Diversity and Distribution of β-Lactamase Genes Circulating in Indian Isolates of Multidrug-Resistant Klebsiella pneumoniae

Klebsiella pneumoniae (Kp) has gained prominence in the last two decades due to its global spread as a multidrug-resistant (MDR) pathogen. Further, carbapenem-resistant Kp are emerging at an alarming rate. The objective of this study was (1) to evaluate the prevalence of β-lactamases, especially carbapenemases, in Kp isolates from India, and (2) determine the most prevalent sequence type (ST) and plasmids, and their association with β-lactamases. Clinical samples of K. pneumoniae (n = 65) were collected from various pathology labs, and drug susceptibility and minimum inhibitory concentrations (MIC) were detected. Whole genome sequencing (WGS) was performed for n = 22 resistant isolates, including multidrug-resistant (MDR) (n = 4), extensively drug-resistant (XDR) (n = 15), and pandrug-resistant (PDR) (n = 3) categories, and genomic analysis was performed using various bioinformatics tools. Additional Indian MDRKp genomes (n = 187) were retrieved using the Pathosystems Resource Integration Center (PATRIC) database. Detection of β-lactamase genes, location (on chromosome or plasmid), plasmid replicons, and ST of genomes was carried out using CARD, mlplasmids, PlasmidFinder, and PubMLST, respectively. All data were analyzed and summarized using the iTOL tool. ST231 was highest, followed by ST147, ST2096, and ST14, among Indian isolates. blaampH was detected as the most prevalent gene, followed by blaCTX-M-15 and blaTEM-1. Among carbapenemase genes, blaOXA-232 was prevalent and associated with ST231, ST2096, and ST14, which was followed by blaNDM-5, which was observed to be prevalent in ST147, ST395, and ST437. ST231 genomes were most commonly found to carry Col440I and ColKP3 plasmids. ST16 carried mainly ColKP3, and Col(BS512) was abundantly present in ST147 genomes. One Kp isolate with a novel MLST profile was identified, which carried blaCTX-M-15, blaOXA-1, and blaTEM-1. ST16 and ST14 are mostly dual-producers of carbapenem and ESBL genes and could be emerging high-risk clones in India.


Introduction
Klebsiella pneumoniae (Kp), a member of the Enterobacteriaceae family, is one of the commensal organisms in the gastrointestinal tract of healthy humans and animals [1]. Since the last two decades, Kp has gained importance because of its worldwide spread

Results
We collected Kp isolates (n = 65) from Gujarat, of which n = 22 isolates were found to be resistant to multiple antimicrobial agents of various classes, and these were used for WGS analysis. Among the total 22 isolates, n = 3, n = 15, and n = 4 isolates were identified as PDR, XDR, and MDR, respectively (Table 1). PDR strains (SBS12, DGL5, DGL8) were resistant to all antimicrobial agents in all classes. All PDR isolates were resistant to colistin and tigecycline, with MIC values of ≥4 µg/mL and ≥2 µg/mL, respectively. Majority of the XDR isolates (DGL6, DGL7, DGL9, DGL10, DGL11, DGGL12, DGL13, DGL14, DGL15, DGL16, DGL17) were resistant to all antibiotics, including tigecycline, with a MIC of ≥2 µg/mL, and only remained susceptible against colistin. Two XDR isolates (SBS3, SBS5) were resistant to all antimicrobial agents except for colistin and tigecycline (Supplementary Table S1). Table 1. List of isolates (strain) with resistance category and genome accession number submitted in NCBI (Bioproject no: PRJNA694019).

Strain
Resistance Category Genome Accession No. Strain Resistance Category Genome Accession No.
Regarding the state-wise distribution of Klebsiella genomes, the highest number of genomes (63.15%, n = 132) was reported from the southern states of India (Tamil Nadu n = 128 and Kerala n = 4), followed by 15.13% from Eastern states (n = 32) (West Bengal n = 21, Assam n = 10, and Odisha n = 1), 12.44% from Western states (n = 26) (Gujarat n = 22 and Maharashtra n = 4), 7.17% from Northern states (n = 15) (New Delhi n = 11, Uttar Pradesh n = 2, and Punjab n = 2), and 1.43% from the central part of India (n = 3) (Madhya Pradesh) ( Figure 2). The geographic location for one isolate (Strain: ME30) was not available. ST231 was observed to be the most prevalent ST among all regions of India. ST147 was detected in Tamil Nadu, Gujarat, West Bengal, and Uttar Pradesh, while ST2096, ST16, and ST42 were detected in Tamil Nadu and Gujarat. Presence of ST14 was detected in Tamil Nadu, West Bengal, Gujarat, and New Delhi, whereas ST307 was only found in Assam. State-wise distribution for STs is shown in Figure 2. Genomic analysis of β-lactamase genes and plasmid replicons circulating in Kp genomes from India. Strains in yellow denote the lab isolates, black denotes retrieved genomes. Circles with filled color (blue-carbapenemase, yellow-ESBLs, purple-BSBLs, and green-other β-lactamases) for beta-lactamase genes denote the presence of genes, unfilled denotes the absence of genes. C inside a circle denotes the location of genes on the chromosome, without C denotes genes on plasmids. Red ring outside the circle denotes 3 copies of the gene, dark red square outside the circle denotes 2 copies of the gene. C and P denote that one is on chromosome and another is on the plasmid. For plasmids, red color-filled square denotes the presence of a plasmid, unfilled denotes the absence. iTOL was used to create this image.

