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PubMed2003

Colonization of Arabidopsis thaliana with Salmonella enterica and enterohemorrhagic Escherichia coli O157:H7 and competition by Enterobacter asburiae.

Enteric pathogens, such as Salmonella enterica and Escherichia coli O157:H7, have been shown to contaminate fresh produce. Under appropriate conditions, these bacteria will grow on and invade the plant tissue. We have developed Arabidopsis thaliana (thale cress) as a model system with the intention of studying plant responses to human pathogens. Under sterile conditions and at 100% humidity, S. enterica serovar Newport and E. coli O157:H7 grew to 10(9) CFU g(-1) on A. thaliana roots and to 2 x 10(7) CFU g(-1) on shoots. Furthermore, root inoculation led to contamination of the entire plant, indicating that the pathogens are capable of moving on or within the plant in the absence of competition. Inoculation with green fluorescent protein-labeled S. enterica and E. coli O157:H7 showed invasion of the roots at lateral root junctions. Movement was eliminated and invasion decreased when nonmotile mutants of S. enterica were used. Survival of S. enterica serovar Newport and E. coli O157:H7 on soil-grown plants declined as the plants matured, but both pathogens were detectable for at least 21 days. Survival of the pathogen was reduced in unautoclaved soil and amended soil, suggesting competition from indigenous epiphytes from the soil. Enterobacter asburiae was isolated from soil-grown A. thaliana and shown to be effective at suppressing epiphytic growth of both pathogens under gnotobiotic conditions. Seed and chaff harvested from contaminated plants were occasionally contaminated. The rate of recovery of S. enterica and E. coli O157:H7 from seed varied from undetectable to 19% of the seed pools tested, depending on the method of inoculation. Seed contamination by these pathogens was undetectable in the presence of the competitor, Enterobacter asburiae. Sampling of 74 pools of chaff indicated a strong correlation between contamination of the chaff and seed (P = 0.025). This suggested that contamination of the seed occurred directly from contaminated chaff or by invasion of the flower or silique. However, contaminated seeds were not sanitized by extensive washing and chlorine treatment, indicating that some of the bacteria reside in a protected niche on the seed surface or under the seed coat.

Arabidopsis↗

PubMed1999

Community-acquired pneumonia in adults: a multicentric observational AIPO study.

The aim of this study was to obtain reliable data about the current aetiology (i.e. the frequency of the individual pathogens) of community-acquired pneumonia (CAP) while surveying the diagnostic and therapeutic behaviour of Italian chest physicians, compared with existing guidelines, and to test the usefulness of the current severity "criteria" or score as a predictor of disease outcome and guide for appropriate hospitalization. A prospective multicentre observational trial was carried out between October 1994 and February 1996 by the Italian Association of Hospital Pneumologists (AIPO) study group on respiratory infections. A total of 613 consecutive patients suffering from CAP were enrolled in 25 centres throughout Italy. Clinical, radiological and microbiological data were collected and patients were followed-up until complete resolution or death. Aetiological tests were not carried out in 204 patients. In the remaining 409 cases, the aetiology was defined by serological and quantitative microbiological tests in 184 (44.9%) patients. A total of 194 strains of pathogen were detected. The most frequently detected micro-organism was Streptococcus pneumoniae (18.5% of pathogen strains) but, unlike in other series of patients, high percentages of intracellular pathogens (32.5%, all with serological confirmation, mostly due to Chlamydia pneumoniae (13.4%) and of Gram-negative enterobacteria and Pseudomonas aeruginosa (12.5%) were also found. Antibiotic treatment differed from that recommended in American Thoracic Society guidelines, with a greater use of third-generation cephalosporins. Overall, a higher rate of hospitalization and a lower death rate than in other comparable studies was observed.

Adolescent↗

PubMed1992

[The pathogenicity factors of bacteria in the genus Acinetobacter].

