Microbiol. Res., Volume 12, Issue 3 (September 2021) – 17 articles

This study aimed to clarify the population-based distributions of Mycobacterium avium and Mycobacterium intracellulare in Japan. We conducted a combined analysis of the national insurance claim and microbiological databases. The incidence rates of M. avium by province were similar throughout the country, with some exceptions, such as in Okinawa, probably because the bathing customs are different from those in mainland Japan. In contrast, M. intracellulare showed a gradual increase from the central part of the country to the southwestern region, with precise gradation, which may indicate infection sources in the natural environment. This study found that assessment of the infection route of M. intracellulare in the natural environment, which is similar to the distribution of M. intracellular patients, is warranted. In conclusion, improvement of the household environment could decrease the incidence of M. avium, while environmental countermeasures will be required to decrease the incidence of M. intracellulare. Full article

Zika virus (ZIKV) is a mosquito-borne flavivirus in which human infection became relevant during recent outbreaks in Latin America due to its unrecognized association with fetal neurological disorders. Currently, there are no approved effective antivirals or vaccines for the treatment or prevention of ZIKV infections. Amantadine and rimantadine are approved antivirals used against susceptible influenza A virus infections that have been shown to have antiviral activity against other viruses, such as dengue virus (DENV). Here, we report the in vitro effectiveness of both amantadine and rimantadine hydrochlorides against ZIKV replication, resulting in a dose-dependent reduction in viral titers of a ZIKV clinical isolate and two different ZIKV reference strains. Additionally, we demonstrate similar in vitro antiviral activity of these drugs against DENV-1 and yellow fever virus (YFV), although at higher drug concentrations for the latter. ZIKV replication was inhibited at drug concentrations well below cytotoxic levels of both compounds, as denoted by the high selectivity indexes obtained with the tested strains. Further work is absolutely needed to determine the potential clinical use of these antivirals against ZIKV infections, but our results suggest the existence of a highly conserved mechanism across flavivirus, susceptible to be blocked by modified more specific adamantane compounds. Full article

Host immunity to Mycobacterium leprae encompasses a spectrum of mechanisms that range from cellular immunity-driven protection to damage associated with humoral immunity as in type-2 leprosy reactions. Although type I interferons (IFNs) participate in eliminating intracellular pathogens, their contribution to the production of antibodies and CD3⁺ FOXP3⁺ regulatory T cells (Tregs) in BCG vaccine-mediated protection in leprosy is unknown. BCGphipps (BCGph) priming followed by intramuscular hIFN-α 2b boost significantly reduced lesion size and Mycobacterium lepraemurium growth in the skin. T follicular regulatory cells (TFR), a subset of Tregs induced by immunization or infection, reside in the germinal centers (GCs) and modulate antibody production. We found impaired Treg induction and improved GCs in draining lymph nodes of BCGph primed and hIFN-α 2b boosted mice. Moreover, these mice elicited significant amounts of IL-4 and IL-10 in serum. Thus, our results support the adjuvant properties of hIFN-α 2b in the context of BCGph priming to enhance protective immunity against skin leprosy. Full article

Brucellosis has a wide range of clinical severity in humans that remains poorly understood. Whole genome sequencing (WGS) analysis may be able to detect variation in virulence genes. We used Brucella melitensis sequences in the NCBI Sequence Read Archive (SRA) database to assemble 248 whole genomes, and additionally, assembled 27 B. melitensis genomes from samples of human patients in Southern Israel. We searched the 275 assembled genomes for the 43 B. melitensis virulence genes in the Virulence Factors of Pathogenic Bacteria Database (VFDB) and 10 other published putative virulence genes. We explored pan-genome variation across the genomes and in a pilot analysis, explored single nucleotide polymorphism (SNP) variation among the ten putative virulence genes. More than 99% of the genomes had sequences for all Brucella melitensis virulence genes included in the VFDB. The 10 other virulence genes of interest were present across all the genomes, but three of these genes had SNP variation associated with particular Brucella melitensis genotypes. SNP variation was also seen within the Israeli genomes obtained from a small geographic region. While the Brucella genome is highly conserved, this novel and large whole genome study of Brucella demonstrates the ability of whole genome and pan-genome analysis to screen multiple genomes and identify SNP variation in both known and novel virulence genes that could be associated with differential disease virulence. Further development of whole genome techniques and linkage with clinical metadata on disease outcomes could shed light on whether such variation in the Brucella genome plays a role in pathogenesis. Full article

