Susceptibility of Campylobacter Strains to Selected Natural Products and Frontline Antibiotics.

Campylobacter species have developed resistance to existing antibiotics. The development of alternative therapies is, therefore, a necessity. This study evaluates the susceptibility of Campylobacter strains to selected natural products (NPs) and frontline antibiotics. Two C. jejuni strains (ATCC® 33560TM and MT947450) and two C. coli strains (ATCC® 33559TM and MT947451) were used. The antimicrobial potential of the NPs, including plant extracts, essential oils, and pure phytochemicals, was evaluated by broth microdilution. The growth was measured by spectrophotometry and iodonitrotetrazolium chloride. Antibiotic resistance genes (tet(O) and gyrA) were characterized at the molecular level. The minimum inhibitory concentrations (MICs) and the minimum bactericidal concentrations (MBCs) ranged from 25 to 1600 µg/mL. Cinnamon oil, (E)-Cinnamaldehyde, clove oil, eugenol, and baicalein had the lowest MIC and MBC values (25-100 µg/mL). MT947450 and MT947451 were sensitive to erythromycin and gentamicin but resistant to quinolones and tetracycline. Mutations in gyrA and tet(O) genes from resistant strains were confirmed by sequencing. The findings show that NPs are effective against drug-sensitive and drug-resistant Campylobacter strains. The resistance to antibiotics was confirmed at phenotypic and genotypic levels. This merits further studies to decipher the action mechanisms and synergistic activities of NPs.

Genetic diversity and risk factors for the transmission of antimicrobial resistance across human, animals and environmental compartments in East Africa: a review.

BACKGROUND: The emergence and spread of antimicrobial resistance (AMR) present a challenge to disease control in East Africa. Resistance to beta-lactams, which are by far the most used antibiotics worldwide and include the penicillins, cephalosporins, monobactams and carbapenems, is reducing options for effective control of both Gram-positive and Gram-negative bacteria. The World Health Organization, Food and Agricultural Organization and the World Organization for Animal Health have all advocated surveillance of AMR using an integrated One Health approach. Regional consortia also have strengthened collaboration to address the AMR problem through surveillance, training and research in a holistic and multisectoral approach. This review paper contains collective information on risk factors for transmission, clinical relevance and diversity of resistance genes relating to extended-spectrum beta-lactamase-producing (ESBL) and carbapenemase-producing Enterobacteriaceae, and Methicillin-resistant Staphylococcus aureus (MRSA) across the human, animal and environmental compartments in East Africa. MAIN BODY: The review of the AMR literature (years 2001 to 2019) was performed using search engines such as PubMed, Scopus, Science Direct, Google and Web of Science. The search terms included 'antimicrobial resistance and human-animal-environment', 'antimicrobial resistance, risk factors, genetic diversity, and human-animal-environment' combined with respective countries of East Africa. In general, the risk factors identified were associated with the transmission of AMR. The marked genetic diversity due to multiple sequence types among drug-resistant bacteria and their replicon plasmid types sourced from the animal, human and environment were reported. The main ESBL, MRSA and carbapenem related genes/plasmids were the blaCTX-Ms (45.7%), SCCmec type III (27.3%) and IMP types (23.8%), respectively. CONCLUSION: The high diversity of the AMR genes suggests there may be multiple sources of resistance bacteria, or the possible exchange of strains or a flow of genes amongst different strains due to transfer by mobile genetic elements. Therefore, there should be harmonized One Health guidelines for the use of antibiotics, as well as regulations governing their importation and sale. Moreover, the trend of ESBLs, MRSA and carbapenem resistant (CAR) carriage rates is dynamic and are on rise over time period, posing a public health concern in East Africa. Collaborative surveillance of AMR in partnership with regional and external institutions using an integrated One Health approach is required for expert knowledge and technology transfer to facilitate information sharing for informed decision-making.

Laboratory confirmed puerperal sepsis in a national referral hospital in Tanzania: etiological agents and their susceptibility to commonly prescribed antibiotics.

