Insect antimicrobial peptide complexes prevent resistance development in bacteria

Insect Antimicrobial Peptide ComplexesPrevent Resistance Development in Bacteria Sergey Chernysh1*, Natalia Gordya1, Tatyana Suborova2 1 Laboratory of Insect Biopharmacology and Immunology, Faculty of Biology, St. Petersburg StateUniversity, St. Petersburg, Russia, 2 Research Center of Kirov Military Medical Academy, St. Petersburg,Russia In recent decades much attention has been paid to antimicrobial peptides (AMPs) as natural antibiotics, which are presumably protected from resistance development in bacteria. How- ever, experimental evolution studies have revealed prompt resistance increase in bacteria to any individual AMP tested. Here we demonstrate that naturally occurring compounds containing insect AMP complexes have clear advantage over individual peptide and small Citation: Chernysh S, Gordya N, Suborova T (2015) molecule antibiotics in respect of drug resistance development. As a model we have used Insect Antimicrobial Peptide Complexes Prevent the compounds isolated from bacteria challenged maggots of Calliphoridae flies. The com- Resistance Development in Bacteria. PLoS ONE 10(7): e0130788. doi:10.1371/journal.pone.0130788 pound isolated from blow fly Calliphora vicina was found to contain three distinct families of cell membrane disrupting/permeabilizing peptides (defensins, cecropins and diptericins), Editor: Axel Cloeckaert, Institut National de laRecherche Agronomique, FRANCE one family of proline rich peptides and several unknown antimicrobial substances. Resis- tance changes under long term selective pressure of the compound and reference antibiot- Received: February 26, 2015 ics cefotaxime, meropenem and polymyxin B were tested using Escherichia coli, Klebsiella Accepted: May 26, 2015 pneumonia and Acinetobacter baumannii clinical strains. All the strains readily developed Published: July 15, 2015 resistance to the reference antibiotics, while no signs of resistance growth to the compound Copyright: 2015 Chernysh et al. This is an open were registered. Similar results were obtained with the compounds isolated from 3 other fly access article distributed under the terms of the species. The experiments revealed that natural compounds containing insect AMP com- plexes, in contrast to individual AMP and small molecule antibiotics, are well protected from unrestricted use, distribution, and reproduction in anymedium, provided the original author and source are resistance development in bacteria. Further progress in the research of natural AMP com- plexes may provide novel solutions to the drug resistance problem.
Data Availability Statement: All relevant data arewithin the paper.
Funding: This work was supported by St.PetersburgState University (URL ), grant number1.39.323.2014 - SC, NG. The funder had no role in study design, data collection and analysis, decision to The global expansion of antibiotic resistant bacteria is a major threat to human health. Despite publish, or preparation of the manuscript.
great progress in better knowledge of the resistance mechanisms, the solution to this problem Competing Interests: The authors of this manuscript remains elusive ]. Many efforts have been made to employ antimicrobial peptides (AMPs) as have the following competing interests: SC and NG anti-infective drugs with protection against resistance development –]. However, the grow- are inventors of Pat № RU № 2 447 896 ing body of evidence demonstrates that therapeutic AMPs have no real advantage over conven- "Antimicrobial material", inventors Sergey Chernyshand Natalia Gordya, patentee Allopharm company.
tional antibiotics since bacteria possess many ways to neutralize AMPs through enzymatic SC and NG are members of Allopharm company. TS: degradation, mutation of target structures, decrease of cell membrane permeability, membrane PLOS ONE DOI:10.1371/journal.pone.0130788 Insect AMP Complexes Prevent Resistance Development in Bacteria none to declare. This does not alter the authors' net charge alteration, active extrusion, etc. [–]. It is no wonder that bacteria, according to the adherence to all PLOS ONE policies on sharing data experimental evolution studies reviewed below, rapidly lose susceptibility towards any individ- and materials. The authors declare that they have no ual AMP tested so far. It is quite amazing that natural AMPs could remain antibacterial to the conflict of interest.
present day.
In our opinion, based on the study of insect immunity, a possible solution to the riddle lies in the fact that the immune system engages not a single AMP but a battery of active moleculesintegrated into a co-adapted antimicrobial peptide complex ]. The capacity for preventingresistance development, from that standpoint, is a feature of the complex as a whole, but notindividual compounds.
