Comparison of automated and conventional microbiological examination of donated human cardiac tissue in heart valve banking

Most tissue banks use the conventional method; however, the automated method has advantages over the conventional method. The aim of this study was to compare the conventional and automated methods of culture in human cardiac tissue using an artificial contamination model. Myocardial samples were contaminated with sequential concentration (10⁴ to 10^?1 CFU/mL) with Pseudomonas aeruginosa, Escherichia coli, Staphylococcus aureus and Candida albicans. Cultures were obtained from solution were the fragment was immersed and minced tissue, before and after the routine decellularization solution, with automated and conventional culture methods. Automated and conventional methods were compared and a p value ≤?0.05 was considered significant. Staphylococcus aureus presented a significantly higher growth in the automated method, as well as faster than the conventional (p?<?0.05). The positivity for growth in the automated method was higher in concentrated inoculum (>?10² CFU/mL) (p?<?0.05). The growth in the automated method was significantly faster than conventional when inoculum concentration was above 10³ CFU/mL. The automated culture method is faster than conventional method with a higher positivity in a contaminated model of myocardial and transport solution used in tissue banks.     

Rapid Detection Device for <Emphasis Type="Italic">Salmonella typhi</Emphasis> in Milk,Juice, Water and Calf Serum

A limit of detection of 200 CFU/mL of Salmonella typhi spiked in various sample matrices were achieved in 30 min. The sample matrices were raw/unprocessed milk, commercially available milk, juice from packed bottles, fresh juice from carts, potable water, turbid water and calf serum. The complete protocol comprised of three steps: (a) cell lysis (b) nucleic acid amplification and (c) an in situ optical detection. The cell lysis was carried out using a simple heating based protocol, while the loop-mediated isothermal amplification of DNA was carried out by an in-house designed and fabricated system. The developed system consists of an aluminum block fitted with two cartridge heaters along with a thermocouple. The system was coupled to a light source and spectrometer for a simultaneous in situ detection. Primers specific for STY2879 gene were used to amplify the nucleic acid sequence, isolated from S. typhi cells. The protocol involves 15 min of cell lysis and DNA isolation followed by 15 min for isothermal amplification and simultaneous detection. No cross-reactivity of the primers were observed at 10⁶ CFU/mL of Escherichia coli, Vibrio cholerae, Salmonella typhimurium, Salmonella paratyphi A, Pseudomonas aeruginosa, Bacillus cereus, Lysteria monocytogenes, Clostridium botulinum, Staphylococcus aureus and Salmonella havana. In addition, the system was able to detect S. typhi of 200 CFU/mL in a concoction of 10⁶ CFU/mL of E. coli, 10⁶ CFU/mL of V. cholerae, and 10⁶ CFU/mL of hepatocyte-derived cellular carcinoma HUH7 cells. The proposed rapid diagnostic system shows a promising future in the field of food and medical diagnostics.     

Evaluation of molecular techniques for identification and enumeration of <Emphasis Type="Italic">Raoultella terrigena</Emphasis> ATCC 33257 in water purifier efficacy testing

Raoultella terrigena ATCC 33257, a representative of the coliform group, is commonly used as a challenge organism in water purifier efficacy testing. In addition to being time consuming, traditional culturing techniques and metabolic identification systems (including automated systems) also fail to accurately differentiate this organism from its closely related neighbors belonging to the Enterobacteriaceae group. Molecular-based techniques, such as real-time quantitative polymerase chain reaction (qPCR) and enterobacterial repetitive intergenic consensus (ERIC)-PCR fingerprinting, are preferred methods of detection because of their accuracy, reproducibility, specificity, and sensitivity, along with shorter turnaround time. ERIC-PCR performed with the 1R primer set demonstrated stable unique banding patterns (~800, ~300 bp) for R. terrigena ATCC 33257 different from patterns observed for R. planticola and R. ornithinolytica. The primer pair developed from gyraseA (gyrA) sequence of R. terrigena for the SYBR Green qPCR assay using the AlleleID^® 7.0 primer probe design software was highly specific and sensitive for the target organism. The sensitivity of the assay was 10¹ colony forming units (CFU)/ml for whole cells and 4.7 fg with genomic DNA. The primer pair was successful in determining the concentration (5.5 ± 0.3 × 10⁶ CFU/ml) of R. terrigena from water samples spiked with equal concentration of Escherichia coli and R. terrigena. Based on these results from the ERIC-PCR and the SYBR Green qPCR assay, these molecular techniques can be efficiently used for rapid identification and quantification of R. terrigena during water purifier testing.     

