Antimicrobial Therapy as a Risk Factor of Multidrug-Resistant Acinetobacter Infection in COVID-19 Patients Admitted to the Intensive Care Unit

Background Multidrug-resistant Acinetobacter (MDR-Ab) is one of the most important pathogens causing superinfections in COVID-19 patients hospitalised in the intensive care unit (ICU). The occurrence of MDR-Ab superinfection significantly impairs the prognosis of patients in the ICU. Overuse of antibiotics in COVID-19 patients might contribute to the risk of developing MDR-Ab infection. Objective The objective was to assess the role of prior antibiotic exposure as an independent predictor of MDR-Ab infection in COVID-19 patients admitted to the ICU. Methods We conducted a retrospective cohort study in 90 patients admitted to the ICU of the Department of Infectology and Geographical Medicine, University Hospital in Bratislava, for respiratory failure due to COVID-19 between 1 September 2021 and 31 January 2022 (delta variant predominance). Patients underwent regular microbial screening. Superinfection was defined as infection occurring ≥48 h after admission. We assessed the role of prior antibiotic exposure and other factors as independent predictors of MDR-Ab isolation. Results Fifty-eight male and 32 female patients were included in the analysis. Multidrug-resistant bacteria were cultured in 43 patients (47.8%), and MDR-Ab was isolated in 37 patients. Thirty-three (36.7%) patients had superinfection caused by MDR-Ab. Fifty-four (60%) patients were exposed to antibiotics prior to MDR-Ab isolation; of those, 35 (64.8%) patients received ceftriaxone. Prior exposure to ceftriaxone (odds ratio (OR) 4.1; 95% confidence interval (CI) 1.4–11.9; P < 0.05), tocilizumab therapy (OR 4.7; 95% CI 1.3–15.0; P < 0.05), and ICU length of stay exceeding 11 days (OR 3.7; 95% CI 1.3–10.3; P < 0.05) were independent predictors of MDR-Ab infection. Conclusions Prior exposure to ceftriaxone increases the risk of MDR-Ab infection in COVID-19 patients admitted to the ICU. Our findings suggest that antibiotic use in COVID-19 patients admitted to the ICU should be restricted to patients with documented bacterial superinfection.


Introduction
Coronavirus disease 2019 (COVID- 19) is caused by the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).In the vast majority of cases, it is a selflimiting disease.Nevertheless, the infection is able to cause interstitial pneumonia, acute respiratory distress syndrome (ARDS), and hypoxemic respiratory failure.Patients with ARDS often require high-fow nasal oxygen (HFNO) or mechanical ventilation and admission to the intensive care unit (ICU) [1].While bacterial or fungal superinfections in COVID-19 patients are rare on presentation to the hospital, they are more common among patients admitted to the ICU [2][3][4].Superinfections in COVID-19 are associated with a higher intubation rate and increased in-hospital mortality and ICU length of stay [5][6][7].Some of the most important pathogens causing superinfections in COVID-19 patients in the ICU are multidrug-resistant Acinetobacter strains (MDR-Ab) [8,9].MDR-Ab may cause outbreaks in ICU and non-ICU wards; they are difcult to control by standard epidemiological measures [10][11][12][13].Te ability of Acinetobacter spp. to cause outbreaks in the ICU setting is based on its ability to survive on various surfaces under dry conditions and its resistance to antimicrobials and standard decontamination procedures [14,15].Te incidence of MDR-Ab superinfections in the ICU was already high in the prepandemic era, and it has increased during COVID-19 pandemic due to the strain on health care systems and the wide usage of broad-spectrum antibiotics and immunosuppressants [15][16][17][18].Opportunistic MDR-Ab infections are further facilitated by immune dysregulation associated with severe COVID-19 [19,20].MDR-Ab superinfections in COVID-19 are associated with worse patient outcomes and greater use of resources [7,[21][22][23].Empiric antimicrobial therapy is abundantly prescribed in COVID-19 patients with no evidence of bacterial superinfection, while current guidelines do not specifcally address this issue [18,24,25].Some of the antimicrobials have been linked to the risk of superinfections by multidrug-resistant bacteria in COVID-19 patients; however, the evidence is still relatively weak [26,27].Overuse of antibiotics in COVID-19 patients is potentially manageable.Terefore, we focused on whether exposure to the most frequently used antibiotics increases the risk of MDR-Ab superinfection in patients with COVID-19 admitted to the ICU.

