Dihydropteroate Synthase Gene Research Articles

High Frequency Mutations in pfdhfr and pfdhps of Plasmodium falciparum in Response to Sulfadoxine-Pyrimethamine: A Cross-Sectional Survey in Returning Chinese Migrants From Africa.

BackgroundSulfadoxine-pyrimethamine (SP) is recommended for intermittent preventive treatment in Africa against Plasmodium falciparum infection. However, increasing SP resistance (SPR) of P. falciparum affects the therapeutic efficacy of SP, and pfdhfr (encoding dihydrofolate reductase) and pfdhps (encoding dihydropteroate synthase) genes are widely used as molecular markers for SPR surveillance. In the present study, we analyzed single nucleotide polymorphisms (SNPs) of pfdhfr and pfdhps in P. falciparum isolated from infected Chinese migrant workers returning from Africa.MethodsIn total, 159 blood samples from P. falciparum-infected workers who had returned from Africa to Anhui, Shangdong, and Guangxi provinces were successfully detected and analyzed from 2017 to 2019. The SNPs in pfdhfr and pfdhps were analyzed using nested PCR. The genotypes and linkage disequilibrium (LD) were analyzed using Haploview.ResultsHigh frequencies of the Asn51Ile (N51I), Cys59Arg(C59R), and Ser108Asn(S108N) mutant alleles were observed, with mutation frequencies of 97.60, 87.43, and 97.01% in pfdhfr, respectively. A triple mutation (IRN) in pfdhfr was the most prevalent haplotype (86.83%). Six point mutations were detected in pfdhps DNA fragment, Ile431Val (I431V), Ser436Ala (S436A), Ala437Gly (A437G), Lys540Glu(K540E), Ala581Gly(A581G), Ala613Ser(A613S). The pfdhps K540E (27.67%) was the most predominant allele, followed by S436A (27.04%), and a single mutant haplotype (SGKAA; 62.66%) was predominant in pfdhps. In total, 5 haplotypes of the pfdhfr gene and 13 haplotypes of the pfdhps gene were identified. A total of 130 isolates with 12 unique haplotypes were found in the pfdhfr-pfdhps combined haplotypes, most of them (n = 85, 65.38%) carried quadruple allele combinations (CIRNI-SGKAA).ConclusionA high prevalence of point mutations in the pfdhfr and pfdhps genes of P. falciparum isolates was detected among Chinese migrant workers returning from Africa. Therefore, continuous in vitro molecular monitoring of Sulfadoxine-Pyrimethemine combined in vivo therapeutic monitoring of artemisinin combination therapy (ACT) efficacy and additional control efforts among migrant workers are urgently needed.

Dihydropteroate Synthase (DHPS) Gene Mutations in Human Pneumocystosis

Extraordinary journey began on June 18, 1981, the Centre for Disease Control and Prevention (CDC) in United States reported a cluster of Pneumocystis carinii pneumonia (now known as Pneumocystis jirovecii (P. jirovecii) pneumonia or Pneumocystis pneumonia in five gay men in Los Angeles.

Genetic diversity of Pneumocystis jirovecii isolates among Turkish population based on mitochondrial large subunit ribosomal RNA and dihydropteroate synthase gene typing.

Pneumocystis jirovecii (P. jirovecii) is an atypical fungus that can cause life-threatening pneumonia in immunocompromised patients. In this study, we investigated the mtLSU-rRNA and DHPS gene polymorphisms in P. jirovecii isolates from both hospital and autopsy cases.

Dihydropteroate Synthase (DHPS) Gene Mutations and Mitochondrial Large Subunit (mtLSU) rRNA Genotype Variations in Pneumocystis jirovecii Strains

