Prognostic Value of Gut Microbiome for Conversion from Mild Cognitive Impairment to Alzheimer's Disease Dementia within 4 Years: Results from the AlzBiom Study.

Alterations in the gut microbiome are associated with the pathogenesis of Alzheimer's disease (AD) and can be used as a diagnostic measure. However, longitudinal data of the gut microbiome and knowledge about its prognostic significance for the development and progression of AD are limited. The aim of the present study was to develop a reliable predictive model based on gut microbiome data for AD development. In this longitudinal study, we investigated the intestinal microbiome in 49 mild cognitive impairment (MCI) patients over a mean (SD) follow-up of 3.7 (0.6) years, using shotgun metagenomics. At the end of the 4-year follow-up (4yFU), 27 MCI patients converted to AD dementia and 22 MCI patients remained stable. The best taxonomic model for the discrimination of AD dementia converters from stable MCI patients included 24 genera, yielding an area under the receiver operating characteristic curve (AUROC) of 0.87 at BL, 0.92 at 1yFU and 0.95 at 4yFU. The best models with functional data were obtained via analyzing 25 GO (Gene Ontology) features with an AUROC of 0.87 at BL, 0.85 at 1yFU and 0.81 at 4yFU and 33 KO [Kyoto Encyclopedia of Genes and Genomes (KEGG) ortholog] features with an AUROC of 0.79 at BL, 0.88 at 1yFU and 0.82 at 4yFU. Using ensemble learning for these three models, including a clinical model with the four parameters of age, gender, body mass index (BMI) and Apolipoprotein E (ApoE) genotype, yielded an AUROC of 0.96 at BL, 0.96 at 1yFU and 0.97 at 4yFU. In conclusion, we identified novel and timely stable gut microbiome algorithms that accurately predict progression to AD dementia in individuals with MCI over a 4yFU period.

Human commensals producing a novel antibiotic impair pathogen colonization.

The vast majority of systemic bacterial infections are caused by facultative, often antibiotic-resistant, pathogens colonizing human body surfaces. Nasal carriage of Staphylococcus aureus predisposes to invasive infection, but the mechanisms that permit or interfere with pathogen colonization are largely unknown. Whereas soil microbes are known to compete by production of antibiotics, such processes have rarely been reported for human microbiota. We show that nasal Staphylococcus lugdunensis strains produce lugdunin, a novel thiazolidine-containing cyclic peptide antibiotic that prohibits colonization by S. aureus, and a rare example of a non-ribosomally synthesized bioactive compound from human-associated bacteria. Lugdunin is bactericidal against major pathogens, effective in animal models, and not prone to causing development of resistance in S. aureus. Notably, human nasal colonization by S. lugdunensis was associated with a significantly reduced S. aureus carriage rate, suggesting that lugdunin or lugdunin-producing commensal bacteria could be valuable for preventing staphylococcal infections. Moreover, human microbiota should be considered as a source for new antibiotics.

Oxidative stress drives the selection of quorum sensing mutants in the Staphylococcus aureus population.

Quorum sensing (QS) is the central mechanism by which social interactions within the bacterial community control bacterial behavior. QS-negative cells benefit by exploiting public goods produced by the QS-proficient population. Mechanisms to keep the balance between producers and nonproducers within the population are expected but have not been elucidated for peptide-based QS systems in gram-positive pathogens. The Agr system of Staphylococcus aureus comprises the secretion and sensing of an autoinducing peptide to activate its own expression via the response regulator AgrA as well as the expression of a regulatory RNAIII and psmα/psmß coding for phenol-soluble modulins (PSMs). Agr mutants can be monitored on blood agar due to their nonhemolytic phenotype. In vitro evolution and competition experiments show that they readily accumulate in a process that is accelerated by ciprofloxacin, while the wild type (WT) is retained in the population at low numbers. However, agr mutants possess a fitness advantage only under aerobic conditions. Under hypoxia, Agr activity is increased but without the expected fitness cost. The Agr-imposed oxygen-dependent fitness cost is not due to a metabolic burden but due to the reactive oxygen species (ROS)-inducing capacity of the PSMs and RNAIII-regulated factors. Thus, selection of mutants is dictated by the QS system itself. Under aerobic conditions, emergence of agr-negative mutants may provide the population with a fitness advantage while hypoxia favors QS maintenance and even affords increased toxin production. The oxygen-driven tuning of the Agr system might be of importance to provide the pathogen with capabilities crucial for disease progression.

