Effect of cooling rate on the survival of cryopreserved rooster sperm: Comparison of different distances in the vapor above the surface of the liquid nitrogen.

The aim of the present trial was to study the effect of different freezing rates on the survival of cryopreserved rooster semen packaged in straws. Slow and fast freezing rates were obtained keeping straws at different distances in the vapor above the surface of the nitrogen during freezing. Adult Lohmann roosters (n=27) were used. Two experiments were conducted. In Experiment 1, semen was packaged in straws and frozen comparing the distances of 1, 3 and 5cm in nitrogen vapor above the surface of the liquid nitrogen. In Experiment 2, the distances of 3, 7 and 10cm above the surfaces of the liquid nitrogen were compared. Sperm viability, motility and progressive motility and the kinetic variables were assessed in fresh and cryopreserved semen samples. The recovery rates after freezing/thawing were also calculated. In Experiment 1, there were no significant differences among treatments for all semen quality variables. In Experiment 2, the percentage of viable (46%) and motile (22%) sperm in cryopreserved semen was greater when semen was placed 3cm compared with 7 and 10cm in the vapor above the surface of the liquid nitrogen. The recovery rate of progressive motile sperm after thawing was also greater when semen was stored 3cm in the vapor above the surface of the liquid nitrogen. More rapid freezing rates are required to improve the survival of rooster sperm after cryopreservation and a range of distances from 1 to 5cm in nitrogen vapor above the surface of the liquid nitrogen is recommended for optimal sperm viability.

Growth performance, carcass characteristics and meat composition of Milanino chickens fed on diets with different protein concentrations.

Milanino is a heavy Italian chicken breed included in a conservation project of the University of Milan and is an important genetic resource for alternative production systems. This research was aimed to study the effect of the dietary protein concentration on growth, slaughter performance and meat composition in free-range reared Milanino chickens. A total of 120 Milanino chickens were fed on different protein concentrations (HP = 20% CP and LP = 16% CP), reared according to a free-range system and slaughtered at 150 and 180 d of age. Growth, slaughter performance and meat (breast and thigh) composition were recorded. The protein concentration of the diet did not affect the overall Milanino mean body weight recorded in the straight-run group in the whole rearing period. However, the growth rate within sex was significantly different between the dietary treatments: heavier females were found in the HP group from 125 d onwards, while no differences were recorded in male body weights. The protein concentration of the diet did not affect carcass weight data or meat composition. The present results suggest the use of a low-protein diet for rearing straight-run Milanino chickens for long rearing periods. However, in females, a high-protein diet is recommended from 125 d of age onwards.

Effect of testicle postmortem storage on goat frozen-thawed epididymal sperm quality as a tool to improve genebanking in local breeds.

The interest to develop assisted reproductive technologies and cryobanking for farm animal genetic resource conservation has recently increased. However, cryopreservation for ex-situ management of genetic diversity sometimes is not routinely feasible, owing to the lack of facilities (AI centres, laboratories) and expertise near the local breed farming area. In these cases, epididymal sperm obtained from slaughtered or castrated animals, associated with the possibility of managing rather long periods between animal death, sperm recovery and freezing, would increase the opportunities to create semen storages. This investigation addresses the pre-freeze/post-thaw quality of goat epididymal sperm as a function of testicle storage temperature (environment or +5°C) and time elapsed between animal's death and sperm recovery (0, 24, 48, 72 h) to establish the optimal protocols for the recovery and cryopreservation of epididymal sperm in this species. Testicles of 50 mature bucks collected at the abattoir were divided in two groups: half of the testicles (n=50) were transported to the laboratory at environment temperature (E), whereas the remaining half (n=50) at a refrigeration temperature (R) of +5°C. In the two groups (E) and (R), one testicle from each pair was processed after slaughter forming the time 0 groups (0E and 0R). The contralateral testicle was processed after 24, 48 or 72 h of storage, at the corresponding temperature. Sperm motility and kinetic parameters, viability and morphology were assessed in pre-freeze and post-thaw samples. Until 48 h postmortem, both E and R temperatures are able to maintain good pre-freeze epididymal sperm quality. After 48 h postmortem, R temperature is fundamental to reduce epididymal sperm quality decay in pre-freeze samples. Moreover, testicle refrigeration also has a positive impact on post-thaw samples, allowing a lower decline through time considering total motility, kinetics parameters, sperm viability and sperm abnormalities. Therefore, when sperm cryopreservation is not immediately practicable, goat testicles should be transported and stored at 5°C up to a maximum of 48 h postmortem to ensure an acceptable sperm quality.

