Discrimination of Arcobacter butzleri isolates by polymerase chain reaction-mediated DNA fingerprinting

AIMS: The objective of this study was to subtype Arcobacter butzleri isolates using RAPD-PCR. METHODS AND RESULTS: Thirty-five A. butzleri isolates obtained from chicken carcasses were examined. PCR-mediated DNA fingerprinting technique with primers of the variable sequence motifs was used to detect polymorphism within the isolates. Eleven distinct DNA profiles were obtained as follows: Of the 35 strains, 10 as profile 4; seven as profile 1; five as profile 3; three as profiles 2 and 9; two as profile 10; one as profiles 5, 6, 7, 8 and 11. CONCLUSIONS: Chicken carcasses sold in markets were found to be contaminated with several different strains of A. butzleri. RAPD-PCR technique was found to be a useful technique for distinguishing A. butzleri isolates. SIGNIFICANCE AND IMPACT OF THE STUDY: The presence of several different A. butzleri strains on chicken carcasses may indicate multiple sources of contamination. The epidemiological role of A. butzleri in human and animal diseases should be investigated further.     

Genome mapping of Arcobacter butzleri

A genome map of the potential food-poisoning pathogen Arcobacter butzleri, strain NCTC 12481, has been constructed. The map was ordered by a combination of one- and two-dimensional pulsed field gel electrophoresis, using the restriction enzymes EagI, SacII, SalI and XhoI, and Southern hybridization with digoxygenin-labelled probes. The size of the genome was estimated at 2.57+/-0.01 Mb; this is 0.9 Mb larger than the genome of the closely related species Campylobacter jejuni. We deduce that there are five copies of the 23S and 16S rRNA genes present in the A. butzleri NCTC 12481 genome and two copies of the gene glyA. A gene homologous to pglF, involved in bacterial glycosylation in C. jejuni, was also located on the genome map. In C. jejuni and Wolinella succinogenes, most glycosylation genes are clustered together and the gene order is similar. In A. butzleri NCTC 12481, no analogous organization was found in the region sequenced.     

Isolation of Arcobacter butzleri from ground water

Arcobacter butzleri was isolated from a contaminated ground water source. These organisms, previously designated as aerotolerant Campylobacter, were capable of surviving in the ground water environment. Specific DNA probes were used to characterize the isolates in the initial identification and survival studies. Arcobacter butzleri was found to be sensitive to chlorine inactivation.     

Susceptibility of sixty-five non-oral clinical isolates of the Streptococcus milleri group to seven antimicrobial agents.

Antimicrobial susceptibilities of sixty-five non-oral Streptococcus milleri group clinical isolates to penicillin, gentamicin, lincomycin, ampicillin, chloramphenicol, tetracycline and erythromycin were determined by an agar dilution method. All strains were penicillin-sensitive (MIC < or = 0.031 microgram/ml) and the majority (64/65) were susceptible to erythromycin (MIC < or = 0.125 microgram/ml). Low-level resistance to gentamicin was observed, and the majority of strains possessed an MIC of 8 micrograms/ml. Lincomycin and ampicillin at 0.5 microgram/ml inhibited 52/65 and 61/65 strains, respectively. Of the isolates 92% were inhibited by chloramphenicol at < or = 2 micrograms/ml. Twenty-two S. milleri group strains (of which thirteen were vaginal isolates) were resistant to tetracycline (MIC > or = 8 micrograms/ml).     

Susceptibility of Pediococcus spp. to antimicrobial agents

AIM: To investigate the susceptibility of Pediococcus species to antimicrobial agents. METHODS AND RESULTS: The susceptibility to 14 antimicrobial agents of 31 genotypically distinct strains of six Pediococcus species was assessed by using Etests on ISO-sensitest agar supplemented with horse blood. The species included were Pediococcus acidilactici, Pediococcus damnosus, Pediococcus dextrinicus, Pediococcus inopinatus, Pediococcus parvulus and Pediococcus pentosaceus. For several antimicrobial agents, some species were more susceptible than others. The two industrially important species, P. acidilactici and P. pentosaceus, differed with respect to erythromycin and trovafloxacin susceptibility, and in general both species had higher minimum inhibitory concentrations than the other species. In an erythromycin-resistant P. acidilactici, an erythromycin resistance methylase B [erm(B)] gene was identified by PCR. Using a plasmid preparation from strain P. acidilactici 6990, a previously erythromycin-sensitive Lactococcus lactis strain was made resistant. Transformants harboured a single plasmid, sized at 11.6 kb through sequence analysis. In addition, the erm(B) gene was identified within the plasmid sequence. CONCLUSIONS: The phenotypic test indicated the absence of acquired antimicrobial resistance genes in 30 of the strains. SIGNIFICANCE AND IMPACT OF THE STUDY: These results will help in selection of the best Pediococcus strains for use as starter cultures.     

