FosA8-producing E. coli ST131: clinical cases in Italy, February 2023.

Fosfomycin-resistant FosA8-producing Enterobacterales are uncommon strains with extremely low incidence in Europe, based on only three reports in the literature. We detected FosA8-producing Escherichia coli ST131 in clinical isolates from two patients admitted in February 2023 to a rehabilitation unit in Italy. The occurrence of rare fosA-like genes in the high-risk clone ST131 is of clinical relevance. The dissemination of FosA-producing E. coli, although still at low levels, should be continuously monitored.

Sequential Induction of Drug Resistance and Characterization of an Initial Candida albicans Drug-Sensitive Isolate.

BACKGROUND: The pathogenic fungus Candida albicans is a leading agent of death in immunocompromised individuals with a growing trend of antifungal resistance. METHODS: The purpose is to induce resistance to drugs in a sensitive C. albicans strain followed by whole-genome sequencing to determine mechanisms of resistance. Strains will be assayed for pathogenicity attributes such as ergosterol and chitin content, growth rate, virulence, and biofilm formation. RESULTS: We observed sequential increases in ergosterol and chitin content in fluconazole-resistant isolates by 78% and 44%. Surface thickening prevents the entry of the drug, resulting in resistance. Resistance imposed a fitness trade-off that led to reduced growth rates, biofilm formation, and virulence in our isolates. Sequencing revealed mutations in genes involved in resistance and pathogenicity such as ERG11, CHS3, GSC2, CDR2, CRZ2, and MSH2. We observed an increase in the number of mutations in key genes with a sequential increase in drug-selective pressures as the organism increased its odds of adapting to inhospitable environments. In ALS4, we observed two mutations in the susceptible strain and five mutations in the resistant strain. CONCLUSION: This is the first study to induce resistance followed by genotypic and phenotypic analysis of isolates to determine mechanisms of drug resistance.

Genomic characterization of ST38 NDM-5-producing Escherichia coli isolates from an outbreak in the Czech Republic.

A 2-year national genomic screening in the Czech Republic identified a notable prevalence of the New Delhi metallo-ß-lactamase 5 (NDM-5)-producing Escherichia coli sequence type 38 (ST38) in the city of Brno. Forty-two ST38 E. coli isolates harbored the blaNDM-5 gene on the chromosome. Virulence factors confirmed the persistence of these isolates through biofilm formation. Single Nucleotide Polymorphisms (SNPs)-based phylogeny and CRISPR assay typing showed minimal genomic variations, implying a clonally driven outbreak. Results suggest that this high-risk clone may impose a nationwide problem.

A novel F type plasmid encoding mcr-10 in a clinical Enterobacter ludwigii strain from a tertiary hospital in the Czech Republic.

OBJECTIVE: Here we describe a novel IncFIA plasmid harbouring mcr-10 gene in a clinical Enterobacter ludwigii strain isolated at the University Hospital in Pilsen in the Czech Republic. METHODS: The strain was subjected to antibiotic susceptibility testing. Whole genome sequencing was performed using Illumina for short-read sequencing and Oxford Nanopore Technologies for long-read sequencing followed by hybrid assembly. The resulting genome was used to detect species using average nucleotide identity, resistance genes, plasmid replicon and MLST (using centre for genomic epidemiology databases; ResFinder, PlasmidFinder and MLST, respectively) and virulence genes using VFDB. RESULTS: Τhe strain showed susceptibility against tetracycline, cefuroxime and chloramphenicol, and it was susceptible to the second and third generation of cephalosporins, carbapenems and colistin. Genome analysis identified the strain as E. ludwigii sequence type ST20 and located the mcr-10 gene on an IncFIA (HI1)/IncFII (Yp) plasmid (pI9455333_MCR10; 129 863 bp). Upon blasting the nucleotide sequence of pI9455333_MCR10 against the NCBI database, no similar plasmid sequence was detected, implying a novel plasmid structure. Nevertheless, it showed a partial similarity with pRHBSTW-00123_3 and FDAARGOS 1432, which were detected in Enterobacter cloacae complex (ECC) strains in wastewater samples in 2017 in UK and in 2021 in the United States, respectively, and pEC81-mcr, which was detected in a clinical Escherichia coli strain in 2020 in China. Moreover, I9455333cz genome carried virulence genes coding for curli fibers, fimbrial adherence determinants, siderophore aerobactin, iron uptake proteins and regulators of sigma factor. CONCLUSION: In conclusion, we identified a novel IncF plasmid harbouring mcr-10 gene in a clinical Enterobacter ludwigii strain. To our knowledge, this is the first clinical report of mcr-10 in the Czech Republic.

