AAC Accepted Manuscript Posted Online 15 June 2015 Antimicrob. Agents Chemother. doi:10.1128/AAC.00177-15 Copyright © 2015, American Society for Microbiology. All Rights Reserved.
1
Impact of ceftiofur injection on gut microbiota and E. coli resistance in pigs
2 3
M. A. Fleury1,2,3, G. Mourand1,2, E. Jouy1,2, F. Touzain1,2, L. Le Devendec1,2, C. de
4
Boisseson1,2, F. Eono1,2, R. Cariolet1,2, A. Guérin1,2,4, O. Le Goff3, S. Blanquet-Diot3, M.
5
Alric3 and I. Kempf1,2
6 7 8 9 10 11 12 13 14 15
1
ANSES, Laboratoire de Ploufragan-Plouzané, F-22440 Ploufragan, France
2
Université Européenne de Bretagne, France
3
Clermont Université, Université d'Auvergne, Centre de Recherche en Nutrition Humaine
Auvergne, EA 4678 CIDAM, Conception, Ingénierie et Développement de l'Aliment et du Médicament, BP 10448, F-63000 Clermont-Ferrand, France 4
INRA, UMR408 Sécurité et Qualité des Produits d'Origine Végétale, F-84000 Avignon,
France
16 17
Abstract
18
Resistance to extended-spectrum cephalosporins (ESCs) is an important health concern. Here,
19
we studied the impact of the administration of a long-acting form of ceftiofur on the pig gut
20
microbiota and ESC resistance in E. coli. Pigs were orally inoculated with an ESC-resistant E.
21
coli M63 strain harboring a conjugative plasmid carrying a gene conferring resistance, blaCTX-
22
M-1.
23
were studied using quantitative PCR analysis of main bacterial groups and quantification of
24
short-chain fatty acids. E. coli and ESC-resistant E. coli were determined by cultural methods
25
and ESC-resistant E. coli isolates were characterized. The copies of the blaCTX-M-1 gene were
26
quantified.
27
After ceftiofur injection, the main change in gut microbiota was the significant, but transitory
28
decrease of the E. coli population. Acetate and butyrate levels were significantly lower in the
29
treated group. In all inoculated groups, E. coli M63 persisted in most pigs and the blaCTX-M-1
30
gene was transferred to other E. coli. Culture and PCR results showed that the ceftiofur-
31
treated group shed significantly more resistant strains one and three days after ESC injection.
32
Thereafter, on most dates, there were no differences between groups, but notably one pig of
33
the non-treated group regularly excreted very high numbers of ESC-resistant E. coli, probably
34
leading to a higher contamination level in its pen. In conclusion, the use of ESCs, but also
35
presence of high-shedder animals, are important features in the spread of ESC-resistance.
On the same day, they were given or not a unique injection of ceftiofur. Fecal microbiota
36 37 38
Running title: Ceftiofur effects on pig gut microbiota and resistance
39 40
Key words: cephalosporin - pig - intestinal microbiota - antimicrobial resistance
41 42
Introduction
43
Resistance to extended-spectrum cephalosporins (ESCs) is a major public health concern.
