Piperacillin-tazobactam Resistance Research Articles

Exploring uncatalogued genetic variation in antimicrobial resistance gene families in Escherichia coli: an observational analysis

Antimicrobial resistance (AMR) in Escherichia coli is a global problem associated with substantial morbidity and mortality. AMR-associated genes are typically annotated based on similarity to variants in a curated reference database, with the implicit assumption that uncatalogued genetic variation within these is phenotypically unimportant. In this study, we evaluated the performance of the AMRFinder tool and, subsequently, the potential for discovering new AMR-associated gene families and characterising variation within existing ones to improve genotype-to-susceptibility phenotype predictions in Ecoli. In this cross-sectional study of international genome sequence data, we assembled a global dataset of 9001 Ecoli sequences from five publicly available data collections predominantly deriving from human bloodstream infections from: Norway, Oxfordshire (UK), Thailand, the UK, and Sweden. 8555of these sequences had linked antibiotic susceptibility data. Raw reads were assembled using Shovill and AMR genes (relevant to amoxicillin-clavulanic acid, ampicillin, ceftriaxone, ciprofloxacin, gentamicin, piperacillin-tazobactam, and trimethoprim) extracted using the National Center for Biotechnology Information AMRFinder tool (using both default and strict [100%] coverage and identity filters). We assessed the predictive value of the presence of these genes for predicting resistance or susceptibility against US Food and Drug Administration thresholds for major and very major errors. Mash was used to calculate the similarity between extracted genes using Jaccard distances. We empirically reclustered extracted gene sequences into AMR-associated gene families (≥70% match) and antibiotic-resistance genes (ARGs; 100% match) and categorised these according to their frequency in the dataset. Accumulation curves were simulated and correlations between gene frequency in the Oxfordshire and other datasets calculated using the Spearman coefficient. Firth regression was used to model the association between the presence of blaTEM-1 variants and amoxicillin-clavulanic acid or piperacillin-tazobactam resistance, adjusted for the presence of other relevant ARGs. The performance of the AMRFinder database for genotype-to-phenotype predictions using strict 100% identity and coverage thresholds did not meet US Food and Drug Administration thresholds for any of the seven antibiotics evaluated. Relaxing filters to default settings improved sensitivity with a specificity cost. For all antibiotics, most explainable resistance was associated with the presence of a small number of genes. There was a proportion of resistance that could not be explained by known ARGs; this ranged from 75·1% for amoxicillin-clavulanic acid to 3·4% for ciprofloxacin. Only 18 199 (51·5%) of the 35 343ARGs detected had a 100% identity and coverage match in the AMRFinder database. After empirically reclassifying genes at 100% nucleotide sequence identity, we identified 1042unique ARGs, of which 126 (12·1%) were present ten times or more, 313 (30·0%) were present between two and nine times, and 603 (57·9%) were present only once. Simulated accumulation curves revealed that discovery of new (100% match) ARGs present more than once in the dataset plateaued relatively quickly, whereas new singleton ARGs were discovered even after many thousands of isolates had been included. We identified a strong correlation (Spearman coefficient 0·76 [95% CI 0·73-0·80], p<0·0001) between the number of times an ARG was observed in Oxfordshire and the number of times it was seen internationally, with ARGs that were observed six times in Oxfordshire always being found elsewhere. Finally, using the example of blaTEM-1, we showed that uncatalogued variation, including synonymous variation, is associated with potentially important phenotypic differences; for example, two common, uncatalogued blaTEM-1 alleles with only synonymous mutations compared with the known reference were associated with reduced resistance to amoxicillin-clavulanic acid (adjusted odds ratio 0·58 [95% CI 0·35-0·95], p=0·031) and piperacillin-tazobactam (0·50 [95% CI 0·29-0·82], p=0·005). We highlight substantial uncatalogued genetic variation with respect to known ARGs, although a relatively small proportion of these alleles are repeatedly observed in a large international dataset suggesting strong selection pressures. The current approach of using fuzzy matching for ARG detection, ignoring the unknown effects of uncatalogued variation, is unlikely to be acceptable for future clinical deployment. The association of synonymous mutations with potentially important phenotypic differences suggests that relying solely on amino acid-based gene detection to predict resistance is unlikely to be sufficient. Finally, the inability to explain all resistance using existing knowledge highlights the importance of new target gene discovery. National Institute for Health and Care Research, Wellcome, and UK Medical Research Council.

Molecular insights into the evolutionary trajectory of a Klebsiella aerogenes clinical isolate with a complex trade-off between resistance and virulence.

