Lower Bacterial Numbers Research Articles

CD14-Dependent Monocyte Isolation Enhances Phagocytosis of Listeria monocytogenes by Proinflammatory, GM-CSF-Derived Macrophages

Macrophages are an important line of defence against invading pathogens. Human macrophages derived by different methods were tested for their suitability as models to investigate Listeria monocytogenes (Lm) infection and compared to macrophage-like THP-1 cells. Human primary monocytes were isolated by either positive or negative immunomagnetic selection and differentiated in the presence of granulocyte macrophage colony-stimulating factor (GM-CSF) or macrophage colony-stimulating factor (M-CSF) into pro- or anti-inflammatory macrophages, respectively. Regardless of the isolation method, GM-CSF-derived macrophages (GM-Mφ) stained positive for CD206 and M-CSF-derived macrophages (M-Mφ) for CD163. THP-1 cells did not express CD206 or CD163 following incubation with PMA, M- or GM-CSF alone or in combination. Upon infection with Lm, all primary macrophages showed good survival at high multiplicities of infection whereas viability of THP-1 was severely reduced even at lower bacterial numbers. M-Mφ generally showed high phagocytosis of Lm. Strikingly, phagocytosis of Lm by GM-Mφ was markedly influenced by the method used for isolation of monocytes. GM-Mφ derived from negatively isolated monocytes showed low phagocytosis of Lm whereas GM-Mφ generated from positively selected monocytes displayed high phagocytosis of Lm. Moreover, incubation with CD14 antibody was sufficient to enhance phagocytosis of Lm by GM-Mφ generated from negatively isolated monocytes. By contrast, non-specific phagocytosis of latex beads by GM-Mφ was not influenced by treatment with CD14 antibody. Furthermore, phagocytosis of Lactococcus lactis, Escherichia coli, human cytomegalovirus and the protozoan parasite Leishmania major by GM-Mφ was not enhanced upon treatment with CD14 antibody indicating that this effect is specific for Lm. Based on these observations, we propose macrophages derived by ex vivo differentiation of negatively selected human primary monocytes as the most suitable model to study Lm infection of macrophages.

Antagonistic effects of probiotic Escherichia coli Nissle 1917 on EHEC strains of serotype O104:H4 and O157:H7

The largest EHEC outbreak up to now in Germany occurred in 2011. It was caused by the non-O157:H7 Shiga-toxinogenic enterohemorrhagic E. coli strain O104:H4. This strain encodes in addition to the Shiga toxin 2 (Stx2), responsible for the hemolytic uremic syndrome (HUS), several adhesins such as aggregative adherence fimbriae. Currently, there is no effective prophylaxis and treatment available for EHEC infections in humans. Especially antibiotics are not indicated for treatment as they may induce Stx production, thus worsening the symptoms. Alternative therapeutics are therefore desperately needed. We tested the probiotic Escherichia coli strain Nissle 1917 (EcN) for antagonistic effects on two O104:H4 EHEC isolates from the 2011 outbreak and on the classical O157:H7 EHEC strain EDL933. These tests included effects on adherence, growth, and Stx production in monoculture and co-culture together with EcN. The inoculum of each co-culture contained EcN and the respective EHEC strain either at a ratio of 1:1 or 10:1 (EcN:EHEC). Adhesion of EHEC strains to Caco-2 cells and to the mucin-producing LS-174T cells was reduced significantly in co-culture with EcN at the 1:1 ratio and very dramatically at the 10:1 ratio. This inhibitory effect of EcN on EHEC adherence was most likely not due to occupation of adhesion sites on the epithelial cells, because in monocultures EcN adhered with much lower bacterial numbers than the EHEC strains. Both EHEC strains of serotype O104:H4 showed reduced growth in the presence of EcN (10:1 ratio). EHEC strain EDL933 grew in co-culture with EcN only during the first 2h of incubation. Thereafter, EHEC counts declined. At 24h, the numbers of viable EDL933 was at or slightly below the numbers at the time of inoculation. The amount of Stx2 after 24h co-incubation with EcN (EcN:EHEC ratio 10:1) was for all 3 EHEC strains tested significantly reduced in comparison to EHEC monocultures.Obviously, EcN shows very efficient antagonistic activity on the EHEC strains of serotype O104:H4 and O157:H7 tested here regarding adherence to human gut epithelial cells, bacterial growth, and Stx2 production in vitro.

