Björn A. Espedido

Predictors of Mortality in Staphylococcus aureus Bacteremia

SUMMARY Staphylococcus aureus bacteremia (SAB) is an important infection with an incidence rate ranging from 20 to 50 cases/100,000 population per year. Between 10% and 30% of these patients will die from SAB. Comparatively, this accounts for a greater number of deaths than for AIDS, tuberculosis, and viral hepatitis combined. Multiple factors influence outcomes for SAB patients. The most consistent predictor of mortality is age, with older patients being twice as likely to die. Except for the presence of comorbidities, the impacts of other host factors, including gender, ethnicity, socioeconomic status, and immune status, are unclear. Pathogen-host interactions, especially the presence of shock and the source of SAB, are strong predictors of outcomes. Although antibiotic resistance may be associated with increased mortality, questions remain as to whether this reflects pathogen-specific factors or poorer responses to antibiotic therapy, namely, vancomycin. Optimal management relies on starting appropriate antibiotics in a timely fashion, resulting in improved outcomes for certain patient subgroups. The roles of surgery and infectious disease consultations require further study. Although the rate of mortality from SAB is declining, it remains high. Future international collaborative studies are required to tease out the relative contributions of various factors to mortality, which would enable the optimization of SAB management and patient outcomes.

blaIMP-4 in Different Genetic Contexts in Enterobacteriaceae Isolates from Australia

ABSTRACT The IMP-4 metallo-β-lactamase, originally recognized in Acinetobacter spp. from Hong Kong, more recently appeared simultaneously in isolates of the family Enterobacteriaceae from Sydney and Melbourne, Australia. The blaIMP-4-qacG2-aacA4-catB3 cassette array was found in isolates from both cities, but in different wider genetic contexts and on different plasmids, suggesting movement of this array by homologous recombination.

The use of whole-genome sequencing for molecular epidemiology and antimicrobial surveillance: identifying the role of IncX3 plasmids and the spread of blaNDM-4-like genes in the Enterobacteriaceae

Aims To characterise the resistome of a multi-drug resistant Klebsiella pneumoniae (Kp0003) isolated from an Australian traveller who was repatriated to a Sydney Metropolitan Hospital from Myanmar with possible prosthetic aortic valve infective endocarditis. Methods Kp0003 was recovered from a blood culture of the patient and whole genome sequencing was performed. Read mapping and de novo assembly of reads facilitated in silico multi-locus sequence and plasmid replicon typing as well as the characterisation of antibiotic resistance genes and their genetic context. Conjugation experiments were also performed to assess the plasmid (and resistance gene) transferability and the effect on the antibiotic resistance phenotype. Results Importantly, and of particular concern, the carbapenem-hydrolysing β-lactamase gene blaNDM-4 was identified on a conjugative IncX3 plasmid (pJEG027). In this respect, the blaNDM-4 genetic context is similar (at least to some extent) to what has previously been identified for blaNDM-1 and blaNDM-4-like variants. Conclusions This study highlights the potential role that IncX3 plasmids have played in the emergence and dissemination of blaNDM-4-like variants worldwide and emphasises the importance of resistance gene surveillance.

Whole Genome Sequencing in Real-Time Investigation and Management of a Pseudomonas aeruginosa Outbreak on a Neonatal Intensive Care Unit.

OBJECTIVE To use whole genome sequencing to describe the likely origin of an outbreak of Pseudomonas aeruginosa in a neonatal unit. DESIGN Outbreak investigation. SETTING The neonatal intensive care unit service of a major obstetric tertiary referral center. PATIENTS Infants admitted to the neonatal unit who developed P. aeruginosa colonization or infection. METHODS We undertook whole genome sequencing of P. aeruginosa strains isolated from colonized infants and from the neonatal unit environment. RESULTS Eighteen infants were colonized with P. aeruginosa. Isolates from 12 infants and 7 environmental samples were sequenced. All but one of the clinical isolates clustered in ST253 and no differences were detected between unmapped reads. The environmental isolates revealed a variety of sequence types, indicating a large diverse bioburden within the unit, which was subsequently confirmed via enterobacterial repetitive intergenic consensus-polymerase chain reaction typing of post-outbreak isolates. One environmental isolate, obtained from a sink in the unit, clustered within ST253 and differed from the outbreak strain by 9 single-nucleotide polymorphisms only. This information allowed us to focus infection control activities on this sink. CONCLUSIONS Whole genome sequencing can provide detailed information in a clinically relevant time frame to aid management of outbreaks in critical patient management areas. The superior discriminatory power of this method makes it a powerful tool in infection control.

Evolutionary dynamics of Enterococcus faecium reveals complex genomic relationships between isolates with independent emergence of vancomycin resistance.

