Pandemic Clones of CTX-M-15 Producing Klebsiella pneumoniae ST15, ST147, and ST307 in Companion Parrots

Psittacine birds are commonly kept as companion birds and the maintenance of these birds in captivity may represent a zoonotic risk and contribute to the propagation of multidrug-resistant and β-lactamase extended-spectrum (ESBLs)-producing pathogens. This study aimed to identify and characterize strains of the Klebsiella pneumoniae complex isolated from diseased psittacine birds, determining virulence and resistance profiles. K. pneumoniae strains were isolated from 16 birds (16/46). All strains carried more than three virulence genes, with a high frequency of fimH and kpn (93.75%), uge (87.52%), and irp-2 (81.25%) genes. The antimicrobial susceptibility revealed that 3/16 strains were ESBL producers. Genomic analysis revealed that CTX-M-15-positive strains belonged to sequence types (STs) ST15, ST147, and ST307, characterized as international clones associated with outbreaks of healthcare-associated infections (HAIs) worldwide.


Introduction
Members of the Klebsiella pneumoniae complex are adapted to survive in environments and have a broad ecological range, including wastewater, human, and domestic animals. The greatest concern about K. pneumoniae is associated with the potential to amplify and spread clinically important antimicrobial resistance genes (AMR) [1]. K. pneumoniae is highlighted as an opportunistic pathogen that causes urinary tract infection, pneumonia, wound infections, hemorrhagic colitis, and sepsis [2]. Symptomatic and asymptomatic individuals can function as a reservoir of an agent, after colonization of the nasopharyngeal and gastrointestinal tract, and represent a risk of transmission of multidrug-resistant strains [1]. Specifically, some international clones of Klebsiella pneumoniae have become a global public health issue due to virulence and/or multidrug-resistant profiles [1]. In this regard, the most common resistant lineages of K. pneumoniae have been associated with the production of extended-spectrum beta-lactamase (most CTX-M-15 variants) and/or KPC-type carbapenemases, being categorized as critical priority pathogens by WHO (2020) [3]. On the other hand, although clones belonging to clonal group CC58 have been predominant in healthcare-associated infections (HAIs) worldwide, novel STs have emerged as high-risk clones, such as ST15, ST147, and ST307, which are associated with high human mortality [4][5][6].
Since most surveillance studies focus on the impact of human nosocomial infections, the pandemic clones of K. pneumoniae have been reported in companion animals, such as dogs, and cats [1,[7][8][9]. Chen et al. (2021) evaluated the dynamics of transmission of K. pneumoniae between dogs and their owners. The authors showed a clonal diverse population of K. pneumoniae in the gastrointestinal tracts of healthy humans, but some dominant clones were present in pets that shared the same house, including mammals and reptile pets [10].
The role of the microbial community of wild birds on the trafficking of AMR genes in one health approach remains unknown [7][8][9]. Free-living psittacine birds are colonized by Gram-positive bacteria, but they acquire the Gram-negative microbiota during captivity in conservation and rescue centers. There they can spread resistant pathogens from stool. Silva et al. (2021) reported the presence of KPC-producing E. coli in Psittaciformes rescued from trafficking in Brazil. The authors highlighted the risks associated with reintroduction or domestic breeding of these birds [11]. In a previous study, we reported the importance of virulent strains of K. pneumoniae isolated from parrots and passerines seized from illegal wildlife trade, which carried plasmid-mediated quinolone resistance, SHV-1, SHV-11, and blaTEM-1 genes [9].
In this study, we report the occurrence and characterization of international clones of CTX-M-15-positive K. pneumoniae causing respiratory diseases in companion parrots, highlighting the zoonotic and anthropozoonotic potential of this critical priority pathogen, within a One Health perspective.
The swabs were incubated in brain heart infusion broth (Difco™) at 37 • C for 18 h and cultured on MacConkey agar (Difco™) at 37 • C for 24 h. Selected colonies were subjected to species identification by MALDI-TOF MS (Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry) [12].

Plasmid Analysis
ESBL-producing isolates were selected to demonstrate the transferability of the plasmids. Conjugation was conducted using Escherichia coli C600 as a receptor strain. Donor and receptor strains (2:1) were inoculated in 5 mL of Luria Bertani broth (Difco™). Transconjugants were selected after 24 h of incubation at 35 • C, 100 µL of the inoculum was plated in MacConkey agar (Difco™) supplemented with 2000 mg/L of streptomycin and 2 mg/L of cefotaxime. For the isolate carrying both bla CTX-M-15 and bla CTX-M-8 , ceftazidime 4 mg/mL was used instead of cefotaxime, to select colonies with only the plasmid carrying bla CTX-M-15 [16]. The plasmid sizes were determined by S1-nuclease PFGE and the plasmids were typed by PCR-based replicon typing and replicon sequence typing of IncF plasmids [17].

