Microbiota of critical areas prior to reopening in an oncology center: Potential uncommon nosocomial pathogens for vulnerable populations

Highlights

• •Data on the microbial populations in critical areas before they are used is scarce.
• •Baseline microbiota of critical areas consisted only of saprophytic microorganisms.
• •Nosocomial saprophytes can become uncommon pathogens for vulnerable populations.
• •Uncommon opportunistic pathogens have been linked to healthcare-associated infections (HAIs) in cancer patients.

Abstract

Healthcare-associated infections are linked with the contamination of inanimate surfaces and the air in occupied hospital areas by recognized pathogens. However, there is limited information about the presence of these microorganisms or other potential pathogens in critical areas prior to their clinical operation. Here, we determined the microbial community in critical areas prior to their validation for hospital care and reviewed the background for the potential pathogenic role of this microbiota for populations susceptible to opportunistic infections. We evaluated environmental samples from operating theatres (OTs) and bone marrow transplant rooms (BMTRs) at the Peruvian National Cancer Center. A total of 164 samples (58 air samples and 106 surface samples) were collected for bacterial and fungal culture. In the OTs, the air conditioning sample yielded the highest microbial isolation from air, with a predominance of the genera Bacillus (5/12 isolates; 41.7%) and Aspergillus (5/8 isolates; 62.5%), including Nigri (2/5) and Flavi (2/5) sections and Aspergillus sp. (1/5). Meanwhile, the surface sample with the highest bacterial isolation came from the shelf in the stock area, where there was a predominance of non-glucose-fermenting Gram-negative bacilli (NF-GNB) (8/15 isolates; 53.3%), including the genera Pseudomonas (4/8), Acinetobacter (2/8) and Stenotrophomonas (2/8). In BMTRs, the only microorganisms isolated from the air were coagulase-negative Staphylococcus species and Penicillium sp. In conclusion, the microbial community composition of the critical areas prior to their reopening was consistent with their unoccupied status, consisting of nosocomial saprophytic microorganisms. Furthermore, the predominant species of the basal microbiota included uncommon hospital pathogens for people susceptible to opportunistic infections, such as cancer patients.

1. Introduction

Healthcare-associated infections (HAIs) cause significant morbidity and mortality, and increase healthcare costs. HAIs primarily result from the invasive procedures, but the nosocomial setting and patient susceptibility also play an important role in their occurrence [1]. The hospital environment harbors microorganisms that remain viable for long periods of time and can be transmitted throughout the environment [2], making it a potential source of HAIs, especially for patients treated in critical areas. In addition, oncology patients are highly susceptible to nosocomial opportunistic infections due to the immunosuppressive therapies administered as part of their antineoplastic treatment [3].

HAIs are mainly caused by bacterial [Staphylococcus aureus (S. aureus), Enterococcus spp., Pseudomonas aeruginosa (P. aeruginosa), Acinetobacter baumannii (A. baumannii), Escherichia coli (E. coli), and Klebsiella pneumoniae (K. pneumoniae)] [4] and fungal pathogens (Aspergillus fumigatus, Candida spp., and Mucorales species) [5]. These microorganisms can contaminate inanimate surfaces and the air in critical hospital areas such as operating theatres and transplant rooms, where patients susceptible to opportunistic infections are present [6]. The isolation of these pathogens has been associated with the occupancy of nosocomial areas [7], but there is limited information on the presence of these pathogens or other microorganisms in critical areas prior to clinical operation. Therefore, it is important to understand the baseline microbiota of these areas, as these could pose a serious threat to vulnerable populations. Our study aimed to identify the bacterial and fungal communities present in critical areas prior to their validation for use in hospital care at a specialized cancer center. Furthermore, we aimed to review the potential of the baseline microbiota as pathogens in a population susceptible to opportunistic infections.

2. Materials and methods

2.1. Study site

A retrospective and cross-sectional study of environmental samples from critical areas at Peru’s National Oncology Reference Center (Instituto Nacional de Enfermedades Neoplásicas, INEN, as per its acronym in Spanish) was conducted between January and February 2022. Records from the microbiology laboratory were used to obtain data. INEN is an institute specializing in the detection, diagnosis, treatment, and rehabilitation of neoplastic diseases. It is located in the city of Lima, Perú, and has nearly 400 hospital beds. INEN patients are often immunosuppressed due to the treatment of their malignancy and are exposed to the invasive medical procedures.

