Cohnii Strain Research Articles

The use of macroalgal hydrolysate as a carbon source in the lipid production of the marine heterotrophic dinoflagellate Crypthecodinium AL–05

Abstract Crypthecodinium cohnii strains can accumulate significant amounts of lipids with a high fraction of docosahexaenoic acid that is used in pharmaceutical and nutraceutical applications. In this study, macroalgal hydrolysate was used as a low-cost carbon source for the growth of the oleaginous strain Crypthecodinium sp. AL–05 to reduce the cost of its growth and lipid production. The Saudi strain was placed in the genus Crypthecodinium with high statistical support based on the sequences of three concatenated genes (SSU, ITS, and LSU rDNA). Crypthecodinium sp. AL-05 was grown on four different glucose concentrations and three different macroalgal hydrolysate concentrations: 20, 40, and 80 g l−1. We also used the same concentrations of macroalgal hydrolysate with 10 g l−1 of four carbon sources: acetic acid, glycerol, glucose, and sugarcane molasses. Glucose (40 g l−1) gave the best results, followed by 80 g l−1 macroalgae alone and with glucose or molasses. One-way ANOVA followed by a Bonferroni correction post-hoc test showed that the dry weight of Crypthecodinium sp. AL–05 produced using the following carbon sources: the four concentrations of the pure glucose, 80 g l−1 macroalgae alone, 40 and 80 g l−1 macroalgae + glucose, 40 and 80 g l1 macroalgae + molasses were significantly higher than the other 10 carbon sources tested. Three carbon sources (20 g l−1 macroalgae with glucose, molasses and acetic acid) produced percentages of lipids that were significantly lower than the other 16 carbon sources. For the first time, our research shows that the dinoflagellate Crypthecodinium AL–05 can be successfully grown on macroalgal hydrolysate, which can be used in industrial processes. Importantly, Crypthecodinium sp. AL–05 grown on 80 g l−1 macroalgae produced only saturated fatty acids, of which caprylic acid represented 43.45 % of the total fatty acids. Caprylic acid is widely used in industry and pharmaceuticals and the Saudi isolate can be used for its commercial production using macroalgae as a carbon source.

IS257-mediated amplification of tet(L) variant as a novel mechanism of enhanced tigecycline resistance in Staphylococcus cohnii

The mechanism of enhanced tigecycline MIC in Staphylococcus cohnii after in vitro tigecycline exposure was investigated. S. cohnii 11-B-312 was exposed to incremental concentrations of tigecycline (2–32 mg/L) and the mutants growing at 8, 16 and 32 mg/L were determined by AST and WGS. Copy number and relative transcription level of the tet(L) gene were determined by quantitative PCR. The fitness cost was evaluated by growth kinetics and competition assays. The results revealed that enhanced tigecycline MIC was identified in S. cohnii mutants. Copy number and relative transcription level of tet(L) in the mutants increased 8-, 20-, and 23-fold and 20-, 34-, and 39-fold in the presence of 8, 16, and 32 mg/L tigecycline, respectively. The read-mapping depth ratio analysis indicated that a multidrug resistance region carrying the tet(L) variant has a gradually increased copy number, correlating with the tigecycline selection pressure. S. cohnii strain 11-B-312_32 had a fitness cost, and enhanced tigecycline MIC can revert to the initial level in the absence of tigecycline. In summary, enhanced tigecycline MIC develops with extensive amplification of an IS257-flanked tet(L)-carrying segment in S. cohnii. IS257 seems to play a vital role in the gain and loss of the amplification product.

P16 Oxazolidinone resistance-associated genes cfr and optrA in MDR CoNS from healthy pigs in Italy