44
ESC-resistant Enterobacteriaceae are often resistant to other families of antimicrobials,
45
leading to therapeutic problems and ESCs are classified as “critically important
46
antimicrobials” in human medicine by the World Health Organization (1). ESC-resistant
47
Enterobacteriaceae are prevalent in food-producing animals in various countries (2) and these
48
resistant strains are sometimes present at alarming rates in the gut microbiota of animals (3,
49
4). In pigs — as in humans — the most frequently reported extended-spectrum β-lactamases
50
that confer resistance are CTX-M (5), encoded by genes present on conjugative plasmids. In
51
some countries, the third- and fourth-generations of cephalosporins are authorized for use in
52
pigs to fight respiratory diseases, associated with Actinobacillus pleuropneumoniae,
53
Pasteurella multocida, Haemophilus parasuis and Streptococcus suis, mastitis- metritis-
54
agalaxia syndrome in sows, exudative epidermitis and meningitis. However, off-label use for
55
other conditions such as “blanket” prophylactic treatments has been reported (6). The
56
summary of product characteristics of Naxcel, a vegetable oil-based suspension of ceftiofur
57
crystalline free acid, reports that approximately 60% and 15% of the dose is excreted in the
58
urine and feces, respectively, within 10 days after administration. The impact of the use of
59
ESC on the selection and dissemination of ESC-resistant Enterobacteriaceae is debated (7-9)
60
and very few data are available concerning the impact of these antimicrobials on the bacterial
61
populations that make up pig microbiota and participate in their metabolism. Using pigs
62
inoculated with ESC-resistant (ESCR) Escherichia coli and housed in controlled conditions,
63
the present study aimed at evaluating the impact of a long-acting form of ESC on gut
64
microbiota composition and metabolism and intestinal E. coli ESC-resistance. Cultural,
65
molecular and biochemical methods were used to monitor the modifications in microbiota
66
composition, the ESC-susceptibility of E. coli and the levels of short-chain fatty acids
67
(SCFA).
68 69
Materials and methods
70
Preparation of the ESCR E. coli strain
71
We prepared the ESCR E. coli strain inoculated in pigs so as to increase the likelihood of
72
bacterial colonization in the study pigs. Thus E. coli were first isolated on MacConkey media
73
from feces of piglets from the specific pathogen-free (SPF) experimental swine herd at the
74
ANSES Ploufragan laboratory (France). After identification par PCR (10), the strains were
75
tested for the presence of K85, K87, K88, K81, K82 and F6(987P) antigens (typical of pig
76
pathogenic E. coli strains) with antisera (Biovac, France) and the antimicrobial susceptibility
77
was determined by disk diffusion assay and interpreted according to the recommendations of
78
the French national antimicrobial susceptibility testing committee (CA-SFM) (11). One
79
randomly chosen pan-susceptible isolate UB12/059-3 was then made resistant to rifampicin
80
by culture in Mueller-Hinton media (MH) containing 250 mg/L rifampicin. The rifampicin-
81
resistant mutant was then used as receptor for in vitro conjugation with an ESCR E. coli 05-
82
M63-1 from our strain collection. This ESCR E. coli originated from fecal material obtained
83
from a healthy pig at the slaughterhouse. Conjugation was performed by mixing equal
84
cultures of the recipient strain UB12/059-3 and the donor strain E. coli 05-M63-1 in MH
85
media containing rifampicin (250 mg/L) and cefotaxime (32 mg/L). One ESCR
86
transconjugant (M63) was obtained and further characterized by determining the minimal
87
inhibition concentrations (MIC) of different antimicrobials by micro-dilution using Sensititre
88
plate (Trek/Biocentric, Bandol, France) following Clinical and Laboratory Standards Institute
89
guidelines (12), identifying its phylogenetic group (13), and screening for the presence of
90
blaCTX-M-1 gene (14). The transconjugant strain was tested for the presence of K85, K87, K88,
91
K81, K82 and F6(987P) antigens. To check the in vivo fitness of the transconjugant strain, a
92
preliminary experiment was performed and confirmed that the transconjugant strain could
93
colonize pigs from the SPF herd and could be transmitted between pigs housed in the same
94
pen (data not shown). The blaCTX-M-1 plasmid contained in the transconjugant was sequenced
95
using Mi-seq Illumina technology (paired-end 2x250 nucleotides). Sequences were cleaned
96
with
97
(ILLUMINACLIP:illumina_oligos_and_reverse_complements:2:30:5:1:true
98
TRAILING:3 MAXINFO:40:0.2 MINLEN:36 options). Two bowtie2 (16) alignments were
99
performed (-non-deterministic -very-sensitive options) on cleaned sequences, to blaCTX-M-1
100
gene (DQ915955) so as to evaluate sequencing depth and to phiX17. This second alignment
101
was made to remove reads matching to phiX174 material which is used in Illumina
102
sequencing in case of very redundant samples. The unaligned reads were downsampled to fit a
103
global coverage estimation of 80 times. Remaining reads were provided to Spades 3.1.1 de
104
novo assembler (17). Redundant or poorly covered contigs were filtered out. The resulting
105
assembly was submitted to ResFinder (18) to identify resistance genes and to the RASTA-
106
Bacteria program version 2.12 (http://genoweb1.irisa.fr/duals/RASTA-Bacteria/) (19) to
107
identify putative toxin-antitoxin systems.