Klebsiella spp. are associated with several nosocomial and opportunistic infections. Increasing antimicrobial resistance of Klebsiella species is aggravated by a number of intrinsic and extrinsic factors. The main aim of this study is to determine antimicrobial resistance due to production of β-lactamase enzymes, extended spectrum beta-lactamase (ESBL), metallo-beta-lactamase (MBL) and AmpC and Klebsiella pneumoniae carbapenemase (KPC) and biofilm formation in Klebsiella isolates. A total of 2197 non-duplicate specimens of urine, sputum and pus were obtained from the National Public Health Laboratory (NPHL), Kathmandu, Nepal, between February and August 2019. Klebsiella species were isolated, identified and screened for antimicrobial susceptibility testing with the disk diffusion method. Phenotypic detection of ESBL, MBL, KPC and AmpC production was observed and biofilm production was detected by the microtiter plate method. Out of a total of 2197 clinical specimens, bacterial growth was detected in 8% (175/2197) of the specimens. Of the total isolates, 86.3% (151/175) were Gram-negative bacteria and 37.7% (57/151) were Klebsiella spp. Of the total Klebsiella spp., 56% (32/57) were multi drug resistant (MDR), 16% (9/57) were ESBL, 26% (15/57) were MBL, 4% (2/57) were KPC (class A carbapenemase), 16% (9/57) were AmpC producers and 95% (54/57) were biofilm producers. Gentamicin was the most effective antibiotic, followed by cotrimoxazole, as 68% (39/57) and 47% (27/57) of the Klebsiella isolates were susceptible towards these drugs, respectively. The study results show evidence of β-lactamase production, high prevalence of MDR and biofilm producing Klebsiella species. Integrating the test parameters for phenotypic confirmation of ESBL, MBL, AmpC β lactamase and KPC in routine diagnostic procedures can help in the early detection and management of these resistant strains. Full article

Livestock products supply about 13 percent of energy and 28 percent of protein in diets consumed worldwide. Diarrhea is a leading cause of sickness and death of beef and dairy calves in their first month of life and also affecting adult cattle, resulting in large economic losses and a negative impact on animal welfare. Despite the usual multifactorial origin, viruses are generally involved, being among the most important causes of diarrhea. There are several viruses that have been confirmed as etiological agents (i.e., rotavirus and coronavirus), and some viruses that are not yet confirmed as etiological agents. This review summarizes the viruses that have been detected in the enteric tract of cattle and tries to deepen and gather knowledge about them. Full article

Chlamydia spp. are intracellular pathogens of humans and animals that cause a wide range of diseases such as blinding trachoma and sexually transmitted infections. According to the World Health Organization (WHO), there are more than 127 million new infections each year worldwide. Chlamydial urogenital infections can cause cervicitis, urethritis, pelvic inflammatory disease and infertility. From within an intracellular niche, termed an inclusion, the Chlamydiae complete their life cycle shielded from host defenses. The host cell defense response used to eliminate the pathogen must subvert this protective shield and is thought to involve the gamma interferon-inducible family of immunity related GTPase proteins and nitric oxide. Typically, azithromycin and doxycycline are the first line drugs for the treatment of chlamydial infections. Although C. trachomatis is sensitive to these antibiotics in vitro, currently, there is increasing bacterial resistance to antibiotics including multidrug-resistant C. trachomatis, which have been described in many instances. Therefore, alternative drug candidates against Chlamydia should be assessed in vitro. In this study, we tested and quantified the activity of plant extracts against Chlamydia-infected HeLa cells with C. trachomatis inclusions. The in vitro results show that post-treatment with Artemisia inculta Delile extract significantly inhibits Chlamydia infection compared to DMSO-treated samples. In conclusion, plant extracts may contain active ingredients with antichlamydial activity potential and can be used as alternative drug candidates for treatment of Chlamydia infection which has significant socio-economic and medical impact. Full article

