Hsp65 Gene Sequencing Research Articles

Challenges in diagnosing bovine tuberculosis through surveillance and characterization of Mycobacterium species in slaughtered cattle in Kolkata

BackgroundTuberculosis in cattle is caused by Mycobacterium tuberculosis complex (MTBC) species. Apart from MTBC, different Nontuberculous Mycobacteria (NTM) species have also been isolated from cattle. The presence of NTM infection in bovines makes the diagnosis of bovine tuberculosis (bTB) a cumbersome task. Therefore, a cross sectional study was conducted to isolate and characterize different Mycobacterium spp. from a slaughterhouse situated in Kolkata, a city in the eastern part of India.ResultsOut of 258 morbid samples, 98 isolates were found to be positive for bacterial growth, and 35% (n = 34) were positive for Mycobacterium. 94% of Mycobacterial cultural isolates were NTM (n = 32), and the rest (n = 2) were found to be MTBC. Species-level identification of the isolates by hsp65 sequencing revealed that out of 32 isolates, 24 were M. fortuitum, three were M. abscessus, two each were M. chelonae and M. parascrofulaceum, and one was M. novocastrense. A phylogenetic tree with partial hsp65 gene sequences was also constructed to determine the relatedness of the unknown isolates to the reference strains.ConclusionBoth NTM species and MTBCs were identified from TB-like lesions in cattle that were slaughtered at the Kolkata abattoir. This discovery may indicate that NTM contributes to the development of lesions in cattle. Also, we recommend implication of more specific diagnostic tests for bTB.

Genotypic and phenotypic characterization of resistance to fenhexamid, carboxin, and, prochloraz, in Botrytis cinerea isolates collected from cut roses in Colombia.

Gray mold, caused by Botrytis sp., is a significant disease in Colombian rose crops and its control depends primarily on the intensive use of chemically synthesized fungicides. Despite the importance of this pathogen, there is limited information in Colombian floriculture about molecular taxonomy of species, fungicide resistance of populations and their genetic mechanism of resistance. In this study, we analyze 12 isolates of this fungus collected from rose-producing crops in the Department of Cundinamarca and conducted phylogenetic analysis using HSP60, G3PDH, and RPB2 gene sequences. Additionally, we realize phenotypic and genotypic characterization of resistance to the fungicides fenhexamid, carboxin, and prochloraz, evaluating the in vitro EC50 and presence of mutations of target genes of each isolate. All isolates were characterized as Botrytis cinerea in the phylogenetic analysis and presents different levels of resistance to each fungicide. These levels are related to mutations in target genes, with predominancy of L195F and L400F in the ERG27 gene to fenhexamid resistance, H272R/Y in the SDHB gene for carboxin resistance, and Y136F in the CYP51 gene for prochloraz resistance. Finally, these mutations were not related to morphological changes. Collectively, this knowledge, presented for the first time to the Colombian floriculture, contribute to a better understanding of the genetic diversity and population of B. cinerea from rose-producing crops in the department of Cundinamarca, and serve as a valuable tool for making informed decisions regarding disease management, future research, and improving crop management and sustainability in the Colombian floriculture industry.

Looking at the potential of HSP70 gene sequence in the germplasm of Indonesian green jungle fowl

Looking at the potential of HSP70 gene sequence in the germplasm of Indonesian green jungle fowl

Helicobacter ibis sp. nov., isolated from faecal droppings of black-faced ibis (Theristicus melanopis).