108
Animals and experimental design
109
Experiments were performed in accordance with the animal welfare experimentation
110
recommendations issued by the Direction Départementale de la Protection des Populations
111
des Côtes d’Armor (ANSES registration no. B-22-745-1), and were approved by the ComEth
112
ANSES/ENVA/UPEC ethics committee (authorization no. 12-003). Six animal rooms were
113
used to house 48 Large White piglets obtained from the SPF herd at the ANSES Ploufragan
114
laboratory. The piglets of 7 weeks of age were the progeny of five different sows and were
115
randomized before the experiment. Each room contained eight piglets placed in two pens of
Trimmomatic
0.32
(15)
software LEADING:3
116
four animals. The experimental groups were as follows: the non-treated control group (8
117
pigs,NT) in room 1A, the ceftiofur-treated group (8 pigs,T) in room 1B, the E. coli M63-
118
inoculated group (M63) in rooms 1C (8 pigs) and 2C (8 pigs), and the E. coli M63-inoculated,
119
ceftiofur-treated group (M63-T) in rooms 1D (8 pigs) and 2D (8 pigs) (Table 2). The animals
120
in rooms 1A to 1D were monitored up to 35 days after inoculation (D35), whereas the animals
121
of rooms 2C and 2D were monitored only up to D8, because they were then included in
122
another study. The animals did not receive any treatment with any antibiotics prior to the
123
assay. The same non-supplemented starter diet was offered to the animals up to D2, then pigs
124
were fed with a non-supplemented standard growing diet until the end of the experiment.
125
These diets were formulated to cover pig nutritional requirements. Strict biosecurity measures
126
were implemented to avoid contamination of the pigs, including the use of an air filtration
127
system and airlocks for each room, the use of unit-specific clothes for each room, and
128
compulsory showers after visiting the pigs.
129
E. coli M63 inoculations were performed on D0. Each piglet from groups M63 and M63-T
130
was orally given a 10 mL suspension prepared from E. coli M63 cultivated on MH agar
131
containing cefotaxime (2 mg/L). The titer of the suspension was determined by spreading
132
tenfold dilutions on MH plates. Piglets from groups NT and T were similarly inoculated with
133
sterile medium.
134
On D0, just after E. coli M63 inoculation, piglets from groups T and M63-T were given a
135
single intramuscular injection of a vegetable oil-based suspension of ceftiofur crystalline-free
136
acid (Naxcel, Pfizer) at the recommended dose of 5 mg/kg of body weight. Animal weights
137
were individually recorded once a week. During the week, daily clinical examinations
138
consisted in looking for general clinical signs and taking rectal temperatures. Pigs were
139
euthanized on days 35 to 37 post-inoculation by intravenous injection of sodium pentobarbital
140
followed by exsanguination, and lesions were observed. Individual fecal samples were
141
collected from all animals from the day before inoculation (D-1) and on D1, D3, D7, D10,
142
D13, D17, D22 and D28. The fecal samples were immediately placed in generators for
143
anaerobic bacteria (GENbag anaer, Biomerieux, Marcy l’Etoile, France), freezed in liquid
144
nitrogen and then stored at -70°C until analysis.
145
Bacteriological analysis
146
Bacteriological analyses were conducted on samples collected from the six animal rooms
147
during the first week and samples from the four animal rooms (rooms 1A to 1D) thereafter.
