Molecular Detection of Carbapenemase Genes in Extensive Drug Resistant Acinetobacter baumannii Clinical Isolates from ICU Patients, Khartoum ()
1. Introduction
Acinetobacter baumannii is a major cause of hospital acquire infections mainly among patients admitted at intensive care units (ICU) in many hospitals [1], regarding its ability to survive for long periods and could easily spread in hospital environment beside developing resistance to multiple antimicrobial agents leading to serious therapeutic problems [2] [3]. These traits could define its propensity for causing extended outbreaks [2]. Throughout last decades, mortality ranging from 5% in general wards to 54% in intensive care units (ICUs) is associated with A. baumannii infections [3] [4]. Carbapenems have been used as the most appropriate choice for treatment of infections due to MDR strains of A. baumannii. Unfortunately, extensive administration of broad-spectrum cephalosporins and/or carbapenems is a significant risk factor for development of colonization or infection with carbapenemase-producing A. baumannii [2] [5]. CPAB in hospital settings ranged from 2.3% to 67.7% in North Africa and from 9% to 60% in sub-Saharan Africa and the major bla genes were OXA-23-like, OXA-58-like, OXA-48-like, NDM-1 and VIM-2 associated with A. baumannii isolates of hospitalized patient years between (2010-2018) [3] [6]. More alarmingly; there was record of extensive drug resistance A. baumannii with intermediate resistance to colistin. This situation leads to limited options for treatment [7]. The lack of systematically collected data on the Sudan area contributes to a poor understanding of antimicrobial resistance and limits an effective response to the problem. The present study was aimed to investigate frequency of carbapenemase producing A. baumannii strains exhibiting extensive drug-resistant (XDR) profile isolated from ICU patients in two large hospitals at Khartoum city of Sudan.
2. Materials and Methods
2.1. Study Design and Setting
This cross-sectional, descriptive study was conducted in Royal Care International Hospital (RCIH) and National Ribat Hospital (NRH) in Khartoum state, in Sudan between 2017 and 2019, from previous isolated Gram-negative coccobacilli isolates of various clinical from intensive care unit (ICU) patients. Those hospitals serve an average of 30 patients per day in ICU as well as the referral hospitals for the Khartoum city.
2.2. Clinical Isolates
A total of 100 nonduplicate Gram-negative coccobacilli strains were purposively collected regarding availability at study time. The nonduplicate Gram-negative coccobacilli obtained from various cultures of sputum, blood, urine, wound swabs, central-line catheter and tips of ICU patients at the RCHI and NRH in the Microbiology Laboratory. This strains were preliminarily re-identified phenotypically using standard microbiological procedures: growth characteristics and Gram-negative identification biochemical set and confirmed by restriction analysis of the 16 s - 23 s using polymerase chain reaction (PCR) amplification of A. baumannii (Table 1). All of the strains were stored in skim milk with 15% glycerol at −80˚C until further use. Before performing the tests strains were cultured on Brain Heart Infusion (BHI) agar plates at 37˚C for 24 h.
2.3. Antimicrobial Susceptibility Testing
Antimicrobial susceptibility test of confirmed A. baumannii isolates was performed by disc diffusion method as per the (CLSI) guidelines [8], on Muller- Hinton agar (Hi-Media, Mumbai) using gentamicin (10μg), amikacin (30 µg), Co-trimoxazole (25 µg), ceftriaxone (30 μg), cefixime (30 μg), ceftazidime (30 μg), cefuroxime (30 μg), ciprofloxacin (5 μg), amoxycillin/clavulanic acid (30 μg), Imipenem (10 µg), meropenem (10 μg), tetraycline (10 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 of the disks were checked by using reference strain A. baumannii strain ATCC 17,978 was used. Interpretation of Multidrug-resistant (MDR) and extensively drug-resistant (XDR) profiles were defined using previously established criteria [9].
