Clinical Microbiology Laboratory Research Articles

A STUDY OF FUNGAL INFECTIONS IN CLINICALLY SUSPECTED TUBERCULOSIS PATIENTS

Objectives: The purpose of the current study is to create awareness among the clinicians on the role of fungal etiological agents in suspected tuberculosis (TB) cases. This would help in preventing unnecessary use of anti-tubercular drugs and in decreasing drug-resistant TB cases. Methods: Prospective study was performed in the clinical microbiology laboratory of Annapoorana Medical College and Hospital, Salem, Tamil Nadu, from January 2013 to February 2017 subsequently getting approval from the institutional ethics committee. 464 sputum samples were collected from both inpatients and outpatients of numerous departments. Signs and symptoms of clinically suspected TB patients were noted for all cases. Statistical Analysis: Categorical variables were summarized by percentages (%). The Fishers exact test (2 × 2) analysis was done. Results: A total of 119 fungal isolates were isolated from the culture. The highest number of fungal isolates were of Candida species (n=61) (30.5%) followed by Cryptococcus neoformans n=38 (19%) and Histoplasma capsulatum n=20 (10%). Amongst the used anti-fungals, Fluconazole was the most effective drug for all the isolated fungi followed by Itraconazole, Amphotericin B, Voriconazole, and Nystatin. Conclusion: Our study findings indicate the significance of considering fungal infections as a prospect however treating disseminated granulomatous infections, even in immune-competent cases, particularly if the reaction to the TB therapy is insufficient.

Molecular epidemiological characterization of hypervirulent carbapenem-resistant Klebsiella pneumoniae in a hospital in Henan Province from 2020 to 2022

Objective: The study investigated the clinical distribution, antimicrobial resistance and epidemiologic characteristics of hypervirulent Carbapenem-resistant Klebsiella pneumoniae (hv-CRKP) in a hospital in Henan Province to provide a scientific basis for antibiotic use and nosocomial infection prevention and control. Methods: A retrospective analysis of the clinical data from the cases was carried out in this study. Clinical data of patients infected with the CRKP strain isolated from the clinical microbiology laboratory of Henan Provincial Hospital of Traditional Chinese Medicine from January 2020 to December 2022 were retrospectively analyzed. A string test, virulence gene screening, serum killing, and a G. mellonella infection model were used to screen hv-CRKP isolates. The clinical characteristics of hv-CRKP and the drug resistance rate of hv-CRKP to twenty-five antibiotics were analyzed using WHONET 5.6. Carbapenemase phenotypic characterization of the hv-CRKP was performed by colloidal gold immunochromatographic assay, and Carbapenemase genotyping, multi-locus sequence typing (MLST) and capsular serotyping of hv-CRKP isolates were performed by PCR and Sanger sequencing. Results: A total of non-duplicate 264 CRKP clinical isolates were detected in the hospital from 2020 to 2022, and 23 hv-CRKP isolates were detected, so the corresponding detection rate of hv-CRKP was 8.71% (23/264). The hv-CRKP isolates in this study were mainly from the intensive care unit (10/23) and neurosurgery department (8/23), and the main sources of hv-CRKP isolates were sputum (10/23) and bronchoalveolar lavage fluid (6/23). The hv-CRKP isolates in this study were highly resistant to β-lactam antibiotics, fluoroquinolones and aminoglycosides, and were only susceptible to colistin, tigecycline and ceftazidime/avibactam. The detection rate of the blaKPC-2 among 23 hv-CRKP isolates was 91.30% (21/23) and none of the class B and class D carbapenemases were detected. Results of MLST and capsular serotypes showed that ST11 type hv-CRKP was the dominant strain in the hospital, accounting for 56.52% (13/23), and K64 (9/13) and KL47 (4/13) were the major capsular serotypes. Conclusion: The hv-CRKP isolates from the hospital are mainly from lower respiratory tract specimens from patients admitted to the intensive care department and the drug resistance is relatively severe. The predominant strains with certain polymorphisms are mainly composed of the KPC-2-producing ST11-K64 and ST11-KL47 hv-CRKP isolates in the hospital.

