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Esophageal Bacteria and Barrett’s Esophagus: A Preliminary Report GLENN L. OSIAS, MD,* MATTHEW Q. BROMER, DO,* REBECCA M. THOMAS, MD,‡ DAVID FRIEDEL, MD,* LARRY S. MILLER, MD,* BYUNGSE SUH, MD, PHD,† BENNETT LORBER, MD,† HENRY P. PARKMAN, MD,* and ROBERT S. FISHER, MD*
The objective of this study was to investigate if esophageal bacteria are associated with Barrett’s esophagus (BE). This study was comprised of a retrospective (Part 1) and a subsequent prospective (Part 2) study. In Part 1, Gram stains were performed on esophageal biopsy specimens obtained in 47 patients. Bacteria were quantitated from 0 to 4. In Part II, Gram stains and cultured bacterial counts of esophageal biopsies were obtained in 18 GERD patients (9 with BE and 9 without BE). Part 1 results were as follows. Bacteria were found in 37 of 47 esophageal biopsies. Quantitative bacterial stain scores for BE (2.5 ± 0.2) were higher than for non-BE (1.5 ± 0.3; P = 0.02). The quantitative bacterial stain scores correlated with increasing severity of dysplasia (r = 0.37, P = 0.028). In Part 2, bacteria were found in 8 of 18 esophageal biopsies by Gram stain (6 of 9 patients with BE vs. 2 of 9 non-BE). The distal esophageal bacterial stain scores in BE patients (1.6 ± 0.5) were higher than in those without BE (0.4 ± 0.3; P = 0.07). Patients on proton pump inhibitors tended to have higher bacterial stain scores (1.2 ± 0.4) than patients who were not (0.7 ± 0.3; P = 0.45). Bacterial colony counts were similar in patients with BE compared to those without BE. In conclusion, bacteria in esophageal biopsies were detected more often in BE than non-BE. Increasing bacterial stain scores were associated with metaplasia and increasing dysplasia. Esophageal bacteria, possibly related to stasis or gastric acid suppression therapy, may play a role in the pathogenesis of BE and dysplasia. KEY WORDS: esophageal bacteria; Barrett’s esophagus; dysplasia.
The annual incidence of esophageal adenocarcinoma has increased dramatically in recent decades (1, 2). Reasons for this increase are unknown. Barrett’s esophagus (BE), considered to be a precursor of adenocarcinoma, is found in approximately 6–12% of patients undergoing endoscopy for symptoms of gastroesophageal reflux (1–3). The annual incidence of esophageal adenocarcinoma among patients with BE is approximately 0.2–0.5% (1, 2). The development of BE and progression to dysplaManuscript received August 23, 2003; accepted November 30, 2003. From the Sections of *Gastroenterology and †Infectious Diseases, Department of Medicine, and ‡Surgical Pathology Section, Department of Pathology, Temple University School of Medicine, Philadelphia, Pennsylvania 19140, USA. Address for reprint requests: Henry P. Parkman, MD, Gastroenterology Section, Department of Internal Medicine, Temple University Hospital, Parkinson Pavilion, 8th Floor, 3401 North Broad Street, Philadelphia, Pennsylvania 19140, USA;
[email protected].
