Genetic determinants of resistance to antimicrobial therapeutics are rare in publicly available Clostridioides difficile genome sequences.

OBJECTIVES: To determine the frequencies and clonal distributions of putative genetic determinants of resistance to antimicrobials applied for treatment of Clostridioides difficile infection (CDI), as documented in the genomic record. METHODS: We scanned 26 557 C. difficile genome sequences publicly available from the EnteroBase platform for plasmids, point mutations and gene truncations previously reported to reduce susceptibility to vancomycin, fidaxomicin or metronidazole, respectively. We measured the antimicrobial susceptibility of 143 selected C. difficile isolates. RESULTS: The frequency of mutations causing reduced susceptibility to vancomycin and metronidazole, respectively, increased strongly after 2000, peaking at up to 52% of all sequenced C. difficile genomes. However, both mutations declined sharply more recently, reflecting major changes in CDI epidemiology. We detected mutations associated with fidaxomicin resistance in several major genotypes, but found no evidence of international spread of resistant clones. The pCD-METRO plasmid, conferring metronidazole resistance, was detected in a single previously unreported C. difficile isolate, recovered from a hospital patient in Germany in 2008. The pX18-498 plasmid, putatively associated with decreased vancomycin susceptibility, was confined to related, recent isolates from the USA. Phenotype measurements confirmed that most of those genetic features were useful predictors of antibiotic susceptibility, even though ranges of MICs typically overlapped among isolates with and without specific mutations. CONCLUSIONS: Genomic data suggested that resistance to therapeutic antimicrobial drugs is rare in C. difficile. Public antimicrobial resistance marker databases were not equipped to detect most of the genetic determinants relevant to antibiotic therapy of CDI.

The best of both worlds: a proposal for further integration of Candidatus names into the International Code of Nomenclature of Prokaryotes.

The naming of prokaryotes is governed by the International Code of Nomenclature of Prokaryotes (ICNP) and partially by the International Code of Nomenclature for Algae, Fungi and Plants (ICN). Such codes must be able to determine names of taxa in a universal and unambiguous manner, thus serving as a common language across different fields and activities. This unity is undermined when a new code of nomenclature emerges that overlaps in scope with an established, time-tested code and uses the same format of names but assigns different nomenclatural status values to the names. The resulting nomenclatural confusion is not beneficial to the wider scientific community. Such ambiguity is expected to result from the establishment of the 'Code of Nomenclature of Prokaryotes Described from DNA Sequence Data' ('SeqCode'), which is in general and specific conflict with the ICNP and the ICN. Shortcomings in the interpretation of the ICNP may have exacerbated the incompatibility between the codes. It is reiterated as to why proposals to accept sequences as nomenclatural types of species and subspecies with validly published names, now implemented in the SeqCode, have not been implemented by the International Committee on Systematics of Prokaryotes (ICSP), which oversees the ICNP. The absence of certain regulations from the ICNP for the naming of as yet uncultivated prokaryotes is an acceptable scientific argument, although it does not justify the establishment of a separate code. Moreover, the proposals rejected by the ICSP are unnecessary to adequately regulate the naming of uncultivated prokaryotes. To provide a better service to the wider scientific community, an alternative proposal to emend the ICNP is presented, which would result in Candidatus names being regulated analogously to validly published names. This proposal is fully consistent with previous ICSP decisions, preserves the essential unity of nomenclature and avoids the expected nomenclatural confusion.

Bacterial genome sequencing tracks the housefly-associated dispersal of fluoroquinolone- and cephalosporin-resistant Escherichia coli from a pig farm.

