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A new biphasic test for the detection of Helicobacter pylori in gastric biopsies
Journal of Microbiological Methods 96 (2014) 19–24
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Journal of Microbiological Methods journal homepage: www.elsevier.com/locate/jmicmeth
A new biphasic test for the detection of Helicobacter pylori in gastric biopsies Sueptrakool Wisessombat a,⁎, Chatruthai Meethai b, Souvalak Hamgo b a b
School of Allied Health Sciences and Public Health, Walailak University, Tha Sala, Nakhon Si Thammarat 80161, Thailand Faculty of Medical Technology, Prince of Songkla University, Hat Yai, Songkhla 90110, Thailand
a r t i c l e
i n f o
Article history: Received 1 July 2013 Received in revised form 16 October 2013 Accepted 16 October 2013 Available online 5 November 2013 Keywords: Biphasic media Helicobacter pylori Gastric biopsy
a b s t r a c t The objective of this study was to create a biphasic cultural method for the detection of Helicobacter pylori in gastric biopsy specimens. The biphasic systems were made by using a urea agar slant with overlaying broth in a single vessel. Initially, three different liquid media including brain–heart infusion broth, Brucella broth, and Bolton broth were tested for their ability to support the growth of H. pylori. Bolton broth with 10% defibrinated horse blood demonstrated a significant increase in the numbers of H. pylori (p b 0.05). The result showed that positive urease was used to concentrate viable H. pylori cells where the numbers of bacteria were 105 cfu. In addition, the reliable incubation time was at least 36 h. In total, 55 biopsies were comparatively studied using commercial rapid urease test and PCR. Seven samples (12.72%) were positive with H. pylori by the biphasic test. With the CLOtest, 6 (10.91%) samples were positive. In conclusion, the Hp biphasic test achieved more positive samples than did the commercial rapid urease test. © 2013 Elsevier B.V. All rights reserved.
1. Introduction Helicobacter pylori, Gram-negative microaerobic bacterium is a major cause of human gastritis and gastric ulcer and is associated with an increased risk of gastric cancer (Farinha and Gascoyne, 2005; McColl, 2010). H. pylori have an estimated prevalence of about half the world's population (Woodward et al., 2000). Diagnosis of H. pylori infections can be performed by various methods. Histology has been considered to be the gold standard for detection of H. pylori (El-Zimaity, 2000). Unfortunately, the detection of H. pylori relies upon the number and size of gastric biopsies, staining methods, and level of experience of the examining pathologist (Cirak et al., 2007). The rapid urease testing identifies active H. pylori infection. There are a number of commercially available rapid urease testing kits including the CLOtest (Kimberly-Clark, USA), HpFast (GI Supply, USA), and PyloriTek (Serim, USA). However, antibiotics, proton pump inhibitors, and acute ulcer bleeding can decrease the sensitivity of the test (Lee et al., 2000; Midolo and Marshall, 2000). Culture is highly specific method for identifying H. pylori infection and allows characterization of antimicrobial sensitivities (HPA, 2012). However, conventional cultural methods are complex and require a special atmosphere-generating system. The recovery of H. pylori from clinical specimens is difficult. Brain– heart infusion broth supplemented with blood or serum usually proves adequate for culture (Goodwin and Worsley, 1993). Shadowen and Sciortino (1989) found that Brucella agar and broth supplemented ⁎ Corresponding author. Tel.: +66 7 567 24 72; fax: +66 7 567 21 06. E-mail address: [email protected] (S. Wisessombat). 0167-7012/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.mimet.2013.10.010
with fetal bovine serum increased growth of H. pylori in biphasic system. However, urease activity for the identification of H. pylori requires additional days. The aim of the present investigation was to develop a novel H. pylori biphasic test for culturing H. pylori in gastric biopsies. This method combines the selective enrichment broth and biochemical test using urea agar in a single vessel. 2. Materials and methods 2.1. Bacterial strains and culture conditions Reference strains including H. pylori NCTC 11637 and H. pylori NCTC 11638 were used for the validation of the developed method. Helicobacter spp. were cultured on Columbia blood agar base (Fluka, Switzerland) with 10% defibrinated horse blood (Oxoid, UK). Plates were incubated at 37 °C for 48 h under microaerobic atmosphere using gas pack system (Mitsubishi, Japan). Other microorganisms for the validation test including Campylobacter jejuni, Campylobacter concisus, Proteus mirabilis, Proteus vulgaris, Klebsiella pneumoniae, Staphylococcus aureus, Streptococcus group D, Enterococcus faecalis, Serratia marcescens, Morganella morganii, Escherichia coli, Candida albicans, and Candida tropicalis were cultured in brain–heart infusion broth (Difco, USA) at 37 °C for 24 h. 2.2. H. pylori biphasic test Biphasic test was considered as two systems incorporated into one container. For solid phase, urea agar (Merck, Germany) was prepared
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according to the manufacturer's directions. 2 mL urea agar slant were prepared in 5 mL sterile screw cap flat tube. Urea agar contains phenol red, a pH indicator and has a pH of 6.5. Urease-producing organisms hydrolyze urea to form ammonia. The medium turns from salmon color to magenta in response to bacterial metabolic reduction. Liquid phase was Helicobacter enrichment broth containing 10% defibrinated horse blood. Before use, the mixture of antimicrobial agents including 10 mg/L of amphotericin B, 1250 U/L polymyxin B, 2 mg/L trimethoprim, and 10 mg/L vancomycin (Sigma-Aldrich, USA) were added. Then, 1.5 mL of this broth was poured over the formed slant. This vessel was a closed system for the isolation of H. pylori without gas-generating sachet. 