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 Table of Contents  
Year : 2022  |  Volume : 10  |  Issue : 1  |  Page : 18-26

Microbiological profile of oxygen humidifiers at a tertiary care centre in the Himalayas: An exploratory study

1 Department of Microbiology, All India Institute of Medical Sciences, Rishikesh, Uttarakhand, India
2 Department of Internal Medicine, All India Institute of Medical Sciences, Rishikesh, Uttarakhand, India

Date of Submission23-Jun-2022
Date of Acceptance28-Sep-2022
Date of Web Publication12-Nov-2022

Correspondence Address:
Dr. Mohit Bhatia
Department of Microbiology, All India Institute of Medical Sciences, Rishikesh, Uttarakhand
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/jpsic.jpsic_27_22

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Background: Reusable humidifiers are often colonised by microbes, the aerosols generated from which are hypothesised to transmit respiratory infections-jeopardising patient safety.
Materials and Methods: In this time-bound cross-sectional study, 10 ml of water was collected in sterile containers from humidifiers installed in selected wards/critical care units/intensive care units and from the source used to refill these humidifiers. These samples were subjected to KOH and gram staining followed by inoculation on blood, MacConkey and Sabouraud dextrose agar and brain heart infusion broth in the aerobic environment at 37°C. Observations were recorded as per standard guidelines and compared against blood and respiratory cultures of patients.
Results: Despite an average of 8.23 days of exposure to oxygen humidified by contaminated water, n = 28 (of n = 39) blood samples reported no growth (NG) and n = 12 reported nonpathogenic organisms (NPO). Among n = 18 available respiratory samples, n = 1 reported Escherichia coli, which was not cultured from the same humidifier indicative of some other source. n = 1 reported NG, n = 6 reported NPO and n = 10 reported normal throat flora. No fungal elements were reported from any humidifier, source, or patient samples. The source-humidifier pathogen pair did not match for any humidifier.
Conclusions: Sterile patient cultures, despite prolonged exposure to oxygen humidified with contaminated water, indicate that humidifier contaminants did not infect patients. The disparity between the source or patient cultures and humidifier contaminants may be attributed to compromised universal precautions due to the exhaustion of health-care professionals during COVID-19. Furthermore, the type of water used to refill (Distilled/RO/Tap water) had no effect on the microbial contamination of humidifiers.

Keywords: Hospital-acquired infection, microbial contamination, oxygen humidifiers, reusable humidifier, ventilator-associated pneumonia

How to cite this article:
Puri O, Udayakumar SR, Bhatia M, Pathania M, Dhar M. Microbiological profile of oxygen humidifiers at a tertiary care centre in the Himalayas: An exploratory study. J Patient Saf Infect Control 2022;10:18-26

How to cite this URL:
Puri O, Udayakumar SR, Bhatia M, Pathania M, Dhar M. Microbiological profile of oxygen humidifiers at a tertiary care centre in the Himalayas: An exploratory study. J Patient Saf Infect Control [serial online] 2022 [cited 2023 Mar 30];10:18-26. Available from: https://www.jpsiconline.com/text.asp?2022/10/1/18/361006

  Introduction Top

It has been postulated that humidifiers used in the intensive care of hospitals are often colonised by microorganisms.[1],[2] Colonisation of ventilator circuits is reported to begin from parts of the circuit nearest to the patient and contaminate 33% of all ventilators as soon as 2 h after installation of new circuitry and 80% of all circuits by the end of 24 h with a median contamination level to be 7 × 104 organisms/ml.[1] Inhalation of oxygen humidified through contaminated humidifiers and the aerosols generated in the process have been hypothesised to transmit respiratory infections to the patient using the humidifier as well as to those in the surroundings.[1],[2] While some studies have reported patients contracting the contaminant microbiological pathogen, others have concluded on the contrary.[1],[2] This might be attributed to the difference in the type of ventilator humidifier, intensity and duration of use and the environmental conditions that govern the service life of the humidifier water. For instance, the number of contaminated humidifiers has been reported to increase with the duration of use,[1] and reusable humidifiers have shown a high percentage of contamination, unlike the disposable systems that reported no contamination.[3] While Goularte et al. concluded that humidifiers with heated water are colonised at a slower rate, Gilmour et al. identified bubble-through humidifiers to rapidly contaminate the circuitry as well as effluent gasses.[4],[5]

