Historically, the environment has been viewed as a passive deposit of antimicrobial resistance mechanisms, where bacteria show biological cost for maintenance of these genes. Thus, in the absence of ...antimicrobial pressure, it is expected that they disappear from environmental bacterial communities. To test this scenario, we studied native IntI1 functionality of 11 class 1 integron-positive environmental strains of distant genera collected in cold and subtropical forests of Argentina. We found natural competence and successful site-specific insertion with no significant fitness cost of both aadB and bla
antimicrobial resistance gene cassettes, in a model system without antibiotic pressure. A bidirectional flow of antimicrobial resistance gene cassettes between natural and nosocomial habitats is proposed, which implies an active role of the open environment as a reservoir, recipient and source of antimicrobial resistance mechanisms, outlining an environmental threat where novel concepts of rational use of antibiotics are extremely urgent and mandatory.
Tuberculous pleurisy allows the study of specific cells at the site of Mycobacterium tuberculosis infection. Among pleural lymphocytes, natural killer (NK) cells are a major source of interferon γ ...(IFN-γ), and their functions are regulated by activating and inhibitory receptors. Programmed death-1 (PD-1), programmed death ligand 1 (PD-L1), and programmed death ligand 2 (PD-L2) are recognized inhibitory receptors in adaptive immunity, but their role during innate immunity remains poorly understood. We investigated the PD-1:PDL1/PD-L2 pathway on NK cell effector functions in peripheral blood and pleural fluid from patients with tuberculosis. M. tuberculosis stimulation significantly up-regulated PD-1, PD-L1, and PD-L2 levels on NK cells. Interestingly, a direct correlation between PD-1 and IFN-γ expression on NK cells was observed. Moreover, blockade of the PD-1 pathway markedly augmented lytic degranulation and IFN-γ production of NK cells against M. tuberculosis. Furthermore, PD-1+ NK cells displayed a diminished IFN-γ mean fluorescence intensity, denoting the relevance of PD-1 on IFN-γ regulation. Together, we described a novel inhibitory role played by PD-1:PD-L interactions in innate immunity in tuberculosis.
Protective immunity against Mycobacterium tuberculosis requires the generation of cell-mediated immunity. We investigated the expression and role of programmed death 1 (PD-1) and its ligands, ...molecules known to modulate T cell activation, in the regulation of IFN-gamma production and lytic degranulation during human tuberculosis. We demonstrated that specific Ag-stimulation increased CD3+PD-1+ lymphocytes in peripheral blood and pleural fluid from tuberculosis patients in direct correlation with IFN-gamma production from these individuals. Moreover, M. tuberculosis-induced IFN-gamma participated in the up-regulation of PD-1 expression. Blockage of PD-1 or PD-1 and its ligands (PD-Ls: PD-L1, PD-L2) enhanced the specific degranulation of CD8+ T cells and the percentage of specific IFN-gamma-producing lymphocytes against the pathogen, demonstrating that the PD-1:PD-Ls pathway inhibits T cell effector functions during active M. tuberculosis infection. Furthermore, the simultaneous blockage of the inhibitory receptor PD-1 together with the activation of the costimulatory protein signaling lymphocytic activation molecule led to the promotion of protective IFN-gamma responses to M. tuberculosis, even in patients with weak cell-mediated immunity against the bacteria. Together, we demonstrated that PD-1 interferes with T cell effector functions against M. tuberculosis, suggesting that PD-1 has a key regulatory role during the immune response of the host to the pathogen.
Mycobacterium tuberculosis expands CD4+IFN‐γ+IL‐17+ lymphocytes in direct correlation with the severity of active disease.
Th1 lymphocytes are crucial in the immune response against Mycobacterium ...tuberculosis. Nevertheless, IFN‐γ alone is not sufficient in the complete eradication of the bacteria, suggesting that other cytokines might be required for pathogen removal. Th17 cells have been associated with M. tuberculosis infection, but the role of IL‐17‐producing cells in human TB remains to be understood. Therefore, we investigated the induction and regulation of IFN‐γ and IL‐17 during the active disease. TB patients were classified as High and Low Responder individuals according to their T cell responses against the antigen, and cytokine expression upon M. tuberculosis stimulation was investigated in peripheral blood and pleural fluid. Afterwards, the potential correlation among the proportions of cytokine‐producing cells and clinical parameters was analyzed. In TB patients, M. tuberculosis induced IFN‐γ and IL‐17, but in comparison with BCG‐vaccinated healthy donors, IFN‐γ results were reduced significantly, and IL‐17 was markedly augmented. Moreover, the main source of IL‐17 was represented by CD4+IFN‐γ+IL‐17+ lymphocytes, a Th1/Th17 subset regulated by IFN‐γ. Interestingly, the ratio of antigen‐expanded CD4+IFN‐γ+IL‐17+ lymphocytes, in peripheral blood and pleural fluid from TB patients, was correlated directly with clinical parameters associated with disease severity. Indeed, the highest proportion of CD4+IFN‐γ+IL‐17+ cells was detected in Low Responder TB patients, individuals displaying severe pulmonary lesions, and longest length of disease evolution. Taken together, the present findings suggest that analysis of the expansion of CD4+IFN‐γ+IL‐17+ T lymphocytes in peripheral blood of TB patients might be used as an indicator of the clinical outcome in active TB.
