Sharing of Clinically Important Antimicrobial Resistance Genes by Companion Animals and Their Human Household Members

Abstract

The aims of this study were to implement a rapid easy methodology, to characterize the antimicrobial resistance gene (AMR) gut content associated with Enterobacteriales and staphylococci; and to evaluate statistical association between AMRs present in fecal samples from healthy companion animals and their human household members. Fecal samples were collected from 27 humans and 29 companion animals living in close contact in 20 households. Nineteen healthy humans without daily contact with companion animals were the control group. After DNA extraction, β-lactamase families and 10 genes of other antimicrobial classes were screened by PCR. Furthermore, third-generation cephalosporin-resistant, carbapenem-resistant, and colistin-resistant Enterobacteriales and methicillin-resistant staphylococci were screened by bacteriological methods. The bla_TEM-1B gene with a P3 promotor was the most frequent β-lactam-resistant gene detected in humans and companion animals from households (33.3%, and 17.2%, respectively). The sul2 was the most frequently shared gene by humans and animals from the same household. In 50% of households at least one AMR was detected simultaneously in companion animal/owner pairs. Healthy humans and companion animals carried several AMRs of clinical importance. To the best our knowledge, this study reports the first detection of the bla_SHV-27 gene in fecal samples from healthy humans in Portugal and in Europe.

Introduction

The spread of antimicrobial resistance is one of the greatest challenges faced nowadays in human and veterinary medicine. Antimicrobial-resistant bacterial pathogens are widespread in humans, animals, and the environment, and antimicrobial-resistant infections in humans account for substantial morbidity and mortality, alongside with staggering economic costs.¹ During the last 50 years, the number of companion animals has substantially increased to the point that in many regions, the majority of people have regular and intensive contact with pets.²

β-Lactams and potentiated sulfonamides are in many cases the first-line empirical antimicrobial therapeutic of choice either in human or veterinary medicine and, therefore, resistance leads to important therapeutic failures.¹ It is known that the use of antimicrobials in therapeutics and agriculture increases the selection of antimicrobial resistance and the risk of the gut colonization by antimicrobial-resistant bacteria.^3,4 The close contact of companion animals with humans provides excellent opportunities for interspecies transmission of resistant bacteria and their resistance genes, in either direction.

The public health risks associated with the transfer of antimicrobial-resistant bacteria from companion animals were reviewed in the European Medicine Agency and in its Antimicrobial Working Party reflection article, warning on the existence of antimicrobial resistance microbiological hazards coming from companion animals to humans.^1,5 There is a gap of knowledge on the dynamics of transmission and selection of antimicrobial resistance genes (AMRs) at the companion animal/human interface. Animals may exchange antimicrobial-resistant bacteria and resistance genes with humans, but the extent to which this happens is unknown. Yet, this information is critical to establish the measures to be implemented to decrease the spread of AMRs.

The objectives of this study were implement a rapid easy methodology, to characterize the antimicrobial-resistant gene gut content associated with Enterobacteriales and staphylococci in healthy companion animals and their human household members and identify which AMRs were shared by both; and to evaluate statistical association between AMRs present in fecal samples. The AMRs studied were responsible for phenotypic resistance to six antimicrobial classes: β-lactams, aminoglycosides, colistin, trimethoprim/sulfamethoxazole, tetracycline, chloramphenicol in Enterobacteriales and in Staphylococcus spp.

Materials and Methods

Sampling and collection of data

During April to November 2016, fecal samples were obtained from households (n = 20) constituted of healthy humans (n = 27) living with healthy companion animals (n = 29, 9 cats and 20 dogs). A control group composed of 19 healthy humans without daily contact with companion animals were also enrolled. Humans and companion animals were not eligible for this study if they had been under antimicrobial treatment or were hospitalized in the previous month; and if they suffered from vomiting or diarrheal disease in the last 3 months before the study. Ethics approval for this study was obtained from the Comissão de Ética e Bem-Estar Animal (CEBEA) from the Faculty of Veterinary Medicine of the University of Lisbon. Informed consent was obtained from all human participants. The pet owners were informed of the procedures and conducted the fecal sample collection from their companion animals with sterile containers, gloves, plastic bags, or for humans themselves with the option of using Feces collection paper Fe-Col^® (Alpha Laboratories Ltd, United Kingdom) that was then transferred to a sterile plastic bag. Humans and companion animals were sampled using noninvasive methods.

A brief epidemiological questionnaire about each animal was filled by the owner containing information about age, gender, origin, contact with hospital environment, antimicrobial use in the last year, hospitalization in the last year, kennel/hotel access and lifestyle (indoor or outdoor). Also, a brief epidemiological questionnaire about each human was filled with age, gender, if employee or student in human or veterinary health care, and prior antimicrobial treatment or hospitalization within the last year. To maintain the anonymity, a code number was given to the questionnaires and to samples. To ensure that inclusion was anonymous, human control group, households, humans, and animals were coded with numbered letters HC, S, H and A, respectively.

Immediately after collection, fecal samples were stored at 4°C until processing and were aliquoted and preserved at −80°C until genomic DNA extraction.

Genomic DNA extraction and purification

Fecal samples were homogenized with appropriate aseptic techniques and avoiding aerosolizing. Undigested food fragments were removed. Genomic DNA extraction was conducted using the NZY Tissue gDNA Isolation Commercial Kit (NZYtech-Genes & Enzymes, Portugal) with some modifications to the manufacturer's instructions. Briefly, ∼250 mg of feces were added to 1 mL of TE buffer (10 mM Tris/HCl; 1 mM EDTA, pH = 8). Samples were mixed by vortex and centrifuged during 15 min at 4,000 g. The supernatant was removed, and the pellet was suspended in 0.5 mL of buffer NT1. To 200 μL of the suspended sample 180 μL of buffer NT1 and 25 μL of Proteinase K solution were added. The samples were then incubated overnight at 65°C followed by 10 min at 95°C. The remaining steps were done according to the manufacturer's recommendations.

PCR amplification for genomic DNA from fecal samples

All PCR mix was prepared to a final volume of 50 μL containing nuclease-free water, DreamTaq 10 × Buffer (according to the manufacturer's instructions), 0.0 to 1 μM of 25 mM MgCl₂, 0.2 mM of 25 mM deoxyribonucleotide triphosphates, 1 to 2 μM of each forward and reverse primer, 10 μg per reaction of bovine serum albumin (Sigma-Aldrich, St. Louis), 2 U DreamTaq (5 U/μL) (Fermentas Thermo Scientific, Chicago), and 5 μL of genomic DNA.

Negative and previously sequenced positive controls were included in all PCRs for quality control. PCR amplification of the 16S ribosomal DNA gene was conducted in all samples as a quality control for genomic DNA quality.⁶ Oligonucleotides used in this study can be found in Supplementary Table S1.

