Initial investigation of small colony variants of Staphylococcus aureus in chronic rhinosinusitis Sarah A. Gitomer, M.D.,1,2 Vijay R. Ramakrishnan, M.D.,1 Kenneth C. Malcolm, Ph.D.,3,4 Jennifer M. Kofonow, M.S.,2 Diana Ir, B.S.,2 and Daniel N. Frank, Ph.D.2,5
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ABSTRACT
Background: Small colony variants (SCVs) are a metabolically inactive form of bacteria that can be difficult to eradicate. To examine whether SCVs contribute to Staphylococcus aureus persistence in chronic rhinosinusitis (CRS), we compared the prevalence of S. aureus SCVs in CRS patients and healthy controls. Methods: Endoscopically guided middle meatus samples were collected from 23 CRS patients and 12 controls. Samples were cultured and screened for the presence of phenotypically small colonies. Candidate SCV isolates were classified by 16S rRNA gene sequencing. To further characterize the capacity of S. aureus isolates to form SCVs when stressed, colonies underwent a gentamicin exposure assay. Results: Among CRS patient samples, 15 were culture positive for S. aureus (65.2%), and of those, two grew putative SCVs on selective media (8.7%). However, neither was genetically confirmed to be S. aureus upon sequencing. In healthy controls, eight specimens were culture positive for S. aureus (66.7%), and of these, two grew putative S. aureus SCVs on selective media (16.7%); but again, neither was confirmed to be S. aureus by 16S analysis. None of the four patients colonized with SCVs had evidence of sinonasal disease at a mean follow-up of eight months. S. aureus isolates from CRS patients and controls were equally likely to form SCVs with gentamicin exposure. Conclusion: S. aureus SCVs were not associated with CRS in the current study. Their role in refractory CRS remains theoretical, and further research is warranted to determine whether S. aureus SCVs may reside in the intracellular compartment. (Am J Rhinol Allergy 29, 29 –34, 2015; doi: 10.2500/ajra.2015.29.4133)
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hronic rhinosinusitis (CRS) is a common and burdensome disease process, in which infection or colonization with specific bacterial pathogens is thought to play a role in at least a subset of patients. Both circumstantial and direct evidence has increasingly implicated Staphylococcus aureus in the pathogenesis of many CRS patients, including both those with and without nasal polyps. S. aureus is the most commonly detected bacterium in sinonasal samples of CRS patients and is more abundant in CRS patients than in controls.1–3 In CRS, S. aureus is not only common but also has been linked to both disease severity and recurrence of disease after aggressive medical and surgical therapies.3,4 For reasons that are not clearly understood, S. aureus is particularly challenging to eradicate using standard treatments.4 The role of biofilms in CRS has been explored as a potential mechanism for S. aureus recalcitrance,2,5,6 but biofilms alone do not fully explain persistent colonization, because patients who are S. aureus positive and biofilm negative often continue to have symptoms despite appropriate treatment. Another proposed mechanism is the presence and persistence of intramucosal and even intracellular S. aureus, which have been identified in patients with CRS.7 Like biofilms, intracellular S. aureus potentially evades the host immune response and antibiotic treatment, thereby persisting despite otherwise adequate treatment. This phenomenon has been demonstrated in other diseases, such as cystic fibrosis and endocarditis, in addition to preliminary findings in CRS.7–10 Intracellular persistence of S. aureus within the sinonasal
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Department of Otolaryngology, 2Division of Infectious Diseases, and 4Division of Pulmonary Medicine, University of Colorado School of Medicine, Aurora, Colorado, 3 National Jewish Health, Division of Pulmonary Medicine, Denver, Colorado, and 5 University of Colorado Microbiome Research Consortium, Denver, Colorado Funded by Department of Otolaryngology and Division of Infectious Diseases, University of Colorado School of Medicine Presented at the American Society of Microbiology Meeting in Denver, Colorado, May 2013 The authors have no conflicts of interest to declare pertaining to this article Address correspondence to Vijay R. Ramakrishnan, M.D., Department of Otolaryngology, 12631 East 17th Avenue, B205, Aurora, CO 80045 E-mail address: [email protected] Copyright © 2015, OceanSide Publications, Inc., U.S.A.
American Journal of Rhinology & Allergy
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epithelium is reported to be a risk factor for recurrent CRS symptoms that are refractory to traditional therapies.11,12 The presence of, or ability to convert to, small colony variants (SCVs) may facilitate persistence of S. aureus in CRS by behavioral characteristics similar to biofilms.11,13 Like biofilms, SCV bacteria are metabolically inactive, slow-growing variants that can develop and persist when the extracellular environment is stressed.13,14 Environmental stress created by antibiotic exposure can induce conversion of S. aureus from morphologically normal variants to SCVs.14–17 Many auxotrophic variants of SCVs have been identified, most commonly in persistent and recurrent infections in cystic fibrosis lung disease and joint infections.15,18 These bacteria are able to revert to the wild-type phenotype and live extracellularly, presumably when external stress is alleviated, which may explain the delayed recurrence of S. aureusmediated CRS after initial treatment success. Therefore, in this prospective cross sectional study, we tested the hypothesis that S. aureus SCV presence is more frequent in CRS given the species’ association with disease presence, recalcitrance, and severity.
MATERIALS AND METHODS This study was approved by the Institutional Review Board of the University of Colorado (COMIRB protocol 11-1442), and informed consent was obtained from all patients.
