CTX-M Genotyping among Cystitis Associated Cefotaxime-Resistant Uropathogenic Escherichia coli (CRUPEC)

Urinary tract infection (UTI) is the second most common bacterial infection and important public health problem of human among all age groups from neonate to geriatric. Uropathogenic Escherichia coli (UPEC) accounts for approximately 85% of community acquired UTIs and 50% of hospital acquired UTIs. The current study aimed to investigate dominant CTX-M genotypes among local UPEC isolated from patients with cystitis. Phenotypic detection of Cefotaxime-Resistant Uropathogenic Escherichia coli (CRUPEC) were performed by KirbyBauer disk diffusion method (CLSI 2016). The DNA were extracted and investigation of four resistance genes blaCTX-M by a multiplex PCR assay using specific primer pairs. The results of phylogenetic subgrouping of UPEC using multiplex PCR to detect chuA (279bp), yjaA (211bp) and TspE4.C2 (152bp) revealed that among 56 CRUPEC 47(83.93%) were ExPEC (37(66.07%) for B23, 6(10.72%) for B22, 3(5.36%) for D1 and 1(1.78%) for D2). InPEC compile 9(16.07%). Genotypic investigation of cefotaxime resistance among 56 CRUPEC were performed using specific four primer pairs to detect the genotypes of blaCTX-M clusters (I, II, III and IV). Among 65 CRUPEC, 36 (64.28%) were positive for for blaCTX-MI, 38(67.85%) for blaCTX-MII 26(46.43%) for blaCTX-MIII and 18(32.14%) for blaCTX-MIV (Figure 9). Concern possessing of isolates for more than one blaCTX-M genotypes were also reported in this study. Our results revealed that blaCTX-M were present in 50 (89.29%) of CRUPEC. Co-existence of more than on genotypes were reported in 38(67.86%). The results displayed that 40(80%) of blaCTX-M positive CRUPEC were belong to group B2, 4(8%) for group D, 3(6%) for group A and 3(6%) for group B1. The current study conclude the presence of blaCTX-M clusters (I, II, III, IV) as a main mechanisim or cefotaxime resistance among CRUPEC and coexistance of multible cluters within same isolates that may leads to emergence of new hybrides of blaCTX-M.

Urinary tract infection (UTI) is the one of the most common bacterial infection in humans and a major cause of morbidity and represent an important public health problem of all ages from neonate to geriatric age group (Mazzariol et al., 2017). Among of the most common infectious diseases, second ranking after respiratory tract infection is urinary tract infection which involve about 250 million people in developing countries annually. (Piranfar et al., 2014) . It is classified to bladder infection (cystitis) and kidney infection (pyelonephritis), which can be either symptomatic or asymptomatic (Prakasam et al., 2012). Although diûerent causative agents can be responsible for UTIs, bacteria are the major cause being responsible for more than 95% of UTI cases, The Escherichia coli (E.coli) accounts for approximately 85% of community acquired UTIs and 50% of hospital acquired UTIs (Ahmad et al., 2015). Clermont and colleagues developed a triplex PCR assay to detect the genes chuA, yjaA, and TspE4.C2 in 2000. Regarding the presence/ absence of these three genes, an E. coli strain could be classified into one of the main phylogroups intestinal pathogenic E. coli (InPEC) (include A, B1 group) while extraintestinal pathogenic E. coli (ExPEC) ( include B2, or D group) (Clermont et al.,  -II F  GCG ACC TGG TTA ACT ACA ATC C  351  55°C  R  CGG TAG TAT TGC CCT TAA GCC  bla CTX-M-III F  CGC TTT GCC ATG TGC AGC ACC  305  55°C  R GCT CAG TAC GAT CGA GCC bla CTX-M-IV F GCT GGA GAA AAG CAG CGG AG 474 62°C  , 2006). Cefotaxime is one of third generation cephalosporins with broad spectrum activity and regards one of the WHO Model List of Essential Medicines, contains the medications considered to be most effective and safe to meet the most important needs in a health system (WHO, 2015). It is widely used as post-surgery infection prophylaxis and also used for UTIs. Cefotaxime is one of the extend spectrum cephalosporin that resist hydrolysis by b-lactamase but it is sensitive to one of the extended spectrum b-lactamases (ESBLs). The name 'CTX' is an abbreviation for 'cefotaximase' and refers to the potent hydrolytic activity of these enzymes against cefotaxime.

Bacterial isolates
Fifty six UPEC isolates with confirmed resistance to cefotaxime (according to CLSI, 2016) were selected to study the phylogenetic subgroups using specific primer pairs to amplify chuA, yjaA and TspE2.C4 genes. Genotyping of blaCTX-M were performed using specific four pairs of primers for blaCTX-M-I, blaCTX-M-II, blaCTX-M-III and blaCTX-M-IV groups.

extraction of Genomic dna
Favor Prep TM Genomic DNA Mini Kit (Favorgen/Taiwan) was used to extract genomic DNA from E. coli isolates following the manufacturer's protocol.

ConClusion
The current study conclude the presence of blaCTX-M clusters (I, II, III, IV) as a main mechanisim or cefotaxime resistance among CRUPEC and coexistance of multible cluters within same isolates that may leads to emergence of new hybrides of blaCTX-M.
aCknowledGeMenT I am warmly thanks Dr. Noor S.K. Al-Khafaji for kind cooperation and many thanks for Dr. Naeem R. Al-Jebori for assistance in isolate collection.