Journal of Food & Industrial Microbiology

Acanthamoeba is a genus of small, free-living amoebae common to most land and freshwater habitats. The body has a life cycle of feeding and replicating trophozoite which, in response to adverse conditions, can form a dormant cyst stage. Its Acanthamoeba spp. Opportunistic human pathogens cause lethal granulomatous encephalitis in the immunocompromised host and, more commonly, potentially blinding keratitis in both non-contact lens and Contact Lens (CL) wearers.

There are currently around 4.1 million CL wearers in the UK and established independent risk factors for developing Acanthamoeba Keratitis (AK) in CL wearers include exposure to tap water at home, swimming or bathing while wearing CL, poor lens hygiene and the use of rigid CLs in orthokeratology. In addition, previous outbreaks of AK in both the United Kingdom and the United States were due to effectiveness issues with some CL disinfection systems. Given the sight-threatening risk with AK, it accounts for less than 5 percent of all CL-related microbial keratitis in most episodes. The published incidence rates for CL users are 1 to 2 per million in the United States and 17 to 20 per million in the United Kingdom. A recent research in a tertiary hospital in the United Kingdom showed an incidence rate of only 2.3 per cent for Acanthamoeba in more than 1500 cases of keratitis over a 12-year period. Due to the small number of patients with AK, many are diagnosed late because they were initially misdiagnosed and treated for bacterial or other forms of keratitis, such as fungal and herpes simplex keratitis. Early diagnosis of AK has a substantial effect on prognosis, and patients are more likely to have worse visual outcomes, longer period of treatment, corneal perforation, and a need for penetrating keratoplasty. Traditional medical treatment for AK is unlicensed and requires topical administration of biguanide, i.e. 0.02 per cent PolyHexaMethylene Biguanide (PHMB) or 0.02 per cent chlorhexidine, either as monotherapy or in conjunction with 0.1 per cent propamidine or 0.1 per cent hexamidine. It has been reported that PHMB and chlorhexidine are the most effective against both trophozoites and Acanthamoeba cysts.

Methods

Amoebicidal and cysticidal assays were performed against both the trophozoites and cysts of Acanthamoeba castellanii (ATCC 50370) and Acanthamoeba polyphagae (ATCC 30461). Testing agents included topical ophtalmic preparations of specific anesthetics, antivirals, antibiotics and biocides. Organisms were exposed to serial two-fold dilution of the test compounds in the microtiter plate wells to examine the effect on Acanthamoeba spp. In addition, the toxicity of each test compound was assessed against the mammalian cell line.

Results

The inhibitory range against Acanthamoeba trophozoites for proxymetacaine, tetracaine and oxybuprocaine anesthetics was 9.75 to 39 μg / ml, while lidocaine provided no growth inhibition until the 312-to 625-μg / ml range for both organisms. In the trophozoite amoebicidal tests, proxymetacaine, tetracaine, and oxybuprocaine were amoebicidal in the 19.5 to 250 μg / ml range, while the amoebicidal activity against A was lidocaine. Polyphage and A. Castellani was 312 and 1250 μg / ml, respectively. For cyst assays, proxy-metacaine, tetracaine, and oxybuprocaine were cysticidal in the 39-to 250-μg / ml range. For lidocaine, anti-A cysticidal operation. Polyphage and A. Castellani were 1.25 and 10 mg / ml respectively. In the mammalian cell line toxicity assay, proxymetacaine, tetracaine, and oxybuprocaine were cytotoxic in the 39-to 156-μg / ml range, while lidocaine was not cytotoxic before 5 mg / ml. Lidocaine Minims contains sodium fluorescein that has been checked independently for regulation and found to be non-toxic at a concentration of 2 per cent. Proxymetacaine, oxybuprocaine, and tetracaine in particular, were all toxic to Acanthamoeba spp. trophozoites and cysts but lidocaine was well tolerated.