The fitness cost associated with antimicrobial resistance has an important influence on evolutionary dynamics. We compared the genomes of three Klebsiella aerogenes isolates recovered from blood samples or deep abscess cultures from the same patient: the wild-type strain (CT_WT), a piperacillin-tazobactam-resistant strain (CT_PENI), and an extended-spectrum-cephalosporin (ESC)-resistant strain (CT_R). Whole-genome sequencing revealed that CT_PENI had acquired a TEM-1 β-lactamase with a mutated promoter, accounting for overproduction. CT_PENI then acquired an E240G substitution in the TEM-1 β-lactamase (resulting in TEM-207) and lost the porin-encoding ompK36 gene to give CT_R. All three strains showed the same virulence in a mouse model of intraperitoneal infection. The results of recombination and transformation assays indicated that when present separately, the TEM-207 overproduction and the ompK36 gene deletion had only small effects on susceptibility to ESCs. However, the combination of the two changes led to a much lower susceptibility to ESCs. Moreover, the levels of fitness in vitro and in vivo in a murine model of gut colonization were significantly lower after TEM-1 β-lactamase overproduction and lower still after E240G substitution and OmpK36 loss. We hypothesize that the chosen courses of antibiotics led to the stepwise emergence of a clone with resistance to penicillins and ESCs and no loss of virulence. However, acquired resistance may have a fitness cost that limits evolutionary success. Our results might explain why the overproduction of extended-spectrum β-lactamases (which should confer a high level of piperacillin-tazobactam resistance) is not observed in clinical practice and why TEM-207 has rarely been detected in clinical isolates.

Antimicrobial Resistance Pattern and Empirical Antibiotic Treatments in Neonatal Sepsis: A Retrospective, Single-Center, 12-Year Study.

Neonatal sepsis is an important cause of morbidity and mortality in neonatal intensive care units (NICUs). Continuous evaluation of antimicrobial resistance (AMR) profiles is advised to implement antimicrobial stewardship (AMS) programs and establish effective empiric antibiotic protocols. AMS may reduce AMR in NICUs and improve sepsis outcomes. In this retrospective observational study, we report data on culture-positive neonatal sepsis, assessing differences after the implementation of an AMS program (2011-2016 vs. 2017-2022). A total of 215 positive bacterial cultures from 169 infants were retrieved, with 79 early-onset (36.7%) and 136 late-onset (63.3%) sepsis episodes. Frequent causative agents for early-onset sepsis were S. agalactiae and E. coli, all susceptible to empiric treatment. Late-onset sepsis was mainly caused by Enterobacterales and S. aureus. Aminoglycosides, cefotaxime, and piperacillin-tazobactam resistance among Enterobacterales was substantially low; S. aureus was mostly susceptible to oxacillin and vancomycin. There were no differences in mortality and multidrug-resistant pathogens rates between the two study periods. There were five episodes of fungal late-onset sepsis, mostly due to C. albicans, of which one was fatal. The microbial distribution pattern and AMR profiles overlapped with other European studies. Because susceptibility patterns are rapidly changing worldwide, with the emerging threat of Methicillin-resistant S. aureus and extended-spectrum beta-lactamases producers, infection prevention and control practices and AMS strategies require continuous optimization to limit selection pressure and AMR escalation.

Role of blaTEM and OmpC in the piperacillin-tazobactam resistance evolution by E. coli in patients with complicated intra-abdominal infection

Piperacillin-tazobactam resistance (P/T-R) is increasingly reported among Escherichia coli isolates. Although in vitro experiments have suggested that blaTEM gene plays a key role in the P/T-R acquisition, no clinical in vivo study has yet confirmed the role of blaTEM or other genes. Therefore, we aimed to identify the mechanisms underlying P/T-R by following up patients with E. coli complicated intra-abdominal infections (cIAI) who experienced P/T treatment failure. Four pairs of strains, clonally related from four patients, were isolated both before and after treatment with P/T dosed at 4g/0.5g intravenously. The P/T MIC was tested using broth microdilution, and β-lactamase activity was determined in these isolates. Whole-genome sequencing (WGS) was performed to decipher the role of blaTEM and other genes associated with P/T-R. Changes in the outer membrane protein (OMP) profile were analyzed using SDS-PAGE, and blaTEM and ompC transcription levels were measured by RT-qPCR. In addition, in vitro competition fitness was performed between each pairs of strains (P/T-susceptible vs. P/T-resistant). We found a higher copy number of blaTEM gene in P/T-R isolates, generated by three different genetic events: (1) IS26-mediated duplication of the blaTEM gene, (2) generation of a small multicopy plasmid (ColE-like) carrying blaTEM, and (3) adaptive evolution via reduction of plasmid size, leading to a higher plasmid copy number. Moreover, two P/T-R strains showed reduced expression of OmpC. This study describes the mechanisms involved in the acquisition of P/T-R by E. coli in patients with cIAI. The understanding of P/T-R evolution is crucial for effectively treating infected patients and preventing the spread of resistant microorganisms.

Multidrug Resistance and Plasmid Profiles of Escherichia coli Isolated from Lebanese Broiler Farms.

