Detection of carbapenemase producing Acinetobacter baumannii ST19 from Georgia and Ukraine carrying bla OXA-23, bla OXA-72, and/or bla NDM-5, December 2019 to June 2023.

In 2003-2023, amid 5,436 Acinetobacter baumannii isolates collected globally through the Multidrug-Resistant Organism Repository and Surveillance Network, 97 were ST19PAS, 34 of which carbapenem-resistant. Strains (n = 32) sampled after 2019 harboured either bla OXA-23, bla OXA-72, and/or bla NDM-5. Phylogenetic analysis of the 97 isolates and 11 publicly available ST19 genomes revealed three sub-lineages of carbapenemase-producing isolates from mainly Ukraine and Georgia, including an epidemic clone carrying all three carbapenemase genes. Infection control and global surveillance of carbapenem-resistant A. baumannii remain important.

Six Extensively Drug-Resistant Bacteria in an Injured Soldier, Ukraine.

Blood and surveillance cultures from an injured service member from Ukraine grew Acinetobacter baumannii, Klebsiella pneumoniae, Enterococcus faecium, and 3 distinct Pseudomonas aeruginosa strains. Isolates were nonsusceptible to most antibiotics and carried an array of antibiotic resistant genes, including carbapenemases (blaIMP-1, blaNDM-1, blaOXA-23, blaOXA-48, blaOXA-72) and 16S methyltransferases (armA and rmtB4).

Identification of an Outbreak Cluster of Extensively Antibiotic-Resistant GC1 Acinetobacter baumannii Isolates in U.S. Military Treatment Facilities.

An outbreak involving an extensively antibiotic-resistant Acinetobacter baumannii strain in three military treatment facilities was identified. Fifty-nine isolates recovered from 30 patients over a 4-year period were found among a large collection of isolates using core genome multilocus sequence typing (MLST). They differed by only 0 to 18 single nucleotide polymorphisms (SNPs) and carried the same resistance determinants except that the aphA6 gene was missing in 25 isolates. They represent a novel sublineage of GC1 lineage 1 that likely originated in Afghanistan. IMPORTANCE A. baumannii is recognized as one of the most important nosocomial pathogens, and carbapenem-resistant strains pose a particularly difficult treatment challenge. Outbreaks linked to this pathogen are reported worldwide, particularly during periods of societal upheaval, such as natural disasters and conflicts. Understanding how this organism enters and establishes itself within the hospital environment is key to interrupting transmission, but few genomic studies have examined these transmissions over a prolonged period. Though historical, this report provides an in-depth analysis of nosocomial transmission of this organism across continents and within and between different hospitals.

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.

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.

Molecular characterization of multidrug-resistant ESKAPEE pathogens from clinical samples in Chonburi, Thailand (2017-2018).

BACKGROUND: ESKAPEE pathogens Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, Enterobacter spp. and Escherichia coli are multi-drug resistant (MDR) bacteria that present increasing treatment challenges for healthcare institutions and public health worldwide. METHODS: 431 MDR ESKAPEE pathogens were collected from Queen Sirikit Naval Hospital, Chonburi, Thailand between 2017 and 2018. Species identification and antimicrobial resistance (AMR) phenotype were determined following CLSI and EUCAST guidelines on the BD Phoenix System. Molecular identification of antibiotic resistant genes was performed by polymerase chain reaction (PCR), real-time PCR assays, and whole genome sequencing (WGS). RESULTS: Of the 431 MDR isolates collected, 1.2% were E. faecium, 5.8% were S. aureus, 23.7% were K. pneumoniae, 22.5% were A. baumannii, 4.6% were P. aeruginosa, 0.9% were Enterobacter spp., and 41.3% were E. coli. Of the 401 Gram-negative MDR isolates, 51% were carbapenem resistant, 45% were ESBL producers only, 2% were colistin resistance and ESBLs producers (2%), and 2% were non-ESBLs producers. The most prevalent carbapenemase genes were blaOXA-23 (23%), which was only identified in A. baumannii, followed by blaNDM (17%), and blaOXA-48-like (13%). Beta-lactamase genes detected included blaTEM, blaSHV, blaOXA, blaCTX-M, blaDHA, blaCMY, blaPER and blaVEB. Seven E. coli and K. pneumoniae isolates showed resistance to colistin and carried mcr-1 or mcr-3, with 2 E. coli strains carrying both genes. Among 30 Gram-positive MDR ESKAPEE, all VRE isolates carried the vanA gene (100%) and 84% S. aureus isolates carried the mecA gene. CONCLUSIONS: This report highlights the prevalence of AMR among clinical ESKAPEE pathogens in eastern Thailand. E. coli was the most common MDR pathogen collected, followed by K. pneumoniae, and A. baumannii. Carbapenem-resistant Enterobacteriaceae (CRE) and extended spectrum beta-lactamases (ESBLs) producers were the most common resistance profiles. The co-occurrence of mcr-1 and mcr-3 in 2 E. coli strains, which did not affect the level of colistin resistance, is also reported. The participation of global stakeholders and surveillance of MDR remain essential for the control and management of MDR ESKAPEE pathogens.