Simultaneous PCR detection of Paenibacillus larvae targeting insertion sequence IS256 and Melissococcus plutonius targeting pMP1 plasmid from hive specimens.

Paenibacillus larvae and Melissococcus plutonius represent the most threatening bacterial diseases of honeybee (Apis mellifera)-American and European foulbrood, respectively. For efficient control of those diseases, rapid and accurate detection of the pathogens is crucial. Therefore, we developed a novel multiplex PCR method simultaneously detecting both pathogens. To design and optimize multiplex PCR reaction, four strains of P. larvae representing four ERIC genotypes I-IV (strain DSM 7030-ERIC I, DSM 25430-ERIC II, LMG 16252-ERIC III, DSM 3615-ERIC IV) were selected. Those strains were fully sequenced using long-read sequencing (Sequel I, Pacific Biosciences). For P. larvae, the multicopy insertion sequence IS256 identified in all genotypes of P. larvae was selected to provide high sensitivity. M. plutonius was detected by plasmid pMP1 sequence and the virulence verified by following detection of ETX/MTX2 toxin responsible for pore formation in the cell membrane. As an internal control, a gene encoding for major royal jelly protein 1 specific for honeybees was selected. The method was validated on 36 clinical specimens collected from the colonies suffering from American and European foulbrood in the Czech Republic. Based on the results, sensitivity of PCR was calculated to 93.75% and specificity to 100% for P. larvae diagnosed from hive debris and 100% sensitivity and specificity for honeybee workers and larval scales as well as for diseased brood infected by M. plutonius.

Targeted long-read sequencing identified a causal structural variant in X-linked nephrogenic diabetes insipidus.

BACKGROUND: X-linked nephrogenic diabetes insipidus (NDI) is a rare genetic renal disease caused by pathogenic variants in the AVPR2 gene. Single nucleotide variants and small insertions/deletions in AVPR2 are reliably detected by routine clinical sequencing. Nevertheless, structural variants involving AVPR2 are challenging to identify accurately by conventional genetic testing. Here, we report a novel deletion of AVPR2 in a Czech family identified for the first time by targeted long-read sequencing (T-LRS). METHODS: A male proband with X-linked NDI underwent clinical sequencing of the AVPR2 gene that failed and thus indicated possible whole-gene deletion. Therefore, PCR mapping and subsequent targeted long-read sequencing (T-LRS) using a Pacific Biosciences sequencer were applied to search for the suspected deletion. To validate the deletion breakpoints and prove variant segregation in the family with X-linked NDI, Sanger sequencing of the deletion junction was performed. Quantitative real-time PCR was further carried out to confirm the carrier status of heterozygous females. RESULTS: By T-LRS, a novel 7.5 kb deletion of AVPR2 causing X-linked NDI in the proband was precisely identified. Sanger sequencing of the deletion junction confirmed the variant breakpoints and detected the deletion in the probands´ mother, maternal aunt, and maternal cousin with X-linked NDI. The carrier status in heterozygous females was further validated by quantitative real-time PCR. CONCLUSIONS: Identifying the 7.5 kb deletion gave a precise molecular diagnosis for the proband, enabled genetic counselling and genetic testing for the family, and further expanded the spectrum of structural variants causing X-linked NDI. Our results also show that T-LRS has significant potential for accurately identifying putative structural variants.

Fosfomycin resistance mechanisms in Enterobacterales: an increasing threat.

