https://imcjms.com Ibrahim Medical College Journal of Medical Science https://imcjms.com/registration/journal_full_text/458 Wed, 12 Apr 2023 12:58:28 +0000 Abstract Background and objectives: Several patients with SARS-CoV-2 infection presents with bacterial co-infection. The aim of the present study was to determine the bacteria responsible for co-infection in Covid-19 infected patients visiting a tertiary care hospital of Maharashtra, India. Material and methods: A cross sectional study was conducted for 3 months at tertiary care center. Covid-19 patients attending the hospital were included in the study. All the specimens were collected either at the time of admission at outdoor or within 24-48 hours of admission. All the specimens were processed for culture and antibiotic susceptibility testing as per institutional policy and standard methods. Results: Total 200 samples were collected out of which 98 (49%) patients were diagnosed with bacterial co-infection. Majority of cases with bacterial co-infection were above 21 years of age. Culture was positive in 80%, 66.7%, 49.2% and 38.8% of tracheal aspirate, pus, blood and urine samples respectively. Out of 98 cases of bacterial co-infection, 62.2% and 37.8% had infection with Gram negative and positive bacteria respectively. Most common organism isolated was Klebsiella pneumoniae (20.4%) followed by Enterococcus species (14.3%). Over 70% of Klebsiella pneumoniae isolates were resistant to aminoglycosides, cephalosporins, fluroquinolones and carbapenems while 100% Acinetobacter was resistant to all antimicrobials tested. Among 57 Of the Gram negative isolates, 5 and 24 isolates were positive for ESBL carbapenemase respectively. Conclusion: The study revealed that bacterial co-infection was present in considerable proportion of Covid-19 patients and the organisms responsible were resistant to several antimicrobial agents. IMC J Med Sci. 2023; 17(2):006. DOI: https://doi.org/10.55010/imcjms.17.016 *Correspondence: Sahjid Mukhida, Department of Microbiology, Dr. D. Y. Patil Medical College, Hospital and Research Centre, Dr. D. Y. Patil Vidyapeeth, Pimpri, Pune, Maharashtra, India-411018. E-mail: drssmukhida@rediffmail.com   Introduction The novel coronavirus first emerged in Wuhan, China, in December 2019 and has led to a global pandemic and as of May, 2022, about 500 million cases and more than 6 million deaths have been recorded around the world [1]. People with underlying morbidities are more susceptible to complications [2]. However, healthy individuals experience a mild flu-like illness or may be asymptomatic, recuperating from the infection even without any particular intervention [3]. Multiple studies have reported a correlation between SARS-CoV-2 infection and bacterial co-infections/superinfections [4-8]. About 20% of patients with SARS-CoV-2 are presented with co-infection, while 41% of superinfections were found among the ICU patients [7]. Therefore, antimicrobial agents are frequently used in cases of Covid-19 disease. The availability of bacterial and antimicrobial resistance profiles is important for rational prescription of antibiotics to treat Covid-19 patients effectively. However, data regarding types of bacteria causing co-infections and their antimicrobial resistance profiles are lacking. So, the present study was undertaken to determine the bacteria responsible for co-infection in Covid-19 patients visiting a tertiary care hospital in Maharashtra, India.   Material and methods This cross sectional study was conducted for 3 months from January to March 2022 at a tertiary care hospital of western Maharashtra. The study was approved by the institutional ethical committee prior to the initiation of the study (Ethical approval letter No. I.E.S.C./31/2022). Study population and sample collection: Covid-19 patient attending the outpatient department or admitted in the hospital were included in the study. Covid-19 was defined if a case was positive for SARS-CoV-2 either by RT-PCR or rapid antigen test or both. Detailed history regarding age, sex, associated conditions, and antibiotic, steroid, or antiviral therapies was taken from the enrolled patients. All the specimens were collected within 24-48 hours of admission. None of the sample was collected after 48 hours of hospital admission to exclude patient with hospital acquired infection in our current study sample. Samples from patients attending the outpatient were collected at the time of visit or admission of the patients. Samples for the culture were collected aseptically only from those who had suspected co-infection(s). Sample processing: All the collected specimens were processed for culture as per institutional policy and standard methods. Specimens