E-ISSN: 2814 – 1822; P-ISSN: 2616 – 0668
ORIGINAL RESEARCH ARTICLE
*1Nuhu T. , 1Olawale S.A. and 1Raji M.I.O.
1Department of Pharmaceutical Microbiology and Biotechnology, Faculty of Pharmaceutical Sciences, Usmanu Danfodiyo University Sokoto, Nigeria
* Corresponding author: Nuhu Tanko, nuhu.tanko@udusok.edu.ng, +2348034985445
The emergence of antimicrobial resistance (AMR) is a major threat to global health. Its effects include high mortality and morbidity rates, treatment failure, and increased treatment costs. This study aimed to evaluate the co-production of colistin-resistant and extended-spectrum beta-lactamase (ESBL) genes among Gram-negative clinical isolates from Usmanu Danfodiyo University Teaching Hospital in Sokoto. Gram-negative bacteria were isolated from clinical specimens, including urine, feces, and wound aspirates. The Double-Disk Synergy Test and the Colistin Agar Test, respectively, were used to phenotypically validate the existence of colistin resistance and ESBL. Polymerase chain reaction (PCR) was used for molecular characterization. Primers were used to target genes linked to colistin resistance (mcr-1 and mcr-2) and ESBL genes (blaCTX-M, CTX-M 1, CTX-M 2, and CTX-M 8). The findings indicated that 13.9% of the isolates displayed co-production of Colistin and ESBL, and of these isolates, 60% had blaCTX-M genes, and 20% had CTX-M 8 linked to ESBL production. However, the presence of colistin resistance genes was not detected by PCR. Therefore, molecular analysis did not confirm the existence of the colistin resistance genes (mcr-1 and mcr-2) in these isolates. Consequently, the findings showed no molecular co-production of the ESBL and colistin resistance genes. This work emphasizes how crucial it is to look into molecular and phenotypic traits to completely comprehend how colistin resistance and ESBL genes coexist in Gram-negative isolates. More research is required to investigate other mechanisms behind the resistance phenotypes identified.
Keywords: Colistin resistance, ESBL genes, Co-occurrence resistance, Gram-negative isolates.
According to Aslam et al. (2018), antimicrobial resistance (AMR) is a major worldwide danger to the health of people, animals, and the environment. This is because "superbugs," or drug-resistant bacteria, have become more prevalent and persistent (Davies and Davies 2010). AMR is a major danger to public health worldwide due to the ongoing rise in antibiotic consumption worldwide (Van Boeckel et al., 2014; Laxminarayan et al., 2016). The World Health Organization (WHO) estimates that AMR causes 700,000 deaths worldwide annually, and if the current trend of rising AMR is not stopped, 10 million people could die each year from resistant bacterial infections by the year 2050 (Kupferschmidt, 2016; O'Neill, 2014).
The emergence of resistance in bacterial pathogens, such as nontyphoidal Salmonella, Staphylococcus aureus, Klebsiella pneumoniae, and Escherichia coli, which cause the majority of nosocomial infections and community-acquired infections globally, is a particular cause for concern (WHO, 2014). The World Health Organization is particularly concerned about the emergence and spread of resistance to antibacterial drugs frequently used to treat infections caused by common bacterial pathogens. Examples of these pathogens include E. coli resistance to third-generation cephalosporins and fluoroquinolones, K. pneumoniae resistance to third-generation cephalosporins and carbapenems, S. aureus resistance to methicillin, S. pneumoniae resistance to penicillin, and nontyphoidal Salmonella resistance to fluoroquinolones (WHO, 2014).
Multidrug resistance (MDR) has increased and is now a major global source of morbidity and mortality (Dadgostar, 2019). Antibiotic therapy for bacterial infections may become more limited in the near future due to resistance to antibiotics used as a last resort, such as colistin and newer-generation cephalosporins (Fair & Tor, 2014). Colistin and polymyxin B have been used for preventive and therapeutic purposes in poultry production (Sun et al., 2018).
Colistin is a last-resort antibiotic for treating infections caused by Multidrug-resistant Gram-negative bacteria (Sharma et al., 2022). However, its effective use is seriously threatened by the emergence of resistance mediated by mobile colistin-resistant (mcr) genes (Anyanwu et al., 2021). Even though the clinical significance of the ESBL and mcr genes is well established, there is a knowledge gap in the prevalence and distribution of mcr genes among colistin and ESBL in low-income countries such as Nigeria (Zhang et al., 2021). Compared to infections produced by non-ESBL-producing bacteria of the same species, Rozenkiewicz et al. (2021) report that ESBL-producing bacteria can increase mortality, length of hospital stays, and healthcare expenses.
Regrettably, colistin-resistant infections have spread throughout the world as a result of the overuse and abuse of colistin in human and veterinary medicine. However, the emergence of bacteria resistant to colistin may also happen without prior colistin exposure, leaving medical professionals ill-prepared to treat patients (Mlynarcik & Kolar, 2019). Because they are sometimes the only antimicrobial drug active against multidrug-resistant Gram-negative bacteria (MDR GNB), polymyxins are now mostly effective against life-threatening Gram-negative infections (Kaye et al., 2016). Numerous investigations revealed a sharp rise in the frequency of colistin resistance in Enterobacteriaceae. Because MDR bacteria can evolve colistin resistance through mutation or adaption processes, clinicians need to be on the lookout for this phenomenon. Colistin should only be prescribed as a last option for treating infections, according to the scientific community, specialists, government authorities, and public-private consortiums, who have called for a decrease in the drug's use (Sharma et al., 2022).
