Insight into the Burden of Antimicrobial Resistance among Bacterial Pathogens Isolated from Patients Admitted in ICUs of a Tertiary Care Hospital in India

Intensive care unit (ICU) patients are prone to develop infections by hospital prevalent organisms. The aim of the study was to determine the bacteriological profiles and their drug resistance pattern among different infections in ICU patients of a tertiary care hospital. The record-based retrospective data of culture reports of the patients admitted to all the ICUs of a tertiary care hospital during the period from January 2020 to May 2022 were analyzed. A total of 3,056 samples were obtained from 2308 patients. The infection rate among ICU patients was found to be 53.40%. Isolates belonged equally to males (50.86%) and females (49.14%). The most common culture-positive clinical specimen received was blood (39.08%) followed by respiratory samples (29.45%). Acinetobacter sp. (33.02%) was the most common organism isolated from various clinical specimens, followed by Klebsiella pneumoniae (20.89%), and Escherichia coli (13.8%). More than 80% of Acinetobacter species were found to be resistant to third-generation cephalosporins, aminoglycosides, and carbapenems, whereas minocycline (56.31% S) and colistin (100% S) were the most effective drugs. Klebsiella sp. was found to be more resistant than E.coli, and the least resistance was observed to be tetracycline (43.97%) and doxycycline (55.84%). Among Staphylococcus aureus, 82.78% of strains were methicillin-resistant (MRSA). Vancomycin-resistant Enterococci (VRE) sp. accounted for 16.67% of the isolates. Evidence-based knowledge regarding the local bacterial organisms and their antimicrobial resistance pattern is pivotal in deciding empirical drug therapy, ultimately leading to the management of antimicrobial resistance (AMR).


Introduction
Microbial infections and antimicrobial resistance have been recognized as a critical issue worldwide, afecting public health, therefore considering the most important causes of mortality and morbidity [1].According to INEbase death statistics, in the year 2020, infectious diseases were the third most common cause of death, accounting for 16.4% of the total, which includes identifed and suspected COVID-19 cases [2].Although intensive care units (ICUs) account for fewer than ten percent of total beds, they serve as a factory for the development and spread of microbial infections [3].In countries where routine infection control measures are implemented extensively, ICUs are still potential sources of nosocomial infections [4].Accommodation of seriously ill patients who are usually immunocompromised, and undergoing invasive procedures in ICUs, results in a fve to seven-fold higher risk of nosocomial infections than other patients [5].Other factors involved are increased duration of stay, use of immunosuppressive drugs, and prolonged or inappropriate use of broad-spectrum antibiotics [5].Tis leads to a huge economic burden on the health system of developing countries.Terefore, we hypothesized to investigate the antimicrobial resistance among bacterial pathogens isolated from patients admitted in ICUs.
Te development of antibiotics has been acknowledged as the greatest medical advance in human history.However, antimicrobial resistance (AMR) has been rising due to misuse of these valuable compounds which has ultimately resulted in some infections becoming efectively untreatable [6].According to a report by the UK government health department, ten million people will die in a year from drugresistant infections by 2050, if urgent action is not taken.Currently, at least 700,000 people lose their lives each year globally, because of drug resistance in illnesses such as bacterial infections, malaria, HIV/AIDS, or tuberculosis [7].In addition, the emergence of highly resistant microorganisms in ICUs has become a major threat to patients, leading to worse outcomes and demand for the last line of antimicrobials [8].
Surveillance of AMR is the frst and foremost essential step towards curtailing the spread of antimicrobial resistance, forming policies, and for infection prevention and control interventions.Importantly, it is the cornerstone for monitoring the impact of local, national, and global strategies.In 2015, WHO launched the Global Antimicrobial Resistance and Use Surveillance System (GLASS), the frst global collaborative efort to standardize AMR surveillance.Similarly, in 2021, the Indian Council of Medical Research (ICMR) also started the Antimicrobial Resistance Surveillance System (i-AMRSS), a promising tool for the collection, management, and analysis of AMR data [9].
AMR surveillance helps to generate baseline data on the pattern of microorganisms in the hospital and their susceptibility profle, which in turn helps in deciding efective and rational empirical treatment.Tese data vary from country to country, hospital to hospital, and even among diferent wards of the same hospital.Terefore, the objective of this study was to determine the bacteriological profles and their drug resistance pattern among diferent infections in ICU patients of a tertiary care hospital.

