In the world of infectious diseases, gram-negative bacilli are emerging and causing serious infections in few patients in both community and hospital based settings [1]. One of the major public health problems is the increasing multi-drug resistant pathogens, these pathogens resulted in a major clinical crisis and comprising therapy [2]. These ESBL and MBL pathogens can spread easily between species and they are coded by plasmid. Globally, the treatment options for ESBL and MBL are limited, as infections are common presentation in our patients and also in ICU patients. Treating of ESBL and MBL is becoming difficult and often resulting in therapeutic failure.

Βeta lactamases enzymes such as extended-spectrum β-lactamse (ESBL), AmpC β-lactamses and carbapenemases are responsible for resistance to β-lactam antibiotics such as penicillin, cephamycin and carbapenem [3]. Antibiotic resistance pathogens are emerging and their importance in public health is a growing concern. As the year’s passes, the spread of AMR bacteria is increases and being detected worldwide. The transmission of these bacteria and emergence of more antibiotic resistance organisms is due to misuse of antibiotics in medical industries and in animal husbandry. In previous years, carbapenems have efficiently of treating ESBL and AmpC organisms efficiently and was using as a last resort of management of threatening infections caused by these resistant pathogens [4,5]. In recent years, carbapenems resistance has emerged due to development of carbapenem resistance in ESBL and AmpC pathogens by selective pressure. Worldwide, metallo beta- lactamase enzymes are responsible for carbapenem resistant organisms [6]. Options to initiate antibiotic therapy is draining, there is a need to prioritize the antimicrobial stewardship program in all health care institutions, a strigent regulations from government authorities is mostly needed to implement this. Within the mean time hospitals can focus on detection of resistant pathogens and plotting a antibiogram quarterly or every 6 months.  Detection of antibiotic resistance pathogens has become crucial in diagnostic laboratories

AIM: The aim of the present study is to isolate the pathogens from various clinical samples and to determine antimicrobial susceptibility pattern of isolates with a special focus on ESBL and MBL resistance mechanisms.

Study design: An observational cross-sectional study was performed at department of Microbiology, Government Medical College, Anantapuram. This study was conducted for a period of one year from June 2023 to June 2024 on clinical samples received to the department. Institutional ethical board committee has approved this study to be undertaken for the benefit of community to get rid of antimicrobial resistance pathogens. Informed consent was taken from all included patients before doing this study.

Inclusion criteria:

Both community and hospital acquired clinical samples of all ages and both sexes.

Exclusion criteria:

Sample with multiple isolates or identified as colonizers after through clinical examination.

Sample size calculation:

Sample size was calculated based on the prevalence estimates of previous studies of ESBL and MBL isolates from clinical samples.

Sample Collection and Preparation:

All clinical samples fallen in acceptable criteria as per the lab policy were collected and received at the lab. Specimens were inoculated on to nutrient agar, 5% sheep blood agar, Macconkey agar and chocolate agar. After incubation at 37⁰C for 24-48 hours colony count was done and expressed as number of colony forming units per ml for BAL, ET aspirates and urine samples. Pathogen identification up to species was performed by colony characterization, biochemical reactions and inoculation on special media.

Antibiotic Susceptibility Testing:

AST is done by modified Kirby bauer disc diffusion method on Mueller Hinton agar based on CLSI guidelines [7,8]. The quality check done with the quality control strains. – Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853 and S.aureus ATCC 25923.

Antibiotic disks used for Gram negative isolates antibiotics were: amoxyclav (30 µg), piperacillin+tazobactum (100/10 µg), ceftazidime (30 µg), ceftriaxone (30 µg), cefipime (30 µg), Ceftazidime+clavulanic acid (30/10 µg), piperacillin+tazobactum (30/6 µg), ciprofloxacin (5 µg), meropenem (10 µg), amikacin (30 µg), tigecycline (15 µg) and colistin (50 µg). Standard Quality Control strains were used as a part of testing. Multi Drug testing was done for all strains isolated according to CLSI guidelines.

ESBL and MBL Detection:

ESBL and MBL detection: Extended spectrum beta lactamases (ESBL) producing strains were confirmed by utilizing a double-disk synergy test with cephalosporin and cephalosporin/ clavulanate combination disks (Ceftazidime and Ceftazidime/Ceftazidime+clavulanic acid) for non fermenters isolated. Metallo beta-lactamases producing strains were confirmed by using disks of imipenem (10µg) and imipenem with ethylene diamine tetraacetic acid (EDTA) (10µg + 750 mg) for non fermenters.

Data Tabulation: All details pertaining to patients including age, sex, type of specimen, ICU admission number, socioeconomic status, previous history of hospitalization, antibiotic intake, organism isolated, sensitivity pattern of antibiotics was collected and entered into Microsoft excel sheet. All descriptive quantitative variables were expressed as numbers and percentages.