Eighty Acinetobacter strains, isolated in Togliatti from patients with purulent inflammatory diseases, were studied to determine their pathogenicity factors. Out of these 80 strains, 32.5% were found to have enterotoxigenic activity and 46.2%, adhesive activity. They were related to adhesins of the human type and to adhesins of the sheep, rabbit, swine and guinea pig types. But the most important phenomenon established in this study was the combination of different pathogenicity factors detected in Acinetobacter bacteria. Analysis of the combination of pathogenicity factors revealed that 7.5% of Acinetobacter strains had adhesive and enterotoxigenic activity, 15.3% of these strains combined adhesive and hemolytic activity and 1.2% of them were found to be enterotoxigenic and hemolytic. Only 5.0% of Acinetobacter strains were found to carry all there pathogenicity factors simultaneously.

Acinetobacter↗

PubMed1990

Prospects for standardized assays in vector biology.

The benefits of technological advances continue to accrue at an ever accelerating rate. In 1987 I presented a brief review of recent advances and applications of biotechnology to the study of arthropods and disease-vector relationships. Since 1987 progress has been made in practically all areas included in that earlier review and I want to give brief mention of just a few significant advances that have appeared in print since that time. An obvious spin-off of modern technology has been the production of continually improved tools to aid in species identification. This is true for the arthropods as well as for pathogenic organisms transmitted by arthropods. Consequently, we can now approach the problems presented by species complexes with an array of techniques, and we find that we are limited only by our access to the necessary reagents, grasp of the technology and funding. Likewise, in studies of arthropod-pathogen relationships, many new tools have been developed for detection of pathogens in the vectors and these tools are applicable to field and laboratory investigations into the many issues of vector competence.

Animals↗

PubMed2006

Quantifying leukocytes in first catch urine provides new insights into our understanding of symptomatic and asymptomatic urethritis.

We quantitatively investigated inflammatory cells in the male urethra. Leukocytes in the first catch urine (FCU) from 87 men with and without urethritis were quantitated using haemocytometer counts and stained with an anti-CD45 pan-leukocyte antibody. An increased number of leukocytes in FCU specimens was associated with urethritis (P > 0.002), the presence of discharge and/or dysuria (P < 0.001), and detection of Chlamydia trachomatis (P < 0.001) and Neisseria gonorrhoeae (P < 0.001). In men with urethritis, higher leukocyte counts were also observed in the above groups (P = 0.07, 0.03 and P < 0.0001, respectively). As leukocyte number increased, the likelihood of detecting either pathogen increased. This study suggests that symptoms and signs are a surrogate marker for the degree of inflammation present, and that as urethral inflammation increases, the likelihood of detecting a sexually transmitted pathogen also increases. This would explain why men with asymptomatic urethritis are less likely to have a sexually transmitted infection detected than those with discharge and/or dysuria.

Adolescent↗

PubMed2004

Detection of bacterial DNA in synovial fluid from horses with infectious synovitis.

Standard culturing techniques are often unrewarding in confirming diagnosis of synovial infection in the equine patient. Several human studies report the use of sensitive polymerase chain reaction (PCR) techniques for the detection of bacterial involvement in acute synovitis. However, successful extraction of bacterial DNA directly from clinical samples from horses without prior culture has not been reported yet. The goal of this study was to develop a sensitive and reliable method for molecular detection and identification of bacterial species in synovial fluid from horses with infectious synovitis. Synovial fluid samples from 6 horses with culture confirmed synovial infection were used for broad range 16S rRNA gene PCR. Synovial aspirates of 2 healthy horses were used as negative controls. Following extraction and purification of synovial fluid DNA, all samples were processed by touchdown PCR. Amplicons were detected by reverse line blot hybridisation and visualised with chemiluminescence. Pathogen-specific detection of 16S rRNA gene sequences was successful in all 6 synovial fluid samples. No bacterial DNA was detected in the aspirates from the negative control horses using touchdown PCR followed by 25 additional cycles of amplification. The identity of the pathogens was confirmed by DNA sequencing of the amplicons. It can be concluded that broad range 16S rRNA gene PCR followed by reverse line blot hybridisation is a promising technique for detection of bacterial DNA in synovial fluid samples. Further research should aim at the detection of bacterial DNA in synovial fluid samples suspected of infection but having negative culture results. When the 16S PCR proves to be reliable and more sensitive than standard culturing techniques, it may become a powerful tool in the diagnosis of synovial infection.

