Whole Genome Sequencing Reveals Presence of High-Risk Global Clones of Klebsiella pneumoniae Harboring Multiple Antibiotic Resistance Genes in Multiple Plasmids in Mwanza, Tanzania

Background: Klebsiella pneumoniae is an important multidrug-resistant (MDR) pathogen, causing both community- and healthcare-associated infections. The resistance is due to the continuous accumulation of multiple antibiotic-resistance-genes (ARGs) through spontaneous genomic mutations and the acquisition of conjugative plasmids. This study presents antibiotics resistance genes, plasmids replicons, and virulence genes of K. pneumoniae isolates from clinical specimens in a tertiary hospital, Mwanza, Tanzania. Methods: Whole genome sequencing (WGS) of 34 K. pneumoniae was performed, using an Illumina NextSeq 500, followed by in silco analysis. Results: A total of 34 extended-spectrum beta-lactamase-producing K. pneumoniae, isolated from blood samples from neonatal units were whole-genome sequenced. Of these, 28 (82.4%) had an identified sequence type (ST), with ST14 (39.3%, n = 11) being frequently identified. Moreover, 18 (52.9%) of the bacteria harbored at least one plasmid, from which a total of 25 plasmid replicons were identified with a predominance of IncFIB(K) 48.0% (n = 12). Out of 34 sequenced K. pneumoniae, 32 (94.1%) were harboring acquired antibiotic/biocides-resistance-genes (ARGs) with a predominance of blaCTX-M-15 (90.6%), followed by oqxB (87.5%), oqxA (84.4%), blaTEM-1B (84.4%) and sul2 (84.4%). Interestingly, we observed the ColRNAI plasmid-replicon (n = 1) and qacE gene (n = 4) for the first time in this setting. Conclusion: Global high-risk clones of K. pneumoniae isolates carry multiple ARGs in multiple plasmid-replicons. Findings from this study warrant genomic-based surveillance to monitor high-risk global clones, epidemic plasmids and ARGs in low- and middle-income countries.


Introduction
Klebsiella pneumoniae is an opportunistic nosocomial pathogen that is responsible for a number of infectious diseases, namely urinary tract infections (UTIs), skin and soft tissue infections (SSTIs), both community-and health-care-associated pneumonia, and bloodstream infections (BSIs) [1][2][3]. Infections by K. pneumoniae are reported to be associated with increased health-care costs from treatment failure and prolonged hospitalization, which in turn results in a high risk of mortality [4]. Recently, K. pneumoniae has been reported as the commonest member of Enterobacterales, with high rates of resistance towards beta-lactams, aminoglycosides, quinolones, and folate-pathway antagonists antibiotics [5]. K. pneumoniae is an important multidrug-resistant (MDR) pathogen, which continuously accumulates for itself multiple antibiotic-resistance-genes (ARGs) through spontaneous genomic mutations and acquisitions of transferable genetic elements, particularly conjugative plasmids [4]. Therefore, K. pneumoniae has been identified as the major source and shuttle for antibiotic resistance genes [4].

Laboratory Procedures
A total of 34 Klebsiella pneumoniae isolated between 2012 and 2015 with resistance towards third-generation cephalosporins were retrieved and whole-genome sequenced. The bacteria were isolated from blood samples of neonates who were admitted to neonatal units (neonatal ICU and premature unit) between 2012 and 2015, presenting with signs and symptoms of bloodstream infections. Conventional bacteriological culture, in-house biochemical-identification testing, and the disk-diffusion method using the Kirby-Bauer technique [19] were used for isolation, identification and antibiotics-susceptibility testing of K. pneumoniae, respectively. The Clinical and Laboratory Standards Institute (CLSI) guidelines were used for the interpretations of the zone of inhibition [20]. The isolates were archived at −80 • C in the Microbiology Research Laboratory at the Catholic University of Health and Allied Sciences (CUHAS).
For DNA extraction prior to sequencing, K. pneumoniae isolates were recovered by plating on plates of Columbia Blood Agar (Becton Dickinson GmbH, D-69126 Heidelberg, Germany) and then incubated aerobically at 35 ± 2 • C, for 20 h.
DNA extraction and purification was carried out using QIAmp®DNA Mini kit (QIA-GEN, Hilden, Germany). Briefly, a loopful of 1 µL was used to transfer bacterial colonies into a 1.5 mL safe-lock microcentrifuge tube containing 500 µL nuclease-free ultra-pure molecular water, followed by vortex-mixing to make homogenous suspensions. The suspensions were centrifuged at 13,000 rpm for 10 min, and then the supernatants were discarded. Cell pellets were re-suspended in 180 µL of ATL buffer. Further procedures of DNA extractions and purifications were conducted as instructed by the manufacturer. NanoDrop was used to determine the quantity and quality of the extracted and purified DNA samples.

