The term biofilm describes the structurally complex bacterial BRIEF COMMUNICATION

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1 CLINICAL GASTROENTEROLOGY AND HEPATOLOGY 2010;8: BRIEF COMMUNICATION Biofilm Demolition and Antibiotic Treatment to Eradicate Resistant Helicobacter pylori: A Clinical Trial GIOVANNI CAMMAROTA,* GIOVANNA BRANCA, FAUSTA ARDITO, MAURIZIO SANGUINETTI, GIANLUCA IANIRO,* ROSSELLA CIANCI,* RICCARDO TORELLI,* GIOVANNA MASALA, ANTONIO GASBARRINI,* GIOVANNI FADDA, RAFFAELE LANDOLFI,* and GIOVANNI GASBARRINI* *Institute of Internal Medicine and Institute of Microbiology, Catholic University of Medicine and Surgery, Rome, Italy; and Molecular and Nutritional Epidemiology Unit, Cancer Research and Prevention Institute, Florence, Italy BACKGROUND & AIMS: Helicobacter pylori attaches to gastric mucosa and grows as a biofilm. This constitutes protection from antimicrobial agents. We assessed the role of a pretreatment with n-acetylcysteine in destroying biofilm and overcoming H pylori antibiotic resistance. METHODS: In an open-label, randomized controlled trial, 40 subjects with a history of at least 4 H pylori eradication failures were evaluated for biofilm presence, antibiotic susceptibility, and H pylori genotypes. Subjects were assigned randomly to receive (group A) or not (group B) n-acetylcysteine before a culture-guided antibiotic regimen. The primary end point was the H pylori eradication rate as assessed by 13 C- labeled urea breath testing. RESULTS: H pylori was eradicated in 13 of 20 (both per-protocol and intention-to-treat analyses, 65%; 95% confidence interval, 44% 86%) group A participants and 4 of 20 (both per-protocol and intention-to-treat analyses, 20%; 95% confidence interval, 3% 37%) group B participants (P.01). Biofilms persisted only in unsuccessfully treated participants. H pylori genotypes did not influence treatment outcome. CONCLUSIONS: N-acetylcysteine pretreatment before a culture-guided antibiotic regimen is effective in overcoming H pylori antibiotic resistance. Keywords: Biofilm; Helicobacter pylori; Rescue Therapy; Resistance. The term biofilm describes the structurally complex bacterial ecosystems that allow bacteria to survive in unfavorable environments by enclosing themselves in an exopolysaccharide matrix. 1 Biofilm-producing bacteria are more resistant to antibiotics presumably owing to slow or incomplete penetration of the antibiotic into the biofilm. 2 Biofilm formation also guarantees decreased metabolic and growth rates, leading to a lower susceptibility to antimicrobials. 2 A reliable mechanism to eradicate these resistant infections therefore consists of eliminating biofilm. 1 Several studies have shown the ability of Helicobacter pylori to form biofilm. 3,4 The possible survival of biofilm outside the gastric niche has been suggested as a probable transmission mode for this organism. 5 Two distinct studies have shown the presence of biofilm on the gastric mucosa of H pylori positive subjects, as well as the absence of biofilm in H pylori negative control subjects. 6,7 Study Aim We tested the hypothesis that a mucolytic pretreatment was able to demolish the biofilm architecture, rendering H pylori resistant strains more vulnerable to antibiotics. Materials and Methods Study Design In the first part of the study, we determined in vitro the ability of N-acetylcysteine (NAC) to interfere with H pylori biofilm formation or to provoke its demolition. In a second phase, we conducted a prospective, open-label, randomized trial to assess the effect of NAC pretreatment on the efficacy of a culture-guided antibiotic regimen in patients with a history of multiple treatment failures against H pylori. Patients gastric mucosa also was investigated for biofilm presence before and after treatment. Finally, genetic studies on antibiotic resistance and vacuolating cytotoxin A/cytotoxin-associated gene-a determinants were performed on cultured H pylori isolates. The primary end point of the study was to evaluate the H pylori eradication rate as assessed by urea breath testing at least 2 months from the end of antibiotic treatment. The study was approved by the ethics committee of our University (Clinical- Trials.gov, number NCT ). Patients provided written informed consent to participate in the study. In Vitro Study Gastric biopsy specimens from 10 patients with a history of at least 4 failed H pylori eradication attempts were cultured for H pylori in accordance with methods described elsewhere. 