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ABSTRACTThe H30Rx subclade of Escherichia coli ST131 is a clinically important, globally dispersed pathogenic lineage that typically displays resistance to fluoroquinolones and extended spectrum β-lactams. Isolates EC233 and EC234, variants of ST131-H30Rx with a novel sequence type (ST) 8196, isolated from unrelated patients presenting with bacteraemia at a Sydney Hospital in 2014 are characterised here. EC233 and EC234 are phylogroup B2, serotype O25:H4A, and resistant to ampicillin, amoxicillin, cefoxitin, ceftazidime, ceftriaxone, ciprofloxacin, norfloxacin and gentamicin and are likely clonal. Both harbour an IncFII_2 plasmid (pSPRC_Ec234-FII) that carries most of the resistance genes on an IS26 associated translocatable unit, two small plasmids and a novel IncI1 plasmid (pSPRC_Ec234-I). SNP-based phylogenetic analysis of the core genome of representatives within the ST131 clonal complex places both isolates in a subclade with three clinical Australian ST131-H30Rx clade-C isolates. A MrBayes phylogeny analysis of EC233 and EC234 indicates ST8196 share a most recent common ancestor with ST131-H30Rx strain EC70 isolated from the same hospital in 2013. Our study identified genomic hallmarks that define the ST131-H30Rx subclade in the ST8196 isolates and highlights a need for unbiased genomic surveillance approaches to identify novel high-risk MDR E. coli pathogens that impact healthcare facilities.

Salmonella enterica displaying resistance to extended-spectrum cephalosporins and fluoroquinolone (FQs) has been deemed a high-priority pathogen by the World Health Organization (WHO). While CTX-M type acquired β-lactamases have been detected in S. enterica serovar Bovismorbificans, DHA enzymes have been rarely reported in S. Bovismorbificans. In this study, we here report for the first time the isolation of two multi-drug resistant (MDR) S. Bovismorbificans strains co-harboring plasmid-encoded AmpC (pAmpC) β-lactamase gene (bla DHA-1) and qnrB gene, 16Sal017 isolated from a chicken meat sample and 16Sal018 from a grass carp fish sample, collected from retail markets in Guangzhou, China. The bla DHA-1 and qnrB genes in these two strains were both located on the same novel 217,773 bp IncHI2 plasmid belonged to ST2. The plasmid contained 16 additional acquired antimicrobial resistance genes encoding resistance to eight antibiotic classes and quaternary ammonium compound. Besides, 16Sal017 contained an additional 10,124 bp Col (pHAD28)-like plasmid harboring qnrS1. The bla DHA-1 and qnrB4 genes were located in an 18,198 bp region, sul1 - qacEΔ1 - ampR - bla DHA-1 - pspABCDF - qnrB4 - sapABC -IS 91 - sul1 - qacEΔ1 , which has been identified in various bacteria species, indicating the high transfer ability of bla DHA-1 and qnrB4 genes within this gene cluster. The IncHI2 plasmid was found to be transferable to Escherichia coli J53 by conjugation and resulted in the acquiring of multiple resistance in the transconjugants. Genome sequence comparisons by cgMLST and MAUVE alignment indicated 16Sal017 and 16Sal018 are highly similar and are not epidemiologically linked with strains from other sources and countries. Our findings suggest S. Bovismorbificans as a new host for conjugative mega-plasmid harboring bla DHA-1 and qnrB4 genes, and highlight the potential transmission opportunity of these S. Bovismorbificans clones through the food chain, which need continuous investigation. • Two foodborne S. Bovismorbificans isolates of different origins were isolated in China. • The two isolates were highly similar and co-resistant to CIP and CTX. • The two isolates harbored a same novel IncHI2-ST2 type conjugative plasmid. • bla DHA-1 and qnrB located gene cluster was firstly reported in S. Bovismorbificans. • One isolate contained one more Col (pHAD28)-like plasmid harboring qnrS1. [ABSTRACT FROM AUTHOR]

