Phenotypic and genetic characterization of Vibrio cholerae O1 clinical isolates collected through national antimicrobial resistance surveillance network in Nepal

Cholera occurs in sporadic cases and outbreaks in Nepal each year. Vibrio cholerae O1 (n = 522) isolated during 2007-2010 from diarrheal patients at 10 different hospital laboratories in Nepal were characterized. Biochemical and serologic identifications showed that all the isolates belonged to serogroup O1, El Tor biotype. Except 72 isolates of Inaba serotype isolated in the year 2007, all the remaining isolates were of Ogawa serotype. All isolates were resistant to nalidixic acid and furazolidone. Resistance to tetracycline, ciprofloxacin, erythromycin and co-trimoxazole were 21, 4, 16 and 90 % respectively. Seventy-seven of these isolates were selected for further characterization for ctxB gene and MLVA typing. Two different variants of classical type cholera toxin were observed. Ogawa strains from 2007 and 2010-Western Nepal outbreak harbored CTX-3 type cholera toxin, whereas Inaba serotypes in 2007 and the remaining Ogawa serotypes in 2008-2010 harbored CTX 3b-type toxin. MLVA analysis showed circulation of four different groups of altered V. cholerae O1 El Tor strains. Two different profiles were seen among 2007 Inaba (9, 3, 6, x, x) and Ogawa (10, 7, 6, x, x) isolates. The MLVA profile of 2008 and 2009 Ogawa isolates were similar to those of Inaba strains of 2007. Isolates from 2010 also showed three different MLVA profiles; profile 9, 3, 6, x, x in 3 isolates, 11, 7, 6, x, x among 2010 Western Nepal outbreak strains and profile 8, 3, 6, x, x among isolates from Butwal and Kathmandu.     

Expression of ActA, Ami, InlB, and listeriolysin O in Listeria monocytogenes of human and food origin

Expression of proteins involved in the adhesion of Listeria monocytogenes to mammalian cells or in the intracellular life cycle of this bacterium, including listeriolysin O (LLO), ActA, Ami, and InlB, was used to compare two populations of L. monocytogenes strains. One of the populations comprised 300 clinical strains, and the other comprised 150 food strains. All strains expressed LLO, InlB, and ActA. No polymorphism was observed for LLO and InlB. Ami was detected in 283 of 300 human strains and in 149 of 150 food strains. The strains in which Ami was not detected were serovar 4b strains. Based on the molecular weights of the proteins detected, the strains were divided into two groups with Ami (groups Ami1 [75% of the strains] and Ami2 [21%]) and into four groups with ActA (groups ActA1 [52% of the strains], ActA2 [18%], ActA3 [30%], and ActA4 [one strain isolated from food]). Logistic regression showed that food strains were more likely to belong to group ActA3 than human strains (odds ratio [OR] = 2.90; P = 1 x 10(-4)). Of the strains isolated from patients with non-pregnancy-related cases of listeriosis, bacteremia was predominantly associated with group Ami1 strains (OR = 1.89; P = 1 x 10(-2)) and central nervous system infections were associated with group ActA2 strains (OR = 3.04; P = 1 x 10(-3)) and group ActA3 strains (OR = 3.91; P = 1 x 10(-3)).     

基于生态效应的水稻籽粒蛋白质含量预测模型研究

在中国、日本、泰国不同生态环境下进行多品种籼型和粳型水稻(Oryza sativa)的区域种植试验,通过分析水稻籽粒蛋白质含量与纬度、海拔、抽穗后温度和太阳辐射等气候生态因子的相互关系,确立了影响水稻籽粒蛋白质积累的主要气候生态因子函数,并使用权重系数来进一步修订各气候生态因子对水稻籽粒蛋白质的作用,构建出基于生态效应(主要气候生态因子函数)的水稻籽粒蛋白质含量预测模型。利用不同年份、不同生态点、不同品种类型的试验资料对所建模型进行了检验,籼稻和粳稻籽粒蛋白质含量的预测误差RMSE平均分别为0.27%和0.24%;籼稻试验点和粳稻试验点的预测误差平均为0.25%和0.22%,表明模型总体上具有较好的预测性和实用性。     

