Veterinary Microbiology 178 (2015) 230–237

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Veterinary Microbiology journal homepage: www.elsevier.com/locate/vetmic

Genetic diversity of Escherichia coli isolates of animal and environmental origins from an integrated poultry production chain Frédérique Pasquali a, * , Alex Lucchi a , Simonetta Braggio a , Davide Giovanardi b , Achille Franchini a , Maurizio Stonfer c , Gerardo Manfreda a a b c

Department of Agricultural and Food Sciences, Alma Mater Studiorum—University of Bologna, Via del Florio 2, 40064 Ozzano dell’Emilia, Italy TRE VALLI Laboratory, Viale Apollinare Veronesi 5, 37132 San Michele Extra (VR), Italy Bayer Health Care, Animal Health Division, Viale Certosa 130, 20156 Milan, Italy

A R T I C L E I N F O

A B S T R A C T

Article history: Received 26 January 2015 Received in revised form 8 May 2015 Accepted 11 May 2015

Escherichia coli is a normal inhabitant of the intestinal tract of chickens, but when an imbalance in the gut microbiota occurs, E. coli may overgrow and cause extraintestinal infections. The aims of this study were to assess the distribution and spread of E. coli isolates with specific phylogenetic groups and antimicrobial resistance characters among asymptomatic breeder flocks and their broiler progenies with early symptoms of colibacillosis. Broiler flocks were treated with lincospectin during the first week of life and sampled at one, 21 and 42 days. The majority of the 363 E. coli isolates belonged to phylogenetic group A (53.17%), followed by groups D (23.14%), B1 (19.28%) and B2 (4.41%). In broilers, group A was the most represented in birds of 21 and 42 days of age whereas group B1 was the most represented phylogroup in one-day old chicks. More than 90.00% of the isolates were resistant to one or more antimicrobials. Along the life-time of broilers, no differences were found on the occurrence of resistant isolates except for the number of E. coli with elevated MIC to spectinomycin, which increased significantly after the lincospectin treatment. According to XbaI-macrorestriction analysis, a high genetic diversity among E. coli isolates was underlined. Four antimicrobial resistant E. coli isolates of phylogroups A, B1 and D collected from breeders showed similar PFGE patterns to five isolates collected from the respective broiler progenies suggesting a potential spread of these isolates from breeders to broilers. ã2015 Elsevier B.V. All rights reserved.

Keywords: Lincospectin Colibacillosis Broiler Breeder

1. Introduction Escherichia coli is a normal inhabitant of the intestinal tract of chickens and is harmless as long as its growth and colonization is inhibited by other commensal intestinal microbial populations. When an imbalance in the gut microbiota occurs, E. coli may overgrow and cause extraintestinal infections. Avian pathogenic E. coli strains (APEC) might cause colibacillosis often associated to extraintestinal diseases such as omphalitis, yolk sac infections, respiratory tract infections as well as pericarditis and aerosacculitis (Barnes et al., 2003). E. coli strains might be categorised into one of four phylogenetic groups A, B1, B2 and D. In Humans, these groups apparently differ from some characteristics including site of infection, pathogenicity and antimicrobial resistance. It is believed that most commensal strains belong to phylogenetic groups A or B1, although a

* Corresponding author. Tel.: +39 051 209 7862; fax: +39 051 209 7852. E-mail address: [email protected] (F. Pasquali). http://dx.doi.org/10.1016/j.vetmic.2015.05.007 0378-1135/ ã 2015 Elsevier B.V. All rights reserved.

comprehensive review examining published data on distribution of phylogenetic groups among commensal E. coli isolates from all over the world revealed large geographic and temporal variations with overall groups A and B2 more abundant than B1 or D in human feces (Duriez et al., 2001; Bailey et al., 2010). Among pathogenic strains, extraintestinal strains of phylogenetic group B2 showed to kill mice at the highest frequency and carried the highest number of virulence determinants (Picard et al., 1999). Moreover, the phylogenetic group B2 was the most abundant group within pathogenic E. coli implicated in urinary tract infections, neonatal meningitis and inflammatory bowel disease (Bingen et al., 1998; Petersen et al., 2009; Basu et al., 2013). In poultry, this association is still under debate. APEC E. coli strains belonging to serogroups O1, O2, O18 were mostly assigned to group B2, whereas APEC strains of O78 serogroup clustered within phylogenetic groups B1 and D (Moulin-Schouleur et al., 2007). However in another study, APEC E. coli strains clustered within group A, B1 and D and only in less than 20% of cases to B2 (Rodriguez-Siek et al., 2005; Johnson et al., 2008; Graziani et al., 2009). As far as commensal E. coli strains are concerned, only few

