Immunochemical studies and gene cluster relationships of closely related O-antigens of Aeromonas hydrophila Pt679, Aeromonas popoffii A4, and Aeromonas sobria K928 strains classified into the PGO1 serogroup dominant in Polish aquaculture of carp and rainbow trout.

The present study included three Aeromonas sp. strains isolated from fish tissues during Motile Aeromonas Infection/Motile Aeromonas Septicaemia disease outbreaks on commercial farms, i.e.: Aeromonas hydrophila Pt679 obtained from rainbow trout as well as Aeromonas popoffii A4 (formerly Aeromonas encheleia) and Aeromonas sobria K928 both isolated from carp, which were classified into the new provisional PGO1 serogroup prevailing among aeromonads in Polish aquaculture. The structure of the O-specific polysaccharides of A4 and K928 has been previously established. Here, immunochemical studies of the O-specific polysaccharide of A. hydrophila Pt679 were undertaken. The O-specific polysaccharide was obtained from the lipopolysaccharide of A. hydrophila Pt679 after mild acid hydrolysis and separation by gel-permeation chromatography. The high-molecular-mass fraction was studied using chemical methods and 1H and 13C NMR spectroscopy, including 1H,1H NOESY, and 1H,13C HMBC experiments. The following structure of the branched repeating unit of the O-polysaccharide from A. hydrophila Pt679 was determined: [Formula: see text] The studies indicated that O-polysaccharides from A. hydrophila Pt679, A. popoffii A4 and A. sobria K928 share similarities but they also contain unique characteristics. Western blotting and an enzyme-linked immunosorbent assay revealed that the cross-reactivity of the related O-antigens is caused by the occurrence of common structural elements, whereas additional epitopes define the specificity of the O-serotypes. For genetic relationship studies, the O-antigen gene cluster was characterized in the genome of the A. hydrophila Pt679 strain and compared with the corresponding sequences of A. popoffii A4 and A. sobria K928 and with sequences available in the databases. The composition of the regions was found to be consistent with the O-antigen structures of Aeromonas strains classified into the same PGO1 serogroup.

Structure and gene cluster annotation of the O-antigen of Aeromonas sobria strain K928 isolated from common carp and classified into the new Aeromonas PGO1 serogroup.

Aeromonas sobria strain K928 was isolated from a common carp during a Motile Aeromonas Infection/Motile Aeromonas Septicaemia disease outbreak on a Polish fish farm and classified into the new provisional PGO1 serogroup. The lipopolysaccharide of A. sobria K928 was subjected to mild acid hydrolysis, and the O-specific polysaccharide, which was isolated by gel-permeation chromatography, was studied using sugar and methylation analyses and 1H and 13C NMR spectroscopy. The following structure of the branched O-specific polysaccharide repeating unit of A. sobria K928 was established. →2)[α-D-Fucp3NRHb-(1→3)]-α-L-Rhap-(1→3)-ß-L-Rhap-(1→4)-α-L-Rhap-(1→3)-ß-D-FucpNAc-(1→ The O-antigen gene cluster was identified and characterized in the genome of the A. sobria K928 strain after comparison with sequences in the available databases. The composition of the O-antigen genetic region was found to be consistent with the O-polysaccharide structure, and its organization was proposed.

Different Algicidal Modes of the Two Bacteria Aeromonas bestiarum HYD0802-MK36 and Pseudomonas syringae KACC10292T against Harmful Cyanobacteria Microcystis aeruginosa.