8 Specimens from 5 patients were cultured with added NAC 2 mg/ml. Both NAC-containing and NAC-lacking trays were tested for biofilm formation as described. 3 NAClacking trays then were tested, in 3 different plates, upon addition of increasing concentration of NAC broth (2, 10, and 20 mg/ml) to assess biofilm quantity. Biofilm formation was quantified as previously described. 3 By subtracting the percentage transmittance (%T) value for each Abbreviations used in this paper: NAC, N-acetylcysteine; %T bloc, percentage transmittance blocked by the AGA Institute /$36.00 doi: /j.cgh

2 818 CAMMAROTA ET AL CLINICAL GASTROENTEROLOGY AND HEPATOLOGY Vol. 8, No. 9 test sample from the %T value of the blank reagent, we calculated the amount of blocked light through the wells (%T bloc ). Biofilm production by each isolate was scored as follows: negative (%T bloc 0 10); biofilm producers (1, %T bloc 10 20; 2, %T bloc 20 35; 3,%T bloc 35 50; or 4,%T bloc 50). Positive and negative controls were a known H pylori biofilm and a brucella broth, respectively. Figure 1. (A) Scanning electron micrograph of gastric mucosal surface covered in a blanket of biofilm before the treatment. (B) Biofilm disappearance after H pylori eradication. Clinical Study Patient recruitment. Consecutive patients who had a history of at least 4 H pylori eradication failures, and were therefore referred to our tertiary endoscopy center for endoscopic examination and H pylori culture, were recruited. Patients affected by serious concomitant illnesses and those with recent or continuing use of antibiotics were excluded. Fourteen patients who had interrupted at least one of the previous eradication treatments because of adverse events were not included. Two eligible patients also were excluded when their H pylori strains did not survive culture processing. H pylori characterization. Two gastric biopsy specimens were used for biofilm demonstration by scanning electron microscopy. As in other reports, 6,7 a biofilm architecture was defined as a dense accumulation of bacteria within an amorphous matrix (Figure 1A). Two additional specimens were used for H pylori culture: antibiotic susceptibility testing, and genetic analysis. For each strain, minimal inhibitory concentrations were obtained for amoxicillin, clarithromycin, metronidazole, tetracycline, and levofloxacin by the epsilometer test method (AB Biodisk, Solna, Sweden). 9,10 The 23S, gyra, and gyrb gene mutations (for clarithromycin and quinolone resistance, respectively), as well as vacuolating cytotoxin A and cytotoxinassociated gene-a genotypes of H pylori isolates, were analyzed as previously described Patient randomization and follow-up evaluation. Patients were assigned randomly, using a computer-assisted allocation method, to 1 of 2 different eradication schedules: group A received a 1-week treatment course with NAC 600 mg once a day before a culture-guided 1-week antibiotic regimen, including a proton pump inhibitor plus 2 antibiotics; and group B (control) received solely a 1-week culture-guided antibiotic treatment, including a proton pump inhibitor plus 2 antibiotics. Sensitive antibiotics were chosen on the basis of minimal inhibitory concentration values. Side effects were recorded 1 week after treatment by a questionnaire. Treatment compliance was measured by pill count, together with a patient interview. At least 2 months after the end of therapy, patients underwent urea breath testing to determine their H pylori status. At that time, patients had to discontinue any acid-suppressive therapy for at least 2 weeks. Patients also were invited to repeat endoscopic examination for biofilm monitoring. Statistical