Resistance phenotype and virulence potential of Leclercia adecarboxylata strains isolated from different sources.

Introduction. Leclercia adecarboxylata is a member of Enterobacterales, often considered an opportunistic pathogen. Recent reports have highlighted L. adecarboxylata as an emerging pathogen harbouring virulence and resistance determinants.Gap statement. Little information exists on virulence and resistance determinants in L. adecarboxylata strains isolated from environmental, food, and clinical samples.Aim. To determine the presence of resistance and virulence determinants and plasmid features in L. adecarboxylata strains isolated from environmental, food, and clinical samples, as well as their phylogenetic relationship.Results. All strains tested showed resistance to ß-lactams and quinolones but were sensitive to aminoglycosides and nitrofurans. However, even though fosfomycin resistance is considered a characteristic trait of L. adecarboxylata, the resistance phenotype was only observed in 50 % of the strains; bla TEM was the most prevalent BLEE gene (70 %), while the quinolone qnrB gene was observed in 60 % of the strains. Virulence genes were differentially observed in the strains, with adhesion-related genes being the most abundant, followed by toxin genes. Finally, all strains carried one to seven plasmid bands ranging from 7 to 125 kbps and harboured several plasmid addiction systems, such as ParDE, VagCD, and CcdAB in 80 % of the strains.Conclusions. L. adecarboxylata is an important emerging pathogen that may harbour resistance and virulence genes. Additionally, it has mobilizable genetic elements that may contribute to the dissemination of genetic determinants to other bacterial genera.

Apirhabdus apintestini gen. nov., sp. nov., a member of a novel genus of the family Enterobacteriaceae, isolated from the gut of the western honey bee Apis mellifera.

A Gram-negative, motile, rod-shaped bacterial strain, CA-0114T, was isolated from the midgut of a western honey bee, Apis mellifera. The isolate exhibited ≤96.43 % 16S rRNA gene sequence identity (1540 bp) to members of the families Enterobacteriaceae and Erwiniaceae. Phylogenetic trees based on genome blast distance phylogeny and concatenated protein sequences encoded by conserved genes atpD, fusA, gyrB, infB, leuS, pyrG and rpoB separated the isolate from other genera forming a distinct lineage in the Enterobacteriaceae. In both trees, the closest relatives were Tenebrionicola larvae YMB-R21T and Tenebrionibacter intestinalis BIT-L3T, which were isolated previously from Tenebrio molitor L., a plastic-eating mealworm. Digital DNA-DNA hybridization, orthologous average nucleotide identity and average amino acid identity values between strain CA-0114T and the closest related members within the Enterobacteriaceae were ≤23.1, 75.45 and 76.04 %, respectively. The complete genome of strain CA-0114T was 4 451669 bp with a G+C content of 52.12 mol%. Notably, the apparent inability of strain CA-0114T to ferment d-glucose, inositol and l-rhamnose in the API 20E system is unique among closely related members of the Enterobacteriaceae. Based on the results obtained through genotypic and phenotypic analysis, we propose that strain CA-0114T represents a novel species and genus within the family Enterobacteriaceae, for which we propose the name Apirhabdus apintestini gen. nov., sp. nov. (type strain CA-0114T=ATCC TSD-396T=DSM 116385T).

Diverse gut pathogens exploit the host engulfment pathway via a conserved mechanism.

Macrophages clear infections by engulfing and digesting pathogens within phagolysosomes. Pathogens escape this fate by engaging in a molecular arms race; they use WxxxE motif-containing "effector" proteins to subvert the host cells they invade and seek refuge within protective vacuoles. Here, we define the host component of the molecular arms race as an evolutionarily conserved polar "hot spot" on the PH domain of ELMO1 (Engulfment and Cell Motility protein 1), which is targeted by diverse WxxxE effectors. Using homology modeling and site-directed mutagenesis, we show that a lysine triad within the "patch" directly binds all WxxxE effectors tested: SifA (Salmonella), IpgB1 and IpgB2 (Shigella), and Map (enteropathogenic Escherichia coli). Using an integrated SifA-host protein-protein interaction network, in silico network perturbation, and functional studies, we show that the major consequences of preventing SifA-ELMO1 interaction are reduced Rac1 activity and microbial invasion. That multiple effectors of diverse structure, function, and sequence bind the same hot spot on ELMO1 suggests that the WxxxE effector(s)-ELMO1 interface is a convergence point of intrusion detection and/or host vulnerability. We conclude that the interface may represent the fault line in coevolved molecular adaptations between pathogens and the host, and its disruption may serve as a therapeutic strategy.

