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Volume: 7, Issue: 1, Jan-Feb, 2019
DOI: 10.7324/JABB.2019.70110

Research Article

Molecular detection and antimicrobial resistance of Clostridium perfringens isolated from diabetic patients and bullet wounds


Aliaa Mohamed Hmood, Maysa S M Al-Shukri, Alaa H Al-Charrakh

  Author Affiliations


Abstract

Clostridium perfringens is a major cause of gas gangrene. The morbidity of C. perfringens is connected with producing toxins. This cross-sectional study was designed to isolate, genetically diagnose, and study the antibiotic susceptibility patterns of C. perfringens isolated from clinical samples. Different wound swabs (from diabetic patients, cellulitis, and bullet wounds) were taken from 140 patients. For isolation of anaerobic bacteria, samples (in thioglycolate broth) were immediately incubated anaerobically then identified according to the cultural properties and biochemical tests. DNA was extracted from all specimens. Polymerase chain reaction was applied for detection of 16SrRNA and internal transcribed spacer (ITS) genes of C. perfringens. The susceptibility of bacterial isolates to different antibiotics was determined using Vitek 2 system and disk diffusion test. Out of 140 clinical samples collected during this study, 3 (2.14%) C. perfringens isolates were recovered of which 2 isolates (1.43%) obtained from diabetic patients and one (0.71%) from bullet wounds. Results also showed that only 7 isolates (5%) were detected by a molecular method using specific primers 16S rRNA and ITS genes of C. perfringens. Results of antibiotic susceptibility testing showed that all isolates were highly susceptible to penicillins and β-lactamase inhibitors, metronidazole, and aminoglycosides. On the other hand, all isolates were highly resistant to tetracycline, levofloxacin, and erythromycin. The susceptibility patterns of C. perfringens isolates showed that all isolates were multidrug resistance. Using the amplification of ITS gene increases specificity and sensitivity (by reducing non-specific annealing and primer dimer formation) which increases the probability of detection of suspected C. perfringens isolates.

Keywords:

Molecular detection, Internal transcribed spacer gene, Antibiotic susceptibility, Clostridium perfringens, Diabetic patients.



Citation: Hmood AM, Al-Shukri MSM, Al-Charrakh AH. Molecular detection and antimicrobial resistance of Clostridium perfringens isolated from diabetic patients and bullet wounds. J App Biol Biotech. 2019;7(01):54-59. DOI: 10.7324/JABB.2019.70110of


Copyright: Author(s). This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License, which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited.

References

Aggelidakis J, Lasithiotakis K, Topalidou A, Koutroumpas J, Kouvidis G, Katonis P. Limb salvage after gas gangrene: a case report and review of literature. World J Emerg Surg 2011; 17(6): 28. https://doi.org/10.1186/1749-7922-6-28

Akhi MT, Asl SB, Pirzadeh T, Naghili B, Yeganeh F, Memar Y, Mohammadzadeh Y. Antibiotic Sensitivity of Clostridium perfringens Isolated From Faeces in Tabriz, Iran. Jundishapur J Microbiol 2015; 8(7):210-54.

Altın C, Kanyılmaz S, Baysal S, Özin B. QT. Interval prolongation due to metronidazole administration. Anadolu Kardiyol Derg 2011; 11:468. https://doi.org/10.5152/akd.2011.120

Baker GC, Smith JJ, Cowan DA. Review and re-analysis of domain-specific 16S primers. J Microbiol 2003; 55:541–55.

Barry T, Colleran G, Glennon M, Dunican LK, Gannonl F. PCR Methods Applications 2015; 51(2):434-67.

Claeys LG, Matamoros R. Anaerobic cellulitis as the result of Clostridium perfringens: a rare cause of vascular access graft infection. J Vascular Surg 2002; 35(6): 1287-88. https://doi.org/10.1067/mva.2002.122026

Clinical and Laboratory Standards Institute. Performance standards or antimicrobial susceptibility testing; Twenty-Fifth Informational Supplement. CLSI document M100-S25. Clinical and Laboratory Standards Institute. Wayne, PA; 2015.

Gamboa-Coronado Mdel M, Mau-Inchaustegui S, Rodriguez-Cavallini E. Molecular characterization and antimicrobial resistance of Clostridium perfringens isolates of different origins from Costa Rica. Rev Biol Trop 2011; 59:1479-85.

Hausmann R, Albert F, Geissdorfer W, Betz P. Clostridiumfallax associated with sudden death in a 16 year-old boy. J Med Microbiol 2004; 53: 581-83. https://doi.org/10.1099/jmm.0.05495-0

Janvilisri T, Scaria J, Gleed R, Fubini S, Bonkosky MM, Gröhn YT, Chang YF. Development of a microarray for identification of pathogenic Clostridium spp. Diagn Microbiol Infect Dis 2010; 66:140-7. https://doi.org/10.1016/j.diagmicrobio.2009.09.014

Javed S, Rafeeq M, Tariq MM, Awan MA, Rashid N, Ali M. Study on in vitro biochemical growth characterization and assessment of hemolytic toxin of C. perfringens type B and D. Pak J Zool 2012; 44:1575–80.

Kitterer D, Braun N, Jehs MC, Schulte B, Alscher MD, Latus J. Gas Gangrene Caused By Clostridium perfringens Involving the Liver, Spleen, and Heart in a Man 20 Years After an Orthotopic Liver Transplant: A Case Report. Exp Clin Transplant 2013; 32 :345-78.