BackgroundOxazolidinones are relatively novel antibiotics used exclusively in human medicine as last resort drugs for resistant pathogens like MRSA, vancomycin-resistant enterococci and penicillin-resistant Streptococcus pneumoniae. However, in the last two decades, oxazolidinone resistance genes cfr and optrA have been sporadically reported worldwide in Staphylococcus spp. of livestock origin.1 These genes can be chromosomal, but they are often transferable through mobile genetic elements, especially plasmids.2 In Italy, they have been recently detected in enterococci of swine origin.3ObjectivesTo uncover the presence of cfr and optrA genes in methicillin-resistant CoNS (MRCoNS) originating from swine nasal swabs sampled in a high farm-density area of northwestern Italy. Healthy pigs were sampled from three productive stages (finishing, weaners and sows). After isolating pure cultures, selected staphylococci (n = 27), resulted methicillin-resistant from previous mecA identification, were phenotypically tested through Kirby–Bauer disc diffusion method for the antibiotics clindamycin, doxycycline, erythromycin, enrofloxacin, florfenicol, gentamicin, linezolid, tetracycline, tiamulin and trimethoprim/sulfamethoxazole (EUCAST v.11.0 guidelines for linezolid disc, CLSI VET08 for the other antibiotics). MIC through Etest (Liofilchem®, Roseto degli Abbruzzi, Teramo, Italy) was used for the antibiotic ceftaroline.ResultsAll the chosen MRCoNS were MDR (MDR CoNS), as they were phenotypically resistant to more than three antibiotic classes. No strain was positive for ceftaroline resistance. Since linezolid resistance was recovered in six samples, we decided to perform PCR for the cfr gene (746 bp), which was detected in Staphylococcus sciuri from a piglet (GenBank accession number OL412394), and optrA (1395 bp), which was recovered in Staphylococcus pasteuri from a finisher, S. sciuri from a sow and Staphylococcus cohnii from a weaner (GenBank accession numbers OM165030, OM165031 and OM165032). Sanger sequencing confirmed PCR result for cfr, with 100% identity with the cfr gene detected from a clinical Italian isolate of MRSA (MH746818), and for optrA gene, which had 100% identity with the optrA previously found in a swine Italian Enterococcus faecium strain (MT723958). As far as we know, this is the first time that a cfr gene has been detected in S. sciuri from a nasal sample of animal origin in Italy. Furthermore, optrA was never detected in S. pasteuri and S. cohnii strains.ConclusionsThese results are relevant from a One Health perspective, as they underline the need for oxazolidinone resistance monitoring, not only in human medicine, but also at farm level. In this way, it will be easier to prevent the dissemination of this resistance to human community and hospitals, where oxazolidinones are considered last-resort antibiotics. Furthermore, they remind the importance of surveillance of antibiotic usage in pigs, as cfr and optrA resistance in staphylococci can be elicited using certain antibiotics, like phenicols, due to cross-resistance to this antibiotic class.

Identification of pathogen bacteria from camel (Camelus dromedarius) mastitis and investigation of antibiotic susceptibility

The scope of this study was to investigate the presence of pathogenic bacteria in milk from female camels with mastitis and to select antibiotics for treatment with antibiotic susceptibility testing. A total of 40 milk samples taken from 20 dromedarian females, after application of CMT test and determination of SCC values, the camels were diagnosed with subclinical mastitis. Milk samples were inoculated into blood agar for identification of bacterial agents leading to mastitis. A total of 4 (12.5%) Staphylococcus aureus, 4 (12.5%) S. auricularis, 2 (6.25%) S. pettenkoperi, 2 (6.25%) S. cohnii spp. cohnii, 2 (6.25%) S. equorum, 2 (6.25%) S. capitis, 2 (6.25%) Streptococcus agalactiae, 2 (6.25%) S. dysgalactiae, 4 (12.5%) Escherichia coli, 2 (%) 6.25) Pseudomonas pseudalcaligenes, 2 (6.25%) Corynebacterium pseudotuberculosis, 2 (6.25%) Aerococcus viridans and 2 (6.25%) Gemella morbillorum were identified. Gram-positive bacteria were sensitive to Levofloxacin, Linezolid and Tetracycline and Daptomycin, resistant to Beta lactam-group antibiotics and macrolides. Vancomycin resistance was determined in S. aureus and S. cohnii spp. cohnii strains. Gram-negative strains are found generally susceptible to Cefepime and Pipersilin; resistant to Trimethoprim-sulfomethoxazole and Amoxicillin-Clavulanic acid. As a result, it is recommended to use antibiotic use to prevent the development of antimicrobial resistance as well as mastitis control methods such as the prevention of infection and monitoring the health status of the mammary of camels.