The Complete Genome Sequence of a Gossypol-Degrading Bacterial Strain, Raoultella sp. YL01.

Cottonseed meal is an important source of plant protein for the meal fodder materials. But its usage in animal breeding industry is limited by a type of toxic phenol, gossypol, that has toxic effects on animal health. Microbial degradation is a promising way to lower down gossypol in cottonseed meal. However, the molecular mechanisms of bio-degradation of gossypol is still unclear. In this study we isolated a gossypol-degrading bacterial strain, YL01, and sequenced its complete genome via Oxford Nanopore sequencing method. There is a chromosome (5,737,005 bp) and a plasmid (136,446 bp) in YL01. 5489 protein coding genes in total were functionally annotated. 16S rRNA analysis showed that YL01 taxonomically belongs to the genus of Raoultella. YL01 is the first published complete genome sequence of microbes capable of gossypol degradation. Gene function annotation showed that 126 protein coding genes may involve in gossypol catabolism. Sequence similarity analysis showed that, as the only gossypol-degrading strain in the genus of Raoultella, YL01 uniquely holds 260 genes that are not possessed by other Raoultella strains. Our work gives a preliminary list for genes responsible for gossypol degradation but further investigations are needed to completely disclose this molecular processes.

De novo sequencing, diploid assembly, and annotation of the black carpenter ant, Camponotus pennsylvanicus, and its symbionts by one person for $1000, using nanopore sequencing.

The black carpenter ant (Camponotus pennsylvanicus) is a pest species found widely throughout North America. From a single individual I used long-read nanopore sequencing to assemble a phased diploid genome of 306 Mb and 60X coverage, with quality assessed by a 97.0% BUSCO score, improving upon other ant assemblies. The mitochondrial genome reveals minor rearrangements from other ants. The reads also allowed assembly of parasitic and symbiont genomes. I include a complete Wolbachia bacterial assembly with a size of 1.2 Mb, as well as a commensal symbiont Blochmannia pennsylvanicus, at 791 kb. DNA methylation and hydroxymethylation were measured at base-pair resolution level from the same reads and confirmed extremely low levels seen in the Formicidae family. There was moderate heterozygosity, with 0.16% of bases being biallelic from the parental haplotypes. Protein prediction yielded 14 415 amino acid sequences with 95.8% BUSCO score and 86% matching to previously known proteins. All assemblies were derived from a single MinION flow cell generating 20 Gb of sequence for a cost of $1047 including consumable reagents. Adding fixed costs for equipment brings the total for an ant-sized genome to less than $5000. All analyses were performed in 1 week on a single desktop computer.

Creating reference animal genomes is typically a large, expensive process. Here I sequenced the genome of the black carpenter ant for only $1000 as a sole researcher in just one week. Along with the nuclear genome, I assembled the mitochondrial genome and two commensal bacteria species living within the ant. Nanopore technology also enabled epigenetic measurements from the same ant and replicated other studies showing very low DNA methylation. The reference genome compared favorably to other ant species in continuity and protein prediction accuracy. This method will allow other low-resource labs to create high quality genome assemblies with a low cost.

Impact of Empirical Antibiotic Therapy on Outcomes of Outpatient Urinary Tract Infection Due to Nonsusceptible Enterobacterales.

