Environmental contamination is a major cause of HAIs, and hospitals are perfect for pathogen transmission [1].   HAIs cause more disease, longer hospital stays, higher healthcare costs, and even mortality worldwide.   Contaminated surfaces, medical equipment, air, and water can spread infectious diseases to susceptible patients and healthcare workers [2].   Bed rails, door knobs, and medical gadgets are often olonized by microbes and spread Multidrug-resistant Organisms (MDROs) such as Staphylococcus aureus, Clostridium difficile, and Pseudomonas aeruginosa.   HAIs highlight the need for effective infection prevention and management, such as environmental monitoring to identify contamination sources.

Figure 1 Microbiological Testing in Monitoring Environmental Contamination [3]

Healthcare facilities must monitor environmental contamination for many reasons.   First, it helps hospitals identify microbe-prone and risky regions for more precise cleaning and disinfection [4].   Environmental surveillance data can also guide infection control policy, ensuring evidence-based and successful operations [5]. The third argument is that environmental monitoring in high-risk areas including neonatal units, surgical wards, and critical care units is essential to prevent opportunistic infections [6]. Through environmental microorganism monitoring, healthcare facilities can assess the long-term efficacy of cleaning, disinfection, and sterilisation treatments.   Systematic microbial contamination surveillance reduces HAIs and improves patient safety [7].  This helps hospitals detect emerging diseases and take preventive actions.

Hospital environmental monitoring relies on microbiological tests.   Traditional culture-based methods are used to detect and quantify microbial contamination on surfaces and in the air.   Microswabs and contact plates capture germs from regularly touched surfaces [8].  Pathogens that cause respiratory disorders are collected by air sampling devices.  Legionella pneumophila, which can spread in hospital water systems and harm immunocompromised people, is also tested in water samples [9].   In addition to culture-based methods, Polymerase Chain Reaction (PCR) and other sophisticated molecular techniques are increasingly used to detect specific infections and drug-resistant genes [10].   PCR can detect slow-growing or hard-to-culture microorganisms due to its high sensitivity and specificity.   Combining conventional and molecular approaches helps hospitals understand the environmental microbial load and make better infection control decisions [11].

Environmental monitoring is necessary, but standardising microbiological testing and interpreting results is difficult. Sampling approach, sample size, testing frequency, and laboratory methods affect infection detection and contamination evaluation. Some environmental bacteria are harmless, while others are only deadly under particular conditions, making it difficult to determine which ones are clinically relevant.   Given this complexity, evidence-based standards are needed to ensure microbiological test accuracy and infection prevention efficacy. Adequate cleaning and environmental monitoring can significantly minimise HAIs [12].   Unfortunately, there is little data on the usefulness of microbiological testing techniques in hospitals, even tertiary care centres like PMCH, which treat many patients with complex conditions.

This study evaluates microbiological tests for environmental pollution monitoring in a hospital setting.   This study uses systematic sampling over six months to assess PMCH microorganism contamination, identify the most common bacteria, and assess environmental hygiene changes.   This study will inform environmental monitoring practices and infection control strategies.   This study also attempts to bridge the gap between microbiological surveillance data and infection prevention strategies by highlighting potential intervention sites and supporting thorough cleaning practices.   The study reveals how routine microbiological testing can protect patients and prevent healthcare workers from spreading infectious diseases.

Environmental contamination influences public health outside healthcare establishments.   Hospitals are often hubs for pathogen transmission by patients, visitors, and staff.   Thus, hospital pollution monitoring and regulation protect patients and reduce community-acquired diseases.   This study evaluates microbiological testing in support of hospital sanitation best practices to reduce HAIs. Healthcare staff need daily cleaning methods, infection control policies that incorporate microbiological data, and ongoing professional training.

Hospital contamination endangers patient safety and healthcare quality. Microbiological testing of ambient microorganisms helps prevent HAIs, improve cleaning, and make informed infection control decisions. Combining culture-based and molecular testing methods can detect, measure, and characterise microbial contamination.   This PMCH study examines how well various testing methods operate in real-world settings over six months with an 80-person sample.   The research team is optimistic that this extensive evaluation of microbial contamination in crucial hospital settings will improve infection control, patient health, and hospital-wide environmental monitoring practices.

Objectives

• To evaluate the effectiveness of microbiological testing methods in detecting and monitoring environmental contamination in key hospital areas at PMCH.
• To identify the predominant microorganisms present on hospital surfaces, in the air, and in water sources.
• To assess temporal trends in environmental contamination over the study period (January 2025 – June 2025).
• To compare contamination levels across different hospital zones, such as wards, ICUs, and high-touch areas.

