Critical analysis of methods to determine growth, control and analysis of biofilms for potential non-submerged antibiofilm surfaces and coatings.

The potential uses for antibiofilm surfaces reach across different sectors with significant resultant economic, societal and health impact. For those interested in using antibiofilm surfaces in the built environment, it is important that efficacy testing methods are relevant, reproducible and standardised where possible, to ensure data outputs are applicable to end-use, and comparable across the literature. Using pre-defined keywords, a review of literature reporting on antimicrobial surfaces (78 articles), within which a potential application was described as non-submerged/non-medical surface or coating with antibiofilm action, was undertaken. The most used methods utilized the growth of biofilm in submerged and static systems. Quantification varied (from most to least commonly used) across colony forming unit counts, non-microscopy fluorescence or spectroscopy, microscopy analysis, direct agar-contact, sequencing, and ELISA. Selection of growth media, microbial species, and incubation temperature also varied. In many cases, definitions of biofilm and attempts to quantify antibiofilm activity were absent or vague. Assessing a surface after biofilm recovery or assessing potential regrowth of a biofilm after initial analysis was almost entirely absent. It is clear the field would benefit from widely agreed and adopted approaches or guidance on how to select and incorporate end-use specific conditions, alongside minimum reporting guidelines may benefit the literature.

Climate change vulnerability of cetaceans in Macaronesia: Insights from a trait-based assessment.

Over the last decades global warming has caused an increase in ocean temperature, acidification and oxygen loss which has led to changes in nutrient cycling and primary production affecting marine species at multiple trophic levels. While knowledge about the impacts of climate change in cetacean's species is still scarce, practitioners and policymakers need information about the species at risk to guide the implementation of conservation measures. To assess cetacean's vulnerability to climate change in the biogeographic region of Macaronesia, we adapted the Marine Mammal Climate Vulnerability Assessment (MMCVA) method and applied it to 21 species management units using an expert elicitation approach. Results showed that over half (62%) of the units assessed presented Very High (5 units) or High (8 units) vulnerability scores. Very High vulnerability scores were found in archipelago associated units of short-finned pilot whales (Globicephala macrorhynchus) and common bottlenose dolphins (Tursiops truncatus), namely in the Canary Islands and Madeira, as well as Risso's dolphins (Grampus griseus) in the Canary Islands. Overall, certainty scores ranged from Very High to Moderate for 67% of units. Over 50% of units showed a high potential for distribution, abundance and phenology changes as a response to climate change. With this study we target current and future information needs of conservation managers in the region, and guide research and monitoring efforts, while contributing to the improvement and validation of trait-based vulnerability approaches under a changing climate.

Intralesional injection of triamcinolone hexacetonide as an alternative treatment for central giant cell lesions: a prospective study.

The aim of this prospective study was to report on the response to treatment of central giant cell lesions (CGCL) with intralesional corticosteroid injections. Consecutive cases of CGCL were treated with a biweekly intralesional injection of 20mg/ml triamcinolone hexacetonide diluted in an anaesthetic solution of 2% lidocaine/epinephrine 1:200 000 at the proportion 1:1. All patients were monitored using cone beam computed tomography. Eleven patients were treated; their ages ranged from 15-34 (mean 22 years); and eight lesions were in the mandible, and three in the maxilla. Three cases were diagnosed as non-aggressive, and eight as aggressive. Six cases presented good results (four aggressive and two non-aggressive); three cases presented a moderate response (two aggressive and one non-aggressive); and two had a poor response to treatment (both aggressive). In four cases with a good response, osteoplasty was done. In all cases with a moderate response, the remaining lesion was curetted. Cases with a poor response were submitted to either curettage or denosumab injections. Corticotherapy, as main or neoadjuvant therapy, may be an option for treatment of CGCL.