Resistance to oral antibiotics commonly used to treat outpatient urinary tract infections (UTIs) is increasing, but the implications of empirical treatment of resistant pathogens are not well described. Using an electronic records database, we reviewed the outcomes of patients >18 years of age who developed an outpatient UTI and had an outpatient urine culture result showing a member of the order Enterobacterales along with prescription data for an oral antibiotic filled on the day before, day of, or day after the culture was collected. Linear probability models were used to estimate partial effects of select clinical and demographic variables on the composite outcome. In all, 4,792 patients had 5,587 oral antibiotic prescriptions. Of 5,395 evaluable episodes, 22% of patients received an antibiotic to which the pathogen was resistant in vitro, and those patients were almost twice as likely to require a second prescription (34% versus 19%) or be hospitalized (15% versus 8%) within 28 days of the initial prescription fill compared to patients who received an antibiotic to which the pathogen was susceptible. Approximately 1% of Enterobacterales isolates were resistant to all commonly available classes of oral antibiotics. A greater risk of treatment failure was seen in patients over 60 years of age, patients with diabetes mellitus, men, and those treated with an antibiotic when prior culture identified an organism resistant to that class. The increasing resistance among members of Enterobacterales responsible for outpatient UTIs is limiting the effectiveness of empirical treatment with existing antibiotics, and consequently, outpatients with UTI are more likely to require additional courses of therapy or be hospitalized. IMPORTANCE Resistance rates for bacteria that cause urinary tract infections (UTIs) have increased dramatically. Regional rates of resistance to commonly prescribed antibiotics now exceed 20%, which is the threshold at which the Infectious Diseases Society of America recommends therapy be guided by culture. Our goals were to describe outcomes for outpatients with UTIs caused by bacteria resistant to empirically chosen antibiotics and to create a simple stratification schema for clinicians to identify UTI patients at increased risk of treatment failure due to antibiotic mismatch. These data are relevant to clinicians, given how common uncomplicated UTIs are, and highlight the need for clinicians to understand local resistance rates and the importance of culture-guided treatment, especially in vulnerable patients. These findings also showed that 1% of bacteria were resistant to all major classes of oral antibiotics, underscoring the need for new antibiotics to treat patients with UTIs due to resistant bacteria.

Tenebrionibacter intestinalis gen. nov., sp. nov., a member of a novel genus of the family Enterobacteriaceae, isolated from the gut of the plastic-eating mealworm Tenebrio molitor L.

A Gram-negative, white-pigmented, motile and rod-shaped strain, BIT-L3T, was isolated from the gut of plastic-eating mealworm Tenebrio molitor L. Its taxonomic position was determined by using a polyphasic approach. A preliminary analysis based on the 16S rRNA gene sequence (1445 bp) revealed that this strain was closely related to the members within the family Enterobacteriaceae. Phylogenetic trees based on the concatenated partial sequences of seven housekeeping genes (atpD, gyrB, infB, rpoB, pyrG, fusA, leuS) and genome sequences further showed that strain BIT-L3T constituted a separate lineage within the family Enterobacteriaceae. In silico DNA-DNA hybridization values and average nucleotide identity values between strain BIT-L3T and its closest related species within the family Enterobacteriaceae were less than 21.8 and 76.7 %, respectively. The major fatty acids (>5 %) of strain BIT-L3T were C16 : 0, C14 : 0, C17 : 0 cyclo, summed feature 8 (comprising C18 : 1 ω7c and/or C18 : 1 ω6c), summed feature 3 (comprising C16 : 1 ω7c and/or C16 : 1 ω6c and/or iso-C15 : 0 2-OH) and summed feature 2 (comprising iso-C16 : 1 I/C14 : 0 3-OH and/or C12 : 0 aldehyde and/or an unknown fatty acid of equivalent chain length 10.9525). Its genomic DNA G+C content was 53.7 mol%. Based on the results of phylogenetic, physiological and biochemical analyses, strain BIT-L3T is considered to represent a novel species of a novel genus within the family Enterobacteriaceae, for which the name Tenebrionibacter intestinalis gen. nov., sp. nov. is proposed. The type strain is BIT-L3T (=CCTCC AB 2020371T=LMG 32222T=TBRC 14825T).

Prior Carriage Predicts Intensive Care Unit Infections Caused by Extended-Spectrum Beta-Lactamase-Producing Enterobacteriaceae.

