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The current status of molecular biological methods to control mycoses was described. New tools for detecting of specific fungal DNA by reaction at a constant temperature with Loop-mediated isothermal amplification of DNA (LAMP, Eiken Chemical Co., Ltd.), and for strain typing by multilocus sequence typing (MLST) will greatly aid in managing complex fungal infection.
BACKGROUND: Acinetobacter non-baumannii (Anb) species are reported worldwide to cause infections in both adults and neonates, although less frequently than Acinetobacter baumannii. However, limited information is available on their genomic diversity, resistance mechanisms, and virulence potential. This study investigates novel Anb isolates causing neonatal septicemia in India to characterize their resistance and pathogenic traits. METHODS: Anb isolates from neonatal blood cultures (2007-2025) were identified by VITEK2 Compact system, MALDI-TOF MS, and Whole-genome sequencing (WGS). Antimicrobial susceptibility was tested by VITEK2. Genomic analysis included MLST, resistome, virulome, plasmid typing, integrons, and core-genome phylogeny analysis. In vitro and in vivo studies assessed pathogenic potential of Anb species. RESULTS: Anb infections were low (11%) among the neonates during the study period. WGS revealed 11 novel Sequence Types (STs) which include A. indicus, A. variabilis, A. schindleri, and A. bereziniae. Six out of these eleven Anbs harbored carbapenemases such as bla NDM-1 and/or bla OXA-58-like genes (bla OXA-58, bla OXA-420). bla NDM-1 was acquired via Tn125 transposon. ISAba125 was located upstream of bla NDM-1, and a conserved structure extending to IS91 family transposase was detected in bla NDM-1-harboring genomes. bla OXA-58-like genes were found to be associated with ISAba3. Most carbapenemases were likely located on chromosome. Class 1 integrons carrying multiple antimicrobial resistance genes (ARGs) and diverse plasmid replicase families were detected in Anbs. Core genome phylogeny showed that the study Anbs were not closely related to the global Anbs. In vitro virulence-associated assays (biofilm formation, surface motility, adherence/invasion, apoptosis) and in vivo lethality in murine infection model showed reduced pathogenicity, reinforcing earlier observations that Anb species are generally less virulent than A. baumannii. Several virulence factors (VFs) were detected; however, no clear correlation was observed between virulence genes, in vitro pathogenicity, and in vivo lethality. CONCLUSION: These results indicate the multifactorial nature of Anb pathogenicity and the current limitations of knowledge of its VFs. However, the presence of numerous VFs suggests a capacity to cause disease, particularly in vulnerable host populations such as neonates. Furthermore, the presence of multiple ARGs indicates a strong potential for persistence and dissemination in hospital environments with high antibiotic pressure. Overall, these findings underscore the importance of continued AMR surveillance, genome characterization and further investigations into Anb pathogenicity.
BACKGROUND: Reduced susceptibility to penicillin in Streptococcus pneumoniae is mediated primarily by alterations in penicillin-binding proteins (PBPs) and often coexists with multidrug resistance within successful lineages. The region-specific genomic characterization of clinically relevant pneumococci with reduced penicillin susceptibility in Southwest China remains limited. METHODS: We performed whole-genome sequencing of 204 clinical S. pneumoniae isolates collected from five institutions in Southwest China (2018-2022) that met our operational screening definition of reduced susceptibility to penicillin (PEN MIC ≥0.12 μg/mL). Molecular serotypes, MLST types, and Global Pneumococcal Sequence Clusters (GPSCs) were assigned; virulence and antimicrobial resistance determinants were profiled; and a core genome phylogeny was reconstructed with international contextualization through the use of PubMLST genomes meeting the same MIC criterion. Amino acid variability in PBP1a/PBP2b/PBP2x was quantified using TIGR4 numbering, and highly variable noncatalytic residues located within 15 Å of catalytic motifs were prioritized via structure-guided screening. RESULTS: The isolates showed a high burden of resistance to non-β-lactam antibiotics (erythromycin, 98.5%; tetracycline, 82.8%; trimethoprim-sulfamethoxazole, 64.7%), while fluoroquinolone susceptibility was largely preserved (≥97%), and vancomycin/linezolid resistance was not detected. Twenty-seven serotypes were identified, among which 19F (23.5%) and 19A (14.2%) were dominant, and the estimated PCV13 coverage was 69.6%. GPSC1 was the dominant lineage (36.8%), and the lineage composition among our isolates differed markedly from those in the PubMLST-USA and PubMLST-Thailand subsets. Virulence and resistance gene carriage differed markedly between GPSC1 and non-GPSC1 isolates, with enrichment of pilus operons, mef(A)/msr(D), and folA/folP in GPSC1. PBP variations were clustered in transpeptidase domains and motif-adjacent regions while essential catalytic residues were conserved; with the structure-guided filter, 12, 11, and 11 motif-proximal noncatalytic candidate sites were prioritized in PBP1a, PBP2b, and PBP2x, respectively. CONCLUSION: Clinical S. pneumoniae isolates with reduced penicillin susceptibility collected in Southwest China demonstrated resistance and accessory gene profiles that were strongly structured by a GPSC-defined lineage background. Our site-resolved, structure-guided PBP analysis provides a regional PBP variability landscape and a compact set of recurrent motif-proximal candidate substitutions to support surveillance and downstream functional validation.
