folP Gene Research Articles

A Hunt for the Resistance of Haemophilus influnezae to Beta-Lactams.

Infections due to Haemophilus influnezae require prompt treatment using beta-lactam antibiotics. We used a collection of 81 isolates obtained between 1940 and 2001 from several countries. Whole genome sequencing showed the high heterogeneity of these isolates but allowed us to track the acquisition of beta-lactamase, which was first detected in 1980. Modifications of the ftsI gene encoding the penicillin-binding protein 3, PBP3, also involved in resistance to beta-lactams, appeared in 1991. These modifications (G490E, A502V, R517H, and N526K) were associated with resistance to amoxicillin that was not relieved by a beta-lactamase inhibitor (clavulanic acid), but the isolates retained susceptibility to third-generation cephalosporins (3GC). The modeling of the PBP3 structure suggested that these modifications may reduce the accessibility to the PBP3 active site. Other modifications appeared in 1998 and were associated with resistance to 3GC (S357N, M377I, S385T, and L389F). Modeling of the PBP3 structure suggested that they lie near the S379xN motif of the active site of PBP3. Overall resistance to amoxicillin was detected among 25 isolates (30.8%) of this collection. Resistance to sulfonamides was predicted by a genomic approach from the sequences of the folP gene (encoding the dihydropteroate synthase) due to difficulties in interpreting phenotypic anti-microbial testing and found in 13 isolates (16.0%). Our data suggest a slower spread of resistance to sulfonamides, which may be used for the treatment of H. influnezae infections. Genomic analysis may help in the prediction of antibiotic resistance, inform structure-function analysis, and guide the optimal use of antibiotics.

Characterization of MultidrugResistant serogroup 19 Streptococcus pneumoniae isolated from healthy children below 5 years of age in Indonesia.

We investigated the resistance genes, pilus islets, biofilm formation ability and sequence types of multidrug-resistant Streptococcus pneumoniae (MDRSP) isolated from healthy children below 5 years of age in Indonesia. In all, 104 archived MDRSP isolates from previous carriage studies in Indonesia in 2016-2019 were screened for the presence of antibiotic resistance genes and the rrgC (pilus islet 1) and pitB (pilus islet 2) genes. Multilocus sequence typing and biofilm formation were determined by PCR sequencing and the ability of cells to adhere to the walls, respectively. Results have shown that the mefA, ermB and tetM genes were found in 93, 52 and 100 % of MDRSP isolates, respectively. Insertions of arginine, proline and Ile-100-Leu were the most common mutations in the folA and folP genes. Pilus islets 1 and 2 were discovered in 93 and 82 % of MDRSP isolates, respectively. The MDRSP isolates showed no biofilm formation ability (64 %), and 5 out of 10 strains of MDRSP strains were ST1464. This finding can be used to provide further considerations in implementing and monitoring pneumococcal vaccination in Indonesia.

Novel mutations found in Mycobacterium leprae DNA repair gene nth from central India

IntroductionThe importance of DNA repair enzymes in maintaining genomic integrity is highlighted by the hypothesis that DNA damage by reactive oxygen/nitrogen species produced inside the host cell is essential for the mutagenesis process. Endonuclease III (Nth), formamidopyrimide (Fpg) and endonuclease VIII (Nei) DNA glycosylases are essential components of the bacterial base excision repair process. Mycobacterium leprae lost both fpg/nei genes during the reductive evolution event and only has the nth (ML2301) gene. This study aims to characterize the mutations in the nth gene of M. leprae strains and explore its correlation with drug-resistance. MethodA total of 91 M. leprae positive DNA samples extracted from skin biopsy samples of newly diagnosed leprosy patients from NSCB Hospital Jabalpur were assessed for the nth gene as well as drug resistance-associated loci of the rpoB, gyrA and folP1 genes through PCR followed by Sanger sequencing. ResultsOf these 91 patients, a total of two insertion frameshift mutations, two synonymous and seven nonsynonymous mutations were found in nth in seven samples. Sixteen samples were found to be resistant to ofloxacin and one was found to be dapsone resistant as per the known DRDR mutations. No mutations were found in the rpoB region. Interestingly, none of the nth mutations were identified in the drug-resistant associated samples. ConclusionThe in-silico structural analysis of the non-synonymous mutations in the Nth predicted five of them were to be deleterious. Our results suggest that the mutations in the nth gene may be potential markers for phylogenetic and epidemiological studies.

