Genome Sequencing Identifies Previously Unrecognized Klebsiella pneumoniae Outbreaks in Neonatal Intensive Care Units in the Philippines.

BACKGROUND: Klebsiella pneumoniae is a critically important pathogen in the Philippines. Isolates are commonly resistant to at least 2 classes of antibiotics, yet mechanisms and spread of its resistance are not well studied. METHODS: A retrospective sequencing survey was performed on carbapenem-, extended spectrum beta-lactam-, and cephalosporin-resistant Klebsiella pneumoniae isolated at 20 antimicrobial resistance (AMR) surveillance sentinel sites from 2015 through 2017. We characterized 259 isolates using biochemical methods, antimicrobial susceptibility testing, and whole-genome sequencing (WGS). Known AMR mechanisms were identified. Potential outbreaks were investigated by detecting clusters from epidemiologic, phenotypic, and genome-derived data. RESULTS: Prevalent AMR mechanisms detected include blaCTX-M-15 (76.8%) and blaNDM-1 (37.5%). An epidemic IncFII(Yp) plasmid carrying blaNDM-1 was also detected in 46 isolates from 6 sentinel sites and 14 different sequence types (STs). This plasmid was also identified as the main vehicle of carbapenem resistance in 2 previously unrecognized local outbreaks of ST348 and ST283 at 2 different sentinel sites. A third local outbreak of ST397 was also identified but without the IncFII(Yp) plasmid. Isolates in each outbreak site showed identical STs and K- and O-loci, and similar resistance profiles and AMR genes. All outbreak isolates were collected from blood of children aged < 1 year. CONCLUSION: WGS provided a better understanding of the epidemiology of multidrug resistant Klebsiella in the Philippines, which was not possible with only phenotypic and epidemiologic data. The identification of 3 previously unrecognized Klebsiella outbreaks highlights the utility of WGS in outbreak detection, as well as its importance in public health and in implementing infection control programs.

Impact on beef cattle productivity of infection with anthelmintic-resistant nematodes.

ABSTRACTAims: The main goal of the current study was to evaluate, on a commercial beef cattle farm, the impact of infection with gastrointestinal nematodes resistant to both ivermectin (IVM) and moxidectin (MXD) on the productivity of calves.Methods: Male Aberdeen Angus calves, aged 9-11 months, with faecal nematode egg counts (FEC) ≥200 epg and body weight ≥190 kg, were allocated to two herds. Herd A (n = 90) grazed a maize-winter forage crop rotation and Herd B (n = 90) grazed a 2-year-old Agropyrum pasture. On Day 0 in each herd, calves were randomly allocated into five groups (n = 18), which were treated with 0.2 mg/kg IVM; 0.2 mg/kg MXD; 3.75 mg/kg ricobendazole (RBZ), both IVM and RBZ, or remained untreated. Faecal samples collected on Days -1 and 19 were used to determine the percentage reduction in FEC, and genera of the nematodes were determined by the identification of the third-stage larvae recovered from faecal cultures. Total weight gain was determined from body weights recorded on Days -1 and 91.Results: Overall mean reduction in FEC was 42% for IVM, 67% for MXD, 97% for RBZ and 99% for IVM + RBZ. The reduction in FEC for Cooperia spp. was ≤78% for IVM and MXD, and for Haemonchus spp. was 0 and 36% for IVM and MXD, respectively, confirming the presence of parasites resistant to both anthelmintics. Only IVM + RBZ treatment resulted in 100% efficacy against Haemonchus spp. The overall estimated mean total weight gain for calves treated with IVM was 15.7 (95% CI = 11.9-19.7) kg and for calves treated with IVM + RBZ was 28.8 (95% CI = 25-32.5) kg (p < 0.001). Mean total weight gain for calves treated with MXD was 23.5 (95% CI = 19.7-27.2) kg.Conclusions and clinical relevance: In calves naturally infected with resistant nematodes, under the production system assessed here, weight gains were lower in calves treated with anthelmintics that were moderately or highly ineffective compared to those treated with highly effective anthelmintics. These results demonstrate to farmers and veterinarians the importance of a sustainable and effective nematode control under field conditions.

Failure of ivermectin efficacy against Psoroptes ovis infestation in cattle: Integrated pharmacokinetic-pharmacodynamic evaluation of two commercial formulations.