To examine this idea we have selected as a model a semi-purified AMP complex of bacteria- challenged blow fly Calliphora vicina (Diptera, Calliphoridae) larvae. C. vicina as well as manyother Calliphoridae flies are synantropic insects living in locations like animal wounds, deadbodies and excretae highly contaminated with human and animal pathogenic microflora [Since animal and human pathogens are obligatory attribute of Calliphoridae flies environment,they must be well adapted to this kind of infection. C. vicina larvae are known to respond tobacterial infection or septic injury by production and accumulation in the hemolymph of AMPcomplex comprising all major families of insect AMPs like defensins, cecropins, diptericins,proline-rich peptides and antiviral peptides alloferons [The antibacterial activityspectrum of the complex covers different groups of human pathogens from Enterobacteriacea,Coccaceae, Enterococcaceae, Pseudomonadaceae, Moraxellaceae and Corynebacteriaceae fami-lies commonly present in the larvae natural habitats [ Antibiotic multi-resistant clinical strains of Escherichia coli, Klebsiella pneumoniae and Aci- netobacter baumannii sensitive to the complex antibacterial activity [] have been employedin antimicrobial selective pressure experiments as model species. Recent efforts to combatthese Gram-negative bacteria have come into particular prominence with regard to the antibi-otic resistance problem. Carbapenem and third-generation cephalosporin resistant strains of E.
coli and K. pneumoniae are recognized as the most urgent threats to human health worldwideA. baumannii is also in the list of the most dangerous pathogens resistant to all or nearlyall antibiotics ].
The overall aim of this study was to elucidate a difference in resistance development under selective pressure of the natural compounds containing insect AMP complexes and conven-tional antibiotics. Based on the results of antimicrobial selective pressure experiments, we pro-pose a novel approach to the prevention of drug resistance development in bacterial pathogens.
Moreover, we suggest use of C. vicina naturally occurring AMP complex as a drug candidateeffective against E. coli, K. pneumoniae and A. baumannii antibiotic multiresistant strains.
Materials and Methods Insect species used in experiments were obtained from the Laboratory of Insect Biopharmacol-ogy and Immunology of the St. Petersburg State University. Experiments were performed witha wild type laboratory strain of C. vicina characterized by stable larval diapause Breed-ing conditions were essentially the same as previously described []. To induce diapause inthe progeny, adult flies were kept under short day conditions (12L:12D). The larvae were fedby fresh beef in not sterile conditions at 12°C, III instar larvae were transferred to 3°C at theend of feeding period, left there for 2 weeks to form diapause and then taken to the experi-ments. In addition to C. vicina we have used three other dipteran species: blue blow fly Calli-phora vomitoria belonging to the same genus, green bottleneck fly Lucilia sericata from thesame Calliphoridae family, and house fly Musca domestica from the evolutionary distant PLOS ONE DOI:10.1371/journal.pone.0130788 Insect AMP Complexes Prevent Resistance Development in Bacteria Muscidae family. Diapausing III instar larvae of C. vomitoria and L. sericata were obtained inaccordance with the same protocol as C. vicina. III instar larvae of M. domestica having no dia-pause in their life cycle were maintained at constant temperature 25°C and used in experimentsshortly after the end of the feeding period.
Preparation of natural compound containing C. vicina AMP complex Mixture of Escherichia coli D31 and Micrococcus luteus A270 cells have been used to induceimmune response in diapausing C. vicina larvae. One-day cell cultures were grown on the sur-face of solid LB agar nutritive medium in sterile conditions, individual colonies were picked up,transferred into flasks with 200 mL of liquid nutritive agar medium (Luria Broth Base, 25 g/L)and incubated overnight at 37°C. Then bacterial cells concentration was calculated using thesuspension optical density measurement and the cells were sedimented by centrifugation(tabletop centrifuge, 3000g, 15 min). Then the supernatant was removed and the cells wereresuspended in the nutritive medium to adjust their concentration to 1011 cells/mL. Finally, E.
coli and M. luteus suspensions were pooled in a 1:1 ratio. The larvae were pricked with a needlepreviously dipped into the suspension and were left overnight at 25°C. Their surface was thensterilized in 70% ethanol, washed with distilled water and dried. Hemolymph (approximately10 μl per animal) was collected in ice-cold tubes through a cuticle puncture. Hemolymph sam-ples were kept at -70°C until use. Thawed hemolymph was acidified with 0.1% trifluoroaceticacid (TFA) to a final concentration of 0.05% and insoluble particles were removed by centrifu-gation (30 min at 8000g at 4°C). The supernatant was applied to reversed-phase Sep-Pak C18cartridges (Waters) stabilized by 0.05% TFA in the amount of 5 mL/g of sorbent. Highly hydro-philic compounds were removed by cartridge washing with 0.05% TFA. Compounds absorbedin the cartridge were eluted with 50% acetonitrile solution acidified with 0.05% TFA, lyophi-lized (FreeZone, Labconco) and stored at -70°C. Prior to use, the lyophilized sample was dis-solved in deionized water (50 mg/mL), sterilized by filtration through a membrane with a poresize 0.22 μm (Milliex-GS, Millipore) and frozen at -70°C.
