John Anthony McCulloch

Molecular techniques for MRSA typing: current issues and perspectives

Staphylococcus aureus has long been recognised as an important pathogen in human disease. Serious staphylococcal infections can frequently occur in inpatients and may lead to dire consequences, especially for therapy with antimicrobial agents. The increase in the frequency of Methicillin-Resistant Staphylococcus aureus (MRSA) as the causal agent of nosocomial infection and the possibility of emergence of resistance to vancomycin demands a quick and trustworthy characterization of isolates and identification of clonal spread within hospitals. Enough information must be generated to permit the implementation of appropriate measures for control of infection, so that outbreaks can be contained. Molecular typing techniques reviewed in this manuscript include: plasmid profile analysis, analysis of chromosomal DNA after enzymatic restriction, Southern blotting, pulsed field gel electrophoresis (PFGE), techniques involving polymerase chain reaction and multilocus sequence typing (MLST). Repetitive DNA Sequence PCR (rep-PCR) may be used for screening due to its practicality, low cost and reproducibility. Because of its high discriminatory power Pulsed-Field Gel Electrophoresis (PFGE) still remains the gold standard for MRSA typing. New techniques with higher reproducibility and discriminatory power, such as Multi-Locus Sequence Typing (MLST), are appearing. These are mostly useful for global epidemiology studies. Molecular typing techniques are invaluable tools for the assessment of putative MRSA outbreaks and so should be extensively used for this purpose.

Evidence for Reductive Genome Evolution and Lateral Acquisition of Virulence Functions in Two Corynebacterium pseudotuberculosis Strains

Background Corynebacterium pseudotuberculosis, a Gram-positive, facultative intracellular pathogen, is the etiologic agent of the disease known as caseous lymphadenitis (CL). CL mainly affects small ruminants, such as goats and sheep; it also causes infections in humans, though rarely. This species is distributed worldwide, but it has the most serious economic impact in Oceania, Africa and South America. Although C. pseudotuberculosis causes major health and productivity problems for livestock, little is known about the molecular basis of its pathogenicity. Methodology and Findings We characterized two C. pseudotuberculosis genomes (Cp1002, isolated from goats; and CpC231, isolated from sheep). Analysis of the predicted genomes showed high similarity in genomic architecture, gene content and genetic order. When C. pseudotuberculosis was compared with other Corynebacterium species, it became evident that this pathogenic species has lost numerous genes, resulting in one of the smallest genomes in the genus. Other differences that could be part of the adaptation to pathogenicity include a lower GC content, of about 52%, and a reduced gene repertoire. The C. pseudotuberculosis genome also includes seven putative pathogenicity islands, which contain several classical virulence factors, including genes for fimbrial subunits, adhesion factors, iron uptake and secreted toxins. Additionally, all of the virulence factors in the islands have characteristics that indicate horizontal transfer. Conclusions These particular genome characteristics of C. pseudotuberculosis, as well as its acquired virulence factors in pathogenicity islands, provide evidence of its lifestyle and of the pathogenicity pathways used by this pathogen in the infection process. All genomes cited in this study are available in the NCBI Genbank database (http://www.ncbi.nlm.nih.gov/genbank/) under accession numbers CP001809 and CP001829.

First Isolation of Metallo-β-Lactamase-Producing Multiresistant Klebsiella pneumoniae from a Patient in Brazil

ABSTRACT A multiresistant Klebsiella pneumoniae isolate was taken from the blood of a 75-year-old patient with nosocomial pneumonia who developed septic shock and failed therapy with imipenem. The isolate presented an MIC of imipenem of 128 μg/ml, and the production of a metallo-β-lactamase was confirmed by phenotypic and genotypic techniques. We here report, for the first time, the detection of a metalloenzyme (IMP-1)-producing K. pneumoniae clinical strain in Latin America. The gene responsible for this phenotype was found to be blaIMP-1, carried in a class 1 integron.

First Report of the Globally Disseminated IncX4 Plasmid Carrying the mcr-1 Gene in a Colistin-Resistant Escherichia coli Sequence Type 101 Isolate from a Human Infection in Brazil

ABSTRACT A colistin-resistant Escherichia coli strain was recovered from a patient with a diabetic foot infection in Brazil. Whole-genome analysis revealed that the E. coli isolate belonged to the widespread sequence type (ST) 101 and harbored the mcr-1 gene on an IncX4 plasmid that was highly similar to mcr-1-bearing IncX4 plasmids that were recently identified in Enterobacteriaceae from food, animal, and human samples recovered on different continents. These results suggest that self-transmissible IncX4-type plasmids may represent promiscuous plasmids contributing to the intercontinental spread of the mcr-1 gene.

Rapid hybrid de novo assembly of a microbial genome using only short reads: Corynebacterium pseudotuberculosis I19 as a case study

Due to the advent of the so-called Next-Generation Sequencing (NGS) technologies the amount of monetary and temporal resources for whole-genome sequencing has been reduced by several orders of magnitude. Sequence reads can be assembled either by anchoring them directly onto an available reference genome (classical reference assembly), or can be concatenated by overlap (de novo assembly). The latter strategy is preferable because it tends to maintain the architecture of the genome sequence the however, depending on the NGS platform used, the shortness of read lengths cause tremendous problems the in the subsequent genome assembly phase, impeding closing of the entire genome sequence. To address the problem, we developed a multi-pronged hybrid de novo strategy combining De Bruijn graph and Overlap-Layout-Consensus methods, which was used to assemble from short reads the entire genome of Corynebacterium pseudotuberculosis strain I19, a bacterium with immense importance in veterinary medicine that causes Caseous Lymphadenitis in ruminants, principally ovines and caprines. Briefly, contigs were assembled de novo from the short reads and were only oriented using a reference genome by anchoring. Remaining gaps were closed using iterative anchoring of short reads by craning to gap flanks. Finally, we compare the genome sequence assembled using our hybrid strategy to a classical reference assembly using the same data as input and show that with the availability of a reference genome, it pays off to use the hybrid de novo strategy, rather than a classical reference assembly, because more genome sequences are preserved using the former.