Pneumocystis jirovecii is an atypical fungus that causes Pneumocystis pneumonia (PCP) in HIV/AIDS and immunocompromised patients. Antibiotics containing sulfa and sulfone groups are widely used in PCP prophylaxis and treatment. Especially, long-term use of trimethoprim sulfamethoxazole (TMP-SMX) is known to cause certain point mutations associated with drug resistance in the P.jirovecii dihydropteroate synthase (DHPS) gene. In addition, DHPS and mitochondrial large subunit (mtLSU) rRNA genotype characterization provides important data on the epidemiology of P.jirovecii. In this study, it was aimed to investigate the DHPS and mtLSU rRNA gene polymorphisms of P.jirovecii strains isolated from immunocompromised patients in Mersin University Hospital. In this study, 16 P.jirovecii positive samples, which isolated from 96 patients samples, between August 2016 and February 2018, were included. P.jirovecii mtLSU rRNA genotypes were determined by sequence analysis according to polymorphisms at the 85th and 248th nucleotide positions. Nested PCR and RFLP method was applied for mutation analysis of DHPS locus, 165th and 171st nucleotide positions. In the DHPS mutation analysis, 12/16 (75%) wild type (W165/W171) and 4/16 (25%) mutant type (M165/W171) were detected. Two mutant types belonged to HIV/AIDS positive patients with PCP and had a history of prophylaxis; the other 2 mutant types belonged to patients with colonization. In the study, a history of prophylaxis in 3 (19%) of the 16 patients were recorded, and mutant type was detected in these 2 of 3 patients. According to mtLSU-rRNA analysis, 3 different genotypes were obtained from 16 P.jirovecii isolates. In our region, genotype 2 (43.75%; n= 7) was the most common genotype, genotype 1 (37.5%; n= 6) was the second common and genotype 3 (18.75%; n= 3) was the least one. Genotype 4 was not detected in our region. When DHPS and mtLSU-rRNA were evaluated as multilocus, five different genotypes were observed. As a result, these findings provided important data on P.jirovecii epidemiology in our region and potential drug-resistant strains showed a risk of transmission in immunosuppressive patients. Multicenter studies involving more P.jirovecii isolates are needed to better define the epidemiology of P.jirovecii in our region and in our country.

The Drug Resistance of Plasmodium falciparum and P. vivax in Iran: A Review Article.

Background:One of the main obstacles to malaria control in the world has been the emergence of resistance in Plasmodium falciparum to chloroquine and other anti-malarial drugs. This study aimed to review studies in Iran on resistance in P. falciparum and P. vivax to drugs, and to reveal the mechanisms and molecular markers of resistance of these two species.Methods:The databases of PubMed, Scopus, Google Scholar, Magiran, and reputable Iranian journals were searched to find published studies on the resistance in P. falciparum and P. vivax to antimalarial drugs in Iran.Results:There is a significant relationship between resistance to chloroquine in P. falciparum and the emergence of K76T mutation in the P. falciparum chloroquine-resistance transporter gene in Iran. Resistance to sulfadoxine-pyrimethamine (SP) in P. falciparum is also significantly associated with the development of mutations in the dihydrofolate reductase and dihydropteroate synthase genes. Resistance to chloroquine in P. vivax has not been reported in Iran and it is used as a first-line treatment for P. vivax malaria.Conclusion:P. falciparum has become resistant to chloroquine in different regions of Iran and is not currently used to treat malaria. Besides, cases have emerged of P. falciparum resistance to SP in different parts of southern Iran, and SP is not administered alone for treating P. falciparum.

Sequencing and Phylogenetic of Dihydropteroate synthase (DHPS) gene in Pneumocystis jirovecii isolated from immunocompromised patients in Iraq

Sequencing and Phylogenetic of Dihydropteroate synthase (DHPS) gene in Pneumocystis jirovecii isolated from immunocompromised patients in Iraq

Increased prevalence of pfdhfr and pfdhps mutations associated with sulfadoxine\u2013pyrimethamine resistance in Plasmodium falciparum isolates from Jazan Region, Southwestern Saudi Arabia: important implications for malaria treatment policy

