Design and application of a 23-gene panel by next-generation sequencing for inherited coagulation bleeding disorders.

INTRODUCTION: Molecular testing of Inherited bleeding coagulation disorders (IBCDs) not only offers confirmation of diagnosis but also aids in genetic counselling, prenatal diagnosis and in certain cases genotype-phenotype correlations are important for predicting the clinical course of the disease and to allow tailor-made follow-up of individuals. Until recently, genotyping has been mainly performed by Sanger sequencing, a technique known to be time consuming and expensive. Currently, next-generation sequencing (NGS) offers a new potential approach that enables the simultaneous investigation of multiple genes at manageable cost. AIM: The aim of this study was to design and to analyse the applicability of a 23-gene NGS panel in the molecular diagnosis of patients with IBCDs. METHODS: A custom target enrichment library was designed to capture 31 genes known to be associated with IBCDs. Probes were generated for 296 targets to cover 86.3 kb regions (all exons and flanking regions) of these genes. Twenty patients with an IBCDs phenotype were studied using NGS technology. RESULTS: In all patients, our NGS approach detected causative mutations. Twenty-one pathogenic variants were found; while most of them were missense (18), three deletions were also identified. Six novel mutations affecting F8, FGA, F11, F10 and VWF genes, and 15 previously reported variants were detected. NGS and Sanger sequencing were 100% concordant. CONCLUSION: Our results demonstrate that this approach could be an accurate, reproducible and reliable tool in the rapid genetic diagnosis of IBCDs.

Determination of lipolytic and proteolytic activities of mycoflora isolated from dry-cured teruel ham.

Fungi play a key role in dry-cured ham production because of their lipolytic and proteolytic activities. In the present study, 74 fungal strains from dry-cured Teruel hams and air chambers were tested for proteolytic and lipolytic activities, with a view to their possible use as starter cultures. Lipolytic activity of fungi was studied against lauric, palmitic, stearic and oleic acids, whereas proteolytic activity was studied against casein and myosin. Of the 74 fungal strains tested, most of them demonstrated lipolytic activity (94.59 %). Lipolytic activity against lauric and oleic acids was stronger than against palmitic and stearic acids. 39 strains (52.70 %) demonstrated proteolytic activity against casein and the 6 highest proteolytic strains were also tested for pork myosin proteolysis. Some strains belonging to Penicillium commune, Penicillium chrysogenum, Penicillium nalgiovense and Cladosporium cladosporioides were selected because of their significant proteolytic and lipolytic activities and could be suitable to use as starters in dry-cured ham.

Efficacy of low-dose tylvalosin for the control of clostridiosis in broilers and its effect on productive parameters.

The study was carried out under field conditions in a commercial farm, and 1,440 as-hatched Ross-308 broilers were included. Broilers were randomly distributed into 24 experimental 4-m(2) pens (60 broilers/pen). Pens were randomized to the 3 treatment groups: a) tylvalosin 10 mg/kg of live BW during 2 d, b) positive control (tylosin during 2 d), and c) negative control (no treatment). The drugs were provided in the water supply. Mortality, individual BW, and feed intake were assessed. Clostridium presence was assessed in fecal and cecal samples, coccidian oocyst counts were assessed in fecal samples, and bacterial diversity was assessed in ileal content. Live BW at 42 d old was significantly better in the tylvalosin group than in tylosin and no-treatment groups, with tylvalosin-treated broilers reaching 80 to 100 g higher final live weight. Average daily gain results mirrored BW findings. The improvement of feed conversion rate with tylvalosin amounted to 0.13 and to 0.10 versus tylosin and no-treatment, respectively, with mortality being similar in all groups. Significantly reduced sulfite-reducing Clostridium and Clostridium perfringens counts in tylvalosin and tylosin groups versus the no-treatment group were observed in cecum content samples. In conclusion, according to the present study results, tylvalosin, at doses substantially lower than registered for poultry in Europe, has proven effective in controlling the colonization of the cecum by Clostridium ssp. in broilers, improving some productive performances.

