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Characterization of the extended-spectrum b-lactamases and determination of the virulence factors of uropathogenic Escherichia coli strains isolated from children

Charakterisierung der extended spectrum b-Lactamasen (ESBL) und Bestimmung der von Kindern isolierten uropathogenen Virulenzfaktoren von Escherichia coli (E. coli) Stämmen

Wiener klinische Wochenschrift volume 124, pages 504–515 (2012)Cite this article

Summary

Background and aim

The aim of the study was to characterize ESBL-producing uropathogenic Escherichia coli (UPEC) strains isolated in children. That included the investigation of virulence factors and the analysis of the types of β-lactamases at the molecular genetic level.

Material and methods

During the 2-year study period, 77 ESBL-producing E. coli strains were recovered from urine samples of febrile children with significant bacteriuria hospitalized at one Croatian hospital. Susceptibility of isolates to bactericidal serum activity was tested by Shiller and Hatch method, while adhesin expression was determined by agglutination methods. Characterization of ESBLs was performed by PCR with specific primers for ESBLs and by sequencing of bla ESBL genes. Genotyping of the E. coli isolates was performed by pulsed-field gel electrophoresis (PFGE).

Results

Twenty-seven (35.1 %) and 50 (64.9 %) ESBL-producing UPEC strains were isolated in neonates and infants, respectively. Of 70 strains investigated for the presence of virulence factors, adhesins were detected in 48.6 % strains (8.6 % in the neonate and 40 % in the infants group) giving a statistically significant difference in adhesin expression between the two groups (p < 0.01). Hemolysin was produced by 84.3 %, whereas 70 % of strains were serum-resistant. The bla TEM gene was detected in 22 (28 %) and bla SHV gene in 57 strains (74 %), whereas bla CTX-M gene was detected in only two isolates (2.5%). In ten isolates, bla TEM and bla SHV were simultaneously detected. Sequencing of bla SHV genes revealed that SHV-5 β-lactamase was by far the most prevalent and was found in 51 strains (66 %). The strains were clonally related as demonstrated by PFGE and assigned into ten clusters.

Conclusions

Infection control measures should be employed and the consumption of expanded-spectrum cephalosporins in the hospital should be restricted.

Zusammenfassung

Hintergrund und Ziel der Studie

Ziel der Studie war es, von Kindern isolierte ESBL produzierende uropathogene E. coli (UPEC) Stämme zu charakterisieren und die Typen der β-Lactamasen auf molekular-genetischem Niveau zu analysieren.

Material und Methoden

Während der 2 jährigen Studienperiode wurden 77 ESBL produzierende E coli Stämme aus Harnproben von an einem kroatischen Spital stationär aufgenommenen Kindern isoliert. Mittels der Shiller – Hatch Methode wurde die Empfindlichkeit der Isolate auf Aktivität von bakterizidem Serum getestet. Die Adhesin Expression wurde mit Agglutinationsmethoden untersucht. Die Charakterisierung der ESBL erfolgte mittels PCR mit spezifischen Primers für ESBL und durch Sequenzierung der bla ESBL Gene. Die Gentypisierung der E. coli Isolate wurde mittels puls-feld-gel- Elektrophorese (PGFE) durchgeführt.

Ergebnisse

ESBL produzierende UPEC Kulturen wurden aus Harnproben von 27 (35,1 %) Neugeborenen und von 50 (64,9 %) älteren Kindern isoliert. Von den 70 auf das Vorhandensein von Virulenzfaktoren untersuchten Kulturen wurden bei 48,6 % Adhesine entdeckt (8,6 % bei den Neugeborenen und 40 % bei den Älteren). Die Adhesin Expression beider Gruppen unterschied sich demnach signifikant (p < 0,01). Hämolysin wurde von 84,3 % produziert. Siebzig Prozent der Kulturen waren Serumresistent. Das bla TEM Gen wurde in 22 (28 %) und das bla SHV Gen in 57 (74 %) Kulturen nachgewiesen. Das bla CTX-M Gen konnte aber nur in zwei Isolaten 2,5 % entdeckt werden. Die Sequenzierung der bla SHV Gene zeigte, dass die SHV – 5 β – Lactamase bei weitem am häufigsten war. Sie wurde bei 51 Kulturen (66 %) gefunden. Durch PFGE konnte gezeigt werden, dass die Kulturen Klon-mäßig mit einander verwandt waren.

