Molecular characterization of colistin-resistant Klebsiella pneumoniae & its clonal relationship among Indian isolates.

Background & objectives: Klebsiella pneumoniae (KP), a common cause of invasive infections, is often extensively drug resistant in India. At present, studies on resistance mechanism and clonal relationship of KP from India are limited. The present study was undertaken to determine the resistance mechanism and clonal relationship of colistin-resistant isolates obtained from various specimens. Carbapenemases were also determined since the isolates were carbapenem resistant.
Methods: Sixty five isolates from blood, exudates and respiratory specimens collected between 2016 and 2017 were studied. Colistin minimum inhibitory concentration (MIC) was performed by broth-micro dilution method. Multiplex PCR was carried out to determine carbapenemases. Targeted sequencing was performed to determine mutations in mgrB, phoP, phoQ and multilocus sequence typing was performed to determine the prevalent clones.
Results: Colistin MIC ranged from 4 to 256 μg/ml. SHV, TEM and CTX-M were co-produced in 60 per cent and OXA48-like in 71 per cent. Thirteen isolates had mutations in mgrB. Mutations included a premature stop codon at 21st amino acid, the presence of insertion sequences such as IS903, IS Kpn 14 and ISK pn 26; and elongation of mgrB. Novel mutations were also observed among phoP and phoQ genes. Colistin resistance due to mcr genes was absent. Fifteen clonal types were seen with ST231, ST14 and ST2096 being predominant. Interpretation & conclusions: This study revealed the changing trend of carbapenem resistance mechanism predominantly to OXA48-like from NDM. Known mgrB mutations and novel mutations in phoP and phoQ were detected. There was no plasmid-mediated colistin resistance. ST14 and ST231 were international clones associated with carbapenem resistance. Colistin-resistant KP was of diverse clones with predominantly ST231, ST14 and ST2096.

Klebsiella pneumoniae (KP) causes a wide spectrum of infections including pneumonia, bacteraemia and skin and soft-tissue infections. The incidence of extensively drug-resistant (XDR, non-susceptible to at least one agent in all classes, but susceptible to ≤2 classes of antimicrobials) isolates have been on the rise1. Colistin resistance is mediated by chromosomal mutations and plasmid-borne genes2. Chromosomal mutations are predominantly reported in the genes encoding two-component systems (mgrB, phoP, phoQ, pmrA, pmrB) involved in lipopolysaccharide modification2. Plasmid-mediated mcr1-5 and its variants encoding phosphoethanolamine transferase contribute to colistin resistance.

At present, studies on molecular characterization of XDR KP (carbapenem and colistin resistant) and clonal relatedness are limited in India. This study was performed to determine the molecular resistance mechanisms and its association with clonal relationship among colistin-resistant KP. Chromosomal colistin resistance contributed by mutations in mgrB, phoP and phoQ and plasmid-mediated mcr-1 and mcr-3 were investigated. Multilocus sequence typing (MLST) to look for clonal types and its relationship with colistin-resistant mechanism were determined in isolates collected from various clinical specimens. Since the study isolates were carbapenem-resistant, carbapenemases were also characterized.

Material & Methods

Totally 65 colistin-resistant KP isolated from clinical specimens including blood and body fluids (n=43), exudates (n=11) and respiratory specimens (n=11) collected between 2016 and 2017 at the department of Clinical Microbiology, Christian Medical College, Vellore, India, were selected retrospectively. The study protocol was approved by the Institutional Review Board. The isolates were identified up to species level as per standard procedures3. First colistin-resistant KP isolate from a patient was included in the study.