Intensive care unit-acquired infection (ICU-AI) and extended-spectrum beta-lactamase-producing Enterobacteriaceae (ESBL-PE) carriage are a major concern worldwide. Our objective was to investigate the impact of ESBL-PE carriage on ICU-AI. Our study was prospective, observational, and noninterventional. It was conducted over a 5-year period (Jan 2013-Dec 2017) in the medical-surgical intensive care unit of the Cayenne General Hospital (French Amazonia). During the study period, 1,340 patients were included, 271 (20.2%) developed ICU-AI, and 16.2% of these were caused by ESBL-PE. The main sites of ICU-AI were ventilator-associated pneumonia (35.8%) and primary bloodstream infection (29.8%). The main responsible microorganisms were Staphylococcus aureus, Pseudomonas aeruginosa, Klebsiella pneumoniae (ESBL-P in 35.8% of isolates), and Enterobacter cloacae (ESBL-P in 29.8% of isolates). Prior ESBL-PE carriage was diagnosed in 27.6% of patients with ICU-AI. In multivariable analysis, the sole factor associated with ESBL-PE as the responsible organism of ICU-AI was ESBL-PE carriage before ICU-AI (P < 0.001; odds ratio: 7.9 95% CI: 3.4-18.9). ESBL-PE carriers (74 patients) developed ICU-AI which was caused by ESBL-PE in 32 cases (43.2%). This proportion of patients carrying ESBL-PE who developed ICU-AI to the same microorganism was 51.2% in ESBL-P K. pneumoniae, 5.6% in ESBL-P Escherichia coli, and 40% in ESBL-P Enterobacter spp. NPV of ESBL-PE carriage to predict ICU-AI caused by ESBL-PE was above 94% and PPV was above 43%. Carriage of ESBL-P K pneumoniae and Enterobacter spp. is a strong predictor of ICU-AI caused by these two microorganisms.

Phylogenomic analysis of the Erwiniaceae supports reclassification of Kalamiella piersonii to Pantoea piersonii comb. nov. and Erwinia gerundensis to the new genus Duffyella gen. nov. as Duffyella gerundensis comb. nov.

To better understand the taxonomy of Erwinia in the context of the Erwiniaceae family, we carried out a taxogenomic analysis of the Erwiniaceae, a family that was created following the taxonomic revision of the family, Enterobacteriaceae. There has been no systematic analysis of this family, including the agriculturally relevant genus, Erwinia. Our analyses focused on 80 strains of Erwinia along with 37 strains representing 7 other genera in the family. We identified 308 common proteins, generated a genome-level phylogeny and carried out Average Nucleotide Identity, Average Amino Acid Identity and Percentage of Conserved Protein analyses. We show that multiple strains of Erwinia cannot be assigned to established species groups and that both Erwinia gerundensis and "Erwinia mediterraneensis" are not members of Erwinia. We propose the creation of the genus Duffyella gen. nov. and the reclassification of Erwinia gerundensis to this genus as the type species, Duffyella gerundensis comb. nov. Furthermore, divergence between other species within Erwinia as measured by Average Amino Acid Identity is greater than the divergence between Erwinia and other genera, supporting the possible subdivision of the genus Erwinia into at least two genera. Our analyses also suggest that there is no basis for the establishment of the genus Kalamiella within the Erwiniaceae or the taxonomic revision of the Pantoea septica lineage. Therefore, we propose reclassifying Kalamiella piersonii as Pantoea piersonii comb. nov. Our study provides new insight into the diversity of the Erwiniaceae and provides a solid foundation for advancing taxonomic revision of this broadly relevant family.

Intestinal gluconeogenesis shapes gut microbiota, fecal and urine metabolome in mice with gastric bypass surgery.

