Three novel Enterobacter cloacae bacteriophages for therapeutic use from Ghanaian natural waters.

Infections caused by multidrug-resistant (MDR) bacteria are a growing global concern. Enterobacter cloacae complex (ECC) species are particularly adept at developing antibiotic resistance. Phage therapy is proposed as an alternative treatment for pathogens that no longer respond to antibiotics. Unfortunately, ECC phages are understudied when compared to phages of many other bacterial species. In this Ghanaian-Finnish study, we isolated two ECC strains from ready-to-eat food samples and three novel phages from natural waters against these strains. We sequenced the genomic DNA of the novel Enterobacter phages, fGh-Ecl01, fGh-Ecl02, and fGh-Ecl04, and assessed their therapeutic potential. All of the phages were found to be lytic, easy to propagate, and lacking any toxic, integrase, or antibiotic resistance genes and were thus considered suitable for therapy purposes. They all were found to be related to T4-type viruses: fGh-Ecl01 and fGh-Ecl04 to karamviruses and fGh-Ecl02 to agtreviruses. Testing of Finnish clinical ECC strains showed promising susceptibility to these novel phages. As many as 61.1% of the strains were susceptible to fGh-Ecl01 and fGh-Ecl04, and 7.4% were susceptible to fGh-Ecl02. Finally, we investigated the susceptibility of the newly isolated ECC strains to three antibiotics - meropenem, ciprofloxacin, and cefepime - in combination with the novel phages. The use of phages and antibiotics together had synergistic effects. When using an antibiotic-phage combination, even low concentrations of antibiotics fully inhibited the growth of bacteria.

Ara h 2 and Ara 6 are the best predictors of severe peanut allergy: a double-blind placebo-controlled study.

BACKGROUND: Component-resolved diagnostics offers a modern tool in peanut allergy, but studies applying consistently double-blind placebo-controlled challenges are lacking. We aimed to optimize diagnostics for moderate-to-severe peanut allergy in a birch-endemic region and to create an oral-peanut challenge with its allergen activity characterized. METHODS: We performed double-blind placebo-controlled peanut challenges for a referred sample of 6- to 18-year-olds with peanut sensitization or a high suspicion of peanut allergy, including anaphylaxis. We measured specific IgE (sIgE) to Ara h 1, 2, 3, 6, 8, and 9. Testing of allergen activity of the challenge products was by IgE microarray inhibition. RESULTS: Of the 102 patients, 69 were challenge positive: 25 (36%) had severe, 36 (52%) moderate, and 8 (12%) mild symptoms; 38 (37%) received adrenalin. SIgE to Ara h 6 AUC 0.98 (95%CI, 0.96-1.00) was the best marker of moderate-to-severe allergy. When sIgE to Ara h 2 and Ara h 6 was measured together, all (100%) severe reactions at low doses were successfully diagnosable. SIgE to Ara h 8 had no diagnostic value, AUC 0.42 (95%CI, 0.30-0.52). Both nonroasted and roasted peanut inhibited 100% of IgE binding to Ara h 1, 2, 3, and 6. Nonroasted peanut inhibited 87% of IgE binding to Ara h 8, roasted inhibited 30%. The products lacked Ara h 9 activity. CONCLUSION: Co-sensitization to Ara h 2 and Ara h 6 was associated with severe reactions distinguishing severe allergy from mild symptoms. SIgE to Ara h 8 added no diagnostic value. Component-resolved diagnostics reduce the need for oral challenges in peanut allergy.

Expression of heterologous O-antigen in Yersinia pestis KIM does not affect virulence by the intravenous route.

All strains of Yersinia pestis examined have been found to lack an O-antigen. In other members of the Enterobacteriaceae, the rough phenotype often results in attenuation. However, Y. pestis is the aetiological agent of bubonic plague. In evolving from the ancestral enteropathogenic Yersinia pseudotuberculosis, and with the development of an arthropod-vectored systemic pathogenesis, smooth LPS production is not necessary for Y. pestis virulence and the metabolic burden has been alleviated by inactivation of the O-antigen biosynthetic operon. To investigate this, Y. pestis strain KIM D27 was transformed with a plasmid carrying the operon encoding the O-antigen of Yersinia enterocolitica O : 3. Expression of the O-antigen could be detected in silver-stained gels. The receptor for bacteriophage phiYeO3-12 has been shown to be O-antigen, and infection by this bacteriophage results in lysis of Y. enterocolitica O : 3. Expression of the O-antigen in Y. pestis conferred sensitivity to lysis by phiYeO3-12. The O-antigen-expressing clone was shown to be as virulent in mice by the intravenous route of challenge as the rough wild-type. Assays showed no alteration in the ability of Y. pestis to resist lysis by cationic antimicrobial peptides, serum or polymyxin.

