In vitro killing of canine strains of Staphylococcus pseudintermedius and Escherichia coli by cefazolin, cefovecin, doxycycline and pradofloxacin over a range of bacterial densities.

BACKGROUND: Bacterial densities likely fluctuate during infection and may exceed the bacterial density used in susceptibility testing. As such, investigation of bacterial killing by antibiotics over a range of varying bacterial densities may provide important differences between compounds and could impact drug selection for therapy. HYPOTHESIS/
OBJECTIVES: To measure killing of clinical isolates of Staphylococcus pseudintermedius and Escherichia coli by cefazolin, cefovecin, doxycycline and pradofloxacin at clinically relevant (minimum inhibitory, mutant prevention, maximum serum and maximum tissue) drug concentrations against varying densities of bacteria. ANIMALS/MATERIALS: Bacterial strains collected from dogs with urinary tract infections were studied. METHODS AND MATERIALS: High bacterial densities ranging from 106 to 109 colony forming units (cfu)/mL were exposed to minimum inhibitory, mutant prevention, blood and tissue drug concentrations, and the percentages (log10 ) of viable cells killed following 30 min, 1, 2, 4, 6, 12 and 24 h of drug exposure were quantified.
RESULTS: Doxycycline exhibited bacteriostatic properties with less killing than the other three agents. For example, at a 107  cfu/mL density of S. pseudintermedius, more cells were killed by pradofloxacin (P < 0.0001) and cefovecin (P = 0.0014) but not cefazolin when compared to doxycycline at the maximum serum drug concentration following 12 h of drug exposure. CONCLUSIONS AND CLINICAL IMPORTANCE: Differences were seen between some drugs in the speed and extent of bacterial killing; this could be clinically important and may impact drug selection and length of therapy.

Introduction

Bacterial eradication is considered important for clinical cure from an infectious disease, even though this concept may be more complex for skin infections. Numerous authors have debated on similarities and differences between bactericidal and bacteriostatic agents, and where they might be best used in clinical medicine and influence clinical outcome.1, 2, 3 Noninferiority clinical trials in humans with mild to moderate community‐acquired infections failed to show differences in outcome in patients treated with bactericidal versus bacteriostatic agents and such observations are not surprising as differences in outcome were not expected (nor were the trials powered to detect differences). In patients with life‐threatening infections, bactericidal agents alone or in combination with bacteriostatic agents have been recommended for therapy.4

Differentiating bactericidal agents from each other and from bacteriostatic agents has involved in vitro kill studies where log10 reductions in viable cells are used to determine ‐cidal versus ‐static activity. A reduction in viable cells of >3 log10 or greater differentiates a bactericidal drug from a bacteriostatic agent where a <2 log10 reduction is seen. Log reduction values between <2 log10 and >3 log10 is considered a grey or indeterminate zone.5, 6

We have previously argued that clinically relevant drug concentrations need to be used in in vitro kill assays and over a range of bacterial densities expected to occur during acute or chronic infections.7, 8 In particular, higher bacterial densities are known to occur in central nervous system,9, 10 respiratory11 and urinary tract infections,12 and likely others. In a previous report,7 we compared killing of canine isolates of Staphylococcus pseudintermedius and Escherichia coli by cefazolin, cefovecin, doxycycline and pradofloxacin using bacterial densities of 105 colony forming units (cfu)/mL – the bacterial density used for in vitro susceptibility testing. Bacterial killing was measured over 180 min and differences in killing by the drugs tested were seen for S. pseudintermedius and E. coli at some of the various time points. In the present report, we extended the design to compare bacterial killing over 24 h and at higher bacterial densities ranging from 106 to 109 cfu/mL7.

Methods and materials

Bacterial strains

The same clinical isolates from dogs as used in a previous study (three each of S. pseudintermedius and E. coli) were tested.7 They had been identified by Matrix‐assisted laser desorption ionization – time of flight (MALDI‐TOF) (BioMerieux; St Laurent, Quebec, Canada) and each isolate had to be susceptible to each agent based on current recommended susceptibility minimum inhibitory concentration (MIC) breakpoints.13

Antimicrobial compounds

Sources of antimicrobial compounds and their preparation and storage were as described previously.7

MIC/mutant prevention concentration (MPC) testing

The MIC/MPC testing using a modified protocol was as summarized in detail in our earlier publication.7, 14, 15 Measured MIC and MPC values for the six strains examined are as reported previously and summarized in Table 1.7

Table 1

Minimum inhibitory (MIC) and mutant prevention (MPC) concentration values for Staphylococcus pseudintermedius and Escherichia coli strains and pharmacological parameters for four antimicrobial agents.7

Drug	Isolates RUH‐CASP1		RUH‐CASP2		RUH‐CASP3		Maximum Serum Concentration	Maximum Tissue (skin) Concentration*
	MIC	MPC	MIC	MPC	MIC	MPC
S. pseudintermedius
Cefazolin	0.125	2	0.063	4	0.063	0.25	74	18.5
Cefovecin	2	4	0.25	0.5	0.25	0.5	90	22.5
Doxycycline	0.063	16	2	64	0.063	8	5.7	2.8
Pradofloxacin	0.031	0.125	0.031	0.125	0.125	0.125	1.4	4.5

CASP companion animal S. pseudintermedius, CAEC companion animal E. coli, RUH Royal University Hospital.

Maximum tissue (skin) concentration were estimated for cefovecin, cefazolin and doxycycline, from skin drug concentrations18, 19 and from serum drug concentrations.37 For pradofloxacin, published data were used.49, 50

Kill studies

Kill studies were a modification to protocols published previously.16, 17 Bacterial isolates were incubated overnight on blood agar (BA) plates for 18–24 h at 35–37°C in O2. Following overnight incubation, an inoculum was transferred to Mueller–Hinton Broth (MHB) and incubated for 2 h in O2 at 35–37°C; then spectrophotometric readings (>0.3) verified cell densities >109 cfu/mL which were subsequently confirmed by colony counts.14 Further adjustment of inocula to achieve cell densities ranging from 106 to 109 cfu/mL were done in MHB; to this, cefazolin, cefovecin, doxycycline or pradofloxacin was added based on the measured drug MIC or MPC values or the C max or Tissuemax drug concentration for each drug tested against each strain. In vitro measurements did not take into account protein binding. A summary of C max and Tissuemax concentrations is presented in Table 1 and is as reported previously.7 For cefovecin, cefazolin and doxycycline, skin drug concentration was estimated from previous publications.18, 19 Measurements of killing (log10 reduction in viable cells and % kill) were recorded at 30 min, 1, 2, 4, 6, 12 and 24 h following drug exposure by culturing aliquots on drug‐free blood agar plates and incubating overnight as described and counting colonies. Colony counts recorded at time 0 at the 106, 107, 108 and 109 cfu/mL densities (respectively) were as follows:

Three separate aliquots were sampled at each time point and results averaged; as such each data point represents nine independent measurements (i.e. three strains x three triplicate samplings). The log10 and percentage kill reduction of viable cells were calculated and recorded.