Intestinal gluconeogenesis (IGN), gastric bypass (GBP) and gut microbiota positively regulate glucose homeostasis and diet-induced dysmetabolism. GBP modulates gut microbiota, whether IGN could shape it has not been investigated. We studied gut microbiota and microbiome in wild type and IGN-deficient mice, undergoing GBP or not, and fed on either a normal chow (NC) or a high-fat/high-sucrose (HFHS) diet. We also studied fecal and urine metabolome in NC-fed mice. IGN and GBP had a different effect on the gut microbiota of mice fed with NC and HFHS diet. IGN inactivation increased abundance of Deltaproteobacteria on NC and of Proteobacteria such as Helicobacter on HFHS diet. GBP increased abundance of Firmicutes and Proteobacteria on NC-fed WT mice and of Firmicutes, Bacteroidetes and Proteobacteria on HFHS-fed WT mice. The combined effect of IGN inactivation and GBP increased abundance of Actinobacteria on NC and the abundance of Enterococcaceae and Enterobacteriaceae on HFHS diet. A reduction was observed in the amounf of short-chain fatty acids in fecal (by GBP) and in both fecal and urine (by IGN inactivation) metabolome. IGN and GBP, separately or combined, shape gut microbiota and microbiome on NC- and HFHS-fed mice, and modify fecal and urine metabolome.

Co-occurrence of gut microbiota dysbiosis and bile acid metabolism alteration is associated with psychological disorders in Crohn's disease.

This study aims to elucidate the relationships between gut microbiota, bile acid metabolism, and psychological comorbidity in Crohn's disease (CD). We profiled the fecal microbiota composition and quantified the bile acid pool of 39 CD patients and 14 healthy controls using 16S rRNA gene sequencing and liquid chromatography-tandem mass spectrometry, respectively. Significant reductions in the secondary bile acids, LCA and DCA, were found in both the feces and serum samples of CD patients, while the concentration of 7-DHCA was particularly higher in the serum of CD patients with psychological disorders. The fecal levels of HDCA and 12-DHCA of the CD patients were inversely correlated with their Self-Rated Depression Scale (SDS) scores, whereas the serum level of 7-DHCA was positively correlated with the SDS scores. In addition, the fecal levels of TDCA, TLCA, and TßMCA showed a positive correlation with the Self-Rated Anxiety Scale (SAS) scores. The fecal microbiota biodiversity was particularly declined in CD patients with psychological disorders. An enrichment of Ruminococcus gnavus in CD patients may cause psychological disorders by affecting the microbiota-gut-brain axis via its ability to degrade the gut barrier, regulate the tryptophan-kynurenine metabolism, and modulate bile acid metabolism. In addition, the overabundant Enterobacteriaceae and Lachnospiraceae in CD patients may contribute to psychological comorbidity via dysregulating their bile acids metabolism. Taken together, changes in the gut microbiota composition may cooperate with alterations in the bile acid metabolism that are involved in the development of psychological disorders in CD.

Appropriateness of empirical antibiotic prescription for bloodstream infections in an emergency department from 2006 to 2018: impact of the spread of ESBL-producing Enterobacterales.

The spread of ESBL producers in the community may impact the management of patients with bloodstream infections (BSI) involving Enterobacterales in emergency departments. Thus, from 2006 to 2018, data for all BSI episodes involving Enterobacterales from the emergency department of a French teaching hospital were retrospectively included. Antimicrobial susceptibility test results and empirical antibiotic regimens were recorded. Treatment was considered as appropriate if all isolates were susceptible in vitro to at least one prescribed antibiotic. A total of 1369 BSI episodes in 1321 patients was included. Urinary tract infection was the main source of BSI (61%). The prevalence of ESBL producers increased from zero to 9.2/100 Enterobacterales BSI cases (p < 0.001), mainly Escherichia coli (6.9 cases/100 BSI in 2018); and no Klebsiella. Third-generation cephalosporins (3GC) were used most frequently (71.8%) and their use as monotherapy increased during the study period (p < 0.001). The rate of appropriate treatment decreased from 95.8 to 89.2% (p = 0.023). Appropriateness of treatment was greater using two drugs vs one (97.3% vs 89.3%, p < 0.001). Treatments with 3GC were appropriate in 92% and 98.3%, when used alone or with another antibiotic, respectively (p < 0.001). Among inappropriate treatments, 45% concerned 3GC, with 74.6% of them attributable to ESBL production. The spread of ESBL producers in the community had a direct impact on the rate of inappropriate empirical treatment. Local antimicrobial resistance monitoring is required to optimize the management of BSI in emergency departments.

