Prevalence of Antibiotic Resistance and Minimum

Global Journal of Gastroenterology & Hepatology, 2013, 1, 000-000

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Prevalence of Antibiotic Resistance and Minimum Inhibitory Concentrations of Helicobacter pylori Isolates from Clinical Gastritis Patients, Saudi Arabia Amer Ibrahim Al-Omar1,2, Maripandi Arjunan1,3 and Ali A. Al-Salamah1,* 1

Unit of Medical Bacteriology, Department of Botany and Microbiology, College of Science, King Saud University, P.O. Box 2455, Riyadh, Saudi Arabia 2

Health Science Colleges, P.O. Box 3761, Dammam 31481, Saudi Arabia

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Department of Microbiology, Government Arts College for Men, Krishnagiri, Tamilnadu, India Abstract: Background: Establishing effective and acceptable treatment regimens for patients infected with Helicobacter pylori continues to pose problems for physician and patient alike in Saudi Arabia. The aim of the study was to assess the antibiotics resistance and to find out the MICs of the drugs which are commonly used against H. Pylori. Methods: A total of one hundred antral biopsy specimens were collected and processed from which seventy-six strains of H. pylori were isolated. All the isolates were performed antibiotics sensitivity and E-test. Results: The antibiotics result revealed that of H. pylori isolates 72% strains were resistant to metronidazole and 8% of isolates were resistant to clarithromycin. On the basis of antibiotics resistant a five different antibiotics phenotypes were found among H. pylori isolates. The distribution of metronidazole MICs was high in the range between 32 to128 mg/L. However all other antibiotics showed the effective MICs range was below 1 mg/L. Conclusions: Our findings suggested that resistance to metronidazole has been increasing in the Saudi population. The resistance may be linked to the frequent use of these antimicrobial agents for treating parasitic infections. The E test results revealed that the most effective antibiotic MICs were amoxicillin and clarithromycin against H. Pylori infection.

Keywords: Peptic ulcer, Helicobacter pylori, Antral biopsy, Antibiotics, Resistance. INTRODUCTION Helicobacter pylori is a capnophilic Gram-negative spiral bacterium. It is one of the most prevalent human pathogens that are closely associated with gastritis and peptic ulcer (PU) diseases. It has been estimated that 50% world population is infected by these organisms [1, 2]. The prevalence of H. pylori infection varies greatly worldwide, with infection rates of more than 80% in some developing countries and below 20% in some developed countries [3]. H. pylori naturally reside in the human stomach. Outside its host, H. pylori are fastidious and can grow only under microaerophilic conditions at 37°C in nutrient-rich media [1]. The drug resistance is a growing problem past few years in several countries including Saudi Arabia [4, 5]. However the current recommended treatment regimens for H. pylori worldwide are combined antibiotics treatment is possible by triple-therapy, which includes application of a proton pump inhibitor (PPI) combined with two antibiotics (claritithromycin and amoxicillin or clarithromycin and metronidazole) [6]. A triple regimen including raberprazole, amoxicillin and gatifloxacin has

*Address correspondence to Department of Botany and University, P.O. Box 2455, Fax: +966 1 4675833; E-mail:

this author at the Unit of Medical Bacteriology, Microbiology, College of Science, King Saud Riyadh, Saudi Arabia; Tel: +966 1 4675817; [email protected], [email protected] E-ISSN: 2308-6483/13

also been recommended for treatment of H. pylori infection with a highly effective eradication rate [7, 8]. On the other hand, the effect of antibiotics on patients living in different geographical area is slightly different. In Japanese patients the combination of a PPI drug with amoxicillin and metronidazole is highly effective [9] report shows that a combination of new antibiotics such as erythromycin and rifamycin derivatives with new polycyclic compounds has a powerful antibacterial activity on H. plyori. In addition, emergence of resistant strains of H. pylori is usual [7]. The resistance rates of H. pylori strains to metronidazole increased from 35.2% to 78.5% between 1987-1988 and 1990-1996, respectively, in Saudi Arabia [10]. Correspondingly, the low H. pylori resistance rate towards quinolones, mainly used for second-line therapy, observed in the past has increased during the last decade, whilst both amoxicillin and tetracycline resistance rates seem to have remained low [6]. Alternative molecules, such as furazolidone, bismuth salts and rifabutin are not available worldwide and they are not free of significant side-effects. All these observations highlight the crucial role of antibiotic resistance in the management of H. pylori infection [10]. Therefore, the knowledge of resistance mechanisms may contribute to elaborate more rational © 2013 Synergy Publishers

