Antibiotic Sensitivity of Proteus mirabilis Urinary Tract Infection in Patients with Urinary Calculi

Background The study's objective was to determine Proteus mirabilis susceptibility in individuals with urinary tract infections and stones to antibiotics and prescribe optimal antimicrobial treatment. Methods Nonrepetitive Proteus mirabilis strains were isolated from urine specimens obtained from 317 patients diagnosed with urinary stones from January, 2018, to December, 2021. A VITEK mass spectrometer was used for species identification, and a VITEK-compact 2 automatic microbial system was used for the antimicrobial susceptibility test (AST). Susceptibility to imipenem and cefoperazone/sodium sulbactam was tested by the disc diffusion method (K-B method). The antibiotic sensitivity of the strains was analyzed by sex and season. Results A total of 317 patients were reviewed: 202 females (63.7%) and 115 males (36.3%). Proteus mirabilis infections were observed during spring (21.8%, n = 69), summer (26.2%, n = 83), autumn (33.8%, n = 107), and winter (18.2%, n = 57). Proteus mirabilis infections in females were diagnosed most often during the fall (24.3%, n = 77) and during the summer in males (11.0%, n = 35) (p = 0.010). Female patients responded best to levofloxacin (p = 0.014), and male patients responded best to sulfamethoxazole (p = 0.023). Seasonal variation in antibiotic sensitivity was confirmed, with significantly higher rates in the winter for cefuroxime (p = 0.002) and sulfamethoxazole (p = 0.002). Significant seasonal increases were also found in levofloxacin sensitivity during the summer (p = 0.005). Conclusions Highly effective antibiotics such as cefoxitin and ceftazidime should be used empirically by considering antibiotic sensitivity changes by sex, season, and year. Regional studies should be conducted frequently.


Introduction
Bacterial resistance is one of the biggest risks to global public health since it is growing more prevalent [1,2]. Urinary tract infections (UTIs) should be taken seriously. Urolithiasis is the most common urological disease. A high percentage of patients with urinary stones develop UTIs, and the two are closely linked. In addition, some infected stones may contain bacteria. Proteus mirabilis, a member of the Enterobacteriaceae family, can cause UTIs and is the second leading pathogen after Escherichia coli [3,4]. Proteus mirabilis is closely associated with complicated UTIs, especially in patients with functional or anatomical abnormalities, such as urinary stones and long-term catheters [5].
Te abuse of antibiotics has led to enhanced bacterial pathogenicity due to resistance [6]. Te resistance of enterobacteria, including Proteus mirabilis, has increased signifcantly, particularly to cephalosporins, which poses a serious challenge for the clinical treatment of UTIs [7]. Te features and sensitivity patterns of urine bacteriology in patients with stones and Proteus mirabilis infection have not been fully researched, particularly in China. Terefore, we conducted this study to investigate the characteristics of Proteus mirabilis associated with UTIs in patients with urinary stones and to guide the correct and efective clinical treatment.

Study Background and Population.
Tis was a retrospective analysis of patients with urinary stones and Proteus mirabilis infection who visited Taizhou Hospital of Zhejiang Province afliated to Wenzhou Medical University, a 1500bed medical center located in Taizhou, Zhejiang Province, from January, 2018, to December, 2021. Two infectious disease specialists reviewed the urine culture and antimicrobial susceptibility test results. Two clinicians collected the following medical records: diagnosis, admission date, age, sex, urine culture results, and in vitro antimicrobial susceptibility. Inclusion criteria were as follows: (1) nonenhanced CT was employed to diagnose urinary calculi. (2) Only patients with stones in which Proteus mirabilis was identifed as a urogenic pathogen were eligible. (3) Te frst Proteus mirabilis isolated from the urine of each stone patient was considered. Exclusion criteria were as follows: patients with multiple positive urine cultures were excluded. Te study protocol was approved by the Medical Ethics Committee of Taizhou Hospital, Zhejiang Province. Te seasons were divided into spring (March-May), summer (June-August), autumn (September-November), and winter (December-February of the following year).

