Coaggregation among nonflocculating bacteria isolated from activated sludge

Thirty-two strains of nonflocculating bacteria isolated from sewage-activated sludge were tested by a spectrophotometric assay for their ability to coaggregate with one other in two-membered systems. Among these strains, eight showed significant (74 to 99%) coaggregation with Acinetobacter johnsonii S35 while only four strains coaggregated, to a lesser extent (43 to 65%), with Acinetobacter junii S33. The extent and pattern of coaggregation as well as the aggregate size showed good correlation with cellular characteristics of the coaggregating partners. These strains were identified by sequencing of full-length 16S rRNA genes. A. johnsonii S35 could coaggregate with strains of several genera, such as Oligotropha carboxidovorans, Microbacterium esteraromaticum, and Xanthomonas spp. The role of Acinetobacter isolates as bridging organisms in multigeneric coaggregates is indicated. This investigation revealed the role of much-neglected nonflocculating bacteria in floc formation in activated sludge.     

Effect of surfactants on stability of Acinetobacter johnsonii S35 and Oligotropha carboxidovorans S23 coaggregates

The effect of anionic (sodium dodecyl sulphate or SDS) and cationic (cetyltrimethylammonium bromide or CTAB) surfactants on the stability of binary bacterial coaggregates comprising Acinetobacter johnsonii S35 and Oligotropha carboxidovorans S23 (both sewage sludge isolates) was studied and compared with that on the complex sewage sludge flocs. Both SDS and CTAB enhanced the bacterial coaggregation at their lower concentrations of 0.2 and 0.07 mg ml(-1), respectively. However, complete deflocculation of coaggregates was observed at 1 mg ml(-1) SDS and 0.3 mg l(-1) CTAB concentrations. Further, sewage sludge flocs did not deflocculate in the presence of CTAB, although a concentration-dependent deflocculation was observed in the presence of SDS. A. johnsonii S35 and O. carboxidovorans S23 cells were separately pretreated (prior to coaggregation) with the surfactants. In spite of the partial (complete) loss of viability during SDS (CTAB) pretreatment, washed cells still retained hydrophobic character and displayed significant coaggregation (aggregation index ranging from 84% to 97% in comparison to 96% in the case of non-treated cells), demonstrating reversibility of the surfactant induced deflocculation. Further, when exposed to lower concentration of surfactants (0.2 mg ml(-1) SDS), coaggregates were more resistant (76% viability) as compared to the individual partner (S35: 52%; S23: 39% viability). Since the coaggregates are stable and provide protection from surfactants at lower concentrations (those normally expected in the sewage treatment plants), their presence as well as a sustained role in the sewage sludge bioflocculation is evident.     

Novel coaggregating microbial consortium: testing strength for field applications

Acinetobacter johnsonii S35 has an ability to coaggregate with Oligotropha carboxidovorans S23 when grown with favorable carbon substrate and nutrient-rich growth media. However, nutrient-deficient situations or poorly accessible carbon substrates in waste streams could trigger changes in the cell surface properties and thus modify the coaggregation behavior. Thus, in the present study, we test the stability of these novel interactions under various stresses that might transpire in the waste streams. The effect of mineral-component deficiency and shortage of substrate on the growth, cell surface hydrophobicity and coaggregation ability of S35 and O. carboxidovorans S23 has been investigated. As compared to the control, growth of S35 was adversely affected in mineral-deficient medium; the cells had lower hydrophobicity but displayed only slightly lower aggregation index (AI, 75-84%) as compared to control (92%). S35 grown on marginally supplemented Sterile Filtrate of Activated Sludge (SFAS) could also grow well and form larger stable coaggregates with S23. S35 grown with mineral-deficient medium and with the supplemented SFAS still posses the ability to coaggregate with S23. Hence the coaggregation ability of S35 strain is constitutive and is not significantly affected by environmental factors. It would be desirable to work out suitable formulations of such robust and environmentally relevant microbes.     

