Multidrug resistance (MDR) refers to the capability of bacterial pathogens to withstand lethal doses of structurally diverse drugs which are capable of eradicating non-resistant strains. MDR has been identified as a major threat to the public health of human being by the World Health Organization (WHO). Among the four general mechanisms that cause antibiotic resistance including target alteration, drug inactivation, decreased permeability and increased efflux, drug extrusion by the multidrug efflux pumps serves as an important mechanism of MDR. Efflux pumps not only can expel a broad range of antibiotics owing to their poly-substrate specificity, but also drive the acquisition of additional resistance mechanisms by lowering intracellular antibiotic concentration and promoting mutation accumulation. Over-expression of multidrug efflux pumps have been increasingly found to be associated with clinically relevant drug resistance. On the other hand, accumulating evidence has suggested that efflux pumps also have physiological functions in bacteria and their expression is subject tight regulation in response to various of environmental and physiological signals. A comprehensive understanding of the mechanisms of drug extrusion, and regulation and physiological functions of efflux pumps is essential for the development of anti-resistance interventions. In this review, we summarize the development of these research areas in the recent decades and present the pharmacological exploitation of efflux pump inhibitors as a promising anti-drug resistance intervention.

Clonorchis sinensis (C. sinensis), an important food-borne parasite that inhabits the intrahepatic bile duct and causes clonorchiasis, is of interest to both the public health field and the scientific research community. To learn more about the migration, parasitism and pathogenesis of C. sinensis at the molecular level, the present study developed an upgraded genomic assembly and annotation by sequencing paired-end and mate-paired libraries. We also performed transcriptome sequence analyses on multiple C. sinensis tissues (sucker, muscle, ovary and testis). Genes encoding molecules involved in responses to stimuli and muscle-related development were abundantly expressed in the oral sucker. Compared with other species, genes encoding molecules that facilitate the recognition and transport of cholesterol were observed in high copy numbers in the genome and were highly expressed in the oral sucker. Genes encoding transporters for fatty acids, glucose, amino acids and oxygen were also highly expressed, along with other molecules involved in metabolizing these substrates. All genes involved in energy metabolism pathways, including the β-oxidation of fatty acids, the citrate cycle, oxidative phosphorylation, and fumarate reduction, were expressed in the adults. Finally, we also provide valuable insights into the mechanism underlying the process of pathogenesis by characterizing the secretome of C. sinensis. The characterization and elaborate analysis of the upgraded genome and the tissue transcriptomes not only form a detailed and fundamental C. sinensis resource but also provide novel insights into the physiology and pathogenesis of C. sinensis. We anticipate that this work will aid the development of innovative strategies for the prevention and control of clonorchiasis.

A new species of Fonsecaea, Fonsecaea nubica, morphologically similar to F. pedrosoi and F. monophora, is described using multilocus molecular data including AFLP profiles, sequences of the ribosomal internal transcribed spacers (ITS), and partial sequences of the cell division cycle (cdc42), beta-tubulin (tub1) and actin (act1) genes. A phylogenetic approach was used to evaluate species delimitation. Topologies of the trees were concordant. Fonsecaea strains could be classified into three major entities, i.e., one representing Fonsecaea pedrosoi isolates, another consisting of strains of F. monophora, and a third, unnamed group comprising isolates mostly recovered from cases of chromoblastomycosis in South America and China. F. nubica is part of this latter group. Based on strains analyzed thus far, we have found that the pathologies of these three Fonsecaea species are somewhat different in that F. pedrosoi and F. nubica are preponderantly associated with chromoblastomycosis, while F. monophora may also act as a systemic opportunist in cases involving brain infections. The latter species is also the most frequently recovered of the three from environmental samples.

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Chromoblastomycosis is a chronic, cutaneous and subcutaneous infection caused by members of the order Chaetothyriales and commonly found in China. Among the etiologic agents, members of the genus Cladophialophora are predominant in northern China. Alternatively, Fonsecaea spp. are particularly common in southern China. However, the identification of Fonsecaea isolates recovered in China is difficult due to the fact that different species lack distinctive morphological characters. Therefore, the identification of 24 Fonsecaea isolates from symptomatic patients were re-evaluated by using morphology, ITS rDNA sequence diversity and partly through the use of randomly amplified polymorphic DNA (RAPD) typing. Twenty strains, including a morphological mutant were found to be Fonsecaea monophora, while four strains corresponded to F. pedrosoi. We have demonstrated that Fonsecaea monophora is the predominant etiologic agent of chromoblastomycosis in southern China and populations showed marked geographic structuring.

Fonsecaea and Cladophialophora are genera of black yeast-like fungi harboring agents of a mutilating implantation disease in humans, along with strictly environmental species. The current hypothesis suggests that those species reside in somewhat adverse microhabitats, and pathogenic siblings share virulence factors enabling survival in mammal tissue after coincidental inoculation driven by pathogenic adaptation. A comparative genomic analysis of environmental and pathogenic siblings of Fonsecaea and Cladophialophora was undertaken, including de novo assembly of F. erecta from plant material. The genome size of Fonsecaea species varied between 33.39 and 35.23 Mb, and the core genomes of those species comprises almost 70% of the genes. Expansions of protein domains such as glyoxalases and peptidases suggested ability for pathogenicity in clinical agents, while the use of nitrogen and degradation of phenolic compounds was enriched in environmental species. The similarity of carbohydrate-active vs. protein-degrading enzymes associated with the occurrence of virulence factors suggested a general tolerance to extreme conditions, which might explain the opportunistic tendency of Fonsecaea sibling species. Virulence was tested in the Galleria mellonella model and immunological assays were performed in order to support this hypothesis. Larvae infected by environmental F. erecta had a lower survival. Fungal macrophage murine co-culture showed that F. erecta induced high levels of TNF-α contributing to macrophage activation that could increase the ability to control intracellular fungal growth although hyphal death were not observed, suggesting a higher level of extremotolerance of environmental species.

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Economic development and urbanization of cities favor a shift of etiological agents from anthroponoses to zoonoses in contemporary China. Pets are becoming the most likely sources of infection in modern lifestyles, replacing the earlier human-to-human transmission mode. However, the latter transmission mode is still prevalent in less developed areas lacking adequate social and public health facilities.