The Changing Pattern of Bacterial and Fungal Respiratory Isolates in Patients with COVID-19 Admitted to Intensive Care Unit

Gianluca Zuglian (  gianluca.zuglian@gmail.com ) Azienda Socio-Sanitaria Territoriale "Papa Giovanni XXIII," Bergamo Diego Ripamonti Azienda Socio-Sanitaria Territoriale "Papa Giovanni XXIII," Bergamo Alessandra Tebaldi Azienda Socio-Sanitaria Territoriale "Papa Giovanni XXIII," Bergamo Marina Cuntrò Azienda Socio-Sanitaria Territoriale "Papa Giovanni XXIII," Bergamo Ivano Riva Azienda Socio-Sanitaria Territoriale "Papa Giovanni XXIII," Bergamo Claudio Farina Azienda Socio-Sanitaria Territoriale "Papa Giovanni XXIII," Bergamo Marco Rizzi Azienda Socio-Sanitaria Territoriale "Papa Giovanni XXIII," Bergamo


Introduction
Severe acute respiratory syndrome 2 (SARS-CoV-2) has spread worldwide since 2019. Patients with SARS-CoV-2 infections may develop a severe form of coronavirus disease (COVID-19) requiring hospitalization and admission to intensive care units (ICU) in approximately 30% of them 1 . A large proportion of COVID-19 patients received antimicrobial therapy for proven or suspected co-bacterial infections during their ICU stay 2 and several studies have highlighted the antibiotic over-exposure in this population, despite the low rate of culture-proven bacterial co-infections 3 . This may be caused by several factors including the severity of COVID-19, the uncertainties about this new disease and the limitations of invasive diagnostic procedures due to the SARS-Cov-2 transmission precautions. Moreover, COVID-19 pandemic has represented an exceptional stress for the hospital setting, especially for the ICU setting, due to the overwhelming number of patients requiring a prolonged ICU stay.
The above-mentioned factors may have affected the local fungal and bacterial epidemiology. The aim of this study is to describe the prevalence of bacterial and fungal species in a cohort of COVID-19 patients admitted to ICUs compared to the patients observed at the same hospital during the previous year (before the COVID-19 pandemic).

Materials And Methods
"ASST Papa Giovanni XXIII" is a large tertiary referral hospital (990 beds) placed in Bergamo, one of the most affected provinces during the COVID-19 pandemic in Northern Italy in 2020. During the COVID-19 pandemic, following the large number of patients admitted to ICU wards, a surveillance program with collection of respiratory specimens [i.e. bronchoalveolar lavages (BAL) or tracheal aspirates (TA)] was performed every 48-72 hours or weekly in all the patients, according the different intensive wards, while such procedures, in not COVID-19 patients, had been previously performed only in selected individuals and according to clinical judgment. We have retrospectively collected the microbiological data from BAL and TA of patients hospitalized in ICUs from 22nd February 2020 to 31st May 2020 (Period 1) and from 22nd February 2019 to 31st May 2019 (Period 2). We compared the prevalence of bacterial and fungal species in the two time periods. Then, we have categorized the most prevalent bacterial species potentially pathogenic of the respiratory tract (Enterobacterales, Pseudomonas spp. and Staphylococcus aureus) on the basis of their antibiotic resistant pro le as multidrug resistant (MDR) for Enterobacterales and Pseudomonas spp., (according to de nitions by Magiorakos at all 4 ) and methicillin resistant (MR) for Staphylococcus aureus (according to cefoxitin screening). In addition, we calculated the odds ratio (OD) whit 95% con dence interval (CI) to evaluate the difference of the prevalence of bacterial and fungal species and the resistant pathogens in the two study periods.

