Fast Antibiotic Susceptibility Testing via Raman Microspectrometry on Single Bacteria: An MRSA Case Study.

Cefoxitin was administered parenterally in doses of 2 g, every six hours for two days, to 20 patients undergoing colorectal surgery. Blood and fecal specimens were obtained for five days for analysis of cefoxitin concentrations. Tissue samples from the gut wall were obtained at surgery. The maximum serum concentrations during surgery varied between 25 and 100 microgram/ml, and the cefoxitin concentrations for the fecal samples were 1.5-35 microgram/g and for the tissue samples, 2.0-50 microgram/g. Fecal samples for cultivation of aerobic and anaerobic bacteria were also obtained daily during the first five days, and after 14 day after operation. Pronounced changes in the microflora occurred during the investigation period. Among the aerobic bacteria, cefoxitin-sensitive Escherichia coli and other enterobacteria decreased markedly while cefoxitin-resistant enterococci, Pseudomonas and Enterobacter proliferated during the antibiotic prophylaxis period. Among the anaerobic bacteria, Bacteroides fragilis and other Gram-negative bacteria decreased significantly. However, after the antibiotic administration period, all cefoxitin-resistant strains decreased while the suppressed E. coli and B. fragilis strains increased. In one patient, a minor postoperative infection caused by a cefoxitin-resistant strain of Pseudomonas aeruginosa was observed.

The rapid and accurate identification of microbial pathogens is crucial for effective management of infectious diseases. Traditional methods, though reliable, are often time-consuming. Recent advancements in molecular diagnostics, mass spectrometry, and next-generation sequencing (NGS) have revolutionized the field by enhancing both the speed and accuracy of microbial identification. Polymerase chain reaction (PCR) and its derivatives, such as real-time PCR, enable rapid amplification of genetic material, significantly reducing diagnostic times. Techniques like nucleic acid sequence-based amplification (NASBA) and loop-mediated isothermal amplification (LAMP) further extend these capabilities to resource-limited settings by operating at constant temperatures. Mass spectrometry, particularly matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF), provides rapid and accurate microbial identification by analyzing protein profiles. This method has high throughput, is cost-effective, and offers the ability to detect antimicrobial resistance, significantly impacting clinical decision-making and patient management. NGS technologies offer comprehensive pathogen detection by sequencing entire microbial genomes. This approach is invaluable in diagnosing complex infections, tracking outbreaks, and identifying antibiotic resistance genes, though its high cost and complexity limit widespread use. The integration of these innovative technologies into clinical practice enhances the ability to diagnose and manage infectious diseases effectively. Molecular diagnostics, mass spectrometry, and NGS each bring unique strengths, from rapid and precise pathogen detection to comprehensive genetic analysis and resistance profiling. These advancements contribute to improved patient outcomes and more effective public health strategies. The continued development and implementation of these technologies are essential to address the evolving challenges in microbial diagnostics, paving the way for more precise and personalized approaches in infectious disease management.

PurposeStudies have shown that multiple genes influence antibiotic susceptibility, but the relationship between genotypic and phenotypic antibiotic susceptibility is unclear. We sought to analyze the concordance between the presence of antibiotic resistance (ABR) genes and antibiotic susceptibility results in urine samples collected from patients with symptomatic urinary tract infection (UTI).Patients and MethodsUrine samples were collected from patients presenting to 37 geographically disparate urology clinics across the United States from July 2018 to February 2019. Multiplex polymerase chain reaction was used to detect 27 ABR genes. In samples containing at least one culturable organism at a concentration of ≥ 104 cells per mL, pooled antibiotic susceptibility testing (P-AST), which involves simultaneous growing all detected bacteria together in the presence of antibiotic and then measure susceptibility, was performed against 14 antibiotics. The concordance rate between the ABR genes and the P-AST results was generated for the overall group. The concordance rates for each antibiotic between monomicrobial and polymicrobial infection were compared using chi-square test.ResultsResults from ABR gene detection and P-AST of urine samples from 1155 patients were included in the concordance analysis. Overall, there was a 60% concordance between the presence or absence of ABR genes and corresponding antimicrobial susceptibility with a range of 49–78% across antibiotic classes. Vancomycin, meropenem, and piperacillin/tazobactam showed significantly lower concordance rates in polymicrobial infections than in monomicrobial infections.ConclusionGiven the 40% discordance rate, the detection of ABR genes alone may not provide reliable data to make informed clinical decisions in UTI management. However, when used in conjunction with susceptibility testing, ABR gene data can offer valuable clinical information for antibiotic stewardship.

