Abstract
Background
Accurately detecting the quantity of microorganisms in hospital purified water is of significant importance for early identification of microbial contamination and reducing the occurrence of water-borne hospital infections. The choice of detection method is a prerequisite for ensuring accurate results. Traditional Plate Count Agar (PCA) belongs to a high-nutrient medium, and there may be limitations in terms of accuracy or sensitivity in detecting microorganisms in hospital purified water. On the other hand, Reasoner’s 2A agar (R2A) has characteristics, such as low-nutrient levels, low cultivation temperature, and extended incubation time, providing advantages in promoting the growth of aquatic microorganisms. This study, through comparing the differences in total colony counts between two detection methods, aims to select the method more suitable for the growth of aquatic microorganisms, offering new practical insights for accurately detecting the total count of heterotrophic bacteria in hospital purified water.
Methods
The most commonly used plate count agar (PCA) method, and the R2A agar culture were adopted to detect microorganisms and determine the total number of bacterial colonies in the water for oral diagnosis and treatment water and terminal rinse water for endoscopes in medical institutions. The two water samples were inoculated by pour plate and membrane filtration methods, respectively. Using statistical methods including Spearman and Pearson correlation, Wilcoxon signed-rank sum test, paired-Chi-square test, and linear regression, we analyze the differences and associations in the bacterial counts cultivated through two different methods.
Results
In 142 specimens of the water, the median and interquartile range of the heterotrophic bacterial colony number under the R2A culture method and under the PCA culture method were 200 (Q1–Q3: 25–18,000) and 6 (Q1–Q3: 0–3700). The total number of heterotrophic bacteria colonies cultured in R2A medium for 7 days was more than that cultured in PCA medium for 2 days (P < 0.05). The linear regression results showed a relatively strong linear correlation between the number of colonies cultured by the R2A method and that cultured by the PCA method (R2 = 0.7264). The number of bacterial species detected on R2A agar medium is greater than that on PCA agar medium.
Conclusion
The R2A culture method can better reflect the actual number of heterotrophic bacterial colonies in hospital purified water. After logarithmic transformation, the number of colonies cultured by the two methods showed a linear correlation.
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1 Introduction
Hospital purified water is widely utilized in clinical medical activities, as well as for the cleaning, disinfection, and sterilization of medical equipment and supplies. Contaminated hospital purified water can pose a risk of nosocomial cross-infections within healthcare facilities [1,2,3,4]. At present, the serious microbial contamination of hospital purified water has become a difficult problem faced by hospitals [5]. The contamination of hospital purified water can increase the risk of iatrogenic infection and the occurrence of related diseases, thus threatening the health of patients and medical staff [6, 7], especially those with basic diseases and low immunity, and even leading to death in severe cases [8, 9]. Research indicates that in a newly opened Neonatal Intensive Care Unit (NICU), there were two outbreaks of Pseudomonas aeruginosa infections over a span of more than 2 years. These outbreaks resulted in a total of 31 patient infections, with 3 fatalities. The primary cause of the outbreaks was contamination of the water supply pipes [10]. Some patients have died from infection with Legionella in the waterway during dental visits; a female patient succumbed to Legionnaires’ disease after receiving dental treatment. Upon investigation, Legionella were detected in the water discharged from the implicated high-speed turbine, and it was confirmed that the Legionella DNA genome found in the water was a complete match with the Legionella strain causing the patient’s infection [11]. Among the top ten medical technology hazards published by the Emergency Care Research Institute (ECRI) in 2016, gastrointestinal endoscopy is one of the medical devices most likely to cause nosocomial cross infection; however, the contamination of the terminal rinse water easily causes endoscope contamination, thus leading to iatrogenic infection [12, 13]. Therefore, it is of great significance to strengthen the microbiological monitoring of hospital purified water and attach importance to the strategy for detecting microorganisms in water for guiding the disinfection and quality control of hospital purified water and timely and effectively finding the source of infection and hidden danger of infection.
So far, the relevant research reports on microorganisms in hospital purified water have been mainly inclined to compare the total number of microbial colonies. However, there are relatively great differences in the detection methods for the total number of microbial colonies and also comparatively little relevant research. Currently, there is yet no uniform international standard for microbiological monitoring methods for hospital purified water [14]. Plate count agar (PCA) is the most commonly used medium for detecting the heterotrophic bacteria number in water in Heterotrophic Plate Counts (HPC). Comprising beef extract, peptone, sodium chloride, agar, and distilled water, PCA which is characterized by high-nutrient content has been used for the longest time in China. Also, it is the most widely applied medium. However, some studies have shown that the evaluation of microbial indexes in water and the cleaning and disinfection effect of waterways and pipe networks based on PCA counting results may not be complete, with the degree of microbial contamination in water possibly beyond people’s expectations [3, 6].
