Genetic determinants of multi-drug resistance in Acinetobacter baumannii at an academic hospital in Pretoria, South Africa
1Department of Microbiological Pathology, Sefako Makgatho Health Sciences University, Pretoria, South Africa
2National Health Laboratory Service, Dr George Mukhari Tertiary Laboratory, Pretoria, South Africa
*Corresponding author: Andrew M Musyoki; E-mail: andrew.musyoki@smu.ac.zaAbstract
Antimicrobial resistance is now globally recognised as the greatest threat to human health. Acinetobacter baumanniis’ (A. baumannii) clinical significance has been driven by its ability to obtain and transmit antimicrobial resistance factors. In South Africa, A. baumannii is a leading cause of healthcare associated infections (HAI). In this study, we investigated the genetic determinants of multi-drug resistant A. baumannii (MDRAB) at a teaching hospital in Pretoria, South Africa.
One hundred non repetitive isolates of A. baumannii were collected for the study at Dr George Mukhari Tertiary Laboratory (DGMTL). Antimicrobial susceptibility testing was performed using the VITEK2 system (bioMerieux, France). The prevalence of common resistance associated genes and AdeABC efflux pump system associated genes were investigated using conventional PCR. Genetic relatedness of isolates was then determined using rep-PCR.
Seventy (70) of 100 isolates collected were confirmed to be multi-drug resistant and were blaOXA51 positive. Phenotypically, the isolates where resistant to almost all tested antibiotics. However, one isolate showed intermediate susceptibility to tigecycline while all were susceptible to colistin. Oxacillinase encoding gene blaOXA-23 was the most detected at 99% and only 1% was positive for blaOXA-40. The PCR results for metallo-betalactamase (MBL) encoding genes showed that MBL blaVIM was the most frequently detected at 86% and blaSIM-1 at 3% was the least detected. Out of 70 isolates, 56 isolates had the required gene combination for an active efflux pump. The most prevalent clone was clone A at 69% of the isolates. Regarding treatment; colistin and tigecycline are the most effective against strains encountered at DGMTL as all tested carbapenems seem to have lost their effectiveness.
The major genotypic determinants for drug resistances are oxacillinases: blaOXA-51 (100%) and blaOXA-23 (99%). The study reports for the first time, blaOXA-40 and blaSIM-1 detection in A. baumannii in South Africa.
Background
Antibiotics are at the forefront of the battle against bacteria and other potentially dangerous infectious agents to human health. The use of antibiotics has changed the outcome of bacterial infections and saved millions of lives [1]. On the 27th of February 2017 in Geneva, World Health Organisation (WHO) released a list of bacteria that have become resistant to multiple classes of antibiotics including carbapenems and third-generation cephalosporins [2]. Multi-drug resistant Acinetobacter baumannii (MDRAB) has been identified as one of the leading clinically relevant multi-drug resistant organism that threatens human health [2]. In South Africa, A. baumannii is the leading cause of nosocomial infections [3] and its prevalence of isolation in various clinical samples has been increasing over time [4; 5; 6]. MDRAB’s clinical importance has been driven by its ability to obtain and transmit antimicrobial resistance genes [5] and to cause outbreaks in health care settings [7]. It is associated with high morbidity, high mortality, prolonged hospitalization and increased cost of hospitalisation [7; 3; 8].
Resistance genes are acquired through various mechanisms and enabling A. baumannii to resist action of several antibiotic families. Combination of mechanisms such as an increased expression of oxacillinase (OXA)-type carbapenemases and non-enzymatic mechanisms, such as decreased cell membrane permeability, overexpression of multi-drug efflux pump proteins and/or alterations in penicillin-binding proteins are reported to induce multiple drug resistance in A. baumannii [9]. For effective infection control and treatment, it is important to investigate and report on the diversity of prevalent strains. In this study, genetic determinant of MDRAB at Dr George Mukhari Tertiary Laboratory (DGMTL) were investigated.
