https://doi.org/10.15446/rfmvz.v69n3.

103805
Investigación _________________________________ Rev Med Vet Zoot. 69(3), septiembre-diciembre 2022: 245-258

Isolation of Escherichia coli and Klebsiella pneumoniae strains producing

extended-spectrum β-lactamases from dog urine of the Metropolitan Area
of the Aburrá Valley (Antioquia, Colombia)

A. M. Ochoa 1 , M. I. García 1 , A.V. Cienfuegos2 , L. Vásquez-Jaramillo1*

Recibido: 05/08/2021. Aprobado: 19/11/2021

ABSTRACT
Escherichia coli and Klebsiella pneumoniae are the most common pathogens causing urinary
tract infections in humans and animals. Close contact between humans and companion
animals can facilitate the spread of multidrug resistant pathogens between both species.
The objective of the research was to characterize extended-spectrum β-lactamases (ESBL)
-producing E. coli and K. pneumoniae isolated from dogs with urinary tract infections
in the metropolitan area of Valle del Aburrá (Antioquia, Colombia). Three-hundred
seventy-one urine samples collected from March 2018 to March 2019 in a veterinary
clinical laboratory were analyzed. E. coli and K. pneumoniae isolates were detected in
chromogenic agar and identified by biochemical tests. Susceptibility testing was perfor-
med by disc diffusion and ESBL production was evaluated by the double disk test in all
isolates. MIC determination of ESBL-positive isolates were performed on the automated
VITEK®2 system. Multiple PCR was used for the detection of CTX-M beta-lactamases
(group 1, 2, 9 and 8/25), SHV, TEM, and AmpC of plasmid origin in ESBL-positive
isolates. In total 22 out 371 isolates were positive for ESBL production by double
disc test, 11 E. coli (ESBL-Ec) and 11 K. pneumoniae (ESBL-Kp). The multiple PCR
detected CTX-M group 1 in the 22 ESBL-positive isolates. Multi-drug resistance was
observed in all ESBL-producing isolates. In conclusion, a high frequency of antibiotic
multi-resistance was found in ESBL-Ec and ESBL-Kp. The main ESBL detected was
CTX-M group 1, which also prevails in human isolates.
Keywords: beta-lactam, resistance, dogs, enterobacteriaceae, public health, urinary tract
infection.

Aislamiento de Escherichia coli y Klebsiella pneumoniae productoras

de betalactamasas de espectro extendido a partir de orina de perros
del Área metropolitana del Valle de Aburrá (Antioquia, Colombia)

RESUMEN
Escherichia coli y Klebsiella pneumoniae son los patógenos más comunes causantes de
infecciones en tracto urinario en humanos y animales. El contacto estrecho con los animales

1
Línea de Epidemiología y Salud Pública Veterinaria, Grupo Centauro, Escuela de Medicina Veterina-
ria, Facultad de Ciencias Agrarias, Universidad de Antioquia.
2
Línea de Epidemiología Molecular Bacteriana, Grupo Microbiología Básica y Aplicada -MICROBA-,
Escuela de Microbiología, Universidad de Antioquia.
*
Correo electrónico: [email protected]

Ochoa et al., Isolation of Escherichia coli and Klebsiella pneumoniae strains producing 245
extended-spectrum β-lactamases from dog urine of the Metropolitan Area of the Aburrá
Valley (Antioquia, Colombia)
Rev Med Vet Zoot. 69(3), septiembre-diciembre 2022: 245-258 _________________________________ Investigación

de compañía puede favorecer la diseminación de patógenos multiresistentes entre ambas

especies. El objetivo de la investigación fue caracterizar E. coli (Ec –BLEE) y K. pneumo-
niae (Kp -BLEE) productores de betalactamasas de espectro extendido provenientes de
aislados de caninos con infecciones del tracto urinario del Área Metropolitana del Valle
de Aburrá. 371 muestras de orina de caninos colectadas entre marzo de 2018 y marzo
2019 en un laboratorio clínico veterinario fueron analizadas. E. coli y K. pneumoniae
se detectaron en agar cromogénico y se identificaron mediante pruebas bioquímicas.
La prueba de susceptibilidad se realizó por difusión en disco y la producción de BLEE
se evaluó por test de doble disco en todos los aislados. La determinación de la CIM en
aislados positivos a BLEE se realizó en el sistema automatizado VITEK®2. Se utilizó
PCR múltiple para la detección de betalactamasas tipo CTX-M (grupo 1, 2, 9 y 8/25),
SHV, TEM y AmpC de origen plasmídico en aislados positivos a BLEE. Un total de 22
de 371 aislados fueron positivos a BLEE por test de doble disco, 11 E. coli (Ec –BLEE)
y 11 K. pneumoniae (Kp-BLEE). La PCR detectó CTX-M grupo 1 en los 22 aislados
positivos a BLEE. Se observó multirresistencia en todos los aislamientos productores de
BLEE. En conclusión, se encontró una alta frecuencia de multirresistencia en Ec-BLEE
y Kp-BLEE. La principal BLEE detectada fue CTX-M grupo 1, que también predomina
en aislados humanos.
Palabras clave: resistencia a betalactámicos, perros, enterobacterales, infección del tracto
urinario, salud pública.

