Multilocus Sequence Typing Method Research Articles

New Species-Specific Real-Time PCR Assays for Colletotrichum Species Causing Bitter Rot of Apple.

Bitter rot of apple is an economically important worldwide disease caused by different Colletotrichum species, depending on many factors such as climate, geography, other hosts, and crop management practices. Culture, morphology, and single-locus sequencing-based methods for identifying the Colletotrichum species are severely limited in effectiveness, while the multilocus sequence typing methods available for delineating species are costly, time-intensive, and require high expertise. We developed species-specific hydrolysis probe real-time PCR assays for the following nine Colletotrichum species causing bitter rot in the Mid-Atlantic U.S.A.: C. fructicola, C. chrysophilum, C. noveboracense, C. gloeosporioides s.s., C. henanense, C. siamense and C. theobromicola from the C. gloeosporioides species complex, and C. fioriniae and C. nymphaeae from the C. acutatum species complex. After searching 14 gene regions, we designed primers and probes in 5 of them for the nine target species. Four primer-probe set pairs were able to be duplexed. Sensitivity tests showed as little as 0.5 pg DNA were detectable. These real-time PCR assays will provide rapid and reliable identification of these key Colletotrichum species and will be critically important for studies aiming to elucidate their biology, epidemiology, and management on apples as the number one produced and consumed tree fruit in the U.S.A.

Molecular epidemiological and clinical infection characteristics analysis of Ralstonia

PurposeThis study was to clarify the molecular epidemiology and clinical infection characteristics of Ralstonia pickettii and establish sequence typing system.Methods48 nonrepetitive Ralstonia pickettii strains were collected from January 2008 to December 2013 at the Chinese People’s Liberation Army General Hospital (PLAGH) and were identified through a specific PCR experiment, 16 S rDNA experiment and VITEK 2 system to compare the identification accuracy. The sequence types of the strains were analyzed by multilocus sequence typing (MLST) method. The antibiotic sensitivity of these strains was determined with disc diffusion tests and broth microdilution method. The clinical data of Ralstonia pickettii infected patients were collected.ResultsAll of the 48 strains were identified as Ralstonia pickettii by VITEK 2 system. 30 and 34 strains were identified as Ralstonia pickettii by PCR and 16 S rDNA experiment respectively. ST9 was the most sequence types (STs) in these 18 STs of 42 strains. 42 strains were divided into 2 groups (A and B) and 18 genotypes. Ralstonia pickettii was sensitive to some cephalosporins, β-lactam/β-lactamase inhibitor, levofloxacin and trimethoprim/sulfamethoxazole. Cough, sputum, shortness of breath and pulmonary rales were the common clinical symptoms of most Ralstonia pickettii infected patients.ConclusionWe established a sequence typing system with a relatively fine resolution and the PCR assay is a faster and more sensitive method for clinical identification of Ralstonia pickettii. ST9 is the most common sequence types of Ralstonia pickettii. The most common clinical characteristics of Ralstonia pickettii infected patients were cough, sputum, shortness of breath and pulmonary rales.

Multilocus sequence typing and antibiotic resistance of Aeromonas isolated from freshwater fish in Hebei Province.

Aeromonas spp. are the opportunistic pathogens that infect both aquatic and terrestrial homeotherms. They were commonly present in aquatic environments, including effluent, tap water, marine, river, and lake, where they are often isolated from aquatic animals, including fish, molluscs, and crustaceans. The Aeromonas infections can cause sepsis, ulcer, and other symptoms, resulting in the death of massive aquatic animals. Therefore, the prevention and control of Aeromonas is of great significance for the healthy development of aquaculture. In this study, we used modern molecular methods to enhance disease control of Aeromonas isolates from freshwater fish in Hebei Province. A total of 130 Aeromonas spp. isolates were isolated from freshwater fish farms in Hengshui, Handan, and Shijiazhuang and all 130 Aeromonas spp. isolates were sequenced for species identification. Of the 130 Aeromonas spp. isolates, 104 isolates were successfully sequenced, and BLAST analysis showed that Aeromonas veronii was predominant in freshwater fish farms in Hebei Province. In addition, 26 antibiotic resistance profiles were obtained from 102 fully cultured isolates among the 104 Aeromonas spp. isolates whose species was primarily identified, and 44 multidrug-resistant bacteria among the 102 isolates were identified using an antibiotic susceptibility test. Using the Multilocus Sequence Typing (MLST) method, 33 out of 44 multidrug-resistant isolates with 14 non-Aeromonas reference strains were selected for phylogenetic and MLST analysis, and all 33 multidrug-resistant isolates were A. veronii. A total of 30 new Sequence Types (STs) were obtained by comparing concatenated sequences (gyrB-groL-gltA-metG-ppsA-recA) on PubMLST website. Furthermore, recombination event analysis detected using RDP5 and ClonalFrameML software 42 and 49 recombination events, respectively, and 22 recombination events were validated by four or more algorithms. Since mutation and recombination events increase clonal diversity and single housekeeping gene sequence alignments are limited for identifying species, we propose the use of multiple concatenated sequence loci to increase discriminatory power. In addition, we propose that the MLST method is an appropriate technique to study and develop the resistance mechanisms of multidrug-resistant Aeromonas and to identify Aeromonas systematically in complex samples obtained from the environment.

