Characterization of Aminoglycoside -Resistant Acinetobacter baumannii in Respiratory Specimens: Phenotypic and Molecular Insights

Background: The global rise of antibiotic-resistant bacteria presents a formidable challenge to healthcare systems, with Acinetobacter baumannii standing out as a particularly resilient pathogen in hospital settings. The propensity of this organism to exhibit multidrug resistance complicates treatment protocols and underscores the need for in-depth research into its resistance mechanisms. Objective: The objective of this study was to characterize the allelotypic and molecular features of aminoglycoside -resistant Acinetobacter baumannii isolates from respiratory specimens and to determine their antibiotic susceptibility profiles to inform better clinical decision-making and treatment approaches. Methods: This cross-sectional study was conducted at the different labs of various institute of Lahore. A total of 50 respiratory specimens were cultured on selective media. The isolates underwent phenotypic characterization through colony morphology and biochemical tests. Antimicrobial susceptibility was assessed using the disc diffusion method, and molecular analysis was performed using polymerase chain reaction (PCR) to identify the armA gene associated with aminoglycoside resistance. Statistical analysis was executed using SPSS Version 25. Results: Of the 50 isolates, 54% were from male patients and 46% from female patients. Antibiotic resistance was alarmingly high, with resistance rates of 92% for TZP, 90% for FEP, CAZ, IPM, MEM, 86% for AK, 74% for CN, 54% for TOB, and 48% for DO. The presence of the armA gene was detected in 86% of the isolates, suggesting a link to the high levels of aminoglycoside resistance observed. Conclusion: The study revealed a high prevalence of multidrug-resistant Acinetobacter baumannii in respiratory specimens, with significant resistance to commonly used antibiotics. These findings highlight the necessity for continuous surveillance of antibiotic resistance patterns and call for innovative approaches to antimicrobial therapy.


INTRODUCTION
Acinetobacter baumannii, a member of the Moraxellaceae family, is recognized as a significant pathogen in the realm of nosocomial infections.This gram-negative, coccobacillary organism is pleomorphic and aerobic by nature and is known for its inability to ferment glucose, lactose, or sucrose (1,2).Its clinical relevance is underscored by its potent virulence factors, such as lipopolysaccharide, phospholipases, and outer membrane protein A (OmpA).The latter binds to epithelial cells and mitochondria, inducing mitochondrial dysfunction and leading to apoptosis, facilitated by the release of cytochrome c oxidase and the consequent formation of the apoptosome (3)(4)(5).
The robust biofilm-producing ability of A. baumannii is particularly noteworthy.Biofilms, which are communities of microbial cells ensconced within an extracellular polymeric substance, are hotspots for genetic exchange and significantly contribute to the organism's resistance phenotype and the spread of resistance genes (6).Furthermore, Acinetobacter species have evolved to exhibit significant antimicrobial resistance, which is especially concerning in the context of hospital-acquired infections caused by ESKAPE pathogens.The group of bacteria known as ESKAPE (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, A. baumannii, Pseudomonas aeruginosa, and Enterobacter species) is notorious for hospital infections and its members demonstrate various mechanisms of resistance, including the expression of different isoforms of enzymes like E4PDH, which is akin to the glycolytic enzyme GAPDH involved in the DXP pathway (7).The intrinsic resistance of Acinetobacter to many antibiotics is partly due to its less permeable outer membrane and its access to a vast pool of genetic resistance mechanisms (8).Beta-lactams, one of the main classes of antibiotics, act by binding to penicillinbinding proteins, disrupting bacterial cell wall biosynthesis (9).Among these, carbapenems are crucial; however, A. baumannii's growing carbapenem resistance poses a formidable challenge in clinical settings.Resistance to colistin, one of the last-resort antibiotics, has also been identified, attributed to structural changes in LPS and the presence of plasmid-borne mcr genes (10).Given its clinical significance, A. baumannii is the focus of intense study, including efforts to elucidate the mechanisms underlying its resistance to aminoglycosides.Aminoglycosides, which bind to the bacterial ribosomal subunits, are known for their ability to interrupt protein synthesis and thus kill the bacteria.Nevertheless, A. baumannii's remarkable aminoglycoside resistance is due to various adaptive strategies, such as modifications to the targets of these antibiotics, enzymatic degradation, and efflux pumps that extrude harmful substances from the cell (8-10).The identification of bacterial pathogens, including A. baumannii, leverages both phenotypic and genotypic methods.Classic microbiological approaches like gram staining are used to differentiate between gram-positive and gram-negative bacteria, while culture techniques allow for the growth and morphological analysis of microbial colonies.Biochemical testing provides further insights into bacterial identity based on metabolic capabilities.To ascertain the most effective treatment, antimicrobial susceptibility patterns are assessed.Meanwhile, molecular techniques such as PCR offer a powerful means to detect resistance genes and understand the genetic makeup of these formidable pathogens.The amalgamation of these methods affords a comprehensive approach to tackling the challenges posed by aminoglycoside-resistant A. baumannii, enabling both the detection and understanding of this critical healthcare-associated pathogen.

