Evaluation of Acinetobacter baumannii, Klebsiella pneumoniae, and Staphylococcus aureus respiratory tract superinfections among patients with COVID-19 at a tertiary-care hospital in Tehran, Iran

Background The emergence of healthcare-associated infections (HAIs) or superinfections in COVID-19 patients has resulted in poor prognosis and increased mortality. Methods In a cross-sectional study, 101 respiratory samples were collected from ICU-admitted COVID-19 patients. The HAI rate, demographics, and antibiotic resistance were assessed. Results The HAI rate was 83.16% (76.62% bacterial and 6.54% fungal). The prevalence of 3 major HAI-causing organisms included Klebsiella pneumoniae (41.5%), Acinetobacter baumannii (20.8%), and Staphylococcus aureus (4.9%). Mortality and intubation ventilation proportions of 90% (p = 0.027) and 92.2% (p = 0.02) were significant among patients with superinfection, respectively. Multiple logistic regression analysis showed SpO2 pressure (odds ratio 0.842; 95% CI 0.750–0.945; p = 0.004) as a predictive factor in the association between antibiotic usage and mortality. More than 50% of patients received carbapenems. The resistance rates to at least one antibiotic of third-generation cephalosporins, aminoglycosides, quinolones/fluoroquinolones, tetracyclines, and β-lactam inhibitors were 95.2%, 95.2%, 90%, 57.1%, and 100% among A. baumannii isolates and 71.4%, 55%, 69%, 61.9%, and 59.5% among K. pneumoniae isolates, respectively. A proportion of 60% was recorded for methicillin-resistant S. aureus isolates. Conclusion As a result, antibiotic treatment should be administered following the microbial resistance profile. Contact isolation and infection control measures should be implemented as needed.


Background
SARS-CoV-2, the newest and seventh member of the Coronaviridae family, is the causative agent of severe respiratory illness in humans, similar to SARS-CoV and MERS-CoV [1], which was discovered in Wuhan's Hubei Province in China at the end of 2019 and was designated coronavirus disease 2019 (COVID- 19) by the WHO on February 11, 2020 [2].Since its first appearance, the COVID-19 virus has infected more than 753 million individuals worldwide, resulting in over 6.8 million fatalities by February 2023 [3].This global pandemic has impacted individuals of all ages, causing various clinical symptoms ranging from the common cold to fatal pneumonia [4].Risk factors and severe clinical COVID-19 cases are usually seen in elderly and immunocompromised individuals who suffer from various concomitant problems, such as cardiovascular diseases, diabetes, hypertension, and chronic obstructive pulmonary disease [5].SARS-CoV-2 virus can predispose immunocompromised hosts to various bacterial infections by exaggerated production of inflammatory cytokines causing uncontrollable immune responses, multiple organ damage, and severe pneumonia [6].The lung histopathologic findings reveal the role of healthcare-associated infections (HAIs) causing pneumonia in tissue damage, which can accompanied by increased mortality [7] (Fig. 1).
HAIs are defined as infections that manifest in hospitalized patients at least 48 h after hospital admission and are classified according to the Centers for Disease Control and Prevention/National Healthcare Safety Network (CDC/NHSN) criteria [8].K. pneumoniae, A. baumannii, and S. aureus isolates are three prevalent microorganisms causing HAI.These organisms are the leading cause of multidrug-resistant nosocomial pathogens, the so-called Enterococcus faecium, S. aureus, K. pneumoniae, A. baumannii, Pseudomonas aeruginosa, and Enterobacter spp.(ESKAPE) group, which account for rising annual healthcare costs and mortality [9].
Carbapenem-resistant K. pneumoniae (CRKP) and A. baumannii (CRAB) isolates contributed to a significant death rate among critically ill COVID-19 patients with a prolonged hospitalization history [10,11].In addition, S. aureus isolates are the second leading source of up to 11.8% of HAIs in the United States.They are responsible for both community and HAIs isolated from the lower respiratory tract of COVID-19 patients receiving ventilation [12] (Fig. 1).Given the apparent complication of MRSA in causing ventilator-acquired superinfections in previous SARS-CoV and influenza outbreaks [13], the Fig. 1 The schematic view of different prognoses of the SARS-CoV-2 virus in predisposing to HAIs prevalence of superinfection caused by this organism should be considered in the current pandemic to account for therapeutic targets against this infection [14].
According to the superinfection rate up to 70.6% [15], there has also been a 46.8% increase trend in inappropriate antibiotic treatment among COVID-19 patients with superinfection [16].
This study aimed to assess the prevalence of healthcare-associated respiratory infections or superinfections, antibiotic resistance profiles against the three HAIs mentioned above, and compliance with clinical outcomes among COVID-19 patients admitted to the ICUs of a major hospital in Tehran, Iran.

