Primary Culture Plates Research Articles

Liquid chromatography mass spectrometry-based proteomics of Escherichia coli single colony

The Escherichia coli proteome is the most extensively characterized and studied of all prokaryotic proteomes. Despite this, large scale bacterial proteomics experiments performed on E. coli cells grown in liquid cultures have failed to identify key virulence factors thought to be important determinants in establishing bacterial infections. It seems likely that many important determinants associated with virulence and host cell adhesion are exclusively expressed during growth in biofilms, which can be crudely mimicked on solid media. This method describes a simple workflow to characterize the unique proteome signature of individual, isolated single colonies, using E. coli K12 strain grown on solid media as a model system. The workflow thus provides a means to explore the proteomes of minimally passaged clinical isolates of bacteria grown on primary culture plates and to identify both unique and differentially expressed proteins contained therein.Value of the method:- Simple mass spectrometry-based proteomics workflow to characterise the proteome of single colony forming units- Enables exploration of the proteomes of minimally passaged clinical isolates from primary culture plates- Identification of virulence factors expressed in true or mimicked biofilms that may be missed in liquid culturesMethod name: E. coli single colony proteome analysis

Antibacterial Susceptibility Pattern of Bacteria Isolated from Retail Fish in Okada, Edo State Nigeria

This study aimed at the microbiological analysis of retail frozen and smoked fish in Okada Edo state Nigeria. Four different retail fish samples were collected from different point of sale in Okada market. Two fresh iced and smoked samples respectively were used for the study. After sub culturing prepared samples into appropriate media, 38 bacterial isolates were obtained from primary culture plates and characterized using standard microbiological methods. Antimicrobial susceptibility tests were performed on the isolates using the Kirby-Bauer disc diffusion method. All the isolates obtained were Gram positive cocci bacteria and had a probable identity of Streptococcus sp, Staphylococcus sp and Coagulase negative Staphylococcus sp. The fresh fish samples had a minimum viable count of 1.7x 106 cfu/g and a maximum count of 4x 106 cfu/g while the smoked fish samples had a minimum viable count of 1.8x 106 cfu/g and a maximum count of 8.5x 106 cfu/g. Results from this study shows that the samples do not meet the acceptable limit (5x 105 cfu/g) of the International Commission of Microbiological Specification for Food (ICMSF). The antibiotic susceptibility test showed the presence of antibiotic resistant organisms among the retail fish samples. The detection of high viable counts of bacteria isolates beyond acceptable limits and their antibiotic resistance pattern elucidates the need for continuous monitoring of retail sea foods in the environment. Prudent use of antibiotics in aquaculture should be promoted to avoid the public health impact caused by antibiotic resistance transferred from retail sea foods to humans.

Development of a simple selection protocol for optimizing the harvest of mesenchymal stem cells from explanted human umbilical cord Wharton’s jelly

Background: Human umbilical cord Wharton's jelly (hUCWJ) derived Mesenchymal stem cells (MSCs) is a non-controversial, easily available source of human tissue which has a close association with embryonic tissue. Explant culture technique of isolating MSCs do not require any proteolytic enzymes giving an edge when compared to enzymatic methods.
 Aims and Objective: This study was designed to develop a suitable protocol to optimize the number of primary cells by transferring the explants after a successful migration and before trypsinizing of MSCs from primary culture plate to new culture plates.
 Materials and Methods: For defining the isolated cells as MSCs, the cells were studied by colony forming assay, semiquantitative two steps reverse transcriptase polymerase chain reaction (RT-PCR) and International Society for Cellular Therapy (ISCT) criteria to determine the purity of the lineage.
 Results: The explant cultured cells fulfil the defining criteria by ISCT. Further, they form colony and RT-PCR confirmed the presence of thy-1, endoglin and β-actin. The incubated explant shows successful cells migration after transferring from one culture plate to another for five consecutive times. Further, it was seen that the migrated cells can be cultured for more than 18 passages. However, a reduction was seen in the migration of the cells after the 3rd transfer of the explants.
 Conclusion: This study found that the transferred explant has the potential to yield cells for 5 consecutive explant transfers. Further, this technique optimized the yield of primary cells and thereby reducing the quantity and frequency of sample collection, processing time and cost.

