Advances in Clinical and Experimental Medicine

Title abbreviation: Adv Clin Exp Med
JCR Impact Factor (IF) – 1.736
5-Year Impact Factor – 2.135
Index Copernicus  – 168.52
MEiN – 70 pts

ISSN 1899–5276 (print)
ISSN 2451-2680 (online)
Periodicity – monthly

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Advances in Clinical and Experimental Medicine

2007, vol. 16, nr 2, March-April, p. 239–247

Publication type: original article

Language: English

Conjugative Transfer of Multiresistance Plasmids from ESBL−positive Escherichia coli and Klebsiella spp. Clinical Isolates to Escherichia coli Strain K12 C600

Transfer koniugacyjny plazmidów wielooporności z ESBL−dodatnich klinicznych szczepów Escherichia coli i Klebsiella spp. do szczepu Escherichia coli K12 C600

Roman Franiczek1,, Izabela Dolna1,, Barbara Krzyżanowska1,, Krzysztof Szufnarowski1,, Beata Kowalska−Krochmal1,

1 Department of Microbiology, Silesian Piasts University of Medicine in Wrocław, Poland

Abstract

Objectives. The aim of this study was to evaluate the transfer frequency of plasmid−borne genes coding for extended−spectrum β−lactamases (ESBLs) from clinical isolates of E. coli and Klebsiella spp. to the E. coli K12 C600 recipient strain. Additionally, the antimicrobial susceptibility of the donor strains and transconjugants obtained in mating experiments were studied.
Material and Methods. A total of 51 ESBL−producing E. coli (n = 32) and Klebsiella spp. (n = 19) clinical strains isolated from children hospitalized in the Medical University Hospital in Wrocław, Poland, were used in this study. Transfer of plasmids carrying ESBL−encoding genes was performed using the conjugational broth method. ESBL production was detected by the double−disk synergy test (DDST). The minimal inhibitory concentrations (MICs) of antimicrobial drugs were determined by an agar dilution technique on Mueller−Hinton agar. The presence of the blaCTX−M gene in donor strains and transconjugants was determined by PCR.
Results. The majority of the isolates studied (92.2%) transferred ESBL−encoding plasmids to the E. coli K12 C600 recipient strain with a frequency of 10−5–10–1 per donor strain. Donor strains and transconjugants displayed resistance patterns typical of ESBL producers. They were resistant to cefotaxime, cefrtiaxone, and aztreonam but susceptible to carbapenems and oxyimino−β−lactams (ceftazidime, cefotaxime, ceftriaxone, and aztreonam) in combination with clavulanic acid. Moreover, the majority of the strains exhibited a high level of resistance to non−β−lactam antimicrobials (gentamicin, amikacin, co−trimoxazole). The MIC values of cefotaxime and cefrtiaxone were significantly higher than those of ceftazidime, suggesting that this resistance may result from CTM−X−type ESBLs. PCR based on primers specific for CTX−M−type β−lactamases confirmed the presence of the blaCTX−M gene in 31 (66%) donor strains and 23 (48.9%) transconjugants.
Conclusion. The majority of the strains tested harbored conjugative plasmids coding for CTX−M−type ESBLs. Additionally, genes conferring resistance to antimicrobial agents other than β−lactams were often co−transferred to the recipient strain in the conjugation process, indicating that these determinants were carried by ESBL−encoding plasmids.

