Prevalence of Extended-spectrum Beta-lactamase (ESBL), Metallo Beta-lactamase (MBL) and Carbapenemase Producing Acinetobacter Species Isolated from Various Clinical Samples in Tertiary Care Hospital

Acinetobacter is an important nosocomial pathogen causing health care associated infections. It is highly antibiotic resistant gram-negative bacilli. The study was done to determine the prevalence of Acinetobacter species isolated from various clinical samples with their antibiotic susceptibility pattern. To determine the antimicrobial susceptibility pattern of the isolated Acinetobacter species and also the multidrug resistant mechanisms by phenotypic characterization. The retrospective study was carried out in patients diagnosed with Acinetobacter infection in the Microbiology Department, Krishna Institute of Medical Sciences,


INTRODUCTION
Acinetobacter are ubiquitous, free livings saprophytes, small aerobic Gram negative coccobacilli that prefer moist environment and can be easily found in soil, water, food and sewage. 1 They are also ubiquitous organisms in the hospital environment, where they play a significant role in the colonization and infection of patients admitted in hospitals. 1Acinetobacter causes wide spectrum of infections, including nosocomial pneumonia, secondary meningitis, surgical wound infections, skin and soft tissue infections, urinary tract infections (UTI) and bacteraemia. 2Acinetobacter spp.are generally considered a part of the normal flora of the skin, 3 mucous membrane or the pharynx, and human respiratory secretions 4 and are accountable for a wide variety of local and systemic infections, including pneumonia, septicaemia and wound infections. 5The main body areas populated by these microorganisms in hospitalized patients are the skin, oropharynx, and digestive tract.Acinetobacter spp.were isolated from various locations of the healthy individuals' body including the forehead, nose, ear, throat, trachea, conjunctiva, hand, vagina and perineum, inhabiting humid areas, such as axillae, the groin and toe webs. 6Acinetobacter spp.are considered to be relatively low-grade bacteria. 7In nature, the bacteria belonging to the genus Acinetobacter, an environmental bacterium, is widely distributed and able to survive on environmental surfaces and linked to numerous nosocomial and opportunistic infections.
The sites in which Acinetobacter nosocomial infections predominantly occur are contingent on the duration and local epidemiological factors present.In the preliminary reports, it has been observed that urinary tract infections (UTIs) are prevalent in intensive care units.However, it is worth nothing that the incidence of UTIs has witnessed a decline, owing to the improved care of urinary catheters.On the contrary, there has been a considerable increase in the occurrence of nosocomial pneumonia. 8f the Acinetobacter spp, A. baumannii is an important pathogen with a high morbidity and mortality, especially in the critically ill patients. 9Acinetobacter spp. is associated with a wide variety of infections -ventilator-associated pneumonia (VAP), blood stream infections (BIs), urinary tract infections, bacteraemia, meningitis, skin and wound diseases, ventriculitis, cholangitis, peritonitis, and infective endocarditis.Colonization of the skin and respiratory tract by the bacteria may occur without causing an infection.The survival of A. baumannii in harsh environmental conditions and its ability to develop multidrug resistance attributes to making infections caused this organism highly lethal, mainly in patients who have undergone major surgeries, the immunocompromised, those with malignancy, prolonged illness and in the extremes of age. 9Antimicrobials are chemical compounds either bactericidal or bacteriostatic, used in medical interventions to actively kill or inhibit the pathogens.
The emergence of antimicrobial-resistant Acinetobacter species is due both to the selective pressure exerted by the use of broad-spectrum antimicrobials and transmission of strains among patients, although the relative contributions of these mechanisms are not yet known. 10nfections caused by antibiotic-susceptible Acinetobacter isolates have usually been treated with broad-spectrum cephalosporins, b-lactam-b-lacta mase inhibitor combinations (e.g., a combination that includes sulbactam, a drug marketed only in combination, in the United States), or carbapenems (e.g., imipenem or meropenem), used alone or in combination with an aminoglycoside. 11The primary goal for the control of Acinetobacter infection is recognizing its presence in a hospital or long-term care facility at an early stage, controlling its spread aggressively and preventing the establishment of endemic strains.Control measures are based almost entirely on experiences from outbreaks of Acinetobacter infection and generally address the organism's major epidemic modes of transmission and the excessive use of broad-spectrum antibiotics.

