Increasing relevance of Gram-positive cocci in urinary tract infections: a 10-year analysis of their prevalence and resistance trends

Urinary tract infections (UTIs) are the third most common types of infection in human medicine worldwide. There is increasing appreciation for the pathogenic role of Gram-positive cocci (GPC) in UTIs, as they have a plethora of virulence factors, maintaining their pathogenicity and high affinity for the epithelial cells of the urinary tract. The study was carried out using microbiological data collected corresponding to the period between 2008 and 2017. Antimicrobial susceptibility testing was performed using the disk diffusion method and E-tests. The age range of patients affected from the outpatient and inpatient groups differed significantly (43 [range 0.7–99] vs. 68 [range 0.4–99] years; p = 0.008). 3962 GPCs were obtained from inpatient and 4358 from outpatient samples, corresponding to 20.5 ± 2.8% (range 17.5–26.8%) and 20.6 ± 2.6% (range 17.8–26.0%) of all positive urine samples (p > 0.05); in both groups, Enterococcus spp. were the most prevalent (outpatients: 79.6%; inpatients: 88.5%). High-level aminoglycoside resistance in enterococci was noted in 31.0–46.6% of cases. A pronounced increase in the number of MRSA was seen in the second half of the study period (0.6–1.9% vs. 9.8–11.6%; p = 0.038). The ratio of VRE isolates was 0.16%, no VISA/VRSA isolates were detected.

To evaluate the demographic characteristics of these infections, limited amount of patient data was also collected (sex, age at sample submission, date of samples submission, inpatient/outpatient status). The study was deemed exempt from ethics review by the Institutional Review Board of the University of Szeged (Szeged, Hungary), and informed consent was not required as data anonymity was maintained.
Identification of isolates. Ten microliters of each uncentrifuged urine sample was cultured on UriSelect chromogenic agar (Bio-Rad, Berkeley, CA, USA) and blood agar (bioMérieux, Marcy-l'Étoile, France) plates with a calibrated loop, according to the manufacturer's instructions, and incubated at 37 °C for 24-48 h, aerobically. In the period between 2008 and 2012, presumptive, biochemical reaction-based methods and VITEK 2 Compact ID/AST (bioMérieux, Marcy-l'Étoile, France) were used for bacterial identification; from 2013 onward, MALDI-TOF MS (Bruker Daltonics, Germany) was introduced to the workflow of the Department of Bacteriology. Mass spectrometry was performed by the Microflex MALDI Biotyper (Bruker Daltonics, Germany) instrument, using the MALDI Biotyper RTC 3.1 software (Bruker Daltonics, Germany) and the MALDI Biotyper Library 3.1 for spectrum analysis. Sample preparation methodology and the technical details of MALDI-TOF MS measurements were described elsewhere 39 .
Methicillin-resistant S. aureus (MRSA) was detected using mannitol-salt agar (MSA) using cefoxitin disks (< 22 mm zone diameter) and PBP2′ Latex Agglutination Test Kit (Thermo Fisher Scientific Hungary Gmbh., Budapest, Hungary) 28 . After 2013, a combined MALDI-TOF MS and PBP2′ latex agglutination protocol was introduced in our laboratory 41 . MRSA-positive isolates were reported as resistant to all β-lactam antibiotics (except for 5th generation cephalosporins). Screening for high-level aminoglycoside resistance (HLAR) was done using gentamicin (30 μg) disks, while verification of positive results was performed using broth microdilution method (with a gentamicin concentration of 500 μg/ml) 42  Statistical analyses. Statistical analyses, including descriptive analysis (means or medians with ranges and percentages to characterize data) and statistical tests (χ 2 -test, Student's t-test and Mann-Whitney U test) were performed with SPSS software version 24 (IBM SPSS Statistics for Windows 24.0, Armonk, NY, USA, IBM Corp.). The normality of variables was tested using Shapiro-Wilk tests. p values < 0.05 were considered statistically significant 43 .