2.3. Comparative efficacy of the different liquid media for growth of H. pylori In preliminary study, three different liquid media were tested in this experiment including brain–heart infusion broth, Brucella broth (Difco) and Bolton broth (Oxoid). Each broth was supplemented with or without 10% defibrinated horse blood to determine whether blood was essential for growth of H. pylori. Reference strains of the H. pylori were inoculated into Columbia blood agar and incubated at 37 °C for 48 h under microaerobic atmosphere. After incubation, a loopful of bacterial was taken and suspended in 1 mL of buffer peptone water (BPW). Cell density was determined by spectrophotometer at 625 nm (OD 0.08– 0.1) to give approximately 2 × 108 cfu/mL. A 10-fold serially diluted culture from 2 × 102 to 2 × 105 cfu/mL was prepared. One hundred fifty microliters of viable inoculum was added to 1.5 mL of each broth to give the final cell numbers at 10–104 cfu/mL then incubated at 37 °C for 12, 24, 36, and 48 h. To determine the numbers of viable H. pylori, 1 mL of each broth was determined by a drop plate method. One hundred microliters of the bacterial suspension in each serial dilution was pipetted into a tube containing 900 μL of sterile BPW. From each dilution, 10 μL of the samples was transferred to Columbia blood agar. Plates were allowed to dry and incubated at 37 °C for 48 h. All measurements were performed in triplicate and all experiments were performed twice. The mean and standard error was calculated from the combined measurements. 2.4. Artificially-inoculated gastric biopsy A fresh gastric biopsy from pig was obtained from a slaughterhouse of Hat Yai municipality, Thailand and placed on ice and immediately transported to the laboratory. The organ was opened and rinsed with cold distilled water. Squares, 2 × 2 cm, were cut from the wall of stomachs. Then, tissues were homogenized in 500 μL of normal saline with a tissue grinder. Inoculum preparation was carried out as described previously. Gastric biopsy with or without 150 μL of the viable bacterial suspension was added to 1.5 mL of liquid media in a sterile container. The initial numbers of artificially-inoculated viable cells per g gastric biopsy were approximately 10–104 cfu. 2.5. Evaluation of selectivity of Hp biphasic test The biphasic test was evaluated with viable H. pylori strains in the presence of mixed microbiota. Each of bacterial inoculum at 104 cfu/mL was pooled together and prepared as described previously to give the final cell numbers at 104 cfu/mL. For a nonviable inoculum control, the bacterial cells were resuspended in 1 mL of saline containing 200 ppm sodium hypochlorite and incubated at room temperature for 4 h to kill all viable cells (Hayashi et al., 2006). Subsequently, the cell suspension was washed three times with saline and finally resuspended in 1 mL of BPW. After the inoculation of cell suspension in the biphasic medium, a lid was loosely fitted over the container. The apparatus was then incubated at 37 °C and was observed for 12, 24, 36, and 48 h.
The numbers of viable H. pylori were determined by the drop plate method as described previously. The identification of H. pylori was carried out by the observation on colony characteristics, motility and morphology under phase-contrast microscopic, and biochemical tests including oxidase reaction, catalase production, and ability to hydrolyze urea (HPA, 2012). 2.6. Comparison study between Hp biphasic test and rapid urease test for the detection of H. pylori in gastric biopsies Gastric biopsies were collected from the Institute of Gastroenterology and Hepatology, Songklanagarind Hospital, Prince of Songkla University, Thailand. A total of 55 dyspeptic patients undergoing upper endoscopy were biopsied and tested for H. pylori infection by a Campylobacter-like organism (CLO) test (Kimberly-Clark, USA) and Hp biphasic test. The CLOtest was performed according to the manufacturer's instructions, and the results were interpreted after 24 h. Biopsy specimens were homogenized and incubated in liquid-overlay of the apparatus at 37 °C for 48 h under normal atmosphere. The identification of H. pylori was confirmed by 16S rRNA with H. pylori specific primers (Tiwari et al., 2006). The forward (5′-TAA GAG ATC AGC CTA TAT GTC C-3′) and the reverse (5′-TCC CAC GCT TTA AGC GCA AT-3′) primers amplified a product of approximately 534 bp. Amplification consisted of initial denaturation at 94 °C for 4 min, followed by denaturation at 94 °C for 30 s, primers annealing at 56 °C for 30 s, and extension at 72 °C for 30 s. The samples were amplified for 40 cycles, with a final extension step at 72 °C for 5 min. PCR cycles were carried out in PTC-100, Peltier thermal cycler (Pegasus Scientific, USA). Two μl amplified products were analyzed by 2% agarose (Gibco-BRL Life Technologies, USA) gel electrophoresis in Tris–acetate– EDTA buffer at 100 V for 35 min. PCR products were visualized after ethidium bromide staining. 2.7. Statistical analysis All experiments were repeated at least three times to ensure their reproducibility. Data are presented as mean ± standard error. Differences between the different types of media were analyzed using the Dunnett's method of One-way analysis of variance (ANOVA). Differences between each liquid media with and without blood were analyzed by One-sample t-test. Statistical analysis was performed using the Statistical Package for Social Sciences package version 13 (SPSS, USA). 3. Results Three different liquid media were tested for their ability to support the growth of H. pylori. With inoculum level of H. pylori NCTC 11637 (104 cfu/mL) in Bolton broth, the highest concentration of cell numbers achieved 105 cfu/mL while they were approximately 104 cfu/mL in brain–heart infusion broth and Brucella broth at 48 h (Fig. 1A, B, C). Bolton broth showed a significant increase in the numbers of viable H. pylori (p b 0.05). The effect of blood on the growth of H. pylori was investigated. With inoculum level of H. pylori (104 cfu/mL), the cell numbers reached 106 cfu/mL in Bolton broth while those in brain–heart infusion broth and Brucella broth were 105 cfu/mL. Following the inoculation as few as 10 cfu/mL in Bolton broth, the numbers of viable cells were approximately 102 cfu/mL after 48 h. Whereas, no difference was found in the growth of H. pylori among other tested media (Fig. 1D, E, F). Bolton broth with 10% defibrinated horse blood demonstrated a significant increase in the numbers of viable H. pylori (p b 0.05). Similar patterns were obtained with H. pylori NCTC 11638 (Fig. 2). Therefore, Bolton broth supplemented with 10% defibrinated horse blood and antimicrobial agents was selected as liquid-overlay for further experiments.