were cultured on blood agar, MacConkey agar, Cystine-Lactose-Electrolytes-Deficient (CLED) agar by streaking methods and incubated at 37°C for 18-24 hours. Blood specimen was collected in automated BacT/Alert blood culture bottle and incubated at 370C for up to 7 days. Positive sample was sub-cultured on blood agar, MacConkey agar and incubate for 18 -24 hours. Suspected colonies were identified by Gram stain, motility, catalase, oxidase, coagulase and other standard biochemical tests [9]. All bacterial isolates were tested for antibiotic susceptibility using Kirby Bauer disc diffusion method on Mueller-Hinton agar plates. Isolated colonies were inoculated by lawn culture and antibiotic discs were placed on the surface and the plates were kept for incubation for 18-24 hours. The zone of inhibition was interpreted following the CLSI 2022 guidelines [10]. Automated Vitek 2C was used for identification of organism and antibiotic susceptibility testing as and when required. Extended spectrum Beta lactamase (ESBL) and carbapenemase production were detected by double disc diffusion method and modified Hodge test respectively [11,12].   Results Total 200 samples were collected and out of which 98 (49%) specimens yielded bacterial growth. Out of 200 cases, Gram negative and positive bacteria were isolated from 61 (30.5%) and 37 (18.5%) cases respectively and 86 (43%) had infection in single site and in 12 (6%) cases bacteria were isolated from more than one anatomical sites. The rate of culture positivity was 45.9% and 55.4% in male and female patients respectively while the rates ranged from 55.7% - 37.7% in samples collected from outdoor, indoor and ICU. Majority of cases belonged to 21 years to above 60 years of age. Culture was positive in 80%, 66.7%, 49.2% and 38.8% of tracheal aspirate, pus, blood and urine samples respectively. Only 30 (15%) patients had comorbidities and of them 6 (20%) had co-infection (Table-1).   Table-1: Detail characteristics of the study population (N=200)     Table-2 shows the pattern of bacteria isolated from different samples of Covid-19 patients. Overall, out of 98 cases of bacterial co-infection, 61 (61/98=62.2%) had infection with Gram negative bacteria while 37 (37/98=37.8%) was infected with Gram positive bacteria. Except pus, other specimens yielded mostly (55%-100%) growth of Gram negative organisms. Out of 14 pus samples, 9 (64.3%) showed growth of Gram positive bacteria. Most common organism isolated from the covid-19 patients was Klebsiella pneumoniae (20.4%) followed by Enterococcus species (14.3%), Staphylococcus aureus and Pseudomonas sp (each 12.2%) Less common isolates were Proteus sp, Elizabethkingia meningoseptica and Aerococcus viridans.   Table-2: Pattern of bacteria isolated from different specimens of Covid-19 patients     Table-3 shows the detail antimicrobial resistance profile of Gram-negative bacteria isolated from Covid-19 patients. As per the antibiotic susceptibility testing, more than 70% of Klebsiella pneumoniae isolates were resistant to aminoglycosides, cephalosporins, fluroquinolones and carbapenems. All (100%) the Acinetobacter sp, Proteus sp and E. meningoseptica were resistant to all antimicrobials tested. Among the gram-positive isolates, major drug resistance was noted against the fluroquinolones, macrolides and ampicillin (Table-4). None of the isolate was resistant to vancomycin and linezolid.   Table-3: Antimicrobial resistance patterns of isolated Gram-negative bacteria     Table-4: Antimicrobial resistance patterns of isolated Gram-positive bacteria.     Among 61 Gram negative isolates, 57 isolates were tested for ESBL and carbapenemase production. Out of 57 isolates 5 (8.8%) and 24 (42.1%) were positive for ESBL and carbapenemase respectively (Table-5). Highest (58.3%) carbapenemase production was detected in Pseudomonas sp.   Table-5: Rate of ESBL and carbapenemase producing bacteria     Discussion In the present study, bacterial co-infection was found in 49% patient. The rate was higher than many reported studies [13-18]. Those studies reported bacterial co-infection from 4% to 20% in Covid-19 patients. However on the contrary, Alshrefy et al [19] and Sreenath et al [20] reported almost similar rate of bacterial co-infection (42.4% and 47.1%) like ours. Covind-19 affects all age group patients which include from pediatric to geriatric age groups but older age group patient is infected more compared to other age group. In the present study, there was no significant increase of bacterial isolation rate with the increase of age. Mean age of the Covid-19 patients in current study was 47.13 years which was lower compared to various studies done on bacterial co-infection in Covid-19 admitted