Patient Selection Adult patients were recruited from a tertiary care rhinology practice from July 2012–September 2012. CRS was defined according to 2007 American Academy of Otolaryngology-Head and Neck Surgery Task Force Criteria:19 healthy control patients undergoing endoscopic orbital or pituitary surgery were recruited when no symptomatic sinonasal disease was noted on history, and normal appearance was confirmed on both nasal endoscopy and sinus computed tomography (CT). Patient demographics, disease history, and treatment information were recorded. Symptom severity was assessed using the SinoNasal Outcomes Test (SNOT)-22 questionnaire,20 severity of clinically apparent inflammation on endoscopic examination was graded using the Lund-Kennedy scale,21 and CT imaging severity of radiographic disease was graded using the Lund-Mackay scoring system.22
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Sample Collection and Bacterial Culture Nylon Liquid Amies Elution Swabs (ESwabs) (Becton Dickinson and Company, Franklin Lakes, NJ) were used to aggressively swab the middle meatus epithelial surface under endoscopic guidance, taking care of not to contaminate the swab by contacting the anterior nasal passage. Samples were immediately transported to the laboratory, where they were vortexed for 60 seconds in the Amies liquid, and 100 and 10 L of liquid were plated in duplicate onto mannitol salt agar and Columbia naladixic acid (CNA) agar, respectively. At the same time, 10 L of undiluted and a 100 L of 1:100 dilution of Amies liquid were plated onto blood agar, based on the optimal amount to grow individual, countable colonies on each type of plate. Bacteria were cultured following recommended culture conditions for the growth of S. aureus SCVs.14 All plates were incubated under aerobic conditions without carbon dioxide at 37°C for 72 hours total, with growth evaluated every 24 hours. Plates were screened for growth of phenotypically small colonies, defined as colonies that were not visible within the first 24 hours of incubation but that appeared between 24 and 72 hours of incubation and remained phenotypically small (Fig. 1). The presence or absence of small colonies was assessed on specific media (mannitol salt and Columbia CNA), and individual colonies were then streaked onto blood agar. Colonies that remained small when grown for 48 hours were identified as SCVs.14 S. aureus SCV positive control samples were obtained from the National Jewish Health (Denver, CO) clinical microbiology laboratory, defined in a similar fashion, and isolated under the same conditions as the middle meatus samples.
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Figure 1. Small colony variants compared with normal colonies. (A) Blood agar plate growing small colony variants at day three (arrow). These colonies were not visible after a 24-hour incubation. (B) Columbia CNA plate growing wild-type S. aureus, with normal phenotypic colonies.
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Gentamicin Challenge
To determine whether bacterial colonies of normal morphology would be likely to convert to an SCV phenotype, a model of exposure to environmental stress was administered using a gentamicin challenge. S. aureus samples were grown from single colonies overnight in tryptic soy broth. After dilution to OD600 ⫽ 1.0, bacteria (⬃1 ⫻ 10∧6 CFU/mL) were incubated in tryptic soy broth containing 1.0-g/mL gentamicin. At one and seven days after inoculation, samples were serially diluted and plated. Plates were visually inspected for SCV formation, and the percentage of SCV to total colonies was determined in a blinded fashion by a single observer (K.C.M.).
Molecular Identification of Putative SCVs DNA was extracted from individual isolates by boiling and beadbeating clonal colonies in TEN buffer [10mM Tris-Cl (pH 8.0), 1mM EDTA, and 1% NP40 nonionic detergent] and approximately 250 mg of zirconium beads (0.1 mm; Biospec Products, Inc., Bartlesville, OK), using methods previously reported.23,24 DNA was amplified using
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Identification of S. aureus After incubation for 24 hours, mannitol and CNA plates were screened for colonies phenotypically consistent with S. aureus. When present, the identity of these presumed S. aureus isolates was confirmed in each sample by latex agglutination test (BBL Staphyloslide latex test; Becton Dickinson). In parallel with bacterial culture, DNA was extracted from the remaining Amies media using the UltraClean Fecal DNA Isolation kit (MoBio Laboratories, Carlsbad, CA) according to the manufacturer protocol. The extracted DNA was used as a template for quantitative polymerase chain reaction (PCR) with S. aureus-specific 16S rRNA gene primers23 and a 6-carboxyfluoresceinlabeled TaqMan Probe. PCR reactions (20 L total volume) included 10 L of Dynamo ColorFlash (Thermo-Fisher Inc) PCR mastermix, 0.2M each of forward, reverse, and TaqMan oligonucleotides and 2 L of purified DNA. The thermocycler conditions were as follows: 10 minutes at 95°C followed by 40 cycles of 15 seconds at 95°C and 1 minute at 60°C with a plate reading step after each cycle. Each quantitative PCR was run in triplicate.
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16S rRNA gene primers 27F and 907R.25 The PCR amplicons were then Sanger sequenced by the University of Colorado DNA Sequencing and Analysis Core. Paired-end sequences were assembled using the software phrap26 and assembled sequences compared with known sequences by web-based BLAST search of the NIH GenBank nonredundant database.
Student’s t-test was used to compare mean age, SNOT-22, endoscopy, and CT scores. The 2 test was used for culture-based comparison of control and CRS patient groups. Unpaired and paired t-tests were used to compare the SCV formation with gentamicin challenge. Statistical tests were performed using JMP Pro 10 (SAS Institute, Inc., Cary, NC), and significance was evaluated at ␣ ⫽ 0.05.
RESULTS Thirty-five middle meatus swabs were collected from 30 patients. Of these, 12 samples were from control patients, and 23 were obtained from 18 CRS patients. Five CRS patients were sampled either bilaterally when the two sides were markedly different in terms of clinical disease severity, or at separate clinical visits. The mean age of the cohort was 50.9 ⫾ 14.9 years, with an 11:24 ratio of males to females (no difference between groups). The mean SNOT-22, Lund-Mackay, and Lund-Kennedy scores were statistically higher in the CRS group and confirm the presence of disease in the CRS group when compared with controls (Table 1).
SCV and Bacterial Identification Positive control samples of S. aureus SCVs identified in the clinical microbiology lab were successfully cultured using the experimental culture methods. Phenotypically normal colonies of S. aureus and small colonies were cultured on mannitol salt and/or Columbia CNA agar plates from both control and disease cohorts. Phenotypically normal S. aureus was confirmed with PCR and latex agglutination, and frequency of isolation was not significantly different between CRS and control groups (65.2% versus 66.6% positive for CRS and controls, respectively; p ⫽ 0.8). Two candidate SCVs were cultured from CRS patient samples on S. aureus selective media and two from non-CRS patients (p ⫽ 0.267). To identify the bacterial species of the candidate SCVs, we performed 16S rRNA gene sequencing of PCR amplicons generated from cell lysates. By this method, two of the isolates were found to be members of the Actinomyces genus and one a species of Corynebacterium. The fourth bacterial colony could not be reliably assigned to a specific bacterial genus, because sequences of this isolate aligned with multiple bacterial genera but did not align with any portion of the S. aureus 16S rRNA gene, effectively defining this isolate as non-S. aureus. Thus, DNA se-
January–February 2015, Vol. 29, No. 1
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Table 1 Demographic characteristics of control and CRS patient populations
Number of samples Number of patients Mean age of patient (years) Females (%) Duration of symptoms (years) Number of smokers (%) Patients with polyps (%) Cystic fibrosis (%) Mean SNOT-22 score Mean Lund-Kennedy score Mean Lund-Mackay score Recent antibiotic use (less than 30 days) Current use of systemic steroids Culture positive S. aureus (%) Presence of SCVs (%)
Overall
CRS
Control
35 30 50.9 ⫾ 14.9 24 (68.6) N/A 7 (20.0) 12 (34.3) 4 (11.4%) 37.4 (32.4) 5 ⫾ 3.65 7.1 ⫾ 6.82 11 (31.4) 3 (8.57) 23 (65.7) 4 (17.4)
23 19 51.1 ⫾ 16.1 17 (73.9) 13.2 5 (21.7) 12 (52.2) 4 (17.4) 51.7 ⫾ 28.0 6.00 ⫾ 3.12 9.31 ⫾ 6.37 7 (30.4) 3 (13.0) 15 (65.2) 2 (8.69)
12 11 51.2 ⫾ 11.3 7 (58.3) N/A 2 (15.4) 0 0 4 ⫾ 4.97 0⫾0 0⫾0 4 (33.3) 0 8 (66.7) 2 (16.7)
p-Values for comparison of CRS to controls. Table 2 Characteristics of patients with samples that grew SCVs SCV Sample
1
2
CRS/control Cystic fibrosis Immunocompromised AERD Asthma Smoker Polyps SNOT-22 score Lund-Mackay score Recent antibiotic use Current systemic steroid use Previous sinonasal surgery Wild type S. aureus present Species of SCV
CRS N Y Y Y N Y 107 2 Y Y Y N Corynebacterium sp.