Collection of Urine Specimens.
After routine perineal disinfection of the patient, approximately, 25 ml of urine was collected from early morning to mid-morning and placed in a sterile cup. Te specimen was sent for examination immediately after collection. If testing could not be performed within 30 minutes of collection, the specimen was stored in a 4°C refrigerator for no more than 24 hours.

Reagents and Instruments.
A VITEK mass spectrometer, VITEK-compact2 automatic microbial system, and supporting antimicrobial sensitivity card GN334 were purchased from bioMerieux, France. Antimicrobial-sensitive discs of imipenem and cefoperazone/sodium sulbactam were purchased from Oxiod, UK.

Quality Control Strain. Escherichia coli (ATCC 25922)
and Pseudomonas aeruginosa (ATCC 27853) were purchased from Guangzhou Dijing Microbiological Technology Co., Ltd. (Guangzhou, Guangdong, China) and used for quality control during the AST.

Antimicrobial Sensitivity
Test. Sensitivity to levofoxacin, cefuroxime sodium, cefoxitin, ceftazidime, ceftriaxone, cefoperazone/sulbactam, cefepime, amicacin, piperacillin/ tazobactam, and sulfamethoxazole was evaluated by a VITEK-compact2 automatic microbial system with supporting reagents. Te K-B method was used to evaluate sensitivity to imipenem and cefoperazone/sulbactam, and the antimicrobial sensitivity test results were measured according to the guidelines of the CLSI M100 31TH standard of 2021 [8].

Sex Distribution of Antibiotic Sensitivity.
According to CLSI guidelines, all 317 strains of Proteus mirabilis cultured from patients with urinary calculi were submitted for routine antibiotic sensitivity tests. Table 2 lists the diferences in the sex distribution of Proteus mirabilis strains when treated with 12 diferent antibiotics. Overall, the distribution of susceptibility to the tested antibiotics (meropenem, imipenem, cefuroxime sodium, cefoxitin, ceftazidime, ceftriaxone, cefoperazone/sulbactam, cefepime, amikacin, and piperacillin/tazobactam) was the same in both male and female patients. However, female patients were more responsive to levofoxacin (62.9% vs. 48.7%, p � 0.014), and male patients were more responsive to sulfamethoxazole (41.7% vs. 29.2%, p � 0.023). Table 3 lists the annual and total susceptibility rates of Proteus mirabilis strains to 12 diferent antibiotics. Overall, the sensitivity rates of the tested antibiotics showed a relatively stable pattern over the approximately three-year study period, although the annual sensitivity rates of levofoxacin in 2021 (54.8%) were higher than those in 2020 (50.8%) (p � 0.028), and the annual sensitivity rates of cefuroxime decreased each year from 58.4% in 2019 to 52.7% in 2020 to 41.4% in 2021 (p � 0.045). Te total sensitivity of Proteus mirabilis strains to antibiotics was the highest for amikacin (99.1%), meropenem (98.1%), imipenem (96.2%), cefoperazone/sulbactam (95.9%), and piperacillin-tazobactam (87.7%) ( Table 3). In contrast, sulfamethoxazole was the least efective antibiotic, with only 33.8% of Proteus mirabilis isolates showing sensitivity. Te overall three-year rates sensitivity to levofoxacin (57.7%), cefuroxime (50.8%), and ceftriaxone (61.8%) ranged from 50 to 65%. In general, the rate of Proteus mirabilis sensitivity to cephalosporin was higher than 50%, and the rate of sensitivity to cefoperazone/sulbactam was the highest (96.9%) (third-generation cephalosporin + βlactamase inhibitor). Seasonal variations in antibiotic susceptibility rates were confrmed. Tere was a signifcant seasonal increase in sensitivity to cefuroxime (65.5%, p � 0.002) and sulfamethoxazole (48.3%, p � 0.002) during winter (December to February). Summer (June-August) was associated with a higher sensitivity to levofoxacin than other seasons (72.3%, p � 0.005). Te rates of sensitivity to meropenem, imipenem, cefoxitin, ceftazidime, ceftriaxone, cefoperazone, sulbactam, cefepime, amikacin, and piperacillin-tazobactam among Proteus mirabilis strains isolated from urinary stones showed no signifcant difference when stratifed by season (Table 4).

