Antimicrobial histones from chicken erythrocytes bind bacterial cell wall lipopolysaccharides and lipoteichoic acids

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hospitals for the past 9 years showed that the number of secondary cases varied significantly depending on the speed and strictness with which the measures were implemented around the index case, including assignment of dedicated nursing staff and setup of simple barrier precautions [4].

INSERM U1135, Centre d’Immunologie et des Maladies Infectieuses, CIMI, team E13 (Bacteriology), F-75013 Paris, France c Assistance Publique–Hôpitaux de Paris, Microbiology, Trousseau Hospital, Paris, France d Assistance Publique–Hôpitaux de Paris, Microbiology, Saint-Antoine Hospital, Paris, France

Acknowledgment The authors are grateful to Sandra Fournier (Infection Control Team, Direction de la Politique Médicale, Assistance Publique–Hôpitaux de Paris, Paris, France) for helpful discussion. Funding: Assistance Publique–Hôpitaux de Paris (Paris, France). Competing interests: None declared. Ethical approval: Not required.

Dominique Decré a,b,c Sorbonne Universités, UPMC Univ Paris 06 CR7, Centre d’Immunologie et des Maladies Infectieuses (CIMI), team E13 (Bacteriology), F-75013 Paris, France b INSERM U1135, Centre d’Immunologie et des Maladies Infectieuses, CIMI, team E13 (Bacteriology), F-75013 Paris, France c Assistance Publique–Hôpitaux de Paris, Microbiology, Saint-Antoine Hospital, Paris, France

References

∗ Corresponding

a

[1] Institut National de Veille Sanitaire. Entérobactéries productrices de carbapénémases [Carbapenemase-producing Enterobacteriaceae]; 2014 May http://www.invs.sante.fr [accessed 11.07.14]. [2] Potron A, Rondinaud E, Poirel L, Belmonte O, Boyer S, Camiade S, et al. Genetic and biochemical characterisation of OXA-232, a carbapenem-hydrolysing class D ␤-lactamase from Enterobacteriaceae. Int J Antimicrob Agents 2013;41:325–9. [3] Doi Y, O’Hara JA, Lando JF, Querry AM, Townsend BM, Pasculle AW, et al. Coproduction of NDM-1 and OXA-232 by Klebsiella pneumoniae. Emerg Infect Dis 2014;20:163–5. [4] Fournier S, Monteil C, Lepainteur M, Richard C, Brun-Buisson C, Jarlier V, et al. Long-term control of carbapenemase-producing Enterobacteriaceae at the scale of a large French multihospital institution: a nine-year experience, France, 2004 to 2012. Euro Surveill 2014;19, pii: 20802. [5] Haut Conseil de la Santé Publique. Commission spécialisée sécurité des patients: infections nosocomiales et autres évènements indésirables liés aux soins et aux pratiques [Screening of faecal carriage and transmission control of multidrug resistant bacteria in patients hospitalised in French hospitals through transfers from another country]. Paris, France: HSCP; May 2010 http://www.hcsp.fr/docspdf/avisrapports/hcspr20100518 bmrimportees.pdf [accessed 11.07.14].

Aurore Bousquet a,b,∗ Marion Duprilot a,b a Sorbonne Universités, UPMC Univ Paris 06 CR7, Centre d’Immunologie et des Maladies Infectieuses (CIMI), team E13 (Bacteriology), F-75013 Paris, France b INSERM U1135, Centre d’Immunologie et des Maladies Infectieuses, CIMI, team E13 (Bacteriology), F-75013 Paris, France Didier Moissenet Assistance Publique–Hôpitaux de Paris, Microbiology, Trousseau Hospital, Paris, France Béatrice Salauze Assistance Publique–Hôpitaux de Paris, Equipe Opérationnelle d’Hygiène, Trousseau Hospital, Paris, France Jérôme Rambaud Assistance Publique–Hôpitaux de Paris, Medical Intensive Care Unit, Trousseau Hospital, Paris, France Nathalie Genel a,b Sorbonne Universités, UPMC Univ Paris 06 CR7, Centre d’Immunologie et des Maladies Infectieuses (CIMI), team E13 (Bacteriology), F-75013 Paris, France b INSERM U1135, Centre d’Immunologie et des Maladies Infectieuses, CIMI, team E13 (Bacteriology), F-75013 Paris, France a

