• 1

    Vargas M, Gascón J, Casals C, Schellenberg D, Urassa H, Ka-higwa E, Ruiz J, Vila J, 2004. Etiology of diarrhea in children less than five years of age in Ifakara, Tanzania. Am J Trop Med Hyg 70 :536–539.

    • Search Google Scholar
    • Export Citation
  • 2

    Bhan MK, Raj P, Levine MM, Kaper JB, Bhandari N, Srivastava R, Kumar R, Sazawal S, 1989. Enteroaggregative Escherichia coli associated with persistent diarrhea in a cohort of rural children in India. J Infect Dis 159 :1061–1064.

    • Search Google Scholar
    • Export Citation
  • 3

    Bouzari S, Jafari A, Azizi A, Oloomi M, Nataro JP, 2001. Characterization of enteroaggregative Escherichia coli isolates from Iranian children. Am J Trop Med Hyg 65 :13–14.

    • Search Google Scholar
    • Export Citation
  • 4

    Fang GD, Lima AA, Martins CV, Nataro JP, Guerrant RL, 1995. Etiology and epidemiology of persistent diarrhea in northeastern Brazil: a hospital-based, prospective, case-control study. J Pediatr Gastroenterol Nutr 21 :137–144.

    • Search Google Scholar
    • Export Citation
  • 5

    Okeke IN, Lamikanra A, Czeczulin J, Dubovsky F, Karper J, Nataro JP, 2000. Characterization of Escherichia coli strains from cases of childhood diarrhea in provincial south-western Nigeria. J Infect Dis 181 :252–260.

    • Search Google Scholar
    • Export Citation
  • 6

    Sarantuya J, Nishi J, Wakimoto N, Erdene S, Nataro JP, Sheikh J, Iwashita M, Manago K, Tokuda K, Yoshinga M, Miyata K, Kawano Y, 2004. Typical Enteroaggregative Escherichia coli is the most prevalent pathotype among E.coli strains causing diarrhea in Mongolian children. J Clin Microbiol 42 :133–139.

    • Search Google Scholar
    • Export Citation
  • 7

    Scaletsky ICA, Fabbricotti SH, Silva SOC, Morais MB, Fagun-des-Neto U, 2002. Hep-2-adherent Escherichia coli strains associated with acute infantile diarrhea, Sao Paulo, Brazil. Emerg Infect Dis 8 :855–858.

    • Search Google Scholar
    • Export Citation
  • 8

    Gascón J, Vargas M, Schellenberg D, Urassa H, Casals C, Ka-higwa E, Aponte JJ, Mshinda H, Vila J, 2000. Diarrhea in children under 5 years of age from Ifakara, Tanzania: a case-control study. J Clin Microbiol 38 :4459–4462.

    • Search Google Scholar
    • Export Citation
  • 9

    Gomes TAT, Blake PA, Trabulsi LR, 1989. Prevalence of Esche-richia coli strains with localized, diffuse and aggregative adherence to HeLa cells in infants with diarrhea and matched controls. J Clin Microbiol 27 :266–269.

    • Search Google Scholar
    • Export Citation
  • 10

    Huang DB, Okhuysen PC, Zhi-Dong Jiang MD, DuPont HL, 2004. Enteroaggregative Escherichia coli: an emerging enteric pathogen. Am J Gastroenterol 99 :383–389.

    • Search Google Scholar
    • Export Citation
  • 11

    Nataro JP, Steiner T, Guerrant RL, 1998. Enteroaggregative Escherichia coli.Emerg Infect Dis 4 :251–261.

  • 12

    Sheikh J, Hicks S, Dall’Agnol M, Phillips AD, Nataro JP, 2001. Roles for Fis and YafK in biofilm formation by enteroaggre-gative Escherichia coli.Mol Microbiol 41 :983–997.

    • Search Google Scholar
    • Export Citation
  • 13

    Wakimoto N, Nichi J, Sheik J, Nataro JP, Sarantuya J, Iwashita M, Manago K, Tozuda K, Yoshinaga M, Kawano Y, 2004. Quantitative biofilm assay using a microtiter plate to screen for enteroaggregative Escherichia coli.Am J Trop Med Hyg 71 :687–690.

    • Search Google Scholar
    • Export Citation
  • 14

    Kjaergarrd K, Schembri MA, Ramos C, Molin S, Klemm P, 2000. Antigen 43 facilitates formation of multispecies biofilms. Environ Microbiol 2 :695–702.

    • Search Google Scholar
    • Export Citation
  • 15

    Schembri MA, Kjaergarrd K, Klemm P, 2003. Global gene expression in Escherichia coli biofilms. Mol Micorbiol 48 :253–267.

  • 16

    Baudry B, Savarino SJ, Vial P, Koper JB, Levine MM, 1990. A sensitive and specific DNA probe to identify enteroaggrega-tive Escherichia coli, a recently discovered diarrheal pathogen. J Infect Dis 161 :1249–1251.

    • Search Google Scholar
    • Export Citation
  • 17

    Schmidt H, Knop C, Franke S, Aleksic S, Heesemenn J, Karch H, 1995. Development of PCR for screening of enteroaggregative Escherichia coli.J Clin Microbiol 33 :701–705.

    • Search Google Scholar
    • Export Citation
  • 18

    Czeczulin JR, Balepur S, Hicks S, Phillips A, Hall R, Kothary MH, Navarro-García F, Nataro JP, 1997. Aggregative adherence fimbria II, a second fimbria antigen mediating adherence in enteroaggregative Escherichia coli.Infect Immun 65 :4135–4145.

    • Search Google Scholar
    • Export Citation
  • 19

    Nataro JP, Deng Y, Maneval DR, German AL, Martin WC, Le-vine MM, 1992. Aggregative adherence fimbria I of enteroag-gregative Escherichia coli mediate adherence to Hep-2 cells and hemagglutination of human erythrocytes. Infect Immun 60 :2297–2304.

    • Search Google Scholar
    • Export Citation
  • 20

    Savarino SJ, Fasano A, Robertson DC, Levine MM, 1991. En-teroaggregative Escherichia coli elaborate a heath-stable en-terotoxin demonstrable in an in vitro rabbit intestinal model. J Clin Invest 87 :1450–1455.

    • Search Google Scholar
    • Export Citation
  • 21

    Vila J, Gene A, Vargas M, Gascon J, Latorre C, Jiménez de Anta MT, 1998. A case-control study of diarrhea in children caused by Escherichia coli producing heat-stable enterotoxin (EAST-1). J Med Microbiol 47 :889–891.