Potencial reprodutivo (PR) de touros Nelore avaliados por parâmetros andrológicos e comportamento sexual. Classificação por pontos: nova proposição / [Reproductive potential of Nelore bulls evaluated by andrological parameters and sexual behavior. Classification by points - a new proposition]

Este estudo teve por objetivo comparar variações de parâmetros andrológicos e comportamentais de touros Nelore de diferentes faixas etárias, calcular seu potencial reprodutivo (PR) e propor uma nova tabela de classificação por pontos, de acordo com as médias atualmente alcançadas por eles nas características estudadas. Foram utilizados dados de 6162 exames andrológicos de touros da raça Nelore, entre 12 e 80 meses de idade, em regime de monta natural. O número de touros classificados como aptos consistiu em 88,9% dos animais avaliados (n=5480), sendo 51,6% desses considerados excelentes (n=2827), 41,2% muito bons (n=2257) e 7,2% considerados bons (n=394). Entre os animais questionáveis (n=682; 11,1%), 79,6% foram classificados como inaptos temporários (n=542) e 20,4% (n=139) como animais descarte, de acordo com o exame andrológico, independentemente do teste da libido. O número de touros classificados como excelentes se reduziu para 752 (12,2%) quando dados de comportamento sexual foram incluídos para definição do seu PR. Concluiu-se que o uso de tabelas de classificação andrológica por pontos com atualizações técnicas beneficia a seleção mais apurada de touros Nelore. O teste da libido é ferramenta importante para a determinação do PR, o qual permite melhor aproveitamento dos reprodutores.(AU)

This study aimed to compare variations of andrological and behavioral parameters from Nelore bulls of different ages, to calculate their reproductive potential (RP) and propose a new classification table by points, considering current averages in each reproductive trait studied. Data were collected from 6162 breeding soundness examinations of Nelore bulls aged between 12 and 80 months, under natural mating. According to andrological parameters, regardless of the libido test, the number of bulls classified as approved was 88.9% (n= 5480), being 51.6% considered as excellent (n= 2827), 41.2% very good (n= 2257) and 7.2% considered as good (n= 394). Among the animals considered as questionable (n= 682; 11.1%), 79.6% were classified as temporarily reproved (n= 542) and 20.4% (n= 139) as discarded animals. The number of bulls classified as excellent decreased to 752 (12.2%) when sexual behavior data were included to define their RP. It was concluded that the use of tables for andrological classification by points with technical updates improves the reproductive selection of Nelore bulls. The libido test is an important tool for RP determination which provides better utilization of the sires.(AU)

In vitro and in vivo pharmacological characterization of the synthetic opioid MT-45.

MT-45 is a synthetic opioid that was developed in the 1970s as an analgesic compound. However, in recent years MT-45 has been associated with multiple deaths in Europe and has been included in the class of novel psychoactive substances known as novel synthetic opioids (NSOs). Little is known about the pharmaco-toxicological effects of MT-45. Therefore, we used a dynamic mass redistribution (DMR) assay to investigate the pharmacodynamic profile of this NSO in vitro compared with morphine. We then used in vivo studies to investigate the effect of the acute systemic administration of MT-45 (0.01-15 mg/kg i.p.) on motor and sensorimotor (visual, acoustic and tactile) responses, mechanical and thermal analgesia, muscle strength and body temperature in CD-1 male mice. Higher doses of MT-45 (6-30 mg/kg i.p.) were used to investigate cardiorespiratory changes (heart rate, respiratory rate, SpO2 saturation and pulse distention). All effects of MT-45 were compared with those of morphine. In vitro DMR assay results demonstrated that at human recombinant opioid receptors MT-45 behaves as a potent selective mu agonist with a slightly higher efficacy than morphine. In vivo results showed that MT-45 progressively induces tail elevation at the lowest dose tested (0.01 mg/kg), increased mechanical and thermal antinociception (starting from 1 to 6 mg/kg), decreased visual sensorimotor responses (starting from 3 to 6 mg/kg) and reduced tactile responses, modulated motor performance and induced muscle rigidity at higher doses (15 mg/kg). In addition, at higher doses (15-30 mg/kg) MT-45 impaired the cardiorespiratory functions. All effects were prevented by the administration of the opioid receptor antagonist naloxone. These findings reveal the risks associated with the ingestion of opioids and the importance of studying these drugs and undertaking more clinical studies of the current molecules to better understand possible therapeutic interventions in the case of toxicity.

