A panel of diverse Klebsiella pneumoniae clinical isolates for research and development.

Klebsiella pneumoniae are a leading cause of healthcare-associated infections worldwide. In particular, strains expressing extended-spectrum ß-lactamases (ESBLs) and carbapenemases pose serious treatment challenges, leading the World Health Organization (WHO) to designate ESBL and carbapenem-resistant Enterobacteriaceae as 'critical' threats to human health. Research efforts to combat these pathogens can be supported by accessibility to diverse and clinically relevant isolates for testing novel therapeutics. Here, we describe a panel of 100 diverse K. pneumoniae isolates that are publicly available to assist the research community in this endeavour. Whole-genome sequencing (WGS) was performed on 3878 K. pneumoniae clinical isolates housed at the Multidrug-Resistant Organism Repository and Surveillance Network. The isolates were cultured from 63 facilities in 19 countries between 2001 and 2020. Core-genome multilocus sequence typing and high-resolution single-nucleotide polymorphism-based phylogenetic analyses captured the genetic diversity of the collection and were used to select the final panel of 100 isolates. In addition to known multidrug-resistant (MDR) pandemic lineages, the final panel includes hypervirulent lineages and isolates with specific and diverse resistance genes and virulence biomarkers. A broad range of antibiotic susceptibilities, ranging from pan-sensitive to extensively drug-resistant isolates, are described. The panel collection, and all associated metadata and genome sequences, are available at no additional cost and will be an important resource for the research community and for the design and development of novel antimicrobial agents and diagnostics against this important pathogen.

Genome Sequence Analysis of a Wohlfahrtiimonas chitiniclastica Strain Isolated from a Septic Wound of a Hospitalized Patient in Uganda.

We report a genome sequence of Wohlfahrtiimonas chitiniclastica strain MUWRP0946, isolated from a hospitalized patient in Uganda. The genome size was 2.08 million bases, and the genome completeness was 94.22%. The strain carries tetracycline, folate pathway antagonist, ß-lactam, and aminoglycoside antibiotic resistance genes.

A one-year genomic investigation of Escherichia coli epidemiology and nosocomial spread at a large US healthcare network.

BACKGROUND: Extra-intestinal pathogenic Escherichia coli (ExPEC) are a leading cause of bloodstream and urinary tract infections worldwide. Over the last two decades, increased rates of antibiotic resistance in E. coli have been reported, further complicating treatment. Worryingly, specific lineages expressing extended-spectrum ß-lactamases (ESBLs) and fluoroquinolone resistance have proliferated and are now considered a serious threat. Obtaining contemporary information on the epidemiology and prevalence of these circulating lineages is critical for containing their spread globally and within the clinic. METHODS: Whole-genome sequencing (WGS), phylogenetic analysis, and antibiotic susceptibility testing were performed for a complete set of 2075 E. coli clinical isolates collected from 1776 patients at a large tertiary healthcare network in the USA between October 2019 and September 2020. RESULTS: The isolates represented two main phylogenetic groups, B2 and D, with six lineages accounting for 53% of strains: ST-69, ST-73, ST-95, ST-131, ST-127, and ST-1193. Twenty-seven percent of the primary isolates were multidrug resistant (MDR) and 5% carried an ESBL gene. Importantly, 74% of the ESBL-E.coli were co-resistant to fluoroquinolones and mostly belonged to pandemic ST-131 and emerging ST-1193. SNP-based detection of possible outbreaks identified 95 potential transmission clusters totaling 258 isolates (12% of the whole population) from ≥ 2 patients. While the proportion of MDR isolates was enriched in the set of putative transmission isolates compared to sporadic infections (35 vs 27%, p = 0.007), a large fraction (61%) of the predicted outbreaks (including the largest cluster grouping isolates from 12 patients) were caused by the transmission of non-MDR clones. CONCLUSION: By coupling in-depth genomic characterization with a complete sampling of clinical isolates for a full year, this study provides a rare and contemporary survey on the epidemiology and spread of E. coli in a large US healthcare network. While surveillance and infection control efforts often focus on ESBL and MDR lineages, our findings reveal that non-MDR isolates represent a large burden of infections, including those of predicted nosocomial origins. This increased awareness is key for implementing effective WGS-based surveillance as a routine technology for infection control.

