Clarithromycin and fluoroquinolone are presently the drugs of choice employed for triple combination therapy in the treatment of H. pylori
]. Resistance to these drugs is emerging and presents a challenge. Different studies all over the world have reported resistance to clarithromycin and fluoroquinolone [8
]. The high resistance rates to these antibiotics, the burden of H. pylori
infection, and its associated disease conditions coupled with the difficulties of rapid diagnosis and management of patients [16
] necessitated the determination of the antibiogram and associated mutation to clarithromycin and fluoroquinolone to H. pylori
strains isolated from the Eastern Cape province known to have a high prevalence of H. pylori
-related morbidities [2
] using the GenoType HelicoDR assay.
was detected in 168/254 (66.1%) of the specimens studied. Clarithromycin is used worldwide as one of the potent antibiotics in the eradication therapy of H. pylori
]. However, resistance to clarithromycin has been increasingly reported in several studies [12
]. This led to the introduction of new treatments such as the fluoroquinolones which seemingly is offering great hope, but unfortunately resistance to them is emerging [7
]. The presence of resistance is often associated with failure of eradication therapy [20
]. Findings from this study revealed moderate rate of resistance to clarithromycin and fluoroquinolone with percentages of 15.38% and 10.26%, respectively. This result corroborates the finding of Kim et al. [12
] who reported resistance to clarithromycin with a range of 7.6% to 18.6% in Korea. Macrolides like clarithromycin are expensive; however, cross-resistance linked with the use of other less expensive macrolides may be responsible for this resistance. Worthy of note is the fact that clarithromycin susceptible and resistant strains have been isolated from patients with no history of exposure to macrolides [21
]. Therefore, it is imperative to guide empiric treatment since administration of clarithromycin can be selected for resistance.
The moderate rate of resistance of 10.26% to fluoroquinolone reported in this study ties with the findings of Wang et al. [13
] who reported 15.6% resistance to ciprofloxacin in their study in Alberta, Canada. However, generally low resistance to the fluoroquinolones has been reported compared to other antibiotics. For example, in our previous study [16
], all strains were susceptible to ciprofloxacin as opposed to the 10.26% resistance generally reported for the fluoroquinolone in the current study. We may relate this to the difference in strains as well as the methods (phenotypic versus molecular) used in both studies. Also, the strips in the GenoType HelicoDR assay are designed to generally target the fluoroquinolones. Hung et al. [3
] also documented 5.7% resistance of their strains to ciprofloxacin, and Kohanteb et al. [22
] reported 4.7% in their study. Isolates from Belgium, France, Italy, and Germany have higher resistance rates to ciprofloxacin or levofloxacin ranging between 16.8% and 23% [3
]. Also, higher resistance rates (ciprofloxacin: 33.8%; levofloxacin: 21.5%) have been observed in Japan [19
]. These disparate rates of resistance could be attributed to geographical region and drug usage differences [9
The prevalence of antibiotic resistance was higher in males than in females in this study reaching statistical significant difference for clarithromycin (P
= 0.006), but not for fluoroquinolone. Importantly, more males were recruited for this study than females and this could account for the difference observed. Noteworthy is the fact that the number of strains resistant to clarithromycin (12) and fluoroquinolone (8) is almost the same, suggesting that these antibiotics could be close in their suitability as drugs of choice for H. pylori
treatment in our environment corroborating our previous finding [16
Mutations associated with resistance to these antibiotics were investigated. Three strains showed 2 or more mutations with the highest number of mutations occurring in 252C (). We observed that the higher the number of mutations per strain, the higher the MIC value of that strain. Strains in our study which were resistant to clarithromycin with mutation A2147G had MIC values that ranged from 32 to 256μ
g/mL. Strains 252A and 252C had MIC of 32μ
g/mL, respectively, while 247A had MIC 256μ
g/mL; they all possessed multiple mutations; 247C and 253C had MIC values of 8μ
g/mL and 16μ
g/mL (), respectively. These variations could be due to strain diversity.
Fluoroquinolone acts by inhibiting DNA gyrase, topoisomerase, and interfering with bacterial DNA replication; since topoisomerase is not found in the H. pylori
genome, mutation in the gyrA
gene which encodes DNA gyrase is considered to be the major cause of resistance to fluoroquinolones [7
]. The codons N87 and D91 are recognised as the most important target sites for ciprofloxacin binding [3
]. The N87H, N87I, N87K, and N87Y as well as D91G, D91N, and D91Y mutations in gyrA
have been reported in fluoroquinolone-resistant H. pylori
]. The assay used in this study was designed to depict N87K, D91N, D91G, and D91Y which have been frequently reported. However, N87K and D91N mutations were the only mutations associated with fluoroquinolone found in this study. N87K was the most prevalent mutation (8/17; 47.05%) associated with fluoroquinolone amongst our strains whilst D91N was found in one strain ().
Our findings are in line with those of Wang et al. [13
] and Hung et al. [3
] who reported the presence of these mutations in their studies, respectively. Furthermore, it confirms the fact that these mutations are the most frequently found mutations [3
]. N87K was found in eight strains. The D91Y and D91G mutations were not found in the current study. This contradicts the finding of Garcia et al. [14
] who reported the presence of these mutations in six and seven strains, respectively. However, occurrence in just five and two strains of their huge sample size may imply a generally low occurrence.
Clarithromycin acts by inhibiting protein synthesis by binding to the peptidyltransferase loop of 23S rRNA which has been shown at residues A2058 and A2059 in the 23S rRNA gene of E. coli
. When mutation occurs in these residues, the binding affinity of clarithromycin to ribosomes is reduced, resulting in clarithromycin resistance [11
]. The assay used in this study was designed to target the presence of A2147G, A2146G, and A2146C associated with clarithromycin resistant strains [18
]; there is a dearth of information in the literature on A2147G, A2146G and A2146C mutation compared to A2142G, and A2143G which are frequently reported [6
] to be associated with clarithromycin resistance [12
Twelve (70.5%) of the 17 strains reported to be resistant in this study had A2147G mutation (Tables and ). This accords with the findings of Cambau et al. [18
] who reported a high prevalence of A2147G mutation amongst their strains. The high occurrence of A2147G mutation suggests its high frequency amongst our local strains as opposed to A2146C which occurred in one (252C) of the 17 strains studied. The sensitivity and specificity of detecting resistance to clarithromycin and fluoroquinolone observed in our current investigation corroborate with those of Cambau et al. [18
], although they specifically used levofloxacin, which is also a member of the fluoroquinolone. They reported values of 94% and 99% for clarithromycin and 87% and 99% for levofloxacin, respectively.
In conclusion, this study revealed a moderate rate of resistance to fluoroquinolone and clarithromycin, with A2147G and N87K being the main mutations associated with clarithromycin and fluoroquinolones, respectively. However, continuous surveillance of fluoroquinolone and clarithromycin resistance in H. pylori is thus relevant in this area to guide empiric treatment.