Abstract: Background and Objective: Throughout the world, antibiotic resistance has been recognized as an emerging major issue of concern. In this study, genes responsible for the production of extended spectrum beta-lactamase (ESBL) in various E. coli isolates were characterized. Materials and Methods: Isolates of E. coli were obtained from clinical and environmental samples. These samples were collected from Cape Coast, Ghana. The isolates were tested for ESBL production. Subsequently, PCR assays of DNA extractions from the isolates were used to determine prevalence of blaTEM and blaSHV genes. Results: Findings revealed that more ESBL producers were phenotypically identified in E. coli isolates recovered from clinical than those recovered from environmental samples. Very low percentages of environmental and clinical isolates that were phenotypically confirmed as non-ESBL producers were found to carry only blaTEM. There was no detection of either gene in majority of the environmental and clinical isolates that were phenotypically confirmed as non-ESBL producers. Conclusion: The environment could be a repository of blaTEM and blaSHV genes thus it is imperative to intensify public health education on the risks associated with the misuse of antibiotics.
INTRODUCTION
In recent decades, the rate at which pathogenic micro-organisms have developed resistance to various drugs is on the ascendency worldwide1,2. Most often, micro-organisms including bacteria acquire resistance through various mechanisms. The release by bacteria of key enzymes known as β-lactamases is a notable example. Essentially, these enzymes break down β-lactam antibiotics. These antibiotics comprise the penicillins, cephalosporins, cephamycins, monobactams and carbapenems. In Gram-negative enteric bacteria, β-lactamase production is a major contributor to resistance to β-lactam antibiotics3. There are over 300 different kinds of β-lactamases that have been well documented. In the past few years, extended spectrum β-lactamases (ESBLs) were identified which can degrade several new β-lactam antibiotics. Examples of such antibiotics with extended spectrum also known as third generation cephalosporins are cefotaxime, ceftriaxone and ceptazidime3. Though ESBLs can degrade the monobactam aztreonam, they are ineffective against cephamycins and carbapenems4. Klebsiella pneumonia, Escherichia coli and to a lesser extent Proteus mirabilis have been found to be the major ESBL producing bacteria worldwide. The ESBLs can mainly be grouped into three: blaTEM, blaSHV and blaCTX-M. Currently, most infections caused by ESBL producing organisms are treated using β-lactam antibiotics in combination with clavulanic acid, an inhibitor of β-lactamases. Most strains of other enteric bacteria including Citrobacter spp. and Enterobacter spp. which are resistant to third-generation cephalosporins produce an AmpC β-lactamase. Typically, AmpC β-lactamases can inactivate cephalosporins and cephamycins, such as Cefotetan and Cefoxitin3. However AmpC β-lactamases are not inactivated by clavulanic acid.
The ESBLs are detected either phenotypically by testing the ability of ESBL enzymes to hydrolyze different cephalosporins or genotypically using molecular techniques to screen for genes responsible for the production of these enzymes5. Phenotypic and genotypic methods are used in many developed countries for routine clinical diagnosis in laboratories and run alongside other tests in automated systems6. In developing countries, such as Ghana however, genotypic tests are yet to be incorporated into antibiotic susceptibility tests that are routinely performed at various hospitals. A few studies however have reported on the prevalence of ESBLs producers among various bacterial isolates recovered from clinical samples in different regions of Ghana7-10. Two key ESBL-producing bacteria: E. coli and Klebsiella spp. have been reported to be multidrug-resistant bacteria to which new treatment options are crucially needed11.
As at now, little information is available on the prevalence of ESBL-producing bacteria isolated from food, water and other environmental samples12. Also, the rising incidence of multi-drug resistance in pathogens such as Staphylococcus aureus, Streptococcus pneumoniae, Pseudomonas aeuroginosa and pathogenic E. coli suggests that antibiotic resistance surveillance must be made as often as possible in Ghana13-16. The present study is a follow-up to an earlier one, which established the presence of E. coli isolates with multidrug-resistance in environmental and clinical samples17. In 2006, a study in Ghana showed high resistance of pathogens to various antibiotics used in the 10 socioeconomic regions of the country18. The rate of emergence of multi-drug resistance in bacteria such as E. coli is on the increase in the Cape Coast Metropolis of Ghana19. Though antibiotics have been very beneficial in the treatment of E. coli infections, the rapid emergence of resistance to these drugs appear to compromise their efficacy. This is what necessitated the present study to determine the occurrence of two of the genes (blaTEM and blaSHV) that confer the extended spectrum β-lactamase-producing trait on E. coli isolates.