Bloodstream infections caused by bacteria can cause potentially fatal sepsis, which needs immediate antibiotic therapy to prevent morbidity and death of patient. Blood culture remains the gold standard procedure that provides the vital information for the diagnosis and guiding appropriate antimicrobial treatment. The study sought to assess the antibiotic susceptibility and bacteriological profile of blood culture isolates in intensive care unit (ICU) settings. Understanding the incidence of diverse bacteria in ICU blood cultures, as well as their antibiotic susceptibility, is crucial for developing effective treatment plans that work. During the study period, 3,594 blood cultures underwent analysis, revealing 388 cases positive for growth. To identify isolates, VITEK 2 GN ID cards were utilized, capable of discerning both fermentative and non-fermentative bacteria. Further, VITEK 2 GP ID was employed for selected Gram-positive cocci. Antibiotic susceptibility testing was conducted using VITEK 2 AST 407 Critical Care cards for fermentative and non-fermentative Gram-negative bacilli, with subsequent testing on VITEK 2 AST 628 cards for Gram-positive cocci. Out of which, 230 (59.2%) were Enterobacteriaceae isolates, 87 (22.4%) were non-fermenters, and 71 (18.2%) were Gram-positive cocci. The majority of the blood culture isolates were multidrug-resistant (MDR), extended-spectrum beta-lactamase (ESBL) producers, carbapenemase producers, and methicillin-resistant Staphylococcus aureus (MRSA). In our study, we observed carbapenem-resistant Klebsiella spp., Escherichia coli (E. coli), Acinetobacter baumannii, Pseudomonas aeruginosa, and Burkholderia cepacia, which is alarming. The results show the diverse range of microorganisms responsible for bloodstream infections in severely ill ICU patients. Understanding the antibiotic susceptibility characteristics of these isolates is crucial for developing effective therapeutic regimens.

Carbapenem-hydrolyzing class D β-lactamases (CHDLs) are on the rise and are a major public health problem worldwide. Pseudomonas aeruginosa is resistant to carbapenem; currently, the most effective treatment option is being increasingly reported. This study aimed to identify blaOXA-145, blaOXA-224, blaOXA-539, and blaOXA-675 genes in CHDL strains. Samples were collected from clinical specimens admitted to the hospital. Antibiotic susceptibility was determined using the disk diffusion methods. CHDL strains were detected using a phenotypic method (disk diffusion). The PCR method was used to identify blaOXA-145, blaOXA-224, blaOXA-539, and blaOXA-675 genes. Piperacillin-resistant strains (n = 9, 17.4%) had the lowest frequency, and cefoxitin-resistant strains (n = 100, 91.7%) had the highest distribution in P. aeruginosa isolates. Also, 29.35%, 12.8%, and 8.2% were multidrug-resistant, extensively drug-resistant, and pan drug-resistant, respectively. MBL-producing P. aeruginosa and KPC-producing P. aeruginosa were detected, respectively, in 47.7% and 37.6% of isolates. Biofilm formation was observed in 63.3% of P. aeruginosa isolates. The frequency of OXA genes was as follows: blaOXA-145 gene in 30 isolates (27.5%), blaOXA-224 in 24 isolates (22.0%), blaOXA-539 in 22 isolates (20.1%), and blaOXA-675 in 13 isolates (11.9%). However, 19 (17.4%) isolates carry all blaOXA-145, blaOXA-224, blaOXA-539, and blaOXA-675 genes. The antimicrobial resistance and OXA genes among biofilm former strains were significantly higher than those of nonbiofilm former strains (p < 0.05). The emergence of carbapenem-resistant isolates of P. aeruginosa has posed serious threats to the community because they exhibit multiple drug resistance, thus limiting the therapeutic options for clinicians.