Molecular Identification and Antimicrobial Sensitivity Profile of Bacteria Isolated from Food Handlers in Akure Nigeria

Hand washing and hand swab samples culminating to a total of 477 samples were collected from different food vending locations in Akure. Bacteria were isolated and identified, after which the sensitivity patterns of the isolates to standard antibiotics and plant extracts were determined using disc diffusion agar and well diffusion methods, respectively. The bacterial isolates include Escherichia coli, Klebsiella oxytoca, Klebsiella pneumoniae, Micrococcus luteus, Proteus mirabilis, Proteusvulgaris, Pseudomonas aeruginosa, Salmonella enteritica, Salmonella typhi, Serratia marcescens, Shigella sonnei, Staphylococcus epidermidis, Staphylococcus aureus and, Streptococcus pyogenes. Staphylococcus aureus was observed to be the most prominent bacteria harboured by the food handlers. The isolated bacteria were subjected to 16s rRNA polymerase chain reaction, DNA gene extraction and sequence blasting using the amplified length of 1500 base pair. The sequence obtained was blasted in National Centre for Biotechnology Information (NCBI) database. Based on the 16s rRNA sequences, the following bacteria were confirmed; Escherichia coli str. K12 substr. DH10B, Klebsiella pneumoniae strain J1, Proteus mirabilis strain HI4320, Pseudomonas fluorescens SBW25, Salmonella enterica subsp.enterica serovar Typhi str. CT18, Salmonella enterica subsp. enterica serovar Infantis, Shigella flexneri 2a str. 2457T and Staphylococcus cohnii subsp. cohnii strain 532 Contig16. Phylogeny was employed in the phylogenetic tree construction due to its robustness, reliability and less-laboriousness. The result of the sequence was aligned and the phylogenetic tree constructed shows the evolutionary relationship between eight bacterial species. Two clusters were generated; Salmonella enterica subsp. enterica serovar Typhi, Salmoenella typhi LN854584.1 Staphylococcus cohnii, and Staphylococcus aureus are in cluster 1, Proteusmirabilis NC0100554.1, Bacillus thuringiensis KY652120.1 and Bacillus subtilis MG231261.1 is incluster 2. However, bacteria in the same cluster were observed to have different phenotypic characteristics. The antibacterial efficacy of the selected medicinal plant extracts (Ocimum gratissimum and Annona muricata) were copared to that of standard antibiotics (Ciprofloxacin, Nitrofurantoin, Ampliclox, and Chloramphenicol) using three different solvents (methanol, water and petroleum ether) at 50 mg/ml, 100 mg/ml, 200mg/ml and 300mg/ml, concentrations. Bioactive characterization of the selected plants revealed the presence ofvarious active components such as Thymol, Eugenol, Caryophyllene, Gamma-terpinene, 8-methyl-4-vanillyl-7-nonenamide, N-4-Hydroxy-3-methoxybenyl 8methyldec 6 enamide, DL-6, 8-tio acidamide and N-4-Hydroxy-3-methoxyphenyl methyl nonanamide. Phytochemical analysis of the plants affirmed the presence of Tannins, Alkaloids, Flavonoids, Anthraquinone and Saponins in O. gratissimum and A. muricata. It can be deduced from this study that food handlers play a major role in the transfer of bacterial contaminants to the edible food. Also, the metabolites present in O. gratissimum and A. muricata render these plants cost effective alternative source of therapy to food borne illnesses.

Methicillin-Resistant and Methicillin-Susceptible Staphylococcus from Vervet Monkeys (Chlorocebus sabaeus) in Saint Kitts

Antimicrobial resistance has been described in all ecosystems, including wildlife. Here we investigated the presence of methicillin-resistant and susceptible staphylococci in both colony-born and wild vervet monkeys (Chlorocebus sabaeus). Through selective isolation, PCR, MALDI-TOF, and whole-genome sequencing, methicillin-resistant and susceptible Staphylococcus spp. isolated from vervet monkeys were characterized. We obtained putatively methicillin-resistant staphylococci from 29 of the 34 nasal samples collected. Strains were identified by MALDI-TOF analysis. Staphylococcus cohnii (n = 15) was the most commonly isolated species, while nine other species were isolated one or two times. PCR analysis indicated that eight [28%] strains were mecA positive. The whole-genome sequencing [WGS] included eight methicillin-resistant strains (S. epidermidis (n = 2), S. cohnii (n = 3), S. arlettae (n = 2) and S. hominis (n = 1)), nine additional S. cohnii strains and two strains that could not be identified by MALDI-TOF, but genetically characterized as one S. cohnii and one S. warneri. Different resistance genes carried by different mobile genetic elements, mainly blaZ (n = 10) and tet(K) (n = 5) were found, while msr(A), cat, fosB, dfrG, erm(C), mph(C) and str were identified in one to three strains. Phylogenetic analysis of the S. cohnii strains based on SNPs indicated four clusters associated with colony born or wild. In addition, one singleton S. cohnii isolated did not form a separate group and clustered within other S. cohnii strains submitted to the NCBI. In this study, we demonstrated the presence of AMR and mobile genetic elements to both colony-born and wild vervet monkeys. We also identified a previously undescribed prevalence of S. cohnii in the nasal flora of these monkeys, which merits further investigation.

