gms | German Medical Science

GMS Hygiene and Infection Control

Deutsche Gesellschaft für Krankenhaushygiene (DGKH)

ISSN 2196-5226

nim gene-independent metronidazole-resistant Bacteroides fragilis in surgical site infections

nim-Gen-unabhängige Metronidazolresistenz von Bacteroides fragilis in Surgical Site Infections

Research Article

  • Mohammad Taghi Akhi - Research Center of Infectious and Tropical Disease, Tabriz University of Medical Sciences, Tabriz, Iran; Department of Bacteriology and Virology, School of Medicine, Tabriz University of Medical Sciences, Iran
  • Reza Ghotaslou - Department of Bacteriology and Virology, School of Medicine, Tabriz University of Medical Sciences, Iran
  • Naser Alizadeh - Department of Bacteriology and Virology, School of Medicine, Tabriz University of Medical Sciences, Iran
  • Mina Yekani - Department of Bacteriology and Virology, School of Medicine, Tabriz University of Medical Sciences, Iran
  • Samad Beheshtirouy - Cardiothoracic Department, Tabriz University of Medical Sciences, Tabriz, Iran
  • Mohammad Asgharzadeh - Laboratory Department, Tabriz University of Medical Sciences, Tabriz, Iran
  • Tahereh Pirzadeh - Department of Bacteriology and Virology, School of Medicine, Tabriz University of Medical Sciences, Iran
  • corresponding author Mohammad Yousef Memar - Research Center of Infectious and Tropical Disease, Tabriz University of Medical Sciences, Tabriz, Iran; Department of Bacteriology and Virology, School of Medicine, Tabriz University of Medical Sciences, Iran

GMS Hyg Infect Control 2017;12:Doc13

doi: 10.3205/dgkh000298, urn:nbn:de:0183-dgkh0002988

Published: August 17, 2017

© 2017 Akhi et al.
This is an Open Access article distributed under the terms of the Creative Commons Attribution 4.0 License. See license information at


Background: Bacteroides fragilis is the most common anaerobic pathogen isolated from surgical site infections (SSIs). Metronidazole resistance is increasing and the mechanisms of resistance are not clear in some isolates. The aim of the present study was to investigate the metronidazole susceptibility prevalence, and detect nim genes in B. fragilis isolates from SSIs.

Methods: This study included 100 surgery patients with signs and symptoms indicative of SSIs. Syringe aspiration of the infected site was used to collect specimens. All specimens were cultured on BBA (Brucella blood agar), KVLB (kanamycin-vancomycin laked blood), and BBE (Bacteroides bile esculin) agar. The MIC (minimum inhibitory concentration) of metronidazole was determined by the agar dilution method according to the Clinical and Laboratory Standard Institute (CLSI). Then the PCR method was used to determine the presence of the nim gene.

Results: In the present study, 26 B. fragilis were isolated from 100 SSIs specimens. Eight isolates were metronidazole resistant; the metronidazole MIC was 32 µg/mL for 7 isolates and 64 µg/mL for one isolate. All isolates were nim gene negative.

Conclusion: The emergence of metronidazole-resistant B. fragilis limits the application of this drug for treatment and prophylaxis of SSIs. Thus, rapid identification of metronidazole-resistant B. fragilis is essential to restrict inappropriate, superfluous administration. In spite of various metronidazole resistance mechanisms other than that depending on the nim gene, detection of nim by PCR is unsuitable for identifying resistant isolates. Therefore, phenotypic methods are better to screen for and identify metronidazole-resistant B. fragilis.

Keywords: Bacteroides fragilis, surgical site infections, metronidazole, minimum inhibitory concentration, nim gene


Hintergrund: Bacteroides fragilis ist der häufigste bei Surgical Site Infections (SSI) isolierte Anaerobier. Da die Resistenz gegen Metronidazol im Anstieg begriffen ist und die Resistenzmechanismen bei einigen Isolaten unklar sind, sollten die Prävalenz Metronidazol-sensibler Erreger und das Vorkommen von nim-Genen bei B. fragilis-Isolaten von SSIs untersucht werden.

