Drosophila mauve mutants reveal a role of LYST homologs late in the maturation of phagosomes and autophagosomes.

Chediak-Higashi syndrome (CHS) is a lethal disease caused by mutations that inactivate the lysosomal trafficking regulator protein (LYST). Patients suffer from diverse symptoms including oculocutaneous albinism, recurrent infections, neutropenia and progressive neurodegeneration. These defects have been traced back to over-sized lysosomes and lysosome-related organelles (LROs) in different cell types. Here, we explore mutants in the Drosophila mauve gene as a new model system for CHS. The mauve gene (CG42863) encodes a large BEACH domain protein of 3535 amino acids similar to LYST. This reflects a functional homology between these proteins as mauve mutants also display enlarged LROs, such as pigment granules. This Drosophila model also replicates the enhanced susceptibility to infections and we show a defect in the cellular immune response. Early stages of phagocytosis proceed normally in mauve mutant hemocytes but, unlike in wild type, late phagosomes fuse and generate large vacuoles containing many bacteria. Autophagy is similarly affected in mauve fat bodies as starvation-induced autophagosomes grow beyond their normal size. Together these data suggest a model in which Mauve functions to restrict homotypic fusion of different pre-lysosomal organelles and LROs.

Rhomboid-7 over-expression results in Opa1-like processing and malfunctioning mitochondria.

Rhomboid-7 (rho-7) is a mitochondrial-specific intramembranous protease. The loss-of-function mutation rho-7 results in semi-lethality, while escapers have a reduced lifespan with several neurological disorders [1]. Here we show that general, or CNS-specific expression of rho-7 can rescue the lethality of rho-7. General, or CNS-specific over-expression of rho-7 in otherwise wild-type animals caused semi-lethality, with approximately 50% of the animals escaping this lethality, developing into adults displaying a shortened life span with larval locomotory problem. On a cellular level, over-expression resulted in severe depression of ATP levels and cytochrome c oxidase subunit II mRNA levels, a lowered number of mitochondria in neurons and aggregation of mitochondria in the brain indicating mitochondrial malfunction. Over-expression of rho-7 in developing eye discs resulted in an elevated apoptotic index. In the CNS, elevated levels of rho-7 were accompanied by both isoforms of Opa1-like, a dynamin-like GTPase, a mitochondrial component involved in regulating mitochondrial dynamics and function, including apoptosis. Most, but not all, of rho-7 over-expression phenotypes were suppressed by introducing a heterozygous mutation for Opa1-like. Our results suggest that rho-7 and Opa1-like function in a common molecular pathway affecting mitochondrial function and apoptosis in Drosophila melanogaster.

Prevalence and transmission of antimicrobial resistance among Aeromonas populations from a duckweed aquaculture based hospital sewage water recycling system in Bangladesh.

In order to investigate the influence of a duckweed aquaculture based hospital sewage water recycling plant on the prevalence and dissemination of antibiotic resistance, we made use of an existing collection of 1,315 Aeromonas isolates that were previously typed by the biochemical fingerprinting PhP-AE system. In these treatment plant, hospital raw sewage water is first collected in a settlement pond (referred to as sewage water in this study) and is then transferred to a lagoon, where the duckweed (Lemnaceae) is grown (referred to as lagoon). The duckweed is harvested and used as feed for the fish in a separate pond (referred to as fish pond). From this collection, representatives of 288 PhP types were subjected to antibiotic susceptibility testing for eight antimicrobials by broth microdilution method. The overall resistance rates among Aeromonas isolates from the treatment plant were highest for ampicillin (87%) and erythromycin (79%) followed by cephalothin (58%), nalidixic acid (52%), streptomycin (51%), tetracycline (31%), chloramphenicol (13%) and gentamicin (8%). A significantly lower prevalence of antibiotic resistance was found in Aeromonas from environmental control water, patient stool samples, duckweed and fish compared to sewage water isolates. The prevalence of resistance in the sewage water was not significantly reduced compared to the lagoon water and fish pond. Throughout the treatment system, the frequencies of resistant strains were found to diminish during the sewage water purification process, i.e. in the lagoon where sewage water is used to grow the duckweed. However, the frequency of resistant strains again increased in the fish pond where sewage grown duckweed is used for aquaculture. Among the selected isolates, two multiresistant clonal groups of Aeromonas caviae HG4 were identified that exhibited indistinguishable PhP and amplified fragment length polymorphism fingerprints and shared a common plasmid of approximately 5 kb. Representatives of both groups were recovered from almost every part of the sewage treatment plant but not in the control ponds nor in human samples, which suggests that specific multiresistant Aeromonas clones are able to persist and spread throughout the entire purification process.

