Molecular characterization of potentially virulent multidrug-resistant Enterococcus faecalis isolated from acquired urinary tract infections in Egyptian patients


  • Heba A. Ahmed Zoonoses Department, Faculty of Veterinary Medicine, Zagazig University, Zagazig City, 44511, Sharkia Governorate, Egypt
  • Nagham Mostafa Department of Botany and Microbiology, Faculty of Science, Zagazig University, Zagazig City 44519, Egypt
  • Eman Y.T. Elariny Department of Botany and Microbiology, Faculty of Science, Zagazig University, Zagazig City 44519, Egypt
  • Rania M. Ahmed Department of Botany and Microbiology, Faculty of Science, Zagazig University, Zagazig 44519, Egypt



Enterococcus faecalis, multidrug-resistance, virulence determinants


Paper description:

  • Enterococcus faecalis was detected in 28% urine samples of hospitalized Egyptian patients with urinary tract infections (UTIs). E. faecalis infection was more frequent in females than males and most common in ages ≥41 with increased frequency of infection in rural regions.
  • The genes encoding the virulence determinants in E. faecalis strains (cylA, cylB, cylM, gelE, esp, efaA, asa, ace) were determined using real-time PCR.
  • Most E. faecalis strains were multidrug resistant and 56% of strains were biofilm-producers.
  • Multidrug resistance of E. faecalis, their virulence potential and biofilm formation ability are a public health risk to UTI patients.

Abstract: In this study, 154 urine samples were collected from urinary tract infections (UTIs) of hospitalized patients, with 43 (28%) positive for the presence of Enterococcus faecalis without detection of Enterococcus faecium. Based on the demographic data associated with UTI patients, the prevalence of E. faecalis among females was higher than among males in the age group ≥41 years; cases from rural areas showed higher infection than from urban localities. Patients not treated with antibiotics were 2.8-fold more likely to be infected with E. faecalis than patients who received antibiotics. At the molecular level, the genes encoding the virulence determinants in E. faecalis, including cytolysins (cylA, cylB and cylM), gelatinase (gelE), E. faecalis antigen A (efaA), extracellular surface protein (esp), aggregation substance (asa) and collagen binding adhesion (ace) were determined using SYBR green real-time PCR. Antibiotic susceptibility testing showed that almost all strains were multidrug-resistant, with an average multiple antibiotic resistance (MAR) index of 0.55. The colorimetric microtiter plate assay showed that 56% of the strains were biofilm producers. A significant correlation was observed between strong biofilm formation and the presence of cylB and cylM genes. Multidrug-resistant E. faecalis and its virulence potential and biofilm formation ability pose a risk to UTI patients.


Download data is not yet available.


Flores-Mireles AL, Walker JN, Caparon M, Hultgren SJ. Urinary tract infections: epidemiology, mechanisms of infection and treatment options. Nat Rev Microbiol. 2015;13(5):269-84.

Heidari H, Hasanpour S, Ebrahim-Saraie HS, Motamedifar M. High incidence of virulence factors among clinical Enterococcus faecalis isolates in Southwestern Iran. Infect Chemother. 2017;49(1):51-56.

Esmail MAM, Abdulghany HM, Khairy RM. Prevalence of multidrug-resistant Enterococcus faecalis in hospital-acquired surgical wound infections and bacteremia: concomitant analysis of antimicrobial resistance genes. Infect Dis (Auckl). 2019;12:1178633719882929.

Hidron AI, Edwards JR, Patel J, Horan TC, Sievert DM, Pollock DA, Fridkin SK; National Healthcare Safety Network Team and Participating National Healthcare Safety Network Facilities. NHSN annual update: Antimicrobial-resistant pathogens associated with healthcare-associated infections: Annual summary of data reported to the National Healthcare Safety Network at the Centers for Disease Control and Prevention, 2006-2007. Infect Control Hosp Epidemiol. 2008;29(11):996-1011. Erratum in: Infect Control Hosp Epidemiol. 2009;30(1):107

Fallah F, Yousefi M, Pourmand MR, Hashemi A, Nazari Alam A, Afshar D. Phenotypic and genotypic study of biofilm formation in enterococci isolated from urinary tract infections. Microb Pathog. 2017;108:85-90.

Dunny GM, Craig RA, Carron RL, Clewell DB. Plasmid transfer in Streptococcus faecalis: production of multiple sex pheromones by recipients. Plasmid. 1979;2(3):454-65.

