Prevalencia de legionella en instalaciones de suministro de agua en España: revisión sistemática y meta-análisis / Prevalence of legionella species in water supply facilities of Spain: a systematic review and meta-analysis

Antecedentes: Las especies de Legionella tienen su hábitat natural o reservorio primario en las aguas dulces superficiales de lagos, ríos, estanques y aguas termales, desde donde a través de los sistemas de suministro colonizan el agua de consumo. El presente estudio pretende investigar la prevalencia de Legionella en instalaciones de agua de consumo de España. Métodos: Se realizó una revisión sistemática mediante la búsqueda en bases de datos electrónicas de información científica de estudios observacionales sobre contaminación de agua de consumo por Legionella, con resultados de análisis de muestras de agua obtenidos entre enero de 2001 y abril de 2021. Se efectuó meta-análisis mediante el software MetaXL© en la hoja de cálculo Microsoft Excel. Resultados: Se seleccionaron un total de 21 estudios. La prevalencia de Legionella en España fue de 21,8 % (IC 95 %: 15,0-29,6). La especie más comúnmente aislada fue L. pneumophila serogrupos 2-15 (44,4 %, IC 95 %: 29,5-59,8). Los valores más altos se hallaron en puntos terminales de las redes de agua caliente sanitaria (31,7 %, IC 95 %: 21,6-42,9), y en las comunidades autónomas de Aragón (24,7 %, IC 95 %: 8,8-44,9) y Cataluña (21,3 %, IC 95%: 4,4-44,8).Conclusiones: La prevalencia global de Legionella obtenida puede compararse con los estudios de otros autores, siendo en líneas generales cercana a la obtenida a nivel mundial, con variaciones en relación a otros países. Las instalaciones con mayor prevalencia han resultado ser las de mayor riesgo de proliferación y dispersión de la bacteria según la normativa vigente, y aquellas situadas en la mitad oriental de España.(AU)

Background: Legionella species have their natural habitat or primary reservoir in the fresh surface waters of lakes, rivers, ponds and hot springs, from where they colonize drinking water through supply systems. The present study aims to investigate the prevalence of Legionella in drinking water facilities in Spain.Methods: A systematic review was performed by searching in electronic databases of scientific information for observational studies on contamination of drinking water by Legionella, with results of analysis of water samples obtained between January 2001 and April 2021. Meta-analysis was carried out using the MetaXL© software in the Microsoft Excel spreadsheet. Results: A total of 21 studies were selected. The prevalence of Legionella in Spain was 21.8 % (95 % CI: 15.0-29.6). The most commonly isolated species were L. pneumophila serogroups 2-15 (44.4 %, 95 % CI: 29.5-59.8). The highest values were found at terminal points of the sanitary hot water networks (31.7 %, 95 % CI: 21.6-42.9), and in the autonomous regions of Aragon (24.7 %, 95 % CI: 8.8-44.9) and Catalonia (21.3 %, 95 % CI: 4.4-44.8). Conclusions: The overall prevalence of Legionella obtained can be compared to studies performed by other authors, being in general terms close to that obtained worldwide, with variations in relation to other countries. The facilities with the highest prevalence have turned out to be those with the highest risk of proliferation and dispersal of the bacteria according to current regulations, and those located in the eastern half of Spain.(AU)

Prevalence of Salmonella enterica serovars and genovars from chicken carcasses in slaughterhouses in Spain.