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studies have been conducted but all agrees in A as the phylogenetic group including the highest number of E. coli strains (Johnson et al., 2006; Carlos et al., 2010; Hussein et al., 2013). Besides pathogenicity, associations of phylogenetic groups and antimicrobial resistance were found in humans. B2 was identified as the phylogenetic group including the least number of E. coli strains resistant to one or more than three antibiotics in community acquired bacteremia (Bukh et al., 2009). Few data are available on the association of phylogenetic groups and antimicrobial resistance in E. coli strains isolated from animals. Within commensal E. coli strains isolated from healthy pigs, a clear association could not be drawn since resistant determinants were found in strains belonging to all four phylogenetic groups (Sabarinath et al., 2011). On avian isolates and ExPEC isolates from retail poultry products, the number of fluoroquinolone resistant strains was found to be significant lower than the number of susceptible ones among B2 and A phylogroups respectively (Johnson et al., 2003; Graziani et al., 2009). The potential persistence of specific E.coli isolates along the poultry production chain as well as the potential transfer of these isolates to humans rises concern on the spread of specific phylogenetic groups associated to specific antimicrobial resistant characters. In a comprehensive study including different European countries, strong correlations between prevalence of resistance to ampicillin, aminoglycosides, third-generation cephalosporins and fluoroquinolones were observed between human blood stream E. coli isolates and poultry commensal E. coli isolates although the transmission of resistant isolates from poultry to humans was not investigated (Vieira et al., 2011). Moreover, the exposure of hatching to resistant clones present on the surface of the egg can potentially result in dissemination of these strains from the breeder flock to progeny (Idris et al., 2006). The aims of this study were to assess: (1) the distribution of E. coli phylogenetic groups among isolates collected from different organs of breeders and their diseased broiler progenies at different ages; (2) the antimicrobial resistance profiles of these isolates in relation to their phylogenetic characterization; (3) the potential spread of these isolates from broiler breeders to their progeny.

2. Materials and methods 2.1. Sampling scheme Twelve broiler flocks and 4 breeder flocks were selected along an integrated poultry production chain located in Northern Italy. All broiler flocks included birds with early symptoms of colibacillosis (i.e., omphalitis, yolk sac infection). Three flocks of broiler progeny per each breeder flock were selected. Broiler progeny P1 included flocks A1, B1 and C1. P1 was hatched by breeder flock R1. Similarly P2 included A2, B2, C2 and was hatched by R2; P3 (A3, B3, C3) by R3 and P4 (A4, B4, C4) by R4. All four breeder flocks where asymptomatic. Each flock of broilers was sampled three times: first day of life before the antibiotic treatment, at 21 days of age and at the end of the productive cycle (42 days of age). Breeder flocks were sampled at 55 weeks of age. For each sampling time 5–10 birds were collected along with three pools of environmental dust. During the first week of life broiler flocks were treated with lincospectin (50 mg of lincomycin hydrochloride and 100 mg of spectinomycin sulfate) in the water supply for 7 days at a rate of 50 mg/bird/day. Overall a total of 289 birds were randomly selected. All birds were humanely euthanized by cervical dislocation and from each of them different organs (yolk sac or gut, liver, heart, lungs, tracheal mucus, brain, marrow, and air sac) were collected.

