Screening, characterization and flocculating property of carbohydrate polymer from newly isolated Enterobacter cloacae WD7

A total of 188 carbohydrate polymer-producing bacterial strains were isolated from recycled sludge of five seafood processing plants. Among three selected isolates, identified as Enterobacter cloacae WD7, Enterobacter agglomerans WD50 and Pseudomonas alcaligenes WD22. E. cloacae WD7 generated a viscous culture broth exhibiting the highest flocculating activity and a crude polymer yield of 2.27 g/L after 3 days cultivation. Partial purification of this polymer was performed by precipitation with 95% ethanol, dialysis and freeze-drying. It was characterized as an acidic heteropolysaccharide, composed of neutral sugars (29.4%), uronic acids (14.2%) and amino sugars (0.93%). The functional group analysis by FT-IR spectroscopy showed the presence of hydroxyl, carboxyl, carbonyl and methoxyl groups. Thermal analysis by DSC showed the crystalline transition and the crystalline melting point (T-m) at 300 degrees C. This polysaccharide was soluble in water and insoluble in any organic solvents tested; gelation occurred under alkaline conditions in the presence of divalent cations in which copper as CuSO4 gave the best result. Studies on the flocculation property revealed that this polysaccharide was stable at 4-60 degrees C and pH 5-7. The optimal concentrations for the flocculating activity were 2 mg/L polysaccharide and 40 mM CaCl2 which played the synergistic effect on kaolin flocculation. Moreover, this polysaccharide could flocculate the kaolin suspension over a wide range of pH (pH 2-8) and temperature (4-50 degrees C) tested in the presence of CaCl2. (c) 2006 Elsevier Ltd. All rights reserved.

Genetic characterization of tigecycline resistance in clinical isolates of Enterobacter cloacae and Enterobacter aerogenes

OBJECTIVES:
The intrinsically encoded ramA gene has been linked to tigecycline resistance through the up-regulation of efflux pump AcrAB in Enterobacter cloacae. The molecular basis for increased ramA expression in E. cloacae and Enterobacter aerogenes, as well as the role of AraC regulator rarA, has not yet been shown. To ascertain the intrinsic molecular mechanism(s) involved in tigecycline resistance in Enterobacter spp., we analysed the expression levels of ramA and rarA and corresponding efflux pump genes acrAB and oqxAB in Enterobacter spp. clinical isolates.

METHODS:
The expression levels of ramA, rarA, oqxA and acrA were tested by quantitative real-time RT-PCR. The ramR open reading frames of the ramA-overexpressing strains were sequenced; strains harbouring mutations were transformed with wild-type ramR to study altered ramA expression and tigecycline susceptibility.

RESULTS:
Tigecycline resistance was mediated primarily by increased ramA expression in E. cloacae and E. aerogenes. Only the ramA-overexpressing E. cloacae isolates showed increased rarA and oqxA expression. Upon complementation with wild-type ramR, all Enterobacter spp. containing ramR mutations exhibited decreased ramA and acrA expression and increased tigecycline susceptibility. Exceptions were one E. cloacae strain and one E. aerogenes strain, where a decrease in ramA levels was not accompanied by lower acrA expression.

CONCLUSIONS:
Increased ramA expression due to ramR deregulation is the primary mediator of tigecycline resistance in clinical isolates of E. cloacae and E. aerogenes. However, some ramA-overexpressing isolates do not show changes in ramR, suggesting alternate pathways of ramA regulation; the rarA regulator and the oqxAB efflux pump may also play a role in tigecycline resistance in E. cloacae.

Efecto de las condiciones de fermentación sobre la producción de expolisacárido por una cepa nativa de Enterobacter cloacae

[Tesis] ( Maestría en Ciencias con Especialidad en Microbiología Industrial) U.A.N.L.

Efecto de las variaciones entre las fuentes de carbono y nitrógeno sobre la capacidad de producción de exopolisacáridos por una cepa nativa de enterobacter cloacae.

Tesis (Maestría en Ciencias con Orientación en Microbiología Industrial) UANL, 2010.

Characterisation and clinical features of Enterobacter cloacae bloodstream infections occurring at a tertiary care university hospital in Switzerland: is cefepime adequate therapy?