C. vicina AMP complex characterization Natural compound containing C. vicina AMP complex was characterized by a combination ofreversed phase HPLC, MS and bacterial growth inhibition assays. 1 mg of the lyophilized com-pound was dissolved in deionized water and applied to Shimadzu LC20 Prominence HPLC sys-tem equipped with analytical column C18 Vydac (4.6 х 250 mm, 5 μm, Grace), equilibratedwith 0.05% TFA. The column was eluted with a linear gradient of acetonitrile (ACN) from 0 to50% in acidified water (0.05% TFA) for 50 min ]. Chromatographic fractions were automat-ically collected with 1 min intervals. The fractions' optical densities were registered by meansof a UV detector at two fixed wavelengths 214 and 280 nm. The fractions were lyophilized, dis-solved in deionized water and tested against M. luteus A270 and E. coli D31 using the plategrowth inhibition assay described below. Active antibacterial fractions were analyzed by MS(MicroTOF ESI, Bruker Daltonics) and experimentally determined masses were comparedwith the previously published characteristics of C. vicina individual AMPs [, The peptideswere sequenced by Edman degradation method as described [].
Escherichia coli D31 and Micrococcus luteus A270 strains routinely used in insect AMP studieswere obtained from the Institute of Genetics and Molecular and Cellular Biology (IGBMC)Clinical strains of E. coli, Klebsiella pneumoniae and Acinetobacter baumannii used in theantibiotic/antimicrobial selective pressure experiments were obtained from infected patients of PLOS ONE DOI:10.1371/journal.pone.0130788 Insect AMP Complexes Prevent Resistance Development in Bacteria Antibiotic resistance spectra of bacterial strains used in selection experiments.
Strain / Antibiotic K. pneumoniae 104.2 Antibiotic abbreviations: Amc—amoxicillin/clavulanic acid, Ami–amikacin, Net–netilmicin, Gen–gentamicin, Ipm–imipenem, Mem–meropenem, Chl–chloramphenicol, Cip–ciprofloxacin, Cfp—cefoperazone, Cfp/sul–cefoperazone, Sul—sulbactam, Caz–ceftazidime, Ctx–cefotaxime, Cpe–cefepime.
*- no data.
the surgery clinic of the Kirov Military Medical Academy (St. Petersburg, Russia). Profiles ofthe strains' antibiotic resistance were determined as recommended (National Committee forClinical Laboratory Standards, 2003. Performance Standards for Antimicrobial Disk Suscepti-bility Tests. Approved standard M2-A8. NCCLS, Wayne, PA). The strains were classified assusceptible (S), intermediate (I) or resistant (R) to the antibiotic tested by disc diffusion method).
The compound containing C. vicina AMP complex was prepared for selection experimentsand characterized in accordance with the protocols described above. For comparison, similarcompounds were isolated from three other insect species: C. vomitoria, L. sericata and M.
domestica. Protocols of the compounds preparation were essentially the same as described forC. vicina compound. MICs for each preparation were determined before selection experimentsusing the microdilution method described below.
Third generation cephalosporin cefotaxime, meropenem from carbopenems' group and polypeptide polymyxin B were applied as reference antibiotics in the selection experiments.
These antibiotics were chosen because of their clinical importance and relevance to the bacteriatested. Carbapenems and third generation cephalosporins are the most important antibioticsfor the treatment of E.coli and K. pneumonia infections however their therapeutic efficacy isdramatically decreased by growing prevalence of beta-lactamase producing strains []. Poly-myxin B is an antibiotic primarily used for resistant Gram-negative infections like beta-lactamresistant E.coli and K. pneumonia and multidrug-resistant A. baumannii ]. A number ofresistance mechanisms to many classes of antibiotics are known to exist in A. baumannii,including beta-lactamases, efflux pumps, aminoglycoside-modifying enzymes, permeabilitydefects, and the alteration of target sites [, ].
The following commercial preparations were used as reference antibiotics in the experi- ments: sodium cefotaxime (ABOLmed, Duckacha str., No. 4, Novosibirsk, 630096, Russia),meropenem trihydrate (AstraZeneca) and naturally occurring polypeptide polymyxin B sulfate(Kievmedpreparat, Saksaganskogo str., No. 139, Kiev, 01033, Ukraine). The antibiotics weredissolved in sterile deionized water in concentration 1 mg/mL, aliquoted in 0.05 mL volumesand kept at -70°C until use.
Antibacterial activity assays Standard plate-growth inhibition assay was employed for identification and relative quantifica-tion of the complex active compounds. The method was essentially the same as the one previ-ously described []. E. coli D31 and M. luteus А270 cultures were grown in LB liquid nutrient PLOS ONE DOI:10.1371/journal.pone.0130788 Insect AMP Complexes Prevent Resistance Development in Bacteria medium (Invitrogen) for 18–20 hours at 37°C. Sterile Petri dishes (9 cm in diameter) were filledwith 7.5 mL of LB medium supplemented with 12g/L agarose (Invitrogen). 4 x 106 CFU/dishtest microorganisms measured by OD were inoculated into the warm medium. The analytes(fractions 1–53 of ) were dissolved in 20 μl of deionized sterile water and 2 μl aliquot ofthe solution was applied onto a solid medium surface. The diameter of the growth inhibitionzone was measured after 24-hour incubation at 37°C and the inhibition zone area was calcu-lated and used for relative quantification of the AMP anti-M. luteus and anti-E. coli activity.