The regular use of antimicrobials in livestock production selects for antimicrobial resistance. The potential impact of this practice on human health needs to be studied in more detail, including the role of the environment for the persistence and transmission of antimicrobial-resistant bacteria. During an investigation of a pig farm and its surroundings in Brandenburg, Germany, we detected abundant cephalosporin- and fluoroquinolone-resistant Escherichia coli in pig faeces, sedimented dust, and house flies (Musca domestica). Genome sequencing of E. coli isolates revealed large phylogenetic diversity and plasmid-borne extended-spectrum beta lactamase (ESBL) genes CTX-M-1 in multiple strains. [Correction added on 28 February 2023, after first online publication: In the preceding sentence, 'and TEM-1' was previously included but has been deleted in this version.] Close genomic relationships indicated frequent transmission of antimicrobial-resistant E. coli between pigs from different herds and across buildings of the farm and suggested dust and flies as vectors for dissemination of faecal pathogens. Strikingly, we repeatedly recovered E. coli from flies collected up to 2 km away from the source, whose genome sequences were identical or closely related to those from pig faeces isolates, indicating the fly-associated transport of diverse ESBL-producing E. coli from the pig farm into urban habitation areas. The observed proximity of contaminated flies to human households poses a risk of transmission of antimicrobial-resistant enteric pathogens from livestock to man.

Low airborne tenacity and spread of ESBL-/AmpC-producing Escherichia coli from fertilized soil by wind erosion.

ESBL-/AmpC-producing Escherichia coli from organic fertilizers were previously detected on soil surfaces of arable land and might be emitted by wind erosion. To investigate this potential environmental transmission path, we exposed ESBL-/AmpC-positive chicken litter, incorporated in agricultural soils, to different wind velocities in a wind tunnel and took air samples for microbiological analysis. No data exist concerning the airborne tenacity of ESBL-/AmpC-producing E. coli. Therefore, we explored the tenacity of two ESBL/AmpC E. coli strains and E. coli K12 in aerosol chamber experiments at different environmental conditions. In the wind tunnel, ESBL/AmpC-producing E. coli were detected in none of the air samples (n = 66). Non-resistant E. coli were qualitatively detected in 40.7% of air samples taken at wind velocities exceeding 7.3 m s-1 . Significantly increased emission of total viable bacteria with increasing wind velocity was observed. In the aerosol chamber trials, recovery rates of airborne E. coli ranged from 0.003% to 2.8%, indicating a low airborne tenacity. Concluding, an emission of ESBL/AmpC E. coli by wind erosion in relevant concentrations appears unlikely because of the low concentration in chicken litter compared with non-resistant E. coli and their low airborne tenacity, proven in the aerosol chamber trials.

Agricultural fertilization with poultry manure results in persistent environmental contamination with the pathogen Clostridioides difficile.

During a field experiment applying broiler manure for fertilization of agricultural land, we detected viable Clostridioides (also known as Clostridium) difficile in broiler faeces, manure, dust and fertilized soil. A large diversity of toxigenic C. difficile isolates was recovered, including PCR ribotypes common from human disease. Genomic relatedness of C. difficile isolates from dust and from soil, recovered more than 2 years after fertilization, traced their origins to the specific chicken farm that had delivered the manure. We present evidence of long-term contamination of agricultural soil with manure-derived C. difficile and demonstrate the potential for airborne dispersal of C. difficile through dust emissions during manure application. Clostridioides genome sequences virtually identical to those from manure had been recovered from chicken meat and from human infections in previous studies, suggesting broiler-associated C. difficile are capable of zoonotic transmission.

Rapid cell division of Staphylococcus aureus during colonization of the human nose.