Methode: Es wurden 100 Patienten mit diagnostizierter SSI untersucht. Die mikrobiologischen Proben wurden mittels Nadelaspiration aus dem Infektionsherd gewonnen. Die Kultivierung erfolgte auf Brucella-Blutagar, Kanamycin-Vancomycin-Laked Blutagar und Bacteroides-Galle-Esculin-Agar. Die minimale Hemmkonzentration (MHK) von Metronidazol wurde im Agarverdünnungstest gemäß Clinical and Laboratory Standard Institute (CLSI) bestimmt. Der Nachweis des nim-Gens erfolgte mittels PCR.

Ergebnisse: In 26 Fällen wurde B. fragilis isoliert. Acht Isolate waren gegen Metronidazol resistent (MHK 32 µg/mL bei 7, 64 µg/mL bei einem Isolat). In keinem Isolat war das nim-Gen nachweisbar.

Schlussfolgerung: Das Vorkommen Metronidazol-resistenter B. fragilis-Stämme kann den Erfolg der perioperativen Antibiotikaprophylaxe und ebenso der Therapie infrage stellen. Daher ist die rasche Identifikation Metronidazol-resistenter B. fragilis-Stämme wichtig, um eine ungeeignete Verabreichung zu verhindern. In Anbetracht verschiedener nim-Gen-unabhängiger Resistenzmechanismen ist der Nachweis des nim-Gens mittels PCR zur Identifikation resistenter Isolate ungeeignet. Stattdessen eignen sich phänotypische Methoden zum Screening Metronidazol-resistenter B. fragilis-Stämme.

Schlüsselwörter: Bacteroides fragilis, Surgical Site Infections, Metronidazol, minimale Hemmkonzentration, nim-Gen


Surgical site infections (SSIs) are mono- and polymicrobial infections caused by anaerobic and aerobic bacteria [1], [2], [3]. Bacteroides fragilis is the most important opportunistic anaerobic pathogen isolated from SSIs [4], [5]. Due to its virulence factors such as adhesions, hemagglutinin, polysaccharide capsule, fimbria, and antibiotic resistance, B. fragilis is considered to be the most virulent pathogen [6]. Some studies indicate a high prevalence of anaerobic Gram-negative bacilli such as B. fragilis in SSIs [7]. Resistance to the most common antimicrobial drugs against Gram-negative anaerobic bacteria, such as β-lactam/β-lactamase inhibitors, fourth-generation fluoroquinolones, carbapenems, metronidazole, clindamycin and other antibiotics, has been reported in B. fragilis [8], [9], [10]. The rates of resistance show clinically considerable differences between countries and may also vary from one hospital to another within a country [9]. Metronidazole, a 5-nitroimidazole agent, is the most commonly administrated antibiotic globally for the treatment of infections caused by B. fragilis and has been the drug of choice for perioperative surgical prophylaxis in combination with cephalosporins of the 2nd or 3rd generation (e.g. for colorectal surgery) [11]. Metronidazole is administered as an inactive form that activates by reduction of its nitro group under anaerobic condition in the cells. This chemical change gives rise to the active, toxic form, which can break single- and double-strand DNA [12]. Because metronidazole is ineffective against aerobic and facultatively anaerobic bacteria, an additional antibiotic effective against these organisms (e.g., fluoroquinolone or a cephalosporin) is necessary when treating a polymicrobial infection such as SSI [13].

The emergence of metronidazole resistance in B. fragilis can lead to a decrease in the efficacy of this drug [14]. Metronidazole-resistant B. fragilis were isolated from patients following long-term therapy with this drug [14]. The B. fragilis group can develop resistance to this antimicrobial drug by different mechanisms [12]. One of these mechanisms is associated with nim A to H genes that encode nitroimidazole reductase, which transforms 4- or 5-nitroimidazole to 4- or 5-aminoimidazole, the nontoxic derivative [8], [12], [14]. Other mechanisms include overexpression of the multidrug efflux pump, overexpression of RecA protein, and deficiency of the ferrous iron transporter FeoAB [11], [12], [15].