Antimicrobial efficacy of Propolis, Morinda citrifolia, Azadirachta indica (Neem) against Actinomyces Radicidentis: Invitro study

Objective: The objective of this in-vitro study was to evaluate and compare the antimicrobial activity of three endodontic herbal irrigating agents, Propolis, Azadirachtaindica (neem) and Morinda citrifiolia (MCJ) against Actinomyces adicidentis. Methods: Disk diffusion method was used. After the preparation of herbal medicine, few colonies were transferred in Trypticase Soy Broth (Hope Bio China) medium contained in a test tube and kept for overnight growth. Culture was centrifuged at 3000 rpm (Cence China) for 10 minutes and 0.5 McFarland standard prepared until optical density of 600 nm was achieved. The samples were collected and 1:10 serial dilution was prepared with it. 100 µL of the suspension was added to Columbia blood agar (Oxoid) plates by using streak method. Primary culture plates were incubated at 37? in 5% CO2 incubator for 24 hours. After 24 hour zone of inhibition was achieved, sample that were analyzed by counting the colony forming units. The ANOVA test was used for statistical analysis of the data. The level of significance was set at 0.05. Result: A quantitative and qualitative analysis showed complete inhibition. Neem showed the highest zone of inhibition against Actinomyces radicidentis followed by sodium hypochlorite, Propolis and MCJ. Neem, propolis, MCJ and NaOCl showed a significant reduction (p <0.05) in the mean CFU counts for bacteria reduction. Conclusion: An in vitro observation of herbs seems promising and is highly satisfactory. It seems herbs have better efficacy, bio compatibility, economics and easily available. NaOCL have several undesirable characteristic.

PAIN AND ANTISEPSIS AFTER OCULAR ADMINISTRATION OF POVIDONE-IODINE VERSUS CHLORHEXIDINE.

To investigate ocular bacterial count before and after antisepsis with aqueous chlorhexidine (AC) or povidone-iodine (PI) and to assess discomfort with each agent. Bacterial swabs were taken from participants' eyes before and after antisepsis. These underwent microscopy, culture, and sensitivity testing. Aqueous chlorhexidine drops were administered to left eyes and PI to right eyes. Participants rated their pain (scale 0-10) for each eye but were blinded to the type of drop. There were 20 participants (17 women, 3 men), and the mean age was 43 years. Pain scores were significantly higher in right (PI) than in left (AC) eyes (mean 7 vs. mean 2, P < 0.001). No abnormalities were detected on specimen microscopy and gram staining. Seven preantisepsis swabs (three left and four right) grew bacteria in culture. Two postantisepsis swabs grew bacteria in primary culture plate (1 after AC and 1 after PI). For an additional one post-PI swab, bacteria were detected in enrichment broth only. The efficacy of AC and PI are similar, and patient discomfort is lower with AC. Aqueous chlorhexidine is a good alternative to PI for antisepsis before intravitreal injection.

Phenotypic identification of periodontal Prevotella intermedia/nigrescens group isolates validated by MALDI-TOF mass spectrometry

The accuracy of a phenotypic scheme to recognize periodontal Prevotella intermedia/nigrescens group clinical isolates on primary isolation culture plates was assessed with matrix-assisted laser desorption-ionization time-of-flight mass spectrometry (MALDI-TOF MS). A total of 84 fresh subgingival isolates from 23 chronic periodontitis patients were presumptively recognized on anaerobically-incubated enriched Brucella blood agar primary isolation plates as P. intermedia/nigrescens based on their dark-pigmented colony morphology, brick-red autofluorescence under long-wave ultraviolet light, and a negative fluorescence test for lactose production. The presumptive P. intermedia/nigrescens clinical isolates were subjected to MALDI-TOF MS analysis using Bruker MALDI Biotyper analytic software containing mass spectra for P. intermedia and Prevotella nigrescens in its reference library of bacterial protein profiles. Using a ≥1.7 log score agreement threshold, 60 (71.4%) of the presumptive P. intermedia/nigrescens clinical isolates were confirmed as either P. intermedia (25 isolates) or P. nigrescens (35 isolates). All isolates with a <1.7 log score were also identified as P. intermedia or P. nigrescens from the top choice designated on the MALDI Biotyper most likely species identification list. These MALDI-TOF MS findings document the ability of the phenotypic scheme to correctly recognize most periodontal P. intermedia/nigrescens group clinical isolates on primary isolation culture plates.

