DISEÑO DE LA RED DE MEDIO, BAJO VOLTAJE Y ALUMBRADO

resistant mutants

Five quinolone-resistant clones of E. coli AB 1157 and of AB 1157 (R46), selected from cultures grown for four days in nutrient broth containing 0.75 x MIC of either nalidixic acid or ciprofloxacin and then plated on nutrient agar containing 4 x MIC of each respective drug, were tested to determine their MICs. Another five clones were tested from cultures that had grown for eight days in the presence of 0.75 x MIC of the drugs, and which had been subject to similar selection procedures. Overnight cultures grown in drug-free nutrient broth were plated directly onto nutrient agar containing 4 x MIC o f drug to give control colonies for testing.

Table 22 shows the MICs of nalidixic acid for the selected colonies. From 20 control clones plated on medium containing nalidixic acid, 11 showed MICs to nalidixic acid of > 512 yUg m L'\ Similarly, from the 20 control clones plated on medium containing ciprofloxacin, 10 had MICs to nalidixic acid of >512 yUg mL \ However, no control clone selected after growth on ciprofloxacin-containing medium exhibited a nalidixic acid MIC of 128 yUg mL'% compared with 4 control clones selected on medium containing nalidixic acid which did record this MIC. Of the 10 more sensitive control clones selected on ciprofloxacin, 6 had nalidixic acid MICs of 64 yUg mL *, and 4 gave MICs o f 32 yUg mL *. Only a single control clone selected on medium containing nalidixic acid gave an MIC for this drug o f 32 yUg mL'*, with four clones having nalidixic acid MICs o f 64 yUg mL * (Table 22).

With the exception of colonies selected after growth o f the plasmidless ABl 157 strain for eight days in broth containing 0.75 x MIC nalidixic acid, clones selected after

growth in the presence of sub-inhibitory concentrations of nalidixic acid gave similar resistance patterns to control strains (Table 22). Four ABl 157 clones selected after eight days growth in nalidixic acid-containing broth had MICs for this drug of only 16 yUg mL'% the same concentration of drug on which the clones were selected. The fifth clone gave a nalidixic acid MIC of 32 ij,g mL"’. Clones of AB 1157 selected after four days in broth containing 0.75 x MIC nalidixic acid gave MICs of 64 ywg mL"’ (3 colonies) and ^512 /^g mL"’ (2 colonies). After four days growth in sub-inhibitory nalidixic acid, selected colonies o f strain ABl 157 (R46) gave a similar pattern of nalidixic acid resistance as clones recovered after eight days growth in the same medium (Table 22). All 10 colonies o f ABl 157 and ABl 157 (R46) selected after four days growth in a sub-inhibitory concentration of ciprofloxacin and plating on medium containing 4 x MIC ciprofloxacin, gave nalidixic acid MICs o f <64 yUg mL"’, whereas 6 colonies selected after eight days growth in the same medium had nalidixic acid MICs o f ^512 yUg mL"’, with 4 recording an MIC of 64 yWg mL "’ (Table 22).

Table 23 shows the ciprofloxacin MICs of the same colonies whose nalidixic acid resistance levels are reported in Table 22. The clones of ABl 157 R" selected after 8 days growth in nalidixic acid and which gave lower nalidixic acid MICs than control clones, also exhibited lower ciprofloxacin MICs than the controls (Table 23). Control clones selected on nalidixic acid gave a similar pattern o f ciprofloxacin resistance as control clones selected on ciprofloxacin. O f the 20 control clones growing on nalidixic acid- containing medium, 4 gave a ciprofloxacin MIC of 0.16 ywg mL"’, 13 an MIC o f 0.08 yUg mL"’ and 3 a ciprofloxacin MIC of 0.04 yUg mL"’. Similarly, of the control clones selected on ciprofloxacin, 4 gave a ciprofloxacin MIC of 0.16 yUg mL"’, 10 an MIC of 0.08 yUg mL "’ and 6 an MIC of 0.04 yWg mL"’ (Table 23). Clones grown in a sub-inhibitory concentration

Table 22. Minimum inhibitory concentrations (MICs) of nalidixic acid for clones selected on nutrient agar containing nalidixic acid (4 x MIC) or ciprofloxacin (4 x MIC). Cultures of Escherichia coli ABl 157 either harbouring or not harbouring plasmid R46, were grown for four or eight days in nutrient broth containing 0.75 x MIC o f nalidixic acid or ciprofloxacin. Control clones were selected from colonies produced by plating drug-free cultures onto plates containing nalidixic acid (4 x MIC) or ciprofloxacin (4 x MIC). Figures refer to the number o f clones with that particular MIC to nalidixic acid.

