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.

Background

OXA carbapenemases are oxacillinases which hydrolyse isoxazolylpenicillins. They have been divided into 12 groups based on amino acid sequences. OXA48-like is the commonly seen group among K. pneumoniae. OXA-181 and OXA-232 are the frequently produced variants of OXA48-like carbapenemases. OXA-181 and OXA-232 differ from each other by four amino acids: T104A; N110D; E168Q; S171A [1]. OXA-232 is a five amino acid variant of OXA-48 (T104A; N110D; E168Q; S171A; R214S). OXA-232 varies from OXA-181 by single amino acid (R214S) [1]. OXA-181 and OXA-232 have been reported with NDM-1 especially in India [2, 3]. Turkey, Morocco, Egypt, Libya and India are considered to be endemic for OXA48-like carbapenemases [4].

The blaOXA48-like genes are always carried on plasmids. Initially, IncL plasmids mediated the spread of blaOXA48-like genes. However, they have now been reported among other plasmid types such as IncH, IncA/C, IncX3 and ColKP3 [5-8]. 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 blaOXA48-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 blaOXA48-like.

Methods

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 blaIMP, blaVIM, blaNDM, blaSPM, blaOXA48-like and blaKPC were performed as described previously [2].

The isolates were subjected to whole genome sequencing using Ion Torrent PGM platform with 400 bp chemistry. Raw reads were assembled using Assembler SPAdes v.5.0 software in Torrent suite server version 4.4.3. The genome was annotated using RAST (Rapid Annotation using Subsystems Technology- http://rast.nmpdr.org/), Patric (Pathosystems Resource Integration Centre - https://www.patricbrc.org/) and the National Centre for Biotechnology Information Prokaryotic Genomes Automatic Annotation Pipeline (NCBI PGAAP) softwares. The resistance genes were identified using ResFinder version 2.1 (https://cge.cbs.dtu.dk/services/ResFinder/) and Multi-locus Sequence typing (MLST) was determined using database at https://cge.cbs.dtu.dk/services/MLST/ . Plasmids present in the genome were identified by PlasmidFinder version 1.3 available at https://cge.cbs.dtu.dk/services/PlasmidFinder/.

The presence of insertion sequences and other mobile genetic elements adjacent to blaOXA-181 and blaOXA-232 were determined by NCBI annotation and further using ISFinder (https://www-is.biotoul.fr/) to confirm the identity of insertion element.

Whole genome single nucleotide polymorphism (SNP) tree was constructed using CSI Phylogeny at https://cge.cbs.dtu.dk/services/CSIPhylogeny/ . For the phylogenetic tree, metadata was labelled using iTOL software at https://itol.embl.de .

Results

The isolates from CMC, Vellore, were distributed over a span of 6 years: 2013 (n = 3), 2014 (n = 5), 2015 (n = 3), 2016 (n = 5), 2017 (n = 6) and 2018 (n = 3). All the isolates were resistant to aminoglycosides, β-lactams, fluoroquinolones and minocycline. Twenty one isolates were colistin resistant with minimum inhibitory concentration (MIC) ranging from 4 to 1024 μg/ml. All isolates except Kp21 and Kp22 were susceptible to tigecycline. The accession numbers for genomes, blaOXA48-like variant, year of isolation, plasmid carrying blaOXA48-like and MLST have been mentioned in Table 1. Among the CMC study isolates, 19 carried blaOXA-232 and six carried blaOXA-181. Three isolates co-expressed blaNDM with blaOXA48-like as mentioned in Table 1.