The detection and identification of fungal DNA from clinical samples is one of the fundamental approaches in biomedicine. The incidence, distribution, and control of dermatophytes has progress significantly and the use of phylogenetic species concepts based on rRNA regions have enhanced the taxonomy of dermatophyte species; however, the use of 28S rDNA genes has certain limitations. This gene has been used in dermatophyte taxonomy with limited enumeration; we appraised the sequence disparity within and among groups of the species, the gene ranking in identification, phylogenetic analysis, and taxonomy of 32 strains of eight dermatophyte species. In this study, a set of primers was adopted to amplify the target followed by a partial sequencing of the rDNA. The utilization of a pairwise nucleotide differentiation, an affinity was observed among eight dermatophyte species, with disparity among species ranging from 0 to 197 base pair (bp). Intra-species bp differences were found within strains of Trichophyton eriotrephon, Trichophyton bullosum, Trichophyton simii (Trichophyton genus), Microsporum audouinii, and Trichophyton tonsurans (Microsporum and Trichophyton genus, respectively); however, only some strains of Trichophyton eriotrephon were found to be invariant having three genotypes. Trichophyton tonsurans exhibited most intra-species variability. The characterization and construction of a phylogenetic tree of 28S rDNA gene on dermatophyte species provide a bedrock of an additional finding of connections between species. However, 28S rRNA capture provides a novel method of effective and sensitive detection of dermatophytes lodged in human skin scale. We report for the first time the emergence of T. eriotrephon, T. bullosum, T. simii, T. benhamiae, and Ctenomyces serratus dermatophytes from Tinea capitis in Nigeria. Full article

Pseudomonas aeruginosa causes chronic infections, such as cystic fibrosis, endocarditis, bacteremia, and sepsis, which are life-threatening and difficult to treat. The lack of antibiotic response in P. aeruginosa is due to adaptive resistance mechanism, which prevents the entry of antibiotics into the cytosol of the cell to achieve tolerance. Among the different groups of antibiotics, aminoglycosides are used as a parenteral antibiotic for the treatment of P. aeruginosa. This study aimed to determine the kinetics of antibiotic tolerance and gene expression changes in P. aeruginosa exposed to amikacin, gentamicin, and tobramycin. These antibiotics were exposed to P. aeruginosa at their MICs and the experimental setup was monitored for 72 h, followed by the measurement of optical density every 12 h. The growth of P. aeruginosa in the MICs of antibiotics represented the kinetics of antibiotic tolerance in amikacin, gentamicin, and tobramycin. The transcriptomic profile of antibiotic exposed P. aeruginosa PA14 was taken from the Gene Expression Omnibus (GEO), NCBI as microarray datasets. The gene expressions of two datasets were compared by test versus control. Tobramycin-exposed P. aeruginosa failed to develop tolerance in MICs of 0.5 µg/mL, 1 µg/mL, and 1.5 µg/mL, whereas amikacin- and gentamicin-treated P. aeruginosa developed tolerance. This illustrated the superior in vitro response of tobramycin over gentamicin and amikacin. Further, in silico transcriptomic analysis of tobramycin-treated P. aeruginosa resulted in differentially expressed genes (DEGs), enriched in 16s rRNA methyltransferase E, B, and L, alginate biosynthesis genes, and several proteins of the type II secretion system (T2SS) and type III secretion system (T3SS). The regulation of mucA in alginate biosynthesis, and gidB in RNA methyltransferases, suggested an increased antibiotic response and a low probability of developing resistance during tobramycin treatment. The use of tobramycin as a parenteral antibiotic with its synergistic combination might combat P. aeruginosa with increased response. Full article

The role of the microbiota–gut–brain axis in maintaining a healthy status is well recognized. In this bidirectional flux, the influence of host hormones on gut bacteria is crucial. However, data on commensal/probiotics are scarce since most reports analyzed the effects of human bioactive compounds on opportunistic strains, highlighting the risk of increased pathogenicity under stimulation. The present investigation examined the modifications induced by 5HT, a tryptophan-derived molecule abundant in the intestine, on the probiotic Enterococcus faecium NCIMB10415. Specific phenotypic modifications concerning the probiotic potential and possible effects of treated bacteria on dendritic cells were explored together with the comparative soluble proteome evaluation. Increased resistance to bile salts and ampicillin in 5HT-stimulated conditions relate with overexpression of specific proteins (among which Zn-beta-lactamases, a Zn-transport protein and a protein involved in fatty acid incorporation into the membrane). Better auto-aggregating properties and biofilm-forming aptitude are consistent with enhanced QS peptide transport. Concerning interaction with the host, E. faecium NCIMB10415 enhanced dendritic cell maturation, but no significant differences were observed between 5HT-treated and untreated bacteria; meanwhile, after 5HT exposure, some moonlight proteins possibly involved in tissue adhesion were found in higher abundance. Finally, the finding in stimulated conditions of a higher abundance of VicR, a protein involved in two-component signal transduction system (VicK/R), suggests the existence of a possible surface receptor (VicK) for 5HT sensing in the strain studied. These overall data indicate that E. faecium NCIMB10415 modifies its physiology in response to 5HT by improving bacterial interactions and resistance to stressors. Full article

