Methods for the genetic manipulation of marine bacteria

Genetic manipulation of bacteria is a procedure necessary to obtain new strains that express peculiar and defined genetic determinants or to introduce genetic variants responsible for phenotypic modifications. This procedure can be applied to explore the biotechnological potential associated with environmental bacteria and to utilize the functional properties of specific genes when inserted into an appropriate host. In the past years, marine bacteria have received increasing attention because they represent a fascinating reservoir of genetic and functional diversity that can be utilized to fuel the bioeconomy sector. However, there is an urgent need for an in-depth investigation and improvement of the genetic manipulation tools applicable to marine strains because of the paucity of knowledge regarding this. This review aims to describe the genetic manipulation methods hitherto used in marine bacteria, thus highlighting the limiting factors of the different techniques available today to increase manipulation efficiency. In particular, we focus on methods of natural and artificial transformations (especially electroporation) and conjugation because they have been successfully applied to several marine strains. Finally, we emphasize that, to avoid failure, future work should be carried out to establish tailored methodologies for marine bacteria.

Síndrome de Sjõgren, más que un ojo seco / Sjõgren syndrome, more than a dry eye

El síndrome de Sjõgren primario (SSp) es una exocrinopatía autoinmune de gran importancia en la actualidad, ya que afecta entre 0.2 al 3 % de la población general. Al SSp se le ha atribuido una estrecha relación con la susceptibilidad genética de cada individuo, debido a la presencia de alelos del HLA DR y HLA DQ y a la influencia ambiental que conllevan al desarrollo de dicha enfermedad.El SSp se caracteriza por pérdida de la tolerancia central, la cual origina una epitelitispor la infiltración de células linfomonocitarias, proceso que tiene como consecuencia atrofia acinar de las glándulas exocrinas. Las manifestaciones clínicas fundamentalmente se dividen en dos grupos: manifestaciones glandulares como xerostomía,xeroftalmia e hipertrofia parotídea y manifestaciones extraglandulares como artralgias,neuropatía, fatiga, entre otras.El SSp es un reto para el clínico, ya que requiere un alto índice de sospecha debido a la amplia gama de manifestaciones, pero apoyándose en una adecuada implementación e interpretación de ayudas diagnósticas y siguiendo los criterios de clasificación se puede realizar un diagnóstico oportuno. El tratamiento depende de la sintomatología y del compromiso que genere. Este se basa en tres pilares fundamentales: medidas generales para evitar la sequedad, estimulantes de secreción y la medicación cuando existe compromiso sistémico.En los últimos años se ha despertado el interés acerca del uso de anticuerpos monoclonales en el tratamiento del SSp, los cuales han llevado a resultados prometedores como el uso de anti CD20...

Resistencia antimicrobiana de bacterias aisladas de clínicas veterinarias de la ciudad de Ibagué, Colombia / Antimicrobial resistance of bacteria isolated from veterinary clinics in the city of Ibague, Colombia

Objetivo: Aislar bacterias que circulan en clínicas veterinarias de la ciudad de Ibagué, conocer su perfil de resistencia a antimicrobianos y en algunas, su capacidad de transferir dicha resistencia a bacterias sensibles. Materiales y métodos: Se tomaron muestras de 10 clínicas a las que se les realizó cultivo bacteriológico, identificación bioquímica, antibiograma y pruebas de conjugación bacteriana para transmitir dicha resistencia. El diseño metodológico fue de tipo cuasi-experimental, el análisis de los resultados se hizo mediante estadística descriptiva. Resultados: En todas las áreas de las 10 clínicas se encontraron bacterias potencialmente patógenas multirresistentes que pertenecían a 8 de 16 especies aisladas. Los microorganismos que aparecieron con mayor frecuencia en los diferentes sitios de las clínicas fueron: Staphylococcus intermedius, Acinetobacter baumannii, Pantoea agglomerans, Klebsiella pneumoniae y Burkhordelia cepacia. Los lugares donde se aislaron microorganismos multirresistentes con más frecuencia fueron el piso de consulta externa y la mesa de examen clínico. La resistencia se presentó principalmente a amoxicilina y cloranfenicol. El estudio muestra la presencia de patógenos potenciales de causar infecciones nosocomiales, que se constituyen en reservorio de genes de resistencia a los antibióticos para las bacterias patógenas no resistentes.