BackgroundDespite significant progress in eliminating malaria from the Kingdom of Saudi Arabia, the disease is still endemic in the southwestern region of the country. Artesunate plus sulfadoxine–pyrimethamine (AS + SP) has been used in Saudi Arabia since 2007 as a first-line treatment for uncomplicated Plasmodium falciparum malaria. This study aimed to investigate the prevalence of mutations associated with resistance to artemisinin and sulfadoxine–pyrimethamine (SP) resistance in P. falciparum parasites circulating in Jazan region, southwestern Saudi Arabia.MethodsA total of 151 P. falciparum isolates were collected between April 2018 and March 2019 from 12 of the governorates in Jazan region. Genomic DNA was extracted from dried blood spots and amplified using nested PCR. Polymorphisms in the propeller domain of the P. falciparum k13 (pfkelch13) gene and point mutations in the P. falciparum dihydrofolate reductase (pfdhfr) and dihydropteroate synthase (pfdhps) genes were identified by sequencing.ResultsNo mutations in the pfkelch13 propeller domain were found in any of the 151 isolates. However, point mutations in the pfdhfr and pfdhps genes were detected in 90.7% (137/151) of the isolates. The pfdhfr double mutations N51I + S108N (i.e. ACICNI haplotype) and triple mutations N51I + C59R + S108N (i.e. ACIRNI haplotype) were detected in 47% and 37.8% of the isolates, respectively. Moreover, the pfdhps single mutation at codon A437G and double mutations A437G + K540E (i.e. SGEAAI haplotype) were observed in 4.6% and 51.7% of the isolates, respectively. Interestingly, 23.8%, 25.1 and 12.6% of the isolates had quintuple, quadruple and triple mutated combined pfdhfr–pfdhps genotypes, respectively. Furthermore, significant associations were found between the prevalence of mutant haplotypes and the age, gender and nationality of the patients (P < 0.05).ConclusionThis study revealed a high prevalence of point mutations in the pfdhfr and pfdhps genes of P. falciparum isolates from Jazan region, with quintuple and quadruple mutant pfdhfr–pfdhps genotypes reported for the first time in Saudi Arabia and the Arabian Peninsula. Despite the absence of the pfkelch13 mutation in the isolates examined, the pfdhfr and pfdhps mutations undermine the efficacy of SP partner drug, thereby threatening the main falciparum malaria treatment policy in Saudi Arabia, i.e. the use of AS + SP. Therefore, the continuous molecular and in-vivo monitoring of ACT efficacy in Jazan region is highly recommended.

Surveillance of molecular markers for antimalarial resistance in Zambia: Polymorphism of Pfkelch 13, Pfmdr1 and Pfdhfr/Pfdhps genes

Antimalarial resistance is an inevitable feature of control efforts and a key threat to achieving malaria elimination. Plasmodium falciparum, the deadliest of several species causing human malaria, has developed resistance to essentially all antimalarials. This study sought to investigate the prevalence of molecular markers associated with resistance to sulfadoxine-pyrimethamine (SP) and artemether-lumefantrine (AL) in Southern and Western provinces in Zambia. SP is used primarily for intermittent preventive treatment during pregnancy, while AL is the first-line antimalarial for uncomplicated malaria in Zambia. Blood samples were collected from household members of all ages in a cross-sectional survey conducted during peak malaria transmission, April to May of 2017, and amplified by polymerase chain reaction (PCR). Amplicons were then analysed by high-resolution melt following PCR to identify mutations associated with SP resistance in the P. falciparum dihydrofolate reductase (Pfdhfr) and P. falciparum dihydropteroate synthase (Pfdhps) genes and lumefantrine resistance in the P. falciparum multi-drug resistance 1 (Pfmdr1) gene. Finally, artemether resistance was assessed in the P. falciparum Kelch 13 (PfK13) gene using nested PCR followed by amplicon sequencing. The results showed a high frequency of genotypic-resistant Pfdhps A437G (93.2%) and Pfdhfr C59R (86.7%), N51I (80.9%), and S108N (80.8%) of which a high proportion (82.4%) were quadruple mutants (Pfdhfr N51I, C59R, S108N +Pfdhps A437G). Pfmrd1 N86Y, Y186F, and D1246Y - NFD mutant haplotypes were observed in 41.9% of isolates. The high prevalence of quadruple dhps/dhfr mutants indicates strong antifolate drug pressure from SP or other drugs (e.g., co-trimoxazole). Three samples contained PfK13 mutations, two synonymous (T478 and V666) and one non-synonymous (A578S), none of which have been associated with delayed clearance. This suggests that artemisinin remains efficacious in Zambia, however, the moderately high prevalence of approximately 40% Pfmdr1 NFD mutations calls for close monitoring of AL.

Clinical characteristics and prevalence of dihydropteroate synthase gene mutations in Pneumocystis jirovecii-infected AIDS patients from low endemic areas of China.

