Clostridial neurotoxins, including tetanus and botulinum neurotoxins, generally target vertebrates. We show here that this family of toxins has a much broader host spectrum, by identifying PMP1, a clostridial-like neurotoxin that selectively targets anopheline mosquitoes. Isolation of PMP1 from Paraclostridium bifermentans strains collected in anopheline endemic areas on two continents indicates it is widely distributed. The toxin likely evolved from an ancestral form that targets the nervous system of similar organisms, using a common mechanism that disrupts SNARE-mediated exocytosis. It cleaves the mosquito syntaxin and employs a unique receptor recognition strategy. Our research has an important impact on the study of the evolution of clostridial neurotoxins and provides the basis for the use of P. bifermentans strains and PMP1 as innovative, environmentally friendly approaches to reduce malaria through anopheline control.
Staphylococcus aureus is a major human pathogen, especially methicillin-resistant S. aureus (MRSA), which causes a wide range of hospital and community-acquired infections worldwide. Conventional testing for detection of MRSA takes 2-5 days to yield complete information of the organism and its antibiotic sensitivity pattern.
The aim of the present study was to assess and compare the antimicrobial susceptibility pattern against a panel of antibiotics and molecular and methicillin resistance-associated genotypes of 120 carriage S. aureus isolates previously isolated from a student population at two isolation events within a one-month interval. The antibiotic susceptibility of isolates was determined using the Kirby-Bauer disc-diffusion method (cefoxitin by Etest). The MRSA was screened using polymerase chain reaction for the presence of the mecA gene. The mecA-positive isolates were subjected to staphylococcal cassette chromosome (SCC) mec typing, multilocus sequence typing (MLST) and eBURST analysis. All isolates were characterized for the presence of the Panton-Valentine leukocidin (PVL) gene, an enterobacterial repetitive intergenic consensus-polymerase chain reaction (ERIC-PCR) pattern and the spa type. For the two occasions where S. aureus was isolated, the highest frequency of resistance was observed for penicillin (70% and 65%, respectively), with a lower rate against erythromycin and tetracycline (<12%). All isolates were susceptible to ciprofloxacin and gentamycin. As for methicillin resistance, eight isolates had minimum inhibitory concentrations (MIC) of resistant categories, but 10 isolates (8.33%) were positive for the mecA gene. The mecA-positive isolates belonged to SCCmec types I (n=9) and V (n=1). MLST was resolved for only three MRSAs, ST508 (n=1), ST88 (n=1) and ST96 (n=1). The results of the eBURST analysis showed that the MRSA isolates analyzed in the present study were potentially related to MRSA identified in other countries. Approximately half of the persistent S. aureus carriers harbored S. aureus of a similar spa type in the respective individuals during both isolation events. A persistent antimicrobial pattern and limited distinct MRSAs were observed over the short study period. The latter frequently exhibited SCCmec type I, commonly associated with hospital-acquired (HA) characteristics, but further delineation is needed to justify the origins of these bacteria.
Patients with diarrhoea during enteral nutrition (EN) have been shown to have low faecal bifidobacteria concentrations. Oligofructose/inulin selectively stimulate the growth of bifidobacteria in healthy humans. This study investigates the effect of additional oligofructose/inulin on the gastrointestinal microbiota, short-chain fatty acids (SCFA) and faecal output in patients receiving EN.
