Whole-Genome Sequencing (WGS) of Carbapenem-Resistant K. pneumoniae Isolated in Long-Term Care Facilities in the Northern Italian Region

K. pneumoniae (KPN) is one of the widest spread bacteria in which combined resistance to several antimicrobial groups is frequent. The most common β-lactamases found in K. pneumoniae are class A carbapenemases, both chromosomal-encoded (i.e., NMCA, IMI-1) and plasmid-encoded (i.e., GES-enzymes, IMI-2), VIM, IMP, NDM, OXA-48, and extended-spectrum β-lactamases (ESBLs) such as CTX-M enzymes. In the present study, a total of 68 carbapenem-resistant KPN were collected from twelve long-term care facilities (LTCFs) in the Northern Italian region. The whole-genome sequencing (WGS) of each KPN strain was determined using a MiSeq Illumina sequencing platform and analysed by a bacterial analysis pipeline (BAP) tool. The WGS analysis showed the prevalence of ST307, ST512, and ST37 as major lineages diffused among the twelve LTCFs. The other lineages found were: ST11, ST16, ST35, ST253, ST273, ST321, ST416, ST1519, ST2623, and ST3227. The blaKPC-2, blaKPC-3, blaKPC-9, blaSHV-11, blaSHV-28, blaCTX-M-15, blaOXA-1, blaOXA-9, blaOXA-23, qnrS1, qnrB19, qnrB66, aac(6′)-Ib-cr, and fosA were the resistance genes widespread in most LTCFs. In this study, we demonstrated the spreading of thirteen KPN lineages among the LTCFs. Additionally, KPC carbapenemases are the most widespread β-lactamase.


Introduction
β-lactams are the most widely prescribed antibiotics, used to treat a wide range of bacterial infections worldwide [1]. Since their introduction in clinical practice, resistance to β-lactams has progressively increased. Gram-negative bacteria resistance to β-lactams involves several factors such as (i) active efflux modification, (ii) decreased outer membrane permeability, (iii) mutations altering PBPs expression or function, and (iv) β-lactamases production, which remains the main mechanism of resistance employed by bacteria [2,3]. β-lactamases are usually divided into four classes, considering the Ambler classification: class A (TEM, SHV, ESBLs, and KPC); class B (MBL, NDM, VIM, and IMP); class C (Amp-C); and class D (OXA). Particularly widespread are the extended spectrum β-lactamases (ESBLs), against which the antibiotic class of carbapenems maintains their activity. However, carbapenemases, β-lactamases capable of hydrolysing even carbapenems, have emerged since 1982 in the Serratia marcescens clinical isolate (SME-1 carbapenemase) [4]. To date, serine-and metallo-carbapenemases are widely spread, especially among Enterobacterales [5]. The data from the European Antimicrobial Resistance Surveillance Network (EARS-Net) displayed that carbapenemase-producing Enterobacterales (CPE) are responsible for the majority of human infections [6]. Among Enterobacterales, Klebsiella pneumoniae, one of the most widely spread bacteria, is listed by the World Health Organization (WHO) as one of the worrisome pathogens in which combined resistance to several antimicrobials (β-lactams, quinolones, and aminoglycosides) is very frequent [7,8]. K. pneumoniae represents an important reservoir for class A (KPC, SHV, GES, IMI, and NMCA), class B (i.e., VIM, IMP, and NDM), class D carbapenemases (OXA- 48), and extended-spectrum β-lactamases (ESBLs) such as CTX-M variants [5,9]. Carbapenem-resistant K. pneumoniae is largely disseminated around the world [10][11][12][13][14]. One reason for the rapid dissemination of K. pneumoniae is the presence of antibiotic resistance genes in mobile genetic elements and the Inc-groups, which belong to plasmids IncF, IncFII(K1), IncR, IncX, IncX3, IncI2, and ColE1 [15][16][17][18][19]. However, carbapenem-resistant K. pneumoniae strains are not only confined to healthcare settings, but also to the community and among residents of LTCFs [20][21][22][23][24]. In Italy, the carbapenem-resistant Enterobacterales from LTCFs have been reported since 2010 in different endemic areas [25][26][27][28]. The aim of the present study was to investigate the dissemination of antibiotic resistance genes in K. pneumoniae isolated from rectal swabs of residents in the LTCFs of the Veneto Region (Northern Italy). The molecular analysis of the WGS of these strains was performed by next-generation sequencing (NGS).

