Carbon : nitrogen : phosphorus ratios influence biofilm formation by Enterobacter cloacae and Citrobacter freundii

AIMS: To test the effects of C : N : P ratio modification of a well-known nutrient medium formulation, the Endo formulation on biofilm formation by Enterobacter cloacae Ecl and Citrobacter freundii Cf1 in both single-species and binary species biofilms. METHODS AND RESULTS: The C : N : P atom : atom ratio of a well-known nutrient medium formulation, the Endo formulation, that has been applied in fermentative biohydrogen studies, was modified to include two different C concentrations, one containing 17.65 g l(-1) and the other 8.84 g l(-1) sucrose, each containing four different C : N : P ratios, two at higher C : N : P ratios (334 : 84 : 16.8 and 334 : 84 : 3) and two at lower C : N : P ratios (334 : 28 : 5.6 and 334 : 28 : 1). Attached cells were enumerated after dislodging the biofilms that had formed on granular activated carbon (GAC). The modified medium containing 17.65 g l(-1) sucrose and having a C : N : P ratio of 334 : 28 : 5.6 resulted in significantly (P < 0.05) higher counts of attached cells for both single-species biofilms at 7.73 log(10) CFU g(-1) GAC and 9.3 log(10)CFU g(-1) GAC for Ent. cloacae Ecl and Cit. freundii Cf1, respectively, and binary species biofilms at 8.2 log(10) CFU g(-1) GAC and 6.34 log(10) CFU g(-1) GAC for Ent. cloacae Ecl and Cit. freundii Cf1, respectively. Scanning electron micrographs showed qualitative evidence that the 334 : 28 : 5.6 ratio encouraged more complex and extensive biofilm growth for both single-species and binary species biofilms. CONCLUSIONS: The differences in the attachment numbers between the different ratios were found not to be a result of the individual actions of the bacterial isolates involved but rather because of the effects of the various C : N : P ratios. The 334 : 28 : 5.6 ratio showed significantly (P < 0.05) higher counts of attached cells for both single-species and binary species biofilms. SIGNIFICANCE AND IMPACT OF THE STUDY: This study indicates that C : N : P ratios should be a key consideration with regard to maximizing biofilm formation in shake flask and fluidized bed bioreactor studies as well as understanding fundamental factors affecting biofilm growth in natural environments.     

Inhibitory activity of natural antimicrobial compounds alone or in combination with nisin against Enterobacter sakazakii

Aim: To determine the antimicrobial activity of natural organic compounds alone and in combination with nisin on the growth of Enterobacter sakazakii in laboratory media. Methods and Results: The minimum inhibitory concentrations (MIC) of five natural organic compounds were determined, and their effects in combination with nisin were evaluated by comparing treatment with each natural organic compound alone and in combination with 25 mg ml^?1 nisin in tryptic soy broth. Among the tested natural organic compounds, the MIC of carvacrol and thymol was 1·25 mmol l^?1 and showed the strongest inhibitory activity against E. sakazakii, whereas the MIC of cinnamic acid was higher than 5 mmol l^?1, and therefore showed the weakest inhibitory activity. However, the combination of each compound with nisin did not result in the enhancement of their antimicrobial activities except when nisin was combined with diacetyl. Conclusions: The order of inhibition attributed to natural organic compounds was carvacrol = thymol > eugenol > diacetyl > cinnamic acid, and only the combination of diacetyl and nisin showed a synergistic effect of inhibiting the growth of E. sakazakii. Significance and Impact of the Study: This study shows the potential of natural organic compounds for controlling E. sakazakii.     

Bioproduction of D-Psicose from Allitol with Enterobacter aerogenes IK7: A New Frontier in Rare Ketose Production

D-Psicose, a new alternative sweetener, was produced from allitol by microbial oxidation of the newly isolated strain Enterobacter aerogenes IK7. Cells grown in tryptic soy broth medium (TSB) supplemented with D-mannitol at 37 °C were found to have the best oxidation potential. The cells, owing to broad substrate specificity, oxidized various polyols (tetritol, pentitol, and hexitol) to corresponding rare ketoses. By a resting cell reaction, 10% of allitol was completely transformed to the product D-psicose, which thus becomes economically feasible for the mass production of D-psicose. Finally, the product was crystallized and confirmed to be D-psicose by analytical methods.     