Study Design This study was conducted as a prospective observational study aimed at evaluating the effectiveness of microbiological testing in monitoring environmental contamination within PMCH. A prospective design was chosen to allow systematic collection of environmental samples over a defined period and to observe temporal changes in microbial load. The study focused on key hospital areas with a high risk of contamination, enabling the assessment of microbiological testing methods under real-world clinical conditions. By adopting an observational approach, the study sought to describe contamination patterns without introducing interventions, ensuring that findings reflect routine hospital practices. Study Setting The study was conducted at Patna Medical College and Hospital (PMCH), a tertiary care teaching hospital in India. Specific areas were selected for sampling based on their clinical significance and potential for microbial contamination. These included intensive care units (ICUs), surgical wards, patient rooms, outpatient areas, and high-touch surfaces such as bed rails, doorknobs, infusion pumps, and medical instruments. The selection of these areas was guided by previous evidence indicating that such zones are common reservoirs for pathogenic microorganisms in hospital environments. Study Duration The study was carried out over a six-month period, from January 2025 to June 2025. This duration allowed for repeated sampling across different times to capture temporal variations in environmental contamination, including potential seasonal influences on microbial load. Sample Size A total of 80 environmental samples were collected during the study period. Sample size selection was based on previous hospital environmental studies and logistical feasibility, ensuring adequate representation across different hospital zones and surfaces while maintaining a manageable workload for laboratory analysis. Sampling Method Environmental samples were collected using standardized procedures to ensure reliability and reproducibility. Surface samples were obtained using sterile swabs moistened with sterile saline or neutralizing buffer, which were rubbed over a defined area (approximately 10 cm²) of high-touch surfaces. Air samples were collected using a volumetric air sampler to capture airborne microorganisms on selective agar plates. Water samples were collected in sterile containers from taps, sinks, and other water sources, with care taken to avoid contamination during collection. All samples were transported to the microbiology laboratory under controlled conditions and processed within two hours of collection to preserve microbial viability. Microbiological Testing Culture Methods: All collected samples were subjected to culture-based microbiological analysis. Surface and water samples were plated on selective and differential media suitable for bacterial and fungal growth. Bacterial cultures included nutrient agar, MacConkey agar, and blood agar, while Sabouraud dextrose agar was used for fungal isolation. Plates were incubated at appropriate temperatures (37°C for bacteria and 25–30°C for fungi) for 24–72 hours, depending on the organism. Identification Techniques: Microbial colonies were identified based on morphological characteristics, Gram staining, and biochemical tests. In addition, standard identification kits such as API strips were used to confirm bacterial species. Fungal isolates were identified using lactophenol cotton blue staining and microscopy, and when required, additional molecular identification methods, such as PCR, were employed for specific pathogens. Quantification: Microbial load was quantified in CFU per unit area (CFU/cm²) for surfaces and CFU per milliliter (CFU/mL) for water samples. Airborne microbial concentration was expressed as CFU per cubic meter of air (CFU/m³). These quantitative measures allowed comparisons across hospital areas and over time, facilitating assessment of contamination trends. Inclusion and Exclusion Criteria Samples were collected from high-touch surfaces and critical zones, including patient care areas, ICUs, surgical theaters, and outpatient units. Areas under renovation, low-risk zones with minimal patient contact, and surfaces with routine disinfection before sampling were excluded to avoid bias and ensure the relevance of findings to infection control practices. Data Collection and Analysis Data from microbiological testing were recorded systematically in pre-designed forms. Descriptive statistics, including mean, standard deviation, and percentages, were calculated to summarize microbial contamination levels. Comparative analysis was performed using chi-square tests or t-tests where applicable to assess differences in contamination across wards, surfaces, and sampling periods. Trend analysis was conducted to observe temporal changes in microbial load over the six-month study period. All statistical analyses were conducted using SPSS software version, with significance set at p < 0.05. Ethical Considerations The study was conducted with permission from the administration of PMCH. As environmental samples do not involve human subjects directly, informed consent was not required. All laboratory procedures were performed in accordance with biosafety protocols to ensure safe handling of potentially pathogenic microorganisms. Confidentiality of hospital areas and staff was maintained throughout the study.

Descriptive Statistics

A total of 80 environmental samples were collected from various hospital areas over the six-month study period. Out of these, 52 samples (65%) were found to be positive for microbial growth, while 28 samples (35%) showed no detectable growth. Surface swabs accounted for the majority of positive samples (35/52, 67.3%), followed by water samples (10/52, 19.2%) and air samples (7/52, 13.5%). Among the microorganisms identified, Staphylococcus aureus was the most prevalent, detected in 18 samples (34.6% of positive samples), followed by Pseudomonas aeruginosa in 12 samples (23.1%), Escherichia coli in 8 samples (15.4%), Klebsiella spp. in 6 samples (11.5%), and fungal species such as Candida albicans in 8 samples (15.4%). These findings indicate a significant presence of both bacterial and fungal pathogens in hospital environments, emphasizing the importance of routine microbiological monitoring.