Phenotypic characterization: Antimicrobial susceptibility testing was performed as per Clinical and Laboratory Standards Institute (CLSI) guidelines and interpreted based on CLSI 2016 and 2017 breakpoints45. The panel of antimicrobials tested included ceftazidime (30 μg), cefotaxime (30 μg), cefepime (30 μg), piperacillin/tazobactam (100/10 μg), imipenem (10 μg), meropenem (10 μg), ciprofloxacin (5 μg), gentamicin (10 μg), netilmicin (30 μg), amikacin (30 μg), minocycline (30 μg) and tigecycline (15 μg). All the isolates were carbapenem resistant which was defined as resistant to meropenem with zone diameter of ≤19 mm45. Escherichia coli ATCC 25922, Staphylococcus aureus ATCC 25923 and Pseudomonas aeruginosa ATCC 27853 were used as control strains for susceptibility testing as recommended by CLSI45. Susceptibility to tigecycline was interpreted with FDA breakpoints (). Colistin minimum inhibitory concentration (MIC) was determined by reference broth-micro dilution method67. mcr-1 positive E. coli (Courtesy: Dr Olga Perovic, National Institute for Communicable Diseases, Johannesburg, South Africa) was used as positive control and E. coli ATCC 25922 and P. aeruginosa ATCC 27853 were used as negative controls. MIC results were interpreted as per European Committee on Antimicrobial Susceptibility Testing (EUCAST) guidelines 2016 and 2017, respectively67.

Molecular characterization

Characterization of β-lactamases: Bacterial DNA was extracted from 18 h cultures by Qiasymphony (Qiagen, Hilden, Germany) as per manufacturer's instructions. Multiplex PCR was performed for the detection of genes encoding β-lactam resistance which included EBSL (blaSHV, blaTEM, blaPER, blaVEB, blaCTX-M-1, blaCTX-M-2, blaCTX-M-8, blaCTX-M-9 and blaCTX-M-25)89, AmpC (blaACT, blaACC, blaCIT, blaDHA and blaFOX)10 and carbapenemases (blaNDM, blaVIM, blaIMP, blaOXA48-like, blaKPC, blaGES and blaSPM)111213. Known positive controls (Courtesy: International Health Management Association Inc., USA) were used.

Characterization of colistin resistance: Chromosomal mutations: PCR for amplification of mgrB, phoP and phoQ genes was performed as described by Jayol et al14. From the amplified product, mutations were determined by targeted sequencing. For analysis of mutations, KP ATCC 35657 was used as reference. To determine insertion sequences present in mgrB, the sequences were run through ISfinder () and the position of insertion was assigned by aligning with reference mgrB. Sequencing was performed using 3500 Genetic Analyzer (Applied Biosystems, USA) as per manufacturer's instructions.

Sorting Intolerant From Tolerant (SIFT) scores for the non-synonymous mutations implicating in the amino acid substitutions were predicted for mgrB, phoP and phoQ genes. A value of <0.05 for a substitution is predicted to be deleterious and affects the function of the protein (). SIFT score calculates the deleterious effect based on the probability of the substituted amino acid being tolerated at a position.

Plasmid-mediated colistin resistance: To determine plasmid-borne colistin resistance, PCR was performed for mcr-1 and mcr-3 genes, as controls were available for these two mcr variants1516.

Multilocus sequence typing (MLST): MLST was performed as described by Diancourt et al17. Seven housekeeping genes used to determine clonal type among KP included gapA (coding for glyceraldehyde-3-phosphate), infB (coding for translation initiation factor-2), mdh (coding for malate dehydrogenase), pgi (coding for phosphoglucose isomerase), phoE (coding for phosphorine E), rpoB (coding for β-subunit of RNA polymerase B) and tonB (coding for periplasmic energy transducer). The sequence type was assigned by determining the allele number for each of the housekeeping genes using the database maintained by Pasteur Institute, Paris, France ().

Genetic analysis: The relatedness of the predicted sequence types was investigated by eBURST V3 software employing the BURST algorithm18. Splits Tree4 programme version 4.14.6 was used to determine the phylogenetic relatedness among the various clonal types based on the seven house-keeping genes that define the MLST19. Using the concatenated sequences of seven housekeeping genes of MLST, phylogenetic tree was constructed using MEGA v.7 () and meta-data were added using iTOL ().

Results

All the 65 isolates were XDR being resistant to aminoglycosides, cephalosporins and fluoroquinolones and susceptible only to tigecycline; 10 blood isolates were susceptible to minocycline and two isolates each from exudates were susceptible to chloramphenicol and trimethoprim-sulphamethoxazole, respectively. The colistin MIC for the isolates ranged from 4 to 256 μg/ml. The mean colistin MIC value was found to be 19±31.9 μg/ml.