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sia and adenocarcinoma may have several etiologies. Gastroesophageal reflux, with its associated inflammation, is accepted as an important factor (4, 5). Patients who undergo ureterosigmoidostomy have a high incidence of colonic adenocarcinoma at the ureterosigmoid anastomosis. It has been suggested that high concentrations of urinary nitrosamines or carcinogens within the urinary and fecal stream, perhaps related to stasis and bacterial overgrowth, may contribute to the development of colonic adenocarcinoma in this setting (6–8). A similar situation may occur with gastroesophageal reflux disease (GERD), BE, and esophageal adenocarcinoma. The prevailing hypothesis has been that inflammation of the distal esophagus and gastric cardia coupled with reflux of acid, pepsin, and/or bile acids may play a role in the development and progression of esophageal metaplasia. Alternatively, inflammation from Digestive Diseases and Sciences, Vol. 49, No. 2 (February 2004)
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acid, pepsin, and/or bile acids coupled with bacterial colonization/overgrowth and gastroesophageal nitrosamines may affect normal healing, leading to development of BE and subsequent dysplasia. The upper gastrointestinal tract is considered to be a relatively sterile environment due to normal motility preventing ascending migration of bacteria and gastric acid preventing bacterial growth (9, 10). Patients with BE have a high prevalence of motility disturbances, such as a weak lower esophageal sphincter (LES) pressure and decreased esophageal contractility, contributing to reflux and delayed esophageal clearance (11, 12). These motility disturbances may predispose to bacterial growth in the distal esophagus. Proton pump inhibitors (PPIs), used for treating acid/peptic disorders, by suppressing gastric acid secretion, may also contribute to the presence of increased bacteria in the upper gastrointestinal tract. The objectives of this preliminary study were to determine whether bacteria are increased in the distal esophagus of patients with BE and to determine whether esophageal bacteria are related to the GERD–Barrett’s metaplasia–dysplasia–adenocarcinoma sequence. The presence of bacteria in the distal esophagus was investigated in patients experiencing gastroesophageal reflux symptoms with BE and without BE (nonBE) in both retrospective and prospective pilot protocols. The role of proton pump inhibition and its effect on bacterial colonization of the distal esophagus was examined. METHODS This study was performed in two parts. Part 1 was a retrospective study of esophageal biopsies previously obtained from patients with GERD symptoms and/or Barrett’s esophagus. Part 2 was a prospective study to assess risk factors and perform quantitative bacterial counts on cultured esophageal biopsy specimens. This study was approved by the Temple University Health Sciences Center Institutional Review Board. Patients Retrospective Study (Part 1). Mucosal biopsy specimens were reviewed from the distal esophagus in 47 patients who had previously undergone upper endoscopy with esophageal biopsy. Biopsies had been taken within the BE segment in patients with BE. In patients without BE, biopsies had been obtained 5 cm above the squamocolumnar junction, a location often biopsied when looking for evidence of microscopic esophagitis. Five patient groups were evaluated: (1) BE with high-grade dysplasia (HGD), (2) BE with low-grade dysplasia (LGD), (3) BE without dysplasia (ND), (4) erosive esophagitis without BE (EE), and (5) no esophageal pathology (NP). Ten consecutive patients were sought for each group by reviewing recent endoscopy and surgical pathology records. Tissue Gram stains were retrospectively performed on archived mucosal biopsy specimens by R.M.T., who was unaware of how the result of tissue Gram stain related to clinical diagnosis. Digestive Diseases and Sciences, Vol. 49, No. 2 (February 2004)
