Characteristics of microbial drug resistance and its correlates in chronic diabetic foot ulcer infections

While virulence factors and the biofilm-forming capabilities of microbes are the key regulators of the wound healing process, the host immune response may also contribute in the events following wound closure or exacerbation of non-closure. We examined samples from diabetic and non-diabetic foot ulcers/wounds for microbial association and tested the microbes for their antibiotic susceptibility and ability to produce biofilms. A total of 1074 bacterial strains were obtained with staphylococci, Pseudomonas, Citrobacter and enterococci as major colonizers in diabetic samples. Though non-diabetic samples had a similar assemblage, the frequency of occurrence of different groups of bacteria was different. Gram-negative bacteria were found to be more prevalent in the diabetic wound environment while Gram-positive bacteria were predominant in non-diabetic ulcers. A higher frequency of monomicrobial infection was observed in samples from non-diabetic individuals when compared to samples from diabetic patients. The prevalence of different groups of bacteria varied when the samples were stratified according to age and sex of the individuals. Several multidrug-resistant strains were observed among the samples tested and most of these strains produced moderate to high levels of biofilms. The weakened immune response in diabetic individuals and synergism among pathogenic micro-organisms may be the critical factors that determine the delicate balance of the wound healing process.

Coliforms in processed mango: Significance and control

The aims of this investigation were to enumerate coliforms in fresh mangoes, puree, cheeks, and cheeks-in-puree in order to determine the source of these organisms in the processed products, to determine methods for their control, and to identify coliforms isolated from cheeks-in-puree to determine whether they have any public health significance. Product from four processors was tested on two occasions. The retail packs of cheeks-in-puree having the highest coliform counts were those in which raw puree was added to the cheeks. Coliform counts in these samples ranged between 1.4 × 103 and 5.4 × 104 cfu/g. Pasteurisation reduced the coliform count of raw puree to < 5 cfu/g. Forty-seven percent of the 73 colonies, isolated as coliforms on the basis of their colony morphology on violet red bile agar, were identified as Klebsiella pneumoniae using the ATB 32E Identification System. Klebsiella strains were tested for growth at 10 °C, faecal coliform response, and fermentation of -melizitose, to differentiate the three phenotypically similar strains, K. pneumoniae, K. terrigena and K planticola. Results indicated that 41% of K. pneumoniae isolates gave reactions typical of K. pneumoniae. A further 44% of strains gave an atypical reaction pattern for these tests and were designated ‘psychrotrophic’ K. pneumoniae. Klebsiella pneumoniae counts of between 2.1 × 103 and 4.9 × 104 cfu/g were predicted to occur in the retail packs of mango cheeks-in-puree produced by the processors who constituted this product with raw puree. In view of the opportunistic pathogenic nature of K. pneumoniae, its presence in these products is considered undesirable and steps, such as pasteurisation of puree, should be taken in order to inactivate it

Induced Resistance: Potential for Control of Postharvest Diseases of Horticultural Crops

Fortunately, plants have developed highly effective mechanisms with which to defend themselves when attacked by potentially disease-causing microorganisms. If not, then they would succumb to the many pathogenic fungi, bacteria, viruses, nematodes and insect pests, and disease would prevail. These natural defence systems of plants can be deliberately activated to provide some protection against the major pathogens responsible for causing severe yield losses in agricultural and horticultural crops. This is the basis of what is known as ‘induced’ or ‘acquired’ disease resistance in plants. Although the phenomenon of induced resistance has been known amongst plant pathologists for over 100 years, its inclusion into pest and disease management programmes has been a relatively recent development, ie. within the last 5 years. This review will discuss very briefly some of the characteristics of the induced resistance phenomenon, outline some of the advantages and limitations to its implementation and provide some examples within a postharvest pathology context. Finally some approaches being investigated by the fruit pathology team at DPI Indooroopilly and collaborators will be outlined.

sopB of Salmonella enterica serovar Typhimurium is a potential DNA vaccine candidate in conjugation with live attenuated bacteria

