RESEARCH LETTER

In vitro effectiveness of the antimicrobial peptide eCATH1 against antibiotic-resistant bacterial pathogens of horses Margot Schlusselhuber1, Kristen Guldbech2, Corinne Sevin3, Matthias Leippe4, Sandrine Petry1, € tzinger5, Steeve Gigue re2 & Julien Cauchard1 Joachim Gro Unit Bacteriology and Parasitology, Dozule Laboratory for Equine Diseases, Anses, Goustranville, France; 2Department of Large Animal Medicine, College of Veterinary Medicine, University of Georgia, Athens, GA, USA; 3Epidemiology, Laboratory for equine diseases, Anses, Goustranville, France; 4Department of zoophysiology, Zoologisches Institut, Christian Albrechts University, Kiel, Germany; and 5Department of Biochemistry, Christian-Albrechts-University of Kiel, Kiel, Germany

1

Correspondence: Julien Cauchard, Unit Bacteriology and Parasitology, Dozule Laboratory for Equine Diseases, Anses, Goustranville, France. Tel.: +33(0) 231792276; fax: +33(0)31392137; e-mail: [email protected] Received 14 October 2013; accepted 17 November 2013. Final version published online 18 December 2013. DOI: 10.1111/1574-6968.12337

MICROBIOLOGY LETTERS

Editor: S eamus Fanning Keywords equine; eCATH1; antimicrobial peptide; bacteria; resistance.

Abstract The equine antimicrobial peptide eCATH1 previously has been shown to have in vitro activity against antibiotic-susceptible reference strains of Rhodococcus equi and common respiratory bacterial pathogens of foals. Interestingly, eCATH1 was also found to be effective in the treatment of R. equi infection induced in mice. The aim of this study was to assess the in vitro activity of eCATH1 against equine isolates of Gram-negative (Escherichia coli, Salmonella enterica, Klebsiella pneumoniae and Pseudomonas spp.) and Gram-positive (R. equi, Staphylococcus aureus) bacteria resistant to multiple classes of conventional antibiotics. A modified microdilution method was used to evaluate the minimum inhibitory concentrations (MICs) of the antimicrobial peptide. The study revealed that eCATH1 was active against all equine isolates of E. coli, S. enterica, K. pneumoniae, Pseudomonas spp. and R. equi tested, with MICs of 0.5–16 lg mL 1, but was not active against most isolates of S. aureus. In conclusion, the activity of the equine antimicrobial peptide eCATH1 appears to not be hampered by the antibiotic resistance of clinical isolates. Thus, the data suggest that eCATH1 could be useful, not only in the treatment of R. equi infections, but also of infections caused by multidrug-resistant Gram-negative pathogens.

Introduction The susceptibility of bacteria to conventional antibiotics has decreased substantially in human and equine medicine, coupled with the emergence of multidrug-resistant (MDR) bacteria. There is an increasing frequency of isolation of enterobacteria producing extended spectrum b-lactamases, fluoroquinolone-resistant Pseudomonas aeruginosa, methicillin-resistant Staphylococcus aureus, macrolide- and rifampin-resistant Rhodococcus equi and other MDR bacteria from horses, raising serious concerns for the future of antimicrobial therapy in equine medicine (Singh et al., 2007; Vo et al., 2007; Singh, 2009; Van den Eede et al., 2009; Giguere et al., 2010; Burton et al., 2013). As a consequence, there is growing interest in anti-infective agents with fundamentally different modes ª 2013 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved

of action than those of traditional antibiotics (Levy & Marshall, 2004). Antimicrobial peptides (AMPs) are virtually found in all living organisms and constitute key factors of the innate immune system of animals. Their mode of action, although not yet fully understood, most often involves electrostatic interactions between positively charged peptides and negative compounds of bacterial membranes, followed by disruption or alteration of membrane integrity (Yeaman & Yount, 2003). Because of their mode of action, AMPs are now emerging as particularly innovative molecules in the antimicrobial drug research area. With more than 30 putative AMPs identified so far in the horse (Bruhn et al., 2011), this animal represents a considerable source of new drugs for future medical applications. eCATH1, a peptide encoded by the equine genome, is a FEMS Microbiol Lett 350 (2014) 216–222

