Microbiological profile of chronic osteomyelitis with special reference to anaerobic osteomyelitis in a tertiary care hospital of coastal Karnataka

Abstract

Chronic osteomyelitis is a relapsing, persistent, low-grade inflammation of bone caused by various infectious agents. The present study, conducted over a two-year period, on specimens received from cases of chronic osteomyelitis was, to determine the frequency of isolation of aerobic and anaerobic bacteria and to analyse their antimicrobial susceptibility pattern. Specimens were processed for Gram stain, aerobic and anaerobic culture, and were identified according to standard techniques. Significant growth was observed in 102/204 specimens, in which aerobic growth was observed in 62 (60.8%) and anaerobic in 40 (39.2%). Resistance to metronidazole and clindamycin was observed in 6.7% and 30% of the anaerobic isolates, respectively. None of these were resistant to meropenem. A significant proportion of anaerobic isolates were found to be resistant to commonly used empirical drugs, such as clindamycin, thus necessitating a need for routine anaerobic susceptibility testing.

Keywords

Anaerobic osteomyelitis microbiological profile clindamycin metronidazole antimicrobial resistance

Introduction

Osteomyelitis is an infection of bone marrow, and may be acute or chronic based on its period of evolution and host immune response.^1,2 Chronic osteomyelitis is a relapsing, persistent, low-grade inflammation of bone characterised by formation of sequestrum (dead bone), new bone opposition and fistulous tracts.² It usually results from contiguous spread persisting for several weeks and it may not necessarily result from haematogenous seeding or from penetrating injury.³

Aerobic organisms are commonly involved in the causation of chronic osteomyelitis and anaerobes are very rarely encountered. Staphylococcus aureus is the frequent isolate among aerobes; however, Gram-negative aerobes, especially members of Enterobacteriaceae family, are recently becoming more frequent.^4,5 Osteomyelitis involving anaerobes is usually polymicrobial in nature with the commonest anaerobes encountered being Bacteroides, Fusobacterium, Peptostreptococcus and Clostridium species.^6,7 Propionibacterium spp. is commonly associated with bone infection associated with prostheses.⁶ Anaerobic osteomyelitis is more common among individuals with diabetes mellitus and any underlying disease leading to vascular insufficiency or in those with a history of trauma, including surgery.^6,8,9

Chronic osteomyelitis remains a therapeutic challenge even in rich countries; its high morbidity and disabling sequelae are common in low- and middle-income countries.¹ Its management involves surgical intervention and, secondarily, antibiotic therapy. The microbiological diagnosis of chronic osteomyelitis depends on the proper collection and transport of good quality specimens combined with an extensive bacteriological workup.¹⁰

The surge in antimicrobial resistance and consequent higher rates of relapse means treating this ailment still demands extensive expertise.^3,4 In view of the paucity of data regarding chronic anaerobic osteomyelitis, our study was conceived.

Materials and methods

A prospective cross-sectional study was conducted from March 2015 to February 2017 after obtaining clearance from Institutional Ethics Committee. The specimens including pus aspirates, infected tissue/biopsy and bone biopsy samples from patients with a clinical diagnosis of chronic osteomyelitis were included in the study. The demographic details and data pertaining to predisposing factors were obtained from medical records. The specimens collected under aseptic precautions during surgical procedures or in the ward were directly inoculated into a sterile wide-mouth container and Robertson’s cooked meat broth (RCM) for aerobic and anaerobic bacterial isolation, respectively. These specimens were transported to our microbiology laboratory immediately for further processing. Aerobic and anaerobic cultures were performed according to standard guidelines.^11,12 The inoculated anaerobic culture plates were incubated in an anaerobic workstation (Don Whitley Scientific, Shipley, UK) and were observed daily for growth for five days. The inoculated RCMs were incubated for 14 days and Gram stain was performed to look for any additional morphotypes. Identification of aerobic and anaerobic isolates was done by automated microbial identification system, matrix-assisted laser desorption ionisation time-of-flight mass spectrometry (MALDI-TOF Vitek MS) (bioMérieux Inc.).

The antimicrobial susceptibility for aerobic isolates was done by Vitek 2 systems (bioMérieux, Inc., Durham, NC, USA), and for anaerobic isolates by E test (bioMerieux, Marcy l’Etoile, France). Minimum inhibitory concentrations (MICs) were interpreted according to Clinical Laboratory Standards Institute (CLSI) guidelines.¹³

The susceptibility testing for anaerobic isolates was performed against metronidazole (range = 0.016–256 µg/mL), clindamycin (range = 0.016–256 µg/mL) and meropenem (range = 0.002–32 µg/mL). The bacterial suspension of 1 McFarland was prepared in normal saline and lawn culture was made on 5% sheep blood agar. The plates were incubated in anaerobic atmosphere (80% N_2, 10% CO_2, 10% H₂) at 37℃ for 48 h. The MICs were read at the point where the elliptical zone intersected with the test strip. B. fragilis ATCC 25285 was used as reference strain for quality control.¹³

Results

Of the various specimens (n = 204) received during the study period, growth of pathogenic bacteria was observed in 102 (50%) (Table 1). Out of these, pure growth of aerobic bacteria was obtained in 62/88 (60.8%) isolates (Table 2, Figure 1a) and anaerobic growth in 40/57 (39.2%) isolates (Figure 1b).