Methods
We conducted a retrospective cohort study to determine possible risk factors of MDR-Ab isolation and infection in the COVID-19 patients admitted to the ICU.We enrolled all patients meeting the inclusion criteria admitted to the ICU of the Department of Infectology and Geographical Medicine, University Hospital in Bratislava, between 1 September 2021 and 31 January 2022 (delta variant predominance in our region).Te inclusion criteria were COVID-19 infection confrmed by polymerase chain reaction for SARS-CoV-2 RNA from the nasopharyngeal swab at the time of admission or a maximum of 14 days preceding admission, acute hypoxemic respiratory failure determined by oxygen saturation below 90% on ambient air, and ICU length of stay of at least 48 hours.
All patients underwent regular microbiological screening during their ICU stay.Sputum or tracheal aspirate, oropharyngeal swab, nasopharyngeal swab, and urine for cultivation were collected three times per week.Blood sampling for galactomannan examination for the presence of invasive aspergillosis was conducted weekly.Sampling for blood cultures was conducted in case of new onset of fever, suspicion of sepsis, or central venous catheter infection.
Te following patient data were collected from the hospital information system: demographics; history of diabetes mellitus; clinical data such as need for oxygen therapy (HFNO and mechanical ventilation), antimicrobial treatment, immunomodulatory treatment, development of bacterial pneumonia, ventilator-associated pneumonia, sepsis, urinary tract infection, and central venous catheterassociated bloodstream infection; microbiological data; ICU length of stay; in-hospital death; any infection during hospitalisation; duration of antibiotic therapy; procedures (insertion of central venous catheter, orotracheal intubation, continuous renal replacement therapy); and anamnestic MDR-Ab colonisation or clinical infection during hospitalisation.Empiric antimicrobial treatment was defned as any treatment with systemic bacteriostatic or bactericidal antibiotics started during hospitalisation prior to identifcation of pathogens of possible superinfection.Te diagnosed infections were classifed as hospital acquired if their frst clinical presentation developed at least 48 hours after admission.Infections in patients with COVID-19 were defned as superinfection if diagnosis occurred ≥48 hours after admission for COVID-19.Only superinfections were included in this study.Te types of superinfections were defned and classifed according to the CDC/NHSN Surveillance Defnitions for Specifc Types of Infections [28].
Isolation and identifcation of Acinetobacter strains and other bacteria were conducted according to local laboratory techniques using blood agar cultivation and matrix-assisted laser desorption/ionisation (MALDI-TOF, MBT Smart, Bruker Daltonics Inc., Billerica, MA, USA).Minimal inhibitory concentrations (MICs) of isolated bacterial strains were assessed using an in-house broth microdilution assay.MICs were established according to the European Committee on Antimicrobial Susceptibility Testing (EUCAST) breakpoints [29].Acinetobacter species isolates and other isolated bacteria were considered MDR in concordance with international expert proposals for interim standard defnitions for acquired resistance [30].
Quantitative variables are expressed as medians and interquartile ranges.According to the Kolmogorov-Smirnov test, the quantitative variables in our cohort were not normally distributed.Hence, medians of quantitative variables were compared between groups using the Mann-Whitney U test.Receiver operating characteristic curve analysis was used to determine the optimal cut-of value for ICU length of stay associated with risk of MDR-Ab development.Associations between MDR-Ab isolation and infection and the selected variables were assessed using a multivariate binary logistic regression model with a forward stepwise procedure, entering all variables with P < 0.1 in univariate analysis.Statistical signifcance was established at P < 0.05.All reported P values are two-tailed.Te odds ratio (OR) and 95% confdence intervals (CIs) were used to quantify the strength of the associations between covariates and dependent variables.SPSS version 20 (IBM Corp., Armonk, NY, USA) was used for statistical analysis.
