An Additional L451G452N453 in the RpoB Protein Suppressed the Synthetic Lethality in Escherichia coli at 37 Degrees Caused by Depletion of DnaK/J and Trigger Factor.

Escherichia coli depletion of chaperone trigger factor and DnaK/J were not viable at 37°C, but viable below 30°C. Among the engineered E. coli depleted of trigger factor and DnaK/J, one strain Z625, exhibited survival at 37°C, while another strain Z629 only survived below 30°C. Comparative analysis of fatty acid profiles of Z625 and Z629 revealed absence of numerous saturated fatty acids in Z625 as compared to the wild-type E. coli BW25113. In addition, increased unsaturated fatty acids were present in Z625, whereas the fatty acids profile of Z629 closely resembled that of BW25113. Whole genome sequencing revealed a 9-bp insertion in rpoB of Z625. Combined structural analysis of simulated RpoB protein bearing the amino acid sequence L451G452N453 insertion and susceptibility analysis to rifampicin suggested that the insertion did not disturb the individual RpoB structure as beta subunit of RNA polymerase. Comparative transcriptomic analysis of Z625 and Z629 suggested that this insertion impacted transcription of the overall RNA polymerase in Z625, leading to potential repression of some genes whose overexpression was toxic to E. coli. Additionally, Z625 exhibited distinctive metabolic adaptations, likely contributing to its survival at 37°C. In summary, our study elucidated one LGN insertion in rpoB that impacts transcriptional regulation in E. coli, thereby explaining the survival of E. coli depletion of trigger factor and DnaK/J at 37°C, and these founding suggested that some simple mutations in critical genes like rpoB might play an important role in driving adaptive evolution.

The mutation rate of rpoB gene showed an upward trend with the increase of MIRU10, MIRU39 and QUB4156 repetitive number.

BACKGROUND: Mycobacterial interspersed repetitive unit-variable number tandem repeat (MIRU-VNTR) is a frequently used typing method for identifying the Beijing genotype of Mycobacterium tuberculosis (Mtb), which is easily transformed into rifampicin (RIF) resistance. The RIF resistance of Mtb is considered to be highly related with the mutation of rpoB gene. Therefore, this study aimed to analyze the relationship between the repetitive number of MIRU loci and the mutation of rpoB gene. METHODS: An open-source whole-genome sequencing data of Mtb was used to detect the mutation of rpoB gene and the repetitive number of MIRU loci by bioinformatics methods. Cochran-Armitage analysis was performed to analyze the trend of the rpoB gene mutation rate and the repetitive number of MIRU loci. RESULTS: Among 357 rifampicin-resistant tuberculosis (RR-TB), 304 strains with mutated rpoB genes were detected, and 6 of 67 rifampicin susceptible strains were detected mutations. The rpoB gene mutational rate showed an upward trend with the increase of MIRU10, MIRU39, QUB4156 and MIRU16 repetitive number, but only the repetitive number of MIRU10, MRIU39 and QUB4156 were risk factors for rpoB gene mutation. The Hunter-Gaston discriminatory index (HGDI) of MIRU10 (0.65) and QUB4156 (0.62) was high in the overall sample, while MIRU39 (0.39) and MIRU16 (0.43) showed a moderate discriminatory Power. CONCLUSION: The mutation rate of rpoB gene increases with the addition of repetitive numbers of MIRU10, QUB4156 and MIRU39 loci.

Rifampin resistance mutations in the rpoB gene of Enterococcus faecalis impact host macrophage cytokine production.