A novel multiplex real-time polymerase chain reaction for the molecular diagnosis of metacestode infections in human patients

ObjectivesThe diagnosis of larval cestodiases in humans primarily depends on using imaging techniques in combination with serological tests. However, in case of atypical imaging results, negative serology results due to immunosuppression, or infection with rare taeniid species, traditional diagnostic tools may not provide a definitive species-level diagnosis. We aimed to validate a rapid, reliable, and cost-effective single-step real-time PCR method that can identify and differentiate larval cestodiases from biopsy material. MethodsWe validated a real-time PCR technique able to distinguish Echinococcus multilocularis, E. granulosus sensu lato (s.l.), and Taenia spp. from biopsy or cytology material in a single-step analysis. Further Sanger sequencing of E. granulosus s.l. and Taenia spp. amplicons enables differentiation of various Echinococcus and Taenia species. The assay was validated on (a) a reference sample collection of 69 clinical and veterinary cases confirmed by imaging, serology, and morphological analysis, (b) 38 routine human patient samples confirmed for aforementioned pathogens by a conventional end-point PCR, and (c) 127 samples from patients with suspected echinococcosis that were submitted to our laboratory for diagnostic analysis. ResultsCompared to a conventional reference end-point PCR approach, the quadruplex real-time PCR exhibited a lower limit of detection in a serial dilution with 5-log dilutions for all three targets (2 log for E. multilocularis, 1 log for E. granulosus s.s., and 1 log for T. saginata). We were able to detect DNA from E. multilocularis, E. granulosus s.l. (E. granulosus s.s., E. canadensis, E. ortleppi, and E. felidis), a wide range of Taenia spp., as well as from non-echinococcal metacestodes such as Hydatigera taeniaformis, Hymenolepis spp., Versteria sp., and Spirometra erinaceieuropaei. DiscussionWe suggest that the presented real-time PCR method is a suitable tool to be routinely used in a clinical microbiology laboratory to rapidly detect and identify larval cestodiases in human tissue.

P20 Rapid 15 min test to identify the three most common Gram-negative uropathogens in urinary tract infections

Abstract Background Every year, 6.2 million cases of urinary tract infection (UTI) are reported in England and Wales and have the second highest antibiotic prescription rate. The absence of fast and sensitive diagnostic tests has increased empirical antibiotic use and has contributed to the rise of antibiotic-resistant infections. To address this challenge, we have developed NANOPLEX™: a rapid 15 min test that uses glycan-functionalized-latex nanoparticles, microscopy and image-analysis software to identify and enumerate samples with bacteria. In this work, we show that NANOPLEX™ can correctly identify the three most common uropathogens using lab reference strains, clinical isolates and clinical urine samples. Materials and Methods The NANOPLEX™ assay was performed as per Vendeville et al. 2021.1 ACS Biomater 2022; 8, 1:242-252. Clinical isolates of Escherichia coli, Klebsiella pneumoniae and Proteus mirabilis were obtained from an NHS clinical microbiology lab waste stream. N=30 isolates (per species) were tested via single-blind study protocols at concentrations of 10³–108 cfu/mL and compared with microbial culture to define performance metrics (sensitivity, specificity, PPV, NPV and accuracy) of each probe. Results A total of 90 isolates were tested against their target probes and demonstrated >90% accuracy (sensitivity 88%–90%, specificity 94%–98%, PPV 93%–98%, NPV 88%–90%, accuracy 91%–94%) for all probe-bacteria combinations at clinically significant concentrations (105 cfu/mL). Conclusions NANOPLEX™ was able to successfully detect the top three common Gram-negative pathogens in UTI at clinically relevant concentrations. A compact hardware and single-use cartridge system is under development to enable urologists to conduct rapid and accurate UTI testing in point-of-care environments. Larger clinical studies are planned to clinically validate these results.

