Journal of Global Antimicrobial Resistance (JGAR)-Letter to the Editor

Letter to the Editor

Professor Stefania Stefani

Increased biofilm-associated Carbapenem-resistant Acinetobacter-calcoaceticus-baumannii complex infections among the hospitalized patients in Kathmandu Model Hospital, Nepal

Shova Bhandari¹, Milan Kumar Upreti¹, Khadga Bikram Angbuhang¹, Basudha Shrestha², Upendra Thapa Shrestha^{3 *}

 

¹GoldenGate International College, Battisputali, Kathmandu Nepal

²Kathmandu Model Hospital, Kathmandu, Nepal

³Central Department of Microbiology, Tribhuvan University, Kirtipur, Kathmandu, Nepal

 

^*Corresponding author: Upendra Thapa Shrestha, Assistant Professor, Central Department of Microbiology, Tribhuvan University, Kirtipur, Kathmandu, Nepal, Email: upendrats@gmail.com / upendra.thapashrestha@cdmi.tu.edu.np

 

Dear Editor

Acinetobacter calcoaceticus-baumannii complex (ACBC), a Gram-negative commensal bacterium, often infects immunocompromised patients or patients with indwelling devices, especially in the intensive care unit, and causes a wide range of hospital-acquired infections, including respiratory tract infections, urinary tract infections, bacteremia, sepsis, endocarditis, meningitis, skin and soft tissue infections, burns, as well as central and nervous system infections. A. baumannii is an emerging pathogen with the ability to produce a biofilm that is mostly associated with ventilator-associated pneumonia and catheter-related infections [1]. The bacteria inside the biofilm are shielded by extracellular polymeric substances, which act as a barrier to antibiotics, leading the bacteria to antibiotic-resistance. Biofilm-forming bacteria show 1000-fold higher drug resistance than planktonic cells, and the infections caused by such bacteria are chronic, prone to relapse, and more difficult to treat. In addition, within biofilm, the bacterial cells are in close proximity and have a high chance of horizontal gene transfer, particularly via conjugation of antibiotic resistance genes, promoting their survival and the spread of antibiotic resistance [2].

Biofilm-related virulence factors involved in A. bauamnnii infections are biofilm-associated protein (Bap), the extended-spectrum beta-lactamase family bla_PER1 gene, and CsuA/BABCDE pilus usher-chaperone assembly system [3]. To address the biofilm-associated carbapenem-resistant A. baumannii infections among hospitalized patients, we conducted a hospital-based cross-sectional study at a tertiary care hospital in Kathmandu, Nepal. We primarily determined the rate of A. baumannii in different clinical specimens, and then we evaluated the association between biofilm formation and carbapenem-resistant ACBC isolates detecting biofilm-forming genes Bap, csuE, and bla_PER1.

 

This study was conducted at Kathmandu Model Hospital, Kathmandu, Nepal, from February 2020 to August 2020 among hospitalized patients of all age groups who gave written consent to be enrolled in the study (IRC 003-2020). A total of 665 different clinical samples, including pus, sputum, tracheal aspirates, blood, endotracheal tips, catheter tips, wound samples, suction tips, and tissue were processed using standard microbiological procedures to isolate and identify the potential bacterial pathogens. The antibiotic susceptibility pattern of ACBC was determined by a modified Kirby-Bauer disk diffusion method following CLSI guidelines. The screening for biofilm formation was done by the microtitre plate method [4]. And the biofilm-related virulence factors were detected by using specific primers; bap-F (5’-TGCTGACAGTGACGTAGAACCACA-3’), bap-R (5’-TGCAACTAGTGGAATAGCAGCCCA-3’), csuE-F (5’-CATCTTCTATTTCGGTCCC-3’), csuE-R (5’-CGGTCTGAGCATTGGTAA-3’), and bla_PER1-F (5’- GCAACTGCTGCAATACTCGG-3’), bla_PER1-R (5’-ATGTGCGACCACAGTACCAG-3’) [3]. The correlation between biofilm formation and carbapenem resistance was analyzed using the Chi-Square test (SPSS version 22).

 

Out of 665 clinical samples, bacterial growth was observed in 281 (42.3%). Escherichia coli (28.8%) was the most predominant pathogen, followed by Staphylococcus aureus (20.3%), Klebseilla pneumoniae (16.4%), ACBC (11.4%), and Pseudomonas aeruginosa (8.1%). A significantly higher incidence of ACBC infection was observed among the male patients (26/32; 81.3%). Similarly, the highest incidence of ACBC infection was reported in patients aged 20-50 which accounts for 59.6%. The highest number of ACBC was isolated from pus samples (n = 12, 37.5%).