BACKGROUND: Staphylococcus aureus is an important opportunistic pathogen and a commensal bacterium, thriving in the nasal cavities of 20% of the human population. Little is known about the dynamics of asymptomatic colonization and the occasional transition to infectious disease. RESULTS: In this study, we inferred that S. aureus cells replicate every one to three hours on average while colonizing the human nose, based on two independent lines of genomic evidence. First, we collected nasal swab samples from human subjects, extracted and sequenced metagenomic DNA, and analyzed the distribution of sequencing coverage along the staphylococcal chromosome. Calibration of this data by comparison to a laboratory culture enabled measuring S. aureus cell division rates in nasal samples. Second, we applied mutation accumulation experiments paired with genome sequencing to measure spontaneous mutation rates at a genome scale. Relating these mutation rates to annual evolutionary rates confirmed that nasal S. aureus continuously pass several thousand cell divisions per year when averaged over large, globally distributed populations and over many years, corresponding to generation times of less than two hours. CONCLUSIONS: The cell division rates we determined were higher than the fastest documented rates during fulminant disease progression (in a mouse model of systemic infection) and much higher than those previously measured in expectorated sputum from cystic fibrosis patients. This paper supplies absolute in-vivo generation times for an important bacterial commensal, indicating that colonization of the human upper respiratory tract is characterized by a highly dynamic equilibrium between bacterial growth and removal.

Identification of the novel class D ß-lactamase OXA-679 involved in carbapenem resistance in Acinetobacter calcoaceticus.

OBJECTIVES: The aim of this study was to characterize the Acinetobacter calcoaceticus clinical isolate AC_2117 with the novel carbapenem-hydrolysing class D ß-lactamase (CHDL) OXA-679. METHODS: Identification of the species and ß-lactamases was verified by genome sequencing (PacBio) and phylogenetic analyses. Antibiotic susceptibility of AC_2117 and transformants harbouring cloned blaOXA-679 was evaluated using antibiotic gradient strips and microbroth dilution. OXA-679 was purified heterologously and kinetic parameters were determined using spectrometry or isothermal titration calorimetry. The impact of OXA-679 production during imipenem therapy was evaluated in the Galleria mellonella infection model. RESULTS: Sequencing of the complete genome of the clinical A. calcoaceticus isolate AC_2117 identified a novel CHDL, termed OXA-679. This enzyme shared sequence similarity of 71% to each of the families OXA-143 and OXA-24/40. Phylogenetic analyses revealed that OXA-679 represents a member of a new OXA family. Cloning and expression of blaOXA-679 as well as measurement of kinetic parameters revealed the effective hydrolysis of carbapenems which resulted in reduced susceptibility to carbapenems in Escherichia coli and A. calcoaceticus, and high-level carbapenem resistance in Acinetobacter baumannii. Infection of larvae of G. mellonella with a sublethal dose of blaOXA-679-expressing A. baumannii could not be cured by high-dose imipenem therapy, indicating carbapenem resistance in vivo. CONCLUSIONS: We identified blaOXA-679 in a clinical A. calcoaceticus isolate that represents a member of the new OXA-679 family and that conferred high-level carbapenem resistance in vitro and in vivo.

Hospital outbreak due to Clostridium difficile ribotype 018 (RT018) in Southern Germany.

Clostridium (Clostridioides) difficile is the main cause of nosocomial diarrhoea. Ribotype 018 (RT018) has been recognized as the predominant strain responsible for C. difficile infection (CDI) in Italy, whereas in most other European countries only sporadic RT018 cases occur. Between August and October 2015, a suspected C. difficile outbreak at two associated hospitals in Southern Germany was investigated by comprehensive molecular typing. Surprisingly, RT018 was detected in 9/82 CDI patients, which has never been described before in a German outbreak. Phenotypic analysis revealed fluoroquinolone and macrolide resistance. Genetic subtyping using multiple-locus variable-number tandem-repeat analysis (MLVA) and whole genome sequencing (WGS) was performed and outbreak isolates were directly compared to sporadic German RT018 isolates and to epidemic ones from Milan, Northern Italy. Molecular typing confirmed a hospital outbreak with closely related RT018 isolates. Both, MLVA and WGS revealed high similarity of outbreak strains with epidemic isolates from Italy, but low similarity to other German isolates. Comparison between both typing strategies showed that ribotyping in combination with MLVA was appropriate to identify related isolates and clonal complexes, whereas WGS provided a better discrimination with more detailed information about the phylogenetic relationship of isolates. This is the first hospital outbreak in Germany presumably caused by cross-national transmission of an Italian epidemic RT018 strain.