Antibiotic-resistant bacteria in the genus Enterococcus are a major cause of nosocomial infections and are an emergent public health concern. Similar to a number of bacterial species, resistance to the antibiotic rifampicin (RifR) in enterococci is associated with mutations in the gene encoding the ß subunit of RNA polymerase (rpoB). In Mycobacterium tuberculosis, RifRrpoB mutations alter mycobacterial surface lipid expression and are associated with an altered IL-1 cytokine response in macrophages upon infection. However, it is not clear if RifR mutations modulate host cytokine responses by other bacteria. To address this question, we utilized Enterococcus faecalis (E. faecalis). Here, we treated human monocyte-derived macrophages with heat-inactivated wild type or RifRrpoB mutants of E. faecalis and found that RifR mutations reduced IL-1ß cytokine production. However, RifR mutations elicited other potent pro- and anti-inflammatory responses, indicating that they can impact other immune pathways beyond IL-1R1 signaling. Our findings suggest that immunomodulation by mutations in rpoB may be conserved across diverse bacterial species and that subversion of IL-1R1 pathway is shared by RifR bacteria.

Insight rifampicin-resistant (rpoB) mutation in Pseudomonas stutzeri leads to enhance the biosynthesis of secondary metabolites to survive against harsh environments.

In this study, a wild-type and five distinct rifampicin-resistant (Rifr) rpoB mutants of Pseudomonas stutzeri (i.e., Q518R, D521Y, D521V, H531R and I614T) ability were investigated against harsh environments (particularly nutritional complexity). Among these, the robust Rifr phenotype of P. Stutzeri was associated only with base replacements of the amino deposits. The use of carboxylic and amino acids significantly increased in various Rifr mutants than that of wild type of P. stutzeri. The assimilation of carbon and nitrogen (N) sources of Rifr mutants' confirmed that the organism maintains the adaptation in nutritionally complex environments. Acetylene reduction assay at different times also found the variability for N-fixation in all strains. Among them, the highest nitrogenase activity was determined in mutant 'D521V'. The assimilation of carbon and nitrogen sources of P. stutzeri and its Rifr mutants ensures that the organism maintains the adaptability in nutritionally complex environments through fixing more nitrogen.

Genetic diversity and distribution of rhizobia associated with soybean in red soil in Hunan Province.

To explore the genetic diversity and distribution of rhizobia in the rhizosphere of soybean grown in red soil, we have collected 21 soil samples from soybean fields across seven counties in Hunan province, China. MiSeq sequencing of rpoB gene was used to determine the intra-species diversity of rhizobia existing in soybean rhizospheres. Soil chemical properties were determined by routine methods. The Principal Coordinates Analysis (PCoA) plot indicated a clear biogeographical pattern characterizing the soybean rhizosphere across different sites. The Mantel test demonstrated that biogeographical pattern was significantly correlated with the geographical distance (Mantel statistic R 0.385, p < 0.001). There were obvious differences in the rhizobial communities among northeastern eco-region, southeastern eco-region and western eco-region. In general, Bradyrhizobium diazoefficiens was the most abundant rhizobial species in the soybean rhizosphere. At an intermediate (10-400 km) spatial scale, the biogeographical pattern of rhizobial communities in soybean rhizosphere is associated with both soil properties and geographical distance. Redundancy analysis (RDA) showed that total potassium (TK), available potassium (AK), soil organic carbon (SOC), and available nitrogen (AN) were the main factors that influenced the α-diversity of rhizobial communities. Canonical correspondence analysis (CCA) showed that pH and exchangeable Ca and Mg had the greatest influence on the ß-diversity of the rhizobial communities in the soybean rhizosphere. These findings characterize the distribution pattern and its influencing factors of soybean rhizobia in rhizosphere in Hunan province, which may be helpful in selecting suitable strains or species as inoculants for soybeans in red soil regions.

Two new rapid PCR-based methods for identification of Acinetobacter baumannii isolated from clinical samples.

