Extended-Spectrum Beta-Lactamase-Producing Klebsiella pneumoniae Isolated from Healthy and Sick Dogs in Portugal

Abstract

Extended-spectrum beta-lactamase (ESBL)- and carbapenemase (CP)-producing Klebsiella pneumoniae isolates are a public health concern at clinical level, mainly in Southern European countries. However, there are scarce data on the role of companion animals in the emergence of resistance to clinically relevant antibiotics. Therefore, our study aimed to determine the presence of K. pneumoniae with relevant beta-lactamases in fecal samples from healthy dogs (kennel and house dogs) and sick dogs in seven different hospitals in Portugal. Fecal samples from 125 healthy dogs and 231 sick dogs (one per animal) were collected during April–August 2017. Samples were screened on MacConkey agar supplemented with meropenem, and matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) was used for K. pneumoniae identification. Genotypic detection of ESBLs or CPs was carried out by PCR/sequencing. Moreover, the presence of other antimicrobial resistance genes and multilocus sequence typing was tested by PCR/sequencing. K. pneumoniae isolates were obtained from 16 tested samples (4.4%), and 3 of them were ertapenem and/or meropenem intermediate/resistant (all of them imipenem susceptible and negative for CP genes). Fifteen K. pneumoniae isolates were ESBL producers, and they carried the following beta-lactamase genes: bla_CTX-M-15+bla_SHV-28 (four isolates, in three cases associated with bla_TEM-1), bla_CTX-M-15+bla_SHV-1 (five isolates, associated with TEM-1 in three cases), and bla_SHV-28+bla_TEM-1 (six isolates). Three ESBL-producing K. pneumoniae isolates of different origins and beta-lactamase genotypes (CTX-M-15+SHV-28, CTX-M-15+SHV-28+TEM-1, or SHV-28+TEM-1) belonged to the lineage ST307, and one isolate was identified as ST15 (CTX-M-15+SHV-1). These findings highlight that dogs are frequent carriers of ESBL-producing K. pneumonia isolates, harboring mostly genes encoding CTX-M-15 or SHV-28, associated in some cases with the high-risk clones ST307 and ST15.

Introduction

The dissemination of multidrug-resistant (MDR) microorganisms is considered a public health concern in medicine. It is a result of the use, overuse, and misuse of antibiotics in humans and animals, which causes the emergence and rapid dissemination of antibiotic resistance.^1–3 The “One Health concept” plays a significant role in the prevention and control of zoonosis combining human, animal, and environmental components to address global health challenges.^3–6 According to the World Health Organization (WHO),⁷ ∼75% of new emerging human infectious diseases are defined as zoonotic, meaning that they are naturally transmitted from vertebrate animals to humans.

Klebsiella pneumoniae, belonging to the Enterobacteriaceae family, is a common bacterium of the gastrointestinal tract of healthy humans and animals.^8,9 This natural inhabitant colonizes the mucosal surfaces of mammals and can also be found in the environment in surface water, food, and soil.^9–11 In the case of pets, Klebsiella spp. can also cause serious infections such as respiratory and urinary tract infections, among others^12,13; of particular relevance is canine cystitis or mastitis insofar it is the second most common pathogen to cause these diseases. Furthermore, K. pneumoniae is also a relevant MDR pathogen, usually spreading in health care environments among immune-compromised patients and is a major source of hospital infections (located in catheters or on ventilator machines). MDR microorganisms are transmitted among pets, owners, and veterinary staff and, in this way, spread to the community.^14,15

The emergence of MDR clones containing relevant resistance genes, especially those encoding extended-spectrum beta-lactamases (ESBLs) or carbapenemases (CPs), is considered an important issue in relation to gram-negative bacteria with public health significance.^1,16–18 Currently, few antibiotics are available for the treatment of MDR gram-negative bacteria, resulting in longer hospitalization, high morbidity and mortality with limited therapeutic options.¹⁹ The worldwide use of carbapenems has increased considerably in recent years due to the dissemination of MDR gram-negative bacteria. Moreover, the frequency of ESBL-producing K. pneumoniae isolates in clinical settings is very high in some countries.¹¹ According to the EARS-Net data for Portugal in 2017, the proportion of invasive K. pneumoniae isolates showing resistance to third-generation cephalosporins and carbapenems was in the range of 25–50% and 5–10%, respectively.²⁰

