Results Of Phenotypic Tests Research Articles

P-2097. Genomic basis of Oxacillin- and Cefoxitin-Susceptible PBP2a-Positive Staphylococcus aureus

Abstract Background Methicillin-resistance Staphylococcus aureus (MRSA) is resistant to all ß-lactam antibiotics except for the fifth-generation cephalosporin drug, ceftaroline. The mecA gene, which encodes an alternative penicillin-binding protein, PBP2a, is responsible for the high-level resistance to methicillin. However, mecA positive, oxacillin- and cefoxitin susceptible MRSA has been reported, with limited understanding of the genomic basis behind this contradictory phenomenon. Here, we studied the molecular base of a “stealth” MRSA from clinical specimen. Methods A MRSA isolate was routinely isolated from a deep wound specimen, which was positive for PBP2a by lateral flow assay but negative for cefoxitin screen test and susceptible to oxacillin (MIC of 0.5 ug/ml) by VITEK 2 AST-GP75 card. It was also susceptible to cefoxitin using disk diffusion method (diameter of 26 mm). The “stealth” MRSA isolate was sequenced on illumina NovaSeq 6000 platform using 150 bp paired end sequencing. Bacteria genome was assembled and annotated using pipelines on BV-BRC (https://www.bv-brc.org/). Results Genomic sequencing revealed a point mutation (G to A) in the mecA gene resulting in a Gly599Ser substitution located in the transpeptidase domain of PBP2a, which is close to the active site of Ser403 on the 3D structure of the protein. Additionally, an insertion in the methicillin resistance regulatory sensor-transducer mecR1 gene was identified, resulting in a frameshift mutation, and predicted to cause premature termination. However, the sequence of frame shift mutation in mecR1 has been described and was not associated with reversed cefoxitin susceptibility. Therefore, the point mutation in mecA gene is most likely responsible for loss of function of PBP2a, and lead to susceptible cefoxitin and oxacillin results despite a positive PBP2a reaction. Conclusion Whole genome sequencing is valuable to understand the genomic basis of unusual phenotypic testing results. The reversion of “stealth” MRSA strain to a high-level oxacillin- and cefoxitin-resistant strain has been reported, which highlights the importance of considering tests for mecA or PBP2a in routine laboratory practice to avoid the risk of missing such strains. Disclosures All Authors: No reported disclosures

Phenotypic Resistance to Rifampicin of Mycobacterium tuberculosis with the rpoB Leu430Pro Mutation

The objective: to determine the role of the rpoB Leu430Pro mutation in the degree of phenotypic resistance of Mycobacterium tuberculosis to rifampicin.Subjects and Methods. The WHO classifies the rpoB Leu430Pro mutation of Mycobacterium tuberculosis as a borderline resistance mutation but of clinical significance. From an array of cultures, we selected and analyzed 14 isolates of M. tuberculosis with discrepancies in the results of molecular genetic and phenotypic testing, carrying only this mutation in rpoB. For all samples, the phenotypic resistance level (MIC) of these isolates to rifampicin was determined using BACTEC MGIT 960 and Middlebrook 7H11 medium.Results. It was found out that 12 of 14 isolates had the rifampicin MIC below the current critical concentration when tested by both methods, thus they were phenotypically sensitive. One isolate was resistant when tested with Middlebrook 7H11 but susceptible when tested with BACTEC MGIT 960. Only one isolate which had the additional rpoB F425L mutation demonstrated high-level phenotypic resistance when tested by the same tests. Our data indicate that the clinical significance of this mutation requires clarification since even a decrease in the critical concentration of rifampicin does not lead to unambiguous results of molecular genetic and phenotypic tests. It is necessary to standardize the use of various molecular genetic tests and principles of their clinical interpretation to optimize strategies for managing patients with tuberculosis caused by M. tuberculosis with the rpoB Leu430Pro mutation.

Antagonistic Effects and the Underlying Mechanisms of Bacillus velezensis and its Antibacterial Peptide LCI Against Aeromonas hydrophila Infection in Largemouth Bass.

