https://orcid.org/0000-0002-2395-6609
Figures
Abstract
This study examined 70 Klebsiella pneumoniae isolates derived from companion animals with urinary tract infections in Taiwan. Overall, 81% (57/70) of the isolates carried extended-spectrum β-lactamase (ESBL) and/or plasmid-encoded AmpC (pAmpC) genes. ESBL genes were detected in 19 samples, with blaCTX-M-1, blaCTX-M-9, and blaSHV being the predominant groups. pAmpC genes were detected in 56 isolates, with blaCIT and blaDHA being the predominant groups. Multilocus sequence typing revealed that sequence types (ST)11, ST15, and ST655 were prevalent. wabG, uge, entB, mrkD, and fimH were identified as primary virulence genes. Two isolates demonstrated a hypermucoviscosity phenotype in the string test. Antimicrobial susceptibility testing exhibited high resistance to β-lactams and fluoroquinolones in ESBL-positive isolates but low resistance to aminoglycosides, sulfonamides, and carbapenems. Isolates carrying pAmpC genes exhibited resistance to penicillin-class β-lactams. These findings provide valuable insights into the role of K. pneumoniae in the context of the concept of One Health.
Citation: Lee MMY, Kuan N-L, Li Z-Y, Yeh K-S (2024) Occurrence and characteristics of extended-spectrum-β-lactamase- and pAmpC-producing Klebsiella pneumoniae isolated from companion animals with urinary tract infections. PLoS ONE 19(1): e0296709. https://doi.org/10.1371/journal.pone.0296709
Editor: Nabi Jomehzadeh, Abadan University of Medical Sciences, ISLAMIC REPUBLIC OF IRAN
Received: September 1, 2023; Accepted: December 17, 2023; Published: January 16, 2024
Copyright: © 2024 Lee et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: All relevant data are within the paper and its supporting information files.
Funding: This work was supported by a grant (NSTC-112-2313-B-002-031) from the National Science and Technology Council, Taiwan to K.-S. Yeh..The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Introduction
Klebsiella pneumoniae is a Gram-negative, nonmotile, encapsulated bacterium belonging to the Enterobacteriaceae family. This bacterium is a crucial opportunistic pathogen that causes infections in humans, particularly in those with a compromised immune system and urinary tract infections (UTIs) [1]. The emergence and spread of multidrug-resistant K. pneumoniae strains pose a challenge for clinical treatment [2]. The World Health Organization has recognized K. pneumoniae and other Enterobacteriaceae resistant to third-generation cephalosporins (3GCs) as a Priority 1 group [3]. 3GCs are commonly used for the treatment of UTIs [4, 5]. Moreover, K. pneumoniae is a common cause of UTIs in pets, with 14% of dogs and 3%–19% of cats estimated to have a UTI during their lifetime [6–9]. K. pneumoniae also causes infections at other sites and can become resistant to 3GCs by producing extended-spectrum beta-lactamases (ESBLs) and AmpC β-lactamases. ESBL genes are mainly located on plasmids of various molecular sizes, which enhance their horizontal transfer, in addition, those plasmids can confer resistance to other antibiotics such as fluoroquinolones and aminoglycosides [10]. AmpC β-lactamases can also be located on plasmids or chromosomally encoded [11, 12].
In clinical settings, K. pneumoniae strains carrying pAmpC are commonly observed to harbor ESBL. These two types of β-lactamase genes can be located on the same plasmid or on different plasmids [13]. In addition to antibiotic resistance, K. pneumoniae has four major virulence factors: fimbriae, capsules, lipopolysaccharides, and iron-acquiring proteins [14]. Several other factors such as OMPs, porins, efflux pumps, iron transport systems, and genes involved in allantoin metabolism have also been described, but some of their function remained to be elucidated [15]. Briefly, fimH and mrkD encode the adhesion molecules of type 1 and type 3 fimbriae, respectively. Fimbrial structures mediate K. pneumoniae binding to host epithelial cells. RmpA is a plasmid-located virulence factor of K. pneumoniae that regulates the synthesis of capsular polysaccharides. LPS production is regulated by the uridine diphosphate galacturonate 4‑epimerase (uge) gene. Without this gene, K. pneumoniae is less able to cause urinary tract infections, pneumonia and sepsis. The Klebsiella ferric iron uptake (kfu) is a regulator gene of the iron transport system involved in iron absorption. Siderophores are small molecules that can compete iron from the host’s iron‑chelating proteins. Siderophores expressed in K. pneumoniae include enterobactin, yersiniabactin and aerobactin. The gene associated with allantoin metabolism (allS) is used by bacteria to obtain carbon and nitrogen from the environment. Some of the genes encoding the aforementioned virulence factors are widely used to evaluate the pathogenicity of K. pneumoniae [16].