Statistical analysis

Statistical analysis of the data was as described previously.7 Values of P < 0.05 were considered significant for all analyses.

Results

Exposure of 106 cfu/mL of S. pseudintermedius (Table 2) to the MIC drug concentration of four drugs tested did not show any statistically significant differences in bacterial killing between the drugs (refer to Table 2 for all log10 reduction comparisons).

Table 2

Log10 reduction in viable cells (percentage of cells killed) over time for suspensions of Staphylococcus pseudintermedius and Escherichia coli (106 cfu/mL) exposed to various concentrations of cefazolin, cefovecin, doxycycline and pradofloxacin

Variable	S. pseudintermedius				E. coli
	Cefazolin	Cefovecin	Doxycycline	Pradofloxacin	Cefazolin	Cefovecin	Doxycycline	Pradofloxacin
Minimum inhibitory concentration (MIC)
0.5 h	–0.03 (–7.55)	–0.05 (–0.66)	–0.01 (–3.06)	–0.17 (–17.81)	–0.09 (–18.13)	0.00 (1.92)	0.05 (19.04)	–0.03 (–1.02)
1 h	0.04 (12.78)	–0.01 (1.01)	0.07 (18.26)	–0.27 (–43.84)	–0.05 (–8.54)	0.00 (–1.10)	0.01 (2.86)	0.02 (5.60)
2 h	0.17 (51.05)	–0.04 (15.34)	0.06 (15.07)	–0.18 (–14.75)	–0.44 (–89.59)	–0.30 (–37.60)	0.09 (4.25)	0.05 (13.15)
4 h	–0.29 (209.45)	–0.46 (–24.17)	0.06 (15.46)	–0.53 (–69.13)	–0.03 (70.14)	–1.33 (–91.67)	0.02 (6.96)	–0.75 (–81.59)
6 h	–0.66 (591.99)	–0.65 (–30.50)	0.04 (10.99)	–0.99 (–88.04)	0.40 (1290.56)	–1.82 (–98.10)	0.09 (35.15)	–0.36 (17.40)
12 h	–0.33 (1324)	–1.37 (–94.76)	0.06 (182.08)	–1.65 (–87.08)	1.23 (2635.74)	–2.26 (–99.23)	0.18 (55.17)	–0.94 (–31.51)
24 h	0.95 (1427.62)	–1.44 (–60.87)	0.68 (772.31)	0.28 (–2.92)	2.24 (17438.95)	–1.37 (–87.32)	–1.79 (196.70)	1.27 (1981.05)
Mutant prevention concentration (MPC)
0.5 h	–0.04 (–7.70)	0.01 (9.16)	–0.02 (–4.93)	0.09 (29.17)	–0.28 (–44.26)	–0.02 (–4.55)	–0.03 (–6.26)	–0.56 (–64.15)
1 h	0.04 (12.18)	–0.03 (–5.53)	–0.09 (–15.50)	–0.03 (–6.34)	–1.37 (–89.51)	–0.09 (–18.21)	–0.09 (–15.53)	–1.98 (–92.73)
2 h	–0.10 (–10.70)	–0.11 (–11.79)	–0.22 (–39.33)	–0.56 (–71.82)	–1.94 (–98.53)	–0.38 (–57.91)	–0.21 (–36.44)	–3.65 (–99.92)
4 h	–1.02 (–85.71)	–0.76 (–51.04)	–0.21 (–36.82)	–1.94 (–98.55)	–3.15 (–99.86)	–1.74 (–97.65)	–0.71 (–76.43)	–4.54 (–99.97)
6 h	–1.67 (96.52)	–1.68 (–91.16)	–0.43 (–47.46)	–2.31 (–99.47)	–4.04 (–99.97)	–2.62 (–99.71)	–1.01 (–86.97)	–5.12 (–99.98)
12 h	–2.23 (–98.76)	–3.12 (–99.85)	–0.72 (–77.86)	–3.20 (–99.89)	–4.89 (–99.99)	–2.90 (–99.70)	–1.09 (–92.27)	–5.94 (–100.00)
24 h	–2.54 (–99.05)	–3.87 (–99.98)	–0.93 (–79.70)	–3.69 (–99.94)	–5.21 (–99.99)	–4.14 (–99.99)	–2.15 (–99.19)	–5.40 (–99.99)
C max
0.5 h	0.11 (31.36)	0.05 (–19.35)	0.04 (10.04)	–1.18 (–87.16)	–0.49 (–67.08)	–0.14 (–27.68)	0.00 (6.07)	–1.53 (–87.07)
1 h	0.02 (6.20)	–0.14 (–40.29)	0.05 (11.67)	–1.46 (–95.32)	–1.22 (–98.29)	–0.33 (–45.14)	0.05 (13.61)	–3.38 (–99.84)
2 h	–0.02 (–1.98)	–0.27 (–48.70)	–0.03 (–7.73)	–2.32 (–98.95)	–2.56 (–99.70)	–0.74 (–74.71)	–0.03 (–5.78)	–4.51 (–99.99)
4 h	–0.20 (–28.82)	–0.86 (–83.03 )	–0.04 (–3.40)	–3.45 (–99.86)	–3.31 (–99.95)	–2.76 (–99.81)	–0.10 (–17.50)	–5.77 (–99.99)
6 h	–1.15 (–88.35)	–1.85 (–96.64)	–0.15 (–24.95)	–3.85 (–99.95)	–4.06 (–99.99)	–3.53 (–99.97)	–0.01 (9.99)	–6.68 (–100.00)
12 h	–2.16 (–98.10)	–2.30 (–99.67)	–0.36 (–55.28)	–4.54 (–99.99)	–5.02 (–99.99)	–3.94 (–99.95)	–0.08 (–9.89)	–6.68 (–100.00)
24 h	–4.68 (–99.99)	–3.68 (–96.43)	–0.87 (–85.27)	–5.67 (–99.99)	–5.57 (–99.99)	–5.71 (–99.99)	–0.50 (–56.00)	–6.68 (–100.00)
Tissuemax
0.5 h	0.08 (21.56)	0.09 (23.60)	–0.03 (–5.91)	–1.39 (–95.24)	–0.28 (–43.46)	–0.10 (–12.48)	–0.09 (–18.23)	–2.85 (–98.88)
1 h	–0.09 (–14.77)	0.11 (61.01)	0.03 (8.58)	–1.65 (–95.26)	–1.06 (–67.31)	–0.13 (–24.14)	–0.10 (–20.78)	–4.02 (–99.99)
2 h	–0.09 (–16.47)	–0.14 (–12.23)	0.02 (4.89)	–2.10 (–98.67)	–1.94 (–97.36)	–0.22 (–37.50)	–0.05 (–3.97)	–4.83 (–99.99)
4 h	–0.75 (–78.94)	–0.83 (–47.54)	–0.09 (–18.53)	–3.21 (–99.61)	–3.00 (–99.68)	–2.35 (–99.24)	–0.21 (–31.18)	–6.17 (–100.00)
6 h	–1.91 (–98.28)	–1.56 (–90.99)	–0.16 (–30.40)	–3.62 (–99.72)	–3.40 (–99.90)	–3.73 (–99.98)	–0.58 (–67.72)	–6.72 (–100.00)
12 h	–3.17 (–96.62)	–2.67 (–99.72)	–0.01 (14.44)	–4.44 (–99.90)	–3.75 (–99.98)	–4.42 (–99.82)	–0.52 (–47.71)	–6.72 (–100.00)
24 h	–4.07 (–99.94)	–4.31 (–99.99)	–0.74 (–81.19)	–5.26 (–99.99)	–4.47 (–99.99)	–4.59 (–99.95)	–1.24 (–91.03)	–6.72 (–100.00)