In Vitro Antimicrobial Activity and Downregulation of Virulence Gene Expression on <Emphasis Type="Italic">Helicobacter pylori</Emphasis> by Reuterin

Helicobacter pylori is an infectious agent commonly associated with gastrointestinal diseases. The use of probiotics to treat this infection has been documented, however, their potential antimicrobial metabolites have not yet been investigated. In the present study, the effect of reuterin produced by Lactobacillus reuteri on H. pylori growth and virulence gene expression was evaluated. It was observed that reuterin caused significant (P < 0.05) H. pylori growth inhibition at concentrations from 0.08 to 20.48 mM, with minimal inhibitory concentrations (MICs) of 20.48 mM for H. pylori ATCC700824 and 10.24 mM for H. pylori ATCC43504. In a reuterin bacterial killing assay, it was observed that half of the MIC value for H. pylori (ATCC700824) significantly (P < 0.01) reduced colony numbers from 5.65 ± 0.35 to 3.78 ± 0.35 Log₁₀ CFU/mL after 12 h of treatment and then increased them to 5.25 ± 0.23 Log₁₀ CFU/mL at 24 h; at its MIC value (20.48 mM), reuterin abrogated (P < 0.01) H. pylori (ATCC700824) growth after 20 h of culture. In addition, reuterin significantly (P < 0.01) reduced H. pylori (ATCC 43504) colony numbers from 5.65 ± 0.35 to 4.1 ± 0.12 Log10 CFU/mL from 12 to 24 h of treatment and abrogated its growth at its MIC value (10.24 mM), after 20 h of treatment. Reuterin did not alter normal human gastric Hs738.St/Int cell viability at the concentrations tested for H. pylori strains. Furthermore, 10 μM reuterin was shown to significantly (P < 0.01) reduce mRNA relative expression levels of H. pylori virulence genes vacA and flaA at 3 h post-treatment, whose effect was higher at 6 h post-treatment, as measured by RT-qPCR. The observed direct antimicrobial effect and the downregulation of expression of virulence genes on H. pylori by reuterin may contribute to the understanding of the mechanisms of action of probiotics against H. pylori.     

Laboratory and simulated-field bioassays for assessing mixed cultures of <Emphasis Type="Italic">Lysinibacillus sphaericus</Emphasis> against <Emphasis Type="Italic">Aedes aegypti</Emphasis> (Diptera: Culicidae) larvae resistant to temephos

Aedes aegypti (L.) is the main vector of tropical diseases such as dengue, chikungunya and Zika. Due to the overuse of insecticides, Ae. aegypti resistant populations have increased. Biological control with Lysinibacillus sphaericus (Ahmed) has been used against Culex sp. and Anopheles sp. Although Ae. aegypti is refractory to the binary toxin of L. sphaericus spores, vegetative cells have been shown to be effective against Ae. aegypti larvae. In this work, the effect of L. sphaericus vegetative cells on Ae. aegypti temephos-resistant larvae was assessed under lab and simulated field conditions. L. sphaericus caused about 90% mortality of insecticide-resistant Ae. aegypti larvae under simulated field conditions. Likewise, Ae. aegypti larvae were more sensitive to mixed cultures of L. sphaericus than to individual strains; then, the most effective mixed culture exhibited an LC₅₀ of 1.21 × 10⁵ CFU/mL with Rockefeller larvae and 8.04 × 10⁴ CFU/mL with field-collected larvae. Additionally, we found that mixed cultures composed of two L. sphaericus strains were more effective than a culture formed by the three strains. Our results suggest that mixed cultures comprising L. sphaericus vegetative cells could be useful for controlling temephos-resistant populations of Ae. aegypti, as evidenced by the effectiveness demonstrated under laboratory and simulated field conditions.     