Enumeration and antibiotic resistance patterns of fecal indicator organisms isolated from migratory Canada geese (Branta canadensis)

Thermotolerant fecal indicator organisms carried by migratory waterfowl may serve as reservoirs of antibiotic resistance. To determine the extent to which such antibiotic resistance markers were present in migratory Canada geese (Branta canadensis) on the Maryland Eastern Shore, we isolated Enterococcus spp. and Escherichia coli from fresh feces and examined the antibiotic resistance profiles of these bacteria. Samples were obtained in October 2002, January 2003, and March 2003. Thermotolerant E. coli counts ranged from 0 to 1.0x10(7) colony forming units (CFU)/0.1g (g-1) wet weight of feces, whereas Enterococcus spp. counts ranged from 1.0x10(2)-1.0x10(7) CFU g-1 wet weight of feces. Primary isolates of each indicator organism were tested against a panel of 10 antibiotics. Greater than 95% of E. coli isolates were resistant to penicillin G, ampicillin, cephalothin, and sulfathiazole; no E. coli were resistant to ciprofloxacin. Enterococcal isolates showed highest resistance to cephalothin, streptomycin, and sulfathiazole; no enterococci were resistant to chloramphenicol. The tetracyclines, streptomycin, and gentamycin provided the greatest discrimination among E. coli isolates; chlortetracycline, cephalothin, and gentamycin resistance patterns provided the greatest discrimination between enterococcal strains. Multiple antibiotic resistance (MAR) profiles were calculated: fall (E. coli=0.499; enterococci=0.234), winter (E. coli=0.487; enterococci=0.389), and spring (E. coli=0.489; enterococci=0.348). E. faecalis and E. faecium, which are recognized human nosocomial pathogens, were cultured from winter (44 and 56%, respectively) and spring (13 and 31%, respectively) fecal samples.     

Effect of repeat copy number on variable-number tandem repeat mutations in Escherichia coli O157:H7

Variable-number tandem repeat (VNTR) loci have shown a remarkable ability to discriminate among isolates of the recently emerged clonal pathogen Escherichia coli O157:H7, making them a very useful molecular epidemiological tool. However, little is known about the rates at which these sequences mutate, the factors that affect mutation rates, or the mechanisms by which mutations occur at these loci. Here, we measure mutation rates for 28 VNTR loci and investigate the effects of repeat copy number and mismatch repair on mutation rate using in vitro-generated populations for 10 E. coli O157:H7 strains. We find single-locus rates as high as 7.0 x 10(-4) mutations/generation and a combined 28-locus rate of 6.4 x 10(-4) mutations/generation. We observed single- and multirepeat mutations that were consistent with a slipped-strand mispairing mutation model, as well as a smaller number of large repeat copy number mutations that were consistent with recombination-mediated events. Repeat copy number within an array was strongly correlated with mutation rate both at the most mutable locus, O157-10 (r2= 0.565, P = 0.0196), and across all mutating loci. The combined locus model was significant whether locus O157-10 was included (r2= 0.833, P < 0.0001) or excluded (r2= 0.452, P < 0.0001) from the analysis. Deficient mismatch repair did not affect mutation rate at any of the 28 VNTRs with repeat unit sizes of >5 bp, although a poly(G) homomeric tract was destabilized in the mutS strain. Finally, we describe a general model for VNTR mutations that encompasses insertions and deletions, single- and multiple-repeat mutations, and their relative frequencies based upon our empirical mutation rate data.     

