Twelve strains of Escherichia coli O157:H7 were isolated from 9 of 25 beef samples purchased from retail stores in Malaysia. These strains produced Shiga toxin 2 with or without Shiga toxin 1 and had the eae gene and a 60-MDa plasmid. The antibiograms and the profiles of the arbitrarily primed PCR of the strains were diverse, suggesting that the strains may have originated from diverse sources.
Molecular characterization of a total of 54 isolates of Salmonella typhi from Santiago, Chile, was performed by pulsed-field gel electrophoresis (PFGE) after digestion of chromosomal DNA with three restriction endonucleases: XbaI (5'-TCTAGA-3'), AvrII (5'-CCTAGG-3'), and SpeI (5'-ACTAGT-3'). Thirteen of the 54 isolates were obtained from environmental sources (sewage and river water), and the rest were isolates from clinical cases of typhoid fever. Considerable genetic diversity was detected among the human isolates obtained in 1994, as evidenced by the presence of 14 to 19 different PFGE patterns among 20 human isolates, with F (coefficient of similarity) values ranging from 0.69 to 1.0 (XbaI), 0.61 to 1.0 (AvrII), and 0.70 to 1.0 (SpeI). A total of eight phage types were detected among these 20 isolates, with 50% possessing the E1 or 46 phage type. There was no correlation between PFGE pattern and phage types. Similar diversity was seen among 21 isolates obtained in 1983, with 17 to 19 PFGE patterns detected and F values of 0.56 to 1.0 (XbaI), 0.55 to 1.0 (AvrII), and 0.67 to 1.0 (SpeI). Comparison of these two groups of human isolates obtained 11 years apart indicated that certain molecular types of S. typhi are shared and are able to persist for considerable periods. A similar degree of genetic diversity was also detected among the environmental isolates of S. typhi, for which 10 to 12 different PFGE patterns were detected among the 13 isolates analyzed, with F values ranging from 0.56 to 1.0 (XbaI), 0.52 to 1.0 (AvrII), and 0.69 to 1.0 (SpeI). Certain molecular types present among the environmental isolates of S. typhi were also found among the human isolates from the same time period, providing evidence for the epidemiological link between environmental reservoirs and human infection.
A new mosquitocidal Bacillus thuringiensis subsp., jegathesan, has recently been isolated from Malaysia. Parasporal crystal inclusions were purified from this strain and bioassayed against fourth-instar larvae of Culex quinquefasciatus, Aedes aegypti, Aedes togoi, Aedes albopictus, Anopheles maculatus, and Mansonia uniformis. The 50% lethal concentration of crystal inclusions for each species was 0.34, 8.08, 0.34, 17.59, 3.91, and 120 ng/ml, respectively. These values show that parasporal inclusions from this new subspecies have mosquitocidal toxicity comparable to that of inclusions isolated from B. thuringiensis subsp. israelensis. Solubilized and chymotrypsin-activated parasporal inclusions possessed low-level hemolytic activity. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that the crystals were composed of polypeptides of 77, 74, 72, 68, 55, 38, 35, 27, and 23 kDa. Analysis by Western blotting (immunoblotting) with polyclonal antisera raised against toxins purified from B. thuringiensis subsp. israelensis reveals that proteins in parasporal inclusions of subsp. jegathesan are distinct, because little cross-reactivity was shown. Analysis of the plasmid content of B. thuringiensis subsp. jegathesan indicates that the genes for toxin production may be located on 105- to 120-kb plasmids. Cry- clones that have been cured of these plasmids are nontoxic. Southern blot analysis of plasmid and chromosomal DNA from subsp. jegathesan showed little or low homology to the genes coding for CryIVA, CryIVB, and CryIVD from B. thuringiensis subsp. israelensis.