S. Wisessombat et al. / Journal of Microbiological Methods 96 (2014) 19–24
A
D Brain-heart infusion broth
8 6 4 2
8 6 4 2 0
0
12
24
36
Brain-heart infusion broth with 10% defibrinated horse blood
10
Cell numbers (Log cfu/ml)
Cell numbers (Log cfu/ml)
10
0
48
0
12
B
E Brucella broth Cell numbers (Log cfu/ml)
8
6
4
2
12
C
36
4
2
F
6
4
2
24
24
36
48
8
*, ** 6
4
2
0 12
12
Bolton broth with 10% defibrinated horse blood
10
*
0
0
Time (h)
8
0
6
48
Bolton broth
10
Cell numbers (Log cfu/ml)
24 Time (h)
48
8
0
0
36
Brucella broth with 10% defibrinated horse blood
10
Cell numbers (Log cfu/ml)
Cell numbers (Log cfu/ml)
10
24
Time (h)
Time (h)
0
21
36
48
Time (h)
0
12
24
36
48
Time (h)
Fig. 1. Effect of various liquid media on the growth of H. pylori NCTC 11637. The initial bacterial concentration of each inoculum was 104 (■), 103 (□), 102 (▲), and 10 (Δ) cfu/mL. After the inoculation of H. pylori into the liquid phase, the apparatus was incubated at 37 °C for 48 h. Viable counts were determined on Columbia blood agar. Asterisk (*, p b 0.05) represents significant difference between three liquid media as analyzed by One way ANOVA. Double asterisks (**, p b 0.05) represent significant difference between each liquid media with and without blood as analyzed using One-sample t-test.
The model of Hp biphasic test was applied for the isolation of H. pylori from artificially-inoculated gastric biopsies. We evaluated this biphasic test with various concentrations of viable H. pylori (104–10 cfu/mL) in the presence of background microbiota (104 cfu/mL). All artificially-inoculated samples with viable H. pylori cells yielded positive results whereas those with nonviable cells and mixed microbiota did not. Following the inoculation of H. pylori NCTC 11637 (104 cfu/mL), the highest concentration of the cells was 106 cfu/mL, after 48 h. When the inoculation was 103 cfu/mL and 102 cfu/mL, the numbers of viable cells were 106 cfu/mL and 105 cfu/mL, respectively (Table 1). As expected, C. jejuni, C. concisus,
and other urease-positive bacteria did not interfere with the legitimate positive test reactions. Similar patterns were observed with H. pylori NCTC 11638 (Table 2). Urea agar was used as a solid phase for the detection of viable H. pylori. The results revealed that positive urease was used to concentrate viable H. pylori cells where the numbers of bacteria were 105 cfu. In addition, the reliable incubation time required for the detection of 105 bacterial cells for H. pylori was at least 36 h irrespective of initial inoculum (Tables 1–2). Additionally, when the biphasic media was inoculated by mixed microbiota, the color of the urease slant did not undergo any change for a long period of incubation time.
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A
10
Cell numbers (Log cfu/ml)
Cell numbers (Log cfu/ml)
D
Brain-heart infusion broth
10 8 6 4 2 0
0
12
24
36
8 6 4 2 0
48
Brain-heart infusion brothwith 10% defibrinated horse blood
0
12
Time (h)
B 8 6 4 2 0
0
12
24
36
6 4 2 0
48
0
12
*
6 4 2
0
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36
48
Cell numbers (Log cfu/ml)
Cell numbers (Log cfu/ml)
F
36
48
Bolton brothwith 10% defibrinated horse blood
10
8
0
24
Time (h)
Bolton broth
10
48
8
Time (h)
C
36
Brucella broth with 10% defibrinated horse blood
10
Cell numbers (Log cfu/ml)
Cell numbers (Log cfu/ml)
E
Brucella broth
10
24
Time (h)
*, **
8 6 4 2 0 0
Time (h)
12
24
36
48
Time (h)
Fig. 2. Effect of various liquid media on the growth of H. pylori NCTC 11638. The initial bacterial concentration of each inoculum was 104 (■), 103 (□), 102 (▲), and 10 (Δ) cfu/mL. After the inoculation of H. pylori into the liquid phase, the apparatus was incubated at 37 °C for 48 h. Viable counts were determined on Columbia blood agar. Asterisk (*, p b 0.05) represents significant difference between three liquid media as analyzed by One way ANOVA. Double asterisks (**, p b 0.05) represent significant difference between each liquid media with and without blood as analyzed using One-sample t-test.
Rapid urease test was used for the comparative evaluation of the biphasic test (Table 3). The results revealed that 7 of 55 (12.72%) biopsy specimens yielded positive by both methods. Hp biphasic method yielded positive results for 5 (9.09%) of gastric biopsies after 24 h incubation. Moreover, 2 (3.63%) specimens were detected as positive by the biphasic test after 48 h incubation. In addition, none of the specimens were positive after 72 h incubation. All positive samples from biochemical identification were confirmed to be H. pylori by PCR. The Hp biphasic test achieved 100% (7/7) specificity after 48 h, compared to 71.43% (5/7) after 24 h. However, it was found that results from rapid urea test were not in agreement with PCR technique. Most isolates gave the same results by both methods,
except for one strain that was initially identified as H. pylori by the CLOtest but was negative by PCR. The result was considered false positive. 4. Discussion Several media containing growth and selective supplements have been proposed to increase the probability to retrieve H. pylori. All media contain peptones, antibiotics and blood, and many include oxygen-quenching agents to overcome the adverse effects of toxic oxygen species (Chomvarin et al., 2006; Morgan et al., 1987; Sainsus et al., 2008; Shadowen and Sciortino, 1989). In this study, the effects of
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Table 1 Evaluation of Hp biphasic test for the detection of H. pylori NCTC 11637 in the presence of background microbiota. Inoculum (cfu/mL)
12 h
24 h
36 h
48 h
Liquid phase
Solid phase
Liquid phase
Solid phase
Liquid phase
Solid phase
Liquid phase
Solid phase
Viable cell (cfu/mL)
Urea agar
Viable cell (cfu/mL)
Urea agar
Viable cell (cfu/mL)
Urea agar
Viable cell (cfu/mL)
Urea agar
− − − −
1.50 3.00 2.03 1.24
− − − −
3.50 2.02 2.22 1.13
+ + − −
2.32 1.41 3.08 2.44
Viable cells 104 103 102 10
2.12 2.41 1.33 2.20
Nonviable cells 104
0
−
0
−
0
−
0
−
Mixed microbiota 104
0
−
0
−
0
−
0
−
± ± ± ±
0.53 0.74 0.22 0.13
× × × ×
104 103 102 10
± ± ± ±
0.25 0.51 0.15 0.08
× × × ×
104 103 102 10
± ± ± ±
0.22 0.32 0.77 0.33
× × × ×
105 105 104 10
± ± ± ±
0.33 0.22 0.55 0.14
× × × ×
106 106 105 102
+ + + −
After the inoculation of H. pylori into liquid phase, the apparatus was incubated at 37 °C for 12, 24, 36, and 48 h. Viable counts were determined on Columbia blood agar. The plates were incubated at 37 °C for 48 h under microaerobic conditions. + = urease positive; − = urease negative; 0 = no growth.
