Dipetalonema viteae: in vitro blastogenesis of hamster spleen and lymph node cells to phytohemagglutinin and filarial antigens

Hamsters of the randomly bred LAKZ and inbred LSH strains were infected with Dipetalonema viteae, and the in vitro responses of lymph node and spleen lymphocytes to male and female worm antigens and phytohemagglutinin (PHA) were measured by a [³H]-thymidine-uptake assay at various times after infection. The PHA response remained unchanged at the level of controls in infected LAKZ hamsters while LSH hamsters showed a depressed response to the mitogen during late infection. Stimulation of lymph node cells by filarial antigens was maximal in both strains of hamsters at Week 4 postinfection, almost reaching values obtained in PHA stimulated cultures. A similar high lymphocyte transformation reaction was measured after the injection of dead third stage larvae. During transient microfilaremia, when antibody titers reached a maximal level, the lymphocyte reactivity to filarial antigens decreased drastically and only occasionally was demonstrated in hamsters 20 and 30 weeks after infection. No correlation between lymphocyte reactivity and parasitological findings (worm load or intensity and duration of microfilaremia) could be demonstrated. The cellular unresponsiveness to filarial antigens was further analyzed in chronically infected LAKZ hamsters. No suppressor cells could be found in lymphocyte suspensions of nonresponding hamsters. A challenge infection did not restore lymphocyte reactivity. Serum of chronically infected hamsters caused marked inhibition when added to filarial antigen-sensitive lymphocytes. Lymphocytes from hamsters with a mixed D. viteae and Schistosoma mansoni infection responded as well to soluble schistosomal egg antigens at Week 30 of a D. viteae infection as lymphocytes from hamsters infected with S. mansoni alone. The humoral immune response to schistosomal antigens, however, was significantly lower in animals with a mixed infection.     

Influence of culture age on the phytochemical content and pharmacological activities of five Scenedesmus strains

Five axenic Scenedesmus strains (MACC-411, MACC-422, MACC-493, MACC-720, and MACC-727) were cultured and harvested after 5 and 10 days in culture. Using colorimetric methods, the concentrations of total phenolic, condensed tannin, and iridoids in 50 % methanol extracts from both 5- and 10-day-old cultures were quantified. Different solvent extracts from the strains were also tested for antioxidant, acetylcholinesterase inhibitory (AChEI), and antimicrobial activities using various in vitro test systems. Phenolic content was highest (3.6?±?0.42 mg GAE g^?1 DW) in 10-day-old MACC-727. This was approximately fourfold and significantly higher than in the 5-day-old cultures of MACC-727. Among the tested Scenedesmus strains, 5-day-old MACC-411 had the highest iridoid content (3.4?±?0.3 mg HE g^?1 DW), and this was significantly higher than the level detected in the 10-day-old MACC-411. Scenedesmus strains showed better antioxidant potential in the β-carotene–linoleic acid model compared to the DPPH free radical scavenging assay. The AChEI activity (IC₅₀?μg mL^?1) in all strains (besides MACC-422) was higher in 10-day-old cultures compared to the 5-day-old cultures. Although a broad-spectrum of antibacterial activity was observed, the tested microalgae strains demonstrated varying degrees of antimicrobial potential depending on the harvest time, strain-type, and extracting solvent. Thus, the Scenedesmus strain and time of harvest played a significant role in determining their phytochemical content and resultant pharmacological activity. The promising bioactivity in the tested Scenedesmus strains indicates their potential as possible sources of novel/alternative antioxidants and AChE inhibitors.     