MATERIALS AND METHODS
Study site and period: The research was carried out in Cape Coast, Ghana and its environs from June, 2010 to May, 2012. Besides being a coastal town, Cape Coast (5°6" 0" N, 1°15' 0" W) is one of the famous tourist destinations in West Africa owing to its numerous sites of attraction.
Ethical considerations: Ethical approval was obtained from the Ghana Health Service Ethical Review Committee on Research Involving Human Subjects, before the entire research began. The consent of all study participants was obtained prior to collection of clinical samples.
Sample collection: Clinical and environmental samples were collected as previously described17. The clinical samples comprised wound aspirates, stool, urine, and high vaginal swabs while environmental samples comprised drainage canals surrounding the Abura, Kotokuraba and Anaafo markets, the Kakum river and the Fosu lagoon. Other environmental samples such as cabbage heads, fresh beef, fresh chicken and smoked fish were also included.
Bacteria isolation and identification: Samples collected were subjected to bacteria isolation followed by identification as previously described17.
Phenotypic detection and confirmation of ESBL producers among E. coli: All E. coli isolates obtained were screened for their sensitivity to the antibiotics ceftriaxone (30 μg) and cefotaxime (30 μg). Details on antibiotics susceptibility testing methods were earlier described17. All E. coli that recorded clear zones with diameters >5 and >27 mm for ceftriaxone and cefotaxime, respectively, were categorized as potential ESBL producers20. The latter were subjected to a phenotypic confirmatory test using Mastdiscs TM ID (Mast Group LTD., Merseyside, UK) following manufacturers’ instructions. As a quality control measure, reference bacterial strains: Escherichia coli ATCC 25922 (β-lactamase negative) and Klebsiella pneumonia ATCC 700603 (β-lactamase positive) were tested once every week.
DNA extraction and confirmation as bacterial DNA: For every E. coli isolate, 1 mL of fresh overnight culture of bacterial cells (OD600 = 1) growing in Luria-Bertani broth was transferred into a clean 1.5 mL microfuge tube. Using a Wizard Genomic DNA Purification Kit (Promega Corporation, USA) and following manufacturers’ instructions DNA was extracted. It was ensured that all DNA samples were pure before using them for subsequent analysis. The concentration of each DNA sample was standardized to 100 ng μL&–1. All DNA samples obtained were subsequently confirmed as bacterial DNA through PCR assays using the 16S rRNA gene, a universal primer for bacterial DNA21. In a single reaction mixture of 50 μL, 1 μL each of 1 pmol of forward and reverse primers for the 16S rRNA (Table 1) was added to a PCR microtube containing Accupower Multiplex PCR premix (Bioneer Corporation, South Korea). In addition, 46 μL of sterile molecular biology grade water (Sigma-Aldrich, Inc. USA) and 2 μL of 100 ng μL&–1 template DNA were added. The PCR amplification was carried out using a 96 well plate of a Techne TC-512 thermal cycler (Bibby Scientific, UK). The amplification process involved an initial denaturation at 94°C for 3 min; 30 cycles of 94°C for 30 sec, annealing at 55°C for 30 sec followed by extension at 72°C for 1 min and a final extension at 72°C for 10 min.
Detection of ESBL Genes (blaSHV and blaTEM) in clinical and environmental isolates of E. coli : Two separate PCR assays were used for the detection of blaSHV and blaTEM genes, primarily, because the primers used in their detection produced very similar amplified products. A list of primers used in this study is presented on Table 1. Consequently, in a reaction mixture of 50 μL, 1 μL each of 1 pmol of forward and reverse primers for either blaSHV or blaTEM genes were added to a PCR microtube containing Accupower Multiplex PCR premix (Bioneer Corporation, South Korea) together with 46 μL of sterile molecular biology grade water and 2 μL of 100 ng μL&–1 template DNA. The amplification process involved an initial denaturation at 95°C for 15 min; 30 cycles of 94°C for 30 sec, annealing at 56°C for blaTEM or 60°C for blaSHV primers, respectively, for 30 sec followed by extension at 72°C for 30 sec and a final extension at 72°C for 7 min22 (Table 1).
For each PCR product, 10 μL were loaded in each of the wells of 1.5 % (w/v) agarose gel containing 0.001% (w/v) ethidium bromide (Sigma-Aldrich, Inc. USA) and a 100 bp DNA ladder (Sigma-Aldrich, Inc. USA) was used as a molecular weight marker. The PCR amplicons were separated by electrophoresis at 100 V for an hour. Each gel was visualised under an ultraviolet trans-illuminator (UVP products, UK) and photographic records were made using a Canon digital camera S110 12.1 Mega Pixels.