Among the ESBL genes, 60 per cent (n=40) of the isolates co-produced blaSHV, blaTEM and blaCTX-M-15. blaOXA48-like was the predominant carbapenemase gene produced in 71 per cent (n=47) followed by co-production of blaNDM and blaOXA48-like in 11 per cent (n=7), blaNDM in seven per cent (n=5) and blaKPC three per cent (n=2) and four isolates did not produce any of the carbapenemases genes tested. Fig. 1 shows the distribution of β-lactamases among the various clinical specimens.

Fig. 1

Distribution of β-lactamases in colistin-resistant Klebsiella pneumoniae among various clinical specimens.

Colistin MIC, mutations in mgrB, phoP and phoQ and associated clonal types among the study isolates are shown in the Table. The mutations in mgrB observed among clinical specimens were among diverse clonal types. Fig. 2A–E shows the association of mgrB with insertion sequences. A total of 41 isolates did not have mutations in mgrB and in 11 isolates mgrB gene was absent. Mutations in phoP and phoQ were present among six and nine isolates, respectively. Screening for mcr-1 and mcr-3 genes by PCR was negative for all the isolates.

Table

Mutations in mgrB, phoP and phoQ genes among the colistin-resistant Klebsiella pneumoniae

Isolate ID	Source	Colistin MIC (µg/ml)	MLST	Carbapenemase	mgrB	phoP	phoQ	Accession number
Kp1	Blood	16	ST86	Nil	Nil	Alanine 114 arginine (0.22)	Leucine 209 cysteine (0.00*)	bPAW96459.1 cNRQZ01000030.1
Kp2	Blood	32	ST11	Nil	Elongated mgrB of 55 amino acids	Nil	Nil	aPBD34554.1
Kp3	Blood	16	ST14	OXA48-like	Nil	Nil	Tryptophan 161 leucine (0.30)	cMH211110
Kp4	Blood	32	ST14	OXA48, NDM	No amplification	Nil	Tryptophan 161 leucine (0.30)	cMH211111
Kp5	Blood	32	ST14	OXA48-like	Truncated by IS1R of IS1 family	Nil	Nil	aMH590725
Kp6	Blood	8	ST231	OXA48-like	Premature stop codon at 21st amino acid and change in the sequence of protein after 10th amino acid	Nil	Nil	aMH807824
Kp7	Blood	8	ST15	KPC	Truncated by IS903B group of IS5 family	Nil	Nil	aMH590724
Kp8	Blood	16	ST231	OXA48-like	Truncated by ISKpn14 of IS1 family	Nil	Nil	aMH590726
Kp9	CSF	16	ST14	OXA48-like	Truncated by ISKpn26 of IS5 family	Nil	Changes from 218 to 239 and 436 to 448 amino acids	aMH590727 cMH807825
Kp10	Exudate	8	ST23	NDM	Nil	Nil	Glycine 117 aspartic acid (1.00)	cMH211112
Kp11	Exudate	4	ST2096	OXA48-like	Nil	Nil	Valine 370 glutamic acid (0.03*)	cMH211113
Kp12	Exudate	32	ST14	OXA48, NDM	Cysteine to glycine at 28th amino acid (0.00*)	Threonine 151 Alanine (0.00*)	Nil	aMH337365 bMH211116
Kp13	Exudate	16	ST147	OXA48, NDM	Nil	Glutamic acid 22 lysine (0.49)	Nil	bMH211117
Kp14	Exudate	32	ST231	OXA48-like	Premature stop codon at 21st amino acid and change in the sequence of protein after 10th amino acid	Nil	Nil	aMH807823
Kp15	Respiratory	32	ST14	NDM	Cysteine to glycine at 28th amino acid (0.00*)	Nil	Leucine 172 glutamine (0.01*); Tryptophan 182 serine (0.04*)	aMH337366 cMH211114
Kp16	Respiratory	8	ST231	OXA48-like	Nil	Nil	Valine444^, phenylalanine 445 glycine (0.00*)	cMH211115
Kp17	Respiratory	8	ST147	OXA48-like	Methionine to arginine first amino acid (0.00*)	Nil	Nil	aMH590728
Kp18	Respiratory	16	ST231	OXA48-like	Premature stop codon at 21st amino acid and change in the sequence of protein after 10th amino acid	Nil	Nil	aMH337364
Kp19	Respiratory	8	ST2957	KPC-9	No mutation; associated with IS102 of IS5 family	Nil	Proline 424 leucine (0.37) V446W (0.00*), change from 448th amino acid	aPAT25546.1 cCJ307_16160