    • Search Google Scholar
    • Export Citation
  • 22

    Nataro JP, Yikang D, Yingkang D, Walker K, 1994. AggR, a transcriptional activator of aggregative adherence fimbrial I expression in enteroaggregative Escherichia coli.J Bacteriol 176 :4691–4699.

    • Search Google Scholar
    • Export Citation
  • 23

    Nataro JP, 2005. Enteroaggragtive Escherichia coli pathogenesis. Curr Opin Gastroenterol 21 :4–8.

  • 24

    Sheikh J, Czeczulin JR, Harrington S, Hicks S, Henderson IR, Le Bouguénec C, Gounon P, Phillips A, Nataro JP, 2002. A novel dispersin protein in enteroaggregative Escherichia coli.J Clin Invest 110 :1329–1337.

    • Search Google Scholar
    • Export Citation
  • 25

    Okeke IN, Nataro JP, 2001. Enteroaggregative Escherichia coli.Lancet Infect Dis 1 :304–313.

  • 26

    Vilaseca JM, Navarro-García F, Mendoza-Hernandez G, Nataro JP, Cravioto A, Eslava C, 2000. Pet toxin from enteroaggre-gative Escherichia coli produces cellular damage associated with fodrin disruption. Infect Immun 68 :5910–5927.

    • Search Google Scholar
    • Export Citation
  • 27

    Navarro-García F, Canizales-Roman A, Luna J, Sears C, Nataro JP, 2001. Plasmid-encoded toxin of enteroaggregative Esche-richia coli is internalized by epithelial cells. Infect Immun 69 :1053–1060.

    • Search Google Scholar
    • Export Citation
  • 28

    Fasano A, Noriega FR, Maneval DR, Chanasongcram S, Russel R, Guandalini S, Levine MM, 1995. Shigella enterotoxin 1: an enterotoxin of Shigella flexneri 2ª active in rabbit small intestine in vivo. J Clin Invest 95 :2853–2861.

    • Search Google Scholar
    • Export Citation
  • 29

    Vargas M, Gascon J, Gallardo F, Jimenez de Anta MT, Vila J, 1998. Prevalence of diarrheagenic Escherichia coli strains detected by PCR in patients with traveler’s diarrhea. Clin Micro-biol Infect 4 :682–688.

    • Search Google Scholar
    • Export Citation
  • 30

    Vila J, Vargas M, Henderson IR, Gascon J, Nataro JP, 2000. Enteroaggregative Escherichia coli virulence factors in traveler’s diarrhea strains. J Infect Dis 182 :1780–1783.

    • Search Google Scholar
    • Export Citation
  • 31

    Ruiz J, Vargas M, Nataro JP, Vila J, Gascón J, 2005. Validación de la técnica de PCR para la detección de Escherichia coli enteroagregativa causante de diarrea del viajero. Enfer Infec Microbiol Clin 23 :479–481.

    • Search Google Scholar
    • Export Citation
  • 32

    Soto SM, Smithson A, Martinez JA, Horcajada JP, Mensa J, Vila J, 2007. Biofilm formation in uropathogenic Escherichia coli strains: relationship with prostatitis, urovirulence factors and antimicrobial resistance. J Urol 177 :365–368.

    • Search Google Scholar
    • Export Citation
  • 33

    Adachi JA, Jiang ZD, Mathewson JJ, Verenkar MP, Thompson S, Martinez-Sandoval F, Steffen R, Ericsson CD, DuPont HL, 2001. Enteroaggregative Escherichia coli is a major etiologic agent in traveler’s diarrhea in 3 regions of the world. Clin Infect Dis 32 :1706–1709.

    • Search Google Scholar
    • Export Citation
  • 34

    Baqui AH, Sack RB, Sack RE, Haider K, Hossain A, Alim AR, Yunus M, Chowdhury HR, Siddique AK, 1992. Enteropatho-gens associated with acute and persistent diarrhea in Bang-ladeshi children <5 years of age. J Infect Dis 166 :792–796.

    • Search Google Scholar
    • Export Citation
  • 35

    Gascon J, Vargas M, Quinto L, Corachan M, Jimenez de Anta MT, Vila J, 1998. Enteroaggregative Escherichia coli strains as a cause of traveler’s diarrhea: a case-control study. J Infect Dis 177 :1409–1412.

    • Search Google Scholar
    • Export Citation
  • 36

    Nataro JP, Koper JB, Robins-Browne R, Prado V, Vial P, Levine MM, 1987. Patterns of adherence of diarrheagenic Escherichia coli to Hep-2 cells. Pediatr Infect Dis J 6 :829–831.

    • Search Google Scholar
    • Export Citation
  • 37

    Piva IC, Pereira AL, Ferrz LR, Silva RSN, Vieira AC, Blanco JE, Blanco M, Blanco J, Giuglano LG, 2003. Virulence markers of enteroaggregative Escherichia coli isolated from children and adults with diarrhea in Brasilia, Brazil. J Clin Microbiol 41 :1827–1832.

    • Search Google Scholar
    • Export Citation
  • 38

    Schultsz CJ, van den Ende J, Cobelens F, Vervoort T, van Gompel A, Wetsteyn JC, Dankert J, 2000. Diarrheagenic Escherichia coli and acute and persistent diarrhea in returned travelers. J Clin Microbiol 38 :3550–3554.

    • Search Google Scholar
    • Export Citation
  • 39

    Snyder JD, Merson MH, 1992. The magnitude of the global problem of diarrhoeal disease: a ten-year update. Bull World Health Organ 70 :705–714.

    • Search Google Scholar
    • Export Citation
  • 40

    Wanke CA, Gerrior J, Blais V, Mayer H, Acheson D, 1998. Successful treatment of diarrheal disease associated with entero-aggregative Escherichia coli in adults infected with immuno-deficiency virus. J Infect Dis 178 :1369–1372.

    • Search Google Scholar
    • Export Citation
  • 41

    Jiang ZD, Greenberg DD, Nataro JP, Steffen R, DuPont HL, 2002. Rate of occurrence and pathogenic effect of enteroag-gregative Escherichia coli virulence factors in international travelers. J Clin Microbiol 40 :4185–4190.

    • Search Google Scholar
    • Export Citation
  • 42

    Kahali S, Sarkar B, Rajendran K, Khanam J, Yamasaki S, Nandy RK, Bhattacharya SK, Ramamurthy T, 2004. Virulence characteristics and molecular epidemiology of enteroaggregative Escherichia coli isolates from hospitalized diarrheal patients in Kolkata, India. J Clin Microbiol 42 :4111–4120.

