Prevalence of subclinical mastitis, associated risk factors and antimicrobial susceptibility pattern of bacteria isolated from milk of dairy cattle in Kajiado Central sub-county, Kenya.

BACKGROUND: Literature is scarce on the occurrence of bovine mastitis and antimicrobial resistance among dairy animals kept by pastoralists in the Kenya. OBJECTIVES: A cross-sectional study was carried out to investigate the prevalence and risk factors of subclinical mastitis (SCM) and evaluate the antibiotic sensitivity of bacteria isolated from dairy cattle kept by farmers in Kajiado Central sub-county, Kenya. METHODS: A total of 202 lactating cows from 40 farms were sampled. Milk from the cows was screened for SCM using the California mastitis test, and the bacteria present in the milk samples were determined using standard bacteriological methods. The sensitivity of the isolated coagulase-negative staphylococci (CNS) and Staphylococcus aureus against antibiotics was tested using the Kirby-Bauer disk diffusion method. RESULTS: The prevalence of SCM at quarter- and cow-level was 31.7% and 53%, respectively. The prevalence of SCM was significantly higher (p < 0.05) in exotic breeds of cattle and those kept under an extensive system of production. A total of 19 bacterial species were isolated with the majority being CNS (40.1%), S. aureus (15.8%) and Micrococcus spp. (10.4%). S. aureus isolates showed varied resistance to the tested antibiotics with the highest resistance being against ceftazidime (75%), amoxycillin (50%) and streptomycin (46.9%). Several S. aureus isolates were resistant to oxacillin (34.4%) and cefoxitin (12.5%). CNSs were more resistant against ceftazidime (79.1%), amoxycillin (34.6%) and oxacillin (32.1%). Majority (92%-100%) of the Staphylococcus spp. were highly sensitive to ciprofloxacin a fluoroquinolone and augmentin. CONCLUSIONS: The high prevalence of SCM and bacteria resistant to antibiotics shows a need for animal health professionals and farmers to develop strategies for the management of mastitis and antibiotic resistance in dairy cows in the study area.

Prevalence of mutations in the cysteine desulfurase IscS (Pfnfs1) gene in recurrent Plasmodium falciparum infections following artemether-lumefantrine (AL) and dihydroartemisinin-piperaquine (DP) treatment in Matayos, Western Kenya.

BACKGROUND: Malaria remains a public health concern globally. Resistance to anti-malarial drugs has consistently threatened the gains in controlling the malaria parasites. Currently, artemether-lumefantrine (AL) and dihydroartemisinin-piperaquine (DP) are the treatment regimens against Plasmodium falciparum infections in many African countries, including Kenya. Recurrent infections have been reported in patients treated with AL or DP, suggesting the possibility of reinfection or parasite recrudescence associated with the development of resistance against the two therapies. The Plasmodium falciparum cysteine desulfurase IscS (Pfnfs1) K65 selection marker has previously been associated with decreased lumefantrine susceptibility. This study evaluated the frequency of the Pfnfs1 K65 resistance marker and associated K65Q resistant allele in recurrent infections collected from P. falciparum-infected individuals living in Matayos, Busia County, in western Kenya. METHODS: Archived dried blood spots (DBS) of patients with recurrent malaria infection on clinical follow-up days after treatment with either AL or DP were used in the study. After extraction of genomic DNA, PCR amplification and sequencing analysis were employed to determine the frequencies of the Pfnfs1 K65 resistance marker and K65Q mutant allele in the recurrent infections. Plasmodium falciparum msp1 and P. falciparum msp2 genetic markers were used to distinguish recrudescent infections from new infections. RESULTS: The K65 wild-type allele was detected at a frequency of 41% while the K65Q mutant allele was detected at a frequency of 22% in the recurrent samples. 58% of the samples containing the K65 wild-type allele were AL treated samples and while 42% were DP treated samples. 79% of the samples with the K65Q mutation were AL treated samples and 21% were DP treated samples. The K65 wild-type allele was detected in three recrudescent infections (100%) identified from the AL treated samples. The K65 wild-type allele was detected in two recrudescent DP treated samples (67%) while the K65Q mutant allele was identified in one DP treated (33%) recrudescent sample. CONCLUSIONS: The data demonstrate a higher frequency of the K65 resistance marker in patients with recurrent infection during the study period. The study underscores the need for consistent monitoring of molecular markers of resistance in regions of high malaria transmission.

