Phylogeny of the genus Mycobacterium: Many doubts,few certainties

The genus Mycobacterium is characterized by very limited interspecies genetic variability and this makes the definition of a robust phylogeny problematic. In this study a twofold phylogenetic approach was adopted. Phylogenetic trees were constructed using as targets the almost complete 16S rRNA gene sequences and the concatenated amino acid sequences coded by fragments of hsp65 and rpoB genes. The comparison of the results made it possible to identify clusters of species sharing common phylogenetic pathway but for the majority of mycobacteria the definition of a robust phylogeny remained unreached.     

聚合酶链反应鉴定结核分枝杆菌常用靶基因序列的研究进展

分枝杆菌种类繁多,至今已发现有150余种,包括结核分枝杆菌复合群、麻风分枝杆菌以及非结核分枝杆菌,而结核分枝杆菌复合群是引起人类结核病的主要病原菌。分子生物学技术的发展为实现结核分枝杆菌的快速鉴定提供了方向,建立了各种以核酸序列为靶基因,如IS6110、16S rRNA、16S-23S rRNA ITS、hsp65、rpoB和gyrB等的快速鉴定方法。本文对聚合酶链反应(PCR)检测方法中鉴定结核分枝杆菌常用靶基因序列鉴定方法的敏感度和特异度研究进展进行综述。     

乳腺炎奶牛双重感染抗热分枝杆菌和象分枝杆菌的鉴定及药敏试验研究

目的 对疑似分枝杆菌感染乳腺炎奶牛进行病原及药敏谱调查。方法 采集1头患乳腺炎奶牛的牛奶,采用4% NaOH预处理,接种于L-J培养基分离培养。阳性培养物利用抗酸染色和多位点PCR方法进行初步鉴定,采用16S rRNA、hsp65、ITS,和SodA基因的多位点序列分析进行种的鉴定,利用Alamar blue显色法对分离菌株进行27种药物的药敏试验。结果 从1头患乳腺炎奶牛的牛奶中同时分离获得2株抗酸染色阳性培养物,经PCR鉴定为非结核分枝杆菌,多位点序列分析鉴定为抗热分枝杆菌和象分枝杆菌。药敏试验表明这2株菌对利福平和异烟肼等大多数抗结核药物耐药,但对阿米卡星、莫西沙星、左氧氟沙星、乙胺丁醇、链霉素、妥布霉素、环丙沙星和利奈唑胺等敏感。结论 乳腺炎奶牛中分离了抗热分枝杆菌和象分枝杆菌,有其独特的药敏谱特征,为其感染防治提供了科学依据。     

The Problem of Mixing up of Leishmania Isolates in the Laboratory: Suggestion of ITS1 Gene Sequencing for Verification of Species

Background

Leishmaniasis is endemic in Iran. Different species of Leishmania (L.) parasites are causative agents of this disease. Correct identification of Leishmania species is important for clinical studies, prevention, and control of the diseases. Mix up of Leishmania isolates is possible in the laboratory, so there is need for verification of species for isolates of uncertain identity. Different methods may be used for this purpose including isoenzyme electrophoresis and molecular methods. The isoenzyme electrophoresis, due to its drawbacks, is feasible only in specialized laboratories while molecular methods may be more feasible. The aim of this research was to study the application of the internal transcribed spacer 1 (ITS1) sequencing method, in comparison to isoenzyme electrophoresis method, for verification of Leishmania species.

Methods

Six Leishmania isolates were received from different research institutions in Iran. The species of these isolates were known by donating institution according to their isoenzyme profile. The species of these isolates were re-identified in Pasteur Institute of Iran by PCR amplification of ITS1 followed by sequencing and comparison of these sequences with Leishmania sequences in GenBank. Isoenzyme electrophoresis was performed for confirmation of the results of ITS1.

Results

ITS1 sequence showed that some isolates were mixed up or contaminated with Crithidia. Isoenzyme electrophoresis confirmed the results of ITS1 sequences.

Conclusion

ITS1 sequencing is relatively more feasible than the traditional isoenzyme electrophoresis method and is suggested for verification of Leishmania species.     

