ESBL Activity, MDR, and Carbapenem Resistance among Predominant Enterobacterales Isolated in 2019

Antimicrobial-resistance in Enterobacterales is a serious concern in Saudi Arabia. The present study retrospectively analyzed the antibiograms of Enterobacterales identified from 1 January 2019 to 31 December 2019 from a referral hospital in the Aljouf region of Saudi Arabia. The revised document of the Centers for Disease Control (CDC) CR-2015 and Magiorakos et al.’s document were used to define carbapenem resistance and classify resistant bacteria, respectively. The association of carbapenem resistance, MDR, and ESBL with various sociodemographic characteristics was assessed by the chi-square test and odds ratios. In total, 617 Enterobacterales were identified. The predominant (n = 533 (86.4%)) isolates consisted of 232 (37.6%), 200 (32.4%), and 101 (16.4%) Escherichia coli, Klebsiella pneumoniae, and Proteus mirabilis, respectively. In general, 432 (81.0%) and 128 (24.0%) isolates were of MDR and ESBL, respectively. The MDR strains were recovered in higher frequency from intensive care units (OR = 3.24 (1.78–5.91); p < 0.01). E. coli and K. pneumoniae resistance rates to imipenem (2.55 (1.21–5.37); p < 0.01) and meropenem (2.18 (1.01–4.67); p < 0.04), respectively, were significantly higher in winter. The data emphasize that MDR isolates among Enterobacterales are highly prevalent. The studied Enterobacterales exhibited seasonal variation in antimicrobial resistance rates towards carbapenems and ESBL activity.


Introduction
Globally, infections with resistant Gram-negative bacteria are recognized as a severe threat to patients' health. Enterobacteriaceae resistant to third-generation cephalosporins and carbapenems are critical microorganisms that require urgent attention [1]. A greater number of patients are at risk of dying from infections caused by carbapenem-resistant, extended-spectrum beta-lactamase (ESBL)-producing, and multidrug-resistant (MDR) Enterobacteriacea [2]. Carbapenemase-producing Enterobacteriaceae infections are associated with a very high case fatality rate with currently available antibiotics [3,4]. Intrinsic and acquired bacterial resistance, international travel, inappropriate antibiotic use, and poor infection control activities are predisposing factors for developing resistance [5,6].
Global dissemination of resistant Enterobacteriaceae is increasing [7,8]. The Kingdom of Saudi Arabia receives numerous pilgrims throughout the year, particularly in the holy month of Hajj, raising the possibility of resistant Enterobacteriaceae transmission. International travel is a serious concern for the transmission of resistant Enterobacteriaceae in the country [9]. Various studies and reports emerging from different regions of the kingdom have highlighted the spread of such resistant strains [10][11][12][13][14]. Furthermore, the expatriate population in the kingdom is a matter of concern considering the transmission of Enterobacteriaceae with diverse resistance mechanisms [15].
The surveillance of resistant strains is a prerequisite for control measures [16]. Insights into antibiotic resistance provide valuable information about significant pathogens

MDR, ESBL, and Associated Sociodemographic Characteristics
The multidrug-resistant organisms were isolated with significantly higher frequency from clinical samples of intensive care units (OR = 3.24 (1.78-5.91); p < 0.01) compared to the samples received from surgical wards and other hospitals (OR = 2.67 (1.29-5.50); p < 0.01). Other sociodemographic characteristics did not reveal any significant association with multidrug strains (Table 2). The ESBL isolates were observed with significantly (p < 0.05) lesser frequency in the samples received in the second, third, and fourth quarters of the year compared to the first quarter (January-March) ( Table 3).

