Enterobacter cloacae is a Gram-negative bacterium associated with high morbidity and mortality in intensive care patients due to its resistance to multiple antibiotics. Currently, therapy against multi-resistant bacteria consists of using colistin, in spite of its toxic effects at higher concentrations. In this context, colistin-resistant E. cloacae strains were challenged with lower levels of colistin combined with other antibiotics to reduce colistin-associated side effects. Colistin-resistant E. cloacae (ATCC 49141) strains were generated by serial propagation in subinhibitory colistin concentrations. After this, three colistin-resistant and three nonresistant replicates were isolated. The identity of all the strains was confirmed by MALDI-TOF MS, VITEK 2 and MicroScan analysis. Furthermore, cross-resistance to other antibiotics was checked by disk diffusion and automated systems. The synergistic effects of the combined use of colistin and chloramphenicol were observed via the broth microdilution checkerboard method. First, data here reported showed that all strains presented intrinsic resistance to penicillin, cephalosporin (except fourth generation), monobactam, and some associations of penicillin and β-lactamase inhibitors. Moreover, a chloramphenicol and colistin combination was capable of inhibiting the induced colistin-resistant strains as well as two colistin-resistant clinical strains. Furthermore, no cytotoxic effect was observed by using such concentrations. In summary, the data reported here showed for the first time the possible therapeutic use of colistin–chloramphenicol for infections caused by colistin-resistant E. cloacae.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.


  1. 1.

    et al. EmmdR, a new member of the MATE family of multidrug transporters, extrudes quinolones from Enterobacter cloacae. Arch. Microbiol. 193, 759–765 (2011).

  2. 2.

    , , & Characterization of ertapenem-resistant Enterobacter cloacae in a Taiwanese university hospital. J. Clin. Microbiol. 50, 223–226 (2012).

  3. 3.

    , & Emergence of VIM-1-carbapenemase-producing Enterobacter cloacae in Tyrol, Austria. J. Med. Microbiol. 61, 567–571 (2012).

  4. 4.

    et al. Emergence of VIM-12 in Enterobacter cloacae. J. Clin. Microbiol. 48, 3414–3415 (2010).

  5. 5.

    et al. Emergence of AcrAB-mediated tigecycline resistance in a clinical isolate of Enterobacter cloacae during ciprofloxacin treatment. Int. J. Antimicrob. Agents 35, 478–481 (2010).

  6. 6.

    & GIsul2, a genomic island carrying the sul2 sulphonamide resistance gene and the small mobile element CR2 found in the Enterobacter cloacaesubspecies cloacae type strain ATCC 13047 from 1890, Shigella flexneri ATCC 700930 from 1954 and Acinetobacter baumannii ATCC 17978 from 1951. J. Antimicrob. Chemother. 66, 2175–2176 (2011).

  7. 7.

    & Antibiotics in phase II and III clinical trials. Handb. Exp. Pharmacol. 211, 167–183 (2012).

  8. 8.

    , & New antimicrobial frontiers. Mini. Rev. Med. Chem. 11, 888–900 (2011).

  9. 9.

    et al. Colistin resistance in Acinetobacter baumannii is mediated by complete loss of lipopolysaccharide production. Antimicrob. Agents Chemother. 54, 4971–4977 (2010).

  10. 10.

    et al. Colistin and polymyxin B: a re-emergence. Indian J. Crit. Care Med. 13, 49–53 (2009).

  11. 11.

    & Introduction of a lysine residue promotes aggregation of temporin L in lipopolysaccharides and augmentation of its antiendotoxin property. Antimicrob. Agents Chemother. 57, 2457–2466 (2013).

  12. 12.

    Antimicrobial peptides for gram-negative sepsis: a case for the polymyxins. Front. Immunol. 3, 252 (2012).

  13. 13.

    , , & A novel negative regulation mechanism of bacterial outer membrane proteins in response to antibiotic resistance. J. Proteome. Res. 9, 5952–5959 (2010).

  14. 14.

    New beta-lactam-beta-lactamase inhibitor combinations in clinical development. Ann. N. Y. Acad. Sci. 1277, 105–114 (2013).

  15. 15.

    et al. Synergistic effect between colistin and bacteriocins in controlling gram-negative pathogens and their potential to reduce antibiotic toxicity in mammalian epithelial cells. Antimicrob. Agents Chemother. 57, 2719–2725 (2013).

  16. 16.

    , , & Physicochemical aspects of the coformulation of colistin and azithromycin using liposomes for combination antibiotic therapies. J. Pharm. Sci. 102, 1578–1587 (2013).