Effect of cultivation time and growth medium on the coaggregation capability of <Emphasis Type="Italic">Acinetobacter johnsonii</Emphasis> S35

The effects of different cultivation periods and growth media on the cell surface hydrophobicity and coaggregation ability of Acinetobacter johnsonii S35 with Oligotropha carboxidovorans S23 (both sewage sludge isolates) were examined. It was observed that when cultivated in polypeptone medium, A. johnsonii S35 shows high hydrophobicity and strong coaggregation (98% aggregation index) with O. carboxidovorans S23, which remain unaltered after a 1-day, 3-day or 7-day cultivation period. When A. johnsonii S35 was cultivated with different carbon substrates, large coaggregates and a high aggregation index were observed using acetate-grown cells (96%), followed by ethanol-(83%) and vegetable oil-grown cells (72%). Coaggregates were small and narrow in ethanol-grown cells and still smaller in case of the vegetable oil-grown cells. Although no direct correlation could be observed, more hydrophobic cells showed a higher aggregation index. None of the culture supernatants of A. johnsonii S35 showed emulsification of hexadecane or a significant ability to aggregate O. carboxidovorans S23 cells, indicating that the coaggregation factor of A. johnsonii S35 is cell-bound. These results demonstrate that the coaggregation ability of A. johnsonii S35 is constitutive and is not lost under diverse cultivation conditions.     

PCR differentiation of seventeen genospecies of Acinetobacter

Abstract In the present study, strains of 17 reference Acinetobacter genospecies were investigated by using the polymerase chain reaction (PCR). We used primers to amplify spacer regions between the 16S and 23S genes in the prokaryotic rRNA genetic loci. When the spacer amplification products were resolved by electrophoresis, the resulting patterns could be used to distinguish all of the tested acinetobacters into 15 groups. The genospecies 5 ( Acinetobacter junii ), 7 ( Acinetobacter johnsonii ) and 10 produced the same characteristic PCR patterns, suggesting the identity of these three genospecies. A preliminary evaluation of the proposed scheme for PCR diagnostics was carried out. Using the proposed scheme, tested clinical strains were identified correctly to the genospecies level, and the identifications confirmed by conventional biochemical tests. On the basis of our results, PCR amplification of the 16S–23S spacer region shows significant promise as a tool for the simple identification of genospecies belonging to Acinetobacter sp. The nucleotide sequences of our primers are sufficiently highly conserved among these organisms as to permit PCR reactions to be carried out with a single set of reaction conditions and amplification parameters irrespective of species or genus.     

Involvement of Acinetobacter sp. in the floc-formation in activated sludge process

The coaggregation behavior of Acinetobacter johnsonii S35 isolate with sewage bacteria was assessed by a spectrophotometric assay using different samples from a municipal wastewater treatment plant and a community plant. A. johnsonii S35 coaggregated well with other free bacteria and microflocs at the mixing ratios of 0.2:1-0.6:1 of A. johnsonii S35 and sewage samples. In addition, the size of coaggregates became larger (100 μm or more) under the same conditions. A. johnsonii S35 cells were highly adsorbed (adsorption=93-99%) onto sludge samples. Microbial adhesion to hydrocarbon (MATH) test and adsorption to octyl-Sepharose CL-4B showed that A. johnsonii S35 cells and sludge samples had a hydrophobic character. The population of Acinetobacter spp. in sewage treatment plants was 2-7% and its role in bioflocculation was discussed. The present study revealed that A. johnsonii S35 isolate can play as a bridging organism and contribute in floc-formation in activated sludge process.     

Pair-dependent co-aggregation behavior of non-flocculating sludge bacteria

Two strains of non-flocculating sewage sludge bacteria (Xanthomonassp. S53 and Microbacterium esteraromaticum S51) showed 91% and 77% co-aggregation, respectively, with Acinetobacter johnsonii S35 using a spectrophometric assay. The co-aggregates in case of Xanthomonas sp. S53 and A. johnsonii S35 were above 100 m and stable against EDTA (2 mM) and a commercial protease (0.2 mg ml^–1). Protease/periodate pretreatment of the partners did not affect this co-aggregation. On the other hand, co-aggregates of M. esteraromaticum S51 and A. johnsonii S35 (50–70 m) were deflocculated by EDTA or protease. Protease pretreatment of M. esteraromaticum S51 and periodate pretreatment of A. johnsonii S35 prevented their co-aggregation with respective untreated partners. The potential co-aggregation mechanisms of A. johnsonii S35 varied depending upon the other partner involved.     

