Abstract
Seventy-four strains representing the four species of the genus Dialister were isolated from various clinical samples. Dialister pneumosintes and Dialister micraerophilus were the two mainly encountered species. Fifty-five isolates were tested against 14 antimicrobial agents. Decreased susceptibilities to piperacillin, metronidazole, macrolides, fluoroquinolones, and rifampin were demonstrated. The clinical impact of these decreased susceptibilities remains to be investigated but should prompt microbiologists to perform antimicrobial susceptibility testing for clinically important Dialister spp.
Four species are currently described in the genus Dialister: Dialister pneumosintes, Dialister invisus, Dialister micraerophilus, and Dialister propionicifaciens, the last three being described since 2003 (5, 11). They are small, anaerobic or microaerophilic gram-negative coccobacilli that grow as small, circular, tiny, and transparent colonies on Columbia blood agar, making their recovery in mixed anaerobic cultures relatively difficult. Besides, strains may be difficult to distinguish from tiny, gram-negative anaerobic cocci. Usually, Dialister spp. could be distinguished from members of the genus Veillonella by both the absence of nitrate reduction and the pattern of susceptibility to special-potency disks. Indeed, they display susceptibility to kanamycin (500 μg), bile (1 mg), and metronidazole (50 μg) and resistance to vancomycin (5 μg) and colistin (10 μg) disks, whereas Veillonella species are susceptible to the colistin disk, except for Veillonella montpellierensis and Veillonella ratti (10, 11). However, due to their biochemical characteristics (i.e., asaccharolytic and nonreactive in conventional biochemical tests), identification of members of the genus Dialister often requires molecular methods such as 16S rRNA gene sequencing (11). The involvement of Dialister spp. in human infections is now clearly established. D. pneu-mosintes and D. invisus have been mainly implicated in oral diseases such as periodontitis, acute necrotizing ulcerative gingivitis, and endodontic infections (2, 3, 5, 15-17, 19). More rarely, D. pneumosintes has been isolated from bite wound infections, during respiratory tract and head and neck infections (7, 9), from amniotic fluid and placenta samples (6), in human brain abscesses (18), and during bacteremia (12, 18). D. invisus has also been identified in urinary tract specimens from renal transplant recipients (4). D. micraerophilus strains have been characterized from various human clinical specimens, including bone and blood cultures, whereas the four D. propionicifaciens isolates currently reported were from cutaneous infections and semen (11). However, the relative significance of Dialister species in human clinical samples remains unknown. In particular, the involvement of D. pneumosintes remains questionable for all of the cases reported before the description of the three other Dialister species and documented by phenotypic means only. Moreover, because our knowledge on the pathogenic role of Dialister spp. has been greatly improved by culture-independent studies, mainly focused on oral flora (2, 15-17, 19), comprehensive microbiological information on Dialister spp. remains relatively scarce and antimicrobial susceptibility data are available for only a few isolates (8, 18).
To our knowledge, we have assembled the largest described collection of Dialister clinical isolates, which has allowed us to evaluate the relative clinical significance and sources of the Dialister species and to report their antimicrobial susceptibility.
(This work was presented during the 8th Congress of the Anaerobe Society of the Americas, Boise, ID, July 2006.)
From March 2002 to June 2004, 74 strains consecutively isolated from 73 patients hospitalized at the University Hospital of Montpellier, France, were identified as one of the four species of the genus Dialister by using 16S rRNA gene sequencing as previously described (1). A 600-bp partial sequence was analyzed for each strain, and the threshold retained for identification to the species level was 99% of sequence identity to the type strain of the most-related species. D. pneumosintes was the predominantly isolated species (n = 46 [62.1%]), and D. micraerophilus, D. invisus, and D. propionicifaciens accounted for 25.7% (n = 19), 6.8% (n = 5), and 5.4% (n = 4) of the total isolates, respectively (Table 1). This underlined that Dialister spp. are not infrequent in human clinical samples, and the relatively high proportion of the species D. micraerophilus is revealed here for the first time. Demographic information showed that the majority of Dialister isolates were identified from male patients (66.2%) and adults (mean age of recovery, 47 years; range, 21 months to 92 years). It is noteworthy that only one strain of D. pneumosintes was isolated in a patient <18 years of age. The isolates were recovered from various clinical samples, including blood cultures, bone, and supra- and infra-diaphragmatic pus (Table 1). The majority of the strains were isolated from cutaneous and soft tissue infections (n = 39 [52.7%]). D. pneumosintes was the only species recovered from blood cultures in this study (n = 2 [2.7%]) and was the predominant species in intra-abdominal (11 out of 13 isolates) and respiratory (7 out of 8 isolates) samples, whereas D. micraerophilus was the main species found in gynecological tract samples (7 out of 8 isolates) (Table 1). Except for one strain from blood, all isolates of Dialister spp. were recovered in mixed flora, mainly associated with anaerobic gram-positive cocci and with Streptococcus spp. The overall pattern of isolation sites is consistent with previous reports recovering Dialister species from oral as well as nonoral sites (6, 8, 12). However, the present study conducted with a large collection of strains gives an overview of their relative significance in human clinical samples and reveals that an unexpectedly high proportion of Dialister strains were recovered from nonoral sites, contrasting with the small number of such strains reported in the literature.