The presence of benzalkonium chloride (BAC) preservatives in levofloxacin has resulted in a high degree of toxicity to trophozoites and cysts. The involvement of BAC in the propamidine drops was responsible for the operation against Acanthamoeba spp. Hexamidine drops without BAC showed strong activity against trophozoites, and the biguanides polyhexamethylene biguanide, chlorhexidine, alexidine, and octenidine all displayed outstanding activity against both types of trophozoites and cysts.

Conclusions

Based on the antibiotic technique used, empiric treatment with fluoroquinolones of third or fourth generation due to their wide-spectrum antimicrobial activity is frequently used as an initial therapy for the treatment of microbial keratitis. We observed that the pure levofloxacin product and the preservative-free preparation of moxifloxacin (Moxeza) did not have a significant antimicrobial effect on the viability of Acanthamoeba, while the modified levofloxacin (Oftaquix), which is modified with BAC, had a much higher antimicrobial activity on both species of Acanthamoeba. This indicates that it is the BAC that causes the antimicrobial effect observed in preserved levofloxacin rather than the drug itself. Thompson and coworkers did not notice any adverse effects on Acanthamoeba PCR amplification with gatifloxacin or moxifloxacin. The gatifloxacin used in their analysis (Zymar; Allergan, Irvine, CA) was maintained with BAC, while moxifloxacin was self-preserved. While BAC has not been tested on its own, the limited inhibitory effect of both antibiotics suggests that the impact of BAC on PCR in the detection of Acanthamoeba DNA may be lower compared to the amoebicidal and cysticidal assay methods used in this report.

The antiamoebic effects of BAC, povidone iodine and tetracaine are higher than current diamidines and slightly lower than those of the biguanides used in the treatment of Acanthamoeba keratitis. In conclusion, the present research indicates that the use of proxymetacaine, oxybuprocaine and tetracaine to reduce pain; ophtalmic preparations containing preservatives such as BAC; and the use of povidone iodine prior to sampling may affect the viability of Acanthamoeba in vivo, resulting in reduced crop yield and inhibition of PCR amplification.

Human noroviruses (HuNoVs) are the main cause of acute non-bacterial gastroenteritis worldwide. In this analysis, a novel in situ mediated viral receptor RT-qPCR (ISC-RT-qPCR) was used to detect HuNoVs in oysters and compared to the standard RT-qPCR system. Ten HuNoVs RT-PCR positive and 5 negative clinical samples from patients with gastroenteritis were used to equate the specificity and sensitivity of the ISC-RT-qPCR with that of the RT-qPCR assay. ISC-RT-qPCR had a one-log and two-log increase in sensitivity relative to the RT-qPCR studies for genotype I (GI) and GII, respectively. HuNoV concentrations in oyster tissues have been studied in artificially inoculated oysters. GI HuNoVs could be found in all tissues of inoculated oysters by both ISC-RT-qPCR and RT-qPCR. GII HuNoVs could only be found in gills and gastrointestinal glands by both methods. The number of viral genomic copies (vgc) calculated by ISC-RT-qPCR was comparable to RT-qPCR for the identification of GI and GII HuNoVs in inoculated oysters. Thirty-six samples of oyster from the local market were checked for both HuNoVs. More HuNoVs could be found in retail oysters by ISC-RT-qPCR. The GI HuNoV detection levels for gills, digestive glands and residual tissues were 33.3, 25.0 and 19.4 per cent for ISC-RT-qPCR; and 5.6, 11.1 and 11.1 per cent for RT-qPCR, respectively. The GII HuNoV detection rate in gills was 2.8 per cent by ISC-RT-qPCR; no GII HuNoV was found in these oysters by RT-qPCR. Overall, all results have shown that ISC-RT-qPCR is a promising method for the detection of HuNoVs in oyster samples.