Objective: To isolate bacteria circulating in veterinary clinics in the city of Ibague for knowing its antimicrobial resistance profile and in some cases, its ability to transfer this resistance to susceptible bacteria. Materials and Methods: Samples of 10 clinics that underwent bacterial culture, biochemical identification, antimicrobial susceptibility testing and bacterial conjugation to transfer this resistance were taken. The methodological design was quasi-experimental and the analysis of the results was made using descriptive statistics. Results: In all areas of the 10 clinical multiresistant potentially pathogenic bacteria which belonged to 8 of 16 species isolated were found. The microorganisms that occurred more frequently in different clinical places were: Staphylococcus intermedius, Acinetobacter baumannii, Pantoea agglomerans, Klebsiella pneumoniae and Burkhordelia cepacia. The places where multiresistant microorganisms were most frequently isolated were the outpatients' floor and the clinical examination table. The resistance occurred mainly to amoxicillin and chloramphenicol. The study shows the presence of potential pathogens causing nosocomial infections, which constitute a reservoir of resistance genes to antibiotics for non-resistant pathogenic bacteria.

Conjugative transfer of resistance determinants among human and bovine Streptococcus agalactiae

Streptococcus agalactiae (GBS) is a major source of human perinatal diseases and bovine mastitis. Erythromycin (Ery) and tetracycline (Tet) are usually employed for preventing human and bovine infections although resistance to such agents has become common among GBS strains. Ery and Tet resistance genes are usually carried by conjugative transposons (CTns) belonging to the Tn916 family, but their presence and transferability among GBS strains have not been totally explored. Here we evaluated the presence of Tet resistance genes (tetM and tetO) and CTns among Ery-resistant (Ery-R) and Ery-susceptible (Ery-S) GBS strains isolated from human and bovine sources; and analyzed the ability for transferring resistance determinants between strains from both origins. Tet resistance and int-Tn genes were more common among Ery-R when compared to Ery-S isolates. Conjugative transfer of all resistance genes detected among the GBS strains included in this study (ermA, ermB, mef, tetM and tetO), in frequencies between 1.10-7 and 9.10-7, was possible from bovine donor strains to human recipient strain, but not the other way around. This is, to our knowledge, the first report of in vitro conjugation of Ery and Tet resistance genes among GBS strains recovered from different hosts.

Incidence and transferability of antibiotic resistance in the enteric bacteria isolated from hospital wastewater

This study reports the occurrence of antibiotic resistance and production of β-lactamases including extended spectrum beta-lactamases (ESβL) in enteric bacteria isolated from hospital wastewater. Among sixty-nine isolates, tested for antibiotic sensitivity, 73.9% strains were resistant to ampicillin followed by nalidixic acid (72.5%), penicillin (63.8%), co-trimoxazole (55.1%), norfloxacin (53.6%), methicillin (52.7%), cefuroxime (39.1%), cefotaxime (23.2%) and cefixime (20.3%). Resistance to streptomycin, chloramphenicol, nitrofurantoin, tetracycline, and doxycycline was recorded in less than 13% of the strains. The minimum inhibitory concentration (MIC) showed a high level of resistance (800-1600 µg/mL) to one or more antibiotics. Sixty three (91%) isolates produced β-lactamases as determined by rapid iodometric test. Multiple antibiotic resistances were noted in both among ESβL and non-ESβL producers. The β-lactamases hydrolyzed multiple substrates including penicillin (78.8% isolates), ampicillin (62.3%), cefodroxil (52.2%), cefotoxime (21.7%) and cefuroxime (18.8%). Fifteen isolates producing ESβLs were found multidrug resistant. Four ESβL producing isolates could transfer their R-plasmid to the recipient strain E. coli K-12 with conjugation frequency ranging from 7.0 x 10-3 to 8.8 x 10-4. The findings indicated that ESβL producing enteric bacteria are common in the waste water. Such isolates may disseminate the multiple antibiotic resistance traits among bacterial community through genetic exchange mechanisms and thus requires immediate attention.

