Microbial Predominance and Antimicrobial Resistance in a Tertiary Hospital in Northwest China: A Six-Year Retrospective Study of Outpatients and Patients Visiting the Emergency Department

Background With the wide use of antibiotics, antimicrobial resistance becomes a serious issue. Timely understanding of microbial pathogen profiles and the change of antimicrobial resistance provide an important guidance for effective and optimized use of antibiotics in local healthcare systems. The aim was to investigate the characteristics of microbial species and their antimicrobial resistances in a tertiary hospital with an Emergency Department and outpatient clinics for a period of six years. Methodology. A retrospective study was conducted using the HIS database of a tertiary hospital between 2013 and 2018. Antimicrobial susceptibility was tested by automated systems and/or the Kirby–Bauer disc diffusion method. The data were analyzed using the WHONET 5.6 software. The Cochran-Armitage test was used to study the trends over the period of research. Results In a total of 19,028 specimens submitted for microbial tests during the period from 49 units of the hospital, only the samples from the Emergency Department and Kidney Transplantation Clinic showed an annually significant increase (P < 0.001). More than 200 species with 46.4% gram-positive cocci and 45.3% gram-negative bacilli were identified in the 3,849 nonrepetitive isolates. The methicillin-resistant S. aureus and S. epidermidis rates were 25.1% and 74.6%, respectively. 60.9% E. coli and 33.5% K. pneumonia samples carried extended-spectrum-β-lactamase. All Staphylococci and Enterococci samples were not resistant to linezolid, vancomycin, and tigecycline. In addition, only 0.01% E. coli, 1.1% K. pneumonia, and 18.7% P. aeruginosa isolates showed resistance to carbapenems. Conclusions Vancomycin, linezolid and tigecycline were the most effective antibiotics for outpatients with gram-positive infection. Carbapenems were the most effective antibiotics for gram-negative infection. There was no significant annual increase of common multidrug resistances.


Introduction
Increased usage of broad-spectrum antimicrobial treatments leads to microbial resistance to the treatments that were originally sensitive. e increased antimicrobial resistance (AMR) limits treatment options and is considered as a global challenge to public health with increased morbidity, mortality, and healthcare costs [1] To accurately estimate the challenge, the surveillance of AMR profiling at local, regional, and national levels has been employed to understand the global trends on the type of predominant pathogens and their respective resistance profiling [2]. In China, the surveillance of AMR is established at the hospital, provincial, and national levels. For example, CHINET is one of well-established national surveillance networks for AMR [3][4][5][6].
is network recently reported that the frequency of gram-negative bacilli among clinical isolates was over twice higher than that of gram-positive cocci [3,4]. In addition, it is critical to recognize that China is a large country with variety of microbial occurrences and AMR profiling. For example, Xiao et al. reported that the percentage of MRSA (methicillin-resistant Staphylococcus aureus) in North Central and South Central China is higher than other regions [7]. e geographic variety is likely associated with regional differences in socioeconomic development [7]. erefore, the microbial predominance and AMR profiling in a specific region and province is not always consistent with the trend at a national level. Indeed, the surveillance of AMR at the hospital and provincial levels is a valuable complement to the national surveillance. Furthermore, the updated AMR profiling at the hospital and regional levels is particularly important for physicians to determine the adequate empiric antimicrobial therapy in clinical practice. erefore, the objectives of this study were to analyze the data from a six-year surveillance of AMR at an university affiliated tertiary hospital and to obtain the characteristics of microbial species and their antimicrobial resistances for both surveillance and clinical practice perspectives. e routine surveillance was mainly focused on hospitalacquired infections, and most specimens were collected from inpatients with severe infections [8], while community-acquired infections from outpatient service were always underreported [2,8]. erefore, in our study, we tried to bridge the gap and studied the microbial predominance and AMR profiling in outpatient clinics and emergency department in a specific region of Northwestern China. Our results were also compared with the findings from the CHINET.