Composition of Fungal Flora in Raw Refinery Effluent, Effluent Retention Pond and a Treated Effluent Recipient River

Investigations were carried out to determine the composition of fungal flora in the studied sites. Samples of the raw effluent were collected along the flow channel and the retention pond. Water samples were also collected at the discharge point and up and down stream of the river from the discharge point. The samples were spinned at a speed of 250rpm for 10minutes and spread inoculated the deposits on potato carrot agar (PCA) and potato agar supplemented with 7.5% Nacl. Inoculated plates were incubated aerobically at room temperature in dark cupboard for 7days. Fungal colonies that emerged on the primary culture plates were distinguished into types. The pure isolates were characterized into genera using standard taxonomic guides. Genera such as Aspergillus, Penicillium, Curvularia, Fusarium, Microsporum, Trichoderma, Rhizoctonia, Nigrospora and Chaetophoma species were detected in the raw effluent. However, Microsporum, Trichoderma, Rhizoctonia, Nigrospora and Chaetophoma species were conspicuously absent in the effluent retention pond. Only Trichoderma and Chaetophoma species were absent in water samples collected at the treated effluent discharge point into the recipient River. Samples of water collected up stream of the discharge point did not contain Geotrichum, Nigrospora and Chaetophoma species. Curvularia, Microsporum, Rhizoctonia and Nigrospora species were not detected in water samples collected downstream of the discharge point. It was therefore concluded that, fungi constitute a significant proportion of the microflora of sites contaminated with the refinery effluent and could be playing an important role in the remediation of sites receiving the effluent. © JASEM Keywords : Fungal flora, Refinery effluent, Retention ponds, Discharge point, Upstream, Downstream

Automation in the Clinical Microbiology Laboratory

The clinical microbiology laboratory has historically been considered “low-tech,” especially when compared to the clinical chemistry laboratory. However, systems are emerging for the clinical microbiology laboratory with the potential to automate almost all areas of testing, including inoculation of primary culture plates, detection of growth on culture media, identification of microorganisms, susceptibility testing, and extraction and detection of nucleic acids in clinical samples. As a result, the workflow in the microbiology laboratory is changing at a rapid pace and microbiologists have the challenge of selecting the most appropriate, clinically useful, and cost-effective automation for their laboratories. We have asked 4 experts in this field, from clinical microbiology laboratories in the US and Europe, as well as from industry, to comment on the feasibility and impact of automation in the clinical microbiology laboratory. Are you currently using or do you anticipate using an automation platform in your microbiology laboratory? If yes, which sections of your laboratory are automated? Robin Patel: Mayo Clinic's clinical microbiology laboratory has been performing testing since 1911. Although select tests today resemble those performed a century ago, we have many examples of automated, state-of-the-art tests. These include blood cultures, infectious disease serologic platforms, and nucleic acid and proteomic diagnostics, to name a few. For over 2 decades, microbiology laboratories have been using automated blood culture instruments that “sense” microbial growth in blood culture bottles and “flag” positive bottles for immediate attention by laboratory technologists. Before the availability of such systems (in the not-so-distant past), laboratory technologists manually evaluated each blood culture bottle on multiple occasions. Technologists today could not fathom returning to the manual approach used a mere 3 decades ago. As with many chemistry tests, a myriad of infectious disease serologic tests are performed on automated platforms. Nucleic acid diagnostics, which have been used in our …

Photo Quiz: A Woman on Hemodialysis for End-Stage Renal Disease with Fever

A 73-year-old woman on hemodialysis for end-stage renal disease was admitted to our institution from a subacute-rehabilitation facility due to a fever of 102°F. The patient reported some nausea, diarrhea, abdominal pain, and weakness but stated that she had been experiencing these symptoms for some time. Other than the above-mentioned gastrointestinal symptoms, the patient denied any cardiopulmonary, upper respiratory, or genitourinary symptoms. No contaminated food items were identified, and no other residents at the facility were presenting with similar symptoms. Physical examination revealed a diffusely tender abdomen without evidence of any other systemic signs. A computed tomography (CT) examination of the abdomen demonstrated possible right-sided colitis, and the patient was begun on ciprofloxacin and metronidazole. Initial blood cultures (Bactec; Becton, Dickinson and Company) collected in the emergency room demonstrated no growth; however, blood cultures collected the following day, as the patient underwent hemodialysis, grew Gram-positive bacilli. Initial Gram stainings performed directly from the aerobic and anaerobic blood culture bottles demonstrated long, chaining, Gram-positive bacilli (Fig. 1). The morphology of the Gram-positive rods was suggestive of lactobacilli. The blood culture plates demonstrated pure growth of small, pale-appearing colonies. The colonies were found to be Gram-positive coccobacilli which were catalase positive and oxidase negative. A definitive identification was made from the primary culture plates. Fig 1 Gram stain of bacilli from a blood culture bottle. (For answer and discussion, see page 1655 in this issue [doi:10.1128/JCM.01126-12].)