Discussion
Proteus mirabilis is a Gram-negativerod-shaped bacterium that frequently causes catheter-associated UTIs that may be associated with urolithiasis due to the bioflmforming ability and invasion of urinary epithelial cells by urease, which catalyzes the hydrolysis of urea leading to alkalinization of urine and the development of bladder or kidney stones [5]. In this regard, Proteus mirabilis is the leading cause of struvite formation with magnesium ammonium phosphate and carbonate apatite [9]. UTIs caused by Proteus mirabilis are generally more severe than those caused by E. coli and are associated with a higher incidence of pyelonephritis [10]. To our knowledge, the characteristics and antibiotic-sensitive patterns of Proteus mirabilis infection in kidney stone patients have not been extensively studied thus far, especially in China; thus, an in-depth understanding of the  patterns of antibiotic sensitivity in Proteus mirabilis is necessary to ensure efective treatment. Terefore, we conducted this study to investigate the characteristics of Proteus mirabilis sensitivity to antibiotics in patients with urinary calculi and to provide evidence for appropriate antimicrobial therapy. Proteus mirabilis may exhibit diferent epidemiological characteristics due to season, sex, age, and regional diferences. Terefore, regional studies conducted during diferent seasons are essential for a better understanding of the disease, effective treatment, and prevention of complications. Our review found that Proteus mirabilis infection is more common in women than men as are infective struvite stones [11]. Tere have been few studies on the susceptibility of Proteus mirabilis to antibiotics associated with urinary tract infection or urinary stones, particularly during various seasons. UTIs caused by E. coli or Klebsiella are more common during summer and less common during spring [12]. Our study found that UTIs caused by Proteus mirabilis in urolithiasis patients were the most common during autumn and the least common during winter. If sex was considered, Proteus mirabilis   infection in females was more common during autumn and less common during spring. Proteus mirabilis infection in males was more common during summer and less common during winter. Terefore, sex, season, and etiological factors should be considered before initiating empiric treatment in light of these fndings. Understanding antibiotic sensitivity in the region where patients live will help in selecting the appropriate empiric antibiotic for the treatment of Proteus mirabilis infection. Over time, however, antibiotic sensitivity patterns in the region are likely to change. Especially in developing countries such as China, antibiotic sensitivity rates are quite low due to the inappropriate use of antibiotics. In a study conducted from 2010 to 2015 in China, the most efective antibiotics for patients with Proteus mirabilis infection and urinary calculi were as follows: furantoin (0.6%), tigecycline (15.4%), sulfamethoxazole (51%), ampicillin (56.5%), cefazolin (55.8%), imipenem (93.4%), meropenem (100%), ciprofoxacin (67.6%), levofoxacin (85.6%), cefoxitin (93.7%), cefoperazone sulbactam (100%), ceftriaxone (93.7%), cefepime (95.6%), and amikacin (97.6%) [13]. A study in Brazil found that efective antibiotics against Proteus mirabilis isolated from patients with communityacquired urinary tract infection were as follows: sulfamethoxazole (78.1%); naphthalinic acid and gentamicin (94.5%); norfoxacin and ciprofoxacin (96.7%); amikacin and amoxicillin + clavulanic acid (99.5%); ampicillin (80.3%); cephalosporin (97.8%); cefuroxime, ceftriaxone and cefepime (98.4%); and ertapenem, meropenem, and piperacillin + tazobactam (100%) [14]. Bandy et al. retrospectively analyzed the antimicrobial spectrum of Enterobacter in a referral hospital in Al Khov, Saudi Arabia, in 2019 and found that the antibiotics used to treat Proteus mirabilis infection that were efective were as follows: furantoin (0.0%), sulfamethoxazole (15.8%), ampicillin (14.0%), cefazolin (55.8%), levofoxacin (13.1%), cefoxitin (78.2%), ceftazidime (25%), ceftriaxone (21.8%), cefepime (23.2%), amikacin (51.4%), and cefuroxime (20%) [15]. Our study