Detection of AMRs

Regarding Enterobacteriales five serine-β-lactamase molecular class A families of genes were screened by PCR: bla_SHV and bla_TEM genes and the bla_CTX-M genes belonging to the bla_CTX-M-1, bla_CTX-M-2, and bla_CTX-M-9 groups and positive amplicons were purified and submitted to nucleotide sequencing.^7–11

The genes encoding the serine-β-lactamase molecular class C family of AmpC β-lactamases bla_MOX-1, _MOX-2, _CMY-1, _CMY-8 to _CMY-11, bla_LAT-1 to _LAT-4, _CMY-2 to _CMY-7, and _BIL-1, bla_DHA-1, _DHA-2, bla_ACC, bla_MIR-1T, _ACT-1, and bla_FOX-1 to _FOX-5b were screened by multiplex PCR.¹² Positive samples for the group bla_LAT-1 to _LAT-4, _CMY-2 to _CMY-7, and _BIL-1 and bla_DHA were submitted to amplification of the bla_CMY-2 and bla_DHA-1 genes and underwent nucleotide sequencing.⁴ Furthermore, the genes encoding the serine-β-lactamase molecular class D family of carbapenemases bla_OXA, bla_BIC, bla_NDM, bla_KPC, bla_IMP, bla_VIM, and bla_SPM also were screened by multiplex PCR.¹³

Sulfonamide (sul1, sul2, and sul3), trimethoprim (dfrIa, targeting dfrA1, dfrA5, dfrA15, dfrA15b, dfrA16, dfrA16b), tetracycline (tet(A)), chloramphenicol (cmlA), and aminoglycoside (aac(6’)-Ib) resistance genes were also screened by PCR.^14–16 Positive amplicons of aac(6’)-Ib gene were purified and submitted to nucleotide sequencing.

Moreover, the presence of five colistin plasmid-mediated resistance genes (mcr-1 to mcr-5) were screened by multiplex PCR.^17,18

Regarding staphylococcal, the following resistance genes were screened by PCR: to β-lactams (blaZ and mecA),⁶ to trimethoprim [dfr(G) and dfr(K)], to tetracyclines [tet(M) and tet(K)], and to chloramphenicol (cat_pC221).^19,20

DNA purification and sequencing

The PCR products were purified using the NZYTech Gel Pure Kit (NZYtech-Genes & Enzymes, Portugal), according to the manufacturer's protocol, and sequencing was performed by a commercial laboratory (Stabvida, Portugal). Sequences were analyzed using the Mega (v.7) software and Nucleotide database and Basic Local Alignment Search Tool (BLAST) from the National Center for Biotechnology Information.

Bacteriological methods

One gram of homogenized fecal sample was added to 10 mL of sterile 0.85% NaCl (Merck, Germany) solution and mixed thoroughly. Ten microliters of fecal suspension was plated onto MacConkey (MCK) agar plates (Scharlau, Spain), with 1.5 μg/mL of cefotaxime (CTX) (Sigma-Aldrich) or meropenem (MEM) (Sigma-Aldrich) supplementation for detection of extended-spectrum β-lactamases (ESBLs) or AmpC and carbapenemases-producing Enterobacteriales. Moreover, SuperPolymyxin medium plates was used to screening colistin plasmid-resistant Enterobacteriales.²¹

Furthermore, Brilliance™ MRSA plates (Oxoid, Spain) were used to detect methicillin-resistant Staphylococcus spp. To improve detection of low numbers of Enterobacteriales and methicillin-resistant Staphylococcus spp., 1 g of feces was added to 5 mL of sterile buffered peptone water (Biokar Diagnostics, France), vortexed, and incubated at 36°C ± 1°C for 18 hr. A negative quality control consisting of buffered peptone water alone was also incubated. Following incubation, 1 μL of buffered peptone water fecal suspension was plated onto the MCK agar, SuperPolymyxin medium, and Brilliance MRSA plates described above. Plates were incubated at 36°C ± 1°C for 18 hr. Furthermore, suspected colonies obtained were quantified and to have presumptive identification, were streaked onto UriSelect agar plates (Bio-Rad). All fecal samples had a high number of colony-forming unit (CFU) after direct plating onto Columbia +5% blood sheep (Biomérieux, France) and MCK agar plates, thus confirming fecal sample viability.

Statistical analysis

Statistical analysis was performed on SAS statistical software package for Windows, version 9.4 (SAS Institute, Inc., Cary, NC). Fisher's exact test was used with a significant p-value of ≤0.05.

Results

Enrollment and questionnaire analyses

Among the 20 households included, the household composition varied in the number of humans and companion animals, 12 households had 1 companion animal and 1 human, 2 households had more than 1 human and only 1 companion animal, 3 households had 1 human and more than 1 animal, and 3 households had multiple humans and companion animals. Companion animal owners (n = 27) presented ages from 8 to 66 years, 20 were females and 7 were males. Fifty-two percent (n = 14/27) were employees or students in human or veterinary health care institutions, 33.3% (n = 9/27) had antimicrobial treatment during the last year and 11.1% (n = 3/27) were hospitalized in the previous year. Regarding the human control group (n = 19), the ages ranged between 22 and 65 years, 18 were females and 2 were males. About 74% (n = 14/19) were employees or students in human or veterinary health care institutions, 31.6% (n = 6/19) underwent antimicrobial treatment in the last year and 5.3% (n = 1/19) were hospitalized in the previous year.

Among companion animals (n = 29), 18 were males (12 dogs and 6 cats) and 11 were females (8 dogs and 3 cats) with ages ranging from 2 months to 17 years of age.

All companion animals lived with their owners for at least 6 months before the sample collection, except one animal that had been adopted 1 month before (S16-cat). About 72% (n = 21/29, 20 dogs and 1 cat) of companion animals had access to outdoors. Hospitalization in the previous year was reported in 20.7% (n = 6/29, 2 dogs and 4 cats) of the companion animals. Only one dog (3.4%, n = 1/29) had stayed in kennel/pet hotel in the last year. Contact with hospital environment was observed only in dogs (10.3% of total sample, n = 3/29). Around 17% (n = 5/29, 3 dogs and 2 cats) from companion animals from four households (S3, S4, S14, and S17) had been under antimicrobial treatment within the last year and amoxicillin/clavulanate was the antimicrobial prescribed. Moreover, the two cats were from the same household (S3) (Supplementary Table S2).

AMRs in fecal samples and bacteriological methods

Ten percent (n = 2/20) and 70% (n = 14/20) of the households concerning Enterobacteriales and staphylococci, respectively, lacked any of the tested AMRs (Tables 1 and 2).

Table 1.

ESBLs/AmpC, Carbapenemase-Producing, and Colistin Plasmid-Resistant Enterobacteriales Screening and Antimicrobial Resistance Genes in Fecal Samples from Companion Animals and Humans from Households

