Role of biofilm formation in Ureaplasma antibiotic susceptibility and development of bronchopulmonary dysplasia in preterm neonates

Abstract

Background: Ureaplasma respiratory tract colonization is a risk factor for bronchopulmonary dysplasia (BPD) in preterm infants, but whether Ureaplasma isolates from colonized infants can form biofilms is unknown. We hypothesized that Ureaplasma isolates vary in capacity to form biofilms that contribute to their antibiotic resistance and ability to evade host immune responses. Study objectives were to (1) determine the ability of Ureaplasma isolates from preterm neonates to form biofilms in vitro; (2) compare the susceptibility of the sessile and planktonic organisms to azithromycin (AZI) and erythromycin; and (3) determine the relationship of biofilm-forming capacity in Ureaplasma isolates and the risk for BPD.

Methods: Forty-three clinical isolates from preterm neonates and 5 American Tissue Culture Collection strains were characterized for their capacity to form biofilms in vitro, and antibiotic susceptibility was performed on each isolate prebiofilm and postbiofilm formation.

Results: Forty-one (95%) clinical and 4 of 5 (80%) American Tissue Culture Collection isolates formed biofilms. All isolates were more susceptible to AZI (minimum inhibitory concentration, MIC50 2 µg/mL) than erythromycin (MIC50 4 µg/mL), and biofilm formation did not significantly affect antibiotic susceptibility for the 2 tested antibiotics. The MIC50 and minimum biofilm inhibitory concentrations (MBIC50) for Ureaplasma urealyticum clinical isolates for AZI were higher than for MIC50 and MBIC50 for Ureaplasma parvum isolates. There were no differences in MIC or MBICs among isolates from BPD infants and non-BPD infants.

Conclusions: Capacity to form biofilms is common among Ureaplasma spp. isolates, but biofilm formation did not impact MICs for AZI or erythromycin.

Figure 1

Comparison of antibiotic susceptibility of planktonic and sessile cultures of ATCC and clinical isolates. Conventional MIC susceptibility testing with erythromycin and azithromycin were performed for each ATCC (N=5) and clinical (N=43) isolate in quadruplicate by the broth microdilution method and the MBIC determined following biofilm-formation as previously described . Data are presented as box plots with the bottom, top, and line through the middle of the box corresponding to the 25th percentile, 75th percentile, and 50th percentile (median), respectively. The whiskers extend from the 10th percentile to the 90th percentile. Erythro, erythromycin, AZI, azithromycin *p=0.006 vs AZI MIC₅₀ ATCC strains by Wilcoxon sign rank test

Figure 2

Comparison of antibiotic susceptibility of planktonic and sessile cultures of U. parvum and U. urealyticum isolates. Conventional MIC susceptibility testing with erythromycin and azithromycin were performed for each U. parvum (N=26) and U. urealyticum (N=17) isolate in quadruplicate by the broth microdilution method and the MBIC determined following biofilm-formation as previously described . MIC₅₀ and MBIC₅₀ for erythromycin (Erythro) and azithromycin (AZI). Data are presented as box plots with the bottom, top, and line through the middle of the box corresponding to the 25th percentile, 75th percentile, and 50th percentile (median), respectively. The whiskers extend from the 10th percentile to the 90th percentile. *p<0.01 compared to U. parvum isolate erythromycin MIC₅₀ and MBIC₅₀ by Wilcoxon sign rank test †p<0.001 compared to azithromycin MIC₅₀ and MBIC₅₀ U. urealyticum isolates by Wilcoxon sign rank test

Figure 3

Comparison of clinical isolates MIC₅₀ and MBIC₅₀ by clinical outcomes. There were no significant differences in antibiotic susceptibility among biofilm-forming isolates from infants with and without BPD/death. Data are presented as box plots with the bottom, top, and line through the middle of the box corresponding to the 25th percentile, 75th percentile, and 50th percentile (median), respectively. The whiskers extend from the 10th percentile to the 90th percentile.