Plasmid Replicons among Dominant STs
A diverse set of plasmid combinations were found to be circulating in Indian genomes and different plasmid combinations were observed to be associated with specific STs (Figures 1 and 5). For instance, most ST231 genomes carried Col440I and ColKP3, while few genomes of ST231 carried ColRNAI. ColRNAI was mainly associated with ST2096 and ST23. ST14 and ST2096 mainly carried ColKP3. Col(BS512) was abundantly present in ST147 genomes, while not frequently carried by ST231, ST14, and ST2096. ColpVC was seen to be associated mainly with ST43 and little with ST147 and ST437. Among prevalent STs, Col440II was carried by ST16, ST23, and ST101. Regarding IncF plasmids, different sets of IncF plasmids were observed to be associated with different STs. IncFIA, IncFIB(pQil), IncFII(K), and IncF(pAMA1167-NDM-5) were abundantly present in ST231 genomes, whereas ST14 harbored only IncFII(K) and IncFIB(K), which were not carried by ST231. A few STs: ST395, ST147, ST43, and ST11, were found to be associated with IncFIB(pQil). IncHI1B(pNDM-MAR) was carried mainly by ST2096, ST14, and ST43. IncFII was carried by the genomes of ST147, ST395, ST14, and ST16. IncFIB(pNDM-Mar) was detected mainly in ST2096 and ST14. IncR was observed to be strongly associated with ST147, while IncFIB(pKPHS1), IncFII(pKPX1), and ColpVC were also detected in genomes of ST147 (Figures 1 and 5).

