The Emerging Biotherapeutic Agent: Akkermansia

The human gastrointestinal tract (GIT) is a well-recognized hub of microbial activities. The microbiota harboring the mucus layer of the GIT act as a defense against noxious substances, and pathogens including Clostridium difficile, Enterococcus faecium, Escherichia coli, Salmonella Typhimurium. Toxins, pathogens, and antibiotics perturb the commensal floral composition within the GIT. Imbalanced gut microbiota leads to dysbiosis, manifested as diseases ranging from obesity, diabetes, and cancer to reduced lifespan. Among the bacteria present in the gut microbiome, the most beneficial are those representing Firmicutes and Bacteroidetes. Recent studies have revealed the emergence of a novel biotherapeutic agent, Akkermansia, which is instrumental in regaining eubiosis and conferring various health benefits.     

Eutrophication and Bacterial Pathogens as Risk Factors for Avian Botulism Outbreaks in Wetlands Receiving Effluents from Urban Wastewater Treatment Plants

Due to the scarcity of water resources in the “Mancha Húmeda” Biosphere Reserve, the use of treated wastewater has been proposed as a solution for the conservation of natural threatened floodplain wetlands. In addition, wastewater treatment plants of many villages pour their effluent into nearby natural lakes. We hypothesized that certain avian pathogens present in wastewater may cause avian mortalities which would trigger avian botulism outbreaks. With the aim of testing our hypothesis, 24 locations distributed in three wetlands, two that receive wastewater effluents and one serving as a control, were monitored during a year. Sediment, water, water bird feces, and invertebrates were collected for the detection of putative avian pathogenic Escherichia coli (APEC), Salmonella spp., Clostridium perfringens type A, and Clostridium botulinum type C/D. Also, water and sediment physicochemical properties were determined. Overall, APEC, C. perfringens, and C. botulinum were significantly more prevalent in samples belonging to the wetlands which receive wastewater. The occurrence of a botulism outbreak in one of the studied wetlands coincided with high water temperatures and sediment 5-day biochemical oxygen demand (BOD₅), a decrease in water redox potential, chlorophyll a, and sulfate levels, and an increase in water inorganic carbon levels. The presence of C. botulinum in bird feces before the onset of the outbreak indicates that carrier birds exist and highlights the risk of botulinum toxin production in their carcasses if they die by other causes such as bacterial diseases, which are more probable in wastewater wetlands.     

Phenotypic and Genotypic Characterization of Bacteriocinogenic Enterococci Against <Emphasis Type="Italic">Clostridium botulinum</Emphasis>

The present study aimed to characterize Enterococcus faecalis (n = ?6) and Enterococcus faecium (n = 1) isolated from healthy chickens to find a novel perspective probiotic candidate that antagonize Clostridium botulinum types A, B, D, and E. The isolated enterococci were characterized based on phenotypic properties, PCR, and matrix-assisted laser desorption/ionization time of flight (MALDI-TOF). The virulence determinants including hemolytic activity on blood agar, gelatinase activity, sensitivity to vancomycin, and presence of gelatinase (gelE) and enterococcal surface protein (esp) virulence genes were investigated. Also, the presence of enterocin structural genes enterocin A, enterocin B, enterocin P, enterocin L50A/B, bacteriocin 31, enterocin AS48, enterocin 1071A/1071B, and enterocin 96 were assessed using PCR. Lastly, the antagonistic effect of the selected Enterococcus spp. on the growth of C. botulinum types A, B, D, and E was studied. The obtained results showed that four out of six E. faecalis and one E. faecium proved to be free from the tested virulence markers. All tested enterococci strains exhibited more than one of the tested enterocin. Interestingly, E. faecalis and E. faecium significantly restrained the growth of C. botulinum types A, B, D, and E. In conclusion, although, the data presented showed that bacteriocinogenic Enterococcus strains lacking of virulence determinants could be potentially used as a probiotic candidate against C. botulinum in vitro; however, further investigations are still urgently required to verify the beneficial effects of the tested Enterococcus spp. in vivo.     

