https://doi.org/10.5812/jjm.20863v2

Antibiotic Sensitivity of Clostridium perfringens Isolated From Faeces in Tabriz, Iran

authors:

avatar Mohammad Taghi Akhi 1 , 2 , * , avatar Saeid Bidar Asl 2 , avatar Tahereh Pirzadeh 2 , avatar Behruz Naghili 3 , avatar Fatemeh Yeganeh 2 , avatar Yousef Memar 2 , avatar Yalda Mohammadzadeh 2

Research Center of Infectious and Tropical Diseases, Tabriz University of Medical Sciences, Tabriz, IR Iran
Department of Bacteriology and Virology, School of Medicine, Tabriz University of Medical Sciences, Tabriz IR Iran
Department of Infectious Disease, Medical Faculty, Tabriz University of Medical Science, Tabriz, IR Iran
Jundishapur Journal of Microbiology: Vol.8, issue 7; e20863
published online: July 25, 2015
article type: Research Article
received: June 1, 2014
revised: August 12, 2014
accepted: August 28, 2014

how to cite: Akhi M T, Bidar Asl S, Pirzadeh T, Naghili B, Yeganeh F, et al. Antibiotic Sensitivity of Clostridium perfringens Isolated From Faeces in Tabriz, Iran. Jundishapur J Microbiol. 2015;8(7):e20863. https://doi.org/10.5812/jjm.20863v2.

Abstract

Background:

Clostridium perfringens, a Gram-positive, anaerobic bacterium that produces at least 16 virulence factors including 12 toxins (α-ν), enterotoxin, hemolysin and neuraminidase, can create variable pathogenic condition, ranging from a food poisoning to life-threatening myonecrosis. Among C. perfringens strains, resistance to the drug choices such as penicillin as well as to alternatives of penicillin like metronidazole and clindamycin has also been observed.

Objectives:

The aim of this study was to determine the resistance of isolated toxigenic and non-toxigenic C. perfringens strains against common antimicrobial agents.

Materials and Methods:

In this descriptive study, a total of 136 stool specimens were collected. At first, cooked meat medium enrichment method was performed on samples at 45°C. Thereafter, a loopful of the enriched culture was transferred to blood agar and incubated anaerobically at 37°C for 24-72 hours. Colonies with double zone of hemolysis were identified by different biochemical tests such as phospholipase C (lecithinase) test, indole and urease production. The Minimum Inhibitory Concentration (MIC) for common antibiotics was determined by Etests (Epsilometer) and duplex Polymerase Chain Reaction (PCR) reaction was performed with specific primers for amplification of cpe (426 bp) and plc (283 bp) Genes.

Results:

Of 136 stool samples including diarrhea [48] and non-diarrhea [88] ones, 83 (61.02%) C. perfringens were cultured. Of these 83, 79 C. perfringens isolates showed the alpha-toxin (phospholipase C) production gene by PCR. Respectively, 3 (9.09%) and 2 (4.34%) cpe genes were present in diarrhea and non-diarrhea samples. Of 79 isolates of C. perfringens, 34 (43.03%) cases showed no resistance, 18 (22.78%) had one resistance and 27 (34.17%) isolates had multiple resistance to imipenem, metronidazole, ceftriaxone, clindamycin, chloramphenicol, and penicillin.

Conclusions:

Periodic evaluation of antimicrobial susceptibility for C. perfringens should be performed. Harboring of enterotoxigenic C. perfringens in individuals not necessarily results in diarrhea.

1. Background

Clostridium perfringens, a Gram-positive anaerobic bacterium that produces spores is widely distributed in nature and normally found in the intestines of animals and humans. Based on the four main toxins produced by C. perfringens species (α, β, ε, ι), they are divided into five groups (A, B, C, D and E). In addition to strong metabolic activities, C. perfringens species also produce at least 16 virulence factors which include 12 toxins (α-ν), enterotoxin, hemolysin, and neuraminidase (sialidase). Alpha-toxin or phospholipase C is the leading cause of death and necrosis as the first virulence factor of this microorganism and is produced by all strains of C. perfringens. C. perfringens creates variable pathogenic conditions, ranging from food poisoning to life-threatening myonecrosis (1).

