In vitro and in vivo activity of contezolid (MRX-I

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(Photoelectric Colorimeter; Manostat Corp. New York, NY). 62. 63. Polystyrene 96-well round-bottom plates (Corning Inc., Corning, NY) were prepared with 50µl.
AAC Accepted Manuscript Posted Online 21 May 2018 Antimicrob. Agents Chemother. doi:10.1128/AAC.00493-18 Copyright © 2018 American Society for Microbiology. All Rights Reserved.

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In vitro and in vivo activity of contezolid (MRX-I) against Mycobacterium tuberculosis

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Carolyn Shoena#, Michelle DeStefanoa, Barry Hafkinb, and Michael Cynamona,c#

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a

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4 Central New York Research Corporation, Syracuse NY MicuRx Pharmaceuticals, Hayward, CA

Veterans Affairs Medical Center, Syracuse, NY

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Address Correspondence to Michael Cyanmon, [email protected] or Carolyn Shoen, [email protected]

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Abstract

25 The in vitro activity of contezolid (MRX-I) against clinical isolates of M. tuberculosis was

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evaluated using a microtiter broth dilution assay. MRX-I was as effective in vitro as linezolid

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(LZD). MRX-I and LZD were subsequently studied in BALB/c mice infected intranasally with

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M. tuberculosis Erdman. MRX-I and LZD at 100mg/kg significantly reduced the bacterial load

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in lungs compared to the untreated early and late controls.

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The emergence of multidrug (MDR) and extensively drug-resistant (XDR) M. tuberculosis has

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severely hampered the control of tuberculosis (TB) infection worldwide. Improved regimens to

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treat these types of infections are needed. Oxazolidininones were found to have promising in

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vitro activity against M. tuberculosis soon after their discovery (1, 2). Linezolid (LZD), the first

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oxazolidinone to be used in humans, was observed to have promising in vitro (3) and in vivo

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activities (4) against M. tuberculosis. LZD has been found to be a useful agent in regimens for

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therapy of drug-resistant TB in humans (5), however, its use has been limited in part due to

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toxicity issues which include myelosuppression, peripheral and optic neuropathy (5, 6, 7). Based

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on phase I clinical trials contezolid (MRX-I), a new oxazolidinone developed to treat Gram

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positive infections, such as methicillin-resistant Staphylococcus aureus and vancomycin-resistant

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Enterococcus, was shown to have decreased toxicity compared to LZD (6, 8). MRX-I may lead

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to a dramatic improvement in ease of use in patients with drug resistant TB. In this study we

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evaluated the in vitro and in vivo activities of MRX-I compared to LZD against M. tuberculosis.

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Isoniazid (INH) was purchased from Sigma-Aldrich Chemical Company (St. Louis, MO).

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Sutezolid (SZD) and tedizolid (TZD) were obtained from NIAID (Bethesda, MD) and Trius

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Pharmaceutical (San Diego, CA), respectively. LZD and MRX-I were provided by MicuRx

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Pharmaceuticals (Hayward, CA). For in vitro testing all drugs were dissolved in 100% dimethyl

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sulfoxide (DMSO) at 5mg/ml and were frozen at -20oC. Drugs were diluted in modified 7H10

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broth (pH 6.6; 7H10 agar formulation with agar and malachite green omitted) with 10% OADC

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(oleic acid, albumin, dextrose, catalase) enrichment (BBL Microbiology Systems, Cockeysville,

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MD) and 0.05% Tween 80.

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M. tuberculosis Erdman (ATCC 35801) and M. tuberculosis H37Rv (ATCC 27294) were

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purchased from the American Type Culture Collection (Manassas, VA). Clinical M.

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tuberculosis isolates were received from SUNY Upstate Medical University, University of

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Stellenbosch, South Africa (Dr. Tommie Victor), National Center of TB and Lung Disease of

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Georgia (Republic of Georgia, Dr. Natalia Shabladze), National Jewish Health (Dr. Leonid

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Heifets), and Public Health Research Institute (Dr. Barry Kreiswirth). The isolates were grown

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in modified 7H10 broth with 10% OADC enrichment and 0.05% Tween 80 on a rotary shaker at

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37oC for 7-10 days. The cultures were diluted to 100 Klett units [equivalent to 5 × 107 CFU/ml]

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(Photoelectric Colorimeter; Manostat Corp. New York, NY).