FEMS Microbiol Lett 350 (2014) 216–222

Uterine cervix swab, NS Uterine cervix swab, NS Bronchoalveolar lavage fluid, foal, evidence of respiratory disease Tracheal wash, NS Tissue, 7-month-old foetus, abortion Lungs, foal, cause of death: R. equi infection Various organs, adult, euthanasia Hoof, adult, euthanasia Various organs, foal, cause of the death: pneumonia

Escherichia coli ATCC 25922 1* Joint aspiration, NS 2 Various organs, NS 3 Various organs, NS 4* Faeces, adult, signs of diarrhoea 5* Faeces, adult, signs of diarrhoea 6* Faeces, adult, signs of diarrhoea 7* Faeces, adult, signs of diarrhoea 8* Faeces, adult, signs of diarrhoea 9* Faeces, adult, signs of diarrhoea 10* Abdomen, NS 11* NS 12* Pus swab, adult, NS 13* Stomach, NS Salmonella enterica serovar Typhimurium ATCC 14028 1* Faeces, adult, signs of diarrhoea 2* Faeces, adult, signs of diarrhoea 3* Faeces, adult, signs of diarrhoea 4* Faeces, adult, signs of diarrhoea 5* Faeces, adult, signs of diarrhoea 6 Sinus swab, adult, NS 7 Tissue, 9 month-old foetus, abortion

7 8 9

4* 5 6

Pseudomonas spp. ATCC 10145 1* 2* 3*

Bacterial strains and isolate number

Historical data of the horse (specimen, age group and available clinical details)

S R R R R R R R R R R R R R S R R R R R S R

S S S S S S S S

R R R

R R R

R R R R

AMP

S S S S R S S S I S S S S S

S S S

S S S

S S S S

AMI

S R R R R R S S

S R R R R S S S I R S R R R

R R R

R R R

R R R R

CHL

S R R R R R S S

S R R R R S S S R R R R R R

R R R

R R R

R R R R

DOX

S S S S S S S S

S R R S S S S S S S R S S S

I I R

R I R

I R I R

ENR

S R R R R R S R

S R R S R R R R R R R S R R

R R R

R R R

R R R R

FAZ

Table 1. Drug resistance spectrums of equine clinical isolates of Gram-negative bacteria and MICs of eCATH1

S R R R R R S S

S R R S R S R R S S R R R R

S S S

S S S

S R S R

GEN

S S S S S S S S

S S S S S S S S S S S S S S

S S S

I S S

S S S S

IMI

S R R R R R S R

S R R R R S R R R S R R R R

R R R

R R R

R R R R

SXT

S R R R R R S R

S R R S R R R R I I S S S S

I R I

I I I

S R I R

TAZ

S R R R R R S R

S R R R R S S S R R R R R R

R R R

R R R

R R R R

TET

S R R R R R S R

S R R R R R R R R R R R R R

R R R

R R R

R R R R

TIC

S R R R R R S S

S R R S I I I I I I R I I R

R R R

R R R

R R R R

TIM

S R R R R R S R

S R R S R R R R R R R S R R

R R R

R R R

R R R R

XNL

2 1–2 2 0.5–2 2 2 2 8–16

4 8–16 1–2 16 1–2 1 1 1–2 1 1 1–2 2 16 2–4

4 2–4 2–4

2–4 4 2–4

4 4 4–8 1–4

MICs of eCATH1 (lg mL 1)

Activity of eCATH1 against antibiotic-resistant bacteria

217

ª 2013 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved

218

4* 5*

ª 2013 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved

ATCC strains are reference strains. MIC, Minimum inhibitory concentration; R, Resistant; I, Intermediate; S, Susceptible; NS, Not specified; AMI, Amikacin; AMP, Ampicillin; CHL, Chloramphenicol; DOX, Doxycycline; ENR, Enrofloxacin; FAZ, Cefazolin; GEN, Gentamicin; IMI, Imipenem; SXT, Trimethoprim–sulphamethoxazole; TAZ, Ceftazidime; TET, Tetracycline; TIC, Ticarcillin; TIM, Ticarcillin + clavulanate; XNL, Ceftiofur. *Bacterial strains isolated from alive horses (unless indicated) collected from animal diagnostic laboratories. All other bacterial strains were isolated from horse necropsies.