Schlussfolgerung

Maßnahmen zur Vermeidung einer Infektion sollten ergriffen werden. Der Einsatz von Cephalosporinen mit erweitertem Spektrum sollte im Spitalsbereich eingeschränkt werden.

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References

  1. Bradford PA. Extended-spectrum b-lactamases in the 21st century: characterization, epidemiology, and detection of this important resistance threat. Clin Microbiol Rev. 2001;14:933–51.

    PubMed  Article  CAS  Google Scholar 

  2. Jacoby GA, Munoz-Price LS. The new b-lactamases. N Engl J Med. 2005;352:380–92.

    PubMed  Article  CAS  Google Scholar 

  3. Bonnet R. Growing group of extended-spectrum b-lactamases: the CTX-M enzymes. Antimicrob Agents Chemother. 2004;48:1–14.

    PubMed  Article  CAS  Google Scholar 

  4. Pitout JD, Nordmann P, Laupland KB, Poirel L. Emergence of Enterobacteriaceae producing extended-spectrum b-lactamases in the community. J Antimicrob Chemother. 2005;56:52–9.

    PubMed  Article  CAS  Google Scholar 

  5. Pallecchi L, Bartoloni A, Fiorelli C, et al. Rapid dissemination and diversity of CTX-M extended-spectrum b-lactamase genes in commensal Escherichia coli isolates from healthy children from low resource settings in Latina America. Antimicrob Agents Chemother. 2007;51:2720–5.

    PubMed  Article  CAS  Google Scholar 

  6. Lartigue MF, Zinsius C, Wenger A, Bille J, Poirel L, Nordman P. Extended-spectrum b-lactamases of the CTX-M type now in Switzerland. Antimicrob Agents Chemother. 2007;51:2855–60.

    PubMed  Article  CAS  Google Scholar 

  7. Edelstein M, Pimkin M, Palagin I, Edelstein I, Stratchounski L. Prevalence and molecular epidemiology of CTX-M b-lactamase-producing Escherichia coli and Klebsiella pneumoniae in Russian Hospitals. Antimicrob Agents Chemother. 2003;47:3724–32.

    PubMed  Article  CAS  Google Scholar 

  8. Pournaras S, Ikonomidis A, Kristo I, Tsakris A, Maniatis A. CTX-M enzymes are the most common extended-spectrum b-lactamases among Escherichia coli in a tertiary Greek hospital. J Antimicrob Chemother. 2004;54:574–5.

    PubMed  Article  CAS  Google Scholar 

  9. Canton R, Oliver A, Coque TM. Epidemiology of extended-spectrum b-lactamase-producing Enterobacter isolates in a Spanish hospital during a 12 year period. J Clin Microbiol. 2002; 40:1237–43.

    PubMed  Article  CAS  Google Scholar 

  10. Yamasaki K, Komatsu M, Yamashita T. Production of CTX-M-3 extended-spectrum b-lactamase and IMP-1 metallo-b-lactamase by five Gram-negative bacilli: survey of clinical isolates from seven laboratories collected in 1998 and 2000, in the Kinki region of Japan. J Antimicrob Chemother. 2003;51:631–8.

    PubMed  Article  CAS  Google Scholar 

  11. Yu WL, Winokur P, Von Stein DL. First description of CTX-M-β-lactamases (CTX-M-14 and CTX-M-3) in Taiwan. Antimicrob Agents Chemother. 2002; 46:1098–1100.

    PubMed  Article  CAS  Google Scholar 

  12. Chanawong A, M’Zalli FH, Heritage J. Three cefotaximases, CTX-M-9, CTX-M-13, and CTX-M-14 among Enterobacteriaceae in the People’s Republic of China. Antimicrob Agents Chemother. 2002;46:630–7.

    PubMed  Article  CAS  Google Scholar 

  13. Quinteros M, Radice M, Gardella N. Extended-spectrum b-lactamases in Buenos Aires, public hospitals. Antimicrob Agents Chemother. 2003;47:2864–7.

    PubMed  Article  CAS  Google Scholar 

  14. Calbo E, Romani V, Xercavins M, et al. Risk factors for community-onset urinary tract infections due to Escherichia coli harbouring extended-spectrum b-lactamases. J Antimicrob Chemother. 2006;57:780–3.

