Nosocomial Outbreak of Carbapenemase-Producing Proteus mirabilis With Two Novel Salmonella Genomic Island 1 Variants Carrying Different blaNDM–1 Gene Copies in China

NDM-1-producing multidrug-resistant Proteus mirabilis brings formidable clinical challenges. We report a nosocomial outbreak of carbapenem-resistant P. mirabilis in China. Six P. mirabilis strains collected in the same ward showed close phylogenetic relatedness, indicating clonal expansion. Illumina and MinION sequencing revealed that three isolates harbored a novel Salmonella genomic island 1 carrying a blaNDM–1 gene (SGI1-1NDM), while three other isolates showed elevated carbapenem resistance and carried a similar SGI1 but with two blaNDM–1 gene copies (SGI1-2NDM). Four new single nucleotide mutations were present in the genomes of the two-blaNDM–1-harboring isolates, indicating later emergence of the SGI1-2NDM structure. Passage experiments indicated that both SGI variants were stably persistent in this clone without blaNDM–1 copy number changes. This study characterizes two novel blaNDM–1-harboring SGI1 variants in P. mirabilis and provides a new insight into resistance gene copy number variation in bacteria.

GR13-type plasmids in Acinetobacter potentiate the accumulation and horizontal transfer of diverse accessory genes

Carbapenem resistance and other antibiotic resistance genes (ARGs) can be found in plasmids in Acinetobacter, but many plasmid types in this genus have not been well-characterised. Here we describe the distribution, diversity and evolutionary capacity of rep group 13 (GR13) plasmids that are found in Acinetobacter species from diverse environments. Our investigation was prompted by the discovery of two GR13 plasmids in A. baumannii isolated in an intensive care unit (ICU). The plasmids harbour distinct accessory genes: pDETAB5 contains blaNDM-1 and genes that confer resistance to four further antibiotic classes, while pDETAB13 carries putative alcohol tolerance determinants. Both plasmids contain multiple dif modules, which are flanked by pdif sites recognised by XerC/XerD tyrosine recombinases. The ARG-containing dif modules in pDETAB5 are almost identical to those found in pDETAB2, a GR34 plasmid from an unrelated A. baumannii isolated in the same ICU a month prior. Examination of a further 41 complete, publicly available plasmid sequences revealed that the GR13 pangenome consists of just four core but 1086 accessory genes, 123 in the shell and 1063 in the cloud, reflecting substantial capacity for diversification. The GR13 core genome includes genes for replication and partitioning, and for a putative tyrosine recombinase. Accessory segments encode proteins with diverse putative functions, including for metabolism, antibiotic/heavy metal/alcohol tolerance, restriction-modification, an anti-phage system and multiple toxin-antitoxin systems. The movement of dif modules and actions of insertion sequences play an important role in generating diversity in GR13 plasmids. Discrete GR13 plasmid lineages are internationally disseminated and found in multiple Acinetobacter species, which suggests they are important platforms for the accumulation, horizontal transmission and persistence of accessory genes in this genus.

A novel plasmid entry exclusion system in pKPC_UVA01, a promiscuous conjugative plasmid carrying the bla KPC carbapenemase gene

Conjugative plasmids are the principal mediator in the emergence and spread of antibiotic resistance genes in Enterobacterales. Plasmid entry-exclusion (EEX) systems can restrict their transfer into the recipient bacteria carrying closely related plasmids. In this study, we have identified and characterized a novel plasmid entry exclusion system in a carbapenem resistance plasmid pKPC_UVA01, responsible for widespread dissemination of the bla KPC carbapenemase gene among Enterobacterales in the United States. The identified eex gene in the recipient strain of different Enterobacterales species inhibits the conjugation transfer of pKPC_UVA01 plasmids at a range of 200-400 fold, and this inhibition was found to be a dose-dependent function of the EEX protein in recipient cells. The C-terminus truncated version of eex or eex with an early termination codon at the C-terminus region alleviates inhibition of conjugative transfer. Unlike the strict specificity of plasmid exclusion by the known EEX protein, the newly identified EEX in the recipient strain can inhibit the transfer of IncP and IncN plasmids. The eex gene from the plasmid pKPC_UVA01 is not required for conjugative transfer but is essential in the donor bacteria for entry exclusion of this plasmid. This is a novel function of a single protein that is essential in both donor and recipient bacteria for entry exclusion of a plasmid. This eex gene is found to be distributed in multi-drug resistance plasmids similar to pKPC_UVA01 in different Enterobacterales species and may contribute to the stability of this plasmid type by controlling its transfer.