Statistical analysis: Chi-square test (SPSS version 16) was performed to analyze all data obtained at a 5% level of significance.
RESULTS
Distribution of ESBL producers in E. coli isolates from clinical and environmental sources: The phenotypic confirmatory test for ESBL producers using a bacterial lawn of the isolates showed clear zones of varying diameters around each 2nd or 3rd generation cephalosporin (Fig. 1).
The distribution of ESBL producers in all E. coli isolates from clinical and environmental sources is presented in Table 2. There was a significantly (p<0.001) lower percentage of ESBL producers in both clinical and environmental sources as compared to non-ESBL producers.
| Table 1: | Primers used for confirmation of bacterial DNA and detection of ESBL genes |
AT: Annealing temperature, ESBL: Extended spectrum β-lactamase, F: Forward primer, R: Reverse primer | |
| Fig. 1: | Phenotypic confirmation of ESBL production by E. coli isolates using three sets of antibiotics with and without clavulanic acid |
CAZ: Ceftazidime (30 μg), CTX: Cefotaxime (30 μg), CPD: Cefpodoxime (30 μg), CV: Clavulanic acid (10 μg) |
|
| Table 2: | Distribution of ESBL-producing and non-ESBL-producing E. coli strains isolated from clinical and environmental samples |
χ2 = 14.2 (p<0.0001), a: p<0.0001-ESBL producers significantly lower than non-ESBL producers for clinical samples, b: p<0.0001- ESBL producers significantly lower than non-ESBL producers for environmental samples, c: p<0.0001-ESBL producers in clinical samples significantly higher than in environmental samples, d: p<0.0001-Non-ESBL producers in environmental samples significantly higher than in clinical samples | |
| Table 3: | Prevalence of blaSHV and blaTEM genes among ESBL producing and non-ESBL producing-E. coli isolates |
ESBL: Extended spectrum β-Lactamase, +ve: Positive;, -ve: Negative | |
The number of ESBL producers in clinical samples was also significantly (p<0.0001) higher than in the environmental samples. A similar trend was recorded for non-ESBL producers (p<0.0001).
Furthermore, 29.9% of E. coli isolates of clinical origin were found to be ESBL producers. A lower proportion of 12.5% of environmental E. coli isolates were found to be ESBL producers.
Characterization of ESBL producing genes in E. coli isolates from clinical and environmental sources: The DNA extraction resulted in 389 DNA samples with 261 and 128 from clinical and environmental E. coli isolates, respectively (Fig. 2a). An agarose gel picture resulting from a confirmation of DNA extract as bacterial DNA is shown on Fig. 2b.
The results obtained from the characterization of ESBL genes in all 389 E. coli isolates are summarized in Table 3. The single detection of the blaTEM gene among phenotypically confirmed non-ESBL producers was recorded in 5.4 and 4.9% of environmental and clinical E. coli isolates respectively (Table 3). However, 10.8 and 18.6% of non-ESBL environmental and clinical E. coli isolates, respectively, were found to carry only blaSHV. There was no detection of either gene for 12.5 and 5.1%, respectively, of the phenotypically confirmed ESBL-producing isolates obtained from environmental and clinical samples. Among the E. coli isolates phenotypically confirmed as ESBL-producing, 21.8% from clinical samples possessed both blaTEM and blaSHV genes while only 1.1% of the phenotypically confirmed non-ESBL producers from clinical Presence of the blaSHV gene in lanes 1, 2, 3, 5, 7, 8, 11, 12, 13 and 14 and absence in lanes 4, 6, 9 and 10, Lane M: 100 bp ladder samples had both ESBL genes and none for environmental isolates (Table 3).
| Fig. 2(a-b): | (a) Ethidium bromide stained 1% agarose gel showing 15 representative genomic DNA samples. Lane 1-15, genomic DNA extracted from E. coli isolates and (b) Ethidium bromide stained 2.5% agarose gel showing PCR products obtained after confirmation of DNA samples as bacterial DNA. Lane 1, 2, 3, 4, 5 and 6, presence of 16 S rRNA gene; Lane 7, absence of 16 S rRNA gene; Lane M, 20 bp molecular marker |
| Fig. 3: | Ethidium bromide-stained PCR amplification products on a 1.5% agarose gel showing the presence or absence of the blaSHV gene among E. coli isolates |
A representative gel photograph obtained during the screening for the presence of the blaSHV gene in E. coli isolates is shown on Fig. 3. Another representative gel photograph obtained during the screening for the presence of the blaTEM gene in E. coli isolates is also shown on Fig. 4.
