antibiotics

Article
Combining Functional Genomics and Whole-Genome
Sequencing to Detect Antibiotic Resistance Genes in Bacterial
Strains Co-Occurring Simultaneously in a Brazilian Hospital
Tiago Cabral Borelli 1,† , Gabriel Lencioni Lovate 1,† , Ana Flavia Tonelli Scaranello 1 , Lucas Ferreira Ribeiro 1 ,
Livia Zaramela 2 , Felipe Marcelo Pereira-dos-Santos 3 , Rafael Silva-Rocha 3,‡ and María-Eugenia Guazzaroni 1, *,‡

1 Department of Biology, Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto,

Universidade de São Paulo, Av. Bandeirantes 3900, Ribeirão Preto, SP 14049-901, Brazil;
[email protected] (T.C.B.); [email protected] (G.L.L.);
[email protected] (A.F.T.S.); [email protected] (L.F.R.)
2 Department of Pediatrics, University of California San Diego, San Diego, CA 92161, USA;
[email protected]
3 Department of Cell and Molecular Biology, Faculdade de Medicina de Ribeirão Preto, University of São Paulo,
Av. Bandeirantes 3900, Ribeirão Preto, SP 14049-900, Brazil; [email protected] (F.M.P.-d.-S.);
[email protected] (R.S.-R.)
* Correspondence: [email protected]



† Both authors contributed equally to this work.

 ‡ Both authors contributed equally to this work.
Citation: Borelli, T.C.; Lovate, G.L.;
Scaranello, A.F.T.; Ribeiro, L.F.; Abstract: (1) Background: The rise of multi-antibiotic resistant bacteria represents an emergent
Zaramela, L.; Pereira-dos-Santos, threat to human health. Here, we investigate antibiotic resistance mechanisms in bacteria of several
F.M.; Silva-Rocha, R.; Guazzaroni, species isolated from an intensive care unit in Brazil. (2) Methods: We used whole-genome analysis
M.-E. Combining Functional to identify antibiotic resistance genes (ARGs) and plasmids in 34 strains of Gram-negative and
Genomics and Whole-Genome Gram-positive bacteria, providing the first genomic description of Morganella morganii and Ralstonia
Sequencing to Detect Antibiotic
mannitolilytica clinical isolates from South America. (3) Results: We identified a high abundance of
Resistance Genes in Bacterial Strains
beta-lactamase genes in resistant organisms, including seven extended-spectrum beta-lactamases
Co-Occurring Simultaneously in a
(OXA-1, OXA-10, CTX-M-1, KPC, TEM, HYDRO, BLP) shared between organisms from different
Brazilian Hospital. Antibiotics 2021,
species. Additionally, we identified several ARG-carrying plasmids indicating the potential for a
10, 419. https://doi.org/10.3390/
antibiotics10040419
fast transmission of resistance mechanism between bacterial strains. Furthermore, we uncovered
two pairs of (near) identical plasmids exhibiting multi-drug resistance. Finally, since many highly
Academic Editor: Cécile Muller resistant strains carry several different ARGs, we used functional genomics to investigate which
of them were indeed functional. In this sense, for three bacterial strains (Escherichia coli, Klebsiella
Received: 24 February 2021 pneumoniae, and M. morganii), we identified six beta-lactamase genes out of 15 predicted in silico
Accepted: 6 April 2021 as those mainly responsible for the resistance mechanisms observed, corroborating the existence of
Published: 11 April 2021 redundant resistance mechanisms in these organisms. (4) Conclusions: Systematic studies similar
to the one presented here should help to prevent outbreaks of novel multidrug-resistant bacteria in
Publisher’s Note: MDPI stays neutral healthcare facilities.
with regard to jurisdictional claims in
published maps and institutional affil-
Keywords: whole-genome analysis; functional genomics; resistome; mobilome; antibiotic resis-
iations.
tance genes

Copyright: © 2021 by the authors. 1. Introduction

Licensee MDPI, Basel, Switzerland.
Microbial resistance to antibiotics is a growing global concern. Established protocols in
This article is an open access article
clinics to fight nosocomial infections include the isolation of microorganisms from patient
distributed under the terms and
conditions of the Creative Commons
samples to allow their identification and to determine antibiotic susceptibility [1]. However,
Attribution (CC BY) license (https://
this process is time-consuming (taking 48 h or more), and prone to pathogen misidentifica-
creativecommons.org/licenses/by/
tion [2]. Therefore, the advent of next-generation sequencing (NGS) tools has allowed the
4.0/). rise of novel approaches to identify microbial pathogens and to fight infection [3]. Thus,

Antibiotics 2021, 10, 419. https://doi.org/10.3390/antibiotics10040419 https://www.mdpi.com/journal/antibiotics

Antibiotics 2021, 10, 419 2 of 19

in the last two decades, whole-genome sequencing (WGS) of microbial pathogens has
moved from being used mainly as a basic research tool to understand the biology and
evolution of pathogens [4,5], to a valuable tool to investigate outbreaks in hospitals and
nosocomial infection pathways [6–9]. For diagnostics purposes, current WGS technologies
can be cost-effective even for slow-growing pathogens such as Mycobacterium tuberculosis,
providing faster and more accurate results even for antibiotic resistance determination [10].
Additionally, culture-independent methods based on clinical metagenomics can be used to
identify several pathogens from nucleic acids extracted from patient samples without the
need for microbial isolation [11–13]. Furthermore, recent progress in the use of artificial
intelligence tools has allowed for the construction of computational models that can predict
with high accuracy the antimicrobial susceptibility of microbial pathogens based on WGS
data [14–16].
While routine WGS analysis for microbial identification is not a worldwide reality, it
has been extensively used to investigate microorganism population structures on different
scales. For example, Arias and coworkers, using WGS analysis from 96 methicillin-resistant
Staphylococcus aureus (MRSA) samples from nine countries in Latin America, demonstrated
a high degree of variation in the genome of different isolates from those countries [17]. The
same study indicated that among sampled hospitals, those in Brazil presented a higher
incidence of MRSA strains (up to 62%). Similarly, recent work by David and coworkers
investigated the pathway of the nosocomial spread of Klebsiella pneumoniae in 244 hospitals
in 32 European countries [18]. Using a well-defined sampling strategy and WGS analysis of
more than 1700 K. pneumoniae strains, the authors could quantify the role of intra-hospital
pathogen dissemination, as well as some potential pathways for the introduction of novel
strains from the United States to Europe. In addition to those examples, large-scale WGS
analysis has been used to investigate the molecular adaptation to different hosts, as in the
work by Arimizu et al., where the authors analyzed Escherichia coli strains from human
versus bovine samples [19].
Another key process playing a significant role in the rise of new microbial threats
is the propagation of mobile virulence and antimicrobial resistance factors within these
populations mediated especially by plasmids and transposons [5,20]. Therefore, the rapid
evolution of plasmids through structural rearrangements, acquisition of virulence genes,
plasmid fusions, and propagation to pathogens can account for the fast dissemination of
super-virulent or super-resistant bacteria [21–23]. Understanding the very dynamic and
complex processes could hold the potential for the design of new drugs aiming at reducing
plasmid propagation between pathogens [24]. While the use of WGS is currently growing
worldwide, most studies (especially in Brazil) have been restricted to some particular
species (such as K. pneumoniae or S. aureus), without considering the importance of other
species as repositories of antibiotic resistance genes (ARGs) in hospital settings. In our
study, we collected clinical bacterial strains to search for ARGs and their association with
mobile genetic elements. We identified ARGs with high spreading potential by combining
whole-genome sequencing and functional genomics. Here, we investigate 34 bacterial
strains at the genomic level from 18 different species (and 11 genera) isolated over the
same two weeks at a public reference hospital in Brazil. WGS analysis indicated that
many strains are only distantly related to those available in public databases. We aimed to
identify ARGs and plasmids harboring these elements, as well as evidence for common
resistance mechanisms shared between strains from the same or different species. We
were able to identify several ARG-harboring plasmids, two of which were present in both
Gram-negative and Gram-positive strains, and seven beta-lactamases located in multiple
hosts with 100% identity at the nucleotide level, two of which were inferred as being active
using functional genomic library screening. In addition, comparative sequence analysis
identified a novel IncFII K. pneumoniae plasmid harboring two ARGs, potentially indicating
a recent introduction from Asia to Brazil.
Antibiotics 2021, 10, 419 3 of 19

2. Materials and Methods

2.1. Sample Collection
Samples were taken at the Ribeirão Preto Clinics Hospital (HCRP, Ribeirão Preto,
Brazil), a tertiary reference hospital in Latin America with 920 beds and 34,000 hospital
admissions per year. Samples were isolated from different patients hospitalized in weeks
44 and 48 of 2018. Strains were obtained from different samples, as indicated in Figure 1A.
Thirty-five strains were randomly selected from a total of the 105 strains that were available
and represent different species, with a major prevalence of the Klebsiella genus, E. coli,
Pseudomonas aeruginosa, and the genus Staphylococcus. After strain characterization by Vitek
2, samples were inactivated and used for genomic DNA extraction and sequencing, as
indicated below.

