Antibiotic resistance in tick-borne bacteria: A One Health approach perspective.

The emergence and re-emergence of tick-borne bacteria (TBB) as a public health problem raises the uncertainty of antibiotic resistance in these pathogens, which could be dispersed to other pathogens. The impact of global warming has led to the emergence of pathogenic TBB in areas where they were not previously present and is another risk that must be taken into account under the One Health guides. This review aimed to analyze the existing information regarding antibiotic-resistant TBB and antibiotic-resistance genes (ARG) present in the tick microbiome, considering the potential to be transmitted to pathogenic microorganisms. Several Ehrlichia species have been reported to exhibit natural resistance to fluoroquinolones and typhus group Rickettsiae are naturally susceptible to erythromycin. TBB have a lower risk of acquiring ARG due to their natural habitat, but there is still a probability of acquiring them; furthermore, studies of these pathogens are limited. Pathogenic and commensal bacteria coexist within the tick microbiome along with ARGs for antibiotic deactivation, cellular protection, and efflux pumps; these ARGs confer resistance to antibiotics such as aminoglycosides, beta-lactamase, diaminopyrimidines, fluoroquinolones, glycopeptides, sulfonamides, and tetracyclines. Although with low probability, TBB can be a reservoir of ARGs.

Mexican Oregano (Lippia berlandieri Schauer and Poliomintha longiflora Gray) Essential Oils Induce Cell Death by Apoptosis in Leishmania (Leishmania) mexicana Promastigotes.

Leishmaniasis is a neglected vector-borne disease; there are different manifestations of the diseases and species involved, and cutaneous leishmaniasis caused by Leishmania (L.) mexicana is the most prevalent in Mexico. Currently, the drugs available for the treatment of leishmaniasis are toxic, expensive, and often ineffective; therefore, it is imperative to carry out research and development of new therapeutic alternatives, with natural products being an attractive option. In particular, oregano is a plant with worldwide distribution; in Mexico, two species: Lippia berlandieri Schauer and Poliomintha longiflora Gray are endemic. Both essential oils (EO's) have been reported to have antimicrobial activity attributed to their main components, thymol and carvacrol. In this research, the leishmanicidal effect and mechanism of cell death induced by L. berlandieri EO, P. longiflora EO, thymol, and carvacrol in L. mexicana promastigotes were determined in vitro. Additionally, the cytotoxic activity in mammalian cells was evaluated. L. berlandieri EO presented higher leishmanicidal activity (IC50 = 41.78 µg/mL) than P. longiflora EO (IC50 = 77.90 µg/mL). Thymol and carvacrol were the major components of both Mexican oregano EO's. Thymol presented higher leishmanial inhibitory activity (IC50 = 22.39 µg/mL), above that of carvacrol (IC50 = 61.52 µg/mL). All the EO's and compounds evaluated presented lower cytotoxic activity than the reference drug; thymol was the compound with the best selectivity index (SI). In all cases, apoptosis was identified as the main mechanism of death induced in the parasites. The leishmanicidal capacity of the Mexican oregano EO is an accessible and affordable alternative that can be further explored.

Detection of Antibodies to Lokern, Main Drain, St. Louis Encephalitis, and West Nile Viruses in Vertebrate Animals in Chihuahua, Guerrero, and Michoacán, Mexico.