Pneumocystis pneumonia (PCP) is an opportunistic and potentially life-threatening infection of AIDS patients caused by the fungus Pneumocystis jirovecii (P. jirovecii). Trimethoprim-sulfamethoxazole (TMP-SMX) is the most commonly used drug combination in the treatment and prophylaxis of PCP. However, with long-term use of this combination, mutations in the dihydropteroate synthase (DHPS) gene of P. jirovecii bring about the development of resistance. Data on the prevalence of P. jirovecii and its DHPS mutants in China, especially in low endemic areas, are still limited. Thus, in the present study, we measured the P. jirovecii infection rate among HIV-positive and AIDS (HIV/AIDS) patients with suspected PCP and investigated the relationship between CD4+ T cell count and PCP occurrence. As well as the polymerase chain reaction (PCR) analysis and sequencing, the restriction fragment length polymorphism (RFLP) method was used to analyze DHPS point mutation in P. jirovecii strains. P. jirovecii was detected in 40.82% of cases. The clinical symptoms and signs of PCP were not typical; with decreasing CD4+ T cell counts, PCP infection in HIV/AIDS patients increased. In only one case (1.67%), the patients’ DHPS gene could not be cut by the Acc I restriction enzyme. Furthermore, mutation at codon 171 was detected in 11 cases and no mutation was found at codon 57. Patients treated with sulfamethoxazole combined with Voriconazole or Caspofungin exhibited favorable results. After treatment, the symptoms of dyspnea were alleviated, and chest computed tomography findings showed the improvement of lung shadows. These indicated that the prevalence of DHPS mutations in P. jirovecii isolates in AIDS-PCP patients in the region was low. Thus, the contribution of gene mutations to treatment failure requires further research.

Analysis of sulphadoxine-pyrimethamine resistance-associated mutations in Plasmodium falciparum isolates obtained from asymptomatic pregnant women in Ogun State, Southwest Nigeria.

Intermittent preventive treatment in pregnancy with sulphadoxine-pyrimethamine (IPTp-SP) is one of the main strategies for protecting pregnant women, fetus, and their new-born against adverse effects of P. falciparum infection. The development of the drug resistance linked to mutations in P. falciparum dihydrofolate reductase gene (pfdhfr) and P. falciparum dihydropteroate synthase gene (pfdhps), is currently threatening the IPTp-SP approach. This study determined the prevalence of pfdhfr and pfdhps mutations in isolates obtained from pregnant women with asymptomatic P. falciparum infection in Nigerian. Additionally, P. falciparum genetic diversity and multiplicity of infection (MOI) was assessed by genotyping the P. falciparum merozoite surface Protein 1 and 2 (pfmsp-1 and pfmsp-2) genes. The pfdhfr and pfdhps were genotyped by direct sequencing, and the pfmsp-1 and pfmsp-2 fragment analysis by polymerase chain reaction was used to determine P. falciparum genetic diversity. Of the 406 pregnant women recruited, 123 had P. falciparum infection by PCR, and of these, 52 were successfully genotyped for pfdhfr and 42 for pfdhps genes. The pfdhfr triple-mutant parasites (N51I, C59R, and S108N) or the IRN haplotype were predominant (98%), whereas pfdhfr mutations C50R and I164L did not occur. For pfdhps gene, the prevalence of A437G, A581G, A436A, and A613S mutations were 98, 71, 55, and 36%, respectively. Nineteen (44%) isolates with quintuple mutations (CIRNI- SGKGA) had the highest combined pfdhfr-pfdhps haplotype. Isolates with sextuple mutants; CIRNI- AGKAS and CIRNI- AGKGA had a prevalence of 29 and 14%, respectively. High genetic diversity (7 pfmsp-1 alleles and 10 pfmsp-2 alleles) and monoclonal infection rate (76%) was observed. This study demonstrated a continuous high prevalence of pfdhfr mutation and an increase in pfdhps mutations associated with SP-resistance in southwest Nigeria. Continuous surveillance of IPTp-SP effectiveness and consideration of alternative IPTp strategies is recommended.