Coagulase-negative staphylococci strains resistant to oxacillin isolated from neonatal blood cultures

Coagulase-negative staphylococci (CoNS) are the microorganisms most frequently isolated from clinical samples and are commonly found in neonatal blood cultures. Oxacillin is an alternative treatment of choice for CoNS infections; however, resistance to oxacillin can have a substantial impact on healthcare by adversely affecting morbidity and mortality. The objective of this study was to detect and characterise oxacillin-resistant CoNS strains in blood cultures of newborns hospitalised at the neonatal ward of the University Hospital of the Faculty of Medicine of Botucatu. One hundred CoNS strains were isolated and the mecA gene was detected in 69 of the CoNS strains, including 73.2% of Staphylococcus epidermidis strains, 85.7% of Staphylococcus haemolyticus strains, 28.6% of Staphylococcus hominis strains and 50% of Staphylococcus lugdunensis strains. Among these oxacillin-resistant CoNS strains, staphylococcal cassette chromosome mec (SCCmec) type I was identified in 24.6%, type II in 4.3%, type III in 56.5% and type IV in 14.5% of the strains. The data revealed an increase in the percentage of CoNS strains isolated from blood cultures from 1991-2009. Furthermore, a predominant SCCmec profile of the oxacillin-resistant CoNS strains isolated from neonatal intensive care units was identified with a prevalence of SCCmec types found in hospital-acquired strains.     

Radiation-induced human chromosome aberrations. II. Human in vitro irradiation compared to in vitro and in vivo irradiation of marmoset leukocytes

Whole blood cultures from humans and from the New World primate, Saguinus fuscicollis, were irradiated with various doses of 250 kV X-rays. The resulting centric ring plus dicentric aberration yields were fitted to the three models, Y = a+bD, Y = a+bD+cD², and Y = a+cD², by least squares regression. In both instances the best fit was to the model Y = a+bD+cD², with coefficients of the one- and two-track components for human and marmoset being: b = (0.78 ± 0.09)·10⁻³, c = (5.92 ± 0.31)·10⁻⁶, and b = (1.11 ± 0.36)·⁻³, c = (7.7 ± 1.7)·10⁻⁶, respectively.     

Disseminated Amphotericin-Resistant Fusariosis in Acute Leukemia Patients: Report of Two Cases

Disseminated fusariosis has emerged as a significant, usually fatal infection in immunocompromised hosts despite antifungal treatment. We describe here two patients with acute leukemia who developed disseminated amphotericin-resistant fusariosis, and review of six studies of cases series in the literature. Two Fusarium solani strains were isolated from blood and skin cultures of one patient, and one strain from the blood culture of the second patient. Both patients died despite antifungal treatment. Strains were identified by sequencing of ITS1 and ITS4 regions. Random amplified polymorphic DNA analysis of the three F. solani isolates showed a low degree of similarity. Screening for Fusarium spp. contaminants within our facility was negative. Using the CLSI M-38-A2 broth dilution method and E tests^®, we found that the MICs were low for voriconazole (0.12 and 0.5 mg/L, respectively), unexpectedly high for amphotericin B (≥8 and ≥32 μg/mL, respectively) and itraconazole (≥16 mg/ml). Patients with leukemia or persistent neutropenia should be assessed for disseminated fungal infections, including biopsy and skin cultures. Antifungal susceptibility tests are important due to the possibility of the strains being amphotericin resistant. Treatments must be aggressive, with high doses of antifungals or combined therapy.     

Pre-existing isoniazid resistance, but not the genotype of Mycobacterium tuberculosis drives rifampicin resistance codon preference in vitro

