Colony-forming Units Per mL Research Articles

An In vitro Comparative Evaluation of Disinfectants on Standard and Clinical Microbial Strains on Heat Cure Resins.

Oral cavity is colonised by numerous micro-organisms that form a biofilm on the acrylic resin. Hence, routine hygiene is essential to prevent oral mucosal inflammation and lesions. Knowledge of appropriate disinfecting agents for acrylic resins is crucial in this context. To compare and evaluate the effectiveness of four commercially available disinfectants on heat cure acrylic resin specimens contaminated with standard and clinical strains of two micro-organisms commonly inhabiting the oral microflora. Two hundred acrylic resin specimens (n=200), 10 in each group were contaminated in vitro with 1x106 cells/ml suspensions of standard and clinical strains of micro-organisms (Candida albicans and Streptococcus mutans) and were immersed in four disinfectants (1% sodium hypochlorite, 2% chlorhexidine digluconate, 2% glutaraldehyde and 3.8% sodium perborate) for 10 minutes. The control group was not subjected to any disinfection process. For collection of clinical strains, oral swab was passed over the buccal mucosa and grown on blood agar culture media. Organism confirmation was done by growing them on selective culture media. Final counts of micro-organisms per ml were performed by plating method for evaluation of microbial level reduction. Results obtained were subjected to ANOVA and Tukey's test. Standard strains of Candida albicans (C) and Streptoccocus mutans (S) subjected to various disinfectants showed varied mean Colony Forming Units per ml (CFU/ml) from <10,000 to 25,000 and <10000 to <50,000 respectively. Clinical strains and of Candida albicans (C) and Streptococcus mutans (S) subjected to various disinfectants showed varied mean CFU/ml from <10,000 to 50,000 and from 10,000 to 50,000 respectively. Control groups showed maximum mean CFU/ml (>105). All intergroup comparisons were highly significant (p<0.001; HS) and intragroup comparisons were significant (p<0.05; S) except the comparison of clinical strains of Streptococcusmutans subjected to 2% chlorhexidine digluconate and 2% glutaraldehyde which was found to be non significant (p>0.05; NS). Almost 1% sodium hypochlorite was found to be the most effective disinfectant for both Candida albicans and Streptococcus mutans. The least effective disinfectant being: 3.8% sodium perborate.

Viability kinetics of Lactobacillus casei Shirota strain in a commercial fermented milk drink during refrigerated storage

Background: One of the most important parameters in a commercial fermented milk drink containing probiotics is the viability of the microorganisms. Aims and Objectives: This study described the viability kinetics of Lactobacillus casei Shirota strain in a commercial fermented milk drink during refrigerated storage using nonlinear models. Materials and Methods: Viability of L. casei Shirota strain was monitored during refrigerated storage using standard bacterial plate count method. Several nonlinear mathematical models such as the zero-order, first-order, and second-order kinetic equations were employed in describing the population dynamics. Best-fit models were selected based on prescribed criteria including sum of squares of the error, p-value, and coefficient of determination. Results: The viable counts of L. casei Shirota strain in the fermented milk samples stored under refrigerated conditions decreased from 3.73 × 108 colony forming units per ml (CFU/ml) to 2.70 × 108 CFU/ml when monitored every 3-4 days interval over 10 different monitoring points within the product’s indicated shelf life. The counts significantly differed between the monitoring points that were at least 14 days apart. The lowest viable count was still within the recommended therapeutic dose. The decrease in the bacterial population behaved under a second-order kinetic relationship. Conclusion: The viability of L. casei Shirota strain in a commercial fermented milk product during refrigerated storage is governed in accordance with the second-order kinetic mechanism.

Antiseptic mouthwash against pharyngeal Neisseria gonorrhoeae: a randomised controlled trial and an in vitro study

BackgroundGonorrhoea is increasing among men who have sex with men (MSM). We aimed to determine whether Listerine, a commercial mouthwash product, has an inhibitory effect against Neisseria gonorrhoeae in a...

Selective detection of viable seed-borne Acidovorax citrulli by real-time PCR with propidium monoazide.

