Programming bacteria for multiplexed DNA detection.

DNA is a universal and programmable signal of living organisms. Here we develop cell-based DNA sensors by engineering the naturally competent bacterium Bacillus subtilis (B. subtilis) to detect specific DNA sequences in the environment. The DNA sensor strains can identify diverse bacterial species including major human pathogens with high specificity. Multiplexed detection of genomic DNA from different species in complex samples can be achieved by coupling the sensing mechanism to orthogonal fluorescent reporters. We also demonstrate that the DNA sensors can detect the presence of species in the complex samples without requiring DNA extraction. The modularity of the living cell-based DNA-sensing mechanism and simple detection procedure could enable programmable DNA sensing for a wide range of applications.

Phylogroup typing and carbapenem resistance of Escherichia coli from agricultural samples in Metro Manila, Philippines.

Primary production environment is considered as reservoir of Escherichia coli contamination of produce. E. coli is classified into eight phylogroups which differ in ecological niches, evolutionary history, and phenotypic properties. To understand the population genetic structure and composition of E. coli in primary production environments in Metro Manila, Philippines, a total of 80 E. coli recovered from irrigation water, soil, vegetables, and feces of cat, carabao, chicken, dog, and goat were allocated into distinct phylogroups based on the presence and absence of genetic markers. Results showed that the most prevalent phylogroup was B1 (71.3%), followed by A (18.6%), D (6.3%), B2 (1.3%), E (1.3%), and an unknown phylogroup (1.3%). The most prevalent genetic marker was arpA, followed by TspE4.C2, yjaA, and chuA. The carbapenem resistance of 24 E. coli isolates representing different phylogroups was also evaluated. Intriguingly, all isolates exhibited uniform susceptibility. This is the first report to provide insights into the phylogroup structure and composition, as well as carbapenem resistance of E. coli from primary production in the Philippines, which highlights possible source of and solution for gastrointestinal and enteric diseases.

A role for ColV plasmids in the evolution of pathogenic Escherichia coli ST58.

Escherichia coli ST58 has recently emerged as a globally disseminated uropathogen that often progresses to sepsis. Unlike most pandemic extra-intestinal pathogenic E. coli (ExPEC), which belong to pathogenic phylogroup B2, ST58 belongs to the environmental/commensal phylogroup B1. Here, we present a pan-genomic analysis of a global collection of 752 ST58 isolates from diverse sources. We identify a large ST58 sub-lineage characterized by near ubiquitous carriage of ColV plasmids, which carry genes encoding virulence factors, and by a distinct accessory genome including genes typical of the Yersiniabactin High Pathogenicity Island. This sub-lineage includes three-quarters of all ExPEC sequences in our study and has a broad host range, although poultry and porcine sources predominate. By contrast, strains isolated from cattle often lack ColV plasmids. Our data indicate that ColV plasmid acquisition contributed to the divergence of the major ST58 sub-lineage, and different sub-lineages inhabit poultry, swine and cattle.

Genetic diversity and multidrug resistance of phylogenic groups B2 and D in InPEC and ExPEC isolated from chickens in Central China.

BACKGROUND: Avian colibacillosis is an infectious bacterial disease caused by avian pathogenic Escherichia coli (APEC). APEC causes a wide variety of intestinal and extraintestinal infections, including InPEC and ExPEC, which result in enormous losses in the poultry industry. In this study, we investigated the prevalence of InPEC and ExPEC in Central China, and the isolates were characterized using molecular approaches and tested for virulence factors and antibiotic resistance. RESULTS: A total of 200 chicken-derived E. coli isolates were collected for study from 2019 and 2020. The prevalence of B2 and D phylogenic groups in the 200 chicken-derived E. coli was verified by triplex PCR, which accounted for 50.53% (48/95) and 9.52% (10/105) in ExPEC and InPEC, respectively. Additionally, multilocus sequence typing method was used to examine the genetic diversity of these E. coli isolates, which showed that the dominant STs of ExPEC included ST117 (n = 10, 20.83%), ST297 (n = 5, 10.42%), ST93 (n = 4, 8.33%), ST1426 (n = 4, 8.33%) and ST10 (n = 3, 6.25%), while the dominant ST of InPEC was ST117 (n = 2, 20%). Furthermore, antimicrobial susceptibility tests of 16 antibiotics for those strains were conducted. The result showed that more than 60% of the ExPEC and InPEC were resistant to streptomycin and nalidixic acid. Among these streptomycin resistant isolates (n = 49), 99.76% harbored aminoglycoside resistance gene strA, and 63.27% harbored strB. Among these nalidixic acid resistant isolates (n = 38), 94.74% harbored a S83L mutation in gyrA, and 44.74% harbored a D87N mutation in gyrA. Moreover, the prevalence of multidrug-resistant (MDR) in the isolates of ExPEC and InPEC was 31.25% (15/48) and 20% (2/10), respectively. Alarmingly, 8.33% (4/48) of the ExPEC and 20% (2/10) of the InPEC were extensively drug-resistant (XDR). Finally, the presence of 13 virulence-associated genes was checked in these isolates, which over 95% of the ExPEC and InPEC strains harbored irp2, feoB, fimH, ompT, ompA. 10.42% of the ExPEC and 10% of the InPEC were positive for kpsM. Only ExPEC isolates carried ibeA gene, and the rate was 4.17%. All tested strains were negative to LT and cnf genes. The carrying rate of iss and iutA were significantly different between the InPEC and ExPEC isolates (P < 0.01). CONCLUSIONS: To the best of our knowledge, this is the first report on the highly pathogenic groups of InPEC and ExPEC in Central China. We find that 50.53% (48/95) of the ExPEC belong to the D/B2 phylogenic group. The emergence of XDR and MDR strains and potential virulence genes may indicate the complicated treatment of the infections caused by APEC. This study will improve our understanding of the prevalence and pathogenicity of APEC.

