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1 Department of Microbiology, Medical University of Sofia, Zdrave Street 2, 1431 Sofia, Bulgaria
2 University Hospital of Maxillofacial Surgery, Sofia, Bulgaria
Correspondence
Lyudmila Boyanova
lboyanova{at}hotmail.com or
l.boyanova{at}lycos.com
Received 8 January 2006
Accepted 31 May 2006
Correspondence should be sent to blvd ‘Evlogui Georgiev’ 76 – B, 1124 Sofia, Bulgaria. 
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INTRODUCTION |
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 TOP INTRODUCTION METHODSRESULTS AND DISCUSSIONREFERENCES |
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The aim of this study was to evaluate the incidence and susceptibility patterns to antibacterial agents of anaerobes in patients with abscesses and cellulitis of the head and neck over a period of 4 years and to assess the influence of the start of empirical treatment on the isolation rates of the anaerobes.
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METHODS |
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TOPINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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Strain isolation and culture. After skin disinfection with 70 % ethanol and iodophor, pus aspirates were taken by needle aspiration or during incision. The specimens were placed in Stuart transport medium (Merck) and processed within 2 h of collection. The specimens were inoculated onto Brucella agar (Becton Dickinson) enriched with haemin, vitamin K (Becton Dickinson) and 5 % sheep blood (Jousimies-Somer et al., 2002). Part of each specimen was placed in Komkova anaerobic broth [National Centre of Infectious and Parasitic Diseases (NCIPD)], which was boiled for 5–10 min and cooled prior to use. Komkova broth is a cooked-meat medium, containing glucose, gelatin and 0.3 % agar (Tiagunenko & Marina, 1990). After inoculation, the Komkova anaerobic broth was overlaid with 1–2 ml sterile liquid paraffin and incubated at 37 °C. The broth was subcultured after 48–72 h on enriched Brucella blood agar. A direct smear was made and examined after Gram staining with 0.1 % basic fuchsin as a counterstain. The specimens were plated on blood agar plates as an aerobic control. Anaerobic media were incubated using GasPak anaerobic system envelopes (Becton Dickinson) or Anaerobe Pack (NCIPD) at 37 °C for up to 7 or 14 days, when actinomycosis was clinically suspected. Anaerobic strains were identified by Gram stain, colonial morphology, aerobic control, susceptibility to special potency discs, catalase, spot indole and API system Rapid ID 32 A (bioMérieux) (Jousimies-Somer et al., 2002). The special potency discs (Rosco and Becton Dickinson) contained oxgall, kanamycin (1000 µg), vancomycin (5 µg), colistin (10 µg) and metronidazole (5 µg).
Antibacterial susceptibility testing. The antibacterial susceptibility of 151 anaerobic strains was evaluated by using an agar dilution method with two to three consecutive concentrations (National Committee for Clinical Laboratory Standards, 2004). Enriched Brucella blood agar plates containing the following agents were used (µg ml–1): amoxicillin (0.5, 1 and 2), clindamycin (2 and 4), ampicillin/sulbactam (8/4 and 16/8) and metronidazole (8, 16 and 32). Antimicrobial agents were obtained from Sigma (amoxicillin, metronidazole and clindamycin) and Pfizer (ampicillin/sulbactam). The bacterial inoculum corresponded to 0.5 McFarland standard and the final inoculum was about 105 c.f.u. per spot (National Committee for Clinical Laboratory Standards, 2004). When no growth was observed on the plate after 48 h of anaerobic incubation, the isolate was considered to be susceptible to the agent. Breakpoints for intermediate susceptibility and resistance to amoxicillin (for Gram-negative anaerobes), clindamycin, ampicillin/sulbactam and metronidazole were 1 and 2, 4 and 8, 16/8 and 32/16, and 16 and 32 µg ml–1, respectively (National Committee for Clinical Laboratory Standards, 2004). Amoxicillin breakpoints have been considered to be equivalent to ampicillin breakpoints because, according to in vitro data, the MICs for ampicillin and amoxicillin against anaerobes have been reported to be identical (National Committee for Clinical Laboratory Standards, 2004).