On the basis of differences in the accessory genome, K. pneumoniae can be divided into opportunistic, hypervirulent, and multidrug-resistant types [17]. Opportunistic K. pneumoniae still commonly causes hospital-acquired infections and is more likely to infect patients with compromised immune systems. Hypervirulent K. pneumoniae can infect individuals with healthy immune systems, causing various diseases, such as liver abscess, endophthalmitis, meningitis, and septic arthritis, primarily in community-acquired cases. The antibiotic resistance genes of multidrug-resistant K. pneumoniae are often located on transmissible plasmids. Some strains can accumulate more resistance genes through transmission and conjugation, forming extremely drug-resistant strains characterized by a super resistome [18, 19]. In conclusion, K. pneumoniae has evolved beyond merely being an opportunistic pathogen and now exhibits a diverse range of pathogenic behaviors
In contrast to wild animals and humans, pets and humans come in close contact over the long term, creating a relatively high chance for bacterial transmission between them [20]. Some ESBL-producing K. pneumoniae strains isolated from pets have been identified as high-risk bacterial populations that commonly infect humans [21–23]. This finding underscores the importance of K. pneumoniae in zoonotic transmission and public health [24]. According to the concept of One Health, this study focused on K. pneumoniae isolated from pets with UTIs. We determined the proportion of K. pneumoniae strains harboring ESBL, pAmpC, or both. The virulence factors of these resistant K. pneumoniae strains were examined through polymerase chain reaction (PCR). The relationships among these strains were analyzed using multilocus sequence typing (MLST). The findings of this study provide valuable information from a public health perspective.
Materials and methods
K. pneumoniae strain collection
Bacterial strains analyzed in this study were obtained from pet visits (only dogs and cats) at National Taiwan University Veterinary Hospital between January 1, 2014, and December 31, 2018. A total of 171 K. pneumoniae strains were isolated from dogs and cats, and 70 isolates were obtained from the urine samples of animals with UTIs. These collected bacteria were identified to the species level by using a Vitek-2 Compact microbial detection system (bioMérieux, Marcy I’Etoile, France) [25]. The bacterial strains were stored in Microbank System cryovials (Pro-Lab Diagnostics, Richmond Hill, ON, Canada) at −80°C.
Screening and confirmation of ESBL-producing K. pneumoniae
The K. pneumoniae strains were removed from the −80°C freezer, inoculated on tryptone soy agar (Becton Dickison, Franklin Lakes, NJ, USA), and incubated at 37°C for 16–18 hours. After confirming the growth of colonies, we inoculated them on ESBL selective culture medium, CHROMagar ESBL (CHROMagar, Paris, France), and incubated them at 37°C for 16–18 hours for preliminary screening. On this culture medium, ESBL-producing K. pneumoniae yielded deep blue colonies, whereas non-ESBL-producing K. pneumoniae strains did not grow. The strains that screened positive in the initial tests were subjected to phenotypic confirmation. According to the Clinical and Laboratory Standards Institute guidelines, a phenotypic confirmatory test for ESBL production in K. pneumoniae can be performed using the disk diffusion method [26]. This method operates on the principle of inhibiting the activity of ESBL through β-lactamase clavulanic acid, enabling third-generation cephalosporins to kill the bacteria, thus broadening the inhibition zone. The difference in the size of the inhibition zone between the presence and absence of clavulanic acid was used to determine whether the strain produced ESBL. Briefly, the procedure involved aseptically collecting a bacterial colony by using a loop, mixing it with phosphate-buffered saline to adjust to a McFarland concentration of 0.5, and then uniformly applying it to Mueller–Hinton (MH) agar (Neogen, Lansing, MI, USA) using a sterile cotton swab. Four antibiotic discs, namely ceftazidime (CAZ, 30 μg), ceftazidime (30 μg)–clavulanic acid (10 μg, CAZ/CA), cefotaxime (CTX, 30 μg), and cefotaxime (30 μg)–clavulanic acid (10 μg, CTX/CA) were placed on the agar, and the plates were incubated at 37°C for 16 to 18 hours. The discs were purchased from Becton Dickison. The difference in the size of inhibition zones served as the basis for interpretation. If the difference between CAZ/CA and CAZ was greater than or equal to 5 mm or the difference between CTX/CA and CTX was greater than or equal to 5 mm, the strain was considered to be an ESBL-positive strain. K. pneumoniae ATCC70063 and E. coli ATCC25922 served as positive and negative control groups for ESBL, respectively.