Ethics statement
The current study was reviewed and approved by the institutional ethics committee of Tehran University of Medical Sciences with an approved ID: IR.TUMS.MEDI-CINE.REC.1399.1092.Informed consent was obtained from patients or the relatives of unconscious patients.Additionally, due to the probable inconvenience for critical COVID-19 patients and the risk of virus transmission during sampling, we used respiratory samples taken routinely by nursing personnel who were transferred to the microbiology laboratory.

Study design and sample collection
In a cross-sectional study between March 2021 and July 2021, 101 single and nonduplicate respiratory adult patient samples, including endotracheal aspirate (ETA), bronchoalveolar lavage, and sputum, were collected from confirmed critically ill SARS-CoV-2 patients admitted to Imam Khomeini Hospital Complex, a referral teaching hospital of Tehran University of Medical Sciences.The current study was conducted according to guidelines by the Helsinki Declaration per Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) guidelines, [17], and the collection of 101 respiratory patient samples was based on a pilot study on 20 ICU patients with COVID-19, which estimated the prevalence of nosocomial infections to be 77%, with a type I error of 5% and a power of 80%.The population consisted of all ICU patients with SARS-CoV-2, excluding pregnant women and children.

Data collection
Data were gathered from electronic medical and nursing records.These included patient demographics, clinical SARS-CoV-2 symptoms, baseline comorbidities, average length of hospital stay, microbial analysis, radiographic findings of lung involvement, SpO 2 pressure, need for mechanical ventilation, prescribed antibiotic therapy and final disposition (discharged alive or expired).In this study, the severity of coronavirus disease in all confirmed COVID-19 patients was evaluated based on criteria including the need for intubation, respiratory rate > 30 per minute, lung involvement > 50%, and SpO 2 pressure, which was divided into four categories [mild (SpO 2 ≥ 93), moderate (90 ≥ SpO 2 ≥ 92), severe (88.1 ≥ SpO 2 ≥ 89.9), and critical (SpO 2 ≤ 88)].

Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) for detection of confirmed SARS-CoV-2 patients
Nasopharyngeal samples of suspected SARS-CoV-2 patients were tested by multiple one-step quantitative RT-PCR approaches.This experiment was carried out by targeting the E-gene (FAM) and S-gene (ROX) as screening values and detecting the RNaseP (HEX) gene of coronavirus as a confirmatory agent using the Covitech multiplex qPCR kit (ACECR, Iran) according to the manufacturer's instructions.

Detection of bacterial and fungal superinfection
Respiratory specimens were collected after 48 h of admission and transported to the microbiology laboratory to detect bacterial and fungal infection types by culturing the samples on standard culture media such as blood agar, chocolate agar, and Sabouraud dextrose agar.The generated colonies were then subcultured on several differential culture media, such as MacConkey Agar, Mannitol Salt Agar (MSA), and CHROM Agar Candida media, with incubation at 35-37 °C for 18-24 h.The resulting colonies were validated by Gram staining and a series of standard biochemical and diagnostic tests, such as catalase, urease, Simmon's citrate, oxidase, triple sugar iron (TSI) agar, sulfide indole motility (SIM), methyl red/ Voges-Proskauer (MR/VP), DNase, and lysin decarboxylase.A quantitative culture-based method was also performed to discriminate true infection from potential contamination.A colony-forming unit load of at least 10 4 per positive culture was considered a true pathogen [18,19].
To identify the suspected A. baumannii colonies, the 16S-23SrRNA gene intergenic spacer (ITS) region and bla OXA-51-like carbapenemase gene were amplified, as described previously [20].In addition, for confirmation of the Candida albicans complex colonies, the hwp1 gene was also amplified as described previously [21].

