Combining culture and microbead-based immunoassay for the early and generic detection of bacteria in platelet concentrates.

BACKGROUND: Despite current preventive strategies, bacterial contamination of platelets is the highest residual infectious risk in transfusion. Bacteria can grow from an initial concentration of 0.03-0.3 colony-forming units (CFUs)/mL up to 108 to 109 CFUs/mL over the product shelf life. The aim of this study was to develop a cost-effective approach for an early, rapid, sensitive, and generic detection of bacteria in platelet concentrates. STUDY DESIGN AND METHODS: A large panel of bacteria involved in transfusion reactions, including clinical isolates and reference strains, was established. Sampling was performed 24 hours after platelet spiking. After an optimized culture step for increasing bacterial growth, a microbead-based immunoassay allowed the generic detection of bacteria. Antibody production and immunoassay development took place exclusively with bacteria spiked in fresh platelet concentrates to improve the specificity of the test. RESULTS: Antibodies for the generic detection of either gram-negative or gram-positive bacteria were selected for the microbead-based immunoassay. Our approach, combining the improved culture step with the immunoassay, allowed sensitive detection of 1 to 10 CFUs/mL for gram-negative and 1 to 102 CFUs/mL for gram-positive species. CONCLUSION: In this study, a new approach combining bacterial culture with immunoassay was developed for the generic and sensitive detection of bacteria in platelet concentrates. This efficient and easily automatable approach allows tested platelets to be used on Day 2 after collection and could represent an alternative strategy for reducing the risk of transfusion-transmitted bacterial infections. This strategy could be adapted for the detection of bacteria in other cellular products.

Development of a set of multiplex PCR assays for the detection of genes encoding important beta-lactamases in Enterobacteriaceae.

OBJECTIVES: To develop a rapid and reliable tool to detect by multiplex PCR assays the most frequently widespread beta-lactamase genes encoding the OXA-1-like broad-spectrum beta-lactamases, extended-spectrum beta-lactamases (ESBLs), plasmid-mediated AmpC beta-lactamases and class A, B and D carbapenemases. METHODS: Following the design of a specific group of primers and optimization using control strains, a set of six multiplex PCRs and one simplex PCR was created. An evaluation of the set was performed using a collection of 31 Enterobacteriaceae strains isolated from clinical specimens showing a resistance phenotype towards broad-spectrum cephalosporins and/or cephamycins and/or carbapenems. Direct sequencing from PCR products was subsequently carried out to identify beta-lactamase genes. RESULTS: Under optimized conditions, all positive controls confirmed the specificity of group-specific PCR primers. Except for the detection of carbapenemase genes, multiplex and simplex PCR assays were carried out using the same PCR conditions, allowing assays to be performed in a single run. Out of 31 isolates selected, 22 strains produced an ESBL, mostly CTX-M-15 but also CTX-M-1 and CTX-M-9, SHV-12, SHV-5, SHV-2, TEM-21, TEM-52 and a VEB-type ESBL, 6 strains produced a plasmid-mediated AmpC beta-lactamase (five DHA-1 and one CMY-2) and 3 strains produced both an ESBL (two SHV-12, one CTX-M-15) and a plasmid-mediated AmpC beta-lactamase (DHA-1). CONCLUSIONS: We report here the development of a useful method composed of a set of six multiplex PCRs and one simplex PCR for the rapid screening of the most frequently encountered beta-lactamases. This method allowed direct sequencing from the PCR products.

Main intestinal markers associated with the changes in gut architecture and function in piglets after weaning.

We analysed the spatio-temporal sequence of events concerning the morphology, physiology and ecology of the gut of piglets during the 2 weeks following weaning, in order to provide a limited number of variables that could be relevant markers of the gut post-weaning changes. An experiment was conducted on sixty piglets fasted for 2 d, then administered a weaning diet with a moderate or a high content of wheat using controlled gastric feeding, and slaughtered at different time-points post-weaning. Sixty-nine variables were analysed by principal component analysis. The results showed that the temporal changes induced in the gut by weaning can be divided into two periods: an acute period happening immediately after weaning, followed after day 5 by a more progressive adaptative and maturational phase. The main factors of this adaptation were the refeeding process and the time, while the diet per se had little influence. The villus length, lactose activity, macromolecule fluxes across the jejunum and the plasma cholecystokinin were proposed as markers of the acute phase. Ths mass of the jejunum, the weight of the pancrease, the content of stomach, the trypsin activity and the theophyl-line-induced secretion in jejunum were related to the re-feeding. Markers proposed to follow the gut maturation were the maltase activity, the glucose absorption and the basal resistance in the ileum, the lactobacilli and enterococci in the colon, and the pH of colonic and caecal contents. These markers might be helpful to design suitable diets to limit posts-weaning gut disorders in pigs.

High-viscosity carboxymethylcellulose reduces carbachol-stimulated intestinal chloride secretion in weaned piglets fed a diet based on skimmed milk powder and maltodextrin.