Psoroptic mange is an important parasitic disease that mainly affects beef cattle producing marked economic losses. Ivermectin (IVM) is considered one of the most effective treatments against psoroptic mange and is used worldwide to control both endo and ectoparasites in different species. The current work assessed the relationship between pharmacokinetic behavior of IVM and its efficacy against Psoroptes ovis after the subcutaneous administration of two commercial formulations in a cattle feedlot. Aberdeen Angus and Hereford steers were selected based on the presence of active mite infestations. Animals were allocated into 4 experimental groups and treated with a single (day 0) or repeated subcutaneous injection (days 0 and 7) of one of two commercial formulations of IVM (1%) at 0.2 mg/kg. Blood and skin samples were taken from 8 randomly selected animals of each experimental group to measure IVM concentrations by HPLC. Skin scrapings were also collected from six different sites in each animal, mites were counted and ranked based on a density score. Equivalent plasma concentrations of IVM were measured after the administration of IVM formulations under study. The repeated administration of both IVM formulations at day 0 and 7 accounted for a greater plasma drug availability compared with the single administration (P < 0.05). IVM was well distributed from the plasma to the skin without significant differences between both IVM formulations. There was a positive correlation between IVM concentrations in skin and plasma (r: 0.73 P < 0.0001). The mean ratios between IVM availabililty (measured as AUC) in the skin and in plasma were between 1.2 and 2.1. The repeated administration of IVM increased significantly the IVM concentrations in the skin of areas affected by mange. IVM failed to obtain a parasitological cure in the different groups affected by mange. The failure was observed with both formulations administeredat single or repeated doses. Based on the number of animals cured, the range of efficacy was between 0% on day 7 and 60% on day 28 post-treatment. No significant differences in the P. ovis density scores were observed after the IVM treatment at single or repeated doses. Additional studies are needed to confirm the presence of resistant strains of P.ovis and to establish the appropriate measures to control these parasitic infestations in feedlot cattle.

Pharmaco-parasitological evaluation of the ricobendazole plus levamisole nematodicidal combination in cattle.

The goals of the current study were to evaluate the potential pharmacokinetic (PK) interactions and the clinical efficacy occurring after the subcutaneous (s.c.) administration of ricobendazole (RBZ) and levamisole (LEV) given both separately and co-administered to calves naturally infected with susceptible gastrointestinal nematodes. The clinical efficacy was shown in two seasons, winter and spring, with predominance of different nematode populations. Groups of 15 calves were treated with RBZ alone, LEV alone and RBZ + LEV combination, and an untreated group was kept as a Control. RBZ and LEV plasma concentrations were quantified by HPLC. The clinical efficacy was determined by the faecal egg count reduction test. RBZ and LEV have similar plasma persistence, being detected in plasma over 24 hr post-treatment. No PK interactions were observed after the combined treatment, with similar PK parameters (p > .05) obtained for the single-drug and the combination-based strategy. In winter, the observed clinical efficacies were 96%, 99% and 100% for groups treated with RBZ, LEV and RBZ + LEV, respectively; however, in spring, the efficacies were 95%, 93% and 96% for the same groups. Remarkably, the combination was the only treatment that achieved 100% clinical efficacy against both Haemonchus spp and Ostertagia spp in winter; but the increased presence of Ostertagia spp. in spring (28% in untreated group) determined a tendency to reduced efficacies compared to winter time (only 10% of Ostertagia spp. in untreated group), even for the combined treatment. Overall, in a scenario where the nematode population is susceptible, the RBZ + LEV treatment may be a valid combination in cattle to delay the development of resistance, especially in winter when this combination achieved 100% of efficacy. Thus, selection of anthelmintic resistance will never occur. In fact, this is one of the greatest challenges for the whole cattle production system: to be one step ahead of anthelmintic resistance.

An attempt to replace an ivermectin-resistant Cooperia spp. population by a susceptible one on grazing pastures based on epidemiological principles and refugia management.