Discussion
β-lactams are widely prescribed antibiotics for treating Klebsiella infections, and carbapenems are one of the last-resort drugs used to treat highly resistant strains. Public health is currently under immediate threat from the advent of Kp that is resistant to carbapenems. To track infections and resistance quickly and affordably, WGS is being employed more and more in research and public health labs. In the present study, 72.24% (n = 151) of isolates were detected with at least one carbapenemase gene (either bla OXA or bla NDM ), and 19.20% (n = 29) of isolates were detected with dual carbapenemase genes in their genome. The two major types of carbapenemase genes, namely bla OXA-48-like and bla NDM1/5 , were detected, and these were mainly associated with ST231 and ST147, respectively. To date (9 February 2023), 48 variants of bla OXA-48-like and 48 variants of bla NDM have been reported (https://www.ncbi.nlm.nih.gov/pathogens/refgene, accessed on 9 February 2023) so far, but fortunately only 4 variants of bla NDM and 3 variants of bla OXA-48-like were detected in Kp genomes circulating in India.
Recently, Kp genomic surveillance studies from India were reported mostly from South India [15,16]. To the best of our knowledge, in this report, Klebsiella genomes from Western India were included for the first time in a surveillance study. Our finding regarding the most common STs, ST231, followed by ST147, corroborates with these studies; however, the third most common ST we found was ST2096, while it was ST14 in the earlier studies. Next, we found bla OXA-232 as the most prevalent carbapenemase and it was mostly associated with ST231. Similar observations have been reported from South and North India [15,17]. The rapid dissemination of bla OXA-232 in ST231 can be correlated with the diverse set of mobile genetic elements found neighboring bla OXA-232/181 , which include numerous insertion sequences and transposons of the Tn3 family [18].
bla OXA-232 was first identified in Kp and E. coli isolated from three patients who had been transported from India to France in 2011 [19]. Since then, outbreaks of bla OXA-232 Kp have been reported worldwide and diverse STs have been identified, including ST14 and ST15 in China [20], ST16 in Thailand [21], ST147 in Germany [22], ST231 and ST2096 in France [23], ST307 and ST101 in The Netherlands [24], and ST437 and ST395 in India. For bla OXA-232 and bla OXA-181 , both belonging to the bla OXA-48-like group, we found 24 genomes that contained bla OXA-181 and 2 genomes of ST101 with bla OXA-48 . bla OXA-48 -producing Kp from Europe and Africa have been found to belong to ST395 [19]. bla OXA-181 is currently considered the second most common global bla OXA-48-like derivative after bla OXA-48 [25]. Until 2007, bla OXA-181 was considered endemic in India as it was reported as the most common bla OXA-48-like carbapenemase. However, it is possible that bla OXA-181 was misreported because of a biased pool of samples from a few centers that had molecular diagnosis facilities. From the current scenario, it is evident that Kp with bla OXA-232 and bla OXA-181 are endemic in India with a higher prevalence of bla OXA-232 (as observed in the present study). bla OXA-48 is currently the most common bla OXA-48-like enzyme globally, followed by bla OXA-181 [25]. It is interesting to find that though bla OXA-181 was first reported in India, its prevalence is currently less compared to bla OXA-232 . bla OXA-232 differs from bla OXA-181 by a single amino acid substitution, and the genetic environment surrounding the bla OXA-232 was initially very similar to the environment surrounding bla OXA-181 [26]. The similarities of the genes, transposons, and plasmids between bla OXA-181 and bla OXA-232 suggested a common origin and transposition followed by the subsequent evolution of bla OXA-232 from bla OXA-181 . However, in the last decade, the genetic environment of bla OXA-232 (especially from India) has attained vast diversity, as suggested by the MGEs found associated with it [18]. Future studies using long-read sequencing are warranted for a greater number of isolates from India to give a detailed understanding of the exchanges occurring that lead to the successful dissemination of bla OXA-232 (particularly in ST231) and not bla OXA-181/48 in India.
Another important observation in the present study was the selected number (n = 29) of isolates that co-harbored bla OXA-48-like and bla NDM-1/5 . The combination of bla NDM-5 + bla OXA-232 was highest (n = 11) among 29 dual-producers. Only 2 genomes (B35725 and SBS12) of ST147 with the bla OXA-181 + bla NDM-5 combination were found to carry both carbapenemase genes on the chromosome, and the rest of the dual-producers had both genes on plasmids. Genomes of ST16 in this study with bla OXA-181 were seen to co-exist with bla NDM-5 . Dual carbapenemase producers have also been reported in South Korea [27], Italy [28], Saudi Arabia [29], Iran [30], and Algeria [31]. bla NDM-5 was mostly carried by ST147 genomes that belonged to bloodstream infections from Tamil Nadu, while other genomes of ST147 were found to carry bla KPC-2 that belonged to respiratory tract infections from West Bengal. The plasmid replicon IncFIB(K)(pCAV1099-114) was present only in those bla KPC-2 -producing isolates of ST147. A total of 65.78% (n = 25/38) of bla NDM-5 co-existed with bla OXA-48-like . Patients from South Korea, The United States, and Nepal who had traveled to India or the Indian subcontinent were reported to have bla OXA-48-like and bla NDM-5 [27,32,33]. The presence of carbapenemase duplex (bla OXA-48-like and bla NDM-1/5 ) among the genomes could lead to pan-carbapenem-resistant isolates, and hence conditions leading to the origination of such duplexes need to be addressed for tackling them. A group evaluated the worldwide spread and genotype distribution of human clinical isolates of bla NDM -producing Kp and found that bla NDM was present in all 5 continents and dispersed among numerous STs [34]. The lack of any dominating lineages suggests that there are not any bla NDM -positive Kp clones that are obviously high risk. bla NDM -positive Kp strains are frequently reported to be associated with ST14 [35][36][37]. Another prevalent sequence type in many investigations is ST11 [35,36]. It should be noted that ST11, which is the most common ST of carbapenemresistant Kp in China, mostly carries bla KPC-2 rather than bla NDM [38]. Even though there is not enough evidence to prove that ST11, ST14, ST15, and ST147 are epidemic clones that are mediating the global spread of bla NDM , their prevalence across several nations calls for more research.
Although bla NDM has been found on bacterial chromosomes [14,39], the majority of carriage occurs on plasmids, which are essential for dissemination. Several different plasmid replicon types have been identified to carry bla NDM , and the Enterobacteriaceae include 20 different replicon types of bla NDM -carrying plasmids, including the IncC, IncB/O/K/Z, IncFIA, IncFIB, IncFIC, IncFIII, IncHI1, IncHI2, IncHI3, IncN, IncN2, IncL/M, IncP, IncR, IncT, IncX1, IncX3, IncX4, IncY, and ColE10 types [25,37,39]. This indicates that different plasmids have acquired bla NDM on several occasions, and it also emphasizes the worrisome fact that many different plasmids are involved in the horizontal transfer of bla NDM . We also found ambiguity regarding the location of bla CTX-M-15 among genomes that is still unclear, while other β-lactamase genes were dominantly found on either the plasmid or chromosome.
There are a few shortcomings of this study; first, the antibiotic susceptibility data of publicly available genomes were not included for the analysis as the data were not available; second, this is a biased population of isolates that were randomly selected by the respective research/clinical lab, and third, majority of the isolates belonged to South India, especially Tamil Nadu. Hence, there is a need for genome-based surveillance from other parts of the country. Further, the exact correlation as well as copy number of bla genes with respective plasmids/chromosomes can only be achieved by long-read sequencing, and this is warranted for future work.

