Ingenuity pathways analysis of urine metabonomics phenotypes toxicity of gentamicin in multiple organs

We introduce the use of Ingenuity Pathway Analysis to analyzing global metabonomics in order to characterize phenotypically biochemical perturbations and the potential mechanisms of the gentamicin-induced toxicity in multiple organs. A single dose of gentamicin was administered to Sprague Dawley rats (200 mg/kg, n = 6) and urine samples were collected at -24-0 h pre-dosage, 0-24, 24-48, 48-72 and 72-96 h post-dosage of gentamicin. The urine metabonomics analysis was performed by UPLC/MS, and the mass spectra signals of the detected metabolites were systematically deconvoluted and analyzed by pattern recognition analyses (Heatmap, PCA and PLS-DA), revealing a time-dependency of the biochemical perturbations induced by gentamicin toxicity. As result, the holistic metabolome change induced by gentamicin toxicity in the animal's organisms was characterized. Several metabolites involved in amino acid metabolism were identified in urine, and it was confirmed that gentamicin biochemical perturbations can be foreseen from these biomarkers. Notoriously, it was found that gentamicin induced toxicity in multiple organs system in the laboratory rats. The proof-of-knowledge based Ingenuity Pathway Analysis revealed gentamicin induced liver and heart toxicity, along with the previously known toxicity in kidney. The metabolites creatine, nicotinic acid, prostaglandin E2, and cholic acid were identified and validated as phenotypic biomarkers of gentamicin induced toxicity. Altogether, the significance of the use of metabonomics analyses in the assessment of drug toxicity is highlighted once more; furthermore, this work demonstrated the powerful predictive potential of the Ingenuity Pathway Analysis to study of drug toxicity and its valuable complementation for metabonomics based assessment of the drug toxicity.

SNP diversity of Enterococcus faecalis and Enterococcus faecium in a South East Queensland waterway, Australia, and associated antibiotic resistance gene profiles

BACKGROUND: Enterococcus faecalis and Enterococcus faecium are associated with faecal pollution of water, linked to swimmer-associated gastroenteritis and demonstrate a wide range of antibiotic resistance. The Coomera River is a main water source for the Pimpama-Coomera watershed and is located in South East Queensland, Australia, which is used intensively for agriculture and recreational purposes. This study investigated the diversity of E. faecalis and E. faecium using Single Nucleotide Polymorphisms (SNPs) and associated antibiotic resistance profiles. RESULTS: Total enterococcal counts (cfu/ml) for three/six sampling sites were above the United States Environmental Protection Agency (USEPA) recommended level during rainfall periods and fall into categories B and C of the Australian National Health and Medical Research Council (NHMRC) guidelines (with a 1-10% gastrointestinal illness risk). E. faecalis and E. faecium isolates were grouped into 29 and 23 SNP profiles (validated by MLST analysis) respectively. This study showed the high diversity of E. faecalis and E. faecium over a period of two years and both human-related and human-specific SNP profiles were identified. 81.8% of E. faecalis and 70.21% of E. faecium SNP profiles were associated with genotypic and phenotypic antibiotic resistance. Gentamicin resistance was higher in E. faecalis (47% resistant) and harboured the aac(6')-aph(2') gene. Ciprofloxacin resistance was more common in E. faecium (12.7% resistant) and gyrA gene mutations were detected in these isolates. Tetracycline resistance was less common in both species while tet(L) and tet(M) genes were more prevalent. Ampicillin resistance was only found in E. faecium isolates with mutations in the pbp5 gene. Vancomycin resistance was not detected in any of the isolates. We found that antibiotic resistance profiles further sub-divided the SNP profiles of both E. faecalis and E. faecium. CONCLUSIONS: The distribution of E. faecalis and E. faecium genotypes is highly diverse in the Coomera River. The SNP genotyping method is rapid and robust and can be applied to study the diversity of E. faecalis and E. faecium in waterways. It can also be used to test for human-related and human-specific enterococci in water. The resolving power can be increased by including antibiotic-resistant profiles which can be used as a possible source tracking tool. This warrants further investigation.