  17. 17.

    et al. Combination therapy with intravenous colistin for management of infections due to multidrug-resistant Gram-negative bacteria in patients without cystic fibrosis. Antimicrob. Agents Chemother. 49, 3136–3146 (2005).

  18. 18.

    & Optimizing drug exposure to minimize selection of antibiotic resistance. Clin. Infect. Dis. 45(suppl 2), S129–S136 (2007).

  19. 19.

    & A method for estimating the number of bacteria in liquids and tissues. J. Bacteriol. 64, 837–845 (1952).

  20. 20.

    CLSI. in Approved Standard - 8th ed. CLSI document M07-A8 (Clinical and Laboratory Standards Institute, Wayne, PA, 2009).

  21. 21.

    et al. Proteomic analysis of the sarcosine-insoluble outer membrane fraction of Pseudomonas aeruginosa responding to ampicilin, kanamycin, and tetracycline resistance. J. Proteome. Res. 4, 2257–2265 (2005).

  22. 22.

    et al. Identification of Borrelia species after creation of an in-house MALDI-TOF MS database. PLoS One. 9, e88895 (2014).

  23. 23.

    et al. High interlaboratory reproducibility of matrix-assisted laser desorption ionization-time of flight mass spectrometry-based species identification of nonfermenting bacteria. J. Clin. Microbiol. 47, 3732–3734 (2009).

  24. 24.

    CLSI. in Twentieth Informational SupplementCLSI document M100-S20 (Clinical and Laboratory Standards Institute, Wayne, PA, 2010).

  25. 25.

    , & Potent synergy and sustained bactericidal activity of a vancomycin-colistin combination versus multidrug-resistant strains of Acinetobacter baumannii. Antimicrob. Agents Chemother. 54, 5316–5322 (2010).

  26. 26.

    et al. Functional characterization of a synthetic hydrophilic antifungal peptide derived from the marine snail Cenchritis muricatus. Biochimie 94, 968–974 (2012).

  27. 27.

    et al. Cn-AMP1: a new promiscuous peptide with potential for microbial infections treatment. Biopolymers 98, 322–331 (2012).

  28. 28.

    , , , & End-tagging of ultra-short antimicrobial peptides by W/F stretches to facilitate bacterial killing. PLoS ONE 4, e5285 (2009).

  29. 29.

    et al. Analysis of the matrix-assisted laser desorption ionization-time of flight mass spectrum of staphylococcus aureus identifies mutations that allow differentiation of the main clonal lineages. J. Clin. Microbiol. 51, 1809–1817 (2013).

  30. 30.

    & Prevalences of the Enterobacter cloacae complex and its phylogenetic derivatives in the nosocomial environment. Eur. J. Clin. Microbiol. Infect. Dis. 31, 2951–2955 (2012).

  31. 31.

    et al. Genomic diversity within the Enterobacter cloacaecomplex. PLoS ONE 3, e3018 (2008).

  32. 32.

    et al. Comparison of MALDI TOF with conventional identification of clinically relevant bacteria. Swiss Med. Wkly. 140, w13095 (2010).

  33. 33.

    , , , & Proteomic applications in the clinical microbiology laboratory. Enferm. Infecc. Microbiol. Clin. 30, 383–393 (2012).

  34. 34.

    , & Enterobacter cloacae complex: clinical impact and emerging antibiotic resistance. Future Microbiol. 7, 887–902 (2012).

  35. 35.

    et al. Differentiation of cfiA-negative and cfiA-positive Bacteroides fragilis isolates by matrix-assisted laser desorption ionization-time of flight mass spectrometry. J. Clin. Microbiol. 49, 1961–1964 (2011).

  36. 36.

    et al. MALDI-TOF MS fingerprinting allows for discrimination of major methicillin-resistant Staphylococcus aureus lineages. Int. J. Med. Microbiol. 301, 64–68 (2011).

  37. 37.

    , , , & Application of matrix-assisted laser desorption ionization-time of flight mass spectrometry for discrimination of laboratory-derived antibiotic-resistant bacteria. Biol. Pharm. Bull. 35, 1841–1845 (2012).

  38. 38.

    et al. MALDI-ToF mass-spectrometry in analysis of genetically determined resistance of Streptococcus pneumoniae to fluoroquinolones. Antibiot. Khimioter. 52, 10–17 (2007).

  39. 39.

    et al. Characterisation and clinical features of Enterobacter cloacae bloodstream infections occurring at a tertiary care university hospital in Switzerland: is cefepime adequate therapy? Int. J. Antimicrob. Agents 41, 236–249 (2013).

