In Vitro and in Vivo Evaluation of Antibiotic Combination against Multidrug Resistant Enterobacter Species Isolated From Patients of a Tertiary Care Hospital, Bangladesh

Nazmun Nahar Munny^1, , SM Shamsuzzaman² and Tamzeed Hossain³

¹Department of Microbiology, East West Medical College, Dhaka, Bangladesh

²Department of Microbiology, Dhaka Medical College, Dhaka, Bangladesh

³Department of Internal Medicine, Dhaka Medical College Hospital, Dhaka, Bangladesh

Cite this paper:
Nazmun Nahar Munny, SM Shamsuzzaman and Tamzeed Hossain. In Vitro and in Vivo Evaluation of Antibiotic Combination against Multidrug Resistant Enterobacter Species Isolated From Patients of a Tertiary Care Hospital, Bangladesh. American Journal of Infectious Diseases and Microbiology. 2021; 9(3):98-105. doi: 10.12691/ajidm-9-3-4

Abstract

The emergence of multidrug-resistant (MDR) Enterobacter as a worrying resistant pathogen is an important health concern, especially when there is scarcity of new antibiotics active against Gram-negative bacteria. However, currently no defined therapies available for MDR Enterobacter infections. In this study, in vitro and in vivo efficacy of different antimicrobial combinations were assessed. This cross-sectional study was carried out in the department of Microbiology of Dhaka medical college hospital, Bangladesh from July, 2018 to June, 2019. Multidrug resistance among isolated Enterobacter species were detected phenotypically by disk diffusion method. PCR and sequencing of fosfomycin resistance genes were done. In vitro activity of fosfomycin, amikacin, imipenem, piperacillin-tazobactam and their combinations were evaluated using agar dilution method and synergy was assessed by Fractional inhibitory concentration index. Mice models were made by using the MDR Enterobacter strain. We evaluate the efficacy of fosfomycin, amikacin, imipenem and their combination against multi-drug resistant Enterobacter infection in experimental mice models. Among 28 isolated Enterobacter spp. 53.33% were multidrug-resistant. Among the fosfomycin resistant Enterobacter spp. 70% , 50%, 40% were positive for fosA. foA₅ and fosB₂ respectively. The fractional inhibitory concentration index indicated that combining antibiotics resulted 2 to 8 fold reduction of MIC compared to single therapy. The ratio of synergy observed in imipenem-amikacin, fosfomycin-amikacin, fosfomycin-imipenem 16.67%, 87.33%, 50.0% respectively in vitro. No synergy observed in imipenem-piperacillin tazobactam combination. In mice model, compared to single antibiotic therapy, fosfomycin-amikacin, imipenem-amikacin, fosfomycin-imipenem showed increased sterile blood culture (100%, 60%, 80%). Fosfomycin plus amikacin or fosfomycin plus imipenem may be alternative treatment option against multidrug-resistant Enterobacter infection.

Keywords:
Enterobacter species multidrug-resistance combination therapy in vitro and in vivo efficacy fosfomycin resistance gene

This work is licensed under a Creative Commons Attribution 4.0 International License. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/

References:

[1]	Davin-Regli A, Lavigne JP, Pages JM. Enterobacter spp.: update on taxonomy, clinical aspects, and emerging antimicrobial resistance. Clin Microbiol Rev 32, 2019; 32(4).
[2]	Davin-Regli A, PagèsJM.. Enterobacter aerogenes and Enterobacter cloacae; versatile bacterial pathogens confronting antibiotic treatment. Front Microbiol 2015; 6: 392.
[3]	Davin-Regli A, Masi M, Bialek S, Nicolas-Chanoine MH, Pagès JM. Antimicrobial resistance and drug efflux pumps in Enterobacter and Klebsiella. In Li X-Z, Elkins CA, Zgurskaya HI (eds), Efflux-Mediated Drug Resistance in Bacteria: Mechanisms, Regulation and Clinical Implications. Springer International Publishing Switzerland, Adis Springer Nature. 2016.
[4]	Mezzatesta ML, Gona F, Stefania S. Enterobacter cloacae complex: clinical impact and emerging antibiotic resistance. Future Microbiol, 2012; 7(7): 887-902.
[5]	Susan W, Simmonds AG, Annavajhala MK, Wang Z, Macesic N, Hu Y et al. Genomic and geographic context for the evolution of high-risk carbapenem-resistant Enterobacter cloacae complex clones ST171 and ST78. mBio, 2018; 9.
[6]	Gallet AVC, Ocampo AM, Chavda K, Chen L, Barry N. Kreiswirth BN et al. Molecular epidemiology of carbapenem-resistant Enterobacter cloacae complex infections uncovers high frequency of non-carbapenemase-producers in five tertiary care hospitals from Colombia. bioRxiv preprint first posted online, 2018.
[7]	Zhang R, Huang L, Hu YY. Prevalence of fosfomycin resistance and plasmid-mediated fosfomycin-modifying enzymes among carbapenem-resistant Enterobacteriaceae in Zhejiang, China. J Med Microbiol, 2017; 66(9): 1332-1334.
[8]	Sastry S, Doi Y. Fosfomycin: Resurgence of An Old Companion. J Infect Chemother 2016; 22(5):273-80.
[9]	Nakamura G, Wachino J, Sato N, Kimura K, Yamada K, Jin Wet al. Practical agar-based disk potentiation test for detection of fosfomycin-nonsusceptible Escherichia coli clinical isolates producing glutathione S-transferases. J Clin Microbiol, 2014; 52(9): 3175-3179.
[10]	Chessbrough M. Microscopial techniques used in Microbiology, culturing bacterial pathogens, biochemical test to identify bacteria. In: Chessbrough M, ed. District Laboratory Practice in Tropical Countries, Part 2, 2nd edn: Cambridge University Press, India; 2009: pp. 35-195.
[11]	Clinical Laboratory Standards Institute. Performance Standard for Antimicrobial Susceptibility Testing: Twenty-seventh Informational Supplement M200-S28. Wayne, PA, USA: CLSI; 2018.
[12]	European Committee on Antimicrobial Susceptibility Testing. Breakpoint tables for interpretation of MICs and zone diameters. Version 8.0, valid from 2018-01-01. EUCAST: Clinical breakpoints www.eucast.org/clinicalbreakpoints/(accessed on13.02.2019).
[13]	Performance Standards for Antimicrobial Susceptibility Testing; Twenty-Fourth Informational Supplement. Wayne, USA: CLSI; 2014.
[14]	Franco MR, Caiaffa-Filho HH, Burattini MN, Rossi F. Metallo-beta-lactamases among imipenem-resistant Pseudomonas aeruginosa in a Brazilian university hospital. Clinics (Sao Paulo), 2010; 65(9): 825-829.
[15]	Ho PL, Chan J, Lo WU, Lai EL, Cheung YY, Chow KH et al. Prevalence and molecular epidemiology of plasmid mediated fosfomycin resistance genes among blood and urinary Escherichia coli isolates. J Med Microbiol, 2013; 62(Pt 11): 1707-1713.
