Genomic characterization of an emerging Enterobacteriaceae species: the first case of co-infection with a typical pathogen in a human patient.

BACKGROUND: Opportunistic pathogens are important for clinical practice as they often cause antibiotic-resistant infections. However, little is documented for many emerging opportunistic pathogens and their biological characteristics. Here, we isolated a strain of extended-spectrum ß-lactamase-producing Enterobacteriaceae from a patient with a biliary tract infection. We explored the biological and genomic characteristics of this strain to provide new evidence and detailed information for opportunistic pathogens about the co-infection they may cause. RESULTS: The isolate grew very slowly but conferred strong protection for the co-infected cephalosporin-sensitive Klebsiella pneumoniae. As the initial laboratory testing failed to identify the taxonomy of the strain, great perplexity was caused in the etiological diagnosis and anti-infection treatment for the patient. Rigorous sequencing efforts achieved the complete genome sequence of the isolate which we designated as AF18. AF18 is phylogenetically close to a few strains isolated from soil, clinical sewage, and patients, forming a novel species together, while the taxonomic nomenclature of which is still under discussion. And this is the first report of human infection of this novel species. Like its relatives, AF18 harbors many genes related to cell mobility, various genes adaptive to both the natural environment and animal host, over 30 mobile genetic elements, and a plasmid bearing blaCTX-M-3 gene, indicating its ability to disseminate antimicrobial-resistant genes from the natural environment to patients. Transcriptome sequencing identified two sRNAs that critically regulate the growth rate of AF18, which could serve as targets for novel antimicrobial strategies. CONCLUSIONS: Our findings imply that AF18 and its species are not only infection-relevant but also potential disseminators of antibiotic resistance genes, which highlights the need for continuous monitoring for this novel species and efforts to develop treatment strategies.

Production of molybdenum blue by two novel molybdate-reducing bacteria belonging to the genus Raoultella isolated from Egypt and Iraq.

AIM: The aim of this study was to isolate potential molybdate-reducing bacteria to be used for bioremediation. METHODS AND RESULTS: Two molybdate-reducing bacteria (Mo1 and MoI) were isolated from polluted soil samples from Ismailia Canal, Egypt and Sallah Elddin Governorate, Iraq respectively. The isolates exhibited dark blue colonies when grown on solid medium containing sodium molybdate which indicated the reduction of molybdate to molybdenum (Mo) blue. The absorbance values at 865 nm were 0·743 ± 0·007 and 0·453 ± 0·005 for Mo1 and MoI respectively. The Mo blue produced showed characteristic absorption spectrum peak at 865 nm and a shoulder at 700 nm. The isolates were identified by 16S rRNA genes sequencing and were submitted to GenBank as Raoultella ornithinolytica strain Mo1 and Raoultella planticola strain MoI. The optimum conditions were glucose as electron donor, initial pH of 6 and incubation temperature of 30°C. Scanning electron micrographs were taken for both isolates in the presence and absence of molybdate source. CONCLUSION: To the best of our knowledge, this is the first recordation of molybdate reduction by Raoultella sp. isolated from Egypt and Iraq. SIGNIFICANCE AND IMPACT OF THE STUDY: The isolated bacteria belonging to the Raoultella could be used in in situ bioremediation.

Green Synthesis of Silver Nanoparticles with Culture Supernatant of a Bacterium Pseudomonas rhodesiae and Their Antibacterial Activity against Soft Rot Pathogen Dickeya dadantii.

Bacterial stem and root rot disease of sweet potato caused by Dickeya dadantii recently broke out in major sweet potato planting areas in China and calls for effective approaches to control the pathogen and disease. Here, we developed a simple method for green synthesis of silver nanoparticles (AgNPs) using bacterial culture supernatants. AgNPs synthesized with the cell-free culture supernatant of a bacterium Pseudomonas rhodesiae displayed the characteristic surface plasmon resonance peak at 420-430 nm and as nanocrystallites in diameters of 20-100 nm determined by transmission electron microscopy, scanning electron microscopy, and X-ray diffraction spectroscopy. Functional groups associated with proteins in the culture supernatant may reduce silver ions and stabilize AgNPs. The AgNPs showed antibacterial activities against D. dadantii growth, swimming motility, biofilm formation, and maceration of sweet potato tubers whereas the culture supernatant of P. rhodesiae did not. AgNPs (12 µg∙ml-1) and AgNO3 (50 µg∙ml-1) showed close antibacterial activities. The antibacterial activities increased with the increase of AgNP concentrations. The green-synthesized AgNPs can be used to control the soft rot disease by control of pathogen contamination of sweet potato seed tubers.