2.2. DNA Extraction and Genome Sequencing

Total genomic DNA was extracted using Wizard Genomic DNA Purification Kit
(Promega, Madison, WI, USA) following the manufacturer’s instructions. Figure 1B
schematically represents the overall strategy used for WGS analysis. The DNA concentra-
tions were measured fluorometrically (Qubit® 3.0, kit Qubit® dsDNA Broad Range Assay
Kit, Life Technologies, Carlsbad, CA, USA). Purified DNA from 34 isolates was prepared
for sequencing using the Nextera XT DNA Library Prep Kit (Illumina, San Diego, CA,
USA). Libraries were assessed for quality using the 2100 Bioanalyzer (Agilent Genomics,
Santa Clara, CA, USA) and subsequently sequenced using HiSeq 2 × 150 bp cycle kits
(Illumina, San Diego, CA, USA). On average, 5.5 million reads were generated per library.
Adapters were trimmed using Trimmomatic v0.36. Samples were filtered for possible
human contamination by aligning the trimmed reads against reference databases using
Bowtie2 v2-2.2.3 with the following parameters: (-D 20 -R 3 -N 1 -L 20 very-sensitive-local).
Overlapped reads were merged using Flash version 1.2.11. Merged and unmerged reads
were assembled using Spades v3.12.0 with the following parameters: (-k 21, 33, 55, 77,
99, 127 –merge). Genome quality (completeness and contamination) was evaluated using
CheckM v1.0.7 and QUAST. Genome annotations were performed using Prokka v1.11
with default parameters. Amino acid sequences of all genes identified using Prokka were
aligned to the National Database of Antibiotic Resistant Organisms (NDARO) database
obtained from NCBI (March 2020). The alignment was performed using Diamond v0.8.24
with the following parameters: (blastx -k 5 -f 6 –E value 0.001). Alignments with ≥60 simi-
larity score were selected for further analysis. Quality assessment of sequenced genomes is
provided in Figure S1. All genomes are available at the NCBI under the BioProject number
PRJNA641571.

2.3. Identification of Antibiotic Resistance Genes, Plasmids, and Phylogenomic Analysis

The identification of antibiotic resistance genes was performed using the ABRicate
pipeline by searching annotated genes using reference databases (ARG-ANNOT, NCBI
AMRFinderPlus (https://www.ncbi.nlm.nih.gov/bioproject/PRJNA313047, accessed on 1
December 2020), CARD, and ResFidner), as well as DeepARG [25]. The identification of
plasmids was performed using Plasmidfinder, and contigs harboring ARGs and plasmid-
related genes were further analyzed in detail. For genomic comparison, reference and
assembly genomic data were downloaded from the NCBI databank. Phylogenomic analy-
ses were performed using Parsnp and Gingr [26] and phylogenetic trees were visualized
using iTOL [27].

2.4. Genomic Library Construction

For the cloning of the genomic DNAs in the pSEVA232 [28–30] vector, 2 µg of genomic
DNA from each strain were digested with Sau3AI. Meanwhile, pSEVA232 digestion using
BamHI and further dephosphorylation were performed. Genomic fragments from 1.5
to 6 kb and the linearized pSEVA232 vector were selected and incubated with the T4
DNA Ligase enzyme in a 2:1 insert/vector ratio. Then, ligations were transformed in the
Antibiotics 2021, 10, 419 4 of 19

electrocompetent E. coli DH10B [31] with a MicroPulser electroporator (Bio-Rad, Hercules,

CA, USA). The resulting libraries were analyzed for the percentage of plasmids carrying
genomic DNA and the average size of the insert they contained.

2.5. Determination of Minimum Inhibitory Concentrations (MICs)

The MICs were determined in the same culture medium (solid LB) and conditions
of the screenings, employing serial dilution of the test antibiotics (amoxicillin, oxacillin,
or penicillin G). Solid medium plates supplemented with kanamycin (50 µg mL−1 ), IPTG
(100 µM), and dilutions of each beta-lactam antibiotic were inoculated with approximately
2.5 × 106 colony forming units (CFUs) of E. coli DH10B harboring the pSEVA232 vector.
We performed dilutions of antibiotics and culture media according to the CLSI M100
supplement [32].

2.6. Screening and Phenotype Confirmation

Three pools of clones (2.5 × 106 clones per plate) were plated from each library on
solid LB supplemented with kanamycin (50 µg µL−1 ), IPTG (100 µM), and inhibitory
concentrations of each beta-lactam antibiotic (8 µg mL−1 for amoxicillin, 32 µg mL−1 for
penicillin G, or 256 µg mL−1 for oxacillin). Positive clones were cultured in liquid LB
medium supplemented with kanamycin (50 µg mL−1 ) for extraction of plasmid DNA
with the Wizard Plus SV Minipreps DNA Purification System Kit (Promega, Madison,
WI, USA). Plasmids with unique EcoRI/HindIII restriction patterns were subjected to
re-transformation and phenotypic confirmation by streaking the clones in solid LB medium
supplemented with kanamycin (50 µg mL−1 ), IPTG (100 µM), and the test antibiotics.

2.7. Extraction of Insert Sequence from Assembled Genomes

Plasmid DNA was sequenced on both strands by primer walking using the ABI
PRISMDye Terminator Cycle Sequencing Ready Reaction kit (PerkinElmer, Waltham, MA,
USA) and an ABI PRISM 377 sequencer (Perkin-Elmer, Waltham, MA, USA) according to the
manufacturer’s instructions. A python script was developed to extract and annotate inserts
from Sanger reads, which is available on GitHub (https://github.com/tiagocabralborelli/
Borelli-et-al-insert-finder, accessed on 1 December 2020). First, the algorithm converted
ab1 files to the fasta format and searched for the read sequence in the assembled genome
using BLAST+ [33]. We then extracted the inter-read region to a fasta file, which was then
annotated by PROKKA [34].

3. Results and Discussion

3.1. WGS Analysis of Clinical Strains Isolated Over the Same Two Weeks
We selected 34 bacterial strains isolated from different patient samples and performed
WGS as represented in Figure 1. While well-studied pathogens such as K. pneumoniae, E. coli,
P. aeruginosa, and S. aureus were well represented in the sampling, we were able to analyze
pathogens with very little representative genomic information in January 2021. For exam-
ple, two Ralstonia mannitolilytica strains were sequenced, but only nine complete or draft
genomes were available at the NCBI. Other relatively underrepresented strains were Strepto-
coccus gallolyticus (28 genomes available) and Morganella morganii (96 genomes). These num-
bers contrast with those of K. pneumoniae, E. coli, or S. aureus, where 8000–20,000 genome
sequences are available. This evidence indicates that many clinically relevant pathogens
have been underrepresented in WGS analysis efforts worldwide. For instance, phylogenetic
analysis of Burkholderia cepacia 540A against all 166 available genomes in NCBI (Figure S2)
indicates this strain is also very divergent from other strains but belongs to a branch formed
by strains isolated from a patient with cystic fibrosis in the United Kingdom and endo-
phytic bacteria isolated from Australia [35]. Therefore, some of the new genomes generated
here demonstrate a significant diversity of some underrepresented microorganisms and
should serve as reference sequences for future studies on clinical isolates in Brazil and
South America.
 some of the new genomes generated here demonstrate a significant diversity of some un-
Antibiotics 2021, 10, 419 derrepresented microorganisms and should serve as reference sequences for 5future
of 19 stud-
ies on clinical isolates in Brazil and South America.

A A. pittii 466A
B. cepacia 540A
I E. coli 508A
A. pittii 112M3 E. coli 537B
K. pneumoniae 112M3 E. coli 522B
E. coli 456A
E. asburiae 790C
K. variicola 659A
K. pneumoniae 98M3 II K. quasipneumoniae 614A
P. aeruginosa 155M3
K. pneumoniae 764A
P. aeruginosa 104M3
K. pneumoniae 508B
IV K. oxytoca 457A
E. coli 92M3 M. morganii 538A
E. coli 126M3 III R. mannitolityca 451A
E. roggenkampii 91M3 R. mannitolityca 547A
K. pneumoniae 125M3 S. aureus 481A
P. aeruginosa 140M3 S. aureus 468A
S. epidermidis 452B
S. haemolyticus 637C
S. warneri 732B
S. warneri 579A
S. maltophilia 599B
S. maltophilia 685B
S. gallolyticus 471B

B De novo assembly
Sample collection (Spades)

Strain characterization (Vitek 2) Annotation (Prokka)

Antibiotic resistance genes (ARG-
DNA extraction ANNOT, NCBI-beta, CARD,
ResFinder, DeepARG)
Illumina sequencing
Virulence genes (VFBD)
Raw reads
Plasmid identification
(Plasmidfinder)
Trimmed reads (Trimmomatic)
Core-genome alignment and
analysis (Parsnp, Gingr)
Remove human reads (Bowtie)

Merge overlapping reads (Flash) Tree visualization (iTOL)

Figure 1. Overall strategy used for the whole-genome analysis of clinical strains. (A) In total, 34 bacterial strains were
Figure 1. Overall strategy used for the whole-genome analysis of clinical strains. (A) In total, 34 bacterial strains were
isolated from several samples, such as cerebrospinal fluid (I, 2 strains), bronchoalveolar lavage (II, 3 strains), ascitic fluid
isolated from several samples, such as cerebrospinal fluid (I, 2 strains), bronchoalveolar lavage (II, 3 strains), ascitic fluid
(III, 5 strains), and blood (IV, 25 strains). The four most common bacterial groups (Klebsiella pneumoniae, Escherichia coli,
(III, 5 strains), and blood (IV, 25 strains). The four most common bacterial groups (Klebsiella pneumoniae, Escherichia coli,
Pseudomonas aeruginosa, and Staphylococcus spp.) are colored. (B) Schematic representation of the main bioinformatic pipeline
Pseudomonas aeruginosa, and Staphylococcus spp.) are colored. (B) Schematic representation of the main bioinformatic pipe-
used for genome sequencing, assembly, annotation, and identification of antibiotic resistance genes (ARGs) and virulence
line used for genome sequencing, assembly, annotation, and identification of antibiotic resistance genes (ARGs) and viru-
factor and genomic analysis.
lence factor and genomic analysis.
Antibiotics 2021, 10, 419 6 of 19

