Structural investigation of a polysaccharide from the mycelium of Enterobacter cloacae and its antibacterial activity against extensively drug-resistant E. cloacae producing SHV-12 extended-spectrum β-lactamase.

In this study, a polysaccharide (ECP) was isolated from the mycelium of Enterobacter cloacae and was found to exhibit strong antibacterial activities against E. cloacae producing SHV-12 ESBL with the increase of the inhibition zone diameter. Its minimum inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) were 12.5 mg/L and 25 mg/L, respectively. ECP at these concentrations immediately inhibited planktonic growth of the bacteria especially at the time from 2 to 10 h. Flow cytometry analysis further revealed that almost all the bacterial cells were damaged following ECP treatment. The permeability of the cytoplasmic membrane of E. cloacae was increased when ECP concentrations increasing, as evidenced by an influx of Na and an efflux of K, P or S, the leakage of intracellular ATP and the UV-absorbing substances, as well as the depolarization of the cytoplasmic membrane, indicating that bactericidal activity of ECP was achieved by inducing cell membrane damage.

[1]  R. Hancock,et al.  Interactions of Bacterial Cationic Peptide Antibiotics with Outer and Cytoplasmic Membranes ofPseudomonas aeruginosa , 2000, Antimicrobial Agents and Chemotherapy.

[2]  Huanjun Chen,et al.  Structural characterization of a polysaccharide from Chrysanthemum morifolium flowers and its antioxidant activity. , 2015, Carbohydrate polymers.

[3]  W. Zhou,et al.  Mode of action of pentocin 31-1 : An antilisteria bacteriocin produced by Lactobacillus pentosus from Chinese traditional ham , 2008 .

[4]  A. Yousef,et al.  The Lipopeptide Antibiotic Paenibacterin Binds to the Bacterial Outer Membrane and Exerts Bactericidal Activity through Cytoplasmic Membrane Damage , 2014, Applied and Environmental Microbiology.

[5]  H. Abou-Shleib,et al.  Membrane permeability alteration of some bacterial clinical isolates by selected antihistaminics , 2011 .

[6]  H. Nikaido Molecular Basis of Bacterial Outer Membrane Permeability Revisited , 2003, Microbiology and Molecular Biology Reviews.

[7]  J. R. Sachs Na+/K+ Pump , 2003 .

[8]  Jicheng Liu,et al.  Structural elucidation and immunological activity of a polysaccharide from the fruiting body of Armillaria mellea. , 2009, Bioresource technology.

[9]  Y. Nitzan,et al.  Low-Intensity Photosensitization May Enhance RecA Production , 2006, Current Microbiology.

[10]  Li Lan-juan Mohnarin report of 2011:monitoring of bacterial resistance in northwest China , 2012 .

[11]  Jun Liu,et al.  Association of antibiotic resistance with SHV-12 extended-spectrum β-lactamase in Enterobacter cloacae. , 2016, Experimental and therapeutic medicine.

[12]  M. Jin,et al.  Polysaccharides produced by Enterobacter cloacae induce apoptosis in cervical cancer cells. , 2015, International journal of biological macromolecules.

[13]  Mary Jane Ferraro,et al.  Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically : approved standard , 2000 .

[14]  Y. Zhao,et al.  The dissemination mode of drug-resistant genes in Enterobacter cloacae , 2015, Indian journal of medical microbiology.

[15]  A. Bauer,et al.  Antibiotic susceptibility testing by a standardized single disk method. , 1966, American journal of clinical pathology.

[16]  P. Fey,et al.  Extended spectrum beta-lactamase (ESBL)-producing Enterobacteriaceae: considerations for diagnosis, prevention and drug treatment. , 2003, Drugs.

[17]  Zhiyong Luo,et al.  A polysaccharide from Huaier induced apoptosis in MCF-7 breast cancer cells via down-regulation of MTDH protein. , 2016, Carbohydrate polymers.

[18]  J. Skou The Na,K-pump. , 1992, Methods in enzymology.

