Prevalence of multidrug-resistant and extensively drug-resistant phenotypes of Gram-negative bacilli isolated in clinical specimens at Centre Hospitalo-Universitaire Ibn Rochd, Morocco

Introduction antimicrobial resistance in gram-negative bacilli is one of the major concerns in public health. We aimed to evaluate gram-negative bacilli epidemiology, antimicrobial profiles, and the resistance´s mechanism for Enterobacteriaceae isolated from specimens of hospitalized patients in wards of University Hospital Center Ibn Rochd of Casablanca, Morocco. Methods a prospective study of the patient's specimens, collected from December 2016 to 31st March 2017. Isolation and identification were performed using conventional biochemical tests. According to the European Committee on Antimicrobial Susceptibility Testing guidelines, antibiotic susceptibility was determined. Polymerase Chain Reaction (PCR) was used to detect β-lactamase and carabapenemase genes: CTX-M, SHV, TEM, OXA-48, NDM, and VIM among the Enterobacteriaceae. Results according to inclusion criteria, 38 Enterobacteriaceae, 25 Acinetobacter baumannii (A. baumannii), and 10 Pseudomonas aeruginosa (P. aeruginosa) were included during the study period; these bacteria were mainly responsible for bacteremia. Fifty-five percent of enterobacteria were extended-spectrum β-lactamase (ESBL), 42% EBSL and carbapenemase, and 3% carbapenemase, with high coresistances. Eighty-four percent of A. baumannii were XDR. All P. aeruginosa were MDR; amikacin showed the best activity (70% susceptibility). The genotypic approach revealed the presence of blaCTX-M, blaSHV, blaTEM in 68%, 22%, and 11% respectively. Of the 22 carbapenemase-producers, 41% were blaOXA-48 and 18% blaNDM; none had blaVIM. Furthermore, various genes coexistence were detected: blaCTX-M+blaOXA-48; blaCTX-M+blaNDM; blaCTX-M+blaSHV+blaOXA-48; and blaSHV+blaOXA-48. Conclusion findings revealed highly resistance rate among isolates. This raises the need to control antibiotics and regular screening to identify dynamics promoting resistance. Thus, we recommend developing antimicrobial stewardship programs and improving hygiene systems to prevent the nosocomial spreading of these phenotypes in our center.


Introduction
Bacteria are one of the primary sources of infectious diseases in humans. Nowadays, gramnegative bacilli, including Enterobacteriaceae, Acinetobacter baumannii (A. baumannii), and Pseudomonas aeruginosa (P. aeruginosa), have been identified as the primary cause of infections. Therefore, these pathogens are becoming challenging in developing and developed countries significantly among hospitalized and nonhospitalized patients. Furthermore, they are increasingly isolated in different hospital wards from respiratory tract infections, urinary tract infections, intra-abdominal infections, septic, postsurgical infections, and pneumonia, a severe menace, especially in critically ill patients [1,2].
The injudicious misuse and accessibility of antibiotics have led to the emergence of antimicrobial-resistant strains to commonly prescribed antibiotics, a significant challenge in medical practice. Antimicrobial resistance (AMR) in this group has been recognized by World Health Organization (WHO) as a threat to the public health [3]. Studies conducted in Morocco have reported the alarming rate of β-lactamase and carbapenemase-producing bacterial pathogens and the increasing prevalence of resistance to other antibiotics classes [4,5]. This problem is crucial in fermenting and non-fermenting bacteria because of their ubiquity and ability to acquire genes that confer resistance to other antibiotics classes. This situation is associated with therapeutic impasses leading to high morbidity and mortality rates and an extended hospital stays. It has been predicted that if appropriate control and prevention measures are not taken, in 2050, AMR would be responsible for a death every 3 seconds [6]. Accordingly, in the current study, we established the period prevalence and antimicrobial sensitivity profile of the following phenotypes ESBL and carbapenemaseproducing of the main pathogenic bacteria Enterobacteriaceae, A. baumannii P. aeruginosa imipenem resistants. Then we detected the presence of β-lactamase and carbapenemase encoding genes: bla CTX-M , bla SHV , bla TEM , bla OXA-48 , bla NDM , and bla VIM among the Enterobacteriaceae isolated from various clinical specimens from hospitalized patients at University Hospital Center, Ibn Rochd of Casablanca, Morocco. Antimicrobial sensitivity testing and ESBL production: the antimicrobial susceptibility profile of isolates was determined using the disc diffusion method, and the results were interpreted according to the European Committee on Antimicrobial Susceptibility Testing (EUCAST) guidelines [7]. The following antibiotics were used; ampicillin (AMP) (10 μg), amoxicillin/clavulanic acid (AMC) (20/10 μg), piperacillin/tazobactam (PTZ) ( All Enterobacteriaceae isolates with an inhibition diameter ≤ to 22 mm with CAZ and ≤ to 27 mm with CTX were subjected to the ESBL confirmatory test using the double-disc synergy [8]. In addition, Escherichia coli ATCC 25922 and P. aeruginosa ATCC 27953 were used as quality control for media and antibiotics performance.

