First Report of Whole-Genome Sequence of Colistin-Resistant Klebsiella quasipneumoniae subsp. similipneumoniae Producing KPC-9 in India

Introduction

Klebsiella pneumoniae carbapenemase (KPC) is the chief enzyme causing carbapenem resistance in the United States, China, South America, and Europe ¹ but is rarely reported from India, where NDM and OXA48-like carbapenemases are endemic. The outcome for patients with KPC K. pneumoniae infections is poor with a reported mortality rate of up to 56%. To date, 24 KPC variants have been described at Lahey Health website. KPC-2 has been commonly reported from various regions,^2–4 whereas there have been only two reports of KPC-9 to date, both from Israel.^5,6

K. pneumoniae is composed of three phylogenetic groups: KpI, KpII, and KpIII. KpI comprises K. pneumoniae subsp. pneumoniae, K. pneumoniae subsp. rhinoscleromatis, and K. pneumoniae subsp. ozaenae. KpII, which is designated Klebsiella quasipneumoniae, has recently been further classified as KpIIa, which is K. quasipneumoniae subsp. quasipneumoniae, and KpIIb, which is K. quasipneumoniae subsp. similipneumoniae. The group KpIII comprises Klebsiella variicola.^7–9 The three groups are based on nucleotide sequences, as accurate phenotypic identification is impossible. The classification of the three phylogenetic groups was carried out based on nucleotide sequences of five protein-coding genes fusA, gapA, gyrA, leuS, and rpoB. ¹⁰ In this study we document the first report of colistin-resistant K. quasipneumoniae subsp. similipneumoniae producing KPC-9 from a respiratory catheter tip.

Materials and Methods

A 41-year-old woman with traumatic brain injury after poly trauma in a road traffic accident presented to the hospital. She was unresponsive and was dependent for all activities of daily living. In the hospital she developed a low-grade fever with no rigors. She had a tracheostomy in situ but there were no significant purulent secretions. Her vitals were stable (pulse, 84/min; blood pressure, 110/70; and respiratory rate, 16/min). She had a blanching rash that was most prevalent over the chest and back. Her Glasgow Coma Score was 7/15. Cultures from the tracheal aspirate and respiratory catheter tip were sent for analysis; these cultures grew carbapenem-resistant K. pneumoniae. The culture was polymicrobial growing carbapenem-resistant K. pneumoniae and Pseudomonas aeruginosa. The patient did not have any symptoms and the chest X-ray was clear. Blood cultures drawn during this time were negative and blood counts were normal. Hence the isolate was considered to be a colonizer and was not treated.

Isolate from the culture were identified as K. pneumoniae using conventional biochemical methods. ¹¹ The antimicrobial susceptibility testing for cefotaxime (30 μg), ceftazidime (30 μg), piperacillin/tazobactam (100/10 μg), imipenem (10 μg), meropenem (10 μg), ciprofloxacin (5 μg), levofloxacin (5 μg), amikacin (30 μg), gentamicin (10 μg), minocycline (30 μg), and tigecycline (15 μg) was performed using the Kirby–Bauer disk diffusion method as recommended by the Clinical and Laboratory Standards Institute (CLSI) and interpreted according to CLSI 2016 guidelines. ¹¹ Escherichia coli ATCC 25922 and P. aeruginosa ATCC 27853 were used as the control strain for susceptibility testing. In-house CarbaNP test as recommended by CLSI for the detection of carbapenemase activity was performed. Minimum inhibitory concentration (MIC) was determined for first- and second-line antimicrobials with Vitek2 AST system (bioMerieux). String test was performed to screen for hypermucoviscous phenotype.

DNA was extracted from 18- to 24-hr-old culture using Qiasymphony (Qiagen) as per the manufacturer's instructions. The isolate was 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), Patric (Pathosystems Resource Integration Centre), and the National Centre for Biotechnology Information Prokaryotic Genomes Automatic Annotation Pipeline (PGAAP) software. Resistance genes were identified using ResFinder version 2.1. Multilocus sequence typing (MLST) was determined using the following database. Plasmids present in the genome were identified by PlasmidFinder version 1.3. Mutations in chromosomal genes contributing to colistin resistance including mgrB, phoP, phoQ, pmrA, pmrB, eptA, and arnT were determined. The CRISPR finder program was used to determine the CRIPRs present in the genome.

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

Results

The isolate was found to be resistant to all antimicrobials except tigecycline by disk diffusion. The string test was negative for hypermucoviscousity. The isolate was positive with in-house CarbaNP. Using the Vitek2 AST system (bioMerieux), MIC values were ≥16 μg/mL for meropenem, ≥16 μg/mL for minocycline, 1 μg/mL for tigecycline, and 8 μg/mL for colistin.

The isolate was identified as K. quasipneumoniae subsp. similipneumoniae by the presence of bla_OKP-B gene using ResFinder. The isolates possessed the following genes coding for antimicrobial resistance: aminoglycoside resistance encoded by aac(3)-Ild and aac(6′)-Ild; β-lactam resistance by bla_OKP-B-3, bla_KPC-9, bla_CTX-M-15, bla_OXA-1, and bla_TEM-1A; fluoroquinolone resistance by aac(6′)-Ild, oqxA, oqxB, and qnrB1; fosfomycin resistance by fosA; phenicol resistance by catB3; tetracycline resistance by tetD, and trimethoprim resistance by dfrA14. Colistin resistance was chromosomally mediated and mcr-1 to mcr-5 and their variants were not present. It belonged to a novel sequence type, ST2957, containing five plasmids namely IncFII, IncFII (pKPX1), IncHI1B, IncR and IncFIB. The genome coverage was 38.8X. The isolate was deposited at GenBank with accession number NQYU00000000 and the version described in this report is NQYU01000000.

