Microbiological methods

2.4.1 Bacterial strains and culture techniques

2.4.1.1 Culture of tracheoesophageal speech valve isolates

Micro-organisms liberated from the surface of used TESVs (see 2.10.1) were cultured on Sabouraud Dextrose Agar (Oxoid) and Nutrient Agar (Oxoid). Plates were incubated aerobically at 37°C and checked for growth every 24 h for at least 7 days. Sabouraud Dextrose Agar was used as a medium on which to grow fungi, including Candida spp., that often foul TESVs. Nutrient Agar was used as a non-selective medium for the culture of both bacteria and fungi.

2.4.1.2 Isolation and culture of chronic rhinosinusitis micro-organisms

A variety of growth media were employed for the isolation and routine culture of CRS isolates. Blood agar contained 37 g/L Brain Heart Infusion (Oxoid), 5 g/L Yeast Extract (Merck), and 15 g/L Agar (Merck). After sterilization, 5% (v/v) Defibrinated Horse Blood (TCS Biosciences) was added. Chocolate agar was prepared using the same recipe except that, after the addition of horse blood, the medium was heated to 70°C for 10 min. Fastidious Anaerobe Agar (FAA) was purchased from LabM and Sabouraud Dextrose Agar was from Oxoid.

For isolation of micro-organisms, a portion of sinus aspirate from each patient was homogenized in sterile phosphate buffered saline (PBS) and inoculated onto blood agar, chocolate agar, FAA and two plates of Sabouraud Dextrose Agar. Blood and chocolate

53 agar plates were incubated in 5% CO2 at 37°C. Pre-reduced FAA plates were incubated

at 37°C anaerobically (Ruskinn, Bugbox Plus) in a gas mix consisting of 10% CO2,

10% H2 and 80% N2. The Sabouraud Dextrose Agar plates were incubated aerobically,

one at 37°C and another at 30°C. Plates were examined every 24-48 h for at least seven days. Individual colonies were picked and sub-cultured three times to obtain pure isolates. Strains were stored at -80°C in BHY medium (brain heart infusion 37 g/L and yeast extract 5 g/L) diluted to 50% strength by the addition of glycerol (Sigma Aldrich).

2.4.1.3 Routine culture of oral bacteria

Streptococcus gordonii DL1, Actinomyces oris MG1 and Streptococcus mutans GS-5

were routinely cultured in THYE medium containing 30 g/L BactoTM Todd Hewitt Broth (Becton Dickinson and Co.) and 5 g/L yeast extract at 37°C anaerobically (10% CO2, 10% H2 and 80% N2). Fusobacterium nucleatum subsp. nucleatum ATCC 25586

was grown in BHIG comprising 37 g/L brain heart infusion, 5 g/L yeast extract, and 2.5 g/L L-glutamic acid (Sigma Aldrich) anaerobically at 37°C.

2.4.1.4 Culture of Escherichia coli

Strains of Escherichia coli (see table 2.2) were used for cloning and overexpression of proteins. The strains were grown in Luria Bertani (LB) Broth (Melford Laboratories Ltd.) at 35°C, whilst shaking at 200 RPM in aerobic conditions. Escherichia coli were also grown aerobically on LB agar (LB broth plus 15 g/L agar) at 37°C. When required, the appropriate antibiotics were added (all Sigma Aldrich); ampicillin (50 μg/mL), kanamycin (50 μg/mL), erythromycin (400 µg/mL) and chloramphenicol (33 µg/mL).

2.4.1.5 Biofilm formation in microtiter plates

For biofilm assays, microbial stock cultures (5 µL) were added to triplicate wells of sterile polystyrene microtiter-well plates containing growth media (200 µL). Plates were wrapped in parafilm and incubated without shaking aerobically or anaerobically at 37°C for various time periods. Following overnight growth (20 h), non-adherent planktonic cells were removed and transferred to a clean 96-well plate (Greiner Bio-One) and the OD600 was read in a microplate reader (BioTek Synergy HT) to quantify growth in the

54 planktonic phase. Biofilm extent was quantified using the crystal violet assay (see section 2.10.2).

2.4.2 Strains used in this study

Table 2.2 Strains used in this study

Strain Description Source or Reference

E. coli strains

TOP10 F– mcrA Δ(mrr-hsdRMS-

mcrBC) Φ80lacZΔM15

ΔlacX74 recA1 araD139 Δ(ara leu)

7697 galU galK rpsL (StrR) endA1 nupG

Invitrogen

GEXssnA ampr, camr, expresses SsnA-

GST

This thesis (NSJ)a

DH5α F– Φ80lacZΔM15

Δ(lacZYA-argF) U169 recA1 endA1 hsdR17

(rK–, mK+) phoA supE44 λ– thi-1 gyrA96 relA1

Invitrogen

BL21(DE3)pLysS F- ompT hsdSB (rB-mB-) gal

dcm (DE3) pLysS (camr, ampr)

G. Scholefield, CBCB, UK

B. subtilis strains

ATCC6633 Produces subtilin ATCC

NZ8900 pNZ8901 (Nijland et al., 2010)