INTRODUCTION Most ESBLs can be classified into

Bacterial urinary tract infections (UTIs) three groups: (i) TEM and (ii) SHV—de-
are a common cause of disease in dogs. rived from gene mutations in the classic
The most frequently isolated bacteria in narrow-spectrum β-lactamases TEM-1,
both humans and dogs are Escherichia coli, TEM-2, and SHV-1-, and (iii) CTX-M
followed by Staphylococcus, Proteus, and group which predominates over the other
Klebsiella species (Byron 2019; Gómez ESBL-types globally. CTX-M enzymes
Beltrán et al. 2020; Sierra González and possess preferential hydrolytic activity
Arango Uribe 2017). UTIs and other bac- over cefotaxime and have been classified
terial infections are frequently treated with into five groups (CTX-M-1, CTX-M-2,
β-lactam antibiotics, however resistance CTX-M-8, CTX-M-9, and CTX-M-25),
to this antibiotic group has increased in based on amino acid identities (Bonnet
the last decade. The main mechanism of 2004). ESBL-type enzymes are usually
resistance to β-lactam in Enterobacteriaceae encoded in plasmids, facilitating their
is the production of extended-spectrum rapid dissemination between different
β-lactamases (ESBL). These enzymes confer species through horizontal transfer by
resistance to penicillins, cephalosporins conjugation (Morejón García 2013).
(including oximinocephalosporins), and In addition, the same plasmid can carry
aztreonam. On the contrary, ESBL are genes that encode resistance to quinolo-
unable to hydrolyze cefamycins (cefoxitin nes, aminoglycosides, tetracyclines, and
and cefotetan) and carbapenems (Paterson trimethoprim/sulfamethoxazole, which
and Bonomo 2005). contribute to antibiotic multi-resistance

246 Ochoa et al., Isolation of Escherichia coli and Klebsiella pneumoniae strains producing
extended-spectrum β-lactamases from dog urine of the Metropolitan Area of the Aburrá
Valley (Antioquia, Colombia)
Investigación _________________________________ Rev Med Vet Zoot. 69(3), septiembre-diciembre 2022: 245-258

(Tafur et al. 2011). Due to their impact food-producing animals that are also used
on morbidity, mortality, and frequent in companion ones (Cabrera García 2010).
resistance and limited therapeutic options, The widespread and excessive use
carbapenem-resistant and ESBL-producing of antibiotics, mainly third-generation
Enterobacteriaceae are included in the cephalosporins have usually been attributed
priority pathogens list on antibiotic resis- as a selection pressure that drives ESBL
tance of the World Health Organization evolution (Paterson and Bonomo 2005).
(Tacconelli et al. 2017). Thus, inappropriate use of antibiotics in
Close contact between humans (owners, animals can contribute to the spread of
vets, animal caretakers, etc.) and pets, as resistant bacteria that cause complicated
well as direct contact with animal secretions infections in both animals and humans, and
during the performance of routine actions that are associated with higher morbidity,
related to their care (feeding, grooming, mortality, and treatment costs in humane
cleaning living spaces, petting), implies a medicine (Alós 2015). Therefore, unders-
potential transmission risk of pathogens tanding the phenomenon of antimicrobial
between both species, as well as the transfer resistance from a veterinary medicine
of resistance genes. Moreover, since it is perspective is important to determine its
very common that dogs urinate in public impact on public health.
areas, the contamination of the soil and Although urinary infections in dogs
water sources could represent a problem are very common, as well as the use of
for the community and the environmental antibiotics in companion animals is high
health. and contaminated urine can be discharged
In Colombia, in the clinical practice in into the environment, information about
companion animals the use of antibiotics the phenomenon of antimicrobial resistance
that are also used in human medicine is in companion animals in Colombia is
frequent (Cabrera García 2010), mainly limited. Therefore, this study aimed to
β-lactams (Astaiza Martínez et al. 2016; characterize isolates of ESBLs producing
Gutiérrez et al. 2002). In addition, it is E. coli and K. pneumoniae, obtained from
common in daily practice the recognition urine samples of dogs from the metropo-
of pathologies based on presumptive litan area of Valle del Aburrá (Antioquia,
diagnoses and the scarce use of diagnostic Colombia).
aids, such as culture with antibiogram,
resulting in the establishment of empirical
antibiotic therapies is common in the MATERIALS AND METHODS
daily practice (Gómez Beltrán et al. 2021;
Sánchez et al. 2015). Location
Estimating the use of antimicrobials in In this study urine samples from cani-
companion animal species in Colombia nes collected at TESTLAB© Veterinary
is also difficult. Usually, its magnitude is Clinical Laboratory were included. This
assessed in reference to the sales reported laboratory performs ESBL-detection test
by the manufacturing laboratories, without routinely in E. coli and K. pneumoniae
considering the dispensing of authorized urine isolates. In addition, the laboratory
antimicrobials for human use, as well as is certified under the Colombian law (ICA
injectable antimicrobials designed for ISO 17025 standard). The laboratory