IFN-γ induced antimicrobial response against M. leprae in human macrophages (117.12)

Abstract The role of IFN-γ in inducing an antimicrobial response in macrophages was investigated in leprosy, a disease caused by intracellular bacterium M. leprae. Tuberculoid leprosy (T-lep) is characterized by few skin lesions with low bacterial numbers, whereas lepromatous leprosy (L-lep) is characterized by disseminated skin lesions and increased bacilli numbers. The host immune response in T-lep is characterized by Th1 cytokines and expression of the vitamin D dependent antimicrobial pathway, in contrast to L-lep, which is predominately Th2. We have linked expression of the Th1 cytokine IFN-γ and antimicrobial response in leprosy by induction of vitamin D dependent antimicrobial response genes CAMP and DEFB4. IFN-γ is present in T-lep patients, where the disease is self-limited, however, the role of IFN-γ in antimicrobial activity is unclear. IFN-γ mediates antimicrobial activity against M. tuberculosis, a related pathogen, in the presence of vitamin D sufficient serum, but not in the presence of vitamin D deficient serum. Our preliminary data shows that M. leprae infected macrophages pretreated with IFN-γ prior to infection in combination with post-infection IFN-γ treatment in vitamin D sufficient serum reduced bacterial viability by approximately 80% compared to media control. Our studies suggest that IFN-γ can trigger a vitamin D dependent antimicrobial response in human macrophages as well as antimicrobial activity against M. leprae.

A comprehensive microbiological evaluation of fifty-four patients undergoing revision surgery due to prosthetic joint loosening

The diagnosis of a chronic prosthetic joint infection (PJI) is challenging, and no consensus exists regarding how best to define the criteria required for microbiological identification. A general view is that culture of periprosthetic biopsies suffers from inadequate sensitivity. Recently, molecular analyses have been employed in some studies but the specificity of molecular analyses has been questioned, mainly due to contamination issues. In a prospective study of 54 patients undergoing revision surgery due to prosthetic joint loosening, we focused on two aspects of microbiological diagnosis of chronic PJI. First, by collecting diagnostic specimens in a highly standardized manner, we aimed at investigating the adequacy of various specimens by performing quantitative bacteriological culture. Second, we designed and performed real-time 16S rRNA gene PCR analysis with particular emphasis on minimizing the risk of false-positive PCR results. The specimens analysed included synovial fluid, periprosthetic biopsies from the joint capsule and the interface membrane, and specimens from the surface of the explanted prosthesis rendered accessible by scraping and sonication. No antibiotics were given prior to specimen collection. Based on five diagnostic criteria recently suggested, we identified 18 PJIs, all of which fulfilled the criterion of ≥2 positive cultures of periprosthetic specimens. The rate of culture-positive biopsies from the interface membrane was higher compared to specimens from the joint capsule and synovial fluid, and the interface membrane contained a higher bacterial load. Interpretational criteria were applied to differentiate a true-positive PCR from potential bacterial DNA contamination derived from the reagents used for DNA extraction and amplification. The strategy to minimize the risk of false-positive PCR results was successful as only two PCR results were false-positive out of 216 negative periprosthetic specimens. Although the PCR assays themselves were very sensitive, three patients with low bacterial numbers in periprosthetic specimens tested negative by real-time PCR. This overall lowered sensitivity is most likely due to the reduced specimen volume used for PCR analysis compared to culture and may also be due to interference from human DNA present in tissue specimens. According to the protocol in the present study, 16S rRNA gene real-time PCR did not identify more cases of septic prosthetic loosening than did culture of adequate periprosthetic biopsies.