Nontuberculous Mycobacteria in Household Plumbing as Possible Cause of Chronic Rhinosinusitis

Symptoms of chronic rhinosinusitis (CRS) often persist despite treatment. Because nontuberculous mycobacteria (NTM) are resistant to commonly used antimicrobial drugs and are found in drinking water that patients may use for sinus irrigation, we investigated whether some CRS patients were infected with NTM in New York, New York, USA, during 2001–2011. Two approaches were chosen: 1) records of NTM-infected CRS patients were reviewed to identify common features of infection and Mycobacterium species; 2) samples from plumbing in households of 8 NTM-infected patients were cultured for NTM presence. In 3 households sampled, M. avium sharing rep-PCR and pulsed field gel electrophoresis fingerprints identified M. avium isolates clonally related to the patients’ isolates. We conclude that patients with treatment-resistant CRS may be infected with NTM and should have cultures performed for NTM so appropriate therapy can be instituted. In addition, the results suggest that CRS patients can be infected by NTM in their household plumbing.     

Polyphasic characterization of the genus Anabaena from the Gulf of Gdańsk (Southern Baltic Sea). Application of DGGE in taxonomic studies on environmental samples

The aim of this paper was a taxonomic verification of cyanobacteria of the genus Anabaena that occur in the Gulf of Gda??sk. Classical taxonomic methods were combined with modern molecular taxonomic methods (polyphasic approach). Analysis of the species diversity of cyanobacteria from the genus Anabaena was based on the microscopic analysis and on the analysis of 16S rRNA and ITS region. Comparison of the obtained results with sequences in GenBank showed 97.8?C100% similarity for 16S rRNA and 98.8-100% similarity in the case of the ITS fragment. Similarity of the 16S rRNA and ITS sequence of 98.5% seems to be sufficient to determine the species.     

Molecular Identification and Differentiation of Fasciola Isolates Using PCR- RFLP Method Based on Internal Transcribed Spacer (ITS1, 5.8S rDNA,ITS2)

Background

In this study, we used both ITS1 and ITS2 for molecular identification of Fasciola species.

Methods

The region between 18S and 28S of ribosomal DNA was used in PCR-RFLP method for molecular identification of Fasciola species. Ninety trematodes of Fasciola were collected during abattoir inspection from livers of naturally infected sheep and cattle from Khorasan, East Azerbaijan, and Fars provinces in Iran. After DNA extraction, PCR was performed to amplify region ITS1, 5.8S rDNA, ITS2. To select a suitable restriction enzyme, we sequenced and analyzed the PCR products of F. hepatica and F. gigantica samples from sheep and cattle. Tsp509I fast digest restriction enzyme was selected for RFLP method that caused the separation specifically of Fasciola species.

Results

The fragment approximately 1000bp in all of the Fasciola samples was amplified and then digested with the Tsp509I restriction endonuclease. Seventy F. hepatica and 20 F. gigantica were identified of total 90 Fasciola isolates.

Conclusion

The new PCR-RFLP assay using Tsp509I restriction enzyme provides a simple, practical, fast, low cost, and reliable method for identification and differentiation of Fasciola isolates.     

Genetic characterization of human-pathogenic Cyclospora cayetanensis parasites from three endemic regions at the 18S ribosomal RNA locus

Cyclospora cayetanensis is an apicocomplexan parasite that infects the gastrointestinal tract and causes acute diarrheal disease in humans. In recent years, this human-pathogenic parasite has led to several foodborne outbreaks in the United States and Canada, mostly associated with imported produce. Understanding the biology and epidemiology of C. cayetanensis is difficult because little is known about its origin, possible zoonotic reservoirs, and genetic relationships with other coccidian parasites. Recently, we developed a 70 kDa heat shock protein (HSP70) gene based nested PCR protocol for detection of C. cayetanensis parasite and sequenced the PCR products of 16 human isolates from Nepal, Mexico, and Peru. In this study, we have characterized the regions of 18S ribosomal RNA (rRNA) gene of 17 human C. cayetanensis isolates for molecular detection, and also to ascertain the genetic diversity of this parasite. The 18S rRNA primer sets were further tested by PCR amplification followed by nucleotide sequencing of the PCR amplified products of previously characterized C. cayetanensis isolates from three endemic regions at HSP70 locus. Although no genetic polymorphism was observed at the regions of HSP70 locus characterized in our previous study, the data analysis of this study revealed a minor genetic diversity at the 18S rRNA locus among the C. cayetanensis isolates. The 18S rRNA gene-based nested PCR protocol provides a useful genetic marker for the detection of C. cayetanensis parasite and confirms it as a genetically distinct species in genus Cyclospora. The results also supported lack of geographic segregation and existence of genetically homogeneous population for the C. cayetanensis parasites both at the HSP70 as well as at the18S rRNA loci.     