AIMS: To determine the prevalence of Salmonella enterica serovars in chicken carcasses in slaughterhouses in Spain and to examine genotypic relations among these serovars. METHODS AND RESULTS: A total of 336 chicken carcasses were collected from six slaughterhouses in Northwestern Spain. Salmonellae were isolated (ISO-6579-1993), serotyped, phage-typed, ribotyped and antibiotyped against 20 antibiotics. Salmonella strains were detected in 60 (17.9%) carcasses. Isolates belonged to nine different serotypes, with Salm. Enteritidis being the most common. Three strains (5%) were resistant to one antibiotic and 24 (40%) were multi-resistant (to more than one antibiotic). The most frequently encountered resistances were to sulphamides, fluoroquinolones and tetracycline. Ribotyping was able to differentiate isolates of the same serotype and phage type. CONCLUSIONS: The Salmonella serotypes and phage types detected are among those most frequently associated with human diseases in Spain. The large percentage of antimicrobial resistant strains is a matter for concern. A high genetic relationship between strains from different slaughterhouses was found. SIGNIFICANCE AND IMPACT OF THE STUDY: This study provides detailed information about Salmonella isolates from poultry in Spain. It emphasizes the importance of controlling this pathogen in poultry products, and suggests the need for more prudent use of antibiotics.

Artificial neural network based identification of Campylobacter species by Fourier transform infrared spectroscopy.

Two prototypes of artificial neural network (ANN), multilayer perceptron (MLP), and probabilistic neural network (PNN), were used to analyze infrared (IR) spectral data obtained from intact cells belonging to the species Campylobacter coli and Campylobacter jejuni. In order to establish a consistent identification and typing procedure, mid infrared spectra of these species were obtained by means of a Fourier transform infrared (FT-IR) spectroscope. FT-IR patterns belonging to 26 isolates subclassified into 4 genotypes were pre-processed (normalized, smoothed and derivatized) and grouped into training, verification and test sets. The two architectures tested (PNN, MLP) were developed and trained to identify or leave unassigned a number of IR patterns. Two window ranges (w(4), 1200 to 900 cm(-1); and w(5), 900 to 700 cm(-1)) in the mid IR spectrum were presented as input to the ANN models functioning as pattern recognition systems. No matter the ANN used all the training sets were correctly identified at subspecies level. For the test set, the four-layer MLP network was found to be specially suitable to recognize FT-IR data since it correctly identified 99.16% of unknowns using the w(4) range, and was fully successful in detecting atypical patterns from closely related Campylobacter strains and other bacterial species. The PNN network obtained lower percentages in assignation and rejection. Overall, ANNs constitute an excellent mathematical tool in microbial identification, since they are able to recognize with a high degree of confidence typical as well as atypical FT-IR fingerprints from Campylobacter spp.

Discrimination of enterobacterial repetitive intergenic consensus PCR types of Campylobacter coli and Campylobacter jejuni by Fourier transform infrared spectroscopy.

Fourier transform infrared spectroscopy (FT-IR) has been used together with pattern recognition methodology to study isolates belonging to the species Campylobacter coli and Campylobacter jejuni and to compare FT-IR typing schemes with established genomic profiles based on enterobacterial repetitive intergenic consensus PCR (ERIC-PCR). Seventeen isolates were cultivated under standardized conditions for 2, 3, and 4 days to study variability and improve reproducibility. ERIC-PCR profiles and FT-IR spectra were obtained from strains belonging to the species Campylobacter coli and C. jejuni, normalized, and explored by hierarchical clustering and stepwise discriminant analysis. Strains could be differentiated by using mainly the first-derivative FT-IR spectral range, 1,200 to 900 cm(-1) (described as the carbohydrate region). The reproducibility index varied depending on the ages of the cultures and on the spectral ranges investigated. Classification obtained by FT-IR spectroscopy provided valuable taxonomic information and was mostly in agreement with data from the genotypic method, ERIC-PCR. The classification functions obtained from the discriminant analysis allowed the identification of 98.72% of isolates from the validation set. FT-IR can serve as a valuable tool in the classification, identification, and typing of thermophilic Campylobacter isolates, and a number of types can be differentiated by means of FT-IR spectroscopy.

Characterization of Staphylococcus aureus isolated from poultry meat in Spain.

