Correlation between the presence of symptoms and the Giardia duodenalis genotype.

Clinical manifestations of Giardia duodenalis infection vary from asymptomatic infection to chronic diarrhoea. We study the correlation between the presence of symptoms and the G. duodenalis genotype in 108 patients with giardiasis. Patient age ranged from 2 to 72 years old. We found a correlation between assemblage AII and symptomatic infections, and between assemblage B and asymptomatic infections in the overall patient group and in patients less than five years of age. Nevertheless, if only patients of more than five years of age were considered, no statistically significant relationship between assemblage and symptomatic or asymptomatic Giardia infections was found. In these patients, host factors may affect the presence of clinical manifestations more than Giardia assemblage.

Combination of analytical and microbiological techniques to study the antimicrobial activity of a new active food packaging containing cinnamon or oregano against E. coli and S. aureus.

The aim of this work is the optimization and application of a group of analytical and microbiological techniques in the study of the activity of essential oils (EOs) incorporated in a new antimicrobial packaging material and the research in depth of the interaction between the microbial cells and the individual compounds present in the active material. For this purpose the antimicrobial activity of the active packaging containing cinnamon or oregano was evaluated against E. coli and S. aureus. The vapour phase activity and the direct contact between the antimicrobial agents themselves, or once incorporated in the packaging material, and the microbial cells have been studied. The direct contact was studied using a broth dilution method. The vapour phase was evaluated by using a new method which involves the use of a filter disk containing the EOs. Furthermore, the kill time assay was used to determine the exposure time for the maximum efficiency in packaging, and transmission electron microscopy was used to investigate the antimicrobial activity and the possible mechanism of action against E. coli and S. aureus. Finally, the compounds absorbed by cells were identified. The results showed that the techniques used provide relevant information about the antibacterial activity of cinnamon and oregano in direct contact as well as in the vapour phase. The antimicrobial packaging showed a fast efficiency which supports its likely application as a food packaging material. Bacteria treated with EOs exhibit a wide range of significant abnormalities; these include formation of blebs, coagulation of cytoplasmatic constituents, collapse of the cell structure and lack of cytoplasmatic material. Some of these observations are correlated to the ability of some of these substances to disrupt envelop structure, especially the inner membrane. After an extraction from dead cells, cinnamaldehyde was detected by GC-MS in E. coli exposed to the active packaging containing cinnamon.

Genetic relatedness and antimicrobial resistance determinants among clinical isolates of enterococci from Cuba.

This study describes the genetic relationships and antimicrobial resistance determinants found among 99 clinical isolates of enterococci from 15 different hospitals in Cuba. Pulsed-field gel electrophoresis SmaI analysis demonstrated a high degree of genetic diversity. A limited number of multiresistant Enterococcus faecalis clones, showing resistance to three or more families of antimicrobial agents, were detected simultaneously in different institutions, suggesting inter-hospital circulation of selected clones, and/or selection of particular clones following their introduction into the hospital environment. Antimicrobial resistance determinants, including erm(B), aac(6')-aph(2'), aph(3'), ant(6), vanB (E. faecalis) and vanA (Enterococcus faecium) were detected by PCR in various isolates.

In vitro activity of telithromycin, quinupristin/dalfopristin, linezolid and comparator antimicrobial agents against Staphylococcus aureus clinical isolates.

We have studied the prevalence of the different macrolide, lincosamide, streptograminB (MLS(B)) phenotypes among clinical Staphylococcus aureus isolates erythromycin- and/or oxacillin-resistant; and also the activity of other antimicrobial agents including telithromycin, quinupristin/dalfopristin, linezolid, aminoglycosides, chloramphenicol and vancomycin. We found that 64.86% of S. aureus were oxacillin-resistant. While the most prevalent MLS(B) phenotype among methicillin-resistant S. aureus (MRSA) was constitutive MLS(B) (cMLS) (83%), among methicillin-susceptible S. aureus (MSSA) it was inducible MLS(B) (iMLS(B)) (90%). Kanamycin resistance was more frequent than resistance to other aminoglycosides, being 100% for MRSA. Telithromycin was only active against iMLS(B), MS and erythromycin-susceptible isolates, although resistance rates were found among iMLS(B) MSSA (2.78%). Quinupristin/dalfopristin showed greater activity, with resistance rates of 2.5% for MRSA and 1.53% for MSSA. Both vancomycin and linezolid were fully active against all the isolates tested, with the highest MIC value being 2 microg/ml and 4 microg/ml, respectively. Among MRSA strains, 81.67% displayed resistance to five or more antimicrobials. This multiresistance was more frequently found among cMLS(B) strains (96.38% MRSA resistant to 6-9 agents).

