Evaluation of novel beta-ribosidase substrates for the differentiation of Gram-negative bacteria

AIMS: To synthesize novel substrates for the detection of beta-ribosidase and assess their potential for the differentiation of Gram-negative bacteria. METHODS AND RESULTS: Two novel chromogenic substrates, 3',4'-dihydroxyflavone-4'-beta-D-ribofuranoside (DHF-riboside) and 5-bromo-4-chloro-3-indolyl-beta-D-ribofuranoside (X-riboside) were evaluated along with a known fluorogenic substrate, 4-methylumbelliferyl-beta-D-ribofuranoside (4MU-riboside). A total of 543 Gram-negative bacilli were cultured on media containing either DHF-riboside or X-riboside. Hydrolysis of DHF-riboside or X-riboside resulted in the formation of clearly distinguishable black or blue-green colonies, respectively. Hydrolysis of 4MU-riboside was evaluated in a liquid medium in microtiter trays and yielded blue fluorescence on hydrolysis which was measured using fluorimetry. beta-Ribosidase activity was widespread with 75% of strains, including 85.6% of Enterobacteriaceae, showing activity with at least one substrate. Genera that demonstrated beta-ribosidase activity included Aeromonas, Citrobacter, Enterobacter, Escherichia, Hafnia, Klebsiella, Morganella, Providencia, Pseudomonas, Salmonella and Shigella. In contrast, strains of Proteus spp., Acinetobacter spp., Yersinia enterocolitica, Vibrio cholerae and Vibrio parahaemolyticus generally failed to demonstrate beta-ribosidase activity. CONCLUSIONS: The novel substrates DHF-riboside and X-riboside are effective for the detection of beta-ribosidase in agar-based media and may be useful for the differentiation and identification of Gram-negative bacteria. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report describing the application and utility of chromogenic substrates for beta-ribosidase. These substrates could be applied in chromogenic media for differentiation of Gram-negative bacteria.     

Development of a simple and rapid fluorogenic procedure for identification of vibrionaceae family members

We describe a simple colony overlay procedure for peptidases (COPP) for the rapid fluorogenic detection and quantification of Vibrionaceae from seawater, shellfish, sewage, and clinical samples. The assay detects phosphoglucose isomerase with a lysyl aminopeptidase activity that is produced by Vibrionaceae family members. Overnight cultures are overlaid for 10 min with membranes containing a synthetic substrate, and the membranes are examined for fluorescent foci under UV illumination. Fluorescent foci were produced by all the Vibrionaceae tested, including Vibrio spp., Aeromonas spp., and Plesiomonas spp. Fluorescence was not produced by non-Vibrionaceae pathogens. Vibrio cholerae strains O1, O139, O22, and O155 were strongly positive. Seawater and oysters were assayed, and 87 of 93 (93.5%) of the positive isolates were identified biochemically as Vibrionaceae, principally Vibrio vulnificus, Vibrio parahaemolyticus, Aeromonas hydrophila, Photobacterium damselae, and Shewanella putrefaciens. None of 50 nonfluorescent isolates were Vibrionaceae. No Vibrionaceae were detected in soil, and only A. hydrophila was detected in sewage. The COPP technique may be particularly valuable in environmental and food-testing laboratories and for monitoring water quality in the aquaculture industry.     

Tolerance to acid in pH 5.0-grown organisms of potentially pathogenic Gram-negative bacteria

S.A. NOJOUMI, D.G. SMITH AND R.J. ROWBURY. 1995. A wide range of potentially pathogenic species of Gram-negative bacteria were far more resistant to extreme acidity (pH 2.0–3.5) when cultured at pH 5.0 (habituated to acid) than after pH 7.0 culture. The differences were particularly great for Citrobacter spp., Enterobacter spp., Klebsiella spp. and for Vibrio parahaemolyticus ; substantial habituation was also observed for Proteus mirabilis and Aeromonas formicans but the effect was less marked for Serratia marcescens and Acinetobacter calcoaceticus . Growth at pH 5.0 was substantially poorer than at pH 7.0 for most of the above species and also for Salmonella typhimurium and Salm. enteritidis but phosphate markedly enhanced growth at pH 5.0 for many of these species without affecting growth at pH 7.0.     

