Multilocus Sequence Typing Reveals Extensive Genetic Diversity of the Emerging Fungal Pathogen Scedosporium aurantiacum

Scedosporium spp. are the second most prevalent filamentous fungi after Aspergillus spp. recovered from cystic fibrosis (CF) patients in various regions of the world. Although invasive infection is uncommon prior to lung transplantation, fungal colonization may be a risk factor for invasive disease with attendant high mortality post-transplantation. Abundant in the environment, Scedosporium aurantiacum has emerged as an important fungal pathogen in a range of clinical settings. To investigate the population genetic structure of S. aurantiacum, a MultiLocus Sequence Typing (MLST) scheme was developed, screening 24 genetic loci for polymorphisms on a tester strain set. The six most polymorphic loci were selected to form the S. aurantiacum MLST scheme: actin (ACT), calmodulin (CAL), elongation factor-1α (EF1α), RNA polymerase subunit II (RPB2), manganese superoxide dismutase (SOD2), and β-tubulin (TUB). Among 188 global clinical, veterinary, and environmental strains, 5 to 18 variable sites per locus were revealed, resulting in 8 to 23 alleles per locus. MLST analysis observed a markedly high genetic diversity, reflected by 159 unique sequence types. Network analysis revealed a separation between Australian and non-Australian strains. Phylogenetic analysis showed two major clusters, indicating correlation with geographic origin. Linkage disequilibrium analysis revealed evidence of recombination. There was no clustering according to the source of the strains: clinical, veterinary, or environmental. The high diversity, especially amongst the Australian strains, suggests that S. aurantiacum may have originated within the Australian continent and was subsequently dispersed to other regions, as shown by the close phylogenetic relationships between some of the Australian sequence types and those found in other parts of the world. The MLST data are accessible at http://mlst.mycologylab.org. This is a joined publication of the ISHAM/ECMM working groups on “Scedosporium/Pseudallescheria Infections” and “Fungal Respiratory Infections in Cystic Fibrosis”.

Application of simplified MLST scheme for direct typing of clinical samples from human leptospirosis cases in a tertiary hospital in the Philippines

Despite the major threat of leptospirosis to public health in the Philippines, its epidemiologic data remain scarce. Multilocus sequence typing (MLST) is a method often used for identification of circulating Leptospira species and disease surveillance. Unfortunately, molecular typing of Leptospira isolates is not routinely done in most hospital settings. A simplified MLST scheme targeting three loci (adk, lipL41, mreA) was performed for rapid direct typing of Leptospira in clinical specimens. Blood samples from suspected or clinically diagnosed cases (n = 50) were initially screened via polymerase chain reaction (PCR) targeting 23S rRNA, 16S rRNA (rrs2), and lipL32 genes. From the nine positives, seven had interpretable data from MLST. Allelic profiles identified L. interrogans in all positive samples. Six were assigned to ST12 of serovar Manilae (serogroup Pyrogenes) while one sample cannot be clearly differentiated between two serovars/serogroups, Bataviae/Losbanos (serogroup Bataviae) or Australis (serogroup Australis), indicating possibility of a new ST. Phylogenetic analysis confirmed that the application of simplified MLST scheme produces consistent results with the seven-loci genetic profile of published Leptospira MLST schemes. Reduced scheme addressed the challenges often encountered in the amplification of full MLST genetic profile of Leptospira. The approach is a potential alternative to serological tests for rapid typing of clinical specimens and can also aid in investigations on disease epidemiology specifically to monitor occurrence, pathogen transmission, host specificity and susceptibility, and other factors that could lead to potential outbreaks.

Rapid high-resolution melting genotyping scheme for Escherichia coli based on MLST derived single nucleotide polymorphisms

Routinely used typing methods including MLST, rep-PCR and whole genome sequencing (WGS) are time-consuming, costly, and often low throughput. Here, we describe a novel mini-MLST scheme for Eschericha coli as an alternative method for rapid genotyping. Using the proposed mini-MLST scheme, 10,946 existing STs were converted into 1,038 Melting Types (MelTs). To validate the new mini-MLST scheme, in silico analysis was performed on 73,704 strains retrieved from EnteroBase resulting in discriminatory power D = 0.9465 (CI 95% 0.9726–0.9736) for mini-MLST and D = 0.9731 (CI 95% 0.9726–0.9736) for MLST. Moreover, validation on clinical isolates was conducted with a significant concordance between MLST, rep-PCR and WGS. To conclude, the great portability, efficient processing, cost-effectiveness, and high throughput of mini-MLST represents immense benefits, even when accompanied with a slightly lower discriminatory power than other typing methods. This study proved mini-MLST is an ideal method to screen and subgroup large sets of isolates and/or quick strain typing during outbreaks. In addition, our results clearly showed its suitability for prospective surveillance monitoring of emergent and high-risk E. coli clones’.

