Changes In Lower Respiratory Tract Bacterial Isolates In ICU Patients Before and After the COVID-19 Pandemic

Summary

Here, a retrospective microbiological protocol is presented to analyze lower respiratory tract specimens in intensive care unit patients, including culture processing, MALDI-TOF MS identification, and comparison of bacterial pathogen distributions in COVID-19–negative patients before and after the COVID-19 pandemic.

Abstract

To compare the distribution of bacterial pathogens isolated from lower respiratory tract specimens in COVID-19–negative intensive care unit patients before and after the COVID-19 pandemic, culture results from patients admitted to a university hospital chest diseases intensive care unit were retrospectively analyzed. Patients without clinical or radiological suspicion of COVID-19 and with negative PCR results were included and classified as pre-COVID-19 or post-COVID-19 based on March 2020. Specimens were cultured on 5% sheep blood agar, eosin methylene blue agar, and chocolate agar, and evaluated after 24–48 h incubation at 37 °C. Only dominant microorganisms demonstrating growth in at least the second streaking sector were considered clinically significant. A total of 1,665 patients were included, with microbial growth detected in 48.9% of samples, the majority originating from the pre-COVID-19 period. Acinetobacter baumannii was the most frequently isolated organism in both periods. Logistic regression analysis showed reduced detection rates of Klebsiella pneumoniae, Pseudomonas aeruginosa, and Staphylococcus aureus (odds ratio ≈0.3), and Escherichia coli (odds ratio ≈0.4) in the post-COVID-19 period. Mortality rates were also lower in the post-COVID-19 period, although no significant difference in discharge type was observed. Overall pathogen diversity remained similar across periods; however, decreases in selected bacterial detections were observed following the onset of the pandemic. These findings may reflect changes in infection control practices during the pandemic period, although causality cannot be established due to the retrospective study design and absence of direct measurements of contributing factors.

Introduction

Lower respiratory tract infections (LRTIs) encompass inflammatory conditions of the airways, including bronchitis, bronchiectasis, bronchiolitis, pulmonary abscess, pleural effusion, and pneumonia¹. These infections represent a major cause of morbidity and mortality in hospitalized patients and pose a significant risk in high-acuity settings such as intensive care units (ICUs)². LRTIs account for approximately 10%–25% of hospital-acquired infections, with reported mortality rates ranging from 22% to 71% among ICU patients^3,4. They may arise from bacterial, viral, or fungal pathogens, with common bacterial agents including Klebsiella pneumoniae, Pseudomonas aeruginosa, Acinetobacter baumannii, Streptococcus pneumoniae, and Staphylococcus aureus⁵. Gram-negative bacilli such as Escherichia coli, K. pneumoniae, Serratia marcescens, P. aeruginosa, Acinetobacter spp., and Enterobacter spp. are frequently associated with late-onset hospital-acquired LRTIs, often linked to prolonged hospitalization and increased oropharyngeal colonization⁶.

The management of hospital-acquired pneumonia has become increasingly challenging due to the emergence of multidrug-resistant pathogens. Clinical outcomes are influenced not only by patient-related factors but also by the causative organism and its antimicrobial susceptibility profile^7,8. Consequently, surveillance of pathogen distribution in ICU settings remains essential for guiding empirical therapy and improving patient outcomes⁹.

The coronavirus disease 2019 (COVID-19) pandemic has substantially altered healthcare systems worldwide, leading to significant changes in infection control practices, patient management strategies, and antimicrobial use^10–12. Increased implementation of infection prevention measures and the widespread use of broad-spectrum antimicrobials during this period may have influenced the epidemiology of respiratory pathogens in ICU settings.

This study aimed to identify microorganisms responsible for lower respiratory tract infections in COVID-19–negative ICU patients and to compare pathogen distributions before and after the COVID-19 pandemic using standardized microbiological methods.

Protocol

The study protocol was approved by the University of Harran’s Medical Faculty Ethics Committee (decision no. HRU/23.12.23; July 10, 2023). Due to the retrospective design, informed consent was waived. All procedures were conducted in accordance with institutional laboratory standards. The research tools used in this protocol are listed in the Table of Materials.

