Microbiology Laboratory Research Articles

Biofilm Formation and Antibiotic Resistance Profiles in Carbapenemase-Producing Gram-Negative Rods-A Comparative Analysis between Screening and Pathological Isolates.

(1) Background: Carbapenem-resistant (CR) bacteria pose a significant global public health challenge due to their ability to evade treatment with beta-lactam antibiotics, including carbapenems. This study investigates the biofilm-forming capabilities of CR clinical bacterial isolates and examines the impact of serum on biofilm formation. Additionally, the study evaluates the resistance profiles and genetic markers for carbapenemase production. (2) Methods: Bacterial isolates were collected from the microbiology laboratory of Mures County Clinical Hospital between October 2022 and September 2023. Pharyngeal and rectal swabs were screened for carbapenem-resistant bacteria using selective media. Lower respiratory tract samples were also analyzed for CR Gram-negative bacteria. The isolates were tested for their ability to form biofilms in the presence and absence of fetal bovine serum at 24 and 48 h. Carbapenemase production was detected phenotypically and confirmed via PCR for relevant genes. (3) Results: Out of 846 screened samples, 4.25% from pharyngeal swabs and 6.38% from rectal swabs tested positive for CR bacteria. Acinetobacter baumannii and Klebsiella pneumoniae were the most common species isolated. Biofilm formation varied significantly between clinical isolates and standard strains, with clinical isolates generally showing higher biofilm production. The presence of serum had no significant effect on biofilm formation in Klebsiella spp., but stimulated biofilm formation for Acinetobacter spp. Carbapenemase genes blaKPC, blaOXA-48-like, and blaNDM were detected in various isolates, predominantly in Klebsiella spp., but were not the main determinants of carbapenem resistance, at least in screening isolates. (4) Conclusions: This study highlights the variability in biofilm formation among CR clinical isolates and underscores the differences between the bacteria found as carriage versus infection. Both bacterial species and environmental factors variably influence biofilm formation. These insights are crucial for the development of effective treatment and infection control strategies in clinical settings.

Point-of-Care Testing Biosafety Decisions: An Investigation Summary Illustrating Current Decision-Making Process in Ontario, Canada.

Point-of-care testing (POCT) is increasingly being used in healthcare, including hospitals, and POCT-style tests are also used within some laboratories. The principles of biosafety, including risk assessment and containment of biohazardous agents, can be utilized as a foundation to establish policies and procedures guiding safe performance of POCT. However, specific biosafety guidelines for POCT are generally lacking, particularly for those performed outside laboratories by healthcare workers. This study aims to explore POCT biosafety program decision-making infrastructure and oversight in Ontario. The Institute of Quality Management in Healthcare distributed a survey to 249 laboratories in Ontario. There were 11 questions on POCT biosafety practices. The survey had a high response rate of 88.7%. How POCT biosafety decisions were made was variable among respondents. For POCT-style tests conducted within laboratories, the biosafety officer (BSO) and/or the microbiologist were involved in biosafety decisions in 95% of microbiology labs or 55% of other labs. Only 27% of the respondents reported that biosafety decisions were made by BSOs and/or microbiologists when POCT was conducted outside the laboratory. When POCT is performed outside the laboratory, biosafety decisions were made largely by Infection Prevention and Control (IPAC) and POCT laboratory staff. Similarly, training and auditing of staff who perform POCT were mainly done by IPAC and POCT laboratory staff. The survey showed that a wide variety of POCT was being conducted for COVID-19 patients during the pandemic.

Progresses Toward Polio Eradication in Asian Countries: Its History and Japan's Contributions.

Japan experienced a large outbreak of wild poliovirus in the 1960s. The government made an exceptional decision to import oral polio vaccines (OPVs) from the Soviet Union and Canada while bypassing the usual approval process for medical products. Mass vaccination and subsequent, routine immunization successfully contained the wild poliovirus; the last case in Japan was reported in 1980. Domestic OPV had been used to sustain Japan's polio-free state. In 2012, the world's first inactivated polio vaccine developed from the Sabin vaccine replaced OPVs. Domestic vaccines combined with Sabin-derived inactivated polio vaccine are currently used in Japan. When the World Health Assembly announced the Global Polio Eradication Initiative in 1988, the Japanese government made a commitment to support endemic countries. The Japan International Cooperation Agency supported the establishment of microbiological laboratories, surveillance, distribution of polio vaccines and capacity building. Vaccine-derived poliovirus emerged as a new, international risk in the early 2000s. Vaccine-derived poliovirus was also detected in several Asian countries and required an outbreak response with additional vaccinations and strengthened surveillance. Genetically stable, novel, oral polio vaccine type 2 became available for use in outbreak responses and was used in Indonesia. Japan maintains its commitment to work toward the eradication of the poliovirus.

