George Filioussis

High efficiency electrotransformation of Lactococcus lactis spp. lactis cells pretreated with lithium acetate and dithiothreitol

BackgroundA goal for the food industry has always been to improve strains of Lactococcus lactis and stabilize beneficial traits. Genetic engineering is used extensively for manipulating this lactic acid bacterium, while electropolation is the most widely used technique for introducing foreign DNA into cells. The efficiency of electrotransformation depends on the level of electropermealization and pretreatment with chemicals which alter cell wall permeability, resulting in improved transformation efficiencies is rather common practice in bacteria as in yeasts and fungi. In the present study, treatment with lithium acetate (LiAc) and dithiothreitol (DTT) in various combinations was applied to L. lactis spp. lactis cells of the early-log phase prior to electroporation with plasmid pTRKH3 (a 7.8 kb shuttle vector, suitable for cloning into L. lactis). Two strains of L. lactis spp. lactis were used, L. lactis spp. lactis LM0230 and ATCC 11454. To the best of our knowledge these agents have never been used before with L. lactis or other bacteria.ResultsElectrotransformation efficiencies of up to 105 transformants per μg DNA have been reported in the literature for L. lactis spp.lactis LM0230. We report here that treatment with LiAc and DDT before electroporation increased transformation efficiency to 225 ± 52.5 × 107 transformants per μg DNA, while with untreated cells or treated with LiAc alone transformation efficiency approximated 1.2 ± 0.5 × 105 transformants per μg DNA. Results of the same trend were obtained with L. lactis ATCC 11454, although transformation efficiency of this strain was significantly lower. No difference was found in the survival rate of pretreated cells after electroporation. Transformation efficiency was found to vary directly with cell density and that of 1010 cells/ml resulted in the highest efficiencies. Following electrotransformation of pretreated cells with LiAc and DDT, pTRKH3 stability was examined. Both host-vector systems proved to be reproducible and highly efficient.ConclusionThis investigation sought to improve still further transformation efficiencies and to provide a reliable high efficiency transformation system for L. lactis spp. lactis. The applied methodology, tested in two well-known strains, allows the production of large numbers of transformants and the construction of large recombinant libraries.

Stenotrophomonas maltophilia isolated from the airways of animals with chronic respiratory disease.

Stenotrophomonas maltophilia (S. maltophilia) is a nonfermentative bacterium, which is naturally resistant against a panel of commonly-used antibiotics. It is frequently isolated from humans with chronic respiratory disease, e.g. cystic fibrosis or chronic obstructive pulmonary disease. In veterinary medicine S. maltophilia is perceived to be a mere coloniser. We herewith report 7 strains of S. maltophilia isolated from animals, of which 5 strains were harvested from 3 horses, a dog and a cat with chronic respiratory disease. The dog isolate showed resistance to trimethoprim / sulphamethoxazole, which was confirmed by detection of the sul 1 gene. Analysis with pulsed field gel electrophoresis revealed that 2 horses, which were boarded in the same clinic but two years apart, harboured the same strain of S. maltophilia. This is indicative of a hospital acquired colonisation / infection, which contradicts involvement in the pre-existing chronic disease.

Reproductive performance of sows was improved by administration of a sporing bacillary probiotic (Bacillus subtilis C-3102).

This field study assessed the efficacy of a probiotic based on viable spores of Bacillus subtilis C-3102 (Calsporin; Calpis Co. Ltd., Japan) on the health status and productivity of sows and their litters through 2 full, sequential reproductive cycles from service of the first cycle to weaning of the second cycle. Fifty-six sows were allocated to 2 experimental groups, an untreated control (T1) group and a probiotic-treated (T2) group that received the same basal feed as the T1 group plus the probiotic at an approximate allowance of 30 g/t of feed (3 × 10(5) cfu/g). The offspring of T1 and T2 sows were offered basal and T2 creep feed (3 × 10(5) cfu/g), respectively. Health and zootechnical parameters of sows and piglets were recorded. Feeding the probiotic to sows and piglets resulted in significant benefits, observed in both cycles: 1) improved sow body condition during pregnancy (P < 0.05), 2) increased sow feed consumption, 3) reduced sow weight loss during lactation (P < 0.05), 4) reduced sow weaning-estrus interval (P < 0.05), and 5) higher BW of piglets at weaning (P < 0.05). Additionally, a significant (P < 0.05) improvement in piglet birth weight and in the number of piglets weaned was observed in the second cycle of T2 sows, while a significant improvement of mean daily gain of piglets from birth to weaning was observed in the first cycle of T2 sows. Microbiological examination of fecal samples showed that probiotic treatment significantly reduced both Escherichia coli and Clostridium spp. in piglet feces, particularly during the second cycle. The data suggested that continuous feed supplementation with the probiotic is beneficial for both sows and piglets, since zootechnical benefits were observed in both cycles.