Restriction fragment length polymorphism and virulence analyses were used to evaluate the population structure of Xanthomonas oryzae pv. oryzae, the rice bacterial blight pathogen, from several rice-growing countries in Asia. Two DNA sequences from X. oryzae pv. oryzae, IS1112, an insertion sequence, and avrXa10, a member of a family of avirulence genes, were used as probes to analyze the genomes of 308 strains of X. oryzae pv. oryzae collected from China, India, Indonesia, Korea, Malaysia, Nepal, and the Philippines. On the basis of the consensus of three clustering statistics, the collection formed five clusters. Genetic distances within the five clusters ranged from 0.16 to 0.51, and distances between clusters ranged from 0.48 to 0.64. Three of the five clusters consisted of strains from a single country. Strains within two clusters, however, were found in more than one country, suggesting patterns of movement of the pathogen. The pathotype of X. oryzae pv. oryzae was determined for 226 strains by inoculating five rice differential cultivars. More than one pathotype was associated with each cluster; however, some pathotypes were associated with only one cluster. Most strains from South Asia (Nepal and India) were virulent to cultivars containing the bacterial blight resistance gene xa-5, while most strains from other countries were avirulent to xa-5. The regional differentiation of clusters of X. oryzae pv. oryzae in Asia and the association of some pathotypes of X. oryzae pv. oryzae with single clusters suggested that strategies that target regional resistance breeding and gene deployment are feasible.
A Mo -reducing bacterium (strain 48), which grew on medium supplemented with 200 mM Mo, was isolated from stream water obtained from Chengkau, Malaysia. The chemical properties of strain 48 conform to the characteristics of Enterobacter cloacae. Under anaerobic conditions in the glucose-yeast extract medium containing phosphate ion (2.9 mM) and Mo (10 mM), the bacterium reduced Mo to form molybdenum blue. Approximately 27% of Mo added to the medium was reduced after 28 h of cultivation. The reduction of Mo with glucose as an electron donor was strongly inhibited by iodoacetic acid, sodium fluoride, and sodium cyanide, suggesting an involvement of the glycolytic pathway and electron transport in Mo reduction. NADH and N,N,N',N' -tetramethyl-p-phenylenediamine served as electron donors for Mo reduction. When NADH was used as an electron donor, at first cytochrome b in the cell extract was reduced, and then molybdenum blue was formed. Sodium cyanide strongly inhibited Mo reduction by NADH (5 mM) but not the reduction of cytochrome b in the cell extract, suggesting that the reduced component of the electron transport system after cytochrome b serves as an electron donor for Mo reduction. Both ferric and stannous ions strongly enhanced the activity of Mo reduction by NADH.
Trace (microgram liter) quantities of either toluene or benzene injected into an amino-acid-limited continuous culture of Pseudomonas sp. strain T2 were utilized immediately with affinities of 2.6 and 6.8 liters g of cells h, respectively, and yielded large amounts of organic products, carbon dioxide, and cells. The immediate utilization of hydrocarbons by hydrocarbon-deprived organisms helps to establish the nutritional value of nonpolar substrates in the environment. The observation of small Michaelis constants for toluene transport led to tests of metabolic competition between hydrocarbons; however, competitive inhibition of toluene metabolism was not found for benzene, naphthalene, xylene, dodecane, or amino acids. Benzene and terpenes were inhibitory at milligram liter concentrations. Toluene was metabolized by a strongly inducible system when compared with benzene. The capacity of toluene to effect larger affinity values increased with exposure time and concentration. The kinetics of induction suggested saturation phenomena, resulting in an induction constant, K(ind), of 96 mug of toluene liter. Maximal induction of amino-acid-grown cells required about 80 h, with the affinity reaching 317 liters g of cells h.