Genotypes, exotoxin gene content, and antimicrobial resistance of Staphylococcus aureus strains recovered from foods and food handlers.

Staphylococcal food poisoning, one of the most common food-borne diseases, results from ingestion of one or more staphylococcal enterotoxins (SEs) produced by Staphylococcus aureus in foods. In the present study, 64 S. aureus isolates recovered from foods and food handlers, associated or not associated with food-poisoning outbreaks in Spain, were investigated. They were assigned to 31 strains by spa typing, multilocus sequence typing (MLST), exotoxin gene content, and antimicrobial resistance. The strains belonged to 10 clonal complexes (CCs): CC5 (29.0%), CC30 (25.8%), CC45 (16.1%), CC8, CC15 (two strains each), CC1, CC22, CC25, CC59, and CC121 (one strain each). They contained hemolysin genes (90.3%); lukED (77.4%); exfoliatin genes eta, etd (6.5% each), and etb (3.2%); tst (25.8%); and the following enterotoxin or enterotoxin-like genes or clusters: sea (38.7%), seb (12.9%), sec (16.1%), sed-selj with or without ser (22.9%), selk-selq (6.5%), seh, sell, selp (9.7% each), egc1 (32.3%), and egc2 (48.4%). The number of se and sel genes ranged from zero to 12. All isolates carrying tst, and most isolates with genes encoding classical enterotoxins (SEA, SEB, SEC, and SED), expressed the corresponding toxin(s). Two CC5 isolates from hamburgers (spa type t002, sequence type 5 [ST5]; spa type t2173, ST5) were methicillin resistant and harbored staphylococcal cassette chromosome mec (SCCmec) IVd. Six (19.4%) were mupirocin resistant, and one (spa type t120, ST15) from a food handler carried mupA (MIC, 1,250 µg/ml). Resistance to ampicillin (blaZ) (61.3%), erythromycin (ermA-ermC or ermC) (25.8%), clindamycin (msrA-msrB or msrB) (16.1%), tetracycline (tetK) (3.2%), and amikacin-gentamicin-kanamycin-tobramycin (aphA with aacA plus aphD or aadD) (6.5%) was also observed. The presence of S. aureus strains with an important repertoire of virulence and resistance determinants in the food chain represents a potential health hazard for consumers and merits further observation.

Gender Differences in Perceived Family Involvement and Perceived Family Control during Emerging Adulthood: A Cross-Country Comparison in Southern Europe.

The aim of the present study was to explore gender differences in perceived parental involvement and perceived psychological and behavioral control during emerging adulthood in two Southern European countries (Spain and Portugal). Data were collected from 491 Portuguese and 552 Spanish undergraduate emerging adults (53.7 % women and 46.3 % men) aged between 18 and 29 years (M = 20.24 and SD = 2.12). Results indicated that women perceived higher levels of parental involvement than men in both countries, and men perceived more behavioral control than women in Portugal. Furthermore, gender was found to moderate the association between perceived parental involvement and perceived psychological and behavioral control differently in each country. Taken together, our findings suggest that gender-differentiated socialization patterns persist during emerging adulthood and that these patterns may be affected by the sociocultural context.

Guillain-Barré syndrome associated with SARS-CoV-2 infection: A case series from 4 Colombian cities during the pandemic.

SARS-CoV-2 infection has been associated with multiple neurological manifestations. One such manifestation, which has been described since the early stages of the COVID-19 pandemic and is relevant for current neurological practice, is Guillain-Barré syndrome (GBS). The literature describes neurotoxic mechanisms of the virus itself and the possible pathways by which it may affect the peripheral nerves in experimental studies; however, we still lack information on the mechanisms causing the immune response that gives rise to GBS in the context of SARS-CoV-2 infection. Colombia is one of the Latin American countries worst affected by the pandemic, with the third-highest number of cases in the region; thus, it is essential to recognise GBS, as this potential postinfectious complication may severely compromise the patient's functional status in the absence of timely diagnosis and treatment. We present a series of 12 cases of GBS associated with SARS-CoV-2 infection from hospitals in 4 different Colombian cities and describe the clinical presentation, laboratory and electrophysiological study findings, and treatment.