Ochoa et al., Isolation of Escherichia coli and Klebsiella pneumoniae strains producing 247
extended-spectrum β-lactamases from dog urine of the Metropolitan Area of the Aburrá
Valley (Antioquia, Colombia)
Rev Med Vet Zoot. 69(3), septiembre-diciembre 2022: 245-258 _________________________________ Investigación

receives samples from veterinary clinics Bacterial isolation and identification

located in the metropolitan area of Va- The BBL™ CHROMagar™ Orienta-
lle del Aburrá (Province of Antioquia, tion (Becton, Dickinson and Company,
Colombia), where Medellin is the main Sparks, USA) was used for the isolation,
city surrounded by the municipalities of characterization, differentiation, and
Barbosa, Girardota, Copacabana, Bello, enumeration of the bacteria present in
Itagüí, Sabaneta, Envigado, La Estrella, the urine samples. In the cultures with
and Caldas. It is estimated that the study significant growth of uropathogens, one
region had a population of 480,659 dogs colony compatible with E. coli and K.
in 2018 (Área Metropolitana del Valle de pneumoniae in each positive culture was
Aburrá 2018). selected for genus and species confirmation
by biochemical oxidase tests, Simmons
Data collection citrate, TSI agar, lysine decarboxylation,
Information on dogs’ characteristics and urease, indole production, and mobility
clinical practices related to the presence of the bacteria.
of ESBL producing E. coli (ESBL-Ec) and
ESBL producing K. pneumoniae (ESBL-Kp) Antimicrobial susceptibility testing
in urine was retrieved from clinical records. and ESBL evaluation
Information on the use of antibiotics, Antimicrobial susceptibility testing by disk
infectious diseases, hospitalizations, and diffusion and ESBL evaluation by double
surgeries during the last 12 months, as well disc test was performed simultaneously
as the method used to obtain the urine in accordance with the Clinical and
sample and cohabits with other animals Laboratory Standards Institute (CLSI)
were obtained through the medical history recommendations (CLSI M100-S25,
and the attending veterinarian. 2015 y CLSI VET08, 2018). Briefly,
the bacterial colonies were suspended in
Ethics committee saline solution, adjusted to 0.5 McFar-
The study was approved by the Ethics land turbidity standard and plated on
Committee for Animal Experimentation Mueller-Hinton agar. According to the
(CEEA) of the Universidad de Antioquia request of the attending veterinarian,
(act number 120, October 9th, 2018). different antibiotics were evaluated for
each isolate, including amoxicillin 25 µg
Samples (AML), ampicillin (AMP), amoxicillin/
All urine samples from dogs (n=371, one clavulanic acid 30 µg (AMC), ampicillin/
sample from each dog) analyzed at the sulbactam 20 µg (SAM), cefotaxime 30
laboratory for microbiological culture µg (CTX), ceftazidime 30 µg (CAZ),
and antibiogram between March 2018 ceftriaxone 30 µg (CRO), imipenem
and March 2019 were included in the 10 µg (IPM), ertapenem 10 µg (ETP),
study. The urine sample was considered as nitrofurantoin 300 µg (F), trimethoprim/
the sampling unit, and the dogs and the sulfamethoxazole 25 µg (SXT), enrofloxacin
isolates as analysis units. A urine sample 5 µg (ENR), ciprofloxacin 5 µg (CIP),
was considered positive when the growth chloramphenicol 30 µg (C), norfloxacin
of at least one (1) isolate of ESBL-Ec or 10 µg (NOR), amikacin 30 µg (AK),
ESBL-Kp was confirmed. gentamicin 10 µg (CN), doxycycline

248 Ochoa et al., Isolation of Escherichia coli and Klebsiella pneumoniae strains producing
extended-spectrum β-lactamases from dog urine of the Metropolitan Area of the Aburrá
Valley (Antioquia, Colombia)
Investigación _________________________________ Rev Med Vet Zoot. 69(3), septiembre-diciembre 2022: 245-258

