Multidrug-resistant mcr-1 gene-positive Klebsiella pneumoniae ST307 causing urinary tract infection in a cat

Resumo

The Klebsiella pneumoniae (K. pneumoniae) bacterium is responsible for many opportunistic infections such as sepsis, and a multidrug-resistant (MDR) clone sequence type (ST) 307 has recently begun to spread. The objective of this study was to report the first occurrence of this virulent genotype, which was found in the context of a urinary infection in a domestic feline in Brazil. The K. pneumoniae isolate was identified from the urine of a 6-month-old male crossbreed cat using 16S rRNA sequencing. It was then subjected to antimicrobial susceptibility testing, followed by multilocus sequence typing analysis, and PCR detection of virulence and resistance genes. The antimicrobial susceptibility profile demonstrated that the isolate was MDR and associated with the presence of the colistin resistance gene (mcr-1). Genotyping allowed us to classify the isolate as K. pneumoniae ST307 with the presence of wabG, uge, and entB genes. MDR K. pneumoniae is important in human and veterinary medicine because it causes many types of infections. Clonal propagation of virulent or MDR genotypes such as K. pneumoniae ST307 is a global concern. This report of ST307 isolation from a urine sample in a domestic feline is the first in Brazil.

Klebsiella pneumoniae is a bacterium that causes infections, including bacteremia, sepsis, urinary tract infections, and soft tissue, lung, and intestinal diseases in hospital settings [1, 2]. Increased resistance to antimicrobials has recently been noted [3], which worsens the prognosis for infected patients, particularly those with weakened immune systems [4, 5]. The “One Health” concept includes animals and the environment as an important part of the process of antimicrobial resistance development [6].

Due to the severity of K. pneumoniae infections and scarce animal research supporting the virulence and treatment of such infections, this study aimed to report the occurrence of the virulent multidrug resistance (MDR) K. pneumoniae ST307 strain in a case of urinary tract infection in a domestic feline in Brazil. The information on the dispersal of this clone is also essential.

A sample of feline urine was collected and cultured on 8% sheep blood agar (Sigma-Aldrich, Darmstadt, Germany) and MacConkey agar (Neogen Corporation, São Paulo, Brazil) at 37 °C in aerobiosis. Isolated colonies were morphologically and biochemically characterized according to Quinn et al. (2013) [7].

Antimicrobial susceptibility testing was performed using the disk diffusion agar method as described by Bauer and Kirb (1966) [8]. Nine antimicrobial classes including 26 antibiotics were tested: penicillins (ampicillin, amoxicillin, amoxicillin with clavulanic acid, piperacillin-tazobactam, ticarcillin – clavulanate, and ampicillin-sulbactam), cephalosporins (cefuroxime, cefoxitin, ceftazidime, cefepime, cefazolin, and ceftriaxone), carbapenems (imipenem, meropenem, and ertapenem), aminoglycosides (amikacin, gentamicin, netilmicin, and tobramycin), quinolones (ciprofloxacin), phenicol (chloramphenicol), tetracyclines (doxycycline and tetracycline), nitrofurans (nitrofurantoin), and sulfonamides (sulfonamides and trimethoprim sulfonamides). Antimicrobial susceptibility testing results were classified according to the Brazilian Committee on Antimicrobial Susceptibility Testing (BrCAST) and Clinical and Laboratory Standards Institute (CLSI) criteria [9–11].

The results of antimicrobial susceptibility tests that have been classified according to BrCAST [9] are amoxicillin, amoxicillin with clavulanic acid, cefuroxime, cefoxitin, ceftazidime, cefepime, ceftriaxone, imipenem, meropenem, ertapenem, tobramycin, and ciprofloxacin. Human CLSI breakpoints were used for piperacillin-tazobactam, ticarcillin – clavulanate, ampicillin-sulbactam, netilmicin, and sulfonamides [10]. Veterinary CLSI breakpoints were used for ampicillin, cefazolin, amikacin, gentamicin, chloramphenicol, doxycycline, tetracycline, nitrofurantoin, and trimethoprim sulfonamides [11].

The minimum inhibitory concentration (MIC) was determined for colistin (susceptible, ≤2 mg/L; resistant, >2 mg/L) using the method EUCAST microdilution cation adjusted (Polymyxin Sigma) according to BrCAST standards [9]. Isolates were also classified according to the resistance profile as described by Magiorakos et al. (2012) [12], which defines MDR bacteria as having resistance to one or more agents in three or more antimicrobial drug categories.

Genomic DNA extraction from the isolate was performed using the phenol-chloroform method as described by Sambrook and Russel (2004) [13] before amplifying the 16S rRNA based on oligonucleotide sequences 27F [14] and 1492R [15]. The PCR product was purified and used in the sequencing reaction on an ABI 3500 Genetic Analyzer (Applied Biosystems). The sequence was compared to the GenBank database using BLAST (http://www.ncbi.nlm.nih.gov/BLAST). PCR was performed to detect the virulence genes wabG [16], entB [17], uge [18], and the colistin resistance gene (mcr-1) [19]. The products were analyzed using an agarose gel.

Oligonucleotides were used as described by Diancourt et al. (2005) [20] and Institute Pasteur (http://bigsdb.pasteur.fr/klebsiella/klebsiella.html) to perform the MLST technique. Amplification products were purified and sequenced as described above. The sequences were processed using the CLC DNA Workbench 6.0 Program and compared to alleles deposited in the MLST database (http://www.pasteur.fr/recherche/genopole/PF8/mlst/Kpneumoniae.html) as described by Diancourt et al. (2005) [20].