The beta-lactamases known as Extended Spectrum Beta-lactamases (ESBLs) provide resistance against oxyimino "second- and third-generation" cephalosporins, such as aztreonam and cefotaxime (CTX), ceftriaxone, cefpodoxime, and ceftazidime (Moawad et al., 2018). Soon after the third-generation cephalosporins, CTX and ceftiofur (CEF), were introduced into clinical settings in 1980, there were reports of ESBL-producing bacteria (Yamasaki et al., 2017). Around 350 ESBL genes have been identified; these genes are primarily produced by point mutations of the classical SHV-1 and TEM-1 Beta-lactamases, with the CTX-M kinds becoming more and more dominant (Brolund & Sandegren, 2016). Over the past ten years, animal-origin E. coli isolates that produce ESBL have made blaCTX-M-55, one of the CTX-M enzymes, the most common kind (Abbas et al., 2019). However, among isolates of human origin, blaCTX-M-15 appears to be the most prevalent type (Woerther et al., 2013).
Additionally, ESBLs are beta-lactamase enzymes that can hydrolyze β-lactam antibiotics, such as Penicillins, Aztreonam, and first to fourth-generation Cephalosporins, excluding Cephamycins, Moxalactam, and Carbapenems, (Rajivgandhi et al., 2021). However, β-lactamase inhibitors such as clavulanic acid, tazobactam, and sulbactam suppress the activity of beta-lactamase enzymes. The most worrisome thing is that, in addition to β-lactams, ESBL production can also lead to resistance to other antibiotics like aminoglycosides, fluoroquinolones, trimethoprim-sulfamethoxazole, and chloramphenicol (Taghizadeh et al., 2018).
Globally, uropathogens that produce ESBLs are becoming an issue (Mahmoud et al., 2020). Consequently, there are significant therapeutic ramifications from co-producing ESBL resistance genes and colistin. Treatment options are severely limited by co-producing isolates, which frequently show resistance to numerous types of antibiotics. This increases the likelihood of treatment failure and unfavorable patient outcomes, posing a serious challenge in managing infections brought on by these strains. Also, understanding the underlying genetic mechanisms of colistin resistance and its spread requires the molecular characterization of colistin and ESBL-resistant genes (Cao et al., 2018). To improve infection control procedures, antimicrobial stewardship, and treatment approaches, it is important to look at the frequency, genetic diversity, and possible transmission pathways of mcr genes in colistin-resistant isolates (Shen et al., 2018). Thus, the need to carry out this research to evaluate the co-production of Colistin resistance and ESBL genes among Gram-negative clinical isolates from Usmanu Danfodiyo University Teaching Hospital, Sokoto, to enhance management and control of drug-resistant infections at the center with the understanding of the incidence and genetic properties of mcr and ESBL genes in Gram-negative isolates.
Gram-negative isolates were obtained from the Medical Microbiology Laboratory, Usmanu Danfodiyo University Teaching Hospital (UDUTH) in Sokoto, Nigeria. The bacteria stock was transferred to the Pharmaceutical Microbiology Laboratory, Faculty of Pharmaceutical Sciences, Usmanu Danfodiyo University, Sokoto, Nigeria, for further analysis.
AST was carried out using Kirby-Bauer’s disk diffusion method. Fifty (50) Mueller Hinton agar plates were prepared by suspending 38g of Mueller Hinton agar in 1000ml of distilled water, then boiled on a hot plate until it dissolved completely, after which it was sterilized in an autoclave at 121°C for 15 minutes. The agar mixture was allowed to cool, after which approximately 20ml each was poured into fifty (50) sterile Petri dishes using an aseptic technique and then left to solidify at room temperature.
Fifty (50) inoculums were prepared using a sterile loop to transfer 2 to 3 isolated colonies of the organism from each quadrant of the subcultured agar plates to test the tubes containing sterile saline. The turbidity was adjusted to match a 0.5 McFarland standard. The agar plates were then inoculated by dipping a sterile swab stick into the broth, and excess moisture was expelled by pressing the swab against the side of the tube. The surface of the agar was then swabbed completely, ensuring that no areas were left un-swabbed. It was then turned 90 degrees, and the swabbing process was repeated, after which the swab stick was run around the plate's circumference before discarding it in the discard bag. This was done for each of the isolates.
The surfaces were then allowed to dry for about 5 minutes, after which a pair of sterile forceps were used to remove the combined antibiotic disk from the dispenser and placed on the agar ensuring it maintained good contact with the agar surfaces. The multi-disk antibiotic containing Amoxicillin Clavulanate (30µg), Cefotaxime (25µg), Imipenem/cilastatin (10/10µg), Nitrofurantoin (30µg), Cefuroxime (30µg), Ceftriaxone sulbactam (45µg), Ofloxacin (5µg), Gentamycin (10µg), Nalidixic acid (30µg), Ampiclox (10µg), Cefixime (5µg), and Levofloxacin (5µg)) was then placed at the surface of the inoculated agar plates. The plates were allowed for a pre-diffusion time of 15 minutes and then incubated upside down at 37°C for 18 – 24 hours. The inhibition zones were measured and interpreted according to CLSI (2021) standards.
