Carbapenem MICs in Escherichia coli and Klebsiella Species Producing Extended-Spectrum ß-Lactamases in Critical Care Patients from 2001 to 2009.

Extended-spectrum ß-lactamase (ESBL)-producing Enterobacteriaceae strains are increasing in prevalence worldwide. Carbapenem antibiotics are used as a first line of therapy against ESBL-producing Enterobacteriaceae We examined a cohort of critical care patients for gastrointestinal colonization with carbapenem-resistant ESBL-producing strains (CR-ESBL strains). We cultured samples from this cohort of patients for ESBL-producing Klebsiella spp. and Escherichia coli and then tested the first isolate from each patient for susceptibility to imipenem, doripenem, meropenem, and ertapenem. Multilocus sequence typing was performed on isolates that produced an ESBL and that were carbapenem resistant. Among all patients admitted to an intensive care unit (ICU), 4% were positive for an ESBL-producing isolate and 0.64% were positive for a CR-ESBL strain on surveillance culture. Among the first ESBL-producing E. coli and Klebsiella isolates from the patients' surveillance cultures, 11.2% were carbapenem resistant. Sequence type 14 (ST14), ST15, ST42, and ST258 were the dominant sequence types detected in this cohort of patients, with ST15 and ST258 steadily increasing in prevalence from 2006 to 2009. Patients colonized by a CR-ESBL strain were significantly more likely to receive antipseudomonal and anti-methicillin-resistant Staphylococcus aureus (anti-MRSA) therapy prior to ICU admission than patients colonized by carbapenem-susceptible ESBL-producing strains. They were also significantly more likely to have received a cephalosporin or a carbapenem antibiotic than patients colonized by carbapenem-susceptible ESBL-producing strains. In conclusion, in a cohort of patients residing in intensive care units within the United States, we found that 10% of the isolates were resistant to at least one carbapenem antibiotic. The continued emergence of carbapenem-resistant ESBL-producing strains is of significant concern, as infections due to these organisms are notoriously difficult to treat.

Rapid testing using the Verigene Gram-negative blood culture nucleic acid test in combination with antimicrobial stewardship intervention against Gram-negative bacteremia.

Rapid identification of microorganisms and antimicrobial resistance is paramount for targeted treatment in serious bloodstream infections (BSI). The Verigene Gram-negative blood culture nucleic acid test (BC-GN) is a multiplex, automated molecular diagnostic test for identification of eight Gram-negative (GN) organisms and resistance markers from blood culture with a turnaround time of approximately 2 h. Clinical isolates from adult patients at the University Maryland Medical Center with GN bacteremia from 1 January 2012 to 30 June 2012 were included in this study. Blood culture bottles were spiked with clinical isolates, allowed to incubate, and processed by BC-GN. A diagnostic evaluation was performed. In addition, a theoretical evaluation of time to effective and optimal antibiotic was performed, comparing actual antibiotic administration times from chart review ("control") to theoretical administration times based on BC-GN reporting and antimicrobial stewardship team (AST) review ("intervention"). For organisms detected by the assay, BC-GN correctly identified 95.6% (131/137), with a sensitivity of 97.1% (95% confidence interval [CI], 90.7 to 98.4%) and a specificity of 99.5% (95% CI, 98.8 to 99.8%). CTX-M and OXA resistance determinants were both detected. Allowing 12 h from Gram stain for antibiotic implementation, the intervention group had a significantly shorter duration to both effective (3.3 versus 7.0 h; P < 0.01) and optimal (23.5 versus 41.8 h; P < 0.01) antibiotic therapy. BC-GN with AST intervention can potentially decrease time to both effective and optimal antibiotic therapy in GN BSI.

Characterization of Klebsiella sp. strain 10982, a colonizer of humans that contains novel antibiotic resistance alleles and exhibits genetic similarities to plant and clinical Klebsiella isolates.