Larval susceptibility of the diamondback moth, Plutella xylostella (Lepidoptera: Plutellidae), to Bacillus thuringiensis H serovars isolated in Japan

A total of 1700 Japanese strains of Bacillus thuringiensis, belonging to at least 47 H serogroups, were examined for insecticidal activity against larvae of the diamondback moth, Plutella xylostella. The high-level toxicity was associated with 612 isolates (36.0%). Of these, 608 isolates (99.3%) fell into 13 H serogroups belonging to the low-numbered H serotypes, H1-H10. Conversely, most isolates belonging to the high-numbered serotypes (>H10) had little or no larvicidal activity; only one isolate of the serovar japonensis H23 was active. P xylostella larvae were susceptible to 89.8% of the serovar morrisoni H8a:8b strains and 85.7% of galleriae H5a:5b strains. High values of 60-80% were also obtained in six serovars (thuringiensis H1, alesti H3a:3c, kurstaki H3a:3b:3c, kenyae H4a:4c, aizawai H7, and tolworhi H9), while relatively low values of <60% in two other common serovars, sotto H4a:4b and darmstadiensis H10a:10b. Five selected isolates, belonging to H serovars other than kurstaki and aizawai, were 10-60 times less toxic than the reference strain HD-1 (serovar kurstaki). Parasporal inclusion proteins of these strains were immunologically unrelated to those of the strain HD-1 and the aizawai type strain.     

Isolation of four aquatic streptomycetes strains capable of growth on organochlorine pesticides

Ninety-three wild-type isolates identified as actinomycetes were tested against 11 organochlorine pesticides (OPs): aldrin, chlordane, DDD, DDE, DDT, dieldrin, heptachlor, and heptachlor epoxides, lindane, and methoxychlor. Qualitative screening agar assays displayed 62-78% tolerance of strains to OPs. Four strains designed M4, M7, M9 and M15 were selected based on multi-OP-tolerance, and identified as members of the streptomycetes group. Different growth profiles were observed in cultures of the four selected streptomycetes cultured in synthetic medium containing 5-50 microg x l(-1) aldrin or chlordane or lindane. Increase of aldrin removal by the selected microorganisms was concomitant with the 4.8-36.0 microg x l(-1) pesticide concentration range. After 72 h of streptomycete M7 growth in synthetic medium containing 48.0 microg x l(-1) aldrin, the remaining OP concentration in the supernatant was approximately 10% of the initial concentration. Also, in stationary growth phase less than 2.5 microg x l(-1) aldrin residual concentration was detected in the medium.     

香港养殖海鲷弧菌致病菌药物敏感性及耐药质粒研究

从发病海鲷(Sparus sarba)中共分离到51株弧菌(\%Vibrio)\%,经API20E细菌快速鉴定系统及Alsina和Blanch关键生理生化特性分析鉴定为7个种,它们分别是：溶藻胶弧菌(\%V.alginolyticus)(24株),创伤弧菌(V.vulnificus)(12株)和副溶血弧菌(V.parahaemolyticus)(7株),火神弧菌(V.logei)(4株),远洋弧菌Ⅱ菌(V.pelagius Ⅱ)(2株),河弧菌(V.fluvialis)(1株)和地中海弧菌(V.mediterranei)(1株)\%。其中3种优势菌溶藻胶弧菌创伤弧菌和副溶血弧菌证实对海鲷有致病性。另外采用平板稀释法检测了51株菌对16种抗菌素的敏感性。发现所有菌株对ceftriaxone,链霉素,萘啶酮酸和利福霉素敏感,几乎所有菌株对ceftazidime, netilimicin,氯霉素和sulfamethoxazole敏感.大部分菌株对氨苄青霉素 (60.8%),cefuroxime(667%),丁胺卡那霉素(55%),卡那霉素(588%)和三甲氧苄氨嘧啶(765%)等具有较强的耐药性。通过对菌株中所含有的耐药质粒进行分析,发现15株菌株含有1～4个质粒,分子量范围为9～123kb之间,对12株既含有较大分子量质粒又具有耐药性的菌株进行了质粒转化试验,结果其中9株菌的质粒具有转化能力,转化率为１０－１１～１０－９,表明所分离的菌株的抗药性是由于细菌染色体相关突变造成的。     

Phenotypic and genetic characterization of bacteria isolated from diseased cultured sea cucumber Apostichopus japonicus in northeastern China

