Characteristics of Listeria monocytogenes Strains Isolated from Milk and Humans and the Possibility of Milk-Borne Strains Transmission

SKOWRON, KRZYSZTOF; WAŁECKA-ZACHARKSA, EWA; GRUDLEWSKA, KATARZYNA; WIKTORCZYK, NATALIA; KACZMAREK, AGNIESZKA; GRYŃ, GRZEGORZ; KWIECIŃSKA-PIRÓG, JOANNA; JUSZCZUK, KLAUDIA; PALUSZAK, ZBIGNIEW; KOSEK-PASZKOWSKA, KATARZYNA; GOSPODAREK-KOMKOWSKA, EUGENIA

doi:10.33073/pjm-2019-038

Figures & Tables

Genetic similarity of tested Listeria monocytogenes strains.

Electrophoregram presenting the occurrence of virulence genes among the strains tested (M – marker, K+ – positive control (IW41), line 4, 143, 185, 199, 344, 267, 112, 317, 368 – L. monocytogenes strains from milk, K (−) – negative control).

Prevalence of virulence genes among L. monocytogenes strains isolated from milk and blood.

Differences in biofilm formation among clinical L. monocytogenes strains and the strains isolated from milk.

The intensity of biofilm formation by the selected L. monocytogenes strains.A – weak biofilm, B – moderate biofilm, C – strong biofilm, green – live cells, red – death cells.

Multiple correlations between the intensity of biofilm formation, the number of virulence genes, and the drug resistance of L. monocytogenes strains isolated from milk.

Multiple correlations between the intensity of biofilm formation, the number of virulence genes, and the drug resistance of the clinical L. monocytogenes strains.

The profiles of drug resistance/susceptibility of strains of L_ monocytogenes isolated from milk and the blood_

Profile	Drug resistance/susceptibility	Number of strains		Total (n = 28)
Profile	Drug resistance/susceptibility	Strains from milk (n = 18) (the strain identification number)	Strains from the blood (n = 10) (the strain identification number)	Total (n = 28)
A	R: -S: P, AM, MEM, E, SXT	8 (44.44%)(7, 29, 63, 199, 231, 282, 317)	4 (40.0%)(3, 4, 8, 10)	12 (42.86%)
B	R: PS: AM, MEM, E, SXT	3 (16.67%)(76, 185, 251)	0 (0.0%)	3 (10.71%)
C	R: ES: P, AM, MEM, SXT	1 (5.56%)(267)	4 (40.0%)(1, 2, 7, 9 )	5 (17.86%)
D	R: P, ES: AM, MEM, SXT	1 (5.56%)(143)	0 (0.0%)	1 (3.57%)
E	R: E, SXTS: P, AM, MEM	1 (5.56%)(344)	0 (0.0%)	1 (3.57%)
F	R: P, AM, MEM, SXTS: E	1 (5.56%)(368)	0 (0.0%)	1 (3.57%)
G	R: P, AM, MEM, E, SXTS: - - -	3 (16.67%)(112, 372, 375)	0 (0.0%)	3 (10.71%)
H	R: MEM, ES: P, AM, SXT	0 (0. 0%)	2 (20.0%)(5, 6)	2 (7.14%)

The intensity of biofilm formation by L_ monocytogenes strains derived from cow’s milk and the blood_

Strains origin	The strain identification number	Mean number of bacteria [log CFU/cm⁻²]	STD
Milk	372	6.87	± 6.12^*
Milk	375	6.79	± 6.09
Milk	112	6.71	± 6.13
Milk	368	6.64	± 6.17
Milk	344	6.05	± 5.21
Milk	143	5.95	± 4.99
Milk	267	5.81	± 5.07
Milk	251	5.18	± 4.80
Milk	185	4.99	± 4.06
Milk	7m	4.94	± 4.00
Milk	199	4.94	± 3.76
Milk	317	4.89	± 3.78
Milk	4m	4.85	± 3.67
Milk	282	4.81	± 3.81
Milk	76	4.77	± 3.65
Milk	29	4.76	± 3.74
Milk	231	4.74	± 3.96
Milk	63	4.64	± 3.72
ATCC	19111	5.80	± 5.04
Blood	5	5.70	± 4.87
Blood	6	5.67	± 4.76
Blood	3	4.18	± 3.83
Blood	1	4.01	± 2.87
Blood	8	4.00	± 3.27
Blood	2	3.97	± 3.61
Blood	9	3.92	± 3.83
Blood	7	3.72	± 3.63
Blood	10	3.71	± 3.72
Blood	4	3.61	± 3.71

