Regulation of energy metabolism by the extracytoplasmic function (ECF) σ factors of Arcobacter butzleri.

The extracytoplasmic function (ECF) σ factors are fundamental for bacterial adaptation to distinct environments and for survival under different stress conditions. The emerging pathogen Arcobacter butzleri possesses seven putative pairs of σ/anti-σ factors belonging to the ECF family. Here, we report the identification of the genes regulated by five out of the seven A. butzleri ECF σ factors. Three of the ECF σ factors play an apparent role in transport, energy generation and the maintenance of redox balance. Several genes like the nap, sox and tct genes are regulated by more than one ECF σ factor, indicating that the A. butzleri ECF σ factors form a network of overlapping regulons. In contrast to other eubacteria, these A. butzleri ECF regulons appear to primarily regulate responses to changing environments in order to meet metabolic needs instead of an obvious role in stress adaptation.

The complex interplay between stress and bacterial infections in animals.

Over the past decade, an increasing awareness has arisen of the role of neuroendocrine hormones in the susceptibility of mammalian hosts to a bacterial infection. During a stress response, glucocorticoids, catecholamines and neuroendocrine factors are released into the circulation of the host. For a long time the effects of stress on the course of an infection have been exclusively ascribed to the direct effect of stress-related hormones on the immune system and the intestinal barrier function. Chronic stress is known to cause a shift from T helper 1-mediated cellular immunity toward T helper 2-mediated humoral immunity, which can influence the course of an infection and/or the susceptibility to a microorganism. Bacteria can however also respond directly to stress-related host signals. Catecholamines can alter growth, motility, biofilm formation and/or virulence of pathogens and commensal bacteria, and as a consequence influence the outcome of infections by these bacteria in many hosts. For some bacteria, such as Salmonella, Escherichia coli and Pseudomonas aeruginosa it was shown that this influence is regulated by quorum sensing mechanisms. In this manuscript an overview of how and when stress influences the outcome of bacterial infections in animals is provided.

Pythium insidiosum: an overview.

Pythium insidiosum is an oomycete pathogenic in mammals. The infection occurs mainly in tropical and subtropical areas, particularly in horses, dogs and humans. Infection is acquired through small wounds via contact with water that contains motile zoospores or other propagules (zoospores or hyphae). The disease, though described as emerging has in fact already been described since 1884. Depending on the site of entry, infection can lead to different forms of pythiosis i.e. a cutaneous, vascular, ocular, gastrointestinal and a systemic form, which is rarely seen. The infection is not contagious; no animal-animal or animal-human transmission has been reported so far. Therapy includes radical surgery, antifungal drugs, immunotherapy or a combination of these therapies. The prevention to contract the disease in endemic areas is difficult. Avoiding stagnant waters could be of help, although the presence of P. insidiosum on grass and soil in enzootic areas renders this practice useless.

Antimicrobial resistance, class 1 integrons, and genomic island 1 in Salmonella isolates from Vietnam.

BACKGROUND: The objective was to investigate the phenotypic and genotypic resistance and the horizontal transfer of resistance determinants from Salmonella isolates from humans and animals in Vietnam. METHODOLOGY/PRINCIPAL FINDINGS: The susceptibility of 297 epidemiologically unrelated non-typhoid Salmonella isolates was investigated by disk diffusion assay. The isolates were screened for the presence of class 1 integrons and Salmonella genomic island 1 by PCR. The potential for the transfer of resistance determinants was investigated by conjugation experiments. Resistance to gentamicin, kanamycin, chloramphenicol, streptomycin, trimethoprim, ampicillin, nalidixic acid, sulphonamides, and tetracycline was found in 13 to 50% of the isolates. Nine distinct integron types were detected in 28% of the isolates belonging to 11 Salmonella serovars including S. Tallahassee. Gene cassettes identified were aadA1, aadA2, aadA5, bla(PSE-1), bla(OXA-30), dfrA1, dfrA12, dfrA17, and sat, as well as open reading frames with unknown functions. Most integrons were located on conjugative plasmids, which can transfer their antimicrobial resistance determinants to Escherichia coli or Salmonella Enteritidis, or with Salmonella Genomic Island 1 or its variants. The resistance gene cluster in serovar Emek identified by PCR mapping and nucleotide sequencing contained SGI1-J3 which is integrated in SGI1 at another position than the majority of SGI1. This is the second report on the insertion of SGI1 at this position. High-level resistance to fluoroquinolones was found in 3 multiresistant S. Typhimurium isolates and was associated with mutations in the gyrA gene leading to the amino acid changes Ser83Phe and Asp87Asn. CONCLUSIONS: Resistance was common among Vietnamese Salmonella isolates from different sources. Legislation to enforce a more prudent use of antibiotics in both human and veterinary medicine should be implemented by the authorities in Vietnam.