Diversity of culturable bacterial communities in the intestinal tracts of goldfish (<Emphasis Type="Italic">Carassius auratus</Emphasis>) and their ability to produce <Emphasis Type="Italic">N</Emphasis>-acyl homoserine lactone

Intestinal bacteria isolated from goldfish (Carassius auratus) were identified based on 16 ribosomal RNA (rRNA) gene sequences and screened for their ability to produce N-acyl homoserine lactone (AHL), an autoinducer of the quorum sensing (QS) system. The 230 aerobes/facultative anaerobes that were isolated comprised members of the genera Aeromonas (184 isolates), Citrobacter (11), Enterobacter (2), Shewanella (28), Vagococcus (1), and Vibrio (4). Among these genera, the two most abundant species were Aeromonas veronii (163 isolates) and Shewanella xiamenensis (27). In addition, 142 obligate anaerobes consisting of Cetobacterium somerae (139 isolates), Clostridium frigidicarnis (2), and Cetobacterium sp. (1) were also isolated. One hundred seventy isolates (74.2%) belonging to the genera Aeromonas, Citrobacter, Enterobacter, Shewanella, and Vibrio produced AHL, while 155 (67.7%) and 91 (39.7%) isolates possessed the luxR and luxI gene homologs, respectively. None of the obligate anaerobes produced AHL or possessed luxRI homologs. Total viable counts ranged from 1.2 × 10⁷ to 2.2 × 10⁹ CFU/g, which were accounted for 0.8 to 15.2% of direct counts. Aeromonas veronii, S. xiamenensis, and C. somerae were detected from five goldfish at densities ranging from 4.0 × 10⁶ to 1.7 × 10⁹ CFU/g, indicating that these bacteria are dominant components of the culturable gut flora in goldfish. In addition, members of the genera Aeromonas and Shewanella appeared to communicate with each other by using the QS system to some extent when the concentration of AHL reaches a certain threshold. It is therefore suggested that bacteria with the ability to disrupt AHL secretion in intestinal environments are potential candidates for probionts for preventing opportunistic infections in freshwater fish such as goldfish.     

Evaluation of Potential Probiotics Isolated from Human Milk and Colostrum

Several studies have demonstrated a diversity of bacterial species in human milk, even in aseptically collected samples. The present study evaluated potential probiotic bacteria isolated from human milk and associated maternal variables. Milk samples were collected from 47 healthy women and cultured on selective and universal agar media under aerobic and anaerobic conditions. Bacterial isolates were counted and identified by Biotyper Matrix-Assisted Laser Desorption Ionization–Time of Flight mass spectrometry and then tested for probiotic properties. Total bacteria in human milk ranged from 1.5 to 4.0 log₁₀ CFU/mL. The higher bacterial counts were found in colostrum (mean = 3.9 log₁₀ CFU/mL, 95% CI 3.14–4.22, p = 0.00001). The most abundant species was Staphylococcus epidermidis (n = 76). The potential probiotic candidates were Lactobacillus gasseri (n = 4), Bifidobacterium breve (n = 1), and Streptococcus salivarius (n = 4). Despite the small sample size, L. gasseri was isolated only in breast milk from mothers classified into a normal weight range and after a vaginally delivered partum. No potential probiotics showed antagonism against pathogens, but all of them agglutinated different pathogens. Nine bacterial isolates belonging to the species L. gasseri, B. breve, and S. salivarius were selected as potential probiotics. The present study confirms the presence in breast milk of a bacterial microbiota that could be the source of potential probiotic candidates to be used in the formula of simulated maternal milk.     

Rapid and simultaneous detection of <Emphasis Type="Italic">Salmonella</Emphasis> spp., <Emphasis Type="Italic">Escherichia coli</Emphasis> O157, and <Emphasis Type="Italic">Listeria monocytogenes</Emphasis> by magnetic capture hybridization and multiplex real-time PCR

