PREVALENCE OF  BLATEM, BLASHV, AND BLACTX-M GENES AMONG ESBL-PRODUCING ESCHERICHIA COLI ISOLATED FROM THE BLOOD SAMPLES OF ICUS PATIENTS OF UNIVERSITY HOSPITALS IN SANA'A CITY, YEMEN

Ghamdan Ahmed Ali Al-Tahish1 , Eshtiaq A  Al-Yosaffi2 , Arwa Mohammed Othman2 , Hassan Abdulwahab Al-Shamahy2 , Ahmed Mohammed Al-Haddad3 ,

Khaled Abdulkarim Al-Moyed2,4 , Abdul-Al-Raoof Mohammad Al-Shawkany5 

1Microbiology and Immunology Department , Faculty of Laboratory Medicine, 21 September University. 

2Medical Microbiology and Clinical Immunology Department, Faculty of Medicine and Health Sciences, Sana’a University.

3Department of Medical Laboratories, College of Medicine and Health Sciences, Hadhramout University, Al-Mukalla.

4University of 21 September for Medical and Applied Sciences, Sana’a, Yemen.

5Molecular Genetics Department, College of Veterinary Medicine, Sana`a University, Yemen.

 

ABSTRACT

Aim and Objective: With the emergence of organisms such as Enterobacteriaceae that produce extended-spectrum β-lactamase (ESBL), which are resistant to multiple medications (multidrug-resistance), concerns about how best to treat infections have significantly increased. The current study examined the molecular features of ESBL in clinical isolates of Escherichia coli that resulted in bloodstream infections as well as the pattern of antibiotic resistance to gather useful data on the infection's epidemiology among Yemeni ICU patients.

Subjects and methods: A cross-sectional study was conducted on sepsis patients admitted in intensive care units at four hospitals in Sana'a, Yemen, between January, 2021 and April, 2022. Blood cultures were used on patients suspected of having sepsis. Standard laboratory procedures were then used to isolate and identify possible bacterial infections, and the disk diffusion method was used to test for microbial susceptibility.  All strains were tested for Extended Spectrum Beta-Lactamase (ESBL) production using the Modified Double Disc Synergy Test (MDDST). Following analysis, polymerase chain reaction, β-lactamase genes (blaTEM, blaSHV, and blaCTX-M) were identified.

Results: The results of the conventional PCR experiment revealed that 33.3% of the blaCTX-M genes, 0.0% of blaSHV, and 100% of blaTEM were present in the strains of ESBL-producing E. coli that were collected. It was discovered that the E. coli isolates' patterns of antibiotic resistance to 23 different antibiotics differed greatly. The bulk of the E. coli isolates were found to be multi-drug resistant (MDR). Furthermore, MDR characteristics were observed in 85% of E. coli isolates.

Conclusion: Control and surveillance of antibiotic resistance depend on an understanding of the resistance genes and patterns of antimicrobial resistance of bacterial pathogens within a given geographic area. The current study's findings showed that MDR was very common. According to the current study's findings, TEM was significantly more common than other ESBL gene types.

Keywords: blaCTX-M, blaSHV, blaTEM, Blood stream infections (BSIs), ESBL, Escherichia coli, ICUs, Multi-drug resistant (MDR).

 

INTRODUCTION

 

Numerous nosocomial illnesses worldwide have been documented to have bacteria from the Enterobacte-riaceae family as their causative cause.  Given the limited therapy options resulting from the organisms' ongoing rise in antibiotic resistance, infections caused by bacilli Enterobacteriaceae are challenging to control. Indeed, one of the most well-known resistance mechanisms in Gram-negative bacilli was first described by Ojdana et al.1, and involves ESBLs.  A class of enzymes known as ESBLs increases the resistance to cephalosporins, penicillins, related β-lactams, ceftazidime, and acetaminophen-olactams, however clavulanic acid inhibits ESBLs1. The three primary categories of ESBLs are TEM, SHV, and CTX-M. The rapidly growing CTX-M family, which is now more common than SHV and TEM, is found in a wide variety of clinically significant bacteria and over large geographic regions2.  Treatment plans become more complicated because organisms that produce ESBL frequently show resistance to antibiotics from other classes, such as quinolones, aminoglycosides, and sulfonamides3. Furthermore, members of the Enterobacteriaceae family, like Escherichia coli, frequently produce ESBLs; yet, it has recently been shown that some additional enzymes may be present in other Enterobacteriaceae family genera. It was initially discovered that patients in European intensive care units who had extended hospital stays exhibited a greater degree of resistance in these organisms. Nevertheless, isolates were found in South and North America, Africa, Asia, the Middle East, and Africa, and ESBL GNB quickly spread around the world4.  