Seasonal Occurrence and Distribution of Microbial Indicators and Pathogens in the Rhode River of Chesapeake Bay

The seasonal incidence and occurrence of indicator organisms and pathogens were studied at four sites in the Rhode River, a subestuary of Chesapeake Bay. The highest frequency of occurrence of total and fecal coliforms and fecal streptococci was in Muddy Creek, a marsh area receiving pasture land runoff. Second highest frequency of occurrence of these bacteria was in Cadle Creek, a populated area. Lowest measurements of these parameters were obtained at stations in the central portion of the Rhode River. No Salmonella spp. were detected by the methods employed in this study. However, it is concluded that if these organisms are present, the concentrations are ≤1 organism per liter. The presence of Clostridium botulinum was detected in 12% of the samples tested.     

Enteric bacteria isolated from acute diarrheal patients in the Republic of Korea between the year 2004 and 2006

In an epidemiological survey of human enterobacterial infections in the Republic of Korea during three years from 2004 to 2006, we isolated 1,784 (6.2%, isolation rate of enteropathogens from stool samples) in 2004, 2,547 (9.5%) in 2005 and 3,506 bacteria (12.3%) from people who visited clinics. Among the isolated bacteria, pathogenic Escherichia coli, especially, EAEC was the most frequently identified pathogen in both urban and rural regions followed by Staphylococcus aureus, Salmonella species, Bacillus cereus, Vibrio parahaemolyticus, Campylobacter jejuni, Clostridium perfringens, and Shigella species. Distinct seasonality was found in V. parahaemolyticus species, while this pathogen showed no age-specific patterns. However, other bacteria, i.e., pathogenic E. coli, S. aureus, Salmonella spp., and B. cereus showed similar seasonality throughout the year, showing a slight increase in the infection rate during the summer months and high prevalence among children under 10 years of age and elder-age people. The antibiotic susceptibility patterns of pathogenic E. coli, Salmonella spp., and S. aureus showed high resistance to penicillins. However, both pathogenic E. coli and Salmonella spp. were susceptible to several cephems, imipenem, and amikacin. Moreover, S. aureus strains resistant to vancomycin were not found. In conclusion, these surveillances can play an important role for the control and prevention to the diseases originated by enteritis bacteria.     

Focus: Zoonotic Disease: Bacterial Infections in Humans and Nonhuman Primates from Africa: Expanding the Knowledge

The close phylogenetic relationship between humans and other primates creates exceptionally high potential for pathogen exchange. The surveillance of pathogens in primates plays an important role in anticipating possible outbreaks. In this study, we conducted a molecular investigation of pathogenic bacteria in feces from African nonhuman primates (NHPs). We also investigated the pathogens shared by the human population and gorillas living in the same territory in the Republic of Congo. In total, 93% of NHPs (n=176) and 95% (n=38) of humans were found to carry at least one bacterium. Non-pallidum Treponema spp. (including T. succinifaciens, T. berlinense, and several potential new species) were recovered from stools of 70% of great apes, 88% of monkeys, and 79% of humans. Non-tuberculosis Mycobacterium spp. were also common in almost all NHP species as well as in humans. In addition, Acinetobacter spp., members of the primate gut microbiota, were mainly prevalent in human and gorilla. Pathogenic Leptospira spp. were highly present in humans (82%) and gorillas (66%) stool samples in Congo, but were absent in the other NHPs, therefore suggesting a possible gorillas-humans exchange. Particular attention will be necessary for enteropathogenic bacteria detected in humans such as Helicobacter pylori, Salmonella spp. (including S. typhi/paratyphi), Staphyloccocus aureus, and Tropheryma whipplei, some of which were also present in gorillas in the same territory (S. aureus and T. whipplei). This study enhances our knowledge of pathogenic bacteria that threaten African NHPs and humans by using a non-invasive sampling technique. Contact between humans and NHPs results in an exchange of pathogens. Ongoing surveillance, prevention, and treatment strategies alone will limit the spread of these infectious agents.     

Hermetia illucens L. larvae–associated intestinal microbes reduce the transmission risk of zoonotic pathogens in pig manure

Black soldier fly (BSF) larvae are considered a promising biological reactor to convert organic waste and reduce the impact of zoonotic pathogens on the environment. We analysed the effects of BSF larvae on Staphylococcus aureus and Salmonella spp. populations in pig manure (PM), which showed that BSF larvae can significantly reduce the counts of the associated S. aureus and Salmonella spp. Then, using a sterile BSF larval system, we validated the function of BSF larval intestinal microbiota in vivo to suppress pathogens, and lastly, we isolated eight bacterial strains from the BSF larval gut that inhibit S. aureus. Results indicated that functional microbes are essential for BSF larvae to antagonise S. aureus. Moreover, the analysis results of the relationship between the intestinal microbiota and S. aureus and Salmonella spp. showed that Myroides, Tissierella, Oblitimonas, Paenalcalignes, Terrisporobacter, Clostridium, Fastidiosipila, Pseudomonas, Ignatzschineria, Savagea, Moheibacter and Sphingobacterium were negatively correlated with S. aureus and Salmonella. Overall, these results suggested that the potential ability of BSF larvae to inhibit S. aureus and Salmonella spp. present in PM is accomplished primarily by gut‐associated microorganisms.

