Antimicrobial resistance burden pre and post-COVID-19 pandemic with mapping the multidrug resistance in Egypt: a comparative cross-sectional study

Overuse of antibiotics during coronavirus disease 2019 (COVID-19) in an attempt to reduce COVID-19 mortality in the short term may have contributed to long-term mortality from antimicrobial resistance (AMR). The aim of this study was to evaluate the impact of the COVID-19 pandemic on AMR in Egypt and map the distribution of multidrug-resistant (MDR) and extensive drug-resistant (XDR) across Egypt. Through a multicenter cross-sectional study 2430 culture results were collected in 2019 and 2022 pre and post-COVID-19 pandemic in Egypt, including 400 Klebsiella pneumoniae, 760 Escherichia coli, 650 Acinetobacter baumannii, and 620 Methicillin-resistant staphylococcus aureus (MRSA) culture results. MDR and XDR culture results distribution across Egypt was highlighted through the geographic information system. Mixed effect logistic regression models and sub-group analysis were performed according to the type of specimens to test the impact of COVID-19 on resistance. Adjusted analysis demonstrated K. pneumoniae resistance has increased against quinolones and carbapenems (P < 0.001). Resistance of E. coli has increased significantly against imipenem and meropenem. While E.coli susceptibility has increased to cefoxitin, levofloxacin, and ciprofloxacin. A. baumannii resistance has increased more than double against ceftazidime, cefepime, and piperacillin-tazobactam (P < 0.001). MRSA reserved its susceptibility to vancomycin and linezolid. MDR K. pneumoniae and A. baumannii have increased post-COVID-19 from 67% to 94% and from 79% to 98%, respectively (P < 0.001). XDR K. pneumoniae and A. baumannii have increased from 6% to 46%, and from 47% to 69%, respectively (P < 0.001). COVID-19 has changed the profile of AMR in Egypt so that urgent action is required to mitigate this threat and preserve our capacity to face infections in future decades.


Methods
According to the latest census in 2024, Egypt's population has increased by 1.57% to reach 114,484,252.They reside in 27 governorates across the country 14 .Through a multicenter cross-sectional study, culture results were collected from the Mabaret Al-Asafra laboratories database, one of the largest laboratories across Egypt, and other laboratories dealing with Mabaret Al-Asafra laboratories (lab to lab) in sixteen different governorates across Egypt.Each laboratory receives more than 30,000 cultures per year.On February 14, 2020 Egypt reported the first COVID-19 case.Positive culture results were collected over six months before and after the COVID-19 pandemic, throughout 2019 and 2022.We excluded the polymicrobial cultures and positive fungal cultures results.Culture results were collected over different seasons and different types of specimens; respiratory, blood, pus, and urine cultures.Antimicrobial susceptibility testing followed The Clinical and Laboratory Standards Institute (CLSI) guidelines 15 .The most prevalent bacteria in Egypt; K. pneumoniae, E.coli, A. baumannii, and MRSA were selected to evaluate AMR 16,17 .A total of 2430 culture results were collected over the two periods (pre and post COVID-19) including 400 culture results of K. pneumoniae, 650 culture results of A. baumannii, 760 culture results of E.coli, and 620 culture results of MRSA, divided according to 1:1 ratio before and after COVID-19 pandemic.

Outcomes
The main objective of this study was to compare AMR pre-and post-COVID-19 and exploring the MDR, XDR, and PDR distribution post COVID-19 in Egypt.We also described the temporal and geospatial patterns of AMR. 18Multidrug resistant bacteria (MDR) are bacteria resistant to at least one agent in three or more antimicrobial categories.

Operational definition
• Extensively drug-resistant (XDR): this means that bacteria are only susceptible to one or two antimicrobial categories.• Pan drug-resistant (PDR): are bacteria that are resistant to all agents in all antimicrobial categories.

Ethical approval
The ethical approval was received from the Ethical Committee of the Faculty of Medicine, Alexandria University (IRB number: 00012098, serial number: 0305804).Informed consent was waived, as the culture results were collected anonymously, without identifying any patient characteristics, from the laboratory electronic database.All methods were performed in accordance with the relevant guidelines and regulations of the ethical committee of the Faculty of Medicine, Alexandria University.

