Oral Presentation on International Conference-2024, Chitwan, Nepal

Interlinkage of β-Lactam Resistant Bacterial Genes from Clinical, Environmental, and Poultry Isolates from Kathmandu; A Big Threat of AMR

 

 

Vulvovaginal Candidiasis, an increasing burden to women in the tropical regions attending Bharatpur Hospital, Chitwan

Anisha Subedi^1#, Milan Kumar Upreti^1#, Jid Chani Rana², Ram Prasad Sapkota², Upendra Thapa Shrestha^{3 *}                                              

¹ Department of Microbiology, Goldengate International College, Battisputali, Kathmandu

² Bharatpur Hospital, Chitwan

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

^# These authors equally contributed to the study

^* Corresponding author: Upendra Thapa Shrestha, Assistant Professor, Central Department of Microbiology, Tribhuvan University, Kirtipur, Kathmandu, Nepal. Email: upendrats@gmail.com

ABSTRACT

 

Vulvovaginal candidiasis is a yeast infection commonly caused by the overgrowth of Candida species in and around the vulva and vagina. Abnormal vaginal discharge, itching and irritation, swelling and redness of the vaginal area, pain during sexual intercourse, and dyspareunia are important clinical findings of the infection. Currently, the infection is one of the growing burdens to married women. Moreover, the infection with antifungal-resistant Candida species adds challenges to managing the disease. Hence, this study was conducted to identify the different Candida species causing vulvovaginal candidiasis and to determine its susceptibility pattern against different antifungal drugs. A hospital-based cross-sectional and quantitative study was conducted for the period of six months from September 2022 to March 2023 among symptomatic married women in the Gynecology and Obstetrics Department of Bharatpur Hospital, Chitwan. A total of 300 symptomatic cases were enrolled in the study. Candida species were isolated from vaginal swabs following standard microbiological procedures and antifungal susceptibility testing was performed with different antifungal agents. The total prevalence of vulvovaginal candidiasis was found to be 37.3% (112/300). Among different isolates, Candida albicans was found to be the most predominant (52.6%), followed by Candida glabrata (29.3%) among non-albicans. Women from the age group 25-35 years were found to be more infected (47.3%) and the relationship between contraceptive use and vulvovaginal candidiasis was found to be statistically significant (p<0.05). Candida species showed higher susceptibility toward Amphotericin-B (68.1%), followed by Fluconazole (51.7%), and Clotrimazole (50.9%). Whereas the least susceptibility was observed to Voriconazole (27.6%) and Itraconazole (35.30%). Candida albicans was comparatively more susceptible to different antifungal drugs than non-albicans species. Candida parapsilosis was only susceptible to Amphotericin-B and the increasing incidence of vaginal candidiasis due to non-albicans Candida indicates the need for routine speciation of Candida.

 

Keywords:    Vulvovaginal candidiasis, HiCrome agar, Candida albicans, Candida non-albicans Antifungal Susceptibility testing

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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

1.     1. Rosalino V, Georgina S, Andr LAMM, Nabil E, Vega L, Franyuti-kelly G, Abelardo D, Moncaleano V, Ernesto J, Felix M, Antonio J. Acinetobacter baumannii Resistance : A Real Challenge for Clinicians. Antibiotics. 2020;9(205):1–22.

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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Dahal et al. 2023, Antimicrobial Activity of Traditional Medicinal Plants Available at Banepa and Bhaktapur against Uropathogens, TUJM 10(1): 8-20

 

Antimicrobial Activity of Traditional Medicinal Plants Available at Banepa and Bhaktapur against Uropathogens

Susma Dahal^1#, Renuka Thapa^1#, Anisha Suwal^1#, Dinesh Dhakal¹, Alina Singh², Milan Kumar Upreti³, Upendra Thapa Shrestha^{4 *}

¹Sainik Awasiya Mahavidhyalaya (affiliated to Tribhuvan University) Sallaghari, Bhaktapur, Nepal

²Department of Laboratory Medicine, Shree Birendra Hospital, Chhauni, Kathmandu, Nepal

³Department of Microbiology, GoldenGate International College, Battisputali, Kathmandu, Nepal

⁴Central Department of Microbiology, Tribhuvan University, Kirtipur, Kathmandu, Nepal

 

^# All authors have equally contributed in the research work.

 

*Corresponding author: Upendra Thapa Shrestha, Central Department of Microbiology, Tribhuvan University, Kirtipur, Kathmandu, Nepal, Email: upendra.thapashrestha@cdmi.tu.edu.np

                                                            

ABSTRACT

Objectives: The study was aimed to determine the antimicrobial activity of traditional medicinal plants against the uropathogens.

Methods: Overall, 360 urine samples were collected from both outpatient and inpatient for culture and antimicrobial susceptibility testing. All the isolates were processed and identified following standard microbiological procedure and subjected to antibiotic susceptibility testing at Microbiology laboratory of Shree Birendra Hospital following CLSI guidelines. All the three plant extracts were processed by agar well diffusion method and Tube dilution method for antimicrobial activity against Escherichia coli, Klebsiella. pneumoniae, Pseudomonas aeruginosa and Enterobacter spp. at Microbiology laboratory of Sainik Awasiya Mahavidhyalaya following standard laboratory techniques.