Macrolide resistance phenotypes of commensal viridans group streptococci and Gemella spp. and PCR detection of resistance genes.

One hundred and sixty viridans group streptococci (VGS) and 26 Gemella spp. resistant to erythromycin were studied to detect macrolide lincosamide and streptogramin B (MLS(B)) phenotypes and to investigate resistance rates to other antibiotics. The M phenotype was most prevalent in both bacterial groups (59.6% in VGS, 69.2% in gemellae) and the iMLS(B) phenotype was found least often (9.3 and 13.9%, respectively). All isolates with M phenotype had the mef(A/E) gene, being prevalent the mef(E) subclass. cMLS(B) and iMLS(B) strains contained the erm(B) gene, alone or in combination with the mef(A/E) gene. Thirteen isolates were intermediately resistant to quinupristin/dalfopristin and 11 strains showed low susceptibility to telithromycin. Linezolid was active against all the isolates tested and tetracycline resistance was the major one in VGS (41.6%) and Gemella spp. (46.2%).

Antibiotic resistance and epidemiological typing of Staphylococcus aureus strains from ovine and rabbit mastitis.

Mastitis is a serious problem for sheep and rabbit farms, Staphylococcus aureus being the main causal agent. Fifty strains of S. aureus isolated from sheep and rabbits from farms located in diverse geographical regions of Spain were studied. Their resistance pattern and plasmid profile was related to the pulsotypes obtained by pulsed-field gel electrophoresis (PFGE). The results showed great heterogeneity in staphylococci isolated from sheep, both in pulse-type and plasmid profile. We found in addition, antibiotic-resistant strains and aminoglycoside-modifying enzyme (AGMEs) producer strains. The genotypes corresponding to staphylococci isolated from rabbits were less heterogeneous, although they also could be subdivided by plasmid profile and resistance patterns. Resistance to antibiotics such as methicillin or AGMEs production could indicate possible human origin of the strains or a possible source of resistant strains for human beings.

Characterization of the molecular mechanisms of quinolone resistance in Yersinia enterocolitica O:3 clinical isolates.

OBJECTIVES: The aim of this study was to determine the roles of mutations in the gyrA and parC genes and the overexpression of efflux pump(s) as mechanisms of resistance to quinolones. Forty-five Yersinia enterocolitica O:3 clinical isolates (41 nalidixic acid-resistant, three nalidixic acid-susceptible and one nalidixic acid-resistant strain obtained in vitro) were analysed. RESULTS: All the nalidixic acid-resistant strains showed mutations in the gyrA gene and none in the parC gene. The presence of the inhibitor produced decreases in the MIC values of nalidixic acid by two to six serial dilution steps in 37 of the 41 nalidixic acid-resistant strains. Meanwhile, the MIC value of ciprofloxacin was affected in two strains whose values diminished three serial dilution steps. The nalidixic acid-resistant mutant obtained in vitro was also affected by the inhibitor decreasing the MIC value of nalidixic acid three serial dilutions steps whereas the MICs for the nalidixic acid-susceptible strains were not affected. CONCLUSIONS: Our results show that the high level of resistance to nalidixic acid is likely due to an overexpression of an efflux pump plus a mutation in the gyrA gene, whereas decreased susceptibility to ciprofloxacin is only associated with the presence of a mutation in the gyrA gene.

Epidemiological study of resistance to nalidixic acid and other antibiotics in clinical Yersinia enterocolitica O:3 isolates.

Forty-six Yersinia enterocolitica O:3 clinical isolates resistant to nalidixic acid were studied. The use of molecular typing techniques, other indicators of resistance patterns, the plasmid profile, and the presence of genes that encode aminoglycoside-modifying enzyme production suggested to us a clonal dissemination of the studied strains.

