Search

Background Analytical methods applied in diagnostic microbiology laboratories are usually performed manually and have several drawbacks, the long time-to-results (48–72 h) being the major one. Rapid detection methods may overcome the disadvantages of traditional microbiological methods and achieve fast detection, which will help preventing the spread of waterborne pathogens and outbreaks of waterborne diseases. The objective of the MICROALARM project is to validate a platform for monitoring the quantification of the total bacteria present in water samples. Methods The MICROALARM system integrates a microfluidic cartridge where the microorganisms from the sample are labelled to facilitate the detection and measurement by a fluorescence sensor. The prototype system has been validated according to ISO 16140-2 : 2016 in the laboratory through the comparison with conventional (culture isolation) and alternative methods (flow cytometry) to determine whether the method is suitable for quantifying microorganisms in water samples. Results The system will be capable of processing, labelling and quantifying automatically the total bacteria of the water sample using a membrane-permeable fluorescence marker. The system has been validated at the laboratory scale using a wide range of water matrices, then installed and validated in different facilities with satisfactory results. Conclusion The MICROALARM system is designed to be portable, thus suitable for on-site applications. It will be a low-cost solution for rapid microbiological analysis. The system will avoid water sampling and transport to the laboratory while enabling on-site quantification of the total bacteria in a reduced timeframe for early decision-making.

We introduce an image cytometer (I-CYT) for the analysis of phytoplankton in fresh and marine water environments. A linear quantification of cell numbers was observed covering several orders of magnitude using cultures of Tetraselmis and Nannochloropsis measured by autofluorescence in a laboratory environment. We assessed the functionality of the system outside the laboratory by phytoplankton quantification of samples taken from a marine water environment (Dutch Wadden Sea, The Netherlands) and a fresh water environment (Lake Ijssel, The Netherlands). The I-CYT was also employed to study the effects of two ballast water treatment systems (BWTS), based on chlorine electrolysis and UV sterilization, with the analysis including the vitality of the phytoplankton. For comparative study and benchmarking of the I-CYT, a standard flow cytometer was used. Our results prove a limit of detection (LOD) of 10 cells/ml with an accuracy between 0.7 and 0.5 log, and a correlation of 88.29% in quantification and 96.21% in vitality, with respect to the flow cytometry results.

We introduce a Fourier imaging cytometer for ballast water analysis. We demonstrate detection of Tetraselmis and Nannochloropsis by autofluorescence, and E. faecalis and V. cholerae by brightfield scattering. Achieving sensitivity of ~95% and specificity of ~80%.

Standard operating procedures used for the detection of bacteria in environmental samples are primarily based on bacterial growth on specific culture media and confirmation by biochemical and/or immunological tests. In the case of Legionella, isolation on BCYE-α medium is the standard method, although it presents a number of drawbacks, and for this reason, the implementation of molecular methods, mainly those based on PCR, has increased over the last years. Following the ISO/IEC 17025, laboratories need an external evaluation of their work to assure the quality of the results they are producing, and the participation in proficiency testing (PT) schemes is compulsory. For those water-testing laboratories using PCR methods for Legionella, we have developed a PT scheme accredited according to ISO/IEC 17043. The preparation and the statistical analysis of the results are performed following this standard and the ISO 13528. The used samples have a very rapid and easy to use format, consisting of tablets with inactivated freeze-dried Legionella cells or freeze-dried Legionella DNA. In this PT scheme, participants evaluate both Legionella pneumophila and Legionella spp. detection systems and control the whole PCR process from the water sample concentration until the PCR results.