Several infectious disease outbreaks with high mortality in humans have been attributed to viruses that are thought to have evolved from bat viruses. In this study from Luxembourg, the genetic diversity and epidemiology of paramyxoviruses and coronaviruses shed by the bat species Rhinolophus ferrumequinum and Myotis emarginatus were evaluated. Feces collection (n = 624) was performed longitudinally in a mixed-species colony in 2015 and 2016. In addition, feces (n = 254) were collected cross-sectionally from six Myotis emarginatus colonies in 2016. By use of degenerate primers in a nested format, overall prevalences of 1.1% (10/878) and 4.9% (43/878) were determined for paramyxoviruses and coronaviruses. Sequences of the partial RNA-dependent RNA polymerase and spike glycoprotein genes of coronaviruses, as well as sequences of the partial L gene of paramyxoviruses, were obtained. Novel paramyxovirus and Alphacoronavirus strains were identified in different Myotis emarginatus colonies, and severe acute respiratory syndrome (SARS)-related Betacoronavirus strains were shed by Rhinolophus ferrumequinum Logistic regression revealed that the level of Alphacoronavirus shedding was highest in July (odds ratio, 2.8; P < 0.01), probably due to periparturient stress. Phylogenetic analyses point to close virus-host coevolution, and the high genetic similarity of the study strains suggests that the Myotis emarginatus colonies in Luxembourg are socially connected. Most interestingly, we show that bats also host Betacoronavirus1 strains. The high similarity of the spike gene sequences of these viruses with mammalian Betacoronavirus 1 strains may be of concern. Both the SARS-related and Betacoronavirus 1 strains detected in bats in Luxembourg may cross the species barrier after a host adaptation process.IMPORTANCE Bats are a natural reservoir of a number of zoonotic pathogens. Several severe outbreaks in humans (e.g., a Nipah virus outbreak in Malaysia in 1998, and the almost global spread of severe acute respiratory syndrome in 2003) have been caused by bat-borne viruses that were transmitted to humans mostly after virus adaptation (e.g., in intermediate animal hosts). Despite the indigenousness of bat species that host viruses with suspected zoonotic potential and despite the zoonotic transmission of European bat 1 lyssavirus in Luxembourg, knowledge about the diversity and epidemiology of bat viruses remains limited in this country. Moreover, in contrast to other European countries, bat viruses are currently not included in the national surveillance activities of this land-locked country. We suggest that this gap in disease surveillance should be addressed, since we show here that synanthropic bats host viruses that may be able to cross the species barrier.
Gastrointestinal (GI) or gut microbiotas play essential roles in host development and physiology. These roles are influenced partly by the microbial community composition. During early developmental stages, the ecological processes underlying the assembly and successional changes in host GI community composition are influenced by numerous factors, including dispersal from the surrounding environment, age-dependent changes in the gut environment, and changes in dietary regimes. However, the relative importance of these factors to the gut microbiota is not well understood. We examined the effects of environmental (diet and water sources) and host early ontogenetic development on the diversity of and the compositional changes in the gut microbiota of a primitive teleost fish, the lake sturgeon (Acipenser fulvescens), based on massively parallel sequencing of the 16S rRNA gene. Fish larvae were raised in environments that differed in water source (stream versus filtered groundwater) and diet (supplemented versus nonsupplemented Artemia fish). We quantified the gut microbial community structure at three stages (prefeeding and 1 and 2 weeks after exogenous feeding began). The diversity declined and the community composition differed significantly among stages; however, only modest differences associated with dietary or water source treatments were documented. Many taxa present in the gut were over- or underrepresented relative to neutral expectations in each sampling period. The findings indicate dynamic relationships between the gut microbiota composition and host gastrointestinal physiology, with comparatively smaller influences being associated with the rearing environments. Neutral models of community assembly could not be rejected, but selectivity associated with microbe-host GI tract interactions through early ontogenetic stages was evident. The results have implications for sturgeon conservation and aquaculture production specifically and applications of microbe-based management in teleost fish generally.IMPORTANCE We quantified the effects of environment (diet and water sources) and host early ontogenetic development on the diversity of and compositional changes in gut microbial communities based on massively parallel sequencing of the 16S rRNA genes from the GI tracts of larval lake sturgeon (Acipenser fulvescens). The gut microbial community diversity declined and the community composition differed significantly among ontogenetic stages; however, only modest differences associated with dietary or water source treatments were documented. Selectivity associated with microbe-host GI tract interactions through early ontogenetic stages was evident. The results have implications for lake sturgeon and early larval ecology and survival in their natural habitat and for conservation and aquaculture production specifically, as well as applications of microbe-based management in teleost fish generally.
