Pyogenic liver abscess caused by an atypical hypervirulent Klebsiella pneumoniae K1-ST23 in Mexico.

Hypervirulent K. pneumoniae infection has been raising worldwide and is one of the major causes of community-acquired pyogenic liver abscess. We described a case report of pyogenic liver abscess caused by an atypical hypervirulent (non-hypermucoviscous) K. pneumoniae K1 ST23 in a diabetic Asian patient who resided in Mexico. The susceptibility to antimicrobials, pathogenicity, molecular and genomic analysis were determined. A man from Guangdong (China) with a recent diagnosis of diabetes mellitus was admitted to the hospital, and he denied traveling in the last 3 months. A computed tomography revealed a right lobe liver abscess. On the third day after admission a Klebsiella pneumoniae isolate (14652) was obtained. The isolate corresponded to a susceptible K. pneumoniae with capsular type K1 and ST23 (CG23) and exhibited a non-hypermucoviscous phenotype. The isolate 14652 was genetically related to the globally distributed lineage ST23-KL1. This study describes the first case in Mexico of K. pneumoniae capsular type K1 and ST23 with an atypical hypervirulent phenotype.

Comprehensive study reveals phenotypic heterogeneity in Klebsiella pneumoniae species complex isolates.

Here, we conducted a comprehensive analysis of 356 Klebsiella pneumoniae species complex (KpSC) isolates that were classified as classical (cl), presumptive hypervirulent (p-hv) and hypermucoviscous-like (hmv-like). Overall, K. pneumoniae (82.3%), K. variicola (2.5%) and K. quasipneumoniae (2.5%) were identified. These isolates comprised 321 cl-KpSC, 7 p-hv-KpSC and 18 hmv-like-KpSC. A large proportion of cl-KpSC isolates were extended-spectrum-ß-lactamases (ESBLs)-producers (64.4%) and 3.4% of isolates were colistin-resistant carrying carbapenemase and ESBL genes. All p-hv-KpSC showed an antibiotic susceptible phenotype and hmv-like isolates were found to be ESBL-producers (8/18). Assays for capsule production and capsule-dependent virulence phenotypes and whole-genome sequencing (WGS) were performed in a subset of isolates. Capsule amount differed in all p-hv strains and hmv-like produced higher capsule amounts than cl strains; these variations had important implications in phagocytosis and virulence. Murine sepsis model showed that most cl strains were nonlethal and the hmv-like caused 100% mortality with 3 × 108 CFUs. Unexpectedly, 3/7 (42.9%) of p-hv strains required 108 CFUs to cause 100% mortality (atypical hypervirulent), and 4/7 (57.1%) strains were considered truly hypervirulent (hv). Genomic analyses confirmed the diverse population, including isolates belonging to hv clonal groups (CG) CG23, CG86, CG380 and CG25 (this corresponded to the ST3999 a novel hv clone) and MDR clones such as CG258 and CG147 (ST392) among others. We noted that the hmv-like and hv-ST3999 isolates showed a close phylogenetic relationship with cl-MDR K. pneumoniae. The information collected here is important to understand the evolution of clinically important phenotypes such as hypervirulent and ESBL-producing-hypermucoviscous-like amongst the KpSC in Mexican healthcare settings. Likewise, this study shows that mgrB inactivation is the main mechanism of colistin resistance in K. pneumoniae isolates from Mexico.

Genome sequence of a virulent and hypermucoviscous-like Klebsiella michiganensis clinical isolate.

OBJECTIVES: The hypermucoviscous-like phenotype has been described in Klebsiella pneumoniae species complex (KpSC) and was described as a contributor of increased virulence. This study described the characterization and whole-genome sequencing of an antibiotic susceptible and hypermucoviscous-like Klebsiella michiganensis 9273 clinical isolate. DATA DESCRIPTION: Here, we report the genome sequence of a K. michiganensis clinical isolate obtained from a urinary tract infection exhibiting the hypermucoviscous-like phenotype. The draft genome sequence consisted of 145 contigs and ~ 6.6 Mb genome size. The annotation revealed 6648 coding DNA sequences and 56 tRNA genes. The strain belongs to the sequence type (ST) 50, and the OXY-1 beta-lactam resistance gene, aph(3')-Ia gene for aminoglycoside resistance and multidrug efflux pumps were identified. The fyuA siderophore receptor of yersiniabactin siderophore was identified. Increased virulence was observed in Galleria mellonella larvae model and increased capsule production was determined by uronic acid quantification. The clinical implications of this phenotype are unknown, but the patient outcome might worsen compared to susceptible- or MDR-classical K. michiganensis isolates.

