Lipopolysaccharide from Burkholderia thailandensis E264 provides protection in a murine model of melioidosis

Burkholderia thailandensis is a less virulent close relative of Burkholderia pseudomallei, a CDC category B biothreat agent. We have previously shown that lipopolysaccharide (LPS) extracted from B. pseudomallei can provide protection against a lethal challenge of B. pseudomallei in a mouse model of melioidosis. Sugar analysis on LPS from B. thailandensis strain E264 confirmed that this polysaccharide has a similar structure to LPS from B. pseudomallei. Mice were immunised with LPS from B. thailandensis or B. pseudomallei and challenged with a lethal dose of B. pseudomallei strain K96243. Similar protection levels were observed when either LPS was used as the immunogen. This data suggests that B. thailandensis LPS has the potential to be used as part of a subunit based vaccine against pathogenic B. pseudomallei.     

Comparison of the protective effects of killed Burkholderia pseudomallei and CpG oligodeoxynucleotide against live challenge

Melioidosis is a fatal disease caused by Burkholderia pseudomallei. Currently there is no vaccine available. Synthetic oligodeoxynucleotides with unmethylated CpG dinucleotide motifs (CpG ODN) can stimulate vertebrate immune cells and clear certain pathogens that are susceptible to a strong Th1 response. In our previous study, pretreatment with CpG ODN alone or CpG-ODN with cationic liposomes for 2–10 or 30 days before B. pseudomallei infection in mice conferred 80–100% protection. In the present study we investigated the protective effect of CpG-ODN together with heat-killed (HK) or paraformaldehyde-killed B. pseudomallei (PP). HK or PP were used to immunize BALB/c mice twice at 15-day intervals before intra-peritoneal challenge with 5LD50 of B. pseudomallei and observed for 30 days. We found that PP could significantly protect mice (60%) with an increased survival time (24.8 ± 11.63 days) while in the HK and PBS groups, all infected mice died within 6 days. Although either CpG ODN or PP conferred significant protection, giving them in combination did not enhance it further. Serum IFN-γ levels on day-5 (before challenge) of the PP and PP + CpG ODN groups were significantly higher than those of the PBS control group. The results further support the importance of IFN-γ in host protection against B. pseudomallei and suggest further study on paraformaldehyde-killed bacteria as a component of a future B. pseudomallei vaccine.     

Use of Whole-Genome Sequencing to Link Burkholderia pseudomallei from Air Sampling to Mediastinal Melioidosis,Australia

The frequency with which melioidosis results from inhalation rather than percutaneous inoculation or ingestion is unknown. We recovered Burkholderia pseudomallei from air samples at the residence of a patient with presumptive inhalational melioidosis and used whole-genome sequencing to link the environmental bacteria to B. pseudomallei recovered from the patient.     

Improved culture-based detection and quantification of Burkholderia pseudomallei from soil

Environmental surveillance of the Gram-negative soil bacterium Burkholderia pseudomallei, the aetiological agent of melioidosis, is important in order to define human populations and livestock at risk of acquiring the infection. This study aimed to develop a more sensitive method for the detection of B. pseudomallei from soil samples in endemic areas compared with the currently used culture method based on soil dispersion in water. We report the development of a new protocol that involves soil dispersion in a polyethylene glycol (PEG) and sodium deoxycholate (DOC) solution to increase the yield of viable B. pseudomallei from soil samples. Comparative testing of soil samples from Northeast Thailand covering a wide range of B. pseudomallei concentrations demonstrated a significantly higher recovery (P < 0.0001) of B. pseudomallei colony-forming units by the new method compared with the conventional method. The data indicate that using the detergents PEG and DOC not only results in a higher recovery of viable B. pseudomallei but also results in a shift in the bacterial species recovered from soil samples. Future studies on the geographical distribution and population structure of B. pseudomallei in soil are likely to benefit from the new protocol described here.     