Molecular characterisation of environmental enterococci derived from water samples and assessment of associated public health hazards

Enterococci are versatile Gram-positive bacteria that can survive under extreme conditions. Most enterococci are non-virulent and found in the gastrointestinal tract of humans and animals. Other strains are opportunistic pathogens that contribute to a large number of nosocomial infections globally. Epidemiological studies demonstrated a direct relationship between the density of enterococci in surface waters and the risk of swimmer-associated gastroenteritis. The distribution of infectious enterococcal strains from the hospital environment or other sources to environmental water bodies through sewage discharge or other means, could increase the prevalence of these strains in the human population. Environmental water quality studies may benefit from focusing on a subset of Enterococcus spp. that are consistently associated with sources of faecal pollution such as domestic sewage, rather than testing for the entire genus. E. faecalis and E. faecium are potentially good focal species for such studies, as they have been consistently identified as the dominant Enterococcus spp. in human faeces and sewage. On the other hand enterococcal infections are predominantly caused by E. faecalis and E. faecium. The characterisation of E. faecalis and E. faecium is important in studying their population structures, particularly in environmental samples. In developing and implementing rapid, robust molecular genotyping techniques, it is possible to more accurately establish the relationship between human and environmental enterococci. Of particular importance, is to determine the distribution of high risk enterococcal clonal complexes, such as E. faecium clonal complex 17 and E. faecalis clonal complexes 2 and 9 in recreational waters. These clonal complexes are recognized as particularly pathogenic enterococcal genotypes that cause severe disease in humans globally. The Pimpama-Coomera watershed is located in South East Queensland, Australia and was investigated in this study mainly because it is used intensively for agriculture and recreational purposes and has a strong anthropogenic impact. The primary aim of this study was to develop novel, universally applicable, robust, rapid and cost effective genotyping methods which are likely to yield more definitive results for the routine monitoring of E. faecalis and E. faecium, particularly in environmental water sources. To fullfill this aim, new genotyping methods were developed based on the interrogation of highly informative single nucleotide polymorphisms (SNPs) located in housekeeping genes of both E. faecalis and E. faecium. SNP genotyping was successfully applied in field investigations of the Coomera watershed, South-East Queensland, Australia. E. faecalis and E. faecium isolates were grouped into 29 and 23 SNP profiles respectively. This study showed the high longitudinal diversity of E. faecalis and E. faecium over a period of two years, and both human-related and human-specific SNP profiles were identified. Furthermore, 4.25% of E. faecium strains isolated from water was found to correspond to the important clonal complex-17 (CC17). Strains that belong to CC17 cause the majority of hospital outbreaks and clinical infections globally. Of the six sampling sites of the Coomera River, Paradise Point had the highest number of human-related and human-specific E. faecalis and E. faecium SNP profiles. The secondary aim of this study was to determine the antibiotic-resistance profiles and virulence traits associated with environmental E. faecalis and E. faecium isolates compared to human pathogenic E. faecalis and E. faecium isolates. This was performed to predict the potential health risks associated with coming into contact with these strains in the Coomera watershed. In general, clinical isolates were found to be more resistant to all the antibiotics tested compared to water isolates and they harbored more virulence traits. Multi-drug resistance was more prevalent in clinical isolates (71.18% of E. faecalis and 70.3 % of E. faecium) compared to water isolates (only 5.66 % E. faecium). However, tetracycline, gentamicin, ciprofloxacin and ampicillin resistance was observed in water isolates. The virulence gene esp was the most prevalent virulence determinant observed in clinical isolates (67.79% of E. faecalis and 70.37 % of E. faecium), and this gene has been described as a human-specific marker used for microbial source tracking (MST). The presence of esp in water isolates (16.36% of E. faecalis and 19.14% of E. faecium) could be indicative of human faecal contamination in these waterways. Finally, in order to compare overall gene expression between environmental and clinical strains of E. faecalis, a comparative gene hybridization study was performed. The results of this investigation clearly demonstrated the up-regulation of genes associated with pathogenicity in E. faecalis isolated from water. The expression study was performed at physiological temperatures relative to ambient temperatures. The up-regulation of virulence genes demonstrates that environmental strains of E. faecalis can pose an increased health risk which can lead to serious disease, particularly if these strains belong to the virulent CC17 group. The genotyping techniques developed in this study not only provide a rapid, robust and highly discriminatory tool to characterize E. faecalis and E. faecium, but also enables the efficient identification of virulent enterococci that are distributed in environmental water sources.