METHODOLOGY
"Prevalence of Extended-spectrum betalactamase (ESBL), Metallo beta-lactamase (MBL) and Carbapenemase producing Acinetobacter species isolated from various clinical sample in tertiary care hospital'' study was carried out in the Microbiology department at Krishna Institute of Medical Sciences and Krishna Hospital and Medical Research Centre, Karad over a 2 years period from November 2020 to November 2022.

Inclusion criteria
Isolates of Acinetobacter species, from clinical samples of patients admitted with clinical infection to the IPD/OPD.Patients with both sexes involved.

Exclusion criteria
To avoid duplication, isolates from the same patients and specimens were excluded.

Statistical analysis
Data were filled in the MS Excel Software.Then, analyzed results were expressed as percentage and p values, by Chi square test using Graph Pad Instant software.If the probability is less than 0.05, the association or difference is said to be significant.

Sample collection
The various clinical samples from which Acinetobacter species were isolated includes -Pus, Sputum, Urine, Blood, ETT secretions, Body fluids, Wound swab, CSF and others, from all age groups and both gender of patients.Appropriate sterile containers were used for collection of the samples and then transported to the laboratory.

Bacterial identification
The clinical samples were cultured on appropriate culture media and the organisms isolated were identified using standard Microbiology procedures. 12Nutrient agar, MacConkey agar, (Figure 1a and 1b), Blood agar, Chocolate agar were used for inoculation of the clinical samples and incubated at 37°C for 24 hours.The isolates were then identified based on colony morphology on agar and Gram stain of the smear of colonies (Figure 2).Oxidase, Catalase reactions were performed (Figure 3).Further Biochemical reactions carried out for identification of the organisms (Figure 4a, 4b and Figure 5). 12

Metallo-b-lactamase detection (MBL) testing
Imipenem-EDTA combined disc diffusion test: The screening test for the detection and confirmation was tested by Imipenem-EDTA combined disc diffusion test.Test and control organism inoculum was prepared and matched with 0.5 McFarland turbidity standard.The bacterial strains cultured on Mueller Hinton agar plates, as per CLSI guidelines.Imipenem 10 µg disc, placed 20 mm apart, center from an Imipenem + EDTA disc containing 0.5 µl of 0.5 mg EDTA (750 µg per disk).Plates incubated at 37°C for 16-18 hours.
On overnight incubation, an increase in zone size ≥ 7 mm around the Imipenem-EDTA disc compared to the Imipenem disc alone was recorded positive for MBL (Figure 7b). 16

Carbapenemase production detection testing Modified Hodge Test (MHT)
Meropenem 10 µg disc placed in the center of the Muller Hinton agar plate and 10 µl of 50 mg Zinc sulfate solution added to the Meropenem disc and incubated at 37°C overnight.Zone around Meropenem disc with clover leaflike indentation, was interpreted as positive for Carbapenemase detection by the Modified Hodge Test (Figure 7c). 17