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2.2. Escherichia coli isolation Ten grams of dust or organ were diluted 1:10 in Ringer’s solution (Oxoid, Hampshire, UK). An aliquot was plated on Eosin Methylene Blue agar (EMB) and incubated at 37  C for 24 h in duplicate. From each plate, two colonies showing characteristic E. coli morphology were selected for confirmation through biochemical tests by API system (BioMérieux, Marcy L’Etoile, Lyon, France). A total of 363 E. coli isolates were collected. 2.3. Phylogenetic typing The phylogenetic group of E. coli strains was determined by a PCR-based method as described previously (Clermont et al., 2000). Briefly, a triplex PCR was performed on 5 ml of DNA template consisting of boiled lysate of overnight culture. The primer pairs used were ChuA.1 50 -GACGAACCAACGGTCAGGAT-30 , ChuA.2 50 TGCCGCCAGTACCAAAGACA-30 , YjaA.1 50 -TGAAGTGTCAGGAGACGCTG-30 , YjaA.2 (50 -ATGGAGAATGCGTTCCTCAAC-30 , TspE4C2.1 50 -GAGTAATGTCGGGGCATTCA-30 and TspE4C2.2 50 -CGCGCCAACAAAGTATTACG-30 . The PCR steps were as follows: denaturation for 4 min at 94  C, 30 cycles of 5 s at 94  C and 10 s at 59  C, and a final extension step of 5 min at 72  C. The data of the three amplifications resulted in assignment of the strains to phylogenetic groups as follows: chuA +, yjaA –, group D; chuA +, yjaA +, group B2; chuA –, TspE4.C2 –, group A; chuA –, TspE4.C2 +, group B1 (Clermont et al., 2000). In case of yjaA –, chuA –, TspE4.C2 –, the multiplex PCR was repeated with the addition of a fourth primer pair (GadA.1 50 -GATGAAATGGCGTTGGCGCAAG-30 , GadA.2 50 GGCGGAAGTCCCAGACGATATCC-30 ) targetting a 373 bp sequence within the E. coli glutamate decarboxylase-alpha gene, gadA. This target was included as internal amplification control (IAC) to rule out potential false negative results leading to false assignment of the isolates to phylogenetic group A as previously described (Doumith et al., 2012). The optimized protocol included 0.1 mM of each primer ChuA.1, ChuA.2, TspE4C2.1, TspE4C2.2, GadA.1 and GadA.2 and 0.5 mM of each primer YjA.1 and YjA.2. 2.4. Macrorestriction analysis All E. coli isolates were molecular characterised by genomic DNA macrorestriction analysis followed by pulsed-field gel electrophoresis (PFGE), using the PulseNet standardised protocol with XbaI as restriction enzyme (Ribot et al., 2006). Salmonella enterica serovar Braenderup H9812 was used as the molecular size marker in the PFGE experiment. In case of degraded DNA, the PFGE was repeated in 1X HEPES buffer as previously described using the following running protocol: initial switch time: 2.16 s; final switch time: 54.17 s; voltage: 4 V; included Angle: 120 , run time: 26 h (Koort et al., 2002). The XbaI-macrorestriction profiles were imported into Bionumerics 7.1 software (Applied Maths, Saint-Martens-Latem, Belgium) and the normalized profiles were compared using the Dice similarity index, and the dendrogram was constructed with the unweighted-pair group method using average linkage algorithm (UPGMA); 1.0% optimization setting and 1.2% band position tolerance. The discriminatory index (D) of the typing method was calculated as previously described (Hunter and Gaston, 1988). 2.5. Antimicrobial susceptibility testing The antimicrobial susceptibility of E. coli isolates toward amoxicillin (AMX) (Sigma, Milan, Italy), tetracycline (TET) (Sigma), spectinomycin (SP) (Sigma), enrofloxacin (ENR) (Bayer HealthCare, Milan, Italy) and ciprofloxacin (CIP) (Bayer HealthCare) was investigated by the minimum inhibitory concentration (MIC)

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following standard procedures of the microdilution method reported in the document VET01-A4 of the clinical laboratory standard institute (CLSI) (CLSI, 2013a). E. coli ATCC 25922 was used as quality control. Resistance of E. coli poultry isolates was evaluated using chicken-specific derived MIC breakpoints for enrofloxacin and human derived MIC breakpoints for amoxicillin and tetracycline (CLSI document VET01-S2) (CLSI, 2013b). For interpretation of ciprofloxacin MIC values, clinical breakpoints reported in the CLSI document M100-S24 were used in combination to CLSI document M07-A9 (CLSI, 2012, 2014). Breakpoints were as follow: AMX: S  8, I = 16, R  32; TET: S  4, I = 8, R  16; ENR: S  0,25, I = 0,5-1, R  2; CIP: S  1, I = 2, R  4. Due to the absence of spectinomycin MIC breakpoints for E. coli, a spectinomycin elevated MIC value was considered equal or higher than 128 mg/ml.