The screening test was conducted similarly to the antimicrobial susceptibility test, as described. Disks containing the third-generation cephalosporins ceftazidime (30µg) and cefotaxime (30µg) were used for the screening process, unlike AST.
The double disc synergy test (DDST) was used for clinical samples that passed the screening test to confirm the ESBL phenotype. Amoxicillin/clavulanic acid (20/10µg) and two third-generation cephalosporins, ceftazidime (30µg) and cefotaxime (30µg) were used. Ceftazidime and cefotaxime were added to Mueller Hinton Agar with a sterile needle and positioned 15 mm apart from the center of amoxicillin/clavulanic acid (CLSI, 2021). Following a 24-hour incubation period at 37°C, the plates were inspected for the development of a zone of inhibition extended toward the amoxicillin/clavulanic acid. The development of ESBL is indicated by an enhancement of the zone of inhibition of one or both ceftazidime and cefotaxime towards the amoxicillin/clavulanic acid disc (CLSI, 2021).
The colistin agar dilution method was used for phenotypic detection of colistin resistance. The CLSI (2021) procedure was adopted at 3μg/ml of colistin sulfate. Using a poison balance, a 3 mg powder of colistin sulfate was aseptically weighed and then added to 500 milliliters of distilled water. After suspending 38g of Mueller Hinton agar powder in 500ml of distilled water and boiling it on a hot plate until it dissolved entirely, the mixture was autoclaved for 15 minutes at 121°C before being allowed to cool. After carefully mixing the colistin sulfate stock solution into the agar mixture, 20 milliliters of each was aseptically poured into sterile Petri dishes and allowed to harden at room temperature. Spots measuring about 20 mm in diameter were created on the agar surface using sterile swab sticks dipped into bacterial inoculums that had been prepared and adjusted to the 0.5 McFarland standard. After 18 to 20 hours of upside-down incubation at 35°C, the growth of one or more colonies was regarded as resistant.
Table 1: Primer sequences of mcr and CTX-M
|
|
|
|---|---|---|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
Multiplex PCR was utilized to amplify the DNA. In an Eppendorf tube, 90µl of nuclease-free water and 10µl of the previously reconstituted primers (Table 1) were combined to create working primers. Next, 100 µl of a master mix, 5µl of each working primer, and 60µl of nuclease-free water were combined to create the cocktail. Ultimately, 18µl of the cocktail and 2µl of each extracted DNA were combined to create a final volume of 20µl, which was used to prepare the PCR reaction mixes. "At 94 °C: initial denaturation for 5 min and 25 cycles of denaturation for 30 seconds, then annealing at 52 °C for 30 s, and finally at 72 °C: extension for 30 s and a final extension for 5 min" was the protocol followed when it was transported into the thermocycler for amplification. In order to evaluate the amplicons, agarose gel electrophoresis was used.
A 1.5g agarose powder was weighed, dissolved in 100 ml of Tris base, acetic acid, and EDTA (TAE) buffer, and then put into the water bath for full dissolution after ensuring the electronic digital balance was calibrated. After letting the melted agar cool to around 50°C, 5µl of ethidium bromide was added and mixed with a swirl. After the gel was put into an agarose gel tank and 500 milliliters of 0.5x TBE buffer were added, it was left for 30 minutes to solidify in the broad Bio-rad gel casting device. Subsequently, a 6x loading dye was combined with 10µl of the standard and added to each designated well. The electrophoresis power pack was set to operate at 10 volts for 30 minutes after the tank was closed, and the Biorad gel imager was used to view the gel's results. The outcome was determined by referring to the forward and reverse base pair base pairs.
Of the fifty isolates that were obtained, 45 (90.0%) were from urine samples, 03 (6.0%) from wound swabs, and 02 (4.0%) from stool cultures as shown in Fig. 1. In the meantime, Escherichia coli 39 (78%) was the most isolated Gram-negative pathogen, followed by Pseudomonas spp. 10 (20%) and only one isolate (2%) of Klebsiella spp., according to the frequency and distribution of different bacterial isolates as shown in Fig. 2.
Fig 1: Distribution of the sources of Isolates
Fig 2: Occurrence of the identified Organisms
Gram-negative bacterial isolates have varying degrees of antibiotic resistance, as demonstrated by the antibiotic susceptibility test (AST) shown in Figure 3. Remarkably, Klebsiella species showed complete resistance to gentamicin, nalidixic acid, and cephalosporin. E. coli showed varying degrees of resistance, ranging from 100% to 78% to tested β-lactam antibiotics in Augmentin. Even so, resistance to the remaining antibiotics was least noticeable regarding gentamicin (44%) and the tested fluoroquinolones (54%). Compared to gentamicin, which only showed 40% resistance, imipenem, a β-lactam, revealed an 80% resistance in Pseudomonas species. Concurrently, ceftriaxone plus sulbactam showed increased cephalosporin sensitivity; this was explained by the possibility that sulbactam inhibits beta-lactamase. Compared to other antibiotics, cephalosporins with beta-lactamase inhibitors, like ceftriaxone with sulbactam and fluoroquinolones, showed greater susceptibility.
Fig. 3: Resistant profiles of the isolates from different sources
Screening for ESBLs Producers
Of the 30 isolates, 38.8% (14) were found to be resistant to both cephalosporins and another 77.7% (n=28) to only cefotaxime, while 44.4% (n=16) isolates show resistance to only ceftatxidime. The screening test revealed that 83.3% (30) were resistant to at least one of the 2 third generation cephalosporins and are potential ESBL producers (Fig. 4).