A unique Klebsiella species strain, 10982, was cultured from a perianal swab specimen obtained from a patient in the University of Maryland Medical Center intensive care unit. Klebsiella sp. 10982 possesses a large IncA/C multidrug resistance plasmid encoding a novel FOX AmpC ß-lactamase designated FOX-10. A novel variant of the LEN ß-lactamase was also identified. Genome sequencing and bioinformatic analysis demonstrated that this isolate contains genes associated with nitrogen fixation, allantoin metabolism, and citrate fermentation. These three gene regions are typically present in either Klebsiella pneumoniae clinical isolates or Klebsiella nitrogen-fixing endophytes but usually not in the same organism. Phylogenomic analysis of Klebsiella sp. 10982 and sequenced Klebsiella genomes demonstrated that Klebsiella sp. 10982 is present on a branch that is located intermediate between the genomes of nitrogen-fixing endophytes and K. pneumoniae clinical isolates. Metabolic features identified in the genome of Klebsiella sp. 10982 distinguish this isolate from other Klebsiella clinical isolates. These features include the nitrogen fixation (nif) gene cluster, which is typically present in endophytic Klebsiella isolates and is absent from Klebsiella clinical isolates. Additionally, the Klebsiella sp. 10982 genome contains genes associated with allantoin metabolism, which have been detected primarily in K. pneumoniae isolates from liver abscesses. Comparative genomic analysis of Klebsiella sp. 10982 demonstrated that this organism has acquired genes conferring new metabolic strategies and novel antibiotic resistance alleles, both of which may enhance its ability to colonize the human body.

Comparative genomics of an IncA/C multidrug resistance plasmid from Escherichia coli and Klebsiella isolates from intensive care unit patients and the utility of whole-genome sequencing in health care settings.

The IncA/C plasmids have been implicated for their role in the dissemination of ß-lactamases, including gene variants that confer resistance to expanded-spectrum cephalosporins, which are often the treatment of last resort against multidrug-resistant, hospital-associated pathogens. A bla(FOX-5) gene was detected in 14 Escherichia coli and 16 Klebsiella isolates that were cultured from perianal swabs of patients admitted to an intensive care unit (ICU) of the University of Maryland Medical Center (UMMC) in Baltimore, MD, over a span of 3 years. Four of the FOX-encoding isolates were obtained from subsequent samples of patients that were initially negative for an AmpC ß-lactamase upon admission to the ICU, suggesting that the AmpC ß-lactamase-encoding plasmid was acquired while the patient was in the ICU. The genomes of five E. coli isolates and six Klebsiella isolates containing bla(FOX-5) were selected for sequencing based on their plasmid profiles. An ∼ 167-kb IncA/C plasmid encoding the FOX-5 ß-lactamase, a CARB-2 ß-lactamase, additional antimicrobial resistance genes, and heavy metal resistance genes was identified. Another FOX-5-encoding IncA/C plasmid that was nearly identical except for a variable region associated with the resistance genes was also identified. To our knowledge, these plasmids represent the first FOX-5-encoding plasmids sequenced. We used comparative genomics to describe the genetic diversity of a plasmid encoding a FOX-5 ß-lactamase relative to the whole-genome diversity of 11 E. coli and Klebsiella isolates that carry this plasmid. Our findings demonstrate the utility of whole-genome sequencing for tracking of plasmid and antibiotic resistance gene distribution in health care settings.

End-to-end model-based trajectory prediction for ro-ro ship route using dual-attention mechanism.

