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BioMed Research International
Volume 2014, Article ID 430581, 5 pages
http://dx.doi.org/10.1155/2014/430581
Research Article

The Bacterial Contamination of Allogeneic Bone and Emergence of Multidrug-Resistant Bacteria in Tissue Bank

1Department of Biochemistry and Microbiology, School of Life Sciences, North South University, Dhaka 1229, Bangladesh
2Department of Microbiology, Gono Bishwabidyalay, Savar 1344, Bangladesh

Received 16 March 2014; Revised 17 June 2014; Accepted 23 June 2014; Published 8 July 2014

Academic Editor: Gundlapally S. Reddy

Copyright © 2014 Fahmida Binte Atique and Md. Masudur Rahman Khalil. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Present study was carried out for the microbiological evaluation of allogeneic bone processed from femoral heads. A total 60 bacterial isolates comprising five different species including Streptococcus spp., Staphylococcus spp., Klebsiella spp., Bacillus spp., and Pseudomonas spp. were characterized based on their cultural and biochemical characteristics. Average bioburden was ranged from to  cfu/gm. The majority (81.7%) of the microbial contaminants were detected as Gram positive with the predominant organism being skin commensal coagulase negative Staphylococci (43.3%). Antimicrobial resistance was evaluated by the activities of 14 broad and narrow spectrum antibiotic discs. Comparing the overall pattern, marked resistance was noted against Penicillin and Amoxicillin 100% (60/60). The most effective single antibiotics were Gentamicin, Tobramycin, and Ofloxacin which were bactericidal against 100% (60/60) isolates. Multidrug resistance (MDR) was confirmed in 70% (42/60) of the samples. Among them, the most prevalent antibiotypes were Penicillin, Amoxicillin, Oxacillin, Polymyxin, and Cefpodoxime (80% of total MDR). The study results revealed higher contamination rate on bone allografts and recommend the implementation of good tissue banking practices during tissue procurement, processing, and storage in order to minimize the chances of contamination.

1. Introduction

Human bone is the second most transplanted tissue after blood which has the unique ability to heal itself perfectly. It is estimated that more than 2.2 million bone grafting procedure annually take place worldwide in order to revise skeletal defects by replacement or augmentation [1]. In addition, bone grafts are also used to repair the defects in bone caused by birth defects, maxillofacial defects, traumatic injury, infections or enbloc resection of malignant tumours and in reinforcement of host bone prior to implantation of prosthesis [26].

Disease transmission and bacterial contamination are always a risk in allograft transplantation [7]. Thorough donor screening for the presence of transmissible diseases, bacterial testing, and aseptic processing practices can substantially reduce the risk but do not completely eliminate all the possible microbial contaminants from allograft [8]. So, for the safety of allogeneic tissue grafts, complete eradication of microorganisms is essential.

The risk of infectious disease transmission emphasizes the need of appropriate sterilization technique in tissue banking practice [9]. But the alteration in the biomechanical properties of particular tissues made it obvious that all forms of sterilization technique are not applicable [10]. Antibiotics has for long time been used to control infectious diseases. Even potentially fatal infections are now curable with the courses of antibiotics. But one of the alarming matters is that despite the development of new antibiotics with novel mechanism of action, it has become difficult to control the local bacterial prevalence and emergence of infectious diseases due to their resistance to the common antibiotics. Bacteria can defend themselves from the action of antibiotics by producing various metabolites which either degrade antibiotics or help bacteria to survive by various mechanisms.

2. Materials and Methods

2.1. Tissue Sample Collection

Tissue samples were collected through the donation of femoral heads removed during hip replacement, hemiarthroplasty, and traumatic limb amputation surgery from eleven hospitals of the Dhaka city including BDM Hospital, Center for Rehabilitation of Paralyzed Hospital, National Institute of Traumatology, Orthopaedic and Rehabilitation Hospital, Bangabandhu Sheikh Mujib Medical University Hospital, Ibn Sina Hospital, Bangladesh Medical College Hospital, Islami Bank Hospital, Central Hospital, Shikdar Medical college Hospital, and Al-Markajul Hospital and Trauma Center.

2.2. Tissue Donor Identification and Screening

All the tissues were collected by the written consent of the donor or next of kin by following “Human Organ/Tissue Donation and Transplantation Act” that has been passed by the National Parliament of the People’s Republic of Bangladesh. The ages of donors were ranged from 40 to 75 years and all the donors were prescreened for the presence of transmissible diseases (e.g., HIV, HBV, and VDRL).