The standard microdilution method was carried out for MIC determination with LB broth (Invitrogen), as recommended (National Committee for Clinical Laboratory Standards, 1997.
Methods for dilution antimicrobial susceptibility test for bacteria that grow aerobically.
Approved standard M7-A4. NCCLS, Wayne, PA).
Antibacterial activity, chromatographic, mass spectrometric and structural characteristics of active AMPs present in C. vicina AMPcomplex.
Known AMPs characteristics (Chernysh, Gordja, 2011) found in the sample, PLOS ONE DOI:10.1371/journal.pone.0130788 Insect AMP Complexes Prevent Resistance Development in Bacteria Selection experiments Individual wells of a 96-well tissue culture plate (Sarstedt AG & Co., Newton, NC) containing100 μl of liquid nutrient medium LB (Invitrogen) with doubling antibiotic dilutions were inoc-ulated with approximately 5 x 105 CFU/mL of test bacteria at antibiotic concentrations rangingfrom 3 doubling dilutions above to 3 doubling dilutions below the MIC of each agent for eachstrain. The initial inoculum was grown on the solid LB agar nutritive medium (Invitrogen),individual colonies were picked up, transferred into liquid medium (Luria broth base, 25 g/L)and incubated overnight at 37°C. The plates were incubated at 35°C for 24 hours. For each sub-sequent daily transfer, 1 μl inoculum was taken from the first well containing a sub-inhibitorydrug concentration and sub-cultured into the next passage wells containing each diluted drug.
The number of transfers in the presence of antibiotic varied from 15 to 35 depending on theMIC changes monitored in the course of each experiment. Typically, the experiment finishedwhen the MIC value in the control antibiotic treated population reached plateau and remainedunchanged during next transfers whereas no changes in the AMP complex treated populationwere registered. The experiment was continued over the next 15 transfers if MIC value of AMPcomplex demonstrated a small variability in the course of selection. MICs of the preparationand reference antibiotics were tested in three independent repetitions before and after selec-tion. MIC value was also monitored after each transfer.
MICs before and after selection were measured in three repetitions and analyzed by ANOVA.
MIC raw data were transformed into log10 MIC to approximate a normal distribution prior tostatistical analysis as recommended [, ]. MICs after each transfer were measured in onerepetition, numbers of paired timing points varied from 15 to 35 depending on the transfernumbers. The statistical significance of MIC changes in reference antibiotic and C. vicina AMPcomplex treated bacteria was evaluated by means of a nonparametric Wilcoxon paired differ-ence test and paired measures ANOVA test. The methods applied for each experiment statisti-cal analysis are specified in relevant places of the Results section. Calculations were made bymeans of the Primer of Biostatistics software, version 4.03.
C. vicina AMP complex characterization To characterize the composition of antimicrobial compounds, 1 mg was fractionated by HPLC). 53 fractions collected with 1 min intervals were lyophilized and their antibacterial activ-ities were quantified through plate growth inhibition assay using Gram-negative E. coli D31and Gram-positive M. luteus A270 bacteria as test-organisms (The majority of anti-M. luteus activity was present in fractions 27 to 30, whereas compounds active against E. coliwere found in a broad range of fractions starting from 24 to 43. MS analysis of fractions 27 to33 revealed masses precisely corresponding to the masses of defensin, P-peptide, 4 diptericinsand cecropin previously isolated from C. vicina and structurally characterized , ]. Profilesof the peptides' antibacterial activity (prevalence of anti-M. luteus or anti-E. coli activity) andchromatographic mobility were also consistent with known characteristics of the peptides. Rel-ative quantification of the peptides' activity based on the growth inhibition zone calculationshows that defensin is a leading anti-M. luteus constituent while proline-rich peptideseems to take second place. Diptericins are responsible for the most part of the complex anti-E.
coli activity complemented by cecropin and a series of unidentified compounds. Summarizingthe data of MS, chromatography and bioassays, we conclude that the complex contains four PLOS ONE DOI:10.1371/journal.pone.0130788 Insect AMP Complexes Prevent Resistance Development in Bacteria Chromatographic characteristics of naturally occurring compound containing C. vicina AMP complex. 1 mg of purified complex isolated frombacteria challenged C. vicina larvae were subjected to reversed-phase HPLC fractionation with 1 min intervals as described in Materials and Methodssection. Optical density of the fractions was measured in mAU units at 214 nm wave length. 53 fractions were individually collected, lyophilized and stored at-70°C until further antimicrobial activity and mass spectrometry analyses summarized in .