DNA extraction
The DNA of the test strains was extracted through boiling [27]. Briefly, the tested K. pneumoniae strains were cultured for 16–18 h at 37°C on tryptic soy agar plates (Becton Dickinson). A loopful of cells was collected and added to a microcentrifuge tube supplemented with 200 μL of double-distilled H2O (ddH2O), and this mixture was boiled for 10 min. The supernatant was collected after centrifugation at 12,000 × g for 10 min and stored at −20°C. This was used as the template in subsequent PCR experiments.
β-lactamase genotype detection and sequencing analysis
PCR was performed to amplify target ESBL genes, including seven gene groups: blaTEM, blaSHV, and blaCTX-M (CTX-M-1, CTX-M-2, CTX-M-8, CTX-M-9, and CTX-M-25). The amplified genes were confirmed through DNA electrophoresis. The PCR mixture was prepared by combining 5 μL of DNA template, 1 μL each of 10 μM forward and reverse primers (Tri-I Biotech, New Taipei, Taiwan), 18 μL of sterile water, and 25 μL of 2× MasterMix (Ampliqon, Odense M, Denmark) in a 200-μL microcentrifuge tube, which was then placed in a PCR thermocycler (SensoQuest, Göttingen, Germany). The PCR conditions were as follows: an initial cycle for 5 min at 95°C, followed by 35 cycles at 30 s each at 95°C for denaturation, 40 s of annealing at a primer-determined temperature, and 60 s of extension at 72°C, and finally a cycle at 72°C for 10 min. The PCR products were stored at 4°C. The product was examined through DNA electrophoresis to confirm ESBL gene amplification results. If the expected outcome was obtained, the product of the desired size was excised from the agarose gel. Then, the complete nucleic acid sequence was assembled using DNASTAR Lasergene-Seqman software (DANSTAR, Madison, WI, USA) based on forward and reverse primer sequences obtained from sequencing (Tri-I Biotech, Taipei, Taiwan). The results were uploaded to the β-lactamase database Beta-Lactamase DataBase (BLDB)-Structure and Function website (http://www.bldb.eu/) for β-lactamase genotype determination.
PCR was performed to detect commonly recognized pAmpC genes, namely blaCIT, blaDHA, blaMOX, blaEBC, and blaFOX, following the same procedure as used for ESBL gene detection. The primers used to detect these β-lactamase genes are listed in Table 1. All 70 K. pneumoniae isolates were included in the PCR test.
Antimicrobial susceptibility test
The disc agar diffusion method was used for antimicrobial susceptibility testing [26], and the selected drug discs were as follows: amoxicillin/clavulanate (20/10 μg), ampicillin (10 μg), augmentin (5 μg), cefixime (5 μg), cefotaxime (30 μg), cefovecin (30 μg), cefoxitin (30 μg), cefpodoxime (10 μg), cephalothin (30 μg), ciprofloxacin (30 μg), doxycycline (30 μg), enrofloxacin (5 μg), gentamicin (10 μg), imipenem (10 μg), tetracycline (30 μg), and trimethoprim/sulfamethoxazole (25 μg).
