Resistome of a carbapenemase-producing novel ST232 Klebsiella michiganensis isolate from urban hospital effluent in South Africa

Abstract

Objectives:

Klebsiella michiganensis is an emerging pathogen implicated in nosocomial infections. Here we report on the resistome, virulome and mobilome of a carbapenemase-producing K. michiganensis isolate from urban hospital effluent in Pietermaritzburg, KwaZulu-Natal, South Africa. Klebsiella sp. isolate KP124 was originally isolated from the final effluent of an urban tertiary hospital in Pietermaritzburg, KwaZulu-Natal, South Africa.

Methods:

Following phenotypic characterization and antibiotic susceptibility testing, the genome of the carbapenemase-producing isolate KP124 was sequenced using an Illumina MiSeq platform, de novo assembled and analysed using established bioinformatics tools.

Results:

The draft genome of strain KP124 was 6 544 586 bp long, comprising of 203 contigs >200bp. Following confirmation of isolate KP124 as K. michiganensis by using reference genomes, the carbapenemase gene bla_OXA-181 as well as 11 additional genes encoding resistance against β-lactam, aminoglycoside, fluoroquinolone, and sulfonamide antibiotics were detected. Virulence factors enabling iron acquisition and cell adherence, capsule locus type and plasmid replicon types were identified.

Conclusions:

This study represents the first report of a bla_OXA-181 carbapenemase-producing K. michiganensis isolate from hospital effluent in South Africa. The presence of such a strain in the environment due to the absence of hospital effluent treatment presents a potential risk to informal communities that may use contaminated surface water domestically.

Introduction

South Africa currently has no guidelines regulating the treatment of hospital effluent before release into the municipal sewage system. Such a practice allows for the transfer of antibiotic resistance genes between clinical strains originating from hospitals and commensal bacteria present in wastewater treatment systems [1]. Unless wastewater treatment systems completely eliminate antibiotic resistant bacteria, water bodies such as rivers receiving wastewater treatment plant (WWTP) effluent might contain such bacteria [2]. This is a potential public health risk as surface water is still utilized for irrigation, recreation, and domestic use in South Africa [3]. Additionally, extended spectrum β-lactamase (ESBL) producing Enterobacteriaceae such as Klebsiella spp. have been highlighted by the World Health Organization as priority pathogens for research and surveillance [4]. This paper describes the genome analysis of a carbapenemase-producing K. michiganensis isolate from the final effluent of an urban hospital in Pietermaritzburg in the uMgungundlovu district of the province of KwaZulu-Natal, South Africa.

Methods

Klebsiella sp. strain KP124 was initially isolated from the final effluent of an urban tertiary hospital in Pietermaritzburg, KwaZulu-Natal, South Africa. This isolate was phenotypically characterized using the API20E system and establishing antibiotic resistance profiles using the EUCAST disk diffusion method and breakpoints as reported [5]. This ESBL and carbapenemase-producing isolate was selected for whole genome analysis. Genomic DNA was extracted from cells grown in nutrient broth after incubation for 24 hours at 37 °C, using a commercial kit (MasterPure DNA Purification Kit, Lucigen, USA), and its purity and concentration analysed using the Qubit ds-HS DNA assay kit (Life Technologies, USA). Illumina sequencing libraries were created using 2 μl of pooled and normalized library DNA (0.2 ng/μl) using the Nextera XT DNA Library Preparation and Nextera XT v2 index kit C (Illumina, USA) as per manufacturer’s instructions. The normalized libraries were sequenced using a MiSeq version 2-reagent kit with a paired-end read length of 2 x 250 bp using the Illumina MiSeq platform (Illumina, USA).

A total of 1 360 596 reads were obtained, resulting in an estimated sequencing coverage of 113X. FastQC was utilized to assess the quality metrics of the reads (v0.11.8 http://www.bioinformatics.babraham.ac.uk/projects/fastqc), which were then de novo assembled using SPAdes (v3.12 - Galaxy server, https://usegalaxy.eu/). The genome assembly metrics were analysed and verified by Quast (v5.0 - http://quast.sourceforge.net/). The draft genome was analysed using orthoANI (OAT platform v0.93.1 - https://www.ezbiocloud.net/tools/orthoani) and the Type (Strain) Genome Server (TYGS - https://tygs.dsmz.de) [6] at default program parameters to verify the identity of isolate KP124. Genome annotations were completed using the Rapid Annotations Subsystems Technology tool kit (RASTtk v2.0) at PATRIC [7]. The multi-locus sequence typing allele sequence and profile data were obtained using the MLST 2.0 tool at the Centre for Genomic Epidemiology, while the Kaptive tool (http://kaptive.holtlab.net/) was used to establish the capsule synthesis locus of KP124 [8]. Plasmids and resistance genes were elucidated using PlasmidFinder 2.1 and ResFinder 3.2 at the Centre for Genomic Epidemiology: https://cge.cbs.dtu.dk/services/, using an ID threshold of 95% and a minimum length of 60%. VF analyser at VFDB (http://www.mgc.ac.cn/VFs/) was used to predict clinically relevant virulence factors.