Occurrence and Characteristics of Toxigenic Staphylococcus aureus in Retail Foods in Iran.

Staphylococcus aureus causes various toxigenic and invasive diseases in humans worldwide. This study examined the prevalence, virulence genes, and antibiotic resistance of S. aureus isolates collected from 894 retail food samples in Ardabil, Iran. Staphylococcal cassette chromosome mec (SCCmec), spa, and multilocus sequence typing methods were employed to further investigate the molecular characteristics of methicillin-resistant S. aureus (MRSA) isolates. The results revealed that 11.18% (n = 100) of food samples exhibited contamination with S. aureus (10.50% methicillin-sensitive S. aureus [MSSA] and 0.67% MRSA). Notably, raw minced meat (29.41%), Faloodeh (25%), and Olivier salad (21.42%) emerged as the most frequently contaminated food items. Among the 100 isolates of S. aureus, 94% were characterized as MSSA, with the remaining 6% identified as MRSA. The highest resistance was observed for penicillin (12%). MRSA isolates exhibited significantly higher resistance rates. Seventy-nine percent of the isolates were positive for sea, 14% for seb, 8% for a sec, and 0% for sed enterotoxin-encoding genes. Sixteen percent of isolates harbored two or more staphylococcal enterotoxin genes, simultaneously. Moreover, 97%, 94%, 24%, and 22% of isolates were positive for hla, hld, tst, and pvl virulence-encoding genes, respectively. No isolate was positive for the exfoliative toxins encoding eta and etb genes. MRSA isolates belonged to CC8 (n = 4) and CC22 (n = 2). Isolates in CC8 belonged to lineage ST239-MRSA-III and spa type t030; the isolates in CC22 belonged to ST22-MRSA-IV and spa types t310 and t223. In conclusion, a relatively high proportion of our retail food samples were contaminated with S. aureus. The high incidence of isolates with toxigenic genes raises serious health concerns. Furthermore, the presence of MRSA lineages linked to humans suggests that retail foods may be contaminated with human origin.

Genetic diversity and biofilm formation ofinvasive and noninvasive Streptococcus agalactiae isolates: Emergence of hypervirulentCC19 strains in Tehran, Iran.

The prevalence of Streptococcus agalactiae infections in adult populations is increasing. The current study aimed to characterize the genetic features of S. agalactiae strains responsible for different infections. Across-sectional study was performed on 65 S. agalactiae strains (30 invasive and 35 noninvasive) isolated from non-pregnant women. All S. agalactiae isolates were confirmed by atr and dltS PCR assays. Antibiotic susceptibility patterns were determined using the disk diffusion method. Biofilm production was investigated by microtiter plate assay. PCR was done to detect resistance determinants. Isolates were characterized using the multilocus sequence typing (MLST) method. cMLSB, iMLSB, and M phenotypes accounted for 47.7%, 30.8%, and 6.2%, respectively. MDR was detected in 15.4% of noninvasive and 44.6% of invasive isolates. MtP assay indicated that 80% of isolates were biofilm producers. Biofilm formation was common among noninvasive compared with invasive strains (94.3% versus 66.7%). tet (M) (46.2%) and erm (B) (69.2%) were the most prevalent tetracycline and macrolide-resistance genes. The most prevalent serotype was type III (50.8%), followed by Ia (18.4%), II (15.4%), V (12.3%), and IV (3.1%). The frequency of serotype III among biofilm producer strains (81.8%) was found to be significantly higher than that of non-producer isolates (18.2%) (P < 0.05). S. agalactiae was resolved within four clonal complexes, including CC19 (46.2%; in both invasive and noninvasive), followed by CC23 (30.8%; only noninvasive isolates), CC1 (15.4%; only noninvasive isolates) and CC17 (7.6%; only invasive isolates). Themain sequence types (STs) found were ST19 (27.7%), ST17 (7.7%), ST27 (6.2%), and ST28 (4.6%) linked with invasive infections and ST23 (18.4%), ST933 (12.3%), ST644 (9.2%), ST19 (7.7%), ST1 (6.2%) found in noninvasive infections. The high prevalence of CC19 and CC23 clones among S. agalactiae strains reflects the emergence of these lineages as successful clones in Iran.