Streszczenie

Cel pracy. Określenie częstości przekazywania genów plazmidowych kodujących β−laktamazy o rozszerzonym spektrum substratowym (ESBL) z klinicznych szczepów Escherichia coli i Klebsiella spp. do szczepu biorcy E. coli K12 C600. Oznaczono ponadto wrażliwość na leki przeciwbakteryjne szczepów dawców oraz uzyskanych w krzyżówkach transkoniugantów.
Materiał i metody. W badaniach zastosowano 51 szczepów klinicznych wytwarzających ESBL: E. coli (n = 32) oraz Klebsiella spp. (n = 19), wyizolowanych od dzieci hospitalizowanych w Akademickim Szpitalu Klinicznym we Wrocławiu (Polska). Przekazywanie plazmidów zawierających geny kodujące ESBL przeprowadzono za pomocą metody koniugacji w podłożu bulionowym. Wytwarzanie ESBL wykrywano testem synergizmu dwóch krążków (DDST). Minimalne stężenia hamujące (MIC) leków przeciwbakteryjnych oznaczono metodą seryjnych rozcieńczeń w podłożu agarowym Mueller−Hintona. Występowanie genu blaCTX−M w szczepach dawców i transkoniugantach oznaczono metodą PCR.
Wyniki. Większość badanych szczepów (92,2%) przekazywała plazmidy kodujące ESBL do szczepu biorcy E. coli K12 C600 z częstością 10–5–10–1 w przeliczeniu na komórkę dawcy. Szczepy dawców oraz transkoniuganty wykazywały typowe dla producentów ESBL wzorce oporności. Charakteryzowały się opornością na cefotaksym, ceftriakson, aztreonam oraz wrażliwością na karbapenemy i oksyimino−β−laktamy (ceftazydym, cefotaksym, ceftriakson i aztreonam) skojarzone z kwasem klawulanowym. Większość szczepów wykazywała ponadto wysoki poziom oporności na nie−β−laktamowe leki przeciwbakteryjne (gentamycynę, amikacynę, kotrimoksazol). Wartości MIC dla cefotaksymu i ceftriaksonu były znacznie wyższe w porównaniu z wartościami MIC dla ceftazydymu. Wyniki te mogą sugerować oporność wynikającą z wytwarzania ESBL typu CTX−M. Metoda PCR z wykorzystaniem sekwencji starterowych swoistych dla β−laktamaz typu CTX−M potwierdziła obecność genu blaCTX−M u 31 (66%) szczepów dawców i 23 (48,9%) transkoniugantów.
Wnioski. Większość badanych szczepów zawierała plazmidy koniugacyjne kodujące β−laktamazy ESBL typu CTX−M. Geny warunkujące oporność na inne niż β−laktamy leki przeciwdrobnoustrojowe były ponadto często przekazywane w procesie koniugacji do szczepu biorcy, co wskazuje na ich umiejscowienie w obrębie plazmidów kodujących ESBL.

Key words

ESBL, CTX−M, plasmids, multiresistance

Słowa kluczowe

ESBL, CTX−M, plazmidy, wielooporność

References (28)