Carriage of distinct blaKPC-2 and blaOXA-48 plasmids in a single ST11 hypervirulent Klebsiella pneumoniae isolate in Egypt

Background Carbapenem-resistant hypervirulent K. pneumoniae (CR-hvKP) causes serious infections with significant morbidity and mortality. However, the epidemiology and transmission mechanisms of CR-hvKP and the corresponding carbapenem-resistant plasmids require further investigation. Herein, we have characterized an ST11 K. pneumoniae strain EBSI041 from the blood sample encoding both hypervirulence and carbapenem resistance phenotypes from a patient in Egypt. Results K. pneumoniae strain EBSI041 showed multidrug-resistance phenotypes, where it was highly resistant to almost all tested antibiotics including carbapenems. And hypervirulence phenotypes of EBSI041 was confirmed by the model of Galleria mellonella infection. Whole-genome sequencing analysis showed that the hybrid plasmid pEBSI041-1 carried a set of virulence factors rmpA, rmpA2, iucABCD and iutA, and six resistance genes aph(3′)-VI, armA, msr(E), mph(E), qnrS, and sul2. Besides, blaOXA-48 and blaSHV-12 were harboured in a novel conjugative IncL-type plasmid pEBSI041-2. The blaKPC-2-carrying plasmid pEBSI041-3, a non-conjugative plasmid lacking the conjugative transfer genes, could be transferred with the help of pEBSI041-2, and the two plasmids could fuse into a new plasmid during co-transfer. Moreover, the emergence of the p16HN-263_KPC-like plasmids is likely due to the integration of pEBSI041-3 and pEBSI041-4 via IS26-mediated rearrangement. Conclusion To the best of our knowledge, this is the first report on the complete genome sequence of KPC-2- and OXA-48-coproducing hypervirulent K. pneumoniae from Egypt. These results give new insights into the adaptation and evolution of K. pneumoniae during nosocomial infections.

Recurrent emergence of carbapenem resistance in Klebsiella pneumoniae mediated by an inhibitory ompK36 mRNA secondary structure

Outer membrane porins in Gram-negative bacteria facilitate antibiotic influx. In Klebsiella pneumoniae (KP), modifications in the porin OmpK36 are implicated in increasing resistance to carbapenems. Analysis of large KP genome collections, encompassing major healthcare-associated clones, revealed the recurrent emergence of a synonymous cytosine to thymine transition at position 25 (25c>t) in ompK36. We show that the 25c>t transition increases carbapenem resistance through depletion of OmpK36 from the outer membrane. The mutation attenuates KP in a murine pneumonia model, which accounts for its limited clonal expansion observed by phylogenetic analysis. However, in the context of carbapenem treatment, the 25c>t transition tips the balance towards treatment failure, thus accounting for its recurrent emergence. Mechanistically, the 25c>t transition mediates an intramolecular mRNA interaction between a uracil encoded by 25t and the first adenine within the Shine-Dalgarno sequence. This specific interaction leads to the formation of an RNA stem structure, which obscures the ribosomal binding site thus disrupting translation. While mutations reducing OmpK36 expression via transcriptional silencing are known, we uniquely demonstrate the repeated selection of a synonymous ompK36 mutation mediating translational suppression in response to antibiotic pressure.