2–4 4 R R R R R R S I R R S S R R R R S S R R R R S S

R R

R R R R

8 9 Klebsiella pneumoniae ATCC 13883 1 2 3

Various organs, NS Intestine, NS Various organs, foal, cause of the death: pneumonia Pus swab, adult, NS Faeces, foal, NS

S S S S

S R R S

S R R S

S S S S

S R R S

S R R S

S S S S

S R R S

S R R S

R R

2 2–4 2–4 4 S R R S S R R S

2 2

S I R S

R R R R

XNL

R S R S

TIM

R S S S

TIC TET TAZ

R R

SXT IMI

S S R S

GEN FAZ

R S S S

ENR DOX

R S R S

CHL

R S S S Blood, newborn foal, NS NS, adult, NS

AMP AMI Historical data of the horse (specimen, age group and available clinical details) Bacterial strains and isolate number

Table 1. Continued

R S

MICs of eCATH1 (lg mL 1)

M. Schlusselhuber et al.

promising candidate for the treatment of equine bacterial infections because it exhibits a high degree of antimicrobial activity against antibiotic-susceptible reference strains of Escherichia coli, Salmonella enterica, P. aeruginosa, Klebsiella pneumoniae, Streptococcus zooepidemicus, some strains of Staphylococcus spp. and R. equi (Skerlavaj et al., 2001; Schlusselhuber et al., 2012). Moreover, a recent study demonstrated its intracellular antirhodococcal properties in mice (Schlusselhuber et al., 2013). In the present study, the investigation of the in vitro activity of eCATH1 was extended to a large panel of clinically relevant equine Gram-negative and Gram-positive bacterial isolates mostly resistant to conventional antibiotics.

Materials and methods Reference strains of bacteria (n = 6) were obtained from the American Type Culture Collection (ATCC), while other strains, all isolated from horses, were collected from necropsies performed at Dozule laboratory for equine diseases (France) or from animal diagnostic laboratories from various countries. All bacterial strains, from France (n = 38), the Netherlands (n = 5) and the USA (n = 22), were identified according to standard procedures and confirmed with commercial galleries (API systems, AES; bioMerieux). Minimum inhibitory concentrations (MICs) of antibiotics against R. equi were determined using the Etest concentration gradient test (bioMerieux), applying MIC breakpoints for Staphylococcus spp., as no breakpoints are defined for R. equi (Giguere et al., 2010). Antibiotic susceptibilities of other strains were determined by a microdilution method using the commercial Sensititre equine 96-well microtitre plate (TREK Diagnostic Systems). All assays were evaluated in accordance with the guidelines of the Clinical and Laboratory Standards Institute (CLSI, 2008). To test the antimicrobial activity of eCATH1, a microdilution assay was performed in triplicate in roundbottom polypropylene 96-well microtitre plates (Costar catalogue number 3879, Sigma) using a protocol previously described (Schlusselhuber et al., 2012).

Results The activity of eCATH1 against Gram-negative bacteria is shown in Table 1. The MICs of eCATH1 for Gram-negative reference strains, consistent with previous findings, were in the same range than those of the antibiotic-resistant isolates (Skerlavaj et al., 2001; Schlusselhuber et al., 2012). Indeed, eCATH1 was active against all equine isolates of E. coli, S. enterica, K. pneumoniae and Pseudomonas spp. tested, with MICs of 0.5–16 lg mL 1. Interestingly, Pseudomonas spp., known to be naturally FEMS Microbiol Lett 350 (2014) 216–222

Staphylococcus aureus ATCC 29213 1* Nasal swab, NS 2* Nasal swab, NS 3* Nasal swab, NS 4 Tissue, 8 monthold foetus 5 Various organs, foal, cause of the death: heart attack 6 Various organs, newborn foal, cause of the death: influenzaassociated pneumonia 7 Various organs, newborn foal, NS 8 NS, newborn foal, cause of the death: S. Equisimilis and S aureus associated septicaemia 9 Muscle, adult, cause of the death:cachexy 10 Various organs, adult, euthanasia 11 Perianal abscess, NS

Bacterial strains and isolate number

Historical data of the horse (specimen, age group and available clinical details) R R S R S R