Evaluation of the Zybio EXS3000 mass spectrometry in routine identification of Clinical isolates

The matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) has been widely applied in routine clinical microbiology laboratories as an efficient and reliable technique for diagnostic purpose. In this work, we evaluated the performance of the newly developed Zybio EXS3000 (Zybio Inc., China) in microbial identification and compared it with VITEK MS (bioMérieux, France). For this study, a total of 1340 isolates from various clinical specimens were collected. These isolates were analyzed simultaneously on both EXS3000 and VITEK MS. The inconsistent or unidentifiable data were further identified using the help of either 16S rRNA gene or ITS region sequencing. During the study, we observed that EXS3000 and VITEK MS provided positive confirmatory diagnostics for 95.0% and 96.5% of the isolates, respectively, which were consistent with the sequencing results. However, it is worth noting that the EXS3000 system needs to improve the identification performance of Candida albicans in the follow-up. There are no significant differences between the two devices in terms of microbial identification performance. The advantage of EXS3000 over VITEK MS is in its ability to perform in significantly lesser time period. In conclusion, the results of this investigation showed that EXS3000 can be used to identify microorganisms in clinical microbiology laboratories.

Cefepime in vivo activity against carbapenem-resistant Enterobacterales that test as cefepime susceptible or susceptible-dose dependent in vitro: implications for clinical microbiology laboratory and clinicians.

Carbapenem-resistant Enterobacterales (CRE) are a public health concern. Among these isolates, there are reports of isolates that test as cefepime susceptible or susceptible-dose dependent (SDD) in vitro despite presence of a carbapenemase. This study aimed to evaluate the pharmacokinetic/pharmacodynamic profile of cefepime against carbapenemase-producing (CP-CRE) and non-producing (non-CP-CRE) isolates with a range of cefepime MICs. Reference broth microdilution and modified carbapenem inactivation method (mCIM) were performed on genotypically characterized clinical CRE isolates. Ultimately, CP-CRE (n = 21; blaKPC) and non-CP-CRE (n = 19) isolates with a distribution of cefepime MICs (≤0.5 to >256 mg/L) were utilized in the murine thigh infection model. Mice were treated with cefepime human-simulated regimens (HSRs) representative of a standard dose (1 g q12h 0.5 h infusion) or the SDD dose (2 g q8h 0.5 h infusion). Efficacy was assessed as the change in bacterial growth at 24 h compared with 0 h control, where ≥1 log bacterial reduction is considered translational value for clinical efficacy. Among both cohorts of CRE isolates, i.e. CP-CRE and non-CP-CRE, that tested as SDD to cefepime in vitro, 1 log bacterial reduction was not attainable with cefepime. Further blunting of cefepime efficacy was observed among CP-CRE isolates compared with non-CP-CRE across both susceptible and SDD categories. Data indicate to avoid cefepime for the treatment of serious infections caused by CRE isolates that test as cefepime susceptible or SDD. Data also provide evidence that isolates with the same antibiotic MIC may have different pharmacokinetic/pharmacodynamic profiles due to their antimicrobial resistance mechanism.

BACTERIOLOGICAL PROFILE OF WOUND INFECTION AND ANTIBIOTIC SUSCEPTIBILITY PATTERNS IN A PUBLIC HOSPITAL IN SURABAYA, INDONESIA

