Susceptibility Test Results Research Articles

Antibiogram of Pseudomonas aeruginosa Clinical Isolates Tested for Pan, Extensive and Multidrug Resistances

Background: Pseudomonas aeruginosa usually cause nosocomial infections with concurrent morbidity and mortality and is generally resistant to many antibiotics.
 Aim: This study was aimed to determine the proportion of pan-drug-resistant (PDR), extensively drug-resistant (XDR), and multidrug-resistant (MDR) P. aeruginosa strains recovered from human samples.
 Methodology: The retrospective study was conducted in the University of Nigeria Teaching Hospital Enugu in 2023. Clinical samples obtained from patients between October 2022 and April 2023 were analysed. A total of 100 Pseudomonas aeruginosa isolates recovered from 780 clinical samples were used. Standard microbiological techniques were used to identify and categorize the isolates. Antibiotic susceptibility pattern was determined using the Kirby Bauer disc diffusion method.
 Results: Isolates recovered from wound was 38%, voided urine, catheter tip urine, ear swab, and high vaginal swab samples recorded 29%, 11%, 10% and 4%, respectively. Ceftazidime recorded the highest level of resistance (70.0%) and the least was Colistin (20%). Resistance patterns showed that 32(32.0%) bacterial strains were MDR, 68(68.0%) were XDR and no PDR was recorded.
 Conclusions: For the best selection of empirical therapy, P. aeruginosa susceptibility monitoring is essential due to the high prevalence of antibiotic resistance. The resistance pattern raises the possibility of misuse of broad-spectrum antibiotics. Treatment for bacterial infections should be directed by the results of antimicrobial susceptibility tests.

EMERGENCE OF CEFTAZIDIME-AVIBACTAM RESISTANCE IN ENTERO-BACTERALES AND PSEUDOMONAS AERUGINOSA

Objective: The objective of this study is to determine the emergence of resistance to Ceftazidime-Avibactam (CAZ-AVI) by Enterobacterales and Pseudomonas aeruginosa in clinical isolates. Material and Methods: In this cross-sectional study (6-months) March-August 2022 Carbapenem resistant Enterobacterales were tested for Ceftazidime-Avibactam (30/20 µg, Oxoid Pvt Ltd) using Disk diffusion technique and enzymes were identified in resistant strains by Carbapenem Inactivation Method (mCIM, eCIM) as per CLSI M100 Guidelines 2022. Results: CAZ-AVI effectiveness has greatly decreased among Enterobacterales and P. aeruginosa isolates in recent past. Antimicrobial susceptibility testing results were interpreted using CLSI M100 document. Resistance against CAZ-AVI in Enterobacterales was found to be 80.8 % in E. coli and 72.1% in Klebsiella pneumonia isolates. This higher emergence is associated with CRE isolates majorly comprising MBLs in our country. Moreover, it has been observed that Metallo- β-lactamases mediated enzyme resistance is one of the major resistance patterns followed by serine carbapenemases. Conclusion: The high frequency of resistance 77% was observed against CAZ-AVI in CRE and in CRPA, the resistance is 80.1% respectively. In our country this tremendously increase in CAZ-AVI resistance is attributed to the existence of NDM in the region. Keywords: Carbapenem Resistant Enterobacterales (CRE), Metallo beta lactamases (MBLs), Carbapenem Resistant Pseudomonas aeruginosa (CRPA) Ceftazidime-Avibactam (CAZ-AVI), Modified Carbapenem Inactivation Method (mCIM), EDTA-modified Carbapenem Inactivation Method(eCIM), Multiple Drug Resistance (MDR)

Impact of a hospital sepsis management protocol on the selection of empirical antibiotics in infectious disease consultations