Bacteriophage phiYeO3-12, specific for Yersinia enterocolitica serotype O:3, is related to coliphages T3 and T7.

Bacteriophage phiYeO3-12 is a lytic phage of Yersinia enterocolitica serotype O:3. The phage receptor is the lipopolysaccharide O chain of this serotype that consists of the rare sugar 6-deoxy-L-altropyranose. A one-step growth curve of phiYeO3-12 revealed eclipse and latent periods of 15 and 25 min, respectively, with a burst size of about 120 PFU per infected cell. In electron microscopy phiYeO3-12 virions showed pentagonal outlines, indicating their icosahedral nature. The phage capsid was shown to be composed of at least 10 structural proteins, of which a protein of 43 kDa was predominant. N-terminal sequences of three structural proteins were determined, two of them showing strong homology to structural proteins of coliphages T3 and T7. The phage genome was found to consist of a double-stranded DNA molecule of 40 kb without cohesive ends. A physical map of the phage DNA was constructed using five restriction enzymes. The phage infection could be effectively neutralized using serum from a rabbit immunized with whole phiYeO3-12 particles. The antiserum also neutralized T3 infection, although not as efficiently as that of phiYeO3-12. phiYeO3-12 was found to share, in addition to the N-terminal sequence homology, several common features with T3, including morphology and nonsubjectibility to F exclusion. The evidence conclusively indicated that phiYeO3-12 is the first close relative of phage T3 to be described.

Detection and characterization of atopic allergens.

The aim of the research of atopic allergens is to gather knowledge in order to be able to identify and to characterize environmental allergens and to develop better allergen preparations for diagnosis and treatment of IgE-mediated hypersensitivity diseases. Allergens or allergen activity can be detected and characterized with several in vivo and in vitro methods. In vivo tests measure the biological allergen activity, which is the most important characteristic of allergen preparations. Chemical and some immunochemical methods do not directly measure allergen activity, unless human IgE antibodies have been utilized. Since all these methods have their advantages and disadvantages, more than one method would be favourable in characterization of an allergen or an allergen preparation.

Banana allergy in patients with immediate-type hypersensitivity to natural rubber latex: characterization of cross-reacting antibodies and allergens.

BACKGROUND: An association between allergy to latex and banana has been reported. Even though cross-reacting IgE antibodies have been demonstrated, in no study has the existence of structurally similar allergens been confirmed. In the present study banana allergy was studied in a large series of patients with latex allergy. Specific IgE antibodies were characterized for cross-reactivity and compared with pollen RAST results. Latex and banana extracts were investigated for common antigens and allergens. METHODS: Latex-, banana-, and pollen-specific (birch, timothy, mugwort) IgE were measured in 47 sera from patients with latex allergy. Thirty-one patients were skin prick tested with banana and questioned for possible reactions after eating bananas. Several RAST inhibition and immunospot inhibition studies were used to characterize cross-reacting IgE antibodies. Structurally similar antigens and allergens were evaluated with crossed-line immunoelectrophoresis and crossed-line radioimmunoelectrophoresis, respectively. RESULTS: Latex RAST results were positive in 31 (66%) and banana RAST results were positive in 26 (55%) of the 47 sera. Of the 31 latex RAST-positive sera, 25 (81%) were also banana RAST-positive. Results from latex RAST correlated significantly with results from banana RAST (p < 0.001), but not with those from pollen RAST (p > 0.05). Banana skin prick test results were positive in 11 (35%) of the 31 patients tested. Symptoms after eating bananas were reported by 16 (52%) of the 31 patients. In inhibition studies the binding of IgE antibodies to solid-phase banana and to several latex preparations was inhibited by latex and banana, respectively. In crossed-line immunoelectrophoresis at least one antigen from banana fused with an antigen from latex, which also bound IgE antibodies in autoradiography (crossed-line radioimmunoelectrophoresis). CONCLUSIONS: Patients with latex allergy have symptoms caused by banana and show positive skin test and specific IgE test results. Cross-reacting IgE antibodies were confirmed by several inhibition techniques. For the first time, a structurally similar antigen/allergen was demonstrated.

