Analysis of Antimicrobial Susceptibility in Bacterial Pathogens Associated with Urinary Tract Infections from Beijing Teaching Hospital in China, 2009–2017

Objective Since a urinary tract infection (UTI) is easy to relapse and difficult to treat, the antibiotic resistance rate has increased year by year in recent years. This study was to analyze the characteristics of the common pathogenic bacteria and the changes of antibiotic resistance in urinary system infection, so as to guide the standard use of antibiotics in a clinical urinary tract infection and control nosocomial infection effectively. Methods A total of 5,669 strains of a urinary tract infection in the hospital from January 2009 to December 2017 were retrospectively analyzed. Bacterial identification and the antibiotic sensitivity test (AST) were analyzed by using a VITEK-2 Compact system. Results Of the 5669 pathogens, 3,256 (57.44%) of the strains were Gram-negative bacteria (GNB), 1,474 (26%) were Gram-positive bacteria (GPB), and 939 (16.56%) were fungi. Resistant rates of ESBL-producing strains were all significantly different from non-ESBL-producing strains in Escherichia coli (p < 0.05). The resistance rate of ESBL-producing strains to β-lactam antibiotics was all higher than that of non-ESBL-producing strains in Klebsiella pneumoniae (p < 0.05). The detection rate of vancomycin-resistantEnterococcus faecium and Enterococcus faecalis was 37.3% and 3.1%, respectively, and the detection rate of linezolid-resistantEnterococcus faecium and Enterococcus faecalis was 0.68% and 0%, respectively. The drug resistance rate of candida sp. to fluconazole, itraconazole, and voriconazole was 1.7%, 8.5%, and 3.4%, respectively. No amphotericin B-resistant strains were detected in the research. Conclusions Among the 5669 strains isolated from urinary tract infection patients, GNB were the main pathogens. Escherichia coli was the major pathogen. The resistance rate of ESBLs-producingEscherichia coli was higher than that of non-ESBLs-producingEscherichia coli in general; meanwhile, β-lactam/β-lactamase inhibitors and carbapenems maintained good antimicrobial activity against Escherichia coli. The resistance rate of non-ESBLs-producingKlebsiella pneumoniae strains was significantly higher than that of ESBLs-producingKlebsiella pneumoniae strains, and drug resistance was more prominent; most of the antibiotic resistance rates were over 50%. The antimicrobial resistance rate of Enterococcus faecium was significantly higher than that of Enterococcus faecalis. There were rare linezolid-resistant strains. The antimicrobial resistance rate of imidazole to fungi was controlled less than 10%.


Introduction
A urinary tract infection (UTI) is one of the most common infectious diseases following the upper respiratory tract infection and is the main cause of morbidity and mortality in humans [1]. Pathogens can grow in the urinary tract and invade the urinary tract mucosa or tissue. 150 million people are infected with UTI each year worldwide [2]. A urine culture is currently the key standard for the diagnosis of a urinary tract infection [3]. A catheterassociated urinary tract infection is the most common nosocomial infection. With the prolongation of catheter placement time, the risk of urinary tract infections gradually increases. Some data indicate that the incidence of the urinary tract infection increases gradually with age, and most patients are bedridden due to underlying diseases such as cerebrovascular diseases, which is due to the frequent need for indwelling urinary catheters, thus signifcantly increasing the risk of urinary tract infections. Escherichia coli was the most frequent microbial agent of UT [4]. Uropathogenic Escherichia coli (UPEC) strains are the main pathogenic bacteria that cause urinary tract infections (UTIs), due to the presence of fmbrial adhesins in the pathogen [5]. One of the major difculties with diagnosis of UTI in older people is a high prevalence of asymptomatic bacteriuria (ASB): the presence of bacteria in urine of people without attributable symptoms [6]. For this group of patients, monitoring urine cultures is an efective method to diagnose the disease. Urinary tract candidiasis is the most common hospital fungal infection worldwide, and this phenomenon cannot be ignored [7]. Efective drugs can help doctors treat urinary tract infections more efectively [8]. Te abuse or overuse of antibiotics worldwide and antimicrobial resistance (AMR) are considered one of the top ten threats to global health [9]. For pathogens causing urinary tract infections, it is of great signifcance to analyze the infection characteristics for clinical diagnosis and treatment. Te classifcation and analysis of urinary data for this hospital from 2009 to 2017 could help understand the distribution characteristics and trends of nosocomial pathogens and help doctors standardize drug use.

