IncF Plasmids Research Articles

Risk Factors for Community-Acquired Urinary Tract Infections Caused by Multidrug-Resistant Enterobacterales in Thailand.

The dissemination of multidrug-resistant Enterobacterales (MDRE) in community settings is becoming a great concern. This study aimed to assess the incidence and risk factors associated with community-acquired urinary tract infections (CA-UTIs) caused by MDRE. A prospective case–control study was undertaken among patients with UTIs visiting an outpatient department in Phitsanulok Province, Thailand. Urine samples were collected and screened to include only patients with Enterobacterales infections. Risk factors were analyzed by multivariate logistic regression analysis. Of the 284 patients with CA-UTIs, 25.7% (n = 73) and 74.3% (n = 211) were positive for MDRE (case) and non-MDRE (control), respectively. Being a farmer was identified as an independent risk factor for MDRE-associated CA-UTIs (adjusted odds ratio = 3.101; 95% confidence interval = 1.272–7.564; p = 0.013). A total of 309 Enterobacterales isolates were recovered, and Escherichia coli was the most frequently detected (86.4%). The highest resistance rate was observed for ampicillin (67.0%), followed by ciprofloxacin (34.0%) and cotrimoxazole (32.7%), while resistance to third-generation cephalosporins (cefotaxime, ceftriaxone) and levofloxacin remained <20%. Resistance to ampicillin–gentamicin–cotrimoxazole was the most common pattern among MDRE isolates. Interestingly, we detected a colistin-resistant Enterobacter cloacae harboring mcr-9 (colistin MIC = 16 µg/mL). mcr-9 was transferable at high frequency (4.5 × 10−4) and resided on IncF plasmid. This study demonstrates that being a farmer is a risk factor for MDRE-associated CA-UTIs. Interestingly, this is the first report to identify mcr-9-positive E. cloacae from a Thai patient in the community.

Research Note: Longitudinal fecal shedding patterns and characterization of Salmonella enterica and mcr-positive Escherichia coli in meat-type ducks raised in an open-house system

This longitudinal study aimed to determine the fecal shedding pattern and characterize Salmonella enterica and mcr-positive Escherichia coli from meat-type ducks raised in an open-house system in Thailand. Fecal samples (n = 1,475) were collected from ducks over a 6-month period. Overall, the detection rate of S. enterica was 5.4% and the highest fecal shedding rate was noted in 4-day-old ducklings (28.8%). Then, S. enterica shedding decreased to 10, 8, 4.7, and 0.7% when ducks reached the ages of 10 d, 17 d, 3 wk, and 4 wk, respectively. Seventy-nine isolates were recovered and Salmonella Amsterdam was the predominant serovar (79.7%). With respect to colistin-resistant E. coli, mcr-positive E. coli (colistin MICs = 8–16 μg/mL) was noted in ducks at the ages of 16 wk (6.0%) and 24 wk (18.7%). mcr-1 was the most common (75.7%), followed by mcr-3 (13.5%), and mcr-1 and mcr-3 co-carriage (10.8%). Most S. enterica isolates were susceptible to antibiotics and multidrug resistant (MDR) was found in only a single isolate. However, as many as 89.2% of mcr-positive E. coli were defined as MDR. Almost all S. enterica isolates (97.5–100%) carried several virulence genes involving in invasion, intracellular survival, and iron metabolism. Pulsed Field Gel Electrophoresis revealed that several mcr-positive E. coli isolates were clonally unrelated. Conjugative transfer of mcr-1, mcr-3 as well as co-transfer of mcr-1 and mcr-3 was observed with the frequencies ranging from 10−8 to 10−3. All mcr-1 resided on IncI2, while mcr-3 was associated with IncF and IncX4 plasmids. Our study provides the evidence of fecal shedding pattern of S. enterica and mcr-positive E. coli from meat-type ducks, highlighting the importance of duck farming in the dissemination of pathogenic bacteria that are potentially hazardous to human.

Antimicrobial Resistance Genes, Virulence Genes, and Associated Mobile Genetic Elements of Eight Multidrug-Resistant Enterobacterales Isolated from Hospital-Acquired Urinary Tract Infections in Sri Lanka.

The study describes the first isolation of multidrug-resistant (MDR) Klebsiella pneumoniae ST16, Escherichia coli ST131 (Esc), and Enterobacter hormaechei subsp. steigerwaltii ST93 (Enterobacter cloacae complex [ECC]) in Sri Lanka. Eight MDR strains of uropathogenic Enterobacterales isolated from hospital acquired urinary tract infections (UTIs) were analyzed using genomic sequencing and comparative genomics. Isolates carried multiple carbapenemase, AmpC, and ESBL (extended-spectrum β-lactamase) genes. ECC manifested both blaNDM-4 and blaOXA-181. The K. pneumoniae strains harbored fimbrial genes that facilitate pathogenesis of UTI. Several extraintestinal pathogenic E. coli associated virulence genes were identified in Esc. The efflux pump gene, acrA, and the T6SS gene cluster were detected in ECC. Many antimicrobial resistance (AMR) and virulence genes were identified associated with mobile genetic elements. ISEcp1 flanked upstream of blaCTX-M-15. The carbapenemase genes were carried on ColKP3 plasmids and were associated with ISEcp1. In Esc, the AMR gene blaTEM-1B and virulence gene traT were found on an IncF plasmid replicon. In K. pneumoniae the AMR genes sul1 and tetB present on IncR plasmid replicons and were associated with the insertion sequence IS6100. In Kp5, blaLAP-2 and qnrS1 coexisted and were flanked by ISEcl. AMR gene clusters, conferring resistance to multiple antimicrobial classes, flanked by mobile elements were identified in seven isolates.