Genomic analysis of three Clostridioides difficile isolates from urban water sources.

We investigated inflow of a wastewater treatment plant and sediment of an urban lake for the presence of Clostridioides difficile by cultivation and PCR. Among seven colonies we sequenced the complete genomes of three: two non-toxigenic isolates from wastewater and one toxigenic isolate from the urban lake. For all obtained isolates, a close genomic relationship with human-derived isolates was observed.

glnA Truncation in Salmonella enterica Results in a Small Colony Variant Phenotype, Attenuated Host Cell Entry, and Reduced Expression of Flagellin and SPI-1-Associated Effector Genes.

Many pathogenic bacteria use sophisticated survival strategies to overcome harsh environmental conditions. One strategy is the formation of slow-growing subpopulations termed small colony variants (SCVs). Here we characterize an SCV that spontaneously emerged from an axenic Salmonella enterica serovar Typhimurium water culture. We found that the SCV harbored a frameshift mutation in the glutamine synthetase gene glnA, leading to an ∼90% truncation of the corresponding protein. Glutamine synthetase, a central enzyme in nitrogen assimilation, converts glutamate and ammonia to glutamine. Glutamine is an important nitrogen donor that is required for the synthesis of cellular compounds. The internal glutamine pool serves as an indicator of nitrogen availability in Salmonella In our study, the SCV and a constructed glnA knockout mutant showed reduced growth rates, compared to the wild type. Moreover, the SCV and the glnA mutant displayed attenuated entry into host cells and severely reduced levels of exoproteins, including flagellin and several Salmonella pathogenicity island 1 (SPI-1)-dependent secreted virulence factors. We found that these proteins were also depleted in cell lysates, indicating their diminished synthesis. Accordingly, the SCV and the glnA mutant had severely decreased expression of flagellin genes, several SPI-1 effector genes, and a class 2 motility gene (flgB). However, the expression of a class 1 motility gene (flhD) was not affected. Supplementation with glutamine or genetic reversion of the glnA truncation restored growth, cell entry, gene expression, and protein abundance. In summary, our data show that glnA is essential for the growth of S. enterica and controls important motility- and virulence-related traits in response to glutamine availability.IMPORTANCESalmonella enterica serovar Typhimurium is a significant pathogen causing foodborne infections. Here we describe an S Typhimurium small colony variant (SCV) that spontaneously emerged from a long-term starvation experiment in water. It is important to study SCVs because (i) SCVs may arise spontaneously upon exposure to stresses, including environmental and host defense stresses, (ii) SCVs are slow growing and difficult to eradicate, and (iii) only a few descriptions of S. enterica SCVs are available. We clarify the genetic basis of the SCV described here as a frameshift mutation in the glutamine synthetase gene glnA, leading to glutamine auxotrophy. In Salmonella, internal glutamine limitation serves as a sign of external nitrogen deficiency and is thought to regulate cell growth. In addition to exhibiting impaired growth, the SCV showed reduced host cell entry and reduced expression of SPI-1 virulence and flagellin genes.

Nannocystis konarekensis sp. nov., a novel myxobacterium from an Iranian desert.