Conjugation test
A conjugation test was conducted to determine whether the ESBL/pAmpC-producing K. pneumoniae strain isolated in the experiment could horizontally transfer its antibiotic resistance genes. Since 16 of the total 57 ESBL/pAmpC-producing K. pneumoniae strains were naturally resistant to sodium azide because they could grow on Mueller–Hinton agar supplemented with sodium azide for unknown reason; Therefore, these strains were excluded from the conjugation test. The number of strains used for the conjugation test was 41. The aforementioned K. pneumoniae strain was used as the donor, and E. coli J53 (ATCC BAA-2730TM), which is resistant to sodium azide, was used as the recipient. The two strains were co-cultivated and screened for successful transconjugants by using a double-selective MH agar medium containing cefotaxime (2 mg/L) and sodium azide (150 mg/L) (Sigma-Aldrid, Burlington, MA, USA) [34]. The presence of an antibiotic resistance gene in the transconjugant E. coli J53 was confirmed through PCR. The procedure involved the following steps: the donor K. pneumoniae strain and E. coli J53 strain were retrieved from frozen tubes and cultured in test tubes containing 5 mL of Luria–Bertani (LB) broth. The tubes were incubated at 37°C for 16–18 hours, Subsequently, 0.5 mL of each bacterial suspension was mixed with 4.5 mL of fresh LB broth and incubated at 37°C for 4 hours. Then, 0.5 mL of the donor solution and 0.5 mL of the recipient solution were mixed, 4 mL of fresh LB broth was added, and the mixture was incubated at 37°C for 16–18 hours. A 0.1-mL aliquot of the cultured cells was spotted and evenly spread on the agar surface of MH agar supplemented with sodium azide (150 mg/L) and cefotaxime (2 mg/L) [34]. If transconjugant colonies were observed, a lysate was prepared from the colony to serve as a DNA template. PCR was performed on the transconjugant strains using primers specific for the antibiotic resistance gene present in the donor K. pneumoniae strain to confirm the transfer of antibiotic resistance genes.
Detection of virulence genes
The following virulence genes of K. pneumoniae were detected through PCR: fimbriae (fimH: type 1 fimbriae, mrkD: type 3 fimbriae) [16, 35], iron uptake (entB: iron-chelating agent, iutA: iron-chelating agent, kfu: iron transport and phosphotransferase function) [35, 36], lipopolysaccharides (wabG: synthesis of the lipopolysaccharide core, uge: UDP-galacturonate-4-epimerase) [35], capsule (rmpA: a regulator of mucoid phenotype A) [37], and metabolism (allS: allantoin metabolism) [35]. The primers used, annealing temperature, and the predicted sizes of PCR products are listed in Table 2.
Hypermucoviscosity string test
A string test was performed on K. pneumoniae isolates inoculated on MHA and incubated at 37°C overnight. The test involved stretching the mucoid thread of the colony by using a loop, and a positive string test was defined as a stretch greater than 5 mm [38].
Multilocus sequence typing (MLST)
To understand the prevalence and evolutionary trends of K. pneumoniae included in this study, we examined the relationship between the strains through MLST. Seven housekeeping genes of K. pneumoniae were amplified through PCR following the method reported by Diancourt et al. [39]. These 7 genes were rpoB (a beta subunit of RNA polymerase), gapA (glyceraldehyde 3-phosphate dehydrogenase), mdh (malate dehydrogenase), pgi (phosphoglucose isomerase), phoE (phosphorine E), infB (translation initiation factor 2), and tonB (periplasmic energy transducer) [39]. The PCR reaction conditions were as follows: initial denaturation at 95°C for 2 min, followed by 35 cycles of denaturation at 94°C for 20 s, annealing at 50°C (gapA 60°C, tonB 45°C) for 30 s, extension at 72°C for 30 s, and finally extension at 72°C for 5 min. The reaction was maintained at 4°C. The primers used are listed in Table 3. The product was electrophoresed to confirm the results of gene amplification. If a PCR product of the correct molecular weight was observed, DNA was excised from the gel by using a blade, placed in a microcentrifuge tube, and sequenced. The sequencing results were uploaded to the Institut Pasteur K. pneumoniae MLST database (https://bigsdb.pasteur.fr/klebsiella/) for comparison and sequence type (ST) identification. The similarities between these ESBL/pAmpC-producing K. pneumoniae strains were analyzed using BioNumerics version 7.0 (Applied Maths, Sint-Martens-Latem, Belgium).
