Molecular characterization of multidrug-resistant Gram-negative pathogens in three tertiary hospitals in Cairo, Egypt.

High rates of antimicrobial resistance (AMR) among Gram-negative pathogens (GNP) have been reported in Egypt. Antimicrobial surveillance and identifying the genetic basis of AMR provide important information to optimize patient care. In this study, we aimed to identify the beta-lactam resistance phenotypes and genotypes of multidrug-resistant (MDR) non-repetitive GNP from 3 tertiary hospitals in Egypt. WZe studied 495 non-repetitive MDR Gram-negative isolates from patients with complicated intra-abdominal infections (cIAI), complicated urinary tract infection (cUTI), and lower respiratory tract infection (LRTI), collected as part of the "Study for Monitoring Antimicrobial Resistance Trends" (SMART) conducted in 3 tertiary hospitals in Cairo, Egypt, from 2015 to 2016. Identification and susceptibility testing of GNP to antimicrobials were tested in each hospital laboratory and confirmed in a reference laboratory (International Health Management Associates (IHMA), Inc., Schaumburg, IL, USA). Molecular identification of extended-spectrum beta-lactamases (ESΒLs), AmpC, and carbapenem resistance genes was conducted in IHMA. Among the 495 MDR isolates, Klebsiella pneumoniae (K. pneumoniae) and Escherichia coli (E. coli) were the most common (52.7% and 44.2%). K. pneumoniae was most susceptible to colistin, amikacin, ertapenem, and imipenem (92.7%, 72.7%, 69.3%, and 64%, respectively). E. coli was most susceptible to colistin (100%), amikacin (94.1%), imipenem (90.4%), and ertapenem (83.6%). ESBL was detected in 96.2% and ESBL genotypes included blaCTX-M-15 (70.1%), blaTEM-OSBL (48.5%), blaSHV-OSBL (27.9%), and blaCTX-M-14 (10.7%). AmpC resistance genes were identified in 9.7% of the isolates, dominated by blaCMY-2 (5.7%). Carbapenem resistance genes were detected in 45.3% of the isolates. In K. pneumoniae, blaOXA-48 dominated (40.6%), followed by blaNDM-1 (23.7%) and blaOXA-232 (4.5%). In E. coli, the most frequent genes were blaNDM-5 (9.6%), blaOXA-181 (5.5%), blaOXA-244 (3.7%), and blaNDM-1 (3.7%). blaKPC-2 was identified in 0.4% of isolates. Notably, 32.3% of isolates carried more than one resistance gene. Our findings emphasize the continued need for molecular surveillance of MDR pathogens, implementation of strict infection control measures, and antimicrobial stewardship policies in our hospitals.


Introduction
Antimicrobial resistance (AMR) among Gram-negative pathogens (GNP) increased worldwide. A high rate of AMR has been reported in Egypt since more than 20 years, among GNP causing nosocomial infections and outbreaks [1][2][3]. AMR rates have increased especially among nosocomial GNP, probably due to widespread abuse of antimicrobials including carbapenems in Egyptian hospitals and poor compliance with infection control practices [4,5]. In small-scale studies, bla OXA , bla NDM , bla VIM , bla IMP , and bla KPC carbapenemase genes were detected in Egypt [6][7][8]. As the genetic basis of beta-lactam resistance was not yet studied at a large scale in Egypt, we aimed to molecularly characterize multidrugresistant (MDR) GNP.

Study sites and strains
This study was conducted in 3 major tertiary care Egyptian hospitals participating in the "Study for Monitoring Antimicrobial Resistance Trends" (SMART) from 2015 to 2016. The hospitals were Ain Shams University Hospital, Ain Shams Specialized University Hospital, and Dar Al-Fouad Hospital. Isolates were collected according to SMART protocol as previously reported [9][10][11]. Briefly, the participating hospitals collected 1070 non-repetitive consecutive GNP isolates from lower respiratory tract specimens, urine and pus or abdominal fluid of hospitalized patients with lower respiratory tract infections (LRTI), complicated urinary tract infections (cUTI), and complicated intra-abdominal infections (cIAI) during the study period. Of these, we studied 495 isolates that showed phenotypic resistance to third-generation cephalosporins or carbapenems. The identification of GNP, susceptibility testing, and detection of resistance phenotypes were conducted in hospital laboratories according to the methods of the Clinical and Laboratory Standards Institute [12] and confirmed in a reference laboratory (International Health Management Associates (IHMA), Inc., Schaumburg, IL, USA), where susceptibility and extended-spectrum β-lactamase (ESBL) phenotype were determined using the CLSI broth microdilution method with custom dehydrated panels manufactured by Trek Diagnostic Systems (Thermo Scientific, Independence, OH) in 2015-2016. MIC interpretive criteria followed the 2017 M100-S27 guidelines of the CLSI [13]. EUCAST breakpoints were used only for colistin against Enterobacteriaceae, because no CLSI breakpoints exist [14].The susceptibility of all Gramnegative isolates combined was calculated using breakpoints appropriate for each species.

