Fatih Büyük

The Use of Germinants to Potentiate the Sensitivity of Bacillus anthracis Spores to Peracetic Acid

Elimination of Bacillus anthracis spores from the environment is a difficult and costly process due in part to the toxicity of current sporicidal agents. For this reason we investigated the ability of the spore germinants L-alanine (100 mM) and inosine (5 mM) to reduce the concentration of peracetic acid (PAA) required to inactivate B. anthracis spores. While L-alanine significantly enhanced (p = 0.0085) the bactericidal activity of 500 ppm PAA the same was not true for inosine suggesting some form of negative interaction. In contrast the germinant combination proved most effective at 100 ppm PAA (p = 0.0009). To determine if we could achieve similar results in soil we treated soil collected from the burial site of an anthrax infected animal which had been supplemented with spores of the Sterne strain of B. anthracis to increase the level of contamination to 104 spores/g. Treatment with germinants followed 1 h later by 5000 ppm PAA eliminated all of the spores. In contrast direct treatment of the animal burial site using this approach delivered using a back pack sprayer had no detectable effect on the level of B. anthracis contamination or on total culturable bacterial numbers over the course of the experiment. It did trigger a significant, but temporary, reduction (p < 0.0001) in the total spore count suggesting that germination had been triggered under real world conditions. In conclusion, we have shown that the application of germinants increase the sensitivity of bacterial spores to PAA. While the results of the single field trial were inconclusive, the study highlighted the potential of this approach and the challenges faced when attempting to perform real world studies on B. anthracis spores contaminated sites.

The effect of prolonged storage on the virulence of isolates of Bacillus anthracis obtained from environmental and animal sources in the Kars Region of Turkey.

The stability of the plasmid-mediated virulence factors of Bacillus anthracis, a tripartite toxin located on pXO1 and an antiphagocytic capsule encoded by genes located on pXO2, following long-term storage was investigated. A collection of 159 isolates of B. anthracis were collected from the Kars region of Turkey between 2000 and 2013 and stored at -20°C in Brucella broth supplemented with 20% glycerine. A total of 142 isolates were recovered of which one failed to express a capsule upon primary culture. A further 35 isolates yielded a mixture of mucoid and non-mucoid colonies; the majority of which had lost the pXO2 plasmid as determined by PCR analysis. Results would suggest that pXO2 is more unstable than pXO1 and that this instability increases with the length of storage. It is possible that the pXO2-deficient isolates of B. anthracis described here could be developed into a vaccine to treat at risk animals in the Kars region as many animal vaccines are based upon pXO2 deficiency.

Topical use of enilconazole (18%) in the treatment of bovine dermatophytosis: clinical, mycological, and histopatological findings.

Bu çal[şma, yağ bazl[ %10’luk Enilconazole’ün topikal kullan[m[n[n s[ğ[r dermatofitozisinde tedavi etkinliğinin araşt[r[lmas[ amac[yla planlanm[şt[r. Çal[şma materyalini, Kars ve çevre köylerinden sağlanan, 2-10 ayl[k yaşta, klinik ve mikrobiyolojik olarak dermatofitozis teşhisi konulan 18 deneme ve 10 kontrol olmak üzere, toplam 28 s[ğ[r oluşturdu. Her iki gruptaki hayvanlar[n klinik durumlar[; lezyonlar[n lokalizasyonu, büyüklüğü, say[lar[ ve hayvanlar[n kondüsyonlar[na göre, hafif (+), orta (++) ve şiddetli (+++) olarak skorland[r[ld[. Çal[şma süresince hayvanlar[n bak[m, besleme ve bar[nak koşullar[nda herhangi bir değişiklik yap[lmad[. Klinik muayene skorland[rmas[na göre; deneme grubu 5’i hafif, 8’i orta ve 5’i şiddetli; kontrol grubu 3’ü hafif, 5’i orta ve 2’si şiddetli olarak skorland[r[lan s[ğ[rlardan oluştu. Deneme grubundaki hayvanlar[n derilerindeki lezyonlar[n üzerine %10 Enilconazole içeren yağ bazl[ formülasyonu 3 gün ara ile 3 kez sürüldü. Kontrol grubundaki hayvanlar[n lezyonlar[ için hiçbir uygulama yap[lmad[. İyileşme süreçleri ilaç uygulamas[n[ takiben birer hafta ara ile 3 kez kontrol edildi. Deneme grubundaki hayvanlarda 2. uygulamay[ takiben, lezyonlardaki keratinize dokular[n büyük oranda azald[ğ[, 3. uygulama sonunda ise tamamen kaybolduğu görüldü. Lezyonlu bölgelerde 2-3. haftalarda k[llanman[n başlad[ğ[, 5-6. haftalarda lezyonun tamamen iyileştiği tespit edildi. Herhangi bir uygulama yap[lmayan kontrol grubundaki hayvanlarda dermatofitoz lezyonlar[nda hiç bir değişiklik olmad[ğ[ görüldü. Sonuç olarak, yağ bazl[ %10’luk Enilconazole formülasyonu, kullan[m[n[n kolay olmas[ ve k[sa sürede tedavi edici etki göstermesi nedeniyle, s[ğ[rlarda dermatofitozis olgular[n[n sağalt[m[nda önemli bir avantaj sağlayacağ[ kan[s[na var[ld[.