Evaluation of the Bio-Speedy Meningitis/Encephalitis Panel in the Diagnosis of Central Nervous System Infections

Infections of the central nervous system (CNS) can lead to severe outcomes if not accurately diagnosed and treated. The broad spectrum of pathogens involved in CNS infections can make diagnosis challenging. Polymerase chain reaction (PCR) -based multiplex molecular diagnostic panels can rapidly and simultaneously detect multiple neuropathogens in cerebrospinal fluid (CSF). This study was aimed to assess the Bio-Speedy Meningitis/Encephalitis RT-PCR MX-17 panel (Bioeksen, İstanbul, Türkiye), a novel multiplex PCR test, in diagnosing CNS infections. The panel can detect a range of pathogens, including Escherichia coli K1, Haemophilus influenzae, Listeria monocytogenes, Neisseria meningitidis, Streptococcus pneumoniae, Streptococcus agalactiae, enterovirus (EV), herpes simplex virus (HSV) 1 and 2, HHV-6, HHV-7, HHV-8, human parechovirus (HPeV), varicella zoster virus (VZV), cytomegalovirus (CMV) and Cryptococcus gatti/neoformans in CSF samples. This retrospective study included 128 CSF samples from 128 patients sent to Bursa Uludağ University Health Application and Research Center Microbiology Laboratory between June 2022 and July 2023 to search for CNS infectious agents. Patient clinical, radiological and laboratory data were collected from the Hospital Information Record System (HIRS). Bacterial pathogens were identified through culture, while viral pathogens were detected in CSF samples using the Fast Track Diagnostics (FTD) multiplex RT-PCR panel (Fast Track Diagnostics Ltd., Luxembourg) for HSV-1, HSV-2, VZV, EV, mumps virus and HPeV. The stored CSF samples were then tested using the BioSpeedy panel and the results were compared with those of the culture and the FTD panel. Pathogens that were detected were considered positive if they were consistent with the patient's symptoms and CSF characteristics according to infectious disease and pediatric infectious disease specialists. Pathogens detected but not supported by the patient's symptoms and CSF characteristics were classified as uncertain clinical relevance (UCR). Out of the 128 patients tested for CNS infectious agents, 44 (34.4%) were diagnosed with a CNS infection. The overall pathogen detection rate with all methods was 43.2% (19/44). The Bio-Speedy panel identified pathogens in 29.5% (13/44) of the patients, followed by the FTD panel (20.5%, 9/44) and culture (9.1%, 4/44). Four bacteria were identified with culture, three of which were also detected by the Bio-Speedy panel. Additionally, six bacteria were identified with Bio-Speedy panel, that were not identified by culture. The FTD panel identified nine viruses, four of which were also identified by Bio-Speedy. In total, the Bio-Speedy panel detected 13 of the 19 positive pathogens (nine bacteria and four viruses: [S.pneumoniae (n= 3), VZV (n= 3), N.meningitidis (n= 2), H.influenzae (n= 2), L.monocytogenes (n= 1), E.coli (n= 1) ve EV (n= 1)]. However, the Bio-Speedy panel identified 15 pathogens [S.pneumoniae (n= 1), E.coli (n= 1), C.gatti/neoformans (n= 1), CMV (n= 8), HHV-6 (n= 3) ve HHV-7 (n= 1)] considered as UCR. The Bio-Speedy identified the causative pathogens in the highest percentage (29.5%) of patients with confirmed CNS infections. Nevertheless, test results should be interpreted based on patient characteristics to ensure appropriate patient management. Using multiple methods and multiplex tests may improve diagnostic accuracy for CNS infections.

Performance of Flow Cytometry-Based Rapid Assay in Detection of Carbapenemase-Producing Enterobacterales.