Listeria monocytogenes is a Gram-positive zoonotic pathogen responsible for listeriosis, a severe foodborne disease with high mortality in humans and animals. This study aimed to investigate the molecular epidemiology and genomic characteristics of L. monocytogenes isolated from raw yak meat in Nyingchi, Xizang, China. A total of 231 yak-related samples were collected in Nyingchi, consisting of 214 retail raw yak meat samples, 14 farm environmental samples, and 3 nearby water source samples. L. monocytogenes isolates were identified and characterized using culture-based methods, PCR serotyping, and whole-genome sequencing (WGS). Bioinformatic analyses were performed for virulence, antimicrobial resistance, and functional gene annotation using KEGG and COG databases. The overall contamination rate of Lm was 13.08% (28/214) for retail raw yak meat samples, whereas no isolates were recovered from 14 farm environmental samples (0.00%, 0/14) and 3 nearby water source samples (0.00%, 0/3). The serotypes of isolates were 1/2a (9/28, 32.14%), 1/2b (7/28, 25.00%), and 1/2c (12/28, 42.86%). These 28 isolates exhibited varied antimicrobial resistance profiles, with universal resistance to trimethoprim-sulfamethoxazole, high resistance to erythromycin and clindamycin, and low resistance to vancomycin. MLST analysis revealed seven sequence types (STs): ST9 (12/28, 42.86%), ST619 (6/28, 21.43%), ST8 (6/28, 21.43%), ST7 (1/28, 3.57%), ST87 (1/28, 3.57%), ST121 (1/28, 3.57%), ST297 (1/28, 3.57%). ST619 isolates harbored multiple virulence genes, including those located on Listeria pathogenicity islands LIPI-1, LIPI-3, and LIPI-4, indicating high genomic potential for virulence. Representative isolate Y2 (ST619) possessed a 3,009,858 bp genome with 3036 coding genes, four genomic islands, and two prophages. Functional annotation revealed enrichment of genes involved in carbohydrate transport and metabolism and amino acid biosynthesis pathways. Our findings provide the first genomic insight into L. monocytogenes contamination in yak meat from Nyingchi, Xizang, China, highlighting the urgent need to strengthen food safety monitoring and hygiene management in this region.
Molecular typing and fingerprinting of bacterial pathogens has become a major part of epidemiology, disease monitoring, intervention and food safety research. This treatise will briefly address the aims of molecular typing and the criteria that are important to choose the optimal method of typing. However, since the method of choice will depend on a number of factors, including available facilities and research goals, no particular methods are recommended; instead, theoretical considerations are presented as a guide line for the best decision. Since molecular typing is examining the nature of the population genetics of a particular organism, it is important to understand how the degree of clonality of that particular organism will influence outcome and interpretation of results. The mechanisms that lead to non-clonality are briefly outlined. The problems with typing of clonal, non-clonal, and weakly clonal populations are briefly treated. The merits and myths of multilocus sequence typing (MLST) are discussed.
OBJECTIVES: Development of extended polymerase chain reaction (PCR) for non-culture detection of Nesseria meningitidis, Haemophilus influenzae and Streptococcus pneumonie from invasive infections. MATERIALS AND METHODS: A method of PCR was optimalised on strains of Nesseria meningitidis, Haemophilus influenzae b and Streptococcus pneumonie. Detection of pathogens was evaluated on 230 samples from patiens with invasive infection. RESULTS: Positive results of PCR were found in 103 samples of 230 (44.7 %). The percentage of positivity was higher in CSF samples (57.0 %) than in serum (33.8 %) or blood (33.3 %) samples. CONCLUSION: PCR method enables etiological diagnostics in cases, where antibiotic treatment was started. PCR results are available earlier than the results of cultivation. Multilocus sequence typing (MLST) of PCR products enables clonal analysis of etiological agents even in cases with negative results of cultivation.
The present study proposed use of modified multilocus sequence typing (MLST) approach, for subspecies differentiation of Enterococcus faecalis isolates. We disgesed with SspI endonuclease product of amplification of three E. faecalis antigen-encoding genes (ace, encoding a collagen and laminin adhesin; efaA, encoding an endocarditis antigen; and salA, encoding a cell wall associated antigen) and one housekeeping gene (pyrC) of 32 E. faecalis isolates MLRA analysis of polymorphic regions of these four genes identified 23 distinct types 21.4 to 100% identity among the 32 isolates. Further studies are needed to evaluate the value of proposed method.
Using multilocus sequence typing (MLST), 22 Helicobacter pylori isolates from Russia have been characterized. All of the Russian strains were assigned to a single population, hpEurope.