2.2. Description of the critical areas assessed

Operating theaters (OTs) are used for breast, head and neck, gynecological, abdominal, emergency, orthopedic, thoracic, urological, and laparoscopic surgeries. All rooms have positive air pressure, high-efficiency particulate air (HEPA) filters, controlled airflow, and temperature and humidity controls. In addition to the operating theatres, adjacent environments with different functions were evaluated, including a storage area for individual surgical supplies, a sterile area for staff clothing, and a laparoscopic area for all materials used in laparoscopic surgery.

Bone marrow transplant rooms (BMTRs) comprise two beds and a bathroom, negative air pressure, and HEPA filters. Two types of patients are accommodated in this environment: pre-transplant (approximately 17 days) and post-transplant (20 days for autologous and 30 to 40 days for allogeneic procedure).

2.3. Motivation for the microbiological study of critical areas

OTs and BMTRs were assessed for validation in the medical care following infrastructure improvements. In the operating theatres, doors were replaced with sensors for automatic touchless opening, while in the transplant rooms, HEPA filters were replaced. These environments were closed and covered during the refurbishment to avoid the dust generated from reaching nearby operating areas. On completion of the work, terminal cleaning and disinfection were performed before sampling for microbiological testing.

2.4. Collection of environmental samples

Air samples were collected and seeded using the RCS® High Flow Touch Microbial Air Sampler (Merck Millipore) and HYCON® agar strips (Merck Millipore). Blood agar 5 % strips were used for the bacteriological study, and Sabouraud with chloramphenicol (0.05 g/L) agar strips were used for the mycological study. The sampler was programmed for a volume of 100 L/min with a sampling time of 10 min. Surface samples were collected using one nylon swab for each microbiological study and placed in transport media (FLOQSwabs™ COPAN Diagnostics Inc., USA). A 10 cm² surface was rubbed with the swab using rotary movements in 3 directions (vertical, horizontal, and diagonal) and at an angle of 30 degrees. The samples were transported to the laboratory, where seeding was performed by swabbing the entire surface of the agar in three directions with the sampling swab. To ensure homogeneous seeding, the swab was rotated on the Petri dishes containing 5 % blood agar and Sabouraud with chloramphenicol (0.05 g/L) agar.

2.5. Isolation and identification of microorganisms

Blood agar 5 % strips and Petri dishes were incubated at 37 °C for up to 72 h, while Sabouraud with chloramphenicol (0.05 g/L) agar strips and plates were incubated at 25 °C for up to 7 days. Growth was checked daily and the number of colonies forming unit (CFU) observed was recorded. The number of bacteria and fungi isolated from air samples was reported in CFU/m³ and from surface samples in CFU/cm². Bacterial identification was performed using a protein profile study with an automated MALDI-TOF MS mass spectrometry system (Bruker Microflex LT; Biotyper v3.1 software; and Biotyper v5.0 5898 library) (Bruker Daltonics, Germany). The direct smear method was used, yielding a validity identification score of 2.0 for genus and species and 1.7–1.9 for genus only. Fungal identification was based on macroscopic and microscopic characteristics [8]. Sampling and microbiological processing were carried out according to the recommendations of the Spanish Society of Infectious Diseases and Clinical Microbiology for Environmental Microbiological Control [9].

The identification and quantification data of the isolated microorganisms according to the evaluated critical area were recorded on data collection sheets and compiled in an Excel 2010 database.

3. Results

A total of 164 samples (58 air samples and 106 surface samples) were collected for bacterial and mycological culture during the study period. The samples analyzed were from OTs (152: 46 air samples from 15 environments and 106 surface samples from 13 environments) and BMTRs (12 air samples from 6 rooms, 2 samples per room).

3.1. Composition and quantification of the microbial community

Of the 164 samples evaluated, 35 (21.3 %) were positive for bacterial and/or fungal culture. The proportions of samples positive for bacteria and/or fungi were BMTRs (6/12; 50.0 %) and OTs (29/152; 19.1 %).