Both the probability of a mutation occurring and the ability of the mutant to persist will influence the distribution of mutants that arise in a population. We studied the interaction of these factors for the in vitro selection of rifampicin (RIF)-resistant mutants of Mycobacterium tuberculosis. We characterised two series of spontaneous RIF-resistant in vitro mutants from isoniazid (INH)-sensitive and -resistant laboratory strains and clinical isolates, representing various M. tuberculosis genotypes. The first series were selected from multiple parallel 1 ml cultures and the second from single 10 ml cultures. RIF-resistant mutants were screened by Multiplex Ligation-dependent Probe Amplification (MLPA) or by sequencing the rpoB gene. For all strains the mutation rate for RIF resistance was determined with a fluctuation assay. The most striking observation was a shift towards rpoB-S531L (TCG→TTG) mutations in a panel of laboratory-generated INH-resistant mutants selected from the 10-ml cultures (p<0.001). All tested strains showed similar mutation rates (1.33×10⁻⁸ to 2.49×10⁻⁷) except one of the laboratory-generated INH mutants with a mutation rate measured at 5.71×10⁻⁷, more than 10 times higher than that of the INH susceptible parental strain (5.46–7.44×10⁻⁸). No significant, systematic difference in the spectrum of rpoB-mutations between strains of different genotypes was observed. The dramatic shift towards rpoB-S531L in our INH-resistant laboratory mutants suggests that the relative fitness of resistant mutants can dramatically impact the distribution of (subsequent) mutations that accumulate in a M. tuberculosis population, at least in vitro. We conclude that, against specific genetic backgrounds, certain resistance mutations are particularly likely to spread. Molecular screening for these (combinations of) mutations in clinical isolates could rapidly identify these particular pathogenic strains. We therefore recommend that isolates are screened for the distribution of resistance mutations, especially in regions that are highly endemic for (multi)drug resistant tuberculosis.     

The MAK16 Gene of Entamoeba histolytica and Its Identification in Isolates from Patients

To identify sequences of Entamoeba histolytica associated with the development of amebic liver abscess (ALA) in hamsters, subtractive hybridization of cDNA from E. histolytica HM-1:IMSS under 2 growth conditions was performed: 1) cultured in axenic medium and 2) isolated from experimental ALA in hamsters. For this procedure, 6 sequences were obtained. Of these sequences, the mak16 gene was selected for amplification in 29 cultures of E. histolytica isolated from the feces of 10 patients with intestinal symptoms and 19 asymptomatic patients. Only 5 of the 10 isolates obtained from symptomatic patients developed ALA and amplified the mak16 gene, whereas the 19 isolates from asymptomatic patients did not amplify the mak16 gene nor did they develop ALA. Based on the results of Fisher''s exact test (P<0.001), an association was inferred between the presence of the mak16 gene of E. histolytica and the ability to develop ALA in hamsters and with the patient''s symptoms (P=0.02). The amplification of the mak16 gene suggests that it is an important gene in E. histolytica because it was present in the isolates from hamsters that developed liver damage.     

The effects of Lactobacillus-fermented milk on lipid metabolism in hamsters fed on high-cholesterol diet

The objective of this study was to evaluate the effects of local Lactobacillus strains (NTU 101 and 102) on cholesterol-lowering effects in vivo. Thirty male hamsters were housed, divided into five groups, and fed on a cholesterol diet (5 g/kg diet) to induce hypercholesterolemia. Milk fermented by Lactobacillus paracasei subsp. paracasei NTU 101, Lactobacillus plantarum NTU 102, and Lactobacillus acidophilus BCRC 17010 was administrated for this study. After treatment with different fermented milk, blood was taken and liver was removed for the determination of lipoproteins, including total cholesterol, high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), and triglyceride. Lactobacilli and bifidobacteria decreased (10⁵) in the control group; when hamsters were fed on fermented milk, the number of lactobacilli (10⁷–10⁸) and bifidobacteria (10⁵–10⁷) was increased. Serum and liver total cholesterol levels were significantly reduced by about 26.4, 23.5, and 30.1% and by about 17.7, 15.9, and 13.4% when hamsters were given fermented milk. However, serum HDL-C and LDL-C were also reduced. The results of this study showed that the hypocholesterolemic effect of local Lactobacillus strains was attributed to its ability to lower serum and liver total cholesterol levels. Thus, local Lactobacillus strains could significantly increase probiotic count.     

In vitro development of a primary antibody response with lymph node cells.