Results
Demographic characteristics of affected patients, sample types. During the study period, the median age of outpatients affected by UTIs caused by Gram-positive cocci was 43 years, which showed the following variability during the two parts of the study period: age range for outpatients was 0.7-99 years, whereas the median age for the first half of the study period was 35, while for the second half was 54 years (p = 0.038) (see Fig. 1. for detailed age distribution). In contrast, for the inpatients, the median age overall was 68 years; the age rage range was 0.4-99 years, with a median age for the first half of the study period was 64, while for the second half was 70 years (p < 0.05). The female-to-male ratio of the outpatient group was 2.03 (that is 67.1% female), and 1.15 (that is 53.6% female) in the inpatient group, respectively. The observed difference in age distribution of the two patient groups (inpatients and outpatients) was statistically significant (43 vs. 68 years; p = 0.008). Patients under 10 (outpatients: 18.7%, inpatients: 19.1%) and over 60 years of age (outpatients: 35.6%, inpatients: 61.4%) were predominantly affected. The sample distribution among relevant urine samples was the following: among samples from outpatient clinics, the overwhelming majority was midstream urine (98.8%), with a minority of first-stream urine (1.2%); on the other hand, the distribution from inpatient departments was more variable: midstream urine (29.2%), first-stream urine (6.3%), catheter-specimen urine (63.6%) and suprapubic bladder taps (0.9%).
Distribution of Gram-positive cocci in urine samples. Between  were in a minority. The epidemiology and detailed species distribution of Gram-positive cocci in both patient groups are presented in Fig. 2 (outpatients) and Fig. 3 (inpatients). There was an obvious seasonal trend in the isolation of Gram-positive cocci in the outpatient group (24.7% was isolated in the June-July periods, 22.9% in the December-January periods), while no such tendency was noted in the inpatient group. In the inpatient group, 14 different species of Gram-positive urinary pathogens were isolated (median 7; range 5-9), while in the outpatient group, the species distribution was less diverse, with 12 different species (median 6; range 5-8) detected.
Antibiotic resistance trends among Gram-positive cocci isolated from UTIs. Antibiotic resistance data of the isolated enterococci, staphylococci and streptococci in the 10-year study period is presented in Tables 1, 2 and 3, respectively. To identify temporal developments in resistance trends, the 10-year study period was divided into two 5-year periods (2008-2012 and 2013-2017). The level of resistance in Enterococcus species was significantly higher in isolates originating from inpatients in both periods regarding ciprofloxacin, but not other antibiotics. Apart from intrinsic resistance, resistance rates against ciprofloxacin and HLAR did not show relevant differences among E. faecalis and non-faecalis enterococci (p > 0.05). Likewise, there was no significant increase noted in the ratio of resistance strains in either patient groups between the two 5-year periods. HLAR was detected in 31.0-46.6% of isolates overall and there was a numerical, but not significant increase in the second half of the study period (p = 0.067). Very few VRE isolates were recovered (0.16%; n = 11 from inpatients and n = 4 from outpatients, exclusively from E. faecium), while no linezolid-resistant isolates were detected (Table 1.). The resistance levels in inpatient Staphylococcus samples were significantly higher for amikacin, gentamicin, azithromycin (in the 2008-2012 period), ciprofloxacin, doxycycline, nitrofurantoin (in the 2013-2017 period) and trimethoprim-sulfamethoxazole (SMX/TMP). There was no significant increase in the resistance of any tested antibiotics between 2008-2012 and 2013-2017, however, a numerical tendency was found for azithromycin (p = 0.071). The difference in the number of MRSA isolates among inpatient and outpatients was not significant, however, a pronounced increase in the number of MRSA was seen in the second half of the study period (0.6-1.9% vs. 9.8-11.6%; p = 0.038). No VISA/VRSA strains were found, in addition, none of the Staphylococcus strains were resistant to the supplementary antibiotics (quinpristin/dalfopristin, tigecycline, linezolid, fusidic acid) ( Table 2.). In the case of streptococci, significant differences were observed in the resistance levels of ciprofloxacin and SMX/TMP, but not in the case of other antibiotics. Additionally, no significant temporal changes were noted between the two study periods. No vancomycin or linezolid-resistant strains were detected.

