Clinical Microbiology: Open Access

A Molecular Diagnosis and Identification of Carbapenemase Producing Acinetobacter Baumannii among ICU Patients, In Khartoum State-Sudan

Shirehan M Ibrahim^1*, Elamin M Ibrahim², Omer A Ibrahim¹, Enas Awad³, Omnia M Hamid⁴ and Hassan A Alaziz^{5 1}Department of Medical Microbiology, Faculty of Medical Laboratory Sciences, National Ribbat University, Khartoum, Sudan
²Department of Medical Microbiology, Faculty of Medical Laboratory Sciences, University of Khartoum. Khartoum, Sudan
³Department of Medical Microbiology, Faculty of Medical Laboratory Sciences, Ibn Sena University Khartoum, Sudan
⁴Department of Medical Microbiology, Faculty of Medical Laboratory sciences, University of Medical Sciences & Technology, UMST-Khartoum, Sudan
⁵Faculty of Medicine, National Ribbat University, Sudan
^*Correspondence: Shirehan M Ibrahim, Department of Medical Microbiology, Faculty of Medical Laboratory Sciences, National Ribbat University, Khartoum, Sudan, Tel: 0024911333300, Email:

Received: 07-Dec-2021 Published: 27-Dec-2021

List of Abbreviations

ICU Intensive Care Unit

A. baumannii Acinetobacter baumannii

Introduction

Acinetobacter baumannii is a major cause of hospital acquire infections mainly among patients admitted at intensive care units (ICU) in many hospitals, regarding its ability to develop resistance to multiple antimicrobial agents is difficult to control and treat leading to serious therapeutic problems [1]. Carbapenems are thus often considered "last-resort antibiotics" for treating multi-drug resistant (MDR) bacteria and for treating A. baumanniiserious infections [2]. However, reports of carbapenem resistant A. baumannii (CRAB) strains have been rising steadily during the past few years, and these isolates are often multidrug-resistant [3]. This emergence of CRAB has become a worldwide problem especially, multiresistant strains seriously challenges the treatment of these infections [4].The carbapenem resistance, most commonly caused by carbapenemases which are β-lactamases capable of hydrolysing carbapenems [7]. The Ambler classification allows differentiation of carbapenemases into class A (e.g., KPC, SME, IMI, and GES), class B (e.g., NDM, VIM, and IMP), and class D (OXA-48-like). The carbapenemases of class A and class D are serine β-lactamases, and the carbapenemases of class B are metallo-β-lactamases (MBLs) [8]. The emergence of CPAB is of special concern in developing countries, since antibiotic prescription rates and intake without prescription is markedly higher [9].

Antibiotic resistance causing increased morbidity, mortality, and economic impacts on health services especially among ICU patients as they are critically ill or debilitated patients at high risk [4]. A. baumannii has the ability to survive for long periods and could easily spread in hospital environments [5]. These traits could define its propensity for causing extended outbreaks [5, 6]. Consequently, there is a critical need to conduct research to estimate the burden of carbapenemase producing A. baumanii underlying the attributed resistance. In our region lack of systematically identification of A. baumannii infection among ICU patients and environments in hospital which is contributes to a poor understanding of antimicrobial resistance and limits an effective response to the problem. The present study was carried out toisolates and evaluates the antibiotic resistance of A. baumannii isolated from ICU patients and to evaluate the frequency of common carbapenemase-encoding genes in isolated carbapenem resistant A. baumannii by polymerase chain reaction (PCR).

Materials and Methods

One hundred Gram-negative coccobacilli isolates were collected in 2019 from Microbiology laboratory department at Royal Care International Hospital (RCIH) and National Ribat Hospital (NRH) as identified by both microbiology laboratories, both hospitals located in Khartoum city, Sudan. The clinical specimens were sputum, blood, urine, wound swab, central-line catheter and tips then plated out on MacConkey agar.

Isolation and identification of A. baumannii: was done phenotypically based on cultural characteristics, Gram stains, oxidase test and conventional gram negative biochemical set following standard assay of gram negative rods at microbiology laboratory at both hospitals, and by molecular identification of A. baumanii was further confirmed by restriction analysis of the 16s-23s using polymerase chain reaction (PCR) amplification (Table 1). Confirmed A. baumannii isolates were tested for antimicrobial susceptibility and likewise screened for the presence of most common carbapenemase-encoding blagenes.