Data Source.
is study was performed at the First Affiliated Hospital of Xi'an Jiaotong University, which is one of the largest hospitals in northwest China. It provides medical and surgical care to the residents of Shaanxi Province with a total population 37.33 million. e institutional review board at the First Affiliated Hospital approved this study (No: XJTU1AF2017LSK-83) and written informed consent was not required because the laboratory tests for microbiology are part of standard care and the patient records were excluded prior to this analysis. Archived laboratory data between 2013 and 2018 were retrieved from the HIS database of the hospital for analysis. A total of 19,028 specimens were collected from the emergency department and 49 outpatient clinics. A full list of these clinics is provided in Table 1. e specimens included urine, blood, prostatic fluid, sputum, pleural effusion and ascites, stool, dialysate, pus, secretion, drainage, cerebrospinal fluid, and bronchoalveolar lavage fluid.

Isolate Identification.
e clinical specimens were processed according to the recommended microbiological procedures as previously described [9][10][11][12]. Species were identified through colony morphology, conventional biochemical reactions and/or the use of an automated system (bioMerieux, Marcy l'Etoile, France).

Statistical Analyses.
Data from the isolates and the susceptibility testing were analyzed using the WHONET 5.6 software provided by the World Health Organization. e Cochran-Armitage test was used to study the trends of the specimen numbers submitted from different outpatient clinics, different specimen types, and the antibiotic resistance percentages over time.
is statistical analysis was performed using R 3.6.1 (R Foundation, Vienna, Austria) and a two-sided P-value 0.05 was considered as statistically significant.

Specimens' Information.
From 2013 to 2018, a total of 19,028 specimens were submitted to the clinical microbiology laboratory from 49 outpatient units. e total number of specimens increased from 2,303 in 2013 to 5,378 in 2018. As observed in Table 1, the number of specimens submitted from each department showed an increase over time. e top three units with the largest number of submissions were Emergency (30.7%), Urology (18.9%), and Nephrology (16.0%). However, only the Emergency and Kidney Transplantation Clinic showed significant increases in the percentage of specimens submitted annually (P < 0.001).
Notably, the data from the Breast Surgery Clinic were incomplete and therefore were not included in the analysis.
ere were no significant differences for the percentages of specimen submission from the Peritoneal Dialysis Clinic. In contrast, all other departments showed a significant decrease in the annual percentage of specimen submission (Table 1). Table 2, the number of submitted specimens increased annually among different types of specimens, which was consistent with the increase of total number of specimen submission each year in Table 1. Sputum, whole blood, and pus showed a significant increase of specimen submission annually (P < 0.001). In contrast, urine, prostatic fluid, and other types of specimens showed a significant decrease of specimen submission annually (P < 0.001). e submissions of dialysate, peritoneal drainage, and pleural effusion and ascites had no significant changes over the period (P > 0.05).

Common Microbial Isolates and Species.
In a total of 4,719 isolates of bacteria and fungi identified, after excluding duplicate isolates obtained from the same patient (each patient was sampled only once), there were 3,849 nonrepetitive isolates. ese isolates belong to 211 different species, including 1,786 Gram-negative isolates (44.6%), 1,744 Gram-positive isolates (45.3%), 150 fungus isolates (3.9%), and 169 isolates containing other species (4.4%). e most frequently identified Gram-negative species were Escherichia coli, Klebsiella pneumonia and Pseudomonas  Table 3 shows the number and percentage (%) of bacterial isolates from the outpatient specimens annually between 2013 and 2018. e percentage of S. aureus (P � 0.002) and E. cloacae (P � 0.033) demonstrated a significant increase annually, while S. epidermidis (P < 0.001) and S. haemolyticus (P < 0.001) showed a decrease. Candida albicans (78 isolates) and Aspergillus fumigatus (24 isolates) were the major fungi isolated, followed by Aspergillus flavus (9 isolates), Genus of Mucor and Fusarium (9 isolates), and     (Table 4) without significant changes annually (Table 5). In contrast, the CHINET reported that the prevalence of MRSA decreased from 69.0% in 2005 to 35.2% in 2017 [3]. e difference between our study and the CHINET is likely due to the lower prevalence of MRSA in community-acquired infections over the period in our study, suggesting the infiltration of MRSA isolates from hospitals to the community is very limited. As shown in Table 4, less than 30% of S. aureus isolates were resistant to trimethoprim/sulfamethoxazole (29.5%), gentamicin (23.3%), ciprofloxacin (17.6%), levofloxacin (17.6%), and moxifloxacin (6.6%), indicating a minor resistance to fluoroquinolones and aminoglycosides. In comparison, more than 30% of S. aureus isolates were resistant to erythromycin (70.5%), clindamycin (41.4%) and tetracycline (41.4%). Like S. aureus, almost all of S. epidermidis isolates (96.6%) in our community-acquired infections were resistant to penicillin ( Table 4). Strains of S. epidermidis that were resistant to oxacillin and methicillin were historically termed methicillin-resistant S. epidermidis (MRSE). Our study showed that the percentage of MRSE isolates was 74.6% during the study period (Table 4) without significant changes annually (Table 5). Less than 30% of S. epidermidis isolates were resistant to clindamycin (22.0%), tetracycline (20.1%), gentamicin (8.6%) and moxifloxacin (7.5%), whereas more than 30% of S. epidermidis isolates were resistant to erythromycin (82.1%), compound sulfamethoxazole (64.6%), levofloxacin (40.7%), and ciprofloxacin (32.1%).
ese findings indicate a moderate resistance of S. epidermidis to fluoroquinolones and a minor resistance to aminoglycosides. Significantly, all of S. epidermidis and S. aureus isolates collected from our study were sensitive to vancomycin, linezolid, and tigecycline.

