Ampicillin Susceptibility Research Articles

P-1349. Multi-hospital Dissemination of Ampicillin Resistance Associated with a Frame-shift Deletion in pbp4 of Vancomycin-Resistant Enterococcus faecalis (VREfs)

Abstract Background Penicillin (PEN) and ampicillin (AMP) resistance is rare in E. faecalis, and may arise via increased expression of penicillin-binding protein 4 (PBP4), mutations affecting PBP4’s affinity to β-lactams, and rarely β-lactamase production. We recently identified 23 vanA vancomycin-resistant E. faecalis (VREfs), recovered from 19 patients in 4 hospitals in Chile (2020-2023) that exhibited PEN resistance and unusually elevated AMP MICs (4-16 µg/mL). Here, we characterize the molecular basis of the reduced β-lactam susceptibility in these VREfs isolates.Table 1.AMP susceptibility after inducible expression of pbp4SCL10298 in the pbp4-defective and AMP hypersusceptible strain JH2-2Δpbp4 Methods We analyzed 23 VREfs including sequence type (ST), resistome, PBP sequences and pbp4 promoter. PBP4 production was assessed by ELISA and Western blot using goat anti-rPBP4 sera, in the Chilean VREfs isolate SCL10298 and using pbp4-defective strains (JH2-2Δpbp4 and OG117Δpbp4). We recreated the pbp4 changes of SCL10298 by allelic replacement of E. faecalis OG117 (OG117 Δpbp4SCL10298) using CRISPR-Cas9. Expression of pbp4SCL10298 was also assessed in-trans in a JH2-2 background (JH2-2Δpbp4) using a nisin-inducing vector (pMSP3535).Figure 1.Schematic representation of predicted open reading frames (ORFs) on the JH2-2 and SCL10298 Black line represents the nucleotide sequence for the PBP4 promoter region, followed by the predicted ORFs indicated with grey arrows. Text in red represents the amino acid sequence sharing 100% identity between JH2-2 ORF1 and SCL10298 ORF1-p1 and ORF1-p2. Results All 23 isolates belonged to ST1518 (an ST6 single-locus variant). No β-lactamase-encoding genes or changes in pbppromoter regions were detected. A frameshift deletion of 100 nt was observed in pbp4 in all isolates, resulting in 2 open reading frames, including a potential transcript of a shorter pbp4 (ORF1-p1, fig1). All the other PBP sequences were identical to those of E. faecalis JH2-2. PBP4 was detected in SCL10298 by whole-cell ELISA. Further, western blots revealed no differences in PBP4 amounts between SCL10298 and control strains. Allelic replacement of OG117Δ did not result in decreased AMP susceptibility as compared to the parental OG117 strain (AMP MIC 0.5 µg/mL) nor did the inducible expression of the truncated version of pbp4 in JH2-2Δpbp4 (0.19 µg/ml, table1). Conclusion We document the emergence of a new lineage of VREfs in Chile with alteration in PBP4 that exhibits decreased susceptibility to B-lactams (akin E. faecium). Our molecular experiments suggest that an alteration of PBP4 is necessary, but not sufficient to increase the MICs of AMP. Therefore, we propose the strain background might be key for the full expression of the B-lactam-resistant phenotype. Disclosures William R. Miller, M.D., Merck: Grant/Research Support|UptoDate: Royalties José M Munita, MD, MSD: Grant/Research Support|Pfizer: Grant/Research Support

Diverse genomic and epidemiological landscapes of redundant pbp5 genes in Enterococcus spp.: Insights into plasmid mobilization, ampicillin susceptibility, and environmental interactions.

Genetic redundancy in bacteria plays a crucial role in enhancing adaptability and accelerating evolution in response to selective pressures, particularly those associated with rapid environmental changes. Aminopenicillins like ampicillin are important therapeutic options for Enterococcus infections in both humans and animals, with resistance mostly associated with pbp5 gene mutations or overexpression. While the occurrence of redundant pbp5 genes has been occasionally reported, the advantages for the host bacteria have not been explored in detail. During a whole-genome sequencing project of Enterococcus faecium from bacteremic patients, we identified an ST592 strain (Efm57) with redundant pbp5 genes. This presented an opportunity to investigate the prevalence and implications of multiple pbp5 acquisitions in diverse Enterococcus species across various sources, geographical regions, and timeframes. The analysis of 618 complete Enterococcus genomes from public databases revealed that 3.2% harbored redundant pbp5 genes, located on chromosomes or plasmids across different species from diverse epidemiological backgrounds. The proteins encoded by these genes showed homologies ranging from 51.1% to 97.5% compared to native copies. Phylogenetic analysis grouped redundant PBP5 amino acid sequences into three distinct clades, with insertion sequences (mostly IS6-like) facilitating their recent spread to diverse plasmids with varying genetic backbones. The presence of multiple antibiotic resistance genes on pbp5-plasmids, including those conferring resistance to linezolid, underscores their involvement in co-selection and recombination events with other clinically-relevant antibiotics. Conjugation experiments confirmed the transferability of a specific 24kb pbp5-plasmid from the Efm57 strain. This plasmid was associated with higher minimum inhibitory concentrations of ampicillin and conferred bacteria growth advantages at 22°C. In conclusion, the widespread distribution of redundant pbp5 genes among Enterococcus spp. highlights the complex interplay between genetic mobility, environmental factors, and multidrug resistance in overlapping ecosystems emphasizing the importance of understanding these dynamics to mitigate antibiotic resistance spread within the One Health framework.