Editorial: assessing the antimicrobial susceptibility of bacteria obtained from animals.

The accurate performance of antimicrobial susceptibility testing of bacteria from animal sources and the correct presentation of the results is a complex matter. A review of the published literature revealed a number of recurring errors with regard to methodology, quality control, appropriate interpretive criteria, and calculation of MIC(50) and MIC(90) values. Although more subjective, there is also no consensus regarding the definition of multiresistance. This Editorial is intended to provide guidance to authors on how to avoid these frequently detected shortcomings.

Capnocytophaga canimorsus.

Capnocytophaga canimorsus is a commensal bacterium in the oral flora of dogs and cats. The bacterium is a zoonotic agent and has been isolated from humans, infected by dog or cat bites, scratches, licks or simply exposure to dogs or cats. Here the infectious agent, its pathogenicity and potential virulence factors, infection in animals and humans, diagnostic methods, prevalence, therapy and prevention are described. Suggestions for future research are given.

Rat bite fever.

Rat bite fever (RBF) is a bacterial zoonosis for which two causal bacterial species have been identified: Streptobacillis moniliformis and Spirillum minus. Haverhill fever (HF) is a form of S. moniliformis infection believed to develop after ingestion of contaminated food or water. Here the infectious agents, their host species, pathogenicity (virulence factors and host susceptibility), diagnostic methods, therapy, epidemiology, transmission and prevention are described. Special emphasis is given on information from the field of laboratory animal microbiology and suggestions for future research.

The introduction of Arcobacter spp. in poultry slaughterhouses.

Despite the presence high levels of Arcobacter spp. on chicken carcasses, the source of arcobacter contamination in slaughterhouses still remains unclear. It has been hypothesised in the literature that Arcobacter species that contaminate carcasses originate in in-plant slaughterhouses and/or supply water. The present study aimed to determine the source of Arcobacter contamination in two poultry slaughterhouses in The Netherlands. Carcasses and intestinal tracts from 3 hen flocks and 2 broiler flocks were collected. Water draining off carcasses during processing in 2 slaughterhouses and supply water in one slaughterhouse were also taken. For one flock, cloacal swabs and faecal samples were taken on the farm before slaughtering. ERIC-PCR was applied to study the genetic diversity and relationship among the isolates. No Arcobacter spp. were found in the supply water but on almost all of the sampled carcasses and in carcass-draining-off water arcobacters were identified. Arcobacter spp. were detected in the gut systems of chickens, ranging from 20% to 85% in hens and 3.3% and 51% in broilers. Similar ERIC-PCR genotypes were detected in gut contents as well as on carcasses from the same flock. The present study demonstrated that Arcobacter spp. can be detected in chicken intestines at slaughter and could be brought in this way into slaughterhouses where the bacteria contaminate carcasses during processing.

Arcobacter spp. possess two very short flagellins of which FlaA is essential for motility.

Like Campylobacter and Helicobacter spp., Arcobacter spp. possess two flagellin genes (flaA and flaB) located adjacent to each other. The aim of this study was to characterize the flagellin proteins of Arcobacter spp., because these proteins are known virulence factors in the Epsilonproteobacteria, to which these three species belong. With the exception of Arcobacter nitrofigilis, Arcobacter flagellins are almost half the size of those in other Epsilonproteobacteria. Arcobacter flagellin proteins lack a large part of the variable central region. The low homology observed among flagellins of different Arcobacter species indicates genetic heterology between the members of this genus. Unlike in other Epsilonproteobacteria, the transcription of flagellin genes is not regulated by sigma 28- or sigma 54-dependent promoters, which suggests that transcription must be regulated in a different way in Arcobacter spp. Mutational studies revealed that only FlaA is needed for the motility of Arcobacter spp. Quantitative PCR analysis showed that transcription of flaB is higher at 30 degrees C than at 37 degrees C. Mutation of flaB had no effect on motility or on flaA transcription while mutation of flaA abolished motility and increased the transcription of flaB. These results underline that the genus Arcobacter is an unusual taxon in the epsilon subdivision of the Proteobacteria.

