Human pandemic K27-ST392 CTX-M-15 extended-spectrum β-lactamase-positive Klebsiella pneumoniae: A one health clone threatening companion animals

Extended spectrum β-lactamase (ESBL)-producing Klebsiella pneumoniae is a medically important pathogen that commonly causes human nosocomial infections. Since veterinary emergency and critical care services have also significantly progressed over the last decades, there are increasing reports of ESBL-producing K. pneumoniae causing hospital-associated infections in companion animals. We present microbiological and genomic analysis of a multidrug-resistant ESBL-positive K. pneumoniae (LCKp01) isolated from a fatal infection in a dog admitted to a veterinary intensive care unit. LCKp01 strain belonged to the sequence type ST392 and displays a KL27 (wzi-187) and O-locus 4 (O4). A broad resistome and presence of the blaCTX-M-15 ESBL gene were predicted. SNP-based phylogenomic analysis, using an international genome database, clustered LCKp01 (60–80 SNPs differences) with K. pneumoniae ST392 from human and animal infections, isolated at 4-year interval, whereas phylogeographical analysis confirmed successful expansion of ST392 as a global clone of One Health concern.


Introduction
Klebsiella pneumoniae is a clinically relevant pathogen frequently associated with antimicrobial resistance, being considered an important cause of community-acquired and nosocomial infections in humans [1]. In this regard, after the global spread of extended spectrum β-lactamase (ESBL)-producing K. pneumoniae causing human nosocomial infections, these bacteria are currently emerging as worrisome causes of infections in pets worldwide [2,3]. Due to their clinical and epidemiological importance, the World Health Organization (WHO) has recently classified ESBL-producing K. pneumoniae isolates as critical priority pathogens [4]. In view of the wide diversity of ESBL-positive K. pneumoniae being recovered from humans, animals, and the environment, the One Health approach has been encouraged for a better understanding of the clonal spread of these strains [5].
Analogously to human medicine, small animal patients hospitalized in intensive care units (ICUs) are mostly affected by life-threatening infections, including those caused by multidrug-resistant (MDR) pathogens [6]. Indeed, since veterinary emergency and critical care services have progressed meaningfully in recent times, increasing reports of ESBL-producing pathogens causing hospital-associated infections in companion animals could be expected [6][7][8]. Therefore, the use of invasive devices, antimicrobial prescribing practices, and increased hospital stay could be predisposing factors for the acquisition of ESBLproducing K. pneumoniae infections by pets [6,7], which poses a substantial challenge for veterinary clinicians, being also a One Health issue.

Materials and methods
In 2018, a 2-year-old mixed breed female dog was admitted to a veterinary ICU after gastrointestinal surgery procedures. Veterinary medical records revealed that the dog was subjected to two laparotomies ten days before ICU admission. In the first procedure, gastrostomy and enterostomy were performed for foreign body removal. During the postoperative period, the dog developed surgical wound dehiscence and a significant amount of intra-abdominal free fluid was observed during ultrasonography. On account of these complications, a second laparotomy procedure for enterectomy and anastomosis was performed. Following 3 days of the second procedure, the dog was referred to the ICU due to significant hemodynamic instability, abdominal pain, peritoneal fluid escaping from the abdominal cavity, decreased consciousness, and inappetence. A peritoneal fluid sample was collected and subjected to bacteriological culture and antimicrobial susceptibility testing. Antibiotic therapy with enrofloxacin and metronidazole was started, and support treatment was immediately performed, however, the animal died 36 h following ICU admission.

Results and discussion
The genome size of LCKp01 was calculated at 5,782,901 bp, comprising 5701 total genes, 3rRNAs and80 tRNAs and 10 non-coding RNA, with 322× coverage. Multilocus sequence typing analysis showed that K. pneumoniae LCKp01 strain was assigned to ST392. Interestingly, this K. pneumoniae lineage has been observed so far in humans in Africa, Asia, Europe, Oceania, and in the Americas (Fig. 1); being frequently recovered from nosocomial infections with the production of CTX-M-type ESBLs and/or carbapenemases [10,11]. To the best of our knowledge, there is a single K. pneumoniae ST392 strain isolated from animals, and it was recovered from a cat suffering from urinary tract infection in China, in 2018.
There are few reports of K. pneumoniae ST392 in the literature; however, some studies suggest the emergence of ST392 isolates with the potential to become a lineage of clinical relevance [11,12]. A recent investigation focused on phenotypical and molecular assessment of the virulence potential of K. pneumoniae ST392 clinical isolates revealing that most of the strains were highly resistant to human sera and were also strong biofilm producers, showing strong levels of adhesion to the HT-29 epithelial intestinal cell line [11]. These phenotypic behaviors were associated to the presence of genes involved in serum resistance (aroE and traT) and adhesion (pgaA) [11]. Although, in silico analysis of K. pneumoniae LCKp01 revealed the presence of aroE, traT, and pgaA genes, the existence of these genes alone is not sufficient to infer that LCKp01 is a strong biofilm producer or highly resistant to serum. Therefore, in vitro serum resistance and biofilm assays must be investigated, in order to elucidate whether these genes could contribute with persistence of LCKp01 in medical devices and healthcare-associated infections in human and veterinary medicine [13][14][15].
While plasmid replicons IncHI1B, IncFIB, and IncFII were identified in the K. pneumoniae LCKp01 strain, plasmidome analysis showed that Col-like and IncF-type replicons have been the most frequent plasmids carried by K. pneumoniae strains belonging to ST392, followed by IncHI1, IncL, IncC, IncN, IncR, IncI1, IncX, IncHI2, IncM2, and IncQ1 replicons ( Fig. 2A and [16], Tanzania [17], and South Africa [18]. In summary, we report genomic data of K. pneumoniae ST392 harboring bla CTX-M-15 and other clinically important antimicrobial resistance genes, isolated from an infected companion animal in South America. Currently, WGS-based studies on ESBL-positive K. pneumoniae from companion animals remain scarce [19]. Additionally, phylogenomic information on human-associated clones of K. pneumoniae recovered from dogs and cats is also poorly investigated [19]. Considering that K. pneumoniae ST392 has been so far predominantly reported in human patients, our findings suggest that this K. pneumoniae clone Fig. 1. Worldwide distribution and sources of Klebsiella pneumoniae of ST392 circulating at the One Health interface. Data were retrieved from PubMed database via the National Center for Biotechnology Information (NCBI) interface and NCBI RefSeq database (http://www.ncbi.nlm.nih.gov/RefSeq/) (accessed on April 27, 2022). Duplicates (i.e., the same K. pneumoniae ST392 strain in both databases) were excluded. may spread beyond human hospital settings and affecting now hospitalized pets. Last but not least, our data could be helpful for comparative genomic analyses of K. pneumoniae ST392 strains that could emerge at the human-animal-environment interface, since our early phylogeographical analysis confirmed successful expansion of ST392 as a global clone of One Health concern.

Data availability
The data that support the findings of this study are available from the corresponding author upon reasonable request. This Whole Genome Shotgun project has been deposited at DDBJ/ENA/GenBank under the accession JAEDYP000000000. The version described in this paper is version one. In addition, genomic information of K. pneumoniae LCKp01 strain is available on the OneBR platform under the number ID ONE207 (http://onehealthbr.com/).

Ethics statement
The authors confirm that the ethical policies of the journal, as noted on the journal's author guidelines page, have been adhered to. No ethical approval was required for this specific study.

Declaration of Competing Interest
All authors declare no conflicts of interest.
Brazil) and CEFAP-GENIAL facility for kindly supplying antibiotic discs for susceptibility testing and Illumina sequencing, respectively.