Prevalence and Potential Risk Factors for the Acquisition of Antibiotic-Resistant Staphylococcus spp. Bacteria Among Pastoralist Farmers in Kajiado Central Subcounty, Kenya.

Antimicrobial resistance (AMR) is a growing health problem globally. To address this challenge, there is a need to generate baseline data on the prevalence and AMR profile of the main disease-causing bacteria. Here, we interrogated the prevalence of bacteria in the nasal cavity of healthy pastoralists in Kajiado Central Subcounty, Kenya, and the occurrence of AMR in Staphylococcus isolates among the study subjects. Nasal swabs from 176 pastoralists were cultured, and the bacteria isolates identified using standard phenotypic and biochemical bacteriological methods. Among the obtained 195 isolates, the most prevalent isolates were coagulase-negative Staphylococcus (CoNS) (44.9%), followed by Enterococci spp. (43.2%) while Staphylococcus aureus prevalence was 8%. Antimicrobial sensitivity of the Staphylococcus spp. isolates to 14 antibiotics representing six antibiotic groups was undertaken using the Kirby-Bauer disk diffusion method. Among the CoNS, the highest resistance was reported in amoxicillin (78.7%) and ceftazidime (76%), while the most resistance for S. aureus was reported in ceftazidime (100%), amoxicillin (71.4%), and streptomycin (71.4%). From an administered questionnaire looking at gender, animal contact frequency, history of hospital visitation and antibiotic usage, and habitual intake of raw milk, the study showed that male participants had a higher risk of carrying multiple drug resistant (MDR) bacteria than females (p = 0.02, OR = 1.3). Likewise, habitual intake of raw milk was significantly associated MDR acquisition (p = 0.02, OR = 1.82). This study reveals a high prevalence of AMR Staphylococcus isolates in the study area laying a foundation for further analysis of molecular characterization of the observed resistance as well as the development of interventions that can reduce the occurrence of AMR in the study area.

Distinct kinetics of antibodies to 111 Plasmodium falciparum proteins identifies markers of recent malaria exposure.

Strengthening malaria surveillance is a key intervention needed to reduce the global disease burden. Reliable serological markers of recent malaria exposure could improve current surveillance methods by allowing for accurate estimates of infection incidence from limited data. We studied the IgG antibody response to 111 Plasmodium falciparum proteins in 65 adult travellers followed longitudinally after a natural malaria infection in complete absence of re-exposure. We identified a combination of five serological markers that detect exposure within the previous three months with >80% sensitivity and specificity. Using mathematical modelling, we examined the antibody kinetics and determined that responses informative of recent exposure display several distinct characteristics: rapid initial boosting and decay, less inter-individual variation in response kinetics, and minimal persistence over time. Such serological exposure markers could be incorporated into routine malaria surveillance to guide efforts for malaria control and elimination.

Artemisinin Binds and Inhibits the Activity of Plasmodium falciparum Ddi1, a Retroviral Aspartyl Protease.

Reduced sensitivity of the human malaria parasite, Plasmodium falciparum, to Artemisinin and its derivatives (ARTs) threatens the global efforts towards eliminating malaria. ARTs have been shown to cause ubiquitous cellular and genetic insults, which results in the activation of the unfolded protein response (UPR) pathways. The UPR restores protein homeostasis, which otherwise would be toxic to cellular survival. Here, we interrogated the role of DNA-damage inducible protein 1 (PfDdi1), a unique proteasome-interacting retropepsin in mediating the actions of the ARTs. We demonstrate that PfDdi1 is an active A2 family protease that hydrolyzes ubiquitinated proteasome substrates. Treatment of P. falciparum parasites with ARTs leads to the accumulation of ubiquitinated proteins in the parasites and blocks the destruction of ubiquitinated proteins by inhibiting the PfDdi1 protease activity. Besides, whereas the PfDdi1 is predominantly localized in the cytoplasm, exposure of the parasites to ARTs leads to DNA fragmentation and increased recruitment of the PfDdi1 into the nucleus. Furthermore, we show that Ddi1 knock-out Saccharomycescerevisiae cells are more susceptible to ARTs and the PfDdI1 protein robustly restores the corresponding functions in the knock-out cells. Together, these results show that ARTs act in multiple ways; by inducing DNA and protein damage and might be impairing the damage recovery by inhibiting the activity of PfDdi1, an essential ubiquitin-proteasome retropepsin.

ß-lactam resistance in bacteria associated with subclinical mastitis in goats in Thika Subcounty, Kenya.