DISCUSSION
In the present study, a total of 150 Acinetobacter was identified from 450 non-lactose fermenting bacteria.Our study is comparable with other studies, 18 wherein maximum males were affected than females as given in the Table 1.The most commonly isolated species was Acinetobacter baumannii, followed by Acinetobacter lwoffii and Acinetobacter hemolyticus (Table 2).This finding can be corelated with the other study, 19 wherein they have been reported maximum number of isolates from pus, followed by blood, endotracheal aspirate, urine, sputum, BAL (Bronchoalveolar lavage), swab (gluteal abscess), throat swab, CVP tip (Table 3).Similarly, other study 20 reported that majority of isolates were from pus sample (Table 4).The other study 21 has reported a similar observation of maximum isolates, from ICU (Figure 8 and 9).Other study 22 reported maximum isolates from ICU.
The most commonly isolated species was Acinetobacter baumannii, followed by Acinetobacter lwoffii and Acinetobacter hemolyticus (Figure 3).Similar findings have been observed from the other study 23 reporting maximum isolate  x 2 = 3.376, p value = 0.4970,Not significant.Of the Acinetobacter species isolated from various OPD departments, maximum isolates were from pus 7 (25.3%)followed by sputum 3 (11%), urine 1 (19%) from Acinetobacter baumannii (Figure 10).In our study, we observed that the antibiotic sensitivity pattern showed maximum sensitivity to Colistin and Tigecycline (Table 5).Similarly, a study 20 showed that most of Acinetobacter, were Colistin sensitive, and other study 24 reported maximum susceptibility of Acinetobacter to Colistin.
Acinetobacter showed extremely high degree of resistant to Ampicillin, Nalidixic acid, Ceftazidime, Cefepime, Ciprofloxacin, Piperacillin, Gentamicin, Amikacin, Meropenem, Imipenem correlating with the other studies by Guckan and Peymani    4 and Figure 11).Other study 23 reported high carbapenem resistance; resistant to Imipenem and Meropenem.The findings of our study showed ESBL production, comparable to the other study 28 documenting same ESBL production (Table 6 and Figure 12).I n t h e p r e s e n t s t u d y, M B L production was similar to the findings by other study 29 documenting MBL production (Table 7 and Figure 13).
T h e p r e s e n t s t u d y s h o w e d Carbapenemase production in Acinetobacter baumannii isolates, by Modified Hodge Test (Table 8 and Figure 14).As compared to the other study, 30 present study exhibited less positive percentage for Carbapenemase detection.

CONCLUSION
At present, Acinetobacter is common threat in health care associated infections particularly in critically ill ICU patients.Maximum percentage of Acinetobacter isolates were from pus sample followed by tracheal aspirates.Acinetobacter baumannii was the most common bacterial isolate among the Acinetobacter species.Our study showed that Acinetobacter species were resistant to most of the commonly used antibiotics such as Ampicillin, Nalidixic acid and Ceftazidime.
It thus proves that extensive use of carbapenems has created a selective antibiotics pressure resulting in increased prevalence of carbapenems resistant Acinetobacter (CRA).Acinetobacter, reported as MDR, showed susceptibility to Colistin and Tigecycline, which remains the drug of choice in the treatment for patients.All isolates were sensitive to Colistin 113 (75.3%) and resistant towards Ampicillin 130 (87%).Ceftazidime has been proposed as the indicator of ESBL production as compared to other antibiotics.The remaining isolates showed resistance to Ceftazidime.
Thus, Ceftazidime is a superior indicator for the detection of ESBL production.Imipenem has been suggested as the indicator of MBL production in comparison to others.So, Imipenem is a better indicator for the detection of MBL production.The study showed that ESBL production in Acinetobacter is 22(15%) and MBL 80(53%) and is on the ascent world over, in this way making these infections challenging to treat.ESBL and MBL production detection would be important for decreasing death rate and spread of multidrug resistant organisms.Acinetobacter species isolates were resistant to carbapenems such as imipenem 106(71%) and meropenem 112(75%).Modified Hodge test is a simple and easy test to be performed to identify carbapenems producing organisms.In our study, detection of Modified Hodge test by carbapenemase was positive in 11 (7%) isolates.

Table 1 .
Age and gender wise distribution of Acinetobacter

Table 2 .
Speciation of Acinetobacter isolates

Table 3 .
Acinetobacter species distribution in various IPD departments

Table 4 .
Acinetobacter species distribution in various OPD departments

Table 5 .
Antibiotic susceptibility pattern of Acinetobacter species isolated from various clinical samples

Table 6 .
Distribution of ESBL among the Acinetobacter species

Table 7 .
Distribution of MBL among the Acinetobacter species