Screening and Characterization of Multidrug-Resistant Enterobacterales among Hospitalized Patients in the African Archipelago of Cape Verde.

This study aimed to investigate, for the first time, the occurrence and characteristics of extended-spectrum β-lactamase (ESBL)- and carbapenemase-producing Enterobacterales in Cape Verde. A total of 98 inpatients hospitalized at Hospital Universitário Agostinho Neto were screened for rectal colonization. All ESBL- and carbapenemase-producing isolates were tested for antimicrobial susceptibility and characterized by multilocus sequence typing. Mating-out assay followed by PCR-based replicon typing were performed to characterize the plasmids harboring carbapenemase encoding genes. A large proportion of patients carried ESBL- or carbapenemase-producing Enterobacterales (56% and 6%, respectively). Among 93 ESBL-producing isolates, there were mainly Klebsiella pneumoniae (58%) and Escherichia coli (37%). Five different ESBLs were detected, with CTX-M-15 being highly predominant (92%). Six carbapenemase-producing isolates (five E. coli and one K. pneumoniae) were recovered, and all of the OXA-48-like type (four OXA-181, one OXA-48, and one OXA-244). The blaOXA-48 gene was located on an IncFI-type plasmid, the blaOXA-181 gene on IncFI or IncX3 plasmids, and the blaOXA-244 gene was found to be chromosomally located. The five carbapenemase-producing E. coli isolates belonged to five distinct sequence types. This study overall showed a very high prevalence of ESBL-producing Enterobacterales, as well as the emergence of carbapenemase producers in this hospital.

A Newly Identified Group of P-like (PL) Fimbria Genes from Extraintestinal Pathogenic Escherichia coli (ExPEC) Encode Distinct Adhesin Subunits and Mediate Adherence to Host Cells.

Fimbrial adhesins promote bacterial adherence and biofilm formation. Sequencing of avian pathogenic Escherichia coli (APEC) strain QT598 identified new fimbriae belonging to the π group, which we named PL (P-like) fimbriae since the genetic organization and sequence are similar to those of P and related fimbriae. Genes encoding PL fimbriae located on IncF plasmids are present in diverse E. coli isolates from poultry, human systemic infections, and other sources. As with P fimbriae, PL fimbriae exhibit divergence in adhesin-encoding genes and could be divided into 5 classes based on sequence differences in the PlfG adhesin. plf genes from two predominant PlfG adhesin classes, PlfG class I (PlfGI) and PlfGII, were cloned. PL fimbriae were visualized by electron microscopy, associated with increased biofilm, demonstrated distinct hemagglutination profiles, and promoted adherence to human bladder and kidney epithelial cells. The genes encoding hybrid fimbriae were comprised of genes from plfQT598, wherein plfG was replaced by papG; the adhesin-encoding genes were also functional and mediated adherence to epithelial cells, demonstrating compatibility between the components of these two types of fimbriae. Deletion of plf genes did not reduce colonization of the mouse urinary tract in a single-strain infection model. In contrast, loss of plf genes significantly reduced competitive colonization in the mouse kidneys. Furthermore, plf gene expression was increased over 40-fold in the bladder compared to during in vitro culture. Overall, PL fimbriae represent a new group of fimbriae demonstrating both functional differences from and similarities to P fimbriae, which mediated adherence to host cells and improved competitive colonization of the mouse kidney. IMPORTANCE Fimbriae are important colonization factors in many bacterial species. The identification of a new type of fimbriae encoded on some IncF plasmids in E. coli was investigated. Genomic sequences demonstrated these fimbrial gene clusters have genetic diversity, particularly in the adhesin-encoding plfG gene. Functional studies demonstrated differences in hemagglutination specificity, although both types of Plf adhesin under study mediated adherence to human urinary epithelial cells. A plf mutant also showed decreased colonization of the kidneys in a mouse competitive infection model. PL fimbriae may represent previously unrecognized adhesins that could contribute to host specificity and tissue tropism of some E. coli strains.

Profiling the plasmid conjugation potential of urinary Escherichia coli.