Black soldier fly larvae can inhibit zoonotic pathogens in pig manure. Intestinal microbes are essential for black soldier fly larvae to antagonise zoonotic pathogens. Black soldier fly larvae and their intestinal microbes can reduce zoonotic pathogens transmission risk in pig manure.     

In vitro evaluation of the antimicrobial activity of a range of probiotics against pathogens: Evidence for the effects of organic acids

The aim of this study was to investigate the antimicrobial properties of fifteen selected strains belonging to the Lactobacillus, Bifidobacterium, Lactococcus, Streptococcus and Bacillus genera against Gram-positive and Gram-negative pathogenic bacteria.In vitro antibacterial activity was initially investigated by an agar spot method. Results from the agar spot test showed that most of the selected strains were able to produce active compounds on solid media with antagonistic properties against Salmonella Typhimurium, Escherichia coli, Enterococcus faecalis, Staphylococcus aureus and Clostridium difficile. These results were also confirmed when cell-free culture supernatants (CFCS) from the putative probiotics were used in an agar well diffusion assay. Neutralization of the culture supernatants with alkali reduced the antagonistic effects. These experiments are able to confirm the capacity of potential probiotics to inhibit selected pathogens. One of the main inhibitory mechanisms may result from the production of organic acids from glucose fermentation and consequent lowering of culture pH. This observation was confirmed when the profile of organic acids was analysed demonstrating that lactic and acetic acid were the principal end products of probiotic metabolism.Furthermore, the assessment of the haemolytic activity and the susceptibility of the strains to the most commonly used antimicrobials, considered as basic safety aspects, were also studied.The observed antimicrobial activity was mainly genus-specific, additionally significant differences could be observed among species.     

Gut Microbiome of an 11th Century A.D. Pre-Columbian Andean Mummy

The process of natural mummification is a rare and unique process from which little is known about the resulting microbial community structure. In the present study, we characterized the microbiome of paleofeces, and ascending, transverse and descending colon of an 11^th century A.D. pre-Columbian Andean mummy by 16S rRNA gene high-throughput sequencing and metagenomics. Firmicutes were the most abundant bacterial group, with Clostridium spp. comprising up to 96.2% of the mummified gut, while Turicibacter spp. represented 89.2% of the bacteria identified in the paleofeces. Microbiome profile of the paleofeces was unique when compared to previously characterized coprolites that did not undergo natural mummification. We identified DNA sequences homologous to Clostridium botulinum, Trypanosoma cruzi and human papillomaviruses (HPVs). Unexpectedly, putative antibiotic-resistance genes including beta-lactamases, penicillin-binding proteins, resistance to fosfomycin, chloramphenicol, aminoglycosides, macrolides, sulfa, quinolones, tetracycline and vancomycin, and multi-drug transporters, were also identified. The presence of putative antibiotic-resistance genes suggests that resistance may not necessarily be associated with a selective pressure of antibiotics or contact with European cultures. Identification of pathogens and antibiotic-resistance genes in ancient human specimens will aid in the understanding of the evolution of pathogens as a way to treat and prevent diseases caused by bacteria, microbial eukaryotes and viruses.     