families of insect AMPs known in C. vicina: defensins, cecropins, diptericins and proline-richpeptides. Moreover, the data obtained demonstrate the presence of some additional antimicro-bial substances in the complex, which remain to be structurally characterized, and compoundshaving no direct antimicrobial activity (and Resistance development to reference antibiotics and the compoundcontaining C. vicina A MP complex Four clinical strains were used in antibiotic/antimicrobial selective pressure experiments: anti-biotic sensitive E. coli 774.1, antibiotic resistant E. coli 863.1, K. pneumoniae 104.2 and A. bau-mannii 882.2. Experiments with E. coli 774.1 strain were repeated twice, using cefotaxime and polymyxin B ) as reference antibiotics. 16-fold and 8-fold increases in MIC val-ues were registered in bacteria subjected to selection by cefotaxime and polymyxin B, corre-spondingly. Detectable MIC changes became visible after the first 3 to 5 transfers. MICincrease reached maximum value after the 9-th and 17-th transfers in polymyxin B and cefo-taxime treated populations, correspondingly. Subsequently, MICs remained at the maximumlevel until the end of the experiments. In contrast to the reference antibiotics, MIC of C. vicinacompound demonstrated no changes in the course of the experiments.
The antibiotic multi-resistant meropenem sensitive E. coli 863.1 strain demonstrated essen- tially the same results: rapid growth of meropenem resistance up to 64-fold level under selec-tive pressure from the antibiotic and no detectable MIC changes in the compound treatedpopulation ( The antibiotic multi-resistant meropenem sensitive K. pneumoniae 104.2 strain demon- strated a similar response to selective pressure from the antibiotic and the complex: dramatic128-fold MIC increase in the meropenem treated population and no MIC changes in the com-pound treated bacteria as we have reported previously ].
Polymyxin B resistance development in the A. baumannii strain 882.2 was limited to stable 4-fold growth while no regular MIC changes were found in the population experiencing thecompound repeated treatments ). It is notable that differences between MIC changes inantibiotic and the complex treated populations were highly significant in all experimentsaccording to Wilcoxon test for paired samples as indicated in the figures' footnotes.
PLOS ONE DOI:10.1371/journal.pone.0130788 Insect AMP Complexes Prevent Resistance Development in Bacteria MIC changes in bacterial strains exposed to selection by the compounds containing C. vicinaAMP complex or conventional antibiotics. (A) E. coli 774.1 (reference antibiotic cefotaxime). E. coliantibiotic sensitive strain 774.1 was exposed to selection by the AMP complex or cefotaxime in the course of25 daily transfers as explained in Materials and Methods section. Resistance rate is expressed as foldchange in MICs. 1 MIC unit is equal to the MIC value at transfer 1 (250 mg/L for the compound and 0.125 mg/L for cefotaxime, correspondingly). Selection by cefotaxime caused 16-fold increase of MIC while no signs ofMIC change were found in the compound treated population. Difference in the compound versus cefotaximeeffects on the resistance development was highly significant according to Wilcoxon test statistics (W = 276,n = 23, P<0.001). (B) E. coli 774.1 (reference antibiotic polymyxin B). The strain was exposed to selection bythe compound or polymyxin B in the course of 15 daily transfers. 1 MIC unit is equal to the MIC value attransfer 1 (250 mg/L for the compound and 8.0 mg/L for polymyxin B, correspondingly). Difference in thecompound versus polymyxin B effects on the resistance development was highly significant according toWilcoxon test statistics (W = 91, n = 13, P<0.022). (C) E. coli 863.1 (reference antibiotic meropenem). E. coliantibiotic multiresistant meropenem sensitive strain 863.1 was exposed to selection by the compound ormeropenem in the course of 15 daily transfers. 1 MIC unit is equal to the MIC value at transfer 1 (500 mg/L forthe compound and 0.125 mg/L for meropenem, correspondingly). Difference in the compound versusmeropenem effects on the resistance development was highly significant according to Wilcoxon test statistics(W = 78, n = 12, P<0.020). (D) A. baumannii 882.2 (reference antibiotic polymyxin B). A. baumannii antibioticmultiresistant strain 882.2 was exposed to selection by the AMP complex or polymyxin B in the course of 35daily transfers. 1 MIC unit is equal to the MIC value at transfer 1 (500 mg/L for the compound and 2 mg/L forpolymyxin B, correspondingly). Difference in the compound versus polymyxin B effects on the resistancedevelopment were highly significant according to Wilcoxon test statistics (W = 561, n = 33, P<0.001).
Data characterizing the compound and reference antibiotics MICs before and after selection are summarized in Results were essentially the same as the results of the MIC monitor-ing described above. Selective pressure of reference antibiotics caused statistically significantincrease of MIC values (P equal or below 0.001, according to ANOVA test). Maximum MICvalues varied from 3- to 128-fold depending on the antibiotic and bacterial strain. At the sametime, no statistically significant changes in the compound MICs before and after selection wereregistered. An analysis of summary data covering all five experiments also found no significantdifferences in the complex MICs before and after selection (ANOVA, Wilcoxon test).
Properties similar to C. vicina compound were demonstrated in the compounds obtainable from other insect species: C. vomitoria, L. sericata and M. domestica ). Selective pres-sure of the compounds in the course of 25 consecutive transfers equal to 150–175 generationsof E. coli did not cause statistically significant changes in MIC values.