Results
Occurrence of K. pneumoniae carrying ESBL and/or pAmpC genes
The 70 Klebsiella pneumoniae isolates used in this study (52 from dogs and 18 from cats) were obtained from the urine samples of 52 dogs and 18 cats at National Taiwan University Veterinary Hospital between 2014 and 2019. These 70 isolates were initially screened with CHROMagar ESBL. Then, 39 of the 70 isolates that exhibited dark blue colonies on the agar surface were tested for ESBL-producing K. pneumoniae by using the combination disc test as a phenotypic confirmation method. From the results of this test, 21 isolates were assumed to produce ESBL. However, sequencing results from the PCR amplification of ESBL bla genes indicated that only 19 isolates expressed ESBL bla genes. In addition, the PCR of pAmpC genes revealed that 56 of the 70 isolates carried pAmpC bla genes. Furthermore, 13 K. pneumoniae (12 from dogs and one from cat) did not carry ESBL or pAmpC genes (Table 4). In the 7 ESBL genes that were tested, only blaSHV, blaCTX−M−1, and blaCTX−M−9 groups were detected. Among the 5 pAmpC genes tested, only genes from the blaCIT and blaDHA groups were detected. Table 5 lists the presence of the ESBL and pAmpC genes of the 57 ESBL and/or pAmpC-producing K. pneumoniae. In summary, a total of 57 K. pneumoniae isolates carried ESBL and/or pAmpC bla genes, with one isolate containing only ESBL genes, 38 isolates containing only pAmpC genes, and 18 isolates containing both ESBL and pAmpC genes.
Antimicrobial susceptibility test
Fig 1 presents the resistance rates of the 57 isolates to various classes of antimicrobial drugs. With the exception of cefotaxime and imipenem, all these isolates exhibited a resistance rate of more than 50%.
Orange, yellow, and green color bars indicate susceptible (S), intermediate (I), and resistant (R), respectively. The numbers on the Y-axis represent the resistant percentage resistant, and the antimicrobials tested are listed on the X-axis.
Conjugation test
The transferability of ESBL and pAmpC genes from the K. pneumoniae isolates to the E. coli J53 strain was evaluated by performing a bacterial conjugation test. Sixteen strain that were naturally resistant to sodium azide was excluded from testing. Of the remaining isolates tested, 43.9% (18/41) could successfully transfer ESBL and/or pAmpC genes to the E. coli J53 strain. Among these 18 isolates, 15 contained both ESBL and pAmpC genes, whereas the remaining 3 isolates carried only pAmpC genes. The PCR results revealed that all genes from the blaCTX−M−1 and blaCTX−M−9 groups were transferred to E. coli J53, whereas the blaCIT genes of 3 K. pneumoniae isolates (isolate number 2555, 2561, and 2851) were not transferred. A comparison between the original isolate and transconjugant strain is presented in Table 6.
Detection of virulence genes
The prevalence of virulence genes in K. pneumoniae was determined using PCR. The genes wabG and entB were detected in all the 57 isolates. The mrkD and fimH genes were detected in 98.2% (56/57) of the isolates, whereas the uge gene was present in 96.5% (55/57) of the isolates. However, the kfu gene was detected in only 33.3% (19/57) of the isolates, and the iutA gene was present in only 3.5% (2/57) of the isolates. The rmpA and allS genes were identified in only 1.8% (1/57) of the isolates each. The most commonly detected virulence gene combination was wabG, uge, entB, mrkD, and fimH (61.4%, 35/57), followed by wabG, uge, entB, kfu, mrkD, and fimH (31.6%, 18/57). Other combinations were observed only once. Table 7 lists the frequency of the virulence gene combinations.
Hypermucoviscosity string test
The string test was positive for two K. pneumoniae isolates. Fig 2 presents the result of isolate number 2517 that exhibited positivity in the string test.
The K. pneumoniae isolate number 2517 can be pulled into threads longer than 5 mm, indicating a positive result for the hypermucoviscosity thread test.