30 µg (DO), erythromycin 15 µg (E), gentamycin (CN), ciprofloxacin (CIP),

tetracyclines 30 µg (TE), oxytetracycline tigecycline (TGC), and colistin (CT).
30 µg (OT), minocycline 30 µg (MH),
oxacillin (OX), azithromycin 15 µg (AZM), Molecular detection of ESBL genes by
and neomycin 30 µg (N). multiple polymerase chain reaction
ESBL production was evaluated by (PCR)
double-disk test as recommended by CLSI Bacterial DNA extraction from positive
(CLSI M100-S25, 2015; Papich 2018). ESBL-producing isolates was performed
Briefly, a bacterial suspension with a turbi- using the Wizard® Genomic DNA Purifica-
dity pattern of 0.5 McFarland was plated tion kit (Promega Corporation, Madison,
on Mueller-Hinton agar. Ceftazidime/ USA) and according to the manufacturer’s
clavulanic acid (30/10μg), cefotaxime/ instructions. Subsequently, bla genes of the
clavulanic acid (30/10 μg), ceftazidime (30 TEM, SHV, and CTX-M subtypes (groups
μg), and cefotaxime (30 μg) disks were used. 1, 2, 8, 9, and 25) were amplified using
Antibiotic disks were placed at a 25 mm primers and conditions previously described
center-to-center distance and incubated (Dallenne et al. 2010). A non-ESBL- Ec
at 37° C for 18-24 h. An increase of ≥5 strain (ATCC 25922) was used as negative
mm in the halo zone diameter for cepha- control. The PCR products were visualized
losporin disks combined with clavulanic by ethidium bromide in 2% agarose gel
acid compared to the antibiotic alone was electrophoresis (AMRESCO Inc, Solon
considered positive for ESBL-production. Ohio, USA ISO 9002). The size of the
E. coli ATCC 25922 and K. pneumoniae bands was evaluated by comparison with
ATCC 700603 were used as negative and a 100 bp molecular weight DNA ladder
positive controls, respectively. Positive (Gene Ruler- Qiagen, Germantown, USA).
isolates for the ESBL-production were
stored in cryobeads (CRYOBANK™) for Statistical analysis
preservation and subsequent determination The information collected was stored in
of the minimum inhibitory concentration Excel 2010 (Microsoft, Redmond, USA).
(MIC) and molecular analysis. The data were analyzed using descriptive
statistics. Absolute and relative frequencies
Determination of the minimum were calculated for the qualitative variables,
inhibitory concentration (MIC) and summary and central tendency measures
The MIC for ESBL-Ec and ESBL-Kp for the quantitative ones, according to the
isolates was determined using the VITEK®2 normality assumption of the data. STATA
automated system (bioMérieux, Marcy 15.1 (Statacorp, Texas, USA) software was
l’Étoile, France). A total of 14 antibiotics used for the statistical analysis.
were evaluated and interpreted according
to the standards (CLSI M100-S25, 2015
y CLSI VET08, 2018), including ampici- RESULTS
llin/sulbactam (SAM), ceftoxitin (FOX), A total of 371 urine samples were sent
ceftazidime (CAZ), ceftriaxone (CRO), to the laboratory for urine culture and
cefepime (FEP), doripenem (DOR), antibiogram during March 2018 and
ertapenem (ETP), imipenem (IPM), March 2019. In 190 samples E. coli and
meropenem (MEM), amikacin (AK), K. pneumoniae were identified. E. coli was

Ochoa et al., Isolation of Escherichia coli and Klebsiella pneumoniae strains producing 249
extended-spectrum β-lactamases from dog urine of the Metropolitan Area of the Aburrá
Valley (Antioquia, Colombia)
Rev Med Vet Zoot. 69(3), septiembre-diciembre 2022: 245-258 _________________________________ Investigación