Parâmetros reprodutivos de touros Nelore (Bos taurus indicus) criados a pasto, em de diferentes faixas etárias / Reproductive parameters of pasture-raised Nelore bulls (Bos taurus indicus) in different age

Este estudo teve o objetivo de demonstrar o efeito da idade sobre as características de circunferência escrotal, cor de pelagem e qualidade seminal, desde a puberdade até após a maturidade sexual. Foram utilizados dados de 6607 exames andrológicos de touros da raça Nelore criados a pasto. Os animais eram de diferentes faixas etárias, variando de 12 até 80 meses. O exame andrológico consistiu em exame clínico reprodutivo, perímetro escrotal (PE), avaliação do sêmen e nota para cor do pelame (COR; 1-4). Estabeleceram-se quatro faixas etárias, que foram comparadas pelo teste de Bonferroni. Os parâmetros seminais PE e COR variaram (P<0,05) conforme a faixa etária dos animais: A) 12-18m: COR=1,45±0,64a, PE=31,63±3,51cma, motilidade total (Mot)=67,73±17,99%a, total de defeitos espermáticos (TDE)=16,22±16,95%a; B) 18-24m: COR=1,50±0,57b, PE=32,00±3,47cma, Mot=69,60±29,13%a, TDE=14,49±15,00%b; C) 24-36m: COR=1,51±0,66b, PE=33,56±3,91cmb, Mot=69,46±15,52%a, TDE=12,29±12,92%c; D) 36-48m: COR=1,60±0,57c, PE=36,66±3,50cmc, Mot=71,04±16,19%b, TDE=10,87±12,97%d; E) >48m: COR=1,64±0,72c, PE=38,00±3,22d, Mot=71,54±15,30b, TDE=9,70±16,95d. Concluiu-se que a faixa etária influencia o tamanho testicular, a cor da pelagem e os parâmetros de qualidade seminal. Com o avançar da idade, ocorre escurecimento do pelo, aumento do perímetro escrotal, da motilidade e do vigor, e redução dos defeitos espermáticos de touros Nelores criados a pasto, avaliados a partir de 12 meses de idade.(AU)

This study aimed to demonstrate the effect of age on bull traits such as scrotal circumference, pelage color, and semen quality, from puberty to post sexual maturity. Data from 6607 breeding soundness examinations of pasture raised Nelore bulls were used. The animals presented different age groups ranging from 12 to 80 months. The andrological examination consisted in reproductive clinical evaluation, assessment of scrotal perimeter (PE). In addition, color of pelage (COR; 1-4) was recorded for each animal. Four age groups were established, which were compared by Bonferroni test. Semen parameters, scrotal circumference (PE) and color of the pelage (COR) varied (P< 0.05) according to the age range: A) 12-18m: COR=1.45±0.64 a , PE=31.63±3.51cm a , Total Motility (Mot)=67.73±17.99% a , Total os sperm defects (TDE)=16.22±16.95% a ; B) 18-24m: COR=1.50±0.57 b , PE=32.00±3.47cm a , Mot=69.60±29.13% a , TDE=14.49±15.00% b ; C) 24-36m: COR=1.51±0.66 b , PE=33.56±3.91cm b , Mot=69.46±15.52% a , TDE=12.29±12.92% c ; D) 36-48m: COR=1.60±0.57 c , PE=36.66±3.50cm c , Mot=71.04±16.19% b , TDE=10.87±12.97% d ; E) >48m: COR=1.64±0.72 c , PE=38.00±3.22 d , Mot=71.54±15.30 b , TDE=9.70±16.95 d . It was concluded that age influences testicular size, pelage color, and semen quality parameters. As the age progresses, there is an increase in scrotal perimeter, hair darkening, sperm motility and vigor, and reduction of sperm morphological defects of pasture raised Nelore bulls, assessed as from as 12 months of age.(AU)

Pharyngeal airway space changes after maxillomandibular advancement: a five-year retrospective study.