As part of a larger study on Epsilonproteobacteria carried by wild birds in the city of Valdivia (southern Chile), two curved rod-shaped Gram-stain-negative strains (A82T and WB-40) were recovered from faecal samples and subjected to a taxonomic study. Results of a genus-specific PCR showed that these isolates belonged to the genus Helicobacter. Further identification by 16S rRNA and hsp60 (60 kDa heat-shock protein) gene sequence analysis revealed that they formed a separate phylogenetic clade, different from other known Helicobacter species with 'Helicobacter burdigaliensis' CNRCH 2005/566HT and Helicobacter valdiviensis WBE14T being the most closely related species. This was confirmed by core-genome phylogeny as well as digital DNA-DNA hybridization and average nucleotide identity analyses between the genomes of strains A82T and WB-40 and all other Helicobacter species. The draft genome sequences of A82T and WB-40, obtained by Illumina NextSeq 2000 sequencing, consisted of 1.6 Mb with a G+C content of 31.9-32.0 mol%. The results obtained from the phylogenetic and genomic characterization, together with their different morphological and biochemical features, revealed that these two strains represent a novel species, for which we propose the name Helicobacter ibis sp. nov. with A82T (=LMG 32718T=CCCT 22.04T) as the type strain.

Identification of Significant Pathogenic Nontuberculous Mycobacteria Species from Presumptive TB Patients Using Partial hsp65 Gene Sequencing

Identification of Significant Pathogenic Nontuberculous Mycobacteria Species from Presumptive TB Patients Using Partial hsp65 Gene Sequencing

Antimicrobial Susceptibility Distributions of Clinical Isolates of Nontuberculous Mycobacteria in Israel.

There is a scarcity of data regarding the antimicrobial susceptibility testing profiles of nontuberculous mycobacterial (NTM) in Israel and other Middle Eastern countries. We aimed to describe the antimicrobial susceptibility profiles of NTM in Israel. A total of 410 clinical isolates of NTM, identified to the species level using matrix-assisted laser desorption ionization-time of flight mass spectrometry or hsp65 gene sequencing, were included. Minimum inhibitory concentrations for slowly growing mycobacteria (SGM) and rapidly growing mycobacteria (RGM) for 12 and 11 drugs were determined using the Sensititre SLOMYCOI and RAPMYCOI broth microdilution plates, respectively. Mycobacterium avium complex (MAC) was the most frequently isolated species (n = 148; 36%), followed by Mycobacterium simiae (n = 93; 23%), Mycobacterium abscessus group (n = 62; 15%), Mycobacterium kansasii (n = 27; 7%), and Mycobacterium fortuitum (n = 22; 5%) accounting together for 86% of isolates. The most active agents against SGM were amikacin (98%/85%/100%) and clarithromycin (97%/99%/100%), followed by moxifloxacin (25%/10%/100%) and linezolid (3%/6%/100%) for MAC, M. simiae, and M. kansasii, respectively. For RGM, the most active agents were amikacin (98%/100%/88%) followed by linezolid (48%/80%/100%) and clarithromycin (39%/28%/94%) for M. abscessus group, M. fortuitum, and M. chelonae, respectively. These findings can assist in guiding the treatment of NTM infections.

Identification of Mycobacterium chelonae from Lined Seahorse Hippocampus erectus and Histopathological Analysis

The lined seahorse (Hippocampus erectus) is an aquacultural species in China and has important economic and ornamental value. However, the species is affected by disease, which restricts their large-scale cultivation. In 2021, a disease was observed in cultured seahorses in Tianjin, China, with a cumulative mortality rate of 20%. The symptoms observed in the moribund seahorse included a weak swimming capacity, discolored body surface, enlarged liver and kidneys, and numerous white nodules in the parenchymatous organs. The strains HM-2021-1 and HM-2021-2 were isolated from diseased seahorses and were identified as being responsible for the disease. It demonstrated the potential to infect seahorse, and the cumulative mortalities of the seahorses artificially infected with strains HM-2021-1 and HM-2021-2 were 93.3% and 90.0%. The pathogen was identified as Mycobacterium chelonae based on physiological and biochemical tests, and 16S rDNA, rpoB, and Hsp65 gene sequence analysis. To our knowledge, this study is the first report of M. chelonae associated with diseased seahorses in China.

