Healthcare-associated infection (HAI) includes nosocomial infections occurring in hospitals, out-of-hospital infections resulting from discharge, and outbreaks in the community (1). According to a study in the United States, HAI caused by multidrug-resistant bacteria infections occur in more than two million people each year. It was confirmed to account for 1% of all deaths (2). HAI includes surgical wound infections, bloodstream infections, urinary tract infections, and respiratory infections. Patients with significantly reduced immunity, such as the elderly, chronically ill, and organ transplant patients, are particularly vulnerable to HAI. Among the various treatments of patients, renal patients undergoing hemodialysis are treated with reduced immunity and are easily exposed to HAI, including injection needle accidents and multidrug-resistant bacteria, so infection control for hemodialysis is important (3, 4). According to the Korean Nephrological Association’s report “2018 Korea’s Renal Replacement Therapy,” the number of patients undergoing renal replacement therapy in Korea is 98,746, of whom 73,059 are undergoing hemodialysis. In particular, the number of renal failure patients per one million population was reported as 1,907. The number of people with renal failure is increasing every year, and the mortality rate is high, so many academics and the media are interested. Chronic kidney disease is generally associated with old age, diabetes, hypertension, obesity, and cardiovascular disease, and hemodialysis is a major treatment for these kidney diseases (5). Most patients with CKD receive hemodialysis three times a week, and the dialysis regimen is targeted to eliminate about two-thirds of urea during each treatment (6). Waste products, such as urea, diffuse through a thin membrane that separates the dialysate flowing in the opposite direction from the blood. Most bacteria and viruses present in dialysate have a high molecular weight and cannot pass through a thin membrane. However, toxins and the like have a small molecular weight, so they can pass through the membrane, requiring special attention in the use of the dialysate (7). The quality management of dialysis water used as dialysate is important for patients exposed to large amounts of water (8). Abnormalities in the water purification facility of dialysis water cause chemical and microbiological contamination, and dialysis water contaminated with bacteria causes not only inflammatory reactions but also various diseases due to the influx of toxins into the body (9). Fever from bacterial infection is caused by bacterial inflammatory substances and stimulates the secretion of interleukin-1 (IL-1) and cytokines by peripheral blood mononuclear cells (PBMC) (10). IL-1 causes an increase in C-reactive protein (CRP), and cytokine mediates the host response to infection and is involved in acute and chronic inflammation of bacterial ions (11). Therefore, even if there is no sign of fever, increased cytokine production due to the contamination of dialysis water causes chronic inflammatory conditions, resulting in dialysis-related amyloidosis, hypoalbuminemia, atherosclerosis, hypotension, and coma (12). In medical institutions that perform dialysis, HAI should be prevented through the regular monitoring of dialysis water. Detailed guidelines for managing dialysis water are provided by organizations such as the Association for the Advancement of Medical Instrumentation (AAMI), the American National Standards Institute (ANSI), the International Organization for Standardization (ISO), and the European Best Practice Guidelines (EBPG). According to the AAMI guidelines (13), the quality control of dialysis water includes a chemical test (Table 1) to confirm the presence of heavy metals and a microbiological test (Table 2) to confirm the presence or absence of endotoxins and microorganisms. According to the AAMI guidelines, the acceptance criteria for dialysis water are less than 100 CFU/ml of bacteria and less than 0.25 EU/ml of endotoxins. In dialysis water, the action level (the concentration of bacterial contamination that must be taken before the maximum limit) is 50 CFU/ml of bacteria and 0.125 EU/ml of endotoxins, and corrective action must be taken quickly to drop below this level. As for the enforcement standards for each test, chemical tests should be conducted at least once a year, endotoxin tests must be conducted once a quarter, and microbial culture tests must be conducted once a month. For microbial culture tests, Trypticase Soy Agar (TSA) is recommended by the AAMI in the United States, and Reasoner’s 2a Agar (R2A) is recommended by the International Organization for Standardization (ISO) (14). R2A and TSA studies reported that microbial cultures using R2A showed higher microbial detection compared to TSA cultures (12, 15). However, according to a study published in 2016, there was no significant distinction in microbial cultures using TSA and R2A (16). Looking at the above foreign cases, differences were confirmed in R2A and TSA. In addition, foreign research data did not include studies on environmental factors such as climate, season, and water quality in the country. In Korea, studies related to microbial culture tests and environmental factors for R2A and TSA in hemodialysis water are insufficient, and studies suitable for the domestic environment have been judged necessary to control infection and prevent HAI in hemodialysis water.