Characterization of bacterial communities at heavy-metal-contaminated sites

The microbial community in soil samples from two long-term contaminated sites was characterized by using culture-dependent and culture-independent methods. The two sites investigated contained high amounts of heavy metals and were located in the upper Silesia Industrial Region in southern Poland. The evaluation of the aerobic soil microbial population clearly demonstrated the presence of considerable numbers of viable, culturable bacteria at both sites. A high fraction of the bacterial population was able to grow in the presence of high amounts of metals, i.e. up to 10mM Zn2+, 3mM Pb2+ or 1mM Cu2+. Site 1 contained significantly (P<0.05) lower bacterial numbers growing in the presence of 10mM Zn2+ than site 2, while the opposite was observed for bacteria tolerating 1mM Cu2+. This coincided with the contents of these two metals at the two sites. Ecophysiological (EP) indices for copiotrophs (r-strategists) and oligotrophs (K-strategists) pointed to high bacterial diversity at both sites. Fluorescence in situ hybridization (FISH) analysis indicated that Actinobacteria and Proteobacteria represent the physiologically active fraction of bacteria at the two sites. Shannon diversity (H′) indices for FISH-detected bacterial phylogenetic groups were not significantly different at the two sites.

Survival of Brucella spp. in mineral water, milk and yogurt

Knowledge of the number of organisms in a food product at the time of consumption is crucial to assess the risk from a deliberate contamination of food samples with Brucella. To date, very little data on the survival times of Brucella in different food matrices is available. This study was conducted to assess the survival times of Brucella spp. in water, milk and yogurt. These food products were inoculated with bacteria, serial dilutions of the food samples plated and the number of surviving bacteria counted. Under normal storage conditions Brucella survived in UHT milk for 87 days, for 60 days in water and less than a week in yogurt. Also, when milk was inoculated with low bacterial numbers, Brucella multiplied by five log units within three weeks. Further we could not confirm that a high fat content in food has a protective effect on Brucella survival. Brucella survived in 3.5% and 10.0% fat yogurt for four and two days, respectively. These results show that appropriate methods for the rapid detection of this pathogen from food matrices are required.

AtolCMutant ofFrancisella tularensisIs Hypercytotoxic Compared to the Wild Type and Elicits Increased Proinflammatory Responses from Host Cells

The highly infectious bacterium Francisella tularensis is a facultative intracellular pathogen and the causative agent of tularemia. TolC, which is an outer membrane protein involved in drug efflux and type I protein secretion, is required for the virulence of the F. tularensis live vaccine strain (LVS) in mice. Here, we show that an LVS DeltatolC mutant colonizes livers, spleens, and lungs of mice infected intradermally or intranasally, but it is present at lower numbers in these organs than in those infected with the parental LVS. For both routes of infection, colonization by the DeltatolC mutant is most severely affected in the lungs, suggesting that TolC function is particularly important in this organ. The DeltatolC mutant is hypercytotoxic to murine and human macrophages compared to the wild-type LVS, and it elicits the increased secretion of proinflammatory chemokines from human macrophages and endothelial cells. Taken together, these data suggest that TolC function is required for F. tularensis to inhibit host cell death and dampen host immune responses. We propose that, in the absence of TolC, F. tularensis induces excessive host cell death, causing the bacterium to lose its intracellular replicative niche. This results in lower bacterial numbers, which then are cleared by the increased innate immune response of the host.

Safe drinking water still a dream in rural areas of South Africa. Case Study: The Eastern Cape Province

From October to November 2004, and from July to September 2005, a survey of 55 plants was conducted in five District Municipalities (Cacadu, Chris Hani, Amathola, Ukhahlamba and O.R. Tambo) of the Eastern Cape Province, with the aim of examining the disinfection practices used in small rural systems of the Eastern Cape and their effect on the quality of drinking water they supply to the communities.Eighty six percent of the visited plants were treating surface water while 7% were treating groundwater and another 7% were treating both ground- and surface water. The majority of plants surveyed employed some variation of conventional treatment (coagulation, flocculation, sedimentation, filtration, chlorination). Turbidity values in 55% of the plants were within the acceptable SABS limits while the other 45% showed high values. Out of the 55 surveyed plants only 18% complied with the SABS recommended limits in terms of microbiological quality. The major factors that contributed to high bacterial numbers were high turbidity and inefficient chemical (coagulant and chlorine) dosing, which led to low chlorine residuals. It was also noted that although some plants had low bacterial numbers at the point of treatment, bacterial re-growth occurred in the distribution system, thereby compromising the quality of water at the point of use. The worst technical problems encountered tended to be at the newly commissioned or upgraded plants where properly trained operators had yet to be appointed or where the existing staff lack proficiency in the use of the upgraded systems or why certain modifications to the treatment process were being made. Operators were lacking in the required technical knowledge such as flow rate and chemical dosing to effectively execute their duties. Lack of communication between consultants, operators and municipal officials on technical issues relating to plant operation appeared to be a problem. The results of this study gave conclusive evidence that rural water treatment plants are still failing to produce safe drinking water.