Dissemination and characteristics of carbapenem-resistant Klebsiella pneumoniae in nine district hospitals in southwestern China.

Carbapenem-resistant Klebsiella pneumoniae (CRKP) is epidemically transmitted globally, but few studies focused on the prevalence in district-level hospitals. In this study, we investigated CRKP strains collected from nine district hospitals from September 2019 to September 2020, aiming to determine the resistance mechanisms, virulence profiles, and molecular epidemiological characteristics of CRKP in district hospitals in Southwest China. A total of 51 CRKP strains were collected from 9 district-level hospitals. Matrix-assisted laser desorption/ionization-time of flight mass spectrometer was used for strain identification review, and the micro-broth dilution method was used for antibiotic sensitivity detection. Molecular epidemiological investigation of strains was performed by multilocus sequence typing (MLST) and pulsed-field gel electrophoresis (PFGE) methods. PCR and efflux pump inhibition tests were used to detect CRKP resistance mechanisms. PCR and serum killing tests were used to detect capsular serotype, virulence-related genes, and virulence validation. The CRKP strains in district hospitals presented high levels of MIC50 and MIC90 in carbapenem antibiotics especially ertapenem and meropenem. A total of 90.2% (46/51) CRKP strains were detected as carbapenemase producers, and the proportion of strains co-expressing carbapenemases was 11.8% (6/51). All CRKP strains were grouped into eight MLST types, and ST11 was the most prevalent genotype. A total of 11.8% (6/51) CRKP isolates were positive for the string test, and three strains of hypervirulent and carbapenem-resistant K. pneumoniae (HV-CRKP) were positive in serum killing test. The molecular typing of all the CRKP isolates was grouped into 29 different PFGE patterns, and 40 ST11 isolates belonged to 20 different PFGE clusters. CRKP strains showed high-level antibiotic resistance and virulence phenotype in district hospitals in Southwest China, which suggested that we should immediately pay attention to the rapid dissemination of the CRKP in regional hospitals. Our study will provide new insights into the epidemiology of CRKP in regional hospitals, which will help regional hospitals develop nosocomial infection prevention and control policies tailored to local conditions.

The Genotyping Diversity and Hemolytic Activity of Cronobacter spp. Isolated from Plant-Based Food Products in Poland.

The present study aimed to determine the genotyping diversity and hemolytic properties of 24 strains of Cronobacter spp. (15 Cronobacter sakazakii, 6 Cronobacter malonaticus, 2 Cronobacter turicensis, and 1 Cronobacter condimenti) isolated from commercial ready-to-eat leaf vegetables, sprouts, nuts, and dried fruits. The multilocus sequence typing (MLST) method was used to determine the sequence types (ST) and clonal complexes (CC) of these strains. The study demonstrated the high genotypic diversity of the Cronobacter genus bacteria isolated from plant-based foods. Five novel sequence types (804, 805, 806, 807, and 808) and the presence of novel alleles in the ppsA, gltB, gyrB, and infB loci were detected. In total, 16 of the 24 strains were assigned to the sequence types ST99, ST258, ST17, ST648, ST21, ST494, and ST98. One C. sakazakii strain (s12) isolated from alfalfa sprouts was assigned to the clonal complex CC4, which encompasses strains often associated with severe infections leading to meningitis in infants. In addition, 87.5% and 16.7% of the Cronobacter spp. strains showed β-hemolysis of equine and sheep red blood cells, respectively. The presence of the pathogenic species C. sakazakii, C. malonaticus, and C. turicensis in ready-to-eat plant-derived food products shows they are potential sources of infection, especially to those with compromised immunity, which substantiates their further multi-faceted characterization. The significance of this study may prove useful not only in epidemiological investigations, but also in assessing the risk of infections caused by the presence of Cronobacter.

