MATERIALS AND METHODS

3.2.1 Bacterial Strains

The C. difficile isolates used for this study have been described in chapter 2.1.1. Control strains used included; E4 (PCR ribotype 010), which was previously characterised as being reduced susceptible to metronidazole(Brazier et al., 2001), p62 (PCR ribotype 001) as a clindamycin- resistant control (Fawley et al., 2003), Bacteroides fragilis (B. fragilis) ATCC 25285 as a metronidazole-susceptible control, and Staphylococcus aureus (S. aureus) ATCC 29213 was included as an indicator for aerobic contamination. Escherichia coli (E. coli) NCTC 9001 was used as a nitrofurantoin-susceptible control.

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3.2.2 Antimicrobials Susceptibility testing and Minimum Inhibitory

Concentration (MIC) Determination

Antimicrobial susceptibility testing was carried out according to methods described in 2.2. The MIC was determined as the lowest antimicrobial concentration where an absence or marked reduction of growth (multiple tiny colonies, haze or fine film of growth or one or two colonies) compared with the growth control was observed. The MIC values for individual strains were categorised as susceptible, intermediate and resistant based on the CLSI/EUCAST/BSAC MIC interpretative breakpoint values (Table 3-1). However, there were no approved breakpoints for the following antimicrobials: nitrofurantoin, clarithromycin, and trimethoprim against C. difficile; therefore, MIC breakpoint values for these antibiotics antimicrobials against S. aureus were used.

3.2.3 Clindamycin inducible resistance

Resistance to clindamycin was investigated by the clindamycin Inducible resistance (‘D test’) test. This test has been previously used to identify strains of Staphylococcus species that are inducible resistant to clindamycin(Prabhu et al., 2011). The aim of the test is to induce the expression of erm-mediated resistance through the exposure of isolates to both clindamycin and erythromycin, adjacent to one another on an agar plate. This test was performed on C. difficile isolates with MICs ≥8 mg/L, which according to CLSI guidelines were resistant to clindamycin. One hundred microliters of C. difficile overnight culture in Schaedler’s anaerobe broth was inoculated onto pre-dried Wilkins Chalgren agar (WCA). Using a sterile swab, the bacterial suspension was evenly spread out on the WCA agar plates. Subsequently, a 30µg disc of erythromycin (CT0021B, Oxoid) was placed at 15mm from a 10µg clindamycin (CT0015B, Oxoid) using an Oxoid disc dispenser and incubated anaerobically for 48h at 37°C.

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Following incubation, plates were retrieved from the anaerobic cabinet and examined for flattening of inhibition zone (D-shaped) around the clindamycin disc in the area between two discs (Figure 3.1). This phenomenon is an indication of inducible clindamycin resistance.

A -Positive ‘D’- test B- Negative ‘D’- test

Figure 3-1 Schematic diagram showing clindamycin inducible resistance: A) is a representation of a

positive D- test (D+ Phenotype). B) Is representation of a negative D- test phenotype E- Erythromycin, C-Clindamycin.

3.2.4 Beta-Lactamase Activity Assay

Beta-lactamase activity was assayed based on the hydrolysis of nitrocefin, a chromogenic cephalosporin that results in the generation of a coloured product. All isolates were investigated for β-Lactamase activity, using the broth method. Four drops of rehydrated nitrocefin (SR0112, ThermoFisher) solution was added to 1mL of C. difficile culture grown in Wilkins Chalgren broth. β- Lactamase activity was monitored by a visible colour change from yellow to red within 30 minutes of incubation.

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3.2.5 Molecular analysis of mechanisms of resistance

Molecular analysis of the mechanisms of resistance to erythromycin, clindamycin, rifampicin, ciprofloxacin and moxifloxacin were investigated using a PCR assay. PCR products were purified using Wizard SV gel and PCR clean-up system (A9281, Promega, USA), and sequencing was done by Eurofins Genomics Germany (Eurofins GATC, Ebersberg, Germany), and Source BioScience (Cambridge, UK). Pairwise alignments of DNA sequences were carried out using the BLAST server and Clustal W Omega as described in 2.6.6.