Identification of Drug Resistance Determinants in a Clinical Isolate of Pseudomonas aeruginosa by High-Density Transposon Mutagenesis.

With the aim to identify potential new targets to restore antimicrobial susceptibility of multidrug-resistant (MDR) Pseudomonas aeruginosa isolates, we generated a high-density transposon (Tn) insertion mutant library in an MDR P. aeruginosa bloodstream isolate (isolate ID40). The depletion of Tn insertion mutants upon exposure to cefepime or meropenem was measured in order to determine the common resistome for these clinically important antipseudomonal ß-lactam antibiotics. The approach was validated by clean deletions of genes involved in peptidoglycan synthesis/recycling, such as the genes for the lytic transglycosylase MltG, the murein (Mur) endopeptidase MepM1, the MurNAc/GlcNAc kinase AmgK, and the uncharacterized protein YgfB, all of which were identified in our screen as playing a decisive role in survival after treatment with cefepime or meropenem. We found that the antibiotic resistance of P. aeruginosa can be overcome by targeting usually nonessential genes that turn essential in the presence of therapeutic concentrations of antibiotics. For all validated genes, we demonstrated that their deletion leads to the reduction of ampC expression, resulting in a significant decrease in ß-lactamase activity, and consequently, these mutants partly or completely lost resistance against cephalosporins, carbapenems, and acylaminopenicillins. In summary, the determined resistome may comprise promising targets for the development of drugs that may be used to restore sensitivity to existing antibiotics, specifically in MDR strains of P. aeruginosa.

Description of Citrobacter cronae sp. nov., isolated from human rectal swabs and stool samples.

Nine independent Gram-negative bacterial strains were isolated from rectal swabs or stool samples of immunocompromised patients from two different wards of a university hospital. All isolates were phylogenetically analysed based on their 16S rRNA gene sequence, housekeeping gene recN, multilocus sequence analysis of concatenated partial fusA, leuS, pyrG and rpoB sequences, and by whole genome sequencing data. The analysed strains of the new species cluster together and form a separate branch with Citrobacter werkmanii NBRC105721T as the most closely related species. An average nucleotide identity value of 95.9-96% and computation of digital DNA-DNA hybridization values separate the new species from all other type strains of the genus Citrobacter. Biochemical characteristics further delimit the isolates from closely related Citrobacter type strains. As a result of the described data, a new Citrobacter species is introduced, for which the name Citrobacter cronae sp. nov. is proposed. The type strain is Tue2-1T with a G+C DNA content of 52.2 mol%.

Distinct impact of antibiotics on the gut microbiome and resistome: a longitudinal multicenter cohort study.

BACKGROUND: The selection pressure exercised by antibiotic drugs is an important consideration for the wise stewardship of antimicrobial treatment programs. Treatment decisions are currently based on crude assumptions, and there is an urgent need to develop a more quantitative knowledge base that can enable predictions of the impact of individual antibiotics on the human gut microbiome and resistome. RESULTS: Using shotgun metagenomics, we quantified changes in the gut microbiome in two cohorts of hematological patients receiving prophylactic antibiotics; one cohort was treated with ciprofloxacin in a hospital in Tübingen and the other with cotrimoxazole in a hospital in Cologne. Analyzing this rich longitudinal dataset, we found that gut microbiome diversity was reduced in both treatment cohorts to a similar extent, while effects on the gut resistome differed. We observed a sharp increase in the relative abundance of sulfonamide antibiotic resistance genes (ARGs) by 148.1% per cumulative defined daily dose of cotrimoxazole in the Cologne cohort, but not in the Tübingen cohort treated with ciprofloxacin. Through multivariate modeling, we found that factors such as individual baseline microbiome, resistome, and plasmid diversity; liver/kidney function; and concurrent medication, especially virostatic agents, influence resistome alterations. Strikingly, we observed different effects on the plasmidome in the two treatment groups. There was a substantial increase in the abundance of ARG-carrying plasmids in the cohort treated with cotrimoxazole, but not in the cohort treated with ciprofloxacin, indicating that cotrimoxazole might contribute more efficiently to the spread of resistance. CONCLUSIONS: Our study represents a step forward in developing the capability to predict the effect of individual antimicrobials on the human microbiome and resistome. Our results indicate that to achieve this, integration of the individual baseline microbiome, resistome, and mobilome status as well as additional individual patient factors will be required. Such personalized predictions may in the future increase patient safety and reduce the spread of resistance. TRIAL REGISTRATION: ClinicalTrials.gov, NCT02058888 . Registered February 10 2014.