    • Search Google Scholar
    • Export Citation
  • 43

    Zamboni A, Fabbricotti SH, Fagundes-Neto U, Scaletsky IC, 2004. Enteroaggregative Escherichia coli virulence factors are found to be associated with infantile diarrhea in Brazil.J Clin Microbiol 42 :1058–1063.

    • Search Google Scholar
    • Export Citation
  • 44

    Czeczulin JR, Whittam TS, Henderson IR, Navarro-Garcia F, Nataro JP, 1999. Phylogenetic analysis of enteroaggregative and diffusely adherent Escherichia coli.Infect Immun 67 :2692–2699.

    • Search Google Scholar
    • Export Citation
  • 45

    Mohamed JA, Huang DB, Jiang ZD, DuPont HL, Nataro JP, Belkind-Gerson J, Okhuysen PC, 2007. Association of putative enteroaggregative Escherichia coli virulence genes and biofilm production in isolates from travelers to developing countries. J Clin Microbiol 45 :121–126.

    • Search Google Scholar
    • Export Citation
  • 46

    Guyer DM, Henderson IR, Nataro JP, Mobley HL, 2000. Identification of sat, an autotransporter toxin produced by uro-pathogenic Escherichia coli.Mol Microbiol 38 :53–66.

    • Search Google Scholar
    • Export Citation
  • 47

    Henderson IR, Meehan M, Owen P, 1997. Antigen 43, a phase-variable bipartite outer membrane protein, determines colony morphology and autoaggregation in Escherichia coli K–12. FEMS Microbiol Lett 149 :115–120.

    • Search Google Scholar
    • Export Citation
  • 48

    Ruiz J, Navia MM, Vila J, Gascon J, 2002. Prevalence of the Sat gene among clinical isolates of Shigella spp. causing traveler’s diarrhea: geographical and specific differences. J Clin Micro-biol 40 :1565–1566.

    • Search Google Scholar
    • Export Citation
  • 49

    Johnson JR, Stell AL, 2000. Extended virulence genotypes of Escherichia coli strains from patients with urosepsis in relation to phylogeny and host compromise. J Infect Dis 181 :261–272.

    • Search Google Scholar
    • Export Citation
  • 50

    Vila J, Simon K, Ruiz J, Horcajada JP, Velasco M, Barranco M, Moreno A, Mensa J, 2002. Are quinolone-resistant uropatho-genic Escherichia coli less virulent? J Infect Dis 186 :1039–1042.

    • Search Google Scholar
    • Export Citation

 

 

 

 

 

Prevalence of Different Virulence Factors and Biofilm Production in Enteroaggregative Escherichia coli Isolates Causing Diarrhea in Children in Ifakara (Tanzania)

View More View Less
  • 1 Servei de Microbiologia, Centre de Diagnostic Biomèdic; Servei de Malalties Infeccioses and Centre de Salut Intenacional and IDIBAPS, Hospital Clínic, Facultat de Medicina, Universitat de Barcelona, Villarroel 170 08036-Barcelona, Spain; Ifakara Health Research and Development Centre, National Institute for Medical Research, Ifakara, Tanzania

This study investigated the prevalence of 19 virulence factors and biofilm production in 86 EAEC isolates causing diarrhea in children less than 5 years of age from Ifakara, Tanzania. Virulence factors were detected by PCR, whereas biofilm production was determined using a microtiter plate assay. No virulence factor, with the exception of the aat gene used to identify EAEC, was detected in 11/86 isolates (12.8%). The most frequently detected virulence factor was the aggR gene in 53 (61.6%) EAEC, followed by antigen 43 in 33.7%, dispersin in 26.7%, yersiniabactin in 22.1%; autrotransporter Sat in 20.9%; Shigella enterotoxin-1 in 16.3%, and heat-stable toxin-1 in 15.1%. Biofilm was produced in 66/86 (76%) isolates. AggR was the most prevalent virulence factor in the biofilm-forming group (65% versus 38%, P = 0.032). These results again show the high heterogeneity of virulence factors among EAEC isolates causing diarrhea in children, and that biofilm may be an important virulence factor, strongly associated with the presence of AggR.

INTRODUCTION

Diarrheal diseases are a leading cause of morbidity and mortality among children in low-income countries. Although oral rehydration has been shown to reduce early childhood mortality, diarrhea-specific mortality in children less than 5 years of age in Africa has been estimated at about 10.6 per 1,000.1

Many reports have demonstrated the association of entero-aggregative E.coli (EAEC) with diarrhea in children in developing countries.27 However, in some studies EAEC was not related to diarrhea.8,9 Overall, even in studies where EAEC isolates were statistically associated with diarrhea, the percentage of controls with this enteropathogen was high. Some early epidemiologic reports implicated EAEC as a cause of persistent (≥ 14 days) diarrhea and it is currently recognized as an important cause of both acute and persistent diarrhea.10,11 EAEC have been reported to produce bio-film12,13 and this can explain, at least in part, the high number of EAEC isolates in controls and the persistence of EAEC. Antigen 43 is a surface protein involved in bacterial aggregation and it has been found that expression of Ag43 dramatically enhances biofilm formation in bacteria.14,15

In addition to biofilm formation, a large number of putative virulence factors have been identified in EAEC strains. However, no single factor appears to be consistently present in all pathogenic strains. Most of the genes encoding these potential virulence factors are located in the 60–65 MDa pAA plas-mid, from which a probe and also primers used for detection of the EAEC by PCR have been designed.16,17 In this plas-mid, genes encoding adherence fimbria (AAFI and AAFII) as well as the heat-stable enterotoxin-1 (EAST-1, encoded in the astA gene) have been detected.1821 Nataro and colleagues22 described a transcriptional factor encoded by the aggR gene, which controls not only the expression of the adherence factors (AAFI and AAFII), but also chromosomal genes.23

The pAA plasmid also carries the aap gene, previously called aspU, which encodes the dispersin. Dispersin is a secreted low-molecular weight protein that promotes dispersal of EAEC on the intestinal mucosa to establish new foci of infection and facilitates efficient colonization.24

Other factors, such as a pAA plasmid-encoded toxin (Pet), which is a serine protease autotransporter protein able to cleave spectrin, a component of the membrane cytoskele-ton,2527 or ShET-1, an enterotoxin first identified in Shigella flexneri 2a,28 have been referred as virulence factors present in EAEC isolates.