Antimicrobial susceptibility testing: Antimicrobial susceptibility test of the A. baumannii confirmed isolates was performed by disc diffusion method as per the (CLSI) guidelines [7], on Muller-Hinton agar (Hi-Media, Mumbai) using Ceftazidim (30 μg), ccefuroxime (30 μg), gentamicin (10 μg), cefixime (30 μg), ciprofloxacin (5 μg), amoxiclav (30 μg), meropenem (10 μg), ceftriaxone (30 μg) and colistin (10 μg) (bioanalyse, Turkey and Hi-Media, Mumbai).

The diameter of inhibition zones was measured and reported as susceptible or resistant. For quality control, standard strain of E. coli (ATCC 25922) was used.

Detection carbapenemase-encoding blagenes: A. baumannii isolates were screened for 6 common carbapenemase-encoding genes including blaNDM[8] blaIMP, blaKPC, blaVIM, blaOXA, blaGES [9] and forOXA-type carbapenemase-encoding genes including blaOXA-23, blaOXA-24, blaOXA-143 and blaOXA-51[10] by PCR amplification with specific sets of primers (Table 1).

Table 1: Primers set used in the sequence and amplicon size (bp).

PCR amplification: The deoxyribonucleic acid (DNA) was extracted by boiling technique as follow; a loopful of each A. baumannii isolate was emulsified in200 μl of distilled water then boiled for 15 min and centrifugation at 13,000 rpm for 10 min. Supernatant was used for PCR amplification. All PCR-Reaction conditions were prepared by using ready master mix (APSLABS, India), 0,5 μl of each primer and 1 μl of template DNA (about 10 mg) in a total 25 μl.The PCR cycling conditions were as follows: for blaNDM comprised of initial denaturation at 94° Cfor 10 min followed by 30 cycles of 1 min denaturation at 94° C, 1 min annealing at 60°C and 1 min extension at 72°C and final extension step of 10 min at 72°C.

For multiplex (VIM, IMP and KPC) and (GES and OXA) carried out as a following: initial denaturation at 94°C for 10 min; 30 cycles of 94°C for 40 s, 55°C for 40 s and 72°C for 1 min; and a final elongation step at 72°C for 7 min. The annealing temperature of multiplex GES and OXA was optimal at 57°C instead of 55°C. PCR products were assessed by electrophoresis using 1.5% (w/v) agarose gel and visualized by using an ultraviolet (UV) trans illuminator.

Statistical analysis: all data were analyzed using the Statistical Package for the Social sciences for Windows software package version 21.0 (SPSS-IBM, Armonk, NY).

Results were presented using frequency and percentages for qualitative variables. Categorical variables were compared by Chi-square test and all tests were two-sided, and differences with P-value <0.05 were considered statistically significant.

Results

Detection of A. baumanii: A total of 39 A. baumaniiout of 100 gram-negative coccobacilli isolates were recovered from different clinical specimens; sputum (n=29), urine (n=4), blood, central line and tip (n=3) for each, wound and bed sore (n=1) for each, collected from the microbiology laboratory at Royal Care International Hospital (RCIH) and National Ribat Hospital (NRH) were included in the study (Table 2).

Table 2: Distribution pattern of A. baumanii and CP-AB isolates by their respective sources of specimens from ICU patients at Khartoum state selected hospitals.

Table 3: Antimicrobial susceptibilities profiles of A. baumanii isolates and carbapenem resistant A. baumaniifrom ICU patients at Khartoum state selected hospitals.

Antimicrobial susceptibility of A. baumannii isolates: 100% (39/39) of A. baumannii isolates from ICU patient’s samples were resistant to ciprofloxacin, cefixime, ceftazidime, ceftriaxone, cefuroxime amoxacillin/clavulanic acid and gentamicin followed by 97.4% (38/39) meropenem. In other words, most of A. baumannii isolates were multidrug resistant. However, 59.0% (23/39) of the isolates were resistant to colistin.

Carbapenemase-encoding genes in A. baumannii isolates: 56.4% (22/39) of A. baumannii were positive for one or more carbapenemase blagenes. The most prevalent single blagenes detected were OXA (n=5) followed by NDM (n=4) and GES (n=1). Whereas twelve A. baumanii isolates were co-produced carbapenemase blagenes blaND +OXA(n=5)blaOXA-23/51(n=4), blaNDM-1+OXA-23/51/143(n=2), whileblaNDM-1+ blaOXA-51 was detected in only one (n=1) isolate (Figure 1-3).

