Introduction

ESBL-producing Enterobacterales (ESBLE) present a significant clinical challenge due to their capacity to generate extended-spectrum β-lactamases, enzymes capable of breaking down a broad spectrum of β-lactam antibiotics. These bacteria, containing species like Escherichia coli, Klebsiella pneumoniae, and Proteus mirabilis, have acquired genes encoding ESBLs, resulting in resistance to commonly utilized antibiotics such as penicillins and cephalosporins [1]. Multidrug-resistant Enterobacterales (MDRE) strains commonly show resistance to widely used antibiotics, including penicillin’s, cephalosporins, fluoroquinolones, aminoglycosides, and sometimes carbapenems, which are considered last-resort antibiotics [2]. The rise of colistin resistance in Enterobacterales constitutes a growing concern in healthcare settings worldwide. Colistin resistance mechanisms often involve chromosomal mutations and the acquisition of mobile genetic elements carrying resistance genes [3,4,5].

Infections caused by ESBLE and MDRE are linked to increased morbidity, mortality, and healthcare spending, causing prolonged hospitalizations and the use of more costly antibiotics [6].

ESBLE contains various genes conferring resistance to antimicrobial agents, with common ones including CTXm, TEM, and SHV. MDRE strains frequently carry genes encoding resistance mechanisms against other antibiotic classes, such as aminoglycosides, fluoroquinolones, and carbapenems. Examples include genes encoding aminoglycoside-modifying enzymes (e.g., AAC, ANT), fluoroquinolone resistance determinants (e.g., qnr genes), and carbapenemases (e.g., KPC, NDM, OXA) [7].

On a global scale, ESBL-positive E. coli (23.7%) and K. pneumoniae (35.1%) predominantly contained the CTXm-15 variant (E. coli: 53.9%; K. pneumoniae: 80.0%), with the highest incidence observed in the Africa/Middle East (AfME) region [8].

The prevalence of MDRE, particularly those carrying extended-spectrum β-lactamases, constitutes a significant challenge in the West Bank, Palestine. In the Gaza Strip, ESBL prevalence ranges from 35 to 54% [9]. The prevalent genes include CTXm, found in 45–60% of cases, followed by TEM and SHV genes, with prevalence rates ranging from 16.8 to 57.6% and 5.2–38.3%, respectively [9]. Conversely, research on ESBL prevalence in the other region of Palestine, the West Bank, is limited. One study reported a prevalence of 38% [10], while another found that ESBL-producing E. coli accounted for 62.8% of healthcare-acquired multidrug-resistant gram-negative bacilli [5]. Moreover, there have been no studies characterizing the phenotypic and genotypic traits of Enterobacterales in the West Bank. Despite efforts to combat antimicrobial resistance, the exact burden and genetic composition of ESBL-producing strains remain poorly understood. This research aims to address this knowledge gap by conducting a comprehensive phenotypic and genotypic assessment of ESBL-producing and multidrug-resistant Enterobacterales in the West Bank.

Materials and methods

Study design and setting

A cross-sectional study was conducted from October 1st to October 31st, 2023, on clinical bacterial isolates collected from five governmental Palestinian hospitals situated throughout the West Bank. These hospitals include Martyr Khalil Suleiman Hospital (MKSH) in Jenin, Rafidia Surgical Hospital (RSH) in Nablus, both located in the northern region of the West Bank. Palestinian Medical Complex (PMC) is positioned in the city of Ramallah, centrally within the West Bank. Additionally, Al-Hussain Hospital is situated in Beit Jala, and Alia Governmental Hospital (AGH) is located in Hebron, situated in the southern part of the West Bank.

Sources of bacterial isolates

The research was conducted on 600 isolates chosen at random from a pool of 1380 isolates collected from patients during October 2023. Bacterial isolates were collected from both female and male patients (367(61%) and 233/600 (39%), respectively). The patients from whom the samples were collected range in age from one to 92 years old. Bacterial isolates were cultivated from different patient samples: 364 urine samples, 120 wound samples, 68 blood samples, 45 sputum samples and 3 CSF samples.