To determine the pattern of blood stream infections and their antibiotic susceptibility profile with infectivity predictors in a neonatal setting. The descriptive cross-sectional study was conducted at the Armed Forces Institute of Pathology, Rawalpindi, Pakistan, from December 1, 2016,to April 30, 2018, and comprised blood culture samples received in Bactec/BactAlert paediatric bottles from neonates aged 0-30 days admitted in the neonatal intensive care unit. The samples were processed as per the standard guidelines. Antibiotic susceptibility was checked as per guidelines of the Clinical and Laboratory Institute. VITEK 2 system was used for rapid identification and minimum inhibitory concentrations of the drugs. SPSS 24 was used for data analysis. Out of 640 samples, 172(27%) were culture-positive. Among them, 98(57%) were gramnegative rods, 50(29%) gram-positive cocci and 24(14%) were fungi. Of the 172 pathogens identified, Klebsiella pneumoniae was 39(22.7%) followed by Candida species 24(14%) and methicillin-resistant Coagulase-negative staphylococci 20(11.6%). Of Klebsiella pneumoniae isolates, 26(58%) were extended spectrum -lactamase producers. Among Acinetobacterbaumanii, 11(58%) were extensively drug resistant and Carbapenem-resistant strains were 20(91%). Also, 4(8%) isolates of Enterococcus faecium were vancomycin-resistant. Majority of the isolates causing blood stream infections in neonatal intensive care unit were multi drug resistant, posing a therapeutic challenge for the neo natologists .

The goal of translational research is to advance basic research and new technology toward clinical utility. Molecular diagnostics focuses primarily on nucleic acids. Rapid advances in molecular diagnostics both enable basic research and result in practical diagnostic tests. This central position of molecular diagnostics in translational research has led us to bring you this special edition of Clinical Chemistry, Molecular Diagnostics: At the Cutting Edge of Translational Research. We start the issue with a reminder that basic research is the foundation for advances in molecular diagnostics through an interview with Dr. Elizabeth Blackburn, a pioneer in telomere biology. This area of research holds promise in improving our understanding of aging, stem cell biology, and diseases like cancer and has stimulated the development of new molecular diagnostic tests. Basic research …

Objective To study pathogen aetiology and distribution of drug resistance in neonatal septice-mia. Methods A retrospective study of positive organisms of blood cultures and the results of antimicrobial suscep-tibility testing was conducted from January 2000 to December 2006 in 1293 cases of neonatal septicemia;The results were divided into three groups:the earlier group,the middle group and the later group. The strains were identified by VITEK-AMS and antibiotics susceptibility was tested by K-B method, results were interpreted according to NCCLS. inant in three groups(76.3% ,74.1% and 65.7% ) (P <0.05). Among Gram-pesitive coccus,the most prevalent strains was staphylococcus, Gram-negative bacilli increased gradually,accounting for 21.3% ,21.3% and 28.4% in cpidermids to erythromycin and sulfamethoxazole/trimethoprim were rather higher:93.3% ,73.7% vs 87.8% vs 100.0%, respectively, the resistance -rates to penicillin were less,but to susceptible to vancomyein,piperacill/tazo-batam,amikacinand ciprofloxacin;gram-negative bacilli were only sensitible to imipenem, cefepime, and cefopera zone/sulbactam;multiresistances of drug strains was commonly seen. Condusions It is important for using antibi-otics rationally according to drug sensitivity test, because multiresistant strains and opportunistic pathogen are com-monly seen. Key words: Neonate; Septicemia; Blood culture; Drug resistance

