Rrs Sequences Research Articles

Controlling genome topology with sequences that trigger post-replication gap formation during replisome passage: the E. coli RRS elements.

We report that the Escherichia coli chromosome includes novel GC-rich genomic structural elements that trigger formation of post-replication gaps upon replisome passage. The two nearly perfect 222 bp repeats, designated Replication Risk Sequences or RRS, are each 650 kb from the terminus sequence dif and flank the Ter macrodomain. RRS sequence and positioning is highly conserved in enterobacteria. At least one RRS appears to be essential unless a 200 kb region encompassing one of them is amplified. The RRS contain a G-quadruplex on the lagging strand which impedes DNA polymerase extension producing lagging strand ssDNA gaps, $ \le$2000 bp long, upon replisome passage. Deletion of both RRS elements has substantial effects on global genome structure and topology. We hypothesize that RRS elements serve as topological relief valves during chromosome replication and segregation. There have been no screens for genomic sequences that trigger transient gap formation. Functional analogs of RRS could be widespread, possibly including some enigmatic G-quadruplexes in eukaryotes.

Influence of Hydrodynamic Forces on Electroactive Bacterial Adhesion in Microbial Fuel Cell Anodes.

This investigation examined the role of shear stress on the dynamic development of microbial communities within anodic biofilms in single-chamber microbial fuel cells (MFCs). Bacterial attachment to surfaces, often regarded as a crucial step in biofilm formation, may significantly contribute to the selection of electroactive bacteria (EAB). It is well established that hydrodynamic forces, particularly shear forces, have a profound influence on bacterial adhesion. This study postulates that shear stress could select EAB on the anode during the adhesion phase by detaching non-EAB. To examine this hypothesis, MFC reactors equipped with a shear stress chamber were constructed, creating specific shear stress on the anode. The progression of adhesion under various shear stress conditions (1, 10, and 50 mPa) was compared with a control MFC lacking shear stress. The structure of the microbial community was assessed using 16S rRNA gene (rrs) sequencing, and the percentage of biofilm coverage was analyzed using fluorescence microscopy. The results indicate a significant impact of shear stress on the relative abundance of specific EAB, such as Geobacter, which was higher (up to 30%) under high shear stress than under low shear stress (1%). Furthermore, it was noted that shear stress decreased the percentage of biofilm coverage on the anodic surface, suggesting that the increase in the relative abundance of specific EAB occurs through the detachment of other bacteria. These results offer insights into bacterial competition during biofilm formation and propose that shear stress could be utilized to select specific EAB to enhance the electroactivity of anodic biofilms. However, additional investigations are warranted to further explore the effects of shear stress on mature biofilms.

Genetic Evidence for a Potentially New Pathogenic Leptospira sp. Circulating in Bats from Brazilian Amazon

Leptospirosis is a neglected zoonosis frequently reported worldwide, caused by a spirochete of the genus Leptospira. It is capable of infecting domestic animals, free-living animals, and humans. Among wild animals, the role of bats in the epidemiology of leptospirosis has been investigated but is far from being elucidated. The Amazonian biome has the ideal conditions for maintaining and disseminating leptospires and, despite efforts that have been made to better understand leptospires’ occurrence in wild animals in the region, few studies aimed to explore and genetically characterize leptospires in bats. Based on this, the aim of the present study is to deeper investigate and genetically characterize leptospires detected in bats from the southwest Amazon. Animals were trapped by mist netting at five sites in the state of Acre, Brazil. Kidney samples were obtained and stored for molecular analysis. Polymerase chain reaction (PCR) was conducted first based on the LipL32 gene, and positive samples were submitted to rrs and secY-PCR and sequencing. Sequences were then submitted to phylogenetic analyses through multiple bioinformatic tools. The rrs sequences from the present study formed one single haplotype, different from any other previously deposited, grouped in a highly supported cluster with sequences from bats from Madagascar and China. The initial secY screening revealed no identity with previously deposited sequences. The phylogenetic trees revealed the sequences from the present study in an isolated branch, clearly separated from all previously known pathogenic Leptospira spp., suggesting the existence of a potentially undescribed species. The haplotype network including only leptospires from the Amazon region confirmed two new haplotypes from the same taxon unity, isolated from the others, with a probable origin of the species from L. noguchii. The characterization of this potentially new species in bats reinforces the complexity of the transmission dynamics of leptospires, including wild, periurban, and urban environments, emphasizing the need for an integrative look at leptospirosis vigilance within the context of One Health.

