Distribution of mcr-1 Harboring Hypervirulent Klebsiella pneumoniae in Clinical Specimens and Lytic Activity of Bacteriophage KpnM Against Isolates

Figures & data

Figure 1 (A) purified purple colonies of K. pneumoniae on Klebsiella selective agar. (B) Pink mucoid colonies of K. pneumoniae on MacConkey agar. (C) Identification of hypervirulent phenotype through string test. (D) Results of Antimicrobial Susceptibility testing (AST) by disk diffusion method.

Table 1 Distribution of Virulence Genes and ARGs Among hvKp Isolates Along with the Details of the Primer Used in the Study

Name of the Gene	Primer’s Sequence	Annealing Temp (°C)	Percentage Prevalence Out of 28 hvKp
Virulence Genes
wzy (K1)	GGTGCTCTTTACATCATTGC	48	09 (32%)
	GCAATGGCCATTTGCGTTAG
rmpA	ACTGGGCTACCTCTGCTTCA	49	08 (28%)
	CTTGCATGAGCCATCTTTCA
fimH	GCTCTGGCCGATACCTACCGACGG	48	24 (85%)
	GCGAATAGATAACGTCGCCTGACGG
ARGs
blaTEM	TCAACATTTCCGTGTCG	42	25 (89%)
	CTGACAGTTACCAATGCTTA
blaSHV	ATGCGTTATATTCGCCTGTG	47	26 (93%)
	AGATAAATCACCACAATGCGC
PANCTX-M	GGATATCGTTGGTGGTGCCATA	60	25 (89%)
	TTTGCGATGTGCAGTACCAGTAA
blaKPC	TGCAGAGCCCAGTGTCAGTTT	52	08 (28%)
	CGCTCTATCGGCGATACCA
blaIMP	GGAATAGAGTGGCTTAATTCTC	55	02 (7%)
	CCAAACCACTACGTTATC
blaVIM	GATGGTGTTTGGTCGCATA	52	03 (10%)
	CGAATGCGCAGCACCAG
blaGIM	TCGACACACCTTGGTCTGAA	52	Not Detected
	AACTTCCAACTTTGCCATGC
blaNDM-1	TGCCCAATATTATGCACCCGG	60	19 (68%)
	CGAAACCCGGCATGTCGAGA
blaOXA-48	TTGGTGGCATCGATTATCGG	56	20 (71%)
	GAGCACTTCTTTTGTGATGGC
qnrA	ATTTCTCACGCCAGGATTTG	52	02 (7%)
	GATCGGCAAAGGTTAGGTCA
qnrB	GATCGTGAAAGCCAGAAAGG	50	17 (61%)
	ACGATGCCTGGTAGTTGTCC
qnrS	ACGACATTCGTCAACTGCAA	52	15 (53%)
	TAAATTGGCACCCTGTAGGC
tetA	GTGAAACCCAACATACCCC	52	13 (46%)
	GAAGGCAAGCAGGATGTAG
tetB	CCTTATCATGCCAGTCTTGC	52	14 (50%)
	ACTGCCGTTTTTTCGCC
sul1	CGGCGTGGGCTACCTGAACG	67	16 (57%)
	GCCGATCGCGTGAAGTTCCG
sul2	GCGCTCAAGGCAGATGGCATT	67	15 (53%)
	GCGTTTGATACCGGCACCCGT
Mobilized Colistin Resistance Gene
mcr-1	AGTCCGTTTGTTCTTGTGGC	61	8 (28%)
	AGATCCTTGGTCTCGGCTTG

Table 2 Sample-Wise Distribution of Mcr-1 Harboring K. Pneumoniae (hvKp/Non-hvKp)

Sample Site	No of Sample	Positive Isolates	hvKp ISOLATES	No. MDR Among Positive Isolates	MDR hvKp Isolates	mcr-1 Harboring (hvKp/non-hvKp)
Pus exudates	38	18	8	9	4	1 hvKp 1 non-hvKp
Sputum	20	7	2	2	0
Catheter tips	35	17	9	8	4	1 hvKp 1 non-hvKp
Infectious wounds	40	17	6	10	3	2 hvKp 2 non-hvKp
Tracheal Aspirates	17	8	3	3	1
Total	150	67		32	12	8

Figure 2 (A) Comparative analysis of resistance against different antibiotics maximum resistance in (Gray) against ampicillin. While least was against colistin shown in (Blue). (B) Determination of colistin resistance through rapid polymyxin NP test. (C) Detection of PCR product for mcr-1 gene through gel electrophoresis.

Table 3 Comparative Analysis of Colistin Resistance with PCR Results of Mcr-1 Gene, Spot Test, and hvKp Isolates

Sr. No.	Isolate ID	mcr-1 Gene	Colistin Resistance	hvKp	Spot test
1	S32	+Ve	+Ve	+Ve	+Ve
2	S3	+Ve	+Ve	+Ve	+Ve
3	S29	+Ve	+Ve	+Ve	+Ve
4	S4	+Ve	+Ve	+Ve	+Ve
5	S9	+Ve	+Ve	-Ve	+Ve
6	S17	+Ve	+Ve	-Ve	+Ve
7	S6	+Ve	+Ve	+Ve	+Ve
8	S21	+Ve	+Ve	-Ve	+Ve
9	S24	-Ve	+Ve	-Ve	+Ve
10	S19	-Ve	+Ve	-Ve	+Ve
11	S13	-Ve	+Ve	-Ve	+Ve
12	S23	-Ve	+Ve	-Ve	+Ve

Figure 3 (A) Plaque assay after bacteriophage purification. (B) Detection of bacteriophages in wastewater sample through spot test. (C) Determination of host range by spot test. (D) TEM analysis of isolated bacteriophage shows icosahedral-isometric head with neck connected to a long, rigid and contractile tail as located in Myoviridae family. (E) Plaque Assay for Colistin resistant MDR hvKp isolates. (F) Comparative analysis of phage titer against colistin resistant MDR Klebsiella pneumoniae isolates.

Figure 4 (A) Comparative analysis of phage titer at various ranges pH. (B) Comparative analysis of phage titer at various ranges temperatures. (C) Comparative analysis of phage titer at various concentrations of chloroform.

Figure 5 (A) Determination of one step growth curve through plaque assay. (B) Graphical analysis of phage titer at several time periods of one step growth curve.

Figure 6 (A) Comparative analysis of bacteriophage susceptibility between mcr-1 negative (in red) and mcr-1 positive (in blue) isolates. (B) Biofilm assay (BA) in blue represents OD₆₀₀ of biofilm assay. Biofilm inhibition assay (BIA) in red represents OD₆₀₀ of Biofilm inhibition assay and biofilm eradication assay (BEA) in green represents OD₆₀₀ of biofilm eradication assay. The comparative analysis of means at absolute right shows significance decrease in biofilm production.