amgrB accession number; bphoP accession number; cphoQ accession number. Numbers in parenthesis indicate the SIFT score; values with *<0.05 show that the mutation affects the protein function. #Position of insertion in mgrB gene. ^Indicates Valine deletion at 444th position in phoQ. MLST, multilocus sequence typing; SIFT, sorting intolerant from tolerant; MIC, minimum inhibitory concentration; IS, insertion sequence

Fig. 2

(A) Insertion sequence (IS) associated with mgrB in Kp2; (B) IS1R inserts at 132nd position in mgrB in Kp7; (C) Insertion of ISKpn26 between 66th and 67th bases of mgrB in Kp14 with repeats of 9 bases; (D) Insertion of IS903B between 34th and 35th bases of mgrB in Kp19 with repeats of 29 bases; (E) Insertion of ISKpn14 at 93rd position of mgrB in Kp20.

SIFT score was calculated to predict the effect of amino acid substitutions identified in this study isolates. In mgrB, both the substitutions Met1Ala and Cys28Gly were found deleterious. In phoP, among the three substitutions observed, Ala114Arg, Glu22Lys showed a score of 0.22 and 0.49, while Thr151Ala scored zero and found significant and deleterious. Similarly, in phoQ, mutations such as Leu205Gln, Leu209Cys, Trp215Ser, Val403Glu, Val446Trp and Phe478Gly were predicted to affect the function of the protein. Non-significant mutations of Trp194Leu (phoQ) in Kp7, Kp8 and Glu22Lys (phoP) in Kp13 were also noted.

Totally, 15 different sequence types were obtained among the 65 isolates and these included ST231 (n=23), ST14 (n=13), ST2096 (n=9), ST 147 (n=5), ST78 (n=3), ST15 (n=2), ST16 (n=2) and one isolate each of ST11, ST17, ST23, ST38, ST43, ST86, ST395 and ST2957. Fig. 3 shows the eBURST analysis for the clonal types among the study isolates. ST17 and ST16 belonged to the same clonal complex; while ST14, ST15, ST78 and ST2096 form another clonal complex, CC14. Other sequence types such as ST11, ST23, ST43, ST147 and ST231 were found as singletons.

Fig. 3

eBURST of colistin-resistant Klebsiella pneumoniae. The clonal types identified in the present study are highlighted. ST17 and ST16 belonged to the same clonal complex; ST14, ST15, ST78 and ST2096 formed a clonal complex. Other sequence types such as ST11, ST23, ST38, ST43, ST86, ST147, ST231 and ST395 were found as singletons.

Among the study isolates, KP harbouring carbapenemases were of diverse sequence types. Overall, ST14 was the most predominant. Among the STs, OXA48-like producers were distributed among diverse clonal types such as ST14, ST16, ST17, ST38, ST43, ST231, ST395 and ST2096. Co-producers of blaNDM with blaOXA48 (n=6) belonged to ST14, ST78 and ST147. Isolates producing blaNDM (n=6) alone belonged to ST14, ST15, ST23 and ST78. Two of the study isolates were blaKPC producers, each belonging to ST15 and ST2957.

Fig. 4 shows the Splits Tree diagram for the sequence types. All the clonal types were closely related except ST2957 which was an outlier. The outlier was identified as K. quasipneumoniae belonging to phylogenetic group II of KP. Fig. 5 shows the maximum likelihood tree constructed based on the concatenated sequences of seven housekeeping genes used for MLST. As shown, isolates of ST231 were OXA48-like carbapenemase producers and was the predominant clone. The isolates of CC14 were more diverse in carbapenemases producing New Delhi metallo-β-lactamase (NDM) and/ or OXA48-like and one isolate with KPC. Fig. 5 also shows the clones present among different specimen sources. The isolates with chromosomal mutations contributing to colistin resistance (Table) are also indicated and the distribution of carbapenemases among these isolates is evident from Fig. 5.