Microbiological methods

Bacterial identification and antimicrobial susceptibility testing

All collected isolates (600 isolates from1380 patients) were analyzed at the microbiology labs of the participating hospitals. The VITEK® 2 Compact system from bioMérieux, France, was used to identify all isolates. Bacterial identification utilized VITEK 2 GN cards, while susceptibility testing for both beta-lactam and non-beta-lactam antibiotics was carried out using VITEK 2 GN AST 204 and 417 cards. These antibiotic panels are suitable for testing Enterobacterales isolates in line with the 2023 guidelines of the Clinical Laboratory Standards Institute (CLSI) [11].

All bacterial isolates were sent to the microbiology laboratory of An-Najah National University for confirmation of ESBLE and CPE phenotyping, genotyping, and testing for colistin resistance. The Double Disc Synergy Test (DDST)was employed to retest all ESBL-producing bacteria identified by the VITEK 2 Compact system. Antibiotics were used for DDST namely: Amoxicillin-Clavulanic acid (20/10 µg), Ceftriaxone (30 µg), and Cefotaxime (30 µg). At center Amoxicillin-Clavulanic acid disc was placed and these discs were placed at a distance of 1.5 cm. Development of the zone of inhibition towards the Clavulanate disc at 37 °C after 24 h. incubation was indicative of a potential ESBL-positive organism, this involved placing antibiotic discs containing ceftriaxone 30 µg and cefotaxime 30 µg, with and without amoxicillin clavulanic acid 20/10µg, on a Mueller Hinton agar plate [12].

We classified bacterial isolate as MDRE when they showed resistance to at least one antibiotic in three distinct classes of antibiotics. Results were interpreted following the CLSI 2023 guideline [11], and E. coli American type culture collection (ATCC) 25,922 was utilized as a quality control for antibiotic disc potency.

Phenotypic carbapenemases detection

The modified carbapenem inactivation method (mCIM) was conducted on all CRE and ESBLE isolates to test for carbapenemase production. We conducted mCIM on ESBLE isolates to assess for the absence or presence of slow carbapenemase enzyme activity among these isolates.

This assay operates on the principle that when a 10-µg meropenem disk is exposed to a cation adjusting Mueller Hinton broth (CAMHB) of a carbapenemase-producing microorganism for 4 h, the carbapenemase degrades the carbapenem in the disk. Conversely, if the test microorganism does not produce carbapenemase, meropenem maintains its antimicrobial activity after being incubated in the bacterial suspension. Subsequently, the disk is removed from the suspension and transferred onto a Mueller-Hinton agar (MHA) plate that has been inoculated with a suspension of a carbapenem-susceptible indicator organism (E. coli ATCC 25922). After overnight incubation, the zone of inhibition is measured to determine whether the meropenem has been hydrolyzed (indicated by the growth of the indicator organism close to the disk) or remains active (evidenced by a large zone of inhibition around the disk). CRE isolates were classified into carbapenemase producer and non-producer Enterobacterales (CPE and none CPE).

Colistin minimum inhibitory concentration (MIC)

Colistin susceptibility test was conducted using the agar dilution method (ADM) where all isolates were streaked on four plates of Mueller Hinton agar containing 0, 1, 2, 4 µg/ml of colistin sulfate (Thero Fisher Scientific). Results were interpreted as sensitive or resistant according to CLSI 2023 guidelines [11].

Colistin resistance by agar dilution method (MIC > 4 µg/ml) was confirmed using two other methods: broth microdilution method (BMD) and disk elution method.

BMD was conducted according to CLSI 2017 M52-Ed 1 guidelines [13], briefly:

  1. A)

    Preparation of Colistin Solutions: Colistin sulfate powder and CAMHB were used to prepare 5120 µg/ml stock solutions. From this stock solution, a 512 µg/ml concentration was prepared by mixing 1 ml of the stock solution with 9 ml of CAMHB. Using the 512 µg/ml concentration, subsequent concentrations of 256, 128, and 64 µg/ml were prepared in separate test tubes. From the 64 µg/ml test tube, further dilutions of 32, 16, and 8 µg/ml were made. Using the 8 µg/ml tube, additional concentrations of 4, 2, and 1 µg/ml were prepared. From the 1 µg/ml tube, further dilutions of 0.5, 0.25, and finally 0.125 µg/ml were made. We then dispensed 100 µl of each of the following concentrations into separate wells: 64, 32, 16, 8, 4, 2, 1, 0.5, 0.25, and 0.125 µg/ml.