Pretreatment of waste-activated sludge (WAS) is an effective way to destabilize sludge floc structure and release organic matter for improving sludge digestion efficiency. Nonetheless, information on the impact of WAS pretreatment on digestion sludge microbiomes, as well as mechanistic insights into how sludge pretreatment improves digestion performance, remains elusive. In this study, a genome-centric metagenomic approach was employed to investigate the digestion sludge microbiome in four sludge digesters with different types of feeding sludge: WAS pretreated with 0.25 mol/liter alkaline/acid (APAD), WAS pretreated with 0.8 mol/liter alkaline/acid (HS-APAD), thermally pretreated WAS (thermal-AD), and fresh WAS (control-AD). We retrieved 254 metagenome-assembled genomes (MAGs) to identify the key functional populations involved in the methanogenic digestion process. These MAGs span 28 phyla, including 69 yet-to-be-cultivated lineages, and 30 novel lineages were characterized with metabolic potential associated with hydrolysis and fermentation. Interestingly, functional populations involving carbohydrate digestion were enriched in APAD and HS-APAD, while lineages related to protein and lipid fermentation were enriched in thermal-AD, corroborating the idea that different substrates are released from alkaline/acid and thermal pretreatments. Among the major functional populations (i.e., fermenters, syntrophic acetogens, and methanogens), significant correlations between genome sizes and abundance of the fermenters were observed, particularly in APAD and HS-APAD, which had improved digestion performance.IMPORTANCE Wastewater treatment generates large amounts of waste-activated sludge (WAS), which consists mainly of recalcitrant microbial cells and particulate organic matter. Though WAS pretreatment is an effective way to release sludge organic matter for subsequent digestion, detailed information on the impact of the sludge pretreatment on the digestion sludge microbiome remains scarce. Our study provides unprecedented genome-centric metagenomic insights into how WAS pretreatments change the digestion sludge microbiomes, as well as their metabolic networks. Moreover, digestion sludge microbiomes could be a unique source for exploring microbial dark matter. These results may inform future optimization of methanogenic sludge digestion and resource recovery.
Vancomycin-resistant enterococci (VRE) have been reported to be present in humans, chickens, and pigs in Malaysia. In the present study, representative samples of VRE isolated from these populations were examined for similarities and differences by using the multilocus sequence typing (MLST) method. Housekeeping genes of Enterococcus faecium (n = 14) and Enterococcus faecalis (n = 11) isolates were sequenced and analyzed using the MLST databases eBURST and goeBURST. We found five sequence types (STs) of E. faecium and six STs of E. faecalis existing in Malaysia. Enterococcus faecium isolates belonging to ST203, ST17, ST55, ST79, and ST29 were identified, and E. faecium ST203 was the most common among humans. The MLST profiles of E. faecium from humans in this study were similar to the globally reported nosocomial-related strain lineage belonging to clonal complex 17 (CC17). Isolates from chickens and pigs have few similarities to those from humans, except for one isolate from a chicken, which was identified as ST203. E. faecalis isolates were more diverse and were identified as ST4, ST6, ST87, ST108, ST274, and ST244, which were grouped as specific to the three hosts. E. faecalis, belonging to the high-risk CC2 and CC87, were detected among isolates from humans. In conclusion, even though one isolate from a chicken was found clonal to that of humans, the MLST analysis of E. faecium and E. faecalis supports the findings of others who suggest VRE to be predominantly host specific and that clinically important strains are found mainly among humans. The infrequent detection of a human VRE clone in a chicken may in fact suggest a reverse transmission of VRE from humans to animals.
Bacterial attachment onto materials has been suggested to be stochastic by some authors but nonstochastic and based on surface properties by others. We investigated this by attaching pairwise combinations of two Salmonella enterica serovar Sofia (S. Sofia) strains (with different physicochemical and attachment properties) with one strain each of S. enterica serovar Typhimurium, S. enterica serovar Infantis, or S. enterica serovar Virchow (all with similar physicochemical and attachment abilities) in ratios of 0.428, 1, and 2.333 onto glass, stainless steel, Teflon, and polysulfone. Attached bacterial cells were recovered and counted. If the ratio of attached cells of each Salmonella serovar pair recovered was the same as the initial inoculum ratio, the attachment process was deemed stochastic. Experimental outcomes from the study were compared to those predicted by the extended Derjaguin-Landau-Verwey-Overbeek (XDLVO) theory. Significant differences (P < 0.05) between the initial and the attached ratios for serovar pairs containing S. Sofia S1296a for all different ratios were apparent for all materials. For S. Sofia S1635-containing pairs, 7 out of 12 combinations of serovar pairs and materials had attachment ratios not significantly different (P > 0.05) from the initial ratio of 0.428. Five out of 12 and 10 out of 12 samples had attachment ratios not significantly different (P > 0.05) from the initial ratios of 1 and 2.333, respectively. These results demonstrate that bacterial attachment to different materials is likely to be nonstochastic only when the key physicochemical properties of the bacteria were significantly different (P < 0.05) from each other. XDLVO theory could successfully predict the attachment of some individual isolates to particular materials but could not be used to predict the likelihood of stochasticity in pairwise attachment experiments.
