In Silico Analysis and PCR Characterization of non-Tn4401 Transposable Elements in Pseudomonas aeruginosa ^†

Abstract

The multi-resistance presented by 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 Tn4401, the main mobile genetic element that carries bla_KPC in its structure. However, some non-Tn4401 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 elements, a search algorithm was performed using NCBI databases; sequences were filtered and pair-aligned to describe the bla_KPC genetic environment. Additionally, a PCR-based strategy was designed to target Tn4401 variants and NTE_KPC groups and assessed in 61 Colombian clinical isolates. Using an in silico approach, 51 bla_KPC-positive entries longer than 3kb (in the bla_KPC upstream region) were found; from these, 72.7% carried an NTE_KPC. On the PCR assay, Tn4401 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.

1. Introduction

The 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 multi-resistant bacteria that have become a public health problem around the world [1]. The carbapenemase-encoding bla_KPC 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 bla_KPC dissemination toward new genetic structures is Tn4401, a transposon commonly associated with high-risk plasmids and clones that facilitate the propagation of this gene, such as ST258 in K. pneumoniae [6,7,8,9,10]. Nonetheless, in recent years different elements unrelated to Tn4401 surrounding bla_KPC have been identified. These elements are known as NTE_KPC (non-Tn4401 elements) [7], and may play a relevant role in the spread of bla_KPC [7,11]. Based on the bla_KPC upstream structure, NTE_KPC 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 bla_KPC 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 bla_KPC in P. aeruginosa, according to reports presented in the GenBank, to contribute information that allows us to elucidate the dissemination mechanisms of this resistance determinant. Subsequently, a method was designed for the identification by PCR of the Tn4401 variants and NTE_KPC groups and used to determine the elements and frequency in a cohort of Colombian clinical isolates.

2. Methods

This research was divided into two phases: an in silico phase, which aims to analyze and characterize the genetic environment of bla_KPC-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 bla_KPC-positive isolates of P. aeruginosa, to characterize the region that flanks upstream the bla_KPC gene and establish the frequency of circulation of NTE_KPC elements in this species in Bogotá, Colombia.

2.1. In Silico Phase

Exploration of the bla_KPC Genetic Environment for P. aeruginosa in the GenBank

Initially, a database was created for compiling information of the bla_KPC 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 bla_KPC were included. General information of the entries such as country, length, replicon type (linear or circular), bla_KPC variant, position in the genome, isolate 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 NTE_KPC and its different subgroups (I, II and III), whose classification criteria are based on the region upstream of bla_KPC.

In the case of no association with previously reported genetic environments, the entry was characterized by manual curation using the 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 [17], showing identity between pairs in a window of 300 bp.

2.2. PCR Essay for Tn4401 or NTE_KPC Identification

Primer Design

Using default parameters in the NCBI Primer BLAST platform [14], several primer sets were designed, which aimed to amplify bla_KPC upstream regions and differentiate, by amplicon size, the Tn4401 subtypes or NTE_KPC subgroups. Briefly, to amplify the different upstream regions, the reverse primer must align with the bla_KPC 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 has not been reported on the NTEs. However, for the NTE_KPC subgroups, the primers were designed to align with the ISKpn27 (initially misreported, like ISKpn8) (NTE_KPC-I), the resistance gene bla_TEM (NTE_KPC-II) and the Tn5563 resolvase—genes that are unique for each subgroup.

For the PCR assays, bacterial isolates were cultured in 3 mL of LB broth;later, total DNA was extracted by phenol-chloroform method [18] and purified with 70% ethanol. Finally, the DNA was resuspended in 50 µL of molecular-biology-grade water. Using the designed oligonucleotides, PCRs were performed to identify and classify bla_KPC upstream surroundings as NTE_KPC (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 µg/mL). With the results, the frequency of circulation of these genetic structures in the Colombian P. aeruginosa was reported.

3. Results and Discussion

3.1. In Silico Analysis of the Collected Reports

Using an in silico approach, 60 bla_KPC-positive P. aeruginosa sequences longer than 3 Kb in the upstream flanking region were retrieved, of which, 73.3% (n = 44) carried bla_KPC in an NTE_KPC 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 bla_KPC [19]. These results suggest the dynamics of dissemination of this resistance gene in P. aeruginosa present different behavior to that observed in Klebsiella pneumoniae. Additionally, all the sequences were associated with the bla_KPC-2 isoform, which historically is the predominant variant in the world [8]. In P. aeruginosa, NTE_KPC elements were identified in three countries, in South America and Asia, whilst Tn4401 was identified in North America, South America and Asia (

Table 1. KPC distribution based on the collected reports to date (21 May 2022).

MGE	Genetic Landmark	Location	ST
NTEKPC-I n = 32	ISKpn27	China (n = 30)	Chromosome (n = 5)
			Plasmid (n = 25)
		Brazil (n = 2)	Plasmid (n = 2)
NTEKPC-II n = 9	blaTEM	China (n = 5)	Plasmid (n = 5)
		Brazil (n = 1)	Plasmid (n = 1)
		Colombia (n = 3)	Plasmid (n = 3)
NTEKPC-III n = 0	Tn5563 resolvase	Not reported	Not reported
NTEKPC n = 3	ND	China (n = 1)	Plasmid (n = 1)
		Brazil (n = 1)	Plasmid (n = 1)
		France (n = 1)	Plasmid (n = 1)
Tn4401 n = 16	ISKpn7	France (n = 2)	Plasmid (n = 2)
		Argentina (n = 2)	Plasmid (n = 2)
		Colombia (n = 2)	Chromosome (n = 1)
			Plasmid (n = 1)
		USA (n = 2)	Plasmid (n = 2)
		Brazil (n = 1)	Plasmid (n = 1)
		Chile (n = 2)	Plasmid (n = 2)
		China (n = 4)	Plasmid (n = 4)
		Nepal (n = 1)	Not reported

).