Despite many years of clinical experience with cefepime, data regarding the outcome of patients suffering from bloodstream infections (BSIs) due to Enterobacter cloacae (Ecl) are scarce. To address the gap in our knowledge, 57 Ecl responsible for 51 BSIs were analysed implementing phenotypic and molecular methods (microarrays, PCRs for bla and other genes, rep-PCR to analyse clonality). Only two E. cloacae (3.5%) were ESBL-producers, whereas 34 (59.6%) and 18 (31.6%) possessed inducible (Ind-Ecl) or derepressed (Der-Ecl) AmpC enzymes, respectively. All isolates were susceptible to imipenem, meropenem, gentamicin and ciprofloxacin. Der-Ecl were highly resistant to ceftazidime and piperacillin/tazobactam (both MIC₉₀≥256 μg/mL), whereas cefepime retained its activity (MIC₉₀ of 3 μg/mL). rep-PCR indicated that the isolates were sporadic, but Ecl collected from the same patients were indistinguishable. In particular, three BSIs initially due to Ind-Ecl evolved (under ceftriaxone or piperacillin/tazobactam treatment) into Der-Ecl because of mutations or a deletion in ampD or insertion of IS4321 in the promoter. These last two mechanisms have never been described in Ecl. Mortality was higher for BSIs due to Der-Ecl than Ind-Ecl (3.8% vs. 29.4%; P=0.028) and was associated with the Charlson co-morbidity index (P=0.046). Using the following directed treatments, patients with BSI showed a favourable treatment outcome: cefepime (16/18; 88.9%); carbapenems (12/13; 92.3%); ceftriaxone (4/7; 57.1%); piperacillin/tazobactam (5/7; 71.4%); and ciprofloxacin (6/6; 100%). Cefepime represents a safe therapeutic option and an alternative to carbapenems to treat BSIs due to Ecl when the prevalence of ESBL-producers is low.

Padrão de sensibilidade de cepas de Enterobacter spp isoladas na região de Presidente Prudente - SP

Enterobacter spp. are considered important causing agents of infection, specially in hospitalized individuals. The natural resistance of these microorganisms and the great facility to develop resistance to new antimicrobial agents make this genus an important object of study. In this work, 176 strains isolated from various clinical samples were used from hospitalized patients (University Hospital Domingos Leonardo Cerávolo) and from clinic patients (Clinical Laboratory from Unoeste), both situated in Presidente Prudente - SP. E. cloacae (78.9%), E. aerogenes (7.9%) and E. (pantoea) agglomerans (3.9%) were the ones more frequently isolated. Eleven antimicrobial agents were tested by the disk diffusion method and around 90% of the strains presented resistance to the cephalotin, ampicillin and cefaclor. Strains of E. (pantoea) agglomerans presented wide profile of sensibility However one strain of E. cloacae presented resistance to all the antimicrobial agents. The antimicrobial agents with greater inhibitory activity were imipenem and cefepime, for this reason these antimicrobial agents could be the treatment of choice in emergencial therapeutic. This emergencial therapy can be applied with relative security, whereas the data obtained in this study show homogeneity in the profile of sensibility to these antimicrobial agents, independent of the infection site and from the isolated species. The ESBL enzyme could not be detected in no one of the strains by the double diffusion test.

Photocatalytic disinfection of bacterial pollutants using suspended and immobilized TiO(2) powders

The photocatalytic disinfection of Enterobacter cloacae and Enterobacter coli using microwave (MW), convection hydrothermal (HT) and Degussa P25 titania was investigated in suspension and immobilized reactors. In suspension reactors, MW-treated TiO(2) was the most efficient catalyst (per unit weight of catalyst) for the disinfection of E. cloacae. However, HT-treated TiO(2) was approximately 10 times more efficient than MW or P25 titania for the disinfection of E. coli suspensions in surface water using the immobilized reactor. In immobilized experiments, using surface water a significant amount of photolysis was observed using the MW- and HT-treated films; however, disinfection on P25 films was primarily attributed to photocatalysis. Competitive action of inorganic ions and humic substances for hydroxyl radicals during photocatalytic experiments, as well as humic substances physically screening the cells from UV and hydroxyl radical attack resulted in low rates of disinfection. A decrease in colony size (from 1.5 to 0.3 mm) was noted during photocatalytic experiments. The smaller than average colonies were thought to occur during sublethal (•) OH and O(2) (•-) attack. Catalyst fouling was observed following experiments in surface water and the ability to regenerate the surface was demonstrated using photocatalytic degradation of oxalic acid as a model test system

