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Indian Journal for the Practising Doctor

Original Paper: Bacteriological Profile of Osteomyelitis with Special Reference to Staphylococcus aureus

Author(s): Kaur J, Gulati VL, Aggarwal A, Gupta V

Vol. 4, No. 6 (2008-01 - 2008-02)

Kaur J, Gulati VL, Aggarwal A, Gupta V

ISSN: 0973-516X


Dr Jaspal Kaur, Dr V L Gulati and Dr Aruna Aggarwal are from the Department of Microbiology, Government Medical College, Amritsar.
Dr Varsha Gupta is from the Department of Microbiology, Government Medical College, Chandigarh.

Address for correspondence: Dr Jaspal Kaur , 908-A, Sec-43, Chandigarh.
[Ph: 9872179579; E-mail: jasmicro (at) rediffmail.com]


Abstract

Osteomyelitis continues to be an important cause of morbidity. Advances in the identification of infections and early diagnosis of osteomyelitis have lead to improved management of osteomyelitis. Only a few studies of osteomyelitis have been conducted in India to find out aetiology, diagnostic criteria and treatment options used here. Thus, this study was undertaken to determine the bacteriological profile of osteomyelitis and the antibiotic resistance pattern of various isolates obtained.

A total of 100 patients of osteomyelitis either attending the outpatient department or admitted in the wards of a teaching and tertiary care hospital in North India from Aug 2002 to Jan 2005 were included in the study. All those patients who were clinically and/or radiologically suspected of having osteomyelitis were enrolled as cases. Pus/pus swabs or sequestrum samples taken aseptically were cultured aerobically at 37°C for 18-24hrs on Blood and MacConkey agar plates. Culture isolates were identified by a series of standard biochemical reactions. Antibiotic susceptibility was tested on Mueller Hinton agar by Kirby Bauer disc diffusion method. Betalactamase production of S. aureus strains was verified by iodometric filter paper and acidometric agar plate methods. S. aureus strains were screened for methicillin resistance by using conventional microbiological methods.

S. aureus turned out to be the most comm organism isolated (43%). Other organisms isolated were P. aeruginosa (10%), Proteus spp. (6%), Klebsiella spp. (5%), E.coli (5%), Enterobacter spp. (3%), S. epidermidis (4%), Streptococcus pyogenes (2%) and Enterococcus spp. (2%). 20% of the cultures were sterile.

Beta-lactamase production and methicillin resistance was seen in 81.4% and 27.9% of S. aureus strains respectively. Multidrug resistance was observed in 55.8% of S. aureus strains, 50% S. epidermidis, 50% Enterococcus spp., 60% E.coli, 60% Klebsiella spp., 66.7% Enterobacter spp., 50% Proteus spp. and 80% of the Pseudomonas strains. Infections caused by Methicillin-resistant S. aureus (MRSA) and multidrug resistant organisms are posing a major challenge in the treatment of osteomyelitis. So, appropriate drug selected by antibiotic sensitivity testing should be used to treat osteomyelitis.

Key words: Aetiology, antibiotic resistance, osteomyelitis


Introduction

Although with the advent of antibiotics and chemotherapeutic agents, incidence of osteomyelitis has decreased to some extent, yet it continues to be a major problem in our country. In the absence of early diagnosis and prompt treatment or failure of antibiotic therapy due to development of drug resistance, osteomyelitis is still an important cause of high morbidity.1 With the increased prevalence of Methicillin-resistant S. aureus (MRSA) in many hospitals, infections caused by antibiotic-resistant strains are becoming more common. Of particular concern is the increasing resistance to routine antibiotics among pathogenic organisms ever since the introduction of first β-lactam antibiotic, penicillin, in 1940.2 The increasing prevalence of multidrug resistance and β-lactamase production among pathogenic bacteria is posing a major challenge by limiting the number of treatment options which can be used.