Table 1

Details of study isolates including accession numbers

Centre	Isolate no.	Accession no/ Bioproject ID	blaOXA-48 variant	Plasmid	Insertion sequence flanking blaOXA-48 variant	MLST
CMC, Vellore	Kp1	MPCT00000000	OXA-232	ColKP3	ISKpn26, IS5 family; IS110 family	ST231
	Kp2	MOXL00000000	OXA-232	ColKP3/ IncFII	None	ST231
	Kp3	PUXB0000000	OXA-181 NDM-5	unidentified	None ISAba125	ST147
	Kp4	MEBR00000000	OXA-232 NDM-1	unidentified	ISKpn26, IS5 family None	ST14
	Kp5	MDZG00000000	OXA-232	ColKP3	TnAs3, Tn3 family	ST231
	Kp6	MOXN00000000	OXA-232	ColKP3	ISX4, IS1 family; ISRaq1, IS3 family	ST231
	Kp7	MOXM00000000	OXA-232	ColKP3	None	ST14
	Kp8	MIEJ00000000	OXA-232	ColKP3	IS1A and IS1F, IS1 family	ST14
	Kp9	LZYN00000000	OXA-181	ColKP3	None	ST147
	Kp10	MCFO00000000	OXA-232	ColKP3	None	ST231
	Kp11	MCFP00000000	OXA-181	unidentified	None	ST43
	Kp12	NTHQ00000000	OXA-232	ColKP3	None	ST231
	Kp13	PJOP00000000	OXA-232	ColKP3	None	ST16
	Kp14	PKMV00000000	OXA-181	unidentified	None	ST147
	Kp15	PETC00000000	OXA-232	ColKP3	None	ST231
	Kp16	PKOL00000000	OXA-232	ColKP3	None	ST231
	Kp17	PKOK00000000	OXA-232	unidentified	ISKpn26, IS5 family; ISSpu2, IS630 family	ST14
	Kp18	NSCV00000000	OXA-232	unidentified	None	ST231
	Kp19	NRSU00000000	OXA-232	ColKP3	None	ST231
	Kp20	PKOM00000000	OXA-181	IncA/C2	None	ST231
	Kp21	PPXS00000000	OXA-232	ColKP3	None	ST395
	Kp22	PPXT00000000	OXA-232	unidentified	ISKpn1, IS3 family; IS4321R, IS110 family	ST570
	Kp23	PUIG00000000	OXA-181	unidentified	None	ST14
	Kp24	PYSM00000000	OXA-232	unidentified	None	ST231
	Kp25	PUIF00000000	OXA-232 NDM-5	ColKP3 unidentified	None ISAba125	ST147
AIIMS Trauma Centre, New Delhi	Kp26	PWAF00000000	OXA-181	unidentified	ISKox1, partial, IS66family	ST43
	Kp27	PWAD00000000	OXA-181	unidentified	ISKox1, partial, IS6family	ST43
	Kp28	MNPB00000000	OXA-232	ColKP3	ISPa38, Tn3 family; IS4321R, IS110 family	ST11
	Kp29	MNPC00000000	OXA-232	ColKP3	ISKpn25, ISL3 family	ST11
	Kp30	MNPG00000000	OXA-232	ColKP3	ISPa38, Tn3 family	ST11
	Kp31	MNPH00000000	OXA-232	unidentified	ISKpn26, IS5 family	ST14
	Kp32	PRJNA494951	OXA-232	unidentified	TnAs3, Tn3 family	ST14
	Kp33	PRJNA494951	OXA-232	ColKP3	Tn2, Tn3 family	ST2040
	Kp34	MNPA00000000	OXA-232	unidentified	IS26, IS6 family; IS903, IS5 family; ISPa38, Tn3 family	ST231
	Kp35	PYUL00000000	OXA-181	unidentified	ISKox1 IS66 family; ISEc36 IS3 family; ISKpn42 IS110 family	ST43
	Kp36	PRJNA494951	OXA-181	unidentified	ISKpn1, IS3 family	ST43
	Kp37	PRJNA494951	OXA-181	unidentified	ISKpn1 partial, IS3 family	ST11
	Kp38	PRJNA494951	OXA-232	unidentified	ISKpn1, partial, IS3 family; IS5075, IS110 family	ST11
	Kp39	PWAH00000000	OXA-232	unidentified	None	ST101
	Kp40	PWAE00000000	OXA-232	ColKP3	None	ST231
	Kp41	PRJNA494951	OXA-232	ColKP3	None	ST231
	Kp42	PRJNA494951	OXA-181	ColKP3	None	ST16
	Kp43	PRJNA494951	OXA-181	IncA/C2	None	ST231
	Kp44	PRJNA494951	OXA-232	ColKP3	None	ST15
	Kp45	PRJNA494951	OXA-232	ColKP3	None	ST15
	Kp46	PRJNA494951	OXA-181	ColKP3	None	ST15
	Kp47	PRJNA494951	OXA-232	ColKP3	None	ST231
	Kp48	PRJNA494951	OXA-232	ColKP3	None	ST231
	Kp49	PRJNA494951	OXA-232	ColKP3	None	ST231