With the rapid increase of economic globalization, the significant expansion of shipping volume has resulted in shipping route congestion, causing the necessity of trajectory prediction for effective service and efficient management. While trajectory prediction can achieve a relatively high level of accuracy, the performance and generalization of prediction models remain critical bottlenecks. Therefore, this article proposes a dual-attention (DA) based end-to-end (E2E) neural network (DAE2ENet) for trajectory prediction. In the E2E structure, long short-term memory (LSTM) units are included for the task of pursuing sequential trajectory data from the encoder layer to the decoder layer. In DA mechanisms, global attention is introduced between the encoder and decoder layers to facilitate interactions between input and output trajectory sequences, and multi-head self-attention is utilized to extract sequential features from the input trajectory. In experiments, we use a ro-ro ship with a fixed navigation route as a case study. Compared with baseline models and benchmark neural networks, DAE2ENet can obtain higher performance on trajectory prediction, and better validation of environmental factors on ship navigation.

In situ stimulus-responsive self-assembled nanomaterials for drug delivery and disease treatment.

The individual motifs that respond to specific stimuli for the self-assembly of nanomaterials play important roles. In situ constructed nanomaterials are formed spontaneously without human intervention and have promising applications in bioscience. However, due to the complex physiological environment of the human body, designing stimulus-responsive self-assembled nanomaterials in vivo is a challenging problem for researchers. In this article, we discuss the self-assembly principles of various nanomaterials in response to the tissue microenvironment, cell membrane, and intracellular stimuli. We propose the applications and advantages of in situ self-assembly in drug delivery and disease diagnosis and treatment, with a focus on in situ self-assembly at the lesion site, especially in cancer. Additionally, we introduce the significance of introducing exogenous stimulation to construct self-assembly in vivo. Based on this foundation, we put forward the prospects and possible challenges in the field of in situ self-assembly. This review uncovers the relationship between the structure and properties of in situ self-assembled nanomaterials and provides new ideas for innovative drug molecular design and development to solve the problems in the targeted delivery and precision medicine.

Application of a Random Forest Algorithm in Natural Landscape Animation Design.

Natural landscape simulation is one of the most popular research contents in computer graphics in the field of research simulation system. The natural landscape animation scene can immerse viewers in the scene, and it is widely used in visual simulation systems. Simulating natural scenery on a computer is a powerful method for studying the rules of the scenery's growth process as well as the mystery of life. The simulation of natural scenery is of particular importance and has far-reaching implications. The most important aspect of optimizing natural landscape design is creating a natural landscape animation that users enjoy. This article proposes a natural landscape animation design method with a self-learning function based on this concept. The random forest model (RF) is introduced in this method and applied to the entire animation design process. RF can generate a learning model with user evaluation as the classification result to guide the automatic design of natural landscape animation, resulting in user-satisfying animations. Simultaneously, the RF-based natural landscape animation design can continuously update the learning model based on user needs and is self-learning. The experimental part of this article verifies the effectiveness of the natural landscape animation design proposed in this article by comparing the selection rate of user satisfaction and dissatisfaction scenes, and further demonstrates that the method in this article can improve the natural landscape. The market application value of user satisfaction generated by animation is high.

Effect of mupirocin for Staphylococcus aureus decolonization on the microbiome of the nose and throat in community and nursing home dwelling adults.

OBJECTIVE: To characterize the microbial communities of the anterior nares (nose) and posterior pharynx (throat) of adults dwelling in the community and in nursing homes before and after treatment with intranasal mupirocin. METHODS: Staphylococcus aureus-colonized adults were recruited from the community (n = 25) and from nursing homes (n = 7). S. aureus colonization was confirmed using cultures. Participants had specimens taken from nose and throat for S. aureus quantitation using quantitative PCR for the nuc gene and bacterial profiling using 16S rRNA gene sequencing over 12 weeks. After two baseline study visits 4 weeks apart, participants received intranasal mupirocin for 5 days with 3 further visits over a 8 week follow-up period. RESULTS: We found a decrease in the absolute abundance of S. aureus in the nose for 8 weeks after mupirocin (1693 vs 141 fg/ul, p = 0.047). Mupirocin caused a statistically significant disruption in bacterial communities of the nose and throat after 1 week, which was no longer detected after 8 weeks. Bacterial community profiling demonstrated that there was a decrease in the relative abundance of S. aureus (8% vs 0.3%, p<0.01) 8 weeks after mupirocin and a transient decrease in the relative abundance of Staphylococcus epidermidis in the nose (21% vs 5%, p<0.01) 1 week after mupirocin. CONCLUSIONS: Decolonization with mupirocin leads to a sustained effect on absolute and relative abundance of S. aureus but not for other bacteria in the nose. This demonstrates that a short course of mupirocin selectively decreases S. aureus in the nose for up to 8 weeks.