Animals↗

PubMed2005

Detection of Mycoplasma pneumoniae in serum specimens from patients with mycoplasma pneumonia by PCR.

There are few data on detection of Mycoplasma pneumoniae from blood, serum or plasma, and systematic studies on this diagnostic approach in community-acquired pneumonia (CAP) are scarce. Compared to testing respiratory specimens, this approach has the advantages that it is less dependent on proper specimen collection, serum is easily stored and handled, and the pathogen is detected in a primary sterile site, where colonization can be ruled out. In this study, acute-phase serum specimens from 29 patients of Vienna University Hospital (treated between 11/1994 and 6/2004; female: 14, male: 15; median age: 31 years, range: 15-66 years) with CAP and serologically verified M. pneumoniae infection, who had not received anti-mycoplasma therapy prior to serum collection, were tested for M. pneumoniae by conventional PCR and real-time PCR. Conventional PCR yielded negative results for all specimens, but real-time PCR detected M. pneumoniae in 15/29 patient sera (52%). These findings indicate that M. pneumoniae is present in the bloodstream of a substantial proportion of patients with mycoplasma pneumonia. Despite the possible adherence of M. pneumoniae to human erythrocytes, the pathogen can be detected from serum, if a method with enhanced sensitivity is applied. However, the negative predictive value of PCR from serum with regard to etiological diagnosis is low. With regard to the potential clinical benefit of blood-based PCR diagnosis of mycoplasma pneumonia the diagnostic accuracy of this approach using either serum or whole-blood specimens should be addressed by large-scale studies.

Adult↗

PubMed1999

Detection of Lawsonia intracellularis in faeces of swine from the main producing regions in Brazil.

Swine proliferative enteropathy is an enteric disease caused by Lawsonia intracellularis which affects animals between 6 and 20 weeks of age, causing diarrhea, anorexia, and poor growth. The presence of L. intracellularis was evaluated in the faecal samples of 636 swine from 75 randomly chosen herds in the main swine-producing regions of Brazil. The pathogen was detected by the polymerase chain reaction method (PCR) using L. intracellularis specific primers. A 319-bp DNA fragment specific for L. intracellularis was produced on amplification of DNA from the faeces of pigs with proliferative enteropathy. Equal amounts of DNA extracted from the faeces of animals from the same herd were pooled together and, once L. intracellularis was detected, the faecal material of each animal was analyzed separately. The incidence of L. intracellularis was 33.4% in the state of Santa Catarina, 29.4% in Paraná, 26.3% in Minas Gerais, 16.7% in Mato Grosso, and 7.1% in São Paulo. The presence of the pathogenic agent was detected in samples from 15 farms, representing a total incidence of 20%. Although 46 animals (7.2%) were shown to be infected, 11% did not present any symptoms of swine proliferative enteropathy. The use of PCR allowed the detection of L. intracellularis in swine farms and the evaluation of the incidence of proliferative enteropathy in different regions of Brazil.

Animals↗

PubMed1997

Sensitive detection of viable Listeria monocytogenes by reverse transcription-PCR.

