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BackgroundMycoplasma pneumoniae (MP) is a leading cause of community-acquired pneumonia in children (CAP). Rising macrolide resistance and variability in clinical severity have become growing concerns. This study aimed to describe the molecular epidemiology, resistance patterns, and risk factors for severe M. pneumoniae pneumonia (SMPP) in children from Liaocheng, Shandong Province, China.MethodsWe retrospectively enrolled 421 children hospitalized with MP pneumonia (MPP) between January 2023 and December 2024 in this study. Clinical and laboratory data were collected. M. pneumoniae-DNA-positive specimens were analyzed by 23S rRNA gene sequencing to identify macrolide resistance mutations, and by Multilocus Variable-Number Tandem Repeat Analysis (MLVA) for molecular typing. Univariate and multivariate logistic regression analyses were used to identify risk factors for SMPP.ResultsOf the 421 patients, 212 (50.4%) were diagnosed with SMPP. Children with SMPP were significantly older than those with general MPP (GMPP) (P = 0.001). Elevated D-dimer and lactate dehydrogenase (LDH) levels were identified as independent risk factors for SMPP. Among 148 successfully sequenced specimens, 144 (97.30%) carried macrolide resistance mutations, with A2063G being the most common (96.62%). MLVA typing identified 14 distinct subtypes, the most prevalent were M2-4-5-7-2, M5-4-5-7-2, M3-4-5-7-2, and M4-3-5-6-2. The subtype M4-3-5-6–2 was associated with a lower risk of SMPP (OR = 0.155, 95% CI: 0.037-0.654), while the subtype M3-4-5-7–2 carried the highest risk (OR = 14.0, P = 0.001).ConclusionThis study reveals a high rate of macrolide-resistant MP in Liaocheng, largely caused by the A2063G mutation. Elevated D-dimer and elevated LDH are independent predictors of SMPP. Certain MLVA subtypes are significantly associated with disease severity, suggesting they may help in risk assessment and clinical decision-making.

Background Q fever, caused by Coxiella burnetii , is a zoonosis that presents a worldwide distribution and affects both humans and animals. The route of dispersal of the pathogen by ruminants into the environment usually involves stages of abortion and parturition, nevertheless the agent can, also, be detected in other animal samples. Therefore it is considered as important in terms of proper diagnosis, as well as, for epidemiology and surveillance purposes, to genotype the pathogen. The aim of the current study was to investigate the presence of different genotypes of the agent in animals that had suffered from abortion during a two-year survey in Greece. Results Sixty nine tissue samples (37 stomach contents, 11 liver samples, 21 cotyledons) were collected from 59 abortion cases in sheep ( N  = 45) and goats ( N  = 14) from 65 farms at eight different areas of Greece. Samples were screened by qPCR and positive ones were further genotyped using a 10-locus multiple loci (ms 1, 3, 7, 12, 20, 21, 22, 26, 30 and 36) variable number of tandem repeat analysis (MLVA) method. Three genotypes were identified in sheep (A, B, C). Samples representing each of the obtained MLVA profile were further used for MST genotyping. Ten spacers (Cox 2, 5, 6, 18, 20, 22, 37, 51, 56 and 57) were amplified. A close relatedness among the identified MLVA genotypes was confirmed since they all belonged to MST group 32. Conclusions The current study introduces into the aspect of genotyping of C. burnetii in Greece. Further studies are needed to explore the presence of more genotypes, to associate the genotypes circulating in the animal and tick population with those causing human disease in order to further expand on the epidemiological aspects of the pathogen.

is a potentially deadly human pathogen associated with high morbidity, mortality and global economic burden. The universally used bacterial genotyping methods are multilocus sequence typing and pulsed field gel electrophoresis. However, another highly discriminatory, rapid and less expensive genotyping technique, multilocus variable number of tandem repeat analysis (MLVA), has been developed. Unfortunately, no universal MLVA protocol exists, and some MLVA protocols do not amplify certain loci for all pneumococcal serotypes, leaving genotyping profiles incomplete. A number of other genotyping or characterization methods have been developed and will be discussed. This review examines the various protocols for genotyping and highlights the current direction technology and research is heading to understand this bacterium.