Setting
Between July 2018 and June 2019, we conducted a point-prevalence survey among the residents of 27 LTCFs in the Veneto Region, Northern Italy. Participation was on a voluntary basis. The study-specific data were collected on a single day for each LTCF involved; in the LTCFs with a high number of beds, data collection was spread over two or more consecutive days. However, all the beds in one ward were surveyed on the same day. The surveys were not conducted simultaneously in all 27 LTCFs, but at different periods for each facility in accordance with the availability of both the local staff to collaborate with researchers in the collection of clinical data and study-specific biological samples, and of the reference microbiology laboratory to accept samples and perform analysis.
Only subjects housed in the facility for at least 48 h were asked for consent to participate. A total of 118 variables, such as hospitalization and surgery during the previous year, antibiotics within the last three months, and the presence of medical devices (i.e., urinary catheter; peripheral vascular catheter; central venous catheter; nasogastric tube; and percutaneous endoscopic gastrostomy) were collected from each enrolled participant. Additionally, the enteric carriage of ESBL and carbapenemase-producing Gram-negative bacteria was assessed collecting a rectal swab from every enrolled resident.

Strains Selection
The rectal swabs were collected and inoculated onto ChromID ESBL agar (bioMerieux, Marcy l'Etoile, France) with an Ertapenem disk (10 µg) and on Mac Conkey agar with a Meropenem disk (10 µg). The plates were incubated at 35 ± 2 • C under aerobic conditions for 24 h. The isolates were identified at the species level using an automated Vitek2 System (bioMerieux, Marcy l'Etoile, France). Resistance to carbapenems were interpreted according to the EUCAST criteria and confirmed with an immunochromatographic lateral flow assay Carba5 (NG Biotech, Guipry, France). A Vitek2 system (version 9.02, bioMérieux, Marcy l'Etoile, France) was used to confirm carbapenem resistance and to perform antimicrobial susceptibilities for other substances.

DNA Extraction and Whole-Genome Sequencing (WGS)
Total nucleic acid was extracted using a MagMAX Microbiome Ultra Nucleic Acid Isolation kit (Applied Biosystems and ThermoFisher Scientific, Monza, Italy). The DNA concentrations were measured using a Qubit fluorometer (ThermoFisher Scientific) to Microorganisms 2021, 9,1985 3 of 13 determine DNA input. The genomic libraries were prepared using a Swift 2S Turbo DNA Library kit (Swift Biosciences, Ann Arbor, MI, USA) and the WGS was performed on an Illumina MiSeq platform using v3 reagent kits generating 2 × 300 bp paired-end reads (Illumina, San Diego, CA, USA).

Bioinformatic Analysis
Raw data from the paired-end sequencing were quality checked with the FastQC tool (v.0.11.6, BaseSpaceLabs, Illumina, San Diego, CA, USA) and assembled with Velvet (v.1.2.10, BaseSpaceLabs, Illumina, San Diego, CA, USA) [29]. Velvet is incorporated as an assembler in a multiple-tool workflow, the CGE Bacterial Analysis Pipeline (BAP) (BaseSpaceApps, Illumina, San Diego, CA, USA). The BAP application predicts the species of bacterial input genomes using a k-mer-based approach [30]. These acquired antimicrobial resistance genes were identified using a BLAST-based approach, where the nucleotide sequence of the input genome was compared to the genes in the ResFinder database [31]. Multilocus sequence typing (MLST) was performed also using a BLAST-based approach [32]. BLAST was used to search for plasmid replicons using the PlasmidFinder database [33]. Identified plasmids of the IncF, IncH1, IncH2, IncI1, IncN, or IncA/C type were subtyped by plasmid MLST [33]. KmerFinder, ResFinder, and PlasmidFinder databases were synchronized with databases from the Center for Genomic Epidemiology (http://www.genomicepidemiology.org/) on 2 June 2017. MLST and pMLST data were downloaded from pubmlst.org on 17 May 2017.

Results
In total, 1933 rectal swabs in 2890 residents were performed. Overall, of 159 K. pneumoniae (KPN), isolated on a selective medium, only 68 of them were selected for this study for their carbapenem resistance profile. As shown in Table 1, all strains were resistant to at least two different class of antibiotics. Indeed, they exhibited a high resistance profile to βlactams/β-lactamase inhibitors (amoxicillin-clavulanic acid and piperacillin-tazobactam), oxyiminocephalosporins (cefotaxime and ceftazidime), carbapenems (meropenem and ertapenem), and ciprofloxacin. Of 68 KPN, 36 (52.9%) were resistant to trimethoprim-sulfamethoxazole, respectively. All strains, analysed by Vitek2 system, were susceptible to colistin.
For legal aspects, we used only the acronym of the twelve LTCFs.

WGS of K. pneumoniae
Whole-genome sequencing was carried out on 68 KPN isolates, and bioinformatic analysis was performed with a BAP tool that gave information about genome size, MLST, plasmid replicons, pMLST, and antimicrobial resistance genes. The genome size of the 68 KPN ranged from 5.1 to 5.74 Mb.