Severe co-infection caused by difficult-to-diagnose hypermucoviscous Klebsiella pneumoniae K1-ST82 in a patient with COVID-19: a case report

BackgroundCo-infection with Klebsiella pneumoniae presents a significant concern in hospitalized patients with coronavirus disease (COVID-19), increasing the risk of severe disease progression. Hypervirulent (hv) and hypermucoviscous (hm) K. pneumoniae (Kp) has gained prominence in Asia due to its capacity to cause invasive community-acquired infections. However, recognition of hvKp/hmKp co-infections in the context of COVID-19 remains limited. We report a severe case of rapidly progressing co-infection with hmKp exhibiting “difficult-to-diagnose” phenotypes in a hospitalized patient with COVID-19.Case presentationA 61-year-old woman with COVID-19 initially exhibited mild symptoms resembling the common cold. However, her condition rapidly deteriorated over 7 days, leading to hospital admission with the development of dyspnea. The patient required supplemental oxygen, antibiotic treatment, and mechanical ventilation. Gram-negative bacteria with atypical phenotypes were isolated from alveolar lavage fluid and blood cultures. Both strains formed small, glossy, non-lactose-fermenting colonies on clinically relevant media and were susceptible to ampicillin. Conventional biochemical tests failed to identify the Enterobacteriales strains owing to the urease-negative phenotype. Consequently, the identification of K. pneumoniae was difficult until matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) analysis was performed. A positive string test indicated mucoviscosity, but with variability in the material used for stretching colonies. Whole-genome sequencing performed on the MiSeq and GridION platforms revealed the blood-derived strain JARB-RN-0063 as belonging to serotype K1 and sequence type (ST) 82. The hvKp-associated genes rmpA and iroCD were located on a 5.0-Mb chromosome, and iucABCD-iutA was identified on a 217.9-kb IncFIB(K)/IncR-type plasmid. Therefore, JARB-RN-0063 was genetically classified as hvKp with a Kleborate virulence score of 3. The intrinsic penicillinase gene blaSHV was defective owing to an IS1F element insertion, resulting in the strain being atypically susceptible to ampicillin.ConclusionsThis is the first case of severe COVID-19-associated co-infection with a difficult-to-diagnose K. penummoniae strain. Notably, co-infection by the hmKp K1-ST82 clone exhibited atypical phenotypes, including stunted growth, non-lactose fermentation, urease-negative reaction, ampicillin susceptibility, and abnormal mucoviscosity, posing diagnostic challenges for clinical laboratories and impedes the identification of hvKp/hmKp. Delayed identification may worsen patient outcomes, highlighting the need for increased clinical awareness of such difficult-to-diagnose clones to prevent deterioration.

Isolation and Preliminary Characterization of Probiotic Isolates from Healthy Thai Adult Feces

Probiotics are known for health-promoting microorganisms, for examples, assisting gastrointestinal digestion and short chain fatty acid producers. Several current oral human probiotics were derived from human samples; yet, of various ethnic (genetics) and dietary patterns. As probiotics from similar ethnic (genetics) and dietary pattern might confer certain advantages (e.g., more compatibility with hosts), this study isolated and characterized probiotics from healthy Thai adults. To screen for potential probiotic isolates from healthy Thai adult feces, and preliminary characterize these probiotic isolates by biochemical tests and 16S rRNA gene sequencing, following the established Thailand’s Food and Drug Administration (FDA) and National Center for Genetic Engineering and Biotechnology (BIOTEC) guidelines. Twenty fecal samples with clinically verified healthy Thai males and females aged 22-42 years were cultivated on MRS (or M17) media along the broad probiotic screenings (pH 4.0 and 0.1% oxgall) for bacterial probiotic propagation. All derived colonies of different morphologies were continued colony PCR using universal probiotic primers (specific for genera Bifidobacterium, Enterococcus, Lactobacillus, Pediococcus and Streptococcus thermophilus), and only PCR-positive colonies from any of these probiotic primer pair were further cultivation for isolates. Then, these isolates were screened and characterized general probiotic properties that included acid tolerance (pH 2.0), bile salt tolerance (0.3% bile salt), hydrophobicity, antibiotic (ampicillin, ciprofloxacin, erythromycin and vancomycin) susceptibility, no hemolysis activity, catalase, and potassium hydroxide (KOH). The final passing isolates represented potential probiotic isolates that could perhaps maintain in gastrointestinal tract with safety, and were full-length 16S rRNA gene sequenced to identify the genus and species by BLASTN against non-redundant GenBank database. A total of 295 bacterial isolates were obtained from the cultivated on MRS (or M17) media along the broad probiotic screenings from 20 fecal samples. Then, following the general probiotic property characterizations and the full-length 16S rRNA gene sequence analysis, 7 potential isolates were finally derived: 2 Bifidobacterium animalis subsp. lactis, 1 Lactobacillus (new genus name as Limosilactobacillus) reuteri, 1 L. reuteri or L. vaginalis, and 3 Pediococcus pentosaceus. These isolates exhibited strong acid and bile salt tolerances, and relatively high cell surface hydrophobicity. These isolates were susceptible to ampicillin and erythromycin, similar to a reference probiotic control Lactobacillus casei strain Shirota; and the CU13-450 and CU13-451 were also susceptible to vancomycin, similar to a reference control Bifidobacterium breve. Catalase-negative consistent with these bacteria in intestinal tract as anaerobes or facultative anaerobes, and KOH-negative result inferred the Gram-positive bacteria consistent with the full-length 16S rRNA gene sequence identification (Bifidobacterium, Lactobacillus, and Pediococcus are Gram-positive bacteria). No hemolysis activity was observed for these isolates. These 7 probiotic isolates had general probiotic properties and with confirmed the 16S rRNA gene sequences, that follow the Thailand’s FDA and BIOTEC guidelines. However, additional beneficial function investigations, such as in vitro epithelial cell adhesion and in vivo animal model, and whole genome sequencing, should be performed to better validate their possible human health benefits and safety.