isolated from 165 samples, of which 6.6% addition to the resistance to cephalosporins,
(11/165) were ESBL positive by double a high resistance to ciprofloxacin (78%,
disc test. K. pneumoniae was isolated from 14/18), enrofloxacin (88%, 15/17), and
25 samples, of which 44% (11/25) were trimethoprim-sulfamethoxazole (85%,
ESBL positive by double disc test. 11/13) was found.
The mean age of the 22 dogs with The MICs of the 14 antibiotics, obtained
ESBL-positive isolates was 9.4 ± 3.1 years through the broth microdilution method
and most of the dogs 59.09% (13/22) using the VITEK®2 system for the 22
were males. 81.8% (18/22) were purebred isolates of E. coli and K. pneumoniae, are
dogs (i.e., Jack Russell Terrier, Yorkshire shown in tables 1 and 2, respectively. These
Terrier, Labrador Retriever, Pitbull, Cocker results showed all isolates were susceptible
Spaniel, Pug, Schnauzer, Beagle, Shih-Tzu, to carbapenems (ertapenem, imipenem,
German Shepherd, Dachshund, Golden meropenem and doripenem), and most
Retriever, French Bulldog, and English of them were susceptible to amikacin,
Bulldog), and 18.2% (4/22) were mixed- and tigecycline. In addition, resistance
breed. Fifty percent (11/22) of dogs had to gentamicin was more frequent in K.
undergone a previous sterilization process pneumoniae than E. coli.
more than 6 months ago and 50% (11/22) The 22 isolates harbored group 1
lived with other dogs. A high frequency CTX-M genes, based on the multiple
of hospitalization (77.3%, 17/22) and the PCR analysis. The CTX-M group (2, 8,
use of antibiotic-based treatments (77.3%, 9, and 25 genes) were not detected in any
17/22) were found in the last year. isolate. Two of the 22 isolates were also
Regarding the ESBL-Ec isolates, resis- positive to TEM and SHV genes, carrying
tance to enrofloxacin and ciprofloxacin was all three genes simultaneously.
found in 87.5% (7/8) and 70% (7/10),
respectively. The highest susceptibility
was found to nitrofurantoin (70%, 7/10). DISCUSSION
Two of the isolates were resistant to all the In this study, a general frequency of
antibiotics evaluated. 11,6% (22/190) of E. coli and K. pneumo-
In addition, 82% (9/11) of the K. niae producing ESBL was found. When
pneumoniae isolates were resistant to all the compared to similar studies where both
antibiotics evaluated, being nitrofurantoin bacteria were analyzed in dog urine, this
100% (9/9), trimethoprim/sulfamethoxa- frequency was slightly higher than the one
zole 100% (6/6), enrofloxacin 88,8% found in Brazil of 9,6% (2/62) (Melo et
(8/9), and ciprofloxacin 88% (7/8) the al. 2018) but lower than that reported
antibiotics with the highest proportion of in Switzerland of 25% (39/156) (Zogg
resistance. One of the other two isolates et al. 2018). However, if we consider
was susceptible to enrofloxacin and cipro- the difference in the length of the study
floxacin and the other one to amikacin, of those researches (two and four years
imipenem, ertapenem, and meropenem. respectively) compared with this one-year
In general, all isolates showed resistance study, our findings may represent a major
to three or more antibiotics groups by the concern by showing a greater number
Kirby Bauer (disk diffusion) method. In of diseased dogs carrying E. coli and

250 Ochoa et al., Isolation of Escherichia coli and Klebsiella pneumoniae strains producing
extended-spectrum β-lactamases from dog urine of the Metropolitan Area of the Aburrá
Valley (Antioquia, Colombia)
 TABLE 1. Minimum inhibitory concentration (MIC) values for Escherichia coli isolates obtained from urine samples of dogs from the metropolitan area of Valle
del Aburrá (Province of Antioquia, Colombia), 2018-2019

ISOLATE SAM TZP FOX CAZ CRO FEP DOR ETP IPM MEM AMK CN CIP TGC
2-241447-KP >=32 8 <=4 4 >=64 4 <=0,12 <=0,5 1 <=0,25 <=2 <=1 2 <=0,5

3-237353-KP >=32 <=4 <=4 4 >=64 2 <=0,12 <=0,5 <=0,25 <=0,25 <=2 >=16 <=0,25 <=0,5

Valley (Antioquia, Colombia)

4-242407-KP >=32 16 <=4 16 >=64 >=64 <=0,12 <=0,5 <=0,25 <=0,25 4 >=16 >=4 <=0,5

7-243889-KP >=32 16 <=4 8 >=64 4 <=0,12 <=0,5 <=0,25 <=0,25 <=2 >=16 2 1

8-247697-KP >=32 16 <=4 16 >=64 32 <=0,12 <=0,5 <=0,25 <=0,25 4 >=16 >=4 <=0,5

14-255779-KP >=32 16 <=4 8 >=64 4 <=0,12 <=0,5 <=0,25 <=0,25 <=2 >=16 >=4 1

15-259003-KP >=32 16 <=4 4 >=64 4 <=0,12 <=0,5 <=0,25 <=0,25 <=2 <=1 2 <=0,5

16-259320-KP >=32 32 >=64 >=64 >=64 32 <=0,12 <=0,5 0,5 <=0,25 >=64 >=16 2 2

20-271234-KP >=32 16 <=4 8 >=64 2 <=0,12 <=0,5 <=0,25 <=0,25 <=2 >=16 1 1

21-274461-KP >=32 >=128 8 16 >=64 32 <=0,12 <=0,5 0,5 <=0,25 <=2 >=16 >=4 2

27-295328-KP >=32 64 >=64 >=64 >=64 2 <=0,12 <=0,5 <=0,25 <=0,25 >=64 >=16 2 2

SAM (ampicillin/sulbactam), TZP (piperacillin/tazobactam), FOX (cefoxitin), CAZ (ceftazidime), CRO (ceftriaxone), FEP (cefepime), DOR (doripenem), ETP (ertapenem), IPM

