Lower Bacterial Numbers Research Articles

The performance of tongue swabs for detection of pulmonary tuberculosis.

Oral and/or tongue swabs have demonstrated ability to detect Mycobacterium tuberculosis (Mtb) in adults with pulmonary tuberculosis (TB). Swabs provide useful alternative specimens for diagnosis of TB using molecular assays however, the diagnostic pickup by culture requires further improvement and development. Several studies identified the presence of differentially culturable tubercle bacilli (DCTB) populations in a variety of clinical specimens. These organisms do not grow in routine laboratory media and require growth factors in the form of culture filtrate (CF) from logarithmic phase cultures of Mtb H37Rv. Herein, we compared the diagnostic performance of sputum and tongue swabs using Mycobacterial Growth Indicator Tube (MGIT) assays, Auramine smear, GeneXpert and DCTB assays supplemented with or without CF. From 89 eligible participants, 83 (93%), 66 (74%) and 79 (89%) were sputum positive by MGIT, smear and GeneXpert, respectively. The corresponding tongue swabs displayed a lower sensitivity with 39 (44%), 2 (2.0%) and 18 (20%) participants respectively for the same tests. We aimed to improve the diagnostic yield by utilizing DCTB assays. Sputum samples were associated with a higher positivity rate for CF-augmented DCTB at 82/89 (92%) relative to tongue swabs at 36/89 (40%). Similarly, sputum samples had a higher positivity rate for DCTB populations that were CF-independent at 64/89 (72%) relative to tongue swabs at 26/89 (29%). DCTB positivity increased significantly, relative to MGIT culture, for tongue swabs taken from HIV-positive participants. We next tested whether the use of an alternative smear stain, DMN-Trehalose, would improve diagnostic yield but noted no substantial increase. Collectively, our data show that while tongue swabs yield lower bacterial numbers for diagnostic testing, the use of growth supplementation may improve detection of TB particularly in HIV-positive people but this requires further interrogation in larger studies.

Liver-resident CD44hiCD27− γδT Cells Help to Protect Against Listeria monocytogenes Infection

Gamma delta (γδ) T cells are heterogeneous and functionally committed to producing interferon (IFN)-γ and interleukin (IL)-17. γδT cells are defined as tissue-resident lymphocytes in barrier tissues. Among them, IL-17-producing γδT cells are relatively abundant in the liver. However, a systematic and comprehensive understanding of the residency characteristics and function of hepatic IL-17A+ γδT cells is lacking. We undertook a single-cell analysis of γδT17 cells derived from murine livers. A parabiosis model was used to assess tissue residency. Fluorescence-activated cell sorting and adoptive transfer experiments were used to investigate the response and protective role of liver-resident CD44hiCD27- γδT cells in Listeria monocytogenes infection. Transwell assay was used to assess the role of macrophages in the chemotaxis of liver-resident CD44hiCD27- γδT cells. We identified hepatic IL-17A-producing γδT cells as CD44hiCD27- γδT cells. They had tissue-resident characteristics and resided principally within the liver. Vγ6+ T cells also exhibited liver-resident features. Liver-resident CD44hiCD27- γδT cells had significantly increased proliferation capacity, and their proportion rapidly increased after infection. Some CD44hiCD27- γδT cells could produce IL-17A and IFN-γ simultaneously in response to Lm infection. Adoptive transfer of hepatic CD44hiCD27- γδT cells into Lm-infected TCRδ-/- mice led to markedly lower bacterial numbers in the liver. Hepatic macrophages promoted the migration and accumulation of liver-resident CD44hiCD27- γδT cells into infection sites. Liver-resident CD44hiCD27- γδT cells protect against Lm infection. Hepatic macrophages coordinate with liver-resident CD44hiCD27- γδT cells and contribute to the clearance of Lm at the early stage of infection corporately.