Correlation between the development of phage resistance and the original antibiotic resistance of host bacteria under the co-exposure of antibiotic and bacteriophage

Bacteriophages (phages) are viruses capable of regulating the proliferation of antibiotic resistant bacteria (ARB). However, phages that directly cause host lethality may quickly select for phage resistant bacteria, and the co-evolutionary trade-offs under varying environmental conditions, including the presence of antibiotics, remains unclear as to their impact on phage and antibiotic resistance. Here, we report the emergence of phage resistance in three distinct E. coli strains with varying resistance to β-lactam antibiotics, treated with different ampicillin (AMP) concentrations. Hosts exhibiting stronger antibiotic resistance demonstrated a higher propensity to develop and maintain stable phage resistance. When exposed to polyvalent phage KNT-1, the growth of AMP-sensitive E. coli K12 was nearly suppressed within 18 h, while the exponential growth of AMP-resistant E. coli TEM and super-resistant E. coli NDM-1 was delayed by 12 h and 8 h, respectively. The mutation frequency and mutated colony count of E. coli NDM-1 were almost unaffected by co-existing AMP, whereas for E. coli TEM and K12, these metrics significantly decreased with increasing AMP concentration from 8 to 50 μg/mL, becoming unquantifiable at 100 μg/mL. Furthermore, the fitness costs of phage resistance mutation and its impact on initial antibiotic resistance in bacteria were further examined, through analyzing AMP susceptibility, biofilm formation and EPS secretion of the isolated phage resistant mutants. The results indicated that acquiring phage resistance could decrease antibiotic resistance, particularly for hosts lacking strong antibiotic resistance. The ability of mutants to form biofilm contributes to antibiotic resistance, but the correlation is not entirely positive, while the secretion of extracellular polymeric substance (EPS), especially the protein content, plays a crucial role in protecting the bacteria from both antibiotic and phage exposure. This study explores phage resistance development in hosts with different antibiotic resistance and helps to understand the limitations and possible solutions of phage-based technologies.

Characterization of Antibiotic Resistance in Select Tertiary Hospitals in Uganda: An Evaluation of 2020 to 2023 Routine Surveillance Data.

Antimicrobial resistance (AMR) is a public health concern in Uganda. We sought to conduct an extended profiling of AMR burden at selected Ugandan tertiary hospitals. We analyzed routine surveillance data collected between October 2020 and March 2023 from 10 tertiary hospitals. The analysis was stratified according to the hospital unit, age, gender, specimen type, and time. Up to 2754 isolates were recovered, primarily from pus: 1443 (52.4%); urine: 1035 (37.6%); and blood: 245 (8.9%). Most pathogens were Staphylococcus aureus, 1020 (37%), Escherichia coli, 808 (29.3%), and Klebsiella spp., 200 (7.3%). Only 28% of Escherichia coli and 42% of the other Enterobacterales were susceptible to ceftriaxone, while only 44% of Staphylococcus aureus were susceptible to methicillin (56% were MRSA). Enterococcus spp. susceptibility to vancomycin was 72%. The 5-24-year-old had 8% lower ampicillin susceptibility than the >65-year-old, while the 25-44-year-old had 8% lower ciprofloxacin susceptibility than the >65-year-old. The 0-4-year-old had 8% higher ciprofloxacin susceptibility. Only erythromycin susceptibility varied by sex, being higher in males. Escherichia coli ciprofloxacin susceptibility in blood (57%) was higher than in urine (39%) or pus (28%), as was ceftriaxone susceptibility in blood (44%) versus urine (34%) or pus (14%). Klebsiella spp. susceptibility to ciprofloxacin and meropenem decreased by 55% and 47%, respectively, during the evaluation period. During the same period, Escherichia coli ciprofloxacin susceptibility decreased by 40%, while Staphylococcus aureus gentamicin susceptibility decreased by 37%. Resistance was high across the Access and Watch antibiotic categories, varying with time, age, sex, specimen type, and hospital unit. Effective antimicrobial stewardship targeted at the critical AMR drivers is urgently needed.

Ampicillin susceptibility testing of Haemophilus influenzae in the routine clinical laboratory by the EUCAST methodology compared to broth microdilution and the presence of ftsI gene mutations

ObjectivesTo investigate the prevalence of ampicillin resistance in Haemophilus influenzae and the diagnostic accuracy of the EUCAST recommended disc diffusion method to detect the increasingly prevalent ampicillin resistance due to the presence of PBP3 alterations based on mutations in the ftsI gene. MethodsDuring a 6-month period all consecutive non-duplicate H. influenzae isolates were prospectively collected and stored. MICs of ampicillin were determined by broth microdilution (BMD). PCR was performed to detect mutations in the ftsI gene. Results of routine disc diffusion susceptibility testing, including the penicillin screening test in accordance with the current EUCAST methodology, as well as additional Etest results, were compared to the BMD as the reference method. ResultsIn 102 isolates, the prevalence of ampicillin resistance was 28% (29/102) by BMD. There was a good correlation between MICs of ampicillin and the presence of a β-lactamase and/or an ftsI gene mutation. The prevalence of ampicillin resistance was overestimated using the EUCAST method (33% (34/102)) and underestimated when an additional Etest was used (24% (24/102)) (not significant). The sensitivity and specificity of the EUCAST methodology for the detection of ampicillin resistance were 97% ((28/29); 95% CI, 82–100%) and 92% ((67/73); 95% CI, 83–97%), respectively. ConclusionsThe prevalence of ampicillin resistance was 28%, as determined by BMD. Although the overall diagnostic accuracy of the EUCAST ampicillin disc diffusion was high, misclassification of ampicillin susceptibility may still occur.