Egg related parameters affecting fertility and hatchability in the Italian bantam breed Mericanel della Brianza.

Local chicken breeds are a vital reservoir of gene resources and their conservation has a technical role related to the future development of the productive system, as well as a social-cultural role. The aim of this study was to evaluate the effects of egg weight, egg storage period and egg weight loss on hatchability of fertile eggs in the Italian bantam breed Mericanel della Brianza. Fourteen females and eight males were kept in floor pens and divided in 8 families (1M:1 or 2F) during the reproductive season (March-June). Birds received a photoperiod of 14L:10D and were fed ad libitum. Egg production and egg weight were recorded daily. Eggs were divided in 4 weight groups: EW1 =< 33 g, EW2 = 33-36 g, EW3 = 36-39 g and EW4 =≥ 39 g. Eggs were stored at 18 °C and classified in 3 egg storage groups: ES1 = 0-4, ES2 = 5-9 and ES3 = 10-15 days. Egg weight loss was recorded and distributed in 5 different classes: EWL1 =< 10%, EWL2 = 10-15%, EWL3 = 16-20%, EWL4 = 21-25%, EWL5 => 25%. Fertility, embryo mortality and hatchability were recorded. The mean values during the reproductive season were 82% fertility and 50% hatchability of fertile eggs. The best combination of fertility and hatchability values were recorded in EW2 and lower fertility was recorded in EW1 (P < 0.05). Hatchability decreased under 50% after 10 day storage period before incubation and the best hatchability was recorded in EWL1. The present results contribute to the knowledge on reproductive parameters necessary to improve the reproductive efficiency of this Italian breed within a conservation plan.

Influence of recovery methods and extenders on bull epididymal spermatozoa quality.

The aim of this study was to compare the effects of two extraction methods in combination with two different extenders in bull epididymal sperm collection. Testes from 23 sexually mature Limousine bulls were collected at the abattoir. Epididymal sperm recovery was performed using both the float-up (FL) and the retrograde flushing (RF) technique. Within extraction methods, half testes were processed with a Tris egg yolk extender and half with a Tris egg yolk-free extender. Sperm concentration, motility, viability and morphology were evaluated. Sperm concentration was not significantly different between methods. Flushing technique was significantly better than the FL method in terms of sperm quality, considering total motility (80.3 ± 2.3% vs 71.6 ± 2.0%, p < 0.001, respectively) and viability (84.5 ± 1.5% vs 77.2 ± 1.3%, p < 0.001, respectively). Egg yolk influenced positively motility and morphology in the FL method, whereas decreased viability in flushed samples. Results suggest the use of the RF technique to collect cattle epididymal sperm.

Differences in semen freezability and intracellular ATP content between the rooster (Gallus gallus domesticus) and the Barbary partridge (Alectoris barbara).

