Abstract
The emergence and dissemination of high-risk clones of Klebsiella pneumoniae producing extended-spectrum β-lactamases (ESBLs) in animal infections is a critical issue. We report the detection and genomic features of a multidrug-resistant (MDR) ESBL (CTX-M-15)-producing K. pneumoniae infecting a domestic cat. Whole-genome sequencing analysis identified the international ST340 (clonal group CG258), and genes and mutations conferring resistance to β-lactams, aminoglycosides, macrolides, phenicols, fosfomycin, sulfonamides, tetracycline, trimethoprim, and fluoroquinolones. In addition, the presence of genes encoding resistance to disinfectant and heavy metals hazardous to humans was also confirmed. The MDR profile exhibited by the strain contributed to treatment failure and death of the companion animal. Therefore, active surveillance of critical priority lineages of K. pneumoniae should not only focus on human infections but also on veterinary infections.
Introduction
Extended-spectrum β-lactamase (ESBL)-producing Klebsiella pneumoniae is a leading cause of hospital-acquired infections, and recently has been included in the priority World Health Organization's (WHO) list of antibiotic-resistant pathogens. 1 In this regard, most high-risk clones of K. pneumoniae have been clustered within the international clonal group CG258, which include the sequence types ST258, ST11, ST340, and ST437. 2 Worryingly, some lineages belonging to CG258 have begun to be identified in animal hosts and in aquatic environments, constituting a serious public health problem.3,4
CTX-M-15 type enzymes are considered the most common ESBLs identified worldwide in humans, and nowadays they are increasingly reported not only in food-producing animals but also in domestic animals raising some important concerns. 5
In this study, we report the detection and genomic characterization of a high-risk clone of K. pneumoniae ST340 of the clonal group CG258, producing a CTX-M-15-type ESBL, isolated from a male household cat presenting lower urinary tract symptoms. In this regard, these data can contribute for the study of epidemiology, prevention, and management of infections caused by critical priority WHO pathogens, 6 circulating at the human–animal interface.
Materials and Methods
Case presentation
In July 2016, a 2-year-old mixed breed male cat was admitted to a private veterinary clinic with signs of acute dysuria, mild dehydration, moderate colic, anorexia, and prostration. During the physical examination, it was evidenced moderate to severe dehydration, congested mucous membranes, fever, abdominal discomfort, bladder distension, enlarged kidneys, moderate colic, and weakness. Based on the anamnesis and initial physical examination, the animal was diagnosed with lower urinary tract obstruction. Therefore, it was established emergency stabilization with sedation morphine (0.1 mg/kg), acepromazine (0.03 mg/kg), and telazol (4 mg/kg), and lower urinary tract obstruction was relieved with a urethral catheter. Aggressive fluid therapy (lactated ringer solution) was started, and dexamethasone (0.5 mg/kg/intravenous [IV]/once a day [SID]/3 days) and tramadol (3 mg/kg/IV/three times a day [TID]/3 days) were administered to reduce inflammation and pain. Finally, vitamin C (200 mg/mL = 1 mL/orally [PO]/TID/14 days) was administered as a supportive measure, whereas bladder lavage (two times a day [BID]/1 week) and enrofloxacin (2.5 mg/kg/IV/BID/7 days) were scheduled for infection control. A week later, the animal underwent perineal urethrostomy. Although, clinical condition improved for 1 week, the animal presented re-obstruction, fever, anorexia, apathy, leading to the death.
