Structures and functions of the gut microbial lipidome.

Microbial lipids provide signals that are responsible for maintaining host health and controlling disease. The differences in the structures of microbial lipids have been shown to alter receptor selectivity and agonist/antagonist activity. Advanced lipidomics is an emerging field that helps to elucidate the complex bacterial lipid diversity. The use of cutting-edge technologies is expected to lead to the discovery of new functional metabolites involved in host homeostasis. This review aims to describe recent updates on functional lipid metabolites derived from gut microbiota, their structure-activity relationships, and advanced lipidomics technologies.

The interaction of secreted phospholipase A2-IIA with the microbiota alters its lipidome and promotes inflammation.

Secreted phospholipase A2-IIA (sPLA2-IIA) hydrolyzes phospholipids to liberate lysophospholipids and fatty acids. Given its poor activity toward eukaryotic cell membranes, its role in the generation of proinflammatory lipid mediators is unclear. Conversely, sPLA2-IIA efficiently hydrolyzes bacterial membranes. Here, we show that sPLA2-IIA affects the immune system by acting on the intestinal microbial flora. Using mice overexpressing transgene-driven human sPLA2-IIA, we found that the intestinal microbiota was critical for both induction of an immune phenotype and promotion of inflammatory arthritis. The expression of sPLA2-IIA led to alterations of the intestinal microbiota composition, but housing in a more stringent pathogen-free facility revealed that its expression could affect the immune system in the absence of changes to the composition of this flora. In contrast, untargeted lipidomic analysis focusing on bacteria-derived lipid mediators revealed that sPLA2-IIA could profoundly alter the fecal lipidome. The data suggest that a singular protein, sPLA2-IIA, produces systemic effects on the immune system through its activity on the microbiota and its lipidome.

Multiple-locus variable-number of tandem-repeats analysis distinguishes Vibrio parahaemolyticus pandemic O3:K6 strains.

A specific serotype of Vibrio parahaemolyticus, O3:K6, has recently been linked to epidemics of gastroenteritis in Southeast Asia, Japan, and North America. These pandemic O3:K6 strains appear to have recently spread across continents from a single origin to reach global coverage, based on profiling of strains by several molecular typing methods. In this study, variable-number tandem repeats (VNTR)-based fingerprinting was applied to clinical and environmental V. parahaemolyticus O3:K6 strains in an attempt to develop a molecular method with increased sensitivity for discriminating strains; the relative discriminatory powers were compared with ribotyping and pulsed-field gel electrophoresis (PFGE). All clinical strains tested were independent human isolates obtained from different outbreaks or from sporadic cases in Tokyo during the period from 1996 to 2003. Multiple-locus VNTR analysis (MLVA) was shown to have high resolution and reproducibility for typing of V. parahaemolyticus clones. MLVA analysis of 28 pandemic V. parahaemolyticus O3:K6 strains isolated from human cases produced 28 distinct VNTR patterns. The VNTR loci displayed between 2 and 15 alleles at each of eight loci with Nei's diversity index ranging from 0.35 and 0.91. These data demonstrated that MLVA is useful for individual strain typing of new O3:K6 strains, which appear to be closely related by other molecular methods.

[The risk of pertussis and diphtheria infections among pediatric healthcare workers in Japan].

For infection control in pediatric hospitals, we investigated the risk of pertussis and diphtheria infections among pediatric healthcare workers. Forty-nine Japanese pediatric healthcare workers in 12 general hospitals were screened for antibodies of pertussis toxin (PT), filamentous hemagglutinin (FHA), and diphtheria toxin (DT). The seropositive rates of anti-PT IgG (protective level, > 10 U/mL), anti-FHA IgG (> 10 U/ mL), and anti-DT (> 0.11 U/mL) were 50, 82, and 59%, respectively. During this survey period (Oct. 2003-Feb. 2004), 16 (33%) of the healthcare workers were in contact with pertussis-infant (s). However, all culture and PCR tests for Bordetella pertussis were negative. One of the 16 exposed healthcare workers, a male pediatrician, had serological evidence of a pertussis infection, but no disease symptomatic of pertussis. Our observations indicate that i) 50 and 41% of Japanese pediatric healthcare workers were seronegative for pertussis (anti-PT IgG) and diphtheria antibodies, respectively, and ii) although the healthcare workers had a high rate of contact with pertussis-infant (s), the infection rate was low. For pertussis and diphtheria infection control in pediatric hospitals, it is important for healthcare workers to be aware of their own protection levels against these diseases.

[Comparison of DNA sequencing analysis with phenotyping test for identification of yeasts isolated from foods].