Table 1 Sample-wise Microbial Growth

Comparative Analysis

Comparing contamination levels across different hospital wards revealed notable differences. ICUs exhibited the highest contamination rate, with 22 of 28 samples (78.6%) testing positive. Surgical wards showed a moderate level of contamination (18/30, 60%), while outpatient areas had the lowest positivity rate (12/22, 54.5%). High-touch surfaces, including bed rails, infusion pumps, and door handles, were consistently more contaminated compared to low-touch areas such as walls or ceilings.

Temporal analysis over the six months indicated fluctuations in microbial load, with the highest contamination observed in March 2025 (10/12 samples positive, 83.3%), followed by May 2025 (9/12, 75%). The lowest contamination rate occurred in January 2025 (7/14, 50%), suggesting potential seasonal or operational influences on microbial presence. Overall, the trend suggests that periodic monitoring is essential to detect temporal spikes in environmental contamination and implement timely interventions.

Table 2 Microorganism Distribution Across Samples

The data further highlighted differences in contamination by sample type. Surface swabs consistently showed the highest microbial load, with mean CFU ranging from 15–120 CFU/cm², whereas water samples exhibited CFU counts of 5–50 CFU/mL, and air samples showed 10–70 CFU/m³. This quantification emphasizes that surface contamination remains the primary concern in hospital infection control, followed by water and air sources.

These results collectively demonstrate that microbiological testing provides valuable insights into the extent, type, and distribution of environmental contamination in hospital settings. By identifying high-risk areas, common pathogens, and temporal trends, the study underscores the importance of implementing targeted cleaning protocols, frequent surveillance, and effective infection control measures to reduce hospital-acquired infection risk.

This study found microbial growth in 65% of samples, indicating that healthcare facilities are still concerned about environmental contamination. Surface swabs showed the highest positive rates, suggesting that high-touch surfaces can house dangerous microorganisms.   Staphylococcus aureus and Pseudomonas aeruginosa are common in ICUs and Surgical Wards, supporting previous results that these bacteria cause HAIs. March and May are peak contamination months.  This implies that microorganism levels can alter over time due to factors including patient numbers, cleaning frequency, and seasons. These findings underline the importance of constant monitoring to spot anomalies and make modifications quickly.

Significance for Infection Control

The study's findings support PMCH infection prevention efforts.   Identifying high-risk areas like intensive care units and frequently touched surfaces might help hospital staff prioritise cleaning and disinfection.   Since bacterial and fungal diseases have been discovered, infection control must address multiple microbial types.   This study reveals that regular environmental monitoring helps policymaking, cleaning resource optimisation, and infection control program evaluation.   When hospitals combine microbiological monitoring data with infection control programs, HAIs can be reduced and patient safety improved.

Comparison with Previous Studies

The observed contamination patterns are consistent with prior studies conducted in tertiary care hospitals globally. Research from India and other countries has reported similar predominance of Staphylococcus aureus, Pseudomonas aeruginosa, and Candida albicans in hospital environments, particularly on frequently touched surfaces. Studies have also documented higher contamination rates in ICUs compared to general wards, reflecting the vulnerability of critically ill patients and the intensity of medical procedures performed in these areas. While some previous research included advanced molecular techniques for pathogen detection, our study primarily relied on culture-based methods, which remain standard for routine surveillance. Despite methodological differences, the overall trends observed in our study corroborate the importance of regular environmental monitoring in infection control programs.

Table 3 Comparison of Present Study with Existing Studies

Strengths of the Study

This study possesses several strengths that enhance the validity of its findings. Systematic sampling across multiple hospital zones ensured a comprehensive assessment of environmental contamination. The six-month duration allowed observation of temporal trends, capturing variations in microbial load over time. The inclusion of multiple sample types, including surface swabs, air, and water, provided a holistic view of the hospital environment, highlighting areas requiring targeted intervention. These strengths support the study’s relevance and practical applicability in guiding infection prevention policies at PMCH.

