We analyzed the kinetic properties of the metagenomic class B3 β-lactamase LRA-12, and determined its crystallographic structure in order to compare it with prevalent metallo-β-lactamases (MBLs) ...associated with clinical pathogens. We showed that LRA-12 confers extended-spectrum resistance on E. coli when expressed from recombinant clones, and the MIC values for carbapenems were similar to those observed in enterobacteria expressing plasmid-borne MBLs such as VIM, IMP or NDM. This was in agreement with the strong carbapenemase activity displayed by LRA-12, similar to GOB β-lactamases. Among the chelating agents evaluated, dipicolinic acid inhibited the enzyme more strongly than EDTA, which required pre-incubation with the enzyme to achieve measurable inhibition. Structurally, LRA-12 contains the conserved main structural features of di-zinc class B β-lactamases, and presents unique structural signatures that differentiate this enzyme from others within the family: (i) two loops (α3-β7 and β11-α5) that could influence antibiotic entrance and remodeling of the active site cavity; (ii) a voluminous catalytic cavity probably responsible for the high hydrolytic efficiency of the enzyme; (iii) the absence of disulfide bridges; (iv) a unique Gln116 at metal-binding site 1; (v) a methionine residue at position 221that replaces Cys/Ser found in other B3 β-lactamases in a predominantly hydrophobic environment, likely playing a role in protein stability. The structure of LRA-12 indicates that MBLs exist in wild microbial populations in extreme environments, or environments with low anthropic impact, and under the appropriate antibiotic selective pressure could be captured and disseminated to pathogens.
We analyzed the kinetic properties of the metagenomic class B3 beta-lactamase LRA-12, and determined its crystallographic structure in order to compare it with prevalent metallo-beta-lactamases ...(MBLs) associated with clinical pathogens. We showed that LRA-12 confers extended-spectrum resistance on E. coli when expressed from recombinant clones, and the MIC values for carbapenems were similar to those observed in enterobacteria expressing plasmid-borne MBLs such as VIM, IMP or NDM. This was in agreement with the strong carbapenemase activity displayed by LRA-12, similar to GOB beta-lactamases. Among the chelating agents evaluated, dipicolinic acid inhibited the enzyme more strongly than EDTA, which required pre-incubation with the enzyme to achieve measurable inhibition. Structurally, LRA-12 contains the conserved main structural features of di-zinc class B beta-lactamases, and presents unique structural signatures that differentiate this enzyme from others within the family: (i) two loops (alpha3-beta7 and beta11-alpha5) that could influence antibiotic entrance and remodeling of the active site cavity; (ii) a voluminous catalytic cavity probably responsible for the high hydrolytic efficiency of the enzyme; (iii) the absence of disulfide bridges; (iv) a unique Gln116 at metal-binding site 1; (v) a methionine residue at position 221that replaces Cys/Ser found in other B3 beta-lactamases in a predominantly hydrophobic environment, likely playing a role in protein stability. The structure of LRA-12 indicates that MBLs exist in wild microbial populations in extreme environments, or environments with low anthropic impact, and under the appropriate antibiotic selective pressure could be captured and disseminated to pathogens.
Isolation of colistin- and carbapenem-resistant Klebsiella pneumoniae (CCR-Kp) is increasing in hospital settings worldwide, which is related to increased morbidity, mortality and healthcare costs. ...The aim of this work was to perform whole-genome sequencing (WGS), genomic and phylogenetic analysis, and conjugation assays of an extensively drug-resistant (XDR) CCR-Kp isolate from Argentina.
WGS of strain KpS26 isolated from a bloodstream infection was performed using Illumina MiSeq-I, and de novo assembly was achieved using SPAdes v.3.11. A maximum likelihood tree was created using MEGA7 based on core genome single nucleotide polymorphisms from whole-genome alignment of K. pneumoniae isolates identified in silico as sequence type 15 (ST15). The resistome, plasmids and integrons were analysed using ResFinder, AMRFinderPlus, ISfinder, plasmidSPAdes, PlasmidFinder and IntegronFinder. Standard conjugation was performed.
KpS26 belonged to ST15, which is less common than ST258, ST25 and ST11 that are globally reported as responsible for CCR-Kp outbreaks. Fourteen transferable antimicrobial resistance genes (ARGs), including blaKPC-2 in a novel genetic platform transferable by conjugation, were detected contributing to the XDR phenotype. The amino acid substitution T157P in the protein encoded by the pmrB gene of KpS26, previously reported as being responsible for resistance to colistin in K. pneumoniae lineages globally disseminated, was also identified in this strain.
The XDR CCR-Kp isolate analysed here shows that ST15 is also disseminating blaKPC-2 in Argentina alongside other ARGs, evidencing that KPC epidemiology continues to be shaped by intricate and assorted ways of lateral gene transfer.
Isolation of colistin- and carbapenem-resistant Klebsiella pneumoniae (CCR-Kp) is increasing in hospital settings worldwide, which is related to increased morbidity, mortality and healthcare costs. ...The aim of this work was to perform whole-genome sequencing (WGS), genomic and phylogenetic analysis, and conjugation assays of an extensively drug-resistant (XDR) CCR-Kp isolate from Argentina.
WGS of strain KpS26 isolated from a bloodstream infection was performed using Illumina MiSeq-I, and de novo assembly was achieved using SPAdes v.3.11. A maximum likelihood tree was created using MEGA7 based on core genome single nucleotide polymorphisms from whole-genome alignment of K. pneumoniae isolates identified in silico as sequence type 15 (ST15). The resistome, plasmids and integrons were analysed using ResFinder, AMRFinderPlus, ISfinder, plasmidSPAdes, PlasmidFinder and IntegronFinder. Standard conjugation was performed.