  2. B)

    Preparation of Bacterial Isolates: Isolates were prepared to a 0.5 McFarland standard and then diluted to a concentration of 1:1000 (100,000 cfu/ml). Subsequently, 100 µl of the diluted bacteria were dispensed into all wells except for the well dedicated to negative growth control.

The plates were incubated at 35 °C ± 2 for 18–24 h, and the results were interpreted as either sensitive or resistant.

In the disk elution method (DE), all isolates demonstrating resistance to colistin by ADM (MIC > 4 µg/ml) underwent retesting with DE. Specifically, four 10 ml tubes were filled with 5 ml of CAMHB, and 50 µl of 0.5 McFarland of the isolates were introduced into each of the four tubes. Subsequently, commercially available 1 µg colistin disks from Thermo Fisher Scientific were placed into the tubes. The first tube contained no colistin disc (0 µg/ml CT), the second tube contained one colistin disc (1 µg/ml CT), the third tube contained two colistin discs (2 µg/ml CT), and the fourth tube contained four colistin discs (4 µg/ml CT).

Following this, all tubes were incubated at 35 °C ± 2 for 18–24 h, and the results were interpreted as either sensitive or resistant following the CLSI 2023 guidelines [11].

Antibiogram of all ESBLE isolates with non-beta lactam antibiotics and CRE combination antibiotics

Susceptibility testing of all ESBLE isolates was examined by VITEK 2 compact system from bioMerieux, using AST N204 (ESBL test, ampicillin, amoxicillin-clavulanic acid, piperacillin-tazobactam, cefotaxime, ceftazidime, cefepime, meropenem, ertapenem, imipenem, ciprofloxacin, gentamycin, amikacin, nitrofurantoin, fosfomycin, and trimethoprim-sulfamethoxazole), AST N417(amoxicillin-clavulanic acid, piperacillin-tazobactam, cefazolin, cefuroxime, ceftriaxone, ceftazidime, cefepime, meropenem, ertapenem, imipenem, ciprofloxacin, gentamycin, amikacin, nitrofurantoin, fosfomycin, and trimethoprim-sulfamethoxazole), and AST NX20 cards (ampicillin-sulbactam, Aztreonam, cefotaxime, cefoxitin, ceftazidime-avibactam, colistin, doripenem, levofloxacin, meropenem-vaborbactam, tetracycline, doxycycline, minocycline, tigecycline, and tobramycin). Disk Diffusion Method (DDM) was used to determine antibiotic susceptibility against the following antibiotic categories, Aminoglycosides (30-µg amikacin, 10-µg gentamicin, 10-µg tobramycin), fluoroquinolones (5-µg ciprofloxacin, 5-µg levofloxacin, 5-µg moxifloxacin), other categories (1.25/23.75 µg trimethoprim/sulfamethoxazole, 200 µg fosfomycin and 300 µg nitrofurantoin) and CRE combinations antibiotics (30/20-µg ceftazidime-Avibactam, 20/10-µg meropenem-vaborbactam, 30-µg cefiderocol).

Molecular characterization of ESBLE and CPE

DNA extraction was performed using a column-based DNA isolation kit (DNA mini kit, Qiagen, Germany). The DNA from all ESBLE isolates was tested for the presence of ESBL genes (blaCTXm, blaSHV, and blaTEM) and the presence of CRE genes (blaKPC, blaOXA48, blaNDM, blaVIM). Additionally, the DNA from CRE isolates was tested for the presence of CRE genes (blaKPC, blaOXA48, blaNDM, blaVIM). The primers used for the detection of the ESBL and CRE genes are listed in Table 1. The presence of ESBL genes and carbapenemase genes was examined using a Quanta Stadium 5 real-time PCR system with primer sets and a specialized program designed for the detection of carbapenemases, as recommended by the European Committee on Antimicrobial Susceptibility Testing (EUCAST), as shown in Table 2. (Accessible at www.ecdc.europa.eu/en.)