Aeromonas hydrophila is one of the most prevalent pathogenic bacteria in largemouth bass. The use of antibiotics to inhibit A. hydrophila poses a significant threat to fish and environmental safety. Bacillus velezensis, a safe bacterium with probiotic and antibacterial characteristics, is an ideal candidate for antagonizing A. hydrophila. This study explored the antagonistic effects of B. velezensis FLU-1 on A. hydrophila in vivo and in vitro. In addition, we explored the antimicrobial peptides (AMPs) produced by strain FLU-1 and clarified the underlying antibacterial mechanisms. The results showed that strain FLU-1 could inhibit a variety of fish pathogens, including A. hydrophila. The challenge test showed that dietary supplementation with B. velezensis FLU-1 significantly improved the survival rate of largemouth bass and reduced the bacterial load in liver. Subsequently, the AMP LCI was isolated from B. velezensis FLU-1 and was found to be effective against A. hydrophila in vitro and in vivo. Transcriptomic analysis revealed that LCI downregulated the genes associated with flagellar assembly and peptidoglycan synthesis in A. hydrophila. Phenotypic test results showed that LCI disrupted the membrane integrity, markedly reduced the biofilm biomass and diminished the swimming motility of A. hydrophila. Furthermore, the results showed that LCI bound to the genomic DNA of A. hydrophila and destroyed the DNA structures. Overall, these findings elucidated the mechanism of action of LCI against A. hydrophila at the phenotypic and physiological levels. This study suggests that B. velezensis FLU-1 and its AMP LCI could serve as antibiotic alternatives for controlling pathogens in aquaculture.

Evaluation of Whole Genome Sequencing-Based Predictions of Antimicrobial Resistance to TB First Line Agents: A Lesson from 5 Years of Data.

Phenotypic susceptibility testing of the Mycobacterium tuberculosis complex (MTBC) isolate requires culture growth, which can delay rapid detection of resistant cases. Whole genome sequencing (WGS) and data analysis pipelines can assist in predicting resistance to antimicrobials used in the treatment of tuberculosis (TB). This study compared phenotypic susceptibility testing results and WGS-based predictions of antimicrobial resistance (AMR) to four first-line antimicrobials-isoniazid, rifampin, ethambutol, and pyrazinamide-for MTBC isolates tested between the years 2018-2022. For this 5-year retrospective analysis, the WGS sensitivity for predicting resistance for isoniazid, rifampin, ethambutol, and pyrazinamide using Mykrobe was 86.7%, 100.0%, 100.0%, and 47.8%, respectively, and the specificity was 99.4%, 99.5%, 98.7%, and 99.9%, respectively. The predictive values improved slightly using Mykrobe corrections applied using TB Profiler, i.e., the WGS sensitivity for isoniazid, rifampin, ethambutol, and pyrazinamide was 92.31%, 100%, 100%, and 57.78%, respectively, and the specificity was 99.63%. 99.45%, 98.93%, and 99.93%, respectively. The utilization of WGS-based testing addresses concerns regarding test turnaround time and enables analysis for MTBC member identification, antimicrobial resistance prediction, detection of mixed cultures, and strain genotyping, all through a single laboratory test. WGS enables rapid resistance detection compared to traditional phenotypic susceptibility testing methods using the WHO TB mutation catalog, providing an insight into lesser-known mutations, which should be added to prediction databases as high-confidence mutations are recognized. The WGS-based methods can support TB elimination efforts in Canada and globally by ensuring the early start of appropriate treatment, rapidly limiting the spread of TB outbreaks.

Faecalibacterium taiwanense sp. nov., isolated from human faeces.

Two Gram-stain-negative, straight rods, non-motile, asporogenous, catalase-negative and obligately anaerobic butyrate-producing strains, HLW78T and CYL33, were isolated from faecal samples of two healthy Taiwanese adults. Phylogenetic analyses of 16S rRNA and DNA mismatch repair protein MutL (mutL) gene sequences revealed that these two novel strains belonged to the genus Faecalibacterium. On the basis of 16S rRNA and mutL gene sequence similarities, the type strains Faecalibacterium butyricigenerans AF52-21T(98.3-98.1 % and 79.0-79.5 % similarity), Faecalibacterium duncaniae A2-165T(97.8-97.9 % and 70.9-80.1 %), Faecalibacterium hattorii APC922/41-1T(97.1-97.3 % and 80.3-80.5 %), Faecalibacterium longum CM04-06T(97.8-98.0% and 78.3 %) and Faecalibacterium prausnitzii ATCC 27768T(97.3-97.4 % and 82.7-82.9 %) were the closest neighbours to the novel strains HLW78T and CYL33. Strains HLW78T and CYL33 had 99.4 % both the 16S rRNA and mutL gene sequence similarities, 97.9 % average nucleotide identity (ANI), 96.3 % average amino acid identity (AAI), and 80.5 % digital DNA-DNA hybridization (dDDH) values, indicating that these two strains are members of the same species. Phylogenomic tree analysis indicated that strains HLW78T and CYL33 formed an independent robust cluster together with F. prausnitzii ATCC 27768T. The ANI, AAI and dDDH values between strain HLW78T and its closest neighbours were below the species delineation thresholds of 77.6-85.1 %, 71.4-85.2 % and 28.3-30.9 %, respectively. The two novel strains could be differentiated from the type strains of their closest Faecalibacterium species based on their cellular fatty acid compositions, which contained C18 : 1 ω7c and lacked C15 : 0 and C17 : 1 ω6c, respectively. Phenotypic, chemotaxonomic and genotypic test results demonstrated that the two novel strains HLW78T and CYL33 represented a single, novel species within the genus Faecalibacterium, for which the name Faecalibacterium taiwanense sp. nov. is proposed. The type strain is HLW78T (=BCRC 81397T=NBRC 116372T).