As the isolate was also resistant to colistin, mutations in the genes responsible for lipidA modification including mgrB, phoP, phoQ, pmrA, pmrB, eptA, and arnT were compared with the genes of K. quasipneumoniae ATCC 700603 to determine mutations responsible for colistin resistance. No mutations were identified in mgrB, pmrA, and arnT genes. A few nucleotide variations were identified in phoP, pmrB, and eptA but these were nonsynonymous. Multiple nucleotide variations were found in phoQ. These included a change in amino acid from proline to leucine at position 424, deletion of guanine at position 1334 resulting in a change in amino acid sequence from amino acid 445 onward, and a change from cytosine to thymidine at position 1270 resulting in an amino acid change from proline to leucine. The mutations responsible for colistin resistance are given in Table 1. The substitution of P 424 L was predicted to score 0.37 and so was not deemed significant. However, frameshift from 1334th nucleotide would affect function of the protein. The isolate did not carry any mcr variant as determined by ResFinder. The isolate carried one CRISPR region of 516 bp with 8 spacer regions and direct repeat length of 29. Figure 1 depicts the flanking regions of bla_KPC-9 in K. quasipneumoniae.

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FIG. 1.

Flanking regions of bla_KPC-9 in Klebsiella quasipneumoniae. KPC, Klebsiella pneumoniae carbapenemase.

KPC, first isolated from K. pneumoniae, belongs to classA serine carbapenemase with 24 variants reported to date, bla_KPC1 to bla_KPC24. KPC was first described in 1996, and is the predominant carbapenemase in Europe and the United States but is rare in India. ¹² It is now seen among other bacteria including E. coli, E. aerogenes, Citrobacter freundii, P. aeruginosa, and Acinetobacter baumannii.^13–16 The sole KPC K. pneumoniae reported from India was isolated from urine specimen (Kumarasamy and Kalyanasundaram). This isolate produced KPC-2 along with β-lactamases such as NDM-1, CTX-M-15, SHV-12, TEM-1, and OXA-1. ¹⁷ Another study in India reported the coexistence of bla_KPC-2 and bla_NDM-1 among clinical isolates of P. aeruginosa. ¹⁸ KPC K. pneumoniae is not prevalent in India unlike the NDM and OXA-48-like enzymes that are endemic.^19,20

There have been two reports of KPC-9 in K. pneumoniae from Israel and Greece.^5,6 KPC-9 is a variant of KPC-3 differing from the latter by a single amino acid substitution at position 239 where valine has been substituted by alanine. In both the studies, K. pneumoniae harboring KPC-9 belonged to ST258, which is an international epidemic clone.^5,6 In this study, KPC-9 is reported from K. quasipneumoniae for the first time. This highlights the importance of accurate molecular identification of the three phylogenetic groups, K. pneumoniae, K. quasipneumoniae, and K. variicola; they cannot be distinguished by biochemical tests. The isolate was extensively drug resistant and resistant to colistin. There have been a few reports of multiresistant and extensively drug-resistant K. quasipneumoniae from geographically varied regions such as Mexico, Chicago, and Malaysia.^21–23 However, all previously reported were susceptible to colistin. Our center has previously reported an isolate of K. quasipneumoniae subsp. similipneumoniae belonging to a novel clonal type of ST2320. ²⁴ This isolate was susceptible to all antimicrobials in contrast to the isolate described in the present study. As resistance to antimicrobials is largely plasmid mediated in K. pneumoniae, the chances of acquiring resistance is high especially in settings such as our own where 60% of invasive K. pneumoniae are extended spectrum β-lactamase (ESBL) producers and 30% are carbapenemase producers. ²⁵ Approximately 37% of the carbapenem-resistant isolates are also colistin resistant (unpublished data).

A single report has previously described bla_KPC-9. ⁶ In that report, bla_KPC-9 was found on Tn4401 and was flanked by ISKpn6 and ISKpn7 similar to bla_KPC-2. However, in the present study, bla_KPC-9 was not carried on Tn4401 and both ISKpn6 and ISKpn7 were absent. Interestingly, no insertion element was found adjacent to bla_KPC-9. These insertions are frequently associated with bla_KPC genes. This can also be attributed to the clonal types among the isolates. Most reports of bla_KPC are among K. pneumoniae of ST258 clone, whereas in this study we report a novel clone ST2957. The genetic environment of bla_KPC is very diverse associated with several isoforms of Tn4401 and non-Tn4401 elements.^26,27

Although KPC K. pneumoniae is associated with high mortality rates of 50% to 66%,^25,28,29 our patient did not succumb to infection, probably as this organism was causing colonization rather than infection.

Misidentification of K. quasipneumoniae as K. pneumoniae can be overcome by the use of molecular methods for identification. Fonseca and colleagues have developed a multiplex PCR for the identification of K. pneumoniae, K. quasipneumoniae, and K. variicola using the intrinsic β-lactamase genes bla_SHV, bla_OKP, and bla_LEN, respectively. ³⁰ Accurate identification is important for epidemiology and phylogenetic studies.

Conclusion

This is the first report of colistin-resistant K. quasipneumoniae subsp. similipneumoniae harboring bla_KPC-9. This novel clone of K. quasipneumoniae signifies evolution of the species with acquisition of antimicrobial resistance. Accurate molecular identification of K. pneumoniae, K. quasipneumoniae, and K. variicola is essential to understand the epidemiology of drug resistance.

Availability of Data

The isolate was deposited at GenBank with accession number NQYU00000000 and the version described in this report is NQYU01000000.