Genetic diversity of endophytic diazotrophs of the wild rice, Oryza alta and identification of the new diazotroph, Acinetobacter oryzae sp. nov

Thirty-three endophytic diazotrophs were isolated from surface-sterilized leaves, stem, and roots of wild rice Oryza alta. The SDS-PAGE profile of total protein and insertion sequence-based polymerase chain reaction (IS-PCR) fingerprinting grouped the isolates into four clusters (I-IV). The 16S rRNA gene sequence homology of the representative strains B21, B31, B1, and B23 of clusters I, II, III, and IV were assigned to Pseudomonas oleovorans (99.2% similarity), Burkholderia fungorum (99.4% similarity), Enterobacter cloacae (98.9% similarity), and Acinetobacter johnsonii (98.4% similarity), respectively. The results showed wide genetic diversity of the putative diazotrophic strains of the wild rice, O. alta, and the strains of cluster IV are the first report of nitrogen-fixing Acinetobacter species. The cell size, phenotypic characters, total protein profile, genomic DNA fingerprinting, DNA-DNA hybridization, and antibiotic resistance differentiated strain B23(T) from its closest relatives A. johnsonii LMG999(T) and Acinetobacter haemolyticus LMG996(T). The DNA-DNA hybridization also distinguished the strain B23(T) from the closely related Acinetobacter species. Based on these data, a novel species, Acinetobacter oryzae sp. nov., and strain B23(T) (=LMG25575(T)?=?CGMCC1.10689(T)) as the type strain were proposed.     

ARDRA联合RAPD对不动杆菌基因型鉴定的研究

收集多重耐药的不动杆菌10株,以标准参照株作对照,采用扩增核糖体DNA限制性酶切(ARDRA)DNA指纹技术联合随机扩增多态性DNA(RAPD)技术对其基因亚型进行分析;以非加权组间平均法(UPG-MA)进行聚类分析。该法可以有效地鉴定不动杆菌基因亚型;并从10株不动杆菌中鉴定出1株琼氏不动杆菌及9株鲍曼不动杆菌。ARDRA联合RAPD基因指纹分型技术有良好的互补性,可准确鉴定不动杆菌基因型。     

Coaggregation between Prevotella nigrescens and Prevotella intermedia with Actinomyces naeslundii strains

Abstract Using a visual coaggregation assay, 43% (6 of 14) of Prevotella nigrescens and 50% (4 of 8) of Prevotella intermedia strains coaggregated with Actinomyces naeslundii strains which represented the six Actinomyces coaggregation groups (A to F). For both species, coaggregation occurred most frequently with A. naeslundii strains from coaggregation groups C, D and E. No coaggregation was observed with Actinomyces israelii , Actinomyces odontolyticus or six oral Streptococcus species. Coaggregation was not inhibited by lactose, saliva or serum. Pretreatment of Prevotella strains with heat, SDS and proteinase K abolished coaggregation when the treated cells were added to untreated Actinomyces strains. The same pretreatment of the Actinomyces strains had no effect on their ability to coaggregate with untreated Prevotella strains. Pretreatment of all coaggregating P. nigrescens strains with trypsin abolished coaggregation, whereas the coaggregation ability of the P. intermedia and Actinomyces strains was resistant to trypsin pretreatment. Pretreatment of the strains of both Prevotella species and the Actinomyces with periodate abolished coaggregation in all cases. These results suggest that the Prevotella strains each possess a protein coaggregation adhesin, which for the P. intermedia strains is resistant to trypsin, that interacts with a non-protein receptor on the A. naeslundii strains.     

Combination of ARDRA and RAPD genotyping techniques in identification of Acinetobacter spp. genomic species

A total of 10 non-repetitive multi-drug-resist-ant Acinetobacter strains were collected. With reference to A. calcoaceticus (ATCC23055), A. baumannii (ATCC19606), A. lwoffii (ATCC17986), and A. junii (NCTC5866), DNA fingerprint technique, amplified ribo-somal DNA restriction analysis (ARDRA), and random amplified polymorphism DNA (RAPD) were carried out to identify the genomic species of Acinetobacter spp. The distances between them were calculated by the unweighted pair group method with arithmetic (UPGMA). Genotypes ofAcinetobacter spp. were effectively classified and an A. junii together with nine A. baumannii isolates was genomically identified. The combination of ARDRA and RAPD DNA-fingerprint technique shows high com-plementarity, and could be a useful tool in Acinetobacter genomic species identification.     