A Multicenter Assessment of the Outcomes and Toxicities of Foscarnet for Treatment of Acyclovir-Resistant Mucocutaneous Herpes Simplex in Immunocompromised Patients.

Acyclovir-resistant mucocutaneous herpes simplex virus (HSV) infection is an uncommon problem typically seen in immunocompromised hosts. Systemic treatment options are limited. The performance of foscarnet and its toxicities in this population are poorly characterized. This was a multicenter retrospective study of adults treated with foscarnet for HSV infection between January 2012 and December 2017. Relevant data were collected including demographics, baseline conditions, previous anti-HSV medications, concomitant medications, HSV outcomes, and adverse events. Acyclovir-resistant HSV infection was defined based on genotypic or phenotypic testing results; refractory infection was defined as infection not improving after 5 days of treatment-dosed antiviral therapy in those not tested for resistance. Twenty-nine patients had 31 episodes of HSV (15/18 resistant; among episodes without resistance testing, 7/10 refractory; 3 not evaluable) treated with foscarnet. All patients were immunocompromised including 19 (66%) with hematologic malignancy and 9 (31%) with HIV. Median duration of foscarnet was 16 days (range, 6-85 days). Fifteen episodes (48%) healed by the end of or after foscarnet. Median time to healing among those with resolution was 38 days (range, 9-1088 days). At least 1 adverse event during therapy was reported in 26 (84%) treatment episodes including 23 (74%) that were considered drug related. Common adverse events were electrolyte disturbance (20 [65%]) and kidney dysfunction (13 [42%]). Foscarnet was discontinued in 10 episodes (32%) due to an adverse event, including 6 due to kidney dysfunction. Among 31 episodes of HSV treated with foscarnet, only half resolved with treatment, and adverse events were common.

Phenotypic detection of carbapenem-resistant Enterobacterales in clinical isolates at a tertiary care hospital

Abstract Background: In the past decade, there has been a global emergence of carbapenem-resistant Gram-negative bacilli, especially Enterobacterales. Carbapenem resistance is attributed to the ability of the bacteria to produce carbapenemases. The aim of the study is to detect carbapenem-resistant Enterobacterales (CRE) in different clinical isolates and study carbapenemase production by phenotypic methods in CRE. Materials and Methods: A total of 379 Enterobacterales were isolated from different clinical samples from patients attending outpatient departments and admitted in wards and intensive care units (ICUs). They were tested for carbapenem resistance by Kirby–Bauer disk diffusion method and then tested for carbapenemase production by ethylenediaminetetraacetic acid (EDTA)–disk synergy test and Modified Carbapenem Inactivation Method (mCIM). Results: This study was conducted from February 2021 to August 2022. Out of 379 Enterobacterales, 70 (18.47%) were CRE, out of which maximum carbapenem resistance of 23.53% was shown by Klebsiella pneumoniae isolates. The maximum carbapenem resistance was seen in the age group of 16–45 years and the most number of CRE isolates were from ICUs. Phenotypic test results indicated that 54.28% (38/70) of isolates were positive for carbapenemase production by either of the phenotypic methods. Conclusion: About one-fifth of the Enterobacterales isolates were carbapenem resistant. This study highlights the use of phenotypic methods to detect carbapenemase production in CRE, which is responsible for multidrug resistance. This information is relevant for surveillance, to implement infection prevention and control practices and antibiotic policies.

National monitoring of antibiotic resistance of pathogens causing community-acquired urinary tract infections in Russia: results of the multicenter epidemiological study «DARMIS -2023»

