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The SARS-CoV-2 main protease (Mpro) is a medicinal chemistry target for COVID-19 treatment. Given the clinical efficacy of β-lactams as inhibitors of bacterial nucleophilic enzymes, they are of interest as inhibitors of viral nucleophilic serine and cysteine proteases. We describe the synthesis of penicillin derivatives which are potent Mproinhibitors and investigate their mechanism of inhibition using mass spectrometric and crystallographic analyses. The results suggest that β-lactams have considerable potential as Mproinhibitors via a mechanism involving reaction with the nucleophilic cysteine to form a stable acyl–enzyme complex as shown by crystallographic analysis. The results highlight the potential for inhibition of viral proteases employing nucleophilic catalysis by β-lactams and related acylating agents.

Maria Rus,1,2 Monica Licker,1– 3 Corina Musuroi,2 Edward Seclaman,4 Delia Muntean,1– 3 Natalia Cirlea,2 Alina Tamas,2 Silvana Vulpie,2 Florin George Horhat,1,3 Luminita Baditoiu3,5 1Department of Microbiology, “Victor Babes” University of Medicine and Pharmacy, Timisoara, Romania; 2“Pius Brinzeu” County Clinical Emergency Hospital, Timisoara, Romania; 3Multidisciplinary Research Center on Antimicrobial Resistance, “Victor Babes” University of Medicine and Pharmacy, Timisoara, Romania; 4Department of Biochemistry, “Victor Babeş” University of Medicine and Pharmacy, Timişoara, Romania; 5Department of Epidemiology, “Victor Babeş” University of Medicine and Pharmacy, Timişoara, RomaniaCorrespondence: Monica LickerDepartment of Microbiology, “Victor Babes” University of Medicine and Pharmacy, 2 E. Murgu, Timisoara 300041, RomaniaTel +40 0256-707513Email licker.monica@umft.roCorina MusuroiPius Brinzeu” County Clinical Emergency Hospital, No. 156 L.Rebreanu, Timisoara 300723, RomaniaTel +40745050232Email cormus1@yahoo.comBackground: Carbapenem-resistant Proteeae (CRP) is a group of multidrug-resistant (MDR) microorganisms that raise special treatment problems due to their intrinsic resistance to colistin. In this study, our aim is to provide a phenotypic and molecular characterization of the carbapenemases secreted by CRP strains isolated from inpatients from an intensive care unit (ICU) and surgical wards, as well as the identification of the risk factors involved in their acquisition.Methods: An observational, cross-sectional study was performed which included all Proteeae strains isolated in samples from inpatients on high-risk wards of the largest university hospital in Western Romania, from July 2017 to April 2019. Meropenem-resistant strains (N=65) with MIC ≥ 16 μg/mL were subjected to a singleplex PCR assay for the detection of blaNDM, blaVIM and blaCTX-M genes. The analysis of risk factors was performed by logistic regression.Results: Out of 8317 samples that were processed, 400 Proteeae strains were isolated: 64% belonging to the genus Proteus, 26.75% to the genus Providencia and 9.25% to the genus Morganella. Most CRP strains (N=56) were of MBL type, and 55 had the blaNDM gene as the prevalent gene substrate. P. stuartii was the main species that provided the circulating MDR strains. Most CRP strains came from patients admitted to ICU, being isolated mainly from bronchial aspirates and blood cultures. Multivariate analysis revealed 3 independent risk factors – mechanical ventilation> 96h (HR: 40.51 [13.65– 120.25], p < 0.001), tracheostomy (HR: 2.65 [1.14– 6.17], p = 0.024) and prolonged antibiotic therapy (HR: 1.01 [1.00– 1.02], p = 0.03).Conclusion: There is a significant increase in the incidence of CR P. stuartii strains, the MBL-blaNDM type being predominant. These strains presented various other resistance mechanisms, being often extremely difficult to treat and led to an excess of lethality of 27.16%.Keywords: carbapenem-resistant Proteeae, intensive care unit, P. stuartii