Fig. 4: Assessment of suspected ESBL producers
Phenotypic Detection of ESBL Producers
According to the phenotypic test, 23.3% (n=7) of the suspected ESBL producers had negative phenotypic results, whereas 76.7% (n=23) of the suspected ESBL producers were found to be positive for ESBL production (Fig. 5). 23 Gram-negative bacterial isolates were shown to be ESBL producers phenotypically out of all the isolated organisms (Fig. 5).
Fig. 5: Assessment of suspected ESBL producers for ESBL production
Phenotypic Detection of Colistin Resistance
As illustrated in Fig. 6, of the 36 isolates, 22.2% (n=8) were resistant to colistin. P. aeruginosa did not exhibit any resistance, and all resistant isolates (100%) were E. coli.
Fig. 6: Phenotypic result for colistin resistance
Evaluation of clinical isolates for phenotypical co-production of colistin and ESBL resistance
Based on the colistin and ESBL phenotypic data, 5.9% of the isolates coproduced colistin and ESBL resistance, whereas 86.1% (31) did not coproduce. (Figure 7).
Fig. 7: Percentage of phenotypic co-producers
Gel image of PCR product for CTX-M
As shown in Fig. 8, four of the five isolates, or eighty percent (80%), have ESBL genes (three CTX-M at 550 bp and one CTX-M 8 at 490 bp).
Fig. 8: Gel image showing the presence of three CTX-M and one CTX-M 8 genes
Amplification bands (500 bp) were obtained from only three of the five isolates (60%), and these bands did not match the predicted sizes for the mcr-1 (307) and mcr-2 (1747) genes, respectively (Fig. 9).
Fig. 9: Gel image of colistin-resistant DNA showing bands at 500bp
A high occurrence of resistance was noted based on the results at different points in the investigation. According to Abbas et al. (2019), the majority of Gram-negative bacteria pathogens in this investigation were E. coli (78%), which is consistent with several earlier results from Nigeria, including those from Sokoto state (Nuhu et al., 2015, Tanko et al., 2020). The antibiotic susceptibility test (AST) results indicated that resistance to aminoglycoside, cephalosporins, fluoroquinolones, and penicillin was highly prevalent. This resistance pattern is in line with findings from a number of studies, including those by Omoya and Ajayi (2016), Nyandwi et al. (2017), and Udoh et al. (2020), which reported high resistance rates to Augmentin, cephalosporin, and fluoroquinolones among bacterial isolates, particularly E. coli and Klebsiella species. It is also compatible with research conducted in Sokoto, Northeastern Nigeria, by Adeluola et al. (2018) and Olowo-Okere et al. (2018), as well as Nuhu et al. (2015).
According to Mohammed et al. (2016), Ugwu et al. (2020), and Olufunke et al. (2014), there was a high prevalence of ESBL producers (76.7%) in this study as well, which is in contrast to research done in Kano, Northwestern Nigeria, where ESBL prevalence was 9.25%, 27.7% in Southeastern Nigeria, and 26.1% in Southwestern Nigeria. This is, however, in line with studies by Peculiar-Onyekere et al. (2019) in Nnewi, Nigeria, which revealed a high frequency of 67.7% for ESBL-mediated UTIs in pregnant women.
Additionally, among ESBL-producing Enterobacteriaceae isolates, CTX-M-2, CTX-M-8, and CTX-M-9 groups were revealed to be the most often discovered CTX-M-type enzymes (Chagas et al., 2011). This is consistent with the study, which found that the most common subtypes were CTX-M-2 and CTX-M-8. However, the CTX-M, CTX-M-1, CTX-M-2, and CTX-M-8 subtypes are the only ones we address in this study. Additionally, phenotypic results for colistin revealed that 22% of the isolates were resistant to the drug. Vounba et al. (2019), Arif et al. (2022), and Ngbede et al. (2020) reported similar results in Vietnam, where P. aeruginosa had a 20% resistance rate to colistin, 22.8% in Pakistan among Gram-negative uropathogens, and 17.0% in Nigeria. This, however, differs from a study by Li et al. (2023) that was carried out in China and found a prevalence of 53.47%.
Furthermore, a study conducted in Oyo State, Nigeria, discovered that 84% of clinical isolates of E. coli and Klebsiella spp. Obtained from blood samples of outpatients and inpatients were colistin-resistant (Hassen et al., 2022), whereas 43.6% of resistance was reported from Lagos (Egwuatu et al., 2021). It was observed phenotypically that there was colistin and ESBL resistance co-production, as 13.9% (N=5) of the isolate were co-producers. Nevertheless, there was no co-production at the molecular level after molecular characterization. Only three of the five isolates produced 500 bp amplification bands for colistin, and those bands did not match the predicted sizes for the mcr-1 (307) and mcr-2 (1747) genes. Four of the five isolates (80%) displayed the presence of ESBL genes (three CTX-M and one CTX-M 8). The differences that have been found show that the bacterial community is genetically diverse. It's possible that the primers' targets, the mcr-1, mcr-2, and CTX-M genes, are absent from the two isolates lacking bands in colistin and one in ESBL. There could be other resistance genes or genetic variations in these isolates that were missed by the particular primers used. Resistance can develop through a variety of mechanisms. Reports of isolates resistant to colistin but lacking the mcr genes have surfaced. For instance, colistin-resistant Enterobacteriaceae isolates from several sources were examined in a study by Olaitan et al. (2014). Several isolates lacked the known mcr genes yet exhibited phenotypic resistance to colistin. Rather, whole-genome sequencing identified mutations in genes related to the manufacture or modification of LPS, resulting in changed outer membrane characteristics and decreased susceptibility to colistin.