The standard methodology used for the detection of bacteria in environmental samples and food is primarily based on bacterial growth on specific culture media and confirmation by biochemical and/or immunological tests of all presumptive colonies. However, this methodology presents a number of drawbacks, such as low sensitivity and specificity, and the long time needed to obtain results. For this reason, the implementation of molecular methods in diagnostic laboratories has increased over the past several years. Nevertheless, most of these newer methods have not been included in current legislation, and, in most of cases, they have not yet been normalized. In this sense, the availability of appropriate reference materials (RMs) can help to overcome these deficiencies. The aim of this study was to develop and validate, following ISO Guide 34, a new RM, in a tablet format, for the quantification of Legionella pneumophila and Salmonella spp. by quantitative PCR (qPCR). This new RM can be used as a work reference sample in internal quality control, in the organization of proficiency testing schemes (PTS), as well as for the validation of molecular methods based on qPCR.

This research found evidence of an association between occupational exposure to bioaerosols in composting plants and health outcome occurrence in exposed workers. An occupational exposure assessment in six composting plants was performed to better characterize personal exposure levels and evaluate associated health risk in workers. Sampling results showed large ranges of concentrations of dust, bacteria, molds, and endotoxin in ambient air and in personal samples, both when driving a front-end loader and when cleaning, monitoring, and performing maintenance tasks. Mean personal exposure levels were high at 100 to more than 10 000 times higher than outdoor background levels and fully consistent with occurrence of inflammatory and allergic respiratory outcomes among workers. Engineering control, personal protection, and education and training programs for employees, health, and safety officials, and occupational physicians are being developed and implemented.

Aims: A new real-time PCR assay that simultaneously amplifies a 102-bp fragment of the cagE gene from Helicobacter pylori and a new internal positive control containing a specific sequence of the gyrB gene from Aeromonas hydrophila, was developed and validated for the detection of H. pylori in environmental samples. Methods and Results: The specificity, limits of detection and quantification, repeatability, reproducibility, and accuracy of the method were calculated. The resulting values confirmed the applicability of the method for the quantitative detection of H. pylori. The feasibility of the method was also evaluated by testing 13 pyloric antrum-positive biopsies and 69 water samples, including potable (10), surface (19) and wastewater (40) matrices. The results showed that all the biopsies and 3 of the 40 wastewater samples analysed were positive. Conclusions: This real-time PCR method provides a sensitive, specific, and accurate method for the rapid quantification of H. pylori in environmental samples. Significance and Impact of the Study: The PCR diagnostic system proposed in this work, provides a suitable tool for the quantitative detection of H. pylori in environmental samples and can be useful for verifying the role of water as a potential route of its transmission.

Airborne transmission is an important route for many microbial pathogens in outdoor and indoor environments, including hospitals. A 2-year-long survey of bioaerosol quality in operating theatres (OT), hospital rooms (HR) and maternity wards (MW) at a hospital in Murcia, Spain, was performed. Total aerobic counts (TAC) and fungal load (FL) were assessed using a microbiological air sampler (MAS-100 single-stage impactor). While fungal levels were below 1 cfu/m(3) (0-7.33 cfu/m(3)) in OT, they were higher in MW (mean, 6.9 cfu/m(3); range 0.44-44.67 cfu/m(3)) and in HR (mean, 10.6 cfu/m(3); range, 0-266 cfu/m(3)). In OT the aerobic counts were considerably higher, with a mean of 25.6 cfu/m(3) (range, 1.67-157 cfu/m(3)). MW and HR also showed higher means for total aerobic counts compared to OT. Seasonal changes were not detected in mould and bacteria levels in OT. Hospital renovation occurred during this study and OT adjacent to renovated areas were closed. A survey of TAC and FL in OT resumed when renovation was completed. We observed an outstanding increase in FL (more than 100 cfu/m(3)), particularly Aspergillus spp., during this period, but no significant changes in TAC were observed after renovation.