  40. 40.

    et al. Activity of cecropin A-melittin hybrid peptides against colistin-resistant clinical strains of Acinetobacter baumannii: molecular basis for the differential mechanisms of action. Antimicrob. Agents Chemother. 50, 1251–1256 (2006).

  41. 41.

    , & In vitrosynergy of colistin combinations against colistin-resistant Acinetobacter baumannii,Pseudomonas aeruginosa, and Klebsiella pneumoniae isolates. Antimicrob. Agents Chemother. 56, 4856–4861 (2012).

  42. 42.

    et al. Colistin and fusidic acid, a novel potent synergistic combination for treatment of multidrug-resistant Acinetobacter baumannii infections. Antimicrob. Agents Chemother. 59, 4544–4550 (2015).

  43. 43.

    et al. Cefepime and amikacin synergy in vitro and in vivo against a ceftazidime-resistant strain of Enterobacter cloacae. J. Antimicrob. Chemother. 41, 367–372 (1998).

  44. 44.

    , , & Synergistic activities of moxifloxacin combined with piperacillin-tazobactam or cefepime against Klebsiella pneumoniae,Enterobacter cloacae, and Acinetobacter baumannii clinical isolates. Antimicrob. Agents Chemother. 48, 1055–1057 (2004).

  45. 45.

    , & Characterization of two clinical, multiple-drug-resistant isolates of Enterobacter cloacae. Chemotherapy 30, 308–321 (1984).

  46. 46.

    , & Synergistic antibacterial activity of Salvia officinalis and Cichorium intybus extracts and antibiotics. Acta. Pol. Pharm. 69, 457–463 (2012).

  47. 47.

    et al. Synergism of Leu-Lys rich antimicrobial peptides and chloramphenicol against bacterial cells. Biochim. Biophys. Acta. 1764, 24–32 (2006).

  48. 48.

    et al. Cefepime and amikacin synergy against a cefotaxime-susceptible strain of Enterobacter cloacae in vitro and in vivo. J. Antimicrob. Chemother. 39, 363–369 (1997).

Download references


This study was supported by grants from CNPq, CAPES, FUNDECT, FAPDF, EMBRAPA, UCB and UCDB.

Author information

Author notes

    • Thais Bergamin Lima
    • , Osmar Nascimento Silva
    •  & Keyla Caroline de Almeida

    These authors contributed equally to this work.


  1. Department of Biotechnology, Centro de Análises Proteômicas e Bioquímicas, Universidade Católica de Brasília, Brasilia, Brazil

    • Thais Bergamin Lima
    • , Keyla Caroline de Almeida
    • , Dielle de Oliveira Motta
    • , Simone Maria-Neto
    • , Michelle Brizolla Lara
    • , Carlos Roberto Souza Filho
    • , Alicia Simalie Ombredane
    • , Nadia Skorupa Parachin
    • , Beatriz Simas Magalhães
    •  & Octávio Luiz Franco
  2. UDF Centro Universitario, Brasilia, Brazil

    • Thais Bergamin Lima
  3. Department of Biotechnology, S-Inova Biotech, Universidade Católica Dom Bosco, Campo Grande, Brazil

    • Osmar Nascimento Silva
    • , Suzana Meira Ribeiro
    •  & Octávio Luiz Franco
  4. Department of Cell Biology, Universidade de Brasília, Campus Asa Norte, Brasília, Brazil

    • Keyla Caroline de Almeida
    •  & Octávio Luiz Franco
  5. Department of microbiology, Núcleo de Bacteriologia, Laboratório Central de Saúde Pública Distrito Federal, Brasília, Brazil

    • Celio de Faria Junior


  1. Search for Thais Bergamin Lima in:

  2. Search for Osmar Nascimento Silva in:

  3. Search for Keyla Caroline de Almeida in:

  4. Search for Suzana Meira Ribeiro in:

  5. Search for Dielle de Oliveira Motta in:

  6. Search for Simone Maria-Neto in:

  7. Search for Michelle Brizolla Lara in:

  8. Search for Carlos Roberto Souza Filho in:

  9. Search for Alicia Simalie Ombredane in:

  10. Search for Celio de Faria Junior in:

  11. Search for Nadia Skorupa Parachin in:

  12. Search for Beatriz Simas Magalhães in:

  13. Search for Octávio Luiz Franco in:

Competing interests

The authors declare no conflict of interest.

Corresponding author

Correspondence to Octávio Luiz Franco.

Supplementary information

About this article

Publication history






Supplementary Information accompanies the paper on The Journal of Antibiotics website (

Further reading