CEPHALOSPORINASES IN PROTEUS MIRABILIS ISOLATES FROM LONG-TERM CARE FACILITIES AND THE COMMUNITY

Proteus mirabilis (P. mirabilis) je sve značajniji uzročnik bolničkih i izvanbolničkih infekcija – uključujući i infekcije u domovima za starije i nemoćne osobe. Prethodna istraživanja rezistencije u P. mirabilis u Hrvatskoj su pokazala dominaciju TEM-52 beta-laktamaze proširenog-spektra (ESBL) i pojavu plazmidnih AmpC beta-laktamaza. Cilj ovog istraživanja bio je utvrditi evoluciju i dinamiku pojavljivanja cefalosporinaza u P. mirabilis u domovima umirovljenika te usporediti izolate iz domova s onima u izvanbolničkoj populaciji. Ukupno je prikupljen 41 izolat rezistentan na cefalosporine treće generacije iz dva doma za starije i nemoćne te od izvanbolničkih pacijenata u razdoblju od ožujka 2015. do rujna 2017. godine. Testiranje osjetljivosti na antibiotike provedeno je metodom dilucije u bujonu. ESBL i plazmidne AmpC beta-laktamaze su detektirane fenotipskim testovima s inhbitorima te metodom lančane reakcije polimerazom (PCR). Plazmidi su karakterizirani konjugacijom, transformacijom i replikon-tipizacijom. Svi izolati su pokazivali visoki stupanj rezistencije na amoksicilin te na cefalosporine prve, druge i treće generacije. Tri izolata su pokazivala pozitivan test dvostrukog diska i kombiniranih diskova s klavulanskom kiselinom, što je upućivalo na produkciju ESBL. Trideset-osam izolata je bilo negativno fenotipski na ESBL ali su imali pozitivan Hodgeov test i test kombiniranih diskova s fenilboroničnom kiselinom, što je upućivalo na produkciju AmpC beta-laktamaze. PCR-om su dokazane CTX-M i TEM beta-laktamaze u ESBL-pozitivnih izolata i CMY u izolata pozitivnih na AmpC. U CTX-M pozitivnim izolatima identifi ciran je plazmid iz IncK grupe, dok AmpC-pozitivni sojevi nisu posjedovali plazmid. Ovim istraživanjem dokazali smo perzistenciju AmpC beta-laktamaze iz CMY porodice u izolatima ove bakterijske vrste u jednom domu za starije i nemoćne osobe, ali i diseminaciju takvih izolata u drugom domu te u izvanbolničkoj populaciji. Kao i u nekim drugim istraživanja, uočen je trend skretanja beta-laktamaza od TEM varijanti prema CTX-M i CMY tipovima. Shodno tome, pravilna i brza laboratorijska identifi kacija tipa cefalosporinaze postaje sve važniji preduvjet za odabir adekvatne terapije.
Proteus mirabilis (P. mirabilis) is increasingly recognized as a causative agent of hospital and community acquired infections, including those in long-term care facilities (LTCFs). Previous studies on P. mirabilis from Croatia showed the predominance of TEM-52 extended spectrum beta-lactamase (ESBL) and the emergence of plasmid AmpC beta-lactamases. The aim of the present study was to investigate the evolution and dynamics of cephalosporinases in P. mirabilis from LTCFs and to compare the isolates found in the LTCFs with those from a community setting. A total of 41 isolates of P. mirabilis resistant to third-generation cephalosporins were collected from two nursing homes and from outpatients from March 2015 until September 2017. Antibiotic susceptibility testing was performed by the broth microdilution method. ESBLs and plasmid-mediated AmpC beta-lactamases were detected by phenotypic inhibitor-based tests and by polymerase chain reaction (PCR). Plasmids were characterized by utilizing conjugation and transformation experiments, as well as PCR-based replicon typing. All isolates exhibited high level of resistance to amoxicillin alone and in combination with clavulanic acid, as well as to fi rst-, second- and third-generation cephalosporins. Three isolates tested positive in inhibitor-based test with clavulanic acid, and 38 in Hodge test and combined disk test with phenylboronic acid, indicating the production of ESBLs and plasmid-mediated AmpC beta-lactamases, respectively. Two ESBL-positive organisms yielded amplicons with primers specifi c for CTX-M beta-lactamase of group 1 and one for TEM. All AmpC-positive organisms were identifi ed by PCR as CMY. CTX-M positive strains harbored IncK plasmid, whereas AmpC-positive strains were negative for known plasmid types. This study demonstrated persistence of CMY beta-lactamases in one LTCF, but also dissemination of the aforementioned resistance determinants to another nursing home and to the community setting. Akin to other studies, there was a trend of cephalosporinase dynamic switch from TEM variants to CTX-M and CMY. Therefore, accurate and swift laboratory identifi cation of cephalosporinase type is becoming pivotal for appropriate choice of treatment.