AIM: This study determined the resistance pattern to ß-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 ß-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 ß-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 ß-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.

Prevalence of mutations in Plasmodium falciparum genes associated with resistance to different antimalarial drugs in Nyando, Kisumu County in Kenya.

Resistance to the mainstay antimalarial drugs is a major concern in the control of malaria. Delayed Plasmodium falciparum parasite clearance has been associated with Single Nucleotide Polymorphisms (SNPs) in the kelch propeller region (K13). However, SNPs in the Pf-adaptor protein complex 2 mu subunit (Pfap2-mu), Pfcrt and Pfmdr1 are possible markers associated with multi-drug resistance. Here, we explored the prevalence of SNPs in the K13, Pfap2-mu, Pfcrt, and Pfmdr1 in 94 dried blood spot field isolates collected from children aged below 12 years infected with P. falciparum during a cross-sectional study. The samples were collected in 2015 during the peak malaria transmission season in the Nyando region of Western Kenya before treatment with Artemether-Lumefantrine, the first-line artemisinin-based combination therapy (ACT) in Kenya. However, 47 of the 94 samples had recurrent parasitemia and were interrogated for the presence of the SNPs in K13 and Pfap2-mu. We used PCR amplification and sequencing to evaluate specific regions of K13 (codons 432-702), Pfap2-mu (codons 1-350), Pfmdr1 (codons 86, 1034-1246), and Pfcrt (codons 72-76) gene(s). The majority of parasites harbored the wild type K13 sequence. However, we found a unique non-synonymous W611S change. In silico studies on the impact of the W611S predicted structural changes in the overall topology of the K13 protein. Of the 47 samples analyzed for SNPs in the Pfap2-mu gene, 14 (29%) had S160 N/T mutation. The CVIET haplotype associated with CQ resistance in the Pfcrt yielded a 7.44% (7/94), while CVMNK haplotype was at 92.56%. Mutations in the Pfmdr1 region were detected only in three samples (3/94; 3.19%) at codon D1246Y. Our data suggest that parasites in the western part of Kenya harbor the wildtype strains. However, the detection of the unique SNP in K13 and Pfap2-mu linked with ACT delayed parasite clearance may suggest slow filtering of ACT-resistant parasites.

Deciphering the targets of retroviral protease inhibitors in Plasmodium berghei.

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.

Amodiaquine resistance in Plasmodium berghei is associated with PbCRT His95Pro mutation, loss of chloroquine, artemisinin and primaquine sensitivity, and high transcript levels of key transporters.

Background: The human malaria parasite Plasmodium falciparum has evolved complex drug evasion mechanisms to all available antimalarials. To date, the combination of amodiaquine-artesunate is among the drug of choice for treatment of uncomplicated malaria. In this combination, a short acting, artesunate is partnered with long acting, amodiaquine for which resistance may emerge rapidly especially in high transmission settings. Here, we used a rodent malaria parasite Plasmodium berghei ANKA as a surrogate of P. falciparum to investigate the mechanisms of amodiaquine resistance. Methods: We used serial technique to select amodiaquine resistance by submitting the parasites to continuous amodiaquine pressure. We then employed the 4-Day Suppressive Test to monitor emergence of resistance and determine the cross-resistance profiles. Finally, we genotyped the resistant parasite by PCR amplification, sequencing and relative quantitation of mRNA transcript of targeted genes. Results: Submission of P. berghei ANKA to amodiaquine pressure yielded resistant parasite within thirty-six passages. The effective dosage that reduced 90% of parasitaemia (ED 90) of sensitive line and resistant line were 4.29mg/kg and 19.13mg/kg, respectively. After freezing at -80ºC for one month, the resistant parasite remained stable with an ED 90 of 18.22mg/kg. Amodiaquine resistant parasites are also resistant to chloroquine (6fold), artemether (10fold), primaquine (5fold), piperaquine (2fold) and lumefantrine (3fold). Sequence analysis of Plasmodium berghei chloroquine resistant transporter revealed His95Pro mutation. No variation was identified in Plasmodium berghei multidrug resistance gene-1 (Pbmdr1), Plasmodium berghei deubiquitinating enzyme-1 or Plasmodium berghei Kelch13 domain nucleotide sequences. Amodiaquine resistance is also accompanied by high mRNA transcripts of key transporters; Pbmdr1, V-type/H+ pumping pyrophosphatase-2 and sodium hydrogen ion exchanger-1 and Ca 2+/H + antiporter. Conclusions: Selection of amodiaquine resistance yielded stable "multidrug-resistant'' parasites and thus may be used to study common resistance mechanisms associated with other antimalarial drugs. Genome wide studies may elucidate other functionally important genes controlling AQ resistance in P. berghei.