3.2. Identification of Resistance Genes in Clinical Strains

We next analyzed the existence of ARGs in the sequenced genomes. Analysis of ARGs
using the ARG-ANNOT database indicated a higher prevalence of beta-lactamase coding
genes, followed by amino-glycosidases (Figure 2A), while analysis with the DeepARG
tool [25] showed the multidrug category to be the most abundant on the genomes analyzed
(Figure S3). We next analyzed the ARG distribution in the two most abundant groups, E.
coli and Klebsiella. In the first case, each of the six E. coli strains displayed a unique set of
ARGs from different categories, and five particular beta-lactamases (blaTEM-105 , blaOXA-1 ,
blaKPC-2 , blaCTX-M-15 , and blaCMY-111 ) were found in at least one genome (Figure 2B). Only
one strain (E. coli 456A) did not present any of these five bla genes, and this strain was
sensitive to all antibiotics tested by Vitek 2 (Figures 2B and 3). Next, we investigated which
beta-lactamase genes were conserved in the strains analyzed. For this, we compared all
of the ~125 bla genes identified in Figure 2 and searched for those coding proteins with
100% identity at the amino acid sequence (Figure 3). Using this approach, we identified
seven bla genes (blaOXA-1 , blaOXA-10 , blaCTX-M-1 , blaKPC , blaTEM , blaHYDRO , and blaBLP ) which
have 100% aa identity between two or more strains. Notably, blaOXA-1 , blaCTX-M-1 , blaKPC ,
and blaTEM were present in three to six strains from different species. Interestingly, both
blaOXA-1 and blaCTX-M-1 were found in E. coli, K. pneumoniae, and M. morganii, which could
reveal horizontal gene transfer between these strains in the past. From these seven genes,
two (blaOXA-1 and blaKPC ) were found to be active in the functional screening presented
below. The ARG patterns identified in E. coli and Klebsiella are quite common and are
often associated with widespread mobile genetic elements (MGE) with different levels of
conservation, as seen in our work (Figure 3) [36–39].

3.3. Intrinsic Resistome and Hospital-Associated Resistome

Classically, antimicrobial resistance has been seen from a hospital-associated resistome
point of view which comprises genes originated by selective pression due to antibiotic
overuse and/or acquired from horizontal gene transfer (HGT). Their hosts are capable
of thriving under treatment with antimicrobial substances above a clinically established
concentration threshold (MIC). However, recent works found that some genes from differ-
ent ontologies and not directly involved in antimicrobial response can provide resistance
(the so-called intrinsic resistome) in environments apart from hospitals [40]. For instance,
ABC-system efflux pumps extrude a myriad of harmful substances from within the bacte-
rial cell and, by doing so, they reduce the inner antibiotic concentration [40,41]. Since the
identification of the intrinsic resistome, new definitions such as the ecological threshold
(ECOFF) arrived to classify whether a species is intrinsically resistant. In this work we
chose ABRicate to search for ARGs since this tool only identifies acquired ARGs, therefore
excluding the intrinsic resistome. Furthermore, resistance phenotypic testing in hospital
conditions matches with the ARGs found using the ARG-ANNOT database. A similar
strategy was applied by Zankari and colleagues [42], who were responsible for the Res-
Finder database. Furthermore, according to the Beta-Lactamase DataBase (BLDB) [43], only
two beta-lactamases detected in this study belong to the intrinsic resistome of their hosts
(Table S1) (blaOXY2-1 and blaSHV-187 from K. oxyteca and K. pneumoniae, respectively). Finally,
further experimental validations confirmed the increased MIC in the presence of the ARGs
that we found in heterologous hosts, as discussed below.
Antibiotics 2021, 10, 419 7 of 19
Antibiotics 2021, 10, 419 7 of 20

Figure 2. Identification of ARGs in sequenced

Figure genomes. (A)
2. Identification of Heatmap
ARGs inshowing
sequenced the presence
genomes.of ARGs
(A)identified
Heatmap byshowing
ARG- the presence of
ANNOT indicating the number of each resistance gene per genome. Data were clustered using hierarchical mapping with
ARGs identified by ARG-ANNOT indicating the number of each resistance gene per genome. Data
Euclidian distance. The blue to red scale indicates the number of ARGs for each strain in each category, as indicated in the
were clustered
legend. The nucleotide sequences in ARG-ANNOTusingfrom
hierarchical mappingclasses
different antibiotics with Euclidian distance.
are abbreviated The blue to red scale indicates
as follows—AGly:
the number
aminoglycoside resistance genes; of ARGs for
Bla: beta-lactamases; each
Fcyn: strain in
fosfomycin; each
Flq: category, asGly:
fluoroquinolones; indicated in the MLS:
glycopeptides; legend. The nucleotide
macrolide–lincosamide–streptogramin;
sequencesPhe: phenicols; Rif: rifampin;
in ARG-ANNOT Sul: sulfonamides;
from different Tet: classes
antibiotics tetracyclines; and Tmt: tri- as follows—AGly:
are abbreviated
Aminoglycoside resistance genes; Bla: Beta-lactamases; Fcyn: Fosfomycin; Flq: Fluoroquinolones;
Gly: Glycopeptides; MLS: Macrolide–lincosamide–streptogramin; Phe: phenicols; Rif: rifampin; Sul:
sulfonamides; Tet: tetracyclines; and Tmt: trimethoprim. (B) Distribution of different ARGs per
genome of E. coli, colored by antibiotic category. The maximum likelihood phylogeny for the strains
was based on the core genome. (C) Distribution of different ARGs per genome of K. pneumoniae,
following the scheme in (B).
 Antibiotics 2021, 10, 419 8 of 20

methoprim.
Antibiotics 2021, 10, 419 (B) Distribution of different ARGs per genome of E. coli, colored by antibiotic category. The maximum likeli- 8 of 19
hood phylogeny for the strains was based on the core genome. (C) Distribution of different ARGs per genome of K. pneu-
moniae, following the scheme in (B).