The aim of this study was to compare the alterations in three regions of the airway-nasopharynx, oropharynx, and hypopharynx-in relation to the area of the midsagittal plane, volume, and minimal axial area after maxillomandibular advancement (MMA) surgery. Thirty patients who had undergone MMA surgery were evaluated at four time points: preoperative (T0), immediately postoperative (T1), 1year postoperative (T2), and ≥5 years postoperative (T3). All measurements were performed using computed tomography, analyzed in Dolphin Imaging 11.0 Premium 3D software. The area in the midsagittal plane presented a mean increase of 22.0% between T0 and T3 (P<0.001), with the highest increase in the oropharynx (24.1%, P<0.001). The total volumetric increase at T3 was 16.7% (P<0.001), with the highest increase in the nasopharynx (15.7%; P<0.001). The lowest minimal axial area was found for the oropharynx at all time points, and the highest increase in minimal axial area was found for the nasopharynx (114.9%; P<0.001). MMA surgery showed the highest increase in upper posterior airway between T0 and T1, and this was followed by a progressive reduction until T3, but with a statistically significant increase at T3 compared with T0 in all cases.

Impact of the systematic use of the Gugging Swallowing Screen in patients with acute ischaemic stroke.

BACKGROUND AND PURPOSE: Post-stroke dysphagia occurs in up to three quarters of patients with acute stroke and is associated with a higher risk of respiratory infections and poor outcome. Systematic screening of dysphagia in the acute stroke unit is essential to identify patients at risk of aspiration and to provide dietary recommendations. Our study aimed to assess the impact of the systematic application of the Gugging Swallowing Screen (GUSS) in patients with acute ischaemic stroke. METHODS: This was a retrospective study of consecutive patients with acute ischaemic stroke admitted to an acute stroke unit in two time periods: pre-GUSS (February 2014-July 2015), when the 10-mL water-swallowing test was systematically administered, and GUSS (August 2015-October 2016), when the GUSS test was systematically administered. Groups were compared with regard to baseline and stroke characteristics, and the occurrence of stroke-associated pneumonia (SAP), in-hospital death and 3-month outcome. RESULTS: Of the 344 patients who were included in the study (median age 71 years), 51.7% were male with a median National Institutes of Health Stroke Scale score of 11. A total of 204 patients were included during the pre-GUSS period and 140 during the GUSS period. Patients in the GUSS period more frequently had diabetes and partial anterior circulation syndromes, and were more frequently treated with thrombectomy. There was no difference in the occurrence of SAP between the two groups (pre-GUSS, 12.5%; GUSS, 15.1%; P = 0.490) and no differences were found concerning in-hospital mortality (P = 0.996), 3-month functional independence (P = 0.647) or 3-month mortality (P = 0.598). CONCLUSIONS: The systematic administration of GUSS in a population of patients with acute ischaemic stroke did not reduce the occurrence of SAP, mortality or 3-month functional dependence when compared with the systematic administration of the 10-mL water-swallowing test.

Eco-friendly non-biocide-release coatings for marine biofouling prevention.

