The impact of COVID-19 on respiratory tract microbiota pattern in patients with severe pneumonia

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In December 2019, the coronavirus infection was first reported in Wuhan, China. On March 11 2020, the World Health Organization (WHO) declared a COVID-19 pandemic.

The causative agent of the infection is the severe acute respiratory syndrome virus SARS-CoV-2. SARS-CoV-2 has a tropism for lung tissue, where it causes diffuse alveolar damage, leading to extensive lung lesions. The pathogen binds to angiotensin-converting enzyme 2 receptors, penetrating the cell, triggers pattern recognition receptors responsible for the innate antiviral response. Start producing Interferons (IFN) types I, IFN III and proinflammatory cytokines [6]. The virus produced factor inhibition of IFN, which leads to dysregulation of the immune system and excessive release of proinflammatory cytokines, chemokines, and factors migration of immune cells (macrophages, neutrophils, monocytes and lymphocytes). Development of hyperactivation of the immune response (cytokine storm) leads to depletion of the immune system and an increase in the pathological process. SARS-CoV-2 also suppresses the cellular immune response impaired lymphopoiesis and induction of apoptosis of CD4⁺ T cells, causing lymphocyte depletion [2].

Bacterial complications develop in 6.9% patients with COVID-19 [3]. Bacterial co-infections are developing within 4–14 days from infection and leads to severe pneumonia. Complications occur significantly more often in patients undergoing treatment in intensive care unit (ICU). They reach 14–40%, and fatal outcomes in ICU patients occur in 50% or more cases [4, 10].

The development of bacterial pneumonia in COVID-19 patients is associated with a disruption of the mucous barrier and mucociliary clearance, as well as a weakening of specific defense factors. As a result, the respiratory tract favorable conditions are created for bacterial colonization [8]. The aggressive synergism of bacteria and viruses increases the hyperactivation of the immune response and leads to a “cytokine storm”, which negatively affects the outcome of the disease [11].

Before COVID-19 pandemic the most common pathogens of bacterial pneumonia were Streptococcus pneumoniae and Haemophilus influenzae. S. pneumoniae is isolated in 30–50% of cases, and H. influenzae — up to 25%. The role of gram-negative bacteria is also increasing, including in patients undergoing inpatient treatment [7, 13].

During the pandemic, there was a change in the structure of microorganisms isolated from the sputum of COVID-19 patients. In the first months of the pandemic, there was a decrease in the number of pneumonias caused by S. pneumoniae. In the spring of 2021, their number reached pre-pandemic levels. This was due to the circulation of seasonal viral infections, such as influenza [12].

During pandemic, there was also an increase in bacterial concomitant complications caused by Klebsiella pneumoniae and Acinetobacter baumannii [1, 5]. K. pneumoniae was mainly isolated from patients with severe pneumonia and comorbidities. It has been es

 

Table 1. Changes in the frequency of isolation of gram-positive and gram-negative microorganisms during the study periods

Microorganisms	Presence in clinical material	Periods			р
		Before COVID-19	COVID-19	After COVID-19
Gram-positive	Not found	45 (72.6%)	112 (91.8%)	84 (70.0%)	< 0.001
	Found	17 (27.4%)	10 (8.2%)	36 (30.0%)
Gram-negative	Not found	20 (32.3%)	11 (9.0%)	36 (30.0%)	< 0.001
	Found	42 (67.7%)	111 (91.0%)	84 (70.0%)

 

lished that coinfection of pneumonia caused by K. pneumoniae and COVID-19 is accompanied by a high 28-day mortality rate [9]. The highest mortality was observed in the group of patients with hospital-acquired pneumonia — 39.6%. Mortality from community-acquired pneumonia was 27.9%. The addition of bacterial pathogens worsens the condition of patients and increases the risk of death [14].

After pandemic changes in the etiological structure of pneumonia pathogens are possible. This could be due to the uncontrolled use of antibiotics during the pandemic, as well as the effect of SARS-CoV-2 on the immune system and lung tissue.

The aim of this study was to analyze the dynamic change in the structure of the respiratory tract microbiota in patients with severe pneumonia from 2019 to 2023 years.

Materials and methods

We analyzed the results of microbiological examination of sputum from 304 patients treated in the ICU with a diagnosis of pneumonia from January 2019 to December 2023 inclusive. The study stages were divided into three periods: before (January 2019–March 2020), during (April 2020–June 2022) and after (July 2022–December 2023) the COVID-19 pandemic. The diagnosis of COVID-19 was confirmed by polymerase chain reaction. In the post-COVID period, all patients had negative test results of SARS-CoV-2. Pneumonia was confirmed by X-ray diagnostic methods.