Co-Infection by Leptospira montravelensis and Leptospira interrogans Serovar Pomona in Urine Samples of Donkeys and Pigs in Sardinia, Italy.

Saprophytic leptospires are spirochetes enclosed within the non-pathogenic clade of the genus Leptospira, which in turn is subdivided into two subclades S1 and S2. To date, the microorganisms included in these subclades have been isolated from the environment in various parts of the world, and are believed to have no known animal reservoirs. After a case of Leptospira interrogans serovar Pomona was notified to the owner of a farm in Sardinia, all of the farm animals (11 pigs and 3 donkeys) were examined for the presence of Leptospira. Sera of all tested animals resulted positive for antibodies to Leptospira using a microscopic agglutination test (MAT). Moreover, nine (82%) kidney samples from pigs and three urine samples collected from donkeys (100%) tested positive for Leptospira DNA after qPCR. Results obtained after MLST analysis and sequencing of rrs, rpoB, and secY genes, performed on six Leptospira strains isolated in culture, revealed the presence of the genomospecies L. interrogans serovar Pomona in the kidney samples. Conversely, whole-genome sequencing combined with mean nucleotide identity revealed the presence of the saprophytic L. montravelensis in the urine samples. Our results report, for the first time, the isolation of a saprophytic species from mammalian urine, suggesting a new ecological specialization for these bacteria, with a possible transition from free-living to a symbiotic lifestyle. Further studies will have to be conducted to understand the evolution of virulence of these bacteria, potential infectivity, and possible public health implications.

A novel Rickettsia species closely related to Rickettsia felis in Anopheles mosquitoes from Yingkou City, Northeast China.

Mosquitoes are generally recognized as the most important vector of many zoonotic pathogens. In this study, seven mosquitoes species were identified (Anopheles pullus, Anopheles sinensis, Anopheles lesteri, Anopheles kleini, Ochlerotatus dorsalis, Aedes koreicus and Culex inatomii) in samples collected from Yingkou City, Liaoning Province, Northeastern China. A novel Rickettsia species was detected in Anopheles sinensis (two of 71, 2.82%) and Anopheles pullus (one of 106, 0.94%) mosquitoes. Genetic analysis indicated that the rrs and ompB genes have highest 99.60% and 97.88%-98.14% identities to Rickettsia felis, an emerging human pathogen of global concern mainly harboured by fleas, mosquitoes and booklice. The gltA sequences of these strains have 99.72% of nucleotide similarity with Rickettsia endosymbiont of Medetera jacula. The groEL sequences have 98.37% similarity to both Rickettsia tillamookensis and Rickettsia australis. The htrA sequences have 98.77% similarity to Rickettsia lusitaniae. In the phylogenetic tree based on concatenated nucleotide sequences of rrs, gltA, groEL, ompB and htrA genes, these strains are closely related to R. felis. Herein, we name it 'Candidatus Rickettsia yingkouensis'. Its human pathogenicity to humans and animals is still to be determined.

Novel Borrelia Genotypes from Brazil Indicate a New Group of Borrelia spp. Associated with South American Bats.

The bacterial genus Borrelia comprises vector-borne spirochetes that have been classified into three major groups: the relapsing fever group (RFG), the Borrelia burgdorferi Johnson, Schmid, Hyde, Steigerwalt & Brenner sensu lato group (Bbsl), and the reptile-monotreme group (RMG). All three groups have been associated mainly with ticks and wild animals, especially rodents, birds, and reptiles. Here, we searched for Borrelia infection among 99 vampire bats [Desmodus rotundus (É. Geoffroy)] (Chiroptera: Phyllostomidae) from the Brazilian semiarid region. Through molecular investigation of bat internal organs, haplotypes of a potentially novel Borrelia organism were detected in 5% (5/99) of the bats. Borrelia DNA was detected in the liver, blood, spleen, kidney and brain, suggesting a systemic infection. Phylogenetic analyses inferred from partial sequences of the borrelial rrs and flaB genes indicated that the vampire bat-associated Borrelia sp. of this study form a monophyletic group with a newly reported Borrelia associated with a Colombia bat, distinct from the three main currently recognized groups of Borrelia spp., Bbsl, RFG, and RMG. These novel bat-associated Borrelia spp. from South America might have arisen through an independent event along the borrelial evolutionary history, since previous molecular reports of Borrelia organisms in bats or bat-associated ticks from Africa, Europe, and North America were all classified in the RFG.