Detection of Amino Acid Mutations in Dihydrofolate Reductase &amp; Dihydro Pteroate Synthase Gene in Plasmodium Vivax Isolates

Detection of Amino Acid Mutations in Dihydrofolate Reductase &amp; Dihydro Pteroate Synthase Gene in Plasmodium Vivax Isolates

Complete genome sequence and genome-scale metabolic modelling of Acinetobacter baumannii type strain ATCC 19606

Multidrug-resistant (MDR) Acinetobacter baumannii is a critical threat to global health. The type strain ATCC 19606 has been widely used in studying the virulence, pathogenesis and mechanisms of antimicrobial resistance in A. baumannii. However, the lack of a complete genome sequence is a hindrance towards detailed bioinformatic studies. Here we report the generation of a complete genome for ATCC 19606 using PacBio sequencing. ATCC 19606 genome consists of a 3,980,848-bp chromosome and a 9,450-bp plasmid pMAC, and harbours a chromosomal dihydropteroate synthase gene sul2 conferring resistance to sulphonamides and a plasmid-borne ohr gene conferring resistance to peroxides. The genome also contains 69 virulence genes involved in surface adherence, biofilm formation, extracellular phospholipase, iron uptake, immune evasion and quorum sensing. Insertion sequences ISCR2 and ISAba11 are embedded in a 36.1-Kb genomic island, suggesting an IS-mediated large-scale DNA recombination. Furthermore, a genome-scale metabolic model (GSMM) iATCC19606v2 was constructed using the complete genome annotation. The model iATCC19606v2 incorporated a periplasmic compartment, 1,422 metabolites, 2,114 reactions and 1,009 genes, and a set of protein crowding constraints taking into account enzyme abundance limitation. The prediction of bacterial growth on 190 carbon and 95 nitrogen sources achieved a high accuracy of 85.6% compared to Biolog experiment results. Based upon two transposon mutant libraries of AB5075 and ATCC 17978, the predictions of essential genes reached the accuracy of 87.6% and 82.1%, respectively. Together, the complete genome sequence and high-quality GSMM iATCC19606v2 provide valuable tools for antimicrobial systems pharmacological investigations on A. baumannii.

Polymorphisms involving the Pneumocystis jirovecii-related genes in AIDS patients in eastern China

ObjectiveTo investigate the genetic polymorphisms of mitochondrial large ribosomal subunit (mtLSU)-rRNA, dihydrofolate reductase (DHFR), dihydropteroate synthase (DHPS), cytochrome b (CYB), and superoxide dismutase (SOD) genes and its correlation with clinical outcomes of Pneumocystis jirovecii pneumonia in acquired immune deficiency(AIDS) patients. MethodsEighty AIDS patients with P. jirovecii pneumonia that were admitted to our hospital from 2016 to 2018 were included in this study. Their demographic information and clinical data were collected, as well as corresponding saliva specimens for PCR and sequencing of mtLSU-rRNA, DHFR, DHPS, CYB, and SOD genes to analyze genetic polymorphisms, different polymorphic combinations, and clinical outcomes. ResultsOf the 80 saliva specimens, mtLSU-rRNA was successfully amplified and sequenced in 30 cases; CYB was successfully amplified and sequenced in 26 cases; and SOD, DHFR, and DHPS were successfully amplified and sequenced in 18 cases. These results indicate that The mtLSU-rRNA, CYB, and SOD genes were highly polymorphic. mt85T and CYB1 were the variants dominantly detected at the mtLSU-rRNA and CYB loci, respectively. The SOD1 and SOD2 variants (each in 50% of the cases) were detected at the SOD locus. Among the 18 cases that were successfully amplified and sequenced for DHFR and DHPS, three DHFR nonsense mutations and no DHPS mutation were observed. The mt85C, CYB1, SOD1, and DHFR312T genes harbored common polymorphisms (n = 4; 22.22%) and the mt85T, CYB1, SOD1, DHFR312T genes were associated with poor clinical outcomes. ConclusionThe types of genetic polymorphisms and polymorphic combinations of mtLSU-rRNA, DHFR, DHPS, CYB, and SOD in P. jirovecii were related to the clinical outcomes of patients with P. jirovecii pneumonia in Zhejiang Province, China.

Plasmids of Shigella flexneri serotype 1c strain Y394 provide advantages to bacteria in the host