Environmental concerns have been changing the way of looking for solutions to problems. The hydrosphere, together with its biosphere, has been feeling the impact of many pollutants, used for instance in the marine industry for economic reasons or lack of knowledge of their effects. In particular biocides, applied as coatings in paints, are released into the waters becoming toxic and persistent extending their action to an area far beyond the initial coated surface they should protect. In order to minimize these side effects, two biocides, Irgarol (I) and Econea (E), were covalently attached to polyurethane (PU) and foul-release silicone based (PDMS) marine paints through an isocyanate linker. Their antifouling bioactivity was better in PDMS coatings, both for single (Econea) and combined biocides (E/I ratio = 1.5) with contents lower than 0.6 wt%. The treated samples remained almost clean after more than one year immersion in the Portuguese shore of the Atlantic Ocean, and after about 24 weeks under the tropical conditions of Singapore (Fouling rate < 1%). Complementary biofilm adhesion susceptibility tests against Pseudoalteromonas tunicata D2 showed adhesion reduction higher than 90% for PU formulations containing single biocides and close to 100% for PDMS with combined biocides. The eco-toxicity assessment evidenced a low environmental impact, in accordance with the European standards. In addition, shipping field trial tests showed the best antifouling performance for the Econea-based PDMS formulations (E = 0.6 wt%), which remained clean for about nine months in open seawaters, proving the efficacy of this non-release strategy, when applied under dynamic conditions.

Tongue pressure measurement in children with mouth-breathing behaviour.

The tongue plays an important role in oral functions. Reduced tongue strength is often noted among children with mouth-breathing behaviour. The purposes of this study were to measure the tongue pressure in children with mouth-breathing behaviour, to compare these values to those of children with nasal-breathing behaviour and to analyse the relationship between age and tongue pressure in children with a mouth-breathing pattern and in children with a nasal-breathing pattern. In this cross-sectional analytical observational study, we enroled 40 children aged 5-12 years who either exhibited mouth-breathing behaviour (n = 20) or nasal-breathing behaviour (gender- and age-matched [±2 years] controls; n = 20). Tongue pressure was evaluated using the Iowa Oral Performance Instrument; 3 measurements were recorded for each participant, with a 30-seconds rest interval. The average tongue pressure in the mouth-breathing group was lower than that in the nasal-breathing group. There was no difference in tongue pressure between genders. There was a strong and direct correlation between tongue pressure and age in the nasal-breathing group. The breathing pattern impacts tongue pressure development.

Surface modifications for antimicrobial effects in the healthcare setting: a critical overview.

The spread of infections in healthcare environments is a persistent and growing problem in most countries, aggravated by the development of microbial resistance to antibiotics and disinfectants. In addition to indwelling medical devices (e.g. implants, catheters), such infections may also result from adhesion of microbes either to external solid-water interfaces such as shower caps, taps, drains, etc., or to external solid-gas interfaces such as door handles, clothes, curtains, computer keyboards, etc. The latter are the main focus of the present work, where an overview of antimicrobial coatings for such applications is presented. This review addresses well-established and novel methodologies, including chemical and physical functional modification of surfaces to reduce microbial contamination, as well as the potential risks associated with the implementation of such anticontamination measures. Different chemistry-based approaches are discussed, for instance anti-adhesive surfaces (e.g. superhydrophobic, zwitterions), contact-killing surfaces (e.g. polymer brushes, phages), and biocide-releasing surfaces (e.g. triggered release, quorum sensing-based systems). The review also assesses the impact of topographical modifications at distinct dimensions (micrometre and nanometre orders of magnitude) and the importance of applying safe-by-design criteria (e.g. toxicity, contribution for unwanted acquisition of antimicrobial resistance, long-term stability) when developing and implementing antimicrobial surfaces.

Detection of Escherichia coli O157 by peptide nucleic acid fluorescence in situ hybridization (PNA-FISH) and comparison to a standard culture method.