The application of rapid, specific, and sensitive methods for pathogen detection and quantification is very advantageous in diagnosis of human pathogens in several applications, including food analysis. The aim of this study was the evaluation of a method for the multiplexed detection and quantification of three significant foodborne pathogenic species (Escherichia coli O157, Salmonella spp., and Listeria monocytogenes). The assay combines specific DNA extraction by multiplex magnetic capture hybridization (mMCH) with multiplex real-time PCR. The amplification assay showed linearity in the range 10⁶–10 genomic units (GU)/PCR for each co-amplified species. The sensitivity corresponded to 1 GU/PCR for E. coli O157 and L. monocytogenes, and 10 GU/PCR for Salmonella spp. The immobilization process and the hybrid capture of the MCH showed good efficiency and reproducibility for all targets, allowing the combination in equal amounts of the different nanoparticle types in mMCH. MCH and mMCH efficiencies were similar. The detection limit of the method was 10 CFU in samples with individual pathogens and 10² CFU in samples with combination of the three pathogens in unequal amounts (amount’s differences of 2 or 3 log). In conclusion, this multiplex molecular platform can be applied to determine the presence of target species in food samples after culture enrichment. In this way, this method could be a time-saving and sensitive tool to be used in routine diagnosis.     

Comparative analysis of prokaryotic diversity in solar salterns in eastern Anatolia (Turkey)

The prokaryotic communities of four salterns (Bingöl, Fadlum, Kemah, and Tuzlagözü) in Turkey were examined and compared using the cultivation and cultivation-independent methods [fluorescence in situ hybridization (FISH) and 454 pyrosequencing]. FISH analysis with universal probes revealed that feeding waters carried 1.6 × 10²–1.7 × 10³ cells mL^?1, while crystallization ponds carried 3.8 × 10⁶–2.0 × 10⁷ cells mL^?1 that were mostly haloarchaea, including square cells (except for Kemah). High-throughput 16S rRNA-based gene sequencing showed that the most frequent archaeal OTUs in Bingöl, Fadlum, Tuzlagözü, and Kemah samples were affiliated with Haloquadratum (76.8 %), Haloarcula (27.8 %), Halorubrum (49.6 %), and Halonotius (59.8 %), respectively. Bacteroidetes was the dominant bacterial phylum in Bingöl and Fadlum, representing 71.5 and 79.5 % of the bacterial OTUs (respectively), while the most abundant bacterial phylum found in the Kemah saltern was Proteobacteria (79.6 %). The majority of the bacterial OTUs recovered from Tuzlagözü belonged to the Cyanobacteria (35.7 %), Bacteroidetes (35.0 %), and Proteobacteria (25.5 %) phyla. Cultivation studies revealed that the archaeal isolates were closely related to the genera Halobacterium, Haloarcula, and Halorubrum. Bacterial isolates were confined to two phyla, Proteobacteria (Alphaproteobacteria and Gammaproteobacteria classes) and Bacteroidetes. Comparative analysis showed that members of the Euryarchaeota, Bacteroidetes, Proteobacteria, and Cyanobacteria phyla were major inhabitants of the solar salterns.     

Detection and Quantification of <Emphasis Type="Italic">Vibrio cholerae,Vibrio parahaemolyticus</Emphasis>, <Emphasis Type="Italic">and Vibrio vulnificus</Emphasis> in Coastal Waters of Guinea-Bissau (West Africa)

V. cholerae, V. parahaemolyticus, and V. vulnificus are recognized human pathogens. Although several studies are available worldwide, both on environmental and clinical contexts, little is known about the ecology of these vibrios in African coastal waters. In this study, their co-occurrence and relationships to key environmental constraints in the coastal waters of Guinea-Bissau were examined using the most probable number-polymerase chain reaction (MPN-PCR) approach. All Vibrio species were universally detected showing higher concentrations by the end of the wet season. The abundance of V. cholerae (ISR 16S-23S rRNA) ranged 0–1.2 × 10⁴ MPN/L, whereas V. parahaemolyticus (toxR) varied from 47.9 to 1.2 × 10⁵ MPN/L. Although the presence of genotypes associated with virulence was found in environmental V. cholerae isolates, ctxA+ V. cholerae was detected, by MPN-PCR, only on two occasions. Enteropathogenic (tdh+ and trh+) V. parahaemolyticus were detected at concentrations up to 1.2 × 10³ MPN/L. V. vulnificus (vvhA) was detected simultaneously in all surveyed sites only at the end of the wet season, with maximum concentrations of 1.2 × 10⁵ MPN/L. Our results suggest that sea surface water temperature and salinity were the major environmental controls to all Vibrio species. This study represents the first detection and quantification of co-occurring Vibrio species in West African coastal waters, highlighting the potential health risk associated with the persistence of human pathogenic Vibrio species.     