Background: After Aspergillus fumigatus, A. flavus is the second leading cause of invasive and non-invasive aspergillosis. These fungi are of significant epidemiological importance in provinces with dry and hot climates. Objectives: In the present study, antifungal susceptibility testing (AFST) and genotyping of sixty-five A. flavus clinical isolates originating from patients in Mazandaran and Tehran were performed. Methods: Antifungal susceptibility testing of 65 clinical isolates of A. flavus was conducted against amphotericin B (AMB), itraconazole (ITR), voriconazole (VOR), posaconazole (POS), isavuconazole (ISA), luliconazole (LUL), lanoconazole (LAN), and 5-fluorocytosine according to the Clinical Laboratory Standards Institute (CLSI) method (M38-A2). The minimum inhibitory concentrations (MICs) were determined for each antifungal drug against all strains. Additionally, microsatellite typing using six variable number tandem repeat (VNTR) markers was performed to assess the genetic diversity and potential relationships among the clinical strains. Results: Luliconazole had the lowest geometric mean MIC (0.020 μg/mL), followed by LAN (0.021 μg/mL), POS (0.089 μg/mL), ISA (0.115 μg/mL), ITR (0.220 μg/mL), VOR (0.244 μg/mL), AMB (0.870 μg/mL), and 5-fluorocytosine (58.76 μg/mL). Microsatellite typing revealed sixty-five distinct sequence genotypes. Statistically, there was no significant relationship between genotypes and AFST profiles (P ≥ 0.05). Conclusions: Luliconazole and lanoconazole demonstrated the greatest in vitro activity among all tested antifungals. However, most A. flavus strains exhibited reduced sensitivity to AMB. Microsatellite genotyping indicated no genetic similarity among the clinical strains, revealing high genetic diversity among A. flavus isolates obtained from clinical samples.

Background: Cockroaches, recognized as mechanical vectors, play a crucial role in transmitting microbial pathogens. Staphylococcus aureus (S. aureus), particularly antibiotic-resistant strains, poses a significant threat as a nosocomial path­ogen. This study aimed to investigate the resistance profiles to gentamicin, vancomycin, and antiseptics in S. aureus strains isolated from cockroaches in hospitals affiliated with Babol University of Medical Sciences. Methods: In this cross-sectional study, 60 S. aureus strains were isolated from 376 cockroaches in three university-affiliated hospitals. Antibiotic susceptibility to gentamicin and vancomycin was tested by disk diffusion and agar dilu­tion. PCR was used to detect resistance and antiseptic genes, and MLVA typing determined the genetic relatedness of resistant isolates. Results: Among the 60 bacterial isolates, 46.7% (28) displayed resistance to gentamicin. The frequencies of aminogly­coside resistance coding genes (AMEs) for the aac(6')-Ie+aph(2″), ant(4')-Ia, aph(3')-IIIa and ant(6)-Ia genes were 64.3%, 42.8%, 17.8%, and 46.4%, respectively. Only 3.3% (2 isolates) exhibited vancomycin resistance, with one iso­late (1.7%) carrying the vanA gene. The frequencies of genes encoding the antiseptic resistance genes qacA/B, qacC, qacD, psmA, sasX, and smr were 5%, 20%, 18.3%, 26.4%, 1.2%, and 31.7%, respectively. Analysis of agr gene types showed that agr type I was the most prevalent. In addition, the multiple-locus variable number tandem-repeat analysis (MLVA) identified 29 unique type sequences among the identified antibiotic-resistant isolates. Conclusion: The high genetic diversity among antibiotic-resistant S. aureus isolates, as revealed by MLVA, under­scores the importance of controlling hospital cockroach populations to curb the spread of antibiotic resistance.  