In six isolates from CMC, blaOXA-232 was associated with insertion sequences as depicted on Fig. 1. Figure 1 also shows the genetic backbone among two isolates in which blaOXA232 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).

Fig. 1

Mobile genetic elements flanking blaOXA-48 like among K. pneumoniae from CMC, Vellore. a and b: blaOXA-48 like without insertion sequences in Kp2 and Kp10 respectively. c to h: blaOXA-48 like associated with insertion sequences and transposon in Kp1, Kp4, Kp5, Kp6, Kp17, KP22 respectively

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.

The isolates from AIIMS, New Delhi, were obtained during 2016 and 2017. The isolates belonged to diverse sequence types including ST231 (n = 7), ST11 (n = 5), ST43 (n = 4), ST14 (n = 2), ST15 (n = 3), ST16 (n = 1), ST101 (n = 1), and ST2040 (n = 1). CC11 including ST11, ST14, ST15 and ST2040, was predominant in north India. ST231 is predominantly present in both the study centres. Among the 24 isolates from AIIMS, eight were OXA-181 producers and 16 were OXA-232 producers. The genetic backbone among these isolates from New Delhi seems to be very diverse despite the clonality. Genetic backbone of isolates with blaOXA48-like associated with mobile genetic elements is shown in Fig. 2.

Fig. 2

Genetic environment of blaOXA48-like among K. pneumoniae from AIIMS, New Delhi, India Kp27, Kp28, Kp29, Kp30, Kp31, Kp32, Kp33, Kp34, Kp35, Kp36, Kp37, Kp38, Kp39

As seen from Table 1, isolates from northern and southern India belonging to same clone had diverse genetic backbone for blaOXA48-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 blaOXA-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 blaOXA-232 and blaOXA-181 belonging to blaOXA48-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.

Fig. 3

Whole genomes SNP based phylogenetic tree of the study isolates

Discussion

The commonest variants of blaOXA48-like reported among K. pneumoniae are blaOXA-181 and blaOXA-232. In the present study, significantly, 80% of the isolates were blaOXA-232 producers. In 14 of the study isolates, blaOXA-232 was associated with mobile genetic elements such as insertion sequences (IS) and transposons. Interestingly, among the isolates with IS, the regions flanking blaOXA-232 were diverse. No two isolates had the same genetic environment even among the isolates in which blaOXA-232 was not flanked by IS. ISKpn26 was found with blaOXA-232 in four isolates.

Tn1999 and its isoforms have been frequently described carrying blaOXA-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 absent and significantly different from global isolates. Also, IncL/M type of plasmids are frequently found carrying blaOXA48-like gene [16]. However, in the present study, none of the isolates harboured IncL/M plasmid. In contrast, in most of the isolates blaOXA48-like gene was present on ColKP3 plasmid and on IncA/C2 in one of the isolates. IncA/C harbouring blaOXA48-like gene has been previously reported [7]. A recent study in the US reported blaOXA-232 in all the study isolates to be present on ColKP3 plasmid [17].

In two of the study isolates, along with blaOXA48-like, blaNDM-5 was also present. blaNDM-5 was flanked by ISAba125 which is frequently associated with blaNDM [18, 19]. Both these isolates were of ST147 isolated during 2013 and 2018. blaOXA-181 and blaNDM-5 has been previously reported in USA and South Korea [17, 20]. Similar to the present study, coexistence of blaOXA-181 and blaNDM-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 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. Among isolates belonging to same ST, diverse MGEs were observed associated with blaOXA48-like. Monitoring the genetic backbone of OXA48-like carbapenemase is essential to better understand the transmission dynamics of XDR K. pneumoniae.