ASAP ECMO : antibiotic, sedative and analgesic pharmacokinetics during extracorporeal membrane oxygenation : a multi-centre study to optimise drug therapy during ECMO

BACKGROUND: Given the expanding scope of extracorporeal membrane oxygenation (ECMO) and its variable impact on drug pharmacokinetics as observed in neonatal studies, it is imperative that the effects of the device on the drugs commonly prescribed in the intensive care unit (ICU) are further investigated. Currently, there are no data to confirm the appropriateness of standard drug dosing in adult patients on ECMO. Ineffective drug regimens in these critically ill patients can seriously worsen patient outcomes. This study was designed to describe the pharmacokinetics of the commonly used antibiotic, analgesic and sedative drugs in adult patients receiving ECMO. METHODS: This is a multi-centre, open-label, descriptive pharmacokinetic (PK) study. Eligible patients will be adults treated with ECMO for severe cardiac and/or respiratory failure at five Intensive Care Units in Australia and New Zealand. Patients will receive the study drugs as part of their routine management. Blood samples will be taken from indwelling catheters to investigate plasma concentrations of several antibiotics (ceftriaxone, meropenem, vancomycin, ciprofloxacin, gentamicin, piperacillin-tazobactum, ticarcillin-clavulunate, linezolid, fluconazole, voriconazole, caspofungin, oseltamivir), sedatives and analgesics (midazolam, morphine, fentanyl, propofol, dexmedetomidine, thiopentone). The PK of each drug will be characterised to determine the variability of PK in these patients and to develop dosing guidelines for prescription during ECMO. DISCUSSION: The evidence-based dosing algorithms generated from this analysis can be evaluated in later clinical studies. This knowledge is vitally important for optimising pharmacotherapy in these most severely ill patients to maximise the opportunity for therapeutic success and minimise the risk of therapeutic failure

Antibiotic resistance and virulence traits in clinical and environmental Enterococcus faecalis and Enterococcus faecium isolates

This study compared virulence and antibiotic resistance traits in clinical and environmental E. faecalis and E. faecium isolates. E. faecalis isolates harboured a broader spectrum of virulence determinants compared to E. faecium isolates. The virulence traits Cyl-A, Cyl-B, Cyl-M, gel-E and esp were tested and environmental isolates predominantly harboured gel-E (80% of E. faecalis and 31.9% of E. faecium) whereas esp was more prevalent in clinical isolates (67.79% of E. faecalis and 70.37 % of E. faecium). E. faecalis and E. faecium isolated from water had different antibiotic resistance patterns compared to those isolated from clinical samples. Linozolid resistance was not observed in any isolates tested and vancomycin resistance was observed only in clinical isolates. Resistance to other antibiotics (tetracycline, gentamicin, ciprofloxacin and ampicillin) was detected in both clinical and water isolates. Clinical isolates were more resistant to all the antibiotics tested compared to water isolates. Multi-drug resistance was more prevalent in clinical isolates (71.18% of E. faecalis and 70.3 % of E. faecium) compared to water isolates (only 5.66 % E. faecium). tet L and tet M genes were predominantly identified in tetracycline-resistant isolates. All water and clinical isolates resistant to ciprofloxacin and ampicillin contained mutations in the gyrA, parC and pbp5 genes. A significant correlation was found between the presence of virulence determinants and antibiotic resistance in all the isolates tested in this study (p<0.05). The presence of antibiotic resistant enterococci, together with associated virulence traits, in surface recreational water could be a public health risk.

Typing early Australian healthcare-associated MRSA : confirmation of major clones and emergence of ST1-MRSA-IV and novel ST2249-MRSA-III

AIMS: To investigate the evolutionary origins of Australian healthcare-associated (HCA) methicillin-resistant Staphylococcus aureus (MRSA) strains from a panel of historical isolates typed using current genotyping techniques. METHODS: Nineteen MRSA isolates from 1965 to 1981 were examined and antibiotic susceptibility profiles determined. Genetic characterisation included real-time (RT) polymerase chain reaction (PCR) assays to identify single nucleotide polymorhpism (SNP) clonal complexes (SNP CC) and sequence type (SNP ST), multi locus sequence typing (MLST) and staphylococcal chromosomal cassette mec typing. RESULTS: All SNP CC30 isolates belonged to a novel sequence type, ST2249. All SNP CC239 isolates were confirmed as ST239-MRSA-III, except for a new single locus variant of ST239, ST2275. A further new type, ST2276, was identified. CONCLUSIONS: The earliest MRSA examined from 1965 was confirmed as ST250-MRSA-I, consistent with archaic European types. Identification of ST1-MRSA-IV in 1981 is the earliest appearance of this clinically important lineage which manifested in Australia and the United States in the 1990s. A previously unknown multi-resistant clone, ST2249-MRSA-III, was identified from 1973. Gentamicin resistance first appeared in this novel strain from 1976 and not ST239 as previously suspected. Thus, ST2249 was present in the earliest phase of the HCA MRSA epidemic in eastern Australia and was perhaps related to the emergence of the globally epidemic strain ST239.