There are still scarce data available useful to evaluate the role of companion animals in the emergence of Enterobacteriaceae resistant to clinical “last resort antibiotics.”^21,22 However, these antimicrobials are increasingly administrated not only in human medicine but also to animals in veterinary hospitals, particularly to pets in Europe.^18,23,24 Thus, the prevalence of beta-lactamases in pets has already been brought to the attention of the scientific community.

In general, pets live in close contact with humans, increasing the possibility of the spread of MDR bacteria between humans and animals. Therefore, this study aimed to detect and characterize K. pneumoniae isolates with relevant beta-lactamase genes isolated in healthy dogs (kennel and house dogs) and sick dogs from seven different hospitals in Portugal.

Materials and Methods

Animals and sampling

A total of 361 fecal samples were recovered from different dogs (healthy and hospitalized) between April and August 2017 in Portugal. This study was carried out on 235 hospitalized dogs in seven different veterinary hospitals or clinics and on 126 healthy dogs (31 local kennel dogs and 95 local house dogs, all of them from Vila Real). The hospitals and clinics were in Vila Real, Aveiro, and Lisbon. All samples were obtained with the owner's permission (healthy and hospitalized dogs) or with kennel collaboration (kennel dogs). One fecal sample per animal was obtained rectally using a sterile cotton swab immediately after the dogs defecated, giving a total of 361 samples, which were dispatched immediately to the Microbiology Laboratory of the University of Trás-os-Montes and Alto-Douro (UTAD), located in Vila Real (Portugal).

K. pneumoniae isolation

The fecal samples were inoculated onto MacConkey agar plates supplemented with 0.8 mg/L of meropenem and were incubated for 24 hr at 37°C to recover β-lactam-resistant K. pneumoniae isolates (potential carbapenem-resistant isolates). Isolates with K. pneumoniae morphology were subjected to standard biochemical methods named IMViC (Indole, Methyl-red, Voges–Proskauer, and Citrate). The matrix-assisted laser desorption/ionization time-of-flight mass spectrometry method (MALDI-TOF MS; Bruker) was applied in this study to confirm the bacterial species identification. One K. pneumoniae isolate per positive sample was kept and further characterized.

Susceptibility testing

Antimicrobial susceptibility testing was performed using Kirby–Bauer disk diffusion method on Mueller–Hinton agar, according to the Clinical and Laboratory Standards Institute (CLSI) guidelines.²⁵ K. pneumoniae isolates were tested against the following antimicrobial agents (μg/disk): amoxicillin–clavulanic acid (20 + 10), cefoxitin (30), cefotaxime (30), ceftazidime (30), aztreonam (30), tobramycin (10), streptomycin (10), ciprofloxacin (5), gentamicin (10), amikacin (30), trimethoprim–sulfamethoxazole (1.25 + 23.75), tetracycline (30), and chloramphenicol (30). In addition, minimum inhibitory concentration (MIC) for meropenem, ertapenem, and imipenem was determined by Etest, following the manufacturer's instructions and the CLSI breakpoints.²⁵ The screening of phenotypic ESBL production was carried out by the double disk synergy test using cefotaxime, ceftazidime, and amoxicillin–clavulanic acid disks.²⁵ Furthermore, the immunochromatographic assay NG-Test CARBA 5 (NG Biotech) was used to detect the five main CP enzymes (OXA-48-like, NDM, KPC, VIM, and IMP) in isolates showing carbapenem resistance/intermediate susceptibility.