The accurate identification of Acinetobacter spp. is challenging due to their high phenotypic and biochemical similarities. Because clinical relevance and antibiotic susceptibility are significantly different among different genomic species of Acinetobacter, the exact identification of A. baumannii is necessary and it can help us prevent inappropriate antibiotic use and inferior clinical care. This project employed a sequence-specific PCR assay for the rpoB region in A. baumannii to distinguish it from non-Acinetobacter baumannii Acinetobacter species. Moreover, a duplex PCR assay was used to detect blaOXA-51-like and gluconolactonase genes as a second identification method. In this study, 210 isolates of Acinetobacter spp. were considered and identified by PCR-sequencing of rpoB gene as a reference test. PCR-sequencing of rpoB revealed that 179 isolates were A. baumannii and 31 were non- A. baumannii Acinetobacter strains. PCR amplification targeting the rpoB gene as the first method, detected 182 isolates of A. baumannii, while duplex PCR assay confirmed 163 isolates as A. baumannii. Data analysis indicated that the sensitivities of sequence-specific PCR of the rpoB gene and duplex PCR assay were 100% and 91.06%, respectively, while specificities were 91.18% and 100%, respectively. Given the data, it was revealed that these two methods showed a reasonable potential for the accurate identification of A. baumannnii from non- A. baumannii species. Sequence-specific PCR assay for the rpoB gene and duplex PCR assay for blaOXA-51-like and gluconolactonase genes are rapid, reliable and cost-effective methods which can be used in clinical laboratories for the accurate identification of A. baumannii.

First case report of prosthetic valve endocarditis caused by Mycobacterium wolinskyi.

To date, only 26 cases of Mycobacterium wolinskyi infections have been reported in humans. We herein report a first case of prosthetic valve endocarditis due to this organism after cardiovascular surgery. An 82-year-old man presented with repeat episodes of syncope and fever after aortic valve replacement, mitral valve replacement, left atrial appendage closure, and pulmonary vein isolation. Blood cultures maintained in aerobic bottles were repeatedly positive after 90-100 hours, and Gallium scan revealed abnormal accumulations in the sternum and left testis. While colonies formed by culturing the fluid of the parasternal area and blood cultures revealed gram-positive rods, we could not analyze the colony using matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF). M. wolinskyi was finally identified on 16S rRNA, hsp65, and rpoB gene sequencing. We treated the patient with multiple antimycobacterial drugs, i.e., amikacin, imipenem, and clarithromycin for 6 weeks, which was changed to oral ciprofloxacin and minocycline for 12 months. This case highlights the need to consider rapidly growing mycobacteria, including M. wolinskyi, if chronic fever persists from weeks to months after surgery, the blood culture is positive, and the organism is not identified. In addition, sequencing the 16S rRNA, hsp65, and rpoB genes is essential for diagnosis.

Development of a semi-quantitative PCR assay for the detection of Francisella halioticida and its application to field samples.

The current study describes the development and application of a TaqMan® real-time PCR assay for the detection of the bacterium Francisella halioticida. Previously, detection of F. halioticida is relied on bacterial culture and conventional PCR; however, the real-time PCR provides many advantages because it is faster, less labour-intensive and reduces the risk of cross-contamination. DNA samples from mussels collected in April 2020 from seven sites in northern Brittany (France) were tested using the newly developed real-time PCR assay. The objective was to screen for the presence of F. halioticida during spring mortality events. The bacterium was detected in 71.4% of the samples tested and was present at all sites except for Saint-Brieuc and Mont-Saint-Michel, two sites which were not concerned by mortality at the time of sampling. Less than a month later, Saint-Brieuc was affected by unusual mortalities and F. halioticida was detected in almost all mussels (81.25%). The findings from this study provide further evidence indicating that F. halioticida may be contributing to mussel mortalities; however, a direct causal relationship has not yet been established. The real-time PCR assay developed in this study allows for rapid, specific and sensitive detection of F. halioticida which should prove useful for future studies concerning the involvement of this bacterium with shellfish mortalities.

Mutational Frequencies in Mycobacterial rpoB gene using GeneXpert/MTB Rif Assay in Rifampicin Resistant patients at a tertiary care setting in Urban Sindh, Pakistan: Analysis from a Five-Year Period.