Table 2 Evaluation of Hp biphasic test for the detection of H. pylori NCTC 11638 in the presence of background microbiota. Inoculum (cfu/mL)
12 h
24 h
36 h
48 h
Liquid phase
Solid phase
Liquid phase
Solid phase
Liquid phase
Solid phase
Liquid phase
Solid phase
Viable cell (cfu/mL)
Urea agar
Viable cell (cfu/mL)
Urea agar
Viable cell (cfu/mL)
Urea agar
Viable cell (cfu/mL)
Urea agar
− − − −
3.33 2.72 1.52 1.62
− − − −
1.72 1.19 2.04 2.33
+ − − −
2.88 1.19 2.25 1.55
Viable cells 104 103 102 10
2.00 2.22 1.11 2.32
Nonviable cells 104
0
−
0
−
0
−
0
−
Mixed microbiota 104
0
−
0
−
0
−
0
−
± ± ± ±
0.24 0.35 0.73 0.32
× × × ×
104 103 102 10
± ± ± ±
0.55 0.89 0.61 0.18
× × × ×
104 103 102 10
± ± ± ±
0.49 0.33 0.43 0.25
× × × ×
105 104 103 10
± ± ± ±
0.36 0.33 0.05 0.94
× × × ×
106 105 105 103
+ + + −
After the inoculation of H. pylori into liquid phase, the apparatus was incubated at 37 °C for 12, 24, 36, and 48 h. Viable counts were determined on Columbia blood agar. The plates were incubated at 37 °C for 48 h under microaerobic conditions. + = urease positive; − = urease negative; 0 = no growth.
various liquid media on growth of H. pylori were investigated. Bolton broth supplemented with antibiotics was employed for the selective enrichment broth of Campylobacter (Hunt et al., 2001). Our results confirmed that Bolton broth was also suitable for growth of H. pylori. Enzymatic digest of animal tissues, lactalbumin hydrolysate, and yeast extract provide essential growth nutrients like vitamins (Bolton and Robertson, 1982). The addition of blood, sodium metabisulphite, and sodium pyruvate quenches toxic compounds and increases the recovery rate of the culture (Bolton et al., 1984). The results confirmed that competing bacteria did not cause false positive test reactions in the biphasic test. The antibiotics including vancomycin, polymyxin B, and trimethoprim present in the supplement inhibit the growth of Gram-positive and Gram-negative bacteria. Yeast may cause an alkaline environment causing the false positive results. Therefore, Hp biphasic test also contains amphotericin B that can inhibit the growth of contaminant yeast. We concluded that this broth
Table 3 Comparison of the numbers of H. pylori positive samples by Hp biphasic test, CLOtest and PCR from 55 gastric biopsies. Methods
Hp biphasic test CLOtest PCR n/a: not applicable.
represents the best overall compromise between growth and inhibition of contaminants for the isolation of H. pylori. The urease reaction can be used as an indicator test for the presence of urease-positive H. pylori in the culture. In comparison with CLOtest, the Hp biphasic test is found to be comparable in results. Interestingly, the results demonstrated that urease positivity depends on bacterial density. With the biphasic test, minimal concentration of organism needed to show a positive urease reaction was approximately 105 cfu/mL. Previous studies demonstrated that 104 cfu/mL is necessary to observe urease positivity in gel preparation (Deltenre et al., 1989). Nevertheless, using this new culture method and using the CLOtest to determine the prevalence of H. pylori in gastric biopsies demonstrated that H. pylori were detected in 12.72%. False positive urea test has been reported by other studies (Schrader et al., 1993; van Doorn et al., 2000; Vaira et al., 2010). CLOtest showed 90–95% sensitivity and 100% specificity after 24 h (Schrader et al., 1993). However, other urease-positive bacteria can decrease the specificity and give false-positive results (Vaira et al., 2010). In conclusion, the advantages of Hp biphasic test are the ability of culturing and simultaneously identifying H. pylori directly from gastric biopsy. Furthermore, it can be used under an aerobic condition and allows antimicrobial susceptibility testing.
Numbers of positive samples (%) 24 h
48 h
72 h
Total
5 (9.09) 7 (12.72) n/a
2 (3.63) n/a n/a
0 (0) n/a n/a
7 (12.72) 7 (12.72) 7 (12.72)
Acknowledgments This work was supported by Prince of Songkla University, Research Development Fund, Fiscal Year 2011–2012, Thailand. We also thank Dr. Brian Hodgson for their valuable suggestions.