A gel-free quantitative proteomics approach to investigate temperature adaptation of the food-borne pathogen Cronobacter turicensis 3032.

The opportunistic food-borne pathogen Cronobacter sp. causes rare but significant illness in neonates and is capable to grow at a remarkably wide range of temperatures from 5.5 to 47 degrees C. A gel-free quantitative proteomics approach was employed to investigate the molecular basis of the Cronobacter sp. adaptation to heat and cold-stress. To this end the model strain Cronobacter turicensis 3032 was grown at 25, 37, 44, and 47 degrees C, and whole-cell and secreted proteins were iTRAQ-labelled and identified/quantified by 2-D-LC-MALDI-TOF/TOF-MS. While 44 degrees C caused only minor changes in C. turicensis growth rate and protein profile, 47 degrees C affected the expression of about 20% of all 891 identified proteins and resulted in a reduced growth rate and rendered the strain non-motile and filamentous. Among the heat-induced proteins were heat shock factors, transcriptional and translational proteins, whereas proteins affecting cellular morphology, proteins involved in motility, central metabolism and energy production were down-regulated. Notably, numerous potential virulence factors were found to be up-regulated at higher temperatures, suggesting an elevated pathogenic potential of Cronobacter sp. under these growth conditions. Significant alterations in the protein expression profile and growth rate of C. turicensis exposed to 25 degrees C indicate that at this temperature the organism is cold-stressed. Up-regulated gene products comprised cold-shock, DNA-binding and ribosomal proteins, factors that support protein folding and proteins opposing cold-induced decrease in membrane fluidity, whereas down-regulated proteins were mainly involved in central metabolism.

Chromosomal location and evolutionary rate variation in enterobacterial genes.

The basal rate of DNA sequence evolution in enterobacteria, as seen in the extent of divergence between Escherichia coli and Salmonella typhimurium, varies greatly among genes, even when only "silent" sites are considered. The degree of divergence is clearly related to the level of gene expression, reflecting constraints on synonymous codon choice. However, where this constraint is weak, among genes not expressed at high levels, divergence is also related to the chromosomal location of the gene; it appears that genes furthest away from oriC, the origin of replication, have a mutation rate approximately two times that of genes near oriC.

Huge symbiotic organs in giant scale insects of the genus Drosicha (Coccoidea: Monophlebidae) harbor flavobacterial and enterobacterial endosymbionts.

Giant scale insects (Drosicha: Coccoldea: Monophlebidae) were investigated for their symbiotic organs and bacterial endosymbionts. Two types of bacterial 16S rRNA gene sequences, flavobacterial and enterobacterial, were consistently detected in D. corpulenta and D. pinicola. The former sequences formed a compact clade in the Bacteroidetes, allied to the symbionts of cushion and armored scales. The latter sequences formed a robust clade in the gamma-Proteobacteria, allied to enteric bacteria like Enterobacter aerogenes and Escherichia coli. Another type of 16S sequence derived from Wolbachia was also detected in D. pinicola. In-situ hybridization demonstrated that the flavobacterial and enterobacterial symbionts were localized in a pair of huge bacteriomes in the abdomen, the former in uninucleated peripheral bacteriocytes and the latter in syncytial central bacteriocytes. Electron microscopy confirmed the endocellular locations of the pleomorphic flavobacterial symbiont and the rod-shaped enterobacterial symbiont, and also revealed the location and fine structure of the Wolbachia symbiont in D. pinicola. Infection frequencies of the flavobacterial and enterobacterial symbionts were consistently 100% in populations of D. corpulenta and D. pinicola, while the Wolbachia symbiont exhibited 0% and 100% infection frequencies in D. corpulente and D. pinicola, respectively. Neither the flavobacterial symbiont nor the enterobacterial symbiont exhibited AT-biased nucleotide composition or accelerated molecular evolution. The huge bacteriomes of Drosicha giant scales would provide a useful system for investigating biochemical, physiological, and genomic aspects of the host-symbiont and symbiont-symbiont interactions.