Carbapenemase-producing Enterobacterales are increasingly being recognized in nosocomial infections. The performance of a flow cytometry-based rapid assay for their detection and differentiation was evaluated. This is a disruptive phenotypic technology, phenotypic and growth-independent, that searches for the lesions produced by drugs acting on cells after a short incubation time. Overall, 180 Gram-negative bacteria were studied, and results were compared with those obtained molecularly by PCR and phenotypically by 'KPC, MBL and OXA-48 Confirm Kit'. This phenotypic method was used as reference for comparison purposes. Susceptibility to carbapenems (imipenem, meropenem, and ertapenem) was determined by standard broth microdilution. Overall, 112 isolates (62.2%) were carbapenemase producers, 41 KPCs, 36 MβLs, and 31 OXA-48, and 4 strains were KPC + MβL co-producers. Sixty-eight isolates were carbapenemase-negative. The percentage of agreement, sensitivity, and specificity were calculated according to ISO 20776-2:2021. The FASTinov assay showed 97.7% agreement with the reference method for carbapenemase detection. Discrepant flow cytometry results were obtained in four isolates compared with both reference and PCR results. The sensitivity and specificity of this new technology were 95.3% and 98.5%, respectively, for KPCs, 97.6% and 99.3% for MβLs, and 96.9% and 98% for OXA-48 detection. In conclusion, we describe a rapid flow cytometry assay with high accuracy for carbapenemase detection and the differentiation of various carbapenemases, which should impact clinical microbiology laboratories and patient management.

Comparative Analysis of Methicillin-Resistant Staphylococcus pseudintermedius Prevalence and Resistance Patterns in Canine and Feline Clinical Samples: Insights from a Three-Year Study in Germany.

The emergence of methicillin-resistant Staphylococcus pseudintermedius (MRSP) presents a significant public health concern globally, particularly within veterinary medicine. MRSP's resistance to multiple antibiotics is limiting treatment options and potentially leading to severe infections in companion animals. This study aimed to understand antimicrobial resistance in dogs and cats, focusing on MRSP resistance patterns and its prevalence in Germany. We analyzed results of bacterial diagnostic samples from canines and felines, sourced from a German veterinary diagnostic microbiology laboratory between 2019 and 2021. This dataset included samples from 3491 veterinary practices, covering 33.1% of veterinary practices and clinics in Germany. MRSP rates were detailed by host species, sample types and co-resistance patterns. Analysis of 175,171 bacterial examination results revealed S. pseudintermedius in 44,880 samples, yielding a 25.6% isolation rate. S. pseudintermedius was more prevalent in dogs (35.0%) than cats (3.6%). Methicillin resistance was found in 7.5% of all S. pseudintermedius isolates. MRSP prevalence was higher in feline samples (16.1%, 95% CI 14.4-17.8) compared to canine samples (7.1%, 95% CI 6.8-7.0). S. pseudintermedius showed high resistance rates to ampicillin (cats: 48.6%, dogs: 67.6%) and clindamycin (cats: 37.2%, dogs: 32.7%), while MRSP exhibited high co-resistance to clindamycin (cats: 82.8%, dogs: 85.4%) and sulfamethoxazole + trimethoprim (cats: 66.4%, dogs: 66.2%). Our study revealed distinct resistance patterns of MRSP in cats compared to dogs, highlighting the need for tailored treatment approaches and the importance of antimicrobial resistance surveillance.

Duration of surgical antibiotic prophylaxis and surgical site infection in orthopaedic surgery: A prospective cohort study.

The optimal duration for surgical antibiotic prophylaxis (SAP) for preventing surgical site infection (SSI) in orthopaedic surgeries remains poorly supported by high-level evidence. This study aimed to assess the association between SAP duration and the occurrence of SSI within one year postoperatively. This prospective cohort study was based on the database from Surgical Site Infection Surveillance and Improvement Project (SISIP) of a tertiary orthopaedic university hospital from October 2014 to December 2020. The main outcome was SSI, defined according to the CDC/NHSN criteria, determined by review of index hospitalization medical records, microbiology laboratory reports, and readmission records for SSI treatment within one-year after discharge. Adjusted Generalized additive models (GAMs) were performed to assess the relationships between SAP duration and SSI, determined the cut-off point of SAP duration, and estimate the relative contribution of each included variable, across the overall cohort and the three subgroups (open fracture, closed fracture, and non-traumatic group). Multivariable logistic regression models were used to estimate the association between prolonging SAP duration based on the cut-off point and SSI. There were 37,046 patients (55.1% male) included, with the overall SSI incidence of 2.35% (871/37,046). In adjusted GAMs, no statistically significant relationships were observed in overall cohort and open or closed group (P>0.05), but a nonlinear relationship was exhibited non-traumatic group (P=0.03); the cut-off point were 2.4 days for overall cohort and 3.6 days (open), 2.6 days (closed), 1.1 days (non-trauma) for three subgroups. In adjusted logistic regression, prolonging SAP duration did not demonstrate a statistically significant protective effect in overall cohort (aOR=0.868; 95% CI, 0.746-1.011) and three groups (open: aOR=0.867; 95% CI, 0.668-1.124; closed: aOR=0.925; 95% CI, 0.754-1.135; non-trauma: aOR=1.184; 95% CI, 0.832-1.683). The relative contribution ranks of SAP duration were 21st overall among 34 factors, 14th for open fractures, 28th for closed fractures, and 3rd for non-traumatic group among 33 factors. Prolonged postoperative SAP duration has no protective effect against SSI in orthopaedic surgery. Our findings support current guidelines against the practice of continuing SAP postoperatively.