MATERIAL AND METHODS
In the execution of this study, meticulous preparation of all media and reagents was undertaken using distilled water.Glassware, essential in the process, underwent a rigorous cleaning protocol: professionally cleansed, heat-dried, and sterilized to ensure the absence of any contaminants that could potentially compromise the integrity of the results.The sterilization process, a pivotal step in the preparation, was carried out for a precise duration of 15 minutes at the stringent conditions of 121°C and 15psi, a standard set to ensure complete sterility (REF: CM0055B, CM0337B, CM0277, NCM0277A, CM0155).Doxycycline (DO) 30 µg The cross-sectional study design was selected to provide a snapshot of the current situation within the microbiology landscape.This observational study type allowed for the analysis of data from a population subset at a specific point in time.The Institutional Review Board's approval marked the commencement of the data collection phase, which spanned three months, situated within the wellequipped confines of the Life Sciences Laboratories at Lahore College for Women University.Respiratory specimens, encompassing naso-pharyngeal swabs, tracheal washings, bronchial brushings, and sputum, were meticulously collected following a stringent inclusion criterion only isolates of Acinetobacter baumannii from respiratory patients were considered, while specimens not meeting this criterion were excluded (1-4).
The isolated strains underwent initial culturing on various media, including nutrient agar, MacConkey agar, and sheep blood agar, meticulously incubated at 37°C.After 24 hours, the colonies that had developed were subject to a detailed morphological assessment, with their features documented for comparative analyses in subsequent research phases.The subsequent isolation involved the use of fastidious media to ensure the purity of the colonies, which, after a day, exhibited unadulterated growth suitable for further examination (5).The morphological characteristics, critical in the initial identification stages, were observed on the culture media.A range of colony features, including color, size, and texture, were evaluated, aiding in the classification of the infections.The slide preparation entailed transferring a minute amount of bacterial growth onto a glass slide, followed by careful drying to prevent any damage or alteration to the sample.Gram staining, a crucial step in differentiating bacterial types, was meticulously executed, adhering to the timehonored protocol involving crystal violet, iodine solution, decolorizer, and safranin, each component serving its unique purpose in this classic microbiological technique (6).Diagnostic tests to identify specific pathogens were employed post initial microbial identification.Catalase and oxidase tests, alongside citrate utilization and urease tests, provided insight into the enzymatic activities of the bacteria.For movement analysis, motility tests were conducted using both wet mount preparation and inoculation in a motility medium.Furthermore, the sulfurindole-motility test was instrumental in identifying the production of sulfide, indole, and motility traits, essential in the characterization of bacterial isolates (7).Antimicrobial susceptibility testing was another cornerstone of the study, utilizing the disc diffusion method in line with the guidelines set out by the Clinical and Laboratory Standards Institute.The zones of inhibition around antibiotic discs placed on Mueller-Hinton agar plates were measured post incubation, establishing the susceptibility profile of each bacterial strain.The analysis of resistance patterns against a spectrum of antibiotics including β-lactams and aminoglycosides was crucial in understanding the antimicrobial resistance landscape of Acinetobacter baumannii (8,9,10,11,12,13).DNA extraction, a fundamental molecular technique, was executed using the Gene Jet Genomic DNA Purification Kit.This was followed by polymerase chain reaction (PCR), a pivotal step in amplifying specific DNA sequences, and gel electrophoresis, which

