The lion's share of bacteria in various environments cannot be cloned in the laboratory and thus cannot be sequenced using existing technologies. A major goal of single-cell genomics is to complement gene-centric metagenomic data with whole-genome assemblies of uncultivated organisms. Assembly of single-cell data is challenging because of highly non-uniform read coverage as well as elevated levels of sequencing errors and chimeric reads. We describe SPAdes, a new assembler for both single-cell and standard (multicell) assembly, and demonstrate that it improves on the recently released E+V-SC assembler (specialized for single-cell data) and on popular assemblers Velvet and SoapDeNovo (for multicell data). SPAdes generates single-cell assemblies, providing information about genomes of uncultivatable bacteria that vastly exceeds what may be obtained via traditional metagenomics studies. SPAdes is available online ( http://bioinf.spbau.ru/spades ). It is distributed as open source software.

SPAdes, a new genome assembly algorithm and its applications to single-cell sequencing ( 7th Annual SFAF Meeting, 2012)

Summary Background Until now, polymyxin resistance has involved chromosomal mutations but has never been reported via horizontal gene transfer. During a routine surveillance project on antimicrobial resistance in commensal Escherichia coli from food animals in China, a major increase of colistin resistance was observed. When an E coli strain, SHP45, possessing colistin resistance that could be transferred to another strain, was isolated from a pig, we conducted further analysis of possible plasmid-mediated polymyxin resistance. Herein, we report the emergence of the first plasmid-mediated polymyxin resistance mechanism, MCR-1, in Enterobacteriaceae. Methods The mcr-1 gene in E coli strain SHP45 was identified by whole plasmid sequencing and subcloning. MCR-1 mechanistic studies were done with sequence comparisons, homology modelling, and electrospray ionisation mass spectrometry. The prevalence of mcr-1 was investigated in E coli and Klebsiella pneumoniae strains collected from five provinces between April, 2011, and November, 2014. The ability of MCR-1 to confer polymyxin resistance in vivo was examined in a murine thigh model. Findings Polymyxin resistance was shown to be singularly due to the plasmid-mediated mcr-1 gene. The plasmid carrying mcr-1 was mobilised to an E coli recipient at a frequency of 10 −1 to 10 −3 cells per recipient cell by conjugation, and maintained in K pneumoniae and Pseudomonas aeruginosa . In an in-vivo model, production of MCR-1 negated the efficacy of colistin. MCR-1 is a member of the phosphoethanolamine transferase enzyme family, with expression in E coli resulting in the addition of phosphoethanolamine to lipid A. We observed mcr-1 carriage in E coli isolates collected from 78 (15%) of 523 samples of raw meat and 166 (21%) of 804 animals during 2011–14, and 16 (1%) of 1322 samples from inpatients with infection. Interpretation The emergence of MCR-1 heralds the breach of the last group of antibiotics, polymyxins, by plasmid-mediated resistance. Although currently confined to China, MCR-1 is likely to emulate other global resistance mechanisms such as NDM-1. Our findings emphasise the urgent need for coordinated global action in the fight against pan-drug-resistant Gram-negative bacteria. Funding Ministry of Science and Technology of China, National Natural Science Foundation of China.

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Emergence of plasmid-mediated colistin resistance mechanism MCR-1 in animals and human beings in China: A microbiological and molecular biological study