Expansion of Vancomycin-Resistant Enterococcus faecium in an Academic Tertiary Hospital in Southwest Germany: a Large-Scale Whole-Genome-Based Outbreak Investigation.

Vancomycin-resistant Enterococcus faecium (VREfm) is a frequent cause of nosocomial outbreaks. In the second half of 2015, a sharp increase in the incidence of VREfm was observed at our university medical center. Next-generation sequencing (NGS) was used to analyze the first isolates of VREfm recovered from patients between 2010 and 2016 (n = 773) in order to decipher epidemiological change, outbreak dynamics, and possible transmission routes. VREfm isolates were analyzed using whole-genome sequencing followed by sequence type extraction and phylogenetic analysis. We examined epidemiological data, room occupancy data, and patient transferals and calculated an intensity score for patient-to-patient contact. Phylogenetic analysis revealed the presence of 38 NGS clusters and 110 single clones. The increase of VREfm was caused mainly by the expansion of two newly introduced NGS clusters, comprising VanB-type strains determined by multilocus sequence typing (MLST) as sequence type 80 (ST80) and ST117. By combining phylogenetic information with epidemiological data, intrahospital transmission could be demonstrated, however to a lesser extent than initially expected based solely on epidemiological data. The outbreak clones were continuously imported from other hospitals, suggesting a change in the epidemiological situation at a regional scale. By tracking intrahospital patient transferals, two major axes could be identified that contributed to the spread of VREfm within the hospital. NGS-based outbreak analysis revealed a dramatic change in the local and regional epidemiology of VREfm, emphasizing the role of health care networks in the spread of VREfm.

Colistin susceptibility test evaluation of multiple-resistance-level Pseudomonas aeruginosa isolates generated in a morbidostat device.

Objectives: Colistin is a last-resort antibiotic against the critical-status pathogen Pseudomonas aeruginosa. There is still uncertainty regarding how to accurately measure colistin susceptibility in P. aeruginosa. Evaluation of antimicrobial susceptibility testing (AST) methods is largely hampered by the lack of resistant isolates and those around the susceptibility breakpoint. The aim of this study was to generate such strains in a morbidostat device for use in AST method evaluation. Methods: A morbidostat device was used to cultivate susceptible clinical strains into isolates with a wide range of colistin MICs. Subsequently, five commercial AST methods were compared against the gold standard broth microdilution (BMD) method: MICRONAUT-S, SensiTest, Sensititre, Rapid Polymyxin Pseudomonas and Etest. Results: A total of 131 P. aeruginosa isolates were used for colistin susceptibility test evaluation (100 colistin susceptible and 31 colistin resistant). The 31 colistin-resistant isolates evolved resistance in the morbidostat to different MIC ranges (4-512 mg/L, 100% resistance generation efficacy). The categorical agreement (CA) rates for MICRONAUT-S, SensiTest and Rapid Polymyxin Pseudomonas were 94.7%, 93.9% and 92.4%, respectively. The Sensititre achieved the highest CA score (96.9%), whereas the Etests had the lowest CA score (84%). The very major discrepancy (VMD) rates for all tests were between 3.2% and 67.7%. Conclusions: The morbidostat device can efficiently provide laboratories with colistin-resistant strains for test evaluation. Although CA rates were high for commercial AST methods except for Etests, none met the ≤1.5% CLSI limit for VMD rates. Performance was generally inferior when using isolates with low-level resistance.

Genomic characterisation of clinical and environmental Pseudomonas putida group strains and determination of their role in the transfer of antimicrobial resistance genes to Pseudomonas aeruginosa.