Pattern of Bacterial Flora and Effect of Disinfection on Fingerprinting Biometric Devices at a Tertiary Care Health Facility: An Interventional Study.

Introduction Recently, many public and private sector institutions and hospitals have installed biometric fingerprint devices for attendance purposes. This step is taking us toward modernization but biometric devices have their cons and pros; if not sterilized at regular intervals, then it may be a potent cause of transmission of various infections. Many studies have reported the presence of coagulase-negative Staphylococcus aureus (CONS), methicillin-resistant Staphylococcus aureus (MRSA), Escherichiacoli, Pseudomonas, and others. Aim To study the pattern of bacterial flora and the effect of disinfection on fingerprinting biometric devices at a tertiary care health facility. Materials andmethods A total of 138 biometric devices were used, out of which 105 were frequently (at least 50 uses per day) used and functional. So, 105 samples were collected on day zero (baseline), of which 43 and 62 were from clinical and non-clinical groups, respectively. The devices were disinfected with isopropyl alcohol (w/v 70%) and subsequent samples were taken on day 1 (after 24 hours) and day 7. The samples were collected and transported to the microbiology lab for culture and incubation. Statistical analysis was performed usingSPSS software version 25 (IBM Corp., Armonk, NY) employing chi-square, Cochran's Q test, and post hoc test. A p-value ≤0.05 at a 95% confidence interval was considered to be statistically significant. Results At baseline (day 0), bacterial growth was observed in 13 (38%) devices from the clinical group and 10 (20%) from the non-clinical group. After disinfection with 70% isopropyl alcohol, bacterial growth was reduced by 83% on day 1 but increased by 82% on day 7. These changes were statistically significant (p ≤ 0.05). Conclusion The present study concluded the definite presence of bacterial flora on the biometric fingerprint devices which are prone to carry and transmit microorganisms indirectly from person to person. The surface of biometric fingerprinting devices should be disinfected daily. If not possible, it should be done on an average of every third day to control and minimize the transmission of microorganisms.

Evaluation of Fosfomycin in-vitro susceptibility among gram negative isolates from urine culture in a tertiary care hospital in Lucknow

The rise of bacteria that are Multidrug- resistant or Extended Drug Resistant (MDR or XDR) and the lack of novel antibiotics have wreaked havoc on patient treatment options. Fosfomycin has just recently come to light because of the development of resistance to existing drugs. This study aims to evaluate the Fosfomycin susceptibility of Gram- negative isolates in vitro. A prospective study done in the Department of Microbiology, Integral Institute of Medical Sciences of Research, Lucknow, over a period of six months from April 2023 to September 2023. A total of 1328 urine samples were processed in microbiology lab and after identification of urinary isolates, antibiotic susceptibility testing was performed, and results were interpreted following the Clinical and Laboratory Standards Institute guidelines (CLSI). Fosfomycin sensitivity was tested by the Kirby-Bauer disc diffusion method. 353 of the 1328 processed urine samples showed a growth of pathogens. 184 (52.1%) of the isolates were Gram-negative, while 169 (47.8%) were Gram-positive. Fosfomycin (7%) and Nitrofurantoin (13%) showed lower rates of resistance among these isolates. Relatively higher rates of resistance were observed for Doripenem (17%), Meropenem (17%), Imipenem (18%), Amikacin (18%), Gentamicin (27.9%). Norfloxacin, Aztreonam, Cefepime, Ciprofloxacin and Levofloxacin and Cotrimoxazole showed a high resistance rate of 46.2%, 48.8%, 50%, 60.4%, 61% and 70% respectively. Fosfomycin's low resistance (7%) to the antibiotic indicates that it works well as a urinary antibiotic. Uropathogenic Enterobacteriaceae isolates exhibited an excellent level of in vitro susceptibility to Fosfomycin. These in vitro results highlight the unexplored possibilities of Fosfomycin as a superior therapeutic option for the treatment of UTIs, and it has a good chance of expanding into a safe and effective oral alternative treatment that can be used in inpatient as well as outpatient settings.