The data in the interpretive tables in this supplement are valid only if the methodologies in the following Clinical and Laboratory Standards Institute (CLSI)–approved standards are followed: M02-A12—Performance Standards for Antimicrobial Disk Susceptibility Tests; Approved Standard—Twelfth Edition; M07-A10—Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria That Grow Aerobically; Approved Standard—Tenth Edition; and M11-A8—Methods for Antimicrobial Susceptibility Testing of Anaerobic Bacteria; Approved Standard—Eighth Edition. The standards contain information about both disk (M02) and dilution (M07 and M11) test procedures for aerobic and anaerobic bacteria. Clinicians depend heavily on information from the microbiology laboratory for treatment of their seriously ill patients. The clinical importance of antimicrobial susceptibility test results demands that these tests be performed under optimal conditions and that laboratories have the capability to provide results for the newest antimicrobial agents. The tabular information presented here represents the most current information for drug selection, interpretation, and QC using the procedures standardized in the most current editions of M02, M07, and M11. Users should replace the tables published earlier with these new tables. (Changes in the tables since the previous edition appear in boldface type.) Clinical and Laboratory Standards Institute (CLSI). Performance Standards for Antimicrobial Susceptibility Testing. 27th ed. CLSI supplement M100 (ISBN 1-56238-804-5 [Print]; ISBN 1-56238-805-3 [Electronic]). Clinical and Laboratory Standards Institute, 950 West Valley Road, Suite 2500, Wayne, Pennsylvania 19087 USA, 2017. The Clinical and Laboratory Standards Institute consensus process, which is the mechanism for moving a document through two or more levels of review by the health care community, is an ongoing process. Users should expect revised editions of any given document. Because rapid changes in technology may affect the procedures, methods, and protocols in a standard or guideline, users should replace outdated editions with the current editions of CLSI documents. Current editions are listed in the CLSI catalog and posted on our website at www.clsi.org. If you or your organization is not a member and would like to become one, and to request a copy of the catalog, contact us at: Telephone: +1.610.688.0100; Fax: +1.610.688.0700; E-Mail: customerservice@clsi.org; Website: www.clsi.org. M100S, 26th ed. January 2016 Replaces M100-S25 Performance Standards for Antimicrobial Susceptibility Testing Jean B. Patel, PhD, D(ABMM) Franklin R. Cockerill III, MD George M. Eliopoulos, MD Stephen G. Jenkins, PhD, D(ABMM), F(AAM) James S. Lewis II, PharmD Brandi Limbago, PhD David P. Nicolau, PharmD, FCCP, FIDSA Robin Patel, MD M ir Pow ll, MD, FRCP, FRCPath Sandra S. Richter, MD, D(ABMM) Jana M. Swenson, MMSc Maria M. Traczewski, BS, MT(ASCP) John D. Turnidge, MD Melvi P. Weinstein, MD Barbara L. Zimmer, PhD

Performance standards for antimicrobial susceptibility testing

Extended-spectrum β-lactamases (ESBLs) are a rapidly evolving group of β-lactamases which share the ability to hydrolyze third-generation cephalosporins and aztreonam yet are inhibited by clavulanic acid. Typically, they derive from genes for TEM-1, TEM-2, or SHV-1 by mutations that alter the amino acid configuration around the active site of these β-lactamases. This extends the spectrum of β-lactam antibiotics susceptible to hydrolysis by these enzymes. An increasing number of ESBLs not of TEM or SHV lineage have recently been described. The presence of ESBLs carries tremendous clinical significance. The ESBLs are frequently plasmid encoded. Plasmids responsible for ESBL production frequently carry genes encoding resistance to other drug classes (for example, aminoglycosides). Therefore, antibiotic options in the treatment of ESBL-producing organisms are extremely limited. Carbapenems are the treatment of choice for serious infections due to ESBL-producing organisms, yet carbapenem-resistant isolates have recently been reported. ESBL-producing organisms may appear susceptible to some extended-spectrum cephalosporins. However, treatment with such antibiotics has been associated with high failure rates. There is substantial debate as to the optimal method to prevent this occurrence. It has been proposed that cephalosporin breakpoints for the Enterobacteriaceae should be altered so that the need for ESBL detection would be obviated. At present, however, organizations such as the Clinical and Laboratory Standards Institute (formerly the National Committee for Clinical Laboratory Standards) provide guidelines for the detection of ESBLs in klebsiellae and Escherichia coli. In common to all ESBL detection methods is the general principle that the activity of extended-spectrum cephalosporins against ESBL-producing organisms will be enhanced by the presence of clavulanic acid. ESBLs represent an impressive example of the ability of gram-negative bacteria to develop new antibiotic resistance mechanisms in the face of the introduction of new antimicrobial agents.

Extended-spectrum beta-lactamases: a clinical update.

Infectious Diseases Society of America.

The precautions described by Emma have only been put into place after it became evident that certain strains of the Burkholderia cepaciacomplex (Bcc) not only cause life-threatening lung damage in Cystic Fibrosis (CF), but may be transmissible between patients. CF is a multisystem, inherited genetic disorder caused by an abnormality in sodium and chloride ion transport across cell membranes. The Bcc consists of Gram-negative, oxidase-positive bacilli and currently comprises some closely related species, formerly known as genomovars. It is thought that most Bcc infections in CF patients are acquired independently from the environment, although siblings typically share the same strain, and certain strains have been associated with a more widespread cross-infection risk. Transient infections with these organisms occur rarely, with the majority of patients becoming chronically infected. Lung transplant is a potential treatment for some patients with CF lung disease but is associated with a poor outcome in patients infected with Bcc.