It is well-established that Infectious Diseases consultation (IDC) enhances the prognosis of bloodstream infections. However, it is unclear if adoption of an institutional sepsis protocol would lead to any further improvement in a setting where IDC and infectious diseases approval (IDA) - available throughout 7 days/24 hours -are mandatory for administering broad spectrum antibiotics. We aimed to evaluate the influence of the institutional sepsis protocol developed by Department of Infectious Diseases and Clinical Microbiology on the selection of appropriate empirical antibiotics by IDC through focusing on patients who had bloodstream infections caused by Extended-spectrum β-lactamase (ESBL) producing Escherichia coli and Klebsiella pneumoniae, which poses a therapeutic challenge. One hundred and fifty-three adult patients (58 patients in the pre-protocol period and 95 patients in the post-protocol period), who received empirical antibiotic treatment for ESBL-producing E. coli and K. pneumoniae, in whom at least one systemic antibiotic was started either on the day blood cultures were drawn or not later than 24 hours were included in the study, retrospectively. The primary outcome was whether the empirical treatment regimen included a carbapenem that was accepted as the appropriate treatment based on the results of the MERINO trial. Secondary outcomes included empirical treatment based on pre-defined risk factors suggesting multidrug resistance (MDR), 30-day inpatient mortality, and appropriate antibacterial treatment according to antimicrobial susceptibility test (AST) results. The median age (Interquartile range) was 61 (48-70.5) years and 76 (49.7%) out of 153 patients were male. The patients in the post-protocol period were older compared to the pre-protocol period (54 years vs 64 years, p = 0.045). The Charlson Comorbidity Index was higher during the post-protocol period compared to the pre-protocol period (4 vs 5, p=0.038). At least one risk factor for MDR bacteria infection was present in 147 (96.1%) of the 153 patients. While the rate of risk factors for MDR bacteria infections did not differ significantly between the pre-protocol and post-protocol periods, the post-protocol period showed a significantly higher level of appropriate antibiotic treatment according to the presence of MDR risk factors compared to the pre-protocol period (44.8% vs 64.2%, p=0.019). There was a significant increase in the use of carbapenems in the post-protocol period compared to the pre-protocol period (34.5% vs. 56.8%, p=0.007). When the subgroup of patients who were likely to have infection caused by ESBL-producing bacteria is taken into consideration, the carbapenem use was more frequent in the post-protocol period (37.8% vs 68.9%, p=0.002). The rate of appropriate empirical treatment according to AST was not statistically different between pre-protocol and post-protocol period. The 30-day mortality rates were similar in both periods (24.1% vs 31.5, p=0.33). However, the rate of susceptibility to piperacillin-tazobactam was statistically higher in the pre-protocol period (82.6% vs 46.2%, p=0.016) when 39.7% of the patients received piperacillin-tazobactam as the empirical treatment. This study highlights the significance of using a structured protocol to attain appropriate empirical treatment for patients suspected of sepsis, even in a setting where IDC is readily available.

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.

Prevalence and Antimicrobial Resistance Pattern of Bacterial Strains Isolated from Patients with Urinary Tract Infection in Al-Wahda Hospital in Derna City/Libya

Background: Urinary tract infection (UTI) is One of the most prevalent infectious diseases in human, where the study aimed to identify the most uropathogen that caused UTIs ,and its the antimicrobial resistance pattern. Methods: the samples were collected from January and December 2021 using a standard data collection form, where the patient outcome records were mined for information on the patients' gender, age, isolated organisms, and susceptibility test results, then statistical analysis was done. Results: 266 from 463 samples were identified as S. aureus 14.3% , in addition, the antibacterial resistance rats showed that the highest resistance was against ampicillin/Sulbactam, ceftazidine, and meropenem 79%, while, the lowest was against ciprofloxacin 40%. Conclusion: The study showed that S. aureus is the primary cause of UTIs in Derna city, as a result of consuming antibiotics randomly, which resulted in an increase in spread and resistance of S. aureus.

Assessment of in vitro colistin susceptibility of carbapenem-resistant clinical Gram-negative bacterial isolates using four commercially available systems & Whole-genome sequencing: A diagnostic accuracy study

AimTo analyze the diagnostic utility of commercially available platforms and Whole-genome sequencing (WGS) for accurate determination of colistin susceptibility test results. Material & methodsAn exploratory diagnostic accuracy study was conducted in which sixty carbapenem-resistant Gram-negative bacteria were subjected to identification and AST using MALDI-TOF MS & MicroScan walkaway 96 Plus. Additional AST was performed using the BD Phoenix system and Mikrolatest colistin kit. The test isolates were subjected to Vitek-2 and WGS at CRL, Bengaluru. ResultsThere was no statistically significant agreement between the colistin susceptibility results obtained by WGS, with those of commercial phenotypic platforms. The MicroScan 96 Plus had the highest sensitivity (31 %) & NPV (77 %), and the BD Phoenix system had the highest specificity (97 %) and PPV (50 %), respectively, for determining colistin resistance. ConclusionThe utility of WGS as a tool in AMR surveillance and validation of phenotypic AST methods should be explored further.