The epidemiology of Salmonella Infantis is complex in terms of its distribution and transmission. The continuous collection and analysis of updated data on the prevalence and antimicrobic resistance are essential. The present work aimed to investigate the antimicrobial resistance and the correlation among S. Infantis isolates from different sources through the multiple-locus variable-number of tandem repeat (VNTR) analysis (MLVA). A total of 562 Salmonella strains isolated from 2018 to 2020 from poultry, humans, swine, water buffalo, mussels, cattle, and wild boar were serotyped, and 185 S. Infantis strains (32.92%) were identified. S. Infantis was commonly isolated in poultry and, to a lesser extent, in other sources. The isolates were tested against 12 antimicrobials, and a high prevalence of resistant strains was recorded. S. Infantis showed high resistance against fluoroquinolones, ampicillin, and tetracycline, which are commonly used in human and veterinary medicine. From all S. Infantis isolates, five VNTR loci were amplified. The use of MLVA was not sufficient to understand the complexity of the epidemiological relationships between S. Infantis strains. In conclusion, an alternative methodology to investigate genetic similarities and differences among S. Infantis strains is needed.

Background. Previous studies have suggested that asymptomatic carriers of toxigenic Clostridium difficile are a source of hospital-associated (HA) infections. Multilocus variable number of tandem repeats analysis (MLVA) is a highly discriminatory molecular subtyping tool that helps to determine possible transmission sources. Methods. Clostridium difficile isolates were recovered from perirectal swabs collected for vancomycin-resistant Enterococcus (VRE) surveillance as well as from clinical C. difficile toxin–positive stool samples from July to November 2009 at the University of Pittsburgh Medical Center Presbyterian (UPMC). MLVA was performed to determine the genetic relationships between isolates from asymptomatic carriers and patients with HA C. difficile infection (HA-CDI). Asymptomatic carriage and HA-CDI isolates were considered to be associated if the carriage isolate was collected before the HA-CDI isolate and if the MLVA genotypes had a summed tandem-repeat difference of ≤2. Results. Of 3006 patients screened, 314 (10.4%) were positive for toxigenic C. difficile, of whom 226 (7.5%) were detected only by VRE surveillance cultures. Of 56 incident cases of CDI classified as HA at UPMC during the study with available isolates, 17 (30%) cases were associated with CDI patients, whereas 16 (29%) cases were associated with carriers. Transmission events from prior bed occupants with CDI (n = 2) or carriers (n = 2) were identified in 4 of 56 cases. Conclusions. In our hospital with an established infection control program designed to contain transmission from symptomatic CDI patients, asymptomatic carriers appear to have played an important role in transmission. Identification and isolation of carriers may be necessary to further reduce transmission of C. difficile in such settings.

Background This study was aimed to evaluate the antibiotic resistance, biofilm formation, and genetic diversity of carbapenem-resistant Pseudomonas aeruginosa (CRPA) strains isolated from four types of nosocomial infections (NIs) including urinary tract infection (UTI), ventilator-associated pneumonia (VAP), surgical site infection (SSI), and bloodstream infection (BSI). Methods and results In total, 115 isolates of NIs-causing P. aeruginosa were collected from NIs. Antibiotic susceptibility testing (AST) was performed using disk diffusion method and minimum inhibitory concentrations. Biofilm formation was tested on 96-well polystyrene microtiter plates (MTP). CRPA isolates were genotyped using multiple-locus variable number of tandem repeat analysis (MLVA). The most resistance and susceptibility rates were observed to amikacin (70.6%) and colistin (96.1%), respectively. Colistin and meropenem were the most active antimicrobial agents in VAP, SSI, and BSI. While, colistin and cefepime were the most active in UTIs. In total, 52.2% ( n  = 60/115) of P. aeruginosa isolates were carbapenem resistant, of which 95.0%, 55.0%, and 5.0% were multidrug-resistant, extensively drug-resistant, and pandrug-resistant, respectively. There was a significant association between resistance to carbapenem and resistance to other antibiotics except for piperacillin/tazobactam. The biofilm production of CRPA isolates was 95.0%, of which 23.3% were strong biofilm producers. Based on MLVA, there were 34 different types of CRPA isolates classified into three main clusters and 5 sub clusters. Conclusion The association of CRPA with other antibiotic resistance, the high rates of biofilm production, and the high genetic diversity of the isolates may be a warning of the need for a careful surveillance program.

