Molecular Characterization and Mutational Analysis of Fluoroquinolones and Tetracycline Resistant Genes of <i>Escherichia coli</i> Isolated from UTI Patients

Antibiotic resistance is more challenging in third world countries due to irrational use of antibiotics and poor antimicrobial resistance surveillance. The current study is aimed to determine the molecular characterization and mutational analysis of antibiotic resistant genes of E. coli isolated from UTIs patients. A total of 112 E. coli isolates were recovered from UTI suspected patients from Khyber Teaching Hospital (KTH), Peshawar. The collected samples were identified phenotypically by API-10S strips and confirmed by Polymerase Chain Reaction. The selected antibiotic resistant genes were detected by PCR and subsequently sequenced by Next Generation Sequencing. The results of antibiogram revealed that the E. coli isolates were resistant to various antibiotics; Ampicillin, Cotrimoxazole and Ciprofloxacin while showed good results against Tigecycline, Meropenem and Cefoperazone-Sulbactam. Molecular analysis showed that 72 isolates were positive for GyrA gene, GyrB gene 46, tetB 28 and tetR gene 6. The results of mutational analysis revealed that gyrA gene have 3 amino acid substitutions (S83L, D87N and A828S), gyrB have 2 amino acid substitutions (E185D and Q554K), tetB also have 2 amino acid substitutions (H204R and R334S) while only one amino acid substitution in tetR gene (L108P) was observed. The current study reported the novel mutation in gyrB gene at codon554, due to which Glutamine (Q) is substituted by Lysine (K). The high frequency of GyrA gene was observed in 72 E. coli isolates followed by GyrB gene 46, tetB 28 and tetR gene 6 while novel mutation was detected in gyrB gene at codon554.

Keywords:
E.coli; antibiogram; USP; GyrA; GyrB; tetB and tetR genes; NGS; novel mutation.

Targeted Genes	Specific Primers	Product Size (bp)	Annealing Temperature	Cycles
USP	F: ATGCTACTGTTTCCGGGTAGTGTGT R: CATCATGTAGTCGGGGCGTAACAAT	884	56^OC for 1 min	35
Tet R	F: AGAATCGGTTATTGATGCGGC R: TCAGCAAAAGGGGATGATAAGT	640 bp	53^OC for 30 sec	35
Tet B	F: CCTCAGCTTCTCAACGCGTG R: GCACCTTGCTCATGACTCTT	634 bp	49^OC for 30 sec	35
Gyr A	F: ATGAGCGACCTTGCGAGAG R: TCCACTTCCGGAGCGATTTC	2600 bp	59 ^OC for 30 sec	35
Gyr B	F: GTAAGCGCCCGGGTATGTAT R: TCGATATTCGCCGCTTTCAG	2350 bp	52^OC for 30 sec	35

Antibiotics Names	Symbols	Sensitive (n)	Percentage (%)	Resistant (n)	Percentage (%)
Amoxicillin - Clavulanate	AMC	80	71.5	32	28.5
Ampicillin	AMP	14	12.5	98	87.5
Cefoperazone-sulbactam	SCF	93	83.0	19	17.0
Piperacillin-Tazobactam	TZP	86	76.8	26	23.2
Cefepime	FEP	88	78.6	24	21.4
Cefotaxime	CTX	70	62.5	42	37.5
Ceftazidime	CAZ	72	64.3	40	35.7
Aztreonem	ATM	74	66.1	38	33.9
Meropenem	MEM	95	84.8	17	15.2
Amikacin	AK	87	77.7	25	22.3
Doxycycline	DO	40	35.7	72	64.3
Ciprofloxacin	CIP	38	33.9	74	66.1
Levofloxacin	LEV	44	39.3	68	60.7
Cotrimoxazole	SXT	23	20.5	89	79.5
Chloramphenicol	C	64	57.1	48	42.9
Fosfomycin	FOS	80	71.4	32	28.6
Tigecycline	TGC	112	100	0	0

Antibiotic resistant genes	Frequency (n)	Percentage (%)
Tet R	6	5.4
Tet B	28	25.0
Gyr A	72	64.3
Gyr B	46	41.1

Nucleotide position	Reference amino acid	Altered amino acid	Amino acid position
Non synonymous mutation of gene GyrA
248	S (TCG)	L (TTG)	83
259	D (GAC)	N (AAC)	87
2482	A (GCG)	S (TCG)	828
Non synonymous mutation of gene GyrB
555	E (GAA)	D (GAC)	185
1660	Q (CAG)	K (AAG)	554
Non synonymous mutation of gene TetB
611	H (CAC)	R (CGC)	204
1002	R (AGA)	S (AGT)	334
Synonymous mutation of gene TetB
942	CGA - CGG	R	314
Non synonymous mutation of gene TetR
323	L (CTT)	P (CCT)	108
Synonymous mutation of gene TetR
279	TCT - TCC	S	93

Instituto de Tecnologia do Paraná - Tecpar Rua Prof. Algacyr Munhoz Mader, 3775 - CIC, 81350-010 Curitiba PR Brazil, Tel.: +55 41 3316-3052/3054, Fax: +55 41 3346-2872 - Curitiba - PR - Brazil
E-mail: babt@tecpar.br

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[1] Editor-in-Chief: Alexandre Rasi Aoki