Pan-drug resistant Providencia rettgeri contributing to a fatal case of COVID-19.

Following prolonged hospitalization that included broad-spectrum antibiotic exposure, a strain of Providencia rettgeri was cultured from the blood of a patient undergoing extracorporeal membrane oxygenation treatment for hypoxic respiratory failure due to COVID-19. The strain was resistant to all antimicrobials tested including the novel siderophore cephalosporin, cefiderocol. Whole genome sequencing detected ten antimicrobial resistance genes, including the metallo-ß-lactamase bla NDM-1, the extended-spectrum ß-lactamase bla PER-1, and the rare 16S methyltransferase rmtB2.

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.

Colistin-resistant Klebsiella pneumoniae bloodstream infection: old drug, bad bug.

Multi-drug-resistant (MDR) Enterobacteriaceae pose a global threat to hospitalized patients. We report a series of colistin-resistant Klebsiella pneumoniae blood isolates from Israel and explore their resistance mechanisms using whole genome sequencing (WGS). Patients with colistin-resistant K. pneumoniae bloodstream infection (BSI) were identified during the period between 2006 and 2018. Demographic and clinical data were collected, and antibiotic susceptibility testing (AST) was performed using three commercial platforms. Long and short read sequencing were performed on a PacBio RS II (Pacific Biosciences) and an Illumina Miseq (Illumina), respectively. Thirteen patients with colistin-resistant K. pneumoniae BSI were identified, and seven isolates from seven different patients were successfully revived. Patient records indicated that five of the patients were previously treated with colistin. AST indicated that six of the seven isolates were colistin resistant and four of these isolates were resistant to carbapenems. WGS assigned the isolates to four distinct clusters that corresponded to in silico-derived multi-locus sequence types (MLST). Three isolates carried blaKPC-3 on two different plasmids and one carried blaOXA-48 on a novel IncL/M plasmid. All colistin-resistant isolates carried a variety of different mutations that inactivated the mgrB gene. We report the first comprehensive analysis of a series of colistin-resistant K. pneumoniae from Israel. A diverse set of isolates were obtained and colistin resistance was found to be attributed to different mechanisms that ablated the mgrB gene. Notably, carbapenemase genes were identified in four isolates and were carried on novel plasmids.

A Diverse Panel of Clinical Acinetobacter baumannii for Research and Development.

Over the past two decades, Acinetobacter baumannii has emerged as a leading cause of nosocomial infections worldwide. Of particular concern are panresistant strains, leading the World Health Organization (WHO) to designate carbapenem-resistant A. baumannii as a priority 1 (critical) pathogen for research and development of new antibiotics. A key component in supporting this effort is accessibility to diverse and clinically relevant strains for testing. Here, we describe a panel of 100 diverse A. baumannii strains for use in this endeavor. Whole-genome sequencing was performed on 3,505 A. baumannii isolates housed at the Multidrug-Resistant Organism Repository and Surveillance Network. Isolates were cultured from clinical samples at health care facilities around the world between 2001 and 2017. Core-genome multilocus sequence typing and high-resolution single nucleotide polymorphism (SNP)-based phylogenetic analyses were used to select a final panel of 100 strains that captured the genetic diversity of the collection. Comprehensive antibiotic susceptibility testing was also performed on all 100 isolates using 14 clinically relevant antibiotics. The final 100-strain diversity panel contained representative strains from 70 different traditional Pasteur scheme multilocus sequence types, including major epidemic clones. This diversity was also reflected in antibiotic susceptibility and antimicrobial resistance (AMR) gene content, with phenotypes ranging from pansensitive to panresistant, and over 100 distinct AMR gene alleles identified from 32 gene families. This panel provides the most diverse and comprehensive set of A. baumannii strains for use in developing solutions for combating antibiotic resistance. The panel and all available metadata, including genome sequences, will be available to industry and academic institutions and federal and other laboratories free of charge.