Clinical Whole Genome Sequencing for Clarithromycin and Amikacin Resistance Prediction and Subspecies Identification of Mycobacterium abscessus

Mycobacterium abscessus infections are an emerging health care concern in patients with chronic pulmonary diseases, leading to high morbidity and mortality. One major challenge is resistance to clarithromycin, a cornerstone antibiotic with high efficacy. Therefore, treatment is primarily guided by phenotypic susceptibility results of clarithromycin, which requires extended incubation to assess for inducible resistance. Resistance mechanisms for clarithromycin include induction of erm(41) and mutations in the 23S rRNA gene (rrl). In addition, mutations in the 16S rRNA encoding gene (rrs) can confer high-level amikacin resistance, another essential drug in the treatment of M. abscessus infections. Herein, we developed a clinical whole genome sequencing (WGS) assay for clarithromycin resistance based on rrl and erm(41) gene sequences and amikacin resistance based on the rrs sequence in M. abscessus, as well as subspecies identification. Genotypic-based predictions were determined for 104 isolates from 68 patients. The overall accuracy of genotypic prediction for clarithromycin compared with phenotypic susceptibility results was 100% (95% CI, 96.45%-100%). For amikacin, we also obtained 100% accuracy (95% CI, 96.52%-100%). The high concordance between the genotypic and phenotypic results demonstrates that a WGS-based assay can be used in a clinical laboratory for determining resistance to clarithromycin and amikacin in M. abscessus isolates. WGS can also provide subspecies identification and high-definition phylogenetic information for more accurate M. abscessus strain typing.

Distribution and molecular characterization of rickettsiae in ticks in Harbin area of Northeastern China.

Tick-borne rickettsioses are world-spreading infectious zoonoses. Ticks serve as reservoirs and vectors for Rickettsia and play a key role in transmission of rickettsioses. Most of the Chinese rickettsiosis patients are reported from Northeastern China but the distribution of tick and tick-borne Rickettsia species in Northeastern China remain poorly studied. In this study, a total of 1,286 ticks were captured from the seven counties of Harbin, an area in Northeastern China, and the tick-borne Rickettsia species were identified by PCR and sequencing of rrs, gltA, groEL, ompA and 17-kDa antigen-encoding genes. Of the 5 identified tick species, Haemaphysalis longicornis and Ixodes persulcatus were the predominant tick species in the livestock and vegetation, respectively. Rickettsia raoultii and “Candidatus Rickettsia tarasevichiae” were the two detectable Rickettsia species in the ticks with a 28.8% positive rate but no rickettsiae were found in ticks of Haemaphysalis concinna. R. raoultii detected in 37.6% of the Dermacentor nuttalli, Dermacentor silvarum and H. longicornis ticks while “Ca. R. tarasevichiae” was only present in 22.8% of the I. persulcatus ticks. In particular, the positive rate of both R. raoultii and “Ca. R. tarasevichiae” in ticks from the livestock (40.7%) was significantly higher than that from the vegetation (19.5%). The results indicate that the tick and tick-borne Rickettsia species are diverse in different regions of Harbin due to geographic difference and the ticks from livestock may play a more important role in transmission of rickettsioses to human.

Extensive genetic diversity of Anaplasma bovis in ruminants in Xi’an, China

Anaplasma bovis is an organism significant to cattle and buffalo since it is one of the causative agents of bovine anaplasmosis. Previous studies have shown the worldwide distribution of A. bovis. However, most of these studies about its genetic diversity only focused on the rrs gene. In this study, DNA of A. bovis was detected in blood samples of cattle and goats in Xi’an city, China by nested-PCR. Near full-length rrs, groEL, and gltA genes were amplified successfully from the positive samples. Genetic analysis showed that specific genetic marker (an insertion and a deletion) was found in the rrs sequences in some strains, as well as clone 88 from monkeys in previous study. Phylogenetic analysis based on the rrs, groEL, and gltA genes revealed that A. bovis circulating in Xi’an exhibited great genetic diversity. Our results also indicated that variants outside China presented geographic clustering, and all A. bovis isolates based on the groEL or gltA gene also showed a host origin clustering. Also of note was that the phylogenetic analyses of the groEL and gltA genes suggested that both frequent dispersals over long distances in recent years and local adaptation over long evolutionary timescales played important roles in the distribution and evolution of A. bovis in China. Finally, a potential recombination event in the genome of Zhouzhi-cattle-10 based on inconsistent positions in the groEL and gltA trees was also observed. These results also reinforce the need for assessing the pathogenicity to humans of A. bovis variants with specific marker in the rrs gene.