Methods
The definition of Magiorakos et al. [9] was used to classify bacterial isolates into MDR, extensively drug-resistant XDR, and pan drug-resistant pandrig resistance (PDR). MDR=resistant to at least one agent in three or more antimicrobial classes, XDR= susceptible to two or fewer antimicrobial classes, and PDR=resistant to all antimicrobial agents in all antimicrobial classes.
Preparation of deoxyribonucleic acid (DNA) and PCR: polymerase chain reaction technique was used to target the bla CTX-M [10], bla SHV [11], bla TEM [12], bla OXA-48 [13], bla NDM [14], and bla VIM [14] among all Enterobacteriaceae ESBL and carbapenemase-producing included. All Enterobacteriaceae ESBL and carbapenemaseproducing were grown on Mueller-Hinton (MH) agar plates (Bio-Rad®) for 18-24 hours at 37°C. Bacterial cells were suspended in 400 μL of ultrapure water. The suspension was heated at 100°C for 10 minutes and immediately frozen at 0°C for 5 minutes. One hundred and eighty μL were then recovered after centrifugation of 12000 g for 10 min. The supernatant containing DNA was stored at -20°C until further use.
Extended-spectrum β-lactamase (ESBL)-producing isolates were screened by PCR simplex for the following β-lactamase-encoding genes: bla CTX-M , bla SHV , and bla TEM as described by [12]. Also screened by PCR for the following carbapenemaseencoding genes: bla OXA-48 , bla NDM , and bla VIM as described by [13] with slight modifications. The primers used for PCR are listed in Table 1. Data analysis and interpretation: the data was collected and entered into Microsoft Excel 16.59. The frequency and percentages of MDR, carbapenemase, and ESBL-producing gramnegative bacteria were calculated using SPSS®-26. Tables were used for data presentation.
Ethical considerations: in order to ensure the confidentiality of patient´s personal information, data of isolates recovered during the diagnosis routine were collected and anonymity was maintained. So, no personal information of patients was taken into consideration in this study.

Molecular
resistance analysis of Enterobacteriaceae isolates: after amplifying the target genes, PCR showed that all isolates harbored genetic support of the resistance. Of the 37 ESBL-producer, bla CTX-M were the mainly detected gene (65% 25/37), eight strains harbored bla SHV (22%; 8/37) and (11%; 4/37) carried bla TEM . Of the 22 carbapenemase-producers, 9 were positive to bla OXA-48 , 4 for bla NDM , VIM gene was not detected in any of our isolates. Furthermore, the coexistence of bla CTX-M with bla OXA-48 and bla CTX-M with bla NDM was detected in 2 strains, the coexistence of bla CTX-M with bla SHV and bla OXA-48 was detected in one strain, 4 strains harbored bla SHV with bla OXA-48 . Of the 9 cases positive to bla OXA-48 , six have been reported in an isolate of K. pneumoniae, two in E. coli and one of E. cloacae. This distribution of the mentioned genes among the enterobacteria species is shown in Table 5.

Discussion
Due to the overuse of antibiotics, invasive procedures, and devices expose patients to a high risk of comorbidity and mortality. Therefore, in developing countries such as Morocco, investigating and controlling bacterial infections' epidemiology and resistance patterns is critical, especially in hospitals, for clinicians and decision-makers on health to implement adequate measures and effective management of these diseases [15].