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Lee, J.M., Breslin, N.P., Fallon, C., O'Morain, C.A., 2000. Rapid urease tests lack sensitivity in Helicobacter pylori diagnosis when peptic ulcer disease presents with bleeding. Am. J. Gastroenterol. 95, 1166–1170. McColl, K.E., 2010. Helicobacter pylori infection. N. Engl. J. Med. 362, 1597–1604. Midolo, P., Marshall, B.J., 2000. Accurate diagnosis of Helicobacter pylori. Urease tests. Gastroenterol. Clin. N. Am. 29, 871–878. Morgan, D.R., Freedman, F., Depew, C.E., Kraft, W.G., 1987. Growth of Campylobacter pylori in liquid media. J. Clin. Microbiol. 25, 2123–2125. Sainsus, N., Cattori, V., Lepadatu, C., Hofmann-Lehmann, R., 2008. Liquid culture medium for the rapid cultivation of Helicobacter pylori from biopsy specimens. Eur. J. Clin. Microbiol. Infect. Dis. 27, 1209–1217. Schrader, J.A., Peck, H.V., Notis, W.M., Shaw, P., Venezia, R.A., 1993. A role for culture in diagnosis of Helicobacter pylori-related gastric disease. Am. J. Gastroenterol. 88, 1729–1733. Shadowen, R.D., Sciortino, C.V., 1989. Improved growth of Campylobacter pylori in a biphasic system. J. Clin. Microbiol. 27, 1744–1747. Tiwari, S.K., Khan, A.A., Ibrahim, M., Habeeb, M.A., Habibullah, C.M., 2006. Helicobacter pylori and other Helicobacter species DNA in human bile samples from patients with various hepato-biliary diseases. World J. Gastroenterol. 12, 2181–2186. Vaira, D., Gatta, L., Ricci, C., Perna, F., Saracino, I., Fiorini, G., Castelli, V., Holton, J., 2010. A comparison amongst three rapid urease tests to diagnose Helicobacter pylori infection in 375 consecutive dyspeptic. Intern. Emerg. Med. 5, 41–47. van Doorn, L.J., Henskens, Y., Nouhan, N., Verschuuren, A., Vreede, R., Herbink, P., Ponjee, G., van Krimpen, K., Blankenburg, R., Scherpenisse, J., Quint, W., 2000. The efficacy of laboratory diagnosis of Helicobacter pylori infections in gastric biopsy specimens is related to bacterial density and vacA, cagA, and iceA genotypes. J. Clin. Microbiol. 38, 13–17. Woodward, M., Morrison, C., McColl, K., 2000. An investigation into factors associated with Helicobacter pylori infection. J. Clin. Epidemiol. 53, 175–181.
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Journal of Microbiological Methods journal homepage: www.elsevier.com/locate/jmicmeth
A new biphasic test for the detection of Helicobacter pylori in gastric biopsies Sueptrakool Wisessombat a,⁎, Chatruthai Meethai b, Souvalak Hamgo b a b
School of Allied Health Sciences and Public Health, Walailak University, Tha Sala, Nakhon Si Thammarat 80161, Thailand Faculty of Medical Technology, Prince of Songkla University, Hat Yai, Songkhla 90110, Thailand
a r t i c l e
i n f o
Article history: Received 1 July 2013 Received in revised form 16 October 2013 Accepted 16 October 2013 Available online 5 November 2013 Keywords: Biphasic media Helicobacter pylori Gastric biopsy
a b s t r a c t The objective of this study was to create a biphasic cultural method for the detection of Helicobacter pylori in gastric biopsy specimens. The biphasic systems were made by using a urea agar slant with overlaying broth in a single vessel. Initially, three different liquid media including brain–heart infusion broth, Brucella broth, and Bolton broth were tested for their ability to support the growth of H. pylori. Bolton broth with 10% defibrinated horse blood demonstrated a significant increase in the numbers of H. pylori (p b 0.05). The result showed that positive urease was used to concentrate viable H. pylori cells where the numbers of bacteria were 105 cfu. In addition, the reliable incubation time was at least 36 h. In total, 55 biopsies were comparatively studied using commercial rapid urease test and PCR. Seven samples (12.72%) were positive with H. pylori by the biphasic test. With the CLOtest, 6 (10.91%) samples were positive. In conclusion, the Hp biphasic test achieved more positive samples than did the commercial rapid urease test. © 2013 Elsevier B.V. All rights reserved.
1. Introduction Helicobacter pylori, Gram-negative microaerobic bacterium is a major cause of human gastritis and gastric ulcer and is associated with an increased risk of gastric cancer (Farinha and Gascoyne, 2005; McColl, 2010). H. pylori have an estimated prevalence of about half the world's population (Woodward et al., 2000). Diagnosis of H. pylori infections can be performed by various methods. Histology has been considered to be the gold standard for detection of H. pylori (El-Zimaity, 2000). Unfortunately, the detection of H. pylori relies upon the number and size of gastric biopsies, staining methods, and level of experience of the examining pathologist (Cirak et al., 2007). The rapid urease testing identifies active H. pylori infection. There are a number of commercially available rapid urease testing kits including the CLOtest (Kimberly-Clark, USA), HpFast (GI Supply, USA), and PyloriTek (Serim, USA). However, antibiotics, proton pump inhibitors, and acute ulcer bleeding can decrease the sensitivity of the test (Lee et al., 2000; Midolo and Marshall, 2000). Culture is highly specific method for identifying H. pylori infection and allows characterization of antimicrobial sensitivities (HPA, 2012). However, conventional cultural methods are complex and require a special atmosphere-generating system. The recovery of H. pylori from clinical specimens is difficult. Brain– heart infusion broth supplemented with blood or serum usually proves adequate for culture (Goodwin and Worsley, 1993). Shadowen and Sciortino (1989) found that Brucella agar and broth supplemented ⁎ Corresponding author. Tel.: +66 7 567 24 72; fax: +66 7 567 21 06. E-mail address: [email protected] (S. Wisessombat). 0167-7012/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.mimet.2013.10.010
with fetal bovine serum increased growth of H. pylori in biphasic system. However, urease activity for the identification of H. pylori requires additional days. The aim of the present investigation was to develop a novel H. pylori biphasic test for culturing H. pylori in gastric biopsies. This method combines the selective enrichment broth and biochemical test using urea agar in a single vessel. 2. Materials and methods 2.1. Bacterial strains and culture conditions Reference strains including H. pylori NCTC 11637 and H. pylori NCTC 11638 were used for the validation of the developed method. Helicobacter spp. were cultured on Columbia blood agar base (Fluka, Switzerland) with 10% defibrinated horse blood (Oxoid, UK). Plates were incubated at 37 °C for 48 h under microaerobic atmosphere using gas pack system (Mitsubishi, Japan). Other microorganisms for the validation test including Campylobacter jejuni, Campylobacter concisus, Proteus mirabilis, Proteus vulgaris, Klebsiella pneumoniae, Staphylococcus aureus, Streptococcus group D, Enterococcus faecalis, Serratia marcescens, Morganella morganii, Escherichia coli, Candida albicans, and Candida tropicalis were cultured in brain–heart infusion broth (Difco, USA) at 37 °C for 24 h. 2.2. H. pylori biphasic test Biphasic test was considered as two systems incorporated into one container. For solid phase, urea agar (Merck, Germany) was prepared