The present study was undertaken to determine the antimicrobial resistance patterns and plasmid fingerprints of commensal Escherichia coli isolated from Lebanese broiler chickens. To that end, a total of 30 E. coli isolates were collected from 15 semi-open broiler farms from North Lebanon and Bekaa Valley. Results showed that all the isolates were resistant to at least nine out of 18 evaluated antimicrobial agents. The best-performing antibiotic families were Carbapenems (Imipenem) and Quinolones (Ciprofloxacin and Norfloxacin) to which only 0.0 and 8.3% of the isolates were resistant, respectively. Fifteen various plasmid profiles were depicted, and all the isolates were found to possess one or multiple plasmids. The plasmid sizes varied from 1.2 to 21.0 kbp, and the most commonly detected plasmid had a size of 5.7 kbp (23.3% of the isolates). There was no significant association between the number of plasmids per isolate and resistance to a particular drug. Nevertheless, the presence of specific plasmids, namely, the 2.2 or 7.7 kbp sized ones, was strongly correlated to Quinolones or Trimethoprim resistance, respectively. Both the 7.7 and 6.8 kbp plasmids showed mild correlation to Amikacin resistance, and the 5.7 kbp plasmid was mildly correlated to Piperacillin-Tazobactam resistance. Our findings highlight the need to revise the list of antimicrobials currently used in Lebanese poultry and associate the presence of specific plasmids to antimicrobial resistance patterns in E. coli isolates. The revealed plasmid profiles could also serve any future epidemiological investigation of poultry disease outbreaks in the country.

Changing Trends in Antimicrobial Susceptibility Patterns of Bloodstream Infection (BSI) in Secondary Care Hospitals of India.

Introduction Bloodstream infection (BSI) and subsequent sepsis are life-threatening medical conditions. The onset of antimicrobial resistance and subsequent multi-drug resistant organisms (MDRO) significantly increase healthcare-associated expenditure with adverse clinical outcomes. The present study was undertaken to identify the trends of BSI in community settings in secondary care hospitals (smaller private hospitals and district hospitals) in the state of Madhya Pradeshin Central India with the support of the Indian Council of Medical Research (ICMR) and National Health Mission, Madhya Pradesh. Methodology The present study was a prospective, longitudinal observational chart review type of study. The study was carried out at 10 secondary care hospitals (eight smaller private hospitals and two government district hospitals) nominated by the State Government as part of the ICMR Antimicrobial Resistance Surveillance and Research Network (AMRSN). The hospitals were nominated depending on the availability of a microbiology laboratory and a full-time microbiologist. Result A total of 6202 blood samples were received from patients with suspected BSI, out of which 693 samples were positive for aerobic culture. Among these, 621 (89.6%) showed bacterial growth and 72 (10.3%) grew Candida species (spp). Out of the 621 bacterial growth samples, Gram-negative bacteria were 406 (65.3%) and Gram-positive bacteria were 215 (34.6%). Among the Gram-negative isolates (406), the predominant isolate was Escherichia coli (115; 28.3%) followed by Klebsiella pneumoniae(109; 26.8%), Pseudomonas aeruginosa(61; 15%), Salmonella spp. (52; 12.8%), Acinetobacter spp. (47; 11.6%) and the other Enterobacter spp. (22; 5.4%). Among the Gram-positive isolates (215), the predominant isolate was Staphylococcus aureus(178; 82.8%) followed by Enterococcus spp. (37; 17.2%). Among the Escherichia coli, third-generation cephalosporin resistance was identified in 77.6%, piperacillin-tazobactam resistance in 45.2%, carbapenem resistance in 23.5% and colistin resistance in 16.5% of cases. Among the Klebsiella pneumoniae, third-generation cephalosporin resistance was identified in 80.7%, piperacillin-tazobactam resistance in 72.8%, carbapenem resistance in 63.3% and colistin resistance in 14% of cases. Among the Pseudomonas aeruginosa, ceftazidime resistance was identified in 61.2%, piperacillin-tazobactam resistance in 55%, carbapenem resistance in 32.8%, and colistin resistance in 38.3% of cases. Among the Acinetobacter spp., piperacillin-tazobactam resistance was identified in 72.7%, carbapenem resistance in 72.3%, and colistin resistance in 9.3% cases. While analyzing the antibiogram for Staphylococcus aureus isolates, methicillin resistance (MRSA) was seen in 70.3% of cases, followed by vancomycin resistance (VRSA) in 8% of cases and linezolid resistance in 8.1%. Among the Enterococcus spp. isolates, linezolid resistance was found in 13.5%, vancomycin resistance (VRE) in 21.6%, and teicoplanin resistance in 29.7% of cases. Conclusion In conclusion, the first-ever study to identify the risk of high-end antibiotics causing significant drug resistance in secondary and tertiary care settings has highlighted the urgent need for more randomized control studies and proactive measures from healthcare authorities and serves as a beacon for future research efforts and underscores the importance of implementing antibiograms to combat the growing threat of antibiotic resistance.

Retrospective analysis of bacteraemia due to extended-spectrum beta-lactamase-producing Enterobacterales: the challenge of healthcare-associated infections