3. Shared
FigureFigure beta-lactamases
3. Shared beta-lactamaseswith 100%
with 100%identity atat
identity the
theprotein
proteinlevel.
level. Seven beta-lactamasecoding
Seven beta-lactamase coding genes
genes were
were found as
found
sharedasamong
sharedgram-negative
among gram-negative
strainsstrains analyzed.
analyzed. Connected
Connected circles
circles indicate
indicate thatthe
that thegenes
genes are
are presented
presented ininthose
thosestrains.
strains. On
On the
theright, the antibiotic resistance
profileprofile
of theofanalyzed
the analyzed strains is shown.Genes
Genes for
for bla
blaOXA-1 and blaKPC are highlighted
the right, antibiotic resistance strains is shown. OXA-1 and blaKPC are highlighted
(red triangles) since these genes were identified in the functional screening carried out in this study. On the right, antibiotic
(red triangles) since these genes were identified in the functional screening carried out in this study. On the right, antibiotic
resistance levels are indicated, with numbers indicating the resistance levels in mg/mL. Red indicates resistance to the
resistance levelswhile
antibiotic, are indicated,
blue denotes with numbers
sensitivity. indicating
In the the itresistance
final column is indicatedlevels
if thein mg/mL.
strain Red indicates ß-lactamase
is extended-spectrum resistance to the
antibiotic, while blueordenotes
(ESBL)-positive -negative.sensitivity. In the final
AMP: ampicillin; APS: column it is indicated
ampicillin/sulbactam; if the
PIT: strain is extended-spectrum
piperacillin/tazobactam; ß-lactamase
CFX: cefuroxime,
CFX-A: cefuroxime
(ESBL)-positive axetil;AMP:
or -negative. CTX:ampicillin;
cefotaxime; APS:
CAZ:ampicillin/sulbactam;
ceftazidime; CPM: cefepime; CFO: cefoxitin; ERT: ertapenem;
PIT: piperacillin/tazobactam; IMP:
CFX: cefuroxime,
imipenem; MER: meropenem; CIP: ciprofloxacin; GEN: gentamicin.
CFX-A: cefuroxime axetil; CTX: cefotaxime; CAZ: ceftazidime; CPM: cefepime; CFO: cefoxitin; ERT: ertapenem; IMP:
imipenem; MER: meropenem; CIP: ciprofloxacin;
3.4. Identification of GEN:
ARGs gentamicin.
Located in Plasmids
We next aimed to identify ARGs with potential mobilization through plasmids in the
3.4.analyzed
Identification of For
species. ARGs Located
this, in Plasmids
we crossed the data from ARG-ANNOT with the prediction of
We next
plasmid aimedgenerated
elements to identifyby ARGs with potential
PlasmidFinder. mobilization
Using this approach, we through
were ableplasmids
to iden- in the
tify ninespecies.
analyzed potential plasmids
For this, wefrom seventhe
crossed species
data associated with at leastwith
from ARG-ANNOT one the
ARG. As
prediction
shown inelements
of plasmid Figure 4A,generated
a ~42kb plasmid (pKP98M3N42)
by PlasmidFinder. harboring
Using a blaKPC-2 and
this approach, weawere
sat-2Aable to
resistance
identify ninedeterminant were identified
potential plasmids in K. pneumoniae
from seven 98M3, andwith
species associated this plasmid
at least also
one car-
ARG. As
ries transposases, recombinases, and type IV secretion system genes.
shown in Figure 4A, a ~42 kb plasmid (pKP98M3N42) harboring a blaKPC-2 and a sat-2AAn identical plasmid
(pKP125M3N44; 100% nucleotide sequence identity) was also found in K. pneumoniae
resistance determinant were identified in K. pneumoniae 98M3, and this plasmid also carries
125M3 (Figures 4A and S4A). As mentioned before, this blaKPC-2 gene was identical in these
transposases, recombinases, and type IV secretion system genes. An identical plasmid
two K. pneumoniae strains and in E. coli 126M3, but it was not possible to locate the ARG
(pKP125M3N44;
in a plasmid in the100%
latternucleotide sequence
case. This might be dueidentity) was in
to limitations also
ourfound in K. pneumoniae
draft assemblies. In
125M3 (Figure 4A and Figure S4A). As mentioned before,
two recent studies, the authors demonstrated ARG-carrying plasmid this bla transfer inwas
KPC-2 gene identical
clinical
in these two
settings K. pneumoniae
between bacteria ofstrains
the same E. coli 126M3,
andorindifferent species, but it was notthe
emphasizing possible to locate
significance of the
ARG in a plasmid in the latter case. This might be due to limitations in our
revealing potential plasmid-mediated outbreaks to efficiently track horizontal ARG trans- draft assemblies.
In two recent
mission studies, [44,45].
in hospitals the authors
Once demonstrated ARG-carrying
phages could transfer ARGs among plasmid transfer
their hosts we in clinical
also
employed Phaster [46], a web-service for phage identification, to search
settings between bacteria of the same or different species, emphasizing the significance for the co-occur-
rence of phages
of revealing and ARGs,
potential but ARGS were found
plasmid-mediated in phage-related
outbreaks regions
to efficiently of the
track genomes ARG
horizontal
transmission in hospitals [44,45]. Once phages could transfer ARGs among infor-
(Table S2). A comprehensive HCRP phagesphere analyses would provide more their hosts
wemation on this topic.
also employed Phaster [46], a web-service for phage identification, to search for the
Another strain, K. pneumoniae 508B, harbors two plasmids with ~136kb
co-occurrence of phages and ARGs, but ARGS were found in phage-related regions of the
(pKP508BN15) and ~62kb (pKP508BN34). Both plasmids harbor transposon elements,
genomes (Table S2). A comprehensive HCRP phagesphere analyses would provide more
with the larger one harboring a sul2 resistance gene and the smaller one with two ARGs
information on this topic.
Another strain, K. pneumoniae 508B, harbors two plasmids with ~136 kb (pKP508BN15)
and ~62 kb (pKP508BN34). Both plasmids harbor transposon elements, with the larger
one harboring a sul2 resistance gene and the smaller one with two ARGs (qnr-S1 and
blaLAP-2 , Figure 4B,C). In general, the coexistence of ARGs with transposon elements was
also observed for two plasmids identified in E. coli strains (Figure S4B,C) and Staphylococcus
warneri 732B (Figure S5A). Finally, two almost identical small plasmids (~2.3 kb; 99.94%
nucleotide identity) were identified in S. warneri 732B and Staphylococcus epidermidis 452B,
which harbor aadC and ermC resistance determinants (Figure S5B). Taken together, these
data demonstrate the potential for dissemination of many ARGs genes identified here,
 (qnr-S1 and blaLAP-2, Figure 4B,C). In general, the coexistence of ARGs with transposon el-
ements was also observed for two plasmids identified in E. coli strains (Figure S4B,C) and
Antibiotics 2021, 10, 419 Staphylococcus warneri 732B (Figure S5A). Finally, two almost identical small plasmids 9 of 19
(~2.3kb; 99.94% nucleotide identity) were identified in S. warneri 732B and Staphylococcus
epidermidis 452B, which harbor aadC and ermC resistance determinants (Figure S5B). Taken
together, these data demonstrate the potential for dissemination of many ARGs genes
and the existence
identified ofthe
here, and identical or of
existence near-identical plasmids between
identical or near-identical different
plasmids between species could
different
species could
indicate indicate
that these that these
elements haveelements have been among
been mobilizing mobilizing
someamong some
of these of theseWe
species. spe-
then
cies. We then
confirmed the confirmed
presence ofthe presence
IncC of IncC
and IncX3 and IncX3
replicons replicons
in the plasmidsin the
of K.plasmids
pneumoniaof K.
508B
pneumonia
and 98M3, 508B and 98M3,
respectively, respectively,
with bacWGSTdb with[47,48]
bacWGSTdb
(Table [47,48]
S3), but(Table S3),replicons
no new but no newwere
replicons
found were foundwith
in association in association
antibioticwith antibiotic
resistance resistance genes.
genes.

Figure 4. Schematic representation of genes of three K. pneumoniae plasmids. (A) Plasmid pKP98M3N42 (43 kb) from
K. pneumoniae 98M3. This plasmid carries two ARGs (blaKPC-2 and sat-2A), elements of a type IV secretion system, two
resolvases, and a Tn3 transposase. This plasmid is very similar to pKP125M3N44 from K. pneumoniae 125M3 (Figure S4A).
Whole-plasmid visualization was performed using a python module for prokaryotic genome analyses (DnaFeaturesView)
and a matplotlib module combined, as well as ARG-ANNOT and Prokka’s results [49]. (B) Plasmid pKP508BN15 (136.8 kb)
from K. pneumoniae 508B transports sul2 and an rRNA adenine n-6-methyltransferase (ermC) resistance marker. A transposase,
a xerC recombinase, and a type IV secretion system virB8 protein are also found in this plasmid. (C) Plasmid pKP508BN34
(63 kb) is also from K. pneumoniae 508B and carries several transposases and two resistance determinants, qnr-S1 and blaLAP-2 .
Legends represent the colors code for the identified genes.
Antibiotics 2021, 10, 419 10 of 19

3.5. Experimental Validation of Functional Beta-Lactamases from High-Resistance Bacteria

Once we distinguished several ARGs in the genomes analyzed, we decided to perform
a functional screening to potentially discover unknown ARGs in the strains while also
identifying which of these genes could confer resistance to a heterologous host. For this,
we selected three strains (E. coli 126M3, K. pneumoniae 508B, and M. morganii 538A) to
construct genomic libraries into laboratory E. coli DH10B (Table 1). The libraries were
constructed into the broad host range vector pSEVA232, which contains a kanamycin
resistance marker, a broad host range oriV with a medium-copy number, and a variant
of the lacZα multiple cloning site with a Plac promoter [28–30]. In this way, the libraries
generated during this study or individual plasmids of interest can be transferred to other
bacterial strains for another screening or functional evaluation (Figure 5A). The use of a
medium copy-number plasmid allows a closer assessment of the natural genetic context of
ARGs, in contrast to other studies that use high-copy plasmids [50]. Accordingly, plasmids
with lower copy-number and monomeric states also tend to be more stably inherited
throughout bacterial populations [51]. We screened ~750,000 clones of each library against
each antibiotic (amoxicillin, oxacillin, and penicillin G) and obtained 44 clones with unique
sequences containing ARGs (Figure 5B and Table 2).
Clones containing the blaKPC-2 were by far more abundant in the screening (Table 2 and
Figure 6). The genomic context of identified blaKPC-2 indicates that it is prone to horizontal
transfer once it is flanked by transposases. Martínez et al. [52] described a framework to
prioritize the risk of ARGs, the Resistance Readiness Condition (RESCon). The RESCon
algorithm considers the similarity of an ARG to known genes, functional evaluation, the
clinical relevance of the antibiotic, the presence of a mobile genetic element, and presence
in a human pathogen. The characteristics of this ARG would categorize it as RESCon
1, an ARG with the highest possibility of thriving in a clinical setting [52]. Although a
framework better describing the impacts of gene transfer to prioritize risk is needed [53],
the RESCon classification indicates the clinical relevance of this ARG. With ARGs found in
all three functional screenings, we were able to identify with high frequency six different
bla genes using the functional approach presented here, with at least two of them being
present in plasmids—blaKPC-2 and blaLAP-2 (see next section (Figure 6)).
We propose that functional genomics can be combined with current approaches based
on large-scale sequencing in order to better understand the functional aspects of ARGs.
Recent developments in machine learning [25] have provided tools to find ARGs that can
be used to detect ARGs that would not be otherwise identified with sequence similarity
tools. Still, the databases used to train those tools are biased for specific antibiotic resistance
classes such as beta-lactam, bacitracin, macrolide–lincosamide–streptogramin (MLS), and
efflux pumps. Indeed, we could only find just a fraction of ARGs annotated through
bioinformatics tools with our functional approach. This paradox could be due to the
other ARGs not being functional in the experimental conditions used here or because our
screening was not exhaustive enough to cover those sequences.

Table 1. Features of the generated genomic libraries.

Genomic Library E. coli 126M3 K. pneumoniae 508B M. morganii 538A

Total number of clones 13,442 25,693 11,804
Percentage of clones with insert (%) 90 90 90
Number of clones with insert 12,122 23124 10,624
Insert size variation (kb) 1.0–10.5 0.2–2.2 0.2–6.5
Average insert size (kb) 5.2 1.4 3.0
Total genomic library size (mb) 62.8 32.4 32.1
Estimated genome coverage 11.9× 5.8× 7.8×
 Antibiotics 2021, 10, 419 11 of 19
Antibiotics 2021, 10, 419 11 of 20

FigureFigure
5. Experimental
5. Experimentaldesign
design forforfunctional
functional genomics analysis.
genomics analysis. (A)(A) Strains
Strains and and backbone
backbone used toused to construct
construct the
the library library
and
and features
features of
of the obtainedlibraries.
the obtained libraries. (B)(B) Functional
Functional screening
screening for beta-lactam
for beta-lactam resistant
resistant clones, clones, phenotypic
phenotypic confirmation,
confirmation, and
and sequence
sequence identification. CFU:Colony
identification. CFU: colonyforming
forming unit;
unit; RFLP:
RFLP: restriction
Restriction fragment
fragment length
length polymorphism;
polymorphism; KmR: kanamy-
KmR: Kanamycin
cin resistance
resistance marker; pBBR1: A broad host range oriV; lacZα: lacZα gene with multiple cloning site; Plac: Plac Plac
marker; pBBR1: a broad host range oriV; lacZα: lacZα gene with multiple cloning site; Plac: promoter;
promoter;
pFGRnnnn: plasmid naming schema.
pFGRnnnn: Plasmid naming schema.
Antibiotics 2021, 10, 419 12 of 19

Table 2. Comparison of ARG identification using in silico and functional approaches.