Table 1 Primers used for the detection of the ESBL and CRE genes with their amplicon size
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Table 2 The details of the programs used for detection of carbapenemases and ESBL -encoding genes as recommended by EUCAST
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Ethical considerations and consent to participate

Ethical approval (Ref. Mas. Oct. 2023/7) was obtained from the Institutional Review Board (IRB) of An-Najah National University, which approved all aspects of the study design, including the collection of bacterial isolates from microbiology laboratories, and the patient demographic data. The need for informed consent was waived by the IRB of An-Najah National University. Approval to collect demographic data and bacterial isolates from the governmental hospitals was also obtained from the Palestine Ministry of Health (MOH). The data collected were used exclusively for research, kept confidential, and not used for any other purpose.

Statistical analysis

Data were coded, categorized, and entered using Microsoft Office Excel (2010). Descriptive statistics were conducted with frequencies and percentages for categorical variables.

Results

Distribution of Enterobacterales isolates across different sources

Five government Palestinian hospitals spread around the West Bank were the source of bacterial isolates. 367/600 (61%) were from female and 233/600 (39%) were from male patients. The patients’ ages went from one day to 92 years with a median age of 41.5 years.

As shown in Table 3, there is a diversity in the distribution of Enterobacterales isolates across different sources. Urine samples accounted for the highest proportion of Enterobacterales isolates, with 364 (60.7%) isolates identified. This was followed by isolates from wounds (120, 20%) and blood (68, 11.3%). Among the Enterobacterales isolates, K. pneumoniae and E. coli were the most prevalent species across all sources. However, their distribution varied depending on the sample source. For instance, K. pneumoniae was predominant in sputum (82%) and wound (68%) samples, while E. coli was more prevalent in urine samples (58%).

Table 3 Sources of bacterial isolates
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Phenotypic and genotypic characterization of ESBL among isolated Enterobacterales

Table 4 shows the distribution of ESBL phenotypes and genotypes among different species of gram-negative bacteria. Among the total isolates tested, 287 (47.8%) exhibited the ESBL phenotype. The most prevalent species showing the ESBL phenotype were K. pneumoniae (43.2%) followed by E. coli (41.5%).

Table 4 Phenotypic and genotypic characteristics of ESBLE isolates
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The majority of ESBL-producing isolates demonstrated resistance to various antibiotics, including cefotaxime (100%), ceftriaxone (100%), amoxicillin-clavulanic acid (63.4%), ceftazidime (48.8%), cefepime (41.8%), and piperacillin-tazobactam (18.8%). All ESBL isolates tested negative with mCIM.

The prevalence of ESBL genotypes varied among species; ESBL genotypes identified were blaTEM, blaSHV, and blaCTXm. Notably, blaCTXm was the predominant genotype across all species.

Each species showed specific patterns of ESBL genotype distribution. For instance, E. coli predominantly carried blaTEM and blaCTXm, while K. pneumoniae exhibited a higher prevalence of blaTEM and blaSHV. E. cloacae had a notable presence of blaCTXm, and Salmonella group showed a unique pattern with only blaCTXm detected.

Phenotypic and genotypic characteristics of carbapenem-resistant Enterobacterales (CRE) isolates

The total number of CRE isolates phenotyping based on carbapenem resistance (at least resistant or intermediate to one agent of the four carbapenems used in the study) was 102 (17%), while out of them 85 (83.3%) isolates, based on the mCIM were identified as CPE. As shown in Table 5, among the total CRE isolates, K. pneumoniae had the highest proportion with 63 (61.7%) isolates, followed by E. coli with 25 (24.5%) isolates and E. cloacae with 13 (12.5%) isolates. P. mirabilis had the lowest proportion with only 1 (1%) isolate. Among the CRE genotypes, blaNDM was the most prevalent, with 30 (35%) isolates, followed by OXA48 with 24 (28%) isolates and KPC with 23 (27%) isolates. Some isolates also harbored multiple carbapenemase genes, such as KPC and NDM, KPC and OXA48, and NDM and OXA48, as shown in Table 5.

Table 5 Phenotypic and genotypic characteristics of carbapenem-resistant Enterobacterales (CRE) isolates
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Frequency of multidrug-resistant Enterobacterales

The total number of MDRE was 424 isolates (70.67%) of them 396 isolates (66%) were eligible for colistin testing.