BACKGROUND: In most developing countries, puerperal sepsis is treated empirically with broad spectrum antibiotics due to lack of resources for culture and antibiotics susceptibility testing. However, empirical treatment does not guarantee treatment success and may promote antimicrobial resistance. We set to determine etiological agents and susceptibility pattern to commonly prescribed antimicrobial agents, among women suspected of puerperal sepsis, and admitted at Muhimbili National Hospital. METHODS: Hospital based cross-sectional study conducted at tertiary hospital from December 2017 to April 2018. The study recruited post-delivery women suspected with puerperal sepsis. Socio- demographic, clinical and obstetric information were collected using structured questionnaire. Blood and endocervical swab samples were collected for aerobic culture. Blood culture bottles were incubated in BACTEC FX40 (Becton-Dickinson, Sparks, MD, USA). Positive blood cultures and cervical swabs were inoculated onto sheep blood agar, MacConkey agar, chocolate agar and Sabouraud's dextrose agar, incubated aerobically at 37 °C for 18-24 h. Antimicrobial susceptibility was determined by Kirby-Bauer disc diffusion method. RESULTS: A total of 197women were recruited, of whom 50.3% had spontaneous vaginal delivery, while 49.2% had caesarean section. Bacteraemia was detected in 22 (11.2%) women, along with 86 (43.6%) isolated from endocervical swabs. Gram-negative bacilli were the predominant isolates detected in 92(46.7%) cases. Majority of the isolates were E. coli 68(61.8%) followed by Klebsiella spp. 22(20.0%). E. coli were highly susceptible to meropenem (97.0%), while resistance to ceftriaxone, ampicillin and ceftazidime was 64.7, 67.6 and 63.2%, respectively. Klebsiella spp. were susceptible to meropenem (86.4%) and resistant to ceftriaxone (77.3%), gentamicin (86.4%), ampicillin (81.8%) and ceftazidime (86.4%). Staphylococcus aureus isolates were 100% susceptible to clindamycin. The proportion of extended spectrum beta lactamase producers among gram-negative bacilli was 64(69.6%) and 53.8% of S. aureus isolates were resistant to methicillin. CONCLUSION: In this study puerperal sepsis was mostly caused by E. coli and Klebsiella spp. Causative agents exhibited very high levels of resistance to most antibiotics used in empiric treatment calling for review of treatment guidelines and strict infection control procedures.

Sialic acid levels in breast milk from HIV-positive Tanzanian women and impact of maternal diet.

OBJECTIVE: To quantify total sialic acid in milk from HIV-positive Tanzanian mothers and to determine the impact of maternal diet on milk sialic acid levels. DESIGN: Milk samples were analyzed from 74 HIV-positive, Tanzanian women enrolled in a randomized, controlled clinical study of a dietary macronutrient supplement. Women were provided with a daily protein-calorie supplement and a micronutrient supplement or micronutrient supplement only during the last trimester of pregnancy and up to the first 6 months of breastfeeding. METHODS: Milk samples were collected at approximately 2 weeks and at least 3 months postpartum and assayed for total sialic acid. Milk sialic acid was assessed relative to maternal macronutrient intake, age, BMI, CD4+ cell count and infant birth weight. RESULTS: The mean concentration of milk sialic acid was highest in the first 2 weeks postpartum (6.89 ±â€Š2.79 mmol/l) and declined rapidly by 3 months (2.49 ±â€Š0.60 mmol/l). Sialic acid content in milk was similar between both treatment arms of the study, and did not correlate with maternal macronutrient intake. No correlation was found between maternal age, BMI, CD4+ cell count or infant birth weight and total milk sialic acid concentration. CONCLUSION: Milk sialic acid levels in HIV-positive, Tanzanian women without malnutrition are comparable with reported values for women of European descent and show a similar temporal decline during early lactation. These findings suggest that total milk sialic acid is maintained despite macronutrient deficiencies in maternal diet and support a conserved role for milk sialic acid in neonatal development.

Pre- and post-natal macronutrient supplementation for HIV-positive women in Tanzania: Effects on infant birth weight and HIV transmission.