Hoang Vu-Thien Assistance Publique–Hôpitaux de Paris, Microbiology, Trousseau Hospital, Paris, France Guillaume Arlet a,b,c,d Sorbonne Universités, UPMC Univ Paris 06 CR7, Centre d’Immunologie et des Maladies Infectieuses (CIMI), team E13 (Bacteriology), F-75013 Paris, France a

author at: Sorbonne Universités,UPMC Univ Paris 06 CR7, Centre d’Immunologie et des Maladies Infectieuses (CIMI), team E13 (Bacteriology), F-75013, Paris, France. Tel.: +33 6 18 03 00 16. E-mail address: [email protected] (A. Bousquet) 20 June 2014 http://dx.doi.org/10.1016/j.ijantimicag.2014.06.004

Antimicrobial histones from chicken erythrocytes bind bacterial cell wall lipopolysaccharides and lipoteichoic acids Sir Extensive use of antibiotics has selected for a variety of multidrug-resistant bacteria, which is a pressing concern owing to their increasing prevalence. From 1995 to 2007, Canadian hospitals experienced a 17-fold increase in the incidence of meticillin-resistant Staphylococcus aureus (MRSA), of which >75% were thought to be acquired in a hospital or healthcare setting [1]. The gravity of the situation was recently highlighted by the World Health Organization (WHO) [2]. Moreover, 12% of hospitalacquired infections in the USA are from the species Enterococcus, of which 80–90% are Enterococcus faecalis [3]. Promising alternatives to antibiotics are cationic antimicrobial peptides (CAMPs), key players in the innate immune defence system of many species which target pathogen components that cannot be easily mutated. Histones are an archetypal CAMP. This study reports the minimum inhibitory concentrations (MICs) and minimum bactericidal concentrations (MBCs) of histones purified from chicken erythrocytes against Gram-negative and Gram-positive bacterial strains, including E. faecalis and MRSA (Fig. 1A–G). The most susceptible Gram-positive bacterial species was Bacillus subtilis, with a MIC of 3 ± 1 ␮g/mL; the least susceptible was E. faecalis, requiring 700 ± 100 ␮g/mL to inhibit bacterial growth (MIC) and 1100 ± 200 ␮g/mL to cause bacterial cell death (MBC) (Fig. 1A and B). The MICs of S. aureus and MRSA were not significantly different from each other, demonstrating similar susceptibilities at MICs of 6 ± 1 ␮g/mL and 8 ± 2 ␮g/mL, respectively (Fig. 1C and D). The histone mixture was significantly more potent in inhibiting the growth of Gram-negative Salmonella enterica serovar Typhimurium and Pseudomonas aeruginosa compared with Escherichia coli (Fig. 1E–G). E. coli, S. Typhimurium and P. aeruginosa have MICs of 21 ± 3, 3.6 ± 0.4 and 5 ± 1 ␮g/mL, respectively, which are not statistically different from their MBCs. Therefore, histones exert potent antimicrobial activity both towards Gramnegative and Gram-positive bacteria, with E. faecalis demonstrating markedly lower susceptibility.

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Fig. 1. (A–G) Dose-dependent growth inhibition of Gram-positive and Gram-negative bacteria treated with a histone mixture from chicken erythrocytes as well as minimum inhibitory concentrations (MICs) determined by a microbroth dilution assay: four Gram-positive bacterial strains (A–D) and three Gram-negative bacterial strains (E–G) were tested, with H2 O and kanamycin as negative and positive controls for inhibition, respectively. Results are representative of three independent trials, each in triplicate. (H–M) Mobility shift assays of histone–LTA and histone–LPS complexes in non-denaturing conditions. Purified histones (5 ␮g) were incubated with increasing amounts of LTA from B. subtilis (H), S. aureus (I) and E. faecalis (J) or LPS from E. coli (K), S. enterica serovar Typhimurium (L) and P. aeruginosa (M). Controls include 5 ␮g of histones and 10 ␮g of LPS/LTA loaded individually on each gel. LTA: lipoteichoic acid; LPS: lipopolysaccharide.