Burden of perianal Staphylococcus aureus colonization in nursing home residents increases transmission to healthcare worker gowns and gloves.

OBJECTIVE: To evaluate the effect of the burden of Staphylococcus aureus colonization of nursing home residents on the risk of S. aureus transmission to healthcare worker (HCW) gowns and gloves. DESIGN: Multicenter prospective cohort study. SETTING AND PARTICIPANTS: Residents and HCWs from 13 community-based nursing homes in Maryland and Michigan. METHODS: Residents were cultured for S. aureus at the anterior nares and perianal skin. The S. aureus burden was estimated by quantitative polymerase chain reaction detecting the nuc gene. HCWs wore gowns and gloves during usual care activities; gowns and gloves were swabbed and then cultured for the presence of S. aureus. RESULTS: In total, 403 residents were enrolled; 169 were colonized with methicillin-resistant S. aureus (MRSA) or methicillin-sensitive S. aureus (MSSA) and comprised the study population; 232 were not colonized and thus were excluded from this analysis; and 2 were withdrawn prior to being swabbed. After multivariable analysis, perianal colonization with S. aureus conferred the greatest odds for transmission to HCW gowns and gloves, and the odds increased with increasing burden of colonization: adjusted odds ratio (aOR), 2.1 (95% CI, 1.3-3.5) for low-level colonization and aOR 5.2 (95% CI, 3.1-8.7) for high level colonization. CONCLUSIONS: Among nursing home patients colonized with S. aureus, the risk of transmission to HCW gowns and gloves was greater from those colonized with greater quantities of S. aureus on the perianal skin. Our findings inform future infection control practices for both MRSA and MSSA in nursing homes.

[Effect of yiqi wenyang huoxue recipe on bone marrow stem cells' mobilization and its influence on heart function in patients with myocardial infarction].

OBJECTIVE: To observe the effect of Yiqi Wenyang Huoxue Recipe (YWHR) on bone marrow stem cells' mobilization and heart function of myocardial infarction (MI) patients. METHODS: Sixty acute MI patients were equally assigned to the treatment group and the control group randomly according to the numeration table. Standard therapy composed of subcutaneous injection of 0.4 mL low molecular heparin every 12 h for 7 successive days combined with continuously orally taken of enteric-coated aspirin tablets 0.1 g, Perindopril 4 mg and Atorvastatin 10 mg once a day (if there was no contraindication), was given to all patients. But to the treatment group, one dose of YWHR was given every day additionally, which consisted of Panax ginseng 10 g, Radix Astragali 15 g, Radix Aconiti lateralis preparata 5 g, Radix Ilex pubesceus 30 g and Herbal Leonuri 10 g for 10 successive days. The change of CD34+ cells' count was observed. The infarcted area size was estimated with QRS scoring system at the end of the 3rd week after onset, and the heart function parameters, including left ventricular ejection fraction (LVEF), left ventricular end-systolic volume index (LVESVI), left ventricular end-diastolic volume index (LVEDVI) and wall motion score index (WMSI), were measured with ultrasonic cardiogram. RESULTS: On the fifth and tenth day, the peripheral CD34+ cell count in the treatment group (48.26 +/- 5.74 and 34.18 +/- 6.70) were significantly higher than that on the day of admission (20.33 +/- 6.01), also higher than the corresponding count in the control group (22.45 +/- 5. 31 and 19.89 +/- 5.93), showing statistical difference (P < 0.01). The QRS score (2.68 +/- 0.41) and infarcted area (22.54 +/- 2.49)% in the treatment group were lower than those in the control group (3.96 +/- 0. 34 and 29.38 +/- 2.59) respectively. Comparison of heart function parameters between groups showed that LVEF (0.59 +/- 0.07 vs. 0.50 +/- 0.07) was higher LVEDVI (68.92 +/- 11.52 vs. 78.46 +/- 13.16), LVESVI (27.47 +/- 7.86 vs. 36.71 +/- 8.85), and WMSI (2.10 +/- 0.11 vs. 2.98 +/- 0.12) were lower in the treatment group than those in the control group respectively, also showing statistical difference (all P < 0.01). CONCLUSION: YWHR has a good effect on bone marrow stem cell mobilization, it can reduce the infarcted area and improve the heart function of patients with acute MI.

