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Concept: Enterobacteriaceae

208

Disulfides from Allium stipitatum, commonly known as Persian shallot, were previously reported to possess antibacterial properties. Analogues of these compounds, produced by S-methylthiolation of appropriate thiols using S-methyl methanethiosulfonate, exhibited antimicrobial activity, with one compound inhibiting the growth of Mycobacterium tuberculosis at 17 µM (4 mg L-1) and other compounds inhibiting Escherichia coli and multi-drug-resistant (MDR) Staphylococcus aureus at concentrations ranging between 32-138 µM (8-32 mg L-1). These compounds also displayed moderate inhibitory effects on Klebsiella and Proteus species. Whole-cell phenotypic bioassays such as the spot-culture growth inhibition assay (SPOTi), drug efflux inhibition, biofilm inhibition and cytotoxicity assays were used to evaluate these compounds. Of particular note was their ability to inhibit mycobacterial drug efflux and biofilm formation, while maintaining a high selectivity towards M. tuberculosis H37Rv. These results suggest that methyl disulfides are novel scaffolds which could lead to the development of new drugs against tuberculosis (TB).

Concepts: Microbiology, Antibiotic resistance, Tuberculosis, Mycobacterium, Mycobacterium tuberculosis, Enterobacteriaceae, Inhibitor, Disinfectant

164

Carbapenem-resistant Enterobacteriaceae (CRE) are among the most severe threats to the antibiotic era. Multiple different species can exhibit resistance due to many different mechanisms, and many different mobile elements are capable of transferring resistance between lineages. We prospectively sampled CRE from hospitalized patients from three Boston-area hospitals, together with a collection of CRE from a single California hospital, to define the frequency and characteristics of outbreaks and determine whether there is evidence for transfer of strains within and between hospitals and the frequency with which resistance is transferred between lineages or species. We found eight species exhibiting resistance, with the majority of our sample being the sequence type 258 (ST258) lineage of Klebsiella pneumoniae There was very little evidence of extensive hospital outbreaks, but a great deal of variation in resistance mechanisms and the genomic backgrounds carrying these mechanisms. Local transmission was evident in clear phylogeographic structure between the samples from the two coasts. The most common resistance mechanisms were KPC (K. pneumoniae carbapenemases) beta-lactamases encoded by blaKPC2, blaKPC3, and blaKPC4, which were transferred between strains and species by seven distinct subgroups of the Tn4401 element. We also found evidence for previously unrecognized resistance mechanisms that produced resistance when transformed into a susceptible genomic background. The extensive variation, together with evidence of transmission beyond limited clonal outbreaks, points to multiple unsampled transmission chains throughout the continuum of care, including asymptomatic carriage and transmission of CRE. This finding suggests that to control this threat, we need an aggressive approach to surveillance and isolation.

Concepts: Sample, Hospital, Antibiotic resistance, Enterobacteriaceae, Klebsiella pneumoniae, Klebsiella, Klebsiella oxytoca

160

To date there are no clear criteria to determine whether a microbe is susceptible to biocides or not. As a starting point for distinguishing between wild-type and resistant organisms, we set out to determine the minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) distributions for four common biocides; triclosan, benzalkonium chloride, chlorhexidine and sodium hypochlorite for 3319 clinical isolates, with a particular focus on Staphylococcus aureus (N = 1635) and Salmonella spp. (N = 901) but also including Escherichia coli (N = 368), Candida albicans (N = 200), Klebsiella pneumoniae (N = 60), Enterobacter spp. (N = 54), Enterococcus faecium (N = 53), and Enterococcus faecalis (N = 56). From these data epidemiological cut-off values (ECOFFs) are proposed. As would be expected, MBCs were higher than MICs for all biocides. In most cases both values followed a normal distribution. Bimodal distributions, indicating the existence of biocide resistant subpopulations were observed for Enterobacter chlorhexidine susceptibility (both MICs and MBCs) and the susceptibility to triclosan of Enterobacter (MBC), E. coli (MBC and MIC) and S. aureus (MBC and MIC). There is a concern on the potential selection of antibiotic resistance by biocides. Our results indicate however that resistance to biocides and, hence any potential association with antibiotic resistance, is uncommon in natural populations of clinically relevant microorganisms.