Whole Genome Sequencing and In-Silico Analysis
Whole genome sequencing (WGS) was performed on an Illumina NextSeq 500/550 instrument (Illumina, San Diego, CA, USA,) using an Illumina Nextera XT library with 2 × 150 bp paired-end reads. Sequences data (mean length: 5084 kb) were quality checked before being assembled for multi-locus sequence typing (MLST) and phylogenetic-tree construction using Neighbor Joining tree (NJ-tree) algorithm(Center for Demographic and Population Genetics, University of Texas Health Science Center, Houston 77225), and analyzed for virulence genes (employing VFDB platform) on SeqSphere + v.8.4.0 (Ridom GmbH, Münster, Germany) pipelines [21]. Genes coding for virulence factors with an identity of ≥95% and an alignment of 100% with the reference sequence were considered present in our sequenced isolates. The detection and typing of plasmid replicons [22] and acquired antibiotics resistance genes (ARGs) were performed on Center for Genomic Epidemiology database (https://www.genomicepidemiology.org/services/; accessed on 9 September 2022).

Systematic Review
For the comparison of changes and/or persistence of sequence types (STs), acquired ARGs, virulence factors (VRFs) and plasmid replicons, we systematically reviewed our own studies between 2011 and 2022 in Mwanza, Tanzania. We included studies that established sequence types (STs), acquired ARGs, virulence factors (VRFs), and plasmid replicons among Enterobacterales from humans, animals and the environment both colonizing and causing invasive infections.

General Overview of Genome-Sequenced Klebsiella pneumoniae
A total of 34 Klebsiella pneumoniae isolated from blood samples from neonatal units were whole-genome sequenced. All isolates were extended spectrum beta-lactamaseproducing isolates. These isolates were multidrug resistant (MDR), showing resistance to third-and fourth-generation cephalosporin, gentamicin, tetracycline and trimethoprimsulfamethoxazole. Regarding ciprofloxacin, only 3 (8.8%) isolates were sensitive to this antibiotic, and all isolates were sensitive to meropenem.

Sequence Types (STs), Antibiotic Resistance Genes (ARGs), Virulence Genes (VRGs) and Plasmid Replicons Circulating between 2011 and 2022 in Mwanza among Enterobacterales
In the nine previous studies reviewed, the majority involved humans (n = 6 studies), working with blood samples (n = 4 studies). These studies focused on E. coli (n = 8 studies) and K. pneumoniae complex (n = 7 studies). In characterizing the isolates, conventional PCR and sequencing was used in five studies, while WGS was used in four studies.

Sequence Types (STs), Antibiotic Resistance Genes (ARGs), Virulence Genes (VRGs) and Plasmid Replicons Circulating Between 2011 and 2022 in Mwanza Among Enterobacterales
In the nine previous studies reviewed, the majority involved humans (n = 6 studies), working with blood samples (n = 4 studies). These studies focused on E. coli (n = 8 studies) and K. pneumoniae complex (n = 7 studies). In characterizing the isolates, conventional PCR and sequencing was used in five studies, while WGS was used in four studies.
K. pneumoniae from Blood (n = 61), pus/wound swabs (n = 19) and urine (n = 12), isolated between 2009 and 2010 using PCR and sequencing showed the predominance of ST14. Other STs detected were ST101, ST48, ST348 and ST147. These isolates were found to carry predominantly blaCTX-M-15 in conjugative plasmids of IncFII and IncFIA. Other resistance genes detected were blaSHV-11, blaTEM-10 and blaTEM-170. Using WGS, the three K. pneumoniae from fish isolated in 2015 were typed as ST37 (n = 2) and ST280. These isolates were found to carry blaCTX-M-15 and other resistance genes such as strA/B, qnrS1, aac(3)-IIa Regarding the STs, ARG, plasmid replicons and virulence genes from other Enterobacterales in the study setting, a similar picture was observed as that for K. pneumoniae. Using WGS, E. coli from humans, the environment and animals, global clones such as ST131, ST38, ST617, ST648 and ST10 were observed circulating in humans, animals and the environment. E.coli strains harbored multiple acquired ARGs similar to those observed in K. pneumoniae. These genes were located in multiple plasmids of replicon types IncI2, Col156, IncFIA, IncFIB, IncFII, IncQ1, and IncY.

Plasmids Harboring Genes Encoding for Antibiotics Resistance
We observed that extracted plasmid-contigs of the sequenced K. pneumoniae were harboring ARGs, conferring resistance to beta-lactams, aminoglycosides, sulfamethoxazole, trimethoprim, tetracyclines, and quinolones, and the biocides, ammonium quaternary compounds (Table 3).