Phenotypic and Genotypic Characterization of Streptomycin-Resistant Multidrug-Resistant Mycobacterium tuberculosis Clinical Isolates in Southern China.

Streptomycin (STR) is the first antibiotic used in the treatment of tuberculosis (TB) and the earliest antituberculosis drug with acquired resistance developed by Mycobacterium tuberculosis. The high prevalence of such resistance in many parts of the world limits its use for treating multidrug-resistant (MDR) TB. The aims of this study are to characterize of mutations in rpsL, rrs, and gidB genes in MDR M. tuberculosis isolates originating from southern China and to investigate possible relationship between mutations and strain genotypes for precise diagnosis and treatment. Sequences of rpsL, rrs, and gidB genes and the resistance profiles were analyzed for 218 MDR M. tuberculosis isolates. Our study showed that 68.35% of MDR M. tuberculosis isolates were resistant to STR and 89.91% of STR-resistant (STRR) isolates were Beijing lineage strains. Mutations were observed in STRR MDR M. tuberculosis isolates at the following rates: 72.48% in rpsL, 36.91% in rrs, and 15.44% in gidB. Compared with the phenotypic data, the combination of mutations in rpsL, rrs, and gidB has sensitivity and specificity of 96.64% and 100.00%, respectively. The most common mutations in STRR isolates were rpsL128,263 and rrs514,1401, of which rpsL128 showed association with Beijing lineage (p < 0.001). It is noteworthy that a1401g mutation was present in rrs, while MDR M. tuberculosis isolates were resistant to both STR and amikacin. Twenty two novel mutations were found in STRR isolates. These findings could be helpful to develop rapid molecular diagnostic methods and understand STR resistance in China for developing TB precision medicine and disturbance of drug-resistant TB transmission.

Bosea caraganae sp. nov. a new species of slow-growing bacteria isolated from root nodules of the relict species Caragana jubata (Pall.) Poir. originating from Mongolia.

Two Gram-stain-negative strains, RCAM04680T and RCAM04685, were isolated from root nodules of the relict legume Caragana jubata (Pall.) Poir. originating from the south-western shore of Lake Khuvsgul (Mongolia). The 16S rRNA gene (rrs) sequencing data showed that these novel isolates belong to the genus Bosea and are phylogenetically closest to the type strains Bosea lathyri LMG 26379T, Bosea vaviloviae LMG 28367T, Bosea massiliensis LMG 26221T and Bosea lupini LMG 26383T (the rrs-similarity levels were 98.7-98.8 %). The recA gene of strain RCAM04680T showed the highest sequence similarity to the type strain B. lupini LMG 26383T (95.4 %), while its atpD gene was closest to that of B. lathyri LMG 26379T (94.4 %). The ITS, dnaK and gyrB sequences of this isolate were most similar to the B. vaviloviae LMG 28367T (86.8 % for ITS, 90.4 % for the other genes). The most abundant fatty acid was C18 : 1ω7c (40.8 %). The whole genomes of strains RCAM04680T and RCAM04685 were identical (100 % average nucleotide identity). The highest average nucleotide identity value (82.8 %) was found between the genome of strain RCAM04680T and B. vaviloviae LMG 28367T. The common nodABC genes required for legume nodulation were absent in both strains; however, some other symbiotic nol, nod, nif and fix genes were detected. Based on the genetic study, as well as analyses of the whole-cell fatty acid compositions and phenotypic properties, a new species, Boseacaraganae sp. nov. (type strain RCAM04680T (=LMG 31125T), is proposed.