Tis study was carried out in accordance with the Code of Ethics of the World Medical Association (Declaration of Helsinki) for experiments involving humans and was approved by the local Ethical Committee of University Hospital in Bratislava.Written informed consent for participation was obtained from all participants before enrolment in the study.No administrative permission to access the raw data used in this study was required by local authorities or the University Hospital.Te raw data were fully anonymised before use.We have preserved the full anonymity of all participants.
Forty-three (47.8%) patients were colonised or infected by MDR bacteria; Table 1 provides the ADR isolates.Most of the MDR isolates belonged to the Acinetobacter genus.Tirtythree patients (36.7%) had superinfection caused by MDR-Ab.MDR-Ab was the most prevalent cause of MDR bacterial superinfection (76.7% of patients with MDR bacterial superinfection).Acinetobacter baumannii was isolated in 22 cases, and all isolates except two were carbapenem resistant.In the remaining cases, Acinetobacter junii, Acinetobacter johnsonii, Acinetobacter dijkshoorniae, or Acinetobacter pittii was isolated.All these isolates were carbapenem susceptible but met the criteria for MDR pathogens.Te antimicrobial resistance pattern of Acinetobacter isolates is provided in Table 2. Te most common MDR-Ab superinfection was ventilator-associated pneumonia in 16 patients, followed by pneumonia not associated with mechanical ventilation in 10 patients.Four patients had an MDR-Ab bloodstream infection, and three had a urinary tract infection.Four patients with MDR-Ab isolates did not meet the criteria for superinfection and were regarded as colonisation.Fifty-four (60%) patients were exposed to antibiotic therapy during hospital stay.Ceftriaxone was used in 35 (38.9%) patients, which means that 64.8% of patients exposed to antibiotics received ceftriaxone.Meropenem was the second most utilised antibiotic used in 15 (16.7%) patients.Two patients were treated with cefoperazone-sulbactam, one with moxifoxacin and one with azithromycin.
In 42 (45.6%)patients, the ICU length of stay exceeded 11 days.Te baseline patient characteristics are provided in Tables 3 and 4. Patients with MDR-Ab isolates had a signifcantly longer ICU length of stay (Table 2).In univariate analysis, ICU length of stay, exposure to ceftriaxone and tocilizumab, and mechanical ventilation were signifcantly associated with MDR-Ab isolation (Table 4).In the multivariate analysis, previous exposure to ceftriaxone (OR 4.1, 95% CI 1.4-11.9,P < 0.05), tocilizumab therapy (OR 4.7, 95% CI 1.3-15.0,P < 0.05), and ICU length of stay exceeding 11 days (OR 3.707, 95% CI 1.332-10.317,P < 0.05) were positively associated with the risk of MDR-Ab superinfection.Te observed trend of increased risk of MDR-Ab isolation in patients on mechanical ventilation was not statistically signifcant (Table 5).Of 37 patients with MDR-Ab isolates, 17 (51.5%)died compared with 21 (36.8%)deaths in patients without MDR-Ab superinfection.However, the observed trend was not signifcant.
We created a simple scoring system to stratify the risk of MDR-Ab using the variables that were signifcantly associated with the risk of MDR-Ab in multivariate analysis (tocilizumab therapy, ICU length of stay exceeding 11 days, and previous exposure to ceftriaxone).Each risk factor represented 1 point of the risk score.When adding previous exposure to ceftriaxone as a risk factor to the cohort of patients with no other risk factors, one or two other risk factors increase the proportion of patients with MDR-Ab in all cohorts.Moreover, the proportion of patients with MDR-Ab incrementally rose with the risk score (Table 6).