Type A C. perfringens is a major factor distributed in the environment and is the cause of diseases such as Clostridium myonecrosis, food poisoning, necrotic enterocolitis infection in children, necrotic bowel disease (Pig Bel), and antibiotic-associated diarrhea (1, 2). Enterotoxin is produced about 1-5% by type A C. perfringens. The toxin is a single polypeptide chain of about 35 kDa which unlike other toxins is produced and released in the sporulation stage (1, 2). Following food poisoning caused by Salmonella and Staphylococcus, the enterotoxin of C. perfringens is the third leading cause of food-borne diseases worldwide (3). Enterotoxin-producing genes are located on a transposon called Tn5565.

Several studies have shown that the risk of acquiring antibiotic resistance in C. perfringens is high (4). Because of the importance of the treating diseases caused by C. perfringens, extensive researches have been conducted in this area (5, 6). In some of these studies, resistance to the drugs of choice such as penicillin was observed. Resistance to alternative medicines of penicillin like metronidazole and clindamycin has also been observed (5, 6). Despite extensive studies in the world, there are no particular guidelines regarding the management of C. perfringens infections in Tabriz, Iran.

2. Objectives

The aims of this study were culturing and isolation of toxigenic and non-toxigenic C. perfringens, determination of the Minimum Inhibitory Concentration (MIC) for selected antibiotics by the Etest method and molecular study of the enterotoxin gene.

3. Materials and Methods

3.1. Sampling

In a descriptive study, a total of 136 stool specimens, including 56 specimens from hospitalized patients and 40 out-patients as well as 40 samples from healthy individuals of Imam Reza Educational and Medical Center were consecutively collected. Fecal samples of healthy people were collected from people referred to the laboratory for other purposes. These healthy people were justified orally or in some cases were acquired written consent.

3.2. Culturing and Identifying Methods for Clostridium perfringens

At first, cooked meat medium (Que Lab Inc. European division, UK) enrichment method was performed on samples at 45°C. Thereafter, a loopful of the enriched culture was transferred into blood agar and incubated anaerobically (H2 = 10%, N2 = 80%, CO2 = 10%) using Anoxomat and Mart jar at 37°C for 24 - 72 hours. The obtained typical colonies with double zone of hemolysis were identified after conducting the anaerobic tolerance test by different biochemical tests such as Gram staining, phospholipase C (lecithinase) test, indole and urease production, gelatin hydrolysis, and fermentation of sugars like glucose, lactose and mannitol (7).

3.3. The Evaluation of Sensitivity to Antibiotics

MICs for imipenem, metronidazole, ceftriaxone, clindamycin, chloramphenicol, and penicillin were determined by Etests (AB Biomerieux, Sweden). Since in some anaerobic bacteria the growth on Mueller-Hinton agar (Liofilchem Ltd. Italy) was not sufficient, to determine the antibiotic susceptibility of the isolates, enriched brucella agar medium (Fluka Chemie AG CH-9471 Buchs, Switzerland) with vitamin K and hemin were used. All the plates were incubated anaerobically for 24 hours at 37°C and manufacturer’s instructions and Clinical and Laboratory Standards Institute (CLSI) guidelines were considered for testing and reading the obtained results (8).

3.4. Detection of Enterotoxigenic Clostridium perfringens by Duplex Polymerase Chain Reaction

The C. perfringens isolates were cultured on Columbia agar (Himedia Laboratories Pvt. Ltd. India) and the suspension was prepared in distilled water with a net absorption of 0.09-1 at a 600 nm. Afterwards, the suspension was diluted to 1:10 and mixed well. After boiling it for 10 minutes, it was centrifuged with 13000 × g rpm for five minutes and the supernatants were used as templates in the duplex PCR reaction performed with the following specific primers for amplification of 283 bp and 426 bp fragments:

F: ATA GAT ACT CCA TAT CAT CCT GCT

R: AAG TTA CCT TTG CTG CAT AAT CCC (phospholipase C 283 bp)

F: GAA AGA TCT GTA TCT ACA ACT GCT GGT CC

R: GCT GGC TAA GAT TCT ATA TTT TTG TCC AGT (enterotoxin, 426 bp) (9, 10)

The PCR mixture (30 µL) consisted of 3 µL of 10x PCR buffer (the 1x buffer included 10 mM Tris-HCl, pH 8.3, 50 mM KCl, 1.5 mM MgCl2 and 0.01% gelatin), 0.6 µL of 10 mM dNTP mixture, 1.2 µL (50 pmoles) for CPE and 0.6 µL (25 pmoles) for PLC genes, 1 µL (1 U) of Taq polymerase (Fermentas), 10 µL of the DNA template, and distilled water to make a total volume of 30 µL.

The amplification was performed in a DNA thermal cycler (Eppendorf, Germany), programmed for a primary denaturation, (95°C, 10 minutes), followed by 35 cycles of denaturation (94°C, one minute), annealing (55°C, one minute), elongation (72°C, one minute), and then extension (72°C, 10 minutes). A negative control without template was included in each PCR run (11). The amplified products were visualized by electrophoresis on 2% agarose gel in 1x TBE buffer (1 M Tris, 0.9 M boric acid, 0.01 M EDTA, pH = 8.4), at 80 V, for two hours. A 100-bp DNA ladder was used as a molecular mass marker. The gels were stained with ethidium bromide (0.5 µg mL-1) and photographed on a gel documentation system (UVP, USA) for the analysis of the bands (9-11) (all the PCR materials including primers were provided by CinnaGen; Nedayeh Fan Co., Iran).

4. Results

Of 136 stool samples including diarrhea [48] and non-diarrhea [88] ones, 83 (61.02%) C. perfringens strains were cultured and identified by the above mentioned tests; 33 (68.75%) and 50 (56.81%) of the isolates were obtained from diarrhea and non-diarrhea stool samples, respectively. Of the 83 C. perfringens isolates, 79 showed alpha-toxin (phospholipase C) production gene by PCR; so, were confirmed them as C. perfringens. Totally, 3 (9.09%) and 2 (4.34%) cpe genes were present in diarrhea and non-diarrhea samples, respectively (Figure 1). Of the 79 isolates of C. perfringens, 34 (43.03%) showed no resistance, 18 (22.78%) had one resistance and 27 (34.17%) isolates had multiple resistance to the tested antibiotics. The antimicrobial resistance patterns of the tested isolates are given in Table 1.

Duplex Polymerase Chain Reaction Amplification of cpe (426 bp) and plc (283 bp) Genes of Enterotoxigenic Clostridium perfringens
M, 100 bp DNA ladder; 5 is representative of cpe and plc negative isolates; 1, 2, 4, 5, 7, 8 are representative of non-enterotoxigenic C. perfringens isolates and 3, 6, 9 are representative of the cpe gene-producing isolates.

M, 100 bp DNA ladder; 5 is representative of cpe and plc negative isolates; 1, 2, 4, 5, 7, 8 are representative of non-enterotoxigenic C. perfringens isolates and 3, 6, 9 are representative of the cpe gene-producing isolates.

Figure 1.

Duplex Polymerase Chain Reaction Amplification of cpe (426 bp) and plc (283 bp) Genes of Enterotoxigenic Clostridium perfringens

Table 1.

Minimum Inhibitory Concentration of the Tested Antibiotics to Clostridium perfringens Isolated From Stools of Diarrhea and Non-Diarrhea Patients a

AntibioticsAntibiotic Content of Etests Strips, µg/mLObtained MIC by Etests, µg/mLCLSI Interpretative MIC, µg/mLObtained Results by Etests
S, %I, %R, %S, No. (%)R, No. (%)
Imipenem0.002 - 320.5 - 32481649 (62.02)30 (37.97)
Metronidazole0.016 - 2560.2 - 644163270 (88.60)9 (11.39)
Ceftriaxone0.016 - 2560.016 - 12816326477 (97.46)2 (2.53)
Clindamycin0.016 - 256≤ 2 - 3224866 (83.54)13 (16.46)
Chloramphenicol0.016 - 2562 - 648163275 (94.93)4 (5.06)
Penicillin0.016 - 2560.25 - 40.51272 (91.13)7 (8.86)

aAbbreviations: CLSI, Clinical and Laboratory Standards Institute; I, intermediate; MIC, minimum inhibitory concentration; R, resistant; S, sensitive.