Detection of pathogens in contaminated food products by PCR can result in false-positive data due to the amplification of DNA from nonviable cells. A new method based on reverse transcription-PCR (RT-PCR) amplification of mRNA for the specific detection of viable Listeria monocytogenes was developed. The expression of three L. monocytogenes genes, iap, hly, and prfA, was examined to determine a suitable target for amplification of RT-PCR. Total RNA from L. monocytogenes was isolated, and following DNase treatment, the RNA was amplified by both RT-PCR and PCR with primers specific for the three genes. Amplicon detection was accomplished by Southern hybridization to digoxigenin-labeled gene probes. The levels of expression of these three genes differed markedly, and the results indicated that the iap gene would provide a good target for development of a specific method for detection of viable L. monocytogenes based on RT-PCR amplification. After a 1-h enrichment, the 371-bp iap-specific product was detected with a sensitivity of ca. 10 to 15 CFU/ml from pure culture. Detection of the 713-bp hly-specific amplicon was ca. 4,000 times less sensitive after 1 h, whereas detection of the 508-bp prfA product showed the lowest level of sensitivity, with detection not observed until after a 5-h enrichment period. The amplification of the iap mRNA was specific for L. monocytogenes. Overall, the assay could be completed in ca. 54 h. The use of RT-PCR amplification for the detection of viable L. monocytogenes was validated in artificially contaminated cooked ground beef. Following a 2-h enrichment incubation, the iap-specific amplification product could be detected in a cooked meat sample that was originally inoculated with ca. 3 CFU/g. These results support the usefulness of RT-PCR amplification of mRNA as a sensitive method for the specific detection of viable L. monocytogenes and indicate that this method may prove useful in the detection of this pathogen in ready-to-eat, refrigerated meat products.

Bacterial Proteins↗

PubMed2006

Lemierre's syndrome and genetic polymorphisms: a case report.

BACKGROUND: Lemierre's syndrome presents a classic clinical picture, the pathophysiology of which remains obscure. Attempts have been made to trace genetic predispositions that modify the host detection of pathogen or the resultant systemic reaction. CASE PRESENTATION: A 17-year old female, with no previous medical history, was admitted to the intensive care unit for septic shock, acute respiratory distress syndrome and Lemierre's syndrome. Her DNA was assayed for single nucleotide polymorphisms previously incriminated in the detection of the pathogen, the inflammatory response and the coagulation cascade. We observed functional variations in her Toll like 5 receptor (TLR 5) gene and two coagulation variations (Tissue Factor (TF) 603 and Plasminogen-Activator-Inhibitor-1 (PAI-1) 4G-4G homozygosity) associated with thrombotic events. CONCLUSION: The innate immune response and the prothrombogenic mutations could explain, at least in part, the symptoms of Lemierre's syndrome. Genomic study of several patients with Lemierre's syndrome may reveal its pathophysiology.

Adolescent↗

PubMed2002

Astrovirus acute gastroenteritis among children in Madrid, Spain.

BACKGROUND: Human astroviruses cause infantile gastroenteritis worldwide, but the prevalence of disease varies greatly by setting. Since 1997 we have conducted a survey to determine the causes of diarrhea among Spanish children attending an emergency room in Madrid and to characterize the clinical features of viral-associated gastroenteritis. OBJECTIVES: To define the epidemiologic role of astrovirus-associated gastroenteritis in Spanish children, to review its clinical features and to compare these illnesses with those caused by rotavirus. To assess the sensitivity of two methods of detection [enzyme-linked immunosorbent assay (EIA) and reverse transcriptase (RT)-PCR]. METHODS: Fecal specimens from 822 children with acute diarrhea treated at an emergency room were screened by EIA assays. Random astrovirus-positive samples were characterized by RT-PCR and nucleotide sequencing for their phylogenetic grouping. RESULTS: Astrovirus was detected in 44 (5.3%) of 822 specimens tested by EIA. No pathogens were detected in fecal specimens from 238 (29%) children; however, in 137 of those with adequate remaining specimens, we found an additional 50 (6.1%) that were positive by RT-PCR. HAstV-1 was the most prevalent type followed by HAstV-2. The gastroenteritis associated with astrovirus alone was slightly less severe and had a lower score or risk of hospitalization than that associated with rotavirus (P < 0.05). CONCLUSIONS: Astrovirus was found in 11.4% of all children whom we tested for enteric viral and bacterial pathogens, making it the second most common cause of acute gastroenteritis among Spanish children. True prevalence of astrovirus could be underestimated if only EIAs were used for detection.

Acute Disease↗

PubMed2006

Quantitative and simultaneous detection of four foodborne bacterial pathogens with a multi-channel SPR sensor.