The immune response against Salmonella is multi-faceted involving both the innate and the adaptive immune system. The characterization of specific Salmonella antigens inducing immune response could critically contribute to the development of epitope based vaccines for Salmonella. We have tried to identify a protective T cell epitope(s) of Salmonella, as cell mediated immunity conferred by CD8+ T cells is the most crucial subset conferring protective immunity against Salmonella. It being a proven fact that secreted proteins are better in inducing cell mediated immunity than cell surface and cytosolic antigens, we have analyzed all the genbank annotated Salmonella pathogenicity island 1 and 2 secreted proteins of Salmonella enterica serovar Typhimurium (S. typhimurium) and S. enterica serovar Typhi (S. typhi). They were subjected to BIMAS and SYFPEITHI analysis to map MHC-I and MHC-II binding epitopes. The huge profile of possible T cell epitopes obtained from the two classes of secreted proteins were tabulated and using a scoring system that considers the binding affinity and promiscuity of binding to more than one allele, SopB and SifB were chosen for experimental confirmation in murine immunization model. The entire SopB and SifB genes were cloned into DNA vaccine vectors and were administered along with live attenuated Salmonella and it was found that SopB vaccination reduced the bacterial burden of organs by about 5-fold on day 4 and day 8 after challenge with virulent Salmonella and proved to be a more efficient vaccination strategy than live attenuated bacteria alone.

Quantitative assessment of total and Gram-positive aerobic bacteria in fresh and ambient-temperature-stored sub-tropical marine fish

The current study was undertaken to enumerate Gram-positive bacteria in fresh sub-tropical marine fish and determine the effect of ambient storage (25°C) on the Gram-positive bacterial count. Total and Gram-positive bacteria were enumerated in the muscles, gills and gut of fresh and stored Pseudocaranx dentex, Pagrus auratus and Mugil cephalus on tryptone soya agar (TSA) and TSA with 0.25% phenylethyl alcohol (PEA), respectively. Initial studies indicated that PEA significantly reduced total aerobic bacterial count (TABC) whereas control Gram-positive bacteria were not affected by 0.25% PEA. TABC significantly increased in all fish body parts, whereas Gram-positive aerobic bacterial count (GABC) significantly increased only in the muscles and gills during ambient storage for 15 h. The TABC of the fish species increased from 4.00, 6.13 and 4.58 log cfu g-1, respectively in the muscles, gills, and gut to 6.31, 7.31 and 7.23 log cfu g-1 by the end of storage. GABC increased from 2.00, 3.52 and 2.20 log cfu g-1 to 4.70, 5.85 and 3.36 log cfu g-1. Within each species, TABC were significantly higher in the gills compared to that of muscles and gut; however, no significant differences were found in GABC between muscles and gills. This study demonstrated the potential importance of Gram-positive bacteria in sub-tropical marine fish and their spoilage.

Identification and characterization of non-pathogenic Fusarium oxysporum capable of increasing and decreasing Fusarium wilt severity

Fusarium wilt of banana is a potentially devastating disease throughout the world. Options for control of the causal organism, Fusarium oxysporum f.sp. cubense (Foc) are limited. Suppressive soil sites have previously been identified where, despite the presence of Foc, Fusarium wilt does not develop. In order to understand some aspects of this disease suppression, endophytic Fusarium oxysporum isolates were obtained from banana roots. These isolates were genetically characterized and compared with an isolate of Fusarium oxysporum previously identified as being capable of suppressing Fusarium wilt of banana in glasshouse trials. Three additional isolates were selected for glasshouse trials to assess suppression of Fusarium wilt in two different cultivars of banana, Cavendish and Lady Finger. One isolate (BRIP 29089) was identified as a potential biocontrol organism, reducing the disease severity of Fusarium wilt in Lady Finger and Cavendish cultivars. Interestingly, one isolate (BRIP 45952) increased Fusarium wilt disease severity on Cavendish. The implications of an isolate of Fusarium oxysporum, non-pathogenic on banana, increasing disease severity and the potential role of non-pathogenic isolates of Fusarium oxysporum in disease complexes are discussed.