3.2. Bacteriological study

3.2.1. Operating theatre

The air sample with the highest bacterial isolation was the conditioning air sample (4/8 positive samples; 50.0 %, isolating only Gram-positive bacteria with a predominance of the genera Bacillus (5/12 isolates; 41.7 %), which represented the highest bacterial load (30/80 CFU; 37.5 %). Meanwhile, the surface sample with the highest bacterial isolation was the shelf in the storage area (8/15 positive samples; 53.3 %), where there was a predominance of non-glucose-fermenting Gram-negative bacilli (NF-GNB) (8/15 isolates; 53.3 %), mainly of the genera Pseudomonas (4/8), followed by Acinetobacter (2/8) and Stenotrophomona (2/8). The genus Pseudomonas had the highest bacterial load of the NF-GNB (400/552 CFU; 72.5 %).

3.2.2. Bone marrow transplant room

The only bacterial agent isolated from the air was coagulase-negative Staphylococci (CoNS), with S. caprae being the species with the highest microbial load (7/13 CFU; 53.8 %). Overall, 4/15 (26.7 %) OTs failed to meet the proposed values allowed for isolation of aerobic bacteria in the air of very clean areas (<10 CFU/m³) (34), all of them Gram-positive bacilli. Room 2 was the only environment that presented the same genus and species, Staphylococcus hominis, in isolates from air and surfaces (Table 1).

Table 1.

Location and number of bacterial isolates from air and surfaces in critical areas.

Area	Sample	Location	Positive item	Count (CFU)	Isolates
OTs	Air	Room 1	Extraction air	8	Bacillus megaterium
				1	Staphylococcus vitulinus
			Conditioning air	10	Clostridium cadaveris
		Room 2	Conditioning air	15	Bacillus muralis
				11	Staphylococcus hominis
		Room 3	Conditioning air	3	Bacillus cereus
				3	Bacillus pumilus
			Injection air	10	Staphylococcus hominis
				2	Lactobacillus salivarius
		Room 4	Conditioning air	15	Tsukamurella tyrosinosolvens
			Injection air	1	Micrococcus luteus
		Room 7	Room center air	1	Bacillus cereus
	Surface	Room 2	Air injector	2	Staphylococcus hominis
			Air extractor	20	Staphylococcus hominis
		Room 5	Air extractor	5	Acinetobacter iwoffi
		Room 6	Air extractor	2	Staphylococcus hominis
			Ledge	3	Staphylococcus epidermidis
		Room 8	Ledge	14	Stenotrophomonas maltophilia
			Laparoscopy column	33	Stenotrophomonas maltophilia
			Doorknob	˃100	Acinetobacter johnsonii
		Room 10	Laparoscopy arm	1	Staphylococcus epidermidis
		Stock area	Ledge 1	˃ 100	Pseudomonas xanthomonas
			Ledge 2	˃ 100	Pseudomonas xanthomonas
			Ledge 4	˃ 100	Pseudomonas stuzeri
			Ledge 9	˃ 100	Pseudomonas xanthomonas
		Sterile area	Ledge 3	30	Aerococcus viridans
			Ledge 8	40	Aerococcus viridans
BMTRs	Air	Room 306–308	Room center air	1	Staphylococcus epidermidis
				2	Staphylococcus hominis
		Room 314–316	Room center air	3	Staphylococcus haemolyticus
		Room 318–320	Room center air	7	Staphylococcus caprae

Abbreviations: OTs, operating theaters; CFU, colonies forming unit; BMTRs, bone marrow transplant rooms.

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3.3. Mycological study

3.3.1. Operating theatre

The air sample with the highest fungal isolation was the conditioning air sample (3/6 positive samples; 50.0 %), where the genera Aspergillus predominated (5/8 isolates; 62.5 %), including Nigri (2/5) and Flavi (2/5) sections and Aspergillus sp. (1/5). In addition, Aspergillus spp. had the highest fungal load (36/42 CFU; 85.7 %). No fungal isolates were found in the surface samples.

3.3.2. Bone marrow transplant room

The only fungus isolated from the air was Penicillium sp. with a fungal load of 8 CFU. Overall, 3/15 (20.0 %) OTs and 3/6 (50.0 %) BMTRs were above the proposed values allowed for the isolation of filamentous fungi from air in very clean environments (0 CFU/m³) (34), mainly by Aspergillus species and Penicillium sp., respectively (Table 2).

Table 2.

Location and number of fungal isolates from air of critical areas.