ObjectivesHealthcare-associated bacteraemia is defined as bacteraemia diagnosed ≤48 h after hospital admission in patients recently exposed to healthcare procedures or settings. It differs from hospital-acquired bacteraemia, which is diagnosed >48 h after hospital admission. Healthcare-associated bacteraemia is reported increasingly, often due to resistant pathogens including extended-spectrum beta-lactamase (ESBL) producers, representing a challenge to empirical treatment. This study aimed to assess the appropriateness of empirical treatment for ESBL bacteraemia at the authors’ centre, to perform a descriptive analysis according to the mode of infection acquisition (community-acquired, healthcare-associated, hospital-acquired), and to assess the risk factors for mortality. MethodsA retrospective study on patients with ESBL bacteraemia was undertaken. ResultsIn total, 129 consecutive cases of bacteraemia due to ESBL producers were included in this study. Compared with community- and hospital-acquired bacteraemia, healthcare-associated bacteraemia affected older patients (P=0.001) and patients with higher Charlson Comorbidity Index scores (P=0.007), and was more frequently associated with piperacillin-tazobactam resistance (P=0.025) and multi-drug resistance (P=0.026). Overall, ineffective empirical treatment was common (42%). Factors associated with 30-day mortality were septic shock [odds ratio (OR) 7.096, 95% confidence interval (CI) 2.58–24.58], high Pitt score (OR 6.636, 95% CI 1.71–23.62) and unknown source of bacteraemia (OR 19.28, 95% CI 2.80–30.70). ConclusionsAntimicrobial stewardship interventions focusing on both in-hospital and community settings are advocated to better manage healthcare-associated infections due to ESBL producers.

423. Molecular Epidemiology of ESBL E. coli Over a 2 Year Period in Worcester Massachusetts USA

Abstract Background Extended-spectrum beta-lactamase (ESBL) producing Escherichia coli are an increasing health burden contributing to a pandemic of resistant bacteria. Methods To better understand the population genomics of ESBL E. coli, 129 phenotypically identified ESBL isolates collected between 2017-04 to 2018-11 from a single academic medical center underwent whole genome sequencing with the presence of antibiotic resistance genes identified by screening against the CARD database. The odds of phenotypic resistance to beta-lactam agents were calculated for each known resistance gene. A novel approach based on k-mer-enrichment among antibiotic resistance gene groups was developed to predict genes associated with phenotypic resistance. Results Genomic analysis revealed that 60% of the ESBL isolates collected were pandemic strain ST131. All isolates contained at least 1 gene in the beta-lactamase family with a median of 3 different beta-lactamase genes being present. Ninety-five percent of isolates contained a CTX-M gene with 52% CTX-M-15, 43% CTX-M-27, 5% other CTX-M; 24% had OXA and 34% had TEM. The presence of CTX-M-15 significantly increased the odds of resistance to ampicillin-sulbactam (95% confidence interval 5.9-116.5, p-value &amp;lt; 1e-7), ceftazidime (4.2-25.9, p&amp;lt; 1e-6) and cefepime (3.6-20.1, p&amp;lt; 1e-5) but not to piperacillin-tazobactam (0.9-6.3, p 1). CTX-M-27 decreased the odds of resistance to these 4 beta-lactams (0.003-0.07, p&amp;lt; 1e-13; 0.04-0.3, p&amp;lt; 1e-05; 0.06-0.4, p&amp;lt; 0.001; 0.03-0.6, p 0.11). TEM-1 significantly increased the odds for ampicillin-sulbactam resistance (3.8-1036.7, p&amp;lt; 1e-3) but not for other beta-lactams. Our de novo sequence approach was able to identify the known genes listed above and also identified a 683 bp open reading frame with unknown function that had increased odds of resistance to piperacillin-tazobactam resistance (2.1-43.6, p&amp;lt; 1e-3). This 683 bp open reading frame did not share any homology with known beta-lactamases. Conclusion The majority of phenotypic ESBL isolates contain multiple beta-lactamase genes. The presence of CTX-M-15 appears to increase the odds of resistance to key beta-lactam agents. Our novel k-mer-enrichment approach identified a novel marker of piperacillin-tazobactam resistance. Disclosures All Authors: No reported disclosures.

Evaluation of Piperacillin-Tazobactam ETEST for the Detection of OXA-1 Resistance Mechanism among Escherichia coli and Klebsiella pneumoniae.

Globally, piperacillin-tazobactam resistance among Escherichia coli and Klebsiella pneumoniae is driven by OXA-1 beta-lactamases. Expression of blaOXA-1 yields piperacillin-tazobactam MICs of 8 to 16 μg/mL, which straddle the susceptible/susceptible-dose dependent breakpoint set by the Clinical and Laboratory Standards Institute in 2022. Variability of the reference broth microdilution method (BMD) was evaluated by manufacturing BMD panels using 2 brands of piperacillin, 2 brands of tazobactam and 2 brands of cation-adjusted Mueller-Hinton broth. In addition, ETEST, which harbors an intermediate dilution of 12 μg/mL was evaluated for the ability to differentiate isolates with and without blaOXA-1. A collection of 200 E. coli and K. pneumoniae, of which 82 harbored a blaOXA-1 gene, were tested. BMD variability was on average 1.3-fold, within the accepted 2-fold variability of MICs. However, categorical agreement (CA) between BMD reads was 74.0% for all isolates and 63.4% for those with a blaOXA-1 gene and 81.3% for those without blaOXA-1 detected (P = 0.004, Pearson's Chi Square). ETEST overall CA with the BMD mode was 68.0% and essential agreement (EA) was 80.5%. For isolates with blaOXA-1, CA was 50.0% and EA was 69.5%, versus 80.5% and 88.1%, respectively, for isolates without blaOXA-1 (P < 0.0001 for both comparisons). All ETEST errors were major errors (false resistance) compared to BMD mode. However, the negative predictive value of the ETEST for the presence of blaOXA-1 was 94.1%, compared to only 74.2% negative predictive value for BMD. Clinicians and microbiologists should be aware of the challenges associated with testing piperacillin-tazobactam in regions where blaOXA-1 is prevalent.