Background Multidrug-resistant organisms (MDRO) pose a significant threat to public health worldwide. The ability to identify antimicrobial resistance determinants, to assess changes in molecular types, and to detect transmission are essential for surveillance and infection prevention of MDRO. Molecular characterization based on long-read sequencing has emerged as a promising alternative to short-read sequencing. The aim of this study was to characterize MDRO for surveillance and transmission studies based on long-read sequencing only. Methods Genomic DNA of 356 MDRO was automatically extracted using the Maxwell-RSC48. The MDRO included 106 Klebsiella pneumoniae isolates, 85 Escherichia coli , 15 Enterobacter cloacae complex, 10 Citrobacter freundii , 34 Pseudomonas aeruginosa , 16 Acinetobacter baumannii , and 69 methicillin-resistant Staphylococcus aureus (MRSA), of which 24 were from an outbreak. MDRO were sequenced using both short-read (Illumina NextSeq 550) and long-read (Nanopore Rapid Barcoding Kit-24-V14, R10.4.1) whole-genome sequencing (WGS). Basecalling was performed for two distinct models using Dorado-0.3.2 duplex mode. Long-read data was assembled using Flye, Canu, Miniasm, Unicycler, Necat, Raven, and Redbean assemblers. Long-read WGS data with > 40 × coverage was used for multi-locus sequence typing (MLST), whole-genome MLST (wgMLST), whole-genome single-nucleotide polymorphisms (wgSNP), in silico multiple locus variable-number of tandem repeat analysis (iMLVA) for MRSA, and identification of resistance genes (ABRicate). Results Comparison of wgMLST profiles based on long-read and short-read WGS data revealed > 95% of wgMLST profiles within the species-specific cluster cut-off, except for P. aeruginosa . The wgMLST profiles obtained by long-read and short-read WGS differed only one to nine wgMLST alleles or SNPs for K. pneumoniae , E. coli , E. cloacae complex, C. freundii , A. baumannii complex, and MRSA. For P. aeruginosa , differences were up to 27 wgMLST alleles between long-read and short-read wgMLST and 0–10 SNPs. MLST sequence types and iMLVA types were concordant between long-read and short-read WGS data and conventional MLVA typing. Antimicrobial resistance genes were detected in long-read sequencing data with high sensitivity/specificity (92–100%/99–100%). Long-read sequencing enabled analysis of an MRSA outbreak. Conclusions We demonstrate that molecular characterization of automatically extracted DNA followed by long-read sequencing is as accurate compared to short-read sequencing and suitable for typing and outbreak analysis as part of genomic surveillance of MDRO. However, the analysis of P. aeruginosa requires further improvement which may be obtained by other basecalling algorithms. The low implementation costs and rapid library preparation for long-read sequencing of MDRO extends its applicability to resource-constrained settings and low-income countries worldwide.

Assess the best approach to type methicillin-resistant (MRSA), ( ) typing, multiple-locus variable number tandem repeat analysis (MLVA) or both. Discriminatory power of typing and MLVA was determined using 20,771 MRSA isolates. There were twice as many MLVA types (MTs) as types present in the collection. Among the top 70% of the isolates, 37 types and 139 MTs were found. MLVA diversity among the top-10 types was high (diversity index 0.96), while diversity among the top-10 MTs was much lower (diversity index 0.83). The probability that two MRSA isolates with the same type also had the same MT was low (Wallace's coefficient 0.27). By contrast, most MRSA isolates yielding the same MT also had the same type (Wallace's coefficient 0.90). MLVA is superior to typing and will suffice to characterize MRSA isolates for surveillance.