Strain In Silico (Argannot) Number of Sequenced Resistant Clones

E. coli 126M3 Penicillin_Binding_Protein_Ecoli -
AmpC1_Ecoli -
blaKPC-2 28
AmpC2_Ecoli 1
blaOXA-1 -
ampH_Ecoli -
K. pneumoniae 508B blaSHV-11 -
Penicillin_Binding_Protein_Ecoli -
blaLAP-2 2
ampH -
blaCTX-M-15 8
blaOXA-1 1
M. morganii 538A blaCTX-M-15 -
blaOXA-1 -
blaMOR-2 4

3.6. Mapping of Plasmids in the Brazilian Territory

To evaluate the biogeographic distribution of functional ARGSs from K. pneumo-
niae, we performed genomic comparative analysis including 50 plasmids deposited in
the NCBI data bank and two of the plasmids identified in this study (pKP98M3N42,
pKP508BN34). Distance tree analysis showed the presence of two distinguishable groups.
Plasmid pKP98M3N42 (identical to plasmid pKP125M3N44, also identified in this work) is
located in the first group (comprising seven strains, all reported in Brazilian cities from 2009
to 2015 except for one from the United States), which indicates that pKP98M3N42 should
share structural features with the other plasmids positioned in this group (Figure 7A).
Additionally, pKP98M3N42 shares 99.9–100% sequence identity with IncX3 plasmids of
these seven strains, which have previously been shown to play an important function in
mediating the horizontal transmission of blaKPC-2 genes among hospital-associated mem-
bers of the Enterobacteriaceae family [54,55]. Moreover, IncX3 plasmids carrying blaKPC-2
genes were also reported in countries that are very distant geographically, such as the
United States [56], Brazil [57,58], Australia [59], Italy [60], France [61], South Korea [62,63],
China [64], and Israel and Greece [54] (to cite a few). Interestingly, although IncX3 self-
transmissible plasmids are widespread globally, the evident diversification of plasmids in
the first separated branch of the tree (Figure 7A) could indicate that plasmids from strains
isolated in clinically relevant bacteria in Brazilian ground undergo their own structural
reorganization. The group formed with K. pneumonia 98M3 is mostly related to isolates
previously found in Brazil, as well as one strain from Japan and another from Thailand
(Table S4), which means that both the host and its derivative plasmid are established
in Brazil.
On the other hand, distance tree representation of the 50 close plasmid sequences
related to pKP508BN34 did not show the presence of evident different groups. BLAST
analysis showed that plasmid pKP508BN34 is widely distributed in K. pneumoniae strains,
with 99.9–100% sequence identity and query coverage ranging from 84% to 100% to the
18 closest related plasmids, with some associated to hypervirulent strains, such as strain
KP58 bearing plasmid pKP58-3 (Figure 7B). The plasmid pKP508BN34, which is 63.067 bp
in size and harbors various mobile elements that contain antimicrobial resistance genes
including qnrS1 and blaLAP-2 , belongs to the IncFII plasmid group. As seen in the branch
highlighted in Figure 7B, most of the strains carrying the closest related plasmids to
pKP508BN34 were described in different cities of China, as well as one in Germany, one
in Japan, and three in Thailand, and were associated with diverse host diseases such as
pneumoniae, pulmonary infection, urinary tract infection, intestinal infection, and diarrhea,
according to the data deposited in the bioprojects of the NCBI. In some cases, related strains
Antibiotics 2021, 10, 419 13 of 19

were reported in asymptomatic patients (strains TH164 carrying plasmid pTH164-3 and
strain TH114 bearing plasmid pTH114-3, both from Thailand), which is reasonable since
K. pneumoniae is also a member of the gut microbiota [65]. Mortality due to infection of
K. pneumoniae has been very rare in the past. However, this pathogen’s fast evolution
due to the gaining of hypervirulence plasmids has allowed this bacterium to cause severe
community-transmitted infections in relatively young and healthy hosts since the late
1980s (54, 55). To the best of our knowledge, this is the first time that this plasmid has been
reported in Brazil. Remarkably, in contrast to K. pneumoniae 98M3, the group formed with K.
pneumonia 508B3 is mostly related to isolates previously found in places outside Brazil such
as the United Kingdom, Austria, and the Czech Republic (Table S5), which contrasts with
the origin of the plasmids. Those findings show that plasmid and host came from different
Antibiotics 2021, 10, 419 13 of 20
regions on the globe, suggesting acquirement of KP508BN3 by K. pneumoniae 508B.

Figure 6. Features found in identified clones conferring resistance to antibiotics. (A) ampC-2 gene identified from E. coli
126M3.Figure Features
6. gene
(B) blaKPC-2 found
identified from E.incoliidentified clones
126M3. (C) bla conferring resistance to antibiotics. (A) ampC-2 gene
LAP-2 gene identified from pKP508BN34 plasmid from K. pneu-

moniaeidentified from E. gene

508B. (D) blaCTX-M-15 coli 126M3.
identified from blapneumoniae
(B) K. KPC-2 gene
508B.identified from
(E) blaOXA-1 gene E. colifrom
identified 126M3. (C) blaLAP-2 gene
K. pneumoniae
508B. (F) blaMOR-2 gene identified from M. morganii 538A. ORFs are colored according to their function: red—ORFs directly
identified from pKP508BN34 plasmid from K. pneumoniae 508B. (D) blaCTX-M-15
related to antibiotic resistance; orange—ORFs related to virulence; yellow—ORFs related to horizontal transfer of the
gene identified
from K. pneumoniae 508B. (E) blaOXA-1 gene identified from K. pneumoniae 508B. (F) blaMOR-2 gene
ARG; blue—ORFs with no identified relation to pathogenesis. The cloned region represents contig regions that were iden-
tified in our screenings. Overlapping cloned regions are darker, while dim regions have fewer overlapping identified
clones.
identified from M. morganii 538A. ORFs are colored according to their function: red—ORFs directly
related to antibiotic resistance; orange—ORFs related to virulence; yellow—ORFs related to horizontal
transfer of the ARG; blue—ORFs with no identified relation to pathogenesis. The cloned region
represents contig regions that were identified in our screenings. Overlapping cloned regions are
darker, while dim regions have fewer overlapping identified clones.
Antibiotics 2021, 10, 419 15 of 20

Antibiotics 2021, 10, 419 14 of 19

Figure 7. Distance relationship analysis for two plasmids from K. pneumoniae. (A) Distance relationship of the seven closest
Figure 7. Distance relationship analysis for two plasmids from K. pneumoniae. (A) Distance relationship of the seven closest
plasmids’ nucleotide sequences to pKP98M3N42, showing an E-value of less than 0.0 and a minimal sequence cover of 70%
plasmids’ nucleotide sequences to pKP98M3N42, showing an E-value of less than 0.0 and a minimal sequence cover of
in BLAST analysis. The tree was produced using pairwise alignments by means of the fast-minimum evolution method.
70% in BLAST analysis. The tree was produced using pairwise alignments by means of the fast-minimum evolution
The year denotes the collection data, and the asterisk indicates the data reported in the public database. (B) Distance
method. The year denotes the collection data, and the asterisk indicates the data reported in the public database. (B) Dis-
relationship of the 18 closest plasmids’ nucleotide sequences to pKP508BN34, showing an E-value of less than 0.0 and a
tance relationship of the 18 closest plasmids’ nucleotide sequences to pKP508BN34, showing an E-value of less than 0.0
minimal sequence cover of 50% in BLAST analysis. The tree was produced using pairwise alignments by means of the
and a minimal sequence cover of 50% in BLAST analysis. The tree was produced using pairwise alignments by means of
fast-minimum evolution method. The year indicates the collection data and the asterisk indicates the data reported in the
the fast-minimum evolution method. The year indicates the collection data and the asterisk indicates the data reported in
public database, provided when the collection data were not available. iTOL (https://itol.embl.de accessed on 1 December
the public database, provided when the collection data were not available. iTOL (https://itol.embl.de accessed on 1 De-
2020) was used for tree visualization.
cember 2020) was used for tree visualization.
As shown in Figure 8A,B, plasmids pKP98M3N42 and pKP508BN34 are highly struc-
turally conserved between K. pneumoniae strains available in the databank. Plasmids
pKP98M3N42 and pKP1253N44 (Figure 8A) are most similar to plasmids found in K. pneu-
moniae and E. coli, which have been frequently reported in Brazil (as in Figure 7A). Plasmid
pKP508BN34 (Figure 8B) was most similar to plasmids only isolated from K. pneumonia,
mostly identified in Asia (shown in Figure 7B) with a high degree of conservation. How-
ever, plasmid pKP508BN15 (which is present in K. pneumoniae 508B) presented a strong
structural diversification in the region close to the antibiotic markers, and these changes
seem to be related to the activity of the ISSpu21 transposon element located in this region
(Figure 8C). Plasmid pKP508BN15 (Figure 8C) was most similar to plasmids found in
several different bacterial species (K. pneumoniae, Photobacterium damselae subsp. piscicida,
Vibrio alginolyticus, and Vibrio cholerae) reported in Asia, Africa, and North America. Both
sul2 and rRNA adenine n-6-methyltransferase (ermC) resistance markers are located in
a highly divergent region of the plasmid and are not present in most related plasmids.
Interestingly, most related sequences available in the database are from worldwide strains,
including some isolated from Asia, Africa, North America, and Europe, but no sequences
were found from South America.
Antibiotics 2021, 10, 419 16 of 20
Antibiotics 2021, 10, 419 15 of 19

