We have selected piperaquine (PQ) and lumefantrine (LM) resistant Plasmodium berghei ANKA parasite lines in mice by drug pressure. Effective doses that reduce parasitaemia by 90% (ED(90)) of PQ and LM against the parent line were 3.52 and 3.93 mg/kg, respectively. After drug pressure (more than 27 passages), the selected parasite lines had PQ and LM resistance indexes (I(90)) [ED(90) of resistant line/ED(90) of parent line] of 68.86 and 63.55, respectively. After growing them in the absence of drug for 10 passages and cryo-preserving them at -80 degrees C for at least 2 months, the resistance phenotypes remained stable. Cross-resistance studies showed that the PQ-resistant line was highly resistant to LM, while the LM-resistant line remained sensitive to PQ. Thus, if the mechanism of resistance is similar in P. berghei and Plasmodium falciparum, the use of LM (as part of Coartem) should not select for PQ resistance.

Retroviral protease inhibitors (RPIs) such as lopinavir (LP) and saquinavir (SQ) are active against Plasmodium parasites. However, the exact molecular target(s) for these RPIs in the Plasmodium parasites remains poorly understood. We hypothesised that LP and SQ suppress parasite growth through inhibition of aspartyl proteases. Using reverse genetics approach, we embarked on separately generating knockout (KO) parasite lines lacking Plasmepsin 4 (PM4), PM7, PM8, or DNA damage-inducible protein 1 (Ddi1) in the rodent malaria parasite Plasmodium berghei ANKA. We then tested the suppressive profiles of the LP/Ritonavir (LP/RT) and SQ/RT as well as antimalarials; Amodiaquine (AQ) and Piperaquine (PQ) against the KO parasites in the standard 4-day suppressive test. The Ddi1 gene proved refractory to deletion suggesting that the gene is essential for the growth of the asexual blood stage parasites. Our results revealed that deletion of PM4 significantly reduces normal parasite growth rate phenotype (P = 0.003). Unlike PM4_KO parasites which were less susceptible to LP and SQ (P = 0.036, P = 0.030), the suppressive profiles for PM7_KO and PM8_KO parasites were comparable to those for the WT parasites. This finding suggests a potential role of PM4 in the LP and SQ action. On further analysis, modelling and molecular docking studies revealed that both LP and SQ displayed high binding affinities (-6.3 kcal/mol to -10.3 kcal/mol) towards the Plasmodium aspartyl proteases. We concluded that PM4 plays a vital role in assuring asexual stage parasite fitness and might be mediating LP and SQ action. The essential nature of the Ddi1 gene warrants further studies to evaluate its role in the parasite asexual blood stage growth as well as a possible target for the RPIs.

The evolution of drug-resistant parasites is a major hindrance to malaria control, and thus understanding the behaviour of drug-resistant mutants is of clinical relevance. The study aimed to investigate how resistance against lumefantrine (LU) and piperaquine (PQ), anti-malarials used as partner drugs in artemisinin-based combination therapy (ACT), impacts parasite fitness. This is important since resistance to ACT, the first-line anti-malarial regimen is increasingly being reported. The stability of Plasmodium berghei ANKA strain that was previously selected for LU and PQ resistance was evaluated using the 4-day assay and established infection test in mice. Fitness cost of resistance was determined by comparing parasites proliferation rates in absence of drug pressure for the drug-exposed parasites between day 4 and 7 post-infection (pi), relative to the wild-type. Statistical analysis of data to compare mean parasitaemia and growth rates of respective parasite lines was carried out using student’s t-test and one-way analysis of variance, with significance level set at p<0.05. During serial passaging in the absence of the drug, the PQ-resistant parasite maintained low growth rates at day 7 pi (mean parasitaemia, 5.6% ± 2.3) relative to the wild-type (28.4% ± 6.6), translating into a fitness cost of resistance of 80.3%. Whilst resistance phenotype for PQ was stable, that of LU was transient since after several serial passages in the absence of drug, the LU-exposed line assumed the growth patterns of the wild-type. The contrasting behaviour of PQ- and LU-resistance phenotypes support similar findings which indicate that even for drugs within the same chemical class, resistance-conferred traits may vary on how they influence parasite fitness and virulence. Resistance-mediating polymorphisms have been associated with less fit malaria parasites. In the absence of drug pressure in the field, it is therefore likely that the wild-type parasite will out-compete the mutant form. This implies the possibility of reintroducing a drug previously lost to resistance, after a period of suspended use. Considering the recent reports of high failure rates associated with ACT, high fitness cost of resistance to PQ is therefore of clinical relevance as the drug is a partner in ACT.

Aim: This study determined the resistance pattern to β-lactam antibiotics of bacteria isolated from goats with subclinical mastitis in Thika subcounty, Kenya. We also administered a questionnaire to assess the risk factors associated with the occurrence of resistance to commonly used antibiotics. Materials and Methods: We collected milk samples from 110 lactating dairy goats in Thika subcounty to screen for subclinical mastitis using the California mastitis test. Bacterial isolation and identification were performed according to colony morphology, the hemolytic pattern on sheep blood agar, lactose fermentation on MacConkey plates, Gram staining, and standard biochemical tests. The antibiotic susceptibility of the isolates was determined by the agar disk diffusion method using penicillin G, cephalexin, cefoxitin, and cefotaxime antibiotic disks. The double-disk synergy test using amoxicillin-clavulanic acid was employed as a confirmatory test for extended-spectrum β-lactamase (ESBL) production. Fisher's exact test was used to determine the risk factors associated with the occurrence of antibiotic resistance (p≤0.05 was considered significant). Results: Of the 110 dairy goats sampled, 72.7% (80) were positive for subclinical mastitis. Isolation and identification of the bacteria from the positive samples yielded 149 bacteria isolates, including Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter spp., Yersinia spp., coagulase-negative staphylococci, and Escherichia coli. A high percentage (76.5%, 114/149) of the bacterial isolates was resistant to at least one of the tested antibiotics. At least 56/106 isolates (52.8%) showing cross-resistance to the β-lactam antibiotics were resistant to all four of the tested antibiotics, while only one isolate was resistant to three antibiotics (penicillin G, cephalexin, and cefoxitin). The double-disk synergy test confirmed that none of the isolates possessed ESBLs. Pre- and post-milking practices (p=0.0336) were found to be significantly associated with the occurrence of antibiotic resistance. Conclusion: A large proportion of the goats in our study cohort were infected with β-lactam-resistant bacteria associated with subclinical mastitis. Because the identified bacteria are of zoonotic importance, further studies should be undertaken to determine the transmission dynamics between humans and livestock and to identify novel intervention strategies.