The aim of this study was to investigate the prevalence of 19 virulence factors, some of which have been previously investigated in EAEC and some not, and biofilm production in EAEC isolates causing diarrhea in children less than 5 years of age with acute diarrhea from Ifakara, Tanzania.

MATERIALS AND METHODS

Subjects and strains.

The study was carried out in the town of Ifakara, in the Kilombero district, in South-western Tan-zania. This town has 40,000 inhabitants, most of whom are small-scale farmers. All children less than 5 years of age who were admitted to St. Francis Hospital in Ifakara, Tanzania because of diarrhea were recruited during the period from July to September (dry season). Informed consent was obtained from parents and/or close relatives. The study was reviewed and approved by the Ethical Committees of the Hospital Clinic (Barcelona, Spain) in a shared authorization with the St. Francis Hospital.

E.coli strains were isolated from stool samples of 348 children using conventional methods.1,8 Diarrhea was defined as three or more watery or loose stools over a 24-hour period prior to admission to the hospital.

Characterization of EAEC isolates and detection of virulence genes.

The EAEC strains were identified by PCR amplification of the aat gene (previously referred to as CVD432 or AA probe), following primers and conditions previously described,29,30 and have been validated by comparison with the adherence assay.31 The presence of the aggR gene was detected by PCR using primers designed in this study. The primers were evaluated using the E. coli 042 strain as a control. All the primers used are shown in Table 1. In both cases, the PCR conditions were identical to those described below for the amplification of different virulence factors.

The genes encoding the following virulence factors were detected by PCR: heat-stable toxin (ST), heat-labile toxin (LT), enteroaggregative heat-stable enterotoxin-1 (EAST), Shigella enterotoxins 1 and 2 (ShET1 and ShET2), adherence factors AAF/I and AAF/II, the plasmid encoded toxin (Pet), plasmid-encoded cryptic secreted protein (Aap), autotrans-ported toxin Sat, cytolethal-distending toxin (Cdt), yersinio-bactin (Fyu), Antigen 43 (Ag43), enteropathogen adherence factor (EAF), intimin (eae), the bundle-forming pili (bfp), and the verotoxins VT1 and VT2.

The agn43 gene, encoding antigen 43 was amplified using the primers Ag43-F 5′-ACGCACAACCATCAATAAAA-3′ and Ag43-R 5′-CCGCCTCCGATACTGAATGC-3′. Briefly, one colony of each isolate was suspended in 25 μL of sterile water and boiled for 10 minutes. Twenty-five μL of reaction mixture containing 20 mM Tris-HCL (pH 8.8), 100 mM KCL, 3.0 Mm MgCl2, 0.1% gelatin, 400 μM dNTPs, and 1 μM of each primer were added, together with 2.5 units of Taq polymerase. The reaction mixture was overlaid with a drop of mineral oil and subjected to the following program: 30 cycles at 94°C for 1 minute, 55°C for 1 minute and 72°C for 1 minute, except for amplification of cdtB, which was amplified with an annealing temperature of 59°C. The PCR products were detected by electrophoresis on 2% agarose gel and stained with ethidium bromide (0.5 mg/L). At least one amplified product of each different PCR performed was sequenced, to verify the correctness of the amplified products. Controls carrying the specific genes were included in each PCR.

Quantitative biofilm assay.

Biofilm assay was carried out using minimal glucose medium (M63). The strains were grown overnight in LB medium at 37°C without shaking. An aliquot (1.25 μL) of the overnight culture was subcultured in 125 μL of M63 medium with 1% of LB in each well of a polystyrene microtiter plate and incubated at 30°C overnight without shaking. A 1.25 μL of each culture was subcultured again in 125 μL of M63 medium in a new polystyrene micro-titer plate, and incubated as cited above. After 24 hours, the culture was removed turning the plate upside down and the biofilm was stained with 175 μL of violet crystal for one minute, washed with 1 × PBS, and air dried for about 1 hour. The colorant was solubilized in dimethyl sulfoxide (DMSO) to measure the absorbance at λ of 550 nm in an automatic spectrophotometer (Anthos Reader 2001, Innogenetics, Spain). The result was considered positive when the absorbance was > 4-fold the value obtained in the well containing bacteria-free medium. All the assays were carried out in duplicate using positive and negative controls. The controls were uropathogenic E. coli strains that had been investigated in depth regarding the production of biofilm.32

Statistical analysis.

To analyze the data the Fisher’s exact and the χ2 tests were used.

RESULTS AND DISCUSSION

Different reports have demonstrated the association of EAEC with diarrhea in children in developing countries, international travelers, or immunocompromised patients.2,4,6,9,3340 However, the pathogenic mechanisms of EAEC infections are not fully understood. There appears to be a significant heterogeneity of virulence factors among EAEC isolates.30,41 The prevalence of cases of diarrhea with a known etiology in children in Ifakara, Tanzania was 71.8% in the dry season, with the most commonly isolated entero-pathogens being diarrheagenic E.coli (37.4%).1 Eighty-six of 348 (24.7%) E.coli isolates recovered from stools of children with diarrhea were EAEC.1 In 21 (24%) of the 86 children included in this study, enteropathogens other than EAEC were found. These enteropathogens were: Rotavirus in 12 (14%) children, Shigella spp. in 5 (6%) children (4 S. flexneri, 1 Shigella dysenteriae), 2 (2.3%) children had Entamoeba hys-tolytica, and 2 children had Salmonella thyphimurium and Campylobacter jejuni each. In the present study we investigated the prevalence of different virulence factors and biofilm production in these EAEC isolates.

Of these 86 EAEC isolates, no virulence factor was detected in 11 (12.8%), similar to the figures presented by other reports analyzing the presence of different virulence factors in EAEC isolates.15,35,4143 For instance, Sarantuya and others6 found that 38.8% of the EAEC isolates causing diarrhea did not show any virulence factor. The most frequently detected virulence factor was the transcriptional factor AggR in 53 (61.6%) of the EAEC, followed by dispersin in 26.7%, yer-siniabactin in 22.1%; autrotransporter Sat in 20.9%; Shigella enterotoxin-1 in 16.3%, and heat-stable toxin-1 in 15.1%. The remaining virulence factors were detected in less than 10% of the strains, whereas Eaf, AAF/I, VT-1, and VT-2 were not detected (Table 2). The distribution of the different virulence factors was very heterogeneous with only 8 EAEC isolates having greater than or equal to 4 virulence factors (Table 3). A large number of putative virulence factors have been identified in EAEC strains, yet no single factor appears to be consistently present in all pathogenic strains.23 In our study the most frequent gene combinations were aggR/agn43 (18.6%), aggR/aap (17.4%), aggR/sat (14%), and aggR/fyuA (12.8%).