Material and Methods

A total of 100 patients with osteomyelitis either attending the outpatient department or admitted in the wards of a teaching and tertiary care hospital in North India from Aug 2002 to Jan 2005 were included in the study. All those patients who were clinically and/or radiologically suspected of having osteomyelitis were taken as cases. Important parameters like age, sex, site of involvement, signs and symptoms, duration of illness, and any predisposing factors were taken into consideration. The specimens which comprised of sequestrum or pus/pus swabs taken from the depth of the wound under strict aseptic conditions were transported to the laboratory in Stuart’s transport media. Direct smear examination of the samples was done. The specimens were inoculated on to Brain Heart Infusion broth and Blood and MacConkey agar plates and incubated aerobically at 37°C for 18-24hrs. The isolates were identified by culture characteristics, gram staining and series of standard biochemical reactions.3

Antibiotic sensitivity was put on Mueller Hinton agar by Kirby Bauer disc diffusion method.4 The antibiotic discs used were – penicillin (10IU), cephalexin (30μg), cefotaxime (30μg), ciprofloxacin (5μg), erythromycin (5μg), gentamicin (10μg), amikacin (30μg), piperacillin (30μg) vancomycin (30μg) and linezolid (30μg) for gram-positive isolates and cefotaxime (30μg), ciprofloxacin (5μg), gentamicin (10μg), amikacin (30μg), piperacillin (30μg) and cefoperazonesulbactum (50μg) for gram-negative isolates. All the antibiotic discs used were obtained from Hi- Media Laboratories Pvt. Ltd, Mumbai. For screening S. aureus strains for methicillin resistance, methicillin (5μg) discs were used on Mueller-Hinton agar supplemented with 4% NaCl and plates were incubated at 35°C for 24hrs. All methicillin-resistant S.aureus strains were also confirmed by oxacillin screening using 6μg/ml of oxacillin in Mueller-Hinton agar supplemented with 4% NaCl5. Beta-lactamase production of the S. aureus strains was studied by filter paper iodometric and acidometric agar plate method.3

Results

The highest incidence of osteomyelitis (36%) was seen in the 16-30 yr age group 16-30yrs followed, in order, by age groups of 31-45yrs (34%), 0-15yrs (17%), and > 46yrs (13%). Male patients outnumbered the female patients by a ratio of 3.3:1. The commonest predisposing factor was compound fractures followed by post-operative and prosthetic infections (Table 1).

Table 1: Predisposing factors in 100 cases of osteomyelitis

S. N. Predisposing factors No. of cases
1. Compound fractures due to road side
and other accidents
41 (41)
2. Post-operative/Prosthetic infections 15 (15)
3. Diabetes mellitus 15 (15)
4. Soft tissue injuries & infection 11 (11)
5. Blunt trauma 9 (9)
6. Others -otitis media, intra-abdominal abscess,
sinusitis, tonsillitis, peritonsillar abscess,
dental abscess or no significant
predisposing factor
9 (9)

Figures in parenthesis indicate percentages.

Long bones of the lower extremity were involved in 60% cases, while those of upper extremity were involved in 14% cases. The bones involved in rest of the cases included short bones of hands and feet (10%), pelvic bones (8%),

Table 2: Total number and percentages of various isolates obtained in 100 cultures.

S.N. Organism No. of
isolates
1. S. aureus 43 (43)
2. Pseudomonas aeruginosa 10 (10)
3. Proteus spp. 6 (6)
4. Klebsiella spp. 5 (5)
5. E.coli 5 (5)
6. S. epidermidis 4 (4)
7. Enterobacter spp. 3 (3)
8. Streptococcus pyogenes 2 (2)
9. Enterococcus spp. 2 (2)

Figures in parenthesis indicate percentages.

vertebral bones (4%) and bones of face (4%). Tibia was the most commonly involved bone (32% cases) followed by femur (23%). In all the 17 pediatric patients (0-15 yrs), bones involved were long bones and in 11 of them, bones of lower extremity were affected.

Twenty five percent patients, mostly in the pediatric age group (15 yr), presented with acute osteomyelitis with fever, lethargy, tenderness at the affected site, and decreased range of motion. Adults mostly presented with sub-acute or chronic form of osteomyelitis with deformity, instability and draining sinus tracts.