A sensitive enzyme-linked immunosorbent assay for determination of bovine beta-lactoglobulin in infant feeding formulas and in human milk.

We developed a sensitive sandwich-type ELISA for measuring low levels of cow's milk (CM) beta-lactoglobulin. Purified anti-beta-lactoglobulin was used as coating antibody and also as second antibody conjugated with alkaline phosphatase. Polyethylene glycol 6000 was added to the incubation buffers to improve sensitivity. The detection limit of the assay was 0.002 microgram/l, which is much better than sensitivities reported for other beta-lactoglobulin assays. The sensitivity was not impaired by the presence of other CM proteins. The recovery from breast milk was 93% and from the diluting buffer 127%. The coefficient of variation within day was 5-15% and between days 10%. One hour after oral intake of milk, beta-lactoglobulin could be detected in the breast milk of three mothers at concentrations of about 1-2 micrograms/l. Widely different concentrations of beta-lactoglobulin were measured in two protein hydrolysates based on CM whey and casein proteins; the observed concentrations were 200 and 0.0056 micrograms beta-lactoglobulin/g dry weight, respectively.

Bovine beta-lactoglobulin levels in hydrolysed protein formulas for infant feeding.

A bovine milk protein, beta-lactoglobulin (beta LG), was measured by an enzyme-linked immunosorbent assay (ELISA) in seven different infant formulas based on protein hydrolysates from cows' milk whey or casein, and from bovine collagen and soy. beta LG levels in the formulas were 1/100 to 1/4,800,000 lower than in cows' milk (CM). There was a great difference in the beta LG level between the partly and the extensively hydrolysed formulas; the amount of beta LG was 40,000-fold higher in the partial hydrolysates vs the extensively hydrolysed formulas. Residual beta LG may have been responsible for the allergic reactions described in some children with cows' milk allergy (CMA) receiving these formulas.

Occupational asthma and rhinoconjunctivitis caused by senna.

Occupational sensitization to senna is seldom reported. We describe a 21-year-old male atopic factory worker who developed IgE-mediated asthma and rhinoconjunctivitis 5 months after exposure to senna while he was working for a company manufacturing hair dyes. In the bronchial challenge test with senna, he exhibited a strong reaction. The skin prick test gave a 12-mm wheal reaction. The radioallergosorbent test (RAST) with senna was highly positive (33.6 PRU/ml, class 4), and could not be inhibited with pollens or foods. In protein staining with SDS-PAGE, two heavy bands were detected in senna at about 14 and 25 kDa, and two faint bands were visible at 19 and 21 kDa, In IgE immunoblotting with the patient's serum, one heavy band was detected at about 16 and four faint bands at 23, 25, 28, and 38 kDa. The patient became symptom free after he had changed his job within the same company.

Complete genomic sequence of the lytic bacteriophage phiYeO3-12 of Yersinia enterocolitica serotype O:3.

phiYeO3-12 is a T3-related lytic bacteriophage of Yersinia enterocolitica serotype O:3. The nucleotide sequence of the 39,600-bp linear double-stranded DNA (dsDNA) genome was determined. The phage genome has direct terminal repeats of 232 bp, a GC content of 50.6%, and 54 putative genes, which are all transcribed from the same DNA strand. Functions were assigned to 30 genes based on the similarity of the predicted products to known proteins. A striking feature of the phiYeO3-12 genome is its extensive similarity to the coliphage T3 and T7 genomes; most of the predicted phiYeO3-12 gene products were >70% identical to those of T3, and the overall organizations of the genomes were similar. In addition to an identical promoter specificity, phiYeO3-12 shares several common features with T3, nonsubjectibility to F exclusion and growth on Shigella sonnei D(2)371-48 (M. Pajunen, S. Kiljunen, and M. Skurnik, J. Bacteriol. 182:5114-5120, 2000). These findings indicate that phiYeO3-12 is a T3-like phage that has adapted to Y. enterocolitica O:3 or vice versa. This is the first dsDNA yersiniophage genome sequence to be reported.