2.6. Statistical analysis Statistical analysis of data was performed by Fisher exact test or One-way ANOVA using the software STATISTICA 10 (StatSoft, Padova, Italy). Data were considered statistically different with P  0.05. Multiple correspondence analysis (MCA) was performed in order to look simultaneously for associations among the traits (origin of isolation, phylogenetic group, antimicrobial resistance and age of birds).

3. Results and discussion The present study investigated the genetic diversity and antimicrobial resistant character of E.coli isolates along an integrated poultry production chain. Three hundred sixty-three isolates of E.coli were collected from different organs of birds as well as from environmental samples of 12 broiler flocks and their respective breeder flocks. All broiler flocks were selected for diseased chicks with early symptoms of colibacillosis and treated with lincospectin during the first week of life.

3.1. Phylogenetic groups The phylogenetic group assignment was performed by PCR (Clermont et al., 2000). In case of a negative result for all three target sequences yjA, chuA and TspE4.C2, PCR was repeated with the inclusion of a couple of primers specific for the amplification of an internal amplification control: the 367 bp sequence of the gadA gene. All 100 isolates negative for yjA, chuA and TspE4.C2 were positive for the target gadA confirming the assignment to phylogroup A (Fig. S1 of Supplementary data). The majority of the E. coli isolated from breeders of 55 weeks of age belonged to phylogenetic group A (43.90%), followed by groups B1 (41.46%), D (9.760%) and B2 (4.88%). Phylogenetic group A was the most abundant also within broiler isolates (54.35%), followed by groups D (24.84%), B1 (16.46%) and B2 (4.35%). B2 was the least represented in both breeders and broilers with only 16 isolates in total (Table 1). In relation to the age of broilers, one-day-old chicks showed a different distribution of phylogenetic groups in comparison to 21 and 42 days old birds with group B1 as the most represented in chicks (52.50%), followed by A (27.50%), D (20.00%) and B2 (0.00%) (Table 1). In the present study, as the broiler chickens aged from 1 to 21 days, the number of E. coli isolates of group A increased and of group B1 decreased. As the birds age, the gut microbiota community changes in composition with two different community structures at 7–21 days of age and between 21 and 28 days of age (Lu et al., 2003). Further studies are required to verify whether different E. coli phylogenetic groups harbor different colonisation abilities along the lifetime of birds. Regarding the site of isolation, group A was equally distributed among sampled organs. Group B1 was frequently isolated from the gut whereas group B2 to extraintestinal sites (Table S1 of Supplementary data). Group D was mainly found in the gut and respiratory tract (Table S1). The abundance of phylogenetic groups A and D in E. coli from broilers and the observation of B2 more represented in E. coli isolates from extraintestinal sites is in agreement with previous studies on commensal and ExPEC E. coli isolated from broilers, broiler carcasses and retail poultry products all over the world (Johnson et al., 2003, 2006; Carlos et al., 2010;

Table 1 Distribution of phylogenetic groups within E. coli isolates of breeders at 55 weeks of age and their broiler progenies at 1, 21 and 42 days of age. Phylogenetic group

Breeders Group

Broilers Age (weeks)

Group

55 (N = 41) A

B1

B2

D

R1 R2 R3 R4 TOT R1 R2 R3 R4 TOT R1 R2 R3 R4 TOT R1 R2 R3 R4 TOT

11 2 3 2 18 (43.90)ab 3 5 4 5 17 (41.46)a 0 0 0 2 2 (4.88) 2 0 1 1 4 (9.76)b

P1 P2 P3 P4 TOT P1 P2 P3 P4 TOT P1 P2 P3 P4 TOT P1 P2 P3 P4 TOT

TOTAL Age (days)

TOTAL

(Breeders + Broilers)

1 (N = 40)

21 (N = 170)

42 (N = 112)

(N = 322)

(N = 363)