Ciprofloxacin and Azithromycin Susceptibility in Salmonella Causing Bloodstream Infection in a Tertiary Care Hospital in South India

Enteric fever is a major health concern worldwide, especially in tropical countries. Enteric fever caused by Salmonella enterica is a gastrointestinal tract disease to begin with and later involving many systems,. After the advent of multidrug resistant Salmonellae, ciprofloxacin became the focus of its treatment. Azithromycin is widely being used for the prevention and treatment of Enteric fever. Of late, there are reports in literature to support that Salmonella have regained susceptibility to Ampicillin, Cotrimaxazole and Chloramphenicol. Therefore, this study undertaken aims to find out the antimicrobial susceptibility of Salmonella isolates to conventional drugs like Ampicillin, Cotrimaxazole and Chloramphenicol and find out susceptibility pattern of Ciprofloxacin that is over used for treatment of enteric fever and Azithromycin, most often used in children. As there is no disc diffusion method as per CLSI 2019 guidelines to detect the susceptibility to ciprofloxacin, ciprofloxacin resistance was determined by minimum inhibitory concentration(MIC)by E-test.Kirby-Bauer disk diffusion technique was used to monitor Azithromycin resistance among Salmonella entericaserovarTyphi as per CLSI 2019 guidelines. METHODS:The Prospective study was conducted over a period of 8 months (May 2019- December 2019) with the clinical samples yielding the growth of Salmonella from blood. These strains were screened for Ciprofloxacin susceptibility by E-test. Azithromycin,Ampicillin, Co-Trimaxazole and Chloramphenicol susceptibility were detected by Kirby-Bauer disc diffusion technique. RESULTS: Of the 80 isolates that were screened, 52 (65%) were resistant, 24 (30%) were intermediate susceptible and remaining 4(5%) were sensitive to ciprofloxacin by MIC detection. 69 (86.25%) of the isolates were sensitive and 11 (13.75%) were resistant to Azithromycin by disc diffusion technique. All isolates (100%)were found to be susceptible to Ampicillin, Co-Trimaxazole and Chloramphenicol CONCLUSION:The current study shows a high prevalence of resistance to ciprofloxacin in Salmonella species isolated in and around Mysuru district in southern India. Detection of ciprofloxacin resistance by the Kirby Bauer disk diffusion method is less effective, so it is advised that MIC of ciprofloxacin must be regularly done in the diagnostic laboratories for all strains. The findings from our study also showed low level resistance (13.75%) to Azithromycin. It is advised that Azithromycin can be used as a therapeutic agent only if it is reported to be sensitive by the susceptibility test. Conventional agents like Ampicillin, Co-Trimaxazole and Chloramphenicol can be reconsidered for treatment of Enteric fever.

184. Implications of Enterococcus faecalis penicillin susceptibility in patients with bacteremia

Abstract Background Enterococcus faecalis isolates with an elevated penicillin minimum inhibitory concentration (MIC) ≥ 4 mg/L have decreased in vitro ampicillin plus ceftriaxone bactericidal and synergistic activity, despite ampicillin susceptibility. This phenotype’s implication on patient outcomes and prevalence in the US is unknown due to limited penicillin susceptibility reporting. Methods This was a retrospective cohort study of adult patients with E. faecalis bacteremia between August 20, 2018 and May 13, 2021 from the Mount Sinai Health System in New York City. Patients were excluded if they were discharged or deceased within 48 hours of index blood culture, did not receive active definitive therapy, or the blood culture was determined to be contamination. The primary outcome was differences in 30-day and 90-day mortality based on the penicillin MIC and the secondary outcome was microbiological relapse. Multivariable logistic regression was used to identify risk factors for 90-day mortality. Corresponding blood isolates were tested for penicillin susceptibility by broth microdilution in accordance with CLSI. Results A total of 124 patients were included, with 27% having an elevated penicillin MIC ≥ 4 mg/L. Differences in baseline characteristics (Table 1) as well as source of infection and treatments received (Table 2) were similar between groups. Endocarditis infected 18.5% of patients and was less common in cases with an elevated penicillin MIC. Thirty-day and 90-day mortality, and microbiological relapse were higher in cases with an elevated penicillin MIC (14.4% vs. 20.6%, p=0.58; 20% vs. 26.5%, p=0.59; and 4.4% vs. 8.8%, p=0.39, respectively). Pitt bacteremia score and prior ceftriaxone usage had a higher odds of 90-day mortality (OR 1.2 [95% CI 1.03-1.4], p=0.02; OR 2.8 [95% CI 1.02-7.9], p=0.04, respectively), whereas definitive treatment with an aminopenicillin compared to piperacillin/tazobactam was protective of 90-day mortality (OR 0.21 [95% CI 0.05-0.93], p=0.04). Conclusion E. faecalis with an elevated penicillin MIC ≥ 4 mg/L is present in over a quarter of patients across New York City. Larger studies are warranted to determine the impact on patient outcomes. E. faecalis bacteremia treatment with piperacillin/tazobactam may be associated higher mortality. Disclosures Jaclyn A. Cusumano, PharmD, BCIDP, Shionogi: Advisor/Consultant

Ampicillin-susceptible Enterococcus faecium infections: clinical features, causal clades, and contribution of MALDI-TOF to early detection.