En el año 2020 se declaro la pandemia ocasionada por la infección por el virus SARSCoV-2, virus de la familia del coronavirus, adoptándose el nombre de COVID-19 a la enfermedad 1. En Bogotá, Colombia, se confirmó el primer caso de COVID-19 el 6 de marzo de 2020 (2). Los principales síntomas reportados en la infección por SARSCoV-2 son fiebre (43.8% en la admisión y 88.7% durante la hospitalización) y tos (67.8%) (3). Otros síntomas encontrados son fatiga (38.1%), producción de esputo (33.7%) y cefalea (13.6%). Los principales signos neurológicos reportados en los pacientes con infección severa por SARS-Cov-2 son agitación (69%), compromiso en tracto corticoespinal (67%) y delirium (65%) (4). Las principales complicaciones neurológicas descritas asociadas a Covid 19 son: anosmia, disgeusia, encefalopatia, Síndrome de Guillain Barre, complicaciones cerebrovasculares y daño en musculo esquelético (5­8).En el presente articulo se presenta una serie de casos de pacientes con síndrome de Guillain-Barré asociado a infección por SARS-CoV-2. Se recolectaron casos de diferentes instituciones medicas de Colombia.

Virulence and resistance determinants of German Staphylococcus aureus ST398 isolates from nonhuman sources.

A series of 100 Staphylococcus aureus isolates ascribed to sequence type 398 (ST398) and recovered from different sources (healthy carrier and diseased pigs, dust from pig farms, milk, and meat) in Germany were investigated for their virulence and antimicrobial resistance genetic background. Antimicrobial resistance was determined by the disk diffusion method. Virulence and resistance determinants (37 and 31 genes, respectively) were tested by PCR. Only two virulence profiles, including the accessory gene regulator agrI and three or four hemolysin-encoding genes, were detected. In contrast, 33 resistance profiles were distinguished (only 11 were shown by more than one isolate). Fifty-nine isolates were multiresistant (four or more antimicrobial classes), and 98 were methicillin resistant (mecA positive). All of the ST398 isolates showed resistance to tetracycline [encoded by tet(M) alone or together with tet(K) and/or tet(L)]. In addition, 98% were resistant to other antimicrobials, including macrolide-lincosamine-streptogramin B (70%, encoded by ermA, ermB, and ermC, alone or in combination), trimethoprim (65%, mostly due to dfrK and dfrG), kanamycin and gentamicin [29% and 14%, respectively, mainly related to aac(6')-Ie-aph(2″)-Ia and/or ant(4')-Ia but also to aph(3')-IIIa], chloramphenicol (9%, fexA or cfr), quinupristin-dalfopristin (9%), ciprofloxacin (8%), and trimethoprim-sulfamethoxazole (4%). The heterogeneity of the resistance profiles underlines the ability of the ST398 clone to acquire multiple antimicrobial resistance genes. However, the virulence gene content of the tested isolates was low. Continuous surveillance is needed to clarify whether its pathogenicity potential for animals and humans will increase over time.

Stability of the Antimicrobial Capacity of Human Milk Against Cronobacter Sakazakii During Handling.

