Journal of Medical Sciences and Health

Acinetobacter baumannii which is an aerobic, catalase - positive, oxidase - negative, gram negative coccobacilli, although first described in 1911, taxonomy was published in 1986 after several studies according to which there are 54 species of the genus Acinetobacter in which Acinetobacter baumannii is one of the most virulent species in the genus causing a bulk of human infections including nosocomial infections as this bacterium can survive in environment for months and has simple nutritional requirements/not fastidious and also has the ability to grow in diverse pH and temperature 1, 2. They are also resistant to commonly used disinfectants and antiseptics and also have the ability to form biofilm in biotic and abiotic surfaces. Acinetobacter baumannii also exhibit increased antimicrobial resistance due to their ability to incorporate resistance determinants along with virulence determinants. Apart from Acinetobacter baumannii other species like Acinetobacter pittii, Acinetobacter nosocomialis, Acinetobacter lwoffii and Acinetobacter radioresistens are also identified as significant nosocomial pathogens 3.

Health care associated infections are most common infections encountered by Acinetobacter species due to hospitalization of patients leading to colonisation, improper disinfection procedures and unnecessary antibiotic usage like third generation cephalosporins, fluoroquinolones and carbapenems, lack of hand hygiene, spray of contaminated fluids due to pulsatile lavage of wounds or bronchoscopy etc., which lead to heavy environmental contamination leading to transmission 4, 5.

In the healthcare setting Acinetobacter species is mainly associated with Ventilator associated pneumonia (VAP) accounting for 3-7% of cases who require mechanical ventilation for more than five days and also implicated in 26% of respiratory infections. They are also responsible for other nosocomial infections like blood stream infections associated with intravascular catheters, Surgical site infections (SSI’s), Urinary tract infections (UTI’s), wound infections, meningitis after neurosurgery procedure and soft tissue infections in burns patients 6.

Acinetobacter species are also responsible for community-associated infections like pneumonia in tropical regions during summer season and bacteremia which is often associated with respiratory infection. Meningitis is rare but they are associated with invasive soft tissue infections especially during warfare and natural disasters 7.

Acinetobacter is well known for its antimicrobial resistance mechanisms which are clustered on their resistance islands where Insertion sequences called IS Abal present in its genome is responsible for several key resistance mechanisms. Acinetobacter exerts its antibiotic resistance by expression of beta-lactamases in which group-1 AmpC beta-lactamases are chromosomally encoded and are cephalosporinases responsible for hydrolysis of penicillins and 1^st, 2^nd & 3^rd generation cephalosporins like ceftazidime, cefotaxime and ceftriaxone 8.

Emergence and rapid spread of Acinetobacter species with group-2be Ambler class A Extended-spectrum β-lactamases (ESBL’s) lead to global spread of Multidrug Resistant (MDR) Acinetobacter species which hydrolyse penicillins and all cephalosporins which lead to the usage of carbapenems. However, resistance to carbapenems has increased in recent years due to acquisition of group 2d Ambler class D oxacillinases through Insertion sequence IS Abal resulted in increase in carbapenem resistance successfully spreading the International Clonal Complexes (ICC’s) 9.

Acinetobacter also expresses resistance through efflux pumps AdeABC which are chromosomal and in the Resistance-Nodulation-Division (RND) which are chromosomal encodes resistance to antibiotics like β-lactams, chloramphenicol, macrolides, tetracyclines, tigecycline, aminoglycosides, polymyxins etc. 10

Other mechanisms of resistance include expression of porins (β-lactams and carbapenems), mutations (fluoroquinolones, polymyxins) 11, 12.

Carbapenem Resistant Acinetobacter baumannii is now a global health threat where therapeutic options available for infections with carbapenem resistant strains are limited. Minocycline, which is a second generation tetracycline introduced in 1960’s is proven to have antibacterial activity against Acinetobacter baumannii which is available as oral and IV formulation. It acts by inhibiting bacterial protein synthesis by binding to 30s ribosomal subunit of bacteria which inhibits the tRNA to ribosomal acceptor site thus preventing peptide chain elongation leading to inhibition of protein synthesis 13.

Minocycline is more lipophilic counterpart of tetracycline which will help the drug for increased tissue penetration, enhanced antibacterial activity, longer half-life and increase the spectrum of activity against many bacteria including Acinetobacter baumannii 14.