The role of c-di-GMP signaling in an Aeromonas veronii biovar sobria strain.

Aeromonas is a ubiquitous gram-negative bacterium that persists in the environment. It is shown that all isolates of persistent Aeromonas clones show strong biofilm formation ability. C-di-GMP regulates biofilm formation in many bacteria. To investigate the impact of c-di-GMP signaling, we introduced heterologous GGDEF and EAL domain proteins from Salmonella Typhimurium to an Aeromonas veronii biovar sobria strain. Overexpression of the GGDEF domain protein AdrA increased c-di-GMP concentration and biofilm formation and reduced motility. Production of the quorum-sensing signaling molecule C4-homoserine lactone and adhesion to aquatic plant duckweed and amoeba surfaces were enhanced. On the other hand, overexpression of the EAL domain protein YhjH decreased biofilm formation and increased motility.

Visual neurotransmission in Drosophila requires expression of Fic in glial capitate projections.

Fic domains can catalyze the addition of adenosine monophosphate to target proteins. To date, the function of Fic domain proteins in eukaryotic physiology remains unknown. We generated genetic models of the single Drosophila Fic domain­containing protein, Fic. Flies lacking Fic were viable and fertile, but blind. Photoreceptor cells depolarized normally following light stimulation, but failed to activate postsynaptic neurons, as indicated by the loss of ON transients in electroretinograms, consistent with a neurotransmission defect. Functional rescue of neurotransmission required expression of enzymatically active Fic on capitate projections of glia cells, but not neurons, supporting a role in the recycling of the visual neurotransmitter histamine. Histamine levels were reduced in the lamina of Fic null flies, and dietary histamine partially restored ON transients. These findings establish a previously unknown regulatory mechanism in visual neurotransmission and provide, to the best of our knowledge, the first evidence for a role of glial capitate projections in neurotransmitter recycling.

Persistence, transmission, and virulence characteristics of Aeromonas strains in a duckweed aquaculture-based hospital sewage water recycling plant in Bangladesh.

The persistence and transmission of Aeromonas in a duckweed aquaculture-based hospital sewage water treatment plant in Bangladesh was studied. A total of 670 samples from different sites of the hospital sewage water treatment plant, from feces of hospitalized children suffering from diarrhea, from environmental control ponds, and from feces of healthy humans were collected over a period of three years. In total, 1,315 presumptive Aeromonas isolates were biochemically typed by the PhenePlate rapid screening system (PhP-AE). A selection of 90 representative isolates was further analyzed with PhenePlate (PhP) extended typing (PhP-48), fatty acid methyl ester analysis, and amplified fragment length polymorphism (AFLP) fingerprinting. In addition, the prevalence of the putative virulence factors hemolysin and cytotoxin and the presence of the cytolytic enterotoxin gene (AHCYTOEN) were analyzed. Aeromonas was found at all sites of the treatment plant, in 40% of the samples from environmental control ponds, in 8.5% of the samples from hospitalized children suffering from diarrhea, and in 3.5% of samples from healthy humans. A significantly high number of Aeromonas bacteria was found in duckweed, which indicates that duckweed may serve as a reservoir for these bacteria. PhP-AE typing allowed identification of more than 192 distinct PhP types, of which 18 major PhP types (MTs) were found in multiple sites and during several occasions. AFLP fingerprinting revealed the prevalence of genotypically indistinguishable Aeromonas isolates among certain PhP MTs recovered from different sampling occasions and/or at multiple sites. Hemolytic and cytotoxic activities were observed in 43% of the tested strains, whereas 29% possessed the cytolytic enterotoxin gene AHCYTOEN. Collectively, two specific MTs associated with diarrhea were shown to exhibit high cytotoxicity. Furthermore, all tested isolates of these major types were positive for the cytolytic enterotoxin gene. In conclusion, our data indicate that certain phenotypically and genotypically stable clonal lineages of Aeromonas have persisted in the treatment system for a prolonged period and might spread from the hospitalized children suffering from diarrhea to fish produced for human consumption through the sewage water treatment system.

Biochemical fingerprinting of Vibrio parahaemolyticus by the PhenePlate system: comparison between pandemic and non-pandemic serotypes.