Development of a New Rapid Identification Method for <i>Bifidobacterium globosum</i> and <i>B. pseudolongum</i>Using Published Genome Data

Bifidobacterium globosum is expected to be applied to foods as a probiotic, and a method to rapidly distinguish it from the related Bifidobacterium pseudolongum is necessary for product development and quality control. In this study, we evaluated existing methods of bacterial species classification for B. globosum/pseudolongum with the published whole-genomes. The results showed that the Polymerase Chain Reaction (PCR) - Restriction Fragment Length Polymorphism (RFLP) method by the restriction enzyme HaeIII for hsp60 genes and the sequence analysis of hsp60 and rpoB gene sequences could not accurately distinguish B. globosum/pseudolongum, and both species could be accurately distinguished by the sequence analysis of clpC gene sequences. The clpC gene sequence similarity was at least 97.9% in the same species and no more than 95.4% in different species. Therefore, we developed a novel method of RFLP by restriction enzyme BspT104I on PCR products of clpC genes to identify B. globosum/pseudolongum. This method was considered to be faster and more accurate than previously proposed methods for identification of B. globosum/pseudolongum. Thus, the method presented in this study enables efficient species classification and it is very useful in utilization of B. globosum/pseudolongum for foods.

Phylogenetic evidence for a clade of tick-associated trypanosomes

BackgroundTrypanosomes are protozoan parasites of vertebrates that are of medical and veterinary concern. A variety of blood-feeding invertebrates have been identified as vectors, but the role of ticks in trypanosome transmission remains unclear.MethodsIn this study, we undertook extensive molecular screening for the presence and genetic diversity of trypanosomes in field ticks.ResultsExamination of 1089 specimens belonging to 28 tick species from Europe and South America led to the identification of two new trypanosome strains. The prevalence may be as high as 4% in tick species such as the castor bean tick Ixodes ricinus, but we found no evidence of transovarial transmission. Further phylogenetic analyses based on 18S rRNA, EF1-α, hsp60 and hsp85 gene sequences revealed that different tick species, originating from different continents, often harbour phylogenetically related trypanosome strains and species. Most tick-associated trypanosomes cluster in a monophyletic clade, the Trypanosoma pestanai clade, distinct from clades of trypanosomes associated with transmission by other blood-feeding invertebrates.ConclusionsThese observations suggest that ticks may be specific arthropod hosts for trypanosomes of the T. pestanai clade. Phylogenetic analyses provide further evidence that ticks may transmit these trypanosomes to a diversity of mammal species (including placental and marsupial species) on most continents.Graphical

Novel and rare species of nontuberculous mycobacteria by Hsp-65 gene sequencing.

Nontuberculosis mycobacterium (NTM) is the emerging group of organisms being recognized as etiological agents for diverse clinical conditions such as lymphadenitis, cutaneous, and pulmonary or disseminated lesions. Diverse background patients can acquire these infections such as immunocompetent, immunocompromised patients, or postoperative settings. Rapid addition of newer strains to this group necessitates heightened suspicion in the clinical settings. Specific requirements for cultures, biochemical testing, and molecular methods are needed to diagnose these organisms. The prospective study conducted at Nizam's Institute of Medical Sciences from January 2019 to December 2021 using various clinical samples using molecular techniques such as line probe assay and hsp-65 gene sequencing to discover new NTM species. The management is challenging since it requires prolonged treatment, multiple drugs, drug resistance, and individualization of treatment in the combination of surgery if needed. In this article, we describe three different NTM species which were not reported in India and highlight to consider these organisms in adequate clinical situation. Mycobacterium iranicum is a rare strain with quick growth and scotochromogenic colonies that are orange-colored. Eight distinct strains were discovered in clinical samples from six different countries: Two each from Iran, Italy, Greece, the Netherlands, Sweden, and the United States. Two of the strains were recovered from cerebrospinal fluid, which is unusual. Mycobacterium species AW6 is an unidentified and unclassified Mycobacterium according to NCBI taxonomy. Mycobacteria malmoense has been linked to lymphadenitis, notably cervical adenitis in children, and pulmonary infection in the majority of cases. Using Line Probe Assay and hsp-65 gene sequencing, novel and uncommon species of NTM were detected from a clinical samples, including sputum and tissue. We report three unusual species of NTMs: M. iranicum, M. species-AW6, and M. malmoense for the first time in India. Novel and rare emerging species of NTMs need to be considered in diverse clinical situations for appropriate therapy and good clinical outcomes.