Discussion
In this study, we reported the microbial predominance and AMR profiling in a tertiary hospital using retrospective data from outpatient clinics and emergency department in a specific region of Northwestern China over six years. We will discuss our findings in the following five aspects.

e Medical Value to Study the Distribution of Specimen Submitted between Different Outpatient Clinics and Emergency Department.
ere are limited publications that compared the number and percentage of specimens submitted to clinical microbiology laboratories between different outpatient clinics. Different from other countries, patients in China can directly visit healthcare services without referral requirements from primary care doctors. Our study showed a trend of increasing number of specimen submissions in each clinic (Table 1), likely associated with the increased population in the communities. e departments of Emergency, Urology, and Nephrology were among the top three clinics that submitted specimens for microbiological studies. Urinary tract infection has been considered the major reason for outpatients to visit the Urology and Nephrology departments [15] and is also one of the major concerns for the Emergency visits [16]. More importantly, our study revealed that only two departments, the Emergency Department and Kidney Transplantation Clinic, demonstrated a significant increase of specimen submitted annually (P < 0.001).
is indicates that these two departments, particularly the Emergency Department  [17], are in the frontline of infection control. e increased importance of the Emergency Department is likely associated with enhanced medical functions of the emergency service. ere is a trend for the Emergency Department to handle more previously inpatient cares. e increased importance of the Kidney Transplantation Department indicates that Kidney Transplantation is becoming a mainstream medical service and repeated infection after kidney transplantation is an urgent medical issue in outpatient clinics [18].

Relatively Lower Ratio of Gram-Negative Isolates in Our
Community-Acquired Infections. Among 3,849 nonrepetitive isolates, the ratio of Gram-negative to Grampositive was nearly 1:1 (45.3% vs. 46.4%) in our study while the ratio reported by the CHINET was roughly 2:1 with approximately 70% of Gram-negative bacteria [3,4]. is difference is probably because that the CHINET data were significantly derived from inpatient service and our data were from a mixture of outpatient and inpatient services. Studies from US hospitals reported that Gram-negative bacteria were more common in cases with ventilator-associated pneumonia and urinary tract infections [19] and Gram-negative bacteria were the dominant type of infection at the intensive care units [20]. ese results suggest that there is a likely association between the higher ratio of Gram-negative infections and the inpatients experiencing invasive medical devices and surgical procedures. Indeed, the Gram-negative bacteria contain highly efficient mechanisms of antibiotic drug resistance [21]. By comparing Tables 4 and 6 in this study, Gram-negative bacteria, particularly E. coli, appeared more resistant to the antibiotics and required more complicated therapeutic regimens. Overall, relatively lower ratio of Gram-negative isolates was likely a general feature in outpatient service.