Molecular epidemiology of Leptospira interrogans in Rattus norvegicus in Hanoi, Vietnam

Leptospirosis is a zoonotic disease that is caused by pathogenic spirochaetes of Leptospira spp. and it has become a public health concern in urban localities in the tropics. Rats are important reservoir animals for the transmission of leptospirosis in urban areas. Leptospirosis is considered endemic in Vietnam. However, information on the causative Leptospira genotypes and serotypes in the country is limited. We investigated the carrier status of Leptospira spp. in rats captured in Hanoi by culturing and DNA detection. Isolates were characterized using a serological method and multiple-locus variable-number tandem repeat analysis (MLVA).We captured 144 rats (1 Rattus argentiventer, 135 R. norvegicus, and 8 R. rattus) and obtained 17 L. interrogans, determined by rrs sequencing, from R. norvegicus (12.6%). Sixteen of the isolates were serogroup Bataviae. Five of the 16 isolates exhibited an MLVA type identical to that of the serovar Bataviae reference strain Van Tienen, while there were nine repeats for the other 11 isolates at VNTR31 compared with the reference strain. The remaining isolate grew poorly, and we were unable to determine its serogroup. However, it had an MLVA type matching those of serogroup Pomona strains isolated from R. norvegicus in Japan. Three different flaB sequences were detected in 23 out of 81 R. norvegicus kidney tissue samples (28.4%) using nested PCR followed by DNA sequencing. Two of the sequences were identical with those of serogroups Bataviae and Pomona, and no strain with another sequence was detected in the present study.The present study reveals a high prevalence rate of L. interrogans among R. norvegicus in Hanoi, Vietnam, indicating a potential risk of rat-borne leptospirosis in the area. The present study also demonstrates that a fastidious L. interrogans strain circulates among rats and that molecular detection is crucial in facilitating the accurate determination of reservoir animals.

Leptospirainterrogans serogroup Pomona serovar Kennewicki infection in two sheep flocks with acute leptospirosis in Uruguay.

Acute leptospirosis is an infrequent disease in sheep that can cause jaundice, haemolysis, haemoglobinuria, hepatitis and nephritis. In most reports the diagnoses have been made by clinical, pathological or serological evidence without isolation or direct identification of the agent. Here, we report one confirmed and one presumptive outbreak of acute leptospirosis in suckling lambs from two unrelated sheep farms in Uruguay with mortalities of 9/60 (15%) and 9/163 (5.5%) lambs. Both outbreaks occurred in Sep-Oct 2017 after heavy rainfall and flooding events. The main gross and histologic pathological findings in two autopsied lambs, one from each farm, included severe diffuse jaundice, haemoglobinuria, acute necrotizing hepatitis with cholestasis and interstitial nephritis. Leptospirainterrogans serogroup Pomona serovar Kennewicki was isolated from sheep in both flocks and the same genotype was identified directly in clinical samples from infected animals, including one of the deceased lambs subjected to autopsy, by amplification and partial sequencing of rrs and secY genes. This serovar has recently been identified in infected cattle and humans in Uruguay. The impact of Leptospira spp. infection in ovine health, and the epidemiologic role of sheep as reservoirs of leptospirosis for humans and animals need further investigation.

Clay beads as artificial trapping matrices for monitoring bacterial distribution among urban stormwater infiltration systems and their connected aquifers.

Stormwater infiltration systems (SIS) have been developed to limit surface runoff and flooding in urban areas. The impacts of such practices on the ecological and biological quality of groundwater ecosystems remain poorly studied due to the lack of efficient methodologies to assess microbiological quality of aquifers. In the present study, a monitoring method based on the incubation of artificial matrices (clay beads) is presented to evaluate microbial biomass, microbial activities, and bacterial community structure. Four microbial variables (biomass, dehydrogenase and hydrolytic activities, bacterial community structures) were measured on clay beads incubated in three urban water types (stormwater surface runoffs, SIS-impacted and non-impacted groundwaters) for six SIS. Analyses based on next-generation sequencing (NGS) of partial rrs (16S rRNA) PCR products (V5-V6) were used to compare bacterial community structures of biofilms on clay beads after 10days of incubation with those of waters collected from the same sampling points at three occasions. Biofilm biomass and activities on clay beads were indicative of nutrient transfers from surface to SIS-impacted groundwaters. Biofilms allowed impacts of SIS on groundwater bacterial community structures to be determined. Although bacterial communities on clay beads did not perfectly match those of waters, clay beads captured the most abundant bacterial taxa. They also captured bacterial taxa that were not detected in waters collected at three occasions during the incubation, demonstrating the integrative character of this approach. Monitoring biofilms on clay beads also allowed the tracking of bacterial genera containing species representing health concerns.