In this report we describe the results of astudy conducted to better estimate airbornemicroorganisms present in wastewater treatmentplants (WWTP) environments. Air samples weretaken with the Air Sampler MAS 100 impactor(Merck) at each stage of an activated-sludgeWWTP (pre-treatment, primary clarifiers,aeration basins and sludge processors), inorder to determine levels of contaminationaccording to work stations. Culture methodswere used to investigate heterotrophic platecount, moulds and yeasts, total and fecalcoliforms, Pseudomonas aeruginosa andspecies belonging to Mycobacteriumtuberculosis complex, whereas these lastmicroorganisms were also analysed by PolymeraseChain Reaction (PCR) methods. Statisticalanalysis showed that pretreatment and primaryclarifiers were the stages with the highestemission of bioaerosols, and that bioaerosolconcentration at each stage depended on windspeed and daily inflow at the WWTP. Lowconcentrations of P. aeruginosa werefound isolated at pretreatment and primaryclarifiers; however, species of M.tuberculosis complex were not detected at anystage of the treatment plant.

Most studies focusing on detecting microorganisms in air by polymerase chain reaction (PCR) have used a liquid impinger to sample bioaerosols, mainly because a liquid sample is easy to be processed by PCR analysis. Nevertheless, the use of multiple-hole impactors for the analysis of bioaerosols by PCR has not been reported despite its great utility in culture analysis. In this study we have modified the impaction onto an agar surface sampling method to impaction onto a liquid medium using the MAS-100 air sampler (Merck) (single-stage multiple-hole impactor). To evaluate the recovery of airborne microorganisms of both sampling methods, a suspension containing Escherichia coli was artificially aerosolized and bioaerosols were collected onto Tergitol-7 agar and phosphate-buffered saline (PBS) with the MAS-100. A linear regression analysis of the results showed a strong positive correlation between both sampling methods (r = 0.99, slope 0.99, and y intercept 0.07). Afterwards, the method of impingement into a liquid medium was used to study airborne Legionella pneumophila by PCR. A total of 64 samples were taken at a wastewater treatment plant, a chemical plant, and an office building and analyzed by culture and PCR. Results showed that three samples were positive both by PCR and plate culture, and that nine samples negative by plate culture were positive by PCR, proving that L. pneumophila was present in bioaerosols from these three different environments. The results demonstrate the utility of this single-stage multiple-hole impactor for sampling bioaerosols, both by culture and by PCR.Key words: Legionella pneumophila, bioaerosols, PCR, multiple-hole impactor.

Most studies focusing on detecting microorganisms in air by polymerase chain reaction (PCR) have used a liquid impinger to sample bioaerosols, mainly because a liquid sample is easy to be processed by PCR analysis. Nevertheless, the use of multiple-hole impactors for the analysis of bioaerosols by PCR has not been reported despite its great utility in culture analysis. In this study we have modified the impaction onto an agar surface sampling method to impaction onto a liquid medium using the MAS-100 air sampler (Merck) (single-stage multiple-hole impactor). To evaluate the recovery of airborne microorganisms of both sampling methods, a suspension containing Escherichia coli was artificially aerosolized and bioaerosols were collected onto Tergitol-7 agar and phosphate-buffered saline (PBS) with the MAS-100. A linear regression analysis of the results showed a strong positive correlation between both sampling methods (r = 0.99, slope 0.99, and y intercept 0.07). Afterwards, the method of impingement into a liquid medium was used to study airborne Legionella pneumophila by PCR. A total of 64 samples were taken at a wastewater treatment plant, a chemical plant, and an office building and analyzed by culture and PCR. Results showed that three samples were positive both by PCR and plate culture, and that nine samples negative by plate culture were positive by PCR, proving that L. pneumophila was present in bioaerosols from these three different environments. The results demonstrate the utility of this single-stage multiple-hole impactor for sampling bioaerosols, both by culture and by PCR.