Table 6 Frequency of MDR enterobacterales among isolates eligible for colistin testing
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Among the isolates eligible for colistin testing 396 (66%), K. pneumoniae isolates exhibiting the highest percentage of MDRE (50.5%), followed by E. coli (37%) and then E. cloacae (12.5%) as depicted in Table 6.

Colistin resistance among multidrug-resistant Enterobacterales

As discussed in the previous sections and shown in Table 7, of our 600 selected isolates, 396 (66%) were MDRE, with 47 of these (11.9%) being colistin-resistant. Additionally, 287 isolates (48%) were ESBL, of which 9 (3.1%) were colistin-resistant. Furthermore, 102 isolates (17%) were CRE, with 38 of them (37.6%) were colistin-resistant.

Table 7 Colistin resistance among Enterobacterales
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Colistin resistance among carbapenem-resistant Enterobacterales phenotypes

Both the broth microdilution and disc elution methods were utilized to evaluate colistin resistance phenotypically. Thirty-eight out of 101 (38%) isolates demonstrated resistance to colistin using both techniques, while 84 (83%) were identified as carbapenemase-producing Enterobacterales by the mCIM. Among the total 101 CRE isolates, K. pneumoniae had the highest proportion with 63 (62%) isolates, followed by E. coli with 25 (25%) isolates, and E. cloacae with 13 (13%) isolates, as depicted in Table 8.

Table 8 Colistin resistance among CRE phenotypes
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Detection of silent carbapenemases genes among ESBLE

Sixty-five isolates were chosen at random from the pool of 287 ESBLE isolates for CRE genotyping. Despite the absence of any CRE phenotypes among them, eleven ESBLE isolates were discovered to have CRE genes. Within this group, 6 (54%) contained the NDM gene, 4 (36%) harbored the OXA48 gene in conjunction with NDM, and 1 (9%) carried only the OXA48 gene. These genetic markers were identified in 6 isolates of ESBL-producing K. pneumoniae and 5 isolates of ESBL-producing E. cloacae. As shown in Table 9.

Table 9 Detection of silent genes among ESBLE
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Colistin resistance among ESBL-positive Carbapenems sensitive and bacterial isolates

Nine isolates (3.14%) were found to be ESBL-producing Enterobacterales that were resistant to colistin but sensitive to carbapenems. All of these isolates were members of the K. pneumoniae species. However, one K. pneumoniae ESBL producer that was resistant to colistin according to broth microdilution (BMD) testing was found to be sensitive to colistin according to disk diffusion (DE) testing as shown in Table 10.

Table 10 Colistin resistance among ESBL-positive Carbapenems sensitive enterobacterales
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Antibiogram of ESBL-producing Enterobacterales

Table 11 shows the varying resistance rates of ESBL-producing Enterobacterales to non-beta-lactam antibiotics, as well as to combinations of beta-lactam antibiotics with beta-lactamase inhibitors.

Antibiogram of ESBL-producing E. Coli isolates

ESBL-producing E. coli isolates exhibited notably high resistance rates for fluoroquinolones with 32% resistance to ciprofloxacin, levofloxacin, and moxifloxacin. Additionally, a high resistance rate of 48% was observed against trimethoprim/sulfamethoxazole.

Antibiogram of ESBL-producing K. pneumoniae isolates

Resistance rates of ESBL-producing K. pneumoniae isolates were high against trimethoprim/sulfamethoxazole 54%. Extremely high resistance to nitrofurantoin was noted, affecting 90% of isolates. Resistance to fosfomycin was prevalent, with 70% of isolates showing resistance.

Antibiogram of ESBL-producing E. Cloacae isolates

ESBL-producing E. cloacae isolates exhibited resistance rate of 35% against trimethoprim/sulfamethoxazole. Notably, all isolates demonstrated total resistance to both nitrofurantoin and fosfomycin.

Antibiogram of ESBL-producing P. mirabilis isolates

ESBL-producing P. mirabilis isolates showed resistance to gentamicin with 50% resistance rat. High resistance rates to fluoroquinolones were observed for ciprofloxacin, levofloxacin, and moxifloxacin, each at 83%. All isolates exhibited total resistance to trimethoprim/sulfamethoxazole, nitrofurantoin and fosfomycin.

