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J Clin Microbiol. Sep 2007; 45(9): 3053–3057.
Published online Jun 27, 2007. doi:  10.1128/JCM.00156-07
PMCID: PMC2045271

Characterization of Aerococcus viridans Isolates from Swine Clinical Specimens[down-pointing small open triangle]

Abstract

We present here the biochemical and genetic characterization and antimicrobial susceptibility of 58 isolates of Aerococcus viridans isolated in pure culture from different clinical specimens of normally sterile body sites of pigs. A. viridans isolates were commonly susceptible to β-lactam antimicrobials and exhibited a great genetic heterogeneity as determined by pulsed-field gel electrophoresis typing. The results indicate that A. viridans might be included in the list of possible etiological agents causing disease in pigs.

The genus Aerococcus was initially described including a single species, Aerococcus viridans. Five new species of Aerococcus have been further identified: Aerococcus urinae, Aerococcus sanguinicola, Aerococcus christensenii, Aerococcus urinaeequi, and Aerococcus urinaehominis (6; http://www.bacterio.net). The clinical significance of these species has been clearly established for A. urinae and A. viridans. A. urinae is a rarely reported human pathogen that has been identified as responsible for urinary tract infections, septicemia, sepsis, endocarditis, or lymphadenitis (22-24, 32). A. viridans has been associated with different human infections such as endocarditis, urinary tract infections, arthritis, or meningitis (7, 10, 13, 20). Moreover, this species is a pathogen for crustaceans, causing gaffkemia in marine lobsters (1, 26), and it has been associated with septicemia in sea turtles (27). A. viridans has also been isolated from the milk of cows with subclinical mastitis (5) and has been associated with septicemia in immunodeficient mice (3); there have been no reports, as far as we know, of the isolation of this microorganism from other animal species. In the present study we report the isolation, biochemical and genetic characterization, and antimicrobial susceptibility of 58 isolates of A. viridans from different clinical specimens of pigs.

According to the source of isolation, 30 isolates were obtained from the joints of pigs with arthritis, 14 isolates were obtained from the brains of pigs with meningitis, and 14 isolates were obtained from the lungs of pigs with pneumonia. All isolates were recovered in pure culture from individual animal samples in moderate to high numbers, which is indicative of their clinical significance and therefore likely to be associated with bacterial infection. Details of the clinical background of the A. viridans isolates included in the study are given in Table Table1.1. Isolation of A. viridans isolates from clinical specimens was carried out on Columbia agar containing 5% of defibrinated sheep blood (bioMérieux España, s.a.) after incubation at 37°C for 24 h under aerobic and anaerobic conditions. All isolates were gram-positive catalase-negative cocci arranged in single cells, in pairs, in tetrads, or in small groups. The organisms were facultatively anaerobic and produced unpigmented, alpha-hemolytic, circular, colonies <1 mm in diameter after 24 h of incubation at 37°C on blood agar. Biochemical identification of A. viridans isolates was carried out by using the commercial identification system API Rapid ID32 Strept (bioMérieux España, s.a.) according to the manufacturer's instructions. Identification was achieved after 24 h by using the corresponding identification software (version 2.0). A total of 75.9% (n = 44) of the isolates examined in the present study were accurately identified (excellent or very good identification) by using this identification system, although a considerable phenotypic diversity was observed with 36 different biochemical profiles. In addition, two isolates (3.4%) yielded an unacceptable biochemical profile for A. viridans, five isolates (8.6%) were doubtfully identified as A. viridans, and seven isolates (12.1%) were identified with a low discrimination level between A. viridans and Streptococcus acidominimus. The failure of the API Rapid ID32 system to correctly identify A. viridans was not unexpected because it has been shown that commercial available phenotypic identification systems are not completely reliable for the accurate identification of aerobic gram-positive catalase-negative cocci (2). The phenotypic misidentification of A. viridans with S. acidominimus agrees also with previous reports (8). Most of the isolates that were not correctly identified as A. viridans with the API Rapid ID32 system failed to produce pyroglutamic acid arylamidase and failed to produce acid from mannitol, lactose, and trehalose. Details of the numerical profiles and discrepant tests obtained with these A. viridans isolates are given in Table Table22.

