Prevalence of extended-spectrum beta-lactamase-producing Enterobacteriaceae among clinical isolates in Turaif general hospital, northern borders-Saudi Arabia

Introduction: Enterobacteriaceae that produce extended-spectrum beta-lactamase (ESBL) are quickly spreading, posing a threat to world healthcare. Methodology: 138 gram-negative bacteria were collected from different samples (stool, urine, wound, blood, tracheal aspirate, catheter tip, vaginal swab, sputum, and tracheal aspirate) from hospitalized patients. Samples were subcultured and identified in accordance with their biochemical reactions and culture characteristics. Against all the isolated Enterobacteriaceae , an antimicrobial susceptibility test was performed. VITEK®2 system, phenotypic confirmation, and Double-Disk Synergy Test (DDST) had been utilized to identify the ESBLs. Results: Of the 138 samples studied, the prevalence of ESBL-producing infections among the clinical samples of the present study was 26.8 % (n = 37). E. coli was the commonest ESΒL producer at 51.4% (n = 19) followed by K. pneumoniae at 27% (n = 10). The potential risk factors for the ESBL development that produces bacteria were as follows, patients with the presence of indwelling devices, previous history of hospital admission, and usage of antibiotics. ESBL is statistically ( p ≤ 0.05) higher among the patients with indwelling devices, ICU admission, who had a previous hospital admission in the last 6 months as well as who was given antibiotics (quinolones and/or cephalosporins) in the last 6 months. One hundred thirty-two (95.7%) of ESBL isolates were resistant to amoxicillin, while the lowest resistance was for fosfomycin (15.2%). Conclusions: ESBL -producing Enterobacteriaceae are highly prevalent in Turaif General Hospital setting with some potential risk factors. A strict policy to be made available on the usage of antimicrobials in hospitals and clinics should be established.


Introduction
Bacterial antimicrobial resistance (AMR) is a serious threat to public health. Several reports about the increasing risk of infection caused by antibioticresistant bacteria could increase the mortality rate by 2050 worldwide [1,2].
A high level of bacterial resistance which is responsible for different and frequent types of infections in several countries had been reported by The World Health Organization (WHO) Global Antimicrobial Surveillance System (GLASS) [3]. A group of bacteria recently identified and reported by the WHO including AMR bacteria are divided into three main categories according to the impact of these bacteria on the health of human beings and the urgent need for new antimicrobials to face this problem. These categories are critical, high, and medium priority [4][5][6].
Extended-spectrum β lactamase (ESBL) producing Enterobacteriaceae are a bacteria group that lies under the critical category. Their infection is associated with severe infections and even may be fatal [2]. ESBLproducing Enterobacteriaceae spread rapidly, which poses a challenge to global healthcare. ESBL has over three hundred variants, making it easy to spread to many world regions with a continued upward trend [7,8]. Healthcare workers are highly concerned with the significant upward trend of ESBL organisms caused by gene mutation [9].
The ESBL-producing feature is the ability of the microorganism to synthesize a B-lactamases. This is a family of enzymes that have efficient hydrolytic activity against B lactam class of antibiotics such as penicillin and cephalosporins. This resistance is acquired by plasmid-mediated mutation encoding for the parent enzymes, either by amino acid substitution in the active site such as the case for Temonieara (TEM; named after patient Temoniera) or sulfhydryl reagent variable (SHV; class A) enzymes or by inter bacteria gene transfer like the case of cephalosporinases (class C enzymes) [10,11].
The Enterobacteriaceae family's TEM-1, TEM-2, and SHV-1 genes are highly mutant, altering the configuration of amino acids [12]. Thus, this confines the enzyme's capability to hydrolyze a wider spectrum of beta-lactam antibiotics, such as penicillin monobactams and oxyimino cephalosporins. Additionally, the enzymes mediated by plasmids are susceptible to beta-lactamase inhibitors. [13].
There are extensive reports of ESBL-producing organisms globally. A 1983 report by Haller et al. [5] confirmed the first outbreak of ESBL made in Europe, particularly Germany, the United Kingdom, and Sweden. The role of ESBL-producing organisms as primary agents in transmitting nosocomial infections has been demonstrated by statistics. The widespread of the ESBL enzymes like CTX-M beta-lactamases had resulted in an endemic situation in the Middle East, South America, and Europe. Confirmed by evidence that ESBL causes primary infections in Saudi Arabia associated with high mortality and morbidity rates [6,7].
In Saudi Arabia, ESBL-producing bacteria are spreading at an alarming rate. Statistics show that ESBL has a 38% upward projection yearly of ESBLproducing Enterobacteriaceae in Saudi Arabia. The central region reported a high frequency of ESBL, and the eastern region reported the lowest frequency rate [9]. A study by Alqasim et al. indicates that ESBLproducing E coli in Riyadh resulted in a high spread of urinary tract infections (UTIs) at the rate of 7% in 2020 [9]. Sfeir et al. added that 20% of Enterobacteriaceae resist beta-lactam antibiotics when treating ESBL infections.
Ciprofloxacin, gentamicin, and trimethoprim-sulfamethoxazole are the least effective drugs in treating ESBL. It is necessary to change ESBL treatment because of the highly resistant nature of ESBL-producing Enterobacteriaceae . This report provides an in-depth analysis of the prevalence of ESBL-producing Enterobacteriaceae and recommends efficient control measures for its prevention [14]. This study aimed to determine the prevalence of Enterobacteriaceae in clinical samples obtained from Turaif General Hospital in Northern Saudi Arabia, screening the antibiotics profile against the most used antimicrobials, and calculating the prevalence rates of ESBL among isolated samples.

