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. 2023 Apr 26;13(5):139. doi: 10.1007/s13205-023-03556-5

NDM-5-carrying Klebsiella pneumoniae ST437 belonging to high-risk clonal complex (CC11) from an urban river in eastern India

Saubhagini Sahoo 1, Rajesh Kumar Sahoo 1, Sangita Dixit 1, Dibyajyoti Uttameswar Behera 1, Enketeswara Subudhi 1,✉
PMCID: PMC10133422  PMID: 37124981

Abstract

In this study, we described the carbapenem blaNDM-5-carrying extensive drug-resistant (XDR) K. pneumoniae ST437 from an urban river water Kathajodi in Odisha, India. The presence of carbapenem and co-occurrence of other resistance determinants (blaNDM-5, blaCTX-M, blaSHV, and blaTEM), virulence factors (fimH, mrkD, entB, irp-1, and ybtS), and capsular serotype (K54) represent its pathogenic potential. The insertion sequence ISAba125 and the bleomycin resistance gene bleMBL at upstream and downstream, respectively, could play a significant role in the horizontal transmission of the blaNDM-5. Its biofilm formation ability contributes toward environmental protection and its survivability. MLST analysis assigned the isolate to ST437 and clonal lineage to ST11 (CC11) with a single locus variant. The ST437 K. pneumoniae, a global epidemic clone, has been reported in North America, Europe, and Asia. This work contributes in understanding of the mechanisms behind the spread of blaNDM-5 K. pneumoniae ST437 and demands extensive molecular surveillance of river and nearby hospitals for better community health.

Supplementary Information

The online version contains supplementary material available at 10.1007/s13205-023-03556-5.

Keywords: Klebsiella pneumoniae, Carbapenem resistant, XDR, BlaNDM-5, Virulence factors, Urban river

Antibiotics are used prophylactically and as growth promoters in food additives in cattle, poultry, aquaculture, and crop plants (Manyi-Loh et al. 2018). Consequently, antibiotics are discovered in anthrophonic areas such as sewage and urban wastewater treatment plants. Discharge of untreated or incompletely treated waste into the natural environment leads to a paradigm shift in selection of antimicrobial resistance (AMR) in non-clinical environments. European Union has identified the environment as one of the primary driving forces behind the brief appearance and transmission of antimicrobial resistance genes (ARGs) and antimicrobial-resistant bacteria (AMB) soon after discovering the horizontal transfer of resistance genes in aquatic organisms this decade (Smith et al. 2016). Therefore, monitoring and surveillance of the water body have been launched as one of five strategic objectives to strengthen the knowledge of the spread of AMR in the Global action plan by the World Health Organization (WHO 2015) (https://www.who.int/publications/i/item/9789241509763).

The latest threat to public health by New Delhi metallo β-lactamase (blaNDM-1) has received global attention because of its high resistance to the majority of the β-lactam antibiotics and ability to get transmitted to different geographic locations (Thomas et al. 2011). Since the discovery of blaNDM-1, 41 distinct blaNDM variants have been detected in Gram-negative bacteria all over the world (http://www.bldb.eu/BLDB.php?prot=B1#NDM). Among the blaNDM variants, blaNDM-5, first reported in the UK in 2011, gained much attention due to its enhanced resistance to carbapenems and broad-spectrum cephalosporins (Li et al. 2018). So far, reports of blaNDM-5 from the non-human sources have been restricted only to the wastewater treatment plant in Japan (Sekizuka et al. 2019), urban rivers in France (Almakki et al. 2017), and China (Li et al. 2021). In India, blaNDM-5 was discovered in medical wastewater (Parvez and Khan 2018) and sewage treatment plants (Akiba et al. 2016).

The blaNDM-5 is mostly found among the members of Enterobacteriaceae, specifically Klebsiella pneumoniae, the major inhabitant of human and animal gut flora (Zhang et al. 2015). Moreover, carbapenem-resistant K. pneumoniae (CR-KP) has been emerged as one of the most threatening pathogens in recent years due to its ability to cause many fatal diseases, such as pneumonia and bloodstream infections. Management of the infections caused by the blaNDM-5-carrying CR-KP is even more challenging as these pathogens are associated with a high rate of mortality, transmission, and left with limited treatment options (Paczosa and Mecsas 2016).