IS26-mediated plasmid reshuffling results in convergence of toxin-antitoxin systems but loss of resistance genes in XDR Klebsiella pneumoniae from a chronic infection.

Carbapenem-resistant Enterobacterales pose an urgent threat to human health worldwide. Klebsiella pneumoniae sequence type (ST) 14, initially identified in the Middle East and South-Asia and co-harbouring the carbapenemase genes bla OXA-232 and bla NDM-1, is now emerging globally. One such strain was detected in the USA in 2013 from a patient initially treated in India that also carried armA, a 16S rRNA methyltransferase that confers resistance to all clinically relevant aminoglycosides. Genetic and phenotypic changes were observed in 14 serial isolates collected from this chronically infected patient. The index isolate carried five plasmids, including an IncFIB-IncHI1B (harbouring armA and bla NDM-1), an IncFIA (bla CTX-M-15) and a ColE-like (bla OXA-232), and was extensively resistant to antibiotics. Four years later, a subsequent isolate had accumulated 34 variants, including a loss-of-function mutation in romA, resulting in tigecycline non-susceptibility. Importantly, this isolate now only carried two plasmids, including a large mosaic molecule made of fragments, all harbouring distinct toxin-antitoxin systems, from three of the canonical plasmids. Of the original acquired antibiotic resistance genes, this isolate only retained bla CTX-M-15, and as a result susceptibility to the carbapenems and amikacin was restored. Long-read sequencing of a subset of five representative isolates, collected between 2013 and 2017, allowed for the elucidation of the complex plasmid patterns and revealed the role of IS26-mediated plasmid reshuffling in the evolution of this clone. Such investigations of the mechanisms underlying plasmid stability, together with global and local surveillance programmes, are key to a better understanding of plasmid host range and dissemination.

First Report: Colistin Resistance Gene mcr-3.1 in Salmonella enterica Serotype Choleraesuis Isolated from Human Blood Sample from Thailand.

This study describes the first finding of Salmonella enterica serotype Choleraesuis (Salmonella Choleraesuis) isolate harboring mobile colistin resistance (mcr)-3.1 obtained from human blood sample. The clinical relevant blood sample was collected during October 2018. The phenotypic identification and antimicrobial susceptibility testing (AST) were studied by using automate microbiology platform (Phoenix M50, BD), and in-depth characterization by whole genome sequencing. The phenotypic identification was reported Salmonella Choleraesuis. AST result demonstrated that this isolate had high minimum inhibitory concentrations (MICs) against colistin, fluoroquinolone, and cephalosporin III and IV, which are first-line antibiotic treatment choices for Gram-negative bacterial pathogen infections. This Salmonella Choleraesuis is harboring mcr-3.1 and presented a diversity carbapenemase including blaTEM and blactx-m-55. Regarding the multilocus sequence typing result, this Salmonella presented ST139 that related to the Choleraesuis variant sensu stricto. Swine is not the host specific for the Salmonella Choleraesuis since it also causes enteric and other diseases in human. Hence, the presence of the mobile plasmid colistin mcr-3.1 resistant gene in human sample is resulting to the public health concerns due to the fact that it is enable to transmit to other hosts and distribute into an environment.

Characterization of KPC-82, a KPC-2 Variant Conferring Resistance to Ceftazidime-Avibactam in a Carbapenem-Nonsusceptible Clinical Isolate of Citrobacter koseri.

KPC-82 is a KPC-2 variant identified in a carbapenem-nonsusceptible Citrobacter koseri that confers high-level resistance to ceftazidime-avibactam. Genomic analysis revealed that blaKPC-82 is carried by a chromosomally integrated Tn4401 transposon (disrupting porin gene phoE) and evolved by a 6-nucleotide tandem repeat duplication causing a two-amino-acid insertion (Ser-Asp) within the Ala267-Ser275 loop. Similar to related KPC variants, KPC-82 showed decreased carbapenemase activity when expressed in a heterologous background and remained susceptible to carbapenem/ß-lactamase inhibitor combinations.