This study aimed to compare viability, ATP content, and DNA integrity of rooster (Gallus gallus domesticus) and Barbary partridge (Alectoris barbara) fresh and frozen spermatozoa in order to identify factors possibly related to differences in semen freezability. Ejaculates were obtained from March to May by the abdominal massage method from 3 adult roosters and 12 adult Barbary partridges. Semen was frozen with different cryoprotectants using Lake's diluents as a base medium: 1) glycerol 11%; 2) glycerol 11% and trehalose 70 mmol/L; 3) dimethylacetamide (DMA) 6%; 4) DMA 6% and trehalose 70 mmol/L. Both fresh and frozen semen showed a lower viability and higher intracellular ATP concentrations in the Barbary partridge compared with the rooster (P < 0.05). In the Barbary partridge, semen viability after thawing did not differ among the 4 media used, but glycerol showed positive effects in avoiding a significant loss of ATP after thawing, compared with DMA containing media (P < 0.05). On the other hand, in the rooster a higher viability was recorded when semen was frozen in glycerol containing media compared to DMA (P < 0.0001), while ATP values significantly decreased after thawing (P < 0.05) without showing any differences among the semen frozen in the 4 different media. DNA integrity, as evaluated by the comet assay, was assessed only in frozen semen. In the Barbary partridge, mean scored parameter did not differ significantly among semen frozen in the 4 different media. In the rooster DNA fragmentation was higher in DMA ctr medium compared with the other media and with values found in Barbary partridge semen frozen in the same medium (P < 0.001). In both species, the addition of trehalose did not show any positive effects on viability, ATP levels and DNA integrity after thawing. In conclusion, species-related differences in semen features exist between the rooster and the Barbary partridge and the wide variation observed in ATP levels may account for differences in semen freezability between the two species.

[Identification of Legionella spp. by means of cultural and molecular methods in a public hospital]. / Isolamento di Legionella spp. mediante metodi colturali e tecniche molecolari in matrici ambientali prelevate in una struttura ospedaliera pubblica.

An epidemiologic study on the isolation of Legionella spp from the sanitary water of a public Hospital in Cagliari (Italy) has been performed. The aim of the study was the comparison between the isolation of various Legionella spp from different hospital sources and the real hazard of Legionella infection of the inpatients. Two test methods were used for Legionella detection: a) the culture on selective media, that has the disadvantage of being quite time-consuming and of isolating also other bacterial species. Furthermore, the culture method often fails the isolation of vital but not culturable bacteria (VBNC); b) the PCR molecular method, which is rapid and precise and recognizes also VBNC cells. The most relevant result of this work was that, in spite of the isolation of a considerable number of Legionella spp (even Legionella pneumophila), no case of infection was detected in the Hospital during the period of the study.

Recovery of COCs from ovaries with high follicle numbers enhances in vitro embryo yield in sheep.

The aim of the present study was to investigate the correlation between the number of ovarian follicles and in vitro embryo development and quality in sheep. Sarda ewe ovaries were classified according to the number of follicles: or=8 (High). IVM, IVF and IVC were performed under standard conditions. Cleavage rate and blastocyst development were assessed 48 h after fertilization and on Days 6, 7 and 8 of culture, respectively. Expanded blastocysts were vitrified; blastocoel re-expansion and hatching rates were assessed at 8, 16 and 72 h post-thawing and hatched blastocysts were analyzed with the TUNEL assay. In a subset of thawed blastocysts the incorporation of amino acids was evaluated. The proportion of ovaries varied significantly among the three groups (ANOVA F=12.20, P=0), and more ovaries (59%) were assigned to the Low group than to the Intermediate (28%; ANOVA F=8.19, P=0.009) and High group (13%; ANOVA F=18.63, P=0), (High vs. Intermediate F=6.31, P=0.020). The three groups statistically differed in the proportion of total blastocysts (chi(2)(2)=22.616, P=0.00), of blastocysts produced on Days 6 (chi(2)(2)=6.829, P=0.033) and 7 (chi(2)(2)=6.810, P=0.033), while no difference was found in the proportion of blastocysts obtained on Day 8 (chi(2)(2)=3.874, P=0.144) of culture after fertilization. A higher proportion of total blastocysts was obtained from the High (44%) compared with the other two groups (Low: 28%, chi(2)(2)=22.629, P=0; Intermediate: 33%, chi(2)(2)=7.266, P=0.007), while the Low and Intermediate groups did not statistically differ either in the total blastocyst output (chi(2)(2)=3.384, P=0.066), nor in the number of blastocysts produced on Days 6 (Low: 7%, Intermediate: 9%; chi(2)(2)=0.874, P=0.35), 7 (Low: 14%, Intermediate: 16%, chi(2)(2)=1.256, P=0.26) and 8 (Low: 6%, Intermediate: 7% chi(2)(2)=0.554, P=0.45) of culture. The High group produced a significantly higher percentage of embryos on Days 6 (High: 13%, Low: 7%; chi(2)(2)=6.840, P=0.009) and 7 (High: 21%, Low: 14%; chi(2)(2)=6.806, P=0.009) of culture post-insemination than the Low group. The three categories did not differ in the blastocoel re-expansion (chi(2)(2)=0.095, P=0.95) and hatching rates (chi(2)(2)=0.754, P=0.68) after 72 h post-warming, in the total number of cells per blastocyst (ANOVA F=1.12, P=0.337) and in the (F=0.46, P=0.639) incorporation of amino acids. The number of TUNEL-positive cells per embryo was higher (ANOVA F=4.32, P=0.022) in the Low group compared to the other groups. In conclusion, high ovarian follicle number enhances in vitro embryo output in sheep, but has no effect on blastocyst quality.