BackgroundShigella flexneri has an extremely complex genome with a significant number of virulence traits acquired by mobile genetic elements including bacteriophages and plasmids. S. flexneri serotype 1c is an emerging etiological agent of bacillary dysentery in developing countries. In this study, the complete nucleotide sequence of two plasmids of S. flexneri serotype 1c strain Y394 was determined and analysed.ResultsThe plasmid pINV-Y394 is an invasive or virulence plasmid of size 221,293 bp composed of a large number of insertion sequences (IS), virulence genes, regulatory and maintenance genes. Three hundred and twenty-eight open reading frames (ORFs) were identified in pINV-Y394, of which about a half (159 ORFs) were identified as IS elements. Ninety-seven ORFs were related to characterized genes (majority of which are associated with virulence and their regulons), and 72 ORFs were uncharacterized or hypothetical genes. The second plasmid pNV-Y394 is of size 10,866 bp and encodes genes conferring resistance against multiple antibiotics of clinical importance. The multidrug resistance gene cassette consists of tetracycline resistance gene tetA, streptomycin resistance gene strA-strB and sulfonamide-resistant dihydropteroate synthase gene sul2.ConclusionsThese two plasmids together play a key role in the fitness of Y394 in the host environment. The findings from this study indicate that the pathogenic S. flexneri is a highly niche adaptive pathogen which is able to co-evolve with its host and respond to the selection pressure in its environment.

High prevalence of Pfdhfr\u2013Pfdhps quadruple mutations associated with sulfadoxine\u2013pyrimethamine resistance in Plasmodium falciparum isolates from Bioko Island, Equatorial Guinea

BackgroundSulfadoxine–pyrimethamine (SP) is recommended for intermittent preventive treatment of malaria in Africa. However, increasing SP resistance (SPR) affects the therapeutic efficacy of the SP. As molecular markers, Pfdhfr (dihydrofolate reductase) and Pfdhps (dihydropteroate synthase) genes are widely used for SPR surveillance. This study aimed to assess the prevalence of Pfdhfr and Pfdhps genes mutations and haplotypes in Plasmodium falciparum isolates collected from Bioko Island, Equatorial Guinea (EG).MethodsIn total, 180 samples were collected in 2013–2014. The single nucleotide polymorphisms (SNPs) of the Pfdhfr and Pfdhps genes were identified with nested PCR and Sanger sequencing. The genotypes and linkage disequilibrium (LD) tests were also analysed.ResultsSequences of Pfdhfr and Pfdhps genes were obtained from 92.78% (167/180) and 87.78% (158/180) of the samples, respectively. For Pfdhfr, 97.60% (163/167), 87.43% (146/167) and 97.01% (162/167) of the samples carried N51I, C59R and S108N mutant alleles, respectively. The prevalence of the Pfdhps S436A, A437G, K540E, A581G, and A613S mutations were observed in 20.25% (32/158), 90.51% (143/158), 5.06% (8/158), 0.63% (1/158), and 3.16% (5/158) of the samples, respectively. In total, 3 unique haplotypes at the Pfdhfr locus and 8 haplotypes at the Pfdhps locus were identified. A triple mutation (CIRNI) in Pfdhfr was the most prevalent haplotype (86.83%), and a single mutant haplotype (SGKAA; 62.66%) was predominant in Pfdhps. A total of 130 isolates with 12 unique haplotypes were found in the Pfdhfr and Pfdhps combined haplotypes, 65.38% (85/130) of them carried quadruple allele combinations (CIRNI-SGKAA), whereas only one isolate (0.77%, 1/130) was found to carry the wild-type (CNCSI-SAKAA). For LD analysis, the Pfdhfr N51I was significantly associated with the Pfdhps A437G (P < 0.05).ConclusionBioko Island possesses a high prevalence of the Pfdhfr triple mutation (CIRNI) and Pfdhps single mutation (SGKAA), which will undermine the pharmaceutical effect of SP for malaria treatment strategies. To avoid an increase in SPR, continuous molecular monitoring and additional control efforts are urgently needed in Bioko Island, Equatorial Guinea.

Discovery of Novel Antibiotic Resistance Determinants in Forest and Grassland Soil Metagenomes.