Evaluation of an immunochromatographic dip-strip test for the detection of Cryptosporidium oocysts in stool specimens.

The study presented here examined the efficacy of a commercially available qualitative immunochromatographic assay for detecting Cryptosporidium oocysts in stool samples. A total of 75 samples were tested, including 50 positive for Cryptosporidium spp. by acid-fast stain, 20 positive for other parasites ( Blastocystis hominis, Endolimax nana, Entamoeba coli, Giardia lamblia, Ascaris lumbricoides, Strongyloides stercoralis and Trichuris trichiura), and five negative samples. The observed sensitivity was 98%, while specificity was 100%; the detection threshold was near 1000 oocysts/ml. Correctly diagnosed positive samples included Cryptosporidium parvum genotypes 1 and 2, whereas the single false-negative sample corresponded to a Cryptosporidium meleagridis infection.

Western blot applied to the diagnosis and post-treatment monitoring of human hydatidosis.

The serologic diagnosis of hydatidosis (caused by Echinococcus granulosus) can be made by different techniques, although the lack of standardization of the antigens affects the sensitivity, specificity and concordance among the different tests. We have applied the Western-Blot (WB) technique, associated with a purified antigen from sheep hydatid fluid, at 60 samples of serum from 14 patients suffering echinococcosis in different bodily locations, monitored for 3 years. The WB test enabled the detection of antibodies in the pre-surgical samples for proteins of 12-14, 16, 20, 24-26, 34, 39 and 42 kDa in molecular weight in 15-96% of the patients. The combination involving 2 of the 3 proteins of 20, 39 and 42 kDa has made it possible to diagnose 100% of the cases. The antibodies specific to proteins 39 and 42 kDa disappeared in less than one year in the patients cured after surgery, while in patients with persistent or recurrent parasitism the bands present before surgery persisted or other new ones appeared. The WB with purified antigens proved to be highly useful in the diagnosis and post-surgical monitoring of hydatidosis patients. The antigen used is proposed as a standard antigen for the diagnosis and follow-up of pre- and postsurgical hydatidosis.

Comparative in vitro bacteriostatic and bactericidal activity of trovafloxacin, levofloxacin and moxifloxacin against clinical and environmental isolates of Legionella spp.

The susceptibility of 140 Legionella spp isolates (106 clinical and 34 environmental isolates) to trovafloxacin (TRFX), levofloxacin (LEVX), moxifloxacin (MOFX), ciprofloxacin (CIPX), ofloxacin (OFLX), erythromycin (ERY), azithromycin (AZI) and rifampicin (RIF) was studied using a standard microdilution method and buffered yeast extract broth (BYE) supplemented with 0.1% alpha-ketoglutarate. The post-antibiotic effects (PAEs) of the study drugs against 10 clinical isolates of Legionella pneumophila sg.1 were compared. The MIC inhibiting 90% of strains tested on BYEalpha broth were 0.008, 0.016, 0.016, 0.06, 0.125, 0.5, 0.5, and 0.004 mg/l for TRFX, LEVX, MOXX, CIP, OFLX, ERY, AZI, and RIF, respectively. The MBC/MIC ratios ranged from one to eight depending on the antibiotic tested: TRFX [1x-2 x MIC], LEVX, MOFX, CIPX and OFLX [1x-4 x MIC], RIF [2x-4 x MIC], ERY and AZI [2x-8 x MIC]. TRFX, RIF, LEVX, MOFX, CIPX, OFLX, ERY and AZI showed similar activity against Legionella species other than L. pneumophila. One-hour exposures to the study antimicrobial agents at a concentration of 4 x MIC resulted in PAEs as follows (average in hours): TRFX: 2.68 h; RIF: 2.63 h; CIPX: 2.62 h; MOFX: 2.56 h; LEVX: 2.41 h; OFLX: 2.25 h; AZI: 1.65 h; and ERY: 1.54 h. In conclusion, our in vitro data confirm that trovafloxacin, levofloxacin, moxifloxacin and rifampicin have excellent bacteriostatic and bactericidal activity against Legionella spp and show significant post-antibiotic effect.

