Advances in Clinical and Experimental Medicine

Title abbreviation: Adv Clin Exp Med
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ISSN 1899–5276 (print)
ISSN 2451-2680 (online)
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Advances in Clinical and Experimental Medicine

2010, vol. 19, nr 3, May-June, p. 313–322

Publication type: original article

Language: English

Conjugative Transfer of Plasmid-Mediated CTX-M-Type β-Lactamases from Clinical Strains of Enterobacteriaceae to Salmonella enterica Serovars

Transfer koniugacyjny kodowanych plazmidowo β-laktamaz CTX-M ze szczepów klinicznych Enterobacteriaceae do serowarów Salmonella enterica

Roman Franiczek1,, Barbara Krzyżanowska1,, Izabela Dolna1,, Grażyna Mokracka-Latajka1,

1 Department of Microbiology, Wroclaw Medical University, Poland

Abstract

Objectives. The aim of the study was to evaluate the transfer frequency of plasmid-mediated extended-spectrum β-lactamases (ESBLs) from clinical isolates of Enterobacteriaceae to Salmonella enterica and Escherichia coli K12 C600 recipient strains. Moreover, the susceptibility to selected antibiotics and chemotherapeutics of the donor strains and transconjugants obtained in the mating experiments was estimated.
Material and Methods. Ten ESBL-positive clinical isolates, including Escherichia coli, Klebsiella pneumoniae, Citrobacter freundii, Enterobacter cloacae, and Serratia marcescens (two strains of each species) were used as donor strains. Salmonella enterica serovar Enteritidis (S. Enteritidis), S. Virchow, S. Hadar, and E. coli K12 C600 were used as recipient strains. ESBL production in donor strains and transconjugants was detected by the double disk synergy test (DDST). Transfer of ESBL-encoding plasmids was performed by a liquid conjugational method. The minimal inhibitory concentrations (MICs) of antibacterial 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. A total of 40 conjugation crosses between donor and recipient strains were performed. Transconjugants were obtained in twenty-seven (67.5%) of them. E. coli K12 C600 strain was found to be the best recipient. It acquired plasmid-mediated ESBL from all of the donor strains tested. Among Salmonella enterica recipients, S. Enteritidis and S. Infantis acquired ESBL-encoding genes from 9 and 7 donor strains respectively, whereas S. Hadar acquired this gene from a single donor strain only. The effectiveness of conjugational transfer ranged from 10–6 to 10–1 per donor cell. The donor strains and their transconjugants displayed resistance patterns typical of ESBL producers. They were uniformly resistant to cefotaxime and ceftriaxone but susceptible to carbapenems, tigecycline and oxyimino-β-lactams in combination with clavulanic acid. In addition, resistance to gentamicin, amikacin and co-trimoxazole was, in many cases, co-transferred with oxyimino-β-lactam resistance to recipients by means of conjugation. The MIC values of cefotaxime and ceftriaxone were higher than those of ceftazidime. PCR results revealed the presence of the blaCTX-M gene in all donor strains and their transconjugants.
Conclusion. The results of the study demonstrated the differences in conjugational acquisition of the blaCTX-M gene among the Salmonella enterica serovars studied. Of the S. enterica strains, Salmonella enterica serovar Enteritidis was found to be the best recipient of plasmid-mediated CTX-M-type β-lactamases.

Streszczenie

Cel pracy. Określenie częstości przekazywania plazmidowo kodowanych β-laktamaz o rozszerzonym spektrum substratowym (ESBL) z klinicznych szczepów Enterobacteriaceae do szczepów biorców Salmonella enterica i Escherichia coli K12 C600. Określono ponadto wrażliwość na wybrane antybiotyki i chemioterapeutyki szczepów dawców i transkoniugantów uzyskanych w krzyżówkach.
Materiał i metody. W badaniach zastosowano 10 izolatów klinicznych wytwarzających ESBL w charakterze dawców: Escherichia coli, Klebsiella pneumoniae, Citrobacter freundii, Enterobacter cloacae i Serratia marcescens (2 szczepy z każdego gatunku). Szczepy Salmonella enterica serowar Enteritidis (S. Enteritidis), S. Virchow, S. Hadar i E. coli K12 C600 użyto w charakterze biorców. ESBL wykrywano testem synergizmu dwóch krążków (DDST). Przekazywanie plazmidów kodujących ESBL przeprowadzono za pomocą metody koniugacji w podłożu płynnym. Minimalne stężenia hamujące (MIC) leków przeciwbakteryjnych oznaczono metodą seryjnych rozcieńczeń w podłożu agarowym Mueller-Hintona. Występowanie genu blaCTX-M w szczepach dawców i transkonjugantach oznaczono metodą PCR.
Wyniki. Ogółem wykonano 40 krzyżówek koniugacyjnych między szczepami dawców i biorców. Transkoniuganty otrzymano w 27 (67,5%) krzyżówkach. Najlepszym biorcą okazał się szczep E. coli K12, który nabył plazmidowo kodowane ESBL od wszystkich badanych szczepów dawców. Wśród biorców Salmonella enterica, serowary S. Enteritidis and S. Infantis nabyły geny kodujące ESBL odpowiednio od 9 and 7 szczepów dawców, a serowar S. Hadar nabył ten gen tylko od jednego szczepu donorowego. Skuteczność koniugacji wynosiła od 10–6 do 10–1 w przeliczeniu na komórkę dawcy. Szczepy dawców oraz ich transkoniuganty odznaczały się typowymi dla producentów ESBL wzorcami oporności. Były one oporne na cefotaksym i ceftriakson, wrażliwe natomiast na karbapenemy, tigecyklinę i oksyimino-β-laktamy skojarzone z kwasem klawulanowym. Ponadto, w wielu przypadkach oporność na gentamycynę, amikacynę i kotrimoksazol była przekazywana na drodze koniugacji wraz z opornością na oksyimino-β-laktamy. Wartości MIC dla cefotaksymu i ceftriaksonu były większe w porównaniu z wartościami MIC dla ceftazydymu. Wyniki badań PCR wykazały obecność genu blaCTX-M u wszystkich szczepów dawców oraz ich transkoniugantów.
Wnioski. Wyniki badań wykazały różnice w koniugacyjnym nabywaniu genu blaCTX-M wśród badanych serowarów Salmonella enterica. Spośród badanych szczepów S. enterica najlepszym biorcą plazmidowo kodowanych β-laktamaz CTX-M okazał się szczep Salmonella enterica serowar Enteritidis.

Key words

Salmonella, ESBL, CTX-M

Słowa kluczowe

Salmonella, ESBL, CTX-M

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