Decrease in Pneumococcal Co-Colonization following Vaccination with the Seven-Valent Pneumococcal Conjugate Vaccine

Understanding the epidemiology of pneumococcal co-colonization is important for monitoring vaccine effectiveness and the occurrence of horizontal gene transfer between pneumococcal strains. In this study we aimed to evaluate the impact of the seven-valent pneumococcal conjugate vaccine (PCV7) on pneumococcal co-colonization among Portuguese children. Nasopharyngeal samples from children up to 6 years old yielding a pneumococcal culture were clustered into three groups: pre-vaccine era (n = 173), unvaccinated children of the vaccine era (n = 169), and fully vaccinated children (4 doses; n = 150). Co-colonization, serotype identification, and relative serotype abundance were detected by analysis of DNA of the total bacterial growth of the primary culture plate using the plyNCR-RFLP method and a molecular serotyping microarray-based strategy. The plyNCR-RFLP method detected an overall co-colonization rate of 20.1%. Microarray analysis confirmed the plyNCR-RFLP results. Vaccination status was the only factor found to be significantly associated with co-colonization: co-colonization rates were significantly lower (p = 0.004; Fisher's exact test) among fully vaccinated children (8.0%) than among children from the pre-PCV7 era (17.3%) or unvaccinated children of the PCV7 era (18.3%). In the PCV7 era there were significantly less non-vaccine type (NVT) co-colonization events than would be expected based on the NVT distribution observed in the pre-PCV7 era (p = 0.024). In conclusion, vaccination with PCV7 resulted in a lower co-colonization rate due to an asymmetric distribution between NVTs found in single and co-colonized samples. We propose that some NVTs prevalent in the PCV7 era are more competitive than others, hampering their co-existence in the same niche. This result may have important implications since a decrease in co-colonization events is expected to translate in decreased opportunities for horizontal gene transfer, hindering pneumococcal evolution events such as acquisition of antibiotic resistance determinants or capsular switch. This might represent a novel potential benefit of conjugate vaccines.

Clostridium difficile infection: monoclonal or polyclonal genesis?

Clostridium difficile is considered to be a leading cause of hospital-acquired diarrhea. C. difficile (CDI) infection shows a high rate of recurrence. There would have to be a predominantly monoclonal mechanism of CDI within individual patients in order for molecular epidemiologic tools such as polymerase chain reaction (PCR) ribotyping to be useful in outbreak investigation or differentiation between infection relapse versus re-infection. It was the aim of our study to determine whether CDI is of monoclonal or of polyclonal genesis. Between December 2009 and June 2010, 11 patients with nosocomial CDI were chosen arbitrarily. Five individual colonies of C. difficile were picked from each of the primary culture plates. Of 55 isolates gained, 47 were available for PCR ribotyping (eight isolates failed attempts to re-culture). Among these 47 isolates, eight different PCR ribotypes were identified. Only one of the 11 patients had a stool sample that yielded more than one ribotype (PCR ribotypes 438 and 232); this 67-year-old female cancer patient was already suffering from recurring diarrhea prior to the fatal episode of colitis which was subsequently investigated. We conclude that polyclonal infections may occasionally occur in patients with CDI. Our findings of predominantly monoclonal origin of CDI within patients suggest that molecular epidemiologic investigations can be used reliably for outbreak investigations or discrimination between relapse and re-infection.