Abdelkareem MZ, Sayed M, Hassuna NA, Mahmoud MS, Abdelwahab SF. Multi-drug-resistant Enterococcus faecalis among Egyptian patients with urinary tract infection. J Chemother. 2017;29:74-82.

Aleksandrovna ZE, Sergeevna KT, Aleksandrovna ME, Mikhailovich SA, Nikolaevna LV. Phenotypic and genetic diversity of uropathogenic Enterococcus faecalis strains isolated in the Primorsky region of Russia. In: Behzadi P, editor. Microbiology of Urinary Tract Infections - Microbial Agents and Predisposing Factors. London: IntechOpen; 2019.

Wardal E, Markowska K, Zabicka D, Wróblewska M, Giemza M, Mik E, Polowniak-Pracka H, Wozniak A, Hryniewicz W, Sadowy E. Molecular analysis of vanA outbreak of Enterococcus faecium in two Warsaw hospitals: the importance of mobile genetic elements. Biomed Res Int. 2014;575367.

Limoli D, Jones CJ, Wozniak D. Bacterial extracellular polysaccharides in biofilm formation and function. Microbiol Spectr. 2015;3(3):0011.

Holmberg A, Rasmussen M. Mature biofilms of Enterococcus faecalis and Enterococcus faecium are highly resistant to antibiotics. Diagn Microbiol Infect Dis. 2016;84:19-21.

Khairy RM, Mahmoud MS, Esmail MAM, Gamil AN. First detection of vanB phenotype-vanA genotype vancomycin-resistant enterococci in Egypt. J Infect Dev Ctries. 2019;13(9):837-42.

Moemen D, Tawfeek D, Badawy W. Healthcare-associated vancomycin resistant Enterococcus faecium infections in the Mansoura University Hospitals intensive care units, Egypt. Braz J Microbiol. 2015; 46(3):777-83.

Osman K, Zolnikov TR, Badr J, Naim H, Hanafy M, Saad A, Elbehiry A. Vancomycin and florfenicol resistant Enterococcus faecalis and Enterococcus faecium isolated from human urine in an Egyptian urban-rural community. Acta Trop. 2020;201:105209.

Said HS, Abdelmegeed ES. Emergence of multidrug resistance and extensive drug resistance among enterococcal clinical isolates in Egypt. Infect Drug Resist. 2019;12:1113-25.

Murray PR, Baron EJ, Pfaller MA, Tenover FC, Yolken RH. Manual of clinical microbiology 7th ed. Washington: ASM Press,; 1999.

Matsuda K, Tsuji H, Asahara T, Matsumoto K, Takada T, Nomoto K. Establishment of an analytical system for the human fecal microbiota, based on reverse transcription-quantitative PCR targeting of multicopy rRNA molecules. Appl Environ Microbiol. 2009;75:1961-9.

Zoletti GO, Siqueira JF Jr, Santos KR. Identification of Enterococcus faecalis in root-filled teeth with or without periradicular lesions by culture-dependent and-independent approaches. J Endod. 2006;32(8):722-6.

Jackson CR, Fedorka-Cray PJ, Barrett JB. Use of a genus- and species-specific multiplex PCR for identification of enterococci. J Clin Microbiol. 2004;42:3558.

Eaton TJ, Gasson MJ. Molecular screening of Enterococcus virulence determinants and potential for genetic exchange between food and medical isolates. Appl Environ Microbiol. 2001;67(4):1628-35.

Lowe AM, Lambert PA, Smith AW. Cloning of an Enterococcus faecalis endocarditis antigen: homology with adhesins from some oral streptococci. Infect Immun. 1995;63(2):703-6.

Sillanpää J, Xu Y, Nallapareddy SR, Murray BE, Höök M. A family of putative MSCRAMMs from Enterococcus faecalis. Microbiology (Reading). 2004;150(Pt7):2069-78.

Patel R, Uhl JR, Kohner P, Hopkins MK, Cockerill FR 3rd. Multiplex PCR detection of vanA, vanB, vanC-1, and vanC-2/3 genes in enterococci. J Clin Microbiol. 1997;(3):703-7.

Kariyama R, Mitsuhata R, Chow JW, Clewell DB, Kumon H. Simple and reliable multiplex PCR assay for surveillance isolates of vancomycin-resistant enterococci. J Clin Microbiol. 2000;38(8):3092-5.

CLSI. Clinical and Laboratory Standards Institute. Performance standards for antimicrobial susceptibility testing. 26th edn. CLSI supplement M100S, Wayne, PA.; 2016.