Household	Species	MCK+CTX bacteria (CFU/g)	MCK+MEM bacteria (CFU/g)	SuperPolymyxin medium	β-lactam-resistance genes	Sulfonamide-resistance genes	Trimethoprim-resistance genes	Aminoglycoside-resistance genes	Tetracycline-resistance genes	Chloramphenicol-resistance genes	Colistin plasmid-resistance genes
S1	Human1	No growth	No growth	No growth	—	—	—	—	—	—	—
	Human2	No growth	No growth	No growth	bla _TEM-1B	sul3	—	—	—	cmlA	—
	Human3	No growth	No growth	No growth	bla _TEM-1B	sul2	—	—	—	cmlA	—
	Dog1	No growth	No growth	No growth	—	—	—	aac(6′)-Ib-cr	—	—	—
	Dog2	No growth	No growth	No growth	—	—	—	—	—	—	—
S2	Human1	No growth	No growth	Intrinsic bacterial species^a	—	—	dfrIa	—	—	—	—
S2	Cat1	No growth	No growth	No growth	—	—	—	—	—	—	—
S3	Human1	No growth	No growth	No growth	bla _TEM-1B	—	—	—	tet(A)	—	—
	Cat1	No growth	No growth	Intrinsic bacterial species^a	bla _TEM-1B	sul2	dfrIa	—	—	—	—
	Cat2	No growth	No growth	Intrinsic bacterial species^a	—	sul2	—	—	—	—	—
S4	Human1	No growth	No growth	Intrinsic bacterial species^a	—	sul2	—	—	—	—	—
S4	Dog1	No growth	No growth	No growth	bla _TEM-1B	sul2	—	—	tet(A)	—	—
S5	Human1	No growth	No growth	No growth	bla _SHV-1	sul2	—	—	—	—	—
S5	Cat1	No growth	No growth	No growth	—	—	—	—	—	—	—
S6	Human1	No growth	No growth	Intrinsic bacterial species^a	bla _TEM-1B	sul2, sul3	—	—	tet(A)	cmlA	—
S6	Cat1	No growth	No growth	Intrinsic bacterial species^a	bla _TEM-1B	sul2	—	—	tet(A)	—	—
S7	Human1	E. coli (5 × 10³)	No growth	Intrinsic bacterial species^a	bla _SHV-33, bla _CMY-2	sul1	—	—	tet(A)	—	—
	Human2	No growth	No growth	Intrinsic bacterial species^a	bla _TEM-1B, bla _SHV-33	sul1	—	—	tet(A)	—	—
	Human3	E. coli (6 × 10³)	No growth	Intrinsic bacterial species^a	bla _TEM-1B, bla _CMY-2	sul1, sul2	—	—	tet(A)	—	—
	Cat1	No growth	No growth	No growth	—	sul2	—	—	—	—	—
S8	Human1	No growth	No growth	Intrinsic bacterial species^a	bla _TEM-1B	sul1, sul2	dfrIa	aac(6’)-Ib-cr	—	—
S8	Dog1	No growth	No growth	No growth	—	sul2	—	—	—	—	—
S9	Human1	No growth	No growth	Intrinsic bacterial species^a	—	sul2	—	—	—	—	—
S9	Cat1	No growth	No growth	No growth	—	sul2	—	—	—	cmlA	—
S10	Human1	No growth	No growth	Intrinsic bacterial species^a	—	—	—	—	—	—	—
S10	Dog1	Klebsiella spp.^b	No growth	Intrinsic bacterial species^a	bla _TEM-1B, bla _DHA-1	sul3	—	—	tet(A)	—	—
S11	Human1	No growth	No growth	Intrinsic bacterial species^a	—	—	—	—	—	—	—
S11	Dog1	No growth	No growth	Intrinsic bacterial species^a	—	—	—	—	—	—	—
S12	Human1	No growth	No growth	Intrinsic bacterial species^a	—	sul2	—	—	tet(A)	—	—
S12	Dog1	No growth	No growth	Intrinsic bacterial species^a	—	sul2	—	—	—	—	—
S13	Human1	No growth	No growth	Intrinsic bacterial species^a	—	sul1	—	—	—	—	—
	Dog1	No growth	No growth	Intrinsic bacterial species^a	—	sul1	—	—	—	—	—
	Dog2	No growth	No growth	Intrinsic bacterial species^a	—	—	—	—	—	—	—
S14	Human1	No growth	No growth	No growth	—	—	—	—	—	—	—
	Human2	No growth	No growth	Intrinsic bacterial species^a	—	—	—	—	—	—	—
	Dog1	No growth	No growth	No growth	—	—	—	—	—	—	—
	Dog2	No growth	No growth	No growth	bla _TEM-1B	—	—	—	—	—	—
	Dog3	No growth	No growth	Intrinsic bacterial species^a	—	—	—	—	—	—	—
S15	Human1	No growth	No growth	Intrinsic bacterial species^a	—	—	—	—	—	—	—
	Dog1	No growth	No growth	No growth	—	—	—	—	—	—	—
	Dog2	No growth	No growth	No growth	—	—	—	—	—	—	—
	Cat1	No growth	No growth	No growth	—	—	—	—	—	—	—
S16	Human1	No growth	No growth	Intrinsic bacterial species^a	bla _TEM-1B	—	—	—	—	—	—
	Human2	No growth	No growth	Intrinsic bacterial species^a	—	sul2	—	—	tet(A)	—	—
	Dog1	No growth	No growth	Intrinsic bacterial species^a	—	—	—	aac(6’)-Ib-cr	—	—
	Cat1	No growth	No growth	No growth	—	—	—	—	—	—
S17	Human1	No growth	No growth	Intrinsic bacterial species^a	—	sul2	—	—	—	—	—
S17	Dog1	No growth	No growth	Intrinsic bacterial species^a	—	—	—	—	—	—	—
S18	Human1	No growth	No growth	Intrinsic bacterial species^a	—	sul1	—	—	—	—	—
S18	Dog1	No growth	No growth	Intrinsic bacterial species^a	—	—	—	—	—	—	—
S19	Human1	No growth	No growth	Intrinsic bacterial species^a	bla _TEM-1B, bla _SHV-1	—	dfrIa	—	—	—	—
	Human2	No growth	No growth	Intrinsic bacterial species^a	bla _SHV-1	—	—	—	—	—	—
	Dog1	No growth	No growth	No growth	—	—	—	—	—	—	—
S20	Human1	No growth	No growth	No growth	bla _SHV-1	—	—	—	—	—	—
	Dog1	No growth	No growth	No growth	—	—	—	—	—	—	—
	Dog1	No growth	No growth	Intrinsic bacterial species^a	—	—	—	—	—	—	—

Proteus spp. and/or Serratia spp. (polymyxin resistance intrinsic bacterial species).²¹

Obtained from the enrichment medium.

CFU, colony-forming unit; ESBLs, extended-spectrum β-lactamases; MCK+CTX, MacConkey agar plates with 1.5 μg/mL of cefotaxime; MCK+MEM, MacConkey agar plates with 1.5 μg/mL of meropenem; —, not detected.

Table 2.

Methicillin-Resistant Staphylococcus spp. Screening and Antimicrobial Resistance Genes Detected from Staphylococcus spp. in Fecal Samples from Companion Animals and Humans from Households