BACKGROUND: Pseudomonas putida is a Gram-negative, non-fermenting bacterium frequently encountered in various environmental niches. P. putida rarely causes disease in humans, though serious infections and outbreaks have been reported from time to time. Some have suggested that P. putida functions as an exchange platform for antibiotic resistance genes (ARG), and thus represents a serious concern in the spread of ARGs to more pathogenic organisms within a hospital. Though poorly understood, the frequency of ARG exchange between P. putida and the more virulent Pseudomonas aeruginosa and its clinical relevance are particularly important for designing efficient infection control strategies, such as deciding whether high-risk patients colonized with a multidrug resistant but typically low pathogenic P. putida strain should be contact isolated or not. RESULTS: In this study, 21,373 screening samples (stool, rectal and throat swab) were examined to determine the presence of P. putida in a high-risk group of haemato-oncology patients during a 28-month period. A total of 89 P. putida group strains were isolated from 85 patients, with 41 of 89 (46.1%) strains harbouring the metallo-beta-lactamase gene bla VIM. These 41 clinical isolates, plus 18 bla VIM positive environmental P. putida isolates, and 17 bla VIM positive P. aeruginosa isolates, were characterized by whole genome sequencing (WGS). We constructed a maximum-likelihood tree to separate the 59 bla VIM positive P. putida group strains into eight distinct phylogenetic clusters. Bla VIM-1 was present in 6 clusters while bla VIM-2 was detected in 4 clusters. Five P. putida group strains contained both, bla VIM-1 and bla VIM-2 genes. In contrast, all P. aeruginosa strains belonged to a single genetic cluster and contained the same ARGs. Apart from bla VIM-2 and sul genes, no other ARGs were shared between P. aeruginosa and P. putida. Furthermore, the bla VIM-2 gene in P. aeruginosa was predicted to be only chromosomally located. CONCLUSION: These data provide evidence that no exchange of comprehensive ARG harbouring mobile genetic elements had occurred between P. aeruginosa and P. putida group strains during the study period, thus eliminating the need to implement enhanced infection control measures for high-risk patients colonized with a bla VIM positiv P. putida group strains in our clinical setting.

Rapid and Consistent Evolution of Colistin Resistance in Extensively Drug-Resistant Pseudomonas aeruginosa during Morbidostat Culture.

Colistin is a last-resort antibiotic commonly used against multidrug-resistant strains of Pseudomonas aeruginosa To investigate the potential for in situ evolution of resistance against colistin and to map the molecular targets of colistin resistance, we exposed two P. aeruginosa isolates to colistin using a continuous-culture device known as a morbidostat. As a result, colistin resistance reproducibly increased 10-fold within 10 days and 100-fold within 20 days, along with highly stereotypic yet strain-specific mutation patterns. The majority of mutations hit the pmrAB two-component signaling system and genes involved in lipopolysaccharide (LPS) synthesis, including lpxC, pmrE, and migA We tracked the frequencies of all arising mutations by whole-genome deep sequencing every 3 to 4 days to obtain a detailed picture of the dynamics of resistance evolution, including competition and displacement among multiple resistant subpopulations. In 7 out of 18 cultures, we observed mutations in mutS along with a mutator phenotype that seemed to facilitate resistance evolution.

Protracted Regional Dissemination of GIM-1-Producing Serratia marcescens in Western Germany.

The metallo-beta-lactamase GIM-1 has been found in various bacterial host species nearly exclusively in western Germany. However, not much is known about the epidemiology of GIM-1-positive Serratia marcescens Here we report on a surprisingly protracted regional dissemination. In-hospital transmission was investigated by using conventional epidemiological tools to identify spatiotemporal links. Strain typing was performed using pulsed-field gel electrophoresis (PFGE) and whole-genome sequencing (WGS). Bayesian phylogeny was used to infer the time axis of the observed occurrence. Thirteen S. marcescens strains from 10 patients from 6 different German hospitals were investigated. Suspected in-hospital transmissions were confirmed by molecular typing at a higher resolution by WGS than by PFGE. A detailed sequence analysis demonstrated the spread of one predominant strain variant but also provided evidence for transfer of the blaGIM-1 gene cassette between different strains. A Bayesian phylogenetic analysis showed that the most recent common ancestor of the identified clonal cluster could be dated back to April 1993 (95% highest posterior density interval, January 1973 to March 2003) and that this strain might have already harbored the blaGIM-1 at that time and, therewith, years before the first detection of this resistance gene in clinical specimens. This study shows a long-standing clonal and plasmid-mediated expansion of GIM-1-producing S. marcescens that might have gone unnoticed in the absence of a standardized and effective molecular screening for carbapenemases. The systematic and early detection of resistance is thus highly advisable, especially for the prevention of potentially long-term dissemination that may progress beyond control.

