Activity of host antimicrobials against multidrug resistant. Acinetobacter baumannii acquiring colistin resistance through loss of. lipopolysaccharide

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1 AAC Accepts, published online ahead of print on 24 February 2014 Antimicrob. Agents Chemother. doi: /aac Copyright 2014, American Society for Microbiology. All Rights Reserved. 1 SHORT FORM PAPER Activity of host antimicrobials against multidrug resistant Acinetobacter baumannii acquiring colistin resistance through loss of lipopolysaccharide Meritxell García-Quintanilla, Marina R. Pulido, Patricia Moreno-Martínez, Reyes Martín-Peña, Rafael López-Rojas, Jerónimo Pachón and Michael McConnell Biomedical Institute of Seville (IBiS), University Hospital Virgen del Rocío/CSIC/ University of Sevilla, 41013, Sevilla, Spain. Author to whom correspondence should be addressed: Michael J. McConnell Biomedical Institute of Seville Hospital Universitario Virgen del Rocío Avenida Manuel Siurot s/n, Sevilla, Spain mcconnell.mike75@gmail.com Phone: Running Title: LPS deficient A. baumannii and host antimicrobials 23 Key Words: host cationic peptides, antibiotic resistance, human lysozyme, LL-37

2 Abstract Acinetobacter baumannii can acquire resistance to the cationic peptide antibiotic colistin through complete loss of lipopolysaccharide expression. The activity of the host cationic antimicrobials LL-37 and human lysozyme against multidrug resistant clinical isolates of A. baumannii that acquired colistin resistance through lipopolysaccharide loss was characterized. We demonstrate that LL-37 has similar activity against strains lacking lipopolysaccharide compared to their colistin sensitive parent strains, whereas human lysozyme has increased activity against colistin resistant strains lacking LPS. Downloaded from on August 31, 2018 by guest 2

3 Colistin is a cationic peptide antibiotic and one of the few antimicrobials that retains activity against many isolates of multidrug resistant Acinetobacter baumannii. Unfortunately, colistin resistant clinical isolates of A. baumannii have been reported (1-3). Two mechanisms for acquiring colistin resistance have been described in A. baumannii, ethanolamine addition to the lipid A moiety of lipopolysaccharide (LPS) resulting from mutations in the PmrAB two-component system (4, 5), and complete loss of LPS expression due to mutation in the lpxa, lpxc and lpxd genes, which encode the enzymes that catalyze the first steps in LPS biosynthesis (6, 7). Both mechanisms are thought to reduce the activity of colistin by decreasing the electrostatic interactions between positively charged colistin molecules and the bacterial membrane due to negatively charged LPS. Host cationic antimicrobials play an important role in the innate immune response to infections caused by Gram-negative bacteria. LL-37 is an antimicrobial peptide primarily expressed by epithelial cells and neutrophils (8), and has been shown to have antibacterial activity against various pathogens, including A. baumannii (9, 10). Human lysozyme is present in mucosal secretions, and produces membrane damage in bacteria by hydrolyzing components of the peptidoglycan layer (11). The cationic C- terminal domain has been shown to have antibacterial activity produced via membrane permeabilization (12, 13). Importantly, a recent study has demonstrated that A. baumannii clinical isolates that acquired colistin resistance through mutations in the PmrAB system developed cross-resistance to LL-37 and human lysozyme, suggesting that a potential risk associated with the use of colistin could be the selection of strains that have reduced susceptibility to effectors of the host innate immune system (10). In this context, and given that LPS is thought to play an important role in mediating the activity of cationic antimicrobials due to its negative charge, the objective of the present 3

4 study was to assess the activity of LL-37 and human lysozyme on multidrug resistant clinical isolates of A. baumannii that acquired colistin resistance through complete loss of LPS. Isolation of LPS deficient strains. Colistin resistance was selected for in four previously characterized, clonally distinct multidrug resistant clinical isolates of A. baumannii (14) by plating overnight cultures of each strain on Mueller-Hinton agar containing 10 mg/l of colistin, as described previously (7). In all cases, colistin resistant colonies appeared after incubation for 24 hours at 37 ºC. Sequencing of the lpxa, lpxc and lpxd genes of these isolates identified derivatives that harbored mutations in these genes that produced either amino acid changes or a premature stop codon (Table 1). Derivatives were confirmed as colistin resistant by determining colistin MICs by broth microdilution according to Clinical Laboratory Standards Institute guidelines (15), and all strains showed high level resistance to colistin (Table 1). The loss of LPS was confirmed by using the QCL-1000 Limulus Amebocyte Assay (Lonza) according to the manufacturer s instructions to quantify endotoxin levels in three independent cultures of each strain (Table 1). Two previously described strains that acquired colistin resistance due to mutations in the PmrAB system, RC64 (16, 17) and CR17 (1), and their colistin susceptible parental counterparts were included for comparison (Table 1). Antimicrobial activity of LL-37 on LPS deficient strains. The activity of LL- 37 against each strain included in the study was determined based on a previously described assay (10). Bacterial cells were resuspended in 10 mm sodium phosphate buffer (ph 6.5) and adjusted to an OD 600 of 0.5. The bacterial suspensions were diluted 1:100 and 100 µl were mixed with 100 µl 10 mm sodium phosphate buffer containing the indicated concentrations of LL-37 (Sigma) and incubated for 3 hours at 37ºC. After incubation, cell viability was measured by plating serial dilutions on blood agar. 4