S. pseudintermedius

Negative values indicate a reduction in viable cells and positive values indicate growth.

MPC drug concentration pradofloxacin versus doxycycline at 24 h

Tissue

E. coli

MIC drug concentration at 6 h pradofloxacin versus cefovecin

Tissue

When 106 cfu/mL were exposed to MPC drug concentrations of the four drugs, pradofloxacin (P = 0.0001) and cefovecin (P = 0.0001) killed more cells than doxycycline did following 24 h of drug exposure.

Exposure of 106 cfu/mL to the C max drug concentration showed statistically significant differences in kill following 4 h of exposure to pradofloxacin versus doxycycline (P < 0.0006) or cefazolin (P = 0.0042), pradofloxacin versus doxycycline (P < 0.0001) or cefazolin (P = 0.0007). Following 12 h of drug exposure, more cells were killed by pradofloxacin than by doxycycline (P < 0.0001). Following 24 h of drug exposure, more cells were killed by pradofloxacin than by doxycycline (P < 0.0001) and by cefovecin (P = 0.007) and cefazolin (P = 0.0005) than doxycycline.

Exposure of 106 cfu/mL to the Tissuemax drug concentration showed a statistically significant difference in killing by pradofloxacin versus doxycycline (P = 0.0054) following 6 h of drug exposure, pradofloxacin versus doxycycline (P < 0.0001) following 12 h, cefazolin versus doxycycline (P = 0.0312) following 12 h, pradofloxacin versus doxycycline (P < 0.0001) at 24 h and cefovecin versus doxycycline (P = 0.0004) and cefazolin versus doxycycline (P = 0.0018) following 24 h of drug exposure.

Exposure of 107 cfu/mL of S. pseudintermedius (Table 3) to the MIC drug concentration of the four drugs tested showed a significant difference between cefovecin and cefazolin (P = 0.0116) following 12 h of drug exposure. At the MPC drug concentration, significantly more bacterial cells were killed by pradofloxacin than doxycycline (P = 0.0123) and by cefovecin (P = 0.0118) and cefazolin (P = 0.0105) than doxycycline following 12 h of drug exposure. Cefovecin killed more cells than doxycycline (P = 0.0013) following 24 h of drug exposure. At the C max drug concentration, pradofloxacin killed more cells by 4 and 6 h than doxycycline did (P = 0.0999 and P = 0.0081, respectively) and showed a tendency toward more killing than cefazolin (P = 0.06) at 4 h. Following 12 h of drug exposure, pradofloxacin killed more cells than doxycycline did (P < 0.0001) as did cefovecin (P = 0.0014). At 24 h of drug exposure, more cells were killed by pradofloxacin (P < 0.0001), cefovecin (P = 0.0002) and cefazolin (P = 0.0045) than doxycycline. At the Tissuemax drug concentration, more bacterial cells were killed by pradofloxacin than by doxycycline following 6 h (P = 0.0001) and 12 h (P < 0.0001) of drug exposure. Additionally, at 12 h, more cells were killed by cefovecin (P < 0.0001) and cefazolin (P = 0.0017) than by doxycycline (growth). Pradofloxacin (P < 0.0001), cefovecin (P < 0.0001) and cefazolin (P = 0.0001) killed more cells following 24 h of drug exposure than doxycycline did.

Table 3

Log10 reduction in viable cells (percentage of cells killed) over time for suspensions of Staphylococcus pseudintermedius and Escherichia coli (107 cfu/mL) exposed to various concentrations of cefazolin, cefovecin, doxycycline and pradofloxacin.