Design and setting
This cross-sectional study was conducted in the microbiology lab of the clinical analysis department at Turaif General Hospital in Northern Saudi Arabia from the period between January to June 2021.

Subjects
A non-probability sequential sample was taken from various clinical sites of infection from hospitalized patients in various wards considering the following measures; age, sex sample types, infection clinical signs, hospital admission duration, antibiotic therapy history) in the last 6 months (cephalosporins and quinolones), indwelling devices, such as an intravenous catheter(IV), urinary catheter, wound drains, orthopedic prosthesis, central venous pressure(CVP) catheter and endotracheal tubes (ventilator), causes of disturbed systemic or local immune status, such as old age (> 50 years), malignancy, diabetes mellitus, burn, presence of surgical sutures or pressure ulcers, ICU admission and previous hospital admission in the last six months.

Inclusion criteria
The infection signs and symptoms became apparent after > 48 hours following hospital admission, such as purulent discharge, turbid urine, and Chest X-ray (CXR) consolidation; especially for patients who had indwelling medical devices, such as an IV catheter, urinary catheter, wound drains, orthopedic prosthesis, central venous pressure (CVP) catheter and endotracheal tubes (Ventilator); with local or systemic manifestations of infections associated with the indwelling devices.
Exclusion criteria 1. Pre-admission infection presence (proved by the history and clinical examination on admission).

Infection symptoms appeared within the first 48
hours after the hospital admission. 3. Patients who were not willing to cooperate.

Ethical approval
Bioethical approval was obtained from the local committee of bioethics (LCBE) of Jouf University, Saudi Arabia, (LCBE No: 02-07-42), and from the Saudi Ministry of Health, National Ethics Committee, Northern Borders Region No (H-09-A-51).

Sample collection and transport
Several samples were collected from different clinical sites of infection, such as mid-stream urine, suction tip, pus, and blood specimens.
All the participants provided written consent. In accordance with the standard microbiological methods in the microbiology laboratory of Turaif General Hospital, all the samples were collected, and in accordance with the manufacturer's instructions, all the media included in this study were prepared.
On nutrient, blood, MacConkey's medium, and Cystine-lactose-electrolyte-deficient agar (CLED) agar (Oxoid Ltd, Basingstoke, Hampshire, UK), all the samples were directly cultured. Media were prepared according to the manufacturer's instructions. Blood samples were cultured overnight at 37 °C in brain heart infusion broth (Oxoid Ltd Basingstoke, Hampshire, UK). A drop of the inoculated broth was then cultured on MacConkey and blood agar and incubated at 37 °C. Colony morphology, gram-stained films, and biochemical reactions had been utilized to identify grown colonies.