Phylogenetic analysis of K. pneumoniae isolates
A total of 26 STs were identified among the 57 ESBL- and/or pAmpC-producing K. pneumoniae isolates: ST11 (n = 7), ST15 (n = 6), ST655 (n = 6), ST485 (n = 5), ST37 (n = 3), ST3393 (n = 3), ST1825 (n = 3), ST709 (n = 2), ST147 (n = 2), ST967 (n = 1), ST1995 (n = 1), ST846 (n = 1), ST265 (n = 1), ST273 (n = 1), ST592 (n = 1), ST469 (n = 1), ST29 (n = 1), ST966 (n = 1), ST950 (n = 1), ST45 (n = 1), ST198 (n = 1), ST2643 (n = 1), ST1431 (n = 1), ST3216 (n = 1), ST636 (n = 1), ST2817 (n = 1), and unknown STs (n = 3). Among the 7 ST11 isolates, 42.9% (3/7) were obtained from cats and 57.1% (4/7) from dogs. All the six ST15 and ST655 isolates were obtained only from dogs and cats, respectively. Fig 3 displays a visual representation of the MLST results of the 57 isolates, illustrating the relatedness of each ST based on the degree of allele sharing between strains.
Each circle indicates a sequence type (ST), divided into one sector for each isolate, surrounded by the ST number. The numbers on the connecting line between STs within the MSTree indicate the number of different alleles. Solid lines represent an allelic difference of 3 or less, whereas dotted and faint lines indicate an allelic difference of 4 or more.
Discussion
In this study, we observed a lower prevalence of ESBL genes alone at 1.8% (1/57) than in East Asian countries such as China (75%, 15/20) [40], Japan (82.9%, 29/35) [22], and Korea (42.8%, 12/28) [41]. However, the prevalence of pAmpC genes was significantly higher in our study. We detected pAmpC genes in 66.7% (38/57) of the isolates examined in this study. However, these genes were present in only 10% (2/20) and 11.4% (4/35) of isolates examined in China and Japan, respectively [22, 40]. In Korea, none of the 28 isolates contained pAmpC genes [41]. Additionally, 31.6% (18/57) of the isolates examined in this study harbored both ESBL and pAmpC genes. This proportion was higher than that observed in China (15%, 3/20) and Japan (5.7%, 2/35) but lower than that in Korea (57.1%, 16/28) [22, 40, 41]. In the aforementioned previous studies and the present study, CTX-M type ESBLs, particularly those in the blaCTX−M−1 and blaCTX−M−9 groups, were the most prevalent ESBLs. Similarly, blaDHA was the dominant gene among pAmpC-containing isolates. However, in our study, one SHV- and no TEM-type ESBLs were detected. Although SHV and TEM type β-lactamases were less common than CTX-M type ESBLs in other studies, most of the SHV and all the TEM genes detected in our study were β-lactamases, not ESBLs. CTX-M type ESBLs are widely prevalent in both humans and companion animals. Among K. pneumoniae populations, blaCTX−M−1 group enzymes are predominantly found in African and European populations, whereas blaCTX−M−9 group enzymes are more common in Asia [42]. In the Taiwan Surveillance of Antibiotic Resistance (TSAR) study conducted between 2002 and 2012, clinical isolates were collected from 25–28 hospitals and medical centers across Taiwan [43]. These isolates were obtained from patients of various age groups and included blood and urine samples. Among the 138 aztreonam-, ceftazidime-, and cefotaxime-resistant isolates analyzed in the study, 54 (39.1%) contained only ESBL genes, 34 (24.6%) contained only AmpC genes, and 27 (19.6%) isolates contained both ESBL and AmpC genes. Within ESBL genes, blaCTX-M genes were predominant (52/81, 64%), followed by blaSHV (24/81, 30%) and blaTEM genes (5/81, 6%). The AmpC genes were predominantly represented by blaDHA (55/61, 90%), a finding consistent with that of the present study.