Additionally, we compared rates of resistance development to cefotaxime, polymyxin B and a combination of antibiotics in E. coli 774.1 antibiotic sensitive strain Antibioticsapplied one at a time and in a combination caused similar growth of the selection agents'MICs. Thus, combining two conventional antibiotics did not prevent resistance developmentin contrast to the insect AMP complexes.
PLOS ONE DOI:10.1371/journal.pone.0130788 Insect AMP Complexes Prevent Resistance Development in Bacteria Resistance development under selective pressure of cefotaxime, meropenem, polymyxin B and C. vicina AMP complex.
K. pneumoniae 104.2 A. baumannii 882.2 *KR−ratio of MIC after selection to MIC before selection.
Moreover, results of C. vicina compound combination with cefotaxime have been analyzed in the same model system ). The complex sub-inhibitory concentration could not pre-vent cefotaxime resistance development though it distinctly delayed the process.
Animal AMPs combine many favorable properties as potential antimicrobial drugs ].
However, experimental evolution studies revealed prompt resistance growth to any individualAMP tested so far. Pseudomonas aeruginosa became resistant to cecropin P1, indolicidin,magainin II, nisin or ranalexin after seven daily cycles of exposure (168 total hours) []. Melit-tin and gramicidin D resistant clones of Mycoplasma pulmonis were obtained in two rounds ofselection [Seven overnight passages with pexiganan caused MIC increase in 6 of 7 bacterialspecies tested ]. The increase was not considered by the authors as noteworthy, nonethelessfurther experiments with P. aeruginosa and E. coli confirmed that pexiganan selects for sharpMIC increase after longer exposure ]. Resistance development to pexiganan, melittin andiseganan was registered in Staphylococcus aureus after two weeks of exposure []. Cross- Resistance before and after selection by Calliphora vomitoria, Lucilia sericata and Musca domestica AMP complexes in E. coli 774.1strain.
Source of AMP complex *KR–ratio of MIC after selection to MIC before selection PLOS ONE DOI:10.1371/journal.pone.0130788 Insect AMP Complexes Prevent Resistance Development in Bacteria MIC changes in the course of selection by the combinations of antimicrobial agents. (A)Cefotaxime and polymyxin B combination. E. coli antibiotic sensitive strain 774.1 was exposed to selection bycefotaxime, polymyxin B or a mixture of polymyxin B and cefotaxime in the course of 15–20 daily transfers.
Resistance level is expressed as fold change in MICs. 1 MIC unit is equal to 8 mg/L for polymyxin B, 0.125mg/L for cefotaxime and 1.0 mg/L for a mixture containing cefotaxime and polymyxin B in ratio 1:32,correspondingly. Selection by cefotaxime, polymyxin B or a mixture of the antibiotics caused identical 8-foldincrease of MIC. Differences in the mixture versus cefotaxime (W = 19, n = 6, P = 0.062) and polymyxin B(W = 19, n = 6, P = 0.062) effects on the rate of resistance development were statistically insignificantaccording to Wilcoxon test. (B) The compound containing C. vicina AMP complex and cefotaximecombination. E. coli strain 774.1 was exposed to selection by cefotaxime alone or cefotaxime in combinationwith the compound (50 mg/L) in the course of 15 daily transfers. Resistance level is expressed as cefotaximefold change in MICs. 1 MIC unit corresponds to MIC value of cefotaxime at transfer 1 (0.125 mg/L). Delay ofcefotaxime resistance development in presence of the compound sub-inhibitory concentration wasstatistically significant according to Wilcoxon test (W = 78, n = 12, P<0.02) and repeated measures ANOVAtest (F = 16.465, η = 29, P = 0.001).
resistance development to host AMPs under selective pressure of therapeutic AMP is particu-larly alarming [–]. It is notable that not only animal AMPs, but also their microbial coun-terparts like colistin may induce cross-resistance to the host AMPs [From this perspective,the AMP-based platform of antimicrobial drug discovery is debatable now. Taking into consid-eration the lingering crisis in small molecule antibiotics discovery, it makes the future of anti-bacterial chemotherapy especially worrisome [, Here we tested the capacity of resistance development towards naturally occurring com- pounds containing insect AMP complexes in comparison with reference antibiotics: polypep-tide polymyxin B and beta-lactam antibiotics cefotaxime and meropenem. Eight independentselection experiments with four clinical strains of E. coli, K. pneumoniae and A. baumanniiclearly demonstrated that the bacteria readily developed resistance to any individual antibiotictested. The first signs of the resistance growth were seen after 3 to 5 daily transfers. Taking intoaccount that one daily transfer in similar conditions covers 6 to 7 bacterial generations [],the growth in our experiments became evident after 18 to 35 generations continuously affectedby the antibiotics. It is notable that these data do not allow discriminating genetic (selection ofresistant mutants, horizontal transfer of mobile genetic elements) and epigenetic (increasedexpression of factors that aid resistance) mechanisms of antibiotic resistance development.