Table 11 Antibiogram of ESBL producing Enterobacterales
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Discussions

Infections caused by ESBLE, CRE, and colistin-resistant Enterobacterales pathogens, are becoming a top noticeable issue worldwide, this is due to the dramatically increased isolation of these pathogens from clinical samples, and their threat to human wellbeing [18]. Our study found that the overall percentage of multidrug-resistant Enterobacterales (MDRE) among the tested clinical isolates was 70.67% (424 out of 600). Our results agree with a 2021 report from Nablus, Palestine by Aiesh et al. which concluded that infections caused by ESBL-producing and carbapenem-resistant E. coli and K. pneumoniae have resulted in increased morbidity and mortality [5].

The biological differences, social inequities and restrictive cultural norms, make women more vulnerable to infectious diseases than men, this is highlighted in a study conducted by Cataldo, C. 2023, who found that women were at higher risk for many infectious diseases, they had more chance to infections than men [19]. In addition to that, most of the samples used in this study were urine samples, since females are more vulnerable to UTIs than males, (because the normal female urinary tract has a comparatively short urethra) therefore, carring an inherent predisposition to proximal seeding of bacteria. This anatomy increases the frequency of infections. Due to the fact that, females use more antibiotics than males especially third-generation cephalosporin like cefixime, ceftriaxone, and cefotaxime. As a consequence they become more vulnerable to selective pressure and harboring MDRE [20]. Approximately two-thirds of the bacterial isolates (61%) came from female patients, and the patients’ age was distributed from 1 day to 92 years old, similar to a study from the same region regarding infections with ESBLE at an institutional level that showed female predominance with 51.6%, and the median age of patients was 53 years [5]. Consistently, other studies from India showed that ESBL E. coli was predominate in female patients [12, 21].

Many reports demonstrated variations in the distribution of ESBLE isolates across different sources worldwide, a study conducted in India showed that ESBLE were most commonly isolated from urine (40%) followed by pus samples (22%) [21]. Another study conducted in south Asia regions, Bangladesh, and India, 2023, found that ESBLE were commonly isolated from urine samples (80%) in Bangladesh, and from skin and soft tissues in India (70%) [6]. Another study conducted in Palestine, Gaza Strip showed that ESBLE were commonly isolated from pus samples (55.2%) followed by urine samples (53.3%) [9]. Our study found that wound swabs were a major source of ESBLE (68/120) (56.6%), followed by urine samples (194/364) (53%).

A study conducted in the Gulf Cooperation Council countries showed that, among Gram-negative bacterial infections, E. coli (33%) was the most isolated organism followed by K. pneumoniae (19.2%) [22],, this is consistent with our study which found E. coli and K. pneumoniae were major causative agents of infections among Enterobacterales..

This study highlights the challenges between the detection of ESBLE and CRE by phenotypic and genotypic methods, it also highlights the prevalence rate of MDRE, ESBLE, CRE, and colistin resistance, in Palestine, and provide an antibiograms for Enterobacterales species with beta-lactam and none beta-lactam antibiotics.

Identifying ESBLE isolated from clinical samples is imperative for disease monitoring and the provision of efficacious treatments, although phenotypic method such as antimicrobial susceptibility profiling is still the most commonly used method to detect MDRE, ESBLE, CRE, and colistin-resistant bacteria. Some ESBLE harboured slow activity CRE genes called cryptic or silent genes, that thought to be a problematic issue, that lead to under-reporting or misdiagnosis of CRE as carbapenems sensitive bacteria. This highlights the importantce of the integration between phenotypic and genotypic to characterize MDRE [23]. Palestine like other developing countries, mainly depends on phenotyping characterizations for the detection of MDRE, ESBL and CRE, this is considered a challenge that increases the chance of missing the detection of Enterobacterales with cryptic mobile genomic elements, and could lead to failed treatment with prophylactics or drugs of choices among hospitalized patients. Our study found that among the randomly selected 65 ESBLE isolates from clinical samples, 11(17%) isolates harbored cryptic CRE genes, this finding will not only highlight the prevalence of both ESBLE and CRE in Palestine, but also will impair the infection control activities, therapeutic plan for infected patients, and the appropriate detection of CRE. Phenotypic detections alone could lead to failure in the treatment of patients with serious infections, that might increase the morbidity and mortality rates.

