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Article

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

by
Suraj Shukla
1,†,
Siddhi Desai
1,†,
Ashutosh Bagchi
2,
Pushpendra Singh
3,
Madhvi Joshi
4,*,
Chaitanya Joshi
4,
Jyoti Patankar
5,
Geeti Maheshwari
6,
Ekadashi Rajni
7,
Manali Shah
8 and
Devarshi Gajjar
1,*
1
Department of Microbiology and Biotechnology Centre, Faculty of Science, The Maharaja Sayajirao University of Baroda, Vadodara 390002, Gujarat, India
2
Amity Institute of Biotechnology, Amity University of Noida, Noida 201313, Uttar Pradesh, India
3
ICMR-National Institute of Research in Tribal Health, Jabalpur 482003, Madhya Pradesh, India
4
Gujarat Biotechnology Research Centre, Department of Science and Technology, Government of Gujarat, Gandhinagar 382011, Gujarat, India
5
Sterling Hospital, Vadodara 390007, Gujarat, India
6
Toprani Advanced Lab Systems, Vadodara 390020, Gujarat, India
7
Department of Microbiology, Mahatma Gandhi University of Medical Sciences & Technology, Jaipur 302015, Rajasthan, India
8
Desai Metropolis Health Service Pvt. Ltd., Surat 395001, Gujarat, India
*
Authors to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Antibiotics 2023, 12(3), 449; https://doi.org/10.3390/antibiotics12030449
Submission received: 16 January 2023 / Revised: 19 February 2023 / Accepted: 21 February 2023 / Published: 23 February 2023
(This article belongs to the Special Issue Antimicrobial Resistance and Drug Design—a Themed Issue in Honor of Professor Kim Lewis)
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Abstract

:
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.

1. 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 as a multidrug-resistant (MDR) pathogen. Further, Kp poses a great concern since the acquisition of plasmids and transposons carrying antibiotic resistance genes are not only restricted to horizontal transfer to other Kp strains but also other enteric bacteria [1]. The broad host range plasmids (IncX3, IncA/C, IncN, and IncL) acquired by Kp have made it the harbinger of resistance determinants among other enterobacteria [2]. Consequently, Kp was declared as a “Priority 1: CRITICAL” pathogen by the World Health Organization (WHO) in 2017 [3], while it was listed in the Indian priority pathogen list (IPPL) in 2021 [4], due to the increasing antibiotic resistance (ABR), including last-resort antibiotics such as carbapenems, colistin, and tigecycline. Further, carbapenem-resistant Enterobacteriaceae (CRE) are emerging at an alarming rate; hence, surveillance studies of MDRKp have become highly important.
The epidemiological features of carbapenem-resistant Kp have been previously reviewed [5]. The global presence of carbapenem resistance in Kp is mainly due to the presence of isolates containing class A-type β-lactamase (blaKPC), class B-type metallo-β-lactamase (blaNDM), and class D-type oxacillinase (blaOXA-48), with geographical variation. For example, Greece, Taiwan, Columbia, the USA, Canada, and China have many more strains that produce blaKPC and blaNDM than strains that produce blaOXA-48, which are far less common in those nations. While in the Arabian Peninsula and India, blaOXA-48 and blaNDM producers are common while blaKPC producers are rare.
The resistance rate against carbapenems and extended-spectrum β-lactamases (ESBLs) has been worryingly increasing in India in the past few years. A report from India has manifested an increase in carbapenem resistance rates from 9% in 2008 to 44% in 2010 [6]. Another report from a tertiary hospital in India reported 24.6% resistance to ESBLs in 2007 [7]. In 2017, carbapenem resistance was reported as high as 44% in India [8]. In 2018, a significant rise in the resistance level of ESBLs (45.1–93.1%) was reported in India, and the highest resistance of 84.9% was reported against cephalosporins [9]. A report from North India suggested a 29.4% resistance rate against carbapenems [10]. Recently, the overall prevalence of multidrug resistance (MDR: isolate that has developed resistance to at least one antimicrobial agent from three or more antimicrobial categories [11]) among Indian Kp isolates was reported at 58.0%. Further, it was also reported that tigecycline and colistin were the most effective drugs so far [12]. However, extensively drug-resistant (XDR: resistant to at least one agent in all antimicrobial groups except two or fewer, i.e., bacterial isolates remain susceptible to only one or two categories [11]) isolates with co-resistance against carbapenem and colistin [8,13] and pandrug-resistant (PDR: resistant to any antimicrobial agent [11]) isolates have also recently been reported in India [14].
Genome analysis of multidrug-resistant organisms provides us with important information about the phylogroup and the number of genes and plasmids in MDR bacteria at the same time. The multi-locus sequence typing (MLST) aids in identifying region-specific sequence types (STs) and their association with AMR genes. In recent times, extensive sequencing of pure genomes of Kp has led to huge momentum in providing a consolidated snapshot of resistance. Here, we report a comparison of all whole-genome sequences of Kp genomes from India. We leveraged a collection of 209 genomes including genomes of our 22 isolates available in the PATRIC database (https://www.patricbrc.org, accessed on 25 September 2021) and reported the most prevalent MLST, plasmid replicons, and AMR genes and their location in genomes for Indian isolates.

