To test the validity of the use of median mutation frequency from five replica cultures in this analysis, six isolates with low,medium or high mutation frequencies were subject to further analysis.Mutation frequencies were determined from 5 to 20 further cultures, using standardised starter cultures for inoculation and monitoring growth to ensure consistent cell density at the time mutants were selected (this is critical due to the rapid autolysis of pneumococci at stationary phase). The combined data sets for each isolate were used to calculate the mutation rate (number of mutations per cell division) by the Jones median estimator,which accommodates the plating of only part of a culture, as described by Foster.Mutation rates for laboratory strain R6 and hex-deficient CP1015 were also calculated.Inclusion of additional cultures did, as would be expected from the stochastic nature of mutation, alter the median mutation frequencies somewhat (Table 2), but no isolate would move up or down bymore than one category of mutation frequency as defined in Fig. 1. Mutation rates to rifampicin resistance for clinical isolates ranged from 6.0×10−10 to 1.5×10−8; laboratory strain R6 fell around the middle of this range, whilst known mutator CP1015 had a rate of 2.2×10−8, only slightly higher than the most mutable clinical isolate PN02/1325.
As the mutation frequency determinations described above are time consuming to conduct, a rapid method to screen large numbers of clinical isolates for putative mutators was developed.Nine 10L spots of late log-phase broth cultures inoculated from overnight plates were placed on rifampicin agar. In 12 repeat
experiments with the hexB-deficient strain CP1015, a median of 7 colonies per spot was found, with a range of 4 to >12 (Fig. 2). In contrast the wild-type strain R6 gave a median of zero colonies per spot, with a range of 0–4 (Fig. 2).
Isolates that produced a median of three or more colonies per spot (and confirmed in a repeat test) were subject to full mutation frequency determina-tion. Of 180 clinical isolates examined (63 resistant, 19 susceptible and 98 undetermined),
two were potential mutators producing a comparable number of rifampicin-resistant colonies per spot to the hypermutable control.However, the mutation frequencies of these two isolates were determined to be 1.34×10−8 and 4.99×10−9 when tested by the standard method,
well within the normal range of mutability
- 1 0 年前最佳解答
為了測試使用的有效性正中突變頻率從五副本文化在這種分析中，六株低，中或高突變頻率均有待進一步 analysis.Mutation頻率決定從5日至20進一步培養，使用標準化發酵對接種和監測，以確保一致細胞生長密度在時間選擇突變體（這是關鍵的原因是快速自溶的肺炎雙球菌在固定相）。合併後的數據集被用來隔離每個計算的突變率（每數突變細胞分裂）由瓊斯中位數估計，可容納電鍍只有一種文化的一部分，所描述的Foster.Mutation率應變 R6的實驗室和六角缺陷CP1015也calculated.Inclusion沒有更多的文化，正如所預期的隨機突變的性質，改變有點突變頻率中位數（表2），但沒有孤立會向上或向下移動 bymore超過一類突變頻率定義圖。 1。對利福平耐藥突變率，為臨床分離株介於6.0 ×1.5×10-810-10，實驗室應變 R6的下跌約中間這個範圍，而已知的突變率CP1015有2.2 ×10-8，僅略高於比最易變的臨床分離 PN02/1325。
實驗與 hexB-缺陷株CP1015，中位數為7殖民地當場被發現，射程為 4至>12（圖2）。與此相反的野生型菌株R6的中位數了每點零殖民地，射程0-4（圖2）。
菌株產生的中位數或更多的殖民地，每三個點（並證實了重複測試）都受到全突變頻率 Determina的- tion。 180株臨床研究（63耐，19和98易感待定），二是潛在的存取器產生了相當數量的利福平抗性菌落每點的高突變 control.However，突變頻率，這兩個菌株被確定為1.34×4.99×10-8和10-9進行測試時按標準方法，以及在正常範圍內的可變性。