S. gordonii strains

DL1 Wild type Newcastle Dental

Hospital Isolate

ccpA::ermAM ∆ccpA, ermr This thesis

malR::ermAM ∆malR, ermr This thesis

ssnA::ermAM ∆ssnA, ermr This thesis (NSJ)a

55 Other oral bacteria

A. oris MG1 Wild type J. Cisar, NIDCR,

USA

F. nucleatum 22586 Wild type ATCC

S. mutans GS-5 Wild type Newcastle Dental

Hospital Isolate

S. mutans NG8 Wild type L. C. Dutton, Bristol

University, UK

S. mutans UA140 Wild type Unknown

S. mutans UA159 Wild type ATCC

CRS Isolates

C. propinquum FH1 Isolated from patient 8 This thesis

C. pseudodiphtheriticum FH2 Isolated from patient 6 This thesis

M. catarrhalis FH3 Isolated from patient 3 This thesis

M. catarrhalis FH4 Isolated from patient 6 This thesis

S. aureus FH5 Isolated from patient 3 This thesis

S. aureus FH6 Isolated from patient 5 This thesis

S. aureus FH7 Isolated from patient 7 This thesis

S. epidermidis FH8 Isolated from patient 1 This thesis

S. epidermidis FH10 Isolated from patient 4 This thesis

S. epidermidis FH11 Isolated from patient 5 This thesis

S. lugdunensis FH12 Isolated from patient 6 This thesis

S. lugdunensis FH13 Isolated from patient 8 This thesis

S. lugdunensis FH14 Isolated from patient 15 This thesis

S. warneri FH15 Isolated from patient 7 This thesis

S. warneri FH17 Isolated from patient 14 This thesis

S. anginosus FH18 Isolated from patient 3 This thesis

S. anginosus FH19 Isolated from patient 17 This thesis

S. constellatus FH20 Isolated from patient 4 This thesis

S. constellatus FH21 Isolated from patient 7 This thesis

S. intermedius FH22 Isolated from patient 4 This thesis

S. pneumoniae FH26 Isolated from patient 10 This thesis

S. salivarius FH27 Isolated from patient 1 This thesis

56

S. salivarius FH29 Isolated from patient 17 This thesis

a

Unpublished strain constructed by Nick Jakubovics as part of this project.

2.4.3 Identification of micro-organisms

2.4.3.1 Routine microbiological identification

Whilst culturing micro-organisms, they were often checked for the presence of contaminant microbes using standard microbiological techniques. These included the Gram stain, the catalase test, and checking cell morphology using light microscopy. The appearance of colonies on agar was also used as a general guide for culture of the correct microbe.

2.4.3.2 Microbiology of chronic rhinosinusitis

All isolates were initially characterized by Gram staining, inspection of colony morphology, testing for catalase production, haemolysis, DNase production and ability to grow aerobically or anaerobically. The majority of clinical isolates were further identified to species level using a Matrix Assisted Laser Desorption/Ionization Time-of- Flight (MALDI-TOF) Mass Spectrometer (Bruker, Microflex). Isolates were streaked onto blood agar, incubated under 5% CO2 or in the absence of oxygen, at 37°C for 24 h

and transferred to the Pathology Department, Freeman Hospital, Newcastle upon Tyne, for identification.

In cases where MALDI-TOF analysis yielded ambiguous results, for example the majority of α-haemolytic streptococci, bacterial identification was confirmed by analysis of the 16S rRNA gene. The gene was amplified using the oligonucleotide primers 0063F and 1387R (Marchesi et al., 1998) in a ReddyMix™ reaction. PCR reactions were run using a GeneAmp PCR System 9700 (Applied Biosystems). PCR products were checked on a 1% agarose gel, and fragments of the expected size were sequenced by MWG Eurofins. Forward and reverse sequences were aligned and sequence matched using the Ribosomal Database Project website (http://rdp.cme.msu.edu/).

57 Bacterial growth was calculated by measuring the turbidity of a bacterial population, growing in culture medium. Using a spectrophotometer the absorbance at 600 nm of 1 mL of suspension was determined, using unconditioned medium to measure background absorbance.

This method of optical density measuring was used to calculate the growth rate of organisms. Isolates were cultured overnight, subcultured the following day into pre- warmed media, and grown for a period of 6-8 h, with optical density measurements taken every hour. To determine the doubling time the number of generations (g) was calculated using the following equation:

g = (Log10Nt - Log10N0)/Log102

Nt = A600 at time exiting the exponential growth phase

N0 = A600 at time beginning the exponential growth phase

After g was calculated, the value was divided by the number of min the exponential growth phase lasted, thereby giving a doubling time.

2.4.5 DNase agar test for extracellular nuclease activity

DNase agar is a nutrient rich medium that contains DNA. If an organism produces an extracellular enzyme that hydrolyses DNA it is possible to observe this activity by staining the agar. All chronic rhinosinusitis isolates, oral bacteria and mutants of S.

gordonii DL1 were tested for nuclease activity in this way. A single thick streak of each

bacterium was plated onto DNase Test Agar (Oxoid). Plates were incubated aerobically or anaerobically at 37°C for between 24-96 h. Once colonies had grown, plates were flooded with 4 mL of 0.1% (w/v) toluidine blue (Sigma Aldrich) or 4 mL of 1N HCl, to highlight nuclease production. HCl precipitates the DNA, creating an opaque medium except for areas of nuclease activity, whereas toluidine blue appears blue in the presence of intact DNA and pink when complexed with nucleotides.