Low microbial biomass within the reproductive tract of mid-lactation dairy cows: A study approach

The microbiome from the reproductive tract is being investigated for its putative effect on fertility, embryo development, and health status of the human or animal host postpartum. Besides the presence of a vaginal microbiome, recent studies have claimed the existence and putative role of the uterine microbiome. Yet, the extremely low bacterial numbers and high eukaryotic/prokaryotic DNA ratio make this a highly challenging environment to study with next-generation sequencing (NGS) techniques. Here, we describe the methodological challenges that are typically encountered when performing an accurate analysis of low microbial biomass samples, illustrated by data of our own observational study. In terms of the research question, we compared the microbial composition throughout different parts of the reproductive tract of clinically healthy, mid-lactation Holstein-Friesian cows. Samples were collected from 5 dairy cows immediately after killing. Swabs were taken from the vagina, and from 4 pre-established locations of the uterine endometrium. In addition to the conventional DNA extraction blank controls, sterile swabs rubbed over disinfected disposable gloves and the disinfected surface of the uterus (tunica serosa) before incision were taken as sampling controls. The DNA extraction, DNA quantification, quantitative PCR of the 16S rRNA genes, and 16S rRNA gene sequencing were performed. In terms of NGS data analysis, we performed prevalence-based filtering of putative contaminant operational taxonomic units (OTU) using the decontam R package. Although the bacterial composition differed between the vagina and uterus, no differences in bacterial community structure (α and β diversity) were found among the different locations in the uterus. At phylum level, uterine samples had a greater relative abundance of Proteobacteria, and a lesser relative abundance of Firmicutes than vaginal samples. The number of shared OTU between vagina and uterus was limited, suggesting the existence of bacterial transmission routes other than the transcervical one to the uterus. The mid-lactation bovine genital tract is a low microbial biomass environment, which makes it difficult to distinguish between its constitutive versus contaminant microbiome. The integration of key controls is therefore strictly necessary to decrease the effect of accidentally introduced contaminant sequences and improve the reliability of results in samples with low microbial biomass.

A comparison of scanning electron microscopy preparation methods to observe anatomical features and microbial changes during the vase life of Acacia and Rosa

There are several sample preparation methods used to observe plant material in scanning electron microscopy (SEM) and choosing the most appropriate method can be difficult. This choice is especially critical when investigating bacterial colonisation of cut stem ends during vase life, in case some preparation methods remove microbial material. When critical point drying (CPD) was used to compare bacterial colonisation at days 0, 1, 3 and 5 of vase life in Acacia amoena (Wendl.), few bacteria were observed by 3 d. Colonisation only became evident after 5 d. Because our previous study’s enumeration of bacteria revealed high numbers (103 cfu mL−1) by 3 d, it was hypothesised that the numerous changes of solution during CPD had removed bacteria. To test this, three SEM preparation methods: air drying (AD); CPD; and freeze-drying (FD), were compared, based on their ability to detect microbial changes and observe anatomical features in Rosa hybrida L. ‘Sonia’, a well-studied plant. The choice of CPD, AD or FD did not markedly affect bacterial colonisation in Rosa, implying that CPD was not the cause of lower bacterial numbers in Acacia. Amorphous deposits found in both plant genera were likely to be the bacterial biofilms seen by others. Vestures and warts were observed on inner xylem vessel walls and pits in Acacia, but not definitively in Rosa. All SEM preparation methods provided clear images of plant anatomy and microbial colonisation in Rosa, suggesting that they can also be used validly in the study of other genera like Acacia, although AD preserved bacterial biofilms more clearly. The easiest preparation method in terms of equipment required and labour intensity was AD but, overall, CPD provided the highest quality images with no surface distortion. When choosing the most appropriate SEM preparation method, it is also important to consider the purpose of the observations.

Extracellular Pneumococcal Serine Proteases Affect Nasopharyngeal Colonization.