Research indicates that using R2A agar medium to detect the quantity of microorganisms in water yields good results [15,16,8]. The findings of our study contribute to understanding the bacterial species in hospital purified water in develo** countries. In this investigation, the diversity of heterotrophic bacteria detected on R2A agar exceeded that on PCA agar. While Corynebacterium and Burkholderiaceae were identified in PCA agar samples, heterotrophic bacteria on R2A agar were primarily classified into Pseudomonadaceae, Burkholderiaceae, Ralstonia spp, Acetobacteraceae, and Herbaspirillum. This aligns with common bacteria isolated and identified in DUWLs, as reported in the other study [33].
The presence of heterotrophic bacteria in hospital water poses certain biosecurity risks, threatening patient health and leading to infections. Burkholderia contamination in dentures may potentially result in pneumonia [34]. Burkholderia gladioli, a non-fermenting Gram-negative rod-shaped aerobe, has been reported in the literature primarily in immunocompromised adults and neonates in cases of gladiolus infection [35]. Ralstonia pickettii is not considered a major pathogen, exhibiting relatively low virulence. However, infections caused by this bacterium have been reported, including instances of bloodstream infection [36], meningitis [37], and contamination in Plasmodium in vitro cultures [38]. Brevundimonas vesicularis, a Gram-negative rod, rarely causes human infections, but there are studies reporting cases of chronic peritoneal dialysis patients develo** Sphingomonas paucimobilis peritonitis [39]. As a nitrogen-fixing bacterium, Herbaspirillum huttiense has been implicated in the first reported case of sepsis in Korea caused by this organism [40]. Gluconacetobacter and Sphingomonas paucimobilis have limited literature reporting human infections. Future investigations can employ more detailed analyses, such as colony counts and strain ty**, to gain deeper insights into the microbial diversity and abundance in hospital purified water.
The R2A culture method had higher sensitivity for the detection of microorganisms in hospital purified water. This will provide reference and guidance for medical institutions to carry out more accurate microbial detection on hospital purified water, with great significance for the timely and effective detection of hidden dangers of water-borne infection. There were the following limitations in this study. First, the acquisition time of oral samples was all within 7 days after the pipes of the oral comprehensive treatment table were disinfected. Therefore, the total heterotrophic bacterial colony number was relatively low compared with other studies. Second, this study, which was carried out in the provincial capital of Hubei Province, China, was not verified in other cities. Therefore, it was not ruled out that the detection rates under the R2A culture method could be all affected due to regional differences, the concentration of organic substances in water bodies, and the characteristics of the category, physiology, biochemistry, etc. of the primary contaminated bacteria. The research needs to be confirmed in more regional studies in the future.
5 Conclusion
Compared to the PCA culture method, the R2A culture method exhibits higher sensitivity, providing a more accurate reflection of the actual colony counts of heterotrophic bacteria in hospital purified water. It is recommended to conduct parallel specimens using the R2A method for key water departments in medical institutions (such as dental departments, endoscopy centers, disinfection supply centers, operating rooms, etc.). In circumstances where feasible, parallel testing with the PCA method should be carried out simultaneously, with a more detailed examination of colony counts, strain ty**, and other aspects. This comprehensive approach aims to thoroughly understand the contamination of heterotrophic bacteria in medical institution water sources, gradually establishing an evaluation system and quality control standards for the R2A detection method.
The selected subjects in this study are highly representative, and the comparison of detection results under different inoculation methods enhances the generalizability of the research findings. Additionally, the study identifies a linear correlation between the total colony counts of heterotrophic bacteria on PCA and R2A agar, providing a basis for comparing data from different detection methods across regions. However, due to the diversity of physiological and biochemical characteristics of bacteria, it is necessary to expand the scope of future research and further investigate the applicable scenarios and target bacteria for the R2A culture method.
Data Availability
The datasets during and/or analyzed during the current study are available from the corresponding author on reasonable request.
Abbreviations
- PCA:
-
Plate count agar
- ECRI:
-
Emergency care research institute
- HPC:
-
Heterotrophic plate counts
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Acknowledgements
The authors deeply appreciate the contribution to this thesis made in various ways by all members in the Department of Nosocomial Infection Management, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology. In addition, the authors would like to thank the anonymous reviewers for their helpful remarks.
Funding
This study was supported by the funds from the National Natural Science Foundation of China (Grant No.72004068).
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XC, HX, and YF contributed equally to this work. XC took responsibility for the integrity of the data and the accuracy of the data analysis. XC and HX drafted the manuscript. HX and YF conducted the statistical analyses. LX managed the project and provided guidance. All authors read and approved the final manuscript.
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Cao, X., **ong, H., Fan, Y. et al. Comparing the Effects of Two Culture Methods to Determine the Total Heterotrophic Bacterial Colony Count in Hospital Purified Water. J Epidemiol Glob Health 14, 184–192 (2024). https://doi.org/10.1007/s44197-023-00186-1
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DOI: https://doi.org/10.1007/s44197-023-00186-1