Targeted elimination of Vancomycin resistance gene vanA by CRISPR-Cas9 system

ObjectiveThe purpose of this study is to reduce the spread of the vanA gene by curing the vanA-harboring plasmid of vancomycin-resistant using the CRISPR-Cas9 system.MethodsTwo specific spacer sequence (sgRNAs) specific was designed to target the vanA gene and cloned into plasmid CRISPR-Cas9. The role of the CRISPR-Cas system in the plasmid elimination of drug-resistance genes was verified by chemically transformation and conjugation delivery methods. Moreover, the elimination efficiency in strains was evaluated by plate counting, PCR, and quantitative real-time PCR (qPCR). Susceptibility testing was performed by broth microdilution assay and by Etest strips (bioMérieux, France) to detect changes in bacterial drug resistance phenotype after drug resistance plasmid clearance.ResultsIn the study, we constructed a specific prokaryotic CRISPR-Cas9 system plasmid targeting cleavage of the vanA gene. PCR and qPCR results indicated that recombinant pCas9-sgRNA plasmid can efficiently clear vanA-harboring plasmids. There was no significant correlation between sgRNA lengths and curing efficiency. In addition, the drug susceptibility test results showed that the bacterial resistance to vancomycin was significantly reduced after the vanA-containing drug-resistant plasmid was specifically cleaved by the CRISPR-Cas system. The CRISPR-Cas9 system can block the horizontal transfer of the conjugated plasmid pUC19-vanA.ConclusionIn conclusion, our study demonstrated that CRISPR-Cas9 achieved plasmid clearance and reduced antimicrobial resistance. The CRISPR-Cas9 system could block the horizontal transfer of plasmid carrying vanA. This strategy provided a great potential to counteract the ever-worsening spread of the vanA gene among bacterial pathogens and laid the foundation for subsequent research using the CRISPR-Cas9 system as adjuvant antibiotic therapy.

2810. A synergy test to assess the activity of the Ceftazidime-Avibactam and Aztreonam combination: An experience from a single centre in Malaysia

Abstract Background Southeast Asia has a high prevalence of metallo-β-lactamase-producing carbapenem-resistant Enterobacterales (CRE). Ceftazidime-avibactam (CZA) and Aztreonam (ATM) combination is one of the preferred therapeutic options against them. Methods A prospective study was performed at a university hospital in Kuala Lumpur, Malaysia, involving CRE isolates resistant to CZA and ATM. Susceptibility testing results were interpreted using the Clinical and Laboratory Standards Institute (CLSI) breakpoints. A synergy test for both agents [as in Falcone et al. study (CID 2021:72)] was carried out using the double disk synergy (DDS) and the gradient test superposition (GTS) methods. Synergy was considered present when the inhibition zone between the two disks became evident and if CZA reduced the ATM minimum inhibitory concentration (MIC) below its CLSI susceptibility cut-off (≤4 mg/L). Results Eleven isolates (8 K. pneumoniae, 2 E. coli, and 1 K. aerogenes) from 10 patients were tested. Concordant results were seen for both methods for all the isolates. Synergy was demonstrated with 8 isolates; for 2 of the 3 isolates with no synergy, the MIC of ATM reduced to an intermediate result after GTS. The most common sites of infection were bloodstream infection (4) and intra-abdominal infection (3). Five patients received the CZA-ATM combination (one received two courses of CZA-ATM). Five isolates from four patients demonstrated synergy, and one showed intermediate synergy. The latter received additional IV tigecycline. The median time to CZA-ATM initiation from the index culture was 8 (3-13) days. The median duration of therapy was 21 (6-28) days. Four patients responded favorably to the agents (survived the infection), while the remaining patient died. For those that did not receive CZA-ATM, one received polymyxin-based therapy, one received high-dose, extended-infusion meropenem, and one received no treatment. Only the patient that received meropenem survived the infection. Please refer to Table 1.0 for more detail. Conclusion Based on this small sample size, only a 73% synergy rate between CZA and ATM was seen for our CRE isolates resistant to CZA and ATM. Favorable treatment response was seen in those receiving the CZA-ATM combination therapy when synergy was demonstrated Disclosures All Authors: No reported disclosures

Phenotypic drug susceptibility characterization and clinical outcomes of tuberculosis strains with A-probe mutation by GeneXpert MTB/RIF