Identification of the 4-hydroxycinnamate decarboxylase (PAD) gene of Klebsiella oxytoca

A 7.1-kbp DNA fragment isolated from a wild strain of Klebsiella oxytoca was sequenced, leading to the identification of 10 open-reading frames (ORFs), including a 504-bp Pad gene. The Pad gene of the Gram-negative bacterium was subsequently expressed in Escherichia coli as a chimeric Pad. The deduced amino acid (AA) sequence of the Pad gene from wild-type K. oxytoca showed approximately 50% homology to those of other bacterial PADs from Gram-positive bacilli plus a coccus. These data and a genomic library search of some gamma-proteobacteria, including E. coli and Vibrio sp., indicated that PAD of K. oxytoca is a member of the bacterial PAD family characteristic of Gram-negative bacteria. Using Pad-specific PCR primers designed from the Gram-negative bacterial Pad of K. oxytoca, Pad genes of two further strains of K. oxytoca, another wild isolate and JCM 1665 and two PAD-positive Enterobacter spp. were successfully amplified for specific Pad detection.     

Isolation of Aeromonas spp. from water by using anaerobic incubation

A membrane filtration method incorporating a combination of anaerobic and aerobic incubation has been developed for the enumeration of Aeromonas spp. in drinking water. The use of anaerobic incubation improved the detection of Aeromonas spp. by reducing the growth of nonaeromonads. The confirmation rate of presumptive Aeromonas spp. identified on the initial isolation agar exceeded 92%.     

Isolation of Aeromonas spp. from water by using anaerobic incubation.

A membrane filtration method incorporating a combination of anaerobic and aerobic incubation has been developed for the enumeration of Aeromonas spp. in drinking water. The use of anaerobic incubation improved the detection of Aeromonas spp. by reducing the growth of nonaeromonads. The confirmation rate of presumptive Aeromonas spp. identified on the initial isolation agar exceeded 92%.     

Eradication of methicillin-resistant Staphylococcus aureus with an antiseptic soap and nasal mupirocin among colonized patients – an open uncontrolled clinical trial

Resistance to β-lactam antibiotics in enteric Gram-negative bacilli may be difficult to detect using standard methods of either Kirby-Bauer disc diffusion (KBDD) or broth dilution for minimal inhibitory concentration (MIC). This difficulty is due to genetic differences in resistance determinants, differences in levels of gene expression, and variation in spectra of enzymatic activity against the substrate β-lactams used for susceptibility testing. We have examined 95 clinical isolates reportedly susceptible to ceftazidime and ceftriaxone, as originally determined by either KBDD or MIC methods. The organisms studied here were isolated in 2002 from two pediatric hospital centers (Seattle, USA and Shanghai, China). They belong to the inducible β-lactamase producing Gram-negative bacilli, such as Enterobacter spp., Citrobacter spp., Serratia spp., Morganella spp., Providencia spp., and Proteus vulgaris. A Kirby-Bauer disc approximation (KBDA) method identified inducible phenotypes of third-generation cephalosporin resistance in 76% of isolates, which would otherwise be considered susceptible by standard KBDD methods.     

Production and specificity of poly- and monoclonal antibodies raised against Shewanella putrefaciens

B. FONNESBECH, H. FRØKIAER, L. GRAM AND C. MOSBY JESPERSEN. 1993. Polyclonal antibodies were raised in rabbits and mice against Shewanella putrefaciens. Murine monoclonal antibodies were produced against the type strain (ATCC 8071) as well as wild type strains isolated from fish products. The specificities of four polyclonal and 12 monoclonal antibodies were tested by dot-blotting, an indirect and a competitive ELISA against 16 Gram-negative strains; including six strains of S. putrefaciens and one strain of Pseudomonas rubescens (NC 10695). All polyclonal antibodies reacted strongly with S. putrefaciens and with Ps. rubescens and cross-reacted with the nine other bacteria ( Pseudomonas spp., Aeromonas spp. and Vibrio anguillarum ). The monoclonal antibodies could be divided into three groups with different patterns of specificity. The largest group (8 monoclonal antibodies) reacted strongly with S. putrefaciens and with Ps. rubescens and showed only weak reactions with the other strains. The results confirm that Ps. rubescens should be classified as S. putrefaciens.     

