According to the European Food Safety Authority (EFSA) report in 2017 all tested milk samples included in the RTE (ready-to-eat) products category were in line with Food Safety Criteria (FSC). One of 85 samples (1.2%) from two member countries, in the category “Raw cow’s milk for direct consumption” in the retail trade, was positive for
Despite the improvement in the hygiene of the production process,
Although most
This study aimed was to determine the multiple correlations between drug susceptibility, virulence genes and biofilm formation on silicone teat cups from milking machines of milk-borne and human
Then, multiplex PCR was performed. For the genus
DNA was isolated using the Genomic Mini AX Bacteria Spin kit (A&A Biotechnology, Poland), according to the manufacturer’s instructions. Amplification was performed in the mixture of 25 μl, containing: 1 × PCR buffer (Promega, United States), 2.0 mM MgCl2 (ABO, Poland), 1.25 mmol dNTPs (Promega, United States), 0.5 μM of each of the primers (Oligo.pl, Poland), 1.0 U Taq DNA polymerase (Promega, United States), ultrapure water (Merck, Poland) and 2.0 μl DNA. The amplification conditions and primer sequences are presented in Table I.
Primers used for identification of
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 |
To determine the degree of genetic relationship between the isolates, the phylogenetic dendrogram was drawn in the program Phoretix 1D Pro (TotalLab). Data clustering was performed using the UPGMA hierarchic grouping technique with the Dice coefficient.
Two multiplex-PCR reactions were optimized to detect these virulence genes. The first included the detection of
Primers used for the detection of virulence genes in
Primer | Primer sequence (5’ - 3’) | Size of PCR product (bp) |
---|---|---|
CGC CGC GGA AAT TAA AAA AAG A | 839 | |
ACG AAG GAA CCG GGC TGC TAG | ||
GCA GTT GCA AGC GCT TGG AGT GAA | 456 | |
GCA ACG TAT CCT CCA GAG TGA TCG | ||
ACA AGC TGC ACC TGT TGC AG | 131 | |
TGA CAG CGT GTG TAG TAG CA | ||
CAG GCA GCT ACA ATT ACA CA | 2 341 | |
ATA TAG TCC GAA AAC CAC ATC T | ||
AGG AGA GGA TAG TGT GAA | 1 905 | |
TTA TTT CTG TGC CCT TAA | ||
CTG CTT GAG CGT TCA TGT CTC ATC CCC C | 1 484 | |
ATG GGT TTC ACT CTC CTT CTA C | ||
GCA AGT GTT CTA GTC TTT CCG G | 794 | |
ACC TGC CAA AGT TTG CTG TGA | ||
CAT GAA CGC TCA AGC AGA AG | 706 | |
AAT TTT CCC AAG TAG CAG GA |
The intensity of biofilm formation was determined using a quantitative method by Kwiecińska-Piró (g et al. 2011) with some modifications. The study was conducted on one clinical strain, one strain isolated from milk, and the reference strain ATCC 19111. The sterile fragments of rubber (3 replications for each strain) were placed in tubes containing 3 ml of the bacterial suspensions in the Brain Heart Infusion Broth (BHI) (Merck, Poland) (0.5 of the McFarland scale). Incubation was conducted in the aerobic atmosphere at 37°C for 72 h, and the medium was replaced with a sterile one every 24 h. At each exchange of the medium, the rubber fragments were rinsed with sterile PBS (Phosphate Buffered Saline) (BTL, Poland). The fragments of rubbers incubated in the sterile BHI medium (Merck, Poland) were used as negative controls. After incubation, the samples were rinsed with PBS, placed in a tube containing 3 ml of PBS, and sonicated for 10 minutes (30 kHz, 150W) with the sonicator Ultrasonic DU-4 (Nickel-Electro Ltd.). Then, the samples were shaken for 10 minutes (400 rpm), and the serial 10-fold dilutions were prepared and inoculated on the Columbia Agar medium with 5% sheep blood (Becton Dickinson, United States). After 24-hours incubation at 37°C, the number of colonies per 1 cm2 of the fragment’s surface (CFU/cm-2) was calculated.
