Gram-negative Bacterial Growth Research Articles

Owner-collected swabs of pets: a method fit for the purpose of zoonoses research

As part of the preparation of a large cohort study in the entire German population, this study examined the feasibility of cat and dog owners collecting nasal and oral swabs of their animals at home as a method of assessing exposure to zoonoses. In veterinary clinics in Hannover, Germany, 100 pet owners were recruited. Nasal and oral swabs of pets were taken by a veterinarian at the clinic and owners took swabs at home. Swabs were analysed regarding bacterial growth and compared (owner vs. vet) using Cohen's kappa and McNemar's test. The return rate of kits was 92%, and 77% of owners thought it unnecessary to have veterinarian assistance to swab the mouth. McNemar's test results: oral swabs 78% agreement with Gram-positive bacterial growth, 87% agreement with Gram-negative bacterial growth; with similar results for nasal swabs. Although sample quality differed, this method allowed the receipt of swabs from pets in order to obtain information about colonization with zoonotic pathogens.

Non-Ribosomal Halogenated Protease Inhibitors from Cyanobacterial Isolates as Attractive Drug Targets

Cyanobacteria possess the ability to produce compounds with remarkable biological activity, and have thus attracted the attention of the pharmaceutical industry. Cyanopeptides acting as protease inhibitors have shown potential in the field of pharmacotherapy through regulation of abnormal physiological processes in the human body. Despite the already described cyanopeptide protease inhibitors, the search for new congeners is of considerable interest which may pave the way for more efficient molecules. In this study, the presence of the protease inhibitors aeruginosin and cyanopeptolin with non-, mono- and dichlorination and also genes coding for their synthetases was investigated in 90 cyanobacterial strains. Mass spectrometry analyses highlighted production of 91, 19 and 3 non-, mono- and dichlorinated congeners, respectively. The purified extract of Microcystis botrys SPC759 inhibited 61% of pepsin protease. PCR amplifications of aeruginosin and cyanopeptolin synthetase gene regions were observed in 41 and 28% of evaluated strains, respectively. The sequences obtained for the aerA-aerB (aeruginosin) and mcnC-mcnE (cyanopeptolin) gene regions grouped together with their homologues found in other cyanobacterial strains in the phylogenetic analyses with high bootstrap support. Antimicrobial activity assays performed using all intracellular extracts inhibited 31 and 26% of Gram-negative and Gram-positive pathogenic bacterial growth, respectively. The results of this study showed the production of aeruginosin and cyanopeptolin and the presence of their genes in several cyanobacterial genera for the first time besides the discovery of novel congeners.

Alanine scanning analysis and structure–function relationships of the frog‐skin antimicrobial peptide temporin‐1Ta

The increasing resistance of bacteria and fungi to the available antibiotic/antimycotic drugs urges for a search for new anti-infective compounds with new modes of action. In line of this, natural CAMPs represent promising and attractive candidates. Special attention has been devoted to frog-skin temporins, because of their short size (10-14 residues long) and their unique features. In particular, temporin-1Ta has the following properties: (i) it is mainly active on Gram-positive bacteria; (ii) it can synergize, when combined with temporin-1Tl, in inhibiting both gram-negative bacterial growth and the toxic effect of LPS; (iii) it preserves biological activity in the presence of serum; and (iv) it is practically not hemolytic. Rational design of CAMPs represents a straightforward approach to obtain a peptide with a better therapeutic index. Here, we used alanine scanning analogs to elucidate the contribution of the side chains of each amino acid residue to the peptide's antimicrobial and hemolytic activity. Beside providing insight into the biophysical attributes and the critical positions within the peptide sequence, which govern the antimicrobial/hemolytic activity of this temporin isoform, our studies assist in optimizing the design of temporin-based lead structures for the production of new anti-infective agents.

Characterization and expression of HLysG2, a basic goose-type lysozyme from the human eye and testis

Lysozyme plays an important role in human innate immunity by causing bacterial cell lysis. We describe for the first time, the actual performance of human lysozyme g-like 2 (HLysG2), a mammalian g-type lysozyme. RT-PCR revealed that the HLysG2 gene was transcribed in eye and testis tissues. A spot was detected from human tears using 2D gel electrophoresis and was identified as HLysG2 using MALDI–TOF/TOF MS and a MASCOT search with a matching score of 140 and 27% sequence coverage of the whole amino acid sequence. To gain insight into the in vitro antimicrobial activities of HLysG2, the mature peptide-coding region was cloned into Pichia pastoris for heterogeneous expression. Recombinant HLysG2, had an optimal at pH 6.0 and 30°C, reached the peak activity of 1.2×104U/mg at the sodium ion concentration of 75mM and showed a higher salt tolerance than human c-type lysozyme (HLysC). Recombinant HlysG2 inhibited Gram-positive bacterial growth and did not inhibit Gram-negative bacterial and Candida albicans growth. Results indicated that HLysG2 is a potent antibacterial protein that may play a role in the innate immunity of the human eye.