The identification of novel single nucleotide polymorphisms to assist in mapping the spread of Bacillus anthracis across the Southern Caucasus

Anthrax is common as a zoonotic disease in the southern Caucasus area including parts of Turkey and Georgia. In this region, population genetics of the etiological agent Bacillus anthracis comprises, where known, the major canonical single nucleotide polymorphism (canSNP) groups A.Br.Aust94 and A.Br.008/009 of the pathogen’s global phylogeny, respectively. Previously, isolates of B. anthracis from Turkey have been genotyped predominantly by multi locus variable number of tandem repeat analysis (MLVA) or canSNP typing. While whole genome sequencing is the future gold standard, it is currently still costly. For that reason we were interested in identifying novel SNPs which could assist in further distinguishing closely related isolates using low cost assay platforms. In this study we sequenced the genomes of seven B. anthracis strains collected from the Kars province of Eastern Anatolia in Turkey and discovered new SNPs which allowed us to assign these and other geographically related strains to three novel branches of the major A-branch canSNP-group (A.Br.) Aust94. These new branches were named Kafkas-Geo 1–3 and comprised isolates from the Kars region and the neighboring republic of Georgia suggesting a common ancestry. The novel SNPs identified in this study connect the population genetics of B. anthracis in the South Caucasus and Turkey and will likely assist efforts to map the spread of the pathogen across this region.

Caenorhabditis elegans Predation on Bacillus anthracis: Decontamination of Spore Contaminated Soil with Germinants and Nematodes

Remediation of Bacillus anthracis-contaminated soil is challenging and approaches to reduce overall spore levels in environmentally contaminated soil or after intentional release of the infectious disease agent in a safe, low-cost manner are needed. B. anthracis spores are highly resistant to biocides, but once germinated they become susceptible to traditional biocides or potentially even natural predators such as nematodes in the soil environment. Here, we describe a two-step approach to reducing B. anthracis spore load in soil during laboratory trials, whereby germinants and Caenorhabditis elegans nematodes are applied concurrently. While the application of germinants reduced B. anthracis spore load by up to four logs depending on soil type, the addition of nematodes achieved a further log reduction in spore count. These laboratory based results suggest that the combined use of nematodes and germinants could represent a promising approach for the remediation of B. anthracis spore contaminated soil. Originality-Significance Statement: This study demonstrates for the first time the successful use of environmentally friendly decontamination methods to inactivate Bacillus anthracis spores in soil using natural predators of the bacterium, nematode worms.

The mismatched isolation of Brucella strains from nomic hosts

Bulgular: Onaltı koyun örneğinin 15’i B. melitensis ve 14 sığır örneğinin 13’ü B. abortus olarak identifiye edildi. İlginçtir ki, koyun aborte fötusten izole edilen bir suş B. abortus ve sığırdan izole edilen bir suş B. melitensis olarak tanımlandı. Bununla birlikte, koyun fötusundan elde edilen bir suş bütün fenotipik özellikleri yönünden Brucella cinsine benzer karakterde olmasına rağmen genotipik olarak (ne Real Time PCR ne de Bruce-Ladder PCR) Brucella spp. olarak doğrulanamadı ve ileride karakterize edilmek üzere Ochrobactrum spp. şüpheli olarak saklandı.