During the winter-spring from 2004 to 2006 in northeastern China cultured Japanese sea cucumber Apostichopus japonicus suffered from a serious disease. Clinical signs included swollen mouth, skin ulceration and massive mortality. Clinical samples taken during this period were studied. Thirty-one bacterial samples were isolated from diseased sea cucumbers and identified through biochemical tests, 16S rRNA gene sequence analysis and PCR amplification, followed by pathogenicity determination. The results showed that the 31 isolates belonged to the genera Vibrio (64.5%), Shewanella (12.9%), Serratia (12.9%), Pseudoalteromonas (6.4%) and Flavobacterium (3.2 %). The 3 prominent strains were Vibrio splendidus (41.9%), Shewanella (12.9%) and Serratia odorifera biogroup I (12.9%). Pathogenicity tests demonstrated that 13 out of 31 isolates were pathogenic, including 8 strains of V splendidus, 3 strains of Shewanella sp. and 2 strains of Pseudoalteromonas tetraodonis. The pathogenic V splendidus showed the highest frequency of appearance. Median lethal dose (LD50) values (14 d) of V splendidus, Shewanella sp. and P. tetraodonis were 1.74 x 10(7), 7.76 x 10(6), 7.24 x 10(7) CFU g(-1) body weight of sea cucumber, respectively. The virulences differed by species: Shewanella sp. > V splendidus> P. tetraodonis. This is the first report of Shewanella sp. virulence in sea cucumber.     

Halomonas aidingensis sp. nov., a moderately halophilic bacterium isolated from Aiding salt lake in Xinjiang, China

Two Gram-negative moderately halophilic bacterial strains, designated Ad-1(T) and C-12, were isolated from Aiding salt lake of Xinjiang in China. The novel isolates were subjected to a polyphasic taxonomic study. Cells of these strains were cocci or short rods and motile with polar flagella. Colonies produced brown-red pigment. The isolates grew in the range of 0.5-25% (w/v) NaCl, pH 5.5-10.5 and 4-45°C. Analysis of their 16S rRNA gene sequences indicated that strains Ad-1(T) and C-12 belonged to the genus Halomonas showing 92.7-98.4% similarity with the type species. The isoprenoid quinones of the isolates were Q-9 and Q-8. The major cellular fatty acids were C18:1ω7c, C16:1ω7c/6c, C16:0, C12:0-3OH and C10:0. The DNA G + C contents of strains Ad-1(T) and C-12 were 64.6 and 63.9 mol%, respectively. The DNA relatedness between the two isolates was 89.2%. The similarities of these newly isolated strains with closely related type strains were lower than 35% at the genetic level. Based on phenotypic, chemotaxonomic and genetic characteristics, the representative strain Ad-1(T) is considered to be a novel species of the genus Halomonas, for which the name Halomonas aidingensis sp. nov. is proposed, with Ad-1(T) (= CGMCC 1.10191(T) = NBRC 106173(T)) as the type strain.     

Recent emergence of serogroup C meningococcal disease in Greece

The number of cases of meningococcal disease reported to the Meningitis Reference Laboratory in Athens rose dramatically in 1996-1997. The aims were (1) to determine if the increase was due to introduction of new strains, (2) to assess the geographic and age distribution of the cases, (3) to compare antibiotic sensitivity patterns of the current isolates with strains from the early 1990s. In 1993-1994, 15/19 (74%) of the cases for which information on age was available were in children < or = 5 years; in 1995-1997, 80/179 (45%) of cases were in children < or = 5 years and 99 (55%) in the older age range (P < 0.02). From 593 cases in 1993 1997, 214 (36%) isolates were available for characterisation. Serogroup B was predominant in the early 1990s, but by 1997, serogroup C accounted for 46/72 (64%) of isolates and serogroup B for 25/72 (35%). Serogroup B was predominant in children < or = 5 years (44/78, 56%) but only 19/99 (18%) of older children and adults (P=0.0000005). Sulfonamide resistance decreased from 10/22 (45%) in 1993-1994 to 27/192 (14%) in 1995-1997 (P<0.01). Multilocus enzyme electrophoresis of 70 strains obtained during this period identified the epidemic ET-15 clone in 24 (34.3)%. The profiles of the Greek ET-15 isolates were identical to C:2a:P1.2(P1.5) strains responsible for the epidemic in the Czech Republic which began in 1993. This genotype was not found in Greek strains isolated prior to 1993. We conclude that the increase in meningococcal disease is due to introduction of the epidemic serogroup C:2a:P1.2(P1.5) strain responsible for disease in the Czech Republic and Canada.     