Cannatelli et al. (2013) found isolates of Klebsiella pneumoniae resistant to colistin and did not have the mcr-1 gene in another investigation. These isolates' genome sequencing revealed gene changes linked to the two-component regulatory system pmrAB, which changed LPS and boosted colistin resistance (Olowo-Okere & Yacouba, 2020). These instances demonstrate the presence of mechanisms for colistin resistance that go beyond the identified mcr genes. When examining isolates that show phenotypic resistance but lack the mcr genes, it is crucial to consider other resistance mechanisms. When the primers used in the PCR reaction bind to unwanted DNA sequences, non-specific amplification may result. Larger bands that might not be connected to the important colistin resistance genes may be produced due to the amplification of non-target areas of the genome.
High primer concentrations, poor primer design, or non-specific primer binding due to sequence similarities with other genome parts can all contribute to non-specific amplification. Contamination during the PCR process may introduce extraneous DNA templates, which could lead to the formation of unexpected bands. A number of things, including reagents, equipment, and past PCR reactions, can contaminate an area. The bands that were seen might have been caused by contamination with 500 base pairs or fewer DNA fragments.
Additionally, research has shown that colistin resistance genes, such as mcr-1 and mcr-2, exhibit genetic diversity. For instance, Cannatelli et al. (2013) discovered an allelic variation of the PmrB sensor kinase in an Escherichia coli strain that causes colistin resistance. When employing particular primers, this genetic diversity can lead to varied amplicon sizes, particularly if insertions or deletions are in the target area. Olaitan et al. (2016) found that one isolate of Escherichia coli carried the mcr-1.2 gene variant, which is a variation of the mcr-1 gene. A 484-base pair insertion resulted in the mcr-1.2 gene having a greater amplicon size than the original mcr-1 gene. This demonstrates how different gene variants can exist within the same family of colistin-resistance genes. Numerous researches have also reported on gene rearrangements or insertions involving colistin resistance genes.
For example, Olaitan et al. (2014) found that the inactivation of the PhoP/PhoQ regulator mgrB led to the global establishment of colistin resistance in Klebsiella pneumoniae. Genetic insertions and rearrangements involving mgrB were identified as the cause of the inactivation. When PCR primers target certain genetic events, the amplicon sizes may increase. In a study published in, Borowiak et al. 2017 found that isolates of Escherichia coli carried a novel colistin resistance gene called mcr-3.3. Compared to the mcr genes that were previously reported, such as mcr-1 and mcr-2, the amplicon size of the mcr-3.3 gene was greater. This discovery indicates more colistin resistance genes with various amplicon sizes. Therefore, the bigger bands seen in these isolates may also point to the existence of novel, as yet uncharacterized, colistin resistance genes. These genes have extra genetic elements or mutations that lead to a bigger amplicon size, yet they may have some sequence similarity with mcr-1 or mcr-2. It would take more research to locate and describe these new genes, such as whole-genome sequencing or gene-specific PCR. In another study, Xavier et al. (2016) looked at colistin-resistant Escherichia coli isolates that exhibited resistance traits but lacked the mcr-1 or mcr-2 genes. After more investigation, they discovered mcr-3, a novel plasmid-mediated colistin resistance gene that shared tight ties with mcr-1. This indicates that there are more colistin-resistance genes that traditional PCR techniques might overlook.
It's possible that the bacterial strains under study separately acquired the genes causing ESBL and colistin resistance. This implies that diverse processes, such as horizontal gene transfer from several sources or mutations happening in various genetic locations, may have contributed to the acquisition of the resistance genes. According to this hypothesis, colistin and ESBL resistance do not directly result from co-production; their co-occurrence is accidental.
In the absence of co-production, a number of investigations have documented the presence of ESBL resistance genes and colistin. For instance, a study by Smith et al. (2017) examined the genetic profiles of Enterobacteriaceae that produced ESBL and discovered that certain strains had colistin resistance and ESBL genes, but the genes were not co-produced or physically connected. The lack of co-production at the molecular level may also be explained by genetic variations between the strains or populations that are being studied. The co-occurrence or co-expression of these resistance genes may not be encouraged by the genetic backgrounds of the ESBL- and colistin-resistant bacteria. Variations in the genetic background, regulatory components, environmental factors, or other factors influencing the expression of these resistance determinants may cause this. The coexistence of colistin and carbapenem resistance in clinical isolates of Klebsiella pneumoniae was examined in a study by Liakopoulos et al. (2016). They discovered that although there was evidence of both resistance mechanisms, co-production was not evident. According to the researchers, the lack of co-production was likely driven by variations in the strains' genetic backgrounds. It is feasible for isolates to show phenotypic co-production resistance to both ESBL and colistin without disclosing the known colistin resistance genes, according to the literature that is currently available. Further research must determine whether the 500 base pair bands are real or PCR artifacts. It is also important to consider the potential that novel colistin resistance genes or gene variants/subtypes could explain the bigger bands seen in this study.