The synthesis of bacterial polyhydroxyalkanoates (PHA) is very much dependent on the expression and activity of a key enzyme, PHA synthase (PhaC). Many efforts are being pursued to enhance the activity and broaden the substrate specificity of PhaC. Here, we report the identification of a highly active wild-type PhaC belonging to the recently isolated Chromobacterium sp. USM2 (PhaC(Cs)). PhaC(Cs) showed the ability to utilize 3-hydroxybutyrate (3HB), 3-hydroxyvalerate (3HV), and 3-hydroxyhexanoate (3HHx) monomers in PHA biosynthesis. An in vitro assay of recombinant PhaC(Cs) expressed in Escherichia coli showed that its polymerization of 3-hydroxybutyryl-coenzyme A activity was nearly 8-fold higher (2,462 ± 80 U/g) than that of the synthase from the model strain C. necator (307 ± 24 U/g). Specific activity using a Strep2-tagged, purified PhaC(Cs) was 238 ± 98 U/mg, almost 5-fold higher than findings of previous studies using purified PhaC from C. necator. Efficient poly(3-hydroxybutyrate) [P(3HB)] accumulation in Escherichia coli expressing PhaC(Cs) of up to 76 ± 2 weight percent was observed within 24 h of cultivation. To date, this is the highest activity reported for a purified PHA synthase. PhaC(Cs) is a naturally occurring, highly active PHA synthase with superior polymerizing ability.
We investigated the temporal variation of bacterial production, respiration, and growth efficiency in the tropical coastal waters of Peninsular Malaysia. We selected five stations including two estuaries and three coastal water stations. The temperature was relatively stable (averaging around 29.5 degrees C), whereas salinity was more variable in the estuaries. We also measured dissolved organic carbon and nitrogen (DOC and DON, respectively) concentrations. DOC generally ranged from 100 to 900 microM, whereas DON ranged from 0 to 32 microM. Bacterial respiration ranged from 0.5 to 3.2 microM O2 h(-1), whereas bacterial production ranged from 0.05 to 0.51 microM C h(-1). Bacterial growth efficiency was calculated as bacterial production/(bacterial production + respiration), and ranged from 0.02 to 0.40. Multiple correlation analyses revealed that bacterial production was dependent upon primary production (r2 = 0.169, df = 31, and P < 0.02) whereas bacterial respiration was dependent upon both substrate quality (i.e., DOC/DON ratio) (r2 = 0.137, df = 32, and P = 0.03) and temperature (r2 = 0.113, df = 36, and P = 0.04). Substrate quality was the most important factor (r2 = 0.119, df = 33, and P = 0.04) for the regulation of bacterial growth efficiency. Using bacterial growth efficiency values, the average bacterial carbon demand calculated was from 5.30 to 11.28 microM C h(-1). When the bacterial carbon demand was compared with primary productivity, we found that net heterotrophy was established at only two stations. The ratio of bacterial carbon demand to net primary production correlated significantly with bacterial growth efficiency (r2 = 0.341, df = 35, and P < 0.001). From nonlinear regression analysis, we found that net heterotrophy was established when bacterial growth efficiency was <0.08. Our study showed the extent of net heterotrophy in these waters and illustrated the importance of heterotrophic microbial processes in coastal aquatic food webs.
Blastocystis cysts were detected in 38% (47/123) (37 Scottish, 17 Malaysian) of sewage treatment works. Fifty percent of influents (29% Scottish, 76% Malaysian) and 28% of effluents (9% Scottish, 60% Malaysian) contained viable cysts. Viable cysts, discharged in effluent, provide further evidence for the potential for waterborne transmission of Blastocystis.