Biochemical characteristics and virulence of environmental group F bacteria isolated in the United States.

Bacteria phenotypically resembling Aeromonas hydrophila, but requiring NaCl for growth, have been isolated form the New York Bight. The bacteria proved to be identical to group F organisms isolated from cases of human diarrhea in Indonesia and Bangladesh. Anaerogenic strains initiated responses in Y-1 tissue culture and rabbit ileal loop, consistent with those associated with cytotoxin- and enterotoxin-producing Aeromonas spp. strains. Separation on the basis of production of gas from glucose by group F strains was correlated with differences in mean guanine-plus-cytosine deoxyribonucleic acid base composition and in deoxyribonucleic acid relative reassociation. Both aerogenic and anaerogenic strains reassociated to a significantly greater extent with Vibrio spp. than with Aeromonas spp. and indeed should be considered a new species of the genus Vibrio.     

SGAP-10C agar for the isolation and quantification of Aeromonas from water

Glutamate starch penicillin (GSP) medium was used for the simultaneous isolation of Pseudomonas and Aeromonas. Modifications to reduce the number of Pseudomonas and background flora and to improve the recovery of Aeromonas from water samples are described. The original medium was modified by adding glucose and ampicillin. The addition of 10 micrograms/l of C-glucose to the medium (SGAP-10C) permitted better recuperation of stressed cells of aeromonads and the ampicillin reduced the numbers of Pseudomonas. The best temperature for the recovery of aquatic aeromonads was 28 degrees C. The recovery of different species of Aeromonas on SGAP-10C was 93%. The selectivity of the medium was validated because 95.5% of 28 colonies tested with an Aeromonas-like morphology belonged to the genus Aeromonas. Moreover, when 45 strains of different genera were cultured on the medium, only Vibrio alginolyticus presented a confusing morphology. When the SGAP-10C was compared with GSP with 45 river samples, the new medium gave a significantly better recovery of Aeromonas spp., especially when large numbers of Pseudomonas spp. were present. SGAP-10C used at 28 degrees C and 48 h was an efficient selective medium for the isolation of Aeromonas from fresh waters.     

SGAP-10C agar for the isolation and quantification of Aeromonas from water

Glutamate starch penicillin (GSP) medium was used for the simultaneous isolation of Pseudomonas and Aeromonas . Modifications to reduce the number of Pseudomonas and background flora and to improve the recovery of Aeromonas from water samples are described. The original medium was modified by adding glucose and ampicillin. The addition of 10 μg/l of C-glucose to the medium (SGAP-10C) permitted better recuperation of stressed cells of aeromonads and the ampicillin reduced the numbers of Pseudomonas . The best temperature for the recovery of aquatic aeromonads was 28°C. The recovery of different species of Aeromonas on SGAP-10C was 93%. The selectivity of the medium was validated because 95·5% of 28 colonies tested with an Aeromonas -like morphology belonged to the genus Aeromonas . Moreover, when 45 strains of different genera were cultured on the medium, only Vibrio alginolyticus presented a confusing morphology. When the SGAP-10C was compared with GSP with 45 river samples, the new medium gave a significantly better recovery of Aeromonas spp., especially when large numbers of Pseudomonas spp. were present. SGAP-10C used at 28°C and 48 h was an efficient selective medium for the isolation of Aeromonas from fresh waters.     

Identification of Vibrio cholerae by Pyrolysis Gas-Liquid Chromatography

Cholera-like vibrios examined by pyrolysis gas-liquid chromatography could be distinguished from other common aerobic gram-negative bacilli, including oxidase-positive organisms, e.g., Aeromonas. Vibrios in Heiberg group I were subdivided into three types on the basis of differences in one complex in the chromatogram, and these closely corresponded with the identification as classical, El Tor, or "intermediate" biotypes of Vibrio cholerae by conventional methods.     