The Resistance Mechanisms and Clinical Impact of Resistance to the Third Generation Cephalosporins in Species of Enterobacter cloacae Complex in Taiwan.

Enterobacter cloacae complex (ECC) is ubiquitous in the environment and is an important pathogen causing nosocomial infections. Because routine methods used in clinical laboratories cannot identify species within ECC, the clinical significance of each species within ECC is less known. We applied hsp60 gene sequencing to identify the species/clusters of ECC and detected β-lactamase genes and class 1 integrons with PCR for 184 clinical ECC isolates in Taiwan from 2013 to 2014 to investigate the clinical impact of species within ECC. The four most common clusters were E. hormaechei subsp. steigerwaltii (cluster VIII) (29.9%), E. hormaechei subsp. oharae (cluster VI) (20.1%), E. cloacae subsp. cloacae (cluster XI) (12%), and E. kobei (cluster II) (10.3%). E. hormaechei, which consisted of four clusters (clusters III, VI, VII, and VIII), is the predominant species and accounted for 57.1% of the isolates. The ceftazidime resistance rate was 27.2%, and the ceftriaxone resistance rate was 29.3%. Resistance to third generation cephalosporin was associated with a higher 30-day mortality rate. In total, 5 (2.7%), 24 (13.0%), and 1 (0.5%) isolates carried ESBL, AmpC, and carbapenemase genes, respectively. Class 1 integrons were present in 24.5% of the isolates, and most of the cassettes pertain to antibiotic resistance. Resistance to third generation cephalosporins, multidrug resistance, and class 1 integrons were significantly more in E. hormaechei (clusters III, VI, VII, and VIII) than in the other species. The 30-day mortality rate and 100-day mortality did not differ significantly between patients with E. hormaechei and those with infections with the other species. In conclusion, the distribution of third generation cephalosporin resistance, multidrug resistance, and class 1 integrons were uneven among Enterobacter species. The resistance to third generation cephalosporins possessed significant impact on patient outcome.

Clinical manifestations and antimicrobial susceptibility of Nocardia species at a tertiary hospital in Taiwan, 2011–2020

BackgroundThe study aimed to assess the clinical characteristics of patients with nocardiosis, to evaluate the in vitro susceptibility of antimicrobial agents against Nocardia species, and to explore changes in antimicrobial susceptibilities in this era of multidrug resistance. MethodsNocardia isolates were identified to the species level using matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) and 16S rRNA, hsp65, and secA1 gene sequencing, and minimum inhibitory concentrations (MICs) of 15 antimicrobial agents were assessed with the broth microdilution method. ResultsEighty-nine isolates from 68 patients were identified to species level. The most common species were Nocardia brasiliensis (n = 28, 31.5%), followed by N. farcinica (n = 24, 27%) and N. cyriacigeorgica (n = 16, 18%). Skin and soft tissue were the most common sites of nocardiosis. In multivariate analysis, cutaneous infection (OR, 0.052; p = 0.009), immunosuppressant use (OR, 16.006; p = 0.013) and Charlson combidity index (OR, 1.522; p = 0.029) were significant predictors for death. In total, 98.9% isolates were susceptible to trimethoprim-sulfamethoxazole and linezolid. Further, the MIC range and resistance rate of all Nocardia species to ceftriaxone, imipenem, and amoxicillin-clavulanic acid were found to generally increase over time. ConclusionConsidering that trimethoprim-sulfamethoxazole is effective against most Nocardia species, it is the antibiotic of choice in Taiwan. Besides, amikacin, tigecycline, and linezolid showed high activity against Nocardia species and are thus good alternatives or additional therapies to treat nocardiosis, depending on patient's underlying conditions and site of infection.

Campylobacter bilis sp. nov., isolated from chickens with spotty liver disease.