Broad Genome Sequencing of Environmental and Clinical Strains and Genotyping.

The Next-Generation Sequencing revolution had a great impact on the genomics of Pseudomonas aeruginosa. Since the first release of the P. aeruginosa PAO1 genome, there are more than 5700 genomes published. This wealth of information has been accompanied by the development of bioinformatic tools for handling genomic and phenotypic data. Bioinformatics, indeed,become de facto a big data science. In this chapter, we give a brief historical overview of the knowledge gained from P. aeruginosa genome sequencing, then we describe the wet-lab procedure to extract the DNA and prepare the library for broad genome sequencing using Illumina MiSeq technology. As last, we describe three user-friendly bioinformatics procedures to infer the P. aeruginosa genotype, starting from NGS data, with the Multi-Locus Sequence Typing method, and visualize it as a minimum spanning tree.

Genetic characterization of Bartonella henselae samples isolated from stray cats by multi-locus sequence typing

BackgroundBartonella henselae is one of the most commonly identified Bartonella species associated with several human diseases. Although B. henselae was detected in humans and cats in Turkey, they have not been genotyped previously. Therefore, this study aimed to genotype B. henselae samples (n = 44) isolated from stray cats using the multi-locus sequence typing (MLST) method. For this aim, eight different housekeeping markers were amplified by nested PCR and then sequenced to reveal sequence types (STs) of B. henselae samples.ResultsAllelic profiles obtained from 40 B. henselae isolates (90.9%) were compatible with available allelic profiles in the MLST online database. However, allelic profiles obtained from the remaining 4 B. henselae isolates (9.1%) were incompatible with the database. Among B. henselae isolates with compatible allelic profiles, 5 different STs including ST1, ST5, ST9, ST35 and ST36 were identified according to the B. henselae MLST online database. ST35 was the most prevalent ST with a prevalence rate of 29.5% (13/44), followed by ST36 with a prevalence rate of 22.7% (10/44). In addition, ST5 (16%, 7/44) and ST9 (18.2%, 8/44) were also among the prevalent STs. The prevalence of ST1 was 4.5% (2/44). For B. henselae isolates with incompatible allelic profiles, we recommended a new ST called ST38.ConclusionThe present study genotyped B. henselae samples isolated from stray cats in Turkey for the first time and ST1, ST5, ST9, ST35, and ST36 as well as a new sequence type named ST38 were identified among these B. henselae isolates.

Decoding the enigma: unveiling the molecular transmission of avian-associated tet(X4)-positive E. coli in Sichuan Province, China

Tigecycline is considered one of the "last resort antibiotics" for treating complex infections caused by multidrug-resistant (MDR) bacteria, especially for combating clinical resistant strains that produce carbapenemases. However, the tet(X4) gene, which carried by different plasmids can mediate high levels of bacterial resistance to tigecycline, was first reported in 2019. Here, we report the emergence of the plasmid-mediated tet(X4) in avian environment of Sichuan Province. A total of 21 tet(X4)-positive Escherichia coli (E. coli) strains were isolated and identified from avian samples in selected regions, with an isolation rate of 1.6% (21/1,286), and all of them were MDR strains. Multilocus Sequence Typing (MLST) method was used to classify the 21 tet(X4)-positive E. coli into the ST206, ST761, ST155, ST1638, ST542, and ST767 types, which also belong to the 3 phylogenetic subgroups A, B1, and C. Tet(X4) is located on mobile plasmids that can be efficiently and stably propagated. The results of fitness cost experiments showed that tet(X4)-positive plasmids may incur some fitness cost to host bacteria, but different tet(X4)-positive plasmids bring about differential fitness costs. Whole-genome sequencing further confirmed the tet(X4) gene can be located on IncX1-type plasmids and the core genetic structures are ISVsa3-rdmc-tet(X4) or rdmc-tet(X4)-ISVsa3, the former is a 7 copies tandem repeat structure. In this study, we isolated and identified tet(X4)-positive E. coli from the avian origin in Sichuan, analyzed the mobility of the tet(X4) by conjugational transfer and S1-PFGE, and evaluated the biological characteristics of the tet(X4)-positive plasmid using the results of conjugational frequency, plasmid stability, and fitness costs. Finally, combined with the third-generation whole-genome sequencing analysis, the molecular transmission characteristics of the tet(X4) were preliminarily clarified, providing a scientific basis for guiding veterinary clinical use in this area, as well as risk assessment and prevention of the transfer and spread of tigecycline resistant strains or genes.

