ANTIBIOTIC RESISTANCE

PROFILE IN CO-INFECTING
BACTERIA ISOLATED FROM
SHEEP DISLAYING ORF LESIONS
 Elisabeta PASTOR1, Crinu MESTEȘANU2 Petru POJAR1,
Marina SPÎNU1
1
Faculty of Veterinary Medicine ,University of Agricultural Sciences and
Veterinary Medicine, Cluj-Napoca, Romania

Veterinary doctor, Santău, Romania

2

*
Corresponding author, e-mail: mestesanu.elisabeta-sm@ansvsa.ro
Introduction

- Antibiotic resistance in veterinary medicine poses

lately increasing risks in therapeutic success and
threatens the life of patients with various bacterial
infections. Environmental bacteria are very likely
to develop on the initial lesions induced by orf
virus in sheep, worsening the development and
prognosis of the disease. The research aimed at
verifying the hypothesis that the persistence of the
bacteria and thus, increased severity of the orf
lesions is residing in the high antibiotic resistance
of the co-infecting bacteria.
-Orf virus belongs to the genus Parapoxvirus, family Poxviridae and it is
causative agent of ovine and caprine pustulo vesicular dermatitis also
known as contagious ecthyma, soremouth. Usually evolution of the
lesions in orf is from the stage of erythema to macula, papules, vesicles,
pustules and crusts, which are located mostly around the mouth, on the
nipples of lactating sheep or around the hooves (Abbas and col., 2019).
-Secondary bacterial infections involve Staphylococcus aureus and
Arcanobacterium pyogenes, Streptococcus spp., Pseudomonas aeruginosa,
bacteria that can create severe complications of the disease due to the
phenomen of antibiotic resistance often encountered in orf evolution.
-The widespread and unwise use of antibiotics can lead to their use in the
treatment of resistant bacteriosis. These resistant bacteria cannot only
become predominant species in a population, but they can also transfer
their genetic characteristics to other bacterial species, making them
resistant to antibiotics (Quinn et al., 2016). Resistance to an antibacterian
agent often leads to cross-resistance to other agents in the same class.
-The alarming increase in antibiotic resistance is one of the main threats
in modern medicine.
Materials and methods
-perilesional exudate was collected using sterile
swabs soaked in sterile saline solution and
stored in sterile containers.
-direct smear , examinated at the microscope
(Gram staining, capsule coloring, spore
evidentiation)
-inoculation, incubation, examination of
cultural charateristics.
-moving colonies onto special and selective
media, studying biochemical particularities.
-innocultaing and redaing API galleries
-After identifying the infectious agent, Kirby Bauer-discdiffusion test,
for antibiotic susceptibility on each strain. 50 strains of bacteria
isolated.
-Antibiotics were: gentamicin (10µg), amoxicillin with clavulanic acid
(20 / 10µg), cefuroxime (30µg), enrofloxacin (10µg), ciprofloxacin (5µg),
penicillin ( 10µg) and trimethoprim (5µg), the interpretation of the
results being performed according to CLSI 2015.
-Depending on the diameter of the inhibition zone, the tested bacterial
strain can be interpreted as ”sensitive”, ”moderately sensitive” (or
intermediate) or ”resistant” to the action of antiinfectious substances.
-MAR index calculation according to Krumperman´s method
(raportation of the number of antibiotics with resistant testing to the
total number of antibiotics used), intermedieate sensitivity being
considered resistance .
-a value greater than 0.2 indicates multiple antibiotic resistance.
-GraphPad Prism used to statistical determinations.
Resuslts and discussions
Following bacterioscopical and bacteriological
investigations, bacteria from species
Staphylococcus aureus (30 strains),
Corynebacterium pseudotuberculosis (6 strains),
Bacillus Cereus (7 strains) and Pseudomonas
aeruginosa (9 strains) were identified.
Secondary infections percentage