Switching Shiga Toxin (Stx) Type from Stx2d to Stx2a but Not Stx2c Alters Virulence of Stx-Producing Escherichia coli (STEC) Strain B2F1 in Streptomycin (Str)-Treated Mice.

Shiga toxin (Stx)-producing Escherichia coli (STEC) strain B2F1 produces Stx type 2d, a toxin that becomes more toxic towards Vero cells in the presence of intestinal mucus. STEC that make Stx2d are more pathogenic to streptomycin (Str)-treated mice than most STEC that produce Stx2a or Stx2c. However, purified Stx2d is only 2- or 7-fold more toxic by the intraperitoneal route than Stx2a or Stx2c, respectively. We hypothesized, therefore, that the toxicity differences among Stx2a, Stx2c, and Stx2d occur at the level of delivery from the intestine. To evaluate that hypothesis, we altered the toxin type produced by stx2d+ mouse virulent O91:H21 clinical isolate B2F1 to Stx2a or Stx2c. Because B2F1 encodes two copies of stx2d, we did these studies in a derivative of B2F1 in which stx2d1 was deleted. Although the strains were equivalently virulent to the Str-treated mice at the 1010 dose, the B2F1 strain that produced Stx2a was attenuated relative to the ones that produced Stx2d or Stx2c when administered at 103 CFU/mouse. We next compared the oral toxicities of purified Stx2a, Stx2c, and Stx2d. We found that purified Stx2d is more toxic than Stx2a or Stx2c upon oral administration at 4 µg/mouse. Taken together, these studies suggest that Stx2 toxins are most potent when delivered directly from the bacterium. Furthermore, because Stx2d and Stx2c have the identical amino acid composition in the toxin B subunit, our results indicate that the virulence difference between Stx2a and Stx2d and Stx2c resides in the B or binding subunit of the toxins.

Emergence of colistin-resistance in extremely drug-resistant Acinetobacter baumannii containing a novel pmrCAB operon during colistin therapy of wound infections.

BACKGROUND: Colistin resistance is of concern since it is increasingly needed to treat infections caused by bacteria resistant to all other antibiotics and has been associated with poorer outcomes. Longitudinal data from in vivo series are sparse. METHODS: Under a quality-improvement directive to intensify infection-control measures, extremely drug-resistant (XDR) bacteria undergo phenotypic and molecular analysis. RESULTS: Twenty-eight XDR Acinetobacter baumannii isolates were longitudinally recovered during colistin therapy. Fourteen were susceptible to colistin, and 14 were resistant to colistin. Acquisition of colistin resistance did not alter resistance to other antibiotics. Isolates had low minimum inhibitory concentrations of an investigational aminoglycoside, belonged to multi-locus sequence type 94, were indistinguishable by pulsed-field gel electrophoresis and optical mapping, and harbored a novel pmrC1A1B allele. Colistin resistance was associated with point mutations in the pmrA1 and/or pmrB genes. Additional pmrC homologs, designated eptA-1 and eptA-2, were at distant locations from the operon. Compared with colistin-susceptible isolates, colistin-resistant isolates displayed significantly enhanced expression of pmrC1A1B, eptA-1, and eptA-2; lower growth rates; and lowered fitness. Phylogenetic analysis suggested that colistin resistance emerged from a single progenitor colistin-susceptible isolate. CONCLUSIONS: We provide insights into the in vivo evolution of colistin resistance in a series of XDR A. baumannii isolates recovered during therapy of infections and emphasize the importance of antibiotic stewardship and surveillance.

Bacterial peritonitis due to Acinetobacter baumannii sequence type 25 with plasmid-borne new delhi metallo-ß-lactamase in Honduras.

A carbapenem-resistant Acinetobacter baumannii strain was isolated from the peritoneal fluid of a patient with complicated intra-abdominal infection and evaluated at the Multidrug-resistant Organism Repository and Surveillance Network by whole-genome sequencing and real-time PCR. The isolate was sequence type 25 and susceptible to colistin and minocycline, with low MICs of tigecycline. blaNDM-1 was located on a plasmid with >99% homology to pNDM-BJ02. The isolate carried numerous other antibiotic resistance genes, including the 16S methylase gene, armA.