Background: Pyogenic infections that occur can be caused by direct bacterial contamination of the wound, such as infection in a post-surgical wound or infection after trauma. Efforts to control infection can be done with the use of antibiotics. Purpose: To determine the profile of bacteria and antibiotic sensitivity in wound culture in hospitalized patients in Hajj General Hospital Surabaya, East Java in 2021. Method: This study used the Chi-square test and descriptive analysis in the form of distribution tables and percentages using secondary data based on the results of examination of pus culture at the Clinical Microbiology Laboratory in Hajj General Hospital Surabaya, East Java in 2021. Result: The results of research on wound culture samples showed as many as 113 patients (56.22%) with positive culture results, which were predominantly female as many as 59 patients (50.9%) and the highest age category namely at the age of 46-55 years as many as 39 patients (33.6%). Based on bacteria, the most dominant type of bacteria in the Gram-negative group was Escherichia coli ESBL with 21 isolates (26.6%), while Gram-positive bacteria, Staphylococcus aureus, was found in 16 isolates (43.3). The results of antibiotic sensitivity on Gram-negative bacteria were Piperacillin/Tazobactam and Meropenem, and on Gram-positive bacteria Vancomycin and Linezolid were obtained. Conclusion: E. coli and S. aureus are the most important causes of wound infections and the suppressor organisms in this study. Antibiotic susceptibility testing of all isolates showed that the antibiotics amikacin and meropenem were sensitive to Gram-negative bacteria especially ESBL-producing bacteria, and the antibiotics vancomycin and linezolid were sensitive to Gram-positive bacteria especially MRSA.

Klebsiella pneumoniae, an important Uropathogen: Characteristics and antimicrobial susceptibility pattern at Prof. Dr. I.G.N.G. Ngoerah general hospital, period February – September 2020

Background: Klebsiella pneumoniae (K. pneumoniae) caused urinary tract infections (UTIs) are the rise all over the world and have emerged as a significant issue for public health. K. pneumoniae's characteristics as a uropathogen and their antimicrobial susceptibility pattern will be examined in this study at a tertiary care hospital. Method: The Clinical Microbiology laboratory at Prof. Dr. I. G. N. G. Ngoerah General Hospital was where this descriptive retrospective cross-sectional study was carried out. All urine cultures were included in this study, and Vitek-2 Compact (BioMerieux®) was used to identify and test for bacterial susceptibility. Data were collected over a period February-September 2020. Result: The results show 60 isolates K. pneumoniae was isolated from urine specimens. This study included outpatients and inpatients of both sex with different age. The male (51.7%) has the highest rate of infection compared to the female (48.3%), with adult patients (76.7%) and child patients (23.3%), whereas Extended-Spectrum β-Lactamase (ESBL) producing K. pneumoniae (53.3%) and non-ESBL (46.7%). The antimicrobial susceptibility test showed that K. pneumoniae mostly sensitive to meropenem (96.7%) and amikacin (93.3%) and resistance to nitrofurantoin (73.3%) and ciprofloxacin (65%). Conclusion: This study found that the percentage of ESBL producing K. pneumoniae was 53.3%. Antibiotics that are often used for the treatment of UTIs, ciprofloxacin and trimethoprim/sulfamethoxazole, have high resistance rate of 65% and 51.7%, respectively. In addition, Ertapenem, meropenem, and amikacin had the highest antimicrobial sensitivity rates.

Beyond the usual suspects: emerging uropathogens in the microbiome age

The advent of sensitive enhanced culture (metaculturomic) and culture-independent DNA-based (metagenomic) methods has revealed a rich collection of microbial species that inhabit the human urinary tract. Known as the urinary microbiome, this community of microbes consists of hundreds of distinct species that range across the entire phylogenetic spectrum. This new knowledge clashes with standard clinical microbiology laboratory methods, established more than 60 years ago, that focus attention on a relatively small subset of universally acknowledged uropathogens. Increasing reports support the hypothesis that this focus is too narrow. Single uropathogen reports are common in women with recurrent urinary tract infection (UTI), although wider disruption of their urinary microbiome is likely. Typical “UTI” symptoms occur in patients with “no growth” reported from standard culture and sometimes antibiotics improve these symptoms. Metaculturomic and metagenomic methods have repeatedly detected fastidious, slow growing, and/or anaerobic microbes that are not detected by the standard test in urine samples of patients with lower urinary tract symptoms. Many of these microbes are also detected in serious non-urinary tract infections, providing evidence that they can be opportunistic pathogens. In this review, we present a set of poorly understood, emerging, and suspected uropathogens. The goal is to stimulate research into the biology of these microbes with a focus on their life as commensals and their transition into pathogens

Role of Clinical Microbiology Laboratory for Prevention of Infection in Hospital Settings: Bangladesh Perspective

Abstract not available Bangladesh Journal of Medical Microbiology, July 2022;16(2):36-37

Development of a Polymicrobial Checkerboard Assay as a Tool for Determining Combinatorial Antibiotic Effectiveness in Polymicrobial Communities.