We conducted serologic surveillance for flaviviruses and orthobunyaviruses in vertebrate animals in Mexico in 2018-2019. Sera were collected from 856 vertebrate animals, including 323 dogs, 223 horses, and 121 cows, from 16 species. The animals were from 3 states: Chihuahua in northwest Mexico (704 animals) and Guerrero and Michoacán on the Pacific Coast (27 and 125 animals, respectively). Sera were assayed by plaque reduction neutralization test using four flaviviruses (dengue type 2, St. Louis encephalitis, West Nile, and Zika viruses) and six orthobunyaviruses from the Bunyamwera (BUN) serogroup (Cache Valley, Lokern, Main Drain, Northway, Potosi, and Tensaw viruses). Antibodies to West Nile virus (WNV) were detected in 154 animals of 9 species, including 89 (39.9%) horses, 3 (21.4%) Indian peafowl, and 41 (12.7%) dogs. Antibodies to St. Louis encephalitis virus (SLEV) were detected in seven animals, including three (0.9%) dogs. Antibodies to Lokern virus (LOKV) were detected in 22 animals: 19 (8.5%) horses, 2 (1.7%) cows, and a dog (0.3%). Antibodies to Main Drain virus (MDV) were detected in three (1.3%) horses. WNV and LOKV activity was detected in all three states, SLEV activity was detected in Chihuahua and Michoacán, and MDV activity was detected in Chihuahua. None of the animals was seropositive for Cache Valley virus, the most common and widely distributed BUN serogroup virus in North America. In conclusion, we provide serologic evidence that select flaviviruses and BUN serogroup viruses infect vertebrate animals in Chihuahua, Guerrero, and Michoacán. We also provide the first evidence of LOKV and MDV activity in Mexico.

Antibiotics, Multidrug-Resistant Bacteria, and Antibiotic Resistance Genes: Indicators of Contamination in Mangroves?

Multidrug-resistant bacteria and antibiotic resistance genes can be monitored as indicators of contamination in several environments. Mangroves are among the most productive ecosystems, and although they can be resilient to the action of climate phenomena, their equilibrium can be affected by anthropogenic activities. Regarding the presence and persistence of multidrug-resistant bacteria in mangroves, it is common to think that this ecosystem can function as a reservoir, which can disperse the antibiotic resistance capacity to human pathogens, or serve as a filter to eliminate drug-resistant genes. The possible impact of anthropogenic activities carried out near mangroves is reviewed, including wastewater treatment, food production systems, leisure, and tourism. Adverse effects of antibiotic resistance genes or multidrug-resistant bacteria, considered as emerging contaminants, have not been reported yet in mangroves. On the contrary, mangrove ecosystems can be a natural way to eliminate antibiotics, antibiotic-resistant bacteria, and even antibiotic-resistant genes from the environment. Although mangroves' role in decreasing antibiotics and antibiotic resistance genes from the environment is being proposed, the mechanisms by which these plants reduce these emerging contaminants have not been elucidated and need further studies. Additionally, further evaluation is needed on the effects of antibiotics and antibiotic-resistant bacteria in mangroves to generate an analysis of the human contribution to the degradation of this specific ecosystem as well as to define if these contaminants can be used as indicators of contamination in mangrove ecosystems.

Demonstrating the presence of Ehrlichia canis DNA from different tissues of dogs with suspected subclinical ehrlichiosis.

BACKGROUND: Nowadays, Ehrlichia canis receives increasing attention because of its great morbidity and mortality in animals. Dogs in the subclinical and chronic phases can be asymptomatic, and serological tests show cross-reactivity and fail to differentiate between current and past infections. Moreover, there could be low parasitaemia, and E. canis might be found only in target organs, hence causing results to be negative by polymerase chain reaction (PCR) on blood samples. METHODS: We evaluated by PCR the prevalence of E. canis in blood, liver, spleen, lymph node and bone marrow samples of 59 recently euthanised dogs that had ticks but were clinically healthy. RESULTS: In total, 52.55% of the blood PCRs for E. canis were negative, yet 61.30% yielded positive results from tissue biopsies and were as follows: 63.15% from bone marrow; 52.63% from liver; 47.36% from spleen; and 15.78% from lymph node. In addition, 33% had infection in three tissues (spleen, liver and bone marrow). CONCLUSIONS: Our results show the prevalence of E. canis from tissues of dogs that were negative by blood PCR. Ehrlichia canis DNA in tissue was 30% lower in dogs that tested negative in PCR of blood samples compared to those that were positive. However, it must be taken into account that some dogs with negative results were positive for E. canis in other tissues.