Area	Sample	Location	Positive item	Count (CFU)	Isolates
OTs	Air	Room 1	Extraction air	1	Penicillium sp.
			Conditioning air	3	Penicillium sp.
		Room 3	Conditioning air	16	Aspergillus section Nigri
				2	Penicillium sp.
			Injection air	3	Aspergillus section Nigri
				14	Aspergillus sp.
		Room 4	Conditioning air	1	Aspergillus section Flavi
			Injection air	2	Aspergillus section Flavi
BMTRs	Air	Room 302–304	Room center air	4	Penicillium sp.
		Room 306–308	Room center air	1	Penicillium sp.
		Room 318–320	Room center air	3	Penicillium sp.

Abbreviations: OTs, operating theaters; CFU, colonies forming unit; BMTRs, bone marrow transplant rooms.

4. Discussion

The composition of the nosocomial microbiota varies according to environmental factors such as building design, ventilation, operational characteristics, but most importantly, the state of occupation [10]. The presence of patients and healthcare workers increases the microbial load in occupied environments and favors the emergence of common pathogens, as humans are considered their main reservoir [[11], [12], [13]]. Studies conducted in critical areas in several countries during routine medical care, analyzing samples from patients, air and surfaces, have isolated S. aureus, K. pneumoniae, E. coli, Enterococcus spp. and P. aeruginosa, among others [[14], [15], [16], [17], [18]]. These studies have all documented the isolation of recognized nosocomial pathogens in occupied critical areas, which contrasts with our findings.

We also identified a bacterial distribution pattern, with only Gram-positive in the air of OTs and BMTRs and a predominance of Gram-negative on OTs surfaces. This may be related to the peptidoglycan composition of the cell wall and the humidity levels. Gram-positive bacteria with a higher peptidoglycan composition can survive in dry environments, such as air [19]. Whereas, Gram-negative bacteria with a lower peptidoglycan composition found the moisture they needed to survive on surfaces [20].

Although the microbiota isolated in our study are considered non-pathogenic, they pose a potential risk to patients in critical hospital areas, whose immunological status makes them highly vulnerable to saprophytic microorganisms [21]. Here, we present briefly the background of the predominant saprophytic microorganisms found in our study as opportunistic pathogens.

Operating theatres temporarily expose patients to environmental pathogens associated with surgical site infections (SSIs) [22]. On one hand, the high microbial load of NF-GNB found on the shelves in the storage area qualifies this furniture as a potential source of cross-contamination to the adjacent operating room. Stenotrophomonas maltophilia (S.maltophilia) is a nosocomial pathogen whose colonization is associated with predisposing factors such as immunosuppression and malignant neoplasms [23]. The presence of S. maltophilia in surgical devices has been linked to its isolation from patients after ocular surgery [24]. Similarly, Pseudomonas stutzeri (P. stutzeri) can cause nosocomial infections in immunocompromised patients with chronic underlying disease, history of surgery, exposed trauma, or infected skin abrasions [25]. Cases of P. stutzeri brain abscesses acquired in a hospital setting have been reported following the implantation of a subdural grid prior to surgery for refractory epilepsy [26]. Finally, Acinetobacter iwoffii and Acinetobacter johnsonii are common commensals of the skin, oropharynx, and perineum, but they are also emerging as nosocomial pathogens in immunocompromised individuals [27].

On the other hand, the presence of opportunistic pathogenic mold in the air conditioning samples from operating rooms suggests that these rooms can potentially concentrate and disperse these microorganisms into the surgical environment. The isolation of Aspergillus spp. in these areas deserves singular attention, as they are the second most common cause of nosocomial invasive mycoses, with high mortality rates [28]. Aspergillus species are ubiquitous molds that can cause disease in humans from allergic reactions to invasive disease [29]. These fungi can be found in indoor hospital environments, posing a potentially life-threatening risk to immunocompromised patients who have undergone bone marrow or solid organ transplants, and those receiving chemotherapy for cancer [30]. Aspergillus fumigatus is the most common human pathogen and is responsible for most cases of invasive aspergillosis [31]. These hospital-acquired infections occur more frequently during or after construction or renovation activities as this cause dust contamination and disperse large amounts of spores. Additionally, nosocomial outbreaks of invasive aspergillosis have been documented, which can result from one or more Aspergillus species [32,33]. We isolated species belonging to the Aspergillus sections Nigri and Flavi, both of which cause invasive aspergillosis [34]. Conversely, Penicillium spp. is not a common nosocomial pathogen, but it has been associated with probable cases of nosocomial surgical wound infections and skin lesions in immunocompromised patients [35,36].