Characterization of Genetic Elements Carrying mcr-1 Gene in Escherichia coli from the Community and Hospital Settings in Vietnam.

Colistin is widely used in agriculture and aquaculture as prophylaxis, particularly in Asia. Recently, mcr-1 and other mobilizable genes conferring colistin resistance have spread globally in community and hospital populations. Characterizing mcr-1 mobile genetic elements and host genetic background is important to understand the transmission of this resistance mechanism. We conducted whole-genome sequencing of 94 mcr-1-positive Escherichia coli isolates (Mcr1-Ec isolates) from human and animal feces, food, and water in a community cohort (N = 87) and from clinical specimens from a referral hospital (N = 7) in northern Vietnam. mcr-1 was plasmid-borne in 71 and chromosomally carried in 25 (2 isolates contain one copy on chromosome and one copy on a plasmid) of 94 E. coli isolates from the community and hospital settings. All seven clinical isolates carried mcr-1 on plasmids. Replicon types of mcr-1-carrying plasmids included IncI2, IncP, IncX4, and IncFIA single replicons and combinations of IncHI2, IncN, and IncX1 multireplicons. Alignment of a long-read sequence of an IncI2 plasmid from animal feces with short-read sequences of IncI2 plasmids from a healthy human, water, and hospitalized patients showed highly similar structures (query cover from 90% to 98%, overall identity of >81%). We detected the potential existence of multireplicon plasmids harboring mcr-1 regardless of sample setting, confirming 10/71 with long-read sequencing. An intact/conserved Tn6330 transposon sequence or its genetic context variants were found in 6/25 Mcr1-Ec isolates with chromosomally carried mcr-1. The dissemination of mcr-1 is facilitated by a high diversity of plasmid replicon types and a high prevalence of the chromosomal Tn6330 transposon. IMPORTANCE The article presented advances our understanding of genetic elements carrying mcr-1 in Escherichia coli in both community and hospital settings. We provide evidence to suggest that diverse plasmid types, including multireplicon plasmids, have facilitated the successful transmission of mcr-1 in different reservoirs. The widespread use of colistin in agriculture, where a high diversity of bacteria are exposed, has allowed the selection and evolution of various transmission mechanisms that will make it a challenge to get rid of. Colocalization of mcr-1 and other antibiotic resistance genes (ARGs) on multireplicon plasmids adds another layer of complexity to the rapid dissemination of mcr-1 genes among community and hospital bacterial populations and to the slow pandemic of antimicrobial resistance (AMR) in general.

Outbreak of NDM-1-Producing Escherichia coli in a Coronavirus Disease 2019 Intensive Care Unit in a Mexican Tertiary Care Center.