Genotypic characterization of Mycobacterium leprae strains resistant to rifampicin and ofloxacin in three health districts in Chad

Antimicrobial surveillance and identification of the genetic basis of antimicrobial resistance provides important information to optimize patient care. The present study was an analytical cross-sectional study aimed at determining the prevalence of rifampicin and ofloxacin resistance genes among Mycobacterium leprae strains in three health districts in Chad.
 The determination of the folP1, rpoB and gyrA resistance genes was carried out by PCR-RLEP and confirmed by sequencing from 80 biopsy samples taken from patients with multibacillary leprosy, including 12 relapsed patients and 68 new cases. In the whole cohort, 1/80 (1.2%) showed resistance to rifampicin and 1/80 (1.2%) to ofloxacin. No mutations were detected for dapsone. The presence of M. leprae mutation associated with rifampicin resistance was observed in a relapsed patient and the mutation associated with ofloxacin resistance was observed in a patient with multibacillary leprosy who had not been sensitized by ofloxacin but should have used other quinolones. Both mutant strains revealed the emergence of secondary resistance.
 This study, the first to highlight the emergence of resistance to rifampicin and ofloxacin in Chad. It raises the need to implement a robust surveillance system to detect resistance of Mycobacterium leprae in Chad and even in Central Africa.
 Keywords: Mycobacterium leprae, resistance, Chad

Molecular detection of some Gram-negative bacterial species using folp gene sequences

In this study, the folp gene encoding DHPS was tested as a possible alternative phylogenetic marker for more closely related Gram-negative bacterial species. In this new method, 854 bp were implemented for classification instead of 1435 mostly used in 16S gene detection. Phylogenetic analysis was performed based on DNA sequences obtained from the GenBank including the most important Gram-negative bacterial species. The 16S rRNA-based tree openly portrayed three distinct clusters where cluster 1

Molecular Detection of some Staphylococcal species with the newly developed folp gene sequences

16SrRNA gene sequencing, universally accepted for bacterial identification could not effectively discriminate some closely related Staphylococcal species. For this reason, for bacterial species identification, other DNA sequencing genes are required. In this study, folp gene, encoding DHPS, was tested as a possible and alternative phylogenetic marker for more closely related Staphylococcal species. In this new method, 813 bp were implemented for classification instead of 1447 of 16S rRNA mostly used. Phylogenetic analysis was performed based on DNA sequences obtained from the GenBank including most important Staphylococcal species. As a result, the 16S rRNA-based tree clearly delineated 7 distinct clusters with high bootstrap values (1000) at distance adjusted to 5. Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus hominis, Staphylococcus haemolyticus and Staphylococcus saprophyticus were identified in separate clusters. However, 2 mixed clusters were resolved including Staphylococcus epidermidis and Staphylococcus warneri in one cluster, while Staphylococcus pasteuri and Staphylococcus warneri in the other one. Comparatively, the folp gene-based tree yielded 9 clusters with at distance adjusted to 5 and 1000 bootstrap value. Staphylococcus haemolyticus, Staphylococcus hominis, Staphylococcus haemolyticus, Staphylococcus ludunensis, Staphylococcus epidermidis, Staphylococcus aureus, Staphylococcus saprophyticus, Staphylococcus pasteuri were resolved in separate clusters, however, Staphylococcus warneri and Staphylococcus epidermidis were identified in one mixed cluster. In addition, DNA-DNA relatedness studies indicated high sequence divergence of folp gene exhibiting 61.58-96.48 % interspecies homology compared to 16S rRNA with sequences similarities of 97.09-99.32 %. At the intraspecies level, the nucleotide substitution rates were ranged between 0-25%, mostly at low level for most Staphylococcal species compared to 98.65-99.93 identified in 16S rRNA gene. Moreover, in blind tests, this method was able for correct identification of 13 bacteria isolates of different Staphylococcal species. As a conclusion, folp gene sequences provide better resolution compared to the 16S rRNA gene sequences for both interspecies and interspecies DNA analysis of Staphylococci.

Lactobacillus Strains for Vegetable Juice Fermentation-Quality and Health Aspects.

