Anatomy of an extensively drug-resistant Klebsiella pneumoniae outbreak in Tuscany, Italy.

A protracted outbreak of New Delhi metallo-ß-lactamase (NDM)-producing carbapenem-resistant Klebsiella pneumoniae started in Tuscany, Italy, in November 2018 and continued in 2020 and through 2021. To understand the regional emergence and transmission dynamics over time, we collected and sequenced the genomes of 117 extensively drug-resistant, NDM-producing K. pneumoniae isolates cultured over a 20-mo period from 76 patients at several healthcare facilities in southeast Tuscany. All isolates belonged to high-risk clone ST-147 and were typically nonsusceptible to all first-line antibiotics. Albeit sporadic, resistances to colistin, tigecycline, and fosfomycin were also observed as a result of repeated, independent mutations. Genomic analysis revealed that ST-147 isolates circulating in Tuscany were monophyletic and highly genetically related (including a network of 42 patients from the same hospital and sharing nearly identical isolates), and shared a recent ancestor with clinical isolates from the Middle East. While the blaNDM-1 gene was carried by an IncFIB-type plasmid, our investigations revealed that the ST-147 lineage from Italy also acquired a hybrid IncFIB/IncHIB-type plasmid carrying the 16S methyltransferase armA gene as well as key virulence biomarkers often found in hypervirulent isolates. This plasmid shared extensive homologies with mosaic plasmids circulating globally including from ST-11 and ST-307 convergent lineages. Phenotypically, the carriage of this hybrid plasmid resulted in increased siderophore production but did not confer virulence to the level of an archetypical, hypervirulent K. pneumoniae in a subcutaneous model of infection with immunocompetent CD1 mice. Our findings highlight the importance of performing genomic surveillance to identify emerging threats.

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.

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.

Chromosomally Encoded mcr-5 in Colistin-Nonsusceptible Pseudomonas aeruginosa.

Whole-genome sequencing (WGS) of historical Pseudomonas aeruginosa clinical isolates identified a chromosomal copy of mcr-5 within a Tn3-like transposon in P. aeruginosa MRSN 12280. The isolate was nonsusceptible to colistin by broth microdilution, and genome analysis revealed no mutations known to confer colistin resistance. To the best of our knowledge, this is the first report of mcr in colistin-nonsusceptible P. aeruginosa.

Detecting 16S rRNA Methyltransferases in Enterobacteriaceae by Use of Arbekacin.

16S rRNA methyltransferases confer resistance to most aminoglycosides, but discriminating their activity from that of aminoglycoside-modifying enzymes (AMEs) is challenging using phenotypic methods. We demonstrate that arbekacin, an aminoglycoside refractory to most AMEs, can rapidly detect 16S methyltransferase activity in Enterobacteriaceae with high specificity using the standard disk susceptibility test.

Genomic and transcriptomic differences in community acquired methicillin resistant Staphylococcus aureus USA300 and USA400 strains.

BACKGROUND: Staphylococcus aureus is a human pathogen responsible for substantial morbidity and mortality through its ability to cause a number of human infections including bacteremia, pneumonia and soft tissue infections. Of great concern is the emergence and dissemination of methicillin-resistant Staphylococcus aureus strains (MRSA) that are resistant to nearly all ß-lactams. The emergence of the USA300 MRSA genetic background among community associated S. aureus infections (CA-MRSA) in the USA was followed by the disappearance of USA400 CA-MRSA isolates. RESULTS: To gain a greater understanding of the potential fitness advantages and virulence capacity of S. aureus USA300 clones, we performed whole genome sequencing of 15 USA300 and 4 USA400 clinical isolates. A comparison of representative genomes of the USA300 and USA400 pulsotypes indicates a number of differences in mobile genome elements. We examined the in vitro gene expression profiles by microarray hybridization and the in vivo transcriptomes during lung infection in mice of a USA300 and a USA400 MRSA strain by performing complete genome qRT-PCR analysis. The unique presence and increased expression of 6 exotoxins in USA300 (12- to 600-fold) compared to USA400 may contribute to the increased virulence of USA300 clones. Importantly, we also observed the up-regulation of prophage genes in USA300 (compared with USA400) during mouse lung infection (including genes encoded by both prophages ΦSa2usa and ΦSa3usa), suggesting that these prophages may play an important role in vivo by contributing to the elevated virulence characteristic of the USA300 clone. CONCLUSIONS: We observed differences in the genetic content of USA300 and USA400 strains, as well as significant differences of in vitro and in vivo gene expression of mobile elements in a lung pneumonia model. This is the first study to document the global transcription differences between USA300 and USA400 strains during both in vitro and in vivo growth.