patients. The reported mean age of patients in other studies ranged from 56 to 74 years [14,16,17,21-24]. Majority of the Covid-19 patients need ventilator support as well as urinary catheterization during their ICU stay. Due to use of various immunosuppressive drugs, ICU patients have more chance to develop the bacterial infections. Several studies reported 28% to 83% bacterial infection in ICU admitted Covid-19 patients [14,15,21]. In the present study, 37.7% specimens from ICU patients had positive bacterial growth. Several studies reported Gram negative bacteria as predominant infecting agents in Covid-19 patients. Bacterial co-infection in Covid-19 patients due to Gram negative organism varied from 75% to about over 90% [22,16,15] while it was around 40% by Gram positive bacteria [16,22]. Similarly, in the current study we also found Gram negative bacteria as the predominant (62.2%) offending agents for causing co-infection in our Covid-19 cases. Only, 37.4% cases were infected by Gram positive bacteria. Among the Gram negative bacteria, K. pneumoniae was the most commonly isolated bacteria followed by Pseudomonas and Acinetobacter species. Same types of bacteria were most commonly isolated from Covid-19 cases by others [15,16,19,24]. As found by other studies [16,20], we also observed enterococci and S. aureus as the most commonly isolated Gram positive bacteria. Resistance against antimicrobial agents is a global health burden in current time. With the extensive use of antimicrobials, multi-drug resistant isolates have arisen globally. During the covid-19 pandemic, antibiotics are extensively used by the clinicians. Over 70% of Klebsiella pneumonia, the most commonly isolated bacteria in our series, was resistant to all the antimicrobial agents tested. Similar high rate of resistance was exhibited by our isolated Acinetobacter sp and Gram positive bacteria to several antibiotics tested. This could be due to prevalence of such drug resistant bacteria in the local community. The increasing exposure to healthcare environments and invasive procedures, as well as increased antibiotic usage, raises the potential for emergence of multidrug resistant bacteria [15,22,24]. The present study had some limitations. The study was conducted at a single center over a short period and the sample size was small. The present study has demonstrated that about half of the Covid-19 cases suffer from bacterial co-infections and many of those are caused by multidrug resistant bacteria. However, it is still unclear what exact roles co-infections and/or super infections play in patients with COVID-19 cases. Accurate and quick detection of bacterial co-infection with antibiotic susceptibility testing, particularly for severe infections, can assist clinicians to effectively treat Covid-19 patients with better clinical outcomes.   Acknowledgement The authors would like to acknowledge the contributions of staff and laboratory personnel of the Department of Microbiology, Dr. D.Y. Patil Medical College, Hospital and Research Center, Pimpri, Pune, India.   Source of Funding All the tests and procedures were carried out from institutional funds. Funds were not received from any external agency.   Conflict of Interest None of the author has any conflict of interest.   References 1.     World Health Organization. WHO Coronavirus (COVID-19) Dashboard. Geneva: World Health Organization; 2022. 2.     Silva D, Lima C, Magalhães V, Baltazar L. Peres N, Caligiorne R, et al. Fungal and bacterial coinfections increase mortality of severely ill COVID-19 patients. J Hosp Infect. 2021; 113: 145–154.  3.    Jose M, Desai K. Fatal superimposed bacterial sepsis in a healthy Coronavirus (COVID-19) patient. Cureus. 2020; 12(5): e8350. Doi: 10.7759/cureus.8350. 4.     Phua J, Weng L, Ling L, Egi M, Lim CM, Divatia JV, et al. Intensive care management of coronavirus disease 2019 (COVID-19): challenges and recommendations. Lancet Respir Med. 2020; 8: 506–517. doi: 10.1016/S2213-2600(20)30161-2. 5.     Elabbadi A, Turpin M, Gerotziafas GT, Teulier M, Voiriot G, Fartoukh M. Bacterial coinfection in critically ill COVID-19 patients with severe pneumonia. Infection. 2021; 49: 559–562. doi: 10.1007/s15010-020-01553-x. 6.     Garcia-Vidal C, Sanjuan G, Moreno-García E, Puerta-Alcalde P, Garcia-Pouton N, Chumbita M, et al. Incidence of co-infections and superinfections in hospitalized patients with COVID-19: a retrospective cohort study. Clin Microbiol Infect. 2020; 27: 83–88. doi: 10.1016/j.cmi.2020.07.041. 7.     Musuuza JS, Watson L, Parmasad V, Putman-Buehler N, Christensen L, Safdar N. Prevalence and outcomes of co-infection and superinfection with SARS-CoV-2 and other pathogens: a systematic review and meta-analysis. PLoS ONE. 2021; 16: e0251170. doi: org/10.1371/journal.pone.0251170. 