5. Discussion

In cases of serious anaerobic infections, not only should the clinical laboratory be able to accurately identify C. perfringens isolates but it is also important that the laboratory perform susceptibility tests on individual isolates from patients. The precise identification of C. perfringens by different methods is always necessary because identification by biochemical tests in some cases is not satisfactory. In this study, to achieve this goal, in addition to biochemical tests, genes coding production of lecithinase were also detected by PCR, which reduced false positives. Other researchers used commercially available kits such as RapID ANA II, AnIDent, and ATB 32A (12). PRAS biochemical fermentation tests, end product analysis by gas-liquid chromatography and other reactions are also able to confirm the identification of the strains (13, 14).

Clostridia neither invade healthy cells nor multiply within them. They are able to enter the host organisms by two ways, the oral route and wounds, but their proliferation in the intestinal content or in wounds require the presence of risk factors (15). Toxins as the main virulence factors are responsible for all the symptoms and lesions observed in clostridial diseases. Consequently, toxin-producing C. perfringens strains are the main targets for diagnosis of C. perfringens diseases. Enterotoxin-positive strains of C. perfringens are recognized as the causes of food-borne diarrhea as well as several non-food-related diarrheas, including sporadic diarrhea and antibiotic-associated diarrhea (16, 17). In Tabriz, few data are available on the detection of this organism. The analysis of food-borne anaerobic bacteria is not routinely performed due to difficulties in their isolation and identification. The procedures require special techniques which may be expensive, time-consuming and labor intensive. For this reason, the outbreaks of C. perfringens are often not recognized. In addition, the knowledge of antimicrobial susceptibility of this organism isolated from human, animals, foods and environment is limited.

In this study, the enterotoxigenic and non-enterotoxigenic C. perfringens strains were isolated from human feces and identified using biochemical tests and a duplex PCR procedure, which enables C. perfringens species identification and differentiation between enterotoxigenic and non-enterotoxigenic strains by detection of the plc and cpe genes (9, 18). Our results showed that only 79 out of 83 C. perfringens isolates were alpha-toxin (phospholipase C) producers and the presence of the plc gene by PCR confirmed the isolates of C. perfringens (Figure 1). These results indicated that identification only by biochemical tests is not satisfactory. In addition, the MICs of six antimicrobial agents commonly used in human medicine for all the C. perfringens isolated strains were determined using the Etest method. In this study, imipenem was the least active agent to C. perfringens with an overall rate of resistance equal to 37.97%. While this finding is nearly in agreement with another research (19), Camacho et al. reported sensitivity of all his isolates to imipenem (6). Of the three antimicrobials including metronidazole, ceftriaxone and chloramphenicol, which were highly active against C. perfringens with low MIC values, ceftriaxone could inhibit the most C. perfringens strains tested with an overall rate of resistance of 2.53%. These findings are also consisting of Camacho et al. report (6). Penicillin G is still an effective drug based upon its low MIC values and low rate of resistance (8.86%) and may be considered when choosing an antimicrobial agent for prophylaxis or treatment of C. perfringens in humans. In agreement with this, there are several reports (6, 19). The isolates of C. perfringens in this study were susceptible to clindamycin (%83.54), while cases of clindamycin-resistance have been described (20, 21).