OBJECTIVES: To assess the mutational frequencies in Mycobacterial rpoB gene using GeneXpert/MTB Rif Assay in rifampicin resistant patients during 2013-2017 at a tertiary care setting in Urban Sindh, Pakistan. METHODS: This Retrospective Descriptive Cross-Sectional Study was conducted at the TB laboratories, Ojha Institute of Chest Diseases, Dow University of Health Sciences. The record of 713 positive cases of Rifampicin Resistant Tuberculosis from January 2013 to December 2017 were analysed. These were diagnosed using GeneXpert® that detects mutations in the 81 base pair region of rpoB gene with the help of five molecular probes A, B, C, D and E. All invalid and extra pulmonary samples were excluded. RESULTS: In total, 713 cases were found to be rifampicin resistant during the five-year period, among which 374 (52.45%) were males while 339 (47.55%) were females. Among the five standard probes A, B, C, D and E, 97.48% of the cases had a single mutation. Among these, mutations in Probe E (66.48%) were the most common, followed by Probe B (14.3%) and Probe D (11.08%). Only 13 cases (1.82%) of double mutations and five cases (0.7%) of triple mutations were detected. CONCLUSION: The rpoB gene Probe E region 529-533 appears the most potent site for a mutation and development of rifampicin resistance in the rpoB gene in Mycobacterium tuberculosis, that encodes the ß-subunit of RNA polymerase. The most affected age-group in both males and females is 19-45 Years.

Paenibacillus ottowii sp. nov. isolated from a fermentation system processing bovine manure.

Strain MS2379T was isolated from a pasteurized solution sample from a predominantly anaerobic fermentation system processing bovine manure in Pilot Point, Texas. Phylogenetic analyses based on both 16S rRNA gene and rpoB gene sequences showed that MS2379T was most closely related to Paenibacillus polymyxa (DSM 36T), P. jamilae (DSM 13815T), and P. peoriae (DSM 8320T), yet DNA-DNA relatedness through DNA-DNA hybridization revealed only 22.6, 32.0 and 24.7 % relatedness to these three species respectively. Rod-shaped cells of strain MS2379T are Gram-stain variable with sub-terminal, ellipsoidal, deforming endospores. The peptidoglycan contains meso-diaminopimelic acid (mDAP) and the predominant fatty acids are anteiso-C15 : 0 (61.9 %) and anteiso-C17 : 0 (11.6 %), confirming that strain MS2379T has diagnostic features of other Paenibacillus species. The G+C content of MS2379T is 45.9 mol%. Fermentation of glucose yields acid and gas end-products. The polar lipids found were diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, and glycolipids, but also included some unidentified lipids, aminolipids, aminoglycolipid, and phosphatidylmethylethanolamine. The growth range of MS2379T was observed from 10-45 °C with optimal growth temperature at 30 °C. Growth was observed between pH 6-10 and up to 3 % NaCl. Unlike the most closely related Paenibacillus species, strain MS2379T was negative in the Voges-Proskauer reaction. Nucleic acid, chemotaxonomic and biochemical features support the distinctiveness of strain MS2379T. Thus, strain MS2379T represents a novel species of the genus Paenibacillus for which the name Paenibacillus ottowii sp. nov. is proposed with the type strain MS2379T (=DSM 107750T=ATCC TSD-165T).

Description of three bacterial strains belonging to the new genus Novipirellula gen. nov., reclassificiation of Rhodopirellula rosea and Rhodopirellula caenicola and readjustment of the genus threshold of the phylogenetic marker rpoB for Planctomycetaceae.