A spectrophotometric method for determination of fluorophore-to-protein ratios in conjugates of the blue fluorophore 7-amino-4-methylcoumarin-3-acetic acid (AMCA)

7-Amino-4-methylcoumarin-3-acetic acid (AMCA) has been found to be a useful fluorophore for immunofluorescence. The present study describes a spectrophotometric method for determining the ratio of moles AMCA to moles protein (or the f/p ratio) in an AMCA-conjugated IgG. The concentration of a substance absorbing light can be determined spectrophotometrically using Beer's Law: Absorbance = Concentration x Extinction coefficient. From Beer's law, one can derive the following formula for determining the f/p ratio of AMCA-IgG conjugates: f/p = (epsilon 280IgG).A350 - (epsilon 350IgG).A280/(epsilon 350AMCA).A280 - (epsilon 280AMCA).A350 where A is the optical density of the conjugate at the given wavelength and epsilon is the extinction coefficient of a substance at the wavelength specified. Using conjugates of model proteins, it was found that the extinction coefficients of the AMCA moiety of AMCA-conjugated protein were 1.90 x 10(4) at 350 nm and 8.29 x 10(3) at 280 nm. Similarly, it was found that the extinction coefficients of swine IgG were 1.56 x 10(3) at 350 nm and 1.26 x 10(5) at 280 nm. Thus, for AMCA-conjugated swine IgG: f/p = (1.26 x 10(5)).A350 - (1.56 x 10(3)).A280/(1.47 x 10(4)).A280 - (6.42 x 10(3)).A350 [corrected]. Based on this formula, the f/p ratios of some AMCA-IgG conjugates useful for immunohistochemistry have been found to range between 6 and 24.     

Antimicrobial susceptibility and clonal relatedness between community- and hospital-acquired methicillin-resistant Staphylococcus aureus from blood cultures

We compared the antimicrobial resistance and clonal relationships among the community-acquired (CA) and hospital-acquired (HA) methicillin-resistant Staphylococcus aureus (MRSA) strains that were isolated from blood cultures in a university hospital over a 4-year period. A total of 131 MRSA isolates, including 28 CA-MRSA and 103 HA-MRSA strains, were identified; antimicrobial susceptibility testing indicated that the CA-MRSA isolates were more susceptible to erythromycin (21% vs 6%; P=0.02), clindamycin (46% vs 12%; P=0.01), ciprofloxacin (43% vs 11%; P=0.01), and gentamicin (43% vs 6%; P=0.01) than were the HA-MRSA isolates. Pulsed-field gel electrophoresis (PFGE) typing and antimicrobial resistance profiles separated the 20 CA-MRSA isolates into 14 and 10 different patterns, respectively, and the 53 HA-MRSA isolates were separated into 24 and 7 different patterns, respectively. Twenty-one (40%) of the 53 HA-MRSA isolates belonged to two predominant PFGE types, and most of them showed multi-drug resistant patterns. Four (20%) of the 20 CA-MRSA and 10 (19%) of the 53 HA-MRSA isolates fell into two common PFGE patterns, and each of them showed the same multi-drug resistant pattern. This study suggests that, although the CA-MRSA blood isolates showed diverse PFGE and antimicrobial resistance patterns, some of these isolates may have originated from the HA-MRSA strains.     

Prostaglandins potentiate U-46619-induced pulmonary microvascular dysfunction.