Candidatus Bartonella mayotimonensis and Endocarditis

We describe a new Bartonella species for which we propose the name Candidatus Bartonella mayotimonensis. It was isolated from native aortic valve tissue of a person with infective endocarditis. The new species was identified by using PCR amplification and sequencing of 5 genes (16S rRNA gene, ftsZ, rpoB, gltA, and internal transcribed spacer region).     

Nontuberculous mycobacteria: Insights on taxonomy and evolution

Seventy years have passed since Ernest H. Runyon presented a phenotypic classification approach for nontuberculous mycobacteria (NTM), primarily as a starting point in trying to understand their clinical relevance. From numerical taxonomy (biochemical testing) to 16S rRNA gene sequencing to whole genome sequencing (WGS), our understanding of NTM has also evolved. Novel species are described at a rapid pace, while taxonomical relationships are re-defined in large part due to the accessibility of WGS. The evolutionary course of clonal complexes within species is better known for some NTM and less for others. In contrast with M. tuberculosis, much is left to learn about NTM as a whole.     

Association of non-tuberculous mycobacteria with Mycobacterium leprae in environment of leprosy endemic regions in India

Non-tuberculous mycobacteria (NTM) are environmental mycobacteria found ubiquitously in nature. The present study was conducted to find out the presence of various species of NTM in leprosy endemic region along with Mycobacterium (M) leprae. Water and wet soil samples from the periphery of ponds used by the community were collected from districts of Purulia of West Bengal and Champa of Chhattisgarh, India. Samples were processed and decontaminated followed by culturing on Lowenstein Jensen (LJ) media. Polymerase chain reaction (PCR) was performed using 16S rRNA gene target of mycobacteria and species was confirmed by sequencing method. Indirect immune-fluorescent staining of M. leprae from soil was performed using M. leprae-PGL-1 rabbit polyclonal antibody. The phylogenetic tree was constructed by using MEGA-X software. From 380 soil samples 86 NTM were isolated, out of which 34(40%) isolates were rapid growing mycobacteria (RGM) and 52(60%) isolates were slow growing mycobacteria (SGM). Seventy-seven NTM isolates were obtained from 250 water samples, out of which 35(45%) were RGM and 42(55%) were SGM. Amongst all the RGM, we isolated M. porcinum, M. psychrotolerans, M. alsenase, M. arabiense and M. asiaticum from Indian environmental samples. M. fortuitum was the most commonly isolated species of all RGM. Out of all SGM, M. holsaticum, M. yongonense, M. seoulense, M. szulgai, M. europaeum, M. simiae and M. chimaera were isolated for the first time from Indian environment. M. intracellulare was the commonest of all isolated SGM. Presence of M. leprae was confirmed by indirect immunofluorescent microcopy and PCR method from the same environmental samples. Phylogenetic tree was showing a close association between these NTMs and M. leprae in these samples. Several NTM species of pathogenic and nonpathogenic in nature along with M. leprae were isolated from soil and pond water samples from leprosy endemic regions and these might be playing a role in causing disease and maintaining leprosy endemicity in India.     