2.4. Detection Carbapenemase-Encoding Blagenes
The deoxyribonucleic acid (DNA) was extracted by boiling technique as follow; a loopful of each A. baumannii isolate was emulsified in 200μl of distilled water then boiled for 15 min and centrifugation at 13,000 rpm for 10 min. Supernatant was used for PCR amplification. A. baumannii isolates were screened for 6 common carbapenemase-encoding genes including blaNDM [10], blaIMP, blaKPC, blaVIM, blaOXA-48-like, blaGES [11] and for OXA-type carbapenemase-encoding genes including blaOXA-23-like, blaOXA-24-like, blaOXA-143-like and blaOXA-51-like [12] by PCR amplification with specific sets of primers (Table 1). All PCR-Reaction conditions were prepared by using ready master mix (APSLABS, India), 0.5 μl of each primer
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Table 1. List of primers used for PCR amplification with sequence and amplicon size (bp).
and 1 μl of template DNA (about 10 ng) in a total 25 μl. The PCR cycling conditions were as follows [10] [11] [12]: PCR products were assessed by electrophoresis using 1.5% (w/v) agarose gel and visualized by using an ultraviolet (UV) transilluminator.
2.5. Statistical Analysis
All data were analysed 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.
3. Results
3.1. Detection of A. baumannii
A total of 38 A. baumannii were identified out of 100 gram-negative coccobacilli isolates using PCR, from different clinical specimens. The demographic characteristics of the patients with A. baumannii infection investigated in our study presented a slight male preponderance of 63.2% females versus 36.8% males). The age of the overall patients ranged between 28 - 73 years (mean 55.6 years, SD ± 11.4 years). A. baumannii were isolated highest from sputum (n = 28), urine (n = 2), 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.
3.2. Antimicrobial Susceptibility
All strains of A. baumannii (n = 38) showed resistance for all antimicrobials tested except 39.5% (15/38) of the isolates were susceptible to colistin. However, according to the susceptibility testing results (Table 2). Besides, 60.5% of A. baumannii isolated strains were categorized as XDR strains.
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Table 2. Antimicrobial susceptibilities profiles of 38 A. baumannii strains.
3.3. Carbapenemase-Encoding Genes in A. baumannii Isolates
57.9% (22/38) of XDR A. baumannii were positive for one or more carbapenemase blagenes. The most prevalent single blagenes detected were blaOXA-48-like (n = 5) in Figure 1, followed by blaNDM (n = 4) and blaGES (n = 1). Whereas twelve A. baumannii isolates were co-produced carbapenemase blagenes blaNDM+OXA-48-like (n = 5), blaOXA-23-like/OXA-51-like (n = 4) in Figure 2(a), blaNDM-1+OXA-23/51/143-likes (n = 2), while blaNDM-1+ blaOXA-51-like (Figure 2(b)) was detected in only one (n = 1) isolate (Table 3).
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Table 3. Distribution of carbapenemase (blagenes) in 38 XDR A. baumannii strains.
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Figure 1. Gel-electrophoresis result of blaOXA-48-likeamplification: lane 1: DNA ladder 100 bp; lane 2: blaOXA-48-like positive control 281 bp; lane 3: negative control; lanes 4, 6 & 8: Positive blaOXA-48-like A. baumannii isolates.
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Figure 2. Gel-electrophoresis result of: (a) blaOXA specific amplification: lane 1: DNA ladder; lane 5 & 6: blaOXA-143-like; lane 3: blaOXA-51-like enzymes; lane 1, 2, 4, 7 & 8: blaOXA-51-like and blaOXA-23-like. The molecular size marker (lane 1) is a 123 bp ladder (Invitrogen, Paisley, UK) and (b) blaNDM-1amplification: lane 1: DNA ladder; lane 4: blaOXA-51-like & blaNDM-1 positive.