Enterococcus faecium, a major cause of hospital-acquired infections, remains problematic because of its propensity to acquire resistance to vancomycin, which currently is considered first-line therapy. Here, we assess the evolution and resistance acquisition dynamics of E. faecium in a clinical context using a series of 132 bloodstream infection isolates from a single hospital. All isolates, of which 49 (37 %) were vancomycin-resistant, underwent whole-genome sequencing. E. faecium was found to be subject to high rates of recombination with little evidence of sequence importation from outside the local E. faecium population. Apart from disrupting phylogenetic reconstruction, recombination was frequent enough to invalidate MLST typing in the identification of clonal expansion and transmission events, suggesting that, where available, whole-genome sequencing should be used in tracing the epidemiology of E. faecium nosocomial infections and establishing routes of transmission. Several forms of the Tn1549-like element–vanB gene cluster, which was exclusively responsible for vancomycin resistance, appeared and spread within the hospital during the study period. Several transposon gains and losses and instances of in situ evolution were inferred and, although usually chromosomal, the resistance element was also observed on a plasmid background. There was qualitative evidence for clonal expansions of both vancomycin-resistant and vancomycin-susceptible E. faecium with evidence of hospital-specific subclonal expansion. Our data are consistent with continuing evolution of this established hospital pathogen and confirm hospital vancomycin-susceptible and vancomycin-resistant E. faecium patient transmission events, underlining the need for careful consideration before modifying current E. faecium infection control strategies.

Carriage of an ACME II Variant May Have Contributed to Methicillin-Resistant Staphylococcus aureus Sequence Type 239-Like Strain Replacement in Liverpool Hospital, Sydney, Australia

ABSTRACT Approximately 39% of methicillin-resistant Staphylococcus aureus (MRSA) sequence type 239 (ST239)-like bloodstream isolates from Liverpool Hospital (obtained between 1997 and 2008) carry an arginine catabolic mobile element (ACME). Whole-genome sequencing revealed that an ACME II variant is located between orfX and SCCmec III, and based on pulsed-field gel electrophoresis patterns and temporal relationships of all ST239-like isolates (n = 360), ACME carriage may have contributed to subpulsotype strain replacement.

Can molecular DNA-based techniques unravel the truth about diabetic foot infections?

Diabetes foot infections are a common condition and a major causal pathway to lower extremity amputation. Identification of causative pathogens is vital in directing antimicrobial therapy. Historically, clinicians have relied upon culture‐dependent techniques that are now acknowledged as both being selective for microorganisms that thrive under the physiological and nutritional constraints of the microbiology laboratory and that grossly underestimate the microbial diversity of a sample. The amplification and sequence analysis of the 16S rRNA gene has revealed a diversity of microorganisms in diabetes foot infections, extending the view of the diabetic foot microbiome. The interpretation of these findings and their relevance to clinical care remains largely unexplored. The advent of molecular methods that are culture‐independent and employ massively parallel DNA sequencing technology represents a potential ‘game changer’. Metagenomics and its shotgun approach to surveying all DNA within a sample (whole genome sequencing) affords the possibility to characterize not only the microbial diversity within a diabetes foot infection (i.e. ‘which microorganisms are present’) but the biological functions of the community such as virulence and pathogenicity (i.e. ‘what are the microorganisms capable of doing’), moving the focus from single species as pathogens to groups of species. This review will examine the new molecular techniques for exploration of the microbiome of infected and uninfected diabetic foot ulcers, exploring the potential of these new technologies and postulating how they could translate to improved clinical care. Copyright

Metallo-β-Lactamase or Extended-Spectrum β-Lactamase: a Wolf in Sheep's Clothing

ABSTRACT Diagnostic algorithms in commonly used automated bacterial identification systems fail to reliably identify a metallo-β-lactamase in the Enterobacteriaceae. Misidentification as an extended-spectrum β-lactamase may result in inappropriate dismissal of drugs such as aztreonam in favor of carbapenems, which may in turn select for a highly carabapenem resistant phenotype.

Immediate Appearance of Plasmid-Mediated Resistance to Multiple Antibiotics upon Antibiotic Selection: an Argument for Systematic Resistance Epidemiology

ABSTRACT We describe a conjugative plasmid appearing in a bacteremic clone of Escherichia coli immediately upon exposure to the antibiotics for which it encoded resistance. Effective antibiotic choice was made possible by prior screening for this plasmid. Surveillance for transmissible resistance plasmids may be clinically important.

Citrobacter freundii carrying blaKPC-2 and blaNDM-1: characterization by whole genome sequencing

A carbapenem-resistant Citrobacter freundii strain WCHCF65 was recovered from hospital sewage and was characterized by genome sequencing and conjugation experiments. The strain carried nine genes encoding β-lactamases including two carbapenemase genes, blaNDM-1 and blaKPC-2. blaNDM-1 was carried on an IncX3 plasmid, which was identical to a plasmid found in a local Escherichia coli, suggesting interspecies horizontal transfer. blaKPC-2 was bracketed by two copies of insertion sequence ISKpn19, which could form a composite transposon with the potential to mobilize blaKPC-2, on a new type of plasmid. The coexistence of blaNDM-1 and blaKPC-2 conferred higher levels of resistance to carbapenems compared with blaNDM-1 or blaKPC-2 alone. The coexistence of these carbapenemase genes, on two different plasmids, in one strain may allow new genetic platforms to be generated to mediate their spread.