The global spread of CR-KP is primarily due to the proliferation of a few effective clones. The global spread of blaKPC-type CR-KP is reported to be influenced by the high-risk fast-spreading clonal group CC11/CC258 (Navon-Venezia et al. 2017). More particularly, blaKPC-2-producing K. pneumoniae ST437 is of global epidemiological significance since it is widely distributed among nosocomial infections in humans (Tolentino et al. 2019). Sporadically, the ST437 strain harbors carbapenem resistance determinants other than blaKPC-2 such as blaOXA-48, blaNDM-types (specifically blaNDM-1, blaNDM-7, and blaNDM-23), and the coexistence of other antibiotic resistance genes. In addition, virulence characteristics and resistance determinants harboring in ST437 strains are likely to be the major contributors to the ecological success of these strains.

In this study, we first isolated the blaNDM-5-producing K. pneumoniae ST437 from Kathajodi river water in Odisha, which has already been reported as one of the most polluted rivers in India (Mallick 2012). Then we performed the genetic characterization of blaNDM-5, blaCTX-M, and virulence factors. This work will contribute to a better understanding of the mechanisms behind the spread of blaNDM-5 among aquatic bacterial communities.

The carbapenem-resistant K. pneumoniae was isolated from the Kathajodi river in Cuttack, eastern India and has been referred as SS1 throughout the text. The water sample was collected in January 2020, as part of AMR surveillance research designed to track the presence of carbapenemase-producing Gram-negative bacteria in the urban river of Odisha, India. Kathajodi river is a vital water source for the Cuttack city and numerous villages located downstream, and it receives the daily flow of untreated wastewater from domestic, industries, and hospitals. About 500 ml of water samples were taken from three locations within 100 m of the wastewater discharge point at the bank of the river. These samples were then combined to make a single representative sample. The water sample was membrane filtered and serially diluted with 0.85% NaCl solution (10–5 dilution) and 100 µl of dilutant spread on a HiCromeTM Klebsiella Selective Agar (HiMedia) plate with meropenem (4 µg/ml) and incubated at 37 °C for 24 h. The dark purple colonies from meropenem-supplemented media were chosen and passaged for 10 days on meropenem-supplemented Luria Bertani agar plates to obtain stable carbapenemase producers (Parvez and Khan 2018).

Using Kirby Bauer’s method, the antibiotic susceptibility of carbapenem-resistant colonies from meropenem-supplemented Luria Bertani agar plates was performed by employing antibiotic discs from Himedia Laboratories Pvt Ltd, Mumbai. In this study, we used 21 antibiotics from 16 different antimicrobial classes—β-lactam: piperacillin (PI; 100 µg); β-lactam/β-lactamase inhibitor: amoxicillin–clavulanic acid (AMC; 20/10 µg), piperacillin–tazobactam (PIT; 100/10 µg); third-generation cephalosporin: ceftriaxone (CTR; 30 µg), ceftazidime (CAZ; 30 µg); fourth generation cephalosporin: cefepime (CPM; 50 µg); carbapenem: meropenem (MRP; 10 µg), imipenem (IMP; 10 µg); aminoglycosides: amikacin (AK; 10 µg), gentamicin (GEN; 10); tetracycline: tetracycline (TE; 30 µg); fluoroquinolone: ciprofloxacin (CIP; 5 µg), levofloxacin (LE; 5 µg); sulphonamides: co-trimoxazole (COT; 25 µg); phenicol: chloramphenicol (C; 30 µg); nitrofurans: nitrofurantoin (NIT; 300 µg); fosfomycin: fosfomycin (FOS; 200 µg); monobactams: aztreonam (AT; 30 µg); macrolide: azithromycin (AZM; 15 µg). The broth microdilution method was performed for the susceptibility analysis of colistin and tigecycline through minimum inhibitory concentration determination. The results were interpreted by Clinical and Laboratory Standards Institute (CLSI) guidelines except for tigecycline (CLSI 2020). The MIC breakpoint of tigecycline against K. pneumoniae was unavailable in CLSI, so the MIC breakpoint of E. coli mentioned in the EUCAST guideline was used as a reference for K. pneumoniae. The reference strain E. coli ATCC 25922 was used as quality control.