Streptococcus pneumoniae has evolved versatile strategies to colonize the nasopharynx of humans. Colonization is facilitated by direct interactions with host cell receptors or via binding to components of the extracellular matrix. In addition, pneumococci hijack host-derived extracellular proteases such as the serine protease plasmin(ogen) for ECM and mucus degradation as well as colonization. S. pneumoniae expresses strain-dependent up to four serine proteases. In this study, we assessed the role of secreted or cell-bound serine proteases HtrA, PrtA, SFP, and CbpG, in adherence assays and in a mouse colonization model. We hypothesized that the redundancy of serine proteases compensates for the deficiency of a single enzyme. Therefore, double and triple mutants were generated in serotype 19F strain EF3030 and serotype 4 strain TIGR4. Strain EF3030 produces only three serine proteases and lacks the SFP encoding gene. In adherence studies using Detroit-562 epithelial cells, we demonstrated that both TIGR4Δcps and 19F mutants without serine proteases or expressing only CbpG, HtrA, or PrtA have a reduced ability to adhere to Detroit-562 cells. Consistent with these results, we show that the mutants of strain 19F, which preferentially colonizes mice, abrogate nasopharyngeal colonization in CD-1 mice after intranasal infection. The bacterial load in the nasopharynx was monitored for 14 days. Importantly, mutants showed significantly lower bacterial numbers in the nasopharynx two days after infection. Similarly, we detected a significantly reduced pneumococcal colonization on days 3, 7, and 14 post-inoculations. To assess the impact of pneumococcal serine proteases on acute infection, we infected mice intranasally with bioluminescent and invasive TIGR4 or isogenic triple mutants expressing only CbpG, HtrA, PrtA, or SFP. We imaged the acute lung infection in real-time and determined the survival of the mice. The TIGR4lux mutant expressing only PrtA showed a significant attenuation and was less virulent in the acute pneumonia model. In conclusion, our results showed that pneumococcal serine proteases contributed significantly to pneumococcal colonization but played only a minor role in pneumonia and invasive diseases. Because colonization is a prerequisite for invasive diseases and transmission, these enzymes could be promising candidates for the development of antimicrobials to reduce pneumococcal transmission.

Brucella: Reservoirs and Niches in Animals and Humans.

Brucella is an intracellular bacterium that causes abortion, reproduction failure in livestock and leads to a debilitating flu-like illness with serious chronic complications if untreated in humans. As a successful intracellular pathogen, Brucella has developed strategies to avoid recognition by the immune system of the host and promote its survival and replication. In vivo, Brucellae reside mostly within phagocytes and other cells including trophoblasts, where they establish a preferred replicative niche inside the endoplasmic reticulum. This process is central as it gives Brucella the ability to maintain replicating-surviving cycles for long periods of time, even at low bacterial numbers, in its cellular niches. In this review, we propose that Brucella takes advantage of the environment provided by the cellular niches in which it resides to generate reservoirs and disseminate to other organs. We will discuss how the favored cellular niches for Brucella infection in the host give rise to anatomical reservoirs that may lead to chronic infections or persistence in asymptomatic subjects, and which may be considered as a threat for further contamination. A special emphasis will be put on bone marrow, lymph nodes, reproductive and for the first time adipose tissues, as well as wildlife reservoirs.

In vitro performance and in vivo fertility of antibiotic-free preserved boar semen stored at 5\u2009\xb0C