    PubMed  Article  CAS  Google Scholar 

  15. Delinska-Galinska A, Kurlenda J, Kozielska E, Borkowska A, Luczak G, Plata-Nazar K. Prevalence of ESBL strains in urinary tract infections in children in 1996 and 2004. Przegl Epidemiol. 2006;60:59–64.

    PubMed  Google Scholar 

  16. Uzunović-Kamberović S, Bedenić B, Vraneš J. Predominance of SHV-5 b-lactamase in community acquired urinary tract infections. Clin Microbiol Infect. 2007;13:820–3.

    PubMed  Article  Google Scholar 

  17. Hryniewicz K, Szczypa K, Sulikowska A, Jankowski K, Betlejewska K, Hryniewicz W. Antibiotic susceptibility of bacterial strains isolated from urinary tract infections in Poland. J Antimicrob Chemother. 2007;47:773–80.

    Article  Google Scholar 

  18. Vranes J, Kruzic V, Sterk-Kuzmanovic N, Schoenwald S. Virulence characteristics of Escherichia coli strains causing asymptomatic bacteriuria. Infection 2003;31:216–20.

    PubMed  CAS  Google Scholar 

  19. Vranes J. Hemagglutination ability and adherence to the Buffalo green monkey kidney cell line of uropathogenic Escherichia coli. APMIS. 1997;105:831–7.

    PubMed  Article  CAS  Google Scholar 

  20. Schiller NL, Hatch RA. The serum sensitivity, colony morphology, serogroup specificity, and outer membrane protein of Pseudomonas aeruginosa strains isolated from several clinical sites. Diagn Microbiol Infect Dis. 1983;1:145–7.

    PubMed  Article  CAS  Google Scholar 

  21. CLSI. Performance standards for antimicrobial susceptibility testing methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically; Eighteen informational supplement. CLSI document M100-S18. Wayne: Clinical and Laboratory Standards Institute; 2008.

    Google Scholar 

  22. Rasheed JK, Anderson GJ, Yigit H, et al. Characterization of the extended-spectrum b-lactamase reference strain, Klebsiella pneumoniae K6, (ATCC 700603) which produces the novel enzyme SHV-18. Antimicrob Agents Chemother. 2000;44:2382–8.

    PubMed  Article  CAS  Google Scholar 

  23. Jarlier V, Nicolas MH, Fournier G, Philippon A. Extended broad-spectrum b-lactamases conferring transferable resistance to newer b-lactam agents in Enterobacteriaceae: hospital prevalence and susceptibility patterns. Rev Infect Dis. 1988;10:867–78.

    PubMed  Article  CAS  Google Scholar 

  24. Elwell LP, Falkow S. The characterization of R plasmids and the detection of plasmid-specified genes. In: Lorian V editor. Antibiotics in laboratory medicine. 2nd edn. Baltimore: Williams and Wilkins; 1986. p. 683–721.

    Google Scholar 

  25. Nüesch-Inderbinen MT, Hächler H, Kayser FH. Detection of genes coding for extended-spectrum SHV b-lactamases in clinical isolates by a molecular genetic method, and comparison with the E test. Eur J Clin Microbiol Infect Dis. 1996;15:398–402.

    PubMed  Article  Google Scholar 

  26. Arlet G, Brami G, Decre D, Flippo A, Gaillot O, Lagrange PH, Philippon, A. Molecular characterization by PCR restriction fragment polymorphism of TEM b-lactamases. FEMS Microbiol Lett. 1995;134:203–8.

    PubMed  CAS  Google Scholar 

  27. Woodford N, Ward ME, Kaufmann ME, et al. Community and hospital spread of Escherichia coli producing CTX-M extended-spectrum b-lactamases in the UK. J Antimicrob Chemother. 2006;54:735–43.

    Article  Google Scholar 

  28. Pagani L, Mantengoli E, Migliavacca R, et al. Multifocal detection of multidrug-resistant Pseudomonas aeruginosa producing PER-1 extended-spectrum b-lactamase in Northern Italy. J Clin Microbiol. 2004;42:2523–9.