Since it is a common practice in health-care setups, especially in our country, to use the same humidifier until the water is used up or the patient is discharged, concerns have been raised regarding microbial infections through inhalation of oxygen humidifier by contaminated reusable humidifiers, thus compromising patient safety.[3] The disparity in the results of the existing evidence and the paucity of literature from Indian health-care setups necessitates further investigation into humidifiers as a possible source of infection. While the posed risk of infection is still explored, disposable humidifiers are being studied as contamination-proof humidifier systems with a lower risk of infection associated with them. While disposable humidifiers might be a safer, replacing a disposable humidifier after every patient might not be feasible for resource-deficient settings like our country. Thus, bridging the gap in the evidence on humidifier contamination and patient safety is even more important for a country like India.

While the effect of this contamination is being understood, studies are also being conducted to identify the source of these microbial contaminants. While some studies have compared the source of the water itself, others have compared the purification techniques that should be used to pre-treat water to be used in humidifiers. The current evidence suggests that distilled water and water used at a higher temperature are better alternatives to clean/refill the humidifiers limiting the growth of microbial flora.

There is a need for comprehensive data from India on the microbial profile of ventilator humidifiers, the transmission of pathogens to the patients, and the possible source of this contamination. To the best of our knowledge, this study is the first of its kind from the northern hilly terrain of India. The objectives of this study were to generate preliminary data on the microbiological profile of water samples obtained from oxygen humidifier systems installed in critical care units (CCUs), medical intensive care unit (MICU), paediatric intensive care unit (PICU), high-dependency unit (HDU) and general medicine wards and to compare the microbiological profile of water samples obtained from oxygen humidifier systems to culture reports of the patients and of the source water used to clean/refill the humidifier.

  Materials and Methods Top

An exploratory study was conducted with due Ethical Clearance from the Institutional Ethical Committee (Regd No-EC/NEW/Inst/2020/1046) received via Letter No.-IEC/21/399 Dated-16/07/2021, at a tertiary care teaching hospital in Uttarakhand, India, during August 2021.

Inclusion criteria

Water samples were collected from oxygen humidifier systems installed in CCUs 2, 4 and 7, MICU, PICU, HDU and General Medicine Wards 1 and 2, respectively.

Exclusion criteria

Humidifier systems installed in other CCUs/ICUs/HDUs/wards, etc., in the hospital were excluded from the study.

Sample collection was done in accordance with protocols followed in existing evidence reported by La Fauci et al.[3] 5–10 ml of water samples were collected from all bubbling humidifiers installed in CCU - 2, 4 and 7, MICU, PICU, HDU and General Medicine Wards 1 and 2 in sterile universal containers after a period of 5 or more days from the initial use of humidifiers. The source of water used to clean/refill oxygen humidifier systems was also sampled through the same protocol. Sampling was done only once per CCUs/ICU/HDU/Wards. These samples were sent to the bacteriology laboratory for culture. All samples were subjected to preliminary KOH and gram stain microscopic examination. This was followed by inoculation of these samples on blood agar, MacConkey agar, Sabouraud dextrose agar plates and brain heart infusion broth, respectively. The inoculated culture media were subjected to aerobic incubation at 37 degrees Celsius and observations were recorded as per standard microbiological guidelines. Cultures that reported growth were then subject to identification and antimicrobial susceptibility testing (as applicable) using the MicroScan WalkAway 96 Plus ID/AST system. A record of the patient's symptoms, diagnosis and the report of the microbiological investigations (blood culture and respiratory culture) done for the patients whose humidifiers were samples was also collected from the hospital database. The following outcomes were considered as indicators for the results of the study:

  1. The patient's clinical symptoms and diagnosis
  2. Culture reports of blood and respiratory samples of the patients
  3. Microbial profile and antimicrobial sensitivity of water samples collected from the patient's humidifiers
  4. Microbial profile and antimicrobial sensitivity of water samples collected from the source used to clean and refill the humidifiers.