More research is required to discover and describe these genes and their possible involvement in colistin resistance, such as functional studies and genetic sequencing. These findings further demonstrate the intricacy and diversity of colistin resistance pathways and their possible implications for clinical practice and public health.
The study reveals high-level resistance to fluoroquinolones, cephalosporins, aminoglycosides, and penicillin by Gram-negative bacteria isolates from Usmanu Danfodiyo University Teaching Hospital, Sokoto, and a high percentage (76.7%) of the isolates produced ESBL and had several CTX-M genes (CTX-M and CTX-8). The study detected colistin and ESBL co-production phenotypically; however, PCR did not detect co-production. These results imply that novel or alternate co-existing resistance mechanisms facilitate the isolates' co-production of resistance.
Abbas, G., Khan, I., Mohsin, M., Sajjad-ur-Rahman, Younas, T., & Ali, S. (2019). High rates of CTX-M group-1 extended-spectrum Beta-lactamases producing Escherichia coli from pets and their owners in Faisalabad, Pakistan. Infection and Drug Resistance, 571–578. [Crossref].
Adeluola AO, Oyedeji KS, Mendie UE, Johnson JR, Porter JR. (2018). Detection, inhibition and molecular analysis of multidrug resistant aerobic gram-negative clinical isolates from a tertiary hospital in Nigeria. Afr J Biomed Res., 21(1):15–21. https://www.ajol.info/index.php/ajbr/article/view/165960
Anyanwu, M., Okpala, C., Chah, K., & Shoyinka, V. (2021). Prevalence and traits of mobile colistin resistance gene harbouring isolates from different ecosystems in africa. Biomed Research International, 2021, 1-20. [Crossref].
Arif A., Ullah I., Ullah O., and Zaman R. (2022). Identification of colistin resistance and its bactericidal activity against uropathogenic gram negative bacteria from Hayatabad Medical Complex Peshawar. Pak J Med Sci. 38(4Part-II): 981–986. [Crossref]
Aslam, B., Wang, W., Arshad, M.I., Khurshid, M., Muzammil, S., Rasool, M.H., Nisar, M.A., Alvi, R.F., Aslam, M.A., Qamar, M.U., Salamat, M.K.F., and Baloch, Z. (2018). Antibiotic resistance: a rundown of a global crisis. Infection and Drug Resistance, 11: 1645–1658. [Crossref]
Borowiak, M., Fischer, J., Hammerl, J. A., Hendriksen, R. S., Szabo, I., and Malorny, B. (2017). Identification of a novel transposon-associated phosphoethanolamine transferase gene, mcr-5, conferring colistin resistance in d-tartrate fermenting Salmonella enterica subsp. enterica serovar Paratyphi B. Journal of Antimicrobial Chemotherapy, 72(12), 3317-3324. [Crossref]
Brolund, A., and Sandegren, L. (2016). Characterization of ESBL disseminating plasmids. Infectious Diseases, 48(1), 18–25. [Crossref]
Cannatelli A, Giani T, Aiezza N, et al., 2013. An allelic variant of the PmrB sensor kinase responsible for colistin resistance in an Escherichia coli strain of clinical origin. Antimicrob Agents Chemother. 57(10): 4900-4902.
Cao, L., Li, X., Xu, Y., and Shen, J. (2018). Prevalence and molecular characteristics of mcr-1 colistin resistance in Escherichia coli: Isolates of clinical infection from a Chinese University Hospital. Infection and Drug Resistance, 1597–1603. [Crossref]
Chagas T.P.G., Alves R.M., Vallim D.C., Seki L.M., Campos L.C. and Asensi M.D. (2011). Diversity of genotypes in CTX-M-producing Klebsiella pneumoniae isolated in different hospitals in Brazil. The Brazilian Journal of Infectious Diseases; 15(5): 420-425. [Crossref]
CLSI - Clinical and Laboratory Standards Institute (2021). Performance Standards for Antimicrobial Susceptibility Testing. 31st ed. CLSI Supplement M100, Wayne, PA., USA.