Environmental monitoring of bacterial contamination and antibiotic resistance patterns of the fecal coliforms isolated from Cauvery River, a major drinking water source in Karnataka, India

The present study focuses prudent elucidation of microbial pollution and antibiotic sensitivity profiling of the fecal coliforms isolated from River Cauvery, a major drinking water source in Karnataka, India. Water samples were collected from ten hotspots during the year 2011-2012. The physiochemical characteristics and microbial count of water samples collected from most of the hotspots exhibited greater biological oxygen demand and bacterial count especially coliforms in comparison with control samples (p <= 0.01). The antibiotic sensitivity testing was performed using 48 antibiotics against the bacterial isolates by disk-diffusion assay. The current study showed that out of 848 bacterial isolates, 93.51 % (n=793) of the isolates were found to be multidrug-resistant to most of the current generation antibiotics. Among the major isolates, 96.46 % (n=273) of the isolates were found to be multidrug-resistant to 30 antibiotics and they were identified to be Escherichia coli by 16S rDNA gene sequencing. Similarly, 93.85 % (n=107), 94.49 % (n=103), and 90.22 % (n=157) of the isolates exhibited multiple drug resistance to 32, 40, and 37 antibiotics, and they were identified to be Enterobacter cloacae, Pseudomonas trivialis, and Shigella sonnei, respectively. The molecular studies suggested the prevalence of blaTEM genes in all the four isolates and dhfr gene in Escherichia coli and Sh. sonnei. Analogously, most of the other Gram-negative bacteria were found to be multidrug-resistant and the Gram-positive bacteria, Staphylococcus spp. isolated from the water samples were found to be methicillin and vancomycin-resistant Staphylococcus aureus. This is probably the first study elucidating the bacterial pollution and antibiotic sensitivity profiling of fecal coliforms isolated from River Cauvery, Karnataka, India.

Expressão de fatores de virulência, mecanismos de resistência aos agentes antimicrobianos e análise molecular da resistência aos beta-lactâmicos de enterobactérias isoladas de bolsas periodontais

Em estudo anterior, as espécies de enterobactérias apresentando perfis variados de resistência aos antimicrobianos foram detectadas em 20% dos sítios com lesões periodontais de pacientes sadios do ponto de vista sistêmico. Tais cepas microbianas foram submetidas a investigações com o intuito de determinar à expressão de enzimas hidrolíticas para substratos diversos, a multirresistência aos agentes antimicrobianos e os mecanismos de resistência aos antimicrobianos da classe dos β lactâmicos. A maioria das amostras expressou atividade de gelatinase (65%), caseinase (30%) e elastase (10%). Lipase, lecitinase e DNase foram observadas apenas para Serratia marcescens. A multirresistência (considerado como a resistência a pelo menos dois agentes antimicrobianos de famílias diferentes) foi observada em 56% das amostras isoladas. A maioria das cepas foi resistentes à ampicilina (93,75%) e amoxicilina/ácido clavulânico (81,25%). Investigações sobre a resistência aos antibióticos β-lactâmicos mostraram que três amostras resistentes à cefalosporinas de 2 geração, apresentaram perfis plasmidiais de diferentes pesos moleculares. A expressão fenotípica de β-lacatamases, foi detectada nas cepas de Enterobacter cloacae (PcOM46 e PcOM5) e S. marcescens (PcOM63). No entanto, na análise molecular, não foi possível confirmar a expressão fenotípica de diferentes β-lactamases, com exceção do E. cloacae PcOM46, que apresentou amplificação para AmpC e blaTEM. Embora sensível à maioria dos antibióticos β-lactâmicos (exceção feita à ampicilina e amoxicilina / ácido clavulânico), amostra de S. marcescens PcOM68 apresentou amplificação para o gene blaSHV. Os experimentos de conjugação não detectaram a transferência de plasmídios para uma cepa de Escherichia coli K12 sensívei aos β-lactâmicos, o mesmo ocorreu nos procedimentos de transformação por eletroporação e por CaCl2, sugerindo uma resistência dependente de genes cromossomiais. A expressão de diferentes atividades enzimáticas, juntamente com a resistência aos antimicrobianos, aponta estes grupos de bactérias como agentes patogênicos potenciais capazes de contribuir para a patogênese e resposta à quimioterapia antimicrobiana nas doenças periodontais, além da disseminação sistêmica para outros locais do corpo, especialmente em indivíduos imunocomprometidos. A colonização prévia de lesões periodontais por espécies resistentes aos β-lactâmicos, pode contribuir para a disseminação destes genes relacionados à resistência aos antimicrobianos em ambientes hospitalares.