2.3. Initial Laboratory Processing and Bioburden Estimation

Fresh bones were collected under aseptic/sterile condition. During collection each container was labeled with donor ID and hospital registration number and kept at freezer (below −20°C). The plastic container with bone is placed in a cool box and transported immediately to the tissue banking laboratory. In the tissue banking laboratory the bones were preserved in freezer at −40°C. For the isolation, tissue samples were weighed by digital balance and taken into a sterile beaker containing 150 mL sterile normal saline and/or sterile distilled water. After using the orbital shaker the beaker containing the sample was gently shaken. 10 mL of suspension was taken by sterile pipette, which was sterilized by a sterilizer (at 180°C for 1 hour) into a test tube from the beaker. Then the sample was serially diluted up to 10−4. If discrete colonies were not detected in 10−4 dilution, further dilutions were prepared and the tests were then repeated. All the plates were incubated at 37°C for 24 hours. The bacterial colonies were counted after 24–72 hours.

2.4. Cultural Characterization and Biochemical Studies of Microbial Contaminants

The bacterial isolates, obtained from the selective and differential media, were characterized on the basis of their morphology (size, shape, and arrangement) by following Gram staining procedure. Cultural characteristics of the bacterial isolates were studied after 24–48 hours of incubation using freshly prepared reagents. According to Bargey’s Manual of Determinative Bacteriology [11], several biochemical tests were performed to identify the biochemical characteristics of the bacterial isolates. The tests were Oxidase test, Catalase test, Indole production test, Methyl Red test, Voges-Proskauer test, Urease test, Citrate utilization test, Triple Sugar Iron test, and Carbohydrate (Lactose, Sucrose, and Dextrose) fermentation tests.

2.5. Antimicrobial Susceptibility Testing

Total 60 bacterial isolates were selected for antibiotic susceptibility test by Kirby-Bauer disc diffusion method described by Bauer et al. [12] using 14 broad and narrow spectrum antibiotic discs. Muller-Hinton agar plates were used to determine the antibiotic susceptibility of the bacterial isolates. A 0.5 McFarland was used as a standard tool to maintain the perfect turbidity. After swabbing with the bacterial suspension, antibiotic disks were placed aseptically over the inoculated media surface and at the same time spatial arrangement was maintained by means of sterile needle within a distance of 5 mm. Then the plates were incubated for 24 hours at 37°C. After the completion of incubation period, the plates were examined and the diameters of the clear zones were measured by a ruler in mm. The zone diameters were translated into susceptible (S), intermediate (I), and resistant (R) categories according to the National Committee for Clinical Laboratory Standards (NCCLS) [13].

3. Results

3.1. Determination of Bioburden in Bone

Microbial evaluation of bone allograft was carried out. A total 60 bacterial isolates obtained from 4 different batches of allograft processing. The bioburden varied from 0.57 to 3.94 Log cfu/gm. Maximum count was recorded for the first batch of processing, ranged from 3.23 to 3.94 Log cfu/gm. The lowest microbial levels from 0.93 to 1.92 Log cfu/gm were observed for the fourth batch. Microbial load of bone allografts from different batches of processing is presented in Figure 1.

fig1
Figure 1: Microbial load of bone allografts from different batches of processing.
3.2. Characterization of Bacterial Isolates

Characterization of the bacterial isolates was performed based on their colony morphology. According to the Gram staining, majority (81.7%) of the microbial contaminants found as Gram positive, in which 67.8% were Gram positive cocci. The second most frequently isolated group was Gram positive bacilli as 13.9%. On the contrary, 18.3% of the microbial contaminants were Gram negative rods. No fungi or yeast were found. Types of microbial contaminants are presented in Figure 2.