Materials and methods
Sample collection
Ethical clearance to conduct the study was granted by Sefako Makgatho Health Sciences University Research and Ethics Committee. A hundred non repetitive isolates of MDRAB identified by VITEK2 system (bioMerieux, France) were collected at Dr George Mukhari Tertiary Laboratory (DGMTL) located at Dr George Mukhari Academic Hospital; from March 2017 to August 2017 and February 2018 to April 2018. Strains of A. baumannii were considered multi-drug resistant when resistant to at least one antibiotic in three different antibiotic classes [10].
Antimicrobial susceptibility testing
Antimicrobial susceptibility of isolates was established using the VITEK2 system (bioMerieux, France). Piperacillin + tazobactam, ceftazidime, cefepime, cefotaxime/ceftriaxone, imipenem, meropenem, trimethoprim/sulfamethoxazole, gentamycin, ciprofloxacin, tigecycline and colistin were tested.
Confirming multi-drug resistance and genotypic identification of A. baumannii isolates
Multi-drug resistance profile was confirmed using the disk diffusion method according to Machanda et al., [10]. A standardized inoculum of the isolate was prepared using normal saline (Diagnostic media product (DMP), NHLS, South Africa) and adjusted to 0.5 McFarland using a turbidity meter (DensiCHEK plus bioMerieux, France). The suspension was lawned on to Muller Hinton (MH) (Diagnostic media product, NHLS, South Africa (DMP)) agar plate. Susceptibility to one of each of the different classes of antibiotics was tested as follow: Gentamicin 10μg for aminoglycosides; Ciprofloxacin 5μg for quinolones and colistin 10μg for polypeptides. For beta-lactams, Piperacillin + Tazobactam 110μg for penicillins combined with inhibitors, ceftriaxone 30μg and ceftazidime 30μg for cephalosporins, meropenem 10μg for carbapenems were used. Antibiotic disks were placed onto the inoculated agar plate. The process was carried out according to CLSI guidelines (2017; CLSI Document M100-S27). The OXA-51 is a naturally occurring oxacillinase in A. baumannii that can be used to reliably identify this organism genotypically [11; 12; 13]. Therefore, it was used to confirm the genotypic identification of the A. baumannii isolates in this study in addition to the phenotypic methods.
Determining the prevalence of common antimicrobial resistance genes
DNA extraction
DNA extraction was performed using the boiling method as described by Olive and Bean [14] with a slight modification. Briefly, from each fresh overnight culture, a loopful of bacteria was taken from MH agar plates and suspended in 1mL of saline (Normale Saline G121721, DMP; South Africa) in an eppendorf tube, then centrifuged (MIKRO 200, Labotec, South Africa) at 7500rpm for 30 minutes. The supernatant was discarded and the pellet was re-suspended in 200μL of PCR water (Water for Molecular Biology, BioConcept Ltd, Switzerland). The obtained suspension was centrifuged at 7500rpm for 20 minutes. Thereafter, the suspension was boiled at 95 °C for 20 minutes using a thermomixer (Thermomixer Compact Eppendorf 5350 MERCK Chemical Pty. Ltd, South Africa), then centrifuged for 5 minutes at 7500rpm. The supernatant was used as the template for PCR immediately or stored at −20 °C until use.
Master Mix preparation
PCR master mix was prepared by using MyTaqTM HS ready mix (Bioline, UK) to detect genes of interest in A. baumannii. All PCR primers used in this study were synthesized by a commercial vendor in South Africa. Primers are listed in Supplimentary1 Table (S1). The Bioline protocol (Bioline; UK) was followed to prepare multiplex PCR assays using primer pairs with similar melting temperatures and monoplex PCR for primer pairs with different melting temperatures. PCR was performed in a reaction mixture of a total volume of 25μL; 12.5μL of My Taq ™ Red Mix (Bioline; UK), 0.5μL of each primer (forward and backward) and PCR grade water (Water for Molecular Biology, BioConcept Ltd, Switzerland) was added to make up to 20μL and 5μL of DNA template was added to constitute a 25μL reaction mix. The thermocycling conditions used for the detection of drug resistance associated genes are listed in supplementary 2 Table (S2).
