Investigation of the effects of some processing conditions on the fate of oxytetracycline and tylosin antibiotics in the making of commonly consumed cheeses from the East Mediterranean.

BACKGROUND AND AIM: Transfer of antibiotics from raw milk to derived products is directly related to the processes involved in the manufacturing of dairy products, including East Mediterranean cheeses, since these have particular flow diagrams of production. The aim of this study was to assess the effects of skimming, pasteurization, curding, pressing, salting, cheese boiling, and whey acidification/heating on two widely used antibiotics in Lebanon, oxytetracycline (OTC) and tylosin (TYL), in the manufacture of commonly consumed cheeses in the East Mediterranean. MATERIALS AND METHODS: Four hundred and fifty kilograms of full-fat bovine milk were spiked with OTC and TYL, then skimmed and pasteurized using holder and high-temperature short-time (HTST) methods. Milk was then processed to make cheeses (23 kg Baladi, 20 kg Akkawi, 20 kg Halloum, and 18 kg Double Cream). Liquid chromatography-mass-spectrometry was used to measure antibiotics. Analysis was performed using Statistical Package for the Social Sciences v25. RESULTS: Skimming significantly (p=0.015) decreased TYL concentration by 68.6%. OTC degradation during holder (41-54%) proved to be significant (p=0.015). HTST had a significant (p=0.012) effect on TYL with 32% degradation. Curding step in making Baladi had a significant (p=0.028) effect on OTC only with the concentration increasing by 1.5-fold. Acidification and heating of whey to produce Double Cream decreased significantly (p=0.037) OTC concentration (14.7-46.3%), while TYL concentration increased significantly (p=0.000) by 300%. Pressing and salting in making Akkawi did not have any significant effect, while cheese boiling in making Halloum significantly decreased both antibiotics. CONCLUSION: OTC is transferred to Baladi and Akkawi (curd based) mainly, while double cream (whey based) has a high level of TYL transfer. Hence, people who consume these cheeses excessively could be exposed to high amounts of both antibiotics and thus be prone to their detrimental effect on health.

Effectiveness of purified methylene blue in an experimental model of Mycobacterium ulcerans infection.

Mycobacterium ulcerans is responsible for Buruli ulcer, characterised by extensive, disabling ulcers. Standard treatment combining rifampicin and streptomycin exposes patients to toxicity and daily painful injections. In this study, the in vitro susceptibilities of 3 M. ulcerans strains, 1 Mycobacterium marinum strain and 18 strains representative of eleven other Mycobacterium species and subspecies to methylene blue were determined. Whilst growth of M. ulcerans was inhibited by 0.0125 g/L methylene blue, growth of all other tested strains was not inhibited by 1 g/L methylene blue. The effectiveness of methylene blue in a murine model of M. ulcerans infection was then tested. Topical treatment by brushing a methylene blue solution on the skin lesion, systemic treatment by intraperitoneal injection of methylene blue, and a combined treatment (topical and systemic) were tested. The three treatment groups exhibited a significantly lower clinical score compared with the non-treated control group (P <0.05). Moreover, subcutaneous nodules were significantly smaller in the systemic treatment group (excluding males) (3 ± 0.7 mm) compared with the other groups (P <0.05). The M. ulcerans insertion sequence IS2404 and the KR-B gene were detected in all challenged mice, but not in negative controls. The density of M. ulcerans (mycobacteria/cell) was significantly lower in the combined treatment group compared with the other groups. These data provide evidence for the effectiveness of purified methylene blue against the initial stage of Buruli ulcer.

Draft Genome Sequence of Mycobacterium acapulcensis Strain CSURP1424.

Mycobacterium acapulcensis is a rapidly growing scotochromogenic acid-fast bacillus. The draft genome of M. acapulcensis CSURP1424 comprises 5,290,974 bp, exhibiting a 66.67% G+C content, 4,870 protein-coding genes, and 71 predicted RNA genes.

Draft Genome Sequence of Mycobacterium houstonense Strain ATCC 49403T.

Mycobacterium houstonense is a nontuberculous species rarely responsible for human infection. The draft genome of M. houstonense ATCC 49403(T) comprises 6,451,020 bp, exhibiting a 66.96% G+C content, 5,881 protein-coding genes, and 65 predicted RNA genes.

Draft Genome Sequence of Mycobacterium interjectum Strain ATCC 51457T.