Genetic diversity and spatiotemporal dynamics of DENV-1 and DENV-2 infections during the 2012-2013 outbreak in Mexico.

Dengue fever is caused by four related dengue virus serotypes, DENV-1 to DENV-4, where each serotype comprises distinct genotypes and lineages. The last major outbreak in Mexico occurred during 2012 and 2013, when 112,698 confirmed cases were reported (DENV-1 and DENV-2 were predominant). Following partial E, NS2A and NS5 gene sequencing, based on the virus genome variability, we analyzed 396 DENV-1 and 248 DENV-2 gene sequences from serum samples from dengue acute clinical cases from 13 Mexican states, Mutations were identified, and their genetic variability estimated, along with their evolutionary relationship with DENV sequences sampled globally. DENV-1 genotype V and DENV-2 Asian-American genotype V were the only genotypes circulating during the outbreak. Mutations in NS2A and NS5 proteins were widely disseminated and suggested local emergence of new lineages. Phylogeographic analysis suggested viral spread occurred from coastal regions, and tourist destinations, such as Yucatan and Quintana Roo, which played important roles in disseminating these lineages.

Prediction of neuropeptide precursors and differential expression of adipokinetic hormone/corazonin-related peptide, hugin and corazonin in the brain of malaria vector Nyssorhynchus albimanus during a Plasmodium berghei infection.

Insect neuropeptides, play a central role in the control of many physiological processes. Based on an analysis of Nyssorhynchus albimanus brain transcriptome a neuropeptide precursor database of the mosquito was described. Also, we observed that adipokinetic hormone/corazonin-related peptide (ACP), hugin and corazonin encoding genes were differentially expressed during Plasmodium infection. Transcriptomic data from Ny. albimanus brain identified 29 pre-propeptides deduced from the sequences that allowed the prediction of at least 60 neuropeptides. The predicted peptides include isoforms of allatostatin C, orcokinin, corazonin, adipokinetic hormone (AKH), SIFamide, capa, hugin, pigment-dispersing factor, adipokinetic hormone/corazonin-related peptide (ACP), tachykinin-related peptide, trissin, neuropeptide F, diuretic hormone 31, bursicon, crustacean cardioactive peptide (CCAP), allatotropin, allatostatin A, ecdysis triggering hormone (ETH), diuretic hormone 44 (Dh44), insulin-like peptides (ILPs) and eclosion hormone (EH). The analysis of the genome of An. albimanus and the generated transcriptome, provided evidence for the identification of myosuppressin neuropeptide precursor. A quantitative analysis documented increased expression of precursors encoding ACP peptide, hugin and corazonin in the mosquito brain after Plasmodium berghei infection. This work represents an initial effort to characterize the neuropeptide precursors repertoire of Ny. albimanus and provides information for understanding neuroregulation of the mosquito response during Plasmodium infection.

Expression of Heat shock protein 70 (Hsp70-1) in Plasmodium berghei ookinetes and its participation in midgut mosquito infection.

The heat shock protein family 70 (Hsp70) comprises chaperone proteins that play major multiple roles in Plasmodium asexual and sexual development. In this study, we analyzed the expression of Hsp70-1 in gametocytes, gametes, zygotes, and its participation in ookinete formation and their transition into oocysts. A monoclonal antibody against recombinant Hsp70-1 revealed its presence in zygotes and micronemes of ookinetes. Compared to wild type parasites, Hsp70-1 knockout ookinetes produced fewer oocysts in Plasmodium-susceptible Anopheles albimanus mosquitoes. This may indicate a defective transformation of ookinetes into oocysts in the absence of Hsp70-1. The presence of this protein in micronemes suggests its participation in mosquito infection, probably aiding to the adequate structural conformation of proteins in charge of motility, recognition and invasion of the insect midgut epithelium.

De Novo DNA Synthesis in Aedes aegypti Midgut Cells as a Complementary Strategy to Limit Dengue Viral Replication.