The identification of 20 strains of yeasts isolated from foods by means of DNA sequence analysis with two kinds of universal primers for the rDNA region was examined, and the results were compared with those of the conventional phenotyping test using API 20C AUX. In the analysis of the 26S region, all 20 yeast strains tested were identified at the species level. In the ITS1 region, 16 strains were also classified at the species level. In addition, all results of DNA sequence analysis were consistent with those of the phenotyping test at the genus level. Furthermore, DNA sequence analysis was able to identify causative yeasts observed in two suspect foods, though phenotyping tests alone failed to identify them.

[Effects of handwashing on Feline Calicivirus removal as Norovirus surrogate].

Viral gastroenteritis caused by Norovirus (NV) mainly appears during the winter season. In fact, outbreaks and patients with NV gastroenteritis are the major cause of community disease in the winter. Strategies to avoid gastroenteritis caused by NV are thus needed. No effective method for evaluating virus inactivation and removal exists for of NV, which cannot be cultured using cell-lines. Trials using Feline Calici Virus (FCV; a member of the calicivirus family) as a NV surrogate have been conducted by culturing FCV in CRFK cells. By washing one's hands, about 99% of the viruses can be removed, compared with simply rinsing one's hands in running water. Washing one's hands with alcohol, chlorhexidine, quaternary ammonium, or 3 other kinds of hand soaps (containing povidone-iodine, triclosan, and isopropylmethyl phenol, respectively), was also effective for removing viruses. These results suggest that washing one's hands may be an effective method of preventing viral gastroenteritis.

Modeling Staphylococcus aureus growth and enterotoxin production in milk.

Staphylococcus aureus growth and its enterotoxin production in sterilized milk were modeled with a modification of a new logistic model recently developed by us. The modified model and the Baranyi model described the early exponential phase of a growth curve more accurately than the previous model, at constant temperatures from 14 to 36.5 degrees C. The amount of toxin in milk increased linearly with time from the time the cell population reached about 10(6.5) cfu/ml. The rate of toxin production linearly increased at temperatures between 14 and 32 degrees C. From parameter values obtained at the constant temperatures, the model successfully predicted bacterial growth in the milk at a varying temperature. For toxin level estimation, we postulated that the rate of toxin production might be regulated with the temperature after the cell concentration reached 10(6.5) cfu/ml; the time point when the cell concentration reached that value was predicted with the modified growth model. Introduction of a correction factor in the toxin estimation successfully predicted the toxin level in milk at a varying temperature. These results showed that this prediction system consisting of the modified model and the toxin production algorithm might be a useful tool for modeling bacterial growth and its metabolite production in liquid foods.

[Isolation of thermostable direct hemolysin producing Vibrio parahaemolyticus from food using screening by PCR in food-borne outbreaks].

The producibility of thermostable direct hemolysin (TDH) is the most important pathogenic factor in Vibrio parahaemolyticus. TDH (+) V. parahaemolyticus is usually isolated from patients having V. parahaemolyticus food-borne disease. TDH (+) V. parahaemolyticus is, however, very difficult to isolate from food and environmental samples. In the 5 years from 2000 to 2004 in Tokyo, V. parahaemolyticus was isolated from food samples related to 67 of 227 V parahaemolyticus food-borne outbreaks. In these outbreaks, TDH (+) strains were also tried to isolate using PCR as the screening methods. TDH (+) V. parahaemolyticus strains were able to isolate from enrichment broth in which toxR and tdh genes become positive in PCR. TDH (+) strains of the same serotype with patients were able to be isolated from 23 food samples related to 11 outbreaks (16.4%); 3 outbreaks in 2000, 2 in 2001, 2 in 2002, 1 in 2003, and 3 in 2004. The serotypes of V. parahaemolyticus isolated from food were O3 : K6 (10 samples), O3 : K5 (6 samples), O1 : K25 (4 samples), O3 : K29 (2 samples), O4 : K 8 (1 sample), and O4 : K11 (1 sample). The isolation rate of the TDH (+) strain from enrichment broth differed with samples. In several samples TDH (+) strains were isolated easily only by examining 3 colonies, hence no TDH (+) strains were isolated in spite of the examination of 250 colonies. No correlation was seen between the number of V. parahaemolyticus and the isolation rate of TDH (+) strains in food samples. Screening using PCR is very effective method for isolating TDH (+) V. parahaemolyticus from food samples.

Characteristics and modeling of Escherichia coli growth in pouched food.