Bacterial Isolates.
Te clinical data on patients with urinary tract infections in Beijing Shijitan Hospital from January 2009 to December 2017 were collected. A total of 5,669 strains of pathogenic bacteria were isolated from urine samples. Strict aseptic procedures were carried out during specimen collection. Laboratory data requirements do not allow multiple isolates from the same patient to be counted twice. We take a 10 μl urine sample and inoculate it tightly onto a Columbia blood plate. Plates were placed in a 5% CO 2 incubator and incubated at 35°C for 24 hours. Te number of bacterial colonies was counted on the blood plate ( Figure 1).

Antimicrobial Susceptibility
Testing. Te bacterial culture and the antibiotic susceptibility test (AST) were performed according to the National Guide to Clinical Laboratory Procedures (4 th edition). VITEK-2 Compact was used for bacteria land fungus identifcation. VITEK-2 Compact was used in bacterial drug susceptibility experiments. Te bacterial's AST results were judged according to Performance Standards for Antimicrobial Susceptibility Testing M100 (31 st Edition). Antifungal susceptibility testing was determined using an ATB FUNGUS 3(BIOMERIEUX) strip following the manufacturer's instructions. Antifungal susceptibility testing results were judged according to Performance Standards for Antimicrobial Susceptibility Testing M60.
Ceftazidime/ceftazidime-clavulanate was used to validate the production of ESBLs for isolates according to CLSI. Te ESBL-producing strain was judged to have increased ≥5 mm after the addition of clavulanic acid in the inhibition zone compared with the inhibition zone without clavulanic acid. E coli ATCC25922 and K. pneumonia ATCC 700603 were used as controls in the ESBL test.

Gender Distribution of Pathogenic Bacteria in the Urinary
System. During the past 9 years, the proportion of male and female urinary tract infections caused by pathogenic bacteria was 48.69% in female and 51.31% in male ( Table 2).

Age Distribution of Pathogenic Bacteria in the Urinary
System. As shown in Table 3, the top three age groups in urinary tract infections during the past 9 years were 81∼90 age group, 71∼80 age group, and 61∼70 age group, with 44.26%, 26.27%, and 10.23%, respectively. Te population with urinary tract infections was mainly distributed among elderly people over 60 years old, with the highest proportion being those aged 81-90.

Susceptibility of E.coli to Antimicrobial Agents.
Tere were 1495 E.coli isolates from the urinary system. It contained 651 ESBL-producing E. coli strains and 844 non-ESBL-producing E. coli strains, with a composition ratio of 43.55% and 56.45%, respectively. Te drug resistance of ESBL-producing E. coli was higher than that of non-ESBL-producingE. coli, and the resistance rate was signifcantly diferent (p < 0.05). As shown in Table 4, ESBL-producing strains were more resistant to multiple drugs and had better efects on piperacillin/tazobactam and imipenem. Te non-ESBL-producing strains had high resistance rates of ampicillin and quinolones.

Susceptibility of Klebsiella pneumoniae to Antimicrobial
Agents. As shown in Table 5, a total of 544 strains of Klebsiella pneumoniae were isolated from the urinary system, including 126 strains of ESBL-producing Klebsiella pneumoniae and 418 non-ESBL-producing strains of Klebsiella pneumoniae, with a composition ratio of 23.16% and 76.84%, respectively. Non-ESBL-producing strains were signifcantly more than ESBL-producing strains in the resistance rate of β-lactam drugs (p < 0.05), whereas the drug resistant rate of aminoglycosides, quinolones, and furans showed no signifcant diference (p < 0.05). ESBL-producing strains were highly resistant to most β-lactams. Piperacillin/ tazobactam and imipenem had high resistance rates and better drug sensitivity. For non-ESBL-producing strains, the  Canadian Journal of Infectious Diseases and Medical Microbiology form of imipenem resistance was not optimistic, and most of the beta-lactam drugs were not efective. Te resistance rate of quinolone drugs and furan drugs was higher.
3.6. Susceptibility of the Gram-Positive Bacteria. As shown in Table 6, the Gram-positive bacteria of urinary tract infections mainly consisted of Enterococcus faecium and Enterococcus faecalis. Among them, 362 strains of Enterococcus faecalis and 733 strains of Enterococcus faecium had a composition ratio of 33.06% and 66.94%, respectively. Enterococcus faecium was signifcantly more resistant than Enterococcus faecalis. Linezolid-resistant strains were not found, the VRE detection rate of Enterococcus faecalis was below 10%, and the quinolone resistance rate fuctuated around 50% in Enterococcus faecalis. Te resistance rate of Enterococcus faecium to ampicillin was higher, the resistance rate of ciprofoxacin and erythromycin was higher, clinically linezolid-resistant strains were few, and the resistance rate of vancomycin-resistant Enterococcus faecium was 37.3%; meanwhile, the resistance rate of quinupristin/davapride was 3.9%. Te bold values are the sum of the classifed and summarized data, indicating emphasis.

