Assessing the molecular basis of transferable quinolone resistance in Escherichia coli and Salmonella spp. from food-producing animals and food products.

Enterobacteriaceae resistant to quinolones frequently arise in animals, being easily disseminated through the food-chain. The aim of this study was to investigate the presence of plasmid-mediated quinolone resistance (PMQR) determinants in Salmonella spp. (n=183) and Escherichia coli (n=180) isolates, collected from food-producing animals and food products among swine, poultry, rabbits and cattle. All isolates were subjected to antimicrobial susceptibility testing and molecular screening of PMQR determinants. ß-Lactamase-encoding genes, and the quinolone resistance determining region (QRDR) of gyrA, gyrB, parC and parE genes were also investigated in PMQR-positive isolates. Plasmid characterization was performed by conjugation, followed by replicon-typing. Genetic relatedness of PMQR-positive E. coli was examined by Multilocus Sequence Typing, while Salmonella was previously serotyped. The association of mobile genetic elements and PMQR was investigated through PCR mapping assays. Overall, 4.1% (15/363) isolates harbored qnrB2 (n=3), qnrB19 (n=3), and qnrS1 (n=9) genes. All but one isolate presented one to four mutations in QRDR of gyrA or parC genes, which is consistent with the range of MIC values detected (0.19-64 mg/L) for ciprofloxacin; 60% (9/15) of qnr-harboring isolates were non-susceptible to ß-lactam antibiotics which was justified by the presence of ß-lactamases from TEM (TEM-1, n=8; TEM-135, n=1) and SHV (SHV-108, n=1) families. Analysis of mobile genetic elements revealed that qnr genes were detected nearby relevant genetic elements like intI1, ISEcl2, IS26 and ISCR1 and enclosed in diverse Inc. type plasmids. This study illustrated the existence of Qnr-producing E. coli and Salmonella from food-producing animals, associated to specific mobile elements that might mediate their transference between species and among distinct settings.

Genetic diversity and clonal evolution of carbapenem-resistant Acinetobacter baumannii isolates from Portugal and the dissemination of ST118.

In this study, 116 multidrug-resistant Acinetobacter baumannii (MDR-Ab) isolates recovered in various regions of Portugal were studied. All isolates were non-susceptible to tigecycline; one isolate was also non-susceptible to colistin, making it a step closer to pandrug resistance. Among 72 isolates tested by PFGE, 98.6% carried bla(OXA-66), 1.4% bla(OXA-104), 77.8% bla(OXA-23), 23.6% bla(OXA-24), 18.1% bla(TEM-1) and 1.4% bla(CTX-M-15-like) genes. No OXA-58 or metallo-ß-lactamase-encoding genes were detected. ISAba1 was found in 58/72 isolates (80.6%). Among these, ISAba1 was found upstream of bla(OXA-51-like) in 54 isolates. All but two of these isolates also carried ISAba1-bla(OXA-23), highlighting the coexistence of ISAba1-bla(OXA-51-like) and ISAba1-bla(OXA-23) genetic platforms, emphasising the importance of mobile genetic elements in the dissemination of carbapenem-hydrolysing class D ß-lactamase genes. Tn2006-like and Tn2008-like, found within ST92 and ST118, may reflect either multiple genetic structures in the origin of bla(OXA-23) acquisition or interclonal complex evolution. These results indicate that there may exist different genetic origins for carbapenem resistance among MDR-Ab isolates. Six PFGE profiles were associated with three major sequence types, with ST118 (OXA-23- or OXA-24-producer) being widely disseminated since 2009. ST98 (described so far as endemic in Portugal) and ST92 (which co-existed with ST98 before 2009) appeared to have been gradually replaced by ST118. The new ST188 (OXA-104-producer) was detected for the first time in this country. Identification of an extensively drug-resistant ST118 and carbapenem-resistant ST92, ST98 and ST118 isolates, both in community and healthcare facilities, demonstrates the menace of A. baumannii-associated infections.

First Report of Tomato infectious chlorosis virus in Spain.