This study revealed a moderate prevalence of antimicrobial-resistant C. perfringens strains. Among 56.95% of antimicrobial-resistant strains, 22.78% were single drug resistant and 34.17% were Multidrug Resistant (MDR). This result also has been confirmed by a set of results obtained from Germany (22). We found the presence of the cpe gene among C. perfringens strains isolated from diarrhea stool (9.09%) more than the non-diarrhea samples (4.34%), indicating the role of toxin production in diarrhea (P ≤ 0.05). The C. perfringens strains isolated from various origins carry nearly 6% cpe gene, but this percentage is higher (59%) among the strains isolated from confirmed outbreaks of food poisoning (23). This could be the reason for our results not being in agreement with the results of Tansuphasiri et al. (4.8%) which was much lower than our findings (18).

The fecal samples obtained from the hospitalized patients with non-diarrheal diseases might harbor a small number of these enterotoxigenic strains in their intestines. However, it suggests the need of more studies to evaluate the role of enterotoxigenic C. perfringens in patients with diarrhea as well as non-diarrhea stools. These organisms must be looked for routinely and a periodic evaluation of antimicrobial susceptibility should be performed.

Acknowledgements

References

  • 1.

    Cavalcanti MTH, Porto T, Porto A, Brandi IV, Lima Filho J, Pessoa Junior A. Large scale purification of Clostridium perfringens toxins: a review. Brazil J Pharm Scie. 2004;40(2):151-64. https://doi.org/10.1590/S1516-93322004000200004.

  • 2.

    Rood JI, Cole ST. Molecular genetics and pathogenesis of Clostridium perfringens. Am oc Microbiol. 1991;55(4):621-48.

  • 3.

    Brynestad S, Sarker MR, McClane BA, Granum PE, Rood JI. Enterotoxin plasmid from Clostridium perfringens is conjugative. Infect Immun. 2001;69(5):3483-7. [PubMed ID: 11292780]. https://doi.org/10.1128/IAI.69.5.3483-3487.2001.

  • 4.

    Brynestad S, Granum PE. Evidence that Tn5565, which includes the enterotoxin gene in Clostridium perfringens, can have a circular form which may be a transposition intermediate. FEMS Microbiol Lett. 1999;170(1):281-6. https://doi.org/10.1111/j.1574-6968.1999.tb13385.x.

  • 5.

    Koch CL, Derby P, Abratt VR. In-vitro antibiotic susceptibility and molecular analysis of anaerobic bacteria isolated in Cape Town, South Africa. J Antimicrob Chemother. 1998;42(2):245-8. [PubMed ID: 9738844].

  • 6.

    Camacho N, Espinoza C, Rodriguez C, Rodriguez E. Isolates of Clostridium perfringens recovered from Costa Rican patients with antibiotic-associated diarrhoea are mostly enterotoxin-negative and susceptible to first-choice antimicrobials. J Med Microbiol. 2008;57(Pt 3):343-7. [PubMed ID: 18287298]. https://doi.org/10.1099/jmm.0.47505-0.

  • 7.

    Fader RC. Anaerobes of clinical importance. In: Mahon CR, Lehman DC, Manuselis G, editors. Textbook of diagnostic microbiology. 5th ed. China: W.B.Saunders Company; 2015. p. 495-528.

  • 8.

    Performance standards for Antimicrobial susceptibility Testing: Document M10. S15. Wayne, PA, USA: CLSI; 2005.

  • 9.

    Fach P, Popoff MR. Detection of enterotoxigenic Clostridium perfringens in food and fecal samples with a duplex PCR and the slide latex agglutination test. Appl Environ Microbiol. 1997;63(11):4232-6. [PubMed ID: 9361409].

  • 10.

    Popoff MR. Detection of Toxigenic Clostridia. In: Sachse. K, Frey J, editors. Methods in Molecular Biology: Methods and Protocols. Totowa, NJ, USA: Humana Press Inc; 2003. p. 137-52.

  • 11.

    Tansuphasiri U, Chanyasanha C, Cheaochantanakij N. An enrichment broth culture-duplex PCR combination assay for the rapid detection of enterotoxigenic Clostridium perfringens in fecal specimens. Southeast Asian J Trop Med Public Health. 2005;36(5):1229-38. [PubMed ID: 16438150].