Analysis We calculated the sample size by considering the proportion of patients successfully treated as the primary outcome measure. Based on our experience, we predicted an eradication rate in the standard group (group B) in the range of 15% to 20%. Therefore, setting an alpha value of.05, with 20 patients allocated to the treatment arm (group A) and 20 patients allocated to the control arm (group B, standard treatment), we had a power of 80% to observe the following: an eradication rate of 40% in the treatment arm with an expected eradication rate of 15% for the control group (eradication rate difference, 25%), or an eradication rate of 47% in the treatment arm with an expected eradication rate of 20% for the control group (eradication rate difference, 27%). The success rate was calculated as the percentage of patients whose H pylori infection was cured. Both per-protocol and intention-to-treat analyses were performed. The 95% confidence interval for a binomial variable was calculated. The Fisher exact test was performed using SAS statistical software (SAS/STAT version 9.1; SAS Institute Inc, Cary, NC). Treatment results also were compared in light of the genetic findings. Results In Vitro Study None of the 5 H pylori isolates cultured with NAC 2 mg/ml developed biofilm (%T bloc negative). Biofilm did develop in the other 5 plates, in which cultures had not been enriched with NAC. In these NAC-lacking cultures, NAC supplementation determined a progressive biofilm reduction until its disappearance (Table 1). Clinical Study A total of 40 patients were recruited for the study treatment: 20 in group A (8 men; mean age, 49 y) and 20 in group B (10 men; mean age, 47 y). All patients completed the treatment. Thirteen group A subjects were eradicated (both

3 September 2010 H PYLORI BIOFILM AND RESCUE TREATMENT 819 Table 1. Results of the In Vitro Study Patient number Developed biofilm NAC, 2 mg/ml NAC, 10 mg/ml NAC, 20 mg/ml Positive control (H pylori biofilm) Brucella broth (negative control) Negative Negative 4 Negative Negative Negative 4 Negative Negative Negative 4 Negative Negative Negative 4 Negative Negative Negative 4 Negative NOTE. H pylori isolates from 5 patients were cultured without NAC and all developed biofilm. Thereafter, each isolate was challenged, in 3 different plates, with increasing concentrations of NAC. The amount of blocked light through the wells (%T bloc ) was calculated and compared with control cultures. per-protocol and intention-to-treat analyses, 65%; 95% confidence interval, 44% 86%), whereas only 4 group B patients were treated successfully (both per-protocol and intention-to-treat analyses, 20%; 95% confidence interval, 3% 37%) (P.01). Overall, 14 (35%) subjects (6 in group A and 8 in group B) reported mild adverse effects (diarrhea, abdominal pain, stomatitis). At recruitment, scanning electron microscopy analysis documented biofilm in all recruited patients (100%). A total of 24 patients (60%) (14 in group A and 10 in group B) agreed to repeat endoscopic examination after treatment. Ten of these patients were eradicated, whereas 14 were still H pylori positive. Scanning electron microscopy analysis showed the disappearance of biofilm in all patients (100%) in whom the microorganism was eradicated (Figure 1B). H pylori cytotoxin-associated gene-a and vacuolating cytotoxin A status did not affect eradication outcome or biofilm status. Antibiotic susceptibility testing data and genetic findings are detailed in the Supplementary information (see Supplementary Tables 1 and 2). Discussion A standard rescue therapy against H pylori is still lacking, and antimicrobial sensitivity testing currently is recommended for patients who have failed eradication therapy. 14 In Figure 2. Representation of the findings of this study. (A) NAC was effective both in inhibiting the biofilm formation and in destroying developed biofilms in cultured H pylori isolates. (B) A NAC pretreatment course before a culture-guided antibiotic regimen is more effective in eradicating resistant H pylori infection than antibiotics alone.