430581.fig.002
Figure 2: Types of microbial contaminants enumerated from bone.
3.3. Physiological and Biochemical Studies of the Bacterial Isolates

Several physicobiochemical tests were performed to identify the selected bacterial isolates up to genus level (Table 1). Based on the physiobiochemical characteristics, Twenty-one Gram positive cocci (B1, B5, B7, B14, B17, B19, B31, B32, B33, B34, B35, B39, B41, B42, B44, B45, B48, B50, B52, B58, and B59) were identified as Staphylococcus spp. and twelve Gram positive cocci (B3, B10, B11, B15, B21, B22, B26, B28, B29, B49, B53, and B55) were identified as Streptococcus spp. On the other hand, sixteen isolates of Gram positive rods (B2, B8, B16, B20, B25, B24, B30, B36, B38, B40, B43, B46, B51, B54, B60, and B63) were identified as Bacillus spp. Among the eleven Gram negative rods, eight of the bacterial isolates were Pseudomonas spp. (B4, B12, B18, B23, B27, B47, B56, and B57) and only three of the isolates were Klebsiella spp. (B6, B9, and B13).

tab1
Table 1: Summary of the biochemical tests of bacterial isolates.
3.4. Antibiogram Profile of the Bacterial Isolates

The bacterial isolates () were subjected to antibiotic susceptibility test against 14 antibiotics from different groups including, Penicillin (P), Oxacillin (OX), Gentamicin (G), Erythromycin (E), Clindamycin (DA), Tobramycin (TOB), Ofloxacin (OXF), Polymyxin (PB), Azithromycin (AZM), Levofloxacin (LEV), Imipenem (IPM), Cefpodoxime (CPD), Amoxicillin (AML), and Meropenem (MEM) (Table 2). These antibiotics were selected upon the consideration of two facts: which antibiotics are the commonly prescribed by the physicians and which antibiotics are susceptible against bone contaminants. Disc diffusion method was used to frequently observe the antibiotic effects among the strains.

tab2
Table 2: Antimicrobial susceptibility pattern of the bacterial isolates from bone allograft.

Among the 14 drugs, Penicillin and Amoxicillin were 100% () resistant. On the contrary, Gentamicin, Tobramycin, and Ofloxacin were 100% sensitive. Apart from this, other drugs showed different level of resistance such as Oxacillin (80%), Polymyxin (70%), Cefpodoxime (60%), Imipenem (45%), Meropenem (40%), and Erythromycin (30%). Individual resistance and sensitivity pattern of the bacterial isolates is presented below (Figure 3).

430581.fig.003
Figure 3: Percentages of antimicrobial resistance on bacterial isolates.

Among the 60 bacterial isolates, 70% () were multidrug resistant (MDR). The highest prevalent antibiotic resistance pattern was P, AML, OX, PB, CPD, IPM, E, MEM, and DA showed by bacterial isolates of batch-I. On the other hand, the lowest prevalent antibiotic resistance pattern was showed by batch-III as P, AML, OX, PB, and IPM (Table 3).

tab3
Table 3: MDR pattern of different bacterial isolates.

4. Discussion

The primary focus of our study was to determine the bioburden level of allogeneic bone. Study results showed that most of the samples were contaminated with Gram positive cocci specifically coagulase negative Staphylococci. Cultures were also positive for Streptococcus spp., Pseudomonas spp., Bacillus spp., and Klebsiella spp., respectively. The bacterial isolates found in our study are comparable with the previous reported studies. According to Saegeman et al. [14] in 36–38% cases infection of cadaveric bone and soft tissue allograft occurs due to coagulase negative Staphylococci especially that Staphylococcus epidermidis is the causative agent of disease. Deijkers et al. [15] analysed the bacterial contamination of bone allograft under aseptic operating condition and divided the organisms into low and high pathogenicity in which they considered organisms of low pathogenicity to be skin commensals and microorganisms of high pathogenicity were thought to be originated from endogenous sources in the donor, which more likely to cause infection in the recipient. Though Streptococcus spp. are not usually associated with graft infections, a survey study of tissue bank conducted by Vangsness et al. [16] reported about the invasive bacterial disease in which a 17-year-old male was found to be infected with Streptococcus pyogenes after reconstructive knee surgery. Ibrahim et al. [17] also reported that twelve of their bone allografts were contaminated with streptococci. Emergence of Bacillus subtilis and Micrococcus spp. was also summarized by many authors [18, 19]. Besides bacterial contaminations, environmental exposure, underlying diseases, and host defense mechanism can also contribute to the graft contamination in ratio between 2 and 5% [20].

We think that disease transmission can occur mainly in two ways: either through an infected donor or during tissue procurement, processing, even at the time of surgery in the operating theatre, as it has already been reported with surgical needles and suckers [21]. Bacterial transmission might be occurring from infected donor to recipient (tuberculosis and syphilis) or through viral transmission from infected donor (HIV and Hepatitis) or through bacterial contamination during procurement, processing, and storage of the bone allograft [22].