BackgroundGeneXpert MTB/RIF (Xpert) assay was applied widely to detect Mycobacterium tuberculosis (MTB) and rifampicin resistance.MethodsRetrospectively investigated the association among treatment histories, phenotypic drug susceptibility testing (pDST) results, and clinical outcomes of patients infected with probe A absent mutation isolate confirmed by Xpert.Results63 patients with only probe A absent mutation and 40 with additional pDST results were analyzed. 24 (60.0%) patients had molecular-phenotypic discordant rifampicin (RIF) susceptibility testing results, including 12 (12/13, 92.3%) new tuberculosis (TB) patients and 12 (12/27, 44.4%) retreated ones. 28 (28/39, 71.8%) retreated patients received first-line treatment regime within two years with failed outcomes. New patients had better treatment outcomes than retreated ones (successful: 83.3% VS. 53.8%; P value = 0.02). The clinical results of RIF-susceptible TB confirmed by pDST were not better than RIF-resistant TB (successful: 62.5% VS. 50.0%; P value = 0.43). INH-resistant TB and INH-susceptible TB had similar treatment outcomes too (successful: 61.5% VS. 50.0%; P value = 0.48). 11 (11/12, 91.7%) new patients treated with the short treatment regimen (STR) had successful outcomes.ConclusionsMore than half of mono probe A absent isolates had RIF molecular-phenotypic discordance results, especially in new patients. Probe A mutations were significantly associated with unsuccessful clinical outcomes, whether the pDST results were RIF susceptible or not. STR was the best choice for new patients.Trial registrationretrospectively registered in Wuhan Jinyintan Hospital (No. 2021-KY-16).

193. Standard of Care versus Alternative Beta-lactams for Treatment of Infections due to Serratia marcescens

Abstract Background The threat of inducible resistance and treatment failure positioned cefepime and carbapenems as the standard of care (SOC) treatment agents for infections due to AmpC-producing Enterobacterales regardless of antimicrobial susceptibility testing results. Recently published Infectious Diseases Society of America Guidance (IDSA) classifies the overall risk of clinically relevant AmpC expression for Serratia marcescens as low (< 5%). We aimed to compare the clinical outcomes of patients treated with a SOC antibiotic (carbapenem or cefepime) to those treated with an alternative beta-lactam agent for S. marcescens bloodstream infection (BSI). Methods This multisite, retrospective study included patients from five hospitals in the Atlanta-Metropolitan area with S. marcescens BSIs between January 1, 2018 and December 31, 2022. Patients were included if they received at least 72 hours of therapy with a beta-lactam antibiotic whose in vitro susceptibility was confirmed. Patients with polymicrobial bacteremia, those receiving combination therapy for > 72 hours, or those with a diagnosis of endovascular infection were excluded. We used a desirability of outcome ranking (DOOR) analysis to determine the probability of having a more desirable outcome receiving SOC antibiotics compared to receiving an alternative beta-lactam agent. The DOOR analysis includes counting undesirable events (Figure 1) and was performed using an online calculator (https://methods.bsc.gwu.edu).Figure 1:Primary endpoint - Desirability of outcome ranking. Abbreviations: AKI = acute kidney injury; CDI = Clostridioides difficile infection; EMR = electronic medical record; SCr = serum creatinine Results Of the 175 S. marcescens blood cultures reviewed, 66 patients met criteria for study inclusion, 43 in the SOC group and 23 in the alternative group. Baseline characteristics were comparable in both groups (Figure 2). The DOOR distribution between treatment groups was similar (Figure 3) and there was no significant difference in the probability of a more desirable outcome for patients who received an alternative beta-lactam compared to SOC (54.2% [95% CI, 40.3 - 67.5%]; Figure 4).Figure 2:Baseline patient characteristics. Abbreviations: BMT = bone marrow transplant within previous 12 months; Corticosteroid = ≥15 mg prednisolone daily equivalents; HIV = human immunodeficiency virus; ICU = intensive care unit; IQR = interquartile range; NS = non-significant; SOC = standard of care; SOT = any history of solid organ transplantFigure 3:Desirability of Outcome Ranking Distribution Summary Desirability of outcome ranking distribution for the standard of care and alternative beta-lactam treatment groups; SOC = standard of careFigure 4:Desirability of Outcome Ranking Probability Summary Forest plot of desirability of outcome ranking probabilities by overall treatment group and individual ranking components; CI = confidence interval, DOOR = desirability of outcome ranking, SOC = standard of care Conclusion The overall outcome of patients receiving alternative beta-lactam antibiotics was similar to those receiving SOC antibiotics in this study. These results support use of alternative beta-lactams for treatment of susceptible S. marcescens BSIs as recommended by IDSA guidance. Disclosures Joseph Torrisi, PharmD, BCIDP, Clinical Care Operations: Honoraria Ahmed Babiker, MBBS, Roche: Advisor/Consultant

Molecular Detection of Multidrug Resistance and Characterizations of Mutations in Mycobacterium Tuberculosis Using Polycarbonate Track-Etched Membrane Based DNA Bio-Chip.