Both studies involving clinical samples were conducted in the BSL-2 laboratory. Live stool samples were diluted in a 1:20 suspension in phosphate-buffered saline (PBS, pH 7.2, NaCl 137.0 mmol / L, KCl 2.7 mmol / L, Na2HPO4 10.0 mmol / L, KH2PO4 2.0 mmol / L), clarified by low-speed centrifugation (3,000 rpm) for 5 min, and processed as viral stocks at −80 ° C. Each sample was tested using RT-PCR with JV12/13 primers. The RT-PCR products have been sequenced by the Genewiz Bio-Technology Co. Ltd (Suzhou, China, USA). Two chosen samples were used to compare the ISC-RT-qPCR and RT-qPCR sensitivities for GI and GII HuNoV and used for oyster inoculation. RT-qPCR was performed with extracted viral RNA followed by reverse transcription and qPCR amplification with the same primer sample sets used for ISC-RT-qPCR as mentioned above. For the RNA extraction process, RNA was extracted from 100.0 μL of clinical and oyster samples using the manufacturer's protocol RNA extraction package. The extracted RNA was air-dried and dissolved in 10.0 μL of diethyl-pyrocarbonate (DEPC) treated water prior to the reverse transcription response. The tissues of each oyster were divided into gills (G), digestive glands (D), including stomach, digestive diverticula, and residual tissues (O). PBS was applied to each sample (approximately 2.0 g) at a ratio of 4:1 followed by homogenization with the homogenizer (AES Chemunex, France) at 12,000 rpm for 1 min. Homogenized samples were combined with equivalent quantities of glycerol (50.0%) and processed at −80 ° C for potential use.

The viral stocks in the clinical samples were 1:100 diluted in PBS and calculated by RT-PCR using JV12/13. The RT-PCR results showed that 5 were negative and 10 were positive for HuNoVs. Both amplified productions have been sequenced and sent to GenBank. Detailed information was defined as follows. Both clinical samples were measured using both ISC-RT-qPCR and RT-qPCR. All 5 identified negatives registered as negatives by both ISC-RT-qPCR and RT-qPCR. All 10 known positive values were positive for ISC-RT-qPCR, while only 8 were positive for RT-qPCR when measured at the initial 1:100 diluted viral stocks (1:2000 dilution from raw stool samples). Such two samples (samples 1028 and 3134) were further screened positive when viral stocks were 1:10 diluted (1:200 dilution from raw stool samples) and retested. There was no important difference between these two methods in the titers (in vgc / mL in log10). For the identification of HuNoVs in oysters obtained from retail markets, the ISC-RT-qPCR was significantly more sensitive than the RT-qPCR (p<0,05). Thirty-six oyster samples from retail markets in Shanghai were tested using both methods. For the detection of GI HuNoVs in tissues G, D and O, the detection levels were 33.3, 25.0 and 19.4 per cent respectively by ISC-RT-qPCR and 5.6, 11.1 and 11.1 per cent respectively by RT-qPCR. The GII HuNoV detection rate was 2.8% by ISC-RT-qPCR; no GII HuNoV was detected by RT-qPCR in these oysters. Of GI HuNoVs in oysters tested by ISC-RT-qPCR, the corresponding D and G tissues were also positive while O tissues were positive. If the D tissue was positive, the corresponding G tissue was positive. G tissue was thus the chosen tissue for the ISC-RT-qPCR test. D and O tissues were not needed to be tested to determine if the oyster was infected by the ISC-RT-qPCR process. Nevertheless, the target tissue for the RT-qPCR assay was not clear. If G tissue was healthy, the corresponding D and O tissues were also healthy for RT-qPCR. At the other hand, some samples were only positive in D tissue or O tissue tested by RT-qPCR.

The identification of HuNoVs from food samples other than oysters has been difficult. Many infected food samples contained far fewer HuNoVs than oysters. Complicated processes are needed to concentrate viruses, release viral genomes, and extract RT-PCR inhibitors from different food matrixes. The ISC-RT-qPCR method could simplify steps in the concentration of viruses, viral extraction, removal of RT-PCR inhibitors with a higher sensitivity than the conventional RT-qPCR assay and have a high potential for use in food samples other than oysters. We are currently in the process of checking whether this approach could be used for the identification of HuNoVs in industrial and environmental samples.