The first step in the antimicrobial mechanism of CAMPs is the electrostatic interaction between the positive charges of the antimicrobial and the negatively charged molecules located on the surface of the bacterial cell wall. We investigated the mechanism for this interaction using a mobility shift assay to determine the affinity of histones for S. aureus, E. faecalis and B. subtilis lipoteichoic acids (LTAs) as well as for E. coli, S. Typhimurium and P. aeruginosa lipopolysaccharides (LPS) (Fig. 1H–M). Fig. 1H–J demonstrates that histones bind strongly to B. subtilis and S. aureus LTAs,

with saturated binding at 5 ␮g of LTA, compared with E. faecalis that required 7.5 ␮g of LTA. Similarly, 5 ␮g of P. aeruginosa LPS completely bound to and neutralised the positive charges of the histone molecules (Fig. 1M). However, the lowest histone–LPS binding affinities observed were for S. Typhimurium LPS, requiring 10 ␮g of LPS to saturate the charge on histone molecules, whilst >10 ␮g of E. coli LPS was necessary (Fig. 1K and L). Overall, these results suggested that histones target pathogens via conserved negatively charged components imbedded in their cellular

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membranes. A correlation between the antimicrobial activity of histones towards Gram-positive bacteria and LTA–histone binding affinity was observed. A haemolytic assay was used to determine whether the histone mixture displayed toxicity towards mammalian cells over a wide range of histone concentrations (0.005–1.25 mg/mL). Histones did not damage the red blood cell membranes and were nonhaemolytic even at the highest concentration tested (1.25 mg/mL). Chicken erythrocytes are an ideal source of purified histones. The ease of harvest and lack of connective tissue allow for rapid and simple large-scale acid extraction from nuclei [4]. Proteomic analysis revealed that the histone mixture assessed in the present study contains histones H1, H2B, H2A, H3, H4 and H5. Neutrophil extracellular traps (NETs), which are key players in the innate immune response in a variety of vertebrate species, also contain a variety of histones (H1, H2A, H2B, H3 and H4) [5]. NETs are composed of the neutrophil nuclear contents that have been released extracellularly to create an elevated concentration of antimicrobial proteins at the site of infection. In addition to a bactericidal function, they also prevent further microbial spreading within the organism [5]. The current results clearly indicate that histones possess antimicrobial activity, potentially of a synergistic nature, and could play a role in pathogen recognition in the vertebrate immune system. Development of antimicrobial peptides is essential to combat emerging antibiotic-resistant bacterial strains that have forced exploration of alternatives for inhibition of microbial growth. These results demonstrate that histone-derived molecules have the possibility for development of novel antibiotics. Acknowledgments The authors would like to thank Dr Sattar and Richard Kibbee from the University of Ottawa’s Centre for Research on Environmental Microbiology (Ottawa, ON, Canada) for the gift of the bacterial species used in this study. They are also grateful to Tom Henderson of Tom Henderson Custom Meat (Chesterville, ON, Canada) and Robert Laplante from Laplante Poultry Farms Ltd. (Monkland, ON, Canada) for assisting with collection of fresh chicken blood. Funding: This study was funded by the Poultry Industry Council (PIC) and the Agriculture and Agri-Food Canada (AAFC)–Canadian Poultry Research Council (CPRC) poultry cluster. M.R.-M. would also like to thank the CPRC for a student scholarship. Competing interests: None declared. Ethical approval: All procedures involving chickens were in accordance with Canadian Food Inspection Agency (CFIA) guidelines and regulations, and all procedures involving rats were carried out in accordance with the University of Ottawa Animal Care Committee guidelines. References [1] Simor AE, Gilbert NL, Gravel D, Mulvey MR, Bryce E, Loeb M, et al. Canadian Nosocomial Infection Surveillance Program Methicillin-resistant Staphylococcus aureus colonization or infection in Canada: National Surveillance and Changing Epidemiology, 1995–2007. Infect Control Hosp Epidemiol 2010;31: 348–56. [2] Reardon S. WHO warns against ‘post-antibiotic’ era. Nat News 2014:15135, http://dx.doi.org/10.1038/nature.2014. [3] Fisher K, Phillips C. The ecology, epidemiology and virulence of Enterococcus. Microbiology 2009;155:1749–57. [4] Shechter D, Dormann HL, Allis CD, Hake SB. Extraction, purification and analysis of histones. Nat Protoc 2007;2:1445–57. [5] Brinkmann V, Reichard U, Goosmann C, Fauler B, Uhlemann Y, Weiss DS, et al. Neutrophil extracellular traps kill bacteria. Science 2004;303:1532–5.