The intensity of biofilm formation by
To evaluate the transmission of bacteria from the biofilm formed on the teat rubbers to the udder skin (according to our method), a piece of skin was rubbed with the contaminated rubber in two directions. This simulated the insertion and removal of the teat into the milking cup. For each strain, eight skin fragments were used in six replicates. After swabbing, each piece of skin (1–3 replicates) was placed in sterile PBS (BTL, Poland) and subjected to a 10-minute sonication. Subsequently, the serial 10-fold dilutions were made and plated onto Columbia Agar with 5% sheep blood (Becton Dickinson, United States). After 24 hours at 37°C, the number of bacteria in 1 cm2 of the udder skin was calculated. Also, to test the proliferation of
To evaluate the transmission of
The bacterial suspensions in sterile PBS (BTL, Poland) (0.5 McF) were prepared and 50 μl of the suspension (six replicates) was poured on the sterile skin fragments. After drying at room temperature, the sterile teat rubber piece was rubbed in two directions with the contaminated skin. Then, the rubber pieces were placed (repeats 1–3) in sterile PBS (BTL, Poland) and sonicated. The number of bacteria per 1 cm2 of rubber was determined by plating the sample into Columbia Agar with 5% sheep blood (Becton Dickinson, United States), and incubation at 37°C for 24 hours. The remaining contaminated teat rubber fragments (repeats 4–6) were used to rub six sterile fragments of the udder skin. The number of bacteria transferred to them as well as proliferation on the udder skin was determined as described previously (“
The frequency of the genes encoding virulence factors in
The number of bacteria re-isolated from biofilm was averaged separately for both groups of strains and compared with each other and with the reference strain using the analysis of variance ANOVA and the post hoc Bonferroni test, at the significance level α = 0.05.
The multiple correlations between antibiotic resistance, virulence genes, and intensity of biofilm formation among clinical strains and milk isolates were tested. Single correlations between antibiotic resistance and prevalence of virulence genes, antibiotic resistance and intensity of biofilm formation, and incidence of virulence genes and the intensity biofilm formation were also evaluated. Correlation coefficients were evaluated according to Guilford’s scale.
Of the 380 milk samples tested, 21 (5.5%) were positive for
Among
The profiles of virulence genes in strains of
Profile | Virulence genes | Number of strains | The strain identification number |
---|---|---|---|
Strains isolated from milk | |||
I | 2 | 4, 63 | |
II | 2 | 7, 29 | |
III | 2 | 199, 231 | |
IV | 2 | 282, 317 | |
V | 2 | 76, 185 | |
VI | 2 | 143,368 | |
VII | 2 | 372, 375 | |
VIII | 1 | 267 | |
IX | 1 | 344 | |
X | 1 | 251 | |
XI | 1 | 112 | |
Strains isolated from the blood | |||
I | 5 | 1, 2, 5, 6, 9 | |
II | 1 | 4 | |
III | 1 | 10 | |
IV | 1 | 3 | |
V | 1 | 8 | |
VI | 1 | 7 |
The profiles of drug resistance/susceptibility of strains of
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: - | 8 (44.44%) | 4 (40.0%) | 12 (42.86%) |
B | R: P | 3 (16.67%) | 0 (0.0%) | 3 (10.71%) |
C | R: E | 1 (5.56%) | 4 (40.0%) | 5 (17.86%) |
D | R: P, E | 1 (5.56%) | 0 (0.0%) | 1 (3.57%) |
E | R: E, SXT | 1 (5.56%) | 0 (0.0%) | 1 (3.57%) |
F | R: P, AM, MEM, SXT | 1 (5.56%) | 0 (0.0%) | 1 (3.57%) |
G | R: P, AM, MEM, E, SXT | 3 (16.67%) | 0 (0.0%) | 3 (10.71%) |
H | R: MEM, E | 0 (0. 0%) | 2 (20.0%) | 2 (7.14%) |
R - resistant
S - susceptible
P - penicillin, AM - ampicillin, MEM - meropenem, E - erythromycin, SXT - cotrimoxazole
The intensity of biofilm formation by
Strains origin | The strain identification number | Mean number of bacteria [log CFU/cm−2] | STD |
---|---|---|---|
Milk | 372 | 6.87 | ± |
Milk | 375 | 6.79 | ± |
Milk | 112 | 6.71 | ± |
Milk | 368 | 6.64 | ± |
Milk | 344 | 6.05 | ± |
Milk | 143 | 5.95 | ± |
Milk | 267 | 5.81 | ± |
Milk | 251 | 5.18 | ± |
Milk | 185 | 4.99 | ± |