With the widespread use of rifampicin (RMP) and isoniazid (INH), multidrug resistance (MDR) in Mycobacterium tuberculosis (M.tb) poses a threat to the success of tuberculosis (TB) control programs. We have developed a new polycarbonate track-etched membranes(PC-TEM) based DNA bio-chip designed for rapid detection of mutations conferring MDR inM.tbculture isolates. Bio-chips were designed to contain 14 specific probes for wild type and mutated allele of selected codons within 80bprifampicin resistance determining region of rpoB gene, katGgene andmabA-inhAregulatory region. RMP-resistance-associated gene mutation pointsrpoB 516, 526, 531and533, and the INH-resistance-associated gene mutation pointskatG315andinhA-15were targeted. Bio-chip signal was detected using enhanced chemiluminescence. A total of 50 culture isolates that were sensitive or resistant to RMP and/or INH were analyzed by bio-chip. The results of culture-based drug susceptibility testing (DST)were used as the gold standard and gene sequencing was performed to resolve the discordance. Amongst 50 culture isolates, we have detected 18 MDR, 9 RMP mono-resistant, 6 INH mono-resistant, and 17 fully susceptible isolates. The developed DNA bio-chip has a sensitivity of 90% for RMP and MDR and 100% for INH resistance. The bio-chip has a specificity of 100% for RMP and MDR and 88.8% for INH detection. The identification of mutations using the DNA bio-chip was 100% concordant with the sequencing data for the probes covered by the bio-chip. The detection ofrpoB, katGand inhA gene mutation points by a DNA bio-chip may be used as a rapid, accurate, and economical, clinical detection method for MDR detection in M.tb. This is very valuable for the control of TB epidemics.

Isolation and Identification of Microorganisms and Antibiotic Resistance Microorganisms from Beehives Located in Palm, Corn and Longan Plantations, Thailand.

This study aims to determine the prevalence of microorganisms and antibiotic-resistant microorganisms in beehives located on different plantations in Thailand. Seventeen swabs immersed in transport media were utilized for samples from different zones within beehives. Traditional microbial culture-based methods, biochemical tests, MALDI-TOF MS (VITEK® MS, bioMerieux, Marcy-l'Étoile, France), and antibiotic drug susceptibility (disk-diffusion) tests were used to detect microorganism and antimicrobial resistance bacteria. The results from 16 beehive swabs found Gram-positive bacteria at 59.5%, Gram-negative bacteria at 35.1%, and fungi (yeast) at 5.4%. These organisms are classified as 11, 11, and 2 types of Gram-positive bacteria, Gram-negative bacteria, and fungi (yeast), respectively. Furthermore, no organism showed resistance to vancomycin or cefoxitin for antibiotic drug susceptibility testing. In contrast, all Acinetobacter spp. were susceptible to ciprofloxacin, levofloxacin, ceftazidime, cefotaxime, imipenem, and meropenem, except for Acinetobacter schindleri, which was resistant to ceftazidime and cefotaxime. For other organisms, due to the limitations of tests to identify some environmental microbial species, the antimicrobial susceptibility test results cannot be interpreted as resistant or susceptible to the drug for these organisms. The study's findings will support prevention, healthcare services, and public health systems.

Antimicrobial resistance of E. coli strains in ready-to-eat red meat products in Nakuru County, Kenya

The demand for Ready-To-Eat (RTE) meat products is rising especially to the busy urban dwellers. Handling, processing, and selling methods of RTE contaminate them with pathogens. However, there is lack of evidence of the risk of antimicrobial resistance (AMR) of the pathogens. In this study, 105 RTE meat products were sampled across Nakuru County and assessed for Escherichia coli load, antimicrobial resistance (AMR), and presence of AMR genes. All the sampled meat products were contaminated with coliforms and E. coli but levels differed significantly (p < 0.05) with location of sample collection and type of RTE. Antimicrobial susceptibility test results of 30 E. coli isolates indicated high prevalence of the isolates’ resistance to co-trimoxazole, tetracycline, streptomycin, and ampicillin at 80%, 73%, 67%, and 67%, respectively. Antibiotic susceptibility of the isolates was highest towards ciprofloxacin and amikacin at 100% and 80%, respectively. All the 30 E. coli isolates showed 14 different AMR patterns, where each isolate was resistant to at least 2 antibiotics; a multiple antibiotic resistance (MAR) index of greater than 0.2. Meat samples collected from one sampling location, Salgaa had the highest number of multi-drug resistant E. coli isolates (88.9%). Molecular identification of AMR genes in four MAR isolates showed that tetA (for tetracycline resistance) and sul1 (for co-trimoxazole resistance) genes were present in all (100.0%) the isolates, while blaTEM (for ampicillin resistance) and strA (for streptomycin) genes were both present in two isolates. The four isolates were not E. coli O157 as confirmed by PCR for the O157 lipopolysaccharide O-antigen synthesis gene rfbE-O157. This study demonstrated the presence of MAR E. coli in RTE red meat products sold in Nakuru County, which is a human and animal public health concern, hence a need to urgently regulate and monitor the use of antimicrobials in animals.