Characteristics of the growth kinetics of Escherichia coli cells in pouched mashed potatoes under various conditions were studied with a mathematical model. Bacterial cells were inoculated in sterile mashed potatoes and then sealed in vinyl pouches, in which a very small amount of air was included. The growth curves of cells in the pouched mashed potatoes at constant temperature (12-34 degrees C) were sigmoidal with time on a semi-logarithmic plot and were successfully described with a new logistic model recently developed by us. The rate constant of growth showed a highly linear relationship to the temperature with the square-root model, and the lag period was longer at lower temperatures. The growth curve in glass tubes containing a large volume of air was similar to that in pouches, showing that the rate of growth was not affected by the volume of the surrounding air. The growth curves in pouched mashed potatoes were very similar to those in nutrient broth or on the surface of nutrient agar, which we previously reported. These results suggested that the growth kinetics of the bacterial cells under various conditions of rich nutrition might be almost identical, and can be described with a simple growth model like ours.

Model comparison for Escherichia coli growth in pouched food.

We recently studied the growth characteristics of Escherichia coli cells in pouched mashed potatoes (Fujikawa et al., J. Food Hyg. Soc. Japan, 47, 95-98 (2006)). Using those experimental data, in the present study, we compared a logistic model newly developed by us with the modified Gompertz and the Baranyi models, which are used as growth models worldwide. Bacterial growth curves at constant temperatures in the range of 12 to 34 degrees C were successfully described with the new logistic model, as well as with the other models. The Baranyi gave the least error in cell number and our model gave the least error in the rate constant and the lag period. For dynamic temperature, our model successfully predicted the bacterial growth, whereas the Baranyi model considerably overestimated it. Also, there was a discrepancy between the growth curves described with the differential equations of the Baranyi model and those obtained with DMfit, a software program for Baranyi model fitting. These results indicate that the new logistic model can be used to predict bacterial growth in pouched food.

[Development of effective detection method for Coxiella burnetii in mayonnaise by real-time PCR and investigation of C. burnetii contamination in commercial mayonnaise in Tokyo].

A PCR method for the effective detection of Coxiella burnetii in commercially available mayonnaise was developed. Sample preparations were isolated from 50 g portions of each mayonnaise product by four successive extraction steps in phosphate buffer with 2.0 M NaCl. These extracts were then centrifuged at 20,000 x g for 60 min. DNA was isolated from the solution containing the precipitate with a commercial kit, and amplified quantitatively using real-time PCR that targeted the com1 region of C. burnetii. The recoveries of C. burnetii from 2 kinds of commercial mayonnaise specimens, with a baseline control of 1 x 10(7) particles of the Nine Mile phase II strain, were 85.0 +/- 6.0% and 72.0 +/- 0.4%, respectively. The determination limit of this method was 500 C. burnetii particles per 50 g of mayonnaise. The DNA specimens isolated from 50 different commercial mayonnaise samples sold in Tokyo using this method were amplified using both nested PCR and real-time PCR. No contamination by C. burnetii was detected in any of the mayonnaise samples.

Multiple viral infections and genomic divergence among noroviruses during an outbreak of acute gastroenteritis.

An epidemic outbreak of both norovirus (NV) and astrovirus (ASV) occurred on a research ship surveying Tokyo Bay, causing acute gastroenteritis in 26 of its 37 crew members. The presence of viral pathogens in fecal specimens was analyzed, and noroviruses were identified by reverse transcription-PCR in 18 (48.6%) of these specimens. In addition, astroviruses were identified in 14 (37.8%) of the fecal samples from the affected crew members, and multiple viral infections of both NV and ASV were observed in 6 cases. The genogrouping of the NV-positive samples was then examined by dot blot hybridization, and it was determined that all of the isolates were from genogroup II (GII). No bacterial pathogens were subsequently isolated from fecal specimens. Furthermore, a variety of NV strains were identified by sequencing and single-stranded conformational polymorphism (SSCP) analyses of PCR products from the fecal samples. One recombinant NV isolate, Minato/14, was identified as a recombinant NV strain of GII/6 and GII/1. The other NV isolates from this outbreak were classified into three NV genotypes (GII/1 [Minato/10], GII/4 [Minato/33], and GII/5 [Minato/6]). Furthermore, ASVs in positive samples were determined to belong to serotypes 1 and 2 by sequencing analysis. Our findings thus indicate that coinfections with NV and ASV, including a number of NV genotypes, persisted during an outbreak of gastroenteritis in a closed environment.

[Development of a predictive program for microbial growth under various temperature conditions].