Utilizing a variety of lymphoid tissues from three common laboratory species, comparative studies were performed to investigate the competence of the dissociated cells to respond to a heterologous erythrocyte with the development of specific plaque-forming cells. Dissociated spleen cells harvested from BDF1 mice consistently developed specific plaque-forming cells (PFC) to sheep red blood cells (SRBC), while hamster spleen cells inconsistently developed specific antibody-forming cells to SRBC. Under identical conditions, guinea pig spleen cells did not develop significant numbers of PFC to SRBC. However, lymph node cell cultures of all three species tested yielded specific PFC. In the mouse and hamster lymph node cell cultures, the yield of PFC per culture or per 10⁶ recovered viable cells was always greater than the yield from companion spleen cell cultures. Guinea pig mesenteric lymph node cell cultures developed the major PFC response to SRBC, while both mesenteric and peripheral lymph node cell cultures from hamsters were equivalent in their response to SRBC. The data demonstrate that it is possible to develop a primary antibody response to SRBC in vitro utilizing normal endogenous hamster or guinea pig lymphoid cells, if lymph nodes are the source of cells.     

Pathogenicity of Five Strains of Toxoplasma gondii from Different Animals to Chickens

Toxoplasma gondii is a protozoan parasite with a broad range of intermediate hosts. Chickens as important food-producing animals can also serve as intermediate hosts. To date, experimental studies on the pathogenicity of T. gondii in broiler chickens were rarely reported. The objective of the present study was to compare the pathogenicity of 5 different T. gondii strains (RH, CN, JS, CAT2, and CAT3) from various host species origin in 10-day-old chickens. Each group of chickens was infected intraperitoneally with 5×10⁸, 1×10⁸, 1×10⁷, and 1×10⁶ tachyzoites of the 5 strains, respectively. The negative control group was mockly inoculated with PBS alone. After infection, clinical symptoms and rectal temperatures of all the chickens were checked daily. Dead chickens during acute phage of the infection were checked for T. gondii tachyzoites by microscope, while living cases were checked for T. gondii infection at day 53 post-inoculation (PI) by PCR method. Histopathological sections were used to observe the pathological changes in the dead chickens and the living animals at day 53 PI. No significant differences were found in survival periods, histopathological findings, and clinical symptoms among the chickens infected with the RH, CN, CAT2, and CAT3 strains. Histopathological findings and clinical symptoms of the JS (chicken origin) group were similar to the others. However, average survival times of infected chickens of the JS group inoculated with 5×10⁸ and 1×10⁸ tachyzoites were 30.0 and 188.4 hr, respectively, significantly shorter than those of the other 4 mammalian isolates. Chickens exposed to 10⁸ of T. gondii tachyzoites and higher showed acute signs of toxoplasmosis, and the lesions were relatively more severe than those exposed to lower doses. The results indicated that the pathogenicity of JS strain was comparatively stronger to the chicken, and the pathogenicity was dose-dependent.     

Prevention of Clostridium difficile -induced ileocecitis with Bacteriophage

A bacteriophage specific for Clostridium difficile was examined for its ability to prevent ileocecitis in a hamster model. This species- and strain-specific bacteriophage was isolated from a lysogenic strain of C. difficile . Hamsters were maintained in sterile isolation cages to prevent the acquisition of C. difficile from the environment. Bicarbonate neutralization of gastric acidity was necessary for bacteriophage survival in the hamster's gastrointestinal tract. Bacteriophage recovery from the hamster cecum was 2×10⁴plaque forming units/mL of cecal contents 24 h after orogastric challenge with 10⁸plaque forming units/mL of bacteriophage. However, there was no bacteriophage recovery 48 h post challenge, indicating dissipation of bacteriophage from the hamster intestinal tract within this time frame. Twenty-four hours after being challenged with clindamycin, one group of hamsters was challenged with C. difficile followed by a single dose of bacteriophage (10⁸plaque forming units/mL). Two additional groups of hamsters received phage doses immediately after C. difficile challenge and subsequently thereafter every 8 h up to 48 and 72 h, respectively. The gastric acidity was neutralized with bicarbonate buffer preceding every bacteriophage treatment. Control animals that received only clindamycin and C. difficile died within 96 h after challenge while the majority of bacteriophage treated hamsters survived. Two weeks after stopping bacteriophage treatment, the surviving hamsters were re-challenged with clindamycin and C. difficile . All the hamsters died within 96 h indicating susceptibility of the surviving hamsters to C. difficile disease in the absence of bacteriophage treatment.     