BackgroundHypothermic preservation of boar semen is considered a potential method for omitting antibiotics from insemination doses, thereby contributing to the global antibiotic resistance defence strategy. The main challenges are chilling injury to spermatozoa and bacterial growth during semen storage leading to reduced fertility.ObjectivesTo examine chilling injury and the number and type of bacteria in boar semen stored at 5 °C in the absence of antibiotics, and to assess the applicability of hypothermic semen storage under field conditions.Material and methodsBoar ejaculates were extended with AndroStar® Premium, stored at 17 °C with and at 5 °C without antibiotics and tested for functional sperm parameters by flow cytometry. Raw semen and extended samples were investigated bacteriologically. Fertility was evaluated after once-daily inseminations of 194 sows in a field study.ResultsLethal sperm damage assessed by motility and membrane integrity was low throughout storage in both experimental groups. Sublethal chilling effects based on the decrease of viable spermatozoa with low membrane fluidity were higher (P < 0.05) up until 72 h in sperm stored at 5 °C compared to 17 °C but did not differ after 144 h. After 72 h, incubation in capacitating medium for 60 min induced a similar decrease in viable sperm with high mitochondria membrane potential and low cytosolic calcium in both groups. In semen stored at 5 °C, bacteria counts were below 103 CFU/mL and the bacteria spectrum was similar to that of raw semen. In 88% of 34 boars, cooled semen fulfilled the requirements for insemination. Fertility was high and did not differ (P > 0.05) between sow groups inseminated with semen stored antibiotic-free at 5 °C and semen stored at 17 °C with antibiotics.ConclusionDespite subtle chilling effects and low bacterial numbers, antibiotic-free hypothermic storage of boar semen offers the possibility to reduce the use of antibiotics in pig insemination. However, strict sanitary guidelines must be maintained and further evidence of efficiency under field conditions is considered desirable.

Vibrational Spectroscopy as a Sensitive Probe for the Chemistry of Intra-Phase Bacterial Growth.

Bacterial growth in batch cultures occurs in four phases (lag, exponential/log, stationary and death phase) that differ distinctly in number of different bacteria, biochemistry and physiology. Knowledge regarding the growth phase and its kinetics is essential for bacterial research, especially in taxonomic identification and monitoring drug interactions. However, the conventional methods by which to assess microbial growth are based only on cell counting or optical density, without any insight into the biochemistry of cells or processes. Both Raman and Fourier transform infrared (FTIR) spectroscopy have shown potential to determine the chemical changes occurring between different bacterial growth phases. Here, we extend the application of spectroscopy and for the first time combine both Raman and FTIR microscopy in a multimodal approach to detect changes in the chemical compositions of bacteria within the same phase (intra-phase). We found a number of spectral markers associated with nucleic acids (IR: 964, 1082, 1215 cm−1; RS: 785, 1483 cm−1), carbohydrates (IR: 1035 cm−1; RS: 1047 cm−1) and proteins (1394 cm−1, amide II) reflecting not only inter-, but also intra-phase changes in bacterial chemistry. Principal component analysis performed simultaneously on FTIR and Raman spectra enabled a clear-cut, time-dependent discrimination between intra-lag phase bacteria probed every 30 min. This demonstrates the unique capability of multimodal vibrational spectroscopy to probe the chemistry of bacterial growth even at the intra-phase level, which is particularly important for the lag phase, where low bacterial numbers limit conventional analytical approaches.

Zur-regulated lipoprotein A contributes to the fitness of Acinetobacter baumannii.

Acinetobacter baumannii is a notorious nosocomial pathogen that commonly infects severely ill patients. Zinc (Zn) is essential to survive and adapt to different environment and host niches in A. baumannii. Of the Zinc uptake regulator (Zur)-regulated genes in A. baumannii, the A1S_3412 gene encoding a Zur-regulated lipoprotein A (ZrlA) is critical for cell envelope integrity and overcoming antibiotic exposure. This study investigated whether ZrlA contributes to the fitness of A. baumannii in vitro and in vivo using the wildtype A. baumannii ATCC 17978, ΔzrlA mutant, and zrlAcomplemented strains. The ΔzrlA mutant showed reduced biofilm formation, surface motility, and adherence to and invasion of epithelial cells compared to the wild-type strain. In a mouse pneumonia model, the ?zrlA mutant showed significantly lower bacterial numbers in the blood than the wildtype strain. These virulence traits were restored in the zrlAcomplemented strain. Under static conditions, the expression of csuCDE, which are involved in the chaperone-usher pili assembly system, was significantly lower in the ΔzrlA mutant than in the wild-type strain. Moreover, the expression of the bfmR/S genes, which regulate the CsuA/BABCDE system, was significantly lower in the ΔzrlA mutant under static conditions than in the wild-type strain. Our results indicate that the zrlA gene plays a role in the fitness of A. baumannii by regulating the BfmR/S two-component system and subsequently the CsuA/BABCDE chaperone-usher pili assembly system, suggesting it as a potential target for anti-virulence strategies against A. baumannii.