The aim of this study was to assess the prevalence of B. fragilis in SSIs, its susceptibility to metronidazole and the presence of the nim gene in these isolates.

Materials and methods


This descriptive cross-sectional study was conducted at Imam Reza Hospital in Tabriz, Iran. The study was carried out for a period of 10 months from October 2013 to July 2014 and involved 100 hospitalized patients. SSIs are wound infections that occur within 30 days after the surgery. The patients were selected according to isolation of pathogenic microorganisms and clinical signs of SSIs (inflammation, irritation, fever, and discharge). For sampling, first the skin was disinfected by povidone iodine, and then specimens were collected by syringe aspiration of material deep within the infected site. The syringe was immediately sealed and transported to the laboratory within 20 min. Inoculation generally took place at the latest 1 hour after collection.

Bacterial isolation

The cytology assessment and detection of bacteria in specimens was performed by the Gram stain smear. Pre-reduced media including vitamin K-enriched Brucella blood agar, kanamycin-vancomycin laked blood agar (KVLB, the basal medium is Brucella agar; Fluka Chemie AG, Buchs, Switzerland) and Bacteroides bile esculin agar (BBE, Himedia Laboratories Pvt. Ltd, India) were inoculated for the isolation of B. fragilis [16], [17]. The plates were incubated under 80% N2, 10% CO2, 10% H2 and 0% O2 in an anaerobic jar using the Anoxomat system (MART microbiology B.V., the Netherlands), and these plates were examined at 48, 72, and 96 h. The aerotolerance test, biochemical tests (such as reaction in the BBE agar, catalase production, indole, and sugar fermentation) and the MAST ID MID8 ANAEROBE ID RING (MAST CO.) were used to identify B. fragilis isolates [17], [18].

Determination of metronidazole MIC

MIC was determined by the agar dilution method on Brucella agar (supplemented with 5% sheep blood, vitamin K (1 µg/mL) and hemin (5 µg/mL), and 1×104 CFU were inoculated on agar according to the recommendation of the CLSI [19]. Ten dilution steps ranging from 0.25 to 128 µg/ml (0.25, 0.5, 1, 2, 4, 8, 16, 32, 64, and 128 µg/mL) were tested for metronidazole susceptibility testing. The plates were incubated at 37°C and read at 48 h after incubation in anaerobic conditions. The lowest antibiotic concentration at which growth of bacteria was inhibited was determined as the MIC. The results were interpreted according to the CLSI guideline [19].

DNA extraction and detection of nim genes

One loopful of cultured B. fragilis was suspended in 400 µL of TE buffer (10 mM Tris-HCL, 1 mM EDTA, PH 8.0) and kept at 80°C for 30 min to kill the bacteria. DNA was extracted by CTAB, SDS, and proteinase K. After sedimentation with isopropanol and washing with ethanol 70%, extracted DNA was solved in 100 µL TE buffer [20].

For the PCR reaction, specific primers of 458-bp fragment NIM-3 (5'-ATG TTC AGA GAA ATG CGG CGT AAG CG-3') and NIM-5 (5'-GCT TCC TTG CCT GTC ATG TGC TC-3') were used [20]. Each PCR assay was performed in 25 µL of reaction mixture containing 2 µL of DNA sample, 2 µL of each primer, 1.25 µL of MgCl2 (50 mM), 0.5 µL d NTP (10 mM), 2.5 µL of PCR Buffer (10X), 0.5 µL of bovine serum albumin (10 mg/mL), 1 µL of Taq polymerase (2.5 u/µL) and 13.25 µL of distilled water [20]. Target DNA was amplified using a thermal cycler (Gradient Eppendorf). After an initial denaturation step at 94°C for 10 min, the reaction mixture was subjected to 32 cycles of amplification consisting of denaturation at 94°C for 30 s, annealing at 62°C for 1 min, extension at 72°C for 1 min, and a final extension step at 72°C for 10 min [20]. The PCR products were analyzed by electrophoresis on 1.2% agarose gels in TBE buffer (89 mM Tris base, 89 mM boronic acid, 2 mM Na2, EDTA, pH 8.25). The agarose gel was stained with 0.5 µg/mL ethidium bromide and visualized under UV light. A 100 bp ladder was used as molecular size marker [20].


