Isolation, Identification and Antibiotic Resistance of Different Clinical Isolates in Savar Area, Bangladesh

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Introduction
Medications used to treat and cure bacterial infections are known as antibiotics.Microorganisms that were previously vulnerable to antimicrobial drugs develop resistance to them, a phenomenon known as antimicrobial resistance (AMR) [1].A particular evolutionary push to create a counterattack mechanism against a particular antimicrobial or class of antimicrobials led to the principal mechanism of antimicrobial resistance [2].Due to the rise in AMR bacteria, hospital-acquired infections and other medical issues are on the rise globally, but the healthcare industries have not given the problem much attention [3].Antimicrobial resistance is a widespread issue with a long history in underdeveloped nations like Bangladesh where there are significant infrastructure and regulatory obstacles [4].In isolates from urine, stools, throats, throat swabs, and blood samples it has been reported that AMR is higher in gastrointestinal pathogens as well as increased rates of resistance to antibiotics such as erythromycin, amoxicillin, or tetracycline.Antimicrobials are threatened by the emergence of resistance as well as other forms of resistance.Resistance has also been transmitted among individuals and particularly among healthcare facilities.Transmission may be due to the day's interrelations between humans or the movement of animals or different types of packed foods and drinks.In countries such as Bangladesh, where the hygiene situation is so bad that bacteria can be easily transmitted, this problem will only get worse.Most bacterial infections are primarily treated empirically in Bangladesh, where etiological agents have rarely been identified.Therefore, optimizing treatment and reducing morbidity and mortality related to the disease would be of great value if it were possible to identify the more common bacterial pathogens and their corresponding AMR profile.Thus, for the purpose of being aware and helping people to cope with this issue, a study has been conducted at Lab One Hospital, Savar, Dhaka where pathogenic bacteria isolates are assessed and their antimicrobial resistance profiles analyzed from different types of clinical samples.

Ethical Consideration
The Gono Bishwabidyalay, Savar, Dhaka, Bangladesh Institutional Ethical Grant Committee agreed to this proposal.

Study Population and Biological samples
The data were collected during the period from January 2021, to January 2022, for both indoor and outdoor patients at Lab One Hospital in Savar, Bangladesh.Randomly, 403 samples, i.e., urine (87), sputum (105), throat swabs (35), stool (65), and blood (111), were collected aseptically from suspected patients and then transferred to the Department of Microbiology at Gono Bishwabidyalay by a cool chain system.

Sample Collection and Processing
Blood samples: From suspected bloodstream infection patients of all ages, blood samples have been collected aseptically.Only aerobic culture has been used for this study.For children aged 0 to 12 years, 1 to 5 ml of blood samples were properly inoculated into peds plus vials/F, and for adults older than 12 years, 8 to 10 ml of blood samples were correctly inoculated into aerobic vials/F.After that, the clinical samples were incubated fast at 35°C for a maximum of 72 hours in the automated BD Bactec TM FX40 system, unless the outcome was indicated as positive.Following regular CLSI microbiological procedures, the vials that produced a positive result were then subculture on Blood Agar and MacConkey Agar [5].
Urine samples: Urine samples were obtained from patients who were instructed to collect midstream clean catch urine in a wide, sterile container supplied by the laboratory.All patients were advised to maintain a proper aseptic procedure before urine collection.

Isolation and identification of uropathogens
By phenotypically examining the organisms on culture media designed for uropathogens, bacteria were identified.One loop of urine samples was inoculated onto blood agar, MacConkey agar, and chromogenic UTI agar media that had been sterilized and solidified.These media were then incubated aerobically for 24 hours at 37°C and counted the colonies [6].Criteria for significant bacterial numbers [7] The estimation of the number of bacteria, or colony-forming units (CFU), per milliliter of urine was performed.The following are reports on the bacterial counts: 1) Less than 10 4 organisms per milliliter, insignificant; 2) Doubtful significant, 10,000-100,000 organisms/mL (10 4 -10 5 /mL), 3) Significant bacteriuria, >105 organisms/mL (more than 100,000 organisms/mL).