Coaggregation among Nonflocculating Bacteria Isolated from Activated Sludge

Thirty-two strains of nonflocculating bacteria isolated from sewage-activated sludge were tested by a spectrophotometric assay for their ability to coaggregate with one other in two-membered systems. Among these strains, eight showed significant (74 to 99%) coaggregation with Acinetobacter johnsonii S35 while only four strains coaggregated, to a lesser extent (43 to 65%), with Acinetobacter junii S33. The extent and pattern of coaggregation as well as the aggregate size showed good correlation with cellular characteristics of the coaggregating partners. These strains were identified by sequencing of full-length 16S rRNA genes. A. johnsonii S35 could coaggregate with strains of several genera, such as Oligotropha carboxidovorans, Microbacterium esteraromaticum, and Xanthomonas spp. The role of Acinetobacter isolates as bridging organisms in multigeneric coaggregates is indicated. This investigation revealed the role of much-neglected nonflocculating bacteria in floc formation in activated sludge.     

Identification of New Delhi metallo-β-lactamase gene (NDM-1) from a clinical isolate of Acinetobacter junii in China

New Delhi metallo-β-lactamase-1 (NDM-1) is a novel type of metallo-β-lactamase (MBL) responsible for bacterial resistance to β-lactam antibiotics. Acinetobacter junii was previously shown to possess a MBL phenotype; however, the genes responsible for this phenotype were not identified. In this study, we reported the identification of NDM-1 gene in a clinical isolate of A. junii from a child patient in China, which was resistant to all β-lactams except aztreonam but sensitive to aminoglycosides and quinolones. The cloned NDM-1 gene contained an open reading frame of 813 bp and had a nucleotide sequence 99.9% identical (812/813) to reported NDM-1 genes carried by Acinetobacter baumannii , Enterococcus faecium , Escherichia coli , and Klebsiella pneumoniae . Recombinant NDM-1 protein was successfully expressed in E.?coli BL21, and antibiotic sensitivities of the NDM-1-producing E.?coli were largely similar to the A.?junii 1454 isolate. The findings of this study raise attention to the emergence and spread of NDM-1-carrying bacteria in China.     

琼氏不动杆菌FM 208850产果胶酶的发酵条件优化

目的:优化琼氏不动杆菌FM208850发酵生产果胶酶的培养基组成及发酵条件,以提高其产量。方法:在研究碳源、氮源、无机盐的种类及量的单因素实验基础上,选取香蕉皮、牛肉膏、NaCl和CaCl2做4因素3水平的正交试验。结果:FM208850产果胶酶的最适培养基组成为:香蕉皮2%,牛肉膏0.25%,NaCl 0.2%,CaCl20.3%;发酵条件为:初始pH7.0、32℃、2%接种量,培养12h。最优条件下酶活可达160.7U/mL。结论:琼氏不动杆菌FM208850作为新型果胶酶产生菌及罗布麻脱胶菌种,利用香蕉皮作为其发酵产果胶酶的碳源和诱导物是可行的,为其综合利用开辟了新思路。     

Characterization of vaginal lactobacilli coaggregation ability with Escherichia coli

The coaggregation abilities of probiotic strains might enable it to form a barrier that prevents colonization by pathogenic bacteria. In the present study, the characterization of the coaggregation ability of 19 vaginal lactobacilli was studied. Coaggregation ability of all lactobacilli with Escherichia coli ATCC 11229 was positive. Only the highest coaggregation percentage of Lactobacillus acidophilus S1 was obtained with E. coli ATCC 11229 under both aerobic (71%) and anaerobic conditions (62%). The coaggregation abilities of strains occurred higher at acidic pH than at basic pH values. Moreover, the coaggregation abilities of tested strains against E. coli decreased after heat treatment (70 or 85 °C). Also, the relationship between hydrophobicity and coaggregation of strains was found to be significant. The effect of sonication, some enzymes (lipase and pepsin) and sodium periodate on coaggregation ability of L. acidophilus S1, which is one of the highest potentials on coaggregation ability, was investigated. Sodium periodate did not have a significant effect on coaggregation ability of L. acidophilus S1. The sonicated cell showed lower coaggregation than the control, the supernatant fluid of this sonicated cells showed similar coaggregation ability to the control. Coaggregation abilities of bacteriotherapeutic lactobacilli with pathogenic bacteria can be used for preliminary screening in order to identify potentially probiotic bacteria suitable for human use against urogenital tract infections.     