2. 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).

2.1. Distribution of Sequence Types (STs)

We investigated the genomic diversity of Klebsiella pneumoniae genomes circulating in the Indian subcontinent using genomes (n = 209) reported from 2012 to early 2021 (Figure 1). The analysis revealed that 50 different sequence types were circulating in India, of which ST231 (33.49%, n = 70) is the most prevalent, followed by ST147 (10.04%, n = 21), ST2096 (7.17%, n = 15), and ST14 (5.74%, n = 12). Other STs, such as ST43, ST395, ST16, ST11, ST15, ST23, ST35, ST48, ST101, ST307, ST437, ST515, ST420, and ST42, were found in at least 2 genomes, and 32 different STs were found individually in single genomes. SBS4 had unique combinations of 7 housekeeping genes (gapA:198, infB:1, mdh:2, pgi:1, phoE:10, rpoB:4, tonB:764), which was submitted to the Institut Pasteur MLST database and identified as novel ST5438. Regarding the origin of the genomes, majority of the genomes have human origin (92.82%, n = 194), of which 49.28% (n = 103) of isolates belonged to blood stream infections (BSIs), followed by respiratory tract infections (RTIs) (27.75%, n = 58), urinary tract infections (UTIs) (8.61%, n = 18), and the central nervous system (CNS) (0.95%, n = 2), while a single isolate was found for each infection, such as nosocomial, wound, sepsis, and implant infection. Here, 6.69% were of environmental origin (n = 14). The origins for 9 genomes were not found and these are mentioned in Figure 1 as not available.
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.

2.2. Distribution of Carbapenemases

The percentage of strains harboring the β-lactamase genes and categorization of β-lactamase genes (carbapenemases, ESBLs, BSBLs, and other β-lactamases) was performed based on their functional properties available in the Beta-Lactamase Database (http://bldb.eu/, accessed on 15 May 2022) (Figure 3). Distribution of carbapenemase genes was observed to vary with different STs (Figure 1 and Figure 4). The carbapenemase genes, blaOXA-48-like (blaOXA-48, blaOXA-232, and blaOXA-181), were found to be the most circulating among Indian isolates, where blaOXA-232 was the most prevalent carbapenemase, found in 46.41% (n = 97) of the total genomes. blaOXA-232 was most frequently carried by ST231 genomes (61.85%, n = 60). Besides ST231, ST2096 (12.37%, n = 12) and ST14 (8.24%, n = 8) genomes also carried blaOXA-232 and the remaining 18% of blaOXA-232 were carried by ST147, ST395, ST23, and ST437 genomes. Four variants of blaNDM were detected, namely, blaNDM-1, blaNDM-4, blaNDM-5, and blaNDM-7. Among these, blaNDM-5 was observed to be most prevalent (18.18%, n = 38), followed by blaNDM-1 (5.74%, n = 12). blaNDM-4 and blaNDM-7 were each found only in a single isolate, belonging to ST11 and ST711, respectively. A total of 28.94% (n = 11) of blaNDM-5 was carried by ST147, one of the clinically prevalent STs found in India. Besides this, blaNDM-5 was also detected in other clinically less prevalent STs: ST395, ST437, ST16, ST14, and ST101. blaNDM-1 was detected mainly in ST14 and ST11. Single isolates of ST231, ST147, ST273, ST624, and ST2816 also carried blaNDM-1, whereas ST231 genomes were not found to carry any blaNDM variants except for one genome with blaNDM-1. blaKPC-2 was also found to be present only in 4 genomes of ST147 and 1 genome of ST101. blaNDM and blaKPC were not observed to be as prevalent as blaOXA-232 in India. blaOXA-181 (11.48%, n = 24) was found mainly in ST43, ST147, and ST16. blaOXA-48 was found in only 2 genomes of ST101. Five different combinations of dual carabapenemase genes were also detected in a few genomes. blaNDM-5 + blaOXA-232 was detected in ST147 (n = 4), ST437 (n = 3), ST2096 (n = 2), ST395 (n = 2), and ST14 (n = 1), blaNDM-5 + blaOXA-181 was detected in ST16 (n = 5) and ST147 (n = 4), blaNDM-5 + blaOXA-48 was detected in ST101 (n = 2), blaNDM-1 + blaOXA-232 was detected in ST14 (n = 3), and blaNDM-1 + blaOXA-181 was seen in ST14 (n = 1), ST11 (n = 1), and ST42 (n = 1) (Figure 1).
Regarding the location of carbapenemase genes, all blaOXA-232 genes were located on plasmids except 2 genomes (EN5338 and DGL15). Two copies of the blaOXA-232 gene were detected in n = 2 genomes (C18 and SBS3). In n = 2 genomes (B35725 and SBS12), blaOXA-181 was located on the chromosome, while the rest were detected on plasmids. All blaNDM-5 were detected on plasmids among genomes except 2 genomes (B35725 and SBS12). BlaOXA-48 and blaKPC were present on plasmids in all genomes. All blaNDM-1 was detected on plasmids, while one genome (JNM8C2) carried 3 copies of blaNDM-1 on the plasmid.