Aminoglycoside-resistant strains of Staphylococcus and Enterococcus, approximately 500 of each, were screened by dot blot hybridization for the presence of genes encoding aminoglycoside-modifying enzymes. The MICs of various aminoglycosides for the strains were determined, and the enzyme contents of the cells were inferred from the resistance phenotypes. The agreements (in percent) of the hybridization results with the deduced enzyme contents for Staphylococcus and Enterococcus species were, respectively, 80 and 87.6 for ANT(6) (aminoglycoside nucleotidyltransferase), 99.8 and 100 for both APH(3') (aminoglycoside phosphotransferase) and APH(2")-AAC(6') (aminoglycoside acetyltransferase), and 100 and 100 for ANT(4'). The weak correlation obtained with the probe for ANT(6) was due to the fact that gram-positive cocci can also be streptomycin resistant by synthesis of APH(3") or ANT(3")(9) and by ribosomal mutation. The remaining probes appeared to be specific: they hybridized with all the resistant clinical isolates but not with the susceptible strains. These results indicate that, except for streptomycin, nucleic acid hybridization is a valid approach for the detection and characterization of aminoglycoside resistance in gram-positive cocci.

BackgroundPeriprosthetic joint infections are mainly caused by Gram-positive cocci. Leuconostoc mesenteroides is a rare microorganism mainly causing bloodstream infections. At times, it might be confused with another type of cocci and give rise to misdiagnosed infections. Molecular diagnosis and biofilm production comprise important techniques to guide antibiotic treatment.Case presentationA 68-year-old Hispanic female with a previous history of bilateral knee arthroplasty presented with acute right-knee inflammation and gait impairment. Blood tests showed inflammatory response and knee x-ray revealed no prosthesis loosening. Irrigation and debridement was performed. Gram-positive cocci were obtained from cultures, and then biochemical and molecular identification revealed L. mesenteroides. Susceptibility and biofilm production were performed. The patient was treated with IntraVenous (IV) Ceftriaxone for ten days and was then switched to Amoxicillin-Clavulanate for 3 months with clinical and laboratory success.ConclusionsMicrobiology diagnosis of fastidious microorganisms is mandatory to treat periprosthetic joint infections adequately. L. mesenteroides may infect non-immunocompromised persons; however, treatment guidelines are lacking.

Infectious disease diagnostics has been transformed by metagenomic next-generation sequencing (mNGS), an unbiased approach that detects bacteria, viruses, fungi, and parasites in a single assay. By sequencing all nucleic acids in a sample, mNGS overcomes the narrow detection scope and slow turnaround of conventional tests, substantially improving pathogen detection. In conditions such as meningitis/encephalitis, sepsis, and pneumonia, mNGS frequently identifies etiologies missed by routine diagnostic tests, thereby facilitating earlier pathogen-directed therapy and, in selected settings, improving clinical management and outcomes. This approach is particularly valuable for immunocompromised, pediatric, and intensive care unit (ICU) patients with atypical infections. Currently, clinical mNGS workflows primarily rely on short-read sequencing platforms (e.g., Illumina), whereas long-read platforms (e.g., Nanopore, PacBio) offer advantages for rapid or high-resolution applications. Optimized bioinformatics and stringent quality control are essential for reliable results. Beyond clinical diagnostics, mNGS provides valuable genetic data on antimicrobial resistance (AMR) and pathogen phylogeny, supporting public health and outbreak surveillance (e.g., wastewater monitoring and variant tracking). Current challenges include distinguishing colonization from infection, interpreting sequencing data quantitatively, and reducing cost and turnaround time. Looking ahead, emerging strategies such as targeted panels, rapid automated workflows, and host‑response integration are expected to further shorten time‑to‑result and improve diagnostic specificity. Parallel progress in ethical and regulatory frameworks remains essential to ensure responsible implementation. To support clinical adoption, a standardized framework for clinical interpretation of mNGS results, together with associated training, has been developed and implemented. Overall, mNGS is likely to become an increasingly important component of infectious disease diagnostics, with ongoing innovations expected to broaden its clinical and epidemiological impact.