Identification and Characterization of the Antigens Expressed On the Surface of Human Erythrocytes Infected With Plasmodium falciparum

Background

Molecules expressed on the surface of infected erythrocytes (IE) with Plasmodium falciparum play important roles in malaria pathogenesis and immune evasion. Some of these molecules are specific adhesive ligands mediating adhesion of IE to the vascular endothelium. In the current study, the antigens exposed on the surface of IE with different isolates and various binding subpopulations of P. falciparum were studied.

Methods

A pooled hyper immune serum (HIS) from Malawian adults and eluted antibodies from the surface of the homologous and heterologous parasites were used. The parasite surface molecules were analyzed by Immuno-Gold-Silver enhancement (IGSE) and Western blotting. Mini-column cytoadherence method was used to select various parasite-binding subpopulations.

Results

Surface antigens of all the isolates were recognized by HIS and high recognition of antigens was observed in all isolates with homologous eluted antibodies. Western blot analysis showed that the eluted antibodies reacted with a small subset of antigens compared with HIS. Three bands, PfEMP-1, were detected in the Triton X- insoluble fraction of the ICAM-1 binding subpopulation. Another interesting band was ∼ 52-55 kDa in various isolates of P. falciparum. This molecule as defined by its low molecular weight, Triton X-100 solubility, surface location and sensitivity to 1 mg/ml trypsin.

Conclusion

The IE''s surface antigens differed in parental population compared with the selected subpopulations. These molecules could induce isolate-specific immunity. Antibodies purified from the surface of IE can be used as specific reagents to investigate parasite-derived proteins expressed on the surface of IE.     

Bats as Reservoir Hosts of Human Bacterial Pathogen,Bartonella mayotimonensis

A plethora of pathogenic viruses colonize bats. However, bat bacterial flora and its zoonotic threat remain ill defined. In a study initially conducted as a quantitative metagenomic analysis of the fecal bacterial flora of the Daubenton’s bat in Finland, we unexpectedly detected DNA of several hemotrophic and ectoparasite-transmitted bacterial genera, including Bartonella. Bartonella spp. also were either detected or isolated from the peripheral blood of Daubenton''s, northern, and whiskered bats and were detected in the ectoparasites of Daubenton''s, northern, and Brandt''s bats. The blood isolates belong to the Candidatus-status species B. mayotimonensis, a recently identified etiologic agent of endocarditis in humans, and a new Bartonella species (B. naantaliensis sp. nov.). Phylogenetic analysis of bat-colonizing Bartonella spp. throughout the world demonstrates a distinct B. mayotimonensis cluster in the Northern Hemisphere. The findings of this field study highlight bats as potent reservoirs of human bacterial pathogens.     

Molecular Characterization of Echinococcus granulosus from Hydatid Cysts Isolated from Human and Animals in Golestan Province,North of Iran

Background

The aim of the present study was to determine the molecular characteristics of Echinococcus granulosus from paraffin-embedded tissues of hydatid cysts isolated from human and protoscoleces of hydatid cysts from sheep, cattle and camel isolates using PCR- RFLP of ITS1- rDNA analysis in Golestan Province, northern Iran.

Methods

E. granulosus isolates from human patients infected with hydatid cyst and protoscoleces from hydatid cysts of sheep, cattle and camel isolates were collected from different hospitals and the abattoir throughout the Golestan Province. In all, 60 E. granulosus genomic DNA were extracted and examined by PCR - ITS1 of rDNA and amplified using BD1 / 4S and EGF1 / EGR2 primers, followed by RFLP using Alu1, Msp1 and TaqI restriction enzymes.

Results

The PCR-ITS1 products obtained from sheep, cattle and human isolates were similar to sheep strain (1000 bp and 391 bp). Majority of the camel samples yielded 295 bp DNA bands. RFLP -ITS1 of E. granulosus with Taq1 in human, sheep and cattle isolates showed similar patterns in the number and size of DNA. RFLP methods in camel isolates showed a different genotype, using Taq1, whereas no DNA bands were observed using Alu1 in camel and human isolates. Therefore, two clearly distinguishable banding patterns of E. granulosus were obtained with the three enzymes, which separating human, sheep and cattle isolates from the camel origin.

