The First Egyptian Report Showing the Co-Existence of bla_NDM-25, bla_OXA-23, bla_OXA-181, and bla_GES-1 Among Carbapenem-Resistant K. pneumoniae Clinical Isolates Genotyped by BOX-PCR

Figures & data

Table 1 Primers and Cyclic Conditions Used in PCR Targeting Three Classes of Carbapenemases and BoxA Region Among CRKP Clinical Isolates

Primer	Target Gene	Primer Sequence	Amplicon Size (bp)	Cycling Condition	Reference
KL_Iden	phoE	F: TGGCCCGCGCCCAGGGTTCGAAA R: GATGTCGTCATCGTTGATGCCGAG	368	Initial denaturation at 95°C for 15 min, then 30 cycles of 95°C for 1 min, 60°C for 1min and 72°C for 5 min and one cycle of final elongation at 72°C.	^Citation16
BOX-AIR	BoxA region	CTACGGCAAGGCGACGCTGACG	-	Initial denaturation at 95°C for 15 min, then 35 cycles of 95°C for 1 min, 40°C for 1min and 72°C for 5 min and one cycle of final elongation at 72°C.	^Citation54
KPC	blaKPC	F: GTATCGCCGTCTAGTTCTGC R: GGTCGTGTTTCCCTTTAGCC	637	Initial denaturation at 95°C for 15 min, then 30 cycles of 95°C for 1 min, 55°C for 1min and 72°C for 5 min and one cycle of final elongation at 72°C.	^Citation55
GES	blaGES	F: GCTTCATTCACGCACTATT R: CGATGCTAGAAACCGCTC	323	Initial denaturation at 95°C for 15 min, then 30 cycles of 95°C for 1 min, 59°C for 1min and 72°C for 5 min and one cycle of final elongation at 72°C.	^Citation55
SME	blaSME	F: ACTTTGATGGGAGGATTGGC R: ACGAATTCGAGCATCACCAG	551	Initial denaturation at 95°C for 15 min, then 30 cycles of 95°C for 1 min, 66.7°C for 1min and 72°C for 5 min and one cycle of final elongation at 72°C.	^Citation56
IPM -Family	blaIPM	F: GGAATAGRRTGGCTTAAYT R: GGTTTAAYAAARCAMCCACC	233	Initial denaturation at 95°C for 15 min, then 30 cycles of 95°C for 1 min, 63°C for 1min and 72°C for 5 min and one cycle of final elongation at 72°C.	^Citation55
VIM-Family	blaVIM	F: GTTTGGTCGCATATCGCAAC R: GAGCAAKTCYAGACCGCCC	591	Initial denaturation at 95°C for 15 min, then 30 cycles of 95°C for 1 min, 55°C for 1min and 72°C for 5 min and one cycle of final elongation at 72°C.	^Citation55
NDM	blaNDM	F: GGTTTGGCGATCTGGTTTTC R: CGGAATGGCTCATCACGATC	621	Initial denaturation at 95°C for 15 min, then 30 cycles of 95°C for 1 min, 55°C for 1min and 72°C for 5 min and one cycle of final elongation at 72°C.	^Citation55
OXA-23	blaOXA-23	F: GATCGGATTGGAGAACCAGA R: ATTTCTGACCGCATTTCCAT	501	Initial denaturation at 95°C for 15 min, then 30 cycles of 95°C for 1 min, 66.6°C for 1min and 72°C for 5 min and one cycle of final elongation at 72°C	^Citation55
OXA-40	blaOXA-40	F: GGTTAGTTGGCCCCCTTAAA R: AGTTGAGCGAAAAGGGGATT	249	Initial denaturation at 95°C for 15 min, then 30 cycles of 95°C for 1 min, 66.6°C for 1min and 72°C for 5 min and one cycle of final elongation at 72°C	^Citation55
OXA-48	blaOXA-48	F: GCTTGATCGCCCTCGATT R: GATTTGCTCCGTGGCCGAAA	281	Initial denaturation at 95°C for 15 min, then 30 cycles of 95°C for 1 min, 60.5°C for 1min and 72°C for 5 min and one cycle of final elongation at 72°C.	^Citation55
OXA-51	blaOXA-51	F: TAATGCTTTGATCGGCCTTG R: TGGATTGCACTTCATCTTGG	353	Initial denaturation at 95°C for 15 min, then 30 cycles of 95°C for 1 min, 60.5°C for 1min and 72°C for 5 min and one cycle of final elongation at 72°C.	^Citation55
OXA-58	blaOXA-58	F: AAGTATTGGGGCTTGTGCTG R: CCCCTCTGCGCTCTACATAC	599	Initial denaturation at 95°C for 15 min, then 30 cycles of 95°C for 1 min, 60.5°C for 1min and 72°C for 5 min and one cycle of final elongation at 72°C.	^Citation55
OXA-181	blaOXA-181	F: ATGCGTGTATTAGCCTTATCG R: AACTACAAGCGCATCGAGCA	888	Initial denaturation at 95°C for 15 min, then 30 cycles of 95°C for 1 min, 55°C for 1min and 72°C for 5 min and one cycle of final elongation at 72°C.	^Citation55

Notes: R=(A⁄G), Y=(C⁄T), K=(G⁄T) and M=(A⁄C). Boldface in this table indicates to forward and reverse primers and annealing temperature.