Staphylococcus borealis – A newly identified pathogen of bovine mammary glands

Twelve Staphylococcus borealis strains, isolated in Canada and Poland from milk of cows with intramammary infections, were characterized phenotypically (biochemical reactions on ID 32 STAPH and Biolog Phenotype MicroArrays™ PM1 and PM2A, ability of biofilm production) and genotypically (random amplified polymorphic DNA). In addition, a genomic comparison was done with S. borealis strains of human and porcine origin using the multilocus sequence typing (MLST) technique. The bovine isolates showed a high degree of phenotypic and genotypic diversity, however, they could be differentiated from human strains by the negative test for urease (found in all but one bovine isolate examined with ID 32 STAPH) and positive reaction for D-galactose (on Biolog phenotype microarray PM1) and D-lactose (on both commercial systems). The MLST method, utilizing six concatenated genes of the total length of ∼2930 bp, revealed that bovine strains (irrespective of the country of origin) show a distinctly greater degree of mutual relationship than to the strains of human and porcine origin, suggesting that S. borealis has evolved independently in these hosts. In conclusion, bovine-specific S. borealis can be involved in intramammary infections in cattle.

A multilocus sequence typing method of Staphylococcus aureus DNAs in a sample from human skin.

The skin and mucous membranes are the primary sites of Staphylococcus aureus colonization, particularly those of health care personnel and patients in long-term care centers. We found that S. aureus colonized with a higher abundance ratio on skins which had recovered from pressure injury (PI) than on normal skins in our earlier research on the skin microbiota of bedridden patients. Multilocus sequence typing (MLST) is a useful tool for typing S. aureus isolated from clinical specimens. However, the MLST approach cannot be used in microbiota DNA owing to the contamination from other bacteria species. In this study, we developed a multiplex-nested PCR method to determine S. aureus MLST in samples collected from human skins. The seven pairs of forward and reverse primers were designed in the upstream and downstream regions, which were conserved specifically in S. aureus. The first amplifications of the seven pairs were conducted in a multiplex assay. The samples were diluted and applied to conventional PCR for MLST. We confirmed that the method amplified the seven allele sequences of S. aureus specifically in the presence of untargeted DNAs from human and other skin commensal bacteria. Using this assay, we succeeded in typing sequence types (STs) of S. aureus in the DNA samples derived from the skins healed from PI. Peaks obtained by Sanger sequencing showed that each sample contained one ST, which were mainly categorized into clonal complex 1 (CC1) or CC5. We propose that this culture-free approach may be used in detecting S. aureus in clinical specimens without isolation.

Epidemiological investigations and multilocus sequence typing of Mycoplasma gallisepticum collected in China

Mycoplasma gallisepticum (MG) is one of the important pathogens in poultry industry and has led to major economic losses. Understanding the epidemiology is crucial to improve the control and eradication program of MG. This study collected 1,250 chicken samples, including trachea and lung, from China in 2022 to investigate the epidemiology of MG. Among the collected samples, 938 samples were positive for MG infection, resulting in an average positive rate of 75.04%. Additionally, 570 samples were positive for both MG and Mycoplasma synoviae (MS) coinfection, with an average positive rate of 45.60%. A total of 183 MG infection positive samples in this study were selected for genotyping, and the multilocus sequence typing (MLST) method based on 7 housekeeping genes was used. As a result, 183 samples belonged to 11 sequence types (STs), with ST-78 being the most prevalent. After BURST analysis, all 183 sequences were divided into group 3. Besides, 119 reference sequences from database and 183 sequences of this study were selected to construct the phylogenetic tree using the neighbor-joining method. The results revealed that the sequences from China, total 196 sequences, were classified into 4 branches. The findings suggest that the MG strains in China exhibit diverse genotypes, which may be related to international trade and the use of live vaccines. Furthermore, we detected the drug susceptibility of 10 isolated strains randomly, which may be helpful to guide the clinical use of drugs to control MG infection.