Molecular Epidemiology of Escherichia coli Clinical Isolates from Central Panama

Escherichia coli represents one of the most common causes of community-onset and nosocomial infections. Strains carrying extended spectrum β-lactamases (ESBL) are a serious public health problem. In Central America we have not found studies reporting the molecular epidemiology of E. coli strains implicated in local infections, so we conducted this study to fill that gap. Materials and Methods: We report on an epidemiological study in two reference hospitals from central Panama, identifying the susceptibility profile, associated risk factors, and molecular typing of E. coli strains isolated between November 2018 and November 2019 using Pasteur’s Multilocus Sequence Typing (MLST) scheme. Results: A total of 30 E. coli isolates with antimicrobial resistance were analyzed, 70% of which came from inpatients and 30% from outpatients (p < 0.001). Two-thirds of the samples came from urine cultures. Forty-three percent of the strains were ESBL producers and 77% were resistant to ciprofloxacin. We identified 10 different sequence types (STs) with 30% of the ESBL strains identified as ST43, which corresponds to ST131 of the Achtman MLST scheme—the E. coli pandemic clone. Thirty-eight percent of the E. coli strains with the ESBL phenotype carried CTX-M-15. Conclusions: To the best of our knowledge, this is the first report confirming the presence of the pandemic E. coli clone ST43/ST131 harboring CTX-M-15 in Central American inpatients and outpatients. This E. coli strain is an important antimicrobial-resistant organism of public health concern, with potential challenges to treat infections in Panama and, perhaps, the rest of Central America.

Molecular Epidemiology, Genetic Diversity, and Antifungal Susceptibility of Major Pathogenic Dermatophytes Isolated From Human Dermatophytosis

BackgroundDermatophytes are a homogeneous group of species with low genetic diversity, and there are still many uncertainties about the boundaries among species.ObjectivesAiming at clarifying the relationships among species in the genus and introducing suitable genes for multilocus sequence typing (MLST), a new MLST scheme approach was developed to characterize the major pathogenic dermatophytes.MethodsWe performed maximum parsimony (MP), MrBayes, RAxML, and eBURST analyses, based on the MLST scheme to scrutinize the evolution within 95 clinical isolates and four reference strains belonging to the four major dermatophytes species. Then, the discriminatory power, pairwise genetic distances, ratio dN/dS, and sequence types (STs) of these isolates were determined. Also, to study taxonomy, sequences of the internal transcribed spacer (ITS), Beta-tubulin (BT2), and translation elongation factor 1-α (TEF-1α) genes of other dermatophytes species available in the GenBank were analyzed.ResultsFindings of the present study indicated that three genes: BT2, ITS, and TEF−1α, which showed the greatest diversity among dermatophyte species, were suitable for MLST. The most prevalent STs were seen among the species of Trichophyton interdigitale. Also, two new genotypes, i.e., XXVII and XXVIII, were introduced for T. interdigitale and Trichophyton mentagrophytes. The least informative sites were found in Epidermophyton floccosum, Trichophyton rubrum, and T. mentagrophytes, while the most informative sites were observed in T. interdigitale. Furthermore, the most informative locus was TEF-1α. The phylogenetic tree, constructed by the combination of the three genes, shows a new topological pattern that confirms the derivation of the anthropophilic and zoophilic genera from the geophilic genus. Also, the phylogenetic analyses and pairwise distances of the combination of the three loci showed that Trichophyton tonsurans and Trichophyton equinum were a species complex, where T. equinum is derived from T. tonsurans.ConclusionsResults of this study showed that MLST is very effective in determining the boundaries between species and taxonomy. Considering that there is no database for MLST dermatophytes, further studies are needed to determine the suitable genes for MLST. Also, the determination of STs in epidemiological studies and raising epidemiological information are helpful. This study was a new starting point to determine the ST and a foundation for a dermatophyte MLST database.