Identification of the isolate and antimicrobial susceptibility testing
A urinary sample was aseptically collected and sent to the laboratory for microbiological investigation. The sample was cultured on blood and MacConkey agar plates being incubated at 37°C overnight. Bacteria were identified by conventional biochemical tests, whereas antimicrobial susceptibility testing was performed by the disk diffusion method on Mueller–Hinton agar plates.7,8 Initially, amoxicillin, doxycycline, enrofloxacin, and gentamicin were tested for clinical diagnostic purposes, since these antibiotics are available for treating routine infections in small animals in the local veterinary clinic. In addition, human and veterinary antibiotics, including amoxicillin-clavulanic acid, ceftazidime, cefotaxime, ceftriaxone, ceftiofur, cefepime, cefoxitin, aztreonam, ertapenem, meropenem, imipenem, ciprofloxacin, trimethoprim/sulfamethoxazole, and amikacin, were tested for research purposes. The results were interpreted according to Clinical and Laboratory Standards Institute.7,8 ESBL production was screened by the double-disk synergy test (DDST). 9
Whole genome sequencing analysis
Whole genomic DNA was extracted (PureLinkTM; Invitrogen) and used to prepare a library that was sequenced using the NextSeq550 platform (2 × 75-bp paired-end) (Illumina). The sequence reads were assembled de novo using SPAdes 3.13.0. 10 Quality filters were applied and a Phred20 quality score was used. The sequence assembly was curated using Geneious version R10 (Biomatters Ltd., Auckland, New Zealand). Draft genome sequence was automatically annotated using the NCBI Prokaryotic Genome Annotation Pipeline v.3.2. Multilocus sequence type (MLST) was predicted using MLST 2.0. The resistome was analyzed using ResFinder 2.2 and the comprehensive antibiotic resistance database.* Plasmid classification was performed in silico using the PlasmidFinder 1.3 database. K-locus (KL) and O-locus classification was executed using Kleborate. The presence of heavy metal (HM) genes was predicted using BIGSdb database; whereas for detection of mercury, arsenic, and disinfectant resistance genes (quaternary ammonium compounds), we aligned sequence reads against our in-house database.11,12 Genomic similarity analysis of K. pneumoniae ST340 from human and nonhuman origin was estimated using FastANI, 13 and genomic sequences freely available on GenBank database.
Results
Identification of the isolate and antibiotic susceptibility profile
Bacterial cultures yielded growth of a K. pneumoniae designated C4 strain, exhibiting a multidrug-resistant (MDR) phenotype, 14 against amoxicillin, ceftazidime, cefotaxime, ceftriaxone, cefepime, aztreonam, ceftiofur, ciprofloxacin, enrofloxacin, trimethoprim-sulfamethoxazole, doxycycline, and gentamicin; remaining susceptible to amoxicillin/clavulanate, cefoxitin, ertapenem, meropenem, imipenem, and amikacin. ESBL production in K. pneumoniae C4 strain was confirmed by DDST.
Genomic background of ESBL-producing K. pneumoniae C4 strain
Whole genomic sequencing produced 9,965,376 paired-end reads with a ∼140 × total coverage. Quality filters were applied and a Phred20 quality score was used. The draft genome sequence of K. pneumoniae C4 generated 157 contigs. In addition, 5,384 coding sequences (CDSs), 6 rRNAs, 41 tRNAs, 9 ncRNAs, and 131 pseudogenes were identified (Table 1). The K. pneumoniae strain was assigned to ST340 (clonal group, CG258), whereas capsule locus was classified as KL15 and O-locus as O4. Using FastANI we have found that K. pneumoniae C4 has the closest genetic relationship (average nucleotide identity of 99.1058) with a human K. pneumoniae (strain B29, GenBank accession number, NZ_NTHW00000000.2) isolated from urinary tract infection, in Brazil.
Genomic Characteristics of a High-risk Clone of CTX-M-15-Producing Klebsiella pneumoniae Strain Isolated from an Infected Companion Animal
CDSs, coding sequences; ST, sequence type; QAC, quaternary ammonium compound; MLST, multilocus sequence type.
Antimicrobial resistome included genes conferring resistance to β-lactams (blaCTX-M-15, blaTEM-1B, blaSHV-11), aminoglycosides [aph(3′)-Ia, aadA2, aph(6)-Id, aph(3′′)-Ib, aac(3)-IId], fluoroquinolones (qnrB19, oqxA, oqxB), macrolides (mphA), phenicols (catA2), fosfomycin (fosA), sulfonamides (sul, sul2), tetracycline (tetD), and trimethoprim (dfrA12). In addition, sequence analysis of mutations in the quinolone resistance-determining region of chromosomal gyrA and parC indicated that K. pneumoniae C4 contained the ParC-80I and GyrA-83I mutations.15,16 Presence of disinfectant (qacEΔ) and HM resistance genes (pcoA-E, pcoR-S, silE, silR, silS, arsH) was also confirmed (Table 1). Finally, three plasmids belonging to IncFIB, IncFII (pMLST, K1:A-:B-) and IncR incompatibility groups were detected.