High-viscosity carboxymethylcellulose (CMC) promotes gastrointestinal disorders, tissue alterations and bacterial overgrowth in pigs. The impact of CMC on intestinal absorptive and secretory physiology is not known. We hypothesised that CMC consumption alters intestinal Na-dependent glucose absorption and stimulates electrogenic chloride secretion. For testing this hypothesis, twenty-four piglets were weaned at 21 d of age and pair-fed for 13 d a starter diet based on skimmed milk powder and maltodextrin containing cellulose (control) or CMC. Body weight and faecal total aerobe and coliform counts were measured kinetically. At slaughter, digesta were weighed and characterised for viscosity and pH. Gastrointestinal tissues were weighed and sampled for physiology in Ussing chambers, morphometry and enzymology. Glucose absorption tended to be higher (P = 0.08) and carbachol-stimulated chloride secretion was lower (P = 0.01) with CMC in the small intestine, without changes in the colon. Aerobes were transiently higher at day 7 (P < 0.05) but coliform counts remained unchanged (P = 0.78) and beta-haemolitic Escherichia coli were virtually absent. Stomach and small-intestinal segments were heavier, and viscosity higher with CMC (0.001 < P < 0.05). The pH in the stomach was higher, and in the caecum and proximal colon lower with CMC (0.001 < P < 0.05). Jejunal villus area was slightly reduced with CMC (P < 0.05) without effects on enzyme activities (P > 0.10). In conclusion, CMC supplementation had pro-absorptive effects on the small intestine, possibly due to the absence of pathogenic E. coli in the present study.

Knockout of the alanine racemase gene in Lactobacillus plantarum results in septation defects and cell wall perforation.

A stable mutant of Lactobacillus plantarum deficient in alanine racemase (Alr) was constructed by two successive homologous recombination steps. When the mutant was supplemented with D-alanine, growth and viability were unaffected. Surprisingly, deprivation of d-alanine during exponential growth did not result in a rapid and extensive lysis as observed in Alr-deficient strains of Escherichia coli or Bacillus subtilis. Rather, the starved mutant cells underwent a growth arrest and were gradually affected in viability with a decrease in colony forming units over 99% in less than 24 h. Additionally, fluorescent techniques demonstrated a loss of cell envelope integrity in the starved cells. Prolonged d-alanine starvation resulted in cells with an aberrant morphology. Scanning and transmission electron microscopy analyses revealed an increase in cell length, deficiencies in septum formation, thinning of the cell envelope and perforation of the cell wall in the septum region. We discuss the involvement of peptidoglycan hydrolases in these phenotypic defects in the context of the crucial role played by D-alanine in peptidoglycan biosynthesis and teichoic acids substitution.

Development of bacterial and bifidobacterial communities in feces of newborn babies.

Microbial 16S rDNA from babies' fecal samples were amplified by PCR, and analysed by denaturing gradient gel electrophoresis (DGGE), cloning and sequencing. PCR-DGGE profiles were used to follow in time the colonization of the intestine by bacteria. Four healthy babies, one baby who received antibiotics and their parents participated to the present study to determine the extent to which administration of antibiotics can modify the bacterial colonization of neonatal human gut and verify the influence of parental factors on the formation of the fecal bacterial community. In the healthy babies, Escherichia coli or bacteria belonging to Clostridium spp. were the initial colonizers rapidly followed by Bifidobacterium, Bacteroides, Clostridium, Streptococcus, Enterococcus and Actinomyces. Bifidobacterium species appeared already after five days in the breast-fed babies while there was a delay in the baby who received a formula based diet during only one day after birth. In each baby two or three bifidobacterial species including B. infantis were found. The observed variations in species were not associated with the feeding changes. The comparison of DGGE profiles of the babies and their parents patterns showed bands with equal migration suggesting a vertical transmission determined by genetic and environmental factors. The brief appearance of pioneer bacteria determined as being E. coli and Enterococcus spp. in the profile from the baby under antibiotic therapy, was succeeded by a small stable community consisting of Ruminococcus species. No Bifidobacterium sequences were detectable in this antibiotic-treated baby in spite of a partly breast-milk diet.

Molecular monitoring of succession of bacterial communities in human neonates.

The establishment of bacterial communities in two healthy babies was examined for more than the first 10 months of life by monitoring 16S ribosomal DNA (rDNA) diversity in fecal samples by PCR and denaturing gradient gel electrophoresis (DGGE) and by analyzing the sequences of the major ribotypes. DGGE profiles of the dominant populations in the intestines of the infants were obtained by analyzing daily or weekly fecal samples. After delivery, the germfree infant gastrointestinal tracts were rapidly colonized, and the succession of bacteria in each ecosystem was monitored. During the first few days of life the profiles were simple, but they became more complex as the bacterial diversity increased with time in both babies. Clone libraries of amplified 16S rDNA fragments from baby feces were constructed, and these libraries allowed identification of the bacterial types by comparative DNA sequence analysis; the bacteria identified included members of the genera Bifidobacterium, Ruminococcus, Enterococcus, Clostridium, and Enterobacter: Species most closely related to the genera Bifidobacterium and Ruminococcus in particular dominated the intestinal microbiota based on the stability over time and the numbers, as estimated by the intensities of the bands. However, 19 of the 34 cloned rDNA sequences exhibited less than 97% identity with sequences of known bacteria or cloned sequences in databases. This study showed that using PCR-DGGE and 16S rDNA sequence analysis together resulted in a dynamic description of bacterial colonization in the infant intestinal ecosystem and allowed visualization of bacteria that are difficult to cultivate or to detect by other methods.