A new selection criterion to assess good quality ovine blastocysts after vitrification and to predict their transfer into recipients.

The feasibility to accurately select viable embryos would be valuable for improving pregnancy rates and avoiding futile transfer attempts. The aim of our study was to assess if in vitro-produced embryo quality could be determined by the timing of blastocoelic cavity re-expansion after vitrification, warming, and in vitro culture using sheep as a model. Blastocysts were produced in vitro, vitrified/warmed, and cultured in TCM-199 plus 10% FCS for 72 hr. Embryos were divided into two groups: re-expanded within 8 hr (A) and from 8 to 16 hr (B) of IVC after warming. Fast re-expanded blastocysts showed higher in vitro hatching rates and total cell number calculated on the hatched blastocysts compared with slow re-expanded ones (P < 0.01). Peroxide status evaluation (P < 0.01) and TUNEL test (P < 0.05) revealed a higher number of positive cells in group B compared with group A. The quantitative analysis of protein synthesis revealed a higher synthesis in fast compared with slow re-expanded embryos (P < 0.05). Quantitative RT-PCR showed that 90-kDa Heat Shock Protein beta was more expressed in group A than in group B (P < 0.05), while the quantity of P34(cdc2), Cyclin b, Aquaporin 3, Na/K ATPase, and Actin did not differ between the two groups. Pregnancy rates after transfer to synchronized recipients were higher in fast compared to slow re-expanded blastocysts (P < 0.05). Our results evidenced that timing of blastocoelic cavity re-expansion after vitrification/warming and in vitro culture can be considered as a reliable index of in vitro produced embryo quality and developmental potential.

Effect of vitrification solutions and cooling upon in vitro matured prepubertal ovine oocytes.

The vitrification procedure effects on molecular and cytoskeletal components and on developmental ability of in vitro matured prepubertal ovine oocytes were evaluated. MII oocytes were divided into three groups: (1) vitrified in cryoloops (VTR); (2) exposed to vitrification solutions and rehydrated without being plunged into liquid nitrogen (EXP); (3) without further treatment as a control (CTR). Two hours after treatment, membrane integrity, assessed by propidium iodide/Hoechst staining, was lower in VTR and EXP than in CTR (70.6%, 88.5% and 95.2%, respectively). Cleavage rate after fertilization was statistically different among all groups (21.4%, 45.4% and 82.8% for VTR, EXP and CTR groups respectively; P<0.01). Blastocyst rate in VTR (0.0%) and EXP (2.8%) groups was lower (P<0.01) than in CTR (22.8%). Maturation promoting factor activity was lower (P<0.01) in VTR and EXP groups compared with CTR at both 0 h (82.2%, 83.6% and 100%, respectively) and 2 h (60% and 53.9% and 100%, respectively) after warming. Immediately after warming VTR and EXP oocytes showed a lower rate of normal spindle and chromosome configuration compared to CTR (59.1%, 48.0% and 83.3%, respectively; P<0.01). After 2 h of culture in standard conditions the percentage of oocytes with normal spindle and chromosome organization decreased in both VTR and EXP groups compared to CTR (36.4%, 42.8% versus 87.5%, respectively). In conclusion the exposition to the tested cryoprotectant solution and the vitrification in cryoloops modified cytoskeletal components and alter biochemical pathways that compromise the developmental capacity of prepubertal in vitro matured ovine oocytes.