Isolation of bacteria at different points of Pleurotus ostreatus cultivation and their influence in mycelial growth

Pleurotus ostreatus is one of the most cultivated edible mushrooms worldwide and few approaches have been done to analyze bacterial influence during its cultivation. Therefore, bacteria from commercial spawn, mycelial-colonized straw and fruiting bodies from healthy productive samples were counted, isolated and tested for their mycelial growth promoting ability. Bacterial cell numbers at different steps of the process showed low bacterial cell numbers in spawn and in fruiting bodies inner tissue compared to the high concentration in mycelial-colonized straw. The majority of the 38 isolates belonged to phyla Firmicutes and Actinobacteria were identified as Bacillus, Paenibacillus and Micromonospora species. Similarly, 16S rRNA gene bacterial clones obtained from mycelial biomass DNA samples showed bacterial presence of various genera including Bacillus and Paenibacillus.In the mycelial growth promoting ability tests, 30 isolates negatively affected mycelial growth, two isolates showed no effect on mycelial growth, and six isolates promoted mycelial growth. Moreover, mycelial thickness was influenced in different ways by the bacterial growth. In general, nearly all isolates growth-preventing were isolated from healthy spawn and mycelial-colonized straw, whereas fruiting bodies were the best source for isolation of mycelial growth-promoting bacteria. Characterization of bacterial isolates revealed that growth-preventing isolates exhibited various enzymatic activities in comparison with positive influencing bacteria that exhibited none or weak enzymatic activities. In addition, the influence of volatile compounds being present in the headspace of bi-plate co-cultures on P. ostreatus mycelial growth was demonstrated.The effect of isolates, that promoted mycelial growth in co-cultivation, to reduce P. ostreatus spawn running time, was evaluated on sterilized rye seeds. Results showed that not all mycelial promoted isolates were able to significantly promote P. ostreatus colonization. However, isolate M46F identified as Micromonospora lupini significantly reduce spawn running time. This is one of few studies to estimate cultivable bacteria from healthy samples of P. ostreatus cultivation, to evaluate a bacterial effect on mycelial growth, to show that fruiting bodies are a good source for mycelia growth-promoting isolates, and the first to report a shorter P. ostreatus spawn running time due to bacterial inoculation.

Improving Proteome Coverage for Small Sample Amounts: An Advanced Method for Proteomics Approaches with Low Bacterial Cell Numbers

Proteome analyses are often hampered by the low amount of available starting material like a low bacterial cell number obtained from in vivo settings. Here, the single pot solid-phase enhanced sample preparation (SP3) protocol is adapted and combined with effective cell disruption using detergents for the proteome analysis of bacteria available in limited numbers only. Using this optimized protocol, identification of peptides and proteins for different Gram-positive and Gram-negative species can be dramatically increased and, reliable quantification can also be ensured. This adapted method is compared to already established strain-specific sample processing protocols for Staphylococcus aureus, Streptococcus suis, and Legionella pneumophila. The highest species-specific increase in identifications is observed using the adapted method with L. pneumophila samples by increasing protein and peptide identifications up to 300% and 620%, respectively. This increase is accompanied by an improvement in reproducibility of protein quantification and data completeness between replicates. Thus, this protocol is of interest for performing comprehensive proteomics analyses of low bacterial cell numbers from different settings ranging from infection assays to environmental samples.