Fungal diversity and drug susceptibility of the oral mycobiome of domestic dogs.

The purpose of this study was to characterize the variety and diversity of the oral mycobiome of domestic dogs and to identify the commensal and potentially pathogenic fungi present. Two hundred fifty-one buccal swabs from domestic dogs were obtained and struck onto a chromogenic fungal growth medium that distinguishes between fungal species based on colony color and morphology. After isolating and harvesting single colonies, genomic DNA was extracted from pure cultures. PCR was used to amplify a fungal-specific variable rDNA region of the genome, which was then sent for sequencing. Sequencing results were input into the NCBI BLAST database to identify individual components of the oral mycobiome of tested dogs. Of the 251 dogs swabbed, 73 had cultivable fungi present and 10 dogs had multiple fungal species isolated. Although the dogs did not show signs of oral infections at the time, we did find fungal species that cause pathogenicity in animals and humans. Among fungal isolates, Malassezia pachydermatis and species from the genus Candida were predominant. Following fungal isolate identification, antifungal drug susceptibility tests were performed on each isolate toward the medically important antifungal drugs including fluconazole, ketoconazole, and terbinafine. Drug susceptibility test results indicated that a large number of isolates had high MIC values for all three drugs. Exploring the oral mycobiome of dogs, as well as the corresponding drug susceptibility profiles, can have important implications for canine dental hygiene, health, and medical treatment. Identifying the microorganisms within the canine mouth can illustrate a common pathway for fungal pathogens of One Health concern to spread from our canine companions to humans.

The Impact of Antimicrobial Stewardship in Treating Patients with Escherichia coli Bacteremia in a Small Single Center Community Hospital.

Purpose: There are many challenges that pharmacist led antimicrobial stewardship programs can encounter including lack of resources, costs, and inaccurate antimicrobial susceptibility testing (AST) results. The COVID-19 pandemic has led to increased resistance especially with gram negative infections. At a small single center community hospital, gram negative infections, particularly Escherichia coli infections, predominately occur. Therefore, this study aims to address gram negative bacteremia burden and its impact on antimicrobial stewardship efforts for combatting Escherichia coli and ESBL organisms with such barriers during the pandemic. Methods: In a retrospective cohort study, patients ≥ 18 years of age from a small community hospital were reviewed. Patients were excluded if their blood cultures were not positive for Escherichia coli and if antibiotics were not initiated during hospital stay. The primary endpoint was to determine the length of hospital stay. Critical secondary endpoints include antibiotic de-escalations, duration of antibiotics, time to definitive antibiotic therapy, serum procalcitonin levels, blood culture availabilities, MIC breakpoints, co-infection of COVID-19, and Clostridioides difficile occurrences. Results: Out of 74 patients with gram negative bacteremia, 41 patients specifically had Escherichia coli bacteremia. The primary endpoint results showed that patients with Escherichia coli bacteremia that stayed in the ICU had a length of stay of 13.6 days. Patients with Escherichia coli bacteremia in the Non-ICU setting has a length of stay of 7.3 days, and patients with ESBL bacteremia in the Non-ICU setting had a length of stay of 6.8 days. Conclusions: Despite the various challenges that antimicrobial stewardship programs (ASP) face in a single center small community hospital, the ASP at this small community hospital utilizes various policies and tools to increase appropriate antibiotic use and decrease hospital length of stay in patients with Escherichia coli bacteremia.

Impact of Prior Tuberculosis Treatment With New/Companion Drugs on Clinical Outcomes in Patients Receiving Concomitant Bedaquiline and Delamanid for Multidrug- and Rifampicin-Resistant Tuberculosis.