A novel species of Campylobacter was isolated from bile samples of chickens with spotty liver disease in Australia, making it the second novel species isolated from chickens with the disease, after Campylobacter hepaticus was isolated and described in 2016. Six independently derived isolates were obtained. They were Gram-stain-negative, microaerobic, catalase-positive, oxidase-positive and urease-negative. Unlike most other species of the genus Campylobacter, more than half of the tested strains of this novel species hydrolysed hippurate and most of them could not reduce nitrate. Distinct from C. hepaticus, many of the isolates were sensitive to 2,3,5-triphenyltetrazolium chloride (0.04%) and metronidazole (4 mg ml-1), and all strains were sensitive to nalidixic acid. Phylogenetic analysis using 16S rRNA and hsp60 gene sequences demonstrated that the strains formed a robust clade that was clearly distinct from recognized Campylobacter species. Whole genome sequence analysis of the strains showed that the average nucleotide identity and the genome blast distance phylogeny values compared to other Campylobacter species were less than 86 and 66%, respectively, which are below the cut-off values generally recognized for isolates of the same species. The genome of the novel species has a DNA G+C content of 30.6 mol%, while that of C. hepaticus is 27.9 mol%. Electron microscopy showed that the cells were spiral-shaped, with bipolar unsheathed flagella. The protein spectra generated from matrix-assisted laser desorption/ionization time of flight analysis demonstrated that they are different from the most closely related Campylobacter species. These data indicate that the isolates belong to a novel Campylobacter species, for which the name Campylobacter bilis sp. nov. is proposed. The type strain is VicNov18T (=ATCC TSD-231T=NCTC 14611T).

Macrococcus armenti sp. nov., a novel bacterium isolated from the skin and nasal cavities of healthy pigs and calves.

Gram-positive coccoid bacteria were isolated from the nasal cavities of pigs and calves as well as from axillar and inguinal skin regions of pigs. Phylogenetic analysis of seven strains based on complete genome, 16S rRNA, hsp60, dnaJ, rpoB and sodA gene sequences and MALDI-TOF MS profiles revealed that they belonged to the genus Macrococcus with the closest relatedness to Macrococcus canis, Macrococcus caseolyticus subsp. caseolyticus and Macrococcus caseolyticus subsp. hominis. DNA relatedness of the type strain JEK37T with the type strains of M. canis, M. caseolyticus subsp. caseolyticus and M. caseolyticus subsp. hominis was 23.4, 23.1 and 23.0 % by digital DNA-DNA hybridization and 80.39, 80.45 and 80.87 % by average nucleotide identity (ANI) calculations, confirming that they do not belong to the same species. The DNA G+C content of JEK37T was 35.65 mol%. The novel strains can be differentiated from M. canis KM 45013T by the ability to fermentate d-ribose and by the absence of DNAase production and haemolysis, from M. caseolyticus subsp. caseolyticus CCUG 15606T by the ability to fermentate sucrose and from both species by the inability to grow in 9 and 12% NaCl. They differ from M. caseolyiticus subsp. hominis by the presence of α-glucosidase. The most common fatty acids of JEK37T were C14 : 0, C18 : 1 ω9c and C18 : 0. Known polar lipids consisted of diphosphatidylglycerol, phosphatidylglycerol, aminolipid, aminoglycolipid, aminophospholipid, glycolipid and phospholipid. Cell-wall peptidoglycan of JEK37T was of type A3α l-Lys-Gly2-L-Ser-Gly (similar to A11.3) and the respiratory quinolone was menaquinone 6. Based on their genotypic and chemotaxonomic characteristics, these strains represent a novel species of the genus Macrococcus, for which we propose the name Macrococcus armenti sp. nov. The type strain is JEK37T (=DSM 112712T=CCOS 1982T).