CRS N N N Y N Y 61 15 Y Y Y Y Actinomyces sp.
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quence-based classification demonstrated that these four potential SCVs, although grown on S. aureus selective media, were in fact fastidious non-S. aureus species.
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Disease Severity in Patients Colonized with SCVs Candidate SCVs were isolated from two CRS patients and two control patients (Table 2). None of these patients had cystic fibrosis, but one CRS patient had underlying immunodeficiency (agammaglobulinemia). Both CRS patients had asthma and polyps, one of whom met diagnostic criteria for aspirin-exacerbated respiratory disease, and both were taking antibiotics and systemic steroids at the time of sampling. The two CRS patients both had higher SNOT and CT scores than the average scores in the CRS cohort, but their endoscopy scores were less than average. One control patient was a current smoker. After a mean follow-up of 8.2 months, none of the four patients colonized with candidate SCVs reported continued sinus symptoms or required further use of antibiotics for sinus disease. This supports the likelihood that putative SCVs identified by traditional culture technique were likely fastidious species that grew as phenotypically small colonies on S. aureus selective media.
p-Value1
0.392 0.169
Y P 0.956
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0.008 0.005 0.027 0.766 ⬍0.001 0.883 0.267
3
4
Control N N N Y Y N 11 0 N N N Y Actinomyces sp.
Control N N N N N N 0 0 N N Y Y Unknown, not S. aureus
the intramucosal or intracellular environment and would revert to their wild-type extracellular phenotype when external stress is alleviated. If this were the case, colonies isolated from CRS patients should revert to the SCV phenotype under in vitro pharmacologic stress.14 To determine whether S. aureus samples from patients with CRS were more likely to form SCVs under stressed conditions, we used gentamicin to induce growth of SCVs in control and CRS sinonasal S. aureus isolates, as well as a commercially available S. aureus strain (ATCC 43300, Manassas, VA) as previously described.27 Six isolates from CRS patients and six from control subjects were evaluated. On day one, half of the control patients’ and all of the CRS patients’ S. aureus samples had at least some conversion to the SCV phenotype, but only one colony had more than 20% conversion on day one. By day seven, all of the sample and control strains had at least some conversion to SCVs, with both groups exhibiting greater than 50% conversion to SCV by day seven. Both groups showed potential to convert to phenotypic SCVs. However, CRS patients were not more likely than controls to form SCVs (Fig. 2). Both groups demonstrated higher SCV formation with longer duration of antibiotic exposure, although there was no significant intergroup difference.
Induction of SCV Growth With Gentamicin Neither CRS nor control patients grew S. aureus SCVs under routine conditions. A potential explanation for the negative results in the SCV screen is that S. aureus SCVs in CRS patients in vivo are protected in
American Journal of Rhinology & Allergy
DISCUSSION The demographics of our CRS patients were congruent with those reported in the literature in terms of age and sex.28 The prevalence of
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Figure 2. Induction of SCV formation by incubation with gentamicin. S. aureus from control patients (group one), CRS patients (group two), and an ATCC control were incubated with gentamicin (1 g/mL), and the conversion into SCVs is reported as a percent of the total colonies at day one and day seven (A and B). There was no difference between the three groups, but it was noted that there was significantly more SCV formation after a seven-day incubation than after a one-day incubation in both clinical groups (C).
S. aureus in our CRS patient population was also similar to the prevalence of S. aureus that our group has previously described using molecular detection methods in a larger cohort.1 However, our control patient population had a higher than previously reported frequency of S. aureus detection. This may be due to sample size, older age,29 recent or frequent antibiotic use, or tertiary care in the hospitalized setting. For the purposes of the current study, a control group of healthy patients with high S. aureus prevalence would be favorable to potentially detect a difference in SCV formation with disease state. We did not distinguish between polyp and nonpolyp CRS, because both populations have a high prevalence of S. aureus that is difficult to eradicate, and both would likely have been subjected to similar standard medical therapies (e.g., frequent antibiotics) over their course of disease. Despite rigorous screening, following established protocols for identification and culture of SCVs,14 we were unable to detect S. aureus SCVs in any member of our cohort of CRS and control patients. Furthermore, S. aureus isolates from CRS and control patients were equally susceptible to experimentally induced transformation from normal to SCV morphology. These findings suggest that the routine microbiologic laboratory screening for S. aureus SCVs is not warranted. However, there still remains a theoretical potential for therapeutic resistance in S. aureus-related rhinosinusitis due to the SCV phenomenon. We noted that longer antibiotic exposure increased S. aureus SCV formation in vitro, regardless of the origin of the strain. This implies that frequent and/or prolonged exposure to antibiotics, as is often the case in CRS patients, may elevate the frequency of SCV formation in vivo, as well. This correlates with results from previous studies of S. aureus SCVs from other anatomic sites, which suggest that antibiotic use induces SCV formation.13,14 We used aggressive middle meatal swabs with a bristled sampling brush to sample intramucosal bacteria as well as surface bacteria, but it is conceivable that the intracellular compartment may harbor different microbes, or may offer an environment suitable for SCV persistence.13 However, our results are supported by those of Niederfuhr et al.,30 who did not culture any SCVs in their 65 sinus lavage and tissue samples. Our results were also similar to those published by Kim et al.,12 in which two presumptive SCVs were found in ethmoid and anterior sphenoid tissue samples, but neither was S. aureus, as determined by PCR for the nuc gene. The theory of intracellular or intramucosal reservoirs of S. aureus suggests that bacterial repopulation could occur from such locations. We would expect that the ability to revert to SCV phenotypes would be preserved if this were the case but did not observe this phenomenon.