Emergency department areas were repurposed as intensive care units (ICUs) for patients with acute respiratory distress syndrome during the initial months of the coronavirus disease 2019 (COVID-19) pandemic. We describe an outbreak of New Delhi metallo-ß-lactamase 1 (NDM-1)-producing Escherichia coli infections in critically ill COVID-19 patients admitted to one of the repurposed units. Seven patients developed infections (6 ventilator-associated pneumonia [VAP] and 1 urinary tract infection [UTI]) due to carbapenem-resistant E. coli, and only two survived. Five of the affected patients and four additional patients had rectal carriage of carbapenem-resistant E. coli. The E. coli strain from the affected patients corresponded to a single sequence type. Rectal screening identified isolates of two other sequence types bearing blaNDM-1. Isolates of all three sequence types harbored an IncFII plasmid. The plasmid was confirmed to carry blaNDM-1 through conjugation. An outbreak of clonal NDM-1-producing E. coli isolates and subsequent dissemination of NDM-1 through mobile elements to other E. coli strains occurred after hospital conversion during the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic. This emphasizes the need for infection control practices in surge scenarios. IMPORTANCE The SARS-CoV-2 pandemic has resulted in a surge of critically ill patients. Hospitals have had to adapt to the demand by repurposing areas as intensive care units. This has resulted in high workload and disruption of usual hospital workflows. Surge capacity guidelines and pandemic response plans do not contemplate how to limit collateral damage from issues like hospital-acquired infections. It is vital to ensure quality of care in surge scenarios.

Antibiotic resistance, virulence, and phylogenetic analysis of Escherichia coli strains isolated from free-living birds in human habitats.

Wild birds can be colonized by bacteria, which are often resistant to antibiotics and have various virulence profiles. The aim of this study was to analyze antibiotic resistance mechanisms and virulence profiles in relation to the phylogenetic group of E. coli strains that were isolated from the GI tract of wildfowl. Out of 241 faecal samples, presence of E. coli resistant to a cephalosporin (ESBL/AmpC) was estimated for 33 isolates (13,7%). Based on the analysis of the coexistence of 4 genes encoding ESBLs/AmpC (blaCTX-M, blaTEM, blaSHV, blaAmpC) and class 1 and 2 integrons genes (intI1, intI2) a subset of two resistance profiles was observed among the investigated E. coli isolates carrying blaAmpC, blaSHV, and blaCTX-M, blaTEM, class 1 and 2 integrons, respectively. The E. coli isolates were categorized into 4 phylogenetic groups A (39.4%), B2 (24.25%), D (24.25%) and B1 (12.1%). The pathogenic B2 and D groups were mainly typical for the Laridae family. Among the 28 virulence factors (Vfs) detected in pathogenic phylogenetic groups B2 and D, 7 were exclusively found in those groups (sfa, vat, tosA, tosB, hly, usp, cnf), while 4 VFs (fecA, fyuA, irp2, kspMTII) showed a statistically significant association (P≤0.05) with phylogroups A and B1. Our results indicated that strains belonging to commensal phylogroups A/B1 possess extensive iron acquisition systems (93,9%) and autotransporters (60,6%), typical for pathogens, hence we suggest that these strains evolve towards higher levels of virulence. This study, which is a point assessment of the virulence and drug resistance potential of wild birds, confirms the importance of taking wild birds as a reservoir of strains that pose a growing threat to humans. The E. coli analyzed in our study derive from different phylogenetic groups and possess an arsenal of antibiotic resistance genes and virulence factors that contribute to their ability to cause diseases.

Hackflex: low-cost, high-throughput, Illumina Nextera Flex library construction.

We developed a low-cost method for the production of Illumina-compatible sequencing libraries that allows up to 14 times more libraries for high-throughput Illumina sequencing to be generated for the same cost. We call this new method Hackflex. The quality of library preparation was tested by constructing libraries from Escherichia coli MG1655 genomic DNA using either Hackflex, standard Nextera Flex (recently renamed as Illumina DNA Prep) or a variation of standard Nextera Flex in which the bead-linked transposase is diluted prior to use. In order to test the library quality for genomes with a higher and a lower G+C content, library construction methods were also tested on Pseudomonas aeruginosa PAO1 and Staphylococcus aureus ATCC 25923, respectively. We demonstrated that Hackflex can produce high-quality libraries and yields a highly uniform coverage, equivalent to the standard Nextera Flex kit. We show that strongly size-selected libraries produce sufficient yield and complexity to support de novo microbial genome assembly, and that assemblies of the large-insert libraries can be much more contiguous than standard libraries without strong size selection. We introduce a new set of sample barcodes that are distinct from standard Illumina barcodes, enabling Hackflex samples to be multiplexed with samples barcoded using standard Illumina kits. Using Hackflex, we were able to achieve a per-sample reagent cost for library prep of A$7.22 (Australian dollars) (US $5.60; UK £3.87, £1=A$1.87), which is 9.87 times lower than the standard Nextera Flex protocol at advertised retail price. An additional simple modification and further simplification of the protocol by omitting the wash step enables a further price reduction to reach an overall 14-fold cost saving. This method will allow researchers to construct more libraries within a given budget, thereby yielding more data and facilitating research programmes where sequencing large numbers of libraries is beneficial.