A survey of current activities and technologies used to detect carbapenem resistance in bacteria isolated from companion animals at veterinary diagnostic laboratories—United States, 2020

Carbapenems are antimicrobial drugs reserved for the treatment of severe multidrug-resistant Gram-negative bacterial infections. Carbapenem-resistant organisms (CROs) are an urgent public health threat and have been made reportable to public health authorities in many jurisdictions. Recent reports of CROs in companion animals and veterinary settings suggest that CROs are a One Health problem. However, standard practices of U.S. veterinary diagnostic laboratories (VDLs) to detect CROs are unknown. We assessed the capacity of VDLs to characterize carbapenem resistance in isolates from companion animals. Among 74 VDLs surveyed in 42 states, 23 laboratories (31%) from 22 states responded. Most (22/23, 96%) include ≥1 carbapenem on their primary antimicrobial susceptibility testing panel; approximately one-third (9/23, 39%) perform phenotypic carbapenemase production testing or molecular identification of carbapenemase genes. Overall, 35% (8/23) of VDLs across eight states reported they would notify public health if a CRO was detected. Most (17/21, 81%) VDLs were not aware of CRO reporting mandates; some expressed uncertainty about whether the scope of known mandates included CROs from veterinary sources. Although nearly all surveyed VDLs tested for carbapenem resistance, fewer had capacity for mechanism testing or awareness of public health reporting requirements. Addressing these gaps is critical to monitoring CRO incidence and trends in veterinary medicine, preventing spread in veterinary settings, and mounting an effective One Health response. Improved collaboration and communication between public health and veterinary medicine is critical to inform infection control practices in veterinary settings and conduct public health response when resistant isolates are detected.

Predominance of Acinetobacter spp., Harboring the blaIMP Gene, Contaminating the Hospital Environment in a Tertiary Hospital in Mwanza, Tanzania: A Cross-Sectional Laboratory-Based Study

Data on colonization and hospital contamination of carbapenem-resistant Gram-negative bacteria (CR-GNB) are limited in low- and middle-income countries. We designed this study to determine the prevalence and co-existence of carbapenemase genes among CR-GNB isolated from clinical, colonization, and hospital environmental samples at a tertiary hospital in Mwanza, Tanzania. The modified Hodge test (MHT), the combined disk test (CDT), and the double-disk synergy test (DDST) were used for the phenotypic detection of carbapenemases. A multiplex PCR assay was used to detect blaIMP and blaKPC, and a singleplex PCR assay was used to detect blaOXA-48. Data were analyzed by STATA version 13.0. Overall, 68.8% (44/64) of the CR-GNB had at least one phenotype by phenotypic methods, whereby 60.9% (39/64) were both CDT and DDST positive and 31.3% (20/64) were MHT positive. A total of 23/64 (35.9%) had at least one of the genes tested with the predominance of blaIMP (91.3%; 21/23). In addition, 47.7% (21/44) of the CR-GNB phenotypes had at least one gene. Around 47.8% (11/23) of the CR-GNB carried multiple genes encoding for carbapenem resistance, with the maximum co-existence of blaIMP/blaKPC/blaOXA-48 (45.5%; 5/11). The majority of carbapenem-resistant genes were detected in Acinetobacter spp. (82.6%; 19/23) and isolated from bed swabs (69.6%; 16/23). Acinetobacter spp. carrying the blaIMP gene predominantly contaminated the hospital environment. Therefore, we recommend routine decontamination of inanimate hospital surfaces, including patient beds.

Proteomic and Transcriptomic Analyses Indicate Reduced Biofilm-Forming Abilities in Cefiderocol-Resistant Klebsiella pneumoniae