Concepts: Bacteria, Pneumonia, Staphylococcus aureus, Antibiotic resistance, Escherichia coli, Linezolid, Enterobacteriaceae, Enterococcus

151

Presumptive identification of different Enterobacteriaceae species is routinely achieved based on the biochemical properties. Traditional practice includes manual comparison of each biochemical property of the unknown sample with known reference samples and inference of its identity based on the maximum similarity pattern with the known samples. This process is labor-intensive, time-consuming, error-prone, and subjective. Therefore, automation of sorting and similarity calculation would be advantageous. Here we present a MATLAB-based graphical user interface (GUI) tool named BioCluster. This tool was designed for automated clustering and identification of Enterobacteriaceae based on biochemical test results. In this tool, we used two types of algorithms, i.e., traditional hierarchical clustering (HC) and the Improved Hierarchical Clustering (IHC), a modified algorithm that was developed specifically for the clustering and identification of Enterobacterioceae species. IHC takes into account the variability in result of 1-47 biochemical tests within this Enterobacterioceae family. This tool also provides different options to optimize the clustering in a user-friendly way. Using computer-generated synthetic data and some real data, we have demonstrated that BioCluster has high accuracy in clustering and identifying enterobacterial species based on biochemical test data. This tool can be freely downloaded at http://microbialgen.du.ac.bd/biocluster/.

Concepts: Algorithm, Enterobacteriaceae, Computer, Computer program, Automation, Identification, User interface, Graphical user interface

148

Pyrosequencing analysis of intestinal microflora from healthy Thai vegetarians and non-vegetarians exhibited 893 OTUs covering 189 species. The strong species indicator of vegetarians and non-vegetarians were Prevotella copri and Bacteroides vulgatus as well as bacterium closed to Escherichia hermanii with % relative abundance of 16.9 and 4.5-4.7, respectively. Core gut microbiota of vegetarian and non-vegetarian group consisted of 11 and 20 different bacterial species, respectively, belonging to Actinobacteria, Firmicutes and Proteobacteria commonly found in both groups. Two species of Faecalibacterium prausnitzii and Gemmiger formicilis had prevalence of 100% in both groups. Three species of Clostridium nexile, Eubacterium eligens and P. copri showed up in most vegetarians while more diversity of Collinsella aerofaciens, Ruminococcus torques, various species of Bacteroides, Parabacteroides, Escherichia, different species of Clostridium and Eubacterium were found in most non-vegetarians. Considering the correlation of personal characters, consumption behavior and microbial groups, the age of non-vegetarians showed strong positive correlation coefficient of 0.54 (p=0.001) to Bacteroides uniformis while exhibited a moderate ones to Alistipes finegoldii and B. vulgatus. Only positive moderate correlation of body mass index (BMI) and Parabacteroides distasonis appeared. Based on significant abundance of potential pathogens, the microbiota of non-vegetarian group showed the abundance of potential pathogen varieties of Bilophila wadsworthia, Escherichia coli and E. hermannii while the one of vegetarian served for only Klebsiella pneumonia. These results implied that the microbiota of vegetarian with high abundance of P. copri and low potential pathogen variety would be a way to maintain healthy in Thai.