Sputum samples were collected in sterile containers and delivered to the laboratory within two hours. The material was plated on nutrient media including 5% blood agar with defibrinated sheep blood (HiMedia, India), chromogenic agar for urinary tract infections (CondaLab, Spain) and Mueller-Hinton agar (CondaLab, Spain). The grown cultures were identified by MALDI-ToF mass spectrometry on a Microflex LT mass spectrometer (Bruker, Germany). During the study, 304 strains of microorganisms were identified.

Statistical analysis was performed using StatTech v. 4.7.2 (developer: StatTech LLC, Russia). Comparisons of percentages in multifield contingency tables were performed using the Pearson chi-square test. Post hoc comparisons were performed using the Pearson chi-square test with Holm’s correction. Differences were considered statistically significant at p < 0.05.

Results

The dynamics of changes in microorganisms isolated from clinical material are presented in Fig. In the pre-COVID-19 period, 62 samples were examined. The most frequently detected bacteria were K. pneumoniae and Stenotrophomonas maltophilia, isolated in 21.0% and 17.7% of cases, respectively. A. baumannii and Staphylococcus aureus were detected with the same frequency of 14.5%. Streptococcus pneumoniae was isolated in 8.1% of cases. Order Enterobacterales, excluding the genus Klebsiella, was isolated in 9.7%.

 

Figure. Structure of isolated microorganisms

 

During the coronavirus pandemic 122 sputum samples were evaluated. K. pneumoniae was detected in half of all samples. The second most common pathogen was A. baumannii — 23.8%. Order Enterobacterales (except K. pneumoniae) were identified in 9.0%. Pneumococcus was isolated in 1.6% of cases, Stenotrophomonas maltophilia — 2.5%.

In post-COVID period, 120 patients were examined. K. pneumoniae was isolated in 31.7% of cases. Also dominated S. aureus (23.3%) and A. baumannii (18.3%). Order Enterobacterales, except for K. pneumoniae, were isolated in 10.0% of cases, S. pneumoniae — 4.2%. The share of Stenotrophomonas maltophilia was 1.7%.

Discussion

Our study revealed a statistically significant change in the frequency of detection of microorganisms in ICU patients (Table 1). Before the COVID-19 pandemic, the proportion of gram-negative microorganisms was 67.7%. During the pandemic, it reached 91.0%, and in the post-pandemic period it approached the pre-COVID-19 values and amounted to 70.0% (p < 0.001). Despite the decrease in the frequency of isolation of gram-positive microorganisms during the pandemic, in the post-pandemic period their proportion increased and began to correspond to pre-COVID-19 values.

The following results were obtained when analyzing the frequency of isolation of various microorganisms (Table 2). Before the COVID-19 pandemic, a high frequency of detection of Stenotrophomonas maltophilia was observed. However, the frequency of isolation of the pathogen decreased from 17.7% in the pre-COVID-19 period to 1.7% in the post-pandemic period (p < 0.001).

 

Table 2. Changes in the frequency of pathogen isolation

Microorganisms	Presence in clinical material	Periods			p-test value
		Before COVID-19	COVID-19	After COVID-19
Staphylococcus aureus	Not discovered	53 (85.5%)	114 (93.4%)	92 (76.7%)	< 0.001
	Found	9 (14.5%)	8 (6.6%)	28 (23.3%)
Streptococcus pneumoniae	Not discovered	57 (91.9%)	120 (98.4%)	115 (95.8%)	0.105
	Found	5 (8.1%)	2 (1.6%)	5 (4.2%)
Stenotrophomonas maltophilia	Not discovered	52 (83.9%)	119 (97.5%)	118 (98.3%)	≤ 0.001
	Found	10 (16.1%)	3 (2.5%)	2 (1.7%)
Klebsiella pneumoniae	Not discovered	48 (77.4%)	60 (49.2%)	82 (68.3%)	≤ 0.005
	Found	14 (22.6%)	62 (50.8%)	38 (31.7%)
Acinetobacter baumannii	Not discovered	53 (85.5%)	93 (76.2%)	98 (81.7%)	0.291
	Found	9 (14.5%)	29 (23.8%)	22 (18.3%)
Escherichia coli	Not discovered	57 (91.9%)	117 (95.9%)	113 (94.2%)	0.536
	Found	5 (8.1%)	5 (4.1%)	7 (5.8%)
Pseudomonas aeruginosa	Not discovered	60 (96.8%)	117 (95.9%)	112 (93.3%)	0.513
	Found	2 (3.2%)	5 (4.1%)	8 (6.7%)

 

K. pneumoniae was detected with high frequency in the pre-COVID-19 period. During the COVID-19 period, the proportion increased from 21.0% to 50.8%, but in post-COVID-19 period, it decreased to 31.7%. The changes in the frequency of K. pneumoniae isolation were statistically significant (p ≤ 0.005).