Discussion
The study presents the epidemiological trends and resistance levels of Gram-positive cocci in urinary tract infections (UTIs) in the southern part of Hungary, over a long surveillance period (10 years 6.0%) were present in lesser numbers. In contrast, these pathogens represented ~ 20% of the etiological agents in UTIs, both for inpatients and outpatients, therefore, their epidemiological significance should not be disregarded. Among the group of Gram-positive cocci, E. faecalis was the predominant species (~ 80% of isolates), which is not surprising, in light of global epidemiological reports on the causative agents for UTIs 36 . To the best of our knowledge, this is the longest-spanning study reporting on the prevalence and susceptibility of this group of uropathogens in Hungary. In contrast to previous studies, dating back some 20-30 years (where the reported prevalence of Enterococcus spp., S. aureus and S. saprophyticus was 2-20%, 0.2-6% and 0.5-8%, respectively), based on current literature results, their prevalence is around 8-35%, 0.5-13% and 5-20%, respectively [48][49][50][51][52][53][54][55] . This increase is prevalence is especially notable in patients affected by recurrent UTIs (recurrence 3 or more times in 6 months) 44 . The increased prevalence of these pathogens may be attributed to the increase in patients with lifestyle diseases (kidney diseases, diabetes), immunosuppression, patients undergoing surgical interventions 56 . Additionally, some reports suggest that Enterococcus spp. may be considered an indicator of more severe pathologies (e.g., diabetes, abnormality of the genitourinary tract) [18][19][20][21][22]49,56 . An overview of the literature published in the last 20 or so years, regarding the prevalence of UTIs caused by Gram-positive cocci outside Hungary is presented in Table 4. In contrast to the present report, most of these studies found that the prevalence of Gram-positive cocci was higher in inpatients (hospital-acquired infections). Of note, the seasonal occurrence/accumulation of these bacteria (particularly S. saprophyticus) was also verified by our results in the Southern Great Plain of Hungary. Although the susceptibility-reporting for some of the antibiotics (e.g., fusidic acid, rifampicin, erythromycin, clindamycin, doxycycline and tigecycline) might seem frivolous in the context of the therapy of UTIs (as these drugs are not used in the therapy of these infections), the reporting of these results for epidemiological purposes is of interest, especially because not many studies are available regarding GPCs as uropathogens from Europe 66    S. saprophyticus is a common agent in UTIs, however, regarding its resistance patterns, it has proven so far to be mostly sensitive to the relevant antibiotics. Among the tested antibiotics, the highest levels of resistance were detected for ciprofloxacin and SMX/TMP, which could a consequence of their prevalent use, due to their broad-spectrum activity against both Gram-positive and Gram-negative bacteria. Out of the agents effective against Gram-positive bacteria, azithromycin and clindamycin had the highest resistance levels. Enterococci are a therapeutic challenge in general, because of their intrinsic resistance mechanisms against many antibiotics (aminoglycosides, cephalosporins), and due to their genetic plasticity, they can easily acquire additional resistance determinants against other antimicrobial drugs. This is especially concerning, as these bacteria normally live in the gastrointestinal tract, where they can pick up resistance plasmids from other members of the commensal flora [69][70][71][72] . Vancomycin resistance in Enterococcus species is therefore a severe therapeutic issue [19][20][21][22][23]25 .
High-level aminoglycoside-resistance (HLAR) in Enterococcus spp. was detected in > 40% in the second part of the study period. This resistance is usually mediated by aminoglycoside-modifying enzymes (e.g., acetyltransferases, phospho-transferases and nucleotidyl-transferases). The detection of HLAR is relevant in antimicrobial therapy, for the combined use of a cell wall-acting agent (ampicillin, imipenem) and the aminoglycoside for their pharmacological synergy 71,72 . Gram-positive cocci have a plethora of virulence determinants, maintaining their high affinity for the epithelial cells of the urinary tract, allowing for their survival. These virulence factors include fibrillar proteins (Ssp) mediating cell-cell interactions, fibronectin-binding proteins, elastin-binding protein, adhesins, hemagglutinin, elastase and lipase. In addition, most of S. saprophyticus and > 90% of S. aureus strains produce urease, breaking down carbamide (urea) in the urine [11][12][13][14][15][16][17][18][19][20][21][22][23] . Staphylococci may colonize the rectum, while Enterococcus spp. are present in fecal matter, therefore their anatomical proximity to the urinary tract may additionally enhance their UTI-causing capabilities 20,28 . Biofilm-production in these species is an another important factor for the emergence and the persistence of UTIs, with some reports suggesting that some 80% of uropathogenic Gram-positive cocci are biofilm-producers 48 . The presence of biofilm in urethral stents and catheters may lead to obstruction; furthermore, microorganisms embedded in biofilm may survive 1000-times higher concentrations of antibiotics, compared to non-embedded (i.e. planktonic) cells 48,73,74 . The diversification and time-dependent use of these virulence determinants allows for the infectivity and survival of these bacteria. At the onset of infection (i.e. low population density), the expression levels of adhesins is more significant, while if high population densities are achieved, genes corresponding for toxin secretion are activated 74 .
Several limitations of this present study need to be acknowledged. In addition to the retrospective study design, the authors were unable to access the charts of the individual patients affected, therefore the correlation between the existence of clinically relevant risk factors (apart from age, inpatient/outpatient status, and catheterization) could not be assessed. The clear differentiation between Gram-positive asymptomatic bacteriuria and clinically significant (symptomatic) urinary tract infection in the elderly is very difficult. Furthermore, the molecular characterization and genotyping of the isolates species (which could have provided us with important data, especially in case of MRSA or S. saprophyticus isolates) was not performed, due to financial constraints. Also, the selection bias of publication should also be noted, as most studies describing the prevalence of infectious diseases are tertiary-care centers or specialized centers, corresponds to patients with more severe conditions or underlying illnesses 75 . Nevertheless, the information presented in this report should be useful in both national and international comparisons for epidemiological purposes; additionally, the resistance trends presented here may aid clinicians in the selection of appropriate antimicrobial therapy 76 .

Conclusions
Although urinary tract infections are principally caused by Gram-negative bacteria, Gram-positives have emerged as important causative agents of UTIs, particularly among elderly patients, often associated with co-morbidities, pregnant women and catheterized patients, both in low-and high-income countries. In our study, corresponding to the southern region of Hungary, their prevalence was found to be around 20%, with Enterococcus spp. in highest numbers. While there was no relevant difference is their prevalence among inpatients and outpatients, the emergence of drug resistance in these pathogens to commonly used antibiotics is a worrisome finding, compromising therapeutic options in the clinical practice and leading to the use of agents with less advantageous side effect profiles, further contributing to the selection pressure on these microorganisms. In our local settings, the resistance rates for fluoroquinolones are particularly concerning (these agents are not recommended to be used empirically), in addition, the same goes for the use of most aminoglycosides for hospitalized patients. In contrast, the use of nitrofurantoin for staphylococci may still be regarded as safe in our settings, and the tested isolates are almost uniformly susceptible to the available last-resort antibiotics.