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Polystyrene 96-well round-bottom plates (Corning Inc., Corning, NY) were prepared with 50µl

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of modified 7H10 broth per well. Drugs were diluted in modified 7H10 broth to four-times the

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maximum concentration tested (64μg/ml for the oxazolidinones and 8μg/ml for INH) and 50µl

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was added to the first well and serial diluted, leaving the last well with broth only (positive

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growth control). Organisms were diluted in 7H10 broth to a final concentration of approximately

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1 X 105 CFU/ml (inoculum range was 6 X 104 – 2.4 X 106 CFU/ml). Fifty microliters of the

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inoculum was added to each well. Plates were sealed and incubated at 37oC in ambient air for

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14-21 days prior to reading. The minimal inhibitory concentration (MIC) was defined as the

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lowest concentration of drug required to visually inhibit growth of M. tuberculosis. The MIC

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assays were run in duplicate. The MIC50 and MIC90 are defined as the concentration at which

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50% or 90% of the clinical isolates tested were inhibited.

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The MICs of MRX-I compared to the three other oxazolidinones and INH against M.

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tuberculosis are presented in Table 1. We tested the compounds against both susceptible and

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MDR or XDR M. tuberculosis isolates. Eight M. tuberculosis isolates were resistant to INH

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(MIC >1µg/ml). MRX-I was as effective as LZD against all M. tuberculosis isolates. There was

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no appreciable difference in MIC90 (range 0.5µg/ml - 1µg/ml) between the oxazolidinones

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evaluated. The MIC50 and MIC90 for MRX-I, LZD, SZD, TZD were 0.5 µg/ml and 1 µg/ml, 0.5

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µg/ml and 1 µg/ml, 0.5 µg/ml and 0.5 µg/ml, and 0.125µg/ml and 0.5µg/ml respectively.

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Based on the promising in vitro results the drug was evaluated in vivo. Six-week old female

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BALB/c mice were purchased from Charles River Laboratories (Wilmington, DE) and

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maintained in the Syracuse Veterans Affairs Medical Center Veterinary Medical Unit in an

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animal Biosafety Level 3 facility. Mice were housed six to a cage in micro-isolator cages. The

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mice ingested water and Prolab® RMH 3000 rodent chow (PMI Nutrition International,

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Brentwood, MO.) ad libitum throughout the course of the studies. All animal protocols were

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approved by the Subcommittee for Animal Studies (SAS), Veterans Affairs Medical Center,

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Syracuse, NY. M. tuberculosis Erdman (ATCC 35801), grown in modified 7H10 broth with

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10% OADC and 0.05% Tween 80, was diluted to 5 X 107 CFU/ml and frozen at -70oC. On the

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day of infection the organism was thawed, sonicated, and further diluted to approximately 2.5 X

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105 CFU/ml. The actual inoculum was determined by titration and plating in triplicate on 7H10

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agar plates (BBL Microbiology Systems, Cockeysville, MD) supplemented with 10% OADC

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enrichment. The plates were incubated at 37°C in ambient air for 4 weeks prior to counting.

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Mice were anesthetized by intramuscular delivery of a telazol (45 mg/kg)/xylazine (7.5 mg/kg)

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cocktail (Lederle Parenterals, Carolina, Puerto Rico and Bayer Corp., Shawnee Mission, KS,

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respectively) and subsequently infected intranasally with 8.2 X 103 CFU of M. tuberculosis

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Erdman in a 20μl volume. Mice were randomly assigned to one of the following 6 groups:

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Untreated early controls (EC) for determination of the infection load at the initiation of therapy,

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late controls (LC) to determine the infection load at the completion of therapy, LZD 100mg/kg,

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MRX-I 100mg/kg once daily, MRX-I 50mg/kg twice daily, or MRX-I 25mg/kg twice daily.

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LZD was dissolved in 20% ethanol: 80% ddH2O to deliver 100mg/kg in a 0.2ml volume and

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was dosed once daily by gavage. MRX-I was dissolved in 20% DMSO : 20% hydroxypropyl β

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cyclodextrin: 60% ddH2O to deliver either 100mg/kg, 50mg/kg, or 25mg/kg in a 0.2ml volume.

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MRX-I was dosed at 100mg/kg once daily, 50mg/kg twice daily, or 25mg/kg twice daily by

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gavage.

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Treatment was initiated one week post-infection and was administered five days per week for

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four weeks. At the initiation of therapy the EC group was euthanized by CO2 asphyxiation as

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were the mice at the completion of therapy. Right lungs from all mice were aseptically removed

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and ground in a sealed tissue homogenizer (IdeaWorks! Laboratory Devices, Syracuse, NY).