<Emphasis Type="Italic">Aeromonas</Emphasis> and <Emphasis Type="Italic">Plesiomonas</Emphasis> species from scarlet ibis (<Emphasis Type="Italic">Eudocimus ruber</Emphasis>) and their environment: monitoring antimicrobial susceptibility and virulence

The present study aimed at evaluating the role of captive scarlet ibises (Eudocimus ruber) and their environment as reservoirs of Aeromonas spp. and Plesiomonas spp., and analyzing the in vitro antimicrobial susceptibility and virulence of the recovered bacterial isolates. Thus, non-lactose and weak-lactose fermenting, oxidase positive Gram-negative bacilli were recovered from cloacal samples (n = 30) of scarlet ibises kept in a conservational facility and from water samples (n = 30) from their environment. Then, the antimicrobial susceptibility, hemolytic activity and biofilm production of the recovered Aeromonas spp. and Plesiomonas shigelloides strains were assessed. In addition, the virulence-associated genes of Aeromonas spp. were detected. Ten Aeromonas veronii bv. sobria, 2 Aeromonas hydrophila complex and 10 P. shigelloides were recovered. Intermediate susceptibility to piperacillin-tazobactam and cefepime was observed in 2 Aeromonas spp. and 1 P. shigelloides, respectively, and resistance to gentamicin was observed in 4 P. shigelloides. The automated susceptibility analysis revealed resistance to piperacillin-tazobactam and meropenem among Aeromonas spp. and intermediate susceptibility to gentamicin among P. shigelloides. All Aeromonas isolates presented hemolytic activity, while 3 P. shigelloides were non-hemolytic. All Aeromonas spp. and 3/10 P. shigelloides were biofilm-producers, at 28 °C, while 10 Aeromonas spp. and 6/10 P. shigelloides produced biofilms, at 37 °C. The most prevalent virulence genes of Aeromonas spp. were asa1 and ascV. Scarlet ibises and their environment harbour potentially pathogenic bacteria, thus requiring monitoring and measures to prevent contamination of humans and other animals.     

New approaches to modeling <Emphasis Type="Italic">Staphylococcus aureus</Emphasis> inactivation by ultrasound

Ultrasound (US) is an effective technology to inactivate vegetative microorganisms in foods. In this study, the effect of amplitude levels (0.4, 7.5, and 37.5), duty cycles (0.3:0.7 s, 0.7:0.3 s, and 0.9: 0.1 s) and time (0, 2, 4, 6, 8, 10, 12, and 14 days) of US on inactivation of Staphylococcus aureus were investigated. In addition, genetic algorithm-artificial neural network (GA-ANN) and adaptive neuro-fuzzy inference system (ANFIS) models were used to predict inactivation of S. aureus. The GA-ANN and ANFIS were fed with three inputs of amplitude levels, duty cycles, and time. The inactivation rate of S. aureus was increased by increasing the amplitude levels, and the best inactivation was obtained at a 37.5 μm amplitude for which the S. aureus population was reduced to 2.59 CFU/mL. The high inactivation of S. aureus was achieved under a duty cycle of 0.7:0.3 s with reduction of the population to 1.49 CFU/mL. The developed GA-ANN, which included 17 hidden neurons, could predict the S. aureus population with a coefficient of determination of 0.986. The overall agreement between ANFIS predictions and experimental data was also very good (R ² ?=?0.979). Sensitivity analysis results showed that the amplitude level was the most sensitive factor for prediction of S. aureus.     

Assessment of the antifungal activity of <Emphasis Type="Italic">Lactobacillus</Emphasis> and <Emphasis Type="Italic">Pediococcus</Emphasis> spp. for use as bioprotective cultures in dairy products