Household	Species	Brilliance™ MRSA plates	β-Lactam-resistance genes	Chloramphenicol-resistance genes	Tetracycline-resistance genes	Trimethoprim-resistance genes
S1	Human1	No growth	—	—	tet(M)	—
	Human2	No growth	blaZ	cat _pC221	—	dfr(K)
	Human3	No growth	—	—	tet(M)	—
	Dog1	No growth	—	—	—	—
	Dog2	No growth	blaZ	cat _pC221	tet(M)	dfr(K)
S2	Human1	No growth	—	—	—	—
S2	Cat1	No growth	—	—	—	—
S3	Human1	No growth	—	—	—	—
	Cat1	No growth	—	—	—	—
	Cat2	No growth	—	—	—	—
S4	Human1	No growth	—	—	—	—
S4	Dog1	No growth	—	—	—	—
S5	Human1	No growth	—	—	—	—
S5	Cat1	No growth	blaZ	cat _pC221	—	dfr(K)
S6	Human1	No growth	—	—	—	—
S6	Cat1	No growth	—	—	—	—
S7	Human1	No growth	—	—	—	—
	Human2	No growth	—	—	—	—
	Human3	No growth	—	—	—	—
	Cat1	No growth	—	—	—	—
S8	Human1	No growth	—	—	—	—
S8	Dog1	No growth	—	—	—	—
S9	Human1	No growth	—	—	—	—
S9	Cat1	No growth	—	—	—	—
S10	Human1	No growth	—	—	—	—
S10	Dog1	No growth	—	—	—	—
S11	Human1	No growth	—	—	—	—
S11	Dog1	No growth	—	—	—	—
S12	Human1	No growth	—	—	—	—
S12	Dog1	No growth	—	—	—	—
S13	Human1	No growth	blaZ	—	tet(M)	dfr(G)
	Dog1	No growth	blaZ	—	tet(M)	dfr(G)
	Dog2	No growth	blaZ	—	tet(M)	dfr(G)
S14	Human1	No growth	—	—	—	—
	Human2	No growth	—	—	—	—
	Dog1	No growth	—	—	—	—
	Dog2	No growth	—	—	—	—
	Dog3	No growth	—	—	—	—
S15	Human1	No growth	—	—	—	—
	Dog1	No growth	—	—	—	—
	Dog2	No growth	—	—	—	—
	Cat1	No growth	—	—	—	—
S16	Human1	No growth	—	—	—	—
	Human2	No growth	blaZ	cat _pC221	tet(M)	dfr(G)
	Dog1	No growth	—	—	—	—
	Cat1	No growth	—	—	—	—
S17	Human1	No growth	—	—	tet(M)	—
S17	Dog1	No growth	—	—	—	—
S18	Human1	No growth	—	—	—	—
S18	Dog1	No growth	—	—	—	—
S19	Human1	No growth	—	—	tet(M)	—
	Human2	No growth	blaZ	—	tet(M)	dfr(G), dfr(K)
	Dog1	No growth	—	—	—	—
S20	Human1	No growth	—	—	—	—
	Dog1	No growth	—	—	—	—
	Dog1	No growth	—	—	—	—

Regarding humans from the control group (HC) in 15.8% (n = 3/19) and in 47.4% (n = 9/19) no AMRs were detected in Enterobacteriales and staphylococci, respectively (Tables 3 and 4).

Table 3.

ESBLs/AmpC, Carbapenemase-Producing, and Colistin Plasmid-Resistant Enterobacteriales Screening and Antimicrobial Resistance Genes from Fecal Human Samples Without Daily Contact With Companion Animals

Human	MCK+CTX bacteria (CFU/g)	MCK+MEM bacteria (CFU/g)	SuperPolymyxin medium	β-Lactam-resistance genes	Sulfonamide-resistance genes	Trimethoprim-resistance genes	Aminoglycoside-resistance genes	Tetracycline-resistance genes	Chloramphenicol-resistance genes	Colistin plasmid-resistance genes
H1	No growth	No growth	Intrinsic bacterial species^a	—	sul2	dfrIa	—	—	—	—
H2	E. coli ^b	No growth	Intrinsic bacterial species^a	bla _CMY-2	sul2, sul3	—	—	—	—	—
H3	No growth	No growth	Intrinsic bacterial species^a	bla _TEM-1B	sul2	—	—	—	cmlA	—
H4	No growth	No growth	No growth	bla _TEM-1B	—	—	—	—	—	—
H5	No growth	No growth	No growth	—	sul1	—	—	—	—	—
H6	No growth	No growth	No growth	bla _TEM-1B	sul2	—	—	—	—	—
H7	Klebsiella spp. (3 × 10³)	No growth	Intrinsic bacterial species^a	bla _TEM-1B, bla _SHV-1, bla _DHA-1	sul1, sul3	dfrIa	—	tet(A)	—	—
H8	No growth	No growth	Intrinsic bacterial species^a	—	sul1, sul2, sul3	—	—	tet(A)	cmlA	—
H9	No growth	No growth	Intrinsic bacterial species^a	bla _TEM-1B	sul2	—	—	tet(A)	—	—
H10	No growth	No growth	Intrinsic bacterial species^a	—	sul1	—	—	—	—	—
H11	No growth	No growth	Intrinsic bacterial species^a	bla _TEM-1B, bla _SHV-186	—	—	—	—	—	—
H12	Klebsiella spp. (1 × 10³)	No growth	Intrinsic bacterial species^a	bla _TEM-1B, bla _SHV-27	sul3	—	—	—	—	—
H13	No growth	No growth	Intrinsic bacterial species^a	bla _TEM-1B	—	—	—	—	—	—
H14	No growth	No growth	Intrinsic bacterial species^a	—	—	—	aac(6’)-Ib-cr	—	—	—
H15	No growth	No growth	Intrinsic bacterial species^a	—	—	—	—	—	cmlA	—
H16	No growth	No growth	Intrinsic bacterial species^a	—	sul1	—	—	—	—	—
H17	No growth	No growth	Intrinsic bacterial species^a	—	—	—	—	—	—	—
H18	No growth	No growth	Intrinsic bacterial species^a	—	—	—	—	—	—	—
H19	No growth	No growth	Intrinsic bacterial species^a	—	—	—	—	—	—	—

Proteus spp. and/or Serratia spp. (polymyxin resistance intrinsic bacterial species).²¹

Obtained from the enrichment medium.

Table 4.

Methicillin-Resistant Staphylococcus spp. Screening and Antimicrobial Resistance Genes Detected from Staphylococcus spp. in Fecal Human Samples Without Daily Contact With Companion Animals

Human	Brilliance™ MRSA plates	β-Lactam-resistant genes	Chloramphenicol resistant genes	Tetracycline-resistant genes	Trimethoprim-resistant genes
H1	No growth	blaZ	cat _pC221	tet(M)	dfr(K)
H2	No growth	blaZ	cat _pC221	tet(M)	dfr(K)
H3	No growth	—	—	tet(M)	—
H4	No growth	—	—	—	—
H5	No growth	—	—	—	—
H6	No growth	—	—	—	—
H7	No growth	—	—	tet(M)	—
H8	No growth	—	—	tet(M)	—
H9	No growth	—	—	—	—
H10	No growth	—	—	—	—
H11	No growth	—	—	tet(M)	—
H12	No growth	—	—	—	—
H13	No growth	—	—	—	dfr(K)
H14	No growth	—	—	tet(M)	—
H15	No growth	—	—	tet(M)	—
H16	No growth	—	—	tet(M)	—
H17	No growth	—	—	—	—
H18	No growth	—	—	—	—
H19	No growth	—	—	—	—

Regarding Enterobacteriales and staphylococci genes, companion animals were positive for bla_TEM-1 (17.2%, n = 5/29), bla_DHA-1 (3.4%, n = 1/29), sul1 (3.4%, n = 1), sul2 (27.6%, n = 8), sul3 (3.4%, n = 1), cmlA (3.4%, n = 1), tet(A) (10.3%, n = 3), aac(6’)Ib-cr (10.3%, n = 3), blaZ (13.8%, n = 4), cat_pC221 (6.9%, n = 2), tet(M) (10.3%, n = 3), dfr(G), and dfr(K) (6.9%, n = 2, both) genes (Table 5).

Table 5.