Volatile Emissions from Mycobacterium avium subsp. paratuberculosis Mirror Bacterial Growth and Enable Distinction of Different Strains

Control of paratuberculosis in livestock is hampered by the low sensitivity of established direct and indirect diagnostic methods. Like other bacteria, Mycobacterium avium subsp. paratuberculosis (MAP) emits volatile organic compounds (VOCs). Differences of VOC patterns in breath and feces of infected and not infected animals were described in first pilot experiments but detailed information on potential marker substances is missing. This study was intended to look for characteristic volatile substances in the headspace of cultures of different MAP strains and to find out how the emission of VOCs was affected by density of bacterial growth. One laboratory adapted and four field strains, three of MAP C-type and one MAP S-type were cultivated on Herrold’s egg yolk medium in dilutions of 10^-0, 10^-2, 10^-4 and 10^-6. Volatile substances were pre-concentrated from the headspace over the MAP cultures by means of Solid Phase Micro Extraction (SPME), thermally desorbed from the SPME fibers and separated and identified by means of GC-MS. Out of the large number of compounds found in the headspace over MAP cultures, 34 volatile marker substances could be identified as potential biomarkers for growth and metabolic activity. All five MAP strains could clearly be distinguished from blank culture media by means of emission patterns based on these 34 substances. In addition, patterns of volatiles emitted by the reference strain were significantly different from the field strains. Headspace concentrations of 2-ethylfuran, 2-methylfuran, 3-methylfuran, 2-pentylfuran, ethyl acetate, 1-methyl-1-H-pyrrole and dimethyldisulfide varied with density of bacterial growth. Analysis of VOCs emitted from mycobacterial cultures can be used to identify bacterial growth and, in addition, to differentiate between different bacterial strains. VOC emission patterns may be used to approximate bacterial growth density. In a perspective volatile marker substances could be used to diagnose MAP infections in animals and to identify different bacterial strains and origins.     

Effect of Gonadectomy on the Androgen-Dependent Behavior of Ganglion Cell-Like Cells in Djungarian Hamsters (Phodopus sungorus)

Ganglion cell-like (GL) cells reside in the dermis of the ventral skin of mature male Djungarian hamsters (Phodopus sugorus) and express androgen receptor (AR). To assess whether GL cells have androgen-dependent behavior, we evaluated the histologic changes of GL cells after gonadectomy. Five male and 5 female hamsters were gonadectomized at the age of 4 wk and necropsied 14 wk later. The number, distribution, and proliferative activity of GL cells in the thoracoabdominal and dorsal skins were evaluated histologically and compared with those of corresponding intact animals. GL cells were more numerous, were distributed throughout the skin more widely, and had higher proliferative activity in the intact male hamsters than in their gonadectomized counterparts. Similar trends regarding these 3 parameters were seen in ovariectomized compared with intact female hamsters and between intact male and intact female hamsters. These results suggest that the GL cells of Djungarian hamsters demonstrate sex-associated differences in their distribution and proliferative activity and that androgen may be involved in the development of these cells.Abbreviations: AR, androgen receptor; GL cell, ganglion cell-like cellGanglion cell-like (GL) cells reside in the dermis of the abdominal and thoracic skin of mature male Djungarian hamsters (Phodopus sungorus).^1,7 GL cells have a small round nucleus with an apparent nucleolus and abundant basophilic foamy cytoplasm. These cells usually aggregate to form nests accompanied by various volumes of stromal collagen fibers. The nests increase in size and number with maturation. The nuclei of GL cells have a positive reaction for androgen receptor (AR), and the cytoplasm is positive for vimentin.^1,7 Although the morphologic characteristics and various immunophenotypes of GL cells have been documented, their behavior and role remain almost unclear. Some authors speculate that increased levels of testosterone may influence GL cell proliferation and the oncogenesis of atypical skin fibromas preferentially arising in this species.¹The current study aims to elucidate the androgen-dependent behavior of GL cells and compare the histologic changes of GL cells in gonadectomized Djungarian hamsters with those in intact control animals.     