In this paper we describe a simple method, noncorrosive to pipes, for the eradication of Legionella pneumophila from potable water systems. This method is based on the systematic purging of the pipe networks with cold water containing 1 – 1.5 mg residual chlorine/L. In the hot water system, a new pipe bypassing the water heater was installed, whereas in the air conditioning system, the circuit is purged with water from the tap water system. The feasibility of this method was studied in two hotels in which the presence of Legionella was detected despite treatment of the water by the hyperchlorination method. The evolution of the presence of Legionella was studied by culture and polymerase chain reaction. Eighty samples from hotel A and sixty-seven samples from hotel B were analyzed during the time that the eradication method was applied. Our results showed that this method permitted the effective elimination of L. pneumophila after 5 months in hotel A and 7 months in hotel B.Key words: Legionella pneumophila, eradication.

Legionella pneumophila infects humans to produce legionellosis and Pontiac fever only from environmental sources. In order to establish control measures and study the sources of outbreaks it is essential to know extent and distribution of strain variants of this bacterium in the environment. Sporadic and outbreak-related cases of legionellosis have been historically frequent in the Comunidad Valenciana region (CV, Spain), with a high prevalence in its Southeastern-most part (BV). Environmental investigations for the detection of Legionella pneumophila are performed in this area routinely. We present a population genetics study of 87 L. pneumophila strains isolated in 13 different localities of the BV area irrigated from the same watershed and compare them to a dataset of 46 strains isolated in different points of the whole CV. Our goal was to compare environmental genetic variation at two different geographic scales, at county and regional levels. Genetic diversity, recombination and population structure were analyzed with Sequence-Based Typing data and three intergenic regions. The results obtained reveal a low, but detectable, level of genetic differentiation between both datasets, mainly, but not only, attributed to the occurrence of unusual variants of the neuA locus present in the BV populations. This differentiation is still detectable when the 10 loci considered are analyzed independently, despite the relatively high incidence of the most common genetic variant in this species, sequence type 1 (ST-1). However, when the genetic data are considered without their associated geographic information, four major groups could be inferred at the genetic level which did not show any correlation with sampling locations. The overall results indicate that the population structure of these environmental samples results from the joint action of a global, widespread ST-1 along with genetic differentiation at shorter geographic distances, which in this case are related to the common watershed for the BV localities.

One of the greatest challenges of implementing fast molecular detection methods as part of Legionella surveillance systems is to limit detection to live cells. In this work, a protocol for sample treatment with propidium monoazide (PMA) in combination with quantitative PCR (qPCR) has been optimized and validated for L. pneumophila as an alternative of the currently used time-consuming culture method. Results from PMA-qPCR were compared with culture isolation and traditional qPCR. Under the conditions used, sample treatment with 50 μM PMA followed by 5 min of light exposure were assumed optimal resulting in an average reduction of 4.45 log units of the qPCR signal from heat-killed cells. When applied to environmental samples (including water from cooling water towers, hospitals, spas, hot water systems in hotels, and tap water), different degrees of correlations between the three methods were obtained which might be explained by different matrix properties, but also varying degrees of non-culturable cells. It was furthermore shown that PMA displayed substantially lower cytotoxicity with Legionella than the alternative dye ethidium monoazide (EMA) when exposing live cells to the dye followed by plate counting. This result confirmed the findings with other species that PMA is less membrane-permeant and more selective for the intact cells. In conclusion, PMA-qPCR is a promising technique for limiting detection to intact cells and makes Legionella surveillance data substantially more relevant in comparison with qPCR alone. For future research it would be desirable to increase the method's capacity to exclude signals from dead cells in difficult matrices or samples containing high numbers of dead cells.

We report a new seminested PCR method for detection of Legionella pneumophila based on the simultaneous amplification of a 387 bp fragment of the dotA gene and a 827 bp recombinant fragment that serves as an internal positive control. This new detection system was validated and its specificity and detection limit were determined. Parallel analysis of 90 environmental samples to compare this method, a real-time PCR method and the standard culture isolation method, demonstrated that this seminested method is useful for the study of L. pneumophila.