Figure 1.

(a) Frequency of isolation of aerobic bacteria from cases of chronic osteomyelitis. (b) Frequency of isolation of anaerobes from cases of chronic osteomyelitis.

Table 1.

Details of isolation of organisms from various clinical specimens (n = 204).

Specimen type	Specimens processed	Specimens with bacterial yield
Soft tissue	146 (71.5)	77 (52.7)
Pus aspirates	43 (21.1)	19 (44.2)
Bone specimens	15 (7.4)	6 (40)

Values are given as n (%).

Table 2.

Nature of microbial growth in various clinical specimens.

Type of microbial growth
Monomicrobial aerobic growth	59 (57.8)
Monomicrobial anaerobic growth	16 (15.7)
Polymicrobial aerobic growth	3 (2.9)
Polymicrobial anaerobic growth	7 (6.9)
Mixed aerobic and anaerobic growth	17 (16.7)
Total	102 (100)

Values are given as n (%).

Klebsiella pneumoniae and S. aureus were the aerobes most commonly associated with anaerobic isolates among mixed infections. Among the 16 monomicrobial anaerobic isolates, Clostridium spp. (n = 6) were the predominant anaerobes followed by Peptostreptococcus anaerobius (n = 4), B. fragilis (n = 3) and others (n = 3).

We found the most commonly affected age group was 41–60 years (n = 31, 29.8%) and a predominance in male gender (n = 82, 78.8%) was observed. Tibial bone involvement (Table 3) was common (n = 33, 32.4%). Diabetes mellitus and trauma were found in more than half of the study population (58%). Clostridium spp. were encountered from cases of trauma (n = 18).

Table 3.

Distribution of various infection sites.

Infection site
Tibia	33 (32.4)
Femur	22 (21.6)
Humerus	8 (7.8)
Radius	8 (7.8)
Metatarsals	8 (7.8)
Metacarpals	7 (6.9)
Ulna	6 (5.9)
Malleolus	4 (3.9)
Vertebra	3 (2.9)
Fibula	2 (2)
Clavicle	1 (1)
Total	102 (100)

Values are given as n (%).

Antimicrobial susceptibility testing was performed randomly on 30 anaerobic isolates, due to financial constraints, which included, F. magna (n = 7), Clostridium spp. (n = 7), B. fragilis group (n = 6), P. anaerobius (n = 5), Prevotella spp. (n = 3), Fusobacterium nucleatum (n = 1) and Peptoniphilus asacharolyticus (n = 1).

Metronidazole resistance was observed among 2 (6.7%) isolates of the B. fragilis group. Significant resistance was observed against clindamycin (n = 9, 30%), among Peptostreptococcus anaerobius (n = 2, 6.7%), B. fragilis group (n = 2, 6.7%), Prevotella spp. (n = 2, 6.7%), Clostridium sporogenes (n = 2, 6.7%) and F. magna (n = 1, 3.3%). Among the resistant anaerobic isolates, two isolates of the B. fragilis group were found to be resistant to both metronidazole and clindamycin. However, all anaerobic bacterial isolates were susceptible to meropenem.

Discussion

Chronic osteomyelitis is a catastrophic sequel of bone and joint infection that arises on account of delay in diagnosis and management of the acute stage of the disease.^10,14 Contiguous spread from exogenous or endogenous foci is common.^3,15 Prior exposure to antibiotic therapy, tubercular and fungal osteomyelitis that demand specialised culture techniques, the presence of biofilms and failure to recognise small colony variants can all result in negative bacteriological culture reporting.¹⁶

Aerobic organisms are the major culprits in chronic cases of osteomyelitis, with S. aureus being the most frequent pathogen.^1,3,4,10 However, anaerobic bacteria are reported with frequency rates in the range of 2%–29% of infections worldwide.^2,8,17–19 Nevertheless, there are reports of anaerobes being aetiological agents in osteomyelitis in up to 77% of cases.²⁰ In the present study, a high frequency of isolation (39.2%) was observed, which may be the result of stringent specimen collection (intraoperative and aseptic) practices, incorporation of appropriate anaerobic media (RCM) for transport and culture workup performed in anaerobic workstation, which allows for daily inspection of growth and further workup towards identification.

Collection of appropriate and good-quality specimens avoiding contamination with resident flora remains an elementary component for laboratory diagnosis of bone and joint infection.²¹ Bone biopsy and deep tissue culture specimens are preferred.^{15,19,21–23} Incorporating appropriate anaerobic transport media can also improve rates of anaerobe isolation.^12,20

The management of chronic osteomyelitis requires long-lasting arrest of the infection which almost inevitably means surgical debridement, dead space management and restoration of bone stability in addition to systemic targeted antimicrobial therapy.^24,25 Biofilm must be countered to avoid treatment failure.²⁶

Regional resistance profile data of prevalent pathogens is essential for the effective management of chronic osteomyelitis cases.

Declaration of conflicting interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) received the following financial support for the research, authorship, and/or publication of this article: This study was conducted as a part of Manipal Academy Higher Education (MAHE) Seed Money Research (2016).

ORCID iDs

Padmaja Ananth Shenoy https://orcid.org/0000-0001-6781-5579 Shyamasunder N Bhat https://orcid.org/0000-0001-9545-4838

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