2.1.
Settings.Tis retrospective observational study was carried out in the department of microbiology of a tertiary care hospital in North India from January 2020 to May 2022.

Subjects.
Te study was undertaken based on reports of bacterial isolates of various clinical specimens from diferent ICUs, such as medical ICUs (MICUs), surgical ICUs (SICUs), and paediatric ICUs (PICUs), which were submitted to the microbiology laboratory for culture and sensitivity during the study period.

Inclusion Criteria.
All the patients who were admitted to various ICUs (medical, surgical, and paediatric ICUs) during the study period and whose reports were retrieved from the laboratory were included in the study.Various sources of clinical specimens included blood, urine, pus, cerebrospinal fuid (CSF), catheter tips, endotracheal tips, drainage fuids (trauma pleural and ascitic), bronchial aspirates (BALs), central venous catheters (CVCs), sputum, and gastric aspirate.Only bacterial isolates were included.

Exclusion Criteria
(1) Mixed growths (three or more isolates) per specimen in urine culture only (2)  Colistin susceptibility testing was performed using the microbroth dilution test as per CLSI guidelines.Te test for extended-spectrum beta-lactamase (ESBL) production was performed using the combined disk difusion method with antibiotic disks of ceftazidime/clavulanic acid (30/10 mcg) and ceftazidime (30 mcg) (as per CLSI).Cefoxitin disk difusion (30 μg) was used to detect MRSA.Vancomycin susceptibility testing for Staphylococcus aureus was done using vancomycin screen agar method (BHI agar with 6μg/ mL of vancomycin).
Statistical analysis: data were analyzed statistically with categorical variables like the proportion of bacterial infections across diferent ICUs, sample type, age groups, and gender.Patterns of microorganisms, their susceptibility profles, and sites of infections were also analyzed and expressed as percentages.SPSS software was used for statistical analysis.

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Canadian Journal of Infectious Diseases and Medical Microbiology

Results
In the present study, a total of 3056 samples were received from 2308 patients admitted to ICUs and were used for data analysis.Of 3056 samples, bacterial pathogens were isolated from 1632 samples (53.40%).

Distribution of Bacteriological
Isolates.Acinetobacter sp.(33.02%) was the most common organism isolated from various clinical specimens, followed by Klebsiella pneumoniae (20.89%), and Escherichia coli (13.8%).Among the Gram-positive organisms, Staphylococcus aureus (16.78%) was the most common organism followed by Enterococcus sp.(3.73%).Details of the distribution of bacterial isolates are shown in Table 2.
Almost half of the isolates of Pseudomonas aeruginosa (P.aeruginosa) were resistant to β-lactam antimicrobials, quinolones, and carbapenems.Least resistance was seen in aztreonam (21.82%) and piperacillin-tazobactam (29.89%) as shown in Figure 2. All the Gram-negative isolates were susceptible to colistin (100% S).Among GNB isolated from urine specimens, E.coli was most susceptible to nitrofurantoin followed by Klebsiella sp.Acinetobacter spp.and Pseudomonas spp.were extremely resistant to nitrofurantoin.