A total of 948 clinical samples were received from various department of government general hospital, Anantapuram. Among these, 63.8% urine samples, 13.8% sputum samples, 4.08% pus samples, 4.9% blood samples, 8.12% bronchioalveolar lavage (BAL) samples, 3.69% fluid samples such as ascitic fluid, pericardial fluid, peritoneal fluid, cerebrospinal fluid, synovial fluid, pleural fluid, and 2.21% other samples were assessed in this particular study to analyze the ESBL and MBL pathogen with its antibiotic susceptibility pattern (Fig 1).

Fig 1. Various samples analyzed for ESBL and MBL pathogens

Different organisms were isolated from 948 clinical samples, among these gram negative organisms were 253 (26.6%). Out of 253 gram negative pathogens, with the commonest being 37.1% (94 out of 253) Escherichia coli, followed by 31.6% (80/253) Klebsiella species, 13.4% (34/253) Pseudomonas species, 7.5% (19/253) Citrobacter species, 3.5% (9/253) Acinetobacter species, 2.7% (7/253) Proteus species, 2.7% (7/253) Enterobacter species and 1.1% (3/253) Morganella species were detected (Table 1).

Among Urine samples, Escherichia coli (44.8%) was the commonest followed by Klebsiella species (20.5%) and Pseudomonas species (9.09%). Among all respiratory samples, the most common pathogen isolated was Klebsiella (43.7%) followed by Pseudomonas species (18.7%) and Acinetobacter species (16.6%). Blood samples showed Escherichia coli (42.8%) and Klebsiella species (42.8%) as the predominant pathogen. Fluids yielded the Escherichia coli (60%) and Klebsiella species (40%) (Table 1).

Table 1. Distribution various pathogens in different clinical samples

23.7% of pathogens are ESBL producers, most common being Klebsiella species and Escherichia coli.

Table 2. ESBL producing organisms

9.09% of pathogens are ESBL producers, most common being Klebsiella species and Escherichia coli.

Table 3. MBL producing organisms

On assessing the sensitivity pattern of ESBL producers, around 90% of isolates were sensitive to Amikacin, gentamicin, ciprofloxacin, imipenem, meropenem, 81 % were sensitive to tetracycline, 63% of isolates sensitive to cotrimoxazole, 70% were sensitive to Piperacillin+ tazobactum and 50% were sensitive to Amoxyclav.

Table 4. Antibiotic susceptibility pattern of ESBL Producers

The sensitivity pattern of MBL producers showed that >60% of isolates were sensitive to Amikacin, gentamicin, ciprofloxacin, around 50 % were sensitive to tetracycline, Piperacillin+ tazobactum.

Table 5. Antibitoic susceptibility pattern of ESBL Producers

Extended-spectrum β-lactamases, enzymes capable of hydrolyzing a broad range of β-lactam antibiotics, and metallo β-lactamases, known for their ability to confer resistance to carbapenems, they play a pivotal role in the world of antimicrobial resistance. Knowing about these pathogens demographic distribution and its susceptibility pattern is important to prevent the spread and treatment of infections.

Out of 253 gram negative pathogens, with the commonest being 37.1% (94 out of 253) Escherichia coli, followed by 31.6% (80/253) Klebsiella species, 13.4% (34/253) Pseudomonas species, 7.5% (19/253) Citrobacter species, 3.5% (9/253) Acinetobacter species, 2.7% (7/253) Proteus species, 2.7% (7/253) Enterobacter species and 1.1% (3/253) Morganella species were detected in the present study. In similar to our study Shrestha A et al [9] did a study on ESBL and MBL pathogens in Nepal, they observed 4.6% of culture positives among them Escherichia coli (46.7%) was noted as the predominant pathogen followed by Klebsiella species (25.4%), Pseudomonas species (13.7%), Citrobacter species (6.6%), Proteus species (4.06%), Acinetobacter (1.5%), Morganella species (1.01%), Enterobacter (0.5%), and Burkholderia species (0.5%). Nepal K et al [10] observed E.coli and K.pneumoniae as predominant pathogens in their study on all clinical samples tested.