In order to avoid infection or diminish its incidences in bone allograft, strategies like careful donor selection, aseptic processing, proper use of disinfectants, and application of sterilization procedure with bacterial cultures need to be taken [23]. Even all the procedures are followed carefully, but what should be done if the culture from an implanted allograft is positive. The perioperative administration of systemic antibiotics is the choice to limit the infection which can occur after graft implant. This method is highly effective against bacteria while the effectiveness is depending on the constituents of antibiotics [24]. One of the feared complications is that, in our study, most of the bacterial isolates enumerated from bone showed multidrug resistance (more than one antibiotic) to the supplied antibiotics, as an explanation of such resistance might be the subsequent external contamination of the allograft. To prevent the endovascular graft infections, antibiotics are recommended to be used in the initial postoperative stage of bacterial seeding [25].

5. Conclusion

Bone allografts were found to be contaminated and about 80% of the contaminants were Gram positive. Study results also revealed the growing antimicrobial resistance of pathogens associated with the bone allografts. To minimize the contamination rate and to reduce the risk of dissemination of antibiotic resistant bacteria through the tissue allografts, it is suggested to use aseptic techniques in all the steps of allograft procurement, processing, and storage.

Conflict of Interests

The authors declare that they have no conflict of interests.

Acknowledgments

This study is the part of a thesis work in partial fulfilment of the requirements for the degree of Master of Science in Microbiology, performed by the direct collaboration of North South University, Gono Bishwabidyalay, and Atomic Energy Research Establishment (AERE), Bangladesh. The authors would like to thank Dr. S. M. Asaduzzaman, who has supervised the whole research work. They would also like to thank Naznin Akhtar for her cordial help and assistance throughout the work.