How to? My first experience as a sports doctor (GOTS young academy presentation)

Non-typhoidal Salmonella (NTS) is the major cause of foodborne infections globally, with considerable morbidity and mortality. The accurate identification of Salmonella serovars is important in disease management and public health surveillance. However, traditional serotyping methods are laborious, time-consuming and may produce ambiguous results. In this study, we evaluated traditional serotyping and seven gene-based multilocus sequence typing (MLST) methods to determine the serogroups of Salmonella strains. This study analysis suggests that MLST based serotyping is accurate in serogroup identification and discrimination of Salmonella serovars compared to the traditional serotyping method and can be implemented in routine clinical practice.

Is it time to move on to gene-based Salmonella typing: Evidence and implications

Non-typhoidal Salmonella (NTS) is the major cause of foodborne infections globally, with considerable morbidity and mortality. The accurate identification of Salmonella serovars is important in disease management and public health surveillance. However, traditional serotyping methods are laborious, time-consuming and may produce ambiguous results. In this study, we evaluated traditional serotyping and seven gene-based multilocus sequence typing (MLST) methods to determine the serogroups of Salmonella strains. This study analysis suggests that MLST based serotyping is accurate in serogroup identification and discrimination of Salmonella serovars compared to the traditional serotyping method and can be implemented in routine clinical practice.

Hospital-Related Lineage of USA300 Methicillin-Resistant Staphylococcus aureus (MRSA) to Cause Bacteremia in Iran.

Staphylococcus aureus is an important pathogen that causes bloodstream infections. This study is aimed at assessing the genotypic characteristics of S. aureus strains responsible for bloodstream infections. An epidemiological study was conducted using 85 S. aureus strains isolated from bloodstream infections. Susceptibility was tested using the broth microdilution method and disk diffusion. All detected methicillin-resistant S. aureus (MRSA) isolates were confirmed by mecA PCR assays. S. aureus isolated from bacteremia were characterized using SCCmec, spa, and multilocus sequence typing methods. The prevalence of S. aureus strains responsible for bloodstream infections was 38.8%. All isolates were MRSA. Multidrug resistance (MDR) was present in 84.7% of isolates. MRSA isolated categorized within six clonal complexes including CC8 (60%), CC22 (22.4%), CC5 (5.9%), CC30 (4.7%), CC45 (4.7%), and CC59 (2.3%). The main lineages found were USA300/CC8-MRSA-IV/t008 (41.2%), followed by ST22-SCCmecIV/t790 (9.4%), ST239-SCCmecIII/t037 (7.1%), ST22-SCCmecIV/t032 (7.1%), ST239-SCCmecIII/t631 (5.9%), ST239-SCCmecIII/t860 (5.9%), ST22-SCCmecIV/t852 (5.9%), ST5-SCCmecIV/t002 (4.7%), ST45-SCCmecIV/t038 (4.7%), ST30-SCCmecIV/t318 (4.7%), ST59-SCCmecIV/t437 (2.3%), and ST225-SCCmecII/t045 (1.1%). Resistance to vancomycin amounted to 5.9% of isolates that belonged to ST239-SCCmecIII/t037 (80%) and ST8-SCCmecIV/t008 (20%). The emergence of USA300 strains in bloodstream infections in our country is a serious alarm and highlights the significant invasion of this lineage into the healthcare system. MDR patterns among these strains appear to be becoming the biggest problem in healthcare treatment.

Study on the features of Clostridioides difficile infection among diarrhea patients in Kunming from 2018 to 2020

Objective: We analyze the characteristics of Clostridioides difficile (C. difficile) infection among diarrhea patients in Kunming from 2018 to 2020 and provide evidence for follow-up surveillance and prevention. Methods: A total of 388 fecal samples of diarrhea patients from four sentinel hospitals in Yunnan Province from 2018 to 2020 were collected. Real-time quantitative PCR was used to detect the fecal toxin genes of C. difficile. The positive fecal samples isolated the bacteria, and isolates were identified by mass spectrometry. The genomic DNA of the strains was extracted for multi-locus sequence typing (MLST). The fecal toxin, strain isolation, and clinical patient characteristics, including co-infection with other pathogens, were analyzed. Results: Among the 388 fecal samples, 47 samples with positive reference genes of C. difficile were positive, with a total positive rate of 12.11%. There were 4 (8.51%) non-toxigenic and 43 (91.49%) toxigenic ones. A total of 18 strains C. difficile were isolated from 47 positive specimens, and the isolation rate of positive specimens was 38.30%. Among them, 14 strains were positive for tcdA, tcdB, tcdC, tcdR, and tcdE. All 18 strains of C. difficile were negative for binary toxins. The MLST results showed 10 sequence types (ST), including 5 strains of ST37, accounting for 27.78%; 2 strains of ST129, ST3, ST54, and ST2, respectively; and 1 strain of ST35, ST532, ST48, ST27, and ST39, respectively. Fecal toxin gene positive (tcdB+) results were statistically associated with the patient's age group and with or without fever before the visit; positive isolates were only statistically associated with the patient's age group. In addition, some C. difficile patients have co-infection with other diarrhea-related viruses. Conclusions: The infection of C. difficile in diarrhea patients in Kunming is mostly toxigenic strains, and the high diversity of strains was identified using the MLST method. Therefore, the surveillance and prevention of C. difficile should be strengthened.

