Gender differences in the microbial spectrum and antibiotic sensitivity of uropathogens isolated from patients with urinary stones

Abstract Purpose The characteristics and resistance patterns of urine bacteriology urolithiasis patients between male and female have not been extensively studied. This study aims to investigate the gender differences in microbial spectrum and antibiotic susceptibility of uropathogens isolated from urolithiasis patients and provide insights for appropriate antimicrobial therapies. Materials and Methods We retrospectively collected clinical microbiology data from urine culture in urolithiasis patients between March 2014 and December 2018 in Xiangya Hospital. Then the patients were divided into male and female groups. The microbial spectrum and frequency of susceptibility to antibiotics were compared. Results A total of 359 uropathogen isolates were collected from 335 patients, including 144 males (43.0%) and 191 females (57.0%). E. coli dominated in both groups, indicating higher frequency in females (53.2%) than in males (26.6%, p < 0.001), followed by E. faecalis, with higher frequency in males (15.6%) than in females (2.9%, p < 0.001). Major Gram‐negative (E. coli and K. pneumoniae) bacteria showed high sensitivity to cefoperazone/sulbactam, cefotetan, piperacillin/ tazobactam, and amikacin. In contrast, the resistance level was high to penicillin, tetracycline, and vancomycin in both groups. Gram‐positive (E. faecalis and E. faecium) isolates demonstrated high sensitivity to gentamicin and vancomycin in both groups. Furthermore, uropathogens isolated from female urolithiasis patients were more susceptible to antimicrobials than males. Conclusions Uropathogen microbial spectrum in female urolithiasis patients is different from males. High susceptibility antibiotics should be used empirically according to gender to avoid multidrug‐resistant bacteria increase.


| INTRODUC TI ON
Urolithiasis has a high prevalence and recurrence rate and is a significant public health issue with a high socioeconomic cost. 1 Urinary calculi patients achieve a higher frequently associated with urinary tract infections (UTIs), which is a complication that precedes or follows urinary stone treatment. 2,3 Also, it causes a urinary stone formation, particularly Proteus spp, mainly formation of struvite crystals is associated with urease-producing bacteria. 4 Moreover, it is a source of recurrent UTIs, and stones may contain bacteria on their matrix or surface. 5 Urinary calculi-associated infections may cause severe complications due to endourological removal procedures, leading to renal insufficiency, urosepsis, and even life-threatening. 6 The high-pressure irrigation system increases urinary tract pressure, causing the spread of bacteria. Therefore, it is essential to choose appropriate antibiotics to manage patients with symptomatic UTIs and prophylaxis prior to surgical procedures.
Studies showed that Gram-negative bacteria Escherichia coli is the most frequent uropathogens. 3,7 Local antibiotic susceptibility and microbiological flora are extremely helpful planning adequate pre-and post-operative empirical antibiotic therapy when microbiological assessment results are temporarily unavailable. However, urine bacteriology in patients with urinary stones appears to have a complex pattern. 8,9 It is unclear whether men and women with UTI stones have the same antimicrobial efficacy.
So far, there has not been extensive research on differences in urine bacteriology characteristics and susceptibility patterns between males and females with urinary stones. Therefore, we conducted this study to investigate the antimicrobial sensitivity of the main species isolates of bacteria associated with UTIs in urolithiasis patients and to get fundamental appropriate antimicrobial therapies.

| Patients
We retrospectively collected clinical microbiology data of positive midstream urine culture in urolithiasis patients from clinical laboratory between March 2014 and December 2018. Then the patients were divided into male and female groups. Non-enhanced CT was employed to diagnose urinary calculi. Patients who prior used antibiotics within the last four weeks or with pregnancy, diabetes, chronic urine retention, neurogenic bladder, or immunosuppressive conditions were excluded. Urine was sampled before antibacterial treatment.

| Urine culture
A clean midstream urine sample was routinely collected into a sterile container and sent to clinical for culture-based microbiology. 10 μl of the urine sample with suspected bacterial infection was inoculated onto a blood agar plate and incubated at 37°C 18-24 h (cultured for seven days at 28°C with suspected fungal infection). If there is no bacterial growth, the culture will be extended to 48 h. Stainability and morphology of colonies were analyzed under a microscope and by mass spectrometry.

| Drug sensitivity test
Furthermore, drug sensitivity and resistance testing were conducted using the microbroth dilution method. The antimicrobial susceptibility standards were followed when determining the pathogenic bacterial colonies MIC reference range. A sample was considered positive if a single micro-organism was isolated with a concentration of >10 5 CFU/ml and related to microscopy findings of >5 leucocytes per high power field.
The main reagents in the drug sensitivity test include yeast-like fungal drug-sensitive reaction strip (biomerieux Company) and a drug-sensitive reaction card (biomerieux Company). 10

| Statistical analysis
Continuous data were presented as means ± SDs. The chi-square test and Mann-Whitney U test were employed to compare uropathogen distribution and susceptibility between two groups. Where a chi-square test was not suitable, Fisher's exact probability test was used. p < 0.05 was considered statistically significant. SPSS version 21.0 software was utilized for data analysis.