2.3. Distribution of Extended-Spectrum β-Lactamases

Among various ESBLs, blaCTX-M-15 (80.38%, n = 168) was found to be the most abundantly present ESBL. The majority of ST231, ST147, ST2096, ST14, ST16, ST43, ST395, ST11, ST15, ST23, ST45, ST48, ST307, and ST437 genomes carried blaCTX-M-15. Only the less prevalent STs such as ST515, ST420, and ST101 did not carry blaCTX-M-15. blaCTX-M-15 was detected on both plasmids and chromosomes. In ST16 genomes, blaCTX-M-15 was located on plasmids, whereas in ST231 (n = 9), two copies of blaCTX-M-15 were located on the chromosomes. Further, one genome of ST231 (B28484) had three copies of blaCTX-M-15 located on the chromosome. A total of n = 4 genomes belonged to ST15, ST23, and ST152 carried a double copy of blaCTX-M-15 gene on their plasmids. Co-existence of blaCTX-M-15 and blaOXA-232 was observed in 39.7% (n = 83) of genomes. Mainly, genomes of ST231 (77.14%, n = 54/70) followed by ST2096 (66.66%, n = 10/15), ST14 (50%, n = 6/12), ST437 (100%, n = 3/3), and ST23 (66.66%, n = 2/3) exhibited co-existence of blaCTX-M-15 and blaOXA-232. Apart from blaCTX-M-15, another ESBL, blaOXA-1 (28.70%, n = 60), was also found to be predominant in genomes. blaOXA-1 was not detected in genomes of the prevalent ST231, and it was detected highest in ST2096 (21.66%, n = 13), followed by ST14 (18.33%, n = 11), ST147 (10%, n = 6), and ST395 (10%, n = 6). BlaOXA-1 was also detected in a few genomes of other prevalent STs, such as ST16, ST11, ST15, ST35, ST48, and ST307. One genome each of ST2096, ST48, and ST11, and three genomes of ST14 had the blaOXA-1 gene on the chromosome, while the rest was present on plasmids among STs.