WITHDRAWN: Antimicrobial resistance, class 1 integrons and a novel variant of Genomic Island 1 in Salmonella isolates from Vietnam.

This manuscript has been withdrawn.

Dogs as vectors of Streptobacillus moniliformis infection?

Rat bite fever is a bacterial zoonosis transmitted through the bite of rats. One of the two etiological agents that cause rat bite fever is Streptobacillus moniliformis. Rat bite fever is rare and very likely under diagnosed but occurs worldwide. Other animals, like dogs and cats that have mouthed a rat are often mentioned in the literature as potential risks for the attraction of rat bite fever. However, rat bite fever caused by the bite of a dog or cat has very seldom been documented. Therefore, to identify the possible risk for humans to become infected with S. moniliformis after having been bitten by a dog that has been in contact with rats, the presence of S. moniliformis in the mouth of these dogs was tested with molecular methods. Swabs taken from the mouth of 18 dogs with proven contacts with rats were tested for the presence of S. moniliformis DNA by PCR. An amplicon of the right size was obtained in 10 of the 18 dogs. Nucleotide sequencing of five amplicons of PCR positive samples demonstrated the presence of S. moniliformis DNA in the mouth of three dogs. A bite by these dogs therefore might infect humans with S. moniliformis and cause rat bite disease.

Carvacrol induces heat shock protein 60 and inhibits synthesis of flagellin in Escherichia coli O157:H7.

The essential oils of oregano and thyme are active against a number of food-borne pathogens, such as Escherichia coli O157:H7. Carvacrol is one of the major antibacterial components of these oils, and p-cymene is thought to be its precursor in the plant. The effects of carvacrol and p-cymene on protein synthesis in E. coli O157:H7 ATCC 43895 cells were investigated. Bacteria were grown overnight in Mueller-Hinton broth with a sublethal concentration of carvacrol or p-cymene, and their protein compositions were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and confirmed by Western blotting. The presence of 1 mM carvacrol during overnight incubation caused E. coli O157:H7 to produce significant amounts of heat shock protein 60 (HSP60) (GroEL) (P < 0.05) and inhibited the synthesis of flagellin highly significantly (P < 0.001), causing cells to be aflagellate and therefore nonmotile. The amounts of HSP70 (DnaK) were not significantly affected. p-Cymene at 1 mM or 10 mM did not induce HSP60 or HSP70 in significant amounts and did not have a significant effect on flagellar synthesis. Neither carvacrol (0.3, 0.5, 0.8, or 1 mM) nor p-cymene (0.3, 0.5, or 0.8 mM) treatment of cells in the mid-exponential growth phase induced significant amounts of HSP60 or HSP70 within 3 h, although numerical increases of HSP60 were observed. Motility decreased with increasing concentrations of both compounds, but existing flagella were not shed. This study is the first to demonstrate that essential oil components induce HSP60 in bacteria and that overnight incubation with carvacrol prevents the development of flagella in E. coli O157:H7.

Characteristics of extended-spectrum cephalosporin-resistant Escherichia coli and Klebsiella pneumoniae isolates from horses.

The aim of the present study was to contribute to the knowledge on extended-spectrum beta-lactamases (ESBL's), AmpC beta-lactamases and integrons in Enterobacteriaceae isolated from horses, which is still limited. The susceptibility of 1581 clinical isolates from animals to ceftiofur was tested. Most of these isolates (n=1347) originated from horses. Seven ceftiofur-resistant equine isolates (four Escherichia coli and three Klebsiella pneumoniae) were identified and all seven were multidrug-resistant. These isolates were further studied for the presence of ESBL's, AmpC beta-lactamases and class 1 integrons. The potential for the horizontal transfer of resistance genes among these clinical isolates was also studied. ESBL-type resistance genes were found in five isolates, AmpC-type genes in one isolates and integrons in six isolates. Nucleotide sequence analysis revealed that the isolates carried the bla(CTX-M-1), bla(CMY-2), bla(TEM-1) and/or bla(SHV-1) genes. This is the first report describing the in vitro conjugal transfer of the bla(CTX-M-1) genes from a clinical E. coli isolate to Salmonella isolates. Gene cassettes encoding resistance to aminoglycosides (aadA1, aadA2 and aadA5), and trimethoprim (dfrA1, drfA12 and dfrA17) were found on the integrons present in the isolates. The cassette arrays of the dfrA17-aadA5 and dfrA1-aadA1 genes in the two integrons of a single E. coli isolate have not yet been described before. To our knowledge this is the first report on ESBL's and AmpC beta-lactamases in equine E. coli and Klebsiella isolates.