Ninety-six Staphylococcus aureus isolates from retail chicken carcasses in Spain were characterized using cultural and biochemical tests. The strains were phage typed with the international bacteriophage set for typing S. aureus of human origin. Eighty-eight (91.7%) strains were of the poultry ecovar. Strains of human ecovar were not found. These facts are congruent with findings of other authors. Ninety (93.7%) strains were phage typeable. Lysis by phages of Group III was the most frequent with 66 (68.7%) sensitive strains. Twenty-eight (29.2%) strains were sensitive at 100 routine test dilution (RTD) and only 16 (16.7%) at RTD. By using reversed phage typing, we managed to increase the number of phage typeable strains by 46 (47.9%). More than one S. aureus phage type was detected in 14 (35%) carcasses, which emphasizes the convenience of subtyping several S. aureus isolates from the same food sample in epidemiological studies. Two phage patterns (75/84 and 6/1030/ W57) were the most common. The S. aureus isolates were closely related, as 78 strains showed the most common or indistinguishable (<2 phage reaction differences) phage patterns.

Evaluation of the international phage typing set and some experimental phages for typing of Listeria monocytogenes from poultry in Spain.

AIMS: The validity of the international phage set and 13 experimental phages for subtyping Listeria monocytogenes strains isolated from poultry in Spain was investigated. METHODS AND RESULTS: Ninety-six L. monocytogenes strains (52 from serogroup 1/2 and 44 from serogroup 4) were phage-typed using the international phage set, 10 experimental phages for typing serogroup 1/2 strains (seven isolated in France: 1313, 9425, 1807, 351, 881, 717 and 586-, and three from Denmark: 5775, 12682 and 6223-) and three experimental phages isolated in France for typing serogroup 4 strains (2425 A, 4286 and 197). Percentages of serogroup 1/2, serogroup 4 and total phage-typeable strains were 57.7%, 52.3% and 55.2%, respectively. Important differences in the behaviour of the phages tested were found. The typeability rate, the specificity index and the percentage of strong reactions were greater in the phages of international set than in the experimental phages. The number of phage typeable strains and the number of phage types (42) were not modified by the use of experimental phages. CONCLUSIONS: The phage set used was not effective for typing L. monocytogenes strains from poultry in Spain, because a low typeability rate was found. SIGNIFICANCE AND IMPACT OF THE STUDY: Our results suggest the importance of the availability of new phages specific to a geographical area in order to improve the typeability of the system.

Comparison of the acidifying activity of Lactococcus lactis subsp. lactis strains isolated from goat's milk and Valdeteja cheese.

AIMS: This work was carried out to study the acid production by Lactococcus lactis subsp. lactis strains isolated from goat's milk and goat cheese (Valdeteja variety) in order to select a suitable starter culture for industrial goat cheese manufacturing. METHODS AND RESULTS: The titrable acidity of 45 Lactococcus lactis subsp. lactis strains isolated from a home-made batch of Valdeteja cheese with excellent sensory characteristics was measured over a period of 18 h. The strains were divided into two groups depending on the acid production rate: 20 fast acid producer (F) strains and 25 slow acid producer (S) strains. The kinetic parameters (lag phase, maximum acid production rate and value of upper asymptote curve) of the acid production curves for F and S strains were significantly (P < 0.001) different. CONCLUSIONS: Significant (P < 0.001) differences between titrable acidity of F and S strains were observed after the second hour of incubation. SIGNIFICANCE AND IMPACT OF THE STUDY: An F strain acetoin producer (Lactococcus lactis subsp. lactis 470Ch2) was selected as autochthonous starter culture for industrial Valdeteja goat cheese manufacturing.

Efficacy of trisodium phosphate solutions in reducing Listeria monocytogenes populations on chicken skin during refrigerated storage.