Electrophoretic Studies of Alkaline Proteinases from Strains of Aspergillus flavus Group

Entevobacter sp. G-1 which produces chitinolytic and chitosanolytic enzymes, was previously isolated in our laboratory. One major chitinase, designated ChiA, was purified 42.9-fold from a culture filtrate of Entevobacter sp. G-L To purify the chitinase, ammonium sulfate fractionation, DEAE-Sephadex A-50 column chromatography, and gel filtration on Sephadex G-100 column chromatography were used. The ChiA protein had a molecular weight of 60,000 estimated by SDS polyacrylamide gel electrophoresis and an isoelectric point of 6.6. The optimal pH and optimal temperature of ChiA against colloidal chitin were pH 7.0, and 40°C, respectively. The purified ChiA degraded colloidal chitin mainly to GlcNAc₂ with a small amount of GlcNAc₃ and GlcNAc₄. ChiA hydrolyzed flaked chitin, colloidal chitin, and ethylenglycol chitin, but did not hydrolyze carboxymethyl cellulose (CMC), nor >90% deacetylated flaked chitosan. The chitinase activity was 42% inhibited by 10mm EDTA, but was not inhibited by Ca²⁺ (<50 mm) or NaCl (<400 mm). The purified ChiA hydrolyzed colloidal chitin and chitin-related compounds in an endo splitting manner.     

Polymerase chain reaction amplification length-dependent ethidium monoazide suppression power for heat-killed cells of Enterobacteriaceae

The polymerase chain reaction (PCR) can confirm the presence of bacteria, but it is unable to differentiate between live and dead bacteria. Although ethidium monoazide (EMA)- and propidium monoazide (PMA)-based PCR have been evaluated, a quantity of ≥ 10(3)cells/ml dead cells produces a false-positive reading at 40 to 50 cycles (K. Rudi et al., Appl. Environ. Microbiol. 71 (2005) 1018-1024). After confirming the precision of real-time PCR of a long DNA target (16S or 23S ribosomal RNA [rRNA] gene, 1490 or 2840 bp), we evaluated the degree of suppression of an EMA treatment on the 16S/23S PCR using various amplification lengths (110-2840 bp) with heat-killed cells of Enterobacteriaceae (e.g., Salmonella enteritidis). We found that the inhibition rate was proportional to the PCR amplification length; short DNA (110 bp) amplification slightly delayed the threshold cycle (C(T)) of heat-killed cells of Enterobacteriaceae when compared with no EMA treatment. Regardless of the amplification length, the C(T) delay using live cells of Enterobacteriaceae with EMA was negligible. Thus, our real-time PCR of a long DNA (16S or 23S) template following EMA treatment is a rapid viable bacterial assay, which can potentially target all genera, for testing pasteurized milk that may have originally been contaminated with high levels of dead bacteria.     

庆大霉素耐药阴沟肠杆菌16S rRNA甲基化酶基因的研究

目的了解庆大霉素耐药阴沟肠杆菌中16S rRNA甲基化酶基因的分布情况。方法筛选临床分离的庆大霉素耐药的阴沟肠杆菌30株。采用聚合酶链反应（PCR）方法扩增16S rRNA甲基化酶五种相关耐药基因armA、rmtA、rmtB、rmtC和rmtD,PCR产物进行电泳分析和测序,抗菌药物的药物敏感性试验采用纸片扩散法进行,Whonet 5.4软件进行数据分析。结果 30株庆大霉素耐药阴沟肠杆菌中16S rRNA甲基化酶基因的检出率为56.7%（17/30）,其中30.0%（9/30）检出armA基因、26.7%（8/30）检出rmtB基因,未检出rmtA、rmtC和rmtD基因。30株庆大霉素耐药阴沟肠杆菌对妥布霉素、阿米卡星耐药率分别为93.3%,83.3%;对其他抗菌药物除亚胺培南较敏感外,其余抗菌药物耐药率大于60.0%。结论庆大霉素耐药阴沟肠杆菌中16S rRNA甲基化酶基因主要是armA和rmtB基因,碳青霉烯类对该菌有较好体外抗菌活性。     

Development of sandwich enzyme-linked immunosorbent assay for the detection of Cronobacter muytjensii (formerly called Enterobacter sakazakii)