Biodegradation of aliphatic waste components of oil sludge used micro symbiont of Sponge Niphates sp.

Investigation has been carried out using a sponge Niphates sp. as biodegradation agent of the aliphatic hydrocarbon component of crude oil sludge. The experiment was carried out by colonizing and making suspension of Bacillus pumilus strains GLB197 and Bacillus cohnii strains of DSM 6307, isolated from sponge Niphates sp. Both types of micro symbiont suspension were mixed with sludge waste for 30 days. There are five biodegradation parameters observed, carried out every five days of incubation. Parameters that observed during incubation were pH, gas bubbles and fermentation odor. Meanwhile, the optical density of the media was measured using spectrometer-D20+, Aliphatic components degradation was measured using GC-MS. Observation of biodegradation parameters showed there was a change in pH from 7 to 6, formed gas bubbles, resulting in fermentation odors and showed an increased optical density by an average of 35%. These four parameters occur during the interaction of 10-20 days of interaction in the media. There were 20 types of aliphatic components identified in the crude oil sludge waste. Analysis of four aliphatic components, namely Tridecane, Octadecane, Tricosane, and Nonacosane showed a decrease in the average concentration of 48.11 %.

336 Mutualistic skin bacteria protect against dermatitis via the induction of steroid biosynthesis pathways

The skin microbiota plays a role in homeostasis and inflammatory diseases such as atopic dermatitis. While the pathogenic potential of several skin microbiota members has been well-characterized, an emerging body of evidence suggests certain commensals possess properties that benefit the host. To elucidate novel host-commensal relationships that endow protection against inflammation, we established a preventable, microbiota-driven dermatitis mice model using gnotobiotic techniques and transgenic mice. Specifically, we used Tmem79 knockout mice which, under specific pathogen-free conditions, develop spontaneous dermatitis accompanied by an increase in both Staphylococcus aureus colonization density and number of IL-17-expressing T cells in the skin. In contrast, we found that dermatitis does not develop when the mice are kept under germ-free conditions. We examined the influence of mouse-origin skin microbiota on the development of dermatitis by monocolonizing germ-free mice with various bacterial isolates. Gnotobiotic mice monocolonized with S. aureus or Corynebacterium mastitidis exhibited dermatitis, whereas those monocolonized with other Staphylococci strains did not. We also found that Staphylococcus cohnii, a mutualistic bacterium on human and murine skin, was negatively correlated with the development of dermatitis. We monocolonized the mice with one of several Staphylococci strains prior to inoculation with S. aureus in order to characterize the preventive effect. Strikingly, some but not all S. cohnii strains were able to prevent S. aureus-driven dermatitis without reducing S. aureus colonization density. RNA-seq analysis revealed that monocolonization with protective S. cohnii strains activates steroid biosynthesis pathways in the skin. These results indicate that S. cohnii may exert anti-inflammatory effects directly on the host via the induction of steroid biosynthesis pathways, and thus has the potential to be used as a probiotic in the clinic.

Integration of a dark fermentation effluent in a microalgal-based biorefinery for the production of high-added value omega-3 fatty acids