Dadgostar, P. (2019). Antimicrobial resistance: Implications and costs. Infection and Drug Resistance, 3903–3910. [Crossref]
Davies, J., and Davies, D. (2010). Origins and evolution of antibiotic resistance. Microbiol Mol Biol Rev. 74(3):417–433. [Crossref]
Egwuatu T.O., Ishola O.D. and Oladele O.E. (2021). The distribution of extended-spectrum Beta-lactamase genes in fomites, healthcare workers, and patients from two hospitals in Lagos State, Nigeria. Ife Journal of Science; 23(2): 015-024. [Crossref]
Fair, R. J. and Tor, Y. (2014). Antibiotics and bacterial resistance in the 21st century. Perspectives in Medicinal Chemistry, 6, PMC-S14459. [Crossref]
Hassen B., Hammami S., Hassen A., and Abbassi M.S. (2022). Molecular mechanisms and clonal lineages of colistinresistant bacteria across the African continent: a scoping review. Letters in Applied Microbiology; 75: 1390-1422. [Crossref]
Kaye, K. S., Pogue, J. M., Tran, T. B., Nation, R. L., and Li, J. (2016). Agents of last resort: Polymyxin resistance. Infectious Disease Clinics, 30(2), 391–414. [Crossref]
Kupferschmidt, K. (2016). Resistance fighters. Science, 352(6287): 758–761. [Crossref]
Laxminarayan, R., Matsoso, P., Pant, S., Brower, C., Røttingen, J, Klugman, K, Davies, S. (2016). Access to effective antimicrobials: a worldwide challenge. The Lancet, 387(10014): 168-175. [Crossref]
Li, D., Ge, Y., Wang, N., Shi, Y., Guo, G., Zou, Q., & Liu, Q. (2023). Identification and characterization of a novel major facilitator superfamily efflux pump, sa09310, mediating tetracycline resistance in Staphylococcus aureus. Antimicrobial Agents and Chemotherapy, 67(4). [Crossref]
Liakopoulos, A., Mevius, D. J., Olsen, B., and Bonnedahl, J. (2016). The Colistin Resistance Mcr-1 Gene is Going Wild. J. Antimicrob. Chemother. 71(8): 2335–2336. [Crossref]
Mahmoud AT, Salim MT, Ibrahem RA, Gabr A and Halby HM. (2020). Multiple Drug Resistance Patterns in Various Phylogenetic Groups of Hospital-Acquired Uropathogenic E. coli isolated from Cancer Patients. Antibiotics, 9(3): 108. [Crossref]
Mlynarcik, P., and Kolar, M. (2019). Molecular mechanisms of polymyxin resistance and detection of mcr genes. Biomedical Papers of the Medical Faculty of Palacky University in Olomouc, 163(1). [Crossref]
Moawad, A. A., Hotzel, H., Neubauer, H., Ehricht, R., Monecke, S., Tomaso, H., Hafez, H. M., Roesler, U., and El-Adawy, H. (2018). Antimicrobial resistance in Enterobacteriaceae from healthy broilers in Egypt: Emergence of colistin-resistant and extended-spectrum β- lactamase-producing Escherichia coli. Gut Pathogens, 10, 1–12. [Crossref]
Mohammed Y., Gadzama GB., Zailani SB. and Aboderin AO. (2016). Characterization of Extended-Spectrum Beta-lactamase from Escherichia coli and Klebsiella Species from North Eastern Nigeria. J Clin Diagn Res.; 10(2): DC07–DC10. [Crossref]
Ngbede E.O, Poudel A., Kalalah A., Yang Y., Adekanmbi F., Adikwu A.A., Adamu A.M., Mamfe L.M., Daniel S.T., Useh N.M., Kwaga J.K.P., Adah M.I., Kelly P., Butaye P., and Wang C. (2020). Identification of mobile colistin resistance genes (mcr-1.1, mcr-5 and mcr-8.1) in Enterobacteriaceae and Alcaligenes faecalis of human and animal origin, Nigeria. International Journal of Antimicrobial Agents; 56(3): 1-24. [Crossref]
Nuhu, T., Bolaji, R. and Olayinka, B. (2015). Prevalence and antimicrobial susceptibility of extended Spectrum Beta-lactamase (ESBL) producing gram-negative uropathogens in Sokoto, Nigeria. Nigerian Journal of Pharmaceutical Research, 11, 59-65.
Nyandwi, E., Veldkamp, A., Amer, S. Karema C. andUmulisa I. (2017). Schistosomiasis mansoni incidence data in Rwanda can improve prevalence assessments, by providing high-resolution hotspot and risk factors identification. BMC Public Health 17 (845): 1-14. [Crossref]
Olaitan AO, Diene SM, Kempf M, et al., 2014. Worldwide emergence of colistin resistance in Klebsiella pneumoniae from healthy humans and patients in Lao PDR, Thailand, Israel, Nigeria, and France owing to inactivation of the PhoP/PhoQ regulator mgrB: an epidemiological and molecular study. Int J Antimicrob Agents. 44(6):500-507. [Crossref]
Olaitan, A. O., Chabou, S., Okdah, L., Morand, S., and Rolain, J. M. (2016). Dissemination of the mcr-1 colistin resistance gene. The Lancet Infectious Diseases, 16(2), 147-149. [Crossref]
Olowo-Okere, A. and Yacouba, A. (2020). Molecular mechanisms of colistin resistance in Africa: a systematic review of literature. Germs, 10(4), 367-379. [Crossref]
Olowo-Okere, A., Ibrahim, Y. K. E. and Olayinka, B. O. (2018). Molecular characterisation of extended-spectrum β-lactamase-producing Gram-negative bacterial isolates from surgical wounds of patients at a hospital in North Central Nigeria. Journal of global antimicrobial Resistance, 14, 85-89. [Crossref].
Olufunke O. A., Aregbesola O.A., and Fashina C.D. (2014). Extended spectrum betalactamase- producing uropathogenic Escherichia coli in pregnant women diagnosed with urinary tract infections in southwestern Nigeria, J. Mol. Bio. Res., 4(1): 34–42. [Crossref].
Omoya FO and Ajayi KO. (2016). Synergistic Effect of Combined Antibiotics against Some Selected Multidrug Resistant Human Pathogenic Bacteria Isolated from Poultry Droppings in Akure, Nigeria. Advances in Microbiology, 6(14): 1075-1090. [Crossref].