Selection with the compound containing C. vicina AMP complex had quite different conse- quences. Five independent selection experiments comprising 15 to 35 daily transfers (90 to 245generations, correspondingly) demonstrated remarkable stability of the complex sensitivityrates in all bacterial strains tested. That strongly distinguishes the compound from conven-tional antibiotics including therapeutic AMPs. Similar results were obtained in experimentswith similar compounds containing AMP complexes of other insects C. vomitoria, L. sericataand M. domestica. Although none of the bacterial strains was able to acquire resistance againstthe compounds in our experimental conditions, one cannot exclude that some bacteria may PLOS ONE DOI:10.1371/journal.pone.0130788 Insect AMP Complexes Prevent Resistance Development in Bacteria evolve in this way in long-term perspective by means of enhanced production of proteases oranother mechanism neutralizing AMPs activity.
Studies of naturally occurring AMPs are mainly focused on the discovery and mode of action analysis of individual peptides, and a little research is dedicated to their interplay in thekilling of bacteria []. The experiments described in this paper prove for the first time thecompounds containing animal AMPs in their naturally occurring combination disable resis-tance development in bacteria. The mechanism of this phenomenon needs further elucidationincluding the role of individual major AMPs already characterized, as well as characterizationof the structure and functions of other entities present in the compound. Nonetheless, theauthors express their willingness to discuss here some ideas based on the available knowledge.
The simplest mechanistic explanation is that the simultaneous action of several agents, affect-ing different targets in bacterial cells minimizes the probability of preexistence of adequatemutations in the population. From that point of view, the multiplicity of AMPs could be suffi-cient for resistance delay or prevention. To verify the hypothesis, we conducted two additionalselection experiments. Firstly, an antibiotic sensitive E. coli 774.1 strain was exposed to selec-tion by cefotaxime and polymyxin B, applied individually or combined. The rate of the resis-tance development was about the same in all three experimental groups. Thus, the artificialcombination of two different agents did not cause evident delay in the adaptation process inbacteria. In the next experiment cefotaxime MIC changes were measured in the course of selec-tion by the antibiotic alone or in combination with C. vicina compound. Although the sub-inhibitory concentration of the compound reliably delayed cefotaxime resistance growth, itwas not able to restrain it for a long time. Similarly, a combination of two AMPs originatingfrom evolutionary distant organisms, pexiganan from amphibians and mellitin from honey beevenom demonstrated slightly decreased resistance growth in S. aureus as compared to the indi-vidual constituents but was not able to block it Thus, the multiplicity of antimicrobialsalone seems to be insufficient for resistance prevention although it may potentially be usefulfor expanding the life span of conventional antibiotics.
Furthermore, the phenomenon may hypothetically be attributed to specific features of the individual AMPs constituting the complex. Peschel and Sahl suggested several mechanismsthat may help cationic antimicrobial peptides to maintain their functionality during host-path-ogen co-evolution []. Particularly, the suggestion is illustrated by the "smart" lantibiotic nisincombining five different antimicrobial activities in one molecule [However, selection exper-iments and the growing prevalence of nisin resistant strains in nature demonstrated that evensuch a "smart" molecule cannot prevent resistance acquisition ]. The publications refer-enced above and the experiments with polymyxin B described here confirm that resistancedevelopment is a general rule for any individually applied AMP.
We suggest that both the multiplicity of AMPs and the specific mechanisms of action of the complex constituents are equally important for resistance prevention. C. vicina AMP complexcomprises four major cationic AMP families, which kill bacteria directly: defensins, cecropins,diptericins and proline-rich peptides. Calliphora defensin, as well as defensins of other insectsand vertebrates, is a peptide with a 3D structure containing α-helix/β-sheet elements coordi-nated by 3 disulfide bridges and is predominantly active against Gram-positive bacteria. Alldefensins cause bacterial cell wall disruption/permeabilization although inhibition of the cellwall biosynthesis was demonstrated as well [, ]. Calliphora cecropin is a linear amphi-pathic α-helical peptide particularly active towards Gram-negative bacteria. All insect cecro-pins are known to have pore-forming and cell membrane permeabilizing activity Calliphora diptericins are members of a glycine-rich AMP family selectively toxic to someGram-negative Enterobacteria like E. coli by means of cell wall disruption Calliphora pro-line-rich peptides belong to the family of proline/arginine-rich AMPs. In contrast to defensins, PLOS ONE DOI:10.1371/journal.pone.0130788 Insect AMP Complexes Prevent Resistance Development in Bacteria cecropins and diptericins, proline/arginine-rich AMPs are known to kill bacteria by damagingDNA and/or protein synthesis and, therefore, must penetrate inside the target cell []. Thus,the C. vicina AMP complex comprises three structurally distinct groups of cell wall disruptingAMPs targeted predominantly to the membranes of Gram-negative (cecropins, diptericins) orGram-positive (defensins) bacteria and one group affecting intracellular targets (proline-richpeptides). Since the activity of intracellularly targeted toxin is inevitably dependent of penetra-tion through the cell membrane, a synergy of the complex proline-rich peptides and membranepermeabilizing constituents looks quite plausible. A synergy of proline-rich AMP and defen-sins has been confirmed with the example of the oyster Crassostrea gigas antimicrobials Differences in structure, antibacterial activity spectrum and toxicity mechanisms of defensins,cecropins and diptericins also prerequisite their synergetic or at least additive interaction. It ispossible that combination of these four peptide families was formed in the course of flesh flyevolution in order to both increase the immune response's immediate efficacy and protect itfrom resistance development. However, theoretical modeling proved by antibiotic selectivepressure experiments shows that synergetic antibiotic combinations (unlike antagonistic ones)tend to speed up the antibiotic resistance formation instead of preventing it It shouldbe taken into account, that the compound used in our experiments contains other constituentsalongside with these major AMPs that may take a part in the resistance prevention.