Resistance to these tetracyclines is mediated by a variety of mechanisms like efflux pumps, ribosomal protection proteins, target modification etc.; where nearly 20 efflux pump mechanisms are identified in Gram-negative organisms in which common is Tet A which leads to decreased intracellular concentration by exchanging a proton for the tetracycline cation complex. Invitro efflux pumps present in Acinetobacter species are effective in transporting out other drugs in this group but not Minocycline 15.

Materials & Methods

This study was a cross sectional observational study conducted between February 2024 to January 2025 (1 year) in the department of microbiology, Dr Pinnamaneni Siddhartha institute of medical sciences and research foundation, Andhra Pradesh which is a tertiary care centre after obtaining approval from institutional ethics committee (IEC) – Reg no: faculty/1028/23 dated 01/12/2023 (CDSCO Reg no- ECR/804/Inst/AP/2016-RR-19 and DHR Reg no- EC/NEW/INST/2021/2140.

Inclusion criteria: All the culture specimens sent to microbiology laboratory from various OPD’s and IPD’s during the study period showing the growth of Acinetobacter baumannii and were resistant to carbapenems (Imipenem and Meropenem) were included in the study.

Exclusion criteria: Repetitive samples and samples showing growth of bacterial isolates other than Acinetobacter baumannii and Acinetobacter baumannii isolates which were sensitive to carbapenems were excluded in the study.

Sample design: A total of 50 non-duplicate strains of Acinetobacter baumannii isolates from various samples like pus, urine, wound swabs, ET secretions, central line tips etc. which were identified by Gram’s stain (Gram negative coccobacilli), catalase test (positive), oxidase (negative), motility (non-motile) and other conventional biochemicals and sugar fermentation tests were collected and further processed by antibiotic susceptibility testing by Kirby Bauer disc diffusion test 16, 17 on Mueller Hinton agar (MHA) by routine antibiotics recommended by CLSI 16 and the strains which were resistant to imipenem and meropenem in disc diffusion method were considered as carbapenem resistant Acinetobacter baumannii and these were further subjected to E test on Mueller Hinton agar and were interpretated as sensitive (S) (≤4µg/ml), intermediate (I) (8µg/ml) and resistant (R) (≥16µg/ml) according to CLSI guidelines 16.

Study variables: The MIC (Minimum Inhibitory Concentration) of minocycline against carbapenem resistant Acinetobacter baumannii (CRAB) isolates cut-off range is based on CLSI guidelines and interpretated as sensitive (≤4µg/ml), intermediate (8µg/ml) and resistant (≥16µg/ml) 16.

MIC was marked at the point where ellipse intersects the scale and the MIC value at complete inhibition of all growth was also marked. If there is any presence of variation in the intersect on either side of the strip, the greater value of MIC was marked and if any growth was observed at the edge of the strip, it was ignored.

Study procedure: A total of 50 non-duplicate strains of Acinetobacter baumannii isolated from different samples like pus, urine, wound swabs, ET-secretions, central-line tips etc which were identified by standard phenotypical tests like Gram’s stain, catalase test, oxidase test, motility test , Indole test, Nitrate test and other standard biochemical and sugar fermentation tests as per CLSI guidelines 16, 17, 18 were included in the study and these strains were subjected to antibiotic susceptibility testing by Kirby Bauer disc diffusion method on Mueller Hinton agar (MHA) by using antibiotic discs (Himedia Labs, Mumbai) of Ampicillin/Sulbactam (10/10µg), Piperacillin/Tazobactam (100/10µg), Ceftazidime (30µg), Ceftriaxone (30µg), Ciprofloxacin (5µg), Imipenem (10µg), Meropenem (10µg), Co-trimoxazole (1.25/23.75µg), Amikacin (30µg), Gentamicin (10µg), Tigecycline (15µg), Doxycycline (30µg) and interpretated as Sensitive(S), Intermediate (I) and Resistant(R) according to zone diameter as per CLSI guidelines 16.

The strains which were resistant to imipenem, meropenem were considered as carbapenem resistant strains and were subjected to susceptibility testing by minocycline (30 µg) disc (Himedia Labs, Mumbai) by Kirby-Bauer Disc diffusion test using Mueller- Hinton agar (MHA) and were interpretated according to zone diameter as Sensitive (≥16mm), Intermediate (13-15mm), Resistant (≤12mm) 16. All these strains were also subjected to Epsilometer (E-test) on Mueller- Hinton agar plate by using E-strips of minocycline (Himedia Labs, Mumbai) with MIC’s ranging from 0.016 to 256mcg/ml according to CLSI guidelines and manufacture recommendation 16.

