Concurrent titration and determination of antibiotic resistance in ureaplasma species with identification of novel point mutations in genes associated with resistance

Abstract

Antibiotic resistance determination of Ureaplasma spp. (Ureaplasma parvum and Ureaplasma urealyticum) usually requires predetermination of bacterial titer, followed by antibiotic interrogation using a set bacterial input. This 96-well method allows simultaneous quantification of bacteria in the presence and absence of antibiotics. A method for determining precise MICs and a method for screening against multiple antibiotics using breakpoint thresholds are detailed. Of the 61 Ureaplasma-positive clinical isolates screened, one (1.6%) was resistant to erythromycin (MIC, >64 mg/liter) and clarithromycin (MIC, 4 mg/liter), one to ciprofloxacin (1.6%), and one to tetracycline/doxycycline (1.6%). Five isolates were also consistently found to have an elevated MIC of 8 mg/liter for erythromycin, but this may not represent true antibiotic resistance, as no mutations were found in the 23S rRNA operons or ribosome-associated L4 and L22 proteins for these strains. However, two amino acids (R66Q67) were deleted from the L4 protein of the erythromycin-/clarithromycin-resistant strain. The tetM genetic element was detected in the tetracycline-resistant clinical isolate as well as in the positive control Vancouver strain serotype 9. The tetM gene was also found in a fully tetracycline-susceptible Ureaplasma clinical isolate, and no mutations were found in the coding region that would explain its failure to mediate tetracycline resistance. An amino acid substitution (D82N) was found in the ParC subunit of the ciprofloxacin-resistant isolate, adjacent to the S83L mutation reported by other investigators in many ciprofloxacin-resistant Ureaplasma isolates. It is now possible to detect antibiotic resistance in Ureaplasma within 48 h of positive culture without prior knowledge of bacterial load, identifying them for further molecular analysis.

FIG. 1.

Photographs of antibiotic breakpoint investigation showing an erythromycin (Erythro)-resistant strain (UHWO10) (A), a ciprofloxacin (Cipro)-resistant strain (HPA18) (B), and a tetracycline (Tet)-resistant strain (HPA23) (C). Dark red wells indicating growth of ureaplasma appear dark gray, while orange-yellow wells representing no ureaplasma growth appear light gray in the grayscale photograph. Columns containing 10⁴ CCU are identified by a dotted box, and comparison of growth in the absence of antibiotics (control) was used to determine resistance to 4 mg/liter erythromycin, 4 mg/liter ciprofloxacin, or 2 mg/liter tetracycline.

FIG. 2.

Full-plate determination of MIC for erythromycin for a susceptible SV1 isolate (A) and a resistant (UHWO10) isolate (B) in plates containing a gradient of antibiotic from 64 mg/liter to 0.06 mg/liter. Dark red wells indicating growth of ureaplasma appear dark gray, while orange-yellow wells representing no ureaplasma growth appear light gray in the grayscale photograph. Columns representing 10⁴ CCU growth in the absence of antibiotic are shown by the dotted box, and for the susceptible strain, a circle shows the first concentration of erythromycin to inhibit growth (MIC = 1 mg/liter). Since the resistant isolate (UHWO10) grew even in the presence of 64 mg/liter erythromycin, the MIC is determined to be >64 mg/liter.