Antimicrobial Resistance Genes Frequency in Fecal Samples from Companion Animals and Humans

Genes	Human (control, n = 19)	Human (owners, n = 27)	Companion animals (n = 29)	Household ^a human/animal sharing	Household human/human sharing	Household animal/animal sharing
Genes	n (%)	n (%)	n (%)	n (%)	n (%)	n (%)
bla_SHVgroup	3 (15.8)	6 (22.2)	0 (0.0)	0 (0.0)	2 (10.0)	0 (0.0)
bla_TEMgroup	7 (36.8)	9 (33.3)	5 (17.2)	2 (10.0)	2 (10.0)	0 (0.0)
bla _DHA-1	1 (5.3)	0 (0.0)	1 (3.4)	0 (0.0)	0 (0.0)	0 (0.0)
bla _CMY-2	1 (5.3)	2 (7.4)	0 (0.0)	0 (0.0)	1 (5.0)	0 (0.0)
sul1	6 (31.6)	6 (22.2)	1 (3.4)	1 (5.0)	1 (5.0)	0 (0.0)
sul2	7 (36.8)	12 (44.4)	8 (27.6)	6 (30.0)	1 (5.0)	1 (5.0)
sul3	4 (21.1)	2 (7.4)	1 (3.4)	0 (0.0)	0 (0.0)	0 (0.0)
dfrIa	2 (10.5)	4 (14.8)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)
cmlA	4 (21.1)	3 (11.1)	1 (3.4)	0 (0.0)	1 (5.0)	0 (0.0)
tetA	4 (21.1)	7 (25.9)	3 (10.3)	1 (5.0)	1 (5.0)	0 (0.0)
aac(6’)Ib-cr	2 (10.5)	0 (0.0)	3 (10.3)	0 (0.0)	0 (0.0)	0 (0.0)
blaZ	2 (10.5)	4 (14.8)	4 (13.8)	2 (10.0)	1 (5.0)	1 (5.0)
cat pC221	2 (10.5)	2 (7.4)	2 (6.9)	1 (5.0)	0 (0.0)	0 (0.0)
tet(M)	9 (47.4)	7 (25.9)	3 (10.3)	2 (10.0)	2 (10.0)	1 (5.0)
dfr(G)	0 (0.0)	3 (11.1)	2 (6.9)	1 (5.0)	0 (0.0)	1 (5.0)
dfr(K)	3 (15.8)	2 (7.4)	2 (6.9)	1 (5.0)	0 (0.0)	0 (0.0)

Total number of households is 20.

The presence of the bla_TEM-1 gene, was detected in 33.3% (n = 9/27) of fecal samples from companion animal owners and in 42.1% (n = 8/19) of humans from the control group. Interestingly, two households (S3 and S6) had positive bla_TEM-1B companion animals and humans simultaneously (Table 1). All bla_TEM-1 gene sequences were identical to the bla_TEM-1B gene framework in the coding and promoter (P3) regions.

The bla_SHV family of genes was only detected in human participants (Table 5). Companion animal owners were positive for bla_SHV-1 (n = 3) and bla_SHV-33 (n = 2), while humans from the control group were positive for bla_SHV-1 (n = 1), bla_SHV-186 (n = 1), and bla_SHV-27 (n = 1). Of note, bla_SHV-33 and bla_SHV-1 were shared between humans of the same household (S7 and S19, respectively) (Table 1). The AmpC β-lactamase family of genes, bla_CMY-2 and bla_DHA-1, were detected in humans from both groups (Table 5). There was no statistical difference between β-lactamase carriage in companion animal owners and humans from the control group (p > 0.05).

The dfraI gene responsible for trimethoprim resistance was only detected in human samples, whereas the aminoglycoside aac(6’)-Ib-cr resistance gene was mostly detected in companion animals (Table 5). However, aac(6’)-Ib-cr and dfraI resistance gene detection had no statistical significance between groups (companion animals vs. owners and all humans vs. companion animals) (p > 0.05). Companion animals and humans of both groups had similar (p > 0.05) carriage of tetracycline tet(A) and chloramphenicol cmlA resistance genes (Table 5). Detection of sulfonamide resistance genes (sul1, sul2, and sul3) were higher in humans from both groups. Detection of sul1 gene was higher (p = 0.048) in owners than in companion animals (Table 5). The sul2 gene was the gene that more humans and companion animals shared within the same household (30%, n = 6/20) (Table 1). Moreover, blaZ, cat_pCC221, tet(M), dfr(K), and dfr(G) genes were shared between companion animals and humans from the same household. The Besides the sul2 genes, the blaZ and tet(M) were the genes that more humans and companion animals shared within the same household (10.0%, n = 2/20; both) (Tables 2 and 5). Yet, no statistical significance for the resistance genes from Staphylococcus spp. were detected between fecal samples from humans without daily contact with humans versus owners and companion animals and humans from the same household.

Humans and companion animals from 10 out of the 20 households (50%) shared at least one AMR (Tables 1 and 2). Yet, fecal samples from owners showed higher AMR frequency and diversity than companion animals (Table 5).

About 17% (n = 5/29) of fecal samples from companion animals were positive for AMRs associated with Enterobacteriales and staphylococci against at least to three different classes of antimicrobials (Tables 1 and 2).

The most common AMR combination in pets regarding Enterobacteriales was bla_TEM-1B-sul2 (n = 2, one dog, one cat) from different households. These pets were one dog that had been under antimicrobial treatment in the last year and also both dog and cat that were hospitalized in the previous year. Regarding companion animal owners, 14.8% (n = 4/27) had the presence of AMRs to at least three different antimicrobial classes regarding Enterobacteriales and Staphylococcus spp. (Tables 1 and 2).

The remaining AMRs studied, namely, carbapenemases, and colistin plasmid-resistant mecA and tet(K) genes were not detected in any fecal sample from humans or companion animals.

Furthermore, third-generation cephalosporin (3GC)-resistant, carbapenem-resistant, and colistin-resistant Enterobacteriales and methicillin-resistant staphylococci (MRS) were screened by bacteriological methods and no positive growth occur for carbapenem-resistant and colistin-resistant Enterobacteriales and MRS (Tables 1–4). Moreover, fecal samples that were positive for (3GC)-resistant Enterobacteriales, ESBLs/or cephalosporinase AmpC β-lactamases were detected by direct Genomic DNA extraction and amplification from fecal samples (Tables 1–4).

Regarding the epidemiological survey, no statistical associations were found with the AMRs present on the fecal samples from humans and companion animals.

Discussion

In the present study, we report the detection of 3GC-resistant Enterobacteriales and the presence of several AMRs in the fecal samples of healthy companion animals and their human household members as well as of humans without daily contact with companion animals.

The relationship between humans and companion animals has changed over the years. Nowadays, companion animals live in a “relationship of mutualism” with their owners.² The anthropomorphizing of companion animals has led to changes in the behavior of owners toward them, with increasing conducts like kissing, licking, sharing food, and sharing beds. Considering the shared environment of humans and companion animals, their close relationship, and the increased frequency of antimicrobial-resistant bacteria detected in humans and companion animals, new opportunities are created for interspecies transfer of AMRs.^1,2

Antimicrobials are used extensively in human medicine, veterinary medicine, food-producing animals, and agriculture.²² In Portugal, β-lactams, such as penicillins, are the most prescribed antimicrobials in humans followed by macrolides, lincosamides, streptogramins, quinolones, tetracyclines, and sulfonamide–trimethoprim.²³ In Veterinary medicine, penicillins are also the most commonly prescribed antimicrobials in companion animals (dogs and cats), namely, amoxicillin and amoxicillin–clavulanate.²⁴ Yet, lincosamides, quinolones, macrolides, tetracyclines (doxycycline), nitroimidazoles, and sulfonamide–trimethoprim are also used in small animal practice.^5,24 Several antimicrobial classes are used in humans and companion animals are the same, leading to an overlap of the detected AMRs.^1,25 Enterobacteriales’ resistance to β-lactams is increasing in humans and in companion animals and there are no specific β-lactamases that are restricted only to animals or humans.¹⁹ This seems to be in line with the results from this study. The β-lactamases that are disseminated in the Enterobacteriales family, especially the ESBLs and cephalosporinases of AmpC type are of particular clinical relevance.