Over the previous decade, there has been an adjustment in the study of disease transmission of Oral Cavity Squamous Cell Cancer growth (OC-SCC). Numerous new instances of OC-SCC come up short on the perceived hazard variables of smoking, liquor and human papilloma infection. The point of this examination was to decide whether the oral microbiome might be related with OC-SCC in non-smoking HPV negative patients. We looked at the oral microbiome of HPV-negative nonsmoker OC-SCC ( n=18), premalignant lesions(PML) (n=8) and ordinary control patients (n=12). We report that the periodontal microbes Fusobacterium, Prevotella, Alloprevotella were improved while commensal Streptococcus exhausted in OC-SCC. In view of the four genera in addition to a marker variety Veillonella for PML, we ordered the oral microbiome into two kinds. Quality/pathway examination uncovered a dynamic increment of qualities encoding HSP90 and ligands for TLRs along the controls→PML→OC-SCC movement succession. Our discoveries propose a relationship between periodontal microbes and OC-SCC in non-smoking HPV negative patients.

Smoking and liquor are the two primary hazard factors for oral malignancy. Different components are additionally ensnared in the etiology of squamous cell head and neck malignant growth, for example, helpless oral cleanliness, diet, infections, word related operators, poisons, hereditary impacts, yet not many case-controlled epidemiological investigations have been done. Since 1990, there has been a consistent increment in oral malignant growth in patients in the USA who don't smoke. Regardless of a decrease in the pervasiveness of cigarette smoking in the USA since 1975 (from ~40% to 20%) the rate of oral malignant growth has remained essentially unaltered. The predominance of overwhelming liquor utilization in the USA has just marginally expanded from 7% to 8.2% somewhere in the range of 2005 and 2029. Oral SCC can be partitioned into oropharyngeal SCC and oral hole SCC (OC-SCC). The pervasiveness of oropharyngeal SCC identified with high-chance Human Papilloma Viruses (HPV) has expanded from 40.5% in 2000 to 72.2% in 201010. The acknowledgment of HPV etiology in oropharyngeal SCC has improved the clinical results and prompted explicit counteraction for HPV disease by immunization. Conversely, the predominance of HPV in OC-SCC is muddled and significantly differed over numerous investigations with a normal of 23.3%. Along these lines, a critical extent of recently analyzed OC-SCC in the USA has no realized hazard factor. It is conceivable this might be straightforwardly identified with helpless oral cleanliness. There are currently a few investigations demonstrating a relationship between helpless oral cleanliness and oral malignant growth. These investigations show a relationship with rare tooth brushing, gum draining and periodontitis. Helpless oral cleanliness will bring about an adjustment in the oral microbiome of such patients. Recently, a few examinations announced change of microbiome in oral malignant growth.

A case-control study was endorsed by the Institutional Review Board of Memorial Sloan Kettering Cancer Center. Composed educated assent was gotten from every member. The cases included two gatherings: oral depression squamous cell carcinoma (OC-SCC) and premalignant injuries (PML) in light of histopathological assessment. All premalignant sores had leukoplakia with dysplasia affirmed on obsessive investigation. The negative controls contained patients with thyroid knobs. To test the oral microbiome, the members washed the mouth enthusiastically with 10 ml clean saline for 30 seconds and spit, and microorganisms were recuperated from the flush fluid by centrifugation at 3,220g for 20min. The pellets were moved into 2ml cylinder and put away at - 80° cooler for additional examination. At long last, we recognized the blends accomplishing the pinnacle exactness. All genera that prompted top characterization precision were chosen for factual correlations among and between the case controls gatherings while those caused decrease in the exactness after the pinnacle were prohibited. Correlation of middle relative bounty of a specific taxon among the three case controls bunches was finished with nonparametric Kruskal Wallis test and between two gatherings with Mann-Whitney U test. The adjustment in plenitude of a taxon along the succession of negative controls→PML→OC-SCC was dissected utilizing Jonckheere's pattern test. Every measurable test was two-sided, with p esteem <0.05 considered of ostensible factual centrality. A bogus disclosure rate (FDR)- balanced p esteem (q esteem) <0.10 was utilized as the edge for criticalness, after change for numerous examinations for Kruskal Wallis test and Mann-Whitney U test in the subsequent investigation.