Microcalorimetric assessment of microbial activity in long-term fertilization experimental soils of Southern China

Microcalorimetry, plate count and PCR-denaturing gradient gel electrophoresis (DGGE) were employed to investigate microbial diversity and activity in soils from the Red Soil Experimental Station of the Chinese Academy of Agricultural Sciences, Hunan Province, China, where a wheat-corn rotation with 12 fertilization treatments was established in 1990. Fertilization greatly increased microbial biomass carbon (C) and nitrogen (N) (C(mic) and N(mic)) as well as the activities of phosphatase, urease, invertase, protease, catalase and dehydrogenase. Manure alone (M) enhanced the number of denitrifying and aerobic bacteria by 54.4% and 20.5%, respectively, whereas fallow (H) increased the number of aerobic cellulose decomposing bacteria by 31.4%. Fallow and soils amended with mineral fertilizers plus pig manure or straw increased both the DGGE band patterns and the Shannon index compared with mineral fertilizers or the control. Mineral treatments with lower bacterial numbers enhanced the values of the peak time (t(max)) more than did organic treatments. The peak height (P(max)) was positively correlated (P<0.01), with soil enzymes, C(mic) and N(mic), and the number of microorganisms, whereas the peak time (t(max)) was negatively connected (P<0.01) with these parameters. The microbial growth rate constant (k) was linked to bacteria (P<0.01), actinomycetes (P<0.05) and catalase (P<0.05). The total heat evolution (Q) had no relationships with any soil microbial properties (except for catalase). We propose that P(max) and t(max) could be used as indices of soil microbial activity, while the values of k and Q are poor indicators.

Lactoferrin as a Natural Regimen for Selective Decontamination of the Digestive Tract: Recombinant Porcine Lactoferrin Expressed in the Milk of Transgenic Mice Protects Neonates from Pathogenic Challenge in the Gastrointestinal Tract

Nosocomial infection with antibiotic-resistant strains is a major threat to critical care medicine. Selective decontamination of the digestive tract (SDD) is one of the strategies used to reduce ventilator-associated pneumonia and sepsis in critically ill patients. In the present study, we performed pathogenic challenges of the digestive tract in a transgenic milk-fed animal model to test whether porcine lactoferrin (pLF) is an effective SDD regimen. Transgenic mice expressing recombinant pLF in their milk at a mean+/-SD concentration of 120+/-13.6 mg/L during the lactation stage fed normal CD-1 mice pups for 4 weeks. The pups were subsequently challenged with pathogenic Escherichia coli, Staphylococcus aureus, and Candida albicans. Compared with the control groups fed wild-type (normal) milk, the groups fed pLF-enriched milk demonstrated statistically significant improvements in weight gain; lower bacterial numbers in intestinal fluid, blood, and liver; healthier microvilli in the small intestine; and alveoli in the lungs. Our results showed that oral administration of pLF-enriched milk to mice led to broad-spectrum antimicrobial activity in the digestive tract and protected the mucosa of the small intestine from injury, implying that pLF can be used as an effective SDD regimen.

Host colonization differences between citrus and coffee isolates of Xylella fastidiosa in reciprocal inoculation

