Alexander Tomasz

Genome sequencing in microfabricated high-density picolitre reactors

The proliferation of large-scale DNA-sequencing projects in recent years has driven a search for alternative methods to reduce time and cost. Here we describe a scalable, highly parallel sequencing system with raw throughput significantly greater than that of state-of-the-art capillary electrophoresis instruments. The apparatus uses a novel fibre-optic slide of individual wells and is able to sequence 25 million bases, at 99% or better accuracy, in one four-hour run. To achieve an approximately 100-fold increase in throughput over current Sanger sequencing technology, we have developed an emulsion method for DNA amplification and an instrument for sequencing by synthesis using a pyrosequencing protocol optimized for solid support and picolitre-scale volumes. Here we show the utility, throughput, accuracy and robustness of this system by shotgun sequencing and de novo assembly of the Mycoplasma genitalium genome with 96% coverage at 99.96% accuracy in one run of the machine.

CD14 Is a pattern recognition receptor

Septic shock caused by a diverse group of bacterial pathogens is a serious human disease. Recognition of bacterial envelope constituents is one mechanism used by mammalian cells to initiate responses leading to bacterial killing or, unfortunately, responses that also cause fatal septic shock. Here we show that CD14 plays a key role in initiating cell activation by a group of bacterial envelope components from Gram-negative and Gram-positive microorganisms, as well as mycobacteria. We propose that CD14 is a receptor used by mammalian cells to recognize and signal responses to a diverse array of bacterial constituents. This finding defines the molecular basis for innate microbial immunity; implicit in these findings are new possibilities for therapeutics.

Rapid Pneumococcal Evolution in Response to Clinical Interventions

Streptococcus pneumonia evades vaccines and drugs by high levels of recombination and rapid adaptation. Epidemiological studies of the naturally transformable bacterial pathogen Streptococcus pneumoniae have previously been confounded by high rates of recombination. Sequencing 240 isolates of the PMEN1 (Spain23F-1) multidrug-resistant lineage enabled base substitutions to be distinguished from polymorphisms arising through horizontal sequence transfer. More than 700 recombinations were detected, with genes encoding major antigens frequently affected. Among these were 10 capsule-switching events, one of which accompanied a population shift as vaccine-escape serotype 19A isolates emerged in the USA after the introduction of the conjugate polysaccharide vaccine. The evolution of resistance to fluoroquinolones, rifampicin, and macrolides was observed to occur on multiple occasions. This study details how genomic plasticity within lineages of recombinogenic bacteria can permit adaptation to clinical interventions over remarkably short time scales.

Tackling antibiotic resistance

The development and spread of antibiotic resistance in bacteria is a universal threat to both humans and animals that is generally not preventable but can nevertheless be controlled, and it must be tackled in the most effective ways possible. To explore how the problem of antibiotic resistance might best be addressed, a group of 30 scientists from academia and industry gathered at the Banbury Conference Centre in Cold Spring Harbor, New York, USA, from 16 to 18 May 2011. From these discussions there emerged a priority list of steps that need to be taken to resolve this global crisis.

Nomenclature of major antimicrobial-resistant clones of Streptococcus pneumoniae defined by the pneumococcal molecular epidemiology network

ABSTRACT The emergence of disease caused by penicillin-resistant and multidrug-resistant pneumococci has become a global concern, necessitating the identification of the epidemiological spread of such strains. The Pneumococcal Molecular Epidemiology Network was established in 1997 under the auspices of the International Union of Microbiological Societies with the aim of characterizing, standardizing, naming, and classifying antimicrobial agent-resistant pneumococcal clones. Here we describe the nomenclature for 16 pneumococcal clones that have contributed to the increase in antimicrobial resistance worldwide. Guidelines for the recognition of these clones using molecular typing procedures (pulsed-field gel electrophoresis, BOX-PCR, and multilocus sequence typing) are presented, as are the penicillin-binding profiles and macrolide resistance determinants for the 16 clones. This network can serve as a prototype for the collaboration of scientists in identifying clones of important human pathogens and as a model for the development of other networks.

Secrets of success of a human pathogen: molecular evolution of pandemic clones of meticillin-resistant Staphylococcus aureus

The first European isolate of meticillin-resistant Staphylococcus aureus (MRSA) was detected in 1960. Since then MRSA has become a leading cause of nosocomial infections worldwide. Using molecular typing techniques--primarily pulsed-field gel electrophoresis (PFGE)--we identified five major MRSA clones that accounted for almost 70% of the over 3000 MRSA isolates recovered in hospitals mainly in southern and eastern Europe, South America, and the USA. Most of our surveillance studies were done in these areas. Multilocus sequencing typing (MLST) of representative isolates of this collection showed that these five pandemic MRSA clones have evolved from only two distinct ancestral genetic backgrounds, one of which can be traced back to the very first European MRSA isolates and also to meticillin susceptible S aureus strains circulating in Danish hospitals during the mid to late 1950s--i.e., shortly before the introduction of meticillin into therapy. The second lineage with a completely different MLST profile included MRSA frequently recovered in the USA, Japan, and among paediatric isolates from several parts of the world. A few isolates with a third distinct MLST type corresponding to that of EMRSA-16 were also detected in the early Danish isolates. The four structural types of mec element, the heterologous DNA segment containing the meticillin resistance determinant mecA, were present in unique combinations with the MRSA clonal types. Our findings establish evolutionary associations in the most widely spread pandemic clones of MRSA. The epidemiological factors that contributed to the massive dissemination of a few MRSA clones are not well understood. We suggest, however, that the secrets of effectiveness of MRSA could be hidden in the unique genetic background of a surprisingly few lineages of S aureus particularly well able to cope with the contemporary clinical environment.

