Table I
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 | ||
| LM1 CCT AAG ACG CCA ATC GAA | 750 | |
| LM2 AAG CAC TTG CAA CTG CTC |
Table II
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 | |
| hlyA - F | GCA GTT GCA AGC GCT TGG AGT GAA | 456 |
| hlyA - R | GCA ACG TAT CCT CCA GAG TGA TCG | |
| iap - F | ACA AGC TGC ACC TGT TGC AG | 131 |
| iap - R | TGA CAG CGT GTG TAG TAG CA | |
| inlA - F | CAG GCA GCT ACA ATT ACA CA | 2 341 |
| inlA - R | ATA TAG TCC GAA AAC CAC ATC T | |
| inlB - F | AGG AGA GGA TAG TGT GAA | 1 905 |
| inlB - R | TTA TTT CTG TGC CCT TAA | |
| plcA - F | CTG CTT GAG CGT TCA TGT CTC ATC CCC C | 1 484 |
| plcA - R | ATG GGT TTC ACT CTC CTT CTA C | |
| plcB - F | GCA AGT GTT CTA GTC TTT CCG G | 794 |
| plcB - R | ACC TGC CAA AGT TTG CTG TGA | |
| prfA - F | CAT GAA CGC TCA AGC AGA AG | 706 |
| prfA - R | AAT TTT CCC AAG TAG CAG GA |
Fig. 1.
Genetic similarity of tested Listeria monocytogenes strains.
Fig. 2.
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).
Fig. 3.
Prevalence of virulence genes among L. monocytogenes strains isolated from milk and blood.
Table III
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 |
Table IV
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) | |
|---|---|---|---|---|
| Strains from milk (n = 18) (the strain identification number) | Strains from the blood (n = 10) (the strain identification number) | |||
| 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: P S: AM, MEM, E, SXT | 3 (16.67%) (76, 185, 251) | 0 (0.0%) | 3 (10.71%) |
| C | R: E S: P, AM, MEM, SXT | 1 (5.56%) (267) | 4 (40.0%) (1, 2, 7, 9 ) | 5 (17.86%) |
| D | R: P, E S: AM, MEM, SXT | 1 (5.56%) (143) | 0 (0.0%) | 1 (3.57%) |
| E | R: E, SXT S: P, AM, MEM | 1 (5.56%) (344) | 0 (0.0%) | 1 (3.57%) |
| F | R: P, AM, MEM, SXT S: E | 1 (5.56%) (368) | 0 (0.0%) | 1 (3.57%) |
| G | R: P, AM, MEM, E, SXT S: - - - | 3 (16.67%) (112, 372, 375) | 0 (0.0%) | 3 (10.71%) |
| H | R: MEM, E S: P, AM, SXT | 0 (0. 0%) | 2 (20.0%) (5, 6) | 2 (7.14%) |
Fig. 4.
Differences in biofilm formation among clinical L. monocytogenes strains and the strains isolated from milk.
Table V
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 |
Fig. 5.
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.
Fig. 6.
Multiple correlations between the intensity of biofilm formation, the number of virulence genes, and the drug resistance of L. monocytogenes strains isolated from milk.
Fig. 7.
Multiple correlations between the intensity of biofilm formation, the number of virulence genes, and the drug resistance of the clinical L. monocytogenes strains.
Table VI
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 | |||||||||||
| 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 | ||
Table VII
Transmission of L. monocytogenes from a biofilm on silicone teat cups to milk.
Table VIII
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 | |||||||
| 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 | 3.54 3.63 | 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 | 1.81 1.71 | 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 |