Epidemiology and Resistance Levels of Enterobacteriaceae Isolates from Urinary Tract Infections Expressed as Multiple Antibiotic Resistance (MAR) Indices

Aims: To assess the epidemiology of UTIs affecting inpatients and outpatients and the antibiotic resistance levels, expressed as multiple antibiotic resistance (MAR) indices from the isolated species at a tertiary-care out to be positive for a significant urinary pathogen. Out of the positive urine samples, E. coli represented the overwhelming majority of all positive urine samples. The resistance levels in inpatient isolates were higher than in the outpatient isolates (average MAR indices: 0.347 vs. 0.410, 0.267 vs. 0.435 and 0.318 vs. 0.473 for the E. coli/Klebsiella , CES and Proteae group, respectively). Conclusion: As the therapeutic options are becoming increasingly limited in the current antibiotic resistance climate, more effort should be put into the prudent use of antibiotics and the development of novel antimicrobial agents.


INTRODUCTION
Urinary tract infections (UTIs) are some of the most common infections in human healthcare, both in community (10-30% of infections in primary healthcare) and nosocomial settings (30-40%) [1,2]. UTIs are often associated with recurrence, complications and sequelae, leading to a decrease in the quality of life for the affected patients [3]. UTIs are most frequently caused by members of the Enterobacteriaceae family (or more recently, the Enterobacterales order): typical pathogens include uropathogenic Escherichia coli and Klebsiella spp., however, the Proteus-Providencia-Morganella species (the Proteae tribe), Citrobacter-Enterobacter-Serratia species (so-called CES pathogens) have now emerged as increasingly relevant Gramnegative pathogens [4][5][6][7]. Some reports suggest that 80% of UTIs are caused by various serotypes of E. coli (there are more than 1650 different serotypes of E. coli, if O, H and K antigens are considered). E. coli and Proteus spp. possess fimbria, allowing for attachment to epithelial cells; the resulting production of IL-6 and IL-8 causes epithelial cell desquamation. On the other hand, Klebsiella spp. produce extracellular polysaccharides, and have a role in stone formation, due to their urease production. Among the Gram-positive bacteria, Staphylococcus aureus from urine may indicate bacteremia or infection of the kidney, Enterococcus spp. may cause uncomplicated cystitis in women, while S. saprophyticus may cause asymptomatic UTI in young females [1][2][3][4][5][6][7][8][9]. The therapy of UTIs is an increasingly complex challenge for clinicians, due to the increasing levels of antibiotic resistance [8,9]. The emergence of multidrug resistance (MDR) and extensive drug resistance (XDR) in urinary pathogens, together with pre-existing, genetically encoded resistance mechanisms means that these pathogens may be resistant to a broad range of antibiotics [10,11].
Since the beginning of the 21 st century, many surveillance reports have been published regarding the resistance trends of Gram-negative bacteria [12]. Nevertheless, the epidemiology and antibiotic susceptibilitypatterns of urinary tract pathogens vary greatly by region, and, therefore, the assessment of local data is essential to evaluate trends over time and to reflect on the national situation compared to international data. In addition, the knowledge of the relevant antibiotic susceptibility patterns of the major bacterial pathogens for UTIs is critically important for the optimal choice for antibiotic therapy. With this in mind, the aim of this study was to assess the epidemiology of UTIs affecting inpatients and outpatients and the antibiotic resistance levels, expressed as multiple antibiotic resistance (MAR) indices from the isolated species at the Albert Szent-Györgyi Clinical Center (Szeged, Hungary) retrospectively, during a 10-year study period.

Study Design, Data Collection
The present retrospective study was carried out using microbiological data collected from the 1 st of January 2008 and 31 st of December 2017 at the University of Szeged, which is affiliated with the Albert Szent-Györgyi Clinical Center, a primary-and tertiary-care teaching hospital in the Southern Great Plain of Hungary. The Clinical Center has a bed capacity of 1820-beds and annually serves more than 400,000 patients in the region, according to the data of the Hungarian National Health Insurance Fund (NEAK), including GP-level care, all the way to specialized medical interventions [13]. Electronic search in the records of the MedBakter laboratory information system (LIS) for positive urine samples was conducted by the author.
Samples with clinically significant colony counts for the abovementioned bacteria (10 5 < colony forming units [CFU]/mL; however, this was subject to interpretation, based on the information provided on the request forms for microbiological analysis and relevant international guidelines, e.g., presence of underlying conditions in the genitourinary tract) were included in the data analysis. Only the first isolate per patient was included in the study, however, isolates with different antibioticsusceptibility patterns were considered as different individual isolates [14].