Co-metabolic degradation of dimethoate by Raoultella sp. X1.

A bacterium Raoultella sp. X1, based on its 16S rRNA gene sequence, was isolated. Characteristics regarding the bacterial morphology, physiology, and genetics were investigated with an electron microscopy and conventional microbiological techniques. Although the isolate grew and degraded dimethoate poorly when the chemical was used as a sole carbon and energy source, it was able to remove up to 75% of dimethoate via co-metabolism. With a response surface methodology, we optimized carbon, nitrogen and phosphorus concentrations of the media for dimethoate degradation. Raoultella sp. X1 has a potential to be a useful organism for dimethoate degradation and a model strain for studying this biological process at the molecular level.

Physicochemical properties of a high molecular weight levan from Brenneria sp. EniD312.

A high molecular weight levan was produced by a novel levansucrase and some properties of this polymer were investigated. The levan exhibited a poroid microstructure as well as series of individual ellipsoidal or spheroidal particles. The weight-average molecular weight (M¯w) of the levan was determined to be 1.41×108Da. In a 0.1% solution, the levan showed a mean diameter of 176nm, while in a 1% solution the diameter was 182nm. The decomposition temperature was determined to be 216.67°C, with an endothermic peak at 147.41°C and a melting enthalpy of 76.9J/g. The small angle X-ray diffraction pattern showed a distinctive peak pattern between 15° and 40° (2q). The levan solution showed a shear-thinning behaviour. These results suggest this levan could be a good additive in the food processing industry, as well as an important bio-based material in the medicinal or chemical industry.

Plasmid-mediated resistance to antibiotic synergism in enterococci.

Mating experiments have shown that high-level resistance (minimal inhibitory concentration greater than 2,000 microgram/ml) to streptomycin and kanamycin, and resistance to penicillin-streptomycin and penicillin-kanamycin synergism are transferable by conjugation from resistant clinical isolates of enterococci to a sensitive recipient strain. Cesium chloride-ethidium bromide ultracentrifugation revealed a satellite (plasmid) band in resistant clinical isolates and the transconjugant strains but not in the sensitive recipient. Examination of these satellite bands by agarose gel electrophoresis and electron microscopy demonstrated a common plasmid with a weight of 45 megadaltons. Novobiocin treatment of a resistant clinical isolate produced simultaneous loss of high-level resistance to streptomycin and kanamycin, and of resistance to penicillin-aminoglycoside synergism. These results suggest that (a) high-level resistance to streptomycin and kanamycin among some clinical isolates of enterococci is associated with a 45 megadalton plasmid, and (b) the same plasmid is also responsible for the resistance to penicillin-aminoglycoside synergism observed in these strains.

Influence of subinhibitory concentrations of amikacin and ciprofloxacin on morphology and adherence ability of uropathogenic strains.

The influence of subinhibitory concentrations (1/2, 1/4, 1/8, 1/16 and 1/32 MIC) of amikacin and ciprofloxacin on the morphology and adherence of uropathogenic strains was studied. Intensity of morphological changes was proportional to the concentrations of these antibiotics. Morphological changes were the most prominent after bacterial exposure to sub-MICs of ciprofloxacin. These concentrations, especially 1/2 MIC of ciprofloxacin, induced the formation of filaments of E. coli, K. pneumoniae, K. oxytoca, E. cloacae and A. calcoaceticus biotype anitratus. No morphological changes were observed in P. aeruginosa, S. epidermidis and S. aureus cells after exposure to subinhibitory concentrations of both antibiotics. Sub-MICs of amikacin affected the changes in cell shape only slightly. The exposure of bacterial strains to 1/2 MIC of ciprofloxacin induced increased vacuolation of the cells. We observed shrinkage of the protoplasm and the pleated cell walls in comparison with control cells. The greatest loss of adherence ability occurred at 1/2 MIC of ciprofloxacin after a 1-d incubation.

Characterization of two polyvalent phages infecting Enterobacteriaceae.