Summary Legionella pneumophila is associated to recurrent outbreaks in several Comunidad Valenciana (Spain) localities, especially in Alcoi, where social and climatic conditions seem to provide an excellent environment for bacterial growth. We have analysed the nucleotide sequences of three loci from 25 environmental isolates from Alcoi and nearby locations sampled over 3 years. The analysis of these isolates has revealed a substan- tial level of genetic variation, with consistent patterns of variability across loci, and comparable to that found in a large, European-wide sampling of clinical isolates. Among the tree loci studied, fliC showed the highest level of nucleotide diversity. The analysis of isolates sampled in different years revealed a clear differenti- ation, with samples from 2001 being significantly distinct from those obtained in 2002 and 2003. Furthermore, although linkage disequilibrium mea- sures indicate a clonal nature for population structure in this sample, the presence of some recombination events cannot be ruled out.

Aim: In this study, we evaluated, the use of universal primers, specific for the 16S-23S rRNA intergenic region, to detect and identify nine species that are of high interest for the microbiological control of water. Methods and Results: The analysis of the fragments was carried out using a High Resolution acrylamide/bisacrylamide gels in a fluorescent automated DNA sequencer. The results showed specific profiles for each of the nine species but this technique failed to detect simultaneously micro-organisms in samples containing a mixed population. Conclusion: Nevertheless, the electrophoretic profiles obtained provided a very useful tool for the rapid and specific identification of water isolates. Significance and impact of the study: A possible new methodology for a rapid identification of pathogens in water.

The genus Legionella is represented by 48 species and Legionella pneumophila includes 15 serogroups. In this work, we have studied the intergenic 16S-23S spacer region (ITS) in L. pneumophila to determine the feasability of using amplification polymorphisms in this region, to establish intraspecies differences and to discriminate Legionella species. The amplification of this region, using 16S14F and 23S0R primers, and the analysis of amplicons by the analysis of fragments technique showed that all the L. pneumophila serogroups studied presented the same electrophoretic pattern. Moreover, the analysis of different Legionella species showed that the amplification polymorphisms obtained were species-specific. In order to study the sequence variability of this region, the existence in L. pneumophila of three ribosomal operons was determined by pulsed field gel eletrophoresis (PFGE). Two of the 16S-23S rRNA ITS presented a tRNA Ala and the third one a tRNA Ile. Nevertheless, the variability expected in this region of the operon was not found and the rest of the ITS contained only punctual mutations.

To analyze the information provided by computed tomography (CT) in Fournier's gangrene, particularly in regard to the early diagnosis of the disease, and its utility as a complementary diagnostic imaging technique.A retrospective study identified five cases of Fournier's gangrene. These patients had been evaluated by CT at least twice. The radiological, clinical and epidemiological aspects are analyzed.Fournier's gangrene was diagnosed in one patient with no clinical suspicion of this disease. Gas was demonstrated in the scrotal region in five patients and in the perineal region in four patients. The abdominal wall was the most frequent extragenito-perineal location. A good correlation was found between the CT and surgical findings in all cases.CT evaluation prior to surgery is recommended in patients with Fournier's gangrene, particularly in order to determine the extent of the disease.

Presentation of one case of CRE (crossed renale ectopia) with triplicity (trifidity) of the ortho and ectopic collector systems, due to the existence in both cases of pyelourethral bifidity and a third cranial blind ureter. This case represents a casual finding during a routine study of a female patient with orthostatic and exertion urinary incontinence (EUI). The case idiosyncrasies, which initially elicited the possibility of urethral ectopia, are analyzed. Incidence and variants of this unusual congenital anomaly are commented upon.

Case report of a pubertal female patient with clinical signs of hypogastric pain initiated with the menarche, dysmenorrhoea and a palpable pelvic mass. The suspected clinical signs, ultrasound scans, urographies and CAT, confirmed the existence of an uterovaginal duplicity with right hematocolpos due to imperforate septum and pool of menstrual blood, as well as ipsilateral renal agenesis. The vagina examination and resection of the septum under sedation solved the case.