Rapid and sensitive detection of Avibacterium paragallinarum in the presence of other bacteria using a 5' Taq nuclease assay: A new tool for diagnosing infectious coryza

A 5' Taq nuclease assay specific for Avibacterium paragallinarum was designed and optimized for use in diagnosing infectious coryza. The region chosen for assay design was one of known specificity for Av. paragallinarum. The assay detected Av. paragallinarum reference strains representing the three Page and the eight Kume serovars, and field isolates from diverse geographical locations. No cross-reactions were observed with other Avibacterium species, with other bacteria taxonomically related to Av. paragallinarum nor with bacteria and viruses likely to be present in swabs collected from suspected infectious coryza cases. The detection limit for the assay was 6 to 60 colony-forming units per reaction. Twenty-two out of 53 swabs collected from sick birds reacted in the 5' Taq nuclease assay, whereas Av. paragallinarum was not isolated from any of the swabs. All of the 22 swabs yielded other bacteria in culture. The presence of Av. paragallinarum in the swabs was also demonstrated by sequencing, thereby confirming the ability of the assay to detect Av. paragallinarum in the presence of other bacteria. The ability to quantify bacterial load in the swabs using the 5' Taq nuclease assay was demonstrated.

Fluorescence in situ hybridization analysis of hindgut bacteria associated with the development of equine laminitis

In the article 'Fluorescence in situ hybridization analysis of hindgut bacteria associated with the development of equine laminitis' (Milinovich et al., 2007), it is found that with reference to Horse 1, the histological signs of laminitis were first observed at 12 h post-oligofructose administration, and not 30 h as was indicated in the Results section under the subheading 'Induction of Laminitis' and in Fig. 1.

Hydrogen utilising bacteria from the forestomach of eastern grey (Macropus giganteus) and red (Macropus rufus) kangaroos

Reductive acetogenesis is an alternative to methanogenesis for removing hydrogen produced during enteric fermentation. In Australia, kangaroos have evolved an enlarged forestomach analogous to the rumen of sheep and cattle. However, unlike sheep and cattle, kangaroos produce very little methane from enteric fermentation. From samples of gut contents from five eastern grey and three red kangaroos, we were not able to detect methanogens using a PCR protocol, but did detect the formyltetrahydrofolate synthetase (FTHFS) gene (likely to be used for reductive acetogenesis) in all animals. Isolations to recover acetogens resulted in two different classes of hydrogen consuming bacteria being isolated. The first class consisted of acetogens that possessed the FTHFS gene, which except for Clostridium glycolicum, were not closely related to any previously cultured bacteria. The second class were not acetogens but consisted of enterobacteria (Escherichia coli and Shigella) that did not possess FTHFS genes but did utilise hydrogen and produce acetate. Enumeration of the acetogens containing the FTHFS gene by real-time PCR indicated that bacteria of the taxa designated YE257 were common to all the kangaroos whereas YE266/YE273 were only detected in eastern grey kangaroos. When present, both species occurred at densities above *106 cell equivalents per mL. C. glycolicum was not detected in the kangaroos and, unlike YE257 and YE266/273, is unlikely to play a major role in reductive acetogenesis in the foregut of kangaroos.

In vitro detection and primary cultivation of bacteria producing materials inhibitory to ruminal methanogens.

A novel method for screening bacterial isolates for their potential to inhibit the growth of ruminal methanogenic Archaea was developed using a modification of the soft agar overlay technique, formally used for the isolation of lytic bacteriophages. This method may be used in the specific, hydrogen-rich conditions required for the growth of ruminal methanogenic Archaea.

Speciation of Gram-positive bacteria in fresh and ambient-stored sub-tropical marine fish.