Development of serological proteome analysis of mastitis by Staphylococcus aureus in ewes.

Staphylococcus aureus is a major agent of mastitis in ruminants worldwide. So far, efficient measures for its prophylaxis (including vaccination) have proven to be unsuccessful and there is a need for a better understanding of the host response to udder infection by S. aureus. Serological proteome analysis (SERPA) is a promising technique that can be used to identify S. aureus immuno-dominant determinants providing that bacterial culture conditions used to grow S. aureus strains for protein sample preparation mimic the context of mastitis. A S. aureus strain was used in experimental mastitis to generate sheep serum used to determine the best growth conditions for SERPA. Sera collected in the field from different ewes suffering from mastitis by S. aureus were used to confirm experimental observations. Three different culture media (BHI, whey and iron-depleted RPMI) were tested. The influence of aeration and growth phase on protein production was also evaluated by immuno-detection of protein samples prepared from cultures grown in different conditions and obtained from different culture fractions (supernatant, cell wall, and total lysates). Our results showed that culturing in iron-depleted RPMI with (secreted proteins, prepared from stationary phase) or without aeration (cell wall proteins, prepared from early stationary phase, and total proteins, prepared from exponential phase) is the condition that best mimics growth in vivo during mastitis and this in vitro growth condition is to be used henceforth in experiments involving SERPA.

Isolation of KPC-2-producing Klebsiella pneumoniae strains belonging to the high-risk multiresistant clonal complex 11 (ST437 and ST340) in urban rivers

Department of Microbiology, Institute of Biomedical Sciences, Universidade de Sao Paulo, Sao Paulo, SP, Brazil; Department of Clinical Analysis, School of Pharmacy, Universidade de Sao Paulo, Sao Paulo, SP, Brazil; Institute of Biological Sciences, Universidade Federal do Para, Belem, PA, Brazil; School of Public Health, Universidade de Sao Paulo, Sao Paulo, SP, Brazil; Environmental Company of Sao Paulo State (CETESB), Sao Paulo, SP, Brazil

Escherichia coli carrying IncX4 plasmid-mediated mcr-1 and blaCTX-M genes in infected migratory Magellanic penguins (Spheniscus magellanicus).

Department of Internal Medicine, School of Veterinary Medicine and Animal Science, University of S~ ao Paulo, S~ ao Paulo, Brazil; Department of Clinical Analysis, Faculty of Pharmaceutical Sciences, University of S~ ao Paulo, S~ ao Paulo, Brazil; Department of Pathology, School of Veterinary Medicine and Animal Science, University of S~ ao Paulo, S~ ao Paulo, Brazil; Veterinary Unit of Santos Aquarium, Santos, Brazil; Department of Microbiology, Institute of Biomedical Sciences, University of S~ ao Paulo, S~ ao Paulo, Brazil

Type IV SCCmec found in decade old Brazilian MRSA isolates

Methicillin-resistant Staphylococcus aureus (MRSA) commonly causes infection in hospitalized patients. Since its appearance in the 1960s, the SCCmec has evolved throughout the years into 5 different types (I-V), each bearing a different set of genes. Infection with MRSA SCCmec types I, II or III is almost exclusively restricted to hospitalised patients. However, recently, community acquired MRSA (CA-MRSA) infections have been reported with increasing frequency, usually caused by a type IV SCCmec MRSA in nosocomial settings. We studied the prevalence of SCCmec types in 50 nosocomial strains collected from 1995 to 1999. The SCCmec complex type and presence of Panton-Valentine leukocidin (PVL) were determined by PCR. Strains had been previously typed by PFGE and were now typed by MLST. We found that 3 of the isolates studied bore a type IVc SCCmec all having different PFGE and MLST profiles (ST3, ST5 and ST88). All strains bearing a type III SCCmec belonged to MLST ST239 (Brazilian/Iberian clone). Only the strain which presented the ST5 profile bore the pvl gene. The type IVc SCCmec strains presented relatively lower levels of resistance to oxacillin in comparison to the type III SCCmec strains. The pattern of dissemination of the type IV SCCmec remains to be elucidated. The finding of strains carrying a type IV SCCmec in the present study among strains isolated at least 7 years ago indicates that clones bearing a type IV SCCmec have been present in Brazil for quite some time, and must have gone by undetected.

Molecular characterization of the Corynebacterium pseudotuberculosis hsp60-hsp10 operon, and evaluation of the immune response and protective efficacy induced by hsp60 DNA vaccination in mice

BackgroundHeat shock proteins (HSPs) are important candidates for the development of vaccines because they are usually able to promote both humoral and cellular immune responses in mammals. We identified and characterized the hsp60-hsp10 bicistronic operon of the animal pathogen Corynebacterium pseudotuberculosis, a Gram-positive bacterium of the class Actinobacteria, which causes caseous lymphadenitis (CLA) in small ruminants.FindingsTo construct the DNA vaccine, the hsp60 gene of C. pseudotuberculosis was cloned in a mammalian expression vector. BALB/c mice were immunized by intramuscular injection with the recombinant plasmid (pVAX1/hsp60).ConclusionThis vaccination induced significant anti-hsp60 IgG, IgG1 and IgG2a isotype production. However, immunization with this DNA vaccine did not confer protective immunity.