A total of 100 SSI specimens were processed; B. fragilis was isolated from 26 of them (in 14 male and 12 female patients) (Table 1 [Tab. 1]). All B. fragilis was isolated from polymicrobial infections. Overall, the greatest number of B. fragilis isolates were recovered from gastrointestinal tracts (23 cases), followed by respiratory tracts (2 cases) and orthopedic (1 case) surgery procedure. The metronidazole MIC values for these isolates ranged from 0.5 to 64 µg/mL. According to the agar dilution method, 8 (30.76%) of 26 isolates were found to be metronidazole resistant. The metronidazole MIC for 7 isolates was 32 µg/mL and for one isolate 64 µg/mL. When these isolates were subjected to PCR to screen for the presence of the nim gene, all isolates were negative. The components of PCR reactions were cross checked for the confidence of action and repeated 3 times. Interestingly, all of the metronidazole-resistant isolates were recovered from patients receiving metronidazole as a prophylaxis before surgery. On the other hand, there was a history of metronidazole use in metronidazole-susceptible B. fragilis as well.


SSIs are largely polymicrobial in nature, involving both aerobic and anaerobic bacteria [4]. These bacterial pathogens cause infection and delay healing. Anaerobic bacterial pathogens remain a significant cause of morbidity and mortality in surgical patients [4]. In some studies, B. fragilis was the most common anaerobic pathogen isolated from SSIs [5], [7].

In our study, this organism was isolated from 26% of the patient sample. By way of comparison, Brook [5] isolated B. fragilis from 36% of patients, while Giacometti [1] isolated B. fragilis from 1.3% of patients. Results of a study by Wollcot et al. [7] which assessed microbial diversity in SSIs using molecular methods indicate the high prevalence of anaerobic Gram-negative rods including B. fragilis in these infections. Since anaerobic bacteria require certain conditions for specimen collection, transport and culture media for in vitro growth, some studies on SSIs may underestimate the prevalence of anaerobic bacteria [4], [7].

In recent years, decreasing susceptibility has been found to a wide range of antimicrobial agents, including metronidazole among B. fragilis strains, which may limit their therapeutic efficiency in treating infections caused by this organism during the course of metronidazole therapy [14]. In 8 patients (30.8%) of this study, we isolated metronidazole-resistant B. fragilis by the agar dilution method, but nim genes were not detectable in these isolates. In previous studies, metronidazole-resistant B. fragilis strains were isolated from 3.9% (Spain, 2010) to 15% (UK, 2004) of patients examined [21], [22].

This study was nosocomial, and B. fragilis were isolated from hospitalized patients with metronidazole administration for prophylaxis and/or treatment of SSIs. The literature describes an association between isolation of metronidazole-resistant B. fragilis and long-term metronidazole therapy [8], [14], [21]. Increasing numbers of clinical metronidazole-resistant B. fragilis that do not possess nimA-H genes are being reported. Resistance to metronidazole could be induced in nim-negative strains by exposure to sub-MICs of metronidazole; the mechanisms behind the increased MICs are not obvious. However, it is clear that there is also a non-nim-based mechanism of resistance to metronidazole [11], [13]. In addition, exposure to sub-inhibitory concentrations of metronidazole may enhance the pathogenicity of the non-susceptible strains of B. fragilis [23], [24].

In our study, no correlation was found between resistance to metronidazole and nim gene presence. In one study [14], metronidazole-resistant B. fragilis were identified from 55% of clinical isolates, while the nim gene was detected from 38.9% isolates. Some studies indicated no correlation between Nim protein levels and metronidazole MIC, and no evidence was found that nim protects B. fragilis from metronidazole. Indeed, the contribution of the nim gene to high metronidazole MICs seen in clinical resistance is unclear [25].

In our study, resistance to metronidazole may be due to non-nim-based mechanisms, such as overexpression of the multidrug efflux pump, overexpression of Rec A, deficiency of feo AB, or some other, as yet unknown mechanism. Regardless of the mechanism, development of metronidazole-resistant B. fragilis may lead to treatment and prophylaxis failure during the course of metronidazole therapy in patients with SSIs caused by B. fragilis. Thus, rapid identification of metronidazole-resistant isolates is essential for early initiation of appropriate antimicrobial therapy and to limit the inappropriate use of antimicrobial agents.