Comparison of resistance development under selective pressure of the compound and con- ventional antibiotics demonstrates doubtless advantage of the compound in respect of theresistance prevention. None bacterial strain tested was able to develop resistance to the com-pound whereas resistance to the antibiotics was rapidly elevated. Prospects of the indicated andsimilar natural compounds as a platform for antimicrobial drug discovery look very attractivewhen they placed to the global context of antibiotic resistance problem under review []. Torefine the compound prospects we have used four clinical strains characterized by differentprofiles of antibiotic resistance summarized in : antibiotic sensitive E. coli 774.1, antibi-otic multiresistant strains E. coli 863.1, K. pneumoniae 104.2 and A. baumannii 882.2.
Antibiotic sensitive E. coli 774.1 rapidly developed resistance under selective pressure of third generation cephalosporin cefotaxime but not the compound. Third generation cephalo-sporin resistant strains of E. coli are one of most frequent forms of antibiotic resistant patho-gens, which require urgents measures for their expansion counteraction Third-generationcephalosporins replacement, when possible, by the compound-based medication would help toslow down the cephalosporins resistance expansion.
An example of E. coli 863.1 strain demonstrate other probable field of the compound appli- cation. The strain is characterized by broad spectrum of antibiotic resistance. Particularly, it isresistant to third generation cephalosporins. Since the strain sensitivity to the cephalosporincefoperazone was recovered by beta-lactamase inhibitor sulbactam, resistance to this kind ofantibiotics may be attributed to the beta-lactamase activity. The strain remains sensitive toanother group of beta-lactam antibiotics, carbapenems, which are often considered as antibiot-ics of last resort for E. coli treatment []. However, it rapidly develops high level of carbape-nem resistance under selective pressure of meropenem ) and may become practicallyuntreatable by carbapenems, third generation cephalosporins and many other antibiotics. Useof the compound instead of carbapenems could allow avoiding this dangerous situation.
Antibiotic resistance profile of K. pneumoniae 104.2 strain is quite similar to the profile of E.
coli 863.1. It belongs to the third generation cephalosporin resistant strains and retains sensitiv-ity to the carbapenems. The carbapenems are the main remaining treatment option for thiskind of K. pneumoniae infections However, experiments with meropenem demonstratedthat it can easily develop high level of carbapenem resistance as well. Carbapenem resistant K.
pneumoniae is considered among most important pathogens [The compound is active PLOS ONE DOI:10.1371/journal.pone.0130788 Insect AMP Complexes Prevent Resistance Development in Bacteria against K. pneumoniae 104.2 strain and can be potentially used as an alternative to carbape-nems. It may help to decrease the carbapenem resistant strains expansion and save carbape-nems for systemic life-threatening K. pneumoniae infections.
Prevalence of multidrug resistant strains typical to A. baumannii puts it in the forefront of the most dangerous pathogens []. The strain 882.2 remains sensitive to polymyxin B, how-ever it decrease efficacy in the course of selection (Since polymyxin B therapeutic dos-age is strongly limited by the antibiotic toxicity, even small increase of resistance would make itpractically unusable. The compound looks prospective as alternative treatment of A. bauman-nii multidrug-resistant infections both in terms of antibacterial activity and prevention of resis-tance development.
Thus, the results of the experimental studies demonstrate prospects of naturally occurring AMP complexes in real clinical situations described above as well as in a broader context ofdrug resistance prevention and fighting of antibiotic resistant bacteria.
The authors are grateful to the research resource center «Molecular and cell technologies» andthe center for Chemical Analysis and Material Research of Saint Petersburg State Universityfor advice in the MS studies. We are also indebted to Nina Simonenko, Alexandr Nesin andAndrei Yakovlev for technical assistance in the AMP complexes production.
Author Contributions Conceived and designed the experiments: SC NG. Performed the experiments: NG TS. Ana-lyzed the data: SC NG TS. Contributed reagents/materials/analysis tools: TS. Wrote the paper:SC NG TS.
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