Actinobaculum schaalii: A truly emerging pathogen?: Actinobaculum schaalii: un pathogène réellement émergent?

Actinobaculum schaalii is a Gram-positive facultative anaerobe bacillus. It is a commensal organism of the genitourinary tract. Its morphology is nonspecific. Aerobic culture is tedious, and identification techniques have long been inadequate. Thus, A. schaalii has often been considered as a nonpathogen bacterium or a contaminant. Its pathogenicity is now well described in urinary tract infections, and infections in other sites have been reported. This pathogen is considered as an emerging one following the growing use of mass spectrometry identification. In this context, the aim of our study was to evaluate the number of isolations of A. schaalii before and after the introduction of mass spectrometry in our hospital and to study the clinical circumstances in which isolates were found.

Introduction

Actinobaculum schaalii is a Gram-positive bacillus that was described for the first time in 1997 [1]. Other species described in the same genus include Actinobaculum suis, Actinobaculum urinale and Actinobaculum massiliense. A. suis has been reported as a veterinary pathogen [2], [3]. A. urinale is rarely found in human infections but has been coisolated with A. schaalii [4]. Recent changes in the classification were made, reclassifying A. schaalii as Actinotignum schaalii [5]. It is a nonsporulated bacillus. Its morphology is nonspecific, and it sometimes looks like immobile corynebacteria. It is facultative anaerobic, which implies a possible anaerobic culture and culture in atmosphere supplemented with 5% CO2. Its aerobic culture is tedious and is facilitated by the use of blood-enriched media. These characteristics explain the difficulty of isolation, especially from urine, which is routinely incubated aerobically on unsupplemented media, such as chromogenic ones. A. schaalii is described as a commensal of the urogenital tract also found on the skin [6]. Many cases of infections have been reported, and urinary tract infections (UTIs) seem to predominate, particularly among elderly patients [7], [8]. However, other types of infections exist. Since its discovery, the number of reported isolates and clinical cases is increasing. It is thus considered to be an emerging pathogen.

The aim of our study was to assess retrospectively the frequency and circumstances of isolation of A. schaalii over 10 years in CH Versailles (France) and the role of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF) in the identification of this bacterium.

Materials and Methods

Using SIRScan software (I2A), we extracted all A. schaalii strains between July 2004 and February 2015. Strains were identified by sequencing of the 16S ribosomal DNA from colonies using the primers A2 (5′-AGAGTTTGATCATGGCTCAG-3′) and S15 (5′-GGGCGGTGTGTACAAGGCC-3′) [9] until November 2011. From December 2011, MALDI-TOF (Microflex; Bruker Daltonics, Wissembourg, France) was used (databases DB 4613 and 5627). Retrospective mass spectrometry (MS) identification was carried out for stored isolates initially identified by sequencing.

For urine samples, microscopic examination (Gram coloration) was carried out in case of leukocyturia >104 leucocytes/mL. Ten microlitres was routinely seeded onto a chromogenic agar incubated aerobically. If microscopic examination showed only Gram-positive bacilli, blood agar was also seeded under atmosphere enriched in CO2 for 48 hours.

Clinical observations of all cases were reviewed to determine the pathogenic role of the isolate. The following informations were collected: age, sex, infection risk factors, site and isolation circumstances. We considered that A. schaalii was a pathogen when it was clearly mentioned in the actual benefit account and/or when it was the only bacteria isolated from the infectious site.

Results

Between July 2004 and February 2015, a total of 24 A. schaalii isolates were collected from 24 patients. Six isolates were initially identified by sequencing, and the other 18 were identified by MALDI-TOF. Among the six isolates initially identified by sequencing, three were further confirmed by MS. The results of the different scores obtained are shown in Table 1. For isolates identified by MS, the statistical averages of the scores obtained for each of the first three proposed identifications are shown in Fig. 1.

Table 1

Retrospective mass spectrometry identification of three Actinobaculum schaalii isolatesa

Isolate no.	Identification (score):
First choice	Second choice	Third choice
1	A. schaalii (2.03)	A. schaalii (2)	A. schaalii (1.8)
2	A. schaalii (2.15)	A. schaalii (1.75)	A. schaalii (1.63)
3	A. schaalii (1.93)	A. schaalii (1.57)	A. schaalii (1.54)

Isolates were initially identified by sequencing.