Inhibitory effect of the natural product betulin and its derivatives against the intracellular bacterium Chlamydia pneumoniae

Chlamydia pneumoniae is a universal pathogen that has been indicated to play a part in the development of asthma, atherosclerosis and lung cancer. The complete eradication of this intracellular bacterium is in practice impossible with the antibiotics that are currently in use and studies on new antichlamydial compounds is challenging because Chlamydia research lacks the tools required for the genetic modification of this bacterium. Betulin is a natural lupane-class triterpene derived from plants with a wide variety of biological activities. This compound group thus has wide medical potentials, and in fact has been shown to be active against intracellular pathogens. For this reason, betulin and its derivatives were selected to be assayed against C. pneumoniae in the present study. Thirty-two betulin derivatives were assayed against C. pneumoniae using an acute infection model in vitro. Five promising compounds with potential lead compound characteristics were identified. Compound 24 (betulin dioxime) gave a minimal inhibitory concentration (MIC) of 1 μM against strain CWL-029 and showed activity in nanomolar concentrations, as 50% inhibition was achieved at 290 nM. The antichlamydial effect of 24 was confirmed with a clinical isolate CV-6, showing a MIC of 2.2 μM. Previous research on betulin and its derivatives has not identified such a remarkable inhibition of Gram-negative bacterial growth. Furthermore, we also demonstrated that this antichlamydial activity was not due to PLA 2 (EC 3.1.1.4) inhibition caused by the betulin derivatives.

Evaluation of the antibacterial activity and toxicity of Myrciaria caulifloria methanolic leaf and fruit extracts

Methanol extracts from M. caulifloria leaves and fruit were tested for antimicrobial activity and toxicity in vitro. M. caulifloria leaf extractinhibited the growth of 9 of the 14 bacteria tested (64%) whilst the fruit extract inhibited the growth of 11 of the 14 bacteria tested (79%). Both Gram-positive and Gram-negative bacterial growth were inhibited by M. caulifloria leaf and flower extracts. 7 of the 10 Gram-negative bacteria (70%) and 2 of the 4 Gram-positive bacteria (50%) tested had their growth inhibited by M. caulifloria leaf extract whereas the fruit extract inhibited 7 of the 10 Gram-negative bacteria (70%) and 100% of the Gram-positive bacteria tested. M. caulifloria leaf extract proved to be toxic in the Artemia fransiscana bioassay with 24, 48 and 72h LC50's of 232.9 ᠳ6.3 姯ml, 169.1 ᠱ1.5 姯ml and 128.1 ᠵ.6 姯ml respectively, making it more toxic than Mevinphos (24, 48 and 72h LC50's 1346.0 ᠷ8.2 姯ml, 505.3 ᠳ7.7 姯ml, 103.9 ᠱ2.8) at all time points except 72h but less toxic than potassium dichromate (24, 48 and 72h LC50's 86.3 ᠵ.1 姯ml, 80.4 ᠴ.3 姯ml, 77.9 ᠴ.9). M. caulifloria fruit extract was non-toxic in the Artemia fransiscana bioassay indicating its potential as an antibacterial agent for medicinal use.

Cecal Ligation and Incision: An Acute Onset Model of Severe Sepsis in Rats

Sepsis is a leading cause of death among critically ill patients. Up to now, severe sepsis with acute onset in animals has been induced mainly through injection of single bacteria species or endotoxin and not through a surgical procedure, which might adequately mirror the situation in septic patients. We therefore aimed to establish a surgical model of severe sepsis in rodents fulfilling international sepsis criteria. Twenty-eight anesthetized/ventilated Sprague Dawley rats underwent laparotomy and cecal mobilization. The cecum was either replaced into the abdomen (SHAM, n = 14) or the cecum and the mesenteric blood vessels were ligated, and the cecum was opened through a 1.5 cm blade incision (cecal ligation and incision, CLI, n = 14). Within 390 min, mortality was 0% (SHAM) and 50% (CLI), respectively. Compared with SHAM, CLI resulted in a 43% reduction of mean arterial blood pressure and in severe metabolic acidosis as measured by arterial base excess and pH. CLI led to a 15-fold increase in mononuclear cell population and to a 5-fold accumulation of nitrite in peritoneal lavage. Abdominal swabs from the Douglas cavity in CLI-animals showed gram-positive and gram-negative bacterial growth on agar compared with sterile swabs from SHAM-animals. In CLI-animals, plasma IL-1beta level was increased to 435 pg/mL (SHAM: 10 pg/mL) and plasma IL-6 level to 19718 pg/mL (SHAM: 832 pg/mL). CLI causes bacterial peritonitis with subsequent systemic inflammation and organ dysfunction. Thus, CLI mimics clinical sepsis and provides a surgical short term model of severe sepsis in rodents.