Antimicrobial resistance is well-known to be a global health and development threat. Due to the decrease of effective antimicrobials, re-evaluation in clinical practice of old antibiotics, as fosfomycin (FOS), have been necessary. FOS is a phosphonic acid derivate that regained interest in clinical practice for the treatment of complicated infection by multi-drug resistant (MDR) bacteria. Globally, FOS resistant Gram-negative pathogens are raising, affecting the public health, and compromising the use of the antibiotic. In particular, the increased prevalence of FOS resistance (FOSR) profiles among Enterobacterales family is concerning. Decrease in FOS effectiveness can be caused by i) alteration of FOS influx inside bacterial cell or ii) acquiring antimicrobial resistance genes. In this review, we investigate the main components implicated in FOS flow and report specific mutations that affect FOS influx inside bacterial cell and, thus, its effectiveness. FosA enzymes were identified in 1980 from Serratia marcescens but only in recent years the scientific community has started studying their spread. We summarize the global epidemiology of FosA/C2/L1-2 enzymes among Enterobacterales family. To date, 11 different variants of FosA have been reported globally. Among acquired mechanisms, FosA3 is the most spread variant in Enterobacterales, followed by FosA7 and FosA5. Based on recently published studies, we clarify and represent the molecular and genetic composition of fosA/C2 genes enviroment, analyzing the mechanisms by which such genes are slowly transmitting in emerging and high-risk clones, such as E. coli ST69 and ST131, and K. pneumoniae ST11. FOS is indicated as first line option against uncomplicated urinary tract infections and shows remarkable qualities in combination with other antibiotics. A rapid and accurate identification of FOSR type in Enterobacterales is difficult to achieve due to the lack of commercial phenotypic susceptibility tests and of rapid systems for MIC detection.

Clinically relevant antibiotic resistance in Escherichia coli from black kites in southwestern Siberia: a genetic and phenotypic investigation.

Wild birds including raptors can act as vectors of clinically relevant bacteria with antibiotic resistance. The aim of this study was to investigate the occurrence of antibiotic-resistant Escherichia coli in black kites (Milvus migrans) inhabiting localities in proximity to human-influenced environments in southwestern Siberia and investigate their virulence and plasmid contents. A total of 51 E. coli isolates mostly with multidrug resistance (MDR) profiles were obtained from cloacal swabs of 35 (64%, n = 55) kites. Genomic analyses of 36 whole genome sequenced E. coli isolates showed: (i) high prevalence and diversity of their antibiotic resistance genes (ARGs) and common association with ESBL/AmpC production (27/36, 75%), (ii) carriage of mcr-1 for colistin resistance on IncI2 plasmids in kites residing in proximity of two large cities, (iii) frequent association with class one integrase (IntI1, 22/36, 61%), and (iv) presence of sequence types (STs) linked to avian-pathogenic (APEC) and extra-intestinal pathogenic E. coli (ExPEC). Notably, numerous isolates had significant virulence content. One E. coli with APEC-associated ST354 carried qnrE1 encoding fluoroquinolone resistance on IncHI2-ST3 plasmid, the first detection of such a gene in E. coli from wildlife. Our results implicate black kites in southwestern Siberia as reservoirs for antibiotic-resistant E. coli. It also highlights the existing link between proximity of wildlife to human activities and their carriage of MDR bacteria including pathogenic STs with significant and clinically relevant antibiotic resistance determinants. IMPORTANCE Migratory birds have the potential to acquire and disperse clinically relevant antibiotic-resistant bacteria (ARB) and their associated antibiotic resistance genes (ARGs) through vast geographical regions. The opportunistic feeding behavior associated with some raptors including black kites and the growing anthropogenic influence on their natural habitats increase the transmission risk of multidrug resistance (MDR) and pathogenic bacteria from human and agricultural sources into the environment and wildlife. Thus, monitoring studies investigating antibiotic resistance in raptors may provide essential data that facilitate understanding the fate and evolution of ARB and ARGs in the environment and possible health risks for humans and animals associated with the acquisition of these resistance determinants by wildlife.

Whole Genome-Based Characterization of Multidrug Resistant Enterobacter and Klebsiella aerogenes Isolates from Lebanon.