Throat swabs, sputum, and stool samples
These samples were collected in secure containers with a tight lid and leak-resistant containers to decrease specimen loss and healthcare worker exposure to the specimen.The containers were clean and free of particles and interfering substances to protect the specimen from contaminants in the sterile and transported containers.Prior to the start of the antibiotic treatment, all samples were taken.Blood agar (7% sheep blood) and MacConkey agar plates were used to aseptically inoculate samples of sputum, throat, and stool, respectively.XLD agar, SS agar, and MacConkeysorbitol agar were used to inoculate stool samples [8].The CLSI recommendations were followed when performing the isolation, detection, characterization, Gram staining, microscopic properties, colony properties, and biochemical assays.

Results
Microbiological infection is now exceedingly difficult and can even be fatal.Because of this, prompt detection, identification, and testing of the antibiotic sensitivity of bacterial pathogens in the diagnostic microbiology laboratory are crucial.Antimicrobials are prescribed by doctors in impoverished nations like Bangladesh at a rate that exceeds their real necessity.Every type of antibiotic is freely accessible at any pharmacy, and anyone can purchase it without a prescription.These medications are mostly to blame for the emergence of bacterial resistance as well as for the unfavourable outcomes of cultures [11].

Isolation of Microorganisms from Different Specimens
For urine, females (18, 72%) were the most affected by the infection.Among the 25 samples, 23 (92%) were likely to suffer from a bacterial infection, and 2 (8%) were likely to suffer from a fungal infection.Among the 25 specimens, adults (17, 68%) were most likely to suffer, followed by the elderly (5, 20%) and children (3, 12%).The most abundant isolated pathogen from urine is E. coli 11 (44%).

DISCUSSION
The emergence and spread of multi-drug resistant infections is one of the main obstacles to the delivery of high-quality healthcare in hospitals in the majority of resource-constrained situations.Identification of bacterial pathogens and wise selection of antimicrobials that are effective against the organisms are essential for the successful therapy of patients with various infectious disorders.This investigation was carried out at a Lab One Hospital in Bangladesh to determine the distribution of bacterial pathogens and, consequently, to analyze their antibiotic resistance profile from various clinical specimens, such as urine, blood, throat, stool, and septum.
In our current study, 91 (22.6%) outcomes were culture-positive.Gram-negative isolates made up 81 (89.01%) more of all isolates, which is understandable given that they frequently cause serious illness and are a significant source of nosocomial infections (sepsis, pneumonia, and meningitis).
The bulk of the clinical isolates were found in throat, blood, urine, stool, and sputum samples.

CONCLUSION
Infectious diseases are getting alarmingly dangerous because of the significant emergence of multidrug-resistant (MDR) pathogenic microorganisms.Finding the most suitable antimicrobial therapeutics against infectious diseases is not well practiced in most cases in third-world countries like Bangladesh, which accelerates frequent drug-resistant strain development.Most of the isolates showed significant resistance to Ciprofloxacin, Ceftazidime, and Levofloxacin, which should not be prescribed as therapeutics against such infections by physicians and practitioners.Such investigations are needed to be accomplished regularly to guide the health care service in prescribing proper antimicrobial medications to treat infectious diseases effectively.

Figure 1 :
Graphical representation of identified pathogens from different clinical samples.

Figure 4 :
Figure 4: Frequency of bacterial isolates in suspected sputum samples

Figure 5 :
Figure 5: Frequency of bacterial isolates in suspected throat swab samples

Figure 6 :
Figure 6: Frequency of bacterial isolates in suspected stool samples

Table 1 :
Biochemical tests of the isolated bacterial species from different specimens A/A,G, H 2 S Glucose and lactose and/or sucrose fermentation, Gas produced, H 2 S produced.8.Red/Yellow with black precipitate K/A, H 2 S Glucose fermentation only, H 2 S produced.9.Yellow/Yellow with black precipitateA/A, H 2 S Glucose and lactose and/or sucrose fermentation, H 2 S produced.

Table 2 :
Bacterial cultures and its frequency

Table 3 :
Gender and age-wise distribution of infected patients (n=93).

Table 4 :
The diversity of bacterial pathogens that have been found in various clinical samples