This study identified Gram-positive bacteria in three sub-tropical marine fish species: Pseudocaranx dentex (silver trevally), Pagrus auratus (snapper) and Mugil cephalus (sea mullet). It further elucidated the role played by fish habitat, fish body part and ambient storage on the composition of the Gram-positive bacteria. A total of 266 isolates of Gram-positive bacteria were identified by conventional biochemical methods, VITEK, PCR using genus- and species-specific primers and/or 16S rRNA gene sequencing. The isolates were found to fall into 13 genera and 30 species. In fresh fish, Staphylococcus epidermidis and Micrococcus luteus were the most frequent isolates. After ambient storage, S. epidermidis, S. xylosus and Bacillus megaterium were no longer present whereas S. warned, B. sphaericus, Brevibacillus borstelensis, Enterococcus faecium and Streptococcus uberis increased in frequency. Micrococcus luteus and S. warned were the most prevalent isolates from P. dentex, while E. faecium and Strep. uberis were the most frequent isolates from P. auratus and M. cephalus. With respect to different parts of the fish body. E. faecium, Strep. uberis and B. sphaericus were the most frequent isolates from the muscles, E. faecium, Strep. uberis from the gills and M. luteus from the gut. This study showed a diversity of Gram-positive bacteria in sub-tropical marine fish; however, their abundance was affected by fish habitat, fish body part and ambient storage.

Controlled exposure as a management tool for Glasser's disease.

In this study, nasal swabs taken from multiparous sows at weaning time or from sick pigs displaying symptoms of Glasser's disease from farms in Australia [date not given] were cultured and analysed by polymerase chain reaction (PCR). Within each genotype detected on a farm, representative isolates were serotyped by gel diffusion (GD) testing or indirect haemagglutination (IHA) test. Isolates which did not react in any of the tests were regarded as non-typable and were termed serovar NT. Serovars 1, 5, 12, 13 and 14 were classified as highly pathogenic; serovars 2, 4 and 15 being moderately pathogenic; serovar 8 being slightly pathogenic and serovars 3, 6, 7, 9 and 11 being non-pathogenic. Sows were inoculated with the strain of Haemophilus parasuis (serovars 4, 6 and 9 from Farms 1, 2 and 4, respectively) used for controlled challenge 3 and 5 weeks before farrowing. Before farrowing the sows were divided into control and treatment groups. Five to seven days after birth, the piglets of the treatment group were challenged with a strain from the farm which had were used to vaccinate the sows. The effectiveness of the controlled exposure was evaluated by number of piglets displaying clinical signs possibly related to infection, number of antibiotic treatments and pig mortality. Nasal swabs of sick pigs were taken twice a week to find a correlation to infection. A subsample of pigs was weighed after leaving the weaning sheds. The specificity of a realtime PCR amplifying the infB gene was evaluated with 68 H. parasuis isolates and 36 strains of closely related species. 239 samples of DNA from tissues and fluids of 16 experimentally challenged animals were also tested with the realtime PCR, and the results compared with culture and a conventional PCR. The farm experiments showed that none of the controlled challenge pigs showed any signs of illness due to Glasser's disease, although the treatment groups required more antibiotics than the controls. A total of 556 H. parasuis isolates were genotyped, while 150 isolates were serotyped. H. parasuis was detected on 19 of 20 farms, including 2 farms with an extensive history of freedom from Glasser's disease. Isolates belonging to serovars regarded as potentially pathogenic were obtained from healthy pigs at weaning on 8 of the 10 farms with a history of Glasser's disease outbreaks. Sampling 213 sick pigs yielded 115 isolates, 99 of which belonged to serovars that were either potentially pathogenic or of unknown pathogenicity. Only 16 isolates from these sick pigs were of a serovar known to be non-pathogenic. Healthy pigs also had H. parasuis, even on farms free of Glasser's disease. The realtime PCR gave positive results for all 68 H. parasuis isolates and negative results for all 36 non-target bacteria. When used on the clinical material from experimental infections, the realtime PCR produced significantly more positive results than the conventional PCR (165 compared to 86).