Molecular characterization of Haemaphysalis longicornis-borne rickettsiae, Republic of Korea and China

Haemaphysalis longicornis, the cattle tick or bush tick, has an extended distribution throughout Asia and the Pacific region, including China, Russia, the Republic of Korea (ROK), Japan, Australia, New Zealand, and the South Pacific islands. It is an obligate ectoparasite found commonly on medium to large sized wild and domestic animals, with humans as an accidental host. Haemaphysalis longicornis transmits a number of pathogens, including severe fever with thrombocytopenia syndrome and tick-borne encephalitis viruses, bacteria, helminths, and protozoans, that impact on veterinary (wild and domestic animals) and human health. Surveys of rickettsial pathogens associated with H. longicornis from China, the ROK, and Japan have resulted in the discovery of more than 35 incompletely characterized molecular isolates of Rickettsia. In response to the increased global threat of tick-borne rickettsial diseases, H. longicornis collected in the ROK and China were assessed in our laboratory and two additional Rickettsia spp. isolates (ROK-HL727 and XinXian HL9) were identified. These agents were fully characterized by multilocus sequence typing using partial gene fragment sequences of rrs, gltA, ompA, ompB, and sca4. Phylogenetic comparisons of these Rickettsia isolates with known Rickettsia species and other molecular isolates identified from H. longicornis were performed to better understand their interrelationships. Phylogenetic analysis of the sequences from these 5 gene fragments showed that ROK-HL727 was closely related to rickettsial isolates of H. longicornis previously reported from China, the ROK and Japan, but distinct from any currently recognized Rickettsia species. It therefore qualifies genetically as a new species, introduced herein as Candidatus Rickettsia longicornii. The XinXian-HL9 isolate detected from China was determined to be genetically similar to the human pathogen Rickettsia heilongjiangensis. People living and working in areas where H. longicornis is endemic should be aware of the potential for rickettsial diseases.

Taxonomically Different Co-Microsymbionts of a Relict Legume, Oxytropis popoviana, Have Complementary Sets of Symbiotic Genes and Together Increase the Efficiency of Plant Nodulation.

Ten rhizobial strains were isolated from root nodules of a relict legume Oxytropis popoviana Peschkova. For identification of the isolates, sequencing of rrs, the internal transcribed spacer region, and housekeeping genes recA, glnII, and rpoB was used. Nine fast-growing isolates were Mesorhizobium-related; eight strains were identified as M. japonicum and one isolate belonged to M. kowhaii. The only slow-growing isolate was identified as a Bradyrhizobium sp. Two strains, M. japonicum Opo-242 and Bradyrhizobium sp. strain Opo-243, were isolated from the same nodule. Symbiotic genes of these isolates were searched throughout the whole-genome sequences. The common nodABC genes and other symbiotic genes required for plant nodulation and nitrogen fixation were present in the isolate Opo-242. Strain Opo-243 did not contain the principal nod, nif, and fix genes; however, five genes (nodP, nodQ, nifL, nolK, and noeL) affecting the specificity of plant-rhizobia interactions but absent in isolate Opo-242 were detected. Strain Opo-243 could not induce nodules but significantly accelerated the root nodule formation after coinoculation with isolate Opo-242. Thus, we demonstrated that taxonomically different strains of the archaic symbiotic system can be co-microsymbionts infecting the same nodule and promoting the nodulation process due to complementary sets of symbiotic genes.

Molecular characterization of mutations associated with resistance to second-line tuberculosis drug among multidrug-resistant tuberculosis patients from high prevalence tuberculosis city in Morocco