References

  1. M. Farrington, I. Matthews, J. Foreman, K. M. Richardson, and E. Caffrey, “Microbiological monitoring of bone grafts: two years' experience at a tissue bank,” Journal of Hospital Infection, vol. 38, no. 4, pp. 261–271, 1998. View at Publisher · View at Google Scholar · View at Scopus
  2. D. J. Berry, H. P. Chandler, and D. T. Reilly, “The use of bone allografts in two-stage reconstruction after failure of hip replacements due to infection,” The Journal of Bone and Joint Surgery A, vol. 73, no. 10, pp. 1460–1468, 1991. View at Google Scholar · View at Scopus
  3. G. R. Buttermann, P. A. Glazer, and D. S. Bradford, “The use of bone allografts in the spine,” Clinical Orthopaedics and Related Research, no. 324, pp. 75–85, 1996. View at Google Scholar · View at Scopus
  4. H. T. Aro and A. J. Aho, “Clinical use of bone allografts,” Annals of Medicine, vol. 25, no. 4, pp. 403–412, 1993. View at Publisher · View at Google Scholar · View at Scopus
  5. H. Kienapfel, D. R. Sumner, T. M. Turner, R. M. Urban, and J. O. Galante, “Efficacy of autograft and freeze-dried allograft to enhance fixation of porous coated implants in the presence of interface gaps,” Journal of Orthopaedic Research, vol. 10, no. 3, pp. 423–433, 1992. View at Publisher · View at Google Scholar · View at Scopus
  6. P. V. Giannoudis, H. Dinopoulos, and E. Tsiridis, “Bone substitutes: an update,” Injury, vol. 36, supplement 3, pp. S20–S27, 2005. View at Google Scholar · View at Scopus
  7. N. S. Das, J. Dey, M. M. Rahman, H. M. Zahid, A. Nessa, and Ashraf, “Bioburden of human amniotic membranes and inhibition of the associated bacteria using antibiotics and gamma-radiation,” Global Journal of Medical Research, vol. 13, no. 2, article 1.0, 2013. View at Google Scholar
  8. R. Singh and D. Singh, “Gamma irradiated bone allografts processed from femoral heads,” Frontiers in Science, vol. 2, no. 5, pp. 119–126, 2012. View at Publisher · View at Google Scholar
  9. G. Dziedzic and W. Stachowicz, “Sterilization of tissue allografts,” in Advances in Tissue Banking, pp. 261–275, World Scientific Publishing, 1997. View at Google Scholar
  10. V. Saegeman, D. Lismont, B. Verduyckt, N. Ectors, J. Stuyck, and J. Verhaegen, “Antimicrobial susceptibility of coagulase-negative staphylococci on tissue allografts and isolates from orthopedic patients,” Journal of Orthopaedic Research, vol. 75, pp. 543–848, 2006. View at Google Scholar
  11. J. G. Holt, N. R. Krieg, H. A. Sneath, J. T. Staley, and S. T. William, Bergey’s Manual of Determinative Bacteriology, Williams and Wilkins, Baltimore, Md, USA, 9th edition, 1994.
  12. A. W. Bauer, W. M. Kirby, J. C. Sherris, and M. Turck, “Antibiotic susceptibility testing by a standardized single disk method,” The American Journal of Clinical Pathology, vol. 45, no. 4, pp. 493–496, 1966. View at Google Scholar · View at Scopus
  13. NCCLS, “Performance standards for antimicrobial susceptibility testing,” in Proceedings of the 10th Informational Supplement (aerobic dilution), M100-S10 (M7), NCCLS, Wayne, Pa, USA, 2000.
  14. V. Saegeman, D. Lismont, B. Verduyckt, N. Ectors, J. Stuyck, and J. Verhaegen, “Antimicrobial susceptibility of coagulase-negative staphylococci on tissue allografts and isolates from orthopedic patients,” Journal of Orthopaedic Research, vol. 25, no. 4, pp. 543–573, 2006. View at Publisher · View at Google Scholar
  15. R. L. M. Deijkers, R. M. Bloem, P. L. C. Petit, R. Brand, S. B. W. Vehmeyer, and M. R. Veen, “Contamination of bone allografts: analysis of incidence and predisposing factors,” Journal of Bone and Joint Surgery B, vol. 79, no. 1, pp. 161–166, 1997. View at Publisher · View at Google Scholar · View at Scopus
  16. C. T. Vangsness Jr., P. P. Wagner, T. M. Moore, and M. R. Roberts, “Overview of safety issues concerning the preparation and processing of soft-tissue allografts,” Arthroscopy, vol. 22, no. 12, pp. 1351–1358, 2006. View at Publisher · View at Google Scholar · View at Scopus
  17. T. Ibrahim, H. Stafford, C. N. A. Esler, and R. A. Power, “Cadaveric allograft microbiology,” International Orthopaedics, vol. 28, no. 5, pp. 315–318, 2004. View at Publisher · View at Google Scholar · View at Scopus
  18. R. A. Dunsmuir and G. Gallacher, “Microwave sterilization of femoral head allograft,” Journal of Clinical Microbiology, vol. 41, no. 10, pp. 4755–4757, 2003. View at Publisher · View at Google Scholar · View at Scopus
  19. N. Akhtar, F. B. Atique, M. M. Miah, and S. M. Asaduzzaman, “Radiation response of bacteria associated with human cancellous bone,” IOSR Journal of Pharmacy and Biological Sciences, vol. 6, no. 2, pp. 79–84, 2013. View at Google Scholar
  20. E. Russu, A. Muresun, and B. Grigorescu, “Vascular graft infection management,” Management in Health, vol. 15, no. 3, pp. 16–19, 2011. View at Google Scholar
  21. N. Davis, A. Curry, A. K. Gambhir et al., “Intraoperative bacterial contamination in operations for joint replacement,” Journal of Bone and Joint Surgery B, vol. 81, no. 5, pp. 886–889, 1999. View at Publisher · View at Google Scholar · View at Scopus
  22. T. Eastlund and D. M. Strong, “Infectious disease transmission through tissue transplantation,” in Advances in Tissue Banking, G. O. Phillips, Ed., vol. 7, pp. 51–131, World Scientific, Singapore, 2004. View at Google Scholar
  23. H. Turgut, S. Sacar, I. Kaleli et al., “Systemic and local antibiotic prophylaxis in the prevention of Staphylococcus epidermidis graft infection,” BMC Infectious Diseases, vol. 5, article 91, 2005. View at Publisher · View at Google Scholar · View at Scopus
  24. P. J. Adds, C. Hunt, and S. Hartley, “Bacterial contamination of amniotic membrane,” British Journal of Ophthalmology, vol. 85, no. 2, pp. 228–230, 2001. View at Publisher · View at Google Scholar · View at Scopus
  25. L. Kirksey, B. J. Brener, S. Hertz, and V. Parsonnet, “Prophylactic antibiotics prior to bacteremia decrease endovascular graft infection in dogs,” Vascular and Endovascular Surgery, vol. 36, no. 3, pp. 171–178, 2002. View at Publisher · View at Google Scholar · View at Scopus