Objective. To study in vitro activity of antimicrobials against clinical isolates obtained from patients with community-acquired urinary tract infections (UTIs) in various regions of Russia in 2023. Materials and Methods. The study included 1098 isolates obtained from the urine of children and adults of both sexes of all age groups with acute and exacerbation of recurrent community-acquired UTIs, including pregnant women with asymptomatic bacteriuria. Isolates were collected in 29 centers in 21 cities of Russia in 2023 as part of a multicenter prospective epidemiological study of the dynamics of antibiotic resistance of pathogens causing community-acquired UTIs in different subsets of patients («DARMIS-2023»). The categories of susceptibility of isolates to antimicrobial drugs were based on the breakpoint values of minimum inhibitory concentrations (MIC) in accordance with the Russian recommendations «Determination of susceptibility of microorganisms to antimicrobial drugs» (Version 2024-02) and the updated EUCAST criteria (v. 14.0, 2024). Results. Enterobacterales accounted for a total of 88.1% of all isolated bacterial pathogens (89.6% in the adult subpopulation; 82.8% in the pregnant subpopulation and 89.6% in the subpopulation of children and adolescents under 18 years of age). The most prevalent species were Escherichia coli (72.2% in the adult subpopulation; 72.8% in the pregnant women subpopulation and 68.9% in the children and adolescents under 18 years of age subpopulation) and Klebsiella pneumoniae (10.8% in the adult subpopulation; 4.8% in the pregnant women subpopulation and 9.8% in the children and adolescents under 18 years of age subpopulation). Of the oral drugs, the minimal resistance in E. coli was demonstrated for nitrofurantoin (0.4% of isolates in the adult subpopulation; 0.0% in the pregnant women subpopulation and 1.6% in the children and adolescents under 18 years of age subpopulation) and fosfomycin (9.6% of isolates in the adult subpopulation; 4.4% in the pregnant women subpopulation and 1.6% in the children and adolescents under 18 years of age subpopulation). Of the parenteral agents, meropenem and amikacin had the highest activity: 0.8% and 1.0% resistant E. coli in the adult subpopulation; no resistant E. coli in the pregnant subpopulation; 0.0% and 0.8% in the children and adolescents under 18 years of age subpopulation, respectively. For each patient subpopulation, antimicrobial resistance of E. coli to ampicillin, amoxicillin/clavulanic acid, cefotaxime, cefixime, and trimethoprim-sulfamethoxazole was more than 20%. Rates of E. coli resistance to ciprofloxacin were 36.7% in the adult subpopulation; 22.1% in the pregnant subpopulation; and 22.2% in the children and adolescents under 18 years of age subpopulation. The rate of extended-spectrum beta-lactamase production based on phenotypic test results was 29.6% in the adult subpopulation; 23.6% in the pregnant subpopulation and 33.3% in the children and adolescents under 18 years of age subpopulation. Conclusions. In community-acquired UTIs the main clinical problem is the persistent high rate of E. coli resistance to cephalosporins, fluoroquinolones, aminopenicillins/beta-lactamase inhibitors, as well as the increase of the extended-spectrum beta-lactamases production. Remaining high in vitro activity of fosfomycin and nitrofurans allows to consider them as drugs of choice in uncomplicated lower urinary tract infections.

Pseudomonas cucumis sp. nov., isolated from the rhizosphere of crop plants.

Two bacterial strains, FP1935T and FP1962, were isolated from the rhizosphere soil of cucumber and Chieh-qua plants, respectively, in Jilin Province, PR China. These strains were Gram-stain-negative, aerobic, rod-shaped and motile with one or two polar flagella. Analysis of the 16S rRNA gene sequences revealed that they represented members of the genus Pseudomonas, with the highest similarity to Pseudomonas silesiensis A3T (99.45 %), Pseudomonas frederiksbergensis JAJ28T (99.45 %), Pseudomonas mandelii NBRC 103147T (99.38 %), Pseudomonas piscium P50T (99.27 %) and Pseudomonas meliae CFBP 3225T (99.18 %). The DNA G+C contents of FP1935T and FP1962 were 58.99 mol% and 58.98 mol%, respectively. The results of in silico genome-based analyses indicated that these strains were distinct from other species in the genus Pseudomonas, as the average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) values were below the recommended thresholds of 95 % (ANI) and 70 % (dDDH) for prokaryotic species delineation, with no values exceeding 94.1 and 55.8 %, respectively, compared with any other related species. The results of phenotypic and chemotaxonomic tests confirmed their differentiation from their closest relatives. The fatty acid profiles of both strains mainly consisted of summed feature 3 (C16 : 1ω6c and/or C16 : 1ω7c), summed feature 8 (C18 : 1ω7c and/or C18 : 1ω6c), C12 : 0 and C16 : 0. The predominant respiratory quinone was Q-9. Polar lipids include phosphatidylethanolamine, unidentified aminophospholipids, unidentified lipids and an unidentified phospholipid. On the basis of these phenotypic and genotypic results, we propose the name Pseudomonas cucumis sp. nov. for these novel strains. The type strain is FP1935T (=ACCC 62445T=JCM 35690T).