[19]  Jian-ping Luo,et al.  Structural identification and sulfated modification of an antiglycation Dendrobium huoshanense polysaccharide. , 2014, Carbohydrate polymers.

[20]  A. Mcheik,et al.  Antibacterial and antibiofilm activities of polysaccharides, essential oil, and fatty oil extracted from Laurus nobilis growing in Lebanon. , 2014, Asian Pacific journal of tropical medicine.

[21]  T. Gavan,et al.  A microdilution method for antibiotic susceptibility testing: an evaluation. , 1970, American journal of clinical pathology.

[22]  S. Stefani,et al.  Enterobacter cloacae complex: clinical impact and emerging antibiotic resistance. , 2012, Future microbiology.

[23]  H. Goossens,et al.  Antibiotic therapy for Klebsiella pneumoniae bacteremia: implications of production of extended-spectrum beta-lactamases. , 2004, Clinical infectious diseases : an official publication of the Infectious Diseases Society of America.

[24]  Robert A. Bonomo,et al.  Extended-Spectrum (cid:2) -Lactamases: a Clinical Update , 2005 .

[25]  Guifeng Zhang,et al.  Extraction, purification and antioxidant activities of the polysaccharides from maca (Lepidium meyenii). , 2014, Carbohydrate polymers.

[26]  P. Bradford Extended-Spectrum β-Lactamases in the 21st Century: Characterization, Epidemiology, and Detection of This Important Resistance Threat , 2001, Clinical Microbiology Reviews.

[27]  Wei Chen,et al.  Antimicrobial activity and partial characterization of bacteriocin-like inhibitory substances (BLIS) produced by Bifidobacterium infantis BCRC 14602 , 2009 .

[28]  S. Honda,et al.  Analysis of the monosaccharide compositions of total non-dialyzable urinary glycoconjugates by the dithioacetal method. , 1981, Journal of chromatography.

[29]  P. Nordmann,et al.  Carbapenem resistance in Enterobacteriaceae: here is the storm! , 2012, Trends in molecular medicine.

[30]  P. Fey,et al.  Extended Spectrum β-Lactamase (ESBL)-Producing Enterobacteriaceae , 2012, Drugs.

[31]  K. Laupland,et al.  Emergence of Enterobacteriaceae producing extended-spectrum beta-lactamases (ESBLs) in the community. , 2005, The Journal of antimicrobial chemotherapy.

[32]  J. Ghigo,et al.  Antibiofilm polysaccharides. , 2013, Environmental microbiology.

[33]  B. Tan,et al.  Immunomodulatory and antimicrobial effects of some traditional chinese medicinal herbs: a review. , 2004, Current medicinal chemistry.

[34]  Jicheng Liu,et al.  A water-soluble polysaccharide (EFP-AW1) from the alkaline extract of the roots of a traditional Chinese medicine, Euphorbia fischeriana: Fraction and characterization , 2012 .

[35]  F. Smith,et al.  COLORIMETRIC METHOD FOR DETER-MINATION OF SUGAR AND RELATED SUBSTANCE , 1956 .

[36]  Ronald N. Jones,et al.  Regional variation in the prevalence of extended-spectrum beta-lactamase-producing clinical isolates in the Asia-Pacific region (SENTRY 1998-2002). , 2005, Diagnostic microbiology and infectious disease.

[37]  P. Budd,et al.  Characterization of Anacardium occidentale exudate polysaccharide , 1998 .

[38]  Fengqin Wang,et al.  Selenium‑enriched exopolysaccharides produced by Enterobacter cloacae Z0206 alleviate adipose inflammation in diabetic KKAy mice through the AMPK/SirT1 pathway. , 2014, Molecular medicine reports.

[39]  T. Montville,et al.  Enterocin P Selectively Dissipates the Membrane Potential of Enterococcus faeciumT136 , 2001, Applied and Environmental Microbiology.