Soil represents a significant reservoir of antibiotic resistance genes (ARGs), which can potentially spread across distinct ecosystems and be acquired by pathogens threatening human as well as animal health. Currently, information on the identity and diversity of these genes, enabling anticipation of possible future resistance development in clinical environments and the livestock sector, is lacking. In this study, we applied functional metagenomics to discover novel sulfonamide as well as tetracycline resistance genes in soils derived from forest and grassland. Screening of soil metagenomic libraries revealed a total of eight so far unknown ARGs. The recovered genes originate from phylogenetically diverse soil bacteria (e.g., Actinobacteria, Chloroflexi, or Proteobacteria) and encode proteins with a minimum identity of 46% to other antibiotic resistance determinants. In particular forest soil ecosystems have so far been neglected in studies focusing on antibiotic resistance. Here, we detected for the first time non-mobile dihydropteroate synthase (DHPS) genes conferring resistance to sulfonamides in forest soil with no history of exposure to these synthetic drugs. In total, three sulfonamide resistant DHPSs, differing in taxonomic origin, were discovered in beech or pine forest soil. This indicates that sulfonamide resistance naturally occurs in forest-resident soil bacterial communities. Besides forest soil-derived sulfonamide resistance proteins, we also identified a DHPS affiliated to Chloroflexi in grassland soil. This enzyme and the other recovered DHPSs confer reduced susceptibility toward sulfamethazine, which is widely used in food animal production. With respect to tetracycline resistance, four efflux proteins affiliated to the major facilitator superfamily (MFS) were identified. Noteworthy, one of these proteins also conferred reduced susceptibility toward lincomycin.

Novel dihydropteroate synthase gene mutation in Pneumocystis jirovecii among HIV-infected patients in India: Putative association with drug resistance and mortality.

Pneumocystis pneumonia (PCP) remains a debilitating cause of death among HIV-infected patients. The combination trimethoprim/sulfamethoxazole (SXT) is the most effective anti-Pneumocystis treatment and prophylaxis. However, long-term use of this combination has raised alarms about the emergence of resistant organisms. This study was performed to investigate mutations in the dihydropteroate synthase (DHPS) gene and their clinical consequences in HIV-infected patients with PCP. A total of 76 clinically suspected cases of PCP among HIV-seropositive adult patients from March 2014 to March 2017 were included. Clinical samples (bronchoalveolar lavage fluid and sputum) were investigated for the detection of Pneumocystis jirovecii using both microscopy and nested PCR. DHPS genotyping and mutational analyses were performed and the data were correlated with clinical characteristics. Among the 76 enrolled HIV-positive patients, only 17 (22.4%) were positive for P. jirovecii. DHPS gene sequencing showed a novel nucleotide substitution at position 288 (Val96Ile) in three patients (3/12; 25.0%). Patients infected with the mutant P. jirovecii genotype had severe episodes of PCP, did not respond to SXT and had a fatal outcome (P=0.005). All three patients had a CD4+ T-cell count <100 cells/μL, and two also had co-infections. This study suggests that the emergence of a mutant P. jirovecii genotype is probably associated with drug resistance and mortality. The data also suggest that DHPS mutational analyses should be performed in HIV-seropositive patients to avoid treatment failure and death due to PCP. However, the role of underlying disease severity and co-morbidities should not be underestimated.

Prevalence of chloroquine and antifolate drug resistance alleles in Plasmodium falciparum clinical isolates from three areas in Ghana.

Background: The emergence and spread of resistance in Plasmodium falciparum to chloroquine (CQ) necessitated the change from CQ to artemisinin-based combination therapies (ACTs) as first-line drug for the management of uncomplicated malaria in Ghana in 2005. Sulphadoxine-pyrimethamine (SP) which was the second line antimalarial drug in Ghana, was now adopted for intermittent preventive treatment of malaria in pregnancy (IPTp). Methods: To examine the prevalence of molecular markers associated with CQ and antifolate drug resistance in Ghana, we employed restriction fragment length polymorphism polymerase chain reaction to genotype and compare single nucleotide polymorphisms (SNPs) in the P. falciparum chloroquine resistance transporter ( pfcrt, PF3D7_0709000), multidrug resistance ( pfmdr1, PF3D7_0523000), bifunctional dihydrofolate reductase-thymidylate synthase ( pfdhfr, PF3D7_0417200) and dihydropteroate synthase ( pfdhps, PF3D7_0810800) genes. Parasites were collected from children with malaria reporting to hospitals in three different epidemiological areas of Ghana (Accra, Kintampo and Navrongo) in 2012-2013 and 2016-2017. Results: The overall prevalence of the CQ resistance-associated pfcrt 76T allele was 8%, whereas pfmdr1 86Y and 184F alleles were present in 10.2% and 65.1% of infections, respectively. The majority of the isolates harboured the antifolate resistance-associated pfdhfr alleles 51I (83.4%), 59R (85.9 %) and 108N (90.5%). Pfdhps 437G and 540E were detected in 90.6% and 0.7% of infections, respectively. We observed no significant difference across the three study sites for all the polymorphisms except for pfdhps 437G , which was more common in Accra compared to Kintampo for the 2016-2017 isolates. Across both pfdhfr and pfdhps genes, a large proportion (61%) of the isolates harboured the quadruple mutant combination ( I 51 R 59 N 108/ G 437). CQ resistance alleles decreased during the 12 years after CQ withdrawal, but an mediate SP resistance alleles increased. Conclusion: Surveillance of the prevalence of resistance alleles is necessary in monitoring the efficacy of antimalarial drugs.