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Our study differed from previous reports in that we identified the bacterial genus of presumptive SCVs. The bacteria identified in this study as phenotypic SCVs were Corynebacter and Actinomyces spp. Their phenotypic small appearance likely resulted from growth under selective conditions that were less ideal for these species. Two presumptive SCVs were grown on mannitol agar, which is generally staphylococcal selective. However, these nonstaphylococcal species have some ability to survive and replicate in environments with high salt concentrations as small colonies and, therefore, may not be representative of colony phenotype in vivo. The other two colonies were grown on CNA agar, which is selective for gram-positive bacteria (including Corynebacter and Actinomyces). After subbactericidal aminoglycoside exposure, S. aureus from CRS patients was able to form SCVs but not more frequently than isolates from healthy subjects. Although some individual variability was observed, these results provide evidence that even in dynamic growth conditions with selective pressure, S. aureus strains that are involved in CRS are not more likely to form SCVs than S. aureus that colonizes the sinonasal mucosa of healthy controls. However, as we know, patients with CRS symptoms are more likely to receive frequent antibiotics,31–33 and the local dose to the sinuses may be subtherapeutic.33,34 Consequently, there may still be a potential physiologic role for antibioticinduced formation of S. aureus SCVs in human disease.35,36 Intramucosal microcolonies of S. aureus have been reported in both control and CRS patients and could in theory be missed even with aggressive surface swabs using a bristled brush, such as the Copan ESwab used in the current study.7,16,37 Consequently, it is tempting to conclude that intramucosal microcolonies isolated from within nasal epithelial cells are SCVs.10,12 Although these colonies have been imaged with fluorescence microscopy, their phenotypic colony appearance has not yet been determined. Wood et al.16 used fluorescence microscopy to study ethmoid and sphenoid mucosa in CRS (some known to be colonized with S. aureus and some not) and control patients undergoing endoscopic sinus surgery. They reported 10/18 CRS patients studied were colonized with subepithelial S. aureus.16 If over half of CRS patients harbor intramucosal SCVs, we would expect to have identified at least an occasional S. aureus SCV in this study. Much of the literature linking S. aureus SCVs with disease severity has come from the cystic fibrosis population. SCVs have been cultured in lower airways disease in patients with cystic fibrosis, and their presence has been correlated with lung disease severity.15,18 The prevalence of SCVs in CF S. aureus carriers is reported to be as high as 17% on analysis of sputum cultures.18 We intentionally included patients in our CRS group with cystic fibrosis, but no phenotypic
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5.
Table 3 Severity of disease in CRS patients with and without S. aureus colonization Patient characteristics
S. aureus ⴙ
S. aureus ⴚ
p-Value
Number of samples Number of patients Mean age of patient (years) Females (%) Number of smokers (%) Cystic fibrosis (%) Immunocompromise (%) Severity of disease Duration of symptoms (years) Patients with polyps (%) Samter’s triad Constant symptoms Mean SNOT-22 score Mean Lund-Kennedy score Mean Lund-Mackay score Treatments Recent antibiotic use (less than 30 days) Current use of systemic steroids Current use of nasal steroids Previous surgery Presence of presumptive SCVs (%)
15 13 53.1 ⫾ 16.1 9 (60.0) 3 (20.0) 2 (13.3) 2 (13.3)
7 6 51.7 ⫾ 13.6 6 (85.7) 3 (42.9) 1 (14.3) 2 (28.6)
0.952 0.015 0.061 0.915 0.160
13.3 ⫾ 8.8 7 (46.7) 1 (6.67) 8 (53.3) 37.3 ⫾ 16.5 5.6 ⫾ 3.0 9.3 ⫾ 6.1
13.9 ⫾ 8.51 5 (71.4) 2 (28.6) 5 (71.4) 78.0 ⫾ 20.6 5.4 ⫾ 3.6 8.6 ⫾ 7.3
0.045 0.032 0.139 0.015 0.879 0.736
6 (40.0)
2 (28.6)
0.343
1 (6.67)
3 (42.9)
0.002
8 (53.3) 11 (73.3)
5 (71.4) 4 (57.1)
0.139 0.193
1 (6.67)
1 (14.3)
0.354
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CONCLUSION
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0.892
T
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SCVs were cultured from these patients. Because only two CF patients were colonized with S. aureus, it is possible that SCVs would be identified in CF sinuses if additional CF S. aureus positive patients were sampled. Overall, in our tertiary care population with advanced CRS and a high rate of S. aureus presence, we did not identify any phenotypical S. aureus SCVs, nor did we observe that S. aureus from CRS patients were more likely to convert to the small colony size with in vitro antibiotic exposure.
Although it is plausible that S. aureus SCVs are one mechanism of S. aureus persistence after treatment, based on the results above, we conclude that further evidence is necessary to implicate S. aureus SCVs as a source of recurrent disease in CRS. This study, along with others,12,30 did not identify a single patient colonized with S. aureus SCVs, despite a large population of wild-type S. aureus-colonized patients. Future study of the role of S. aureus SCVs may require more sophisticated techniques to evaluate intracellular bacteria, or may seek to pursue clinical and phenotypic alterations of host mucosa and normal flora from prolonged or repeated antibiotic administration (Table 3).
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29.
Ramakrishnan VR, Feazel LM, Gitomer SA, et al. The microbiome of the middle meatus in healthy adults. PLoS One 8:e85507, 2013. 30. Niederfuhr A, Kirsche H, Deutschle T, et al. Staphylococcus aureus in nasal lavage and biopsy of patients with chronic rhinosinusitis. Allergy 63:1359–1367, 2008. 31. Piromchai P, Thanaviratananich S, and Laopaiboon M. Systemic antibiotics for chronic rhinosinusitis without nasal polyps in adults. Cochrane Database Syst Rev (5):CD008233, 2011. 32. Suh JD, and Kennedy DW. Treatment options for chronic rhinosinusitis. Proc Am Thor Soc 1:132–140, 2011. 33. Adelson RT, and Adappa ND. What is the proper role of oral antibiotics in the treatment of patients with chronic sinusitis? Curr Op Otolaryngol Head Neck Surg 1:61–68, 2013.