Spiramycin resistance in human periodontitis microbiota

Purpose The occurrence of in vitro resistance to therapeutic concentrations of spiramycin, amoxicillin, and metronidazole was determined for putative periodontal pathogens isolated in the United States. Materials and methods Subgingival plaque specimens from 37 consecutive adults with untreated severe periodontitis were anaerobically cultured, and isolated putative periodontal pathogens were identified to a species level. In vitro resistance to spiramycin at 4 μg/ml, amoxicillin at 8 μg/ml, and/or metronidazole at 16 μg/ml was noted when putative periodontal pathogen growth was noted on the respective antibiotic-supplemented primary culture plates. Results A total of 18 (48.7%) subjects yielded antibiotic-resistant putative periodontal pathogens with spiramycin at 4 μg/ml in drug-supplemented primary isolation plates, as compared to 23 (62.2%) subjects with amoxicillin at 8 μg/ml, and 10 (27.0%) subjects with metronidazole at 16 μg/ml. Spiramycin in vitro resistance occurred among species of Fusobacterium nucleatum (44.4% of organism-positive subjects), Prevotella intermedia/nigrescens (11.1%), Parvimonas micra (10.8%), Streptococcus constellatus (10%), Streptococcus intermedius (10%), Porphyromonas gingivalis (6.7%), and Tannerella forsythia (5.3%). Amoxicillin in vitro resistance was found in P. intermedia/nigrescens (55.5%), T. forsythia (15.8%), S. constellatus (10%), F. nucleatum (5.6%), and P. micra (2.7%). Only S. constellatus (70%) and S. intermedius (40%) exhibited in vitro resistance to metronidazole. When subject-based resistance data for spiramycin and metronidazole were jointly considered, all isolated putative periodontal pathogens were inhibited in vitro by one or the other of the antibiotic concentrations, except for one strain each of S. constellatus and S. intermedius from one study subject. Similarly, either amoxicillin or metronidazole at the drug concentrations tested inhibited in vitro all recovered putative periodontal pathogens, except S. constellatus in one subject. Conclusions In vitro spiramycin resistance among putative periodontal pathogens of United States origin occurred in approximately one-half of severe periodontitis patients evaluated, particularly among subgingival F. nucleatum species. In vitro resistance patterns also suggest that therapeutic concentrations of spiramycin plus metronidazole may have potential antimicrobial efficacy in non- Aggregatibacter actinomycetemcomitans-associated periodontitis similar to amoxicillin plus metronidazole, which may be beneficial, where spiramycin is clinically available, for patients hypersensitive to amoxicillin or other beta-lactam antibiotics.

Burkholderia pseudomallei Is Genetically Diverse in Agricultural Land in Northeast Thailand

BackgroundThe soil-dwelling Gram-negative bacterium Burkholderia pseudomallei is the cause of melioidosis. Extreme structuring of genotype and genotypic frequency has been demonstrated for B. pseudomallei in uncultivated land, but its distribution and genetic diversity in agricultural land where most human infections are probably acquired is not well defined.MethodsFixed-interval soil sampling was performed in a rice paddy in northeast Thailand in which 100 grams of soil was sampled at a depth of 30 cm from 10×10 sampling points each measuring 2.5 m by 2.5 m. Soil was cultured for the presence of B. pseudomallei and genotyping of colonies present on primary culture plates was performed using a combination of pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST).Principal Findings B. pseudomallei was cultured from 28/100 samples. Genotyping of 630 primary colonies drawn from 11 sampling points demonstrated 10 PFGE banding pattern types, which on MLST were resolved into 7 sequence types (ST). Overlap of genotypes was observed more often between sampling points that were closely positioned. Two sampling points contained mixed B. pseudomallei genotypes, each with a numerically dominant genotype and one or more additional genotypes present as minority populations.ConclusionsGenetic diversity and structuring of B. pseudomallei exists despite the effects of flooding and the physical and chemical processes associated with farming. These findings form an important baseline for future studies of environmental B. pseudomallei.

Propionibacterium acnes is a common colonizer of intravascular catheters

Propionibacterium spp. are common flora of human skin. Nevertheless, currently recommended culture procedures do not include anaerobic processing with the result that this organism may go undetected on a colonized catheter. To determine the rate of catheter colonization by Propionibacterium spp., a sample of 1000 vascular catheters was processed by the roll-plate technique and, after conventional aerobic processing, all primary culture plates were reincubated in an anaerobic atmosphere. Propionibacterium acnes was detected in significant counts in the vascular catheters of 39 patients. This represents 14.7% (95% CI, 12.5-16.9) of all positive catheters. Propionibacterium is the second most frequent genus-colonizing catheter tips after Staphylococcus spp. Methodological shortcomings impair the detection and proper adscription of P. acnes as a potential cause of catheter-related infections.