8.     He S, Liu W, Jiang M, Huang P, Xiang Z, Deng D, et al. Clinical characteristics of COVID-19 patients with clinically diagnosed bacterial co-infection: a multi-center study. PloS ONE. 2021; 16: e0249668. doi.org/10.1371/journal.pone.0249668.  9.    Winn W, Allen S, Janda W, Koneman E, Procop G, Schreckenberger P, Woods, G. Koneman’s Color Atlas And Textbook Of Diagnostic Microbiology. 7th Edition, Lippincott Williams and Wilkins, New York. 2016; 804-807. 10.  Clinical and Laboratory Standards Institute. Performance standards for antimicrobial susceptibility testing. 32nd Ed. CLSI Supplement M100. Wayne, PA: Clinical Laboratory Standard Institute; 2022. 11.  Clinical and Laboratory Standards Institute. 2012. Performance standards for antimicrobial susceptibility testing. Twenty second informational supplement update. CLSI document M100-S22 U. Wayne, PA: Clinical and Laboratory Standards Institute; 2012. 12.  Clinical and Laboratory Standards Institute. Screening and confirmatory tests for suspected carbapenemases production. 20 supplemental table 2A-S2. Wayne, PA:Clinical Laboratory Standard Institute;2010. 13.  Westblade LF, Simon MS, Satlin MJ. Bacterial coinfections in Coronavirus Disease 2019. Trends Microbiol. 2021; 29(10): 930-941. Doi: 10.1016/j.tim.2021.03.018. 14.  Russell CD, Fairfield CJ, Drake TM, Turtle L, Seaton RA, Wootton DG, et al. Co-infections, secondary infections, and antimicrobial use in patients hospitalised with COVID-19 during the first pandemic wave from the ISARIC WHO CCP-UK study: a multicentre, prospective cohort study. The Lancet Microbe. 2021; 2(8): E354-E365. doi.org/10.1016/S2666-5247(21)00090-2. 15.  Pourajam S, Kalantari E, Talebzadeh H, Mellali H, Sami R, Soltaninejad F, et al. Secondary bacterial infection and clinical characteristics in patients with COVID-19 admitted to two intensive care units of an academic hospital in Iran during the first wave of the pandemic. Front Cell Infect Microbiol. 2022; 12: 784130. doi: 10.3389/fcimb.2022.784130. 16.  Shafran N, Shafran I, Ben-Zvi H, Sofer S, Sheena L, Krause I, et al. Secondary bacterial infection in COVID-19 patients is a stronger predictor for death compared to influenza patients. Sci Rep. 2021; 11(1): 12703. doi.org/10.1038/s41598-021-92220-0. 17.  d’Humières C, Patrier J, Lortat-Jacob B, Tran-dinh A, Chemali L, Maataoui N, et al. Two original observations concerning bacterial infections in COVID-19 patients hospitalized in intensive care units during the first wave of the epidemic in France. PloS ONE. 2021; 16(4): e0250728. doi.org/10.1371/journal.pone.0250728. 18.  Soltani S, Faramarzi S, Zandi M, Shahbahrami R, Jafarpour A, AkhavanRezayat S, et al. Bacterial coinfection among coronavirus disease 2019 patient groups: an updated systematic review and meta-analysis. New Microbes New Infect. 2021; 43:100910. doi: 10.1016/j.nmni.2021.100910. 19.  Alshrefy AJ, Alwohaibi RN, Alhazzaa SA, Almaimoni RA, AlMusailet LI, AlQahtani SY, Alshahrani MS. Incidence of bacterial and fungal secondary infections in COVID-19 patients admitted to the ICU. Int J Gen Med. 2022; 15: 7475-7485. doi.org/10.2147/IJGM.S382687. 20.  Sreenath K, Batra P, Vinayaraj EV, Bhatia R, SaiKiran K, Singh V, et al. Coinfections with other respiratory pathogens among patients with COVID-19. Microbiol Spectr. 2021; 9(1): e0016321. doi: 10.1128/Spectrum.00163-21. 21.  Bazaid AS, Barnawi H, Qanash H, Alsaif G, Aldarhami A, Gattan H, et al. Bacterial coinfection and antibiotic resistance profiles among hospitalised COVID-19 patients. Microorganisms. 2022; 10(3): 495. doi.org/10.3390/microorganisms10030495. 22.  Saeed NK, Al-Khawaja S, Alsalman, J, Almusawi S, Albalooshi NA, Al-Biltagi M. Bacterial co-infection in patients with SARS-CoV-2 in the Kingdom of Bahrain. World J Virol. 2021; 10: 168–181. 23.  Moreno-García E, Puerta-Alcalde P, Letona L, Meira F, Dueñas G, Chumbita M, et al. Bacterial co-infection at hospital admission in patients with COVID-19. Int J Infect Dis. 2022; 118: 197-202. doi: 10.1016/j.ijid.2022.03.003. 24.  Li J, Wang J, Yang Y, Cai P, Cao J, Cai X, Zhang Y. Etiology and antimicrobial resistance of secondary bacterial infections in patients hospitalized with COVID-19 in Wuhan, China: a retrospective analysis. Antimicrob Resist Infect Control. 2020; 9(1): 153. doi: 10.1186/s13756-020-00819-1          Cite this article as: Patil R, Pandey R, Gandham N, Mirza S, Vyawahare C, Khan S, Ajagunde J, Das NK, Mukhida S. Bacterial co-infection in Covid-19  patients visiting a tertiary care hospital in Maharashtra. IMC J Med Sci. 2023; 17(2): 006. DOI: https://doi.org/10.55010/imcjms.17.016 2026 Ibrahim Medical College. All rights reserved.