Access to axenic cultures of Planctomycetes is crucial for further investigating their complex lifestyle, uncommon cell biology and primary and secondary metabolism. As a contribution to achieve this goal in the future, we here describe three strains belonging to the novel genus Novipirellula gen. nov. The strains were isolated from biotic and abiotic surfaces in the Baltic Sea and from the island Heligoland in the North Sea. Colony colours range from white to light pink. Cells are acorn-shaped and grew optimally at neutral pH and temperatures between 27 and 30 °C. Phylogenetic analyses revealed that the isolated strains represent three novel species belonging to a new genus, Novipirellula gen. nov. Beyond that, our analysis suggests that Rhodopirellula rosea LHWP3T, Rhodopirellula caenicola YM26-125T and Rhodopirellula maiorica SM1 are also members of this novel genus. Splitting the current genus Rhodopirellula into a more strictly defined genus Rhodopirellula and Novipirellula also allowed readjusting the genus threshold value for the gene rpoB, encoding the RNA polymerase ß-subunit, which is used as phylogenetic marker for Planctomycetales. A threshold range of 75.5-78% identity of the analysed partial rpoB sequence turned out to be reliable for differentiation of genera within the family Planctomycetaceae.

Isolation, identification and characterization of Staphylococcus sp. from Indian ethnic fermented fish product.

Staphylococci from Sheedal of Northeast India was isolated, identified and characterized. All the isolated staphylococci were found to be coagulase negative. Based on the rpoB gene sequences followed by analysis using NCBI-BLAST software, seven species of Staphylococcus namely, S. piscifermentans, S. condimenti, S. arlettae, S. sciuri, S. warneri, S. nepalensis and S. hominis were recognized. Phylogenetic analyses revealed three major cluster groups. All the seven Staphylococcus showed their NaCl tolerance from 2 to 8%. No species was able to grow at 55°C. Except S. arlettae and S. sciuri, all the isolated staphylococcal species exhibited growth at pH 4-8. No isolated species was able to ferment mannitol, sucrose and arabinose. All the species exhibited moderate to maximum proteolytic and lipolytic activities. All the seven species were found to be sensitive to the antibiotics, namely, erythromycin, norfloxacin, ampicillin, streptomycin and vancomycin, whereas all were resistant to co-trimoxazole. Only S. piscifermentans was found antagonist to Salmonella enterica, Escherichia coli and Bacillus subtilis, although the clear zone was minimal. All the staphylococcal species except S. arlettae and S. sciuri exhibited hydrophobicity ranging from 25 to 66%. The observed characteristics of isolated Staphylococci from Sheedal revealed their role in fish fermentation.

Proteus faecis sp. nov., and Proteus cibi sp. nov., two new species isolated from food and clinical samples in China.

Eight swarming motile bacteria were isolated from food and clinical samples in China. Cells were Gram-stain-negative, facultatively anaerobic and rod-shaped (0.5-0.8×1.0-3.0 µm) with hairlike pili and flagella. The 16S rRNA and partial rpoB housekeeping gene sequence analyses indicated that the strains belong to the genus Proteusin the family Enterobacteriaceae. Of the eight strains studied, seven and a single isolate formed two separate clades in the phylogeny of Proteusspecies, indicating two separate species. Both the in silico DNA-DNA hybridization and the average nucleotide identity values between these two groups and to the type strains of the genus Proteuswere below the recommended threshold for signifying their candidature as two separate species. The DNA G+C contents of strains TJ1636T and FJ2001126-3T were 37.8 and 38.1 mol%, respectively. The major cellular fatty acids of the two novel type strains were C16:0, cyclo C17:0, summed feature 3 and summed feature 8. The results supported that the strains belong to different taxonomic positions in the genus Proteus. The isolates were named Proteus faecis sp. nov., with type strain TJ1636T (=DSM 106180T=GDMCC 1.1245T), and Proteuscibi sp. nov., with type strain FJ2001126-3T (=DSM 106178T =GDMCC 1.1244T).

Rhodobacter alkalitolerans sp. nov., isolated from an alkaline brown pond.

