In most gram-negative bacteria, acquired multiresistance is conferred by large plasmids compiling numerous antimicrobial resistance genes. Here, we show an evolutionary alternative strategy used by Pasteurella multocida to become resistant to multiple clinically relevant antibiotics. Thirteen β-lactam-resistant clinical isolates, concomitantly resistant to tetracyclines and/or streptomycin as well as to sulfonamides, were studied. Pulsed-field gel electrophoresis analysis revealed different profiles among the isolates, showing that clonal dissemination was not the sole event responsible for the spread of multiresistance. Each P. multocida strain carried two or three small plasmids between 4 and 6 kb in size. A direct association between resistance profile and plasmid content was found. Complete nucleotide sequencing of all plasmids revealed seven different replicons, six of them belonging to the ColE1 superfamily. All plasmids carried one, or a maximum of two, antimicrobial resistance determinants. Plasmids pB1000 and pB1002 bore bla _ROB-1 , pB1001 carried tet (B), pB1003 and pB1005 carried sul2 and strA , pB1006 harbored tet (O), and p9956 bore the tet (H) gene. All plasmids except pB1002 and pB1006 were successfully transformed into Escherichia coli . pB1000, also involved in β-lactam resistance in Haemophilus parasuis (A. San Millan et al., Antimicrob. Agents Chemother. 51:2260-2264, 2007), was mobilized in E. coli using the conjugation machinery of an IncP plasmid. Stability experiments proved that pB1000 was stable in P. multocida but highly unstable in E. coli . In conclusion, bla _ROB-1 is responsible for β-lactam resistance in P. multocida in Spain. Coexistence and the spread of small plasmids are used by P. multocida to become multiresistant.

Zoonotic hepatitis E, mainly caused by hepatitis E virus (HEV) genotype (gt) 3, is a foodborne disease that has emerged in Europe in recent decades. The main animal reservoir for genotype 3 is domestic pigs. Pig liver and liver derivates are considered the major risk products, and studies focused on the presence of HEV in pig muscles are scarce. The objective of the present study was to evaluate the presence of HEV in different organs and tissues of 45 apparently healthy pigs from nine Spanish slaughterhouses (50% national production) that could enter into the food supply chain. Anti-HEV antibodies were evaluated in serum by an ELISA test. Ten samples from each animal were analyzed for the presence of HEV RNA by reverse transcription real-time PCR (RT-qPCR). The overall seroprevalence obtained was 73.3% (33/45). From the 450 samples analyzed, a total of 26 RT-qPCR positive samples were identified in the liver (7/45), feces (6/45), kidney (5/45), heart (4/45), serum (3/45), and diaphragm (1/45). This is the first report on detection of HEV RNA in kidney and heart samples of naturally infected pigs. HEV RNA detection was negative for rib, bacon, lean ham, and loin samples. These findings indicate that pig meat could be considered as a low risk material for foodborne HEV infection.

Brucellosis is endemic in most parts of Egypt, where it is caused mainly byBrucella melitensisbiovar 3, and affects cattle and small ruminants in spite of ongoing efforts devoted to its control. Knowledge of the predominantBrucellaspecies/strains circulating in a region is a prerequisite of a brucellosis control strategy. For this reason a study aiming at the evaluation of the phenotypic and genetic heterogeneity of a panel of 17Brucellaspp. isolates recovered from domestic ruminants (cattle, buffalo, sheep, and goat) from four governorates during a period of five years (2002–2007) was carried out using microbiological tests and molecular biology techniques (PCR, MLVA-15, and sequencing). Thirteen strains were identified asB. melitensisbiovar 3 while all phenotypic and genetic techniques classified the remaining isolates asB. abortus(n=2) andB. suisbiovar 1 (n=2). MLVA-15 yielded a high discriminatory power (h=0.801), indicating a high genetic diversity among theB. melitensisstrains circulating among domestic ruminants in Egypt. This is the first report of the isolation ofB. suisfrom cattle in Egypt which, coupled with the finding ofB. abortus, suggests a potential role of livestock as reservoirs of several zoonoticBrucellaspecies in the region.

Leishmaniasis is one of the most important neglected zoonosis and remains endemic in at least 88 developing countries in the world. In addition, anthropogenic environmental changes in urban areas are leading to its emergency world wide. Zoonotic leishmaniasis control might only be achieved by an integrated approach targeting both the human host and the animal reservoirs, which in certain sylvatic cycles are yet to be identified. Recently, hares have been pointed out as competent reservoirs ofLeishmania infantumin Spain, but the role of other lagomorphs has not been clarified. Here, 69 rabbits (Oryctolagus cuniculus) from a natural area in Madrid in which a high density was present were analyzed using indirect (immunofluorescence antibody test, IFAT) and direct (PCR, culture) techniques. Fifty-seven (82.6%) of the animals were positive to at least one technique, with IFAT yielding the highest proportion of positive samples.L. infantumwas isolated in 13% animals demonstrating the occurrence of infection in this setting. Our results suggest that rabbits could play a role of competent reservoir ofL. infantumand demonstrate that the prevalence of infection is high in the analyzed area.