Rapid and simultaneous detection of the chlorhexidine and mupirocin resistance genes qacA/B and mupA in clinical isolates of methicillin-resistant Staphylococcus aureus.

We describe a real-time PCR-based assay capable of simultaneously detecting femA (Staphylococcus aureus-specific), mecA (methicillin resistance), qacA/B (chlorhexidine tolerance), and mupA (high-level mupirocin resistance) from bacterial cells in less than 90 minutes. The assay was validated with 1968 clinical MRSA submitted to a surveillance network.

Detection of bacterial 16S rRNA and identification of four clinically important bacteria by real-time PCR.

Within the paradigm of clinical infectious disease research, Acinetobacter baumannii, Escherichia coli, Klebsiella pneumoniae, and Pseudomonas aeruginosa represent the four most clinically relevant, and hence most extensively studied bacteria. Current culture-based methods for identifying these organisms are slow and cumbersome, and there is increasing need for more rapid and accurate molecular detection methods. Using bioinformatic tools, 962,279 bacterial 16S rRNA gene sequences were aligned, and regions of homology were selected to generate a set of real-time PCR primers that target 93.6% of all bacterial 16S rRNA sequences published to date. A set of four species-specific real-time PCR primer pairs were also designed, capable of detecting less than 100 genome copies of A. baumannii, E. coli, K. pneumoniae, and P. aeruginosa. All primers were tested for specificity in vitro against 50 species of Gram-positive and -negative bacteria. Additionally, the species-specific primers were tested against a panel of 200 clinical isolates of each species, randomly selected from a large repository of clinical isolates from diverse areas and sources. A comparison of culture and real-time PCR demonstrated 100% concordance. The primers were incorporated into a rapid assay capable of positive identification from plate or broth cultures in less than 90 minutes. Furthermore, our data demonstrate that current targets, such as the uidA gene in E.coli, are not suitable as species-specific genes due to sequence variation. The assay described herein is rapid, cost-effective and accurate, and can be easily incorporated into any research laboratory capable of real-time PCR.

A novel brain heart infusion broth supports the study of common Francisella tularensis serotypes.

Francisella tularensis Schu S4, LVS and U112 have become model organisms for the study of Francisella pathogenesis, and represent a cross section of the different F. tularensis subspecies. Both Schu S4 and LVS are fastidious organisms, requiring medium fortified with supplements and nutrients for enhanced growth. Chamberlains defined medium, Tryptone Soy Broth supplemented with cysteine (TSBc), and cation-adjusted Mueller-Hinton broth (CAMHB) supplemented with 2% IsoVitaleX are typically used in the cultivation of these bacteria. In this report, we describe a simple brain heart infusion broth formulation that can be used to obtain superior growth characteristics in all of these model organisms, and can support bacterial growth from low inoculum. Surprisingly, CAMHB, which is favored in the literature for culturing Schu S4 and LVS, induced the worst growth characteristics of the four formulations studied. To expand on these observations, an additional seven strains of F. tularensis, representing types A.I, A.II, and B were selected from the Department of Defense United Culture Collection (UCC) and a comparative analysis of their growth characteristics performed in the four broth formulations. Results demonstrate differences in the growth characteristics of Francisella species that are significantly influenced by both strain type and the choice of growth medium. Though four of the five additional Type A strains displayed superior growth characteristics in Chamberlain's defined medium, growth characteristics of all three model organisms, as well the Type B strains, were enhanced by the new BHI-based broth formulation. We conclude that this medium represents the optimal choice for cultivation of the three model organisms used for Francisella research.