BACTERIOLOGICAL PROFILE AND ANTIMICROBIAL SUSCEPTIBILITY PATTERN OF SURGICAL SITE INFECTIONS IN A TEACHING HOSPITAL

Objective: Surgical site infections (SSI) are defined as infections occurring within 30 or 90 d after surgical operation or within 1 y, if an implant is left in place after procedure and affecting either incision or deep tissues at the operation site. SSI is one of the quality indicators of the healthcare system. Due to advent of newer antibiotics and emergence of multidrug-resistant bacteria, SSI requires reappraisal of the microbiological flora and in vitro antibiotic sensitivity pattern. The aim of the present study is to determine the incidence etiology of SSI, and determine their Antibiogram. Methods: Cross-sectional study conducted for a period of three months from January 2024 to March 2024. A total of fifty swabs/pus specimens from various types of surgical sites suspected to be infected on clinical grounds were collected from the post-operative wards of surgical departments and processed in Microbiology laboratory. Isolation and identification of bacterial agents were done as per standard protocols. Antimicrobial susceptibility testing was performed by Kirby-Bauer’s disc diffusion method. Results: Out of 50 samples, 32 were culture-positive. 87.50% showed monomicrobial growth and 12.50% showed polymicrobial growth. Analysis of bacterial profile shows Staphylococcus aureus (27.77%) was the predominant isolate, followed by Pseudomonas aeruginosa (19.44%) and Escherichia coli (19.44%). Antibiogram of gram-positive isolates showed sensitivity to Teicoplanin, Vancomycin, and linezolid, whereas gram-negative isolates were sensitive to Meropenem, Piperacillin-tazobactam and levofloxacin. Conclusion: Status of SSI is the main quality indicators of hospital infection control and prevention measures. Continuous surveillance on etiology and antibiogram of SSI is necessary to monitor antimicrobial resistance and guide in empirical treatment.

BACTERIOLOGICAL PROFILE AND ANTIBIOGRAM OF GRAM-NEGATIVE BACILLI ISOLATED FROM ENDOTRACHEAL TUBE SECRETIONS AND TRACHEAL ASPIRATES FROM PATIENTS ON MECHANICAL VENTILATION: A HOSPITAL-BASED CROSS-SECTIONAL STUDY

Objective: Nosocomial infections have become a serious threat to mortality and morbidity among hospitalized patients. Major risk factor among critically ill patients is excessive use of invasive devices like Endotracheal tube (ET) and tracheal intubation. The development of biofilm and their subsequent dislodgement aids in lung colonization and may cause Ventilator-associated pneumonia (VAP) [1]. Methods: Present study was conducted on ET tube tips and Tracheal aspirates received in microbiological laboratory from various ICU’s (Medical, surgical and pediatric) in Andhra Medical College, Visakhapatnam over a period of one year from September 2022 to August 2023. A total of 116 samples were processed according to standard protocols and Antibiotic Susceptible Testing (AST) was performed by Kirby-Bauer disc diffusion method on Mueller-Hinton agar as per CLSI guidelines. Results: In total, 116 samples were processed. ET tube tips were 76/116 and tracheal aspirates were 40/116, with males (69/116) outnumbered females (47/116). Out of a total of 116 samples, 72 (62%) were culture-positive and 44 (38%) were sterile. Acinetobacter spp. was the most common pathogen among 72 culture positives (31/72, 43%), followed by Klebsiella spp. (20/72, 28%), Pseudomonas spp. (13/72, 18%) and Escherichia coli (8/72, 11%). A total of 11 (11/72, 15%) GNB were found as multi-drug resistant. The multi-drug resistant GNB isolated were Acinetobacter (6/72), Klebsiella (3/72) and Pseudomonas (2/72). Meropenem is the most susceptible antibiotic, followed by Piperacillin-Tazobactam. Conclusion: It was concluded from this study that most commonly isolated pathogen was Acinetobacter spp. Most of these isolates were sensitive to Carbapenem and BLBLI combination drugs. Microbial persistence and impaired response to the treatment were more frequent, when multi-drug resistant organisms were present. Hence a local combined antibiotic approach, based on bacteriological profile and AST is essential to initiate empirical therapy which will minimize the bacterial colonization and prevent the incidences of VAP.