[40]  Ming Huang,et al.  Effects of Se-enriched polysaccharides produced by Enterobacter cloacae Z0206 on alloxan-induced diabetic mice. , 2012, International journal of biological macromolecules.

[41]  L. Kokoska,et al.  Screening for antimicrobial activity of some medicinal plants species of traditional Chinese medicine , 2018 .

[42]  P. Hsueh,et al.  Relationships between antimicrobial use and antimicrobial resistance in Gram-negative bacteria causing nosocomial infections from 1991–2003 at a university hospital in Taiwan , 2005, International Journal of Antimicrobial Agents.

[43]  K. Becker,et al.  Plant secondary metabolites in some medicinal plants of Mongolia used for enhancing animal health and production. , 2009 .

[44]  H. Nikaido,et al.  Porin channels in Escherichia coli: studies with liposomes reconstituted from purified proteins , 1983, Journal of bacteriology.

[45]  D. Mukhopadhyay,et al.  In vivo toxicity studies of europium hydroxide nanorods in mice. , 2009, Toxicology and applied pharmacology.

[46]  N. Blumenkrantz,et al.  New method for quantitative determination of uronic acids. , 1973, Analytical biochemistry.

[47]  S. S. Islam,et al.  Structural investigation of a water-soluble glucan from an edible mushroom, Astraeus hygrometricus. , 2004, Carbohydrate research.

[48]  K. Baek,et al.  Antibacterial mode of action of Ginkgo biloba leaf essential oil: Effect on morphology and membrane permeability , 2015 .

[49]  R. Selvendran,et al.  An improved methylation procedure for the analysis of complex polysaccharides including resistant starch and a critique of the factors which lead to undermethylation , 1993 .

[50]  R. Shapiro,et al.  Quantitative analysis by various g.l.c. response-factor theories for partially methylated and partially ethylated alditol acetates☆ , 1975 .

[51]  Cristina Mora,et al.  Actividad inhibitoria de dihidroxifenil propenona sobre betalactamasas de Enterobacter cloacae: estudio preliminar en el desarrollo de fármacos para enfrentar la resistencia bacteriana , 2013 .

[52]  C. Sanders,et al.  Enterobacter spp.: pathogens poised to flourish at the turn of the century , 1997, Clinical microbiology reviews.

[53]  Zhiyan Chen,et al.  Elucidation and biological activities of a new polysaccharide from cultured Cordyceps militaris. , 2014, Carbohydrate polymers.

[54]  R. D. Paula,et al.  Isolation and characterization of soluble sulfated polysaccharide from the red seaweed Gracilaria cornea , 2002 .

[55]  L. Yue,et al.  Purification and identification of one glucan from golden oyster mushroom (Pleurotus citrinopileatus (Fr.) Singer). , 2012, Carbohydrate polymers.

[56]  M. Huang,et al.  Preparation and biological activities of an exopolysaccharide produced by Enterobacter cloacae Z0206 , 2010 .

[57]  Y. Z. Wang,et al.  Preparation, characterization and immunomodulatory activity of selenium-enriched exopolysaccharide produced by bacterium Enterobacter cloacae Z0206. , 2009, Bioresource technology.

[58]  C H Wang,et al.  Studies on the mechanism by which cyanine dyes measure membrane potential in red blood cells and phosphatidylcholine vesicles. , 1974, Biochemistry.

[59]  Martin J T Milton,et al.  Analytical techniques for trace element analysis: an overview , 2005 .

[60]  O. H. Lowry,et al.  Protein measurement with the Folin phenol reagent. , 1951, The Journal of biological chemistry.

[61]  J. Kennedy,et al.  Purification, structural analysis and hydroxyl radical-scavenging capacity of a polysaccharide from the fruiting bodies of Russula virescens , 2010 .

[62]  Xia Li,et al.  Purification, structure and immunobiological activity of a new water-soluble polysaccharide from the mycelium of Polyporus albicans (Imaz.) Teng. , 2008, Bioresource technology.