Two metallo-β-lactamase-producing
Klebsiella pneumoniae
(HA30 and HA31) were isolated in a hospital in Argentina during 2018.
K. pneumoniae
HA30 was isolated from a rectal swab during the ...epidemiological surveillance for carbapenemase-producing strains, while
K. pneumoniae
HA31 was collected from the same patient 4 days after hospitalization. The aim of the present study was to identify the clonal relationships and resistome of these two NDM-producing
K. pneumoniae
strains isolated from a patient with a fatal outcome. Whole-genome sequencing (WGS) was performed using Illumina MiSeq-I, and subsequent analysis involved genome assembly, annotation, antibiotic resistance gene identification, multilocus sequence typing (MLST), and plasmid characterization using bioinformatics tools. Conjugation assays to
E. coli
J53 was conducted as previously described.
K. pneumoniae
HA30 exhibited extensively drug-resistant phenotype, while HA31 was multidrug-resistant as defined by Magiorakos et al., including both resistance to carbapenems, aminoglycosides and ciprofloxacin with
bla
NDM-5
,
bla
CTX-M-15
and
rmtB
genes found in both strains. MLST analysis showed that both strains belonged to ST11, differing by only 4 cgSNPs, indicating that
K. pneumoniae
HA30 and HA31 were the same strain. Conjugation assays revealed that
K. pneumoniae
HA31 strain possessed a transferable plasmid to
E. coli
J53. Bioinformatics studies identified that the same strain colonizing an inpatient during hospital admission subsequently caused the infection leading to a fatal outcome, being the first report of
bla
NDM-5
,
rmtB
and
bla
CTX-M-15
genes in a
K. pneumoniae
ST11 strain from Latin America. Our results also highlighted the importance of focusing on epidemiological surveillance programs.
•AMR and multidrug resistance associated with the animal and the production system.•Higher prevalence of AMR in swine and chicken, and intensive production.•Lower prevalence of AMR in cattle, and in ...extensive production/family farming.•There is a research gap regarding prevalence of AMR in beef-cattle from feedlot.•Need for restriction of highest priority antimicrobials in food-producing animals.
Abuse and misuse of antimicrobial agents has accelerated the spread of antimicrobial-resistant bacteria. The association between antimicrobial-resistant infections in humans and antimicrobial use in agriculture is complex, but well-documented. This study provides a systematic review and meta-analysis of the dissemination of antimicrobial resistance (AMR) to antimicrobials defined as critically important by the WHO, in swine, chicken, and cattle from intensive and extensive production systems in Argentina. We conducted searches in electronic databases (MEDLINE-PubMed, Web of Science, SciELO, the National System of Digital Repositories from Argentina) as well as in the gray literature. Inclusion criteria were epidemiological studies on AMR in the main food-transmitted bacteria, Salmonella spp., Campylobacter spp., Escherichia coli and Enterococcus spp., and mastitis-causing bacteria, isolated from swine, chicken, dairy and beef cattle from Argentina. This study gives evidence for supporting the hypothesis that AMR of common food-transmitted bacteria in Argentina is reaching alarming levels. Meta-analyses followed by subgroup analyses confirmed the association between the prevalence of AMR and (a) animal species (p<0.01) for streptomycin, ampicillin and tetracycline or (b) the animal production system (p<0.05) for streptomycin, cefotaxime, nalidixic acid, ampicillin and tetracycline. Moreover, swine (0.47 0.29; 0.66) and intensive production (0.62 0.34; 0.83) showed the highest pooled prevalence of multidrug resistance while dairy (0.056 0.003; 0.524) and extensive production (0.107 0.043; 0.240) showed the lowest. A research gap regarding beef-cattle from feedlot was identified. Finally, there is an urgent need for political measures meant to coordinate and harmonize AMR surveillance and regulate antimicrobial use in animal production.