Proficiency testing for drug susceptibility testing of Mycobacterium tuberculosis complex using commercial broth microdilution plate in China in 2021

The characteristic and performance of Broth microdilution (BMD) plates for drug susceptibility of Mycobacterium tuberculosis have not been systematically evaluated in China. This study was designed to review the key information and assess the performance of BMD plates by analysis of proficiency testing results. We retrospectively analysed the proficiency testing results of phenotypic drug susceptibility testing (PT-DST) of 45 laboratories using BMD plates in China in 2021. Critical information, such as drug layout, concentration range of each drug, plate storage conditions and duration, operating procedures, and interpretation criteria for binary results were compared. The performance was also analysed. Eight types of BMD plates produced by four manufactures were reported. The drug layout, number of drugs on plates, and concentration range varied a lot between different plates. The total sensitivity and specificity of BMD plates for drug susceptibility of Mycobacterium tuberculosis to ten drugs (isoniazid (INH), rifampin (RIF), kanamycin (KAM), amikacin (AM), levofloxacin (LFX), moxifloxacin (MFX), bedaquiline (BDQ), linezolid (LZD), clofazimine (CFZ), and delamanid (DLM)) were 93.9% (95% CI 92.-94.9) and 99.1% (95% CI 98.8-99.3), respectively. The lowest sensitivity was 84.8% (95% CI 80.3-88.4) for LFX and 86.4% (95% CI 82.5-89.6) for MFX, or 87.5% (95% CI 84.2-90.2) for Y1 plate and 87.9% (95% CI 83.5-91.1) for T plate. The lowest specificity was 94.4% (95% CI 91.4-96.4) for DLM, or 97.9% (95% CI 96.8-98.7) for B3 plate. Commercial BMD plates in China showed varied drug layouts and operational procedures, indicating the urgency of standardization. The lower performance for some drugs showed the low quality of the plates utilized or lack of proficiency of lab staffs in operating and interpreting results.

The Comparisons of Phenotypic and Genotypic Resistance of Staphylococcus aureus Isolates Against β-lactam and Tetracycline Antibiotics

Staphylococcus aureus (S. aureus) is a Gram-positive bacteria that causes various diseases in humans and animals. Treatment of S. aureus infection generally uses antibiotics, but what happens is, S. aureus resistance to antimicrobial agents is an increasing global problem. This study aims to compare the phenotypic and genotypic characters of S. aureus from animals isolates against β-lactam and tetracycline antibiotics class. Determination of resistance characteristics of 8 S. aureus isolates was carried out phenotypically by Kirby Bauer and Minimum Inhibitory Concentration (MIC) methods, while genotypically by detecting the blaZ and tet genes. The results of the study showed a relationship between resistance genes and phenotypic test results using the Kirby Bauer and MIC methods. Isolates C, D, F, and TLK with resistance gene characters blaZ-, tetK-, and tetM- showed a sensitive interpretation of the results of the Kirby Bauer and MIC tests. On the other hand, DMG, J, PGT, and KRG isolates were resistant to one or two antibiotics tested, both phenotypic and genotypic (blaZ+, tetK+/tetM+). It found that the MIC levels of some respective isolates were relatively high, isolates J and KRG had an ampicillin MIC level of 32 μg/ml, while isolates J and PGT had 256 μg/ml for oxytetracycline. Although the identification of resistance genes and Kirby Bauer can determine the character of bacterial resistance, MIC determination is necessary to provide qualitative and quantitative information for the benefit of making therapeutic decisions and infection control strategies.

Comparison of Blood-Based Shotgun and Targeted Metagenomic Sequencing for Microbiological Diagnosis of Infective Endocarditis.

Shotgun and targeted metagenomic sequencing have been shown in separate studies to be potentially useful for culture-free pathogen identification in blood and/or plasma of patients with infective endocarditis (IE). However, the 2 approaches have not been directly compared. The aim of this study was to compare shotgun metagenomic sequencing with targeted metagenomic sequencing (tMGS) for organism identification in blood or plasma of patients with IE. Patients with possible or definite IE were prospectively enrolled from October 2020 to July 2021. Shotgun metagenomic sequencing was performed with the Karius test, which uses microbial cell-free DNA (mcfDNA) sequencing to detect, identify, and quantitate DNA-based pathogens in plasma. tMGS was performed using a 16S ribosomal RNA (rRNA) polymerase chain reaction assay targeting the V1 to V3 regions of the 16S rRNA gene. Results were compared using the McNemar test of paired proportions. Samples from 34 patients were investigated. The Karius test was positive in 24/34 (71%), including 3/6 (50%) with blood culture-negative endocarditis (BCNE), which was not significantly different from the positivity rate of tMGS (P = .41). Results of the Karius test were concordant with tMGS in 75% of cases. The Karius test detected 2 cases of methicillin-resistant Staphylococcus aureus among the 7 S. aureus detections, in accordance with results of phenotypic susceptibility testing. The combination of blood cultures, the Karius test, and tMGS found a potential causative pathogen in 33/34 (97%), including 5/6 with BCNE. The Karius test and tMGS yielded comparable detection rates; however, beyond organism identification, the Karius test generated potentially useful antibiotic resistance data.

Resistance patterns and transmission of mono- and polyresistant TB: clinical impact of WGS.