5 Differences in bacterial survival between groups were determined using the Student s t test, and a P value 0.05 was considered significant. The antimicrobial activity of LL- 37 on the clinical isolates and their colistin resistant mutants was characterized using concentrations ranging from 0 to 3.13 µg/ml on the basis that these concentrations are similar to concentrations of LL-37 encountered in human plasma and in mucosal secretions (18, 19). As can be seen in Figure 1A, LL-37 demonstrated antimicrobial activity against all four of the parental clinical isolates in the dose range tested, with survival between 0.1% and 0.01% at a concentration of 3.13 µg/ml. In general, the antimicrobial activity of LL-37 against the LPS deficient derivatives of the clinical isolates showed a similar pattern of susceptibility, indicating that a lack of LPS had little effect on the activity of LL-37 on A. baumannii. It is interesting to note, however, that the 167R strain appeared to show approximately 100-fold less susceptibility to LL-37 at a concentration of 1.56 µg/ml. Characterization of the A. baumannii strains that had acquired colistin resistance due to mutations in the PmrAB system (RC64 and CR17; Figure 1A) demonstrated that had similar antimicrobial activity against these strains and their colistin-susceptible parent strains. Antimicrobial activity of human lysozyme on LPS deficient strains. The susceptibility assay for human lysozyme (Sigma) was performed similarly as for LL-37 except that a one hour incubation time was employed. The antimicrobial activity of human lysozyme at concentrations of 0 to 1.56 mg/ml was assessed on the basis that these concentrations are similar to those encountered in the upper respiratory tract (20). Interestingly, in contrast to the results obtained with LL-37, LPS deficient derivatives showed dramatically increased susceptibility to human lysozyme compared to their parental counterparts (Figure 1B). Even at the low concentrations of lysozyme tested (0.05 to 0.1 mg/ml) differences of between 100- and 10,000-fold were seen between the 5

6 derivatives and the parental strains. These results indicate that LPS loss increases the susceptibility of A. baumannii to human lysozyme at physiological concentrations. Characterization of strains acquiring resistance to colistin via mutations in the PmrAB system demonstrated that the RC64 strain was more sensitive to lysozyme than its corresponding parental strain at the concentrations tested, whereas CR17 showed modestly increased resistance to lysozyme at the lower concentrations tested (Figure 1B). These results may indicate that the effect of LPS modification on lysozyme susceptibility is, at least in part, strain specific. Resistance to colistin in A. baumannii via LPS modification or complete loss of LPS expression is thought to result from decreased electrostatic attraction between positively charged colistin molecules and the bacterial membrane due to negatively charged LPS. This raises the possibility that other cationic antimicrobial agents, such as LL-37 and human lysozyme, may have decreased activity against A. baumannii strains that acquire resistance to colistin. Data supporting this idea were recently reported in which A. baumannii clinical isolates with mutations in the pmrb gene, and thus presumably ethanolamine modifications of LPS, showed decreased susceptibility to LL- 37 and human lysozyme compared to their colistin susceptible parent strains (10). These results are in contrast to the results presented here in that the present study provides evidence that complete loss of LPS expression during the acquisition of colistin resistance in A. baumannii minimally affects the antimicrobial activity of LL- 37, and increases susceptibility to human lysozyme. The fact that the antimicrobial activity of LL-37 was minimally affected by LPS loss is in some ways surprising given that, like colistin, LL-37 is a cationic antimicrobial peptide and it has been proposed that the initial target of LL-37 in Gram-negative bacteria is LPS (21). This finding may suggest that the mechanism used by LL-37 to achieve antimicrobial activity is, at least 6