Despite the emergence of non-O157 Shiga toxin-producing Escherichia coli (STEC) infections, E. coli serotype O157 is still the most commonly identified STEC in the world. It causes high morbidity and mortality and has been responsible for a number of outbreaks in many parts of the world. Various methods have been developed to detect this particular serotype, but standard bacteriological methods remain the gold standard. Here, we propose a new peptide nucleic acid fluorescence in situ hybridization (PNA-FISH) method for the rapid detection of E. coli O157. Testing on 54 representative strains showed that the PNA probe is highly sensitive and specific to E. coli O157. The method then was optimized for detection in food samples. Ground beef and unpasteurized milk samples were artificially contaminated with E. coli O157 concentrations ranging from 1 × 10(-2) to 1 × 10(2) CFU per 25 g or ml of food. Samples were then preenriched and analyzed by both the traditional bacteriological method (ISO 16654:2001) and PNA-FISH. The PNA-FISH method performed well in both types of food matrices with a detection limit of 1 CFU/25 g or ml of food samples. Tests on 60 food samples have shown a specificity value of 100% (95% confidence interval [CI], 82.83 to 100), a sensitivity of 97.22% (95% CI, 83.79 to 99.85%), and an accuracy of 98.33% (CI 95%, 83.41 to 99.91%). Results indicate that PNA-FISH performed as well as the traditional culture methods and can reduce the diagnosis time to 1 day.

Rapid detection of urinary tract infections caused by Proteus spp. using PNA-FISH.

We developed a fluorescence in situ hybridization (FISH) method for the rapid detection of Proteus spp. in urine, using a novel peptide nucleic acid (PNA) probe. Testing on 137 urine samples from patients with urinary tract infections has shown specificity and sensitivity values of 98 % (95 % CI, 93.2-99.7) and 100 % (95 % CI, 80,8-100), respectively, when compared with CHROMagar Orientation medium. Results indicate that PNA-FISH is a reliable alternative to traditional culture methods and can reduce the diagnosis time to approximately 2 h.

Detection of Salmonella enterica serovar Enteritidis using real time PCR, immunocapture assay, PNA FISH and standard culture methods in different types of food samples.

Several methods for the rapid and specific detection of Salmonella in food samples have been described. Here, we compare 4 of those methods in terms of assay time, procedure complexity, detection limit, sensitivity, specificity and accuracy. Milk, eggs and mayonnaise samples were artificially contaminated with Salmonella enterica serovar Enteritidis cell concentrations ranging from 1×10(-2) to 1×10(2) CFU per 25 g or ml of food. Samples were then pre-enriched and analyzed by either: i) real-time PCR, using the iQ-Check Salmonella kit; ii) immunocapture, using the RapidChek SELECT Salmonella; iii) a peptide nucleic acid fluorescence in situ hybridization (PNA FISH) method and iv) the traditional bacteriological method ISO 6579:2002. All methods were able to detect Salmonella in the different types of food matrixes and presented a similar detection level of 1CFU per 25 g or ml of food sample. The immunocapture and the PNA FISH methods proved to be very reliable, as their results were 100% in agreement with the ISO method. However, real-time PCR presented a significant number of false positives, which resulted in a specificity of 55.6% (CI 95%, 31.3-77.6) and an accuracy of 82.2% (CI 95%, 63.2-91.4) for this method. Sensitivity was 100% since no false negative results were observed. In conclusion, the implementation of these molecular techniques, mainly the immunocapture and PNA-FISH methods, provides a reliable and less time-consuming alternative for the detection of Salmonella spp. in food samples.

Optimization of culture conditions to improve Helicobacter pylori growth in Ham's F-12 medium by response surface methodology.

Helicobacter pylori is a gastroduodenal pathogen that colonizes the human stomach and is the causal agent of gastric diseases. From the clinical and epidemiological point of view, enhancing and improving the growth of this bacterium in liquid media is an important goal to achieve in order to allow the performance of accurate physiological studies. The aim of this work was to optimize three culture conditions that influence the growth of H. pylori in the defined medium Ham s F-12 supplemented with 5 percent fetal bovine serum by using response surface methodology as a statistical technique to obtain the optimal conditions. The factors studied in this experimental design (Box-Behnken design) were the pH of the medium, the shaking speed (rpm) and the percentage of atmospheric oxygen, in a total of 17 experiments. The biomass specific growth rate was the response measured. The model was validated for pH and shaking speed. The percentage of atmospheric oxygen did not influence the growth for the range of values studied. At the optimal values found for pH and shaking speed, 8 and 130 rpm, respectively, a specific growth rate value of 0.164 h-1, corresponding to a maximal concentration of approximately 1.5x108 CFU/ml, was reached after 8 h. The experimental design strategy allowed, for the first time, the optimization of H. pylori growth in a semi-synthetic medium, which may be important to improve physiological and metabolic studies of this fastidious bacterium.