Characteristics and prognostic risk factors of patients with sequence type 5 lineage-associated cryptococcosis in China

ObjectivesCryptococcus neoformans sequence type 5 (ST5) lineage could infect immunocompetent hosts and cause a significant medical burden. We sought to identify characteristics and prognostic risk factors of ST5 lineage-associated cryptococcosis. MethodsMultilocus sequence typing and antifungal susceptibility testing were conducted for Cryptococcus isolates. The clinical and laboratory characteristics of cryptococcosis patients were investigated. The multivariable logistic regression identified variables independently associated with 30-day mortality in patients with ST5 lineage-associated cryptococcosis without HIV. ResultsThe infection rate of the ST5 isolates was 89.4% (370/414) in China. The proportion of ST5 isolates with nonwild-type minimum inhibitory concentrations to amphotericin B, 5-flucytosine, voriconazole, posaconazole, itraconazole, and fluconazole were 0%, 5.4%, 0.3%, 1.4%, 0.3%, and 8.1%, respectively. The ST5 lineage-infected group exhibited significantly higher blood platelet count, lower blood cryptococcal antigen (CrAg) titer, lower cerebrospinal fluid (CSF) CrAg titer than the non-ST5 lineage-infected group, and lower hemoglobin and lower CSF CrAg titer than the Cryptococcus gattii isolates-infected group. Seven baseline parameters, including underlying disease, dyskinesia, anemia, high peripheral blood neutrophils, low platelet count, high CSF fungal burden, and high CSF opening pressure, were associated independently with the 30-day mortality of patients with ST5 lineage-associated cryptococcosis without HIV. ConclusionOur study has provided an understanding of the ST5 lineage associated with cryptococcosis.

Characteristics of Streptococcus agalactiae causing urinary tract infections: Emergence of new sequence types ST74 and ST38 in Iran.

Difficult-to-treat Streptococcus agalactiae infections are increasingly described in patients with urinary tract infections (UTIs). This occurrence could be due to the production of virulence determinants. This study aimed to characterize the molecular features of S. agalactiae responsible for UTIs. In this cross-sectional study, 70 S. agalactiae isolated from UTIs were examined. Antibiotic susceptibility testing was performed using the disk diffusion method. All S. agalactiae isolates were confirmed by atr and dltS PCR assays. Virulence, alpha protein-like, and pilus island genes were detected by PCR. Isolates were characterized using the multilocus sequence typing method. Multidrug resistance was observed in 80% of isolates. Five virulence profiles were detected, wherein cylE, lmb, bca, rib (35.7%), cylE, lmb, alp3 (27.1%), and cylE, lmb, bac, rib, alp2 (21.4%) were the most frequent detected profiles. S. agalactiae was isolated and categorized within three clonal complexes (CCs) including CC22 (40%), CC17 (25.7%), and CC23 (20%). The main sequence types (STs) found were ST22 (27.1%), ST23 (17.1%), ST17 (12.9%), ST31 (8.7%), ST40 (8.7%), ST74 (7.1%), ST48 (4.3%), ST890 (4.3%), ST189 (2.8%), ST38 (2.8%), ST52 (2.8%), and ST155 (1.4%). ST74 and ST38 were reported for the first time in Tehran-Iran. This study highlights the predominance of the CC22 lineage among S. agalactiae strains isolated from UTIs in Tehran, Iran, and highlights the significant penetration of this lineage into hospitals. MDR patterns among these strains appear to be becoming a major concern in the management of infections.

Study of Genetic Diversity of Acne-associated Bacterial Isolates in Thailand Using the Multi-Locus Sequence Typing (MLST) Method

Study of Genetic Diversity of Acne-associated Bacterial Isolates in Thailand Using the Multi-Locus Sequence Typing (MLST) Method