This study aimed to produce a polyclonal antibody against Cronobacter muytjensii (C. muytjensii, formerly called Enterobacter sakazakii) and to develop an immunoassay for its detection. The optimum production of rabbit anti-C. muytjensii immunoglobulin G (IgG) and chicken anti-C. muytjensii IgY was reached in weeks 8 and 9, respectively. Purification of rabbit anti-C. muytjensii IgG from immunized rabbit sera was accomplished using the caprylic acid and ammonium sulfate precipitation method. As a result, sodium dodecyl sulfate-polyacrylamide gel electrophoresis produced two bands around 25 and 50 kDa, corresponding to a light and a heavy chain, respectively. The optimized conditions for sandwich enzyme-linked immunosorbent assay were using rabbit anti-C. muytjensii IgG (1 μg/mL) as a detection antibody and chicken anti-C. muytjensii IgY (10 μg/mL) as a capture antibody. In this assay, no cross-reactivity was observed with the other genera of pathogenic bacteria tested, which included Escherichia coli O157:H7, Salmonella typhimurium, Staphylococcus aureus, Bacillus cereus and Listeria monocytogenes. The developed assay did not show cross-reactivity with other tested species of Cronobacter and Enterobacter genera such as C. turicensis, C. sakazakii, E. aerogenes, E. pulveris and E. helveticus. The detection limit of sandwich ELISA for C. muytjensii was found to be 2.0 × 10(4) colony forming units (CFU)/mL. In addition, detection of C. muytjensii in infant formula powder showed a low matrix effect on the detection curve of sandwich ELISA for C. muytjensii, the detection limit being found to be 6.3 × 10(4) CFU/mL. These findings demonstrate that the developed method is able to detect all strains of C. muytjensii. Hence, this ELISA technique has potent application for the rapid and accurate detection of C. muytjensii in dietary foods.     

Occurrence of Cronobacter spp. in retail foods

Aims: To study the occurrence of Cronobacter spp. in foods and to investigate the phenotypic properties of the strains isolated. Methods and Results: A total of 53 strains of Cronobacter spp. isolated from 399 food samples were identified using conventional biochemical methods and MALDI‐TOF mass spectrometry. Foods of plant origin were the most frequently contaminated samples. No Cronobacter spp. were found in infant milk formula, wheat‐based infant food, pasteurized and raw cow milk, mincemeat, chicken, chickpea and potato dumpling powder. The individual species were identified as Cronobacter sakazakii (54·7%), Cronobacter malonaticus (28·4%), Cronobacter dublinensis (7·5%), Cronobacter muytjensii (7·5%) and Cronobacter turicensis (1·9%). Cronobacter sakazakii and C. malonaticus belong to biotype 1, 2, 2a, 3, 4 and 5, 5a, respectively. Cronobacter dublinensis strains were subdivided into biotypes 6 and 12. All strains were resistant to erythromycin and two of them were resistant to both erythromycin and tetracycline. Conclusions: Cronobacter spp. were isolated from various food samples pre‐eminently of plant origin and dried food ingredients. Significance and Impact of the Study: These findings will increase and detail our knowledge of the presence and diversity of Cronobacter spp. in foods.     

Biodegradation Kinetics of Four Substituted Chlorobenzoic Acids by Enterobacter aerogenes

Enterobacter aerogenes is generally found in soil, sewage plants, and human gastrointestinal tract. Thus, this study was conducted to evaluate the ability of Enterobacter aerogenes to degrade four chlorobenzoic acid compounds (2-chlorobenzoic acid (2-CBA), 3-chlorobenzoic acid (3-CBA), 4-chlorobenzoic acid (4-CBA), and 3,4-dichlorobenzoic acid (3,4-dCBA)) in minimal salt medium. Enterobacter aerogenes was partially able to degrade and dechlorinate these CBAs at concentration of 3.5 mM within 72 h of incubation. According to Haldane single-substrate model, the values of maximum predicted growth rate (μ_max), half saturation constant (K _s), and inhibition constant (K _i) fell in the range of 0.2–0.8 h^?1, 8–41 mM, and 5–53 mM, respectively. Based on the estimated values of both α, a growth-associated constant, and β, a non–growth-associated constant, the production of chloride was predominantly growth associated, since negligible values of the β were determined. Haldane model gave a good prediction of the CBA substrate utilization and degradation, and was in a very good agreement with the experimental data. Because of the capability of Enterobacter aerogenes to utilize these aromatic compounds as carbon and energy sources, this microorganism can be a valuable and promising candidate for use in the biotreatment of wastewater and soil samples contaminated with mixtures of chlorobenzoates.     

E.aero高产1.3-丙二醇菌株发酵条件的研究(Ⅱ)

建立了1.3-丙二醇高产菌株(Enterobacter aerogenes简写为E.aero-N-56)1.3-PD厌氧发酵最适pH值、温度、时间、接种量分别为7.0、30℃、48h、9％;在最适发酵条件下,30L发酵罐中E.aero-N-56菌株1.3-PD产量为47.36g/L,生产率为23.68g/L·d。