Variable	S. pseudintermedius				E. coli
	Cefazolin	Cefovecin	Doxycycline	Pradofloxacin	Cefazolin	Cefovecin	Doxycycline	Pradofloxacin
Minimum inhibitory concentration (MIC)
0.5 h	0.07 (19.57)	0.00 (1.00)	–0.02 (–4.39)	0.06 (14.49)	0.08 (4.68)	–0.01 (–2.52)	0.11 (39.07)	–0.05 (–12.15)
1 h	0.09 (25.67)	0.01 (10.52)	–0.03 (–0.89)	0.00 (0.41)	–0.05 (–10.85)	0.00 (–0.88)	0.01 (9.74)	–0.06 (–12.54)
2 h	0.19 (72.56)	–0.01 (2.05)	0.14 (39.74)	0.08 (21.90)	–0.27 (–43.94)	–0.21 (–30.93)	0.09 (22.58)	–0.05 (–9.29)
4 h	0.29 (124.33)	–0.22 (7.75)	–0.03 (–3.76)	–0.07 (–15.11)	–0.08 (76.36)	–0.73 (–62.94)	0.10 (24.58)	0.12 (36.02)
6 h	0.44 (355.49)	–0.12 (–8.43)	–0.06 (–12.01)	–0.20 (–30.13)	–0.16 (177.90)	–0.34 (–51.95)	0.18 (23.70)	0.08 (29.81)
12 h	0.45 (296.69)	–1.11 (–64.79)	0.12 (34.00)	–0.25 (–32.07)	0.71 (905.53)	–0.62 (61.40)	0.34 (127.55)	–0.02 (5.50)
24 h	0.61 (313.62)	–1.14 (–89.32)	0.60 (363.71)	0.00 (2.99)	1.22 (1704.31)	–0.22 (38.34)	–1.44 (149.08)	–0.15 (1.86)
Mutant prevention concentration (MPC)
0.5 h	0.01 (9.37)	–0.02 (–4.88)	0.00 (0.66)	0.01 (14.77)	–0.48 (–64.62)	–0.07 (–12.58)	0.03 (13.16)	–0.55 (–55.40)
1 h	0.06 (17.33)	–0.10 (–19.07)	–0.04 (–6.68)	–0.10 (–19.26)	–1.57 (–95.04)	–0.05 (–9.96)	–0.04 (–8.11)	–2.17 (–95.07)
2 h	–0.09 (–12.89)	–0.15 (–24.27)	–0.06 (–9.52)	–0.48 (–61.47)	–2.21 (–99.34)	–0.45 (–62.43)	–0.10 (–20.71)	–3.32 (–99.65)
4 h	–0.55 (–63.21)	–0.57 (–33.46)	–0.11 (–10.80)	–1.18 (–87.57)	–3.20 (–99.90)	–1.30 (–92.57)	–0.45 (–63.45)	–3.91 (99.93)
6 h	–1.51 (–90.03)	–1.20 (–65.90)	–0.29 (–46.80)	–1.64 (–93.76)	–3.88 (–99.97)	–2.08 (–98.61)	–0.91 (–86.98)	–4.13 (–99.98)
12 h	–2.40 (–99.52)	–2.48 (–99.35)	–0.34 (–53.56)	–2.36 (–99.31)	–3.75 (–99.71)	–2.73 (–99.53)	–1.32 (–94.94)	–5.08 (–99.97)
24 h	–2.46 (–89.36)	–4.06 (–99.98)	–0.83 (–83.72)	–3.20 (–96.69)	–5.45 (–99.99)	–4.19 (–99.99)	–3.05 (–99.18)	–4.50 (–99.99)
C max
0.5 h	–0.03 (–2.00)	–0.03 (–13.40)	–0.03 (–18.28)	–0.79 (–82.66)	–0.36 (–55.00)	–0.16 (–29.05)	0.00 (1.16)	–2.30 (–97.04)
1 h	–0.05 (–6.91)	–0.09 (–17.16)	–0.04 (–6.72)	–1.34 (–93.51)	–1.68 (–97.81)	–0.01 (0.94)	–0.12 (–20.57)	–2.74 (–98.95)
2 h	–0.11 (–18.18)	–0.30 (–39.17)	–0.04 (–0.44)	–2.21 (–98.57)	–2.48 (–99.58)	–1.10 (–89.23)	–0.12 (–21.90)	–3.96 (–99.98)
4 h	–0.58 (–65.09)	–0.75 (–47.20)	–0.07 (–8.76)	–2.97 (–99.69)	–3.31 (–99.95)	–2.81 (–99.80)	–0.27 (–44.12)	–4.57 (–99.990
6 h	–1.20 (–91.10)	–1.57 (–90.15)	–0.18 (–31.68)	–3.40 (–99.88)	–3.73 (–99.99)	–3.25 (–99.94)	–0.16 (–41.24)	–5.61 (–99.99)
12 h	–3.11 (–99.91)	–3.49 (–99.91)	0.10 (218.22)	–4.19 (–99.96)	–4.92 (–99.99)	–3.52 (–99.97)	–0.41 (–55.87)	–5.97 (–99.99)
24 h	–4.69 (–99.99)	–4.80 (–99.99)	–1.09 (–91.84)	–5.09 (–99.99)	–5.52 (–99.99)	–5.08 (–99.99)	–0.54 (–70.56)	–7.39 (–100.00)
Tissuemax
0.5 h	–0.01 (–24.80)	0.05 (13.45)	0.00 (4.68)	–1.34 (–91.04)	–0.30 (–47.280	–0.11 (–16.45)	–0.07 (–16.31)	–3.39 (–99.95)
1 h	–0.03 (–6.44)	–0.10 (–18.33)	–1.49 (16.01)	–1.67 (–93.69)	–1.44 (–76.60)	–0.12 (–24.52)	–0.09 (–18.65)	–3.81 (–99.98)
2 h	–0.11 (–22.90)	–0.18 (–26.34)	–1.27 (24.71)	–2.25 (–97.58)	–1.89 (–98.30)	–0.63 (–65.78)	–0.08 (–17.10)	–4.03 (–99.98)
4 h	–0.63 (–72.09)	–0.45 (–51.48)	6.40 (25.64)	–3.09 (–99.49)	–2.84 (–99.74)	–2.80 (–99.69)	–0.14 (–26.89)	–5.28 (–99.99)
6 h	–1.43 (–94.36)	–1.44 (–89.46)	0.10 (38.43)	–3.87 (–99.92)	–3.14 (–99.80)	–2.88 (–99.60)	–0.23 (–41.27)	–6.58 (–99.99)
12 h	–2.84 (–99.83)	–2.87 (–99.60	0.01 (4.82)	–4.72 (–99.97)	–2.82 (–99.77)	–3.54 (–99.96)	–0.13 (–25.42)	–7.30 (–100.00)
24 h	–4.42 (–99.99)	–4.23 (–99.99)	–0.70 (–65.65)	–5.78 (–99.99)	–3.03 (–85.93)	–4.61 (–99.93)	–0.45 (–62.30)	–7.79 (–100.00)

S. pseudintermedius

MIC drug concentration following 12 h of drug exposure cefazolin versus cefovecin

MPC drug concentration pradofloxacin versus doxycycline at 12 h

Tissue

E. coli

Tissue

Exposure of 108 cfu/mL and 109 cfu/mL to the MIC, MPC, C max and Tissuemax drug concentrations of the four drugs tested did not yield any significant differences in organism killed by individual drugs (Tables 4 and 5).

Table 4

Log10 reduction in viable cells (percentage of cells killed) over time for suspensions of Staphylococcus pseudintermedius and Escherichia coli (108 cfu/mL) exposed to various concentrations of cefazolin, cefovecin, doxycycline and pradofloxacin.