Prevalence and antibiotic resistance of Klebsiella pneumoniae in a tertiary hospital in Hangzhou, China, 2006-2020.

ObjectiveThis study analyzed the characteristics and tendencies of resistance to common antibiotics for Klebsiella pneumoniae to provide a basis for clinical treatment and prevention.MethodsA total of 71,743 isolates were collected from hospital clinical specimens following standard procedures from 2006 to 2020. Statistical analyses were conducted on laboratory test results.ResultsA total of 3054 isolates of K. pneumoniae were mainly isolated from sputum (53.77%), urine (14.70%), and blood (8.42%). Isolation rates of strains in the AIDS, hepatology, and intensive care wards were 9.72%, 12.52%, and 16.45%, respectively. Resistance rates of imipenem, cefazolin, gentamicin, tobramycin, ciprofloxacin, and ceftazidime respectively increased from 2.33%, 27.91%, 16.28%, 13.95%, 18.60%, and 9.30% to 12.83%, 40.82%, 21.57%, 25.07%, 44.61%, and 17.78%, while piperacillin–tazobactam resistance decreased from 13.95% to 13.70%. Differences in resistance rates to seven antibiotics were significant among specimen types. Detection rates of carbapenem-resistant K. pneumoniae were significantly different among blood, sputum, and urine specimens, and between wards.ConclusionsThe prevalence and drug resistance of K. pneumoniae showed an upward trend over time, and resistance varied according to ward and specimen source. The prevention of nosocomial infections and rational drug use must be emphasized to reduce antimicrobial resistance.

Antibiotic Resistance Patterns of Enterobacteriaceae Isolated from Patients with Healthcare-Associated Infections.

BackgroundThere have been recent proposals to categorize healthcare-associated infections (HCAIs) separately from community-acquired infections (CAIs). The aim of this study was to compare the antibiotic resistance of pathogens causing CAIs, HCAIs, and hospital-acquired infections (HAIs) in Korea, and to investigate the need for different empirical antibiotics therapy for CAIs and HCAIs.Materials and MethodsThis prospective study was conducted in a university hospital between March and December 2019. Inpatients who underwent a bacterial culture within 2 days of hospitalization, with a Enterobacteriaceae strain identified at the infection site and available antibiotic susceptibility results, were included in the analysis. Infections were classified as CAIs, HCAIs or HAIs, depending on the source.ResultsOf the 146 patients included in the analysis, the prevalence of fluoroquinolone-resistant Enterobacteriaceae was 18.8%, 38.5%, and 55.0%; the prevalence of pathogens showing third-generation cephalosporins resistance was 8.3%, 50.0%, and 60.0%; and the prevalence of pathogens showing piperacillin-tazobactam resistance was 8.3%, 7.7%, 15.0% in the CAIs, HCAIs, and HAIs groups, respectively. The prevalence of extended-spectrum beta-lactamase-positive pathogens was 6.3%, 47.3%, and 55.0% in the CAIs, HCAIs, and HAIs group, respectively, with no significant difference between the HCAIs and HAIs groups. Resistance patterns of the HCAIs group more closely resembled those of the HAIs group than those of the CAIs group.ConclusionThe pathogens isolated from patients with HCAIs showed resistance patterns that were more similar to those of patients with HAIs than those with CAIs. Thus, CAIs and HCAIs should be distinguished from each other when selecting antibiotic agents.

646. Adapting the modified Carbapenem Inactivation Method to assess for possible beta-lactamase mediated resistance in Piperacillin-Tazobactam resistant/ Ceftriaxone susceptible Escherichia. coli and Klebsiella pneumoniae

BackgroundA cluster of piperacillin-tazobactam resistant/ceftriaxone susceptible Escherichia coli and Klebsiella pneumonaie bacteremias were noted at our institution. A review of the literature suggested this resistance phenotype was mediated by a beta-lactamase. We sought to further corroborate this phenotypically.MethodsWe adapted the “carbapenem inactivation method” utilizing piperacillin-tazobactam and ceftriaxone discs on all E. coli and K. pneumoniae isolated from blood and demonstrating piperacillin-tazobactam resistance but with ceftriaxone susceptibility. We utilized pan-susceptible and carbapenem resistance Enterobacteriaceae reference strains as well as third generation cephalosporin resistant, piperacillin-tazobactam susceptible isolates as controls.Results96% of the piperacillin-tazobactam resistant, ceftriaxone susceptible strains demonstrated the capacity to degrade the piperacillin-tazobactam discs while 100% spared the ceftriaxone discs. 75% of the piperacillin-tazobactam susceptible, ceftriaxone resistant control strains spared the piperacillin-tazobactam discs while degrading the ceftriaxone discs.ConclusionThe resistance phenotype observed is due to beta-lactamase production and the modified carbapenem inactivation method can be adapted to probe for other beta-lactamases. Further study is required to definitively identify which beta-lactamase is responsible.Disclosures All Authors: No reported disclosures

Healthcare-Associated Infections: Enterobacteriaceae Bloodstream Infections in the ICU Settings