Milk | 7m | 4.94 | ± |
Milk | 199 | 4.94 | ± |
Milk | 317 | 4.89 | ± |
Milk | 4m | 4.85 | ± |
Milk | 282 | 4.81 | ± |
Milk | 76 | 4.77 | ± |
Milk | 29 | 4.76 | ± |
Milk | 231 | 4.74 | ± |
Milk | 63 | 4.64 | ± |
ATCC | 19111 | 5.80 | ± |
Blood | 5 | 5.70 | ± |
Blood | 6 | 5.67 | ± |
Blood | 3 | 4.18 | ± |
Blood | 1 | 4.01 | ± |
Blood | 8 | 4.00 | ± |
Blood | 2 | 3.97 | ± |
Blood | 9 | 3.92 | ± |
Blood | 7 | 3.72 | ± |
Blood | 10 | 3.71 | ± |
Blood | 4 | 3.61 | ± |
- Standard deviation
The biofilm formation intensity by
Transmission of
Strain | Number of re-isolated | |||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|
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* | 4.33 | 3.88 | 3.71 | 3.45 | 2.92 | 2.66 | 2.09 | 1.85 | 2.92 | ||
M375 | 6.79 | 4.54 | 3.99 | 3.65 | 3.10 | 2.83 | 2.51 | 1.92 | 1.61 | 2.79 | ||
M231 | 4.74 | 3.87 | 3.76 | 3.47 | 2.79 | 2.52 | 1.93 | 1.67 | 1.08 | 1.99 | ||
M63 | 4.64 | 3.96 | 3.80 | 3.34 | 2.70 | 2.33 | 1.86 | 1.59 | n.d. | 1.87 | ||
ATCC 19111 | 5.80 | 3.64 | 3.02 | 2.79 | 2.62 | 2.14 | 1.79 | 1.53 | n.d. | 2.01 | ||
B5 | 5.70 | 3.56 | 2.85 | 2.63 | 2.48 | 1.96 | 1.76 | 1.43 | n.d. | 1.81 | ||
B6 | 5.67 | 3.47 | 2.91 | 2.76 | 2.39 | 1.92 | 1.64 | 1.26 | n.d. | 1.72 | ||
B10 | 3.71 | 2.85 | 2.57 | 1.88 | 1.49 | n.d.*** | n.d. | n.d. | n.d. | 1.63 | ||
B4 | 3.61 | 2.92 | 2.73 | 1.84 | 1.20 | n.d. | n.d. | n.d. | n.d. | 1.38 |
- Mean
- Standard deviation
- Not detected
The obtained results showed that even if
All
Transmission of
Strain | Number of re-isolated | |
---|---|---|
Silicone teat cup | Milk | |
M372 | 6.87* | 3.37 |
M375 | 6.79 | 3.29 |
M231 | 4.74 | 2.75 |
M63 | 4.64 | 2.71 |
ATCC 19111 | 5.80 | 2.97 |
B5 | 5.70 | 2.94 |
B6 | 5.67 | 2.84 |
B10 | 3.71 | 2.63 |
B4 | 3.61 | 2.57 |
- Mean
- Standard deviation
Strain | Number of re-isolated | ||||||||
---|---|---|---|---|---|---|---|---|---|
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* | 3.81 | 3.77 | 3,. 0 | 3.00 | 2.81 | 2.81 | 1.92 | 2.83 |
M375 | 5.84 | 3.68 | 3.61 | 2.68 | 2.68 | 1.87 | 1.87 | n.d.*** | 1.66 |
M231 | 5.57 | 3.71 | 3.66 | 2.68 | 2.68 | 2.00 | 2.00 | 1.11 | 2.37 |
M63 | 5.52 | 3.54 | 3.47 | 1.47 | 1.47 | 1.00 | 1.00 | n.d | 1.26 |
ATCC 19111 | 5.66 | 3.76 | 3.72 | 2.74 | 2.74 | 2.26 | 2.26 | 1.64 | 2.57 |
B5 | 5.71 | 3.62 | 3.56 | 2.57 | 2.57 | 1.84 | 1.84 | n.d. | 1.46 |
B6 | 5.69 | 3.79 | 3.73 | 2.91 | 2.91 | 2.72 | 2.72 | 1.81 | 2.72 |
B10 | 5.32 | 3.59 | 3.53 | 1.62 | 1.62 | 1.11 | 1.11 | n.d. | 1.20 |
B4 | 5.13 | 3.58 | 3.51 | 1.52 | 1.52 | 0.90 | 0.90 | n.d. | 1.08 |
- Mean
- Standard deviation
- Not detected
To confirm
Since
In the present study,
Our studies showed the presence of the
To date, many studies evaluating the drug susceptibility of
We found also three (16.7%) strains isolated from milk resistant to all of the antibiotics tested. The strain is described as MDR if is resistant to at least three various groups of antibiotic. According to this definition, we found four (22.2%) MDR strains among strains isolated from milk, and none MDR strain was isolated from blood samples. Similar results were shown by (Garedew et al. 2015) and (Pesavento et al. 2010), who isolated four (16.7%) and 11 (27.5%) multidrug-resistant strains from food, respectively. In contrast, in the study by (Jamali et al. 2013) as much as 71.4% of multidrug-resistant strains of
In Poland, among 471
Monitoring of the occurrence of
Bacteria within biofilms are much more resistant to antimicrobial agents than floating counterparts (Gong et al. 2013). To understand the relationship between biofilms and antimicrobial resistance in
Our study also revealed a positive correlation between the number of virulence genes, drug resistance, and biofilm formation ability. This is in agreement with the study by (Soni et al. 2013), which indicated that multi-drug resistant
Our study showed that teat cups, contaminated during milking might contribute to the transmission of
A better understanding of the epidemiology of
In the available literature, there are few data on the bacterial transmission