Milk causes a rapid urticarial reaction on the skin of children with atopic dermatitis and milk allergy.

Open skin challenge test with whole milk and its large and small molecular fractions was performed on intact skin of children with atopic dermatitis and suspicion of milk allergy. Of the 51 children challenged with milk 35 reacted within minutes with contact urticaria. The large molecular (m.w. greater than 10,000 d) fraction gave an urticarial reaction as often as whole milk, whereas the small molecular fraction gave only a few positive reactions. These were obviously caused by alpha-lactalbumin which was present only in small amounts in the small molecular fraction. These findings indicate that immediate contact allergy to relevant food allergens can be very common in children with atopic dermatitis and that the large molecular antigens readily penetrate children's skin.

Characterization of latex antigens and allergens in surgical gloves and natural rubber by immunoelectrophoretic methods.

Allergy to natural rubber (latex) products is increasingly frequent among both medical personnel and lay subjects. Although individual antigens and allergens in latex products have not been fully characterized, they are believed to derive from the sap of the rubber tree. Rabbit IgG and human IgE antibodies against rubber proteins were used to characterize antigens and allergens in surgical latex gloves and natural rubber by crossed immunoelectrophoresis and its modifications. The results from crossed-line immunoelectrophoresis demonstrated that the surgical latex gloves had at least 10 antigens in common with natural rubber sap and ammoniated latex. In crossed radioimmunoelectrophoresis, at least six of the 10 protein antigens in the surgical glove extract and natural rubber were allergens since they bound IgE antibodies from the latex-allergic patients' sera. The surgical glove extract also demonstrated one allergen not found in natural rubber, suggesting that rubber proteins may be altered during glove manufacture.

Occupational allergic contact urticaria to yucca (Yucca aloifolia), weeping fig (Ficus benjamina), and spathe flower (Spathiphyllum wallisii).

BACKGROUND: Occupational contact urticaria (CU) from plants is often reported, but it is less often attributed to decorative houseplants. We present an atopic gardener and caretaker of plants who developed CU when occupationally exposed to weeping fig, spathe flower, and yucca. METHODS: Sensitization was evaluated by skin prick tests (SPT) and analyses for IgE antibodies. RESULTS: SPT were positive to all three plants, and IgE antibodies were found to weeping fig and spathe flower. SPT were also performed with several decorative houseplants in more than 600 patients. Positive SPT was found to weeping fig (12%), African milk tree (8.3%), yucca (5.8%), Chinese rose (4.7%), massangana (4.6%), bird's nest fern (3.2%), and spathe flower (3.2%). CONCLUSION: Our study indicates that SPT and tests for IgE antibody are useful in detecting occupational CU caused by houseplants.

Occupational allergy caused by spathe flower (Spathiphyllum wallisii).

Occupational allergy caused by plants is seldom reported although it is probably relatively common. We report on a 22-year-old male atopic caretaker of plants who developed IgE-mediated allergic rhinitis, laryngitis, pharyngitis, and contact and generalized urticaria caused by exposure to spathe flower (Spathiphyllum wallisii) while he was working for a firm that supplied plants to offices. He also had an asthmatic attack at work, but in bronchial provocation tests conducted 8 months after he had stopped doing the work in question, he developed rhinoconjunctivitis, pharyngitis, and laryngitis, but exhibited neither bronchial reaction nor fall in PEF values. Prick tests with spathe flower gave 3+ reactions for exudates from the flower, pollen, stem, and leaves. He also had several positive reactions to fruits, vegetables, and spices, but not to natural rubber latex. The radioallergosorbent test with spathe flower was positive (3.4 PRU/ml, close to class 3). In protein staining with SDS-PAGE, one heavy band was detected at about 14 kDa, and other faint bands were visible on both sides. Six faint bands were detected at the mol. mass range of 30-67 kDa. In IgE immunoblotting, one heavy band was detected at about 14 kDa. The patient became symptomless after he had ceased working with plants.

Beta-lactoglobulin secretion in human milk varies widely after cow's milk ingestion in mothers of infants with cow's milk allergy.