TABLE 1.
Details of the 58 strains of A. viridans examined
TABLE 2.
Numerical codes and discrepant biochemical tests for the 14 A. viridans isolates that were not accurately identified with the API Rapid ID32 Strept system

The identification of the 58 isolates was later confirmed by PCR and sequencing of their 16S rRNA gene. A 540-bp 16S rRNA gene fragment was amplified by PCR using the A. viridans species-specific primers AC2 (5′-GTGCTTGCACTTCTGACGTTAGC-3′) and AC4 (5′-TGAGCCGTGGGCTTTCACAT-3′) (12). All of the isolates gave the expected amplification product of 540 bp, which is specific for A. viridans. For sequencing of the 16S rRNA gene, each isolate was amplified by PCR and further sequenced as described previously (29). The determined 16S rRNA sequences consisted of about 1,400 nucleotides and were compared to the sequences of other gram-positive, catalase-negative species available in the GenBank and EMBL databases by using the FASTA program. Sequencing results resulted in 99.4 to 100% 16S rRNA gene sequence similarity with A. viridans ATCC 11536T (accession no. M58797).

Limited data are available on the antimicrobial susceptibility of A. viridans clinical isolates. Thus, the antimicrobial susceptibility to different commonly used antimicrobials was determined by the disk diffusion method using commercially (Oxoid, Ltd.) prepared antimicrobial disks (Table (Table3).3). Inocula were prepared from a 48-h Columbia blood agar plate by suspending four colonies in 5 ml of Mueller-Hinton broth and adjusted to a 0.5 McFarland standard. The disk diffusion test was performed as described by the Clinical and Laboratory Standards Institute (CLSI; formerly the National Committee for Clinical Laboratory Standards) (16) using Mueller-Hinton plates (Oxoid) supplemented with 5% defibrinated sheep blood. The agar plates were examined after 24 h of incubation at 37°C. Staphylococcus aureus ATCC 25923 and Escherichia coli ATCC 25922 were included as quality controls. Since no specific inhibition zone diameter (IZD) breakpoints for aerococci are available, the IZD breakpoints for resistance to clindamycin, tetracycline, ceftiofur, and gentamicin were those recommended by the CLSI (17) for testing veterinary gram-positive microorganisms. For penicillin, vancomycin, ampicillin, and erythromycin, the IZD breakpoints used were those recommended for testing animal enterococci, and the breakpoints for amoxicillin-clavulanic acid and chloramphenicol were those recommended for testing animal staphylococci and streptococci, respectively (17). For ciprofloxacin the IZD breakpoint used was that recommended previously for testing Staphylococcus (15), and for streptomycin the IZD breakpoint used was the one recommended by the CLSI (14). The results of the susceptibility testing of the clinical isolates of A. viridans as distribution of the IZDs values and percentage of resistant isolates are shown in Table Table3.3. Although β-lactam-resistant A. viridans strains can been isolated from human clinical specimens (31), all isolates included in the present study were susceptible to β-lactam antimicrobials (penicillin, ampicillin, amoxicillin-clavulanic acid, and ceftiofur), as well as to vancomycin. These results are in agreement with the susceptibility pattern observed for cow isolates (19). On the other hand, a high frequency of resistance was found to tetracycline, clindamycin, and streptomycin, with more than 79% of the A. viridans isolates being resistant to these antimicrobials. The resistance of A. viridans to the other antimicrobials tested was variable, ranging from 20.7% (ciprofloxacin) to 58.6% (erythromycin). Resistance to ampicillin, chloramphenicol, gentamicin, or fluoroquinolones has also been reported occasionally for human clinical strains (31).