Enterococcus faecium, a common resident of the human gastrointestinal tract, is also a major pathogen. Prompt initiation of appropriate treatment is essential to improve patient outcome in disseminated E. faecium infections. However, ampicillin resistance is frequent in this species, rendering treatment difficult. We used a comprehensive approach, including clinical data review, whole-genome sequencing, and mass spectrometry, to characterize ampicillin-susceptible (EFM-S) and ampicillin-resistant (EFM-R) isolates. We included all patients with culture-confirmed E. faecium infection attending our hospital over a 16-month period. A comparison of 32 patients infected with EFM-S strains and 251 patients infected with EFM-R strains revealed that EFM-R isolates were strongly associated with a longer hospital stay, history of prior hospitalization, and the carriage of multidrug-resistant organisms. An analysis of the genomes of 26 EFM-S and 26 EFM-R isolates from paired patients revealed a population structure almost perfectly matching ampicillin susceptibility, with resistant isolates in clade A1, and susceptible isolates in clades A2 and B. The clade B and A2 isolates mostly came from digestive or biliary tract samples, whereas clade A1 isolates were mostly obtained from urine and blood. Finally, we built a custom database for matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), which differentiated between clade B and clade A1/A2 strains with high-positive and high-negative predictive values (95.6% and 100%, respectively). This study provides important new insight into the clinical features and clades associated with EFM-S and EFM-R isolates. In combination with MALDI-TOF MS, these data could facilitate the rapid initiation of the most appropriate treatment.IMPORTANCEEnterococcus faecium is an important human pathogen in which the prevalence of ampicillin resistance is high. However, little is known about the clinical characteristics of patients infected with ampicillin-resistant and ampicillin-susceptible strains. Indeed, current knowledge is based on genus-wide studies of Enterococcus or studies of very small numbers of susceptible isolates, precluding robust conclusions. Our data highlight specific clinical features related to the epidemiology of EFM-S and EFM-R strains, such as length of hospital stay, history of prior hospitalization, carriage of multidrug-resistant organisms, and type of sample from which the isolate was obtained. The use of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry with a custom-built database may make it possible to distinguish clade B isolates, which are typically susceptible to ampicillin, from clade A1/A2 isolates (A1 being typically resistant), thereby facilitating the management of these infections.

Genetic determinants underlying the progressive phenotype of β-lactam/β-lactamase inhibitor resistance in Escherichia coli.

Currently, whole-genome sequencing (WGS) data have not shown strong concordance with Escherichia coli susceptibility profiles to the commonly used β-lactam/β-lactamase inhibitor (BL/BLI) combinations: ampicillin-sulbactam (SAM), amoxicillin-clavulanate (AMC), and piperacillin-tazobactam (TZP). Progressive resistance to these BL/BLIs in the absence of cephalosporin resistance, also known as extended-spectrum resistance to BL/BLI (ESRI), has been suggested to primarily result from increased copy numbers of blaTEM variants, which is not routinely assessed in WGS data. We sought to determine whether addition of gene amplification could improve genotype-phenotype associations through WGS analysis of 147 E. coli bacteremia isolates with increasing categories of BL/BLI non-susceptibility ranging from ampicillin (AMP) susceptibility to being fully resistant to all three BL/BLIs. Consistent with a key role of blaTEM in ESRI, 112/134 strains (84%) with at least ampicillin non-susceptibility encoded blaTEM. Evidence of blaTEM amplification (i.e., blaTEM gene copy number estimates > 2×) was present in 40/112 (36%) strains. There were positive correlations between blaTEM copy numbers with minimum inhibitory concentrations of AMC and TZP (P < 0.05) but not for SAM (P = 0.09). The diversity of β-lactam resistance mechanisms, including non-ceftriaxone hydrolyzing blaCTX-M variants, blaOXA-1, and ampC and blaTEM strong promoter mutations, was greater in AMC- and TZP-non-susceptible strains but rarely observed within SAM- and AMP-non-susceptible isolates. Our study indicates that comprehensive analysis of WGS data, including β-lactamase-encoding gene amplification, can help categorize E. coli with AMC or TZP non-susceptibility but that discerning the transition from SAM susceptibility to SAM non-susceptibility using genetic data requires further refinement. IMPORTANCE The increased feasibility of whole-genome sequencing has generated significant interest in using such molecular diagnostic approaches to characterize difficult-to-treat, antimicrobial-resistant (AMR) infections. Nevertheless, there are current limitations in the accurate prediction of AMR phenotypes based on existing AMR gene database approaches, which primarily correlate a phenotype with the presence/absence of a single AMR gene. Our study utilized a large cohort of cephalosporin-susceptible Escherichia coli bacteremia samples to determine how increasing the dosage of narrow-spectrum β-lactamase-encoding genes in conjunction with other diverse β-lactam/β-lactamase inhibitor (BL/BLI) genetic determinants contributes to progressively more severe BL/BLI phenotypes. We were able to characterize the complexity of the genetic mechanisms underlying progressive BL/BLI resistance including the critical role of β-lactamase encoding gene amplification. For the diverse array of AMR phenotypes with complex mechanisms involving multiple genomic factors, our study provides an example of how composite risk scores may improve understanding of AMR genotype/phenotype correlations.

Model-Informed Clinical Practice - Determining an Appropriate Ampicillin-Sulbactam Redosing Regimen in Surgical Patients by Utilizing Population Pharmacokinetics and Target Attainment Analysis.

In the current study, population pharmacokinetic (PK) of ampicillin-sulbactam was performed based on the clinical pharmacokinetics data collected from a prospective study conducted in 40 surgical patients undergoing prolonged surgery where antibiotic redosing was implemented. A population PK model was successfully developed to characterize the disposition of ampicillin and sulbactam. The final models were two-compartment models for both drugs, with creatinine clearance and heart failure affecting clearance and body surface area having an impact on the central volume of distribution of both ampicillin and sulbactam. Comprehensive Monte Carlo simulations were performed to evaluate the probability of target attainment (PTA) of 24 different redosing scenarios. Simulation results indicated that the ampicillin-sulbactam 2-h redosing scheme recommended by ASHP guidelines is likely too conservative given that 3-g dose (2-g ampicillin/1-g sulbactam) with 4-h redosing interval can reach the breakpoint of 2 mg/L for ampicillin in all populations even with the aggressive pharmacokinetic/pharmacodynamic (PK/PD) target of 100% fT > MIC. With the target 50% fT > MIC, all redosing schemes evaluated, including the 8-h redosing scenario, are predicted to be able to reach the breakpoint of 64 mg/L in all patients. According to our findings, redosing of ampicillin-sulbactam should be every 4 h instead of the currently recommended 2-h redosing schedule. Our PTA results should inform future updates to existing general antibiotic redosing guidelines; and, when used in combination with the availability of institution- and/or unit-specific ampicillin susceptibility patterns, our PTA results may be used to customize SSI prophylaxis redosing recommendations for ampicillin-sulbactam at individual hospitals.