In recent years, use of the DNA-intercalating dye propidium monoazide (PMA) in real-time PCR has been reported as a novel method to detect viable bacteria in different types of samples, such as food, environmental, and microbiological samples. In this study, viable cells of Acidovorax citrulli, the causal agent of bacterial seedling blight and fruit blotch, were selectively detected and differentiated from dead cells by real-time fluorescent polymerase chain reaction amplification after the bacterial solution was treated with the DNA-binding dye PMA. The primers and TaqMan probe were based on the A. citrulli genome (Aave_1909, Gene ID: 4669443) and were highly specific for A. citrulli. The detection threshold of this assay was 103 colony-forming units per mL (CFU/mL) in pure cell suspensions containing viable and dead cells and infected watermelon seeds. Application of this assay enables the selective detection of viable cells of A. citrulli and facilitates monitoring of the pathogen in watermelon and melon seeds.

Antibacterial and anti-adherence effects of a plant extract mixture (PEM) and its individual constituent extracts (Psidium sp., Mangifera sp., and Mentha sp.) on single- and dual-species biofilms.

BackgroundPlant extracts mixture (PEM) and its individual constituent plant extracts(Psidium sp., Mangifera sp., Mentha sp.) are known to have an anti-adhering effect towards oral bacteria in the single-species biofilm. To date, the adhering ability of the early and late plaque colonisers (Streptococcus sanguinis and Streptococcus mutans) to PEM-treated experimental pellicle have not been investigated in dual-species biofilms.MethodsFresh leaves of these plants were used in the preparation of the respective aqueous extract decoctions. The minimum inhibitory concentration (MIC) of the extracts towards S. sanguinis ATCC BAA-1455 and S. mutans ATCC 25175 was determined using a two-fold serial microdilution method. The sum of fractional inhibitory concentration (ΣFIC) index of PEM and its constituent plant extracts was calculated using the MIC values of the plants. The minimum bactericidal concentration (MBC) of the plant extracts was also determined. The anti-adherence effect of the plant extracts (individually and mixed) was carried out by developing simulated S. sanguinis and S. mutans respectively in single- and dual-species of biofilms in the Nordini’s Artificial Mouth (NAM) model system in which the experimental pellicle was pretreated with the plant extract before bacterial inoculation. The bacterial population in the respective biofilms was quantified using ten-fold serial dilutions method and expressed as colony forming unit per ml (CFU/ml). The bacterial population was also viewed using Scanning Electron Microscope (SEM). All experiments were done in triplicate.ResultsThe PEM compared with its respective constituent plants showed the lowest MIC towards S. sanguinis (3.81 mg/ml) and S. mutans (1.91 mg/ml) and exhibited a synergistic effect. The Psidium sp. (15.24 mg/ml) and, PEM and Psidium sp. (30.48 mg/ml) showed the lowest MBC towards S. sanguinis and S. mutans respectively. The anti-adherence effect of the PEM and its respective constituent plants (except Psidium sp.) was different for the two bacteria in the single-species biofilm. In the dual-species biofilms, PEM demonstrated similar anti-adherence effect towards S. sanguinis and S. mutans. The proportions of the bacterial population viewed under SEM appeared to be in agreement with the quantified population.DiscussionThe combination of the active constituents of the individual plant extracts in PEM may contribute to its low MIC giving rise to the synergistic effect. The different anti-adherence effect towards S. sanguinis and S. mutans in both single- and dual-species biofilms could be due to the different proportion of the active constituents of the extracts and the interaction between different bacteria. The better adhering ability of S. sanguinis towards the PEM-treated pellicle when present together with S. mutans in the dual-species biofilms may suggest the potential of PEM in controlling the balance between the early and late colonisers in biofilms.

Microbiological evaluation of different reprocessing methods for cuffed and un-cuffed tracheostomy tubes in home-care and hospital setting.

Background: Manufacturers’ recommendations on cleaning of tracheostomy tubes focus on general warning information and non-specific manual cleaning procedures. The aim of this experimental study was to evaluate different reprocessing methods and to determine the mechanical integrity and functionality of tracheostomy tubes following reprocessing.Methods: Sixteen cuffed or un-cuffed tracheostomy tubes obtained from hospital in-patients were reprocessed using one of the following reprocessing methods: a) manual brushing and rinsing with tap water, b) manual brushing followed by disinfection with a glutaraldehyde solution, c) manual brushing followed machine-based cleaning in a dishwasher, and d) manual brushing followed by ultrasound cleaning in a commercially available ultrasound device. Microbial burden of the tubes before and after reprocessing was assessed by measurement of microbial colony-forming units per mL (CFU/mL) of rinsing fluid. After cleaning, tracheostomy tubes were investigated for loss of functionality. Findings: Manual brushing and rinsing with tap water reduced microbial colonization in average by 102 CFU/mL, but with poor reproducibility and reliability. Complete microbial reduction was achieved only with additional chemical or machine-based thermal disinfection. Ultrasound sonification yielded no further microbial reduction after manual brushing. Conclusion: Manual brushing alone will not result in complete eradication of microorganism colonising cuffed or un-cuffed tracheostomy tubes. However, manual cleaning followed by chemical or thermal disinfection may be regarded as safe and reproducible reprocessing method. If a machine-based reprocessing method is used for cuffed tubes, the cuffs’ ventilation hose must be secured in a safe position prior to thermal disinfection.