Magiorakos A, Srinivasan P, Carey RB, Carmeli Y, Falagas ME, Giske CG, Harbath S, Hindler JF, Kahlmeter G, Olsson-Lilijequist B, Paterson DL, Rice LB, Stelling, MJ, Struelens MJ, Vatopoulos A, Weber JT, Monnet DL. Multidrug-resistant, extensively drug –resistant and pan drug resistant bacteria: an international expert proposal for interim standard definitions for acquired resistance. Clin Microbial Infect 2012; 18:268-81. https://doi.org/10.1111/j.1469-0691.2011.03570.x

Maynard C, Berthiaume F, Lemarchand K, Harel J, Payment P, Bayardelle P, Masson L, Brousseau R. Waterborne pathogen detection by use of oligonucleotide-based microarrays. Appl Environ Microbiol 2005; 71(85): 8548–57. https://doi.org/10.1128/AEM.71.12.8548-8557.2005

M\éndez MB, Go-i A, Ramirez W, Grau RR. Sugar inhibits the production of the toxins that trigger clostridial gas gangrene. Microb Pathog 2012; 52(1):85-91. https://doi.org/10.1016/j.micpath.2011.10.008

Mingeot-Leclercq MP, Glupczynski Y, Tulkens PM. Aminoglycosides: activity and resistance. Antimicrob Agents Chemother 1999; 43 (4): 727–37.

Ott SJ, Musfeldt M, Ullmann U, Hampe J, Schreiber S. Quantification of intestinal bacterial populations by real-time PCR with a universal primer set and minor groove binder probes: a global approach to the enteric flora. J Clin Microbiol 2004; 422: 566-72. https://doi.org/10.1128/JCM.42.6.2566-2572.2004

Petti CA, Polage CR, Schreckenberger P. The role of 16S rRNA gene sequencing in identification of microorganisms misidentified by conventional methods. J Clin Microbiol 2005; 43:6123–25. https://doi.org/10.1128/JCM.43.12.6123-6125.2005

Piatti RM, Ikuno AA, Baldassi L. Detection of bovine Clostridium perfringens by polymerase chain reaction. J Venom Anim Toxins incl Trop Dis 2004;10(2):1678-99. https://doi.org/10.1590/S1678-91992004000200005

Rechner PM, Agger WA, Mruz K, Cogbill TH. Clinical Features of Clostridial Bacteremia. Clin Infect Dis 2001; 33 (3):349-53. https://doi.org/10.1086/321883

Roberts SA, Shore KP, Paviour SD, Holland D, Morris AJ. Antimicrobial susceptibility of anaerobic bacteria in New Zealand. J Antimicrob Chemother 2006; 57:992-8. https://doi.org/10.1093/jac/dkl052

Rönner SGE, Stackebrandt E. Identification of Clostridium perfringens by 16S and 23S rRNA Oligonucleotide Probes. Syst App Microbiol 1994;17(3): 425-32. https://doi.org/10.1016/S0723-2020(11)80060-3

Stevens DL, Aldape MJ, Bryant AE. Life-threatening clostridial infections. Anaerobe 2012; 18 (2): 254-9. https://doi.org/10.1016/j.anaerobe.2011.11.001

Stevens DL, Aldape MJ, Bryant AE. Necrotizing fasciitis, gas gangrene, myositis, and myonecrosis. In: Infectious Diseases. Editors; Cohen J, Powderly, Opal S M., Mosbey Elsevier Inc 2010; 44: 119-27.

Stevens DL, Bisno AL, Chambers HF, Everett ED, Dellinger P, Goldstein EJC, Gorbach SL, Hirschmann JV, Kaplan EL, Montoya JG, Wade JC. Practice guidelines for the diagnosis and management of skin and soft-tissue infections. Clin Infect. Dis 2005; 41(10):1373-06. https://doi.org/10.1086/497143

Takazawa K, Otsuka H, Nakagawa Y, Inokuchi S. Clinical Features of Non-clostridial Gas Gangrene and Risk Factors for In-hospital Mortality. Tokai J Exp Clin Med 2015; 40 (3):124-9.

Tansuphasiri U, Matra W, Sangsuk L. Antimicrobial resistance among Clostridiumperfringens isolated from various sources in Thailand. Southeast Asian J Trop Med Public Health. 2005; 36(4): 954-961.

Vary JC, O'Connor KM. Common Dermatologic Conditions. The Medical clinics of North America 2014; 98 (3): 445–85. https://doi.org/10.1016/j.mcna.2014.01.005

Wang Y, Lu B, Hao P, Yan MN, Dai KR. Comprehensive treatment for gas gangrene of the limbs in earthquakes. Chin Med J (Engl) 2013; 126(20):3833-39.

Wang Y, Qian P. Conservative fragments in bacterial 16S rRNA genes and primer design for 16S ribosomal DNA amplicons in metagenomic studies. PLoS ONE 2009; 4(10): e7401. https://doi.org/10.1371/journal.pone.0007401

Wu J1, Zhang W, Xie B, Wu M, Tong X, Kalpoe J, Zhang D. Detection and Toxin Typing of Clostridium perfringens in Formalin-Fixed, Paraffin-Embedded Tissue Samples by PCR. J Clin Microbiol 2009; 47(3): 807–10. https://doi.org/10.1128/JCM.01324-08

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