Table 3: Organisms isolated by various workers from cases of osteomyelitis

Worker Year Culture +vity S. aureus E.coli Klebsiella
spp.
Proteus
spp.
P. aeruginosa Enterobacter
spp.
Waldvoge17 1970 95.3% 59.4% ٭ ٭ ٭ ٭ ٭
Bhattacharya17 1974 95.2% 48% 3.4% 3.7% 8.7% 15% 3.2%
Dich18 1975 85% 59% ٭ ٭ ٭ ٭ ٭
Arora15 1977 95 42% ٭ ٭ 27.3% ٭ ٭
Henry et al19 1990 ٭ 42.2% ٭ 6.9% 3.5% 17.25% 11.8%
Perry et al20 1991     3.3% ٭ 8.5% 20% 5%
Dormans and Drummond21 1994 85-90% ٭ ٭ ٭ ٭ ٭ ٭
Present study 2004 80% 43 5% 5% 6% 10% 3%

٭ Data not available

Table 4: Antibiotic resistance pattern of Gram positive isolates

Antibiotic S. aureus S. epidermidis Enterococcus spp. Strep pyogenes
n=43 n=4 n=2 n=2
Penicillin 39(90.7%) 3(75%) 2(100%) 0
Methicillin 12(27.9%) 1(25%) -
Cephalexin 26(60.5%) 3(75%) 2(100%) 0
Cefotaxime 18(41.9%) 2(50%) 2(100%) 0
Ciprofloxacin 19(44.2%) 3(75%) 1(50%) 0
Erythromycin 18(41.9%) 2(50%) 1(50%) 0
Amikacin 11(25.6%) 1(25%) 1(50%) 0
Gentamicin 32(74.4%) 3(75%) 1(50%) 0
Piperacillin 14(32.6%) 2(50%) 1(50%) 0
Linezolid 1(2.3%) 1(25%) 0 0
Vancomycin 0 0 0 0
MDR٭ 24(58.8%) 2(50%) 1(50%) 0

٭Multi-drug resistant

Table 5: Antibiotic resistance pattern of Gram negative isolates

Antibiotic E.coli Klebsiella spp. Enterobacter spp. Proteus spp. P. aeruginosa
n=5 n=5 n=3 n=6 n=10
Ampicillin 4(80%) 5(100%) 2(66.7%) 3(50%) -
Cefotaxime 3(60%) 3(60%) 2(66.7%) 4(66.7%) 9(90%)
Ciprofloxacin 4(80%) 3(60%) 2(66.7%) 5(83.3%) 7(70%)
Amikacin 1(20%) 2(40%) 1(33.3%) 3(50%) 5(50%)
Gentamicin 4(80%) 4(80%) 3(100%) 4(66.7%) 9(90%)
Piperacillin 2(40%) 2(40%) 1(33.3%) 5(83.3%) 5(50%)
Sulbactum-cefoperazone 1(20%) 1(20%) 0 0 2(20%)
MDR 3(60%) 3(60%) 2(66.7%) 3(50%) 8(80%)

Out of 100 cultures put up, growth of organisms was obtained in 80 (80%); 20% cultures were sterile. S. aureus was the commonest organism isolated (43%) (Table 2). Age-wise distribution of organisms showed S. aureus to be the commonest aetiological agent in children (0-15yrs). In 12 out of 17 children affected, the organism isolated was S. aureus while in the higher age groups (>15 yr) gram-negative organisms were predominantly obtained.

Beta-lactamase production was seen in 35 (81.4%) of S. aureus strains and 12 (27.9%) strains were MRSA (Table 4). All MRSA strains were found to produce β-lactamase and were multidrug resistant. Multidrug resistance (resistance to ≥ 3 drugs) was seen in 24 (55.8%) S. aureus strains (Table 4). There was marked difference in antibiotic sensitivity pattern of MRSA vs MSSA isolates (Fig 1). High level of resistance was also observed in gram-negative organisms with majority of the isolates showing multi-drug resistance (Table 5).

antibiotic sensitivity pattern of MRSA vs MSSA isolates

Discussion

Osteomyelitis remains a vexing illness despite major advances made in surgery and antimicrobial therapy. The advent of prosthetic joints has added new dimensions to the challenges of septic arthritis and osteomyelitis, as these are prone to become infected by a wide range of organisms including low-grade pathogens.1 Widespread use of antibiotics has altered aetiological pattern of infections and antibiotic susceptibility.