Prospective Study (Part 2). Mucosal biopsy specimens were obtained prospectively from the distal esophagus in 18 patients with GERD—9 consecutive consenting patients with BE (BE) and 9 consecutive consenting patients without BE (nonBE). Patients identified were those with GERD symptoms and/or known BE who were undergoing upper endoscopy during the study period March 2001 to January 2002. Biopsies were obtained at 1 cm and 5 cm above the gastroesophageal junction for bacterial culture (see below). Biopsies were also obtained within the BE segment in BE patients and 5 cm above the squamocolumnar junction for non-BE patients for histology. Patients were also subdivided into two groups according to whether or not they were taking a PPI at the time of obtaining the endoscopic biopsies: on PPI (12 patients) and non-PPI (6 patients). Histology In both Part 1 (retrospective) and Part 2 (prospective) studies, the tissue block of each biopsy specimen was obtained. A 5-µm section from the biopsy specimen from each patient was Gram stained (crystal violet with safranine as counterstain). Gram-positive bacteria appeared blue–black and Gram-negative bacteria appeared red. A pathologist (R.M.T.), unaware of the patient’s clinical status, interpreted each Gram stain for the presence of bacteria (13). A quantitative bacterial score was assigned to each biopsy specimen as follows: 0 = none, 1 = rare bacteria, 2 = a single cluster or a few scattered bacteria, 3 = a few (i.e., two to four) clusters or a single cluster with numerous scattered bacteria, and 4 = more than four clusters or numerous scattered bacteria. Bacterial Counts In Part 2 (prospective study), an attempt was undertaken to quantify bacterial counts of esophageal biopsy specimens using a serial dilution and pour plate technique routinely used for culturing aerobic organisms (14). Esophageal biopsy specimens obtained at the time of endoscopy were promptly placed in sterile saline in a sterile container and then delivered to the laboratory for culture. Excess moisture was blotted off with sterile filter paper. Specimens were accurately weighed and then minced using a tissue grinder. One milliliter of sterile saline was added to the tissue grinder to make a suspension. Specimen solutions of 0.1, 0.2, and 0.3 ml were then directly inoculated by the pour plate method. Serial dilutions were also prepared and inoculated similarly for accurate bacterial counts on a nutrient agar medium. The number of colony forming units was counted 2 days later. Results are expressed as bacterial colonies per milliliter and, to normalize for the tissue biopsy size, as bacterial colonies per milligram of tissue. Data Analysis Results are expressed as mean ± SE. Mean bacterial scores colony counts were calculated for each group and compared using the Student t-test, ANOVA, chi-square analysis, and Spearman’s correlation.
RESULTS Part 1: Retrospective Study In this retrospective review, 47 patients were identified for the five study group categories. Only seven patients
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Age (years) Gender (M/F) Hiatal hernia Comorbid conditions Diabetes mellitus Scleroderma Regular tobacco use Regular alcohol use Helicobacter pylori status Positive Negative Unknown Medications Acid suppression PPI alone H2RA alone Both PPI and H2RA None Prokinetic agent Immunosuppressives
Barrett’s esophagus
No pathology (n = 10)
Erosive esophagitis (n = 10)
None (n = 10)
Low grade (n = 10)
High grade (n = 7)
50 ± 17 5/5 2
46 ± 15 1/9 3
50 ± 17 10/0 4
57 ± 19 7/3 5
63 ± 24 4/3 3
1 0 5 0
1 1 2 1
0 0 1 2
1 0 0 1
0 1 0 1
2 6 2
2 5 3
1 5 4
0 7 3
1 3 3
5 3 1 1 1 0
7 1 1 1 1 0
8 1 1 0 2 1
7 0 1 2 0 0
5 0 1 2 1 1
could be identified with BE with HGD. Study groups evaluated were as follows: (1) BE with HGD, 7 patients; (2) BE with LGD, 10 patients; (3) BE without dysplasia (ND), 10 patients; (4) erosive esophagitis without BE (EE), 10 patients; and (5) no esophageal pathology (NP), 10 patients. The patient demographic characteristics are listed in Table 1. Bacteria, predominantly Gram-positive cocci, were found in 37 of 47 Gram-stained specimens obtained from esophageal biopsies. Representative Gram-stained biopsy specimens are shown in Figure 1. The quantitative histologic bacterial score for all BE (2.5 ± 0.2; n = 20) was significantly higher than that for non-BE (1.5 ± 0.3; n = 27; P = 0.019) (Figure 2a). The histologic quantitative bacterial scores were 1.6 ± 0.5 for NP, 1.4 ± 0.4 for EE, 2.3 ± 0.4 for ND, 2.4 ± 0.4 for LGD, and 2.9 ± 0.4 for HGD (Figure 2b). The quantitative histologic bacterial scores correlated weakly, but significantly, with the increasing severity of the spectrum from esophagitis without Barrett’s metaplasia to BE with HGD (r = 0.367, P = 0.028). Part 2: Prospective Study Eighteen patients with GERD symptoms and/or known BE who were undergoing upper endoscopy participated in this prospective study. Patient demographic characteristics are listed in Table 2. There were nine patients with BE (BE) and nine patients without BE (non-BE). In the patients with BE, the length of BE was 2 to 5 cm.