DNA extraction and quantification

Genomic DNA from MDR strains was extracted using the InstaGene Matrix (Bio-Rad) according to the manufacturer's instructions. To quantify the DNA concentration and level of purity, the absorbance readings were taken at 260 and 280 nm (Spectrophotometer ND-100; NanoDrop).

Antibiotic resistance genes

The genetic basis of resistance was investigated using PCR methodology and subsequent sequencing of the obtained amplicons. The primers and PCR conditions for specific detection of the different resistance genes are listed in Supplementary Table S1. Positive and negative controls from the University of La Rioja (Logroño, Spain) were used in this work. ESBL-positive isolates as well as other isolates showing resistance to carbapenem and/or broad-spectrum cephalosporins were screened for the presence of beta-lactamase genes (bla_TEM, bla_SHV, bla_{OXA-23/24/48/58}, bla_CTX-M, bla_CMY-2, bla_PER, bla_VEB, bla_IMP, bla_VIM, bla_GIM, bla_SPM, bla_SIM, bla_KPC, bla_GES, bla_IMI, bla_SME, and bla_NDM) by PCR/sequencing. The presence of other genes such as tetA and tetB was analyzed for tetracycline-resistant isolates, as well as mcr-1 gene (implicated in colistin resistance). The intI1 gene, encoding the integrase of class 1 integrons, was also analyzed by PCR.²⁶

Multilocus sequence typing of Klebsiella spp. strains

The multilocus sequence typing (MLST) with seven housekeeping genes (gapA, infB, mdh, pgi, phoE, rpoB, and tonB) was carried out in four selected isolates (representative of different beta-lactamase combinations) according to the protocol on the Institute Pasteur website. The allele combination was determined after sequencing the seven genes, and the sequence type (ST) and clonal complex (CC) were identified.

Results

K. pneumoniae isolates were recovered in meropenem-supplemented MacConkey agar plates from 16 of the 361 fecal samples from dogs tested (4.4%): 3 samples from house healthy dogs and 13 from sick dogs. The 13 positive hospitalized dogs came from veterinary clinics/hospitals located in Vila Real (2 isolates from Transmonvete and 7 isolates from Hospital Veterinário de Trás-os-Montes), Aveiro (1 isolate from Clínica Veterinária do Vouga), and Lisbon (3 isolates from Hospital Veterinário de São Bento). The 16 Klebsiella-carrier dogs (10 males and 6 females) corresponded to 12 mixed-breed dogs, 1 Serra da Estrela, 1 Chihuahua, 1 Galgo, and 1 Labrador Retriever.

All isolates were resistant to third-generation cephalosporins (cefotaxime and/or ceftazidime), aztreonam, and ciprofloxacin, and 88% of isolates were tetracycline-resistant (Table 1). All isolates showed MICs in the range of susceptibility for imipenem (0.125–0.25 μg/mL) and meropenem (0.032–0.75 μg/mL; except one isolate with an MIC of 2 μg/mL, in the intermediate category). Moreover, three isolates showed MICs of ertapenem in the range 1–8 μg/mL (intermediate or resistant category according to the CLSI, 2018). The ESBL phenotype was detected in 15 of the 16 recovered K. pneumoniae isolates (Table 1).

Table 1.

Phenotypic and Molecular Features of the 15 Extended-Spectrum Beta-Lactamase-Producing Klebsiella pneumoniae Isolates Recovered from Dogs in Portugal