Aedes aegypti is the main vector of Dengue Virus, carrying the virus during the whole mosquito life post-infection. Few mosquito fitness costs have been associated to the virus infection, thereby allowing for a swift dissemination. In order to diminish the mosquito population, public health agency use persistent chemicals with environmental impact for disease control. Most countries barely use biological controls, if at all. With the purpose of developing novel Dengue control strategies, a detailed understanding of the unexplored virus-vector interactions is urgently needed. Damage induced (through tissue injury or bacterial invasion) DNA duplication (endoreplication) has been described in insects during epithelial cells renewal. Here, we delved into the mosquito midgut tissue ability to synthesize DNA de novo; postulating that Dengue virus infection could trigger a protective endoreplication mechanism in some mosquito cells. We hypothesized that the Aedes aegypti orthologue of the Drosophila melanogaster hindsight gene (not previously annotated in Aedes aegypti transcriptome/genome) is part of the Delta-Notch pathway. The activation of this transcriptional cascade leads to genomic DNA endoreplication. The amplification of the genomic copies of specific genes ultimately limits the viral spreading during infection. Conversely, inhibiting DNA synthesis capacity, hence endoreplication, leads to a higher viral replication.

Ecdysis-related pleiotropic neuropeptides expression during Anopheles albimanus development.

OBJECTIVE: To analyze the transcription pattern of neuropeptides in the ontogeny of a malaria vector, the mosquito Anopheles albimanus. MATERIALS AND METHODS: The transcription pattern of Crustacean CardioActive peptide (CCAP), corazonin, Ecdysis Triggering Hormone (ETH), allatostatin-A, orcokinin, Insulin Like Peptide 2 (ILP2), Insulin Like Peptide 5 (ILP5) and bursicon was evaluated using qPCR on larvae (1st - 4th instar), pupae and adult mosquitoes. RESULTS: Unlike in other insects, transcripts of CCAP (70.8%), ETH (60.2%) and corazonin (76.5%) were expressed in 4th instar larvae, probably because these three neuropeptides are associated with the beginning of ecdysis. The neuropeptide ILP2 showed higher transcription levels in other stages and orcokinin decreased during the development of the mosquito. CONCLUSIONS: The CCAP, corazonin and ETH neuropeptidesare potential targets for the design of control strategies aimed at disrupting An. albiamnus larval development.

Aedes aegypti antiviral adaptive response against DENV-2.

Priming is the conceptual term defining memory phenomenon in innate immune response. Numerous examples of enhanced secondary immune response have been described in diverse taxa of invertebrates; which naturally lacks memory response. In mosquitoes, a previous non-lethal challenge with some specific pathogens modify their immune response against the same microorganism; developing an improved antimicrobial reaction. In this work, we explore the ability of Aedes aegypti to mount a higher antiviral response upon a second oral DENV challenge. When previously challenged with inactive virus, we observed that the posterior infection showed a diminished number of DENV infectious particles in midguts and carcasses. In challenged tissues, we detected higher de novo midgut DNA synthesis than control group, as determined by DNA incorporation of 5-bromo-2-deoxyuridine. We demonstrated that inactive DENV particle are capable to induce DNA synthesis levels comparable to infective DENV. We considered the Drosophila melanogaster hindsight and Delta-Notch mosquitoes orthologues as potential de novo DNA synthesis pathway components (as observed in fly oocyte development and midgut tissue renewal). We showed that Aedes aegypti hindsight transcript relative expression levels were higher than control during DENV infection and inactive DENV particle alimentation. Also, Aedes aegypti second challenge with active DENV induced higher hindsight, Delta and Notch transcriptions in the primed mosquitoes (compared with the primary infection levels). Considering that the mosquito de novo DNA synthesis is concomitant to viral particle reduction, this finding opens a new perspective on the mechanisms underlying the vector antiviral immune response and the effector molecules involved.