According to the manufacturer’s instructions, genomic DNA was extracted using a DNeasy kit (Qiagen, India). Plasmid DNA isolation was performed by Kado and Liu (1981) method (Kado and Liu 1981) and further identified through PCR-based replicon typing (Carattoli et al. 2005). The conjugation experiment was performed using E. coli J53AzR. The isolate was identified through 16S rRNA gene PCR amplification using universal primer UNI16S-L(5′-ATTCTAGAGTTTGATCATGGCTCA-3′) and UNI16S-R(5′-ATGGTACCGTGTGACGGGCGGTGTGTA-3′) (Sahoo et al. 2014) and sequencing at Agrigenome, Kochi, Kerala. The sequence was analyzed through NCBI-BLAST tools and submitted to GenBank Database with accession number ON556568. Carbapenem resistance genes (blaNDM, blaKPC and blaOXA-48), co-resistance genes (blaCTX-M, blaTEM and blaSHV), virulence factors {type 1 and type 3 adhesins (fimH and mrkD), enterobactin biosynthesis (entB), yersiniabactin biosynthesis (irp-1), (ybtS)}, capsular serotypes (K1, K2, K5, K20, and K54) and outer membrane porin-coding genes (OmpK35 and OmpK36) were searched through PCR-based method. The genetic environment of blaNDM at upstream and downstream genes has been analyzed using PCR. The primer details used in this study have been described in Table S1.

The phenotypic detection of biofilm production was determined based on the formation of black crystalline mucoid colonies on brain heart infusion agar supplemented with 5% (w/v) sucrose and 0.08% (w/v) Congo red as described by Das et al. (2019). Multi-locus sequence typing (MLST) analysis was conducted by PCR amplification of seven housekeeping genes (rpoB, gapA, mdh, pgi, phoE, infB, and tonB) as indicated by the Institute Pasteur MLST Database. The PCR conditions and primers were followed from the MLST website (https://bigsdb.pasteur.fr/klebsiella/). The PCR products were sequenced, the outcome was uploaded to the MLST database, and the allele number and sequence type (ST) were determined. To identify the K. pneumoniae ST 437 reported worldwide, we searched PubMed, Google Scholar, and Scopus with no language restrictions that contained the terms “Klebsiella pneumoniae”; “ST437”; “Clinical”; “Environmental”; “River water”; “wastewater”, “wastewater treatment plant”, and carbapenem-resistant “genes”. The relevant data were extracted and analyzed using QGIS software version 3.16.2, https://qgis.org/en/site/forusers/download.html) and site of emergences is mapped worldwide.

Antibiotic susceptibility test showed that the strain SS1 is resistant to β-lactam, β-lactam/β-lactamase inhibitor, a broad spectrum of cephalosporin, carbapenem, aminoglycosides, tetracycline, fluoroquinolone, sulphonamides, phenicol, nitrofurantoin, fosfomycin, aztreonam, azithromycin (Table S2). The MIC for colistin and tigecycline was < 0.5 µg/ml and considered sensitive. This strain SS1 also showed biofilm production in Congo red plate. Based on the resistance phenotype, the SS1 was classified as extremely drug resistant (XDR) (Magiorakos et al. 2012). The molecular detection could reveal carbapenem resistance and co-resistance genes such as blaNDM, blaCTX-M, blaTEM, and blaSHV. The point mutation Val/Leu at position 88 and Met/Leu at position 154 (GenBank accession number: ON572245) in blaNDM gene is ascribed to be blaNDM-5 variant. The truncated insertion sequence ISAba125 and the bleomycin resistance gene bleMBL were discovered upstream and downstream of the blaNDM-5 gene, respectively, as previously described for the genetic environment of blaNDM-1 (Nordmann et al. 2011). The blaNDM-5 was typically embedded in plasmid; however, in this study, plasmid was absent. The absence of plasmid confirmed through unsuccessful outcome of PCR-based replicon typing and conjugation assay, which suggested that blaNDM-5 could be present in the chromosome. The virulence-associated genes fimH, mrkD, entB, irp-1, and ybtS, and outer membrane porin-coding genes OmpK35 were identified; however, OmpK36 was absent. Similarly, the K54 capsular serotype was detected in this isolate. Multi-locus sequence typing analysis showed that the strain SS1 belonged to ST437 {gapA (3); infB (3); mdh (1); pgi (1); phoE (1); rpoB (1); and tonB (31)}. The ST437 K. pneumoniae (clonal complex CC11) in association with blaKPC producer and other resistance determinants has been reported to be globally disseminated in different countries of North America, Europe, and Asia as discovered from the published data (Fig. 1; Table S3).