Figure 1:Distribution pattern of Carbapenemase producing A. baumanii (blagenes) from ICU patients at Khartoum State in selected hospitals.

Figure 2:Gel-electrophoresis result of blaOXA amplification: lane 1: DNA ladder 100 bp; lane 2: OXA positive control 281 bp; lane 3: negative control; lanes 4, 6 &8: Positive OXA Acinetobacter isolates.

Figure 3:Gel-electrophoresis result of: (A) blaOXA specific amplification: lane 1: DNA ladder; lane 6: OXA-143 enzymes; lane 12: OXA-51 enzymes; lane 14: OXA-51/2. The molecular size marker (lane 1) is a 123 bp ladder (Invitrogen, Paisley, UK) and (B) blaNDM-1 amplification: lane 1: DNA ladder; lane 5: NDM-1 enzyme.

Discussion

A. baumanii is an important opportunistic pathogen that is responsible for health-care infection specially the MDR. A.baumannii is highly resistant to commonly used antibiotics such as penicillins, cephalosporins, aminoglycosides and fluoroquinolones by intrinsic and acquired mechanisms. They are also gradually becoming resistant to carbapenems. The isolation of MDR A. baumannii from ICU samples had been reported earlier by researchers [12].

Hospitals have long served as reservoirs for the transmission of pathogenic bacteria, and. Among the source of the isolates in these study the vast majority of positive cultures were from respiratory specimens (74.4%) followed by urine and tip specimens, consistent with other studies [13,14,15]. Infections with A. baumannii affecting mainly the respiratory tract, urinary tract, wound infections and sometimes local infections may develop bacteraemia as almost all cases received (mechanical ventilation or endotracheal tube and catheters during their ICU stay, all these findings may support by [16].

The results of the present study show that there was an extreme increase in the resistance rate of A. baumannii to meropenem, from 89% in 2015 to 100% in 2019 [17]. In addition, the resistance rate of A. baumannii to colistin was 59%, which is higher than in previous reports in Khartoum state and other studies [18,19,20]. The present study showed 100% resistant rates of the most clinically applicable antibiotics for the treatment of infections caused by A. baumannii, except for colistin, which may be used as the final options in the management of infections caused by this bacterium. In this study, the high resistance rate of A. baumannii against carbapenems may indicate the outcome of overuse and misuse of carbapenems in our hospital.

Overall, blaOXA-51 genes were the most prevalent subgroup, which is consistent with the view that they are intrinsic to A. baumannii [21]. These genes were detected in 7 of 11 isolates, irrespective of levels of carbapenem susceptibility or resistance, these alleles does not correlate with the level of carbapenem resistance of the host isolate. Thus, resistance to carbapenems cannot be inferred from detection of blaOXA-51-like alleles. In contrast, alleles encoding OXA-23-like, OXA-24-like and OXA-58-like enzymes were consistently associated with resistance or, at least, with reduced susceptibility.

The blaOXA-23 carbapenemase-producing A. baumannii are becoming widespread globally in Europe, South America, and Asia [22]. In this study, blaOXA-23 carbapenemase was detected in 6 (15.4%) of the 39 carbapenem-resistant isolates and as in terms of carbapenem non-susceptibility, an alarmingly high rate of 75.0% over 2 years was detected, this high rate is similar to that reported by Perez et al [23] This rate, however; is much higher than that reported for other African countries [24] revealing a worrisome situation in this country. Alleles encoding OXA-24 (OXA-40)-like enzymes were not detected in any of the Sudanese clinical isolates; these enzymes are most often found in Portugal, Spain, Poland, Iran, the United States and Asia [15]. In Saudi Arabia, the blaOXA-24 gene was detected at a rate of 4–45% in Acinetobacter species isolates [25], we first report blaNDM-1-positive A. baumanii isolates in Sudan. In contrast to in other countries where blaNDM-1 was mostly carried by Enterobacteriaceae; all the blaNDM-1-positive A. baumannii isolates, which suggests that this species, which has a robust survival capability, can easily acquire foreign resistance genes such as blaNDM-1[26].