In comparison with other studies performed with EAEC isolated from children in developing countries, our data show slight differences and similarities. Piva and others35 found a high percentage (73% and 82%) of EAEC isolates, causing diarrhea in children from Brazil, positive for EAST and aero-bactin, respectively. On the other hand, AAF/I and AAF/II were found in a minority of EAEC isolates. It is important to highlight that in our study AAF/II was detected in only 3 (3.5%) of the EAEC and that these 3 isolates were also AggR+, in agreement with another study that found that many isolates carrying the aggR gene expressed neither AAFI or AAFII adhesins.44 However, these results are different to data presented in other studies showing a high number of EAEC carrying the aafI and aafII genes.5,11,42 In fact, Okeke and others5 have suggested that the presence of the AAF/II may be a marker for pathogenic EAEC strains. Our study confirms that AggR transcriptional factor, dispersin, heat-stable toxin-1, and Shigella flexneri enterotoxin-1 are among the most prevalent virulence factors found in EAEC.

The ability to form biofilms is a trait that is closely associated with bacterial persistence and virulence, and many persistent and chronic bacterial infections are now believed to be linked to the formation of biofilm.12,23,45 In our study, 66 (76%) of 86 EAEC isolates produced biofilm. When the presence of the different virulence factors was determined in the biofilm-forming EAEC group versus non-biofilm–forming EAEC group, AggR was the most prevalent virulence factor in the first EAEC group. Although ShET-1 was found to be more prevalent in the biofilm-forming group the values did not reach statistical significance. However, the prevalence of CdtB and Pet in the non-biofilm–forming EAEC group was statistically significant (Table 4). On analyzing 57 EAEC isolates, Mohamed and others45 recently found that 53% of the EAEC were biofilm producers, which is a slightly lower value than that found in our study. This difference may be explained by the origin of the EAEC isolates. In our study the EAEC isolates were from a limited geographic area in Tan-zania whereas the isolates in the Mohamed study were from different countries. On the other hand, among our EAEC isolates there may have been a predominant EAEC clone that produces a bias in our data. However, the heterogeneity in the virulence factors found and the non-genetic relationship observed when the isolates were analyzed by REP-PCR (data not shown) ruled out this possibility. In the same study, the production of biofilm was associated with the virulence genes aggR, set1A, aatA, and irp2. This finding partially agrees with our results because we also found aggR to be associated with biofilm production and a trend to significance for the set1A gene. However, we did not find any difference between the biofilm-forming EAEC group and non-biofilm–forming EAEC group regarding the presence of yersiniabactin.

The Ag43 and the Sat proteins were found in 33.7% and 20.9% of the EAEC isolates, respectively. To our knowledge, the present study is the first report on Sat and Ag 43 in EAEC isolates. Both of these are autotransporter proteins, possessing different functions. Thus, while Sat acts as a cytotoxic toxin, Ag43 acts as an autoaggregation factor.46,47 Sat toxin was first described as a virulence factor of uropathogenic E.coli,46 but further studies have established its presence among diarrheogenic pathogens such as Shigella spp.,48 and diffusely adherent E. coli.1 Similarly, Ag43 has been reported in both in uropathogenic and diarrheogenic E. coli.1,29 Ag43 is a self-recognizing molecule capable of conferring bacterial autoaggregation. Deletion in the agn43 gene generates cells with a very limited ability to form biofilm.14,15 In our study we did not find an association between the presence of Ag43 in EAEC isolates and biofilm formation.

In summary, these results have again shown the high heterogeneity of virulence factors among EAEC isolates causing diarrhea in children, and that the biofilm may be considered as an important virulence factor, strongly associated with the presence of AggR.

Table 1

Primers used to identify different Escherichia coli virulence factors

GeneVirulence factorPrimer sequenceAmplicon size (bp)Source or reference
aatAntiaggregation protein transporter5′-CTGGCGAAAGACTGTATCAT-3′ 
 5′-AATGTATAGAAATCCGCTGTT-3′62929
aapDispersin5′-CTTTTCTGGCATCTTGGGT-3′ 
 5′-GTAACAACCCCTTTGGAAGT-3′23244
aggRTranscription factor5′-AGCGGTGTCCCAGTATGTATGC-3′ 
 5′-TTATGTTCCCGCTGCTGTCGT-3′699This study
agn43Antigen 435′-ACGCACAACCATCAATAAAA-3 
 5′-CCGCCTCCGATACTGAATGC-3′600This study
aggAFimbria AAF/I5′-TTAGTCTTCTATCTAGGG-3′ 
 5′-AAATTAATTCCGGCATGG-3′45730
aafAFimbria AAF/II5′-TGCGATTGCTACTTTATTAT-3′ 
 5′-ATTGACCGTGATTGGCTTCC-3′24230
astAHeat stable toxin-15′-ATGCCATCAACACAGTATAT-3′ 
 5′-GCGAGTGACGGCTTTGTAGT-3′11030
fyuAYersiniabactin5′-TGATTAACCCCGCGACGGGAA-3′ 
 5′-CGCAGTAGGCACGATGTTGTA-3′88049
eafEnteropathogen adherence factor5′-CAGGGTAAAAGAAAGATGATAA-3′ 
 5′-TATGGGGACCATGTATTATCA-3′39729
cdtBCytolethal distending toxin5′-GAAAATAAATGGAACACACATGTCCG-3′ 
 5′-AAATCTCCTGCAATCATCCAGTTTA-3′43049
BfpBundle-forming pili5′-ACAAAGATACAACAAACAAAAA-3′ 
 5′-TTCAGCAGGAGTAAAAGCAGTC-3′26029
eaeIntimin5′-CCCGGACCCGGCACAAGCATAAGC-3′ 
 5′-TTGAAATAGTCTCGCCAGTATTCG-3′88129
ltHeat labile toxin5′-TCTCTATGTGCATACGGAGC-3′ 
 5′-CCATACTAGTTGCCGCAAT-3′32229
stHeat stable toxin5′-TTAATAGCACCCGGTACAAGCAGG-3′ 
 5 ′-CTTGACTCTTCAAAAGAGAAAATTAC-3′14729
petPlasmid encoded toxin5′-ACTGGCGGACTCATTGCTGT-3′ 
 5 ′-GCGTTTTTCCGTTCCCTATT-3′83230
set1AShigella enterotoxin-15′-TCACGCTACCATCAAAGA-3′ 
 5 ′-TATCCCCCTTTGGTGGTA-3′30930
senShigella enterotoxin-25′-ATGTGCCTGCTATTATTTAT-3′ 
 5 ′-CATAATAATAAGCGGTCAGC-3′79930
sxt-1Verotoxin-15′-GAAGAGTCGTGGGATTACG-3′ 
 5′-AGCGATGCAGCTATTAATAA-3′13029
sxt-2Verotoxin-25′-TTAACCACACCCACGGCAGT-3′ 
 5′-GCTCTGGATGCATCTCTGGGT-3′34629
satAutotransporter Sat5′-ACTGGCGGACTCATGCTGT-3′ 
 5′-AACCCTGTAAGAAGACTGAGC-3′38750
Table 2