58.00% S.aureus
10.00% C.pseudotuberculosis
14.00% B.cereus
18.00% P.aeruginosa
S.Aureus
Sample no. Genta. Cef. Amoxiclav Enro. Cipro. Pen Trim MAR index
1 17S 25S 18R 23S 23S 21R 12R 0,43
3 19S 18R 17R 22S 22S 23R 18S 0,43
4 18S 23S 23S 25S 25S 20R 17S 0,14
6 17S 26S 25S 22S 26S 19R 12R 0,29
9 19S 21S 26S 25S 25S 21R 20S 0,14
12 20S 23S 14R 25S 25S 21R 21S 0,29
15 19S 23S 21S 24S 16R 20R 17S 0,29
16 20S 23S 22S 21S 27S 22R 20S 0,14
21 19S 25S 24S 14R 14R 16R 9R 0,57
35 18S 21S 14R 19S 26S 10R 20S 0,29
42 18S 21S 24S 14R 14R 21R 21S 0,43
45 20S 22S 27S 17I 13R 31S 11R 0,43
49 19S 24S 23S 14R 14R 25R 18S 0,43
52 19S 23S 15R 21S 25S 12R 19S 0,29
56 18S 25S 25S 20S 27S 20R 18S 0,14
66 20S 21S 25S 17R 17R 21R 18S 0,43
71 21S 27S 25S 20S 25S 22R 17S 0,14
73 19S 23S 21S 22S 22S 18R 17S 0,14
75 13R 11R 21S 25S 25S 17R 18S 0,43
76 20S 22S 21S 25S 25S 20R 18S 0,14
77 21S 25S 23S 16R 16R 19R 20S 0,43
78 18S 13R 26S 21S 27S 18R 21S 0,29
79 19S 18R 17R 23S 24S 19R 20S 0,43
80 20S 22S 17R 23S 23S 23R 19S 0,29
82 19S 21S 23S 20S 25S 21R 9R 0,29
85 19S 22S 21S 21S 23S 23R 10R 0,29
87 18S 22S 25S 22S 22S 23R 9R 0,29
88 20S 21S 26S 23S 25S 23R 13R 0,29
91 18S 25S 23S 21S 23S 26R 19S 0,14
93 18S 25S 24S 24S 24S 21R 19S 0,14
 Graphical representation of antibiotic sensitivity and resistance of S.aureus

22
23
24
29

27
40

23
Gentamicin
Cefuroxime
30

8
Amoxicillin+clavulanic acid

7
Enrofloxacin
6
7

1
20 Ciprofloxacina

Resistant
Sensitive
3

Resistant
Sensitive
Resistant
Sensitive
Penicilina
Resistant
Sensitive
Resistant
Sensitive

10
Resistant
Sensitive

Trimethoprime

Amoxiclav Enro. Cipro Pen. Trim.

Genta. Cef.

96.67% S 80.00% S 76.67% S

90.00% S 23.33% R 3.33% S
3.33% R 10.00% R 76.67% S 20.00% R 23.33% R 73.33% S
96.67% R 26.67% R
 Table no.2 Descriptive statistics on the diameters of the zones of inhibition and MAR indices in the case of
S.aureus

Resistant to 1 antibiotic 9

Multiresistant 21
30.00% Resistant to 1 antibiotic
70.00% Multiresistant
0 5 10 15 20 25

Genta
Parameters Cef. Amoxiclav Enro. Cipro. Pen. Trim. MAR index
.

Minimum 13 11 14 14 13 10 9 0,14
Maximum 21 27 27 25 27 31 21 0,57
Mean 19 22 22 21 22 21 17 0,3

Std. Deviation 1,5 3,4 3,7 3,4 4,4 3,9 4 0,13

Std. Error of Mean 0,27 0,63 0,68 0,61 0,81 0,71 0,72 0,023

Lower 95% CI of mean 18 21 20 20 21 19 15 0,25

Upper 95% CI of mean 19 23 23 22 24 22 18 0,34

Coefficient of variation 8,0% 16% 17% 16% 20% 19% 24% 42%
 C.pseudotuberculosis
Sample no. Genta. Cef. Amoxiclav Enro. Cipro. Pen Trim MAR index

29 15I 25S 28S 7R 11R 30S 30S 0,29

45 18S 22S 27S 17I 13R 31S 28S 0,14

55 20S 22S 28S 9R 9R 30S 27S 0,29

73 19S 23S 21S 22S 22S 18R 17S 0,14

84 5R 21S 19S 10R 16I 32S 20S 0,29

96 6R 16I 20S 9R 14R 31S 20S 0,43
 Graphical representation of C.pseudotuberculosis sensitivity to tested antibiotics
8
Gentamicin

0
Cefuroxime
6 1
Amoxicillin+

Sensitive
1

Resistant
Sensitive clavulanic acid
Resistant
Sensitive
Resistant

4 Enrofloxacin
Resistant

Ciprofloxacina
2 Penicilina
Trimethoprime

Genta. Cef. Amoxiclav Enro. Cipro. Pen. Trim.