Bacteriophages display remarkable genetic diversity and host specificity. In this study, we explore phages infecting bacterial strains of the Enterobacteriaceae family because of their ability to infect related but distinct hosts. We isolated and characterized two novel virulent phages, SH6 and SH7, using a strain of Shigella flexneri as host bacterium. Morphological and genomic analyses revealed that phage SH6 belongs to the T1virus genus of the Siphoviridae family. Conversely, phage SH7 was classified in the T4virus genus of the Myoviridae family. Phage SH6 had a short latent period of 16 min and a burst size of 103 ± 16 PFU/infected cell while the phage SH7 latent period was 23 min with a much lower burst size of 26 ± 5 PFU/infected cell. Moreover, phage SH6 was sensitive to acidic conditions (pH < 5) while phage SH7 was stable from pH 3 to 11 for 1 hour. Of the 35 bacterial strains tested, SH6 infected its S. flexneri host strain and 8 strains of E. coli. Phage SH7 lysed additionally strains of E. coli O157:H7, Salmonella Paratyphi, and Shigella dysenteriae. The broader host ranges of these two phages as well as their microbiological properties suggest that they may be useful for controlling bacterial populations.

The All-Rounder Sodalis: A New Bacteriome-Associated Endosymbiont of the Lygaeoid Bug Henestaris halophilus (Heteroptera: Henestarinae) and a Critical Examination of Its Evolution.

Hemipteran insects are well-known in their ability to establish symbiotic relationships with bacteria. Among them, heteropteran insects present an array of symbiotic systems, ranging from the most common gut crypt symbiosis to the more restricted bacteriome-associated endosymbiosis, which have only been detected in members of the superfamily Lygaeoidea and the family Cimicidae so far. Genomic data of heteropteran endosymbionts are scarce and have merely been analyzed from the Wolbachia endosymbiont in bed bug and a few gut crypt-associated symbionts in pentatomoid bugs. In this study, we present the first detailed genomic analysis of a bacteriome-associated endosymbiont of a phytophagous heteropteran, present in the seed bug Henestaris halophilus (Hemiptera: Heteroptera: Lygaeoidea). Using phylogenomics and genomics approaches, we have assigned the newly characterized endosymbiont to the Sodalis genus, named as Candidatus Sodalis baculum sp. nov. strain kilmister. In addition, our findings support the reunification of the Sodalis genus, currently divided into six different genera. We have also conducted comparative analyses between 15 Sodalis species that present different genome sizes and symbiotic relationships. These analyses suggest that Ca. Sodalis baculum is a mutualistic endosymbiont capable of supplying the amino acids tyrosine, lysine, and some cofactors to its host. It has a small genome with pseudogenes but no mobile elements, which indicates middle-stage reductive evolution. Most of the genes in Ca. Sodalis baculum are likely to be evolving under purifying selection with several signals pointing to the retention of the lysine/tyrosine biosynthetic pathways compared with other Sodalis.

Photoelectrocatalytic inactivation of fecal coliform bacteria in urban wastewater using nanoparticulated films of TiO2 and TiO2/Ag.

Photocatalysis has shown the ability to inactivate a wide range of harmful microorganisms with traditional use of chlorination. Photocatalysis combined with applied bias potential (photoelectrocatalysis) increases the efficiency of photocatalysis and decreases the charge recombination. This work examines the inactivation of fecal coliform bacteria present in real urban wastewater by photoelectrocatalysis using nanoparticulated films of TiO2 and TiO2/Ag (4%w/w) under UV light irradiation. The catalysts were prepared with different thicknesses by the sol-gel method and calcined at 400°C and 600°C. The urban wastewater samples were collected from the sedimentation tank effluent of the university sewage treatment facility. The rate of bacteria inactivation increases with increasing the applied potential and film thicknesses; also, the presence of silver on the catalyst surface annealed at 400°C shows better inactivation than that at 600°C. Finally, a structural cell damage of Escherichia coli (DH5α), inoculated in water, is observed during the photoelectrocatalytic process.

Rapid antibiotic sensitivity testing and trimethoprim-mediated filamentation of clinical isolates of the Enterobacteriaceae assayed on a novel porous culture support.