Comparative study of the prevalence and clinical profiles of diarrheas due to Aeromonas and other enteric pathogens

The prevalence of Aeromonas spp. and other enteric pathogens in stool specimens from diarrheic and non-diarrheic patients was studied over a 12 month period (January to December, 1986). Except for the absence of fever, all the clinical features in Aeromonas diarrhea were comparable to those associated with other diarrheagenic agents. These features included abdominal pain (30%), vomiting (24.5%), fever (31.5%), dehydration (9.5%) and hematochezia (19.5%). Aeromonas spp. were more frequently isolated from patients with gastroenteritis (2.5%) than from control patients (1.0%) (P less than 0.05). Isolates were recovered more often during the dry months (66.7%), than during the wet months (33.3%). Among the enteric pathogens isolated, Aeromonas spp. (2.5%) ranked next to Esch. coli (14.5%) and Shigella spp. (6.3%) in prevalence. Other bacterial isolates included Plesiomonas shigelloides (1.5%) Vibrio spp. (1.0%), Yersinia enterocolitica (1.0%) and Salmonella spp. (1.8%).     

Characterization of Aeromonas and Vibrio species isolated from a drinking water reservoir

AIMS: To study the phenotypic and chemotaxonomic (i.e. phospholipid and cellular fatty acid composition) characteristics of environmental Aeromonas spp. and Vibrio spp. isolated from a drinking water reservoir near Vladivostok City, and the application of some chemotaxonomic markers for discrimination of the two genera and species. METHODS AND RESULTS: Presumptive Aeromonas species were dominant in surface water samples (up to 25% of the total number of bacteria recovered). These strains were consistent with respect to the cultural and biochemical properties used to define the species Aeromonas sobria (seven strains) and Aer. popoffii (three strains). Vibrio mimicus (two strains) and Vibrio metschnikovii (one strain) were identified according to phenotypic features and cellular fatty acid composition. CONCLUSION: Environmental Aer. sobria isolates were atypical in their ability to grow at 42 degrees C, and were haemolytic, proteolytic and cytotoxic. Although it was present in a high proportion in the water samples, atypical Aer. sobria is not an indicator of polluted water. SIGNIFICANCE AND IMPACT OF THE STUDY: The incidence of Aeromonas in the drinking water reservoirs in the Far East of Russia is reported for the first time.     

滴水湖沉积物中可培养优势微生物种群初探

于滴水湖湖心采集底泥样品,对底泥中可培养优势菌种进行分离、纯化,并利用Biolog微生物自动分析系统进行鉴定。结果显示,滴水湖沉积物中菌落总数为2.43×104CFU/g,分离纯化后的8株优势菌种中,革兰氏阴性菌占87.5%,其中7株为GN-NENT(革兰氏阴性非肠道菌)、1株为GP-ROD SB(革兰氏阳性芽孢杆菌)。鉴定结果显示,8株菌种分别为:鳗鱼气单孢菌(Aeromonas encheleia)、乙酸钙不动杆菌/基因型1(Acinetobacter calcoaceticus/genospecies1)、舒氏气单胞菌(Aeromonas schubertiiDNA group12)、腐败希瓦氏菌B(Shewanella putrefaciens B)、维罗纳/温和气单胞菌(Aeromonas veronii/sobria DNA group8)、坎氏弧菌(Vibrio campbelli)、蕈状芽孢杆菌(Bacillus mycoides)和梅氏弧菌(Vibrio metschnikovii)。     

Diluent composition for use of API 20E in characterizing marine and estuarine bacteria.

Nine chemically defined inoculation diluents, with compositions ranging from 0.85% NaCl to 35% marine salts, were used to evaluate the influence of diluent composition on the biochemical profiles of 30 marine and estuarine bacterial strains, including species of Vibrio, Aeromonas, Allomonas, and Photobacterium. Results demonstrated that a 20% marine salts diluent enabled the characterization of halophilic strains normally nonreactive by the API 20E system. Furthermore, the use of 20% marine salts showed that certain environmental isolates, identifiable as Vibrio parahaemolyticus by the recommended clinical inoculation procedure, were Vibrio vulnificus. An analysis of the profiles provided by the nine diluents indicates that the API 20E system, modified by the use of a diluent composed of 20% marine salts and incubated at 22 degrees C, can provide a reliable tool for the rapid characterization of marine and estuarine bacterial isolates.     