Prevalence of molecular markers of sulfadoxine\u2013pyrimethamine and artemisinin resistance in Plasmodium falciparum from Pakistan

BackgroundIn Pakistan, artesunate (AS) in combination with sulfadoxine–pyrimethamine (SP) is the recommended treatment for uncomplicated Plasmodium falciparum malaria. Monitoring molecular markers of anti-malarial drug resistance is crucial for early detection and containment of parasite resistance to treatment. Currently, no data are available on molecular markers of artemisinin resistance (K13 mutations) in P. falciparum isolates from Pakistan. In this study, the prevalence of mutations associated with SP and artemisinin resistance was estimated in different regions of Pakistan.MethodsA total of 845 blood samples that were positive for malaria parasites by microscopy or rapid diagnostic test were collected from January 2016 to February 2017 from 16 different sites in Pakistan. Of these samples, 300 were positive for P. falciparum by PCR. Polymorphisms in the P. falciparum dihydrofolate reductase (pfdhfr) and dihydropteroate synthase (pfdhps) genes were identified by pyrosequencing while polymorphisms in the propeller domain of the pfk13 gene were identified by Sanger sequencing.ResultsThe prevalence of the PfDHFR 108N and 59R mutations was 100% and 98.8%, respectively, while the prevalence of PfDHFR 50R and 51I mutations was 8.6%. No mutation was observed at PfDHFR position 164. In PfDHPS, the prevalence of mutations at positions 436, 437, and 613 was 9.9%, 45.2%, and 0.4%, respectively. No mutations were found at PfDHPS positions 540 and 581. The prevalence of double PfDHFR mutants (59R + 108N) ranged from 93.8% to 100%, while the prevalence of parasites having the PfDHFR 59R + 108N mutations in addition to the PfDHPS 437G mutation ranged from 9.5% to 83.3% across different regions of Pakistan. Nine non-synonymous and four synonymous mutations were observed in the PfK13 propeller domain, none of which correspond to mutations validated to contribute to artemisinin resistance.ConclusionThe absence of the highly resistant PfDHFR/PfDHPS quintuple mutant parasites and the lack of PfK13 mutations associated with artemisinin resistance is consistent with AS + SP being effective in Pakistan.

Detection of the sul2-strA-strB gene cluster in an ice core from Dome Fuji Station, East Antarctica.

Bacteria harbouring antimicrobial resistance genes (ARGs) have been isolated from various locations, including ancient microbiomes, indicating that these genes pre-date the discovery of antibiotics. To gain further information regarding ARGs in the pre-antibiotic era, ice samples derived from Dome Fuji Station, Eastern Antarctica, were examined. DNA was extracted from firn or ice core samples (n=3; 1200-1400ybp, 1700-2100ybp and 2200-2800ybp, respectively) under sterile conditions. Whole-genome amplification and PCR analyses were utilised to detect ARGs. A 2764-bp gene cluster containing the type II dihydropteroate synthase gene sul2 and the aminoglycoside phosphotransferase genes strA and strB was detected in the 1200-1400-year-old Antarctic ice core (DF-63.5). The sul2-strA-strB gene cluster is frequently associated with plasmid RSF1010 and transposon Tn5393; however, these elements were not detected in sample DF-63.5. The gene cluster exhibited a high level of sequence identity to sequences harboured in present-day bacteria, although there were sequence polymorphisms in the strA gene. Furthermore, expression of this gene cluster in Escherichia coli resulted in reduced susceptibility to dihydrostreptomycin and sulfamethoxazole. The results of this study provide further evidence that certain ARGs existed in the pre-antibiotic era. Because the sul2 gene confers resistance to the synthetic compound sulfamethoxazole, these findings suggest that ARGs against synthetic antimicrobials emerged in bacteria during the pre-antibiotic era.