A
pKP125M3N44
K. pneumoniae 1194/11 - pKP1194a
K. pneumoniae A64477 – pKP64477d sat-2A
K. pneumoniae K89 – pKP89
K. pneumoniae Kp13 – pKP13d blaKPC-2
E. coli BR25-DEC – unnamed3
K. pneumoniae KP30835 – unnamed5
E. coli ECO037 – ECO37P1
pKP98M3N42

C
rRNA adenine N-6-methyltransferase

sul2

Figure
Figure8. 8.
Structural
Structuralcomparison
comparison ofofplasmids
plasmids from
from K. pneumoniae
K. pneumoniae strains.
strains. Identified
Identified plasmidsplasmids wereusing
were analyzed analyzed
blast, using
and blast,
andthe
the
best hits were used for comparison using BLAST Ring Image Generator (BRIG) [66]. For simplification, only divergent only di-
best hits were used for comparison using BLAST Ring Image Generator (BRIG) [66]. For simplification,
vergent
regionsregions
betweenbetween the plasmids
the plasmids are(A)
are shown. shown. (A) pKP98M3N42
pKP98M3N42 (black) and (black) and pKP1253N44
pKP1253N44 (magenta). (B)(magenta).
pKP508BN34(B)(black).
pKP508BN34
(black). (C) pKP508BN15
(C) pKP508BN15 (black).(black).

4.4. Conclusions
Conclusions
Here, we
Here, wesampled
sampledclinical bacterial
clinical strains
bacterial to investigate
strains the existence
to investigate of ARGs
the existence ofand
ARGs and
their association with mobile genetic elements. We identified several ARGs shared between
their association with mobile genetic elements. We identified several ARGs shared be-
strains from different species, and some of these ARGs were associated with large plasmids,
tween
mostlystrains
endowed from different
with species,
transposable and some
elements. of these
Instead ARGs were
of focusing associated
on strains with large
from the
plasmids, mostly endowed with transposable elements. Instead of focusing
same species or genus, our approach considered strains co-occurring simultaneously into on strains
from the same
a hospital species
setup, aimingortogenus, our
identify approachresistance
circulating considered strains co-occurring
mechanisms simultane-
that could have
ously into a hospital
been mobilizing in this setup, aimingWhile
environment. to identify circulating
our analysis does notresistance mechanisms that
provide unequivocal
evidence
could havethatbeen
thesemobilizing
resistance mechanisms are being mobilized
in this environment. While ouramong the analyzed
analysis strains,
does not provide un-
equivocal evidence that these resistance mechanisms are being mobilized among the an-
alyzed strains, we found strongly conserved ARGs located in plasmids and associated
with transposon elements that could represent potential mechanisms for the dissemina-
tion of antibiotic resistance among clinical strains.
Antibiotics 2021, 10, 419 16 of 19

we found strongly conserved ARGs located in plasmids and associated with transposon
elements that could represent potential mechanisms for the dissemination of antibiotic
resistance among clinical strains.
Additionally, by using functional genomics, it was possible to investigate which of
the ARG candidates identified in silico could be expressed in E. coli and associated with
the resistance to beta-lactam antibiotics in a different genomic context. Furthermore, for
many of the bacterial species analyzed here, we found a low number of available complete
genome sequences in the NCBI database. Therefore, while 1000 genome sequences are
available for classical pathogens (E. coli, K. pneumoniae, P. aeruginosa, etc.), other clinically
relevant pathogens such as M. morganii and B. cepacia are underrepresented, which makes
it challenging to track genomic events associated with the acquisition of pathogenicity
elements or resistance mechanisms in hospital-associated infections. Finally, an analysis
of plasmids from Klebsiella strains allowed for the identification of both well-known circu-
lating variants in Brazil as well as new variants that seem to have recently been acquired
from Asia. Thus, we argue that more systematic efforts should be made to monitor the
introduction and propagation of mobile genetic elements harboring ARGs, especially in
South America, in order to inform and guide policies to minimize and prevent outbreaks
and respond properly.

Supplementary Materials: The following are available online at https://www.mdpi.com/article/

10.3390/antibiotics10040419/s1, Figure S1: General features of genomes sequenced in this study,
Figure S2: Phylogenetic tree of B. cepacia 540A and total number of available B. cepacia genomes (166)
in NCBI at March 2020, Figure S3: Heatmap showing the presence of ARGs identified by DeepARG
in nearly sequenced genomes, Figure S4: Schematic representation of genes in identified plasmids
exposing the coexistence of ARGs with transposon elements, Figure S5: Schematic representation
of plasmids identified in Staphylococcus strains showing the coexistence of ARGs with transposon
elements, Table S1: Comparison between ARG-ANNOT and susceptibly tests, Table S2: Phage-
related contigs identified by Phaster, Table S3: Replicons identified by bacWSGTdb for K. pneumonia
508B and 98M3 genomes, Table S4: Most similar strains identified by bacWSGTdb SNP strategy
for K. pneumonia 98M3, Table S5: Most similar strains identified by bacWSGTdbSNP strategyfor K.
pneumonia 508B.
Author Contributions: M.-E.G. and R.S.-R. conceived and designed the study. L.F.R., G.L.L. and
A.F.T.S. performed the experiments. T.C.B., L.Z. and F.M.P.-d.-S. performed the bioinformatic analysis.
M.-E.G. and R.S.-R. wrote the manuscript. All authors read and approved the final version. All
authors have read and agreed to the published version of the manuscript.
Funding: This work was supported by the São Paulo State Foundation (FAPESP, award num-
bers 2015/04309-1 and 2019/15675-0). TCB, GLL, LFR, MHAC and FMPS were supported by
FAPESP fellowships (award numbers 2019/00390-0, 2018/18158-3, 2016/18827-7, 2019/06672-7, and
2018/1160-0).
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: All genomes are available at the NCBI under the BioProject number
PRJNA641571.
Acknowledgments: The authors are thankful to their laboratory colleagues for their insightful
comments and suggestions throughout the course of this study. They also thank Murilo Henrique
Anzoline Cassiano for his initial support in genome assembly. The authors are thankful to Roberto
Martinez for providing the bacterial strains used in this study.
Conflicts of Interest: The authors declare no conflict of interest.
Antibiotics 2021, 10, 419 17 of 19