Chronic airway colonization by bacteria and fungi is very common in CF patients, causing irreversible lung damage. It is known that rates of fungal infections are much lower than those of bacterial infections, however they can worsen the medical condition of CF patients. In this study, we identify the most common fungal species isolated from 31 adult CF patients in Qatar and analyze their correlation with lung function, pulmonary exacerbations, bronchial asthma, and pancreatic insufficiency. Mycological evaluation, as well as medical records, were reviewed for the patients regularly under the adult CF service at Hamad General Hospital in the period between 2017–2019. All CF patients included in this study carry rare CFTR mutations. The majority of those patients (n = 25) carried the c.3700A>G; I1234V mutation, whereas three patients carried the heterozygous mutation (c.1657C>T and c.1115A>T) and the remaining three carried the homozygous mutation (c.920G>A). Twenty-two of the adult CF participants (70.9%) were colonized with fungal species regardless of the type of the CFTR mutation. Candida and Aspergillus species were the most common, colonizing 81% and 45% of the patients, respectively. For Candida colonized patients, Candida dubliniensis was the most frequently reported species (55.6%), whereas Aspergillus fumigatus colonization was the most common (50.0%) among Aspergillus colonized patients. These identified fungal pathogens were associated with poor lung function, pancreatic insufficiency, and asthma in this cohort. Such colonization could possibly aggravate the most known CF complications, notably pulmonary exacerbations, asthma, and pancreatic insufficiency. Full article

African swine fever (ASF) is a highly contagious viral infection of domestic and wild pigs with high mortality. First reported in East Africa in the early 1900s, ASF was largely controlled in domestic pigs in many countries. However, in recent years ASF outbreaks have been reported in several countries in Europe and Asia. The occurrence of ASF in China, the largest pork producer in the world, in 2018 and in India, the country that surrounds and shares open borders with Nepal, has increased the risk of ASF transmission to Nepal. Lately, the pork industry has been growing in Nepal, overcoming traditional religious and cultural biases against it. However, the emergence of viral infections such as ASF could severely affect the industry’s growth and sustainability. Because there are no effective vaccines available to prevent ASF, the government should focus on preventing entry of the virus through strict quarantine measures at the borders, controls on illegal trade, and effective management practices, including biosecurity measures. Full article

Bacteria of the genus Wolbachia are widely distributed in arthropods, particularly in ants; nevertheless, it is still little explored with the Multilocus Sequence Typing (MLST) methodology, especially in the genus Solenopsis, which includes species native to South America. Ants from this genus have species distributed in a cosmopolitan way with some of them being native to South America. In Brazil, they are widely spread and preferentially associated with areas of human activity. This study aimed to investigate the diversity of Wolbachia in ants of the genus Solenopsis through the MLST approach and their phylogenetic relationship, including the relationship between mtDNA from the host and the related Wolbachia strain. We also tested the geographic correlation between the strains to infer transmission and distributional patterns. Fifteen new strains and eleven previously unknown alleles were obtained by sequencing and analyzing the five genes that make up the MLST. The phylogenetic relationship between the strains showed a polyphyletic pattern, indicative of the complexity of the evolutionary history of these bacteria in the analyzed species. We detected the correlation of host’s mitochondrial DNA with Wolbachia diversity which imply that related strains exist in related hosts, strongly suggesting the occurrence of vertical transfer. We found no specificity of the Wolbachia strain for a given geographic region that could indicate either that there is no horizontal transfer of the strain from the environment for the host or that the human action could be shuffling the distribution of the Solenopsis ants and the endosymbiont Wolbachia, as well. Our study highlights the complexity and novelty of Wolbachia diversity with this specific group of ants and the need for further studies that focus on understanding of this intricate relationship. Full article