Abbreviations: A, adenine; G, guanine; C, cytosine; T, thymine; CRKP, carbapenem-resistant K. pneumoniae.

Figure 1 Phenotypic tests for detection and characterization of carbapenemase production. (A); Modified Hodge test (K02 to K17 denotes to isolate code), (B); Carba NP test (row A; denotes to solution A, row B; denotes to solution B, row C; denotes to solution A containing tested isolates, rows D & E; denote to solution B containing tested isolates, red color indicates negative result, yellow color indicates positive result) (C); Combined disk synergy test (disk A; meropenem, disk B; meropenem/clavulanic acid, disk C; meropenem/sulbactam, disk D; meropenem/tazobactam, disk E; meropenem/phenylboronic acid, disk F; meropenem/EDTA, disk G; meropenem/mercaptopropionic acid, disk H; meropenem/sodium chloride).

Abbreviations: NP, Nordmann/Poirel; EDTA, ethylenediaminetetraacetic acid.

Table 2 Antimicrobial Susceptibility Pattern of CRKP Clinical Isolates

Susceptibility Pattern							MIC (µg/mL)
Antibiotic	Sensitive		Intermediate		Resistant		^MIC50	^MIC90
	No.	%	No.	%	No.	%
AM	–	0.00	–	0.00	19	100.00	>1024	>1024
AMS	–	0.00	–	0.00	19	100.00	>1024	>1024
AMX	–	0.00	–	0.00	19	100.00	>1024	>1024
AMC	–	0.00	–	0.00	19	100.00	>1024	>1024
CEZ	–	0.00	–	0.00	19	100.00	1024	>1024
CE	–	0.00	–	0.00	19	100.00	>1024	>1024
CXM	–	0.00	–	0.00	19	100.00	1024	>1024
CTX	–	0.00	–	0.00	19	100.00	1024	>1024
CAZ	1	5.26	1	5.26	17	89.48	>1024	>1024
CPZ	–	0.00	–	0.00	19	100.00	>1024	>1024
CPZS	–	0.00	–	0.00	19	100.00	1024	>1024
CRO	1	5.26	–	0.00	18	94.74	512	>1024
FEP	–	0.00	–	0.00	19	100.00	256	512
ATM	1	5.26	–	0.00	18	94.47	128	1024
IPM	1	5.26	2	10.53	16	84.21	4	16
MEM	–	0.00	1	5.26	18	94.74	16	64
ERT	–	0.00	–	0.00	19	100.00	NA	NA
TET	5	26.32	1	5.26	13	68.42	32	1024
TEG	18	94.74	–	0.00	1	5.26	≤0.5	≤0.5
CN	4	21.05	–	0.00	15	78.95	128	>1024
AK	6	31.58	–	0.00	13	68.42	>1024	>1024
THI	1	5.26	–	0.00	18	94.74	>1024	>1024
CIP	–	0.00	1	5.26	18	94.74	64	128
LEV	2	10.53	1	5.26	16	84.21	32	128

Abbreviations: AM, ampicillin; AMS, ampicillin/sulbactam; AMX, amoxicillin; AMC, amoxicillin/clavulanic acid; CEZ, cefazolin; CE, cephradine; CXM, cefuroxime; CTX, cefotaxime; CAZ, ceftazidime; CPZ, cefoperazone; CPZS, cefoperazone/sulbactam; CRO, ceftriaxone; FEP, cefepime; ATM, aztreonam; IPM, imipenem; MEM, meropenem; ERT, ertapenem; TET, tetracycline; TEG, tigecycline; CN, gentamicin; AK, amikacin; THI, thiamphenicol CIP, ciprofloxacin; LEV, levofloxacin. Boldface in this table indicates %.