Sterilizing elastomeric chains without losing mechanical properties. Is it possible?

OBJECTIVE: To investigate the effects of different sterilization/disinfection methods on the mechanical properties of orthodontic elastomeric chains. METHODS: Segments of elastomeric chains with 5 links each were sent for sterilization by cobalt 60 (Co60) (20 KGy) gamma ray technology. After the procedure, the elastomeric chains were contaminated with clinical samples of Streptococcus mutans. Subsequently, the elastomeric chains were submitted to sterilization/disinfection tests carried out by means of different methods, forming six study groups, as follows: Group 1 (control - without contamination), Group 2 (70°GL alcohol), Group 3 (autoclave), Group 4 (ultraviolet), Group 5 (peracetic acid) and Group 6 (glutaraldehyde). After sterilization/disinfection, the effectiveness of these methods, by Colony forming units per mL (CFU/mL), and the mechanical properties of the material were assessed. Student's t-test was used to assess the number of CFUs while ANOVA and Tukey's test were used to assess elastic strength. RESULTS: Ultraviolet treatment was not completely effective for sterilization. No loss of mechanical properties occurred with the use of the different sterilization methods (p > 0.05). CONCLUSION: Biological control of elastomeric chains does not affect their mechanical properties.

English

Ingredients of animal origin are important for the animal feed industry because they contain significant amounts of nutrients, minerals, and vitamins. However, the use of these raw materials is a problem due to pathogenic bacterial contamination, especially Clostridium perfringens and Salmonella spp. One way to control contamination is the addition of chemical products during ingredient production. Thus, the objective of this study was to evaluate a formaldehyde and organic acid-based product (SALMEX®) for two periods of action after experimental challenge with C. perfringens in two poultry feed ingredients. Microbiological analyses to enumerate the pathogen were conducted using colony-forming units per mL (CFU/mL) after incubation on SPS agar at 37°C for 48 h in anaerobic jars using the GasPak® system. The results show that there were significant differences among the dosage treatments and ingredients. With respect to the action time of the product, there were no significant differences observed between 24 h and 5 days, but there was a reduction in bacterial count with doses above 3 kg/t. This reduction was greater in the five-day SALMEX® treatment when compared to the 24-hour period. Thus, we can conclude that a higher product dose and a longer incubation time leads to more efficient product action. Key words: Animal health, microbiology, nutrition, poultry industry, pathogen.

Effectiveness of ozonated water in the reprocessing of blood dialyzers

INTRODUCTION: Ozone is a potent antibacterial agent. Because ozone oxidizes organic material, it directly attacks microorganisms resulting in safe, fast and economical sterilization at low temperatures. This study evaluated the efficacy of ozonated water in the reprocessing of dialyzers obtained from a hemodialysis service in Sao Jose do Rio Preto. METHODS: A total of 23 blood dialyzers were collected at the conclusion of the morning hemodialysis shift. The equipment was collected after the maximum number of reuses, with the last disinfection performed with purified water (obtained by reverse osmosis and subsequent reverse ultrafiltration). The number and species of microorganisms in the dialyzers were evaluated before and after treatment with ozonated water. The ozonation of sterile deionized water was achieved by direct contact between the water and the ozone generating equipment. RESULTS:The mean number of microorganisms before sterilization was 1,47.109 colony forming units per ml (CFU/ml). After treatment with ozonated water, the number of microorganisms was 4,80.101 CFU/ml. CONCLUSION: Ozonated water is an effective decontaminant for most blood dialyzers.