Sepsis frequently leads to multiple organ failure and is a major cause of morbidity and mortality in critically ill patients. Although intensive care protocols and antibiotic therapy have improved sepsis treatment, specific management is lacking with respect to efficient protection from tissue damage and long-term outcomes. Probiotics are live microbes that modulate the immune system and inflammation and colonize the gut. In this narrative review, we have traced the evolution of the administration of probiotics in an animal model of sepsis and treatment alternatives in the intensive care unit setting. First, probiotics are categorized by species before describing their modulation of the microbiota, repair of tissue-specific damage, immune response, and molecular pathways to prevent complications. The impact on therapy for infant and adult patients is also addressed. Finally, we have emphasized the challenges and gaps in current studies as well as future perspectives for further investigation. The present review can open up avenues for new strategies that employ promising probiotic strains for the treatment of sepsis and discusses their ability to prevent disease-associated long-term complications. Full article

The spliced human papillomavirus 16 (HPV16) E1C RNA is associated with high-grade precursor lesions and cervical cancer. This qualifies E1C as a biomarker for high-grade lesions in HPV-based cervical cancer precursor screening. Here, we aimed to characterize the biological activity of HPV16 E1C RNA. In HEK-293T cells overexpressing HPV16 E1C RNA, we detected 9 kDa E1C protein in the cytoplasm using immunological assays with a newly generated E1C-specific monoclonal antibody or in mass spectrometry only after proteasome inhibition with MG132, indicating instability of the E1C protein. In HPV16-transformed cervical cancer cell lines in which the level of endogenous E1C RNA is much lower, E1C protein was not detected even after proteasome inhibition. Transient E1C overexpression in HEK-293T cells, co-transfected with a firefly luciferase reporter gene under the control of the HPV16 upstream regulatory region (URR), activated the HPV16 URR by 38%. This activation was also present when E1C translation was abolished by mutation. However, a construct expressing a random RNA sequence with similar GC content and 45% homology to the E1C RNA sequence also stimulated URR activity, indicating that special E1C RNA motifs might be responsible for the activation. In HPV16-transformed cell lines W12-episomal (W12-epi), W12-integrated HPV (W12-int), CaSki and SiHa stably overexpressing E1C RNA from lentiviral transduction, levels of endogenous HPV16 RNAs E6*I and E7 remained unchanged, while E1^E4 levels were significantly reduced by 20–30% in W12-epi, W12-int and CaSki cells. Overall, our study shows that E1C RNA is active and might contribute to transformation independent of the E6*I or E7 pathways. However, E1C overexpression resulted in only subtle changes in HPV16 RNA expression and very low copies of endogenous E1C RNA were detected in cervical cancer cell lines. This could weigh towards a less prominent role of E1C RNA in natural HPV transformation. Full article

A study was conducted to test the pathogenicity of an endophytic fungus associated with asymptomatic cacao leaves, to determine its identity through cultural, morphological and molecular characterization, and to evaluate itsantagonistic ability vs. Phytophthora palmivora causing cacao black pod rot disease. Experiments were carried out under laboratory and shade house conditions. Homogeneity of variances and normal data distribution were determined using Bartlett’s and Shapiro–Wilk tests, respectively. Inoculation of the endophyte in healthy cacao seedlings and pods at 5 × 10⁵ to 1 × 10⁶ conidia per mL by spraying resulted in asymptomatic infections. The endophyte was recovered from artificially inoculated tissues 14 and 26 days after inoculation (DAI) (UF18 seedlings), and at 10 (K9 seedlings) and 14 DAI from cacao pods. The endophyte was identified as Colletotrichum siamense based on its cultural, morphological and molecular characteristics. In vitroanti-pathogen assays showed that C. siamense had the potential to limit pathogen growth by antibiosis. At 3, 5 and 7 days after incubation period (DAIP), growth of the pathogen in co-cultivation with the endophyte measured 60.0, 70.0 and 71.0 mm, respectively, which wasconsiderably lower than the growth of the pathogen alone. Full article