Burkholderia Cepacia Complex (BCC) in Cystic Fibrosis

Introduction. Klebsiella species are some of those most implicated in neonatal sepsis. However, many isolates from infections appear unremarkable; they are generally susceptible to antibiotics and often of sporadic types not associated with virulence.Hypothesis/Gap Statement. Investigation is needed to identify if such isolates have virulence characteristics.Aim. To sequence multiple isolates of a range of types from cases of neonatal invasive disease to identify elements that may explain their virulence, and to determine if such elements are more common among these isolates than generally.Methodology. In total, 14 isolates of K. pneumoniae/K. variicola belonging to 13 distinct types from blood or CSF from neonatal infections were sequenced using long-read nanopore technology. PCR assays were used to screen a general set of isolates for heavy metal resistance genes arsC, silS and merR.Results. Overall, 12/14 isolates carried one or more plasmids. Ten carried a large plasmid (186 to 310 kb) containing heavy metal resistance genes associated with hypervirulence plasmids, with most (nine) carrying genes for resistance to copper, silver and one other heavy metal (arsenic, tellurite or mercury), but lacking the genes encoding capsule-upregulation and siderophores. Most isolates (9/14) lacked any additional antibiotic resistance genes other than those intrinsic in the species. However, a representative of an outbreak strain carried a plasmid containing bla CTX-M-15, qnrS1, aac3_IIa, dfrA17, sul1, mph(A), tet(A), bla TEM1B and aadA5, but no heavy metal resistance genes. arsC, silS and merR were widely found among 100 further isolates screened, with most carbapenemase-gene-positive isolates (20/27) carrying at least one.Conclusion. Plasmids containing heavy metal resistance genes were a striking feature of isolates from neonatal sepsis but are widely found. They share elements in common with virulence and antibiotic resistance plasmids, perhaps providing a basis from which such plasmids evolve.

Do plasmids containing heavy metal resistance genes play a role in neonatal sepsis and invasive disease caused by Klebsiella pneumoniae and Klebsiella variicola?

182 genes associated with virulence were sought from the whole genome sequences of all non-duplicate isolates of Escherichia coli received by the UK Health Security Agency’s Antimicrobial Resistance and HealthCare Associated Infections laboratory for typing between June 2019 and March 2021 from hospitals in the United Kingdom and Republic of Ireland (n=593). These were from healthcare associated investigations and were not associated with diarrhoeal disease. Genes that were very common or very rare were excluded from further analysis. The frequency of detection of genes was compared among isolates from invasive infection, screening, urine samples and from neonates. cnf1 (coding for cytotoxic necrotising factor), clbK (coding for colibactin), focCDF (coding for F1 fimbriae), kpsM_K1, neuBD (associated with the K1 capsule), mchBC, mcmA (coding for microcins), papG_alleleIII (part of a cluster encoding P fimbriae), pic (protein involved in intestinal colonization), sfaE/sfafoCDE (coding for S fimbriae), tcpC (encoding TLR domain containing-protein C) and vat (encoding toxin vacuolating autotransporter) were 4 to 28 times more prevalent among isolates from invasive infections than among those from carriage. Representatives of sequence types (ST) 12, 73, 998 and 127 carried multiple of these virulence factors, no matter whether they were from screening swabs or from blood or other infection sites. Isolates carrying multiple virulence factors were more prevalent from neonatal screens than those from general screens. Genes associated with the K1 capsule (ibeA, neuBD, kpsM_K1) were particularly found in STs 1193, 10, 998, 538, 80 and 141. Nanopore sequencing of 14 isolates representing 10 different STs showed that the virulence elements sought were largely carried in chromosomal genomic islands, which were mosaic in nature. Some 9 % of isolates carried more than six of the main virulence genes/gene sets sought, highlighting the potential for significant numbers of carriage isolates to cause extraintestinal infections.

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Virulence factors among isolates of extraintestinal Esherichia coli (ExPEC) from hospitals in the United Kingdom

Investigating Pseudomonas aeruginosa population structure and frequency of cross-infection in UK cystic fibrosis clinics - a reference laboratory perspective.

Jane F. Turton

Papers

Burkholderia Cepacia Complex (BCC) in Cystic Fibrosis

Do plasmids containing heavy metal resistance genes play a role in neonatal sepsis and invasive disease caused by Klebsiella pneumoniae and Klebsiella variicola?

Virulence factors among isolates of extraintestinal Esherichia coli (ExPEC) from hospitals in the United Kingdom

Investigating Pseudomonas aeruginosa population structure and frequency of cross-infection in UK cystic fibrosis clinics - a reference laboratory perspective.