Susceptibility of Fungi to Antimicrobial Agents.
Amphotericin B-resistant strains were not found among the main yeasts in urinary tract infections. Te average resistance rate of the imidazole antifungal drug was below 10% (Table 7).

Discussion
Tis article makes statistics on pathogens of urinary tract infections in our hospital and provides reasonable and effective data support for clinicians in the treatment of diseases. Te existence of resistance mechanisms such as β-lactamases, efux pumps, membrane permeability, and change in the sequence of penicillin-binding proteins (PBPs) in bacteria has made the treatment of multidrug-resistant bacteria a very challenging issue globally [10]. Te relationship between antimicrobial phenotypes and bioflm formation ability has always been a topic of widespread concern in the biomedical community. Scientists believe that these two factors may have a signifcant impact on the outcome of infection [11]. Studies have shown that a novel bioflm preventive agent (BPA) can inhibit the formation of bioflms by pathogens on the catheter [12]. ESBL-producing Escherichia coli isolates and ESBLnegative isolates had signifcant diferences in drug resistance rates (p < 0.05) in ampicillin, piperacillin/tazobactam, ceftazidime, aztreonam, imipenem, amikacin, ciprofoxacin, levofoxacin, and so on. Te drug resistance rate of ESBL-producing Escherichia coli isolates was signifcantly higher than that of ESBL-negative isolates. Imipenem and piperacillin/tazobactam maintained higher antibacterial activity. No imipenem-resistant strain was found in EBSL-producing strains. Te resistance rate of Escherichia coli to amikacin was the lowest among aminoglycoside drugs. Aminoglycoside antibiotics mainly act on G-bacilli and have strong and long-lasting antibacterial activity. However, due to its toxicity to the ear and kidney, its usage rate is decreasing year by year. Comprehensive evaluation of renal function is recommended for patients with urinary tract infections. Te resistance rate of Escherichia coli to quinolones was high, and drug resistance situation was not optimistic. Te drug is widely used due to the wide antimicrobial spectrum of quinolones. Te resistance mechanism of quinolone is the carrier of quinoloneresistant genes, such as qnr (qnrA, qnrB, and qnrS), aac(6′)-Ib-cr, qepA, and oqxAB [13]. It required further study. At present, quinolones are not recommended to use. Uropathogenic Escherichia coli (UPEC) is the most important bacteria causing urinary tract infections (UTIs) in patients worldwide. Scholars have proved that DNA microarray was a suitable diagnostic technique for detecting and identifying diseases in UPEC [14]. It has been suggested to design a suitable long oligonucleotide microarray probe for the detection and identifcation of diferent pathogenic Escherichia coli, especially urinary pathogenic Escherichia coli (UPEC) [15]. A UPEC pathogen is considered to be an important microorganism with multiple virulence genes. Understanding the bacterial virulence gene map and related functional mechanisms enables us to design efective preventive approaches for the treatment of urinary tract infections [16]. Te study from Iran showed that cnf1, upaH, hlyA, ibeA, and cdtB were important virulence genes in UPEC pathogens isolated from urine. Te virulence gene of cnf1 in UPEC strains contributes to the occurrence of various types of urinary tract infections [17].
Te resistance rates of ESBL-producing Klebsiella pneumonia isolates to piperacillin/tazobactam, cefazolin, aztreonam, and imipenem were signifcantly diferent from those of ESBL-negative isolates (p < 0.05). Te drug resistance rates were not signifcantly diferent (p < 0.05) in amikacin and ciprofoxacin. Te current statistical results indicated that the resistance rate of ESBL-negative isolates strains was signifcantly higher than that of ESBL-producing Klebsiella pneumonia. Te resistance rate of ESBL-producingKlebsiella pneumonia to piperacillin/tazobactam was 13.5%; the result was similar to the previous report [18]. While the drug resistance rate to imipenem was only 2.4%, the main resistance mechanism was carbapenemase.