3 3 0 5 11 (27.50)b 7 10 0 4 21 (52.50)a 0 0 0 0 0 (0.00) 3 1 0 4 8 (20.00)ab

18 26 28 28 100 (58.82)a 11 3 2 0 16 (9.41)b 6 2 0 3 11 (6.47) 13 10 11 9 43 (25.29)a

13 18 14 19 64 (57.14)a 5 7 4 0 16 (14.29)b 0 1 2 0 3 (2.68) 2 11 6 10 29 (25.89)a

34 47 42 52 175 (54.35) 23 20 6 4 53 (16.46) 6 3 2 3 14 (4.35) 18 22 17 23 80 (24.84)

45 49 45 54 193 (53.17) 26 25 10 9 70 (19.28) 6 3 2 5 16 (4.41) 20 22 18 24 84 (23.14)

Values within a raw with different superscript letters are statistically different with P  0.05

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Hussein et al., 2013; Aslam et al., 2014; Bagheri et al., 2014). Further studies on pathogenicity need to be performed on B2 isolates of the present study in order to verify whether these isolates are pathogenic similarly to extraintestinal B2 isolates described in humans (Bingen et al., 1998, Petersen et al., 2009; Basu et al., 2013). In general, the distribution of phylogenetic groups of isolates included in the present study, does not match the one on human isolates among which groups A and B2 were found to be the most abundant in human feces (Duriez et al., 2001; Bailey et al., 2010). This observation suggests that poultry might not represent the principle reservoir of B2 virulent E. coli strains identified in humans. 3.2. Antimicrobial resistance Overall, 363 E. coli isolates were tested for resistance against amoxicillin, enrofloxacin, ciprofloxacin, spectinomycin and tetracycline (Table 2 and Table S2 of Supplementary data). Among these, 351 isolates (96.7%) were resistant to at least one of the tested antimicrobials. In particular 293 (80.72%), 130 (35.81%), 74 (20.39%), 237 (65.29%) and 190 (52.34%) isolates were resistant or showed elevated MICs to amoxicillin, enrofloxacin, ciprofloxacin, spectinomycin and tetracycline respectively (Table 2). Excluding isolates with elevated MICs to spectinomycin, the percentage of resistant isolates to the other tested antimicrobials were similar in breeders and broilers and among broilers of different ages. The percentage of isolates with elevated spectinomycin MICs were significantly lower in breeders and one-day old broiler chicks in comparison to 21 and 42 days old broilers (Table 2). This observation might be linked to the use of lincospectin as antimicrobial treatment of colibacillosis diseased broiler flocks within the first week of life. Our data differ from other previously published data according to which a significant decreasing trend on the occurrence of antimicrobial resistance was registered in E. coli isolates as the birds aged (Diarra et al., 2007). The reasons of

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the different result in the present study might be due to the antimicrobial treatment applied which might act as a selective pressure for the persistence of antimicrobial resistance bacteria. Moreover Diarra and colleagues performed the study in an experimental poultry house in controlled conditions, whereas we collected samples in a commercial poultry house with a long history of antimicrobial treatments. In our commercial poultry house antimicrobial resistance characters might have persisted in the poultry house environment which ultimately act as a reservoir of antimicrobial resistant character for bacteria colonising birds at different ages and different production cycles. In this regard among the 76 E. coli isolates collected from dust, occurrence of antimicrobial resistance was comparable or higher than that found among isolates collected from birds. In particular 90.80% of E. coli isolates from dust were resistant or showed elevated MICs to amoxicillin, 57.90% to enrofloxacin, 60.50% to ciprofloxacin, 72.40% to spectinomycin and 50.00% to tetracycline (data not shown). Regarding antimicrobial resistance in relation to the phylogenetic group, B1 grouped the highest percentage of enrofloxacin and ciprofloxacin resistant E. coli isolates (51.43% and 38.57% respectively) in comparison to the other three groups (Table S3 of Supplementary data). No significant differences were observed in the distribution of specific antimicrobial resistances among phylogroups (Table S3). Regarding multiresistance, within A and B2 groups the number of E. coli isolates resistant to 3 or 4 different antimicrobial classes was significantly lower than the number of isolates resistant to 0–2 (P = 0.007) (Table S4 of Supplementary data). B2 has been already described as the group including the highest number of susceptible isolates (Bukh et al., 2009; Graziani et al., 2009). Additionally, specific focus was driven on multiresistance involving ciprofloxacin as a critical important antimicrobial for human medicine. Recently, concern has been arisen on increasing percentages of ciprofloxacin resistance and reduced susceptibility in zoonotic pathogens (EFSA, 2015). In particular for Salmonella,