We report the quantitative and simultaneous detection of four species of bacteria, Escherichia coli O157:H7, Salmonella choleraesuis serotype typhimurium, Listeria monocytogenes, and Campylobacter jejuni, using an eight-channel surface plasmon resonance (SPR) sensor based on wavelength division multiplexing. Detection curves showing SPR response versus analyte concentration were established for each species of bacteria in buffer at pH 7.4, apple juice at native pH 3.7, and apple juice at an adjusted pH of 7.4, as well as for a mixture containing all four species of bacteria in buffer. Control experiments were performed to show the non-fouling characteristics of the sensor surface as well as the specificity of the amplification antibodies used in this study. The limit of detection (LOD) for each of the four species of bacteria in the tested matrices ranges from 3.4 x 10(3) to 1.2 x 10(5) cfu/ml. Detection curves in buffer of an individual species of bacteria in a mixture of all four species of bacteria correlated well with detection curves of the individual species of bacteria alone. SPR responses were higher for bacteria in apple juice at pH 7.4 than in apple juice at pH 3.7. This difference in sensor response could be partly attributed to the pH dependence of antibody-antigen binding.

Bacteria↗

PubMed1990

[Investigation of pathogens in the foci of pulmonary and osteo-articular tuberculosis].

The tubercle bacilli detection rate was determined by direct bacterioscopy and the culture plate method immediately in the disease foci in 123 patients with pulmonary tuberculosis and 78 patients with tuberculosis of bones and joints. The culture plate method was shown to have significant advantages over bacterioscopy. However, in some cases with negative responses to the culture plate test, bacterioscopy was the only procedure that detected the pathogen in resected lung tissues. Parallel use of 2 different media in the culture plate test provided higher frequency of the pathogen isolation. Cultures isolated from the lung foci were showed drug resistance in 1/4 of the cases. Cultures from the bone foci were in all the cases but one sensitive to all the drugs used. By their biological properties, all the isolates belonged to human tubercle bacilli. The necessity of microbiological investigation of pathogenes in tuberculosis foci is grounded.

Antitubercular Agents↗

PubMed2002

Species-specific detection of three human-pathogenic microsporidial species from the genus Encephalitozoon via fluorogenic 5' nuclease PCR assays.

This study describes fluorogenic 5' nuclease PCR assays suitable for rapid, sensitive, high-throughput detection of the human-pathogenic microsporidial species Encephalitozoon hellem, E. cuniculi and E.intestinalis. The assays utilize species-specific primer sets and a genus-specific dual fluorescent-labeled probe that anneals to a region within the Encephalitozoon 16S rRNA gene. The assay design theoretically permits the probe to be used either with one set of primers for species-level determination or with a combination of all three primer sets for a genus-level screening of samples. The linear range of all three species-specific calibration curves that were developed using serial ten-fold dilutions of genomic DNA isolated from hemacytometer counted spores was determined to be between 10(4) and 10(-1) spores per PCR sample. The coefficients of variation were < or =5.2% over the entire 5-log span of each calibration curve. When DNA isolated from flow cytometric enumerated spores from each of the three Encephalitozoon species was used to evaluate the quantitative capability of the species' respective calibration curves, the results from 34 out of 36 (94%) samples were within 2 standard deviations. The species-specificity of each assay was confirmed using DNA isolated from 10(4) spores from each of the other two Encephalitozoon species as well as DNA extracted from numerous other protozoa, algae and bacteria.

Animals↗

PubMed2002

Commercial molecular diagnostics in the U.S.: The Human Genome Project to the clinical laboratory.

Molecular diagnosis is the detection of pathogenic mutations in DNA and RNA samples to aid in detection, diagnosis, subclassification, prognosis, and monitoring response to therapy. Principles underlying nucleic-based diagnosis originate from localization, identification, and characterization of genes responsible for human disease. Clinical molecular genetics is now part of the mainstream of medical care in the United States. All commercial clinical reference laboratories now have a molecular genetic diagnostic unit, many of which are in contractual agreement with third party payers to provide services. Gene discovery provides valuable insight into the mechanisms of disease processes and gene-based markers will enable clinicians to study disease predisposition, as well as improved methods for diagnoses, prognosis, and monitoring of therapy. The broad range of mutation spectrum and type performed in the clinical laboratory requires the use of multiple technologies rather than a single typing platform. Platform choice depends on such diverse factors as local expertise, test volume, economies of scale, R&D budget, and royalties. Test validation is a major hurdle and positive control samples are often not readily available. Oversight and the regulatory environment for clinical molecular genetics laboratories in the United States are evolving rapidly. Several government agencies and private organizations are currently involved in revision of specific laboratory standards, including the Secretary's Advisory Committee on Genetic Testing (SACGT), Food and Drug Administration (FDA), Center for Disease Control (CDC), College of American Pathologists (CAP), American College of Medical Genetics (ACMG), and the individual states.