Therefore, this study aims to analyze the microbial culture and sensitivity of R2A, and TSA used in microbiological tests of hemodialysis water, identify seasonal microorganisms, and use them as basic data for improving hemodialysis infection control.

The influx of microorganisms into the body is a risk factor that threatens human health by causing inflammatory reactions and various diseases. The rapid cultivation and identification of microorganisms is required for the treatment and prognosis of diseases. Consequently, various media have been developed, and there have been changes in the culture environment and conditions. In particular, the selection of the medium is the most important factor in increasing the detection rate of microorganisms. In this study, microbiological tests of hemodialysis water were performed using R2A and TSA to confirm the positive rate for each medium, the positive rate for each season, and the results of microbial identification. This contributed greatly to the prevention of medical-related infections and the management of artificial kidney center infections implemented in medical institutions. It is the first in the world to reflect climate change and seasonal specificity, and it is a meaningful study conducted over a long time period. The positive rates of microorganisms in hemodialysis water were confirmed in 99 cases (23.5%) in the R2A medium and 47 cases (11.1%) in the TSA medium. Therefore, the R2A medium was found to be more sensitive than the TSA medium. In the Netherlands (15), in a study conducted on 229 samples of hemodialysis water and reverse osmosis water, the R2A medium showed a higher positive rate than the TSA medium, confirming results similar to this study. In Thailand (12), a study conducted on 143 samples of reverse osmosis water, also confirmed that the R2A medium was more sensitive than the TSA medium. The positive rate of bacteria counts of 50 CFU/mL or higher was found to be 2.8% in R2A and 4.2% in TSA in this study, which was higher than that of 1.5% in R2A and 1.3% in TSA, the results of the US study. When comparing the results with those of advanced countries, it is thought that a more active management of hemodialysis water is necessary. R2A is a low-nutrient agar used with lower incubation temperatures and longer incubation times (17). However, TSA is a universal medium containing two peptones to support the growth of various microorganisms (18). Hence, the identification and positive rates of microorganisms cultured in the two media are considered different. The 188 strains of 30 species isolated in this study were classified into 33.5% Gram-positive microorganisms, 59.0% Gram-negative microorganisms, and 7.4% yeast-like fungi. Aeromonas sp., rarely cultured in TSA, is a bacterium that exists in fresh water and has been reported as a zoonotic pathogen that causes corneal inflammation in a recent domestic study (19). Moraxella sp. is also known as the causative agent of infectious diseases of otolaryngology (20). In Japan, bloodstream infections caused by Methylobacterium sp. have been reported in patients undergoing hemodialysis (21). Especially Sphingomonas paucimobilis, Acinetobacter lwoffii, and Oligella ureolytica showed high separation rates among Gram-negative microorganisms. In a Thai study, Pseudomonas spp. was 40%, Moraxella spp. 23%, Acinetobacter spp. 16%, Staphylococcus spp. 16%, Alcaligenase spp. 14%, Gram-negative rod 7%, Corynebacterium spp. 3%, Micrococcus spp. 3%, Bacillus spp. 1%, Chromobacterium spp. 1%, Gram-positive rod 1%, Rhodococcus spp. 1%, and Streptococcus spp. 1% was isolated, which was like this study in that the separation rate of Gram-negative microorganisms was high (12). The species of microorganisms isolated from hemodialysis water differed from season to season, and Acinetobacter lwoffii and Sphingomonas paucimobilis were identified as bacteria isolated throughout the year from R2A. Sphingomonas paucimobilis is an opportunistic pathogen that causes meningitis, sepsis, bacteremia, and peritonitis in people with reduced immunity (22). Acinetobacter lwoffii is a potential opportunistic pathogen in patients with impaired immune systems, and it has been identified as a cause of healthcare-associated infections (23). It is necessary to manage infections more effectively for hemodialysis water by monitoring bacteria that are separated year-round and bacteria that are separated by season. In foreign countries, there have been no studies on the detection of microorganisms in hemodialysis water related to season or climate. However, in Korea, the four seasons are distinct, and the temperature change varies greatly from season to season. The cultivation of microorganisms is closely related to the temperature and growth of bacteria, as well as the components of the medium and cultivation time. Therefore, if the R2A medium and the TSA medium are used together for the microbiological testing of hemodialysis water, it is believed that they can contribute to patient safety and public health safety infections.