Mycobacterium interjectum is a nontuberculosis species rarely responsible for human infection. The draft genome of M. interjectum ATCC 51457(T) comprises 5,927,979 bp, exhibiting 67.91% G+C content, 5,314 protein-coding genes, and 51 predicted RNA genes.

Inverse correlation between salt tolerance and host-adaptation in mycobacteria.

BACKGROUND: The genus Mycobacterium includes host-adapted organisms regarded as obligate and opportunistic pathogens and environmental organisms. Factors contributing to this wide range of adaptations are poorly known. RESULTS: We studied the salt tolerance of 46 Mycobacterium species of medical interest. Representative strains of the Mycobacterium tuberculosis complex, Mycobacterium avium complex, Mycobacterium chelonae-abscessus complex, Mycobacterium ulcerans, Mycobacterium marinum, Mycobacterium lentiflavum, Mycobacterium fortuitum and Mycobacterium conceptionense were inoculated on Middlebrook 7H10 medium supplemented with 0-10% sodium chloride. Colonies were counted after 2-4 week incubation at the appropriate 30-37 °C temperature depending on the tested strain. Further comparative genomics was done on 15 Mycobacterium strains representing the spectrum of salt-tolerance of mycobacteria. Based on the results the different species were grouped according to their salt tolerance into a "salt-sensitive" group (growth up to ≤3% salt) containing the M. tuberculosis complex, Mycobacterium chelonae, Mycobacterium lentiflavum, Mycobacterium ulcerans and Mycobacterium marinum; a "salt-intermediate" group (growth between 4 and 6% salt) comprising Mycobacterium avium, Mycobacterium intracellulare, Mycobacterium chimaera and a "salt-resistant" group (growth up to >6%) comprising Mycobacterium homonissuis, Mycobacterium bolettii, Mycobacterium fortuitum and Mycobacterium conceptionense. Genomic analysis revealed that 290 genes were unique to species belonging to the salt-sensitive group; and that 15% were annotated as being functionally associated with the ESX secretion systems Pro-Glu and Pro-Pro-Glu family proteins. CONCLUSIONS: In this work we found an inverse correlation between salt tolerance and host adaptation. We thus propose that salinity is one of the multiple factors determining the ecological niches of mycobacteria.

Mycobacterium icosiumassiliensis sp. nov., a New Member in the Mycobacterium terrae Complex Isolated from Surface Water in Algeria.

An acid-fast, rapidly growing, rod-shaped microorganism designated 8WA6 was isolated from a lake in Algiers, Algeria. The lake water was characterized by a temperature of 18 °C, a pH of 7.82, a copper concentration of 8.6 µg/L, and a cadmium concentration of 0.6 µg/L. First-line molecular identification confirmed the 8WA6 isolate to be a member of the Mycobacterium terrae complex, sharing 99.4 % 16S rRNA gene sequence similarity with M. arupense AR-30097, 98.2 % partial hsp65 gene sequence similarity with M. terrae 28K766, and 97.1 % partial rpoB gene sequence similarity with Mycobacterium sp. FI-05396. Its 4.89-Mb genome exhibits a 66.8 GC % and an average nucleotide identity of 64.5 % with M. tuberculosis, 70.5 % with M. arupense, and 75 % with M. asiaticum. In the M. terrae complex, Mycobacterium 8WA6 was unique in exhibiting growth at 42 °C, negative reaction for nitrate reduction, urease activity and Tween 80 hydrolysis, and a positive reaction for α-glucosidase and ß-glucosidase. Its protein profile determined by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry revealed a unique spectrum similar to M. arupense and M. terrae, exhibiting eleven specific peaks at 3787.791, 4578.019, 6349.630, 6855.638, 7202.310, 8149.608, 8775.257, 10,224.588, 10,484.116, 12,226.379, and 12,636.871 m/z. Minimal inhibitory concentrations (MIC) for antibiotics, determined by microdilution, indicated a broad spectrum resistance, except for rifabutin (MIC, 0.5 g/L) and cefoxitin (MIC, 16 g/L). We concluded that the 8WA6 isolate is a representative isolate of a previously undescribed species in the M. terrae complex, which was named M. icosiumassiliensis sp. nov. with strain 8WA6 (Collection de Souches de l'Unité des Rickettsies, CSUR P1561, Deutsche Sammlung von Mikroorganismen und Zellkulturen, DSM 100711) as the type strain.