Application of flow cytometry for the identification of Staphylococcus epidermidis by peptide nucleic acid fluorescence in situ hybridization (PNA FISH) in blood samples.

Staphylococcus epidermidis is considered to be one of the most common causes of nosocomial bloodstream infections, particularly in immune-compromised individuals. Here, we report the development and application of a novel peptide nucleic acid probe for the specific detection of S. epidermidis by fluorescence in situ hybridization. The theoretical estimates of probe matching specificity and sensitivity were 89 and 87%, respectively. More importantly, the probe was shown not to hybridize with closely related species such as Staphylococcus aureus. The method was subsequently successfully adapted for the detection of S. epidermidis in mixed-species blood cultures both by microscopy and flow cytometry.

Fluorescence in situ hybridization method using a peptide nucleic acid probe for identification of Salmonella spp. in a broad spectrum of samples.

A fluorescence in situ hybridization (FISH) method for the rapid detection of Salmonella spp. using a novel peptide nucleic acid (PNA) probe was developed. The probe theoretical specificity and sensitivity were both 100%. The PNA-FISH method was optimized, and laboratory testing on representative strains from the Salmonella genus subspecies and several related bacterial species confirmed the predicted theoretical values of specificity and sensitivity. The PNA-FISH method has been successfully adapted to detect cells in suspension and is hence able to be employed for the detection of this bacterium in blood, feces, water, and powdered infant formula (PIF). The blood and PIF samples were artificially contaminated with decreasing pathogen concentrations. After the use of an enrichment step, the PNA-FISH method was able to detect 1 CFU per 10 ml of blood (5 x 10(9) +/- 5 x 10(8) CFU/ml after an overnight enrichment step) and also 1 CFU per 10 g of PIF (2 x 10(7) +/- 5 x 10(6) CFU/ml after an 8-h enrichment step). The feces and water samples were also enriched according to the corresponding International Organization for Standardization methods, and results showed that the PNA-FISH method was able to detect Salmonella immediately after the first enrichment step was conducted. Moreover, the probe was able to discriminate the bacterium in a mixed microbial population in feces and water by counter-staining with 4',6-diamidino-2-phenylindole (DAPI). This new method is applicable to a broad spectrum of samples and takes less than 20 h to obtain a diagnosis, except for PIF samples, where the analysis takes less than 12 h. This procedure may be used for food processing and municipal water control and also in clinical settings, representing an improved alternative to culture-based techniques and to the existing Salmonella PNA probe, Sal23S10, which presents a lower specificity.

Effect of chlorine on incorporation of Helicobacter pylori into drinking water biofilms.

The use of a specific peptide nucleic acid (PNA) probe demonstrated that Helicobacter pylori persisted inside biofilms exposed to low concentrations of chlorine (0.2 and 1.2 mg liter(-1)) for at least 26 days, although no culturable cells were recovered. Coupled with data obtained using viability stains in pure culture, this result suggests that H. pylori can survive chlorination but remain undetectable by culture methods, which can be effectively replaced by PNA hybridization.

Development and application of a novel peptide nucleic acid probe for the specific detection of Cronobacter genomospecies (Enterobacter sakazakii) in powdered infant formula.

Here, we report a fluorescence in situ hybridization (FISH) method for rapid detection of Cronobacter strains in powdered infant formula (PIF) using a novel peptide nucleic acid (PNA) probe. Laboratory tests with several Enterobacteriaceae species showed that the specificity and sensitivity of the method were 100%. FISH using PNA could detect as few as 1 CFU per 10 g of Cronobacter in PIF after an 8-h enrichment step, even in a mixed population containing bacterial contaminants.