Concepts: Bacteria, Gut flora, Microbiology, Pneumonia, Escherichia coli, Enterobacteriaceae, Firmicutes, Human flora

147

Crohn’s disease (CD) results from a complex interplay between host genetic factors and endogenous microbial communities. In the current study, we used Ion Torrent sequencing to characterize the gut bacterial microbiota (bacteriome) and fungal community (mycobiome) in patients with CD and their nondiseased first-degree relatives (NCDR) in 9 familial clusters living in northern France-Belgium and in healthy individuals from 4 families living in the same area (non-CD unrelated [NCDU]). Principal component, diversity, and abundance analyses were conducted, and CD-associated inter- and intrakingdom microbial correlations were determined. Significant microbial interactions were identified and validated using single- and mixed-species biofilms. CD and NCDR groups clustered together in the mycobiome but not in the bacteriome. Microbiotas of familial (CD and NCDR) samples were distinct from those of nonfamilial (NCDU) samples. The abundance of Serratia marcescens and Escherichia coli was elevated in CD patients, while that of beneficial bacteria was decreased. The abundance of the fungus Candida tropicalis was significantly higher in CD than in NCDR (P = 0.003) samples and positively correlated with levels of anti-Saccharomyces cerevisiae antibodies (ASCA). The abundance of C. tropicalis was positively correlated with S. marcescens and E. coli, suggesting that these organisms interact in the gut. The mass and thickness of triple-species (C. tropicalis plus S. marcescens plus E. coli) biofilm were significantly greater than those of single- and double-species biofilms. C. tropicalis biofilms comprised blastospores, while double- and triple-species biofilms were enriched in hyphae. S. marcescens used fimbriae to coaggregate or attach with C. tropicalis/E. coli, while E. coli was closely apposed with C. tropicalis Specific interkingdom microbial interactions may be key determinants in CD.

Concepts: Genetics, Bacteria, Gut flora, Microbiology, Fungus, Yeast, Escherichia coli, Enterobacteriaceae

147

Colonization and infection by multidrug-resistant gram-negative bacilli (MDR GNB) in neonatal intensive care units (NICUs) are increasingly reported.We conducted a 5-year prospective cohort surveillance study in a tertiary NICU of the hospital “Paolo Giaccone,” Palermo, Italy. Our objectives were to describe incidence and trends of MDR GNB colonization and the characteristics of the most prevalent organisms and to identify the risk factors for colonization. Demographic, clinical, and microbiological data were prospectively collected. Active surveillance cultures (ASCs) were obtained weekly. Clusters of colonization by extended spectrum β-lactamase (ESBL) producing Escherichia coli and Klebsiella pneumoniae were analyzed by conventional and molecular epidemiological tools.During the study period, 1152 infants were enrolled in the study. Prevalences of colonization by MDR GNB, ESBL-producing GNB and multiple species/genera averaged, respectively, 28.8%, 11.7%, and 3.7%. Prevalence and incidence density of colonization by MDR GNB and ESBL-producing GNB showed an upward trend through the surveillance period. Rates of ESBL-producing E coli and K pneumoniae colonization showed wide fluctuations peaking over the last 2 years. The only independent variables associated with colonization by MDR GNB and ESBL-producing organisms and multiple colonization were, respectively, the days of NICU stay (odds ratio [OR] 1.041), the days of exposure to ampicillin-sulbactam (OR 1.040), and the days of formula feeding (OR 1.031). Most clusters of E coli and K pneumoniae colonization were associated with different lineages. Ten out of 12 clusters had an outborn infant as their index case.Our study confirms that MDR GNB are an increasing challenge to NICUs. The universal once-a-week approach allowed us to understand the epidemiology of MDR GNB, to timely detect new clones and institute contact precautions, and to assess risk factors. Collection of these data can be an important tool to optimize antimicrobials use and control the emergence and dissemination of resistances in NICU.