  Results Top

The current time-bound study considered one-time humidifier water samples of 52 patients admitted to different wards within the hospital. Patients enrolled in the study used the humidifier for an average of 8.23 days before sampling. The symptoms, diagnosis and blood and respiratory culture reports of the included patients are summarised in [Table 1]
Table 1: Clinico-microbiological profile of the study participants

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The Source of water used to clean/refill humidifiers in CCUs, MICU, PICU, HDU and General Medicine Wards of the institute was then sampled, the relevant microbiological profile of which is summarised in [Table 2].
Table 2: Microbiological profile of sources of water used to clean and refill humidifiers in different areas of our institute

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  Discussion Top

In the present study, 40 humidifier samples reported growth of one or more pathogenic organisms, the most frequent being Pseudomonas aeruginosa (33.33%), followed by Acinetobacter baumannii (11.1%) and Acinetobacter lwoffii (11.1%), among Gram-negative bacteria and Coagulase Negative Staphylococcus spp. and Bacillus spp.(3.7%) amongst Gram-positive organisms. Jadhav et al., through a similar study conducted in a different part of the country, also identified P. aeruginosa and Acinetobacter spp. as the predominant microbial contaminants of humidifiers.[6] While the major Gram-negative contaminants were similar to those of the current study, they identified methicillin-resistant Staphylococcus aureus and methicillin-sensitive S. aureus as the major Gram-negative microbial contaminants.[6] La Fauci et al. also reported that P. aeruginosa was the most frequently identified Gram-negative bacteria, but identified S. aureus most commonly among Gram-positive organisms.[3] Moreover, organisms such as Serratia spp., Proteus spp., Citrobacter spp., Chryseobacterium spp., S. aureus and Enterococcus spp. identified in medical, surgical and emergency humidifiers in the aforementioned study were not cultured from humidifier samples collected from our centre.[3] Another study conducted by Malecka-Griggs et al. also reported Pseudomonas spp. (P. aeruginosa and Pseudomonas cepacia) as the predominant microbial contaminants amongst all humidifiers, but they also reported several other microbial contaminants such as Coagulase-positive Staphylococci, Streptococcus spp. Proteus and Candida were not identified from samples in our set-up.[7]

The microbiological examination of patients, humidifiers and source samples did not report any fungal species in the present study. The results are strikingly different from Jadhav et al. who reported 75.71% of all humidifiers to be contaminated with fungal species most commonly Aspergillus spp., Fusarium spp., Alternaria spp., Chaetomium spp. and a few more.[6] The disparity in the results can be attributed to the difference in the sampling technique, where the present study used water samples that were first screened under the microscope, Jadhav et al. used swabs collected from the inner surface of the chamber and directly inoculated them on SDA agar.[6] Since moist warm surfaces, better favour fungal growth compared to water, and direct inoculation might support microscopically unidentifiable infective dose to also grow, the disparity might be due to over or under-reporting.[6] In contrast, Nakipoglu et al. reported microbial contamination from only 20% of all humidifiers that too after direct inoculation of centrifuged water samples.[8] This further suggests that reporting of fungal species varied significantly based on sampling and evaluation techniques.

The variety of organisms isolated from different health-care settings suggests that although Pseudomonas spp. continues to be the predominant nosocomial contaminant of humidifiers, the overall microbial flora of humidifiers in a healthcare set-up varies considerably. Epidemiological parameters such as distribution of various microbial species and strains geographically, logistic differences such as source and purification of water used in different health-care setups, methodological differences such as sampling and evaluation, duration of use, types of patient and diagnosis especially preexisting infections might underlie this disparity.