Dark fermentation is an anaerobic digestion process of biowaste, used to produce hydrogen- for generation of energy- that however releases high amounts of polluting volatile fatty acids, such as acetic acid, in the environment. In order for this biohydrogen production process to become more competitive, the volatile fatty acids stream can be utilized through conversion to high added-value metabolites, such as omega-3 fatty acids. The docosahexaenoic acid is one of the two most known omega-3 fatty acids and has been found to be necessary for a healthy brain and proper cardiovascular function. The main source is currently fish, which obtain the fatty acid from the primary producers, microalgae, through the food chain. Crypthecodinium cohnii, a heterotrophic marine microalga, is known for accumulating high amounts of docosahexaenoic acid, while offering the advantage of assimilating various carbon sources, such as glucose, ethanol, glycerol and acetic acid. The purpose of this work was to examine the ability of a C. cohnii strain to grow on different volatile fatty acids, as well as, on a pretreated dark fermentation effluent and accumulate omega-3. The strain was found to grow well on relatively high concentrations of acetic, butyric or propionic acid as main carbon source in a fed-batch pH-auxostat. Most importantly, C. cohnii totally depleted the organic acid content of an ultra-filtrated dark fermentation effluent after 60 h of fed-batch cultivation, therefore offering a bioprocess not only able to mitigate environmental pollutants, but also to provide a solution for a sustainable energy production process. The accumulated docosahexaenoic acid content was as high as 29.8% (w/w) of total fatty acids.

Metabolomic analysis and lipid accumulation in a glucose tolerant Crypthecodinium cohnii strain obtained by adaptive laboratory evolution

Adaptive laboratory evolution (ALE) was commonly used for strain improvement. Crypthecodinium cohnii is known to accumulate lipids with a high fraction of docosahexaenoic acid (DHA). In order to improve DHA production under high substrate concentration, a glucose-tolerant C. cohnii strain was firstly obtained by ALE after 260 cycles for 650days with gradually increased glucose concentration. The results of lipids content showed that DHA-rich lipids accumulation in the evolved strain could increase by 15.49% at 45g/L glucose concentrations. To reveal mechanisms related to glucose tolerance of C. cohnii through ALE, metabolic profiles were then compared and the results showed that hub metabolites including glycerol, glutamic acid, malonic acid and succinic acid were positively regulated during ALE. The study demonstrated that metabolomic analysis complemented with ALE could be an effective and valuable strategy for basic mechanisms of molecular evolution and adaptive changes in C. cohnii.

Molecular Identification of Selected Multiple Antibiotic Resistance Bacteria Isolated from Poultry Droppings in Akure, Nigeria

Molecular techniques have the advantage that, they are rapid, less laborious, and more sensitive, specific and efficient compared to the conventional method of bacteria identification. This work aimed at Molecular Identification of Selected Multiple Antibiotic Resistance Bacteria Isolated from Poultry Droppings in Akure, Nigeria. Samples were plated on selective and differential media. Isolation and identification of bacteria were carried out using standard microbiological method, Antibiotics susceptibility test was determined by the disk diffusion method, Plasmid DNA extraction, profiling and curing, Genomic DNA extraction, Polymerase Chain Reaction (PCR), agarose gel electrophoresis, 16S rRNA gene sequence and phylogenetic analysis were carried out by standard method. Eight bacteria were selected; <i>Proteus mirabilis, Salmonella typhi, Pseudomonas aeruginosa, Staphylococcus aureus, Salmonella</i> <i>sp. Escherichia coli, Shigella</i> sp. and <i>Klebsiella pneumonia</i>, they were resistant to at least seven antibiotics. The result revealed that the plasmid size ranged from 8.60 to 128.00 kilobase pair (kbp) and the number of plasmid carried by bacteria ranged from 1 to 3 plasmids. Based on the 16s rRNA sequences, the following bacteria were identified <i>Salmonella enterica subsp.enterica</i> serovar Typhi str. CT18, <i>Proteus mirabilis</i> strain HI4320, <i>Pseudomonas fluorescens</i> SBW25, <i>Staphylococcus cohnii</i> subsp. cohnii strain 532 Contig16, <i>Salmonella enterica</i> subsp. <i>enterica</i> serovar Infantis, <i>Escherichia coli</i> str. K12 substr. DH10B, <i>Shigella flexneri</i> 2a str. 2457T and <i>Klebsiella pneumoniae</i> strain J1. The presence of multiple resistant bacteria and plasmid mediated resistance of human pathogenic bacteria isolated from poultry droppings in Akure is of great public health importance.