Saccharomyces cerevisiae expressing bacteriophage endolysins reduce Lactobacillus contamination during fermentation

One of the challenges facing the fuel ethanol industry is the management of bacterial contamination during fermentation. Lactobacillus species are the predominant contaminants that decrease the profitability of biofuel production by reducing ethanol yields and causing “stuck” fermentations, which incur additional economic losses via expensive antibiotic treatments and disinfection costs. The current use of antibiotic treatments has led to the emergence of drug-resistant bacterial strains, and antibiotic residues in distillers dried grains with solubles (DDGS) are a concern for the feed and food industries. This underscores the need for new, non-antibiotic, eco-friendly mitigation strategies for bacterial contamination. The specific objectives of this work were to (1) express genes encoding bacteriophage lytic enzymes (endolysins) in Saccharomyces cerevisiae, (2) assess the lytic activity of the yeast-expressed enzymes against different species of Lactobacillus that commonly contaminate fuel ethanol fermentations, and (3) test the ability of yeast expressing lytic enzymes to reduce Lactobacillus fermentum during fermentation. Implementing antibiotic-free strategies to reduce fermentation contaminants will enable more cost-effective fuel ethanol production and will impact both producers and consumers in the farm-to-fork continuum. Two genes encoding the lytic enzymes LysA and LysA2 were individually expressed in S. cerevisiae on multi-copy plasmids under the control of a galactose-inducible promoter. The enzymes purified from yeast were lytic against Lactobacillus isolates collected from fermentors at a commercial dry grind ethanol facility including Lactobacillus fermentum, Lactobacillus brevis, and Lactobacillus mucosae. Reductions of L. fermentum in experimentally infected fermentations with yeast expressing LysA or LysA2 ranged from 0.5 log10 colony-forming units per mL (CFU/mL) to 1.8 log10 (CFU/mL) over 72 h and fermentations treated with transformed yeast lysate showed reductions that ranged from 0.9 log10 (CFU/mL) to 3.3 log10 (CFU/mL). Likewise, lactic acid and acetic acid levels were reduced in all experimentally infected fermentations containing transformed yeast (harboring endolysin expressing plasmids) relative to the corresponding fermentations with untransformed yeast. This study demonstrates the feasibility of using yeast expressing bacteriophage endolysins to reduce L. fermentum contamination during fuel ethanol fermentations.

The Use of Antibiotics in Cryopreservation of Ram Sperm

Antibiotics are used in extenders for sperm cryopreservation, however no specific regulation has been established for ram sperm. This study evaluates the effects of antibiotics in ram sperm extenders for bacterial control and its effect on sperm viability. Sperm from five rams was collected, cooled and frozen using extenders with the following antibiotics: 100,000 IU/mL penicillin and 100 µg/mL streptomycin (PES); 500 µg/mL gentamycin, 100 µg/mL tylosin, 300 µg/mL lincomycin and 600 µg/mL spectinomycin (GTLS); 50 µg/mL ceftiofur sodium (CEF); and 1,000 µg/mL enrofloxacin (ENR). Bacillus sp., Corynebacterium sp., Klebsiella sp. and Staphylococcus sp. were isolated from fresh sperm and perpetual area. For cooled sperm, the antibiotic PES, GTLS and ENR were able to reduce the number of colony forming units per mL (CFU/mL), however, CEF was inefficient in all tested concentrations. Motility of cooled sperm was reduced when GTLS and ENR were used (P 0.05), however, motility was reduced with ENR treatment at doses greater than 50% (P < 0.05). The antibiogram revealed resistance of Staphylococcus sp . and Klebsiella sp. to the penicillin/streptomycin association and to tylosin. Both PES and GTLS were able to control bacterial growth on cooled sperm. However, attention should be given to the antibiotics added to the extenders, which may impair sperm motility.

Survival of Brucella Melitensis Rev - 1 in Experimentally Contaminated and Refrigerated Camel and Goat Milk