Arcobacter butzleri isolated from a diarrhoeic non-human primate

The bacteriological examination of a faecal specimen from a 20-year-old female rhesus macaque (Macaca mulatta) with diarrhoeal illness revealed the presence of a large number of a relatively new enteric pathogen, Arcobacter butzleri. The animal was from a closed colony of about 60 females, some of them were showing intermittent diarrhoea possibly related to Giardia spp. Conditions for the isolation and identification of A. butzleri are reported, as well as discussions about its role as a primary pathogen and its zoonotic potential.     

Arcobacter butzleri,Arcobacter cryaerophilus,and Arcobacter skirrowii Circulation in a Dairy Farm and Sources of Milk Contamination

Even though dairy cows are known carriers of Arcobacter species and raw or minimally processed foods are recognized as the main sources of human Arcobacter infections in industrialized countries, data on Arcobacter excretion patterns in cows and in milk are scant. This study aimed to identify potentially pathogenic Arcobacter species in a dairy herd and to investigate the routes of Arcobacter transmission among animals and the potential sources of cattle infection and milk contamination. A strategy of sampling the same 50 dairy animals, feed, water, and milk every month for a 10-month period, as well as the sampling of quarter milk, animal teats, the milking environment, and animals living on the farm (pigeons and cats), was used to evaluate, by pulsed-field gel electrophoresis (PFGE), the characteristic patterns in animals, their living environment, and the raw milk they produced. Of the 463 samples collected, 105 (22.6%) were positive for Arcobacter spp. by culture examination. All the matrices except quarter milk and pigeon gut samples were positive, with prevalences ranging from 15 to 83% depending on the sample. Only three Arcobacter species, Arcobacter cryaerophilus (54.2%), A. butzleri (34.2%), and A. skirrowii (32.3%), were detected. PFGE analysis of 370 isolates from positive samples provided strong evidence of Arcobacter circulation in the herd: cattle likely acquire the microorganisms by orofecal transmission, either by direct contact or from the environment, or both. Water appears to be a major source of animal infection. Raw milk produced by the farm and collected from a bulk tank was frequently contaminated (80%) by A. butzleri; our PFGE findings excluded primary contamination of milk, whereas teats and milking machine surfaces could be sources of Arcobacter milk contamination.     

Induction and resuscitation of viable nonculturable Arcobacter butzleri cells

Two strains of Arcobacter butzleri, ATCC 49616 and an environmental isolate, became nonculturable in seawater microcosms at 4 degrees C by 20 days and at room temperature by 14 days. Nonculturable cells were viable for up to 270 days of incubation in microcosms. Resuscitation of A. butzleri cells from microcosms at both temperatures was achieved 9 days after nutrient addition.     

Susceptibility of clinical isolates of yeasts to anti-fungal agents

The antimicrobial activity of amphotericin B, 5-fluorocytosine, nystatin, clotrimazole and miconazole were compared in vitro against 244 strains of yeasts that had been isolated from clinical specimens. The yeasts used in this study included 20 species of Candida, Cryptococcus, Saccharomyces Geotrichum, Rhodotorula, Torulopsis and Trichosporon. The majority of the strains (78%) had an MIC of 0.5 g/ml for amphotericin B, 81% an MIC of 1 g/ml for 5-fluorocytosine, 99% 8 g/ml for nystatin, 91%, 8.0 g/ml for clotrimazole and 98% had an MIC of 4.0 for miconazole. Of the anti-fungal agents tested, 5-fluorocytosine and nystatin were found to have the greatest antifungal activity.     