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Global Journal of Gastroenterology & Hepatology, 2013 Vol. 1, No. 2

antibiotic combinations with the aim of improving treatment success. The aims of this study were to evaluate the prevalence of resistance to metronidazole, tetracycline amoxicillin, rifampicin, and clarithromycin in H. pylori isolates from Saudi Arabian patients and to compare disc diffusion method with E-test. MATERIALS AND METHODS Patients and Processing of Biopsy Specimens The study was conducted at Armed Forces Hospital, Riyadh for a period of one year. Three antrum biopsy specimens were taken from each patient who reported with the clinical gastritis, manifested by recurrent abdominal pain for at least 3 months, nausea, and vomiting. Before entering the study, each patient reviewed and signed an informed consent form approved by the Local Ethics Committee. Standards of Good Clinical Practice and the declaration of Royal Decree were followed. Informed consent for gastroscopy and biopsy was obtained from all patients. The overall age groups of patient positive cases were 46 years old. The specimens were obtained from the anterior and posterior gastric wall of the gastric antrum within 2 cm of the pylorus. The biopsy specimens were transported in 0.85% (wt/vol) sterile saline to the laboratory for processing within 8 h. The biopsy specimens were smeared onto the surface of a selective Columbia blood agar (Oxoid.co.uk, CM331) and an enrichment medium brain heart infusion agar with 7% sheep blood. The selective medium was supplemented with cefsulodin and vancomycin 10ml/L, trimethoprim 5ml/L and amphotericin B 5ml/L. The plates were incubated microaerobically at 37°C in a humidified incubator with 10% CO2 for up to 5 days. The culture plates were checked for the presence of H. pylori on a daily basis.

Al-Omar et al.

Biodisk, Solna, Sweden) was applied for the determination of minimum inhibitory concentrations (MICs) of the antibiotics such as amoxicillin (0.016-256 mg/L), tetracycline (0.016-256 mg/L) clarithromycin (0.016-256 mg/L), rifampicin (0.002-32mg/L) and metronidazole (0.016-256 mg/L) [13]. H. pylori isolates were grown for 2 days on Columbia blood agar plates; growth was suspended in Columbia broth to achieve McFarland’s standard opacity of 3 and spread on blood agar plates. The antimicrobial drug strip was placed on the plate and incubated at 37°C for 72 h in an incubator with a 5% O2, 85% N2 and 10% CO2 in an anaerobic jar (gas generating kits, oxoid.co.UK, BR 60). The MIC of all antibiotics was performed and compared with We obtained H. pylori quality control strain number 43579 from ATCC. The breakpoint of MIC was set according to CLSI standards [13] at metronidazole, 32 mg/L; clarithromycin, 1 mg/L; amoxicillin, 0.5 mg/L; rifampicin, 1mg/L and tetracycline, 1 mg/L and to confirm results, each strain was tested at least twice. RESULTS A total of 76 H. pylori were isolated from100 antral biopsy specimen. The culture positive in male and female were 78 and 74% respectively. The overall percentage of incidence was 76%. All the H. Pylori isolates were identified on the basis of rapid urease test. In antibiotics susceptible result revealed that more than forty five isolates were resistant to single dominant drugs metronidazole (72.4%) and less than 10% isolates were resistant to clarithromycin, rifampicin and tetracycline. In amoxicillin there is no resistant was observed against H.pylori (Figure 1). In antibiotic resistance to H.pylori there were five resistant phenotypes were observed MZ; CH; RIMZ; CHMZ; TCMZ (Table 1).

The bacterial strains isolated were confirmed to be H. pylori by Gram staining and the urease, oxidase, and catalase tests. The biopsy specimens were also examined by the rapid urease test (RUT) by the methods described [11]. Antimicrobial Susceptibility and Mic Assay The susceptibilities of the H. pylori isolates were tested for selected antibiotics such as amoxicillin (25g), tetracycline (30g), clarithromycin (15g), rifampicin (5g) and metronidazole (5g) (Oxoid, England) by disc diffusion method [12]. The commercially available Epsilometer test (E test; AB

Figure 1: Helicobacter pylori isolated from peptic ulcer patients- antibiotic resistance percentage.

Prevalence of Antibiotic Resistance and Minimum Inhibitory


Table 1: No. of H. pylori Isolates and Antibiotic Resistant Phenotypes

Total

No. of isolates

TC

RI

AC

CH

MZ

19

S

S

S

S

S

45

S

S

S

S

R

02

S

S

S

R

S

04

S

R

S

S

R

04

S

S

S

R

R

02

R

S

S

S

R

76

2

4

0

6

55

S, Sensitive; R, Resistant; TC, Tetracycline; RI, Rifampicin; AC, Amoxicillin; CH, Clarithromycin; MZ, Metronidazole.

A. MICs break point of Metronidazole is 32mg/L

B. MICs break point of Amoxicillin is 0.016 mg/L

C. MICs break point of Tetracycline is 0.125mg/L

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Al-Omar et al.