Occurrence, antibiogram, high-level vancomycin and aminoglycosides resistance and potential virulence factors of enterococci in dogs in Nigeria

AbstractThis study was conducted to isolate enterococci from dogs in Nigeria, and to determine the potential virulence, antibiotic susceptibility, phenotypic vancomycin (VAN), high-level ampicillin (AMP) and aminoglycosides susceptibility profile of the isolates. Rectal swabs were collected from 295 randomly-selected, clinically-healthy dogs. The isolation of enterococci was done using Slanetz and Bartley enterococcal selective medium. The resistance of 150 non-repetitive isolates was determined using disc diffusion method. VAN resistance was assessed by high-level disc diffusion and agar-screening methods. High-level AMP and aminoglycosides (gentamicin and streptomycin) resistance was determined by agar-screening method. Potential virulence factors were assayed using phenotypic methods. Out of 295 samples, 234 (80.7%) gave positive growth. From these, 250 enterococcal isolates comprised 229 (91.6%) non-pigmented and 21 (8.4%) pigmented strains, were obtained. Resistance of the isolates was 89% to erythromycin, 92% to rifampicin, 77% to chloramphenicol, 83% to tetracycline, 64% to ciprofloxacin, 32.7% to VAN, 24.7% to high-level streptomycin (HLS) and 6% to high-level gentamicin (HLG). Among 150 non-repetitive resistant isolates, 144 (96%), including all the VAN-, HLS- and HLG-resistant strains, exhibited resistance to at least 3 classes of antibiotics. The mean multiple antibiotic resistance index was 0.54 (range = 0.22 – 0.89). Of these 150 isolates, 94 (62.7%), including all the VAN-, HLS- and HLG-resistant strains, displayed virulence potentials as biofilm (44.7%), surface-layer (13.8%), haemolysin (21.3%), gelatinase (40.4%), caesinase (10.6%) and deoxyribonuclease (12.8%) activities. This study showed that dogs in Nigeria are potential reservoirs and disseminators of potentially-virulent, multidrug-, VAN- and high-level aminoglycosides-resistant enterococci.

Antibiotic Resistance of Hemophilus influenzae Isolated from Children in Southwest China.

Hemophilus influenzae (Hi) is one of the major pediatric bacterial pneumonia pathogens that heavily threatens children's lives and global health. With widespread usage as first-line treatment, the prevalence of β-lactam-resistant strains is increasing sharply. In order to treat Hi more effectively, a systematic study on the antibiotic resistance profiles, β-lactamase-negative ampicillin-resistant (BLNAR) strains isolation rate, and potential BLNAR resistance mechanism in our region is needed. This study analyzed antimicrobial susceptibility of Hi, and clinical data of Hi-infected patients retrospectively. BLNAR and β-lactamase-positive ampicillin-clavulanate resistant strains (BLPACR) were confirmed by the Kirby-Bauer method and β-lactamase test. ftsI gene in BLNAR was sequenced to find out whether resistance was induced by penicillin-binding protein mutation. Ampicillin susceptibility test with or without efflux pump inhibitors were done to assess efflux pump contribution in BLNAR. RT-PCR was performed to evaluate the efflux pump genes' transcription levels. A total of 2,561 Hi strains were isolated in our hospital from January 2016 to December 2019. Male to female ratio was 1.52:1. Median age was 10 months. Infant (< 3 years old) infection accounted for 83.72%. Hi resistance rates to sulfamethoxazole-trimethoprim, ampicillin, cefathiamidine, cefaclor, cefuroxime, cephalothin, amoxicillin-clavulanate, tetracycline, chloramphenicol, ofloxacin, cefotaxime, and rifampin were 84.28%, 78.01%, 49.80%, 41.98%, 36.58%, 33.64%, 4.55%, 4.1%, 3.37%, 1.77%, 0.99%, and 0.12%, respectively, while 1.33% were BLNAR. BLNARs were classified into four groups by mutation patterns in ftsI gene and most strains were divided to Group Ⅲ/Ⅲ-like. EmrB, ydeA and norM transcription levels in some ampicillin-resistant strains were higher than their sensitive counterparts. Ampicillin is not sufficiently effective as a first-line Hi infection treatment. However, ampicillin-clavulanate and cefotaxime may be a better choice. Efflux pumps, emrB, ydeA and norM play roles in the high resistance to ampicillin.