Results and Discussion

The assembled genome of Klebsiella sp. strain KP124 comprised of 203 contiguous sequences (> 200 bp), with a total length of 6 544 586 bp and a GC content of 55.01%. The corresponding N₅₀,N₇₅ and L₅₀ values were 147 324, 96 301 and 14 respectively, and the longest contig was 421 049 bp long. In total, 6682 protein-coding sequences were predicted, as well as 7 ribosomal RNA (rRNA) genes and 74 transfer RNA (tRNA) genes (Table 1).

Table 1.

Genome metrics, antibiotic resistance genes, plasmids, virulence factors and sequence type of KP124 isolated from urban hospital effluent in South Africa.

Identity	Klebsiella michiganensis KP124
	Urban hospital effluent, Pietermaritzburg, KwaZulu-Natal, South Africa
Genome metrics
Genome length, # contigs (>200 bp)	6 544 586 bp, 203
GC content	55.01 %
Estimated coverage	~ 113 X
# protein coding sequences	6 682
# tRNA, # rRNA genes	74, 7
MLST profile	ST 232 1
	β-lactamases/carbapenemases	blaOXY-5-ι, blaOXA-181
	Fluoroquinolones	qnrB6, qnrS1
	Aminoglycosides	aadA16, aph(6)-Id
Antibiotic resistance genes2	Fluoroquinolone/Aminoglycoside	aac(6’)Ib-cr
	Rifampicin	arr-3
	Trimethoprim	dfrA14, dfrA27
	Sulfonamides	sul1, sul2
Plasmids2	IncFIA, IncFII(pBK30683), IncHI1, IncHI2, IncX3, Col440I
Virulence factors3
	Type I fimbriae	fimA-D, F-K
Adherence	Type III fimbriae	mrkA-D, F, H-J
	Type IV pili	pilW
Antiphagocytosis	Capsule	Type KL704
	Enterobactin	entA-F, S; fepA-D, G
Iron acquisition	Salmochelin	iroE
	Aerobactin	iutA
Secretion systems	T6SS-I, II & III	tssA-D, tssH, J, ompA

¹Novel sequence type assigned by the K.oxytoca/michiganensis pubMLST database

²Genes and plasmids detected at CGE using an ID threshold of ≥ 95% and length ≥ 60%

³Clinically relevant virulence factors predicted by VFDB

⁴Closest capsule type as defined by the Kaptive tool at http://kaptive.holtlab.net/ (98.34% Identity)

As reported previously, strain KP124 was initially identified as Klebsiella oxytoca using the API20E system [5]. However, orthologous Average Nucleotide Identity analysis of the assembled genome, which met the quality criteria stipulated by Chun et al. [9], against curated Enterobacteriaceae genomes (https://www.ezbiocloud.net/ - Table S1) reclassified KP124 as Klebsiella michiganensis based on 97.71% identity to K. michiganensis KCTC 1686. Additionally, the identity of KP124 was confirmed as K. michiganensis by digital DDH (DNA:DNA hybridization) pairwise comparisons of KP124 and type strain genomes using TYGS (Table S2), based on the dDDH value of 79.5%. Phylogenetic analysis based on selected type strain genomes confirmed that isolate KP124 clustered closely with K. michiganensis (Figures S1, S2).

The assembled genome was submitted to the PubMLST K. oxytoca michiganensis database for the assignment of a new ST (https://pubmlst.org/koxytoca/), and the novel sequence type was assigned as ST232.