A Rapid Total Bacterial Count Method for Food Samples using Syringe Filters and Lectin-Conjugated Semiconductor Nanorods.

Total bacterial count in food is one of important food safety criteria. The current plate count method (Heterotrophic Plate Count) for food analysis requires microbiology lab facilities and at least 2 days turnover time. We developed a rapid fluorescence-based total bacterial count method that utilises semiconductor nanorods (SNRs) conjugated with a lectin Griffonia simplicifolia II (GSII-SNRs) to stain bacterial cells captured on syringe filters, via the common N-acetylglucosamine molecules on bacterial cell wall. This "Filter-and-Stain" detection method has a rapid turnover time of 20 min. The fluorescence emission can be seen under UV light with minimum interference from food sample background. The fluorescence intensity quantified through image analysis is proportional to the bacterial concentration with a limit of detection of 1000 CFU/mL, for total bacterial count assessment in food safety. Moreover, the GSII-SNRs do not bind to heat inactivated bacterial cells, and thus can differentiate live and dead bacteria. Our method has been validated with representative food (coffee powder, raw spinach leaves, and ready-to-eat tomato salsa) to demonstrate its high potential for on-site food safety assessment, especially in places with no immediate access to microbiology labs.

Candida spp.: the burden of a microorganism in a microbiology department.

We believe that this work is of interest because at present, there is no really accurate information available on the total workload involved in isolating a specific microorganism in a clinical microbiology laboratory. The costs related to this have also not been described. We have described the unrestricted workload of Candida spp. in all types of samples for all types of species and patients. We believe that this information would be necessary to collect and share this information as well as to collect it in a standardized way to know the current situation of Candida spp. workload in all clinical microbiology laboratories.

Activity Test of Catalase Enzyme in Rhizospheric Soil Bacteria

This research was conducted at the Microbiology Laboratory of UIN Walisongo Semarang to identify and analyze the catalase enzyme in rhizosphere bacteria around the Laboratory of Campus 2 of UIN Walisongo Semarang. This research was carried out in several stages, namely isolation of rhizosphere bacteria, characterization of rhizosphere bacteria, gram staining of rhizosphere bacteria, and catalase test of rhizosphere bacteria. The data analysis technique used is the descriptive data analysis technique. The results showed that 9 isolates of rhizosphere bacteria produced catalase enzyme in the presence of gas bubbles when reacted with H2O2.

Isolation and identification of hoylesella marshii causing pleural infection: a case report

BackgroundHoylesella marshii can be isolated from human oral cavities affected by dental pulp and periodontal infections, as well as from the dental plaque of healthy individuals, making it a common bacterium within the oral microbiota. However, its role in causing pleural infections in humans is rare.Case presentationA case of purulent pleural effusion occurred shortly after discharge in an elderly patient who had undergone surgery for gastric cancer. The infection was identified as being caused by an obligate anaerobe through laboratory culture, and was further identified as Hoylesella marshii causing pleural infection through 16 S rRNA gene sequence analysis. Susceptibility testing guided precise treatment with cefoperazone-sulbactam and metronidazole. The patient’s clinical symptoms improved rapidly, laboratory test indicators gradually returned to normal, and the patient ultimately recovered.ConclusionHoylesella marshii can cause pleural infections in humans. Clinical microbiology laboratories should pay special attention to the cultivation of obligate anaerobes when routine aerobic cultures do not show bacterial growth but bacteria are visible on smear staining, and when conventional identification methods fail to identify the bacterium, analysis based on the highly conserved 16 S rRNA gene sequence can accurately and specifically identify the bacterium, guiding clinicians in formulating precise anti-infection strategies.