Variable	S. pseudintermedius				E. coli
	Cefazolin	Cefovecin	Doxycycline	Pradofloxacin	Cefazolin	Cefovecin	Doxycycline	Pradofloxacin
Minimum inhibitory concentration (MIC)
0.5 h	0.00 (0.47)	0.01 (4.02)	0.04 (8.37)	–0.03 (–3.29)	–0.04 (–7.15)	–0.03 (5.79)	0.08 (34.16)	0.08 (21.68)
1 h	–0.01 (–1.30)	0.09 (25.82)	0.05 (11.210	0.01 (–4.83)	–0.17 (–27.56)	0.07 (23.33)	0.02 (13.14)	0.18 (54.39)
2 h	0.06 (15.51)	–0.01 (–0.16)	–0.01 (–0.66)	–0.05 (–8.30)	–0.14 (–25.14)	–0.06 (–8.31)	–0.0 (12.91)	0.09 (25.43)
4 h	0.13 (36.57)	0.07 (20.02)	0.07 (17.27)	0.07 (18.76)	–0.04 (23.27)	0.08 (26.12)	0.14 (43.25)	0.15 (43.49)
6 h	0.11 (32.13)	–0.08 (–12.33)	0.11 (32.85)	0.02 (3.68)	0.03 (24.48)	–0.15 (–27.88)	0.23 (80.77)	0.11 (32.32)
12 h	0.11 (27.75)	–0.07 (–27.92)	0.12 (34.16)	–0.02 (–3.56)	0.37 (176.68)	0.00 (3.16)	0.20 (63.02)	0.10 (31.13)
24 h	–0.09 (–15.89)	0.29 (424.16)	0.17 (50.35)	–0.12 (–16.37)	0.37 (140.46)	–0.18 (–32.09)	0.16 (57.97)	–0.01 (3.87)
Mutant prevention concentration (MPC)
0.5 h	–0.01 (–1.73)	–0.02 (–5.08)	0.04 (10.62)	–0.05 (–11.27)	–0.45 (–60.34)	–0.13 (–24.48)	0.11 (32.19)	–0.53 (–64.26)
1 h	0.03 (6.13)	–0.14 (–25.10)	0.11 (31.94)	–0.01 (–1.37)	–1.33 (–90.58)	–0.01 (–0.30)	–0.01 (–0.06)	–1.25 (–80.57)
2 h	–0.02 (–3.84)	0.02 (13.22)	0.03 (9.61)	–0.11 (–21.50)	–2.01 (–98.60)	–0.05 (4.89)	0.09 (54.88)	–1.99 (–92.73)
4 h	–0.11 (–19.17)	0.01 (1.59)	0.06 (24.79)	–0.23 (–37.70)	–2.61 (–99.54)	–0.26 (–43,26)	–0.18 (–31.46)	–2.11 (–96.13)
6 h	–0.20 (–29.09)	0.11 (31.80)	0.07 (21.17)	–0.16 (–31.62)	–2.95 (–99.81)	–0.34 (–47.76)	–0.23 (–29.18)	–2.47 (–98.15)
12 h	–0.24 (–32.59)	–0.08 (–15.06)	0.06 (15/53)	–0.09 (–15.57)	–3.17 (–99.85)	0.00 (3.26)	–0.50 (–57.47)	–2.92 (–99.57)
24 h	–0.67 (–13.93)	–0.40 (–58.38)	–0.47 (–60.08)	–0.31 (–47.77)	–1.44 (–85.53)	–0.02 (–3.37)	–0.59 (–64.83)	–3.10 (–99.65)
C max
0.5 h	0.02 (7.93)	–0.02 (–3.27)	0.01 (0.74)	–0.50 (–66.46)	–0.31 (–45.13)	–0.14 (–22.37)	–0.12 (–23.56)	–2.07 (–98.45)
1 h	0.02 (4.84)	–0.06 (–12.95)	–0.03 (–7.48)	–0.82 (–83.62)	–1.12 (–92.19)	–0.17 (–30.03)	–0.09 (–17.05)	–3.10 (–99.88)
2 h	0.03 (10.71)	–0.14 (–30.35)	–0.13 (–24.48)	–1.39 (–94.44)	–2.20 (–99.20)	–0.64 (–74.22)	0.02 (6.40)	–3.59 (–99.97)
4 h	–0.10 (–9.71)	–0.10 (–19.20)	–0.01 (–2.76)	–1.72 (–96.11)	–2.51 (–99.64)	–0.83 (–78.70)	–0.09 (–13.50)	–3.87 (–99.98)
6 h	–0.12 (–20.47)	–0.07 (–41.19)	0.03 (–5.94)	–1.82 (–96.22)	–2.67 (–99.75)	–1.52 (–94.16)	–0.02 (10.78)	–4.09 (–99.97)
12 h	–0.40 (–51.29)	–0.16 (–24.48)	0.02 (7.07)	–1.61 (–92.52)	–2.46 (–99.11)	–2.56 (–99.52)	0.02 (11.00)	–4.81 (–99.99)
24 h	–1.45 (80.42)	–0.34 (–38.86)	0.15 (6.44)	–2.40 (–98.26)	–1.52 (–88.19)	–4.54 (–99.99)	–0.04 (7.54)	–5.51 (–99.99)
Tissuemax
0.5 h	–0.06 (–16.35)	0.00 (4.47)	0.00 (–0.73)	–1.08 (–94.30)	–0.27 (–39.99)	–0.23 (–40.36)	0.03 (52.73)	–2.76 (–90.85)
1 h	–0.01 (–1.33)	–0.02 (–1.27)	0.03 (6.56)	–1.47 (–97.86)	–0.89 (–61.19)	–0.19 (–16.64)	–0.10 (8.07)	–3.20 (–99.94)
2 h	–0.09 (–14.91)	–0.04 (–6.15)	–0.12 (–23.94)	–1.79 (–98.82)	–1.93 (–97.42)	–0.61 (–62.96)	0.00 (6.38)	–3.65 (–99.99)
4 h	–0.15 (–27.75)	–0.39 (–030.35)	0.04 (15.59)	–2.12 (–99.33)	–2.34 (–99.15)	–1.36 (–84.62)	0.05 (38.98)	–3.98 (–99.96)
6 h	–0.26 (–37.79)	–0.17 (–27.77)	–0.01 (0.78)	–2.27 (–99.54)	–2.61 (–99.20)	–1.01 (–80.31)	–0.04 (53.83)	–4.44 (–99.98)
12 h	–0.90 (–57.97)	–0.18 (–30.02)	0.03 (9.40)	–2.06 (–99.29)	–2.21 (–98.27)	–2.34 (–95.97)	0.36 (229.89)	–4.81 (–99.99)
24 h	–1.72 (–57.91)	–1.51 (–85.59)	–0.02 (–3.47)	–1.96 (–99.12)	–1.25 (–35.85)	–3.74 (–96.02)	0.02 (56.97)	–6.54 (–99.99)

E. coli

MPC drug concentration at 4 h pradofloxacin versus cefovecin

Tissue

Table 5

Log10 reduction in viable cells (percentage of cells killed) over time for suspensions of Staphylococcus pseudintermedius and Escherichia coli (109 cfu/mL) exposed to various concentrations of cefazolin, cefovecin, doxycycline and pradofloxacin.