148
The titers of putative E. coli (red/pink colonies surrounded by hazy medium) for D-1, D1, D3,
149
D10 and D22 and of ESCR Enterobacteriaceae on all sampling times of the individual fecal
150
samples were determined by spreading 100 µL of tenfold dilutions on MacConkey agar plates
151
with or without 2 mg/L cefotaxime in triplicate. After incubation, the colonies were
152
enumerated and the titers were calculated for each pig and each day. The detection limit was
153
100 CFU/g of feces. From the cefotaxime-supplemented plate, five isolates per pig per day
154
were tested for susceptibility to cefotaxime and to rifampicin by restreaking on MacConkey
155
plates containing either 2 mg/L cefotaxime or 250 mg/L rifampicin. All cefotaxime-resistant
156
and rifampicin-susceptible isolates as well as one cefotaxime- and rifampicin-resistant isolate
157
per group and per day were stored for further analysis. They were identified with E. coli-
158
specific PCR (10, 20). The presence of the blaCTX-M-1 gene was screened by PCR (14) and the
159
phylogenetic groups of the E. coli isolates were determined (13). After digestion with SmaI,
160
pulsed field gel electrophoresis (PFGE) profiles of a few isolates were compared to the profile
161
of the inoculated E. coli M63 strain (21).
162
Molecular analysis
163
DNA extracts were prepared from 0.2 g of individual fecal samples according to Yu and
164
Morrison (22), followed by Qiagen’s DNA Stool Kit (Qiagen, Courtaboeuf, France). Each
165
DNA extract was quantified using a Nanodrop 2000 spectrophotometer (Thermo Scientific,
166
Courtaboeuf, France) and was then adjusted to a concentration of 10 ng/µL. For all samples
167
collected from pigs in rooms 1A to 1D, previously validated quantitative PCR (qPCR)
168
analyses, all targeting 16S rRNA gene fragments, were carried out to evaluate the change in
169
abundance of the total bacterial population and of the numbers of Bacteroides/Prevotella,
170
Bifidobacterium, E. coli, Enterococcus and Lactobacillus/Leuconostoc/Pediococcus (23).
171
qPCR for quantification of blaCTX-M-1 gene copies was performed (24) for samples collected
172
from the six animal rooms during the first week and from the rooms 1A to 1D thereafter; copy
173
numbers were determined by comparison with decimal dilutions of plasmid DNA prepared
174
from the blaCTX-M-1 gene previously cloned in the plasmid pCR®4-TOPO® in the One Shot®
175
TOP10 E. coli strain (Life Technologies, St Aubin, France), according to the manufacturer's
176
instructions.
177
Short-chain fatty acids assay
178
Short-chain fatty acids (SCFA), considered as fermentation markers, were analyzed by gas
179
chromatography as described by Gerard-Champod et al. (25). Only samples collected from
180
the four animal rooms 1A to 1D were analyzed. Acetic, propionic, isobutyric, butyric,
181
isovaleric, valeric, caprilyc acids and heptanoate concentrations were determined. Assays
182
were performed after a deproteinization step with phosphotungstic acid and using 2-
183
ethylbutyric acid as an internal standard.
184
Statistical analysis
185
For body weight gains, culture results and numbers of blaCTX-M-1 copies, all 48 pigs in rooms
186
1 to 6 were included to study the effect of the ceftiofur treatment during the first week. For the
187
following days, or for quantification of bacterial groups by qPCR and metabolites, only
188
animals from rooms 1A to 1D were included.
189
Differences between weight gains of animals or culturable E. coli titers from the different
190
groups were analyzed using the Kruskal-Wallis test followed by the Wilcoxon test. The
191
individual titers of culturable E. coli and ESCR Enterobacteriaceae and gene copy numbers
192
were log10-transformed and significant differences between M63 and M63-T groups were
193
compared using the Wilcoxon test.
194
For data regarding quantification of bacterial populations by qPCR and SCFA analysis, a non-
195
parametric Mann-Whitney test was applied to ensure that the four groups were similar at the
196
beginning of the experiment on D-1. After normalization (Δlog=log(Dx) – log(D-1)), values
197
were grouped per period (first week: D1, D3 and D7; second week: D10 and D13 and third
198
period: D17, D22 and D28) and the normalized data of the different groups for the different
199
periods were compared using a non-parametric Mann-Whitney test.