The maintenance of anthelmintic-susceptible parasite refugia to delay the onset of anthelmintic resistance is an almost impossible effort in many grazing livestock production countries given that current refugia consist of already resistant parasites. Rather, efforts could be focused on replacing the resistant parasite refugia by susceptible parasite ones and implementing sustainable parasite control measures from then on. To this purpose, a trial was conducted to attempt to establish a new population of ivermectin-susceptible Cooperia sp. on a beef cattle farm with proven problems of ivermectin-resistant Cooperia. During two consecutive years, 82 (Year 1) and 100 (Year 2) recently weaned and parasite-free heifers were inoculated with 40,000 or 30,000 susceptible Cooperia L3, respectively, at a time when levels of resistant parasite refugia were normally low. The animals were subsequently allowed to graze on the problem pastures during autumn until the end of spring. Levels of parasitism in the animals and on pasture were monitored monthly and animals were treated with levamisole when needed. The combination of parasitological monitoring and local epidemiological knowledge was essential to determine when treatments were to be administered. No clinical signs of gastrointestinal parasitosis in the herd were observed throughout the study and unnecessary treatments were avoided. Faecal egg counts reduction tests (FECRT) and controlled efficacy tests (CET) employing worm counts were carried out at different times throughout the study to determine the clinical efficacy (FECRT) and the absolute efficacy (CET) of ivermectin, respectively. The clinical efficacy of ivermectin increased from an initial 73% to 99.4%, while the absolute efficacy increased from 54.1% to 87.5% after just two animal production cycles. The switch from a resistant parasite population to a susceptible one requires knowledge of parasitological epidemiology, especially in relation to seasonal variations of parasite populations in both the host and in refugia.

Observations on the free-living stages of cattle gastrointestinal nematodes.

A 4-year study on the free-living stages of cattle gastrointestinal nematodes was conducted to determine (a) the development time from egg to infective larvae (L3) inside the faecal pats, (b) the pasture infectivity levels over time, and (c) the survival of L3 on pasture. Naturally infected calves were allowed to contaminate 16 plots on monthly basis. Weekly monitoring of eggs per gram of faeces (epg) values and faecal cultures from these animals provided data for the contamination patterns and the relative nematode population composition. At the same time, faecal pats were shaped and deposited monthly onto herbage and sampled weekly to determine the development time from egg to L3. Herbage samples were collected fortnightly over a 16-month period after deposition to evaluate the pasture larval infectivity and survival of L3 over time. The development time from egg to L3 was 1-2 weeks in summer, 3-5 weeks in autumn, 4-6 weeks in winter, and 1-4 weeks in spring. The levels of contamination and pasture infectivity showed a clear seasonality during autumn-winter and spring, whilst a high mortality of larvae on pasture occurred in summer. Ostertagia spp., Cooperia spp. and Trichostrongylus spp. were predominant and a survival of L3 on pasture over a 1-year period was recorded in this study.

Reproductive disruption in Fasciola hepatica associated with incomplete efficacy of a new experimental formulation of triclabendazole.

The objective of the present study was to analyse the reproductive viability (using histopathologic studies) of Fasciola hepatica from cattle artificially infected and treated subcutaneously with a new experimental formulation of triclabendazole (8mg/kg b.w.). The results of the efficacy controlled test, which only takes into account the presence of live adult flukes, indicated that, whilst in the control group (n=7) 533 live specimens were recovered, in the test groups (doses of 8 and 12mg/kg b.w.) only 195 and 47 adults were recovered, respectively. These numbers indicate efficacies of 69% and 95.6%, respectively. It was observed in that dose of 8mg/kg b.w. some specimens remained viable, but they were infertile, which severely compromises the biological cycle of the trematode. In the testis tubules of flukes treated with the low dose of TCBZ (8mg/kg), very few cells were present and the vitelline follicles were markedly reduced in size and each follicle contained very few cells. This would have direct implications for the pathogenesis of the parasitosis since the remaining parasites would produce little clinical-productive manifestations, would stimulate the immune response and would find it difficult to establish future re-infestations/re-infections. Consequently, these observations will also prompt a review of certain methodological and interpretative aspects related to efficacy tests, where the only discriminative factor is the reduction of the adult parasite load. On one hand, histopathological studies could be complementary to the efficacy controlled test for TCBZ or other BZD formulations.

Environmental impact of ivermectin excreted by cattle treated in autumn on dung fauna and degradation of faeces on pasture.