Effects of trehalose co-incubation on in vitro matured prepubertal ovine oocyte vitrification.

Our aim was to evaluate if loading prepubertal ovine oocyte with trehalose would impact on their further developmental potential in vitro and if it would improve their survival to vitrification procedures. COCs matured in vitro with (TRH) or without (CTR) 100mM trehalose were tested for developmental potential after in vitro fertilization and culture. Trehalose uptake was measured by the antrone spectrophotometric assay. No differences were recorded between the two experimental groups in fertilization rates (91.1 CTR vs 92.5% TRH), cleavage rates calculated on fertilized oocytes (96.1 CTR vs 95.4% TRH), first cleavage kinetic (56.1 CTR vs 51% TRH), and blastocyst rates (14.3 CTR vs 13.0% TRH). Anthrone assay revealed that in TRH group trehalose concentration/oocyte was 2.6microM. MII oocytes were then vitrified using cryoloops in TCM 199 containing 20% FCS, sucrose 0.5M, 16.5% Me(2)SO, 16.5% EG and plunged in LN(2). After warming, oocytes from TRH and CTR groups were tested for membrane integrity using the propidium iodide (PI)/Hoechst differential staining, and for developmental ability after in vitro fertilization. Trehalose in maturation medium affected membrane resistance (P<0.01) to vitrification/warming but not fertilization and cleavage rates. The differential staining showed a lower number of PI positive cells in TRH group compared to CTR one (14.3 vs 24.7%, respectively). Fertilization rates and cleavage rates did not differ between the two groups (55.3 and 41% for TRH and 47.7 and 41.7% for CTR, respectively). In conclusion trehalose in maturation medium stabilizes cell membranes during vitrification/warming of prepubertal ovine oocytes but does not affect fertilization and cleavage rates after warming.

Cryopreservation of European Mouflon (Ovis Gmelini Musimon) semen during the non-breeding season is enhanced by the use of trehalose.

The influence of trehalose on European mouflon spermatozoa cryopreservation during the non-breeding season was tested. Semen was frozen in two different extenders: (a) recommended Tris-based ram extender (CTR); (b) CTR extender supplemented with trehalose 0.147 mm (TRH). Sperm viability and acrosome integrity were assessed using propidium iodide and fluorescein isothiocynate labelled Pisum Sativum agglutinin. Trehalose significantly enhanced sperm viability after thawing compared with CTR extender (62.7% vs 51.8%; p < 0.05), whereas no differences were observed on acrosome integrity (42.9% vs 42.1%). Trehalose influence was also evidenced in the in vitro fertility test performed with sheep oocytes matured in vitro. Both fertilization rates (60.9% TRH vs 43.6% CTR; p < 0.05) and cleavage rates (58% TRH vs 39.8% CTR; p < 0.001) were higher for trehalose frozen semen compared with control extender frozen semen. A higher percentage of zygotes resulting from fertilization with trehalose cryopreserved semen presented the first cleavage earlier if compared with the group fertilized with control semen (48.7% vs 31.5%, respectively; p < 0.01). This result was confirmed by embryo kinetic development. Fertilization with trehalose cryopreserved semen leaded to an higher percentage of blastocysts (40.2% vs 27.8% CTR; p < 0.05), and enhanced in particular the number of blastocysts that developed on the day 6th of culture (28.6% vs 17% CTR; p < 0.05). Our data demonstrated that, during mouflon non-breeding season, trehalose extender enhances spermatozoa viability and its in vitro fertilizing capacity, allowing the production of an higher number of blastocysts.

Vitrification devices affect structural and molecular status of in vitro matured ovine oocytes.