BACKGROUND: Neonatal infections with Cronobacter sakazakii have recently been associated with the consumption of expressed human milk. STUDY AIMS: (1) To evaluate whether human milk has antimicrobial capacity against C. sakazakii and (2) to determine the stability of its capacity when it is subjected to various treatments. METHODS: The antimicrobial capacity of human milk against C. sakazakii was evaluated using an observational, cross-sectional, comparative design. Mature human milk samples (N = 29) were subjected to different treatments. After incubation at 37°C for 72 hr, samples were compared with fresh milk on the stability of their antimicrobial capacity. Two-way analysis of variance (ANOVA) was performed. RESULTS: In fresh milk, counts of C. sakazakii were reduced by 47.26% (SD = 6.74) compared to controls. In treated milk, reductions were: refrigeration at 4°C for 72 hr (M = 33.84, SD = 13.84), freezing at -20°C for 1, 2, and 3 months (M = 40.31, SD = 9.10; M = 35.96, SD = 9.39; M = 26.20, SD = 13.55, respectively), Holder pasteurization (M = 23.56, SD = 15.61), and human milk bank treatment with (M = 14.37, SD = 18.02) and without bovine fortifier (M = 3.70, SD = 23.83). There were significant differences (p < .05) between fresh and treated milk. CONCLUSIONS: Human milk has antimicrobial capacity against C. sakazakii. However, its capacity is negatively influenced by common preservation and hygienization methods. Milk should be stored refrigerated for a maximum of 72 hr or frozen for a short period of time.

High heterogeneity within methicillin-resistant Staphylococcus aureus ST398 isolates, defined by Cfr9I macrorestriction-pulsed-field gel electrophoresis profiles and spa and SCCmec types.

During recent years, the animal-associated methicillin-resistant Staphylococcus aureus clone ST398 has extensively been studied. The DNA of these isolates turned out to be refractory to SmaI restriction, and consequently, SmaI is unsuitable for subtyping this clone by standard pulsed-field gel electrophoresis (PFGE). Very recently, ST398 DNA was shown to be digested by Cfr9I, a neoschizomer of SmaI. In the present study, we employed Cfr9I PFGE on 100 German and 5 Dutch ST398 isolates and compared their PFGE profiles, protein A gene variable repeat regions (spa types), and types of the staphylococcal cassette chromosome mec (SCCmec). The isolates (from healthy carrier pigs, clinical samples from pigs, dust from farms, milk, and meat) were assigned to 35 profiles, which were correlated to the SCCmec type. A dendrogram with the Cfr9I patterns assigned all profiles to two clusters. Cluster A grouped nearly all isolates with SCCmec type V, and cluster B comprised all SCCmec type IVa and V* (a type V variant first identified as III) carriers plus one isolate with SCCmec type V. Both clusters also grouped methicillin-susceptible S. aureus isolates. The association of the majority of isolates with SCCmec type V in one large cluster indicated the presence of a successful subclone within the clonal complex CC398 from pigs, which has diversified. In general, the combination of Cfr9I PFGE with spa and SCCmec typing demonstrated the heterogeneity of the series analyzed and can be further used for outbreak investigations and traceability studies of the MRSA ST398 emerging clone.

Clonal complexes and diversity of exotoxin gene profiles in methicillin-resistant and methicillin-susceptible Staphylococcus aureus isolates from patients in a Spanish hospital.

Molecular epidemiology studies have allowed the identification of the methicillin (meticillin)-resistant (MRSA) and methicillin-susceptible (MSSA) clonal complexes (CCs) and clones of Staphylococcus aureus circulating in a Spanish hospital recently. Of 81 isolates tested, 32.1% were MRSA. Most of them carried staphylococcal cassette chromosome mec (SCCmec) IVc (88.5%) and belonged to CC5 (88.5%; multilocus sequence typing types ST125 [mainly associated with spa type t067], ST5, and ST228). A higher diversity was found among MSSA isolates (67.9%). Eighty percent shared the genetic background of major MRSA lineages (CC5 [38.2%; ST125 and ST5], CC30 [25.5%; ST30], CC45 [14.5%; ST45 and ST47], and CC8 [1.8%; ST8]), but CC12, CC15, CC51, and CC59 were also detected. Many exotoxin genes were present in each of the 81 isolates, independent of whether they were involved in sepsis (11 to 22) or other types of infections (13 to 21), and they appeared in 73 combinations. The relevant data are that (i) all isolates were positive for hemolysin and leukotoxin genes (98.8% for lukED and 25.9% for lukPV); (ii) all contained an enterotoxin gene cluster (egc with or without seu), frequently with one or more genes encoding classical enterotoxins; (iii) about half were positive for tst and 95% were positive for exfoliatin-encoding genes (eta, etb, and/or etd); and (iv) the four agr groups were detected, with agrII (55.6%) and agrIII (23.5%) being the most frequent. Taken together, results of the present study suggest a frequent acquisition and/or loss of exotoxin genes, which may be mediated by efficient intralineage transfer of mobile genetic elements and exotoxin genes therein and by eventual breakage of interlineage barriers.