Comparison of molecular typing methods for the analyses of Acinetobacter baumannii from ICU patients.

Acinetobacter baumannii has emerged as an important cause of healthcare-associated infections causing great morbidity and mortality. Despite its clinical importance, it is still unknown which molecular typing method is the best to determine or confirm institutional outbreaks as well as to identify epidemiologically related isolates from different geographical areas. To determine the most discriminatory molecular typing method, we isolated A. baumannii from perianal swabs collected from intensive care unit (ICU) patients in a cohort study during 2002 and 2008. Strains from each year were analyzed by pulsed-field gel electrophoresis (PFGE), multi-locus sequence typing (MLST), and multi-locus variable-number tandem repeat analysis (MLVA). Genetic relatedness of the isolates was consistent between PFGE and MLST as well as between analyses of loci containing MLVA and MLST. Our data show that PFGE and MLVA are similar when discriminating between isolates and are both good methods to use when questioning whether two isolates are indistinguishable.

Mucosal immunization with attenuated Salmonella enterica serovar Typhi expressing protective antigen of anthrax toxin (PA83) primes monkeys for accelerated serum antibody responses to parenteral PA83 vaccine.

Salmonella enterica serovar Typhi vaccine strain CVD 908-htrA was genetically engineered for stable plasmid-based expression of protective antigen of anthrax toxin (PA83) fused with the export protein ClyA (ClyA-PA83). The priming potential of CVD 908-htrA expressing ClyA-PA83 was assessed in 12 rhesus and 20 cynomolgus macaques that were immunized mucosally (i.e., intranasally) on days 0 and 14. A parenteral booster with purified PA83 plus alum was given to rhesus macaques on days 42 and 225; cynomolgus monkeys received a booster with either PA or licensed anthrax vaccine (BioThrax; Emergent Biosolutions) only one time, 3 months after priming. Monkeys primed with S. Typhi expressing ClyA-PA83 developed high levels of serum toxin-neutralization activity (TNA) antibodies (50% effective dose [ED50], >1.3x10(3)), 7 days after receipt of the booster, whereas unprimed controls lacked serum TNA (ED50, 0). In nonhuman primates, the success of this anthrax vaccine strategy based on heterologous mucosal priming followed by a parenteral subunit vaccine booster paves the way for clinical trials.

Virulence-defective strains of Salmonella enterica serovar Typhi as candidates for education at level 2 facilities.

The use of biosafety level 3 pathogens is an essential element of education and training at medical schools. We previously reported on invasion-defective strains of Salmonella enterica serovar Typhi, GTC 3P408 (DeltainvA, DeltasipB) and GTC 3P409 (DeltainvA, DeltasipB, and DeltaviaB), as candidates for use in educational programs. Vi negative strains of S. enterica serovar Typhi became extremely sensitive to complement attack but showed increased invasiveness. Therefore, this study was conducted to construct two virulencedefective strains, GTC 3P460 (DeltainvA, DeltasipB, and DeltarpoS) and GTC 3P461 (DeltainvA, DeltasipB, DeltaviaB, and DeltarpoS), of S. enterica serovar Typhi by deleting rpoS from the GTC 3P409 and GTC 3P408 strains. Stress tests demonstrated that GTC 3P460 and GTC 3P461 are sensitive to conditions of starvation, acid stress and oxidative stress. These results suggest that these virulence-defective strains have difficulty surviving in the gastric environment and in macrophages, characteristics that make them ideal candidates for education at level 2 facilities. Colony morphology and conventional biochemical features of these strains are identical to the parent strain S. enterica serovar Typhi GIFU 10007.