Integrative taxonomy of Mesocriconema onoense (Tylenchida: Criconematidae) from Vietnam highly suggests the synonymization of Mesocriconema brevistylus and related species

The genus Mesocriconema is one of the most diverse genera within the family Criconematidae, known as ring nematodes, with more than 90 species. Although species in this genus usually show distinct morphological characterizations, the identification based only on morphology can lead to misidentification in many studies resulted in a number of synonymizations in the genus over time. In this study, an integrated approach has been applied in characterizing Mesocriconema onoense from Vietnam. The molecular data of 28S rRNA, ITS, 18S rRNA regions were analyzed and discussed to confirm the correct names on GenBank. Besides, phylogenetic analyses of 28S rRNA, ITS, and 18S rRNA regions of Mesocriconema species revealed that Mesocriconema brevistylus should be considered as a junior synonym of M. onoense. Consequently, M. helicus, M. onostris, and M. paronostris should also be considered as the synonyms of M. onoense.     

First report of New Delhi metallo-β-lactamase-6 (NDM-6) among Klebsiella pneumoniae ST147 strains isolated from dialysis patients in Iran

There has been an alarming health-related concern about the growth of New Delhi metallo-β-lactamase. The aims of this study include the phenotypic detection of β-lactamases and molecular characterization of NDM in Klebsiella pneumoniae isolates in Tehran, Iran. A total of 120 K. pneumoniae isolates were collected from hospitalized haemodialysis patients, Tehran, Iran from March 2014 to February 2017. Antibiotic susceptibility tests were conducted using Kirby-Bauer disc diffusion and Broth Microdilution methods according to Clinical and Laboratory Standards Institute guidelines. Metallo-β-lactamase was detected using the Combined Disc Diffusion Test (CDDT), and production of carbapenemase was screened using the Modified Hodge Test. NDM-producing K. pneumoniae strains were screened for the presence of mcr-1 gene, β-lactamase genes, and 16S rRNA methylase genes by Polymerase Chain Reaction and sequencing. Molecular typing of the strains was determined using Repetitive Sequence Based-PCR and Multilocus Sequence Typing. The bla_NDM-6 gene was detected in 3 (2.5%) out of 120 isolates from dialysis patients. Also, the three isolates were positive for bla_CTX-M-15,bla_TEM extended-spectrum β-lactamase genes, armA type plasmid-mediated 16S rRNA methylase and CMY-type plasmid-mediated AmpC β-lactamase.The isolates were identified as MLST sequence type 147 (ST147). This is the first report of bla_NDM-6 in K. pneumoniae strains, isolated in Iran.     

Genetic characterization and phylogenetic relationships based on 18S rRNA and ITS1 region of small form of canine Babesia spp. from India

Canine babesiosis is a vector borne disease caused by intra-erythrocytic apicomplexan parasites Babesia canis (large form) and Babesia gibsoni (small form), throughout the globe. Apart from few sporadic reports on the occurrence of B. gibsoni infection in dogs, no attempt has been made to characterize Babesia spp. of dogs in India. Fifteen canine blood samples, positive for small form of Babesia, collected from northern to eastern parts of India, were used for amplification of 18S rRNA gene (∼1665 bp) of Babesia sp. and partial ITS1 region (∼254 bp) of B. gibsoni Asian genotype. Cloning and sequencing of the amplified products of each sample was performed separately. Based on sequences and phylogenetic analysis of 18S rRNA and ITS1 sequences, 13 were considered to be B. gibsoni. These thirteen isolates shared high sequence identity with each other and with B. gibsoni Asian genotype. The other two isolates could not be assigned to any particular species because of the difference(s) in 18S rRNA sequence with B. gibsoni and closer identity with Babesia occultans and Babesia orientalis. In the phylogenetic tree, all the isolates of B. gibsoni Asian genotype formed a separate major clade named as Babesia spp. sensu stricto clade with high bootstrap support. The two unnamed Babesia sp. (Malbazar and Ludhiana isolates) clustered close together with B. orientalis, Babesia sp. (Kashi 1 isolate) and B. occultans of bovines. It can be inferred from this study that 18S rRNA gene and ITS1 region are highly conserved among 13 B. gibsoni isolates from India. It is the maiden attempt of genetic characterization by sequencing of 18S rRNA gene and ITS1 region of B. gibsoni from India and is also the first record on the occurrence of an unknown Babesia sp. of dogs from south and south-east Asia.     