This study reveals that hospital environmental contamination requires microbiological testing. Eighty PMCH samples were taken throughout six months. Microbial growth was found in 65% of ambient samples. High-touch surfaces and intensive care units were most contaminated. The most common pathogens were Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, Klebsiella spp., and Candida albicans, highlighting the risk of hospital-acquired infections. Microbiological testing, notably systematic surface swabs, air sampling, and water analysis, can identify high-risk regions, point to infection control measures, and discover microbial contamination. These findings suggest that healthcare facilities should implement systematic and focused environmental monitoring protocols, focussing on high-touch areas and intensive care units. Regular surveillance can inform cleaning, disinfection, and disease transmission. Future research should evaluate treatment impacts on contamination patterns, expand to multi-center studies, and include molecular detection approaches for fastidious or resistant microorganisms. Hospital infection prevention still relies on microbiological monitoring for patient safety and clinical requirements.

[1] J. D. O. Mota, G. Boué, H. Prévost, A. Maillet, E. Jaffres, T. Maignien et al., “Environmental monitoring program to support food microbiological safety and quality in food industries: A scoping review of the research and guidelines,” Food Control, vol. 130, p. 108283, 2021.

[2] C. W. Huang, C. Lin, M. K. Nguyen, A. Hussain, X. T. Bui, and H. H. Ngo, “A review of biosensor for environmental monitoring: principle, application, and corresponding achievement of sustainable development goals,” Bioengineered, vol. 14, no. 1, pp. 58–80, 2023.

[3] M. Ferone, A. Gowen, S. Fanning, and A. G. Scannell, “Microbial detection and identification methods: Bench top assays to omics approaches,” Compr. Rev. Food Sci. Food Saf., vol. 19, no. 6, pp. 3106–3129, 2020.

[4] X. Wen et al., “Microbial indicators and their use for monitoring drinking water quality—A review,” Sustainability, vol. 12, no. 6, p. 2249, 2020.

[5] D. A. Holcomb and J. R. Stewart, “Microbial indicators of fecal pollution: recent progress and challenges in assessing water quality,” Curr. Environ. Health Rep., vol. 7, no. 3, pp. 311–324, 2020.

[6] M. W. Starolis, “The contamination monitoring toolbox: Best practices for molecular microbiology testing,” Clin. Microbiol. Newsl., vol. 47, pp. 21–27, 2024.

[7] V. Kumar, K. Singh, M. P. Shah, A. K. Singh, A. Kumar, and Y. Kumar, “Application of omics technologies for microbial community structure and function analysis in contaminated environment,” in Wastewater Treatment, Elsevier, 2021, pp. 1–40.

[8] A. O. Olatunji, J. A. Olaboye, C. C. Maha, T. O. Kolawole, and S. Abdul, “Environmental microbiology and public health: Advanced strategies for mitigating waterborne and airborne pathogens to prevent disease,” Int. Med. Sci. Res. J., vol. 4, no. 7, pp. 756–770, 2024.

[9] C. Li et al., “Effects of heavy metals on microbial communities in sediments and establishment of bioindicators based on microbial taxa and function for environmental monitoring and management,” Sci. Total Environ., vol. 749, p. 141555, 2020.

[10] S. Datta, K. N. Rajnish, M. S. Samuel, A. Pugazlendhi, and E. Selvarajan, “Metagenomic applications in microbial diversity, bioremediation, pollution monitoring, enzyme and drug discovery: A review,” Environ. Chem. Lett., vol. 18, no. 4, pp. 1229–1241, 2020.

[11] A. Kassem et al., “Applications of Fourier Transform-Infrared spectroscopy in microbial cell biology and environmental microbiology: advances, challenges, and future perspectives,” Front. Microbiol., vol. 14, p. 1304081, 2023.

[12] S. Some et al., “Microbial pollution of water with special reference to coliform bacteria and their nexus with environment,” Energy Nexus, vol. 1, p. 100008, 2021.

[13] L. Sheng and L. Wang, “The microbial safety of fish and fish products: Recent advances in understanding its significance, contamination sources, and control strategies,” Compr. Rev. Food Sci. Food Saf., vol. 20, no. 1, pp. 738–786, 2021.

[14] A. M. Jawad, N. M. Aljamali, S. M. Jwad, A. MJ, and S. MJ, “Development and preparation of ciprofloxacin drug derivatives for treatment of microbial contamination in hospitals and environment,” Indian J. Forensic Med. Toxicol., vol. 14, no. 2, pp. 1115–1122, 2020.

[15] M. A. Borchardt, A. B. Boehm, M. Salit, S. K. Spencer, K. R. Wigginton, and R. T. Noble, “The environmental microbiology minimum information (EMMI) guidelines: qPCR and dPCR quality and reporting for environmental microbiology,” Environ. Sci. Technol., vol. 55, no. 15, pp. 10210–10223, 2021.