Analysis of migration of pathogenic drug-resistant bacteria to soils and groundwater after fertilization with sewage sludge.

The paper discusses the analysis of the effect of using sewage sludge for fertilization on the level of soil and groundwater contamination with drug-resistant bacteria. Other sanitary contaminants in these environments were also analysed. Composted sewage sludge was introduced into the sandy soil over a period of 6 months. The examinations were conducted under conditions of a lysimetric experiment with the possibility of collecting soil leachates (in natural conditions). The following doses of sewage sludge were used: 0, 10, 20, 30 and 40 t/ha calculated per experimental object containing 10 kg of sandy soil. The research were carried out within the time frame of one year. Dactylis glomerata grass was grown on the fertilized soils. In soils and leachates from soils (which may have polluted groundwater) collected from fertilized experimental objects, the sanitary condition and quantity of drug-resistant bacteria (mainly from the families Enterobacteriaceae and Enterococcus) were analysed one year after fertilization. Their drug resistance to selected antibiotics was also analysed based on current recommendations. The study showed that fertilization with sewage sludge (even after stabilization and hygienization) results in contamination of soil and infiltrating waters with many species of drug-resistant pathogenic bacteria. The lowest level of contamination of soil and water environment was found after the application of sewage sludge at a dose of 10 t/ha. The isolated drug-resistant strains of intestinal bacteria were less sensitive to older generations of antibiotics including cefazolin, ampicillin, and co-amoxiclav.

The molecular mechanisms of fluoroquinolone resistance found in rectal swab isolates of Enterobacterales from men undergoing a transrectal prostate biopsy: the rationale for targeted prophylaxis.

BACKGROUND: Transrectal ultrasound-guided prostate biopsy (TRUS-Bx) is considered an essential urological procedure for the histological diagnosis of prostate cancer. It is, however, considered a "contaminated" procedure which may lead to infectious complications. Recent studies suggest a significant share of fluoroquinolone-resistant rectal flora in post-biopsy infections. METHODS: The molecular mechanisms of fluoroquinolone resistance, including PMQR (plasmid-mediated quinolone resistance) as well as mutation in the QRDRs (quinolone-resistance determining regions) of gyrA, gyrB, parC and parE, among Enterobacterales isolated from 32 of 48 men undergoing a prostate biopsy between November 2015 and April 2016 were investigated. Before the TRUS-Bx procedure, all the patients received an oral antibiotic containing fluoroquinolones. RESULTS: In total, 41 Enterobacterales isolates were obtained from rectal swabs. The MIC of ciprofloxacin and the presence of common PMQR determinants were investigated in all the isolates. Nine (21.9%) isolates carried PMQR with qnrS as the only PMQR agent detected. DNA sequencing of the QRDRs in 18 Enterobacterales (E. coli n = 17 and E. cloacae n = 1) isolates with ciprofloxacin MIC ≥ 0.25 mg/l were performed. Substitutions in the following codons were found: GyrA-83 [Ser → Leu, Phe] and 87 [Asp → Asn]; GyrB codon-605 [Met → Leu], ParC codons-80 [Ser → Ile, Arg] and 84 [Glu → Gly, Met, Val, Lys], ParE codons-458 [Ser → Ala], 461 [Glu → Ala] and 512 [Ala → Thr]. Six isolates with ciprofloxacin MIC ≥ 2 mg/l had at least one mutation in GyrA together with qnrS. CONCLUSIONS: This study provides information on the common presence of PMQRs among Enterobacterales isolates with ciprofloxacin MIC ≥ 0.25 mg/l, obtained from men undergoing TRUS-Bx. This fact may partially explain why some men develop post-TRUS-Bx infections despite ciprofloxacin prophylaxis.

Psychological stress impairs IL22-driven protective gut mucosal immunity against colonising pathobionts.