Epigenética e doenças humanas / Epigenetics and human diseases

O termo epigenética é definido pela alteração herdável na expressão gênica, sem que haja mudança na sequência primária de DNA, sendo a metilação do DNA e a modificação de histonas, importantes mecanismos envolvidos. A metilação do DNA influencia a organização da cromatina, e leva à repressão de genes e elementos transponíveis. As modificações pós-traducionais que podem ocorrem em proteínas histonas são muitas e podem ocorrer em diferentes aminoácidos e diferentes posições, e resultam em uma multiplicidade de combinações em um verdadeiro “código de histona”, e elas são interpretadas por diferentes fatores celulares. As marcas epigenéticas atuam simultaneamente para regular a transcrição gênica em um processo complexo, e pequenas falhas no estabelecimento ou manutenção desses podem desencadear o desenvolvimento de patologias, como a encontrada em síndromes genéticas e no câncer. Devido ao grande número de doenças, muitas pesquisas têm sido realizadas na busca de drogas capazes de reverter esses defeitos epigenéticos. Atualmente, já foram descritos alguns agentes capazes de interferir na ação de enzimas que regulam os processos, porém, existem ainda muitas dúvidas na aplicatividade desses tratamentos, se fazendo necessário maiores estudos dos mecanismos epigenéticos para, no futuro, serem mapeadas vias de interferências específicas e um melhor controle de efeitos.

The term epigenetics defines heritable changes in gene expression that do not involve DNA sequence changes. DNA methylation and histone modifi cations are two important related mechanisms. DNA methylation alters chromatin structure and has been shown to have a direct effect on the silencing of genes and transposons. There are many post-traductional modifications on histones and an extra complexity comes from many sites and amino acids that may be modifi ed, what results in multiple matches in a real “histone code” decrypted by different cell factors. The epigenetics mechanisms are complex, and minor failing in the establishment and maintenance causes diseases, as genetics syndromes and cancer. Due to several diseases, an increasing amount of research has been realized to find drugs able to suppress these defects. Nowadays, there have already been described some medicines able to interfere in the action of the enzymes which regulate the epigenetic processes, however, there is still doubt in effectiveness of theses treatment. It is necessary additional research about epigenetics mechanism to, in the future, understand specific pathways and control the effects of these drugs.

Diseño de estrategias para controlar la resistencia a los antimicrobianos utilizando herramientas de bioinformática y modelado molecular / Design of strategies to control antimicrobial resistance using bioinformatics tools and molecular modeling

El éxito del tratamiento de las enfermedades infecciosas se ha visto comprometido en los últimos años debido a la diseminación de genes de resistencia a antibióticos entre las bacterias patógenas. Estos genes de resistencia a antibióticos son transportados por plásmidos, los cuáles son transferidos de una bacteria a otra mediante el proceso de conjugación. La reducción del uso inadecuado de los antibióticos y la búsqueda de inhibidores de la conjugación bacteriana son estrategias que podrían contribuir a la solución de este grave problema de salud pública. Basándose en la primera de esta estrategias, en enero de 2006 se regulo la dispensación de un grupo de antibióticos a fin de controlar su consumo. El análisis realizado en este trabajo seǹala que esta medida ha resultado ineficaz, puesto que el consumo y la resistencia bacteriana total a estos antimicrobianos se incrementó significativamente durante el periodo posterior a su promulgación. La resistencia bacteriana a muchas de las familias de antibióticos estudiadas esta solo parcialmente influenciada por su consumo, destacando la participación de otros factores, como la transferencia de genes de resistencia a antibióticos, en la prevalencia de cepas bacterianas resistentes. La identificación de proteínas del cito-cromo P450 de estructura y ligados conocidos, que tenían una similitud significativa en su secuencia de aminoácidos con la proteína de acoplamiento TRAG de los plásmidos R27 y R478, permitió identificar a los medicamentos diclofenac y ketoprofeno como potenciales inhibidores de la transferencia por conjugación de estos plásmidos. El modelado por homología de TRAG revelo que su dominio de solo hélices alfa podría ser el blanco de estos medicamentos. El ingreso de diclofenac o ketoprofeno a una cavidad en este dominio podría interferir en la interacción con el DNA portador de genes de resistencia a antibióticos que esta siendo transferido mediante el proceso de conjugación.