The checkerboard assay is a well-established tool used to determine the antimicrobial effects of two compounds in combination. Usually, data collected from the checkerboard assay use visible turbidity and optical density as a readout. While helpful in traditional checkerboard assays, these measurements become less useful in a polymicrobial context as they do not enable assessment of the drug effects on the individual members of the community. The methodology described herein allows for the determination of cell viability through selective and differential plating of each individual species in a community while retaining much of the high-throughput nature of a turbidity-based analysis and requiring no specialized equipment. This methodology further improves turbidity-based measurements by providing a distinction between bacteriostatic versus bactericidal concentrations of antibiotics. Herein, we use this method to demonstrate that the clinically used antibiotic combination of ceftazidime and gentamicin works synergistically against Pseudomonas aeruginosa in monoculture but antagonistically in a polymicrobial culture also containing Acinetobacter baumannii, Staphylococcus aureus, and Enterococcus faecalis, highlighting the fundamental importance of this methodology in improving clinical practices. We propose that this method could be implemented in clinical microbiology laboratories with minimal impact on the overall time for diagnosis.

CRISPR-Cas13a-Based Assay for Accurate Detection of OXA-48 and GES Carbapenemases.

Carbapenem-resistant pathogens have been recognized as a health concern as they are both difficult to treat and detect in clinical microbiology laboratories. Researchers are making great efforts to develop highly specific, sensitive, accurate, and rapid diagnostic techniques, required to prevent the spread of these microorganisms and improve the prognosis of patients. In this context, CRISPR-Cas systems are proposed as promising tools for the development of diagnostic methods due to their high specificity; the Cas13a endonuclease can discriminate single nucleotide changes and displays collateral cleavage activity against single-stranded RNA molecules when activated. This technology is usually combined with isothermal pre-amplification reactions in order to increase its sensitivity. We have developed a new LAMP-CRISPR-Cas13a-based assay for the detection of OXA-48 and GES carbapenemases in clinical samples without the need for nucleic acid purification and concentration. To evaluate the assay, we used 68 OXA-48-like-producing Klebsiella pneumoniae clinical isolates as well as 64 Enterobacter cloacae complex GES-6, 14 Pseudomonas aeruginosa GES-5, 9 Serratia marcescens GES-6, 5 P. aeruginosa GES-6, and 3 P. aeruginosa (GES-15, GES-27, and GES-40) and 1 K. pneumoniae GES-2 isolates. The assay, which takes less than 2 h and costs approximately 10 € per reaction, exhibited 100% specificity and sensitivity (99% confidence interval [CI]) for both OXA-48 and all GES carbapenemases. IMPORTANCE Carbapenems are one of the last-resort antibiotics for defense against multidrug-resistant pathogens. Multiple nucleic acid amplification methods, including multiplex PCR, multiplex loop-mediated isothermal amplification (LAMP) and multiplex RPAs, can achieve rapid, accurate, and simultaneous detection of several resistance genes to carbapenems in a single reaction. However, these assays need thermal cycling steps and specialized instruments, giving them limited application in the field. In this work, we adapted with high specificity and sensitivity values, a new LAMP CRISPR-Cas13a-based assay for the detection of OXA-48 and GES carbapenemases in clinical samples without the need for RNA extraction.