Ochoa et al., Isolation of Escherichia coli and Klebsiella pneumoniae strains producing
(imipenem), MEM (meropenem), AK (amikacin), CN (gentamicin), CIP (ciprofloxacin), TGC (tigecycline). Gray shade indicate intermediate or resistance to the antibiotic.
Source: self-made.

extended-spectrum β-lactamases from dog urine of the Metropolitan Area of the Aburrá
251
Investigación _________________________________ Rev Med Vet Zoot. 69(3), septiembre-diciembre 2022: 245-258
 TABLE 2. Minimum inhibitory concentration (MIC) values for Klebsiella pneumoniae isolates obtained from urine samples of dogs from the metropolitan area

252
of Valle del Aburrá (Province of Antioquia, Colombia), 2018-2019

ISOLATE SAM TZP FOX CAZ CRO FEP DOR ETP IPM MEM AK CN CIP TGC
5-242857-EC 16 4 4 16 >=64 2 <=0,12 <=0,5 <=0,2 <=0,25 <=2 >=16 >=0,4 <=0,5
6-243280-EC 4 4 4 8 >=64 2 <=0,12 <=0,5 <=0,2 <=0,25 <=2 <=1 <=0,25 <=0,5
9-248019-EC 4 4 4 4 >=64 2 <=0,12 <=0,5 <=0,2 <=0,25 <=2 <=1 <=0,25 <=0,5
10-253735-EC 8 4 8 16 >=64 2 <=0,12 <=0,5 <=0,2 <=0,25 <=2 <=1 1 <=0,5
11-253824-EC 4 4 4 4 >=64 2 <=0,12 <=0,5 <=0,2 <=0,25 <=2 <=1 1 <=0,5
18-267995-EC 16 4 4 16 >=64 2 <=0,12 <=0,5 <=0,2 <=0,25 <=2 >=16 >=4 <=0,5
19-268858-EC 8 4 8 16 >=64 2 <=0,12 <=0,5 <=0,2 <=0,25 <=2 <=1 1 <=0,5
22-276305-EC 4 4 4 4 >=64 2 <=0,12 <=0,5 <=0,2 <=0,25 <=2 <=1 <=0,25 <=0,5
24-278733-EC 4 4 4 4 >=64 2 <=0,12 <=0,5 <=0,2 <=0,25 <=2 <=1 <=0,25 <=0,5
25-281253-EC 4 4 4 4 >=64 2 <=0,12 <=0,5 <=0,2 <=0,25 <=2 <=1 <=0,25 <=0,5
26-294656-EC 4 5 5 4 >=64 4 <=0,12 <=0,5 <=0,2 <=0,25 <=2 <=1 <=0,25 <=0,5
28-300603-EC 4 4 4 4 >=64 2 <=0,12 <=0,5 <=0,2 <=0,25 <=2 <=1 >=4 <=0,5

SAM (ampicillin/sulbactam), TZP (piperacillin/tazobactam), FOX (cefoxitin), CAZ (ceftazidime), CRO (ceftriaxone), FEP (cefepime), DOR (doripenem), ETP
(ertapenem), IPM (imipenem), MEM (meropenem), AK (amikacin), CN (gentamicin), CIP (ciprofloxacin), TGC (tigecycline), MIC (minimum inhibitory concentration).
Gray shade indicates intermediate or resistance to the antibiotic.
Source: self-made.
Rev Med Vet Zoot. 69(3), septiembre-diciembre 2022: 245-258 _________________________________ Investigación

Valley (Antioquia, Colombia)

extended-spectrum β-lactamases from dog urine of the Metropolitan Area of the Aburrá
Ochoa et al., Isolation of Escherichia coli and Klebsiella pneumoniae strains producing
Investigación _________________________________ Rev Med Vet Zoot. 69(3), septiembre-diciembre 2022: 245-258