Delia Muntean,1 Monica Licker,1 Ersilia Alexa,2 Iuliana Popescu,2 Calin Jianu,3 Valentina Buda,4 Cristina Adriana Dehelean,5 Roxana Ghiulai,6 Florin Horhat,1 Delia Horhat,7 Corina Danciu8 1Department of Microbiology, “Victor Babes” University of Medicine and Pharmacy, Timisoara 300041, Romania; 2Department of Food Control, Faculty of Food Processing Technology, Banat’s University of Agricultural Sciences and Veterinary Medicine “King Michael I of Romania”, Timisoara 300645, Romania; 3Department of Food Tehnologies, Faculty of Food Engineering, Banat’s University of Agricultural Sciences and Veterinary Medicine “King Michael I of Romania”, Timişoara 300645, Romania; 4Department of Pharmacology and Clinical Pharmacy, Faculty of Pharmacy, “Victor Babes” University of Medicine and Pharmacy, Timisoara 300041, Romania; 5Department of Toxicology, Faculty of Pharmacy, “Victor Babes” University of Medicine and Pharmacy, Timisoara 300041, Romania; 6Department of Pharmaceutical Chemistry, Faculty of Pharmacy, “Victor Babes” University of Medicine and Pharmacy, Timisoara 300041, Romania; 7Department of ENT, “Victor Babes” University of Medicine and Pharmacy, Timisoara 300041, Romania; 8Department of Pharmacognosy, “Victor Babes” University of Medicine and Pharmacy, Timisoara 300041, RomaniaCorrespondence: Valentina Buda; Delia Horhat“Victor Babes” University of Medicine and Pharmacy, 2nd EftimieMurgu Square, Timisoara 300041, RomaniaTel +40 25 649 4804Fax +40 25 649 4804Email buda.valentina.oana@gmail.com; horhat.ioana@umft.roBackground: Bacterial multidrug resistance currently poses an increasingly serious threat, with important clinical consequences regarding treatment options. In 2017, the WHO released a global list of resistant bacteria, identifying multidrug-resistant (MDR) Gram-negative bacteria such as carbapenem-resistant Enterobacteriaceae, Pseudomonas aeruginosa or Acinetobacter baumannii, extended-spectrum cephalosporin-resistant Enterobacteriaceae as critical priorities for developing new strategies of treatment.Purpose: The novelty presented in this study refers to the evaluation of the volatile oil obtained from the leaves of Mentha×piperita L., on MDR strains from hospitalized patients.Material and methods: The essential oil was extracted by steam distillation and tested on six reference bacterial strains and also on the MDR strains collected from patients of the “Pius Brînzeu” Emergency Clinical County Hospital Timișoara. The in vitro antibacterial activity was evaluated by agar disk diffusion method and microdilution method.Results: Testing the antibacterial activity of peppermint oil on both reference strains and isolated MDR strains from hospitalized patients demonstrated its bactericidal effect. Minimum inhibitory concentration (MIC) was lower (20 mg/mL) for Staphylococcus aureus, Escherichia coli and Proteus mirabilis and higher (40 mg/mL) for Klebsiella pneumoniae, Pseudomonas aeruginosa and Acinetobacter baumannii strains. Minimum bactericidal concentration (MBC) was equal to MIC, with the exception of Pseudomonas aeruginosa strains, where MBC was the double of MIC.Conclusion: The present study highlights the bactericidal activity of Mentha×piperita L. essential oil on all tested MDR or extensively drug-resistant Gram-positive and Gram-negative strains of Staphylococcus aureus, Escherichia coli, Klebsiellapneumoniae, Proteus mirabilis, Pseudomonas aeruginosa and Acinetobacter baumannii. This oil may be a therapeutic option in the near future for many infectious diseases produced by MDR bacteria.Keywords: bacterial multidrug resistance, peppermint, essential oil, bactericidal effect

Delia Muntean,1,2 Monica Licker,1,2 Florin Horhat,1 Victor Dumitrașcu,1 Dorel Săndesc,1,2 Ovidiu Bedreag,1,2 Dorina Dugăeșescu,1 Dan A Coșniță,1 Anca Krasta,2 Luminița Bădițoiu1 1Victor Babeș University of Medicine and Pharmacy, Timisoara, Romania; 2Pius Brînzeu Emergency Clinical County Hospital, Timisoara, Romania Purpose: The purpose of this study was to identify risk factors for extensively drug-resistant (XDR) Acinetobacter baumannii (AB) and XDR Proteeae association in the largest intensive care unit (ICU) in Western Romania. Materials and methods: This retrospective case-controlled study was conducted between January 2016 and December 2016 in the ICU of the “Pius Brînzeu” County Emergency Clinical Hospital of Timișoara. Data were collected, in strict confidentiality, from the electronic database of the Microbiology Laboratory and the hospital’s electronic medical records. Risk factors were investigated by logistic regression. Independent variables with P≤0.05 and OR >1 (95% CI >1) in the univariate analysis were entered into multivariate sequenced analysis. Findings: The incidence density of coinfection with XDR AB and XDR Proteeae was 5.31 cases per 1,000 patient-days. Independent risk factors for the association of XDR AB and XDR Proteeae were represented by the presence of tracheostomy and naso-/orogastric nutrition ≥ 8 days. In addition, pressure ulcers were independent predictive factors for infections with all three infection types. Previous antibiotic therapy was an independent risk factor for the acquisition of XDR-AB strains, alone or in association, while the prolonged hospitalization in the ICU, blood transfusion, and hemodialysis appear as independent risk factors for single infections. Conclusion: This association of XDR AB and XDR Proteeae may well not be limited to our hospital or our geographical area. Keywords: extensive drug resistance, ICU, infections, risk factors