Krumperman PH. Multiple antibiotic resistance indexing of Escherichia coli to identify high-risk sources of fecal contamination of foods. Appl Environ Microbiol. 1983;46(1):165-70.

Magiorakos AP, Srinivasan A, Carey RB, Carmeli Y, Falagas ME, Giske CG, Harbarth S, Hindler JF, Kahlmeter G, Olsson-liljequist, Paterson DL, Rice LB, Stelling J, Stuelens MJ, Vatopoulos A, Weber JT, Monnet DL.Multidrug-resistant, extensively drug-resistant and pandrug-resistant bacteria: an international expert proposal for interim standard definitions for acquired resistance. Clin Microbiol Infect. 2012;18(3):268-81.

O’Toole GA. Microtiter dish biofilm formation assay. J Vis Exp. 2011;(47):2437.

Saxena S, Banerjee G, Garg R, Singh M. Comparative study of biofilm formation in Pseudomonas aeruginosa isolates from patients of lower respiratory tract infection. J Clin Diagn Res. 2014;8(5):DC09-DC11.

Kolawole AS, Babatunde, SK, Kandaki-Olukemi YT, Durowade K. Prevalence of urinary tract infections (UTI) among patients attending Dalhatu Araf Specialist Hospital, Lafia, Nasarawa State, Nigeria. Int J Med Sci. 2009;1:163-7.

Švec P, Devriese LA. Enterococcus. In: Whitman WB, editor. Bergey’s Manual of Systematics of Archaea and Bacteria. John Wiley & Sons; 2015. p. 1-25.

Abamecha A, Wondafrash B, Abdissa, A. Antimicrobial resistance profile of Enterococcus species isolated from intestinal tracts of hospitalized patients in Jimma, Ethiopia. BMC Res Notes. 2015;8:213.

Kafil HS, Mobarez AM. Assessment of biofilm formation by enterococci isolates from urinary tract infections with different virulence profiles. J King Saud Univ Sci. 2015;27:312-7.

Gonzalez TDJ, Pham P, Top J, Willems RJL, van Schaik W, van Passel MWJ, Smidt H. Characterization of Enterococcus isolates colonizing the intestinal tract of intensive care unit patients receiving selective digestive decontamination. Front Microbiol. 2017;8:1596.

Hussain A, Sohail M, Abbas Z. Prevalence of Enterococcus faecalis mediated UTI and its current antimicrobial susceptibility pattern in Lahore, Pakistan. J Pak Med Assoc. 2016;66:10:1232-6.

Kau AL, Martin SM, Lyon W, Hayes E, Caparon MG, Hultgren SJ. Enterococcus faecalis tropism for the kidneys in the urinary tract of C57BL/6J mice. Infect Immun. 2005;73(4):2461-8.

Venkatesan KD, Chander S, Loganathan K, Victor K. Study on asymptomatic bacteriuria in diabetic patients. Int J Contemp Med Res. 2017;4:480-3.

de Lastours V, Foxman B. Urinary tract infection in diabetes: epidemiologic considerations. Curr Infect Dis Rep. 2014;16(1):389.

Geerlings SE, Stolk RP, Camps MJ, Netten PM, Collet TJ, Hoepelman AI. Diabetes women asymptomatic bacteriuria utrecht study group. risk factors for symptomatic urinary tract infection in women with diabetes. Diabetes Care. 2000;23(12):1737-41.

Duh RW, Singh KV, Malathum K, Murray BE. In vitro activity of 19 antimicrobial agents against enterococci from healthy subjects and hospitalized patients and use of an ace gene probe from Enterococcus faecalis for species identification. Microb Drug Resist. 2001;7:39-46.

Strateva T, Atanasova D, Savov E, Petrova G, Mitov I. Incidence of virulence determinants in clinical Enterococcus faecalis and Enterococcus faecium isolates collected in Bulgaria. Braz J Infect Dis. 2016;20:127-33.

Udo EE, Al-Sweih N. Frequency of virulence-associated genes in Enterococccus faecalis isolated in Kuwait hospitals. Med Princ Pract. 2011;20:259-64.

De Vuyst L, Foulquié Moreno MR, Revets H. Screening for enterocins and detection of hemolysin and vancomycin resistance in enterococci of different origins. Int J Food Microbiol. 2003;84(3):299-318.