To look for new hazard variables of OC-SCC in non-smokers, we led a case control concentrate with 18 instances of OC-SCC, 8 instances of PML and 12 negative controls. The cases and the controls varied essentially by age however not by sex and race. All members never smoked or were liberated from tobacco use for in any event 11 years. Gatherings fluctuated however didn't measurably vary in liquor utilization. All malignancy patients were negative for high hazard HPV. Their oral microbiome was tested by oral wash and characterized by 16S rRNA quality sequencing. This test of the oral microbiome recognized 12 phyla, 21 classes, 35 requests, 66 families, 116 genera and 172 species. Expanded information demonstrating the relationship of the microbiome with malignant growth has activated enthusiasm for the investigation of the oral microbiome in oral disease 39. This examination demonstrated the affiliation was associated to expanded utilization of cigarette smoking, betal quid use and furthermore helpless oral cleanliness. Our discoveries propose periodontal microbes are related with OC-SCC in patients who need chance elements of HPV and smoking. Microbiome-interceded aggravation might be answerable for OC-SCC in these patients. Our examination proposes that further investigations are expected to decide if bacterial HSP90 or TLR ligands add to OC-SCC.

In pharmaceutical manufacturing, it is a serious matter to work in controlled environments, cleanrooms and some cases in completely sterile zones. At one point, a cleanroom or clean zone is simply an area that is free of particles counts and microbial counts. However, EU GMP or the FDA guidelines outline other regulatory criteria for cleanrooms which depend on the use of defined cleaning techniques along with the application of detergents and disinfectants as an important step towards achieving microbial control within a cleanroom. To destroy microorganisms, the detergents and disinfectants used in cleanrooms of pharmaceutical grade need to be of high quality and efficiency. Good product selection and cleaning techniques are important, particularly with regard to some of the latest cleanroom technologies.

In pharmaceutical industries, Pharmacopeia is the reference that offers guidelines for cleaning and sanitization systems required for regulated environments to prevent microbial contamination. It also discusses everything included in sterile drug products: pharmaceutical ingredients, process water, packaging components, manufacturing environment, processing equipment, and manufacturing operators. Current Good Manufacturing Practices (cGMPs) emphasize on the scale, design, and location of buildings and building materials, and the correct material flow to promote the cleaning, repair, and proper drug product manufacturing operations. Where disinfectants are used in manufacturing environments, where care should be taken to prevent contamination of the drug product with chemical disinfectants that may result inherent toxicity of the disinfectants. The types of detergents and disinfectants used are of significant decision for the pharmaceutical manufacturer as there are multiple types with specific activity spectrums and mode of action. There have been a number of advances in clean room technologies over the past few years which have helped in reducing the risk of contamination and streamline process operations. The final assessment of cleaning products and techniques is revealed in terms of the number and types of microorganisms recovered through environmental monitoring programs.

Dettol is commonly used in homes and healthcare facilities for a range of purposes, including disinfection of bodies, artifacts and appliances, as well as environmental surfaces. The number of microorganisms colonizing the skin and surfaces is significantly reduced with prior cleaning prior to application. Antimicrobial properties of chloroxylenol, the main chemical constituent of Dettol and other chlorinated phenols have been extensively examined. Antimicrobial properties of the disinfectant to some pathogenic bacteria have been recorded earlier.