Ecdysis-related pleiotropic neuropeptides expression during Anopheles albimanus development / Expresión de neuropéptidos pleiotrópicos asociados con ecdisis durante el desarrollo del mosquito Anopheles albimanus

Abstract: Objective: To analyze the transcription pattern of neuropeptides in the ontogeny of a malaria vector, the mosquito Anopheles albimanus. Materials and methods: The transcription pattern of Crustacean CardioActive peptide (CCAP), corazonin, Ecdysis Triggering Hormone (ETH), allatostatin-A, orcokinin, Insulin Like Peptide 2 (ILP2), Insulin Like Peptide 5 (ILP5) and bursicon was evaluated using qPCR on larvae (1st - 4th instar), pupae and adult mosquitoes. Results: Unlike in other insects, transcripts of CCAP (70.8%), ETH (60.2%) and corazonin (76.5%) were expressed in 4th instar larvae, probably because these three neuropeptides are associated with the beginning of ecdysis. The neuropeptide ILP2 showed higher transcription levels in other stages and orcokinin decreased during the development of the mosquito. Conclusion: The CCAP, corazonin and ETH neuropeptides are potential targets for the design of control strategies aimed at disrupting An. albiamnus larval development.

Resumen: Objetivo: Describir la expresión de neuropéptidos durante la ontogenia del mosquito vector de la malaria Anopheles albimanus. Material y métodos: Se midió la expresión de CCAP, corazonina, ETH, allatostatina, orcokinina, ILP2, ILP5 y bursicon en larvas de primer (2mm), segundo (4mm), tercer (5mm) y cuarto (6mm) estadio, pupas y mosquitos adultos, mediante qPCR. Resultados. A diferencia de otros insectos en donde, CCAP, corazonina y ETH se expresan principalmente en estadios pupales, en An. albimanus se expresaron mayoritariamente en larvas de cuarto estadio, CCAP tuvo 70.8% de expresión relativa, corazonina 76.5% y ETH 60.2%. ILP2 fue el neuropéptido que más se expresó en el primer, segundo y tercer estadio y orcokinina disminuyó durante el desarrollo del mosquito. Conclusión. Los péptidos estudiados se expresaron en todos los estadios de desarrollo del mosquito. Sin embargo, su expresión varió en cada uno de ellos. Los neuropéptidos CCAP, corazonina y ETH, que son esenciales para la transformación de lavas a pupas, pueden ser blancos potenciales para el diseño de estrategias de control dirigidas a interrumpir el desarrollo larvario de An. albimanus.

Infection with Plasmodium berghei ookinetes alters protein expression in the brain of Anopheles albimanus mosquitoes.

BACKGROUND: The behaviour of Anopheles spp. mosquitoes, vectors for Plasmodium parasites, plays a crucial role in the propagation of malaria to humans. Consequently, it is important to understand how the behaviour of these mosquitoes is influenced by the interaction between the brain and immunological status. The nervous system is intimately linked to the immune and endocrine systems. There is evidence that the malaria parasite alters the function of these systems upon infecting the mosquito. Although there is a complex molecular interplay between the Plasmodium parasite and Anopheles mosquito, little is known about the neuronal alteration triggered by the parasite invasion. The aim of this study was to analyse the modification of the proteomic profile in the An. albimanus brain during the early phase of the Plasmodium berghei invasion. RESULTS: At 24 hours of the P. berghei invasion, the mosquito brain showed an increase in the concentration of proteins involved in the cellular metabolic pathway, such as ATP synthase complex alpha and beta, malate dehydrogenase, alanine transaminase, enolase and vacuolar ATP synthase. There was also a rise in the levels of proteins with neuronal function, such as calreticulin, mitofilin and creatine kinase. Concomitantly, the parasite invasion repressed the expression of synapse-associated proteins, including enolyl CoA hydratase, HSP70 and ribosomal S60 proteins. CONCLUSIONS: Identification of upregulated and downregulated protein expression in the mosquito brain 24 hours after Plasmodium invaded the insect midgut paves the way to better understanding the regulation of the neuro-endocrine-immune system in an insect model during parasite infection.

Plasmodium berghei induced priming in Anopheles albimanus independently of bacterial co-infection.