The long-term usefulness of Bacillus thuringiensis Cry toxins, either in sprays or in transgenic crops, may be compromised by the evolution of resistance in target insects. Managing the evolution of resistance to B. thuringiensis toxins requires extensive knowledge about the mechanisms, genetics, and ecology of resistance genes. To date, laboratory-selected populations have provided information on the diverse genetics and mechanisms of resistance to B. thuringiensis, highly resistant field populations being rare. However, the selection pressures on field and laboratory populations are very different and may produce resistance genes with distinct characteristics. In order to better understand the genetics, biochemical mechanisms, and ecology of field-evolved resistance, a diamondback moth (Plutella xylostella) field population (Karak) which had been exposed to intensive spraying with B. thuringiensis subsp. kurstaki was collected from Malaysia. We detected a very high level of resistance to Cry1Ac; high levels of resistance to B. thuringiensis subsp. kurstaki Cry1Aa, Cry1Ab, and Cry1Fa; and a moderate level of resistance to Cry1Ca. The toxicity of Cry1Ja to the Karak population was not significantly different from that to a standard laboratory population (LAB-UK). Notable features of the Karak population were that field-selected resistance to B. thuringiensis subsp. kurstaki did not decline at all in unselected populations over 11 generations in laboratory microcosm experiments and that resistance to Cry1Ac declined only threefold over the same period. This finding may be due to a lack of fitness costs expressed by resistance strains, since such costs can be environmentally dependent and may not occur under ordinary laboratory culture conditions. Alternatively, resistance in the Karak population may have been near fixation, leading to a very slow increase in heterozygosity. Reciprocal genetic crosses between Karak and LAB-UK populations indicated that resistance was autosomal and recessive. At the highest dose of Cry1Ac tested, resistance was completely recessive, while at the lowest dose, it was incompletely dominant. A direct test of monogenic inheritance based on a backcross of F1 progeny with the Karak population suggested that resistance to Cry1Ac was controlled by a single locus. Binding studies with 125I-labeled Cry1Ab and Cry1Ac revealed greatly reduced binding to brush border membrane vesicles prepared from this field population.
Of 97 strains of Vibrio cholerae isolated from various seafoods in Malaysia in 1998 and 1999, 20 strains carried the ctx gene and produced cholera toxin. Fourteen, one, and five of these toxigenic strains belonged to the O139, O1 Ogawa, and rough serotypes, respectively. The rough strains had the rfb gene of the O1 serotype. The toxigenic strains varied in their biochemical characteristics, the amount of cholera toxin produced, their antibiograms, and the presence or absence of the pTLC plasmid sequence. DNA fingerprinting analysis by arbitrarily primed PCR, ribotyping, and a pulsed-field gel electrophoresis method classified the toxigenic strains into 3, 7, and 10 types, respectively. The relatedness of these toxigenic strains to clinical strains isolated in other countries and from international travelers was examined by using a dendrogram constructed from the pulsed-field gel electrophoresis profiles. The results of the examination of the antibiogram and the possession of the toxin-linked cryptic plasmid were consistent with the dendrogram-based relatedness: the O139 strains isolated from Malaysian seafoods could be separated into two groups that appear to have been introduced from the Bengal area independently. The rough strains of Malaysian seafood origin formed one group and belonged to a cluster unique to the Thailand-Malaysia-Laos region, and this group may have persisted in this area for a long period. The single O1 Ogawa strain detected in Malaysian seafood appears to have an origin and route of introduction different from those of the O139 and the rough strains.
Hepatitis A virus (HAV) is an important pathogen which has been responsible for many food-borne outbreaks. HAV-excreting food handlers, especially those with poor hygienic practices, can contaminate the foods which they handle. Consumption of such foods without further processing has been known to result in cases of infectious hepatitis. Since quantitative data on virus transfer during contact of hands with foods is not available, we investigated the transfer of HAV from artificially contaminated fingerpads of adult volunteers to pieces of fresh lettuce. Touching the lettuce with artificially contaminated fingerpads for 10 s at a pressure of 0.2 to 0.4 kg/cm(2) resulted in transfer of 9.2% +/- 0.9% of the infectious virus. The pretreatments tested to interrupt virus transfer from contaminated fingerpads included (i) hard-water rinsing and towel drying, (ii) application of a domestic or commercial topical agent followed by water rinsing and towel drying, and (iii) exposure to a hand gel containing 62% ethanol or 75% liquid ethanol without water rinsing or towel drying. When the fingerpads were treated with the topical agents or alcohol before the lettuce was touched, the amount of infectious virus transferred to lettuce was reduced from 9.2% to between 0.3 and 0.6% (depending on the topical agent used), which was a reduction in virus transfer of up to 30-fold. Surprisingly, no virus transfer to lettuce was detected when the fingerpads were rinsed with water alone before the lettuce was touched. However, additional experiments with water rinsing in which smaller volumes of water were used (1 ml instead of 15 ml) showed that the rate of virus transfer to lettuce was 0.3% +/- 0.1%. The variability in virus transfer rates following water rinsing may indicate that the volume of water at least in part influences virus removal from the fingerpads differently, a possibility which should be investigated further. This study provided novel information concerning the rate of virus transfer to foods and a model for investigating the transfer of viral and other food-borne pathogens from contaminated hands to foods, as well as techniques for interrupting such transfer to improve food safety.
