UNIVERSITY OF PISA
Research Doctorate School in Biological and Molecular Sciences
Program: Biotechnology for Health
(BIO/19)
PhD Thesis
Synergistic activity of the N-terminus of human lactoferricin with
various antibiotics against carbapenemase producing
Klebsiella pneumoniae strains
PhD Student Supervisor
Paola Morici Prof. Antonella Lupetti
President of the PhD course
Prof. Generoso Bevilacqua
PhD course: 2012-2014 Cycle XXVII
INDEX
1. INTRODUCTION ... 1
1.1 Antimicrobial resistance in Enterobacteriaceae ... 1
1.2 The carbapenems ... 2 1.2.1 Carbapenem resistance ... 3 1.2.2 Carbapenemase enzymes ... 3 1.3Klebsiella pneumoniae ... 5 1.3.1 KPC Klebsiella pneumoniae ... 5 1.3.2 Epidemiology ... 6 1.3.3 Treatment... 7 1.3.4 Combination therapy ... 8
1.4 New therapeutic approaches: antimicrobial peptides ... 10
1.4.1 Human lactoferrin ... 12
1.4.2 The human lactoferrin-derived antimicrobial peptide hLF1-11 ... 13
2. STUDY PROPOSALS ... 16
3. MATERIALS AND METHODS ... 17
3.1 Bacteria ... 17
3.2 Phenotypic and genotypic characterization ... 17
3.3 Synthetic peptide and antibiotics ... 19
3.4 Killing assay ... 20
3.5 Synergy studies... 20
3.5.1 Checkerboard assay ... 20
3.5.2 Time-kill assay ... 21
3.6 Hemolysis assay ... 22
3.7 Ethidium bromide (EtBr) permeability assay ... 23
3.8 Effect of CCCP on the antibacterial activity of hLF1-11 ... 24
3.9 Cytoplasmic membrane depolarization ... 24
Statistical analysis ... 25
4. RISULTS ... 26
4.2 Phenotypic and genotypic characterization ... 26
4.3 Killing assay ... 26
4.4 Synergy studies... 28
4.4.1 Checkerboard assay ... 28
4.5 Hemolytic activity ... 31
4.6 Time-kill assay for the synergistic combination of hLF1-11 and rifampicin... 33
4.7 Ethidium bromide (EtBr) permeability assay ... 35
4.8 Effect of CCCP on the hLF1-11 antibacterial activity ... 37
4.9 Cytoplasmic membrane depolarization ... 39
5. DISCUSSION ... 41
Abstract
The worldwide spread of carbapenem-resistant Klebsiella pneumoniae strains has become a major source of concern for public health.
In light of the need to develop new drugs, cationic antimicrobial peptides have gained attention as possible therapeutic candidates. The antibacterial activity of the antimicrobial synthetic peptide representing the first cationic domain of human lactoferricin, further referred to as hLF1-11, was evaluated against sensitive and resistant K. pneumoniae strains. In addition, the antibacterial activity of hLF1-11 in combination with several antimicrobial drugs (rifampicin, clarithromycin, clindamycin, gentamycin and tigecycline) was evaluated against clinical isolates of carbapenemase-producing K. pneumoniae harboring different resistance genes. The results revealed that the hLF1-11 peptide exhibited antimicrobial activity in a dose dependent manner. This antibacterial activity was significantly more effective against a sensitive than against a resistant K. pneumoniae strain. In addition, the results revealed a synergistic effect between hLF1-11 and all the tested antimicrobial drugs against several multidrug (MDR) KPC K. pneumoniae strains.
Another important finding pertains to the mechanism of action of the synergistic effect induced by hLF1-11 and rifampicin. When these cells were exposed to rifampicin for 30 or 60 min, washed and then treated with a sub-inhibitory concentration of hLF1-11 up to 2 h, were significantly (P < 0.05) killed than untreated cells, while no antibacterial activity was observed when they were first incubated with hLF1-11 and then with rifampicin. CCCP, which is an uncoupler of oxidative phosphorylation, was used to dissipate the proton motive force (PMF). The effect of CCCP on the antibacterial activity of these compounds revealed that the antibacterial activity of hLF1-11 as well as the synergistic effect between hLF1-11 and rifampicin were significantly reduced by CCCP, while no effect was obtained on the antibacterial activity of rifampicin. In agreement, EtBr accumulation increased upon exposure to hLF1-11 dose dependently as well as upon exposure to the combination of hLF1-11 and
rifampicin, while no increase in fluorescence intensity was induced by treatment with rifampicin. Moreover, hLF1-11 was able to induce cytoplasmic membrane depolarization, as measured by the anionic dye DiBAC4(3), which enters the depolarized cells. All together,
these data suggest that the rifampicin molecules accumulated by Klebsiella pneumoniae during the 30 min preincubation were not taken away by washing. The subsequent addition of hLF1-11, by inducing a transient loss of membrane potential, could have facilitated the entrance and retention of rifampicin into the cytoplasm. In agreement, both cell membrane permeabilization, as well as membrane depolarization, increased upon exposure to hLF1-11, thus supporting our hypothesis.
In conclusion, hLF1-11 induces sensitization of KPC K. pneumoniae to rifampicin, an otherwise impermeable hydrophobic antibiotic, by facilitating access of rifampicin to and subsequent retention of rifampicin in the cytoplasm.
1. INTRODUCTION
1.1 Antimicrobial resistance in Enterobacteriaceae
Ever since the discovery and subsequent clinical use of antimicrobials, resistance to these agents has been observed, with a commensurate negative impact on the treatment of infectious diseases.
The concept of antimicrobial resistance is better portrayed within Gram negative bacteria, above all within species belonging to the Enterobacteriaceae family. Following the onset of multidrug resistant strains, such as bacteria producing extended-spectrum beta-lactamases (ESBL), carbapenems became the first line therapy. In the last decades, the extensive use of these antibiotics has led to a dramatic increase of the prevalence of carbapenem-resistant Gram-negative pathogens. Among Enterobacteriaceae, the emergence and worldwide spread of carbapenem-resistant Klebsiella pneumoniae strains is one of the most recent developments in antimicrobial resistance and a major source of concern for public health. Moreover, high rates of mortality are recorded.
The need to prevent the diffusion of these microorganisms is actually contrasted by the lack of new antimicrobial drugs that are highly effective.
Since these microorganisms are resistant to all conventional antimicrobials, there is a pressing need to develop alternative strategies. In recent years, particular interest has been addressed to cationic antimicrobial peptides as promising candidates to overcome the multidrug resistance, having broad-spectrum antimicrobial activities against a wide variety of pathogens and low toxicity.
1.2 The carbapenems
Carbapenems are an important class of β-lactam antibiotics with a broad spectrum antibacterial activity and are considered the last resort drugs to treat serious nosocomial infections caused by multidrug resistant Gram negative bacteria, such as microorganisms producing ESBL. Carbapenems differ from other β-lactam antibiotics in that they possess a carbon instead of sulphur in the four-position of the thyazolidinic moiety of the β-lactam ring. They were originally developed from a naturally derived product of Streptomyces cattleya, named Thienamycin. Like all beta-lactam agents, carbapenems inhibit bacterial cell wall synthesis by binding to and inactivating penicillin-binding proteins (PBPs), enzymes that catalyze cross-linking of peptidoglycans (Merck, 2010). They are not easily diffusible through the bacterial cell wall, but cross the outer membrane of Gram-negative bacteria by porins. The main four members of clinically used carbapenems are imipenem, meropenem, ertapenem, and doripenem. The first carbapenem, imipenem, was developed in 1985. It is indicated for the treatment of serious and complicated infections of the respiratory tract, skin as well as ginaecological and intrabdominal infections. Compared with the other carbapenems, imipenem exhibits increased toxicity in adults with impaired renal function (Merck, 2010).