During recent years a pandemic clone of Vibrio parahaemolyticus has emerged. Isolates of this clone are distributed among several serotypes, but are genotypically related. In the present study, a phenotyping method (biochemical fingerprinting) was used to characterize pandemic and non-pandemic isolates belonging to V. parahaemolyticus. It was found that the pandemic isolates showed a high level of phenotypic homogeneity and a majority of the pandemic isolates belonged to the same biochemical phenotype, whereas non-pandemic V. parahemolyticus isolates were more heterogeneous. In conclusion, biochemical fingerprinting of V. parahaemolyticus can be used as a first screening method to differentiate between pandemic and non-pandemic isolates of V. parahaemolyticus.

Evaluation of a scanner-assisted colorimetric MIC method for susceptibility testing of gram-negative fermentative bacteria.

We describe the ScanMIC method, a colorimetric MIC method for susceptibility testing of gram-negative fermentative bacteria. The method is a slight modification of the National Committee for Clinical Laboratory Standards (NCCLS) recommended broth microdilution method that uses a redox indicator 2,3,5-triphenyltetrazolium chloride (TTC) to enhance the estimate of bacterial growth inhibition in a microplate and a flatbed scanner to capture the microplate image. In-house software was developed to transform the microplate image into numerical values based on the amount of bacterial growth and to generate the MICs automatically. The choice of indicator was based on its low toxicity and ease of reading by scanner. We compared the ScanMIC method to the NCCLS recommended broth microdilution method with 197 coliform strains against seven antibacterial agents. The interpretative categorical agreement was obtained in 92.4% of the assays, and the agreement for MIC differences (within +/-1 log(2) dilution) was obtained in 96% for ScanMIC versus broth microdilution and 97% for a two-step incubation colorimetric broth microdilution versus the broth microdilution method. The method was found to be labor-saving, not to require any initial investment, and to show reliable results. Thus, the ScanMIC method could be useful for epidemiological surveys that include susceptibility testing of bacteria.

Identification and characterization of pathogenic Aeromonas veronii biovar sobria associated with epizootic ulcerative syndrome in fish in Bangladesh.

Sparse information is available on the virulence factors of Aeromonas strains isolated from diseased fish, from the environment, and from humans. In the present study, 52 Aeromonas isolates obtained from epizootic ulcerative syndrome (EUS) lesions in fish, from the aquatic environment, and from children with diarrhea in Bangladesh were identified by biochemical phenotyping (i.e., PhenePlate [PhP] typing) and DNA fingerprinting and then characterized with respect to certain putative virulence factors. The isolates from the fish exhibiting EUS symptoms were identified to be Aeromonas veronii biovar sobria by fatty acid methyl ester analysis and amplified fragment length polymorphism fingerprinting. Biochemical phenotyping revealed that all EUS-associated isolates belonged to a unique phenotype which was not identified among more than 1,600 environmental and diarrheal isolates in a previously collected database of PhP types of Bangladeshi Aeromonas isolates. The 52 Aeromonas isolates were investigated for the production of hemolysin and cytotoxin; for hemagglutination with erythrocytes from fish, human, and rabbit sources; for the presence of a cytolytic enterotoxin gene; and for adhesion to and invasion into fish cell lines. All of the EUS isolates produced all of the virulence factors investigated, as did also some of the environmental isolates, but the isolates from EUS were unique in their ability to agglutinate fish erythrocytes. Our results suggest that a clonal group of A. veronii biovar sobria is associated with, and may be a causative agent of, EUS in fish in Bangladesh.

Evidence for transmission between humans and the environment of a nosocomial strain of Enterococcus faecium.

An ampicillin- and ciprofloxacin-resistant Enterococcus faecium (ARE) strain, named FMSE1, with a characteristic biochemical phenotype, was in a recent study found to dominate among faecal ARE isolates from patients in several Swedish hospitals. In the present study, the prevalence of this strain among 9676 enterococcal isolates from healthy children, hospital sewage, urban sewage, surface water, slaughtered animals (broilers, pigs and cattle) and pig faeces and manure was investigated. Enterococcal isolates having the same biochemical phenotype as the FMSE1 were most common in samples of hospital sewage (50%), surface water (35%), treated sewage (28%) and untreated sewage (17%), but rare in samples from healthy children (0.8%) and animals (2%). PFGE typing of FMSE1-like isolates from hospital sewage indicated that they were closely related to the nosocomial FMSE1 strain. Thus, this study indicated a possible transmission route for nosocomial E. faecium from patients in hospitals to hospital sewage and urban sewage, and further via treatment plants to surface water and possibly back to humans. This proposed route of circulation of drug-resistant enterococci might be further amplified by antibiotic usage in human medicine. In contrast, such transmission from food animals seems to play a negligible role in Sweden.