Salmonella spp., Vibrio spp., Clostridium perfringens, and Plesiomonas shigelloides in Marine and Freshwater Invertebrates from Coastal California Ecosystems

The coastal ecosystems of California are highly utilized by humans and animals, but the ecology of fecal bacteria at the land–sea interface is not well understood. This study evaluated the distribution of potentially pathogenic bacteria in invertebrates from linked marine, estuarine, and freshwater ecosystems in central California. A variety of filter-feeding clams, mussels, worms, and crab tissues were selectively cultured for Salmonella spp., Campylobacter spp., Escherichia coli-O157, Clostridium perfringens, Plesiomonas shigelloides, and Vibrio spp. A longitudinal study assessed environmental risk factors for detecting these bacterial species in sentinel mussel batches. Putative risk factors included mussel collection near higher risk areas for livestock or human sewage exposure, adjacent human population density, season, recent precipitation, water temperature, water type, bivalve type, and freshwater outflow exposure. Bacteria detected in invertebrates included Salmonella spp., C. perfringens, P. shigelloides, Vibrio cholerae, Vibrio parahaemolyticus, and Vibrio alginolyticus. Overall, 80% of mussel batches were culture positive for at least one of the bacterial species, although the pathogens Campylobacter, E. coli-O157, and Salmonella were not detected. Many of the same bacterial species were also cultured from upstream estuarine and riverine invertebrates. Exposure to human sewage sources, recent precipitation, and water temperature were significant risk factors for bacterial detection in sentinel mussel batches. These findings are consistent with the hypothesis that filter-feeding invertebrates along the coast concentrate fecal bacteria flowing from land to sea and show that the relationships between anthropogenic effects on coastal ecosystems and the environmental niches of fecal bacteria are complex and dynamic.     

Changes in bacterial populations in the ileum of pigs fed low-phosphorus diets supplemented with different sources of fermentable carbohydrates

An experiment was conducted to evaluate the effects of differently fermentable carbohydrates on changes in bacterial populations in the ileum of growing pigs fed low-phosphorus (P) diets. Eight barrows (mean surgery BW 36 ± 0.9 kg) were fitted with simple T-cannulae at the distal ileum and were assigned to one of four dietary treatments: maize-soybean meal based control diet (CD), or 0.75 of CD supplemented with 0.25 lignocellulose, maize starch and high-methylated apple-pectin, respectively. Total bacterial cell counts as well as cell counts of Lactobacillus spp., Lactobacillus reuteri, Lactobacillus amylovorus/Lactobacillus sobrius, Lactobacillus mucosae, Enterococcus spp., Enterococcus faecium, Enterococcus faecalis, bifidobacteria, Clostridium coccoides cluster, Clostridium leptum cluster, Bacteroides–Prevotella–Porphyrmonas group and Enterobacteriaceae were determined by quantitative realtime PCR in DNA extracts of ileal digesta. Denaturing gradient gel electrophoresis (DGGE) of DNA fragments, generated by PCR targeting total or Lactobacillus spp. 16S rDNA, was used to estimate the bacterial diversity in the ileum. Lignocellulose supplementation tended (P<0.1) to increase cell counts of total bacteria in faeces compared with the control. Ileal bacterial populations responded differently to carbohydrate addition. Maize starch supplementation strongly stimulated the growth of total lactobacilli and Lactobacillus species (P≤0.05). Lignocellulose, in turn, enhanced the numbers of bifidobacteria, but reduced those of L. amylovorus compared with the control (P<0.05). Finally, pectin tended to increase the cell numbers of L. amylovorus/L. sobrius and the Bacteroides–Prevotella–Porphyrmonas group compared with the control (P<0.1). DGGE analysis revealed increased band numbers for total bacteria in the ileum of animals fed the lignocellulose and maize starch supplemented diets, while pectin reduced total bacterial (P<0.1) and Lactobacillus spp. diversity (P<0.05) compared with the control, as determined with the Shannon's index. Ileal VFA concentrations were decreased by pectin, while lignocellulose decreased faecal VFA concentrations. In conclusion, ileal bacterial populations and diversity are susceptible to changes in the carbohydrate composition of the diet. However, these changes were not related to major differences in the number of total bacteria in ileal digesta and faeces, but rather to changes in the bacterial species composition and their metabolic activity.     