Purpose of ReviewMajor technologic advances in two main areas of molecular infectious disease diagnostics have resulted in accelerated adoption or ordering, outpacing implementation, and clinical utility studies. Physicians must understand the limitations to and appropriate utilization of these technologies in order to provide cost-effective and well-informed care for their patients.Recent FindingsRapid molecular testing and, to a lesser degree, clinical metagenomics are now being routinely used in clinical practice. While these tests allow for a breadth of interrogation not possible with conventional microbiology, they pose new challenges for diagnostic and antimicrobial stewardship programs. This review will summarize the most recent literature on these two categories of technologic advances and discuss the few studies that have looked at utilization and stewardship approaches. This review also highlights the future directions for both of these technologies.SummaryThe appropriate utilization of rapid molecular testing and clinical metagenomics has not been well established. More studies are needed to assess their prospective impacts on patient management and antimicrobial stewardship efforts as the future state of infectious disease diagnostics will see continued expansion of these technologic advances.

Background and aims:The genotypic method could significantly shorten the time needed to obtain antibiotic susceptibility data for Helicobacter pylori. The aim of this study was to explore the profile of H. pylori from gastric biopsies and strains with antibiotic-induced resistance.Methods:A total of 124 gastric biopsies were used to perform gene sequencing and to perform bacterial culture and susceptibility testing. Seven susceptible strains were selected to develop resistance to clarithromycin, levofloxacin, and metronidazole. Four susceptible strains were selected to transfer candidate mutations. The genotype profiles of these groups were analyzed by sequencing analysis. The antibiotic susceptibility of these strains was detected using the E-test method.Results:Phenotypic resistance to clarithromycin, levofloxacin, and metronidazole was observed in 35.5%, 40.0%, and 79.8% strains, respectively. Point mutations in 23 S rRNA, gyrA, and rdxA genes were observed in 39.5%, 38.7%, and 86.3% of gastric biopsies, respectively. The A2143G mutation in the 23S rRNA occurs in most clarithromycin-resistant samples. The A2142C point mutation showed a higher efficacy than A2142G and A2143G for inducing clarithromycin resistance. The D91N and N87K mutations in gyrA occurs in most levofloxacin-resistant samples, and double point mutations showed a higher efficacy than single mutations for inducing levofloxacin resistance. Phenotypic resistance and mutations in rdxA lacked consistency.Conclusion:Genotype-based gastric biopsy analysis was reliable for determining clarithromycin and levofloxacin resistance. A2143G in 23S rRNA and N87K/D91N in the gyrA gene occurred in most resistant strains. Mutations in the rdxA gene were not good indicators of metronidazole resistance.

Pneumococcal bloodstream infections in pediatric patients rose from 3.6 to 14.8 episodes per 100,000 from 1986 to the year 2000 (P < 0.001) in a population of approximately 650,000 inhabitants (16% <16 years of age). Incidence in the group <24 months of age increased from 7.4 to 78.6 episodes per 100,000 per year (P < 0.001). Overall 75% of the serogroups are included in the new heptavalent vaccine.

Worldwide data show that there is increasing resistance among urinary tract pathogens to conventional drugs. The aim of this study was to obtain data on susceptibility patterns of pathogens responsible for urinary tract infections (UTIs) in Poland to currently used antimicrobial agents. A multicentre study of 141 pathogens from hospital-acquired infections and 460 pathogens from community-acquired infections was carried out between July 1998 and May 1999. The most prevalent aetiological agent was Escherichia coli (73.0%), followed by Proteus spp. (8.9%) and other species of Enterobacteriaceae (9.6%). Few community infections were caused by Gram-positive bacteria (2.2%). Gram-positive cocci were isolated more frequently from a hospital setting (14.1%) and the most common were Enterococcus spp. (8.5%). Pseudomonas aeruginosa was found only among hospital isolates and was responsible for 10.7% of infections. E. coli isolates from both community and hospital infections were highly susceptible to many antimicrobial agents with the exception of those isolates producing extended-spectrum beta-lactamases (ESBLs). Of all Enterobacteriaceae tested, 38 strains (6.9%) were capable of producing ESBLs.