7 in part, different from that used by colistin. This idea is supported by reports describing the antimicrobial activity of LL-37 on Gram-positive bacteria and fungi, which lack LPS (22). Interestingly, a recent study showed that an LPS deficient derivative of the ATCC strain of A. baumannii was modestly more sensitive to LL-37 than the parent strain, suggesting that the effect of LPS loss on LL-37 activity may be strain specific (9), although it is important to note that the four strains used in the present study are clonally distinct. The data presented here demonstrate that LPS loss in A. baumannii results in increased susceptibility to human lysozyme. One possible explanation is that the loss of LPS in the outer membrane increases the ability of lysozyme to access its peptidoglycan target. A second possibility is that the loss of membrane structural support provided by LPS results in strains that are unable to withstand hydrolysis of peptidoglycan components by lysozyme. These data may suggest that the lpxa, lpxc and lpxd genes could serve as targets for the development of novel antimicrobials since their inhibition may lead to increased bacterial susceptibility to certain host antimicrobials, and therefore increased bacterial clearance by the innate immune response. This idea is supported by a recent study in which an LpxC inhibitor was able to increase bacterial clearance and reduce mortality in mouse model of A. baumannii infection in spite of the fact that the inhibitor did not demonstrate bactericidal activity (23). Taken together with previously reported data demonstrating the reduced activity of LL-37 and human lysozyme on strains with mutations in the PmrAB system (10), the results presented here indicate that the activity of the host cationic antimicrobials LL-37 and lysozyme on colistin resistant strains of A. baumannii is dependent upon the mechanism of colistin resistance. 7

8 Acknowledgements This work was funded by the Ministerio de Economía y Competitividad, Instituto de Salud Carlos III - co-financed by European s Development Regional Fund "A way to achieve Europe" ERDF, Spanish Network for the Research in Infectious Diseases (REIPI RD06/0008/0000) and a grant from the European Community s 7 th Programme Framework (MagicBullet; Grant Agreement Number: ). MJM is supported by the Subprograma Miguel Servet from the Ministerio de Economía y Competitividad of Spain (CP11/00314). Transparency Declaration JP and MJM own stock in Vaxdyn, S.L., a biotechnology company developing vaccines for multidrug resistant bacteria, including A. baumannii. The other authors declare no potential conflicts of interest. Downloaded from on August 31, 2018 by guest 8

9 References 1. López-Rójas R, Jiménez-Mejías ME, Lepe JA, Pachón J Acinetobacter baumannii resistant to colistin alters its antibiotic resistance profile: a case report from Spain. J Infect Dis 204: Taccone FS, Rodríguez-Villalobos H, De Backer D, De Moor V, Deviere J, Vincent JL, Jacobs F Successful treatment of septic shock due to panresistant Acinetobacter baumannii using combined antimicrobial therapy including tigecycline. Eur J Clin Microbiol Infect Dis 25: Valencia R, Arroyo LA, Conde M, Aldana JM, Torres MJ, Fernández- Cuenca F, Garnacho-Montero J, Cisneros JM, Ortiz C, Pachón J, Aznar J Nosocomial outbreak of infection with pan-drug-resistant Acinetobacter baumannii in a tertiary care university hospital. Infect Control Hosp Epidemiol 30: Adams MD, Nickel GC, Bajaksouzian S, Lavender H, Murthy AR, Jacobs MR, Bonomo RA Resistance to colistin in Acinetobacter baumannii associated with mutations in the PmrAB two-component system. Antimicrob Agents Chemother 53: Beceiro A, Llobet E, Aranda J, Bengoechea JA, Doumith M, Hornsey M, Dhanji H, Chart H, Bou G, Livermore DM, Woodford N Phosphoethanolamine modification of lipid A in colistin-resistant variants of Acinetobacter baumannii mediated by the pmrab two-component regulatory system. Antimicrob Agents Chemother 55: Moffatt JH, Harper M, Adler B, Nation RL, Li J, Boyce JD Insertion sequence ISAba11 is involved in colistin resistance and loss of 9

10 lipopolysaccharide in Acinetobacter baumannii. Antimicrob Agents Chemother 55: Moffatt JH, Harper M, Harrison P, Hale JD, Vinogradov E, Seemann T, Henry R, Crane B, St Michael F, Cox AD, Adler B, Nation RL, Li J, Boyce JD Colistin resistance in Acinetobacter baumannii is mediated by complete loss of lipopolysaccharide production. Antimicrob Agents Chemother 54: Vandamme D, Landuyt B, Luyten W, Schoofs L A comprehensive summary of LL-37, the factotum human cathelicidin peptide. Cell Immunol 280: Moffatt JH, Harper M, Mansell A, Crane B, Fitzsimons TC, Nation RL, Li J, Adler B, Boyce JD Lipopolysaccharide-deficient Acinetobacter baumannii shows altered signaling through host Toll-like receptors and increased susceptibility to the host antimicrobial peptide LL-37. Infect Immun 81: Napier BA, Burd EM, Satola SW, Cagle SM, Ray SM, McGann P, Pohl J, Lesho EP, Weiss DS Clinical use of colistin induces cross-resistance to host antimicrobials in Acinetobacter baumannii. MBio 4:e Callewaert L, Van Herreweghe JM, Vanderkelen L, Leysen S, Voet A, Michiels CW Guards of the great wall: bacterial lysozyme inhibitors. Trends Microbiol 20: During K, Porsch P, Mahn A, Brinkmann O, Gieffers W The nonenzymatic microbicidal activity of lysozymes. FEBS Lett 449:

11 Ibrahim HR, Thomas U, Pellegrini A A helix-loop-helix peptide at the upper lip of the active site cleft of lysozyme confers potent antimicrobial activity with membrane permeabilization action. J Biol Chem 276: Fernández-Cuenca F, Pascual A, Ribera A, Vila J, Bou G, Cisneros JM, Rodríguez-Baño J, Pachón J, Martínez-Martínez L [Clonal diversity and antimicrobial susceptibility of Acinetobacter baumannii isolated in Spain. A nationwide multicenter study: GEIH-Ab project (2000)]. Enferm Infecc Microbiol Clin 22: Clinical Laboratory Standards Institute Performance standards for antimicrobial susceptibility testing; Twenty-third informational supplement. CLSI document M100-S23. Wayne, PA. Clinical Laboratory Standards Institute. 16. Fernández-Reyes M, Rodríguez-Falcon M, Chiva C, Pachón J, Andreu D, Rivas L The cost of resistance to colistin in Acinetobacter baumannii: a proteomic perspective. Proteomics 9: Schaub IG, Hauber FD A biochemical and serological study of a group of identical unidentifiable gram-negative bacilli from human sources. J Bacteriol 56: Overhage J, Campisano A, Bains M, Torfs EC, Rehm BH, Hancock RE Human host defense peptide LL-37 prevents bacterial biofilm formation. Infect Immun 76: Sorensen O, Cowland JB, Askaa J, Borregaard N An ELISA for hcap-18, the cathelicidin present in human neutrophils and plasma. J Immunol Methods 206:

12 Cole AM, Liao HI, Stuchlik O, Tilan J, Pohl J, Ganz T Cationic polypeptides are required for antibacterial activity of human airway fluid. J Immunol 169: Sochacki KA, Barns KJ, Bucki R, Weisshaar JC Real-time attack on single Escherichia coli cells by the human antimicrobial peptide LL-37. Proc Natl Acad Sci U S A 108:E Durr UH, Sudheendra US, Ramamoorthy A LL-37, the only human member of the cathelicidin family of antimicrobial peptides. Biochim Biophys Acta 1758: Lin L, Tan B, Pantapalangkoor P, Ho T, Baquir B, Tomaras A, Montgomery JI, Reilly U, Barbacci EG, Hujer K, Bonomo RA, Fernández L, Hancock RE, Adams MD, French SW, Buslon VS, Spellberg B Inhibition of LpxC protects mice from resistant Acinetobacter baumannii by modulating inflammation and enhancing phagocytosis. MBio 3. 12

13 261 Table 1. Colistin MIC values and endotoxin levels of strains used in this study. Colistin Strain Description MIC (mg/l) EU/10 6 cells a Reference Ab-84 Multidrug resistant clinical isolate ,78 ± 3,54 14 Ab-108 Ab-167 Ab-176 Ab-84R Ab-108R Ab-167R Ab-176R ATCC RC64 Multidrug resistant clinical isolate Multidrug resistant clinical isolate Multidrug resistant clinical isolate Strain Ab-84 containing a 40 nucleotide insertion at nucleotide 321 of lpxc generating a premature stop codon Strain Ab-108 with a T614A mutation in lpxa producing an I205N amino acid substitution Strain Ab-167 containing an ISAba1 insertion at nucleotide 321 of lpxc Strain Ab-176 with a G739T substitution at nucleotide 739 of the lpxd gene producing a premature stop codon Colistin-susceptible A. baumannii type strain. Derivative of ATCC containing R134C and A227V mutations in pmrb > 128 > 128 > 128 > ,53 ± 4,80 20,02 ±2,36 42,97 ±21,52 < 1 < 1 < 1 < 1 ND ND This study This study This study This study CS01 Colistin susceptible A. baumannii clinical isolate 0.25 ND CR17 Colistin-resistant derivative of 64 ND 1 CS01 containing a M12K mutations in pmra a Data represent the mean ± the standard error of the mean of three independent assays. EU; Endotoxin Units, ND; not determined 13

14 Figure Legend Figure 1. Activity of LL-37 and human lysozyme against A. baumannii strains. Graphs represent the percent survival of the strains after incubation with the indicated concentrations of LL-37 (A) and human lysozyme (B). Error bars represent the standard deviations of three independent cultures. * p < 0.05; Student s t test. Downloaded from on August 31, 2018 by guest 14

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