(Figure 2). Continued.

D. MICs break point of Clarithromycin is 0.016mg/L

E. MICs break point of Rifampicin is 0.125mg/L Figure 2: Distribution of metronidazole, amoxicillin, tetracycline, clarithromycin and rifampicin MICs of H. pylori strain (N=76).

The distribution of metronidazole MICs demonstrated a continuous spectrum ranging between 0.5 to 256 mg/L. The MICs of metronidazole ranged from 0.5 to 0.6mg/L in 27.63% (n = 21) of H. pylori strains whereas about 40% isolates showed high MICs i.e. ranged was 32 mg/L (Figure 2A). In case of amoxicillin, 64.47% (n =49) of H. pylori isolates showedthe susceptible range of MICs i.e. between 0.016 to 0.094 mg/L whereas 23.68% isolates the MICs range was 0.125 to 0.75mg/L (Figure 2B). In tetracycline, 77.53% (n =59) of H. pylori isolates showed the susceptible range of MICs i.e. between 0.125 to 0.75 mg/L whereas 14.47% (n= 11) isolates the MICs i.e. range was 1 to 2mg/L (Figure 2C). In clarithromycin, 60.52% (n =46) of H. pylori isolates showed the susceptible range of MICs i.e. between 0.016 to 0.094 mg/L whereas 28.94% (n= 22) isolates the MICs i.e. range was 0.125 to 0.75mg/L, 3.94% (n= 3) isolates the MICs i.e. range was 1mg to 2mg/L and 5.26% (n= 4) isolates the MICs range was 3mg to 6mg/L respectively (Figure 2D). Where as in case of rifampicin, 23.68% (n =18) of H. pylori isolates showed the susceptible range of MICs i.e. between 0.016 to 0.094 mg/L and 77.53% (n =59) isolates the MICs i.e. range was 0.125 to 0.75mg/L (Figure 2E).

DISCUSSION H. pylori were more concerned about common stomach disease and peptic ulcer problems in human beings. It must be treated with suitable antibiotics. For the correct management of peptic ulcer diseases as well as obtaining information on a wide range of diseases associated with H. pylori infection, for the effective diagnostic methods including susceptibility testing are mandatory to control further complication in infected persons [14]. In developed countries, metronidazole resistance rates range from 10 to 50%, whereas, in developing countries, higher rates are usually observed [14, 7]. This may be linked to the frequent use of metronidazole for treating parasitic infections which, besides selecting resistant strains, may induce resistance due to its mutagenic effect [15]. In our studies Metronidazole MICs showed a continuous distribution spectrum across the drug concentrations ranging between 1 and 256 mg/L and showed a definite shift to high concentrations [16]. Metronidazole has been widely prescribed for infections such as parasitic or female genital infections in Saudi and the growing use or abuse of this inexpensive drug may have contributed to

Prevalence of Antibiotic Resistance and Minimum Inhibitory

these increased MICs, of H. pylori in adults. Similar results were reported [15, 17], the MICs ranged from 0.03 to 128 mg/L. The MICs at which 50 and 90% of the isolates were inhibited were 4 and 64 mg/L, respectively. The risk factor for clarithromycin resistance was a previous consumption of macrolides, and therefore, a trend for an increased prevalence was observed. In most of the countries like United States, a prevalence of 10 to 15% was found based on strains isolated during clinical trials, regardless of the region studied [18]. A multicenter European study carried out in 1998 in 17 countries found a global prevalence of 9.9% (95% confidence interval, 8.3 to 11.7) with a significant difference between Northern Europe (9.3%) and Southern Europe (18%) [13]. Tetracycline resistance in H. pylori was 6% in Italy [19]. The present study showed that all H. pylori isolates were sensitive to amoxicillin and no resistant isolates were found. The official breakpoint for amoxicillin resistance has not been designated for H. pylori isolates by the CLSI. Therefore, we cannot assign any provisional breakpoint to amoxicillin. However, H. pylori strains for which the amoxicillin MICs was below 0.1 mg/L were found. On the other hand amoxicillin MICs were lower than 1 mg/L [20]. Resistance to amoxicillin is very infrequent, despite the wide use of this antibiotic, either alone or combined with clavulanic acid, to treat H. pylori infections in both children and adults.


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In conclusion, we present that metronidazole resistance is an emerging problem for the treatment of H. pylori-infected patients in Saudi Arabia. However, the other drugs such as amoxicillin and tetracycline have good responses to the pathogens which should be choice for controlling peptic ulcer and gastritis in human beings. The present study also demonstrates the need for continuous monitoring of the antimicrobial susceptibility in H. pylori for determination of optimal treatment regimens for the patients. ACKNOWLEDGEMENTS

There is no conflict of interest between us.

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