Genetic and morphological characterization of Ixodes apronophorus from Western Siberia,Russia

Genetic variability of I. apronophorus from Western Siberia, Russia was examined using the nuclear internal transcribed spacer 2 (ITS2) and mitochondrial 16S rRNA and cytochrome c oxidase subunit 1 (cox1) genes and compared to those of Ixodes persulcatus and Ixodes trianguliceps from the same site. The I. apronophorus sequences demonstrated the highest nucleotide and haplotype diversity for both mitochondrial genes, whereas I. persulcatus was more variable in the nuclear ITS2. Phylogenetic analysis of the molecular sequence data showed that I. apronophorus differed from other Ixodes species, including Romanian I. apronophorus. The level of identity between 16S rRNA gene sequences of Siberian and Romanian I. apronophorus was only 91%; these sequences did not form a monophyletic group, indicating that I. apronophorus from Siberia and Romania could be different tick species. The analysis of morphological features of the Siberian I. apronophorus confirmed their consistency with those for the previously described I. apronophorus species. Based on the 16S rRNA and ITS2 sequences, Siberian I. apronophorus clustered together with Ixodes kazakstani and Ixodes scapularis, which are the recognized members of the Ixodes ricinus-I. persulcatus species complex within the subgenus Ixodes, and can be assigned to this complex.     

Genetic diversity of Pneumocystis jirovecii in colonized Cuban infants and toddlers

Pneumocystis jirovecii is a leading cause of opportunistic infections among immunocompromised patients. The aim of this study was to determine the genetic diversity of P. jirovecii from colonized Cuban infants and toddlers by analysis of four genetic loci: mitochondrial large subunit (mtLSU) rRNA, cytochrome b (CYB), superoxide dismutase (SOD) and β-tubulin (β-tub). We determined the multilocus profiles based on concatenated genotype data (multilocus genotype; MLG) and nucleotide sequences (multilocus sequence analysis; MLSA) respectively, calculated the discriminatory power of each analysis, and investigated possible associations with demographic and clinical data. Sixteen of 51 PCR-positive nasopharyngeal swab specimens (years 2010–2013) with high P. jirovecii load were selected for downstream analysis. In mixed allelic profiles all genotypes/nucleotide sequence patterns were considered separately. All samples could be genotyped based on mtLSU, CYB and β-tub locus. However, the SOD locus could be successfully amplified in only 7/16 (44%) specimens. Eight different P. jirovecii MLGs were identified among the 16 cases and eight samples presented identical MLG (MLG 1). Seventeen MLSA profiles were distinguished. No statistical association between genotypes or MLGs and demographic or clinical data could be identified. For MLSA the higher discriminatory power (S = 0.976) was observed. The combination of mtLSU, CYB and β-tub loci proved to be useful for molecular epidemiology studies of P. jirovecii. A total of 17 different MLSA profiles observed in 16 specimens indicated high genetic variability of P. jirovecii circulating in colonized Cuban infants and toddlers.     

Antimicrobial susceptibility testing and genotyping of Mycobacterium avium isolates of two tertiary tuberculosis designated hospital,China

Background: Mycobacterium avium is frequently isolated from clinical samples, while the bacteriological features of M. avium clinical isolates from China have never been well defined.Methods: A total of 50 M. avium isolates were recruited from two tertiary tuberculosis designated hospitals, one located in Beijing whereas another in Fujian Province, which are northern and southern parts of China, respectively. Subspecies identification was conducted by sequencing the variable 3′ end of the hsp65 gene. The susceptibility against 15 antimicrobial agents, widely administered for the treatment of non-tuberculosis mycobacteria (NTM) infections, was tested by broth microdilution assay. Variable number of tandem repeats (VNTR) assay was also performed using the 16-loci genotyping method.Results: All of the 50 M. avium isolates were identified as M. avium subsp. hominissuis. The drug susceptibility test revealed that clarithromycin (98%, 49/50) and moxifloxacin (86%, 43/50) had the best antimicrobial activities in vitro against the M. avium isolates. The overall Hunter–Gaston Discriminatory Index (HGDI) value for the VNTR typing was 0.95. However, the genotyping method yielded much greater discriminative power for isolates of northern China than that of southern China (1.00 V.S. 0.86, P < 0.05).Conclusion: M. avium subsp. hominissuis is the dominate subspecies among M. avium clinical isolates in China. The 16-loci VNTR genotyping method is more discriminative in Beijing than in Fujian Province. The bacteriological features of M. avium isolates from different regions of China demonstrated dramatic variations, and stressed the importance of building up knowledge from the local isolates.     