Ciprofloxacin and moxifloxacin resistant isolates (MIC ≥8mg/L) were investigated for the mutations in the DNA gyrase genes (gyrA & gyrB). The quinolone resistance determining region (QRDR) of both gyrA and gyrB genes was amplified using two different pairs of primers, listed in Table 2-6. PCR amplification consisted of 30 cycles of denaturation at 94°C for 30 s, annealing at 54°C (gyrB)/ 58°C (gyrA) for 30 s, and extension at 72°C for 30s(Spigaglia et al., 2011). Using agarose gel electrophoresis,described in 2.8.4, the resulting PCR products were assessed for the presence of a 390bp internal fragment of the gyrB and gyrA (Figure 3-5) by using standard molecular weight markers; Hyper ladder1-kb (BIO-33053, Bioline, London UK).

Erythromycin and clindamycin-resistant isolates (MIC ≥ 8mg/L) were investigated for the presence of the erm (B) gene by using the primers pair ermBf and ermBr listed in Table 2-6, (Sutcliffe et al., 1996). Strain p62 (PCR ribotype 001), previously described as resistant to clindamycin (Fawley et al., 2003), was included as a positive control for erm (B) gene. According to published methods by Solomon et al, PCR amplification consisted of 35 cycles of denaturation at 94°C for 30s, annealing at 52°C for 60s, and elongation at 72°C for 60s. The

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last elongation step was performed at 72°C for 5 minutes(Solomon et al., 2011). Using agarose gel electrophoresis as described in 2.8.4, the resulting PCR product were assessed for the presence of a 639bp internal fragment of the erm(B) (Figure 3-5) by using standard molecular weight markers; 1kb DNA ladder (D3937, Sigma).

To detect the presence of other classes of erm (A, C, F and Q) in ermB negative isolates, primers described by Patterson et al (2007) as listed in Table 2-5 were used. PCR amplifications consisted of 35 cycles of denaturation at 94°C for 30 s, annealing at 48°C (ermA), 43°C (ermC), and 52°C (ermQ) for 1 min, and elongation at 72°C for 2 mins. For ermF, PCR amplification consisted of 35 cycles of denaturation at 94°C for 30 s, annealing at 50°C for 30 s, and elongation at 72°C for 2 min. Using agarose gel electrophoresis as described in 2.8.4, the resulting PCR products were assessed for the presence of 645bp, 642bp, 466bp, and 771bp internal fragments of erm (A, C, F and Q respectively) (Figure 3-5) by using standard molecular weight markers; 1kb DNA ladder (D3937, Sigma).

Rifampicin resistant isolates with an MIC ≥16mg/L were investigated for mutations in rpo (B) gene using previously published primers CDrpoB2F and CDrpoB2R (Curry et al., 2009) as listed in Table 2-6. PCR amplifications consisted of 35 cycles of denaturation at 95°C for 1 min, annealing at 49.9°C for 1 min, and elongation at 72°C for 1min. Using agarose gel electrophoresis as described in 2.8.4, the resulting PCR product were assessed for the presence of 200bp, internal fragments of rpo (B) (Figure 3-5) by using standard molecular weight markers; 100bp DNA ladder (D3687, Sigma).

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3.2.6 Statistical analysis

Statistical analysis was performed using GraphPad Prism 6 software (GraphPad Software, La Jolla, CA). In order to determine the appropriate statistical test to use, data were assessed for normality of distribution using both D’Agostino-Pearson and Shapiro-Wilk normality test. Statistically significant differences were tested using Kruskal-Wallis tests and differences were considered significant at a P-value of <0.05. An additional post hoc testing using Dunn’s multiple comparison tests was used to determine the significant differences that existed between groups.