Table 2 Distribution of E. coli isolates resistant to amoxicillin (AMX), enrofloxacin (ENR), ciprofloxacin (CIP), spectinomycin (SP) and tetracycline (TET) within breeders at 55 weeks of age and their broiler progenies at 1, 21 and 42 days of age. Antimicrobial Resistance

Breeders Group

Broilers Age (weeks)

Group

55 (N = 41) AMX

ENR

CIP

SP

TET

R1 R2 R3 R4 TOT R1 R2 R3 R4 TOT R1 R2 R3 R4 TOT R1 R2 R3 R4 TOT R1 R2 R3 R4 TOT

11 7 4 7 29 (70.73) 5 5 0 2 12 (29.27) 0 4 0 0 4 (9.76) 1 5 0 4 10 (24.39)a 12 2 5 6 25 (60.98)

P1 P2 P3 P4 TOT P1 P2 P3 P4 TOT P1 P2 P3 P4 TOT P1 P2 P3 P4 TOT P1 P2 P3 P4 TOT

TOTAL Age (days)

TOTAL

(Breeders + Broilers)

1 (N = 40)

21 (N = 170)

42 (N = 112)

(N = 322)

(N = 363)

9 14 0 13 36 (90.00) 3 10 0 1 14 (35.00) 1 9 0 0 10 (25.00) 5 11 0 0 16 (40.00)a 10 5 0 0 15 (37.50)

39 32 31 27 129 (75.88) 27 18 9 8 62 (36.47) 20 7 2 5 34 (20.00) 42 27 20 33 122 (71.76)b 33 5 32 28 98 (57.65)

19 31 23 26 99 17 13 6 6 42 9 10 4 3 26 18 24 21 26 89 16 4 20 12 52

67 77 54 66 264 (81.99) 47 41 15 15 118 (36.65) 30 26 6 8 70 (21.74) 65 62 41 59 227 (70.50)b 59 14 52 40 165 (51.24)

78 84 58 73 293 (80.72) 52 46 15 17 130 (35.81) 30 30 6 8 74 (20.39) 66 67 41 63 237 (65.29) 71 16 57 46 190 (52.34)

Values within a raw with different superscript letters are statistically different with P  0.05.

(88.39)

(37.50)

(23.21)

(79.46)b

(46.43)

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Table 3 Antimicrobial resistant patterns of E. coli isolates according to ciprofloxacin MIC value. Ciprofloxacin MIC (mg/ml)

Resistant (N = 74) (R  4)

Antimicrobial resistant pattern AMX

ENR

R R R

R R R R R R

R Intermidiate (N = 28) (I = 2)

Decreased susceptible (94) (DS = 0.12–1)

R R R R R

SP R R R

R R R

R R R R R

R R R

R

R R R R

R R

R R R R R R

R R R R R R

R R R R

Susceptible (167) (S  0.06)

R R R R R R R R

R R R R R R

R R R R R

Isolates N (%)

TET

R R R

4 (5.41) 14 (18.92) 2 (2.70) 1 (1.35) 53 (71.62) 1 (3.57) 1 (3.57) 3 (10.71) 1 (3.57) 12 (42.86) 10 (35.71) 10 (10.64) 7 (7.45) 7 (7.45) 1 (1.06) 2 (2.13) 1 (1.06) 6 (6.38) 2 (2.13) 25 (26.60) 4 (4.26) 2 (2.13) 1 (1.06) 8 (8.51) 18 (19.15) 20 (11.98) 5 (2.99) 6 (3.59) 31 (18.56) 27 (16.17) 29 (17.37) 14 (8.38) 34 (20.36) 1 (0.60)

AMX: amoxicillin; ENR: enrofloxacin; CIP: ciprofloxacin; SP: spectinomycin; TET: tetracycline.