Fungi are commonly involved in dairy product spoilage and the use of bioprotective cultures can be a complementary approach to reduce food waste and economic losses. In this study, the antifungal activity of 89 Lactobacillus and 23 Pediococcus spp. isolates against three spoilage species, e.g., Yarrowia lipolytica, Rhodotorula mucilaginosa and Penicillium brevicompactum, was first evaluated in milk agar. None of the tested pediococci showed antifungal activity while 3, 23 and 43 lactobacilli isolates showed strong antifungal activity or total inhibition against Y. lipolytica, R. mucilaginosa and P. brevicompactum, respectively. Then, the three most promising strains, Lactobacillus paracasei SYR90, Lactobacillus plantarum O_VI9 and Lactobacillus rhamnosus BIO_III28 at initial concentrations of 10⁵ and 10⁷ CFU/ml were tested as bioprotective cultures against the same fungal targets in a yogurt model during a 5-week storage period at 10 °C. While limited effects were observed at 10⁵ CFU/ml inoculum level, L. paracasei SYR90 and L. rhamnosus BIO_III28 at 10⁷ CFU/ml respectively retarded the growth of R. mucilaginosa and P. brevicompactum as compared to a control without selected cultures. In contrast, growth of Y. lipolytica was only slightly affected. In conclusion, these selected strains may be good candidates for bioprotection of fermented dairy products.     

Increased riboflavin production by knockout of 6-phosphofructokinase I and blocking the Entner–Doudoroff pathway in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Objectives

To construct an Escherichia coli strain capable of producing riboflavin with high titer and yield.

Results

A low copy number plasmid pLS01 containing a riboflavin operon under the control of a constitutive promoter was constructed and introduced into Escherichia coli MG1655. Subsequently, the pfkA, edd and ead genes were disrupted, and the resulting strain LS02T produced 667 mg riboflavin/l in MSY medium supplied with 10 g glucose/l in flask cultivation. In a fed-batch process, riboflavin production of the strain reached 10.4 g/l with a yield of 56.8 mg riboflavin/g glucose.

Conclusion

To our knowledge, this is the first report of engineered E. coli strains that can produce more than 10 g riboflavin/l in fed-batch cultivation, indicating that E. coli has potential for riboflavin production.

     

<Emphasis Type="Italic">Plectosphaerella cucumerina</Emphasis> as a bioherbicide for <Emphasis Type="Italic">Cirsium arvense</Emphasis>: proof of concept

Plectosphaerella cucumerina (Lindf.) W. Gams was evaluated as a bioherbicide for Cirsium arvense L. (Scop.) using a Canadian and a New Zealand isolate. Both isolates defoliated C. arvense when applied at 10¹³ conidia ha^?1 in water volumes ranging from 250 to 6400 l ha^?1 with a rapid decline in effect with declining conidial dose. Repeat application and the addition of the adjuvant Pulse^® penetrant to the conidial suspension increased the disease severity in C. arvense. Maximum disease occurred at 20 °C with a 48 h post-application dew period. The experiments demonstrate that P. cucumerina can defoliate C. arvense under the environmental conditions of temperate pastures where the weed is problematic. The results also show that modifications to formulation and strategic application may reduce the 48 h dew period requirement and risk to non-target species respectively, supporting the conclusion that the fungus has potential as a bioherbicide for C. arvense.     

Development of a novel engineered antibody targeting <Emphasis Type="Italic">Neisseria</Emphasis> species

Objectives

A Neissaria bacterial pilus sugar, bacillosamine, was synthesized and, for the first time, used as a probe to screen a single-chain variable fragment (scFv).

Results

Four Neisseria, Neisseria gonorrhoeae, Neisseria meningitidis, Neisseria sicca and Neisseria subflava, and two negative controls, Streptococcus pneumoniae and Escherichia coli, were tested through ELISA, immunostaining and gold nanoparticle immunological assay. All results indicated that the selected scFv is feasible for the specific detection of Neisseria species via the recognition of bacillosamine.

Conclusions

The recombinant scFv could detect Neisseria strains at 10⁶ CFU/ml.

     

Viability and Stress Response of Putative Probiotic <Emphasis Type="Italic">Lactobacillus plantarum</Emphasis> Strains in Honey Environment