This research work aimed to examine the survival of Brucella melitensis Rev-1 in refrigerated camel and goat milk. Fresh camel and goat milk was experimentally contaminated with different concentrations of colony forming units per ml (CFU/ml) of Brucella melitensis Rev-1 and refrigerated for different periods of time. Brucella melitensis Rev-1 was detected in the refrigerated contaminated milk by isolation in culture medium and by PCR. Brucella melitensis Rev-1 was isolated from camel milk samples refrigerated for up to 8 hours. Camel milk samples refrigerated for 24 hours were negative for bacterial isolation. Brucella melitensis Rev-1 was isolated from camel milk samples containing 65 (CFU/ml) while samples containing less than that were negative for culture. The minimum content of the bacterium to be detected by PCR in contaminated camel milk was 65 CFU/ml. The higher the bacterial content of milk the better is the isolation results and whole milk was superior to cream for isolation. Brucella melitensis Rev-1 was isolated from goat milk refrigerated for up to 72 hrs. Isolation was positive for samples containing as low as 3 CFU/ml refrigerated for 4 hrs. Brucella melitensis Rev-1 was isolated from contaminated goat milk samples containing 30 to 300 CFU/ml refrigerated for 24 and 48 hrs, respectively. Samples containing higher bacterial concentrations were positive for isolation after 72 hrs of refrigeration. PCR was positive for Brucella melitensis Rev-1-contaminated goat milk containing as low as 3 CFU/ml. These results show that both isolation and PCR are sensitive for the detection of milk contamination with Brucella melitensis Rev-1, and that Brucella melitensis Rev-1 will not survive in camel milk for more than 8 hrs. the difference found here may be attributed to the nature of milk itself.

A Rabbit Osteomyelitis Model to Simulate Multibacterial War Wound Infections

A challenge facing military caregivers is the presence of multidrug-resistant infection in extremity wounds. Most frequently identified resistant strains are methicillin-resistant Staphylococcus aureus (MRSA), Klebsiella pneumoniae (KP), Pseudomonas aeruginosa (PA), and Acinetobacter baumannii (AB). We adapted an existing osteomyelitis model to simulate an infected extremity wound for antibiotic testing. New Zealand White Rabbits (n = 95) were divided into 6 inoculation groups for infection with MRSA, KP, PA, and AB alone, and in multibacteria infections. Sodium morrhuate was injected into the left tibia to simulate blast wound trauma, then the respective bacteria or combination of pathogens, and finally sterile saline were injected. Colony-forming units for the mono-organism groups showed that AB, KP, or PA alone at 10(7) colony-forming units per mL (CFUs/mL) was effective for rabbit osteomyelitis induction. Colony-forming units for the multiorganism groups showed that the combination of AB (10(7) CFUs/mL)/KP (10(7) CFUs/mL)/PA (10(7) CFUs/mL)/MRSA (10(5) CFUs/mL) yielded a 100% osteomyelitis induction rate. At 8 weeks, however, only one mono-bacterial group and one multibacterial group showed significant radiographic improvement (p < 0.05). The rabbit model of osteomyelitis can be adapted to study infected blast wounds typical of those seen in veterans. To our knowledge, this is the first demonstration of the model simulating multibacterial infections with multidrug-resistant organisms.

Management of Group B Streptococcal Bacteriuria in Pregnancy

ObjectiveTo provide information regarding the management of group B streptococcal (GBS) bacteriuria to midwives, nurses, and physicians who are providing obstetrical care. OutcomesThe outcomes considered were neonatal GBS disease, preterm birth, pyelonephritis, chorioamnionitis, and recurrence of GBS colonization. EvidenceMedline, PubMed, and the Cochrane database were searched for articles published in English to December 2010 on the topic of GBS bacteriuria in pregnancy. Bacteriuria is defined in this clinical practice guideline as the presence of bacteria in urine, regardless of the number of colony-forming units per mL (CFU/mL). Low colony counts refer to < 100 000 CFU/mL, and high (significant) colony counts refer to ≥ 100 000 CFU/mL. Results were restricted to systematic reviews, randomized controlled trials, and relevant observational studies. Searches were updated on a regular basis and incorporated in the guideline to February 2011. Grey (unpublished) literature was identified through searching the websites of health technology assessment and health technology assessment-related agencies, clinical practice guideline collections, clinical trial registries, and national and international medical specialty societies. ValuesRecommendations were quantified using the evaluation of evidence guidelines developed by the Canadian Task Force on Preventive Health Care (Table). Benefits, Harms, and CostsThe recommendations in this guideline are designed to help clinicians identify pregnancies in which it is appropriate to treat GBS bacteriuria to optimize maternal and perinatal outcomes, to reduce the occurrences of antibiotic anaphylaxis, and to prevent increases in antibiotic resistance to GBS and non-GBS pathogens. No cost-benefit analysis is provided.

