In Silico Analysis and PCR Characterization of non-Tn 4401 Transposable Elements in Pseudomonas aeruginosa †

: The multiresistance presented by in P. aeruginosa has greatly increased due to the presence of genes for carbapenemases such as bla KPC . The dissemination of this gene has been associated with the Tn 4401 , the main mobile genetic element that carries bla KPC in its structure. However, some non-Tn 4401 elements (NTE KPC ) associated with bla KPC have been found in different bacteria. Here we characterized in silico and in vitro bla KPC -associated elements in P. aeruginosa . To identify these elemgeents, a search algorithm was performed using NCBI databases, sequences were filtered, and pair-aligned to describe the bla KPC genetic environment. Also, a PCR-based strategy was designed to target Tn 4401 variants and NTE KPC groups and assessed in 61 Colombian clinical isolates. By the in-silico approach it was found 51 bla KPC -positive entries longer than 3kb (in the bla KPC upstream region), from these, 72.7% carried an NTE KPC . On the PCR assay, Tn 4401 was the most frequent element among the P. aeruginosa in Colombia, however NTE KPC -IIf was presented on 29.5% of the isolates, in different genetic lineages and at least in four hospitals. These results show high NTE KPC prevalence in P. aeruginosa .


Introduction
Circulation of bacteria carrying beta lactamases genes is increasing, since these genes are continuously transmitted by horizontal transfer, which has caused the emergence of new classes of bacteria multi resistant that have become a public health problem around the world [1]. The carbapenemase encoding blaKPC gene was initially reported in K. pneumoniae [2]. However, this gene has been transmitted to other enterobacteria, such as Escherichia coli or Salmonella enterica and more recently (in 2007) to Pseudomonas aeruginosa, the first record in non-enterobacteria organisms [3][4][5].
The main mobile genetic element (MGE) associated with blaKPC dissemination toward new genetic structures is Tn4401, a transposon commonly associated with high-risk plasmids and clones that facilitate propagation of this gene, like ST258 in K. pneumoniae [6][7][8][9][10]. Nonetheless, in recent years different elements unrelated to Tn4401 surrounding blaKPC have been identified. These elements are known as NTEKPC (Non-Tn4401 elements) [7], and may play a relevant role in the spread of blaKPC [7,11]. Based on the blaKPC upstream structure NTEKPC can be classified in at least three subgroups (I, II and III) [7]. In P. aeruginosa, the information is mostly focused on anti-biotic resistance, so the relevance of these new elements for blaKPC dissemination has not been deeply studied, yet this gene is showing a rapid expansion around the world in this species [12].
Therefore, the aim of this work was to characterize in silico, the transposable elements associated with blaKPC in P. aeruginosa according to reports presented in the GenBank, to contribute with information that allows elucidating the dissemination mechanisms of this resistance determinant. Subsequently, a method was designed for the identification by PCR of the Tn4401 variants and NTEKPC groups and used to determine the elements and frequency in a cohort of Colombian clinical isolates.

Methods
This research was divided into two phases; an in-silico phase, which aims to analyze and characterize the genetic environment of blaKPC positive isolates in P. aeruginosa that have been reported in the GenBank. And an in vitro phase, which consisted of the experimental analysis of a cohort of blaKPC positive isolates of P. aeruginosa, to characterize the region that flanks upstream the blaKPC gene, to establish the frequency of circulation of NTEKPC elements in this species in Bogotá, Colombia.

In Silico Phase
2.1.1. Exploration of the blaKPC Genetic Environment for P. aeruginosa in the GenBank Initially, a database was created for compiling information of the blaKPC genetic environments in P. aeruginosa collected in the GenBank (reviewed until 13 October 2021). All partial or fully sequenced nucleotide entries with more than 3000-bp upstream blaKPC were included. General information of the entries such as country, length, replicon type (linear or circular), blaKPC variant, position in the genome, isolate's name, and access information (GenBank and PMCID access numbers) were also registered. Nucleotide sequence for all entries was exported and compared against reference sequences of the Tn4401 and its variants (a-i) and NTEKPC and its different subgroups (I, II and III), whose classification criteria are based on the region upstream of blaKPC.
In case of no association with previously reported genetic environments, the entry was characterized by manual curation using Artemis Comparison Tool (ACT), BLASTn and BLASTp [13,14]; and specialized databases for mobile genetic elements (TnRegistry and ISFinder) and resistance genes (CARD) [15,16]. Paired alignments were developed and plotted using Easyfig [18], showing identity between pairs in a window of 300-bp.