Blooms of harmful cyanobacteria Microcystis aeruginosa lead to an adverse effect on freshwater ecosystems, and thus extensive studies on the control of this cyanobacteria's blooms have been conducted. Throughout this study, we have found that the two bacteria Aeromonas bestiarum HYD0802-MK36 and Pseudomonas syringae KACC10292T are capable of killing M. aeruginosa. Interestingly, these two bacteria showed different algicidal modes. Based on an algicidal range test using 15 algal species (target and non-target species), HYD0802-MK36 specifically attacked only target cyanobacteria M. aeruginosa, whereas the algicidal activity of KACC10292T appeared in a relatively broad algicidal range. HYD0802-MK36, as a direct attacker, killed M. aeruginosa cells when direct cell (bacterium)-to-cell (cyanobacteria) contact happens. KACC10292T, as an indirect attacker, released algicidal substance which is located in cytoplasm. Interestingly, algicidal activity of KACC10292T was enhanced according to co-cultivation with the host cyanobacteria, suggesting that quantity of algicidal substance released from this bacterium might be increased via interaction with the host cyanobacteria.

Using Matrix-Assisted Laser Desorption/Ionization Time of Flight Spectra To Elucidate Species Boundaries by Matching to Translated DNA Databases.

A method has been established to map a bacterial colony to the ever-expanding database of publicly available bacterial genomes by means of matrix-assisted laser desorption/ionization (MALDI) spectra. To accomplish this, spectra are mapped to the predicted masses of ∼65 families of mostly ribosomal proteins. Each of the ∼40 000 bacterial strains in the database receives scores, together with tables listing identified protein sequences and how the highest ranking strains are related to one another. The approach was first confirmed with 16 distinct species of bacteria from the Vibrionales whose genome had been sequenced. Identifications of a few species of bacteria from environmental samples from compost, lakes, and streams in Massachusetts are also reported. Most of these organisms map to known species in the Gammaproteobacteria and Firmicutes. The clades of bacteria deducible from shared ribosomal protein sequences do not always correspond well to named bacterial species. Instead, the identifications made by this methodology indicate groupings of organisms that can readily be distinguished by MALDI-TOF and indicate which polymorphisms in highly conserved proteins demarcate the groupings. Successful identifications highlight organism interrelationships that can be deduced from the available genomes, sorting together genomes into new proposed clades typically consistent with relationships deduced from DNA sequence analysis. In contrast, if for a high-quality spectrum from a fresh colony, no group of related organisms receives high scores, one might infer that no closely related genome has yet been deposited into the database.

Proteomic characterization and discrimination of Aeromonas species recovered from meat and water samples with a spotlight on the antimicrobial resistance of Aeromonas hydrophila.

Aeromonas is recognized as a human pathogen following ingestion of contaminated food and water. One major problem in Aeromonas identification is that certain species are phenotypically very similar. The antimicrobial resistance is another significant challenge worldwide. We therefore aimed to use mass spectrometry technology for identification and discrimination of Aeromonas species and to screen the antimicrobial resistance of Aeromonas hydrophila (A. hydrophila). A total of 150 chicken meat and water samples were cultured, and then, the isolates were identified biochemically by the Vitek® 2 Compact system. Proteomic identification was performed by MALDI-TOF MS and confirmed by a microchannel fluidics electrophoresis assay. Principal component analysis (PCA) and single-peak analysis created by MALDI were also used to discriminate the Aeromonas species. The antimicrobial resistance of the A. hydrophila isolates was determined by Vitek® 2 AST cards. In total, 43 samples were positive for Aeromonas and comprised 22 A. hydrophila, 12 Aeromonas caviae (A. caviae), and 9 Aeromonas sobria (A. sobria) isolates. Thirty-nine out of 43 (90.69%) Aeromonas isolates were identified by the Vitek® 2 Compact system, whereas 100% of the Aeromonas isolates were correctly identified by MALDI-TOF MS with a score value ≥2.00. PCA successfully separated A. hydrophila, A. caviae and A. sobria isolates into two groups. Single-peak analysis revealed four discriminating peaks that separated A. hydrophila from A. caviae and A. sobria isolates. The resistance of A. hydrophila to antibiotics was 95.46% for ampicillin, 50% for cefotaxime, 45.45% for norfloxacin and pefloxacin, 36.36% for ceftazidime and ciprofloxacin, 31.81% for ofloxacin and 27.27% for nalidixic acid and tobramycin. In conclusion, chicken meat and water were tainted with Aeromonas spp., with a high occurrence of A. hydrophila. MALDI-TOF MS is a powerful technique for characterizing aeromonads at the genus and species levels. Future studies should investigate the resistance of A. hydrophila to various antimicrobial agents.