Citrus variegated chlorosis (CVC) and coffee stem atrophy (CSA) are important diseases in Brazil associated with closely-related strains of Xylella fastidiosa, but little is know about host aoverlappingnd importance of citrus and coffee as inoculum sources of these strains. In this study, reciprocal-inoculation experiments were performed to determine if CVC and CSA isolates are biologically similar within citrus and coffee plants. These two hosts were mechanically inoculated with a CVC and a CSA isolate of X. fastidiosa at four concentrations ranging between10³ and 10(9) colony forming units CFU mL-1. At two, four and eight months after inoculation, the infection efficiency and bacterial populations of the isolates in each host were determined by culturing. The CVC isolate infected both citrus and coffee plants, but developed lower populations in coffee. The CSA isolate did not colonize citrus. Inoculation of coffee plants with the CVC isolate resulted in low rates of infection and required an inoculum concentration ten-fold higher than that necessary to obtain a similar (25%) rate of infection in citrus. The relatively low infection rates and bacterial numbers of the CVC isolate in coffee plants compared with those observed in citrus suggest that coffee is not a suitable host to serve as a source of inoculum of the CVC strain for primary spread to citrus or within coffee plantations.

Quantification of gene expression of Listeria monocytogenes by real-time reverse transcription PCR: Optimization, evaluation and pitfalls

In the current study, various steps in the real-time reverse transcription PCR (real-time RT-PCR) method for determination of RNA expression levels starting from different numbers of Listeria monocytogenes cells were evaluated and optimized. Our results showed that the RNA isolation method as well as the cDNA synthesis may influence the sensitivity of the procedure. For high bacterial cell numbers (10 9 bacterial cells), the RNAqueous kit and the RNeasy Mini kit were equally useful, whereas for low bacterial cell numbers (≤ 10 7 bacterial cells) the RNAqueous-Micro kit was found to be the most sensitive RNA isolation kit. For cDNA synthesis, the use of random hexamers and an incubation time of 90 min with the Multiscribe RT-enzyme resulted in the highest efficiency of conversion of RNA into cDNA. To compare RNA levels of different L. monocytogenes strains, it is necessary to analyse the expression levels at the same point in the growth phase and to have a 100% matching of the primers for all tested strains to obtain reliable results. In general, our results showed that real-time RT-PCR needs to be optimized to obtain reliable and accurate data and that many factors can influence the outcome of the real-time RT-PCR data.

Microbiological assessments of compost toilets: In situ measurements and laboratory studies on the survival of fecal microbial indicators using sentinel chambers

Compost toilet systems were assessed for their ability to reduce microbial indicators and pathogens. Bacterial pathogens were not detected in any samples indicating a low survival rate in composting feces and/or an initial low occurrence. Indicator bacteria showed large variations with no clear trend of lower bacterial numbers after longer storage. In controlled composting experiments, thermophilic conditions were only reached when amendments were made (grass and a sugar solution). Even then it was impossible to ensure a homogenous temperature in the composting fecal material and therefore difficult to achieve a uniform reduction and killing of indicator organisms. Presumptive thermotolerant coliforms, Salmonella typhimurium Phage 28B and eggs of Ascaridia galli, proved useful as indicators. However, regrowth was detected for enterococci and total numbers of bacteria grown at 36 °C. These indicator parameters may therefore overestimate the level of other (pathogenic) bacteria present in the material and can not be recommended for use as reliable indicator organisms in composting toilet systems. The addition of indicator bacteria to fecal material contained in semi-permeable capsules proved to be a useful technique to ensure that microorganisms were contained in a small test volume.

An Assessment of Sampling Methods and Microbiological Hygiene Indicators for Process Verification in Poultry Slaughterhouses

Studies to determine the appropriateness of the use of populations of indicator bacteria on poultry carcasses for process verification were undertaken in commercial slaughterhouses. Samples were collected from neck skin by excision or from whole carcass rinses and were examined for a range of presumptive process hygiene indicator bacteria. Coefficients of variation were calculated for each bacterial indicator and were significantly lower in excised samples, indicating more reproducible bacterial recovery by this sampling method. Total viable counts of aerobic bacteria, Enterobacteriaceae, and Pseudomonas in samples collected by excision had the lowest coefficients of variation when compared with other indicators and were therefore used for further study. The uncertainties associated with the quantification of each bacterial indicator were calculated and were lowest overall for total viable counts of aerobic bacteria. In general, uncertainty was higher for lower bacterial numbers. Results of microbiological testing on pooled excised neck skin samples were not significantly different from the mean of individually analyzed samples. Bacterial numbers increased by 1 log unit when cultures were stored under chilled conditions typical of those used for transporting samples to external laboratories, but the increases were not significant for Pseudomonas and aerobic bacteria when storage time was less than 17 h. Weak relationships were identified between bacterial indicator numbers and duration of processing, although cleanliness of the processing environment diminished visibly during this time. In the plants visited for this study, there was a poor relationship between presumptive bacterial indicator numbers and process hygiene. Consequently, bacterial analyses for process verification purposes may be of limited value.