The patient was a 6-month-old male crossbreed cat with feline lower urinary tract disease showing clinical signs of a partial urinary tract obstruction; the material was sent in 2017 for culture and antibiogram. After growth and biochemical tests, the isolate was identified as Klebsiella sp. and confirmed as K. pneumoniae by 16S rRNA sequencing (GenBank accession number MN646892). By the antimicrobial susceptibility test, a high percentage of antimicrobial resistance was observed based on the BrCAST and CLSI criteria [9–11]. Therefore, the isolate was tested for MDR, and thus presented resistance to seven antimicrobial classes (piperacillin-tazobactam, ticarcillin – clavulanate, ampicillin-sulbactam, cefuroxime, ceftazidime, cefepime, cefazolin, ceftriaxone, ertapenem, gentamicin, netilmicin, tobramycin, ciprofloxacin, tetracycline, doxycycline, and sulfonamides). The MIC for colistin, 0.5 mg/L, is considered sensitive. Analysis of the isolate by the MLST technique characterized it as genotype 307 (ST307), belonging to Clonal Complex 307 (CC307). The virulence genes wabG, uge, and entB and the colistin resistance gene (mcr-1) were detected.

No antibiotics had been used recently before the patient’s consultation; the animal was hospitalized for 4 days using ceftriaxone; after discharge, the animal was medicated with amoxicillin and clavulanate acid showing an improvement in the condition.

Antimicrobial resistance has been a major concern worldwide. The diversity of MDR bacterial pathogens may be related to infections. Within this broad list, the ESKAPE pathogens (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp.) are considered priorities according to the World Health Organization. Here, the occurrence of a highly virulent genotype (ST307) and MDR in cat urine has been described. The urinary tract is a common location for infections caused by K. pneumoniae as observed in this study, and the ST307 genotype carried virulence factor genes that facilitate its adhesion to the urinary tract [21, 22].

As observed in this case, the isolate was resistant to ciprofloxacin and doxycycline, which are widely used antibiotics in clinical practice [23]. In a study by Marques et al. (2018) [24], the absence of carbapenem resistance was observed in clinical samples of cat urine and in this study’s isolate, and this is due to the infrequent use of this class of antimicrobials in veterinary medicine [25, 26].

The most highly recommended method by CLSI and EUCAST to test colistin susceptibility is the broth microdilution method, because it has a lower error rate compared to other methods; A microdilution susceptibility test comparison in commercially available colistin broth for Enterobacteriaceae was performed according to Chew et al. (2017) [27], and a very serious error rate of 36% (9/25) was demonstrated by Vitek2. Some results in this study were mcr-1-positive and did not exhibit phenotypic resistance, as determined by the current cutoff point.

Heteroresistance confounds the interpretation of colistin susceptibility tests in Gram-negative bacteria, which is caused by a K. pneumoniae concession [28, 29]. The acquisition of the mcr-1 gene and the impact of heteroresistance on phenotypic susceptibility tests still must be resolved to have an adequate cutoff point, requiring further studies. The limitation of this study is the lack of sequencing to confirm the mcr-1 gene. There are still few genotyping studies of K. pneumoniae strains isolated from animal origin. In our study, we identified ST307, which is of great epidemiological importance worldwide because it is widely disseminated among nosocomial infection isolates in humans [30]. This ST has been described in Japan in three companion animal samples: clinical dog and cat urine samples and a clinical cat secretion sample [31]. In Brazil, a CTX-M-15 positive K. pneumoniae ST307 MDR strain was recently found to cause urinary tract infections in dogs [32].

Recent epidemiological analyses have indicated that ST307 is dispersed in different locations around the world, posing a high risk of becoming a major clone responsible for a future epidemic [22] and overtaking CC258 [33]. Animals may be reservoirs of ST307, which has already been described in the urban rat population in West Africa [34]. Other studies have also shown that some K. pneumoniae clones can be shared between humans and domestic animals [24]. The isolate in this study presented with wabG genes, which perform lipopolysaccharide biosynthesis; uge, which constitutes the urease operon; and enterobactin entB, which performs iron rescue for bacterial growth. According to Villa et al. (2017) [22], genome sequencing showed genetic adaptation characteristics in the hospital and host environment, revealing new virulence factors located in the plasmid in ST307. The presence of the mcr-1 resistance gene is very concerning because polymyxin is currently being used as a therapeutic option in human medicine for infections caused by MDR bacteria, especially Gram-negative bacteria [19]. Klebsiella pneumoniae MDR isolates from animals are concerning because they have clinically relevant antimicrobial resistance genes, highlighting the roles of animals as reservoirs of major resistance determinants. This raises great concern in veterinary medicine, as previous studies have shown that K. pneumoniae is prone to nosocomial spread [35].

Klebsiella pneumoniae (ST307) MDR associated with urinary tract infection was first described in a domestic cat in Brazil. In this case, companion animals can act as a reservoir for MDR mcr-1 K. pneumoniae strains and can be transferred to the environment and humans. Thus, it is essential to survey K. pneumoniae strains and associated resistance genes found in animals.

Author contribution

ATHIS, MTSC, HM, LN, and VD: Conceptualization of the study, methodology, interpretation of the data, and writing of the manuscript

ATHIS, MTSC, SLC, HM, IGM, NL: Investigation, sample collection, and laboratory work

ATHIS, MTSC, HM: Data analysis

LN, VD: Funding acquisition

LN, VD: Supervision

All authors read and approved the final manuscript.

Funding

This work was supported by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (Capes).

Declarations

Ethical approval

The project was approved by the Animal Research Ethics Committee (CEUA) under protocol number 23108.236834/2017-13.

Conflict of interest

The authors declare no competing interests.