TABLE 1.
Source of the 74 clinical strains of Dialister spp. in this study
| Origina | No. (%) of strains
|
Total | |||
|---|---|---|---|---|---|
| D. pneumosintes | D. micraerophilus | D. invisus | D. propionicifaciens | ||
| Cutaneous/soft tissues | 24 (52.2) | 10 (52.6) | 2 (40) | 3 (75) | 39 |
| Intra-abdominal | 11 (23.9) | 2 (40) | 13 | ||
| Respiratory | 7 (15.2) | 1 (20) | 8 | ||
| Gynecological | 1 (2.2) | 7 (36.9) | 8 | ||
| Blood | 2 (4.3) | 2 | |||
| Bone | 2 (10.5) | 2 | |||
| Semen | 1 (25) | 1 | |||
| Gastric fluid | 1 (2.2) | 1 | |||
| Total | 46 | 19 | 5 | 4 | 74 |
Cutaneous and soft tissue strain origins include pressure ulcer (3 strains), cellulite (3 strains), fistula (2 strains), and abscess/phlegmon/cyst/wound (31 strains), broken down as pilonidal cyst (4 strains), rectum/anus (4 strains), groin (2 strains), tonsil (2 strains), buttock (2 strains), cervical wound (2 strains), face (1 strain), cheek (1 strain), scrotum (1 strain), abscess of dental origin (1 strain), and origin not available (11 strains). Intra-abdominal strain origins include peritoneal fluid (6 strains), pancreas abscess (1 strain), intra-abdominal abscess (1 strain), false membrane (1 strain), appendix (1 strain), and drain (3 strains). Respiratory strain origins include sinus (7 strains) and pleural fluid (1 strain). Gynecological strain origins include amniotic fluid (4 strains), bartholin gland (1 strain), vaginal collection (1 strain), vaginal swab (1 strain), and pouch of Douglas (1 strain).
Among the 74 Dialister isolates, 55 representatives of the four species could be subcultured for antibiotic susceptibility testing and were tested against a panel of 14 antimicrobial agents. Moreover, 26 randomly selected strains were tested against a panel of six additional drugs (Tables 2 and 3). MICs were determined by the reference agar dilution method according to CLSI (formerly NCCLS) standard M11-A6 (14). The control strains tested included Bacteroides fragilis ATCC 25285, Bacteroides thetaiotaomicron ATCC 29741, and Eggerthella lenta ATCC 43055. Results and susceptibility breakpoints from CLSI and the Antibiogram Committee of the French Society for Microbiology (CA-SFM) (20) are given in Tables 2 and 3 for clinical samples; control strains gave MIC results within the expected range (results not shown). The 55 Dialister isolates were susceptible to all of the antimicrobial agents tested according to the CLSI, whereas 33 strains showed decreased susceptibility to one or several agents according to the CA-SFM. Decreased susceptibility was demonstrated to metronidazole (n = 27 isolates [20 D. pneumosintes and 7 D. micraerophilus isolates]), erythromycin (n = 9 [8 D. pneumosintes isolates and 1 D. micraerophilus isolate]), pristinamycin (n = 9 [all D. pneumosintes]), rifampin (n = 11 [10 D. pneumosintes isolates and 1 D. micraerophilus isolate]), piperacillin (n = 9 [6 D. pneumosintes, 1 D. micraerophilus, and 2 D. invisus isolate]), levofloxacin (n = 1 [D. pneumosintes]), and ciprofloxacin (n = 1 [D. micraerophilus]). None of the strains with MICs of metronidazole of 4 μg/ml (n = 20) or 8 μg/ml (n = 7) harbored nim genes when tested by PCR, as previously described (13, 21). Strains with MICs of piperacillin of 16 μg/ml (n = 6) or 32 μg/ml (n = 3) were tested using nitrocefin disks (BD Diagnostic Systems) and were not β-lactamase producers. Decreased susceptibility to four or five antibiotics was observed for nine strains, including a D. micraerophilus strain isolated from three bone samples in a patient with hip arthritis and eight D. pneumosintes strains of diverse origins.