Chicken skin inoculated with l0(8) CFU/ml of Listeria monocytogenes was dipped for 15 min in sterile water (control) and in 8, 10, or 12% trisodium phosphate (TSP) solutions. Skin samples were stored at 2 degrees C for 5 days, with microbial monitoring on days 0, 1, 3, and 5 after treatment. Compared to the water dip, all TSP treatments significantly (P < 0.05) reduced L monocytogenes populations on chicken skin. The concentration of the TSP was a significant factor in reducing the populations of the bacteria at days 0, 1, 3, and 5 of refrigerated storage. For all sampling times, the best outcomes were attained with the highest TSP concentration studied (12%). Bacterial reductions in counts during the first day of storage were between 1.52 and 2.70 log10 cycles for 8 and 12% TSP-treated samples, respectively. Significantly greater reductions were observed from the third day of refrigerated storage onward. This occurred largely because populations of L. monocytogenes on control samples increased somewhat, but on TSP-treated samples the pathogen remained practically constant. Differences between L monocytogenes counts in skin samples immersed in water and those treated with TSP ranged from 2.10 (8% TSP-treated samples) and 3.63 (12% TSP-treated samples) log10 cycles on day 5 of storage. These results indicated that TSP is effective against L. monocytogenes in chicken meat, especially after several days of refrigerated storage.

Comparison of the efficacy of different techniques, culture media, and sources of blood in determining the hemolytic activity of Listeria spp..

Hemolytic activity is a fundamental criterion for the differentiation of Listeria species; therefore, a simple and inexpensive procedure to clearly distinguish hemolytic strains from each other and from nonhemolytic strains would be of great aid. We compared the efficacy of several techniques, culture media, and types of blood in demonstrating the hemolysis of Listeria spp. The hemolytic activities of Listeria monocytogenes and Listeria seeligeri were more easily detected with a red blood cell top-layer (RBCTL) technique and with a microplate technique than when the strains were streaked on blood agar (BA). Listeria ivanovii produced a marked hemolysis regardless of the technique employed. In general, the hemolytic activity of these three species was stronger on media containing brain heart infusion (BHI) agar and (or) potassium tellurite (PT). However, Listeria innocua produced questionable hemolytic reactions when nonselective culture media with BHI and PT were utilized, limiting the advantages gained by employing the two compounds. The RBCTL and the BA techniques disclosed greater hemolytic activity for L. monocytogenes, L. seeligeri, and L. ivanovii with sheep and guinea pig blood than with horse and human blood. When the microplate technique was used, all four kinds of blood were equally effective.

Influence of strain and trisodium phosphate concentration on growth parameters of Listeria monocytogenes in vitro.

AIMS: The present study was conducted to determine the influence of strain and trisodium phosphate (TSP) concentration in the growth of Listeria monocytogenes in vitro. METHODS AND RESULTS: Three strains (ATCC 11916, 64d, isolated from chicken meat, and M2-5b, a clinical animal isolate) were inoculated in broth with 0, 0.5, 1 and 1.5% (w/v) of TSP. The shortest lag phase and highest maximum rate of growth (mu) were obtained in the presence of 0.5% TSP. In contrast, the highest lag phase and lowest mu were obtained with 1.5% TSP. For each TSP concentration, significant differences (P < 0.05) in lag phase and mu of the three L. monocytogenes strains were observed. CONCLUSION: The behaviour of L. monocytogenes is significantly influenced by both the origin of the strain and the salt concentration. SIGNIFICANCE AND IMPACT OF THE STUDY: Our results indicate the importance of choosing an adequate TSP concentration for the decontamination of foods, because low concentrations could favour the growth of L. monocytogenes.

Occurrence of Listeria species in retail poultry meat and comparison of a cultural/immunoassay for their detection.

The prevalence of Listeria spp. on the skin of a hundred fresh chicken carcasses purchased from 20 retail stores in León was investigated using the routine test procedure recommended by the United States Department of Agriculture (USDA). PALCAM and Modified Oxford agar (MOX) were used for isolation. Listeria spp., Listeria monocytogenes, L. innocua, L. welshimeri, L. grayi and L. ivanovii were present in 95%, 32%, 66%, 7%, 4% and 2% of the samples, respectively. Next, an immunoassay test (Listeria Rapid Test; Oxoid, Unipath) and the routine test procedure (USDA) were compared for their ability to detect Listeria spp. on 40 chicken carcasses. When we used MOX for the isolations, the sensitivity of the immunoassay test was 100% and the specificity 85.7%. When we used PALCAM medium the sensitivity and specificity of the test was 94.29% and 80%, respectively.