The highest incidence of osteomyelitis was observed in the 16-30 yr age group, which is attributable to the greater likelihood of trauma and compound fractures at this age; this turned out to be the commonest predisposing factor in our study (Table 1). Trauma results in hemorrhage and cell destruction in the region of epiphyseal cartilage followed by diminished tissue resistance.6 Postoperative and prosthesis-related infections, which formed the second most important predisposing factor in our study, arise from exogenous and endogenous sources. The patient’s skin is the most common source of contamination even though sporadic outbreaks traced to the operating or ancillary personnel have been reported. Metastatic source of infection may be involved as well, e.g. percutaneous sutures, suction drains, intravenous catheters and indwelling urinary catheters.6

Our study contrasts with Waldvoge17 and Okoroma8 who reported maximum incidence of osteomyelitis in the age group of 3-15 yrs but is in accordance with Esperson9 who described a fall in the incidence of infection in pediatric age group. The decreased incidence observed among children could be due to improved standards of living and early administration of broad-spectrum antibiotics.

In children, bones of lower extremity are more often affected and of these, upper end of tibia and lower end of femur are more liable to infection because of greater amount of growing bones in these areas. Detailed studies by Hobo10 of the vasculature adjacent to the metaphyseal side of growth plate have provided the most satisfactory explanation for the involvement of growing end of long bones. Adult osteomyelitis may occur at any site. In our study long bones were more commonly involved, particularly those of the lower extremity.

In children, S. aureus was the commonest organism isolated because in them the infection is mostly hematogenous while in adults, gram negative organisms are commonly isolated because infection is commonly secondary to contiguous focus of infection. Male patients outnumbered the females. This has been attributed to more exposure to trauma in males.

Although bone and joint infections caused by gram-negative organisms have significantly increased but S. aureus remains the most common cause of osteomyelitis till date (Table 3) and MRSA strains are further complicating the situation. Osteomyelitis caused by MRSA often follows multiple trauma, and is usually hospital acquired. There was a marked difference in the antibiotic sensitivity pattern of MRSA vs MSSA isolates (Fig.1). Beta-lactam antibiotics like penicillin and cephalexin were not effective against MRSA. A high resistance to quinolones (75%) was seen which correlates with studies from other parts of the country11,12. An increase of gentamicin resistance among MRSA strains from 0% prior to 1996 to 80% after 1996 has been reported.13 In our study MRSA showed 83.3% resistance to gentamicin. Beta-lactamase production was seen in 35 (81.4%) of S. aureus strains. All MRSA strains were found to produce β-lactamase and were multi-drug resistant. Multidrug resistance among MRSA strains has serious implications as far as treatment of MRSA infections is concerned. It also highlights the need to test newer group of antibiotics routinely like linezolid and vancomycin. Linezolid and vancomycin showed 91.7% and 100% sensitivity to MRSA respectively. Alternative drugs like teicoplanin and quinu+pristine-dalfopristine have also shown promising results.14

For gram-negative organisms, cefoperazone-sulbactum combination was the most effective drug followed by amikacin. Ampicillin was found to be highly ineffective drug against gram negative infections with 100% resistance in Klebsiella spp. and 80% in E.coli. Gentamicin once found to be most effective drug for the treatment of osteomyelitis has now become a resistant drug.15 In our study, gentamicin showed only 16.2% sensitivity among gram negative isolates.

P. aeruginosa was found to be highly resistant gram-negative organism with resistance to gentamicin, ciprofloxacin and piperacillin, as high as 90%, 70%, and 50% respectively. In various studies investigating the resistance of P. aeruginosa to ciprofloxacin, the resistance was reported to be 0 to 89%.16

Multi-drug resistance among pathogenic organisms poses a major challenge in the treatment of infections and increase the morbidity and mortality associated with these infections. A finding of greater concern is the progressively developing resistance to cefoperazone-sulbactum (beta-lactam + beta-lactamase inhibitor) combination among gram-negative isolates since antibiotics of choice in the treatment of infections due to β-lactamase producing microorganisms are either limited to these combinations or to the carbapenems.

Conclusion

As S. aureus is the most common etiological agent of osteomyelitis, any first line antibiotic should be primarily directed against this pathogen. For coverage of gram-negative bacteria, beta-lactam+beta-lactamase inhibitor combination would be more useful. Use of monodrug therapy needs to be guided by sensitivity report. Lastly, continuous monitoring of susceptibility pattern needs to be carried out in individual setting so as to detect the true burden of antibiotic resistance among organisms and prevent their further emergence by judicious use of drugs.

References

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