Evaluation of the Accelerate Pheno System for Fast Identification and Antimicrobial Susceptibility Testing from Positive Blood Cultures in Bloodstream Infections Caused by Gram-Negative Pathogens.

Bloodstream infections (BSI) are an important cause of morbidity and mortality. Increasing rates of antimicrobial-resistant pathogens limit treatment options, prompting an empirical use of broad-range antibiotics. Fast and reliable diagnostic tools are needed to provide adequate therapy in a timely manner and to enable a de-escalation of treatment. The Accelerate Pheno system (Accelerate Diagnostics, USA) is a fully automated test system that performs both identification and antimicrobial susceptibility testing (AST) directly from positive blood cultures within approximately 7 h. In total, 115 episodes of BSI with Gram-negative bacteria were included in our study and compared to conventional culture-based methods. The Accelerate Pheno system correctly identified 88.7% (102 of 115) of all BSI episodes and 97.1% (102 of 105) of isolates that are covered by the system's identification panel. The Accelerate Pheno system generated an AST result for 91.3% (95 of 104) samples in which the Accelerate Pheno system identified a Gram-negative pathogen. The overall category agreement between the Accelerate Pheno system and culture-based AST was 96.4%, the rates for minor discrepancies 1.4%, major discrepancies 2.3%, and very major discrepancies 1.0%. Of note, ceftriaxone, piperacillin-tazobactam, and carbapenem resistance was correctly detected in blood culture specimens with extended-spectrum beta-lactamase-producing Escherichia coli (n = 7) and multidrug-resistant Pseudomonas aeruginosa (n = 3) strains. The utilization of the Accelerate Pheno system reduced the time to result for identification by 27.49 h (P < 0.0001) and for AST by 40.39 h (P < 0.0001) compared to culture-based methods in our laboratory setting. In conclusion, the Accelerate Pheno system provided fast, reliable results while significantly improving turnaround time in blood culture diagnostics of Gram-negative BSI.

Prevotella colorans sp. nov., isolated from a human wound.

A strain of obligately anaerobic, Gram-stain-negative and non-spore-forming rod-shaped bacterium was isolated from a human wound and characterized both phenotypically and genotypically. The strain was moderately saccharolytic and proteolytic. Phylogenetic analysis was based on full-length 16S rRNA gene sequence analysis and revealed the strain to represent a member of the genus Prevotella, but to be different from the described species, with the closest relationship to Prevotella bergensis and Prevotella multisaccharivorax. The genomic DNA G+C content was 43.2 mol%. The most abundant cellular long-chain fatty acids were 3-OH iso-C17 : 0, anteiso-C15 : 0 and iso-C15 : 0. In view of phenotypical and biochemical characteristics as well as gene sequencing, strain A1336T is considered to represent a novel species within the genus Prevotella, for which the name Prevotella colorans sp. nov. is proposed. The type strain is A1336T (=DSM 100333T =CCUG 67421T =CCOS 902T).

Antibiotic Selection Pressure Determination through Sequence-Based Metagenomics.