Vibrio fluvialis: An Emerging Pathogen for Intestinal and Extraintestinal Infections

The genus Vibrio is a ubiquitous group of bacteria belonging in the family Vibrionaceae. More than 100 species have been discovered in the genus Vibrio and about 14 of them have been reported to cause several human infections. Vibrio fluvialis, a member of this group is widely distributed in the aquatic environment, mostly in the seas, brackish waters and coastal zones.1 V. fluvialis is a pathogenic bacterium more commonly found in coastal areas. It causes diarrhea and enterocolitis and a significant number of extra intestinal infections. Due to increasing number of infections, it is considered as an emerging pathogen.1 Though this pathogen can be easily isolated by conventional staining and culture methods along with the biochemical properties, its identification is considered as a challenging problem due to its close phenotypic similarity of this species either with other vibrios and Aeromonas spp. By using automated BD Phoenix M50 techniques, it has become easier to identify V. fluvialis from clinical and different environmental samples. In this system biochemical properties of different 45 substrates are used to observe their fermentation reactions. Automated identification and antimicrobial susceptibility test systems are essential for modern clinical microbiology laboratories for rapid identification and proper selection of effective antibiotics. Several virulence factors of V. fluvialis have been identified.2 The rule and mechanisms of action of these virulent factors causing pathogenesis and infection are yet to be clarified. In this review article, the epidemiology, identification, pathogenicity, intra- and extra-intestinal clinical features and management of V. fluvialis infections have been focused. KYAMC Journal Vol. 14, No. 01, April 2023: 48-53

16S rRNA Gene PCR/Sequencing of Heart Valves for Diagnosis of Infective Endocarditis in Routine Clinical Practice.

Sequencing is increasingly used for infective endocarditis (IE) diagnosis. Here, the performance of 16S rRNA gene PCR/sequencing of heart valves utilized in routine clinical practice was compared with conventional IE diagnostics. Subjects whose heart valves were sent to the clinical microbiology laboratory for 16S rRNA gene PCR/sequencing from August 2020 through February 2022 were studied. A PCR assay targeting V1 to V3 regions of the 16S rRNA gene was performed, followed by Sanger and/or next-generation sequencing (NGS) (using an Illumina MiSeq), or reported as negative, depending on an algorithm that included the PCR cycle threshold value. Fifty-four subjects, including 40 with IE, three with cured IE, and 11 with noninfective valvular disease, were studied. Thirty-one positive results, 11 from NGS and 20 from Sanger sequencing, were generated from analysis of 16S rRNA gene sequence(s). Positivity rates of blood cultures and 16S rRNA gene PCR/sequencing of valves were 55% and 75%, respectively (P = 0.06). In those with prior antibiotic exposure, positivity rates of blood cultures and 16S rRNA gene PCR/sequencing of valves were 11% and 76%, respectively (P < 0.001). Overall, 61% of blood culture-negative IE subjects had positive valve 16S rRNA gene PCR/sequencing results. 16S rRNA gene-based PCR/sequencing of heart valves is a useful diagnostic tool for pathogen identification in patients with blood culture-negative IE undergoing valve surgery in routine clinical practice.

Less Is More: When to Repeat Antimicrobial Susceptibility Testing.

This study investigated the frequency of change of the antimicrobial susceptibility pattern when the same isolate was found in the same patient in various situations. We used laboratory data collected over a period of 8 years (January 2014 to December 2021) at the clinical microbiology laboratory of a tertiary hospital for Escherichia coli, Klebsiella pneumoniae, Enterobacter spp., Pseudomonas aeruginosa, and Staphylococcus aureus. Antimicrobial susceptibility tests (AST) were performed using Vitek 2 automated system. We determined essential agreement and categorical agreement, and introduced the new terms essential MIC increase and change from nonresistant to resistant to present changes in antimicrobial susceptibility over time. During the study period, 18,501 successive AST were included. The risk for S. aureus to be resistant to any antibiotic upon repeated culture was <10% during a follow-up of 30 days. For Enterobacterales, this risk was approximately 10% during a follow-up of 7 days. For P. aeruginosa, this risk was higher. The longer the follow-up period, the higher the risk that the bacteria would show phenotypic resistance. We also found that some drug-bug combinations were more likely to develop phenotypical resistance (i.e., E. coli/amoxicillin-clavulanic acid and E. coli/cefuroxime). A potential consequence of our finding is that if we regard a risk of resistance below 10% as acceptable, it may be feasible to omit follow-up AST within 7 days for the microorganisms investigated in this study. This approach saves money, time, and will reduce laboratory waste. Further studies are needed to determine whether these savings are in balance with the small possibility of treating patients with inadequate antibiotics.