A Chlorhexidine- Agar Plate Culture Medium Protocol to Complement Standard Broth Culture of Mycobacterium tuberculosis.

The culture of Mycobacterium tuberculosis using parallel inoculation of a solid culture medium and a liquid broth provides the gold standard for the diagnosis of tuberculosis. Here, we evaluated a chlorhexidine decontamination-MOD9 solid medium protocol versus the standard NALC-NaOH-Bactec 960 MGIT protocol for the diagnosis of pulmonary tuberculosis by culture. Three-hundred clinical specimens comprising 193 sputa, 30 bronchial aspirates, 10 broncho-alveolar lavages, 47 stools, and 20 urines were prospectively submitted for the routine diagnosis of tuberculosis. The contamination rates were 5/300 (1.7%) using the MOD9 protocol and 17/300 (5.7%) with the Bactec protocol, respectively (P < 0.05, Fisher exact test). Of a total of 50 Mycobacterium isolates (48 M. tuberculosis and two Mycobacterium abscessus) were cultured. Out of these 50, 48 (96%) isolates were found using the MOD9 protocol versus 35 (70%) when using the Bactec protocol (P < 0.05, Fisher exact test). The time to positivity was 10.1 ± 3.9 days versus 14.7 ± 7.3 days, respectively, (P < 0.05, Student's t-test). These data confirmed the usefulness of parallel inoculation of a solid culture medium with broth for the recovery of M. tuberculosis in agreement with current recommendations. More specifically, chlorhexidine decontamination and inoculation of the MOD9 solid medium could be proposed to complement the standard Bactec 960 MGIT broth protocol.

Draft Genome Sequence of Mycobacterium peregrinum Strain CSUR P2098.

Mycobacterium peregrinum is a nonpigmented rapid growing nontuberculosis species belonging to the Mycobacterium fortuitum group. The draft genome of M. peregrinum type I CSUR P2098 comprises 7,109,836 bp exhibiting a 66.23% G+C content, 6,894 protein-coding genes, and 100 predicted RNA genes. Its genome analysis suggests this species differs from Mycobacterium senegalense.

Draft Genome Sequence of Mycobacterium neworleansense Strain ATCC 49404T.

Mycobacterium neworleansense is a rapid growing nontuberculosis species belonging to the Mycobacterium fortuitum complex. The draft genome of M. neworleansense ATCC 49404(T) comprises 6,287,317 bp exhibiting a 66.85% G+C content, 5,997 protein-coding genes, and 89 predicted RNA genes.

Draft Genome Sequence of Mycobacterium mucogenicum Strain CSUR P2099.

Mycobacterium mucogenicum is a rapid-growing, nontuberculosis Mycobacterium species. The draft genome of M. mucogenicum CSUR P2099 comprises 6,210,127 bp exhibiting a 67.2% G+C content, 6,003 protein-coding genes, and 91 predicted RNA genes.

Rapid culture-based diagnosis of pulmonary tuberculosis in developed and developing countries.

Culturing Mycobacterium tuberculosis remains the gold standard for the laboratory diagnosis of pulmonary tuberculosis, with 9 million new cases and 1.5 million deaths mainly in developing countries. Reviewing data reported over 20 years yields a state-of-the-art procedure for the routine culture of M. tuberculosis in both developed and developing countries. Useful specimens include sputum, induced sputum, and stools collected in quaternary ammonium preservative-containing sterile cans. The usefulness of other non-invasive specimens remains to be evaluated. Specimens can be collected in a diagnosis kit also containing sampling materials, instructions, laboratory requests, and informed consent. Automated direct LED fluorescence microscopy after auramine staining precedes inoculation of an egg-lecithin-containing culture solid medium under microaerophilic atmosphere, inverted microscope reading or scanning video-imaging detection of colonies and colonies identification by recent molecular methods. This procedure should result in a diagnosis of pulmonary tuberculosis as fast as 5 days. It may be implemented in both developed and developing countries with automated steps replaceable by manual steps depending on local resources.

Draft Genome Sequence of Mycobacterium bohemicum Strain DSM 44277T.

The Mycobacterium bohemicum strain is a nontuberculosis species mainly responsible for pediatric cervical lymphadenitis. The draft genome of M. bohemicum DSM 44277(T) comprises 5,097,190 bp exhibiting a 68.64% G+C content, 4,840 protein-coding genes, and 75 predicted RNA genes.

Chlorhexidine decontamination of sputum for culturing Mycobacterium tuberculosis.