ciprofloxacin CLSI breakpoints were revised following observations of treatment failures on decreased ciprofloxacin susceptibility isolates (MIC 0.12-1) (Humphries et al., 2012). In Table 3, E. coli isolates showing susceptibility (MIC Cip  0.006), decreased susceptibility (MIC value 0.12–1 mg/ml), intermediate (MIC value 2 mg/ml) and resistance to ciprofloxacin (MIC Cip  4 mg/ml) are reported along with their antimicrobial resistant pattern. With MIC Cip  4 mg/ml, 74 E. coli isolates out of 363 (20.39%) were resistant to ciprofloxacin in combination with other antimicrobials. Among them, 70 isolates (94.59%) were multiresistant (resistant to three or more antimicrobials belonging to different antimicrobial classes) and 68 (91.89%) showed elevated MICs to spectinomycin, the antimicrobial used for the treatment of broilers for early symptoms of colibacillosis. The percentage of multiresistance was very high also among isolates with intermediate and decreased susceptible MICs for ciprofloxacin (96.43 and 70.21 respectively) (Table 3). The group of decreased ciprofloxacin susceptible isolates is of particular concern. These isolates often carry gyrA, and/or parC mutations and/or plasmid mediated fluoroquinolone resistance genes which might increase the mutant prevention concentration and consequently boost the development of resistance in case of a continued use of fluoroquinolones (Baudry-Simner et al., 2012). These results support the recent focus on epidemiological cut off values specifically implemented to differentiate the wild-type population to the mutated population with descreased susceptibility or resistance to antimicrobials (CLSI, 2014).

More than 86.00% of E. coli isolates showing resistance to tetracycline or elevated MIC to spectinomycin, were co-resistant to amoxicillin (data not shown). Further studies on co-resistant isolates are required in order to investigate the potential molecular bases of this co-resistances. A possible hypothesis is that the genetic determinants of these resistances are located on mobile genetic elements physically linked (Pasquali et al., 2005). 3.3. Macrorestriction analysis According to XbaI-macrorestriction analysis, genomic DNA of isolates possessed a number of recognition sites for XbaI enzyme yielding between 14 and 24 DNA bands with a molecular mass between 29 and 785 kb. 26 E. coli isolates did not show a distinct pattern and therefore were untypable following the PulseNet standard protocol. The discriminatory index of the standard protocol was 0.984. When the 0.5 X TBE running buffer, recommended by the standard protocol, was replaced by 1X HEPES buffer, all 26 isolates showed distinct patterns and turned out to be typable (Fig. S2 of Supplementary data). Due to substantial different running conditions in HEPES and TBE buffer the 26 PFGE-HEPES profiles were not included in the same analysis for the comparison of the Dice similarity coefficient of the other 337 PFGE-TBE prifiles. Considering a cut off of 80%, the 337 isolates were grouped into 102 clusters including 1 to 20 isolates demonstrating the high genetic diversity among isolates (Fig. 1). This high level of genetic diversity was observed both within and between flocks (Fig. 1). Similar findings confirmed previous studies on the lack of clonal dissemination of E. coli in broiler flocks (Kemmett et al., 2013). Interestingly with a cut off value higher than 93%, two E. coli isolates collected from breeder flock R1 (173 and 169), one collected from breeder flock R3 (3) and one from breeder R4 (85), shared similar XbaI pattern and phylogenetic group of E. coli isolates collected from their respective broiler progenies suggesting the potential spread of these isolates along the poultry production chain (Fig. S3 of Supplementary data). In particular the two breeder R1 isolates (173 and 169) were collected from the gut and belonged to phylogroups B1 and D respectively. Breeder isolate (173) had similar PFGE profile and belongs to the same phylogroup to broiler isolates 187 and 191 collected from the yolk sac of oneday old chicks. Similarly the breeder isolate 169 shared a similar PFGE pattern and the same phylogroup than broiler isolate 192 collected from the yolk sac of one-day old chick. The R3 breeder isolate 3 was collected from respiratory tract, belonged to phylogroup A and shared a similar PFGE pattern and phylogroup of broiler isolate 70 collected from the gut of a 35 days old broiler (Fig. S3). The R4 breeder isolate (85) was collected from dust, belonged to phylogroup B1 and shared the same PFGE pattern and phylogroup of broiler isolate 97 collected from the yolk sac of oneday old chick (Fig. S3). Interestingly some E. coli broiler isolates showed additional resistances and/or elevated MICs to tetracycline, amoxicillin and spectinomycin in comparison to the corresponding breeder isolates. These findings might suggest a potential spread of E. coli isolates from breeders to broilers. Occasional spread of antimicrobial resistant E. coli was already described. An occasional transmission of APEC E. coli resistant to amoxicillin, enrofloxacin and tetracycline was observed from breeders to broiler progeny in Italy (Giovanardi et al., 2005). However, for the E. coli isolates which were reisolated exclusively from the yolk sac of chicks and in any other organs, as well as in any of the older broilers, the transmission cannot be confirmed. Moreover three out of four of the chick isolates showed low MICs values to spectinomycin. The lack of re-isolation of these isolates in older broilers can therefore be linked to the lincospectin treatment which might have affected