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Bacteria were found in 8 of 18 esophageal biopsies by Gram stains (6 of 9 BE vs. 2 of 9 non-BE; χ 2 = 2.025; P = 0.08; see Table 3). Most of the bacteria were Grampositive cocci; some were both Gram-positive rods and cocci. The distal esophageal histologic bacterial scores in BE (1.6 ± 0.5; n = 9) were higher compared to non-BE (0.4 ± 0.3; n = 9; P = 0.067). Eight of nine patients with BE were on PPI therapy; six of these patients had bacteria present on Gram stains. Patients on PPIs tended to have higher bacterial histologic scores (1.2 ± 0.4; n = 12) compared to those not on PPIs (0.7 ± 0.4; n = 6; P = 0.45). Bacteria were detected by culture in 15 of 18 (83%) esophageal biopsies from 1 cm above the gastroesophageal junction and were similar in patients with BE (8 of 9 [89%]) and patients without BE (7 of 9 [78%]). Bacterial colony counts obtained 1 cm above the gastroesophageal junction were similar in patients with BE (3.2 ± 1.4 colonies/ml) and those without BE (4.7 ± 1.7 colonies/ml). Esophageal biopsies averaged 11 ± 3 mg wet weight, with a range from 0.5 to 46 mg. After normalizing the bacterial counts for differences in biopsy size, there was also no significant difference in bacterial counts between patients with BE (0.5 ± 0.2 colonies/mg tissue) and those without BE (1.1 ± 0.4 colonies/mg tissue). There were no differences in the bacterial colony counts in patients on PPIs (3.8 ± 1.2 colonies/ml and 0.8 ± 0.3 colonies/mg tissue) and patients not on PPIs (4.3 ± 2.4 colonies/ml and 0.8 ± 0.3 colonies/mg tissue). The data for patients with BE and without BE stratified Digestive Diseases and Sciences, Vol. 49, No. 2 (February 2004)
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Fig 1. Examples of photomicrographs of Gram stain of esophageal biopsy specimens. (a) Bacterial score of 1 (rare bacteria seen) in esophagitis without Barrett’s. (b) Bacterial score of 2 (a few scattered bacteria seen) in Barrett’s with low-grade dysplasia. (c) Bacterial score of 3 (a single cluster with several scattered bacteria seen) in Barrett’s with high-grade dysplasia. (d) Bacterial score of 4 (numerous scattered bacteria) in a patient with Barrett’s with high-grade dysplasia. Digestive Diseases and Sciences, Vol. 49, No. 2 (February 2004)
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Fig 1. Continued.