Taxonomic composition of bacteria associated with cultivated mollusks Crassostrea lugubris and Perna viridis and with the water of the Gulf of Nha Trang Lagoon, Vietnam

One hundred and four strains of heterotrophic bacteria have been isolated and characterized from two species of bivalve mollusks cultivated in the Gulf of Nha Trang (Vietnam) and from the water of a mariculture farm. The isolates have been identified on the basis of morphological, physiological, biochemical, and chemotaxonomic properties, as well as by the content of G+C bases in DNA. In the microflora of mollusks, Vibrio alginolyticus was predominant; the pathogenic species V. harveyi and V. splendidus were found as well. Staphylococci and bacilli occupied the second place in abundance after vibrios. In addition, coryneforms and enterobacteria, as well as Pseudomonas spp. and Pseudoalteromonas spp., were revealed. The composition of the water microflora was more diverse as compared with the microflora of mollusks. In the water, Bacillus spp., Vibrio spp., and Pseudomonas spp. were predominant. Brevibacterium spp. and other coryneform bacteria, as well as enterobacteria, occurred in significant amounts. In addition, Pseudoalteromonas spp., Marinococcus sp., Halobacillus sp., Shewanella sp., Sulfitobacter sp., and bacteria of the CFB cluster were noticed. The presence of pathogenic and conditionally pathogenic bacterial species in the water and mollusks is probably the reason for the high death rate of cultivated animals at the mariculture farm.     

Detection of aerolysin gene in <Emphasis Type="Italic">Aeromonas hydrophila</Emphasis> isolated from fish and pond water

Aerolysin is a hemolytic toxin encoded by aerolysin gene (1482 bp) that plays a key role in the pathogenesis of Aeromonas hydrophila infection in fish. New speciesspecific primers were designed to amplify 326 bp conserved region of aerolysin gene for A. hydrophila. Twenty-five isolates of A. hydrophila recovered from fish and pond water were studied for detection of aerolysin gene. Aerolysin gene was detected in 85% of the isolates during the study. The designed primers were highly specific and showed no cross reactivity with Escherichia coli, Aeromonas veronii, Vibrio cholerae, Flavobacterium spp., Chyseobacterium spp. and Staphylococcus aureus. The sensitivity limit of primers for detection of aerolysin gene in the genomic DNA of A. hydrophila was 5 pg.     

Aeromonas detection and characterization using genus-specific PCR and single-strand conformation polymorphism (SSCP)

Based on sequence alignment, oligonucleotide primers targeting the Aeromonas extracellular lipase gene were developed for PCR detection of member of the genus. A pair of primers designed for conserved regions of the gene amplified a 276?bp sequence in all Aeromonas species and tested strains, but did not have a positive result with other Gram-positive and Gram-negative bacteria, showing high specificity and sensitivity. Selective enrichment in alkaline peptone water, followed by centrifugation, and direct usage of cells suspension as template, detected initial populations of 10?c.f.u.?ml(-1). Single-strand conformation polymorphism analysis of the PCR products allowed the characterization of Aeromonas strains with a high discriminatory power (Simpson's index?=?0.988). The method presented here provides a useful tool for the rapid detection of Aeromonas and the characterization of Aeromonas isolates.     

Diversity and characterization of antagonistic bacteria from tropical estuarine habitats of Cochin, India for fish health management

Mortalities due to pathogenic bacteria are a major problem in aquaculture, especially in larval rearing systems. Use of antibiotics to overcome this problem is not an option any more due to the increasing antibiotic resistance among pathogens. The present study aims to understand the diversity of bacteria with antagonistic properties in the tropical estuarine habitats of Cochin, located along the southwest coast of India, and to use them as an alternative to antibiotics in aquaculture. Among the 4,870 isolates screened, approximately 1 % showed significant antibacterial activity against six common aquaculture pathogens belonging to the genera Aeromonas and Vibrio. The antagonistic bacteria were identified as Bacillus (81 %) and Pseudomonas (19 %) using biochemical and 16S rRNA gene sequence homology. The isolates showing stable and higher levels of antibacterial activity were subjected to enzymatic expression profile, antibiotic resistance pattern and abiotic stress tolerance assays. As a result, five Pseudomonas spp. and four Bacillus spp., were identified as promising antagonistic isolates that could be exploited as probionts or microbial products (MP's), to control bacterial diseases in aquaculture rearing systems.