Fig. 1.

Fig. 1

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Worldwide distribution and status of dissemination of most clinically important carbapenemase genes in Klebsiella pneumoniae ST-437. The letter E represents environment and n represents number of publications. Gene KPC, Klebsiella pneumoniae carbapenemase; NDM, New Delhi metallo-β-lactamase; OXA, Oxacillinase. The name of the country was written in two official letter code

This study describes the first case of blaNDM-5-producing K. pneumoniae ST437 isolated from a river sample in India, although blaNDM-5 in K. pneumoniae ST437 was detected once in a tertiary-care hospital in India in the year 2020 (NCBI-GenBank Assembly No. ASM1174251v2, 2020). Our strain SS1 had an extremely drug resistance profile that remained susceptible to colistin and tigecycline, which aligns with studies from countries (Brazil and China) other than India (Campana et al. 2017; Hu et al. 2017). The presence of genes (blaNDM-5, blaCTX-M-15, blaTEM, and blaSHV) conferred resistance to a wide range of β-lactam, β-lactam / β-lactamase inhibitors, cephalosporin, and carbapenem antimicrobial group. In addition, the absence of the OmpK36 gene could be attributed to its resistance to various antibiotics such as cefoxitin, carbapenem, ciprofloxacin, and chloramphenicol (Indrajith et al. 2021), although the contribution of other resistance mechanisms like efflux pump upregulation, and membrane protein modification for such XDR phenotype may not be ruled out.

The ST437 is of high epidemiological significance worldwide due to its widespread distribution among nosocomial infections in humans (Andrey et al. 2020). This ST is related to dominant epidemic multidrug-resistant clonal complex CC11and was first reported as blaKPC-2-producing K. pneumoniae in Brazil (Azevedo et al. 2019; Boszczowski et al. 2019; Andrey et al. 2020) but currently predominant in America (USA, Nicaragua and Canada) (Wang et al. 2013; Tijet et al. 2014), European (Spain, Slovenia, Serbia, UK, Switzerland, Italy, Netherland, France) (Richter et al. 2012; Wyres et al. 2015; Marti et al. 2017; Benulič et al. 2020; Fuster et al. 2020; Palmieri et al. 2021; Szymankiewicz et al. 2021), and Asian countries (Sri Lanka, China, Saudi Arabia, Singapore, Japan, and India) (Compain et al. 2017; Hernández-García et al. 2018; Zhu et al. 2018; Shankar et al. 2019; Zhang et al. 2020) (Fig. 1; Table S3). This ST has been extensively associated with β-lactamase-producing genes, mainly blaKPC-2 and blaCTX-M-15, and more recently associated with blaNDM-type and blaOXA-type genes (Bleriot et al. 2020; Gato et al. 2021). The presence of this ST in several regions yearly provides evidence of its importance, which bears a substantial risk of developing into a large clone that could contribute to a future epidemic.

Clinical blaNDM variants (blaNDM-1 and blaNDM-7) in K. pneumoniae ST-437 have emerged across the globe intermittently in the last decade in Spain (Seara et al. 2015; Fuster et al. 2020), Slovenia (Benulič et al. 2020), Serbia (Palmieri et al. 2021), and Saudi Arabia (Compain et al. 2017) (Fig. 1; Table S3). However, infections due to blaNDM-5 variant harboring K. pneumoniae have been associated with high morbidity and mortality rates compared with the other blaNDM-containing isolates (Hu et al. 2017; Brinkac et al. 2019). As we understand, blaNDM-5 is the first identified in K. pneumoniae ST437 in any Indian river water.