Fig. 1

Statistical scores (maximum, median, minimum) for first three proposals (17 isolates).

The circumstances of isolation and the clinical characteristics of the patients are summarized in Table 2. Strains were predominantly isolated from abscesses and collections (58% of cases, 14 isolates): bone infections and soft tissue infections including cysts and abscesses (wall, pilonidal sinus, breast, ankle etc.). The urine samples corresponded to 33% of the isolates: eight cases, including seven cases after November 2011 (Table 3). Finally, blood cultures represented two isolates. Urine and blood cultures were monomicrobial, while 64% (9/14) abscesses and collections were plurimicrobial.

Table 2

Clinical characteristics of patients

Patient no.	Age/sex	Risk factor	Nature of sampling	Other microorganism(s)	Accountability Actinobaculum schaalii	Diagnosis retained
1	58/F	Cancer	Blood culture	No	No	Staphylococcus epidermidis portacath infection
2	55/M	Monoclonal gammopathy	Blood culture	No	No	Enterobacter cloacae sepsis
3	8/M	Undescended testis, enuresis	Urine	No	Yes	UTI
4	81/M	Age, squamous cell carcinoma	Urine	No	Yes	UTI
5	83/F	Age, diabetes	Urine	No	Yes	UTI
6	83/F	Age, colectomy, villous tumor	Urine	No	Yes	UTI
7	96/F	Age, urinofaecal incontinence	Urine	No	Yes	UTI
8	75/F	Age	Urine	No	Yes	UTI
9	80/M	Age	Urine	No	Yes	UTI
10	66/M	Age	Urine	No	Yes	UTI
11	77/M	Age, diabetes	Collection	No	Yes	Osteoarthritis
12	23/M	None	Collection	No	Yes	Abdominal wall abscess
13	69/M	Age	Collection	No	Yes	Secondarily infected sebaceous cyst
14	20/F	None	Collection	No	Yes	Pilonidal sinus abscess
15	34/F	None	Collection	No	Yes	Breast abscess
16	17/M	None	Collection	Peptostreptococcus spp.	No	Malleolus abscess
17	28/M	None	Collection	Proteus mirabilis, Klebsiella pneumoniae, Enterococcus faecalis	No	Collection of left tibia
18	56/M	Diabetes	Collection	Streptococcus spp., Peptostreptococcus spp.	No	Secondarily infected sebaceous cyst
19	74/F	Age	Collection	Actinomyces spp., Peptoniphilus spp.	No	Lipoma secondarily infected
20	69/M	Age, diabetes	Collection	Finegoldia spp., Anaerococcus spp.	No	Diabetic foot ulcer
21	54/F	Diabetes	Collection	Acinetobacter spp., Helcococcus spp., Anaerococcus spp.	No	Abscess on sternotomy
22	77/M	Age	Collection	P. mirabilis, Morganella morganii	No	Ankle osteitis
23	27/F	None	Collection	Actinomyces turicensis	No	Vaginal abscess
24	88/F	Age	Collection	Staphylococcus aureus, Fascioloides magna, Propionibacterium acnes	No	Shoulder fistula

UTI, urinary tract infection.

Table 3

Sites of isolation of A. schaalii before and after introduction of MS

Introduction of MS	Urine	Abscess/collection	Blood culture	Total
Before	1	5	0	6
After	7	9	2	18

MS, mass spectrometry.

The male/female sex ratio of the 24 patients was 1.2 with a mean age of 58.2 years (range, 8–96 years). In 75% of cases, at least one risk factor for infection was present, including age >65 years, immunosuppression, abnormality of the genitourinary tract and diabetes.

Discussion

Introduction of MALDI-TOF in clinical practice has led to an increase in the frequency of A. schaalii isolates in our laboratory (Table 3). Indeed, between July 2004 and November 2011, six strains of A. schaalii were identified by sequencing, vs. 18 isolates by MS since November 2011. The most likely explanation is that before November 2011, routine bacterial identification was performed by biochemical tests such as API strips (bioMérieux, Marcy l'Etoile, France) or Vitek cards (bioMérieux). These techniques did not permit the identification of A. schaalii because this organism was then not included in the database [10]; the latest 2015 version of the map Vitek anaerobe and Corynebacterium (ANC) now allows its detection.