[Effectivity of diagnostic methods for the detection of human and canine monocytic ehrlichiosis]. / Efectividad de los métodos diagnósticos para la detección de ehrlichiosis monocítica humana y canina.

Ehrlichiosis is a disease transmitted by tick's bite that affect dogs and humans caused by the species Ehrlichia canis and E. chaffeensis, respectively. These bacteria are obligated intracellular gram negatives, with a cocoid to pleomorph aspect and can infect monocytes and trigger symptoms such as high fever, anorexia, thrombocytopenia, hemorrhages, anemia, and some serious problems such as splenomegaly, hepatomegaly and meningitis. There are several diagnostic tests for ehrlichiosis such as the hematological ones that evaluate the morphology of the monocytes in search of morulae; serological tests that includes the search of anti-Ehrlichia antibodies, although they might be limited due to cross reaction with other species. In other hand, the culture of Ehrlichia species is an effective method to obtain antigens and even develop indirect immunofluorescence assays (IFA). The polymerase chain reaction offers a definitive diagnosis associated to the use of genus-specific and species-specific primers, as well as its increased sensibility and specificity, compared to the others methods. Thus, in this review, we will discuss various methods applied to the diagnosis of this disease, as well as the advantages and disadvantages that these present.

Susceptibility of Multidrug-Resistant and Biofilm-Forming Uropathogens to Mexican Oregano Essential Oil.

Antibiotic resistance along with biofilm formation increases the difficulty for antibiotic therapy in urinary tract infections. Bioactive molecules derived from plants, such as those present in essential oils, can be used to treat bacterial infections. Oregano is one of the spices to have antimicrobial activity. Therefore, three Mexican oregano essential oils (two Lippia berlandieri Schauer and one Poliomintha longiflora) were tested for antimicrobial capacity against multidrug-resistant, biofilm-forming bacterial isolates. Clinical isolates from urinary tract infections were tested for antibiotic resistance. Multidrug-resistant isolates were evaluated for biofilm formation, and Mexican oregano antimicrobial effect was determined by the minimal inhibitory (CMI) and minimal bactericidal concentrations (CMB). The selected isolates were identified by molecular phylogenetic analysis. Sixty-one isolates were included in the study; twenty were characterized as multidrug-resistant and from those, six were strong biofilm formers. Three isolates were identified as Escherichia coli, two as Pseudomonas aeruginosa and one as Enterococcus faecalis based on the phylogenetic analysis of 16 S rRNA gene sequences. The antimicrobial effect was bactericidal; E. faecalis was the most susceptible (<200 mg/L CMI/CMB), and P. aeruginosa was the most resistant (>2,000 mg/L CMI/CMB). There was a range of 500-1000 mg/L (CMI/CMB) for the E. coli isolates. Mexican oregano essential oils demonstrated antimicrobial efficacy against multidrug-resistant clinical isolates.

Efectividad de los métodos diagnósticos para la detección de ehrlichiosis monocítica humana y canina / Effectivity of diagnostic methods for the detection of human and canine monocytic ehrlichiosis

Resumen La ehrlichiosis es una enfermedad transmitida por la picadura de garrapatas que afecta a perros y humanos, causada por las especies Ehrlichia canis y E. chaffeensis, respectivamente. Estas bacterias son gramnegativas, intracelulares obligadas, de aspecto cocoide a pleomorfo, que infectan los monocitos y desencadenan síntomas como fiebre elevada, anorexia, trombocitopenia, hemorragias, anemia y problemas graves como esplenomegalia, hepatomegalia y meningitis. Para diagnosticar esta enfermedad existen diversos métodos, entre los que se encuentran los hematológicos que evalúan la morfología de los monocitos en búsqueda de mórulas y la serología, que incluye la búsqueda de anticuerpos anti-Ehrlichia, pero que se encuentra limitado debido a la reactividad cruzada que presenta. Por otra parte, el cultivo de especies de Ehrlichia ha resultado ser un método efectivo para la obtención de antígenos y así desarrollar ensayos por inmunofluorescencia indirecta (IFI). El método por reacción de polimerasa en cadena ofrece un diagnóstico definitivo por tener una mayor sensibilidad y especificidad que los otros métodos, al haberse desarrollado cebadores género-específicos, así como especie-específicos. En esta revisión, se discutirán los diversos métodos aplicados al diagnóstico de esta enfermedad, así como las ventajas y desventajas que estos presentan.