Genome Mining and Comparative Genomic Analysis of Five Coagulase- Negative Staphylococci (CNS) Isolated from Human Colon and Gall Bladder

Coagulase-negative Staphylococci (CNS) are known to cause distinct types of infections in humans like endocarditis and urinary tract infections (UTI). Surprisingly, there is a lack of genome analysis data in literature against CNS particularly of human origin. In light of this, we performed genome mining and comparative genomic analysis of CNS strains Staphylococcus cohnii subsp. cohnii strain GM22B2, Staphylococcus equorum subsp. strain equorum G8HB1, Staphylococcus pasteuri strain BAB3 isolated from gall bladder and Staphylococcus haemolyticus strain 1HT3, Staphylococcus warneri strain 1DB1 isolated from colon. We identified 29% of shared virulence determinants in the CNS strains which involved resistance to antibiotics and toxic compounds, bacteriocins and ribosomally synthesized peptides, adhesion, invasion, intracellular resistance, prophage regions, pathogenicity islands. 10 unique virulence factors involved in adhesion, negative transcriptional regulation, resistance to copper and cadmium, phage maturation were also present in our strains. Apart from comparing the genome homology, size and G + C content, we also showed the presence 10 different CRISPR-cas genes in the CNS strains. Further, KAAS based annotation revealed the presence of CNS genes in different pathways involved in human diseases. In conclusion, this study is a first attempt to unveil the pathogenomics of CNS isolated from two distinct body organs and highlights the importance of CNS as emerging pathogens of health care sector.

High quality draft genome sequence of Staphylococcus cohnii subsp. cohnii strain hu-01.

Staphylococcus cohnii subsp. cohnii belongs to the family Staphylococcaceae in the order Bacillales, class Bacilli and phylum Firmicutes. The increasing relevance of S. cohnii to human health prompted us to determine the genomic sequence of Staphylococcus cohnii subsp. cohnii strain hu-01, a multidrug-resistant isolate from a hospital in China. Here we describe the features of S. cohnii subsp. cohnii strain hu-01, together with the genome sequence and its annotation. This is the first genome sequence of the species Staphylococcus cohnii.

DIVERSITY OF CRYPTHECODINIUM SPP. (DINOPHYCEAE) FROM OKINAWA PREFECTURE, JAPAN

The genus Crypthecodinium (Dinophyceae) currently consists of only one species: C. cohnii, a heterotrophic marine dinoflagellate widely known able to produce prolific amount of DHA. However, previous studies revealed that there are morphological and genetic differences among Crypthedodinium cohnii-like strains, indicating the potential of undiscovered diversity of this dinoflagellate. Attempts of isolating heterotrophic marine dinoflagellate strains were made from submerged mangrove leaves and seaweed. A total of nine strains were established, cultured and maintained using seawater-based GTY medium for taxonomic identification and phylogenetic analyses based on SSU, ITS1, 5.8S, ITS2 and LSU rDNA. Morphological observation revealed all strains shared similar morphology, e.g.: motile cells were delicately thin armored with single or double flagella surrounding the cingulum and protruding along the sulcus posteriorly, while non-motile cell stage (cyst) may retain more than two daughter cells inside the transparent and thick cell wall. Both cell stages varied in sizes and shapes even within the same clonal culture.Numerous lipid granules can be observed in the cytoplasm. Two types of cingulum encirclement were observed: partially or completely surrounding the cell body. All strains indicated close relatedness (＞95% sequence similarity) with previously reported Cryphtecodinium cohnii strains, except for C. cohnii CAAE-CL2 (87.2-87.4% similarity) in SSU and C. cohnii CCMP316 (79.9-80.4% similarity) in ITS. Moreover, molecular phylogenetic tree grouped the Okinawan isolates into two genetically distinct clades with high similarity (＞98%) among members of each clade in SSU and LSU but only shared 80.5% similarity in ITS rDNA. The results of this study showed evidences of undiscovered diversity in the heterotrophic dinoflagellates currently regarded as Crypthecodinium.

