Genomic Characterization of Two NDM-5-Producing Isolates of Klebsiella pneumoniae ST11 from a Single Patient.

Two metallo-ß-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 blaNDM-5, blaCTX-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 blaNDM-5, rmtB and blaCTX-M-15 genes in a K. pneumoniae ST11 strain from Latin America. Our results also highlighted the importance of focusing on epidemiological surveillance programs.

The Acinetobacter baumannii website (Ab-web): a multidisciplinary knowledge hub, communication platform, and workspace.

Acinetobacter baumannii is a Gram-negative bacterium increasingly implicated in hospital-acquired infections and outbreaks. Effective prevention and control of such infections are commonly challenged by the frequent emergence of multidrug-resistant strains. Here we introduce Ab-web (https://www.acinetobacterbaumannii.no), the first online platform for sharing expertise on A. baumannii. Ab-web is a species-centric knowledge hub, initially with 10 articles organized into two main sections, 'Overview' and 'Topics', and three themes, 'epidemiology', 'antibiotic resistance', and 'virulence'. The 'workspace' section provides a spot for colleagues to collaborate, build, and manage joint projects. Ab-web is a community-driven initiative amenable to constructive feedback and new ideas.

Antimicrobial resistance dissemination associated with intensive animal production practices in Argentina: A systematic review and meta-analysis

Resumen 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.

Abstract 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.

Genomic data reveals the emergence of the co-occurrence of blaKPC-2 and blaCTX-M-15 in an Escherichia coli ST648 strain isolated from rectal swab within the framework of hospital surveillance.

OBJECTIVES: 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. METHODS: 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. RESULTS: 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. CONCLUSION: 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 ß-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.

Replacement of KPC-producing pandemic lineages and dissemination of plasmids associated with antimicrobial resistance determinants during inpatient's hospitalization.

OBJECTIVES: 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). METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

Antimicrobial resistance dissemination associated with intensive animal production practices in Argentina: A systematic review and meta-analysis.

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.

Novel insights related to the rise of KPC-producing Enterobacter cloacae complex strains within the nosocomial niche.

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.

New sequence type of an Enterobacter cloacae complex strain with the potential to become a high-risk clone.

OBJECTIVES: Enterobacter cloacae complex (ECC) has awakened interest recently because of its increasing resistance to carbapenems codified by several genes all over the globe. Even though there are some sequence types (STs) 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 Klebsiella pneumoniae carbapenemase (KPC) -producing multidrug-resistant (MDR) ECC isolate from Argentina. METHODS: 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 (Illumina, San Diego, CA). 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. RESULTS: The strain was identified as Enterobacter hormaechei, an important emerging human pathogen. No ST could be assigned; six of seven 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. CONCLUSION: Because of its MLST profile being so close to that of 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.

Emergence of colistin resistance in Klebsiella pneumoniae ST15 disseminating blaKPC-2 in a novel genetic platform.

OBJECTIVES: 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. METHODS: 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. RESULTS: 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. CONCLUSION: 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.

Serratia marcescens SCH909 as reservoir and source of genetic elements related to wide dissemination of antimicrobial resistance mechanisms.

Serratia marcescens SCH909 is a multidrug resistant strain isolated in 1988 harboring three class 1 integrons. We wondered if these integrons were retained over time and if there were other antimicrobial resistant determinants contributing to its multidrug resistant profile. Genomic analysis showed a fourth multidrug resistance integron, a Tn7 transposon with dfrA1-sat2-ybeA-ybfA-ybfB-ybgA gene cassettes in the variable region. Insertion sequences were involved in the genesis of novel composite transposons in the L4 subtype plasmid pSCH909, such as Tn6824 carrying an arsenic regulon and two head to head class 1 integrons surrounded by two complete IS1. Remarkably, a novel chromosomal genomic island, SmaR, was identified, closely related to Multiple Antimicrobial Resistance Regions (MARR), usually found in AbaR0-type and AbGRI2-0 from global clones of Acinetobacter baumannii, and in M-type plasmids circulating in Enterobacteriaceae. Maintenance studies showed that the three class 1 integrons were maintained over 1 month without antimicrobial pressure. Since S. marcescens is considered a relevant nosocomial pathogen that can have a wide range of niches - human, plant, animal, soil and inanimate surfaces, our findings support the ability of this species to capture, maintain and spread a broad variety of antimicrobial resistance elements.

Genomic analysis of the first isolate of KPC-2-producing Klebsiella pneumoniae from Uruguay.

OBJECTIVES: Since KPC-2-producing Klebsiella pneumoniae are associated with successful dissemination of a major clone, defined as sequence type 258 (ST258), the aim of this study was to perform whole-genome sequencing (WGS) of the first colistin-resistant K. pneumoniae strain (Kpn666) carrying blaKPC-2 identified in Uruguay in 2011 in order to identify genomic and phylogenetic traits. METHODS: WGS of strain Kpn666 isolated from an asymptomatic urinary tract infection was performed using Illumina MiSeq, and de novo assembly was performed using SPADES v.3.11. Contigs were re-ordered using the ST258 reference genome NJST258_1 (GenBank CP006923) and were oriented with the MAUVE Contig Mover. Twenty complete genomes of K. pneumoniae identified as ST258 using the Pasteur MLST site were downloaded from GenBank (May 2017). A maximum-likelihood tree was created using MEGA7 based on core single nucleotide polymorphisms (SNPs) from whole-genome alignment obtained with SNP sites (https://github.com/sanger-pathogens/snp-sites). RESULTS: WGS analysis revealed a genome of 5448179bp (5232 CDS, 108 RNAs). Phylogenetic analysis identified that Kpn666 belonged to clade I lineage of ST258. Further studies also identified IncR, IncFIB(K) and IncFII(K) plasmid replicons and 11 transferable associated antimicrobial resistance genes (ARGs) comprising four drug classes. The mgrB gene involved in colistin resistance was shown to be disrupted by insertion of an IS5-like element. CONCLUSIONS: The first isolate of KPC-2-producing K. pneumoniae detected in Uruguay was sequenced and the results confirm the ability of this bacterium to capture several ARGs. The KPC-2 carbapenemase in Uruguay is likely to have been introduced by the high-risk clone ST258.

Lateral Antimicrobial Resistance Genetic Transfer is active in the open environment.

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 VIM-2 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.