Molecular characteristics of Escherichia coli from bulk tank milk in Korea.

BACKGROUND: Escherichia coli, which causes subclinical or clinical mastitis in cattle, is responsible for transmitting antimicrobial resistance via human consumption of raw milk or raw milk products. OBJECTIVES: The objective of this study was to investigate the molecular characteristics of 183 E. coli from bulk tank milk of five different dairy factories in Korea. METHODS: The molecular characteristics of E. coli such as serogroup, virulence, antimicrobial resistance, and integron genes were detected using polymerase chain reaction and antimicrobial susceptibility were tested using the disk diffusion test. RESULTS: In the distribution of phylogenetic groups, group D was the most prevalent (59.6%) and followed by group B1 (25.1%). The most predominant serogroup was O173 (15.3%), and a total of 46 different serotypes were detected. The virulence gene found most often was fimH (73.2%), and stx1, fimH, incC, fyuA, and iutA genes were significantly higher in isolates of phylogenetic group B1 compared to phylogenetic groups A, B2, and D (p < 0.05). Among 64 E. coli isolates that showed resistance to at least one antimicrobial, the highest resistance rate was observed for tetracyclines (37.5%). All 18 integron-positive E. coli carried the integron class I (int1) gene, and three different gene cassette arrangements, dfrA12+aadA2 (2 isolates), aac(6')-Ib3+aac(6')-Ib-cr+aadA4 (2 isolates), and dfrA17+aadA5 (1 isolate) were detected. CONCLUSIONS: These data suggest that the E. coli from bulk tank milk can be an indicator for dissemination of antimicrobial resistance and virulence factors via cross-contamination.

A comparative study on biological activities of different solvent extracts from whole seed, seed coat and cotyledon of two Lathyrus species

Abstract The present study was conducted to assess the phenolic content, and antibacterial and antioxidant activities of Lathyrus L. species. The extraction of phenolic compounds from whole seeds, seed coat and cotyledon of Lathyrus hierosolymitanus Boiss. and Lathyrus annuus L. seeds was performed employing different solvents. Total phenolic content (TPC) was measured by Folin- Ciocalteau assay, while the antioxidant activity was determined by DPPH radical scavenging activity, and reducing power assay. It was found that TPC of extracts ranged from 0.12 mg to 6.53 mg GAE/gdw. For each solvent, seed coat extracts were generally observed to render higher TPC and antioxidant activities. There was a correlation between TPC and antioxidant activity. In addition, all extracts were also examined for their antimicrobial activity against Bacillus cereus, Escherichia coli and Pseudomonas aeruginosa. Methanol extracts showed the highest antibacterial activity which is consistent with TPC, but there was no correlation between TPC and antibacterial activity. Solvents were observed to have effects on gallic acid, caffeic acid, and epicatechin extractions. HPLC analysis results of extracts confirmed methanol and ethanol as preferred solvents for phenolic extraction from Lathyrus sp. Phenolic content in the extracts could be suggested to contribute to their antioxidant and antibacterial activity.

ECTyper: in silico Escherichia coli serotype and species prediction from raw and assembled whole-genome sequence data.

Escherichia coli is a priority foodborne pathogen of public health concern and phenotypic serotyping provides critical information for surveillance and outbreak detection activities. Public health and food safety laboratories are increasingly adopting whole-genome sequencing (WGS) for characterizing pathogens, but it is imperative to maintain serotype designations in order to minimize disruptions to existing public health workflows. Multiple in silico tools have been developed for predicting serotypes from WGS data, including SRST2, SerotypeFinder and EToKi EBEis, but these tools were not designed with the specific requirements of diagnostic laboratories, which include: speciation, input data flexibility (fasta/fastq), quality control information and easily interpretable results. To address these specific requirements, we developed ECTyper (https://github.com/phac-nml/ecoli_serotyping) for performing both speciation within Escherichia and Shigella, and in silico serotype prediction. We compared the serotype prediction performance of each tool on a newly sequenced panel of 185 isolates with confirmed phenotypic serotype information. We found that all tools were highly concordant, with 92-97 % for O-antigens and 98-100 % for H-antigens, and ECTyper having the highest rate of concordance. We extended the benchmarking to a large panel of 6954 publicly available E. coli genomes to assess the performance of the tools on a more diverse dataset. On the public data, there was a considerable drop in concordance, with 75-91 % for O-antigens and 62-90 % for H-antigens, and ECTyper and SerotypeFinder being the most concordant. This study highlights that in silico predictions show high concordance with phenotypic serotyping results, but there are notable differences in tool performance. ECTyper provides highly accurate and sensitive in silico serotype predictions, in addition to speciation, and is designed to be easily incorporated into bioinformatic workflows.