4 820 CAMMAROTA ET AL CLINICAL GASTROENTEROLOGY AND HEPATOLOGY Vol. 8, No. 9 this study, we performed a novel approach to achieve H pylori eradication after multiple treatment failures, based on the hypothesis that microbial biofilm may support the tenacious resistance of H pylori to antibiotics. NAC might act as a biofilmdissolving agent, thus sensitizing H pylori to antibiotic action. These mechanisms already have been hypothesized for bacteria responsible for pulmonary infection. 15,16 In this study, we first proved, in vitro, that NAC was effective both in inhibiting the H pylori biofilm formation and in destroying developed biofilms. Moreover, keeping with data from the published literature, 6,7,17 we documented H pylori biofilm presence at scanning electron microscopy analysis in all patients enrolled for the clinical study. After treatment, we showed the disappearance of biofilm in all patients who repeated endoscopy and were treated successfully. Finally, H pylori was eradicated in 65% of patients treated with NAC before a culture-guided antibiotic regimen, compared with an eradication rate in control group B of only 20% (P.01) (Figure 2). Larger studies will be necessary to confirm our results and clarify whether treatment success can be improved by increasing the dosage of or the exposure to NAC before or during antibiotic treatment. New strategies are being envisaged to design substances that can either inhibit biofilm formation or destabilize biofilms. 18 Supplementary Tables 1, 2 and Figure 1. Supplementary Material Note: To access the supplementary material accompanying this article, visit the online version of Clinical Gastroenterology and Hepatology at and at doi: / j.cgh References 1. Costerton, JW, Stewart PS, Greenberg EP. Bacterial biofilms: a common cause of persistent infections. Science 1999;284: Stewart PS, Costerton JW. Antibiotic resistance of bacteria in biofilms. Lancet 2001;358: Cole SP, Harwood J, Lee R, et al. Characterization of monospecies biofilm formation by Helicobacter pylori. J Bacteriol 2004; 186: Stark RM, Gerwig GJ, Pitman RS, et al. Biofilm formation by Helicobacter pylori. Lett Appl Microbiol 1999;28: Percival SL, Thomas JG. Transmission of Helicobacter pylori and the role of water and biofilms. J Water Health 2009;7: Carron MA, Tran VR, Sugawa C, et al. Identification of Helicobacter pylori biofilms in human gastric mucosa. J Gastrointest Surg 2006;10: Coticchia JM, Sugawa C, Tran VR, et al. Presence and density of Helicobacter pylori biofilms in human gastric mucosa in patients with peptic ulcer disease. J Gastrointest Surg 2006;10: Cammarota G, Martino A, Pirozzi G, et al. High efficacy of 1-week doxycycline- and amoxicillin-based quadruple regimen in a cultureguided, third-line treatment approach for Helicobacter pylori infection. Aliment Pharmacol Ther 2004;19: Osato MS, Reddy R, Reddy SG, et al. Comparison of the E-test and the NCCLS-approved agar dilution method to detect metronidazole and clarithromycin resistant Helicobacter pylori. Int J Antimicrob Agents 2001;17: Megraud F, Lehours P. Helicobacter pylori detection and antimicrobial susceptibility testing. Clin Microbiol Rev 2007; 20: Posteraro P, Branca G, Sanguinetti M, et al. Rapid detection of clarithromycin resistance in Helicobacter pylori using a PCRbased denaturing HPLC assay. J Antimicrob Chemother 2006; 57: Hung KH, Sheu BS, Chang WL, et al. Prevalence of primary fluoroquinolone resistance among clinical isolates of Helicobacter pylori at a University Hospital in Southern Taiwan. Helicobacter 2009;14: Cellini L, Grande R, Di Campli E, et al. Characterization of an Helicobacter pylori environmental strain. J Appl Microbiol 2008; 105: Malfertheiner P, Megraud F, O Morain C, et al. Current concepts in the management of Helicobacter pylori infection: the Maastricht III Consensus Report. Gut 2007;56: Legnani D. Acute bacterial exacerbation of chronic obstructive pulmonary disease and biofilm. Infez Med 2009;17(Suppl 2): Macchi A, Ardito F, Marchese A, et al. Efficacy of N-acetyl-cysteine in combination with thiamphenicol in sequential (intramuscular/ aerosol) therapy of upper respiratory tract infections even when sustained by bacterial biofilms. J Chemother 2006;18: Cellini L, Grande R, Di Campli E, et al. Dynamic colonization of Helicobacter pylori in human gastric mucosa. Scand J Gastroenterol 2008;43: Del Pozo JL, Patel R. The challenge of treating biofilm-associated bacterial infections. Clin Pharmacol Ther 2007;82: Reprint requests Address requests for reprints to: Giovanni Cammarota, MD, A. Gemelli University Hospital, Department of Internal Medicine, Largo A. Gemelli, 8, Roma, Italy. gcammarota@rm.unicatt.it; fax: (39) Conflicts of interest The authors disclose no conflicts. Funding The study was funded by the Italian Ministry for University, Scientific, and Technological Research.