Despite exposure to oxygen humidified by contaminated water for several days, n = 28 blood samples (of the total available n = 39 blood samples) did not show any growth, and no pathogenic organisms (or contaminants) were grown from n = 11 samples. Similarly, amongst the n = 18 available respiratory samples cultured, only 1 sample reported the presence of Escherichia coli but the same was not cultured from the humidifier sample indicative of a non-humidifier source of microbial infection. Amongst the remaining respiratory samples, n = 1 reported no growth (NG), n = 6 reported no pathogenic organism and n = 10 reported a normal throat flora. While the current study did not identify any pathogenic microbe from patient samples whose humidifiers were contaminated, Malecka-Griggs et al. reported heavy infection of Pseudomonas spp. Streptococcus, E. coli, Klebsiella and Candida spp. from several patients included in the study using humidifiers whose water sampling yielded similar pathogens with similar antimicrobial sensitivity results.[7] Another study by the same investigator conducted with patients from the neonatal intensive care units concluded that humidifier contamination resulted in colonisation and intermittent infection of the infants with Klebsiella pneumoniae, S. aureus and P. aeruginosa.[9] Multiple intensive care units outbreak of Acinetobacter spp. in Portland, led to a study being conducted to investigate the source of the outbreak which concluded that contamination of reusable ventilator support equipment was a probable leading cause for the outbreak.[10] The disparity in inpatient infection rate despite similar microbiological contaminants may be attributed to the difference in the infectivity of the contaminant strains, humidifier set-up or the immunity of the patients under consideration. The current study was conducted as a cross-sectional exploratory study, and hence, the patients were not followed for the development of any infection once the sampling was done. Since all patients were admitted and most received a variety of empirical antibiotics at the time of sample collection, the development of infection might have been prevented. Furthermore, the study was conducted soon after the second wave of COVID-19 subsided in the country, thus more precautions such as limited entry of patient attendees in the ward, gloves, masks and PPEs being used by the health-care workers, regular hand hygiene and adequate distancing between ward patient beds were being followed to prevent infection spread. The same might also have contributed to preventing infection spread through humidifier-generated aerosols.

Colonisation of humidifiers, although significant and potentially pathogenic, might not be infecting the patient. Several studies have been conducted to compare the colonisation of disposable and reusable humidifiers, most of which have concluded that disposable humidifiers have significantly lower microbial colonisation compared to reusable humidifiers. Even though disposable humidifiers are a safer alternative for eliminating contamination, the lack of any significant benefit in preventing patient infection or influencing patient outcomes emphasises that the replacement of reusable humidifiers might not be a necessity.[11]

To identify the possible source of contamination of the humidifier water samples, the source samples were profiled for microbial flora and antimicrobial resistance. Microbes such as P. aeruginosa, A. baumannii and Acinetobacter lwoffii, K. pneumoniae, Pseudomonas stutzeri, coagulase-negative Staphylococcus spp. and Bacillus spp. identified in humidifier samples were also identified in source water samples, unlike Staphylococcus epidermidis, which was reported in the source sample but not in the humidifier samples. Although similar microbial species were identified overall, the source-humidifier organism pair was not always the same. For instance, A. baumannii was identified from patient humidifiers in different general Medicine wards, the source water of which yielded coagulase-negative Staphylococcus spp. and Bacillus spp. and S. epidermidis without any growth of A. baumannii. A similar study done to investigate the source of microbial contamination in hospitals in Turkey identified that the major sources of contamination were either refilling of humidifiers without disinfection or using tap water instead of distilled water.[8] The results of the Turkish study, unlike the current one, indicated that even chlorinated tap water or potable water should not be used to fill the humidifiers because microbes tend to multiply rapidly within humidifier environments.[8] Thus, the current study supports the alternate hypothesis postulated by Nakipoglu et al. that the source of microbial contamination in humidifiers is refilling of humidifiers over several weeks without disinfection between successive usage.

It was also observed that the source water used in different wards varied from distilled water and RO water to Tap water. The microbial profile of the source water did not depend on the purification of water, suggesting that the contamination may not be from the source but may happen during transportation or handling by the staff working in the hospital settings. These findings differ from that of Nakipoglu et al. who through their study in a similar setting recommended the use of only sterile water for rinsing and refilling the humidifiers after chemical disinfection.[8] The disparity in the outcome of the two studies may be attributable to differences in sterilisation and purification protocols between the two settings or due to the source of contamination being elsewhere between the purification/sterilisation plant and in-patient supply.[8] In conclusion, the current study does not suggest any significant benefit of using distilled water in humidifiers, as the source of the microbial contaminants of the humidifier is not the source of water but refilling. Moreover, since oxygen delivered from contaminated humidifiers does not infect the patients, this further disproves the advantages distilled water might have over tap water for refilling the humidifiers in hospital settings.