There are scarce data on the clinical outcomes of persons retreated with new/companion anti-tuberculosis (TB) drugs for multidrug- and rifampicin-resistant tuberculosis (MDR/RR-TB). We sought to evaluate the efficacy and safety of bedaquiline and delamanid containing regimens among patients with and without prior exposure to the new/companion drugs (bedaquiline, delamanid, linezolid, clofazimine, and fluoroquinolones). We conducted a retrospective cohort study among patients with pulmonary MDR/RR-TB in Georgia who received bedaquiline and delamanid combination as a part of a salvage regimen from November 2017 to December 2020 in a programmatic setting. Among 106 persons with a median age of 39.5 years, 44 (41.5%) were previously treated with new/companion TB drugs. Patients with prior exposure to new/companion drugs had higher rates of baseline resistance compared to those without exposure to new/companion TB drugs (bedaquiline 15.2% vs 1.8%, linezolid 22.2% vs 16.7%). Sputum culture conversion rates among patients exposed and not exposed to new/companion drugs were 65.9% vs 98.0%, respectively (P < .001). Among patients with and without prior new/companion TB drug use, favorable outcome rates were 41.0% and 82.3%, respectively (P < .001). Treatment adherence in 32 (30.2%) patients was ≤80%. Five of 21 patients (23.8%) who had a baseline and repeat susceptibility test had acquired bedaquiline resistance. QTC/F prolongation (>500 ms) was rare (2.8%). Prior exposure to new/companion TB drugs was associated with poor clinical outcomes and acquired drug resistance. Tailoring the TB regimen to each patient's drug susceptibility test results and burden of disease and enhancing adherence support may improve outcomes.

Clinical and etiological characteristics of infectious vulvovaginitis in children in Zhejiang province from 2009 to 2019

Objective: To explore the clinical characteristics, common pathogens in children with vulvovaginitis. Methods: This was a retrospective cases study. A total of 3 268 children with vulvovaginitis were enrolled, who visited the Department of Pediatric and Adolescent Gynecology, Children's Hospital, Zhejiang University School of Medicine from January 2009 to December 2019. Patients were divided into 3 groups according to the age of <7, 7-<10 and 10-18 years. Patients were also divided in to 4 groups according to the season of first visit. The pathogen distribution characteristics of infective vulvovaginitis were compared between the groups. Their clinical data were collected and then analyzed by χ2 test. Results: The were 3 268 girls aged (6.2±2.5) years. There were 1 728 cases (52.9%) aged <7 years, 875 cases (26.8%) aged 7-<10 years, and 665 cases (20.3%) aged 10-18 years. Of these cases, 2 253 cases (68.9%) were bacterial vulvovaginitis, 715 cases (21.9%) were fungal vulvovaginitis and 300 cases (9.2%) were vulvovaginitis infected with other pathogens. Bacterial culture of vaginal secretions was performed in 2 287 cases, and 2 287 strains (70.0%) of pathogens were detected, of which the top 5 pathogens were Streptococcus pyogenes (745 strains, 32.6%), Haemophilus influenzae (717 strains, 31.4%), Escherichia coli (292 strains, 12.8%), Staphylococcus aureus (222 strains, 9.7%) and Klebsiella pneumoniae (67 strains, 2.9%). Regarding different age groups, H.influenzae was the most common in children under 7 years of age (40.3%, 509/1 263), S.pyogenes (41.9%, 356/849) was predominantly in children aged 7 to 10 years, and E.coli was predominant in children aged 10 to 18 years (26.3%, 46/175). Susceptibility results showed that S.pyogenes was susceptible to penicillin G (610/610, 100.0%), ceftriaxone (525/525, 100.0%), and vancomycin (610/610, 100.0%); the resistance rates to erythromycin and clindamycin were 91.9% (501/545)and 90.7% (495/546), respectively. For H.influenzae, 32.5% (161/496) produced β-elactamase, and all strains were sensitive to meropenem (489/489, 100.0%) and levofloxacin (388/388, 100.0%), while 40.5% (202/499) were resistant to ampicillin. Among E.coli, all strains were sensitive to imipenem(100%, 175/175). The resistance rates of E.coli to levofloxacin and ceftriaxone were 29.1% (43/148) and 35.1% (59/168), respectively. A total of 48 strains of methicillin-resistant Staphylococcus aureus (MRSA) were isolated with a proportion of 28.3% (45/159) in 3 268 patients. The results of drug susceptibility test showed that all MRSA strains were sensitive to linezolid 100.0% (40/40), vancomycin (45/45, 100.0%), and tigecycline (36/36, 100.0%); the resistance rates of MRSA to penicillin G, erythromycin and clindamycin were 100% (45/45), 95.6% (43/45) and 88.9% (40/45), respectively. All methicillin-sensitive Staphylococcus aureus (MSSA) strains were sensitive to oxacillin (114/114, 100.0%), linezolid (94/94, 100.0%), vancomycin (114/114, 100.0%), and tigecycline (84/84, 100.0%); it's resistance rates to penicillin G, erythromycin and clindamycin were 78.1% (89/114), 59.7% (68/114) and 46.5% (53/114), respectively. The drug resistance rate of MSSA to penicillin G, erythromycin and clindamycin were lower than those of MRSA (χ²=11.71,19.74,23.95, respectively, all P<0.001). Conclusions: The age of consultation for pediatric infectious vulvovaginitis is mainly around 6 years. The most common pathogens are S.pyogenes, H.influenzae and Escherichia coli. Third generation cephalosporins can be used as the first choice of empirical anti-infection drugs. However, the results of drug susceptibility should be considered for targeted treatment.