Molecular subtyping for source tracking of Escherichia coli using core genome multilocus sequence typing at a food manufacturing plant.

When harmful bacteria are detected in the final product at a food manufacturing plant, it is necessary to identify and eliminate the source of contamination so that it does not occur again. In the current study, the source of contamination was tracked using core genome multilocus sequence typing (cgMLST) analysis in cases where Escherichia coli was detected in the final product at a food manufacturing plant. cgMLST analysis was performed on 40 strains of E. coli collected from the environment [floor (26 strains), drainage ditch (5 strains), container (4 strains), post-heating production line (1 strain)] and products [final product (3 strains) and intermediate product (1 strain)]. In total, 40 E. coli isolates were classified into 17 genogroups by cgMLST analysis. The 4 E. coli strains isolated from the intermediate and final products were classified into two genogroups (I and II). Certain isolates collected from the environment also belonged to those genogroups, it was possible to estimate the transmission of E. coli in the manufacturing plant. Thus, the dynamics of E. coli in the food manufacturing location were clarified by using cgMLST analysis. In conclusion, our results indicate that cgMLST analysis can be effectively used for hygiene management at food manufacturing locations.

Cluster-specific gene markers enhance Shigella and enteroinvasive Escherichia coli in silico serotyping.

Shigella and enteroinvasive Escherichia coli (EIEC) cause human bacillary dysentery with similar invasion mechanisms and share similar physiological, biochemical and genetic characteristics. Differentiation of Shigella from EIEC is important for clinical diagnostic and epidemiological investigations. However, phylogenetically, Shigella and EIEC strains are composed of multiple clusters and are different forms of E. coli, making it difficult to find genetic markers to discriminate between Shigella and EIEC. In this study, we identified 10 Shigella clusters, seven EIEC clusters and 53 sporadic types of EIEC by examining over 17000 publicly available Shigella and EIEC genomes. We compared Shigella and EIEC accessory genomes to identify cluster-specific gene markers for the 17 clusters and 53 sporadic types. The cluster-specific gene markers showed 99.64% accuracy and more than 97.02% specificity. In addition, we developed a freely available in silico serotyping pipeline named Shigella EIEC Cluster Enhanced Serotype Finder (ShigEiFinder) by incorporating the cluster-specific gene markers and established Shigella and EIEC serotype-specific O antigen genes and modification genes into typing. ShigEiFinder can process either paired-end Illumina sequencing reads or assembled genomes and almost perfectly differentiated Shigella from EIEC with 99.70 and 99.74% cluster assignment accuracy for the assembled genomes and read mapping respectively. ShigEiFinder was able to serotype over 59 Shigella serotypes and 22 EIEC serotypes and provided a high specificity of 99.40% for assembled genomes and 99.38% for read mapping for serotyping. The cluster-specific gene markers and our new serotyping tool, ShigEiFinder (installable package: https://github.com/LanLab/ShigEiFinder, online tool: https://mgtdb.unsw.edu.au/ShigEiFinder/), will be useful for epidemiological and diagnostic investigations.

Genomic insights into the circulation of pandemic fluoroquinolone-resistant extra-intestinal pathogenic Escherichia coli ST1193 in Vietnam.