Background: Bloodstream infections (BSIs) are one of the most frequently observed hospital-acquired infections (HAIs). Objectives: We aimed to describe the epidemiology and drug resistance of hospital-acquired Enterobacteriaceae BSIs and to check for any correlation with the type of hospital care. Methods: In 2015–2018, 333 Enterobacteriaceae isolates were collected from hospitalized internal medicine and surgical patients. The drug-resistance testing was conducted according to the EUCAST recommendations, using the disc-diffusion method to determine resistance to penicillin, cephalosporins, carbapenems, aminoglycosides, fluoroquinolones, and sulfamethoxazole with trimethoprim. Tests confirming the presence of extended-spectrum β-lactamases (ESBLs) and KPC, NDM, and OXA-48 carbapenemases were performed. We determined the minimum inhibitory concentration (MIC) values (mg/L) for selected antibiotics. To detect the resistance genes, a single PCR reaction, a multiplex PCR, and a real-time PCR were conducted. Results: The prevalence rate of Enterobacteriaceae bacilli in BSIs was 23.5%. Penicillin resistance remained at a very high level of almost 100%, with only the piperacillin-tazobactam resistance remaining at 19%–22%. The same was true for cephalosporins: the bacilli have only shown a high susceptibility to cefoperazone with sulbactam (4%–14% of them were resistant). Ciprofloxacin (53%–62%) and sulfamethoxazole with trimethoprim (48–55%) have proven highly resistant. Carbapenems were the only antibiotics with susceptibility at 98%–99%. No difference was found between the types of hospital care (surgical vs nonsurgical) and the levels of antimicrobial resistance in the studied Enterobacteriaceae isolates (Table 1). Conclusions: The high prevalence of Enterobacteriaceae bacilli in BSI is particularly worrying, as is the high rate of resistance to cephalosporins and aminoglycosides, which are often used in the empirical therapy. Unfortunately, our results indicate the need to base the empirical therapy on carbapenems.Funding: This work was supported by a grant from Jagiellonian Univerity Medical School (No. N41/DBS/000053)Disclosures: None

Piperacillin-Tazobactam-Resistant/Third-Generation Cephalosporin-Susceptible Escherichia coli and Klebsiella pneumoniae Isolates: Resistance Mechanisms and In vitro-In vivo Discordance

We previously reported the detection of Escherichia coli and Klebsiella pneumoniae that displayed in vitro piperacillin-tazobactam (TZP) resistance but were susceptible to third-generation cephalosporins (TZP-R/Ceph3-S). In this study, we assessed the phenotypic and genotypic profiles of 12 clinical non-clonal TZP-R/Ceph3-S E. coli and K. pneumoniae isolates derived from bloodstream infections. Whole-genome sequencing revealed that most of the TZP-R/Ceph3-S E. coli and K. pneumoniae isolates examined harbored blaTEM-1 and blaSHV-1 genes, respectively, but none harbored extended-spectrum β-lactamase, AmpC β-lactamase or carbapenemase genes. Increasing the tazobactam concentration from 4 mg/L to 16 mg/L restored TZP in vitro susceptibility among E. coli isolates expressing TEM-1, but had minimal impact on the susceptibility of K. pneumoniae to TZP. Real-time qPCR analysis showed that blaTEM-1 expression was amplified in TZP-R E. coli upon incubation with sub-inhibitory TZP concentrations. Using an immunocompetent murine septicemia model, the efficacy of TZP against TZP-R/Ceph3-S isolates was assessed using TZP doses that mimicked human plasma exposures following intravenous (IV) administration of TZP 4.5 g q6h over 0.5 h for 24h. Efficacy was assessed by survival through 96h. There was high mortality in untreated control mice for all tested isolates. Compared with controls, TZP human-simulated exposure significantly improved survival for all TZP-R/Ceph3-S E. coli and K. pneumoniae isolates examined (P<0.05). Thus, TZP was associated with remarkable in vivo activity against TZP-R/Ceph3-S E. coli and K. pneumoniae despite the observed resistance in vitro.

A meta-analysis of antibiotic resistance rates in Pseudomonas aeruginosa isolated in blood cultures in Turkey between 2007 and 2017.