The effect of ivermectin excreted in faeces of treated cattle on dung fauna and dung degradation on pasture during autumn was evaluated. Two groups of calves were used. One group was treated subcutaneously with ivermectin while the other remained as untreated control. Faeces deposited on 1, 3, 7, 14 and 21 days post-treatment (dpt) were removed on 1, 3, 7, 14, 21, 30 and 60 days post-deposition (dpd) and were used to determine the concentration of ivermectin and the percentage of organic matter and for the collection of colonising organisms. Samples from 1 and 3 dpt contained the highest drug concentration and percentage of organic matter compared to the control group (p<0.05). Faeces from the treated group showed lesser abundance and diversity of arthropods (p<0.05) than the control group. A reduction in numbers and diversity of dung fauna in faecal samples from treated animals was most remarkable at 1, 3 and 7 dpt, coinciding with the highest concentration of ivermectin and organic matter percentage.

Changes to oxfendazole chiral kinetics and anthelmintic efficacy induced by piperonyl butoxide in horses.

REASONS FOR PERFORMING THE STUDY: The study of novel pharmacological strategies to control parasitism in horses is required since many parasite species have developed resistance to anthelmintic drugs. OBJECTIVES: To evaluate the effects of piperonyl butoxide (PB) (a metabolic inhibitor) on the plasma availability and enantiomeric behaviour of oxfendazole (OFZ) given orally to horses, and to compare the clinical efficacy of OFZ given either alone or co-administered with PB in naturally parasitised horses. METHODS: Fifteen naturally parasitised crossbred male ponies were allocated into 3 groups (n = 5) and treated orally as follows: Group I (control) received distilled water as placebo; Group II was dosed with OFZ (10 mg/kg bwt); and Group III was treated with OFZ (10 mg/kg bwt) co-administered with PB (63 mg/kg bwt). Jugular blood samples were obtained over 120 h post treatment. Three weeks after treatments, all experimental horses were subjected to euthanasia. RESULTS: The observed maximum plasma concentration (Cmax) and area under the concentration vs. time curve (AUC) values for OFZ increased 3- and 5-fold, respectively, in the presence of PB. The plasma concentration profiles of fenbendazole (FBZ), a metabolite generated from OFZ, were significantly lower after the treatment with OFZ alone (AUC = 0.8 microg x h/ml) compared to those obtained after the OFZ + PB treatment (AUC = 2.7 microg x h/ml). The enhanced pharmacokinetic profiles correlated with increased anthelmintic efficacy. The combination OFZ + PB showed 100% efficacy against mature nematode parasites. The efficacy against cyathostome L3 larvae increased from 94% (Group II) to 98.7% (Group III). Consistently, the number of L4 larvae recovered from OFZ + PB treated horses (Group III) (n = 146) was significantly lower (P<0.05) than that recovered from Group II (n = 1397). CONCLUSIONS: The use of PB as a metabolic inhibitor may be useful to enhance OFZ activity against mature and migrating larvae of different parasite species in horses. POTENTIAL RELEVANCE: Metabolic inhibitors may be used to enhance the activity of benzimidazole anthelmintics and extend the effective lifespan of benzimidazole drugs in the face of increasing resistance.

Arrested development of Ostertagia ostertagi: effect of the exposure of infective larvae to natural spring conditions of the humid pampa (Argentina).

The aim of this study was to determine the effect of environmental conditions and the time of exposure to the conditions required for Ostertagia ostertagi to become inhibited in development at the early fourth larval stage in the host. Two comparable experiments were conducted from September to January, experiment I in 1997-1998 and experiment II in 1999-2000. Twenty-thousand third-stage larvae (L3), freshly obtained from coprocultures, were spread in different parasite-free grass plots at the beginning of September, October and November in each experiment and exposed to environmental conditions throughout spring and early summer. Duplicate plots for each exposure period were grazed for 3 days by two dewormed tracer calves after 1, 2, 3, 4 weeks of exposure during the corresponding month, and the remaining plots were grazed for 3 days at monthly intervals until the end of the experimental period. For each month in both experiments, control animals were inoculated orally with 20,000 L3 newly recovered from coprocultures (week 0 animals; infection controls). The control and tracer calves were sacrificed and their parasite burdens analysed. The time required to obtain greater than 50% inhibited larvae (IeL4) in the tracer animals during September and October was 3 weeks, whereas during November around 60% of the parasites were inhibited after one week of exposure. During the period tested, greater than 50% inhibition was found in concurrence with a photoperiod ranging between 13 and 14 h. The highest proportion of IeL4 (75% average) in the animals was found concomitant with a 14 h 43 min photoperiod. A high correlation between the percentage of inhibition and day length was established (0.870 p < 0.001 and 0.815 p < 0.001 for experiment I and II, respectively). In both years, the capacity for developmental arrest was lost by the end of December, when the photoperiod begins to decrease, suggesting either a disappearance of the induction stimulus, or that an excess of the stimulus could block the mechanism of inhibition. The induction time was extended 2 weeks in all months tested when the coprocultures were maintained in the dark (experiment II), suggesting that accumulation of the light stimulus contributes to shortening of the induction time. The data presented here would suggest that photoperiod is a key environmental factor for the induction of hypobiosis.