Antibacterial Ability and Molecular Characterization of Probionts Isolated from Gut Microflora of Cultured Red Tilapia

Four bacterial isolates, which proved to have the potential to be used as probiotics, were used to study their genotypic characterization using analysis of 16S rRNA gene sequence, antagonistic ability and safety when applied on tilapia via immersion. Gram staining showed that all 4 probionts were Gram positive bacteria. Three probionts were rod in shape and the fourth was cocci in shape and arranged in grape-like cluster. These 4 probionts were also identified using 16S rRNA gene sequencing and their identities were Paenibacillus barcinonensis strain D12, Paenibacillus sp. strain D14, Staphylococcus cohnii strain Bll and Bacillus megaterium strain E28, respectively. All of the probionts were examined for their antagonistic ability against pathogenic bacteria (Vibrio alginolyticus ATCC 33839, Aeromonas salmonicida and Aeromonas hydrophila ATCC 35654) under in vitro conditions by using cross-streaking method. P. barcinonensis strain D12 and Paenibacillus sp. strain D14 have shown stronger antagonistic ability than S. cohnii strain Bll and B. megaterium strain E28 in the antagonism test. P. barcinonensis strain D12 and Paenibacillus sp. strain D14 were chosen to test their safety on tilapia due to their better performance in antagonism test. Both probionts, P. barcinonensis strain D12 and Paenibacillus sp. strain D14, were safe for tilapia.

Phenotypic and technological diversity of lactic acid bacteria and staphylococci isolated from traditionally fermented sausages in Southern Greece

The physicochemical and microbiological characteristics of spontaneously fermented sausages made by two medium-sized enterprises (MSE) located in southern Greece have been studied. A total of 300 lactic acid bacteria and 300 staphylococcal strains have been isolated and identified by their physiological characteristics. Lactobacillus plantarum strains were found to dominate the lactic acid bacteria microbiota in most of the cases with L. sakei strains prevailing in some of them and L. rhamnosus strains occasionally accompanying the dominant lactic acid bacteria microbiota. On the other hand, S. saprophyticus strains were found to dominate the staphylococcal microbiota in all spontaneously fermented sausages with of S. simulans, S. xylosus, S. gallinarum and S. cohnii cohnii strains being sporadically present. Following the identification, an evaluation of their technological properties, namely proteolytic and lipolytic capacities as well as production of biogenic amines and antimicrobial compounds, took place. None of the lactic acid bacteria and staphylococci was found to possess lipolytic activity whereas a total of 6 lactic acid bacteria and 51 staphylococci strains were found to be able to hydrolyse either the sarcoplasic, myofibrillar or both protein fractions. Furthermore, only one L. sakei strain and 185 staphylococci strains were found to possess decarboxylase activity against lysine, tyrosine, ornithine or histidine. Finally none of the staphylococcal microbiota and 3 lactic acid bacteria strains were found to be able to produce antimicrobial compounds of proteinaceous nature against Listeria monocytogenes.

The Effectiveness of Lysostaphin Therapy for Experimental Coagulase-Negative Staphylococcus Endophthalmitis

Purpose: To quantitatively determine the effectiveness of lysostaphin therapy for experimental endophthalmitis mediated by coagulase-negative Staphylococcus species, the leading cause of postsurgical endophthalmitis. Methods: Minimal inhibitory concentrations (MIC) of lysostaphin were determined for 54 isolates representing the following species: S. epidermidis, S. warneri, S. haemolyticus, S. cohnii, S. simulans, and S. capitis. The effectiveness of lysostaphin therapy was tested in an experimental model of endophthalmitis by intravitreally injecting log phase bacteria (100 colony-forming units; cfu) into rabbit eyes (n ≥ 3 eyes per group). At 8 hr postinfection (PI), lysostaphin (250 μg) was injected intravitreally, and the number of cfu/ml of vitreous was determined at 24 hr PI. Results: Average MIC for S. epidermidis was 0.7 μ g/ml for 90% of the 33 strains tested. Six methicillin-resistant strains of S. epidermidis (MRSE) had an average MIC of 0.74 μ g/ml. All other species had MIC values of ≤1.1 μg/ml, except for one strain of S. capitis (MIC = 15.6 μ g/ml) and one S. haemolyticus (MIC = 1.41 μ g/ml). At 24 hr PI, all untreated eyes had between 5.7 and 8.08 log cfu/ml vitreous humor. Treatment with lysostaphin significantly reduced the cfu/ml as compared with untreated eyes for 13 strains tested in vivo (p ≤ 0.020), but not for two S. haemolyticus strains (p ≥ 0.13), two MRSE strains (p ≥ 0.544), or one S. cohnii strain (p = 0.1366). Treatment with lysostaphin reduced the cfu/ml of methicillin-sensitive S. epidermidis strains by 6 logs; for S. warneri, there was a 2 log reduction; and for the other species a 4 log reduction in cfu/ml relative to untreated eyes. Conclusions: Lysostaphin was mostly effective in treating coagulase-negative staphylococcal experimental endophthalmitis.