We evaluated the effect of three different cryodevices on membrane integrity, tubulin polymerization, maturation promoting factor (MPF) activity and developmental competence of in vitro matured (IVM) ovine oocytes. IVM oocytes were exposed during 3 min to 7.5% DMSO and 7.5% ethylene glycol (EG) in TCM199 and 25 sec to 0.5 M sucrose, 16.5% DMSO and 16.5% EG, loaded in open pulled straws (OPS), cryoloops (CL) or cryotops (CT) and immersed into liquid nitrogen. Untreated (CTR) or exposed to vitrification solutions but not cryopreserved (EXP) oocytes were used as controls. After warming, double fluorescent staining evidenced a lower membrane integrity in vitrified groups compared to the controls (P < 0.01). After in vitro fertilization and culture OPS and CL groups evidenced a lower cleavage rate than CT and controls (P < 0.01) while blastocysts were obtained only in CL and EXP, at a lower rate than CTR (P < 0.01). All vitrified groups showed alterations in spindle conformation, which were partially recovered in OPS and CT groups. MPF activity was lower in treated compared to CTR and CT showed the lowest value (P < 0.01). After 2 hr culture MPF activity was restored in all groups except CT. Parthenogenetic activation was higher in treated compared to CTR and CT evidenced the highest value. Our results indicate that cryodevice influences not only the ability to survive cryopreservation but is also associated with molecular alterations which affect developmental competence.

Rapid detection of Listeria monocytogenes in foods, by a combination of PCR and DNA probe.

Listeria monocytogenes is a frequent contaminant of water and foods. Its rapid detection is needed before some foods can be prepared for marketing. In this work L. monocytogenes has been searched for in foods, by a combination of polymerase chain reaction (PCR) and a DNA probe. Both PCR and the probe were prepared for recognizing a specific region of the internalin gene, which is responsible for the production of one of the most important pathogenic factors of Listeria. The combined use of PCR and the DNA probe was used for the detection of L. monocytogenes in over 180 environmental and food samples. Several detection methods were compared in this study, namely conventional culture methods; direct PCR; PCR after an enrichment step; a DNA probe alone; a DNA probe after enrichment and another commercially available gene-probe. Finally PCR and the DNA probe were used in series on all the samples collected. When the DNA probe was associated with the PCR, specific and accurate detection of listeria in the samples could be obtained in about a working-day. The present molecular method showed some advantages in terms of rapidity and specificity in comparison to the other aforementioned tests. In addition, it resulted as being easy to handle, even for non-specialized personnel in small diagnostic microbiology laboratories.

Gene correction of integrin beta4-dependent pyloric atresia-junctional epidermolysis bullosa keratinocytes establishes a role for beta4 tyrosines 1422 and 1440 in hemidesmosome assembly.

The cytoplasmic domain of beta4 integrin contains two pairs of fibronectin-like repeats separated by a connecting segment. The connecting segment harbors a putative tyrosine activation motif in which tyrosines 1422 and 1440 are phosphorylated in response to alpha6beta4 binding to laminin-5. Primary beta4-null keratinocytes, obtained from a newborn suffering from lethal junctional epidermolysis bullosa, were stably transduced with retroviruses carrying a full-length beta4 cDNA or a beta4 cDNA with phenylalanine substitutions at Tyr-1422 and Tyr-1440. Hemidesmosome assembly was evaluated on organotypic skin cultures. beta4-corrected keratinocytes were indistinguishable from normal cells in terms of alpha6beta4 expression, the localization of hemidesmosome components, and hemidesmosome structure and density, suggesting full genetic and functional correction of beta4-null keratinocytes. In cultures generated from beta4(Y1422F/Y1440F) keratinocytes, beta4 mutants as well as alpha6 integrin, HD1/plectin, and BP180 were not concentrated at the dermal-epidermal junction. Furthermore, the number of hemidesmosomes was strikingly reduced as compared with beta4-corrected keratinocytes. The rare hemidesmosomes detected in beta4(Y1422F/Y1440F) cells were devoid of sub-basal dense plates and of inner cytoplasmic plaques with keratin filament insertion. Collectively, our data demonstrate that the beta4 tyrosine activation motif is not required for the localization of alpha6beta4 at the keratinocyte plasma membrane but is essential for optimal assembly of bona fide hemidesmosomes.

Studies on the distribution of high-level gentamicin-resistant Enterococcus faecalis using ribotyping and restriction analysis of the 16S-23S rRNA intergenic spacer sequences.