Detection of Salmonella enterica serovar Typhimurium with pUO-StVR2-like virulence-resistance hybrid plasmids in the United Kingdom.

The aim of this study was to investigate the presence in the United Kingdom (UK) of Salmonella enterica serovar Typhimurium isolates carrying pUO-StVR2-like virulence-resistance hybrid plasmids that originated from pSLT. One hundred and fifty ampicillin-resistant isolates of S. Typhimurium, collected in different regions of the UK during 2006, were screened for the presence of bla (OXA-1) carried by an InH-like integron (2000 bp/bla (OXA-1)-aadA1) characteristic of pUO-StVR2. Positive isolates were tested for the presence of a large plasmid that hybridised with probes specific for the bla (OXA-1) and spvC genes, used as resistance and virulence markers of the hybrid plasmid, respectively. Eleven out of the 150 isolates fulfilled both criteria and were assigned to the S. Typhimurium pUO-StVR2 group. Nine were resistant to ampicillin, chloramphenicol, streptomycin/spectinomycin, sulfonamides and tetracycline, encoded by bla (OXA-1), catA1, aadA1-like, sul1 and tet(B), respectively, and carried a pUO-StVR2-like plasmid of ca. 130 kb. Two contained hybrid plasmids of smaller size and lacked resistance(s) to chloramphenicol or chloramphenicol and tetracycline. The eleven isolates, which showed five and six closely related XbaI and BlnI profiles, respectively, were resistant to nitrofurantoin. In conclusion, multidrug-resistant S. Typhimurium isolates of the pUO-StVR2 group, which are endemic in Spain, were also detected in the UK, albeit with a low frequency (7.3%).

Genetic basis of antimicrobial drug resistance in clinical isolates of Salmonella enterica serotype Hadar from a Spanish region.

The genetic bases of antimicrobial drug resistance (R) of 79 Salmonella enterica serotype Hadar clinical isolates (recovered during 1995-2001 in a Spanish region) was investigated. The isolates showed a limited genomic variation, as demonstrated by PFGE analysis using XbaI (three profiles, S>or=0.77) and BlnI (seven profiles, S>or=0.49; with 95% of the isolates falling into two clusters, S>or=0.75). Thirteen R-profiles, ranging from susceptible to multidrug resistant, were recognized. All susceptible isolates (14%) were recovered before or during 1998, when multidrug resistance (MDR) was still uncommon (20% from 1995-1998). In later years, the percentage of MDR increased considerably (92% in 2001). Resistance to nalidixic acid, tetracycline, streptomycin and ampicillin-cefalotin, encoded by gyrA-Asp87/Asn, tet(A), strA/B, and bla (TEM) genes, respectively, were the most common, appearing together in 38% of the isolates. In all tetracycline- and streptomycin-resistant isolates, strA/B and tet(A) were chromosomally located, whereas bla (TEM) was plasmid-born. Five different bla (TEM) plasmids (pUO-ShR1 to pUO-ShR5, of about 9.4, 23, 30, 45, and 95 kb, respectively) were identified. pUO-ShR3 and pUO-ShR5 harbored additional R-genes: [dfrA1] and [acc(3)IV-strA/B], respectively. pUO-Sh2, pUO-Sh3, pUO-ShR4, and pUO-Sh5 were self-transferable, and the latter could also mobilize pUOShR1. The reported data constitute a useful background for further epidemiological studies of MDR in S. Hadar.