Megan Rose-Martel Maxwell T. Hincke ∗ Department of Cellular and Molecular Medicine, University of Ottawa, 451 Smyth Road, Ottawa, Ontario, Canada K1H 8M5 ∗ Corresponding

author. Tel.: +1 613 562 5800x8193; fax: +1 613 562 5434. E-mail address: [email protected] (M.T. Hincke) 22 May 2014

http://dx.doi.org/10.1016/j.ijantimicag.2014.07.008

Comparison of the reduction in the antibacterial potency of a fluoroquinolone conferred by a single mutation in the quinolone resistancedetermining region or by the inoculum size effect Sir, Resistance to fluoroquinolones occurs by random mutations so that any high bacterial inoculum must contain a small proportion of mutant bacteria. To overcome this heterogeneity, different strategies such as targeting the mutant prevention concentration (MPC) have been identified to prevent mutant amplification during fluoroquinolone treatment [1]. However, the clinical efficacy of fluoroquinolones can also be reduced without any change in the minimum inhibitory concentration (MIC) by high inocula [2] and, to the best of our knowledge, no strategy has been proposed to date to take into account the reduction of fluoroquinolone potency conferred by this inoculum effect. In this study, we evaluated whether the fluoroquinolone concentrations needed to eradicate high inocula of susceptible bacteria should be higher or lower than the MPC, i.e. whether the inoculum size effect could be distinguished, or not, from the emergence of resistant mutants. We compared the reduction of fluoroquinolone activity conferred on one hand by acquired genetic mutations, and on the other hand by high inoculum sizes, by exposing a wild-type Escherichia coli strain or its isogenic mutant carrying one mutation in the gyrA gene to marbofloxacin. By testing seven different inoculum sizes from 5 × 102 to 5 × 108 CFU/mL of each bacterium, we showed that the antibacterial activity of marbofloxacin dramatically decreased for inoculum sizes higher than 5 × 106 CFU/mL. The concentrations of marbofloxacin needed to eradicate all of the bacterial population increased from 1- or 2-fold the MIC for low inocula to 128- or 256-fold the MIC for the 5 × 107 and 5 × 108 CFU/mL inocula. In this study, the reduced marbofloxacin activity on high inocula cannot be attributed to the emergence of resistant bacteria by spontaneous mutation since the surviving bacteria showed no difference in the MIC after exposure to marbofloxacin. The lower marbofloxacin potency on high inocula could most likely be explained by the phenotypic heterogeneity of the bacterial population leading to the presence of resistant persisters in such inocula. We then assessed marbofloxacin activity on mixed bacterial populations containing a majority (5 × 107 CFU/mL) of wild-type bacteria and a minority (5 × 103 CFU/mL) of mutant bacteria to simulate the proportion that could be encountered in vivo after spontaneous mutation [3]. We first showed that when mutants were mixed with wild-type bacteria, they did not grow (Fig. 1b),