Methylene blue inhibits lumefantrine-resistant Plasmodium berghei.

INTRODUCTION: Chemotherapy still is the most effective way to control malaria, a major public health problem in sub-Saharan Africa. The large-scale use of the combination therapy artemether-lumefantrine for malaria treatment in Africa predisposes lumefantrine to emergence of resistance. There is need to identify drugs that can be used as substitutes to lumefantrine for use in combination therapy. Methylene blue, a synthetic anti-methemoglobinemia drug, has been shown to contain antimalarial properties, making it a candidate for drug repurposing. The present study sought to determine antiplasmodial effects of methylene blue against lumefantrine- and pyrimethamine-resistant strains of P. berghei. METHODOLOGY: Activity of methylene blue was assessed using the classical four-day test on mice infected with lumefantrine-resistant and pyrimethamine-resistant P. berghei. A dose of 45 mg/kg/day was effective for testing ED90. Parasitemia and mice survival was determined. RESULTS: At 45 mg/kg/day, methylene blue sustained significant parasite inhibition, over 99%, for at least 6 days post-treatment against lumefantrine-resistant and pyrimethamine-resistant P. berghei (p = 0.0086 and p = 0.0191, respectively). No serious adverse effects were observed. CONCLUSIONS: Our results indicate that methylene blue at a concentration of 45 mg/kg/day confers over 99% inhibition against lumefantrine- and pyrimethamine-resistant P. berghei for six days. This shows the potential use methylene blue in the development of antimalarials against lumefantrine- and pyrimethamine-resistant parasites.

Fitness cost of resistance for lumefantrine and piperaquine-resistant Plasmodium berghei in a mouse model.

BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

Piperaquine and Lumefantrine resistance in Plasmodium berghei ANKA associated with increased expression of Ca2+/H+ antiporter and glutathione associated enzymes.

We investigated the mechanisms of resistance of two antimalarial drugs piperaquine (PQ) and lumefantrine (LM) using the rodent parasite Plasmodium berghei as a surrogate of the human parasite, Plasmodium falciparum. We analyzed the whole coding sequence of Plasmodium berghei chloroquine resistance transporter (Pbcrt) and Plasmodium berghei multidrug resistance gene 1(Pbmdr-1) for polymorphisms. These genes are associated with quinoline resistance in Plasmodium falciparum. No polymorphic changes were detected in the coding sequences of Pbcrt and Pbmdr1 or in the mRNA transcript levels of Pbmdr1. However, our data demonstrated that PQ and LM resistance is achieved by multiple mechanisms that include elevated mRNA transcript levels of V-type H(+) pumping pyrophosphatase (vp2), Ca(2+)/H(+) antiporter (vcx1), gamma glutamylcysteine synthetase (ggcs) and glutathione-S-transferase (gst) genes, mechanisms also known to contribute to chloroquine resistance in P. falciparum and rodent malaria parasites. The increase in ggcs and gst transcript levels was accompanied by high glutathione (GSH) levels and elevated activity of glutathione-S-transferase (GST) enzyme. Taken together, these results demonstrate that Pbcrt and Pbmdr1 are not associated with PQ and LM resistance in P. berghei ANKA, while vp2, vcx1, ggcs and gst may mediate resistance directly or modulate functional mutations in other unknown genes.

Methotrexate and aminopterin lack in vivo antimalarial activity against murine malaria species.

The antifolate anticancer drug methotrexate (MTX) has potent activity against Plasmodium falciparum in vitro. Experience of its use in the treatment of rheumatoid arthritis indicates that it could be safe and efficacious for treating malaria. We sought to establish a murine malaria model to study the mechanism of action and resistance of MTX and its analogue aminopterin (AMP). We used Plasmodium berghei, Plasmodium yoelii yoelii, Plasmodium chabaudi and Plasmodium vinckei. None of these species were susceptible to either drug. We have also tested the efficacy of pyrimethamine in combination with folic acid in P. berghei, and data indicate that folic acid does not influence pyrimethamine efficacy, which suggests that P. berghei may not transport folate. Since MTX and AMP utilise folate receptor/transport to gain access to cells, their lack of efficacy against the four tested murine malaria species may be the result of inefficiency of drug transport.