Crohn's disease is an inflammatory disease of the gastrointestinal tract characterized by an aberrant response to microbial and environmental triggers. This includes an altered microbiome dominated by Enterobacteriaceae and in particular adherent-invasive E. coli (AIEC). Clinical evidence implicates periods of psychological stress in Crohn's disease exacerbation, and disturbances in the gut microbiome might contribute to the pathogenic mechanism. Here we show that stress-exposed mice develop ileal dysbiosis, dominated by the expansion of Enterobacteriaceae. In an AIEC colonisation model, stress-induced glucocorticoids promote apoptosis of CD45+CD90+ cells that normally produce IL-22, a cytokine that is essential for the maintenance of ileal mucosal barrier integrity. Blockade of glucocorticoid signaling or administration of recombinant IL-22 restores mucosal immunity, prevents ileal dysbiosis, and blocks AIEC expansion. We conclude that psychological stress impairs IL-22-driven protective immunity in the gut, which creates a favorable niche for the expansion of pathobionts that have been implicated in Crohn's disease. Importantly, this work also shows that immunomodulation can counteract the negative effects of psychological stress on gut immunity and hence disease-associated dysbiosis.

A small RNA is functional in Escherichia fergusonii despite containing a large insertion.

Bacterial small RNAs (sRNAs) are important regulators of gene expression; however, the impact of natural mutations on sRNA functions has not been studied extensively. Here we show that the sRNA MgrR contains a unique 53 bp insertion in Escherichia fergusonii, a close relative of Escherichia coli and Salmonella enterica. The insertion is a repetitive extragenic palindromic (REP) sequence that could block transcription, but full-length MgrR is produced in E. fergusonii, showing that the insertion has not affected sRNA production. Additionally, despite containing the large insertion, the sRNA appears to be functional because deletion of mgrR made E. fergusonii more susceptible to H2O2. The molecular details of MgrR's roles in H2O2defence are yet to be defined, but our results suggest that having an alternative function allowed the sRNA to be retained in E. fergusonii despite it sustaining a large, potentially disruptive mutation.

Proposal for the creation of a new genus Musicola gen. nov., reclassification of Dickeya paradisiaca (Samson et al. 2005) as Musicola paradisiaca comb. nov. and description of a new species Musicola keenii sp. nov.

The Pectobacteriaceae family of important plant pathogens includes the genus Dickeya. There are currently 12 described species of Dickeya, although some are poorly characterized at the genomic level. Only two genomes of Dickeya paradisiaca, the type strain CFBP 4178T and strain Ech703, have previously been sequenced. Members of this species are mostly of tropical or subtropical origin. During an investigation of strains present in our laboratory collection we sequenced the atypical strain A3967, registered as CFBP 722, isolated from Solanum lycopersicum (tomato) in the South of France in 1965. The genome of strain A3967 shares digital DNA-DNA hybridization and average nucleotide identity (ANI) values of 68 and 96 %, respectively, with the D. paradisiaca type strain CFBP 4178T. However, ANI analysis showed that D. paradisiaca strains are significantly dissimilar to the other Dickeya species, such that less than one third of their genomes align to any other Dickeya genome. On phenotypic, phylogenetic and genomic grounds, we propose a reassignment of D. paradisiaca to the genus level, for which we propose the name Musicola gen. nov., with Musicola paradisiaca as the type species and CFBP 4178T (NCPPB 2511T) as the type strain. Phenotypic analysis showed differences between strain A3967T and CFBP 4178T, such as for the assimilation of melibiose, raffinose and myo-inositol. These results support the description of two novel species, namely Musicola paradisiaca comb. nov. and Musicola keenii sp. nov., with CFBP 4178T (NCPPB 2511T=LMG 2542T) and A3967T (CFBP 8732T=LMG 31880T) as the type strains, respectively.

Novel Quadruplex PCR for detecting and genotyping mobile colistin resistance genes in human samples.