In this study among Urine samples, Escherichia coli (44.8%) was the commonest followed by Klebsiella species (20.5%) and Pseudomonas species (9.09%). Among all respiratory samples, the most common pathogen isolated was Klebsiella (43.7%) followed by Pseudomonas species (18.7%) and Acinetobacter species (16.6%). Blood samples showed Escherichia coli (42.8%) and Klebsiella species (42.8%) as the predominant pathogen. Fluids yielded the Escherichia coli (60%) and Klebsiella species (40%). Shrestha A et al [9] noticed urine specimens were most commonly obtained clinical samples, which was 78.7%. Escherichia coli were a predominant pathogen in urine samples with the percentage of 58.1%. Pseudomonas aeruginosa (40%) was the most common pathogen in sputum, from pus samples Klebsiella species (62.5%) were yielded as the predominant isolate. Acinetobacter was isolated from tracheal aspirate, throat swab and urine. Acinetobacter is a usually hospital acquired pathogen, but we are noticing it common acquired pathogen now-a-days, one of the reason might be mixing of hospital waste and sewage with community environments. Escherichia coli were isolated from urine about 84% and K.pneumoniae isolates were detected in 51.3% urine and 17.9% sputum samples [10].

23.7% and 9.09% of pathogens are ESBL and MBL producers respectively, most common being Klebsiella species and Escherichia coli. In line with this 23.9% were ESBL producers and 8.0% were found to be MBL producers in a study [9]. ESBL production was observed in various studies with the percentage of 22.4% [11], 16.0% [12]. Escherichia coli and Klebsiella were the predominant pathogen able to secrete ESBL in various clinical specimens; it is supported by another study [13]. Different research works in Korea, Taiwan, and Hong Kong noted Escherichia coli ESBL percentage as 4.8%, 8.5%, and up to 12% respectively [14,15]. In similar to this study Bora et al [16] documented the MBL production was 18.9% and 21.0% for E.coli and K.pneumoniae respectively. Nepal K et al [10] noted 34.5% of ESBL producers and 7% of MBL producers among which 22.2% E.coli and 55.6% K.pneumoniae. They reported that 7% of isolates were MBL producers. MBL isolates maximum activity showed in E.coli (38%) followed by Pseudomonas spp. (31%), K.pneumoniae (19%), Proteus spp. [12%] [9]. ESBL and MBL production varies from country to country and regions within the country as it depends on the usage of antibiotics in human medicine, animal husbandry and farming, misuse of antibiotics and spread of antimicrobial resistance. ESBL production of E.coli could be as high as 22 to 75% in Asian countries [17] and it could be as low as <0.1% in studies done by Japan [18] and average being 3% in USA [19]. MBL activity showed high in Shrestha S et al [20] research work which was about 17.43%, whereas we noticed in a study conducted by Mishra S et al was low i.e.,1.3% [21].

On assessing the sensitivity pattern of ESBL producers, around 90% of isolates were sensitive to amikacin, gentamicin, ciprofloxacin, imipenem, meropenem, 81 % were sensitive to tetracycline, 63% of isolates sensitive to cotrimoxazole, 70% were sensitive to Piperacillin+ tazobactum and 50% were sensitive to amoxyclav. Shrestha A et al [9] reported all ESBL positive isolates showed 85.1% ciprofloxacin resistance and 83.0% cotrimoxazole resistance. Sensitivity was high towards imipenem (78.7%), amikacin (73.3%), followed by piperacillin tazobactam (68.1%). Khorvash et al [22] showed resistance to cefotaxime and ceftazidime and sensitivity towards carbapenems (imipenem and meropenem) (96%), and piperacillin+tazobactam (84%).

The sensitivity pattern of MBL producers showed that >60% of isolates were sensitive to Amikacin, gentamicin, ciprofloxacin, around 50 % were sensitive to tetracycline, Piperacillin+ tazobactum. In similar to study, high resistance noted to cefepime (80%), piperacillin+tazobactam (75%), gentamicin (75%), cefoperazone+sulbactam (68%) and were found to be sensitive towards amikacin (44%) and cefoperazone+sulbactam (32%) [9].

The strengths of this study is detection of resistance patterns which can help in framing a antibiogram of the hospital, justification to start empirical therapy depending the clinical condition and also alerting the clinicians how the impact of drug resistant pathogens on patient’s health. We have restricted our study to finding the antibiotic resistance pattern of all clinical samples, may be studies focusing on the antibiotic susceptibility pattern of each samples with individual organism helpful for further management of the clinical condition.

From this study we conclude that Escherichia coli and Klebsiella pneumoniae are common isolates in all clinical samples and they are representing the major ESBL and MBL producers among pathogens. The observation in this study has lead to the know the percentage of antibiotic resistance in ESBL and MBL producers, which showed quite a good number of organisms were sensitive to aminoglycosides, penems, fluoroquinolones, and broad spectrum antibiotics. Antibiotic resistance numbers increase in worldwide is creating a major public health problem, so central and government authorities should strengthen the antimicrobial stewardship program in all health care institutes and should create awareness on drug resistant management to all health care workers and other industries those are using antibiotics.

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