A conjugative plasmid carrying the carbapenem resistance gene blaOXA-23 in AbaR4 in an extensively resistant GC1 Acinetobacter baumannii isolate

OBJECTIVES: To locate the acquired bla(OXA-23) carbapenem resistance gene in an Australian A. baumannii global clone 1 (GC1) isolate. METHODS: The genome of the extensively antibiotic-resistant GC1 isolate A85 harbouring bla(OXA-23) in Tn2006 was sequenced using Illumina HiSeq, and the reads were used to generate a de novo assembly. PCR was used to assemble relevant contigs. Sequences were compared with ones in GenBank. Conjugation experiments were conducted. RESULTS: The sporadic GC1 isolate A85, recovered in 2003, was extensively resistant, exhibiting resistance to imipenem, meropenem and ticarcillin/clavulanate, to cephalosporins and fluoroquinolones and to the older antibiotics gentamicin, kanamycin and neomycin, sulfamethoxazole, trimethoprim and tetracycline. Genes for resistance to older antibiotics are in the chromosome, in an AbaR3 resistance island. A second copy of the ampC gene in Tn6168 confers cephalosporin resistance and the gyrA and parC genes have mutations leading to fluoroquinolone resistance. An 86 335 bp repAci6 plasmid, pA85-3, carrying bla(OXA-23) in Tn2006 in AbaR4, was shown to transfer imipenem, meropenem and ticarcillin/clavulanate resistance into a susceptible recipient. A85 also contains two small cryptic plasmids of 2.7 and 8.7 kb. A85 is sequence type ST126 (Oxford scheme) and carries a novel KL15 capsule locus and the OCL3 outer core locus. CONCLUSIONS: A85 represents a new GC1 lineage identified by the novel capsule locus but retains AbaR3 carrying genes for resistance to older antibiotics. Resistance to imipenem, meropenem and ticarcillin/clavulanate has been introduced into A85 by pA85-3, a repAci6 conjugative plasmid carrying Tn2006 in AbaR4.

The ADMIN-ICU survey: A survey on antimicrobial dosing and monitoring in ICUs

Objectives: There is little evidence and few guidelines to inform the most appropriate dosing and monitoring for antimicrobials in the ICU. We aimed to survey current practices around the world. Methods: An online structured questionnaire was developed and sent by e-mail to obtain information on local antimicrobial prescribing practices for glycopeptides, piperacillin/tazobactam, carbapenems, aminoglycosides and colistin. Results: A total of 402 professionals from 328 hospitals in 53 countries responded, of whom 78% were specialists in intensive care medicine (41% intensive care, 30% anaesthesiology, 14% internal medicine) and 12% were pharmacists. Vancomycin was used as a continuous infusion in 31% of units at a median (IQR) daily dose of 25 (25–30) mg/kg. Piperacillin/tazobactam was used as an extended infusion by 22% and as a continuous infusion by 7%. An extended infusion of carbapenem (meropenem or imipenem) was used by 27% and a continuous infusion by 5%. Colistin was used at a daily dose of 7.5 (3.9–9) million IU (MIU)/day, predominantly as a short infusion. The most commonly used aminoglycosides were gentamicin (55%) followed by amikacin (40%), with administration as a single daily dose reported in 94% of the cases. Gentamicin was used at a daily dose of 5 (5–6) mg/day and amikacin at a daily dose of 15 (15–20) mg/day. Therapeutic drug monitoring of vancomycin, piperacillin/tazobactam and meropenem was used by 74%, 1% and 2% of the respondents, respectively. Peak aminoglycoside concentrations were sampled daily by 28% and trough concentrations in all patients by 61% of the respondents. Conclusions: We found wide variability in reported practices for antibiotic dosing and monitoring. Research is required to develop evidence-based guidelines to standardize practices.