Vegetable juices are new carrier variants for beneficial bacteria, representing an alternative to dairy-fermented products, especially for vegan, strict vegetarian, or allergic consumers. The aim of this study was to characterize several Romanian native lactic acid bacteria (LAB) strains to select valuable nutritional and probiotic strains for vegetable juice fermentation. Nineteen LAB strains were analyzed for antibiotic susceptibility (disc-diffusion method), the presence of antibiotic resistance genes, the presence of functional genes. and the production of organic acids by HPLC. Antibiotic resistant strains were observed only with ampicillin (Amp10) and kanamycin (K30), 79% and 32%, respectively, with results partially confirmed by molecular analysis. Multiplex PCR revealed the presence of LBA1272, dltD, folP, agl, α-amy, malL, and ribA genes, related to stress resistance, starch metabolism, and production of vitamins, except for folK. HPLC analyses were performed on beet roots (SF), tomato (TM), and a mixture of carrots, celery, and beet (MTS) juices. High values of lactic acid were recorded in all cases of LAB fermentation (5034-14,176 µg/mL). The maximum values recorded for acetic acid did not exceed 2.5 mg/mL having a positive influence on the product's taste.

Severe skin and subcutaneous pythiosis in China: Metagenomic identification and characterization of Pythium insidiosum.

Pythium insidiosum is a rare fungus-like pathogen that is known to cause pythiosis in mammals with high morbidity and mortality. Identification of the pathogen is essential for timely treatment and rational use of antibiotics. However, Pythium insidiosum is difficult to detect via conventional microbiological tests. The current gold standard is polymerase chain reaction, which is lacking in most hospitals since human pythiosis is rare in China. In this study, we used metagenomic Next-Generation Sequencing and identified Pythium insidiosum in a 56-year-old Chinese male who was hospitalized due to severe edema in the right lower limb with scattered darkening indurations. The patient had a history of cirrhosis and occupational exposure to swamp water. Serological level of immune biomarkers indicated immunodeficiency, and Proteinase 3-Anti-Neutrophil Cytoplasmic Antibody was positive. Surgical incision of the lesions revealed radiating and reticular cutaneous ulcers. Microbial infections were suspected but conventional tests failed to discover the etiology. Empirical use of penicillin, vancomycin, and ceftriaxone had no effect. As a result, the peripheral blood and tissue biopsies were sent for metagenomic Next-Generation Sequencing, which reported Pythium insidiosum. This finding was corroborated by pathological staining, whole-genome sequencing, and internal transcribed spacer sequencing. Notably, antifungal treatment was ineffective, but the patient responded well to oral trimethoprim–sulfamethoxazole, which may be due to the folp gene found in Pythium insidiosum genome. Our study prompts future studies to determine the optimal treatment of skin pythiosis.

Approach of Persisters Relapse in a Smear-negative Leprosy after Second MB-MDT Completion: An Arduous Case Report

Introduction: Many infectious disease intervention programs, including leprosy, are cautious about the establishment of relapse and drug resistance. This case report reports on a male patient who has had two relapses and had undergone twice MB-MDT treatment as well.
 Case Report: A 31 year-old male, came with the main complaint of new hypopigmented patches appeared on the body since 4 months ago, which arose from his second MB-MDT treatment. He started his first MB-MDT treatment in 2019 and second MD-MDT in 2020. Slit skin smear yielded negative results and histopathological examination showed no signs of leprosy. Serological anti-PGL-1 antibody tests yielded very high IgM (3.163) and normal IgG (2) levels. PCR amplification examination showed positive, but DNA sequencing targeting rpoB, folP, and gyrA genes showed no mutations. He was diagnosed with persisters relapse in a borderline-lepromatous leprosy and was treated with rifampicin 600 mg/day for 2 weeks; combination of minocycline 100 mg/ day, clarithromycin 500 mg/ day, and clofazimine 50 mg/ day for 6 months; and methylprednisolone 32 mg/ day for the neuritis. No new lesions appeared during current treatment and he is also showing gradual improvement of foot drop in his left leg.
 Discussion: The fact of a very high IgM levels after 2 courses of MB-MDT indicated that there is still bacteria that continued to induce antibody formation, thus supporting the persisters bacilli hypothesis in this case. Positive results of PCR amplification conclude that there is still M. leprae bacilli in skin tissues, which may be hidden in deeper tissues. We have to make a more proactive approach considering this was his second times of relapse, and knowing the fact that MB-MDT was consumed religiously in the two previous treatment.
 Conclusion: In an arduous case like this, proactive step must be taken. Whenever possible, the use of whole genome sequencing is strongly advised.
 Keywords: leprosy, persisters bacilli, relapse, drug resistance