Escherichia coli is often associated with urinary tract infection (UTI). Antibiotic resistance in E. coli is an ongoing challenge in managing UTI. Extrachromosomal elements – plasmids – are vectors for clinically relevant traits, such as antibiotic resistance, with conjugation being one of the main methods for horizontal propagation of plasmids in bacterial populations. Targeting of conjugation components has been proposed as a strategy to curb the spread of plasmid-borne antibiotic resistance. Understanding the types of conjugative systems present in urinary E. coli isolates is fundamental to assessing the viability of this strategy. In this study, we profile two well-studied conjugation systems (F-type and P-type) in the draft genomes of 65 urinary isolates of E. coli obtained from the bladder urine of adult women with and without UTI-like symptoms. Most of these isolates contained plasmids and we found that conjugation genes were abundant/ubiquitous, diverse and often associated with IncF plasmids. To validate conjugation of these urinary plasmids, the plasmids from two urinary isolates, UMB1223 (predicted to have F-type genes) and UMB1284 (predicted to have P-type genes), were transferred by conjugation into the K-12 E. coli strain MG1655. Overall, the findings of this study support the notion that care should be taken in targeting any individual component of a urinary E. coli isolate’s conjugation system, given the inherent mechanistic redundancy, gene diversity and different types of conjugation systems in this population.

Hybrid Plasmids Encoding Antimicrobial Resistance and Virulence Traits Among Hypervirulent Klebsiella pneumoniae ST2096 in India.

BackgroundHypervirulent variants of Klebsiella pneumoniae (HvKp) were typically associated with a broadly antimicrobial susceptible clone of sequence type (ST) 23 at the time of its emergence. Concerningly, HvKp is now also emerging within multidrug-resistant (MDR) clones, including ST11, ST15, and ST147. MDR-HvKp either carry both the virulence and resistance plasmids or carry a large hybrid plasmid coding for both virulence and resistance determinants. Here, we aimed to genetically characterize a collection of MDR-HvKp ST2096 isolates haboring hybrid plasmids carrying both antimicrobial resistance (AMR) and virulence genes.MethodsNine K. pneumoniae ST2096 isolated over 1 year from the blood sample of hospitalized patients in southern India that were MDR and suspected to be HvKp were selected. All nine isolates were subjected to short-read whole-genome sequencing; a subset (n = 4) was additionally subjected to long-read sequencing to obtain complete genomes for characterization. Mucoviscosity assay was also performed for phenotypic assessment.ResultsAmong the nine isolates, seven were carbapenem-resistant, two of which carried bla NDM-5 on an IncFII plasmid and five carried bla OXA-232 on a ColKP3 plasmid. The organisms were confirmed as HvKp, with characteristic virulence genes (rmpA2, iutA, and iucABCD) carried on a large (~320 kbp) IncFIB–IncHI1B co-integrate. This hybrid plasmid also carried the aadA2, armA, bla OXA-1, msrE, mphE, sul1, and dfrA14 AMR genes in addition to the heavy-metal resistance genes. The hybrid plasmid showed about 60% similarity to the IncHI1B virulence plasmid of K. pneumoniae SGH10 and ~70% sequence identity with the first identified IncHI1B pNDM-MAR plasmid. Notably, the hybrid plasmid carried its type IV-A3 CRISPR-Cas system which harbored spacer regions against traL of IncF plasmids, thereby preventing their acquisition.ConclusionThe convergence of virulence and AMR is clinically concerning in K. pneumoniae. Our data highlight the role of hybrid plasmids carrying both AMR and virulence genes in K. pneumoniae ST2096, suggesting that MDR-HvKp is not confined to selected clones; we highlight the continued emergence of such genotypes across the species. The convergence is occurring globally amidst several clones and is of great concern to public health.

Phenotypic and Genotypic Characterization of Multidrug-Resistant Enterobacter hormaechei Carrying qnrS Gene Isolated from Chicken Feed in China.

ABSTRACTMultidrug resistance (MDR) in Enterobacteriaceae including resistance to quinolones is rising worldwide. The plasmid-mediated quinolone resistance (PMQR) gene qnrS is prevalent in Enterobacteriaceae. However, the qnrS gene is rarely found in Enterobacter hormaechei (E. hormaechei). Here, we reported one multidrug resistant E. hormaechei strain M1 carrying the qnrS1 and blaTEM-1 genes. This study was to analyze the characteristics of MDR E. hormaechei strain M1. The E. hormaechei strain M1 was identified as Enterobacter cloacae complex by biochemical assay and 16S rRNA sequencing. The whole genome was sequenced by the Oxford Nanopore method. Taxonomy of the E. hormaechei was based on multilocus sequence typing (MLST). The qnrS with the other antibiotic resistance genes were coexisted on IncF plasmid (pM1). Besides, the virulence factors associated with pathogenicity were also located on pM1. The qnrS1 gene was located between insertion element IS2A (upstream) and transposition element ISKra4 (downstream). The comparison result of IncF plasmids revealed that they had a common plasmid backbone. Susceptibility experiment revealed that the E. hormaechei M1 showed extensive resistance to the clinical antimicrobials. The conjugation transfer was performed by filter membrane incubation method. The competition and plasmid stability assays suggested the host bacteria carrying qnrS had an energy burden. As far as we know, this is the first report that E. hormaechei carrying qnrS was isolated from chicken feed. The chicken feed and poultry products could serve as a vehicle for these MDR bacteria, which could transfer between animals and humans through the food chain. We need to pay close attention to the epidemiology of E. hormaechei and prevent their further dissemination.IMPORTANCE Enterobacter hormaechei is an opportunistic pathogen. It can cause infections in humans and animals. Plasmid-mediated quinolone resistance (PMQR) gene qnrS can be transferred intergenus, which is leading to increase the quinolone resistance levels in Enterobacteriaceae. Chicken feed could serve as a vehicle for the MDR E. hormaechei. Therefore, antibiotic-resistance genes (ARGs) might be transferred to the intestinal flora after entering the gastrointestinal tract with the feed. Furthermore, antibiotic-resistant bacteria (ARB) were also excreted into environment with feces, posing a huge threat to public health. This requires us to monitor the ARB and antibiotic-resistant plasmids in the feed. Here, we demonstrated the characteristics of one MDR E. hormaechei isolate from chicken feed. The plasmid carrying the qnrS gene is a conjugative plasmid with transferability. The presence of plasmid carrying antibiotic-resistance genes requires the maintenance of antibiotic pressure. In addition, the E. hormaechei M1 belonged to new sequence type (ST). These data show the MDR E. hormaechei M1 is a novel strain that requires our further research.