OBJECTIVE:The prevalence of Pseudomonas aeruginosa has remained stable in recent years, and resistant strains has increased dramatically. In this meta-analysis, we aimed to analyze the P. aeruginosa strains isolated from blood cultures in Turkey during the last 11 years and to reveal their antimicrobial susceptibility.METHODS:Data collected between 2007 and 2017 were divided into two groups as Group-1; 2007–2011 and Group-2; 2012–2017. The differences in antibiotic resistance rates between Group-1 and Group-2 were analyzed. The study data were included according to PRISMA criteria, then meta-analysis was performed.RESULTS:A total of 30 study data from 25 studies were included in the study. The prevalence rate of meropenem (MEM) resistance in P. aeruginosa in Turkey was 25.1% (95% Cl: 20.65–29.83) according to a meta-analysis of 637 isolates. MEM resistance rates in Group-1 and Group-2 were 23.4% (95% Cl: 18.34–28.99) and 29.3% (95% Cl: 21.23–38.23), respectively. The prevalence rate of imipenem (IMP) resistance in P. aeruginosa in Turkey was 26.8% (%95 Cl: 23.40–30.35) according to a meta-analysis of 1421 isolates. IMP resistance rates in Group-1 and Group-2 were 26.2% (95% Cl: 22.41–30.27) and 28.4% (95% Cl: 21.57–35.88), respectively. Ciprofloxacin (CIP) resistance rate was 27.04% (95% Cl: 21.88–32.52) in 1388 isolates. CIP resistance rates in Group-1 and Group-2 were 30.8% (95% Cl: 24.35–37.56) and 18.6% (95% Cl: 10.72–28.11), respectively. The prevalence rate of piperacillin-tazobactam (TZP) resistance in P. aeruginosa in Turkey was 29.2% (95% Cl: 21.058–38.088) according to a meta-analysis of 1030 isolates. TZP resistance rates in Group-1 and Group-2 were 26.1% (95% Cl: 17.76–35.31) and 38.2% (95% Cl: 18.48–60.27), respectively.CONCLUSION:There is a remarkable increase in resistance rates in P. aeruginosa to MEM and TZP in Turkey due to frequent use. Other antibiotics with antipseudomonal effect should be prioritized in the treatment of these infections.

Which antibiotics should we prefer empirical treatment of urinary tract infections in elderly patients?

Aim: Urinary tract infection (UTI) is a major cause of mortality and morbidity in elderly patients. In this cross-sectional study, we aimed to determine the frequency and antibiotic resistance profile of the bacteria causing UTI and contribute to the empirical treatment options in elderly patients.Methods: This study included urine culture results from 347 elderly outpatients who were referred to Karabuk Training and Research Hospital between January 2018 and June 2019. The identification and antibiotic susceptibilities of microorganisms were determined using the BD-Phoenix 100 fully automated system, and the extended-spectrum beta-lactamase (ESBL) positivity was analyzed using the combined disk diffusion method. The results were retrospectively analyzed. Results: The most common pathogens were Escherichia coli (58%), Enterococcus spp. (18%) and Klebsiella pneumoniae (11%). The rate of resistance to ampicillin, amoxicillin-clavulanic acid, trimethoprim-sulfamethoxazole (TMP-SMX), cefixime, and ciprofloxacin, which are oral antibiotics used in the treatment of UTI, was between 30% and 70%. The rate of resistance to nitrofurantoin was 3%. The gentamicin and piperacillin-tazobactam resistance were 12% and 9%, respectively. The ESBL positivity for E. coli and K. pneumoniae were 29% and 49%, respectively (P=0.03).Conclusion: The rates of resistance to oral antibiotics such as ampicillin, amoxicillin-clavulanic acid, TMP-SMX, cefixime, and ciprofloxacin, which are used treatment of UTI, were more than 20%. Therefore, these antibiotics should not be used in the empirical treatment of UTI. Instead, nitrofurantoin may be preferred in the empirical treatment of uncomplicated UTI, or gentamicin and piperacillin-tazobactam, which are parenteral antibiotics that may be used depending on the patient’s clinical condition.

Pooled prevalence and trends of antimicrobial resistance in Pseudomonas aeruginosa clinical isolates over the past 10 years in Turkey: A meta-analysis.

This meta-analysis aimed to evaluate the current prevalence and trends over the past 10 years of Pseudomonas aeruginosa (P. aeruginosa) antimicrobial resistance. Two researches independently searched two national (ULAKBİM, Türk Medline) and two international databases (PubMed, Web of Science) to identify studies on P. aeruginosa resistance to antimicrobials from 2007 to 2017. Homogeneity across studies was assessed using Cochrane guidelines, and total variability due to between-study variations was reflected in the I2 index. A random effects model was developed to estimate the antimicrobial resistance rates and their corresponding 95% CI. Pooled antibiotic resistance rates between 2007-2011 and 2012-2016 were compared to calculate the change in antibiotic resistance over time. Electronic searches with MeSH terms and text words identified 1017 papers. After applying exclusion and inclusion criteria, 45 articles were selected. Pooled resistance prevalence of P. aeruginosa to piperacillin-tazobactam, ceftazidime, cefepime, meropenem, imipenem, ciprofloxacin, gentamicin, amikacin, tobramycin and colistin were 33.9%, 38.6%, 35.6%, 30.1%, 28.0%, 30.7%, 28.2%, 17.8%, 15.7% and 2.2%, respectively. The resistance rates of piperacillin, piperacillin-tazobactam, imipenem, meropenem, amikacin and colistin significantly increased in the second 5 years (P<0.05); however, gentamicin, tobramycin and ciprofloxacin resistance rates significantly decreased (P<0.05). Comparing the resistance rates between the isolates of intensive care unit (ICU) patients and non-ICU patients, meropenem and piperacillin-tazobactam resistance in ICU isolates were significantly higher than non-ICU (P<0.05). These results suggest that antibiotic resistance is high in P. aeruginosa and the trends in antimicrobial resistance continue to increase, mainly in carbapenems and penicillins, in Turkey.

Extensive Gene Amplification as a Mechanism for Piperacillin-Tazobactam Resistance in Escherichia coli.

