Outbreak of Infections with VIM-Positive <i>Proteus mirabilis</i> in a Hospital in Zagreb

Background/objectives: Proteus mirabilis is a frequent causative agent of urinary and wound in-fections in both community and hospital setting. It develops resistance to expanded-spectrum cephalosporins (ESC) due to the production of extended-spectrum β-lactamases (ESBLs) or plasmid-mediated AmpC β-lactamases (p-AmpC). Recently, carbapenem resistant isolates of P. mirabilis emerged due to production of carbapenemases, mostly belonging to Ambler class B and D. Here we report an outbreak of infections due to carbapenem-resistant P. mirabilis ob-served in a psychiatric hospital in Zagreb, Croatia. The characteristics of ESBL and car-bapenemase produced by P. mirabilis associated with an outbreak were analyzed. Material and methods: The antibiotic susceptibility testing was performed by disk-diffusion and broth dilution method. Double disk synergy test (DDST) and inhibitor- based test with clavulanic and phenylboronic acid were applied to screen for ESBLs and p-AmpC, respectively. Carbapenemases were screened by modified Hodge test (MHT) while carbapenem hydrolysis was investigated by CIM and eCIM test. The nature of ESBL, carbapenemases, and fluoroquin-olone resistance determinants was investigated by PCR. Transferability of cefotaxime and imipenem resistance was determined by conjugation. Plasmids were characterized by PCR-based replicon typing. Selected isolates were subjected to molecular characterization of re-sistome by the Interarray Carba Resist Kit and whole genome sequencing (WGS) Results: In total, 20 isolates were collected and analyzed. All isolates exhibited resistance to amoxicillin alone and combined with clavulanic acid, cefuroxime, ceftazidime, cefotaxime, ceftriaxone, cefepime, imipenem, ceftazidime-avibactam, ceftolozane-tazobactam, gentamicin, amikacin and ciprofloxacin. There was uniform susceptibility to ertapenem, meropenem and cefiderocol. DDST and combined disk test with clavulanic acid tested positive indicating pro-duction of an ESBL. MHT was negative in all isolates while CIM showed moderate sensitivity but only with imipenem as indicator disk. E-CIM tested positive in all CIM positive isolates, consistent with an MBL. PCR and sequencing of selected amplicons identified VIM-1 and VIM-4. The Interarray Carba Resist Kit and whole genome sequencing (WGS) identified β-lactam re-sistance genes: blaVIM, blaCTX-M-15, and blaTEM genes, aminoglycoside resistance genes: aac(3)-IId, aph(6)-Id, aph(3'')-Ib, aadA1, armA, and aac(6')-IIc, and resistance genes for sulphonamides sul1 and sul2, trimethoprim dfr1, chloramfenicol cat and tetracycline tetJ. Conclusions: The study revealed epidemic spread of carbapenemase producing P. mirabilis in two wards in a psychiatric hospital. This is the first report of VIM producing P. mirabilis in Croatia.