The blaNDM-5 was typically discovered in plasmids; however, our study found in the chromosome, with insertion elements ISAba125 and bleMBL situated upstream and downstream of this gene, respectively. Previously, Sakamoto et al (2018) reported that blaNDM-1 was found in K. pneumoniae chromosome. The acquisition of the entire ISAba125 sequence upstream of the blaNDM-5 gene suggests that this element could be playing a significant role in the horizontal transmission of the blaNDM gene among the members of the Enterobacteriaceae family (Poirel et al. 2011). The chromosomal integration of blaNDM was possible based on its genetic environment (Sakamoto et al. 2018). The genetic plasticity of blaNDM suggests that this gene can be transported from plasmids to chromosomes via many processes (Sakamoto et al. 2018). In this study, ISAba125 was found at upstream at blaNDM-5, which suggested that blaNDM-5 can be transported from plasmid to chromosome by site-specific recombination. Similarly, bleMBL is associated with blaNDM, and it might be possible they mobilized together and originated from a common ancestor (Dortet et al. 2012). On the other hand, blaNDM-5-producing ST 437 from Christian Medical College, Vellore, India, is located on a plasmid with IS26 and bleMBL upstream and downstream of this gene, respectively (NCBI-GenBank Assembly No. ASM1174251v2, 2020). This indicates blaNDM-5-producing ST437 of the present study had no proven connection with ST-437 from Christian Medical College, Vellore. It might be possible that the hospital effluents of the city played a significant role in spreading blaNDM-5-producing ST437 into the river. The virulence factors Type-1 (fimH) and Type-3 fimbriae (mrkD), responsible for adhesion to the surface of collagen molecule of the host cell, and siderophore (entB and irp-1), responsible for uptake of the iron from the host cell to inhibit T-cell proliferation, (Sahoo et al. 2019) and capsular serotype K54, responsible for pulmonary infection, represent its pathogenic potential (Hasani et al. 2020). Previously, these virulence factors have also been reported in this ST (Aires et al. 2020) isolated from the clinical setting. In addition, this strain SS1 had phenotypic evidence of biofilm formation, which generally improves bacterial attachment to living or nonliving surfaces, inhibits antibiotic and heavy metal penetration, and provides protection against environmental stress (Nirwati et al. 2019). Earlier evidence of biofilm production of ST437 has been reported by Araujo et al. (2018).

The K. pneumoniae SS1 isolate from Kathajodi river water shared similar genetic characteristics, i.e., extremely drug resistance phenotype, virulence factors, biofilm production, and association of blaNDM type with the previously reported clinical isolates affiliated to ST437. It indicates that ST437 K. pneumoniae recovered from river water is likely to be originated from the infected patients or discharge of untreated hospital wastewater. However, whole metagenomic analysis of such river sample needs to be done, to understand the complete AMR status and presence of global epidemiological clones. The Kathajodi river, from where the water sample was collected, is heavily impacted by poor sanitation, urban wastewater discharge, and unavailable wastewater treatment plants in the city and the neighboring area. Even though it is generally regarded as inappropriate for bathing, drinking, and using in agriculture, it has always available to the public for daily utilization. Thus, it is essential to carry out extensive molecular surveillance of carbapenem-resistant bacteria and genes in environments and nearby hospitals as well as installation of treatment facilities to make the supply water free form such life-threating resistant pathogens.

Supplementary Information

Below is the link to the electronic supplementary material.

Supplementary file1 (DOCX 33 KB) (32KB, docx)
Supplementary file2 (DOCX 17 KB) (17.5KB, docx)
Supplementary file3 (DOCX 42 KB) (41.7KB, docx)

Acknowledgements

The authors are grateful to Prof (Dr.) S.C. Si, Dean, Centre for Biotechnology and Prof (Dr.) M.R. Nayak, President, Siksha ‘O’ Anusandhan University, for providing infrastructure and encouragement throughout.

Author contribution

The first draft of manuscript was written by SS. Conceptualization of the study and review of the manuscript were performed by SS, ES, RKS. Sample collection and laboratory: SS, DUB, RKS; interpretation of data and data analysis: SS, ES, SD; supervision: ES. All authors read and approved the final manuscript.

Funding

This research work was supported by the DST-INSPIRE [DST/INSPIRE/03/2018/000860], New Delhi, India.

Data availability

The data that support the finding of this study are available in the supplementary material of this article.

Declarations

Conflict of interest

On behalf of all authors, the corresponding author states that there is no conflict of interest.

Research involves human or animal participant

The current study does not include any experiments involving human or animal participants conducted by any of the authors.

Contributor Information

Saubhagini Sahoo, Email: saubhagini2015@gmail.com.

Rajesh Kumar Sahoo, Email: rajeshkumarsahoo@soa.ac.in.

Sangita Dixit, Email: sangitadixit2011@gmail.com.

Dibyajyoti Uttameswar Behera, Email: dibya01bioinfo@gmail.com.

Enketeswara Subudhi, Email: enketeswarasubudhi@soa.ac.in.

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Supplementary Materials

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Data Availability Statement

The data that support the finding of this study are available in the supplementary material of this article.

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