Fig. 4

Splits Tree for the clonal types identified among colistin-resistant Klebsiella pneumoniae. Fifteen clonal types identified in the study were closely related forming a cluster while ST2957 was distinct. The tree was constructed based on seven housekeeping genes.

Fig. 5

Maximum likelihood tree for colistin-resistant Klebsiella pneumoniae using concatenated sequences of seven multilocus sequence typing genes.

Discussion

In this study molecular resistance mechanisms were assessed and clonal relatedness of colistin-resistant KP was investigated. SHV, TEM and CTX-M-15 were the prevalent β-lactamases similar to earlier study20. Among the study isolates, OXA48-like was the commonest carbapenemase produced. In this study, colistin resistance was contributed by mutations in mgrB, phoP and phoQ genes in the isolates. Our earlier study identified truncated mgrB of 27 amino acids due to a premature stop codon and also deletion of A at 10th nucleotide2. However, in the present study, these two mutations were absent, but various other mutations were seen. All the mgrB negatives could be due to partial or complete deletion of mgrB as reported previously21. The disruption of mgrB due to insertion elements such as IS1R, IS903, ISKpn14 and ISKpn26 similar to the present study have been reported earlier212223. The mgrB mutations were not associated with specific carbapenemases or specific clones. There was no association between colistin MIC, mgrB mutations and specific clones. Substitutions in the phoP and phoQ genes implicated in colistin resistance have been reported23. However, certain mutations observed in the present study were novel. The significance of these novel mutations were predicted using SIFT scores. These scores revealed that the novel mutations were likely to affect protein function. However, detailed functional properties of mutations need to be determined by cloning studies to substantiate its significance.

Clonal relatedness with antimicrobial resistance profile in colistin-resistant KP was determined. New Delhi metallo-β-lactamase (NDM) producing KP are known to be associated with ST11, ST14, ST147 and ST231 in India and globally22425 while the two KPC KP belong to ST15 and ST2957. International KPC clones such as ST258 and ST512 were absent among the study collection.

The colistin-resistant isolates in the present study were diverse and 15 clones were observed among the isolates. ST14, ST231 and ST2096 were predominant. The truncation of mgrB due to IS5-like elements2122 and ISKpn142326 has been reported among ST258, ST512 and ST101. ISKpn26 has been reported among OXA48-like producer belonging to ST30726. In the present study, these were seen among isolates belonging to ST14, ST15 and ST231. Substitutions previously reported in phoP2327 and phoQ2327 were among isolates of ST147 and ST258.

Isolate belonging to ST23 produced NDM; also carried rmpA and rmpA2 genes indicating hypervirulent KP. Earlier, carbapenem-resistant hypervirulent KP belonging to ST14 and ST23128 has been reported. These two clones are classically associated with carbapenem-resistant isolates in India and other countries220. A novel ST2957 was observed in the present study. Earlier in India, Shankar et al29 have reported pan-susceptible hypervirulent K. quasipneumoniae of novel clone ST2320.

The association between the MLST sequence types and the molecular resistance mechanisms was not observed. This could be due to the limited relatedness information provided by MLST. Whole genome SNP-based phylogeny may provide further insights into the strain-specific lineage within a particular sequence type/clonal complex30. A previous study revealed that 97 per cent of KPC KP isolates sequenced, regardless of country of isolation, belong to a well-supported clade, corresponding to the complex CC258 based on the whole genome phylogeny30. MLST can be used to understand the epidemiology at a large scale while whole genome SNP phylogeny would be discriminatory in local outbreak situations.

The mechanism of colistin resistance in isolates which lacked mutations in mgrB, phoP and phoQ were not determined. This was a limitation of the study since in 70 per cent of study isolates the mechanism of colistin resistance was unidentified.

In conclusion, this study revealed the changing trend of carbapenem resistance mechanism predominantly to OXA48-like from NDM. There is alarming increase in colistin resistance with novel mutations in chromosomal genes such as mgrB, phoP and phoQ. There was no plasmid-mediated colistin resistance. Diverse clones of colistin-resistant isolates were observed with CG14 being predominant. No known international clones such as ST258 and ST512 were observed. It is important to monitor colistin resistance by using appropriate testing method and also limit colistin usage.