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Antunes MB, Feldman MD, Cohen NA, and Chiu AG. Dose-dependent effects of topical tobramycin in an animal model of Pseudomonas sinusitis. Am J Rhinol 21:423–427, 2007. 35. von Eiff C, Bettin D, Proctor RA, et al. Recovery of small colony variants of Streptococcus aureus following gentamicin bead placement for osteomyelitis. Clin Infect Dis 5:1250–1251, 1997. 36. Balwit JM, van Langevelde P, Vann JM, and Proctor RA. Gentamicinresistant menadione and hemin auxotrophic Staphylococcus aureus persist within cultured endothelial cells. J Infect Dis 4:1033–1037, 1994. 37. Sachse F, Becker K, Von Eiff C, et al. Staphylococcus aureus invades the epithelium in nasal polyposis and induces IL-6 in nasal epithelial cells in vitro. Allergy 65:1430–1437, 2010. e
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ABSTRACT
Background: Small colony variants (SCVs) are a metabolically inactive form of bacteria that can be difficult to eradicate. To examine whether SCVs contribute to Staphylococcus aureus persistence in chronic rhinosinusitis (CRS), we compared the prevalence of S. aureus SCVs in CRS patients and healthy controls. Methods: Endoscopically guided middle meatus samples were collected from 23 CRS patients and 12 controls. Samples were cultured and screened for the presence of phenotypically small colonies. Candidate SCV isolates were classified by 16S rRNA gene sequencing. To further characterize the capacity of S. aureus isolates to form SCVs when stressed, colonies underwent a gentamicin exposure assay. Results: Among CRS patient samples, 15 were culture positive for S. aureus (65.2%), and of those, two grew putative SCVs on selective media (8.7%). However, neither was genetically confirmed to be S. aureus upon sequencing. In healthy controls, eight specimens were culture positive for S. aureus (66.7%), and of these, two grew putative S. aureus SCVs on selective media (16.7%); but again, neither was confirmed to be S. aureus by 16S analysis. None of the four patients colonized with SCVs had evidence of sinonasal disease at a mean follow-up of eight months. S. aureus isolates from CRS patients and controls were equally likely to form SCVs with gentamicin exposure. Conclusion: S. aureus SCVs were not associated with CRS in the current study. Their role in refractory CRS remains theoretical, and further research is warranted to determine whether S. aureus SCVs may reside in the intracellular compartment. (Am J Rhinol Allergy 29, 29 –34, 2015; doi: 10.2500/ajra.2015.29.4133)
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hronic rhinosinusitis (CRS) is a common and burdensome disease process, in which infection or colonization with specific bacterial pathogens is thought to play a role in at least a subset of patients. Both circumstantial and direct evidence has increasingly implicated Staphylococcus aureus in the pathogenesis of many CRS patients, including both those with and without nasal polyps. S. aureus is the most commonly detected bacterium in sinonasal samples of CRS patients and is more abundant in CRS patients than in controls.1–3 In CRS, S. aureus is not only common but also has been linked to both disease severity and recurrence of disease after aggressive medical and surgical therapies.3,4 For reasons that are not clearly understood, S. aureus is particularly challenging to eradicate using standard treatments.4 The role of biofilms in CRS has been explored as a potential mechanism for S. aureus recalcitrance,2,5,6 but biofilms alone do not fully explain persistent colonization, because patients who are S. aureus positive and biofilm negative often continue to have symptoms despite appropriate treatment. Another proposed mechanism is the presence and persistence of intramucosal and even intracellular S. aureus, which have been identified in patients with CRS.7 Like biofilms, intracellular S. aureus potentially evades the host immune response and antibiotic treatment, thereby persisting despite otherwise adequate treatment. This phenomenon has been demonstrated in other diseases, such as cystic fibrosis and endocarditis, in addition to preliminary findings in CRS.7–10 Intracellular persistence of S. aureus within the sinonasal
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Department of Otolaryngology, 2Division of Infectious Diseases, and 4Division of Pulmonary Medicine, University of Colorado School of Medicine, Aurora, Colorado, 3 National Jewish Health, Division of Pulmonary Medicine, Denver, Colorado, and 5 University of Colorado Microbiome Research Consortium, Denver, Colorado Funded by Department of Otolaryngology and Division of Infectious Diseases, University of Colorado School of Medicine Presented at the American Society of Microbiology Meeting in Denver, Colorado, May 2013 The authors have no conflicts of interest to declare pertaining to this article Address correspondence to Vijay R. Ramakrishnan, M.D., Department of Otolaryngology, 12631 East 17th Avenue, B205, Aurora, CO 80045 E-mail address: [email protected] Copyright © 2015, OceanSide Publications, Inc., U.S.A.
American Journal of Rhinology & Allergy
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epithelium is reported to be a risk factor for recurrent CRS symptoms that are refractory to traditional therapies.11,12 The presence of, or ability to convert to, small colony variants (SCVs) may facilitate persistence of S. aureus in CRS by behavioral characteristics similar to biofilms.11,13 Like biofilms, SCV bacteria are metabolically inactive, slow-growing variants that can develop and persist when the extracellular environment is stressed.13,14 Environmental stress created by antibiotic exposure can induce conversion of S. aureus from morphologically normal variants to SCVs.14–17 Many auxotrophic variants of SCVs have been identified, most commonly in persistent and recurrent infections in cystic fibrosis lung disease and joint infections.15,18 These bacteria are able to revert to the wild-type phenotype and live extracellularly, presumably when external stress is alleviated, which may explain the delayed recurrence of S. aureusmediated CRS after initial treatment success. Therefore, in this prospective cross sectional study, we tested the hypothesis that S. aureus SCV presence is more frequent in CRS given the species’ association with disease presence, recalcitrance, and severity.
MATERIALS AND METHODS This study was approved by the Institutional Review Board of the University of Colorado (COMIRB protocol 11-1442), and informed consent was obtained from all patients.