  • 12.

    Alexander CJ, Citron DM, Brazier JS, Goldstein EJ. Identification and antimicrobial resistance patterns of clinical isolates of Clostridium clostridioforme, Clostridium innocuum, and Clostridium ramosum compared with those of clinical isolates of Clostridium perfringens. J Clin Microbiol. 1995;33(12):3209-15. [PubMed ID: 8586704].

  • 13.

    Osman KM, Elhariri M. Antibiotic resistance of Clostridium perfringens isolates from broiler chickens in Egypt. Rev Sci Tech. 2013;32(3):841-50. [PubMed ID: 24761735].

  • 14.

    Llanco LA, Nakano V, Ferreira AJP, Avila- Campos MJ. Toxinotyping and antimicrobial susceptibility of C. perfringens isolated from broiler chickens with necrotic entritis. Int J Microbiol Res. 2012;4(7):290-4. https://doi.org/10.9735/0975-5276.4.7.290-294.

  • 15.

    Asha NJ, Tompkins D, Wilcox MH. Comparative analysis of prevalence, risk factors, and molecular epidemiology of antibiotic-associated diarrhea due to Clostridium difficile, Clostridium perfringens, and Staphylococcus aureus. J Clin Microbiol. 2006;44(8):2785-91. [PubMed ID: 16891493]. https://doi.org/10.1128/JCM.00165-06.

  • 16.

    Miyamoto K, Wen Q, McClane BA. Multiplex PCR genotyping assay that distinguishes between isolates of Clostridium perfringens type A carrying a chromosomal enterotoxin gene (cpe) locus, a plasmid cpe locus with an IS1470-like sequence, or a plasmid cpe locus with an IS1151 sequence. J Clin Microbiol. 2004;42(4):1552-8. [PubMed ID: 15071003].

  • 17.

    Bos J, Smithee L, McClane B, Distefano RF, Uzal F, Songer JG, et al. Fatal necrotizing colitis following a foodborne outbreak of enterotoxigenic Clostridium perfringens type A infection. Clin Infect Dis. 2005;40(10):e78-83. [PubMed ID: 15844055]. https://doi.org/10.1086/429829.

  • 18.

    Tansuphasiri U, Wongsuvan G, Eampokalap B. PCR detection and prevalence of enterotoxin (cpe) gene in Clostridium perfringens isolated from diarrhea patients. J Med Assoc Thai. 2002;85(5):624-33. [PubMed ID: 12188395].

  • 19.

    Tansuphasiri U, Matra W, Sangsuk L. Antimicrobial resistance among Clostridium perfringens isolated from various sources in Thailand. Southeast Asian J Trop Med Public Health. 2005;36(4):954-61. [PubMed ID: 16295551].

  • 20.

    Khanna N. Clindamycin-resistant Clostridium perfringens cellulitis. J Tissue Viability. 2008;17(3):95-7. [PubMed ID: 18558488]. https://doi.org/10.1016/j.jtv.2008.04.001.

  • 21.

    Lyras D, Adams V, Ballard SA, Teng WL, Howarth PM, Crellin PK, et al. tISCpe8, an IS1595-family lincomycin resistance element located on a conjugative plasmid in Clostridium perfringens. J Bacteriol. 2009;191(20):6345-51. [PubMed ID: 19684139]. https://doi.org/10.1128/JB.00668-09.

  • 22.

    Holzel CS, Harms KS, Schwaiger K, Bauer J. Resistance to linezolid in a porcine Clostridium perfringens strain carrying a mutation in the rplD gene encoding the ribosomal protein L4. Antimicrob Agents Chemother. 2010;54(3):1351-3. [PubMed ID: 20065060]. https://doi.org/10.1128/AAC.01208-09.

  • 23.

    Van Damme-Jongsten M, Rodhouse J, Gilbert RJ, Notermans S. Synthetic DNA probes for detection of enterotoxigenic Clostridium perfringens strains isolated from outbreaks of food poisoning. J Clin Microbiol. 1990;28(1):131-3. [PubMed ID: 2298871].