References
1. Shetty, N.; Hill, G.; Ridgway, G.L. The Vitek analyser for routine bacterial identification and susceptibility testing: Protocols,
problems, and pitfalls. J. Clin. Pathol. 1998, 51, 316–323. [CrossRef] [PubMed]
2. Pancholi, P.; Carroll, K.C.; Buchan, B.W.; Chan, R.C.; Dhiman, N.; Ford, B.; Granato, P.A.; Harrington, A.T.; Hernandez, D.R.;
Humphries, R.M.; et al. Multicenter Evaluation of the Accelerate PhenoTest BC Kit for Rapid Identification and Phenotypic
Antimicrobial Susceptibility Testing Using Morphokinetic Cellular Analysis. J. Clin. Microbiol. 2018, 56. [CrossRef]
3. Köser, C.U.; Ellington, M.J.; Cartwright, E.J.P.; Gillespie, S.H.; Brown, N.M.; Farrington, M.; Holden, M.T.G.; Dougan, G.; Bentley,
S.D.; Parkhill, J.; et al. Routine Use of Microbial Whole Genome Sequencing in Diagnostic and Public Health Microbiology. PLoS
Pathog. 2012, 8, e1002824. [CrossRef] [PubMed]
4. Adams, M.D.; Goglin, K.; Molyneaux, N.; Hujer, K.M.; Lavender, H.; Jamison, J.J.; Macdonald, I.J.; Martin, K.M.; Russo, T.;
Campagnari, A.A.; et al. Comparative Genome Sequence Analysis of Multidrug-Resistant Acinetobacter baumannii. J. Bacteriol.
2008, 190, 8053–8064. [CrossRef] [PubMed]
5. Wyres, K.L.; Lam, M.M.C.; Holt, K.E. Population genomics of Klebsiella pneumoniae. Nat. Rev. Genet. 2020, 18, 344–359.
[CrossRef] [PubMed]
6. Snitkin, E.S.; Won, S.; Pirani, A.; Lapp, Z.; Weinstein, R.A.; Lolans, K.; Hayden, M.K. Integrated genomic and interfacility
patient-transfer data reveal the transmission pathways of multidrug-resistantKlebsiella pneumoniaein a regional outbreak. Sci.
Transl. Med. 2017, 9, eaan0093. [CrossRef]
7. Snitkin, E.S.; Zelazny, A.M.; Thomas, P.J.; Stock, F.; Henderson, D.K.; Palmore, T.N.; Segre, J.A.; Nisc Comparative Sequencing
NISC Comparative Sequencing Program Group. Tracking a Hospital Outbreak of Carbapenem-Resistant Klebsiella pneumoniae
with Whole-Genome Sequencing. Sci. Transl. Med. 2012, 4, 148ra116. [CrossRef]
8. Quick, J.; Cumley, N.; Wearn, C.M.; Niebel, M.; Constantinidou, C.; Thomas, C.M.; Pallen, M.J.; Moiemen, N.S.; Bamford, A.;
Oppenheim, B.; et al. Seeking the source ofPseudomonas aeruginosainfections in a recently opened hospital: An observational
study using whole-genome sequencing. BMJ Open 2014, 4, e006278. [CrossRef] [PubMed]
9. Didelot, X.; Bowden, R.; Wilson, D.J.; Peto, T.E.A.; Crook, D.W. Transforming clinical microbiology with bacterial genome
sequencing. Nat. Rev. Genet. 2012, 13, 601–612. [CrossRef] [PubMed]
10. Pankhurst, L.J.; Elias, C.D.O.; Votintseva, A.A.; Walker, T.M.; Cole, K.; Davies, J.; Fermont, J.M.; Gascoyne-Binzi, D.M.; Kohl, T.A.;
Kong, C.; et al. Rapid, comprehensive, and affordable mycobacterial diagnosis with whole-genome sequencing: A prospective
study. Lancet Respir. Med. 2016, 4, 49–58. [CrossRef]
11. Chiu, C.Y.; Miller, S.A. Clinical metagenomics. Nat. Rev. Genet. 2019, 20, 341–355. [CrossRef]
12. Naccache, S.N.; Federman, S.; Veeraraghavan, N.; Zaharia, M.; Lee, D.; Samayoa, E.; Bouquet, J.; Greninger, A.L.; Luk, K.-C.; Enge,
B.; et al. A cloud-compatible bioinformatics pipeline for ultrarapid pathogen identification from next-generation sequencing of
clinical samples. Genome Res. 2014, 24, 1180–1192. [CrossRef]
13. Greninger, A.L.; Naccache, S.N.; Federman, S.; Yu, G.; Mbala, P.; Bres, V.; Stryke, D.; Bouquet, J.; Somasekar, S.; Linnen, J.M.; et al.
Rapid metagenomic identification of viral pathogens in clinical samples by real-time nanopore sequencing analysis. Genome Med.
2015, 7, 1–13. [CrossRef]
14. Aytan-Aktug, D.; Clausen, P.T.L.C.; Bortolaia, V.; Aarestrup, F.M.; Lund, O. Prediction of Acquired Antimicrobial Resistance for
Multiple Bacterial Species Using Neural Networks. mSystems 2020, 5. [CrossRef] [PubMed]
15. Rahman, S.F.; Olm, M.R.; Morowitz, M.J.; Banfield, J.F. Machine Learning Leveraging Genomes from Metagenomes Identifies
Influential Antibiotic Resistance Genes in the Infant Gut Microbiome. mSystems 2018, 3, e00123-17. [CrossRef]
16. Macesic, N.; Walk, O.J.B.D.; Pe’Er, I.; Tatonetti, N.P.; Peleg, A.Y.; Uhlemann, A.-C. Predicting Phenotypic Polymyxin Resistance in
Klebsiella pneumoniae through Machine Learning Analysis of Genomic Data. mSystems 2020, 5. [CrossRef] [PubMed]
17. Arias, C.A.; Reyes, J.; Carvajal, L.P.; Rincon, S.; Diaz, L.; Panesso, D.; Ibarra, G.; Rios, R.; Munita, J.M.; Salles, M.J.; et al. A
Prospective Cohort Multicenter Study of Molecular Epidemiology and Phylogenomics of Staphylococcus aureus Bacteremia in
Nine Latin American Countries. Antimicrob. Agents Chemother. 2017, 61, e00816-17. [CrossRef] [PubMed]
18. David, S.; The EuSCAPE Working Group; Reuter, S.; Harris, S.R.; Glasner, C.; Feltwell, T.; Argimon, S.; Abudahab, K.; Goater,
R.; Giani, T.; et al. Epidemic of carbapenem-resistant Klebsiella pneumoniae in Europe is driven by nosocomial spread. Nat.
Microbiol. 2019, 4, 1919–1929. [CrossRef]
19. Arimizu, Y.; Kirino, Y.; Sato, M.P.; Uno, K.; Sato, T.; Gotoh, Y.; Auvray, F.; Brugere, H.; Oswald, E.; Mainil, J.G.; et al. Large-scale
genome analysis of bovine commensal Escherichia coli reveals that bovine-adapted E. coli lineages are serving as evolutionary
sources of the emergence of human intestinal pathogenic strains. Genome Res. 2019, 29, 1495–1505. [CrossRef]
20. Bennett, P.M. Plasmid encoded antibiotic resistance: Acquisition and transfer of antibiotic resistance genes in bacteria. Br. J.
Pharmacol. 2008, 153, S347–S357. [CrossRef]
21. Millan, A.S. Evolution of Plasmid-Mediated Antibiotic Resistance in the Clinical Context. Trends Microbiol. 2018, 26, 978–985.
[CrossRef] [PubMed]
22. Lerminiaux, N.A.; Cameron, A.D. Horizontal transfer of antibiotic resistance genes in clinical environments. Can. J. Microbiol.
2019, 65, 34–44. [CrossRef] [PubMed]
23. Porse, A.; Schønning, K.; Munck, C.; Sommer, M.O. Survival and Evolution of a Large Multidrug Resistance Plasmid in New
Clinical Bacterial Hosts. Mol. Biol. Evol. 2016, 33, 2860–2873. [CrossRef]
Antibiotics 2021, 10, 419 18 of 19

24. Buckner, M.M.C.; Ciusa, M.L.; Meek, R.W.; Moorey, A.R.; McCallum, G.E.; Prentice, E.L.; Reid, J.P.; Alderwick, L.J.; Di Maio, A.;
Piddock, L.J.V. HIV Drugs Inhibit Transfer of Plasmids Carrying Extended-Spectrum β-Lactamase and Carbapenemase Genes.
mBio 2020, 11. [CrossRef] [PubMed]
25. Arango-Argoty, G.; Garner, E.; Pruden, A.; Heath, L.S.; Vikesland, P.; Zhang, L. DeepARG: A deep learning approach for
predicting antibiotic resistance genes from metagenomic data. Microbiome 2018, 6, 1–15. [CrossRef] [PubMed]
26. Treangen, T.J.; Ondov, B.D.; Koren, S.; Phillippy, A.M. The Harvest suite for rapid core-genome alignment and visualization of
thousands of intraspecific microbial genomes. Genome Biol. 2014, 15, 1–15. [CrossRef] [PubMed]
27. Letunic, I.; Bork, P. Interactive Tree Of Life (iTOL) v4: Recent updates and new developments. Nucleic Acids Res. 2019, 47,
W256–W259. [CrossRef]
28. Silva-Rocha, R.; Martínez-García, E.; Calles, B.; Chavarría, M.; Arce-Rodríguez, A.; Heras, A.D.L.; Páez-Espino, A.D.; Durante-
Rodríguez, G.; Kim, J.; Nikel, P.I.; et al. The Standard European Vector Architecture (SEVA): A coherent platform for the analysis
and deployment of complex prokaryotic phenotypes. Nucleic Acids Res. 2012, 41, D666–D675. [CrossRef]
29. Martínez-García, E.; Aparicio, T.; Goñi-Moreno, A.; Fraile, S.; De Lorenzo, V. SEVA 2.0: An update of the Standard European
Vector Architecture for de-/re-construction of bacterial functionalities. Nucleic Acids Res. 2015, 43, D1183–D1189. [CrossRef]
30. Martínez-García, E.; Goñi-Moreno, A.; Bartley, B.; McLaughlin, J.; Sánchez-Sampedro, L.; Del Pozo, H.P.; Hernández, C.P.;
Marletta, A.S.; De Lucrezia, D.; Sánchez-Fernández, G.; et al. SEVA 3.0: An update of the Standard European Vector Architecture
for enabling portability of genetic constructs among diverse bacterial hosts. Nucleic Acids Res. 2019, 48, D1164–D1170. [CrossRef]
31. Drury, L. Transformation of Bacteria by Electroporation. Basic DNA RNA Protoc. 2003, 58, 249–256. [CrossRef]
32. CLSI. Performance Standards for Antimicrobial Susceptibility Testing, 28th ed.; CLSI Supplement M100; Clinical and Laboratory
Standards Institute: Wayne, PA, USA, 2018.
33. Camacho, C.; Coulouris, G.; Avagyan, V.; Ma, N.; Papadopoulos, J.S.; Bealer, K.; Madden, T.L. BLAST+: Architecture and
applications. BMC Bioinform. 2009, 10, 421. [CrossRef] [PubMed]
34. Seemann, T. Prokka: Rapid prokaryotic genome annotation. Bioinformatics 2014, 30, 2068–2069. [CrossRef]
35. Johnson, S.L.; Bishop-Lilly, K.A.; Ladner, J.T.; Daligault, H.E.; Davenport, K.W.; Jaissle, J.; Frey, K.G.; Koroleva, G.I.; Bruce, D.C.;
Coyne, S.R.; et al. Complete Genome Sequences for 59BurkholderiaIsolates, Both Pathogenic and Near Neighbor: TABLE 1.
Genome Announc. 2015, 3, e00159-15. [CrossRef] [PubMed]
36. Reyes, J.A.; Melano, R.; Cárdenas, P.A.; Trueba, G. Mobile genetic elements associated with carbapenemase genes in South
American Enterobacterales. Braz. J. Infect. Dis. 2020, 24, 231–238. [CrossRef] [PubMed]
37. Ferreira, J.C.; Filho, R.A.C.P.; Andrade, L.N.; Junior, A.B.; Darini, A.L.C. Evaluation and characterization of plasmids carrying
CTX-M genes in a non-clonal population of multidrug-resistant Enterobacteriaceae isolated from poultry in Brazil. Diagn.
Microbiol. Infect. Dis. 2016, 85, 444–448. [CrossRef]
38. Partridge, S.R.; Kwong, S.M.; Firth, N.; Jensen, S.O. Mobile Genetic Elements Associated with Antimicrobial Resistance. Clin.
Microbiol. Rev. 2018, 31, 1–61. [CrossRef]
39. Cantón, R.; Novais, A.; Valverde, A.; Machado, E.; Peixe, L.; Baquero, F.; Coque, T. Prevalence and spread of extended-spectrum
β-lactamase-producing Enterobacteriaceae in Europe. Clin. Microbiol. Infect. 2008, 14, 144–153. [CrossRef]
40. Olivares, J.; Ebernardini, A.; Egarcia-Leon, G.; Ecorona, F.; Esanchez, M.B.; Martinez, J.L. The intrinsic resistome of bacterial
pathogens. Front. Microbiol. 2013, 4, 103. [CrossRef]
41. Cox, G.; Wright, G.D. Intrinsic antibiotic resistance: Mechanisms, origins, challenges and solutions. Int. J. Med. Microbiol. 2013,
303, 287–292. [CrossRef]
42. Zankari, E.; Hasman, H.; Cosentino, S.; Vestergaard, M.; Rasmussen, S.; Lund, O.; Aarestrup, F.M.; Larsen, M.V. Identification of
acquired antimicrobial resistance genes. J. Antimicrob. Chemother. 2012, 67, 2640–2644. [CrossRef]
43. Naas, T.; Oueslati, S.; Bonnin, R.A.; Dabos, M.L.; Zavala, A.; Dortet, L.; Retailleau, P.; Iorga, B.I. Beta-lactamase database
(BLDB)–structure and function. J. Enzym. Inhib. Med. Chem. 2017, 32, 917–919. [CrossRef]
44. Peter, S.; Bosio, M.; Gross, C.; Bezdan, D.; Gutierrez, J.; Oberhettinger, P.; Liese, J.; Vogel, W.; Dörfel, D.; Berger, L.; et al. Tracking
of Antibiotic Resistance Transfer and Rapid Plasmid Evolution in a Hospital Setting by Nanopore Sequencing. mSphere 2020, 5.
[CrossRef] [PubMed]
45. Prussing, C.; Snavely, E.A.; Singh, N.; Lapierre, P.; Lasek-Nesselquist, E.; Mitchell, K.; Haas, W.; Owsiak, R.; Nazarian, E.; Musser,
K.A. Nanopore MinION Sequencing Reveals Possible Transfer of blaKPC–2 Plasmid Across Bacterial Species in Two Healthcare
Facilities. Front. Microbiol. 2020, 11. [CrossRef]
46. Arndt, D.; Grant, J.R.; Marcu, A.; Sajed, T.; Pon, A.; Liang, Y.; Wishart, D.S. PHASTER: A better, faster version of the PHAST
phage search tool. Nucleic Acids Res. 2016, 44, W16–W21. [CrossRef] [PubMed]
47. Feng, Y.; Zou, S.; Chen, H.; Yu, Y.; Ruan, Z. BacWGSTdb 2.0: A one-stop repository for bacterial whole-genome sequence typing
and source tracking. Nucleic Acids Res. 2021, 49, D644–D650. [CrossRef]
48. Ruan, Z.; Feng, Y. BacWGSTdb, a database for genotyping and source tracking bacterial pathogens. Nucleic Acids Res. 2016, 44,
D682–D687. [CrossRef] [PubMed]
49. Zulkower, V.; Rosser, S. DNA Features Viewer: A sequence annotation formatting and plotting library for Python. Bioinformatics
2020, 36, 4350–4352. [CrossRef] [PubMed]
50. Rozwandowicz, M.; Brouwer, M.S.M.; Fischer, J.; Wagenaar, J.A.; Gonzalez-Zorn, B.; Guerra, B.; Mevius, D.J.; Hordijk, J. Plasmids
carrying antimicrobial resistance genes in Enterobacteriaceae. J. Antimicrob. Chemother. 2018, 73, 1121–1137. [CrossRef] [PubMed]
Antibiotics 2021, 10, 419 19 of 19