2.4. Distribution of Broad-Spectrum β-Lactamases

For broad-spectrum β-lactamase (BSBL), blaTEM-1 (74.16%, n = 155) was most frequently found in ST231 (42.58%, n = 66), followed by ST147 (9.03%, n = 14), ST2096 (7.74%, n = 12), ST14 (5.80%, n = 9), ST16 (4.51%, n = 7), ST43 (3.87%, n = 6), and ST395 (3.22%, n = 5). The co-existence of blaOXA-232 with blaTEM-1 was found in 41.14% (n = 86) of genomes among different STs, while 65.55% (n = 137) of genomes were found to carry both blaCTX-M-15 and blaTEM-1. Co-occurrence of these three genes (carbapenemase—blaOXA-232, ESBLs—blaCTX-M-15, and BSBLs—blaTEM-1) was detected in 37.79% (n = 79) of the genomes. All blaTEM-1 genes among genomes were detected on plasmids, except 2 genomes each of ST231 and ST43. Apart from blaTEM-1, two other BSBLs, blaSHV-1 (37.79%, n = 79) and blaSHV-11 (25.35%, n = 53), were found to be the most circulating across genomes. blaSHV-1 was majorly carried by genomes of ST231 (72.15%, n = 57), followed by ST16 (5.06%, n = 4), ST101 (3.79%, n = 3), ST515 (3.79%, n = 3), and ST48 (2.53%, n = 2). Interestingly, we did not find any blaSHV-1 in the genomes of other prevalent STs, such as ST147, ST2096, ST14, ST43, ST395, ST11, ST15, ST23, ST35, ST307, and ST437. All blaSHV-1 genes were found to be located on chromosomes, except one genome (BA39950) of ST16 and one genome (BA33875) of ST101 detected with two copies of this gene, where one was on the chromosome while the other was on the plasmid. blaSHV-11 was detected mainly in ST147 (39.62%, n = 21), followed by ST43 (13.20%, n = 7) and ST395 (7.54%, n = 4). Apart from this, some other prevalent STs, such as ST11, ST23, and ST437, also carried blaSHV-11 in their genome, and notably the most prevalent ST231, along with ST2096, ST14, and ST16, did not carry the blaSHV-11 gene, except one genome of ST14 and ST16. blaSHV-11 was found to be located on chromosomes, except DGL12 (ST16), while MRK9 had two copies of blaSHV-11, of which one was present on the chromosome while the other was detected on the plasmid.

2.5. Other β-Lactamases

Apart from the most frequent carbapenemases, ESBLs, and BSBLs, some other β-lactamases such as blaampH, which is a penicillin-binding protein that is related to AmpC, were found highest (85.16%, n = 176) across genomes, and all blaampH genes were located on chromosomes. blaSHV-28 (13.39%, n = 28) was detected in ST2096, ST14, ST15, and ST307. All blaSHV-28 were present on chromosomes, except one genome (B32205) of ST14. Variants of blaSHV (blaSHV-5, blaSHV-12, blaSHV-25, blaSHV-27, blaSHV-31, blaSHV-33, blaSHV-36, blaSHV-60, blaSHV-71, blaSHV-75, blaSHV-187), blaCTX-M (blaCTX-M-163, blaCTX-M-238), blaTEM (blaTEM-214, blaTEM-243), and blaCMY (blaCMY-4, blaCMY-6, blaCMY-59), along with blaOXA-9, blaDHA-1, and blaLAP-2, were also found in a few genomes.

2.6. 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 (Figure 1 and Figure 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 (Figure 1 and Figure 5).