Determination of MDR and ESBL-producing isolates
The prevalence of MDR isolates was assessed.MDR isolates are categorized as resistant or nonsusceptible to at least one antimicrobial agent among three or more antibiotic classes [24].Additionally, the combination disk technique was used to examine the extended-spectrum beta-lactamase (ESBL)-producing isolates.Ceftazidime (CAZ, 30 g) and ceftazidime-clavulanate (CAV, 30/10 g, Mast), cefotaxime (CTX, 30 g), and cefotaxime-clavulanate (CV, 30/10 g, Mast) were used on Muller-Hinton agar inoculated by suspected ESBL-producing isolates [25].The positive ESBL-producing K. pneumoniae isolates were verified by a ≥ 5 mm increase in inhibition zone diameter surrounding cefotaxime-clavulanate and/ or ceftazidime-clavulanate compared to the diameter inhibition zone of ceftazidime and cefotaxime separately [26].

Statistical analysis
Student's t test was used to find the mean (SD) of continuous variables through a comparative analysis.The Chi-square test or Fisher's exact Chi-square test was employed to compare categorical variables.Univariate and multiple logistic regression analyses were conducted to determine the parameters independently associated with antibiotic treatment and mortality in COVID-19 patients with and without HAIs.Initially, univariate logistic regression was conducted by considering all causes of mortality.The predictive variables with a p value ≤ 0.2 were selected and introduced into the multiple logistic regression model to control the effect of confounding variables.P values less than 0.05 were considered statistically significant.All statistical analyses were performed with IBM SPSS Statistics 26.0.

Clinical and demographic features
HAI was diagnosed in 89% (84/101) of confirmed COVID-19 patients.Patients with HAI were older on average, with a mean age of 61 (SD 12.45; IQR 17) years; more than half (49/84) were men (58.3%).Almost all clinical symptoms and severity of COVID-19 increased in individuals with HAI compared to those without HAI.As a result, lung involvement of 50% was only recorded in patients with HAI.The median length of hospital stay duration was 18 (IQR 17) days (p = 0.184).The proportion of in-hospital mortality (90%, 63/70) (p = 0.027) and the need for intubation ventilation (92.2%, 59/64) (p = 0.02) were significantly higher in patients with HAI.Almost 84.3% (59/70) of expired patients were under mechanical ventilation (p = 0.000).Hypertension, diabetes mellitus, immunodeficiency, and coronary heart disease were more prevalent in patients with HAI, as shown in Table 1.