Twenty-five high-level gentamicin resistant (HLGR) Enterococcus faecalis strains were isolated from three different University laboratories in Italy. The resistant strains were variously distributed in the three centers with percentages of prevalence ranging from about 3% up to 14%. Almost all strains shared high-level resistance to streptomycin (23 out of 25). Ribotyping and restriction analysis of the 16S-23S rRNA intergenic spacer sequences were used to genetically differentiate the various strains and to study their spreading in the university hospitals serviced by the three laboratories. At least three ribotypes were identified, which showed a peculiar distribution in the various centers. Only the ribotype B was isolated from the University of Padua. In Cagliari, most strains belonged to ribotype A (4/6), whereas in Genoa there was an equal distribution of the ribotypes A and B. A clonal spreading of some HLGR strains is suggested by these findings. The restriction analysis of the 16S-23S rRNA intergenic-spacer sequences gave comparable results with classical ribotyping and, in addition, was quicker and easier to perform than the latter.

Sattabacins and sattazolins: new biologically active compounds with antiviral properties extracted from a Bacillus sp.

A series of new compounds with antiviral properties were isolated from a Bacillus sp. strain B-60. They were named sattabacin (1), hydroxysattabacin (2), sattazolin (3) and methylsattazolin (4). The biologically active compounds were recovered from fermentation broth by ethyl acetate extraction and silica-gel column fractionation. Their antiviral activity was mainly expressed against the Herpes simplex viruses type 1 and 2. The compound 3 showed a selective inhibition of protein synthesis in Herpesvirus-infected cells.

Identification, clinical distribution, and susceptibility to methicillin and 18 additional antibiotics of clinical Staphylococcus isolates: nationwide investigation in Italy.

A multicentric study of clinical Staphylococcus isolates was performed by seven operative units working in different areas of Italy. Over a 6-month period, a total of 3,226 staphylococci, isolated from in- and outpatients, were identified and tested for antimicrobial susceptibility by a protocol agreed upon by all units. On the basis of their bacteriolytic-activity patterns and other conventional tests, the isolates were identified by lyogroups , which closely correlate with human Staphylococcus species. Lyogroup I (Staphylococcus aureus) and lyogroup III (Staphylococcus capitis) were the most and the least frequently isolated staphylococci, respectively. Significant differences depending on strain origin from in- or outpatients were only observed with lyogroup IV (i.e., novobiocin- resistant staphylococci), whose isolation from outpatients was three times greater than from inpatients. Lyogroup I was predominant among isolates from most clinical sources. Lyogroup IV predominated in strains isolated from the urinary tract; lyogroup V (Staphylococcus epidermidis) predominated in strains from blood, cerebrospinal fluid, and indwelling artificial devices; and lyogroup VI ( Staphylococcus hominis, Staphylococcus haemolyticus, and Staphylococcus warneri ) predominated in strains from bile and the male genital tract. The incidence of methicillin resistance within the different lyogroups varied from unit to unit, suggesting epidemiological differences among different hospitals and different geographical areas. On the whole, methicillin resistance was more frequent in coagulase-negative staphylococci than in S. aureus and ranged from 19% for lyogroups I and III to 30% for lyogroup II (Staphylococcus simulans). Laboratory testing with 18 additional antibiotics suggested the occurrence of some specific differences in susceptibility among the different lyogroups . The rate of organisms resistant to the various antibiotics was greater among methicillin-resistant than among methicillin -susceptible staphylococci; particularly marked differences occurred with cephalosporins, rifampin, gentamicin, and tobramycin. The results suggested an increasing spread in Italy, during the last few years, of staphylococcal resistance to methicillin and to many other antibiotics. Some questions about the actual reliability of laboratory tests for the determination of staphylococcal susceptibility to methicillin and other beta-lactam antibiotics were raised by parallel test performances in which both unsupplemented and 5% NaCl-supplemented Mueller-Hinton agars were used. The presence of NaCl heightened, on the whole, the number of resistant strains detected; however, a few isolates resistant in the unsupplemented medium and susceptible in the salt-supplemented medium were also encountered. This was true not only for methicillin but also for all other beta-lactam antibiotics tested except cefamandole. With cefamandole, the presence of 5% NaCl reduced the number of resistant strains detected.