A significant increasing trend in the prevalence of both ESBL and AmpC genes in K. pneumoniae isolates was observed from 2002 to 2012. The detection rate of AmpC-producing isolates increased from 0% to 9.5%, and the incidence of ESBL producers increased from 4.8% to 11.9% [43]. Comparing the results of the TSAR survey, we determined that the prevalence of isolates containing only pAmpC genes or both ESBL and pAmpC genes was higher in isolates obtained from NTUVH than those from human hospitals. However, a significantly lower number of pure ESBL isolates were detected in dogs and cats at NTUVH compared with human patients in the nationwide survey. Previous investigations have reported that the blaCTX−M−1, blaCTX−M−9, and blaDHA groups of enzymes were distributed across Taiwan and represented the predominant enzymatic groups among K. pneumoniae isolates; this finding is in line with that of this study [44]. The consistency in the key bla genes identified in K. pneumoniae isolated from both humans and animals suggests the potential for the interspecies transmission of K. pneumoniae carrying these genes.
Among the 57 isolates analyzed, 26 STs were identified. The most frequently observed ST was ST11, accounting for 12.3% (7/57) of the isolates. Furthermore, ST15, and ST655 were each detected in 10.5% (6/57) of the isolates, whereas ST485 was detected in 8.8% (5/57) of the samples. Analysis of these STs using the BIGSdb database (https://bigsdb.pasteur.fr/) revealed that all the STs, except for ST265, were originally isolated from human hosts. The source of ST265 remains unknown. ST11, ST15, and ST45, particularly ST11 and ST15, are predominant carbapenem-resistant clones in Asia. These two STs are responsible for up to 60% of carbapenem-resistant K. pneumoniae (CRKP) cases in China [45, 46]. Currently, CRKP accounts for a substantial proportion of clinical carbapenem-resistant Enterobacterales (CRE) infections in Europe, China, and the United States, ranging from 60% to 90% [47–49]. In this study, four isolates were identified to be completely resistant to imipenem, a carbapenem antimicrobial, whereas three isolates exhibited intermediate resistance. The resistant isolates were identified as isolate numbers 2899 (ST15), 2900 (ST11), 2937 (ST655), and 2956 (unknown ST). The strains exhibiting intermediate resistance were isolate numbers 2544 (ST11), 2561 (ST11), and 2755 (ST15). These findings are consistent with those of previous surveys, indicating that the majority of K. pneumoniae isolates with varying levels of carbapenem resistance are predominantly associated with ST11 and ST15 clones. Carbapenem resistance poses a considerable challenge in the field of infectious disease treatment because carbapenems are typically considered as the final line of defense against severe bacterial infections. Thus, caution should be exercised when CRKP is observed in companion animals. Additionally, a crucial K. pneumoniae clone identified in this study is ST147, which accounted for 3.5% (2/57) of the isolates. ST147 is distributed globally and has been reported on all continents, except for Antarctica [50]. Moreover, this clone has been associated with the leading cause of nosocomial outbreaks worldwide [51]. Previous research has demonstrated the widespread presence of ST15 K. pneumoniae in companion animals, whereas ST11 is recognized as a high-risk clonal lineage prevalent in human nosocomial infections [52]. However, ST15 was also detected in human hospital and community populations in Portugal [53, 54]. Similarly, ST11 was detected in companion animals in Taiwan, Japan, and Switzerland, with ST11 displaying the highest prevalence among clinical K. pneumoniae isolates in Taiwan [23, 43, 55, 56]. These findings provide evidence of shared STs that can occur in both humans and animals, indicating potential epidemiological connections between companion animals and humans due to their close proximity. However, in Italy and France, ST101 and ST274 were predominant in companion animals, suggesting that the predominant ST in ESBL-producing K. pneumoniae varies by country [57, 58].
The antimicrobial susceptibility test revealed multidrug-resistant property of ESBL/pAmpC-producing K. pneumoniae. Of the 15 antimicrobials tested in this study, more than 50% of K. pneumoniae isolates were resistant to 13 of them, with the exception of cefotaxime and imipenem. K. pneumoniae strains have been documented to exhibit widespread resistance to aminoglycosides, fluoroquinolones, cephalosporins and carbapenems [59]. Many mobile AMR genes were found in this microorganism before they spread to other pathogens. K. pneumoniae is therefore considered an important amplifier and propagator of clinically important AMR genes [60].