Conclusion

The results indicate the possible of transmission of the G1 and G6 genotypes of E. granulosus between livestock animals and human in Golestan Province.     

Molecular Epidemiology of Cryptosporidiosis in Iranian Children,Tehran, Iran

Background

Cryptosporidium is a worldwide protozoan parasite and one of the most common causes of infection and diarrhea in humans and cattle. The aim of the present study was determination of subtypes of Cryptosporidium among children with diarrhea in Tehran by sequence analysis of the highly polymorphic 60-kDa glycoprotein (GP60) gene.

Methods

Fecal samples were collected from 794 diarrheic children. Initial identification of Cryptosporidium was carried out on stool samples by Ziehl-Neelsen acid-fast staining method. DNA was extracted from positive microscopically samples and Cryptosporidium genotypes and subtypes were determined, accordingly.

Results

Out of 794 collected samples, 19 (2.40%) were positive for Cryptosporidium oocysts. Sequences analysis of GP60 gene showed that 17 (89.47%) of the positive isolates were Cryptosporidium parvum and 2 (10.52%) were C. hominis. All subtypes of C. parvum isolates belonged to allele families IIa (6/17) and IId (11/17). The most common allele in all 17 isolates belonged to IId A20G1a (41.18%). A22G1 (IF) subtype was detected in two C. hominis isolates of the children.

Conclusion

The predominancy of C. parvum species (specially, IId A20G1a subtype) in current study underlines the importance of zoonotic Cryptosporidium transmission in Iran.     

Potential Bio-Control Agent from Rhodomyrtus tomentosa against Listeria monocytogenes

Listeria monocytogenes is an important foodborne pathogen implicated in many outbreaks of listeriosis. This study aimed at screening for the potential use of Rhodomyrtus tomentosa ethanolic leaf extract as a bio-control agent against L. monocytogenes. Twenty-two L. monocytogenes isolates were checked with 16 commercial antibiotics and isolates displayed resistance to 10 antibiotics. All the tested isolates were sensitive to the extract with inhibition zones ranging from 14 to 16 mm. Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) values ranged from 16 to 32 µg/mL and 128 to 512 µg/mL, respectively. Time-kill assay showed that the extract had remarkable bactericidal effects on L. monocytogenes. The extract at a concentration of 16 µg/mL reduced tolerance to 10% NaCl in L. monocytogenes in 4 h. Stationary phase L. monocytogenes cells were rapidly inactivated by greater than 3-log units within 30 min of contact time with R. tomentosa extract at 128 µg/mL. Electron microscopy revealed fragmentary bacteria with changes in the physical and morphological properties. Our study demonstrates the potential of the extract for further development into a bio-control agent in food to prevent the incidence of L. monocytogenes contamination.     

Gene Diversity of Trichomonas vaginalis Isolates

Background

Trichomonas vaginalis is protozoan parasite responsible for trichomoniasis and is more common in high-risk behavior group such as prostitute individuals. Interest in trichomoniasis is due to increase one''s susceptibility to viruses such as herpes, human papillomavirus and HIV. The aim of this study was to find genotypic differences between the isolates.

Methods

Forty isolates from prisoners'' women in Tehran province were used in this study. The random amplified polymorphic DNA (RAPD) technique was used to determine genetic differences among isolates and was correlated with patient''s records. By each primer the banding pattern size of each isolates was scored (bp), genetic differences were studied, and the genealogical tree was constructed by using NTSYS software program and UPGMA method.

Results

The least number of bands were seen by using primer OPD8 and the most by using OPD3. Results showed no significant difference in isolates from different geographical areas in Iran. By using primer OPD1 specific amplified fragment with length 1300 base pair were found in only 8 isolates. All these isolates were belonged to addicted women; however, six belonged to asymptomatic patients and two to symptomatic ones.

Conclusion

There was not much genetic diversity in T vaginalis isolates from three different geographical areas.     

Multiplex Real-Time Polymerase Chain Reaction-Based Method for the Rapid Detection of gyrA and parC Mutations in Quinolone-Resistant Escherichia coli and Shigella spp.