Common ESBL genes that code for E. Coli isolates were identified as TEM (discovered and isolated in the early 1980s from Teminora, a Greek patient), and CTX-M (cefotaximase that preferentially hydrolyzes cefotaxime). There are occasional descriptions of these transposon- plasmid- and chromosome-mediated genes all over the world5.

Every year, the rates of bacterial resistance rise, raising concerns around the world. For this reason, it is crucial to understand susceptibility patterns because improper empirical antimicrobial therapy can lengthen hospital stays and increase mortality rates, both of which can be prevented with the right therapy6.  Acquiring additional PBPs in sensitive to ß-lactam or changing the normal PBPs are known as the commonest cause of resistance in cocci such as MRSA and pneumococci which are gram positive. However, a mixture of endogenous acquired ß-lactamases with natural efflux and up-regulated impermeability is the main reason for resistance in the gram-negative bugs7. It should be mentioned that there are well-written materials demonstrating how standard disc diffusion tests are unable to identify the development of ESBLs. There might not be enough resources in labs to stop the spread of these resistance mechanisms since many clinical laboratories do not completely understand the importance and detection technique of ESBLs8.

There are many different types of ESBLs, such as SHV, TEM, OXA, CTX, AmpC, and so on; however, the majority of them are derived from the SHV, TEM, and CTX-M enzymes, which are most frequently present in E. coli. In light of this, the current work examined the presence of blaSHV, blaCTX-M, and blaTEM genes in isolates of E. coli from the bloodstream of intensive care unit patients at tertiary hospitals in Sana'a, Yemen, in order to ascertain the prevalence of the ESBL phenotype.

 

SUBJECTS AND METHODS 

 

Isolates of bacteria: Twenty consecutive non-duplicate E. coli isolates were recovered from blood culture specimens of ICU patients suffering from sepsis.  A cross-sectional study was conducted on sepsis patients admitted in intensive care units at four hospitals in Sana'a, Yemen, between January, 2021 and April, 2022. Blood cultures were performed on patients suspected of having sepsis, and possible bacterial infections were subsequently isolated and identified using conventional laboratory procedures9.  To identify the isolates, standard microbiological methods were used. Additionally, they were re-identified using the VITEK 2 compact system (BioMerieux, France).

Ethic approval: All of the techniques employed in this study were authorized by the research and ethics committee of the Faculty of Medicine and Health Sciences at Sana'a University, Sana'a, Yemen (Approval No. UGR/SU-223).

Antimicrobial susceptibility testing: The isolates were screened using the disc diffusion method (Kirby-Bauer disc diffusion method) on Mueller-Hinton agar (MHA) plates in accordance with the criteria of the Clinical and Laboratory Standards Institute (CLSI) to determine their antibiotic susceptibility [10].  Amoxicillin+Clavulanic acid (20+10μg), Amikacin (10μg), Azithromycin (15μg), Cefotaxime (30μg), Ceftazidime (30μg), Cefazoline (30μg), Cephradin (30μg), Cefoxitin (30μg), Cefuroxime (30μg), Ceftriaxone (30μg), Cefoperazone (30μg), Cefepime (30μg), Co-Trimoxazole (25μg), Ciprofloxacin (10μg), Imipenem (10μg), Gentamicin (10μg), Meropenem (10μg), Norfloxacin (10μg), Moxifloxacin (10μg), Piperacillin (100μg), and Tobramycin (10μg).

Testing for production of ESBL (MDDST): A disc containing four cephalosporins (Ceftriaxone, 3GC-Cefotaxime, 4GC Cefepime, and Cefpodoxime) and amoxicillin-clavulanate (20/10 μg) was used in the Modified Double Disc Synergy Test (MDDST) to assess each strain's ability to produce Extended Spectrum Beta-Lactamase (ESBL). On a Mueller-Hinton agar plate, a lawn culture of the organisms was established in accordance with CLSI guidelines10.  Placing a disc in the center of the plate held 20/10 μg of amoxicillin-clavulanate. The amoxicillin-clavulanate disc's center was positioned 15 mm and 20 mm from the center of the 3GC and 4GC discs, respectively11.  Any expansion or deformation in the zone toward the disc of amoxicillin and clavulanate was considered indicative of ESBL development. The combined disc test was used to validate ESBL production in accordance with CLSI recommendations.

