacterial isolates were successfully recovered. Staphylococcus aureus was identified in samples 1, 2 and 6. Trueperella pyogenes and A. viridans (Fig. 1) were isolated from samples 2 and 7, respectively. Samples 3 and 5 were negative for bacterial growth. Sample 4 showed an overgrowth by Proteus sp. The two pooled samples were G. parasuis-, M. hyorhinis-, M. hyosynoviae-, Actinobacillus pleuropneumoniae-, Actinobacillus suis- and S. suis- negative in the real-time PCR.

Fig. 1.

Blood agar–cultured Aerococcus viridans isolated from a joint swab taken from the hind leg of a 12-week-old pig with tarsal joint swelling

The results of the antibiotic susceptibility testing for A. viridans are presented in Table 1 as minimal inhibitory concentrations. The isolate obtained in this study was susceptible to beta-lactam antibiotics (amoxicillin-clavulanic acid, ampicillin, ceftiofur and penicillin) and gentamicin. The isolate presented intermediate susceptibility to enrofloxacin and florfenicol, and was resistant to all the other tested antimicrobials.

The conducted analysis of 10 representative nucleotide contigs from the 500 obtained (which ranged in length from 700 base pairs (bp) to 22 kbp) showed similarity ranging from 96.6 % to 97.5% to the reference strains obtained from the GenBank database (Fig. 2).

Fig. 2.

Aerococcus viridans reference sequence nucleotide alignment of 10 representative contigs isolated from a joint swab taken from the hind leg of a 12-week-old pig with tarsal joint swelling. CPnnnnnn – GenBank reference strains of A. viridans

The conducted phylogenetic analysis showed the occurrence of four main groups of A. viridans genomic sequences (Fig. 3). The similarity of the obtained genomic sequences of this bacterium ranged from 96.1% to 100%, indicating the high similarity of all the compared isolates. The first group was formed by the sequences contig_499, contig_19 and contig_443. The second group consisted of the contig_487 and contig_447 sequences, of which the sequence similarity reached 100%. The third group held the contig_483, contig_447 and contig_479 sequences, where the sequence similarity was 97.1–99.0%. The fourth group was comprised of five reference strains obtained from the GenBank database and the contig_12 and contig_462 sequences. The latter A. viridans sequence showed 100% homology to sequences from the GenBank database.

Fig. 3.

Phylogenetic tree showing distances between reference sequences of Aerococcus viridans from the GenBank database and the obtained contig nucleotide sequences isolated from a joint swab taken from the hind leg of a 12-week-old pig with tarsal joint swelling. CPnnnnnn – GenBank reference strains of A. viridans

The phenotypical similarity of A. viridans to streptococci and staphylococci could have led to the misidentification and considerable underestimation of the prevalence of infection with this bacterium in clinical veterinary practice worldwide. Our findings corroborate data indicating that A. viridans should be considered a pathogen which can potentially produce arthritis in pigs. Though the complex mechanisms leading to disease development have not yet been satisfactorily elucidated, the very first isolation of the bacterium from swine herds most of which had been exposed to PRRSV infection seems to support the role of immunosuppressive factors in the pathogenicity of A. viridans (22). The contribution of immunosuppression to the development of clinical symptoms of sepsis has been scientifically proven using a murine model (10). Consequently, some less obvious determinants could have facilitated the development of the A. viridans- associated arthritis in the pigs sampled for our research; nonetheless, the samples were obtained from a high-health-status, PRRSV-negative farm, and identification of other factors which may have triggered or exacerbated the symptoms was beyond the scope of our study.

Most of the members of the Aerococcaceae family are sensitive to beta-lactam antibiotics. Regardless of the documented existence of beta-lactam resistant clinical A. viridans strains in humans (4, 5, 33, 37) and in cattle (31), the isolate obtained in our investigation was susceptible to the antimicrobial class in question. Unfortunately, few data regarding the antibiotic susceptibility of A. viridans recovered from clinically affected pigs have been published to date. Moreover, different techniques and sets of antimicrobials were applied to evaluate their biological properties. The available research works describe the susceptibility patterns of Spanish, Brazilian and Korean isolates obtained from various swine clinical specimens (arthritis, pneumonia and meningitis) (22), urinary tract infection (23) and foetuses (25), respectively. In the first two studies, A. viridans clinical strains were found to be either susceptible to beta-lactams (i.e. amoxicillin-clavulanic acid, ampicillin, ceftiofur and penicillin) using the disc diffusion method (22) or to be inhibited by low MIC values of the same class of antibiotics (23) when the technique was not disc diffusion. The third piece of cited research reported a relatively high incidence of beta-lactam resistance amongst A. viridans isolates using the disc diffusion method, with amoxicillin-clavulanic acid-, ampicillin-, ceftiofur- and penicillin-resistant strains comprising 84.6 %, 76.9 %, 46.2 % and 46.2 % of the total isolates, respectively (25).

Gentamicin had a low MIC against the isolate obtained in our study (1 μg/mL). The result was comparable with the MIC50 of ≤1 μg/mL and MIC90 of 2 μg/mL reported in the Brazilian study for this antibiotic against A. viridans strains (23). The intermediate MIC value obtained for florfenicol of 4 μg/mL was significantly lower than the MIC50 of 8 μg/mL described in the same work. Contrary to the previously reported antimicrobial susceptibility data, enrofloxacin was found to have a lower value against the isolate of 0.5 μg/mL, determining it to have intermediate efficacy. The elevated MIC values for macrolides and tetracycline determined in our work are comparable with those obtained in other studies addressing the problem in livestock (21, 26).

The major issue limiting our investigation may be the samples having been collected from a relatively low number of animals originating from one location only; nevertheless, the main study objective was to report the first isolation of A. viridans from a clinical specimen obtained from a pig farm located in Poland, and, since no other research questions were addressed, the sample size should be considered irrelevant. The isolation described herein might have been directly related to a microbiological contamination, because the bacterium has been described as saprophytic and successfully recovered from environmental samples (14, 38, 39). Nevertheless, since none of the major contagious agents generally associated with the development of infectious arthritis in swine (i.e. M. hyosynoviae, M. hyorhinis, G. parasuis, S. suis, Erysipelothrix rhusiopathiae, Actinobacillus suis, Actinobacillus pleuropneumoniae and T. pyogenes) were detected in the A. viridans- positive samples, and compelling evidence on the role of A. viridans infections in livestock has already been provided by other authors, its recovery from a normally sterile body part of a clinically affected animal can be considered indicative of the clinical relevance of the case being discussed.

To the best of our knowledge, this is the first official report of the identification of A. viridans in a clinically affected pig reared in Poland. The results of our investigation should raise awareness of the importance of uncommon pathogens, including A. viridans, in the development of lameness in pigs. Moreover, the presented research emphasises the vital role of modern diagnostic tools allowing the accurate and rapid identification of swine pathogens. Since no concrete details of the mechanisms of A. viridans infection, its prevalence, pathogenicity or virulence factors have yet been given, further research would prove beneficial.