Rapidly diagnosing drug-resistant TB is crucial for improving treatment and transmission control. WGS is becoming increasingly accessible and has added value to the diagnosis and treatment of TB. The aim of the study was to perform WGS to determine the rate of false-positive results of phenotypic drug susceptibility testing (pDST) and characterize the molecular mechanisms of resistance and transmission of mono- and polyresistant Mycobacterium (M.) tuberculosis. WGS was performed on 53 monoresistant and 25 polyresistant M. tuberculosis isolates characterized by pDST. Sequencing data were bioinformatically processed to infer mutations encoding resistance and determine the origin of resistance and phylogenetic relationship between isolates studied. The data showed the variable sensitivity and specificity of WGS in comparison with pDST as the gold standard: isoniazid 92.7% and 92.3%; streptomycin 41.9% and 100.0%; pyrazinamide 15% and 94.8%; and ethambutol 75.0% and 98.6%, respectively. We found novel mutations encoding resistance to streptomycin (in gidB) and pyrazinamide (in kefB). Most isolates belonged to lineage 4 (80.1%) and the overall clustering rate was 11.5%. We observed lineage-specific gene variations encoding resistance to streptomycin and pyrazinamide. This study highlights the clinical potential of WGS in ruling out false-positive drug resistance following phenotypic or genetic drug testing, and recommend this technology together with the WHO catalogue in designing an optimal individualized treatment regimen and preventing the development of MDR TB. Our results suggest that resistance is primarily developed through spontaneous mutations or selective pressure.

High Prevalence of Plasmid-Mediated Quinolone Resistance among ESBL/AmpC-Producing Enterobacterales from Free-Living Birds in Poland.

In this study, we investigated the occurrence of plasmid-mediated quinolone resistance (PMQR) in extended-spectrum β-lactamase- (ESBL) and/or AmpC-type β-lactamase-producing Enterobacterales isolates from free-living birds in Poland. The prevalence of the qnrB19 gene was 63%, and the distribution of isolates in terms of bacterial species was as follows: 67% (22/33) corresponded to Escherichia coli, 83% (5/6) to Rahnella aquatilis, 44% (4/9) to Enterobacter cloacae and 33% (1/3) to Klebsiella pneumoniae. The qnrB19 gene was also found in a single isolate of Citrobacter freundii. The molecular characteristics of qnrB19-positive isolates pointed to extended-spectrum beta lactamase CTX-M as the most prevalent one (89%) followed by TEM (47%), AmpC (37%) and SHV (16%). This study demonstrates the widespread occurrence of PMQR-positive and ESBL/AmpC-producing Enterobacterales isolates in fecal samples from wild birds. In this work, plasmid pAM1 isolated from Escherichia coli strain SN25556 was completely sequenced. This plasmid is 3191 nucleotides long and carries the qnrB19 gene, which mediates decreased susceptibility to quinolones. It shares extensive homology with other previously described small qnrB19-harboring plasmids. The nucleotide sequence of pAM1 showed a variable region flanked by an oriT locus and a Xer recombination site. The presence of a putative recombination site was detected, suggesting that interplasmid recombination events might have played a role in the development of pAM1. Our results highlight the broad geographical spread of ColE-type Qnr resistance plasmids in clinical and environmental isolates of Enterobacterales. As expected from the results of phenotypic susceptibility testing, no resistance genes other than qnrB19 were identified.

First identification, molecular characterization, and pathogenicity assessment of Lactococcus garvieae isolated from cultured pompano in Taiwan.

Lactococcosis, caused by Lactococcus garvieae, is an acute hemorrhagic septicemia in fish recorded in marine and freshwater aquaculture during the summer months. In 2020-2021, several sea cage Pompano farms recorded sudden fish mortality events. Based on the results of phenotypic and biochemical tests, L. garvieae was predicted to be the cause. PCR with L. garvieae specific primers (pLG1 and pLG2) targeting the 16S rRNA region further confirmed the etiological agent as L. garvieae after amplifying an 1100 base pairs (bp) product. Furthermore, the 16S rRNA sequences of the two representative strains (AOD109-196-2B and AOD110-215-2B) shared 99.81% identity with L. garvieae (GenBank accession number: MT597707.1). The genetic profiles of the strains were classified using pulsed-field gel electrophoresis after digestion with SmaI and ApaI, which clustered our strains under the same pulsotype. Multiplex PCR targeting the capsule gene cluster and serotype-specific PCR collectively showed that the strains were non-capsulated; thus, they belonged to serotype I. An experimental infection was designed to fulfil Koch's postulates by infecting healthy Pompano with case-driven L. garvieae strains (AOD109-196-2B and AOD110-215-2B) with a cumulative mortality of 70%. Overall, L. garvieae infection in Pompano emphasizes the need for better monitoring and control procedures in aquaculture settings.