The human gut forms a dynamic reservoir of antibiotic resistance genes (ARGs). Treatment with antimicrobial agents has a significant impact on the intestinal resistome and leads to enhanced horizontal transfer and selection of resistance. We have monitored the development of intestinal ARGs over a 6-day course of ciprofloxacin (Cp) treatment in two healthy individuals by using sequenced-based metagenomics and different ARG quantification methods. Fixed- and random-effect models were applied to determine the change in ARG abundance per defined daily dose of Cp as an expression of the respective selection pressure. Among various shifts in the composition of the intestinal resistome, we found in one individual a strong positive selection for class D beta-lactamases which were partly located on a mobile genetic element. Furthermore, a trend to a negative selection has been observed with class A beta-lactamases (-2.66 hits per million sample reads/defined daily dose; P = 0.06). By 4 weeks after the end of treatment, the composition of ARGs returned toward their initial state but to a different degree in both subjects. We present here a novel analysis algorithm for the determination of antibiotic selection pressure which can be applied in clinical settings to compare therapeutic regimens regarding their effect on the intestinal resistome. This information is of critical importance for clinicians to choose antimicrobial agents with a low selective force on their patients' intestinal ARGs, likely resulting in a diminished spread of resistance and a reduced burden of hospital-acquired infections with multidrug-resistant pathogens.

Analysis of a long-term outbreak of XDR Pseudomonas aeruginosa: a molecular epidemiological study.

OBJECTIVES: Here we report on a long-term outbreak from 2009 to 2012 with an XDR Pseudomonas aeruginosa on two wards at a university hospital in southern Germany. METHODS: Whole-genome sequencing was performed on the outbreak isolates and a core genome was constructed for molecular epidemiological analysis. We applied a time-place-sequence algorithm to improve estimation of transmission probabilities. RESULTS: By using conventional infection control methods we identified 49 P. aeruginosa strains, including eight environmental isolates that belonged to ST308 (by MLST) and carried the metallo-ß-lactamase IMP-8. Phylogenetic analysis on the basis of a non-recombinant core genome that contained 22 outbreak-specific SNPs revealed a pattern of four dominant clades with a strong phylogeographic structure and allowed us to determine the potential temporal origin of the outbreak to July 2008, 1 year before the index case was diagnosed. Superspreaders at the root of clades exhibited a high number of probable and predicted transmissions, indicating their exceptional position in the outbreak. CONCLUSIONS: Our results suggest that the initial expansion of dominant sublineages was driven by a few superspreaders, while environmental contamination seemed to sustain the outbreak for a long period despite regular environmental control measures.

Clinical and treatment-related risk factors for nosocomial colonisation with extensively drug-resistant Pseudomonas aeruginosa in a haematological patient population: a matched case control study.

BACKGROUND: This study aimed to investigate risk factors for colonisation with extensively drug-resistant P. aeruginosa (XDR-PA) in immunocompromised patients and to build a clinical risk score (CRS) based on these results. METHODS: We conducted a matched case-control study with 31 cases and 93 controls (1:3). Cases were colonised with XDR-PA during hospitalisation. Independent risk factors were determined using a three step conditional logistic regression procedure. A CRS was built with respect to the corresponding risk fraction of each risk factor, and its discriminatory power was estimated by receiver operating characteristic (ROC) analysis. RESULTS: The presence of a central venous catheter (OR 7.41, P = 0.0008), the presence of a urinary catheter (OR 21.04, P < 0.0001), CRP > 10 mg/dl (OR 7.36, P = 0.0015), and ciprofloxacin administration (OR 5.53, P = 0.025) were independent risk factors. The CRS exhibited a high discriminatory power, defining a high risk population with an approximately fourteen times greater risk for XDR-PA colonisation. CONCLUSIONS: Unnecessary use of antibiotics, particularly ciprofloxacin should be avoided, and a high standard of infection control measures must be achieved when using medical devices. A CRS can be used for adaptation of the active screening culture policy to the local setting.

Time series analysis as a tool to predict the impact of antimicrobial restriction in antibiotic stewardship programs using the example of multidrug-resistant Pseudomonas aeruginosa.