 

All ACBC isolates were resistant to amoxicillin, cefotaxime, and ceftazidime, whereas 31 isolates were resistant to amikacin and gentamycin. The majority of ACBC isolates (93.8%) were multidrug resistant. Most of the isolates were susceptible to doxycycline (53.1%), followed by cotrimoxazole (18.7%), levofloxacin (15.6%), and ofloxacin (15.6%). All isolates were susceptible to colistin and polymyxin B. The higher rate of antimicrobial resistance in bacterial pathogens is due to the irrational use of antibiotics, adherence to empirical therapy without proper AST, extensive use of antibiotics in poultry, direct disposal of antimicrobial waste in the environment, etc. The higher antibiotic susceptibility of ACBC isolates towards doxycycline antibiotics was reported, so it can be used to treat multidrug-resistant ACBC infections. Carbapenem resistance in A. baumannii is mainly caused by class B MBL and class D OXA type β-lactamase, which can hydrolyze carbapenem antibiotics [5]. CR-AB infections have a high morbidity and death rate in hospital settings due to their low level of antibiotic susceptibility and subsequent failure of therapy.

 

A significant association was observed between carbapenem resistance and biofilm formation (p-value < 0.05), indicating the role of biofilm in carbapenem resistance. Out of 31 biofilm-positive isolates, 21 isolates were positive for both Bap and csuE genes, and 18 isolates were positive for the bla_PER1 gene (Figure 1). The biofilm-related genes help in biofilm formation, survival in hospital environments and medical devices, and disease pathogenesis in hospital settings [2,3]. No biofilm-related genes were found in carbapenem-sensitive ACBC isolates, and a significant association between carbapenem resistance and biofilm-forming genes bap, csuE, and bla_PER-1 was found. Further, the co-existence of Bap, csuE, and bla_PER1 among positive biofilm isolates was found to be 58%, which may have boosted biofilm formation. The co-existence of Bap and csuE was 9.8%, and no genes were singly present, which also indicates the dependence of genes on biofilm formation, such as csuE is critical for initial attachment and bap for biofilm maturation.

Figure 1: Detection of Biofilm-related genes among ACBC isolates by conventional PCR. 1a: Screening of csuE gene (L1: Ladder, L2 & L3: amplified products from ACBC isolates, L4: positive control, L5: no template control and L6: Ladder); 1b: Screening of Bap gene (L1 & L2: amplified products from ACBC isolates, L3: positive control and L5: no template control), and 1c: Screening of bla_PER1 gene (L1: positive control, L2: no template control,  L3 & L4: amplified products from ACBC isolates, and L5: ladder).

 

Conclusion

The increase in biofilm formation significantly associated with carbapenem resistance adds a big challenge to controlling CR ACBC infections. In addition, this capability of ACBC contributed to antibiotic resistance as well as helped them in environmental survival. Hence, proper sterilization of hospital equipment and the environment should be of primary concern, and a strong policy to prescribe effective antibiotics based on the antibiogram profile should be implemented.

 

Acknowledgements

We express our sincere gratitude to laboratory staff members of GoldenGate International College (GGIC), Kathmandu Model Hospital, and the team of CMDN for their support, in completing this study. We are very much thankful to the participants and their legal guardians for providing samples.

 

Data availability

The data used in this study will be available from the corresponding author (Email: upendrats@gmail.com/upendra.thapashrestha@cdmi.tu.edu.np) upon request.

 

Competing interests

The authors declare no competing interests.

 

References

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2.     2. Roy S, Chowdhury G, Mukhopadhyay AK, Dutta S, Basu S. Convergence of Biofilm Formation and Antibiotic Resistance in Acinetobacter baumannii Infection. Frontiers in Medicine. 2022;9:793615.

3.     3. Yang CH, Su PW, Moi SH, Chuang LY. Biofilm formation in Acinetobacter baumannii: Genotype-phenotype correlation. Molecules. 2019;24(10):1–12.

4.     4. Stepanovic S, Vukovic D, Hola V, Bonaventura GD, Djukic S, Circovic I, Ruzicka F. Quantification of biofilm in microtiter plates. Apmis. 2007;115(8):891–899.

      5. Benmahmod AB, Said HS, Ibrahim RH. Prevalence and mechanisms of carbapenem resistance among Acinetobacter baumannii clinical isolates in Egypt. Microbial Drug Resistance. 2019;25(4):480–488.

 

Citation:     Shova Bhandari, Milan Kumar Upreti, Khadga Bikram Angbuhang, Basudha Shrestha, Upendra Thapa Shrestha*. Increased biofilm-associated carbapenem-resistant Acinetobacter calcoaceticus–baumannii complex infections among hospitalised patients in Kathmandu Model Hospital, Nepal. Journal of Global Antimicrobial Resistance, 2024; 39:1-2. ISSN 2213-7165.

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