Discussion
This study detected a high HAI proportion of 83.2% among ICU-admitted COVID-19 patients.Most patients with HAI were critically ill.Additionally, we were in lacked access to microbiological data during the first admission.Thus, some might have been admitted with coinfections that were classified as superinfections.Furthermore, the length of stay in our study was relatively long (median = 18 days).In numerous other studies, the length of hospital stay was prolonged for patients with various superinfections and was accompanied by increased mortality [27][28][29].
Approximately 92% of patients with HAI were intubated, and of those, 80% had at least one underlying disease, which led to a mortality rate of 90%.Similarly, in some investigations, hypertension, diabetes, and coronary heart disease were found to be most common among patients suffering from comorbidities and HAI [29,30].In addition, 84% of patients with HAI received immunosuppressive therapies more frequently, primarily because of multiple organ damage due to hyperinflammatory immune responses in critically ill COVID-19 patients [31].These patients may have been infected by various bacterial, fungal, and viral superinfections that exacerbated the severity of their symptoms [32].These factors may be justifiable for our high incidence of HAI.
In our study, antibiotic administration was more frequent among patients with HAI than those without it, which had a significant association with mortality.In  In agreement with our findings, Tadesse et al. stated the presence of carbapenemase and many antimicrobial resistance (AMR)-encoding genes, such as ESBLs, on mobile genetic elements to facilitate the accumulation and spread of AMR genes.This emphasizes why most carbapenem-resistant Gram-negative bacteria, such as CRKP and CRAB isolates, are primarily resistant to other antibiotic classes, such as aminoglycosides, fluoroquinolones, and SXT [36].In our study, all for CRKP and 23.5% (3/13) for ESBL-producing K. pneumoniae, which was comparable to our findings [37].
In a cross-sectional study by Qodrati et al., a low rate of MRSA (37.5%, 216/576) was obtained from clinical specimens one year before the pandemic.The antibiotic resistance against MRSA isolates was greater than that against MSSA isolates and was similar against GM, TE, LVX, SXT, CP, CD, and SXT.Given the full susceptibility of VAN and LZD to all S. aureus isolates, the authors considered these drugs as the first treatment priority against MRSA-causing infections [40].Despite the frequent use of VAN in hospitalized COVID-19 patients, low numbers of MSSA (2/5) and MRSA isolates (3/5) were observed in our study.Additionally, Falcon [41] and Punjabi et al. [13] reported a low incidence of S. aureus and culturebased MRSA (5.7%) isolates from respiratory COVID-19 patient samples.In contrast to Qodrati et al., Punjabi et al. believed that continuous VAN treatment might not be more beneficial for superinfections caused by MRSA isolates [13].
Although the prescription of antimicrobial treatment in infections of the ongoing SARS-CoV-2 pandemic is unavoidable, there has been no documented sign of its efficacy in lowering mortality [42].Some reports have shown widespread antibiotic use challenges in body metabolism and could prevent the body from generating proper antibodies against SARS-CoV-2 infections.This could be one of the main reasons for the high mortality in the ongoing COVID-19 pandemic [43].The presence of MDR Gram-negative isolates in our investigation might justify the elevated HAI rate and consequent high incidence of superinfected patient mortality.
The current study had certain limitations.First, all analyses were conducted solely on patients with SARS-CoV-2, and the frequency of HAI in hospitalized patients without COVID-19 was not evaluated.Second, the diagnosis of HAI was based on the microbial culture results.We had some constraints in performing the molecularbased methods to detect additional atypical infectious agents.Last, we did not have access to the antibiotic administration data and preliminary microbiological culture results before hospitalization to evaluate the efficacy of antimicrobial regimens on the type and proportion of HAIs among SARS-CoV-2 patients.

Conclusion
The present study reveals a substantial prevalence of bacterial and fungal superinfections (83.2%) among SARS-CoV-2 ICU-admitted patients, particularly MDR CRKP and CRAB isolates.The superinfections were significantly associated with increased mechanical ventilation (92.2%) and high mortality (90%).To mitigate antimicrobial resistance, patients were treated according to their precise microbial resistance profile using antibiotic regimens based on the clinicians' recommendations and hospital procedures.Additionally, implementing contact isolation and infection control programs is necessary if needed.

Table 1
The main characteristics of ICU-admitted patients with SARS-COV-2 a P value by independent sample T test b P value by Chi-square test c Fisher's exact P value by Chi-square test d Linear by linear P value by Chi-square test 1 Immune deficiency: breast cancer, colon cancer, intestinal cancer, liver transplant, kidney transplant, brain tumor, and HIV 2 Kidney disease: benign bladder tumor, kidney stone, and lupus nephritis 3 Other underlying diseases: epilepsy, Parkinson, depression, and

Table 2
Univariate and multiple logistic regression analysis of the association between antibiotic use and outcome