El abuso y mal uso de los antimicrobianos aceleró la propagación de bacterias resistentes. La asociación entre las infecciones que presentan resistencia a antimicrobianos (RAM) en humanos y el uso de antimicrobianos en la producción agropecuaria es compleja, pero está bien documentada. Proporcionamos una revisión sistemática y metaanálisis sobre la diseminación de la resistencia a antimicrobianos designados como críticamente importantes por la Organización Mundial de la Salud (OMS) en cerdos, aves y bovinos de producción intensiva y extensiva en Argentina. Se buscó información en bases de datos electrónicas (Medline-PubMed, Web of Science, SciELO, Sistema Nacional de Repositorios Digitales de Argentina) y en la literatura gris. Se incluyeron estudios epidemiológicos sobre la RAM en las principales bacterias transmitidas por los alimentos – Salmonella spp., Campylobacter spp., Escherichia coli y Enterococcus spp. – y bacterias causantes de mastitis aisladas de cerdos, pollos y bovinos. Los resultados de este estudio apoyan la hipótesis de que la RAM de las bacterias transmitidas por los alimentos alcanza niveles alarmantes. Los metaanálisis seguidos de análisis por subgrupos mostraron asociación entre la RAM y (a) el animal (p<0,01) para estreptomicina, ampicilina y tetraciclina o (b) el sistema productivo (p<0,05) para estreptomicina, cefotaxima, ampicilina, ácido nalidíxico y tetraciclina. La mayor prevalencia conjunta de multirresistencia se detectó en cerdos (0,47 0,29; 0,66) y producción intensiva (0,62 0,34; 0,83), mientras que la menor correspondió a bovinos de leche (0,056 0,003; 0,524) y producción extensiva (0,107 0,043; 0,240). Se observó un vacío de información respecto de los bovinos de feedlot. Es urgente adoptar medidas políticas para coordinar y armonizar la vigilancia de la RAM y regular el uso de antimicrobianos en animales.
According to the World Health Organization, carbapenem-resistant
Enterobacteriaceae
(CRE) belong to the highest priority group for the development of new antibiotics. Argentina-WHONET data showed ...that Gram-negative resistance frequencies to imipenem have been increasing since 2010 mostly in two CRE bacteria:
Klebsiella pneumoniae
and
Enterobacter cloacae
Complex (ECC). This scenario is mirrored in our hospital. It is known that
K. pneumoniae
and the ECC coexist in the human body, but little is known about the outcome of these species producing KPC, and colonizing or infecting a patient. We aimed to contribute to the understanding of the rise of the ECC in Argentina, taking as a biological model both a patient colonized with two KPC-producing strains (one
Enterobacter hormaechei
and one
K. pneumoniae
) and
in vitro
competition assays with prevalent KPC-producing ECC (KPC-ECC) versus KPC-producing
K. pneumoniae
(KPC-Kp) high-risk clones from our institution. A KPC-producing
E. hormaechei
and later a KPC-Kp strain that colonized a patient shared an identical novel conjugative IncM1 plasmid harboring
bla
KPC-2
. In addition, a total of 19 KPC-ECC and 58 KPC-Kp strains isolated from nosocomial infections revealed that high-risk clones KPC-ECC ST66 and ST78 as well as KPC-Kp ST11 and ST258 were prevalent and selected for competition assays. The competition assays with KCP-ECC ST45, ST66, and ST78 versus KPC-Kp ST11, ST18, and ST258 strains analyzed here showed no statistically significant difference. These assays evidenced that high-risk clones of KPC-ECC and KPC-Kp can coexist in the same hospital environment including the same patient, which explains from an ecological point of view that both species can exchange and share plasmids. These findings offer hints to explain the worldwide rise of KPC-ECC strains based on the ability of some pandemic clones to compete and occupy a certain niche. Taken together, the presence of the same new plasmid and the fitness results that showed that both strains can coexist within the same patient suggest that horizontal genetic transfer of
bla
KPC-2
within the patient cannot be ruled out. These findings highlight the constant interaction that these two species can keep in the hospital environment, which, in turn, can be related to the spread of KPC.
The worldwide dissemination of carbapenemase-producing Escherichia coli lineages belonging to high-risk clones poses a challenging public health menace. The aim of this work was to investigate ...genomic features of a colonizing multidrug-resistant strain of Klebsiella pneumoniae carbapenemase (KPC)-producing E. coli from our institution.