This study was conducted to determine the antibiotic susceptibility pattern and distribution of exoU and exoS among 44 clinical isolates of P. aeruginosa collected from different patients over a 3-month period in 2010 at a major Malaysian hospital. Susceptibility data by disk diffusion method for cefepime (30 microg), ceftazidime (30 microg), gentamicin (10 microg), piperacillin-tazobactam (100/10 microg) and ciprofloxacin (5 microg) were available for 38 isolates. Resistance to ceftazidime and piperacillin-tazobactam was the most common (74%) with five isolates not susceptible to three or more different antibiotics. PCR detection of exoU and exoS of all 44 isolates showed the former gene to be present in 18 and exoS in 41. In analyzing the two genes together, 17 isolates were detected for exoU and exoS with only two being negative for both genes. Only one isolate was detected for exoU alone whereas 24 for exoS alone. Distribution of the genes in relation to antibiotic susceptibility was inapplicable due to the majority of the isolates having similar susceptibility patterns, but the tendency of exoU-carrying isolates to be present in male patients (83%) and respiratory sites (61%) was observed (p < 0.050). The finding warrants further investigation in a larger sample of isolates.\
Experimental evidence has implicated genotoxic Escherichia coli (E. coli) and enterotoxigenic Bacteroides fragilis (ETBF) in the development of colorectal cancer (CRC). However, evidence from epidemiological studies is sparse. We therefore assessed the association of serological markers of E. coli and ETBF exposure with odds of developing CRC in the European Prospective Investigation into Nutrition and Cancer (EPIC) study.Serum samples of incident CRC cases and matched controls (n = 442 pairs) were analyzed for immunoglobulin (Ig) A and G antibody responses to seven E. coli proteins and two isoforms of the ETBF toxin via multiplex serology. Multivariable-adjusted conditional logistic regression analyses were used to estimate odds ratios (ORs) and 95% confidence intervals (CIs) for the association of sero-positivity to E. coli and ETBF with CRC.The IgA-positivity of any of the tested E. coli antigens was associated with higher odds of developing CRC (OR: 1.42; 95% CI: 1.05-1.91). Dual-positivity for both IgA and IgG to E. coli and ETBF was associated with >1.7-fold higher odds of developing CRC, with a significant association only for IgG (OR: 1.75; 95% CI: 1.04, 2.94). This association was more pronounced when restricted to the proximal colon cancers (OR: 2.62; 95% CI: 1.09, 6.29) compared to those of the distal colon (OR: 1.24; 95% CI: 0.51, 3.00) (pheterogeneity = 0.095). Sero-positivity to E. coli and ETBF was associated with CRC development, suggesting that co-infection of these bacterial species may contribute to colorectal carcinogenesis. These findings warrant further exploration in larger prospective studies and within different population groups.
This work was conducted to study the ability of bacterial and fungal isolates from keratitis cases in Upper Egypt to produce enzymes, toxins, and to test the isolated fungal species sensitivity to some therapeutic agents.
The binary toxin from Lysinibacillus sphaericus has been successfully used for controlling mosquito-transmitted diseases. An activation step shortens both subunits BinA and BinB before their interaction with membranes and internalization in midgut cells, but the precise role of this activation step is unknown. Herein, we show conclusively using three orthogonal biophysical techniques that protoxin subunits form only monomers in aqueous solution. However, in vitro activated toxins readily form heterodimers. This oligomeric state did not change after incubation of these heterodimers with detergent. These results are consistent with the evidence that maximal toxicity in mosquito larvae is achieved when the two subunits, BinA and BinB, are in a 1:1 molar ratio, and directly link proteolytic activation to heterodimerization. Formation of a heterodimer must thus be necessary for subsequent steps, e.g., interaction with membranes, or with a suitable receptor in susceptible mosquito species. Lastly, despite existing similarities between BinB C-terminal domain with domains 3 and 4 of pore-forming aerolysin, no aerolysin-like SDS-resistant heptameric oligomers were observed when the activated Bin subunits were incubated in the presence of detergents or lipidic membranes.
The sand fly Phlebotomus papatasi is an important disease-bearing vector. Five entomopathogenic nematodes (EPNs) - Steinernema carpocapsae DD136, Steinernema sp. (SII), S. carpocapsae all, S. abbasi, and Heterorhabditis bacteriophora HP88 - were applied as biocontrol agents against the late third instar larvae of P. papatasi. In addition, the effect of toxin complexes (TCs) of Xenorhabdus nematophila and Photorhabdus luminescens laumondii bacteria was evaluated. Results revealed that S. carpocapsae DD136 was the most virulent species followed by Steinernema sp. (SII) and S. carpocapsae all where LC50 were 472, 565, 962 IJs/ml, respectively. Also, the crude TCs were slightly more active and toxic than their fractionated protein. Histopathological examination of infected larvae with H. bacteriophora HP88 showed negative effect on their midgut cells. In conclusion, EPNs with their symbiotic bacteria are more effective as biocontrol agents than the crude or fractionated TCs against sand fly larvae.