Meropenem, licensed for use in the UK in 1988, has subsequently shown clinical advantages over imipenem, and it has become the most widely used carbapenem in the UK (Edwards S. J.
et al., 2005; Hawkey P. M. and Livermore D. M, 2012). Ertapenem developed in 2002, has a more limited spectrum of activity, showing poor activity particularly against non-fermenting bacteria, e.g. Pseudomonas spp., and Acinetobacter spp. in comparison to the other carbapenems (Livermore et al., 2003). The most recently developed carbapenem, doripenem, has high inhibitory effect especially against Pseudomonas aeruginosa isolates, including multidrug resistant strains. Carbapenems are recommended for the empirical treatment of a variety of severe infections, e.g. nosocomial pneumonia, complicated intra-abdominal
infection, septicemia, complicated skin infection, urinary tract infection, meningitis, and acute exacerbations of cystic fibrosis (Kattan J. N. et al., 2008).
1.2.1 Carbapenem resistance
Carbapenem resistance is often due to the production of enzymes, known as carbapenemases, that hydrolyse all β-lactam antibiotics, including carbapenems, which are considered the last resort for treating enterobacterial infections. Other mechanisms, such as those related to the production of other enzymes, i.e., AmpC-type enzymes, ESBL, or reduced intracellular accumulation of drugs due to decreased permeability and/or enhanced expression of efflux pumps can be involved (Tsakris A. et al., 2010; Walsh T. R., 2010; Nordmann P. et al., 2011).
1.2.2 Carbapenemase enzymes
Carbapenemases have been classified on the base of functional, structural and molecular features. Carbapenemases are commonly classified into three different molecular classes, called Amber’s class A (i.e., KPC type), class B (i.e., VIM type), and class D (i.e., OXA-48 type). These are distinguished by the active site of the hydrolytic mechanism: class A and D carbapenemases require serine at their active site (serine carbapenemases), while class B are zinc dependent, (metallo-β-lactamases, MVLs). Carbapenemase production can be chromosomal (SME, IMI, NMC) or plasmid (KPC, GES) based.
Class A carbapenemases include Klebsiella pneumoniae carbapenemase (KPC), IMI-2, Guaina extended spectrum β-lactamases (GES), which effectively hydrolyze carbapenems and
all other β-lactams. Plasmid encoded KPC enzymes have emerged as the most clinical dominant member of this class.
Class B carbapenemases include Verona integron-encoded metallo β-lactamases (VIM), imipenem hydrolyzing β-lactamases (IMP) and New Dehli metallo β-lactamases (NDM-1). These enzymes hydrolyze all β-lactams, except aztreonam, and are inhibited by EDTA, but not by clavulanic acid.
Finally, class D carbapenemases include oxacillin hydrolyzing metallo β-lactamases (OXA), of which OXA-48 is the most common. The first OXA-48 K. pneumoniae isolate was dectected in 2003 in Turkey. Since then, it has disseminated across Europe, the South-East Mediterranean region and Africa. These enzymes exhibit only a weak ability to hydrolyze carbapenems, broad spectrum cephalosporins and aztreonam. Class D carbapenemase has been found in different members of Enterobacteriaceae, such as Citrobacter freundii,
Providencia rettgeri, Enterobacter cloacae, and even in E. coli (Carrër A. et al., 2008;
1.3 Klebsiella pneumoniae
Klebsiella pneumoniae is a non-motile, rod-shaped, Gram-negative bacterium belonging to the Enterobacteriaceae family. Klebsiella pneumoniae has a prominent polysaccharide capsule, which encases the whole cell surface and provides resistance against many host immune mechanisms (Umeh, 2011). It occupies diverse ecological niches ranging from soil to water, but it is commonly reported as an etiological agent of either community acquired pneumonia or urinary tract infections. However, it can cause any type of infection in hospital settings, including outbreaks in newborns and adults under intensive care, which is likely associated to its ability to spread rapidly in the hospital environment (Podschun R. et al., 1998; Podschun R. et al., 2001).
In the last years, the rapid widespread of multidrug resistant K. pneumoniae strains, producing KPC type carbapenemase, are drawing the attention of clinicians and researchers.
1.3.1 KPC Klebsiella pneumoniae
Among the carbapenemases, previously described, K. pneumoniae carbapenemase (KPC) is currently the most common. Infections caused by KPC isolates are an emerging problem worldwide (Tzouvelekis L. S. et al., 2012). They usually concern critically ill patients and are associated with high morbility, mortality, prolongation of hospitalization and costs (Souli M.
et al., 2008; Daikos G. L. et al., 2009; Maltezou H. C. et al., 2009; Perez et al., 2010, Bratu S. et al., 2005; Gasink L. B. et al., 2009; Zarkotou O. et al., 2011). In a recent epidemiologic study, infections caused by KPC-producing K. pneumoniae strains were associated with significantly higher mortality than infections with carbapenem-susceptible K. pneumoniae strains (32.1% versus 9.9%) (Gasink et al., 2009).
KPC enzymes are encoded by the blaKPC gene, which is located within a Tn3-type transposon
(Tn4401). This transposon has been reported in a variety of transferable plasmids with high frequency between nosocomial Klebsiella pneumoniae isolates and other genera of Enterobacteriaceae. The KPC gene has also been identified in many other bacterial species, such as Escherichia coli, Enterobacter species, Salmonella enterica, Proteus mirabilis,
Citrobacter freundii, and Pseudomonas aeruginosa (Queenan A.M. et al., 2007; Villegas
M.V. et al., .2007; Bush K. et al., 1995). However, to date community-acquired infections have not been reported.
Although eleven variants of the KPC gene, blaKPC1/2 through blaKPC12, were identified, the
global dissemination of KPC is largely associated with KPC 2 and KPC 3 (Arnold R. et al., 2011; Richter S. et al., 2012). The primary mode of KPC spread is via the clonal dissemination of K. pneumoniae. The dominant KPC-producing clone is the sequence type (ST) 258 clone (Cuzon, G. et al., 2010).
1.3.2 Epidemiology
The first KPC-producing K. pneumoniae strain was identified in 1996 in North Carolina (Yigit H. et al., 2001). The discovery of KPC-1 was quickly followed by several reports of a single-amino acid variant, KPC-2, that subsequently was shown to be genetically identical to KPC-1. Within a few years, several outbreaks of KPC strains have been described globally. KPC strains remained rare in the USA until 2005, then KPC-producing Enterobacteriaceae were identified in outbreaks in New York and New Jersey hospitals. A spread was then observed throughout North America (Bratu S. et al., 2005; Landman D. et al., 2007). KPC strains spread via colonized patients and air transportation. Later, KPC strains were isolated almost everywhere in the world: in France for the first time in 2005 in a patient coming back from New York, in Israel, China and Taiwan, South America, Brazil, Argentina, and in most
European countries. The main endemic focus in Europe is located in Greece (Nordmann P., 2014). In Italy, a KPC-type producing K. pneumoniae strain was reported for the first time in the late 2008, likely originated from Israel (Giani T. et al., 2009). Next, KPC-type producing
K. pneumoniae has undergone rapid diffusion also in Scotland, Germany, Belgium, Finland,
Scandinavian countries, and Switzerland.
1.3.3 Treatment
Carbapenemase-producing K. pneumoniae strains, in particular KPC, exhibit resistance to multiple antibiotics, because they are usually also resistant to most other classes of drugs, i.e., fluoroquinolones and aminoglycosides (Nordmann P. et al. 2009; Elemam A. et al., 2010). Indeed, the plasmids that carry the KPC gene may also contain genes encoding aminoglycoside-modifying enzymes and some have also been reported to encode QnrA and QnrB, resulting in reduced susceptibility to fluoroquinolones (Endimiani A. et al., 2009; Chmelnitsky I. et al., 2008). Currently, a few drugs remain active, such as colistin (polimixin E), tigecycline, gentamicin and fosfomycin (Grundmann H. et al., 2010; Hirsch E. B. and Tam V. H., 2010).