WGS of K. pneumoniae
Whole-genome sequencing was carried out on 68 KPN isolates, and bioinformatic analysis was performed with a BAP tool that gave information about genome size, MLST, plasmid replicons, pMLST, and antimicrobial resistance genes. The genome size of the 68 KPN ranged from 5.1 to 5.74 Mb.

Discussion
Carbapenem-resistant K. pneumoniae is designated by the Centres for Disease Control and Prevention (CDC) as one of the microorganisms that poses an urgent threat to public health worldwide. K. pneumoniae can spread rapidly in healthcare settings, and it is responsible for numerous human infections such as urinary, respiratory, and bloodstream infections [34,35]. In Italy, an epidemic spread of K. pneumoniae ST258, as a major contributor of carbapenem-resistant Enterobacterales, has been observed since 2010 [36,37]. In parallel, even though most infections still occur in nosocomial settings, K. pneumoniae emerged as a cause of severe community-acquired infections. The present study investigated the genome of carbapenem-resistant KPN in twelve LTCFs in a Northern Italy region. Among the 68 carbapenem-resistant KPNs, the most widespread clones were represented by ST307 and ST512. In ISRAA_TV LTCF, the ST307 was the unique lineage found, and it harbors the same plasmids but different resistance genes. K. pneumoniae ST307 has been reported from many countries, and it has been responsible for several global nosocomial [38,39] and long-term care center outbreaks [40,41]. Whole-genome sequencing performed by Wyres et al. on 95 K. pneumoniae ST307 revealed the presence of FIB-like plasmids harbouring the bla CTX-M-15 gene adjacent to the ISECp1 element such as the other ST307 isolated in different geographical areas [38,42]. The ST307 harbouring bla CTX-M-15 in association with aac(6 )-Ib-cr and qnrB6 genes, as well as in our strains, was also described in an Italian regional survey (Sicily, Southern Italy) [27].
In the present study, K. pneumoniae ST512, found in four LTCFs, harboured KPC-3 and KPC-9, CTX-M-15, SHV-11, OXA-9, OXA-23, and LEN-12 β-lactamases. The carbapenemresistant K. pneumoniae ST512 has been considered the predominant lineage in isolates, causing severe bloodstream infections in a Northern Italian hospital [43]. For a long time, Italy has been an endemic country for K. pneumoniae ST258/ST512 lineages [36,37,[43][44][45], but recently, other STs emerged [46]. The K. pneumoniae ST37 was the third most spread lineage (11 isolates) but it was detected only in one LTCF (IPABRS_VI). A determination of the correlation between this evidence and the presence of specific risk factors in the reference population is underway. The ST37 has been described in several papers as an ertapenem-resistant K. pneumoniae with a modified outer membrane permeability [47,48].
A wide variety of plasmids were found in each whole KPN genome. The IncF plasmids were predominant in the KPN analysed in this study. This plasmid family is widely diffused in clinically relevant Enterobacterales, especially IncFII(K), which is considered a virulent plasmid because of its ability to co-exist with other plasmids in a single cell [49,50]. Most of these plasmids are conjugative and this facilitates the dissemination of resistance genes among different strains and species. This is the case of IncFIA(pBK30683 plasmid found in ST512 (POCS_VR LTCF), co-harbouring bla OXA-23 , bla OXA-1 , bla CTX-M-15 , and bla LEN-12 . The OXA-23 class D carbapenemase is normally produced by A. baumannii, and it is very rare in K. pneumoniae [51,52]. However, pBK30683 is a plasmid of 139,941 bp that seems to originate from the cointegration of pBK30661 (belonging to IncFIA family plasmids) with a 68 Kb genetic element, harbouring a complete set of genes for plasmid replication, stability, and conjugation [53]. This could explain the "jump" of bla OXA-23 from one species to another. Class A, B, and D β-lactamases were identified in all the KPN strains. The major carbapenemases were the KPC variants (KPC-2, KPC-3, and KPC-9), VIM-1, NDM-1 metallo-β-lactamases, and OXA-23. KPC-9 is a KPC-3 variant with a V239A substitution. CTX-M-15 is the most common ESBL in K. pneumoniae. In our strains, CTX-M-15 was identified in eight different ST lineages (11,16,37,273,307,512,2623, and 3227). The bla CTX-M-15 gene is often flanked by a sequence insertion (IS) such as ISEcp1, which facilitates its mobility. The β-lactamases LEN and OKP-B are chromosomally encoded and are frequently found in K. pneumoniae as well as the oxacillinases (OXA-1 and OXA-9).

Conclusions
This study found that the LTCFs represent an important incubator for class A, B, and D carbapenemases, ESBLs, and other antibiotic resistance genes, reflecting the local hospital trends. In many cases, the total dependence of residents on nurse care for their daily living activities exposes them to both the selection and horizontal transmission of antibioticresistant organisms. It is important for LTCFs to develop effective control measures to prevent outbreaks of antibiotic-resistant strains.