The study being cross-sectional with a small sample size is a limitation for the same since the patients were not followed up for the development of infection once discharged. A longitudinal study design on a similar cohort could be more insightful into the effect of microbial contamination of humidifiers on patient outcomes. Furthermore, this study suggests that the source of the microbial contamination is probably not the patient or the source of water; hence, further investigation into the source of contamination is necessary to design policies and introduce interventions to curb contamination at earlier stages.

  Conclusion Top

The study concludes that while humidifiers used in CCU/ICU/HDU/Wards are contaminated by pathogenic microbes, the contaminant microorganisms may not be infecting patients or influencing patient outcomes in the course of their hospital stay. Moreover, the source of these microbial contaminants lies elsewhere except the patient or the source. More studies should be conducted in different parts of the country to generate concrete evidence on this subject.

Financial support and sponsorship


Conflicts of interest

There are no conflicts of interest.

  References Top

Craven DE, Goularte TA, Make BJ. Contaminated condensate in mechanical ventilator circuits. A risk factor for nosocomial pneumonia? Am Rev Respir Dis 1984;129:625-8.  Back to cited text no. 1
Pendleton N, Cheesbrough JS, Walshaw MJ, Hind CR. Bacterial colonisation of humidifier attachments on oxygen concentrators prescribed for long term oxygen therapy: A district review. Thorax 1991;46:257-8.  Back to cited text no. 2
La Fauci V, Costa GB, Facciolà A, Conti A, Riso R, Squeri R. Humidifiers for oxygen therapy: What risk for reusable and disposable devices? J Prev Med Hyg 2017;58:E161-5.  Back to cited text no. 3
Goularte TA, Manning M, Craven DE. Bacterial colonization in humidifying cascade reservoirs after 24 and 48 h of continuous mechanical ventilation. Infect Control 1987;8:200-3.  Back to cited text no. 4
Gilmour IJ, Boyle MJ, Streifel A, McComb RC. The effects of circuit and humidifier type on contamination potential during mechanical ventilation: A laboratory study. Am J Infect Control 1995;23:65-72.  Back to cited text no. 5
Jadhav S, Sahasrabudhe T, Kalley V, Gandham N. The microbial colonization profile of respiratory devices and the significance of the role of disinfection: A blinded study. J Clin Diagn Res 2013;7:1021-6.  Back to cited text no. 6
Malecka-Griggs B, Kennedy C, Ross B. Microbial burdens in disposable and nondisposable ventilator circuits used for 24 and 48 h in intensive care units. J Clin Microbiol 1989;27:495-503.  Back to cited text no. 7
Nakipoglu Y, Erturan Z, Buyukbaba-Boral O, Aksozek A, Aydin S, Derbentli S. Evaluation of the contaminant organisms of humidifier reservoir water and investigation of the source of contamination in a University hospital in Turkey. Am J Infect Control 2005;33:62-3.  Back to cited text no. 8
Malecka-Griggs B. Microbiological assessment of 24- and 48-h changes and management of semiclosed circuits from ventilators in a neonatal intensive care unit. J Clin Microbiol 1986;23:322-8.  Back to cited text no. 9
Hartstein AI, Rashad AL, Liebler JM, Actis LA, Freeman J, Rourke JW Jr., et al. Multiple intensive care unit outbreak of Acinetobacter calcoaceticus subspecies anitratus respiratory infection and colonization associated with contaminated, reusable ventilator circuits and resuscitation bags. Am J Med 1988;85:624-31.  Back to cited text no. 10
de la Fuente-Sancho I, Romeu-Bordas Ó, Fernández-Aedo I, Vallejo De la Hoz G, Ballesteros-Peña S. Microbiological contamination in high and low flow oxygen humidifiers: A systematic review. Med Intensiva (Engl ed) 2019;43:18-25.  Back to cited text no. 11


  [Table 1], [Table 2]


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