TABLE 2.
In vitro activities of 20 antimicrobial agents against Dialister pneumosintes and Dialister micraerophilus
| Species (no. of strains) and antimicrobial agent | MIC (μg/ml)a
|
Susceptibility breakpoint (μg/ml)b
|
|||
|---|---|---|---|---|---|
| Range | 50% | 90% | CLSI | CA-SFM | |
| D. pneumosintes | |||||
| Amoxicillin (33) | ≤0.06-0.125 | ≤0.06 | ≤0.06 | ≤0.5 | Id. |
| Amoxicillin-clavulanate (33) | ≤0.06 | ≤0.06 | ≤0.06 | ≤4/2 | Id. |
| Ticarcillin (33) | ≤0.06-2 | 0.25 | 2 | ≤32 | ≤16 |
| Ticarcillin-clavulanate (33) | ≤0.06-0.25 | 0.125 | 0.25 | ≤32/2 | ≤16/2 |
| Piperacillin (33) | ≤0.125-32 | 1 | 16 | ≤32 | ≤8 |
| Cephalothin (17) | 0.25-1 | 0.5 | 1 | NA | ≤8 |
| Cefoxitin (33) | 0.5-4 | 0.5 | 1 | ≤16 | ≤8 |
| Imipenem (33) | ≤0.06-0.5 | ≤0.06 | 0.25 | ≤4 | Id. |
| Ertapenem (17) | 0.125 | 0.125 | 0.125 | ≤4 | Id. |
| Erythromycin (17) | 0.125-2 | 1 | 2 | NA | ≤1 |
| Pristinamycin (17) | 0.125-2 | 2 | 2 | NA | ≤1 |
| Clindamycin (33) | 0.125 | 0.125 | 0.125 | ≤2 | Id. |
| Telithromycin (33) | ≤0.06 | ≤0.06 | ≤0.06 | NA | ≤0.5 |
| Levofloxacin (17) | 0.25-2 | 0.25 | 0.5 | NA | ≤1 |
| Ciprofloxacin (33) | ≤0.06-0.25 | ≤0.06 | ≤0.06 | NA | ≤0.5 |
| Moxifloxacin (33) | ≤0.06-1 | ≤0.06 | 0.06 | ≤2 | ≤1 |
| Rifampin (33) | ≤0.06-4 | 2 | 4 | NA | ≤2 |
| Doxycycline (17) | ≤0.06-0.5 | 0.25 | 0.25 | NA | ≤4 |
| Chloramphenicol (33) | 0.25-2 | 0.25 | 0.5 | ≤8 | Id. |
| Metronidazole (33) | 1-8 | 4 | 8 | ≤8 | ≤2 |
| D. micraerophilus (14 or 6) | |||||
| Amoxicillin (14) | ≤0.06 | ≤0.06 | ≤0.06 | ≤0.5 | Id. |
| Amoxicillin-clavulanate (14) | ≤0.06 | ≤0.06 | ≤0.06 | ≤4/2 | Id. |
| Ticarcillin (14) | ≤0.06-1 | ≤0.06 | 0.5 | ≤32 | ≤16 |
| Ticarcillin-clavulanate (14) | ≤0.06-0.125 | 0.125 | 0.125 | ≤32/2 | ≤16/2 |
| Piperacillin (14) | ≤0.125-16 | 0.125 | 1 | ≤32 | ≤8 |
| Cephalothin (6) | 0.25 | NA | ≤8 | ||
| Cefoxitin (14) | 0.5 | 0.5 | 0.5 | ≤16 | ≤8 |
| Imipenem (14) | ≤0.06-0.125 | 0.06 | 0.06 | ≤4 | Id. |
| Ertapenem (6) | 0.125 | ≤4 | Id. | ||
| Erythromycin (6) | 0.125-2 | NA | ≤1 | ||
| Pristinamycin (6) | 0.125 | NA | ≤1 | ||
| Clindamycin (14) | 0.125 | 0.125 | 0.125 | ≤2 | Id. |
| Telithromycin (14) | ≤0.06 | ≤0.06 | ≤0.06 | NA | ≤0.5 |
| Levofloxacin (6) | 0.25-1 | NA | ≤1 | ||
| Ciprofloxacin (14) | ≤0.06-1 | ≤0.06 | ≤0.06 | NA | ≤0.5 |
| Moxifloxacin (14) | ≤0.06-1 | ≤0.06 | 0.25 | ≤2 | ≤1 |
| Rifampin (14) | ≤0.06-4 | ≤0.06 | ≤0.06 | NA | ≤2 |
| Doxycycline (6) | ≤0.06-0.5 | NA | ≤4 | ||
| Chloramphenicol (14) | 0.25-1 | 0.25 | 1 | ≤8 | Id. |
| Metronidazole (14) | 1-8 | 2 | 8 | ≤8 | ≤2 |
50% and 90%, MIC50 and MIC90, respectively. Values are indicated only where there were >10 strains.