Sample Collection, Identification, and Antibiotic Susceptibility Testing
Clinical samples (n = 65) were collected from various pathology labs of Gujarat, India. This included samples from urine, blood, sputum, stool, broncho-alveolar fluid (BAL), wound swab, and endotracheal aspirate (ET). The minimum inhibitory concentration (MIC) of colistin and tigecycline was performed using the broth dilution method. Resistance against other antibiotic classes (penicillins, cephalosporins, carbapenems, monobactams, fluoroquinolones, aminoglycosides, tetracyclines, phenicols, and inhibitors of the folic acid pathway) was detected using the disk diffusion method. Results were interpreted as per Clinical and Laboratory Standards Institute guidelines [40] (data provided in Supplementary Table S1). Isolates were then classified as MDR, XDR, and PDR (Table 1) as per the classification criteria reported by Magiorakos et al. [11]. The criteria for defining MDR, XDR, and PDR are as follows: MDR: non-susceptible to >1 agent in >3 antimicrobial categories, XDR: non-susceptible to >1 agent in all but <2 categories, i.e., isolates remain susceptible to only one or two categories, and PDR: non-susceptible to all antimicrobial agents [11]. To explore the resistome and plasmidome of these resistant isolates from all 3 resistant categories, we performed whole genome sequencing (WGS).  Table 1.

Retrieval and Analysis of Publicly Available Genomes of Klebsiella Pneumoniae
Publicly available genome sequences of MDR Kp (n = 187) deposited from India were downloaded from the PATRIC database (https://www.patricbrc.org/, accessed on 25 September 2021). To access genomes deposited from India with antimicrobial resistance properties, filters such as 'India', 'Antimicrobial resistance', and 'Genome quality: good' were used. Recently, in December 2022, this database was merged with the Bacterial and Viral Bioinformatics Resource Center (https://www.bv-brc.org/, accessed on 8 February 2023). The sequences in FASTA format, which were previously assembled and had good quality, were retrieved and used for the combination analysis with our samples. Strains' information such as collection year, geographic location, sample origin, and host health for each genome was also collected from PATRIC.

Conclusions
Collectively, this is the first surveillance analysis of carbapenem-resistant Kp Pan-India genomes, which includes WGS of 194 clinical, 14 environmental, and 1 unknown strain that were collected from various geographic locations of India between 2012 and early 2021. In this surveillance analysis of MDR Kp circulating in India, ST231 was found to be the most predominant ST. We also identified one novel ST5438 in our isolates. In regards to carbapenemase genes, bla OXA-232 was the most circulating, followed by bla NDM-5 , while bla CTX-M-15 was highest among ESBLs followed by bla SHV-1 , and these genes can be targeted for diagnostic purposes. IncFII(K) was the most frequent plasmid replicon belonging to the Inc type, while ColKP3 was detected as the second most prevalent among Col-type plasmids. ST147 is already known as a high-risk clone globally, while ST16 and ST14 from this study, which are mostly dual-producers of carbapenem and ESBL genes, could be emerging high-risk clones in India. Our study suggests an unmet need of future large-scale, multi-regional genomic surveillance of multidrug-resistant Kp isolates with collaboration across different states of India, especially from Northern and Western India.
Supplementary Materials: The following supporting information can be downloaded at: https:// www.mdpi.com/article/10.3390/antibiotics12030449/s1, Table S1: Antibiotic susceptibility and categorization (MDR, XDR, and PDR) of isolates. Data Availability Statement: Data will be available upon request.