Evaluation of Early Reading of Broth Microdilution Technique for Polymyxin B.

Delay in the results of standard phenotypic susceptibility tests is the main obstacle to adequate antibiotic treatment. For this reason, the European Committee for Antimicrobial Susceptibility Testing has proposed the Rapid Antimicrobial Susceptibility Testing for the disk diffusion method directly from blood culture. However, to date, there are no studies evaluating early readings of polymyxin B broth microdilution (BMD), the only standardized methodology for assessing susceptibility to polymyxins. This study aimed to evaluate modifications in the BMD technique for polymyxin B using fewer antibiotic dilutions and reading after an incubation time of 8-9 hr (early reading) in comparison to 16-20 hr of incubation (standard reading) for isolates of Enterobacterales, Acinetobacter baumannii complex, and Pseudomonas aeruginosa. A total of 192 isolates of gram-negative bacteria were evaluated and the minimum inhibitory concentrations were read after early and standard incubations. The early reading presented 93.2% of essential agreement and 97.9% of categorical agreement with the standard reading of BMD. Only three isolates (2.2%) presented major errors and only one (1.7%) presented a very major error. These results indicate a high agreement between the early and the standard reading times of BMD of polymyxin B.

Assessment of systemic AAV-microdystrophin gene therapy in the GRMD model of Duchenne muscular dystrophy.

Duchenne muscular dystrophy (DMD) is a progressive muscle wasting disease caused by the absence of dystrophin, a membrane-stabilizing protein encoded by the DMD gene. Although mouse models of DMD provide insight into the potential of a corrective therapy, data from genetically homologous large animals, such as the dystrophin-deficient golden retriever muscular dystrophy (GRMD) model, may more readily translate to humans. To evaluate the clinical translatability of an adeno-associated virus serotype 9 vector (AAV9)-microdystrophin (μDys5) construct, we performed a blinded, placebo-controlled study in which 12 GRMD dogs were divided among four dose groups [control, 1 × 1013 vector genomes per kilogram (vg/kg), 1 × 1014 vg/kg, and 2 × 1014 vg/kg; n = 3 each], treated intravenously at 3 months of age with a canine codon-optimized microdystrophin construct, rAAV9-CK8e-c-μDys5, and followed for 90 days after dosing. All dogs received prednisone (1 milligram/kilogram) for a total of 5 weeks from day -7 through day 28. We observed dose-dependent increases in tissue vector genome copy numbers; μDys5 protein in multiple appendicular muscles, the diaphragm, and heart; limb and respiratory muscle functional improvement; and reduction of histopathologic lesions. As expected, given that a truncated dystrophin protein was generated, phenotypic test results and histopathologic lesions did not fully normalize. All administrations were well tolerated, and adverse events were not seen. These data suggest that systemically administered AAV-microdystrophin may be dosed safely and could provide therapeutic benefit for patients with DMD.

Virulence genes and antibiotic resistance profiles of Staphylococcus aureus isolated from bovine mastitis milk samples.

The increasing importance of antibiotic resistance shows the need for determining indices of the epidemiology of infection. This study aimed to determine the virulence genes and antibiotic resistance profiles of Staphylococcus aureus isolated from bovine mastitis cases. A total of 200 cattle were selected based on California Mastitis Test (CMT) results, and the samples were cultured in the laboratory. Grown colonies were examined by conventional phenotypic methods and confirmed using PCR amplification of 16S rRNA gene. The prevalence of the virulence genes was also defined. The results of phenotypic and molecular tests were compared using SPSS software by McNemar test. Then, the confirmed isolates were tested for antibiotic susceptibility using the disc diffusion method. Of the 200 positive CMT cattle, 24 animals were positive for S. aureus and confirmed using 16S rRNA gene amplification. Statistical analysis showed that the phenotypic and genotypic tests of hemolysin genes were not significantly different (P>0.01). PCR analysis revealed the presence of coa and clfa genes in more than half of the cases. Overall, nine genetic profiles of virulence factors were found among S. aureus isolates. The highest and lowest resistance rates were against penicillin and gentamicin, respectively. Our findings showed a high rate of antibiotic resistance. So, accurate and fast diagnosis and antimicrobial susceptibility tests should be considered before prescribing the drugs.

PncA Large Deletion is the Characteristic of Pyrazinamide-Resistant Mycobacterium tuberculosis belonging to the East Asian Lineage.