Group I was the most frequent subgroup (77.7%, n = 32) among the NTE_KPC, which is characterized by having an ISKpn27 upstream bla_KPC. However, majority of these reports (93.75%) come from China, suggesting NTE_KPC-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 bla_KPC gene; therefore, this genetic environment is also considered to be an NTE_KPC, most probably of group I [20]. Interestingly, the only subgroup of NTE_KPC that was presented on the chromosome of a P. aeruginosa (n = 5) was the NTE_KPC-I, which may indicate the vertical transmission of bla_KPC through this type of element.

One entry presented upstream bla_KPC, both a Tn3 resolvase and an unknown resolvase that did not match the current nomenclature [7], preventing it to be classified in any of the established groups. In another case, the upstream region showed an IS26 insertion sequence which is not related to any established group. Lastly, the presence of the ISKpn27 (of NTE_KPC-I) and IS6100 (of NTE_KPC-III) prevented group discrimination in another entry. The remaining seven entries (11.36%) belonged to NTE_KPC-II and were reported in China, Brazil and Colombia [7,21,22].

Sixteen entries reported a sequence type for the P. aeruginosa, and from these, six (37.5%) were ST463—all presented in China. These isolates carried NTE_KPC-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) presented in just one isolate and in different countries; this also suggests the dissemination of local clones. Sequence types 235, ST308 and ST1006 were found in Colombia; ST277 and ST381 in Brazil; and ST463 and ST697 in China. The appearance of the diverse genetic backgrounds associated with NTE_KPC in P. aeruginosa suggests these types of elements may play a preponderant role in bla_KPC dissemination in this species. However, the characteristics of these genetic environments must be studied to elucidate the role they play in the genetic mobility of this resistance gene.

3.2. In Vitro Results

3.2.1. Tn4401 and NTE_KPC 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 bla_KPC and istB genes (Figure 1), as they are part of a conserved region in the different Tn4401 isoforms and is absent in all NTE_KPC 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).

Additionally, a PCR was designed to differentially amplify NTE_KPC groups (I, II and III). For this, the reverse primer is located on the bla_KPC gene as in the specific PCR for Tn4401. However, forward oligonucleotides target group-specific hallmarks; then, in combination with the conserved reverse primer targeting bla_KPC, specific products for the NTE_KPC-I, NTE_KPC-II, and NTE_KPC-III elements (Figure 2) were generated. For NTE_KPC-III amplification, the primer was designed to target the tnpR present on Tn5563, unique to this element. For the differential amplification of NTE_KPC-II, the primer targets the bla_TEM resistance gene and for NTE_KPC-I targets the tnpA of ISKpn27 (Figure 2). The ISKpn27 tnpA gene is found in both NTE_KPC-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 bla_TEM allow for differentiation between them.

This strategy is the first reported that allows us to identify and differentiate groups of NTE_KPC and eight of the nine Tn4401 variants (Tn4401g was not included). This method can be used for the rapid genetic screening of bla_KPC harboring isolates, not only in P. aeruginosa, and in clinical settings or research, to contribute to the surveillance of this resistance gene.

3.2.2. Characterization of the Genetic Environment Associated with bla_KPC in Colombian Clinical Isolates of P. aeruginosa

The different PCRs were standardized and implemented for the characterization of the region upstream of the bla_KPC 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 bla_KPC gene. In the analyzed population, two mobilization platforms associated with bla_KPC were identified—Tn4401 (n = 37, 60.7%) and NTE_KPC-II (n = 19, 31.1%). In addition, five (8.2%) isolates did not amplify for any of the PCRs (Figure 3). Although the primers were designed to determine the NTE_KPC group (either I, II or III), but not the specific variants among them (the product generated for the NTE_KPC-II variants a, b, c and e is the same—371 bp), it is possible to differentiate the NTE_KPC-IIf, since this variant has a TnpA gene inserted between bla_TEM and bla_KPC (Figure 2). Here, of 19 NTE_KPC-II-positive isolates identified, 18 (94.7%) harbored NTE_KPC-IIf elements, whilst the remaining isolate amplified the 371 bp product.

The main platform associated with bla_KPC was the Tn4401 (60.7%). However, 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 NTE_KPC-IIf-positive isolates, and in three of these, it was the most predominant element, with 70% (n = 7), 100 (n = 7) and 100% (n = 3), for institutions one, two and three, respectively. Interestingly, NTE_KPC-IIf circulated among different and 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. The results confirmed the presence of the NTE_KPC-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 a high capacity to acquire antibiotic resistance genes [24]. This clone has been previously described transporting bla_KPC 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 bla_KPC embedded in these novel NTE_KPC elements [2].

4. Conclusions

According to the information from the GenBank, the dissemination of the bla_KPC resistance gene is mainly due to NTE_KPC non-conventional elements. In Colombia, although the Tn4401 was abundant (mostly in one institution), a high frequency of NTE_KPC-II elements could be evidenced, in four different institutions, and even though 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 amplify any of the designed PCRs, which indicates that these isolates do not present a Tn4401, but also suggests the presence of a new NTE_KPC variant. However, additional studies are required to determine the characteristics of this region in these isolates.