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according to the manufacturer's directions. 2 mL urea agar slant were prepared in 5 mL sterile screw cap flat tube. Urea agar contains phenol red, a pH indicator and has a pH of 6.5. Urease-producing organisms hydrolyze urea to form ammonia. The medium turns from salmon color to magenta in response to bacterial metabolic reduction. Liquid phase was Helicobacter enrichment broth containing 10% defibrinated horse blood. Before use, the mixture of antimicrobial agents including 10 mg/L of amphotericin B, 1250 U/L polymyxin B, 2 mg/L trimethoprim, and 10 mg/L vancomycin (Sigma-Aldrich, USA) were added. Then, 1.5 mL of this broth was poured over the formed slant. This vessel was a closed system for the isolation of H. pylori without gas-generating sachet. 2.3. Comparative efficacy of the different liquid media for growth of H. pylori In preliminary study, three different liquid media were tested in this experiment including brain–heart infusion broth, Brucella broth (Difco) and Bolton broth (Oxoid). Each broth was supplemented with or without 10% defibrinated horse blood to determine whether blood was essential for growth of H. pylori. Reference strains of the H. pylori were inoculated into Columbia blood agar and incubated at 37 °C for 48 h under microaerobic atmosphere. After incubation, a loopful of bacterial was taken and suspended in 1 mL of buffer peptone water (BPW). Cell density was determined by spectrophotometer at 625 nm (OD 0.08– 0.1) to give approximately 2 × 108 cfu/mL. A 10-fold serially diluted culture from 2 × 102 to 2 × 105 cfu/mL was prepared. One hundred fifty microliters of viable inoculum was added to 1.5 mL of each broth to give the final cell numbers at 10–104 cfu/mL then incubated at 37 °C for 12, 24, 36, and 48 h. To determine the numbers of viable H. pylori, 1 mL of each broth was determined by a drop plate method. One hundred microliters of the bacterial suspension in each serial dilution was pipetted into a tube containing 900 μL of sterile BPW. From each dilution, 10 μL of the samples was transferred to Columbia blood agar. Plates were allowed to dry and incubated at 37 °C for 48 h. All measurements were performed in triplicate and all experiments were performed twice. The mean and standard error was calculated from the combined measurements. 2.4. Artificially-inoculated gastric biopsy A fresh gastric biopsy from pig was obtained from a slaughterhouse of Hat Yai municipality, Thailand and placed on ice and immediately transported to the laboratory. The organ was opened and rinsed with cold distilled water. Squares, 2 × 2 cm, were cut from the wall of stomachs. Then, tissues were homogenized in 500 μL of normal saline with a tissue grinder. Inoculum preparation was carried out as described previously. Gastric biopsy with or without 150 μL of the viable bacterial suspension was added to 1.5 mL of liquid media in a sterile container. The initial numbers of artificially-inoculated viable cells per g gastric biopsy were approximately 10–104 cfu. 2.5. Evaluation of selectivity of Hp biphasic test The biphasic test was evaluated with viable H. pylori strains in the presence of mixed microbiota. Each of bacterial inoculum at 104 cfu/mL was pooled together and prepared as described previously to give the final cell numbers at 104 cfu/mL. For a nonviable inoculum control, the bacterial cells were resuspended in 1 mL of saline containing 200 ppm sodium hypochlorite and incubated at room temperature for 4 h to kill all viable cells (Hayashi et al., 2006). Subsequently, the cell suspension was washed three times with saline and finally resuspended in 1 mL of BPW. After the inoculation of cell suspension in the biphasic medium, a lid was loosely fitted over the container. The apparatus was then incubated at 37 °C and was observed for 12, 24, 36, and 48 h.
The numbers of viable H. pylori were determined by the drop plate method as described previously. The identification of H. pylori was carried out by the observation on colony characteristics, motility and morphology under phase-contrast microscopic, and biochemical tests including oxidase reaction, catalase production, and ability to hydrolyze urea (HPA, 2012). 2.6. Comparison study between Hp biphasic test and rapid urease test for the detection of H. pylori in gastric biopsies Gastric biopsies were collected from the Institute of Gastroenterology and Hepatology, Songklanagarind Hospital, Prince of Songkla University, Thailand. A total of 55 dyspeptic patients undergoing upper endoscopy were biopsied and tested for H. pylori infection by a Campylobacter-like organism (CLO) test (Kimberly-Clark, USA) and Hp biphasic test. The CLOtest was performed according to the manufacturer's instructions, and the results were interpreted after 24 h. Biopsy specimens were homogenized and incubated in liquid-overlay of the apparatus at 37 °C for 48 h under normal atmosphere. The identification of H. pylori was confirmed by 16S rRNA with H. pylori specific primers (Tiwari et al., 2006). The forward (5′-TAA GAG ATC AGC CTA TAT GTC C-3′) and the reverse (5′-TCC CAC GCT TTA AGC GCA AT-3′) primers amplified a product of approximately 534 bp. Amplification consisted of initial denaturation at 94 °C for 4 min, followed by denaturation at 94 °C for 30 s, primers annealing at 56 °C for 30 s, and extension at 72 °C for 30 s. The samples were amplified for 40 cycles, with a final extension step at 72 °C for 5 min. PCR cycles were carried out in PTC-100, Peltier thermal cycler (Pegasus Scientific, USA). Two μl amplified products were analyzed by 2% agarose (Gibco-BRL Life Technologies, USA) gel electrophoresis in Tris–acetate– EDTA buffer at 100 V for 35 min. PCR products were visualized after ethidium bromide staining. 2.7. Statistical analysis All experiments were repeated at least three times to ensure their reproducibility. Data are presented as mean ± standard error. Differences between the different types of media were analyzed using the Dunnett's method of One-way analysis of variance (ANOVA). Differences between each liquid media with and without blood were analyzed by One-sample t-test. Statistical analysis was performed using the Statistical Package for Social Sciences package version 13 (SPSS, USA). 3. Results Three different liquid media were tested for their ability to support the growth of H. pylori. With inoculum level of H. pylori NCTC 11637 (104 cfu/mL) in Bolton broth, the highest concentration of cell numbers achieved 105 cfu/mL while they were approximately 104 cfu/mL in brain–heart infusion broth and Brucella broth at 48 h (Fig. 1A, B, C). Bolton broth showed a significant increase in the numbers of viable H. pylori (p b 0.05). The effect of blood on the growth of H. pylori was investigated. With inoculum level of H. pylori (104 cfu/mL), the cell numbers reached 106 cfu/mL in Bolton broth while those in brain–heart infusion broth and Brucella broth were 105 cfu/mL. Following the inoculation as few as 10 cfu/mL in Bolton broth, the numbers of viable cells were approximately 102 cfu/mL after 48 h. Whereas, no difference was found in the growth of H. pylori among other tested media (Fig. 1D, E, F). Bolton broth with 10% defibrinated horse blood demonstrated a significant increase in the numbers of viable H. pylori (p b 0.05). Similar patterns were obtained with H. pylori NCTC 11638 (Fig. 2). Therefore, Bolton broth supplemented with 10% defibrinated horse blood and antimicrobial agents was selected as liquid-overlay for further experiments.