Colistin, a cyclic polypeptide antibiotic, is the only one that can be used to treat serious bloodstream infections caused by carbapenemase-producing microorganisms. Nevertheless, the use of polymyxins should be limited due to its neurotoxicity and nephrotoxicity. Colistin exerts bactericidal activity through interactions with lipid A on the outer membrane and is active against most Gram-negative species.
Tigecycline is a glycylcycline antibiotic that is reported to have activity against a wide variety of Gram-positive and Gram-negative pathogens, including multidrug-resistant strains of the Enterobacteriaceae family. Tigecycline is similar to tetracycline antibiotics and acts by binding to the bacterial 30S ribosome, blocking the entry of transfer RNA, thus preventing
protein synthesis by halting the incorporation of amino acids into peptide chains (Wyeth, 2005). However, the drug does not achieve adequate levels in urine or blood, thereby it is not indicated for the treatment of urinary tract infections, serious bacteremia, pneumonia, or central nervous system infections (Falagas M. E. et al., 2011).
Gentamicin is an aminoglycoside antibiotic used to treat most bacterial infections, caused by Gram-negative microorganisms. It inhibits protein synthesis by binding to the 30S subunit of the ribosome.
Fosfomycin acts by inhibiting the early stages of cell wall synthesis and is useful for the treatment of urinary tract infections.
Unfortunately, the increasing use of these antibiotics has induced the emergence of multidrug resistant Gram-negative strains (Matthaiou D. K. et al., 2008; Antoniadou A. et al., 2007; Kontopidou F. et al., 2011). These strains were mainly treated with colistin. Recently, colistin resistance has been described with percentages of resistance varying from 8% to 89% (Bratu
et al., 2005; Cuzon et al., 2010; Elemam et al., 2010; Marchaim et al., 2011; Neuner et al.,
2011).
In addition, no novel classes of agents against Gram-negative bacteria are in clinical development (Livermore D., 2009; Boucher H.W. et al., 2009).
1.3.4 Combination therapy
Currently, the combination therapy seems to be a valid option in the treatment of infections caused by carbapenemase-producing K. pneumoniae. Treatments based on the use of various combinations of colistin, tigecycline, meropenem, fosfomycin, and aminoglycoside have been suggested. Moreover, rifampicin, which is currently used in combination therapy, primarily against mycobacteria and Gram-positive cocci, has also been recently adopted for the treatment of multidrug resistant Gram-negative infections, such as those due to KPC strains
(Chen and Kaye, 2009; Drapeau C. M. J. et al., 2009). Rifampicin is a hydrophobic antibiotic, which exerts its bactericidal activity by inhibiting DNA-dependent RNA polymerase in bacterial cells, thus preventing transcription to RNA and subsequent translation to proteins. The combination of rifampicin and polimixin B, an analogue of colistin, against KPC-producing K. pneumoniae showed synergistic activity in in vitro studies (Pankey G. A. et al., 2011).
Two clinical trials are now being conducted to evaluate the efficacy of this combination therapy in vivo (Endimiani et al., 2009; Temkin E., 2014).
1.4 New therapeutic approaches: antimicrobial peptides
In light of the need to develop new drugs, cationic antimicrobial peptides have gained attention as possible therapeutic candidates.
The first human antimicrobial protein, named lysozyme, was identified by Alexander Fleming at the end of 1920s (Fleming A., 1922). Since then, numerous antimicrobial proteins were discovered, like lactoferrin that was isolated from human and bovine milk in the 1960s (Montreuil J. et al., 1960; Groves M. L. et al., 1965). However, the starting point of the history of antimicrobial peptides is popularly taken to be the 1980s, based on the isolation of cecropins from silk moth pupae (Steiner H. et al., 1981) and magainins from the skin of frogs (Zasloff M. et al., 1987). Further studies, led to characterization of various antimicrobial peptides not only in the animal kingdom, but also in plants and bacteria. Over the past decades, a large number of naturally occurring and/or synthetic peptides have been identified and evaluated as antimicrobial molecules against different pathogens.
Currently, an updated list can be consulted at the Antimicrobial Peptide Database
(http://aps.unmc.edu/AP/main.php), where more than 2.000 molecules have been reported.
In higher organisms, antimicrobial peptides play a major role in innate immunity as a part of the first defense line directly against invading pathogens or by modulating the acquired immune response (Hancock and Scott, 2000). In humans, they are produced by granulocytes, macrophages, epithelial and endothelial cells, and their expression may be either constitutive or induced by proinflammatory cytokines, microorganisms or microbial components, such as lipopolysaccharide (LPS). Bacteria and fungi produce antimicrobial peptides as a defense mechanism and as a mean of gaining competitive advantage against other microorganisms, sometimes of the same species (Sang and Blecha, 2008).
Antimicrobial peptides are small cationic molecules, usually composed of 10-50 amino acid residues and are characterized by a net positive charge (between +2 and +9), hydrophobicity and amphipaticity. Antimicrobial peptides can be categorized in four classes on the base of
their secondary structure: alpha-helical, beta-sheet, extended and looped structures. They show broad-spectrum antimicrobial activities against a wide variety of pathogens, including Gram-positive and Gram-negative bacteria, fungi, viruses and parasites (Hancock and Diamond 2000; Jensen et al., 2006). In addition, many peptides are effective against multi-drug resistant (MDR) microorganisms and possess low propensity for developing resistance. Despite the structural differences, the primary step for the antimicrobial activity of these peptides generally seems to be an electrostatic interaction with the negatively charged molecules of the microbial cell wall. In particular, antimicrobial peptides interact with LPS on the outer membrane of Gram-negative bacteria, and with teichoic acids on Gram-positive bacteria. Unlike procariotic cells, the mammalian membranes have low membrane potentials and are enriched in sterols and zwitterionic phospholipids with neutral net charge, thus protecting cells from peptide-binding (Giuliani et al., 2007; Yeaman and Yount, 2003). Subsequent insertion of peptides into the cytoplasmic membrane under the influence of the transmembrane electrical potential gradient results in membrane permeability, leakage of cellular constituents, thus destroying the proton gradient across the membrane and cell death. Various models have been proposed to describe the action of peptides on the cytoplasmic membrane, including barrel-stave, toroidal pore and carpet models (Wimley and Hristova 2011). However, recent studies have revealed that some antimicrobial peptides can cross the lipid bilayer without provoking any damage to the cell membrane and inhibiting essential cellular processes, thus resulting in cell death (Pushpanathan M. et al., 2013).
1.4.1 Human lactoferrin
Lactoferrin (LF) is a 77 kDa iron-binding glycoprotein belonging to the transferrin family (Gonzales-Chavez SA et al., 2009). It is provided to newborns by breast-feeding and found in most of the exocrine secretions, such as milk, tears, nasal secretions, saliva, urine, uterine secretions, and amniotic fluid. It is a major component of neutrophil-specific granules (Masson P. L. et al., 1969; E.R. Öztaş Yeşim and N. Özgüneş, 2005). It is present at high concentrations (1-10 mg/ml) on many mucosal surfaces, since it is synthesized by mucosal gland epithelial cells (Nuijens JH, van Berkel PH, and Schanbacher, 1996). The role of LF in the mucosal defense is illustrated by the observation that the Candida load of the oral cavity increases with decreasing salivary LF levels (van der Strate B.W. et al., 1999). LF is part of the innate defense system and exerts its activity through different mechanisms of action. For example, lactoferrin plays a role in host defenses by sequestering environmental iron through its two high-affinity ferric iron binding sites, thereby inhibiting microbial growth (Jenssen H. and Hancock RE., 2009). It displays direct antimicrobial activity by interacting with a variety of biologically important, negatively charged molecules, such as LPS in Gram-negative bacteria and reducing the net negative charge of Gram-positive bacteria. Moreover, lactoferrin displays anti-inflammatory activities by reducing pro-inflammatory cytokines and by inducing of IL-10 production (Adlerova L. et al., 2008). LF receptors have been identified on activated human T cells, B cells and NK cells (Mincheva-Nilsson L. et al., 1997) and also on the cell surface of platelets, megakaryocytes, brain capillary endothelial cells (Fillebeen C. et
al.,1999). As a result of these properties, LF exhibits a variety of immunoregulatory activities
(Yoo Y-C et al., 1997; Zhang G.H. et al., 1999; Baveye S. et al., 2000; Cumberbatch M. et
al., 2000; Britigan BE et al., 2001).