A predictive program for microbial growth under various temperature conditions was developed with a mathematical model. The model was a new logistic model recently developed by us. The program predicts Escherichia coli growth in broth, Staphylococcus aureus growth and its enterotoxin production in milk, and Vibrio parahaemolyticus growth in broth at various temperature patterns. The program, which was built with Microsoft Excel (Visual Basic Application), is user-friendly; users can easily input the temperature history of a test food and obtain the prediction instantly on the computer screen. The predicted growth and toxin production can be important indices to determine whether a food is microbiologically safe or not. This program should be a useful tool to confirm the microbial safety of commercial foods.

Modeling surface growth of Escherichia coli on agar plates.

Surface growth of Escherichia coli cells on a membrane filter placed on a nutrient agar plate under various conditions was studied with a mathematical model. The surface growth of bacterial cells showed a sigmoidal curve with time on a semilogarithmic plot. To describe it, a new logistic model that we presented earlier (H. Fujikawa et al., Food Microbiol. 21:501-509, 2004) was modified. Growth curves at various constant temperatures (10 to 34 degrees C) were successfully described with the modified model (model III). Model III gave better predictions of the rate constant of growth and the lag period than a modified Gompertz model and the Baranyi model. Using the parameter values of model III at the constant temperatures, surface growth at various temperatures was successfully predicted. Surface growth curves at various initial cell numbers were also sigmoidal and converged to the same maximum cell numbers at the stationary phase. Surface growth curves at various nutrient levels were also sigmoidal. The maximum cell number and the rate of growth were lower as the nutrient level decreased. The surface growth curve was the same as that in a liquid, except for the large curvature at the deceleration period. These curves were also well described with model III. The pattern of increase in the ATP content of cells grown on a surface was sigmoidal, similar to that for cell growth. We discovered several characteristics of the surface growth of bacterial cells under various growth conditions and examined the applicability of our model to describe these growth curves.

[Comparison of the number of Norovirus genome copies in patients and healthy persons].

In viral gastroenteritis outbreaks occurred by Norovirus (NV), NV was detected not only from patients but also from healthy persons who have taken the same food, and also detected from healthy staff members working at community places such as hospital, school and nursing home. The number of fecal NV genome copies of patients, healthy persons and food handlers are examined by real-time PCR method, to investigate foodborne gastroenteritis and person to person transmission outbreaks. There is no significant difference on the number of NV genome copies in feces between patients, and NV-detected healthy persons. Those result indicate asymptomatic carrier of NV who were working as food handlers or staff members at community places will become an origin of food-borne gastroenteritis or person to person transmission outbreaks.

[Investigation of Coxiella burnetii contamination in commercial milk and PCR method for the detection of C. burnetii in egg].

A total of 244 milk samples collected from supermarkets in Tokyo were examined for contamination with Coxiella burnetii. C. burnetii DNA was detected in 131 (53.7%) of the samples by nested PCR. PCR-positive samples were injected into immunosuppressed A/J strain mice. Of the 22 PCR-positive milk samples tested, none resulted in isolation of C. burnetii from the mice. Heat-treatment was sufficient to inactivate C. burnetii in commercial milk. In addition, a PCR detection method for C. burnetii in chicken egg was developed. Egg yolk was added to an equal volume of 1 mol/L of NaCl phosphate buffer and homogenized for removal of protein and lipid. After centrifugal separation, the supernatant was removed, and template DNA in the precipitate was extracted using SDS, proteinase K and NaI. Using such prepared samples, 3.2 x 10(1) C. burnetii particles in 1 g of egg yolk could be detected by nested PCR. All of 200 chicken egg samples collected from supermarkets in Tokyo were negative for C. burnetii by the nested PCR method.

[Anti-drug pattern of drug-resistant Mycobacterium tuberculosis and analysis of mutation in drug-target genes].

The antimycobacterial susceptibility test was performed and minimal inhibitory concentration (MIC) to drugs was determined in 98 strains of Mycobacteium tuberculosis (MTB) isolated in Tokyo from 2000 to 2003, to find which were resistant to any of the four main anti-MTB drugs, isoniazid (INH), rifampicin (RFP), streptomycin (SM), and ethambutol (EMB). 27strains of them were resistant only to SM, and 16 strains were resistant only to INH. 51 strains of them were resistant to not only INH but also other drugs. 38 strains were resistant to both INH and RFP. 19 strains were resistant to all four drugs, including 7 strains resistant to new quinolon anti-biotics also. Nucleotide or amino-acid mutations in drug resistant MTB genome were determined by DNA sequencing method. Mutation of codon 516, 526, or 531 of rpoB gene was detected in 98% of MTBs resistant to RFP. Deletion or insertion of katG gene or nucleotide mutation at regulatory region of ahpC gene was detected in MTBs highly resistant to INH. Amino acid mutation of katG gene, especially at codon 315, was detected in MTBs resistant to INH intermediate. Nucleotide mutations at regulatory region of inhA gene were detected in MTBs resistant to INH at low level. Amino acid mutation at codon 43 or 88 of rpsL gene was detected in MTBs highly resistant to SM, and nucleotide mutation at 512, 513, or 516 of rrs gene was detected in MTBs resistant to SM at low level. Amino acid mutation at codon 306 of embB gene was detected in 87% of MTBs resistant to EMB.