In this paper, we report the cloning and characterization of three Paenibacillus azotofixans DNA regions containing genes involved in nitrogen fixation. Sequencing analysis revealed the presence of nifB1H1D1K1 gene organization in the 4,607-bp SacI DNA fragment. This is the first report of linkage of a nifB open reading frame upstream of the structural nif genes. The second (nifB2H2) and third (nifH3) nif homologues are confined within the 6,350-bp HindIII and 2,840-bp EcoRI DNA fragments, respectively. Phylogenetic analysis demonstrated that NifH1 and NifH2 form a monophyletic group among cyanobacterial NifH proteins. NifH3, on the other hand, clusters among NifH proteins of the highly divergent methanogenic archaea.
The presence or absence of two DNA modification systems, XorI and XorII, in 195 strains of Xanthomonas oryzae pv. oryzae collected from different major rice-growing countries of Asia was assessed. All four possible phenotypes (XorI+ XorII+, XorI+ XorII-, XorI- XorII+ and XorI- XorII-) were detected in the population at a ratio of approximately 1:2:2:2. The XorI+ XorII+ and XorI- XorII+ phenotypes were observed predominantly in strains from southeast Asia (Philippines, Malaysia, and Indonesia), whereas strains with the phenotypes XorI- XorII- and XorI+ XorII- were distributed in south Asia (India and Nepal) and northeast Asia (China, Korea, and Japan), respectively. Based on the prevalence and geographic distribution of the XorI and XorII systems, we suggest that the XorI modification system originated in northeast Asia and was later introduced to southeast Asia, while the XorII system originated in southeast Asia and moved to northeast Asia and south Asia. Genomic DNA from all tested strains of X. oryzae pv. oryzae that were resistant to digestion by endonuclease XorII or its isoschizomer PvuI also hybridized with a 7.0-kb clone that contained the XorII modification system, whereas strains that were digested by XorII or PvuI lacked DNA that hybridized with the clone. Size polymorphisms were observed in fragments that hybridized with the 7.0-kb clone. However, a single hybridization pattern generally was found in XorII+ strains within a country, indicating clonal maintenance of the XorII methyl-transferase gene locus. The locus was monomorphic for X. oryzae pv. oryzae strains from the Philippines and all strains from Indonesia and Korea.
Minimally processed fresh produce has been implicated as a major source of foodborne microbial pathogens globally. These pathogens must attach to the produce in order to be transmitted. Cut surfaces of produce that expose cell walls are particularly vulnerable. Little is known about the roles that different structural components (cellulose, pectin, and xyloglucan) of plant cell walls play in the attachment of foodborne bacterial pathogens. Using bacterial cellulose-derived plant cell wall models, we showed that the presence of pectin alone or xyloglucan alone affected the attachment of three Salmonella enterica strains (Salmonella enterica subsp. enterica serovar Enteritidis ATCC 13076, Salmonella enterica subsp. enterica serovar Typhimurium ATCC 14028, and Salmonella enterica subsp. indica M4) and Listeria monocytogenes ATCC 7644. In addition, we showed that this effect was modulated in the presence of both polysaccharides. Assays using pairwise combinations of S. Typhimurium ATCC 14028 and L. monocytogenes ATCC 7644 showed that bacterial attachment to all plant cell wall models was dependent on the characteristics of the individual bacterial strains and was not directly proportional to the initial concentration of the bacterial inoculum. This work showed that bacterial attachment was not determined directly by the plant cell wall model or bacterial physicochemical properties. We suggest that attachment of the Salmonella strains may be influenced by the effects of these polysaccharides on physical and structural properties of the plant cell wall model. Our findings improve the understanding of how Salmonella enterica and Listeria monocytogenes attach to plant cell walls, which may facilitate the development of better ways to prevent the attachment of these pathogens to such surfaces.