Discussion
Te rapid development and spread of antimicrobial resistance among bacteria are threatening public health worldwide.Multidrug-resistant infections are one of the major causes of mortality and morbidity among patients admitted to hospitals.According to the World Health Organization (WHO), people living in a low-income country are far more likely to die of a communicable disease than of a noncommunicable disease.Despite the global decline, six of the top ten causes of death in low-income countries are communicable diseases [10].Hence, this study was undertaken to provide insight into the extent of antimicrobial resistance among bacteria isolated from patients admitted to ICUs.
A total of 2308 patients were admitted to ICUs in the study period, from which 3056 samples were taken and sent to the microbiology lab for the bacterial culture.Te infection rate among ICU patients was found to be 53.40%.
Te demographic variables of culture-positive patients in this study revealed that the number of males and females developing infection inside the ICU was almost equal.In many studies, the infection rate in men was found to be higher than the one in women [11,12].
Te majority of the isolates were from 1 to 11 years old children followed by adults from 18 to 45 years of age (28.03%).Bloodstream infections (BSIs) accounted for the most common infection in the ICU setting (39.08%) followed by respiratory infections (29.45%).Tis fnding is similar to that of the study performed by Fahim in 2021 in Egypt, where the highest number of pathogens was isolated from blood cultures (44.84%), followed by urine (41.41%), and then wound swabs (13.75%) [13].However, studies performed by Satyajeet et al. in 2016 and Moolchandani et al. in 2017 in diferent parts of India showed that pneumonia was the most common ICU infection [12,14].
Nonfermenting Gram-negative bacilli (NF-GNB) have emerged as important hospital-acquired pathogens because of an increasingly unreasonable and irrational use of broadspectrum antimicrobials.Usually, these pathogens are inhabitants of nature, particularly in soil and water.In the hospital environment, they may be isolated from instruments such as ventilators, tubing, and from the skin of healthcare workers [12].Also, in the present study,   [12].In Asian countries like India, Pseudomonas spp.have been the most common organism isolated from ICUs [4,5,12,16].
Gram-positive cocci such as Staphylococcus aureus (16.78%) and Enterococcus spp.(3.73%) were also seen to cause infections in the ICU settings in this study.Te global scenario shows that Gram-positive organisms are more common in the western world (North America and Europe) than in Asian countries [17][18][19].
Finally, the resistance patterns of various microorganisms were analyzed in this study.More than 80% of Acinetobacter spp.were found to be resistant to third-  generation cephalosporins, aminoglycosides, and carbapenems.Minocycline (56.31%S) and colistin (100% S) were the most efective drugs for Acinetobacter sp.Te results were similar to those of the study performed by Said et al. in 2021 in Saudi Arabia, where A. baumannii was found to be the most resistant pathogen isolated from clinical specimens and the isolates were fully resistant to almost all antibiotics tested, except for amikacin (61.25%), colistin (5%), and ertapenem (0%) [11].Given the noteworthy prevalence of Acinetobacter species, empirical therapy in the ICU setting may need to include agents efective against this organism depending on local resistance patterns.Consideration may be given to newer combination agents such as imipenemcilastatin, ceftazidime-avibactam, or ceftolozanetazobactam.Colistin in combination with carbapenem would be a potential option for the management of such drug-resistant bugs [20].
A wide range of antibiotics were inefective in the treatment of Enterobacteriaceae GNB.E. coli was highly resistant to β-lactam antibiotics including 3rd generation cephalosporins and fuoroquinolones.Least resistance was recorded in chloramphenicol (33.3%), amikacin (40.74%), and gentamicin (42.14%).Klebsiella sp. was found to be more resistant than E.coli, where least resistance was observed in tetracycline (43.97%) and doxycycline (55.84%).Colistin was again found to be 100% susceptible to both organisms.For this reason, it is imperative to reserve colistin until antimicrobial susceptibility patterns mandate its use.Similar results were seen in a study conducted by Fahim in 2021 in Egypt, where Gram-negative isolates showed the least frequency of resistance against nitrofurantoin (52.5%), amikacin (58.01%), followed by imipenem (59.78%), and meropenem (61.82%) [13].
P. aeruginosa was found to be moderately resistant to anti-pseudomonal cephalosporins (41.5%) and carbapenems (55.2%).Least resistant drugs included piperacillintazobactam (29.89%), aztreonam (21.82%), and colistin (0%).Moolchandani et al. had reported a similar resistance pattern of Pseudomonas sp. to various classes of drugs ranging from 25 to 70% [12].In many studies, P. aeruginosa has been recorded as one of the organisms showing high levels of resistance (over 80% R) to routine drugs used for treatment, including carbapenems [11].
In this study, the production of extended-spectrum β-lactamases (ESBLs) was seen to be 16.0% in E. coli and Klebsiella sp., whereas only 4.10% was seen in Acinetobacter sp.Chakraverti in 2015 in Bihar reported a higher rate of ESBL production (30-50%) [5].While the prevalence of ESBL-producing isolates was reported extremely high (67-84%) in a study conducted by Uc-Cachon in Mexico in 2019 [21].
Te analysis of the antibiotic susceptibility profle of S. aureus revealed that 82.78% strains were methicillinresistant (MRSA).MRSA has been reported to vary from 40 to 57% in various studies [12,15].Methicillin resistance was observed to be higher in coagulase-negative Staphylococcus spp.(CONS) accounting for 82.05%.Tis result is frightening as this might lead to an increase in use of reserve antibiotics such as vancomycin, which increases the possibility of development of vancomycin resistance.However, S. aureus was found to be totally susceptible to vancomycin in this study.Similar results were seen in a study conducted by Faim in 2021 in Egypt, where S. aureus exhibited high resistance rates to many antibiotics including penicillin (97.1%), gentamicin (73.91%), and all beta-lactams.Also, CONS showed comparable β-lactam resistance rates to S. aureus with a slightly higher level of methicillin resistance (77.6%), as well as 100% susceptibility to linezolid and vancomycin [13].
In this study, Enterococci spp.expressed a high level of resistance to erythromycin (81.97%), ciprofoxacin (79.63%), tetracycline (74.58%), penicillin (74.51%), and high-level gentamicin (65.57%).Tis pattern of resistance obviates the synergistic action of β-lactam and aminoglycoside agents.Also, the level of VRE was seen to be 16.67% of total Enterococci spp.isolates.In a study conducted by Pawar et al. in 2016 in Maharashtra, VRE was reported in 12% of isolates [14].VRE has been reported to be as high as 63% by Despotovic et al. in 2020 [22].In developed countries, such as in Europe, the proportions of vancomycin-resistant E. faecium were reported to increase from 8.1% in 2012 to 19.0% in 2018 [23].Te limitations of the study included a lack of adequate data on clinical information and an inability to analyze the rate of coresistance among diferent pathogens.Also, the treatment choices and outcomes of the patients with infections could not be analyzed as it was a retrospective study.
Clinicians could use this evidence-based knowledge directly to tailor antibiotic regimens to the specifc pathogens identifed, optimizing the chances of efective treatment [24].Te fndings can be incorporated into local antibiotic guidelines and protocols for the ICU.Te study results may