An outbreak of CTX-M-15-producing Klebsiella pneumoniae isolates in an intensive care unit of a teaching hospital in Kuwait.

Objective: This study reports an outbreak of Klebsiella pneumoniae infections in 14 patients during a 2-month period (August-September, 2008) in the intensive care unit (ICU) of a teaching hospital in Kuwait. Materials and Methods: The clinical sources were blood (9), urine (3) and respiratory secretions (2) identified by the automated VITEK-2 ID System. Susceptibility testing was performed by the E-test method. Extended-spectrum β-lactamase (ESBL) production was assessed using the ESBL E-test and confirmed by PCR. Carriage of bla genes was determined by PCR and sequence analysis. The transferability of resistance phenotypes was demonstrated by conjugation experiments and clonal relatedness was determined by PFGE. Results: The isolates were susceptible to imipenem, meropenem, and tigecycline and produced ESBL. All isolates yielded an amplicon of 499 bp with universal consensus primers (MA primers). DNA sequence analysis showed that they all harboured blaCTX-M-15 and blaTEM-1 genes. The environmental isolate obtained from a suction machine was also CTX-M-15/TEM-1 producer. The resistance phenotypes were transferrable to the Escherichia coli J53 r strain. PFGE, revealed two clones, A and B, related with a Dice coefficient of >94.1%. A mortality rate of 21.4% was recorded. Conclusion: The outbreak was contained by robust and aggressive infection control measures. This study highlights the first outbreak of CTX-M-15-producing K. pneumoniae associated with high mortality in an adult medical ICU in Kuwait.

Análisis de transferencia horizontal de genes en ensayos de biorremediación con grasas recalcitrantes / Analysing horizontal gene transfer in bioremediation trials using recalcitrant grease

El empleo de microorganismos como herramienta para la mineralización de contaminantes orgánicos es una práctica que ha tomado mucha fuerza gracias a su eficiencia y bajo costo. La transferencia horizontal vía conjugación de genes es un requerimiento básico para optimizar procesos de biorremediación, por esta razón, además de conocer la diversidad metabólica es fundamental entender las interacciones que ocurren en una comunidad bacteriana para potenciar los procesos de biorremediación en campo.En este estudio se busca evaluar el potencial de transferencia horizontal (TH), tanto in vitro como en microcosmos de suelo, de aislamientos de bacterias obtenidas de un pasivo ambiental de grasas provenientes de la explotación carbonífera del Cerrejón. Inicialmente se agruparon los aislamientos de acuerdo con sus patrones de resistencia a antibióticos: ampicilina, cloramfenicol, gentamicina, tetraciclina y kanamicina. El potencial de TH de las cepas Vlf4, Ot3, Ot6, Pgt4, Blf11 y Vlf13 fue evaluado in vitro en medio sólido Luria-Bertani (LB) donde se obtuvieron nuevos fenotipos a partir de los cruces Vlf13xOt6 y Pgt4xOt6, el nuevo fenotipo indica resistencia a los dos marcadores (ampR, kanR) y su morfología sugiere que el receptor, en los dos casos, es la cepa Ot6. Los parentales Vlf13, Pgt4 y Ot6 fueron identificados por amplificación del gen RNAr 16S como Pseudomonas sp., Pseudomonas sp. y Chryseobacterium sp., respectivamente, y los transconjugantes como Chryseobacterium sp. Posteriormente, estos dos cruces fueron sometidos a ensayos de transferencia horizontal en microcosmos de suelos, donde se hizo evidente nuevamente la presencia de TH a una menor tasa. Los resultados obtenidos indican la posibilidad de un potencial de transferencia horizontal de genes entre los aislamientos seleccionados, dando lugar a la probabilidad de formular en futuros estudios un consorcio de bacterias que demuestran tener esta ventaja adaptativa.