A porous inorganic material (Anopore) was employed as a microbial culture and microcolony imaging support. Rapid Anopore-based antibiotic sensitivity testing (AST) methods were developed to assess the growth of clinical isolates, with the primary focus on testing the response of the Enterobacteriaceae to trimethoprim, but with the method supporting a wider applicability in terms of strains and antibiotics. It was possible to detect the growth of Enterobacter aerogenes after 25 min culture and to distinguish a trimethoprim-sensitive from a trimethoprim-resistant strain with 40 min incubation. MIC(90) determinations were made on Anopore; these were in good agreement with the results from the Vitek 2 and E-test methods. The Anopore method correctly identified sensitive (40/40) and resistant (17/17) strains of the Enterobacteriaceae and other Gram-negative rods within only 2-3 h culture. Additionally, a trimethoprim-resistant subpopulation (10 % of population) could be detected by microcolony formation within 2 h, and a smaller subpopulation (1 %) after 3.5 h. These results suggest that this is a viable approach for the rapid AST of purified strains, and that it may be able to deal with mixed populations. The microscopic examination of microcolonies during AST is an advantage of this method which revealed additional information. Filamentation triggered by trimethoprim was discovered in many species of the Enterobacteriaceae for which this phenomenon has not previously been reported. Filamentation was characterized by heterogeneity in terms of cell length, and also uneven nucleic acid distribution and flattening of damaged cells. The development and application of Anopore-based AST within clinical diagnostics is discussed.

Sulcia symbiont of the leafhopper Macrosteles laevis (Ribaut, 1927) (Insecta, Hemiptera, Cicadellidae: Deltocephalinae) harbors Arsenophonus bacteria.

The leafhopper Macrosteles laevis, like other plant sap-feeding hemipterans, lives in obligate symbiotic association with microorganisms. The symbionts are harbored in the cytoplasm of large cells termed bacteriocytes, which are integrated into huge organs termed bacteriomes. Morphological and molecular investigations have revealed that in the bacteriomes of M. laevis, two types of bacteriocytes are present which are as follows: bacteriocytes with bacterium Sulcia and bacteriocytes with Nasuia symbiont. We observed that in bacteriocytes with Sulcia, some cells of this bacterium contain numerous cells of the bacterium Arsenophonus. All types of symbionts are transmitted transovarially between generations. In the mature female, the bacteria Nasuia, bacteria Sulcia, and Sulcia with Arsenophonus inside are released from the bacteriocytes and start to assemble around the terminal oocytes. Next, the bacteria enter the cytoplasm of follicular cells surrounding the posterior pole of the oocyte. After passing through the follicular cells, the symbionts enter the space between the oocyte and follicular epithelium, forming a characteristic "symbiont ball."

The coffee-machine bacteriome: biodiversity and colonisation of the wasted coffee tray leach.

Microbial communities are ubiquitous in both natural and artificial environments. However, microbial diversity is usually reduced under strong selection pressures, such as those present in habitats rich in recalcitrant or toxic compounds displaying antimicrobial properties. Caffeine is a natural alkaloid present in coffee, tea and soft drinks with well-known antibacterial properties. Here we present the first systematic analysis of coffee machine-associated bacteria. We sampled the coffee waste reservoir of ten different Nespresso machines and conducted a dynamic monitoring of the colonization process in a new machine. Our results reveal the existence of a varied bacterial community in all the machines sampled, and a rapid colonisation process of the coffee leach. The community developed from a pioneering pool of enterobacteria and other opportunistic taxa to a mature but still highly variable microbiome rich in coffee-adapted bacteria. The bacterial communities described here, for the first time, are potential drivers of biotechnologically relevant processes including decaffeination and bioremediation.

Diffusion of carbapenems through the outer membrane of enterobacteriaceae and correlation of their activities with their periplasmic concentrations.

