Molecular Characterization of Carbapenemase-Producing Klebsiella pneumoniae Isolated from Egyptian Pediatric Cancer Patients Including a Strain with a Rare Gene-Combination of β-Lactamases

Figures & data

Table 1 List of Primers Used in the Current Study

Class	Target Gene	Sequence	Amplicon Size (bp)	Annealing Temperature (Ta)	Reference
Class A	blaKPC	F 5′-GCGGAACCATTCGCTAAACT-3′ R 5′-GGCGGCGTTATCACTGTATT-3′	312	55°C	This study
Class B	blaNDM	F 5′-GTTTGATCGTCAGGGATGGC-3′ R 5′-CCGTATGAGTGATTGCGGC-3′	558	55°C
	blaIMP	F 5′-GAAGAAGTTAACGGGTGGGG-3′ R 5′-GCATACGTGGGGATGGATTG-3′	227	52°C
	blaVIM	F 5′-GCAAGTCCGTTAGCCCATTC-3′ R 5′-CACCCCACGCTGTATCAATC-3′	208	53°C
Class D	blaOXA-48	F 5′-GGGCGTGGTTAAGGATGAAC-3′ R 5′-CATCTTGCTCATACGTGCCT-3′	166	55°C
ESBLs	blaCTX-M	F 5′-TTTGCGATGTGCAGTACCAGTAA-3′ R 5′-CGATATCGTTGGTGGTGCCATA-3′	544	55°C	This study
	blaSHV	F 5′-TGGTTATGCGTTATATTCGCC-3′ R 5′-GGTTAGGGTTGCCAGTGCT-3′	868	54°C
	blaTEM	F 5′-TCGCCGCATACACTATTCTCAGAATGA-3′ R 5′-ACGCTCACCGGCTCCAGATTTAT-3′	445	59°C
ERIC primers	1- (5′-ATGTAAGCTCCTGGGGATTCAC-3′) 2- (5′-AAGTAAGTGACTGGGGTGAGCG-3′)			45°C	^Citation62

Table 2 Relationship Between Different Carbapenemases and β-Lactamases Among the Klebsiella pneumoniae Isolates and Patient’s Clinical Data

Characteristics	Total	blaOXA-48	p-value	blaNDM	p-value	blaVIM	p-value	blaIMP	p-value	blaSHV	p-value	blaTEM	p-value	blaCTX-M	p-value
Gender
Male	38	16	0.7	5	0.53	1	0.37	1	0.86	11	0.2	16	0.86	14	0.76
Female	30	14		7		0		1		5		12		10
Ward
ICU	39	21	0.32	4	0.01^a	1	0.9	2	0.92	11	0.7	10	0.02^a	16	0.6
Haematology	16	4		5		0		0		3		9		5
Solid tumour	3	1		3		0		0		1		3		1
Surgery	6	3		0		0		0		1		4		2
Stepdown	4	1		0		0		0		0		2		0
Specimen
Blood CVP Urine CSF BAL Wound	40 17 2 1 3 5	18 5 0 0 3 4	0.1	5 4 1 0 1 1	0.94	1 0 0 0 0 0	0.98	1 1 0 0 0 0	0.92	13 3 0 0 0 0	0.35	15 7 2 1 1 2	0.46	16 6 0 0 2 0	0.32

Note: ^aValues with p ≤0.05 are considered significantly different.

Abbreviations: CVP, central venous portal; BAL, bronchoalveolar lavage; CSF, cerebrospinal fluid.

Figure 1 Frequencies of the antimicrobial resistance among the carbapenem-resistant K. pneumoniae isolates. A bar graph presenting the percentages of the resistance to 14 different antibiotics among the 52 carbapenem-resistant K. pneumoniae. The chart was generated using GraphPad Prism v5.

Figure 2 Distribution of carbapenemases and ESBLs encoding genes among the carbapenem-resistant K. pneumoniae isolates A bar graph showing the percentages of carbapenem-resistant K. pneumoniae isolates (n=52) harboring genes encoding carbapenemases (bla_OXA-48, bla_NDM, bla_VIM, and bla_IMP) and ESBLs (bla_CTX-M, bla_TEM, and bla_SHV) either in combination (grey bars) or as single genes (white bars).

Figure 3 ERIC-PCR profiles of carbapenem-resistant K. pneumoniae harboring the bla_OXA-48 gene A dendrogram was generated from the ERIC-PCR typing of 30 K. pneumoniae clinical isolates. The analysis was performed using BioNumerics fingerprint data software v7.62 and the unweighted pair group method with arithmetic means (UPGMA) clustering method and the dice similarity coefficient with 1% optimization and 1.5% position tolerance. The different clusters are designated by Roman numerals (I–III). The numbers written on the branches indicate the similarity percentage of the different isolates. On the right side of the dendrogram detailed results including the number of each isolate, clusters, date of isolation, and hospital wards. The alphabets indicate the type of the hospital wards; a: hematology ward, b: solid tumor ward c: surgery ward.

Abbreviations: IP, in-patient; ICU, intensive care unit; stepdown unit, intermediate care unit.

Figure 4 PFGE typing of the bla_OXA-48-harbouring carbapenem-resistant K. pneumoniae isolates A dendrogram was generated from the PFGE patterns of XbaI digested chromosomal DNA of 30 carbapenem-resistant K. pneumoniae clinical isolates. The comparison was performed with the BioNumerics fingerprint data software v7.62 using the UPGMA clustering method and the dice similarity coefficient with 1% optimization and 1.5% position tolerance. PFGE pulsotypes (P) were assigned to isolates according to Tenover criteria.²⁷ The numbers written on the branches indicate the similarity percentage of the different pulsotypes. The black frame indicates the isolates that were indistinguishable and assumed to be the same clone. The different clusters are designated by letters (A–E). On the right side of the dendrogram detailed results for PFGE including the code of each isolate, pulsotypes, date of isolation, hospital wards, and screened carbapenemases and ESBLs determinants (bla_OXA-48, bla_NDM, bla_IMP, bla_VIM, bla_CTX-M, bla_TEM, and bla_SHV). The (+) and (-) signs indicate present and absent, respectively. The alphabets indicate the type of the hospital wards; a: hematology ward, b: solid tumor ward c: surgery ward.

Abbreviations: IP, in-patient; ICU, intensive care unit; stepdown unit, intermediate care unit.

Versalovic J, Koeuth T, Lupski R. Distribution of repetitive DNA sequences in eubacteria and application to fingerprinting of bacterial genomes. Nucleic Acids Res. 1991;19(24):6823–6831. doi:10.1093/nar/19.24.68231762913

 PubMed Web of Science ®Google Scholar

Tenover FC, Arbeit RD, Goering RV, et al. Interpreting chromosomal DNA restriction patterns produced by pulsed-field gel electrophoresis: criteria for bacterial strain typing. J Clin Microbiol. 1995;33(9):2233–2239. doi:10.1128/jcm.33.9.2233-2239.19957494007

 PubMed Web of Science ®Google Scholar