Te frst carbapenemase-producing Klebsiella pneumoniae was frst reported in the United States in 2001 [19]. Te KPC-type carbapenemase producing Klebsiella pneumoniae has rapidly spread around the world, and outbreaks have been reported in hospitals around the world. Te resistance rate of β-lactam antibiotics was on the rise. Te rate of drug resistance was similar to those reported in many other countries [20,21]. Te resistance of Klebsiella bacteria to carbapenem antibiotics is mainly caused by carbapenemase production (such as production of class A KPC enzymes and class B MBL enzymes). Carbapenem-resistant Klebsiella pneumoniae (CRKP) infection poses a major threat to public health. Understanding the exact mechanism by which CRKP occurs is crucial for selecting the most appropriate antibiotic [22]. Strains may or may not have resistance mechanisms such as ESBL, AmpC β-lactamase, efux pumps, and membrane pore protein mutations [23,24]. Currently, multidrug-resistant bacteria require combination drug treatment, such as tigecycline-based two-drug combination: tigecycline + carbapenems/polymyxins/aminoglycosides or polymyxin-based combination of drugs: polymyxins + carbapenems/fosfomycin/aminoglycosides or three-drug combinations: tigecycline + carbapenems + polymyxins [25].
As shown in Table 6, the drug-resistant rate of Enterococcus faecium was signifcantly higher than that of Enterococcus faecalis. Te resistant rate of vancomycinresistant Enterococcus faecium was 37.3%, which was signifcantly higher than in foreign reports. Since the frst report of vancomycin-resistant enterococci (VRE) in Britain [26] and France [27] in 1988, VRE has become an important drug-resistant strain that seriously threatens human health, and it has caused great difculties for clinical treatment. According to the US CDC report, at least 20,000 cases of medically associated infections caused by VRE occurred each year, and nearly 1,300 died of VRE infection [28]. Te possible causes may be related to the production of resistance genes such as VanA, VanB, VanC, and VanD [29]. Te fuctuation of the VRE detection rate of vancomycin-resistant Enterococcus faecium was higher than that in the US [30], the Netherlands, and Germany [31]. Five strains of linezolid-resistant enterococci were isolated. Although these strains are rare, their resistance mechanism may be related to G2576Tpoint mutation in the 23S rDNA gene and plasmid-mediated resistance of the cfr gene [32]. Te specifc mechanism requires further study. Enterococcus faecalis was signifcantly more sensitive than Enterococcus faecium. Te resistance rate of vancomycin was 3.1%, and no linezolid-resistant strains were found. Quinolone drugs have a high resistance rate in the treatment of urinary tract infections.
Te high rate of resistance to quinolones in the treatment of urinary tract infections is an indisputable fact. Hospital management departments must strictly control and take necessary measures to curb its high-drug resistance.
In urinary tract infections, Candida spp. are the most common fungal cause of urinary tract fungal infections [33]. Among the patients with urinary system fungal infections, 469 were male patients and 470 were female patients, which was basically the same. No amphotericin B-resistant strain is found in any of the three major yeasts in Table 7. Tis is also associated with adverse efects of amphotericin B, which can cause hypokalemia, renal damage, and liver damage. Te resistance rate of fuconazole was the lowest (1.7%). Fluconazole has high efcacy, low toxicity, and mild adverse reactions. It is excreted by the kidney, and the concentration in urine is 10 times that of blood, so it is the frst choice for urinary tract fungal infections. Voriconazole has high drug sensitivity. It is suitable for urinary tract infections caused by fungi.

Conclusions
Among 5,669 strains isolated from patients with urinary tract infections, GNB were the main pathogens. Te drug resistance rate of ESBL-producing E. coli was higher than that of ESBL-negative E. coli. in general. Te resistant rate of ESBL-negative Klebsiella pneumoniae was signifcantly higher than that of ESBL-producing Klebsiella pneumoniae. Te resistance rate of Enterococcus faecium to various antibacterial drugs was signifcantly higher than that of Enterococcus faecalis. Te results of the urine culture are the basis of clinical treatment. Clinicians should keep abreast of the changes in drug resistance of pathogens in hospitals. Rational use of antibiotics is very important in clinical diagnosis and treatment.

Data Availability
Te data used to support the fndings of this study are included within the article.

Ethical Approval
Te study was approved by the Ethics Committees of the Beijing Shijitan Hospital. Tis study was conducted in accordance with the Helsinki Declaration and the relevant national laws and policies.

Conflicts of Interest
Te authors declare that there are no conficts of interest.