DISCUSSION
In this study, among the phenotypically confirmed ESBL producing E. coli isolates, 21.8% of the isolates from clinical samples possessed both blaTEM and blaSHV genes. Phenotypically confirmed non-ESBL producers, however, comprised 1.1% of the clinical isolates whilst none of the Lanes 1, 2, 3, 5, 7, 8, 9, 11, 12, 13 and 14, absence of blaTEM gene, Lanes 4, 6 and 10, presence of blaTEM gene, Lane M: 100 bp ladder environmental isolates had both ESBL genes (Table 3).
| Fig. 4: | Ethidium bromide-stained PCR amplification products on a 1.5% agarose gel showing the presence or absence of the blaTEM gene among E. coli isolates |
The prevalence of ESBL-producing environmental isolates was relatively lower (12.5%) than those of clinical isolates. This indicates clearly that there was some level of multidrug resistance in the environment, suggesting that infections caused by such micro-organisms could be much more difficult to treat. Therefore, the presence of such strains could be a major contributing factor to the rapid emergence of multidrug resistance23. The percentage of the phenotypically confirmed ESBL producers among clinical samples collected in this study was lower (29.9%) than the 44.4% reported in a study conducted in Kumasi9 and 49.3% in Accra8. Even though very useful, the phenotypic methods of detection of ESBL production cannot differentiate between the key enzymes that are responsible5 and this could impede effective health delivery. The expression of extended spectrum β-lactamases (ESBL) as a defense mechanism, by some enteric bacteria, especially Klebsiella spp. and E. coli continues to be a major source of antimicrobial therapy failure23,24 and consequently, has serious repercussions for infection control. There are many types of ESBL including blaTEM, blaSHV, blaCTX and blaOXA. However the majority of them are derivatives of blaTEM and blaSHV enzymes and mostly found in E. coli and K. pneumonia25. Several researchers employ molecular methods for detection of specific genes responsible for ESBL production, which can be used to identify very low levels of resistance that could remain undetected when conventional phenotypic protocols are used26. In the current study, the amplification of the two genes blaTEM and blaSHV using PCR was the method adopted without sequencing of the amplified products. Consequently, 12.5% of environmental and 5.1% of clinical isolates of E. coli phenotypically confirmed were negative for either of the two resistance genes, possibly, because they carried some ESBL genes other than blaTEM and blaSHV. It was also found that 16.2% of environmental isolates and 24.6% of clinical ones phenotypically confirmed ESBL-negative E. coli possessed either blaTEM or blaSHV. However, among the phenotypically confirmed ESBL-negative E. coli isolates, higher proportions of isolates were found to possess blaSHV only than blaTEM only. This suggested that blaSHV was not as good a marker for ESBL detection in E. coli as blaTEM. Similar findings were reported in a study conducted in Pretoria, South Africa, on K. pneumonia isolates27. It is also possible that the phenotypic method used in this study was not sensitive enough to detect the ESBL producers in those cases thereby leading to false negative results as reported in another study28,29. This is a major challenge to phenotypic methods confirming their limitations as diagnostic tools. Thus, phenotypic methods should be backed by the reliable PCR method or other genotypic methods. ESBL genes were identified in environmental E. coli isolates at lower frequencies than in clinical samples. It is however worthy to note that if appropriate measures are not taken, the relatively lower levels of resistance observed in the environment are likely to increase steadily in the next few years. The current study pointed to the urgent need for Ghana to effectively implement the policy on antimicrobial use and resistance signed in 2017. This will go a long way to curb the menace in Ghana as other countries including Germany, have taken pragmatic steps to minimize the rate of emergence of multidrug resistance. Also, for effective diagnosis of bacterial and other microbial infections, there is the need to equip health laboratories with some molecular tools to back phenotypic findings. This should allow for correct prescriptions for effective health delivery. Further research will be conducted to characterize the other types of ESBL prevalent in the country including those encoded by the blaCTX-M gene.
CONCLUSION
The study has demonstrated that, apart from the hospital, the environment could be a reservoir of E. coli carrying blaTEM and blaSHV gene. This calls for the intensification of public education on the risks associated with the misuse of antibiotics.
SIGNIFICANCE STATEMENT
This study reports on the presence of ESBL genes in E. coli naturally found in the environment and also from clinical samples. This is a major public health threat as such genes can get integrated into the community thereby contributing to the rise in cases of unsuccessful treatment and mortality in Cape Coast. The findings form very important contributions, as Cape Coast, the famous coastal town of the West African country Ghana, is a major tourist destination in the sub-region.