[16]	Benzerara Y, Gallah S, Hommeril B, Genel N, Rottman M, Arlet G et al. Emergence of plasmid-mediated fosfomycin-resistance genes among Escherichia coli isolate, France. Emerg Infect Dis, 2017; 23(9): 1564-1567.
[17]	Wang X, Zhang Z, Hao Q, Wu J, Xiao J, Jinga H. Complete Genome Sequence of Acinetobacter baumannii ZW85-1.GenomeAnnounc, 2014; 2(1).
[18]	Gombert M. E, Aulicino T. M. “Synergism of imipenem and amikacin in combination with other antibiotics against Nocardia asteroids,” Antimicrob Agents Chemother, 24(5): 810-811, 1983.
[19]	Kheshti R, Pourabbas B, Mosayebi M, Vazin A.In vitro activity of colistin in combination with various antimicrobials against Acinetobacter baumannii species. a report from South Iran. Infect. Drug Resist. 2019; 12, 129.
[20]	Toledo PV, Tuon FF, Bali L, Manente F, Arruda P, Aranha-Junior AA. Experimental model for treatment of extended spectrum betalactamase producing-Klebsiella pneumoniae. Arq Bras Cir Diag, 2014; 27(3): 168-171.
[21]	Hernandez MJR, Pachon J, Pichard C, Cuberos L, Martinez JI, Curiel AG et al. Imipenem, doxycycline and amikacin in monotherapy and in combination in Acinetobacter baumanniiexperimental pneumonia. J Antimicrob Chemother, 2000; 45(4): 493-501.
[22]	Markou N, Markantonis S.L, Dimitrakis E, Panidis D, Boutzouka E, Karatzas S et al. Colistin serum concentrations after intravenous administration in critically ill patients with serious multidrug-resistant, Gram-negative bacilli infections: A prospective, open-label, uncontrolled study. Clin. Ther. 2008, 30, 143-151.
[23]	Monotherapy versus combination therapy for multidrug-resistant Gram-negative infections: Systematic Review and Meta Analysis. Adrian Schmid, Aline Wolfensberger, Johannes Nemeth, PeterW. Schreiber, Hugo Sax & Stefan P. Kuster.
[24]	Liu CP, Wang NY, Lee CM, Weng LC, Tseng HK, Liu CW et al. Nosocomial and community-acquired Enterobacter cloacae bloodstream infection: risk factors for and prevalence of SHV-12 in multiresistant isolates in a medical centre. J Hosp Infect, 2004; 58(1): 63-77.
[25]	Dimitrova D, Stoeva T, Markovska R, Stankova P, Mihova K, Kaneva R et al. Molecular epidemiology of multi drug resistant Enterobacter Cloacae blood isolates from a university hospital. J of IMAB, 2019; 25(2): 2457-2464.
[26]	THOMAS TÄNGDÉN. Combination antibiotic therapy for multidrug-resistant Gram-negative bacteria, Upsala Journal of Medical Sciences. 2014; 119: 149-153.
[27]	Adhikari RP, Shrestha S, Rai JR, Amatya R. Antimicrobial Resistance Patterns in Clinical Isolates of Enterobacteriaceae from a Tertiary Care Hospital, Kathmandu, Nepal. Nep Med J, 2018; 1: 74-78.
[28]	Gopichand P, Agarwal G, Natarajan M, Mandal J, Deepanjali S, Parameswaran Set al .In vitro effect of fosfomycin on multi-drug resistant gram-negative bacteria causing urinary tract infections. Infect Drug Resist. 2019; 12:2005-2013.
[29]	Wei Yu, Ping Shen, Zhang Bao, Kai Zhou, Beiwen Zheng, Jinru Ji et al. In vitro antibacterial activity of fosfomycin combined with other antimicrobials against KPC-producing Klebsiella pneumoniae. Int J Antimicrob Agents. 2017. Aug; 50(2): 237-241.
[30]	Cai Y, Fan Y, Wang R, An MM, Liang BB. Synergistic effects of aminoglycosides and fosfomycin on Pseudomonas aeruginosa in vitro and biofilm infections in a rat model. J Antimicrob Chemother 2009; 64: 563-6.