Strain	Origin	Phenotype of resistance	Beta-lactamases	Integrons and other resistant genes	MLST^a
C10131	HVTM	FOX, CTX, CAZ, ATM, ETP^a, MER^b, TET, CIP, SXT	CTX-M-15, SHV-28	int1, tetA	ST307
C10134	HVTM	AMC, FOX, CTX, CAZ, ATM, TET, CIP, SXT, S	CTX-M-15, SHV-28, TEM-1	int1, tetA	NT
C10138	Transmon.	FOX, CTX, CAZ, ATM, TET, CIP, SXT, TOB, S	CTX-M-15, SHV-28, TEM-1	int1, tetA	ST307
C10139	HD	AMC, FOX, CTX, CAZ, ATM, TET, CIP, SXT, CN, TOB, S, C	CTX-M-15, SHV-28, TEM-1	int1, tetA	NT
C10141	CV Lisboa	CTX, CAZ, ATM, CIP	CTX-M-15, SHV-1, TEM-1	Not present	NT
C10144	HVTM	AMC, FOX, CTX, CAZ, ATM, TET, CIP, SXT, S	CTX-M-15, SHV-1, TEM-1	int1, tetA	NT
C10145	CV Vouga	AMC, FOX, CTX, CAZ, ATM, TET, CIP, SXT, CN, S	CTX-M-15, SHV-1, TEM-1	int1, tetA	NT
C10140	CV Lisboa	AMC, FOX, CTX, CAZ, ATM, TET, CIP, SXT, CN, S, C	CTX-M-15, SHV-1	tetA	NT
C10142	CV Lisboa	CTX, CAZ, ATM, CIP	CTX-M-15, SHV-1	Not present	ST15
C10143	HD	FOX, CTX, CAZ, ATM, TET, CIP, SXT, S	SHV-28, TEM-1	int1	NT
C10130	HVTM	AMC, FOX, CTX, CAZ, ATM, ETP^a, TET, CIP	SHV-28, TEM-1	tetA	NT
C10132	HD	AMC, FOX, CTX, CAZ, ATM, TET, CIP, SXT, CN, S	SHV-28, TEM-1	int1, tetA	ST307
C10133	HVTM	AMC, FOX, CTX, CAZ, ATM, TET, CIP, SXT, S	SHV-28, TEM-1	tetA	NT
C10135	HVTM	AMC, FOX, CTX, CAZ, ATM, ETP^a, TET, CIP, SXT, CN, TOB, C	SHV-28, TEM-1	int1, tetA	NT
C10136	HVTM	FOX, CTX, ATM, TET, CIP, SXT, TOB, CN, S	SHV-28, TEM-1	int1	NT
C10146	Transmon.	AMC, FOX, CTX, CAZ, ATM, TET, CIP, CN, S	SHV-1, TEM-1	int1, tetA	NT

HVTM, Hospital Veterinário de Trás-os-Montes (Vila Real, Portugal); HD, healthy dogs from their owners; Transmon., Clínica Veterinária Transmonvete (Vila Real, Portugal); CV Lisboa, Hospital Veterinário de São Bento (Lisboa, Portugal); CV Vouga, Clínica Veterinária do Vouga (Sever do Vouga, Portugal).

MIC for ertapenem in the range 1–8 μg/mL, category intermediate or resistant according to the CLSI 2018.

MIC for meropenem of 2 μg/mL, category intermediate according to the CLSI 2018.

AMC, amoxicillin–clavulanic acid; ATM, aztreonam; C, chloramphenicol; CAZ, ceftazidime; CIP, ciprofloxacin; CLSI, Clinical and Laboratory Standards Institute; CN, gentamicin; CTX, cefotaxime; ETP, ertapenem; FOX, cefoxitin; MIC, minimum inhibitory concentration; MLST, multilocus sequence typing; MER, meropenem; NT, not tested; S, streptomycin; SXT, trimethoprim–sulfamethoxazole; TET, tetracycline; TOB, tobramycin.

Nine of the 15 isolates with an ESBL phenotype carried the bla_CTX-M-15 gene, associated with bla_SHV-28 (4 strains) or bla_SHV-1 (5 strains). Moreover, six additional ESBL-positive isolates carried the bla_SHV-28 gene, associated in all cases with bla_TEM-1. None of the K. pneumoniae isolates carried genes encoding class A (bla_KPC, bla_SME, and bla_GES) or class D CPs (bla_{OXA-23/24/48/58}) or metallo-β-lactamase genes (bla_IMP, bla_GIM, bla_VIM, bla_SIM, bla_NDM, and bla_SMP), even though three of these strains showed decreased susceptibility or resistance for meropenem and/or ertapenem.