Placebo-controlled trial of gabapentin in patients with amyotrophic lateral sclerosis. WALS Study Group. Western Amyotrophic Lateral Sclerosis Study Group.

We designed a phase II trial to evaluate the efficacy of gabapentin in slowing the rate of decline in muscle strength of patients with amyotrophic lateral sclerosis (ALS) and to assess safety and tolerability. Gabapentin (800 mg) or placebo was administered t.i.d. in a randomized, double-blinded, placebo-controlled, trial for 6 months. We enrolled 152 patients at eight sites in the United States. The primary outcome measure was the slope of the arm megascore, the average maximum voluntary isometric strength from eight arm muscles standardized against a reference ALS population. A secondary outcome measure was forced vital capacity. Slopes of arm megascores for patients on gabapentin were compared with slopes of those taking placebo using a two-way ANOVA. We observed a nonstatistically significant trend (p = 0.057-0.08) toward slower decline of arm strength in patients taking gabapentin compared with those taking placebo (mean difference 24%, median 37%). We observed no treatment effect on forced vital capacity. Gabapentin was well tolerated by patients with ALS. These results suggest that further studies of gabapentin in ALS are warranted.

Characterization and localization of drug resistance determinants in multidrug-resistant, integron-carrying Salmonella enterica serotype Typhimurium strains.

The genetic background of the antimicrobial resistance of 10 selected multiresistant Salmonella serotype Typhimurium (S. Typhimurium) strains (including the emerging monophasic variant [4,5,12:i:- ]) was investigated. All strains shared class 1 integrons (with seven types of variable regions) and belonged to different lineages (L1-L6) according to their phage types, DNA polymorphisms by XbaI-pulsed-field gel electrophoresis (PFGE), integrons, and/or resistance patterns. The strains were screened for the presence and localization (chromosomal or plasmid) of 32 DNA sequences representing integron-, Tn21-like transposon-, resistance-, and virulence-plasmid genes. Strains belonging to lineage L1 (definitive phage type DT104) carried the 90-kb Salmonella virulence plasmid together with the complete or partial chromosomally located Salmonella Genomic Island 1 (SGI1). All strains belonging to the other five lineages carried their resistance determinants on various resistance plasmids. Two of these strains showed complex plasmid profiles, which included a 95 kb virulence plasmid together with two or four resistance plasmids. Two strains carried a resistance plasmid that lacked the virulence-plasmid-encoding sequences. The remaining two strains carried two different hybrid virulence-resistance plasmids. Twenty-three of the DNA sequences could be assigned to distinct XbaI genomic restriction patterns (PFGE profiles). In this way, the influence of the resistance and virulence plasmids on the PFGE profiles was determined, and several groups of resistance genes could be identified. The data obtained represent a useful epidemiological tool for tracing the emergence and distribution of multiresistant S. Typhimurium worldwide.

Molecular characterisation of emergent multiresistant Salmonella enterica serotype [4,5,12:i:-] organisms causing human salmonellosis.

Salmonella multidrug-resistant clinical organisms identified as serotype [4,5,12:i:-] were typed using selected genetic procedures and compared with typhimurium organisms collected in the same Spanish region. Results showed a low genetic heterogeneity among [4,5,12:i:-] organisms, which generated identical ribotypes and similar but not identical XbaI PFGE, RAPD, and plasmid profiles. Multidrug resistance could be eliminated by curing and seems to be mediated by 140-kb (spvC+) and 120-kb (spvC-) non-self-transferable plasmids. The [4,5,12:i:-] organisms fall into a single genetic lineage, which emerged in 1997 and presents a different degree of genetic relationship with typhimurium lineages.

Evaluation of PCR-based methods and ribotyping performed with a mixture of PstI and SphI to differentiate strains of Salmonella serotype Enteritidis.