Discussion
Our retrospective study has confrmed MDR-Ab superinfection as a common complication of COVID-19 patients admitted to the ICU.In addition, exposure to ceftriaxone is an important modifable risk factor of MDR-Ab superinfection.

Acinetobacter Superinfection in COVID-19.
MDR-Ab is one of the most important causes of hospital-acquired infections in COVID-19 patients [8,17].MDR-Ab was the predominant superinfection in our cohort, afecting more than one third of ICU patients.Moreover, MDR-Ab was    In their study, the carbapenem exposure was much more prevalent (33.3% of all patients) than cephalosporin exposure (18.1% of all patients).Tis factor might have contributed to the results [27].
Based on univariate analyses, we found no association between meropenem exposure and the risk of MDR-Ab superinfection.However, most of our patients who were exposed to antimicrobials were treated by ceftriaxone and only 16% were treated by carbapenems.We hypothesise that the lack of association between MDR-Ab isolation and carbapenem exposure in our study is due to the low number of patients exposed to carbapenems.Study by Falcone et al. identifed previous exposure to piperacillin-tazobactam as a risk factor of MDR superinfections in COVID-19 patients treated in the ICU.In contrast to our results, Falcone et al. found no signifcant association with prior ceftriaxone exposure.Tey even found that doxycycline therapy was associated with a lower risk of MDR superinfection.Doxycycline was the most prescribed antimicrobial drug in their study (47.6% of all patients).Ceftriaxone was prescribed in 46.6% and piperacillin-tazobactam in 14.3% of all patients in their study [26].In our study, antibiotics other than ceftriaxone were used only rarely, so we did not include other antimicrobials in the multivariate analysis.In the study by Falcone et al., the most common isolated MDR pathogens were enterobacteria, not Acinetobacter.Nonfermenting Gram-negative rods were found in only up to 22% of patients with MDR superinfection in their study.Tis factor might also contribute to their diferent results [26].In the prepandemic era, the risk of Acinetobacter infections in the ICU setting was found to be associated with previous exposure to broad-spectrum antimicrobials including cephalosporins and carbapenems [31][32][33].According to this evidence, it is likely that exposure to any broad-spectrum antibiotic increases the risk of Acinetobacter colonisation in COVID-19 ICU patients; however, additional studies are needed to support this hypothesis [18].

Other Risk Factors of Acinetobacter Superinfection in COVID-19
Patients.Monoclonal antibodies against interleukin 6 (IL-6) like tocilizumab are recommended to treat COVID-19 patients requiring HFNO or mechanical ventilation because of its clear mortality benefts [18].In our study, treatment with tocilizumab was associated with a higher risk for MDR-Ab isolation.Falcone et al. found an association between MDR infection and tocilizumab therapy in COVID-19 ICU patients.Tey also identifed baricitinib therapy as a risk factor for MDR infections, which is another potent immunomodulator [26].We were unable to evaluate the efect of baricitinib in our study because all of our ICU patients were treated with baricitinib.For the same reason, we were unable to assess the efect of corticosteroids.We found that ICU length of stay was associated with the risk of MDR-Ab isolation and infection in COVID-19 ICU patients.Falcone et al. also found this association [26].In addition, they found an association between risk of MDR superinfection and mechanical ventilation [26].In our study, there was a signifcantly higher portion of mechanically ventilated patients among those with MDR-Ab superinfection; however, the association was not present in multivariate analysis.Invasive procedures, orotracheal intubation, mechanical ventilation, and the use of corticosteroids and immunomodulators were identifed as possible risk factors for MDR bacterial superinfections in the ICU environment in the prepandemic era [31][32][33].Te presence of risk factors in our study had a cumulative efect on the proportion of patients with MDR-Ab.In the cohort of patients with no risk factors, the prevalence of MDR-Ab superinfection was 12%.However, in the cohort of patients treated with tocilizumab, exposed to ceftriaxone, and with an ICU stay exceeding 11 days, the prevalence of MDR-Ab superinfection was 85%.

Clinical Relevance and Implications.
Clinical guidelines for treatment of critically ill COVID-19 patients state that there are insufcient data to recommend for or against the use of empiric broad-spectrum antimicrobial therapy in the absence of another indication [18].However, there is growing evidence that empiric antibiotic therapy might be harmful even in critically ill COVID-19 patients because it is associated with the risk of MDR bacterial superinfection.Previous studies have found that risk of MDR isolation in COVID-19 patients is associated with broad-spectrum antibiotics like piperacillin-tazobactam and carbapenems, but not with narrower spectrum antibiotics like cephalosporins and aminopenicillins [26,27].We identifed ceftriaxone as an independent and potent risk factor for MDR-Ab superinfection in the setting of COVID-19 ICU patients.Te risk of MDR-Ab superinfection development attributed to