[31]	Vergara-López S, Domínguez MC, Conejo MC, Pascual Á, Rodríguez-Baño J. Prolonged treatment with large doses of fosfomycin plus vancomycin and amikacin in a case of bacteraemia due to methicillin-resistant Staphylococcus epidermidis and IMP-8 metallo-β-lactamase-producing Klebsiella oxytoca. J Antimicrob Chemother 2015; 70: 313-15.
[32]	Chukamnerd A, Pomwised R, Phoo M.T.P, Terbtothakun P, Hortiwakul T, Charoenmak B et al. In vitro synergistic activity of fosfomycin in combination with other antimicrobial agents against carbapenem-resistant Klebsiella pneumoniae isolated from patients in a hospital in Thailand. J. Infect. Chemother. 2020, 27, 507-514.
[33]	Montgomery A.B, Rhomberg P.R, Abuan T, Walters K.A, Flamm R.K. Potentiation effects of amikacin and fosfomycin against selected amikacin-nonsusceptible Gram-negative respiratory tract pathogens. Antimicrob. Agents Chemother. 2014, 58, 3714-3719.
[34]	Wei Yu , Qixia Luo , Qingyi Shi , Chen Huang , Xiao Yu , et al . In vitro antibacterial effect of fosfomycin combination therapy against colistin-resistant Klebsiella pneumoniae. Infect. Drug Resist. 2018, 11, 577-585.
[35]	Jia y. The progress in study of fosfomycin. Infection International; 2017; 6(3): 88-92.
[36]	Ebru Evren , Ozlem Kurt Azap, ŞuleÇolakoğlu, Hande Arslan .In vitro activity of fosfomycin in combination with imipenem, meropenem, colistin and tigecycline against OXA 48–positive Klebsiella pneumoniae strains. Diagn Microbiol Infect Dis. 2013; 76(3): 335-8.
[37]	Samonis G, Maraki S, Karageorgopoulos D. E. Synergy of fosfomycin with carbapenems, colistin, netilmicin, and tigecycline against multidrug-resistant Klebsiella pneumoniae, Escherichia coli and Pseudomonas aeruginosa clinical isolates. Eur J Clin Microbiol Infect Dis 2012; 31: 695-701.
[38]	Giancola SE, Mahoney MV, Hogan MD, Raux BR, McCoy C, Hirsch EB. Assessment of fosfomycin for complicated or multidrug-resistant urinary tract infections: Patient Characteristics and Outcomes. Chemotherapy, 2017; 62(2): 100-104.
[39]	Dundar, D, Otkun, M. In-vitro efficacy of synergistic antibiotic combinations in multidrug resistant Pseudomonas aeruginosa strains. Yonsei Med J 2010; 51: 111-116.
[40]	Kohanski MA, Dwyer DJ, Collins JJ. How antibiotics kill bacteria: from targets to networks. Nat Rev Microbiol.2010; 8(6): 423-435.
[41]	Johnson, D. E., Thompson B, Calia F. M, “Comparative Activities of piperacillin, ceftazidime and Amikacin, Alone and in All possible Combinations, against Experimental pseudomonas aeruginosa Infections in Neutropenic Rats,” Antimicrob Agents chemother,. 27(12): 735-739, 1985.
[42]	Inouye S, Niizato T, Komiya I, Yuda Y, Yamada Y. Mode of protective action of fosfomycin against dibekacin-induced nephrotoxicity in the dehydrated rats. J Pharmacobiodyn, 1982; 5(12): 941-950.
[43]	Zhang R, Huang L, Hu YY. Prevalence of fosfomycin resistance and plasmid-mediated fosfomycin-modifying enzymes among carbapenem-resistant Enterobacteriaceae in Zhejiang, China. J Med Microbiol, 2017; 66(9): 1332-1334.
[44]	Jiang Y, Shen P, Zhou Z, Zhang J, Yu Y, Li L. Complete nucleotide sequence of pKP96, a 67 850 bp multiresistance plasmid encoding qnrA1, aac(6')-Ib-cr and blaCTX-M-24 from Klebsiella pneumoniae. J Antimicrob Chemother, 2008; 62(6): 1252-1256.
[45]	Garcia-Muse T, Aguilera A.Causes of genome instability. Annu Rev Genet, 2013; 47: 1-32.