Limited performance of MALDI-TOF for identification of fish Aeromonas isolates at species level.

The aim of this study was to evaluate the usefulness of the MALDI-TOF MS to identify 151 isolates of Aeromonas obtained mostly from diseased fish. MALDI-TOF MS correctly identified all isolates to the genus level but important differences in the percentage of isolates correctly identified depending on the species were observed. Considering exclusively the first identification option, Aeromonas bestiarum, Aeromonas hydrophila, Aeromonas salmonicida, Aeromonas veronii and Aeromonas sobria were the best identified with results >95%. However, considering the first and second identification options, the only species that showed values >90% was A. hydrophila. Overall, when the database was supplemented with 14 new spectra, the number of accurate identifications increased (41% vs. 55%) and the number of inconclusive identifications decreased (45% vs. 29%), but great differences in the success of species-level identifications were found. Species-distinctive mass peaks were identified only for A. hydrophila and A. bestiarum (5003 and 7360 m/z in 95.5% and 94.1% of their isolates, respectively). This work demonstrates the utility of MALDI-TOF MS for Aeromonas identification to the genus level, but there is no consistency for the accurate identification of some of the most prevalent species implicated in fish disease.

Atomic-Resolution Structure of a Class†C ß-Lactamase and Its Complex with Avibactam.

ß-Lactamases (BLs) are important antibiotic-resistance determinants that significantly compromise the efficacy of valuable ß-lactam antibacterial drugs. Thus, combinations with BL inhibitor were developed. Avibactam is the first non-ß-lactam BL inhibitor introduced into clinical practice. Ceftazidime-avibactam represents one of the few last-resort antibiotics available for the treatment of infections caused by near-pandrug-resistant bacteria. TRU-1 is a chromosomally encoded AmpC-type BL of Aeromonas enteropelogenes, related to the FOX-type BLs and constitutes a good model for class C BLs. TRU-1 crystals provided ultrahigh-resolution diffraction data for the native enzyme and for its complex with avibactam. A comparison of the native and avibactam-bound structures revealed new details in the conformations of residues relevant for substrate and/or inhibitor binding. Furthermore, a comparison of the TRU-1 and Pseudomonas aeruginosa AmpC avibactam-bound structures revealed two inhibitor conformations that were likely to correspond to two different states occurring during inhibitor carbamylation/recyclization.

Synthesis of the repeating unit of O-specific polysaccharide isolated from the water-borne bacteria Aeromonas bestiarum 207.

Aeromonas bestiarum 207 is a bacterial pathogen with severe impact on aquaculture. In a recent study, the structure of OPS antigens from Aeromonas bestiarum was identified as pentasaccharide repeating units. Synthesis of the pentasaccharide repeating unit and its derivative are reported. Stereo- and regio-specific synthesis was achieved under Schmidt glycosylation conditions employing appropriately protected L-rhamopyranosyl and D-glucopyranosylamine building blocks. The pentasaccharide synthesis was achieved using a [3 + 2] strategy with an overall yield of 5.2% through 11 linear steps from the monosaccharide building blocks 10 and 14.

Combining molecular and bioprocess techniques to produce poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) with controlled monomer composition by Burkholderia sacchari.