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.

Elimination of blaKPC−2-mediated carbapenem resistance in Escherichia coli by CRISPR-Cas9 system

ObjectiveThe purpose of this study is to re-sensitive bacteria to carbapenemases and reduce the transmission of the blaKPC−2 gene by curing the blaKPC−2-harboring plasmid of carbapenem-resistant using the CRISPR-Cas9 system.MethodsThe single guide RNA (sgRNA) specifically targeted to the blaKPC−2 gene was designed and cloned into plasmid pCas9. The recombinant plasmid pCas9-sgRNA(blaKPC−2) was transformed into Escherichia coli (E.coli) carrying pET24-blaKPC−2. The elimination efficiency in strains was evaluated by polymerase chain reaction (PCR) and quantitative real-time PCR (qPCR). Susceptibility testing was performed by broth microdilution assay and by E-test strips (bioMérieux, France) to detect changes in bacterial drug resistance phenotype after drug resistance plasmid clearance.ResultsIn the present study, we constructed a specific prokaryotic CRISPR-Cas9 system plasmid targeting cleavage of the blaKPC−2 gene. PCR and qPCR results indicated that prokaryotic CRISPR-Cas9 plasmid transforming drug-resistant bacteria can efficiently clear blaKPC−2-harboring plasmids. In addition, the drug susceptibility test results showed that the bacterial resistance to imipenem was significantly reduced and allowed the resistant model bacteria to restore susceptibility to antibiotics after the blaKPC−2-containing drug-resistant plasmid was specifically cleaved by the CRISPR-Cas system.ConclusionIn conclusion, our study demonstrated that the one plasmid-mediated CRISPR-Cas9 system can be used as a novel tool to remove resistance plasmids and re-sensitize the recipient bacteria to antibiotics. This strategy provided a great potential to counteract the ever-worsening spread of the blaKPC−2 gene among bacterial pathogens and laid the foundation for subsequent research using the CRISPR-Cas9 system as adjuvant antibiotic therapy.

Application of Precision Medicine Concepts in Ambulatory Antibiotic Management of Acute Pyelonephritis.

Acute pyelonephritis (APN) is a relatively common community-acquired infection, particularly in women. The early appropriate antibiotic treatment of this potentially life-threatening infection is associated with improved outcomes. The international management guidelines for complicated urinary tract infections and APN recommend using oral antibiotics with <10% resistance among urinary pathogens. However, increasing antibiotic resistance rates among Escherichia coli and other Enterobacterales to fluoroquinolones, trimethoprim-sulfamethoxazole (TMP-SMX), and beta-lactams has left patients without reliable oral antibiotic treatment options for APN. This narrative review proposes using precision medicine concepts to improve empirical antibiotic therapy for APN in ambulatory settings. Whereas resistance rates to a particular antibiotic class may exceed 10% at the population-based level, the predicted antibiotic resistance rates based on patient-specific risk factors fall under 10% in many patients with APN on the individual level. The utilization of clinical tools for the prediction of fluoroquinolones, TMP-SMX, and third-generation cephalosporin resistance improves the ambulatory antibiotic management of APN. It may also reduce the need to switch antibiotic therapy later based on the in vitro antibiotic susceptibility testing results of bacterial isolates in urinary cultures. This approach may mitigate the burden of increasing antibiotic resistance in the community by ensuring that the initial antibiotic prescribed has the highest likelihood of treating APN appropriately.