Estudo da resistência aos antimicrobianos em enterobactérias isoladas de mãos de manipuladores de alimentos em cantinas permissionárias de uma universidade no Município do Rio de Janeiro

A contaminação dos alimentos e transmissão de patógenos via manipuladores pode ocorrer se as condições de higiene, armazenamento e preparo não estiverem satisfatórios. Procedimentos inadequados de manipulação oferecem um perigo potencial à saúde pública devido ao alto risco de ocorrência de doenças de origem alimentar por microrganismos resistentes aos antimicrobianos. Neste estudo foram coletados swabs das mãos de 49 manipuladores de alimentos, isoladas e identificadas por metodologia convencional 244 enterobactérias de 7 cantinas permissionárias em uma Universidade do Município do Rio de Janeiro. Foram utilizados discos contendo os seguintes antimicrobianos: aztreonam, tobramicina, ceftazidima, cloranfenicol, tetraciclina, sulfa, amicacina, ampicilina e sulbactam, ciprofloxacina, gentamicina, cefalotina, cefepime, cefoxitina, imipenem, ampicilina, cefotaxima. A pesquisa de genes que codificam β-lactamases foi realizada pela Reação em Cadeia da Polimerase para os genes blaTEM, blaSHV, blaCTX, blaOXA-1 e blaCMY-2. As espécies mais prevalentes identificadas foram: Enterobacter cloacae (21,3%), Citrobacter braakii (15,2%), Escherichia coli (12,7%), Klebsiella pneumoniae subesp. Pneumoniae (12,2%). O perfil de resistência aos antimicrobianos revelou que 94 (38,5%) cepas identificadas apresentaram resistência a pelo menos dois antibióticos, 55 cepas (22,5%) a pelo menos um antibiótico, 34 cepas (13,9%) a três antibióticos, 14 cepas (5,7%) mostraram-se resistentes a quatro antibióticos. Uma cepa de E. cloacae mostrou resistência múltipla a dez antibióticos e 140 cepas (57,3%) foram resistentes simultaneamente a cefalotina e cefoxitina. Nenhuma cepa foi resistente ao aztreonam e a sulfa e apenas 01 foi resistente ao ciprofloxacina, 01 ao imipenem e 02 a cefotaxima. Foi identificado a presença do gene blaSHV em uma cepa de K. pneumoniae subsp. pneumoniae. Este dado aponta para uma mudança no perfil dessas bactérias isoladas da comunidade que passam a ter características semelhantes com as de origem hospitalar. Os resultados alertam para um perigo à saúde dos consumidores e conseqüentemente à saúde pública, devido a presença de enterobactérias resistentes aos antimicrobianos nas mãos de manipuladores de alimentos.