S

R

R

R

R S

S

S

I

S

S

I S

AMP

S S S S S

AMI

FEMS Microbiol Lett 350 (2014) 216–222

I

R

I

I

R

I

I

I I I R I

AZI

S

I

R

I

R

I

I

S I S I S

CHL

S

R

S

I

I

S

S

S S S S S

CLA

S

R

S

S

R

S

R

S S S S S

DOX

S

I

S

I

I

S

S

S S S S S

ENR

S

R

I

I

I

S

I

I I S I I

ERY

S

R

S

S

S

S

R

S S S S S

FAZ

Table 2. Drug resistance spectrums of equine clinical isolates of Gram-positive bacteria and MICs of eCATH1

S

R

S

S

S

S

R

S S S S S

GEN

S

R

S

S

S

S

R

S S S S S

IMI

S

R

S

S

S

S

R

S S S S S

OXA

S

R

R

R

R

S

R

R R S R S

PEN

S

R

S

S

S

S

S

S S S S S

RIF

S

R

S

R

R

S

S

S S R S S

SXT

S

R

S

S

S

S

R

I S S S S

TAZ

S

R

S

R

R

S

R

S S S S S

TET

S

R

I

S

S

S

R

S I S I S

TIC

S

R

S

S

S

S

R

S S S S S

TIM

S

R

S

S

S

S

R

S S S S S

XNL

4

> 32

> 32

> 32

> 32

> 32

> 32

> 32 > 32 1 > 32 1–2

MICs of eCATH1 (lg mL 1)

Activity of eCATH1 against antibiotic-resistant bacteria

219

ª 2013 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved

ª 2013 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved

N R

R N S N R R R N N R R R N N R N N R N N

N S S S S S N N N N N S N N N S N S N N

AMP

S S

AMI

S R S S R R R R R R R R R R R R R R R R

S S

AZI

S S S S S I R R R S S I R R R S R I R R

S I

CHL

S R S S R R R R R R R R R R R R R R R R

S S

CLA

N I S I N S N S S N N S I I N S I S S I

S S

DOX

N I I I S I S S S S N I S S S I S I S S

S I

ENR

S R S S R R R R R R R R R R R R R R R R

S S

ERY

R R S R R N N N N R R R N N R R N R N N

I R

FAZ

S S S S S S N S S S S S S S N S S S S S

S S

GEN

R S S S R S S N N R R S N N S S N S N N

S S

IMI

R R R R R R N N N R R R N N N R N R N N

R R

OXA

R R R R R R R N N R R R N N R R N R N N

R R

PEN

R R R R R R R R R R R R R R R R R R R R

S R

RIF

S S S S R R R N N S S R N N R R N S N N

S R

SXT

N N N N N N N N N N N N N N N N N N N N

N N

TAZ

N R N N R R N R R N S N R R R R I R R R

R I

TET

N R N R N N S N N N N R N N R R N I N N

R R

TIC

N N N N N N N N N N N N N N N N N N N N

N N

TIM

N S S S R S S S S S N R S S S S S S S S

S I

XNL

0.5 1–2 2–4 1–2 2 1–2 2–4 2 2 2–4 2–4 2–4 2–4 2 2 2 2 2–4 1–2 2–4

4 4

MICs of eCATH1 (lg mL 1)

ATCC strains are reference strains. MIC, Minimum inhibitory concentration; R, Resistant; I, Intermediate; S, Susceptible; NS, Not specified; N, Not done; AMI, Amikacin; AMP, Ampicillin; AZI, Azithromycin; CHL, Chloramphenicol; CLA, Clarithromycin; DOX, Doxycycline; ENR, Enrofloxacin; ERY, Erythromycin; FAZ, Cefazolin; GEN, Gentamicin; IMI, Imipenem; OXA, oxacillin; PEN, Penicillin; RIF, Rifampin; SXT, Trimethoprim– sulphamethoxazole; TAZ, Ceftazidime; TET, Tetracycline; TIC, Ticarcillin; TIM, Ticarcillin + clavulanate; XNL, Ceftiofur. *Bacterial strains isolated from alive horses (unless indicated) collected from animal diagnostic laboratories. All other bacterial strains were isolated from horse necropsies.