In this study, the bla_TEM gene was the most frequent β-lactam-resistant gene in humans and companion animals, which is in agreement with previous studies.^26–28 In Portugal, the TEM-β-lactamase has also been detected in Enterobacteriales from food-producing animals and from commensal and clinical isolates.^7,27 The bla_TEM genes detected in this study (from companion animals and humans) had similar promotor and coding region polymorphisms as the bla_TEM-1B (according to the Sutcliffe numbering system).²⁹ Furthermore, in two households, the bla_TEM-1B was present in coliving humans and companion animals. This finding may have resulted from a zoonotic transfer of bla_TEM-1B genes harbored in Enterobacteriales. Nevertheless, a common source of colonization could also be hypothesized since this resistance mechanism has been extensively detected in Portugal.^7,27

CTX-M β-lactamases are the current dominant type of ESBLs worldwide, having overpassed the TEM and SHV β-lactamases in Europe, both in humans and animals.^4,30 Furthermore, Portugal is among the European countries with the highest frequency of ESBL detection, mainly TEM and CTX-M.^31,32 However, in this study, only the ESBL SHV-27 gene was detected in a healthy human from the control group. The bla_SHV-27 gene has been previously detected in clinical Klebsiella pneumoniae, Escherichia coli, and Enterobacter cloacae from humans and in clinical K. pneumoniae from dogs of different countries.^33–36 To the best of our knowledge, this is the first detection of bla_SHV-27 gene in fecal samples from healthy humans in Portugal and in Europe. Moreover, in this human fecal sample we detected a 3GC-resistant Klebsiella spp.

Only the CMY-2 and DHA-1 β-lactamase-encoding genes were detected among all the AmpC cephalosporinase genes tested in this study, and these occurred mainly in humans. The bla_CMY-2 gene and the bla_DHA-1 gene were each detected in one healthy human without daily contact with companion animals. The bla_CMY-2 gene was detected in two humans from the same household. The detection of these AmpC genes was already described in Portugal in clinical strains of Enterobacteriales from humans in the community and hospital, but as far as we are aware not in healthy individuals in the community.³⁷

The frequency of sulfonamide resistance sul genes (sul1, sul2, and sul3) reported in this study is in agreement with previous studies.^25,32–35 In Portugal, sul1 and sul2 resistance genes have been detected in commensal Enterobacteriales from fecal samples of healthy humans, food-producing animals, and from clinical isolates.^32,40,41 Likely due to its dissemination, the sul2 gene was the most frequent among humans and companion animals and was frequently detected in members of the same household.

The effect of the low antimicrobial consumption, the household controlled environment, and the possible food-borne dissemination of AMRs should also be considered in this study as an explanation for the shared AMRs.^36–39 The presence of ESBLs/AmpC in this study was lower than previously reported in healthy dogs in Portugal (Lisbon area).⁴ In a previous study from our group, dogs from shelters/breeders were approximately three times more likely to have an ESBL/AmpC-producing E. coli than dogs from private owners.⁴ The results in the present study may be explained by the fact that companion animals included in this study had little contact with kennels and were healthy.

This study showed that humans and companion animals carried and shared several AMRs of clinical importance. Most of these genes are usually associated with mobile genetic elements (plasmids, integrons, and transposons), which are important for antimicrobial resistance transfer between different microbiomes.^42–47

The small sample size of this study is a limitation that may have limited the detection of ESBL/AmpC, carbapenemases, colistin plasmid-resistant genes, and methicillin-resistant Staphylococcus spp., and the detection of statistical associations between the presence of AMRs and specific risk factors.

Moreover, in this study, different dfr genes (conferring resistance to trimethoprim) were detected; regarding Staphylococcus spp. and usually the dfr genes found in coagulase-positive staphylococcus (CoPS) and coagulase-negative staphylococcus (CoNS) are different.⁴⁸ In CoPS, the gene dfr(G) is the most common in Staphylococcus pseudintermedius isolates; whereas the dfr(K) gene is the most common in Staphylococcus aureus.^19,48

Nevertheless, the role of companion animals in the dissemination of clinically relevant AMRs to humans through fecal contamination should not be neglected. Additionally, commensal Enterobacteriales and Staphylococcus spp. of healthy humans may also be a reservoir for antibiotic-resistance determinants.^1,49

Further studies are needed to determine the causality and directionality of resistance gene transfer between human and companion animals, to identify the critical control points at which interventions could substantially prevent the spread of AMRs within households and establish the prevention and intervening measures for controlling resistance.

In this study, were implemented a rapid easy methodology, which easily detected antimicrobial resistant genes that are of particular interest to epidemiological studies. Highly discriminatory universal methods, such as whole-genome sequencing, are expensive to most of the laboratories. We also validated its usefulness in situations requiring rapid MRS, ESBLs/AmpC, carbapenemase-producing and colistin plasmid-resistant Enterobacteriales.

The combination of molecular techniques with culture methods should be pursued in the future to increase the detection of antimicrobial resistance determinants leading to a better understanding of the overlap between the human and companion animal gut resistome.

Footnotes

Acknowledgments

The authors acknowledge the PET-Risk Consortium and all its members: Cátia Marques, Luís Telo Gama, and Rodolfo Leal (Portugal); Stefan Schwarz and Claudia Feudi (Germany); Scott Weese, Joyce Rousseau, and Rebecca Flancman (Canada); Anette Loeffler and Sîan Frosini (United Kingdom); and Vincent Perreten (Switzerland).

Disclosure Statement

No competing financial interests exist.

Funding Information

This work was supported by JPIAMR/0002/2016 Project—PET-Risk Consortium and FEDER funds through the Programa Operacional Factores de Competitividade—COMPETE and by National funds through the FCT—Fundação para a Ciência e a Tecnologia—CIISA Project (UID/CVT/00276/2020). A.B. holds an FCT PhD grant SFRH/BD/113142/2015. J.M. holds an FCT research grant supported by the JPIAMR/0002/2016 Project.

Supplementary Material

References

Pomba

, Rantala

, Greko

, Baptiste

K.E

, Catry

, van Duijkeren

, Mateus

, Moreno

M.A

, Pyörälä

, Ružauskas

, Sanders

, Teale

, Threlfall

E.J

, Kunsagi

, Torren-Edo

, Jukes

, and Törneke

. 2017. Public health risk of antimicrobial resistance transfer from companion animals. J. Antimicrob. Chemother. 72:957–968.

Dotson

M.J.

, and Hyatt

E.M.

. 2008. Understanding dog-human companionship. J. Bus. Res. 61:457–466

Barza

, and Travers

. 2002. Excess infections due to antimicrobial resistance: the attributable fraction. Clin. Infect. Dis. 34:126–130.