Similar to our study, a study conducted in Nigeria 2023, showed that (33/49) (67.35%) of isolates harbored carbapenemase genes, which were primarily blaNDM (60.0%), of which three (3/33) (9%) were susceptible to carbapenems by phenotyping, this could be explained by the fact that carbapenemase were not expressed in vitro but could expressed in vivo [23]. In the same context a study conducted in Egypt 2022, showed that, among the phenotypically carbapenem-sensitive isolates, 42.5% were carrying carbapenem resistance genes, blaNDM (80.5%) was the most prevalent, this finding also, demonstrate the importance of integrations between phenotype and molecular techniques to identify MDRE to CRE and ESBLE, to avoid failure treatment, and impaired infection control [24].

The high prevalence of ESBLE in Palestine and worldwide, lead us to think more if these isolates harbored cryptic multi-drug-resistant genes (MDRG). Our study found that (287/600) 47.8% of Enterobacterales isolates were ESBLE, a study conducted in Gaza Strip 2023, found that the prevalence rate of ESBLE was increasing in the children in pediatric Gaza Strip hospitals with a prevalence rate of 51.6% [9].

Many studies in the early 2000s, suggested that blaCTXm-producing isolates were becoming widespread in Europe, Latin America and the Asia-Pacific region [1]. Our study found that (226/287) 78.7% of ESBLE were positive for blaCTXm, this evidence stresses the importance of the collaboration of epidemiological surveillance, and antimicrobial stewardship to reduce the burden of ESBL gene transmissions from blaTEM, blaSHV, to blaCTXm [9]. A study conducted by Ghenea, A. E.2022, showed that blaCTXm has shown a rapid spread in recent years among Enterobacterales and has become the most prevalent ESBL in many parts of the world [25]. Further investigations need to be addressed in the future to demonstrate the relationships between different ESBLE genes and the detection of slow activity of not only CRE genes, but also MDRG among Enterobacterales.

Confirmation of phenotypic detection of CRE by molecular methods could prevent treatment failure and provide an actual prevalence rate of CRE worldwide. Some carbapenemase genes like blaNDM, and blaOXA48 have a slow activity and cannot be detected by phenotype alone, which lead to under-reporting of CRE to ESBLE, AmpC, or even sensitive strains [26]. Our study found that (102/600) 17% were CRE.

According to a 2022 special report by the Centers for Disease Control and Prevention, during the COVID-19 pandemic the rate of CRE infections in hospitals increased by 35% in 2020 compared with 2019 [27]. In a study conducted in Thailand 72% K. pneumoniae and 22% E. coli were CRE by phenotyping [28]. A study conducted in southern Saudi Arabia found that, out of the 86 tested K. pneumoniae CRE isolates, 64 (74.4%) were CPE isolates [26]. In southern Saudi Arabia K. pneumoniae strain was reported with triple carbapenemase genes, the emergence of such an isolate could threaten patients and healthcare workers and requires great attention to rapid interventions to avoid further dissemination [26]. A similar study conducted in Thailand found that blaNDM was more prevalent in several regions. CRE strains from urine, sputum and blood were collected in Thailand from 2016 to 2018 which were composed of 72% K. pneumoniae and 22% E. coli, 80% of the CRE strains produced carbapenemases, 17% (629/4296) produced more than one carbapenemases, and the most common type of carbapenemase was blaNDM, accounting for 65% (2392/4296) [28]. Our study found that the prevalence of CRE in Palestine increased in the timeline determined during October 2023, (85/102) 83% were CPE, (30/85) 30% were blaNDM further investigations based on our study finding need to be addressed, based on the most updated molecular techniques, way to understand the high prevalence of like these genes among MDRE. Also, the presence of cryptic genes among ESBLE will not only increase the actual prevalence of CRE, but also encourage our hypothesis to think about the presence of other cryptic MDRGs among CRE, to avoid treatment failures and infection control obstacles in the future.