Characterization of genetic diversity and population structure within Staphylococcus chromogenes by multilocus sequence typing

Staphylococcus chromogenes is a common skin commensal in cattle and has been identified as a frequent cause of bovine mastitis and intramammary infections. We have developed a seven locus Multilocus Sequence Typing (MLST) scheme for typing S. chromogenes. Sequence-based typing systems, such as MLST, have application in studies of genetic diversity, population structure, and epidemiology, including studies of strain variation as a factor in pathogenicity or host adaptation. The S. chromogenes scheme was tested on 120 isolates collected from three geographic locations, Vermont and Washington State in the United States and Belgium. A total of 46 sequence types (STs) were identified with most of the STs being location specific. The utility of the typing scheme is indicated by a discrimination power of 95.6% for all isolates and greater than 90% for isolates from each of the three locations. Phylogenetic analysis placed 39 of the 46 STs into single core group consistent with a common genetic lineage; the STs in this group differ by less than 0.5% at the nucleotide sequence level. Most of the diversification in this lineage group can be attributed to mutation; recombination plays a limited role. This lineage group includes two clusters of single nucleotide variants in starburst configurations indicative of recent clonal expansion; nearly 50% of the isolates sampled in this study are in these two clusters. The remaining seven STs were set apart from the core group by having alleles with highly variable sequences at one or more loci. Recombination had a higher impact than mutation in the diversification of these outlier STs. Alleles with hypervariable sequences were detected at five of the seven loci used in the MLST scheme; the average sequence distances between the hypervariable alleles and the common core alleles ranged from 12 to 34 nucleotides. The extent of these sequence differences suggests the hypervariable alleles may be remnants of an ancestral genotype.

Analysis of blaCHDL Genes and Insertion Sequences Related to Carbapenem Resistance in Acinetobacter baumannii Clinical Strains Isolated in Warsaw, Poland

Acinetobacter baumannii is an important cause of nosocomial infections worldwide. The elucidation of the carbapenem resistance mechanisms of hospital strains is necessary for the effective treatment and prevention of resistance gene transmission. The main mechanism of carbapenem resistance in A. baumannii is carbapenemases, whose expressions are affected by the presence of insertion sequences (ISs) upstream of blaCHDL genes. In this study, 61 imipenem-nonsusceptible A. baumannii isolates were characterized using phenotypic (drug-susceptibility profile using CarbaAcineto NP) and molecular methods. Pulsed field gel electrophoresis (PFGE) and multi-locus sequence typing (MLST) methods were utilized for the genotyping. The majority of isolates (59/61) carried one of the following acquired blaCHDL genes: blaOXA-24-like (39/59), ISAba1-blaOXA-23-like (14/59) or ISAba3-blaOXA-58-like (6/59). Whole genome sequence analysis of 15 selected isolates identified the following intrinsic blaOXA-66 (OXA-51-like; n = 15) and acquired class D β-lactamases (CHDLs): ISAba1-blaOXA-23 (OXA-23-like; n = 7), ISAba3-blaOXA-58-ISAba3 (OXA-58-like; n = 2) and blaOXA-72 (OXA-24-like; n = 6). The isolates were classified into 21 pulsotypes using PFGE, and the representative 15 isolates were found to belong to sequence type ST2 of the Pasteur MLST scheme from the global IC2 clone. The Oxford MLST scheme revealed the diversity among these studied isolates, and identified five sequence types (ST195, ST208, ST208/ST1806, ST348 and ST425). CHDL-type carbapenemases and insertion elements upstream of the blaCHDL genes were found to be widespread among Polish A. baumannii clinical isolates, and this contributed to their carbapenem resistance.