RESULTS
The results section of our study meticulously quantifies the distribution of Acinetobacter baumannii across various culture media, gender distribution among study participants, the outcome of biochemical tests, age group prevalence, and antibiotic sensitivity.
The colony morphology of Acinetobacter baumannii varied distinctively across different agar media.On Chocolate Agar, the bacterial colonies were predominantly 1-2mm in size, round, with a dome shape, and notably neither mucoid nor pigmented (Table 1).In contrast, colonies on Blood Agar were nonhemolytic, opaque, and grey, maintaining a consistent circular form.MacConkey Agar, known for differentiating lactose fermenters, revealed non-fermenting colonies of Acinetobacter baumannii that were opaque with circular margins, lacking the characteristic color change associated with lactose fermentation.Gender distribution within the study presented a slight male predominance, with males comprising 54% (n=27) and females 46% (n=23) of the 50 study participants.This distribution is graphically encapsulated in the accompanying bar chart, which elucidates the frequency and percentage of each gender (Figure 1), reinforcing the relatively balanced representation of genders in this study (Table 2)..048.0 Biochemical assays yielded insights into the metabolic capabilities of the bacterial isolates.The Catalase Test resulted in bubbling, indicative of a positive reaction.Conversely, the Oxidase Test was negative, suggesting the absence of cytochrome c oxidase activity.Citrate utilization was positive, implying that Acinetobacter baumannii could utilize citrate as a carbon source, while urease activity was absent, signifying no ammonia production.The SIM test was uniformly negative for sulfur reduction, indole production, and motility, underscoring the non-motile nature of the studied bacterial strain.In the TSI test, both the slant and butt were alkaline, consistent with the organism's known non-fermenting properties.The age distribution of the participants was characterized by a bell-shaped curve with the majority of the subjects falling in the oldage adults category, accounting for 36% of the sample size (n=18).Children and young adults each made up 22% (n=11 for each group), while middle-aged adults represented 20% (n=10) of the population (Figure 2).The histogram shows the frequency distribution of participants across these age groups, reflecting a skew towards the older age range.Antibiotic sensitivity testing revealed that Acinetobacter baumannii exhibited a high resistance to most of the antibiotics tested.Particularly notable was the resistance to TZP, with a staggering 92% of samples exhibiting resistance.Similarly, high resistance rates were observed for FEP, CAZ, IPM, and MEM, all above 90%.AK showed an 86% resistance rate, while CN had a slightly lower resistance rate at 74%.TOB presented a more balanced sensitivity profile with a 54% resistance rate.CIP showed an overwhelming resistance rate of 92%, and LEV shared a similar pattern with 90% of the strains resistant.Notably, DO was the only antibiotic where the sensitivity rate (52%) surpassed the resistance rate (48%) (Figure 3), showcasing its potential effectiveness against Acinetobacter baumannii within this study cohort (Table 5).

