Antimicrobial Drug Resistance in Corynebacterium diphtheriae mitis

To the Editor: Corynebacterium diphtheriae is the agent of pharyngeal and cutaneous diphtheria. We did a retrospective analysis of the antimicrobial drug susceptibilities of 46 C. diphtheriae isolates sent during 1993 through 2010 to the French National Reference Centre of Toxigenic Corynebacteria. The isolates came from metropolitan France and French overseas departments and territories. Only 1 isolate, C. diphtheriae biovar mitis, FRC24, expressed the following antimicrobial drug susceptibility profile: susceptible to penicillin, amoxicillin, ciprofloxacin, clindamycin, erythromycin, gentamicin, imipenem, kanamycin, rifampin, tetracycline, and vancomycin and resistant at an uncommonly high level to trimethoprim, sulfamethoxazole, and co-trimoxazole with Etest (bioMerieux, Marcy l’Etoile, France) MICs of >32, >1,024, and >32 mg/L, respectively. 
 
This FRC24 isolate was isolated in 2008 from a cutaneous wound on a vaccinated 11-month-old child in Mayotte, an overseas department located in the Indian Ocean. Cutaneous carriage of C. diphtheriae is frequent in tropical countries where cutaneous diphtheria is endemic; cutaneous carriage represents a common mode of transmission of the bacterium. FRC24 was identified by using the API Coryne strip (bioMerieux). FRC24 is a toxigenic isolate; toxigenicity was confirmed by both tox gene detection and Elek test (1). Multilocus sequence typing was performed, and the sequence type (ST) of the isolate is ST91. This ST contains only this isolate and is part of lineage II, as are all mitis and gravis biovars (2). 
 
To date, resistance to trimethoprim, sulfamethoxazole, or co-trimoxazole seems to be rare among the C. diphtheriae species, but few data are available (3). As trimethoprim resistance is often encoded by integron-driven dfr determinants, we looked for integrons. Integrons are bacterial genetic elements able to capture and express antimicrobial drug resistance gene cassettes (GCs) (4). GC movements are catalyzed by an integron-encoded integrase IntI. GCs, mainly promoterless, are usually expressed through a common Pc promoter (5). Only rare GCs contain their own promoter (cmlA, qac, ereA1). Three main classes of integrons have been described and are involved in the dissemination of antimicrobial drug resistance; class 1 is the most widely found in clinical isolates. Integrons have been mainly described among gram-negative bacteria; only a few studies have reported integrons in Corynebacterium spp (6,7). 
 
After bacterial genomic DNA extraction (DNeasy Blood & Tissue Kit; QIAGEN, Courtaboeuf, France), a multiplex Taqman-based quantitative PCR approach able to detect the 3 main classes of integrons was performed (8). We found that FRC24 harbored a class 1 integron. Analysis of the GC array showed that this integron harbored 2 GCs: dfrA16 of 588 bp conferring resistance to trimethoprim and qacH of 511 bp conferring resistance to quaternary ammonium compounds (GenBank accession no. {"type":"entrez-nucleotide","attrs":{"text":"FR822749","term_id":"323714042"}}FR822749). To our knowledge, this GC array has not been previously reported, even among reports of other gram-negative isolates. Moreover, a qac determinant has been found only once in a Corynebacterium species, C. pseudogenitalium (which harbors a qacH variant in the chromosome [GenBank accession no. {"type":"entrez-nucleotide","attrs":{"text":"ABYQ02000013","term_id":"311303764"}}ABYQ02000013]), but not in an integron background. GC arrays were followed by the qacEΔ1 (which also confers resistance to quaternary ammonium compounds), sul1 (resistance to sulfamethoxazole), and orf5 determinants as found in most class 1 integrons (4). In class 1 integrons, 13 Pc variants have been described (5). In the FRC24 integron, the dfrA16 expression was mediated through a strong Pc variant (PcWTGN-10) (5) that enables the high-level resistance observed for trimethoprim. As previously demonstrated, the qacH GC possessed its own promoter (9). 
 
Trimethoprim is a commonly prescribed antimicrobial agent used in combination with sulfamethoxazole (co-trimoxazole) for the treatment of diarrheal diseases. This antimicrobial drug might have selected the emergence of such a strain expressing trimethoprim resistance. Furthermore, the FRC24 integron contains the antiseptic (quaternary ammonium compounds) resistance gene qacH. As cutaneous carriage of C. diphtheriae is frequent in tropical countries such as Mayotte, this bacterium could be exposed to quaternary ammonium compounds contained in disinfectants, hygienic hand washes, and cosmetic products. These products exert a selective pressure, which might play a role in selecting qac-containing strains, as has been suggested for Staphylococcus spp (10). For staphylococci, the MICs of quaternary ammonium compounds are >2 mg/L. With FRC24, we tested for the MIC of cetyltrimethylammmonium bromide and found a MIC of 4 mg/L, suggesting that qacH is expressed in FRC24. 
 
The sequencing of the genetic environment of this integron showed that it was framed by 2 copies of the insertion sequence IS6100 disrupting at the left-hand side the intI1 integrase gene (Figure A1). IS6100 has been described in a wide spectrum of host organisms, including Corynebacterium spp (6,7), thus enabling this integron to be efficiently transferred to various bacteria. 
 
Our findings show that C. diphtheriae is able to harbor integrons, which is of clinical relevance. Indeed, this genetic feature would give the isolates the capacity to easily acquire new GCs, such as ere GCs encoding resistance to erythromycin, which is one of the antimicrobial drugs recommended for diphtheria treatment.

