The emergence and spread of carbapenem-resistant gram-negative bacteria poses a serious threat to human health worldwide. Currently, little is known about the molecular mechanisms underlying carbapenem resistance and their prevalence among gram-negative bacteria in Egypt. In this study, we analyzed carbapenemase production in gram-negative bacteria isolated from hospitalized patients in Egypt in 2014. All isolates were subjected to phenotypic and genotypic susceptibility testing for carbapenem resistance. Our results indicated a high level of carbapenem-resistant gram-negative bacteria in Egypt, with 50.8% of the isolates harboring at least one carbapenem resistance gene. OXA-48-like and NDM-1 were the most prevalent carbapenemases, being detected in 49.2%, and 47.7% of carbapenemase-positive isolates, respectively, whereas Verona integron-encoded metallo-β-lactamase (VIM) was detected in only 26.2% of carbapenemase-positive isolates. This study reports for the first time carbapenemase-producing Serratia marcescens, Morganella morganii, and bla-producing Pseudomonas aeruginosa in Egypt. It is also the first demonstration of the coexistence of different carbapenemases, being detected in 21.5% of carbapenemase-positive isolates. Effective antibiotic supervision, regional surveillance, and early detection of carbapenemase producers are imperative to prevent their future spread to epidemic levels.

C olistin is considered a last resort for the treatment of infections caused by carbapenemase-producing members of the family Enterobacteriaceae. Recently, a transferable plasmid conferring resistance to colistin was discovered in Escherichia coli and Klebsiella pneumoniae from China (1) and was then reported in various bacteria from many other countries (2-4). This spread of antibiotic resistance heralds a return to the preantibiotic era, especially with no new antibiotics in the pipeline. The plasmid-mediated colistin resistance gene mcr-1 has been identified mainly in bacterial species from animals and animal products (pork and chicken meat) (1, 2). However, it was also detected in humans, i.e., patients (1-4), asymptomatic people (5), and some people by whom it was community acquired (6). Therefore, we used PCR and DNA sequencing to screen for the presence of mcr-1 in 241 nonduplicate Gram-negative clinical bacterial isolates collected from hospitals in different cities in Egypt during 2015. A universal primer pair, CLR5-F (5=-CGGTCAGTCCGTT TGTTC-3=) and CLR5-R (5=-CTTGGTCGGTCTGTAGGG-3=), was used for screening as previously described (1). In addition, the entire mcr-1 region was amplified and sequenced with MCR-1-F2 (5=-CTCATGATGCAGCATACTTC-3=) and MCR-1-R2 (5=-CGAATGGAGTGTGCGGTG-3=), which were based on the consensus sequences that flank mcr-1 (GenBank accession no. KP347127). Analysis showed that mcr-1 was present in only one E. coli isolate (SP-1), which was from the sputum of a patient with bacteremia who was hospitalized in the intensive care unit of a Cairo City hospital with no history of traveling abroad. The MIC of both colistin and polymyxin B for this isolate was 16 mg/liter. E. coli SP-1 had phenotypic resistance to colistin, ciprofloxacin, nalidixic acid, kanamycin, tetracycline, chloramphenicol, ceftriaxone, ampicillin, and cefotaxime but was susceptible to carbapenems. Multilocus sequence typing (MLST) indicated that E. coli SP-1 belonged to sequence type 1011 (ST1011), which differed from sequence types identified for clinical E. coli isolates harboring mcr-1 in Denmark (ST744) (2) and Cambodia (ST354) (3). According to the MLST database (http://mlst.ucc.ie/mlst/mlst /dbs/Ecoli), E. coli ST1011 was reported in humans in Egypt and in cats and swine in Germany. Of note, E. coli ST1011 was detected previously as an avian fecal E. coli strain in Egypt (7) and it was found to be shared by avian pathogenic E. coli and human extraintestinal pathogenic E. coli by phylogenetic grouping in Brazil (8). Also, it was found as CTX-M-64-harboring E. coli isolates from chickens in China (9). Therefore, E. coli ST1011 may have potential zoonotic importance. Southern hybridization showed that mcr-1 was located on a plasmid of Ͼ90 kb. This plasmid was successfully transferred to E. coli J53 recipient cells through conjugation.

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The plasmid-mediated tet(X7) conferring high-level tigecycline resistance was identified in five mcr-1.1-positive Escherichia coli strains [ST10 (n=3), and ST155 (n=2)] isolated from chickens, Egypt. Two fosfomycin-resistant fosA4-carrying IncFII plasmids (⁓79 kb in size) were detected. Transposase ISCR3 (IS91 family) is syntenic with tet(X7) in all isolates suggesting its role in the mobilization of tet(X7). To our knowledge, this is the first global report of (ST4)-IncHI2 plasmids co-carrying tet(X7) and mcr-1.1 from chickens.

This study describes the first full genomic sequence of an mcr-9 and blaVIM-4-carrying multidrug-resistant Enterobacter hormaechei clinical isolate from Egypt. The strain was isolated in April 2015 from the sputum of a patient in Cairo, Egypt. The mcr-9 and blaVIM-4 genes were identified by PCR screening and DNA sequencing; the isolate was subjected to antimicrobial susceptibility testing, conjugation experiments, and whole genomic sequencing. mcr-9 and blaVIM-4 were carried by an IncHI2 plasmid, pAMS-38a (281,121 bp in size); the plasmid also carried genes conferring resistance against sulfonamides (sul1), quinolones (qnrA1), trimethoprim (dfrA1), β-lactams (blaTEM-1B), aminoglycosides (aac (6’)-II, aadA23, aadA2b, and ant(2’’)-Ia). The strain was susceptible to colistin (MIC, <0.25 μg/mL); this could be due to the absence of the qseC/qseB regulatory system located downstream of mcr-9 in Enterobacterales, which is involved in the induction of colistin-resistance. The genetic context of mcr-9 and blaVIM-4 was identified as IS1-mcr-9-IS903-pcoS-∆pcoE-rcnA and intI1-blaVIM-4—aac (6’)-II-dfrA1-∆aadA23-smr-ISPa21-qacE∆1, respectively. This is the first report of an mcr-9 and blaVIM-4 /IncHI2-carrying multidrug-resistant E. hormaechei clinical isolate from Africa and the Middle East. Plasmids of the IncHI2 group and the two insertion sequences (IS1, and IS903) might be the main vehicles for dissemination of mcr-9. Further screening for mcr-9 is essential for identifying its incidence and to prevent its dissemination.

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