    PubMed  Article  CAS  Google Scholar 

  29. Woodford N, Fagan, EJ, Ellington, MJ. Multiplex PCR for rapid detection of genes encoding CTX-M extended-spectrum b-lactamases. J Antimicrob Chemother. 2006; 57:154–5.

    PubMed  Article  CAS  Google Scholar 

  30. Kaufman ME. Pulsed-field gel electrophoresis. In: Woodfor N, Johnsons A, editors. Molecular bacteriology. Protocols and clinical applications. 1st edn. New York: Humana Inc. Totowa; 1998. p. 33–51.

  31. Tenover F, Arbeit R, Goering RV, et al. Interpreting chromosomal DNA restriction patterns produced by pulsed-field gel electrophoresis: criteria for bacterial strain typing. J Clin Microbio. 1995;2233–9.

  32. Empel J, Baraniak A, Literacka E, et al. Molecular survey of b-lactamases conferring resistance to newer b-lactams in Enterobacteriaceae isolates from Polish hospitals. Antimicrob Agents Chemother. 2008; 52:2449–54.

    PubMed  Article  CAS  Google Scholar 

  33. Lee J, Pai H, Kim Y, et al. Control of extended-spectrum b-lactamase producing Escherichia coli and Klebsiella pneumoniae in a children’s hospital by changing antimicrobial agent usage policy. J Antimicrob Chemother. 2007;60:629.

    PubMed  Article  CAS  Google Scholar 

  34. Tonkić M, Goić-Barišić I, Punda-Polić V. Prevalence and antimicrobial resistance of extended-spectrum b-lactamase producing Escherichia coli and Klebsiella pneumoniae in a University Hospital Split. Int Microbiol. 2005; 8:119–24.

    PubMed  Google Scholar 

  35. Spanu T, Luzzaro F, Perilli M, Amicosante G, Toniolo A, Fadda G. Italian ESBL study group Occurrence of extended-spectrum b-lactamases in members of the family Enterobacteriaceae in Italy: implication for resistance to b-lactams and other antimicrobial drugs. Antimicrob Agents Chemother. 2002;46:196–202.

    PubMed  Article  CAS  Google Scholar 

  36. Goosens H. Mystic program: summary of European data from 1997–2000. Diagn Microbiol Infect Dis. 2001;41:183–9.

    Article  Google Scholar 

  37. Yu Y, Zhou W, Chen Y, Ding Y, Ma Y. Epidemiologic and antibiotic resistance study on extended-spectrum b-lactamase producing Escherichia coli and Klebsiella pneumoniae in Zhejinang Province. Chin Med J. 2002;115:1479–82.

    PubMed  CAS  Google Scholar 

  38. Gales AC, Sader HH, Jones RH. Respiratory tract pathogens isolated from patients hospitalized with suspected pneumonia in Latin America; frequency of occurrence and antimicrobial susceptibility profile: results from SENTRY Antimicrobial Resistance Program (1997–2000). Diagn Microbiol Infect Dis. 2002; 44:301–11.

    PubMed  Article  Google Scholar 

  39. Tash H, Hakki-Bahar I. Molecular characterization of TEM and SHV-derived extended-spectrum b-lactamases in hospital-based Enterobacteriaceae in Turkey. Jpn J Infect Dis. 2005;58:162–7.

    Google Scholar 

  40. Prodinger WM, Fille M, Bauernfeind A, et al. Molecular epidemiology of Klebsiella pneumoniae producing SHV-5 b-lactamase: parallel outbreaks due to multiple plasmid transfer. J Clin Microbiol. 1996;34:564–8.

    PubMed  CAS  Google Scholar 

  41. Bauernfeind A, Rosenthal E, Eberlein E, Holley M, Schweighart S. Spread of Klebsiella pneumoniae producing SHV-5 b-lactamase among hospitalized patients. Infection 1993;21:24–8.

    Article  Google Scholar 

  42. Pragai Z, Koczian Z, Nagy E. Characterization of the extended-spectrum b-lactamases and determination of the antibiotic susceptibilities of Klebsiella pneumoniae isolates in Hungary. J Antimicrob Chemother. 1998;42:401–3.

    PubMed  Article  CAS  Google Scholar 

  43. Gniadkowski M, Schneider I, Jungwirth R, Hryniewicz W, Bauernfeind A. Ceftazidime-resistant Enterobacteriaceae isolates from three Polish hospitals: identification of three novel TEM- and SHV-5 type extended-spectrum b-lactamases. Antimicrob Agents Chemother. 1998;4:514–20.