Tarnished gold—the “standard” urine culture: reassessing the characteristics of a criterion standard for detecting urinary microbes

Diagnosis and treatment of urinary tract infections (UTIs) remains stagnant. The presumption that a patient either has a UTI or does not (binary choice) is inappropriately simplistic. Laboratory diagnostic tests have not advanced for decades. The goal of UTI treatment has not been rigorously defined and may increase the prescription of potentially harmful, inappropriate antibiotics. Despite the high incidence of UTI diagnoses, the high cost of UTI treatment, and increasing concerns associated with antimicrobial resistance, the development of novel and more accurate UTI tests has not been considered a priority, in part due to the general perception that current UTI care is already sufficient. In this review, we discuss the importance of improving UTI diagnostic testing to improve treatment outcomes. We discuss the problems associated with UTI diagnosis. Urinary microbes are alive and exist in both healthy and symptomatic individuals—urine is not sterile. We specifically outline the limitations of standard urine culture methods used by clinical microbiology laboratories, explaining clearly why such methods cannot be considered to be the “gold standard,” as standard culture methods underreport most of the urinary tract microbes, including some acknowledged and many emerging uropathogens. We do not recommend abandonment of this test, as no universally accepted substitute yet exists. However, we strongly encourage the development of new and improved diagnostic tests that can both improve outcomes and preserve antibiotic stewardship.

Uropathogenic Escherichia coli population structure and antimicrobial susceptibility in Norfolk, UK.

Urinary tract infections (UTIs) are a frequent cause for visits to primary care providers. In alignment globally, uropathogenic Escherichia coli (UPEC) are the main aetiological agent for UTIs in Norfolk and are increasingly difficult to treat due to multi-drug resistance. We set out to identify which clonal groups and resistance genes are disseminating in the community and hospitals in Norfolk, the first study of its kind for UPEC in this region. We collected 199 clinical E. coli isolates causing UTIs in the community and hospital from the Clinical Microbiology laboratory at Norfolk and Norwich University Hospital between August 2021 and January 2022. These were whole-genome sequenced using the Illumina and MinION platforms for in silico MLST and antibiotic resistance determinant detection. The isolates were composed of 70 STs; 8 lineages represented 56.7% of this population: ST73, ST12, ST69, ST131, ST404, ST95, ST127 and ST1193. Importantly, primary UTI screening deemed 6.5% of isolates to be multidrug resistant (MDR), with high rates of resistance to ampicillin (52.1%) and trimethoprim (36.2%) in hospitals. Of concern is the probable clonal expansion of MDR groups ST131 and ST1193 in hospitals and community settings with chromosomally encoded blaCTX-M-15, blaOXA-1 and aac(6')-Ib-cr5. The burden of reported UTIs in Norfolk is largely caused by non-MDR isolates and mirrors similar UPEC studies nationally and internationally. Continually monitoring samples with consideration of sources will help reduce burden of disease.

False positive cases in automated blood culture systems due to hyperleukocytosis

Automated blood culture systems are widely used in clinical microbiology laboratories to minimize the workload of laboratory personnel and permit fast turnaround times. However, sometimes false positive signals occur due to leukocytosis, presence of fastidious bacteria, or unexplained causes. We experienced false positive signal in a patient with hyperleukocytosis for the first time since the automated blood culture system was introduced in our hospital over 20 years ago. We present two case reports with literature review and describe the procedure for dealing with false-positive cases in our hospital.