Variable	S. pseudintermedius				E. coli
	Cefazolin	Cefovecin	Doxycycline	Pradofloxacin	Cefazolin	Cefovecin	Doxycycline	Pradofloxacin
Minimum inhibitory concentration
0.5 h	–0.01 (–1.30)	–0.04 (–0.74)	–0.05 (–11.41)	–0.05 (–5.55)	–0.11 (–20.93)	–0.11 (–21.92)	–0.06 (–7.10)	–0.07 (–13.40)
1 h	0.00 (–0.16)	–0.05 (–0.31)	–0.01 (–0.47)	–0.13 (–25.66)	–0.09 (–15.90)	0.04 (11.99)	0.06 (16.70)	–0.02 (–4.80)
2 h	0.00 (–0.49)	0.06 (27.44)	–0.03 (–3.11)	0.08 (1.68)	–0.05 (–11.88)	–0.05 (–10.27)	0.14 (39.75)	–0.03 (–5.86)
4 h	0.07 (17.49)	0.10 (48.24)	–0.09 (–17.05)	0.01 (3.15)	–0.09 (–10.16)	–0.25 (–40.82)	0.38 (166.05)	–0.01 (0.06)
6 h	0.04 (10.66)	–0.01 (11.90)	–0.07 (–12.51)	–0.15 (–24.79)	0.13 (34.86)	–0.15 (–12.64)	0.33 (179.36)	–0.03 (–4.00)
12 h	0.37 (211.77)	0.14 (1.34)	–0.30 (–33.03)	0.05 (13.65)	0.23 (73.20)	–0.10 (4.10)	0.13 (46.85)	–0.10 (–20.24)
24 h	–0.05 (–11.48)	–0.07 (–37.68)	–0.11 (15.89)	–0.13 (–24.28)	–0.04 (–8.02)	–0.18 (–30.73)	0.18 (63.01)	–0.13 (–10.99)
Mutant Prevention concentration
0.5 h	–0.03 (–2.53)	0.14 (48.09)	–0.05 (–2.87)	0.00 (2.69)	–0.02 (–3.03)	–0.04 (–5.85)	0.16 (49.33)	0.18 (58.26)
1 h	–0.03 (–5.22)	0.22 (78.10)	–0.04 (–5.64)	–0.05 (–9.43)	–0.22 (–36.94)	0.13 (48.57)	–0.01 (–2.02)	0.16 (48.53)
2 h	0.00 (3.07)	0.15 (50.59)	–0.03 (0.28)	–0.03 (–3.47)	–0.08 (–13.48)	–0.09 (–18.80)	0.09 (37.83)	0.11 (32.76)
4 h	0.00 (–1.72)	0.20 (71.48)	–0.04 (–4.37)	–0.13 (–15.41)	–0.13 (–20.95)	–0.02 (–2.75)	0.05 (13.00)	–0.07 (–29.66)
6 h	–0.05 (–2.41)	0.18 (56.50)	–0.05 (–10.81)	0.03 (10.49)	0.21 (244.33)	–0.05 (0.55)	–0.07 (–12.38)	–0.14 (–12.01)
12 h	0.08 (–21.51)	0.15 (42.71)	–0.27 (–22.52)	–0.05 (–6.55)	0.09 (30.63)	0.14 (46.57)	–0.04 (1.39)	–0.16 (–21.60)
24 h	–0.15 (–19.57)	0.05 (33.26)	–0.08 (–13.82)	–0.15 (–22.26)	0.05 (15.37)	–0.06 (–12.17)	–0.08 (–15.63)	–0.44 (–59.93)
Cmax
0.5 h	0.05 (20.42)	0.01 (–0.17)	–0.02 (–0.27)	–0.09 (–16.70)	0.06 (14.45)	0.00 (4.30)	0.12 (37.69)	–0.48 (–61.12)
1 h	0.06 (15.06)	–0.14 (–21.34)	0.09 (27.08)	–0.15 (–26.11)	0.03 (10.07)	0.11 (49.33)	0.06 (18.09)	–0.63 (–67.51)
2 h	0.09 (24.65)	–0.01 (–4.07)	0.03 (10.62)	–0.20 (–33.97)	0.03 (11.66)	0.00 (6.64)	0.14 (51.59)	–1.26 (–93.04)
4 h	0.05 (20.74)	0.06 (20.06)	0.04 (11.97)	–0.40 (–56.04)	0.20 (91.82)	0.10 (26.28)	–0.05 (–9.90)	–1.97 (–98.48)
6 h	0.06 (14.76)	0.04 (9.16)	–0.09 (–11.99)	–0.60 (–65.82)	0.07 (19.63)	0.20 (62.89)	–0.04 (–5.76)	–2.83 (–99.80)
12 h	0.15 (41.35)	–0.03 (–4.26)	–0.01 (14.52)	–0.29 (–38.10)	0.15 (55.44)	0.26 (100.51)	0.14 (38.49)	–3.08 (–99.85)
24 h	0.09 (24.00)	–0.04 (4.92)	0.02 (7.55)	–1.04 (–88.54)	0.01 (23.95)	0.10 (57.90)	–0.15 (–11.64)	–3.58 (–99.93)
Tissuemax
0.5 h	–0.04 (–8.21)	0.00 (18.55)	0.04 (14.59)	–0.30 (–47.54)	–0.10 (–19.87)	–0.08 (–24.16)	0.08 (20.01)	–0.87 (–85.73)
1 h	–0.02 (–3.91)	–0.05 (13.93)	0.04 (15.76)	–0.65 (–70.34)	–0.10 (–18.48)	–0.09 (–17.22)	0.00 (0.91)	–1.40 (–94.79)
2 h	–0.05 (–10.73)	–0.03 (9.38)	0.06 (22.88)	–0.91 (–85.87)	0.10 (28.61)	–0.06 (–12.95)	0.13 (36.43)	–2.20 (–99.66)
4 h	0.05 (13.21)	–0.16 (–2.88)	0.09 (29.31)	–0.86 (–77.41)	0.00 (1.51)	–0.11 (–21.61)	0.05 (13.05)	–2.98 (–99.94)
6 h	–0.08 (–17.02)	–0.11 (5.43)	0.11 (54.44)	–1.06 (–82.32)	0.11 (33.71)	–0.18 (–29.80)	0.02 (5.95)	–3.50 (–99.98)
12 h	0.06 (17.82)	–0.11 (3.82)	0.33 (101.77)	–0.74 (–64.43)	0.12 (49.27)	–0.12 (–23.42)	–0.08 (39.97)	–4.00 (–99.99)
24 h	–0.13 (–24.67)	–0.16 (0.57)	0.02 (6.03)	–1.30 (93.90)	0.04 (16.09)	–0.18 (–31.86)	0.06 (20.05)	–4.45 (–99.99)