Colistin is considered the last choice for carbapenem- resistant MDRE and it is often used for treatment of blood stream infections, wound infections, and respiratory infections caused by CRE. In recent years, there has been a marked increase in the incidence of colistin-resistant bacterial infections [4]. A similar study found that the overall resistance to colistin was high (41%) among tested clinical isolates, 61.0% of meropenem-resistant Enterobacteriaceae were resistant to colistin [4]. Another study showed that among 100 CRE isolates, 15% were resistant to colistin [29]. Another study conducted on 196 MDRE isolates, (19.9%) showed reduced susceptibility to colistin, which is an alarming sign of increasing colistin resistance rates among CRE isolates [30]. Our study found that colistin resistance is not only among CRE, but also, we found colistin resistance among ESBLE, this will highlight our hypothesis to test all MDRE, or even the whole Enterobacterales for colistin resistance, by the most appropriate and approved methods, that will reveal the actual colistin-resistant status not only in Palestine but also, worldwide. The phenotyping ADM explained in detail by CLSI 2023 guidelines is considered an excellent option for routine use, as it combines ease of performance with affordable cost [31]. Our study found 9 (3.14%) were colistin-resistant ESBLE producers and carbapenems sensitive Enterobacterales by phenotyping, all of them were K. pneumoniae ESBL producers carbapenems sensitive by phenotyping, one K. pneumoniae was an ESBL producer colistin-resistant by BMD and was colistin-sensitive by DE. DDM is not a recommended method by CLSI and EUCAST guidelines, for detecting colistin susceptibility, we should always depend on susceptibility testing by BMD or DE before releasing the final report even in resource-limited settings. This can preserve the therapeutic value of MDRE infections until we have newer treatment options available in the country [32]. Challenges in the methods of sensitivity testing of colistin along with an increase in the prevalence of colistin-resistant Enterobacterales needs to be addressed , our results showed that colistin performed by DE worked well compared to the reference BMD except for one isolate that was colistin-sensitive by DE (MIC = 2 µg/mL), and colistin-resistant by BMD (MIC = 4 µg/mL), since colistin sensitivity by BMD is considered as a gold standard and more reliable than DE we considered the isolate to be resistant to colistin [3, 33].

The increasing of ESBLE-producing bacteria have become a global concern because of their multi-resistance to most antibiotic classes, which makes the treatment difficult [2]. To achieve appropriate treatment choices, identification of the Enterobacterales species that generate ESBL as well as their antibiotic sensitivity pattern is essential worldwide [34]. A study by Ortiz de la Rosa, 2019 found that the ESBL bacterial isolates, may be a source of resistance to ceftazidime-avibactam [35], another study by Petty, L. A. 2018 found that, vaborbactam combined with meropenem had improved activity compared to meropenem alone across all isolates [36], another study by Yao, J. 2021 found that cefiderocol has demonstrated excellent activity against gram-negative bacilli including ESBLE, and CRE [19]. A study conducted in Iran showed the same results as our study, they found that aminoglycosides were confirmed as the most effective treatment option for ESBLE bacteria [34], while a study in Portugal showed that, a higher prevalence of resistance to fluoroquinolones was observed in ESBLE, 16.7% of ESBLE were resistant to fluoroquinolones [37], a study conducted at An-Najah National university hospital showed that ESBLE producing K. pneumoniae was highly resistant to fosfomycin, trimethoprim/sulfamethoxazole and nitrofurantoin [5]. A study conducted at the institutional level in Switzerland in 2022, found that ESBLE is an important reservoir for mobile colistin-resistant (mcr) genes [35]. Our study highlights that, for epidemiological purposes, the prevalence of colistin resistance should be checked among all Enterobacterales isolates not only among CRE isolates.

Conclusion

In conclusion, our study found a high burden of MDRE in West Bank, Palestine, this will limit and complicate treatment options for infections caused by Enterobacterales, which in turn calls for immediate actions to control and monitor the use of antimicrobials in general, it would be beneficial to incorporate both conventional laboratory techniques and modern molecular techniques for comprehensive characterization of the emergence silent carbapenemase genes among MDRE infections in Palestine, and over all the world. Resistant to colistin is not only among CRE bacteria, but some can also be detected even among ESBLE bacteria, combination of β-lactam beta-lactamases inhibitors with beta-lactam antibiotics is still useful for the treatment of various types of MDRE isolated from clinical samples.