Identification and susceptibility tests
By the usage of the automated biomérieux VITEK® 2 compact system, identification, and susceptibility had been carried out. For the identification of gram-negative bacteria, GN cards with different substrates had been utilized. For detection of antimicrobial susceptibility, antibiotic susceptibility card AST-N91 containing antibiotics for detection of ESBL (cefotaxime, cefotaxime with clavulanic acid, ceftazidime, and ceftazidime with clavulanic acid) had been utilized.
ESBL-producing Enterobacteriaceae detection involves two steps, the first is a screening cephalosporin test that looks for resistance or decreased sensitivity, hence identifying isolates that are likely to host ESBLs, and the second is a confirmation test that evaluates the synergy between an oxyimino cephalosporin and clavulanic acid, allowing isolates with ESBLs to be distinguished from those that are resistant for other reasons.

ESBL Screening
Susceptibility of Enterobacteriaceae isolates to different antimicrobial agents was tested by the method of disk diffusion which was also had been utilized as a screening test for the ESBLs production by noting specific zone diameters (around B lactam disks) that indicate a high level of suspicion for ESBL production (for ceftazidime and cefotaxime zone diameter ≤ 14mm while for aztreonam ≤ 15mm) according to the CLSI guidelines [15].

Phenotypic confirmatory test
For phenotypic confirmation of the presence of ESBL ceftazidime (30 µg) and cefotaxime (30 µg) alone or in combination with clavulanic acid (10 µg) were used. If the increase in zone diameter ≥ 5 mm for either of the cephalosporin discs and their respective cephalosporin / clavulanic acid disc were interpreted as ESBL producers [15].
The Double-Disc Synergy Test (DDST) Disk containing cephalosporins cefotaxime (30μg) and ceftazidime (30μg) was placed next to a disk containing clavulanic acid (amoxicillin/ clavulanic acid). After overnight incubation at 37 °C isolates were considered as ESBL producing bacteria when the inhibition zone of the disk of the 3 rd generation cephalosporins is increased towards the amoxicillin /clavulanic acid disk or when the single antibiotic disc is not inhibitory but inhibitory in case of the two antibiotics are combined [16].

Statistical analysis
On a compatible personal computer, the SPSS program (Computer Statistical Package for Social Scientists) (SPSS, Version 22.0. Inc., Chicago, IL, USA) in Windows 10.0 had been utilized. Descriptive variables were presented as frequency and percentages. To identify the potential risk factors for ESΒL organism development, a regression analysis was performed. An odds ratio (OR) that does not include a null value and a p value is considered statistically significant when it measures less than 0.05.

Results
This study lasted six months, from January to June 2021. During the six months, different samples were collected from 138 patients with clinically suspected nosocomial infections, from different departments of Turaif General Hospital, Northern Borders-Saudi Arabia.
Regarding the potential risk factors for infection with ESBL Enterobacteriaceae in our study, the risk is statistically higher (p ≤ 0.05) among the patients with indwelling devices, ICU admission, who had a previous hospital admission in the last 6 months as well as who were given antibiotics (quinolones and /or cephalosporins) in the last 6 months ( Table 2).
In accordance with the Clinical Laboratory Standard Institute (CLSI) guidelines, the total of the isolated Enterobacteriaceae was screened by the method, disk diffusion. Out of which, 37 (26.8%) were found to suspected ESBL produces, 101 (73.2%) gave negative results. All the isolates also were subjected to a double disk synergy test. 33 (23.9%) were positive and 105 (76.1%) were negative. 37 (26.8%) were positive by the VITEK® 2 compact system and 101  (73.2%) were negative. Only 4 isolates were not detected by the double disk synergy test. The methods for ESBL detection were compared in this study. High agreement was found between VITEK® 2 system and screening by disk diffusion method with a statistically non-significant difference between the three methods used (Table 4).