Patient Selection Adult patients were recruited from a tertiary care rhinology practice from July 2012–September 2012. CRS was defined according to 2007 American Academy of Otolaryngology-Head and Neck Surgery Task Force Criteria:19 healthy control patients undergoing endoscopic orbital or pituitary surgery were recruited when no symptomatic sinonasal disease was noted on history, and normal appearance was confirmed on both nasal endoscopy and sinus computed tomography (CT). Patient demographics, disease history, and treatment information were recorded. Symptom severity was assessed using the SinoNasal Outcomes Test (SNOT)-22 questionnaire,20 severity of clinically apparent inflammation on endoscopic examination was graded using the Lund-Kennedy scale,21 and CT imaging severity of radiographic disease was graded using the Lund-Mackay scoring system.22
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Sample Collection and Bacterial Culture Nylon Liquid Amies Elution Swabs (ESwabs) (Becton Dickinson and Company, Franklin Lakes, NJ) were used to aggressively swab the middle meatus epithelial surface under endoscopic guidance, taking care of not to contaminate the swab by contacting the anterior nasal passage. Samples were immediately transported to the laboratory, where they were vortexed for 60 seconds in the Amies liquid, and 100 and 10 L of liquid were plated in duplicate onto mannitol salt agar and Columbia naladixic acid (CNA) agar, respectively. At the same time, 10 L of undiluted and a 100 L of 1:100 dilution of Amies liquid were plated onto blood agar, based on the optimal amount to grow individual, countable colonies on each type of plate. Bacteria were cultured following recommended culture conditions for the growth of S. aureus SCVs.14 All plates were incubated under aerobic conditions without carbon dioxide at 37°C for 72 hours total, with growth evaluated every 24 hours. Plates were screened for growth of phenotypically small colonies, defined as colonies that were not visible within the first 24 hours of incubation but that appeared between 24 and 72 hours of incubation and remained phenotypically small (Fig. 1). The presence or absence of small colonies was assessed on specific media (mannitol salt and Columbia CNA), and individual colonies were then streaked onto blood agar. Colonies that remained small when grown for 48 hours were identified as SCVs.14 S. aureus SCV positive control samples were obtained from the National Jewish Health (Denver, CO) clinical microbiology laboratory, defined in a similar fashion, and isolated under the same conditions as the middle meatus samples.
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Figure 1. Small colony variants compared with normal colonies. (A) Blood agar plate growing small colony variants at day three (arrow). These colonies were not visible after a 24-hour incubation. (B) Columbia CNA plate growing wild-type S. aureus, with normal phenotypic colonies.
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Gentamicin Challenge
To determine whether bacterial colonies of normal morphology would be likely to convert to an SCV phenotype, a model of exposure to environmental stress was administered using a gentamicin challenge. S. aureus samples were grown from single colonies overnight in tryptic soy broth. After dilution to OD600 ⫽ 1.0, bacteria (⬃1 ⫻ 10∧6 CFU/mL) were incubated in tryptic soy broth containing 1.0-g/mL gentamicin. At one and seven days after inoculation, samples were serially diluted and plated. Plates were visually inspected for SCV formation, and the percentage of SCV to total colonies was determined in a blinded fashion by a single observer (K.C.M.).
Molecular Identification of Putative SCVs DNA was extracted from individual isolates by boiling and beadbeating clonal colonies in TEN buffer [10mM Tris-Cl (pH 8.0), 1mM EDTA, and 1% NP40 nonionic detergent] and approximately 250 mg of zirconium beads (0.1 mm; Biospec Products, Inc., Bartlesville, OK), using methods previously reported.23,24 DNA was amplified using
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Identification of S. aureus After incubation for 24 hours, mannitol and CNA plates were screened for colonies phenotypically consistent with S. aureus. When present, the identity of these presumed S. aureus isolates was confirmed in each sample by latex agglutination test (BBL Staphyloslide latex test; Becton Dickinson). In parallel with bacterial culture, DNA was extracted from the remaining Amies media using the UltraClean Fecal DNA Isolation kit (MoBio Laboratories, Carlsbad, CA) according to the manufacturer protocol. The extracted DNA was used as a template for quantitative polymerase chain reaction (PCR) with S. aureus-specific 16S rRNA gene primers23 and a 6-carboxyfluoresceinlabeled TaqMan Probe. PCR reactions (20 L total volume) included 10 L of Dynamo ColorFlash (Thermo-Fisher Inc) PCR mastermix, 0.2M each of forward, reverse, and TaqMan oligonucleotides and 2 L of purified DNA. The thermocycler conditions were as follows: 10 minutes at 95°C followed by 40 cycles of 15 seconds at 95°C and 1 minute at 60°C with a plate reading step after each cycle. Each quantitative PCR was run in triplicate.
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16S rRNA gene primers 27F and 907R.25 The PCR amplicons were then Sanger sequenced by the University of Colorado DNA Sequencing and Analysis Core. Paired-end sequences were assembled using the software phrap26 and assembled sequences compared with known sequences by web-based BLAST search of the NIH GenBank nonredundant database.
Student’s t-test was used to compare mean age, SNOT-22, endoscopy, and CT scores. The 2 test was used for culture-based comparison of control and CRS patient groups. Unpaired and paired t-tests were used to compare the SCV formation with gentamicin challenge. Statistical tests were performed using JMP Pro 10 (SAS Institute, Inc., Cary, NC), and significance was evaluated at ␣ ⫽ 0.05.
RESULTS Thirty-five middle meatus swabs were collected from 30 patients. Of these, 12 samples were from control patients, and 23 were obtained from 18 CRS patients. Five CRS patients were sampled either bilaterally when the two sides were markedly different in terms of clinical disease severity, or at separate clinical visits. The mean age of the cohort was 50.9 ⫾ 14.9 years, with an 11:24 ratio of males to females (no difference between groups). The mean SNOT-22, Lund-Mackay, and Lund-Kennedy scores were statistically higher in the CRS group and confirm the presence of disease in the CRS group when compared with controls (Table 1).
