Rapidly disseminating blaOXA-232 carrying Klebsiella pneumoniae belonging to ST231 in India: multiple and varied mobile genetic elements

Background Recently, in India, there has been a shift from NDM to OXA48-like carbapenemases. OXA-181 and OXA-232 are the frequently produced variants of OXA48-like carbapenemases. OXA48-like carbapenemases are also known to be carried on transposons such as Tn1999, Tn1999.2 and it is also associated with IS1R carried on Tn1999. In India, there are no previous reports studying the association of mobile genetic elements (MGEs) with OXA48-like carbapenemases. The present study was aimed at determining the genetic backbone of OXA48-like carbapenemases to determine the role of MGEs in its transfer and to investigate the Inc plasmid type carrying blaOXA48-like. Results A total of 49 carbapenem resistant K. pneumoniae which included 25 isolates from South India and 24 isolates from North India, were included in the study. Whole genome sequencing using Ion Torrent PGM was performed to study the isolates. OXA-232 was present in 35 isolates (71%). In 19 isolates (39%), blaOXA48-like was associated with MGEs. Insertion sequences such as ISX4, IS1, IS3, ISKpn1, ISKpn26, ISKpn25, ISSpu2, ISKox1, IS4321R, ISEc36, and ISPa38; and transposons such as TnAs3 and Tn2, were present. Isolates from northern and southern India belonging to same sequence type (ST) had diverse genetic backbone for blaOXA48-like. ST14 isolates from north had IS5 and Tn3 families while from south they had IS1, IS5 and IS630 families. ST231 from north had IS5, IS6 and Tn3 families with blaOXA-232 while from south, IS1, IS3 and IS5 families were observed; with ISKpn26 being present among isolates from both the regions. blaOXA48-like was predominantly found on ColKP3 plasmid. ST231 was the predominant ST in 22 isolates (45%). Conclusion OXA-232 is the predominant variant of OXA48-like carbapenemase with ST231 being the commonest ST of OXA48-like carbapenemase producing K. pneumoniae in India. Diverse MGEs have been associated with both blaOXA-232 and blaOXA-181 which contribute to their spread. The MGEs in the present study are different from those reported earlier. There is no clonal expansion of blaOXA48-like producing K. pneumoniae since diverse STs were observed. Monitoring the genetic backbone of OXA48-like carbapenemase is essential to better understand the transmission dynamics of XDR K. pneumoniae.

The bla OXA48-like genes are always carried on plasmids. Initially, IncL plasmids mediated the spread of bla OXA48-like genes. However, they have now been reported among other plasmid types such as IncH, IncA/C, IncX3 and ColKP3 [5][6][7][8]. OXA48-like carbapenemases are also known to be carried on transposons such as Tn1999, Tn1999.2 and it is also flanked by IS1R carried on Tn1999 [9,10]. In India, there are no previous reports studying the association of mobile genetic elements with OXA48-like carbapenemases. Recently, in India, there has been a shift from NDM to OXA48-like carbapenemases [11]. Hence it is important to understand the role of mobile genetic elements (MGEs) in transfer of bla OXA48-like . The present study was aimed at determining the genetic backbone of OXA48-like carbapenemases in order to determine the role of MGEs in its transfer. The study also investigated the Inc plasmid type carrying bla OXA48-like .

Phenotypic characterisation
A total of 49 K. pneumoniae isolates which included 25 from Christian Medical College (CMC), Vellore, from South India, and 24 isolates from All India Institute of Medical Sciences (AIIMS), New Delhi, from North India, were included in the study. The isolates were identified by conventional biochemical methods as K. pneumoniae [12]. The antimicrobial susceptibility testing for imipenem (10 μg) and meropenem (10 μg) was performed for the isolates by Kirby Bauer disk diffusion method as recommended by Clinical and Laboratory Standards Institute (CLSI) and interpreted according to CLSI guidelines. Escherichia coli ATCC 25922 and Pseudomonas aeruginosa ATCC 27853 were used as the control strains for susceptibility testing. The isolates that were resistant to imipenem and meropenem as determined by CLSI guidelines were included in the study.