200
Distributions were compared using the chi-square test or Fisher’s exact test. For all tests,
201
values of P
1
Impact of ceftiofur injection on gut microbiota and E. coli resistance in pigs
2 3
M. A. Fleury1,2,3, G. Mourand1,2, E. Jouy1,2, F. Touzain1,2, L. Le Devendec1,2, C. de
4
Boisseson1,2, F. Eono1,2, R. Cariolet1,2, A. Guérin1,2,4, O. Le Goff3, S. Blanquet-Diot3, M.
5
Alric3 and I. Kempf1,2
6 7 8 9 10 11 12 13 14 15
1
ANSES, Laboratoire de Ploufragan-Plouzané, F-22440 Ploufragan, France
2
Université Européenne de Bretagne, France
3
Clermont Université, Université d'Auvergne, Centre de Recherche en Nutrition Humaine
Auvergne, EA 4678 CIDAM, Conception, Ingénierie et Développement de l'Aliment et du Médicament, BP 10448, F-63000 Clermont-Ferrand, France 4
INRA, UMR408 Sécurité et Qualité des Produits d'Origine Végétale, F-84000 Avignon,
France
16 17
Abstract
18
Resistance to extended-spectrum cephalosporins (ESCs) is an important health concern. Here,
19
we studied the impact of the administration of a long-acting form of ceftiofur on the pig gut
20
microbiota and ESC resistance in E. coli. Pigs were orally inoculated with an ESC-resistant E.
21
coli M63 strain harboring a conjugative plasmid carrying a gene conferring resistance, blaCTX-
22
M-1.
23
were studied using quantitative PCR analysis of main bacterial groups and quantification of
24
short-chain fatty acids. E. coli and ESC-resistant E. coli were determined by cultural methods
25
and ESC-resistant E. coli isolates were characterized. The copies of the blaCTX-M-1 gene were
26
quantified.
27
After ceftiofur injection, the main change in gut microbiota was the significant, but transitory
28
decrease of the E. coli population. Acetate and butyrate levels were significantly lower in the
29
treated group. In all inoculated groups, E. coli M63 persisted in most pigs and the blaCTX-M-1
30
gene was transferred to other E. coli. Culture and PCR results showed that the ceftiofur-
31
treated group shed significantly more resistant strains one and three days after ESC injection.
32
Thereafter, on most dates, there were no differences between groups, but notably one pig of
33
the non-treated group regularly excreted very high numbers of ESC-resistant E. coli, probably
34
leading to a higher contamination level in its pen. In conclusion, the use of ESCs, but also
35
presence of high-shedder animals, are important features in the spread of ESC-resistance.
On the same day, they were given or not a unique injection of ceftiofur. Fecal microbiota
36 37 38
Running title: Ceftiofur effects on pig gut microbiota and resistance
39 40
Key words: cephalosporin - pig - intestinal microbiota - antimicrobial resistance
41 42
Introduction
43
Resistance to extended-spectrum cephalosporins (ESCs) is a major public health concern.
44
ESC-resistant Enterobacteriaceae are often resistant to other families of antimicrobials,
45
leading to therapeutic problems and ESCs are classified as “critically important
46
antimicrobials” in human medicine by the World Health Organization (1). ESC-resistant
47
Enterobacteriaceae are prevalent in food-producing animals in various countries (2) and these
48
resistant strains are sometimes present at alarming rates in the gut microbiota of animals (3,
49
4). In pigs — as in humans — the most frequently reported extended-spectrum β-lactamases
50
that confer resistance are CTX-M (5), encoded by genes present on conjugative plasmids. In
51
some countries, the third- and fourth-generations of cephalosporins are authorized for use in
52
pigs to fight respiratory diseases, associated with Actinobacillus pleuropneumoniae,
53
Pasteurella multocida, Haemophilus parasuis and Streptococcus suis, mastitis- metritis-
54
agalaxia syndrome in sows, exudative epidermitis and meningitis. However, off-label use for
55
other conditions such as “blanket” prophylactic treatments has been reported (6). The
56
summary of product characteristics of Naxcel, a vegetable oil-based suspension of ceftiofur
57
crystalline free acid, reports that approximately 60% and 15% of the dose is excreted in the
58
urine and feces, respectively, within 10 days after administration. The impact of the use of
59
ESC on the selection and dissemination of ESC-resistant Enterobacteriaceae is debated (7-9)
60
and very few data are available concerning the impact of these antimicrobials on the bacterial
61
populations that make up pig microbiota and participate in their metabolism. Using pigs
62
inoculated with ESC-resistant (ESCR) Escherichia coli and housed in controlled conditions,
63
the present study aimed at evaluating the impact of a long-acting form of ESC on gut
64
microbiota composition and metabolism and intestinal E. coli ESC-resistance. Cultural,
65
molecular and biochemical methods were used to monitor the modifications in microbiota
66
composition, the ESC-susceptibility of E. coli and the levels of short-chain fatty acids
67
(SCFA).