An orange-coloured myxobacterium, MNa11734T, was isolated from desert in Iran. MNa11734T had rod-shaped vegetative cells, moved by gliding and was bacteriolytic. No real fruiting body formation could be observed, but sporangioles were produced on water agar. The strain was mesophilic, strictly aerobic and chemoheterotrophic. 16S rRNA gene analyses revealed that MNa11734T belonged to the family Nannocystaceae, genus Nannocystis and was closely related to Nannocystis pusilla Na p29T (DSM 14622T) and Nannocystis exedens Na e1T (DSM 71T), with 97.8 and 97.6 % 16S rRNA gene sequence similarity, respectively. Laboratory-measured DNA-DNA hybridization showed only 9.5/15.7 % (reciprocal) similarity between the novel strain and N. pusilla Na p29T, and 14.1/20.4 % between the strain and N. exedens Na e1T, whereas DNA-DNA hybridization estimates derived from draft genome sequences were 21.8-23.0 % and 22.2-23.7 %, respectively, depending on the calculation method. The G+C content of DNA from Nannocystis konarekensis MNa11734T was 73.3 mol%, for N. pusilla Nap29T it was 71.8 mol% and for N. exedens Nae1T it was 72.2 mol%. The major fatty acids of the new strain were C16 : 1 (56.2 %), iso-C17 : 0 (14.4 %), C14 : 0 (8.2 %), C16 : 0 (6.6 %) and iso-C15 : 0 (5.9 %). Strain MNa11734T exhibited phylogenetic and physiological similarities to the two other species of Nannocystis, i.e. N. pusilla and N. exedens, but the differences were sufficient enough to represent a novel species, for which the name Nannocystiskonarekensis sp. nov. is proposed. The type strain is MNa11734T (=DSM 104509T=NCCB 100618T).

A polyphasic approach leads to seven new species of the cellulose-decomposing genus Sorangium, Sorangium ambruticinum sp. nov., Sorangium arenae sp. nov., Sorangium bulgaricum sp. nov., Sorangium dawidii sp. nov., Sorangium kenyense sp. nov., Sorangium orientale sp. nov. and Sorangium reichenbachii sp. nov.

Seventy-three strains of Sorangium have been isolated from soil samples collected from all over the world. The strains were characterized using a polyphasic approach and phenotypic, genotypic and chemotype analyses clarified their taxonomic relationships. 16S rRNA, xynB1, groEL1, matrix-assisted laser desorption/ioniziation time-of-flight mass spectrometry and API-ZYM analyses were conducted. In addition, from selected representative strains, fatty acids, quinones and phospholipids were analysed. In silico DNA-DNA hybridization and DNA-DNA hybridization against the current type species of Sorangiumcellulosum strain Soce 1871T (DSM 14627T) completed the analyses. Finally, our study revealed seven new species of Sorangium: Sorangium ambruticinum (Soce 176T; DSM 53252T, NCCB 100639T, sequence accession number ERS2488998), Sorangium arenae (Soce 1078T; DSM 105768T, NCCB 100643T, ERS2489002), Sorangium bulgaricum (Soce 321T; DSM 53339T, NCCB 100640T, ERS2488999), Sorangium dawidii (Soce 362T; DSM 105767T, NCCB 100641T, ERS2489000), Sorangium kenyense (Soce 375T; DSM 105741T, NCCB 100642T, ERS2489001), Sorangium orientale (Soce GT47T; DSM 105742T, NCCB 100638T, ERS2501484) and Sorangium reichenbachii (Soce 1828T; DSM 105769T, NCCB 100644T, ERS2489003).

Rapid and Consistent Evolution of Colistin Resistance in Extensively Drug-Resistant Pseudomonas aeruginosa during Morbidostat Culture.

Colistin is a last-resort antibiotic commonly used against multidrug-resistant strains of Pseudomonas aeruginosa To investigate the potential for in situ evolution of resistance against colistin and to map the molecular targets of colistin resistance, we exposed two P. aeruginosa isolates to colistin using a continuous-culture device known as a morbidostat. As a result, colistin resistance reproducibly increased 10-fold within 10 days and 100-fold within 20 days, along with highly stereotypic yet strain-specific mutation patterns. The majority of mutations hit the pmrAB two-component signaling system and genes involved in lipopolysaccharide (LPS) synthesis, including lpxC, pmrE, and migA We tracked the frequencies of all arising mutations by whole-genome deep sequencing every 3 to 4 days to obtain a detailed picture of the dynamics of resistance evolution, including competition and displacement among multiple resistant subpopulations. In 7 out of 18 cultures, we observed mutations in mutS along with a mutator phenotype that seemed to facilitate resistance evolution.