Immunomodulatory effects of probiotic bacteria in healthy adults

Probiooteilla kantakohtaisia vaikutuksia ihmisen immuunijärjestelmään terveillä aikuisilla Probiooteilla on kantakohtaisia tulehduksen välittäjäaineita vähentäviä vaikutuksia ja probioottien yhdistelmien vaikutukset eroavat yksittäisten kantojen vaikutuksista selviää TtM Riina Kekkosen tuoreesta väitöstutkimuksesta. TtM Riina Kekkonen on selvittänyt väitöskirjassaan eri probioottikantojen vaikutuksia immuunivasteeseen valkosolumallissa sekä terveillä aikuisilla lumekontrolloiduissa kliinisissä tutkimuksissa. Aikaisemmin probioottien vaikutuksia on tutkittu lähinnä allergian ja erilaisten vatsavaivojen ehkäisyssä ja hoidossa. Probiootteja sisältäviä tuotteita käyttävät kuluttajat ovat kuitenkin useimmiten terveitä aikuisia, ja probioottien vaikutus terveiden aikuisten immuunijärjestelmään on ollut puutteellisesti selvitettyä. Valkosolumallissa probioottikantojen havaittiin poikkeavan toisistaan niiden kyvyssä aktivoida immuunivasteen välittäjäaineiden, sytokiinien, tuotantoa. Anti-inflammatorisia, eli tulehdusta lievittäviä vaikutuksia nähtiin lähinnä Bifidobacterium ja Propionibacterium sukuihin kuuluvilla kannoilla. Streptococcus ja Leuconostoc sukuihin kuuluvat kannat puolestaan aktivoivat Th1 tyyppistä, soluvälitteistä immuunivastetta. Eri probioottien kombinaatiot eivät saaneet aikaan voimakkaampaa aktivaatiota yksittäisiin kantoihin verrattuna, joka viittaa probioottien keskinäiseen kilpailuun niiden ollessa kontaktissa ihmisen solujen kanssa. Probioottikantojen valinta kliinisiin tutkimuksiin tehtiin niiden anti-inflammatoristen ominaisuuksien perusteella. Parhaita anti-inflammatorisia kantoja olivat B. lactis ssp. animalis Bb12 ja P. freudenreichii ssp. shermanii JS, joiden lisäksi tutkimuksiin valittiin myös L. rhamnosus GG (LGG) hyvin tutkittuna referenssikantana. Solutöiden tulokset eivät olleet täysin verrannollisia kliinisen työn tuloksiin, koska LGG näytti omaavan parhaat anti-inflammatoriset ominaisuudet kliinisissä tutkimuksissa vaikka solutyössä sen aikaansaamat vasteet olivat melko vaimeita. Kolmen viikon kliinisessä tutkimuksessa terveillä aikuisilla LGG alensi mm. tulehdusta kuvaavan C-reaktiivisen proteiinin ja inflammatoristen sytokiinien määrää. Pidemmässä kolmen kuukauden pituisessa kliinisessä tutkimuksessa LGG:llä ei ollut vaikutusta terveiden aikuisten infektiosairastavuuteen, mutta LGG lyhensi vatsavaivojen kestoa. Probioottien vaikutukset immuunijärjestelmään näyttävät olevan kantakohtaisia ja erityisesti Lactobacillus rhamnosus GG:llä havaittiin anti-inflammatorisia vaikutuksia. Valkosolumallia ei tulisi käyttää ainoana probioottikantojen skriinausmenetelmänä niiden immunologisia vaikutuksia selvitettäessä, koska solutöiden tulokset eivät olleet täysin verrannollisia kliinisten tutkimusten tuloksiin. Sen sijaan veren perifeeristen lymfosyyttien eristäminen ja niiden aktivoitumisen selvittäminen lyhytaikaisessa kliinisessä tutkimuksessa voisi toimia suhteellisen helppona skiinausmenetelmänä.

Infection-induced IL-10 and Jak-Stat: A review of the molecular circuitry controlling immune hyperactivity in response to pathogenic microbes

Generation of effective immune responses against pathogenic microbes depends on a fine balance between pro- and anti-inflammatory responses. Interleukin-10 (IL-10) is essential in regulating this balance and has garnered renewed interest recently as a modulator of the response to infection at the JAK-STAT signaling axis of host responses. Here, we examine how IL-10 functions as the “master regulator” of immune responses through JAK-STAT, and provide a perspective from recent insights on bacterial, protozoan, and viral infection model systems. Pattern recognition and subsequent molecular events that drive activation of IL-10-associated JAK-STAT circuitry are reviewed and the implications for microbial pathogenesis are discussed.