BACKGROUND: Culture of Mycobacterium tuberculosis is the gold standard method for the laboratory diagnosis of pulmonary tuberculosis, after effective decontamination. RESULTS: We evaluated squalamine and chlorhexidine to decontaminate sputum specimens for the culture of mycobacteria. Eight sputum specimens were artificially infected with 10(5) colony-forming units (cfu)/mL Mycobacterium tuberculosis and Staphylococcus aureus, Pseudomonas aeruginosa and Candida albicans as contaminants. In the second step, we tested chlorhexidine-based decontamination on 191 clinical specimens, (Chlorhexidine, 0.1, 0.5 and 0.7 %). In a last step, growth of contaminants and mycobacteria was measured in 75 consecutive sputum specimens using the routine NALC-NaOH decontamination protocol or with 0.7 % chlorhexidine decontamination and an inoculation on Coletsos medium. In the artificially model, contaminants grew in 100 % of the artificially infected sputum specimens decontaminated using 100 mg/mL squalamine, in 62.5 % of specimens decontaminated using N-Acetyl-L-Cysteine-Sodium Hydroxide (NALC-NaOH), and in 0 % of specimens decontaminated using 0.1 %, 0.35 %, or 1 % chlorhexidine (P < 0.05). These specimens yielded <10(2) cfu M. tuberculosis using NALC-NaOH and > 1.4.10(2) cfu M. tuberculosis when any concentration of chlorhexidine was used (P < 0.05). In the second step we found that 0.7 %-chlorhexidine yielded 0 % contamination rate, 3.2 % for 0.5 %-chlorhexidine and 28.3 % for 0.1 %-chlorhexidine. As for the 75 specimens treated in parallel by both methods we found that when using the standard NALC-NaOH decontamination method, 8/75 (10.7 %) specimens yielded M. tuberculosis colonies with a time to detection of 17.5 ± 3 days and an 8 % contamination rate. Additionally, 14 specimens yielded mycobacteria colonies (12 M. tuberculosis, and 2 Mycobacterium bolletii) (18.7 %) (P = 0.25), which has yielded a 100 % sensitivity for the chlorhexidine protocol. Time to detection was of 15.86 ± 4.7 days (P = 0.39) and a 0 % contamination rate (P < 0.05) using the 0.7 %-chlorhexidine protocol. CONCLUSION: In our work we showed for the first time that chlorhexidine based decontamination is superior to the standard NALC-NaOH method in the isolation of M. tuberculosis from sputum specimens. We currently use 0.7 %-chlorhexidine for the routine decontamination of sputum specimens for the isolation of M. tuberculosis and non-tuberculosis mycobacteria on egg-lecithin containing media.

Rapid Diagnosis of Tuberculosis by Real-Time High-Resolution Imaging of Mycobacterium tuberculosis Colonies.

Culture remains the cornerstone of diagnosis for pulmonary tuberculosis, but the fastidiousness of Mycobacterium tuberculosis may delay culture-based diagnosis for weeks. We evaluated the performance of real-time high-resolution imaging for the rapid detection of M. tuberculosis colonies growing on a solid medium. A total of 50 clinical specimens, including 42 sputum specimens, 4 stool specimens, 2 bronchoalveolar lavage fluid specimens, and 2 bronchial aspirate fluid specimens were prospectively inoculated into (i) a commercially available Middlebrook broth and evaluated for mycobacterial growth indirectly detected by measuring oxygen consumption (standard protocol) and (ii) a home-made solid medium incubated in an incubator featuring real-time high-resolution imaging of colonies (real-time protocol). Isolates were identified by Ziehl-Neelsen staining and matrix-assisted laser desorption ionization-time of flight mass spectrometry. Use of the standard protocol yielded 14/50 (28%) M. tuberculosis isolates, which is not significantly different from the 13/50 (26%) M. tuberculosis isolates found using the real-time protocol (P = 1.00 by Fisher's exact test), and the contamination rate of 1/50 (2%) was not significantly different from the contamination rate of 2/50 (4%) using the real-time protocol (P = 1.00). The real-time imaging protocol showed a 4.4-fold reduction in time to detection, 82 ± 54 h versus 360 ± 142 h (P < 0.05). These preliminary data give the proof of concept that real-time high-resolution imaging of M. tuberculosis colonies is a new technology that shortens the time to growth detection and the laboratory diagnosis of pulmonary tuberculosis.