The use of microorganisms as a tool for the mineralization of organic pollutants is a practice that has begun to gain strength due to its efficiency and low cost. Horizontal gene transfer by conjugation is important for the optimization of bioremediation processes. For this reason it is fundamental to study the metabolic diversity of catabolic pathways, but we must also understand the microbial interactions occurring in bioremediation consortia. This will help us improve bioremediation in field.The purpose of this study was to evaluate the horizontal gene transfer (HGT) potential, in vitro and in soil microcosms, of bacterial strains isolated from grease samples obtained from a disposal site situated in the coal mine “Cerrejon”. Initially the isolates were grouped by selective markers: Ampicillin, chloramphenicol, gentamicin, tetracycline and kanamycin. The HGT potential of strains: Vlf4, Ot3, Ot6, Pgt4, Blf11 y Vlf13 was evaluated in vitro on Luria-Bertani LB agar. New phenotypes were obtained from matings between Vlf13xOt6 and Pgt4xOt6. The new phenotype indicates resistances to both antibiotic markers and its morphology suggests that isolate Ogt6 is the receptor in both cases. The parental strains Vlf13, Pgt4 and Ot6 were identified by RNAr 16S as Pseudomonas sp. Pseudomonas sp. and Chryseobacterium sp. respectively and the transconjugants as Chryseobacterium sp. Subsequently soil microcosms were conducted with Vlf13xOt6 and Pgt4xOt6 and new phenotypes were detected at a lower rate again but with the same possible receptor but. These results suggest the possibility of horizontal gene transfer potential within the selected isolates, giving the possibility of formulating, in future studies, a bacterial consortium with an adaptive advantage.

Re-engineering the mitochondrial genomes in mammalian cells / 대한해부학회지

Mitochondria are subcellular organelles composed of two discrete membranes in the cytoplasm of eukaryotic cells. They have long been recognized as the generators of energy for the cell and also have been known to associate with several metabolic pathways that are crucial for cellular function. Mitochondria have their own genome, mitochondrial DNA (mtDNA), that is completely separated and independent from the much larger nuclear genome, and even have their own system for making proteins from the genes in this mtDNA genome. The human mtDNA is a small (~16.5 kb) circular DNA and defects in this genome can cause a wide range of inherited human diseases. Despite of the significant advances in discovering the mtDNA defects, however, there are currently no effective therapies for these clinically devastating diseases due to the lack of technology for introducing specific modifications into the mitochondrial genomes and for generating accurate mtDNA disease models. The ability to engineer the mitochondrial genomes would provide a powerful tool to create mutants with which many crucial experiments can be performed in the basic mammalian mitochondrial genetic studies as well as in the treatment of human mtDNA diseases. In this review we summarize the current approaches associated with the correction of mtDNA mutations in cells and describe our own efforts for introducing engineered mtDNA constructs into the mitochondria of living cells through bacterial conjugation.

