Failure of daptomycin ß-Lactam combination therapy to prevent resistance emergence in Enterococcus faecium.

Daptomycin ß-Lactam combination therapy offers "protection" against daptomycin non-susceptibility (DNS) development in Enterococcus faecium. We report failure of this strategy and the importance of source control. Mutations were detected in the LiaF and cls genes in DNS isolates. A single DNS isolate contained an unrecognized mutation, which requires confirmation.

Polyclonal emergence of vanA vancomycin-resistant Enterococcus faecium in Australia.

Objectives: To investigate the genetic context associated with the emergence of vanA VRE in Australia. Methods: The whole genomes of 18 randomly selected vanA -positive Enterococcus faecium patient isolates, collected between 2011 and 2013 from hospitals in four Australian capitals, were sequenced and analysed. Results: In silico typing and transposon/plasmid assembly revealed that the sequenced isolates represented (in most cases) different hospital-adapted STs and were associated with a variety of different Tn 1546 variants and plasmid backbone structures. Conclusions: The recent emergence of vanA VRE in Australia was polyclonal and not associated with the dissemination of a single 'dominant' ST or vanA -encoding plasmid. Interestingly, the factors contributing to this epidemiological change are not known and future studies may need to consider investigation of potential community sources.

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.

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 ß-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.

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 ß-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.

Ceftaroline fosamil salvage therapy: an option for reduced-vancomycin-susceptible MRSA bacteraemia.

OBJECTIVES: To examine the activity of ceftaroline against reduced-vancomycin-susceptible MRSA isolates. METHODS: One-hundred and three MRSA blood culture isolates (predominantly ST239-MRSA-III), with varying vancomycin phenotypes, had their ceftaroline MICs determined by broth microdilution and MIC Evaluator strip (Oxoid-Thermo Fisher). Statistical analyses were performed that examined relationships with vancomycin and daptomycin MICs. Mutations in mecA were also examined. RESULTS: All 103 isolates (including 60 heteroresistant vancomycin-intermediate Staphylococcus aureus/vancomycin-intermediate S. aureus) were susceptible to ceftaroline, with one isolate displaying heteroresistance that may be related to a mecA mutation. Higher ceftaroline MICs were associated with vancomycin-susceptible S. aureus isolates. CONCLUSIONS: This study highlights that ceftaroline fosamil is an option for salvage therapy based on in vitro activity.

In vivo evolution of antimicrobial resistance in a series of Staphylococcus aureus patient isolates: the entire picture or a cautionary tale?

OBJECTIVES: To obtain an expanded understanding of antibiotic resistance evolution in vivo, particularly in the context of vancomycin exposure. METHODS: The whole genomes of six consecutive methicillin-resistant Staphylococcus aureus blood culture isolates (ST239-MRSA-III) from a single patient exposed to various antimicrobials (over a 77 day period) were sequenced and analysed. RESULTS: Variant analysis revealed the existence of non-susceptible sub-populations derived from a common susceptible ancestor, with the predominant circulating clone(s) selected for by type and duration of antimicrobial exposure. CONCLUSIONS: This study highlights the dynamic nature of bacterial evolution and that non-susceptible sub-populations can emerge from clouds of variation upon antimicrobial exposure. Diagnostically, this has direct implications for sample selection when using whole-genome sequencing as a tool to guide clinical therapy. In the context of bacteraemia, deep sequencing of bacterial DNA directly from patient blood samples would avoid culture 'bias' and identify mutations associated with circulating non-susceptible sub-populations, some of which may confer cross-resistance to alternate therapies.

Whole genome sequence analysis of the first Australian OXA-48-producing outbreak-associated Klebsiella pneumoniae isolates: the resistome and in vivo evolution.

Whole genome sequencing was used to characterize the resistome of intensive care unit (ICU) outbreak-associated carbapenem-resistant K. pneumoniae isolates. Importantly, and of particular concern, the carbapenem-hydrolyzing ß-lactamase gene bla(OXA-48) and the extended-spectrum ß-lactamase gene bla(CTX-M-14), were identified on a single broad host-range conjugative plasmid. This represents the first report of bla(OXA-48) in Australia and highlights the importance of resistance gene surveillance, as such plasmids can silently spread amongst enterobacterial populations and have the potential to drastically limit treatment options. Furthermore, the in vivo evolution of these isolates was also examined after 18 months of intra-abdominal carriage in a patient that transited through the ICU during the outbreak period. Reflecting the clonality of K. pneumoniae, only 11 single nucleotide polymorphisms (SNPs) were accumulated during this time-period and many of these were associated with genes involved in tolerance/resistance to antibiotics, metals or organic solvents, and transcriptional regulation. Collectively, these SNPs are likely to be associated with changes in virulence (at least to some extent) that have refined the in vivo colonization capacity of the original outbreak isolate.

