M. C. Mendoza

Enterotoxin production and DNA fingerprinting in Staphylococcus aureus isolated from human and food samples. Relations between genetic types and enterotoxins

A total of 224 Staphylococcus aureus strains from human carriers (110 strains) and manually handled foods (114 strains) collected in the Principality of Asturias, Spain over 1995-1999 were analysed for the production of enterotoxins (SEs) A, B, C, and D by a reversed passive latex agglutination test and by amplification of ent genes (A, B, C, D, E, and J) using PCR. Sixty-two strains were enterotoxigenic and a good relation between detection of SEs and their ent genes was found. No strain carried entE and all strains producing SED carried entD and entJ genes. Among the enterotoxigenic strains the percentages registered were 29, 8, 35, 18, 2, 2, and 6 for SEA, SEB, SEC, SEDJ, SEAC, SEADJ and SECDJ, respectively. DNA fingerprinting of 77 strains (the SE prototypes, 62 enterotoxigenic and 10 non-enterotoxigenic [NE]) was carried out by randomly amplified polymorphic DNA using two selected primers independently. Combining results from both primers, 10 genetic types were defined, which showed a different degree of relationship (similarity coefficient: 0.9-0.36) and were clustered into three lineages. One lineage clustered five genetic types and a wide diversity of strains, mainly SEA, SEB, SEDJ, and NE. Another lineage clustered only SEC, SECDJ and NE strains. These two lineages showed a low genetic relationship and appeared as endemic in healthy humans living in the Principality of Asturias. The third lineage included only the prototype strains for SEA and SEE.

Virulence and antimicrobial-resistance gene profiles determined by PCR-based procedures for Salmonella isolated from samples of animal origin

Abstract The virulence (V) and antimicrobial resistance (R) gene profiles of Salmonella isolates collected from samples of animal origin in a Spanish Region were determined by PCR-based procedures, and compared with V and R gene profiles of clinical strains from the same region. Some V-profiles were serotype-specific, i.e. S. Panama carried all the V-genes tested (invE/A, phoP/Q, stn, iroB, slyA, hin/H2, and agfA), whereas S. Enteritidis lacked hin/H2, and S. Hadar and all but one of the S. Typhimurium isolates, lacked agfA. Salmonella V-plasmids (spv) were found in serotypes Enteritidis, non-motile [9,12:-:-], Typhimurium and its monophasic variant [4,5,12:i:-]. Twenty-six out of the 56 animal-origin isolates showed some drug resistance. In the nalidixic acid-R isolates, mutations in the Ser-83 and/or Gly 133 codons in the gyrA gene were identified. Seventeen R-genes grouped into different R-profiles were detected, [pse1–aadA2–sul1–tet(G)–floR]-profile being the most frequent. Most of the serogroup B isolates, collected from pork or beef products, displayed multidrug-R and carried class 1-sul1 integrons. The integrons contained the following gene cassettes: pse1, aadA2, dfr17–aadA5, dfr1–aadAla, oxa1–aadA1a and dfr12–aadA2. All of them, except the dfr17–aadA5 gene cassette configuration, were also present in integrons of clinical isolates. Integrons could be plasmid- and/or chromosome-located, and those with oxa1–aadA1a and dfr12–aadA2 gene cassettes were located in spv-plasmids, which also carried the transposon associated genes tpnA, tpnR and merA. Conversely, all S. Enteritidis, S. Tennessee, and non-motile isolates collected from poultry samples showed drug susceptibility to all antibiotics tested. Moreover, among S. Enteritidis clinical isolates both ampicillin-R (encoded by tem1 genes, plasmid- or chromosome-located) and quinolone-R (due to mutations involving gyrA gene) were frequent. The efficiency, rapidity, and flexibility of the PCR-protocols applied was high. All of them can be performed directly on aliquots of a bacterial culture, using sequence-specific primers, the same basic equipment, and the same basic PCR-program.

Usefulness of a two-step PCR procedure for detection and identification of enterotoxigenic staphylococci of bacterial isolates and food samples

A two-step PCR procedure for detection of genes encoding Staphylococcus aureus thermonuclease (nuc) and enterotoxins (ses) was developed. For this, the reaction conditions for multiplex-PCR assays were optimized and generic and specific se-primers designed. In a step detection of enterotoxigenic staphylococci using a set of primers targeting nuc, and group-generic se[a, d, e, j] and se[b, c] sequences, is accomplished. The generated amplicons are 468-, 670- and 540-bp long, respectively. In the second step aliquots of the 670- and/or 540-bp amplicons are analysed by nested-PCR using the following sets of primers: the forward se[adej]—together with the reverse sea, sed, see and sej, and/or the forward se[bc]—plus reverse seb and sec. The six se-genes can be distinguished since each of them yield an amplicon of different size. The procedure was validated by using 163 strains of S. aureus, 15 strains of other species, and 31 cake samples; and also by comparison of the obtained results with conventional-PCR-assays and immunoassays (SET-RPLA). The first PCR-step showed that all S. aureus strains, but none of the strains representing other species, were nuc-positive and 52 of them, and only them, were se-positive. Twelve out of the 31 cake samples were probed to be nuc-positive, but only eight were se-positive, and enterotoxigenic S. aureus were collected from them. The second step confirmed that all se-positive strains carried one or more se-genes. A positive correlation between the presence of se-genes by multiplex-PCR, conventional-PCR and SET-RPLA was found. This procedure allows it to be extended to include other se-genes.