Microbiological quality of retail poultry carcasses in Spain.

A total of 40 eviscerated and refrigerated chicken carcasses were collected from five retail outlets (three supermarkets and two poulterers' shops) in León (Spain). The level of microorganisms on chicken carcasses was assessed using the excised breast-skin technique. Mean counts (log10 CFU/g) of psychrotrophs, pseudomonads, fluorescent pseudomonads, enterococci, Micrococcaceae, Staphylococcus aureus, and yeasts and molds were 4.84, 4.11, 3.32, 2.72, 3.80, 3.67, and 2.99, respectively. A significant correlation coefficient was found between pseudomonads and fluorescent pseudomonad counts (r = 0.827; P < 0.001) and between Micrococcaceae and S. aureus counts (r = 0.915; P < 0.001). Levels of psychrotrophs, pseudomonads, fluorescent pseudomonads, and yeasts and molds were significantly (P < 0.05) higher in supermarkets than in poulterers' shops, possibly due to the longer period of time the carcasses spent in the supermarkets (between 1 and 2 days, as opposed to only 4 to 16 h in the case of poulterers' shops). Carcasses from poulterers' shops showed higher (P < 0.05) counts of enterococci. Micrococcaceae, and S. aureus, which suggests higher storage temperatures in these outlets. Only S. aureus counts (especially those from poulterers' shops) exceeded the established values in the microbiological criteria for poultry meat consulted.

Sensibilidad de listeria monocytogenes frente al fosfato trisódico in vitro / Sensitivity of Listeria monocytogenes to trisodium fosfate in vitro

La carne de pollo presenta un elevado grado de contaminación superficial con microorganismos patógenos para el hombre y alterantes. Con el objeto de reducir dicha contaminación, desde 1992, el fosfato trisódico dodecahidrato (Na3PO4.12H2O) se está empleando en los mataderos de aves de Estados Unidos como un procedimiento de descontaminación. El efecto antimicrobiano de esta sal ha sido sobradamente demostrado frente a algunos microorganismos (fundamentalmente Gram negativos). No obstante, los datos publicados hasta el momento no son concluyentes por lo que se refiere a L. monocytogenes. Este estudio se llevó a cabo para evaluar la sensibilidad de 21 cepas de L. monocytogenes frente al fosfato trisódico in vitro. Para estandarizar la sensibilidad frente al fosfato trisódico utilizamos la concentración mínima inhibitoria, que se consideró como la menor concentración de la sal con la que la absorbancia (al cabo de 24 horas de incubación) no superó el valor 'umbral', que fue definido como el valor medio de las absorbancias de los blancos más tres desviaciones estándar. Los valores de las concentraciones mínimas inhibitorias (1,58-1,68 por ciento) fueron muy similares para las cepas de L. monocytogenes estudiadas. No encontramos ninguna relación entre el valor de la concentración mínima inhibitoria y la procedencia de las cepas (AU)

Effect of trisodium phosphate solutions washing on the sensory evaluation of poultry meat.

The effect of trisodium phosphate (TSP) solutions treatments on the sensory evaluation of poultry meat has been explored since TSP was recently approved by USDA for its usage in poultry processing to eliminate Salmonella contamination. In the present study, fresh chicken thighs were dipped in water (control sample) and in 8, 10 and 12% TSP solutions (treated samples) for 15 min. Raw samples' color, smell and overall acceptability and cooked thighs' color, smell, flavor, texture and overall acceptability were evaluated by consumer panelists. The evaluation of raw pieces after treatment or after 7 days storage at 2°C revealed that, with the exception of 12% TSP solutions treatment, sensory quality was not adversely affected by TSP. The color, smell and overall acceptability hedonic scores for the boiled thigh meat were not different between the treated samples and the control ones. Only the color, flavor and overall acceptability of thighs dipped in 12% TSP were rated significantly lower than the control sample. These results suggested that TSP solutions have good potential as dips to sanitize chickens carcasses.