Discussion
The emergence and spread of ESBL-positive bacteria in companion animals constitute an expanding challenge as the therapeutic failures increase in veterinary practice. 17 In addition, small animals can act as silent carriers of ESBL-producing Enterobacteriaceae, constituting a potential source of dissemination of critical important pathogens and/or their resistance genes to humans, other hosts, and ecosystems. 18
Although the identification of ESBL-producing bacteria in companion animals has been overlooked for a long time, 19 the “One Health” approach has expanded interest in surveillance studies directed to determine the occurrence of these MDR pathogens at the human–animal–environment interface. 20 In this regard, molecular investigation has revealed the presence of international clones of CTX-M-producing Escherichia coli and K. pneumoniae circulating in humans and companion animals.21–25
In Brazil, the occurrence of ESBL (most CTX-M-15) producers in human infections has been higher than in developed countries. 26 More recently, intensive screening procedures in veterinary medicine have identified CTX-M-type-producing Enterobacteriaceae (most E. coli) in companion animal and wildlife, which has epidemiological implications.27–30 Therefore, much information is still needed to better understand the epidemiology of ESBL strains at the animal–human–environment interface.
Regarding K. pneumoniae, the main problem in Brazil is the endemic status of clones (predominantly ST11, ST258, ST340, and ST437) coproducing KPC-2 and CTX-M-15 beta-lactamases, belonging to the clonal group CG258, which are not restricted to hospital settings.3,4,24 In fact, KPC-2- and/or CTX-M-15-positive K. pneumoniae ST11 and ST340 have been reported in swine production and impacted aquatic environments.3,4,24,31,32 In this study, we report for the first time the identification and genomic characterization of an international critical clone of CTX-M-15-producing K. pneumoniae ST340/CG258 causing an infection in a companion animal, in Brazil. Similarly, in Italy the identification of CTX-M-15-producing K. pneumoniae ST340 in infected pets was documented. 25 In contrast, evidence of K. pneumoniae sharing between healthy companion animals and cohabiting humans has been documented. 33 Considering that ST340 are widespread in hospital settings, speculations on a possible link could be raised. In fact, the identification of hospital-associated clone of MDR pathogens in companion animals has suggested a zooanthroponotic transmission in the household, indicating that humans can also transmit clinically relevant MDR pathogens to their companion animals. 34
Specifically, K. pneumoniae C4 was categorized MDR based on absence of susceptibility to ≥1 agent within the broad-spectrum cephalosporins, fluoroquinolones, aminoglycosides, and folate pathway inhibitor antibiotic classes, 14 considered as critically important for human medicine. 35 Worryingly, in K. pneumoniae and others priority pathogens progressive accumulation of resistance genes has favored the selection and spread of high-risk clones. In fact, over the years, K. pneumoniae has acquired ESBL encoding genes (most blaCTX-M-15) followed by carbapenemase genes and subsequently developing resistance to polymyxins. 36 In addition, HM tolerance supported by the presence of specific resistance genes could contribute with rapid adaptation of some lineages to hostile conditions, including anthropogenically impacted environments. 37
Genes conferring resistance to cooper (Cu), arsenic (As), and silver (Ag) were identified in K. pneumoniae C4. In this regard, although Cu has been authorized in animal nutrition, As is considered an undesirable contaminant in animal feed, and a hazard to human health, since it can induce organ damage. 38 Silver (in the ionized form Ag+1) has known antimicrobial properties, which have been promoted in medical and consumer products (such as clothing and household appliances), 39 whereas MDR high-risk clones of K. pneumoniae have been capable of evolving and transmitting high-level resistance to silver. 40
In summary, these data provide important information to be used in epidemiological studies of critical ESBL-producing pathogens in “One Health,” to understand genomic aspects related to adaptation and dissemination of high-risk clones at the human–animal interface.
Nucleotide Accession Number(s)
This Whole Genome Shotgun project has been deposited at DDBJ/ENA/GenBank under the accession SHLW00000000. The version described in this article is SHLW01000000.
Footnotes
Acknowledgments
FAPESP and CNPq research grants are gratefully acknowledged. We thank Cefar Diagnóstica Ltda. (Brazil) for kindly supplying antibiotic disks for susceptibility testing.
Disclosure Statement
No competing financial interests exist.
Funding Information
This study was funded by research grants from Fundacão de Amparo à Pesquisa do Estado de São Paulo (FAPESP 2016/08593-9) and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq 462042/2014-6, 312249/2017-9, and 433128/2018-6).