Emergence and Spread of Antimicrobial Resistance: Recent Insights from Bacterial Population Genomics.

Driven by progress of DNA sequencing technologies, recent population genomics studies have revealed that several bacterial pathogens constitute 'measurably evolving populations'. As a consequence, it was possible to reconstruct the emergence and spatial spread of drug-resistant bacteria on the basis of temporally structured samples of bacterial genome sequences. Based on currently available data, some general inferences can be drawn across different bacterial species as follows: (1) Resistance to various antibiotics evolved years to decades earlier than had been anticipated on the basis of epidemiological surveillance data alone. (2) Resistance traits are more rapidly acquired than lost and commonly persist in bacterial populations for decades. (3) Global populations of drug-resistant pathogens are dominated by very few clones, yet the features enabling such spreading success have not been revealed, aside from antibiotic resistance. (4) Whole-genome sequencing proved very effective at identifying bacterial isolates as parts of the same transmission networks.

A genomic portrait of the emergence, evolution, and global spread of a methicillin-resistant Staphylococcus aureus pandemic.

The widespread use of antibiotics in association with high-density clinical care has driven the emergence of drug-resistant bacteria that are adapted to thrive in hospitalized patients. Of particular concern are globally disseminated methicillin-resistant Staphylococcus aureus (MRSA) clones that cause outbreaks and epidemics associated with health care. The most rapidly spreading and tenacious health-care-associated clone in Europe currently is EMRSA-15, which was first detected in the UK in the early 1990s and subsequently spread throughout Europe and beyond. Using phylogenomic methods to analyze the genome sequences for 193 S. aureus isolates, we were able to show that the current pandemic population of EMRSA-15 descends from a health-care-associated MRSA epidemic that spread throughout England in the 1980s, which had itself previously emerged from a primarily community-associated methicillin-sensitive population. The emergence of fluoroquinolone resistance in this EMRSA-15 subclone in the English Midlands during the mid-1980s appears to have played a key role in triggering pandemic spread, and occurred shortly after the first clinical trials of this drug. Genome-based coalescence analysis estimated that the population of this subclone over the last 20 yr has grown four times faster than its progenitor. Using comparative genomic analysis we identified the molecular genetic basis of 99.8% of the antimicrobial resistance phenotypes of the isolates, highlighting the potential of pathogen genome sequencing as a diagnostic tool. We document the genetic changes associated with adaptation to the hospital environment and with increasing drug resistance over time, and how MRSA evolution likely has been influenced by country-specific drug use regimens.

Comprehensive molecular, genomic and phenotypic analysis of a major clone of Enterococcus faecalis MLST ST40.

BACKGROUND: Enterococcus faecalis is a multifaceted microorganism known to act as a beneficial intestinal commensal bacterium. It is also a dreaded nosocomial pathogen causing life-threatening infections in hospitalised patients. Isolates of a distinct MLST type ST40 represent the most frequent strain type of this species, distributed worldwide and originating from various sources (animal, human, environmental) and different conditions (colonisation/infection). Since enterococci are known to be highly recombinogenic we determined to analyse the microevolution and niche adaptation of this highly distributed clonal type. RESULTS: We compared a set of 42 ST40 isolates by assessing key molecular determinants, performing whole genome sequencing (WGS) and a number of phenotypic assays including resistance profiling, formation of biofilm and utilisation of carbon sources. We generated the first circular closed reference genome of an E. faecalis isolate D32 of animal origin and compared it with the genomes of other reference strains. D32 was used as a template for detailed WGS comparisons of high-quality draft genomes of 14 ST40 isolates. Genomic and phylogenetic analyses suggest a high level of similarity regarding the core genome, also demonstrated by similar carbon utilisation patterns. Distribution of known and putative virulence-associated genes did not differentiate between ST40 strains from a commensal and clinical background or an animal or human source. Further analyses of mobile genetic elements (MGE) revealed genomic diversity owed to: (1) a modularly structured pathogenicity island; (2) a site-specifically integrated and previously unknown genomic island of 138 kb in two strains putatively involved in exopolysaccharide synthesis; and (3) isolate-specific plasmid and phage patterns. Moreover, we used different cell-biological and animal experiments to compare the isolate D32 with a closely related ST40 endocarditis isolate whose draft genome sequence was also generated. D32 generally showed a greater capacity of adherence to human cell lines and an increased pathogenic potential in various animal models in combination with an even faster growth in vivo (not in vitro). CONCLUSION: Molecular, genomic and phenotypic analysis of representative isolates of a major clone of E. faecalis MLST ST40 revealed new insights into the microbiology of a commensal bacterium which can turn into a conditional pathogen.