Since 2016, several mobile colistin resistance (mcr) genes have been identified worldwide. It's worth noting that only mcr-1, mcr-3, mcr-8, and mcr-10 have been reported isolated directly from clinical samples which created greater risk to human health than other mcr gene types. A novel Quadruplex polymerase chain reaction (Quad-PCR) protocol was developed to detect and genotype transferable colistin-resistance genes (mcr-1, mcr-3, mcr-8, mcr-10) in Enterobacteria for clinical laboratory purposes. The protocol was validated by testing 11 clinical isolates of Escherichia coli and 3 clinical isolates of Klebsiella of human origin, each well characterized and prospectively validated. The Quad-PCR assay showed full concordance with whole-genome sequence data and displayed higher sensitivity and 100% specificity. The Quad-PCR assay achieved genotyping of mcr alleles (as singleton and mixture with double or triple gene types) described in one test.

Risk factors for colonization with multiple species of extended-spectrum beta-lactamase producing Enterobacterales: a case-case-control study.

BACKGROUND: Approximately 11% of patients colonized with extended-spectrum beta-lactamase producing Enterobacterales (ESBL-PE) are colonized with more than one ESBL-producing species. We investigated risk factors associated with colonization with multiple ESBL-PE species. METHODS: We performed a case-case-control study at the University Hospital Basel, Switzerland, including hospitalized patients colonized with ESBL-PE between 01/2008 and 12/2018. Patients colonized with multiple species of ESBL-PE during the same hospitalization were assigned to group 1. Group 2 consisted of patients with ESBL-PE and a newly acquired ESBL-PE-species identified during subsequent hospitalization. Controls (i.e., group 3) were patients with only one species of ESBL-PE identified over multiple hospitalizations. Controls were frequency-matched 3:1 to group 2 cases according to time-at-risk (i.e., days between ESBL-PE detection during first and subsequent hospitalizations) to standardize the duration of colonization. ESBL was identified with phenotypic assay and the presence of ESBL genes was confirmed by whole genome sequencing. RESULTS: Among 1559 inpatients, 154 cases met eligibility criteria (67 in group 1, 22 in group 2, 65 in group 3). International travel within the previous 12 months (OR 12.57, 95% CI 3.48-45.45, p < 0.001) and antibiotic exposure within the previous 3 months (OR 2.96, 95% CI 1.37-6.41, p = 0.006) were independently associated with co-colonization with multiple ESBL-PE species. Admission from another acute-care facility was the only predictor of replacement of one ESBL-PE species with another during subsequent hospitalizations (OR 6.02, 95% CI 1.15-31.49, p = 0.003). CONCLUSION: These findings point to strain-related factors being the main drivers of co-colonization with different ESBL-PE and may support stratification of infection prevention and control measures according to ESBL-PE species/strains.

A role for arthropods as vectors of multidrug-resistant Enterobacterales in surgical site infections from South Asia.

Understanding how multidrug-resistant Enterobacterales (MDRE) are transmitted in low- and middle-income countries (LMICs) is critical for implementing robust policies to curb the increasing burden of antimicrobial resistance (AMR). Here, we analysed samples from surgical site infections (SSIs), hospital surfaces (HSs) and arthropods (summer and winter 2016) to investigate the incidence and transmission of MDRE in a public hospital in Pakistan. We investigated Enterobacterales containing resistance genes (blaCTX-M-15, blaNDM and blaOXA-48-like) for identification, antimicrobial susceptibility testing and whole-genome sequencing. Genotypes, phylogenetic relationships and transmission events for isolates from different sources were investigated using single-nucleotide polymorphism (SNP) analysis with a cut-off of ≤20 SNPs. Escherichia coli (14.3%), Klebsiella pneumoniae (10.9%) and Enterobacter cloacae (16.3%) were the main MDRE species isolated. The carbapenemase gene blaNDM was most commonly detected, with 15.5%, 15.1% and 13.3% of samples positive in SSIs, HSs and arthropods, respectively. SNP (≤20) and spatiotemporal analysis revealed linkages in bacteria between SSIs, HSs and arthropods supporting the One Health approach to underpin infection control policies across LMICs and control AMR.