Enriched Brucella blood agar plates without antibacterial agents were used for growth and purity controls for the strains (by anaerobic incubation) and aerobic/facultative contaminant control (by aerobic incubation). The control strains used were two laboratory anaerobic isolates (Prevotella intermedia and Clostridium perfringens) with known antibiotic MICs.
Statistical analysis. Differences between groups were compared by using chi-square test with or without Yates' correction factor. Yates' correction factor for continuity was included in the calculation of chi-square values for 2x2 tables when the expected frequency was <10 in one or more cells.
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RESULTS AND DISCUSSION |
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TOPINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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The predominant anaerobic bacteria were Prevotella (49 strains), Fusobacterium species (22), Actinomyces spp. (21), anaerobic cocci (20) and Eubacterium spp. (18) (Table 1
). Microaerophilic streptococci were found in 28 (23.7 %) of the specimens and, in most cases (89.3 %), were associated with anaerobes. Prevotella intermedia, Fusobacterium nucleatum, Prevotella melaninogenica and the Bacteroides fragilis group were the most common Gram-negative anaerobic species, accounting for 9.2, 9.2, 7.5 and 4 %, respectively, of all anaerobic strains. Bacteroides fragilis group strains included Bacteroides fragilis (two strains), Bacteroides vulgatus (one), Bacteroides distasonis (one) and three other strains. Gram-positive anaerobic cocci (GPAC) were detected in 16 (13.6 %) specimens and Finegoldia magna accounted for 37.5 % of all GPAC strains. About half of the 21 Actinomyces strains belonged to Actinomyces odontolyticus. Among the aerobic/facultative isolates from the patients of the University Hospital of Maxillofacial Surgery in 2002–2005, 68 % were Gram-positive cocci, 30.5 % were Gram-negative bacteria and 1.5 % were Candida species.
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The resistance rate to amoxicillin of Gram-negative anaerobes was 26.9 % (21 of 78 strains). Resistance rates to clindamycin and metronidazole of Gram-negative anaerobes were 5.4 % (4 of 74) and 2.5 % (2 of 79), respectively, and those of Gram-positive species were 4.5 % (3 of 66) and 58.3 % (42 of 72), respectively. Only one strain was not susceptible to ampicillin/sulbactam.
In the present study, the predominant anaerobic species were similar to those reported by Brook (2004); however, isolates of Porphyromonas spp. were relatively rare. The involvement of microaerophilic streptococci was considered as, recently, members of the ‘Streptococcus milleri’ group have been recognized as important pathogens in head and neck abscesses (Han & Kerschner, 2001).
The detection rate of anaerobes from patients with deep-space head and neck infections was relatively lower than that (82–100 %) observed by Jousimies-Somer et al. (2002), but was higher than that (21–59.3 %, according to the sources of infection) reported by Huang et al. (2006). Detection rates of anaerobes were similar in children (75 %, 3 of 4 cases), adults (74.8 %, 77 of 103) and the elderly (72.7 %, 8 of 11; P>0.20). The rate of isolation of anaerobes from empirically treated patients was slightly lower (72.4 %, 63 of 87) than that from non-treated patients (80.6 %, 25 of 31; P>0.20).
The rate of isolation of Fusobacterium species from non-treated patients (32.2 %, 10 of 31) was higher than that from treated patients (13.8 %, 12 of 87, P<0.05), whereas no significant difference (P>0.10) was observed between groups for Prevotella spp. The start of empirical treatment appears to influence the frequency or rate of isolation of Fusobacterium species.
Species of the Bacteroides fragilis group have been detected in single cases of head and neck (0.9 %) and pleuropulmonary (0.3 %) infections (Jousimies-Somer et al., 2002). In the present study, Bacteroides fragilis group species were isolated more often (in 5.9 %, 7 of 118 specimens) and accounted for 4 % of all anaerobic strains. Similarly, these organisms accounted for 5.7 % of anaerobic isolates from the respiratory tract, according to Piérard et al. (2003).