Enterobacter spp. and Klebsiella aerogenes are rod-shaped Gram-negative opportunistic pathogens. This study aimed at the molecular and genomic characterization of multidrug resistant Enterobacter spp. and K. aerogenes isolates recovered from hospitalized patients in a tertiary care hospital in Lebanon. A total of 59 Enterobacter spp. clinical isolates consisting of 41 carbapenem-resistant and 18 susceptible by Etest were included in this study. Genotypic identification through whole-genome sequencing (WGS) was performed and confirmed in silico. Resistance and plasmid profiles were studied using ResFinder4.0 and Plasmid-Finder2.1. Multilocus sequence typing (MLST) was used to determine the isolates' clonality. Using the average nucleotide identity (ANI) we identified and confirmed that 47 (80%) isolates were E. hormaechei, 11 (18%) were Klebsiella aerogenes and 1 (2%) was an E. cloacae. Carbapenem-resistance was detected among 41 isolates all showing an MIC90 of ≥ 32 µg/mL for ertapenem, imipenem, and meropenem. blaNDM-1 (58.5%), blaACT-16 (54%), and blaOXA-1 (54%) were the most common detected ß-lactamases, while blaCTX-M-15 (68%) was the main detected extended-spectrum ß-lactamase (ESBL) encoding gene. Chromosomal ampC, carbapenemase encoding genes, and porin modifications were among the detected carbapenem resistance determinants. The carbapenemase encoding genes were linked to three well-defined plasmid Inc groups, IncFII/IncFIB, IncX3, and IncL. MLST typing revealed the diversity within the studied isolates, with ST114 being the most common among the studied E. hormaechei.: The spread of carbapenem-resistant isolates in clinical settings in Lebanon is a serious challenge. Screening and continuous monitoring through WGS analysis could effectively limit the dissemination of drug-resistant isolates in hospitalized patients. IMPORTANCE Drug resistance is an increasing global public health threat that involves most disease-causing organisms and antimicrobial drugs. Drug-resistant organisms spread in health care settings, and resistance to multiple drugs is common. Our study demonstrated the mechanisms leading to resistance against the last resort antimicrobial agents among members of the Enterobacteriaceae family. The spread of carbapenem-resistant bacteria in clinical settings is a serious challenge. Screening and continuous monitoring could effectively limit the dissemination of drug-resistant isolates in hospitalized patients.

Horsing around: Escherichia coli ST1250 of equine origin harbouring epidemic IncHI1/ST9 plasmid with bla CTX-M-1 and an operon for short-chain fructooligosaccharides metabolism.

The relatedness of the equine-associated Escherichia coli ST1250 and its single- and double-locus variants (ST1250-SLV/DLV), obtained from horses in Europe, was studied by comparative genome analysis. A total of 54 isolates of E. coli ST1250 and ST1250-SLV/DLV from healthy and hospitalized horses across Europe [Czech Republic (n=23), the Netherlands (n=18), Germany (n=9), Denmark (n=3) and France (n=1)] from 2008-2017 were subjected to whole-genome sequencing. An additional 25 draft genome assemblies of E. coli ST1250 and ST1250-SLV/DLV were obtained from the public databases. The isolates were compared for genomic features, virulence genes, clade structure and plasmid content. The complete nucleotide sequences of eight IncHI1/ST9 and one IncHI1/ST2 plasmids were obtained using long-read sequencing by PacBio or MinION. In the collection of 79 isolates, only 10 were phylogenetically close (<8 SNP). The majority of isolates belonged to phylogroup B1 (73/79, 92.4%) and carried bla CTX-M-1 (58/79, 73.4%). The plasmid content of the isolates was dominated by IncHI1 of ST9 (56/62, 90.3%) and ST2 (6/62, 9.7%), while 84.5% (49/58) bla CTX-M-1 genes were associated with presence of IncHI1 replicon of ST9 and 6.9% (4/58) with IncHI1 replicon of ST2 within the corresponding isolates. The operon for the utilization of short chain fructooligosaccharides (fos operon) was present in 55 (55/79, 69.6%) isolates, and all of these carried IncHI1/ST9 plasmids. The eight complete IncHI1/ST9 plasmid sequences showed the presence of bla CTX-M-1 and the fos operon within the same molecule. Sequences of IncHI1/ST9 plasmids were highly conserved (>98% similarity) regardless of country of origin and varied only in the structure and integration site of MDR region. E. coli ST1250 and ST1250-SLV/DLV are phylogenetically-diverse strains associated with horses. A strong linkage of E. coli ST1250 with epidemic multi-drug resistance plasmid lineage IncHI1/ST9 carrying bla CTX-M-1 and the fos operon was identified.

Preferred ß-lactone synthesis can explain high rate of false-negative results in the detection of OXA-48-like carbapenemases.