Two real-time polymerase chain reaction assays were developed to detect mutations in codons 83 and 87 in gyrA and in codons 80 and 91 in parC, the main sites that causes quinolone resistance in pathogenic Escherichia coli and Shigella spp. isolates. These assays can be employed as a useful method for controlling infections caused by quinolone-resistant E coli and Shigella isolates.     

Duck Liver–associated Outbreak of Campylobacteriosis among Humans,United Kingdom, 2011

Campylobacter­ spp.–related gastroenteritis in diners at a catering college restaurant was associated with consumption of duck liver pâté. Population genetic analysis indicated that isolates from duck samples were typical of isolates from farmed poultry. Campylobacter spp. contamination of duck liver may present a hazard similar to the increasingly recognized contamination of chicken liver.     

Alterations of gyrA,gyrB, and parC and Activity of Efflux Pump in Fluoroquinolone-resistant Acinetobacter baumannii

Objectives

This study investigated the fluoroquinolone-resistant mechanism of 56 clinical cases of A baumannii infection from 23 non-tertiary hospitals, collected between 2004 and 2006.

Methods

Susceptibility testing was performed by broth microdilution and Epsilometer test. Analyses of quinolone resistance-determining region (QRDR) were done by sequencing. The activity of the efflux pump was measured using inhibitors.

Results

The sequences from selected 56 isolates were divided into seven groups (I-VII) on the basis of mutations in gyrA (S83L), parC (S80L, S80W and S84K) and gyrB (containing the novel mutations E679D, D644Y and A677V). The 27 isolates with triple mutations in gyrA, gyrB and parC (groups IV-VII) showed higher levels of resistance to ciprofloxacin (minimal inhibitory concentration [MIC] of 16-256 μg/mL) than the 26 isolates with double mutations in gyrA and parC (groups II and III, MIC of 8-64 μ g/mL; p < 0.05). Alterations in the efflux pump were observed in four isolates with the parC S80L mutation (group II) or E84K mutation (group VII), but no effect was observed in an isolate with the parC S80 W mutation (group III).

Conclusion

These results suggest that triple mutations in clinical isolates of A baumannii contribute to the development of high levels of resistance to fluoroquinolones and that mutations in parC S80L or E84K (groups II and VII) may contribute to alterations in efflux pump activity in A baumannii.     

Prevalence of Antibiotic Resistance in Escherichia coli Fecal Isolates From Healthy Persons and Patients With Diarrhea

Objectives

This study aimed to investigate the prevalence of antibiotic resistance in fecal Escherichia coli isolates from healthy persons and patients with diarrhea.

Methods

E. coli isolates (n = 428) were obtained from fecal samples of apparently healthy volunteers and hospitalized patients with diarrhea. Susceptibility patterns of isolates to 16 antimicrobial agents were determined by agar disc diffusion.

Results

Most E. coli isolates exhibited less than 10% resistance against imipenem, cefotetan, aztreonam, cefepime, cefoxitin, amikacin and netilamicin, although greater than 65% were resistant to ampicillin and tetracycline. No significant difference in resistance rates for all tested antibiotics was found between isolates from the healthy-and diarrheal-patient groups, including for multi-drug resistance (p = 0.22). The highest number of resistant antibiotics was 12 antibiotics. No significant differences in antibiotic resistance were found among the sex and age strata for isolates from healthy individuals. However, antibiotic resistance rates to cefoxitin, cefotaxime, amikacin, and netilamicin were significantly higher in the isolates of men than those of women (p < 0.05) in isolates from patients with diarrhea. Furthermore, isolates from patients with diarrhea older than 40-years of age showed higher resistance to cefepime and aztreonam (p < 0.05).

Conclusion

High resistance to the antibiotics most frequently prescribed for diarrhea was found in isolates from patients with diarrhea and apparently healthy individuals without any significant difference.     

Phylogenetic Location of the Spirometra Sparganum Isolates from China,Based on Sequences of 28S rDNA D1

Background

The phylogenetic location of Chinese Spirometra sparganum isolates remains unclear. The aim of this study was to explore the phylogenetic location of the Spirometra sparganum isolates from China.