Recently the Ambler class A of the GES (carbapenemase) types has also been reported for A. baumannii [27]. The blaGES genes are usually carried on integrons found in various species, predominantly Klebsiella pneumoniae, Escherichia coli, and Pseudomonas aeruginosa, and these resistance determinants have been reported in several countries in Europe, Asia, South America, and South Africa [28]. Our data further point out the fact that one of CR-AB is producing GES gene, that might now be emerging independently in different areas in the world and indicate that, were also recently reported in an Acinetobacter isolate from Kuwait [29], as an additional mechanism of resistance to carbapenems in A. baumannii. Only GES-type carbapenemase was reported in Mediterranean countries [28].

Acknowledgements: We are grateful to Royal Care International Hospital (RCIH) and National Ribat Hospital (NRH) for providing us the Gram negative coccobacilli isolates from ICU patients to perform this study.This work was partly of Ph.D. project under supervision of Prof. Hassan A. Alaziz and Prof. Elamin. M. Ibrahim,in the Alribat National University (Khartoum, Sudan);

Ethical clearance: This study was approved by the ethics committee of Alribat national university-graduate collage.Bacterial isolates ethics approval and consent was not applicable as samples obtained from Microbiology Laboratory remaining samples and coded by Laboratory ID.

References

1. Ibrahim S, Al-Saryi N, Al-Kadmy I, Aziz SN. Multidrug-resistant Acinetobacter baumannii as an emerging concern in hospitals. Mol Biol Rep. 2021;1-2.
2. Thet KT, Lunha K, Srisrattakarn A, Lulitanond A, Tavichakorntrakool R, Kuwatjanakul W, et al. Colistin heteroresistance in carbapenem-resistant Acinetobacter baumannii clinical isolates from a Thai university hospital. World J Microbiol Biotechnol. 2020;36(7):1-7.
3. Kuo HY, Chang KC, Kuo JW, Yueha HW, Liouf ML. Imipenem:a potent inducer of multidrug resistance in Acinetobacterbaumannii. Int J Antimicrob Agents. 2012;39:33-38.
4. Al-Agamy MH, Khalaf NG, Tawfick MM, Shibl AM, El Kholy A. Molecular characterization of carbapenem-insensitive Acinetobacter baumannii in Egypt. International Journal of Infectious Diseases. 2014;22:49-54
5. Manenzhe RI, Zar HJ, Nicol MP, Kaba M. The spread of carbapenemase-producing bacteria in Africa: a systematic review. Journal of antimicrobial chemotherapy. 2014;70(1):23-40.
6. de Jager P, Chirwa T, Naidoo S, Perovic O, Thomas J. Nosocomial outbreak of New Delhi metallo-β-lactamase-1-producing Gram-negative bacteria in South Africa: a case-control study. PLoS One. 2015;10(4):e0123337.
7. Hansen GT. Continuous evolution: perspective on the epidemiology of carbapenemase resistance among enterobacterales and other gram-negative bacteria. Infect Dis Ther. 2021;1-8.
8. Hammoudi Halat D, Ayoub Moubareck C. The current burden of carbapenemases: Review of significant properties and dissemination among gram-negative bacteria. Antibiot. 2020;9(4):186.
9. Dallenne C, Da Costa A, Decré D, Favier C, Arlet G. Development of a set of multiplex PCR assays for the detection of genes encoding important β-lactamases in Enterobacteriaceae. J Antimicrob Chemother. 2010;65(3):490-5.
10. Higgins PG, Pérez-Llarena FJ, Zander E, Fernández A, Bou G, Seifert H. OXA-235, a novel class D β-lactamase involved in resistance to carbapenems in Acinetobacter baumannii. Antimicrob agents chemother. 2013;57(5):2121-6.
11. Pritsch M, Zeynudin A, Messerer M, Baumer S, Liegl G, Schubert S, et al. First report on bla NDM-1-producing Acinetobacter baumannii in three clinical isolates from Ethiopia. BMC infect dis. 2017;17(1):180.
12. Josheghani SB, Moniri R, Firoozeh F, Sehat M, Dastehgoli K, Koosha H, et al. Emergence of bla OXA-Carrying Carbapenem Resistance in Multidrug-Resistant Acinetobacter baumannii in the Intensive Care Unit. Ir Red Crescent Med J. 2017;19(5).