KpS26 belonged to ST15, which is less common than ST258, ST25 and ST11 that are globally reported as responsible for CCR-Kp outbreaks. Fourteen transferable antimicrobial resistance genes (ARGs), including bla
in a novel genetic platform transferable by conjugation, were detected contributing to the XDR phenotype. The amino acid substitution T157P in the protein encoded by the pmrB gene of KpS26, previously reported as being responsible for resistance to colistin in K. pneumoniae lineages globally disseminated, was also identified in this strain.
The XDR CCR-Kp isolate analysed here shows that ST15 is also disseminating bla
in Argentina alongside other ARGs, evidencing that KPC epidemiology continues to be shaped by intricate and assorted ways of lateral gene transfer.
Untreated wastewater is a reservoir for multidrug-resistant bacteria, but its role in the spread of antibiotic resistance in the human population remains poorly investigated. In this study, we ...isolated a KPC-2-producing ST2787
Klebsiella quasipneumoniae
subsp.
quasipneumoniae
(WW14A), recovered from raw sewage at a wastewater treatment plant in Argentina in 2018 and determined its complete genome sequence. Strain WW14A was resistant to all β-lactams, ciprofloxacin and amikacin. A core genome phylogenetic analysis indicated that WW14A was closely related to a GES-5-producing Taiwanese strain isolated from hospital wastewater in 2015 and it was clearly distinct from strains isolated recently in Argentina and Brazil. Interestingly,
bla
KPC-2
was harbored by a recently described IncP-6 broad-spectrum plasmid which was sporadically reported worldwide and had never been reported before in Argentina. We investigated the presence of the IncP-6 replicon in isolates obtained from the same sampling and found a novel non-typable/IncP-6 hybrid plasmid in a newly assigned ST1407
Enterobacter asburiae
(WW19C) also harboring
bla
KPC-2
. Nanopore sequencing and hybrid assembly of strains WW14A and WW19C revealed that both IncP-6 plasmids shared 72% of coverage (~20 kb), with 99.99% of sequence similarity and each one also presented uniquely combined regions that were derived from other plasmids recently reported in different countries of South America, Asia, and Europe. The region harboring the carbapenem resistance gene (~11 kb) in both plasmids contained a Tn
3
transposon disrupted by a Tn
3
-IS
Apu
-flanked element and the core sequence was composed by ΔIS
Kpn6
/
bla
KPC-2
/Δ
bla
TEM-1
/IS
Kpn27
. Both strains also carried genes conferring resistance to heavy metals (e.g., arsenic, mercury, lead, cadmium, copper), pesticides (e.g., glyphosate), disinfectants, and several virulence-related genes, posing a potential pathogenic risk in the case of infections. This is the first study documenting
bla
KPC-2
associated with IncP-6 plasmids in
K. quasipneumoniae
and
Enterobacter cloacae
complex from wastewater in Argentina and highlights the circulation of IncP-6 plasmids as potential reservoirs of
bla
KPC-2
in the environment.
Untreated wastewater is a reservoir for multidrug-resistant bacteria, but its role in the spread of antibiotic resistance in the human population remains poorly investigated. In this study, we ...isolated a KPC-2-producing ST2787
Klebsiella quasipneumoniae
subsp.
quasipneumoniae
(WW14A), recovered from raw sewage at a wastewater treatment plant in Argentina in 2018 and determined its complete genome sequence. Strain WW14A was resistant to all β-lactams, ciprofloxacin and amikacin. A core genome phylogenetic analysis indicated that WW14A was closely related to a GES-5-producing Taiwanese strain isolated from hospital wastewater in 2015 and it was clearly distinct from strains isolated recently in Argentina and Brazil. Interestingly,
bla
KPC-2
was harbored by a recently described IncP-6 broad-spectrum plasmid which was sporadically reported worldwide and had never been reported before in Argentina. We investigated the presence of the IncP-6 replicon in isolates obtained from the same sampling and found a novel non-typable/IncP-6 hybrid plasmid in a newly assigned ST1407
Enterobacter asburiae
(WW19C) also harboring
bla
KPC-2
. Nanopore sequencing and hybrid assembly of strains WW14A and WW19C revealed that both IncP-6 plasmids shared 72% of coverage (~20 kb), with 99.99% of sequence similarity and each one also presented uniquely combined regions that were derived from other plasmids recently reported in different countries of South America, Asia, and Europe. The region harboring the carbapenem resistance gene (~11 kb) in both plasmids contained a Tn
3
transposon disrupted by a Tn
3
-IS
Apu
-flanked element and the core sequence was composed by ΔIS
Kpn6
/
bla
KPC-2
/Δ
bla
TEM-1
/IS
Kpn27
. Both strains also carried genes conferring resistance to heavy metals (e.g., arsenic, mercury, lead, cadmium, copper), pesticides (e.g., glyphosate), disinfectants, and several virulence-related genes, posing a potential pathogenic risk in the case of infections. This is the first study documenting
bla
KPC-2
associated with IncP-6 plasmids in
K. quasipneumoniae
and
Enterobacter cloacae
complex from wastewater in Argentina and highlights the circulation of IncP-6 plasmids as potential reservoirs of
bla
KPC-2
in the environment.
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