More importantly, human LF is the source of cationic and hydrophobic peptides with antimicrobial activity (Nibbering PH et al., 2001; Lupetti A. et al., 2000). Human lactoferrin, when subjected to pepsinolysis, releases the antimicrobial peptide lactoferricin H (residues 1
to 47), which contains two cationic domains (residues 2 to 5, and 28 to 31). Both domains show antimicrobial activity, albeit recent studies indicate that synthetic peptides, including the first or the second cationic domain of human lactoferrin, show more potent candidacidal activity (Lupetti A. et al., 2000) than the native protein. Moreover, peptides containing the first cationic domain of human LF are even more effective in microbial killing than peptides containing the second domain as shown in in vitro (Lupetti A. et al., 2000) and in vivo experiments (Nibbering PH et al., 2001).
1.4.2 The human lactoferrin-derived antimicrobial peptide hLF1-11
The synthetic peptide hLF1-11, as its name suggests, is the N-terminal peptide of human lactoferrin comprising the first cationic domain of lactoferricin H. This peptide has been demonstrated to possess effective antibacterial and antifungal activity against a variety of microorganisms, including several multidrug resistant pathogens. Its antibacterial activity was first tested by in vitro killing experiments against both Gram-positive and Gram-negative bacteria, such as Listeria monocytogenes, and Staphylococcus aureus (MRSA), and
Escherichia coli, and Klebsiella pneumoniae, in which the peptide killed these pathogens at
relatively low concentrations (Nibbering P.H. et al., 2001). Afterwards, Dijkshoorn et al. demonstrated the antibacterial activity of hLF1-11 against various multi-drug resistant
Acinetobacter baumannii strains (Dijkshoorn L. et al., 2004). However, as the antimicrobial
activity of hLF1-11 is salt-sensitive, these experiments were all performed in vitro at low salt conditions (~10 mM NaCl). At physiological salt concentrations (~155 mM NaCl), this peptide is ineffective in vitro at similar concentrations against all bacterial strains. Remarkably, it proved to be very active against various bacterial strains when tested in animal models. Possible explanations to these findings include i) synergism between the peptide and other antimicrobial proteins/peptides, such as lysozyme or other local factors, such as pH and
Zn2+ concentrations, and ii) interactions with host cells, leading to enhanced antibacterial activity of the cells (Leith E. C., and M. D. Wilcox, 1999; Myauchi H. et al., 1998). In vitro and in vivo studies demonstrated also antifungal activity against Candida albicans and
Aspergillus fumigatus strains.
Stallmann et al. studied the efficacy of local prophylactic treatment with hLF1-11 in a rabbit model of femur infection and observed that the peptide effectively reduced the development of osteomyelitis (Stallmann H. P. et al., 2004). In addition, hLF1-11 acts synergistically with fluconazole against fluconazole-resistant C. albicans in vitro (Lupetti A. et al., 2003). Besides direct killing of C. albicans, the peptide is also able to reduce the virulence of C. albicans by inhibition of the morphological transition of this yeast from a round-shaped conidial form to a more virulent elongated hyphal shape (Lupetti A. et al., 2007).
The exact mechanism of action of hLF1-11 is not fully known. The potent antimicrobial effect of hLF1-11 was attributed to the first two arginines at the N-terminus of human lactoferrin (Lupetti A. et al., 2000). This conclusion is based on the fact that peptides lacking these residues were found to be less effective in the killing of bacteria and fungi (Lupetti A. et al., 2000; Nibbering PH et al., 2001). Arginine possesses chemical properties that confer antimicrobial activity upon arginine-containing peptides: arginine residues endow peptides with positive charge and hydrogen binding properties necessary for interaction with the abundant negatively charged components of bacterial cell walls (D.I Chan et al., 2006).
According to this view, hLF1-11 could be assigned to the class of loop-antimicrobial peptides (Powers and Hancock, 2003; Fornili S. L. et al., 2010).
It was hypothesized that the candidacidal activity of the peptide is due to the peptide induced mitochondrial damage, with subsequent rise in extracellular ATP, which resulted to be essential but not sufficient to kill C. albicans (Lupetti A. et al., 2000). In later studies, it was found that the uptake of calcium by mitochondria plays an essential role in the peptide-induced killing of C. albicans (Lupetti A. et al., 2004). Furthermore, hLF1-11 displays
immunomodulatory activity by inducing the GM-CSF-driven monocyte differentiation toward macrophages and the production of both pro- and anti-inflammatory cytokines. The cellular target for the immune-modulatory activity seems to be myeloperoxidase, to which hLF1-11 binds and, after entering inhibits the monocytes (van der Does A. M. et al., 2010).
The safety and tolerability of hLF1-11 have been tested both in healthy volunteers and haematopoietic stem cell transplantation recipients. These studies showed that the peptide was safe and well tolerated, although some adverse events were graded mild (van Velden W. J. et
2. STUDY PROPOSALS
Infections caused by KPC K. pneumoniae are typically difficult-to-treat, due to the existence of extended antibiotic resistance phenotypes, the paucity of effective drugs and of new therapeutic options. In light of the need to develop new drugs, cationic antimicrobial peptides have gained attention as possible therapeutic candidates, because they exhibit a broad spectrum activity.
First, the present study aimed at evaluating whether the N-terminus of human lactoferricin, further referred to as hLF1-11, exerts antibacterial activity against clinical sensitive and resistant Klebsiella pneumoniae strains, and whether sub-inhibitory concentrations of hLF1-11 act synergistically with various antimicrobial drugs to kill carbapenemase-producing K.
pneumoniae strains, harboring different resistance genes. In addition, the cytotoxicity of the peptide and antibiotics alone and in combination was assessed by a hemolysis assay.
Second, this study aimed at better understanding the underlying mechanisms of the synergistic antibacterial effect induced by the combination of hLF1-11 and rifampicin.
3. MATERIALS AND METHODS
3.1 Bacteria
Klebsiella pneumoniae strains, isolated from blood cultures from January to June 2012, were
collected at the Unità Operativa di Microbiologia Universitaria of the Azienda Ospedaliero-Universitaria Pisana (Pisa, Italy) and transferred into the Bactec FX. Bacterial identification was performed by the MALDI-TOF spectrometry (Bruker Daltonics) and, the antimicrobial susceptibility testing was evaluated by the Vitek 2 system (bioMeriéux, l’Etoile, France) and confirmed by E-test (bioMeriéux, l’Etoile, France). The K. pneumoniae strains, which resulted to be MDR at the antimicrobial susceptibility testing were further analysed phenotypically and genotypically in order to determine the production of the KPC carbapenemase enzyme and the presence of blaKPC gene, respectively. Among the tested
strains, a colistin-susceptible KPC K. pneumoniae strain, further referred to as KPC K.
pneumoniae S and a colistin-resistant KPC K. pneumoniae strain, further referred to as KPC K. pneumoniae 1R, and 1 K. pneumoniae resulted to be completely sensitive to the
antimicrobial testing were selected.
In this study, four multidrug resistant K. pneumoniae strains (OXA-48, KPC 2, KPC 3 and VIM-1) kindly provided by Professor G. Rossolini (University of Florence) and KPC K.
pneumoniae positive (ATCC® BAA-1705™) and negative control (ATCC® BAA-1706™) were
also used. Bacterial cells grown overnight in Luria Bertani (LB) medium (Sigma-Aldrich, St. Louis, MO, USA) were subcultured until mid-log phase and aliquots of this suspension were supplemented with 20% (vol/vol) glycerol and stored at -80°C.