3. 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 blaOXA or blaNDM), and 19.20% (n = 29) of isolates were detected with dual carbapenemase genes in their genome. The two major types of carbapenemase genes, namely blaOXA-48-like and blaNDM1/5, were detected, and these were mainly associated with ST231 and ST147, respectively. To date (9 February 2023), 48 variants of blaOXA-48-like and 48 variants of blaNDM have been reported (https://www.ncbi.nlm.nih.gov/pathogens/refgene, accessed on 9 February 2023) so far, but fortunately only 4 variants of blaNDM and 3 variants of blaOXA-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 blaOXA-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 blaOXA-232 in ST231 can be correlated with the diverse set of mobile genetic elements found neighboring blaOXA-232/181, which include numerous insertion sequences and transposons of the Tn3 family [18].
blaOXA-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 blaOXA-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 blaOXA-232 and blaOXA-181, both belonging to the blaOXA-48-like group, we found 24 genomes that contained blaOXA-181 and 2 genomes of ST101 with blaOXA-48. blaOXA-48-producing Kp from Europe and Africa have been found to belong to ST395 [19]. blaOXA-181 is currently considered the second most common global blaOXA-48-like derivative after blaOXA-48 [25]. Until 2007, blaOXA-181 was considered endemic in India as it was reported as the most common blaOXA-48-like carbapenemase. However, it is possible that blaOXA-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 blaOXA-232 and blaOXA-181 are endemic in India with a higher prevalence of blaOXA-232 (as observed in the present study). blaOXA-48 is currently the most common blaOXA-48-like enzyme globally, followed by blaOXA-181 [25]. It is interesting to find that though blaOXA-181 was first reported in India, its prevalence is currently less compared to blaOXA-232. blaOXA-232 differs from blaOXA-181 by a single amino acid substitution, and the genetic environment surrounding the blaOXA-232 was initially very similar to the environment surrounding blaOXA-181 [26]. The similarities of the genes, transposons, and plasmids between blaOXA-181 and blaOXA-232 suggested a common origin and transposition followed by the subsequent evolution of blaOXA-232 from blaOXA-181. However, in the last decade, the genetic environment of blaOXA-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 blaOXA-232 (particularly in ST231) and not blaOXA-181/48 in India.
Another important observation in the present study was the selected number (n = 29) of isolates that co-harbored blaOXA-48-like and blaNDM-1/5. The combination of blaNDM-5 + blaOXA-232 was highest (n = 11) among 29 dual-producers. Only 2 genomes (B35725 and SBS12) of ST147 with the blaOXA-181 + blaNDM-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 blaOXA-181 were seen to co-exist with blaNDM-5. Dual carbapenemase producers have also been reported in South Korea [27], Italy [28], Saudi Arabia [29], Iran [30], and Algeria [31]. blaNDM-5 was mostly carried by ST147 genomes that belonged to bloodstream infections from Tamil Nadu, while other genomes of ST147 were found to carry blaKPC-2 that belonged to respiratory tract infections from West Bengal. The plasmid replicon IncFIB(K)(pCAV1099-114) was present only in those blaKPC-2-producing isolates of ST147. A total of 65.78% (n = 25/38) of blaNDM-5 co-existed with blaOXA-48-like. Patients from South Korea, The United States, and Nepal who had traveled to India or the Indian subcontinent were reported to have blaOXA-48-like and blaNDM-5 [27,32,33]. The presence of carbapenemase duplex (blaOXA-48-like and blaNDM-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 blaNDM-producing Kp and found that blaNDM was present in all 5 continents and dispersed among numerous STs [34]. The lack of any dominating lineages suggests that there are not any blaNDM-positive Kp clones that are obviously high risk. blaNDM-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 carbapenem-resistant Kp in China, mostly carries blaKPC-2 rather than blaNDM [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 blaNDM, their prevalence across several nations calls for more research.
Although blaNDM 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 blaNDM, and the Enterobacteriaceae include 20 different replicon types of blaNDM-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 blaNDM on several occasions, and it also emphasizes the worrisome fact that many different plasmids are involved in the horizontal transfer of blaNDM. We also found ambiguity regarding the location of blaCTX-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.

4. Materials and Methods

4.1. 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).

4.2. Genomic DNA Extraction, NGS Library Preparation, and Whole Genome Sequencing

Genomic DNA for 22 isolates of Kp was extracted using the XpressDNA Bacterial kit (MagGenome, Chennai, India). Whole genome sequencing of 6 isolates (SBS1, SBS3, SBS4, SBS5, SBS11, and SBS12) was performed using the Ion Torrent (S5-0083-GGI) NGS platform (ThermoFisher Scientific, Waltham, MA, USA), and another 16 isolates (DGL1, DGL2, DGL5, DGL6, DGL7, DGL8, DGL9, DGL10, DGL11, DGL12, DGL13, DGL14, DGL15, DGL16, DGL17, and DGL18) were sequenced using the Illumina MiSeq platform (Illumina, CA, USA). The Ion Xpress™ Plus gDNA fragment library preparation kit (ThermoFisher Scientific, MA, USA) and the Nextera XT DNA Library Prep Kit (Illumina, San Diego, CA, USA) were used to prepare NGS libraries, respectively, as per the instructions in the manuals. The sequences were submitted to NCBI, and the accession number for each sequence is included in Table 1.