Listeria spp_ transmission system: udder skin – silicone teat cup – udder skin_

Strain	Number of re-isolated L. monocytogenes [log CFU/cm²]
	Silicone teat cup	Udder skin	Udder skin
	Silicone teat cup	Udder skin	Sample 1	Sample 2	Sample 3	Sample 4	Sample 5	Sample 6	Recovery of bacteria (12 h/25°C)
M372	5.91^±5.81^*	3.81±3.61	3.77±3.73	3,. 0±2.85	3.00±2.85	2.81±2.65	2.81±2.65	1.92±1.82	2.83±2.53
M375	5.84±5.63	3.68±3.45	3.61±3.83	2.68±2.50	2.68±2.50	1.87±1.93	1.87±1.93	n.d.^***	1.66±1.76
M231	5.57±5.60	3.71±3.23	3.66±3.54	2.68±2.72	2.68±2.72	2.00±1.90	2.00±1.90	1.11±1.16	2.37±2.48
M63	5.52±5.34	3.543.63	3.47±3.55	1.47±1.66	1.47±1.66	1.00±1.69	1.00±1.69	n.d	1.26±1.03
ATCC 19111	5.66±5.54	3.76±3.57	3.72±3.27	2.74±1.99	2.74±1.99	2.26±1.98	2.26±1.98	1.64±1.47	2.57±2.32
B5	5.71±5.61	3.62±3.20	3.56±3.47	2.57±2.42	2.57±2.42	1.84±1.03	1.84±1.03	n.d.	1.46±1.58
B6	5.69±5.45	3.79±3.86	3.73±3.70	2.91±2.78	2.91±2.78	2.72±2.64	2.72±2.64	1.811.71	2.72±2.63
B10	5.32±5.68	3.59±3.02	3.53±3.37	1.62±1.48	1.62±1.48	1.11±1.46	1.11±1.46	n.d.	1.20±1.68
B4	5.13±5.40	3.58±3.50	3.51±3.58	1.52±1.61	1.52±1.61	0.90±0.86	0.90±0.86	n.d.	1.08±1.71

Primers used for identification of L_ monocytogenes_

Primer Primer sequence (5' - 3')	Size of PCR product (bp)	Amplification conditions
L1 CAG CAG CCG CGG TAA TAC	938	Initial denaturation - 94°C/2 min 30 cycles: denaturation - 94°C/30 s annealing, - 50°C/30 s elongation - 72°C/1 min final elongation - 72°C/5 min
L2 CTC CAT AAA GGT GAC CCT	938
LM1 CCT AAG ACG CCA ATC GAA	750
LM2 AAG CAC TTG CAA CTG CTC	750

Transmission of L_ monocytogenes from a biofilm on silicone teat cups to milk_

Strain	Number of re-isolated L. monocytogenes
Strain	Silicone teat cup[log CFU/cm²]	Milk[log CFU/ml]
M372	6.87^±6.12^*	3.37±3.03
M375	6.79±6.09	3.29±3.37
M231	4.74±3.96	2.75±2.62
M63	4.64±3.72	2.71±2.52
ATCC 19111	5.80±5.04	2.972.85
B5	5.70±4.87	2.94±2.74
B6	5.67±4.76	2.84±2.32
B10	3.71±3.72	2.63±2.51
B4	3.61±3.71	2.57±2.32

The profiles of virulence genes in strains of L_ monocytogenes isolated from milk and from the blood_

Profile	Virulence genes	Number of strains	The strain identification number
Strains isolated from milk
I	hlyA, plcB	2	4, 63
II	hlyA, iap, plcB	2	7, 29
III	hlyA, inlB, plcB	2	199, 231
IV	actA, hlyA, plcB	2	282, 317
V	actA, hlyA, plcB, prfA	2	76, 185
VI	actA, hlyA, iap, plcB, prfA	2	143,368
VII	actA, hlyA, iap, inlA, inlB, plcA, plcB, prfA	2	372, 375
VIII	actA, hlyA, inlA, plcB	1	267
IX	actA, hlyA, inlA, plcA, plcB	1	344
X	actA, hlyA, plcA, plcB, prfA	1	251
XI	actA, hlyA, inlA, inlB, plcA, plcB, prfA	1	112
Strains isolated from the blood
I	actA, hlyA, iap, inlA, inlB, plcA, plcB, prfA	5	1, 2, 5, 6, 9
II	actA, hlyA, iap	1	4
III	actA, hlyA, iap, prfA	1	10
IV	hlyA, inlA, plcA, plcB, prfA	1	3
V	hlyA, inlA, inlB, plcA, plcB, prfA	1	8
VI	actA, hlyA, inlA, inlB, plcA, plcB, prfA	1	7