Analysis of the results of identification by MS (Fig. 1) revealed that the first three proposals correspond to the three spectra of A. schaalii of Bruker base (Table 4) in 17 of 18 cases, with a median identification score of 2.045 (1.82 to 2.26) for the first proposal. The three other Actinobaculum species in the database are never proposed. In addition, retrospective MS performed on three isolates stored and previously identified by 16S ribosomal DNA sequencing was successful for identification. As previously described [10], we confirmed the identification performance by Microflex MALDI-TOF (Bruker) for A. schaalii with the three strains also identified by sequencing. However, it was reported that the MALDI-TOF Vitek (bioMérieux) does not currently allow the identification of A. schaalii [11], [12].

Table 4

Mass spectrometer (Bruker Daltonics) local database for Actinobaculum spp.

Species	Database entry
Actinobaculum schaalii	70710715001 MVC
A. schaalii	DSM 15541T DSM
A. schaalii	RV_BA1_032010_E LBK
Actinobaculum suis	DSM 20639T DSM
A. suis	GD75 GDD
Actinobaculum urinale	DSM 15805T DSM

The pathogenic role of A. schaalii was not accepted by clinicians in two cases of bacteraemia because another recognized pathogen was isolated from blood cultures for each of the two patients. In addition, the presence of this microorganism as a contaminant of blood cultures suggests a possible presence on the skin [6]. Nevertheless, we cannot exclude its participation in the infection.

Urine samples all exhibited significant leukocyturia (>104/mL), a positive direct microscopic examination with Gram-positive bacilli and monomicrobial culture >105 CFU/mL. The isolation in urine requires seeding on a blood agar in atmosphere enriched in CO2. This is routinely carried out in our laboratory on urine samples with leukocyturia (>104/mL) and Gram-positive bacilli on microscopic examination. The collected clinical data are consistent with a diagnosis of infection of the urogenital tract. A. schaalii is thus responsible for UTI in eight cases. One or more risk factors were found in these patients, highlighting the opportunistic nature of the microorganism. Advanced age is one of those risk factors (seven of eight patients were aged >65 years) with urinary infection of A. schaalii. However, infections in young subjects should not be excluded [13], as evidenced by case of acute pyelonephritis in a 8-year-old patient.

To assess the clinical relevance of A. schaalii, its isolation in urine samples must be associated with symptoms of UTI because it is important to remember that it can sometimes be a urinary commensalism. As such, a prospective Danish study showed that A. schaalii could be detected by PCR in urine in more than 22% of patients aged >60 years, despite the absence of clinical signs in certain cases [14]. This may explain why A. schaalii is often reported to exist within polymicrobial urine [15]. In our practice, when there is a dominant uropathogen, the other possibly associated bacteria are not always identified, as they are considered commensal.

For abscesses and collections, we distinguish between mono- and plurimicrobial samples. Accountability is questionable for samples with various microorganisms. It is easier when there are no other related bacteria. This is the case of a 34-year-old patient with recurrent breast abscess, in which A. schaalii was clearly implicated, as specified in the medical report. As in most of the cases we have described, the patients were treated by surgery only without antibiotics, and the outcome was always good, suggesting that, unlike UTIs, antibiotic treatment is not routinely required to treat the infection. We emphasize that compared to urinary isolates, the age of the patient is more important for abscesses and collection. Another difference concerns young patients without risk factors, including an abdominal wall abscess in a 23-year-old patient, a pilonidal sinus abscess in a 20-year-old man and breast abscesses observed in a 34-year-old patient. However, it was suggested that A. schaalii could contribute to the infectious process even when it is isolated in the presence of other bacteria [15]. Cases of endocarditis [16], Fournier gangrene [17], spondylitis [18] and urosepsis [19] have already been reported, underscoring the invasive potential of this microorganism.

Overall, although UTIs are the most frequently reported in the literature [12], our study shows that the isolation of A. schaalii in abscesses and collections is common. The frequency of isolation has increased since the onset of use of MS in our laboratory. This suggests that the emerging nature of infections due to A. schaalii is probably attributable to gaps in methods for identifying in the daily microbiology before the era of MS.