The resistance to carbapenems is usually mediated by enzymes hydrolyzing ß-lactam ring. Recently, an alternative way of the modification of the antibiotic, a ß-lactone formation by OXA-48-like enzymes, in some carbapenems was identified. We focused our study on a deep analysis of OXA-48-like-producing Enterobacterales, especially strains showing poor hydrolytic activity. In this study, well characterized 74 isolates of Enterobacterales resistant to carbapenems were used. Carbapenemase activity was determined by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), liquid chromatography/mass spectrometry (LC-MS), Carba-NP test and modified Carbapenem Inactivation Method (mCIM). As meropenem-derived ß-lactone possesses the same molecular weight as native meropenem (MW 383.46 g/mol), ß-lactonization cannot be directly detected by MALDI-TOF MS. In the spectra, however, the peaks of m/z = 340.5 and 362.5 representing decarboxylated ß-lactone and its sodium adduct were detected in 25 out of 35 OXA-48-like producers. In the rest 10 isolates, decarboxylated hydrolytic product (m/z = 358.5) and its sodium adduct (m/z = 380.5) have been detected. The peak of m/z = 362.5 was detected in 3 strains co-producing OXA-48-like and NDM-1 carbapenemases. The respective signal was identified in no strain producing class A or class B carbapenemase alone showing its specificity for OXA-48-like carbapenemases. Using LC-MS, we were able to identify meropenem-derived ß-lactone directly according to the different retention time. All strains with a predominant ß-lactone production showed negative results of Carba NP test. In this study, we have demonstrated that the strains producing OXA-48-like carbapenemases showing false-negative results using Carba NP test and MALDI-TOF MS preferentially produced meropenem-derived ß-lactone. We also identified ß-lactone-specific peak in MALDI-TOF MS spectra and demonstrated the ability of LC-MS to detect meropenem-derived ß-lactone.

The red thread between methylation and mutation in bacterial antibiotic resistance: How third-generation sequencing can help to unravel this relationship.

DNA methylation is an important mechanism involved in bacteria limiting foreign DNA acquisition, maintenance of mobile genetic elements, DNA mismatch repair, and gene expression. Changes in DNA methylation pattern are observed in bacteria under stress conditions, including exposure to antimicrobial compounds. These changes can result in transient and fast-appearing adaptive antibiotic resistance (AdR) phenotypes, e.g., strain overexpressing efflux pumps. DNA methylation can be related to DNA mutation rate, because it is involved in DNA mismatch repair systems and because methylated bases are well-known mutational hotspots. The AdR process can be the first important step in the selection of antibiotic-resistant strains, allowing the survival of the bacterial population until more efficient resistant mutants emerge. Epigenetic modifications can be investigated by third-generation sequencing platforms that allow us to simultaneously detect all the methylated bases along with the DNA sequencing. In this scenario, this sequencing technology enables the study of epigenetic modifications in link with antibiotic resistance and will help to investigate the relationship between methylation and mutation in the development of stable mechanisms of resistance.

Genomic Characterization of Mutli-Drug Resistant Pseudomonas aeruginosa Clinical Isolates: Evaluation and Determination of Ceftolozane/Tazobactam Activity and Resistance Mechanisms.

Resistance to ceftolozane/tazobactam (C/T) in Pseudomonas aeruginosa is a health concern. In this study, we conducted a whole-genome-based molecular characterization to correlate resistance patterns and ß-lactamases with C/T resistance among multi-drug resistant P. aeruginosa clinical isolates. Resistance profiles for 25 P. aeruginosa clinical isolates were examined using disk diffusion assay. Minimal inhibitory concentrations (MIC) for C/T were determined by broth microdilution. Whole-genome sequencing was used to check for antimicrobial resistance determinants and reveal their genetic context. The clonal relatedness was evaluated using MLST, PFGE, and serotyping. All the isolates were resistant to C/T. At least two ß-lactamases were detected in each with the blaOXA-4, blaOXA-10, blaOXA-50, and blaOXA-395 being the most common. blaIMP-15, blaNDM-1, or blaVIM-2, metallo-ß-lactamases, were associated with C/T MIC >256 µg/mL. Eight AmpC variants were identified, and PDC-3 was the most common. We also determined the clonal relatedness of the isolates and showed that they grouped into 11 sequence types (STs) some corresponding to widespread clonal complexes (ST111, ST233, and ST357). C/T resistance was likely driven by the acquired OXA ß-lactamases such as OXA-10, and OXA-50, ESBLs GES-1, GES-15, and VEB-1, and metallo- ß-lactamases IMP-15, NDM-1, and VIM-2. Collectively, our results revealed C/T resistance determinants and patterns in multi-drug resistant P. aeruginosa clinical isolates. Surveillance programs should be implemented and maintained to better track and define resistance mechanisms and how they accumulate and interact.

Interspecies Transmission of CMY-2-Producing Escherichia coli Sequence Type 963 Isolates between Humans and Gulls in Australia.