Isolation and Identification of the Microbiota of Danish Farmhouse and Industrially Produced Surface-Ripened Cheeses

For studying the microbiota of four Danish surface-ripened cheeses produced at three farmhouses and one industrial dairy, both a culture-dependent and culture-independent approach were used. After dereplication of the initial set of 433 isolates by (GTG)5-PCR fingerprinting, 217 bacterial and 25 yeast isolates were identified by sequencing of the 16S rRNA gene or the D1/D2 domain of the 26S rRNA gene, respectively. At the end of ripening, the cheese core microbiota of the farmhouse cheeses consisted of the mesophilic lactic acid bacteria (LAB) starter cultures Lactococcus lactis subsp. lactis and Leuconostoc mesenteorides as well as non-starter LAB including different Lactobacillus spp. The cheese from the industrial dairy was almost exclusively dominated by Lb. paracasei. The surface bacterial microbiota of all four cheeses were dominated by Corynebacterium spp. and/or Brachybacterium spp. Brevibacterium spp. was found to be subdominant compared to other bacteria on the farmhouse cheeses, and no Brevibacterium spp. was found on the cheese from the industrial dairy, even though B. linens was used as surface-ripening culture. Moreover, Gram-negative bacteria identified as Alcalignes faecalis and Proteus vulgaris were found on one of the farmhouse cheeses. The surface yeast microbiota consisted primarily of one dominating species for each cheese. For the farmhouse cheeses, the dominant yeast species were Yarrowia lipolytica, Geotrichum spp. and Debaryomyces hansenii, respectively, and for the cheese from the industrial dairy, D. hansenii was the dominant yeast species. Additionally, denaturing gradient gel electrophoresis (DGGE) analysis revealed that Streptococcus thermophilus was present in the farmhouse raw milk cheese analysed in this study. Furthermore, DGGE bands corresponding to Vagococcus carniphilus, Psychrobacter spp. and Lb. curvatus on the cheese surfaces indicated that these bacterial species may play a role in cheese ripening.     

Dynamics of Bacterial Communities during the Ripening Process of Different Croatian Cheese Types Derived from Raw Ewe's Milk Cheeses

Microbial communities play an important role in cheese ripening and determine the flavor and taste of different cheese types to a large extent. However, under adverse conditions human pathogens may colonize cheese samples during ripening and may thus cause severe outbreaks of diarrhoea and other diseases. Therefore in the present study we investigated the bacterial community structure of three raw ewe''s milk cheese types, which are produced without the application of starter cultures during ripening from two production sites based on fingerprinting in combination with next generation sequencing of 16S rRNA gene amplicons. Overall a surprisingly high diversity was found in the analyzed samples and overall up to 213 OTU₉₇ could be assigned. 20 of the major OTUs were present in all samples and include mostly lactic acid bacteria (LAB), mainly Lactococcus, and Enterococcus species. Abundance and diversity of these genera differed to a large extent between the 3 investigated cheese types and in response to the ripening process. Also a large number of non LAB genera could be identified based on phylogenetic alignments including mainly Enterobacteriaceae and Staphylococcacae. Some species belonging to these two families could be clearly assigned to species which are known as potential human pathogens like Staphylococcus saprophyticus or Salmonella spp. However, during cheese ripening their abundance was reduced. The bacterial genera, namely Lactobacillus, Streptococcus, Leuconostoc, Bifidobacterium, Brevibacterium, Corynebacterium, Clostridium, Staphylococcus, Thermoanerobacterium, E. coli, Hafnia, Pseudomonas, Janthinobacterium, Petrotoga, Kosmotoga, Megasphaera, Macrococcus, Mannheimia, Aerococcus, Vagococcus, Weissella and Pediococcus were identified at a relative low level and only in selected samples. Overall the microbial composition of the used milk and the management of the production units determined the bacterial community composition for all cheese types to a large extend, also at the late time points of cheese ripening.     

Simultaneous and rapid detection of enteric pathogens from raw milk by multiplex PCR

Enteroinvasive Escherichia coli (EIEC), heat-labile enterotoxin (LT) E. coli, Shigella spp., and Salmonella spp. are common enteric pathogens, which cause food-borne diseases if consumed in contaminated milk products. The rapid and reliable methods for detecting are imperative for reduction in hazard of infection. In this study, we selected primers, optimized the polymerase chain reaction (PCR) conditions, and analyzed the sensitivity and specificity of the multiplex PCR assay to screen raw milk from these enteric bacteria. Furthermore, EIEC, LT-E. coli, Shigella spp., Salmonella spp., and 11 non-targeted pathogenic strains were performed for the specificity of the multiplex PCR. Specific bands showed in EIEC, LT-E. coli, Shigella spp., and Salmonella spp. but no bands showed in other 11 pathogenic strains. The sensitivity of multiplex PCR was relatively high, was rounded to 200 CFU/ml (Shigella spp. and EIEC), 320 CFU/ml (Salmonella spp.), and 100 CFU/ml (LT-E. coli). This method for simultaneous and rapid detection of enteric pathogens (EIEC, LT-E. coli, Shigella spp., and Salmonella spp.) in raw milk showed high sensitivity and specificity, and led to faster track to report results.     