Unified metagenomic method for rapid detection of microorganisms in clinical samples

BackgroundClinical metagenomics involves the genomic sequencing of all microorganisms in clinical samples ideally after depletion of human DNA to increase sensitivity and reduce turnaround times. Current human DNA depletion methods preferentially preserve either DNA or RNA containing microbes, but not both simultaneously. Here we describe and present data using a practical and rapid mechanical host-depletion method allowing simultaneous detection of RNA and DNA microorganisms linked with nanopore sequencing.MethodsThe human cells from respiratory samples are lysed mechanically using 1.4 mm zirconium-silicate spheres and the human DNA is depleted using a nonspecific endonuclease. The RNA is converted to dsDNA to allow the simultaneous sequencing of DNA and RNA.ResultsThe method decreases human DNA concentration by a median of eight Ct values while detecting a broad range of RNA & DNA viruses, bacteria, including atypical pathogens (Legionella, Chlamydia, Mycoplasma) and fungi (Candida, Pneumocystis, Aspergillus). The first automated reports are generated after 30 min sequencing from a 7 h end-to-end workflow. Sensitivity and specificity for bacterial detection are 90% and 100%, respectively, and viral detection are 92% and 100% after 2 h of sequencing. Prospective validation on 33 consecutive lower respiratory tract samples from ventilated patients with suspected pneumonia shows 60% concordance with routine testing, detection of additional pathogens in 21% of samples and pathogen genomic assembly achieve for 42% of viruses and 33% of bacteria.ConclusionsAlthough further workflow refinement and validation on samples containing a broader range of pathogens is required, it holds promise as a clinically deployable workflow suitable for evaluation in routine microbiology laboratories.

Impact of the COVID-19 pandemic on extended-spectrum β-lactamase producing Escherichia coli in urinary tract and blood stream infections: results from a nationwide surveillance network, Finland, 2018 to 2022

BackgroundBefore the COVID-19 pandemic there has been a constant increase in antimicrobial resistance (AMR) of Escherichia coli, the most common cause of urinary tract infections and bloodstream infections. The aim of this study was to investigate the impact of the COVID-19 pandemic on extended-spectrum β-lactamase (ESBL) production in urine and blood E. coli isolates in Finland to improve our understanding on the source attribution of this major multidrug-resistant pathogen.MethodsSusceptibility test results of 564,233 urine (88.3% from females) and 23,860 blood E. coli isolates (58.8% from females) were obtained from the nationwide surveillance database of Finnish clinical microbiology laboratories. Susceptibility testing was performed according to EUCAST guidelines. We compared ESBL-producing E. coli proportions and incidence before (2018–2019), during (2020–2021), and after (2022) the pandemic and stratified these by age groups and sex.ResultsThe annual number of urine E. coli isolates tested for antimicrobial susceptibility decreased 23.3% during 2018–2022 whereas the number of blood E. coli isolates increased 1.1%. The annual proportion of ESBL-producing E. coli in urine E. coli isolates decreased 28.7% among males, from 6.9% (average during 2018–2019) to 4.9% in 2022, and 28.7% among females, from 3.0 to 2.1%. In blood E. coli isolates, the proportion decreased 32.9% among males, from 9.3 to 6.2%, and 26.6% among females, from 6.2 to 4.6%. A significant decreasing trend was also observed in most age groups, but risk remained highest among persons aged ≥ 60 years.ConclusionsThe reduction in the proportions of ESBL-producing E. coli was comprehensive, covering both specimen types, both sexes, and all age groups, showing that the continuously increasing trends could be reversed. Decrease in international travel and antimicrobial use were likely behind this reduction, suggesting that informing travellers about the risk of multidrug-resistant bacteria, hygiene measures, and appropriate antimicrobial use is crucial in prevention. Evaluation of infection control measures in healthcare settings could be beneficial, especially in long-term care.

Characterization of Leuconostoc lactis Bacteremia during a 2-year Study at a Tertiary Care Center in North India-An Observational Analysis.