The nematode Caenorhabditis elegans is hypersusceptible to Burkholderia pseudomallei infection. However, the virulence mechanisms underlying rapid lethality of C. elegans upon B. pseudomallei infection remain poorly defined. To probe the host-pathogen interaction, we constructed GFP-tagged B. pseudomallei and followed bacterial accumulation within the C. elegans intestinal lumen. Contrary to slow-killing by most bacterial pathogens, B. pseudomallei caused fairly limited intestinal lumen colonization throughout the period of observation. Using grinder-defective mutant worms that allow the entry of intact bacteria also did not result in full intestinal lumen colonization. In addition, we observed a significant decline in C. elegans defecation and pharyngeal pumping rates upon B. pseudomallei infection. The decline in defecation rates ruled out the contribution of defecation to the limited B. pseudomallei colonization. We also demonstrated that the limited intestinal lumen colonization was not attributed to slowed host feeding as bacterial loads did not change significantly when feeding was stimulated by exogenous serotonin. Both these observations confirm that B. pseudomallei is a poor colonizer of the C. elegans intestine. To explore the possibility of toxin-mediated killing, we examined the transcription of the C. elegans ABC transporter gene, pgp-5, upon B. pseudomallei infection of the ppgp-5::gfp reporter strain. Expression of pgp-5 was highly induced, notably in the pharynx and intestine, compared with Escherichia coli-fed worms, suggesting that the host actively thwarted the pathogenic assaults during infection. Collectively, our findings propose that B. pseudomallei specifically and continuously secretes toxins to overcome C. elegans immune responses.
Various strains of Bacillus thuringiensis (Bt) have been found to produce parasporal proteins that are cytotoxic to human cancer cells. This study aims to establish the binding affinity of purified Bt 18 toxin for CEM-SS (T lymphoblastic leukaemia cell line), to determine if competition exists between the toxin and commercial anticancer drugs for the binding site on CEM-SS and to localise the binding site of the toxin on CEM-SS.
Staphylococcus aureus aregram positive cocci which colonizethe skin and mucous membranes particularly the anterior nares. Prevalence of nosocomial infections associated with methicillin resistant S. aureus have been reported in hospitals (HA-MRSA) for over five decades. Recently,community-acquired MRSA (CA-MRSA) has emerged as a cause of skin and soft tissue infections in healthy individuals. These strains are sensitive to antimicrobials, carry genes for Panton-Valentine leukocidin (PVL) toxin and belong to the staphylococcal cassette chromosome (SCC) mec type IV or V. The suspected mode of transmission involves close contact with carriers leading to skin or nasal colonization that resultin subsequent active infection. Molecular typing is used to determine the mode of transmission of CA-MRSA in the community.General typing methods such as pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST) and specific methods for Staphylococci such as SCCmec typing and spa typing have the capability to characterize bacterial chromosomes and mobile genetic elements. Combination of these molecular typing methods is necessary as each method has its own advantages with respect to discriminatory power, rapidity, cost effectiveness, reproducibility, and ease of performance.
A gene (cbm71) encoding a 71,128-Da mosquitocidal protein (Cbm71) was obtained by screening a size-fractionated XbaI digest of total genomic DNA from Clostridium bifermentans subsp. malaysia CH18 with two gene-specific oligonucleotide probes. The sequence of the Cbm71 protein, as deduced from the sequence of cbm71, corresponds to that of the 66-kDa protein previously described as one of the mosquitocidal components of C. bifermentans subsp. malaysia. Cbm71 shows limited similarities with Bacillus thuringiensis delta-endotoxins, especially in the four first conserved blocks. However, Cbm71 was not immunologically related to any of the Cry toxins and thus belongs to a novel class of mosquitocidal protein. The cbm71 gene was expressed in a nontoxic strain of B. thuringiensis, and Cbm71 was produced during sporulation and secreted to the supernatant of culture. Trichloroacetic-precipitated supernatant preparations were toxic for mosquito larvae of the species Aedes aegypti, Culex pipiens, and Anopheles stephensi.