Granulomatous Lymphadenitis in a Dog Caused by Mycobacterium intracellulare

Background: Mycobacteriosis is caused by bacteria belonging to the genus Mycobacterium, with considerable zoonotic potential and risk to public health. Infection in dogs is rare and is usually associated with immunosuppression, resulting from eating meat or contact with contaminated soil or fomites. Dogs are also known as potential sources for the spread of atypical tuberculosis in humans and other animals. This paper aims to describe the clinical, cytological, histopathological, and molecular findings of a male canine seen at University Veterinary Hospital of Cuiabá, Mato Grosso, with generalized lymphadenomegaly associated Mycobacterium intracellulare infection.Case: A 2-year-old male Lhasa Apso dog was referred to the University Veterinary Hospital in Cuiabá city, located in the Midwest region of Brazil. The patient had a history of intermittent claudication of the left pelvic limb for approximately 6 months and lymphadenomegaly with progression for approximately 2 months. The dog had wheezing and generalized lymphadenopathy (submandibular, axillary, and popliteal lymph nodes); cryptorchidism was also observed. A complete blood count revealed nonspecific results, and in the serum biochemical profile, the values of urea, creatinine, albumin, and alanine aminotransferase were within the reference range. No changes were observed on the radiography of the femurotibiopatellar joints. Considering the generalised lymphadenopathy, fine needle aspiration cytology and histopathological examination through biopsy of the lymph nodes was performed. On the cytology and histopathology, numerous negative images of moderately refringent bacillary structures distending the cytoplasm from the macrophages was found. The samples were also subjected to special Ziehl-Neelsen staining, which confirmed an accentuated and diffuse granulomatous lymphadenitis associated with alcohol acid-resistant bacilli. The polymerase chain reaction (PCR) was used to amplify the DNA of the lymph node fragment for the hsp65 gene, which was subjected to genetic sequencing and construction of a phylogenetic tree, with 99.77% genetic similarity for the species M. intracellulare. As treatment, doxycycline (10 mg/kg twice a day for 60 days) and enrofloxacin (5 mg/kg once a day for 10 days) were prescribed. However, the canine suffered car trauma leading to a fractured pelvis, which motivated the owner to opt for euthanasia at another veterinary establishment.Discussion: In the reported case, it was not possible to determine the source of infection, as the owners reported that the animal lived inside the house with only sporadic access to the street. The clinical signs manifested by this dog were nonspecific, and only the signs of generalised lymphadenopathy could be correlated with the signs expected in the infection with this mycobacterium. The hematological and biochemical laboratory findings were nonspecific, and it did not demonstrate the involvement of other organs. Considering the findings in cytology and histology, mycobacterial infection can be suspected. The diagnosis was confirmed through pathological and molecular findings. In this case, the PCR technique was used with partial amplification of the hsp65 gene and subsequent genetic sequencing, making it possible to identify a species like M. intracellulare (99.77% similarity). Due to euthanasia for another reason, it was impossible to monitor the dog's treatment and investigate other changes in the post mortem examination, especially the pulmonary lesions frequently described in M. intracellulare infection in humans.Keywords: Mycobacterium avium complex (MAC), canine, infection, non-tuberculous mycobacteriosis.

Molecular Epidemiology, Risk Factors and Clinical Outcomes of Carbapenem-Nonsusceptible Enterobacter cloacae Complex Infections in a Taiwan University Hospital.