K. michiganensis strain KP124 is resistant to amoxicillin-clavulanic acid, piperacillin-tazobactam, cefotaxime, ertapenem, doripenem, ciprofloxacin, norfloxacin, moxifloxacin, tobramycin and produces ESBL and carbapenemase (Table S3). Based on the analysis of the genome using Resfinder, the β-lactamase/carbapenemase resistance genes bla_OXY-5-1 and bla_OXA-181 were predicted. The bla_OXY group of genes encodes β-lactamases conferring high-level resistance to certain extended-spectrum cephalosporins in K. oxytoca and related species, with diversification of such OXY genes observed over time [10]. Primarily nosocomial infections caused by clinical strains of K. pneumoniae and K. oxytoca producing the bla_OXA-181 enzyme have been reported from a tertiary hospital in Cape Town, South Africa [11]. In addition, a K. michiganensis isolate from a district hospital patient in South Africa displayed a similar genotype to the current study, co-producing bla_OXA-181, bla_NDM-1 and bla_OXA-5-1 [12]. While limited data exist regarding ESBL and carbapenemase-producing K. michiganensis strains occurring in the South African environment, a strain of K. pneumoniae carrying the bla_OXA-48 carbapenemase gene was reported for hospital wastewater in Pretoria, South Africa [13], A phenotypic/genotypic correlation occurred for the resistances observed against ciprofloxacin, norfloxacin, moxifloxacin, and tobramycin - as genes encoding for resistance against fluoroquinolones (qnrB6, qnrS1) and aminoglycosides (aadA16, aac(6’)Ib-cr, aph(6)-Id) were detected. Additionally, rifampicin (arr-3), sulfonamide (sul1/sul2) and trimethoprim (dfrA14, dfrA27) resistance genes were identified in K. michiganensis strain KP124.

Seven plasmid replicon sequences were predicted by PlasmidFinder, including two representing IncF type plasmids (IA and II), two IncH types (I1 and I2), IncX3, and Col440I. IncF plasmids are typical carriers of antibiotic resistance genes encoding carbapenemases, plasmid-mediated quinolone genes and aminoglycoside modifying enzymes [14]. Such plasmids have been previously reported in another South African K. michiganensis clinical isolate [12].

Several virulence genes, including those associated with antiphagocytosis, cell adherence, iron acquisition and secretion systems were identified on the genome of strain KP124. The presence of genes involved in capsule biosynthesis (vital in evading host immune systems) was confirmed, and the capsule type was identified as KL70. Furthermore, genes encoding type I (fimABCDFGHIK) and type III fimbriae (mrkABCDFHIJ) were predicted, all of which are responsible for adherence and biofilm formation and contribute to the pathogenicity of Klebsiella spp. [15]. The genes encoding T6SS-I, II, and III secretion systems were present in KP124, which, if expressed, provide the isolate with the ability to secrete toxic effector molecules, targeting and potentially killing competing bacteria in polymicrobial infections [16]. Finally, the presence of genes encoding for iron sequestration factors such as aerobactin (iutA), salmochelin (iroE) and enterobactin type siderophores (ent, fep, fes) aid in promoting survival in blood and consequently contribute to hypervirulence [17].

Conclusions

To the best of our knowledge, this is the first report of a carbapenemase-producing Klebsiella michiganensis isolate from South African hospital effluent. Untreated hospital effluent thus may present a public health risk to vulnerable communities exposed to proximate surface waters.

Supplementary Material

1

Supplementary Table 1. Curated Enterobacteriaceae genomes and corresponding NCBI accession numbers utilized for orthologous average nucleotide identity analysis.

Supplementary Table 2. Digital DNA:DNA hybridization (dDDH) pairwise comparisons of isolate KP124 and seventeen type strain genomes generated by the type strain genome server.

Supplementary Table 3. Phenotypic antibiotic resistance profile, extended spectrum β-lactamase and modified carbapenemase inactivation method results of Klebsiella michiganensis isolate KP124.

Supplementary Figure 1. The obtained Orthologous Average Nucleotide Identity (oANI %) values of KP124 against a collection of curated Enterobacteriaceae reference genomes with corresponding UPGMA dendrogram.

Supplementary Figure 2. Phylogenetic tree inferred with FastME from Genome BLAST Distance Phylogeny (GBDP) distances calculated from type strain genome sequences using the TYGS server.

Highlights.

• Identification of novel ST232 Klebsiella michiganensis from urban hospital effluent in South Africa

• Genome encodes bla_OXA-181 carbapenemase and 11 other antibiotic resistance genes

• Virulence factors enabling iron acquisition and cell adherence detected

• The presence of MDR Klebsiella michiganensis in the environment presents a potential health risk

Data availability

The sequence data for this study can be accessed under the NCBI Bioproject accession number PRJNA604671, with KP124 under Biosample SAMN13978237 and Sequence Read Archive accession number SRR11012664.