Discussion
Antimicrobial resistance is prevalent worldwide, and the mortality due to drugresistant infection is predicted to reach 10 million annually by 2050 [18]. The current treatment options for drug-resistant infections are becoming less effective, thereby leading to treatment failures, increasing treatment cost, and endangering life [1]. Antimicrobial resistance, particularly carbapenem resistance in Gram-negative bacteria, is the latest challenge of immense public health importance [19]. Carbapenemase-producing Enterobacteriaceae such as KPC and New Delhi metallo-β-lactamase NDM have achieved global dissemination and pose a serious threat to human life [8,20]. The Kingdom of Saudi Arabia has reported a rise in carbapenem-resistant Enterobacteriaceae, mostly from Riyadh [15,[21][22][23][24] and Makkah [14,25].
In the present study, K. pneumoniae, E. coli, and P. mirabilis were the major Enterobacterales isolated during the study period. These findings are in accordance with earlier studies from Saudi hospitals and countries in the MENA region [20,26,27]. These microorganisms have achieved remarkable antimicrobial resistance and have been increasingly recovered from various clinical samples [27]. In general, a very high prevalence (82.1%) of MDR was observed in this study. The majority of MDR isolates belonged to K. pneumoniae and E. coli in almost equal proportions. Furthermore, this study reported 128 (24.0%) isolates as extended-spectrum beta-lactamase (ESBL) producers. E. coli was the most common ESBL active, followed by K. pneumoniae. The antimicrobial resistance and prevalence of MDR or ESBL activity among bacteria are difficult to compare and depend on various local factors such as region-and countryspecific guidelines on antimicrobial use, self-medication, infection control practices, and practices affecting antimicrobial prescription and use [28]. Furthermore, study-specific factors such as analysis of clinical samples from inpatients, outpatients, or both; type of clinical sample analyzed (urine, blood, sputum, or all types of samples); treating intermediate resistance as resistant or sensitive categories; and the analysis adopted to measure various rates affect comparison across studies. A recent study evaluating the effectiveness of the Saudi Action Plan in combating antimicrobial resistance identified E. coli as the most frequently isolated Enterobacterale and the predominant bacteria for ESBL activity and MDR prevalence, followed by K. pneumoniae [29]. Similarly, another study from the neighboring country of Sudan reported E. coli and K. pneumoniae as the most commonly isolated Enterobacterales. E. coli was reported as a frequent MDR isolate, whereas ESBL activity was more frequent in K. pneumoniae [27]. These reports are in accordance with our results on the most common bacterial isolation and ESBL activity, with a slight variation in MDR prevalence among the two bacteria. A study from Addis Ababa reported a 42.1% MDR among Enterobacteriaceae [30]; however, another recent study from Saudi Arabia reported an overall prevalence of 60% MDR Enterobacteriaceae, with E. coli and K. pneumoniae as the most frequent isolates [31], but this prevalence was only 6.6% in the USA hospitals [32]. The analysis adopted in the present study revealed higher MDR prevalence, and this prevalence was determined for the three predominant bacteria and not for all Enterobacterales isolates reported during the study period. The extended-spectrum penicillin and cephalosporins are safer than other antimicrobials, resulting in their wide use in clinical settings [33], which might partly explain the increased MDR prevalence and ESBL activity in these bacteria. Of the 101 isolates of P. mirabilis recovered, 95 (94%) were MDR. Our results on the MDR prevalence are consistent with a study from India that reported a >95% MDR rate in P. mirabilis [34]. The biofilms produced by this organism not only help to evade the lethal effect of antimicrobials but also induce bacterial changes to resist and escape host defenses [35,36]. The free availability of antimicrobials and non-adherence, which are frequent in Saudi Arabia, could be attributed to this increased resistance and subsequent MDR prevalence.
The MDR isolates were recovered from intensive care units at a significantly higher frequency, whereas ESBL isolates were significantly higher in winter. Other studies have reported similar results regarding the frequency of MDR in intensive care units [32,37]. The reasons for this observed phenomenon are that patients admitted to intensive care units have multiple pathologies and are frequently treated with empirical antibiotic therapy before culture and sensitivity reports compared to non-intensive care unit patients. The high frequency of ESBL isolation in winter may be a result of higher antimicrobial consumption during these months. Seasonal variation in antimicrobial resistance has been previously reported [38].