Comparative Study of Ability for Growth Support and Species Differentiation by Colony Features on Commercial Chromogenic Agar, Pourmedia Vi Candida and CHROMagar Candida

Ability for growth support and species differentiation by colony features were compared on two commercial chromogenic agars, CHROMagar Candida and newly developed Pourmedia Vi Candida. Eleven strains (ten species) of standard strains and twenty-four isolates (five species) of clinical strains were tested. All isolates were grown on both agar plates in 48 hours at 35 degrees C, however, several species had not matured in 22 hours. Color of the colonies for each strain were stable on both agars. The results show that Pourmedia Vi Candida is equivalent to CHROMagar Candida in its ability to differentiate species as a primary culture plate.

Evaluation of the chromogenic Cica- -Test for detecting extended-spectrum, AmpC and metallo- -lactamases

Extended-spectrum, metallo- and AmpC beta-lactamases usually are sought subsequently to susceptibility testing, meaning that producers are not identified until 72 h after a clinical specimen is taken. Chromogenic tests might usefully shorten this delay, and we investigated the Cica-beta-Test for this purpose. Reference and clinical strains with known beta-lactamases, or controls, were grown with a cefpodoxime disc to promote conservation of resistance. The cultures were then tested with nitrocefin and with the Cica-beta-Test, which examines for hydrolysis of the chromogenic oxyimino-cephalosporin HMRZ-86 with and without specific inhibitors of extended-spectrum, metallo- and AmpC beta-lactamases. were scored, as colour changes from yellow to red, with the tester blinded to the strain identity and the mechanism(s) present. Results Proportions of extended-spectrum, metallo- and AmpC beta-lactamase producers correctly identified by the Cica-beta-Test were 85%, 77% and 72%, respectively. Such performance should be achievable if testing colonies from a primary culture plate, 24 h after a specimen was taken. Greater precision, albeit at more delay, would be achieved if results were read in conjunction with antibiogram data available 48 h after the specimen was taken. Limitations were frequent confusion of Klebsiella oxytoca hyperproducing K1 enzyme with AmpC hyperproducers, and that isolates with NMC-A or KPC carbapenemases were wrongly inferred to have AmpC enzymes. The Cica-beta-Test has the potential to provide useful therapeutic guidance, identifying isolates with potent beta-lactamases and informing early therapy; it will also help to monitor beta-lactamase epidemiology among multiresistant strains.

Recurrent Melioidosis: Possible Role of Infection with Multiple Strains of Burkholderia pseudomallei

We were interested to read the paper by Maharjan et al. suggesting that reinfection is more common than previously realized in melioidosis (1). There is one other possibility that they did not consider, which might account for isolates from recurrent melioidosis being genotypically distinct from isolates in primary infection, namely, initial infection with more than one genotype. We report here studies that suggest that this is a not infrequent occurrence. Eighteen patients with culture-positive melioidosis seen in Ubon Ratchathani, northeastern Thailand, were studied. Fourteen patients yielded the organism from blood, three presented with localized abscesses with no systemic spread, and one had a urinary tract infection. Sets of 10 to 40 colonies (dependent on the total number of colonies per plate) were selected from primary culture plates of specimens from multiple body sites or from subculture of blood culture broths. Isolates were assigned to a BamHI ribotype pattern, and pulsed-field gel electrophoresis (PFGE) of XbaI DNA digests was used to discriminate further within a ribotype (4). Strains were then allocated a ribotype number or PFGE letter as part of a larger study (3). This showed that 13 of 18 patients were infected with a single strain. In contrast, five patients harbored more than one genotype (Table ​(Table1).1). Four patients had two distinct genotypes detected, while one (A/C 383) had three. Different genotypes were detected both within a single specimen and from the same patient in samples collected on different days. Figure ​Figure11 illustrates the DNA profiles of the isolates from patient U1128. All of the 10 colonies from blood cultures were homogeneous, and the same strain was one of two recovered from a swab of a toe wound in this patient. FIG. 1. (A) BamHI ribotype patterns of B. pseudomallei isolates. Lanes 1 and 4, toe wound; lanes 2, 3, and 5, blood. (B) XbaI restriction patterns obtained by PFGE. TABLE 1. Variation in genotypes of B. pseudomallei from 5 of 18 patients with melioidosis A statistical comparison (chi-square test) of the clinical manifestations and outcomes of patients infected with single versus multiple strains revealed that the latter were more associated with an acute presentation (5/13 versus 4/4), septicemia (5/12 versus 4/5), and a fatal outcome (6/13 versus 4/5) (P < 0.001), suggesting the possibility that this reflected an acute exposure to a large inoculum. Infection with B. pseudomallei is thought to result from exposure to organisms in the environment. Relatively little is known about the occurrence of different genotypes of B. pseudomallei in the environment, but it is not unreasonable to propose that several different strains may coexist within the complex ecosystem represented by paddy field mud, for example. If so, then concurrent infection with more than one strain might thus be expected to occur on occasion. Infection with multiple strains of the same species is not unknown and was previously described in melioidosis by Sexton et al. (2), who reported the presence of two different ribotypes at different body sites of a single patient. The fact that the proportion of cases of recurrent melioidosis caused by strains with a genotype different from that of the primary isolate increases as the time between episodes increases, as reported by Maharjan et al. (1), does indeed support their suggestion that such cases represent reinfection rather than relapse. However, the finding here that 27% of patients harbored different strains concurrently accords with the frequency of reinfection (25%) reported by Maharjan et al. (1) and might provide an alternative explanation for the appearance of apparently new strains over time.