During the summer and autumn of 2001, symptoms of interveinal yellowing, bronzing, brittleness, and rolling of lower leaves were observed in greenhouse- and field-grown tomato (Lycopersicon esculentum) plants in Castellon Province in eastern Spain. Symptoms resembled those caused by the whitefly-transmitted criniviruses (1,2). Total RNA was extracted from 28 samples of symptomatic leaves collected in three greenhouses and one field and analyzed by reverse transcription-polymerase chain reaction using primers specific for Tomato chlorosis virus (ToCV) (1) and Tomato infectious chlorosis virus (TICV) (2). The 501-bp TICV-specific DNA fragment was amplified in four samples collected during the summer in three greenhouses and one field, and the 439-bp ToCV-specific DNA fragment was amplified in 15 samples collected during the autumn in the same three greenhouses; no mixed infections were found. The DNA fragments amplified from TICV were sequenced and showed 99 to 100% identity with the TICV isolates (GenBank Accession Nos. U67449 and AY048855) from the United States and Italy, respectively, confirming the diagnosis. One sequence was deposited as GenBank Accession No. AF479662. To our knowledge, this is the first report of TICV in Spain and the second in Europe. References: (1) D. Louro et al. Eur. J. Plant Pathol. 106:539, 2000. (2) A. M. Vaira et al. Phytoparasitica. In Press.

Natural recombination between Tomato yellow leaf curl virus-is and Tomato leaf curl virus.

The complete genome sequences (2791 and 2793 nt) of isolates of Tomato yellow leaf curl virus-Is (TYLCV-Is) from Spain (SP72/97) and Portugal (Port2/95) were determined. These isolates are closely related to TYLCV-Is isolates reported in Japan (Japan-A and Japan-S) and Israel (Israel/Mild). Comparison of all sequenced isolates of TYLCV-Is showed that part of the genome comprising the intergenic region and the 5'-end of the rep gene of the Iran and Israel isolates was not closely related to that of other isolates. Phylogenetic analyses suggest that the Israel and Iran isolates may have chimeric genomes that have arisen by recombination between TYLCV-Is-like and tomato leaf curl virus (ToLCV)-like ancestors. The TYLCV-Is donors of the Iran and the Israel genomes were closely related to each other and to other known TYLCV-Is isolates. However, the ToLCV donors differed from each other, although both were related to ToLCV isolates from India (Bangalore-2 and Bangalore-4).

Cucurbit yellow stunting disorder virus (Genus Crinivirus) Associated with the Yellowing Disease of Cucurbit Crops in Portugal.

During autumn 1998, chlorotic mottling, yellowing, and stunting symptoms were observed on cucumber (Cucumis sativus L.) plants in an experimental plot, in Algarve, southern Portugal. The first symptoms appeared 3 weeks after planting, associated with a heavy infestation of Bemisia tabaci (Gennadius). Plants affected early produced few and small fruits. Analysis of double-stranded RNA (dsRNA) extracted from symptomatic cucumber leaves revealed the presence of two dsRNAs of about 8 and 9 kbp, not present in healthy cucumber plants. Reverse-transcription polymerase chain reaction (RT-PCR) using dsRNA or total RNA extracts as template and the oligonucleotide primers 410L and 410U (1), specifically amplified a fragment of expected size of the HSP70-homolog gene of Cucurbit yellow stunting disorder virus (CYSDV). The RT-PCR-amplified fragment was sequenced (Acc. No. AF287474) and showed 99% sequence identities with the corresponding sequences (GenBank accessions AJ223619 and U67170) from two CYSDV isolates belonging to group I (2), confirming CYSDV detection. CYSDV was also detected in samples of cucumber, melon (Cucumis melo L.) and watermelon (Citrullus lanatus [Thunb.] Matsun.) collected during the summer of 1999 in commercial greenhouses. CYSDV is an emerging and important virus of cucurbits in the Middle East and Mediterranean Europe (2). This is the first report of CYSDV infecting cucurbit crops in Portugal. References: (1) A. Célix et al. Phytopathology 86:1370, 1996. (2) L. Rubio et al. Phytopathology 89:707, 1999.