Whole Genome Sequencing and Analysis of ESBL-Producing K. pneumoniae Strains
All strains exhibiting ESBL phenotype were selected for whole-genome sequencing. Genomic DNA was extracted and purified from overnight pure cultures using the PureLink Genomic DNA purification Kit (Invitrogen) following the manufacturer's recommendations. Nextera XT DNA Library kit (Illumina ® ) was used to generate paired-end libraries (2 × 150 bp) according to the manufacturer's instructions, followed by sequencing in the Illumina NextSeq platform (Illumina, San Diego, CA, USA).
Three strains were ESBL-producers and exhibited a multidrug-resistant (MDR) profile, of which two were positive for the bla CTX-M-15 gene and one was positive for both bla CTX-M- 15 and bla CTX-M-8 genes. These strains were selected for whole-genome sequencing. The genomes ranged between 5,622,065 and 5,540,827 bp, which is according to the genome size of K. pneumoniae (mgh). Genomic features of these strains are shown in Table 1. In these isolates, it was possible to transfer resistance to third-generation cephalosporins to a transconjugant strain through conjugation assays (Table 2).
MDR phenotype in CTX-M-15-producing K. pneumoniae strains was related to genes conferring resistance to phenicols, beta-lactams, aminoglycosides, fosfomycin, quinolones, sulphonamides, sulfamethoxazole-trimethoprim, and tetracyclines. Additionally, chromosomal mutations in the gyrA and parC genes were responsible for elevated levels of resistance to fluoroquinolones (Table 1).
Microorganisms 2022, 10, x FOR PEER REVIEW 4 of 10 discriminatory index of 0.96. Studied strains were from noncontacting birds, located in various places, and were heterogeneous (Figure 1).  Table  1. In these isolates, it was possible to transfer resistance to third-generation cephalosporins to a transconjugant strain through conjugation assays (Table 2).
MDR phenotype in CTX-M-15-producing K. pneumoniae strains was related to genes conferring resistance to phenicols, beta-lactams, aminoglycosides, fosfomycin, quinolones, sulphonamides, sulfamethoxazole-trimethoprim, and tetracyclines. Additionally, chromosomal mutations in the gyrA and parC genes were responsible for elevated levels of resistance to fluoroquinolones (Table 1).     In silico analysis showed that these strains belonged to ST15, ST147, and ST307. ST15 isolate (Kp58.3) was isolated from the oropharynx of a Psittacara leucophtalmus specimen. This isolate presents the bla CTX-M-15 gene in a~250 Kb plasmid belonging to K5:A10: B-replicon sequence type (RST) (Tables 1 and 2). Through BLAST alignments, we found this isolates the presence of the kpi operon. This chaperone-usher pili system is related to the ability to produce remarkable adherent phenotypes with abundant fimbriae structures. Our ST15 isolate has a 6628 bp DNA fragment in the genome that encodes seven genes (kpiA, kpiB, kpiC, kpiD, kpiE, kpiF, kpiG) and showed 100% similarity to the kpi operon of the Kp3380 strain, an MDR ST15 strain K. pneumoniae previously isolated from a hospital outbreak in Spain [20]. The strain belonging to ST307 (Kp137) was isolated from a respiratory secretion of the Amazona aestiva parrot that died of sepsis. This strain harbors three plasmids, with bla CTX-M-15 located in an IncFIB(K) plasmid sizing~145 Kb. On the other hand, the isolate Kp41 was recovered from a nasal secretion of an Amazona aestiva parrot and belonged to ST147. Kp41 strain possesses an~220 kb K9:A-:B-plasmid carrying bla CTX-M-15 , and other plasmids belonging to the IncM1 family, harboring bla CTX-M-8 ( Table 1). The IncM1 plasmid was analyzed in a previous study [21]. The bla CTX-M-8 gene was inserted in an IS26-composite transposon, composed of an IS10 copy and two IS26 copies present in that IncM1 plasmid.
In all three strains, the bla CTX-M-15 gene was found upstream ISEcp1 element, with ORF477 downstream. This module presented intact repeat regions (IRs) of ISEcp1, a classic genetic environment of bla CTX-M-15 . In the isolates belonging to ST15 and ST307, this structure was downstream to a resistance region carrying resistance genes to aminoglycosides (aph(3")-Ib, aph(6)-Id), beta-lactams (bla TEM-1 ), and sulphonamides (sul2) (Figure 2). These CTX-M-15-producing K. pneumoniae strains showed an MDR phenotype. Using in silico analysis it was possible to correlate with many acquired genes conferring resistance to antimicrobial drugs (Table 1). In addition to the acquired resistance genes, it was possible to determine chromosomal mutations in the gyrA and parC genes, which These CTX-M-15-producing K. pneumoniae strains showed an MDR phenotype. Using in silico analysis it was possible to correlate with many acquired genes conferring resistance to antimicrobial drugs (Table 1). In addition to the acquired resistance genes, it was possible to determine chromosomal mutations in the gyrA and parC genes, which confer elevated levels of resistance to fluoroquinolones.