4. Discussion
An emerging rise in the incidence of carbapenemase producing A. baumannii has been increasingly reported worldwide [6] and in Sudan as well [13] [14], leaving behind a significant challenging to treat infections. In our study, for a deeper understanding the molecular mechanisms underlying carbapenem resistance, a collection of accurately characterized 38 A. baumannii clinical isolates was screened for carbapenemase-coding genes by PCR. A total of 57.9% (22/38) A. baumannii isolates were confirmed by molecular amplification (PCR) to be carbapenemase positive in clinical samples recovered from ICU patients with severe infections. Among the source of the isolates in the study, the majority of CPAB were from respiratory specimens (72.7%) followed by tip, blood, central line and bed sore specimens, consistent with other studies [1] [14] [15]. Extensive drug resistant among CPAB infections has become a world-wide contest as this organism is resistant to cephalosporins, Monobactams, aminoglycosides, fluoroquinolones, cephalosporins, carbapenems, and now emergence of colistin resistance in this species is of significant concern, leaving restricted treatment options for ICU infections. In our region, lack of systematically identification of A. baumannii infection among ICU patients and environments in hospital contributes to a poor understanding of antimicrobial resistance and limits an effective response to the problem. In recent years, the global emerge of extensive drug resistant producing carbapenemase A. baumannii, the strains have significantly threatened public health and become a major problem in the intensive care unit (ICU) reported by [1] [4] [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 [14] [17]. In addition, the resistance rate of A. baumannii to colistin was 60.5%, which is higher than in previous reports in Khartoum state and other studies [1] [4] [13] [14] [16]. 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-like genes were the most prevalent subgroup, which is consistent with the view that they are intrinsic to A. baumannii [5] [10] [14] [18]. These genes were detected in 7 of 12 isolates, irrespective of levels of carbapenem susceptibility or resistance, these alleles do 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-like carbapenemase-producing A. baumannii is becoming widespread globally in Europe, South America, and Asia [19] [20]. In this study, blaOXA-48-likewas the most common gene encountered in the study isolates as single carbapenemase encoding gene followed by blaNDM and blaGES. Various studies have noted that only blaGES-type carbapenemase was reported in in an Acinetobacter isolate from Mediterranean countries and Kuwait [21] [22]. blaOXA-23-like carbapenemase was detected in 6 (15.4%) of the 38 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. [19] [23]. This rate, however; is much higher than that reported for other African countries [3] revealing a worrisome situation in this country. Alleles encoding blaOXA-24/40-like enzymes were not detected in any of the A. baumannii ICU strains; these enzymes are reported in Portugal, Spain, Poland, Iran, the United States, Asia and Saudi Arabia [3] [5] [20]. In the current study we report blaNDM-1- producing A. baumannii strains, in contrast to 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 [1] [14] [24]. The coexisted CP-AB was detected in 12 isolated CRAB with (5/22; 22.7%) blaNDM and blaOXA-48-like, followed by (4/22; 18.2%) blaOXA-51-like and blaOXA-23-like and only two strains of A. baumannii produced blaOXA-51,blaOXA-23-like,blaOXA-143-likeand blaNDM-1, coexisted carbapenemase gens surveyed in this study as emergent detection in our region, mainly among ICU patients. As the blaOXA-143-like gene is frequently found in the Southeast region of Brazil, especially in the state of São Paulo. It is important to note that two new variants of this gene were recently described [12].
Here, we detected an emergent OXA subclass identified in two A. baumannii strains blaOXA-143-like which reported as High-Risk Clones among XDR A. baumannii, whereas blaOXA-23, blaOXA-51, blaNDM-1 and blaGES producing in XDR A. baumannii strains were prevalent in the ICU. The coexisted genes (blaOXA-51andblaOXA-23-like andblaOXA-51,blaOXA-23-like,blaOXA-143and blaNDM-1) were also associated with increased virulence as compared to other OXAs. Therefore, some infection control measures should be covered and implementation of whale genome sequence (WGS) as advanced molecular characterization of infectious agents could improve both, identification and genetic characterization including resistance profiles, facilitating outbreak investigations and molecular surveillance.
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 were not applicable as samples obtained from Microbiology Laboratory remaining samples and coded by Laboratory ID.