To conclude, our study emphasizes the role of MALDI-TOF in the accurate identification of A. schaalii. This process, which is less-time consuming and less expensive than molecular biology, advantageously replaces biochemical tests in the identification of this organism. The emergence of this bacterium presumably is the result, at least in part, of an evolution in identification methods. This will permit a better understanding of the epidemiology of A. schaalii. The pathogenic role of A. schaalii is questionable in some cases. Further studies are therefore needed, especially on the cutaneous habitat and virulence of this bacterium, to better interpret its presence in a clinical sample.

Tableau 1

Identification rétrospective par spectrométrie de masse de 3 souches initialement identifiées par séquençage

Isolat	1er choix (score)	2ème choix (score)	3ème choix (score)
1	A. schaalii (2,03)	A. schaalii (2)	A. schaalii (1,8)
2	A. schaalii (2,15)	A. schaalii (1,75)	A. schaalii (1,63)
3	A. schaalii (1,93)	A. schaalii (1,57)	A. schaalii (1,54)

Tableau 2

Caractéristiques cliniques des patients

Cas N°	Age/Sexe	Facteurs de risque	Nature du prélèvement	Autre(s) germe(s)	Imputabilité d'A. schaalii	Diagnostic retenu
1	58/F	Cancer	Hémoculture	Non	Non	Infection de PAC à S. epidermidis
2	55/M	Gammapathie monoclonale	Hémoculture	Non	Non	Septicémie à E. cloacae
3	8/M	Ectopie testiculaire, énurésie	Urine	Non	Oui	Infection urinaire
4	81/M	Age, carcinome épidermoïde	Urine	Non	Oui	Infection urinaire
5	83/F	Age, diabète	Urine	Non	Oui	Infection urinaire
6	83/F	Age, colectomie, tumeur villeuse	Urine	Non	Oui	Infection urinaire
7	96/F	Age, incontinence urino-fécale	Urine	Non	Oui	Infection urinaire
8	75/F	Age	Urine	Non	Oui	Infection urinaire
9	80/M	Age	Urine	Non	Oui	Infection urinaire
10	66/M	Age	Urine	Non	Oui	Infection urinaire
11	77/M	Age, diabète	Collection	Non	Oui	Ostéoarthrite
12	23/M	Aucun	Collection	Non	Oui	Abcès de paroi abdominale
13	69/M	Age	Collection	Non	Oui	Kyste sébacé surinfecté
14	20/F	Aucun	Collection	Non	Oui	Abcès du sinus pilonidal
15	34/F	Aucun	Collection	Non	Oui	Abcès du sein
16	17/M	Aucun	Collection	Peptostreptococcus spp.	Non	Abcès de la malléole
17	28/M	Aucun	Collection	P. mirabilis, K. pneumoniae, E. faecalis	Non	Collection du tibia gauche
18	56/M	Diabète	Collection	Streptococcus spp., Peptostreptococcus spp.	Non	Kyste sébacé surinfecté
19	74/F	Age	Collection	Actinomyces spp., Peptonophilus spp.	Non	Lipome surinfecté
20	69/M	Age, diabète	Collection	Finegoldia spp., Anaerococcus spp.	Non	Mal perforant plantaire
21	54/F	Diabète	Collection	Acinetobacter spp., Helcococcus spp., Anaerococcus spp.	Non	Abcès sur sternotomie
22	77/M	Age	Collection	P. mirabilis, M. morganii	Non	Ostéite de la cheville
23	27/F	Aucun	Collection	A. turisensis	Non	Abcès vaginal
24	88/F	Age	Collection	S. aureus, F. magna, P. acnes	Non	Fistule de l'épaule

Tableau 3

Sites d'isolements d'A. schaalii, avant et après la mise en place de la spectrométrie de masse

	Urine	Abcès/collections	Hémocultures	Total
Avant mise en place de la spectrométrie de masse	1	5	0	6
Après mise en place de la spectrométrie de masse	7	9	2	18

Tableau 4

Base de notre spectromètre de masse (Brüker Daltonics) pour les Actinobaculum spp.

Actinobaculum schaalii 70710715001 MVC

Actinobaculum schaalii DSM 15541T DSM

Actinobaculum schaalii RV_BA1_032010_E LBK

Actinobaculum suis DSM 20639T DSM

Actinobaculum suis GD75 GDD

Actinobaculum urinale DSM 15805T DSM