Three new open reading frames were found downstream from cbm71, a toxin gene from Clostridium bifermentans malaysia (Cbm) strain CH18. The first one (91bp downstream) called cbm72, is 1857bp long and encodes a 71727-Da protein (Cbm72) with a sequence similar to that of Bacillus thuringiensis delta-endotoxins. This protein shows no significant toxicity to mosquito larvae. The two others, cbm17.1 (462bp) and cbm17.2 (459bp), are copies of the same gene encoding Cbm P18 and P16 polypeptides and located 426bp and 1022bp downstream from cbm72, respectively. They encode 17189-Da and 17451-Da proteins with sequences 44.6% similar to that of Aspergillus fumigatus hemolysin; however, they were not hemolytic in the conditions tested.
The toxicity of Clostridium bifermentans serovar malaysia to mosquito larvae is due to protein toxins, belonging to a novel class of insecticidal toxins. Toxic extracts contains three major proteins of 66, 18 and 16 kDa. The 18-kDa and 16-kDa proteins are probably involved in toxicity. They are synthesised during sporulation, concomitant with activity. They are absent from non-toxic strains of C. bifermentans and are present at very low levels in non-toxic C. bifermentans serovar malaysia cultures produced at 42 degrees C. The 66-kDa protein is present throughout the growth phases of C. bifermentans serovar malaysia, and an immunologically related 66-kDa protein is present in non-toxic C. bifermentans strains.
A 61-year-old male presented with community-onset pneumonia not responding to treatment despite given appropriate antibiotics. Computed tomography scan of the thorax showed large multiloculated pleural effusion with multiple cavitating foci within collapsed segments; lesions which were suggestive of necrotising pneumonia. Drainage of the effusion and culture revealed methicillin-resistant Staphylococcus aureus, which had the same antibiotic profile with the blood isolate and PVL gene positive.
Comparative laboratory bioassays of three formulations of Bacillus thuringiensis H-14 (IPS-78, San 402-I and Bactimos) were conducted against late 3rd/early 4th instar larvae of four species of mosquito, viz., Aedes aegypti, Culex quinquefasciatus, Anopheles balabacensis and Mansonia (Mansonioides) indiana, in Malaysia. From the average response of the mosquito larvae to the three formulations of B. thuringiensis H-14, Ae. aegypti was found to be most susceptible, followed by Cx. quinquefasciatus, An. balabacensis and M. (M.) indiana in decreasing order. The LC50 values for Ae. aegypti, Cx. quinquefasciatus, An. balabacensis and M. (M.) indiana after a 48-hour exposure to IPS-78 formulation were 50.9, 129.3, 117.8 and 169.6 International Toxic Unit (ITU) Ae. ae./l; to San 402-I formulation were 54.6, 223.1, 405.1 and 177.6 ITU Ae. ae/l and to Bactimos formulation were 57.2, 175.7, 35.6 and 514.5 ITU Ae. ae./l respectively. The efficacy of the bacterial product was also found to be determined by its formulation in relation to the feeding and resting habits of the mosquito larvae. No delayed pupation or emergence was observed on the larvae exposed to B. thuringiensis H-14 at sub-lethal concentrations.
Infections caused by methicillin-resistant Staphylococcus aureus (MRSA) continue to be a problem for clinicians worldwide. The objective of this study was to determine the changes in antibiograms of MRSA and their genotypic characteristics.
The prevalence of Enterotoxigenic Escherichia coli (ETEC) in 433 stool samples from diarrhoeal cases of all ages was studied using two commercially available test kits for the detection of heat labile toxin (LT) and the infant mouse assay for the heat stable toxin (ST). 16 samples (3.7%) were positive for ETEC, of which nine were producing ST alone, six LT alone and only one was producing both LT and ST. Although the percentage of isolation rate was low, its occurrence was almost as common as the Shigella spp and Salmonella spp in the same study. Of the two test kits examined, the Phadebact ETEC-LT Test 50 (Pharmacia Diagnostics, Uppsala, Sweden) was found to be more suitable for use in a routine diagnostic laboratory. Ten out of 12 (83%) of the strains tested were resistant to one or more antibiotics.