Data management
Culture results were described as the type of specimen (blood, sputum, urine, and pus), seasons, geographical distribution (Metropolitan, Upper Egypt, and Lower Egypt), type of AMR (MDR, XDR, and PDR) in percentages, and the prevalence of resistance and sensitivity of each organism in percentages.

Statistical analysis plan
AMR was compared pre-and post-COVID-19 pandemic using chi-square test for each type of tested antibiotic.Subgroup analysis was performed for each bacterial species according to the type of specimens, with at least 10 cultures in each period.Mixed-effects logistic regression models were used to test the impact of COVID-19 on resistance while controlling the type of specimens, seasons, and geographical distribution.Intermediate sensitivity was excluded from the logistic regression.Geographical distribution was used as a random intercept, and the period pre-or post-COVID-19, type of specimens, and seasons were the fixed effects.Models were tested for logistic regression assumptions and goodness of fit; the best models were selected according to the lowest Akaike information criterion (AIC).Results of logistic regression were represented as odds ratio (OR, 95%CI).Antibiotics were selected for logistic regression according to the IDSA 2023 Guidance and the most commonly used antibiotics in Egypt 19 .Geographic information system (GIS) was used in mapping MDR and XDR, thus providing an insight into the distribution pattern and representing the spatial variations in MDR and XDR pre-and post-COVID-19 pandemic.For this purpose, a geodatabase was developed to integrate relevant data on MDR and XDR before and after COVID-19.Generally, GIS application in mapping MDR and XDR organisms can inform decision-making, resource allocation, and public health interventions.This, consequently, can contribute to controlling the spread of AMR.

Pattern of AMR before and after COVID-19
Results illustrated significant differences in the number of positive cultures pre and post the COVID-19 pandemic for each type of specimen, among the cultures of K. pneumoniae (P < 0.001), A. baumannii (P < 0.001), E.coli (P < 0.001), and MRSA (P = 0.002).K. pneumoniae showed the highest prevalence in urine specimens (56.0%pre-COVID-19 pandemic and 71.0% in blood cultures post-COVID-19 pandemic).A. baumannii showed the highest prevalence in respiratory culture results (59.0%before the COVID-19 pandemic and 65.0% in blood samples after the COVID-19 pandemic).The prevalence of E. coli in urine cultures was high before and after the COVID-19 pandemic (86.0% and 74.0%, respectively).MRSA showed the highest prevalence among pus samples before and after the COVID-19 pandemic (89.0% and 80.0%, respectively) Supplementary Table S1.

Temporal and geographical pattern of AMR
The temporal cultures' distribution among the different seasons pre-and post-COVID-19 pandemic didn't show a significant difference in K. pneumoniae (P = 0.612) or A. baumannii (P = 0.203), although the distribution of E.coli cultures and MRSA were significantly different (P < 0.001).The geographical distribution of the cultures in Egypt was significantly different post-COVID-19 pandemic for A. baumannii (P = 0.024), E.coli (P = 0.012), and MRSA (P < 0.001), and non-significant for K. pneumoniae (P = 0.751) Supplementary Table S1.