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Age (years) Gender (M/F) Hiatal hernia Comorbid conditions Diabetes mellitus Scleroderma Regular tobacco use Regular alcohol use Helicobacter pylori status Positive Negative Unknown Medications Acid suppression PPI alone H2RA alone Both PPI and H2RA None Prokinetic agent Immunosuppressives
No Barrett’s
Barrett’s esophagus
(n = 9)
(n = 9)
54 ± 18 2/7 3
48 ± 16 7/2 6
0 0 0 2
0 0 1 3
1 6 2
1 2 5
3 0 1 5 0 0
6 0 2 2 0 0
according to the use of PPIs are given in Table 3. The bacterial colony counts for the esophageal biopsies obtained 5 cm above the gastroesophageal junction were not different between patients with and those without BE or between patients on and patients not on PPIs (data not shown). DISCUSSION The development of BE with progressive dysplasia and adenocarcinoma has been well documented (1–3). Increased distal esophageal acid exposure with esophagitis and inflammation have been proposed mechanisms for the development of BE. This preliminary pilot study of biopsies from the distal esophagus detected bacteria more frequently in patients with BE than in non-BE. Furthermore, increasing bacterial scores correlated weakly with severity of GERD from erosive esophagitis to BE with increasing degrees of dysplasia. Thus, the putative role of chronic inflammation caused by esophageal bacteria in the development of BE and its progression to dysplasia deserves more study. In both retrospective and prospective studies, the histologic bacterial scores were higher in patients with BE compared to patients without BE. Almost all of the bacteria detected in biopsy specimens from this study were Grampositive cocci. The species of bacteria were not identified. Whether esophageal bacteria are associated with BE and its progression to LGD and HGD has not been investigated previously. There are several confounding factors Digestive Diseases and Sciences, Vol. 49, No. 2 (February 2004)
in Part 1 of this study. First, biopsies were obtained 5 cm above the gastroesophageal junction in patients without BE and within the BE segment in patients with BE. In some patients, the length of BE was less than 5 cm; therefore, in these patients, the biopsies were taken at a lower esophageal level than in the controls, which might influence the number of bacteria in the biopsies obtained from patients with BE. Second, bacteria may adhere to Barrett’s epithelium with a higher affinity than to squamous mucosa. If this were to occur, this could explain the higher bacteria in patients with BE. In Part 2, an attempt was made to quantitate prospectively bacteria in the distal esophagus both by Gram stain techniques as in Part 1 and by determination of bacterial colony counts on esophageal biopsy specimens in patients GERD compared to patients with BE. Bacterial colony counts were not significantly different in patients with BE and non-BE. Although the quantitative culture results might seem to refute the observations made using quantitative Gram staining of esophageal biopsy specimens, the culture methodology employed had some limitations. First, the pour plate cultures only tested for aerobic bacteria; strict anaerobic bacteria were not assessed (14). Second, the protocol design involved per oral passage of an endoscope into the esophagus using biopsy forceps not shielded from oral microflora. The channel of the endoscope may also have harbored bacteria if secretions had inadvertently been suctioned while advancing the endoscope. Thus, differences in esophageal bacterial colony counts between BE and non-BE patients may have been masked (diluted) by oropharyngeal and gastric contamination. Further studies to show a relationship between bacteria and the development of BE should address these limitations. Chlorhexidine or acidified sodium chlorite mouth rinse has been used in other studies to decontaminate the oropharynx (15, 16). A prior study in which small intestinal cultures were obtained used a sterilized, sheathed wash pipe that was passed through the suction channel of the endoscope attempted to eliminate this problem of contamination by collecting samples under sterile conditions (17). The use of PPIs in most patients with BE may be a confounding factor in determining whether BE itself is associated with esophageal bacteria overgrowth. Patients with BE, once diagnosed, are often treated with PPIs. PPIs reduce gastric acid secretion; acid suppression may allow bacteria to survive in the stomach (gastric bacterial overgrowth) (18). Since Barrett’s metaplasia of the esophagus is associated with reflux esophagitis, bacteria in the stomach would likely also be present in gastric material refluxed into the esophagus. These bacteria might adhere to the distal esophageal mucosa. This is a potential explanation for the presence of esophageal bacteria in BE.
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Fig 2. Bacterial Gram stain scores of patients stratified by histologic diagnosis. (a) Patients with Barrett’s esophagus had an increase in bacterial Gram stain scores compared to those without Barrett’s esophagus. (b) There was an increased histologic bacterial score with increasing severity of Barrett’s. Spearman correlation coefficient r = 0.367 (P = 0.028). TABLE 3. ESOPHAGEAL BACTERIA RESULTS IN PATIENTS IN PROSPECTIVE STUDY ACCORDING TO PRESENCE OF BARRETT’S AND PROTON PUMP INHIBITOR USE
Barrett’s On PPI Not on PPI Subtotal No Barrett’s On PPI Not on PPI Subtotal Total
No.