Genomic characterization and antimicrobial resistance profiles of Salmonella enterica serovar Infantis isolated from food, humans and veterinary-related sources in Brazil.

To characterize the genetic relatedness, phenotypic and genotypic antimicrobial resistance and plasmid content of 80 Salmonella Infantis strains isolated from food, humans and veterinary sources from 2013 to 2018 in Brazil. Pulsed-field gel electrophoresis and single-nucleotide polymorphism analysis showed major clusters containing 50% and 38.8% of the strains studied respectively. Multilocus sequence typing assigned all strains to ST32. Disk-diffusion revealed that 90% of the strains presented resistant or intermediate resistant profiles and 38.8% displayed multidrug resistance. Resistance genes for aminoglycosides (aac(6')-Iaa; aadA12; aph(3″-Ib; aph(6)-Id), β-lactams (blaTEM-1 ; blaCTX-M-8 ; blaCMY-2 ), trimethoprim (dfrA8), tetracycline (tet(A)), amphenicols (floR), sulfonamide (sul2), efflux pumps (mdsA; mdsB), chromosomal point mutations in gyrB, parC, acrB and pmrA were detected. Strains harboured IncI, IncF, IncX, IncQ, IncN and IncR plasmids. The presence of a prevalent S. Infantis subtype in Brazil and the high antimicrobial resistance rates reinforced the potential hazard of this serovar for the public health and food safety fields. This is the first study characterizing a large set of S. Infantis from Brazil by whole-genome sequencing, which provided a better local and global comprehension about the distribution and characteristics of this serovar of importance in the food, human and veterinary fields.

IncFIB plasmids carrying the resistance gene blaCTX-M-15 in ESBL-producing Escherichia coli clones from pediatric patients.

The emergence of extended-spectrum β-lactamases (ESBLs)-producing Escherichia coli clones are a public health concern worldwide. Scarce information does exist about the spread of ESBLs-producing E. coli in pediatric patients from developing countries. E. coli strains were analyzed by multilocus-sequence-typing, pulsed-field-gel-electrophoresis and phylogenetic group. The antimicrobial-resistance genes were detected by PCR, and plasmid content by the PCR-based replicon-typing. Horizontal transfer was tested by conjugation and the location of the blaCTX-M-15 gene by Southern blot hybridization. Thirty-two cefotaxime-resistant E. coli were recovered. Eleven of them were ESBL-producing isolates, which were well characterized and ascribed to seven sequence types and five phylogroups. The ESBL CTX-M-15 was the most prevalent enzyme (9 of 11). Plasmids of variable sizes (40-220 kb) were visualized, and the incompatibility (Inc) group FIB plasmid-replicon was detected in the ESBL strains and transferred by conjugation in 45.45% of them. Plasmid-borne toxin-antitoxin systems were the most frequently detected systems, strongly associated to IncF plasmids. The CTX-M-15-encoding gene was located on IncFIB plasmids. Even though a small number of ESBL-producing strains was recovered, we evidenced that IncFIB plasmids carry the blaCTX-M-15 gene, highlighting the role of IncF-type plasmids in facilitating the spread and maintenance of ESBL-encoding genes, which further favors the rapid increase of the antimicrobial resistance dissemination in disease-causing E. coli strains in pediatric patients.

Genome plasticity as a paradigm of antibiotic resistance spread in ESKAPE pathogens.