E. coli

Tissue

Exposure of 106 cfu/mL of E. coli (Table 2) to the MIC drug concentration of the four drugs tested showed statistically significant differences in killing of bacterial cells by cefovecin versus pradofloxacin following 6 h (P = 0.0288), 12 h (P = 0.0261) and 24 h (P < 0.0001) of drug exposure. Statistically significant differences also were seen for cefovecin versus doxycycline following 12 h (P = 0.0180) and for cefovecin versus cefazolin following 12 h (P < 0.0001) and 24 h (P < 0.0001) of drug exposure. For MPC drug concentrations, statistically significant differences were not seen between the study drugs. Exposure to the C max drug concentration yielded statistically significant differences in killing between the compounds: 1 h pradofloxacin versus doxycycline and cefovecin (P < 0.0001 for both comparisons), and cefovecin versus cefazolin (P = 0.0386); 2 h pradofloxacin versus cefovecin and doxycycline (P = 0.0001 for both comparisons), and cefovecin versus cefazolin (P = 0.0002); 4 h pradofloxacin versus cefovecin and doxycycline (P < 0.001 for both), and pradofloxacin versus cefazolin (P = 0.0002), doxycycline versus cefovecin (P = 0.0066) and doxycycline versus cefazolin (P < 0.0001); 6 h pradofloxacin versus doxycycline (P < 0.0001), cefovecin (P < 0.0001), and doxycycline and cefazolin (P < 0.0001) versus doxycycline; 12 h pradofloxacin versus doxycycline (P < 0.0001), and doxycycline versus cefovecin (P < 0.0001) and cefazolin (P < 0.0001); 24 h pradofloxacin versus doxycycline (P < 0.0001), and cefovecin (P < 0.0001) versus doxycycline and cefazolin (P < 0.0001) versus doxycycline. Exposure of 106 cfu/mL of E. coli to the Tissuemax drug concentration showed more killing by pradofloxacin than cefovecin (P = 0.0143) following 30 min of drug exposure and more cells killed by pradofloxacin than cefovecin or doxycycline (P‐values from <0.0001 to 0.02) following 1, 2, 4 and 6 h of drug exposure. Cefovecin killed more cells than doxycycline (P = 0.0015) following 6 h of drug exposure. Statistically significant differences in kill were seen between pradofloxacin and doxycycline (P < 0.0001), cefovecin and doxycycline (P < 0.0001), and cefazolin and doxycycline (P = 0.02) following 12 h of drug exposure. Finally, more cells were killed by pradofloxacin (P < 0.0001) than doxycycline and cefovecin killed more cells than doxycycline (P = 0.0045) following 24 h of drug exposure.

Exposure of 107 cfu/mL of E. coli (Table 3) to the MIC drug concentrations of the four drugs tested did not show statistically significant differences in kill by the four agents. At the MPC drug concentration, an overall effect was seen between pradofloxacin and doxycycline at all time points (P = 0.0139) and with cefovecin (P = 0.0543). At the C max drug concentration statistically significant more cells were killed by pradofloxacin than by doxycycline or cefovecin following 30 min, 1, 2, 4, 6, 12 and 24 h after drug exposure (P‐values ranged from <0.0001 to 0.0441). Cefazolin killed more cells than did doxycycline following 1, 2, 4, 6, 12 and 24 h of drug exposure (P < 0.0001–0.0071). Cefazolin killed more cells than cefovecin did following 1 and 12 h of drug exposure (P‐values 0.001 and 0.0057, respectively) and cefovecin killed more cells than doxycycline following 4, 6, 12 and 24 h of drug exposure (P < 0.0001 for all exposures).

Exposure of 108 cfu/mL of E. coli (Table 4) to the MIC drug concentration of the four drugs tested did not yield significant differences in killing between any of the compounds. At the MPC drug concentration, pradofloxacin killed more cells than cefovecin following 4, 6, 12 and 24 h of drug exposure (P = 0.0004–0.0441). Pradofloxacin killed more cells than doxycycline at 12 and 24 h of drug exposure (P = 0.0083 and 0.0087). Cefazolin killed more cells than doxycycline did at 6 h (P = 0.0015) and 12 h (P = 0.0015), and cefazolin killed more cells than cefovecin did at 6 h (P = 0.0497) and 12 h (P = 0.0422) following drug exposure. At the C max drug concentration, pradofloxacin killed more cells than doxycycline did following 1 h (P = 0.0020), 2 h (P = 0.0002), 4 h (P < 0.0001), 6 h (P < 0.001), 12 h (P < 0.001) and 24 h (P < 0.0001) of drug exposure. At the Tissuemax drug concentration, pradofloxacin killed more cells than doxycycline did following 2, 4, 6, 12 and 24 h of drug exposure (P‐values from <0.0001 to 0.0336). No other comparisons were statistically significant for differences in bacterial killing.

No significant differences in kill were seen between any of the investigated compounds when 109 cfu/mL were exposed to the MIC or MPC drug concentrations of the four agents tested (Table 5). At the C max drug concentration pradofloxacin killed more cells following 4 h of drug exposure than cefovecin did (P = 0.0028). At 6, 12 and 24 h following drug exposure, pradofloxacin killed more cells than doxycycline, cefovecin and cefazolin (P‐values were at <0.0001 for all comparisons). At the Tissuemax drug concentration, pradofloxacin killed more cells than doxycycline, cefovecin or cefazolin (growth) following 2, 4, 6, 12 and 24 h of drug exposure (P‐values were <0.0001 for all comparisons).