In the present study, K. pneumoniae isolates exhibited resistance rates of 62.5%, 62% and 58% for ertapenem, imipenem, and meropenem, respectively. E. coli isolates revealed an overall carbapenem resistance of 10.9%, with the highest rate of 15.8% for imipenem. Moreover, 29.3% and 25.8% resistance to ertapenem and meropenem was observed in P. mirabilis, respectively. Ali Al Bshabshe et al. reported 65.2% and 61.7% resistance in K. pneumoniae towards ertapenem and meropenem, respectively, which is consistent with our results [39]. The slight increase in these resistance rates is because their study analyzed clinical samples from intensive care units only. In another study, 38.4% and 46.1% resistance rates to imipenem and meropenem, respectively, were noted in K. pneumoniae [40]. Carbapenem resistance is a concern as these drugs are the treatment of choice, particularly against ESBL-producing K. pneumoniae. These rates indicate the emergence of carbapenemase-producing strains. Timely identification of carbapenem strains is pivotal for controlling these infections in hospital settings. A previous study reported that 43.5% of antimicrobials were inappropriately prescribed; of these, broad-spectrum activity (44.6%) and use of antimicrobials without culture (32.4%) were the main reasons [41]. Inappropriate prescriptions when coupled with self-medication and improper antibiotic consumption may greatly enhance antimicrobial resistance. Appropriate antibiotic prescription guidelines, regulation of antibiotic dispensing by community pharmacies, and patient education are some of the vital measures in combating the menace of antimicrobial resistance.
In the present study, E. coli and K. pneumoniae presented a significant seasonal variation (winter vs. other seasons) in the resistance rates for imipenem (2.55 (1.21-5.37); p < 0.01) and meropenem (2.18 (1.01-4.67); p < 0.04), respectively. A study from the USA covering hospitalized patients did not find significant seasonal variation in carbapenem resistance among Enterobacteriaceae [42]. Although this phenomenon needs further exploration, this variation can be explained from the fact that both antibiotic consumption and antimicrobial resistance increases in the winter season [38]. Furthermore, the present study observed approximately 30% of the Enterobacteriaceae infections in winter, which may explain the different resistance rates towards carbapenems.
Colistin and tigecycline are used against carbapenem-resistant Enterobacteriaceae (CRE). This study evaluated the antimicrobial activity of these antibiotics against carbapenemresistant (meropenem-resistant) and multidrug-resistant strains identified in this study. Tigecycline exhibited 53% and 45% sensitivity against E. coli (CRE) and K. pneumoniae (CRE), respectively. Similarly, colistin was 100% and 82.1% effective against these two strains, respectively. Both antibiotics were ineffective against P. mirabilis. The treatment options for carbapenem-resistant strains are limited, that is, the current standard treatment for such infections includes tigecycline, polymyxins, fosfomycin, and aminoglycosides, which are administered alone or in combination with other antibiotics, the latter revealing better outcomes. An increase in tigecycline sensitivity from 76% to 82% against E. coli (CRE) and from 33% to 50% for K. pneumoniae (CRE) has been reported in Riyadh. The same study reported a decline in colistin sensitivity from 97% to 86% against E. coli (CRE) and from 80% to 76% against K. pneumoniae (CRE) [43]. The observations from Riyadh complement our findings on these two antibiotics, with subtle variations. Biofilms and intrinsic resistance to colistin and tigecycline are prominent features of P. mirabilis, which explains its resistance towards these antibiotics [44].
The major strength of this study is that it is the first exclusive report on the resistance pattern in Enterobacterales from this region. Furthermore, the study analyzed the recent data of 2019, which is the second strength of the study. One limitation of this study is that it included data from only one referral center out of two operating in the capital city of the Aljouf region, thereby limiting the generalizability of the results; however, the study hospital is the oldest in the region and continues to process the majority of clinical samples in addition to those received from other hospitals. Another limitation is the lack of molecular analysis of this acquired resistance.