Vancomycin, Linezolid, and Tigecycline Were the Most
Effective Antibiotics for Patients with Gram-Positive Infection in Our Community. Vancomycin, linezolid, and tigecycline have been used to treat multidrug resistant bacteria in the community [22]. In our study, Staphylococci and Enterococci (Table 4) were 100% sensitive to vancomycin and linezolid, which was in general consistent with the findings from the CHINET. For example, the CHINET surveillance in 2018 showed that Staphylococci isolated from blood, urine, the lower respiratory tract, and cerebrospinal fluid were 100% sensitive to vancomycin and linezolid except less than 1% of Staphylococci isolated from blood was resistant to linezolid [4]. Moreover, less than 5% of E. faecium and less than 1% of E. faecalis were vancomycin-resistant in the CHINET surveillance from 2005 to 2017 [3]. ese findings indicate that vancomycin and linezolid remain the most effective treatment for Gram-positive infection for both hospital and community acquired infections.
In our study, Staphylococci and Enterococci (Table 4) were also 100% sensitive to tigecycline, which was an expanded broad-spectrum intravenous glycylcycline antibiotic.
is finding suggests that tigecycline is a primary antibiotic treatment for multidrug-resistant bacterial infection in our communities.

Carbapenems Were the Most Effective Treatment for
Patients with Gram-Negative Infections in Our Communities. Table 6 shows higher resistance rates of Gram-negative pathogens, particularly E. coli, to the cephalosporins and quinolones in this study. To overcome the clinical challenge, carbapenems such as imipenem and meropenem have been used for these multidrug-resistant bacterial infections [23]. Table 6 also shows that the percentages of E. coli, K. pneumonia and P. aeruginosa isolates resistant to imipenem were 0.0%, 1.1%, and 18.7%, respectively. Moreover, the annual rate of resistance of E. coli and K. pneumonia to carbapenems showed no significant increase as shown in Table 5. ese findings support that carbapenems should be a primary antibiotic for multidrug-resistance bacterial infections in our communities.

ere Was No Significant Increase in Multidrug-Resistant
Bacteria Observed in Our Communities. Tables 4 and 6 show higher resistance rates between certain bacteria and antibiotics, such as methicillin-resistant Grampositive pathogens (MRSA and MRSE) and extended spectrum β lactamases (ESBLs)-resistant Gram-negative pathogens. By using ceftriaxone and oxacillin, Table 5 indicates an annual change of the resistance rate of multidrug-resistant bacteria in recent years. A significant increase of resistance rate would require immediate intervention to investigate the reason. e results in Table 5 suggest that there is no significant increase of multidrug-resistant bacteria, indicating a reasonable prescription of antibiotics in our outpatient and emergency services and successful physician education on the prevention of antibiotic resistance. e main limitation in this study was that this was a single-center retrospective observational study. It should be cautious to translate our results to other hospitals. However, the quality of clinical sampling procedures and techniques were in general better controlled in a single center than multiple centers. First, they also allowed us to obtain a representative specimen in many clinical departments throughout the hospital. When we repeated the sample collection and analysis annually, we could observe trends over a period of time. In addition, a single-center study, when appropriately performed, is a useful and inexpensive surveilling tool to reflect the regional situation of prevailing microorganisms and their resistance to antimicrobials. Second, outpatient surveillance is an important tool to study community-acquired infections. It should be emphasized that not all outpatient and inpatients visiting the Emergency Department with infections are contracted from the communities. is is the reason we only used the term of "community settings" in this study since the patients seeking medical care in our outpatient and emergency services come directly from the community.

Data Availability
All the data are available upon request.

Ethical Approval
is project was approved by the Institutional Review Board of the First Affiliated Hospital of Xi'an Jiaotong University (No: XJTU1AF2017LSK-83).

Consent
Written informed consents cannot be obtained because the laboratory tests for microbiology are part of standard care and the records of the patient's personal identities were removed prior to data analysis.

Conflicts of Interest
ere are no conflicts of interest.

Authors' Contributions
Caifeng Wang was responsible for study design, funding application, and manuscript writing and managed the study. Jine Lei performed laboratory analysis. Wen Li and Yali Li performed data entry. Juanjuan Gao performed statistical analysis of data. Dancheng Zhang and Fang Li performed data analysis.