The major reason behind the spread of antibiotic resistance genes (ARGs) is persistent selective pressure in the environment encountered by bacteria. Genome plasticity plays a crucial role in dissemination of antibiotic resistance among bacterial pathogens. Mobile genetic elements harboring ARGs are reported to dodge bacterial immune system and mediate horizontal gene transfer (HGT) under selective pressure. Residual antibiotic pollutants develop selective pressures that force the bacteria to lose their defense mechanisms (CRISPR-cas) and acquire resistance. The present study targets the ESKAPE organisms (namely, Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa and Enterobacter spp.) causing various nosocomial infections and emerging multidrug-resistant species. The role of CRISPR-cas systems in inhibition of HGT in prokaryotes and its loss due to presence of various stressors in the environment is also focused in the study. IncF and IncH plasmids were identified in all strains of E. faecalis and K. pneumoniae, carrying Beta-lactam and fluoroquinolone resistance genes, whereas sal3, phiCTX, and SEN34 prophages harbored aminoglycoside resistance genes (aadA, aac). Various MGEs present in selected environmental niches that aid the bacterial genome plasticity and transfer of ARGs contributing to its spread are also identified.

Colistin-resistant Enterobacterales among veterinary healthcare workers and in the Dutch population.

ObjectivesPlasmid-mediated colistin resistance can be transferred from animals to humans. We investigated the prevalence of carriage of mcr-mediated colistin-resistant Escherichia coli and Klebsiella pneumoniae (ColR-E/K) in veterinary healthcare workers and in the general population in the Netherlands.MethodsTwo cross-sectional population studies were performed: one among veterinary healthcare workers and one in the general population. Participants sent in a faecal sample and filled in a questionnaire. Samples were analysed using selective enrichment and culture. Mobile colistin resistance genes (mcr) were detected by PCR and ColR-E/K were sequenced using Illumina and Nanopore technologies.ResultsThe prevalence of mcr-mediated ColR-E/K was 0.2% (1/482, 95% CI 0.04%–1.17%) among veterinary personnel and 0.8% (5/660, 95% CI 0.3%–1.8%) in the population sample. mcr-1 was found in E. coli from four persons, mcr-8 in K. pneumoniae from one person and another person carried both mcr-1 and mcr-8 in a K. pneumoniae isolate. mcr-1 was found on different plasmid types (IncX4, IncI1 and IncI2), while mcr-8 was found on IncF plasmids only.Conclusions mcr-mediated ColR-E/K resistance was uncommon in both populations. Professional contact with animals does not increase the chance of carriage of these bacteria in the Netherlands at present. mcr-8 was found for the first time in the Netherlands. Surveillance of colistin resistance and its underlying mechanisms in humans, livestock and food is important in order to identify emerging trends in time.

WGS-Based Phenotyping and Molecular Characterization of the Resistome, Virulome and Plasmid Replicons in Klebsiella pneumoniae Isolates from Powdered Milk Produced in Germany.

The emergence of Klebsiella pneumoniae (K. pneumoniae) in German healthcare is worrying. It is not well-investigated in the veterinary world and food chains. In the current study, antibiotic susceptibility profiles of 24 K. pneumoniae strains isolated from powdered milk samples produced in Germany were investigated by a microdilution test. Next-generation sequencing (NGS) was applied to identify genomic determinants for antimicrobial resistance (AMR), virulence-associated genes and plasmids replicons. All isolates were susceptible to the majority (14/18) of tested antibiotics. Resistance to colistin, fosfomycin, chloramphenicol and piperacillin was found. The ambler class A ß-lactamase, blaSHV variants were identified in all isolates, of which blaSHV-187 was most prevalent and found in 50% of isolates. Single-nucleotide-variants of oqxA and oqxB conferring resistance to phenicol/quinolone were found in all isolates, and the oqxB17 was the most prevalent found in 46% of isolates. 67% of isolates harbored fosA genes; however, only one was fosfomycin-resistant. Two isolates harbored genes conferring resistance to colistin, despite being susceptible. The majority of identified virulome genes were iron uptake siderophores. Two enterobactins (entB, fepC), six adherence-related genes belonging to E. coli common pilus (ECP) and one secretion system (ompA gene) were found in all isolates. In contrast, yersiniabactin was found in two isolates. One ST23 strain was susceptible to all tested antibiotics, and harbored determinants discriminatory for hypervirulent strains, e.g., aerobactin, salmochelin, yersiniabactin, enterobactin and regulator of mucoid phenotype A genes that are highly associated with hypervirulent K. pneumoniae. The IncF plasmid family was found in all strains, while almost half of the isolates harbored Col440I-type plasmids and nine isolates harbored various Inc-type plasmids. The presence of K. pneumoniae carrying different resistomes and major virulent specific virulomes in powdered milk samples is alarming. This could threaten public health, particularly of neonates and infants consuming dried milk.

First identification of bla NDM-5 producing Escherichia coli from neonates and a HIV infected adult in Tanzania.