Biopolymers as polyhydroxyalkanoates (PHA) composed by different co-monomers 3-hydroxybutyrate and 3-hydroxyhexanoate [P(3HB-co-3HHx)] has attracted interest since its properties are similar to low density polyethylene. Burkholderia sacchari produces this copolymer with a very low 3HHx molar fraction, about 2 mol%. B. sacchari mutant (unable to produce polymer) was engineered to host PHA biosynthesis genes (phaPCJ) from Aeromonas sp. In addition, a two-step bioprocess to increase biopolymer production was developed. The combination of these techniques resulted in the production of P(3HB-co-3HHx) with 3HHx content up to 20 mol%. The PHA content was about 78% of dry biomass, resulting in PHA volumetric productivities around 0.45gl-1h-1. The P(3HB-co-3HHx) containing 20 mol% of 3HHx presented an elongation at brake of 945%, higher than reported before for this PHA composition. Here we have described an approach to increase 3HHx content into the copolymer, allowing the precise control of the 3HHx molar fractions.

Biosynthesis of silver nanoparticles using Aeromonas sp. THG-FG1.2 and its antibacterial activity against pathogenic microbes.

Silver nanoparticles were prepared through green route with the aid of Aeromonas sp. THG-FG1.2 as reductant. Visual observation, ultraviolet-visible spectroscopy, transmission electron microscopy, elemental mapping, energy dispersive X-ray spectroscopy, selected area diffraction pattern (SAED), and X-ray diffraction (XRD) were used to characterize the synthesized silver nanoparticles. UV visible studies indicated the surface plasmon resonance at 400 nm which depicts the formation of silver nanoparticles. The TEM images show spherical silver nanoparticles of 8-16 nm. XRD and SAED fringes revealed the structure of silver nanoparticles as face centered cubic (fcc). These silver nanoparticles also tested for their antimicrobial potential and showed effective antimicrobial activity against tested pathogens and thus applicable as potent antimicrobial agent. Furthermore, the nanoparticles potential has been reconnoitered for their enhanced synergistic effect with antibiotics against multidrug resistant bacteria. Thus, the silver nanoparticles synthesized by Aeromonas sp. THG-FG1.2, were effective in inhibition of pathogenic microbes and also show enhanced antibacterial activity with antibiotics.

Assessment of Chemical Impact of Invasive Bryozoan Pectinatella magnifica on the Environment: Cytotoxicity and Antimicrobial Activity of P. magnifica Extracts.

Pectinatella magnifica, an invasive bryozoan, might significantly affect ecosystem balance due to its massive occurrence in many areas in Europe and other parts of the world. Biological and chemical analyses are needed to get complete information about the impact of the animal on the environment. In this paper, we aimed to evaluate in vitro cytotoxic effects of five extracts prepared from P. magnifica using LDH assay on THP-1 cell line. Antimicrobial activities of extracts against 22 different bacterial strains were tested by microdilution method. Our study showed that all extracts tested, except aqueous portion, demonstrated LD50 values below 100 µg/mL, which indicates potential toxicity. The water extract of P. magnifica with LD50 value of 250 µg/mL also shows potentially harmful effects. Also, an environmental risk resulting from the presence and increasing biomass of potentially toxic benthic cyanobacteria in old colonies should not be underestimated. Toxicity of Pectinatella extracts could be partially caused by presence of Aeromonas species in material, since we found members of these genera as most abundant bacteria associated with P. magnifica. Furthermore, P. magnifica seems to be a promising source of certain antimicrobial agents. Its methanolic extract, hexane, and chloroform fractions possessed selective inhibitory effect on some potential pathogens and food spoiling bacteria in the range of MIC 0.5-10 mg/mL. Future effort should be made to isolate and characterize the content compounds derived from P. magnifica, which could help to identify the substance(s) responsible for the toxic effects of P. magnifica extracts.

Involvement of Acylated Homoserine Lactones (AHLs) of Aeromonas sobria in Spoilage of Refrigerated Turbot (Scophthalmus maximus L.).