纳滤和生物活性炭去除微污染原不中阿特拉津

除草剂阿特拉津的大量生产和广泛使用形成了对饮用水源的严重污染，目前在世界范围内的地表水和地下水中均检出一定浓度的阿特拉津，这对人类健康构成严重威胁。本文研究了采用纳滤技术和生物活性炭技术去除微污染原水中阿特拉津的可行性，对上述两种技术的去除效率、作用机理和影响因素进行了研究，并探讨了它们在未来饮用水深度处理中的优势和不足。本论文获得了以下一些具有重要意义的研究结果：（1）除天然有机物外，原水中无机离子在膜表面或孔径内吸附，引起膜有效孔径减小，使纳滤膜对农药截留率提高并降低水通量；在所研究的两个系列四种纳滤膜中，UTC-20截留阿特拉津达90％，截留其他有机物＞95％，对硝酸盐截留率＞70％，是迄今报道的对硝酸盐去除效率最高的纳滤膜，适于去除原水中阿特拉津和硝酸盐的双重污染；由于水合作用，纳滤膜对分子量相似有机物的截留率与有机物的油水分配系数（LogP）呈显著线性负相关，这一研究结果丰富了对有机物疏水性影响纳滤膜截留性能的认识。（2）筛选到一株能够以阿特拉津为惟一碳源生长的菌株-SYSA，经生理生化特性鉴定和1 6SrRNA基因序列分析，该菌为阴沟肠杆菌（Enterobacter cloacae）。这是首次报道肠杆菌属微生物可降解阿特拉津。对SYSA菌的生物学特性研究表明，SYSA菌可耐”受阿特拉津浓度100mg／L；pH7-8，30℃时，在以阿特拉津（20mg／L）惟一碳源的培养基上经146h培养，降解率为87％，该菌代时为2．gld，生长速率为（R）0.344dsel。（3）将SYsA菌接入活性炭柱，形成生物活性炭去除水中微量阿特拉津。研究结果表明空床接触时间为40min、温度为16℃±4℃、溶解氧为4.3-4.6mg／L时，120天后未接菌对照柱去除率下降至30％，而降解菌柱的去除效率却仍保持在65％～75％，延长了炭的使用时间。DGGE图谱表明未接菌对照柱、土壤悬液柱和降解菌柱上均有自来水中的微生物，但降解菌柱上还是以SYSA菌为优势菌。上述研究结果为纳滤或生物活性炭技术用于处理微污染原水、井直接生产饮用水提供了科学依据。

阿特拉津降解菌SYSA的分离筛选和鉴定

从长期施用阿特拉津的土壤中筛选到1株能够以阿特拉津为惟一碳源生长的菌株SYSA,经生理生化特性鉴定和16S rDNA序列分析,该菌为阴沟肠杆菌(Enterobacter cloacae)。对SYSA菌的生物学特性研究表明,pH 7~8,30℃时,在以阿特拉津(20 mg/L)为惟一碳源的培养基上经146 h培养,降解率为87%。

Characterization of RarA, a Novel AraC Family Multidrug Resistance Regulator in Klebsiella pneumoniae

Transcriptional regulators, such as SoxS, RamA, MarA, and Rob, which upregulate the AcrAB efflux pump, have been shown to be associated with multidrug resistance in clinically relevant Gram-negative bacteria. In addition to the multidrug resistance phenotype, these regulators have also been shown to play a role in the cellular metabolism and possibly the virulence potential of microbial cells. As such, the increased expression of these proteins is likely to cause pleiotropic phenotypes. Klebsiella pneumoniae is a major nosocomial pathogen which can express the SoxS, MarA, Rob, and RamA proteins, and the accompanying paper shows that the increased transcription of ramA is associated with tigecycline resistance (M. Veleba and T. Schneiders, Antimicrob. Agents Chemother. 56:4466-4467, 2012). Bioinformatic analyses of the available Klebsiella genome sequences show that an additional AraC-type regulator is encoded chromosomally. In this work, we characterize this novel AraC-type regulator, hereby called RarA (Regulator of antibiotic resistance A), which is encoded in K. pneumoniae, Enterobacter sp. 638, Serratia proteamaculans 568, and Enterobacter cloacae. We show that the overexpression of rarA results in a multidrug resistance phenotype which requires a functional AcrAB efflux pump but is independent of the other AraC regulators. Quantitative real-time PCR experiments show that rarA (MGH 78578 KPN_02968) and its neighboring efflux pump operon oqxAB (KPN_02969_02970) are consistently upregulated in clinical isolates collected from various geographical locations (Chile, Turkey, and Germany). Our results suggest that rarA overexpression upregulates the oqxAB efflux pump. Additionally, it appears that oqxR, encoding a GntR-type regulator adjacent to the oqxAB operon, is able to downregulate the expression of the oqxAB efflux pump, where OqxR complementation resulted in reductions to olaquindox MICs.