Use of water spray and extended drying time to lower bacterial numbers on soiled flooring from broiler transport coops

Broiler transport coops soiled with Campylobacter-positive feces have been shown to facilitate cross-contamination of broilers. Washing and sanitizing coop surfaces do not always effectively eliminate bacteria. The objective of this study was to examine drying as a means of lowering bacterial numbers on transport coop flooring. Small squares (5 x 5 cm) of fiberglass flooring from transport coops were intentionally contaminated with 1 g of Campylobacter-positive broiler gut contents. Soiled floor squares were sprayed with water and allowed to dry for 15 min, 24 h, or 48 h. Unsprayed squares were examined at each time period as controls. All squares were sampled by cotton-tipped applicators that were cultured for Campylobacter, coliforms, and Escherichia coli. Sampling of unsprayed squares at 15 min yielded 7.3 log cfu of Campylobacter, 6.2 log cfu of coliforms and 5.9 log cfu of E. coli per floor square. Water spray alone resulted in a significantly lower number of organisms recovered: 4.1 log cfu Campylobacter, 3.6 log cfu coliform, and 3.2 log cfu E. coli per floor square. When water spray was followed by a 24-hour drying period, no Campylobacter, coliforms, or E. coli were detected on the floor surface. However, allowing unsprayed soiled flooring to simply dry for 24 or 48 h also resulted in no recovery of Campylobacter and very low numbers of coliforms and E. coli. A 24- or 48-hour drying period for fecal matter on broiler transport cage flooring may be a viable method to lower bacterial numbers on these surfaces.

Water Spray and Immersion in Chemical Sanitizer to Lower Bacterial Numbers on Broiler Transport Coop Flooring

Broiler transport coops become soiled with feces during use. When this fecal matter contains Campylobacter, the result can be cross-contamination of subsequent flocks that were previously free of this important human pathogen. Because washing and sanitizing coops requires large amounts of water and is not always effective enough to justify the expense, few broiler companies wash and sanitize dump coops between flocks. In this lab-scale study, a tap water spray was effective in lowering the numbers of bacteria, including Campylobacter, associated with broiler transport coop flooring. Immersion in a chemical sanitizer after spray washing did not enhance the antibacterial effect. It is possible that sanitizing treatments could be made more effective by using higher concentrations of chemicals, high temperature treatment, or high pressure or repeated applications of water spray. However, such changes would come at a cost. Research is needed to find new and innovative ways to lower bacterial numbers in broiler transport coops without undue use of water and the associated expense.

Host genes affect intestinal colonisation of newly hatched chickens by Campylobacter jejuni

Campylobacter jejuni is the leading cause of food-borne gastro-enteritis and infection can be followed by severe clinical complications, such as the autoimmune neuropathy Guillain-Barré syndrome. Poultry meat is considered to be a common source of infection, with most flocks infected from 2 to 3 weeks of age. We have examined the effect of host genetics on the colonisation levels of C. jejuni in chickens. Chicks from different inbred lines were challenged with 10(7) to 10(8) cfu of C. jejuni 14N or C. jejuni 81-176 on the day of hatch and levels of bacterial colonisation measured over a period of 2-3 weeks. We consistently observed a 10- to 100-fold difference between four inbred lines in the number of C. jejuni organisms present in the cloaca or in the caeca, with the greatest differences detected between line N, which carried relatively high bacterial levels, and line 6(1), which carried relatively low numbers of bacteria. Amongst the four lines studied, major histocompatibility complex did not appear to be a major factor in determining the resistance. The difference in numbers of cloacal bacteria was observed as soon as 24 h after challenge and was still present at the end of the experiment. Lines N and 6(1) were chosen to analyse the mode of inheritance of the genetic differences in response to this infection. Challenge of progeny from reciprocal (6(1) female x N male) and (6(1) female x N male) F1 crosses and from (N female x 6(1) male) F1 female x N male backcrosses with C. jejuni 14N revealed that the difference in bacterial numbers was inherited in a manner consistent with the resistance (low bacterial numbers) controlled by a single autosomal dominant locus. These data suggest that it might be possible to identify the genes responsible by genetic mapping and candidate gene analysis.