PCR amplicon detection
PCR amplicons were separated and detected using ethidium bromide stained agarose gel electrophoresis and visualized under UV light. The expected amplicon sizes for the various primer sets and rep-PCR oligonucleotides are indicated in S1 Table.
Gene burden and statistical analysis
The gene burden of overall 11 targeted genes was established using IBM SPSS Statistics 25 software. Statistical analysis with 95% confidence level and p-value was assessed using IBM SPSS Statistics 25 software.
Summary of research methodology
Results
Antimicrobial susceptibility testing
Eleven antibiotics were tested for susceptibility on all 100 study isolates. All isolates (100%) were resistant to ceftazidime, cefepime and piperacillin + tazobactam; 98% of the isolates were resistant to trimethoprim/sulfamethoxazole and cefotaxime/ceftriaxone; 95% of the isolates were resistant to imipenem and meropenem; 90% of isolates were resistant to gentamycin; 88% of isolates were resistant to ciprofloxacin; 1 isolate showed reduced susceptibility to tigecycline with the rest (99%) of the isolates being susceptible. All the isolates (100%) were susceptible to colistin (Table 1).
Confirming multi-drug resistance and molecular identification of A. baumannii isolates
Seventy (70) of 100 isolates collected were confirmed A. baumannii on the basis of the presence of blaOXA-51 genes and confirmed multi-drug resistant based on Manchanda et al., [10] criteria. These isolates constituted the final study samples. The 30 remaining isolates were excluded from our study; 17 of these were excluded because they were not multi-drug resistant; 9 isolates were not multi-drug resistant and were negative for blaOXA-51 and 4 of them were multi-drug resistant but blaOXA-51 negative.
Determining the prevalence of common resistance associated genes
From the 70 study samples, oxacillinase encoding gene blaOXA-23 was the most frequently detected with 69 (98%) positive isolates and only 1 (1%) positive isolate for each blaOXA-58 and blaOXA-40 Table 2. PCR conducted for metallo-betalactamase (MBL) encoding genes detected 60 (86%) blaVIM and 41 (58%) blaNDM positive isolates, followed by 5 (7%) blaIMP and 2 (3%) blaSIM-1 with positive isolates Table 2. Fifty-six out of 70 isolates tested positive for the required gene combination for an active efflux pump. Nine isolates had the structural gene with no regulatory genes while 2 isolates had the structural gene and only one of the regulatory genes (AdeS). Three isolates did not have any of the 3 genes.
Gene burden in the study population
Of the 11 investigated genes; 1 isolate was positive for 10 genes and another isolate for 8 genes. Eight (8) isolates were positive for 7 genes; 14 isolates were positive for 6 genes; 24 isolates were positive for 5 genes; 7 isolates were positive for 4 genes; 12 isolates were positives for 3 genes and 3 isolates were positive for 2 genes.
Determining genetic relatedness of A. baumannii isolates
The strains of MDR A. baumannii were grouped according to band patterns as described by Santimaleeworagun et al., [15]. The most common clone was clone A with 49 (69%) isolates, followed by clone B and D with 8 (12%) each. Clone C and E were less represented with 2 (3%) and 3 (4%) isolates respectively (Fig 1).
Discussion
Antimicrobial susceptibility testing
Bacteria have developed several resistance mechanisms against antibiotics and are still developing new ways to overcome novel molecules [16]. In this study,100 isolates identified by VITEK2 system as MDRAB reveiled a wide range of resistance to various antibiotics. Colistin was the only agent showing susceptibility among all the study isolates. Only 1 isolates showed intermediate susceptibility to tigecycline (Table 1). Similar results were observed by Lowings et al., [6], during a study conducted in Pretoria, South Africa. The study reported resistance to imipenem at 86%; meropenem at 86%; cefepime at 90% and ceftazidime at 89%. Our study results revealed higher resistance prevalence compared to a similar study by Kock et al., [5]. In their study also conducted in Pretoria (2008), they reported resistance to imipenem at 59 %; meropenem at 63 %; cefepime at 62 % and ceftazidime at 45 %. The prevalence of drug resistance to antimicrobials other than the polymixin class in A. baumannii isolates has increased to 100% over the years [5; 17]. To date, Colistin has remained susceptible in all the A. baumannii isolates collected in this region. Our study noted with concern; the occurrence of intermediate susceptibility to Tigecycline, which is the only other drug with activity against MDRAB isolates in this study. The ability of A. baumannii to acquire and transmit drug resistance genes through several mechanisms such as transfer of integrons, plasmids or transposons and mutation of endogenous genes is associated with among others poor infection control practices in health care settings [18; 5].