No. (%) of bacteria on Gram stain
Bacterial Gram stain score (mean ± SE)
8 1 9
6 of 8 0 of 1 6 of 9 (67%)
1.8 ± 0.5 0 1.6 ± 0.5
4 5 9
0 of 4 2 of 5 2 of 9 (33%)
18
8 of 18 (44%)
No. (%) of patients with positive bacterial cultures 1 cm above GEJ
Bacterial colony counts/mg tissue (mean ± SE)
5 cm above GEJ
1 cm above GEJ
5 cm above GEJ
7 (88%) 1 (100%) 8 (89%)
6 (75%) 0 (0%) 6 (67%)
0.5 ± 0.2 0.5 0.5 ± 0.2
0.3 ± 0.1 0.8 0.3 ± 0.1
0±0 0.8 ± 0.5 0.4 ± 0.3
3 (75%) 4 (80%) 7 (78%)
3 (75%) 4 (80%) 7 (78%)
1.3 ± 0.8 0.9 ± 0.4 1.1 ± 0.4
0.2 ± 0.1 0.6 ± 0.6 0.5 ± 0.3
1.0 ± 0.3
15 (83%)
13 (72%)
0.8 ± 0.2
0.4 ± 0.2
Note. GEJ, gastroesophageal junction.
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Of interest, patients taking PPIs tended to have higher bacterial scores compared to those not on PPIs, but this difference did not reach statistical significance. Freid et al. reported that 14 of 25 patients with reflux esophagitis or peptic ulcer disease who received 40 mg of omeprazole for approximately 5 weeks had bacterial overgrowth in the small bowel (19). In addition, there have been a number of reports showing increased gastric bacterial colony counts during acid suppressive therapy (18, 20, 21). The mechanisms for the development of metaplasia development in the distal esophagus are likely multifactorial (1). The relative increase in bacterial colonization of BE in this study relative to patients with erosive esophagitis (without BE) and controls is provocative and suggests a putative role of bacterial colonization in the development of BE. In addition, the presence of bacteria in the distal esophagus may promote nitrosation of ingested compounds, which may also be involved in the development of BE (22). Esophageal motor dysfunction, as a reason for patients with BE to have an increase in esophageal bacteria, was not investigated in this study. However, patients with BE have been reported to have reduced esophageal contractile amplitude and delayed esophageal clearance compared to normal subjects (11, 12). Delayed esophageal clearance and stasis might also create a milieu for bacterial colonization of the distal esophagus. Patients with BE have significantly increased acid reflux time, reduced distal esophageal body contractility, and reduced resting LES pressure compared to normal subjects and patients with mild and moderate esophagitis but not patients with severe esophagitis (12, 23). The extent of intestinal metaplasia (BE) has been related to the reduction in the resting LES pressure and the increase in esophageal acid exposure (11). These esophageal contractile and clearance abnormalities with esophageal stasis might predispose patients to esophageal bacteria. In summary, this preliminary pilot study has shown that bacteria are detected more frequently in patients with BE than in non-BE patients in distal esophageal biopsies. There was also a tendency toward an increase in bacterial stain scores with dysplasia. PPIs did not readily explain these differences. These provocative findings suggest that bacteria in the distal esophagus may potentially play a role in the pathogenesis of BE and its progression to LGD and HGD. Further studies are needed to confirm and enhance these provocative, albeit preliminary, findings. ACKNOWLEDGMENTS This study was supported in part by an NIH Midcareer Investigator Award in Patient-Oriented Research to H. P. Parkman Digestive Diseases and Sciences, Vol. 49, No. 2 (February 2004)
(NIH DK02921) and by the NIH GCRC Program of the NIRR (RR00349). The results of this study were presented at the 2001 American College of Gastroenterology Meeting, with the abstract appearing in American Journal of Gastroenterology 96:S5, 2001, and the 2002 American College of Gastroenterology Meeting, with the abstract appearing in American Journal of Gastroenterology 97:S14, 2002.
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