SCV and Bacterial Identification Positive control samples of S. aureus SCVs identified in the clinical microbiology lab were successfully cultured using the experimental culture methods. Phenotypically normal colonies of S. aureus and small colonies were cultured on mannitol salt and/or Columbia CNA agar plates from both control and disease cohorts. Phenotypically normal S. aureus was confirmed with PCR and latex agglutination, and frequency of isolation was not significantly different between CRS and control groups (65.2% versus 66.6% positive for CRS and controls, respectively; p ⫽ 0.8). Two candidate SCVs were cultured from CRS patient samples on S. aureus selective media and two from non-CRS patients (p ⫽ 0.267). To identify the bacterial species of the candidate SCVs, we performed 16S rRNA gene sequencing of PCR amplicons generated from cell lysates. By this method, two of the isolates were found to be members of the Actinomyces genus and one a species of Corynebacterium. The fourth bacterial colony could not be reliably assigned to a specific bacterial genus, because sequences of this isolate aligned with multiple bacterial genera but did not align with any portion of the S. aureus 16S rRNA gene, effectively defining this isolate as non-S. aureus. Thus, DNA se-
January–February 2015, Vol. 29, No. 1
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Table 1 Demographic characteristics of control and CRS patient populations
Number of samples Number of patients Mean age of patient (years) Females (%) Duration of symptoms (years) Number of smokers (%) Patients with polyps (%) Cystic fibrosis (%) Mean SNOT-22 score Mean Lund-Kennedy score Mean Lund-Mackay score Recent antibiotic use (less than 30 days) Current use of systemic steroids Culture positive S. aureus (%) Presence of SCVs (%)
Overall
CRS
Control
35 30 50.9 ⫾ 14.9 24 (68.6) N/A 7 (20.0) 12 (34.3) 4 (11.4%) 37.4 (32.4) 5 ⫾ 3.65 7.1 ⫾ 6.82 11 (31.4) 3 (8.57) 23 (65.7) 4 (17.4)
23 19 51.1 ⫾ 16.1 17 (73.9) 13.2 5 (21.7) 12 (52.2) 4 (17.4) 51.7 ⫾ 28.0 6.00 ⫾ 3.12 9.31 ⫾ 6.37 7 (30.4) 3 (13.0) 15 (65.2) 2 (8.69)
12 11 51.2 ⫾ 11.3 7 (58.3) N/A 2 (15.4) 0 0 4 ⫾ 4.97 0⫾0 0⫾0 4 (33.3) 0 8 (66.7) 2 (16.7)
p-Values for comparison of CRS to controls. Table 2 Characteristics of patients with samples that grew SCVs SCV Sample
1
2
CRS/control Cystic fibrosis Immunocompromised AERD Asthma Smoker Polyps SNOT-22 score Lund-Mackay score Recent antibiotic use Current systemic steroid use Previous sinonasal surgery Wild type S. aureus present Species of SCV
CRS N Y Y Y N Y 107 2 Y Y Y N Corynebacterium sp.
CRS N N N Y N Y 61 15 Y Y Y Y Actinomyces sp.
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quence-based classification demonstrated that these four potential SCVs, although grown on S. aureus selective media, were in fact fastidious non-S. aureus species.
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Disease Severity in Patients Colonized with SCVs Candidate SCVs were isolated from two CRS patients and two control patients (Table 2). None of these patients had cystic fibrosis, but one CRS patient had underlying immunodeficiency (agammaglobulinemia). Both CRS patients had asthma and polyps, one of whom met diagnostic criteria for aspirin-exacerbated respiratory disease, and both were taking antibiotics and systemic steroids at the time of sampling. The two CRS patients both had higher SNOT and CT scores than the average scores in the CRS cohort, but their endoscopy scores were less than average. One control patient was a current smoker. After a mean follow-up of 8.2 months, none of the four patients colonized with candidate SCVs reported continued sinus symptoms or required further use of antibiotics for sinus disease. This supports the likelihood that putative SCVs identified by traditional culture technique were likely fastidious species that grew as phenotypically small colonies on S. aureus selective media.
p-Value1
0.392 0.169
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0.008 0.005 0.027 0.766 ⬍0.001 0.883 0.267
3
4
Control N N N Y Y N 11 0 N N N Y Actinomyces sp.
Control N N N N N N 0 0 N N Y Y Unknown, not S. aureus
the intramucosal or intracellular environment and would revert to their wild-type extracellular phenotype when external stress is alleviated. If this were the case, colonies isolated from CRS patients should revert to the SCV phenotype under in vitro pharmacologic stress.14 To determine whether S. aureus samples from patients with CRS were more likely to form SCVs under stressed conditions, we used gentamicin to induce growth of SCVs in control and CRS sinonasal S. aureus isolates, as well as a commercially available S. aureus strain (ATCC 43300, Manassas, VA) as previously described.27 Six isolates from CRS patients and six from control subjects were evaluated. On day one, half of the control patients’ and all of the CRS patients’ S. aureus samples had at least some conversion to the SCV phenotype, but only one colony had more than 20% conversion on day one. By day seven, all of the sample and control strains had at least some conversion to SCVs, with both groups exhibiting greater than 50% conversion to SCV by day seven. Both groups showed potential to convert to phenotypic SCVs. However, CRS patients were not more likely than controls to form SCVs (Fig. 2). Both groups demonstrated higher SCV formation with longer duration of antibiotic exposure, although there was no significant intergroup difference.
Induction of SCV Growth With Gentamicin Neither CRS nor control patients grew S. aureus SCVs under routine conditions. A potential explanation for the negative results in the SCV screen is that S. aureus SCVs in CRS patients in vivo are protected in
American Journal of Rhinology & Allergy
DISCUSSION The demographics of our CRS patients were congruent with those reported in the literature in terms of age and sex.28 The prevalence of
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Figure 2. Induction of SCV formation by incubation with gentamicin. S. aureus from control patients (group one), CRS patients (group two), and an ATCC control were incubated with gentamicin (1 g/mL), and the conversion into SCVs is reported as a percent of the total colonies at day one and day seven (A and B). There was no difference between the three groups, but it was noted that there was significantly more SCV formation after a seven-day incubation than after a one-day incubation in both clinical groups (C).