石鲽病原琼氏不动杆菌形态型Ⅰ的鉴定

从由杀鲑气单胞菌(Aeromonas salmonicida)所引起的石鲽(Stone flounder,Kareius bicoloratus L)细菌性败血感染症病(死)鱼肝、脾、肾及肠内容物中,同时检出了作为继发感染的病原菌。经对6株纯培养菌在形态特征、理化特性等方面较系统的表观分类学指征鉴定及代表菌株DNA中G C mol％的测定,表明为琼氏不动杆菌的一个新形态型并定名为琼氏不动杆菌形态型Ⅰ(Acinetobacter junii morphovar Ⅰ);同时,对该菌进行了血清型、对抗菌类药物的敏感性及致病作用等方面的试验,初步表明此6株菌具有同种的表面(K)及同种的菌体(O)抗原,对供试37种抗菌类药物在不同菌株间的敏感及耐药无明显差异,对供试石鲽及牙鲆均具有较强的致病作用。     

鲍曼不动杆菌对碳青霉烯类抗生素的耐药性分析

目的了解鲍曼不动杆菌的耐药情况,并检测耐碳青霉烯类鲍曼不动杆菌的耐药基因,为指导临床合理用药、控制院内感染提供依据。方法利用K-B法检测45株鲍曼不动杆菌临床分离株的耐药情况,通过改良Hodge试验、Carba NP试验和EDTA协同试验对多重耐药鲍曼不动杆菌的碳青霉烯酶进行表型检测,并采用PCR技术检测鲍曼不动杆菌携带OXA-23和NDM-1型耐药基因的情况。结果 45株鲍曼不动杆菌临床分离株中共筛出42株多重耐药菌株;利用改良Hodge试验和Carba NP试验检出36株碳青霉烯酶阳性菌株;采用PCR扩增出OXA-23,未扩增出NDM-1。结论鲍曼不动杆菌耐药情况严重,且耐药基因OXA-23携带率高,治疗时应根据药敏试验结果合理用药。     

Quorum sensing in metal tolerance of Acinetobacter junii BB1A is associated with biofilm production

Acinetobacter junii strain BB1A, a novel metal-tolerant bacterium, produced biofilm in the presence of added ions such as Ni(2+), AsO(2)(-), Cd(2+) and Hg(2+) on surfaces such as glass and polystyrene. Generation of a metal-sensitive and adhesion-deficient mutant by transposition of Tn5-mob in the A. junii genome has putatively confirmed the association of metal tolerance with the production of biofilm. The requirement of a critical cell density for biofilm formation and presence of acyl-homoserine lactone-like autoinducer molecules in the cell-free supernatant indicated the phenomenon of quorum sensing. Addition of a natural quorum-sensing inhibitor (garlic extract) or synthetic quorum-sensing inhibitor (4-nitro-pyridine oxide) significantly inhibited cell growth and biofilm formation in the presence of metal/metalloid ions.     

In vivo characterization of Lactobacillus johnsonii FI9785 for use as a defined competitive exclusion agent against bacterial pathogens in poultry

AIMS: To test the efficacy of Lactobacillus johnsonii FI9785 in reducing the colonization and shedding of Salmonella enterica serotype Enteritidis, Escherichia coli O78:K80 and Clostridium perfringens in poultry. METHODS AND RESULTS: Specific pathogen-free chicks (1 day old) were dosed with a single oral inoculum of 1x10(9) CFU. Lactobacillus johnsonii FI9785 and 24 h later were challenged in separate experiments with S. Enteritidis (S1400, nalr) and E. coli O78:K80 (EC34195, nalr). There were no significant effects against S. Enteritidis whereas colonization of the small intestine by E. coli O78:K80 was reduced significantly. Both S. Enteritidis and E. coli colonized the caeca and colon to levels equivalent to control birds and there was no reduction in shedding as assessed by a semi-quantitative cloacal swabbing technique. Specific pathogen-free chicks (20 day old) were dosed with a single oral inoculum of 1x10(9) CFU L. johnsonii FI9785 and 24 h later were challenged with C. perfringens. A single oral dose of L. johnsonii FI9785 was sufficient to suppress all aspects of colonization and persistence of C. perfringens. CONCLUSIONS: Lactobacillus johnsonii FI9785 may be given to poultry for use as a competitive exclusion agent to control C. perfringens. SIGNIFICANCE AND IMPACT OF THE STUDY: Lactobacillus johnsonii FI9785 may be a valuable tool to control the endemic disease of necrotic enteritis, thereby reducing economic losses associated with reduced use of antimicrobials in the poultry industry.