ABSTRACTAlthough the TEM-1 β-lactamase (BlaTEM-1) hydrolyzes penicillins and narrow-spectrum cephalosporins, organisms expressing this enzyme are typically susceptible to β-lactam/β-lactamase inhibitor combinations such as piperacillin-tazobactam (TZP). However, our previous work led to the discovery of 28 clinical isolates of Escherichia coli resistant to TZP that contained only blaTEM-1. One of these isolates, E. coli 907355, was investigated further in this study. E. coli 907355 exhibited significantly higher β-lactamase activity and BlaTEM-1 protein levels when grown in the presence of subinhibitory concentrations of TZP. A corresponding TZP-dependent increase in blaTEM-1 copy number was also observed, with as many as 113 copies of the gene detected per cell. These results suggest that TZP treatment promotes an increase in blaTEM-1 gene dosage, allowing BlaTEM-1 to reach high enough levels to overcome inactivation by the available tazobactam in the culture. To better understand the nature of the blaTEM-1 copy number proliferation, whole-genome sequence (WGS) analysis was performed on E. coli 907355 in the absence and presence of TZP. The WGS data revealed that the blaTEM-1 gene is located in a 10-kb genomic resistance module (GRM) that contains multiple resistance genes and mobile genetic elements. The GRM was found to be tandemly repeated at least 5 times within a p1ESCUM/p1ECUMN-like plasmid when bacteria were grown in the presence of TZP.

The effects of antibiotic cycling and mixing on antibiotic resistance in intensive care units: a cluster-randomised crossover trial

Whether antibiotic rotation strategies reduce prevalence of antibiotic-resistant, Gram-negative bacteria in intensive care units (ICUs) has not been accurately established. We aimed to assess whether cycling of antibiotics compared with a mixing strategy (changing antibiotic to an alternative class for each consecutive patient) would reduce the prevalence of antibiotic-resistant, Gram-negative bacteria in European intensive care units (ICUs). In a cluster-randomised crossover study, we randomly assigned ICUs to use one of three antibiotic groups (third-generation or fourth-generation cephalosporins, piperacillin-tazobactam, and carbapenems) as preferred empirical treatment during 6-week periods (cycling) or to change preference after every consecutively treated patient (mixing). Computer-based randomisation of intervention and rotated antibiotic sequence was done centrally. Cycling or mixing was applied for 9 months; then, following a washout period, the alternative strategy was implemented. We defined antibiotic-resistant, Gram-negative bacteria as Enterobacteriaceae with extended-spectrum β-lactamase production or piperacillin-tazobactam resistance, and Acinetobacter spp and Pseudomonas aeruginosa with piperacillin-tazobactam or carbapenem resistance. Data were collected for all admissions during the study. The primary endpoint was average, unit-wide, monthly point prevalence of antibiotic-resistant, Gram-negative bacteria in respiratory and perineal swabs with adjustment for potential confounders. This trial is registered with ClinicalTrials.gov, number NCT01293071. Eight ICUs (from Belgium, France, Germany, Portugal, and Slovenia) were randomly assigned and patients enrolled from June 27, 2011, to Feb 16, 2014. 4069 patients were admitted during the cycling periods in total and 4707 were admitted during the mixing periods. Of these, 745 patients during cycling and 853 patients during mixing were present during the monthly point-prevalence surveys, and were included in the main analysis. Mean prevalence of the composite primary endpoint was 23% (168/745) during cycling and 22% (184/853) during mixing (p=0·64), yielding an adjusted incidence rate ratio during mixing of 1·039 (95% CI 0·837-1·291; p=0·73). There was no difference in all-cause in-ICU mortality between intervention periods. Antibiotic cycling does not reduce the prevalence of carriage of antibiotic-resistant, Gram-negative bacteria in patients admitted to the ICU. European Union Seventh Framework Programme.

Automated direct screening for resistance of Gram-negative blood cultures using the BD Kiestra WorkCell.

Early detection of resistance in sepsis due to Gram-negative organisms may lead to improved outcomes by reducing the time to effective antibiotic therapy. Traditional methods of resistance detection require incubation times of 18 to 48h to detect resistance. We have utilised automated specimen processing, digital imaging and zone size measurements in conjunction with direct disc susceptibility testing to develop a method for the rapid screening of Gram-negative blood culture isolates for resistance. Positive clinical blood cultures with Gram-negative organisms were prospectively identified and additional resistant mock specimens were prepared. Broth was plated and antibiotic-impregnated discs (ampicillin, ceftriaxone, piperacillin-tazobactam, meropenem, ciprofloxacin, gentamicin) were added. Plates were incubated, digitally imaged and zone sizes were measured using the BD Kiestra WorkCell laboratory automation system. Minimum, clinically useful, incubation times and optimised zone size cut-offs for resistance detection were determined. We included 187 blood cultures in the study. At 5h of incubation, >90% of plates yielded interpretable results. Using optimised zone size cut-offs, the sensitivity for resistance detection ranged from 87 to 100%, while the specificity ranged from 84.7 to 100%. The sensitivity and specificity for piperacillin-tazobactam resistance detection was consistently worse than for the other agents. Automated direct disc susceptibility screening is a rapid and sensitive tool for resistance detection in Gram-negative isolates from blood cultures for most of the agents tested.