The mcr-1 determinant, encoding colistin resistance, was studied in all K. pneumoniae isolates of this work, and all of them were mcr-1 negative.

The ESBL-negative strain exhibited a MDR phenotype and carried the bla_SHV-1, bla_TEM-1, and tetA genes, as well as a class 1 integron (intI1).

The MLST was performed for four of the ESBL-producing strains, and three of them were identified as ST307 lineage: (1) one isolate from a healthy dog with SHV-28+TEM-1; (2) two isolates from hospitalized dogs, one of them from a veterinary clinic in Vila Real with CTX-M-15+SHV-28+TEM-1 and the other from a hospital in the same city with CTX-M-15+SHV-28. The ST15 lineage was identified in the last isolate, carrying the genes encoding CTX-M-15+SHV-1 (Table 1).

Discussion

K. pneumoniae is a major pathogen of nosocomial infections, and it is frequently associated with resistance to the highest priority critically important antimicrobials.^15,27 Particularly, the prevalence of ESBL-producing K. pneumoniae has made it one of the most challenging MDR pathogens with public health significance. Very few reports have been produced on dogs concerning the prevalence and characterization of ESBL-producing K. pneumoniae in the fecal microbiota of healthy or sick animals,²⁸ and most of the studies have focused on the ESBL characterization of clinical K. pneumoniae isolates in pets.^15,27,29,30 Furthermore, to the best of our knowledge, this is the first report produced in Portugal on ESBL-producing K. pneumoniae isolated from fecal samples in healthy dogs.

This study initially focused on the detection of CP-producing K. pneumoniae isolates, which, in some cases, display a poor increase in carbapenem MICs. For this purpose, we used MacConkey agar plates supplemented with 0.8 mg/L of meropenem. In these specific conditions, we identified only three fecal samples containing K. pneumoniae with carbapenem MICs in the category of resistance/intermediate susceptibility (related to ertapenem and/or meropenem, but not to imipenem). None of these carbapenem-nonsusceptible K. pneumoniae isolates carried the tested CP genes, and we cannot exclude other mechanisms of resistance (such as porin modification), in addition to ESBL production.³¹ It is important to note that K. pneumoniae resistant to ertapenem can be caused by the presence of subtypes of ESBL genes (e.g., the SHV-28 detected in this study) associated with porin loss (OmpK36).³²

In our study, we detected ESBL-producing K. pneumoniae isolates in 4.2% of tested dogs. Taking into consideration the fact that it would have been more appropriate to use cefotaxime- or ceftazidime-supplemented plates for ESBL-producing K. pneumoniae detection, we cannot exclude an underestimated prevalence in the studied dog population; in any case, the carriage level of ESBL-producing K. pneumoniae already identified in this study is worrying.

ESBL-producing K. pneumoniae isolates are also frequently detected in clinical samples of sick dogs in different countries.^{12,13,29,33,34} Specifically, Harada et al.²⁹ found that 31.5% of K. pneumoniae isolates obtained from clinical specimens from dogs in Japan were ESBL producers. Donati et al.³³ analyzed K. pneumoniae obtained from clinical samples from dogs and cats in Italy and found that 21.4% showed resistance to broad-spectrum cephalosporins, and all of them carried ESBL- and AmpC-encoding genes (including bla_CTX-M-15 and bla_SHV-28). Furthermore, Zogg et al.³⁴ found that 26.5% of K. pneumoniae from sick dogs were ESBL producers (carrying bla_CTX-M-15, bla_CTX-M-1, and bla_SHV-12).