A. baumannii
The subgroup of A. baumannii samples revealed that the sputum and pus culture results showed increased resistance to most antibiotics tested.On the other hand, blood culture results revealed a non-significant increase in resistance except against amikacin from 85.0% to 96.0% (P = 0.002), TZP from 82.0% to 97.0% (P < 0.001), and doxycycline from 79.0% to 90.0% (P = 0.010),Fig.4 Figure 5 shows the distribution of MDR and XDR A. baumannii among the sample.The MDR increased post-COVID-19 in eight governorates but didn't change in Cairo, Qalubia, and Minia, while it decreased in Alsharkia.The XDR increased post-COVID-19 in ten governorates, it only decreased in Fayum.
The subgroup analysis of the E. coli blood culture results showed a significant increase in resistance against most tested antibiotics except cefoxitin, where the E. coli blood culture results have become more susceptible to cefoxitin post COVID-19 pandemic, where resistance against cefoxitin decreased from 89.0% to 46.0% (P < 0.001).The results of the urine culture of E. coli showed a non-significant increase in resistance against most tested antibiotics; however, the E. coli in the urine has become more susceptible to levofloxacin and ciprofloxacin, where resistance decreased from 47.0% to 31.0% and from 48.0% to 31.0%(P < 0.001), respectively.The results of the E. coli pus culture showed a significant increase in resistance against most tested antibiotics and a nonsignificant increase against TZP, imipenem, and meropenem.E. coli in pus species have become more susceptible to cefoxitin, where resistance decreased from 79.0% to 37.0% (P = 0.010).Fig.6 Figure 7 shows the distribution of MDR and XDR E. coli among the sample.It illustrates an increase in MDR E. coli after COVID-19 pandemic in Alexandria, Behera, Gharbia, Port Said, and Minia while decreasing in MDR in Cairo, El Bahr Elahmar, Dakahlia, Monufia, Kafr El-Sheikh, and Matrouh.But there was no change in MDR in Fayum and Qalubia.XDR E. coli increased in Alexandria, Behera, Kafr El-Sheikh, Monufia, and Gharbia.
The subgroup analysis for MRSA revealed that resistance in blood samples increased significantly against clindamycin from 15.0% to 71.0% (P = 0.001) and developed resistance against doxycycline from 0% to 18.0% (P = 0.040).Resistance against SXT decreased from 62.0% to 39.0%, but this decrease was not statistically significant (P = 0.181).There was a slight increase in resistance against levofloxacin, ciprofloxacin, and moxifloxacin, although it was not statistically significant (P = 0.301), (P = 0.292), and (P = 0.303), respectively.The MRSA in the sputum showed an increase in resistance against clindamycin from 15.0% to 46.0% (P = 0.020) and doxycycline from 5.0% to 11.0% (P = 0.002).There was a non-significant increase in resistance against quinolones (P = 0.500)  and SXT (P = 0.621).There was a significant increase in the resistance of MRSA in pus specimens against clindamycin.Resistance increased from 15.0% to 34.0% (P < 0.001).Similarly, doxycycline resistance increased from 30.0% to 69.0% (P < 0.001).However, MRSA resistance in pus did not increase significantly against quinolones and SXT Figure 8.

Discussion
The aim of this study was to detect the impact of COVID-19 on AMR in Egypt and to detect the distribution of MDR and XDR in Egypt as an opportunity to face the threat of AMR.The current study reported a significant difference in the number of positive specimens pre-and post-COVID-19 pandemic for K. pneumoniae, A. baumannii, E. coli, and MRSA cultures.The resistance of K. pneumoniae, A. baumannii, E. coli, and MRSA against various antibiotics increased after COVID-19.The distribution of cultures between different seasons before and after the COVID-19 showed significant differences for positive E. coli cultures and MRSA, but not for K. pneumoniae or A. baumannii.Geographical distribution of cultures in Egypt significantly differed post-COVID-19 pandemic for A. baumannii, E. coli, and MRSA, but not for K. pneumoniae.MDR and XDR distribution maps illustrated changes in resistance patterns across various governorates post-COVID-19.Similar to our findings, multiple studies highlighted the impact of the COVID-19 pandemic on the increase, in AMR.Sulayyim 20 illustrated that AMR has increased during COVID-19, especially for A. baumannii, K. pneumoniae, and Staphylococcus aureus.Serapide 21 also observed a higher AMR among COVID-19 patients, with 67.8% Gram-negative culture results were classified as MDR.
Respecting to impact of climate change on AMR, where heat and humidity are considered risk factors for AMR 22 , In our study, seasonality was considered as a variable in logistic regression, however, COVID-19 had the greatest effect on AMR in our study regardless the seasonality.www.nature.com/scientificreports/

K. pneumoniae
Our study revealed that resistance of K. pneumoniae has increased against 20 antibiotics post COVID-19 pandemic.Previous studies concurred with our results, Nicola et al. 23 described an outbreak of a ceftazidimeavibactam-resistant strain of K. pneumoniae during COVID-19, indicating the emergence of increased resistance against a new approved combination during COVID-19.A study in Egypt reported that K. pneumoniae became 100% resistant to tigecycline and more than 65% resistant to cephalosporins and fluoroquinolones 24 .www.nature.com/scientificreports/
Previous studies agreed with our findings, where Kinross et al. 25 concluded that resistance to A. baumannii has increased during COVID-19.A 57% increase in carbapenem-resistant A. baumannii bloodstream infections was reported in European hospitals from 2020 to 2021 compared to 2018-2019.Moreover, Sayed et al. 26 , reported that the risk of carbapenem resistant A. baumannii infections increased to 92.7% among COVID-19 patients.