Priming in invertebrates is the acquired capacity to better combat a pathogen due to a previous exposure to sub-lethal doses of the same organism. It is proposed to be functionally analogous to immune memory in vertebrates. Previous studies with Anopheles gambiae mosquitoes provide evidence that the inhibitory response to a second challenge by the malaria parasite Plasmodium berghei resulted from a sustained activation of hemocytes by midgut bacteria. These bacteria probably accessed the hemolymph during a first aborted infection through lesions produced by parasites invading the midgut. Since the mosquito immune responses to midgut bacteria and Plasmodium overlap, it is difficult to determine the priming responses of each. We herein document priming induced in the aseptic An. albimanus midgut by P. berghei, probably independent of the immune response induced by midgut bacteria. This idea is further evidenced by experiments with Pbs 25-28 knock out parasites (having an impaired capacity for invading the mosquito midgut) and dead ookinetes. Priming protection against a homologous challenge with P. berghei lasted up to 12 days. There was greater incorporation of 5-bromo-2'-deoxyuridine into midgut cell nuclei (indicative of DNA synthesis without mitosis) and increased transcription of hnt (a gene required for the endocycle of midgut cells) in primed versus unprimed mosquitoes, suggesting that endoreplication was the underlying mechanism of priming. Moreover, the transcription of hnt and antimicrobial peptides related to an anti-Plasmodium response (attacin, cecropin and gambicin) was enhanced in a biphasic rather than sustained response after priming An. albimanus with P. berghei.

Sequence variation of ookinete surface proteins Pvs25 and Pvs28 of Plasmodium vivax isolates from Southern Mexico and their association to local anophelines infectivity.

The polymorphism of Pvs25 and Pvs28 ookinete surface proteins, their association to circumsporozoite protein repeat (CSPr) genotypes (Vk210 and Vk247) and their infectivity to local Anopheles albimanus and Anopheles pseudopunctipennis were investigated in Plasmodium vivax-infected blood samples obtained from patients in Southern Mexico. The pvs25 and pvs28 complete genes were amplified, cloned and sequenced; and the CSPr genotype was determined by PCR amplification and hybridization. The amino acid Pvs25 and Pvs28 polymorphisms were mapped to their corresponding protein structure. Infected blood samples were simultaneously provided through artificial feeders to both mosquito species; the ratio of infected mosquitoes and oocyst numbers were recorded. The polymorphism of pvs25 and pvs28 was limited to few nucleotide positions, and produced three haplotypes: type A/A parasites presented Pvs25 and Pvs28 amino acid sequences identical to that of Sal I reference strain; parasites type B1 presented a mutation 130 Ile-->Thr in Pvs25, while type B2 presented 87 Gln-->Lys/130 Ile-->Thr in the same molecule. Both types B1 and B2 parasites presented changes in Pvs28 at 87 Asn-->Asp, 110 Tyr-->Asn and five GSGGE/D repeat sequences between the fourth EGF-like domain and the GPI. Most P. vivaxparasites from the coastal plains and the overlapping region were Pvs25/28 A/A, CSPrVk210 and were infective only to An. albimanus (p< or =0.0001). Parasites originating in foothills were Pvs25/28 type B1/B or B2/B and CSPrVk210 or Vk247, and were more infective to An. pseudopunctipennis than to An. albimanus (p< or =0.001). These results and the analysis of Pvs25/28 from other parts of the world indicated that non-synonymous variations in these proteins occur in amino acid residues exposed on the surface of the proteins, and are likely to interact with midgut mosquito ligands. We hypothesize that these molecules have been shaped by co-evolutionary adaptations of parasites to their susceptible vectors.

The surface protein Pvs25 of Plasmodium vivax ookinetes interacts with calreticulin on the midgut apical surface of the malaria vector Anopheles albimanus.

Malaria parasite transmission-blocking control strategies within the mosquito vector require an adequate understanding of the parasite mosquito interaction at the molecular level. The ookinete P25-P28 surface proteins are required for the transition from ookinete to oocyst in the mosquito midgut; however, their respective molecular interactions in the mosquito are largely unknown. We used recombinant Pvs25 and Pvs28 as probes for identification of potential Anopheles albimanus midgut ligands. A 50 kDa protein interacted with Pvs25 but not with Pvs28 in blot overlay assays. This protein was identified as calreticulin by LS MS and was detected in membrane, but not in soluble midgut protein extracts. Calreticulin was detected in An. albimanus midgut microvilli by immunofluorescence analysis. The An. albimanus calreticulin cDNA was cloned and recombinant calreticulin was shown to interact with recombinant Pvs25 in overlay and co-immunoprecipitation assays, confirming the interaction of the two proteins. The Pvs25-calreticulin interaction in vivo could represent a potential target for developing transmission blocking strategies based on interfering the parasite-midgut interaction.