Id., identical breakpoints for CA-SFM and CLSI; NA, not available. Breakpoints for moxifloxacin were from CLSI standard M11-A7.
TABLE 3.
In vitro activities of antimicrobial agents against Dialister propionicifaciens and Dialister invisus
| Antimicrobial agent | MIC range in μg/ml (no. of strains)a
|
Susceptibility breakpoint (μg/ml)b
|
||
|---|---|---|---|---|
| D. propionifaciens | D. invisus | CLSI | CA-SFM | |
| Amoxicillin | ≤0.06 (4) | ≤0.06 (4) | ≤0.5 | Id. |
| Amoxicillin-clavulanate | ≤0.06 (4) | ≤0.06 (4) | ≤4/2 | Id. |
| Ticarcillin | ≤0.06-1 (4) | 0.25-1 (4) | ≤32 | ≤16 |
| Ticarcillin-clavulanate | ≤0.06-0.125 (4) | 0.125 (4) | ≤32/2 | ≤16/2 |
| Piperacillin | 0.125-4 (4) | 0.125-16 (4) | ≤32 | ≤8 |
| Cephalothin | 0.25 (3) | ND | NA | ≤8 |
| Cefoxitin | 0.5 (4) | 0.5 (4) | ≤16 | ≤8 |
| Imipenem | ≤0.06-0.06 (4) | 0.06 (4) | ≤4 | Id. |
| Ertapenem | 0.125 (3) | ND | ≤4 | Id. |
| Erythromycin | 0.125 (3) | ND | NA | ≤1 |
| Pristinamycin | 0.125 (3) | ND | NA | ≤1 |
| Clindamycin | 0.125 (4) | 0.125 (4) | ≤2 | Id. |
| Telithromycin | ≤0.06 (4) | ≤0.06 (4) | NA | ≤0.5 |
| Levofloxacin | 0.25 (3) | ND | NA | ≤1 |
| Ciprofloxacin | ≤0.06 (4) | ≤0.06 (4) | NA | ≤0.5 |
| Moxifloxacin | ≤0.06 (4) | ≤0.06 (4) | ≤2 | ≤1 |
| Rifampin | ≤0.06 (4) | ≤0.06 (4) | NA | ≤2 |
| Doxycycline | ≤0.06 (3) | ND | NA | ≤4 |
| Chloramphenicol | 0.25 (4) | 0.25-2 (4) | ≤8 | Id. |
| Metronidazole | 1-2 (4) | 2 (4) | ≤8 | ≤2 |
ND, not determined.
Id., identical susceptibility breakpoint for CA-SFM and CLSI; NA, not available. Breakpoints for moxifloxacin were from CLSI standard M11-A7.
Based on inhibitory concentrations, amoxicillin, amoxicillin-clavulanate, telithromycin, and ciprofloxacin were the agents the most active against the four species, with a MIC90 or MIC range of ≤0.06 μg/ml. Imipenem and moxifloxacin were also very active against Dialister spp., three of the four species showing a MIC90 or MIC range of ≤0.06 μg/ml. D. pneumosintes was the species least susceptible to rifampin, with a MIC90 of 4 mg/liter, while strains of the three other species displayed a MIC90 or MIC range of ≤0.06 μg/ml.
Despite the overall susceptibility of Dialister species to all antimicrobial agents tested, some clinically important strains showing decreased susceptibility to several families of antibiotics were found according to the CA-SFM breakpoints. The decrease in susceptibility to agents widely used in the treatment of anaerobic infections, like metronidazole, or to agents prescribed in some infections that may involve Dialister spp., like pristinamycin in respiratory tract infections or sinusitis and rifampin in bone infections, is important to note and now has to be correlated to clinical studies in order to evaluate its clinical impact. These observations, together with the previous report of strains displaying high levels of resistance to macrolides (17) or fluoroquinolones (8), should also prompt microbiologists to perform antimicrobial susceptibility testing for Dialister strains involved in severe polymicrobial diseases or infections refractory to antibiotic treatment. Finally, the monitoring of the antimicrobial susceptibility of Dialister spp. and its evolution has to be continued.
Acknowledgments
We thank I. Zorgniotti and A. Ramain for excellent technical assistance.
This work was partly supported by the association ADEREMPHA, Montpellier, France.
Footnotes
Published ahead of print on 8 October 2007.
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