Primer Design
Using default parameters in the NCBI Primer BLAST platform [14], several primer sets were designed, which aimed to amplify blaKPC upstream regions, and differentiate by amplicon size the Tn4401 subtypes or NTEKPC subgroups. Briefly, to amplify the different upstream regions reverse primer must align with the blaKPC gene, and forward primers were designed to align with specific regions (for each group) that were absent on the other possible MGEs. For the recognition of the Tn4401 the forward primer was designed to align with the istB gene, which it has not been reported on the NTEs. However, for the NTEKPC subgroups, the primers were design to align with the ISKpn27 (initially misreported like ISKpn8) (NTEKPC-I), the resistance gene blaTEM (NTEKPC-II) and with the Tn5563 resolvase, genes that are unique for each subgroup.
For the PCR assays bacterial isolates were cultured in 3 mL of LB broth and later, total DNA was extracted by phenol-chloroform method [18] and purified with 70% Ethanol. Finally, the DNA was resuspended in 50 µ L of molecular biology grade water. Using the designed oligonucleotides, PCRs were performed to identify and classify blaKPC upstream surroundings as NTEKPC (either I, II, or III) or as a Tn4401 variant. PCR products were evaluated by agarose electrophoresis (1% agarose in 1× TBE buffer) and stained with Ethidium Bromide (0.01 µ g/mL). With the results, the frequency of circulation of these genetic structures in the Colombian P. aeruginosa was reported.

In Silico Analysis of the Collected Reports
As result of the in-silico approach, 60 blaKPC positive P. aeruginosa sequences longer than 3-Kb in the upstream flanking region, were retrieved, of which 73.3% (n = 44) carried blaKPC in an NTEKPC environment, and the remaining 26.6% (n = 16) in a Tn4401 transposon. This is a remarkable result, since around the world, Tn4401 has been reported as the main element associated with the dissemination of blaKPC [19]. These results suggest the dynamics of dissemination of this resistance gene in P. aeruginosa present a different behavior to that observed in Klebsiella pneumoniae. Also, all the sequences were associated with the blaKPC-2 isoform, which historically is the predominant variant in the world [8]. In P. aeruginosa, NTEKPC elements were identified in three countries, in South America and Asia, whilst Tn4401 was identified in North America, South America and Asia (Table 1).  Group I was the most frequent subgroup (77.7%, n = 32) among the NTEKPC, which is characterized by having an ISKpn27 upstream blaKPC. However, majority of these reports (93.75%) come from China suggesting NTEKPC-I is locally disseminated in this country. Unlike the rest of the world, China has reported that its main KPC disseminator, not only for P. aeruginosa but for many different bacteria, is a chimera of Tn3-Tn4401 that presents an ISKpn27 (misreported as ISKpn8) upstream of the blaKPC gene, so this genetic environment is also consider as a NTEKPC, most probably of group I [20]. Interestingly, the only subgroup of NTEKPC that was presented on the chromosome of a P. aeruginosa (n = 5) was the NTEKPC-I, which may indicate the vertical transmission of blaKPC through this type of element.
One entry presented upstream blaKPC a Tn3 resolvase and an unknown resolvase, that did not match to the current nomenclature [7], so could not be classified as any of the stablished groups. In other case, the upstream region showed an IS26 insertion sequence which is not related with any stablished group. Lastly, the presence of the ISKpn27 (of NTEKPC-I), and IS6100 (of NTEKPC-III), prevented group discrimination in another entry. Remaining seven entries (11.36%), belonged to NTEKPC-II, and were reported in China, Brazil and Colombia [7,21,22].
Sixteen entries reported sequence type for the P. aeruginosa, and from these, 6 (37.5%) were ST463 and all of them were presented on China, these isolates carried NTEKPC-I, ST1006 was reported by two isolates (12.5%) and three isolates (18.75%) reported ST235, the rest of the STs ( ST381, ST697, ST316, ST277 and ST308) were presented in just one isolate and in different countries, this also suggests dissemination of local clones. Sequence type 235, ST308 and ST1006 were found in Colombia; ST277 and ST381 in Brazil and ST463 and ST697 in China. The appearance of diverse genetic backgrounds associated with NTEKPC in P. aeruginosa suggests these type of elements may play a preponderant role to the blaKPC dissemination in this species. However, characteristics of these genetic environments must be studied to elucidate the role they play in the genetic mobility of this resistance gene.