    Google Scholar 

  44. Vatopoulos AC, Philippon A, Tzouvelekis LS, Komninou Z, Legakis NJ. Prevalence of a transferable SHV-5 type b-lactamase in clinical isolates of Klebsiella pneumoniae and Escherichia coli in Greece. J Antimicrob Chemother. 1990;26:635–48.

    PubMed  Article  CAS  Google Scholar 

  45. Miranda G, Castro N, Leanos B, Valenzuela A, Garza-Ramos U, Rojas T, et al. Clonal and horizontal dissemination of Klebsiella pneumoniae expressing SHV-5 extended-spectrum b-lactamase in a Mexican Pediatric hospital. J Clin Microbiol. 2004;42:30–5.

    PubMed  Article  CAS  Google Scholar 

  46. Bedenic B, Randegger C, Stobberingh E, Haechler H. Molecular epidemiology of extended-spectrum β-lactamases from Klebsiella pneumoniae strains, isolated in Zagreb, Croatia. Eur J Clin Microbiol Infect Dis. 2001;20:505–8.

    Google Scholar 

  47. Bedenic B, Schmidt H, Herold S, et al. Spread of Klebsiella pneumoniae producing SHV-5 b-lactamase in Dubrava University Hospital, Zagreb. J Chemother. 2005;17:367–75.

    Google Scholar 

  48. Vranić-Ladavac M, Bošnjak Z, Beader N, Barišić N, Kalenić S, Bedenić B. Clonal spread of CTX-M producing Klebsiella pneumoniae in Croatian hospital. J Med Microbiol. 2010;59:1069–78.

    PubMed  Article  Google Scholar 

  49. Bedenić B, Vraneš J, Bošnjak Z, Marijan T, Mlinarić-Džepina A, Kukovec T, Anušić M, Beader N, Barl P, Leskovar V, Kalenić S. Emergence of CTX-M group 1 extended-spectrum b-lactamase-producing Klebsiella pneumoniae strains in the community. Med Glas. 2010;1:32–9.

    Google Scholar 

  50. Payne DJ, Marriot MS, Amyes SGB. Characterization of a unique ceftazidime-hydrolyzing b-lactamase, TEM-E2. J Med Microbiol. 1990;12:131–4.

    Article  Google Scholar 

  51. Essack S. Treatment options for extended-spectrum b-lactamase producers. FEMS Microbiol Lett. 2000;190:181–4.

    PubMed  CAS  Google Scholar 

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Conflict of interest

The authors declare that there is no actual or potential conflict of interest in relation to this article.

Author information

Affiliations

  1. Department of Microbiology, School of Medicine, University of Zagreb, Salata 3, 10000, Zagreb, Croatia

    Branka Bedenić &  Jasmina Vraneš

  2. Clinical Hospital Center Zagreb, Zagreb, Croatia

    Branka Bedenić

  3. IML red, synlab MVZ Gauting, Asklepios Fachkliniken, Gauting, Germany

    Sabine Hofmann-Thiel Ph.D. & Harald Hoffmann

  4. Department of Microbiology, Split University Hospital, Split, Croatia

    Marija Tonkić &  Anita Novak

  5. Department of Chemistry, Faculty of Natural Sciences, Mathematics and Education, University of Split, Split, Croatia

    Viljemka Bučević-Popovic Ph.D.

Authors
  1. Branka Bedenić
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  2. Jasmina Vraneš
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  3. Sabine Hofmann-Thiel Ph.D.
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  4. Marija Tonkić
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Correspondence to Branka Bedenić.

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Bedenić, B., Vraneš, J., Hofmann-Thiel, S. et al. Characterization of the extended-spectrum b-lactamases and determination of the virulence factors of uropathogenic Escherichia coli strains isolated from children. Wien Klin Wochenschr 124, 504–515 (2012). https://doi.org/10.1007/s00508-012-0210-5

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Keywords

  • Escherichia coli
  • Virulence
  • Urinary tract infections
  • Children
  • Extended-spectrum β-lactamases

Schlüsselwörter

  • Beta-laktamasen
  • Escherichia coli
  • Virulenzfaktoren
  • Kinder
  • Harnweginfektionen