Virulence factors in EAEC isolates

Virulence factorEAEC n° (%)
Transcription factor – AggR53 (61.6%)
Antigen 4329 (33.7%)
Dispersin23 (26.7%)
Yersiniabactin19 (22.1%)
Autotransporter Sat18 (20.9%)
Shigella enterotoxin-114 (16.3%)
Heat stable toxin-1 (EAST)13 (15.1%)
Cytolethal distending toxin11 (12.8%)
Plasmid encoded toxin (Pet)4 (4.7%)
Fimbria AAF/II3 (3.5%)
Intimin3 (3.5%)
Heat labile toxin (LT)2 (2.3%)
Shigella enterotoxin-22 (2.3%)
Bundle-forming pili1 (1.2%)
Enteropathogen adherence factor
Fimbria AAF/I
Heat stable toxin (ST)
Verotoxin-1
Verotoxin-2
Table 3

Number of virulence factors in EAEC isolates

N ° virulence factors*EAEC n°EAEC %Cumulative %
* Virulence factors other than the aat gene used to detect EAEC.
01112.812.8
12124.437.2
21719.857
31820.977.9
41517.495.3
522.398.8
722.3100
Table 4

Incidence of biofilm (BF+) formation and the presence of several virulence factors among EAEC isolates from children with diarrhea (N = 86)

Biofilm formation [n° isolates (%)]
Factor virulenceBF+ (N = 65)BF- (N = 21)P *
* χ2 test.
AggR42 (64)8 (38)0.032*
Ag4321 (32)8 (38)0.625
Aspu19 (29)4 (19)0.359
FyuA13 (20)6 (29)0.411
Sat13 (20)5 (24)0.709
ShET113 (20)1 (5)0.100
EAST10 (15)3 (14)0.903
CdtB5 (8)6 (29)0.013
Pet1 (2)3 (14)0.016

*

Address correspondence to Jordi Vila, Servei de Microbiología, Hospital Clinic, Villarroel 170, 08036 Barcelona, Spain. E-mail: jvila@ub.edu

Authors’ addresses: Eva Mendez-Arancibia, Sara Soto, and Jordi Vila, Servei de Microbiología, Hospital Clinic, Villarroel 170, 08036 Barcelona, Spain, Tel: 34-932275522, Fax: 34-932279372, E-mail: jvila@ub.edu. Martha Vargas, Servei de Malalties Infeccioses. Joaquim Ruiz and Joaquim Gascón, Centre de Salut Intenacional and IDIBAPS, Hospital Clínic, Facultat de Medicina, Universitat de Bar-celona, Villarroel 170 08036-Barcelona, Spain. Eliseus Kahigwa and David Schellenberg, Honoraty Urassa, Ifakara Health Research and Development Centre, National Institute for Medical Research, Ifakara, Tanzania.

REFERENCES

  • 1

    Vargas M, Gascón J, Casals C, Schellenberg D, Urassa H, Ka-higwa E, Ruiz J, Vila J, 2004. Etiology of diarrhea in children less than five years of age in Ifakara, Tanzania. Am J Trop Med Hyg 70 :536–539.

    • Search Google Scholar
    • Export Citation
  • 2

    Bhan MK, Raj P, Levine MM, Kaper JB, Bhandari N, Srivastava R, Kumar R, Sazawal S, 1989. Enteroaggregative Escherichia coli associated with persistent diarrhea in a cohort of rural children in India. J Infect Dis 159 :1061–1064.

    • Search Google Scholar
    • Export Citation
  • 3

    Bouzari S, Jafari A, Azizi A, Oloomi M, Nataro JP, 2001. Characterization of enteroaggregative Escherichia coli isolates from Iranian children. Am J Trop Med Hyg 65 :13–14.

    • Search Google Scholar
    • Export Citation
  • 4

    Fang GD, Lima AA, Martins CV, Nataro JP, Guerrant RL, 1995. Etiology and epidemiology of persistent diarrhea in northeastern Brazil: a hospital-based, prospective, case-control study. J Pediatr Gastroenterol Nutr 21 :137–144.

    • Search Google Scholar
    • Export Citation
  • 5

    Okeke IN, Lamikanra A, Czeczulin J, Dubovsky F, Karper J, Nataro JP, 2000. Characterization of Escherichia coli strains from cases of childhood diarrhea in provincial south-western Nigeria. J Infect Dis 181 :252–260.

    • Search Google Scholar
    • Export Citation
  • 6

    Sarantuya J, Nishi J, Wakimoto N, Erdene S, Nataro JP, Sheikh J, Iwashita M, Manago K, Tokuda K, Yoshinga M, Miyata K, Kawano Y, 2004. Typical Enteroaggregative Escherichia coli is the most prevalent pathotype among E.coli strains causing diarrhea in Mongolian children. J Clin Microbiol 42 :133–139.

    • Search Google Scholar
    • Export Citation
  • 7

    Scaletsky ICA, Fabbricotti SH, Silva SOC, Morais MB, Fagun-des-Neto U, 2002. Hep-2-adherent Escherichia coli strains associated with acute infantile diarrhea, Sao Paulo, Brazil. Emerg Infect Dis 8 :855–858.

    • Search Google Scholar
    • Export Citation
  • 8

    Gascón J, Vargas M, Schellenberg D, Urassa H, Casals C, Ka-higwa E, Aponte JJ, Mshinda H, Vila J, 2000. Diarrhea in children under 5 years of age from Ifakara, Tanzania: a case-control study. J Clin Microbiol 38 :4459–4462.