Extra-intestinal pathogenic Escherichia coli (ExPEC) ST1193, a globally emergent fluoroquinolone-resistant clone, has become an important cause of bloodstream infections (BSIs) associated with significant morbidity and mortality. Previous studies have reported the emergence of fluoroquinolone-resistant ExPEC ST1193 in Vietnam; however, limited data exist regarding the genetic structure, antimicrobial resistance (AMR) determinants and transmission dynamics of this pandemic clone. Here, we performed genomic and phylogenetic analyses of 46 ST1193 isolates obtained from BSIs and healthy individuals in Ho Chi Minh City, Vietnam, to investigate the pathogen population structure, molecular mechanisms of AMR and potential transmission patterns. We further examined the phylogenetic structure of ST1193 isolates in a global context. We found that the endemic E. coli ST1193 population was heterogeneous and highly dynamic, largely driven by multiple strain importations. Several well-supported phylogenetic clusters (C1-C6) were identified and associated with distinct blaCTX-M variants, including blaCTXM-27 (C1-C3, C5), blaCTXM-55 (C4) and blaCTXM-15 (C6). Most ST1193 isolates were multidrug-resistant and carried an extensive array of AMR genes. ST1193 isolates also exhibited the ability to acquire further resistance while circulating in Vietnam. There were phylogenetic links between ST1193 isolates from BSIs and healthy individuals, suggesting these organisms may both establish long-term colonization in the human intestinal tract and induce infections. Our study uncovers factors shaping the population structure and transmission dynamics of multidrug-resistant ST1193 in Vietnam, and highlights the urgent need for local One Health genomic surveillance to capture new emerging ExPEC clones and to better understand the origins and transmission patterns of these pathogens.

Genomic comparisons of Escherichia coli ST131 from Australia.

Escherichia coli ST131 is a globally dispersed extraintestinal pathogenic E. coli lineage contributing significantly to hospital and community acquired urinary tract and bloodstream infections. Here we describe a detailed phylogenetic analysis of the whole genome sequences of 284 Australian ST131 E. coli isolates from diverse sources, including clinical, food and companion animals, wildlife and the environment. Our phylogeny and the results of single nucleotide polymorphism (SNP) analysis show the typical ST131 clade distribution with clades A, B and C clearly displayed, but no niche associations were observed. Indeed, interspecies relatedness was a feature of this study. Thirty-five isolates (29 of human and six of wild bird origin) from clade A (32 fimH41, 2 fimH89, 1 fimH141) were observed to differ by an average of 76 SNPs. Forty-five isolates from clade C1 from four sources formed a cluster with an average of 46 SNPs. Within this cluster, human sourced isolates differed by approximately 37 SNPs from isolates sourced from canines, approximately 50 SNPs from isolates from wild birds, and approximately 52 SNPs from isolates from wastewater. Many ST131 carried resistance genes to multiple antibiotic classes and while 41 (14 %) contained the complete class one integron-integrase intI1, 128 (45 %) isolates harboured a truncated intI1 (462-1014 bp), highlighting the ongoing evolution of this element. The module intI1-dfrA17-aadA5-qacEΔ1-sul1-ORF-chrA-padR-IS1600-mphR-mrx-mphA, conferring resistance to trimethoprim, aminoglycosides, quaternary ammonium compounds, sulphonamides, chromate and macrolides, was the most common structure. Most (73 %) Australian ST131 isolates carry at least one extended spectrum ß-lactamase gene, typically blaCTX-M-15 and blaCTX-M-27. Notably, dual parC-1aAB and gyrA-1AB fluoroquinolone resistant mutations, a unique feature of clade C ST131 isolates, were identified in some clade A isolates. The results of this study indicate that the the ST131 population in Australia carries diverse antimicrobial resistance genes and plasmid replicons and indicate cross-species movement of ST131 strains across diverse reservoirs.

Study of the diversity of 16S-23S rDNA internal transcribed spacer (ITS) typing of Escherichia coli strains isolated from various biotopes in Tunisia.

We investigated the 16S-23S rRNA intergenic spacer region (ISR)-PCR and the phylogenetic PCR analyses of 150 Escherichia coli isolates as tools to explore their diversity, according to their sampling origins, and their relative dominance in these sampling sources. These genetic markers are used to explore phylogenetic and genetic relationships of these 150 E. coli isolates recovered from different environmental sources (water, food, animal, human and vegetables). These isolates are tested for their biochemical pattern and later genotyped through the 16S-23S rRNA intergenic spacer PCR amplification and their polymorphism investigation of PCR-amplified 16S-23S rDNA ITS. The main results of the pattern band profile revealed one to four DNA fragments. Distributing 150 E. coli isolates according to their ITS and using RS-PCR, revealed four genotypes and four subtypes. The DNA fragment size ranged from 450 to 550 bp. DNA band patterns analysis revealed considerable genetic diversity in interspecies. Thus, the 450 and 550 bp sizes of the common bands in all E. coli isolates are highly diversified. Genotype I appeared as the most frequent with 77.3% (116 isolates), genotype II with 12% (18 isolates); genotype III with 9.7% (14 isolates), and the IV rarely occurred with 4% (2 isolates). Distributing the E. coli phylogroups showed 84 isolates (56%) of group A, 35 isolates (23.3%) of group B1, 28 isolates (18.7%) of group B2 and only three isolates (2%) of group D.