Predominant staphylococci in the intensive care unit of a paediatric hospital

Coagulase-negative staphylococci cause a significant number of infections, especially in immunocompromised patients, including premature neonates. Nosocomial strains present in the environment create a special risk.We studied staphylococci isolated from the intensive care unit of a paediatric teaching hospital over the period of six months in 1997. Biotyping and species identification were performed; resistance to methicillin and other β-lactam antibiotics and patterns of resistance to antimicrobial agents were determined.Staphylococcus cohnii was the predominant species of 147 isolates of staphylococci recovered from the ward environment. Strains were resistant to several antibiotics and 97% were resistant to methicillin. In isolates from infants (72) methicillin-resistant strains of Staphylococcus epidermidis were predominant. Susceptibility to β-lactams (penicillin, amoxycillin, amoxycillin-clavulanic acid and cephalosporins: cephalothin, cefuroxime and cefotaxime) showed differences between the two species. Some S. cohnii were susceptible to penicillin and amoxycillin despite methicillin-resistance. S. epidermidis were relatively susceptible to amoxycillin-clavulanic acid and cephalosporins. All strains investigated were susceptible to vancomycin, but nearly 30% demonstrated high-level resistance to mupirocin. The search for strains of the same origin showed clones belonging to S. epidermidis, S. hominis and S. saprophyticus but not S. cohnii.A large number of multiresistant, phenotypically different S. cohnii strains surviving in the ward environment may provide a reservoir of antimicrobial resistance genes.

Cyclic expression of a nuclear protein in a dinoflagellate.

Nuclei of the dinoflagellate Crypthecodinium cohnii strain Whd were isolated and nuclear proteins were extracted in three fractions, corresponding to the increasing affinity of these proteins to genomic DNA. One fraction contained two major bands (48- and 46-kDa) and antibodies specific to this fraction revealed two major bands by Western blot on nuclear extracts, corresponding to the 46- and 48-kDa bands. The 48-kDa protein was detected in G1 phase but not in M phase cells. An expression cDNA library of C. cohnii was screened with these antibodies, and two different open reading frames were isolated. Dinoflagellate nuclear associated protein (Dinap1), one of these coding sequences, was produced in E. coli and appeared to correspond to the 48-kDa nuclear protein. No homologue of this sequence was found in the data bases, but two regions were identified, one including two putative zinc finger repeats, and one coding for two potential W/W domains. The second coding sequence showed a low similarity to non-specific sterol carrier proteins. Immunocytolocalization with specific polyclonal antibodies to recombinant Dinap1 showed that the nucleus was immunoreactive only during the G1 phase: the nucleoplasm was immunostained, while chromosome cores and nuclear envelopes were negative.

Characterization of a staphylococcal plasmid related to pUB110 and carrying two novel genes, vatC and vgbB, encoding resistance to streptogramins A and B and similar antibiotics.

We isolated and sequenced a plasmid, named pIP1714 (4,978 bp), which specifies resistance to streptogramins A and B and the mixture of these compounds. pIP1714 was isolated from a Staphylococcus cohnii subsp. cohnii strain found in the environment of a hospital where pristinamycin was extensively used. Resistance to both compounds and related antibiotics is encoded by two novel, probably cotranscribed genes, (i) vatC, encoding a 212-amino-acid (aa) acetyltransferase that inactivates streptogramin A and that exhibits 58.2 to 69.8% aa identity with the Vat, VatB, and SatA proteins, and (ii) vgbB, encoding a 295-aa lactonase that inactivates streptogramin B and that shows 67% aa identity with the Vgb lactonase. pIP1714 includes a 2,985-bp fragment also found in two rolling-circle replication and mobilizable plasmids, pUB110 and pBC16, from gram-positive bacteria. In all three plasmids, the common fragment was delimited by two direct repeats of four nucleotides (GGGC) and included (i) putative genes closely related to repB, which encodes a replication protein, and to pre(mob), which encodes a protein required for conjugative mobilization and site-specific recombination, and (ii) sequences very similar to the double- and single-strand origins (dso, ssoU) and the recombination site, RSA. The antibiotic resistance genes repB and pre(mob) carried by each of these plasmids were found in the same transcriptional orientation.