Discussion
The WHO celebrates "World Antimicrobial Awareness Week" every year from the 18 th -the 24 th of November. This celebration aimed to increase awareness about antibiotic resistance and better use of antibiotics among the public, healthcare workers, and other stakeholders [17]. Also, the WHO has initiated global integrated surveillance and approach to handle the ESBL-producing E. coli [18]. The above actions and approaches of the WHO reinstates the importance of managing ESBL-producing organisms in hospitals and other healthcare settings. The purpose of this study was to determine the ESBL prevalence-generating organisms and their risk factors in Turaif general hospital on the northern border of the KSA. This study also attempted to find the pattern and resistant status of commonly used antimicrobials in the same hospital.
The prevalence and incidence rate of ESBLs producing organisms has vast differences around the world [19][20][21][22]. These differences are attributed to the types of healthcare settings, availability of healthcare facilities, knowledge, attitude, and practice of antibiotics by the public and healthcare workers, and so on [23]. The lowest prevalence is reported in European and North American countries (4.6% to 7.5%), while the highest prevalence is reported in Asian countries, especially Southeast Asian countries (29% to 51.3%) [12,24].
The present study revealed that ESBLs producing organisms were significantly higher among the patients with indwelling devices (IV catheter, urinary catheter, wound drains, orthopedic prosthesis, CVP catheter, and endotracheal tubes), ICU admission, previous history (within last 6 months) of hospital admission and usage of antibiotics (quinolones and cephalosporins). Few studies done around the world in the past also found a similar potential risk factor for the ESBL-producing organism's development [25,26]. In addition to the above risk factors and in contrast to our study, some of the authors have found increasing age, male gender, and disturbed immune status are significantly the potential risk factors [27][28][29][30]. These differences are due to various reasons such as the types of samples included in their study, settings in which the research was conducted, and so on.
The present study revealed E. coli was the commonest ESBLs-producing organism (51.4%) followed by K. pneumoniae (27.0%). Most of the studies around the world also revealed that E. coli is the most common ESBL-producing organism and consist of more than half of all ESBL-producing organism [15,17,18,35,36]. In a study by Hameed et al., a high prevalence rate of B lactamase-producing K. pneumoniae was observed [37]. Different studies reinstate the importance of the WHO's initiation of the global integrated surveillance and approach to handling the ESBL-producing E. coli [11].
The methods for ESBL detection were compared in this study. High agreement was found between VITEK® 2 compact system and screening by disk diffusion method with a statistically non-significant difference between the three methods used. We recommend that screening for ESBL production by disk diffusion should be joined to routine culture and susceptibility testing especially when rapid results and low costs are needed.
Antimicrobial resistance is one of the biggest global challenges for the healthcare system. More than 2.8 million people get antibiotic-resistant infections and about 35 thousand people due to antibiotic-resistant infections in the USA alone, stated by the Centers for Disease Prevention and Control (CDC), USA [38]. This states the importance of the prevention and control of antimicrobial resistance. The present study found that highly resistant bacterial strains were found against a majority of the commonly used antibiotics. The highest resistance was present with amoxicillin (95.7%), while the lowest resistance was present with Fosfomycin (15.2%). Several studies done in the hospital setting around the world and KSA found similar findings [39,40]. But the studies done by some of the authors in the community settings have found lower levels of resistance against these commonly used antibiotics [41,42].
Despite the best efforts and with the use of the standard methodology in the present study, certain limitations need to be considered while interpreting the results. Firstly, this study was a cross-section study design, and it finds only the association between potential risk factors, not the causations. Secondly, some of the details were self-reported. Hence, the limitations related to self-reported data are to be considered such as time distortion, recall bias, and subjective base. Finally, this study was done in a single center (Turaif general hospital) of one province (northern border region) of the KSA. Hence, the findings of this study may not reflect the entire region of the KSA.
The high prevalence and distribution of ESBLs among different strains reflect the rapid dissemination of plasmids encoding ESΒLs among distinct strains and genera, which is generated by antibiotic selection pressure. This prevalence poses a problem that requires urgent application of strict infection control measures, restriction of the use of oxyimino-cephalosporins, and antibiotic cycling and /or switching to different classes of antibiotics

Conclusion and Recommendations
Our study results suggest that ESBL-producing organisms are highly prevalent in Turaif general hospital setting. The potential risk factors for the development of ESBL-producing organisms were patients with the presence of indwelling devices, ICU admission, previous history of hospital admission, and usage of antibiotics. Also, highly resistant bacterial strains were found against the majority of the commonly used antibiotics. Hence, effective programs such as the formation of an active surveillance team, diagnostic, and antibiotic stewardship are to be activated in the hospital.
A strict policy to be made available on the usage of antimicrobials in hospitals and clinics should be established. Continuous review of the progress of the newly instituted programs to be done. This can be done through regular auditing from internal and external stakeholders. Furthermore, a multicentric study should be done in the KSA to find the national-level prevalence of ESBLs producing organisms and their risk factors.
This high prevalence of ESBL-producing Enterobacteriaceae poses a problem that requires the urgent application of strict infection control measures restriction of the use of oxyimino-cephalosporines and antibiotic cycling and /or switching to different classes of antibiotics