Whole-genome sequencing of bacteria accountable for lactational mastitis in humans combined with an examination of their antibiotic resistance profiles.

Lactational mastitis, a common condition affecting nursing mothers, is characterized by mammary gland inflammation during lactation. This inflammatory response typically occurs due to bacterial infection. The discomfort and pain associated with lactational mastitis can significantly impact a mother's ability to breastfeed comfortably and may lead to the cessation of breastfeeding altogether if left untreated. Antibiotics are commonly prescribed to target the bacteria causing the infection and alleviate symptoms, aiming to treat the infection. Nevertheless, a notable worry linked to antibiotic use is the emergence of antibiotic resistance, compounded by the possible persistence of antibiotics in milk. Additionally, lactational mastitis is characterized by its polymicrobial nature. In this study, bacteria were isolated from infected breast milk samples and whole-genome sequencing was performed on eleven isolates to accurately identify the bacteria and assess their antibiotic resistance profiles. Using Galaxy tools and the ResFinder database, we identified Bacillus paraanthracis, Bacillus altitudinis, Staphylococcus aureus, Bacillus cereus, Escherichia coli, Alcaligenes faecalis, and Bacillus licheniformis, along with antibiotic-resistant genes like fosB1, cat86, erm (D), blaZ, and mdf (A). ABRicate aided in antimicrobial resistance (AMR) gene analysis, and CARD visualized their distribution. Our study demonstrates that the severity of infection is directly proportional to an increase in somatic cell count (SCC). This research sheds light on microbial diversity in lactational mastitis milk and provides crucial insights into antibiotic-resistance genes. Utilizing bioinformatics tools, such as those employed in this study, can inform the design of effective treatment strategies for lactational mastitis infections.

Comparative assessment on the prevalence of mutations in the Plasmodium falciparum drug-resistant genes in two different ecotypes of Odisha state, India.

Considering malaria as a local and focal disease, epidemiological understanding of different ecotypes of malaria can help in devising novel control measures. One of the major hurdles in malaria control lies on the evolution and dispersal of the drug-resistant malaria parasite, Plasmodium falciparum. We herewith present data on genetic variation at the Single Nucleotide Polymorphism (SNP) level in four different genes of P. falciparum (Pfcrt, Pfmdr1, Pfdhfr, and Pfdhps) that confer resistance to different antimalarials in two different eco-epidemiological settings, i.e. Hilly-Forest (HF) and Riverine-Plain (RP), in a high malaria endemic district of Odisha state, India. Greater frequency of antimalarial resistance conferring SNPs and haplotypes was observed in all four genes in P. falciparum, and Pfdhps was the most variable gene among the four. No significant genetic differentiation could be observed in isolates from HF and RP ecotypes. Twelve novel, hitherto unreported nucleotide mutations could be observed in the Pfmdr1 and Pfdhps genes. While the Pfdhps gene presented highest haplotype diversity, the Pfcrt gene displayed the highest nucleotide diversity. When the data on all the four genes were complied, the isolates from HF ecotype were found to harbour higher average nucleotide diversity than those coming from RP ecotype. High and positive Tajima's D values were obtained for the Pfcrt and Pfdhfr genes in isolates from both the HF and RP ecotypes, with statistically significant deviation from neutrality in the RP ecotype. Different patterns of Linkage Disequilibrium (LD) among SNPs located in different drug-resistant genes were found in the isolates collected from HF and RP ecotypes. Whereas in the HF ecotype, SNPs in the Pfmdr1 and Pfdhfr were significantly associated, in the RP ecotype, SNPs located in Pfcrt were associated with Pfmdr1, Pfdhfr and Pfdhps. These findings provide a baseline understanding on how different micro eco-epidemiological settings influence evolution and spread of different drug resistance alleles. Our findings further suggest that drug resistance to chloroquine and sulfadoxine-pyrimethamine is approaching fixation level, which requires urgent attention of malaria control programme in India.

DNA sequence polymorphisms of the pfmdr1 gene and association of mutations with the pfcrt gene in Indian Plasmodium falciparum isolates.

Mutations in the Plasmodium falciparum multidrug resistance (pfmdr1) gene are known to provide compensatory fitness benefits to the chloroquine (CQ)-resistant malaria parasites and are often associated with specific mutations in the P. falciparum CQ resistant transporter (pfcrt) gene. Prevalence of the specific mutations in these two genes across different malaria endemic regions was mostly studies. However, reports on mutations in the pfmdr1 gene and their genetic associations with mutations in the pfcrt gene in Indian P. falciparum field isolates are scarce. We have sequenced a 560 bp region of pfmdr1 coding sequence in 64 P. falciparum isolates collected from different malaria endemic populations in India. Twenty out of these 64 isolates were laboratory cultured with known in vitro CQ sensitiveness (10 sensitive and 10 resistant). Three low frequency mutations (two non-synonymous and one synonymous) in the pfmdr1 gene were segregating in Indian isolates in addition to the predominant Y86 and Y184 ones, with high haplotype and nucleotide diversity in the field isolates in comparison to the cultured ones. No statistically significant genetic association between the mutations in the pfmdr1 and pfcrt gene could be detected; almost all observed associations were intragenic in nature. The results on the genetic diversity of the pfmdr1 gene were discussed in term of evolutionary perspectives in Indian P. falciparum, with possible future potential of gaining further insights on this gene in view of evolving malaria parasites resistant to artemisinin partner drugs.

Analyses of genetic variations at microsatellite loci present in-and-around the Pfcrt gene in Indian Plasmodium falciparum.

Evolution and spread of chloroquine resistant (CQR) malaria parasite Plasmodium falciparum have posed great threat in malaria intervention across the globe. The occurrence of K76T mutation in the P. falciparum chloroquine resistance transporter (pfcrt) gene has been widely attributed to CQR with four neighboring mutations providing compensatory fitness benefit to the parasite survival. Understanding evolutionary patterns of the pfcrt gene is of great relevance not only for devising new malaria control measures but also could serve as a model to understand evolution and spread of other human drug-resistant pathogens. Several studies, mainly based on differential patterns of diversities of the microsatellite loci placed in-and-around the pfcrt gene have indicated the role of positive natural selection under the 'hitchhiking' model of molecular evolution. However, the studies were restricted to limited number of microsatellite loci present inside the pfcrt gene. Moreover, comparatively higher level of diversities in microsatellite loci present inside the pfcrt gene than the loci flanking the pfcrt gene are hallmarks of Indian P. falciparum, presenting contrasting evolutionary models to global isolates. With a view to infer evolutionary patterns of the pfcrt gene in Indian P. falciparum, we have adopted a unique sampling scheme of two types of populations (cultured and field collected) and utilized 20 polymorphic microsatellite loci (16 located inside the pfcrt gene and four in the two flanking regions) to disentangle between genetic drift (inbred cultured isolates) and natural selection (field isolates). Data analyses employing different population genetic tests could not straightforwardly explain either the model invoking 'genetic hitchhiking' or 'genetic drift'. However, complex evolutionary models influenced by both demography and natural selection or an alternative model of natural selection (e.g. diversifying/balancing selection) might better explain the observed microsatellite variation in-and-around the pfcrt gene in Indian P. falciparum.