Conclusion
Bacteriological profles and antimicrobial susceptibility data are important to identify emerging drug-resistant pathogens, provide opportunities for new drug development, and form local antimicrobial policy, which further helps in having national data.Our study has shown 100% colistin susceptibility for E. coli, Acinetobacter sp., Klebsiella pneumoniae, and Staphylococcus aureus; therefore, it should be considered as the most efective drugs.Evidence-based knowledge regarding the local bacterial organisms and their antimicrobial resistance pattern is pivotal in deciding empirical drug therapy, ultimately leading to the management of antimicrobial resistance.Te Global Antibiotic Resistance Partnership (GARP) guidelines recommend a multipronged strategy in low-and middle-income countries to optimize the use of antibiotics and reduce antibiotic resistance.Te priority actions recommended national surveillance of antibiotic use and antibiotic resistance, as well as strengthening of infection control committees in hospitals.Hence, studies like this are particularly important in countries like India, where infection control practices and antimicrobial policies need to be strengthened to boost antibiotic stewardship and help in the reduction of antibiotic resistance and patient morbidity and mortality in the long run.

Figure 2 :
Figure 2: Percentage resistance to various antimicrobials among Pseudomonas aeruginosa isolates isolated from the ICU patients at LHMC and its associated hospitals.

Table 1 :
[1]adian Journal of Infectious Diseases and Medical Microbiology Acinetobacter sp.(33.02%) was the most common organism isolated from ICUs.Tis fnding is in concordance with the study conducted by Mehta et al. in 2015 in Ahmedabad,where Acinetobacter sp.(30.92%) was the commonest organism[1].Next to Acinetobacter spp., Enterobacteriaceae GNB such as Klebsiella sp.(20.89%) were the second most Clinical specimen distribution and demographic profle of culture-positive patients (n � 1632).

Table 2 :
Distribution of bacterial isolates in culture-positive patients (n � 1632).Burkholderia cepacia (0.06%), Stenotrophomonas sp.(0.06%), and Sphingomonas sp.(0.06%).NF-GNB were also the most common organism in the study conducted by Moolchandani et al. in 2017 in Puducherry; however, the predominating organisms were Percentage resistance to various antimicrobials among major Gram-negative bacilli isolated from the ICU patients at LHMC and its associated hospitals.

Table 3 :
Antimicrobial susceptibility pattern of predominant Gram-positive cocci.Canadian Journal of Infectious Diseases and Medical Microbiology stimulate further research into new treatment modalities or preventive measures for infections caused by these prevalent organisms.Te study encourages regular surveillance within the ICU and broader healthcare environments to track changes in resistance patterns, allowing for timely adjustments in empirical therapy and infection prevention strategies.