The capacity to differentiate Salmonella serotype Enteritidis strains by PCR ribotyping; RAPD typing with three arbitrary primers and ribotyping with a mixture of PstI and SphI or 'PS ribotyping', was evaluated on a series of 65 strains associated with human infections and 11 reference strains. The series had been analysed previously by phage typing and ribotyping performed with PstI and SphI, separately. All methods typed all the strains; however, only ribotyping showed good reproducibility and sensitivity. Twenty-two PS ribotypes (discrimination index = 0.74) were identified, differentiating strains ascribed to seven phage types (PTs 1, 4, 6, 6a, 7, 8 and RDNC) as well as phage untyped strains. Conversely, some strains of PTs, 1, 4, 5a, 6, 6a, 7, 34 and RDNC showed the most frequent PS ribotype. By PCR ribotyping a single profile was found; while by RAPD typing, one, two or three RAPD types were identified with the primers MK22, OPB6 and OPB17, respectively. All Spanish strains were assigned to a single combined RAPD type, except PT11 strains which showed a different and specific RAPD type with OPB17. The banding patterns defining the PS ribotypes were interpreted more easily and the patterns could be compared more accurately than the banding patterns defining RAPD types. A similarity dendrogram generated from the 22 PS ribotypes was traced and compared with RAPD types and phage types. Data from this work indicated that 'PS ribotyping' was the most useful genetic procedure to differentiate Enteritidis strains, and, therefore, it can be used as a complementary or alternative typing method to phage typing within this serotype.

A three-way ribotyping scheme for Salmonella serotype Typhimurium and its usefulness for phylogenetic and epidemiological purposes.

Ribotyping of Salmonella serotype Typhimurium strains was optimised as a tool for epidemiological and phylogenetic purposes. Of five restriction endonucleases evaluated on a series of 84 isolates, HincII, SalI and PvuII were the most useful, generating 13, 9 and 9 ribotypes with 17, 11 and 18 polymorphic restriction sites, and attaining a discrimination index (DI) of 0.81, 0.53 and 0.59, respectively. The combination of results from tests with the three enzymes provided further discrimination (19 ribotypes, DI=0.84). It proved useful for clonal analysis, defining 19 clonal lines with a remarkable degree of genetic heterogeneity, that were grouped into two major clusters (including 12 and 7 lines, respectively) at a significance level of 0.65. When the attributes of this system were compared with those of phage typing, it was found that ribotyping showed higher typability and sensitivity, supporting its use as an appropriate molecular method. In tracing the molecular epidemiology of Typhimurium strains in Asturias, six lines were found that could be considered endemic and were represented by organisms implicated in salmonellosis throughout the period of study; another four lines included organisms isolated from meat, water or both.

Genetic typing methods applied to the differentiation of clonal lines among Salmonella enterica serogroup G strains causing human salmonellosis.

In Spain, in November 1995, an epidemiological alert recommended the surveillance of Salmonella serogroup G. The nine clinical isolates collected after and the four collected before the alert in Asturias were differentiated into six clonal lines by the combination of results from HincII ribotyping, PCR ribotyping, and RAPD typing using primers named A and S. The seven Gumpensis isolates showed identical DNA fingerprinting with the four typing procedures falling into a line. Six of these were collected during May-August from people living in a single health area suggesting that they could be associated with a community outbreak. The four Worthington isolates fell into three other lines, one Poona isolate into another line and one Havana isolate into another. 100% typeability was shown with all methods. The reproducibility of HincII ribotyping was better than that of PCR-based methods, although these were less time-consuming. The highest discriminatory power was obtained with HincII ribotyping and RAPD typing using primer A.

Evidence for the dispersion and evolution of R-plasmids from Serratia marcescens in a hospital.

Incompatibility tests were used to study the types, evolution and dispersion of R-plasmids from Serratia marcescens in a hospital over the period 1975-82. R-plasmids belonged to incompatibility groups IncM, IncC, IncP or were unclassified, or compatible with all the plasmids tested. In 1982, IncM plasmids, including varieties codifying different resistance patterns, predominated. The oldest members of the IncM group codified fewer R determinants and were of smaller size than the more recent ones. Plasmids of IncM and IncC are dispersed among other genera of Enterobacteriaceae.