Identification of free-living amoebas and amoeba-resistant bacteria accumulated in Dreissena polymorpha

To identify the free-living amoeba (FLA) and amoeba-resistant bacteria (ARB) accumulated in zebra mussels and in the water in which they are found, mussels were collected at two locations in the Ebro river basin (North East Spain). FLAs and bacteria were isolated from mussel extracts and from natural water. PCR techniques were used to identify the FLAs and endosymbiont bacteria (Legionella, Mycobacterium, Pseudomonas and cyanobacteria), and to detect Giardia and Cryptosporidium. The most frequently found FLAs were Naegleria spp. The presence of Legionella, Mycobacterium and Pseudomonas inside the FLA was demonstrated, and in some cases both Legionella and Pseudomonas were found together. Differences between FLAs and ARB identified inside the mussels and in the water were detected. In addition, Escherichia coli, Clostridium perfringens, Salmonella spp. and Enterococcus spp. were accumulated in mussels in concentrations unconnected with those found in water. The results show the ability of the zebra mussel to act as a reservoir of potentially pathogenic FLAs, which are associated with potentially pathogenic ARB, although the lack of association between microorganisms inside the mussels and in the water suggests that they are not useful for monitoring microbiological contamination at a specific time.     

Molecular Characterization of Skin Microbiota Between Cancer Cachexia Patients and Healthy Volunteers

Systemic inflammation contributes to both the development of cancer and of cachexia. The microenvironment of bacterial habitats might be changed during the progression of cancer cachexia. The aim of this study was to quantitatively and qualitatively compare the composition of the skin microbiota between cancer cachexia patients and healthy volunteers. Cutaneous bacteria were swabbed at the axillary fossa of 70 cancer cachexia patients and 34 healthy individuals from China. Nested-PCR-denaturing gradient gel electrophoresis (PCR-DGGE) with primers specifically targeting V3 region and quantitative PCR (qPCR) for total bacteria, Corynebacterium spp., Staphylococcus spp., and Staphylococcus epidermidis were performed on all samples. Barcoded 454 pyrosequencing of the V3–V4 regions was performed on 30 randomly selected samples. By comparing diversity and richness indices, we found that the skin microbiome of cachectic cancer patients is less diverse than that of healthy participants, though these differences were not significant. The main microbes that reside on human skin were divided into four phyla: Firmicutes, Actinobacteria, Proteobacteria, and Bacteroidetes. Staphylococcus spp. and Corynebacterium spp. were the dominant bacteria at the genus level. Significantly fewer Corynebacterium spp. had been observed in cachexia patients compared to healthy subjects. These results suggest that the presence of cancer and cachexia alters human skin bacterial communities. Understanding the changes in microbiota during cancer cachexia may lead to new insights into the syndrome.     

The interaction of the antimicrobial peptide cLEAP-2 and the bacterial membrane

Chicken Liver Expressed Antimicrobial Peptide-2 (cLEAP-2) is known to have killing activities against Salmonella spp., but the mechanism by which killing occurs remains to be elucidated. The ability of cLEAP-2 to disrupt the outer membrane of several Salmonella spp. was assessed using the fluorescent probe N-phenyl-1-naphthylamine (NPN). A rapid dose-dependent permeabilization of the outer membranes of Salmonella enterica serovar Typhimurium phoP, and S. enterica serovar Typhimurium SL1344 was observed although no significant permeabilisation of the S. enteriditis membrane was detected. These data suggested that the ability of the mature cLEAP-2 peptide to permeabilise the Salmonella outer membrane is important in mediating its killing activities. The ability of the peptide to kill Gram-positive bacteria, specifically Streptococcus spp. and Staphylococcus spp. was also investigated using recombinant peptide and a time-kill assay. Of the strains analysed the Streptococcus pyogenes M1 strain appeared the most resistant to LEAP-2 killing although S. pyogenes mutants deficient in Sortase and M1 activities showed increased sensitivity to the mature peptide. This suggested the involvement of specific Streptococcus cell wall proteins including M1 in the resistance of the bacteria to cLEAP-2 killing. cLEAP-2 showed no significant toxicity towards mammalian erythrocytes indicating selectivity for bacterial over eukaryote cell membranes. These data provide further support for mature cLEAP-2 functioning in protecting the chicken against microbial attack.     