The Emergence and Persistence of Candida auris in Western New York With No Epidemiologic Links: A Failure of Stewardship?

Reports of Candida auris infection in patients without epidemiologic links to prior outbreaks are scarce. We describe the genomic epidemiology of such a case in Western New York. Before emergence, the patient received >60 days of excess antibiotics. Candida auris was recovered on near-patient surfaces after enhanced terminal cleanings.

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 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.

Detection of diverse carbapenem and multidrug resistance genes and high-risk strain types among carbapenem non-susceptible clinical isolates of target gram-negative bacteria in Kenya.

Carbapenem-resistant gram-negative bacteria are an increasingly significant clinical threat globally. This risk may be underestimated in Kenya as only four carbapenemase genes in three bacterial species have been described. The study aimed to understand the antibiotic resistance profiles, genes, sequence types, and distribution of carbapenem-resistant gram-negative bacteria from patients in six hospitals across five Kenyan counties by bacterial culture, antibiotic susceptibility testing, and whole-genome sequence analysis. Forty-eight, non-duplicate, carbapenem non-susceptible, clinical isolates were identified across the five counties (predominantly in Nairobi and Kisii): twenty-seven Acinetobacter baumannii, fourteen Pseudomonas aeruginosa, three Escherichia coli, two Enterobacter cloacae, and two Klebsiella pneumoniae. All isolates were non-susceptible to ß-lactam drugs with variable susceptibility to tigecycline (66%), minocycline (52.9%), tetracycline (29.4%), and levofloxacin (22.9%). Thirteen P. aeruginosa isolates were resistant to all antibiotics tested. Eleven carbapenemase genes were identified: blaNDM-1, blaOXA-23, -58, -66, -69, and -91 in A. baumannii (STs 1, 2, 164 and a novel ST1475), blaNDM-1 in E. cloacae (STs 25,182), blaNDM-1, blaVIM-1and -6, blaOXA-50 in P. aeruginosa (STs 316, 357, 654, and1203), blaOXA-181, blaNDM-1 in K. pneumoniae (STs 147 and 219), and blaNDM-5 in E. coli (ST164). Five A. baumannii isolates had two carbapenemases, blaNDM-1, and either blaOXA-23 (4) or blaOXA-58 (1). AmpC genes were detected in A. baumannii (blaADC-25), E. cloacae (blaDHA-1 and blaACT-6, 16), and K. pneumoniae (blaCMY). Significant multiple-drug resistant genes were the pan-aminoglycoside resistance16srRNA methyltransferase armA, rmtB, rmtC, and rmtF genes. This study is the first to report blaOXA-420, -58, -181, VIM-6, and blaNDM-5 in Kenyan isolates. High-risk STs of A. baumannii (ST1475, ST2), E. cloacae ST182, K. pneumoniae ST147, P. aeruginosa (ST357, 654), and E. coli ST167, ST648 were identified which present considerable therapeutic danger. The study recommends urgent carbapenem use regulation and containment of high-risk carbapenem-resistant bacteria.

A panel of diverse Pseudomonas aeruginosa clinical isolates for research and development.