Tracking the in vivo evolution of multidrug resistance in Staphylococcus aureus by whole-genome sequencing

The spread of multidrug-resistant Staphylococcus aureus (MRSA) strains in the clinical environment has begun to pose serious limits to treatment options. Yet virtually nothing is known about how resistance traits are acquired in vivo. Here, we apply the power of whole-genome sequencing to identify steps in the evolution of multidrug resistance in isogenic S. aureus isolates recovered periodically from the bloodstream of a patient undergoing chemotherapy with vancomycin and other antibiotics. After extensive therapy, the bacterium developed resistance, and treatment failed. Sequencing the first vancomycin susceptible isolate and the last vancomycin nonsusceptible isolate identified genome wide only 35 point mutations in 31 loci. These mutations appeared in a sequential order in isolates that were recovered at intermittent times during chemotherapy in parallel with increasing levels of resistance. The vancomycin nonsusceptible isolates also showed a 100-fold decrease in susceptibility to daptomycin, although this antibiotic was not used in the therapy. One of the mutated loci associated with decreasing vancomycin susceptibility (the vraR operon) was found to also carry mutations in six additional vancomycin nonsusceptible S. aureus isolates belonging to different genetic backgrounds and recovered from different geographic sites. As costs drop, whole-genome sequencing will become a useful tool in elucidating complex pathways of in vivo evolution in bacterial pathogens.

The Evolution of Pandemic Clones of Methicillin-Resistant Staphylococcus aureus: Identification of Two Ancestral Genetic Backgrounds and the Associated mec Elements

Previous surveillance studies carried out by our laboratories, primarily in Southern and Eastern Europe, Latin America, and the United States, have characterized 3,067 methicillin-resistant Staphylococcus aureus (MRSA) hospital isolates by a combination of molecular typing methods. Nearly 70% of these isolates could be classified into five clonal types showing extensive geographic spread. Representative isolates of these clonal types were now reexamined for their genetic relatedness by multilocus sequence typing (MLST) and by sequencing the polymorphic region of protein A (spaA typing), and also for the type of the Staphylococcal Chromosomal Cassette (SCCmec) resident in the bacteria. Three of the previously classified clonal types (Iberian, Brazilian, and Hungarian clones) shared a common or closely related genetic background A, which was the same as the background of the earliest European isolates of MRSA from England and Denmark. The Pediatric and New York/Japan clones belonged to a completely different genetic background B. The three recently described SCCmec types were specifically associated with different pandemic clones: types I and III with isolates of genetic background A and type II with isolates of genetic background B. A novel SCCmec related to type I, called SCCmec type IV, was identified in some MRSA strains belonging to genetic background A as well as B. Structural variations in SCCmec types I and III were also observed. The data allow tentative identification of an evolutionary pathway for the emergence of pandemic MRSA clones and also provide evidence for the multiple, yet restricted, numbers of acquisition of the mec element by S. aureus.

The evolution of methicillin resistance in Staphylococcus aureus: Similarity of genetic backgrounds in historically early methicillin- susceptible and -resistant isolates and contemporary epidemic clones

The key genetic component of methicillin resistance, the mecA determinant, is not native to Staphylococcus aureus. Thus, the evolution of methicillin-resistant S. aureus (MRSA) must have begun with the acquisition of the mecA determinant from an unknown heterologous source some time before the first reported appearance of MRSA isolates in clinical specimens in the U.K. and Denmark (in the early 1960s). We compared the genetic backgrounds and phenotypes of a group of methicillin-susceptible S. aureus (MSSA) isolates to the properties of MRSA strains isolated in Denmark and the U.K. during the same time period, and also to the genetic profiles of contemporary epidemic clones of MRSA. All early MRSA isolates resembled a large group of the early MSSA blood isolates in phenotypic and genetic properties, including phage group, antibiotype (resistance to penicillin, streptomycin, and tetracycline), pulsed-field gel electrophoresis pattern, and spaA type and multilocus sequence type, strongly suggesting that the early MSSA examined here represented the progeny of a strain that served as one of the first S. aureus recipients of the methicillin-resistance determinant in Europe. The genetic background of this group of early MSSA isolates was also very similar to that of the widely disseminated contemporary “Iberian clone” of MRSA, suggesting that genetic determinants present in early MSSA and essential for some aspects of the epidemicity and/or virulence of these strains may have been retained by this highly successful contemporary MRSA lineage.

The rate of killing of Escherichia coli by beta-lactam antibiotics is strictly proportional to the rate of bacterial growth.

Nongrowing bacteria evade the bactericidal activity of beta-lactam antibiotics. We sought to determine if slow growth rate also alters bactericidal activity. The bactericidal activity of two beta-lactams on Escherichia coli grown in glucose limited chemostats was compared for generation times ranging from 0.7 to 12 h. The degree of killing varied with drug structure and with E. coli strain. However, all killing rates were a constant function of the bacterial generation time: slowly growing bacteria became progressively more phenotypically tolerant to beta-lactam antibiotics as the generation time was extended.