Due to problem of preservation of dairy products which serve as a matrix for probiotics, it is challenging to use these probiotics as food supplements in many developing countries. To determine the suitability of the Lactobacillus strains for exploitation as probiotics in honey, we investigated the effect of their storage on the viability, functionality, and the mechanism associated with their protective effect. Three isolates obtained from our laboratory collection were identified through amplification of the 16S rRNA gene. The viability of the strains in honey at different storage conditions was studied. Three genes (hdc, gtf, and clpL) responsible for the resistance of bacteria in acidic environments were screened. SDS-PAGE analysis of total protein was performed to observe protein profile changes of the strains after exposure to honey. All the three isolates, namely, GGU, GLA51, and GLP56, were identified as Lactobacillus plantarum strains. After 28 days of storage in honey at 4 °C, viable cell concentrations of the three strains were higher than 2.04?×?10⁶ CFU/ml. During the same period at room temperature, only the Lactobacillus plantarum GLP56 strain remained viable with a cell concentration of 1.86?×?10⁴ CFU/ml. The clpL gene coding for ATPase was detected in all the three strains. The protein of molecular weight ~?50 kDa was absent in the protein profile of Lactobacillus plantarum GGU after 60 days of storage in honey at 4 °C. The Lactobacillus plantarum GLP56, Lactobacillus plantarum GLA51, and Lactobacillus plantarum GGU strains exposed to honey can withstand acidic environmental stress but their viability declines over time.     

Resolving the naturalization strategy of <Emphasis Type="Italic">Solidago</Emphasis> × <Emphasis Type="Italic">niederederi</Emphasis> (Asteraceae) by the production of sexual ramets and seedlings

In this study, the authors aimed to revise the ability of Solidago × niederederi, a hybrid between S. canadensis and S. virgaurea, to produce sexual ramets and seedlings as a part of its naturalization strategy. Based on a two-season garden cultivation experiment, we showed that the hybrid produces more generative ramets than vegetative ones and the number of generative ramets increases from one season to another with an increasing number of stem buds located on the caudices. We also revealed a spontaneous seedling recruitment by the hybrid during cultivation in the garden. Based on the seed germination test under laboratory conditions, we evidenced that the hybrid can reach a higher final germination percentage than S. canadensis but a lower one than S. virgaurea. Based on field studies conducted in 35 populations in Poland, the hybrid formed the largest populations in tree plantations and on abandoned fields, reaching 16.5 and 15.7 ramet clusters on average, respectively. The most abundant populations were found on abandoned fields; however, the mean number of ramets per cluster did not differ remarkably among habitats (H = 6.5, p = 0.163). In all populations, the mean number of vegetative ramets per cluster reached 0.85, while the generative ones achieved 6.43 on average. The statistical analysis proved that the aforementioned differences are significant (t = ?12.6, p = 0.0002). Our results suggest that S. × niederederi is able to generate its own offspring by sexual reproduction and that abandoned fields seem to be the most suitable habitats for its establishment.     

Combined land cover changes and habitat occupancy to understand corridor status of Laljhadi-Mohana wildlife corridor,Nepal

Corridor design is a centripetal conservation tool to facilitate movement between fragmented patches. Increases in anthropogenic activity have caused degradation in forest connectivity, influencing animal movement to a small degree. Laljhadi-Mohana wildlife corridor (LMWC), a corridor between Shuklaphanta National Park (Nepal) and Dudhwa National Park (India) created to be used by Panthera tigris and Elephas maximus in western Nepal, is under pressure of anthropogenic change. Using current knowledge, we analyzed land cover changes (LCC) of LMWC between 2002 and 2012. We used ERDAS IMAGINE 9.2 and Arc GIS 9.2 to process satellite images, and occupancy survey to assess status of corridor. We classified land cover into dense forest, sparse forest, cultivation, water bodies, grassland, expose surfaces, and sand bank as structural attributes of the corridor. Our analysis found dense forest area was reduced by 18.35% in a decade while cultivation and sparse forest increased by 10.15% and 8.89%, respectively. Illegal forest encroachment, resource extraction, grazing pressure, invasive species, and flood were major drivers of forest change. The null occupancy model estimated the highest detection probability of Elephas maximus (0.48 ± 0.08) and the lowest of Axis axis (0.20 ± 0.08). Incorporating site covariates improved occupancy estimates of Sus scrofa (0.82), Axis axis (0.76), Elephas maximus (0.76), Boselaphus tragocamelus (0.66), and Panthera pardus (0.55). Distance to cultivation was the most influential covariate, supported by the expansion of cultivated land in the corridor. LMWC is a functional wildlife corridor despite a decline in forest cover. This decline influenced the number and detection rates of large mammals, instigating crop raiding and conflict. Mitigation measures on LCC drivers, particularly forest encroachment, can improve the functional status of LMWC and raise detection rates of large mammals in future studies.