4.3. Analysis of Whole Genome Sequencing Data

Raw data were analyzed using FastQC (https://www.bioinformatics.babraham.ac.uk/projects/fastqc/, accessed on 2 December 2020) and filtered using the FastX toolkit (http://hannonlab.cshl.edu/fastx_toolkit/, accessed on 2 December 2020). Filtered reads were assembled de novo using SPAdes v3.14.1 (http://cab.spbu.ru/software/spades/, accessed on 4 December 2020) with read error correction and assembling mode and a threads value of 32. Quality assessment of assembled sequences was performed using QUAST v5.0.2 (http://quast.sourceforge.net/quast, accessed on 5 December 2020). Assembled genomes were annotated using Prokka v1.13.3 (https://github.com/tseemann/prokka, accessed on 7 December 2020). The assembly data were deposited on NCBI (Bioproject No: PRJNA694019)

4.4. 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.
MLST profiles for all isolates in the PATRIC database were mentioned in the metadata, except for a few isolates, and sequence types for our (n = 22) isolates and those remaining from the PATRIC database were determined using MLST v2.0.4 of the Centre for Genomic Epidemiology (CGE) toolbox (https://cge.cbs.dtu.dk/services/MLST/, accessed on 17 November 2021). For detection of STs, 7 housekeeping genes (gapA, infB, mdh, pgi, phoE, rpoB, tonB) and their allelic combinations were used to generate STs. All isolates (n = 209) were analyzed to detect the β-lactamase genes as well as the copy numbers (analyzing ORF_ID, Contig, and Start and Stop position in genomes) using the Resistance Gene Identifier (RGI) from The Comprehensive Antibiotic Resistance Database (CARD) (https://card.mcmaster.ca/analyze/rgi, accessed on 17 November 2021). The location of β-lactamase genes in the genome was detected using mlplasmids v2.1.0 (https://sarredondo.shinyapps.io/mlplasmids/, accessed on 5 December 2022) (Arredondo-Alonso et al., 2018). Plasmids were detected using PlasmidFinder v2.0.1 of the CGE toolbox (https://cge.cbs.dtu.dk/services/PlasmidFinder/, accessed on 15 December 2021). The single-nucleotide polymorphism (SNP)-based phylogenetic tree was generated using CSI Phylogeny 1.4, and MGH78578 was used as a reference strain. iTOL v6 (https://itol.embl.de/, accessed on 10 February 2023) was used to visualize the phylogeny and genomic profiles of isolates.

5. 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, blaOXA-232 was the most circulating, followed by blaNDM-5, while blaCTX-M-15 was highest among ESBLs followed by blaSHV-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.

Author Contributions

Conceptualization, S.S., S.D. and D.G.; methodology, S.S., A.B. and D.G.; investigation, S.S., S.D., A.B. and D.G.; resources, M.J., C.J., J.P., G.M., M.S., E.R. and D.G.; data curation, S.S., S.D., P.S. and D.G.; writing—original draft, S.S. and S.D.; writing—review and editing, P.S. and D.G.; supervision, M.J., C.J. and D.G.; project administration, M.J., C.J., and D.G.; funding acquisition, D.G. and M.J. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by Gujarat State Biotechnology Mission (GSBTM), grant number GSBTM/JDRD/584/2018/199 & GSBTM/JD(R&D)/616/21-22/00028386 for Network Program on AMR superbugs and one health: Human health.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Data will be available upon request.

Acknowledgments

We thank Heli Upadhyaya for her assistance in data handling. We also acknowledge GSBTM for Funding and Gujarat Biotechnology Research Centre (GBRC) for providing sequencing facilities. We thank Pranav Desai, Metropolis Lab, and Surat and Tushar Toprani, Toprani Lab, for providing isolates.