This research was supported by a grant from the Infectious and Tropical Disease Research Center of Tabriz University of Medical Sciences (TBZMED). The manuscript was written based on a dataset of the M.Sc. thesis of Mohammad Yousef Memar registered at Tabriz University of Medical Sciences. The authors would like to thank the staff of Imam Reza surgical wards and microbiology department for their help.

Compliance with ethical standards

The Ethics Commission of Tabriz University of Medical Sciences approved this study (Number: 5/4/6256). All procedures performed in studies involving human participants were in accordance with the ethical standards of the institution.

Competing interests

The authors declare that they have no competing interests.


Giacometti A, Cirioni O, Schimizzi AM, Del Prete MS, Barchiesi F, D'Errico MM, Petrelli E, Scalise G. Epidemiology and microbiology of surgical wound infections. J Clin Microbiol. 2000 Feb;38(2):918-22.
Saini S, Gupta N, Aparna, Lokveer, Griwan MS. Surgical infections: a microbiological study. Braz J Infect Dis. 2004 Apr;8(2):118-25. DOI: 10.1590/S1413-86702004000200001  External link
Akhi MT, Ghotaslou R, Alizadeh N, Beheshtirouy S, Memar MY. High frequency of MRSA in surgical site infections and elevated vancomycin MIC. Wound Med. 2017;17:7-10. DOI: 10.1016/j.wndm.2017.01.002 External link
Edmiston CE Jr, Krepel CJ, Seabrook GR, Jochimsen WG. Anaerobic infections in the surgical patient: microbial etiology and therapy. Clin Infect Dis. 2002 Sep;35(Suppl 1):S112-8. DOI: 10.1086/341931 External link
Brook I, Frazier EH. Aerobic and anaerobic microbiology of surgical-site infection following spinal fusion. J Clin Microbiol. 1999 Mar;37(3):841-3.
Patrick S, Blakely GW, Houston S, Moore J, Abratt VR, Bertalan M, Cerdeńo-Tįrraga AM, Quail MA, Corton N, Corton C, Bignell A, Barron A, Clark L, Bentley SD, Parkhill J. Twenty-eight divergent polysaccharide loci specifying within- and amongst-strain capsule diversity in three strains of Bacteroides fragilis. Microbiology (Reading, Engl). 2010 Nov;156(Pt 11):3255-69. DOI: 10.1099/mic.0.042978-0 External link
Wolcott RD, Gontcharova V, Sun Y, Zischakau A, Dowd SE. Bacterial diversity in surgical site infections: not just aerobic cocci any more. J Wound Care. 2009 Aug;18(8):317-23. DOI: 10.12968/jowc.2009.18.8.43630 External link
Löfmark S, Fang H, Hedberg M, Edlund C. Inducible metronidazole resistance and nim genes in clinical Bacteroides fragilis group isolates. Antimicrob Agents Chemother. 2005 Mar;49(3):1253-6. DOI: 10.1128/AAC.49.3.1253-1256.2005 External link
Seifert H, Dalhoff A; PRISMA Study Group. German multicentre survey of the antibiotic susceptibility of Bacteroides fragilis group and Prevotella species isolated from intra-abdominal infections: results from the PRISMA study. J Antimicrob Chemother. 2010 Nov;65(11):2405-10. DOI: 10.1093/jac/dkq321 External link
Boyanova L, Kolarov R, Mitov I. Recent evolution of antibiotic resistance in the anaerobes as compared to previous decades. Anaerobe. 2015 Feb;31:4-10. DOI: 10.1016/j.anaerobe.2014.05.004 External link
