2016 Section 5 Green Book

Bacterial repopulation after surgery & antibiotics

reservoirs within paranasal sinus biofilms, in intramucosal

sites, or within the nasopharynx.

8–10

Up to 75% of post-

ESS infections may be from bacterial isolates not present

at the time of surgery, suggesting that opportunistic de

novo bacteria are responsible for recalcitrant CRS, impli-

cating patient-specific alterations in immunity.

However,

it has also been hypothesized that bacterial repopulation

may arise from an extranasal source, including introduc-

tion from the skin or direct inoculation from water irriga-

tion bottles.

9,12

The goals of this study were to examine

changes in the sinus microbiome following ESS and initial

postoperative medical therapies in order to identify poten-

tial sources for postsurgical microbial repopulation.

Patients and methods

Study design and population

This cross-sectional study was approved by the Institutional

Review Board of the University of Colorado (COMIRB

protocol number 11–1442). The diagnosis of CRS was

made according to the 2007 Adult Sinusitis Guidelines,

and accordingly, CRS patients were initially managed med-

ically with a minimum trial of saline rinses, oral antibiotics,

and topical intranasal steroids.

Those with continued ev-

idence of disease who elected to undergo functional endo-

scopic sinus surgery (FESS)

were enrolled in the study.

The extent of surgery was determined by extent of dis-

ease and ranged from maxillary antrostomy with anterior

ethmoidectomy to complete bilateral surgery addressing

all paranasal sinuses. Meticulous mucosal-sparing surgi-

cal technique was used. Patients less than 18 years of age,

with antibiotic use within 1 month of surgery (systemic

or topical), or with cystic fibrosis, immunodeficiency, or

autoimmune diseases were excluded from the study. Post-

operatively, patients were routinely placed on sinus irriga-

tions and a 2-week course of oral antibiotics (amoxicillin-

clavulanate, or clarithromycin if penicillin-allergic). Bacte-

rial load was hypothesized to be the lowest after treatment

(FESS with irrigation at completion of surgery,

followed

by 2 weeks of postoperative antibiotics and saline rinses),

and then bacterial repopulation was expected to subse-

quently occur in the following weeks. As such, we com-

pared microbiota present at the time of surgery, 2 weeks

postoperatively, and 6 weeks postoperatively.

Sample collection

Subjects were recruited between November 2013 and

May 2014. All swabs were collected using CultureSwabs

(BD, Franklin Lakes, NJ), rotating at least 5 full turns

until fully saturated. Samples were collected at surgery

or in clinic during the 2-week and 6-week postoperative

appointments. At surgery, swabs were endoscopically

guided to the anterior nares and nasopharynx and, once

open, into the ethmoid sinus, with care taken to avoid

contamination by neighboring sites. At postoperative

visits, swabs were endoscopically guided to the ethmoid

cavity and sampled in an identical fashion. CultureSwabs

for DNA extraction were placed on ice upon collection

and frozen at

−

C until DNA extraction.

16S amplicon library construction

DNA was extracted from all samples using the

UltraClean

DNA Isolation Kit (Mo Bio, Carlsbad, CA).

Bacterial profiles were determined by broad-range amplifi-

cation and sequence analysis of 16S rRNA genes following

previously described methods.

16,17

In brief, amplicons were

generated using primers that target approximately 340 base

pairs (bp) of the V1V2 variable region of the 16S rRNA

gene (primers 27FYM [Frank et al.

] and 338R,

modi-

fied by adding dual indexes and Illumina adapter sequences;

Illumina, San Diego, CA, USA). Illumina paired-end se-

quencing was performed on the MiSeq platform with ver-

sion v2.3.0.8 of the MiSeq Control Software and version

v2.3.32 of MiSeq Reporter, using a 600-cycle version 3

reagent kit (all from Illumina).

As described,

Illumina MiSeq paired-end se-

quences were sorted by sample via barcodes in the

paired reads with a Python script (Python Software

Foundation;

https://www.python.org

The sorted

paired reads were merged using the phrap assembly

software.

20,21

Potential chimeras identified with Uchime

(usearch6.0.203_i86linux32)

using the Schloss and

Westcott

Silva reference sequences were removed from

subsequent analyses. This process generated 11,989,194

high-quality sequences for 69 samples (2 anterior nares

failed to amplify with 16S primers), with a median of

105,889 sequences/sample (IQR, 45,883 to 259,155). As-

sembled sequences were aligned and classified with SINA

(1.2.11)

using the 418,497 bacterial sequences in Silva

115NR99 (Quast et al.

) as reference configured to yield

the Silva taxonomy. Operational taxonomic units (OTUs)

were produced by clustering sequences with identical

taxonomic assignments. Relative abundances of OTUs

were calculated for each subject by dividing the sequence

counts observed for each OTU by the total number of

high-quality bacterial 16S rRNA sequences generated for

the subject. All sequence libraries had Good’s coverage

scores 99% at the rarefaction point of 15,000 sequences,

indicating that sequence coverage was excellent.

Statistical analysis

Similarity between the microbiomes of pairs of patient sam-

ples (ie, beta-diversity) was measured using the abundance-

based Morisita-Horn index (using the “vegdist” R com-

mand). Similarity/dissimilarity in community composition

(ie, beta-diversity) was assessed using a permutation-based

multiple analysis of variance (PERMANOVA) test, imple-

mented by the adonis function of the vegan R package.

Values of

were generated using 5000 label permutations,

with beta-diversity measured using the Morisita-Horn in-

dex. The R (v3.0.3,

http://cran.r-project.org)

and Explicet

(v2.9.4,

http://www.explicet.org)

software packages were

used for data display, analysis, and figure generation.

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