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Abstract Background The emergence and wide spread of carbapenemase-producing Enterobacteriaceae (CPE) poses a growing threat to global public health. However, clinically derived carbapenemase-producing Citrobacter causing multiple infections has rarely been investigated. Here we first report the isolation and comparative genomics of two bla NDM-5 carrying Citrobacter freundii (C. freundii) isolates from a patient with bloodstream and urinary tract infections. Results Antimicrobial susceptibility testing showed that both bla NDM-5 carrying C. freundii isolates were multidrug-resistant. Positive modified carbapenem inactivation method (mCIM) and EDTA-carbapenem inactivation method (eCIM) results suggested metallo-carbapenemase production. PCR and sequencing confirmed that both metallo-carbapenemase producers were bla NDM-5 positive. Genotyping and comparative genomics analyses revealed that both isolates exhibited a high level of genetic similarity. Plasmid analysis confirmed that the bla NDM-5 resistance gene is located on IncX3 plasmid with a length of 46,161 bp, and could successfully be transferred to the recipient Escherichia coli EC600 strain. A conserved structure sequence (ISAba125-IS5-bla NDM-5-trpF-IS26-umuD-ISKox3) was found in the upstream and downstream of the bla NDM-5 gene. Conclusions The data presented in this study showed that the conjugative bla NDM-5 plasmid possesses a certain ability to horizontal transfer. The dissemination of NDM-5-producing C. freundii isolates should be of close concern in future clinical surveillance. To our knowledge, this is the first study to characterize C. freundii strains carrying the bla NDM-5 gene from one single patient with multiple infections.

Background Conjugative plasmids play a major role in the dissemination of antibiotic resistance genes. Knowledge of the plasmid characteristics and behaviour can allow development of control strategies. Here we focus on the IncX group of plasmids carrying genes conferring quinolone resistance (PMQR), reporting their transfer and persistence within host bacteria of various genotypes under distinct conditions and levels of induced stress in form of temperature change and various concentrations of ciprofloxacin supplementation. Methods Complete nucleotide sequences were determined for eight qnr -carrying IncX-type plasmids, of IncX1 (3), IncX2 (3) and a hybrid IncX1-2 (2) types, recovered from Escherichia coli of various origins. This data was compared with further complete sequences of IncX1 and IncX2 plasmids carrying qnr genes ( n  = 41) retrieved from GenBank and phylogenetic tree was constructed. Representatives of IncX1 (pHP2) and IncX2 (p194) and their qnrS knockout mutants, were studied for influence of induced stress and genetic background on conjugative transfer and maintenance. Results A high level of IncX core-genome similarity was found in plasmids of animal, environmental and clinical origin. Significant differences were found between the individual IncX plasmids, with IncX1 subgroup plasmids showing higher conjugative transfer rates than IncX2 plasmids. Knockout of qnr modified transfer frequency of both plasmids. Two stresses applied simultaneously were needed to affect transfer rate of wildtype plasmids, whereas a single stress was sufficient to affect the IncX ΔqnrS plasmids. The conjugative transfer was shown to be biased towards the host phylogenetic proximity. A long-term cultivation experiment pointed out the persistence of IncX plasmids in the antibiotic-free environment. Conclusions The study indicated the stimulating effect of ciprofloxacin supplementation on the plasmid transfer that can be nullified by the carriage of a single PMQR gene. The findings present the significant properties and behaviour of IncX plasmids carrying antibiotic resistance genes that are likely to play a role in their dissemination and stability in bacterial populations.