In the current primary descriptive study, we examined the epidemiological spectrum by evaluating the antimicrobial patterns of the gramnegative bacilli clinical isolates of patients hospitalized in various wards of University Hospital Center Ibn Rochd of Casablanca, Morocco. Our study revealed that the most common culturepositive clinical samples were blood followed by urines. Similar findings were obtained in several studies [16,17]. However, in the investigations carried out by Beyene et al. and Mirzaei et al. urinary infections were the predominant, followed by bacteremia [18,19]. It is important to note that our study's demographics data showed that bacteremia and respiratory tract infections affect mainly infant patients <2 months old due to their lack of fully developed immune status. This result differs from published Moroccan and international epidemiology, ranging from 20 to 67 years old [20,21]. The detected difference could be due to the brief period of study, the diversity of specimen type, and specimen size. In the specified period, 38 Enterobacteriaceae MDR were isolated, E. coli and K. pneumonia were the most frequent species, mainly in bacteremia and urinary tract infections. Studies have shown the prevalence of these micro-organisms in these infections in Iran, Brazil, and Nigeria [17,22,23].
On the other hand, non-fermenting gram-negative bacilli are represented essentially with A. baumannii and P. aeruginosa plays a key role in nosocomial infections. At the same time, they were only considered to be contaminants in the past. Our study isolation rate of A. baumannii and P. aeruginosa imipenem resistant was 34% and 14%, respectively, parallel to those reported in the literature [19,20]. In this study, A. baumannii was isolated mainly from blood cultures and medical equipment, from ICU, especially neonatology which is the most alarming. This finding is in agreement with many reports [17,20,21,24,25]. According to a study conducted in the United Kingdom, Acinetobacter spp stains causing bacteremia have increased between 1998 and 2005 from 2 to 18 per 100,000 hospital days [26]. Our study observed a higher incidence of isolates from ICU (47%). Several risk factors account for the high ICU infections. These first ICU houses critically ill patients who are often exposed to extensive use of antibiotics, causing selection pressure for the emergence of resistance. These factors coupled with interventional instrumentations such as mechanical ventilation and invasive procedures commonly used expose the patients to a high risk of infections. The susceptibility profile of the isolates displayed a high level of multi-drug resistance against the firstline drug such as penicillin, cephalosporin, fluoroquinolones due to the unrestricted use by physicians, in addition, to their purchasing ease without prescription in the community pharmacies. Furthermore, isolates showed a high rate of resistance to carbapenem. The antibiotic resistance pattern of Enterobacteriaceae showed 100% of resistance to ampicillin, tobramycin, netilmicin, and chloramphenicol; more than 80% of the strains were resistant to amoxicillin/clavulanic acid, cefepime, cefotaxime, ceftriaxone, and trimethoprim/sulfamethoxazole, which is similar to previous studies [18,22]. Likewise, high-rate resistance against piperacillin/tazobactam (63%), ertapenem (61%), and ciprofloxacin (76%) was observed; this may be due to the coexistence of other mechanisms of resistances as inactivating enzymes like β-lactamases, carbapenemases, and aminoglycosidases. The widespread of these bacterial profiles in Morocco is a common trend in hospitals [20,28], community [29], and even the environment [30]. However, fosfomycin (for E. coli), amikacin, and gentamicin were the most active antibiotics. The resistance to aminoglycosides is usually attributable to enzymes modification, such as amino-glycoside 6´-Nacetyltransferases of type Ib (AAC (6')-Ib). This enzyme can modify amikacin, tobramycin, kanamycin, and netilmicin but not gentamicin. Numerous studies reported the existence of the aac(6')-Ib gene and susceptibility to amikacin and gentamicin [31].
Resistance to antibiotics tested in A. baumannii in the current study reached very alarming levels and were screened as follow: imipenem (100%), meropenem (100%), ciprofloxacin (100%), levofloxacin (100%), gentamicin (100%), and tobramycin (100%). In addition, up to 96%, 88%, and 84% of isolates were resistant to amikacin, netilmicin, and trimethoprim/sulfamethoxazole. The infective of carbapenems, even if they're used as a last resort, poses a challenge. The results present similarity with increasing rates of resistance of A. baumannii around the world, in Iran [19], Nepal [32], and Morocco [20,25], Although this rate seems to increase since 2007 in Moroccan hospitals. Indeed, Lahsoune et al. reported a resistance rate of 17.9% and 35% to netilmicin and amikacin [33]. In 2016, Uwingabiye et al. reported that resistance to the same antibiotic increased to reach 33% and 52% [34]. Overall, the variation in antibiotic resistance proportion may be due to many parameters such as geographical difference, study period, and ward origin.