51. Wein, T.; Hülter, N.F.; Mizrahi, I.; Dagan, T. Emergence of plasmid stability under non-selective conditions maintains antibiotic
resistance. Nat. Commun. 2019, 10, 1–13. [CrossRef]
52. Martínez, J.L.; Coque, T.M.; Baquero, F. What is a resistance gene? Ranking risk in resistomes. Nat. Rev. Genet. 2015, 13, 116–123.
[CrossRef]
53. Bengtsson-Palme, J.; Larsson, D.G.J. Antibiotic resistance genes in the environment: Prioritizing risks. Nat. Rev. Genet. 2015,
13, 396. [CrossRef] [PubMed]
54. Nordmann, P.; Naas, T.; Poirel, L. Global Spread of Carbapenemase-producingEnterobacteriaceae. Emerg. Infect. Dis. 2011, 17,
1791–1798. [CrossRef] [PubMed]
55. Liakopoulos, A.; Van Der Goot, J.; Bossers, A.; Betts, J.; Brouwer, M.S.M.; Kant, A.; Smith, H.; Ceccarelli, D.; Mevius, D. Genomic
and functional characterisation of IncX3 plasmids encoding blaSHV-12 in Escherichia coli from human and animal origin. Sci.
Rep. 2018, 8, 1–13. [CrossRef]
56. Ho, P.-L.; Cheung, Y.-Y.; Lo, W.-U.; Li, Z.; Chow, K.-H.; Lin, C.-H.; Chan, J.F.-W.; Cheng, V.C.-C. Molecular Characterization
of an Atypical IncX3 Plasmid pKPC-NY79 Carrying bla KPC-2 in a Klebsiella pneumoniae. Curr. Microbiol. 2013, 67, 493–498.
[CrossRef] [PubMed]
57. Cerdeira, L.T.; Cunha, M.P.; Francisco, G.R.; Bueno, M.F.C.; Araujo, B.F.; Ribas, R.M.; Gontijo-Filho, P.P.; Knöbl, T.; Garcia,
D.D.O.; Lincopan, N. IncX3 plasmid harboring a non-Tn 4401 genetic element (NTE KPC) in a hospital-associated clone of
KPC-2-producing Klebsiella pneumoniae ST340/CG258. Diagn. Microbiol. Infect. Dis. 2017, 89, 164–167. [CrossRef]
58. Fuga, B.; Ferreira, M.L.; Cerdeira, L.T.; De Campos, P.A.; Dias, V.L.; Rossi, I.; Machado, L.G.; Lincopan, N.; Gontijo-Filho, P.P.;
Ribas, R.M. Novel small IncX3 plasmid carrying the blaKPC-2 gene in high-risk Klebsiella pneumoniae ST11/CG258. Diagn.
Microbiol. Infect. Dis. 2020, 96, 114900. [CrossRef] [PubMed]
59. Partridge, S.R.; Ginn, A.N.; Wiklendt, A.M.; Ellem, J.; Wong, J.S.; Ingram, P.; Guy, S.; Garner, S.; Iredell, J.R. Emergence of blaKPC
carbapenemase genes in Australia. Int. J. Antimicrob. Agents 2015, 45, 130–136. [CrossRef]
60. Fortini, D.; Villa, L.; Feudi, C.; Pires, J.P.D.C.; Bonura, C.; Mammina, C.; Endimiani, A.; Carattoli, A. Double Copies of blaKPC-
3::Tn4401a on an IncX3 Plasmid in Klebsiella Pneumoniae Successful Clone ST512 from Italy. Antimicrob. Agents Chemother. 2016,
60, 646–649. [CrossRef]
61. Kassis-Chikhani, N.; Frangeul, L.; Drieux, L.; Sengelin, C.; Jarlier, V.; Brisse, S.; Arlet, G.; Decré, D. Complete Nucleotide Sequence
of the First KPC-2- and SHV-12-Encoding IncX Plasmid, pKpS90, from Klebsiella pneumoniae. Antimicrob. Agents Chemother.
2013, 57, 618–620. [CrossRef]
62. Kim, J.O.; Song, S.A.; Yoon, E.-J.; Shin, J.H.; Lee, H.; Jeong, S.H.; Lee, K. Outbreak of KPC-2-producing Enterobacteriaceae caused
by clonal dissemination of Klebsiella pneumoniae ST307 carrying an IncX3-type plasmid harboring a truncated Tn4401a. Diagn.
Microbiol. Infect. Dis. 2017, 87, 343–348. [CrossRef] [PubMed]
63. Jeong, S.; Kim, J.O.; Yoon, E.-J.; Bae, I.K.; Lee, W.; Lee, H.; Park, Y.; Lee, K.; Jeong, S.H. Extensively Drug-Resistant Escherichia coli
Sequence Type 1642 Carrying an IncX3 Plasmid Containing the blaKPC-2 Gene Associated with Transposon Tn4401a. Ann. Lab.
Med. 2018, 38, 17–22. [CrossRef] [PubMed]
64. Yang, Q.; Fang, L.; Fu, Y.; Du, X.; Shen, Y.; Yu, Y. Dissemination of NDM-1-Producing Enterobacteriaceae Mediated by the
IncX3-Type Plasmid. PLoS ONE 2015, 10, e0129454. [CrossRef] [PubMed]
65. Acton, Q.A. Klebsiella Pneumoniae: New Insights for the Healthcare Professional, 2011 ed.; Scholarly Brief; Scholarly Editions: Atlanta,
GA, USA, 2012.
66. Alikhan, N.-F.; Petty, N.K.; Ben Zakour, N.L.; Beatson, S.A. BLAST Ring Image Generator (BRIG): Simple prokaryote genome
comparisons. BMC Genom. 2011, 12, 402. [CrossRef]