Leishmania tropica: pathogenicity and in vitro macrophage function in strains of inbred mice.

Of seven strains of inbred mice and one hybrid that were infected intracutaneously with 5, 10, or 20 × 10⁶ active promastigotes of Leishmania tropica major, two strains (CBA/Ca and C₃H/He) recovered from the infection and their lesions healed within 3 to 5 months. The other strains, with the possible exception of C57B1/6 animals, remained infected, carrying large cutaneous ulcers throughout their lives. These included DBA/2, A/Jax, Balb/c, athymic nude mice of Balb/c origin (nu/nu) and the heterozygote Balb/c (nu⁺). The responses of C57B1/6 animals were of intermediate type with a tendency toward nonhealing at higher doses of the parasite. The cutaneous infection of athymic nude mice invariably gave rise to fulminating visceral infections and death. This condition was never observed in the other strains tested. Concanavalin A (Con A)-stimulated syngeneic or allogeneic lymphocytes of intact mice activated peritoneal macrophages of both healer and nonhealer mice, resulting in complete destruction of phagocytosed L. enriettii within 24 to 48 hr. The destruction of ingested L. tropica was confined to macrophages of healer mice and required 72 to 96 hr to reach completion. However, removal of Con A-stimulated lymphocytes from macrophage cultures and regular pulsing of the cells with a lymphokine-rich supernatant produced a state of sustained activation, resulting in destruction of L. tropica inside macrophages of both healer and nonhealer mice. The ability of Con A-stimulated lymphocytes of nonhealer animals to induce effective levels of activation in healer macrophages on one hand, and eventual destruction of L. tropica in macrophages of nonhealer mice under condition of sustained activation on the other, had indicated that so far as the in vitro situation is concerned, there is no inherent defect in lymphocytes or macrophages of nonhealer animals, although the threshold of activation necessary for killing of the parasite seems to be higher for cells of nonhealer origin.     

Leishmania (L.) infantum chagasi isolated from skin lesions of patients affected by non-ulcerated cutaneous leishmaniasis lead to visceral lesion in hamsters

In Central America, Leishmania (L.) infantum chagasi infection causes visceral leishmaniasis (VL) and non-ulcerated cutaneous leishmaniasis (NUCL). The aim of the present study was to evaluate the course of an experimental infection in hamsters caused by L. (L.) infantum chagasi isolated from patients affected by NUCL compared with a strain isolated from a patient with VL. Stationary phase parasites in culture were inoculated through subcutaneous and intraperitoneal routes in hamsters. Following the post-infection times, a histopathological study, parasite load and cytokine determination in skin from the cutaneous inoculation site and viscera were performed. Animals subcutaneously infected with the different strains did not develop macroscopic lesions at the inoculation site, and the histopathological changes in the dermis were very slight. Regarding the histopathological study of the viscera, we observed the portal mononuclear inflammatory infiltrate, the presence of nodules in the hepatic parenchyma and the proliferation of macrophages in the spleen, which increased over the infection course. Overall, the parasite load in the liver and spleen and in the total IgG titres in the sera of infected hamster showed an increase with the time of infection, regardless of the route of inoculation. Regarding cellular immunity, we did not observe an increase or decrease in pro- and anti-inflammatory cytokines compared to the healthy control, except for IL-10, which was evident in the infected animals. The data showed that strains isolated from NUCL cause visceral lesions in the hamsters regardless of the route of inoculation, and they were similar to parasites isolated from VL humans.     