The genus Enterobacter is a member of the ESKAPE group, which contains the major resistant bacterial pathogens. Enterobacter cloacae complex (ECC) has emerged as a clinically significant cause of a wide variety of nosocomial infections. Carbapenem-nonsusceptible Enterobacter cloacae complex (CnsECC) has become an emerging threat to public health but there is still a lack of comprehensive molecular and clinical epidemiological analysis. A total of 157 CnsECC isolates were recovered during October 2011 to August 2017. hsp60 gene sequencing and pulsed-field gel electrophoresis (PFGE) were applied to discriminate the species, genetic clusters and clonal relatedness. All the isolates were subjected to polymerase chain reaction (PCR) analysis for carbapenemase, AmpC-type β-lactamase, and extended spectrum β-lactamase (ESBL) genes. Clinical data were collected on all patients for comparing clinical risks and outcomes between patients with carbapenemase-producing (CP)-CnsECC compared with non-CP-CnsECC infection. The most commonly identified species was E. hormaechei subsp. hoffmannii (47.1%), followed by E. hormaechei subsp. steigerwaltii (24.8%). Different species of CnsECC isolates showed heterogeneity in resistance patterns to piperacillin/tazobactam, cefepime and levofloxacin. In the present study, we observed that E. hormaechei subsp. hoffmannii was characterized with higher cefepime and levofloxacin resistance rate but lower piperacillin/tazobactam resistance rate relative to other species of CnsECC. CP-CnsECC comprised 41.1% (65 isolates) and all of these isolates carried IMP-8. In this study, 98% of patients had antimicrobial therapy prior to culture, with a total of 57/150 (38%) patients being exposed to carbapenems. Chronic pulmonary disease (OR: 2.51, 95% CI: 1.25–5.06), received ventilator support (OR: 5.54, 95% CI: 2.25–12.03), steroid exposure (OR: 3.88, 95% CI: 1.91–7.88) and carbapenems exposure (OR: 2.17, 95% CI: 1.10–4.25) were considered risk factors associated with CP-CnsECC infection. The results suggest that CP-CnsECC are associated with poorer outcomes including in-hospital mortality, 30-day mortality and 100-day mortality. Our study provides insights into the epidemic potential of IMP-8-producing E. cloacae for healthcare-associated infections and underscores the importance of understanding underlying resistance mechanisms of CnsECC to direct antibiotic treatment decisions.

Phylogenetic relationships and genetic diversity of Monilinia spp. isolated in Poland based on housekeeping‐ and pathogenicity‐related gene sequence analysis

AbstractMonilinia spp. fungi, which cause brown rot disease, are of great economic importance for pome and stone fruit production in Poland. To date, four species of this genus, M. fructicola, M. fructigena, M. laxa, and M. polystroma, have been identified in this country. However, little is known about their phylogenetic relationships and genetic diversity. In this research, β‐tubulin and three genes not previously studied in these species, heat shock protein 60 (hsp60), acid protease 1 (acp1), and a zinc finger transcription factor (pac1), were investigated to infer the phylogenetic relationships among these species. Diversity data, as well as recombination analyses of the sequenced regions, were used to assess the genetic variation of Monilinia fungi and the evolutionary forces that are currently acting upon these microorganisms. Based on the topology of the obtained dendrograms, the current study demonstrates the lack of a relationship between the phylogenetic position of the strain, the host plant, and the geographical origin of the strain. Additionally, our study provided evidence that phylogenetic analysis using a combination of tub2, hsp60, acp1, and pac1 gene sequences can be used to effectively resolve the relationships among Monilinia species. For each species analysed in this study, low genetic diversity and a lack of recombination were detected. On the basis of the obtained results, it can be concluded that variation in the population of Monilinia spp. in Poland is largely influenced by mutation rather than recombination processes.

Peritoneal dialysis-associated peritonitis caused by Mycobacteroides massiliense: the first case and review of the literature

BackgroundPeritoneal dialysis (PD)-associated peritonitis caused by nontuberculous Mycobacterium is rare; however, the number of cases has increased over the past decades. Mycobacteroides massiliense is a subspecies of the Mycobacteroides abscessus complex. It has different clinical characteristics compared to the other subspecies of the complex. Previous case reports of PD-associated peritonitis caused by Mycobacteroides abscessus complex have not distinguished the subspecies in detail.Case presentationA 40-year-old man presented with an exit-site and tunnel infection refractory to antibiotic therapy. Peritonitis occurred after simultaneous catheter removal and reinsertion. The Mycobacteroides abscessus complex was detected in the culture of the dialysis effluent. Removal of the PD catheter combined with antibiotics, including macrolides, resulted in a good clinical course. Further analysis of multiplex PCR and the hsp65 gene sequence identified the bacterium as Mycobacteroides massiliense.ConclusionsThe Mycobacteroides abscessus complex is classified into three subspecies; Mycobacteroides abscessus, Mycobacteroides massiliense, and Mycobacteroides bolletii. These have different characteristics, particularly antibiotic susceptibility. Therefore, clear identification of the subspecies of the Mycobacteroides abscessus complex is necessary for definitive treatment.