Selection of Borrelia burgdorferi Promoter Sequences Active During Mammalian Infection Using In Vivo Expression Technology.

In vivo expression technology (IVET) has been applied to a variety of organisms to identify active promoters in specific environments or growth conditions of interest. Here, we describe modifications to employ this genome-wide screening method for Borrelia burgdorferi, the Lyme disease spirochete, during an active murine infection. Utilization of this technique provides valuable insights into the B. burgdorferi transcriptome during infection, despite the low bacterial numbers in the mammalian host environment.

Self-contained chlorine dioxide generation and delivery pods for decontamination of floor drainsce:

SUMMARY Listeria monocytogenes can be present in poultry processing and further processing plants, where it can colonize floor drains. This is a concern as the potential exists for cross-contamination of product contact surfaces or even fully cooked product. Self-contained chlorine dioxide-generating and delivery pods were tested as a means to sanitize uninoculated floor drains and L. monocytogenes-inoculated model floor drains. Free-swimming and attached bacteria were enumerated in drains. Standing water and the inner surface of treated drains had significantly lower bacterial numbers than did untreated drains. Listeria monocytogenes numbers decreased by up to 8.7 log cfu/mL in standing water (>99.999999%) and 6.7 log cfu (>99.9999%) as attached cells. Commercially available chlorine dioxide pods may have practical utility for killing L. monocytogenes during periodic sanitization of floor drains in poultry processing facilities.

Feline leprosy due to Mycobacterium lepraemurium.

This paper, the second in a series of three on 'feline leprosy', provides a detailed description of disease referable to Mycobacterium lepraemurium, the most common cause of feline leprosy worldwide. Cases were sourced retrospectively and prospectively for this observational study, describing clinical, geographical and molecular microbiological data for cats definitively diagnosed with M lepraemurium infection. A total of 145 cases of feline leprosy were scrutinised; 114 'new' cases were sourced from the Victorian Infectious Diseases Reference Laboratory records, veterinary pathology laboratories or veterinarians, and 31 cases were derived from six published studies. Sixty-five cats were definitively diagnosed with M lepraemurium infection. Typically, cats were 1-3 years of age when first infected, with a male gender predilection. Affected cats were generally systemically well. All had outdoor access. Lesions tended to consist of one or more cutaneous/subcutaneous nodules, typically located on the head and/or forelimbs, possibly reflecting the most likely locations for a rodent bite as the site of inoculation for organisms. Nodules had the propensity to ulcerate at some stage in the clinical course. The cytological and histological picture varied from tuberculoid, with relatively low bacterial numbers, to lepromatous with moderate to high bacterial numbers. Treatment was varied, although most cats underwent surgical resection of lesions with adjunctive medical therapy, most often using a combination of oral clarithromycin and rifampicin. Prognosis for recovery was generally good, and in two cases there was spontaneous remission without the requirement for medical intervention. Untreated cats continued to enjoy an acceptable quality of life despite persistence of the disease, which extended locally but had no apparent tendency to disseminate to internal organs. M lepraemurium causes high bacterial index (lepromatous) or low bacterial index (tuberculoid) feline leprosy. The infection typically causes nodules of the skin and/or subcutis (which tend towards ulceration) on the head and/or forelimbs. The disease usually has an indolent clinical course and infected cats have a generally favourable response to therapeutic interventions, with rare cases undergoing spontaneous resolution. Genomic analysis may yield clues as to the environmental niche and culture requirements of this elusive organism. Prospective treatment trials and/or additional drug susceptibility testing in specialised systems would further inform treatment recommendations.

Enhanced control of Mycobacterium tuberculosis extrapulmonary dissemination in mice by an arabinomannan-protein conjugate vaccine.