STUDY OBJECTIVE: The severity of narcolepsy is commonly measured with the Multiple Sleep Latency Test (MSLT), focusing on the tendency to fall asleep. A neglected but perhaps equally important complaint is impaired performance in the waking state. We evaluated the Sustained Attention to Response Task (SART) for the quantification of vigilance in narcolepsy. DESIGN: The SART involves withholding key presses to 1 in 9 target stimuli. In the present study, it was administered prior to each of 5 MSLT sessions in a 1-day study. The Epworth Sleepiness Scale was administered to measure subjective sleepiness. SART and MSLT results (number of errors and sleep latency) were compared using Receiver Operator Curves, sensitivity, and specificity. SETTING: Tertiary narcolepsy referral center in a university hospital. PATIENTS: Fifteen untreated narcoleptics and 15 matched controls. INTERVENTIONS: None. MEASUREMENTS AND RESULTS: The area under the receiver operating curve was 0.97 for the MSLT and 0.95 for the SART. Sensitivity and specificity for the MLST were 80% and 100% using a cutoff point of 5 minutes. For the SART, these values were 87% and 100%, using a 5-error cutoff. The SART and MSLT showed no correlation with each other or with the Epworth Sleepiness Scale. CONCLUSIONS: The SART, measuring attention, was abnormal as often as the MSLT, measuring sleepiness. The inability to remain vigilant during the day may be the most serious complaint in narcolepsy, since it impairs performance. The SART is valid in this respect, is easy to administer, and takes little time.
The problem of nosocomial infections is of increasing importance in veterinary medicine. As an example, this review summarizes current knowledge regarding methicillin-resistant Staphylococcus aureus (MRSA) as a typical example, as these pathogens are the most important agents of nosocomial infections in human medicine worldwide and are being increasingly reported in veterinary medicine. MRSA are classified by their ability to be resistant against oxacillin/methicillin, this feature being confered by mecA, a gene which was acquired by horizontal gene transfer of the staphylococcal gene cassette (SCCmec). It is this genetic information that enables MRSA to be resistant against all penicillins, cehalosporins and carbapenems. In addition, MRSA are often resistant against a variety of other antiinfectives, i.e. aminoglycosides, macrolides, lincosamide, streptomycins, tetracyclin, chloramphenicol, but also against fluorquinolones and rifampicin. Presumably, these highly adapted strains are particularly able to acquire resistance genes located on plasmids or transposons. They are also able to develop point mutations, further leading to resistant phenotypes. If these pathogens are leading to infectious diseases, veterinarians may be confronted with a worst-case scenario, being left without any antiinfective therapeutic. As Staphylococcus aureus is highly tenacid, professional hygiene management is of utmost importance. The increasing number of published sporadic MRSA infections, MRSA-infectious diseases as well as MRSA outbreaks in veterinary medicine justifies their recognition as a "New Emerging Pathogen". So far, horses and dogs are mostly affected by MRSA. Although transmission between humans and animals has been reported occasionally, the sources, routes of transmission or the epidemiological relevance of MRSA infections in animals are far from being understood. Therefore, epidemiological investigations utilizing molecular typing tools are mandatory. Typing tools like multilocus-sequence-typing (MLST), pulsefield-gelelectrophoresis (PFGE), sequence analysis of the gene encoding protein A (spa-typing) as well as SCCmec-typing are all at hand.
The analysis of meningococcal strains of different serogroups, isolated from the liquor of patients in Moscow, which was carried out with the method of multilocus sequencing-typing (MLST), was presented. At the periods of epidemic morbidity rises in Moscow the prevalence of group A meningococcal strains, belonging to subgroups III with sequence-types 5 (in the 1970s) and 7 (in 1996), was noted, and at a period between epidemics strains of genetic subgroups VI and X were isolated. Meningococcal strains, groups B and C, isolated in 1995 - 2002, had, as a rule, unique sequence-types, differing both one from another and from N. meningitidis sequence-types detected in other countries. Among group B meningococci the prevalence of strains belonging to clonal complex ST-18 was noted, while for group C meningococcci strains belonging to clonal complex ST-41/44 were most typical. Such genetic variability of circulating meningococci was regarded as characteristic of the period between epidemics, observed in Moscow since the end of the 1980s.
Due to the major impact of yeasts in human life based on the existence of pathogen yeast species and of species with biotechnological abilities, in the last few years new molecular techniques are performed for an accurate identification of natural isolates. Our study is aimed to review some of these techniques such as electrokariotyping by PFGE (Pulsed Field Gel Electrophoresis), estimation of the molar percentage of guanine and cytosine, the applications of PCR reaction in yeast identification using RAPD (Random amplified polymorphic DNA), UP-PCR (Universally Primed Polymerase Chain Reaction), MLST (Multilocus sequence typing) techniques, mtDNA and rDNA homology studies. Such molecular techniques complete the phenotypical characterization based on classical taxonomical tests allowing thus the polyphasic identification of the microorganisms.