Transmission of L_ monocytogenes from a biofilm on silicone teat cups to the cow’s udder skin_

Strain	Number of re-isolated L. monocytogenes strains [log CFU/cm²]
	Silicone teat cup	Udder skin
	Silicone teat cup			Sample 1	Sample 2	Sample 3	Sample 4	Sample 5	Sample 6	Sample 7	Sample 8	Regrowth on first negative or last positive sample (12 h/25°C)
M372	6.87^±6.12^*	4.33±3.98	3.88±3.77	3.71±3.34	3.45±2.88	2.92±2.71	2.66±2.35	2.09±2.31	1.85±1.84	2.92±2.74
M375	6.79±6.09	4.54±4.37	3.99±3.82	3.65±3.63	3.10±3.45	2.83±2.69	2.51±1.96	1.92±1.82	1.61±1.31	2.79±2.62
M231	4.74±3.96	3.87±3.72	3.76±3.79	3.47±3.55	2.79±2.71	2.52±2.33	1.93±1.20	1.67±1.47	1.08±1.54	1.99±1.85
M63	4.64±3.72	3.96±3.82	3.80±3.33	3.34±3.62	2.70±2.58	2.33±2.32	1.86±1.78	1.59±1.48	n.d.	1.87±1.51
ATCC 19111	5.80±5.04	3.64±3.67	3.02±3.51	2.79±2.39	2.62±2.44	2.14±2.48	1.79±1.51	1.53±1.63	n.d.	2.01±2.31
B5	5.70±4.87	3.56±3.71	2.85±2.70	2.63±2.20	2.48±2.23	1.96±1.86	1.76±1.61	1.43±1.01	n.d.	1.81±1.20
B6	5.67±4.76	3.47±3.17	2.91±2.76	2.76±1.99	2.39±2.61	1.92±1.65	1.64±1.71	1.26±1.33	n.d.	1.72±1.80
B10	3.71±3.72	2.85±2.58	2.57±2.20	1.88±1.57	1.49±1.04	n.d.^***	n.d.	n.d.	n.d.	1.63±1.41
B4	3.61±3.71	2.92±2.88	2.73±2.53	1.84±161	1.20±1.35	n.d.	n.d.	n.d.	n.d.	1.38±1.50

Primers used for the detection of virulence genes in Listeria monocytogenes strains (Franciosa et al_ 2005; Rawool et al_ 2007)_

Primer	Primer sequence (5’ - 3’)	Size of PCR product (bp)
actA - F	CGC CGC GGA AAT TAA AAA AAG A	839
actA - R	ACG AAG GAA CCG GGC TGC TAG	839
hlyA - F	GCA GTT GCA AGC GCT TGG AGT GAA	456
hlyA - R	GCA ACG TAT CCT CCA GAG TGA TCG	456
iap - F	ACA AGC TGC ACC TGT TGC AG	131
iap - R	TGA CAG CGT GTG TAG TAG CA	131
inlA - F	CAG GCA GCT ACA ATT ACA CA	2 341
inlA - R	ATA TAG TCC GAA AAC CAC ATC T	2 341
inlB - F	AGG AGA GGA TAG TGT GAA	1 905
inlB - R	TTA TTT CTG TGC CCT TAA	1 905
plcA - F	CTG CTT GAG CGT TCA TGT CTC ATC CCC C	1 484
plcA - R	ATG GGT TTC ACT CTC CTT CTA C	1 484
plcB - F	GCA AGT GTT CTA GTC TTT CCG G	794
plcB - R	ACC TGC CAA AGT TTG CTG TGA	794
prfA - F	CAT GAA CGC TCA AGC AGA AG	706
prfA - R	AAT TTT CCC AAG TAG CAG GA	706

Characteristics of Listeria monocytogenes Strains Isolated from Milk and Humans and the Possibility of Milk-Borne Strains Transmission

Figures & Tables

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Fig. 7.

The profiles of drug resistance/susceptibility of strains of L_ monocytogenes isolated from milk and the blood_

The intensity of biofilm formation by L_ monocytogenes strains derived from cow’s milk and the blood_

Listeria spp_ transmission system: udder skin – silicone teat cup – udder skin_

Primers used for identification of L_ monocytogenes_

Transmission of L_ monocytogenes from a biofilm on silicone teat cups to milk_

The profiles of virulence genes in strains of L_ monocytogenes isolated from milk and from the blood_

Transmission of L_ monocytogenes from a biofilm on silicone teat cups to the cow’s udder skin_

Primers used for the detection of virulence genes in Listeria monocytogenes strains (Franciosa et al_ 2005; Rawool et al_ 2007)_

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