Whole-genome sequencing-based phylogeny, antibiotic resistance, and invasive phenotype of Escherichia coli strains colonizing the cervix of women in preterm labor.

BACKGROUND: Escherichia coli is a major neonatal pathogen and the leading cause of early-onset sepsis in preterm newborns. Maternal E. coli strains are transmitted to the newborn causing invasive neonatal disease. However, there is a lack of data regarding the phenotypic and genotypic characterization of E. coli strains colonizing pregnant women during labor. METHODS: This prospective study performed at the University of Oklahoma Medical Center (OUHSC) from March 2014 to December 2015, aimed to investigate the colonization rate, and the phylogeny, antibiotic resistance traits, and invasive properties of E. coli strains colonizing the cervix of fifty pregnant women diagnosed with preterm labor (PTL). Molecular analyses including bacterial whole-genome sequencing (WGS), were performed to examine phylogenetic relationships among the colonizing strains and compare them with WGS data of representative invasive neonatal E. coli isolates. Phenotypic and genotypic antibiotic resistance traits were investigated. The bacteria's ability to invade epithelial cells in vitro was determined. RESULTS: We recruited fifty women in PTL. Cervical samples yielded E. coli in 12 % (n=6). The mean gestational age was 32.5 (SD±3.19) weeks. None delivered an infant with E. coli disease. Phenotypic and genotypic antibiotic resistance testing did not overall demonstrate extensive drug resistance traits among the cervical E. coli isolates, however, one isolate was multi-drug resistant. The isolates belonged to five different phylogroups, and WGS analyses assigned each to individual multi-locus sequence types. Single nucleotide polymorphism-based comparisons of cervical E. coli strains with six representative neonatal E. coli bacteremia isolates demonstrated that only half of the cervical E. coli isolates were phylogenetically related to these neonatal invasive strains. Moreover, WGS comparisons showed that each cervical E. coli isolate had distinct genomic regions that were not shared with neonatal E. coli isolates. Cervical and neonatal E. coli isolates that were most closely related at the phylogenetic level had similar invasion capacity into intestinal epithelial cells. In contrast, phylogenetically dissimilar cervical E. coli strains were the least invasive among all isolates. CONCLUSIONS: This pilot study showed that a minority of women in PTL were colonized in the cervix with E. coli, and colonizing strains were not phylogenetically uniformly representative of E. coli strains that commonly cause invasive disease in newborns. Larger studies are needed to determine the molecular characteristics of E. coli strains colonizing pregnant women associated with an increased risk of neonatal septicemia.

Prenatal versus Postnatal Initial Colonization of Healthy Neonates' Colon Ecosystem by the Enterobacterium Escherichia coli.

The human colon is a microbial ecosystem whose initial bacterial colonization in neonates is an important step in establishing a beneficial microbiota for the body's health. This study investigated the occurrence of viable culturable Escherichia coli in first-day meconium versus subsequent days' stool to explore the prenatal versus postnatal initial colonization of the colon by E. coli in healthy neonates. E. coli occurrence was investigated on eosin-methylene blue (EMB) agar, followed by morphological and biochemical characterizations and phylogenetic analysis of 16S rRNA-encoding gene sequences. Viable culturable E. coli was not detected in meconium of healthy male or female neonates delivered either vaginally or by cesarean section. Neonates delivered surgically also showed no E. coli colonization on the second and third days, confirming postnatal colonization of the colon by this enterobacterium. E. coli's initial colonization in the colon of neonates delivered vaginally occurred on the second day, which can be attributed to inoculation from the vaginal canal during delivery and, in comparison to the colonization in neonates delivered surgically, leads to the inference that the bacterium is not originally found in meconium. This study suggests no viability of the meconium microbiome in healthy neonates, possibly due to antimicrobial action in the prenatal colon's meconium protecting babies' gut from infection during delivery. IMPORTANCE The results of this study suggest that the initial postnatal colonization of neonates' colon by beneficial bacteria is a naturally controlled process in which the prenatal colon's meconium might play a role in protecting against infection of the babies' gut during delivery.

Whole genome sequencing and characteristics of Escherichia coli with co-existence of ESBL and mcr genes from pigs.