68 69
Materials and methods
70
Preparation of the ESCR E. coli strain
71
We prepared the ESCR E. coli strain inoculated in pigs so as to increase the likelihood of
72
bacterial colonization in the study pigs. Thus E. coli were first isolated on MacConkey media
73
from feces of piglets from the specific pathogen-free (SPF) experimental swine herd at the
74
ANSES Ploufragan laboratory (France). After identification par PCR (10), the strains were
75
tested for the presence of K85, K87, K88, K81, K82 and F6(987P) antigens (typical of pig
76
pathogenic E. coli strains) with antisera (Biovac, France) and the antimicrobial susceptibility
77
was determined by disk diffusion assay and interpreted according to the recommendations of
78
the French national antimicrobial susceptibility testing committee (CA-SFM) (11). One
79
randomly chosen pan-susceptible isolate UB12/059-3 was then made resistant to rifampicin
80
by culture in Mueller-Hinton media (MH) containing 250 mg/L rifampicin. The rifampicin-
81
resistant mutant was then used as receptor for in vitro conjugation with an ESCR E. coli 05-
82
M63-1 from our strain collection. This ESCR E. coli originated from fecal material obtained
83
from a healthy pig at the slaughterhouse. Conjugation was performed by mixing equal
84
cultures of the recipient strain UB12/059-3 and the donor strain E. coli 05-M63-1 in MH
85
media containing rifampicin (250 mg/L) and cefotaxime (32 mg/L). One ESCR
86
transconjugant (M63) was obtained and further characterized by determining the minimal
87
inhibition concentrations (MIC) of different antimicrobials by micro-dilution using Sensititre
88
plate (Trek/Biocentric, Bandol, France) following Clinical and Laboratory Standards Institute
89
guidelines (12), identifying its phylogenetic group (13), and screening for the presence of
90
blaCTX-M-1 gene (14). The transconjugant strain was tested for the presence of K85, K87, K88,
91
K81, K82 and F6(987P) antigens. To check the in vivo fitness of the transconjugant strain, a
92
preliminary experiment was performed and confirmed that the transconjugant strain could
93
colonize pigs from the SPF herd and could be transmitted between pigs housed in the same
94
pen (data not shown). The blaCTX-M-1 plasmid contained in the transconjugant was sequenced
95
using Mi-seq Illumina technology (paired-end 2x250 nucleotides). Sequences were cleaned
96
with
97
(ILLUMINACLIP:illumina_oligos_and_reverse_complements:2:30:5:1:true
98
TRAILING:3 MAXINFO:40:0.2 MINLEN:36 options). Two bowtie2 (16) alignments were
99
performed (-non-deterministic -very-sensitive options) on cleaned sequences, to blaCTX-M-1
100
gene (DQ915955) so as to evaluate sequencing depth and to phiX17. This second alignment
101
was made to remove reads matching to phiX174 material which is used in Illumina
102
sequencing in case of very redundant samples. The unaligned reads were downsampled to fit a
103
global coverage estimation of 80 times. Remaining reads were provided to Spades 3.1.1 de
104
novo assembler (17). Redundant or poorly covered contigs were filtered out. The resulting
105
assembly was submitted to ResFinder (18) to identify resistance genes and to the RASTA-
106
Bacteria program version 2.12 (http://genoweb1.irisa.fr/duals/RASTA-Bacteria/) (19) to
107
identify putative toxin-antitoxin systems.
