Antimicrobial susceptibility of Actinobacillus pleuropneumoniae isolates from clinical outbreaks of porcine respiratory diseases.

Limited data regarding the susceptibility of Actinobacillus pleuropneumoniae to antimicrobials has been published during recent years. Accordingly, the aim of the present study was to investigate the distribution of MICs for the isolates of A. pleuropneumoniae from diseased pigs in the Czech Republic between 2007 and 2009. A total of 242 isolates were tested for susceptibility to 16 antimicrobial agents by a broth microdilution method. A low degree of resistance was observed for florfenicol (0.8%), amoxicillin and clavulanic acid (0.8%), tilmicosin (1.2%), tiamulin (1.7%) and ampicillin (3.3%), whereas resistance to tetracycline was detected more frequently, 23.9% of isolates. Interestingly, resistance to florfenicol has not yet been reported in any study investigating antimicrobial resistance of A. pleuropneumoniae. By PCR the presence of the floR gene was confirmed in all florfenicol resistant isolates.

Distribution of integrons and SGI1 among antibiotic-resistant Salmonella enterica isolates of animal origin.

In Salmonella enterica, resistance to antibiotics can be caused by the presence of SGI1, transposons or conjugative plasmids. In this study we were interested in the relative contribution of these genetic elements to the antibiotic resistance of S. enterica isolates collected within a single year in the Czech Republic from animal sources. Altogether 123 antibiotic-resistant isolates belonging to 16 different S. enterica serovars were classified into 3 groups according to the presence of SGI1 and the presence of integrons. The first group consisted of 62 strains in which neither SGI1 nor class 1 integron was detected. A high diversity among serovars and resistance phenotypes was found in this group. The second group consisted of 56 strains positive for both the SGI1 and class 1 integron, out of which 55 belonged to serovar Typhimurium and one to a nonmotile serovar [4,12] which harboured the SGI1-B variant. The third group comprised five strains which were positive for class 1 integron but negative for the SGI1. Sequencing of the integrons in these isolates identified integron with sat1 and aadA1 gene cassettes in S. Sandiego and S. Pullorum, dfrA1 and aadA1 gene cassettes in S. Typhimurium integron, and aadA21 gene cassette in S. Braenderub and S. Zanzibar.

Characterization of Salmonella enterica serovar Typhimurium conjugative plasmids transferring resistance to antibiotics and their interaction with the virulence plasmid.

OBJECTIVES: In this study, we analysed field isolates of Salmonella enterica serovar Typhimurium for the presence of conjugative plasmids transferring resistances to antibiotics. METHODS: Altogether 23 strains were analysed for the presence of conjugative R-plasmids. In the case of successful conjugation, the R-plasmids were characterized by PCR for antibiotic resistance genes, integrons and replicon typing. Variable regions of integrons were sequenced. RESULTS: Conjugation and transfer of antibiotic resistance was observed in 12 strains. Conjugative plasmids in these strains belonged to the IncI1 and IncHI1 replicons and four of them transferred antibiotic resistance associated with class I integrons. In two cases, resistance to tetracycline and/or ampicillin was not transferred by conjugation to approximately 10% of the transconjugants. Detailed characterization showed that the loss of both resistances was associated with the loss of Tn3 (bla(TEM)) and Tn1721 [tet(A)] from the conjugative plasmids p9046 and p9134. However, when only the tetracycline resistance was lost, the Tn1721 was replaced with a partial sequence of rck, and with complete coding sequences of srgA, srgB, ORF7 and pefI originating from the Salmonella Typhimurium virulence plasmid. CONCLUSIONS: Two plasmids from our collection were capable of recombination with the virulence plasmid of Salmonella Typhimurium and subsequently spread both antibiotic resistance and virulence genes to the recipient.

Biofilm formation in field strains of Salmonella enterica serovar Typhimurium: identification of a new colony morphology type and the role of SGI1 in biofilm formation.