Table 3 Phenotypic and Genotypic Screening Profiles of CRKP Clinical Isolates

Isolate No. Carbapenemase Profile
	Phenotypic	Genotypic
KP01	MHT, CNP	blaOXA-48
KP02	-	blaOXA-48,blaOXA181
KP03	MHT	blaGES, blaNDM,blaOXA-23blaOXA-48,blaOXA-51,blaOXA181
KP05	MHT	blaOXA-48
KP06	MHT	blaOXA-48
KP07	CNP	blaOXA-48
KP08	MHT	blaOXA-48,blaOXA181
KP09	CNP	blaOXA-48
KP12	MHT, CNP	blaOXA-48
KP13	CNP	blaOXA-48
KP14	CNP	blaNDM,blaOXA-48
KP16	MHT, CNP	blaNDM,blaOXA-48,blaOXA181
KP17	MHT, CNP	blaOXA-48
KP18	MHT, CNP	blaOXA-51
KP19	MHT, CNP	blaNDM
KP21	MHT, CNP	-
KP22	MHT, CNP	blaNDM
KP23	MHT	blaOXA-48
KP24	CNP	-

Abbreviations: MHT, modified Hodge test; CNP, Carba Nordmann/Poirel; CRKP, carbapenem-resistant K. pneumoniae; KP, Klebsiella pneumoniae isolate code No.

Table 4 Carbapenemase Inhibitor Profile of CRKP Clinical Isolates

Carbapenemase Inhibitor Profile
Isolate No.	Class A Inhibitors				Class A and Specific blaKPC Inhibitor	Class B Inhibitor		Class D Inhibitor
	CA	Sul	Taz	PBA	PBA	EDTA	MPA	NaCl
KP01	+	+	+	–	+	+	+	–
KP02	–	+	+	–	–	+	+	–
KP03	+	+	+	–	+	+	+	–
KP05	–	–	–	–	+	–	+	–
KP06	–	–	–	–	–	–	+	–
KP07	+	+	+	–	+	+	+	–
KP08	+	+	+	–	+	+	+	+
KP09	+	+	+	–	–	+	–	–
KP12	–	–	–	–	–	+	–	–
KP13	–	–	–	–	–	+	–	–
KP14	+	+	+	–	+	+	+	–
KP16	–	–	–	–	–	+	–	–
KP17	–	+	+	–	–	+	–	–
KP18	+	+	+	–	+	+	+	–
KP19	–	–	+	–	+	+	+	–
KP21	–	+	+	–	–	+	+	+
KP22	+	+	+	–	–	+	–	–
KP23	–	–	+	–	–	–	+	–
KP24	–	+	+	–	+	+	+	–

Abbreviations: CA, clavulanic acid; Sul, sulbactam; Taz, tazobactam; phenyl boronic acid; EDTA, ethylenediaminetetraacetic acid; MPA, mercaptopropionic acid; NaCl, sodium chloride; PBA, phenyl boronic acid; CRKP, carbapenem resistant K. pneumoniae; KP, Klebsiella pneumoniae isolate code No.