Susceptibility of clinical Staphylococcus aureus isolates to innate defense antimicrobial peptides

Antimicrobial peptides (AMPs) are effector molecules of innate immunity. To determine whether AMP susceptibility of S. aureus varies according to different types of infection, 102 isolates from patients with S. aureus bacteremia or recurrent skin and soft tissue infection, and colonizing isolates were investigated. Using microbroth dilution assays we found a narrow range of MICs of human β-defensin-3, cathelicidin LL-37 and bovine indolicidin without significant differences between the groups. Colony-forming unit (CFU) assays revealed minor differences in bactericidal activity with slightly but not significantly higher CFU reduction in colonizing isolates. These data do not support a role for differential AMP susceptibility in vitro as a major determinant of S. aureus invasive infection.     

The complete genome sequence and analysis of the epsilonproteobacterium Arcobacter butzleri

Background

Arcobacter butzleri is a member of the epsilon subdivision of the Proteobacteria and a close taxonomic relative of established pathogens, such as Campylobacter jejuni and Helicobacter pylori. Here we present the complete genome sequence of the human clinical isolate, A. butzleri strain RM4018.

Methodology/Principal Findings

Arcobacter butzleri is a member of the Campylobacteraceae, but the majority of its proteome is most similar to those of Sulfuromonas denitrificans and Wolinella succinogenes, both members of the Helicobacteraceae, and those of the deep-sea vent Epsilonproteobacteria Sulfurovum and Nitratiruptor. In addition, many of the genes and pathways described here, e.g. those involved in signal transduction and sulfur metabolism, have been identified previously within the epsilon subdivision only in S. denitrificans, W. succinogenes, Sulfurovum, and/or Nitratiruptor, or are unique to the subdivision. In addition, the analyses indicated also that a substantial proportion of the A. butzleri genome is devoted to growth and survival under diverse environmental conditions, with a large number of respiration-associated proteins, signal transduction and chemotaxis proteins and proteins involved in DNA repair and adaptation. To investigate the genomic diversity of A. butzleri strains, we constructed an A. butzleri DNA microarray comprising 2238 genes from strain RM4018. Comparative genomic indexing analysis of 12 additional A. butzleri strains identified both the core genes of A. butzleri and intraspecies hypervariable regions, where <70% of the genes were present in at least two strains.

Conclusion/Significance

The presence of pathways and loci associated often with non-host-associated organisms, as well as genes associated with virulence, suggests that A. butzleri is a free-living, water-borne organism that might be classified rightfully as an emerging pathogen. The genome sequence and analyses presented in this study are an important first step in understanding the physiology and genetics of this organism, which constitutes a bridge between the environment and mammalian hosts.     

The recovery of Arcobacter butzleri NCTC 12481 from various temperature treatments

AIMS: The aim of this study was to determine the growth and survival characteristics for Arcobacter butzleri NCTC 12481. METHODS AND RESULTS: The temperature and pH growth ranges were 15-39 degrees C and pH 6.0-8.0, as determined using impedance microbiology. The maximum specific growth rate was 00.57 h(-1) at 30 degrees C, pH 7.0. Arcobacter butzleri harvested from the exponential phase was more resistant to heat treatment than stationary phase cells (D55 1.1 and 0.4 min, respectively). Fluorescent dye uptake, and the release of UV-absorbing material, increased in heat-treated cells. After 21 d storage at 4 and -20 degrees C, A. butzleri was recovered on blood agar, but not on the isolation media CAT or CCDA. CONCLUSION: Arcobacter butzleri cells from the exponential phase were less heat sensitive than those from the stationary phase. The organism was able to survive cold storage for at least 3 weeks. SIGNIFICANCE AND IMPACT OF THE STUDY: The growth and survival characteristics have been quantified thus providing a greater understanding of this newly emerging pathogen.     

Susceptibility of Pediococcus isolates to antimicrobial compounds in relation to hop-resistance and beer-spoilage

Background  

Though important in the context of food microbiology and as potential pathogens in immuno-compromised humans, bacterial isolates belonging to the genus Pediococcus are best known for their association with contamination of ethanol fermentation processes (beer, wine, or fuel ethanol). Use of antimicrobial compounds (e.g., hop-compounds, Penicillin) by some industries to combat Pediococcus contaminants is long-standing, yet knowledge about the resistance of pediococci to antimicrobial agents is minimal. Here we examined Pediococcus isolates to determine whether antibiotic resistance is associated with resistance to hops, presence of genes known to correlate with beer spoilage, or with ability to grow in beer.     