Strengths and Limitations
Tis study has several strengths.First, the sampling for biological material for cultivation was performed on a regular basis in all patients; therefore, variance in sampling frequency did not afect the results.Second, all patients were hospitalised in the same ICU during a single wave of the COVID-19 pandemic caused predominantly by the delta variant; hence, the study population is homogenous.Te delta variant occurred as dominant variant in Europe in July 2021 and remained dominant in Slovakia until January 2022 when it was outcompeted by omicron variant [33][34][35].Our study also has some limitations.Te frst limitation is its retrospective design.Retrospective studies are inheritably more prone to biases.We tried to minimize the risk of bias by including all patients to mitigate the possible risk of selection bias, and we performed multivariate analysis to mitigate the efects of possible confounders.Second, the sample size is relatively small, and the CIs of some signifcant predictors are quite broad.Tis factor is also probably responsible for some of the predictors of MDR-Ab infection showing a trend but not a signifcant association.Tird, we were unable to assess the association between MDR-Ab risk and exposure to other antibiotics because most of our patients were treated with ceftriaxone and other antimicrobials were used rarely.Terefore, additional studies are needed to describe in more detail the risk associated with antibiotic exposure and MDR superinfections in COVID-19 patients.

Table 1 :
Te prevalence of MDR bacteria in our study population.

Table 2 :
Te resistance profle of Acinetobacter isolates.If more than one strain was isolated in one patient, the more resistant strain was included in the analysis.
[21,23]][12] the etiologic pathogen in more than three quarters of superinfections.A multicentric study by Pascale et al. identifed Acinetobacter as the most common and clinically important cause of MDR superinfections in COVID-19 patients admitted to the ICU in Bologna, Italy[9].Other authors have also reported multiple large outbreaks of MDR-Ab in ICUs during the COVID-19 pandemic[10][11][12]. MDR-Ab superinfection in COVID-19 patients signifcantly worsens the outcome of the disease and complicates its management[21,23].Te ability to elude antimicrobial treatment and decontamination procedures allows Acinetobacter to cause large outbreaks, which are

Table 3 :
Basic characteristics of the patients.

Table 4 :
Univariate analysis of the association between possible risk factors and the risk of MDR-Ab isolation.

Table 5 :
Multivariate analysis of association of possible risk factors with the risk of MDR-Ab isolation.Associations between MDR-Ab isolation and infection and age, diabetes mellitus, ICU length of stay exceeding 11 day, mechanical ventilation, obesity, previous ceftriaxone exposure, previous meropenem exposure, tocilizumab therapy, C-reactive protein, glomerular fltration rate, neutrophil count, lymphocyte count, and CD4+ lymphocyte count were assessed using a multivariate binary logistic regression model with a forward stepwise procedure, entering all variables with P < 0.1 in univariate analysis.Statistical signifcance was established at P < 0.05.Only variables such as ICU length of stay exceeding 11 day, mechanical ventilation, previous ceftriaxone exposure, and tocilizumab therapy were included in the fnal model.Other variables were excluded because they were found to be insignifcant in the forward stepwise procedure.
ICU: intensive care unit; MDR-Ab: multidrug-resistant Acinetobacter; P: probability.4.2.Acinetobacter Superinfection in COVID-19 Patients andAntibiotic Exposure.We found that prior exposure to ceftriaxone is a strong independent risk factor of MDR-Ab infection.Ceftriaxone was the most prescribed antimicrobial drug in our study (38.9% of all patients).A recent study by Ceparano et al. identifed carbapenem exposure but not cephalosporin exposure as a risk factor for A. baumannii isolation in COVID-19 ICU patients.

Table 6 :
Te efects of risk factors and their accumulation on the proportion of patients with multidrug-resistant Acinetobacter superinfection.
Exposure to ceftriaxone signifcantly increases the risk for MDR-Ab superinfection in COVID-19 patients admitted to the ICU.Our fndings suggest that antimicrobial treatment in the COVID-19 ICU should be restricted to patients with clinically apparent bacterial infection.However, additional studies are needed to evaluate the role of antimicrobial therapy in COVID-19 critical care.