Introduction. Carbapenem-resistant members of the family Enterobacteriaceae are emerging as a global public-health threat and cause substantial challenges in clinical practice. Gap Statement. There is a need for increased and continued genomic surveillance of antimicrobial resistance genes globally in order to detect outbreaks and dissemination of clinically important resistance genes and their associated mobile genetic elements in human pathogens. Aim. To describe the resistance mechanisms of carbapenem-resistant Escherichia coli . Methods. Rectal swabs from neonates and newly diagnosed human immunodeficiency virus (HIV) infected adults were collected between April 2017 and May 2018 and screened for faecal carriage of carbapenamases and OXA-48 producing members of the family Enterobacteriaceae. Bacterial isolates were identified using matrix assisted laser desorption ionization time of flight mass spectrometry. Antimicrobial susceptibility testing was performed by E-test. Whole genomes of carbapenem-resistant E. coli were investigated using a hybrid assembly of Illumina and Oxford Nanopore Technologies sequencing reads. Results. Three carbapenem-resistant E. coli were detected, two from neonates and one from an HIV infected adult. All three isolates carried bla NDM-5. Two E. coli from neonates belonged to ST167 and bla NDM-5 co-existed with bla CTX-M-15 and bla OXA-01, and all were carried on IncFIA type plasmids. The E. coli from the HIV infected adult belonged to ST2083, and carried bla NDM-5 on an IncX3 type plasmid and bla CMY-42 on an IncI type plasmid. All bla NDM-5 carrying plasmids contained conjugation related genes. In addition, E. coli from the HIV infected adult carried three more plasmid types; IncFIA, IncFIB and Col(BS512). One E. coli from a neonate also carried one extra plasmid Col(BS512). All three E. coli harboured resistance genes to fluoroquinolone, aminoglycosides, sulfamethoxazole, trimethoprim, macrolides and tetracycline, carried on the IncFIA type plasmid. Furthermore, E. coli from the neonates carried a chloramphenicol resistance gene (catB3), also on the IncFIA plasmid. All three isolates were susceptible to colistin. Conclusion. This is the first report, to our knowledge, from Tanzania detecting bla NDM-5 producing E. coli. The carbapenemase gene was carried on an IncFIA and IncX3 type plasmids. Our findings highlight the urgent need for a robust antimicrobial resistance (AMR) surveillance system to monitor and rapidly report on the incidence and spread of emerging resistant bacteria in Tanzania.

First Outbreak of NDM-1-Producing Klebsiella pneumoniae ST11 in a Portuguese Hospital Centre during the COVID-19 Pandemic.

New Delhi metallo-β-lactamase (NDM) carbapenemase has been considered a global threat due to its worldwide widespread in recent years. In Portugal, a very low number of infections with NDM-producing Enterobacterales has been reported. A total of 52 strains from 40 patients and 1 environmental sample isolated during COVID-19 pandemic were included in this study. Wholegenome sequencing (WGS) was performed on 20 carbapenemase-producing strains, including 17 NDM-1-producing Klebsiella pneumoniae ST11-KL105 lineage strains, one NDM-1-producing Escherichia coli ST58 strain and one KPC-3-producing K. pneumoniae ST147 strain, recovered from a total of 19 patients. Of interest, also one NDM-1-producing K. pneumoniae ST11-KL105 was collected from the hospital environment. Genome-wide phylogenetic analysis revealed an ongoing dissemination of NDM-1-producing K. pneumoniae ST11 strains (n = 18) with the same genetic features seen across multiple wards. Furthermore, the ST58 E. coli strain, collected from a patient rectal swab that was also colonised with a K. pneumoniae strain, also showed the IncFIA plasmid replicon and the blaNDM-1 gene (preceded by IS30 and followed by genes bleMBL, trpF, dsbC, cutA, groES and groEL). The blaNDM-1 is part of Tn125-like identical to those reported in Poland, Italy and India. The blaKPC-3 K. pneumoniae ST147-KL64 strain has the genetic environment Tn4401d isoform. In conclusion, herein we report the molecular epidemiology, resistome, virulome and mobilome of the first NDM-1 carbapenemase outbreak caused by K. pneumoniae ST11-KL105 lineage during the COVID-19 pandemic in Portugal. Moreover, the outbreak strains characterised included seventeen different patients (infected and colonised) and one environmental sample which also emphasises the role of commensal and hospital environment strains in the dissemination of the outbreak.

Resistome and Virulome of Multi-Drug Resistant E. coli ST131 Isolated from Residents of Long-Term Care Facilities in the Northern Italian Region

Long-term care facilities (LTCFs) are important reservoirs of antimicrobial-resistant (AMR) bacteria which colonize patients transferred from the hospital, or they may emerge in the facility as a result of mutation or gene transfer. In the present study, we characterized, from a molecular point of view, 43 E. coli strains collected from residents of LTCFs in Northern Italy. The most common lineage found was ST131, followed by sporadic presence of ST12, ST69, ST48, ST95, ST410 and ST1193. All strains were incubators of several virulence factors, with iss, sat, iha and senB being found in 84%, 72%, 63% and 51% of E. coli, respectively. Thirty of the ST131 analyzed were of the O25b:H4 serotype and H30 subclone. The ST131 isolates were found to be mainly associated with IncF plasmids, CTX-M-1, CTX-M-3, CTX-M-15, CTX-M-27 and gyrA/parC/parE mutations. Metallo-β-lactamases were not found in ST131, whereas KPC-3 carbapenemase was found only in two ST131 and one ST1193. In conclusion, we confirmed the spread of extended-spectrum β-lactamase genes in E. coli ST131 isolated from colonized residents living inside LTCFs. The ST131 represents an incubator of fluoroquinolones, aminoglycosides and other antibiotic resistance genes in addition to different virulence factors.