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Fig. 1. XbaI macrorestriction analysis of the 337 E. coli poultry isolates typable following the PulseNet standard protocol. The dendrogram was constructed by Dice similarity coefficient and UPGMA clustering using PFGE images of XbaI-digested genomic DNA. The scale bar at the top left indicates the correlation coefficient. Then from the left to the right: column 1: age of birds (yellow, 55 weeks-breeders; blue, one day-broilers; light blue, 21 days-broilers; green, 42 days-broilers); column 2: origin (pink, gut; yellow, dust; violet, respiratory tract; blue, other—brain, liver, marrow and heart); column 3: phylogenetic group (blue, A; orange, B1; grey, B2; black, D); columns 4 to 8: resistance phenotypes to each antimicrobial tested (green, susceptible; red, resistant). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

the survival of these isolates in older broilers. Interestingly a spectinomycin susceptible E. coli isolate originally collected from breeder flock R4 appeared to survive to the antimicrobial treatment due to the acquisition of the spectinomycin resistance character along the poultry production chain.

3.4. Multiple correspondence analysis (MCA) Looking simultaneously for potential associations among the different traits, a MCA was conducted (Fig. 2). According to the plot, phylogenetic group B1 corresponded to gut as site of

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Fig. 2. Multiple correspondence analysis (MCA) of the 363 poultry isolates taking into account the age (55 weeks for breeders and one, 21 and 42 days for broilers) (black), origin (green), phylogenetic group (blue) and antimicrobial susceptibility (S) (orange), intermediate (light blue) or resistance (R) (red). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

isolation, whereas groups A and D to dust and respiratory tract and group B2 to other (liver, morrow and spleen). Regarding the potential association between phylogenetic groups and antimicrobial resistance, groups A and D corresponded to amoxicillin, spectinomycin and tetracycline resistances, whereas group B2 to antimicrobial susceptibility to all five tested antimicrobials. Regarding the age of birds, groups A and D corresponded to broilers of 21 and 42 days of age and B1 to one-day old chicks (Fig. 2). 4. Conclusion The results of the present study suggest that: (1) the distribution of phylogenetic groups among E. coli isolates collected from poultry is different from the one described in humans; (2) the phylogenetic distribution might change as the bird ages, with group B1 as the most frequently detected in one-day old chicks and group A in broilers of 21 and 42 days; (3) the occurrence of antimicrobial resistance is high in broiler flocks affected by colibacillosis; (4) antimicrobial resistant E. coli strains of phylogenetic group A, B1 and D might occasionally spread from breeders to their broiler progeny. Conflicts of interest None. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j. vetmic.2015.05.007. References Aslam, M., Toufeer, M., Narvaez Bravo, C., Lai, V., Rempel, H., Manges, A., Diarra, M.S., 2014. Characterization of extraintestinal pathogenic Escherichia coli isolated

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Genetic diversity of Escherichia coli isolates of animal and environmental origins from an integrated poultry production chain.

Escherichia coli is a normal inhabitant of the intestinal tract of chickens, but when an imbalance in the gut microbiota occurs, E. coli may overgrow ...
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