The induction of cyclooxygenase is an important event in the pathophysiology of acute lung injury. The purpose of this study was to examine the synergistic effects of various cyclooxygenase products (PGE(2), PGI(2), PGF(2alpha)) on thromboxane A(2) (TxA(2))-mediated pulmonary microvascular dysfunction. The lungs of Sprague-Dawley rats were perfused ex vivo with Krebs-Henseleit buffer containing indomethacin and PGE(2) (5 x 10(-8) to 1 x 10(-7) M), PGF(2alpha) (7 x 10(-9) to 5 x 10(-6) M), or PGI(2) (5 x 10(-8) to 2 x 10(-5) M). The TxA(2)-receptor agonist U-46619 (7 x 10(-8) M) was then added to the perfusate, and then the capillary filtration coefficient (K(f)), pulmonary arterial pressure (Ppa), and total pulmonary vascular resistance (RT) were determined. The K(f) of lungs perfused with U-46619 was twice that of lungs perfused with buffer alone (P = 0.05). The presence of PGE(2), PGF(2alpha), and PGI(2) within the perfusate of lungs exposed to U-46619 caused 118, 65, and 68% increases in K(f), respectively, over that of lungs perfused with U-46619 alone (P < 0.03). The RT of lungs perfused with PGE(2) + U-46619 was approximately 30% greater than that of lungs exposed to either U-46619 (P < 0.02) or PGE(2) (P < 0.01) alone. When paired measurements of RT taken before and then 15 min after the addition of U-46619 were compared, PGI(2) was found to attenuate U-46619-induced increases in RT (P < 0.01). These data suggest that PGE(2), PGI(2), and PGF(2alpha) potentiate the effects of TxA(2)-receptor activation on pulmonary microvascular permeability.     

Laboratory evaluation of entomopathogenic fungi against larvae and adults of onion maggot (Diptera: Anthomyiidae)

Laboratory experiments were done to measure the susceptibility of larvae and adults of the onion maggot, Delia antiqua (Meigen) (Diptera: Muscidae: Anthomyiidae) to 27 isolates of entomopathogenic fungi from four genera [Beauveria Vuillemin, Lecanicillium (Petch) Zare & W. Gams, Metarhizium Sorokin, and Paecilomyces Bainier]. A novel bioassay was developed for D. antiqua larvae by using a diet based on mixed vegetable powder. When evaluated in a virulence screen, the fungal isolates caused less mortality of D. antiqua larvae than adults. Only three isolates caused > 50% mortality of larvae, whereas 12 isolates caused > 50% mortality of adults. Fungal species was a statistically significant factor affecting the mortality of larvae but not of adults. The fungal isolates causing the most mortality of larvae tended to belong to Metarhizium anisopliae (Metschnikoff) Sorokin. Two M. anisopliae isolates (389.93 and 392.93) were evaluated in dose-response bioassays. The median lethal concentrations of the isolates against larvae were 6.1 x 10(7) conidia ml(-1) for isolate 389.93 and 7.6 x 10(7) conidia ml(-1) for isolate 392.93. The emergence of adult flies from pupae was reduced at high concentrations of conidia (3.0 x 10(8) and 1.0 x 10(8) conidia ml(-1)). The median lethal concentrations of the isolates against adults were 1.7 x 10(7) and 4.0 x 10(7) conidia ml(-1), respectively. Some of the fungal isolates examined may have potential as biological control agents of larvae of D. antiqua and related species.     

Endothelum-dependent and-independent relaxations in aortic rings obtained from hypertensive hooded (Aguti) rats.