Genomic evolution of the globally disseminated multidrug-resistant Klebsiella pneumoniae clonal group 147.

The rapid emergence of multidrug-resistant Klebsiella pneumoniae is being driven largely by the spread of specific clonal groups (CGs). Of these, CG147 includes 7-gene multilocus sequence typing (MLST) sequence types (STs) ST147, ST273 and ST392. CG147 has caused nosocomial outbreaks across the world, but its global population dynamics remain unknown. Here, we report a pandrug-resistant ST147 clinical isolate from India (strain DJ) and define the evolution and global emergence of CG147. Antimicrobial-susceptibility testing following European Committee on Antimicrobial Susceptibility Testing (EUCAST) guidelines and genome sequencing (Illumina and Oxford Nanopore Technologies, Unicycler assembly) were performed on strain DJ. Additionally, we collated 217 publicly available CG147 genomes [National Center for Biotechnology Information (NCBI), May 2019]. CG147 evolution was inferred within a temporal phylogenetic framework (beast) based on a recombination-free sequence alignment (Roary/Gubbins). Comparative genomic analyses focused on resistance and virulence genes and other genetic elements (BIGSdb, Kleborate, PlasmidFinder, phaster, ICEfinder and CRISPRCasFinder). Strain DJ had a pandrug-resistance phenotype. Its genome comprised the chromosome, seven plasmids and one linear phage-plasmid. Four carbapenemase genes were detected: bla NDM-5 and two copies of bla OXA-181 in the chromosome, and a second copy of bla NDM-5 on an 84 kb IncFII plasmid. CG147 genomes carried a mean of 13 acquired resistance genes or mutations; 63 % carried a carbapenemase gene and 83 % harboured bla CTX-M. All CG147 genomes presented GyrA and ParC mutations and a common subtype I-E CRISPR-Cas system. ST392 and ST273 emerged in 2005 and 1995, respectively. ST147, the most represented phylogenetic branch, was itself divided into two main clades with distinct capsular loci: KL64 (74 %, DJ included, emerged in 1994 and disseminated worldwide, with carbapenemases varying among world regions) and KL10 (20 %, emerged in 2002, predominantly found in Asian countries, associated with carbapenemases NDM and OXA-48-like). Furthermore, subclades within ST147-KL64 differed at the yersiniabactin locus, OmpK35/K36 mutations, plasmid replicons and prophages. The absence of IncF plasmids in some subclades was associated with a possible activity of a CRISPR-Cas system. K. pneumoniae CG147 comprises pandrug-resistant or extensively resistant isolates, and carries multiple and diverse resistance genes and mobile genetic elements, including chromosomal bla NDM-5. Its emergence is being driven by the spread of several phylogenetic clades marked by their own genomic features and specific temporo–spatial dynamics. These findings highlight the need for precision surveillance strategies to limit the spread of particularly concerning CG147 subsets.

Characterization of NDM-5-producing Enterobacteriaceae isolates from retail grass carp ( Ctenopharyngodon idella) and evidence of bla NDM-5-bearing IncHI2 plasmid transfer between ducks and fish.

We aimed to characterize NDM-5-producing Enterobacteriaceae from aquatic products in Guangzhou, China. A total of 196 intestinal samples of grass carp collected in 2019 were screened for carbapenemase genes. Characterization of blaNDM-5 positive isolates and plasmids was determined by antimicrobial susceptibility testing, conjugation experiments, Illumina HiSeq, and Nanopore sequencing. One Citrobacter freundii and six Escherichia coli strains recovered from seven intestinal samples were verified as blaNDM-5 carriers (3.57%, 7/196). TheblaNDM-5 genes were located on the IncX3 (n=5), IncHI2 (n=1), or IncHI2-IncF (n=1) plasmids. All blaNDM-5-bearing plasmids were transferred by conjugation at frequencies of ~10−4–10−6. Based on sequence analysis, the IncHI2 plasmid pHNBYF33-1 was similar to other blaNDM-5-carrying IncHI2 plasmids deposited in GenBank from Guangdong ducks. In all IncHI2 plasmids, blaNDM-5 was embedded in a novel transposon, Tn7051 (IS3000-ΔISAba125-IS5-ΔISAba125-blaNDM-5-bleMBL-trpF-tat-∆dct-IS26-∆umuD-∆ISKox3-IS3000), which was identical to the genetic structure surrounding blaNDM-5 found in some IncX3 plasmids. The IncHI2-IncF hybrid plasmid pHNTH9F11-1 was formed by homologous recombination of theblaNDM-5-carrying IncHI2 plasmid and a heavy-metal-resistant IncF plasmid through ∆Tn1721. To the best of our knowledge, this is the first report on the characterization of blaNDM-5-bearing plasmids in fish in China. The IncHI2 plasmid pHNBYF33-1 may be transmitted from ducks, considering the common duck-fish freshwater aquaculture system in Guangdong. Tn7051 is likely responsible for the transfer of blaNDM-5 from IncX3 to IncHI2 plasmids in Enterobacteriaceae, resulting in the expansion of transmission vectors of blaNDM-5.