Adaptation of the endogenous Salmonella enterica serovar Typhi clyA-encoded hemolysin for antigen export enhances the immunogenicity of anthrax protective antigen domain 4 expressed by the attenuated live-vector vaccine strain CVD 908-htrA.

Bacterial live-vector vaccines aim to deliver foreign antigens to the immune system and induce protective immune responses, and surface-expressed or secreted antigens are generally more immunogenic than cytoplasmic constructs. We hypothesize that an optimum expression system will use an endogenous export system to avoid the need for large amounts of heterologous DNA encoding additional proteins. Here we describe the cryptic chromosomally encoded 34-kDa cytolysin A hemolysin of Salmonella enterica serovar Typhi (ClyA) as a novel export system for the expression of heterologous antigens in the supernatant of attenuated Salmonella serovar Typhi live-vector vaccine strains. We constructed a genetic fusion of ClyA to the reporter green fluorescent protein and showed that in Salmonella serovar Typhi CVD 908-htrA, the fusion protein retains biological activity in both domains and is exported into the supernatant of an exponentially growing live vector in the absence of detectable bacterial lysis. The utility of ClyA for enhancing the immunogenicity of an otherwise problematic antigen was demonstrated by engineering ClyA fused to the domain 4 (D4) moiety of Bacillus anthracis protective antigen (PA). A total of 11 of 15 mice immunized intranasally with Salmonella serovar Typhi exporting the protein fusion manifested fourfold or greater rises in serum anti-PA immunoglobulin G, compared with only 1 of 16 mice immunized with the live vector expressing cytoplasmic D4 (P = 0.0002). In addition, the induction of PA-specific gamma interferon and interleukin 5 responses was observed in splenocytes. This technology offers exceptional versatility for enhancing the immunogenicity of bacterial live-vector vaccines.

Properties of haemolysin E (HlyE) from a pathogenic Escherichia coli avian isolate and studies of HlyE export.

Haemolysin E (HlyE) is a novel pore-forming toxin first identified in Escherichia coli K-12. Analysis of the 3-D structure of HlyE led to the proposal that a unique hydrophobic beta-hairpin structure (the beta-tongue, residues 177-203) interacts with the lipid bilayer in target membranes. In seeming contradiction to this, the hlyE sequence from a pathogenic E. coli strain (JM4660) that lacks all other haemolysins has been reported to encode an Arg residue at position 188 that was difficult to reconcile with the proposed role of the beta-tongue. Here it is shown that the JM4660 hlyE sequence encodes Gly, not Arg, at position 188 and that substitution of Gly188 by Arg in E. coli K-12 HlyE abolishes activity, emphasizing the importance of the head domain in HlyE function. Nevertheless, 76 other amino acid substitutions were confirmed compared to the HlyE protein of E. coli K-12. The JM4660 HlyE protein was dimeric, suggesting a mechanism for improving toxin solubility, and it lysed red blood cells from many species by forming 36-41 A diameter pores. However, the haemolytic phenotype of JM4660 was found to be unstable due to defects in HlyE export, indicating that export of active HlyE is not an intrinsic property of the protein but requires additional components. TnphoA mutagenesis of hlyE shows that secretion from the cytoplasm to the periplasm does not require the carboxyl-terminal region of HlyE. Finally, disruption of genes associated with cell envelope function, including tatC, impairs HlyE export, indicating that outer membrane integrity is important for effective HlyE secretion.