Molecular tools confirm natural Leishmania (Viannia) guyanensis/L. (V.) shawi hybrids causing cutaneous leishmaniasis in the Amazon region of Brazil

Seven isolates from patients with American cutaneous leishmaniasis in the Amazon region of Brazil were phenotypically suggestive of Leishmania (Viannia) guyanensis/L. (V.) shawi hybrids. In this work, two molecular targets were employed to check the hybrid identity of the putative hybrids. Heat shock protein 70 (hsp70) gene sequences were analyzed by three different polymerase chain reaction (PCR) approaches, and two different patterns of inherited hsp70 alleles were found. Three isolates presented heterozygous L. (V.) guyanensis/L. (V.) shawi patterns, and four presented homozygous hsp70 patterns involving only L. (V.) shawi alleles. The amplicon sequences confirmed the RFLP patterns. The high-resolution melting method detected variant heterozygous and homozygous profiles. Single-nucleotide polymorphism genotyping/cleaved amplified polymorphic site analysis suggested a higher contribution from L. (V.) guyanensis in hsp70 heterozygous hybrids. Additionally, PCR-RFLP analysis targeting the enzyme mannose phosphate isomerase (mpi) gene indicated heterozygous and homozygous cleavage patterns for L. (V.) shawi and L. (V.) guyanensis, corroborating the hsp70 findings. In this communication, we present molecular findings based on partial informative regions of the coding sequences of hsp70 and mpi as markers confirming that some of the parasite strains from the Brazilian Amazon region are indeed hybrids between L. (V.) guyanensis and L. (V.) shawi.

Mapping Mycobacterium tuberculosis genetic diversity in the gulf of guinea – africa

Aims and objectives: Tuberculosis (TB) worldwide re-emergence constitutes a major public health problem with multi-drug resistant (MDR) strains becoming a greater threat. Genotyping and drug susceptibility of Mycobacterium tuberculosis complex (MTBC) are important in order to improve the control of the disease. This study sought to determine drug susceptibility and genetic diversity of MTBC in three West/Central African countries, namely Cameroon, Nigeria and Ghana located in the Gulf of Guinea. Methods: A collection of 498 isolates (150 from Cameroon, 202 from Nigeria and 146 from Ghana) obtained from culture using Lowenstein–Jensen medium, Middlebrook 7H9 Broth and/or Xpert MTB/RIF and Line Probe Assay (LPA) were included in the study and some isolates tested for drug susceptibility using the proportion method. Isolates were further subjected to IS6110 typing, region of difference 1, 4, 9, 12, 702, 711, spoligotyping and hsp65 gene sequencing analysis. Results: The LAM10_CAM was the most prevalent genetic family [128/202 (63.4%) in Nigeria, 46/123 (37.4%) in Ghana and 54/150 (36%) in Cameroon] and SIT 61 the largest cluster with 111/202 (55%) and 43/150 (27%) isolates in Nigeria and Cameroon respectively. Multidrug resistance (MDR) was observed in 29/202 (14.4%) cases in Nigeria, and HIV positivity [37/202 (18.3%) patients] was associated with rifampicin 9/37 (24.3%) resistance (p=0.012), as well as gender (p=0.009). In Cameroon, 13/147 (8.8 %) were resistant to rifampicin (RIF), with 6 (46.2%) of the 13 RIF resistant isolates associated with UgandaI sub-lineage (p=0.0007). Other sublineages such as Haarlem, Ghana, West African 1 were recorded in the three countries. Conclusion: Rifampicin resistance in TB/HIV co-infected patient and its association with a sublineage calls for a reinforced surveillance strategy to curb drug resistance development in the Gulf of Guinea.