Characterization of Carbapenem-Resistant Enterobacteriaceae Cultured From Retail Meat Products, Patients, and Porcine Excrement in China.

The emergence and dissemination of carbapenem-resistant Enterobacteriaceae (CRE) is a growing concern to animal and public health. However, little is known about the spread of CRE in food and livestock and its potential transmission to humans. To identify CRE strains from different origins and sources, 53 isolates were cultured from 760 samples including retail meat products, patients, and porcine excrement. Antimicrobial susceptibility testing was carried out, followed by phylogenetic typing, whole-genome sequencing, broth mating assays, and plasmids analyses. Forty-three Escherichia coli, nine Klebsiella pneumoniae, and one Enterobacter cloacae isolates were identified, each exhibiting multidrug-resistant phenotypes. Genetically, the main sequence types (STs) of E. coli were ST156 (n = 7), ST354 (n = 7), and ST48 (n = 7), and the dominant ST of K. pneumoniae is ST11 (n = 5). blaNDM–5 (n = 40) of E. coli and blaKPC–2 (n = 5) were the key genes that conferred carbapenem resistance phenotypes in these CRE strains. Additionally, the mcr-1 gene was identified in 17 blaNDM-producing isolates. The blaNDM–5 gene from eight strains could be transferred to the recipients via conjugation assays. Two mcr-1 genes in the E. coli isolates could be co-transferred along with the blaNDM–5 genes. IncF and IncX3 plasmids have been found to be predominantly associated with blaNDM gene in these strains. Strains isolated in our study from different sources and regions tend to be concordant and overlap. CRE strains from retail meat products are a reservoir for transition of CRE strains between animals and humans. These data also provide evidence of the dissemination of CRE strains and carbapenem-resistant genes between animal and human sources.

178. Endemic Carbapenem Resistance Driven By Clonal and Horizontal Spread of blaIMP-4 Across Diverse Enterobacterales: Jumping Genes, Promiscuous Plasmids and Killer Clones

BackgroundCarbapenem-resistant Enterobacterales (CRE) have become endemic and cause significant morbidity and mortality globally. The metallo-beta-lactamase gene blaIMP-4 is a key CRE resistance determinant in Australia and Asia but its genomic context remains unknown. We aimed to determine the genomic epidemiology of blaIMP-4 in clinical and environmental isolates from 2008 – 2020 at our institution.MethodsWe performed whole genome sequencing on 219 blaIMP-4-carrying isolates from 134 patients (219 short-read and 75 long-read). Multi-locus sequence types (MLSTs), resistance determinants and plasmid replicons were assessed. High-quality de novo hybrid assemblies were used to identify location of blaIMP-4 gene. We conducted phylogenetic analysis for key MLSTs and plasmids.Results Bla IMP-4 was noted on a class I integron also harboring aminoglycoside, sulfamethoxazole, chloramphenicol and quaternary ammonium compound resistance genes. This integron was able to migrate over time to 10 bacterial species (42 STs) and 6 different plasmid types (Figure 1 and Figure 2). From 2008-2020, blaIMP-4 was present on IncC plasmids in Serratia marcescens and Klebsiella pneumoniae. We noted small outbreaks of Pseudomonas aeruginosa ST111 with chromosomal integration of blaIMP-4 from 2008-2018 (16 isolates) and Enterobacter cloacae complex ST114 with blaIMP-4 on IncFIB(K)/IncFIA(HI1) plasmids from 2011-2020 (19 isolates). From 2016-2020, there was an explosion of diverse IncHI2 plasmids carrying blaIMP-4. This was driven by clonal expansion of E. cloacae complex ST93/ST190 (79 isolates), with spillover of IncHI2 plasmids to Klebsiella spp (13 isolates), Citrobacter spp (2 isolates), S. marcescens (1 isolate), Escherichia coli (4 isolates). In addition to blaIMP-4, these plasmids carried mcr-9.1, a colistin resistance gene, and resistance determinants to nearly all key classes of Gram-negative antimicrobials.Figure 1. Bacterial species harboring blaIMP-4 2008-2020 BlaIMP-4 was noted in diverse bacterial species over the study period. Serratia marcescens and Klebsiella pneumoniae were present throughout. Outbreaks of Enterobacter cloacae complex ST114, ST190 and ST93 and Pseudomonas aeruginosa ST111 were noted.Figure 2. Diverse plasmids associated with blaIMP-4 carriage determined by de novo hybrid assembly Presence of blaIMP-4 on diverse plasmids that varied through the study period was noted. Plasmids were charaterised by analysing de novo hybrid assembly data and co-location of blaIMP-4 and plasmid replicons on the same contigs.Conclusion Bla IMP-4 spread on a class I integron was responsible for endemic carbapenem resistance at our institution. This mobile genetic element was able to persist due to both clonal spread and entry into diverse plasmids. Concerningly, we noted a large outbreak driven by IncHI2 plasmids harboring colistin resistance genes with spread to multiple bacterial species.Disclosures All Authors: No reported disclosures