Whole-genome sequencing was done by Illumina MiSeq-I, and de novo assembly was achieved using SPAdes. Resistome, mobilome, plasmids, virulome, and integrons were analysed using ResFinder, AMRFinder, ISFinder, PlasmidFinder, MOB-suite, VirulenceFinder, and IntegronFinder. Sequence types (STs) were identified with pubMLST and BIGSdb databases. Conjugation assays were also performed.
Escherichia coli HA25pEc was isolated from a rectal swab sample taken within the framework of the hospital epidemiological surveillance protocol for detection of carbapenemase-producing Enterobacterales. Escherichia coli HA25pEc corresponded to the first report of ST648 co-harbouring blaKPC-2 and blaCTX-M-15 in Latin America from a colonized patient. It had 19 antibiotic resistance genes (ARGs), including blaKPC-2, located on a Tn4401a isoform. Conjugation assays revealed that blaKPC-2 was not transferred by conjugation to E. coli J53 under our experimental conditions.
Escherichia coli ST648 has been detected previously in companion and farm animals as well as in hospital- and community-acquired infections worldwide. Although scarcely reported as KPC-producers, our finding in a culture surveillance with several acquired ARGs, including blaCTX-M-15, alerts the potential of this clone for worldwide unnoticed spreading of extreme drug resistance to β-lactams. These data reinforce the importance of carrying out molecular surveillance to identify reservoirs and warn about the dissemination of new international clones in carbapenemase-bearing patients.
:Enterobacter cloacae complex (ECC) has lately awakened interest due to its increasing resistance to carbapenems codified by several genes all over the globe. Even though there are some sequence ...types (ST) which represent high-risk clones, there is substantial clonal diversity in the ECC. This work aimed to perform whole-genome sequencing (WGS), genomic analysis and phylogenetic studies of a KPC-producing multidrug-resistant (MDR) ECC isolate from Argentina.
: We analysed the genome of an MDR KPC-producing ECC strain isolated from a urine sample from a patient in a hospital in Argentina. The WGS was done by Illumina MiSeq-I. The genome was assembled with SPAdes 3.9.0, and annotated with PROKKA, RAST, and Blast. Plasmids were identified with PlasmidFinder. Antibiotic resistance genes were detected using RESfinder, CARD, and Blastn. STs were identified with pubMLST.
: The strain was identified as Enterobacter hormaechei which is an important emerging human pathogen. No ST could be assigned; 6 out of 7 alleles of multilocus sequence typing (MLST) were the same as for E. hormaechei ST66, which is a high-risk clone. We found multiple acquired antibiotic resistance genes including blaKPC-2 in an IncM1 plasmid, and a secretion system VI, which can favour the prevalence of ECC strains while competing with other bacteria.
: Due to its MLST profile being so close to E. hormaechei ST66, the acquisition of multiple resistance genes, and the presence of the secretion systems, the potential of this strain for becoming a new high-risk clone cannot be discarded.
The emergence of blaKPC-2 within nosocomial settings has become a major public health crisis worldwide. Our aim was to perform whole-genome sequencing (WGS) of three KPC-producing Gram-negative ...bacilli (KPC-GNB) strains isolated from a hospitalized patient to identify acquired antimicrobial resistance genes (ARGs).
WGS was performed using Illumina MiSeq-I, and de novo assembly was achieved using SPAdes. Bioinformatics analysis was done using Resfinder, AMRFinder, ISFinder, plasmidSPAdes, PlasmidFinder, MOB-suite, PLSDB database, and IntegronFinder. Conjugation assays were performed to assess the ability of blaKPC-2 to transfer via a plasmid-related mobilization mechanism.
High-risk clone KPC-producing Klebsiella pneumoniae sequence type (ST) 258 (HA3) was colonizing an inpatient who later was infected by KPC-producing Escherichia coli ST730 (HA4) and subsequently by KPC-producing K. pneumoniae ST11 (HA15) during hospitalization. Although belonging to different species, both strains causing infections harbored the same gene configuration for dissemination of blaKPC-2 in related IncM1 plasmids recently found in other KPC-GNB isolated from Hospital Alemán at Ciudad Autónoma de Buenos Aires. Conjugation assays revealed that only pDCVEA4-KPC from E. coli HA4 was successfully transferred with a conjugation frequency of 3.66 × 101.
Interchange of multidrug-resistant K. pneumoniae lineages ST258 replaced by ST11 in the framework of colonization and infection by KPC-GNB of an inpatient from our institution was found. In addition, the transfer of the gene configuration of blaKPC–2 between infecting strains may have occurred in the nosocomial environment, but we cannot rule out that the event took place in vivo, within the patient, during hospitalization.
PDF