BACKGROUND: Cow's milk proteins secreted in human milk may cause cow's milk allergy (CMA) even during exclusive breast-feeding. We studied beta-lactoglobulin levels in human milk of mothers of infants with CMA. We also studied intestinal absorption of macromolecules in the same mothers to see whether it is related to the secretion of beta-lactoglobulin in human milk. METHODS: CMA was verified with oral challenge in 46 of 55 infants assessed. beta-Lactoglobulin levels were assessed in human milk from 53 of 55 mothers of the infants before (basal sample) and 1 and 2 hours after an oral cow's milk load, which was given after a 24-hour milk-free diet. beta-Lactoglobulin was determined by an ELISA with a detection limit of 0.002 microgram/L. The 6-hour urine recovery of a high-molecular-weight polyethylene glycol (PEG) 3000 was assessed after an oral load of PEG in 45 of 55 mothers. RESULTS: beta-Lactoglobulin was found in the 1- or 2-hour samples in 75% of the mothers. beta-Lactoglobulin levels were increased in the 1- or 2-hour samples as compared with the basal levels in about half of the mothers. The respective levels were decreased in one third of the mothers whose basal beta-lactoglobulin levels were higher than in the others. beta-Lactoglobulin was found in none of the three human milk samples in 15% of the mothers. After an oral load of a high-molecular-weight PEG 3000, the 6-hour urine recovery of PEG was similar in the mothers of the infants with CMA and the mothers of infants without CMA. Neither was the urinary recovery of PEG related to the beta-lactoglobulin levels in human milk. CONCLUSIONS: The results support the view that beta-lactoglobulin in human milk may contribute to, but does not alone explain, the development of CMA in breast-fed infants.

Skin prick tests and in vitro immunoassays with native spices and spice extracts.

BACKGROUND: Skin prick tests of native spices (commercial powdered spices) are common in patients with allergy to birch or mugwort pollen. Clinical symptoms from spices are infrequent but occasionally severe. OBJECTIVE: To compare the skin prick test results with native spices and spice extracts and to determine the clinical relevance of test material. METHODS: Skin prick tests with the native spices coriander, caraway, paprika, cayenne, mustard, and white pepper were made twice at 2-month to 2.9-year intervals in 49 patients. During the latter time, tests were also made with spice extracts and spice-specific serum IgE was measured. RESULTS: The reproducibility of skin test results with native spices was 67% to 100%. Spice extracts, except white pepper, elicited positive skin test reactions in half those with positive reactions to native spices. Higher specific IgE concentrations (> or = 3.5 PRU/mL) were seen in cases where the skin tests were positive to the corresponding spices with 5% extracts of > 8 kD Mw. Three-fourths of the patients with positive skin tests to native spices were positive to birch pollen and one-half to a vegetable. Mild clinical symptoms from spices were reported by one-third. CONCLUSIONS: Spice allergens partly crossreact with those of pollens and vegetables. A minority of spice allergens may give clinical symptoms. The > 8-kD 5% extracts may be relevant skin prick test materials for identifying patients at risk of developing severe symptoms from ingested spices.

Immediate contact allergy from hydrolyzed wheat in a cosmetic cream.

Immediate contact allergy to cosmetics seems to be rare, since only a few case reports on it have been published. We report on a case of IgE-mediated allergic contact urticaria caused by hydrolyzed wheat in a body cream.

Allergy to feathers.

Skin prick test reactivity to commercial and self-made feather-allergen extracts was examined in 269 consecutive adult patients with suspected allergic cutaneous or respiratory symptoms who had been referred to a university clinic. Some 177 subjects reacted to any inhalant allergen. Twenty-four (9% of the whole group and 14% of those positive to any inhalant allergen) reacted to commercial feather extracts from ALK (Hørsholm, Denmark), and 51 to any of the seven feather extracts used. Feather-mix RAST (Pharmacia, Sweden) was positive in three cases only. Skin prick test or CAP-RAST or both to house-dust mite were positive in 16 of those 24 subjects positive to the commercial feather extracts, but in only 23 of the 150 other atopic subjects (P < 0.001). A nasal challenge with a feather extract was made in 20 cases, always with negative result. In immunospot studies, concomitant allergy to feather-allergen extracts and house-dust mite could be demonstrated. Mite allergens in feather extracts were verified in RAST-inhibition studies. A clinically significant feather allergy was found in one patient only. The results suggest that true feather allergy is very rare, and most of the positive reactions seen in skin prick tests to feather extracts are probably caused by mite allergens present in feathers.