The inoculum was prepared using 3-4 test strain colonies which were already subcultured from original plates to sheep blood agar and Mac Conkey agar to obtain pure colonies and were emulsified in a test tube containing normal saline and turbidity was compared to that of 0.5 McFarland standards and the turbidity was adjusted accordingly and inoculation was done on Mueller Hinton agar (MHA) by using a sterile cotton swab dipped into the inoculum and the swab was streaked over the entire surface of Mueller Hinton agar and then E strips of minocycline were applied onto the agar surface and the plates were incubated at 35-37^oC for 18-24 hrs 19 and were then interpretated as Sensitive (≤4µg/ml), Intermediate (8µg/ml) and Resistant (≥16 µg/ml) according to CLSI guidelines 16. The entire procedure was carried out for Quality control strain (Acinetobacter baumannii - 19606).

Results

Discussion

The prevalence of various infections caused by Multidrug Resistant organisms (MDR) has been increasing over past decade due to acquisition of various resistance mechanisms which have made treating infections with such organisms more challenging to clinicians. Treatment of infections with MDR pathogens hence require careful evaluation of various factors including antibiotic susceptibility testing apart from evaluation of pharmacokinetics and pharmacodynamics of the drug well designed clinical trials and implementation of strict infection control practices in order to avoid horizontal spread of antibiotic resistance among pathogens 20, 21.

Acinetobacter species especially Acinetobacter baumannii is the most common pathogen associated with both hospital acquired, and community acquired infections which are associated with high mortality and morbidity. Carbapenems were drug of choice to treat MDR Acinetobacter baumannii infections but widespread use of these group of drugs in recent years has led to increase in frequency of emergence of Carbapenem Resistant Acinetobacter baumannii (CRAB) strains which can be attributed to decrease permeability of outer membrane due to loss or modification of porins, modification of Penicillin binding proteins (PBP’s), increased expression of efflux pumps and production of the enzyme carbapenemase which has led to decreased therapeutic effectiveness of these agents and has led to limitations in available treatment options of infections with such MDR Acinetobacter baumannii strains 22, 23.

Minocycline (both oral and intravenous forms) has been approved by US food and drug administration for treatment of infections caused by MDR Acinetobacter baumannii strains which are resistant to Carbapenems and / Polymyxin classes 24.

Minocycline also has shown to have fewer side effects like dizziness, vertigo, nausea and diarrhea and has favorable pharmacodynamics and pharmacokinetic properties and is also stable to many tetracycline resistant mechanisms 25.

The present study was conducted at our institution to evaluate the invitro Minocycline susceptibility against carbapenem resistant Acinetobacter baumannii isolates from various samples where a total of 50 CRAB isolates were collected and subjected to antibiotic susceptibility testing by Disc diffusion method and E-test and interpretated as Sensitive (S), Intermediate (I), Resistant (R) according to CLSI guidelines 16. In our study 40 (80%) isolates were sensitive to Minocycline by Disc diffusion method and 43 (86%) were sensitive by E-test correlating with the findings of Pengwang et al 82.2% 24, Gunasekaran Santhi et al 87.5% 26, Shahram Shahraki Zahedani et al 81.5% 22. But according to Anup R Warrier et al the sensitivity of CRAB isolates to Minocycline by E-test was low (50%) 20 and Asna Parveen et al showed 40.5 % sensitive 27 and Jia-ling Yang et al showed only 55% of isolates sensitive for Minocycline 28.

Conclusion

This study gives insight into invitro Minocycline susceptibility against Carbapenem Resistant Acinetobacter baumannii isolates from various clinical samples in a tertiary care hospital. In conclusion, the results of the present study have demonstrated that Minocycline is effective against Carbapenem Resistant Acinetobacter baumannii strains that were included in the study. Since most of the CRAB isolates are susceptible to Colistin, Minocycline, Tetracycline and Doxycycline, these antibiotics or their combinations are recommended for treatment of infections caused by resistant strains.

Selection of appropriate antibiotic remains main key for treatment of infections caused by such CRAB strains and as Minocycline shows susceptibility against these strains it can be used as an alternative drug to Colistin. But some isolates show resistance to Tigecycline and Minocycline which is of great concern especially in hospital settings. In order to combat this problem, WHO recommended five infection control strategies namely hand hygiene, surveillance for carbapenem resistant organisms, contact precautions, environmental cleansing and patient isolation and these strategies should be monitored, audited and feedback to healthcare workers and management should be done from time to time.

Disclosure

Funding: None

Conflict of Interest: Nil