| The bacterial spectrum isolated from male and female patients with urinary calculi
The bacterial spectrum between male and female patients is significantly different (Table 1 and Figure 1). Particularly, E. coli dominated in both males and females, and it was significantly more frequent in females (53.2%) than in males (26.6%, p < 0.001). E. faecalis was the second most common uropathogen inferior only to E. coli, with  of the total uropathogens. Thirty-nine bacteria types were isolated from 154 males and 33 types from 205 females. Uropathogen detection rates between males and females are displayed in Figure 2. Gram-negative bacteria dominated in both groups, with a higher rate in females (78.5%) than in males (60.4%, p < 0.001). In Gram-positive bacteria, a higher rate was observed in males than females (28.6% vs. 13.7%, p < 0.001).

| The susceptibility of major gramnegative bacteria (E. coli and K. pneumoniae) to antimicrobial drugs
E. coli demonstrated a higher susceptibility in females than males to ceftriaxone, cefazolin, ceftazidime, cefotetan, gentamicin, piperacillin/tazobactam, aztreonam, imipenem, compound trimethoprim, levofloxacin, amikacin, and ciprofloxacin (p < 0.05). E. coli bacteria showed more than 60% sensitivity in males and females to cefoperazone/sulbactam, cefotetan, piperacillin/tazobactam, nitrofurantoin, and amikacin, whereas the resistance level was high in both groups to penicillin, tetracycline, vancomycin, and ampicillin. Ceftriaxone and cefpodoxime presented a higher susceptibility to K. pneumonia in males than females (p < 0.05). K. pneumonia bacteria exhibited more than 60% sensitivity in males and females to cefotetan, piperacillin/tazobactam, imipenem, compound trimethoprim, and amikacin. In contrast, the resistance level was high in both groups to penicillin, tetracycline, vancomycin, ampicillin, and nitrofurantoin (

| DISCUSS ION
Herein, significant differences were observed in uropathogen microbial spectrum between male and female patients with urinary stones. E. coli was the most common bacteria in both groups, possessing higher frequency in females (53.2%) than in males (26.6%, p < 0.001). It may be explained by colonization of gastrointestinal pathogens around periurethral, since these bacteria are common flora in the gastrointestinal tract. Women have short urethral than men, this anatomical and physiological features increased the risk of bacterial rise from the perianal region into bladder. 11 However, less than 62%-75% of studies reported uncomplicated UTIs. 12,13 This discrepancy might be due to more complex bacterial patterns in stone patients which were mainly reflected in the presence of calculi, invasive procedures, catheter-associated placement, etc. 14 It was notable that E. faecalis was the second most common uropathogen in patients with stones. In contrast to our findings, studies showed that K. pneumonia was the second frequent bacteria in hospital and community-acquired UTIs. 12,13 It may due to the reason that E. faecalis was prevalent in catheter-associated UTIs. 15 Urinary catheters provide a surface for E. faecalis attachment and biofilm formation, promoting E. faecalis persistence in the bladder and further dissemination to the kidneys. 16 Interestingly, E. faecalis was especially higher in males than females (15.6% vs. 2.9%, p < .001), possibly because elderly men typically suffer urodynamic dysfunction owing to prostatic hypertrophy, indwelling or intermittent bladder catheterization as the most effective and commonly used treatment. 17,18 What's more, several explanations have been suggested that males at the end of the lifespan exhibit an increased incidence of UTI. Uncircumcised was clearly a risk factor for UTI than their circumcised counterparts. 19 In addition, various chronic conditions in males (diabetes and spinal cord injury) also promote UTI. 18 According to EAU Urological Infections Guidelines 2020, the  be based on local drug resistance research, and the protocol should be adjusted according to drug susceptibility. 21 The antimicrobial sensitivity and resistance interpret that empirical antibiotic choice should take the patient gender into consider-

| CON CLUS IONS
The uropathogen microbial spectrum in females with urinary stones is different from males. High susceptibility antibiotics should be used empirically according to gender to avoid increased multidrugresistant bacteria.

CO N FLI C T O F I NTE R E S T
All authors declare no conflicts of interest in this study.

AUTH O R CO NTR I B UTI O N S
All authors made contributions to the study, and Jie Gu performed database analysis and drafted the study. Xiaobo Zhang and Yao Bai contributed to all the designs of the study. Xiong Chen, Zhiming Yang, Yao Bai, and Xiaobo Zhang reviewed the draft of study. Jie Gu collected the data.

DATA AVA I L A B I L I T Y S TAT E M E N T
The dataset generated during and/or analyzed during the current study is available from the corresponding author on reasonable request.