Sensitivity of different DNA extraction methods and PCR to detect resistance in patients with leprosy stratified by the bacilloscopic index

IntroductionAntimicrobial resistance in leprosy is an emerging problem, and the quantitative impact of low bacilloscopic indexes (BIs) on the sensitivity of molecular tests is unknown. We aimed to evaluate the sensitivity of gene sequencing for the detection of mutations related to antimicrobial resistance in Mycobacterium leprae in patients with low BIs using an analytical model. MethodsPatients with leprosy were included and divided into two groups depending on their BIs (≥ 2+ and < 2+). The sensitivities of the two DNA extraction methods were compared after amplifying and sequencing the repetitive element (RLEP), folP1, rpoB and gyrA in M. leprae. ResultsWe included 56 patients with leprosy: 35 had BIs less than 2+ (22 had negative slit-skin smear [SSS] results) and 21 patients had BIs greater than or equal to 2+. The sensitivity of the amplification of the RLEP target and the gene sequencing of folP1, rpoB and gyrA was 50 to 70% lower in patients with a BI less than 2+ and was significantly reduced in patients with lower BIs for all targets (p < 0.001). One patient had a mutation in the folP1 gene, and 14 patients had mutations in the gyrA gene, but no mutations related to antimicrobial resistance were found. ConclusionsWe can conclude that the sensitivity of molecular tests is directly related to the BI, but these tests can still detect up to 20% of the targets in patients with BIs < 2+. New strategies to improve the sensitivity for detecting antimicrobial resistance in leprosy patients and reasonable clinical criteria for follow-up and the introduction of alternative treatments must be developed.

The genetic determinants of &lt;i&gt;Mycobacterium leprae&lt;/i&gt; resistance to antimicrobial drugs

The review is devoted to the appearance of resistance of a slowly developing disease leprosy to antimicrobial therapy (AMP), primarily recommended by the World Health Organization. The main danger of drug resistant leprosy is in the difficulty of identifying, since the causative agent of the disease is not cultivated on artificial media, and the methods for diagnosing drug resistance that are currently used take a long time. The drug resistance of the Mycobacterium leprae strain even to individual components of combination drug therapy result to the development of symptoms of the disease despite undergo anti-leprosy therapy, which in turn can cause the patient to become disabled. Currently, in the Russian Federation, there is no approved test for detecting Mycobacterium leprae DNA, and the determination of genetic determinants of resistance is carried out by sequencing genome regions determined by WHO recommendations: small gyrA, folP and rpoB genes loci. At the same time, modern studies in endemic regions reveal an increased level of Mycobacterium leprae strains resistant to individual components of combined drug therapy. The use of next generation sequencing (NGS) has made it possible to identify additional genetic determinants of leprosy resistance to the components of combination drug therapy. The current situation is relevant to antimicrobal drug resistance surveillance by using of quick identification systems for most frequent genetic resistance determinants of Mycobacterium leprae. The literature search was carried out using keywords in the Scopus, PubMed and RSCI databases.

Genomic investigation of a Streptococcus pneumoniae serotype 24F strain causing meningoencephalitis in Hong Kong

Pneumococcal conjugate vaccines (PCVs) successfully decreased the incidence of invasive pneumococcal disease in children. However, many countries have reported serotype replacement and a rebound in diseases from non-vaccine serotypes. Here, we report the genomic investigation of a Streptococcus pneumoniae strain M215 that caused severe meningoencephalitis in an infant in 2019. The strain was assigned to serotype 24F using the bioinformatic pipeline SeroBA and pneumococcal type specific anti-sera. The strain was resistant to cotrimoxazole from mutations in both folA and folP genes. It was susceptible to penicillin and other non-β-lactam antibiotics. Phylogenetically, it belongs to Global Pneumococcal Sequence Cluster (GPSC) 6 and multi-locus sequence type 162. A total of 38 virulence genes were detected in the genome of M215. Upon comparison of the profile of virulence genes, GPSC6 but not non-GPSC6 strains of serotype 24F and related serotypes were found to possess the major virulence determinant, pilus islet-1, comprising genes encoding sortases (srtB, srtC, srtD), pilus proteins (rrgA, rrgB and rrgC) and one transcriptional regulator (rlrA), which was previously described to be characteristic feature of international clones in the pre-PCV era. In our locality, this represented the first detection of serotype 24F and GPSC6/ST162 causing serious pneumococcal disease. The emergence of the non-vaccine serotype 24F GPSC6/ST162 lineage with molecular feature of high virulence is concerning and emphasizes the need for full characterization of strains causing severe disease.