Comparative Evaluation of Five Selective and Differential Media for the Detection and Enumeration of Coagulase-Positive Staphylococci in Foods

Five selective media for the detection and enumeration of coagulase-positive staphylococci were evaluated for their efficiency in the recovery of 17 strains of coagulase-positive staphylococci from foods. They were Staphylococcus Medium 110 (SM-110), tellurite-glycine-agar (TGA), egg-tellurite-glycine-pyruvate-agar (ETGPA), tellurite-egg-agar (TEA), and tellurite-polymyxin-egg yolk-agar (TPEY). Statistical analysis by the rank correlation method of the efficiency with which these media recovered staphylococci from pure 24-hr Brain Heart Infusion cultures revealed the following efficiencies in descending order: (i) TPEY, (ii) ETGPA, (iii) TGA, (iv) TEA, (v) SM-110. Growth of 17 strains of coagulase-negative cocci on these media showed the following approximate descending order of inhibition to these organisms: (i) ETGPA, (ii) TEA, (iii) SM-110, (iv) TGA, (v) TPEY. The appearance of colonies of the various coagulase-negative strains on each medium was studied for the degree to which they could be confused with colonies of coagulase-positive strains. Nineteen food contaminants, including Proteus vulgaris, Bacillus sp., Escherichia coli, Erwinia sp., fecal streptococci, and others, were also studied for similarities in appearance to staphylococci and for ability to grow on the selective media. The influence of five sterile food homogenates (frozen chicken and tuna pies, custard, smoked ham, and raw whole egg) on recovery of 1,500 enterotoxigenic staphylococci (three strains) per milliliter was determined by statistical analysis. Three main effects (culture, media, and food) and three interactions (media with food, food with cultures, and media with culture) were found to be significant. Recovery on TPEY was influenced less by food than the other selective media and showed optimal recovery ability from sterile custard, eggs, and ham. TGA recovered well from sterile chicken pie and custard, SM-110 from sterile custard, and TEA from sterile ham. None of the media was outstanding in recovering staphylococci from tuna pie. The ability of the five selective media to recover 1,500 enterotoxigenic staphylococci (three strains) per ml from three sterile foods in the presence of 10 strains of contaminating bacteria added at the 0, 10⁵, and 10⁶ levels per milliliter was also studied and analyzed statistically. Only three factors were significant under these conditions—cultures, foods, and the interaction of media with the level of added contamination. Efficiency of recovery of TGA, SM-110, and ETGPA was found not to be dependent upon the level of contamination. Recovery on TPEY decreased with increases in the number of contaminants. TEA increased in efficiency at the 10⁵ level, but decreased at the 10⁶ level. When recovery on Trypticase Soy Agar was considered to be 100%, the average percentage of recovery by each of the selective media under all experimental conditions was determined.     

Reduced susceptibility to vancomycin and biofilm formation in methicillin-resistant Staphylococcus epidermidis isolated from blood cultures

This study aimed to correlate the presence of ica genes, biofilm formation and antimicrobial resistance in 107 strains of Staphylococcus epidermidis isolated from blood cultures. The isolates were analysed to determine their methicillin resistance, staphylococcal cassette chromosome mec (SCCmec) type, ica genes and biofilm formation and the vancomycin minimum inhibitory concentration (MIC) was measured for isolates and subpopulations growing on vancomycin screen agar. The mecA gene was detected in 81.3% of the S. epidermidis isolated and 48.2% carried SCCmec type III. The complete icaADBC operon was observed in 38.3% of the isolates; of these, 58.5% produced a biofilm. Furthermore, 47.7% of the isolates grew on vancomycin screen agar, with an increase in the MIC in 75.9% of the isolates. Determination of the MIC of subpopulations revealed that 64.7% had an MIC ≥ 4 μg mL^-1, including 15.7% with an MIC of 8 μg mL^-1 and 2% with an MIC of 16 μg mL^-1. The presence of the icaADBC operon, biofilm production and reduced susceptibility to vancomycin were associated with methicillin resistance. This study reveals a high level of methicillin resistance, biofilm formation and reduced susceptibility to vancomycin in subpopulations of S. epidermidis. These findings may explain the selection of multidrug-resistant isolates in hospital settings and the consequent failure of antimicrobial treatment.     