Strain of ABl 157

Grown in Plated on Period of growth

(days)

Nalidixic acid MIC (yUg mL ’)

16 32 64 128 256 k512 R- - NA 1 2 2 5 R46 - NA 2 2 6 R NA NA 4 8 4 1 3 2 R46 NA NA 4 8 1 1 2 1 2 3 R - Cip 1 4 5 R46 - Cip 3 2 5 R Cip Cip 4 8 3 2 4 1 R46 Cip Cip 4 8 4 1 5 VO U)

of ciprofloxacin before selection on ciprofloxacin generally exhibited higher ciprofloxacin resistance than clones selected from nalidixic acid-containing medium. Only one clone selected after nalidixic acid treatment had a ciprofloxacin MIC of 0.16 yUg mL % whereas 5 clones selected from medium containing ciprofloxacin had a ciprofloxacin MIC o f 0.16 fxg m L '\ No clone was isolated with a ciprofloxacin MIC of greater than 0.16 /^g mL \ No clone selected after exposure to ciprofloxacin exhibited a ciprofloxacin MIC o f less than 0.04 /^g mL ', whereas almost half (8 out of 20) o f the clones selected from nalidixic acid-containing medium gave ciprofloxacin MICs <0.02 yUgmL ' (Table 23).

Table 24 compares the MICs of nalidixic acid and ciprofloxacin for all the clones described in Tables 22 and 23. The results clearly show that colonies exhibiting high nalidixic acid MICs acid also had high ciprofloxacin MICs, and vice versa. Almost half o f all the colonies tested (34 from 80) gave nalidixic acid MICs of 512 yUg mL ' or greater (^ 128 x MIC): all 34 had ciprofloxacin MICs of either 0.08 or 0.16 yUg mL ' (16 or 32 X MIC) (Table 24).

The data in Table 22 show that clones with high levels o f resistance to nalidixic acid (^ 128 X MIC) were readily selected from both control and drug-exposed cultures, regardless of whether nalidixic acid or ciprofloxacin was used for selection. O f the 80 clones reported in Table 22, 34 showed a nalidixic acid MIC o f >512 yUg mL ', an increase of 128-fold or greater over the wild-type MIC. These same clones did not exhibit such high levels of resistance to ciprofloxacin, only 14 out of 80 gave ciprofloxacin MICs of 0.16 ywg mL ' (32 x MIC), with none giving greater than this.

Table 23. Minimum inhibitory concentrations (MICs) of ciprofloxacin for clones selected on nutrient agar containing nalidixic acid (4 x MIC) or ciprofloxacin (4 x MIC). Cultures o f Escherichia coli ABl 157 either harbouring or not harbouring plasmid R46, were grown for four or eight days in nutrient broth containing 0.75 x MIC of nalidixic acid or ciprofloxacin. Control clones were selected from colonies produced by plating drug-free cultures onto plates containing nalidixic acid (4 x MIC) or ciprofloxacin (4 x MIC). Figures refer to the number o f clones with that particular MIC to ciprofloxacin.

Strain of ABl 157

Grown in Plated on Period of growth

(days)

Ciprofloxacin MIC (yUg mL ')

0.005 0.01 0.02 0.04 0.08 0.16 R- - NA 3 6 1 R46 - NA 7 3 R NA NA 4 8 1 1 1 3 2 2 R46 NA NA 4 8 1 1 1 1 3 2 1 R - Cip 4 6 R46 - Cip 2 4 4 R Cip Cip 4 8 1 4 3 1 1 R46 Cip Cip 4 8 1 4 2 3 VO LA

Table 24. Comparison of the minimum inhibitory concentrations (MICs) to nalidixic acid and ciprofloxacin o f the clones described in Tables 22 and 23. Figures refer to numbers of clones.

Nalidixic acid MIC (Mg mL ')

16 32 64 128 256 >512 Ciprofloxacin MIC (Mg m L ') 0.005 1 0.01 1 0.02 3 1 4 1 0.04 1 15 1 0.08 9 3 4 23 0.16 1 1 11 VO o \

These results are in agreement with the currently accepted concept that one mutation in E. coli, commonly in the QRDR of the gyrA gene, is required to produce a high level of resistance to nalidixic acid (MIC >128 /^g m L ’) (Vila et al, 1994) and a low level of resistance to fluoroquinolones (ciprofloxacin MIC < 1 yUg mL ’) (Yoshida et al, 1988; Oram and Fisher, 1991). A second mutation in gyrA is necessary for the development o f high-level resistance to fluoroquinolones, resulting in ciprofloxacin MICs of >2 yUg mL ’ (Everett et al, 1996), >8 yUg mL ’ (Vila et al, 1994) or 128 yUg mL ’ (Heisig et al, 1993). The data presented in Tables 22 and 23 therefore suggest that the selected clones carry only one mutation, leading to a high level of resistance to nalidixic acid but only a low-level resistance to ciprofloxacin. The variation in the nalidixic acid resistance levels o f the selected clones (Table 22), suggests that the mutational site within the E. coli chromosome may vary. High-level resistance to nalidixic acid (> 128 yUg mL'’) results from a single mutation in the QRDR o f the gyrA gene, altering the serine amino acid at position 83 of the GyrA protein (Vila et al, 1994). Whereas, those clones showing only a moderate increase in nalidixic acid MIC may have a wild-type GyrA protein, but exhibit altered expression of outer membrane porins, such as OmpF (Hooper et al, 1986; 1992), induction of the marRAB operon (Cohen et al, 1989; 1993c), or mutation in gyrB (Yoshida 1991).

It was, therefore, predicted that if a low-level ciprofloxacin resistant mutant was given a second period of growth in nutrient broth containing a concentration of quinolone approaching its MIC, the resulting culture would contain high-level ciprofloxacin resistant mutants.