Quantitative PCR for Etiologic Diagnosis of Methicillin-ResistantStaphylococcus aureusPneumonia in Intensive Care Unit

BackgroundVentilator-associated pneumonia (VAP) requires prompt and appropriate treatment. Since methicillin-resistant Staphylococcus aureus (MRSA) is a frequent pathogen in VAP, rapid identification of it, is pivotal. Our aim was to evaluate the utility of quantitative polymerase chain reaction (qPCR) as a useful method for etiologic diagnoses of MRSA pneumonia.MethodsWe performed qPCR for mecA, S. aureus-specific femA-SA, and S. epidermidis-specific femA-SE genes from bronchoalveolar lavage or bronchial washing samples obtained from clinically-suspected VAP. Molecular identification of MRSA was based on the presence of the mecA and femA-SA gene, with the absence of the femA-SE gene. To compensate for the experimental and clinical conditions, we spiked an internal control in the course of DNA extraction. We estimated number of colony-forming units per mL (CFU/mL) of MRSA samples through a standard curve of a serially-diluted reference MRSA strain. We compared the threshold cycle (Ct) value with the microbiologic results of MRSA.ResultsWe obtained the mecA gene standard curve, which showed the detection limit of the mecA gene to be 100 fg, which corresponds to a copy number of 30. We chose cut-off Ct values of 27.94 (equivalent to 1×104 CFU/mL) and 21.78 (equivalent to 1×105 CFU/mL). The sensitivity and specificity of our assay were 88.9% and 88.9% respectively, when compared with quantitative cultures.ConclusionOur results were valuable for diagnosing and identifying pathogens involved in VAP. We believe our modified qPCR is an appropriate tool for the rapid diagnosis of clinical pathogens regarding patients in the intensive care unit.

Natural orifice transluminal endoscopic surgery (NOTES): Assessment of peritoneal bacterial load after intraperitoneal antimicrobial wash and evaluation of hemodynamic changes in a porcine model

Aims. Natural orifice transluminal endoscopic surgery (NOTES) is a promising newly developed procedure; however, it is associated with many complications. The main aim of our study is to assess whether peritoneal wash with antibiotics decreases the bacterial load contamination related to the transgastric approach. Methods. Ten female farm pigs underwent transgastric peritoneoscopy with fallopian tubal ligation. Five pigs were randomized to antibiotic wash of the peritoneal cavity and five to placebo. All animals were given one intravenous dose of antibiotic before the procedure. Hemodynamic variables were continuously monitored throughout the procedure. The next day, peritoneal cultures were taken. The fallopian tubes were inspected to determine the success of ligation and the gastric incision sites were assessed for leakage. Results. No significant difference was noted between the antibiotic peritoneal wash group and the placebo group in terms of peritoneal bacterial load with respective median colony-forming units per ml (CFU/ml) of 0 [0; 1] vs. 0 [0; 4], p = 0.637. No clinically significant hemodynamic changes were noted during the procedure. Conclusions. The results of our study indicate that NOTES carries minimal risk of peritoneal bacterial contamination, regardless of the use of intraperitoneal antibiotics, and is not associated with hemodynamic compromise.

Quantitative PCR for etiologic diagnosis of methicillin-resistant Staphylococcus aureus pneumonia in intensive care unit

identification of MRSA was based on the presence of the mecA and femA-SA gene, with the absence of the femA-SE gene. To compensate for the experimental and clinical condition s, we spiked an internal control in the course of DNA extraction. We estimated number of colony-forming units per mL (CFU/mL) of MRSA samples through a standard curve of a serially-diluted reference MRSA strain. We compared the threshold cycle (Ct) value with the microbiologic results of MRSA. Results: We obtained the mecA gene standard curve, which showed the detection limit of the mecA gene to be 100 fg, which corresponds to a copy number of 30. We chose cut-off Ct values of 27.94 (equivalent to 1×10 4 CFU/mL) and 21.78 (equivalent to 1×10 5 CFU/mL). The sensitivity and specificity of our assay were 88.9% and 88.9% respectively, when compared with quantitative cultures. Conclusion: Our results were valuable for diagnosing and identifying pathogens involved in VAP. We believe our modified qPCR is an appropriate tool for the rapid diagnosis of clinical pathogens regarding patients in the intensive care unit.