High-level tetracycline resistant Neisseria gonorrhoeae isolated in Portugal.

The first high-level tetracycline resistance (MIC > or = 16 mg/l) isolates of Neisseria gonorrhoeae (TRNG) were reported in 1990 from patients attending a Sexual Transmitted Disease (STD) Center in Lisbon. The TRNG prevalence was 4% in 1991, 5.3% in 1992 and 10,8% in 1994, exploding to 52.2% in 1995. The tet M determinant was evaluated by PCR. The digests of PCRP using HpaII produced the restriction pattern 2 for all the strains, except one (pattern 3). 78.3% of the TRNG strains were beta-lactamase producers and the 4.5 MDa penicillinase plasmid was the dominant (83%), 90% and 93.3% of the TRNG strains belonged to the auxotype NR and to the serogroup IA, respectively. The IA-8/NR class represented 58.3% of the TRNG isolates, suggesting a clonal spreading.

[Penicillinase-producing Neisseria gonorrhoeae isolated in Lisbon 1982-1987]. / Neisseria gonorrhoeae productrices de pénicillinase (NGPP) isolées à Lisbonne 1982-1987.

PPNG strains were first detected in Lisbon in 1982. Most of the 88 PPNG strains isolated since then in our laboratory were obtained from prostitutes. In this population, PPNG prevalence underwent a dramatic increase, rising from 1.1% in 1983 to 8.2% in 1984, 19.2% in 1985, 13.1% in 1986 and 27% in 1987 (ten months). MICs determined by agar dilution in a sample of 45 non-PPNG strains were as follows: 0.063 less than MIC mg/l less than 2 for penicillin, 0.125 less than MIC mg/l less than 1 for ampicillin, 8 less than MIC mg/l less than 32 for spectinomycin, 0.125 less than MIC mg/l less than 2 for tetracycline, and MIC less than 0.063 for cefotaxime. In contrast, MICs among PPNG strains were 4 less than MIC mg/l less than 64 for penicillin, 4 less than MIC mg/l less than 64 for ampicillin, 16 less than MIC mg/l less than 32 for spectinomycin, 0.250 less than MIC mg/l less than 2 for tetracycline, and MIC mg/l less than 0.063 for cefotaxime. Two different inocula were used, ie. 2 X 10 CFU/spot, and 10 CFU/spot. A significant increase in MICs was found with the heavier inoculum, especially for beta-lactam antibiotics. Plasmid analysis of the PPNG strains found 44% type AFR-, 27% type Afr+, 13% type Asia-, and 13% type Asia+; one strain isolated in 1987 was similar to the "RIO" type (The Netherlands, 1985) with 3.05 Mdal. Distribution of auxotypes among studied strains was NR pheni, 44%, NR 14%; prol-, 31%; and other types, 11%.

Use of protein A-gold complex for specific labelling of antibodies bound to plant viruses. I. Viral antigens in suspensions.

Colloidal gold particles 5 nm in diameter complexed with protein A (pAg) were bound specifically to rabbit antibodies which had reacted with antigens of 12 different plant viruses previously adsorbed to electron microscope (EM) support films. Conditions for highly specific labelling of antigens combined with a low non-specific background were assessed. In antiserum dilution tests, pAg labelling increased by up to four two-fold dilution steps the sensitivity of detecting low amounts of antibodies on virus particles. The pAg complex allowed detection of small antigens having no distinct morphology. The combination of dense antibody coating and pAg labelling substantially increased virus particle dimensions and contrast. This effect was further increased by double-antibody coating of the particles using rabbit anti-virus IgG followed by goat anti-rabbit IgG, prior to pAg incubation. Screening for elongated virus particles at low concentration in crude sap was then facilitated at very low EM magnifications. The pAg technique could be combined with trapping steps by using F(ab')2 fragments instead of complete antibodies for coating the grids.