OBJECTIVE: To determine if a protein-calorie supplement (PCS) plus a micronutrient supplement (MNS) improves outcomes for HIV-infected lactating women and their infants. DESIGN: Randomized, controlled trial. SETTING: Dar es Salaam, Tanzania. SUBJECTS, PARTICIPANTS: Pregnant HIV-infected women enrolled in PMTCT programs who intended to breastfeed for 6 months. INTERVENTION: Randomization 1:1 to administration of a PCS plus MNS versus MNS alone among 96 eligible women beginning in the third trimester and continuing for 6 months of breast-feeding. MAIN OUTCOME MEASURE(S): Primary: infant weight at 3 months. Secondary: maternal BMI at 6 months. RESULTS: PCS resulted in significant increases in daily energy intake compared to MNS at all time points (range of differences: +388-719 Kcal); and increases in daily protein intake (range of differences: +22-33 gm). Infant birth weight (excluding twins) was higher in the PCS than MNS groups: 3.30 kg vs 3.04 kg (p = 0.04). Infant weight at 3 months did not differ between PCS and MNS groups: 5.63 kg vs 5.99 kg (p = 0.07). Maternal BMI at 6 months did not differ between PCS and MNS groups: 24.3 vs 23.8 kg/m2 (p = 0.68). HIV transmission occurred in 0 infants in the PCS group vs 4 in the MNS group (p = 0.03). CONCLUSIONS: In comparison to MNS the PCS + MNS intervention was well tolerated, increased maternal energy and protein intake, and increased infant birth weight, but not weight at 3 months or maternal BMI at 6 months. Reduced infant HIV transmission in the PCS + MNS group was observed. TRIAL REGISTRATION: Clinical Trials.Gov NCT01461863.

Identification and antimicrobial resistance patterns of bacterial enteropathogens from children aged 0-59 months at the University Teaching Hospital, Lusaka, Zambia: a prospective cross sectional study.

BACKGROUND: Bacterial diarrhoeal disease is among the most common causes of mortality and morbidity in children 0-59 months at the University Teaching Hospital in Lusaka, Zambia. However, most cases are treated empirically without the knowledge of aetiological agents or antimicrobial susceptibility patterns. The aim of this study was, therefore, to identify bacterial causes of diarrhoea and determine their antimicrobial susceptibility patterns in stool specimens obtained from the children at the hospital. METHODS: This hospital-based cross-sectional study involved children aged 0-59 months presenting with diarrhoea at paediatrics wards at the University Teaching Hospital in Lusaka, Zambia, from January to May 2016. Stool samples were cultured on standard media for enteropathogenic bacteria, and identified further by biochemical tests. Multiplex polymerase chain reaction was used for characterization of diarrhoeagenic Escherichia coli strains. Antimicrobial susceptibility testing was performed on antibiotics that are commonly prescribed at the hospital using the Kirby-Bauer disc diffusion method, which was performed using the Clinical Laboratory Standards International guidelines. RESULTS: Of the 271 stool samples analysed Vibrio cholerae 01 subtype and Ogawa serotype was the most commonly detected pathogen (40.8%), followed by Salmonella species (25.5%), diarrhoeagenic Escherichia coli (18%), Shigella species (14.4%) and Campylobacter species (3.5%). The majority of the bacterial pathogens were resistant to two or more drugs tested, with ampicillin and co-trimoxazole being the most ineffective drugs. All diarrhoeagenic Escherichia coli isolates were extended spectrum ß-lactamase producers. CONCLUSION: Five different groups of bacterial pathogens were isolated from the stool specimens, and the majority of these organisms were multidrug resistant. These data calls for urgent revision of the current empiric treatment of diarrhoea in children using ampicillin and co-trimoxazole, and emphasizes the need for continuous antimicrobial surveillance as well as the implementation of prevention programmes for childhood diarrhoea.

Multi drug and other forms of drug resistant tuberculosis are uncommon among treatment naïve tuberculosis patients in Tanzania.

BACKGROUND: Surveillance and effective management of drug resistance is important to sustaining tuberculosis (TB) control efforts. We aimed to determine resistance rates to first line anti tuberculosis drugs and to describe factors associated with the resistance to any of the first line anti tuberculosis drugs in Dar es Salaam Tanzania. MATERIALS: Newly diagnosed, TB patients with neither history of tuberculosis treatment nor isoniazid prophylaxis were included into the study. Sputum specimens were cultured on either mycobacteria growth indicator tube 960 (MGIT 960) or Lowenstein Jenstein (LJ) medium supplemented with either glycerol (GLJ) or pyruvate (PLJ). Drug susceptibility for isoniazid, rifampicin, streptomycin and ethambutol was determined by either Lowenstein-Jensen (LJ) medium or mycobacteria growth indicator tube 960 (MGIT 960). RESULTS: A total of 933 newly diagnosed TB patients, were included into the study. Multi drug resistance (MDR) tuberculosis was detected among 2 (0.2%) patients. Resistance to any of the four tested drugs was detected among 54 (5.8%) patients. Mono-resistance to isoniazid, rifampicin, streptomycin and ethambutol were 21(2.3%), 3 (0.3%), 13 (1.4%), 9 (1.0%) respectively. CONCLUSION: Primary resistance to first line anti tuberculosis drugs is still low in this setting. Continued vigilance including periodic national surveillance of anti-tuberculosis resistance is recommended.