Prevalence of non-tuberculous mycobacteria in a hospital environment

In recent years, non-tuberculous mycobacteria (NTM) have emerged as an important cause of opportunistic nosocomial infections but there is little known about the isolation and identification of NTM in Korea. The aim of this study was to assess the prevalence of NTM in the hospital environment and identify the species. A total of 150 samples were collected from different parts of the hospital. NTM were isolated and identified by restriction fragment length polymorphism analysis of the gene encoding rpoB and partial sequencing analysis of hsp65 and rpoB. In this study, 60 strains of NTM were isolated from 50 of the 150 samples. Half of the tap water samples (50 of 100) were positive for mycobacteria. An estimated 73.3% of the isolates were saprophytic, 21.7% were potentially pathogenic and 5% were unidentified. The presence of NTM in hospital tap water is not uncommon. Such water isolates might cause true nosocomial infection in immunocompromised patients, in addition to the risk of false-positive culture results.     

Phylogenetic Analysis of Theileria annulata Infected Cell Line S15 Iran Vaccine Strain

Background

Bovine theileriosis results from infection with obligate intracellular protozoa of the genus Theileria. The phylogenetic relationships between two isolates of Theileria annulata, and 36 Theileria spp., as well as 6 outgroup including Babesia spp. and coccidian protozoa were analyzed using the 18S rRNA gene sequence.

Methods

The target DNA segment was amplified by PCR. The PCR product was used for direct sequencing. The length of the 18S rRNA gene of all Theileria spp. involved in this study was around 1,400 bp.

Results

A phylogenetic tree was inferred based on the 18S rRNA gene sequence of the Iran and Iraq isolates, and other species of Theileria available in GenBank. In the constructed tree, Theileria annulata (Iran vaccine strain) was closely related to other T. annulata from Europe, Asia, as well as T. lestoquardi, T. parva and T. taurotragi all in one clade.

Conclusion

Phylogenetic analyses based on small subunit ribosomal RNA gene suggested that the percent identity of the sequence of Iran vaccine strain was completely the same as Iraq sequence (100% identical), but the similarity of Iran vaccine strain with other T. annulata reported from China, Spain and Italy determined the 97.9 to 99.9% identity.     

Molecular epidemiology of Theileria annulata and identification of 18S rRNA gene and ITS regions sequences variants in apparently healthy buffaloes and cattle in Pakistan

A molecular epidemiological survey was conducted to determine the prevalence of piroplasms in buffaloes and cattle from Sheikhupura and Okara districts of Punjab, Pakistan using reverse line blot (RLB) hybridization assay. The genetic diversity within 18S rRNA gene and ITS regions sequences of various obtained Theileria species (spp.) was also investigated. Briefly, 102 blood samples from buffaloes and cattle in the study districts were collected on blood collection cards and brought to the laboratory. DNA was extracted; the V4 hypervariable region of 18S rRNA was amplified and analyzed using RLB. Out of total samples analyzed, 61 (59.8%) were hybridized with Babesia/Theileria (B/T) genus-specific probe. Only one species of piroplasm was detected in buffaloes and cattle in study districts, i.e. Theileria (T.) annulata. Six samples only hybridized with B/T genus-specific and Theileria genus-specific probes but not with any species-specific probe indicating the presence of novel species or variants. The sequences of 18S rRNA gene and ITS regions of these six samples revealed the presence of T. annulata variants as confirmed through sequence identity estimation and phylogenetic analyses. Meanwhile, an unexpected sequence variation was observed within the 18S rRNA gene and ITS regions sequences of T. annulata identified in the present study. This is the first report on the simultaneous detection of species of piroplasms infecting buffaloes and cattle in Pakistan and molecular characterization of T. annulata 18S rRNA gene and ITS regions. The present study may address the new insights into the epidemiology of theileriosis which will help researches in designing control strategies and developing various molecular diagnostic tools at national level.