In addition, the overall resistance of P. aeruginosa against aminoglycoside, cephalosporin, and fluoroquinolones was alarming, for netilmicin (100%), tobramycin (100%), cefepime (70%), levofloxacin (80%), and ciprofloxacin (60%). However, it was observed that amikacin and meropenem's resistance rate by these isolates was not that high. Amikacin and Meropenem have been reported to effectively treat diseases caused by P. aeruginosa [35]. In a study conducted at the Children's Hospital of Tehran between 2013 and 2018, showed that the sensitivity of P. aeruginosa has increased from 50% to 76% [17]. Therefore, the susceptibility of amikacin may be due to its use as a last resort in treating severe infections.
In general, the international literature reports that the resistance to extended-spectrum β-lactams in Enterobacteriaceae is mainly mediated via the production of bla CTX-M and to a lesser degree via bla SHV [36]. In most local studies, carbapenem resistance in Enterobacteriaceae is often mediated by the production of OXA-48 and NDM carbapenemases. Therefore, this outspread of the OXA-48 enzyme could be endemic for several Mediterranean countries, such as Morocco's case [37][38][39]. This increasing emergence is a severe epidemiological risk, as it is well known that plasmid-mediated transfer of drug-resistance genes is considered to be one of the most essential mechanisms driving their spread. Also, the strains that carried such genes cause nosocomial infections and outbreaks with a high mortality range. Thus, available choices for appropriate treatments for infections are very limited and that´s why the lack of their detection may enhance their hidden. Thus, it is essential to conduct molecular genetic analyses as well as to characterize the β-lactamase and carbapenemase found in specific geographic areas to outcome of the molecular evolution of both genes and resistant clones.
Limitations: our investigation´s main limitations were, first of all, the short duration of the study period, which led to a small sample size also related to inclusion criteria. Secondly, it is a singlecentered study. However, the scope of this study was to investigate the resistance pattern and prevalence of MDR and XDR among the studied groups of pathogens in our hospital. We would also have wished to detect the genes coding carbapenemase for A. baumannii and P. aeruginosa.

Conclusion
Gram-negative bacteria are one of the leading causes of significant antibiotic resistance. The Enterobacteriaceae, A. baumannii and P. aeruginosa isolated during this study were implicated in various infections. The bacteremia was the most frequently diagnosed, also notably, the infections were common among infants. Our results confirm, on the one hand, the international trend of increasing multi-drug resistance among gram-negative bacilli. Withing the Enterobacteriaceae, 55% were ESBL, 42% were ESBL and carbapenemase producing, while 3% were carbapenemase, in addition to the coexistence profile to the several antibiotic classes. Regarding nonfermenting gram-negative bacilli, 84% of A. baumannii were extensively drugresistant, while all P. aeruginosa were MDR. On the other hand, the study underlines the local spread of bla CTX-M , bla OXA-48 , and bla NDM . Nevertheless, the spread of these genes in our isolates, with the coexistence of genes mediated resistance, could be further than expected and lead to a management impasse. Given that antibiotic resistance can cause severe infections in hospitalized patients and lead to a therapeutic dilemma, these findings highlight the need for timely, accurate epidemiological and susceptibility information to guide the identification and implementation of strict antimicrobial stewardship policies and good microbiological surveillance procedures in the hospitals.   Table 3: distribution of infections among gender and patients' age groups Table 4: percentage antibiotics susceptibility of isolates Table 5: distribution of genes mediated resistance among Enterobacteriaceae species   Medical ICU  -1  ----8  7  Burned  center   -1  -----3   Urology  3  -----1  -Visceral  surgery   1  -------EMD  2  -------M. Pediatrics 1  --1  ----OTC  --1  ---1  -PMFR  --1  ---1  -HGEP  1  -------IDD  1  -------Dermatology ------1  -DHCC  -2 ------EMD: endocrinology and metabolic diseases; M. Pediatrics: medical pediatrics; OTC: orthopedic-trauma center; PMFR: physical medicine and functional rehabilitation; HGEP: hepato-gastro-enterology and proctology; IDD: infectious disease department; DHCC: day hospital collecting center; ICU: intensive care unit