An alkali-tolerant, Gram-stain-negative, motile, rod-to-oval-shaped, yellowish brown-colored, phototrophic bacterium, designated as strain JA916T, was isolated from an alkaline brown pond in Gujarat, India. The DNA G + C content of the strain JA916T was 65.1 mol%. Strain JA916T grew well at pH 10. Respiratory quinone was Q-10 and major fatty acid was C18:1ω7c/C18:1ω6c, with significant quantities of C15:02OH observed. Strain JA916T shared the highest 16S rRNA gene sequence similarity with the type strains of Rhodobacter johrii (98.4%), followed by Rhodobacter megalophilus (98.3%), Rhodobacter sphaeroides (98.3%), Rhodobacter azotoformans (97.9%) and other members of the genus Rhodobacter (< 97%). 16S rRNA gene-based phylogenetic tree shows that strain JA916T formed a distinct sub-clade with Rhodobacter johrii, Rhodobacter megalophilus, Rhodobacter sphaeroides and Rhodobacter azotoformans. Further, rpoB-based phylogenetic analysis showed lower similarity with closely related species (≤ 93.0%) of the genus Rhodobacter, which suggests that JA916T is a novel species of the genus Rhodobacter. DNA-DNA hybridization values between strain JA916T and related type strains were less than 40%. Phenotypic, chemotaxonomical and phylogenetic differences showed that strain JA916T was distinct from other species of the genus Rhodobacter, suggesting strain JA916T represents a new species of the genus for which the name Rhodobacter alkalitolerans sp. nov. is proposed. Type strain is JA916T (= KCTC 15473T = LMG 28749T).

Corynebacterium godavarianum sp. nov., isolated from the Godavari river, India.

A Gram-stain-positive, rod-shaped, non-motile bacterium, strain PRD07T, was isolated from Godavari river, India during the world's largest spiritual and religious mass bathing event 'Kumbh Mela'. Molecular analysis using 16S rRNA gene sequencing and phylogenetic analysis reveals the distinct phylogenetic positioning of strain PRD07T within the genus Corynebacterium. The strain demonstrated highest sequence similarity to Corynebacterium imitans DSM 44264T (97.9 %), Corynebacterium appendicis DSM 44531T (97.1 %) and <96.7 % with all other members of the genus Corynebacterium. The G+C content of PRD07T was 68.5 mol% (Tm) and the DNA-DNA hybridization depicts 61.09 % genomic relatedness with C. imitans DSM 44264T. Chemotaxonomic assessment of strain PRD07T suggested presence of C16 : 0 (31.6 %), C18 : 0 (3.5 %) and C18 : 1ω9c (58.6 %) as the major cellular fatty acids. The major polar lipids of strain PRD07T were phosphatidylglycerol, diphosphatidylglycerol and glycophospholipid. Differentiating molecular, phylogenetic and chemotaxonomic characteristics of strain PRD07T with its closest relatives necessitated the description of strain PRD07T as a novel species of genus Corynebacterium for which the name Corynebacteriumgodavarianum sp. nov., has been proposed. The type strain is PRD07T (=MCC 3388T=KCTC 39803T=LMG 29598T).

Novel PCR-RFLP system based on rpoB gene for differentiation of Cronobacter species.

Bacteria from the genus Cronobacter are opportunistic foodborne pathogens that can cause severe infections. More rapid, cost-effective and reliable methods are still required for the species identification of Cronobacter spp. In this study, we present a novel PCR-RFLP-based method that uses a newly designed pair of primers for the PCR-amplification of a partial rpoB gene sequence (1635 bp). The amplified products of DNA from 80 Cronobacter strains were separately digested with three restriction endonucleases (Csp6I, HinP1I, MboI). Using the obtained restriction patterns, a PCR-RFLP identification system was created to enable differentiation between all seven currently-known Cronobacter species. The functionality of our method was successfully verified on real food samples. Moreover, the relationships between the Cronobacter species were determined via a phylogenetic tree created from the RFLP patterns.

The first cases of human bacteremia caused by Acinetobacter seifertii in Japan.