High-resolution multilocus sequence typing for Chlamydia trachomatis: improved results for clinical samples with low amounts of C. trachomatis DNA

Background Several Multilocus Sequence Typing (MLST) schemes have been developed for Chlamydia trachomatis. Bom’s MLST scheme for MLST is based on nested PCR amplification and sequencing of five hypervariable genes and ompA. In contrast to other Chlamydia MLST schemes, Bom’s MLST scheme gives higher resolution and phylogenetic trees that are comparable to those from whole genome sequencing. However, poor results have been obtained with Bom’s MLST scheme in clinical samples with low concentrations of Chlamydia DNA. Results In this work, we present an improved version of the scheme that is based on the same genes and MLST database as Bom’s MLST scheme, but with newly designed primers for nested-1 and nested-2 steps under stringent conditions. Furthermore, we introduce a third primer set for the sequencing step, which considerably improves the performance of the assay. The improved primers were tested in-silico using a dataset of 141 Whole Genome Sequences (WGS) and in a comparative analysis of 32 clinical samples. Based on cycle threshold and melting curve analysis values obtained during Real-Time PCR of nested-1 & 2 steps, we developed a simple scoring scheme and flow chart that allow identification of reaction inhibitors as well as to predict with high accuracy amplification success. The improved MLST version was used to obtain a genovars distribution in patients attending an STI clinic in Tel Aviv. Conclusions The newly developed MLST version showed great improvement of assay results for samples with very low concentrations of Chlamydia DNA. A similar concept could be applicable to other MLST schemes.

Characterization of genetic diversity and population structure within Staphylococcus chromogenes by multilocus sequence typing

Staphylococcus chromogenes is a common skin commensal in cattle and has been identified as a frequent cause of bovine mastitis and intramammary infections. To better understand the extent of strain diversity within this species and to facilitate study of strain variation as a factor in pathogenicity, we have developed a seven locus Multilocus Sequence Typing (MLST) scheme. The scheme was tested on 120 isolates collected from three geographic locations, Vermont and Washington State in the United States and Belgium. A total of 46 sequence types (STs) were identified with most of the STs being location specific. The utility of the typing scheme is indicated by a discrimination power of 95.6% for all isolates and greater than 90% for isolates from each of the three locations. Phylogenetic analysis placed 39 of the 46 STs into single core group consistent with a common genetic lineage; the STs in this group differ by less than 0.5% at the nucleotide sequence level. Most of the diversification in this lineage group can be attributed to mutation; recombination plays a limited role. This lineage group includes two clusters of single nucleotide variants in starburst configurations indicative of recent clonal expansion; nearly 50% of the isolates sampled in this study are in these two clusters. The remaining seven STs were set apart from the core group by having alleles with highly variable sequences at one or more loci. Recombination had a higher impact than mutation in the diversification of these outlier STs. Alleles with hypervariable sequences were detected at five of the seven loci used in the MLST scheme; the average sequence distances between the hypervariable alleles and the common core alleles ranged from 12 to 34 nucleotides. The extent of these sequence differences suggests the hypervariable alleles may be remnants of an ancestral genotype.

Antibiotic induced biofilm formation of novel multidrug resistant Acinetobacter baumannii ST2121 clone

The aim of this study was to identify antimicrobial resistance and virulence factor genes exhibited by multidrug resistant (MDR) Acinetobacter baumannii, to analyze biofilm formation and to investigate clonal subtypes of isolate. Whole genome sequencing was done by Illumina NovaSeq 6,000 platform and multilocus sequence typing (MLST) was performed by Oxford and Pasteur typing schemes. Influence of imipenem and levofloxacin on biofilm formation was investigated in 96-well plates at 3 replicates. The strain was found to carry OXA-23, OXA-51-like, AmpC and TEM-1 beta-lactamases. The sequence of the blaOXA-51-like gene has been identified as a blaOXA-66. According to Pasteur MLST scheme the strain displayed ST2 allelic profile. However, based on Oxford MLST scheme this strain represents the new ST2121, as the gdhB gene has a single allelic mutation namely, the gdhB-227. It was determined that MDR isolate carried bap, basABCDFGHIJ, csuA/BABCDE, bauABCDEF, plcD, pgaABCD, entE, barAB, ompA, abaIR, piT2EAFTE/AUBl, fimADT, cvaC, bfmR, bfmS virulence genes. In our study imipenem induced the highest biofilm formation at a concentration of 32 µg/ml and levofloxacin at a concentration of 16 µg/ml. In conclusion, we detected a new MDR A. baumannii ST2121 clone harboring blaOXA-66 gene that has been reported for the first time in Turkey.