Methods

The 28S ribosomal DNA (rDNA) D1 sequences of 14 Spirometra sparganum isolates collected from thirteen locations in China were analyzed by using Neighbor-Joining (NJ), maximum parsimony (MP) and Bayesian inference (BI), respectively. To investigate the deep variance of 28S rDNA D1 region among included species, the secondary structure of 28S rDNA D1 region was also calculated using the program RNA structure.

Results

The genus Spirometra as a monophyletic group was evidenced by two inference methods (MP and BI). All sequences within the genus Spirometra had a bulge of a cytosine residue (Bulge C) in the stem 13 of the secondary structure model of 28S rRNA D1 region. Varietal sites in sequences from all thirteen Chinese isolates were appeared in loops. In loops, adenine was the most abundant base (averagely 41.9%) followed by guanine (averagely 30.0%), and cytosine (averagely 15.1%). In stems, the average percentage of G + C (58.3%) was higher than the percentage of A + T (41.7%).

Conclusion

The ‘Bulge C’ in the stem 13 of the 28S rDNA D1 secondary structure could be as a suitable mark to identify the Spirometra species.     

Longitudinal and Cross-Sectional Genetic Diversity in the Korean Peninsula Based on the P vivax Merozoite Surface Protein Gene

Objectives

Vivax malaria has reemerged and become endemic in Korea. Our study aimed to analyze by both longitudinal and cross-sectional genetic diversity of this malaria based on the P vivax Merozoite Surface Protein (PvMSP) gene parasites recently found in the Korean peninsula.

Methods

PvMSP-1 gene sequence analysis from P vivax isolates (n = 835) during the 1996-2010 period were longitudinally analyzed and the isolates from the Korean peninsula through South Korea, the demilitarized zone and North Korea collected in 2008-2010 were enrolled in an overall analysis of MSP-1 gene diversity.

Results

New recombinant subtypes and severe multiple-cloneinfection rates were observed in recent vivax parasites. Regional variation was also observed in the study sites.

Conclusion

This study revealed the great complexity of genetic variation and rapid dissemination of genes in P vivax. It also showed interesting patterns of diversity depending, on the region in the Korean Peninsula. Understanding the parasiteninsula. Under genetic variation may help to analyze trends and assess the extent of endemic malaria in Korea.     

The Problem of Mixing up of Leishmania Isolates in the Laboratory: Suggestion of ITS1 Gene Sequencing for Verification of Species

Background

Leishmaniasis is endemic in Iran. Different species of Leishmania (L.) parasites are causative agents of this disease. Correct identification of Leishmania species is important for clinical studies, prevention, and control of the diseases. Mix up of Leishmania isolates is possible in the laboratory, so there is need for verification of species for isolates of uncertain identity. Different methods may be used for this purpose including isoenzyme electrophoresis and molecular methods. The isoenzyme electrophoresis, due to its drawbacks, is feasible only in specialized laboratories while molecular methods may be more feasible. The aim of this research was to study the application of the internal transcribed spacer 1 (ITS1) sequencing method, in comparison to isoenzyme electrophoresis method, for verification of Leishmania species.

Methods

Six Leishmania isolates were received from different research institutions in Iran. The species of these isolates were known by donating institution according to their isoenzyme profile. The species of these isolates were re-identified in Pasteur Institute of Iran by PCR amplification of ITS1 followed by sequencing and comparison of these sequences with Leishmania sequences in GenBank. Isoenzyme electrophoresis was performed for confirmation of the results of ITS1.

Results

ITS1 sequence showed that some isolates were mixed up or contaminated with Crithidia. Isoenzyme electrophoresis confirmed the results of ITS1 sequences.

Conclusion

ITS1 sequencing is relatively more feasible than the traditional isoenzyme electrophoresis method and is suggested for verification of Leishmania species.     