Genetic characterization of co-trimoxazole non-susceptible Streptococcus pneumoniae isolates from Indonesia.

We investigated the genetic variation of folA and folP genes encoding dihydropteroate synthase (DHPS) and dihydrofolate reductase (DHFR) enzymes amongst trimethoprim/sulfamethoxazole (co-trimoxazole) non-susceptible Streptococcus pneumoniae isolated from the Indonesian population. Archived S. pneumoniae isolates were screened for the presence and analysis of folA and folP genes using the polymerase chain reaction sequencing method. We found that 80 % of co-trimoxazole non-susceptible isolates (n=30/39) showed a 6 bp insertion in the sulphonamide-binding site of DHPS. The Asp-92-Ala and Ile-100-Leu substitutions were more common on DHFR (42 %; 22/53) followed by the Asp-92–Ala, Glu-94–Asp and Ile-100–Leu substitutions (32 %; 17/53). The combination of the Ile-100–Leu substitution at the DHFR region and the 6 bp insertion was the most dominant combination among isolates having both folA and folP genes.

Equal rates of drug resistance in leprosy cases with relapse and recurrent/chronic Type 2 reaction: time to revise the guidelines for drug-resistance testing in leprosy?

Leprosy relapse/recurrence is a serious concern particularly in a leprosy-endemic nation such as India. It is believed that bacilli persisting even after multidrug therapy can cause relapse; recently, however, drug resistance as a cause for recurrences and chronic erythema nodosum leprosum (ENL) has been speculated. To study drug-resistance patterns in cases of leprosy relapse and chronic/recurrent (c/r)ENL. This cross-sectional study conducted over a period of 1 year included patients diagnosed as having leprosy relapse and those with c/rENL. Skin biopsy specimens were examined by conventional PCR for resistance testing for rifampicin, dapsone and ofloxacin, respectively targeting the rpoB, folP and gyrA genes of Mycobacterium leprae. In total, 61 patients (25 smear-negative) were included in the study. Of these, 37 were diagnosed as having leprosy relapse and 24 as having c/rENL. Drug resistance to at least one drug was identified in 10 cases (16.4%). Rates of drug resistance were 5.4% (2 of 37) for dapsone, 10.8% (4 of 37) for rifampicin and 2.7% (1 of 37) for ofloxacin among cases of relapse, whereas it was 12.5% (3 of 24) and 8.3% (2 of 24) for dapsone and rifampicin respectively among those with c/rENL. Multidrug resistance was seen in 3.3% patients (2 of 61). Drug-resistance rate among those with c/rENL was almost equalled that of relapse. Smear-negative leprosy relapse cases also had resistance to bactericidal drugs. These findings call for modifications in criteria for testing under leprosy drug-resistance surveillance and all cases of relapse and those with recalcitrant c/rENL should be tested.

Mycobacterium leprae folP1 gene mutations among dapsone resistant patients in Sudan

Objectives Leprosy is a chronic disease that may cause irreparable deformities. Early diagnosis and proper treatment are crucial. This study examines the reliability of molecular diagnostic methods and their use in monitoring the presence of mutations in the Mycobacterium leprae folP1 gene as an indicator for dapsone resistance among Sudanese leprosy patients. Methods Ninety one skin slit smears were collected from clinically suspected leprosy patients. Modified ZN stain was used as the routine method for diagnosis and as a means to follow-up the response to dapsone therapy among leprosy patients. Two sets of specific primers were used for detecting M. leprae and for amplification of the folP1 gene, followed by sequencing the PCR product. Samples from patients with persistently positive ZN smears were suspected to have dapsone resistance and were therefore examined to study mutations at amino acid codon positions 53′ and 55′ of the folP1 gene. Results Of 91 smears, only 32 (35.2%) were found positive by ZN stain, while 50 (54.9%) were positive by PCR, showing PCR to be a more sensitive diagnostic method compared to ZN. Of the 32 ZN positive smears, five were found repeatedly positive by ZN after completion of dapsone treatment. Those five samples were subjected to amplification of the folP1 gene and the sequence analysis of the folP1 gene showed various mutations. Three samples showed dual mutations that were previously reported; while two samples demonstrated newly discovered, unreported mutations. The impact of such mutations should be considered in future studies of dapsone therapeutic failure in the region. Conclusions The results indicated that M. leprae strains circulating among Sudanese patients harbor mutations previously reported at position 55 (Ser) and position 53 (Glu). Further research is required to elucidate the effect of other mutations.