Molecular typing of toxic shock syndrome toxin-1- and Enterotoxin A-producing methicillin-sensitive Staphylococcus aureus isolates from an outbreak in a neonatal intensive care unit.

Outbreaks of Staphylococcus aureus are common in neonatal intensive care units (NICUs). Usually they are documented for methicillin-resistant strains, while reports involving methicillin-susceptible S. aureus (MSSA) strains are rare. In this study we report the epidemiological and molecular investigation of an MSSA outbreak in a NICU among preterm neonates. Infection control measures and interventions were commissioned by the Local Public Health Authority and supported by the Robert Koch Institute. To support epidemiological investigations molecular typing was done by spa-typing and Multilocus sequence typing; the relatedness of collected isolates was further elucidated by DNA SmaI-macrorestriction, microarray analysis and bacterial whole genome sequencing. A total of 213 neonates, 123 healthcare workers and 205 neonate parents were analyzed in the period November 2011 to November 2012. The outbreak strain was characterized as a MSSA spa-type t021, able to produce toxic shock syndrome toxin-1 and Enterotoxin A. We identified seventeen neonates (of which two died from toxic shock syndrome), four healthcare workers and three parents putatively involved in the outbreak. Whole-genome sequencing permitted to exclude unrelated cases from the outbreak and to discuss the role of healthcare workers as a reservoir of S. aureus on the NICU. Genome comparisons also indicated the presence of the respective clone on the ward months before the first colonized/infected neonates were detected.

Evolution of methicillin-resistant Staphylococcus aureus towards increasing resistance.

OBJECTIVES: To elucidate the evolutionary history of Staphylococcus aureus clonal complex (CC) 8, which encompasses several globally distributed epidemic lineages, including hospital-associated methicillin-resistant S. aureus (MRSA) and the highly prevalent community-associated MRSA clone USA300. METHODS: We reconstructed the phylogeny of S. aureus CC8 by mutation discovery at 112 genetic housekeeping loci from each of 174 isolates, sampled on five continents between 1957 and 2008. The distribution of antimicrobial resistance traits and of diverse mobile genetic elements was investigated in relation to the isolates' phylogeny. RESULTS: Our analyses revealed the existence of nine phylogenetic clades within CC8. We identified at least eight independent events of methicillin resistance acquisition in CC8 and dated the origin of a methicillin-resistant progenitor of the notorious USA300 clone to the mid-1970s. Of the S. aureus isolates in our collection, 88% carried plasmidic rep gene sequences, with up to five different rep genes in individual isolates and a total of eight rep families. Mapping the plasmid content onto the isolates' phylogeny illustrated the stable carriage over decades of some plasmids and the more volatile nature of others. Strikingly, we observed trends of increasing antibiotic resistance during the evolution of several lineages, including USA300. CONCLUSIONS: We propose a model for the evolution of S. aureus CC8, involving a split into at least nine phylogenetic lineages and a subsequent series of acquisitions and losses of mobile genetic elements that carry diverse virulence and antimicrobial resistance traits. The evolution of MRSA USA300 towards resistance to additional antibiotic classes is of major concern.