Tn4401 and NTEKPC Primers Design
Two pairs of primers were designed to identify the Tn4401 and its variants. The first pair of oligonucleotides was designed to detect the blaKPC and istB genes (Figure 1), as they are part of a conserved region in the different Tn4401 isoforms and is absent in all NTEKPC reported structures. The size of the amplicons generated in this PCR depends on the Tn4401 variant, since some of the isoforms of this transposon have deletions in this region and are mostly distinguishable by the size of the deleted bases ( Figure 1). The size of the product generated by primers GN790/GN791 is specific for all variants except "e" and "i", which generate products of the same size, and "d" which do not predict to amplify. Primers GN795/GN796 do not predict to amplify for the variants "e" and "f". * NA = Not amplify. ± The number indicates the size of the deletion between istB and blaKPC. Variants b and f have no deletion.
Also, a PCR was designed to differentially amplify NTEKPC groups (I, II and III). For this, the reverse primer is located on the blaKPC gene as in the specific PCR for Tn4401. However, forward oligonucleotides target group-specific hallmarks, then, in combination with the conserved reverse primer targeting blaKPC, it is generated specific products for the NTEKPC-I, NTEKPC-II, and NTEKPC-III elements (Figure 2). For NTEKPC-III amplification, the primer was designed to target the tnpR present on Tn5563, unique to this element. For the differential amplification of NTEKPC-II, the primer targets the blaTEM resistance gene and for NTEKPC-I targets the tnpA of ISKpn27 (Figure 2). The ISKpn27 tnpA gene is found in both NTEKPC-I and II (Figure 2), so amplification with this oligonucleotide generates a product in both groups. However, the size of the amplicon and the presence of blaTEM allow differentiation between them.
This strategy is the first reported that allows to identify and differentiate groups of NTEKPC and eight of the nine Tn4401 variants (Tn4401g was not included). This method can be used for the rapid genetic screening of blaKPC harboring isolates, not only in P. aeruginosa, and in clinical settings or in research, to contribute to the surveillance of this resistance gene.

Figure 2.
Schematic representation of the location of primer GN790 in conjunction with GN792, GN793 and GN794 for the differential amplification of NTEKPC-III, NTEKPC-II and NTEKPC-I, respectively. Light blue bars between sequences indicate areas of identity. White arrows with colored outlines represent primers; these are below the target sequence.

Characterization of the Genetic Environment Associated with blaKPC in Colombian Clinical Isolates of P. aeruginosa
The different PCRs were standardized and implemented for the characterization of the region upstream of the blaKPC gene in 61clinical isolates, from five hospitals, in Bogota, Colombia. None of the isolates amplified for more than one PCR, suggesting that they did not have multiple copies of the blaKPC gene. In the analyzed population, two mobilization platforms associated with blaKPC were identified, the Tn4401 (n = 37, 60.7%) and the NTEKPC-II (n = 19, 31.1%), also five (8.2%) isolates did not amplify for any of the PCRs (Figure 3). Although the primers were designed to determine the NTEKPC group (either I, II or III), but not the specific variants among them (the product generated for the NTEKPC-II variants a, b, c and e is the same, 371 bp), it is possible to differentiate the NTEKPC-IIf, since this variant has an insertion of a TnpA gene, between blaTEM and blaKPC ( Figure 2). Here, of 19 NTEKPC-II positive isolates identified, 18 (94.7%) harbored NTEKPC-IIf elements, whilst the remaining isolate amplified the 371 bp product.
The main platform associated with blaKPC was the Tn4401 (60.7%). Although most of the isolates harboring this element came from one institution (86.8%, n = 33), suggesting a local spread. In four out of five institutions, there were NTEKPC-IIf positive isolates, and in three of these, was the most predominant element, with 70% (n = 7), 100 (n = 7) and 100% (n = 3), for institutions one, two and three, respectively. Interestingly, NTEKPC-IIf circulated among different unrelated PFGE pulsotypes (Figure 3).
Two representative isolates of the most frequent pulsotype (A), were sequenced with a long-reads strategy to obtain a complete assembly. Results confirmed the presence of the NTEKPC-IIf. Additionally, with the complete genome of these isolates, we performed an MLST analysis, which showed that both correspond to ST235, a globally dispersed clone [23], which has shown high capacity to acquire antibiotic resistance genes [24]. This clone has been previously described transporting blaKPC within the classic Tn4401 transposon [10,25]. However, to the best of our knowledge, this is the first report of the high-risk ST235 clone and fourth report of P. aeruginosa isolates carrying blaKPC embedded in these novel NTEKPC elements [2].

Conclusions
According to the information from the GenBank, the dissemination of the blaKPC resistance gene is mainly due to NTEKPC non-conventional elements. In Colombia, although the Tn4401 was abundant (mostly in one institution), a high frequency of NTEKPC-II elements could be evidenced, in four different institutions, and even thought this genetic environment had not been previously reported in our region, it seems to be endemic to these institutions. Additionally, we found a set of isolates that did not amplified for any of the designed PCRs, which indicates that these isolates do not present a Tn4401, but also suggests the presence of a new NTEKPC variant. However, additional studies are required to determine the characteristics of this region in these isolates.