In the conjugation assay, some strains grew on MH agar supplemented with sodium azide, which prevents these strains from serving as donor strains. This phenomenon occurs due to the inability to differentiate such donor bacteria from the successful transconjugant, which should grow on MH agar supplemented with cefotaxime and sodium azide. Sodium azide inhibits bacterial growth by inhibiting secA [61]. Some K. pneumoniae strains may harbor mutations in secA or other genes that confer resistance to sodium azide. The successful rate of conjugation in this study was 43.9%, which is lower than that reported by Xiang et al. [62]. A meta-analysis indicated that decreasing taxonomic relatedness between donor and recipient bacteria is associated with lower conjugation frequencies in liquid mating [63]. The frequency of successful conjugations below 50% observed in this study can be explained by the use of the liquid mating method and the donor and recipient cells belonging to different genera (K. pneumonia vs E. coli). In addition, temperature and plasmid incompatibility contribute to conjugation frequency [63].
The wabG and entB genes were identified as the most frequently detected virulence genes. Moreover, mrkD, uge, and fimH genes were highly prevalent in the isolates. The abundance of the entB gene in K. pneumoniae isolates was expected because siderophores play a crucial role in the uptake of iron, contributing to bacterial survival and enterobactin production in all K. pneumoniae strains. A study conducted in Spain demonstrated the prevalence of the uge gene in the majority of urine isolates of K. pneumoniae. However, the strains of K. pneumoniae lacking the uge gene exhibited a lower likelihood of causing diseases [36, 64–66].
Hypervirulent K. pneumoniae (HvKP) is a highly virulent strain that can infect even healthy hosts. The prevalence of hvKP isolates in Taiwan was 27.5% between 2017 and 2019, with an annual incidence of K1 serotype bacteria ranging from 11%–15% across the country [67]. The HvKP strain can be identified using a phenotypic hypermucoviscosity string test. However, studies have identified specific virulence genes that may be associated with this variant of K. pneumoniae. Virulence genes, such as iutA and rmpA, have been identified as potential markers for the hvKP variant [68, 69]. Limited knowledge exists regarding the prevalence of hvKP in companion animals, with few studies conducted in China and one study in Guadeloupe. In China, hvKP was more common in cats (70%, 14/20) than dogs (58.8%, 50/85). By contrast, only 1 of 4 (25%) dog isolates in the Guadeloupe study was identified as a hypervirulent strain [70–72]. The presence of hvKP in our study underscores the potential role of companion animals as reservoirs in the spread of such bacteria.
Conclusion
This study sheds light on the prevalence and genetic characteristics of K. pneumoniae isolates derived from companion animals with urinary tract infections in Taiwan. The high occurrence of ESBL and pAmpC genes among the isolates underscores the urgent need for vigilance in monitoring antimicrobial resistance in veterinary settings. The distribution of prevalent sequence types (ST11, ST15, and ST655) suggests the circulation of specific K. pneumoniae strains across different hosts, emphasizing the interconnectedness of animal and human health. Further research is warranted to elucidate the dynamics of K. pneumoniae transmission and resistance dissemination within the One Health framework.
Supporting information
S1 Table. The allele number and sequence types (STs) of ESBL and/or pAmpC-producing K. pneumoniae analyzed by multilocus sequence typing (MLST).
https://doi.org/10.1371/journal.pone.0296709.s001
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S2 Table. Presence of the virulence genes of ESBL and/or pAmpC-producing K. pneumoniae.
https://doi.org/10.1371/journal.pone.0296709.s002
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S3 Table. Antimicrobial susceptibility testing of ESBL and/or pAmpC-producing K. pneumoniae.
https://doi.org/10.1371/journal.pone.0296709.s003
(DOCX)
Acknowledgments
The authors extend their gratitude to Dr. Lee-Jene Teng from the Department of Clinical Laboratory Sciences and Medical Biotechnology, National Taiwan University, for generously providing the Klebsiella pneumoniae ATCC 700603 strain. Additionally, heartfelt thanks to Dr. Chao-Tsai Liao from the Department of Medical Laboratory Science and Biotechnology, Central Taiwan University of Science and Technology, for kindly provision of the Escherichia coli J53 strain.
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