1. Study design and patient selection

Lower respiratory tract culture results from patients admitted to a university hospital chest diseases ICU between March 2017 and March 2023 were retrospectively reviewed. Patients were included if COVID-19 was not clinically or radiologically suspected and SARS-CoV-2 polymerase chain reaction (PCR) test results were negative. Patients younger than 18 years and those with positive PCR results were excluded.

Patients were categorized into pre-COVID-19 (before March 2020) and post-COVID-19 (March 2020 and later) groups based on the date of the first confirmed COVID-19 case in Turkey.

2. Specimen collection

Lower respiratory tract specimens included sputum, deep tracheal aspiration (DTA), and bronchoalveolar lavage (BAL) samples collected under aseptic conditions according to standard ICU clinical practices.

Sputum and DTA samples were obtained from intubated and non-intubated patients using sterile suction techniques. BAL samples were collected during fiberoptic bronchoscopy using sterile saline instillation followed by aspiration.

3. Preliminary specimen assessment

Sputum and DTA specimens were initially evaluated by Gram staining. Samples were considered suitable for culture if they contained at least 25 polymorphonuclear leukocytes and fewer than 10 squamous epithelial cells per low-power field at 100x magnification. Specimens not meeting these criteria were excluded.

4. Culture processing

Sputum specimens were processed using a qualitative culture method. Samples were inoculated onto 5% sheep blood agar, eosin methylene blue agar, and chocolate agar, then incubated aerobically at 37 C and evaluated after 24 and 48 hours. Only predominant microorganisms demonstrating growth extending to at least the second streaking sector were considered clinically significant.

DTA and BAL specimens were processed using quantitative culture methods. Aliquots were inoculated onto the same media and incubated under identical conditions. Growth thresholds of at least 100000 colony-forming units (CFU) per mL for DTA and at least 10000 CFU per mL for BAL specimens were considered significant.

5. Microorganism identification

All isolates meeting predefined criteria were identified using matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) according to standard laboratory procedures.

6. Data recording and analysis

Demographic characteristics, specimen types, culture results, identified microorganisms, and discharge outcomes were systematically recorded. Microorganism distributions were compared between pre- and post-COVID-19 periods.

7. Statistical analysis

Statistical analyses were performed using SPSS (version 15.0) and Minitab software. Normality of continuous variables was assessed using the Shapiro-Wilk test. Categorical variables were compared using the chi-square test. Logistic regression analysis was conducted to evaluate associations between microorganism distribution and mortality. A p value less than 0.05 was considered statistically significant.

Results

The study population ranged in age from 18 to 103 years, with a mean age of 68.95 plus or minus 15.00. Women comprised 34.4% (n = 572) and men 65.6% (n = 1093) of participants. Overall, 42.3% (n = 704) of patients were discharged, while mortality occurred in 57.7%. Microbial growth was detected in 48.9% (n = 815) of cultures, whereas no growth was observed in 51.1% (n = 850).

Specimen types included sputum (45.6%, n = 759), DTA (41.9%, n = 698), and BAL (12.5%, n = 208). The most frequently is...

Discussion

Infections developing in intensive care units are associated with high morbidity and mortality, and identification of causative microbiological agents remains essential for effective prevention and treatment strategies^1,7. This study evaluated lower respiratory tract pathogens isolated in a chest diseases ICU over a six-year period and compared pre- and post-COVID-19 periods. Consistent with previous reports, A. baumannii was the most frequently isolated...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Bronchoalveolar lavage collection set	Not applicable	Not applicable	Used for BAL specimen collection
Chocolate agar	RTA	Not applicable	Culture medium for respiratory specimens
Deep tracheal aspiration catheter	Not applicable	Not applicable	Used for DTA specimen collection
Eosin methylene blue agar	RTA	Not applicable	Selective medium for Gram-negative bacteria
Gram staining reagents	Not applicable	Not applicable	Used for preliminary microscopic evaluation
Incubator (37 ºC)	Not applicable	Not applicable	Used for culture incubation
MALDI-TOF MS system (VITEK MS)	bioMérieux	Not applicable	Used for bacterial identification
Matrix solution for MALDI-TOF MS	bioMérieux	Not applicable	Used according to manufacturer’s instructions
Sheep blood agar (5%)	RTA	Not applicable	Primary culture medium