The extensively drug-resistant NDM-1-producing Klebsiella quasipneumoniae subsp. quasipneumoniae M17277 (Kqsq-M17277) was only susceptible to colistin and fosfomycin. Whole genome sequencing of Kqsq-M17277 was performed through Illumina (short reads) and Oxford Nanopore Technologies (long reads). Hybrid assembly of short and long reads rendered circular contigs of the Kqsq-M17277 chromosome and three plasmids of 477,340 (p17277A_477), 138,998 (p17277C_138, which harbored bla NDM-1) and 123,307 bp (p17277B_123). In silico analysis showed that p17277A_477 was not previously described, belonged to the IncHI2 incompatibility group and contained 51 complete or partial (≥300 bp) insertion sequences and Tn 3 family transposons, which encompassed, as a whole, 14% of the p17277A_477 sequence. In silico search for antimicrobial resistance genes revealed that p17277A_477 harbored the recently described plasmid-encoded mcr-9 gene (colistin resistance), besides other resistance genes. This is the first worldwide report of a K. quasipneumoniae harboring mcr-9 and bla NDM-1 and the first report of mcr-9 in Latin America. This gene was embedded in a genetic structure, i.e. , IS 903B -like/ mcr-9 / wbuC /IS 26 , that was found in 71% of the mcr-9 -harboring sequences of the NCBI Nucleotide Collection database. The qseC and qseB genes involved in mcr-9 expression were not found in Kqsq-M17277, which could explain its colistin susceptibility. Fifteen additional resistance genes were detected in p17277A_477. Ten of them aac(6′)-IIc , aadA2 , aph(3′)-Ia , bla DHA-1 , dfrA12 , ereA2 , mphA , qnrB4 , sul1 and tet (D)] were found clustered within a 44,907-bp-long array of IS 26 -bounded resistance genes. The plasmid-mediated quinolone resistance gene qnrB4 was located in a complex class 1 integron within this IS 26 -bounded resistance gene cluster. p17277A_477 was self-transferable by conjugation to Escherichia coli J53, azide-resistant. The Tra1 and Tra2 conjugative transfer regions of the IncHI2 plasmid R478 were found by in silico search. p17277A_477 is a complex plasmid that provides a large baggage of both resistance genes and genetic resources for plasmid rearrangements and additional resistance gene acquisition. • mcr-9 is present in a NDM-1-producing K. quasipneumoniae. • First report of mcr-9 in Latin America. • mcr-9 is harbored in a 477,340-bp conjugative IncHI2 plasmid (p17277A_477). • mcr-9 was embedded in the IS 903B -like/ mcr-9 / wbuC /IS26 structure. • p17277A_477 contains an IS 26 -bounded resistance gene cluster. [ABSTRACT FROM AUTHOR]

The H 30Rx subclade of Escherichia coli ST131 is a clinically important, globally dispersed pathogenic lineage that typically displays resistance to fluoroquinolones and extended spectrum β-lactams. Isolates EC233 and EC234, variants of ST131- H 30Rx with a novel sequence type (ST) 8196, isolated from unrelated patients presenting with bacteraemia at a Sydney Hospital in 2014 are characterised here. EC233 and EC234 are phylogroup B2, serotype O25: H 4A, and resistant to ampicillin, amoxicillin, cefoxitin, ceftazidime, ceftriaxone, ciprofloxacin, norfloxacin and gentamicin and are likely clonal. Both harbour an IncFII_2 plasmid (pSPRC_Ec234-FII) that carries most of the resistance genes on an IS 26 associated translocatable unit, two small plasmids and a novel IncI1 plasmid (pSPRC_Ec234-I). SNP-based phylogenetic analysis of the core genome of representatives within the ST131 clonal complex places both isolates in a subclade with three clinical Australian ST131- H 30Rx clade-C isolates. A MrBayes phylogeny analysis of EC233 and EC234 indicates ST8196 share a most recent common ancestor with ST131- H 30Rx strain EC70 isolated from the same hospital in 2013. Our study identified genomic hallmarks that define the ST131- H 30Rx subclade in the ST8196 isolates and highlights a need for unbiased genomic surveillance approaches to identify novel high-risk MDR E. coli pathogens that impact healthcare facilities.

This study sought to assess the genetic variability of Escherichia coli isolated from bloodstream infections (BSIs) presenting at Concord Hospital, Sydney during 2013-2016. Whole-genome sequencing was used to characterize 81 E. coli isolates sourced from community-onset (CO) and hospital-onset (HO) BSIs. The cohort comprised 64 CO and 17 HO isolates, including 35 multidrug-resistant (MDR) isolates exhibiting phenotypic resistance to three or more antibiotic classes. Phylogenetic analysis identified two major ancestral clades. One was genetically diverse with 25 isolates distributed in 16 different sequence types (STs) representing phylogroups A, B1, B2, C and F, while the other comprised phylogroup B2 isolates in subclades representing the ST131, ST73 and ST95 lineages. Forty-seven isolates contained a class 1 integron, of which 14 carried bla CTX -M- gene. Isolates with a class 1 integron carried more antibiotic resistance genes than isolates without an integron and, in most instances, resistance genes were localized within complex resistance loci (CRL). Resistance to fluoroquinolones could be attributed to point mutations in chromosomal parC and gyrB genes and, in addition, two isolates carried a plasmid-associated qnrB4 gene. Co-resistance to fluoroquinolone and broad-spectrum beta-lactam antibiotics was associated with ST131 (HO and CO), ST38 (HO), ST393 (CO), ST2003 (CO) and ST8196 (CO and HO), a novel ST identified in this study. Notably, 10/81 (12.3 %) isolates with ST95 (5 isolates), ST131 (2 isolates), ST88 (2 isolates) and a ST540 likely carry IncFII-IncFIB plasmid replicons with a full spectrum of virulence genes consistent with the carriage of ColV-like plasmids. Our data indicate that IncF plasmids play an important role in shaping virulence and resistance gene carriage in BSI E. coli in Australia.