The bla_CTX-M-15 and bla_SHV-28 genes, alone or combined, were detected in all 15 ESBL-producing K. pneumoniae isolates. The first report of the SHV-28 enzyme was made in 2002 in China (GenBank AF538324) from one K. pneumoniae strain isolated from an hospitalized patient, and it was later found in a clinical sample in India,³⁵ with subsequent reports.³³ Several studies reported the detection of bla_CTX-M-15 among K. pneumoniae of dog origin, in both healthy and sick dogs from different countries.^29,30,34,36 CTX-M-15, the dominant beta-lactamase type in this study, is also considered the most common ESBL in K. pneumoniae from patients worldwide, an indication of its global distribution across species.^30,34 Our results support previous findings about the generally greater abundance of CTX-M-15 in companion animal isolates,^15,28,30 whereas CTX-M-1 is more commonly reported for livestock animals.

Furthermore, Maeyama et al.²⁷ detected bla_CTX-M-15 in ESBL-producing K. pneumoniae isolates in urine from dogs and cats, in addition to bla_CTX-M-2 and bla_CTX-M-14 (14%), these last two not detected in our study. In addition, the bla_CTX-M-15 gene has become prevalent in K. pneumoniae isolates from hospitalized patients and is currently the most common ESBL identified worldwide in humans.^37–39

The use of β-lactams in clinical practice of veterinary medicine may be considered one of the reasons for the high incidence of ESBL producers worldwide. Thus, pets can be a significant reservoir of β-lactam resistance genes. Therefore, it is important that veterinarians be aware of the correct antibiotic prescriptions, based on susceptibility tests and, from the results obtained, implement the correct drug treatment for infections, reducing the selection pressure on the pathogens.

The lineage ST307 was identified in three of our ESBL-positive K. pneumoniae isolates, two carrying bla_CTX-M-15+bla_SHV-28 genes and another one bla_SHV-28. We also identified the ST15 lineage in an ESBL-positive K. pneumoniae isolate producer of CTX-M-15+SHV-1 enzymes.

Our study was consistent with other reports in which bla_CTX-M-15 is common in ST307.^40,41 This ST307 clone has been recently reported as a potential high-risk clone in human clinical isolates in different European countries, carriers of ESBL and/or CP genes, as is the case for bla_CTX-M-15 and bla_KPC-2/3.^42,43 Moreover, this ST307 clone has also been identified previously in clinical samples from dogs and cats, associated with CTX-M-15 in Japan.^27,29 It seems that this clone is widely distributed not only among the human population but also among pets.

Additionally, our data indicate the presence of ST15-CTX-M-15 K. pneumoniae, which should be considered a zoonotic agent of high clinical relevance for humans and animals. These results are in line with those of previous studies performed on K. pneumoniae clinical samples from sick companion animals in Germany,³⁰ Portugal,¹⁵ and Switzerland.³⁴ The combination of the genes encoding CTX-M-15 and SHV-28 enzymes was previously detected in K. pneumoniae isolates of the high-risk clone ST15 in human and dog clinical isolates.⁴⁴

Conclusions

This study demonstrated that dogs can be colonized by ESBL-producing K. pneumoniae isolates of high epidemic clones (ST307 and ST15), producing important ESBLs (as CTX-M-15 and SHV-28), which could potentially be transferred to close-contact humans, as has already been found for non-ESBL K. pneumoniae in other studies.⁸ These MDR bacteria should be monitored in the future, mostly in this type of animals that live in such close contact with humans.

Footnotes

Acknowledgment

The authors would like to thank Dr. Carla Oliveira who helped us with the collection of fecal samples from pets in Transmonvete veterinary clinic, located in Vila Real.

Disclosure Statement

No competing financial interests exist.

Funding Information

I.C. and V.S. acknowledge the financial support of Fundação para a Ciência e Tecnologia (FCT, Portugal), through the references SFRH/BD/133266/2017 and SFRH/BD/137947/2018 (Medicina Clínica e Ciências da Saúde), respectively. Experimental work performed in the University of La Rioja was financed by the project SAF2016-76571-R of the Agencia Estatal de Investigación (AEI) and Fondo Europeo de Desarrollo Regional (FEDER) of EU. This work was supported by the Associate Laboratory for Green Chemistry—LAQV, which is financed by national funds from FCT/MCTES (UID/QUI/50006/2019).

Supplementary Material

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