found that levofoxacin had a low efective rate when used to treat Proteus mirabilis infection in female and male patients with urinary calculi, which is worthy of attention. Te American Medical Association recommends that clinicians treat men and women who have pyelonephritis simply with fuoroquinolones (5 to 7 days) for a short period, depending on antibiotic sensitivity. Fluoroquinolones are the frst line of treatment [16]. Clinical practice guidelines for the antibiotic treatment of community-acquired UTIs (CTIS) published in Korea in 2018 indicate that pyelonephritis patients who have urinary tract obstruction (e.g., urolithiasis) should be administered empiric antibiotics according to the treatment regimen of pyelonephritis alone, and fuoroquinolones can be used as early empiric antibiotics [17]. Te Infectious Diseases Society of America (IDSA) clinical practice guidelines for treating simple cystitis and pyelonephritis also recommend fuoroquinolones as frstline treatment for patients with complicated pyelonephritis [18]. However, our study found that fuoroquinolone antibiotics are not suitable for the initial treatment of Proteus mirabilis. In view of the low response rates of Proteus mirabilis strains to sulfamethoxazole, levofoxacin, cefuroxime, and ceftriaxone in China, empiric antibiotic therapy for infections with Proteus mirabilis strains should use the more efective cefoxitin, amikacin, meropenem, imipenem, cefoperazone sulbactam, and piperacillin-tazobactam and should avoid the use of sulfamethoxazole, levofoxacin, and cefuroxime. We also assessed seasonal patterns of urine-isolated Proteus mirabilis sensitivity to antimicrobials. We found that the sensitivity of Proteus mirabilis isolates to cefuroxime and sulfamethoxazole showed a seasonal peak during winter, while levofoxacin sensitivity had a summer and winter peak. Previous studies have demonstrated a temporal association between antibiotic prescription use and enterobacterial susceptibility in the community, although we could not examine this relationship in the current study [19]. Te signifcant seasonal changes in levofoxacin sensitivity observed here are inconsistent with previous studies of Enterobacter isolated from the urine of patients in Australia, where the authors reported no seasonal changes in quinolone sensitivity in subjects from Tasmania [10]. However, Australia is more restrictive in prescribing fuoroquinolones than China, which may account for the conficting results. More recently, Martinez et al. demonstrated an association between ciprofoxacin sensitivity in community Enterobacter urine isolates and ciprofoxacin used during the preceding 3-6 months, suggesting that ciprofoxacin sensitivity is responsive to short-term changes in antibiotic use [20]. Te seasonal variation in sulfamethoxazole sensitivity observed here is inconsistent with previous studies on the seasonal relationship between community antibiotic use and resistance in the United States. Te authors suggest that sulfamethoxazole sensitivity is high during summer unlike during other seasons [20]. Terefore, further investigation is needed to understand the drivers of seasonal variations in Proteus mirabilis susceptibility to sulfamethoxazole.

Conclusions
Patients with Proteus mirabilis-infected urinary calculi in developing nations such as China are infected with strains that have a high rate of antibiotic resistance. Because of the substantial number of female patients with urolithiasis caused by Proteus mirabilis infection, levofoxacin, sulfamethoxazole, and cefuroxime should no longer be the primary options for empiric antibiotic therapy. Cefoxitin, a second-generation cephalosporin, and ceftazidime, a third-generation cephalosporin, should be preferred instead. We feel that primary care physicians should be taught how to choose more suitable antibiotics. Regional studies on UTIs involving urinary stones should be performed more regularly, as antibiotic resistance changes by season and year.

Data Availability
Te data that support the conclusions of this study are accessible upon reasonable request from the corresponding author.

Conflicts of Interest
Te authors declare that they have no conficts of interest.