Quantification of “bacterial load” in late-onset neonatal sepsis using molecular techniques

Premature infants are at increased risk of bacterial sepsis. Few studies of neonatal infection have quantified the degree of “bacterial load”. Traditionally, bacterial numbers have been estimated by measuring colony-forming-units per ml (CFU/ml), using conventional culture methods. Modern molecular techniques allow direct estimation of bacterial numbers in preterm infants with late-onset sepsis. Levels of bacterial 16S ribosomal RNA (rRNA) can potentially be used to quantify metabolically active bacteria in clinical samples. Objective To estimate the “bacterial load” in infants presenting with late-onset bacterial sepsis (>48 h old) using quantitative molecular methods. Methods A single-centred, observational study was conducted in all infants greater than 48 h old with clinically suspected sepsis. Blood samples (0.5 ml) were taken for a calibrated TaqMan reverse transcriptase polymerase chain reaction (RT-PCR) in addition to normal infection investigations. This assay allows quantitative detection of bacterial 16S rRNA and rDNA. Clinical and laboratory data were recorded at the time of sampling. Copy numbers (rRNA and rDNA) were calculated and corrected for background genetic material to estimate bacterial load. Results 100 samples were taken from 125 babies at a median age of 14 days. Median (interquartile) birth weight was 1.09 (0.81–1.45) kg and gestation was 27.5 (23–40) weeks. The rate of clinically diagnosed late-onset infection was 33%. The mean calculated and corrected copy number estimate (bacterial rRNA+rDNA) was 1.51×107/ml of blood (range 8.91×104 to 9.77×1010). Estimated copy number correlated positively with white blood cell count, neutrophil number and C-reactive protein and negatively with platelet count, lymphocyte count, pH and base deficit. There was no correlation with birth weight or gestational age. Conclusion Bacterial load in late-onset sepsis can be estimated by quantitative RT-PCR using a 16S rRNA assay. Direct comparison to estimates of CFU/ml was not possible because of the variability of rRNA numbers per bacterial cell. However, the potential of this target as a quantitative biomarker of bacterial infection was demonstrated. Increasing numbers of bacteria appear to stimulate greater immune response. Further study is necessary to place these values in context with other age groups and disease severity.

Oral streptococci growth on aging and non-aging esthetic restorations after radiotherapy

The aim of this study was to examine Streptococcus mutans biofilm growth on both aged and non-aged restorative dental resins, which were submitted to therapeutic irradiation. Sixty-four disks of an esthetic restorative material (Filtek Supreme) were divided into 2 groups: aged group (AG) and a non-aged group (NAG). Each group was subdivided into 4 subgroups: non-irradiated and irradiated with 10Gy, 35Gy, and 70Gy. The biofilms were produced by Streptococcus mutans UA159 growing on both AG and NAG surfaces. The colony-forming units per mL (CFU/mL) were evaluated by the ANOVA and the Tukey LSD tests (α=0.05). AG presented smaller amounts of CFU/mL than the NAG before irradiation and after 10Gy of irradiation (p<0.05). AG irradiated with 35 and 70Gy showed increased amount of bacterial biofilm when compared to non-irradiated and 10Gy-irradiated disks (p<0.05). The exposure to ionizing radiation at therapeutic doses promoted changes in bacterial adherence of aged dental restorative material.

Capillary zone electrophoresis for enumeration of Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus in yogurt

Enumeration of Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus is a priority due to their importance in yogurt production. Capillary electrophoresis (CE) of both bacteria could be achieved in 7.2 min with a resolution of 3.2 in the background electrolyte (BGE) containing 4.5 mM Tris(hydroxymethyl) amminomethane (TRIS)–4.5 mM boric acid–0.1 mM ethylenediamine tetraacetate (EDTA) (TBE) buffer (pH 8.4) and 0.05% (v/v) polyethylene oxide (PEO), using a capillary of 47.5 cm (effective length) × 100 μm i.d., injection of 50 mbar × 3 s followed by −5 kV × 120 s, a voltage and temperature of 20 kV and 25 °C, respectively. Appropriate amounts of PEO in the BGE, sample preparation (i.e. vortex) and introduction were key factors for their separation. A short hydrodynamic injection followed by applying reversed polarity voltage could compress the bacteria into narrow zones, which were detected as separated single peaks. Method linearity ( r 2 > 0.99), precision (%RSDs < 9.3%), recovery (% R = 91.7–106.7%) and limit of quantitation (1.0 × 10 6 colony forming unit per mL (CFU/mL)) were satisfactory. Results from the CE analysis of both bacteria in yogurt were not statistically different from those of the plate count method ( P > 0.05). The CE method can be used as an alternative for quantitation of L. delbrueckii subsp. bulgaricus and S. thermophilus in yogurt since it was reliable, simple, cost and labor effective and rapid, allowing the analysis of 3 samples/h (comparing to 2d/sample by plate count method).