Integrative Analysis of Whole Genome Sequencing and Phenotypic Resistance Toward Prediction of Trimethoprim-Sulfamethoxazole Resistance in Staphylococcus aureus.

As whole genome sequencing is becoming more accessible and affordable for clinical microbiological diagnostics, the reliability of genotypic antimicrobial resistance (AMR) prediction from sequencing data is an important issue to address. Computational AMR prediction can be performed at multiple levels. The first-level approach, such as simple AMR search relies heavily on the quality of the information fed into the database. However, AMR due to mutations are often undetected, since this is not included in the database or poorly documented. Using co-trimoxazole (trimethoprim-sulfamethoxazole) resistance in Staphylococcus aureus, we compared single-level and multi-level analysis to investigate the strengths and weaknesses of both approaches. The results revealed that a single mutation in the AMR gene on the nucleotide level may produce false positive results, which could have been detected if protein sequence analysis would have been performed. For AMR predictions based on chromosomal mutations, such as the folP gene of S. aureus, natural genetic variations should be taken into account to differentiate between variants linked to genetic lineage (MLST) and not over-estimate the potential resistant variants. Our study showed that careful analysis of the whole genome data and additional criterion such as lineage-independent mutations may be useful for identification of mutations leading to phenotypic resistance. Furthermore, the creation of reliable database for point mutations is needed to fully automatized AMR prediction.

Characterization of Pneumococcal Colonization Dynamics and Antimicrobial Resistance Using Shotgun Metagenomic Sequencing in Intensively Sampled South African Infants.

Background: There remains a significant proportion of deaths due to pneumococcal pneumonia in infants from low- and middle-income countries despite the marginal global declines recorded in the past decade. Monitoring changes in pneumococcal carriage is key to understanding vaccination-induced shifts in the ecology of carriage, patterns of antimicrobial resistance, and impact on health. We longitudinally investigated pneumococcal carriage dynamics in PCV-13 vaccinated infants by collecting nasopharyngeal (NP) samples at 2-weekly intervals from birth through the first year of life from 137 infants. As a proof of concept, 196 NP samples were retrieved from a subset of 23 infants to explore strain-level pneumococcal colonization patterns and associated antimicrobial-resistance determinants. These were selected on the basis of changes in serotype and antibiogram over time. NP samples underwent short-term enrichment for streptococci prior to total nucleic acid extraction and whole metagenome shotgun sequencing (WMGS). Reads were assembled and aligned to pneumococcal reference genomes for the extraction of pneumococcal and non-pneumococcal bacterial reads. Pneumococcal contigs were aligned to the Antibiotic Resistance Gene-ANNOTation database of acquired AMR genes. In silico pneumococcal capsular and multilocus sequence typing were performed.Results: Of the 196 samples sequenced, 174 had corresponding positive cultures for pneumococci, of which, 152 were assigned an in silico serotype. Metagenomic sequencing detected a single pneumococcal serotype in 85% (129/152), and co-colonization in 15% (23/152) of the samples. Twenty-two different pneumococcal serotypes were identified, with 15B/15C and 16F being the most common non-PCV13 serotypes, while 23F and 19A were the most common PCV13 serotypes. Twenty-six different sequence types (STs), including four novel STs were identified in silico. Mutations in the folA and folP genes, associated with cotrimoxazole resistance, were detected in 89% (87/98) of cotrimoxazole-non-susceptible pneumococci, as well as in the pbp1a and pbp2x genes, in penicillin non-susceptible ST705215B/15C isolates.Conclusions: Metagenomic sequencing of NP samples is a valuable culture-independent technique for a detailed evaluation of the pneumococcal component and resistome of the NP microbiome. This method allowed for the detection of novel STs, as well as co-colonization, with a predominance of non-PCV13 serotypes in this cohort. Forty-eight resistance genes, as well as mutations associated with resistance were detected, but the correlation with phenotypic non-susceptibility was lower than expected.