    • Search Google Scholar
    • Export Citation
  • 9

    Gomes TAT, Blake PA, Trabulsi LR, 1989. Prevalence of Esche-richia coli strains with localized, diffuse and aggregative adherence to HeLa cells in infants with diarrhea and matched controls. J Clin Microbiol 27 :266–269.

    • Search Google Scholar
    • Export Citation
  • 10

    Huang DB, Okhuysen PC, Zhi-Dong Jiang MD, DuPont HL, 2004. Enteroaggregative Escherichia coli: an emerging enteric pathogen. Am J Gastroenterol 99 :383–389.

    • Search Google Scholar
    • Export Citation
  • 11

    Nataro JP, Steiner T, Guerrant RL, 1998. Enteroaggregative Escherichia coli.Emerg Infect Dis 4 :251–261.

  • 12

    Sheikh J, Hicks S, Dall’Agnol M, Phillips AD, Nataro JP, 2001. Roles for Fis and YafK in biofilm formation by enteroaggre-gative Escherichia coli.Mol Microbiol 41 :983–997.

    • Search Google Scholar
    • Export Citation
  • 13

    Wakimoto N, Nichi J, Sheik J, Nataro JP, Sarantuya J, Iwashita M, Manago K, Tozuda K, Yoshinaga M, Kawano Y, 2004. Quantitative biofilm assay using a microtiter plate to screen for enteroaggregative Escherichia coli.Am J Trop Med Hyg 71 :687–690.

    • Search Google Scholar
    • Export Citation
  • 14

    Kjaergarrd K, Schembri MA, Ramos C, Molin S, Klemm P, 2000. Antigen 43 facilitates formation of multispecies biofilms. Environ Microbiol 2 :695–702.

    • Search Google Scholar
    • Export Citation
  • 15

    Schembri MA, Kjaergarrd K, Klemm P, 2003. Global gene expression in Escherichia coli biofilms. Mol Micorbiol 48 :253–267.

  • 16

    Baudry B, Savarino SJ, Vial P, Koper JB, Levine MM, 1990. A sensitive and specific DNA probe to identify enteroaggrega-tive Escherichia coli, a recently discovered diarrheal pathogen. J Infect Dis 161 :1249–1251.

    • Search Google Scholar
    • Export Citation
  • 17

    Schmidt H, Knop C, Franke S, Aleksic S, Heesemenn J, Karch H, 1995. Development of PCR for screening of enteroaggregative Escherichia coli.J Clin Microbiol 33 :701–705.

    • Search Google Scholar
    • Export Citation
  • 18

    Czeczulin JR, Balepur S, Hicks S, Phillips A, Hall R, Kothary MH, Navarro-García F, Nataro JP, 1997. Aggregative adherence fimbria II, a second fimbria antigen mediating adherence in enteroaggregative Escherichia coli.Infect Immun 65 :4135–4145.

    • Search Google Scholar
    • Export Citation
  • 19

    Nataro JP, Deng Y, Maneval DR, German AL, Martin WC, Le-vine MM, 1992. Aggregative adherence fimbria I of enteroag-gregative Escherichia coli mediate adherence to Hep-2 cells and hemagglutination of human erythrocytes. Infect Immun 60 :2297–2304.

    • Search Google Scholar
    • Export Citation
  • 20

    Savarino SJ, Fasano A, Robertson DC, Levine MM, 1991. En-teroaggregative Escherichia coli elaborate a heath-stable en-terotoxin demonstrable in an in vitro rabbit intestinal model. J Clin Invest 87 :1450–1455.

    • Search Google Scholar
    • Export Citation
  • 21

    Vila J, Gene A, Vargas M, Gascon J, Latorre C, Jiménez de Anta MT, 1998. A case-control study of diarrhea in children caused by Escherichia coli producing heat-stable enterotoxin (EAST-1). J Med Microbiol 47 :889–891.

    • Search Google Scholar
    • Export Citation
  • 22

    Nataro JP, Yikang D, Yingkang D, Walker K, 1994. AggR, a transcriptional activator of aggregative adherence fimbrial I expression in enteroaggregative Escherichia coli.J Bacteriol 176 :4691–4699.

    • Search Google Scholar
    • Export Citation
  • 23

    Nataro JP, 2005. Enteroaggragtive Escherichia coli pathogenesis. Curr Opin Gastroenterol 21 :4–8.

  • 24

    Sheikh J, Czeczulin JR, Harrington S, Hicks S, Henderson IR, Le Bouguénec C, Gounon P, Phillips A, Nataro JP, 2002. A novel dispersin protein in enteroaggregative Escherichia coli.J Clin Invest 110 :1329–1337.

    • Search Google Scholar
    • Export Citation
  • 25

    Okeke IN, Nataro JP, 2001. Enteroaggregative Escherichia coli.Lancet Infect Dis 1 :304–313.

  • 26

    Vilaseca JM, Navarro-García F, Mendoza-Hernandez G, Nataro JP, Cravioto A, Eslava C, 2000. Pet toxin from enteroaggre-gative Escherichia coli produces cellular damage associated with fodrin disruption. Infect Immun 68 :5910–5927.

    • Search Google Scholar
    • Export Citation
  • 27

    Navarro-García F, Canizales-Roman A, Luna J, Sears C, Nataro JP, 2001. Plasmid-encoded toxin of enteroaggregative Esche-richia coli is internalized by epithelial cells. Infect Immun 69 :1053–1060.

    • Search Google Scholar
    • Export Citation
  • 28

    Fasano A, Noriega FR, Maneval DR, Chanasongcram S, Russel R, Guandalini S, Levine MM, 1995. Shigella enterotoxin 1: an enterotoxin of Shigella flexneri 2ª active in rabbit small intestine in vivo. J Clin Invest 95 :2853–2861.

    • Search Google Scholar
    • Export Citation
  • 29

    Vargas M, Gascon J, Gallardo F, Jimenez de Anta MT, Vila J, 1998. Prevalence of diarrheagenic Escherichia coli strains detected by PCR in patients with traveler’s diarrhea. Clin Micro-biol Infect 4 :682–688.

    • Search Google Scholar
    • Export Citation
  • 30

    Vila J, Vargas M, Henderson IR, Gascon J, Nataro JP, 2000. Enteroaggregative Escherichia coli virulence factors in traveler’s diarrhea strains. J Infect Dis 182 :1780–1783.