3.2 Phenotypic and genotypic characterization
A combination disk test was used to determine the production of the KPC carbapenemase enzyme, employing meropenem disks supplemented with carbapenemase enzyme inhibitors
(boronic acid, dipicolinic acid and cloxacillin) (NeosensitabsTM, Rosco, Italy). The test was performed by inoculating Mueller Hinton agar plates (Becton Dickinson& Co, BD; Milan, Italy) with a bacterial suspension (0.5 McFarland) and placing disks of meropenem with or without the various inhibitors. The plates were incubated overnight at 37°C. The K.
pneumoniae ATCC® BAA-1706™ and ATCC® BAA-1705™ were used as negative and positive control strains, respectively. Next, the diameter of the growth-inhibitory zone was measured. Typically, KPC-producing strains show an increased meropenem inhibition zone in the presence of boronic acid. However, the strains showing both overproduction of AmpC and porin loss give positive results as well. In order to identify the KPC strains, the disk diffusion assay with meropenem and cloxacillin was evaluated, as KPC strains show no inhibitory zone with such an assay. Furthermore, in order to exclude strains producing metallo β-lactamases, the disk diffusion assay with meropenem and dipicolinic acid was performed. The growth of the latter strains is inhibited by such a test (Table 1) (Giske C. G. et al., 2011; Hansen F. et
al., 2012).
Table 1. Interpretation of results of the phenotypic test. Meropenem + boronic acid Meropenem + dipicolinic acid Meropenem + cloxacillin KPC Meropenem (10 µg) ≥ 5 mm <5 mm <5 mm
AmpC+porin loss Meropenem
(10 µg) ≥ 5 mm <5 mm ≥ 5 mm
Metallo β-lactamase
Meropenem
(10 µg) <5 mm ≥ 5 mm <5 mm
The results are interpreted by comparing the diameter of the growth-inhibitory zone of different disks containing inhibitor to that obtained with a disk containing meropenem.
The presence of blaKPC gene was determined using Hy-KPC real time PCR (Hy-Labs, Israel)
by ABI Prism® 7500 instrument (Applied Biosystems & Life Technologies, Monza, Italy). The kit, which was kindly provided by Hy-Labs, includes a ready to use PCR mix and a KPC positive control. For each strain, 2-3 isolated colonies were taken using a sterile 0.2-10 µl pipette tip and placed into a PCR tube, in which 30 µl of PCR-grade water were added. This suspension was incubated for 10 min at 100°C, centrifuged for 1 min at 12,000 rpm, and the supernatant was transferred carefully into a clean tube. The test was performed using a 96 well plate; the reaction mixture was prepared by adding 5 µl of supernatant to 15 µl of PCR reaction mix for each strain/well. The plate was closed with an adhesive strip and placed inside the real time PCR termocycler for 2 h.
3.3 Synthetic peptide and antibiotics
The synthetic peptide corresponding to residues 1-11 of human lactoferrin, hLF1-11 (GRRRRSVQWCA; molecular mass, 1374 Da) was purchased from Peptisyntha (Brussels, Belgium). The purity of this peptide exceeded 99%, as determined by reverse-phase high performance liquid chromatography (RP-HPLC). Stocks of the peptide at a concentration of 10 mM of 0.01% acetic acid (pH 3.7) were stored at -20°C and diluted to desired concentrations before use. Rifampicin, clarithromycin, clindamycin, gentamicin, and tigecycline were purchased from Sigma-Aldrich (St. Louis, MO, USA). Rifampicin and tigecycline were dissolved in dimethyl sulfoxide (Fluka Chemie GmbH, Sigma-Aldrich Chemie BV, Zwijndrecht, The Netherlands) and stored at -80°C. The final concentration of DMSO was <0.1%. The remaining antibiotics were dissolved in sterile distilled water, and stored at -20°C until use.
3.4 Killing assay
Bacterial cells grown overnight in LB at 37°C and subcultured for 2 h on a rotary wheel at 37°C were harvested in mid-log phase by centrifugation at 4,500 x g for 10 min, washed twice in NaPB buffer (10 mM; pH 7.4) and diluted to a concentration of 107 cells/ml of NaPB. Equal volumes of this suspension were mixed with hLF1-11, and then incubated for 1 h at 37°C. Thereafter, the number of viable bacterial cells was determined by plating serial dilutions of each sample on blood agar plates (Becton Dickinson & Co, BD; Milan, Italy).
3.5 Synergy studies
3.5.1 Checkerboard assay
In order to determine whether hLF1-11 and antimicrobial agents can interact and have an enhanced antibacterial effect, synergy analysis was carried out by a checkerboard titration method using 96-well round bottom polystyrene microtiter plates. The ranges of the antibiotic dilutions used were as follows: 0.125-32 µg/ml for rifampicin, 0.25-256 µg/ml for clarithromycin, 0.125-64 µg/ml for clindamycin, 0.0078-1 µg/ml for gentamicin (0.015-16 µg/ml only for KPC 2 strain), and 0.06-16 µg/ml for tigecycline. The range of concentrations of hLF1-11 peptide was 2.7-88 µg/ml.
Briefly, the twofold dilutions of each agent were set up in 100 µl of 1/8 strength Mueller Hinton broth (Oxoid, Milan, Italy) and then, an equal volume of the mid-log bacterial suspension (5x105 CFU/ml) was inoculated per well. Growth control well containing the medium was included in each plate.
After an 18-24 h incubation at 37°C, the MIC of both the peptide and the antibiotics were defined on the basis of the turbidity of the wells, as the lowest concentration of the agent that produced the complete inhibition of visible growth. The fractional inhibitory concentration
(FIC) index for the combinations was calculated using the following formula: FIC index = (MIC drug A in combination)/(MIC drug A alone) + (MIC drug B in combination)/(MIC drug B alone). The FIC indices were interpreted as follows: < 0.5, synergy, 1-4, indifference and > 4, antagonism (Moody J. A., 1992). FIC indexwas reported in this study as the mean of the lowest FIC index of at least three independent experiments.
3.5.2 Time-kill assay
In addition, time–kill synergy studies were performed at sub-inhibitory concentrations. At times earlier than 24 h, synergy was defined as a decrease in CFU/ml of ≥ 2 log10 between the
combination of hLF1-11 and antibiotic, and its most active constituent (Eliopoulos GM et al. 1996, Odds FC 2003). All the tests were performed in triplicate.
To obtain some insight into the contributions of the different agents to the synergistic effect of the combination between hLF1-11 peptide and rifampicin against KPC K. pneumoniae cells, sub-inhibitory concentrations of hLF1-11 or rifampicin were preincubated for 5, 30, 60 and 90 min in diluted MH at 37°C. Then, cells were washed in NaPB at 4°C, and reincubated with the other agent till reaching a 2 h incubation at 37°C. Next, the number of viable cells was determined by plating serial dilutions of each sample on blood agar plates. Results expressed as CFU K. pneumoniae per ml, are means plus standard deviation (SD) of 3-4 independent experiments.
3.6 Hemolysis assay
We used a hemolysis assay to assess the cytotoxicity of the peptide and the antibiotics alone and in combination. Briefly, blood samples from three healthy individuals were collected in vacuum tubes containing citrate (Becton Dickinson). Red blood cells (RBCs) were harvested by centrifugation at 1600 × g for 5 min at room temperature, washed three times with phosphate-buffered saline (PBS, pH 7.4) and re-suspended in PBS to a concentration of 8% (v/v). An aliquot (100 µl) of this suspension was transferred to each well of a 96-well microtiter plate and mixed with 100 µl of peptide or antibiotic solution at twice the desired concentration or 50 µl of 4X of the peptide and the antibiotic for the synergy combinations. After incubation of 1 h at 37°C, the microtiter plate was centrifuged (1600 × g, 5 min) and 100 µl of the supernatants were transferred to a flat-bottom 96-well plate for measurement of the haemoglobin release by reading the absorbance at 450 nm. PBS and 1% Triton X-100 were used to establish 0% and 100% hemolysis. The percentage of hemolysis was calculated by the following formula: (Apeptide/antibiotic─ APBS)/(ATriton X-100─APBS) × 100%.
3.7 Ethidium bromide (EtBr) permeability assay
EtBr is a fluorescent probe, often used to study the effect of antimicrobial agents on membrane permeability and the function of efflux pumps. It is generally known that EtBr crosses the outer membrane of Gram-negative bacteria by passive diffusion, accumulates in the periplasmic space, and is pumped out the cell by efflux pumps. EtBr emits a weak fluorescence in aqueous solution and a high fluorescence intensity when it is intercalated between nucleic bases of DNA. The binding constant is sufficiently strong to keep EtBr inside cells avoiding the efflux pump system (Martins M. et al., 2013). The fluorescence that results from the overall intracellular EtBr content was measured by flow cytometry.
KPC Klebsiella pneumoniae 1R cells in mid-log phase were washed in NaPB buffer and re-suspended in the same buffer at a concentration of 1x107 CFU/ml. Aliquots of this suspension, mixed with various concentrations of the peptide, or with the combination of hLF1-11 and/or rifampicin were incubated with EtBr (final concentration, 1 µg/ml) in diluted MH broth at 37°C for 1 hour at the dark. After incubation, bacterial cells were centrifuged (4500 x g, 5 min), washed with NaPB and fixed in 1 ml of 1% PFA (paraformaldehyde, Sigma Aldrich, St. Louis, MO, USA) at room temperature for 10 minutes, and then overnight at 4°C. CCCP (carbonyl cynide m-chlorophenyl hydrazone, CCCP, 100 µM; Sigma-Aldrich) was used as a positive control since it inhibits the proton–motive force dependent processes, including the efflux pumps. The untreated cells as well as cells treated with EtBr, were used as negative controls. Before measuring the EtBr fluorescence, cells were recovered and re-suspended in PBS buffer (300 µl). The emission of EtBr fluorescence was detected by a flow cytometer BD Accuri™ C6 (BD Biosciences) in the channel FL2. In each sample, at least 5,000 events were acquired and analysed using the BD Accuri C6 software.
3.8 Effect of CCCP on the antibacterial activity of hLF1-11
The influence of proton-motive force on the antibacterial activity of hLF1-11 was determined by the killing assay in the presence of the metabolic uncoupler, CCCP. A stock solution of CCCP (10 mM) was prepared in water. KPC K. pneumoniae 1R cells were pretreated with CCCP (100 µM) for 10 min at 37°C prior to addition of hLF1-11 and/or rifampicin. After incubation for 1 h at 37°C, the cell viability was assessed by plating serial dilutions of each sample on blood agar plates.
3.9 Cytoplasmic membrane depolarization
The anionic bis-1,3-dibutylbarbituric acid trimethine oxonol, DiBAC4(3), is a lipophilic
membrane potential-sensitive dye with a low binding affinity for intact membranes. It accumulates inside the depolarized cells by binding to intracellular proteins and membranes, according to the Nernst equation, thus resulting in an increase of fluorescence signal (Deere et
al., 1995).
Due to safety reasons, this set of experiments was performed on a sensitive K. pneumoniae strain. Cells (1x107 CFU/ml) in mid-log phase were incubated in diluted MH broth with sub-inhibitory concentrations of hLF1-11 for 1 h a 37°C. After incubation, samples were stained with DiBAC4(3) (stock solution at -20°C, 5 mg/ml in DMSO; final concentration, 10 µg/ml;
Sigma-Aldrich) and incubated at 37° for 10 min. Next, the suspensions were centrifuged (4500 x g for 10 min at room temperature) and the bacterial pellets were re-suspended in PBS buffer (300 µl). The concentration of hLF1-11 2X MIC was used to assess the membrane depolarization at different time points (up to 1 h). In addition to untreated cells, a negative and a dead control (cells treated with ethanol 70%, or heat-killed at 70°C for 1 h), CCCP was used as a positive control. The emission maximum of DiBAC4(3) fluorescence (λem 516 nm) was
20,000 events were acquired and analysed using the BD Accuri C6 software. The antimicrobial activity of the peptide was evaluated as the percentage of the depolarized bacteria.
Statistical analysis
Results were evaluated by one-way ANOVA test. Differences between the results of the various treatments were evaluated with the Tukey-Kramer test. The level of significance set at a P value of 0.05.
4. RESULTS
4.1 Antimicrobial peptide susceptibility assay
Ten K. pneumoniae strains were isolated from blood cultures and the antimicrobial susceptibility was assessed. Nine strains resulted resistant to carbapenems (meropenem, imipenem and ertapenem MIC ≥ 32 µg/ml), and showed variable sensitivity patterns to gentamicin, tygecicline and colistin. Among these strains, a MDR strain, further referred to as KPC 1R, was also resistant to colistin with MIC ≥ 16 µg/ml. Strain n. 10 was susceptible to all the tested antimicrobials, except to ampicillin and norfloxacin.
4.2 Phenotypic and genotypic characterization
The phenotypic test revealed that the strain n. 10 did not produce carbapenemase enzymes, and the other strains were able to produce KPC enzymes, but no other types of carbapenemases. The real time PCR amplification detected blaKPC gene in all the collected
isolates, except in the strain n. 10, confirming the results of the phenotypic assay.
4.3 Killing assay
An in vitro killing assay was used to assess the antibacterial activity of hLF1-11 against the following K. pneumoniae selected strains: the sensitive strain, and two KPC strains (colistin-susceptible and colistin-resistant 1R). The results of the dose-effect study, shown in Figure 1, revealed that hLF1-11 is more efficient (P < 0.05) in killing the susceptible K. pneumoniae strain than the KPC 1R strain, since it was necessary to use 8 times more hLF1-11 for a similar level of antibacterial activity. It should be noted that the antibacterial effect exerted by
the highest concentration of hLF1-11 amounted to almost 1.80-log reduction against KPC 1R strain.
Figure 1. Antimicrobial activity of hLF1-11 against three representative Klebsiella
pneumoniae strains.
* Significant difference (P < 0.05) from values obtained with untreated sensitive K. pneumoniae cells.
** Significant difference (P < 0.05) from sensitive K. pneumoniae cells exposed to hLF1-11.
0 22 44 88 176 352 704 C F U /m l 107 106 105 104 103 102 101 100 hLF1-11 concentration (µg/ml) * * * * Sensitive K. pneumoniae Colistin-susceptible KPC Colistin-resistant KPC 1R ** ** ** **
4.4 Synergy studies
4.4.1 Checkerboard assay
Synergy studies were performed by the checkerboard method combining hLF1-11 with various antibiotics. Besides the selected clinical KPC K. pneumoniae 1R strain, other strains harboring different blaKPC genes and producing different types of β-lactamases were
evaluated. The MIC values of hLF1-11 and antibiotics achieved by the checkerboard method, are given in Table 1. The results revealed that all strains were inhibited by hLF1-11 alone (MIC 22-88 µg/ml). The variability of one dilution was tolerated to determine the MIC of hLF1-11 and antibiotics for each strain. Among the antibiotics, gentamicin and tigecycline showed the lowest MIC values, whereas rifampicin, clarithromycin and clindamycin the highest MIC values.
Table 1. The MIC values of the hLF1-11 peptide and various antibiotics against different
carbapenemase-producing K. pneumoniae strains.
RIF, rifampicin; CLR, clarithromycin; CLI, clindamycin; GEN, gentamicin; TGC, tigecycline.
The results of the synergy studies revealed that hLF1-11 exhibits synergistic effect (FIC index ≤0.5) with all the tested antibiotics. A high degree of synergy was observed with rifampicin,
MIC (µg/ml)
Strain Carbapenemase hLF1-11 RIF CLR CLI GEN TGC
K. pneumoniae 1R
(colistin-resistant) blaKPC 44-88 16-32 256 32 0.03 1
K. pneumoniae
ATCC® BAA-1705™ blaKPC-2 22-44 16 256 32 0.03 1
K. pneumoniae blaOXA-48 44 8 128 32 0.03 1
K. pneumoniae blaKPC-2 22 8-16 256 32 8 1
K. pneumoniae blaKPC-3 22-44 8 128 32 0.06 0.5
clarithromycin and clindamycin, which are non-conventional antibiotics clinically used to treat infections caused by members of Enterobacteriaceae (Table. 2).
Table 2. Effect of the combination of the peptide with five antibiotics evaluated as lowest FIC
index.
aMean of the lowest FIC index of at least three independent experiments. b
The numbers in parentheses are the MICs (µg/ml) in combination, with the first number corresponding to the antibiotic named in the heading column and the second one corresponding to the peptide hLF1-11.
The numbers in bold are the lowest FIC index > 0.5 indicating no synergism.
The FIC index of hLF1-11 combinations with rifampicin, clarithromycin and clindamycin ranged 0.22-0.46, 0.18-0.46, and 0.19-0.5, respectively. The addition of hLF1-11 induced a 64-fold reduction of the MIC of rifampicin (from 8 µg/ml to 0.125 µg/ml) for OXA-48, KPC-3 and VIM-1 K. pneumoniae strains, a KPC-32-fold reduction (from 16 µg/ml to 0.5 µg/ml) for K.
pneumoniae ATCC® BAA-1705™ and a 8/16-fold reduction for KPC 1R and KPC-2 strains. Similar results were obtained when the MICs of clarithromycin, another hydrophobic
antibiotic, were determined (Table 2). The FICI ranged from 0.15 to 0.46 and the sensitization factors ranged from 4- to 64. The highest MIC reduction of clarithromycin in combination was observed against KPC-2 and ATCC® BAA-1705™ strains. The combination of hLF1-11 with clindamycin induced a 4- to 32-fold reduction of the MIC value of clindamycin with FIC index ranging from 0.19 to 0.5. The combination of hLF1-11 with gentamycin or tigecycline has shown FIC index ranging from 0.28 to 0.42 or from 0.38 to 0.5, respectively. Overall, the highest degree of sensitization induced by hLF1-11 was obtained towards rifampicin.
No synergism was observed by combining hLF1-11 with tigecycline against the selected clinical KPC 1R strain and K. pneumoniae harboring the blaKPC-3 gene, with FIC index of 0.84
and 0.75, respectively. Time-kill in vitro studies performed against KPC 1R strain confirmed the absence of synergism at 24 h incubation for both the tested combinations of the peptide and antibiotic (TGC, 0.25 µg/ml with hLF1-11 at a concentration of 11 or 22 µg/ml (Figure 2). However, at earlier time points (6 h) only the combination of hLF1-11 22 µg/ml and tigecycline 0.25 µg/ml resulted in a synergistic effect (3 log reduction).
Figure 2. Bactericidal activity of hLF1-11 and tigecycline against KPC K. pneumoniae 1R
4.5 Hemolytic activity
The hLF1-11 peptide and antibiotics used alone did not lyze human erythrocytes (< 1%) at concentrations equal to MICs of the peptide and/or antibiotics and 10X the highest MIC observed among all strains (Figure 3). Rifampicin caused hemolysis (1-5%) at MICs (> 8 and < 32 µg/ml), and was highly hemolytic (37%) at 10X the highest MIC (320 µg/ml). Interestingly, the combination of hLF1-11 with rifampicin exhibited no hemolysis at the MIC value (<1%) and 3% haemolysis at 10X MIC.
Figure 3. Hemolytic activity of the hLF1-11 peptide and antibiotics alone (A) and in
combination (B) at MICs and 10X MICs.
B)
Figure 3. Hemolytic activity of peptide and antibiotics alone (A) and in combination (B) at MICs and 10X
MICs.
The values above the bars indicate the tested concentrations (µg/ml). For the combinations, the concentrations are reported as x/y, where x and y represent the concentrations of antibiotic and peptide, respectively. Peptide and antibiotics were incubated with 8% RBCs suspension. The results are expressed as percent hemolysis. RBCs incubated with 1% Triton X-100 and PBS (untreated) were considered as 100% and 0% of hemolysis, respectively.
The percentage of hemolysis was calculated as follows: (Apeptide/antibiotic─ APBS)/(ATriton X-100─APBS) ×100%.
Standard deviations from three independent experiments are reported.
4.6 Time-kill assay for the synergistic combination of hLF1-11 and rifampicin
In order to gain some insight into the mechanism of action of this synergistic effect, KPC-producing K. pneumoniae 1R cells were incubated for various intervals with a sub-inhibitory concentration of hLF1-11 (22 µg/ml) and, after washing, with rifampicin (4 µg/ml), and vice versa (Figure 4).
Figure 4. Effect of preincubation of hLF1–11 and/or rifampicin on the synergistic effect of
the combination of hLF1-11 and rifampicin against KPC K. pneumoniae 1R strain.
* Significantly different (P < 0.05) from values obtained with KPC 1R K. pneumoniae exposed to hLF1-11 or rifampicin alone;
** Significantly different (P < 0.05) from values obtained with KPC 1R K. pneumoniae exposed for 2 h to the combination of hLF1-11 with rifampicin;
The results revealed that the antibacterial activity obtained upon exposure to rifampicin for 30 or 60 min followed, after washing, by hLF1-11 amounted to about 1.5 log reduction in colony count, although the antibacterial effect was significantly different from co-incubation of hLF1-11 and rifampicin for 2 h (P < 0.05). Interestingly, no antibacterial effect was found when cells were incubated with rifampicin for 5 or 90 min before exposure to hLF1-11. The time-kill studies showed that the co-incubation till 30 min of the peptide with rifampicin, at the tested concentrations, caused no reduction of bacterial cell count. The synergistic effect started to become visible after 60 min of co-incubation (1 log reduction), reaching 3 log reduction at 120 min of co-incubation (Figure 5). Differently, when cells were first incubated with the peptide, washed and then incubated with rifampicin up to 2 h, no antibacterial effect was observed.
Figure 5. Time–kill curves of hLF1-11 in combination with rifampicin against KPC K.
4.7 Ethidium bromide (EtBr) permeability assay
EtBr accumulation in the cell is the result of the interplay between cell-wall permeability and efflux pumps, which are dependent on the proton motive force (PMF) (Rodrigues L. et al., 2011).
The results of the EtBr accumulation are shown in figure 6. As CCCP is an uncoupler of oxidative phosphorylation, it was used to dissipate the PMF. The inhibition of the efflux pumps allows EtBr accumulation inside the bacterial cell, thus resulting in a significant fluorescence (P < 0.001) increase in comparison to cells not exposed to CCCP. The flow cytometric analysis revealed that both the cells treated with the peptide (22 µg/ml) and the cells treated with the peptide/rifampicin combination displayed significantly (P < 0.001) increased EtBr fluorescence intensity, than untreated cells. No increase in the EtBr fluorescence intensity was induced by the treatment with rifampicin (4 µg/ml). Furthermore, the peptide induced a dose dependent increase in EtBr fluorescence intensity (Figure 7).
Figure 6. EtBr accumulation induced by hLF1-11 alone and in combination with rifampicin.
* Significant difference (P < 0.05) from untreated cells.
Data are means + standard deviation (SD) from at least 3 independent experiments.
hLF1-11 (22µg/ml) + RIF (4 µg/ml) Control CCCP hLF1-11 RIF (22 µg/ml) (4 µg/ml) 0 500 1000 1500 2000 2500 3000 3500 4000 4500 5000 * M ed ian F lu or es ce n ce I n te n si ty * *
Figure 7. EtBr accumulation inside the KPC K. pneumoniae 1R cells induced by various
concentrations of hLF1-11.
Control represents bacteria untreated and incubated with EtBr. * Significant difference (P < 0.05) from untreated cells.
Data are means + standard deviation (SD) from at least 3 independent experiments. 0 500 1000 1500 2000 2500 3000 3500 4000 4500 5000 0 2,7 5,5 11 22 44 CCCP * * * hLF1-11 concentration (µg/ml) * M ed ian F lu or es ce n ce I n te n si ty *
4.8 Effect of CCCP on the hLF1-11 antibacterial activity
To better understand the role of the PMF on the bactericidal activity of hLF1-11 or rifampicin, cells were first exposed to CCCP and then treated with a bactericidal concentration of hLF1-11 or rifampicin alone. The concentration of CCCP used was not bactericidal. The results revealed that the bactericidal effect of hLF1-11 was significantly (P < 0.001) decreased by CCCP (Figure 8). No inhibitory effect of CCCP was measured on the bactericidal concentration of rifampicin.
Figure 8. Effect of CCCP on the antibacterial activity of bactericidal concentrations of
hLF1-11 and rifampicin against KPC K. pneumoniae 1R strain.
* Significant difference (P < 0.05) from cells incubated with hLF1-11 in absence of CCCP. Data are means + standard deviation (SD) from at least 3 independent experiments.
- CCCP + CCCP * C F U /m l Control 2 h RIF 32 µg/ml hLF1-11 100 µM 108 107 106 105 104 103
Next, the inhibitory effect of CCCP was evaluated on the synergism induced by the combination of hLF1-11 and rifampicin. The results revealed that CCCP significantly (P < 0.001) reduced the synergistic effect induced by the combination of sub-inhibitory concentration of hLF1-11 and rifampicin (Figure 9). Since hLF1-11 was used at a non-bactericidal concentration, CCCP showed no inhibitory effect on hLF1-11 alone.
Figure 9. Effect of CCCP on the antibacterial activity of the combination of sub-inhibitory
concentrations of hLF1-11 and rifampicin against KPC 1R K. pneumoniae strain.
* Significant difference (P < 0.05) from cells incubated with the combination of hLF1-11with rifampicin in absence of CCCP.
Data are means + standard deviation (SD) from at least 3 independent experiments.
- CCCP + CCCP * C F U /m l Control 2 h hLF1-11 (22 µg/ml) RIF (4 µg/ml) hLF1-11 (22 µg/ml) + RIF (4 µg/ml) 108 107 106 105 104 103
4.9 Cytoplasmic membrane depolarization
In order to evaluate whether hLF1-11 was able to induce cytoplasmic membrane depolarization, the anionic dye DiBAC4(3), which enters the depolarized cells, was used.
The results revealed that hLF1-11 induced cytoplasmic membrane depolarization in a dose-dependent manner (Figure 10). As seen, after incubation of 1 h with the peptide, the percentage of the depolarized cells increased. The hLF1-11 peptide at the MIC value induced depolarization of about 50% of the cells.
Figure 10. Cytoplasmic membrane depolarization induced by various concentrations of
hLF1-11 in the sensitive K. pneumoniae strain.
0 10 20 30 40 50 60 70 80 90 100 2,7 5,5 11 22 44 88 MIC % D ep ol ar iz ed c el ls -D iB A C4 (3 ) hLF1-11 concentration (µg/ml)
To shed light into the molecular basis for these effects, the timing of hLF1-11 induced membrane depolarization was evaluated at 2X MIC.
The results revealed that at early time points (< 5 min) cytoplasmic membrane depolarization was already affecting more than 50% of cells, reaching about 80% after 60 min (Figure 11).
Figure 11. Time-dependent membrane depolarization induced by hLF1-11 (2X MIC) in the
sensitive K. pneumoniae strain.
0 10 20 30 40 50 60 70 80 90 100 0 5 15 30 60 Time (min) % D ep ol ar iz ed c el ls -D iB A C4 (3 )
5. DISCUSSION
Two major findings have been made in the present study on the activity exerted by the combination of the hLF1-11 peptide with several antimicrobial drugs in the treatment of infections caused by MDR KPC K. pneumoniae strains.
First, the hLF1-11 peptide showed high antimicrobial activity in a dose dependent manner against a sensitive K. pneumoniae strain. However, this activity was significantly reduced against resistant KPC K. pneumoniae strains. Some authors reported a correlation between colistin resistance (a polycationic antibiotic) and resistance to antimicrobial peptides (Vila-Farres X. et al., 2012; Napier B. A. et al., 2013). In our study, the hLF1-11 induced antibacterial activity was evaluated against one colistin-sensitive and one -resistant KPC K.
pneumoniae strains. No difference in the hLF1-11-induced antibacterial activity was found
between colistin-sensitive and -resistant KPC K. pneumoniae strains. This finding can be explained on the basis of the several mechanisms of antimicrobial resistance that have been described, i.e., structural modifications of the bacterial lipopolysaccharide (LPS) (Cannatelli M. et al. 2013; Velkov et al. 2013), an increased production of polysaccharide capsule (CPS), active efflux pumps, and proteolytic degradation of antimicrobial peptides (Nizet V. et al., 2006; Peschee A. 2002; Llobet E. et al., 2011; Gruenheid S. et al., 2012).
Second, hLF1-11 showed a synergistic effect (FIC index ≤ 0.5) with all the tested antimicrobial drugs against some representative MDR strains of K. pneumoniae producing KPC carbapenemases, metallo β-lactamases and oxacillinases by checkerboard assay. Despite the low antibacterial activity induced by hLF1-11 against the colistin-resistant KPC 1R strain, the peptide combined with the tested antimicrobial drugs, showed a synergistic effect even against such a strain, suggesting a sensitizing effect of KPC K. pneumoniae strains to antibiotics induced by hLF1-11. Such a sensitizing effect was higher when hLF1-11 was
combined with rifampicin, clarithromycin, and clindamycin. These antimicrobial drugs are hydrophobic, normally not able to permeate Gram-negative bacteria, thereby being ineffective on these microorganisms (Molinari M. L. et al., 1993; Vaara M., 1993). Indeed, the hLF1-11 peptide induced up till a 64-fold reduction in the MIC of these antibiotics. In addition, the combination of hLF1-11 and gentamicin or tigecycline induced a decrease in the MIC of these antibiotics ranging from 2 to 8 fold.
Third, the peptide alone and in combination with the tested antibiotics showed no hemolytic effect on mammalian erythrocytes, implying that hLF1-11 might be used in the treatment of these pathogens.
Another important finding of the present study pertains to the mechanism of action of the synergistic effect induced by hLF1-11 combined with rifampicin.
First, we found that the KPC K. pneumoniae 1R cells exposed to rifampicin for 30 or 60 min, but not for 5 or 90 min, washed and then treated with a sub-inhibitory concentration of hLF1-11 up to 2 h, were significantly (P < 0.05) reduced than untreated cells. However, cells exposed for 2 h to the combination of hLF1-11 and rifampicin were significantly (P < 0.05) reduced than cells exposed to rifampicin for 30 or 60 min, washed and then treated with hLF1-11 up to 2 h. Conversely, the exposure with the peptide, followed by incubation with rifampicin, did not kill effectively the bacterial cells. These data suggest that some of the rifampicin molecules, after a 30 min incubation were probably accumulated by K.
pneumoniae and therefore not taken away by washing. The observation that, 90 min preincubation with rifampicin followed by washing and 30 min hLF1-11 did not significantly (P > 0.05) reduce the number of viable cells than untreated cells suggests that the bactericidal effect obtained by 30 min coincubation of hLF1-11 and rifampicin was not sufficient to significantly reduce the number of viable cells. Indeed, our hypothesis was supported by time kill assays, which showed that a co-incubation of at least 60 min was necessary to observe a significant (P < 0.05) reduction of bacterial cell count. The subsequent addition of hLF1-11