This study aimed to analyze three ESBL-producing E. coli co-harboring mcr and ESBL genes from a healthy fattening pig (E. 431) and two sick pigs (ECP.81 and ECP.82) in Thailand using Whole Genome Sequencing (WGS) using either Illumina MiSeq or HiSeq PE150 platforms to determine their genome and transmissible plasmids. E. 431 carrying mcr-2.1 and mcr-3.1 belonged to serotype O142:H31 with ST29 sequence type. ECP.81 and ECP.82 from sick pigs harboring mcr-1.1 and mcr-3.1 were serotype O9:H9 with ST10. Two mcr-1.1 gene cassettes from ECP.81 and ECP.82 were located on IncI2 plasmid with 98% identity to plasmid pHNSHP45. The mcr-2.1-carrying contig in E. 431 showed 100% identity to plasmid pKP37-BE with the upstream flanking sequence of IS1595. All three mcr-3.1-carrying contigs contained the ΔTnAs2-mcr-3.1-dgkA core segment and had high nucleotide similarity (85-100%) to mcr-3.1-carrying plasmid, pWJ1. The mobile elements i.e. IS4321, ΔTnAs2, ISKpn40 and IS3 were identified in the flanking regions of mcr-3. Several genes conferring resistance to aminoglycosides (aac(3)-IIa, aadA1, aadA2b, aph(3'')-Ib, aph(3')-IIa and aph(6)-Id), macrolides (mdf(A)), phenicols (cmlA1), sulphonamide (sul3) and tetracycline (tet(A) and tet(M)) were located on plasmids, of which their presence was well corresponded to the host's resistance phenotype. Amino acid substitutions S83L and D87G in GyrA and S80I and E62K in ParC were observed. The blaCTX-M-14 and blaCTX-M-55 genes were identified among these isolates additionally harbored blaTEM-1B. Co-transfer of mcr-1.1/blaTEM-1B and mcr-3.1/blaCTX-M-55 was observed in ECP.81 and ECP.82 but not located on the same plasmid. The results highlighted that application of advanced innovation technology of WGS in AMR monitoring and surveillance provide comprehensive information of AMR genotype that could yield invaluable benefits to development of control and prevention strategic actions plan for AMR.

Gene Amplification Uncovers Large Previously Unrecognized Cryptic Antibiotic Resistance Potential in E. coli.

The activation of unrecognized antibiotic resistance genes in the bacterial cell can give rise to antibiotic resistance without the need for major mutations or horizontal gene transfer. We hypothesize that bacteria harbor an extensive array of diverse cryptic genes that can be activated in response to antibiotics via adaptive resistance. To test this hypothesis, we developed a plasmid assay to randomly manipulate gene copy numbers in Escherichia coli cells and identify genes that conferred resistance when amplified. We then tested for cryptic resistance to 18 antibiotics and identified genes conferring resistance. E. coli could become resistant to 50% of the antibiotics tested, including chloramphenicol, d-cycloserine, polymyxin B, and 6 beta-lactam antibiotics, following this manipulation. Known antibiotic resistance genes comprised 13% of the total identified genes, where 87% were unclassified (cryptic) antibiotic resistance genes. These unclassified genes encoded cell membrane proteins, stress response/DNA repair proteins, transporters, and miscellaneous or hypothetical proteins. Stress response/DNA repair genes have a broad antibiotic resistance potential, as this gene class, in aggregate, conferred cryptic resistance to nearly all resistance-positive antibiotics. We found that antibiotics that are hydrophilic, those that are amphipathic, and those that inhibit the cytoplasmic membrane or cell wall biosynthesis were more likely to induce cryptic resistance in E. coli. This study reveals a diversity of cryptic genes that confer an antibiotic resistance phenotype when present in high copy number. Thus, our assay can identify potential novel resistance genes while also describing which antibiotics are prone to induce cryptic antibiotic resistance in E. coli. IMPORTANCE Predicting where new antibiotic resistance genes will rise is a challenge and is especially important when new antibiotics are developed. Adaptive resistance allows sensitive bacterial cells to become transiently resistant to antibiotics. This provides an opportune time for cells to develop more efficient resistance mechanisms, such as tolerance and permanent resistance to higher antibiotic concentrations. The biochemical diversity harbored within bacterial genomes may lead to the presence of genes that could confer resistance when timely activated. Therefore, it is crucial to understand adaptive resistance to identify potential resistance genes and prolong antibiotics. Here, we investigate cryptic resistance, an adaptive resistance mechanism, and identify unknown (cryptic) antibiotic resistance genes that confer resistance when amplified in a laboratory strain of E. coli. We also pinpoint antibiotic characteristics that are likely to induce cryptic resistance. This study may help detect novel antibiotic resistance genes and provide the foundation to help develop more effective antibiotics.