TABLE 3.
In vitro susceptibility as determined by the disk diffusion method for the 58 A. viridans isolates

Isolates of A. viridians, as well as of the type strain of this bacterial species (A. viridans CCUG 4311T), were genetically characterized by pulsed-field gel electrophoresis (PFGE) with the restriction enzyme SmaI (MBI Fermentans, Vilnius, Lithuania) as described previously (28). The PFGE patterns were examined visually, and similarities between restriction endonuclease digestion profiles were analyzed by using BioNumerics software (Applied Maths, Belgium). A similarity matrix was computed and transformed into an agglomerative cluster using the unweighted pair group method with arithmetic averages (UPGMA) (25). All but four isolates (93.2%) were successfully characterized by PFGE after genomic DNA digestion; 53 PGFE profiles were identified. The genetic relationship between the A. viridans isolates on the basis of the SmaI-digested DNA restriction patterns after the UPGMA clustering is shown in Fig. Fig.1.1. Overall, A. viridans exhibited a great genetic heterogeneity, with the 92.7% of PFGE profiles being represented by single isolates. Only two isolates (isolates 1545/02 and 1546/02 from farm 14 and isolates C 345/04 and C 346/04 from farm 18; Table Table1)1) isolated in the same year from two different pigs at the two farms exhibited undistinguishable PFGE profiles. In most cases, the number of A. viridans isolates per herd ranged between 1 and 3 (Table (Table1),1), but in three herds in which more than four isolates were isolated (nine, eight, and four isolates in herds 14, 8, and 3, respectively) at least three different PFGE profiles were identified. A. viridans is a usual commensal microorganism of the normal flora, and it is also widespread in the environment (4, 30), which could explain the great number of different PFGE profiles obtained among the isolates studied.

FIG. 1.
Dendrogram showing the genetic relationship, based on UPGMA cluster analysis, of A. viridans isolates examined in the present study.

All of the A. viridans isolates were isolated from normally sterile body sites. Most of them were isolated from joints (51.7%) with clinical signs of arthritis, followed by brains and lungs (24.1% each) with clinical signs of meningitis and pneumonia, respectively. Our overall frequencies of isolation of A. viridans from pigs with arthritis, pneumonia, and meningitis were about 11.5, 1, and 2.2% of the isolates, respectively (A. I. Vela, unpublished data). Arthritis, meningitis, and pneumonia in pigs can be associated with infections of different microorganisms, such as Streptococcus suis, Haemophilus parasuis, Actinobacillus pleuropneumoniae, Pasteurella multocida, Mycoplasma hyopneumoniae, Mycoplasma hyosinoviae, or E. coli (21), but none of these bacterial pathogens was isolated in our laboratory during the routine microbiology processing of the clinical samples from which A. viridans was recovered. These data and the fact that all of the A. viridans isolates were recovered in pure culture greatly support a causal relationship between this microorganism and lesions. However, the great genetic diversity of this microorganism observed in the present study, as well as the isolation of clinical isolates with different PFGE profiles from different pigs of the same herd, indicate an exposure of animals to multiple A. viridans strains, which is in accordance with its wide distribution (4, 30), and also suggests an opportunistic pathogenic character of this microorganism. The pathogenicity of A. viridans is not clearly understood and immunosuppression seems to contribute or to be necessary for the development of clinical disease (3). Also, it is well known that in most infectious diseases of swine, environmental and management factors play a crucial role in the clinical expression of disease (11). All of the herds from which A. viridans was isolated were managed under “intensive” conditions, and most of them also had previous records of infection by porcine respiratory and reproductive syndrome virus (PRRSV). It has been suggested that significant PRRSV-induced immunosuppression might well occur under practical farm conditions, predisposing the animals to bacterial infections (9, 18). Therefore, it is possible that a previous long-term exposure to PRRSV and/or other predisposing environmental farm conditions, such as overcrowding or poor ventilation, that can cause stress and subsequent immune suppression may have favored the infection with this opportunistic pathogen.

To the best of our knowledge, this is the largest study of isolates of A. viridans from clinical specimens in animals. It would appear from the results of the study that A. viridans is an opportunistic pathogen organism that should be included in the list of possible etiological agents causing disease in pigs, principally as responsible for arthritis.

Acknowledgments

A.I.V. has a fellowship from the Ramon y Cajal Program (Spanish Ministry of Science and Technology/U.C.M.). J.C. was a recipient of a predoctoral grant from the Spanish Ministry of Education. This study was partially funded by project AGL2003-08848-C02-01/GAN of the Spanish Ministry of Science and Technology.

Footnotes

[down-pointing small open triangle]Published ahead of print on 27 June 2007.

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