The association between antimicrobial consumption and resistance in nonfermentative Gram-negative bacteria is well-known. Antimicrobial restriction, implemented in clinical routines by antibiotic stewardship programs (ASPs), is considered a means to reduce resistance rates. Whether and how antimicrobial restriction can accomplish this goal is still unknown though. This leads to an element of uncertainty when designing strategies for ASPs. From January 2002 until December 2011, an observational study was performed at the University Hospital Tübingen, Tübingen, Germany, to investigate the association between antimicrobial use and resistance rates in Pseudomonas aeruginosa. Transfer function models were used to determine such associations and to simulate antimicrobial restriction strategies. Various positive associations between antimicrobial consumption and resistance were observed in our setting. Surprisingly, impact estimations of different antimicrobial restriction strategies revealed relatively low intervention expenses to effectively attenuate the observed increase in resistance. For example, a simulated intervention of an annual 4% reduction in the use of meropenem over 3 years from 2009 until 2011 yielded a 62.5% attenuation (95% confidence interval, 15% to 110%) in the rising trend of multidrug-resistant Pseudomonas aeruginosa (three- and four-class-resistant P. aeruginosa [34MRGN-PA]). Time series analysis models derived from past data may be a tool to predict the outcome of antimicrobial restriction strategies, and could be used to design ASPs.

Effect of metallo-ß-lactamase production and multidrug resistance on clinical outcomes in patients with Pseudomonas aeruginosa bloodstream infection: a retrospective cohort study.

BACKGROUND: Blood stream infections (BSI) with Pseudomonas aeruginosa lead to poor clinical outcomes. The worldwide emergence and spread of metallo-ß-lactamase (MBL) producing, often multidrug-resistant organisms may further aggravate this problem. Our study aimed to investigate the effect of MBL-producing P. aeruginosa (MBL-PA) and various other resistance phenotypes on clinical outcomes. METHODS: A retrospective cohort study was conducted in three German hospitals. Medical files from 2006 until 2012 were studied, and a number of 113 patients with P. aeruginosa BSI were included. The presence of VIM, IMP and NDM genes was detected using molecular techniques. Genetic relatedness was assessed through multilocus sequence typing (MLST). The effect of resistance patterns or MBL production on clinical outcomes was investigated by using multivariate Cox regression models. RESULTS: In-hospital mortality was significantly higher in patients with MBL-PA and multidrug-resistant P. aeruginosa. However, neither BSI with MBL-PA nor BSI with various resistance phenotypes of P. aeruginosa were independently associated with mortality or length of hospital stay. In multivariate models, the SAPS II score (HR 1.046), appropriate definitive treatment (HR range 0.25-0.26), and cardiovascular disease (HR range 0.44-0.46) were independent predictors of mortality. Concomitant infections were associated with an excess length of stay (HR < 1). CONCLUSIONS: Medication with appropriate antimicrobial agents at any time during the course of infection remains the key for improving clinical outcomes in patients with P. aeruginosa BSI and should be combined with a strict implementation of routine infection control measures.

Laboratory markers of disease severity in Plasmodium knowlesi infection: a case control study.

BACKGROUND: Plasmodium knowlesi malaria causes severe disease in up to 10% of cases in Malaysian Borneo and has a mortality rate of 1 - 2%. However, laboratory markers with the ability to identify patients at risk of developing complications have not yet been assessed as they have for other species of Plasmodium. METHODS: A case control study was undertaken in two hospitals in Sarikei and Sibu, Malaysian Borneo. One hundred and ten patients with uncomplicated (n = 93) and severe (n = 17) P. knowlesi malaria were studied. Standardized pigment-containing neutrophil (PCN) count, parasite density and platelet counts were determined and analysed by logistic regression and receiver operating characteristic (ROC) analysis. RESULTS: The PCN count was strongly associated with risk of disease severity. Patients with high parasite density (≥ 35,000/µl) or with thrombocytopaenia (≤ 45,000/µl) were also more likely to develop complications (odds ratio (OR) = 9.93 and OR = 5.27, respectively). The PCN count yielded the highest area under the ROC curve (AUC) estimate among all markers of severity (AUC = 0.8561, 95% confidence interval: 0.7328, 0.9794). However, the difference between all parameter AUC estimates was not statistically significant (Wald test, p = 0.73). CONCLUSION: Counting PCN is labour-intensive and not superior in predicting severity over parasitaemia and platelet counts. Parasite and platelet counts are simpler tests with an acceptable degree of precision. Any adult patient diagnosed with P. knowlesi malaria and having a parasite count ≥ 35,000/µl or ≥ 1% or a platelet count ≤ 45,000/µl can be regarded at risk of developing complications and should be managed according to current WHO guidelines for the treatment of severe malaria.