Currently there are a dozen or so of new vaccine candidates in clinical trials for prevention of tuberculosis (TB) and each formulation attempts to elicit protection by enhancement of cell-mediated immunity (CMI). In contrast, most approved vaccines against other bacterial pathogens are believed to mediate protection by eliciting antibody responses. However, it has been difficult to apply this formula to TB because of the difficulty in reliably eliciting protective antibodies. Here, we developed capsular polysaccharide conjugates by linking mycobacterial capsular arabinomannan (AM) to either Mtb Ag85b or B. anthracis protective antigen (PA). Further, we studied their immunogenicity by ELISA and AM glycan microarrays and protection efficacy in mice. Immunization with either Abg85b-AM or PA-AM conjugates elicited an AM-specific antibody response in mice. AM binding antibodies stimulated transcriptional changes in Mtb. Sera from AM conjugate immunized mice reacted against a broad spectrum of AM structural variants and specifically recognized arabinan fragments. Conjugate vaccine immunized mice infected with Mtb had lower bacterial numbers in lungs and spleen, and lived longer than control mice. These findings provide additional evidence that humoral immunity can contribute to protection against Mtb.

Active bacteriophage biocontrol and therapy on sub-millimeter scales towards removal of unwanted bacteria from foods and microbiomes.

Bacteriophages can be used as antibacterial agents as a form of biological control, e.g., such as phage therapy. With active treatment, phages must “actively” produce new virions, in situ, to attain “inundative” densities, i.e., sufficient titers to eradicate bacteria over reasonable timeframes. Passive treatment, by contrast, can be accomplished using phages that are bactericidal but incapable of generating new phage virions in situ during their interaction with target bacteria. These ideas of active versus passive treatment come from theoretical considerations of phage therapy pharmacology, particularly as developed in terms of phage application to well-mixed cultures consisting of physically unassociated bacteria. Here I extend these concepts to bacteria which instead are physically associated. These are bacteria as found making up cellular arrangements or bacterial microcolonies—collectively, clonal bacterial “clumps”. I consider circumstances where active phage replication would be required to effect desired levels of bacterial clearance, but populations of bacteria nevertheless are insufficiently prevalent to support phage replication to bacteria-inundative densities across environments. Clumped bacteria, however, may still support active treatment at more local, i.e., sub-millimeter, within-clump spatial scales, and potential consequences of this are explored mathematically. Application is to the post-harvest biocontrol of foodborne pathogens, and potentially also to precise microbiome editing. Adequate infection performance by phages in terms of timely burst sizes, that is, other than just adsorption rates and bactericidal activity, thus could be important for treatment effectiveness even if bacterial densities overall are insufficient to support active treatment across environments. Poor phage replication during treatment of even low bacterial numbers, such as given food refrigeration during treatment, consequently could be problematic to biocontrol success. In practical terms, this means that the characterization of phages for such purposes should include their potential to generate new virions under realistic in situ conditions across a diversity of potential bacterial targets.

Experimental infection of chickens by a flagellated motile strain of Salmonella enterica serovar Gallinarum biovar Gallinarum

Salmonella enterica subsp. enterica serovar Gallinarum biovar Gallinarum (SG) causes fowl typhoid (FT), a septicaemic disease which can result in high mortality in poultry flocks. The absence of flagella in SG is thought to favour systemic invasion, since bacterial recognition via Toll-like receptor (TLR)-5 does not take place during the early stages of FT. In the present study, chicks susceptible to FT were inoculated with a wild type SG (SG) or its flagellated motile derivative (SG Fla+). In experiment 1, mortality and clinical signs were assessed, whereas in experiment 2, gross pathology, histopathology, systemic invasion and immune responses were evaluated. SG Fla+ infection resulted in later development of clinical signs, lower mortality, lower bacterial numbers in the liver and spleen, and less severe pathological changes compared to SG. The CD8+ T lymphocyte population was higher in the livers of chicks infected with SG at 4 days post-inoculation (dpi). Chicks infected with SG had increased expression of interleukin (IL)-6 mRNA in the caecal tonsil at 1 dpi and increased expression of IL-18 mRNA in the spleen at 4 dpi. In contrast, the CD4+ T lymphocyte population was higher at 6 dpi in the livers of birds infected with SG Fla+. Therefore, flagella appeared to modulate the chicken immune response towards a CD4+ T profile, resulting in more efficient bacterial clearance from systemic sites and milder infection.