DISCUSSION
The discourse surrounding the allelotypic and molecular characteristics of aminoglycoside-resistant Acinetobacter baumannii isolated from respiratory specimens underpins a broader, global conversation about antibiotic resistance.This study, situated at the different labs of various institute of Lahore, serves to reinforce the urgent call for novel antimicrobial strategies and stringent infection control practices.The study delineated a disconcerting level of resistance to a panoply of antibiotics such as TZP, FEP, CAZ, IPM, MEM, AK, CN, TOB, CIP, LEV, and DO, an observation that aligns with the notorious profile of multidrug resistance associated with Acinetobacter baumannii.Such resistance patterns echo findings from other regions, accentuating the pathogen's ability to withstand varied antibiotic assaults and posing significant clinical challenges in healthcare environments (17).The demographic distribution within this study mirrored the gender ratio reported in similar research conducted in Ismailia, Egypt, on 52 ICU patients, underscoring the gender-indiscriminate nature of Acinetobacter baumannii infections.Through rigorous application of various agar media for culture, complemented by biochemical testing and antibiotic sensitivity assays, the pathogen's profile was scrupulously determined.Moreover, state-of-the-art technologies like VITEK MS provided precision in identification, with biochemical reactions and Gram staining fortifying the characterization process.Species identification and antibiotic susceptibility were ascertained using VITEK 2 Compact (18).A comparative analysis with a study from tertiary care centers in Faisalabad and Lahore, Pakistan, further illuminated the A. baumannii landscape.While both studies procured strains primarily from respiratory samples, methodological variations were notable.Our research employed Gram staining, colony morphology, biochemical tests, VITEK MS, and gel electrophoresis for armA gene detection, whereas the comparative study utilized morphological tests, API 20E, and multiplex PCR alongside CLSI and FDA guidelines for antimicrobial susceptibility testing.The armA gene, commonly associated with aminoglycoside resistance, emerged prominently in our gel electrophoresis findings, with an 86% prevalence among isolates, indicating its potential role in resistance mechanisms (19).Examining antibiotic resistance patterns and genetic markers within the broader context revealed netilmicin as relatively efficacious, whereas ciprofloxacin exhibited the highest resistance.The minimum inhibitory concentration (MIC) assays revealed substantial resistance across aminoglycosides.In our cohort, resistance genes including aphA6, aadB, aadA1, aacA4, armA, and aacC2 were identified, aligning with global resistance trends.This project echoed a study conducted at Agha Khan Labs, which reported an alarming rate of antibiotic resistance among 50 A. baumannii strains from respiratory samples and identified the armA gene in a significant majority of isolates, suggesting its pervasive presence in aminoglycoside resistance (20).This investigation enriches the understanding of Acinetobacter baumannii's antimicrobial resistance profiles and underscores the pressing need for ongoing surveillance and innovative treatment approaches.The findings resonate with the pathogen's established multidrug resistance and emphasize the critical need for further research and effective antimicrobial stewardship.They underscore the imperative for continued antibiotic resistance monitoring, the necessity for targeted interventions, and the development of alternative therapeutic strategies to address the burgeoning challenge of multidrug resistance in bacterial pathogens.The study's insights bolster the case for enhanced infection control measures and reinforce the global health imperative to contain the spread of multidrug-resistant organisms.

CONCLUSION
This study underscores the pervasive challenge posed by multidrug-resistant Acinetobacter baumannii, emphasizing the organism's capacity to thwart an array of antibiotics, with significant human healthcare implications.The findings reiterate the critical need for vigilant antimicrobial stewardship, development of new therapeutic agents, and robust infection control practices to combat the rising tide of antibiotic resistance.The results serve as a clarion call for healthcare systems to prioritize research and integrate targeted strategies to manage and mitigate the risks associated with these resilient bacterial pathogens.

Figure 1
Figure 1 Components OF KITS

Figure 4
Figure 4 Bar chart presentations of ANTIBIOTICS; and Bands of armA on gel electrophoresis

Table 1
Nutrient Agar Composition

Table 3
Blood Agar Base Composition

Table 8
Isolate Report Extract

Table 9
Bacterial Colony Morphology on Various Agar Media Culture Plates, Colonies Morphology and Biochemical Tests and Sensitivity of Anti-Microbial