Concepts: Epidemiology, Medical statistics, Intensive care medicine, Escherichia coli, Enterobacteriaceae, Prevalence, Gram negative bacteria, Gram-negative bacteria

144

We aimed to investigate the prevalence of extended-spectrum β-lactamases (ESBL)-producing Escherichia coli (E. coli) in Beijing Tongren hospital and find the relationship between colonization and infection. The clinical data of 650 inpatients included between March 2012 and July 2012 were retrospectively reviewed. The prevalence of ESBL-producing E. coli among inpatients was 25.7% (167/650), with the highest of 50.0% in rheumatology ward and lowest of 10.0% in intensive care unit. Hospital stay more than 2 years, usage of antibiotics less than 3 months, and use of glucocorticoids or immunosuppressive were found to be significantly associated with ESBL carriage (P < 0.05). Totally, 76 sequence types (ST) were revealed by MLST. ST38 (n = 12, 7.2%) was the most common type, followed by ST10 (n = 10, 6.0%), ST131 and ST167 (n = 9, 5.4%). Among the faecal carriers, only one patient suffered infection, which was resulted by a ST38 strain. In conclusion, in Beijing Tongren hospital, the prevalence of ESBL-producing E. coli was not high. The risk factors for ESBL carriage were hospitalization and usage of antibiotics, glucocorticoids and immunosuppressive. ST38, ST10, ST131 and ST167 were the prominent genotypes, but almost 50.0% ST were dispersedly distributed.

Concepts: Bacteria, Hospital, Antibiotic resistance, Escherichia coli, Enterobacteriaceae, Escherichia, Proteobacteria, Beta-lactamase

82

The rise of multidrug-resistant (MDR) bacteria is a growing concern to global health and is exacerbated by the lack of new antibiotics. To treat already pervasive MDR infections, new classes of antibiotics or antibiotic adjuvants are needed. Reactive oxygen species (ROS) have been shown to play a role during antibacterial action; however, it is not yet understood whether ROS contribute directly to or are an outcome of bacterial lethality caused by antibiotics. We show that a light-activated nanoparticle, designed to produce tunable flux of specific ROS, superoxide, potentiates the activity of antibiotics in clinical MDR isolates of Escherichia coli, Salmonella enterica, and Klebsiella pneumoniae. Despite the high degree of antibiotic resistance in these isolates, we observed a synergistic interaction between both bactericidal and bacteriostatic antibiotics with varied mechanisms of action and our superoxide-producing nanoparticles in more than 75% of combinations. As a result of this potentiation, the effective antibiotic concentration of the clinical isolates was reduced up to 1000-fold below their respective sensitive/resistant breakpoint. Further, superoxide-generating nanoparticles in combination with ciprofloxacin reduced bacterial load in epithelial cells infected with S. enterica serovar Typhimurium and increased Caenorhabditis elegans survival upon infection with S. enterica serovar Enteriditis, compared to antibiotic alone. This demonstration highlights the ability to engineer superoxide generation to potentiate antibiotic activity and combat highly drug-resistant bacterial pathogens.

Concepts: Bacteria, Microbiology, Infection, Antibiotic resistance, Escherichia coli, Enterobacteriaceae, Antibiotic, Salmonella enterica

55

The volumetric heating values of today’s biofuels are too low to power energy-intensive aircraft, rockets, and missiles. Recently, pinene dimers were shown to have a volumetric heating value similar to that of the tactical fuel JP-10. To provide a sustainable source of pinene, we engineered Escherichia coli for pinene production. We combinatorially expressed three pinene synthases (PS) and three geranyl diphosphate synthases (GPPS), with the best combination achieving ∼28 mg/L of pinene. We speculated that pinene toxicity was limiting production; however, toxicity should not be limiting at current titers. Because GPPS is inhibited by geranyl diphosphate (GPP) and to increase flux through the pathway, we combinatorially constructed GPPS-PS protein fusions. The Abies grandis GPPS-PS fusion produced 32 mg/L of pinene, a 6-fold improvement over the highest titer previously reported in engineered E. coli. Finally, we investigated the pinene isomer ratio of our pinene-producing microbe and discovered that the isomer profile is determined not only by the identity of the PS used but also by the identity of the GPPS with which the PS is paired. We demonstrated that the GPP concentration available to PS for cyclization alters the pinene isomer ratio.

Concepts: Protein, Bacteria, Antibiotic resistance, Escherichia coli, Biotechnology, Enterobacteriaceae, Escherichia, Proteobacteria