Interaction of Arcobacter spp. with human and porcine intestinal epithelial cells.

Little is known about the pathogenic mechanisms or potential virulence factors of Arcobacter spp. The aim of the study described here was to obtain more insights in the pathogenicity mechanisms of Arcobacter spp. by testing their ability to adhere to, invade and induce interleukin-8 expression in human Caco-2 and porcine IPI-2I cell lines. Eight Arcobacter strains were tested. Four strains were obtained from a culture collection, and represent the four Arcobacter spp. known to be associated with animals and humans. The other four strains were field isolates from the amniotic fluid of sows and from newborn piglets. All eight Arcobacter strains were able to adhere to both cell lines, and induced interleukin-8 production as early as 2 h after a 1h incubation period. This production was still increased 6 h postinfection. Differences in the cell association of the eight strains were obvious, with A. cibarius showing the highest adhesion ability. Invasion of intestinal epithelial cells was only observed for A. cryaerophilus strains. No correlation between invasiveness or strong adhesion of the tested strains and the level of interleukin-8 induction was observed.

A novel Salmonella genomic island 1 and rare integron types in Salmonella Typhimurium isolates from horses in The Netherlands.

OBJECTIVES: To investigate the genotypic resistance of integron-carrying Salmonella Typhimurium isolates from horses and their genetic relationship. METHODS: Sixty-one Salmonella isolates were screened for the presence of class 1 integrons by PCR. The gene cassettes of integron-positive isolates were detected by PCR, restriction fragment length polymorphism typing, and sequencing. The potential for the transfer of resistance determinants was investigated by conjugation experiments. The presence of Salmonella genomic island 1 (SGI1) or its variants was studied by PCR and nucleotide sequencing. PFGE was used to genotype the isolates. RESULTS: Eight distinct XbaI-PFGE profiles and seven integron types were observed among 26 integron-carrying Salmonella Typhimurium isolates. The gene cassettes detected were dfrA1, dfrA7, dfrA14, aadA1, aadA2, aadB and bla(PSE). A rare type of integron found in nine isolates carried the dfrA14 and aadA1 gene cassettes. Twelve Salmonella Typhimurium DT104 isolates contained SGI1 or one of its variants (SGI1, SGI1-B and SGI1-C). A novel variant of SGI1, designated SGI1-M, was identified in one isolate in which the aadA2 gene of SGI1 was replaced by the aadB gene. Transfer of integrons and antimicrobial resistance determinants to Escherichia coli K12 via conjugation was possible with nine isolates. Resistance to fluoroquinolones in nine isolates was caused by mutations in the gyrA gene leading to the amino acid changes Ser-83 --> Ala and Asp-87 --> Asn. CONCLUSIONS: The integron-positive clinical Salmonella Typhimurium isolates from horses belong to distinct strains. The data demonstrate the capability of Salmonella Typhimurium to acquire additional antibiotic resistance determinants and underline the need for the prudent use of antimicrobials.

Comparison of the in vitro pathogenicity of two Salmonella Typhimurium phage types.

The in vitro pathogenicity of Salmonella enterica serovar Typhimurium phage type (pt) 90 and pt 506 (also known as DT 104) isolates from human and porcine origin was studied in adhesion and invasion assays to the human cell line Caco-2 and the porcine cell line IPI-2. Interleukin-8 (IL-8) production by these two cell lines in response to stimulation by the two Salmonella phage types was also measured. Generally, Salmonella Typhimurium pt 506 and pt 90 adhered to and invaded Caco-2 cells and IPI-2 cells equally well. The release of IL-8 by Caco-2 cells or by IPI-2 cells was similar, independent of the Salmonella phage type used for stimulation of the cells. These data suggest that Salmonella Typhimurium pt 90 has a similar ability to cause Salmonella infections as Salmonella Typhimurium DT 104.