Bacterial Profile and Antimicrobial Susceptibility Pattern of Gram Negative Bacteria Isolated from Skin and Soft Tissue Infections in a Tertiary Care Hospital of Western Uttar Pradesh

Background: Pyogenic infections are one of the most common causes of morbidity and mortality among hospitalized patients. Gram negative bacteria (GNB)are predominantly isolated in Hospital settings across India. A study was planned to determine the profile and susceptibility pattern of Gram negative bacteria isolated from pus samples in a tertiary care Hospital. Material and Methods: This prospective hospital based study was conducted over a period of one year. A total of 1623 pus samples received in Clinical Microbiology Laboratory were subjected to culture and identification of aerobic bacterial pathogen as per standard bacteriological method and antimicrobial susceptibility was carried out by Kirby-Bauer disk diffusion method as per CLSI guidelines 2020.Result: The culture positivity rate was 467(28.77%).There was predominance of GNB (78.59%). Klebsiella species was the predominant GNB isolated. Most of the GNBs showed good susceptibility against imipenem. However, lower susceptibility was observed against cephalosporins, quinolones, aminoglycosides and cotrimoxazole. None of the isolated GNB exhibited resistance to colistin.Conclusion: Klebsiella species was the predominant GNB isolated from pus samples from our hospital. Knowledge of bacterial profile and their antimicrobial susceptibility pattern is important for institution of empirical antimicrobial therapy for better patient outcome.

Surveillance of healthcare-onset clinical cultures using whole-genome sequencing reveals hidden nosocomial transmission

Background: Traditional hospital outbreak-detection methods are typically limited to select multidrug-resistant pathogens in a single unit, which can miss transmission of many medically important healthcare-transmissible pathogens. Whole-genome sequencing (WGS) enables comprehensive genomic resolution for accurate identification of clonal transmission. Previously, lack of scalability limited the use of WGS for hospital surveillance. Methods: We conducted prospective surveillance of select bacteria from all inpatient clinical cultures plus all bacteria from clinical cultures from ICUs and oncology units at the University of California Irvine (UCI) Clinical Microbiology Laboratory from September 2021 to February 2022. Due to pandemic stressors, this pilot test was a prelude to a real-time demonstration project. Its goal was to demonstrate the efficiency and scalability of the WGS platform when receiving samples monthly and analyzing results quarterly without the intent for real-time response. Bacterial isolates slated for discard were collected weekly and sent monthly to Day Zero Diagnostics for sequencing. In total, 1,036 samples from 926 patients were analyzed for genomic relatedness, a scalable and automated analysis pipeline already in use for rapid (days) characterization of genomic-relatedness in small and large sets of isolates. Mapping and SNP calling was performed against high-quality, best-match reference genomes. Sets of samples with pairwise distance of 2 persons with genomically related isolates and were denoted as “clusters.” Separately, we also investigated within-patient diversity by quantifying the genomic relatedness of isolates collected from individual patients. Results: Isolates represented 28 distinct species. We identified 10 Escherichia coli clusters (range, 2–4 patients; median, 2 patients), 2 Klebsiella pneumoniae clusters (range, 2–4 patients), and 1 Enterococcus faecium cluster (3 patients). All but 1 involved genomically matched isolates from multiple hospital locations. There were 4 Escherichia coli ST131 clusters spanning 4 months, including 1 with 4 patients across 3 different hospital locations. At a species level, there were distinct differences between the observed SNP distances between samples isolated from the same versus different patients (Fig. 1). All identified clusters had not been flagged by routine outbreak detection methods used by the UCI infection prevention program. Conclusions: Comprehensive WGS-based surveillance of hospital clinical isolates identified multiple potential transmission events between patients not in the same unit at the time cultures were taken. Combining WGS detection and real-time epidemiologic investigation may identify new avenues of transmission risk and could provide early warnings of clonal transmission to prevent larger outbreaks. High-volume surveillance of hospital isolates can also provide species- and context-specific clonality.Financial support: This study was funded by Day Zero Diagnostics.Disclosures: None