1988. Piperacillin-tazobactam plus Ceftriaxone for Enterococcus faecalis endocarditis: A Single Center Experience

Abstract Background Enterococcus faecalis is a leading cause of Infective Endocarditis (IE). IE guidelines recommend combination of penicillin (PCN) or ampicillin (AMP) with ceftriaxone (CTX) in susceptible strains. OPAT allows for antibiotic continuation at home; however outpatient AMP is limited due to frequent dosing and instability. Piperacillin-tazobactam (PTZ) has similar but not identical activity against penicillin binding proteins (PBP) compared to AMP and PCN, suggesting that PTZ may be a substitute in OPAT. This study compares outcomes in those with E. faecalis IE discharged with either PTZ or PCN with CTX. Results of primary outcomes including death at 90 days and readmission at 90 days for those discharged on either PTZ or PCN in combination with CTX for E. faecalis endocarditis Results of non infectious complications and changes in antibiotic therapy in both groups post discharge Methods This single center retrospective study took place from 2016 - 2020. Adults &amp;gt; 18 years with AMP susceptible E. faecalis IE diagnosed using Duke’s criteria and blood or tissue cultures were included, with cultures confirming PCN susceptibility. Primary endpoint was failure of therapy at 90 days, defined as death due to any cause or infection relapse. Secondary outcomes were duration of antibiotics, change in regimen and non-infectious complications. Results Overall, 34 patients were identified, 17 in each group. Average age was 60–80 years, majority having a high Charleston comorbidity index. One did not have available AMP susceptibility but was PCN susceptible. 19 were managed surgically (65% PTZ group, 47% PCN group). Average total antibiotic course was 8.2 and 7.3 weeks in PTZ and PCN groups, with average OPAT course being 4.5 and 4.4 weeks. Primary endpoint was met in 18% patients in the PTZ group and 24% in the PCN group. Of the PCN group, 1 was admitted 4 months later due to an E. faecalis device infection, outside of primary endpoint. Nearly 80% completed antibiotic therapy with 29% in the PTZ group and 35% in the PCN group readmitted for non-infectious complications, including metabolic imbalances, stroke, volume overload and gastro-intestinal bleed. Hyperkalemia was seen in the PCN group with 3 patients requiring readmission. Seizures were not reported with combined PCN and ceftriaxone. Conclusion Similar outcomes were seen using either PTZ or PCN with CTX for treatment of enterococcal IE. Discharging patients on PCN is not always a feasible option. In these instances, PTZ could be considered as an alternative OPAT agent despite dissimilar mechanism of action compared to AMP and PCN. Disclosures All Authors: No reported disclosures.

Epidemiology, microbiological and clinical characteristics of Enterococcus species bloodstream infections: A 10-year retrospective cohort study from Qatar

Background and objectiveEnterococcus species is one of the leading causes of community and healthcare-associated infections resulting in significant morbidity and mortality. In this study, we aim to evaluate the epidemiology, microbiological and clinical characteristics of Enterococcus Blood Stream Infections (BSIs) over 10 years period in a national secondary care setting. MethodsA retrospective cohort study was conducted on verified cases of enterococcal BSIs in adults from January 2009–December 2018 from specialized care hospitals at Hamad Medical Corporation, Qatar. Epidemiological, microbiological and clinical data were reported and analyzed. ResultsA total of 263 enterococcus BSIs cases were identified, predominant were males (65%) with a median age of 63 (IQR 48–74). E. faecalis and E. faecium were predominate at 93.5% (73.38% and 20.15% respectively). Diabetes was the commonest premorbid condition (54.3%) followed by chronic kidney disease (36.5%). Central lines and genitourinary were the most common sources (18.25%, 14.83% respectively) while no identified source was reported in 45.25% of cases. Ampicillin susceptibility was 82.51% while vancomycin resistance was reported in 10.6% of isolates. Successful bacteremia clearance was achieved in 81.37% of cases at a mean of 4 days (Range 2–5 days) while metastatic complications occurred in 5.3% of cases. Univariate mortality risk analysis was associated with ICU admission, low level of consciousness, high bacteremia scores, and presence of catheters. The 30 days mortality was high at 66.54% with CKD and cancer patients at the highest mortality risks (OR 16.334 (CI 4.2–62.4) and 16 (CI 3–84) respectively. ConclusionSignificant mortality was associated with enterococcus BSI despite low rates for ampicillin and vancomycin resistance necessitating early identification of susceptible patients to instigate suitable preventive measures.

A study on the clinical spectrum, prescription pattern and diagnosis of enteric fever in a tertiary care hospital of North India

Background: Enteric fever is a major public health concern in developing countries. Lack of diagnostics diagnosis and antimicrobial resistance challenges in treatment of enteric fever. Timely diagnosis and proper management of fever are essential to reduce the complications and emergence antibiotic resistance.Methods: A prospective observational study was conducted on 71 subjects between February 2018 and December 2019. Spectrum of the disease, antimicrobial resistance and prescription pattern were observed and performances of various diagnostic methods (like culture, serology and loop mediated isothermal amplification) were determined.Results: The most commonly observed clinical features included gastrointestinal complaints, anaemia, leukopenia, splenomegaly and hepatitis. Blood culture was positive for 49.3% (35/71) of the cases using conventional culture method. All samples positive by LAMP were also culture positive, out of which 80% (28/35) were S. typhi and 20% (7/35) were S. paratyphi A. Antimicrobial susceptibility to ciprofloxacin was 39.3% and 28.6% and ampicillin susceptibility was 32.1 and 28.6% in S. typhi and S. paratyphi A respectively. Only one multidrug resistant isolate was noted, while none was resistant to ceftriaxone or azithromycin. Cefixime and azithromycin combination therapy led to the earliest defervescence of fever.Conclusions: Oral combination therapy may be a good option to treat enteric fever and oral combination therapy may be a good option to treat enteric fever and performance of LAMP seem to be as good as culture with a shorter turnaround time. LAMP can be used to obviate the need of culture in resource limited settings.