Monica Licker,1,2 Teodora Sorescu,3,4 Maria Rus,5,6 Natalia Cirlea,6 Florin Horhat,1 Cristiana Jurescu,5,6 Mircea Botoca,7,8 Alin Cumpănas,7,8 Romulus Timar,3,4 Delia Muntean1,2 1Department of Microbiology, “Victor Babes” University of Medicine and Pharmacy, Timisoara, Romania; 2Clinical Laboratory, “Pius Brînzeu” Emergency Clinical County Hospital, Timisoara, Romania; 3Department of Internal Medicine II, “Victor Babes” University of Medicine and Pharmacy, Timisoara, Romania; 4Diabetes Clinic, “Pius Brînzeu” Emergency Clinical County Hospital, Timisoara, Romania; 5“Victor Babes” University of Medicine and Pharmacy, Timisoara, Romania; 6“Pius Brînzeu” Emergency Clinical County Hospital, Timisoara, Romania; 7Department of Orthopaedics – Traumatology, Urology and Medical Imaging, “Victor Babes” University of Medicine and Pharmacy, Timisoara, Romania; 8Urology Unit, “Pius Brînzeu” Emergency Clinical County Hospital, Timisoara, Romania Purpose: We report an outbreak of urinary tract infections (UTIs) caused by Myroides odoratimimus, which occurred in the largest clinical hospital in western Romania. Patients and methods: From June to August 2017, four strains of M. odoratimimus were isolated from the urine samples of patients hospitalized in the urology, diabetes, and surgery departments. Hospital records of all patients whose urine cultures were positive for M.­odoratimimus were reviewed retrospectively. We also reviewed the cases reported in the literature. Results: All UTIs, except one, were hospital-acquired infections. All patients with M. odoratimimus UTIs were immunocompromised. Three patients underwent urinary catheterization with a Foley’s catheter upon admission in the emergency department and one presented for replacement of ureterostomy tubes. All Myroides isolates were resistant to almost all the tested antibiotics. Two patients were successfully treated with tigecycline and one was receiving antimicrobial treatment for another infection at the time of isolation of the microorganism. Conclusion: Although M. odoratimimus is an uncommon pathogen, clinicians should be aware of its ability to cause UTI outbreaks, especially in the immunocompromised population. Due to its multi-drug resistance, it is important to rapidly identify Myroides spp. in order to choose the best treatment regimen. Keywords: Myroides odoratimimus, urinary tract infection, resistance, outbreak

Teodora Chiţă,1,2 Bogdan Timar,1,2 Delia Muntean,1,2 Luminiţa Bădiţoiu,1,3 Florin Horhat,1,2 Elena Hogea,1 Roxana Moldovan,1,3 Romulus Timar,1,2 Monica Licker1,2 1Victor Babes University of Medicine and Pharmacy Timisoara, 2Pius Brinzeu Emergency Hospital, 3Regional Centre of Public Health, Timisoara, Romania Aim: Patients with diabetes mellitus (DM) have an increased risk of infections, especially urinary tract infections (UTIs). The aim of this study was to assess the prevalence and etiology of UTIs and identify the risk factors for their development in patients with DM. Patients and methods: In this retrospective, noninterventional study, the medical records of 2,465 adult patients with DM who were hospitalized in a Diabetes Clinic were reviewed. Data regarding the presence of UTI and possible associated risk factors were collected and their possible relation was analyzed. The study protocol and procedures were approved by the Ethics Committee of Timişoara Emergency Hospital. All data were collected and analyzed using SPSS v.17 statistical software. Results: The prevalence of UTIs in patients with DM was 12.0% (297 cases), being higher in females than in males and higher in patients with type 2 DM compared with patients with type1 DM. In univariate logistic regression analysis, risk factors associated with UTIs were female gender, age, type2 DM, longer duration of DM, and the presence of chronic kidney disease and coronary artery disease. Multivariate analysis identified age, duration of DM, and metabolic control (hemoglobin A1c levels) as independent risk factors for UTIs. The gram-negative bacilli from the Enterobacteriaceae family were predominant, with Escherichia coli being the most frequent of them (70.4%). Conclusion: UTIs are a frequent condition associated with DM. It is necessary to improve the care and the screening of UTIs in patients with DM to prevent the occurrence of possible associated severe renal complications. Keywords: urinary tract infections, diabetes mellitus, epidemiology, incidence