Veeranagouda Y, Husain F, Boente R, Moore J, Smith CJ, Rocha ER, Patrick S, Wexler HM. Deficiency of the ferrous iron transporter FeoAB is linked with metronidazole resistance in Bacteroides fragilis. J Antimicrob Chemother. 2014 Oct;69(10):2634-43. DOI: 10.1093/jac/dku219 External link
Löfmark S, Edlund C, Nord CE. Metronidazole is still the drug of choice for treatment of anaerobic infections. Clin Infect Dis. 2010 Jan;50 Suppl 1:S16-23. DOI: 10.1086/647939 External link
Brook I, Wexler HM, Goldstein EJ. Antianaerobic antimicrobials: spectrum and susceptibility testing. Clin Microbiol Rev. 2013 Jul;26(3):526-46. DOI: 10.1128/CMR.00086-12 External link
Chaudhary M, Payasi A. Emerging metronidazole resistance in anaerobes and mapping their susceptibility behaviour. Am J Inf Dis. 2014;10(2):56. DOI: 10.3844/ajidsp.2014.56.63 External link
Steffens LS, Nicholson S, Paul LV, Nord CE, Patrick S, Abratt VR. Bacteroides fragilis RecA protein overexpression causes resistance to metronidazole. Res Microbiol. 2010 Jun;161(5):346-54. DOI: 10.1016/j.resmic.2010.04.003 External link
Akhi MT, Shirinzadeh M, Ghotaslou R, Sorous MH, Pirzadeh T, Behzad MN. Determination of Antibiotic Sensitivity of Bacteroid fragilis Isolated from Patients and Healthy Individuals in Imam Reza Center of Medical Teaching and Treatment-Tabriz. Jundishapur J Microbiol. 2013;6(9).
Mahon CR, Lehman DC, Manuselis G. Textbook of diagnostic microbiology. Philadelphia: Saunders; 2000.
Akhi MT, Ghotaslou R, Beheshtirouy S, Asgharzadeh M, Pirzadeh T, Asghari B, Alizadeh N, Ostadgavahi AT, Somesaraei VS, Memar MY. Antibiotic susceptibility pattern of aerobic and anaerobic bacteria isolated from surgical site infection of hospitalized patients. Jundishapur J Microbiol. 2015;8(7).
Clinical and Laboratory Standards Institute. Methods for Antimicrobial Susceptibility Testing of Anaerobic Bacteria: Approved Standard. 8th ed. 2012.
Trinh S, Reysset G. Detection by PCR of the nim genes encoding 5-nitroimidazole resistance in Bacteroides spp. J Clin Microbiol. 1996 Sep;34(9):2078-84.
Gal M, Brazier JS. Metronidazole resistance in Bacteroides spp. carrying nim genes and the selection of slow-growing metronidazole-resistant mutants. J Antimicrob Chemother. 2004 Jul;54(1):109-16. DOI: 10.1093/jac/dkh296 External link
Trevińo M, Areses P, Peńalver MD, Cortizo S, Pardo F, del Molino ML, Garcķa-Riestra C, Hernįndez M, Llovo J, Regueiro BJ. Susceptibility trends of Bacteroides fragilis group and characterisation of carbapenemase-producing strains by automated REP-PCR and MALDI TOF. Anaerobe. 2012 Feb;18(1):37-43. DOI: 10.1016/j.anaerobe.2011.12.022 External link
Diniz CG, Cara DC, Nicoli JR, Farias LD, De Carvalho MA. Effect of metronidazole on the pathogenicity of resistant Bacteroides strains in gnotobiotic mice. Antimicrob Agents Chemother. 2000 Sep;44(9):2419-23. DOI: 10.1128/AAC.44.9.2419-2423.2000 External link
Diniz CG, Arantes RM, Cara DC, Lima FL, Nicoli JR, Carvalho MA, Farias LM. Enhanced pathogenicity of susceptible strains of the Bacteroides fragilis group subjected to low doses of metronidazole. Microbes Infect. 2003 Jan;5(1):19-26. DOI: 10.1016/S1286-4579(02)00052-7 External link
Leitsch D, Sóki J, Kolarich D, Urbįn E, Nagy E. A study on Nim expression in Bacteroides fragilis. Microbiology (Reading, Engl). 2014 Mar;160(Pt 3):616-22. DOI: 10.1099/mic.0.074807-0 External link