Results
A total of 194 patients were admitted to ICU with COVID-19 in period 1 (namely 65% of 297 patients admitted in ICU in that time period), compared to 176 patients who were admitted in period 2. A total of 736 samples (3.8 patient) and 392 (2.2 per patient) were collected during the period 1 and period 2, respectively. The proportion of positive respiratory specimens (for at least 1 pathogen, either bacterial or fungal) was 48% (355/736 samples) and 47.7% (187/392 samples) in period 1 and 2, respectively. Table 1 shows the prevalence of bacterial and fungal species by group. In both periods, the most frequent bacterial isolates were Pseudomonas spp. and Enterobacterales; The prevalence of Pseudomonas spp.
shows a statistically signi cant increase from period 2 to period 1, as well as the prevalence of Enterococcus spp. On the contrary, the prevalence of Gram negative non fermenting bacteria (GN-NFB), Haemophilus in uenzae and Streptococcus pneumoniae showed a signi cant reduction in period 2 versus period 1.

Discussion
We observed a variation of microbiological respiratory isolates before and during COVID-19 pandemic. In period 2, the prevalence of potentially pathogenic bacterial isolates from respiratory samples in ICU patients was aligned with the one of previous studies in the same settings 5 . In period 1, we observed the reduction of several bacterial species, especially Enterobacterales and the parallel increase of Pseudomonas spp. and Enterococcus spp.
In the interpretation of this changing epidemiology, some observations may be useful.
Firstly, the isolation of bacterial and fungal species does not necessary imply an active infection caused by these pathogens and, certainly, the systematic respiratory tract sampling, aimed to early intercept an infectious complication in the context of the SAR-CoV-2 pneumonia, may have led to an overestimation of microbiological events compared to a standard and less aggressive approach.
Secondly, with regard to the changing epidemiology for Enterococcus spp. and Pseudomonas spp., the longer ICU stay for COVID-19 patients compared to the ones hospitalized for other causes 6 represents an important risk factor for colonization and/or infection caused by these bacteria 7,8 .
Thirdly, the tropism for respiratory tract by Pseudomonas spp. is well known, especially in patients with underlying lung disease (such as cystic brosis and chronic obstructive pulmonary diseases 9 ). In a recent surveillance of VAP in COVID-19 patients, Pseudomonas aeruginosa was the most common pathogen responsible for ventilator-associated lower respiratory tract infections 10 and the most common isolate in a population of critically ill patients hospitalized for in uenza-associated ARDS 10 . We can speculate that the combination of the lung impairment by SARS-CoV-2 and the predisposition of Pseudomonas spp. could act synergistically to put these patients at risk for colonization/infection by this pathogen.
Fourthly, most COVID-19 patients during ICU hospitalization received empirical antibiotic therapy 2 . In the ICU departments the antibiotics belonging to beta lactams class are the most widely used 11 . In our study, we observed a high resistance rate for this antibiotic class by Pseudomonas spp. and Enterobacterales in period 2, that was dramatically increased compared to period 1. We can speculate that the antibiotic pressure may have favored the emergence of resistant bacteria 12 .
Fifthly, the decrease of other bacterial species associated to respiratory tract infections is consistent with the low incidence rate of co-bacterial infections in COVID-19 patient 13 and the global reduction of prevalence of other respiratory pathogens, secondary to the public health measures against COVID-19 14 .
Sixthly, the high degree of immunosuppression induced by steroid therapy, used as salvage therapy in COVID-19 patients during ICU stay in the rst wave, may have in uenced the rate and the microbiological pattern of respiratory bacterial/fungal complications compared to period 2 15 .
Lastly, as mentioned above, the clinical signi cance of the colonization of the respiratory tract has not been investigated as the aim of the study was to describe the changing microbiological scenario during the COVID-19 pandemic which may help designing the antimicrobial stewardship programs.

Declarations
Ethic approval and consent to participant All methods were carried out in accordance with Declaration of Helsinki. This study was approved by the ethics committee of The Papa Giovanni XXIII Hospital (Protocol N. 257/2020). To maintain the principle of con dentiality, the data used were anonymized. The need for informed consent was waived by the ethical Committee of the "Papa Giovanni XXIII" Hospital due to retrospective nature of the study.

Consent for publication
Not applicable.

Competing interests
The authors declare that they have no competing interests.