K. pneumoniae producing ESBL in a Marques et al. 2018). The latest suggests

shorter period of time. that ESBL-Kp is frequent in clinical samples
In the same way, the frequency of from companion animals and that it is
isolates ESBL producing E. coli and K. necessary to continue monitoring resistance
pneumoniae observed is also remarkably mechanisms in this species.
higher than those obtained from diseased The predominant gene class detected
companion animals between 2008 and in our study was CTX-M. The CTX-M
2010 in a European surveillance program family of enzymes is grouped based on
(Bogaerts et al. 2015) where only five E. the similarities in amino acid sequences;
coli and one K. pneumoniae were isolated. for example, within the group CTX-M-1
Such a difference can probably be explained are the enzymes CTX-M-1, CTX-M-3,
by the fact that all chronically diseased CTX-M-10, CTX-M-12, CTX-M-15, and
animals, as well as those who had been CTX-M-32 (Galvis and Moreno 2019).
recently treated with antibiotics, were Several studies suggest that CTX-M-1
excluded in that study, while the majority is the most frequent genotype, not only
of the animals in our study had a history in companion animals, but also in food-
of hospitalization and/or use of antibiotics producing animals, food, wild animals,
in the previous year. and veterinarians that work with dogs and
Other studies have also evaluated the cats (Abbas et al. 2019; Baede et al. 2015;
presence of ESBL-Ec in dogs suffering from Cormier et al. 2019; Darwich et al. 2019;
UTI worldwide. Among the isolates of E. Ewers et al., 2012; Irrgang et al. 2018).
coli in this study, the observed frequency of In Colombia, a few studies had analyzed
6,6% (11/22) of ESBL producers is higher the presence of blaCTX-M genes. From the
but not so far than that found previously poultry chain, Castellanos et al. (2017)
in the United States (4%), Switzerland identified Escherichia coli carrying blaCTX−M-2,
(3,4%) and China (3,8%) ( Huber et al. blaCTX−M-8 and blaCTX−M-15 and then, in
2013; Li et al. 2017; O’Keefe et al. 2010), 2018, blaCTX−M−2 group was found to be
although data from those studies were the most prevalent ESBL gene among
single-institution based and may not be Salmonella enterica isolates (Castellanos et
entirely comparable. al. 2018). In addition, CTX−M group 1
As it has been mentioned, research — was detected in enterobacteriaceae isolated
particularly in animals— has been more from bulk tank milk samples (Vásquez
focused on studying ESBLs produced by Jaramillo et al. 2017).
E. coli, yet the predominance of ESBL In addition, the predominant circulation
production in this study was higher in K. of the CTX-M group 1 in dogs described
pneumoniae isolates than in E. coli. Some here is consistent with the epidemiology
studies in Japan, Italy, Portugal, Germany, of CTX-M in E. coli and K. pneumoniae
and other European countries have also causing UTI in humans in Colombia,
reported a high number of multidrug resis- both at hospital and community settings
tance and third-generation cephalosporin (Blanco et al. 2016; Galvis and Moreno
resistant K. pneumoniae strains among 2019; Leal et al. 2013; Martínez et al.
companion animals (Donati et al. 2014; 2012; Rada et al. 2019). They all reported
Ewers et al. 2012; Harada et al. 2012; the presence of the CTX-M group 1, with

Ochoa et al., Isolation of Escherichia coli and Klebsiella pneumoniae strains producing 253
extended-spectrum β-lactamases from dog urine of the Metropolitan Area of the Aburrá
Valley (Antioquia, Colombia)
Rev Med Vet Zoot. 69(3), septiembre-diciembre 2022: 245-258 _________________________________ Investigación

blaCTX-M-1, blaCTX-M-3, blaCTX-M-15 and blaCTX- to fluoroquinolones, aminoglycosides, and

M-32
being the mainly found enzymes. We SXT among other antibiotics (Coque et
also detected TEM and SHV-like genes al. 2018; Wieler et al. 2011 ), as already
in some isolates, but it is important to reported in E. coli and K. pneumoniae
note that these genes could also encode isolates from urine of dogs with UTIs
narrow-spectrum beta-lactamases and (Huber et al. 2013; Zogg et al. 2018). This
confirmation of ESBL-type should be suggests that veterinarians are encouraged
performed by gene sequencing. to improve antibiotic prescriptions, based
The detection of CTX-M type enzymes on susceptibility testing in order to reduce
in dog’s uropathogens increases the risk the selection pressure on the pathogens.
of clinical failure because of the use of Surveillance studies implementing a One
inappropriate empirical treatments and Health approach are required to assess the
therefore results in complicated urinary level of genetic relatedness among resistant
infections. In our study, the highest bacteria from animal and human origins
frequency of resistance was found to as well as to help elucidate the complex
enrofloxacin and ciprofloxacin, while the epidemiology of transmission and the
lowest resistance was observed against role of companion animals in the spread
nitrofurantoin among the E. coli isolates, of multi-resistant bacteria. Additionally,
in addition to resistance to penicillins and these findings contribute to the design of
cephalosporins. policies related to the use of antibiotics
Regarding K. pneumoniae isolates, it in both human and veterinary medicine,
was observed that the vast majority were as well as other factors such as hygiene
multidrug-resistant, highlighting the procedures, that may be associated with the
resistance to nitrofurantoin, trimetho- emergence of the antimicrobial resistance
prim/sulfamethoxazole, enrofloxacin, phenomenon in the country.
and ciprofloxacin. These findings agreed This study has some limitations. First,
with a previous retrospective study carried it was difficult to obtain accurate data from
out in a veterinary diagnostic laboratory the patients’ clinical record, such as the
in Medellin, Colombia, which evaluated type of UTI and details on previous anti-
the susceptibility to antimicrobials of microbial treatment. Because veterinarians
bacterial isolates from dogs and reported request urine culture and antibiogram
that in general, the level of resistance more frequently from complicated than
was high (20-50%) to at least 6 or more uncomplicated cases our results likely
antimicrobials. Specifically for urinary correspond to those of complicated UTI
tract infections caused by E. coli or en- patients. Second, the selection of antibiotics
terobacteriaceae (Klebsiella spp., Proteus to be evaluated in each patient was defined
spp.), amikacin and florfenicol were the by the treating veterinarian rather to being
only drugs that demonstrated 100% in standardized, which makes the comparison
vitro efficacy (Gómez Beltrán et al. 2020). of susceptibility results in all the isolates
The resistance of ESBL-producing difficult. Finally, it will be important
strains to other classes of antimicrobial to include in future studies additional
agents can be explained by the fact that molecular techniques to investigate the
plasmids harboring blaCTX-M-like genes fre- predominant ESBL-positive clones causing
quently carry genes that confer resistance UTI in dogs.