Supporting co-authors: Bakker BM, Bernardi P, Boetker HE, Borsheim E, Borutaitė V, Bouitbir J, Calbet JA, Calzia E, Chaurasia B, Clementi E, Coker RH, Collin A, Das AM, De Palma C, Dubouchaud H, Durham WJ, Dyrstad SE, Engin AB, Fornaro M, Gan Z, Garlid KD, Garten A, Gourlay CW, Granata C, Haas CB, Haavik J, Haendeler J, Hand SC, Hepple RT, Hickey AJ, Hoel F, Jang DH, Kainulainen H, Khamoui AV, Klingenspor M, Koopman WJH, Kowaltowski AJ, Krajcova A, Lane N, Lenaz G, Malik A, Markova M, Mazat JP, Menze MA, Methner A, Neuzil J, Oliveira MT, Pallotta ML, Parajuli N, Pettersen IKN, Porter C, Pulinilkunnil T, Ropelle ER, Salin K, Sandi C, Sazanov LA, Silber AM, Skolik R, Smenes BT, Soares FAA, Sokolova I, Sonkar VK, Swerdlow RH, Szabo I, Trifunovic A, Thyfault JP, Valentine JM, Vieyra A, Votion DM, Williams C, Zischka H, As the knowledge base and importance of mitochondrial physiology to human health expand, the necessity for harmonizing nomenclature concerning mitochondrial respiratory states and rates has become increasingly apparent. Clarity of concept and consistency of nomenclature are key trademarks of a research field. These trademarks facilitate effective transdisciplinary communication, education, and ultimately further discovery. Peter Mitchell’s chemiosmotic theory establishes the link between vectorial and scalar energy transformation and coupling in oxidative phosphorylation. The unifying concept of the protonmotive force provides the framework for developing a consistent theory and nomenclature for mitochondrial physiology and bioenergetics. Herein, we follow IUPAC guidelines on general terms of physical chemistry, extended by considerations on open systems and irreversible thermodynamics. We align the nomenclature and symbols of classical bioenergetics with a concept-driven constructive terminology to express the meaning of each quantity clearly and consistently. In this position statement, in the frame of COST Action MitoEAGLE, we endeavour to provide a balanced view on mitochondrial respiratory control and a critical discussion on reporting data of mitochondrial respiration in terms of metabolic flows and fluxes. Uniform standards for evaluation of respiratory states and rates will ultimately support the development of databases of mitochondrial respiratory function in species, tissues, and cells., We thank M. Beno for management assistance. Supported by COST Action CA15203 MitoEAGLE and K-Regio project MitoFit (E.G.).

Bacterial multidrug-resistance (MDR) to antimicrobials has become an important public health issue all over the world and it involves both hospital and community-acquired strains.A number of 75 Escherichia coli and 77 Klebsiella pneumoniae (K.) strains identified in biological samples collected from community (CA) and hospital-acquired (HA) infections were found to be resistant to the third generation cephalosporins. Of these, 93 MDR strains were subjected to microarray analysis to detect the expression of 31 antimicrobial resistance genes.We found that all HA extended-spectrum β-lactamase (ESBL) producing E. coli strains had at least one resistance gene to third generation cephalosporins, while in 54% of all CA strains genetic substrates justifying their antibiotic resistance were identified. Almost 81% of HA-ESBL (Extended-Spectrum β Lactamase) K. pneumoniae strains had at least one resistance gene to third generation cephalosporins, while in only 6% of the CA strains a similar genotype was identified. In the HA group, the blaCTX-M-15 genotype proved to be most frequent in multidrug-resistant E. coli strains and second most frequent (after ampC) in K. pneumoniae, while in the CA group, this genotype was the fourth most frequent in ESBL E. coli (after ampC, sul1, tet(R)).Overall, in 67% of all ESBL producing Enterobacteriaceae strains a genetic substrate justifying the resistance to beta-lactam antibiotics was identified; most of the remaining 33.33% strains were CA with a predominance of K. pneumoniae, in which a different antibiotic resistance genetic substrate (outside the detection limit of the kit used in this study) might have been involved.