OBJECTIVES: Pseudomonas aeruginosa is a leading cause of community- and hospital-acquired infections. Successful treatment is hampered by its remarkable ability to rapidly develop resistance to antimicrobial agents, primarily through mutation. In response, WHO listed carbapenem-resistant P. aeruginosa as a Priority 1 (Critical) pathogen for research and development of new treatments. A key resource in developing effective countermeasures is access to diverse and clinically relevant strains for testing. Herein we describe a panel of 100 diverse P. aeruginosa strains to support this endeavour. METHODS: WGS was performed on 3785 P. aeruginosa isolates in our repository. Isolates were cultured from clinical samples collected from healthcare facilities around the world between 2003 and 2017. Core-genome MLST and high-resolution SNP-based phylogenetic analyses were used to select a panel of 100 strains that captured the genetic diversity of this collection. Antibiotic susceptibility testing was also performed using 14 clinically relevant antibiotics. RESULTS: This 100-strain diversity panel contained representative strains from 91 different STs, including genetically distinct strains from major epidemic clones ST-111, ST-235, ST-244 and ST-253. Seventy-one distinct antibiotic susceptibility profiles were identified ranging from pan-susceptible to pan-resistant. Known resistance alleles as well as the most prevalent mutations underlying the antibiotic susceptibilities were characterized for all isolates. CONCLUSIONS: This panel provides a diverse and comprehensive set of P. aeruginosa strains for use in developing solutions to antibiotic resistance. The isolates and available metadata, including genome sequences, are available to industry, academia, federal and other laboratories at no additional cost.

Clinical and Genomic Features of the First Cases of Elizabethkingia anophelis Infection in New York, Including the First Case in a Healthy Infant Without Previous Nosocomial Exposure.

Elizabethkingia spp are Gram-negative bacteria associated with neonatal meningitis. In 2015-2016, an outbreak of Elizabethkingia anophelis infection that involved 63 patients and 18 deaths occurred in Wisconsin. Despite a multistate investigation, as of September 2016 the source remained undetermined, and experts warned of reemergence. We describe here the first cases of E anophelis infection in New York, including the case of a healthy infant without previous healthcare contact.

Importation, Mitigation, and Genomic Epidemiology of Candida auris at a Large Teaching Hospital.

OBJECTIVE Candida auris (CA) is an emerging multidrug-resistant pathogen associated with increased mortality. The environment may play a role, but transmission dynamics remain poorly understood. We sought to limit environmental and patient CA contamination following a sustained unsuspected exposure. DESIGN Quasi-experimental observation. SETTING A 528-bed teaching hospital. PATIENTS The index case patient and 17 collocated ward mates. INTERVENTION Immediately after confirmation of CA in the bloodstream and urine of a patient admitted 6 days previously, active surveillance, enhanced transmission-based precautions, environmental cleaning with peracetic acid-hydrogen peroxide and ultraviolet light, and patient relocation were undertaken. Pre-existing agreements and foundational relationships among internal multidisciplinary teams and external partners were leveraged to bolster detection and mitigation efforts and to provide genomic epidemiology. RESULTS Candida auris was isolated from 3 of 132 surface samples on days 8, 9, and 15 of ward occupancy, and from no patient samples (0 of 48). Environmental and patient isolates were genetically identical (4-8 single-nucleotide polymorphisms [SNPs]) and most closely related to the 2013 India CA-6684 strain (~200 SNPs), supporting the epidemiological hypothesis that the source of environmental contamination was the index case patient, who probably acquired the South Asian strain from another New York hospital. All isolates contained a mutation associated with azole resistance (K163R) found in the India 2105 VPCI strain but not in CA-6684. The index patient remained colonized until death. No surfaces were CA-positive 1 month later. CONCLUSION Compared to previous descriptions, CA dissemination was minimal. Immediate access to rapid CA diagnostics facilitates early containment strategies and outbreak investigations. Infect Control Hosp Epidemiol 2018;39:53-57.

Genomic Characterization of Nonclonal mcr-1-Positive Multidrug-Resistant Klebsiella pneumoniae from Clinical Samples in Thailand.