Escherichia coli sequence type 963 (ST963) is a neglected lineage closely related to ST38, a globally widespread extraintestinal pathogenic ST causing urinary tract infections (UTI) as well as sepsis in humans. Our current study aimed to improve the knowledge of this understudied ST by carrying out a comprehensive comparative analysis of whole-genome sequencing data consisting of 31 isolates from silver gulls in Australia along with another 80 genomes from public resources originating from geographically scattered regions. ST963 was notable for carriage of cephalosporinase gene blaCMY-2, which was identified in 99 isolates and was generally chromosomally encoded. ST963 isolates showed otherwise low carriage of antibiotic resistance genes, in contrast with the closely related E. coli ST38. We found considerable phylogenetic variability among international ST963 isolates (up to 11,273 single nucleotide polymorphisms [SNPs]), forming three separate clades. A major clade that often differed by 20 SNPs or less consisted of Australian isolates of both human and animal origin, providing evidence of zoonotic or zooanthropogenic transmission. There was a high prevalence of virulence F29:A-:B10 pUTI89-like plasmids within E. coli ST963 (n = 88), carried especially by less variable isolates exhibiting ≤1,154 SNPs. We characterized a novel 115,443-bp pUTI89-like plasmid, pCE2050_A, that carried a traS:IS5 insertion absent from pUTI89. Since IS5 was also present in a transposition unit bearing blaCMY-2 on chromosomes of ST963 strains, IS5 insertion into pUTI89 may enable mobilization of the blaCMY-2 gene from the chromosome/transposition unit to pUTI89 via homologous recombination. IMPORTANCE We have provided the first comprehensive genomic study of E. coli ST963 by analyzing various genomic and phenotypic data sets of isolates from Australian silver gulls and comparison with genomes from geographically dispersed regions of human and animal origin. Our study suggests the emergence of a specific blaCMY-2-carrying E. coli ST963 clone in Australia that is widely spread across the continent by humans and birds. Genomic analysis has revealed that ST963 is a globally dispersed lineage with a remarkable set of virulence genes and virulence plasmids described in uropathogenic E. coli. While ST963 separated into three clusters, a unique specific clade of Australian ST963 isolates harboring a chromosomal copy of AmpC ß-lactamase encoding the gene blaCMY-2 and originating from both humans and wild birds was identified. This phylogenetically close cluster comprised isolates of both animal and human origin, thus providing evidence of interspecies zoonotic transmission. The analysis of the genetic environment of the AmpC ß-lactamase-encoding gene highlighted ongoing evolutionary events that shape the carriage of this gene in ST963.

Fitness effects of blaCTX-M-15-harbouring F2:A1:B- plasmids on their native Escherichia coli ST131 H30Rx hosts.

OBJECTIVES: To investigate the fitness effects of large blaCTX-M-15-harbouring F2:A1:B- plasmids on their native Escherichia coli ST131 H30Rx hosts. METHODS: We selected five E. coli ST131 H30Rx isolates of diverse origin, each carrying an F2:A1:B- plasmid with the blaCTX-M-15 gene. The plasmid was eliminated from each isolate by displacement using an incompatible curing plasmid, pMDP5_cureEC958. WGS was performed to obtain complete chromosome and plasmid sequences of original isolates and to detect chromosomal mutations in 'cured' clones. High-throughput competition assays were conducted to determine the relative fitness of cured clones compared with the corresponding original isolates. RESULTS: We were able to successfully eliminate the F2:A1:B- plasmids from all five original isolates using pMDP5_cureEC958. The F2:A1:B- plasmids produced non-significant fitness effects in three isolates and moderate reductions in relative fitness (3%-4%) in the two remaining isolates. CONCLUSIONS: We conclude that F2:A1:B- plasmids pose low fitness costs in their E. coli ST131 H30Rx hosts. This plasmid-host fitness compatibility is likely to promote the maintenance of antibiotic resistance in this clinically important E. coli lineage.

OXA-244-Producing ST131 Escherichia coli From Surface and Groundwaters of Pavia Urban Area (Po Plain, Northern Italy).