Experimental hypertension studies are few in the hooded (Aguti) rat. The present study was designed to investigate the usefulness of this rat strain for experimental hypertension studies and to test the hypothesis that the hypertension may be associated with a diminution of endothelium dependent and independent relaxations. Hypertension was induced in inbred hooded rats (n=8 each) by administering 8% salt in the diet and /or 100 mg/kg/day Nomega-nitro-L-arginine-methyl-ester (L-NAME) in the drinking water for six and/or four weeks respectively. The rats were anaesthetized using a 25% urethane and 1% chloralose mixture given intraperitoneally at a dose of 5 mg/kg. Their blood pressure was measured invasively. Thereafter, relaxations of rat aortic preparations to acetylcholine, histamine and sodium nitroprusside (SNP) were assessed using standard organ bath conditions. Probabiliity level of 0.05 was taken as statistically significant. The mean arterial pressure (MAP;mm Hg) rose significantly in all test groups (Salt: 148.3 +/- 4.6; L-NAME: 181.7 +/-8.3; Salt+L-NAME:154.9 +/-8.7) compared with control (94.2 +/-6.8; [P < 0.05]. The MAP was significantly [P < 0.05] higher in the L-NAME group than in all the other groups. The heart rate fell significantly in the salt + L-NAME group compared to control [P <0.05].The IC50 of acetylcholine in aortic rings from L-NAME rats (7.9 x 10(-1) +/- 6.0 x 10(-3)) was significantly higher than in rings from control (9.4 x 10(-8) +/- 2.8 x 10(-8)), salt (7.8 x 10(-7) +/- 4.7 x 10(-7)) and salt + L-NAME (3.3 x 10 (-7) +/- 2.1 x 10(-7)) rats [P < 0.05]. The IC50 of histamine and SNP in the rings from the test groups of rats showed no significant difference from control. Also, endothelium dependent and independent relaxations were preserved in the various forms of hypertension studied except in chronic NOS inhibition where the former was attenuated in response to acetylcholine.     

Examination of Food Chain-Derived Listeria monocytogenes Strains of Different Serotypes Reveals Considerable Diversity in inlA Genotypes,Mutability, and Adaptation to Cold Temperatures

Listeria monocytogenes strains belonging to serotypes 1/2a and 4b are frequently linked to listeriosis. While inlA mutations leading to premature stop codons (PMSCs) and attenuated virulence are common in 1/2a, they are rare in serotype 4b. We observed PMSCs in 35% of L. monocytogenes isolates (n = 54) recovered from the British Columbia food supply, including serotypes 1/2a (30%), 1/2c (100%), and 3a (100%), and a 3-codon deletion (amino acid positions 738 to 740) seen in 57% of 4b isolates from fish-processing facilities. Caco-2 invasion assays showed that two isolates with the deletion were significantly more invasive than EGD-SmR (P < 0.0001) and were either as (FF19-1) or more (FE13-1) invasive than a clinical control strain (08-5578) (P = 0.006). To examine whether serotype 1/2a was more likely to acquire mutations than other serotypes, strains were plated on agar with rifampin, revealing 4b isolates to be significantly more mutable than 1/2a, 1/2c, and 3a serotypes (P = 0.0002). We also examined the ability of 33 strains to adapt to cold temperature following a downshift from 37°C to 4°C. Overall, three distinct cold-adapting groups (CAG) were observed: 46% were fast (<70 h), 39% were intermediate (70 to 200 h), and 15% were slow (>200 h) adaptors. Intermediate CAG strains (70%) more frequently possessed inlA PMSCs than did fast (20%) and slow (10%) CAGs; in contrast, 87% of fast adaptors lacked inlA PMSCs. In conclusion, we report food chain-derived 1/2a and 4b serotypes with a 3-codon deletion possessing invasive behavior and the novel association of inlA genotypes encoding a full-length InlA with fast cold-adaptation phenotypes.     