A novel molecule purified from Staphylococcus aureus mutant produces γδ T cell-mediated IL-17A in mice peritoneum and protects MRSA-derived skin abscess expansion

Abstract Staphylococcus aureus, a commensal bacterium in human, causes a wide range of human diseases from soft tissue infections to lethal systemic infections. But, immune-competent people are highly resistant to these diseases, indicating that unknown host defense response(s) can protect host. An effective immune response to S. aureus requires a rapid activation of innate immunity in the shape of γδ-T cells mediated IL-17 production for bacterial clearance. We hypothesized that an unidentified staphylococcal-cell wall derived molecule(s) that can activate host innate γδ-T cells is generated after S. aureus infection in healthy people. To purify this molecule(s) in mice, we generated 11 different S. aureus mutant strains, in which genes involved in staphylococcal cell wall components bio-syntheses were mutated. The mutant cells were acetone-treated to inactivate and extract toxic molecules, and then injected intra-peritoneal cavity of mice. When peritoneal lavage fluid collected 5 hr after injection was examined which mutant bacteria can produce IL-17A, three mutants produced IL-17A. To purify active fractions capable of γδ T cell-mediated IL-17A production, acetone-treated mutant bacterium was solubilized with two different peptidoglycan hydrolyzing enzymes. Then, active molecules were purified to homogeneity on ion-exchange columns. By pre-injection of purified molecule or acetone-treated mutant bacteria, the expansion of USA 300 MRSA-mediated skin abscess was inhibited, leading to a lower bacterial numbers and decrease of abscess lesion size at the site of infection. These results suggest that a novel molecule producing γδ T cell-derived IL-17A in peritoneum protected MRSA-derived skin abscess expansion in mice.

Liquid crystals as optical amplifiers for bacterial detection

Interactions of bacteria with target molecules (e.g. antibiotics) or other microorganisms are of growing interest. The first barrier for targeting gram-negative bacteria is layer of a Lipopolysaccharides (LPS). Liquid crystal (LC) based sensors covered with LPS monolayers, as presented in this study, offer a simple model to study and make use of this type of interface for detection and screening. This work describes in detail the production and application of such sensors based on three different LPS that have been investigated regarding their potential to serve as sensing layer to detect bacteria. The LPS O127:B8 in combination with a LC based sensor was identified to be most useful as biomimetic sensing surface. This LPS/LC combination interacts with three different bacteria species, one gram-positive and two gram-negative species, allowing the detection of bacterial presence regardless from their viability. It could be shown that even very low bacterial cell numbers (minimum 500 cellml−1) could be detected within minutes (maximum 15min). The readout mechanism is the adsorption of bacterial entities on surface bond LPS molecules with the LC serving as an optical amplifier.

Aspirin plus ticlopidine prevented experimental endocarditis due to Enterococcus faecalis and Streptococcus gallolyticus.

Enterococcus faecalis and Streptococcus gallolyticus cause infective endocarditis (IE), which can originate from the continuous release or translocation of low bacterial numbers into the bloodstream. In this context, IE cannot be prevented with antibiotics. We previously demonstrated that aspirin plus ticlopidine protected rats from IE due to S. gordonii and Staphylococcus aureus. Here we showed that aspirin plus ticlopidine significantly reduced vegetation weight and protected 73 and 64% rats (P < 0.005) from IE due to E. faecalis and S. gallolyticus, respectively. These results further support the potential use of aspirin plus ticlopidine for a global prevention of IE in high-risk patients.