There is a slight change in the frequency of occurrence of other representatives of the order Enterobacterales, except K. pneumoniae. During the COVID-19 period, their number decreased from 9.7% to 9.0%, and in the post-pandemic period, it exceeded pre-pandemic values — 10% (p = 0.966).

The dynamics of A. baumannii detection varied from 14.5% in the pre-COVID-19 period and reached a maximum during the pandemic — 23.8%. At the end of the pandemic, a decrease in the frequency of pathogen isolation to 18.3% was revealed (p = 0.291).

Pseudomonas spp. was isolated from 3.2% in pre-COVID-19 to 4.1% during the pandemic and amounted to 6.7% in the post-COVID-19 period. These changes were not statistically significant (p = 0.513).

During the COVID-19 period, a decrease in the proportion of isolated S. aureus was noted from 14.5% to 6.6% compared to pre-COVID-19 values. In the post-COVID-19 period, the frequency of microorganism isolation exceeded pre-pandemic values and reached 23.3% (p < 0.001).

There was a downward trend in the pneumococcal detection rate (p = 0.105). It decreased from 8.1% in the pre-COVID-19 period, reached a minimum during the pandemic (1.6%), and was 4.2% in the post-COVID-19 period. The findings are consistent with literature data, which also noted a decrease in S. pneumoniae during COVID-19 and an increase in the subsequent period [12].

Thus, in all the studied periods, there was a high frequency of detection of pathogens of nosocomial pneumonia in ICU patients. Before the COVID-19 pandemic, gram-negative pathogens (K. pneumoniae, A. baumannii, S. maltophilia) were most often isolated. Among gram-positive microorganisms, S. aureus and S. pneumoniae were dominated.

During the COVID-19 period, a decrease in the diversity of bacterial pathogens of pneumonia and an increase in the isolation of nosocomial pathogens, especially K. pneumoniae and A. baumannii, were noted. In our opinion, this is due to the infection of patients with COVID-19 with bacterial microflora in hospitals, where gram-negative pathogens dominate. The transmission of S. aureus from staff to patients was difficult, since personal protective equipment was used.

In the post-COVID-19 period, changes in the ratio of pathogen detection rates with preservation of their generic structure have been identified. However, further monitoring of pathogens causing severe pneumonia is required.

Conclusion

Thus, one of the global consequences of COVID-19 may be the spread of gram-negative flora often having antibiotic resistance. To prevent the further spread of these strains, it is necessary to conduct mandatory microbiological testing of clinical material in patients with pneumonia, use modern diagnostic methods, determine the sensitivity of bacteria to antibiotics and the minimum inhibitory concentration for justified targeted therapy.

About the authors

M. O. Zolotov

Samara State Medical University of the Ministry of Health of the Russian Federation

Email: l.r.shafigullina@samsmu.ru

PhD (Medicine), Head of the Laboratory of Interdisciplinary Relations, Research and Educational Professional Center for Genetic and Laboratory Technologies

Russian Federation, Samara

A. V. Kozlov

Samara State Medical University of the Ministry of Health of the Russian Federation

Email: l.r.shafigullina@samsmu.ru

PhD (Medicine), Head of Laboratory of Molecular Pathology, Research and Educational Professional Center for Genetic and Laboratory Technologies

Russian Federation, Samara

Liliya R. Shafigullina

Samara State Medical University of the Ministry of Health of the Russian Federation

Author for correspondence.
Email: l.r.shafigullina@samsmu.ru

Specialist of Laboratory of Molecular Pathology, Research and Educational Professional Center for Genetic and Laboratory Technologies

Russian Federation, Samara

A. V. Lyamin

Samara State Medical University of the Ministry of Health of the Russian Federation

Email: l.r.shafigullina@samsmu.ru

DSc (Medicine), Associate Professor, Director of Research and Educational Professional Center for Genetic and Laboratory Technologies

Russian Federation, Samara

References

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