108
Animals and experimental design
109
Experiments were performed in accordance with the animal welfare experimentation
110
recommendations issued by the Direction Départementale de la Protection des Populations
111
des Côtes d’Armor (ANSES registration no. B-22-745-1), and were approved by the ComEth
112
ANSES/ENVA/UPEC ethics committee (authorization no. 12-003). Six animal rooms were
113
used to house 48 Large White piglets obtained from the SPF herd at the ANSES Ploufragan
114
laboratory. The piglets of 7 weeks of age were the progeny of five different sows and were
115
randomized before the experiment. Each room contained eight piglets placed in two pens of
Trimmomatic
0.32
(15)
software LEADING:3
116
four animals. The experimental groups were as follows: the non-treated control group (8
117
pigs,NT) in room 1A, the ceftiofur-treated group (8 pigs,T) in room 1B, the E. coli M63-
118
inoculated group (M63) in rooms 1C (8 pigs) and 2C (8 pigs), and the E. coli M63-inoculated,
119
ceftiofur-treated group (M63-T) in rooms 1D (8 pigs) and 2D (8 pigs) (Table 2). The animals
120
in rooms 1A to 1D were monitored up to 35 days after inoculation (D35), whereas the animals
121
of rooms 2C and 2D were monitored only up to D8, because they were then included in
122
another study. The animals did not receive any treatment with any antibiotics prior to the
123
assay. The same non-supplemented starter diet was offered to the animals up to D2, then pigs
124
were fed with a non-supplemented standard growing diet until the end of the experiment.
125
These diets were formulated to cover pig nutritional requirements. Strict biosecurity measures
126
were implemented to avoid contamination of the pigs, including the use of an air filtration
127
system and airlocks for each room, the use of unit-specific clothes for each room, and
128
compulsory showers after visiting the pigs.
129
E. coli M63 inoculations were performed on D0. Each piglet from groups M63 and M63-T
130
was orally given a 10 mL suspension prepared from E. coli M63 cultivated on MH agar
131
containing cefotaxime (2 mg/L). The titer of the suspension was determined by spreading
132
tenfold dilutions on MH plates. Piglets from groups NT and T were similarly inoculated with
133
sterile medium.
134
On D0, just after E. coli M63 inoculation, piglets from groups T and M63-T were given a
135
single intramuscular injection of a vegetable oil-based suspension of ceftiofur crystalline-free
136
acid (Naxcel, Pfizer) at the recommended dose of 5 mg/kg of body weight. Animal weights
137
were individually recorded once a week. During the week, daily clinical examinations
138
consisted in looking for general clinical signs and taking rectal temperatures. Pigs were
139
euthanized on days 35 to 37 post-inoculation by intravenous injection of sodium pentobarbital
140
followed by exsanguination, and lesions were observed. Individual fecal samples were
141
collected from all animals from the day before inoculation (D-1) and on D1, D3, D7, D10,
142
D13, D17, D22 and D28. The fecal samples were immediately placed in generators for
143
anaerobic bacteria (GENbag anaer, Biomerieux, Marcy l’Etoile, France), freezed in liquid
144
nitrogen and then stored at -70°C until analysis.
145
Bacteriological analysis
146
Bacteriological analyses were conducted on samples collected from the six animal rooms
147
during the first week and samples from the four animal rooms (rooms 1A to 1D) thereafter.