The study aimed to investigate (i) the occurrence of third-generation cephalosporins and/or carbapenems non-sensitive Enterobacterales in Pavia surface and groundwaters, (ii) their resistance determinants, and (iii) the clonal features of the most relevant strains. During May 13 and 14, 2019, n = 18 water samples from n = 12 sampling sites in the urban/peri-urban area of Pavia (Po Plain, Northern Italy) have been evaluated. At first, hydrochemical analysis and bacterial plate counts were carried out on all the water samples. One milliliter of each water sample was then screened on both MacConkey agar (MC) added with cefotaxime (1 mg/L; 2 mg/L) and MC plus meropenem (0.25 mg/L; 4 mg/L). Species identification and antimicrobial susceptibilities were assessed by MicroScan autoSCAN-4. Double Disk Synergy (DD) test, CT103XL microarray, acc(6')-Ib-cr, qnrS, blaCTX-M-/MOX-/VEB-/OXA-type genes targeted PCR and sequencing, Pulsed-Field Gel Electrophoresis (PFGE), MultiLocus Sequence Typing (MLST), and Whole-Genome Sequencing on selected strains were performed. A total of n = 30 isolates grown on ß-lactams enriched MC: Escherichia coli (n = 21; 70%), Klebsiella spp. (n = 5; 16.6%), Citrobacter freundii (n = 2; 6.7%), and Kluyvera intermedia (n = 2; 6.7%). All E. coli and K. pneumoniae were ESßL-producers by DD. The 66.6, 38.0, and 19.0% of E. coli were ciprofloxacin/levofloxacin, trimethoprim-sulfamethoxazole, and gentamicin resistant (EUCAST 2019 breakpoints), respectively. A blaCTX-M-type determinant was identified in E. coli (n = 20/21; 95.2%) and K. pneumoniae (n = 2/3; 66.7%). The remaining E. coli was blaVEB-1 and blaMOX-2 genes positive. The aac(6')-Ib-cr determinant was found in n = 7 E. coli and n = 1 K. pneumoniae, while qnrS was found in n = 1 E. coli and n = 2 K. pneumoniae. PFGE showed clonal heterogeneity among ESßL-E. coli. Two out of four E. coli detected as blaOXA-244-positive, belonged to the pandemic ST131. One XDR K. pneumoniae from a stream sample, detected as blaKPC-2 positive, resulted of ST258. The epidemiological impact of blaOXA-244 ST131 E. coli and blaKPC-2 ST258 K. pneumoniae presence in surface waters of an urban area in Northern Italy must not be underestimated.

Urban Wildlife Crisis: Australian Silver Gull Is a Bystander Host to Widespread Clinical Antibiotic Resistance.

The Australian silver gull is an urban-adapted species that frequents anthropogenic waste sites. The enterobacterial flora of synanthropic birds often carries antibiotic resistance genes. Whole-genome sequence analyses of 425 Escherichia coli isolates from cloacal swabs of chicks inhabiting three coastal sites in New South Wales, Australia, cultured on media supplemented with meropenem, cefotaxime, or ciprofloxacin are reported. Phylogenetically, over 170 antibiotic-resistant lineages from 96 sequence types (STs) representing all major phylogroups were identified. Remarkably, 25 STs hosted the carbapenemase gene blaIMP-4, sourced only from Five Islands. Class 1 integrons carrying blaIMP and blaOXA alongside blaCTX-M and qnrS were notable. Multiple plasmid types mobilized blaIMP-4 and blaOXA-1, and 121 isolates (28%) carried either a ColV-like (18%) or a pUTI89-like (10%) F virulence plasmid. Phylogenetic comparisons to human isolates provided evidence of interspecies transmission. Our study underscores the importance of bystander species in the transmission of antibiotic-resistant and pathogenic E. coli. IMPORTANCE By compiling various genomic and phenotypic data sets, we have provided one of the most comprehensive genomic studies of Escherichia coli isolates from the Australian silver gull, on media containing clinically relevant antibiotics. The analysis of genetic structures capturing antimicrobial resistance genes across three gull breeding colonies in New South Wales, Australia, and comparisons to clinical data have revealed a range of trackable genetic signatures that highlight the broad distribution of clinical antimicrobial resistance in more than 170 different lineages of E. coli. Conserved truncation sizes of the class 1 integrase gene, a key component of multiple-drug resistance structures in the Enterobacteriaceae, represent unique deletion events that are helping to link seemingly disparate isolates and highlight epidemiologically relevant data between wildlife and clinical sources. Notably, only the most anthropogenically affected of the three sites (Five Islands) was observed to host carbapenem resistance, indicating a potential reservoir among the sites sampled.

Genomic characterisation of three GES-producing Enterobacterales isolated in the Czech Republic.