254 Ochoa et al., Isolation of Escherichia coli and Klebsiella pneumoniae strains producing
extended-spectrum β-lactamases from dog urine of the Metropolitan Area of the Aburrá
Valley (Antioquia, Colombia)
Investigación _________________________________ Rev Med Vet Zoot. 69(3), septiembre-diciembre 2022: 245-258

Alós J I 2015. Resistencia bacteriana a los antibióticos:

CONCLUSIONS
una crisis global. Enfermedades infecciosas y mi-
An overall frequency of 11,6% (22/190) crobiología clínica. 33(10): 692-699. Available in:
ESBL-producing E. coli and K. pneumoniae http://www.scielo.org.co/scielo.php?script=sci_ar
isolates in urine samples from dogs with ttext&pid=S0122-06672015000100013
UTI. CTX-M group 1 was the main ESBL Área Metropolitana del Valle de Aburrá. 2018.
detected, and a multidrug resistance profile Resolución Metropolitana 002274 Septiem-
was identified in K. pneumoniae. The timely bre 5 de 2018. Available in: https://www.
identification of ESBL-producing strains metropol.gov.co/ResolucionesMetropolitanas/
of enterobacteriaceae is essential for eva- Resoluci%C3%B3n_2018_002274.pdf
luating the development of antimicrobial Astaiza Martínez JM, Benavides Melo CJ, Muñoz
García GK, Mora Muñoz MF, Cháves Velasquez
resistance and for establishing successful
CA. 2016. Principales hábitos de medicación por
antibiotic therapies in the small-species los propietarios de caninos que acuden a consulta
daily practice, as well as to strengthen veterinaria en Pasto, Nariño, Colombia. Revista
the implementation of infection control Colombiana de Ciencias Químico–Farmacéuti-
measures and the rational administration cas. 45(1):92-108. Available in: https://revistas.
of antibiotics for the protection of public unal.edu.co/index.php/rccquifa/article/view/58019
health. Baede VO, Wagenaar JA, Broens EM, Duim B,
Dohmen W, Nijsse R, Hordijk J. 2015. Longitu-
dinal study of extended-spectrum-β-lactamase-
CONFLICT OF INTEREST and AmpC-producing Enterobacteriaceae in
household dogs. Antimicrobial A gents and C
The authors declare no conflict of interest.
hemotherapy. 59(6):3117-3124.
Blanco VM, Maya JJ, Correa A, Perenguez M,
Muñoz JS, Motoa G, Villegas MV. 2016. Pre-
FUNDING SOURCES valence and risk factors for extended-spectrum
This study was financed by the RedSIN β-lactamase-producing Escherichia coli causing
Research Seedbed Network of the Uni- community-onset urinary tract infections in
versidad de Antioquia. Colombia. Enfermedades Infecciosas y Micro-
biologia Clinica. 34(9):559-565. Available in:
https://europepmc.org/article/med/26774256
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C, Berhin C, El Garch F, ComPath Study
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Forma de citación del artículo:

Ochoa AM, García MI, Cienfuegos AV, Vásquez-Jaramillo L. 2022. Isolation of
Escherichia coli and Klebsiella pneumoniae strains producing extended spectrum
β-lactamases from dog urine of the Metropolitan Area of the Aburrá Valley (Antio-
quia, Colombia). Rev Med Vet Zoot. 69(3):245-258. https://doi.org/10.15446/
rfmvz.v69n3.103805

258 Ochoa et al., Isolation of Escherichia coli and Klebsiella pneumoniae strains producing
extended-spectrum β-lactamases from dog urine of the Metropolitan Area of the Aburrá
Valley (Antioquia, Colombia)