[Drug susceptibility and analysis using pulsed-field gel electrophoresis of Streptococcus pyogenes strains isolated from the patients with streptococcal toxic shock syndrome (STSS) in Japan].

Previously, we have performed T typing of Streptococcus pyogenes strains isolated from patients with streptococcal toxic shock syndrome (STSS) in Japan, and streptococcal pyrogenic exotoxin (SPE) typing for epidemiological examination. In this study, we conducted a drug sensitivity test using these strains, and investigated the results of gene analysis by pulse-field gel electrophoresis (PFGE) of S. pyogenes strains derived from patients with STSS, the patient's family, and patients other than those with STSS. To clarify the relationship between the host and bacterial factors, we investigated the association between clinical symptoms and T typing of the isolated strains/production of streptococcal pyrogenic exotoxin. There were no strains resistant to beta-lactams, and only 1 strain was resistant to multiple agents other than beta-lactams. The PFGE pattern of T1 type strains was classified into 2 ; the pattern was consistent between the strains derived from patients with STSS and those derived from the patient's family. The PFGE pattern of T3 type strains was classified into 5 (IV) ; Pattern I, which was most frequently observed, was detected in both the strains derived from patients with STSS/non-STSS. However, Patterns II and III were detected only in the strains derived from patients with non-STSS. Patterns IV and V were detected only in the strains derived from patients with STSS. When examining the association between clinical symptoms and bacterial factors, disseminated intravascular coagulation (DIC) was associated with T1-SPE B-producing strains, and pharyngitis was associated with T3-SPE A-producing strains. In the future, the relationship between the host and bacterial factors should be further investigated.

[Two nosocomial outbreaks of sepsis caused by Serratia marcescens, which occurred in July 1999 and January 2002--Tokyo].

Two nosocomial outbreaks of sepsis caused by Serratia marcescens, which occurred in Tokyo were the following cases. CASE A: In July 1999, 10 inpatients admitted to the third floor ward of the General Hospital A, developed sudden onset of high fever, coagulation disorders (disseminated intravascular coagulation), and acute renal failure, of which 5 died. Twenty-one strains of Serratia marcescens were isolated from the inpatient's blood and urine, nurse fingers and environmental samples from floor and cooling tower. Serratia infection was strongly suspected as the cause of sepsis. These cases were defined as "inpatients who developed fever 38 degrees C or more during July 26 to 29 and from whom S. marcescens was isolated by blood culture". Ten isolates were detected from the blood. In order to investigate the background of S. marcescens isolation in the hospital and to compare molecular and biochemical characteristics of S. marcescens, cultures were attempted from samples of other inpatients and staffs and hospital environment. Those were classified into 9 groups by various different typings: biotyping with Api Rapid 20; susceptibility typing of antimicrobial agents tested; pulsed-field gel electrophoresis (PFGE) typing of SpeI- or Xba I-restricted chromosome. All 10 isolates causing sepsis were found to be in the same group. CASE B: In January 2002, 24 inpatients, admitted to Neurosurgical Hospital B, developed sudden onset of high fever, of which 7 died. S. marcescens was isolated from a towel, environmental samples and inpatients. These cases were defined as "inpatients who developed fever of 38.5 degrees C and S. marcescens isolated by blood culture". Twelve strains were isolated from the blood samples in 12 cases. In order to investigate the background of S. marcescens isolation in the hospital, cultures were attempted from other inpatient's urine and environmental samples from medical tape, Tshake and a towel. These isolates were classified into 3 groups by the previous typings; biotyping with Api Rapid 20; susceptibility typing of antimicrobial agents tested; and PFGE typing. All 12 isolates in 12 cases were found to be in the same group. These cases of 2 nosocomial outbreaks of sepsis were defined as "in-patient who developed high fever and S. marcescens isolated by blood culture". However in both cases transmission routes of Serratia infection remain unknown by field investigation.