Improving and evaluating the effectiveness of air filtration systems are preventive measures that can reduce the risk of airborne pathogens, such as Aspergillus species. Using higher-efficiency filters and implementing air purification systems are effective ways to prevent air contamination. However, these practices may not be feasible in low-income hospitals. While monitoring hospital HVAC systems for fungi enables the early detection of mould and the measurement of spore concentrations in the air, providing guidance for further corrective action against air contamination. Though, this practice is only recommended after infrastructure changes or to evaluate the implementation of infection control measures [37]. Therefore, depending on the needs and resources of each institution, these measures should be considered for application in critical hospital areas where the presence of Aspergillus is a particular concern.

Invasive treatments such as stem cell or solid organ transplantation significantly increase the risk of HAIs in immunocompromised patients if BMTRs environment is contaminated with nosocomial pathogens [38]. CoNS, the predominant saprophyte group found in these areas, is the most common cause of bloodstream infections in oncology patients and also is associated with the use of intravascular devices [39]. Although catheter colonization and subsequent infection have traditionally been attributed to the patient's own biota, CoNS from the hospital environment can also cause bloodstream infections [40]. While, the presence of Penicillium species in these environments poses an albeit rare risk of opportunistic infection. Cases of respiratory infection in immunocompromised patients associated with the isolation of Penicillium species have been reported [41].

We recommend a microbiological evaluation of critical care hospital units, such as OTs and BMRTs, as an infection control measure. This evaluation should be conducted prior to the validation of these units, in order to identify potential reservoirs of opportunistic microorganisms. Areas with negative cultures can be opened, while areas with positive cultures should undergo terminal cleaning and disinfection to eliminate Gram-positive and Gram-negative bacteria, as well as fungi. Negative cultures in a follow-up microbiological study will allow the area to be opened. In addition, infection control measures should include improving ventilation, monitoring microbes in other high-risk areas [e.g., intensive care units (ICUs) and emergency rooms], and implementing targeted disinfection protocols.

Finally, hand hygiene is the primary intervention to address HAIs. Healthcare workers’ hands can transmit microorganisms in the hospital environment since they often come into direct contact with patients, contaminated surfaces, and medical equipment [42]. Hand hygiene interrupts this chain by reducing the microbial load, decreasing the likelihood of transmitting contaminated microorganisms, and reducing the risk of infection. Thus, this measure can prevent the transmission of the contaminated microbiota in critical areas such as OTs and BMRTs. Furthermore, current evidence suggests that microbial environmental contamination plays a significant role in the HAIs transmission [43]. To provide a more comprehensive assessment of the HAIs risk, future research should include studying environmental samples from high-risk hospital areas in addition to OTs and BMTRs. Additionally, analyzing HAIs incidence data pre- and post-reopening of critical areas will enable us to evaluate the clinical correlation between environmental contamination by uncommon opportunistic pathogens and current patient infections.

Our study had some limitations that motivate further evaluation to overcome them. We did not perform the gold standard genetic studies for identifying bacteria and fungi. However, proteomic fingerprinting reliably identifies clinically relevant bacterial species. Additionally, the preliminary fungal identification based on morphological characteristics allows us to determine whether an isolate belongs to a group of known airborne pathogens, such as Aspergillus species. Finally, although the culture-based method used is less sensitive to microbial detection than methods that identify microbial nucleic acids, it detects viable microorganisms and is affordable for laboratories with limited resources, such as ours.

5. Conclusions

The microbial community composition of the critical areas prior to their reopening was consistent with their unoccupied status, consisting of nosocomial saprophytic microorganisms in the absence of common pathogens. Furthermore, the predominant species of the baseline microbiota included uncommon hospital pathogens, mainly NF-GNB and Aspergillus species, for people susceptible to opportunistic infections, such as cancer patients. Our research provides insights into the prevention and control of pathogens in healthcare settings.

Ethics Statement

All the information analyzed is part of the environmental microbiology study according to the hospital protocol for the prevention of healthcare associated infections. The study was submitted to the INEN Research Committee and approved for implementation under the reference number INEN 21-59.