5 820.e1 CAMMAROTA ET AL CLINICAL GASTROENTEROLOGY AND HEPATOLOGY Vol. 8, No. 9 Supplementary Table 1. Drug Assumption History of the 20 Patients Treated With NAC Before a Culture-Guided, Antibiotic-Based Regimen (Group A) Patient number PPI- and NAC-associated, culture-guided antibiotics Successful eradication Number of previous failed eradication attempts Previous antibiotic assumption (number of times) 23S/GyrA/GyrB mutation VacA/CagA profile E-test results sensitive/ resistant 1 A-L Yes 6 A (5), L (4), C (3), M (2), T (2) 2 A-L No 4 A (4), M (2), C (2), 3 A-C Yes 4 A (4), M (2), L (2), C (1), T (1) 4 A-L No 7 A (7), C (4), L (4), M (3), T (3) 5 A-T No 4 A (3), C (2), M (1), L (1), T (1) 6 A-T Yes 4 A (4), C (1), M (1), L (1), T (1) 7 A-L No 7 A (6), C (5), M (3), L (3), T (3) 8 A-T Yes 4 A (4), C (2), M 9 A-L Yes 4 A (4), C (2), M 10 A-C Yes 4 A (4), C (1), M (3), L (1), T (1) 11 A-L Yes 5 M (4), A (3), C 12 A-C Yes 5 M (4), A (3), T (2), C (1), L (1) 13 A-T Yes 4 A (4), M (3), C 14 A-T No 6 A (6), C (3), T (3), L (3), M (1) 15 A-T Yes 5 M (4), A (3), T (2), C (1), L (1) 16 A-C No 5 A (4), M (2), L (2), C (2), T (2) 17 A-T Yes 5 A (4), M (3), C (2), T (2), L (1) 18 A-L No 6 A (6), M (3), C (2), 19 A-C Yes 5 A (5), M (2), L (2), C (2), T (2) 20 A-T Yes 4 A (4), C (2), M A2143G/WT/WT s1m2/absent A, T, L, M/C A2143G/WT/WT s2m2/p1p2p3 A, T, L/C, M WT/N87I/WT s2m2/absent A, T, C, M/L A2143G/WT/WT s1m2/absent A, T, L, M/C A2143G, T2182C/ N87K, A97V, E105G/WT s1m1/p1p2p3 A, T/L, C, M A2143G/N133K/WT s1m2/p1p2p3 A, T, L, M/C WT/WT/WT s2m2/absent A, T, L, C, M/none A2142G/D91G/WT s2m2/absent A, T/L, C, M WT/WT/WT s2m1/p1p2p2p3 A, T, C, L/M WT/WT/WT s2m2/absent A, T, L, C, M/none WT/WT/WT s2m2/absent A, T, C, L/M A2143G/N87I/WT S1m1/P1P2P2P3 A, T, M/C, L A2143G, T2182C/ N87K, A97V, E105G/WT s1m1/p1p2p2p3 A, T/C, L, M WT/WT/WT s2m1/p1p2p3 A, T, L, C, M/none WT/N87K/WT s1m2//p1p2p2p3 A, T, C/L, M A2143G/N87I/WT s2m2/absent A, T/C, L, M WT/N87I/WT s1m2/absent A, T, C/L, M A2142C/D91G/WT s2m1/p1p2p3 A, T/C, L, M A, amoxicillin; C, clarithromycin; CagA, cytotoxin-associated gene-a; L, levofloxacin; M, metronidazole; PPI, proton pump inhibitor; T, tetracycline; VacA, vacuolating cytotoxin A.