Urusova DV, Merriman JA, Gupta A, Chen L, Mathema B, Caparon MG, Khader SA. Rifampin resistance mutations in the rpoB gene of Enterococcus faecalis impact host macrophage cytokine production. Cytokine. 2022;151:155788.

Morris JG Jr, Shay DK, Hebden JN, McCarter RJ Jr, Perdue BE, Jarvis W, Johnson JA, Dowling TC, Polish LB, Schwalbe RS. Enterococci resistant to multiple antimicrobial agents, including vancomycin. Establishment of endemicity in a university medical center. Ann Intern Med. 1995;123(4):250-9.

Butt T, Leghari MJ, Mahmood A. In-vitro activity of nitrofurantoin in Enterococcus urinary tract infection. J Pak Med Assoc. 2004;54(9):466-9.

Singh D, Umrao PD, Kaistha SD. Multiple antibiotic resistance and biofilm formation of catheter associated urinary tract infection (CAUTI) causing microorganisms. J Bacteriol Mycol. 2018;6:217-21.

Furtula V, Jackson CR, Farrell EG, Barrett JB, Hiott LM, Chambers PA. Antimicrobial resistance in Enterococcus spp. isolated from environmental samples in an area of intensive poultry production. Int J Environ Res Public Health. 2013;10(3):1020-36.

Rathnayake IU, Hargreaves M, Huygens F. Antibiotic resistance and virulence traits in clinical and environmental Enterococcus faecalis and Enterococcus faecium isolates. Syst Appl Microbiol. 2012;35(5):326-33.

Gagetti P, Bonofiglio L, García Gabarrot G, Kaufman S, Mollerach M, Vigliarolo L, von Specht M, Toresani I, Lopardo HA. Resistance to β-lactams in enterococci. Rev Argent Microbiol. 2019;51(2):179-83.

Ono S, Muratani T, Matsumoto T. Mechanisms of resistance to imipenem and ampicillin in Enterococcus faecalis. Antimicrob Agents Chemother. 2005;49(7):2954-8.

Zhanel GG, Laing NM, Nichol KA, Palatnick LP, Noreddin A, Hisanaga T, Johnson JL, Hoban DJ. Antibiotic activity against urinary tract infection (UTI) isolates of vancomycin-resistant enterococci (VRE): results from the 2002 North American vancomycin resistant enterococci susceptibility study (NAVRESS). J Antimicrob Chemother. 2003;52(3):382-8.

Korten V, Huang WM, Murray BE. Analysis by PCR and direct DNA sequencing of gyrA mutations associated with fluoroquinolone resistance in Enterococcus faecalis. Antimicrob Agents Chemother. 1994;38(9):2091-4.

Nakanishi N, Yoshida S, Wakebe H, Inoue M, Mitsuhashi S. Mechanisms of clinical resistance to fluoroquinolones in Enterococcus faecalis. Antimicrob Agents Chemother. 1991;35(6):1053-9.

Zheng JX, Bai B, Lin ZW, Pu ZY, Yao WM, Chen Z, Li DY, Deng XB, Deng QW, Yu ZJ. Characterization of biofilm formation by Enterococcus faecalis isolates derived from urinary tract infections in China. J Med Microbiol. 2018;67(1):60-7.

Seno Y, Kariyama R, Mitsuhata R, Monden K, Kumon H. Clinical implications of biofilm formation by Enterococcus faecalis in the urinary tract. Acta Med Okayama. 2005;59(3):79-87.

Hufnagel M, Koch S, Creti R, Baldassarri L, Huebner J. A putative sugar-binding transcriptional regulator in a novel gene locus in Enterococcus faecalis contributes to production of biofilm and prolonged bacteremia in mice. J Infect Dis. 2004;189(3):420-30.

Garg S, Mohan B, Taneja N. Biofilm formation capability of enterococcal strains causing urinary tract infection vis-a-vis colonisation and correlation with enterococcal surface protein gene. Indian J Med Microbiol. 2017;35(1):48-52.

Mohamed JA, Murray BE. Lack of correlation of gelatinase production and biofilm formation in a large collection of Enterococcus faecalis isolates. J Clin Microbiol. 2005;43(10):5405-7.




How to Cite

Ahmed H, Mostafa N, Elariny E, Ahmed R. Molecular characterization of potentially virulent multidrug-resistant Enterococcus faecalis isolated from acquired urinary tract infections in Egyptian patients. Arch Biol Sci [Internet]. 2023Oct.26 [cited 2024Feb.29];75(3):351-64. Available from: