The aim of this study was to establish a broth microdilution method for antimicrobial susceptibility testing of Helicobacter cinaedi and to assess the prevalence and mechanisms of fluoroquinolone resistance in Japanese clinical isolates. A broth microdilution method using modified Levinthal broth was developed and compared with the agar dilution method for testing susceptibility to ampicillin, gentamicin, tetracycline and ciprofloxacin. The minimum inhibitory concentrations obtained by these two methods were almost the same for all the antibiotics tested, demonstrating the broth microdilution method to be a suitable and reliable technique for antimicrobial susceptibility testing. A broth microdilution method for antimicrobial susceptibility test for H. cinaedi was established. This method is expected to help improve treatment.
brain heart infusion
colony forming unit
Clinical and Laboratory Standards Institute
- E. coli
- H. cinaedi
- H. pylori
minimal inhibitory concentration
tryptic soy broth
Helicobacter cinaedi was first isolated from homosexual men as a Campylobacter-like organism (CLO) associated with proctitis, proctocolitis, and enteritis. This CLO, which belonged to new genetic group that differed from other known Campylobacter species, was named Campylobacter cinaedi . Later in 1991, on the basis of hybridization data, this organism was reclassified as belonging to the genus Helicobacter . Although H. cinaedi infections previously were reported mainly in immunocompromised patients [3-7], recently increasing numbers of infection have been reported in immunocompetent patients [8, 9]. Examples of the diverse range of infections caused by H. cinaedi include proctocolitis, gastroenteritis, neonatal meningitis, localized pain, rash and bacteremia . Because this organism is difficult to culture, it is hard to isolate compared to other Helicobacter spp.; as a result, its biological and clinical characteristics are less well understood .
Kitamura et al. have documented an outbreak of nosocomial H. cinaedi infections caused by direct person-to-person spread [11, 12]. Since 2004, we have received reports of a growing number of cases of nosocomial H. cinaedi infections in Japan, having received approximately 200 H. cinaedi strains from over 15 hospitals; nine of these cases were suspected to be nosocomial infections. These strains were isolated from patients of both sexes and a broad age range (from neonates to 81 years). Some patients had no underlying immunocompromising conditions and had not received immunosuppressive agents . The hospitals in which the H. cinaedi infections were detected were located throughout Japan. Thus, we surmise that infection is more common in Japanese hospitals than currently recognized. Thus far, there are no recommended guidelines for susceptibility testing and treatment of diagnosed infections with H. cinaedi . H. cinaedi reportedly fails to grow when tested for antimicrobial susceptibility by a broth microdilution method . Therefore the relatively laborious agar dilution method is generally used for susceptibility testing of H. cinaedi strains isolated from clinical specimens . Thus, there is clearly a need for a more easy-to-perform and reliable approach to enable appropriate antimicrobial therapy, which would clearly improve patient treatment and help prevent the emergence of antibiotic resistant strains.
MATERIALS AND METHODS
Culture of Helicobacter cinaedi
The Helicobacter cinaedi strains were isolated from clinical materials in hospitals throughout Japan between July 2004 and November 2011 (Supporting Information). The strains were cultured on 5% defibrinated sheep blood agar (Kohjin Bio, Saitama, Japan) and incubated at 37°C for 3 days under microaerobic conditions (6% O2, 7% CO2, 7% H2, 80% N2) generated by Anoxomat apparatus (Mart Ii, Mart Microbiology, Drachten, the Netherlands). Clinical isolates were identified as H. cinaedi by Gram staining and by a H. cinaedi-specific PCR approach . All cultures were stored at − 80°C in tryptic soy broth (Difco Laboratories, Sparks, MD, USA) supplemented with 15% glycerol. In addition to the Japanese isolates nine reference strains isolated outside Japan were assayed: H. cinaedi PAGU 597T (=CCUG 18818T, isolated in the USA), PAGU 639 (=CCUG 19503, isolated in Canada), PAGU 640 (=CCUG 19504, isolated in Canada), PAGU 641 (=CCUG 15432, isolated in Sweden), PAGU 642 (=CCUG 18819, isolated in the USA), PAGU 1744 (=CCUG 19218, isolated in the USA), PAGU 1749 (=CCUG 38648, isolated in Sweden), PAGU 1752 (=CCUG 43522, isolated in Australia) and PAGU 1753 (=CCUG 44719, isolated in Sweden).
Media used in this study were as follows: tryptic soy broth, brain heart infusion broth (Difco Laboratories), brucella broth (Difco Laboratories), RPMI-1640 medium, Sigma–Aldrich, St Louis, MO, USA) containing 3% FBS , F-12 nutrient mixture Ham with glutamine (Gibco BRL, Grand Island, NY, USA) containing 1% FBS  and modified Levinthal broth. The modified Levinthal broth contained (per liter of medium) 10 mL defibrinated horse blood, 1000 mL Mueller Hinton broth (Difco Laboratories), 25.0 mg Ca2+ and 12.5 mg Mg2+. After heating at 100°C for 5 mins the modified broth was filter-sterilized.
Cultures (72 hrs) of H. cinaedi were adjusted in 0.85% (w/v) saline to a turbidity of optical density at 600 nm (OD600) of 0.1. Amount of growth was compared between eight strains isolated within and outside Japan. This suspension yielded about 108 CFU/mL. Fifty microliter samples of the suspensions were inoculated into 2 mL of each medium and incubated using static culture method at 37°C for 72 hrs in a microaerophilic environment. Control wells contained only broth. Amount of growth of H. cinaedi was measured by the turbidity of the OD measurements. H. pylori PAGU 150T (=NCTC 11637T) was also used in this assessment.
Antibiotic susceptibility testing
Minimal inhibitory concentrations were determined by an established agar dilution method [14, 18] and by a broth microdilution method developed in our laboratory. Susceptibilities to ampicillin (Wako Pure Chemical Industries, Osaka, Japan), gentamicin (Wako Pure Chemical), tetracycline (Wako Pure Chemical), and ciprofloxacin (Wako Pure Chemical) were measured using both methods, and the MICs obtained compared. Moxifloxacin (LKT Laboratories, St Paul, MN, USA) and levofloxacin (LKT Laboratories) were tested for using the broth microdilution method.
The CLSI advises the agar dilution method for H. pylori and closely related species. All antimicrobial agents were dissolved and diluted according to CLSI methods . For the agar dilution method, the appropriate dilutions of antimicrobial agents were prepared with diagnostic sensitivity test agar (pH 7.4; Oxoid, London, UK) with 10% defibrinated horse blood . All antimicrobial agents were tested in a series of twofold dilutions from 0.06 to 64.0 μg/mL. Cultures (72 hrs) of H. cinaedi were suspended in trypticase soy broth to a turbidity of OD600 = 0.1 and diluted 1:20 in 0.85% (w/v) saline. This suspension yielded 106–107 CFU/mL. Twenty microliter samples of the diluted suspensions were inoculated into each plate. Plates containing no antibiotics were inoculated to determine viability and quantitation of growth. The inoculated plates were incubated at 37°C for 72 hrs in a microaerophilic environment. MICs were defined as the minimum concentration of the antimicrobial agent which completely inhibited growth of the strain.
A broth microdilution method using modified Levinthal broth in a 96-well microtiter plate format was developed. Microtiter plates containing 0.05 mL broth + antibiotic per well were inoculated with an equal volume of diluted culture: inocula were diluted in broth to an OD600 of 0.1 and then diluted 1:20 for final antibiotic concentrations in the range 0.06–64.0 μg/mL. Control wells containing only broth served as positive growth controls. The plates were incubated for 72 hrs at 37°C under microaerobic conditions. The MICs were read visually as the lowest concentration of antibiotic causing complete inhibition of growth.
Polymerase chain reaction amplification and nucleotide sequence analysis
Extraction of H. cinaedi genomic DNA was performed to detect gyrA mutations as reported previously . PCR amplification using gyrA primers (5′-AGATGGACTAA AGCCGGTGCATAG-3′ and 5′-TGTGGCGGAATAGAAGTCGCCAT-3′) were carried out. PCR cycling conditions consisted of 35 cycles of 15 s denaturation at 94°C, 15 s annealing at 55°C and 30 s extension at 72°C. After confirming amplicons of gyrA on 1% agarose gels, the sequences were determined using a BigDye Terminator v3.1 Cycle Sequencing kit (Applied Biosystems, Tokyo, Japan). The gyrA gene sequences of both strands of the gene were determined using the 3130 Genetic Analyzer (Applied Biosystems). Multiple sequence alignments of DNA sequences and deduced amino acid sequences of all 79 isolates analyzed were compared with those of the known H. pylori gyrA gene using DNASIS pro software (Hitachi Solutions, Yokohama, Japan).
All eight H. cinaedi strains used in comparative testing grew well in modified Levinthal broth at 37°C (Fig. 1). In contrast, a small amount of growth was detected in BHI or TSB. In RPMI-1640 medium, F-12 nutrient mixture Ham medium or brucella broth, abundant growth was detected. Sisto et al. performed the broth microdilution method with RPMI-1640 for H. pylori . Our results show that RPMI-1640 is suitable for cultivation of H. pylori, but is less conclusive for H. cinaedi. The use of modified Levinthal broth not only results in better growth of the organisms but also makes the microtiter tests easier to read because of improved visual contrast. Our results suggest that modified Levinthal broth is suitable for the broth microdilution method for H. cinaedi.
Minimum inhibitory concentration determination
Minimum inhibitory concentrations were determined by both the agar and broth dilution methods for 79 H. cinaedi strains, including 9 strains isolated from outside Japan. The MICs obtained by these two different methods were almost the same for all antimicrobial agents tested; these included penicillins, aminoglycosides, tetracyclines and fluoroquinolones (Table 1).
|Antimicrobial agent||MIC50 (μg/mL)||MIC90 (μg/mL)||No. (%) of isolates with the stated differences in MICs‡||Agreement %§|
|Ampicillin||8||8||16||16||3 (3.8)||18 (22.8)||58 (73.4)||0||0||96.2|
|Gentamicin||0.5||0.5||1||1||2 (2.5)||17(21.5)||59 (73.4)||1 (1.3)||0||97.5|
|Tetracycline||0.5||0.25||1||1||0||1 (1.3)||43 (54.4)||28 (35.4)||7 (8.9)||91.1|
|Ciprofloxacin||32||16||64||64||0||0||58 (73.4)||21 (26.6)||0||100|
Significantly, all strains isolated in Japan were resistant to ciprofloxacin, regardless of differences in hospital location, patient age and sex. The resistance breakpoints of fluoroquinolones were tentatively defined as >1 μg/mL, as previously suggested for H. pylori [21-23]. Several other fluoroquinolone agents, moxifloxacin and levofloxacin, were examined by the broth microdilution method. The rate of resistance of strains isolated in Japanese hospitals to ciprofloxacin, moxifloxacin or levofloxacin was 100% (70/70). Based on these breakpoints, the seven strains isolated outside Japan were found to be susceptible to ciprofloxacin (MIC < 1.0 μg/mL), moxifloxacin and levofloxacin (Table 2). However, two of the strains isolated outside Japan were resistant to ciprofloxacin and levofloxacin.
|Isolation†||No. of isolates||MIC (μg/mL)||Mutation of GyrA|
|Outside Japan (USA, Canada, Sweden)||7||0.06–0.25||0.06–0.12||0.06||—|
|Outside Japan (Sweden, Australia)||2||16–32||4||0.25||Thr84Ile|
|Japan (Hospital B, E, F, I)||7||8||2–4||0.25–0.5||Thr84Ile|
|Japan (Hospital A, B, C, D, E, F, G H, I)||27||16||4–8||0.25–1.0||Thr84Ile|
|Japan (Hospital A, B, D, E, G, H, I)||19||32||4–64||0.25–8||Thr84Ile|
|Japan (Hospital A, B)||5||64||8–64||0.5–8||Thr84Ile|
|Japan (Hospital B)||1||16||16||4||Thr84Ile, Asp88Gly|
|Japan (Hospital B, G)||2||32||16||8||Thr84Ile, Asp88Asn|
|Japan (Hospital B, G, H, I)||12||≥64||32–64||8–32||Thr84Ile, Asp88Gly, or Asp88Asn‡|
Analyses of mutations of the gyrA genes and their relationship with fluoroquinolone resistance
GyrA gene sequencing was determined for all 79 strains of H. cinaedi. Seven fluoroquinolone-susceptible strains isolated outside Japan had no mutations, whereas 72 resistant strains had point mutations in GyrA at Thr-84 or Asp-88. A Thr-84-Ile mutation was identified in 100% (72 of 72). Additionally, 15 strains had another mutation in Asp-88-Asn (12/15) or Asp-88-Gly (3/15). The relationship between MIC levels and GyrA mutation patterns in fluoroquinolone-resistant strains is shown in Table 2. Strains with substitutions at Thr-84 were observed to have moderate or intermediate levels of moxifloxacin-resistance, whereas 15 resistant strains with double substitutions were found to have high-level resistance.
The number of reports of H. cinaedi infections has been steadily growing and the association of this bacterium with a variety of human infections has received increasing attention in recent years. H. cinaedi is currently the most commonly reported enterohepatic Helicobacter isolated from humans. The increasing clinical significance of this infection necessitates effective therapeutic strategies for its eradication. However, because of its fastidious nature, H. cinaedi is relatively difficult to isolate and identify, which hinders fast and effective antimicrobial therapy. Penicillin, aminoglycosides, carbapenem and fluoroquinolone are reportedly effective . However, there is reportedly a high incidence of recurrence of H. cinaedi cellulitis (bacteremia) [11, 14]. Researchers have also described erythromycin-resistant strains with amino acid substitutions within the 23S rRNA gene . Rimbara et al. documented that susceptibility to clarithromycin was low in all strains isolated in Japan (n = 18; MIC50 value 64 μg/mL) . No guidelines have been established for the use of antibiotics against H. cinaedi; this is clearly an important issue for clinical microbiological laboratories.
In the present study, we developed a broth microdilution susceptibility test and obtained comparable data to that for the established agar dilution method for H. cinaedi. Simplification of determination of MIC levels prior to prescribing antibiotics would improve treatment efficacy. The agar dilution method, which has been the standard susceptibility testing method, is an impractical method for routine laboratories. The E test, which is reportedly as easy to perform as the disk diffusion method and gives MIC results comparable to those of the agar dilution method, is an alternative approach [27-29]. However, because flagellated H. cinaedi have a migratory growth pattern, the E test and disk diffusion methods can be inaccurate. These two methods often fail to allow visualization of resistant bacteria within zones of inhibition. Because H. cinaedi is difficult to culture, these methods often fail to produce clear edges around growth zones. Therefore, we developed a broth microdilution method for susceptibility testing that is practical for use in clinical laboratories. We compared growth of H. cinaedi strains in various media. With H. cinaedi strains, which are known to be difficult to grow in liquid culture, the only medium that resulted in a significant increase in turbidity in 72 hr was modified Levinthal broth.
Table 1 displays the correlations of the MICs of four antimicrobials for 79 isolates generated by the agar dilution and broth microdilution methods. We found a >90% agreement (range 91.1%–100%) between MICs by each method for all antimicrobial agents. In this study, we have shown that the broth microdilution method using modified Levinthal broth is a logical choice for susceptibility testing.
The MICs for ampicillin were 0.5–32 μg/mL for the 79 H. cinaedi isolates tested. Amoxicillin, another penicillin used to treat H. pylori infection, showed similar MIC values (1–16 μg/mL; MIC50, 8 μg/mL; MIC90, 8 μg/mL). The MIC range for gentamicin and tetracycline were 0.12–2 μg/mL and 0.06–4 μg/mL, respectively. Bacteremia caused by gram-negative bacteria is often treated with expanded-spectrum cephalosporins and carbapenems. In our preliminary data, cefepime MICs were 0.5–32 μg/mL (MIC50, 4 μg/mL; MIC90, 8 μg/mL) and imipenem MICs 0.06–4 μg/mL (MIC50, 0.12 μg/mL; MIC90, 1 μg/mL).
We detected fluoroquinolone-resistant H. cinaedi strains amongst those isolated from patients at Japanese hospitals during 2004–2011 (Table 2). Nucleotide sequencing of the gyrA gene revealed that all strains had amino acid substitutions. Because H. cinaedi does not have parC or parE genes that encode the topoisomerase IV , gyrA mutations play an important role in fluoroquinolone resistance.
In our experience, the broth microdilution method is an easy and reliable method for determination of the MICs of antibiotics for H. cinaedi. Our broth microdilution method can be performed using commercially available microtiter plates and automated systems for MIC determination [31, 32]. This method may also be used for dry-plate tests. Therefore the broth microdilution method described here is highly suitable for routine laboratories.
We thank Masaru Baba of Toranomon hospital for helpful discussion and cooperation concerning the E test and disk diffusion method for H. cinaedi. We are grateful to the following individuals for providing the H. cinaedi isolates used in this study: Shunji Takahashi, Sapporo City General Hospital; Masashi Narita, Ohta Nishinouchi Hospital; Yoshihito Otsuka, Kameda Medical Center; Haruki Sawamura and Hiroshige Mikamo, Aichi Medical University; Yoko Kawakami, National Hospital Organization Kyushu Cancer Center; and Toshio Kitamura and Shuichi Higashi, Kumamoto Orthopedic Hospital.
The authors declare that they have no conflicts of interest related to this study.