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The number of viable organisms was determined by serial dilution and titration on 7H10 agar

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plates supplemented with 10% OADC. Plates were incubated at 37°C in ambient air for 4 weeks

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prior to counting.

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The viable cell counts per lung were converted to logarithms, which were then evaluated by

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analysis of variance (ANOVA). Statistically significant effects from the analysis of variance

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were further evaluated by Dunnett’s Multiple Comparisons post-test.

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Lungs from the early control mice had a bacterial load of approximately 4.67 ± 0.17 log10 CFU

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per lung (Table 2). The untreated late control group had significantly more mycobacteria in the

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lungs than the early control group (P < 0.05). LZD and MRX-I dosed at 100mg/kg once daily

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had significantly reduced the CFU recovered from the lungs compared to the early and late

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control mice (P < 0.05). There was no significant difference between the reduction seen with

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LZD or once daily MRX-I at 100mg/kg (P > 0.05). Twice daily MRX-I at 50mg/kg and 25mg/kg

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were significantly better than the late control mice (P < 0.05). Once daily MRX-I at 100mg/kg

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was significantly better than twice daily 50mg/kg and 25mg/kg (P < 0.05). There was no

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statistical difference between twice daily 50mg/kg of MRX-I and 25mg/kg (P > 0.05).

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This study evaluated the in vitro and in vivo activity of MRX-I against M. tuberculosis. The in

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vitro activity of MRX-I was similar to that of LZD against both drug-susceptible and drug-

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resistant M. tuberculosis. Its activity in a murine tuberculosis model was also similar to that of

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LZD. It would be interesting to determine if higher doses of MRX-I in mice result in enhanced

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activity.

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It would also be of interest to determine if the addition of the less toxic oxazolidinone, MRX-I,

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to current MDR TB therapy would result in a shorter course or perhaps would allow for the

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simplification of therapy. The present version of the Bangladesh regimen (9) requires 9 months

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of treatment with moxifoxacin, clofazimine, ethambutol, and pyrazinamide supplemented with

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prothionamide, kanamycin, and high-dose INH during the 4 month initial intensive phase.

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Perhaps MRX-I could substitute for the aminoglycoside thus avoiding the toxicity and

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administration issues associated with an aminoglycoside.

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Acknowledgements

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Partial support to perform these studies was provided by MicuRx Pharmaceuticals. Dr. Barry

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Hafkin is an employee of MicuRx Pharmaceuticals.

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References

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Table 1. MICs (µg/ml) of Contezolid (MRX-I) compared to Isoniazid (INH), Linezolid (LZD),

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Sutezolid (SZD), and Tedizolid (TZD) against 22 isolates of Mycobacterium tuberculosis. The

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MIC50 and MIC90 are defined as the concentration as which 50% or 90% of the clinical isolates

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tested were inhibited. Mtb Isolate Erdman H37Rv 532 277 365 764 AH517 HN878 676 BW9 487 C913 CDC1551 S1863 5 56 265 258 352 C-31 5037 S982

INH 0.06 0.06 0.06 0.06 0.06 0.06 0.03 0.06 0.06 0.06 0.03 0.06 0.03 0.03 1 2 1 2 2 4, 4 >8 8

LZD 1 0.5 0.5 1 0.5 0.5 1 1 1 1 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.25 1 1

SZD 1 0.25 0.5 0.25 0.25 0.25 0.5 0.5 0.5 0.5 0.5 0.5 0.25 0.25 0.5 0.5 0.5 0.5 0.25 0.125 0.5 0.5

TZD 0.25 0.125 0.125 0.125 0.125 0.125 0.25 0.5 0.5 0.5 0.125 0.125 0.125 0.125 0.125 0.125 0.25 0.125 0.125 0.125 0.25 0.25

MRX-I 1 0.5 1 1 1 1 1 2 2 1 1 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.25 1 1

MIC50 MIC90

0.06 4

0.5 1

0.5 0.5

0.125 0.5

0.5 1

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Table 2. Log10 CFU ± Standard Deviation of Mycobacterium tuberculosis in the lungs of mice

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infected and treated with Linezolid (LZD) and Contezolid (MRX-I). Groups Early Control Late Control LZD 100mg/kg MRX-I 100mg/kg MRX-I 50mg/kg twice daily

Number of mice 6 6 6 6 5

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Log CFU ± SD 4.67 ± 0.17 6.03 ± 0.31 3.61 ± 0.42 3.76 ± 0.47 4.55 ± 0.33 10

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5.07 ± 0.26 6 MRX-I 25mg/kg twice daily

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