Defining environmental reservoirs hosting mobile genetic elements that shuttle critically important antibiotic resistance genes is key to understanding antimicrobial resistance (AMR) from a One Health perspective. Gulls frequent public amenities, parklands, and sewage and other waste disposal sites and carry drug-resistant Escherichia coli . Although most of the approximately 94 million annual human cases of gastroenteritis due to Salmonella enterica resolve without medical intervention, antimicrobial therapy is recommended for patients with severe disease. Wild birds can be natural hosts of Salmonella that pose a threat to human health; however, multiple-drug-resistant serovars of S. enterica have rarely been described. In 2012, silver gull ( Chroicocephalus novaehollandiae ) chicks at a major breeding colony were shown to host Salmonella , most isolates of which were susceptible to antibiotics. However, multiple-drug-resistant (MDR) Escherichia coli with resistance to carbapenems, ceftazidime, and fluoroquinolones was reported from this breeding colony. In this paper, we describe a novel MDR Salmonella strain subsequently isolated from the same breeding colony. SG17-135, an isolate of S. enterica with phenotypic resistance to 12 individual antibiotics but only nine antibiotic classes including penicillins, cephalosporins, monobactams, macrolides, fluoroquinolones, aminoglycosides, dihydrofolate reductase inhibitors (trimethoprim), sulfonamides, and glycylcyclines was recovered from a gull chick in 2017. Whole-genome sequence (WGS) analysis of SG17-135 identified it as Salmonella enterica serovar Agona ( S . Agona) with a chromosome comprising 4,813,284 bp, an IncHI2 ST2 plasmid (pSG17-135-HI2) of 311,615 bp, and an IncX1 plasmid (pSG17-135-X) of 27,511 bp. pSG17-135-HI2 housed a complex resistance region comprising 16 antimicrobial resistance genes including bla CTX-M-55 . The acquisition of MDR plasmids by S. enterica described here poses a serious threat to human health. Our study highlights the importance of taking a One Health approach to identify environmental reservoirs of drug-resistant pathogens and MDR plasmids. IMPORTANCE Defining environmental reservoirs hosting mobile genetic elements that shuttle critically important antibiotic resistance genes is key to understanding antimicrobial resistance (AMR) from a One Health perspective. Gulls frequent public amenities, parklands, and sewage and other waste disposal sites and carry drug-resistant Escherichia coli . Here, we report on SG17-135, a strain of Salmonella enterica serovar Agona isolated from the cloaca of a silver gull chick nesting on an island in geographic proximity to the greater metropolitan area of Sydney, Australia. SG17-135 is closely related to pathogenic strains of S . Agona, displays resistance to nine antimicrobial classes, and carries important virulence gene cargo. Most of the antibiotic resistance genes hosted by SG17-135 are clustered on a large IncHI2 plasmid and are flanked by copies of IS 26 . Wild birds represent an important link in the evolution and transmission of resistance plasmids, and an understanding of their behavior is needed to expose the interplay between clinical and environmental microbial communities.

Plasmids are mobile genetic elements, contributing to the spread of resistance determinants by horizontal gene transfer. Plasmid-mediated quinolone resistances (PMQRs) are important determinants able to decrease the antimicrobial susceptibility of bacteria against fluoroquinolones and quinolones. The PMQR gene qnrS1, especially, is broadly present in the livestock and food sector. Thus, it is of interest to understand the characteristics of plasmids able to carry and disseminate this determinant and therewith contribute to the resistance development against this class of high-priority, critically important antimicrobials. Therefore, we investigated all commensal Escherichia (E.) coli isolates, with reduced susceptibility to quinolones, recovered during the annual zoonosis monitoring 2017 in the pork and beef production chain in Germany (n = 2799). Through short-read whole-genome sequencing and bioinformatics analysis, the composition of the plasmids and factors involved in their occurrence were determined. We analysed the presence and structures of predominant plasmids carrying the PMQR qnrS1. This gene was most frequently located on IncX plasmids. Although the E. coli harbouring these IncX plasmids were highly diverse in their sequence types as well as their phenotypic resistance profiles, the IncX plasmids-carrying the qnrS1 gene were rather conserved. Thus, we only detected three distinct IncX plasmids carrying qnrS1 in the investigated isolates. The IncX plasmids were assigned either to IncX1 or to IncX3. All qnrS1-carrying IncX plasmids further harboured a β-lactamase gene (bla). In addition, all investigated IncX plasmids were transmissible. Overall, we found highly heterogenic E. coli harbouring conserved IncX plasmids as vehicles for the most prevalent qnr gene qnrS1. These IncX plasmids may play an important role in the dissemination of those two resistance determinants and their presence, transfer and co-selection properties require a deeper understanding for a thorough risk assessment.