Improvements of weaned pigs barn hygiene to reduce the spread of antimicrobial resistance.

The spread of antimicrobial resistance (AMR) in animal husbandry is usually attributed to the use of antibiotics and poor hygiene and biosecurity. We therefore conducted experimental trials to improve hygiene management in weaned pig houses and assessed the impact on the spread. For each of the two groups examined, the experimental group (EG) and the control group (CG), three replicate batches of piglets from the same pig breeder, kept in pre-cleaned flat decks, were analyzed. In the flat decks of the experimental groups, the hygiene conditions (cleaning, disinfection, dust removal and fly control) were improved, while regular hygiene measures were carried out in the control groups. The occurrence and spread of AMR were determined in Escherichia coli (E. coli; resistance indicator) using cultivation-dependent (CFU) and -independent (qPCR) methods as well as whole genome sequencing of isolates in samples of various origins, including feces, flies, feed, dust and swabs. Surprisingly, there were no significant differences (p > 0.05) in the prevalence of resistant E. coli between the flat decks managed with conventional techniques and those managed with improved techniques. Selective cultivation delivered ampicillin- and sulfonamide-resistant E. coli proportions of up to 100% and 1.2%, respectively. While 0.5% E. coli resistant to cefotaxime and no ciprofloxacin resistance were detected. There was a significant difference (p < 0.01) in the abundance of the blaTEM-1 gene in fecal samples between EG and CG groups. The colonization of piglets with resistant pathogens before arrival, the movement of flies in the barn and the treatment of bacterial infections with antibiotics obscured the effects of hygiene improvement. Biocide tolerance tests showed no development of resistance to the farm regular disinfectant. Managing hygiene alone was insufficient for reducing antimicrobial resistances in piglet rearing. We conclude that the complex factors contributing to the presence and distribution of AMR in piglet barns underscore the necessity for a comprehensive management strategy.

Whole genome sequencing versus traditional genotyping for investigation of a Mycobacterium tuberculosis outbreak: a longitudinal molecular epidemiological study.

BACKGROUND: Understanding Mycobacterium tuberculosis (Mtb) transmission is essential to guide efficient tuberculosis control strategies. Traditional strain typing lacks sufficient discriminatory power to resolve large outbreaks. Here, we tested the potential of using next generation genome sequencing for identification of outbreak-related transmission chains. METHODS AND FINDINGS: During long-term (1997 to 2010) prospective population-based molecular epidemiological surveillance comprising a total of 2,301 patients, we identified a large outbreak caused by an Mtb strain of the Haarlem lineage. The main performance outcome measure of whole genome sequencing (WGS) analyses was the degree of correlation of the WGS analyses with contact tracing data and the spatio-temporal distribution of the outbreak cases. WGS analyses of the 86 isolates revealed 85 single nucleotide polymorphisms (SNPs), subdividing the outbreak into seven genome clusters (two to 24 isolates each), plus 36 unique SNP profiles. WGS results showed that the first outbreak isolates detected in 1997 were falsely clustered by classical genotyping. In 1998, one clone (termed "Hamburg clone") started expanding, apparently independently from differences in the social environment of early cases. Genome-based clustering patterns were in better accordance with contact tracing data and the geographical distribution of the cases than clustering patterns based on classical genotyping. A maximum of three SNPs were identified in eight confirmed human-to-human transmission chains, involving 31 patients. We estimated the Mtb genome evolutionary rate at 0.4 mutations per genome per year. This rate suggests that Mtb grows in its natural host with a doubling time of approximately 22 h (400 generations per year). Based on the genome variation discovered, emergence of the Hamburg clone was dated back to a period between 1993 and 1997, hence shortly before the discovery of the outbreak through epidemiological surveillance. CONCLUSIONS: Our findings suggest that WGS is superior to conventional genotyping for Mtb pathogen tracing and investigating micro-epidemics. WGS provides a measure of Mtb genome evolution over time in its natural host context.