BackgroundThe emergence of extensively drug-resistant tuberculosis (XDR-TB) has raised public health concern for global TB control. Although multi drug-resistant tuberculosis (MDR- TB) prevalence and associated genetic mutations in Morocco are well documented, scarce information on XDR TB is available. Hence, the evaluation of pre-XDR and XDR prevalence, as well as the mutation status of gyrA, gyrB, rrs, tlyA genes and eis promoter region, associated with resistance to second line drugs, is of great value for better management of M/XDR TB in Morocco.MethodsDrug susceptibility testing (DST) was performed by the proportional method for first line drugs, and then the selected MDR isolates were tested for second line drugs (Ofloxacin, Kanamycin, Amikacin and Capreomycin). Along with DST, all samples were subjected to rpoB, katG and p-inhA mutation analysis by PCR and DNA sequencing. MDR isolates as well as 30 pan-susceptible strains were subjected to PCR and DNA sequencing of gyrA, gyrB, rrs, tlyA genes and eis promoter, associated with resistance to fluoroquinolones and injectable drugs.ResultsAmong the 703 analysed strains, 12.8% were MDR; Ser531Leu and Ser315Thr being the most common recorded mutations within rpoB and katG genes associated with RIF and INH resistance respectively. Drug susceptibility testing for second line drugs showed that among the 90 MDR strains, 22.2% (20/90) were resistant to OFX, 2.22% (2/90) to KAN, 3.33% (3/90) to AMK and 1.11% (1/90) to CAP. Genotypic analysis revealed that 19 MDR strains harbored mutations in the gyrA gene; the most recorded mutation being Asp91Ala accounting for 47.6% (10/21), and 2 isolates harbored mutations in the promoter region of eis gene. No mutation was found in gyrB, rrs and tlyA genes. Moreover, none of the pan-susceptible isolates displayed mutations in targeted genes.ConclusionMost of mutations associated with SLD resistance occurred in gyrA gene (codons 90-94) and eis promoter region. These findings highlight the impact of mutations in gyrA on the development of fluroquinolones resistance and provide the first estimates of the proportion of pre-XDR-TB among MDR-TB cases in Morocco.

Molecular characterization of novel mosquito-borne Rickettsia spp. from mosquitoes collected at the Demilitarized Zone of the Republic of Korea.

Rickettsiae are associated with a diverse range of invertebrate hosts. Of these, mosquitoes could emerge as one of the most important vectors because of their ability to transmit significant numbers of pathogens and parasites throughout the world. Recent studies have implicated Anopheles gambiae as a potential vector of Rickettsia felis. Herein we report that a metagenome sequencing study identified rickettsial sequence reads in culicine mosquitoes from the Republic of Korea. The detected rickettsiae were characterized by a genus-specific quantitative real-time PCR assay and sequencing of rrs, gltA, 17kDa, ompB, and sca4 genes. Three novel rickettsial genotypes were detected (Rickettsia sp. A12.2646, Rickettsia sp. A12.2638 and Rickettsia sp. A12.3271), from Mansonia uniformis, Culex pipiens, and Aedes esoensis, respectively. The results underscore the need to determine the Rickettsia species diversity associated with mosquitoes, their evolution, distribution and pathogenic potential.

Bacteriome genetic structures of urban deposits are indicative of their origin and impacted by chemical pollutants

Urban activities generate surface deposits over impervious surfaces that can represent ecological and health hazards. Bacteriome genetic structures of deposits washed off during rainfall events, over an urban industrial watershed, were inferred from 16 S rRNA gene (rrs) sequences generated by high throughput sequencing. Deposits were sampled over a 4 year-period from a detention basin (DB). Major shifts, matching key management practices, in the structure of these urban bacteriomes, were recorded. Correlation analyses of rrs similarities between samples and their respective concentrations in chemical pollutants, markers of human fecal contaminations (HF183) and antimicrobial resistances (integrons), were performed. Harsher environmental constraints building up in the older deposits led to an increase number of rrs reads from extremophiles such as Acidibacter and Haliangium. Deposits accumulating in the decantation pit of the DB showed an increase in rrs reads from warm blooded intestinal tract bacteria such as Bacteroides and Prevotella. This enrichment matched higher concentrations of Bacteroides HF183 genotypes normally restricted to humans. Bacteriomes of urban deposits appeared good indicators of human-driven environmental changes. Their composition was found representative of their origin. Soil particles and rain appeared to be major contributors of the inferred bacterial taxa recovered from recent deposits.

Rouxiella badensis sp. nov. and Rouxiella silvae sp. nov. isolated from peat bog soil and emendation description of the genus Rouxiella.

Four bacterial strains isolated from peat bog soil or swampy meadow in Baden-Württemberg (Germany) and found to have rrs sequences close to that of Rouxiella chamberiensis were compared to this species by using multi-locus sequence analysis and phenotypic tests. The four strains constituted two discrete groups (referred to as the Baden and the Silva groups) belonging to the genus Rouxiella. These groups differed in their ability to grow at 37 °C, reduce nitrate into nitrite, and to produce acid from several carbohydrates. Two novel species are, therefore, proposed: Rouxiella badensis sp. nov. for the Baden group (type strain, 323T=CIP 111153T=DSM 100043T) and Rouxiella silvae for the Silva group (type strain, 213T=CIP 111154T=DSM 103735T). The definition of the genus Rouxiellahas also been emended in order to take these two novel species into account.