  1. Livermore DM: β−lactamases in laboratory and clinical resistance. Clin Microbiol Rev 1995, 8, 557–584.
  2. Medeiros AA: Evolution and dissemination of β−lactamases accelerated by generations of β−lactam antibiotics. Clin Infect Dis 1997, 24, 19–45.
  3. Paterson DL, Bonomo RA: Extended−Spectrum β−Lactamases: a clinical update. Clin Microb Rev 2005, 18, 657–686.
  4. Knothe H, Shah P, Krcmery V, Mitsuhashi AS: Transferable resistance to cefotaxime, cefoxitin, cefamandole and cefuroxime in clinical isolates of Klebsiella pneumoniae and Serratia marcescens. Infection 1983, 11, 315–317.
  5. Jacoby GA: Genetics of extended−spectrum beta−lactamases. Eur J Clin Microbiol Infect Dis 1994, 13 (Suppl 1), 2–11.
  6. Bradford PA.: Extended−spectrum β−lactamases in the 21st Century: characterization, epidemiology, and detection of this important resistance threat. Clin Microbiol Rev 2001, 14, 933–951.
  7. Jacoby GA, Sutton L: Properties of plasmids responsible for production of extended−spectrum β−lactamases. Antimicrob Agents Chemother 1991, 35, 164–169.
  8. National Committee for Clinical Laboratory Standards: Performance Standards for Antimicrobial Susceptibility Testing. Wayne, USA 2001, 11th International Supplement, M100–S11.
  9. Jarlier V, Nicolas MH, Fournier G, Philippon A: Extended broad−spectrum β−lactamases conferring transferable resistance to newer β−lactam agents in Enterobacteriaceae: hospital prevalence and susceptibility patterns. Rev Infect Dis 1988, 10, 867–878.
  10. Gniadkowski M, Schneider I, Pałucha A, Jungwirth R, Mikiewicz B, Bauernfeind A: Cefotaxime−resistant Enterobacteriaceae isolates from a hospital in Warsaw, Poland: identification of a new CTX−M−3 cefotaxime−hydrolyzing β−lactamase that is closely related to the CTX−M−1/MEN−1 enzyme. Antimicrob Agents Chemother 1998, 42, 827–832.
  11. Sirot DL, Sirot J, Labia R, Morand A, Courvalin P, Darfeuille−Michaud A, Perroux R, Cluzel R: Transferable resistance to third−generation cephalosporins in clinical isolates of Klebsiella pneumoniae: identification of CTX−1, a novel β−lactamase. J Antimicrob Chemother 1987, 20, 323–334.
  12. Rice LB, Willey SH, Papanicolaou GA, Medeiros AA, Eliopoulos GM, Moellering RC, Jacoby GA: Outbreak of ceftazidime resistance caused by extended−spectrum β−lactamases at a Massachusetts chronic−care facility. Antimicrob Agents Chemother 1990, 34, 2193–2199.
  13. Ramphal R, Ambrose PG: Extended−spectrum beta−lactamases and clinical outcomes: current data. Clin Infect Dis 2006, 42 (Suppl 4), S164–S172.
  14. Bonnet R: Growing group of extended−spectrum β−lactamases: the CTX−M enzymes. Antimicrob Agents Chemother 2004, 48, 1–14.
  15. Tzouvelekis LS, Tzelepi E, Tassios PT, Legakis NJ: CTX−M−type β−lactamases: an emerging group of extended−spectrum enzymes. Int J Antimicrob Agents 2000, 14, 137–143.
  16. Baraniak A, Fiett J, Hryniewicz W, Nordmann P, Gniadkowski M: Ceftazidime−hydrolysing CTX−M−15 extended−spectrum β−lactamase (ESBL) in Poland. J Antimicrob Chemother 2002, 50, 393–396.
  17. Pagani L, Dell’Amico E, Migliavacca R, D’Andrea MM, Giacobone E, Amicosante G, Romero E, Rossolini GM: Multiple CTX−M−type extended−spectrum β−lactamases in nosocomial isolates of Enterobacteriaceae from a hospital in Northern Italy. J Clin Microbiol 2003, 41, 4264–4269.
  18. Radice M, Power P, Di Conza J, Gutkind G: Early dissemination of CTX−M−derived enzymes in South America. Antimicrob Agents Chemother 2002, 46, 602–604.
  19. Bonnet R, Sampaio JLM, Labia R, De Champs C, Sirot D, Chanal C, Sirot J: A novel CTX−M β−lactamase (CTX−M−8) in cefotaxime resistant Enterobacteriaceae isolated in Brazil. Antimicrob Agents Chemother 2000, 44, 1936–1942.
  20. Pallechi L, Malossi M, Mantella A, Gotuzzo E, Trigoso C, Bartoloni A, Paradisi F, Kronvall G, Rossolini GM: Detection of CTX−M−type β−lactamase genes in fecal Escherichia coli isolates from healthy children in Bolivia and Peru. Antimicrob Agents Chemother 2004, 48, 4556–4561.
  21. Pai H, Choi EH, Lee HJ, Hong JY, Jacoby GA: Identification of CTX−M−14 extended−spectrum beta−lactamase in clinical isolates of Shigella sonnei, Escherichia coli, and Klebsiella pneumoniae in Korea. J Clin Microbiol 2001, 39, 3747–3749.
  22. Yan JJ, Ko WC, Tsai SH, Wu HM, Jin YT, Wu JJ: Dissemination of CTX−M−3 and CMY−2 beta−lactamases among clinical isolates of Escherichia coli in southern Taiwan. J Clin Microbiol 2000, 38, 4320–4325.
  23. Mulvey MR, Bryce E, Boyd D, Ofner−Agostini M, Christianson S, Simor AE, Paton S: Ambler class A extended−spectrum beta−lactamase producing Escherichia coli and Klebsiella spp. in Canadian hospitals. Antimicrob Agents Chemother 2004, 48, 1204–1214.
  24. Franiczek R, Krzyżanowska B, Dolna I, Mokracka G, Szufnarowski K: Extended−spectrum β−lactamase−conferring transferable resistance to different antimicrobial agents in Enterobacteriaceae isolated from bloodstream infections. Folia Microbiol 2005, 50, 119–124.
  25. Tonkic M, Goic Barisic I, Pounda−Polic V: Prevalence and antimicrobial resistance of extended−spectrum β−lactamases−producing Escherichia coli and Klebsiella pneumoniae strains isolated in a university hospital in Split, Croatia. Int Microb 2005, 8, 119–224.
  26. Radosz−Komoniewska H, Gniadkowski M, Rogal−Zawada D: Incidence of extended−spectrum beta−lactamases in clinical isolates of the family Enterobacteriaceae in a pediatric hospital. Pol J Microbiol 2004, 53, 27–34.
  27. Puerto AS, Fernandez JG, del Castillo ID, Pino MJ, Angulo GP: In vitro activity of beta−lactam and non−betalactam antibiotics in extended−spectrum beta−lactamase−producing isolates of Escherichia coli. Diagn Microbiol Infect Dis 2006, 54, 135–139.
  28. Paterson DL: Recommendation for treatment of severe infections caused by Enterobacteriaceae producing extended−spectrum β−lactamases (ESBLs). Clin Microb Infect 2000, 6, 460–463.
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