Vertebral osteomyelitis caused by the novel pathogen Cutibacterium modestum: a case report

BackgroundCutibacterium modestum is one of the five species of the genus Cutibacterium. While C. acnes has been reported as an important pathogen in bone and joint infections, the clinical characteristics of C. modestum infections remain unclear. Moreover, thus far, there has been no clinical case report regarding C. modestum infections.Case presentationAn 82-year-old man with a history of repeated trigger point injections for lumbago at the L4 level presented with fever and an exacerbation of lumbago. Physical examination indicated knocking pain at the L4–L5 levels; magnetic resonance imaging showed irregular bone destruction of the L4 vertebral body, and low T1 and high T2 intensity lesions at the L4–L5 intervertebral disc. Two sets of blood cultures (two aerobic and two anaerobic) were performed. Intravenous cefazolin was administered, considering the common pathogens of vertebral osteomyelitis, such as Staphylococcus aureus. The patient’s condition did not improve; thereafter, anaerobic culture bottles revealed Gram-positive rods on day 11 of incubation. There was no evidence of infective endocarditis upon transthoracic echocardiography. Needle aspiration from the L4–L5 intervertebral disc was performed on day 13 that also showed the presence of Gram-positive rods. The patient was diagnosed with vertebral osteomyelitis caused by C. modestum using a combination of characteristic peak analysis with matrix-assisted laser desorption ionization (MALDI), microbial biochemistry examinations, and 16S rRNA gene sequencing from the blood and pus cultures. He was successfully treated with alternative intravenous ampicillin, followed by oral amoxicillin for 10 weeks, according to the tests for ampicillin susceptibility, with a minimum inhibitory concentration of 0.016 μg/mL using E-test® under aerobic conditions.ConclusionsCutibacterium modestum is a microorganism that is difficult to identify. A combination of characteristic peaks with MALDI, appropriate microbial biochemical examinations, and 16S rRNA gene sequencing may serve as an efficient guide for the identification of C. modestum.

Prevalence and antimicrobial susceptibility of group B streptococci among pregnant women in Ethiopia: A systemic review and meta-analysis study.

Objective:Maternal colonization or infection with drug-resistant Group B streptococcus is a serious disease that affects mother, fetus, and infant. The knowledge of maternal colonization and antimicrobial susceptibility test is substantially needed for a nation to formulate a policy or change the already existing one to reduce maternal, fetus, and infant mortality. As a result, the goal of this review was to determine the pooled prevalence Group B streptococcus colonization and antimicrobial susceptibility among Ethiopian pregnant women.Methods:Literature searches were carried out in the electronic biomedical databases and indexing services such as PubMed/MEDLINE, Scopus, Science Direct, Web of Science, and Google Scholar. Original records of research articles, available online from 2014 to December 2020, addressing prevalence and antimicrobial-resistance pattern of Group B streptococcus in pregnant women were identified and screened. Endnote citation manager software version X9 for windows was utilized to collect and organize search outcomes and for removal of duplicate articles. The relevant data were extracted from included studies using a format prepared in Microsoft Excel and exported to STATA 14.0 software for the outcome measures analyses and subgrouping. The I2 index was used to measure heterogeneity between studies and median, and interquartile (25%, 75%) was used to assess antimicrobial susceptibility rate.Results and conclusion:Sixteen original articles were found in both qualitative and quantitative analyses. Group B streptococcus colonization was recorded in 979 of the 5743 pregnant women, resulting in a 16% overall frequency (95% confidence interval: 13%−20%). The estimated prevalence varied significantly between studies with significant heterogeneity (χ2 = 154.31, p = 0.001, I2 = 90.28). Ampicillin (97.8%; interquartile range = 89.5%−100%), penicillin G (95.5%; interquartile range = 89.5%−100%), and vancomycin (100%; interquartile range = 89.5%−100%) susceptibility were all high in Group B streptococcus, whereas tetracycline (29%; interquartile range = 89.5%−100%) susceptibility was low. Group B streptococcus colonization rates in Ethiopian women during pregnancy were virtually similar to those in many underdeveloped countries, and Group B streptococcus isolates were highly sensitive to ampicillin, penicillin G, and vancomycin.

Trends in the epidemiology of urinary tract infections in pregnancy at a tertiary hospital in Johannesburg: Are contemporary treatment recommendations appropriate?

BackgroundUrinary tract infections (UTIs) are common during pregnancy and are associated with maternal and foetal complications. Empiric antibiotic choices in pregnancy require consideration of efficacy and safety, resulting in limited oral options. With rapidly evolving antibiotic resistance, surveillance to guide empiric treatment recommendations is essential.MethodsA retrospective analysis of urine culture isolates from the Charlotte Maxeke Johannesburg Academic Hospital (CMJAH) Obstetrics Department for 1 January 2015 to 31 December 2020 was performed.ResultsThe top 3 pathogens were Escherichia coli, Enterococcus faecalis and Klebsiella pneumoniae. For E. coli susceptibility to cefuroxime declined (95% to 81%, p < 0.0001). Similarly, the E. coli extended spectrum beta-lactamase rate increased from 5% to 10% (p = 0.04). E. coli susceptibility to nitrofurantoin (93%) and fosfomycin (96%) remained high. In 2019, carbapenem-resistant K. pneumoniae emerged. Ampicillin susceptibility was high amongst the E. faecalis isolates. Amoxicillin-clavulanate demonstrated high levels of activity against the top 3 uropathogens.ConclusionThe Essential Drug List recommended antibiotics for lower UTIs, nitrofurantoin and fosfomycin, are appropriate empiric options for E. coli, the most common uropathogen in the CMJAH obstetric population. The high rate of E. faecalis susceptibility to nitrofurantoin reported from other Gauteng tertiary obstetric patients, suggests that nitrofurantoin will provide adequate empiric cover for a large proportion of UTIs. However, the determination of the E. faecalis nitrofurantoin and fosfomycin susceptibility rates in the CMJAH obstetric population will provide useful data. Periodic surveillance at the various levels of antenatal care in different regions of South Africa and the determination of risk factors for infections with resistant uropathogens is needed.