Acinetobacter seifertii, a novel species of Acinetobacter, was first reported in 2015. A. seifertii strains were isolated from human clinical specimens (blood, respiratory tract, and ulcer) and hospital environments. Here, we report the first cases of bacteremia caused by A. seifertii in patients with catheter-related bloodstream infection in Japan. The patients favorably recovered, without any complications, after removal of the peripheral intravenous catheters and administration of antibiotics. The pathogens were initially identified as Acinetobacter baumannii, using phenotypic methods and the MicroScan Walkaway System; however, rpoB gene sequence analysis indicated 99.54% similarity to A. seifertii. Moreover, antimicrobial susceptibility testing revealed that one of the strains was not susceptible to gentamicin and ceftazidime. Our report shows that Acinetobacter species other than A. baumannii can also cause nosocomial infections and that accurate methods for the identification of causative agents should be developed.

Characterization of representative rpoB gene mutations leading to a significant change in toyocamycin production of Streptomyces diastatochromogenes 1628.

Modification of enzymes involved in transcription- or translation-processes is an interesting way to increase secondary metabolite production in Streptomycetes. However, application of such methods has not been widely described for strains which produce nucleoside antibiotics. The nucleoside antibiotic toyocamycin (TM) is produced by Streptomyces diastatochromogenes 1628. For improving TM production in S. diastatochromogenes 1628, the strain was spread on rifamycin-resistant (Rif(r)) medium. Several spontaneous mutants were obtained with mutations in the rpoB gene which encodes a RNA polymerase ß-subunit. The mutants which showed increased TM production were detected at a frequency of 7.5 % among the total Rif(r) mutants. Mutant 1628-T15 harboring amino acid substitution His437Arg was the best TM producer with a 4.5-fold increase in comparison to that of the wild-type strain. The worst producer was mutant 1628-T62 which also showed a poor sporulation behavior. RT-PCR was performed to study the transcription levels of the TM biosynthetic gene toyG in the parental strain as well as in mutants 1628-T15 and 1628-T62. The transcriptional level of toyG was higher in mutant 1628-T15 than that in parental strain 1628, while much lower in mutant 1628-T62. In mutant strain 1628-T62 the expression of adpA sd gene, which is required for morphological differentiation, was also much lower. Our studies also indicate that the introduction of mutations into rpoB is an effective strategy to improve the production of TM which is an important nucleoside antibiotic.

MOLECULAR IDENTIFICATION AND SEQUENCE CHARACTERIZATION OF MYCOPLASMAS IN FREE-LIVING BIRDS OF PREY.

Mycoplasma spp. have been detected in birds of prey, but their prevalence in free living raptors and their significance to birds' health need further investigation. Molecular techniques have been increasingly used to identify mycoplasmas in various avian species, due to the fastidious nature of these pathogens hampering traditional bacteriologic tests. This study reports the identification of 23 novel mycoplasma sequences during the monitoring of 62 birds of prey on admission to wildlife centers in Sardinia, Italy. Molecular investigation performed on pharyngeal swabs revealed 26 birds positive to Mycoplasma (42%). Sequence analysis based on 16S rRNA, 16S-23S rRNA intergenic spacer, and RNA polymerase ß subunit (rpoB) gene highlighted cluster assignment and phylogenetic relationships among the identified types, classified within the hominis group. Additionally, Ornithobacterium rhinotracheale , associated with respiratory disease in poultry, was identified in 17 birds (27%). Potential coinfection and mycoplasma opportunistic nature present implications for raptor species conservation.

Detection and Characteristics of Rifampicin-Resistant Isolates of Mycobacterium tuberculosis.

Genotyping and analysis the drug resistance of 59 isolates of M. tuberculosis obtained from patients living in Altai Territory were performed using a BACTEC MGIT 960 fluorometric system by means of VNTR typing (variable number tandem repeat), PCR-RFLP analysis, and sequence analysis. The occurrence frequency was highest for isolates of the Beijing family (n=30, 50.8%). Analysis of mutation spectrum in the rpoB gene associated with rifampicin resistance revealed the major mutation (codon 531 of the rpoB gene) in 93% samples, which allows us to use rapid test systems.