One quorum sensing strain was isolated from spoiled turbot. The species was determined by 16S rRNA gene analysis and classical tests, named Aeromonas sobria AS7. Quorum-sensing (QS) signals (N-acyl homoserine lactones (AHLs)) were detected by report strains and their structures were further determined by GC-MS. The activity changes of AHLs on strain growth stage as well as the influence of different culture conditions on secretion activity of AHLs were studied by the punch method. The result indicated that strain AS7 could induce report strains to produce typical phenotypic response. N-butanoyl-dl-homoserine lactone (C4-HSL), N-hexanoyl-dl-homoserine lactone (C6-HSL), N-octanoyl-dl-homoserine lactone (C8-HSL), N-decanoyl-dl-homoserine lactone (C10-HSL), N-dodecanoyl-dl-homoserine lactone (C12-HSL) could be detected. The activities of AHLs were density-dependent and the max secretion level was at pH 8, sucrose culture, 1% NaCl and 32 h, respectively. The production of siderophore in strain AS7 was regulated by exogenous C8-HSL, rather than C6-HSL. Exogenous C4-HSL and C8-HSL accelerated the growth rate and population density of AS7 in turbot samples under refrigerated storage. However, according to the total viable counts and total volatile basic nitrogen (TVB-N) values of the fish samples, exogenous C6-HSL did not cause spoilage of the turbot fillets. In conclusion, our results suggested that QS was involved in the spoilage of refrigerated turbot.

Tandem mass spectrometry determination of the putative structure of a heterogeneous mixture of Lipid As isolated from the lipopolysaccharide of the Gram-negative bacteria Aeromonas liquefaciens SJ-19a.

RATIONALE: We report herein the electrospray ionization mass spectrometry (ESI-MS) and low-energy collision-induced dissociation tandem mass spectrometry analysis (CID-MS/MS) of a mixture of lipid As isolated from the rough lipopolysaccharide (LPS) of the mutant wild strain of the Gram-negative bacteria Aeromonas liquefaciens (SJ-19a, resistant) grown in the presence of phages. The interaction between the phages and the Gram-negative bacteria regulates host specificity and the heterogeneity of the lipid A component of the LPS. METHODS: The heterogeneous mixture of lipid As was isolated by the aqueous phenol method from the LPS of the rough wild strain of Gram-negative bacteria Aeromonas liquefaciens (SJ-19a). Hydrolysis of the LPS was with 1% acetic acid, and purification was by chromatography using Sephadex G-50 and Sephadex G-15. ESI-MS and low-energy CID-MS/MS analyses were performed with a triple-quadrupole (QqQ) and a Fourier transform ion cyclotron resonance (FTICR) mass spectrometer. RESULTS: Preliminary analysis of the lipid As mixture was conducted by ESI-MS in the negative ion mode and the spectrum obtained suggested that the lipid A SJ-19a was composed of a heterogeneous mixture of different lipid A molecules. CID-MS/MS experiments confirmed the identities of the various mono-phosphorylated ß-D-GlcpN-(1→6)-α-D-GlcpN disaccharide entities. This lipid As mixture was asymmetrically substituted with fatty acids such as ((R)-14:0(3-OH)), (14:0(3-(R)-(O-12:0)) and (14:0(3-(R)-O-(14:0)) located on the O-3, O-3', N-2 and N-2' positions, respectively. CONCLUSIONS: Low-energy collision-induced dissociation tandem mass spectrometry in-space (QqQ-MS/MS) and in-time (FTICR-MS/MS) allowed the exact determination of the fatty acid acylation positions on the H2 PO3 →4-O'-ß-D-GlcpN-(1→6)-α-D-GlcpN disaccharide backbones of this heterogeneous mixture of lipid As , composed inter alia of seven different substituted lipid As , formed from the incomplete biosynthesis of their respective LPS.

Identification of threshold body burdens of metals for the protection of the aquatic ecological status using two benthic invertebrates.