Outer Membrane Vesicle Production by Escherichia coli Is Independent of Membrane Instability

It has been long noted that gram-negative bacteria produce outer membrane vesicles, and recent data demonstrate that vesicles released by pathogenic strains can transmit virulence factors to host cells. However, the mechanism of vesicle release has remained undetermined. This genetic study addresses whether these structures are merely a result of membrane instability or are formed by a more directed process. To elucidate the regulatory mechanisms and physiological basis of vesiculation, we conducted a screen in Escherichia coli to identify gene disruptions that caused vesicle over- or underproduction. Only a few low-vesiculation mutants and no null mutants were recovered, suggesting that vesiculation may be a fundamental characteristic of gram-negative bacterial growth. Gene disruptions were identified that caused differences in vesicle production ranging from a 5-fold decrease to a 200-fold increase relative to wild-type levels. These disruptions included loci governing outer membrane components and peptidoglycan synthesis as well as the sigma(E) cell envelope stress response. Mutations causing vesicle overproduction did not result in upregulation of the ompC gene encoding a major outer membrane protein. Detergent sensitivity, leakiness, and growth characteristics of the novel vesiculation mutant strains did not correlate with vesiculation levels, demonstrating that vesicle production is not predictive of envelope instability.

Evaluation of bacterial and protozoal contamination of commercially available raw meat diets for dogs

To evaluate bacterial and protozoal contamination of commercially available raw meat diets for dogs. Prospective longitudinal study. 240 samples from 20 raw meat diets for dogs (containing beef, lamb, chicken, or turkey), 24 samples from 2 dry dog foods, and 24 samples from 2 canned dog foods. Each product was purchased commercially on 4 dates approximately 2 months apart. Three samples from each product at each sampling period were evaluated via bacterial culture for non-type-specific Escherichia coli (NTSEC), Salmonella enterica, and Campylobacter spp. Antimicrobial susceptibility testing was performed on selected isolates. Polymerase chain reaction assays were used to detect DNA from Cryptosporidium spp, Neospora spp, and Toxoplasma spp in samples obtained in the third and fourth sampling periods. One hundred fifty-three of 288 (53%) samples were contaminated with NTSEC. Both raw and prepared foods contained NTSEC during at least 1 culture period. Salmonella enterica was recovered from 17 (5.9%) samples, all of which were raw meat products. Campylobacter spp was not isolated from any samples. In 91 of 288 (31.6%) samples, there was no gram-negative bacterial growth before enrichment and in 48 of 288 (16.7%) samples, there was no aerobic bacterial growth before enrichment. Susceptibility phenotypes were variable. Cryptosporidium spp DNA was detected in 3 samples. Bacterial contamination is common in commercially available raw meat diets, suggesting that there is a risk of foodborne illness in dogs fed these diets as well possible risk for humans associated with the dogs or their environments.

Induction of gram-negative bacterial growth by neurochemical containing banana (Musa x paradisiaca) extracts.

Bananas contain large quantities of neurochemicals. Extracts from the peel and pulp of bananas in increasing stages of ripening were prepared and evaluated for their ability to modulate the growth of non-pathogenic and pathogenic bacteria. Extracts from the peel, and to a much lesser degree the pulp, increased the growth of Gram-negative bacterial strains Escherichia coli O157:H7, Shigella flexneri, Enterobacter cloacae and Salmonella typhimurium, as well as two non-pathogenic E. coli strains, in direct relation to the content of norepinephrine and dopamine, but not serotonin. The growth of Gram-positive bacteria was not altered by any of the extracts. Supplementation of vehicle and pulp cultures with norepinephrine or dopamine yielded growth equivalent to peel cultures. Total organic analysis of extracts further demonstrated that the differential effects of peel and pulp on bacterial growth was not nutritionally based, but due to norepinephrine and dopamine. These results suggest that neurochemicals contained within foodstuffs may influence the growth of pathogenic and indigenous bacteria through direct neurochemical-bacterial interactions.

