Advances in Clinical and Experimental Medicine

Title abbreviation: Adv Clin Exp Med
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ISSN 1899–5276 (print)
ISSN 2451-2680 (online)
Periodicity – monthly

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Advances in Clinical and Experimental Medicine

2007, vol. 16, nr 4, July-August, p. 493–500

Publication type: original article

Language: English

Transferable Resistance to Different Antimicrobials Due to CTX−M−Type β−Lactamases Among Citrobacter freundii, Serratia marcescens, and Enterobacter spp. Clinical Isolates

Koniugacyjna oporność na różne grupy antybiotyków uwarunkowana wytwarzaniem β−laktamaz typu CTX−M u klinicznych izolatów Citrobacter freundii, Serratia marcescens i Enterobacter spp.

Roman Franiczek1,, Izabela Dolna1,, Barbara Krzyżanowska1,

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

Abstract

Objectives. The aim of the study was to evaluate the transfer frequency of plasmid−borne genes coding for extended−spectrum β−lactamases (ESBLs) from clinical isolates of Citrobacter freundii, Serratia marcescens, and Enterobacter spp. to the E. coli K12 C600 recipient strain. Moreover, the susceptibility to selected antibiotics and chemotherapeutics of the donor strains and transconjugants obtained in the mating experiments was determined.
Material and Methods. A total of 32 ESBL−producing clinical isolates, including Enterobacter spp. (n = 21), Serratia marcescens (n = 6), and Citrobacter freundii (n = 5) isolated from children hospitalized in the Medical University Hospital in Wrocław, Poland, were used in this study. ESBL production was confirmed by the doubledisk synergy test (DDST). Transfer of plasmid−mediated genes coding for ESBL was carried out by the conjugational broth method. Susceptibility to antibacterial drugs was performed by an agar dilution technique on MuellerHinton agar. The presence of the blaCTX−M gene in the donor strains and transconjugants was determined by PCR.
Results. Nineteen (59.4%) of the 32 clinical isolates studied transferred plasmid−mediated genes coding for ESBL to the E. coli K12 C600 recipient strain with a frequency of 10–7 to 10–1 per donor cell. The donor strains and their transconjugants displayed resistance patterns typical of ESBL producers. They were resistant to cefotaxime, ceftriaxone, and aztreonam, but susceptible to carbapenems (imipenem and meropenem) and oxyimino−β−lactams (ceftazidime, cefotaxime, ceftriaxone, and aztreonam) combined with clavulanic acid. The MICs of cefotaxime and ceftriaxone were significantly higher than those of ceftazidime, suggesting that this resistance may result from cefotaximase activity (e.g. CTX−M−type β−lactamases). On the basis of PCR, the blaCTX−M gene was identified in 15 donor strains as well as in 15 transconjugants. Additionally, resistance to non−β−lactam antibacterial drugs (particularly to aminoglycosides) was, in many cases, co−transferred to the recipient strain by conjugation.
Conclusion. These data confirm the necessity of detecting the ESBL phenotype in clinical isolates of Gram−negative rods since conjugative plasmids responsible for the production of ESBLs may often encode resistance to antimicrobial agents other than β−lactams.

Streszczenie

Cel pracy. Celem badań było określenie częstości przekazywania genów plazmidowych kodujących β−laktamazy o rozszerzonym spektrum substratowym (ESBL) z klinicznych szczepów Citrobacter freundii, Serratia marcescens i Enterobacter spp. do szczepu biorcy E. coli K12 C600. Oznaczono ponadto wrażliwość na wybrane antybiotyki i chemioterapeutyki szczepów dawców oraz uzyskanych w krzyżówkach transkoniugantów.
Materiał i metody. Wbadaniach zastosowano 32 szczepy kliniczne wytwarzające ESBL: Enterobacter spp. (n = 21), Serratia marcescens (n = 6) oraz Citrobacter freundii (n = 5), wyizolowane od dzieci hospitalizowanych w Akademickim Szpitalu Klinicznym we Wrocławiu (Polska). Wytwarzanie ESBL potwierdzono testem synergizmu dwóch krążków (DDST). Przekazywanie genów plazmidowych kodujących ESBL przeprowadzono za pomocą metody koniugacji w podłożu bulionowym. Wrażliwość na leki przeciwbakteryjne oznaczono metodą seryjnych rozcieńczeń w podłożu agarowym Mueller−Hintona. Występowanie genu blaCTX−M w szczepach dawców i transkoniugantach oznaczono metodą PCR.
Wyniki. 19 (59,4%) spośród 32 badanych izolatów przekazywała geny plazmidowe kodujące ESBL do szczepu biorcy E. coli K12 C600 z częstością 10–7 to 10–1 w przeliczeniu na komórkę dawcy. Szczepy dawców oraz transkoniuganty wykazywały typowe dla producentów ESBL wzorce oporności. Charakteryzowały się opornością na cefotaksym, ceftriakson i aztreonam oraz wrażliwością na karbapenemy i oksyimino−β−laktamy (ceftazydym, cefotaksym, ceftriakson i aztreonam) skojarzone z kwasem klawulanowym. Wartości MIC dla cefotaksymu i ceftriaksonu były znacznie wyższe w porównaniu z wartościami MIC dla ceftazydymu. Wyniki te mogą sugerować oporność wynikającą z wytwarzania cefotaksymaz (np. β−laktamaz typu CTX−M). Za pomocą metody PCR wykryto obecność genu blaCTX−M u 15 szczepów dawców, a także u 15 transkoniugantów. W wielu przypadkach ponadto oporność na nie−β−laktamowe leki przeciwbakteryjne (przede wszystkim na aminoglikozydy) była przekazywana do szczepu biorcy w wyniku koniugacji.
Wnioski. Wyniki przedstawione w pracy potwierdzają konieczność wykrywania fenotypu ESBL wśród klinicznych izolatów pałeczek Gram−ujemnych, gdyż plazmidy koniugacyjne determinujące syntezę ESBL często mogą kodować oporność na inne, nie−β−laktamowe, leki przeciwbakteryjne.

Key words

ESBL, plasmids, conjugation, antimicrobial resistance

Słowa kluczowe

ESBL, plazmidy, koniugacja, oporność na leki przeciwbakteryjne

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