Scarce information is available on the real mechanism by which carbapenemes penetrate in Enterobacteriaceae, although a considerable amount of evidence suggests that in many species of this family the lack of certain outer membrane proteins is associated with the acquisition of resistance to these antibiotics. The existance of specific pathways for the carbapenems has never been demonstrated, although at times it has been postulated in both wild and mutant strains, on the basis of evident discordances between permeability patterns and suceptibility data. By using the Zimmerman and Rosselet technique, which requires the strain under investigation to harbor a suitable beta-lactamase, the permeability of intact Escherichia coli and Enterobacter cloacae cells to meropenem and imipenem was investigated by transferring a constructed vector carrying the carbapenem hydrolyzing CphA metallo-beta-lactamase gene into the parental strains and their porin-deficient mutants. Reduced amounts of nonspecific porins significantly reduced the penetration of both carbapenems. The virtual absence of porins caused the MICs of meropenem to increase, mostly in Enterobacter cloacae, while it did not affected the MICs of imipenem. No evidence of specific porin pathways of the type described in Pseudomonas aeruginosa was found.

Differentiation of Enterobacteriaceae, Pseudomonas aeruginosa, and Bacteroides and Haemophilus species in gram-stained direct smears.

The accuracy of examination of the Gram-stained direct smear to classify presumptively Gram-negative rods into three morphotype groups, that is, (a) Enteric bacteria, (b) Pseudomonas, and (c) Bacteroides or Haemophilus, was evaluated. Randomly selected clinical strains (4-9) each of Escherichia coli, Klebsiella pneumoniae, Proteus mirabilis, Ps. aeruginosa, B. fragilis, and H. influenzae were used to produce peritonitis or subcutaneous abscesses in mice. A Gram-stained direct smear was prepared from exudate collected from each animal. The direct smears were examined to classify bacteria observed into one of the three morphotype groups. The percent accuracy was 82, 56, and 95, respectively, and 76 overall. The assumption was made that classification was based primarily on differences in length and width of the organisms. To test this hypothesis, we prepared scanning electron photomicrographs from each specimen of exudate and measured the lengths and widths of bacteria. Examination of the Gram-stained direct smear was more accurate for classification of enteric bacteria, H. influenzae, or B. fragilis. Electron microscopy was more accurate for classification of Ps. aeruginosa. The higher length-width radio should be helpful in recognizing Ps. aeruginosa in direct smears.

Bacterial adhesion in human upper gastrointestinal tract.

Small bowel biopsy specimens were taken from 21 patients undergoing gastrointestinal endoscopy to detect a possible adhesion of bacteria to the mucous layer of the upper gastrointestinal tract. In 30 control biopsy specimens taken from 10 patients free from gastrointestinal pathology, no associated bacteria were found, whereas in 23 biopsy specimens taken from eight gastrectomized patients an associated bacterial flora including E. coli or Pseudomonas was grown. Bacterial adhesion was confirmed by means of transmission electron microscopy of the eight patients yielding positive cultures. Bacterial adhesion induced local alterations of the brush border membrane. These results suggest that adherent bacteria may be present in hypochlorhydric patients. Pathophysiologic consequences require further studies.

Sulbactam: secondary mechanisms of action.

Light and scanning electron microscopy showed that 0.25, 0.5 and 1 times the minimum inhibitory concentrations (MICs) of sulbactam (SULB) caused filament formation in different species of Enterobacteriaceae, while 2 and 4 times the MICs caused spheroplast formation and subsequent lysis. By using a competitive assay with 125I-penicillin X, SULB showed a primary affinity for the PBP 1a and PBP3 of Escherichia coli, as well as for the PBP1a of Proteus mirabilis. The bactericidal interaction of human polymorphonuclear leukocytes (PMN) and SULB against E. coli K-1 resistant to the bactericidal activity of human serum was studied in vitro; however, SULB concentrations showed variations in the medium according to human kinetic data. Under these conditions, bacterial growth occurred in Hanks balanced salt solution containing SULB, PMN, or SULB-PMN in combination. In addition, bactericidal activity was observed in serum, with a killing rate of 90% for PMN or SULB, and 95% for SULB-PMN in combination. The postantibiotic enhancement of PMN bactericidal function was assessed against E. coli K1 pretreated with 0.5 the MIC of SULB (32 micrograms/ml) for 0.5 hr. The 90% bacterial killing rate of PMN occurred by 1.5 hr for pretreated bacteria and by 2.5 hr for untreated bacteria. Furthermore, the luminol-enhanced chemiluminescence (CL) assay using an E. coli stimulus showed that SULB does not modify PMN activity.