Protection of farm goats by combinations of recombinant peptides and formalin inactivated spores from a lethal Bacillus anthracis challenge under field conditions

BackgroundBacillus (B.) anthracis, the causal agent of anthrax, is effectively controlled by the Sterne live spore vaccine (34F2) in animals. However, live spore vaccines are not suitable for simultaneous vaccination and antibiotic treatment of animals being at risk of infection in an outbreak situation. Non-living vaccines could close this gap.ResultsIn this study a combination of recombinant protective antigen and recombinant Bacillus collagen-like antigen (rBclA) with or without formalin inactivated spores (FIS), targeted at raising an immune response against both the toxins and the spore of B. anthracis, was tested for immunogenicity and protectiveness in goats. Two groups of goats received from local farmers of the Kars region of Turkey were immunized thrice in three weeks intervals and challenged together with non-vaccinated controls with virulent B. anthracis, four weeks after last immunization. In spite of low or none measurable toxin neutralizing antibodies and a surprisingly low immune response to the rBclA, 80% of the goats receiving the complete vaccine were protected against a lethal challenge. Moreover, the course of antibody responses indicates that a two-step vaccination schedule could be sufficient for protection.ConclusionThe combination of recombinant protein antigens and FIS induces a protective immune response in goats. The non-living nature of this vaccine would allow for a concomitant antibiotic treatment and vaccination procedure. Further studies should clarify how this vaccine candidate performs in a post infection scenario controlled by antibiotics.

Virulence plasmid stability in environmentally occurring Bacillus anthracis from North East Turkey

The Bacillus anthracis virulence plasmid pXO2, which encodes for a polypeptide capsule, can be lost during long term laboratory storage. To determine if pXO2 is lost in nature we screened B. anthracis isolates obtained from B. anthracis spores from contaminated animal burial sites in Turkey for their ability to express a capsule upon primary culture. A total of 672 B. anthracis colonies were examined of which ten produced a mixed mucoid (capsule +ve)/non-mucoid (capsule −ve) phenotype and a further one colony yielded non-mucoid colonies upon repeated culture. Screening by PCR using pXO2 specific primers revealed that seven of these isolates had eliminated the plasmid. Of the four colonies which were positive by PCR, one regained the ability to express a capsule upon repeated culture suggesting that the defect was reversible. This is an important observation as capsule expression is a principal marker of virulence and in the absence of PCR serves as a key diagnostic marker. The results of this preliminary study suggest that pXO2 is lost in nature and that further studies are need to determine the mechanisms by which this occurs.

Efficacy of pomades containing different percentages of enilconazole in the treatment of bovine dermatophytosis.

BACKGROUND Enilconazole is a broad-spectrum topical antimycotic agent used for the management of bovine dermatophytosis. HYPOTHESIS/OBJECTIVES To investigate the efficacy of pomades containing different concentrations of enilconazole for the treatment of bovine dermatophytosis. METHODS Dermatophytosis was confirmed in 120 cattle from farm in Gole region of Turkey. Animals were divided into six groups (n = 20 in each). Pomades containing 1%, 2%, 3%, 4% and 5% enilconazole were applied topically to individual lesions in groups I-V, respectively, once a day for 3 days. Group VI animals were used as a control group. Animals were monitored clinically once a week for a two month period. RESULTS Cows treated with pomades containing 4% and 5% enilconazole recovered; adverse topical reactions occurred in 40% and 55% of animals, respectively. The success rate for cows treated with pomades containing 3% enilconazole was 95% and they recovered with no adverse reactions. Success rates for treatment were 25% and 50% for cows treated with pomades containing 1% and 2% enilconazole, respectively. No improvement was observed in the control group. CONCLUSIONS AND CLINICAL IMPORTANCE Pomades containing 3% enilconazole are recommended for the treatment of bovine dermatophytosis.

Use of immunoperoxidase technique in smears prepared from vaginal secretions in early diagnosis of listerial abortions in cattle.

Listeriosis is a sporadic disease of ruminants that causes meningoencephalitis, septicemia and abortion. In this study, the usefulness of immunoperoxidase technique was investigated in early diagnosis of cattle listerial abortions. For this purpose, 96 smears prepared from vaginal swab samples that were collected from aborting cattle were stained for L. monocytogenes by immunoperoxidase technique. Presence of the agent in vaginal swab samples were investigated by bacteriological culture technique and the results were compared to that of immunoperoxidase technique. A total of 7 samples, 4 out 5 bacteriological culture positive smear samples and 3 out of 91 bacteriological culture negative smear samples, were detected to be positive by immunoperoxidase technique. Compared to bacteriological culture technique, sensitivity and specificity of immunoperoxidase technique was calculated as 80% and 96.7%, respectively. In conclusion, immunoperoxidase technique in smears prepared from vaginal swabs can be used in early diagnosis of listerial abortions since it can give results the same day samples collected, however the technique must be supported by bacteriological culture technique which is performed for bacterial isolation and identification of the agent.