Neonatal sepsis at Muhimbili National Hospital, Dar es Salaam, Tanzania; aetiology, antimicrobial sensitivity pattern and clinical outcome.

BACKGROUND: Neonatal sepsis contributes significantly to morbidity and mortality among young infants. The aetiological agents as well as their susceptibility to antimicrobial agents are dynamic. This study determined aetiology, antimicrobial susceptibility and clinical outcome of neonatal sepsis at Muhimbili National Hospital. METHODS: Three hundred and thirty neonates admitted at the Muhimbili National Hospital neonatal ward between October, 2009 and January, 2010 were recruited. Standardized questionnaires were used to obtain demographic and clinical information. Blood and pus samples were cultured on MacConkey, blood and chocolate agars and bacteria were identified based on characteristic morphology, gram stain appearance and standard commercially prepared biochemical tests. Antimicrobial sensitivity testing was performed for ampicillin, cloxacillin, gentamicin, amikacin, cefuroxime and ceftriaxone on Mueller Hinton agar using the Kirby Bauer diffusion method. RESULTS: Culture proven sepsis was noted in 24% (74/330) of the study participants. Isolated bacterial pathogens were predominantly Staphylococcus aureus, Klebsiella spp and Escherichia coli. Klebsiella spp 32.7% (17/52) was the predominant blood culture isolate in neonates aged below seven days while Staphylococcus aureus 54.5% (12/22) was commonest among those aged above seven days. Staphylococcus aureus was the predominant pus swabs isolate for both neonates aged 0-6 days 42.2% (98/232) and 7-28 days 52.3% (34/65). Resistance of blood culture isolates was high to ampicillin 81.1% (60/74) and cloxacillin 78.4% (58/74), moderate to ceftriaxone 14.9% (11/74) and cefuroxime 18.9% (14/74), and low to amikacin 1.3% (1/74). Isolates from swabs had high resistance to ampicillin 89.9% (267/297) and cloxacillin 85.2 (253/297), moderate resistance to ceftriaxone 38.0% (113/297) and cefuroxime 36.0% (107/297), and low resistance to amikacin 4.7% (14/297). Sepsis was higher in neonates with fever and hypothermia (p=0.02), skin pustules (p<0.001), umbilical pus discharge and abdominal wall hyperemia (p=0.04). Presence of skin pustules was an independent predictor of sepsis OR 0.26, 95% CI (0.10-0.66) p=0.004. The overall death rate was 13.9% (46/330), being higher in neonates with sepsis 24.3% (18/74) than those without 10.9% (28/256), p=0.003. CONCLUSIONS: Staphylococcus aureus was predominant isolate followed by Klebsiella and Escherichia coli. There was high resistance to ampicillin and cloxacillin. Mortality rate due to neonatal sepsis was high in our setting. Routine antimicrobial surveillance should guide the choice of antibiotics for empirical treatment of neonatal sepsis.

Evaluation of cytotoxic, genotoxic and CYP450 enzymatic competition effects of Tanzanian plant extracts traditionally used for treatment of fungal infections.

HIV-infected patients in sub-Saharan countries highly depend on traditional medicines for the treatment of opportunistic oral infections as candidiasis. Previous investigations on antifungal activity of medicinal plant extracts utilized by traditional healers in Tanzania have revealed 12 extracts with potent antifungal activity. Although the plants may be good candidates for new treatment opportunities, they can be toxic or genotoxic and could cause pharmacokinetic interactions when used concomitantly with antiretroviral agents. Therefore, we investigated the cytotoxicity, genotoxicity and cytochrome P450 interaction potential of these medicinal plants. Cytotoxicity was tested by Hoechst 33342, Alamar Blue, calcein-AM, glutathione depletion and O(2)-consumption assays and genotoxicity by a Vitotox assay. Competition of the 12 extracts on substrate metabolism by CYP3A4, 2C9, 2C19 and 2D6 was tested with high-throughput CYP inhibition screening. Pregnane X receptor (PXR) activation was tested using Chinese hamster ovary cell lines expressing human PXR. Herbal extracts inducing high human PXR activation were tested for enhanced CYP3A4 mRNA levels with quantitative polymerase chain reaction. Genotoxicity was found for Jatropha multifida, Sterculia africana and Spirostachys africana. All plant extracts showed high cytotoxic effects in almost all tests. Potent competition with CYP3A4, 2D6, 2C9 and 2C19 was found for 75% of the herbal extracts. Spirostachys africana did not affect CYP2D6 and for S. africana and Turraea holstii no effect on CYP2D6 and CYP3A4 (DBF) was found. Nine plant extracts showed significant activation of human PXR, but only Agaura salicifolia, Turraea holstii and S. africana significantly induced CYP3A4 mRNA levels. These results indicate the possibility of potential medicinal plant-antiretroviral interactions.