In this study accumulated concentrations of As, Cd, Cr, Cu, Ni, Pb and Zn in two benthic invertebrate taxa, Chironomus sp. and Tubificidae are related to ecological responses expressed as changes in macro invertebrate communities. In addition critical body burdens were estimated above which ecological quality was always lower than a certain threshold. Data from existing studies on bioaccumulation of the metals in both taxa were combined with different biological water quality indices. For all metal-endpoint combinations threshold values could be calculated above which ecological quality was always low. Safe threshold body burdens could be estimated for both species for all metals although the data set was more extended for Chrionomus sp. with estimated threshold values being 65, 3.2, 10, 57, 6.5, 73 and 490 µg/g dw for As, Cd, Cr, Cu, Ni, Pb and Zn. This study demonstrated that metal accumulation in resistant species such as chironomids and tubificid worms have the potential to be used as predictors of ecological effects in aquatic ecosystems. However, the estimated threshold values have to be validated and supported by more lines of evidence before they can be used by regulators.

Structural Basis for Action of the External Chaperone for a Propeptide-deficient Serine Protease from Aeromonas sobria.

Subtilisin-like proteases are broadly expressed in organisms ranging from bacteria to mammals. During maturation of these enzymes, N-terminal propeptides function as intramolecular chaperones, assisting the folding of their catalytic domains. However, we have identified an exceptional case, the serine protease from Aeromonas sobria (ASP), that lacks a propeptide. Instead, ORF2, a protein encoded just downstream of asp, appears essential for proper ASP folding. The mechanism by which ORF2 functions remains an open question, because it shares no sequence homology with any known intramolecular propeptide or other protein. Here we report the crystal structure of the ORF2-ASP complex and the solution structure of free ORF2. ORF2 consists of three regions: an N-terminal extension, a central body, and a C-terminal tail. Together, the structure of the central body and the C-terminal tail is similar to that of the intramolecular propeptide. The N-terminal extension, which is not seen in other subtilisin-like enzymes, is intrinsically disordered but forms some degree of secondary structure upon binding ASP. We also show that C-terminal (ΔC1 and ΔC5) or N-terminal (ΔN43 and ΔN64) deletion eliminates the ability of ORF2 to function as a chaperone. Characterization of the maturation of ASP with ORF2 showed that folding occurs in the periplasmic space and is followed by translocation into extracellular space and dissociation from ORF2, generating active ASP. Finally, a PSI-BLAST search revealed that operons encoding subtilases and their external chaperones are widely distributed among Gram-negative bacteria, suggesting that ASP and its homologs form a novel family of subtilases having an external chaperone.

Fatty acid profiles of Vibrio parahaemolyticus and its changes with environment.

The fatty acid (FA) profiles of 15 strains representing four genera (Vibrio, Pseudomonas, Aeromonas, and Shewanella) and seven species were compared by capillary gas chromatography with flame ionization detection. FA fingerprints of Vibrio parahaemolyticus were established by similarity calculation and principle component analysis. This provided a simple measure for distinguishing V. parahaemolyticus from other bacteria. The similarity scores indicated by correlation coefficient and vector cosine were divided into three regions and suggested that the strains with scores higher than 0.980 probably belonged to V. parahaemolyticus. However, samples with low scores (<0.850) were classified under other genera. Furthermore, the alterations in FA profiles of V. parahaemolyticus in the presence of various environmental pressures were investigated. The production of saturated FA (SFA) increased gradually concomitant with a decreased proportion of unsaturated FA (UFA) with rising temperature. Similarly, the SFA tended to increase at the expense of UFA with prolonged culture time. In addition, V. parahaemolyticus changed its FA profiles to contain increased short-chained FA to resist an acidic environment, whereas alkaline conditions stimulated high production of long-chained FA. Analysis on FA profile is valuable for the physiological study of V. parahaemolyticus and its rapid identification.

Structure of the O-specific polysaccharide from the lipopolysaccharide of Aeromonas sobria strain Pt312.