DNA Mutational Analysis↗

PubMed1993

Biosensors: applications for dairy food industry.

Biosensors are defined as indicators of biological compounds that can be as simple as temperature-sensitive paint or as complex as DNA-RNA probes. Food microbiologists are constantly seeking rapid and reliable automated systems for the detection of biological activity. Biosensors provide sensitive, miniaturized systems that can be used to detect unwanted microbial activity or the presence of a biologically active compound, such as glucose or a pesticide. Immunodiagnostics and enzyme biosensors are two of the leading technologies that have had the greatest impact on the food industry. The use of these two systems has reduced the time for detection of pathogens such as Salmonella to 24 h and has provided detection of biological compounds such as cholesterol or chymotrypsin. The continued development of biosensor technology will soon make available "on-line quality control" of food production, which will not only reduce cost of food production but will also provide greater safety and increased food quality.

Biosensing Techniques↗

PubMed1979

[Travellers' diarrhoea. I. Field study on frequency and causes in German tourists (author's transl)].

Even after successful localization of dangerous epidemics, infectious diseases represent a troublesome accompaniment of growing tourism to warm countries. Classical pathogens of diarrhoea are subjected to a worldwide control network to prevent this disease at least from being spread. Close surveillance has shown that, additionally, less clearly defined pathogens exist which affect a high percentage of tourists and cause a usually harmless but nevertheless often very annoying diarrhoea. In a tropic insular state a field study was carried out with voluntary assistance of German flight passengers in order to investigate the qualitative and quanitative importance of travellers' diarrhoea and to supplement American observations mainly gained in Mexico. In a first study we found that pathogens were detected only in six out of 173 stool samples: the pathogens were Salmonellae which had caused clinical symptoms in two cases only. With respect to diarrhoea with different origin which affected some 20 per cent of our examinees we will inquire into the importance of E. coli as pathogen, which is generally classified harmless but liable to interfere considerably with holidays and recreation.

Diarrhea↗

PubMed2002

The prevalence and PCR detection of Salmonella contamination in raw poultry.

Contaminated poultry meat has been identified as one of the principal foodborne sources of Salmonella. The development of rapid detection assays for Salmonella would enable official agencies and food industries to identify contaminated foodstuffs in a more timely manner. In addition, these diagnostic tools could allow more 'real time' decisions to be made regarding end product acceptability. In this study, a survey was carried out to determine the prevalence of Salmonella in raw broiler carcasses. A total of 198 neck skin samples were obtained from within 40 flocks at a commercial broiler slaughtering facility. The presence of Salmonella was assessed by traditional culture methods and by a Salmonella-specific polymerase chain reaction (PCR) test. Salmonella was recovered from 32 (16%) of all samples using traditional culture methods. In contrast, the PCR assay proved to be more sensitive and detected Salmonella DNA in 38 (19%) of the samples tested. The pathogen was detected in 45 (23%) of the 198 samples when culture and PCR results were combined. The sensitivity of the PCR test was also greater than culture when detecting Salmonella from within flocks (53% of flocks by PCR, 30% of flocks by culture). The combination of both tests revealed that 55% of the flocks were contaminated with Salmonella. The PCR assay proved to be a highly specific and sensitive method for detecting Salmonella and the incorporation of a routine PCR test in conjunction with standard culture could be effective in providing a more accurate profile of the prevalence of this pathogen in broiler carcasses.

Animals↗