Conflicts of Interest

The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

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Figure 1. 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.
Figure 1. 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.
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Figure 2. Geographic location of genome collection and state-wise ST distribution. ST found in at least 2 genomes are included in this figure. (n) Represents the number of genomes, and colors represent the states: carrot red (n = 128)—Tamil Nadu; mustard yellow (n = 22)—Gujarat; cyan (n = 21)—West Bengal; yellow (n = 11)—New Delhi; pink (n = 10)—Assam; purple (n = 4)—Kerala; sky blue (n = 4)—Maharashtra; pastel green (n = 3)—Madhya Pradesh; orange (n = 2)—Uttar Pradesh; green (n = 2)—Punjab; brown (n = 1)—Orrisa. Digits in red fonts in brackets denote the number of genomes having respective STs. MapChart online tool was used to generate this figure.
Figure 2. Geographic location of genome collection and state-wise ST distribution. ST found in at least 2 genomes are included in this figure. (n) Represents the number of genomes, and colors represent the states: carrot red (n = 128)—Tamil Nadu; mustard yellow (n = 22)—Gujarat; cyan (n = 21)—West Bengal; yellow (n = 11)—New Delhi; pink (n = 10)—Assam; purple (n = 4)—Kerala; sky blue (n = 4)—Maharashtra; pastel green (n = 3)—Madhya Pradesh; orange (n = 2)—Uttar Pradesh; green (n = 2)—Punjab; brown (n = 1)—Orrisa. Digits in red fonts in brackets denote the number of genomes having respective STs. MapChart online tool was used to generate this figure.
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Figure 3. Percentage distribution of β-lactamase genes among strains. Bars in the graph indicate individual β-lactamase genes, in which bars in green indicate the carbapenemase gene, orange indicates ESBLs, blue indicates BSBLs, and purple indicates other β-lactamase genes. The GraphPad Prism 8.4.2 tool was used to create this graph.
Figure 3. Percentage distribution of β-lactamase genes among strains. Bars in the graph indicate individual β-lactamase genes, in which bars in green indicate the carbapenemase gene, orange indicates ESBLs, blue indicates BSBLs, and purple indicates other β-lactamase genes. The GraphPad Prism 8.4.2 tool was used to create this graph.
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Figure 4. Distribution of carbapenemase genes circulating in India. CRP—carbapenem-resistant pattern.
Figure 4. Distribution of carbapenemase genes circulating in India. CRP—carbapenem-resistant pattern.
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Figure 5. Heatmap of percentage distribution of plasmid replicons among STs. (n) Indicates the total number of particular STs. On the scale, dark blue indicates 100%, white indicates 0%. The GraphPad Prism 8.4.2 tool was used to generate this heatmap.
Figure 5. Heatmap of percentage distribution of plasmid replicons among STs. (n) Indicates the total number of particular STs. On the scale, dark blue indicates 100%, white indicates 0%. The GraphPad Prism 8.4.2 tool was used to generate this heatmap.
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Table
Table 1. List of isolates (strain) with resistance category and genome accession number submitted in NCBI (Bioproject no: PRJNA694019).
Table 1. List of isolates (strain) with resistance category and genome accession number submitted in NCBI (Bioproject no: PRJNA694019).
StrainResistance CategoryGenome Accession No.StrainResistance CategoryGenome Accession No.
SBS1XDRJAFFRJ000000000DGL8PDRJAJBAG000000000
SBS3XDRJAFFRI000000000DGL9XDRJAJBAF000000000
SBS4MDRJAFFRH000000000DGL10XDRJAJBAE000000000
SBS5XDRJAFFRG000000000DGL11XDRJAJBAD000000000
SBS11MDRJAFFRA000000000DGL12XDRJAJBAC000000000
SBS12PDRJAFFQZ000000000DGL13XDRJAJBAB000000000
DGL1MDRJAJBAN000000000DGL14XDRJAJBAA000000000
DGL2MDRJAJBAM000000000DGL15XDRJAJAZZ000000000
DGL5PDRJAJBAJ000000000DGL16XDRJAJAZY000000000
DGL6XDRJAJBAI000000000DGL17XDRJAJAZX000000000
DGL7XDRJAJBAH000000000DGL18XDRJAJAZW000000000
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MDPI and ACS Style

Shukla, S.; Desai, S.; Bagchi, A.; Singh, P.; Joshi, M.; Joshi, C.; Patankar, J.; Maheshwari, G.; Rajni, E.; Shah, M.; et al. Diversity and Distribution of β-Lactamase Genes Circulating in Indian Isolates of Multidrug-Resistant Klebsiella pneumoniae. Antibiotics 2023, 12, 449. https://doi.org/10.3390/antibiotics12030449

AMA Style

Shukla S, Desai S, Bagchi A, Singh P, Joshi M, Joshi C, Patankar J, Maheshwari G, Rajni E, Shah M, et al. Diversity and Distribution of β-Lactamase Genes Circulating in Indian Isolates of Multidrug-Resistant Klebsiella pneumoniae. Antibiotics. 2023; 12(3):449. https://doi.org/10.3390/antibiotics12030449

Chicago/Turabian Style

Shukla, Suraj, Siddhi Desai, Ashutosh Bagchi, Pushpendra Singh, Madhvi Joshi, Chaitanya Joshi, Jyoti Patankar, Geeti Maheshwari, Ekadashi Rajni, Manali Shah, and et al. 2023. "Diversity and Distribution of β-Lactamase Genes Circulating in Indian Isolates of Multidrug-Resistant Klebsiella pneumoniae" Antibiotics 12, no. 3: 449. https://doi.org/10.3390/antibiotics12030449

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