Transconjugation and genotyping of the plasmid-mediated AmpC beta-lactamase and extended-spectrum beta-lactamase genes in Klebsiella pneumoniae / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>AmpC beta-lactamases and extended-spectrum beta-lactamases (ESBLs) are becoming predominant causes of resistance to third and forth-generation cephalosporins in Klebsiella pneumoniae (K. pneumoniae). It is very difficult to treat infectious diseases caused by multidrug-resistant K. pneumoniae. The purpose of the present study was to investigate transconjugation and characteristics of beta-lactamase genes in K. pneumoniae producing AmpC beta-lactamases and ESBLs.</p><p><b>METHODS</b>AmpC beta-lactamases were detected by three-dimension test and ESBLs by disc confirmatory test. Minimum inhibitory concentrations (MICs) were determined by agar dilution. Transfer of resistance to EC600 (Rif(r)) was attempted by conjugation in broth and screened on agar containing cefotaxime (2 microg/ml) plus rifampin (1024 microg/ml). The genes encoding AmpC or ESBLs and their transconjugants were detected by PCR and verified by DNA sequencing.</p><p><b>RESULTS</b>The resistant rates to ampicillin and piperacillin were 100% in 18 isolates of K. pneumoniae. However, imipenem was still of great bactericidal activity on K. pneumoniae, and its MIC(50) was 0.5 microg/mL. Eleven beta-lactamase genes, including TEM-1, TEM-11, SHV-13, SHV-28, CTX-M-9, CTX-M-22, CTX-M-55, OXA-1, LEN, OKP-6 and DHA-1, were found from 18 isolates. And at least one beta-lactamase gene occurred in each isolate. To our surprise, there were six beta-lactamase genes in the CZ04 strain. Among 18 isolates of K. pneumoniae, the partial resistant genes in 8 isolates were conjugated successfully, which had 100% homological sequence with donors by sequence analysis. Compared with donors, 8 transconjugants had attained resistance to most beta-lactams, including ampicillin, piperacillin, cefoxitin, cefotaxime and aztreonam, or even amikacin and gentamicin.</p><p><b>CONCLUSIONS</b>R plasmids can be easily transferred between the resistant and sensitive negative bacilli. It is very difficult to block and prevent the spread of antimicrobial resistance. So more attention should be paid to reducing the frequency, times and dosage of antimicrobials, especially third or fourth cephalosporins.</p>

Molecular characterization of nosocomial CTX-M type beta-lactamase producing Enterobacteriaceae from a tertiary care hospital in south India.

CTX-M group of extended spectrum beta lactamases (ESBLs) represents a rapidly emerging problem in many countries. The prevalence of nosocomial bla CTX-M-1 producing Enterobacteriaceae strains has not been reported earlier in Indian hospitals. This study describes molecular subtyping of nosocomial bla CTX-M producing strains of Enterobacteriaceae . Polymerase chain reaction with primers specific for bla CTX-M-1 coding genes was used to identify 95 Enterobacteriaceae strains producing bla CTX-M positive isolates. Of the 95 bla CTX-M producing isolates, 45 strains were positive for bla CTX-M-1 . bla CTX-M-1 was found to be most prevalent in Klebsiella strains.

Assessment of pheromone response in biofilm forming clinical isolates of high level gentamicin resistant Enterococcus faecalis.

Twenty five clinical isolates of high level gentamicin resistant Enterococcus faecalis were tested for their biofilm formation and pheromone responsiveness. The biofilm assay was carried out using microtiter plate method. Two isolates out of the 25 (8%) were high biofilm formers and 19 (76%) and four (16%) isolates were moderate and weak biofilm formers respectively. All the isolates responded to pheromones of E. faecalis FA2-2 strain. On addition of pheromone producing E. faecalis FA2-2 strain to these isolates, seven of 19 (37%) moderate biofilm formers developed into high biofilm formers. Similarly one of the 4 (25%) weak biofilm formers developed into high level biofilm former. Twelve (48%) of the 25 isolates were transconjugated by cross streak method using gentamicin as selective marker. This proves that the genetic factor for gentamicin resistance is present in the pheromone responsive plasmid. Among these twelve transaconjugants, seven isolates and one isolate were high biofilm formers on addition of E. faecalis FA2-2 and prior to its addition respectively. Out of the total 25 isolates, eight transconjugants for gentamicin resistance could turn to high biofilm formers on addition of the pheromone producing strain. All the isolates were resistant to more than two antibiotics tested. All the isolates were sensitive to vancomycin. The results indicate the significance of this nosocomial pathogen in biofilm formation and the role of pheromone responding clinical isolates of E. faecalis in spread of multidrug resistance genes.

Characterization of class 1 integrons with unusual 3' conserved region from Salmonella enterica isolates.