The aim of this study is to determine the efficacy of Dettol in comparison to alcohol if used on Epoxy surfaces in the production environment. The experiment was designed to compare the efficacy of 4 Dettol concentrations (5, 2.5, 1.25 and 0.625%) versus alcohol 70% in time intervals 5, 10, 15 and 30 minutes. Log reduction in bacterial or fungal count was chosen as a parameter of disinfectant effect. Bacillus subtilis ATCC 6633, Escherichia coli ATCC 8739, Staphylococcus aureus ATCC 6538, Pseudomonas aeruginosa ATCC 9027, Aspergillus niger ATCC 16404 and Candida albicans ATCC 10231 were chosen as test microbes. The concentration of Dettol that resulted in the highest log reduction in microbial count in the shortest contact time compared to alcohol 70% on epoxy surfaces would be considered a potential alternative in the production environment. The efficacy represented in log reduction is calculated according to the following equation:

Log10 reduction (R)= log10 pre-value cfu/ plate – log10 post value cfu/ plate

Data represent the mean of 4 replicates with estimated standard deviation (SD) equals to 0.5 for each sub-group using α error equal 0.05 will provide a power of 20%.

The results showed that the most significant concentration of Dettol was 2.5% if used for 5 minutes on epoxy surfaces this concentration reduced the count for Escherichia coli. Staphylococcus aureus, Pseudomonas aeruginosa Aspergillus niger, and Candida albicans by 100, 100, 98, 100 and 100%, respectively, compared with alcohol 70%. Bacillus subtilis maximum log reduction by Dettol was 82.5% in 15 min. This could be attributed to its ability to form spores.

In conclusion, Dettol (2.5% for 15min) can be considered a good alternative as disinfectant in controlled ad clean areas in pharmaceutical industry.

I am pleased to introduce Journal of Food & Industrial Microbiology (JFIM) is an internationally reputed, open access research journal oriented towards exploring the latest happenings topics of the food industry and microbial activities that cause the spoilage of food. The journal extends wide coverage to a broad sections of the topics in this field such as types of microbial activities on the perishable and the dried food, methods of food storage, food toxicity, food poison and contamination, food safety, protocols to determine the food expiry, enzymes, food processing. It is our pleasure to announce that during year 2019, all issues of volume 05 were published online on time and the print issues were also brought out and dispatched within 30 days of publishing the issue online.

All published articles of this journal are included in the indexing and abstracting coverage of CAS Source Index (CASSI), Index Copernicus, Google Scholar, Sherpa Romeo, Academic Journals Database, GenamicsJournalSeek, JournalTOCs, CiteFactor, Electronic Journals Library, RefSeek, Hamdard University, EBSCO A-Z, Directory of Abstract Indexing for Journals, World Catalogue of Scientific Journals, OCLC- WorldCat, Scholarsteer, SWB online catalog, Virtual Library of Biology (vifabio), Publons, Dtufindit, Geneva Foundation for Medical Education and Research.

During the calendar year 2019, Journal of Food & Industrial Microbiology received a total of 30 papers, out of which 6 articles were rejected in the preliminary screening due to plagiarism or being out of the format and peer review process. During 2019 around 10 articles were subjected for publication after they are accepted in the peer review process. In the 2 issues of Volume 5 published during the year 2019, a total of 10 articles were published (at an average of 5 articles per issue of which, articles were published from authors all around the world. A total of 30 research scientists from all over the world reviewed the 10 articles published in volume 4. Average publication period of an article was further reduced to 14-21 days.

During the calendar year 2019, a total of three Editors, ten Reviewers joined the board of JFIM and contributed their valuable services towards contribution as well as publication of articles, and their valuable reviewer comments will beneficial to publish quality of article in the Journal.

I take this opportunity to acknowledge the contribution of Editor-in-chief and Associate Editor during the final editing of articles published and bringing out issues of JFIM in time. I would also like to express my gratitude to all the authors, reviewers, the publisher, language editor, honorary editors, the scientific advisory and the editorial board of JFIM, the office bearers for their support in bringing out the new volume (Volume 6) of JFIM for the calendar year 2020 and look forward to their unrelenting support further to release more issues for Journal of Food & Industrial Microbiology (JFIM) in scheduled time.