Confirming multi-drug resistance and identification of A. baumannii isolates
Thirteen percent (13%) of the isolates were not A. baumannii and 87 (87%) out of 100 isolates were confirmed A. baumannii after detection and amplification of blaOXA-51 Table 2. Our results are similar to those reported by Kock et al., [5]. In their study, 19% of isolates identified by VITEK2 as A. baumannii were negative for blaOXA-51. Consequently, they were considered as misidentified. The oxacillinase blaOXA-51 has been used as a simple and reliable genotypic way of identifying A. baumannii strains [12]. A similar study by Lowings et al., [6] reported that 99% A. baumannii isolates collected were positive for blaOXA-51 and only 1% of isolate was negative for blaOXA-51. In that study, isolates were identified using an alternative method (MALDI TOF-MS) which is known to have higher sensitivity than VITEK2. Six (6) isolates were found to be misidentified as A. baumannii by VITEK2 system. Therefore, our identification method may have included other species outside of the A. baumannii complex. This may explain the 13 (13%) blaOXA-51 PCR negative isolates. Among the 87 (87%) confirmed A. baumannii isolates, 17 were not multi-drug resistant according to the definition criteria proposed by Manchanda et al., [10]. Consequently, they were removed from further analysis.
Determining the prevalence of common drug resistance associated genes
A high prevalence of oxacillinase encoding gene blaOXA-23 was noted in 69 (99%) of the 70 isolates Table 2. A study conducted in Thailand also detected the blaOXA-23 in 42 out of 43 (99%) A. baumannii isolates [15]. A similar report by Liakopoulos et al., [19] in Greece, noted that 120 out of 127 (95 %) A. baumannii isolates collected in 2011 were blaOXA-23 positive. Oxacillinase encoding gene blaOXA-23 was also reported as the most common carbapenamase encoding gene among Acinetobacter spp by Corrêa et al., [20] in Brazil and Santimaleeworagun et al., [15] in Thailand. It has been associated with resistance to carbapenems [21]. This study reported resistance rates of 95% to imipenem and meropenem (Table 1). Our results show higher resistance rates than previously reported in the region: 77% by Lowings et al., in Tshwane region and 59% by Kock et al.,in Pretoria [5]. Clearly, the prevalence of blaOXA-23 in A. baumannii isolates in Pretoria is increasing. This observation is not unique to the Pretoria region. Globally there is an increase in OXA-23-producing A. baumannii [22; 23; 24]. This increased occurrence can be due to the acquisition of mobile genetic elements [25] and/or poor infection control practices [26].
In this study, only 1% of the isolates had the oxacillinase encoding gene blaOXA-58 and blaOXA-40 Table 2. A study conducted on A. baumannii isolates by Lowe et al., [17] in South Africa on 69 isolates, reported the blaOXA-58 gene in 4% of their study samples. In 2015, the same team tested 100 isolates for the blaOXA-58 gene and found no positive isolates [6]. A study in Thailand isolated only 1 sample with blaOXA-40 out of 43 isolates [15]. These genes were found to be low in our area during our study. The blaOXA-58 and blaOXA-40 are also associated with resistance to carbapenems [27; 28]. However, strict infection prevention and control measures are needed to avoid an increase in the prevalence of isolates harbouring these genes.