Predictors of mortality in Staphylococcus aureus Bacteremia.

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.

Chromosomal mutations involved in antibiotic resistance in Staphylococcus aureus.

Staphylococcus aureus is an important pathogen involved in infections in both the community and hospital setting. Strains that are resistant to multiple classes of antibiotics, particularly methicillin-resistant strains (MRSA), are prevalent in nosocomial infections and are associated with high morbidity and mortality rates. Such antibiotic-resistant strains limit the therapeutic options and place a burden on the health care system. In the hospital setting, horizontal gene transfer plays an important role in disseminating antibiotic resistant determinants among S. aureus. However, resistance to all known classes of antibiotics have been attributed to genes found within the S. aureus chromosome or to due to mutation as a result of selection pressure. Spontaneous mutations, in particular, are pivotal in the emergence of novel resistances. Consequently, newer drugs with better activity and/or antibacterial agents with novel targets need to be developed to combat and control the further spread of antibiotic resistance.

Immediate appearance of plasmid-mediated resistance to multiple antibiotics upon antibiotic selection: an argument for systematic resistance epidemiology.

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.

Paradoxical effect of Klebsiella pneumoniae OmpK36 porin deficiency.

AIM: To evaluate the effect of defined mutations in the major OmpK35 and OmpK36 porins in Klebsiella pneumoniae on the activity of two common plasmid-mediated AmpC enzymes. METHODS: Naturally occurring conjugative plasmids containing bla(DHA-1) and bla(CMY-2) were obtained from K. pneumoniae isolates in western Sydney. These were moved into K. pneumoniae ATCC13883 and isogenic porin knockouts Kp885 (DeltaompK35) and Kp886 (DeltaompK36), created by homologous recombination of kanamycin resistance cartridges into the specified genes, and their antimicrobial susceptibilities compared. RESULTS: beta-lactam resistance was greater in the presence of CMY-2-containing plasmids than DHA-1-containing plasmids, and higher in K. pneumoniae than Escherichia coli. Neither cefepime nor imipenem resistance was observed, and DHA-mediated cefotaxime and ticarcillin/clavulanate resistance was unexpectedly reduced from 8-24 (CTX) and >256 (TIM) mg/L in Kp13883 to 1-2 (CTX) and 32-48 mg/L (TIM) in the isogenic DeltaompK36 porin knockout Kp886. CONCLUSIONS: AmpC plasmids in particular are an important cause of transmissible resistance to ticarcillin/clavulanate in K. pneumoniae, but probably not in E. coli. Single knockouts of OmpK35 and OmpK36 porins in K. pneumoniae do not significantly increase antibiotic resistance in K. pneumoniae, and a paradoxical lowering of resistance to CTX and TIM is seen with deletion of ompK36. This has potentially important clinical implications.

bla(IMP-4) in different genetic contexts in Enterobacteriaceae isolates from Australia.

The IMP-4 metallo-beta-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 bla(IMP-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.

Metallo-beta-lactamase or extended-spectrum beta-lactamase: a wolf in sheep's clothing.

Diagnostic algorithms in commonly used automated bacterial identification systems fail to reliably identify a metallo-beta-lactamase in the Enterobacteriaceae. Misidentification as an extended-spectrum beta-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.

Beta-lactam resistance in the gram negatives: increasing complexity of conditional, composite and multiply resistant phenotypes.

The greatest impact of microbiology data on clinical care is in the critically ill. Unfortunately, this is also the area in which microbiology laboratories are most often non-contributive. Attempts to move to rapid, culture-independent diagnostics are driven by the need to expedite urgent results. This is difficult in Gram-negative infection because of the complexity of the antibiotic resistance phenotype. Here, we discuss resistance to modern beta-lactams as a case in point. Recent outbreaks of transmissible carbapenem resistance among Gram-negative enteric pathogens in Sydney and Melbourne serve to illustrate the pitfalls of traditional phenotypical approaches. A better understanding of the epidemiology and mosaic nature of antibiotic resistance elements in the microflora is needed for us to move forward.