Discussion

Previous reviews20, 21 included the use of 1st and 3rd generation cephalosporins, fluoroquinolones and tetracyclines (doxycycline) for the treatment of canine skin infections and, as such, were appropriate to investigate in this report and our previous study.7 Cefovecin was shown to be as effective as amoxicillin/clavulanic acid for the treatment of skin infections in dogs,22 and pradofloxacin was shown to be efficacious for the treatment of superficial and deep pyoderma in dogs.23 A previous study reported on the bactericidal properties of pradofloxacin against veterinary pathogens including S. pseudintermedius and E. coli canine strains.24 Another study reported on the in vitro activity of cefovecin against S. pseudintermedius and E. coli strains with MIC90 values of 0.25 µg/mL and 1 µg/mL, respectively.25 The bactericidal activity of cefovecin also has been reported previously.26 Previous studies27 have reported on the bactericidal activity of cefazolin and commented on the tetracyclines being bacteriostatic.28 The previous determination of bactericidal or bacteriostatic concentrations for the various drugs tested is consistent with the findings in this study. Doxycycline is recommended in humans for staphylococcal and streptococcal bacterial strains and skin structure infections,29 yet randomized control trials in dogs are unavailable.21 Likewise, although cefazolin (and cefalexin) have been investigated for treatment of skin and skin structure infections in humans30, 31, randomized controlled trials are unavailable in companion animals.21 The pharmacokinetics of cefazolin for prophylactic administration in dogs has been studied.32 Comparing antibiotics for bactericidal versus bacteriostatic activity as well as speed of kill has clinical relevance, as commented by others.33, 34

In this report, we performed kill measurements using the same strains as in our previous report and the same clinically relevant drug concentrations. The major differences between this report and our previous publication were the time and intervals over which killing occurred (i.e. 3 h versus 24 h) and the densities of bacteria used in the assays. The varying bacterial densities are important to include as the densities of bacteria present during infection (6, 7, 8, 9, 10, 11) have been shown to exceed 105 cfu/mL, and testing drugs against the higher bacterial densities helps to effectively kill cells with reduced susceptibility, as has been shown to occur in bacterial densities >107 cfu/mL. These differences have been argued previously with MIC versus MPC testing.7, 8, 35, 36

Cefazolin, cefovecin and pradofloxacin are all considered bactericidal agents, whereas doxycycline is considered bacteriostatic based on the classical definition.5, 6 The definition of bactericidal versus bacteriostatic is problematic as it is based on a standard bacterial inoculum of 105 cfu/mL and does not appear to have relevance when higher bacterial densities are tested. Additionally, the differentiation of bactericidal from bacteriostatic drugs based on a >3 log10 reduction in viable cells versus <2 log10 reduction (respectively) is arbitrary. In previous work from our laboratory, agents traditionally considered bacteriostatic showed bactericidal properties when tested against higher bacterial densities.6 Having said that, in this study, doxycycline displayed bacteriostatic properties regardless of bacterial densities and/or drug concentrations tested; cefazolin, cefovecin and pradofloxacin were bactericidal over the densities tested and with killing more pronounced at MPC, C max and Tissuemax drug concentrations for all three drugs. For cefazolin and cefovecin, longer times of drug exposure were needed to achieve substantial reductions in viable cells and this is consistent with time‐dependent drugs.

As reported previously, the drug concentration used in this study was from published reports or estimated from published reports.18, 19, 26, 37, 38 Interestingly, for E. coli at the 106–108 cfu/mL densities and C max drug concentrations, statistically more cells were killed by pradofloxacin than by doxycycline and cefovecin within the first 1–4 h of drug exposure and in most instances, these differences were seen over the 24 h of drug exposure. Statistically significant differences with any comparisons were not seen until 4 h of drug exposure at the 109 cfu/mL density and thereafter at the 6, 12 and 24 h samplings. Statistically significant differences also were seen between agents at the Tissuemax, MPC and MIC drug concentrations depending on the density of bacteria and the time after drug exposure sampling.

Overall, but not exclusively, statistically significant differences in kill were seen more often between doxycycline and cefazolin, cefovecin or pradofloxacin than between cefazolin, cefovecin and pradofloxacin for S. pseudintermedius. At the C max and Tissuemax drug concentrations, statistically significant differences were seen between pradofloxacin and doxycycline at earlier sampling times (i.e. 4–6 h) but by 12–24 h following drug exposure, differences also were seen for cefazolin and cefovecin compared to doxycycline at the 106 cfu/mL and 107 cfu/mL densities. At the MPC drug concentrations, statistically significant differences were not observed between any comparisons until 12–24 h following drug exposure at the 106–107 cfu/mL densities.

In this and our previous report,7 we showed that killing of S. pseudintermedius and E. coli strains was different for the four drugs tested and did vary based on the bacterial density and time following drug exposure – generally being statistically different for pradofloxacin with short drug exposure times. A limitation of this and similar studies is that drug concentration remains constant over the duration of the measurements (i.e. 24 h in this report) and as such, does not truly reflect in vivo drug dynamics where drug elimination occurs; drug degradation over time was not measured. In addition, measurements were not corrected for protein binding; Dalhoff showed that high protein binding is associated with reduced antimicrobial activity,39 but protein binding <80–85% appears to be of slight clinical importance.40 Regardless, measurements as reported herein do allow for comparisons between drugs under controlled conditions which may be important clinically. Those observations are consistent with our previous report showing faster killing with pradofloxacin and also are consistent with cefazolin and cefovecin being time‐dependent antibiotics and pradofloxacin being a concentration‐dependent drug.

In human medicine, the overall trend has been toward shorter duration of antimicrobial therapy for uncomplicated infections, an approach not considered inferior to longer durations of therapy.4, 41, 42, 43, 44 In four guideline publications for recommended therapies for companion animal infections,21, 45, 46, 47 longer durations of therapy are recommended with the acknowledgement that shorter durations of therapy might be possible but for data being limited or unavailable. Longer duration of therapy may be necessary for chronic infections where biofilm formation may be a contributing factor.48

Determining effective durations of therapy involves clinical investigation supplemented with in vitro data. As such, data as reported here and in our previous report add to the in vitro data showing differences and similarities between compounds for killing or inhibition of clinically important pathogens. Such data may contribute to decisions related to therapeutic choices and duration of therapy.

		x106	x107	x108	x 109
cefazolin	S. pseudintermedius	1.4–8.7	1.6–9.7	1.9–8.8	2.1–8.7
	E. coli	1.6–4.1	1.6–5.3x107	1.5–4.6	1.6–5.3
cefovecin	S. pseudintermedius	1.6x106–7.0x107	1.5x107–1.1x108	1.2x108–1.3x109	1.1x109–1.1x1010
	E. coli	3.9–8.4	4.3–7.9	4.8–8.5	3.3x109–1x1012
doxycycline	S. pseudintermedius	3.1–8.4	4.8–7.1	4.7x108–1.2x109	2.9–8.7
	E. coli	2.1–5.7	2.2–6.9	2.9–6.3	3.0–5.9
pradofloxacin	S. pseudintermedius	5.7x106–4.3x107	6.7x107–1.3x108	4.7x108–9x109	7.3x109–1.7x1010
	E. coli	2.3–9.2	5.4–9.4	3.9–9.2	9.4x109–1.2x1010