In this study we examined the extent of biofilm formation in field strains of Salmonella enterica serovar Typhimurium (S. Typhimurium), an important foodborne pathogen. Ninety-four field strains of S. Typhimurium were tested for their ability to form biofilm and components contributing to its formation. Most S. Typhimurium strains were highly capable of biofilm formation except for strains of phage type DT2 originating from pigeons. The most efficient biofilm forming strains were those of phage type DT104 positive for Salmonella genomic island 1 (SGI1). A comparison of SGI1 positive and negative strains indicated that the increased biofilm formation of SGI1 positive strains was associated with the presence of this genomic island. Finally, in five strains we found an alternative strategy of biofilm formation independent of curli fimbriae and cellulose production but solely dependent on an overproduction of capsular polysaccharide. Due to a mucoid and brown appearance on Congo Red agar we designated these strains as belonging to the SBAM (smooth brown and mucoid) morphotype.

Identification of putative ancestors of the multidrug-resistant Salmonella enterica serovar typhimurium DT104 clone harboring the Salmonella genomic island 1.

The origin of multidrug-resistant Salmonella enterica serovar typhimurium (S. typhimurium) harboring the Salmonella Genomic Island 1 (SGI1), which was detected for the first time in the mid-1980s is unknown. In this study, we performed microarray genomotyping of four multidrug-resistant SGI1 positive strains and found that unlike the S. typhimurium LT2 strain, the multidrug-resistant strains lacked genes STM0517-0529 allowing the utilization of allantoin as a sole nitrogen source. We extended this observation by PCR screening of additional 120 S. typhimurium field strains and found that this locus was absent in all SGI1 positive and also in 24% of SGI1 negative strains, which were proposed to be the original recipients of SGI1. To prove this hypothesis, we compared the STM0517-0529 negative strains (with or without the SGI1) by PFGE and PCR prophage typing and found that 8 out of 11 of the SGI1 negative strains and 17 out of 22 SGI1 positive strains were of identical PFGE pattern and PCR prophage pattern, while this specific pattern was never observed among STM0517-0529 positive strains. We therefore propose that a lineage of the S. typhimurium DT104 sensitive strain first lost the ability to metabolize allantoin and then acquired SGI1.

An outbreak of human salmonellosis caused by ampicillin-resistant Salmonella enterica serovar Enteritidis PT13 in the Czech Republic.

In summer 2004, an outbreak caused by Salmonella enterica serovar Enteritidis phage type 13 (S. Enteritidis PT13) was recorded in the Czech Republic. As well being a relatively rare phage type the strain was also ampicillin resistant. Outbreak (n=39) and pre-outbreak isolates (n=13) were characterized by pulsed-field gel electrophoresis (PFGE), beta-lactamase gene polymerase chain reaction and plasmid profile. The majority of outbreak isolates (n=37) were identical in XbaI PFGE profile, and two other outbreak isolates each differed from this profile by one or two fragments respectively. The pre-outbreak isolates were uniform in PFGE profile but distinct from the outbreak strain. Ampicillin resistance was confirmed to be encoded by the blaTEM gene located on the TnA transposon. This gene was readily transferable to a S. Enteritidis recipient strain and was associated with the transfer of a 200-kb plasmid. Our results indicate that all S. Enteritidis PT13 tested from 2004 belonged to a single outbreak strain which prior to 2004 had not been recognized in the Czech Republic.

Distribution and function of plasmids in Salmonella enterica.

Plasmids of Salmonella enterica vary in size from 2 to more than 200 kb. The best described group of plasmids are the virulence plasmids (50-100 kb in size) present in serovars Enteritidis, Typhimurium, Dublin, Cholerae-suis, Gallinarum, Pullorum and Abortus-ovis. They all encode spvRABCD genes involved in intra-macrophage survival of Salmonella. Another group of high molecular weight plasmids are plasmids responsible for antibiotic resistance. Since most of these plasmids are conjugative, besides storage of genetic information, they contribute to the spread of genes in bacterial populations. The low molecular weight plasmids are the last group of plasmids found in S. enterica. Some of them have been shown to increase resistance to phage infection due to the presence of restriction modification systems. Despite limited knowledge on their function, their presence or absence is frequently used for strain differentiation in epidemiological studies.