S. Wisessombat et al. / Journal of Microbiological Methods 96 (2014) 19–24
A
D Brain-heart infusion broth
8 6 4 2
8 6 4 2 0
0
12
24
36
Brain-heart infusion broth with 10% defibrinated horse blood
10
Cell numbers (Log cfu/ml)
Cell numbers (Log cfu/ml)
10
0
48
0
12
B
E Brucella broth Cell numbers (Log cfu/ml)
8
6
4
2
12
C
36
4
2
F
6
4
2
24
24
36
48
8
*, ** 6
4
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0 12
12
Bolton broth with 10% defibrinated horse blood
10
*
0
0
Time (h)
8
0
6
48
Bolton broth
10
Cell numbers (Log cfu/ml)
24 Time (h)
48
8
0
0
36
Brucella broth with 10% defibrinated horse blood
10
Cell numbers (Log cfu/ml)
Cell numbers (Log cfu/ml)
10
24
Time (h)
Time (h)
0
21
36
48
Time (h)
0
12
24
36
48
Time (h)
Fig. 1. Effect of various liquid media on the growth of H. pylori NCTC 11637. The initial bacterial concentration of each inoculum was 104 (■), 103 (□), 102 (▲), and 10 (Δ) cfu/mL. After the inoculation of H. pylori into the liquid phase, the apparatus was incubated at 37 °C for 48 h. Viable counts were determined on Columbia blood agar. Asterisk (*, p b 0.05) represents significant difference between three liquid media as analyzed by One way ANOVA. Double asterisks (**, p b 0.05) represent significant difference between each liquid media with and without blood as analyzed using One-sample t-test.
The model of Hp biphasic test was applied for the isolation of H. pylori from artificially-inoculated gastric biopsies. We evaluated this biphasic test with various concentrations of viable H. pylori (104–10 cfu/mL) in the presence of background microbiota (104 cfu/mL). All artificially-inoculated samples with viable H. pylori cells yielded positive results whereas those with nonviable cells and mixed microbiota did not. Following the inoculation of H. pylori NCTC 11637 (104 cfu/mL), the highest concentration of the cells was 106 cfu/mL, after 48 h. When the inoculation was 103 cfu/mL and 102 cfu/mL, the numbers of viable cells were 106 cfu/mL and 105 cfu/mL, respectively (Table 1). As expected, C. jejuni, C. concisus,
and other urease-positive bacteria did not interfere with the legitimate positive test reactions. Similar patterns were observed with H. pylori NCTC 11638 (Table 2). Urea agar was used as a solid phase for the detection of viable H. pylori. The results revealed that positive urease was used to concentrate viable H. pylori cells where the numbers of bacteria were 105 cfu. In addition, the reliable incubation time required for the detection of 105 bacterial cells for H. pylori was at least 36 h irrespective of initial inoculum (Tables 1–2). Additionally, when the biphasic media was inoculated by mixed microbiota, the color of the urease slant did not undergo any change for a long period of incubation time.
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A
10
Cell numbers (Log cfu/ml)
Cell numbers (Log cfu/ml)
D
Brain-heart infusion broth
10 8 6 4 2 0
0
12
24
36
8 6 4 2 0
48
Brain-heart infusion brothwith 10% defibrinated horse blood
0
12
Time (h)
B 8 6 4 2 0
0
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24
36
6 4 2 0
48
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*
6 4 2
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Cell numbers (Log cfu/ml)
Cell numbers (Log cfu/ml)
F
36
48
Bolton brothwith 10% defibrinated horse blood
10
8
0
24
Time (h)
Bolton broth
10
48
8
Time (h)
C
36
Brucella broth with 10% defibrinated horse blood
10
Cell numbers (Log cfu/ml)
Cell numbers (Log cfu/ml)
E
Brucella broth
10
24
Time (h)
*, **
8 6 4 2 0 0
Time (h)
12
24
36
48
Time (h)
Fig. 2. Effect of various liquid media on the growth of H. pylori NCTC 11638. The initial bacterial concentration of each inoculum was 104 (■), 103 (□), 102 (▲), and 10 (Δ) cfu/mL. After the inoculation of H. pylori into the liquid phase, the apparatus was incubated at 37 °C for 48 h. Viable counts were determined on Columbia blood agar. Asterisk (*, p b 0.05) represents significant difference between three liquid media as analyzed by One way ANOVA. Double asterisks (**, p b 0.05) represent significant difference between each liquid media with and without blood as analyzed using One-sample t-test.