Response of the Rumen Microbiota of Sika Deer (Cervus nippon) Fed Different Concentrations of Tannin Rich Plants

High throughput sequencing was used to examine the rumen microbiota of sika deer fed high (OLH) and low concentration (OLL) of tannin rich oak leaves. The results showed that Prevotella spp. were the most dominant bacteria. The most predominant methanogens were the members of the order Methanoplasmatales. The dominant rumen protozoa were Entodinium longinucleatum, Eudiplodinium maggii, and Epidinium caudatum, and the fungal communities were mostly represented by Piromyces spp. Moreover, the relative abundance of Pseudobutyrivibrio spp. (P=0.026), unidentified bacteria (P=0.028), and Prevotella spp. (P=0.022) was lower in the OLH group than in the OLL group. The concentration of propionate in the OLH group was greater than in the OLL group (P=0.006). Patterns of relationships showed that methanogens belonging to the order Methanoplasmatales were negatively correlated with Treponema spp., Ent. Longinucleatum, and acetate. Methanosphaera stadtmanae was positively correlated to propionate, while Methanobrevibacter ruminantium was negatively associated with Methanobrevibacter thaueri and Methanobrevibacter millerae. Tannins altered the rumen microbes and fermentation patterns. However, the response of the entire rumen microbiota and the relationship between rumen microorganisms and the fermentation parameters were not fully understood.     

Zn finger-based direct detection system for PCR products of <Emphasis Type="Italic">Salmonella</Emphasis> spp. and the Influenza A virus

We have detected PCR products from Salmonella spp. and Influenza A virus using Zn finger protein Zif268 and Sp1, respectively. Previously, we demonstrated a novel method of rapid and specific detection of PCR products from Legionella pneumophila genome using Zn finger protein Sp1. In principle, this methodology might be applied to the detection of most bacteria and viruses using various Zn finger proteins. Here, to demonstrate the wider applicability of our method, we detected PCR products from Salmonella spp. and the Influenza A virus. BLAST data indicated the Zif268 and Sp1 recognition sequence were located on the gyrB gene of Salmonella spp. and the nucleoprotein gene of Influenza A virus, respectively. The PCR products from the oligonucleotide corresponding to the gyrB gene of Salmonella spp. or the nucleoprotein gene of the Influenza A virus could be specifically detected by ELISA or fluorescence depolarization measurement using Zif268 or Sp1. These results indicate the wide applicability of our novel methodology.     

Associations between Vaginal Pathogenic Community and Bacterial Vaginosis in Chinese Reproductive-Age Women

Background

Bacterial vaginosis (BV) is one of the most common urogenital infections among women of reproductive age that represents shifts in microbiota from Lactobacillus spp. to diverse anaerobes. The aim of our study was to evalute the diagnostic values of Gardnerella, Atopobium, Eggerthella, Megasphaera typeI, Leptotrichia/Sneathia and Prevotella, defined as a vaginal pathogenic community for BV and their associations with vaginal pH and Nugent scores.

Methods and Findings

We investigated the vaginal pathogenic bacteria and Lactobacillus spp. with species-specific real-time quantitative PCR (qPCR) in 50 BV-positive and 50 BV-negative Chinese women of reproductive age. Relative to BV-negative subjects, a siginificant decline in Lactobacillus and an obvious increase in bacteria in the vaginal pathogenic community were observed in BV-postive subjects (P<0.05). With the exception of Megasphaera typeI, other vaginal pathogenic bacteria were highly predictable for BV with a better sensitivity and specificity. The vaginal pathogenic community was positively associated with vaginal pH and Nugent scores, while Lactobacillus spp., such as L. iners and L. crispatus was negatively associated with them (P<0.05).

Conclusions

Our data implied that the prevalance of vaginal pathogenic bacteria as well as the depletion of Lactobacillus was highly accurate for BV diagnosis. Vaginal microbiota shifts, especially the overgrowth of the vaginal pathogenic community, showed well diagnostic values in predicting BV. Postive correlations between those vaginal pathogenic bacteria and vaginal pH, Nugent score indicated the vaginal pathogenic community rather than a single vaginal microorganism, was participated in the onset of BV directly.