S. aureus in our CRS patient population was also similar to the prevalence of S. aureus that our group has previously described using molecular detection methods in a larger cohort.1 However, our control patient population had a higher than previously reported frequency of S. aureus detection. This may be due to sample size, older age,29 recent or frequent antibiotic use, or tertiary care in the hospitalized setting. For the purposes of the current study, a control group of healthy patients with high S. aureus prevalence would be favorable to potentially detect a difference in SCV formation with disease state. We did not distinguish between polyp and nonpolyp CRS, because both populations have a high prevalence of S. aureus that is difficult to eradicate, and both would likely have been subjected to similar standard medical therapies (e.g., frequent antibiotics) over their course of disease. Despite rigorous screening, following established protocols for identification and culture of SCVs,14 we were unable to detect S. aureus SCVs in any member of our cohort of CRS and control patients. Furthermore, S. aureus isolates from CRS and control patients were equally susceptible to experimentally induced transformation from normal to SCV morphology. These findings suggest that the routine microbiologic laboratory screening for S. aureus SCVs is not warranted. However, there still remains a theoretical potential for therapeutic resistance in S. aureus-related rhinosinusitis due to the SCV phenomenon. We noted that longer antibiotic exposure increased S. aureus SCV formation in vitro, regardless of the origin of the strain. This implies that frequent and/or prolonged exposure to antibiotics, as is often the case in CRS patients, may elevate the frequency of SCV formation in vivo, as well. This correlates with results from previous studies of S. aureus SCVs from other anatomic sites, which suggest that antibiotic use induces SCV formation.13,14 We used aggressive middle meatal swabs with a bristled sampling brush to sample intramucosal bacteria as well as surface bacteria, but it is conceivable that the intracellular compartment may harbor different microbes, or may offer an environment suitable for SCV persistence.13 However, our results are supported by those of Niederfuhr et al.,30 who did not culture any SCVs in their 65 sinus lavage and tissue samples. Our results were also similar to those published by Kim et al.,12 in which two presumptive SCVs were found in ethmoid and anterior sphenoid tissue samples, but neither was S. aureus, as determined by PCR for the nuc gene. The theory of intracellular or intramucosal reservoirs of S. aureus suggests that bacterial repopulation could occur from such locations. We would expect that the ability to revert to SCV phenotypes would be preserved if this were the case but did not observe this phenomenon.
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Our study differed from previous reports in that we identified the bacterial genus of presumptive SCVs. The bacteria identified in this study as phenotypic SCVs were Corynebacter and Actinomyces spp. Their phenotypic small appearance likely resulted from growth under selective conditions that were less ideal for these species. Two presumptive SCVs were grown on mannitol agar, which is generally staphylococcal selective. However, these nonstaphylococcal species have some ability to survive and replicate in environments with high salt concentrations as small colonies and, therefore, may not be representative of colony phenotype in vivo. The other two colonies were grown on CNA agar, which is selective for gram-positive bacteria (including Corynebacter and Actinomyces). After subbactericidal aminoglycoside exposure, S. aureus from CRS patients was able to form SCVs but not more frequently than isolates from healthy subjects. Although some individual variability was observed, these results provide evidence that even in dynamic growth conditions with selective pressure, S. aureus strains that are involved in CRS are not more likely to form SCVs than S. aureus that colonizes the sinonasal mucosa of healthy controls. However, as we know, patients with CRS symptoms are more likely to receive frequent antibiotics,31–33 and the local dose to the sinuses may be subtherapeutic.33,34 Consequently, there may still be a potential physiologic role for antibioticinduced formation of S. aureus SCVs in human disease.35,36 Intramucosal microcolonies of S. aureus have been reported in both control and CRS patients and could in theory be missed even with aggressive surface swabs using a bristled brush, such as the Copan ESwab used in the current study.7,16,37 Consequently, it is tempting to conclude that intramucosal microcolonies isolated from within nasal epithelial cells are SCVs.10,12 Although these colonies have been imaged with fluorescence microscopy, their phenotypic colony appearance has not yet been determined. Wood et al.16 used fluorescence microscopy to study ethmoid and sphenoid mucosa in CRS (some known to be colonized with S. aureus and some not) and control patients undergoing endoscopic sinus surgery. They reported 10/18 CRS patients studied were colonized with subepithelial S. aureus.16 If over half of CRS patients harbor intramucosal SCVs, we would expect to have identified at least an occasional S. aureus SCV in this study. Much of the literature linking S. aureus SCVs with disease severity has come from the cystic fibrosis population. SCVs have been cultured in lower airways disease in patients with cystic fibrosis, and their presence has been correlated with lung disease severity.15,18 The prevalence of SCVs in CF S. aureus carriers is reported to be as high as 17% on analysis of sputum cultures.18 We intentionally included patients in our CRS group with cystic fibrosis, but no phenotypic
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5.
Table 3 Severity of disease in CRS patients with and without S. aureus colonization Patient characteristics
S. aureus ⴙ
S. aureus ⴚ
p-Value
Number of samples Number of patients Mean age of patient (years) Females (%) Number of smokers (%) Cystic fibrosis (%) Immunocompromise (%) Severity of disease Duration of symptoms (years) Patients with polyps (%) Samter’s triad Constant symptoms Mean SNOT-22 score Mean Lund-Kennedy score Mean Lund-Mackay score Treatments Recent antibiotic use (less than 30 days) Current use of systemic steroids Current use of nasal steroids Previous surgery Presence of presumptive SCVs (%)
15 13 53.1 ⫾ 16.1 9 (60.0) 3 (20.0) 2 (13.3) 2 (13.3)
7 6 51.7 ⫾ 13.6 6 (85.7) 3 (42.9) 1 (14.3) 2 (28.6)
0.952 0.015 0.061 0.915 0.160
13.3 ⫾ 8.8 7 (46.7) 1 (6.67) 8 (53.3) 37.3 ⫾ 16.5 5.6 ⫾ 3.0 9.3 ⫾ 6.1
13.9 ⫾ 8.51 5 (71.4) 2 (28.6) 5 (71.4) 78.0 ⫾ 20.6 5.4 ⫾ 3.6 8.6 ⫾ 7.3
0.045 0.032 0.139 0.015 0.879 0.736
6 (40.0)
2 (28.6)
0.343
1 (6.67)
3 (42.9)
0.002
8 (53.3) 11 (73.3)
5 (71.4) 4 (57.1)
0.139 0.193
1 (6.67)
1 (14.3)
0.354
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CONCLUSION
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0.892
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SCVs were cultured from these patients. Because only two CF patients were colonized with S. aureus, it is possible that SCVs would be identified in CF sinuses if additional CF S. aureus positive patients were sampled. Overall, in our tertiary care population with advanced CRS and a high rate of S. aureus presence, we did not identify any phenotypical S. aureus SCVs, nor did we observe that S. aureus from CRS patients were more likely to convert to the small colony size with in vitro antibiotic exposure.
Although it is plausible that S. aureus SCVs are one mechanism of S. aureus persistence after treatment, based on the results above, we conclude that further evidence is necessary to implicate S. aureus SCVs as a source of recurrent disease in CRS. This study, along with others,12,30 did not identify a single patient colonized with S. aureus SCVs, despite a large population of wild-type S. aureus-colonized patients. Future study of the role of S. aureus SCVs may require more sophisticated techniques to evaluate intracellular bacteria, or may seek to pursue clinical and phenotypic alterations of host mucosa and normal flora from prolonged or repeated antibiotic administration (Table 3).
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