OBJECTIVES: The aim of the study is to characterise the genomic features of three GES-producing Enterobacterales isolates from Czech hospitals. METHODS: In 2020, during a routine screening of the hospital's surfaces in Prague General Hospital, two strains (CZ862 and CZ863) that belonged to the Enterobacter cloacae complex were found to be blaGES positive. Another blaGES positive strain identified as Klebsiella oxytoca was recovered from a patient hospitalised in Pilsen. Antibiotic susceptibility profiling was done with broth microdilution assay. Conjugation/transformation experiments were performed on all three strains. Genomic DNA of the three isolates was subjected to whole genome sequencing using PacBio platform. RESULTS: Multilocus sequence types typing of CZ862 and CZ863 identified the strains as ST837 and a novel ST (ST1622). Both blaGES harbouring plasmids showed high sequence similarity and complete query coverage (100% and 99.98%) with pEcl-35771cz. Both plasmids had two copies of blaGES instead of one copy as found in pEcl-35771cz. The clinical isolate CZ598 belonged to ST180. The plasmid harboured blaGES-7 gene, cat and aac(6')-lb and the novel variant blaOXA-1011. No similar sequences were observed, suggesting a novel plasmid. CONCLUSION: The detection of the two blaGES-positive plasmids in the same hospital environment, the first report after 3 years, suggests a hidden source. This highlights the importance of the hidden sources and evolution of such plasmids on the route of spreading into clinical settings. Also, the detection of the new blaOXA-1011, which is thought in this case to be associated with carbapenem resistance, imposes a health risk if disseminated, limiting therapeutic options.

Implication of different replicons in the spread of the VIM-1-encoding integron, In110, in Enterobacterales from Czech hospitals.

Background: VIM metallo-ß-lactamases are enzymes characterized by the ability to hydrolyze all ß-lactams. Usually, bla VIM-like genes are carried by class 1 integrons. In the Czech Republic, only sporadic cases of VIM-producing Enterobacterales have been reported in which those isolates carried the VIM-1 carbapenemase-encoding integron In110. However, during 2019-2020, an increased number was reported. Therefore, the aim of the current study was to characterize the genetic elements involved in the increased spread of bla VIM genes. Materials and methods: 32 VIM-producing Enterobacterales collected between 2019 and 2020 were subjected to: antimicrobial susceptibility testing, integron analysis, and short reads sequencing. Based on the results, 19 isolates were selected as representative and sequenced using Sequel I platform. Results: The 32 VIM-producing isolates exhibited variations in the MICs of carbapenems. Based on short-read data, 26 of the 32 sequenced isolates harbored the bla VIM-1 allele while six isolates carried the bla VIM-4 gene. The most prevalent was the In110 integron (n = 24) and two isolates carried the In4873 class 1 integron. The bla VIM-4 allele was identified in class 1 integrons In1174 (n = 3), In416 (n = 1), In2143 (n = 1) and In2150. Long reads sequencing revealed that the bla VIM was carried by: pKPC-CAV1193-like (n = 6), HI1 (pNDM-CIT; n = 4), HI2 (n = 3), FIB (pECLA; n = 2) and N (n = 1) incompatibility groups. Two bla VIM-carrying plasmids could not be typed by the database, while another one was integrated into the chromosome. Conclusion: We observed the spread of VIM-encoding integrons, mainly of In110, among Enterobacterales isolated from Czech hospitals, but also an increased number of novel elements underlining the ongoing evolution.

Evidence of an epidemic spread of KPC-producing Enterobacterales in Czech hospitals.

The aim of the present study is to describe the ongoing spread of the KPC-producing strains, which is evolving to an epidemic in Czech hospitals. During the period of 2018-2019, a total of 108 KPC-producing Enterobacterales were recovered from 20 hospitals. Analysis of long-read sequencing data revealed the presence of several types of blaKPC-carrying plasmids; 19 out of 25 blaKPC-carrying plasmids could be assigned to R (n = 12), N (n = 5), C (n = 1) and P6 (n = 1) incompatibility (Inc) groups. Five of the remaining blaKPC-carrying plasmids were multireplicon, while one plasmid couldn't be typed. Additionally, phylogenetic analysis confirmed the spread of blaKPC-carrying plasmids among different clones of diverse Enterobacterales species. Our findings demonstrated that the increased prevalence of KPC-producing isolates was due to plasmids spreading among different species. In some districts, the local dissemination of IncR and IncN plasmids was observed. Additionally, the ongoing evolution of blaKPC-carrying plasmids, through genetic rearrangements, favours the preservation and further dissemination of these mobile genetic elements. Therefore, the situation should be monitored, and immediate infection control should be implemented in hospitals reporting KPC-producing strains.