The unusual 3' conserved sequence region of class 1 integrons was characterized in seven Salmonella isolates from swine and poultry. Three types of gene cassette arrays, aadA2-cmlA1-aadA1, sat-psp-aadA2-cm1A1-aadA1 and drfA12-orf-aadA2-cmlA1-aadA1, were found to be linked to a genetic organization qacH-IS440-sul3. All class 1 integrons were located on a conjugative plasmid that could be transferred to Escherichia coli. The results support the notion that the use of an antibiotic can select for resistance not only to that specific agent, but also to other unrelated antimicrobials including those that are no longer approved for use in food animal production.

Incidence of enteric bacterial pathogens in water found at the bottom of commercial freezers in calabar, southeastern Nigeria.

Bacteriological analysis of water that accumulates at the bottom of freezers in restaurants when the power was cut in Calabar, southeastern Nigeria, was carried out using standard procedures. Mean heterotrophic bacterial counts and Escherichia coli counts ranged from 3.1 +/- 0.02 to 7.1 +/- 0.30 x 10(4) cfu/ml and 0.2 +/- 0.10 to 0.6 +/- 0.50 x 10(4) cfu/ml, respectively, indicating heavy bacterial contamination whose source was mostly fecal. There was no significant difference (p > 0.05, 0.01) in bacterial counts between freezers. Some biochemically identified enteric bacterial pathogens were Salmonella typhi, Shigella sp, enteropathogenic E. coli, Yersinia sp, Klebsiella pneumoniae, Vibrio cholerae O1 and Vibrio parahaemolyticus. This reveals that the hygienic quality of the food items stored in the freezers and the hygienic status of the restaurants are in doubt. Infection could be going on unnoticed and thus endemicity maintained in the area. The pathogens showed alarming antibiotic resistance. The water in the freezers was a "soup" in which different species of the enteric pathogens were close to each other and could transfer drug resistance among themselves. Public health education of restaurant operators in southeastern Nigeria is recommended.

Multidrug resistant Salmonella typhi in asymptomatic typhoid carriers among food handlers in Namakkal district, Tamil Nadu.

PURPOSE: to screen Salmonella typhi in asymptomatic typhoid carriers and to find out drug resistance and ability of the strains to transmit drug resistance to other bacteria. METHODS: Cultural characters, biochemical tests, antibiotic sensitivity test (disc diffusion), agarose gel electrophoresis, and conjugation protocols were done. Thirty five stool samples were collected from the suspected food handlers for the study. RESULTS: Among 35 samples, (17.14%) yielded a positive result. Out of these 4 (20.0%) were women and 2 (13.33%) were men. The isolates were tested with a number of conventional antibiotics viz, amikacin, amoxicillin, ampicillin, chloramphenicol, ciprofloxacin, co-trimaxazole, rifampicin, gentamicin, nalidixic acid, ofloxacin and tetracycline. Five isolates were having the multidrug resistant character. Four (66.66%) multidrug resistant isolates were found to have plasmids, while one (16.66%) multidrug resistant isolate had no plasmid and the chromosome encoded the resistance. Only one strain (16.66%) showed single antibiotic resistance in the study and had no plasmid DNA. The molecular weights of the plasmids were determined and found to be 120 kb.The mechanism of spreading of drug resistance through conjugation process was analyzed. In the conjugation studies, the isolates having R+ factor showed the transfer of drug resistance through conjugation, which was determined by the development of antibiotic resistance in the recipients. CONCLUSION: This study shows that drug resistant strains are able to transfer genes encoding drug resistance.

Occurrence of chromium resistant thermotolerant coliforms in tannery effluent.

Twenty six thermotolerant strains resistant to high levels of chromium (50-250 microg/ml) were isolated from treated tannery effluent. They were also found resistant to multiple heavy metals and antibiotics. Majority of them were resistant to copper and bacitracin. Nine strains representing different resistance patterns were selected for plasmid profile and conjugation studies. Agarose gel electrophoresis results revealed that 6 strains harboured a single plasmid, whereas 3 strains exhibited 2 plasmid bands. Among antimicrobials, co-trimazole and bacitracin and among metals, Cu2+, Cd2+, Zn2+ and Ni2+ resistance were transferred most frequently at variable rates. However, chromium resistance was transferred in 6 strains with a frequency ranging 19-49x10(-2). Resistance to Co2+ and Hg2+ did not transfer under environmental conditions. Among the nine strains, three were found predominantly uropathogenic Escherichia coli (UPEC) serotype 04, whereas two strains were untypable. In addition, 4 transconjugants also showed a positive result after serotyping.