E.coli
Multiple studies show that antibiotic resistance in E. coli culture results has increased over time, especially during the COVID-19 pandemic 24,27 .Wardoyo 27 E. coli, showed resistance against penicillin, ampicillin, and cefixime at different rates of 100%, 97%, and 89%, respectively 24 .Our findings aligned with previous findings, although susceptibility to quinolones and cefoxitin increased.While E. coli resistance in urine culture results increased against fosfomycin by 44% and nitrofurantoin by 28%.

MRSA
Our results illustrated a significant increase in resistance to MRSA against five antibiotics, azithromycin, clindamycin, doxycycline, levofloxacin, and moxifloxacin with reserved susceptibility to vancomycin and linezolid.The adjusted analysis showed a significant increase in resistance against clindamycin, doxycycline, levofloxacin moxifloxacin, but not against ciprofloxacin.In the same vein, Helmy et al. 24 reported that the MRSA culture results had low resistance rates against tigecycline and linezolid.Furthermore, a meta analysis included 64 studies concluded that resistance of MRSA slightly changed post COVID-19 pandemic, with reserved susceptibility to vancomycin, tigecycline, or linezolid 28 .

MDR and XDR
During COVID-19 pandemic, MDR bacteria were implicated in 73.17% of COVID-19 patients with secondary infection.K. pneumoniae, Enterococcus spp.and MRSA were the most common MDR detected in 20.43%, 4.30% and 4.30% of all culture results, respectively 22 .In the current study, MDR and XDR but not PDR have increased significantly in Egypt post COVID-19 pandemic.In the same vein, Pałka et al. 29

Strengths and limitations
To our knowledge, this is the first study to compare AMR before and after the COVID-  For controlling these confounders, mixed-effect linear regression models were used for the most commonly used antibiotics in Egypt as a first-line treatment, aligning with IDSA 2023 guidance 19 .However, there was a limitation in our study as, due to the paucity of some antibiotic kits, not all antibiotics were tested, such as ceftazidime-avibactam, daptomycin, and teicoplanin.The small sample size of K. pneumoniae culture results might hinder the discovery of more resistance patterns.Another limitation is that the study relied primarily on culture results collected from Mabaret Al-Asafra laboratories and affiliated facilities in 16 governorates, potentially introducing sampling bias towards patients seeking medical care within this network.This may not fully represent the diverse healthcare landscape across Egypt, leading to limited generalizability of the findings.Additionally, the selection of specific bacteria (K.pneumoniae, E. coli, A. baumannii, and MRSA) for assessing antimicrobial resistance may not encompass the full spectrum of pathogens encountered clinically.Furthermore, variations in antimicrobial susceptibility testing methodologies across laboratories could affect result consistency.

Conclusions
The study analyzing AMR patterns pre and post the COVID-19 pandemic has led to some important conclusions.First, the COVID-19 pandemic had a noticeable impact on AMR dynamics, as shown by changes in the resistance profiles of bacterial strains such as K. pneumoniae, A. baumannii, E. coli, and MRSA.This rise in resistance highlights the need to implement effective antimicrobial stewardship initiatives and surveillance programs to control the spread of resistant pathogens.The study highlights the significance of acknowledging the variability in AMR patterns over time and geography.Differences in resistance profiles between regions require specific interventions and targeted surveillance to effectively manage AMR.Furthermore, it is crucial to understand the temporal variations in resistance patterns to implement customized and timely interventions to address emerging trends and prevent the spread of resistant strains.The research highlights the importance of continuing to observe and monitor AMR patterns.This helps to keep track of any changes and identify potential threats that may arise in the future.Consistent evaluation of resistance profiles is crucial to inform treatment guidelines, facilitate antimicrobial stewardship initiatives, and ensure that public health is protected from the harmful effects of AMR.

Figure 4 .
Figure 4. Sub-group analysis for A. baumannii resistance according to the type of specimen.(a) blood, (b) sputum, (c) pus

Figure 6 .
Figure 6.Sub-group analysis for E.coli resistance according to the type of specimen.(a) blood, (b) urine, (c) pus

Figure 8 .
Figure 8. Sub-group analysis for MRSA resistance according to the type of specimen.(a) blood, (b) sputum, (c) pus