Comparative development and merozoite production of two isolates of Sarcocystis neurona and Sarcocystis falcatula in cultured cells

The development and merozoite production of Sarcocystis falcatula and 2 isolates (SN6 and SN2) of Sarcocystis neurona were studied in various cultured cell lines inoculated with culture-derived merozoites. All 3 parasites underwent multiple cycles of schizogony in VERO cells, bovine monocytes (M617 cells), and bovine pulmonary artery endothelial cells (CPA). Sarcocystis neurona strains SN6 and SN2 formed schizonts in rat myoblasts (L6) but not in quail myoblasts (QM7); S. falcatula formed schizonts in QM7 cells but not in L6 cells. Merozoites did not develop to sarcocysts in the myoblast cells lines. During a 47-day culture period in VERO cells, SN6 produced substantially more merozoites than did SN2 or S. falcatula. M617 cells produced substantially more merozoites of SN6 than did VERO or CPA cells. During a 17-day culture period of SN6, M617 cells produced mean totals of 4.7 x 10(8) merozoites, VERO cells produced 1.9 x 10(8) merozoites, and CPA cells produced 5.9 x 10(7) merozoites. At 4-12 days after inoculation of cultured cells with SN6, M617 cells cultured in the presence of 10% fetal bovine serum (FBS) produced a mean merozoite total of 5.1 x 10(8) compared to 3.6 x 10(8) for culture medium containing 1% FBS.     

Isolation of endospore-forming bacilli toxic to Culiseta longiareolata (Diptera: Culicidae) in Jordan

Ten of 80 endospore-forming bacilli, isolated from various habitats of Jordan, were found to be highly toxic to the 4th instar larvae of Culiseta longiareolata (Macquart). The bacilli were identified into the following species and strains: Bacillus sphaericus (H6), B. sphaericus (H9a, 9b), B. cereus Frankland and Frankland, B. brevis Migula and B. megaterium Bary. Bacillus cereus comprised 50% of the isolates. The toxic concentrations of these isolates against C. longiareolata ranged between 1.2 x 10(7) and 1.1 x 10(9) viable spores ml-1.     

Reconstituting the barrier properties of a water-tight epithelial membrane by design of leaflet-specific liposomes

To define aspects of lipid composition and bilayer asymmetry critical to barrier function, we examined the permeabilities of liposomes that model individual leaflets of the apical membrane of a barrier epithelium, Madin-Darby canine kidney type 1 cells. Using published lipid compositions we prepared exofacial liposomes containing phosphatidylcholine, sphingomyelin, glycosphingolipids, and cholesterol; and cytoplasmic liposomes containing phosphatidylethanolamine, phosphatidylserine, and cholesterol. The osmotic permeability of cytoplasmic liposomes to water (P(f)), solutes, and NH(3) was 18-90-fold higher than for the exofacial liposomes (P(f(ex)) = 2.4 +/- 0.4 x 10(-4) cm/s, P(f(cy)) = 4.4 +/- 0.3 x 10(-3) cm/s; P(glycerol(ex)) = 2.5 +/- 0.3 x 10(-8) cm/s, P(glycerol(cy)) = 2.2 +/- 0.02 x 10(-6) cm/s; P(NH3(ex)) = 0. 13 +/- 0.4 x 10(-4) cm/s, P(NH3(cy)) = 7.9 +/- 1.0 x 10(-3) cm/s). By contrast, the apparent proton permeability of exofacial liposomes was 4-fold higher than cytoplasmic liposomes (P(H+(ex)) = 1.1 +/- 0. 1 x 10(-2) cm/s, P(H+(cy)) = 2.7 +/- 0.6 x 10(-3) cm/s). By adding single leaflet permeabilities, we calculated a theoretical P(f) for a Madin-Darby canine kidney apical membrane of 4.6 x 10(-4) cm/s, which compares favorably with experimentally determined values. In exofacial liposomes lacking glycosphingolipids or sphingomyelin, permeabilities were 2-7-fold higher, indicating that both species play a role in barrier function. Removal of cholesterol resulted in 40-280-fold increases in permeability. We conclude: 1) that we have reconstituted the biophysical properties of a barrier membrane, 2) that the barrier resides in the exofacial leaflet, 3) that both sphingomyelin and glycosphingolipids play a role in reducing membrane permeability but that there is an absolute requirement for cholesterol to mediate this effect, 4) that these results further validate the hypothesis that each leaflet offers an independent resistance to permeation, and 5) that proton permeation was enhanced by sphingolipid/cholesterol interactions.