Pyrazinamide (PZA) is often used as an add-on agent in the treatment of multidrug-resistant tuberculosis, regardless of phenotypic drug susceptibility testing (pDST) results. However, evaluating the effectiveness of PZA is challenging because of its low pH activity, which can result in unreliable pDST results. This study aimed to investigate the genomic characteristics associated with PZA resistance that can be used to develop genotypic DST. A publicly available whole genome sequencing (WGS) dataset of 10,725 Mycobacterium tuberculosis complex genomes (3,326 phenotypically PZA-resistant and 7,399 phenotypically PZA-susceptible isolates) were analyzed. In total, 2,934 pncA non-silent mutations were identified in 2,880 isolates (26.9%). Detected mutations were found throughout the entire coding region of pncA in a scattered pattern, of which the most frequent mutation was p.Q10P (n = 278), followed by p.H57D (n = 167) and c.-11A>G (n = 122). The sensitivity and specificity of the group 1 or 2 mutations reported by the World Health Organization (WHO) mutational catalogue were 73.0% and 98.9%, respectively. We further identified 18 novel pncA mutations that were significantly associated with phenotypically PZA-resistant. In addition to these mutations, we identified 102 large deletions in the pncA gene, and all but two isolates were phenotypically resistant to PZA isolates. Notably, pncA deletions were mutually exclusive to pncA mutations, and more than half of the isolates with pncA large deletions belonged to the East Asian lineage (67.6%). The sensitivity, specificity, positive predictive value, and negative predictive value of the pooled variants (group 1 or 2 mutations, novel resistance-associated mutations, and large deletions of the pncA gene) were 79.0%, 98.9%, 97.0%, and 91.3%, respectively. The area under the curve (AUC) value for the pooled variants was significantly higher than the AUC value for the group 1 or 2 mutations (P <0.001), indicating that the pooled variants have a better discriminative ability for predicting PZA resistance. Using WGS, we found that the pncA mutations are scattered without specific mutational hotspots, and large deletions associated with PZA resistance are more common in the East Asian lineage of M. tuberculosis isolates. Our data also demonstrated the reliability of group 1 or 2 mutations presented in the WHO mutation catalogue and the need for further investigation on group 3 mutations, contributing to the evaluation of the current knowledge base on mutations associated with the PZA-resistant M. tuberculosis complex.

Development and Assessment of a Novel Whole-Gene-Based Targeted Next-Generation Sequencing Assay for Detecting the Susceptibility of Mycobacterium tuberculosis to 14 Drugs.

Targeted next-generation sequencing (tNGS) has emerged as an alternative method for detecting drug-resistant tuberculosis (DR-TB). To provide comprehensive drug susceptibility information and to address mutations missed by available commercial molecular diagnostics, we developed and evaluated a tNGS panel with 22 whole-gene targets using the Ion Torrent platform to predict drug resistance to 14 drugs, namely, rifampicin (RIF), isoniazid (INH), ethambutol (EMB), pyrazinamide (PZA), moxifloxacin (MFX), levofloxacin (LFX), amikacin (AMK), capreomycin (CM), kanamycin (KM), streptomycin (SM), bedaquiline (BDQ), clofazimine (CFZ), linezolid (LZD), and delamanid (DLM). We selected 50 and 35 Mycobacterium tuberculosis isolates with various DR profiles as the training set and the challenge set, respectively. Comparative variant analyses of the DR genes were performed using Sanger sequencing and whole-genome sequencing (WGS). Phenotypic drug susceptibility testing (pDST) results were used as gold standards. Regarding the limit of detection, the tNGS assay detected 2.9 to 3.8% minority variants in 4% mutant mixtures. The sensitivity and specificity of tNGS were 97.0% (95% confidence interval [CI] = 93.1 to 98.7%) and 99.1% (95% CI = 97.7 to 99.7%), respectively. The concordance of tNGS with pDST was 98.5% (95% CI = 97.2 to 99.2%), which was comparable to that of WGS (98.7%, 95% CI = 97.4 to 99.3%) and better than that of Sanger sequencing (96.9%, 95% CI = 95.3 to 98.0%). The agreement between tNGS and pDST was almost perfect for RIF, INH, EMB, MFX, LFX, AMK, CM, KM, SM, BDQ, and LZD (kappa value = 0.807 to 1.000) and substantial for PZA (kappa value = 0.791). Our customized novel whole-gene-based tNGS panel is highly consistent with pDST and WGS for comprehensive and accurate prediction of drug resistance in a strengthened and streamlined DR-TB laboratory program. IMPORTANCE We developed and validated a tNGS assay that was the first to target 22 whole genes instead of regions of drug resistance genes and comprehensively detected susceptibility to 14 anti-TB drugs, with great flexibility to include new or repurposed drugs. Notably, we demonstrated that our custom-designed Ion AmpliSeq TB research panel platform had high concordance with pDST and could significantly reduce turnaround time (by approximately 70%) to meet a clinically actionable time frame. Our tNGS assay is a promising DST solution for providing needed clinical information for precision medicine-guided therapies for DR-TB and allows the rollout of active pharmacovigilance.