Belas

, Salazar

A.S

, Gama

L.T

, Couto

, and Pomba

. 2014. Risk factors for faecal colonisation with Escherichia coli producing extended-spectrum and plasmid-mediated AmpC β-lactamases in dogs. Vet. Rec. 175:202.

European Medicines Agency (EMA). 2015. Reflection paper on the risk of antimicrobials resistance transfer from companion animals. Committee for Medicinal Products for Veterinary Use. EMA/CVMP/AWP/401740/2013. Available at https://www.ema.europa.eu/en/documents/scientific-guideline/reflection-paper-risk-antimicrobial-resistance-transfer-companion-animals_en.pdf (accessed March 19, 2020).

Pomba

, Hasman

, Cavaco

L.M

, da Fonseca

J.D

, and Aarestrup

F.M.

. 2009. First description of methicillin-resistant Staphylococcus aureus (MRSA) CC30 and CC398 from swine in Portugal. Int. J. Antimicrob. Agents, 34:193–194.

Pomba-Féria

, and Caniça

. 2003. A novel sequence framework (bla_TEM-1G) encoding the parental TEM-1 beta-lactamase. FEMS Microbiol. Lett. 220:177–180.

Mendonça

, Ferreira

, Louro

, and Caniça

. 2006. Occurrence of a novel SHV-type enzyme (SHV-55) among isolates of Klebsiella pneumoniae from Portuguese origin in a comparison study for extended-spectrum β-lactamase-producing evaluation. Diagn. Microbiol. Infect. Dis. 56:415–420.

Pomba

, Mendonça

, Costa

, Louro

, Baptista

, Ferreira

, Correia

J.D

, and Caniça

. 2006. Improved multiplex PCR method for the rapid detection of beta-lactamase genes in Escherichia coli of animal origin. Diagn. Microbiol. Infect Dis. 56:103–106.

10.

Woodford

, Fagan

E.J

, and Ellington

M.J.

. 2006. Multiplex PCR for rapid detection of genes encoding CTX-M extended-spectrum (beta)-lactamases. J. Antimicrob. Chemother. 57:154–155.

11.

Caniça

M.M.

, Lu

C.Y

, Krishnamoorthy

, and Paul

G.C.

. 1997. Molecular diversity and evolution of bla_TEM genes encoding beta-lactamases resistant to clavulanic acid in clinical E. coli. J. Mol. Evol. 44:57–65.

12.

Pérez

, and Hanson

. 2002. Detection of plasmid-mediated AmpC beta-lactamase genes in clinical isolates by using multiplex PCR. J. Clin. Microbiol. 40:2153–2162.

13.

Poirel

, Walsh

T.R

, Cuvillier

, and Nordmann

. 2011. Multiplex PCR for detection of acquired carbapenemase genes. Infect. Dis. 70:119–123.

14.

Sáenz

, Brinãs

, Domíngues

, Ruiz

, Zarazaga

, Vila

, and Torres

. 2004. Mechanisms of resistance in multiple-antibiotic-resistant Escherichia coli strains of human, animal and food origins. J. Antimicrob. Chemother. 48:3996–4001.

15.

Robicsek

, Strahilevitz

, Jacoby

G.A

, Mark

, Abbanat

, Park

C.H

, Bush

, and Hooper

D.C.

. 2006. Fluoroquinolone-modifying enzyme: a new adaptation of a common aminoglycoside acetyltransferase. Nat. Med. 12:83–88.

16.

Perreten

, and Boerlin

. 2003. A new sulfonamide resistance gene (sul3) in Escherichia coli is widespread in the pig population of Switzerland. Antimicrob. Agents Chemother. 47:1169–1172.

17.

Rebelo

A.R.

, Bortolaia

, Kjeldgaard

J.S

, Pedersen

S.K

, Leekitcharoenphon

, Hansen

I.M

, Guerra

, Malorny

, Borowiak

, Hammerl

J.A

, Battisti

, Franco

, Alba

, Perrin-Guyomard

, Granier

S.A

, De Frutos Escobar

, Malhotra-Kumar

, Villa

, Carattoli

, and Hendriksen

R.S.

. 2018. Multiplex PCR for detection of plasmid-mediated colistin resistance determinants, mcr-1, mcr-2, mcr-3, mcr-4 and mcr-5 for surveillance purposes. Euro. Surveill. 6:17–00672.

18.

Borowiak

, Fischer

, Hammerl

J.A

, Hendriksen

R.S

, Szabo

, and Malorny

. 2017. Identification of a novel transposon-associated phosphoethanolamine transferase gene, mcr-5, conferring colistin resistance in d-tartrate fermenting Salmonella enterica subsp. enterica serovar Paratyphi B. J. Antimicrob. Chemother. 72:3317–3324.

19.

Schnellmann

, Gerber

, Rossano

, Jaquier

, Panchaud

, Doherr

M.G

, Thomann

, Straub

, and Perreten

. (2006). Presence of new mecA and mph(C) variants conferring antibiotic resistance in Staphylococcus spp. isolated from the skin of horses before and after clinic admission. J. Clin. Microbiol. 44:4444–4454.

20.

Feβler

, Scott

, and Kadlec

. 2010. Characterization of methicillin-resistant Staphylococcus aureus ST398 from cases of bovine mastitis. J. Antimicrob. Chemother. 65:619–625.

21.

Nordmann

, Jayol

, and Poirel

. 2016. A universal culture medium for screening polymyxin-resistant gram-negative isolates. J. Clin. Microbiol. 54:1395–1399.

22.

Rolain

J.M.

2013. Food and human gut as reservoirs of transferable antibiotic resistance encoding genes. Front Microbiol. 4:173. eCollection 2013.

23.

European Centre for Disease Prevention and Control. 2018. Surveillance of antimicrobial consumption in Europe, 2013–2014. Stockholm: ECDC. Available at https://www.ecdc.europa.eu/sites/default/files/documents/Surveillance-antimicrobial-consumption-Europe-ESAC-Net-2013-14.pdf (accessed March 19, 2020).

24.

European Medicines Agency, European Surveillance of Veterinary Antimicrobial Consumption, 2018. Sales of veterinary antimicrobial agents in 30 European countries in 2016. (EMA/275982/2018). Available at https://www.ema.europa.eu/en/documents/report/sales-veterinary-antimicrobial-agents-30-european-countries-2016-trends-2010-2016-eighth-esvac_en.pdf (accessed March 19, 2020).

25.

Guardabassi

, Schwarz

, and Lloyd

. 2004. Pet animals as reservoirs of antimicrobial-resistant bacteria. J. Antimicrob. Chemother, 54:321–332.

26.

Trott

2012. β-Lactam resistance in gram-negative pathogens isolated from animals. Curr. Pharm. Des. 19:239–249.

27.

Costa

, Poeta

, Saenz

, Coelho

A.C

, Matos

, Vinue

, Rodrigues

, and Torres

. 2007. Prevalence of antimicrobial resistance and resistance genes in faecal isolates recovered from healthy pets. Vet. Microbiol. 127:97–105.

28.

Rodríguez-Baño

, Alcalá

J.C

, Cisneros

J.M

, Grill

, Oliver

, Horcajada

J.P

, Tórtola

, Mirelis

, Navarro

, Cuenca

, Esteve

, Peña

, Llanos

A.C

, Cantón

, and Pascual

. 2008. Community infections caused by extended-spectrum β-lactamase-producing Escherichia coli. Arch. Intern. Med. 168:1897–1902.

29.

Sutcliffe

J.G.