Discussion
Convergence of virulence and multidrug resistance in Klebsiella pneumoniae is considered a major public health concern [22,23]. This species is one of the most common MDR bacteria isolated in nosocomial and community-acquired infections in humans [24]. The reports about infection by K. pneumoniae in veterinary medicine have been associated with cats and dogs [7,[25][26][27][28], and little is known about the impact of this pathogen in companion and wildlife bird species. In a previous study, colonization of passerine and psittacine seized from illegal trade in Brazil, by K. pneumoniae, was reported [9]. In this study, the occurrence of K. pneumoniae among psittacine birds was 34.78%, and most of these strains presented virulence factors, including genes associated with fimbriae (fimH-93.75%, fim1like kpn-93.75% and type 3 fimbriae mrkD-62.50%) and siderophores (irp-2-81.25% and kfu-43.75%). According to Lawlor et al. (2007), yersiniabactin is the most important iron acquisition system of K. pneumoniae during in vivo pulmonary infection [29]. Holt et al. (2015) suggested that the acquisition of iron-scavenging systems increases the risk of severe invasive disease and highlighted the public health concern of the yersiniabactin in the ESBL clones, including ST15 K. pneumoniae [22].
In this study, none of the strains presented genes magA and rpmA, associated with hypermucoviscosity. However, 87.20% were positive for the uridine diphosphate galacturonate 4-epimerase gene (uge), which is associated with the integrity of smooth LPS [30].
Among the multidrug-resistant lineages, some clonal groups (CGs) such as CG258, CG307, CG101, CG147, and CG15 are globally disseminated and responsible for an extensive range of infections in humans [2,31]. In this study, three of these STs were found to cause respiratory disease in parrots. In this regard, ST307 has been a pandemic lineage related to CTX-M-15-production, emerging in the middle of the 1990 decade [1]. Genomic studies reveal that ST307 presents a conserved genome, differing only in the mobilome (i.e., plasmids, phages, and resistance genes) [32]. CG307 has some characteristics that favor the colonization of humans and the hospital environment, such as virulence factors and plasmids. Recently, CTX-M-15-positive ST307 strains were implicated in nosocomial outbreaks in Germany and The Netherlands [33,34]. In Brazil, ST307 strains were recovered from wastewater and a urinary tract infection in cats and dogs [7,35,36].
In addition, our study also detected the lineages ST147 and ST15 in psittacine isolates. These lineages of K. pneumoniae producing CTX-M-15 have been described in companion animals, including dogs with urinary tract infections and horses [37,38]. Our results reinforce the link of transmission between humans and pets, including companion birds.
The three ESBL-producing strains here studied belong to the CTX-M-15 variant. CTX-M-15 is the most common ESBL enzyme in Klebsiella pneumoniae isolated from human nosocomial infections worldwide. Through a wide literature review, Calbo and Garau (2015) showed that the epidemiology of ESBL-producing K. pneumoniae changed in the 2000s, and CTX-M-15 has completely replaced other ESBL enzymes, including TEM, SHV, and other CTX-M variants worldwide [39].
One of the reasons for the successful dissemination of CTX-M-15 in hospitals may be the fact that it is an enzyme that has an increased catalytic activity against ceftazidime, a third-generation cephalosporin produced for the treatment of Plasmids have a key role in the dissemination of CTX-M genes enzymes. IncF and IncN plasmids are associated with the worldwide dissemination of bla CTX-M-15 [40]. In our study, the gene was present in IncF (K) plasmids. The three strains analyzed presented bla CTX-M-15 gene upstream ISEcp1 element, with ORF477 downstream, which is the worldwide reported structure [41,42]. These sequences show 100% similarity to the IncF (K) plasmids described in K. pneumoniae isolates in Colombia, the United States, and Australia (GenBank access CP024566, CP024516, CP016925, and CP10390). Previous studies in Brazil have identified the bla CTX-M-15 gene in plasmids of the IncR family, in K. pneumoniae isolated from foodproducing animals [43].
In our work, the CTX-M-8 variant was inserted in an IS26-composite transposon, composed of an IS10 copy and two IS26 copies. An identical transposon has been identified in plasmids of the Incl1 and IncM1 families in environmental isolates in Brazil [35], and in chicken meat imported from Brazil [44]. In addition to bla CTX-M-8 , pKp41 M plasmid contained a resistance region composed of Tn1331, truncated by a 4,108 pb module containing ISEcp-qnrE1-araJ-∆ahp. The qnrE1 gene, which confers resistance to quinolones, was described in a human clinical strain of K. pneumoniae in Argentina [45]. However, the genetic environmental described by Albornoz [45] was not associated with Tn1331, as in the pKp41 M plasmid.

Conclusions
In conclusion, our results showed that international clones of CTX-M-15-positive K. pneumoniae can cause infectious diseases in companion birds, highlighting the zoonotic and anthropozoonotic potential of this critical priority pathogen, within a One Health perspective.