Leuconostoc species are regarded as important causes for many infections in immunocompromised patients. In this study, we assessed the characteristics of Leuconostoc spp. causing bacteremia in patients at our center. This observational analysis was conducted in the microbiology laboratory of a tertiary care center in northern India from July 2021 to July 2023. Patients in whom blood culture bottles were positive for Leuconostoc lactis were included in the study. Culture isolates were identified by MALDI-ToF MS as L. lactis and tested for antibiotic sensitivity results by Kirby-Bauer disk diffusion method. Demographic and clinical details were collected and analyzed. During the study period, 6,742 blood culture bottles flagged positive. Among these, L. lactis was isolated from 14 (0.21%) patients. The median patient age was 34 years. The male-to-female ratio was 2.5:1. All the patients with L. lactis bacteremia had an underlying condition leading to immunosuppression (e.g., carcinoma and chronic kidney disease). All the patients with L. lactis bacteremia had an intravascular device present at the time of bacteremia. All isolates in the study were sensitive to doxycycline, high level gentamicin, minocycline, ampicillin-sulbactam, and linezolid. Mortality was attributed to bacteremia by L. lactis in five patients. Appropriate and timely identification of the Leuconostoc species is important for the clinician to tailor regimens for the patients.

Formic acid sandwich method is well-suited for filamentous fungi identification and improves turn around time using Zybio EXS2600 mass spectrometry

ObjectivesMatrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is extensively employed for the identification of filamentous fungi on MALDI Biotyper (Bruker Daltonics) and Vitek MS (biomerieux), but the performance of fungi identification on new EXS2600 (Zybio) is still unknow. Our study aims to evaluate the new EXS2600 system's (Zybio) ability to rapidly identify filamentous fungi and determine its effect on turnaround time (TAT) in our laboratory.MethodsWe tested 117 filamentous fungi using two pretreatment methods: the formic acid sandwich (FA-sandwich) and a commercial mold extraction kit (MEK, Zybio). All isolates were confirmed via sequence analysis. Laboratory data were extracted from our laboratory information system over two 9-month periods: pre-EXS (April to December 2022) and post-EXS (April to December 2023), respectively.ResultsThe total correct identification (at the species, genus, or complex/group level) rate of fungi was high, FA-sandwich (95.73%, 112/117), followed by MEK (94.02%, 110/117). Excluding 6 isolates not in the database, species-level identification accuracy was 92.79% (103/111) for FA-sandwich and 91.89% (102/111) for MEK; genus-level accuracy was 97.29% (108/111) and 96.39% (107/111), respectively. Both methods attained a 100% correct identification rate for Aspergillus, Lichtheimia, Rhizopus Mucor and Talaromyces species, and were able to differentiate between Fusarium verticillioides and Fusarium proliferatum within the Fusarium fujikuroi species complex. Notably, high confidence was observed in the species-level identification of uncommon fungi such as Trichothecium roseum and Geotrichum candidum. The TAT for all positive cultures decreased from pre EXS2600 to post (108.379 VS 102.438, P < 0.05), and the TAT for tissue decreased most (451.538 VS 222.304, P < 0.001).ConclusionsThe FA-sandwich method is more efficient and accurate for identifying filamentous fungi with EXS2600 than the MEK. Our study firstly evaluated the performance of fungi identification on EXS2600 and showed it is suitable for clinical microbiology laboratories use.

When less is more: the art of communicating clinical microbiology results.

The clinical microbiology laboratory is capable of identifying microorganisms in clinical specimens faster and more accurately than ever before. At face value, this should enable patient care providers to make better-informed decisions and target antimicrobial therapies to deliver individualized care. Ironically, more complete and specific reporting of microorganisms isolated from specimens may result in overtreatment based on the presence of a pathogen, even in the absence of clear signs of clinical infection. This conundrum calls into question the role of the laboratory in contributing to care through selective or "exception" reporting whereby some results are selectively withheld when there is a low probability that laboratory findings correlate with the clinical infection. In a recent article published in the Journal of Clinical Microbiology, Bloomfield et al. (J Clin Microbiol 62:e00342-24, 2024, https://doi.org/10.1128/jcm.00342-24) examine the impact and safety of an exception reporting strategy applied to wound swab specimens. Canonical pathogens associated with skin and soft tissue infections including S. aureus and beta-hemolytic streptococci are withheld from the laboratory report if certain patient criteria are met that would put them at low risk of adverse outcomes if untreated, or if treated with guideline-recommended empiric therapy. Their central finding was an approximately 50% reduction in post-laboratory report antibiotic initiation without adverse events or increased 30-day admission rate (indicative of infection-related complications, e.g., disseminated disease). While effectively achieving their goal, the premise of exception reporting and other modified reporting strategies raises questions about the potential risk of underreporting and how to ensure that the message is being interpreted, and acted upon, by care providers as was intended by the laboratory.