Identification of bla ESBL genes of the TEM, SHV, and CTX-M types
ESBL production was detected in 96.2% of the MDR isolates ( Table 2) (Table 3).

Discussion
Surveillance for AMR is essential to monitor trends, identify emerging resistance mechanisms, and support the antimicrobial stewardship programs. To our knowledge, this is the most extensive molecular study of AMR in Egypt including 495 MDR Gram-negative isolates. In all hospitals, more than half of infections were present on admission (community acquired or transferred from other hospitals; data not shown). We identified 43 resistance phenotypes distributed among isolates from the 3 hospitals. Typing of nosocomial GNP in each hospital based on the phenotypic resistance patterns showed no clonal spread except for few isolates in each hospital (data not shown).
K. pneumoniae was a common pathogen in all 3 types of infection, and most isolates were MDR. Due to its large accessory genome including plasmids and chromosomal loci, K. pneumoniae isolates may act as opportunistic pathogens. Such strains infect critically ill and immunocompromised patients mostly, whereas other strains of K. pneumoniae (hypervirulent) may even infect healthy people in community  settings. Many of the virulent strains encode carbapenemases [15]. E. coli was a frequently identified pathogen from cIAI and cUTI, which is consistent with previous reports, and was also a frequent pathogen in LRTI. E. coli pneumonia is uncommon and may result from micro-aspiration of colonized upper airway secretions in severely ill patients; hence, it is a well-known cause of nosocomial pneumonia [16]. However, E. coli pneumonia may also be community-acquired in patients who have underlying diseases such as diabetes mellitus, alcoholism, chronic obstructive pulmonary disease, and E. coli UTI [17]. Our results confirm the results of previous studies in Egypt [3,18] that showed high rates of ESΒL and carbapenem resistance among ICU pathogens. For example, in the study of Talaat et al. [18], ESBL and carbapenem resistance were identified in 54% and 13.8% of E. coli isolates compared with 42.5% and 48.1% in K. pneumoniae isolates, respectively. Among the many types of ESΒLs reported, bla CTX-M-15 and bla CTX-M-14 are the most commonly identified worldwide as the genes encoding CTX-M enzymes (bla CTX-M ) can be horizontally mobilized by various genetic elements [19]. This reflects the situation in Egypt as well. AmpC genes were less    AK amikacin, AMC ampicillin/sulbactam, FEP cefipeme, CTX ceftriaxone, FOX cefoxtine, CTZ ceftazidieme, CRO cefotaxime, CST colistin, ETP ertapenem, IMP imipenem, LEV levofloxacin, TZP piperacillin/tazobactam frequently identified (9.7%), usually in combination with other resistance genes. CMY-2-like enzymes were the most predominant. Other detected genes were bla CMY-42 , bla DHA , and bla ACT-like . This is in keeping with recent reports on acquisition of plasmid-mediated cephalosporinase producing Enterobacteriaceae after a travel to the tropics and North Africa including Egypt [20,21]. The high rate of ESΒL in Egypt is essentially due to inappropriate use of antimicrobials in human and animal health care. Of the patients treated in public outpatient health care facilities, 49.8% received antibiotics, and antibiotics are still sold over the counter without prescription [22]. Moreover, there is extensive use of antimicrobials for prevention and treatment of infections in veterinary care, and ESΒL and AmpC resistance mechanisms were detected in veterinary E. coli isolates in Egypt [23]. This is an urgent public health problem especially with a growing body of evidence supporting foodborne transmission of resistance, especially from poultry, as poultry meat exhibits the highest levels of contamination by MDR bacteria [24,25].
The most important mechanism of carbapenem resistance is the production of carbapenemases; therefore, all isolates were investigated to identify the carbapenemase genes. We identified them in 45.4% of tested isolates; bla OXA followed by bla NDM genes dominated, while only 2 isolates of K. pneumoniae harbored bla KPC-2 genes. These results confirm previous small-scale reports that the bla NDM and bla OXA genes are the predominant in Egypt and Middle East [26].
The present study detected massive coexistence of different resistance genes among tested isolates. This coexistence could have contributed to the observed elevated variability in resistance phenotypes and genotypes among GNP in Egypt [15].
In conclusion, our study detected alarming rates of resistance and identified many resistance mechanisms in clinical GNP from Egyptian tertiary care hospitals. These high resistance rates highlight the importance of continuous monitoring of the resistance trends, adherence to infection control policies, and underscore urgently implementing a national antimicrobial stewardship plan in Egypt.
Funding information This study was part of the "Study for Monitoring Antimicrobial Resistance Trends" (SMART), funded by Merck & Co., Inc. in 192 sites in 54 countries. Merck & Co. supported laboratory supplies in hospitals, cost of transportation of isolates to the reference laboratory, and cost of testing in the reference laboratory.
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