Ehrlichiosis is a disease transmitted by tick's bite that affect dogs and humans caused by the species Ehrlichia canis and E. chaffeensis, respectively. These bacteria are obligated intracellular gram negatives, with a cocoid to pleomorph aspect and can infect monocytes and trigger symptoms such as high fever, anorexia, thrombocytopenia, hemorrhages, anemia, and some serious problems such as splenomegaly, hepatomegaly and meningitis. There are several diagnostic tests for ehrlichiosis such as the hematological ones that evaluate the morphology of the monocytes in search of morulae; serological tests that includes the search of anti-Ehrlichia antibodies, although they might be limited due to cross reaction with other species. In other hand, the culture of Ehrlichia species is an effective method to obtain antigens and even develop indirect immunofluorescence assays (IFA). The polymerase chain reaction offers a definitive diagnosis associated to the use of genus-specific and species-specific primers, as well as its increased sensibility and specificity, compared to the others methods. Thus, in this review, we will discuss various methods applied to the diagnosis of this disease, as well as the advantages and disadvantages that these present.

Assessment of antibody-dependent respiratory burst activity from mouse neutrophils on Plasmodium yoelii malaria challenge outcome.

New tools are required to expedite the development of an effective vaccine against the blood-stage infection with the human malaria parasite Plasmodium falciparum. This work describes the assessment of the ADRB assay in a mouse model, characterizing the functional interaction between antimalarial serum antibodies and FcRs upon neutrophils. We describe a reproducible, antigen-specific assay, dependent on functional FcR signaling, and show that ADRB activity is induced equally by IgG1 and IgG2a isotypes and is modulated by blocking FcR function. However, following immunization of mice with the blood-stage vaccine candidate antigen MSP142, no measurable ADRB activity was induced against PEMS and neither was vaccine efficacy modulated against Plasmodium yoelii blood-stage challenge in γ(-/-) mice compared with WT mice. In contrast, following a primary, nonlethal P. yoelii parasite challenge, serum from vaccinated mice and nonimmunized controls showed anti-PEMS ADRB activity. Upon secondary challenge, nonimmunized γ(-/-) mice showed a reduced ability to control blood-stage parasitemia compared with immunized γ(-/-) mice; however, WT mice, depleted of their neutrophils, did not lose their ability to control infection. Thus, whereas neutrophil-induced ADRB against PEMS does not appear to play a role in protection against P. yoelii rodent malaria, induction of ADRB activity after challenge suggests that antigen targets of anti-PEMS ADRB activity remain to be established, as well as further supporting the observation that ADRB activity to P. falciparum arises following repeated natural exposure.

The generation and evaluation of two panels of epitope-matched mouse IgG1, IgG2a, IgG2b and IgG3 antibodies specific for Plasmodium falciparum and Plasmodium yoelii merozoite surface protein 1-19 (MSP1(19)).

Murine immunoglobulin G (IgG) plays an important role in mediating protective immune responses to malaria. We still know relatively little about which IgG subclasses protect against this disease in mouse models, although IgG2a and IgG2b are considered to be the most potent and dominate in successful passive transfer experiments in rodent malarias. To explore the mechanism(s) by which the different mouse IgG subclasses may mediate a protective effect, we generated mouse IgG1, IgG2a, IgG2b and IgG3 specific for the C-terminal 19-kDa region of Plasmodium falciparum merozoite surface protein 1 (PfMSP1(19)), and to the homologous antigen from Plasmodium yoelii (P. yoelii), both major targets of protective immune responses. This panel of eight IgGs bound antigen with an affinity comparable to that seen for their epitope-matched parental monoclonal antibodies (mAbs) from which they were derived, although for reasons of yield, we were only able to explore the function of mouse IgG1 recognizing PfMSP1(19) in detail, both in vitro and in vivo. Murine IgG1 was as effective as the parental human IgG from which it was derived at inducing NADPH-mediated oxidative bursts and degranulation from neutrophils. Despite showing efficacy in in vitro functional assays with neutrophils, the mouse IgG1 failed to protect against parasite challenge in vivo. The lack of protection afforded by MSP1(19)-specific IgG1 against parasite challenge in wild type mice suggests that this Ab class does not play a major role in the control of infection with mouse malaria in the Plasmodium berghei transgenic model.