Rapid urease test was used for the comparative evaluation of the biphasic test (Table 3). The results revealed that 7 of 55 (12.72%) biopsy specimens yielded positive by both methods. Hp biphasic method yielded positive results for 5 (9.09%) of gastric biopsies after 24 h incubation. Moreover, 2 (3.63%) specimens were detected as positive by the biphasic test after 48 h incubation. In addition, none of the specimens were positive after 72 h incubation. All positive samples from biochemical identification were confirmed to be H. pylori by PCR. The Hp biphasic test achieved 100% (7/7) specificity after 48 h, compared to 71.43% (5/7) after 24 h. However, it was found that results from rapid urea test were not in agreement with PCR technique. Most isolates gave the same results by both methods,
except for one strain that was initially identified as H. pylori by the CLOtest but was negative by PCR. The result was considered false positive. 4. Discussion Several media containing growth and selective supplements have been proposed to increase the probability to retrieve H. pylori. All media contain peptones, antibiotics and blood, and many include oxygen-quenching agents to overcome the adverse effects of toxic oxygen species (Chomvarin et al., 2006; Morgan et al., 1987; Sainsus et al., 2008; Shadowen and Sciortino, 1989). In this study, the effects of
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Table 1 Evaluation of Hp biphasic test for the detection of H. pylori NCTC 11637 in the presence of background microbiota. Inoculum (cfu/mL)
12 h
24 h
36 h
48 h
Liquid phase
Solid phase
Liquid phase
Solid phase
Liquid phase
Solid phase
Liquid phase
Solid phase
Viable cell (cfu/mL)
Urea agar
Viable cell (cfu/mL)
Urea agar
Viable cell (cfu/mL)
Urea agar
Viable cell (cfu/mL)
Urea agar
− − − −
1.50 3.00 2.03 1.24
− − − −
3.50 2.02 2.22 1.13
+ + − −
2.32 1.41 3.08 2.44
Viable cells 104 103 102 10
2.12 2.41 1.33 2.20
Nonviable cells 104
0
−
0
−
0
−
0
−
Mixed microbiota 104
0
−
0
−
0
−
0
−
± ± ± ±
0.53 0.74 0.22 0.13
× × × ×
104 103 102 10
± ± ± ±
0.25 0.51 0.15 0.08
× × × ×
104 103 102 10
± ± ± ±
0.22 0.32 0.77 0.33
× × × ×
105 105 104 10
± ± ± ±
0.33 0.22 0.55 0.14
× × × ×
106 106 105 102
+ + + −
After the inoculation of H. pylori into liquid phase, the apparatus was incubated at 37 °C for 12, 24, 36, and 48 h. Viable counts were determined on Columbia blood agar. The plates were incubated at 37 °C for 48 h under microaerobic conditions. + = urease positive; − = urease negative; 0 = no growth.
Table 2 Evaluation of Hp biphasic test for the detection of H. pylori NCTC 11638 in the presence of background microbiota. Inoculum (cfu/mL)
12 h
24 h
36 h
48 h
Liquid phase
Solid phase
Liquid phase
Solid phase
Liquid phase
Solid phase
Liquid phase
Solid phase
Viable cell (cfu/mL)
Urea agar
Viable cell (cfu/mL)
Urea agar
Viable cell (cfu/mL)
Urea agar
Viable cell (cfu/mL)
Urea agar
− − − −
3.33 2.72 1.52 1.62
− − − −
1.72 1.19 2.04 2.33
+ − − −
2.88 1.19 2.25 1.55
Viable cells 104 103 102 10
2.00 2.22 1.11 2.32
Nonviable cells 104
0
−
0
−
0
−
0
−
Mixed microbiota 104
0
−
0
−
0
−
0
−
± ± ± ±
0.24 0.35 0.73 0.32
× × × ×
104 103 102 10
± ± ± ±
0.55 0.89 0.61 0.18
× × × ×
104 103 102 10
± ± ± ±
0.49 0.33 0.43 0.25
× × × ×
105 104 103 10
± ± ± ±
0.36 0.33 0.05 0.94
× × × ×
106 105 105 103
+ + + −
After the inoculation of H. pylori into liquid phase, the apparatus was incubated at 37 °C for 12, 24, 36, and 48 h. Viable counts were determined on Columbia blood agar. The plates were incubated at 37 °C for 48 h under microaerobic conditions. + = urease positive; − = urease negative; 0 = no growth.
various liquid media on growth of H. pylori were investigated. Bolton broth supplemented with antibiotics was employed for the selective enrichment broth of Campylobacter (Hunt et al., 2001). Our results confirmed that Bolton broth was also suitable for growth of H. pylori. Enzymatic digest of animal tissues, lactalbumin hydrolysate, and yeast extract provide essential growth nutrients like vitamins (Bolton and Robertson, 1982). The addition of blood, sodium metabisulphite, and sodium pyruvate quenches toxic compounds and increases the recovery rate of the culture (Bolton et al., 1984). The results confirmed that competing bacteria did not cause false positive test reactions in the biphasic test. The antibiotics including vancomycin, polymyxin B, and trimethoprim present in the supplement inhibit the growth of Gram-positive and Gram-negative bacteria. Yeast may cause an alkaline environment causing the false positive results. Therefore, Hp biphasic test also contains amphotericin B that can inhibit the growth of contaminant yeast. We concluded that this broth
Table 3 Comparison of the numbers of H. pylori positive samples by Hp biphasic test, CLOtest and PCR from 55 gastric biopsies. Methods
Hp biphasic test CLOtest PCR n/a: not applicable.
represents the best overall compromise between growth and inhibition of contaminants for the isolation of H. pylori. The urease reaction can be used as an indicator test for the presence of urease-positive H. pylori in the culture. In comparison with CLOtest, the Hp biphasic test is found to be comparable in results. Interestingly, the results demonstrated that urease positivity depends on bacterial density. With the biphasic test, minimal concentration of organism needed to show a positive urease reaction was approximately 105 cfu/mL. Previous studies demonstrated that 104 cfu/mL is necessary to observe urease positivity in gel preparation (Deltenre et al., 1989). Nevertheless, using this new culture method and using the CLOtest to determine the prevalence of H. pylori in gastric biopsies demonstrated that H. pylori were detected in 12.72%. False positive urea test has been reported by other studies (Schrader et al., 1993; van Doorn et al., 2000; Vaira et al., 2010). CLOtest showed 90–95% sensitivity and 100% specificity after 24 h (Schrader et al., 1993). However, other urease-positive bacteria can decrease the specificity and give false-positive results (Vaira et al., 2010). In conclusion, the advantages of Hp biphasic test are the ability of culturing and simultaneously identifying H. pylori directly from gastric biopsy. Furthermore, it can be used under an aerobic condition and allows antimicrobial susceptibility testing.
Numbers of positive samples (%) 24 h
48 h
72 h
Total
5 (9.09) 7 (12.72) n/a
2 (3.63) n/a n/a
0 (0) n/a n/a
7 (12.72) 7 (12.72) 7 (12.72)
Acknowledgments This work was supported by Prince of Songkla University, Research Development Fund, Fiscal Year 2011–2012, Thailand. We also thank Dr. Brian Hodgson for their valuable suggestions.
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