Resistance of Cooperia to ivermectin treatments in grazing cattle of the Humid Pampa, Argentina.

A field experiment and a controlled efficacy trial were conducted to demonstrate resistance of cattle trichostrongylid nematodes to endectocidal compounds in grazing cattle on the Humid Pampa, Argentina. Male Polled Hereford calves, aged 9-11 months old, with a history of frequent treatments with compounds of the avermectin/milbemycin class, were used for the trial. The field experiment involved six groups of 10 animals each, which were subcutaneously treated with either ivermectin (IVM), long-acting ivermectin (LA-IVM), moxidectin (MXD) or doramectin (DRM) at a dosage of 0.2mg/kg BW. A fifth group was treated orally with fenbendazole at a dosage of 5mg/kg BW; the sixth group of calves served as non-treated control. Faecal samples were collected from each animal on the day of treatment and at 14 days after treatment. Nematode egg counts were performed to estimate the faecal egg count reduction test (FECRT). The FECRT showed that reductions were 65% (IVM), -20% (LA-IVM) and 85% (DRM). Egg counts from calves treated with MXD or fenbendazole were reduced by 95 and 100%, respectively. For the controlled efficacy trial, six animals with the highest egg counts from the IVM, LA-IVM and non-treated control groups, were necropsied on day 18 after treatment to determine numbers and nematode species in the abomasum and small intestine. The results indicated that efficacy against Cooperia oncophora was 62.7 and 48% for IVM and LA-IVM, respectively. Neither of the IVM formulations showed efficacy against Trichostrongylus spp. in the small intestine. However, the absence of Trichostrongylus spp. in some animals of both treated and control groups precluded a valid assessment of efficacy or resistance. It was concluded that resistance of trichostrongylid nematodes to the avermectin/milbemycin class of compounds in grazing cattle of the Humid Pampa, Argentina, may be strongly established on farms where cattle are treated at frequent intervals throughout the year.

Hypobiosis induction alters the protein profile of Ostertagia ostertagi (Nematoda: Trichostrongylidae).

The appearance of variations in the protein profile of Ostertagia ostertagi (Stiles, 1892) infective larvae (L3), which were induced by hypobiosis triggering factors, was evaluated by means of SDS-PAGE and densitometric analysis. Area integration analyses of their protein profiles was carried out between 66 and 77 kDa. Important quantitative variations were identified in the protein levels of the induced larvae, where a 5.25 fold increase compared to the control was observed. Two 75.4 and 70 kDa protein bands were found which exceeded the control profile by 4.5 and 44 fold, respectively. This fact suggests that the changes brought about in the process of hypobiosis induction are restricted. This work demonstrates changes at a molecular level corresponding with biological changes induced by conditions causing O. ostertagi hypobiosis.

Study on the inductive factors of hypobiosis of Ostertagia ostertagi in cattle.

Two experiments were carried out to determine the causes producing the Ostertagia ostertagi hypobiosis phenomenon in cattle. In the first experiment, the effect of time on third-stage larvae in the environment was studied during a 2-year period. Three experimental paddocks contaminated with O. ostertagi eggs at different times of the year were used, and the levels of hypobiosis were recorded by using 'indicator' and 'tracer' calves. The results suggest that time as such is not a hypobiosis-inductive factor. The second experiment was conducted under laboratory conditions, where the effects of temperature and light on infective larvae were studied. Infective larvae were subjected to different conditions of temperature and light during 6 weeks, and then inoculated to parasite-naive calves, which were slaughtered after 4 weeks. Percentages of hypobiotic larvae in these calves varied from 3.5 to 94.8%, depending on the different storage conditions the larvae underwent before inoculation. Results suggest that increasing temperature and increasing time of light exposure simulating spring conditions would be the factors which act upon third-stage larvae inducing them to a later hypobiotic stage in the host.