Multidrug-resistant Klebsiella pneumoniae strains are one of the most prevalent causes of nosocomial infections and pose an increasingly dangerous public health threat. The lack of remaining treatment options has resulted in the utilization of older drug classes, including colistin. As a drug of last resort, the discovery of plasmid-mediated colistin resistance by mcr-1 denotes the potential development of pandrug-resistant bacterial pathogens. To address the emergence of the mcr-1 gene, 118 gram-negative Enterobacteriaceae isolated from clinical samples collected at Queen Sirikit Naval Hospital in Chonburi, Thailand were screened for colistin resistance using automated antimicrobial susceptibility testing and conventional PCR screening. Two K. pneumoniae strains, QS17-0029 and QS17-0161, were positive for mcr-1, and both isolates were sequenced to closure using short- and long-read whole-genome sequencing. QS17-0029 carried 16 antibiotic resistance genes in addition to mcr-1, including 2 carbapenemases, blaNDM-1 and blaOXA-232. QS17-0161 carried 13 antibiotic resistance genes in addition to mcr-1, including the extended-spectrum ß-lactamase blaCTX-M-55. Both isolates carried multiple plasmids, but mcr-1 was located alone on highly similar 33.9 Kb IncX4 plasmids in both isolates. The IncX4 plasmid shared considerable homology to other mcr-1-containing IncX4 plasmids. This is the first report of a clinical K. pneumoniae strain from Thailand carrying mcr-1 as well as the first strain to simultaneously carry mcr-1 and multiple carbapenemase genes (QS17-0029). The identification and characterization of these isolates serves to highlight the urgent need for continued surveillance and intervention in Southeast Asia, where extensively drug-resistant pathogens are being increasingly identified in hospital-associated infections.

Pseudomonas Endocarditis with an unstable phenotype: the challenges of isolate characterization and Carbapenem stewardship with a partial review of the literature.

BACKGROUND: Pseudomonas endocarditis is exceedingly rare, especially in patients without predisposing risks. We present such a case that included unexpected switches in antibacterial resistance profiles in two Pseudomonas aeruginosa (PA) strains with the same whole-genome sequence. The case also involved diagnostic and treatment challenges, such as issues with automated testing platforms, choosing the optimal aminoglycoside, minimizing unnecessary carbapenem exposure, and the need for faster, more informative laboratory tests. CASE PRESENTATION: On hospital day one (HD-1) a cefepime and piperacillin-tazobactam (FEP-TZP)-susceptible P. aeruginosa was isolated from the bloodstream of a 62-year-old man admitted for evaluation of possible endocarditis and treated with gentamicin and cefepime. On HD-2, his antibiotic regimen was changed to tobramycin and cefepime. On HD-11, he underwent aortic valve replacement, and P. aeruginosa was isolated from the explanted valve. Unexpectedly, it was FEP-TZP-resistant, so cefepime was switched to meropenem. On HD-14, in preparation for whole-genome sequencing (WGS), valve and blood isolates were removed from cryo-storage, re-cultured, and simultaneously tested with the same platforms, reagents, and inoculations previously used. Curiously, the valve isolate was now FEP-TZP-susceptible. WGS revealed that both isolates were phylogenetically identical, differing by a single nucleotide in a chemotaxis-encoding gene. They also contained the same resistance genes (blaADC35, aph(3')-II, blaOXA-50, catB7, fosA). CONCLUSION: Repeated testing on alternate platforms and WGS did not definitively determine the resistance mechanism(s), which in this case, is most likely unstable de-repression of a chromosomal AmpC ß-lactamase, porin alterations, or efflux upregulation, with reversion to baseline (non-efflux) transcription. Although sub-culture on specialized media to select for less fit (more resistant) colonies, followed by transcriptome analysis, and multiple sequence alignment, might have revealed the mechanism and better informed the optimal choice of ß-lactam, such approaches are neither rapid, nor feasible for hospital laboratories. In this era of escalating drug resistance and dwindling antibiotics, use of the most potent anti-pseudomonals must be balanced with stewardship. Clinicians need access to validated genomic correlates of resistance, and faster, more informative diagnostics. Therefore, we placed these isolates and their sequences in the public domain for inclusion in the Pseudomonas pan-genome and database projects for further countermeasure development.

Complete Genome Sequence of Staphylococcus epidermidis 1457.

Staphylococcus epidermidis 1457 is a frequently utilized strain that is amenable to genetic manipulation and has been widely used for biofilm-related research. We report here the whole-genome sequence of this strain, which encodes 2,277 protein-coding genes and 81 RNAs within its 2.4-Mb genome and plasmid.