Activation of a Specific Gut Bacteroides-Folate-Liver Axis Benefits for the Alleviation of Nonalcoholic Hepatic Steatosis

A beneficial gut Bacteroides-folate-liver pathway regulating lipid metabolism is demonstrated. Oral administration of a Ganoderma meroterpene derivative (GMD) ameliorates nonalcoholic hepatic steatosis in the liver of fa/fa rats by reducing endotoxemia, enhancing lipid oxidation, decreasing de novo lipogenesis, and suppressing lipid export from the liver. An altered gut microbiota with an increase of butyrate and folate plays a causative role in the effects of GMD. The commensal bacteria Bacteroides xylanisolvens, Bacteroides thetaiotaomicron, Bacteroides dorei, and Bacteroides uniformis, which are enriched by GMD, are major contributors to the increased gut folate. Administration of live B.xylanisolvens reduces hepatic steatosis and enhances the folate-mediated signaling pathways in mice. Knockout of the folate biosynthetic folp gene in B.xylanisolvens blocks its folate production and beneficial effects. This work confirms the therapeutic potential of GMD and B.xylanisolvens in alleviating nonalcoholic hepatic steatosis and provides evidence for benefits of the gut Bacteroides-folate-liver pathway.

Evidence for Mycobacterium leprae Drug Resistance in a Large Cohort of Leprous Neuropathy Patients from India.

Resistance to anti-leprosy drugs is on the rise. Several studies have documented resistance to rifampicin, dapsone, and ofloxacin in patients with leprosy. We looked for point mutations within the folP1, rpoB, and gyrA gene regions of the Mycobacterium leprae genome predominantly in the neural form of leprosy. DNA samples from 77 nerve tissue samples were polymerase chain reaction (PCR)-amplified for M leprae DNA and sequenced for drug resistance-determining regions of genes rpoB, folP1, and gyrA. The mean age at presentation and onset was 38.2 ± 13.4 (range 14-71) years and 34.9 ± 12.6 years (range 10-63) years, respectively. The majority had borderline tuberculoid leprosy (53 [68.8%]). Mutations associated with resistance were identified in 6/77 (7.8%) specimens. Mutations seen were those associated with resistance to rifampicin, ofloxacin, and dapsone. All the six patients were drug-naive. The clinical and pathological manifestations in this group did not differ from the drug-sensitive group. This study highlights the occurrence of resistance to the standard multidrug therapy and ofloxacin in leprosy. Among the entire cohort, 1/77 (1.3%) showed resistance to rifampicin, 2/77 (2.6%) to dapsone, and 5/77 (6.4%) to ofloxacin. Six new patients showing infection by mutant strains indicated the emergence of primary resistance. Resistance to ofloxacin could be due to frequent use of quinolones for many bacterial infections. The results of the study indicate the need for development of a robust and strict surveillance system for detecting drug resistance in leprosy in India.

Report of a Leprosy case in Singapore: an age-old disease not to be forgotten in developed countries with low-prevalence settings

Introduction Leprosy is rarely reported in developed countries with low-prevalence settings. Its diagnosis may be missed due to its low frequency in non-endemic regions, as well as its long incubation period. The report describes an imported leprosy case of a healthcare worker in Singapore.Case presentation A Filipino nursing personnel presented with a persistent non-tender erythematous plaque over his right upper back for many years despite topical treatment. He had the lesion before coming to Singapore but decided to seek medical consultation only after the lesion progressed with new erythematous papules developing over his face, trunk and upper limbs. Punch biopsies of skin lesions revealed fite-positive bacilli, which were identified to be Mycobacterium leprae by GenoType LepraeDR v1 assay (Hain LifeScience, Germany). No mutation was detected at rpoB (rifampicin), gyrA (ofloxacin) and folP1 (dapsone) gene targets. He was started on multi-drug therapy and responded to the treatment. The only prolonged close contact he had was his housemate who was screened and given a single dose of rifampicin as chemoprophylaxis.Conclusion In non-endemic settings, awareness is crucial in diagnosing leprosy. The availability of molecular testing and multi-disciplinary management are essential in the confirmation and control of this disease of public health importance.