Fitness of Streptococcus pneumoniae Fluoroquinolone-Resistant Strains with Topoisomerase IV Recombinant Genes

Abstract

The low prevalence of ciprofloxacin-resistant (Cp r ) Streptococcus pneumoniae isolates carrying recombinant topoisomerase IV genes could be attributed to a fitness cost imposed by the horizontal transfer, which often implies the acquisition of larger-than-normal parE-parC intergenic regions. A study of the transcription of these genes and of the fitness cost for 24 isogenic Cp r strains was performed. Six first-level transformants were obtained either with PCR products containing the parC quinolone resistance-determining regions (QRDRs) of S. pneumoniae Cp r mutants with point mutations or with a PCR product that includes parE-QRDR-ant-parC-QRDR from a Cp r Streptococcus mitis isolate. The latter yielded two strains, T6 and T11, carrying parC-QRDR and parE-QRDR-ant-parC-QRDR, respectively. These first-level transformants were used as recipients in further transformations with the gyrA-QRDR PCR products to obtain 18 second-level transformants. In addition, strain Tr7 (which contains the GyrA E85K change) was used. Reverse transcription-PCR experiments showed that parE and parC were cotranscribed in R6, T6, and T11; and a single promoter located upstream of parE was identified in R6 by primer extension. The fitness of the transformants was estimated by pairwise competition with R6 in both one-cycle and two-cycle experiments. In the one-cycle experiments, most strains carrying the GyrA E85K change showed a fitness cost; the exception was recombinant T14. In the two-cycle experiments, a fitness cost was observed in most first-level transformants carrying the ParC changes S79F, S79Y, and D83Y and the GyrA E85K change; the exceptions were recombinants T6 and T11. The results suggest that there is no impediment due to a fitness cost for the spread of recombinant Cp r S. pneumoniae isolates, since some recombinants (T6, T11, and T14) exhibited an ability to compensate for the cost.

Biological and Epidemiological Features of Antibiotic-Resistant Streptococcus pneumoniae in Pre- and Post-Conjugate Vaccine Eras: a United States Perspective

“…In S. pneumoniae, fluoroquinolone-resistant strains have a low frequency due to the high fitness cost imposed by point mutations in the genes that encode type II topoisomerases (topoisomerase IV and DNA gyrase) (272,273). However, the E85K change in gyrA identified as a high-cost mutation can be compensated for by the presence of a recombinant topoisomerase IV enzyme (273) in some laboratory mutants.…”

“…However, the E85K change in gyrA identified as a high-cost mutation can be compensated for by the presence of a recombinant topoisomerase IV enzyme (273) in some laboratory mutants. These specific alterations acquired by interspecific recombination may be selected as they reduce the fitness cost associated with fluoroquinolone resistance mutations.…”

Reactive Oxygen Species Contribute to the Bactericidal Effects of the Fluoroquinolone Moxifloxacin in Streptococcus pneumoniae

“…The MICs of MOX and LVX (Sigma-Aldrich) for the R6 strain were 0.125 g/ml and 0.250 g/ml, respectively. Two R6-derived strains, T1 and T2 (27), were also used. T1 carried the ParC S79F change and had MICs of 2 g/ml (LVX) and 0.125 g/ml (MOX).…”

The Fluoroquinolone Levofloxacin Triggers the Transcriptional Activation of Iron Transport Genes That Contribute to Cell Death in Streptococcus pneumoniae

“…Synthesis of cDNAs from 5 g of total RNA was performed as previously described (45). These cDNAs were subjected to quantitative qRT-PCR (Chromo 4; Bio-Rad) in 20-l reaction mixtures containing 2 l of cDNA, 0.3 M each specific primer, and 10 l of LightCycler FastStart Universal A SYBR green Master (Roche).…”

“…Amplification was achieved with 42 cycles of a tree-segment program: denaturation (30 s at 94°C), annealing (30 s at 45 to 56°C), and elongation (30 s at 68°C). To normalize the three independent cDNA replicate samples, values were divided by those obtained from the amplification of internal fragments of rpoB (45) and 16S rRNA genes. The oligonucleotides used are shown in Table S1 in the supplemental material.…”