13. Sileem AE, Said AM, Meleha MS. Acinetobacter baumannii in ICU patients: A prospective study highlighting their incidence, antibiotic sensitivity pattern and impact on ICU stay and mortality. Egypt J Chest Dis Tubercul. 2017;66(4):693-8.
14. Reddy D, Morrow BM, Argent AC. Acinetobacter baumannii infections in a South African paediatric intensive care unit. J Trop Pediatr. 2015;61(3):182-7.
15. Ibrahim S, Al-Saryi N, Al-Kadmy I, Aziz SN. Multidrug-resistant Acinetobacter baumannii as an emerging concern in hospitals. Mol Biol Rep. 2021;1-2.
16. Hong KB, Oh HS, Song JS, Lim J-h, Kang DK, Son IS, et al. Investigation and control of an outbreak of imipenem-resistant Acinetobacter baumannii infection in a pediatric intensive care unit. The Pediatric infectious disease journal. 2012;31(7):685-90.
17. Omer MI, Gumaa SA, Hassan AA, Idris KH, Ali OA, Osman MM, et al. Prevalence and resistance profile of acinetobacter baumannii clinical isolates from a private hospital in Khartoum, Sudan. Am J Microbiol Res. 2015;3(2):76-79
18.   Bakour S, Olaitan AO, Ammari H, Touati A, Saoudi S, Saoudi K, et al. Emergence of colistin-and carbapenem-resistant Acinetobacter baumannii ST2 clinical isolate in Algeria: first case report. Microb Drug Resist. 2015;21(3):279-85.
19. Qureshi ZA, Hittle LE, O'Hara JA, Rivera JI, Syed A, Shields RK, et al. Colistin-resistant Acinetobacter baumannii: beyond carbapenem resistance. Clin infect dis. 2015;60(9):1295-303.
20. Machado D, Antunes J, Simões A, Perdigão J, Couto I, McCusker M, et al. Contribution of efflux to colistin heteroresistance in a multidrug resistant Acinetobacter baumannii clinical isolate. J med microbiol. 2018;67(6):740-749.
21. Biglari S, Alfizah H, Ramliza R, Rahman MM. Molecular characterization of carbapenemase and cephalosporinase genes among clinical isolates of Acinetobacter baumannii in a tertiary medical centre in Malaysia. J med microbiol. 2015;64(1):53-58.
22. Cieslinski JM, Arend L, Tuon FF, Silva EP, Ekermann RG, Dalla-Costa LM, et al. Molecular epidemiology characterization of OXA-23 carbapenemase-producing Acinetobacter baumannii isolated from 8 Brazilian hospitals using repetitive sequence–based PCR. Diagnos microbiol infect dis. 2013;77(4):337-340.
23. Tomaschek F, Higgins PG, Stefanik D, Wisplinghoff H, Seifert H. Head-to-head comparison of two multi-locus sequence typing (MLST) schemes for characterization of Acinetobacter baumannii outbreak and sporadic isolates. PLoS One. 2016;11(4):0153014.
24. Sileem AE, Said AM, Meleha MS. Acinetobacter baumannii in ICU patients: A prospective study highlighting their incidence, antibiotic sensitivity pattern and impact on ICU stay and mortality. Egyp J  Chest Dis Tubercul. 2017;66(4):693.
25. Al-Agamy MH, Shibl AM, Ali MS, Khubnani H, Radwan HH, Livermore DM. Distribution of β-lactamases in carbapenem-non-susceptible Acinetobacter baumannii in Riyadh, Saudi Arabia. J glob antimicrobial res. 2014;2(1):17-21.
26. Khan AU, Maryam L, Zarrilli R. Structure, genetics and worldwide spread of New Delhi metallo-β-lactamase (NDM): a threat to public health. BMC microbiol. 2017;17(1):101.
27. Cicek AC, Saral A, Iraz M, Ceylan A, Duzgun A, Peleg A, et al. OXA-and GES-type β-lactamases predominate in extensively drug-resistant Acinetobacter baumannii isolates from a Turkish University Hospital. Clin Microbiol Infect. 2014;20(5):410-5.
28. Djahmi N, Dunyach-Remy C, Pantel A, Dekhil M, Sotto A, Lavigne J-P. Epidemiology of carbapenemase-producing Enterobacteriaceae and Acinetobacter baumannii in Mediterranean countries. Bio Med res int. 2014;2014
29. Bonnin RA, Rotimi VO, Al Hubail M, Gasiorowski E, Al Sweih N, Nordmann P, et al. Wide dissemination of GES-type carbapenemases in Acinetobacter baumannii isolates in Kuwait. Antimicrob agents chemother. 2013;57(1):183-188.