Rhodococcus equi ATCC 33701 P+ 1* Lungs (Bronchoalveolar lavage fluid from foals with evidence of respiratory disease) 2* 3* 4* 5* 6* 7* 8* 9* 10* 11* 12* 13* 14* 15* 16* 17* 18* 19* 20* 21*

Bacterial strains and isolate number

Historical data of the horse (specimen, age group and available clinical details)

Table 2. Continued

220 M. Schlusselhuber et al.

FEMS Microbiol Lett 350 (2014) 216–222

221

Activity of eCATH1 against antibiotic-resistant bacteria

resistant to a wide range of antibiotics due to efficient multidrug efflux pumps and low permeability of their envelope, were inhibited by eCATH1 at MICs ≤ 8 lg mL 1. The activity of eCATH1 against Gram-positive bacteria is shown in Table 2; the peptide was highly active (MIC 0.5–4.0 lg mL 1) against R. equi strains resistant to rifampin and macrolides, the antibiotics currently used for the treatment of infection caused by this pathogen. Staphylococcus aureus strains, however, appear to not be susceptible to eCATH1 as the peptide was only active against 3/10 antibiotic-resistant strains of S. aureus and was not active against the S. aureus reference strain (MIC > 32 lg mL 1).

some strains might lack one or more of the aforementioned mechanisms of AMP mechanisms of resistance. Previous animal studies demonstrated that the equine antimicrobial peptide eCATH1 was effective in the treatment of the facultative intracellular pathogen R. equirelated infection, without inducing detectable deleterious effects (Schlusselhuber et al., 2013). Interestingly, the in vitro activity of eCATH1 appears to not be hampered by the antibiotic resistance of R. equi clinical isolates and Gram-negative clinical isolates. In conclusion, the present data suggest that eCATH1 could be useful, not only in the treatment of R. equi infections, but also of infections caused by MDR Gram-negative pathogens.

Discussion

Acknowledgements

All antibiotics tested in this study target intracellular processes (transcription, translation and cell wall synthesis). To reach their targets, these molecules have to cross the bacterial membrane, some of them by a porin-mediated pathway (Delcour, 2009). To limit the entrance of such molecules into the cell, bacteria may reduce the fluidity and permeability of the cell envelop or produce proteases and drug pump efflux that might hamper the insertion of AMPs into the membrane as well. However, neither resistance of Gram-negative bacteria and R. equi strains to macrolides (erythromycin, azithromycin, clarithromycin), aminoglycosides (amikacin, gentamicin), tetracyclines (doxycycline, tetracycline), fluoroquinolone (enrofloxacin), cephalosporins (cefazolin, ceftazidime, ceftiofur), carbapenem (imipenem), penicillins (ampicillin, ticarcillin, ticarcillin-clavulanate, oxacillin, penicillin), rifampin, chloramphenicol or trimethoprim–sulphamethoxazole did not lead to a detectable change in eCATH1 activity (Tables 1 and 2). Interestingly, the data suggest that no cross-resistance occurs between eCATH1 and conventional antibiotics used in equine medicine. Noticeably, the peptide was not active against the majority of S. aureus strains up to a concentration of 32 lg mL 1 (Table 2). This observation may be explained by the fact that this species is naturally resistant to many AMPs due to different mechanisms. Indeed, staphylokinase (extracellular protein that bind and trap AMPs), aureolysin and V8 (proteases), MprF and dltABCD operon (involved in the modification of the negatively charged lipid phospholipidglycerol and teichoic acids with positively charged amino-acid residues resulting in AMPs repulsion) are all known to be involved in AMP resistance (Nizet, 2006). Interestingly, three strains of S. aureus were highly susceptible to eCATH1. The reason for differences in susceptibility among S. aureus isolates remains to be elucidated, it is conceivable, however, that

This work was supported by grants from the European Regional Development Fund, the Regional Council of Low Normandy, the Institut Francßais du Cheval et de l’Equitation and the French Agency for Food, Environmental and Occupational Health Safety. We thank Jaap A. Wagenaar (Faculty of Veterinary Medicine, Utrecht University, the Netherlands) and Karine Maillard (Laboratoire Frank Duncombe, Caen, France) for kindly providing additional equine bacterial strains for the study. We thank Roland Leclercq and Claire Laugier for critically reading the manuscript. We are grateful to Severine Cauchard for advice.

FEMS Microbiol Lett 350 (2014) 216–222

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