Improved methods for producing outer membrane vesicles in Gram-negative bacteria

Outer membrane vesicle formation occurs during Gram-negative bacterial growth. However, natural production of large amounts of outer membrane vesicles has only been described in a few bacterial genera. The purified vesicles of some bacterial pathogens have shown potential applications in vaccinology and in antibiotic therapy. This study focused on the development of a gene expression system able to induce production of large amounts of outer membrane vesicles. The Tol–Pal system of Escherichia coli, required to maintain outer membrane integrity, is composed of five cell envelope proteins, TolA, TolB, TolQ, TolR and Pal. Tol proteins are parasitized by filamentous bacteriophages and by colicins. The phage infection process and colicin import require, respectively, the N-terminal domain of the minor coat g3p protein and the translocation domain of colicins, with both domains interacting with Tol proteins. In this study, we show that the periplasmic production of either Tol, g3p or colicin domains was able to specifically destabilize the E. coli or Shigella flexneri cell envelope and to induce production of high amounts of vesicles. This technique was further found to work efficiently in Salmonella enterica serovar Typhimurium.

Modulation of Antibiotic Efflux in Bacteria

Bacteria develop resistance to antibiotics by receptor alteration, antibiotic modification and by drug efflux, whereby the antibiotic is removed from intracellular compartments by efflux proteins. This article gives an overview of the chemical and biological properties of the major efflux proteins found within resurgent pathogens, their activity in creating resistance, and the effect of inhibitors of these proteins. The major families of efflux protein inhibitors against bacteria include tetracyclines, indanes, ginsensosides, indoles, reserpine and reserpine analogues, dipeptides, peptidomimetic heterocycles and heterocycles, flavones, and benastatins. Their effect on both Gram-positive and Gram-negative bacteria efflux proteins and bacterial growth is detailed.

Morphine-induced degradation of the host defense barrier: role of macrophage injury.

The effect of morphine on the degradation of the host defense barrier in rats and mice was studied. Mice received either 3 or 11 doses of morphine. Mice receiving 11 doses of morphine showed gram-negative bacteremia and bacterial growth in samples of peritoneal fluid (PF), liver, spleen, kidneys, heart, and lungs; PF and tissue samples from only 1 control mouse showed bacterial growth, and no control mice had bacteremia. Mice receiving 11 doses also had suppressed bone marrow macrophage colony formation. Monocytes and peritoneal macrophages harvested from morphine-treated mice showed greater injury than did those from control mice. Pretreatment of mice with naloxone inhibited morphine-induced macrophage injury and degradation of the host defense barrier. In in vitro studies, morphine attenuated the killing of bacteria phagocytosed by macrophages and also facilitated their escape. This study indicates that morphine-induced monocyte and macrophage injury may be linked to degradation of the host defense barrier.

Induction of Gram-negative bacterial growth by neurochemical containing banana ( Musa x paradisiaca) extracts

Bananas contain large quantities of neurochemicals. Extracts from the peel and pulp of bananas in increasing stages of ripening were prepared and evaluated for their ability to modulate the growth of non-pathogenic and pathogenic bacteria. Extracts from the peel, and to a much lesser degree the pulp, increased the growth of Gram-negative bacterial strains Escherichia coli O157:H7, Shigella flexneri, Enterobacter cloacae and Salmonella typhimurium, as well as two non-pathogenic E. coli strains, in direct relation to the content of norepinephrine and dopamine, but not serotonin. The growth of Gram-positive bacteria was not altered by any of the extracts. Supplementation of vehicle and pulp cultures with norepinephrine or dopamine yielded growth equivalent to peel cultures. Total organic analysis of extracts further demonstrated that the differential effects of peel and pulp on bacterial growth was not nutritionally based, but due to norepinephrine and dopamine. These results suggest that neurochemicals contained within foodstuffs may influence the growth of pathogenic and indigenous bacteria through direct neurochemical-bacterial interactions.

Bloodgroup simulating activity in aerobic gram-negative oral bacteria cultured from fresh corpses

The gram-negative aerobic oral bacterial flora of 100 consecutive corpses was isolated. After the identification and culturing of the isolated organisms, blood grouping was performed by the haemagglutination inhibition technique on dried culture smears, the dried culture medium and a dried ethanol extract of the bacteria. Forty-seven of the samples showed a gram-negative aerobic bacterial growth, giving 58 microorganisms of 14 different species. Positive blood grouping results were found in two of them ( Escherichia coli and Serratia marcescens), both of type B. It is concluded that occasional mistyping of blood groups on saliva and oral material may be caused by the oral gram-negative aerobic flora, especially if the specimens are contaminated or putrefying.