Detection of ESBL genotypes by multiplex PCR amplification:  Multiplex PCR was used to check for the presence of blaSHV, blaCTX-M, and blaTEM genes in the isolates that tested positive for ESBL production in the first screening test13. This approach was somewhat modified from that used by Monstein et al.12.  Using a PrestoTM Mini gDNA bacterial kit, freshly cultivated isolates of bacteria were utilized to prepare template deoxyribonucleic acid (DNA). 0.2 units/μl Ampliqon Taq DNA polymerase, 0.4 mM of each dNTP, 0.4 μM of each primer, and 2 μl DNA template (density of 10 ng/μl) were used in all PCR reactions. The Master Mix included 20 mM Tris-HCl pH 8.5, 0.2% Tween® 20, 3 mM MgCl2, and (NH4)2S04. The following settings were made for the polymerase chain reaction amplification: a 10-minute main denaturation step at 95°C; thirty denaturation cycles at 94°C for 30 seconds; annealing at 60°C for 30 seconds; an extension step at 72°C for two minutes; and a final extension step at 72°C for ten minutes. Size separation PCR amplicons were used in conjunction with agarose gel electrophoresis to identify the corresponding genes.

 

RESULTS 

 

Twenty successive non-duplicate E. coli isolates were recovered in total, and tests were performed on their antimicrobial resistance profile against 23 distinct antimicrobial drugs. The current results revealed that E. coli isolates vary widely to different antimicrobials. The resistance rates of isolates of E. coli against the selected 23 antimicrobial agents obtained from blood of ICU patients. It was found that a majority of the E. coli isolates were resistant to several drugs (multi-drug resistant: MDR) where a total of 17/20 (85%) of E. coli isolates indicated MDR phenotypes. Furthermore, the results of the antimicrobial susceptibility test against E. coli revealed that E. coli showed 45% resistance to Amoxicillin+Clavulanic acid (Table 2), whereas susceptibility to ciprofloxacin decreased to 35%. The highest sensitivity rate of E. coli was for aminoglycosides classes were it was 95% for amikacin and 90% forgentamicin. Whereas, the highest resistant rate was for the 1st, 2nd, 3rd and 4th generations of Cephalosporins β-lactam class (80%, 90%, 95%, 90%, 95%, 85% and 100%, respectively) (Table 2). 

Co-trimoxazole with resistance rates 70%, and 70% respectively.  Out of the 20 E. coli isolates, a total of 6 isolates (30%) showed positive results in initial screening test of ESBL production by MDDST and phenotypic confirmatory test of ESBL production. In PCR detection of ESBL genotypes, it was found that all of the ESBL screening positive E. coli isolates had one or more ESBL genes that were tested in the present study. Overall, 30% (6/20) of E. coli isolates were positive for one or more ESBL genes. The multiplex PCR assay results indicated that 100% blaTEM genes, 33.3% blaCTXM and blaSHV genes were not detected in the E. coli isolates.

 

DISCUSSION

 

Antimicrobial resistance in pathogenic bacteria is a global concern that is associated with elevated rates of morbidity and mortality. Furthermore, infections have been reported to be difficult or impossible to treat with traditional antimicrobials due to multidrug resistance patterns. Antibiotics are widely, generously, and usually needlessly utilized because many healthcare facilities fail to identify the underlying bacteria and their patterns of antimicrobial sensitivity in a timely manner in patients with bacteremia and other serious diseases13. High prevalence of MDR E. coli isolates was found in the blood clinical samples used in the current study. MDR traits were seen in 85% of the E. coli isolates overall. Thirty percent of the E. coli MDR isolates were ESBL producers. Unlike the findings of Bora et al.14, which stated that 75% of E. coli isolates produced ESBL. The findings of the test of antimicrobial susceptibility against E. coli in the current investigation showed that the susceptibilities of the isolated bacteria to the tested antimicrobials varied.

Antimicrobial susceptibilities patterns were identified in all isolates. Similar findings were reported by Tabar et al.3, and Liao et al.15.  Amino glycosides and carbapenems are frequently the last effective treatments for infections caused by MDR Enterobacteriaceae16. Imipenem and Meropenem, which have been reported to be the most effective antibiotic, including the isolates that create ESBLs, showed 100% sensitivity, according to previous investigations. Given that carbapenemes can be used to treat a variety of infections, this is a significant finding of the current study. This outcome may be explained by the fact that these antibiotics are less common in this area due to their higher cost.

According to Paterson et al.17, ESBL producers are inherently resistant to all cephalosporins, even if they exhibit an in vitro susceptibility.  In the current investigation, the percentage of ESBL-producing E. coli from all-isolated samples was 6 (30%), while the percentage of non-ESBL-producing E. coli was 14 (70%). In fact, people in hospitals all across the world are said to struggle with ESBLs. Additionally, it has been noted that the prevalence rates of ESBLs among clinical isolates vary widely across the globe and that these rates rapidly fluctuate over time18. It is imperative to develop laboratory testing methods to properly determine the presence of such enzymes in clinical isolates, given the rising incidence of Enterobacteriaceae that produce ESBLs19. Modified double disc synergy tests were the most sensitive of all the ESBL detection techniques20.  Similar results were found in a study by Khan et al.21, which showed that 6/20 isolates had positive MDDST results and that 6/20 isolates had positive double disk synergy test (DDST) results. Thirty percent of E. coli isolates were screened for ESBL production by adhering to the MDDST screening criteria. The presence of one or more ESBL genes in every isolate that passed screening indicated that E. coli isolates that produce ESBL are quite common in the area that is being studied. Kaur et al.22 found that 63.4% of E. coli isolates from India produced ESBL.