Discussion
The Klebsiella pneumoniae isolated between 2012 and 2015 from blood samples confirmed the existence of global high-risk (HiR)-clones ST14, ST37 and ST340, carrying multidrug-resistance genes in multiple plasmid-replicons in Tanzania, causing invasive infections among neonates. Furthermore, the predominance of the HiR clone ST14, causing invasive infections in neonates was observed in 2009. However, in 2016 other HiR clones, ST 45, ST17, ST20 and ST48 were found to be more predominant in the same units, causing invasive infections and colonizing neonates [11].
The insight from our own studies and the findings from the present study show the persistence of K. pneumoniae  [11]. Furthermore, we observed the persistence of acquired ARGs circulating among HiR clones of K. pneumoniae and E. coli, especially bla CTX-M-15 and other quinolones and aminoglycosides genes. These acquired ARGs were found in similar conjugative-plasmid replicon types, pointing to the horizontal transfer of these genes among Enterobacterales. The persistence of a clone in the hospital setting can be due to poor infection-prevention practices in the presence of carriers of these clones. These HiR K. pneumoniae clones are present beyond the hospital settings, as observed by Moremi et al. in 2015, who found that the HiR clone ST37 was found to colonize the gut of tilapia fish in the same region [27].
As observed in this study and by Marando et al. [11] and another study in Germany, HiR K. pneumoniae clones ST14, ST15, ST17, ST20, ST37, ST48, ST147 and ST307 are multidrug resistant (MDR) and are predominantly colonizing and causing sepsis in both hospital and community settings [28]. These clones of the K. pneumoniae complex have a global distribution and a potential association with outbreaks of health-care-associated infections [28,29].
K. pneumoniae ST14 and ST37 have been found to carry high levels of colistin-resistance and carbapenem-resistance genes such as bla NDM and bla OXA-48 [30,31]; however, these genes were not observed in the current study or in previous studies that used WGS from the same setting [10,11,27]. This fact therefore underscores the need for the continuous genomic surveillance of MDR pathogens in the community and in hospital settings, for the early detection of the changes in clones and the acquisition of other acquired ARGs.
Eighteen out of 34 sequenced K. pneumoniae in the current study were harboring mobile genetic elements, notably plasmids of various replicon types including ColRNAI, which was observed in the clinical isolates of K. pneumoniae at our setting for the first time. Similar common replicon-types were observed among Enterobacterales in the study setting, with a predominance of conjugative IncFII and IncFIB plasmids (Supplementary Material Table S1). The ColRNAI was detected in one K. pneumoniae ST340. K. pneumoniae ST340 with ColRNAI plasmid was also harboring other plasmids, including IncFII(K), IncFIB(K) and IncR, of which altogether were carrying acquired ARGs, namely aac(6 )-Ib-cr, aadA2, aph(3 )-Ia, bla CTX-M-15 , bla OXA-1 , OqxA, OqxB, qacE, sul1 and tet(A). A recent study in Japan reported that K. pneumoniae ST258, harboring ColRNAI plasmid [32] reserves multiple ARGs, as observed in the current study.
We observed some isolates harboring more than one different plasmid-replicon, with one isolate carrying four plasmids, namely ColRNAI, IncR, IncFII(K), and IncFIB(K). As previously described [11,33] and observed in our previous studies (Supplementary Material  Table S1), the commonest replicon types are IncY and IncF i.e., IncFIA, IncFIB and In-cFII. The IncFIB and IncFII(K) replicons are commonly reported in clinical isolates of K. pneumoniae and E.coli, and are highly associated with the dissemination of acquired ARGs [34]. On the other hand, we identified two K. pneumoniae with IncHI2/2A plasmidreplicons, despite the fact that IncHI2/2A replicons have been identified as predominant reservoirs of acquired ARGs in multidrug-resistant Salmonella spp. [32,35]. Our findings are also supported by a recent study which observed IncHI1B as the commonest plasmid among K. pneumoniae isolated from blood samples [36]. Rare plasmids of replicon types IncQ1 and IncR were also identified in the current study and in other previous studies in the same setting (Supplementary Material Table S1). IncQ1 and IncR plasmids have been found to carry multiple resistance-genes, such as bla NDM-1 , bla KPC-2 , bla DHA-1 , bla VIM-1 , qnrS1, or armA [37,38].
Similarly to previous studies from the same setting [10,11], bla CTX-M-15 and bla TEM-1B conferring resistance towards β-lactams, sul2 conferring resistance to sulfamethoxazole; aph(3")-Ib, and aph(6)-Id conferring resistance to aminoglycosides were the commonest acquired ARG genes detected in the sequenced K. pneumoniae. However, this is the first study to report bla LEN10 and qacE genes conferring resistance towards β-lactam antibiotics and quaternary ammonium compound, respectively, in K. pneumoniae, in this setting. Our findings support observations by Damiano and colleagues, who reported phenotypic resistance of Enterobacterales towards antiseptics/biocides, in this setting [39]. The bla LEN10 has been previously reported among the environmental isolates of Klebsiella spp. in Nigeria [40] and the USA [41]. The presence of qacE genes was also reported previously in K. pneumoniae from one study in Iran [42] and another study in the UK [43]. The bla LEN10 gene was identified among two K. pneumoniae of the ST1562 type; however, the only IncR plasmid which was present in these isolates did not carry this gene. The bla LEN has been identified as class A chromosomal Beta-Lactamase gene in Klebsiella pneumoniae previously [44]. On the other hand, the four qacE genes were identified among three K. pneumoniae of STs 340, 5199 and 14 and one K. pneumoniae with an unknown ST.
The acquired virulence-genes in the current study were categorized into five groups, namely adhesins (group A), plasma/protein-resistance factors (group B), iron-acquisition/ sequestering system (group C), capsule-formation factors (group D), and lipopolysaccharide O-antigen (group E) [45][46][47]. However, the majority of our sequenced isolates were harboring fimABCDEFGHIK and mrkABCDFHIJ genes, encoding for adhesion/attachment, an essential first step in developing infectious disease; entABCDEF, fepABCDG, fes, iroE, and ybdA genes, encoding for iron acquisition from host tissues, which ensures the availability of essential micronutrients for bacterial survival as well as the destruction of host tissues; acrAB genes that encode for plasma/protein resistance to facilitate evading the host immune-response; rcsAB and wzi genes, encoding for capsule formation and facilitating the evading of the host immune-response through anti-phagocytosis; and wzm, wzt, and wbbMNO genes, responsible for hypervirulence [45][46][47]. Other virulence-encoding genes such as irp1/2 and fyuA were reported in the same setting by Marando and colleagues; these virulence genes are responsible for iron acquisition from the environment [11], and might indicate that these K. pneumoniae might be from the community in origin [48]. Moreover, acrAB, manBC, wzi, wzt, and wzm genes were reported for the first time among clinical isolates of K. pneumoniae in this setting.
The neighbor-joining tree (NJ tree) algorithm grouped sequenced K. pneumoniae into six MLST clonal-clusters. The large MLST clonal-cluster is made with 11 K. pneumoniae, of which 10 belong to ST14 and 1 is of unidentified ST. Other MLST clonal-clusters are made of three K. pneumoniae (n = 1; ST37); and two K. pneumoniae (n = 2 each; ST37, ST322, ST896 and ST1562). These findings indicate the possible cross-transmission of K. pneumoniae, particularly ST14, ST37, ST322, ST896 and ST1562, causing neonatal sepsis in neonatal units in this setting. Similar findings were reported previously, from the same setting [49]. Therefore, the stringent implementation of IPC measures is mandatory for minimizing the spread of HiR clones, i.e., K. pneumoniae ST14 and ST37, causing neonatal sepsis in this setting.

