The MIC50 and MIC90 (mg/L) for the 64 isolates were as follows: ceftazidime (CAZ) 2 and 16; ciprofloxacin (CIP) 0.5 and 16; tobramycin (TOB) 2 and 64; imipenem (IPM) 1 and 16; meropenem (MEM) 0.5 and 4; respectively. BIC50 and BIC90 (mg/L) for all isolates were as follows: CAZ 8 and 256; CIP 1 and 64; TOB 4 and 64; IPM 16 and 256; MEM 2 and 32, respectively. There was a statistical significant difference between MIC and BIC values of isolates for all antibiotics tested (Table ).
The number of “non-susceptible” (“Resistant” - “R” - plus “Intermediate” - “I”) isolates according to MIC and BIC for each antibiotic was as follows: CAZ 9/64 (14.1%) and 24/64 (37.5%); CIP 19/64 (29.7%) and 23/64 (36%); TOB 13/64 (20.4%) and 30/64 (46.8%); IPM 15/64 (23.4%) and 44/64 (68.8%); MEM 6/64 (9.4%) and 18/64 (28.1%), respectively. There was a statistical significant difference between the susceptibility category of isolates for all antibiotics tested, except for CIP (CAZ: P
0.001, CIP: P
0.234, TOB: P
0.001, IPM: P
0.001, MEM: P
The macrolide MIC values were tested for all isolates. Both azithromycin (AZM) (range 32
4096) and clarithromycin (CLR) (range 128
4096) presented a median MIC of 512 mg/L. MIC50
(mg/L) for all isolates were 512 and 1024 for AZM; 512 and 4096 for CLR, respectively.
The non-suscetible isolates according to BIC results were included in the macrolide combination assay (MCA) with CAZ (28 isolates – median BIC 128 mg/L), CIP (23 isolates – median BIC 16 mg/L), TOB (30 isolates – median BIC 16 mg/L), IPM (44 isolates – median BIC 32 mg/L), and MEM (18 isolates – median BIC 8 mg/L). When 2 mg/L of CLR was associated with the anti-pseudomonal agents, the median BIC values were significantly reduced for CAZ (P
< 0.001) and TOB (P
< 0.001), but not for CIP (P
=1.000), IPM (P
=1.000), and MEM (P
1.000). At higher CLR concentration (8 mg/L), BIC values significantly reduced when associated with CAZ (P
< 0.001), but not when associated with CIP (P=
1.000), TOB (P=
0.108), IPM (P
1.000), and MEM (P=
1.000). In the presence of 2 mg/L of AZM in combination with the anti-pseudomonal agents, the median BIC values were reduced significantly for CAZ (P
0.001), CIP (P=
0.009), and TOB (P=
0.001), but not when associated with IPM (P
1.000) and MEM (P
1.000), while the presence of 8 mg/L of AZM in association with all antibiotics showed reduction in median BIC values for all antibiotics tested (CAZ: P
< 0.001, CIP: P
< 0.001, TOB: P<
0.001, IPM: P<
0.001, MEM: P<
0.001) (Figure ).
Figure 1 Azithromycin and clarithromycin action on biofilm inhibitory concentration (BIC) of non-susceptible P. aeruginosa isolates combined with anti-pseudomonal agents. Detailed legend: CAZ - ceftazidime, CIP - ciprofloxacin, TOB - tobramycin, IPM - imipenem, (more ...)
CLR at 2 mg/L presented strong inhibitory quotient (IQ) when associated with TOB (66.7% of isolates) and CAZ (57.1% of isolates). CLR at 8 mg/L presented strong IQ when associated with CAZ (57.1% of isolates). AZM at 2 mg/L presented a strong IQ when associated with CAZ (50% of isolates), CIP (43.5% of isolates), and TOB (86.7% of isolates). Moreover, 8 mg/L of AZM in combination with all anti-pseudomonal agents tested presented the highest proportion of isolates with strong IQ for all antibiotics tested: CAZ (75%); CIP (73.9%); TOB (70%); IPM (88.6%); and MEM (61.1%) (Figure ).
Figure 2 Inhibitory Quotient (IQ) of combinations of macrolide antibiotics to anti-pseudomonal agents against P. aeruginosa isolates. Detailed legend: CAZ 2AZM – ceftazidime with 2 mg/L of azithromycin, CAZ 8AZM - ceftazidime with 8 mg/L of azithromycin, (more ...)
A total of 19 (29.7%) isolates presented the mucoid phenotype, but no statistical significant differences in the susceptibility profile of mucoid and non-mucoid isolates were found for the antibiotics tested in the different conditions performed in this study (MIC, BIC and MCA).
The repeatability of the assays demonstrated a coefficient of variation (CV) of MIC and BIC for CAZ, CIP, IPM, MEM, and TOB of 10.21 and 9.45, 7.09 and 8.46, 14.74 and 2.13, 7.70 and 3.94, 10.01 and 8.51, respectively. When macrolides were associated, the highest CV was 20.12% for CAZ with 8 mg/L of CLR and the lowest was 0% for TOB with 2 and 8 mg/L of CLR.