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The titers of putative E. coli (red/pink colonies surrounded by hazy medium) for D-1, D1, D3,
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D10 and D22 and of ESCR Enterobacteriaceae on all sampling times of the individual fecal
150
samples were determined by spreading 100 µL of tenfold dilutions on MacConkey agar plates
151
with or without 2 mg/L cefotaxime in triplicate. After incubation, the colonies were
152
enumerated and the titers were calculated for each pig and each day. The detection limit was
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100 CFU/g of feces. From the cefotaxime-supplemented plate, five isolates per pig per day
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were tested for susceptibility to cefotaxime and to rifampicin by restreaking on MacConkey
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plates containing either 2 mg/L cefotaxime or 250 mg/L rifampicin. All cefotaxime-resistant
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and rifampicin-susceptible isolates as well as one cefotaxime- and rifampicin-resistant isolate
157
per group and per day were stored for further analysis. They were identified with E. coli-
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specific PCR (10, 20). The presence of the blaCTX-M-1 gene was screened by PCR (14) and the
159
phylogenetic groups of the E. coli isolates were determined (13). After digestion with SmaI,
160
pulsed field gel electrophoresis (PFGE) profiles of a few isolates were compared to the profile
161
of the inoculated E. coli M63 strain (21).
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Molecular analysis
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DNA extracts were prepared from 0.2 g of individual fecal samples according to Yu and
164
Morrison (22), followed by Qiagen’s DNA Stool Kit (Qiagen, Courtaboeuf, France). Each
165
DNA extract was quantified using a Nanodrop 2000 spectrophotometer (Thermo Scientific,
166
Courtaboeuf, France) and was then adjusted to a concentration of 10 ng/µL. For all samples
167
collected from pigs in rooms 1A to 1D, previously validated quantitative PCR (qPCR)
168
analyses, all targeting 16S rRNA gene fragments, were carried out to evaluate the change in
169
abundance of the total bacterial population and of the numbers of Bacteroides/Prevotella,
170
Bifidobacterium, E. coli, Enterococcus and Lactobacillus/Leuconostoc/Pediococcus (23).
171
qPCR for quantification of blaCTX-M-1 gene copies was performed (24) for samples collected
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from the six animal rooms during the first week and from the rooms 1A to 1D thereafter; copy
173
numbers were determined by comparison with decimal dilutions of plasmid DNA prepared
174
from the blaCTX-M-1 gene previously cloned in the plasmid pCR®4-TOPO® in the One Shot®
175
TOP10 E. coli strain (Life Technologies, St Aubin, France), according to the manufacturer's
176
instructions.
177
Short-chain fatty acids assay
178
Short-chain fatty acids (SCFA), considered as fermentation markers, were analyzed by gas
179
chromatography as described by Gerard-Champod et al. (25). Only samples collected from
180
the four animal rooms 1A to 1D were analyzed. Acetic, propionic, isobutyric, butyric,
181
isovaleric, valeric, caprilyc acids and heptanoate concentrations were determined. Assays
182
were performed after a deproteinization step with phosphotungstic acid and using 2-
183
ethylbutyric acid as an internal standard.
184
Statistical analysis
185
For body weight gains, culture results and numbers of blaCTX-M-1 copies, all 48 pigs in rooms
186
1 to 6 were included to study the effect of the ceftiofur treatment during the first week. For the
187
following days, or for quantification of bacterial groups by qPCR and metabolites, only
188
animals from rooms 1A to 1D were included.
189
Differences between weight gains of animals or culturable E. coli titers from the different
190
groups were analyzed using the Kruskal-Wallis test followed by the Wilcoxon test. The
191
individual titers of culturable E. coli and ESCR Enterobacteriaceae and gene copy numbers
192
were log10-transformed and significant differences between M63 and M63-T groups were
193
compared using the Wilcoxon test.
194
For data regarding quantification of bacterial populations by qPCR and SCFA analysis, a non-
195
parametric Mann-Whitney test was applied to ensure that the four groups were similar at the
196
beginning of the experiment on D-1. After normalization (Δlog=log(Dx) – log(D-1)), values
197
were grouped per period (first week: D1, D3 and D7; second week: D10 and D13 and third
198
period: D17, D22 and D28) and the normalized data of the different groups for the different
199
periods were compared using a non-parametric Mann-Whitney test.
200
Distributions were compared using the chi-square test or Fisher’s exact test. For all tests,
201
values of P