High-level Relatedness among Mycobacterium abscessus subsp. massiliense Strains from Widely Separated Outbreaks

Three recently sequenced strains isolated from patients during an outbreak of Mycobacterium abscessus subsp. massiliense infections at a cystic fibrosis center in the United States were compared with 6 strains from an outbreak at a cystic fibrosis center in the United Kingdom and worldwide strains. Strains from the 2 cystic fibrosis outbreaks showed high-level relatedness with each other and major-level relatedness with strains that caused soft tissue infections during an epidemic in Brazil. We identified unique single-nucleotide polymorphisms in cystic fibrosis and soft tissue outbreak strains, separate single-nucleotide polymorphisms only in cystic fibrosis outbreak strains, and unique genomic traits for each subset of isolates. Our findings highlight the necessity of identifying M. abscessus to the subspecies level and screening all cystic fibrosis isolates for relatedness to these outbreak strains. We propose 2 diagnostic strategies that use partial sequencing of rpoB and secA1 genes and a multilocus sequence typing protocol.     

Lower prevalence of antibiotic-resistant Enterococci on U.S. conventional poultry farms that transitioned to organic practices

Background: In U.S. conventional poultry production, antimicrobials are used for therapeutic, prophylactic, and nontherapeutic purposes. Researchers have shown that this can select for antibiotic-resistant commensal and pathogenic bacteria on poultry farms and in poultry-derived products. However, no U.S. studies have investigated on-farm changes in resistance as conventional poultry farms transition to organic practices and cease using antibiotics.Objective: We investigated the prevalence of antibiotic-resistant Enterococcus on U.S. conventional poultry farms that transitioned to organic practices.Methods: Poultry litter, feed, and water samples were collected from 10 conventional and 10 newly organic poultry houses in 2008 and tested for Enterococcus. Enterococcus (n = 259) was identified using the Vitek_® 2 Compact System and tested for susceptibility to 17 antimicrobials using the Sensititre™ microbroth dilution system. Data were analyzed using SAS software (version 9.2), and statistical associations were derived based on generalized linear mixed models.Results: Litter, feed, and water samples were Enterococcus positive. The percentages of resistant Enterococcus faecalis and resistant Enterococcus faecium were significantly lower (p < 0.05) among isolates from newly organic versus conventional poultry houses for two (erythromycin and tylosin) and five (ciprofloxacin, gentamicin, nitrofurantoin, penicillin, and tetracycline) antimicrobials, respectively. Forty-two percent of E. faecalis isolates from conventional poultry houses were multidrug resistant (MDR; resistant to three or more antimicrobial classes), compared with 10% of isolates from newly organic poultry houses (p = 0.02); 84% of E. faecium isolates from conventional poultry houses were MDR, compared with 17% of isolates from newly organic poultry houses (p < 0.001).Conclusions: Our findings suggest that the voluntary removal of antibiotics from large-scale U.S. poultry farms that transition to organic practices is associated with a lower prevalence of antibiotic-resistant and MDR Enterococcus.     

Molecular Identification and Differentiation of Fasciola Isolates Using PCR- RFLP Method Based on Internal Transcribed Spacer (ITS1, 5.8S rDNA,ITS2)

Background

In this study, we used both ITS1 and ITS2 for molecular identification of Fasciola species.

Methods

The region between 18S and 28S of ribosomal DNA was used in PCR-RFLP method for molecular identification of Fasciola species. Ninety trematodes of Fasciola were collected during abattoir inspection from livers of naturally infected sheep and cattle from Khorasan, East Azerbaijan, and Fars provinces in Iran. After DNA extraction, PCR was performed to amplify region ITS1, 5.8S rDNA, ITS2. To select a suitable restriction enzyme, we sequenced and analyzed the PCR products of F. hepatica and F. gigantica samples from sheep and cattle. Tsp509I fast digest restriction enzyme was selected for RFLP method that caused the separation specifically of Fasciola species.

Results

The fragment approximately 1000bp in all of the Fasciola samples was amplified and then digested with the Tsp509I restriction endonuclease. Seventy F. hepatica and 20 F. gigantica were identified of total 90 Fasciola isolates.

Conclusion

The new PCR-RFLP assay using Tsp509I restriction enzyme provides a simple, practical, fast, low cost, and reliable method for identification and differentiation of Fasciola isolates.