The generation and evaluation of recombinant human IgA specific for Plasmodium falciparum merozoite surface protein 1-19 (PfMSP1 19).

BACKGROUND: Human immunoglobulin G (IgG) plays an important role in mediating protective immune responses to malaria. Although human serum immunoglobulin A (IgA) is the second most abundant class of antibody in the circulation, its contribution, if any, to protective responses against malaria is not clear. RESULTS: To explore the mechanism(s) by which IgA may mediate a protective effect, we generated fully human IgA specific for the C-terminal 19-kDa region of Plasmodium falciparum merozoite surface protein 1 (PfMSP1 19), a major target of protective immune responses. This novel human IgA bound antigen with an affinity comparable to that seen for an epitope-matched protective human IgG1. Furthermore, the human IgA induced significantly higher NADPH-mediated oxidative bursts and degranulation from human neutrophils than the epitope-matched human IgG1 from which it was derived. Despite showing efficacy in in vitro functional assays, the human IgA failed to protect against parasite challenge in vivo in mice transgenic for the human Fcα receptor (FcαRI/CD89). A minority of the animals treated with IgA, irrespective of FcαRI expression, showed elevated serum TNF-α levels and concomitant mouse anti-human antibody (MAHA) responses. CONCLUSIONS: The lack of protection afforded by MSP1 19-specific IgA against parasite challenge in mice transgenic for human FcαRI suggests that this antibody class does not play a major role in control of infection. However, we cannot exclude the possibility that protective capacity may have been compromised in this model due to rapid clearance and inappropriate bio-distribution of IgA, and differences in FcαRI expression profile between humans and transgenic mice.

Inhibition of erythrocyte invasion and Plasmodium falciparum merozoite surface protein 1 processing by human immunoglobulin G1 (IgG1) and IgG3 antibodies.

Antigen-specific antibodies (Abs) to the 19-kDa carboxy-terminal region of Plasmodium falciparum merozoite surface protein 1 (MSP1(19)) play an important role in protective immunity to malaria. Mouse monoclonal Abs (MAbs) 12.10 and 12.8 recognizing MSP1(19) can inhibit red cell invasion by interfering with MSP1 processing on the merozoite surface. We show here that this ability is dependent on the intact Ab since Fab and F(ab')(2) fragments derived from MAb 12.10, although capable of binding MSP1 with high affinity and competing with the intact antibody for binding to MSP1, were unable to inhibit erythrocyte invasion or MSP1 processing. The DNA sequences of the variable (V) regions of both MAbs 12.8 and 12.10 were obtained, and partial amino acid sequences of the same regions were confirmed by mass spectrometry. Human chimeric Abs constructed by using these sequences, which combine the original mouse V regions with human gamma1 and gamma3 constant regions, retain the ability to bind to both parasites and recombinant MSP1(19), and both chimeric human immunoglobulin G1s (IgG1s) were at least as good at inhibiting erythrocyte invasion as the parental murine MAbs 12.8 and 12.10. Furthermore, the human chimeric Abs of the IgG1 class (but not the corresponding human IgG3), induced significant NADPH-mediated oxidative bursts and degranulation from human neutrophils. These chimeric human Abs will enable investigators to examine the role of human Fcgamma receptors in immunity to malaria using a transgenic parasite and mouse model and may prove useful in humans for neutralizing parasites as an adjunct to antimalarial drug therapy.