Conclusions
K. pneumoniae ST14 harboring hypervirulent genes (wzm, wzt, and wbbMNO) and multiple acquired-ARGs is an important and successful disseminated sequence-type among clinical isolates, causing bloodstream infections in our setting between 2009 and 2015, with changes in 2016, whereby the ST45 was predominant. The FIB(K) and FII(K) plasmid replicons carrying bla CTX-M-15 , sul2 and aph(3")-Ib and aph(6)-Id ARGs were predominantly identified in the high-risk global clones of K. pneumoniae from this setting. The less-common plasmid-replicon ColRNAI, and qacE and bla LEN10 genes were also reported for the first time among clinical isolates of K. pneumoniae in this setting. The majority of isolates are harboring genes encoding for the iron-sequestering system that can indicate the community spread of MDR pathogens. The predominance and the persistence of the bla CTX-M-15 gene and other acquired-ARGs among Enterobacterales in our setting might be due to circulating conjugative-plasmids and the spread of HiR-clones of K. pneumoniae in the hospital setting, due to poor IPC-practices. These findings warrant sustained improved IPC-practices and genomic-based sequencing surveillance to monitor K. pneumoniae HiR clones, epidemic plasmids, and virulence and acquired antibiotic-resistance genes among MDR pathogens causing invasive infections in developing countries. Informed Consent Statement: Not applicable because the study used archived clinical isolates and permission to use the isolates was obtained from the joint Catholic University of Health and Allied Sciences and Bugando Medical Centre Research Ethics and Review Committee (CREC) by a letter with reference number BU/36/DRI/001/Vol.1.

Data Availability Statement:
The data presented in this study are available on request from the corresponding author. The sequence data have been deposited in the database with accession numbers ERR829918-ERR829962 (https://www.ebi.ac.uk/ena/browser/view/ERR829918-62, accessed on 22 November 2022).