Salmonella serotype Virchow causing salmonellosis in a Spanish region. Characterization and survey of clones by DNA fingerprinting, phage typing and antimicrobial resistance

The diversity of Salmonella serotype Virchow organisms causing human salmonellosis in a Spanish region over 1990–1996 was studied by genetic and phenotypic procedures. Isolates showing identical DNA fingerprintings (ribotypes, RAPD-, REP- and ERIC-types) were clustered into the same lineage. Eight lineages were defined, of which only one caused diseases throughout the studied period. Eleven phage types (PTs) were represented, the most frequent being PTs 8, 19, 31, throughout the study period, and PT4a only during 1994. Class I integrons with variable regions of 1000-, 1600-, and 2300-bp in size were respectively present in 24, 3 and 5 multiresistant isolates; 43.5% of isolates were susceptible to antimicrobials, the rest were grouped into 17 R-profiles, including from one up to eight resistances. Plasmids could be recovered from 71.5% of isolates and grouped into 25 plasmid profiles (with 1–7 plasmids each); a 3.6 kb cryptic-plasmid and a 60 kb virulence-plasmid were those most frequently found. Phage type, presence and size of integrons, and resistance profile were used to differentiate 39 clones. During the period studied 135 cases of Virchow salmonellosis were identified; 93 were apparently sporadic whereas the remainder were associated with four outbreaks. Infants under 1 year constituted the most frequent age group, with 30 gastroenteritis and two septicaemia episodes. In the four outbreaks, different clones falling into the prevalent lineage were implicated but each clone was involved in only one outbreak.

Plasmid typing of Shigella sonnei epidemic strains and molecular relationship of their R-plasmids

We conducted a surveillance program on epidemic and/or endemic Shigella strains in Asturias (Spain), their frequency and dispersion in our community, and their R-plasmids. We analyzed initial isolates of Shigella sonnei from two epidemic outbreaks using antibiotic resistance patterns and plasmid profile analysis as epidemiological markers. We found that the 2 outbreaks were caused by different S. sonnei strains, which respectively carried one and two R-plasmids together with other plasmids. The molecular relationship among these and three other R-plasmids from two S. sonnei strains isolated during a previous outbreak, were studied by restriction enzyme analysis and DNA-DNA hybridizations. We were able to establish different levels of relationship among the six R-plasmids.

EPIDEMIOLOGICAL, CLINICAL AND MICROBIOLOGICAL FEATURES OF YERSINIA ENTEROCOLITICA INFECTIONS IN A COMMUNITY DURING A FOUR-YEAR PERIOD

Epidemiological and clinical features of 17 cases (13 children) of yersiniosis treated in a hospital over a four-year period, as well as factors associated with virulence, antibiotic resistance and plasmids in Yersinia enterocolitica strains from these patients were studied. The proportion of Y. enterocolitica was 3.57% of all fecal cultures positive for enteric pathogens, with 13 sporadic episodes and one outbreak. In 11 cases the infection was unimicrobial. The clinical presentation was: gastroenteritis (14 cases), pseudoappendicitis (1 case) and asymptomatic (2 cases). Eight cases needed hospital care, and 8 required antimicrobial treatment. None of the isolated bacterial strains produced pyrazinamidase, hemolysins, elastase, fibrinolysine, colicines or aerobactin. Eight strains showed calcium dependence, 7 of them exhibited autoagglutination, serum resistance, crystal violet binding and carried a 42 MDa plasmid related with virulence. Two strains carried a 3.3 MDa R-plasmid that encoded streptomycin-sulfadiazine resistance. The restriction analysis showed that the virulence plasmids constitute one variety and R-plasmids another.

Differentiation of strains from a food-borne outbreak of Salmonella enterica by phenotypic and genetic typing methods.

A combination of typing methods was used to identify the strains and the infection source in a food-borne outbreak ofSalmonella enterica occurring in a summer camp and affecting 25 children. All isolates tested were found to be serovar Enteritidis, with an identical biotype API 20E and ribotype. However, they differed in their plasmid profiles and/or antibiograms, and were grouped into three strains. One strain was found in human stools, another in a hens egg, and the third in both stools and another egg pointing to large Spanish omelettes to be the contaminated food source.

Molecular epidemiology of two genes encoding 3-N-aminoglycoside acetyltransferases AAC(3)I and AAC(3)II among gram-negative bacteria from a Spanish Hospital

The molecular epidemiology of the aacC1 and aacC2 genes, encoding 3-N-aminoglycoside acetyltransferases AAC(3)I and AAC(3)II, respectively, was studied by DNA-DNA hybridization. The sample included 315 gentamicin-resistant Gram-negative bacilli collected over a six-month period from patients attending a Spanish Hospital. The aminoglycoside resistance phenotype of these strains was also determined. The aacC1 probe hybridized with 39 strains, the aacC2 probe with 146 strains and both probes hybridized with 26 strains. The aacC1 gene was most frequently detected in Pseudomonas aeruginosa whereas the aacC2 gene was most frequently detected in enterobacteria and Acinetobacter spp. Strains harbouring aacC genes were isolated from both in-and outpatients with different infectious diseases, mainly urinary tract infections. As inferred from the results of Southern hybridization, both genes showed a wide horizontal dispersion among plasmids and bacteria.