Results: Crude extract of plants viz. Centella asiatica, Cuscuta reflexa and Mentha spicata showed good antimicrobial properties against all clinical isolates. Among all plants, ethanolic extract of C. asiatica was found to be most effective against E. coli with zone of inhibition 16 mm and minimum inhibitory concentration (MIC) value 5 mg/ml. Acetone extract of C. reflexa showed good antimicrobial activity against K. pneumoniae with zone of inhibition 14 mm and MIC value 10 mg/ml.

Conclusion: Our research revealed that the crude plant extracts, particularly the acetone and ethanol extracts, had a considerable amount of efficacy against uropathogens. Based on the study results, these traditionally used medicinal plants can overcome the problems of infections caused by multidrug resistant bacteria.

 

Keywords:  Urinary tract infection, antimicrobial activity, Medicinal plant, uropathogens, multidrug-resistant

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Tribhuvan University Journal of Microbiology (TUJM) Volume 10(1), 2023

 

Editorial

Advanced Molecular Techniques and Diagnosis of Febrile Illness

 

The application of multiplex real-time (Reverse Transcriptase - RT) polymerase chain reaction (PCR) in routine diagnosis of infectious diseases is unavoidable. In addition, the technique urgently might be an alternative tool in the diagnosis of febrile illnesses caused by many different kinds of etiologies including bacteria, viruses, and parasites. In Nepal, arboviral infections mainly dengue and chikungunya, scrub typhus, and leptospirosis are considered as major emerging infections in the last two decades. In addition, these infections are the most predominant neglected tropical diseases in tropical and subtropical regions where more than 51% of the total population in Nepal is living. A few of these infections are responsible for chronic infection causing a high morbidity rate. A study reported up to 40% of acute chikungunya infections may lead to chronic infections. Similarly, secondary infections can be more severe among flaviviruses such as Dengue and Zika due to antibody-dependent enhancement.  Unlike the diagnosis of other neglected tropical diseases, the differential diagnosis of arboviral infections is quite difficult as they all present similar clinical features, especially in Dengue Chikungunya and Zika viral infections.  However, few studies showed a significant difference in clinical manifestations in specific viral infections as well. Some clinical features are significantly associated with bacterial and viral infections. High-grade fevers with longer mean duration of fever, severe musculoskeletal pain, central nervous system (CNS) related symptoms (such as altered mentation, confusion, and loss of consciousness), and elevated liver enzymes such as aspartate aminotransferase (AST) and alanine aminotransferase (ALT) are highly associated with viral infections as compared to bacterial infections Finding such clinical manifestations may be helpful in the differential diagnosis of febrile illnesses as the point of care for future aspects. The other means of diagnosis commonly used in hospital settings of Nepal are serological methods including routinely used rapid diagnostic tests (RDTs) and Enzyme-Linked Immunosorbent Assay (ELISA). However, these methods have a few drawbacks including cross-reactivity, low sensitivity, and specificity making misdiagnosis of infections. Moreover, these methods are not applicable to early diagnosis of infections. An alternative to serological methods is molecular methods which are currently not available for diagnostic purposes in most hospitals and health care centers. Although the conventional PCR method has high sensitivity and specificity, it may take a longer time and detect only a particular pathogen at a time. The molecular method mainly real-time (RT) PCR has many advantages over other conventional methods because of which the applications of this technique for the diagnosis of infections or illnesses can’t be avoided. The techniques have already been introduced in the diagnosis of respiratory viral pathogens to diagnose flu-like syndromes in very few hospital settings. However, many tertiary care hospitals in Nepal have not been facilitated to use this technique in routine diagnosis. In addition, febrile illnesses in tropical and subtropical regions are the most undiagnosed illnesses. As a consequence, a number of patients have been suffering from chronic bacterial and viral infections. Real-time (RT) PCR is the only ultimate tool in the diagnosis of such illnesses.   The method has not only higher sensitivity (> 95%) and specificity (100%) but also detects the possible pathogens as early as the first day of onset of illness. Because of good reproducibility, sensitivity, and specificity, this method is considered a gold standard method for the detection of RNA viruses in clinical specimens. In addition, the multiplex real-time (RT) PCR detects multiple pathogens in a single run making it more economical and less time-consuming in the hospital settings of low and middle-income countries. It seems to be affordable to common people as well.  While talking about the current situation in Nepal, several tertiary care hospitals have upgraded to real-time PCR for diagnosis of COVID-19 during the pandemic. Hence, a similar facility can be extended to detect other pathogens in neglected tropical diseases and febrile illnesses in routine diagnosis.

 

 

Komal Raj Rijal, Editor in Chief

Upendra Thapa Shrestha, Associate Editor

DOI: https://doi.org/10.3126/tujm.v10i1.60644