Specific antibodies contribute to the host protection against strains of Francisella tularensis subspecies holarctica

T cells are crucial to the control and eradication of the facultative intracellular bacterium Francisella tularensis. A contributory role of humoral antibodies in the host defence remains to be assessed. We used B-cell-deficient mice to study the possible contribution of antibodies to the defence against the live vaccine strain (LVS) or a clinical isolate of F. tularensis, both belonging to the subspecies holarctica (type B). When B-cell-deficient (Igμ −/−) mice of the C57BL/10 background were administered immune serum one day before intradermal injection of LVS, they developed lower bacterial numbers in skin, liver, and spleen than did mice receiving normal serum, and survived a challenge inoculum that was lethal for mice given normal serum. Administration of immune serum to C57BL/10 mice afforded protection also against infection with the clinical isolate of F. tularensis subsp. holarctica. Five days after intradermal inoculation of bacteria of the isolate, animals receiving immune serum showed 4 log 10 lower bacterial counts in liver and spleen than mice administered normal serum. In mice primed by LVS infection, T-cell immunity and host protection were strong and only a marginal contribution of immune serum against a secondary intradermal infection was demonstrated. Together, these findings show that specific antibodies contribute to the host defence of mice against F. tularensis subsp. holarctica.

Effects of Pre-, In-, and Post-Rigor Filleted Atlantic Salmon (Salmo salar)on Microbial Spoilage and Quality Characteristics During Chilled Storage

Atlantic salmon fillets processed pre-, in-, and post-rigor were compared for microbial growth, odor, water loss, texture, and gaping in two separate experiments. Salmon were chilled to a core temperature of < 2.5°C prior to slaughter, and stored gutted at 0°C on ice. Salmon were filleted, as pre-rigor (4 hours after slaughtering), in-rigor (2 days), and post-rigor (5 days), and the fillets were stored vacuum packaged or exposed to air on ice at 0°C for up to 14 days. The pre-rigor fillets had lower bacterial numbers (total viable counts, H2S-producing bacteria and psychrotrophic bacteria) when stored in air and for most samples in vacuum. The fresh odor scores were higher, gaping lower, but water loss higher in pre-rigor fillets compared to post-rigor. The results show that pre-rigor filleting of Atlantic salmon is a preferable alternative to traditional post-rigor filleting.

The role of P fimbriae for Escherichia coli establishment and mucosal inflammation in the human urinary tract

Bacterial adhesion to the bladder mucosa is a critical step for the establishment of Escherichia coli bacteriuria. The P-fimbriae, encoded by the pap gene cluster, are considered as virulence factors but the mechanisms have been debated. This study defined the roles for P fimbriation during the early colonization of the human urinary tract. Patients with recurrent UTI were first subjected to deliberate colonization with the non-fimbriated ABU strain E. coli 83972. Bacteriuria was established long term (1-4 years) in patients with dysfunctional bladders, but not in the patients with normal bladder function. Super-infections were transient and asymptomatic. P fimbriated transformants of the ABU strain (E. coli 83972pap+/prs+) reached 105 CFU/ml more rapidly than E. coli 83972 and the vector control. This was demonstrated by group wise and intra-individual analysis in patients colonized on different occasions with E. coli 83972 or the P fimbriated transformants. Higher neutrophil numbers and IL-8 and IL-6 concentrations in urine were obtained after colonization with the P fimbriated transformants. These results demonstrated that transformation of E. coli 83972 with the pap sequences is sufficient to convert it to a more potent host response inducer. The P fimbriae were shown to lower the significant bacteriuria threshold. The P fimbriated transformants needed lower bacterial numbers (103-4 CFU/ml) to predict a positive second urine culture with a >80% accuracy and to trigger a significant host response. These studies show that P fimbriae fulfil the Koch Henles molecular postulates for bacterial establishment and host response induction in the human urinary tract.