50.00% S 83.33% S 16.67% S 83.33% R 83.33% S

50.00% R 16.67% R 100.00% S 83.33% R 16.67% S 16.67% R 100.00% S
 66.67% Multiresistant
33.33% Resistant to1 antibiotic

Resistant to 1 antibiotic 2

Multiresistant 4

0 1 2 3 4 5

Parameters Genta. Cef. Amoxiclav Enro. Cipro. Pen. Trim. MAR Inex

Minimum 5 16 19 7 9 18 17 0,14
Maximum 20 25 28 22 22 32 30 0,43
Mean 14 22 24 12 14 29 24 0,26
Std. Deviation 6,7 3 4,3 5,9 4,5 5,3 5,3 0,11

Std. Error of Mean 2,7 1,2 1,7 2,4 1,9 2,2 2,2 0,045

Lower 95% CI of
6,8 18 19 6,2 9,4 23 18 0,15
mean
Upper 95% CI of
21 25 28 18 19 34 29 0,38
mean
Coefficient of
48% 14% 18% 47% 32% 18% 22% 42%
variation
B.cereus

Nr.probă Genta. Cef. Amoxiclav Enro. Cipro. Pen. Trim. MAR index

4 18S 23S 23S 25S 25S 20R 17S 0,14

6 17S 26S 25S 22S 26S 19R 12R 0,29
12 20S 23S 14R 25S 25S 21R 21S 0,29
15 19S 23S 21S 24S 16R 20R 17S 0,29
33 26S 11R 5R 24S 23S 3R 15I 0,43
71 21S 27S 25S 20S 25S 22R 17S 0,14
74 29S 7R 2R 27S 27S 4R 16I 0,43
 B.cereus sensitivity to tested antibiotics
3

8
Gentamicin

0
Cefuroxime

Sensitive
Resistant
6 Sensitive
0

Resistant Amoxicillin+clavulanic acid

Sensitive
Resistant
Resistant

4 Enrofloxacin
Ciprofloxacina
2 Penicilina
Trimethoprime
0

Genta Cef. Amoxiclav. Enro. Pen. Trim.

Cipro.

71.43% S 57.14% S 85.71% S 57.14% S

100.00% S 28.57% R 42.86% R 100.00% S 14.29% R 100.00% R 42.86% R
 28.57% 1 anibiotic resistant
71.43% Multiresistant
Multiresistant

1 antibiotic resistant

0 2 4 6

Parameters Genta. Cef. Amoxiclav. Enro. Cipro. Pen. Trim. MAR index

Minimum 17 7 2 20 16 3 12 0,14
Maximum 29 27 25 27 27 22 21 0,43
Mean 21 20 16 24 24 16 16 0,29
Std.Deviation 4,4 7,8 9,6 2,3 3,7 8,3 2,7 0,12

Std. Error of Mean 1,7 2,9 3,6 0,86 1,4 3,1 1 0,045
Lower 95% CI of mean 17 13 7,5 22 20 7,9 14 0,18

Upper 95% CI of mean 26 27 25 26 27 23 19 0,4

Coefficient of variation 21% 39% 59% 9,5% 15% 53% 16% 41%
 P.aeruginosa

Sample no. Genta. Cef. Amoxiclav Enro. Cipro. Pen. Trim. MAR index
1 17S 25S 18R 23S 23S 21R 12R 0,43
33 26S 11R 5R 24S 23S 3R 15I 0,57
35 18S 21S 14R 19S 26S 10R 20S 0,29
42 10R 21S 24S 22S 23S 21R 21S 0,29
56 15I 11R 9R 20S 27S 20R 18S 0,57
59 14I 7R 3R 25S 25S 12R 22S 0,57
81 15I 10R 9R 28S 24S 5R 25S 0,57
86 14I 10R 10R 28S 27S 7R 25S 0,57
94 15I 8R 11R 26S 25S 9R 23S 0,57
 P.aeruginosa drug resistance
10 Gentamicin
Cefuroxime
8
Amoxicillin+clavulanic acid
6
Enrofloxacin
4 Ciprofloxacina
2 Penicilina

0
Trimethoprime

Genta Cef Amoxiclav Enro. Cipro. Pen. Trim.