Conclusions
The study concludes that a high prevalence of MDR and carbapenem resistance exist in the study area. Intensive care units majorly contribute to the prevalence of MDR. Seasonal variations in ESBL-producing bacteria and carbapenem resistance were noted. High tigecycline resistance was noted, which has an immense impact on treatment options and patient outcomes. Antimicrobial resistance rates are worrying and require immediate action at the institutional and public health levels. Strict infection control practices and antimicrobial prescription guidelines need to be observed in the intensive care units to counter antimicrobial resistance. The seasonal variation in carbapenem resistance should guide empirical antibiotic therapy in this hospital. Identification of the molecular basis of Antibiotics 2021, 10, 744 9 of 12 the observed resistance and exploring the reasons for the seasonal variation are potential areas for future research.

Material and Methods
The Aljouf region has three governorates of Sakaka, Qurayyat, and Dumat Al-Jandal. The total population of the Aljouf region is 520,737 as per the 2018 census. The Sakaka governorate is the capital city of the Aljouf region with two specialist hospitals of 300 beds each. The study was conducted in one of the two specialist hospitals that serve the population of this region. This hospital is the first specialist hospital built in the region. This retrospective hospital-based study analyzed the data on culture and antibacterial reports of all non-duplicate Enterobacteriaceae isolated from 1 January 2019 to 31 December 2019. The BD Phoenix system (BD Diagnostics, Sparks, MD, USA) processed all the samples and generated a sensitivity report.
The antibiotics used to assess bacterial susceptibility were imipenem, meropenem, ertapenem, amikacin, gentamycin, aztreonam, ampicillin, augmentin, piperacillin/tazobactam, cephalothin, cefoxitin, cefuroxime, ceftazidime, ceftriaxone, cefepime, nitrofurantoin, ciprofloxacin, levofloxacin, tigecycline, colistin, and trimethoprim/sulfamethoxazole. Intermediate resistance presented by the bacterial isolates was interpreted as being resistant. Magiorakos et al.'s criteria were used for classification of antimicrobial resistance into multidrug resistance (MDR), extensive drug resistance (XDR), and pan drug resistance (PDR) [45]. Multidrug resistance (MDR) is defined as when a bacterium is 'non-susceptible to ≥1 agent in ≥3 antimicrobial categories'. Extensive drug resistance (XDR) is defined as when a bacterium is 'non-susceptible to ≥1 agent in all but ≤2 categories (i.e., bacterial isolates remain susceptible to only one or two categories), while pan drug resistance (PDR) is defined as when a bacterium is 'non-susceptible to all antimicrobial agents listed' [45]. Moreover, carbapenem-resistant Enterobacteriaceae (ER) was defined according to the revised document of the Centers for Disease Control and Prevention CR-2015 [46]. The bacterial antimicrobial susceptibility results were interpreted using breakpoints established by the Clinical Laboratory Standards Institute (CLSI) [47]. Information on the phenotypic characterization of ESBL provided by the BD Phoenix system was also recorded. Furthermore, the study collected data on the type of specimen, specimen collection date, information of different units referring the sample, and age and gender of patients.

Statistics
Statistical Package for Social Sciences (SPSS) version 21.0 for Windows (SPSS, Inc., Chicago, IL, USA) was used for data analysis. The entries were manually verified for accuracy. The frequencies and percentages of Enterobacteriaceae distribution and MDR isolates were calculated. Additionally, the association between sociodemographic and distribution characteristics of Enterobacteriaceae was assessed using the chi-square test with or without Yates correction. Fisher's exact test was performed whenever necessary. Furthermore, the distribution characteristics of multidrug-resistant strains and various carbapenem-resistant isolates vis-à-vis sociodemographic risk factors such as sex, age groups, seasonality, referring unit, and type of sample were determined by odds ratios with 95% confidence intervals. Statistical significance was set at p < 0.05. Funding: This work did not receive any funding.
Institutional Review Board Statement: Jouf University's Local Committee on Bio-Ethics approved the research proposal vide 03-04/41, dated 6 January 2020. Informed consent is a standard practice ensured at the time of specimen collection in this hospital. For patients admitted to the intensive care unit, informed consent of the legal guardian is recorded. Research was performed in accordance with relevant guidelines/regulations and in accordance with the Declaration of Helsinki. This study analyzed the existing data on antibiograms. The patients' identifiable data points like name, medical record number, and specimen number were removed at the time of data collection to delink identification.
Informed Consent Statement: Informed consent was obtained from all subjects involved in the study.

Data Availability Statement:
The datasets generated and/or analyzed during the current study are available from the corresponding author on reasonable request. The dataset will be uploaded in the relevant repository after acceptance.