Effects of sub-minimum inhibitory concentration antibiotic levels and temperature on growth kinetics and outer membrane protein expression in Mannheimia haemolytica and Haemophilus somnus

Abstract

The objective of this study was to determine the effects of sub-minimum inhibitory concentrations (sub-MICs) of 2 veterinary antibiotic preparations, chlortetracycline (CTC) and chlortetracycline-sulfamethazine (CTC + SMZ), on growth kinetics and outer membrane protein expression in Mannheimia haemolytica and Haemophilus somnus at normal and febrile body temperatures. Sub-minimum inhibitory concentrations of both antibiotics reduced the growth rates of M. haemolytica and H. somnus. Growth of both species was not inhibited when grown at 41 degrees C compared to 37 degrees C. There was no detectable consistent effect of antibiotic or temperature on outer membrane protein expression for either species. Our study indicates that sub-MIC levels of CTC and CTC + SMZ markedly impair growth of clinical M. haemolytica and H. somnus isolates, potentially allowing more effective host clearance during infection.

Figure 1

Growth kinetics of Mannheimia haemolytica strains 55518 (A), D80 (B), and D152 (C) in the presence of sub-minimum inhibitory concentrations (MICs) of chlortetracycline (CTC) at 37°C. Each value represents the mean optical density (OD) readings from 6 cultures that originated from 2 independent starter cultures. Sub-minimum inhibitory concentration doses used are based on MIC values in Table I.

Figure 2

Growth kinetics of Haemophilus somnus strain 700025 (A), Hs-91 (B), and 2336 (C) in the presence of sub-minimum inhibitory concentrations (MICs) of chlortetracycline (CTC) at 37°C. Each value represents the mean of three independent determinations. Sub-minimum inhibitory concentration doses used are based on MIC values in Table I.

Figure 3

Coomassie stained sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of M. haemolytica D80 with sub-minimum inhibitory concentrations (MICs) of chlortetracycline-sulfamethazine (CTC + SMZ) at 37°C and 41°C showing positions of major and minor outer membrane proteins (OMPs) identified in this study. Sub-minimum inhibitory concentration doses used are based on MIC values in Table I. Lane 1, MW marker (Broad Range standard; BioRad Laboratories); lane 2, no antibiotic at 37°C; lane 3, 1/8 MIC at 37°C; lane 4, 1/16 MIC at 37°C; lane 5, no antibiotic at 41°C; lane 6, 1/4 MIC at 41°C.

Figure 4

Coomassie stained sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of Haemophilus somnus strain 700025 in the presence of sub-minimum inhibitory concentrations (MICs) of chlortetracycline (CTC) at 37°C showing positions of OMPs identified in this study. Sub-minimum inhibitory concentration (MIC) doses used are based on MIC values in Table I. Lane 1, MW marker (Broad Range standard; BioRad Laboratories); lane 2, no antibiotic; lane 3, 1/2 MIC; lane 4, 1/4 MIC; lane 5, 1/8 MIC; lane 6, 1/16 MIC.