Antifungal activity of some Tanzanian plants used traditionally for the treatment of fungal infections.

Using the ethnobotanical approach, some Tanzanian plants reported to be used by traditional healers for the treatment of oral candidiasis and fungal infections of the skin were collected and screened for their antifungal activity against Candida albicans, Candida glabrata, Candida tropicalis, Candida parapsilosis, Candida krusei and Cryptococcus neoformans. A total of 65 crude methanol extracts belonging to 56 plant species and 38 families were screened using the broth microdilution method, according to the guidelines of the Clinical and Laboratory Standard Institute (CLSI) (formerly, National Committee for Clinical and Laboratory Standards) [National Committee for Clinical Laboratory Standards, 2002. Reference Method for Broth Dilution Antifungal Susceptibility Testing of Yeasts. Approved Standard-2nd Edition M27-A2, National Committee for Clinical Laboratory Standards, Wayne, PA, USA]. Among the tested plant species, 45% (25 species) showed antifungal activity against one or more of the test fungi. The most susceptible yeasts were Cryptococcus neoformans, followed by Candida krusei, Candida tropicalis, and Candida parapsilosis. The least susceptible were Candida albicans and Candida glabrata. Strong antifungal activity was exhibited by extracts of Clausena anisata Oliv., Sclerocariya birrea Sond, Turraea holstii Gurk, Sterculia africana (Lour) Fiori, Acacia robusta subsp. Usambarensis (Taub) Brenan, Cyphosterma hildebrandti (Gilg), Desc, Elaeodendron buchannanii (Lows), Acacia nilotica (L.) Wild ex Del, Jatropha multifida L., and Pteridium aquilinum (L.) Kuhn.

Screening of Tanzanian medicinal plants for anti-Candida activity.

BACKGROUND: Candida albicans has become resistant to the already limited, toxic and expensive anti-Candida agents available in the market. These factors necessitate the search for new anti-fungal agents. METHODS: Sixty-three plant extracts, from 56 Tanzanian plant species obtained through the literature and interviews with traditional healers, were evaluated for anti-Candida activity. Aqueous methanolic extracts were screened for anti-Candida activity by bioautography agar overlay method, using a standard strain of Candida albicans (ATCC 90028). RESULTS: Twenty- seven (48%) out of the 56 plants were found to be active. Extracts of the root barks of Albizia anthelmintica and Balanites aegyptiaca, and roots of Plectranthus barbatus showed strong activity. CONCLUSION: The extracts that showed strong anti-Candida activity are worth of further investigation in order to isolate and identify the active compounds.

Brine shrimp toxicity evaluation of some Tanzanian plants used traditionally for the treatment of fungal infections.

Plants which are used by traditional healers in Tanzania have been evaluated to obtain preliminary data of their toxicity using the brine shrimps test. The results indicate that 9 out of 44 plant species whose extracts were tested exhibited high toxicity with LC(50) values below 20 microg/ml. These include Aloe lateritia Engl. (Aloaceae) [19.1 microg/ml], Cassia abbreviata Oliv. (Caesalpiniaceae) [12.7 microg/ml], Croton scheffleri Pax (Euphorbiaceae) [13.7 microg/ml], Hymenodactyon parvifolium Brig (Rubiaceae) [13.4 microg/ml], Kigelia Africana L. (Bignoniaceae) [7.2 microg/ml], and Ocimum suave Oliv. (Labiatae) [16.7 microg/ml]. Twelve plants gave LC(50) values between 21 and 50 microg/ml, 11 plants gave LC(50) values between 50 and 100 microg/ml, and 18 plants gave LC(50) values greater than 100 microg/ml.