Clostridia are unusual isolates in head and neck infections (Jousimies-Somer et al., 2002). In the present study, a metronidazole-resistant Clostridium tertium strain was found in association with Prevotella corporis and Propionibacterium acnes in a treated patient with cellulitis of the floor of the mouth. Metronidazole resistance has been reported in Clostridium tertium and some other clostridial species (Miller et al., 2001; Peláez et al., 2002; Speirs et al., 1988).
For the Gram-negative anaerobes, the rates of non-susceptibility to amoxicillin (32 %, 25 of 78 strains) and clindamycin (13.5 %, 10 of 74) were lower than those to penicillin (81.8 %) and clindamycin (31.1 %) in Greece (Papaparaskevas et al., 2005). Penicillin resistance has been found in 83 % of Prevotella isolates (Aldridge et al., 2001), as well as in 32–35 % of those in odontogenic infections (Kuriyama et al., 2001). In the present study, amoxicillin resistance was present in 10 (21.7 %) of 46 Prevotella strains. One (6.7 %) of 15 Fusobacterium strains was amoxicillin resistant and three (20 %) strains were intermediately susceptible to the agent. ß-Lactam-resistant Porphyromonas species have been reported by Aldridge et al. (2001), but have not been detected in other studies (Bahar et al., 2005; Kuriyama et al., 2001). In the present work, one of three Porphyromonas strains was amoxicillin resistant.
Amoxicillin resistance in Gram-negative anaerobes from patients treated with ß-lactams was slightly more common (34 %, 17 of 50) than in those from other patients (14.3 %, 4 of 28; P>0.10) (Table 2). Low rates of non-susceptibility to both amoxicillin and metronidazole were detected in Gram-negative anaerobes (1.3 %, 1 of 78 strains). However, it is important to stress that ß-lactamase testing of anaerobic organisms is useful and recommended (National Committee for Clinical Laboratory Standards, 2004), because all ß-lactamase-positive Gram-negative anaerobes should be considered as resistant, independently of their ampicillin MIC values. In addition, for Gram-positive anaerobes, there is no ampicillin breakpoint. The susceptibility breakpoint for Gram-positive anaerobes should be higher than that for Gram-negative anaerobes (Dubreuil et al., 1999). Therefore, although in the present study three Gram-positive anaerobic strains exhibited amoxicillin MICs of >1 µg ml–1, they should not be considered as amoxicillin-resistant strains.
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Ampicillin/sulbactam was the most active agent evaluated. Orofacial anaerobes are usually susceptible to ampicillin/sulbactam and amoxicillin/clavulanate (Kuriyama et al., 2000), although recent studies have reported a decreased activity of these agents against 5–8 % of Bacteroides fragilis group strains and some Peptostreptococcus anaerobius isolates (Aldridge et al., 2001; Kato et al., 2000; Koeth et al., 2004). Intermediate susceptibility to amoxicillin/clavulanate has been detected in single Prevotella strains by Wexler et al. (2002). In the present study, one Prevotella oralis strain was both amoxicillin resistant and intermediately susceptible to ampicillin/sulbactam. No resistance to ampicillin/sulbactam was observed in Bacteroides fragilis group strains, although one ampicillin/sulbactam-resistant Bacteroides fragilis group isolate was detected in a patient (not involved in the study) with malignancy and maxillofacial wound infection in 2002.
In conclusion, the wide diversity and susceptibility patterns of anaerobic species motivate the use, wherever possible, of anaerobic microbiology in maxillofacial surgery departments. The start of empirical treatment could influence the frequency or rate of isolation of Fusobacterium species. Involvement of the Bacteroides fragilis group in some severe head and neck infections should be considered.
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REFERENCES |
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TOPINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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