1978. Nucleotide sequence of the ampicillin gene of Escherichia coli plasmid pBR322. Proc. Natl. Acad. Sci. U. S. A., 75:3737–3741.

30.

Ewers

, Grobbel

, Bethe

, Wiler

, and Guenther

. 2011. Extended-spectrum beta-lactamases-producing gram-negative bacteria in companion animals: action is clearly warranted!. Berl. Munch. Tierarztl. Wochenschr. 124:94–101.

31.

Fernandes

, Amador

, Oliveira

, and Prudêncio

. 2014. Molecular characterization of ESBL-producing Enterobacteriaceae in northern Portugal. Sci. World J. 2014:782897.

32.

Machado

, Coque

T.M

, Cantón

, Sousa

J.C

, and Peixe

. 2013. Commensal Enterobacteriaceae as reservoirs of extended-spectrum beta-lactamases, integrons, and sul genes in Portugal. Front Microbiol, 4:80.

33.

Abbassi

M.S.

, Torres

, Achour

, Vinué

, Sáenz

, Costa

, Bouchami

, and Ben Hassen

. 2008. Genetic characterisation of CTX-M-15-producing Klebsiella pneumoniae and Escherichia coli strains isolated from stem cell transplant patients in Tunisia. Int. J. Antimicrob Agents. 32:308–314.

34.

Kiratisin

, Apisarnthanarak

, Laesripa

, and Saifon

. 2008. Molecular characterization and epidemiology of extended-spectrum- β-lactamase-producing Escherichia coli and Klebsiella pneumoniae isolates causing health care-associated infection in Thailand, where the CTX-M family is endemic. Antimicrob. Agents Chemother. 52:2818–2824.

35.

Hammami

, Boubaker

I.B.B

, Saidani

, Lakhal

, Hassen

A.B

, Kamoun

, Ghozzi

, Slim

, and Ben Redjeb

. 2011. Characterization and molecular epidemiology of extended spectrum β-lactamase producing Enterobacter cloacae isolated from a Tunisian hospital. Microb. Drug Resist. 18:59–65.

36.

Zhang

P.L.C.

, Shena

X.G

, Chalmersa

R.J

, Reid-Smitha

D.S

, Dicke

, and Boerlina

. 2018. Prevalence and mechanisms of extended-spectrum cephalosporin resistance in clinical and fecal Enterobacteriaceae isolates from dogs in Ontario, Canada. Vet. Microbiol. 213:82–88.

37.

Ribeiro

T.G.

, Â. Novais, Rodrigues

, Nascimento

, Freitas

, Machado

, and Peixe

. 2019. Dynamics of clonal and plasmid backgrounds of Enterobacteriaceae producing acquired AmpC in Portuguese clinical settings over time. Int. J. Antimicrob. Agents, 53:650–656.

38.

Pitout

J.D.

, Nordmann

, Laupland

K.B

, and Poirel

. 2005. Emergence of Enterobacteriaceae producing extended -spectrum beta-lactamases (ESBLs) in the community. J. Antimicrob. Chemother. 56:52–59.

39.

Gong

, Zhuang

, Zhang

, and Dou

, Wang

. 2018. Establishment of a Multiplex Loop-Mediated Isothermal Amplification Method for Rapid Detection of Sulfonamide Resistance Genes (sul1, sul2, sul3) in Clinical Enterobacteriaceae Isolates from Poultry. Foodborne Pathog. Dis. 15:413–419.

40.

Fernandes

, Centeno

, Belas

, Nunes

, Lopes Alves

, Couto

, and Pomba

. 2012. Immediate after birth transmission of epidemic Salmonella enterica Typhimurium monophasic strains in pigs is a likely event. J. Antimicrob Chemother. 67:3012–3014.

41.

Antunes

, Machado

, Sousa

J.C

, and Peixe

. 2005. Dissemination of sulfonamide resistance genes (sul1, sul2, and sul3) in Portuguese Salmonella enterica strains and relation with integrons. Antimicrob. Agents Chemother. 49:836–839.

42.

Cormican

, Hopkins

, Jarlier

, Reilly

, Simonsen

G.S

, Strauss

, Vandenberg

, Zabicka

, Zarb

, Catchpole

, Heuer

, Iosifidis

, Monnet

, Plachouras

, Weist

, Ricci

, Allende

, Bolton

, Chemaly

, Davies

, Escamez

P.S.F

, Girones

, Herman

, Koutsoumanis

, Lindqvist

, Norrung

, Robertson

, Ru

, Sanaa

, Simmons

, Skandamis

, Snary

, Speybroeck

, Ter Kuile

, Threlfall

, Wahlstrom

, Tenhagen

B.A

, Teale

, Schuepbach

, Beloeil

P.A

, Liebana

, Stella

, Murphy

, Hauser

, Urbain

, Kozhuharov

E.I

, Bozic

, Michaelidou-Patsia

, Bures

, Vestergaard

E.M

, Baptiste

, Tiirats

, Nevalainen

, Rouby

J.C

, Hahn

, Schlumbohm

, Malemis

, Kulcsar

, Lenhardsson

J.M

, Beechinor

J.G

, Breathnach

, Pasquali

, Auce

, Maciulskis

, Schmit

, Spiteri

, Schefferlie

G.J

, Hekman

, Bergen dahl

, Swiezcicka

A.W

, Da Silva

J.P.D

, Taban

L.S.C

, Hederova

, Straus

, Madero

C.M

, Persson

E.L

, Jukes

, Weeks

, Kivilahti-Mantyla

, Moulin

, Wallmann

, Grave

, Greko

, Munoz

, Bouchard

, Catry

, Moreno

M.A

, Pomba

, Rantala

, Ruzauskas

, Sanders

, Schwarz

, Teale

, van Duijkeren

, Wester

A.L

, Ignate

, Kunsagi

, Torren- Edo

. 2017. ECDC, EFSA and EMA joint scientific opinion on a list of outcome indicators as regards surveillance of antimicrobial resistance and antimicrobial consumption in humans and food-producing animals. EFSA J. 15:e05017.

43.

Kummer

2004. Resistance in the environment. J. Antimicrob. Chemother. 54:311–320.

44.

Karami

, Nowrouzian

, Adlerberth

, and Wold

A.E.

. 2006. Tetracycline resistance in Escherichia coli and persistence in the infantile colonic microbiota. Antimicrob. Agents Chemother. 50:156–161.

45.

Jernberg

, Lofmark

, Edlund

, and Jansson

J.K.

. 2013. Long-term ecological impacts of antibiotic administration on the human intestinal microbiota. ISME J. 1:56–66.

46.

Broaders

, Gahan

C.G

, and Marchesi

J.R.

. 2013. Mobile genetic elements of the human gastrointestinal tract: potential for spread of antibiotic resistance genes. Gut Microbes. 4:271–280.

47.

Gillings, M.R. 2014. Integrons: past, present, and future. Microbiol. Mol. Biol. Rev. 78:257–277.

48.

Kadlec

, and Schwarz

. (2012). Antimicrobial resistance of Staphylococcus pseudintermedius. Vet. Dermatol. 23:276–e55.

49.

Bailey

J.K.

, Pinyon

J.L

, Anantham

, and Hall

R.M.

. 2010. Commensal Escherichia coli of healthy humans: a reservoir for antibiotic-resistance determinants. J. Med. Microbiol. 59:1331–1339.

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