Stenotrophomonas Maltophilia as a Causal Agent of Pyogranulomatous Hepatitis in a Buffalo (Bubalus Bubalis)

A 7-year-old female buffalo (Bubalus bubalis) from a local herd in Serres, northern Greece, was presented to a private veterinary clinic with a chronic loss of appetite for 15 days. The clinical examination revealed high fever (41.5°C), lethargy, yellow discoloration of skin and mucous membranes, an abdomen that appeared to be empty, hyperactive rumen motility, and tachypnea. A biochemical profile revealed an elevated total bilirubin concentration and hepatic enzyme activities, whereas globulin, creatinine, and glucose concentrations were within the reference intervals. The animal received a 12-day course of treatment with intramuscular administration of ampicillin and corticosteroids. However, no significant clinical improvement was achieved, and the buffalo was euthanized. Gross necropsy lesions included serous atrophy of adipose tissue and hepatomegaly. Microscopic lesions included necrotizing pyogranulomatous hepatitis with thrombosis, hemorrhages, edema, and fibrosis. Small, nonpigmented, bacterial colonies were harvested in pure culture from the liver and were confirmed as Stenotrophomonas maltophilia by polymerase chain reaction. The bacterium was sensitive to ciprofloxacin, enrofloxacin, colistin, polymyxin, trimethoprim/sulfamethaxazole, and chloramphenicol. In contrast, resistance to ticarcillin, piperacillin, imipenem, ceftazidime, amikacin, gentamicin, tobramycin, and tetracycline was displayed. The bacterial strain carried the L1 metallo-β-lactamase (L1) and tet35 genes, which contribute to high-level resistance to β-lactams and tetracycline, respectively. Although S. maltophilia is widely believed to be a contaminant, the present report suggests that the isolation, identification, and susceptibility testing of this multidrug-resistant bacterium may be of clinical importance in diagnostic samples.

Mortality in adult goats attributed to Mycoplasma capricolum subspecies capricolum

Mycoplasma capricolum subspecies capricolum has been isolated from the oral cavity, external ear canal and milk of healthy goats ([Cottew and Yeats 1982][1], [DaMassa and Brooks 1991][2], [De la Fe and others 2005][3]) but has also been found to be pathogenic, and can cause severe losses in goat

Genome Sequences of Two Pseudorabies Virus Strains Isolated in Greece.

ABSTRACT Pseudorabies virus (species Suid herpesvirus 1) belongs to the genus Varicellovirus, subfamily Alphaherpesvirinae, family Herpesviridae, and is the causative agent of an acute and frequently fatal disease that affects mainly pigs. Here, we report the genome sequences of two strains of this virus isolated in Greece in 2010.

Congenital polyarthritis in goat kids attributed to Mycoplasma agalactiae

CONTAGIOUS agalactia is a disease that is mainly caused by Mycoplasma agalactiae . The disease has a severe impact on the economics of small ruminant herds, as it can cause mastitis, severe hypogalactia, arthritis, keratoconjunctivitis, abortions, pneumonia and, rarely, deaths (Da Massa and others 1992, Madanat and others 2001, Al-Momani and others 2006, De la Fe and others 2007). Mycoplasmatic arthritis is an entity that seems to be common and causes severe losses in adult and young goats (Bolske and others 1988, Giadinis and others 2008, Smith and Sherman 2009). Additionally, a congenital form of arthritis in goat kids attributed to Mycoplasma mycoides subspecies mycoides has been reported in Israel (Bar-Moshe and Rapapport 1981). However, congenital arthritis due to other mycoplasma species has not been reported to date. This short communication describes a case of congenital polyarthritis in neonatal goat kids in Greece that was caused by M agalactiae . In a dairy goat herd of Northern Greece, which six months ago (the previous milking season) suffered from …