The subtypes of ESBL cannot be identified by phenotypic testing, which only validate the production of ESBL. Nüesch-Inderbinen et al.23, observed that although molecular approaches have been shown to be sensitive, they are expensive, time-consuming, and require specialist equipment. Only molecular detection techniques are likely to lead to the final identification. According to a research by Navon-Venezia et al.24, the performance of these phenotypic tests must be regularly assessed because the introduction of new enzymes may alter it.  Grover et al.25, reported that PCR is a dependable technique for ESBL identification in their investigation of phenotypic and genotypic ESBL detection strategies. The multiplex PCR amplification assay was used in this study to identify the blaCTX-M, blaSHV, and blaTEM genes in the E. coli clinical isolates that were recovered. This assay has the advantage of allowing for the quick screening of a large number of clinical isolates, and it can also be used to isolate DNA for use in subsequent molecular epidemiological studies if necessary12. Furthermore, a trustworthy epidemiological study into antibiotic resistance requires the identification of beta-lactamase. In the current investigation, E. Coli isolates taken from the bloodstream of ICU patients clinically suspected of having blood sptecimia in Sana'a, Yemen, were surveyed for antimicrobial drug resistance, ESBL phenotypes, and the identification of blaSHV, blaTEM, and blaCTX-M genes.

Compared to SHV and TEM ESBLs, CTX-M-type ESBLs proved to be the most prevalent kind of ESBL worldwide, with a greater prevalence in most locales26,27.  The study revealed that out of the three ESBL genotypes examined, blaTEM 6/6 (100%) and bla CTX-M 2/6 (33.3%) were the most commonly occurring in E. coli isolates that produced ESBL. The blaSHV was the least common ESBL genotype, with a prevalence rate of 0% in ESBL-producing E. coli isolates. Similar results were found in studies carried out in Iraq by Manoharan et al.28, and Pishtiwan and Khadija26.  However, TEM ESBL was the most common genotype in our investigation, whereas blaSHV-type ESBL was less common. Since just a small sample size of strains from Sana'a city were gathered and analyzed for this study, it is considered that geographical variances are the cause of the discrepancy.

Moreover, earlier research conducted in Yemen has revealed that E. coli resistance to third-generation cephalosporins is common there29-38. Furthermore, the outbreak reached 50% in Egypt and Syria39, over 70% in Iraq26, and so forth.  Moreover, 5-15% of participants in our sample showed susceptibility to all cephalosporins; this incidence is higher than that found in other Arabian countries26,28,39. The study revealed that of the three ESBL genotypes examined, blaTEM [6/6 (100%)] and CTX-M [2/6 (33.3%)] were the most commonly occurring in E. coli isolates that produced ESBL. Naturally, based on the aforementioned findings, a number of investigations by Teawtrakul et al.40, Girmenia et al.41, Ricciardi et al.42, and Devrim et al.43, have demonstrated that different nations have different rates and types of Escherichia strains identified. These results demonstrate the need for organized national programs in the area that focus on antimicrobial supervision and infection prevention.

Limitations of the study

The following were the study's limitations. Initially, we were unable to precisely identify the kinds of isolates and their pattern of antibiotic sensitivity for Yemen since the data only came from one place (Sana'a city). Confirming the true prevalence of bacterial resistance genes requires molecular study on a large sample size of isolates.

 

CONCLUSION

 

Knowledge of the antimicrobial resistance patterns and resistance genes of bacterial pathogens in a geographical area is important for control and surveillance of antibiotic resistance. The results of the present study revealed that MDR was highly prevalent. In addition, the carbapenems and amino glycosides were found to be the most active antimicrobial agents in vitro. Based on the results obtained in the present study, TEM was highly prevalent among other types of ESBLs genes.

 

ACKNOWLEDGEMENTS 

 

For their collaboration and support, we thank and appreciate all the medical staff at Kuwait, Al‑Jumhuri, Al-Sabeen, and Al-Thawra hospitals in Sana'a. We also acknowledge Sana’a University's assistance.

 

CONFLICT OF INTEREST 

 

This work does not include any conflicts of interest. 

 

AUTHOR’S CONTRIBUTIONS

 

Eshtiaq A. Al-Yousafi, the study's second author, conducted the fieldwork as part of his PhD studies at Sana'a University's Faculty of Medicine and Health Sciences' Department of Medical Microbiology. Additional authors contributed to the data analysis, the writing, reviewing, and final approval of the work.

 

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