Table 5 Detailed Resistance and Genetic Profiles of CRKP Clinical Isolates

Isolate No.	Antibiotic Resistance Profile	No. of Resistant Antibiotics	Detected Carbapenemases	No. of Detected Carbapenemases
KP01	AMP, SAM, AMX, AMC, CEZ, CE, CXM, CTX, CAZ, CPZ, CPS, FEP, IPM, MEM, ERT, CN, AK, CIP	18	blaOXA-48	1
KP02	AMP, SAM, AMX, AMC, CEZ, CE, CXM, CTX, CAZ, CPZ, CPS, CRO, FEP, ATM, IPM, MEM, ERT, TET, CN, AK, THI, CIP, LEV	23	blaOXA-48, blaOXA181	2
KP03	AMP, SAM, AMX, AMC, CEZ, CE, CXM, CTX, CAZ, CPZ, CPS, CRO, FEP, ATM, IPM, MEM, ERT, TET, CN, AK, THI, CIP, LEV	23	blaGES,blaNDM,blaOXA-23,blaOXA-48,blaOXA-51,blaOXA181	6
KP05	AMP, SAM, AMX, AMC, CEZ, CE, CXM, CTX, CAZ, CPZ, CPS, CRO, FEP, ATM, MEM, ERT, TET, CN, THI, CIP, LEV	21	blaOXA-48	1
KP06	AMP, SAM, AMX, AMC, CEZ, CE, CXM, CTX, CAZ, CPZ, CPS, CRO, FEP, ATM, IPM, MEM, ERT, THI, CIP, LEV	20	blaOXA-48	1
KP07	AMP, SAM, AMX, AMC, CEZ, CE, CXM, CTX, CAZ, CPZ, CPS, CRO, FEP, ATM, IPM, MEM, ERT, TET, CN, THI, CIP, LEV	22	blaOXA-48	1
KP08	AMP, SAM, AMX, AMC, CEZ, CE, CXM, CTX, CPZ, CPS, CRO, FEP, ATM, IPM, MEM, ERT, TET, CN, AK, THI, CIP, LEV	22	blaOXA-48,blaOXA181	2
KP09	AMP, SAM, AMX, AMC, CEZ, CE, CXM, CTX, CAZ, CPZ, CPS, CRO, FEP, ATM, IPM, MEM, ERT, TET, TEG, CN, AK, THI, CIP, LEV	24	blaOXA-48	1
KP12	AMP, SAM, AMX, AMC, CEZ, CE, CXM, CTX, CAZ, CPZ, CPS, CRO, FEP, ATM, IPM, MEM, ERT, TET, CN, AK, THI, CIP, LEV	23	blaOXA-48	1
KP13	AMP, SAM, AMX, AMC, CEZ, CE, CXM, CTX, CAZ, CPZ, CPS, CRO, FEP, ATM, IPM, MEM, ERT, TET, CN, AK, THI, CIP, LEV	23	blaOXA-48	1
KP14	AMP, SAM, AMX, AMC, CEZ, CE, CXM, CTX, CAZ, CPZ, CPS, CRO, FEP, ATM, IPM, MEM, ERT, TET, CN, AK, THI, CIP, LEV	23	blaNDM,blaOXA-48	2
KP16	AMP, SAM, AMX, AMC, CEZ, CE, CXM, CTX, CAZ, CPZ, CPS, CRO, FEP, ATM, IPM, MEM, ERT, CN, AK, THI, CIP, LEV	22	blaNDM,blaOXA-48,blaOXA181	3
KP17	AMP, SAM, AMX, AMC, CEZ, CE, CXM, CTX, CAZ, CPZ, CPS, CRO, FEP, ATM, IPM, MEM, ERT, CN, AK, THI, CIP, LEV	22	blaOXA-48	1
KP18	AMP, SAM, AMX, AMC, CEZ, CE, CXM, CTX, CAZ, CPZ, CPS, CRO, FEP, ATM, IPM, MEM, ERT, TET, THI, CIP, LEV	21	blaOXA-51	1
KP19	AMP, SAM, AMX, AMC, CEZ, CE, CXM, CTX, CAZ, CPZ, CPS, CRO, FEP, ATM, IPM, MEM, ERT, TET, THI, CIP, LEV	21	blaNDM	1
KP21	AMP, SAM, AMX, AMC, CEZ, CE, CXM, CTX, CAZ, CPZ, CPS, CRO, FEP, ATM, IPM, MEM, ERT, TET, CN, AK, THI, CIP, LEV	23	-	-
KP22	AMP, SAM, AMX, AMC, CEZ, CE, CXM, CTX, CAZ, CPZ, CPS, CRO, FEP, ATM, IPM, MEM, ERT, TET, THI, CIP	20	blaNDM	1
KP23	AMP, SAM, AMX, AMC, CEZ, CE, CXM, CTX, CAZ, CPZ, CPS, CRO, FEP, ATM, IPM, MEM, ERT, TET, CN, AK, THI, CIP, LEV	23	blaOXA-48	1
KP24	AMP, SAM, AMX, AMC, CEZ, CE, CXM, CTX, CAZ, CPZ, CPS, CRO, FEP, ATM, IPM, MEM, ERT, CN, AK, THI, CIP, LEV	22	-	-

Abbreviations: AMP, ampicillin; SAM, ampicillin/sulbactam; AMX, amoxicillin; AMC, amoxicillin/clavulanic acid; CEZ, cefazolin; CE, cephradine; CXM, cefuroxime; CTX, cefotaxime; CAZ, ceftazidime; CPZ, cefoperazone; CPS, cefoperazone/sulbactam; CRO, ceftriaxone; FEP, cefepime; ATM, aztreonam; IPM, imipenem; MEM, meropenem; ERT, ertapenem; TET, tetracycline; TEG, tigecycline; CN, gentamicin; AK, amikacin; THI, thiamphenicol; CIP, ciprofloxacin, LEV, levofloxacin; CRKP, carbapenem resistant; K, pneumoniae, KP, Klebsiella pneumoniae isolate code No.

Figure 2 Multiple sequence alignment of sequenced bla_NDMwith the reference sequence bla_NDM-1 KP036459.1; the arrow (A) is pointing to thymine nucleotide in reference sequence bla_NDM-1 KP036459.1; the arrow (B) is pointing to non-mutated thymine nucleotide at position 558 in the query sequence of investigated bla_NDM-1; the arrow (C) is pointing to mutated guanine nucleotide at position 558 in the query sequence of investigated bla_NDM-25.

Figure 3 Representative DNA fingerprint pattern generated by BOX-PCR for carbapenem-resistant K. pneumoniae clinical isolates. L, 100 bp DNA ladder; lanes 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, and 18 are the code No. of CRKP clinical isolates.

Figure 4 Unweighted pair-group method with arithmetic average (UPGMA) dendrogram based on Dice similarity for banding profile generated by BOX-PCR.

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