In vitro susceptibilities of caprine Mycoplasma agalactiae field isolates to six antimicrobial agents using the E test methodology

The minimum inhibitory concentrations (MICs) of enrofloxacin, ciprofloxacin, spectinomycin, tetracycline, spiramycin and erythromycin against 30 caprine Greek isolates of Mycoplasma agalactiae were determined using E test methodology. The E test strips were placed on Eatons agar medium without antimicrobials and phenol red. MICs were then read by determining where the growth inhibition zone intersected with the MIC scale on the strip. An MIC value of 8 µg/mL was considered as a guide to mycoplasma resistance. All isolates were sensitive to fluoroquinolones (MIC50, 0.19 g/mL; MIC90, 0.38 µg/mL; highest MIC, 0.5 µg/mL), spectinomycin (MIC50, 0.5 µg/mL; MIC90, 1 µg/mL; highest MIC, 1 µg/mL), and spiramycin (MIC50, 1 µg/mL; MIC90, 1.5 µg/mL; highest MIC, 2 µg/mL). Two strains exhibited resistance to tetracycline (MIC 32 µg/mL) but these were not found to carry any of the tet(M), tet(O), and tet(S) resistance genes. Finally all isolates expressed resistance to erythromycin (MIC50, 128 µg/mL; MIC90, >256 µg/mL).

Pyogranulomatous Pneumonia in Goats Caused by an Undescribed Porphyromonas Species, “Porphyromonas katsikii”

ABSTRACT A yet-undescribed bacterial species, tentatively named “Porphyromonas katsikii,” was isolated from individuals of a small goat herd with pyogranulomatous pneumonia during an outbreak of acute respiratory disease. The isolated bacteria grew in the form of black-pigmented colonies after 14 days of incubation under anaerobic conditions at 37°C on a tryptic soy blood agar medium. The bacteria were identified as a yet-undescribed Porphyromonas species by determination of the nucleotide sequence of the rrs 16S rRNA gene, and this species was tentatively named Porphyromonas katsikii. PCR amplification with specific primers for this yet-undescribed species revealed the presence of P. katsikii in the lung tissue of all affected animals, while no PCR signals were evidenced from the lungs of healthy goats or from goats with pasteurellosis caused by Mannheimia haemolytica. These data indicate P. katsikii as the causative agent of acute respiratory distress. P. katsikii is phylogenetically related to Porphyromonas somerae and Porphyromonas levii, which cause pathologies in humans and animals, respectively. P. katsikii was not detected by PCR from samples of the gingival pockets or of the faces of healthy goats.

An outbreak of caprine meningoencephalitis due to Escherichia coli O157:H7

Five 1-month-old kid goats from a local herd in Kozani (northwest Greece) developed neurological disorders characterized by decreased appetite, ataxia, and head pressing. The animals received a 3-day course of treatment with intramuscular administration of enrofloxacin and ketoprofen. However, no significant clinical improvement was achieved, and 2 kids died. The remaining 3 animals were euthanized, and a necropsy was performed within 1 hr. Macroscopic lesions were confined to the central nervous system, with congestion and petechiae in the meninges. Microscopic lesions in all 3 animals revealed multifocal acute meningoencephalitis characterized by infiltrations composed of mononuclear inflammatory cells, lesser numbers of lymphocytes, and occasionally neutrophils and eosinophils. Additionally, in the kidney, there was multifocal expansion of the glomerular tufts by eosinophilic amorphous material, multifocal interstitial hemorrhages, and multifocal glomerular hypercellularity. The above noted lesions are consisted with an acute ongoing nephropathy indicative of a septicemic–toxemic procedure at its primary stages. Small, gray bacterial colonies, 3–4 mm in diameter, were obtained in pure culture from the brain of all 3 necropsied animals and were confirmed as Escherichia coli O157:H7 by use of phenotypic and genotypic methods. The isolates were sensitive to cefuroxime, ceftazidime, and gentamicin. In contrast, resistance to enrofloxacin, trimethoprim–sulfamethoxazole, and tetracycline was displayed. Additionally the bacterial isolates were found to carry a plasmid that harbored qnrS, sulII, and tetB genes that contribute to high-level resistance to fluoroquinolones, co-trimoxazole, and tetracycline, respectively.