The O-specific polysaccharide (OPS) obtained by mild-acid degradation of the lipopolysaccharide from Aeromonas sobria strain Pt312 was studied by sugar and methylation analyses along with 1H and 13C NMR spectroscopy, including 2D 1H,1H COSY, TOCSY, NOESY, 1H-detected 1H,13C HSQC, and HMBC experiments. The sequence of the sugar residues was determined using 1H,1H NOESY and 1H,13C HMBC experiments. It was found that the OPS was built up of disaccharide repeating units composed of GlcpNAc and non-stoichiometrically O-acetylated Rhap residues, and had the structure.

Evaluation of the MALDI-TOF MS profiling for identification of newly described Aeromonas spp.

The genus Aeromonas comprises primarily aquatic bacteria and also serious human and animal pathogens with the occurrence in clinical material, drinking water, and food. Aeromonads are typical for their complex taxonomy and nomenclature and for limited possibilities of identification to the species level. According to studies describing the use of MALDI-TOF MS in diagnostics of aeromonads, this modern chemotaxonomical approach reveals quite high percentage of correctly identified isolates. We analyzed 64 Aeromonas reference strains from the set of 27 species. After extending the range of analyzed Aeromonas species by newly described ones, we proved that MALDI-TOF MS procedure accompanied by Biotyper tool is not a reliable diagnostic technique for aeromonads. We obtained quite high percentage of false-positive, incorrect, and uncertain results. The identification of newly described species is accompanied with misidentifications that were observed also in the case of pathogenic aeromonads.

Depolymerase improves gentamicin efficacy during Klebsiella pneumoniae induced murine infection.

BACKGROUND: Presence of capsule enhances the virulence of bacteria that cause pneumonia, meningitis, cystic fibrosis, dental caries, periodontitis. Capsule is an important virulence factor for Klebsiella pneumoniae and infections due to this pathogen have been associated with high mortality rates. In the present study, use of an Aeromonas punctata derived capsule depolymerase against K. pneumoniae, to reinstate the efficacy of gentamicin during pneumonia and septicemia was investigated. METHODS: Depolymerase was administered in mice intraperitoneally (50 µg) alone as well in combination with gentamicin (1.5 mg/kg), 24 h post infection during acute lung infection and 6 h later during septicemia. Bacterial load, neutrophil infiltration and cytokine levels were estimated. The immunogenicity of protein was also studied. RESULTS: In comparison to groups treated with gentamicin alone, combination treatment with depolymerase and gentamicin significantly reduced (P < 0.01) bacterial titer in the lungs, liver, kidney, spleen and blood of experimental animals. Highly significant reduction in neutrophil infiltration and levels of pro-inflammatory and anti-inflammatory cytokines was also observed. This indicated an efficient capsule removal by the enzyme, that improved gentamicin efficacy in vivo. Although the enzyme was found to be immunogenic, but no significant reduction in treatment efficacy was observed in the preimmunized as well as naïve mice. In addition, as confirmed through flow cytometry, the hyperimmune sera raised against the enzyme did not neutralize its activity. CONCLUSION: The results confirm that administration of enzyme 'depolymerase' along with gentamicin not only checked the virulence of K. pneumoniae in vivo but it also increased its susceptibility to gentamicin at a lower concentration. Such a strategy would help to avoid exposure to higher concentration of gentamicin. Moreover, since this decapsulating protein does not possess a lytic activity therefore there would be no chances of development of bacterial resistance against it. Therefore, it should be studied further for its successful inclusion in our prophylactic/therapeutic regimes.

Matrix-assisted laser desorption ionization-time of flight mass spectrometry can accurately differentiate Aeromonas dhakensis from A. hydrophila, A. caviae, and A. veronii.

Among 217 Aeromonas isolates identified by sequencing analysis of their rpoB genes, the accuracy rates of identification of A. dhakensis, A. hydrophila, A. veronii, and A. caviae were 96.7%, 90.0%, 96.7%, and 100.0%, respectively, by the cluster analysis of spectra generated by matrix-assisted laser desorption ionization-time of flight mass spectrometry.