Distribution of resistance genes tet(M), aph3'-III, catpC194 and the integrase gene of Tn1545 in clinical Streptococcus pneumoniae harbouring erm(B) and mef(A) genes in Spain.

The most prevalent macrolide resistance phenotype and genotype among pneumococcal isolates was the cMLSB phenotype [erm(B) or erm(B)/mef(A)] (91.3%). We studied the distribution of other resistance genes, tet(M), catpC194, aph3'-III, in these strains, seeing evolution at work in that some strains carried different combinations of resistance determinants. The most prevalent patterns associated with resistance to erythromycin [erm(B)] were resistance to tetracycline [tet(M)] and chloramphenicol (catpC194) (48.2%) or resistance to tetracycline [tet(M)] alone (42.2%). In our isolates of Streptococcus pneumoniae there was a strong association of the erm(B) and tet(M) genes with Tn1545-related elements.

[Association between MLS antibiotic and tetracycline resistance genes in Streptococcus pneumoniae]. / Asociación de los genes de resistencia a antibióticos MLS y a tetraciclina en Streptococcus pneumoniae.

In Streptococcus pneumoniae, resistance to macrolide, lincosamide and streptogramin type B (MLS(B)) antibiotics is mediated by erm(B) and mef(A) determinants. Tetracycline resistance is always associated with resistance to minocycline and is due to the presence of the tet(M) gene. The erm(B) determinant is predominant. We demonstrated that the erm(B) gene could be present with mef(A), which is of streptococcal origin, and msr(A), which is of staphylococcal origin, this being an example of genetic promiscuity. The tet(M) determinant was associated with pneumococci harboring the erm(B) gene, while it was not associated with the strains harboring the mef(A) gene. This association is due to the fact that, in most of the cases, erm(B) and tet(M) reside in the same chromosomal conjugative transposon.

Bacteriocin production in vancomycin-resistant and vancomycin-susceptible Enterococcus isolates of different origins.

Bacteriocin production was determined for 218 Enterococcus isolates (Enterococcus faecalis [93] and E. faecium [125]) obtained from different origins (human clinical samples [87], human fecal samples [78], sewage [28], and chicken samples [25]) and showing different vancomycin susceptibility patterns (vancomycin resistant, all of them vanA positive [56], and vancomycin susceptible [162]). All enterococcal isolates were randomly selected except for the vancomycin-resistant ones. A total of 33 isolates of eight different bacterial genera were used as indicators for bacteriocin production. Forty-seven percent of the analyzed enterococcal isolates were bacteriocin producers (80.6% of E. faecalis and 21.6% of E. faecium isolates). The percentage of bacteriocin producers was higher among human clinical isolates (63.2%, 81.8% of vancomycin-resistant isolates and 60.5% of vancomycin-susceptible ones) than among isolates from the other origins (28 to 39.3%). Only one out of the 15 vancomycin-resistant isolates from human fecal samples was a bacteriocin producer, while 44.4% of fecal vancomycin-susceptible isolates were. The bacteriocin produced by the vanA-containing E. faecium strain RC714, named bacteriocin RC714, was further characterized. This bacteriocin activity was cotransferred together with the vanA genetic determinant to E. faecalis strain JH2-2. Bacteriocin RC714 was purified to homogeneity and its primary structure was determined by amino acid sequencing, showing an identity of 88% and a similarity of 92% with the previously described bacteriocin 31 from E. faecalis YI717. The presence of five different amino acids in bacteriocin RC714 suggest that this could be a new bacteriocin. The results obtained suggest that the epidemiology of vancomycin resistance may be influenced by different factors, including bacteriocin production.

The eaeA gene is not found in Hafnia alvei from patients with diarrhea in Aragón, Spain.

A total of 102 Hafnia alvei clinical strains isolated from different patients with diarrhea has been tested, using polymerase chain reaction and dot-blot hybridization, for the enteropathogenic Escherichia coli attaching and effacing A (eaeA) gene to establish their role as a causative agent of diarrhea in our environment. None of them was positive for the eaeA gene. We cannot consider the eaeA gene as the virulence-associated factor implicated in the H. alvei strains isolated from diarrheal feces in our region.