Mini transposon vector mediated foreign gene expression in Mesorhizobium huakuii subsp. rengei.

Among the transposable elements, mini-Tn5 transposon vector has proven to be of greater utility for insertion mutagenesis of variety of Gram negative bacteria. The mini-Tn5 vector containing promoter less egfp gene and gentamycin resistant gene was used for the present study. The transposon vector was introduced to M. huakuii from E. coli S17 by conjugation. The conjugants were screened for stable expression of egfp both in free-living and in nodules of Astragalus sinicus. The result showed that the conjugant #3 showed stable expression of green fluorescent both in free-living and bacteroid stage. The visualization of sym plasmid of wild strain and conjugants showed that conjugant #3 had a fragmentation of large sized plasmid into two but without affecting the nodulating ability. These results clearly indicated that mini-Tn5 vectors (Transposon vectors) the best alternate tools for plasmid vectors for integration of foreign genes in chromosomal DNA or symbiotic plasmid and expression, both in free-living and bacteroid stage of Rhizobium.

Genetics of metal resistance in acidophilic prokaryotes of acidic mine environments.

Acidophilic bacteria inhabiting acidic mine regions cause natural leaching of sulphidic ores. They are now exploited in industrial operations for leaching of metals and beneficiation of low-grade and recalcitrant ores. Recent trends emphasize application of thermoacidophiles and genetic engineering of ore-leaching bacteria for greater success in this area. This requires an in-depth understanding on the molecular genetics of these bacteria and construction of cloning vectors for them. Metal resistance is considered as the most suitable phenotypic trait for cloning vectors of bio-mining chemolithoautotrophic (viz. Acidithiobacillus ferrooxidans) and heterotrophic (Acidiphilium and Acidocella species) bacteria of mine environments. These bacteria take part in ore-leaching either directly or indirectly, exhibit low to high level of resistance/tolerance to various metals under different conditions. Majority of these bacteria contain one or more plasmids--the genetic elements that usually carry metal resistant genes. But none of the At. ferrooxidans plasmids has been definitely proved to harbour metal-resistant genes which have mostly been found in the chromosome of this bacterium. Plasmids of acidophilic heterotrophs of the genera Acidiphilium and Acidocella, on the other hand, carry metal resistant genes. While genes bestowing arsenic resistance in Acidiphilium multivorum are similar to those analyzed from other sources, the metal (Cd and Zn)-resistance conferring cloned plasmid DNA fragments from Acidiphilium symbioticum KM2 and Acidocella GS19h strains were found to have no sequence similarity with the reported Cd- and Zn-resistant genes. Such observations indicate some novel aspects of metal resistance in acidophilic bacteria.

Construction of efficient conjugal plasmids between Escherichia coli and Streptomycetes / 生物工程学报

Conjugal plasmid pGH112 has been developed based on the replicons of Streptomyces coelicolor plasmid SCP2 and E. coli ColE. The plasmid contains ampicilin resistance gene(amp) for selection in E. coli and thiostrepton resistance gene (tsr) for selection in Streptomycetes, and a 0.76 kb oriT fragment of (IncP) RK2. Conjugal transfer of pGH112 was performed from E. coli to S. coelicolor A3(2), S. avermitilis, S. lividans TK54, S. toxytricini NNRL15443, S. venezuelae ISP5230 and Sacc. erythraea by conjugation, results show that the plasmid was able to transfer efficenctly from E. coli to Streptomycetes, was stably inherited in the recipients. pGH113 was constructed from pGH112 by combining the constitutive ermE promoter with green fluorescent protein gene(gfp).