Identification of Isolates
10 µL of each un-centrifuged urine sample was cultured on UriSelect chromogenic agar plates (Bio-Rad, Berkeley, CA, USA) with a calibrated loop, according to the manufacturer's instructions and incubated at 37ºC for 24-48 h, aerobically. If the relevant pathogens presented in significant colony count, the plates were passed on for further processing. Between 2008-2012, presumptive phenotypic (biochemical reactionbased) methods and VITEK 2 ID (bioMérieux, Marcy-l'Étoile, France) were used for bacterial identification, while after 2013, this was complemented by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS; Bruker Daltonik Gmbh. Gr., Bremen, Germany) [5][6][7]. The methodology of sample preparation for MALDI-TOF MS measurements was described elsewhere [15]. Mass spectrometry was performed by the Microflex MALDI Biotyper (Bruker Daltonics, Bremen, Germany) in positive linear mode across the m/z range of 2 to 20 kDa; for each spectrum, 240 laser shots at 60 Hz in groups of 40 shots per sampling area were collected. The MALDI Biotyper RTC 3.1 software (Bruker Daltonics) and the MALDI Biotyper Library 3.1 were used for spectrum analysis.

Antimicrobial Susceptibility Testing
Antimicrobial susceptibility testing (AST) was performed using disk diffusion method and when appropriate, E-tests (Liofilchem, Abruzzo, Italy) on Mueller-Hinton agar (MHA) plates. For the verification of questionable results, VITEK 2 AST (bioMérieux, Marcy-l'Étoile, France) was also used.  [5][6][7]. During data analysis, intermediate-susceptible results were grouped with and reported as resistant. The multiple antibiotic resistance (MAR) index of the isolates was determined using the formula: MAR Index = Number of antibiotics isolate is resistant to/Number of antibiotics tested, as described previously [16]. The MAR index may range between 0.0-1.0.

Statistical Analysis
Descriptive statistical analysis (including means or medians with ranges and percentages to characterize data) was performed using Microsoft Excel 2013 (Redmond, WA, USA, Microsoft Corp.).  The increasing levels of antimicrobial drug resistance in urinary tract infection correspond to both local and international changes in treatment guidelines. MDR Gram-negatives leads to poor prognoses and an increased complication rate, mortality rate, especially in nosocomial settings [17]. The abovementioned urinary pathogens were most frequently resistant to the fluoroquinolones, sulfamethoxazole/trimethoprim and ampicillin therefore; the empiric use of these agents should be discouraged [18]. The therapy of Proteae and CES infections is especially challenging due to the various intrinsic resistance mechanisms these pathogens possess [19,20]. In addition, some last-resort drugs used in clinically relevant infections with Gram-negative pathogens, like tigecycline and colistin are not useful in the therapy of Proteae-associated   pathologies and some beta-lactam antibiotics and colistin are not useful against Serratia spp. Intrinsic non-susceptibility to several drug groups severely limits the number of therapeutic alternatives, especially for outpatient care. Nitrofurantoin and fosfomycin (if susceptibility is confirmed) represents a safe and viable option for the treatment of these infections [9].

CONCLUSION
This study presents a current epidemiological snapshot and resistance levels of Enterobacteriaceae associated with of urinary tract infections (UTIs) in Hungary over a long period (2008)(2009)(2010)(2011)(2012)(2013)(2014)(2015)(2016)(2017). To the best of the authors knowledge, this is the first study in Hungary, reporting on the resistance levels of these pathogens using the MAR index. As the therapeutic options are becoming increasingly limited in the current antibiotic resistance climate, more effort should be put into the prudent use of antibiotics and the development of novel antimicrobial agents.

CONSENT
It is not applicable.

ETHICAL APPROVAL
The study was deemed exempt from ethics review by the Institutional Review Board, and informed consent was not required as data anonymity was maintained.