Prevalence of diarrheagenic Escherichia coli in finns with or without diarrhea during a round-the-world trip.

The incidence of diarrhea and the prevalence of bacterial enteropathogens, viruses, and parasites in feces of subjects with and without diarrhea were evaluated in 204 Finns traveling round the world (from Finland to China, Malaysia, Australia, Fiji, Chile, and Brazil and back to Finland). Special emphasis was placed on the finding of diarrheagenic Escherichia coli (enterotoxigenic, enteropathogenic, Shiga toxin-producing, and enteroaggregative strains) by PCR from growth on primary culture plates. From the PCR-positive samples, corresponding strains were isolated, confirmed as E. coli, and O serotyped. Of all the subjects, 37% experienced a total of 90 episodes of diarrhea. No adenoviruses or rotaviruses were detected, and findings of parasites were insignificant. In contrast, enteropathogenic bacteria were present in 62% of the 65 diarrheal and in 33% of the 127 nondiarrheal samples (P < 0.001); diarrheagenic E. coli strains were found in 35 and 26% of these, respectively (not statistically significant). As a single pathogen, E. coli was found in 20 and 24% of samples (not significant). Of all diarrheagenic E. coli strains, enteropathogenic strains were the most commonly found independently of the clinical picture of the subjects, whereas Salmonella enterica as a single pathogen was the most common non-E. coli organism found in diarrheal samples. Multiple bacterial pathogens were found 10 times more commonly in diarrheal than in nondiarrheal samples (20 versus 2%; P < 0.001).

Receptor Subunit-specific Action of Oncostatin M in Hepatic Cells and Its Modulation by Leukemia Inhibitory Factor

The related cytokines, interleukin-6 (IL-6), oncostatin M (OSM), and leukemia inhibitory factor (LIF) direct the formation of specific heteromeric receptor complexes to achieve signaling. Each complex includes the common signal-transducing subunit gp130. OSM and LIF also recruit the signaling competent, but structurally distinct OSMRbeta and LIFRalpha subunits, respectively. To test the hypothesis that the particularly prominent cell regulation by OSM is due to signals contributed by OSMRbeta, we introduced stable expression of human or mouse OSMRbeta in rat hepatoma cells which have endogenous receptors for IL-6 and LIF, but not OSM. Both mouse and human OSM engaged gp130 with their respective OSMRbeta subunits, but only human OSM also acted through LIFR. Signaling by OSMRbeta-containing receptors was characterized by highest activation of STAT5 and ERK, recruitment of the insulin receptor substrate and Jun-N-terminal kinase pathways, and induction of a characteristic pattern of acute phase proteins. Since LIF together with LIFRalpha appear to form a more stable complex with gp130 than OSM with gp130 and OSMRbeta, co-activation of LIFR and OSMR resulted in a predominant LIF-like response. These results suggest that signaling by IL-6 cytokines is not identical, and that a hierarchical order of cytokine receptor action exists in which LIFR ranks as dominant member.

Simplification of a locus-specific DNA typing method (Vir typing) for Streptococcus pyogenes.

We describe a simplification of a highly discriminatory molecular typing method, called Vir typing, for Streptococcus pyogenes (D. Gardiner, J. Hartas, B. Currie, J. D. Mathews, D. J. Kemp, and K. S. Sriprakash, PCR Methods Appl. 4:288-293, 1995). The procedure can be completed within a day, is reproducible, and can be applied directly to colonies growing on primary culture plates, allowing rapid establishment of strain identity in an outbreak.