2010 had distinct random amplifi ed patterns that differed from those generated from isolates A-G (Figure).
A previous study in Taiwan showed that the incidence (no. cases/100,000 inpatients and outpatients) of all pulmonary disease caused by NTM increased signifi cantly from 2.7 (1.26) in 2000 to 10.2 (7.94) in 2008 (6). The most common organism in localized pulmonary infection and disseminated infection was Mycobacteriam avium cellular complex, and M. abscessus predominated in skin and soft tissue infection and lymphadenitis (6,8). The rise in pulmonary infections or colonization by NTM over recent decades, particularly among immunocompromised populations, is reported to be partly associated with the increased use of showers (3)(4)(5)9). Recently, a few studies have shown a link between pulmonary M. avium complex infections and home showerhead water microbiology (3,4). Although pulmonary disease caused by M. abscessus did not develop in the patient reported here, multiple respiratory specimens showed evidence of pulmonary colonization. The fact that cultures of the swabs taken from the interior surface of 4 showerheads were positive for M. abscessus but that cultures of the shower water were negative for the organism support previous fi ndings that assemblages of NTM can occur inside biofi lm that forms on the interior surface of showerheads (5). The same strains of M. abscessus isolated from different showerheads suggested the possibility of contamination in the environment by the aerosolized microorganism from respiratory secretions of the patient.
The mechanisms of susceptibility to mycobacterial infection in the patient with Sjögren's syndrome remain unknown (1,2). Previous studies suggest that toll-like receptor 2, dectin-1, tumor necrosis factor-α, interferon-γ, leptin, T-cells, and possibly neutrophils are major components in the host defense of HIV-noninfected patients against rapidly growing mycobacterial infections, including those caused by M. abscessus (10).
In summary, we report a case of bacteremic lymphadenitis caused by M. abscessus in a patient with Sjögren syndrome. Our data provide evidence that the interior surface of showerheads may serve as a source of infection by this waterborne and aerosolized microorganism. To date, resistance to trimethoprim, sulfamethoxazole, or co-trimoxazole seems to be rare among the C. diphtheriae species, but few data are available (3). As trimethoprim resistance is often encoded by integron-driven dfr determinants, we looked for integrons. Integrons are bacterial genetic elements able to capture and express antimicrobial drug resistance gene cassettes (GCs) (4). GC movements are catalyzed by an integronencoded integrase IntI. GCs, mainly promoterless, are usually expressed through a common Pc promoter (5). Only rare GCs contain their own promoter (cmlA, qac, ereA1). Three main classes of integrons have been described and are involved in the dissemination of antimicrobial drug resistance; class 1 is the most widely found in clinical isolates. Integrons have been mainly described among gram-negative bacteria; only a few studies have reported integrons in Corynebacterium spp. (6,7).

Yu-Min
After bacterial genomic DNA extraction (DNeasy Blood & Tissue Kit; QIAGEN, Courtaboeuf, France), a multiplex Taqman-based quantitative PCR approach able to detect the 3 main classes of integrons was performed (8). We found that FRC24 harbored a class 1 integron. Analysis of the GC array showed that this integron harbored 2 GCs: dfrA16 of 588 bp conferring resistance to trimethoprim and qacH of 511 bp conferring resistance to quaternary ammonium compounds (GenBank accession no. FR822749). To our knowledge, this GC array has not been previously reported, even among reports of other gram-negative isolates. Moreover, a qac determinant has been found only once in a Corynebacterium species, C. pseudogenitalium (which harbors a qacH variant in the chromosome [GenBank accession no. ABYQ02000013]), but not in an integron background. GC arrays were followed by the qacEΔ1 (which also confers resistance to quaternary ammonium compounds), sul1 (resistance to sulfamethoxazole), and orf5 determinants as found in most class 1 integrons (4). In class 1 integrons, 13 Pc variants have been described (5). In the FRC24 integron, the dfrA16 expression was mediated through a strong Pc variant (PcW TGN-10 ) (5) that enables the high-level resistance observed for trimethoprim. As previously demonstrated, the qacH GC possessed its own promoter (9).
Trimethoprim is a commonly prescribed antimicrobial agent used in combination with sulfamethoxazole (co-trimoxazole) for the treatment of diarrheal diseases. This antimicrobial drug might have selected the emergence of such a strain expressing trimethoprim resistance. Furthermore, the FRC24 integron contains the antiseptic (quaternary ammonium compounds) resistance gene qacH. As cutaneous carriage of C. diphtheriae is frequent in tropical countries such as Mayotte, this bacterium could be exposed to quaternary ammonium compounds contained in disinfectants, hygienic hand washes, and cosmetic products. These products exert a selective pressure, which might play a role in selecting qac-containing strains, as has been suggested for Staphylococcus spp. (10). For staphylococci, the MICs of quaternary ammonium compounds are >2 mg/L. With FRC24, we tested for the MIC of cetyltrimethylammmonium bromide and found a MIC of 4 mg/L, suggesting that qacH is expressed in FRC24.
The sequencing of the genetic environment of this integron showed that it was framed by 2 copies of the insertion sequence IS6100 disrupting at the left-hand side the intI1 integrase gene (online Appendix Figure, wwwnc.cdc.gov/ EID/article/17/11/11-0282-FA1.htm). IS6100 has been described in a wide spectrum of host organisms, including Corynebacterium spp. (6,7), thus enabling this integron to be effi ciently transferred to various bacteria.
Our fi ndings show that C. diphtheriae is able to harbor integrons, which is of clinical relevance. Indeed, this genetic feature would give the isolates the capacity to easily acquire new GCs, such as ere GCs encoding resistance to erythromycin, which is one of the antimicrobial drugs recommended for diphtheria treatment.
This work was supported by grants from Ministère de la Recherche et de l'Enseignement Supérieur, Institut National de la Santé et de la Recherche Médicale, and Institut Pasteur Fondation.