O'Neill, J. (2014). Antimicrobial resistance: tackling a crisis for the health and wealth of nations, Review on antimicrobial resistance, http://archive.wphna.org/wp-content/uploads/2015/06/2014-UK-paper-on-superbugs-projected-to-2050.pdf
Peculiar-Onyekere O.C., Martina C.A. and Emmanuel A.E. (2019). ESBL Mediated Antimicrobial Nonsusceptibility of Uropathogenic Escherichia coli and Klebsiella pneumoniae Isolates from Pregnant Women in Nnewi, Nigeria. Journal of Advances in Microbiology; 18(2): 1-13. [Crossref]
Rajivgandhi GN, Alharbi NS, Kadaikunnan S, Khaled JM, Kanisha CC, Ramachandran G, Manoharan N and Alanzi KF. (2021). Identification of carbapenems resistant genes on biofilm forming K. pneumoniae from urinary tract infection. Saudi Journal of Biological Sciences; 28(3): 1750-1756. [Crossref]
Rozenkiewicz D, Esteve-Palau E, Arenas-Miras M, Grau S, Duran X, Luisa Sorlí L, Montero MM and Horcajada JP. (2021). Clinical and Economic Impact of Community-Onset Urinary Tract Infections Caused by ESBL-Producing Klebsiella pneumoniae Requiring Hospitalization in Spain: An Observational Cohort Study. Antibiotics, 10(585): 1-10. [Crossref]
Sharma, J., Sharma, D., Singh, A., and Kumari, S. (2022). Colistin resistance and management of drug resistant infections. Canadian Journal of Infectious Diseases and Medical Microbiology, 2022, 1-10. [Crossref]
Shen, Y., Wu, Z., Wang, Y., Zhang, R., Zhou, H.-W., Wang, S., Lei, L., Li, M., Cai, J., and Tyrrell, J. (2018). Heterogeneous and flexible transmission of mcr-1 in hospital-associated Escherichia coli. MBio, 9(4), 10–1128. [Crossref]
Smith, M., et al. (2017). Molecular mechanisms of co-resistance to colistin and carbapenem in Klebsiella pneumoniae: An in-depth analysis of a high-risk clone. Scientific Reports, 7(1), 1-11. [Crossref]..
Sun, J., Zhang, H., Liu, Y.H., and Feng, Y. (2018). Towards understanding MCR-like colistin resistance. Trends in Microbiology, 26(9), 794–808. [Crossref].
Taghizadeh E, Abdollahi E and Nikkhah M. (2018). TP53 hotspot mutations in astrocytoma. Health Biotechnology and Biopharma, 2(1): 38-45. [Crossref].
Tanko N., Bolaji R.O., Olayinka A.T., Olayinka B.O. (2020). A systematic review on the prevalence of extended-spectrum beta lactamase-producing Gram-negative bacteria in Nigeria. J Glob Antimicrob Resist; 22: 488-496. [Crossref].
Udoh, R.H., Tahiru, M., Ansu-Mensah, M., Bawontuo, V., Danquah, F.I., and Kuupiel, D. (2020). Women’s knowledge, attitude, and practice of breast self-examination in sub-Saharan Africa: A scoping review. Archives of Public Health, 78(84): 1-10. [Crossref]
Ugwu, M., Shariff, M., Nnajide, C., Beri, K., Okezie, U., Ifeanyichukwu, I., and Esimone, C. (2020). Phenotypic and molecular characterization of Beta-lactamases among enterobacterial uropathogens in southeastern nigeria. Canadian Journal of Infectious Diseases and Medical Microbiology, 2020, 1-9. [Crossref]
Van Boeckel, T.P., Gandra, S., Ashok, A., Caudron, Q., Grenfell, B.T., Levin, S.A., Laxminarayan, R. (2014). Global antibiotic consumption 2000 to 2010: an analysis of national pharmaceutical sales data. Lancet Infect. Dis., 14(8): 742-750. [Crossref]
Vounba P., Rhouma M., Arsenault J., Bada Alambédji R., Fravalo P. and Fairbrother J.M. (2019). Prevalence of colistin resistance and mcr-1/mcr-2 genes in extended-spectrum β-lactamase/AmpC-producing Escherichia coli isolated from chickens in Canada, Senegal and Vietnam. J. Glob. Antimicrob. Resist., 19: 222–227. [Crossref]
WHO (2014). Antimicrobial resistance: Global report on surveillance. WHO press, World Health Organization, Geneva, Switzerland, pp. 1-8.
Woerther, P.L., Burdet, C., Chachaty, E., and Andremont, A. (2013). Trends in human fecal carriage of extended-spectrum Beta-lactamases in the community: Toward the globalization of CTX-M. Clinical Microbiology Reviews, 26(4), 744–758. [Crossref]
Xavier, B. B., et al. (2016). Identification of a novel plasmid-mediated colistin resistance gene, mcr-2, in Escherichia coli. Eurosurveillance, 21(27): 30280. [Crossref]
Yamasaki, S., Le, T. D., Vien, M. Q., Van Dang, C., and Yamamoto, Y. (2017). Prevalence of extended-spectrum β-lactamase-producing Escherichia coli and residual antimicrobials in the environment in Vietnam. Animal Health Research Reviews, 18(2): 128–135. [Crossref]
Zhang, S., Abbas, M., Rehman, M. U., Wang, M., Jia, R., Chen, S., Liu, M., Zhu, D., Zhao, X. and Gao, Q. (2021). Updates on the global dissemination of colistin-resistant Escherichia coli: An emerging threat to public health. Science of The Total Environment, 799, 149280. [Crossref]