ATP-dependent deoxyribonuclease in Bacillus subtilis and a mutant deficient in this activity

ATP-dependent DNAse activity was measured in rec⁺ and several rec strains of B. subtilis 168. One of the strains (marker recE₅) was found to lack this activity. The enzyme from the wild type was partially purified and some of its properties were determined. The pH optimum is 9.5. Activity is higher at 50° but inactivation occurs on standing at this temperature. The enzyme requires Mg²⁺ (10^?2M) or Mn²⁺ (2·10^?4M). ATP is an absolute requirement and the only other nucleoside triphosphate that can partially replace it is dATP. Lack of activity in the mutant does not seem to be due to the presence of an inhibitor. Results so far do not allow us to conclude as to whether or not the mutant produces an altered enzyme.     

Community and hospital acquired methicillin resistant <Emphasis Type="Italic">Staphylococcus aureus</Emphasis> efficiently retain the <Emphasis Type="Italic">Van A</Emphasis> determinant

Dissemination of vancomycin resistance from hospital to community strains is a serious threat to public health. Our study aimed to provide evidence for transmission of Van A type resistance from the hospital to the community. Wild-type community and hospital associated methicillin resistant Staphylococcus aureus strains were studied in vitro and in a model that mimicked a natural environment to ascertain their ability to acquire and maintain the vancomycin resistance determinant (Van A gene) from vancomycin resistant Enterococcus faecalis. Fitness was assessed and the cost of Van A acquisition and retention was estimated. In vitro mating experiments were carried out using a filter mating technique and a model of a natural water body environment. Transfer of resistance was carried out in two different conditions: restricted and favorable. Transconjugants were confirmed by E test and PCR using specific primer sets. Growth kinetics and fitness measurements were done by spectrometric analysis. Using the in vitro filter mating technique, high transfer frequencies that ranged from 0.7 × 10^–3(0.0006) to 3.1 × 10^–4(0.00011) were recorded, with the highest transfer frequencies for CA MRSA (thermosensitively homogenous) (0.7 × 10^–3), and 1.2 × 10^–4 to 2.4 × 10^–6 in the model. HA MRSA (homogenous) showed a greater capacity (3.6 × 10^–4) to receive the Van A gene, while CA MRSA showed a reduced ability to maintain the gene after serial subcultures. CA and HA thermosensitively heterogeneous MRSA transconjugants exhibited higher growth rates. The present study provides evidence for the enhanced ability of CA and HA MRSA clones to acquire and maintain Van A type resistance.     

Dilution of Liquid Rhizobium Cultures To Increase Production Capacity of Inoculant Plants

Experiments were undertaken to test whether peat-based legume seed inoculants, which are prepared with liquid cultures that have been deliberately diluted, can attain and sustain acceptable numbers of viable rhizobia. Liquid cultures of Rhizobium japonicum and Rhizobium phaseoli were diluted to give 10⁸, 10⁷, or 10⁶ cells per ml, using either deionized water, quarter-strength yeast-mannitol broth, yeast-sucrose broth, or yeast-water. The variously diluted cultures were incorporated into gamma-irradiated peat, and the numbers of viable rhizobia were determined at intervals. In all of the inoculant formulations, the numbers of rhizobia reached similarly high ceiling values by 1 week after incorporation, irrespective not only of the number of cells added initially but also of the nature of the diluent. During week 1 of growth, similar multiplication patterns of the diluted liquid cultures were observed in two different peats. Numbers of rhizobia surviving in the various inoculant formulations were not markedly different after 6 months of storage at 28°C. The method of inoculant preparation did not affect the nitrogen fixation effectiveness of the Rhizobium strains.