66.67% R 66.67% R 88.89% R 22.22% R

33.33% S 33.33% S 11.11% S 100.00% S 100.00% S 100.00% R 77.78% S
 -All strains of P.aeruginosa showed multiple antibiotic resistance

Parameters Genta. Cef. Amoxiclav. Enro. Cipro. Pen. Trim. MAR index
Minimum 10 7 3 19 23 3 12 0,29
Maximum 26 25 24 28 27 21 25 0,57
Mean 16 14 11 24 25 12 20 0,49
Std. Deviation 4,4 6,6 6,5 3,2 1,6 7 4,4 0,12

Std. Error of Mean 1,5 2,2 2,2 1,1 0,55 2,3 1,5 0,041

Lower 95% CI of mean 13 8,7 6,5 21 24 6,6 17 0,4

Upper 95% CI of mean 19 19 16 26 26 17 24 0,59

Coefficient of variation 27% 48% 56% 13% 6,6% 58% 22% 25%
 7
9
11
13

0
1
0
2
9

1
1
3
2
3
Multiple drug resistance

20
2
2

Total
0

Staphylococcus
15
Corynebacterium
Bacillus
10 Pseudomonas

0
1 Antibiotic 2 Antibiotics 3 Antibiotics 4 Antibiotics

Multiple antibiotic resistance in all the strains

Total Staphylococcus Corynebacterium Bacillus Pseudomonas

25.00% 1 Antibiotic 30.00% 1 Antibiotic 33.33% 1 Antibiotic 66.67% 4 Antibiotice

36.54% 2 Antibiotics 36.67% 2 Antibiotics 28.57% 1 Antibiotic
13.46% 4 Antibiotics 3.33% 4 Antibiotics 50.00% 2 Antibiotics 42.86% 2 Antibiotice 22.22% 2 Antibiotice
25.00% 3 Antibiotics 30.00% 3 Antibiotics 16.67% 3 Antibiotics 28.57% 3 Antibiotice 11.11% 3 Antibiotice
 Bartletts statistics for MAR indexes 0,149
P 0,9854
P summary Ns
Dev.std. significantly different (P < 0.05)? No

Mean Confidence intervals

Tukeys multiple comparisons test Significant? Adjusted P value
differences between means

Corynebacterium vs.
-0,033 -0,1790 to 0,1130 No 0,9311
Staphylococcus

Bacillus vs. Staphylococcus -0,00919 -0,1462 to 0,1279 No 0,998

Pseudomonas vs. Staphylococcus 0,1959 0,07179 to 0,3200 Yes 0,0006

Bacillus vs. Corynebacterium 0,02381 -0,1579 to 0,2055 No 0,9852

Pseudomonas vs. Corynebacterium 0,2289 0,05680 to 0,4010 Yes 0,0048

Pseudomonas vs. Bacillus 0,2051 0,04053 to 0,3696 Yes 0,0091

Differences Standard error of

Test details Mean 1 Mean 2
between means differences
Corynebacterium vs.
0,2633 0,2963 -0,033 0,05487
Staphylococcus

Bacillus vs. Staphylococcus 0,2871 0,2963 -0,00919 0,0515

Pseudomonas vs. Staphylococcus 0,4922 0,2963 0,1959 0,04663

Bacillus vs. Corynebacterium 0,2871 0,2633 0,02381 0,06826

Pseudomonas vs. Corynebacterium 0,4922 0,2633 0,2289 0,06466

Pseudomonas vs. Bacillus 0,4922 0,2871 0,2051 0,06183

Conclusions
-Based on the MAR indexes, at least two of the investigated
environmental bacteria strains could further complicate orf
lesions and delay the positive effects of antibiotic therapy.
-Staphylococcus Aureus ranked first in terms of the number of
secondary infections
-The biggest challenge is the secondary infection with
P.aeruginosa, being resistant to at least two antibiotics.
-Antibiotic testing and the calculated MAR indices corroborated
with the statistical calculations revealed that a good
percentage of reaction to an antibiotic does not guarantee
the success of the treatment with that certain antibiotic.
-Statistical investigations such as standard deviation and
coefficient of variation revealed the existence of an instability
in the response to the action of a particular antibiotic.
-The groups of antibiotics from the group of β-lactams and
penicillins proved to be ineffective.
-With fluoroquinolones and aminoglycosides, there was a high
variability of the response, the success of the treatment being
ensured by the isolated strain.
-antibiotic susceptibility testing and the calculation of MAR indices
are not reliable information regarding the establishment of a
treatment, so if a rebellious bacterium is found for additional
treatment, it is advisable to test the incriminated strain,
genetically. These genetic tests require a lot of time and high
costs, the most convenient way to prevent multidrug resistance is
the judicious use of antibiotics, strictly following a medical
prescription.
Thank you for your
attention!