6 September 2010 H PYLORI BIOFILM AND RESCUE TREATMENT 820.e2 Supplementary Table 2. Drug Assumption History of the 20 Patients Treated With a Culture-Guided, Antibiotic-Based Regimen Alone (Group B) Patient number PPI-associated culture-guided antibiotics (without NAC) Successful eradication Number of previous failed eradication attempts Previous antibiotic assumption (number of times) 23S/GyrA/GyrB mutation VacA/CagA profile E-test results sensitive/resistant 1 A-T Yes 4 A (4),, C (1), M (1) 2 A-L No 5 A (5), L (3), C (2), 3 A-C Yes 4 A (4), L (2), C (2), 4 A-T No 6 A (6), C (3), L (3), M (3), T (3) 5 A-L No 5 A (5), L (3), C (2), M (1), T (2) 6 A-M No 5 A (5), C (2), M (2), 7 A-L No 4 A (4), C (2), L (2), T (1), M (1) 8 A-T Yes 4 A (4), M (2), C (1), L (1), T (1) 9 A-L Yes 5 A (5), C (2), M (2), 10 A-C No 4 A (4), M (2), L (2), C (1), T (1) 11 A-L No 6 A (5), T (4), L (3), M (2), C (2) 12 A-T No 5 A (5), T (3), L (3), C (2), M (1) 13 A-T No 4 A (4), C (1), L (2), 14 A-L No 6 A (6), C (2), T (3), L (3), M (2) 15 A-T No 5 A (5), L (3), T (2), C (2), M (1) 16 A-T No 4 A (4), L (2), C (1), 17 A-T No 4 A (4), M (2), T (2), L (1), C (1) 18 A-C No 5 A (5), L (2), C (2), T (2), M (2) 19 A-L No 6 A (6), M (3), C (2), 20 A-C No 5 A (5), M (3), C (2), A2142G/D91G/WT s1m1/p1p2p3 A, T/L, C, M WT/H57Y/WT s1m2/absent A, T, C, M/L A2143G/D91Y/WT s2m2/absent A, T/L, C, M A2143G/WT/WT s2m2/absent A, T, L, M/C A2142C/D91G/WT s1m1/p1p2p3 A, T, M/L, C A2143G/D91Y/WT s1m2/p1p2p3 A, T, M/L, C WT/WT/WT s1m1/p1p2p3 A, T, C, L, M/none A2142C/WT/WT s2m2/absent A, T, L/C, M A2143G/N87I/WT s1m1/p1p2p3 A, T/C, L, M WT/D91G/WT s1m1/p1p2p2p3 A, T, C/L, M WT/WT/WT s2m2/absent A, T, L, C/ M A2142G/N87I/WT s2m2/absent A, T, M/L, C A2142C/D91G/WT s1m2/p1p2p2p3 A, T, M/L, C A2143G/D91Y/WT s1m1/p1p2p2p3 A, T/C, L, M WT/H57Y/WT S2m2/absent A, T, C/L, M WT/WT/WT s2m1/p1p2p2p3 A, T, C, L/M WT/WT/WT s1m1/absent A, T, C, L/M A, amoxicillin; C, clarithromycin; CagA, cytotoxin-associated gene-a; L, levofloxacin; M, metronidazole; PPI, proton pump inhibitor; T, tetracycline; VacA, vacuolating cytotoxin A.

7 820.e3 CAMMAROTA ET AL CLINICAL GASTROENTEROLOGY AND HEPATOLOGY Vol. 8, No. 9 Supplementary Figure 1. Consolidated Standards of Reporting Trials (CONSORT) flowchart.