Development of a multiplex and real time PCR assay for the specific detection of Arcobacter butzleri and Arcobacter cryaerophilus

A new multiplex PCR and two specific TaqMan assays were developed to target the emerging pathogens A. butzleri and A. cryaerophilus. The assays also included an internal control to verify the presence of bacterial target DNA and amplification integrity. The multiplex assay used a published primer set (CRY1 and CRY2) for detecting A. cryaerophilus DNA (Houf, K., Tutenel, A., De Zutter, L., Van Hoof, J. and Vandamme, P., 2000. Development of a multiplex PCR assay for the simultaneous detection and identification of Arcobacter butzleri, Arcobacter cryaerophilus and Arcobacter skirrowii. FEMS microbiology letters, 193 (1): 89-94.) and a novel A. butzleri primer set designed to target the rpoB/C gene sequences. To improve sample throughput and assay sensitivity a TaqMan assay for each Arcobacter spp. was developed which again utilised the heterogeneity contained in the rpoB/C and 23s rRNA gene sequences. The two TaqMan assays provided >2 log improvement in detection sensitivity for both Arcobacter spp. compared with the multiplex PCR assay and were able to detect <10 CFU per PCR reaction. To evaluate the effectiveness of the Arcobacter TaqMan assays with field isolates the assays were used to screen DNA samples prepared from faecal, hide and environmental samples obtained from two meat processing plants. In these studies, the TaqMan assays revealed that 2/150 (1.3%) samples were A. butzleri-positive, 11/150 (7.3%) were A. cryaerophilus-positive and the identity of generated amplicons was confirmed by DNA sequencing. Our results show that these TaqMan assays provide improvements in sensitivity and species-representation over other published Arcobacter PCR assays and they are compatible with detecting Arcobacters in sub-optimal matrices.     

Susceptibility of the Lyme disease spirochete to seven antimicrobial agents.

The antimicrobial susceptibility of five Lyme disease spirochete strains (two human and three tick isolates) was determined. A macrodilution broth technique was used to determine on three separate test occasions the minimal inhibitory concentrations (MICs) of seven antibiotics. The Lyme disease spirochete was most susceptible to erythromycin with a MIC of less than or equal to 0.06 micrograms/ml. The spirochete was also found to be susceptible to minocycline, ampicillin, doxycycline, and tetracycline-HCL with respective mean MICs of less than or equal to 0.13, less than or equal to 0.25, less than or equal to 0.63, and less than or equal to 0.79 micrograms/ml. The spirochete was moderately susceptible to penicillin G with a mean MIC of 0.93 micrograms/ml. All strains were resistant to rifampin at the highest concentration tested (16.0 micrograms/ml).     

Identification of Arcobacter isolates by PCR

K.M. HARMON AND I.V. WESLEY. 1996. A polymerase chain reaction (PCR) assay was developed for the identification of the three species of Arcobacter which have been recovered from clinically ill or healthy humans and/or livestock, namely Arcobacter butzleri, Arcobacter skirrowii and Arcobacter cryaerophilus . The assay utilizes primers targeted to the genes encoding 16S rRNA of Arcobacter spp. The assay reduces the amount of time required to positively identify strains of Arcobacter .     

Susceptibility of various purple and green sulfur bacteria to different antimicrobial agents

Abstract Several purple and green sulfur bacteria (genera Chromatium, Thiocapsa and Chlorobium ) were tested for their sensitivity to different antimicrobial agents by a disc diffusion assay, using thioacetamide as a source of hydrogen sulfide for plate growth. Chlorobium limicola strains were more sensitive to amoxicillin, erythromycin and nalidixic acid, whereas gentamicin and netilmicin were more active against the purple bacteria tested. None of the organisms were sensitive to oxacillin and trimethoprim + sulfamethoxazole. The critical concentrations at the edge of the inhibition zone were also calculated for three organisms and the antimicrobials colistin, mitomycin C, penicillin G, rifampicin, and streptomycin. The results obtained suggest that colistin, mitomycin C, penicillin G would provide selective conditions against the growth of Chlorobium limicola strains, while streptomycin and other aminoglycoside antibiotics would select against purple bacteria.