Of the 70 isolates, the most frequently detected MBL genes were: blaVIM and blaNDM at 86% and 58% respectively Table 2; blaIMP and blaSIM were detected in 7% and 3% of the isolates respectively Table 2. In South Africa, since their first report in 2008 [5]; 2011 [29] and 2012 [30]; the occurrence of blaVIM, blaNDM and blaIMP respectively has increased over the years [31]. MBLs have mostly been reported in carbapenemase-producing Enterobacteriaceae [32; 31]. Thus, the ability of A. baumannii to easily acquire resistant genes and their high prevalence in hospital settings may explain the increasing prevalence of these genes [33; 25]. These results are consistent with the national picture on the progression of MBL producing strains. Two isolates (3%) were positive for blaSIM-1. MBL blaSIM-1 is rarely found in A. baumannii [34]. This is the first report on blaSIM-1 in South Africa. The overall increased occurrence of MBL suggests rapid dissemination of these genes once introduced into the environment.
Overexpression of efflux pumps is known to be an A. baumannii virulence factor and is associated with decreased susceptibility to several antimicrobial agents [35]. The adeABC operon encodes AdeA membrane fusion protein, multi-drug transporter protein AdeB and AdeC outer membrane protein. The AdeR-AdeS; a two-component system, are genes that regulate adeABC operon [36]. It has been reported that constitutive overexpression of the adeABC efflux system is due to either upstream insertion of ISaba-1 on adeABC operon or by a single or multiple-points mutation (SNP) in the adeR and adeS genes [37]. These two conditions induce a decreased intracellular concentration of antimicrobials leading to resistance not only to fluoroquinolones but also aminoglycosides, tetracyclines, chloramphenicol and beta-lactams [38]. In a study conducted on 50 A. baumannii isolates by Lari et al., 16 (32%) isolates were reported to have an active efflux pump after phenotypic testing [37]. Higgings et al., [38] suggested that an increased adeB induced resistance to ciprofloxacin. In contrast, Bratu et al., [39] suggested that the increased adeB expression by itself did not play a major role in fluoroquinolone resistance and that additional mechanisms should be investigated. In our study, the suitable combination (AdeB+; AdeS+ and AdeR+) for a potential active efflux pump was present in 80% of the study isolates. This correlates with the phenotypic results reported by VITEK2. Though the ISaba-1 insertion or SNPs were not investigated in our study, the correlation between potential active efflux pumps and resistance to aminoglycosides; quinolones; cephalosporins and beta-lactams (Table 1 and Table 2) supports the suggestion that active efflux pumps may play a role in the antimicrobial resistance in A. baumannii isolates collected at DGMTL.
Gene burden in the study population
The presence of specific genes in isolates is associated with a specific mechanism of resistance. This exercise gives us a picture of an increasing number of strains of bacteria able to express several genes responsible for various resistance mechanisms. Since the 1970s antimicrobial resistance genes in A. baumannii have progressively increased. By 2007, up to 70% of isolates in certain settings were multi-drug resistant [40]. The increasing occurrence of antimicrobial resistance genes in A. baumannii strains reduces treatment options [17; 41]. In this study, 63 over 70 isolates had more than 3 antimicrobial resistance genes. This is consistent with the increase of resistance globally [3; 42] and MDRAB specifically in our hospital setting.
Study limitation
Despite the findings, this study carries some limitations. The demographic data collected did not include the residential area of patients from whom the samples were collected; this limits extrapolation of results to reflect the general population in South Africa. The study only used a genetic fingerprinting method that is relevant for establishing relatedness locally; a global established genetic typing method would improve the global relevance of strain typing for this study.
Conclusion
After evaluating 100 A. baumannii isolates, the study concluded that isolates collected at DGMTL are multi-drug resistant due to various mechanisms. The major genotypic determinants for drug resistances were for oxacillinases: blaOXA-51 (100%), blaOXA-23 (98%). We report the first blaOXA-40 and blaSIM-1 positive A. baumannii isolates in South Africa. Colistin and tigecycline are the most active antimicrobial agents against A. baumannii isolates encountered at DGMTL. Judicious prescription, routine surveillance to promote early detection of resistance to commonly used antibiotics are needed in order to forestall the menace of multiple drug resistant A. baumannii isolates.
Acknowledgements
We would like to acknowledge the support by SMU research development grant and the NHLS staff that assisted in one way or the other during this study.
Supporting information
S1 Table. The oligonucleotides sequences for PCR
S2 Table. Thermocycling condition used in the study