The advent of cefiderocol provides hope for the clinical treatment of multi-drug resistant gram-negative bacteria (GNB), especially those with carbapenem resistance. Resistance of Klebsiella pneumoniae to cefiderocol can be enhanced by acclimatization. In the present study, we collected cefiderocol resistant K. pneumoniae isolates during a 36-day acclimatization procedure while increasing the cefiderocol concentration in the culture medium. Strains were studied for changes in their biological characteristics using proteomics and transcriptomics. A decrease in biofilm formation ability was the main change observed among the induced isolates. Downregulation of genes involved in biofilm formation including hdeB, stpA, yhjQ, fba, bcsZ, uvrY, bcsE, bcsC, and ibpB were the main factors that reduced the biofilm formation ability. Moreover, downregulation of siderophore transporter proteins including the iron uptake system component efeO, the tonB-dependent receptor fecA, and ferric iron ABC transporter fbpA may be among the determining factors leading to cefiderocol resistance and promoting the reduction of biofilm formation ability of K. pneumoniae. This is the first study to investigate cefiderocol resistance based on comprehensive proteomic and transcriptomic analyses.

The Antimicrobial Resistance Characteristics of Imipenem-Non-Susceptible, Imipenemase-6-Producing Escherichia coli

Imipenemase-6 (IMP-6) type carbapenemase-producing Enterobacteriaceae is regarded as dangerous due to its unique lack of antimicrobial susceptibility. It is resistant to meropenem (MEPM) but susceptible to imipenem (IPM). In addition to carbapenemase, outer membrane porins and efflux pumps also play roles in carbapenem resistance by reducing the antimicrobial concentration inside cells. Extended-spectrum β-lactamase (ESBL) is transmitted with IMP-6 by the plasmid and broadens the spectrum of antimicrobial resistance. We collected 42 strains of IMP-6-producing Escherichia coli and conducted a molecular analysis of carbapenemase, ESBL, porin, efflux, and epidemiological characteristics using plasmid replicon typing. Among the 42 isolates, 21 strains were susceptible to IPM (50.0%) and 1 (2.4%) to MEPM. Seventeen strains (40.5%) co-produced CTX-M-2 type ESBL. We found that the relative expression of ompC and ompF significantly correlated with the MIC of IPM (p = 0.01 and p = 0.03, respectively). Sixty-eight% of CTX-M-2-non-producing strains had IncI1, which was significantly different from CTX-M-2-producing strains (p < 0.001). In conclusion, 50.0% of our IMP-6-producing strains were non-susceptible to IPM, which is different from the typical pattern and can be attributed to decreased porin expression. Further studies investigating other types of carbapenemase are warranted.

Characterization of Carbapenem-Resistant Enterobacteriaceae Cultured From Retail Meat Products, Patients, and Porcine Excrement in China

The emergence and dissemination of carbapenem-resistant Enterobacteriaceae (CRE) is a growing concern to animal and public health. However, little is known about the spread of CRE in food and livestock and its potential transmission to humans. To identify CRE strains from different origins and sources, 53 isolates were cultured from 760 samples including retail meat products, patients, and porcine excrement. Antimicrobial susceptibility testing was carried out, followed by phylogenetic typing, whole-genome sequencing, broth mating assays, and plasmids analyses. Forty-three Escherichia coli, nine Klebsiella pneumoniae, and one Enterobacter cloacae isolates were identified, each exhibiting multidrug-resistant phenotypes. Genetically, the main sequence types (STs) of E. coli were ST156 (n = 7), ST354 (n = 7), and ST48 (n = 7), and the dominant ST of K. pneumoniae is ST11 (n = 5). blaNDM–5 (n = 40) of E. coli and blaKPC–2 (n = 5) were the key genes that conferred carbapenem resistance phenotypes in these CRE strains. Additionally, the mcr-1 gene was identified in 17 blaNDM-producing isolates. The blaNDM–5 gene from eight strains could be transferred to the recipients via conjugation assays. Two mcr-1 genes in the E. coli isolates could be co-transferred along with the blaNDM–5 genes. IncF and IncX3 plasmids have been found to be predominantly associated with blaNDM gene in these strains. Strains isolated in our study from different sources and regions tend to be concordant and overlap. CRE strains from retail meat products are a reservoir for transition of CRE strains between animals and humans. These data also provide evidence of the dissemination of CRE strains and carbapenem-resistant genes between animal and human sources.