Molecular characterisation
DNA was extracted from 18 to 24 h old cultures using Qiasymphony (Qiagen, Hilden, Germany) as per manufacturer's instructions. Multiplex PCR for determination of carbapenemases such as bla IMP , bla VIM , bla NDM , bla SPM , bla OXA48-like and bla KPC were performed as described previously [2].
The presence of insertion sequences and other mobile genetic elements adjacent to bla OXA-181 and bla OXA-232 were determined by NCBI annotation and further using ISFinder (https://www-is.biotoul.fr/) to confirm the identity of insertion element.
In six isolates from CMC, bla OXA-232 was associated with insertion sequences as depicted on Fig. 1. Figure 1 also shows the genetic backbone among two isolates in which bla OXA232 is not flanked by insertion sequences. The genetic backbone is diverse among the isolates as shown in Fig. 1 even among isolates belonging to same sequence type. Isolates belonging to ST14 had insertions from IS1, IS5 and IS630 families while those of ST231 had insertions belonging to IS5, IS1, IS3 and Tn3 families (Table 1). Seven sequence types were observed among the South Indian isolates which include ST231 (n = 12), ST14 (n = 5), ST147 (n = 4), ST16 (n = 1), ST43 (n = 1), ST395 (n = 1) and ST570 (n = 1). ST231 has been isolated throughout the study period. ST231 and ST43 belong to the same clonal complex (CC), CC43. ST231 is a triple locus variant of ST43 varying in pgi, phoE and tonB genes with 11SNPs.
As seen from Table 1, isolates from northern and southern India belonging to same clone had diverse genetic backbone for bla OXA48-like . Isolates from North belonging to ST14 had MGEs from IS5 and Tn3 families while from South they had MGEs from IS1, IS5 and IS630 families. A single isolate of ST231 from north had MGEs from IS5, IS6 and Tn3 families with bla OXA-232 while from south, IS1, IS3 and IS5 families were observed. This shows that there is no clonal expansion of OXA48-like producers in India.
Diverse mobile genetic elements have been associated with both bla OXA-232 and bla OXA-181 belonging to bla OXA48-like . This includes: a) insertion sequences such as ISX4, IS1, IS3, ISKpn1, ISKpn26, ISKpn25, ISSpu2, ISKox1, IS4321R, ISEc36, and ISPa38; b) transposons such as TnAs3 and Tn2, belonging to Tn3 family. ISKpn26 has been seen among isolates from Vellore and New Delhi. This indicates the role of diverse MGEs in transmission of OXA48-like carbapenemases in India. Figure 3 shows the phylogenetic tree of OXA48-like carbapenemase producing K. pneumoniae. MLST, variant of OXA48-like carbapenemase and centre from where the isolates were obtained are shown in Fig. 3. Mobile genetic elements associated with OXA48-like has also been indicated.

Discussion
The commonest variants of bla OXA48-like reported among K. pneumoniae are bla OXA-181 and bla OXA-232 . In the present study, significantly, 80% of the isolates were bla OXA-232 producers. In 14 of the study isolates, bla OXA-232 was associated with mobile genetic elements such as insertion sequences (IS) and transposons. Interestingly, among the isolates with IS, the regions flanking bla OXA-232 were diverse. No two isolates had the same genetic environment even among the isolates in which bla OXA-232 was not flanked by IS. ISKpn26 was found with bla OXA-232 in four isolates.
Tn1999 and its isoforms have been frequently described carrying bla OXA-232 along with IS1R [9,10,13]. ISEcp1 was reported among isolates from France and Brunei belonging to ST14 and ST231 [14,15]. However, in the present study these mobile genetic elements were Fig. 3 Whole genomes SNP based phylogenetic tree of the study isolates absent and significantly different from global isolates. Also, IncL/M type of plasmids are frequently found carrying bla OXA48-like gene [16]. However, in the present study, none of the isolates harboured IncL/M plasmid. In contrast, in most of the isolates bla OXA48-like gene was present on ColKP3 plasmid and on IncA/C2 in one of the isolates. IncA/C harbouring bla OXA48-like gene has been previously reported [7]. A recent study in the US reported bla OXA-232 in all the study isolates to be present on ColKP3 plasmid [17].
In two of the study isolates, along with bla OXA48-like , bla NDM-5 was also present. bla NDM-5 was flanked by ISAba125 which is frequently associated with bla NDM [18,19]. Both these isolates were of ST147 isolated during 2013 and 2018. bla OXA-181 and bla NDM-5 has been previously reported in USA and South Korea [17,20]. Similar to the present study, coexistence of bla OXA-181 and bla NDM-5 have been reported among E. coli and K. pneumoniae [20,21].
Totally, 11 sequence types were observed in the present study. These were diverse and the two major clonal complexes were CC11 and CC43. ST14 and ST147 have been frequently reported among OXA48-like producing K. pneumoniae in various regions such as North America and Germany [22,23]. ST14 and ST147 have been described as international high risk clones associated with extensively drug resistant (XDR) K. pneumoniae [24]. ST395 has also been reported among European and African OXA48-like producing K. pneumoniae [15].

Conclusion
OXA-232 is the predominant variant of OXA48-like carbapenemase with ST231 being the commonest ST of OXA48-like carbapenemase producing K. pneumoniae in India. Diverse MGEs have been associated with both bla OXA-232 and bla OXA-181 which contribute to their spread. The MGEs in the present study are different from those reported earlier. There is no clonal expansion of bla OXA48-like producing K. pneumoniae since diverse STs were observed. Among isolates belonging to same ST, diverse MGEs were observed associated with bla OXA48-like . Monitoring the genetic backbone of OXA48-like carbapenemase is essential to better understand the transmission dynamics of XDR K. pneumoniae.