    • Search Google Scholar
    • Export Citation
  • 31

    Ruiz J, Vargas M, Nataro JP, Vila J, Gascón J, 2005. Validación de la técnica de PCR para la detección de Escherichia coli enteroagregativa causante de diarrea del viajero. Enfer Infec Microbiol Clin 23 :479–481.

    • Search Google Scholar
    • Export Citation
  • 32

    Soto SM, Smithson A, Martinez JA, Horcajada JP, Mensa J, Vila J, 2007. Biofilm formation in uropathogenic Escherichia coli strains: relationship with prostatitis, urovirulence factors and antimicrobial resistance. J Urol 177 :365–368.

    • Search Google Scholar
    • Export Citation
  • 33

    Adachi JA, Jiang ZD, Mathewson JJ, Verenkar MP, Thompson S, Martinez-Sandoval F, Steffen R, Ericsson CD, DuPont HL, 2001. Enteroaggregative Escherichia coli is a major etiologic agent in traveler’s diarrhea in 3 regions of the world. Clin Infect Dis 32 :1706–1709.

    • Search Google Scholar
    • Export Citation
  • 34

    Baqui AH, Sack RB, Sack RE, Haider K, Hossain A, Alim AR, Yunus M, Chowdhury HR, Siddique AK, 1992. Enteropatho-gens associated with acute and persistent diarrhea in Bang-ladeshi children <5 years of age. J Infect Dis 166 :792–796.

    • Search Google Scholar
    • Export Citation
  • 35

    Gascon J, Vargas M, Quinto L, Corachan M, Jimenez de Anta MT, Vila J, 1998. Enteroaggregative Escherichia coli strains as a cause of traveler’s diarrhea: a case-control study. J Infect Dis 177 :1409–1412.

    • Search Google Scholar
    • Export Citation
  • 36

    Nataro JP, Koper JB, Robins-Browne R, Prado V, Vial P, Levine MM, 1987. Patterns of adherence of diarrheagenic Escherichia coli to Hep-2 cells. Pediatr Infect Dis J 6 :829–831.

    • Search Google Scholar
    • Export Citation
  • 37

    Piva IC, Pereira AL, Ferrz LR, Silva RSN, Vieira AC, Blanco JE, Blanco M, Blanco J, Giuglano LG, 2003. Virulence markers of enteroaggregative Escherichia coli isolated from children and adults with diarrhea in Brasilia, Brazil. J Clin Microbiol 41 :1827–1832.

    • Search Google Scholar
    • Export Citation
  • 38

    Schultsz CJ, van den Ende J, Cobelens F, Vervoort T, van Gompel A, Wetsteyn JC, Dankert J, 2000. Diarrheagenic Escherichia coli and acute and persistent diarrhea in returned travelers. J Clin Microbiol 38 :3550–3554.

    • Search Google Scholar
    • Export Citation
  • 39

    Snyder JD, Merson MH, 1992. The magnitude of the global problem of diarrhoeal disease: a ten-year update. Bull World Health Organ 70 :705–714.

    • Search Google Scholar
    • Export Citation
  • 40

    Wanke CA, Gerrior J, Blais V, Mayer H, Acheson D, 1998. Successful treatment of diarrheal disease associated with entero-aggregative Escherichia coli in adults infected with immuno-deficiency virus. J Infect Dis 178 :1369–1372.

    • Search Google Scholar
    • Export Citation
  • 41

    Jiang ZD, Greenberg DD, Nataro JP, Steffen R, DuPont HL, 2002. Rate of occurrence and pathogenic effect of enteroag-gregative Escherichia coli virulence factors in international travelers. J Clin Microbiol 40 :4185–4190.

    • Search Google Scholar
    • Export Citation
  • 42

    Kahali S, Sarkar B, Rajendran K, Khanam J, Yamasaki S, Nandy RK, Bhattacharya SK, Ramamurthy T, 2004. Virulence characteristics and molecular epidemiology of enteroaggregative Escherichia coli isolates from hospitalized diarrheal patients in Kolkata, India. J Clin Microbiol 42 :4111–4120.

    • Search Google Scholar
    • Export Citation
  • 43

    Zamboni A, Fabbricotti SH, Fagundes-Neto U, Scaletsky IC, 2004. Enteroaggregative Escherichia coli virulence factors are found to be associated with infantile diarrhea in Brazil.J Clin Microbiol 42 :1058–1063.

    • Search Google Scholar
    • Export Citation
  • 44

    Czeczulin JR, Whittam TS, Henderson IR, Navarro-Garcia F, Nataro JP, 1999. Phylogenetic analysis of enteroaggregative and diffusely adherent Escherichia coli.Infect Immun 67 :2692–2699.

    • Search Google Scholar
    • Export Citation
  • 45

    Mohamed JA, Huang DB, Jiang ZD, DuPont HL, Nataro JP, Belkind-Gerson J, Okhuysen PC, 2007. Association of putative enteroaggregative Escherichia coli virulence genes and biofilm production in isolates from travelers to developing countries. J Clin Microbiol 45 :121–126.

    • Search Google Scholar
    • Export Citation
  • 46

    Guyer DM, Henderson IR, Nataro JP, Mobley HL, 2000. Identification of sat, an autotransporter toxin produced by uro-pathogenic Escherichia coli.Mol Microbiol 38 :53–66.

    • Search Google Scholar
    • Export Citation
  • 47

    Henderson IR, Meehan M, Owen P, 1997. Antigen 43, a phase-variable bipartite outer membrane protein, determines colony morphology and autoaggregation in Escherichia coli K–12. FEMS Microbiol Lett 149 :115–120.

    • Search Google Scholar
    • Export Citation
  • 48

    Ruiz J, Navia MM, Vila J, Gascon J, 2002. Prevalence of the Sat gene among clinical isolates of Shigella spp. causing traveler’s diarrhea: geographical and specific differences. J Clin Micro-biol 40 :1565–1566.

    • Search Google Scholar
    • Export Citation
  • 49

    Johnson JR, Stell AL, 2000. Extended virulence genotypes of Escherichia coli strains from patients with urosepsis in relation to phylogeny and host compromise. J Infect Dis 181 :261–272.

    • Search Google Scholar
    • Export Citation
  • 50

    Vila J, Simon K, Ruiz J, Horcajada JP, Velasco M, Barranco M, Moreno A, Mensa J, 2002. Are quinolone-resistant uropatho-genic Escherichia coli less virulent? J Infect Dis 186 :1039–1042.

    • Search Google Scholar
    • Export Citation
Save