Cefepime versus carbapenems for treatment of AmpC beta-lactamase-producing Enterobacterales bloodstream infections.

PURPOSE: Cefepime is recommended for treating infections caused by AmpC beta-lactamase-producing Enterobacterales (AmpC-PE), though supporting evidence is limited. Therefore, this study compared outcomes associated with cefepime versus carbapenem therapy for bloodstream infections (BSIs) caused by AmpC-PE after phenotypic exclusion of ESBL-co-producing isolates. METHODS: This retrospective cohort study compared definite cefepime versus carbapenem treatment for AmpC-PE BSI in hospitalized patients of the University Hospital Basel, Switzerland, between 01/2015 and 07/2020. Primary outcomes included in-hospital death, renal impairment and neurologic adverse events; secondary outcomes included length of hospital stay and recurrent infection. RESULTS: Two hundred and seventy episodes of AmpC-PE BSI were included, 162, 77 and 31 were treated with a carbapenem, cefepime and other antibiotics, respectively. Patients treated with carbapenems were more likely to be transferred to the ICU on admission and more frequently had central venous catheter as a source of infection. In uni- and multivariable analyses, primary and secondary outcomes did not differ between the two treatment groups, except for more frequent occurrence of neurological adverse events among patients treated with carbapenems and shorter length of hospital stay among survivors treated with cefepime. CONCLUSION: After excluding isolates with phenotypic ESBL-co-production, cefepime was not associated with adverse outcomes compared to carbapenems when used to treat BSIs caused by AmpC-PE. Our study provides evidence to support the use of cefepime as a safe treatment strategy for AmpC-PE BSI, particularly in clinically stable patients without initial renal impairment or increased susceptibility to neurological adverse events.

Whole genome analyses of CMY-2-producing Escherichia coli isolates from humans, animals and food in Germany.

BACKGROUND: Resistance to 3rd-generation cephalosporins in Escherichia coli is mostly mediated by extended-spectrum beta-lactamases (ESBLs) or AmpC beta-lactamases. Besides overexpression of the species-specific chromosomal ampC gene, acquisition of plasmid-encoded ampC genes, e.g. blaCMY-2, has been described worldwide in E. coli from humans and animals. To investigate a possible transmission of blaCMY-2 along the food production chain, we conducted a next-generation sequencing (NGS)-based analysis of 164 CMY-2-producing E. coli isolates from humans, livestock animals and foodstuff from Germany. RESULTS: The data of the 164 sequenced isolates revealed 59 different sequence types (STs); the most prevalent ones were ST38 (n = 19), ST131 (n = 16) and ST117 (n = 13). Two STs were present in all reservoirs: ST131 (human n = 8; food n = 2; animal n = 6) and ST38 (human n = 3; animal n = 9; food n = 7). All but one CMY-2-producing ST131 isolates belonged to the clade B (fimH22) that differed substantially from the worldwide dominant CTX-M-15-producing clonal lineage ST131-O25b clade C (fimH30). Plasmid replicon types IncI1 (n = 61) and IncK (n = 72) were identified for the majority of blaCMY-2-carrying plasmids. Plasmid sequence comparisons showed a remarkable sequence identity, especially for IncK plasmids. Associations of replicon types and distinct STs were shown for IncK and ST57, ST429 and ST38 as well as for IncI1 and ST58. Additional ß-lactamase genes (blaTEM, blaCTX-M, blaOXA, blaSHV) were detected in 50% of the isolates, and twelve E. coli from chicken and retail chicken meat carried the colistin resistance gene mcr-1. CONCLUSION: We found isolates of distinct E. coli clonal lineages (ST131 and ST38) in all three reservoirs. However, a direct clonal relationship of isolates from food animals and humans was only noticeable for a few cases. The CMY-2-producing E. coli-ST131 represents a clonal lineage different from the CTX-M-15-producing ST131-O25b cluster. Apart from the ST-driven spread, plasmid-mediated spread, especially via IncI1 and IncK plasmids, likely plays an important role for emergence and transmission of blaCMY-2 between animals and humans.

Ceftolozane-Tazobactam for the Treatment of Multidrug-Resistant Pseudomonas aeruginosa Infections: Clinical Effectiveness and Evolution of Resistance.

Background: Data on the use of ceftolozane-tazobactam and emergence of ceftolozane-tazobactam resistance during multidrug resistant (MDR)-Pseudomonas aeruginosa infections are limited. Methods: We performed a retrospective study of 21 patients treated with ceftolozane-tazobactam for MDR-P. aeruginosa infections. Whole genome sequencing and quantitative real-time polymerase chain reaction were performed on longitudinal isolates. Results: Median age was 58 years; 9 patients (43%) were transplant recipients. Median simplified acute physiology score-II (SAPS-II) was 26. Eighteen (86%) patients were treated for respiratory tract infections; others were treated for bloodstream, complicated intraabdominal infections, or complicated urinary tract infections. Ceftolozane-tazobactam was discontinued in 1 patient (rash). Thirty-day all-cause and attributable mortality rates were 10% (2/21) and 5% (1/21), respectively; corresponding 90-day mortality rates were 48% (10/21) and 19% (4/21). The ceftolozane-tazobactam failure rate was 29% (6/21). SAPS-II score was the sole predictor of failure. Ceftolozane-tazobactam resistance emerged in 3 (14%) patients. Resistance was associated with de novo mutations, rather than acquisition of resistant nosocomial isolates. ampC overexpression and mutations were identified as potential resistance determinants. Conclusions: In this small study, ceftolozane-tazobactam was successful in treating 71% of patients with MDR-P. aeruginosa infections, most of whom had pneumonia. The emergence of ceftolozane-tazobactam resistance in 3 patients is worrisome and may be mediated in part by AmpC-related mechanisms. More research on treatment responses and resistance during various types of MDR-P. aeruginosa infections is needed to define ceftolozane-tazobactam's place in the armamentarium.

The ß-lactamase gene regulator AmpR is a tetramer that recognizes and binds the D-Ala-D-Ala motif of its repressor UDP-N-acetylmuramic acid (MurNAc)-pentapeptide.

Inducible expression of chromosomal AmpC ß-lactamase is a major cause of ß-lactam antibiotic resistance in the Gram-negative bacteria Pseudomonas aeruginosa and Enterobacteriaceae. AmpC expression is induced by the LysR-type transcriptional regulator (LTTR) AmpR, which activates ampC expression in response to changes in peptidoglycan (PG) metabolite levels that occur during exposure to ß-lactams. Under normal conditions, AmpR represses ampC transcription by binding the PG precursor UDP-N-acetylmuramic acid (MurNAc)-pentapeptide. When exposed to ß-lactams, however, PG catabolites (1,6-anhydroMurNAc-peptides) accumulate in the cytosol, which have been proposed to competitively displace UDP-MurNAc-pentapeptide from AmpR and convert it into an activator of ampC transcription. Here we describe the molecular interactions between AmpR (from Citrobacter freundii), its DNA operator, and repressor UDP-MurNAc-pentapeptide. Non-denaturing mass spectrometry revealed AmpR to be a homotetramer that is stabilized by DNA containing the T-N11-A LTTR binding motif and revealed that it can bind four repressor molecules in an apparently stepwise manner. A crystal structure of the AmpR effector-binding domain bound to UDP-MurNAc-pentapeptide revealed that the terminal D-Ala-D-Ala motif of the repressor forms the primary contacts with the protein. This observation suggests that 1,6-anhydroMurNAc-pentapeptide may convert AmpR into an activator of ampC transcription more effectively than 1,6-anhydroMurNAc-tripeptide (which lacks the D-Ala-D-Ala motif). Finally, small angle x-ray scattering demonstrates that the AmpR·DNA complex adopts a flat conformation similar to the LTTR protein AphB and undergoes only a slight conformational change when binding UDP-MurNAc-pentapeptide. Modeling the AmpR·DNA tetramer bound to UDP-MurNAc-pentapeptide predicts that the UDP-MurNAc moiety of the repressor participates in modulating AmpR function.

Marvelous but Morbid: Infective endocarditis due to Serratia marcescens.

A 46-year-old man with HIV infection and active intravenous drug use presented with approximately two weeks of fevers and body aches. On physical examination he was somnolent, had a new systolic murmur, bilateral conjunctival hemorrhages, diffuse petechiae, and left-sided arm weakness. Echocardiography revealed a large mitral valve vegetation and brain imaging demonstrated numerous embolic infarctions. Blood cultures grew Serratia marcescens. Despite aggressive treatment with meropenem the patient died due to intracranial hemorrhage complicated by herniation. Serratia marcescens is an uncommon cause of infective endocarditis. While this disease has historically been associated with intravenous drug use, more recent reports suggest that it is now largely a consequence of opportunistic infections of the chronically ill. Our case highlights several characteristic features of this infection, including isolation of a non-pigmented strain of the organism, an antibiotic susceptibility profile suggestive of AmpC ß-lactamase production, and rapid clinical deterioration with multiple embolic complications resulting in death. In this review we discuss the history, epidemiology, and management of endovascular infections due to Serratia spp., emphasizing the continued importance of considering this organism in the differential diagnosis of endocarditis among intravenous drug users and as a potential indication for surgical therapy.

Beta-lactamase Enzymes of Clinical Pseudomonas aeruginosa Strains.

OBJECTIVES: In this study, the production of extended spectrum beta-lactamase (ESBL), metallo-betalacatamase (MBL) and AmpC beta-lactamase enzymes of Pseudomonas aeruginosa (P aeruginosa) strains which were isolated from clinical samples were investigated. AmpC gene was also detected by the polymerase chain reaction (PCR) analysis. METHODS: A hundred strains of P aeruginosa were included in the study. The presence of ESBL was investigated with combined disk confirmation test, MBL was investigated with E-test method and AmpC beta-lactamase was investigated with disk induction test. In order to detect the production of AmpC betalactamase genotypically, the PCR method was used. RESULTS: Only one strain was found to be MBL positive. Four per cent of strains were found to be ESBL positive. AmpC beta-lactamase production was positive in 73% of the strains with disk induction test. AmpC gene was detected in 96% of the studied strains with the PCR method. CONCLUSION: While ESBL and MBL rates in this study were significantly lower than those found in other studies, the rate of AmpC beta-lactamase was higher. Although AmpC gene was detected in some strains (23%), they were not found to produce AmpC beta-lactamase with disk induction test.

Rare Plasmid-Mediated AmpC Beta-Lactamase DHA-1 Located on Easy Mobilized IS26-Related Genetic Element Detected in Escherichia coli from Livestock and Food in Germany.

AmpC beta-lactamases cause resistance to third-generation cephalosporins, including beta-lactamase inhibitors. In Escherichia coli from the German food production chain, the majority of AmpC beta-lactamase activity can be attributed to plasmid-mediated CMY-2 or overproduction of chromosomal AmpC beta-lactamase, but occasionally other enzymes like DHA-1 are involved. This study investigated the prevalence of the AmpC beta-lactamase DHA-1 in ESBL/AmpC-producing E. coli (n = 4706) collected between 2016 and 2021 as part of a German antimicrobial resistance monitoring program along the food chain. Eight isolates (prevalence < 0.2%) were detected and further characterized by PFGE, transformation and conjugation experiments as well as short-read and long-read sequencing. All eight strains harbored blaDHA-1 together with qnrB4, sul1 and mph(A) resistance genes on an IS26 composite transposon on self-transferable IncFII or IncFIA/FIB/II plasmids. During laboratory experiments, activation of the translocatable unit of IS26-bound structures was observed. This was shown by the variability of plasmid sizes in original isolates, transconjugants or transferred plasmids, and correspondingly, duplications of resistance fragments were found in long-read sequencing. This activation could be artificial due to laboratory handling or naturally occurring. Nevertheless, DHA-1 is a rare AmpC beta-lactamase in livestock and food in Germany, and its dissemination will be monitored in the future.

Phenotypic and genotypic identification of class C and D ß-lactamases in clinical isolates of Pseudomonas aeruginosa: a cross-sectional study.

Background and Aims: The emergence of Pseudomonas aeruginosa (P. aeruginosa) antibiotic resistance is an important public health problem worldwide that can negatively affect infection control. Therefore, obtaining knowledge about antibiotic resistance mechanisms is necessary for infection control policies. This study aimed to determine the frequency of class C and D ß-lactamases in P. aeruginosa strains isolated from patients referred to Ardabil hospitals using phenotypic and genotypic tests. Methods: Phenotypic detection of ß-lactamases including AmpC cephalosporinase, oxacillinase (OXA)-type extended-spectrum ß-lactamases (ESBLs), and OXA-type carbapenemases were performed using the disk diffusion-based methods. Amplification of genes encoding classes C (ampC and FOX genes) and D (OXA-1, OXA-2, OXA-10, OXA-23, and OXA-48 genes) ß-lactamases was performed using the polymerase chain reaction (PCR) method. A quantitative reverse transcription PCR (qRT-PCR) method was used to determine the expression level of the ampC gene among multiple drug-resistant and imipenem-resistant P. aeruginosa strains. Results: In phenotypic tests, the prevalence of AmpC cephalosporinase, OXA-type ESBLs, and OXA-type carbapenemases were 52.5%, 7.2%, and 95.8%, respectively. In genotypic tests, the prevalence of ampC, FOX, OXA-1, OXA-2, OXA-10, OXA-23, and OXA-48 genes were 100%, 0%, 4.3%, 60.8%, 42%, 29.7%, and 2.9%, respectively. In addition, the ampC gene overexpression was seen in 16 (33.3%) drug-resistant P. aeruginosa clinical isolates. Conclusion: Given the presence of class C and D ß-lactamases in clinical isolates of P. aeruginosa in Ardabil hospitals, early detection of these strains can help prevent the spread of resistant strains in hospital environments and subsequent treatment failure.

Therapeutic challenges of ESBLS and AmpC beta-lactamase producers in a tertiary care center.

BACKGROUND: Resistance to broad-spectrum beta lactams mediated by extended spectrum beta lactamases (ESBLs) and AmpC beta lactamases (AmpC ßLs) enzymes is an increasing problem worldwide. Determination of their prevalence is essential to formulate an effective antibiotic policy and hospital infection control measures. Present study was undertaken to determine the prevalence of ESBL and AmpC ßL producers in ICU of a tertiary care center. METHODS: A total of 262 clinical isolates comprising of Escherichia coli, Klebsiella pneumoniae and Proteus mirabilis that were recovered from various clinical specimens over a one year period, were studied. Antibiogram profile was determined to conventionally used antibiotics, along with recommended tests for detection of ESBL and AmpC ßL production. RESULTS: 40.07% (105/262) were found to be ESBL producers, 14.8% (39/262) were AmpC bL producers. The coexistence of ESBL and AmpC ßL producers was detected in 9.9% (26/262) of the isolates. CONCLUSION: Screening of multidrug resistant bacteria especially belonging to the Enterobacteriaceae poses considerable therapeutic challenges in critical care patients because of the production of ESBL and AmpC ßL. Strategies to keep a check on the emergence of such drug resistant microbes by hospital environmental surveillance and laboratory monitoring should form an important aspect of Hospital Infection control policy guidelines.

Analysis of BlaEC family class C beta-lactamase.

Recent years have witnessed an increased prevalence of intrinsic and acquired beta-lactamase-producing bacteria, severely limiting human and veterinary medicine therapeutic options. The present study aimed to design specific oligonucleotides for rapid PCR detection of the cephalosporinase-encoding gene blaEC (BlaEC family class C beta-lactamase). A total of three primers were designed to detect 2281 variants of the blaEC gene and two sets of primer pairs were also tested against DNA from 11 strains. The study indicates that the proposed primers should be able to detect 100% of all described blaEC genes in different bacterial strains and monitor their spread. After comparing the amino acid sequences, a phylogenetic tree was created based on the presence of conserved amino acids and homologous motifs. More than 24 760 mutations in BlaEC enzymes have been identified. The mutations involving 371 amino acid positions and these hotspots can change the structure and activity of the monitored enzymes. We predicted several BlaEC enzymes with a broadened substrate activity against higher-generation cephalosporins.

Characterization of beta-lactamase producing Enterobacterales isolated from an urban community wastewater treatment plant in Iran.

Background and Objectives: he occurrence and characteristics of Extended Spectrum- and AmpC-ß-lactamase producing Enterobacterales (ESBL-PE and AmpC-PE) in an urban wastewater treatment plant (WWTP) were investigated. Materials and Methods: A total of 30 wastewater samples were collected from all sections of WWTP. Enterobacterales were isolated and identified using standard microbiological tests. The antibiotic resistance profile was determined by the Kirby-Bauer disk diffusion method. Phenotypic screening for ESBL-PE and AmpC-PE isolates was performed by double-disk synergy and boronic acid disk potentiation tests, respectively. The isolates were examined for AmpC- and ESBL-encoding genes by PCR and sequencing methods. Results: Among 146 Enterobacterales isolates, 8.9% (n=13) [ESBL-only; 5.48% (n=8) and ESBL + AmpC; 3.42% (n=5)] were ESBL-producers and 15.75% (n=23) [AmpC-only; 12.33% (n=18) and ESBL + AmpC; 3.42% (n=5)] AmpC-producers. Hafnia spp. with 33.33% (n=1/3) and E. coli with 20.58% (n=7/34) [ESBL-only; 17.64% (n=6/34) and ESBL + AmpC; 2.94% (n=1/34)] were the most common ESBL-producing bacteria. Enterobacter spp. with 37.50% (n=6/16) of isolates were the most common AmpC-producing organisms. ESBL- and/or AmpC-producing isolates were identified in all parts of the WWTP including 80% (n=8/10) of samples taken from effluent. Among ESBL-producing isolates, bla CTX-M , bla TEM, and bla SHV ESBL-encoding genes were found in 61.5% (n=8), 15.3% (n=2), and 7.7% (n=1) of isolates, respectively. All CTX-M-type enzymes belonged to the CTX-M-1 group and CTX-M-15 subgroup. bla TEM and bla SHV type genes belonged to bla TEM-20 and bla HSV-12 subtypes, respectively. bla DHA with 73.9% (n=17/23), and bla CIT and bla FOX with 30.4% (n=7/23) each, were the most common AmpC-encoding genes among AmpC-producing isolates. Overall, 75% of ESBL-producing and 55.5% of AmpC-producing isolates exhibited multi-drug resistance phenotypes. The organisms were most resistant against ampicillin (82.2%) nalidixic acid (43.8%) and cephalexin (41.1%). Conclusion: ESBL- and AmpC-producing Enterobacterales spp. with diverse genetic resistance backgrounds in WWTP effluent poses a significant risk to public health.

Building an International One Health Strain Level Database to Characterise the Epidemiology of AMR Threats: ESBL-AmpC Producing E. coli as An Example-Challenges and Perspectives.

Antimicrobial resistance (AMR) is one of the top public health threats nowadays. Among the most important AMR pathogens, Escherichia coli resistant to extended spectrum cephalosporins (ESC-EC) is a perfect example of the One Health problem due to its global distribution in animal, human, and environmental sources and its resistant phenotype, derived from the carriage of plasmid-borne extended-spectrum and AmpC ß-lactamases, which limits the choice of effective antimicrobial therapies. The epidemiology of ESC-EC infection is complex as a result of the multiple possible sources involved in its transmission, and its study would require databases ideally comprising information from animal (livestock, companion, wildlife), human, and environmental sources. Here, we present the steps taken to assemble a database with phenotypic and genetic information on 10,763 ESC-EC isolates retrieved from multiple sources provided by 13 partners located in eight European countries, in the frame of the DiSCoVeR Joint Research project funded by the One Health European Joint Programme (OH-EJP), along with its strengths and limitations. This database represents a first step to help in the assessment of different geographical and temporal trends and transmission dynamics in animals and humans. The work performed highlights aspects that should be considered in future international efforts, such as the one presented here.

Occurrence, Typing, and Resistance Genes of ESBL/AmpC-Producing Enterobacterales in Fresh Vegetables Purchased in Central Israel.

Beta-lactam resistance can lead to increased mortality, higher healthcare expenses, and limited therapeutic options. The primary mechanism of beta-lactam resistance is the production of extended-spectrum beta-lactamases (ESBL) and AmpC beta-lactamases. The spread of beta-lactamase-producing Enterobacterales via the food chain may create a resistance reservoir. The aims of this study were to determine the prevalence of ESBL/AmpC-producing Enterobacterales in vegetables, to examine the association between EBSL/AmpC-producing bacteria and types of vegetables, packaging, and markets, and to investigate the genetic features of ESBL-producing isolates. The antibiotic susceptibilities were determined using VITEK. Phenotypic ESBL/AmpC production was confirmed using disk diffusion. ESBL-producing isolates were subjected to Fourier-transform infrared (FT-IR) spectroscopy and to whole genome sequencing using Oxford Nanopore sequencing technology. Of the 301 vegetable samples, 20 (6.6%) were positive for ESBL producers (16 Klebsiella pneumoniae and 4 Escherichia coli), and 63 (20.9%) were positive for AmpC producers (56 Enterobacter cloacae complex, 4 Enterobacter aerogenes/cancerogenus, and 3 Pantoea spp., Aeromonas hydrophila, and Citrobacter braakii). The blaCTX-M and blaSHV genes were most common among ESBL-producing isolates. The beta-lactamase genes of the ESBL producers were mainly carried on plasmids. Multilocus sequence typing and FT-IR typing revealed high diversity among the ESBL producers. AmpC producers were significantly more common in leafy greens and ESBL producers were significantly less common in climbing vegetables. The presence of ESBL/AmpC-producing Enterobacterales in raw vegetables may contribute to the dissemination of resistance genes in the community.

Acquisition of extended-spectrum cephalosporin-resistant Gram-negative bacteria: epidemiology and risk factors in a 6-year cohort of 507 severe trauma patients.

OBJECTIVES: Severe trauma patients are at higher risk of infection and often exposed to antibiotics, which could favor acquisition of antimicrobial resistance. In this study, we aimed to assess prevalence, acquisition, and factors associated with acquisition of extended-spectrum cephalosporin-resistant Gram-negative bacteria (ESCR-GNB) in severe trauma patients. METHODS: We conducted a retrospective monocentric cohort study in a French level one Regional Trauma Centre between 01 January 2010and 31 December 2015. Patients admitted for ≥ 7 days, with an Injury Severity Score ≥ 15, and ≥ 1 microbiological sample were included in the analysis. Prevalence and acquisition rate of ESCR-GNB were determined then, factors associated with ESCR-GNB acquisition were assessed using a Cox model. RESULTS: Of 1873 patients admitted during the study period, 507 were included (median Injury Severity Score = 29 [22-34] and median intensive care unit length of stay = 16 days [10-28]). Most of them (450; 89%) had an antimicrobial therapy. Prevalence of ESCR-GNB increased from 13% to 33% during intensive care unit stay, bringing the ESCR-GNB acquisition rate to 29%. Acquisition of ESCR-GNB was mainly related to AmpC beta-lactamase Enterobacterales and was independently associated with mechanical ventilation needs (hazard ratio [HR] = 6.39; 95% confidence interval [CI] [1.51-27.17]; P = 0.01), renal replacement therapy needs (HR = 2.44; 95% CI [1.24-4.79]; P = 0.01), exposure to cephalosporins (HR = 1.06; 95% CI [1.01-1.12]; P = 0.02), and/or combination therapy with non-beta-lactam antibiotics such as vancomycin, linezolid, clindamycin, or metronidazole (HR = 1.03; 95% CI [1.01-1.06]; P = 0.02). CONCLUSIONS: Acquisition of ESCR-GNB was prevalent in severe trauma patients. Our results suggest selecting antibiotics with caution, particularly in the most severely ill.

Early Treatment Outcomes for Bloodstream Infections Caused by Potential AmpC Beta-Lactamase-Producing Enterobacterales with Focus on Piperacillin/Tazobactam: A Retrospective Cohort Study.

The Gram-negative bacilli Serratia spp., Providencia spp., Morganella morganii, Citrobacter freundii complex, Enterobacter spp. and Klebsiella aerogenes are common Enterobacterales that may harbor inducible chromosomal AmpC beta-lactamase genes. The purpose of the present study was to evaluate treatment outcomes and identify predictors of early treatment response in patients with bloodstream infection caused by potential AmpC beta-lactamase-producing Enterobacterales (SPICE-BSI). This cohort study included adult patients with SPICE-BSI hospitalized between 01/2011 and 02/2019. The primary outcome was early treatment response 72 h after the start of active treatment, defined as survival, hemodynamic stability, improved or stable SOFA score, resolution of fever and leukocytosis and microbiologic resolution. Among 295 included patients, the most common focus was the lower respiratory tract (27.8%), and Enterobacter spp. (n = 155) was the main pathogen. The early treatment response rate was significantly lower (p = 0.006) in the piperacillin/tazobactam group (17/81 patients, 21.0%) than in the carbapenem group (40/82 patients, 48.8%). Independent negative predictors of early treatment response (p < 0.02) included initial SOFA score, liver comorbidity and empiric piperacillin/tazobactam treatment. In vitro piperacillin/tazobactam resistance was detected in three patients with relapsed Enterobacter-BSI and initial treatment with piperacillin/tazobactam. In conclusion, our findings show that piperacillin/tazobactam might be associated with early treatment failure in patients with SPICE-BSI.

High Prevalence and Mechanism Associated With Extended Spectrum Beta-Lactamase-Positive Phenotype in Laribacter hongkongensis.

In this study, we reported the prevalence and mechanism associated with the extended-spectrum beta-lactamase (ESBL)-positive phenotype in Laribacter hongkongensis isolated from patients and fish. Using the inhibition zone enhancement test, 20 (95.2%) of the 21 patient strains and 8 (57.1%) of the 14 fish strains were tested ESBL-positive. However, ESBL genes, including SHV, TEM, CTX-M, GES, and PER, were not detected in all of these 28 L. hongkongensis isolates. No ESBL gene could be detected in either the complete genome of L. hongkongensis HLHK9 or the draft genome of PW3643. PCR and DNA sequencing revealed that all the 35 L. hongkongensis isolates (showing both ESBL-positive and ESBL-negative phenotypes) were positive for the ampC gene. When the AmpC deletion mutant, HLHK9ΔampC, was subject to the zone enhancement test, the difference of zone size between ceftazidime/clavulanate and ceftazidime was less than 5 mm. When boronic acid was added to the antibiotic disks, none of the 28 "ESBL-positive" isolates showed a ≥ 5 mm enhancement of inhibition zone size diameter between ceftazidime/clavulanate and ceftazidime and between cefotaxime/clavulanate and cefotaxime. A high prevalence (80%) of ESBL-positive phenotype is present in L. hongkongensis. Overall, our results suggested that the ESBL-positive phenotype in L. hongkongensis results from the expression of the intrinsic AmpC beta-lactamase. Confirmatory tests should be performed before issuing laboratory reports for L. hongkongensis isolates that are tested ESBL-positive by disk diffusion clavulanate inhibition test.

High ß-Lactam and Quinolone Resistance of Enterobacteriaceae from the Respiratory Tract of Sheep and Goat with Respiratory Disease.

During the last decade's increase of antimicrobial resistance (AMR) in animals, animal-human transmission has become a major threat. Therefore, the present study aimed to evaluate the genetic basis of AMR in Gram-negative bacteria recovered from sheep and goats with respiratory disease. Nasal and ocular swabs were collected from 69 diseased animals, and 76 Gram-negative bacterial isolates were identified from 59 animals. All isolates were checked phenotypically for resistance and genotypically for different resistance mechanisms, including ß-lactam, quinolone, and aminoglycoside resistance. Our results demonstrated that 9.2% (95% CI 4.5-17.8%) of the isolates were multidrug-resistant, with high resistance rates to ß-lactams and quinolones, and 11.8% (95% CI 6.4-21%) and 6.6% (95% CI 2.8-14.5%) of the isolates were phenotypically positive for AmpC and ESBL, respectively. Genotypically, blaTEM was the most identified ß-lactamase encoding gene in 29% (95% CI 20-40%) of the isolates, followed by blaSHV (14.5%, 95% CI 8.3-24.1%) and blaCTX-M (4%, 95% CI 1.4-11%). Furthermore, 7.9% (95% CI 3.7-16.2%) of the isolates harbored plasmid-mediated quinolone resistance gene qnrS. Our study revealed for the first time to our knowledge high ß-lactam and quinolone resistance associated with the bacteria recovered from sheep and one goat with respiratory disease. Furthermore, different antimicrobial resistant determinants were identified for the first time from animals in Africa, such as blaLEN-13/55, blaTEM-176 and blaTEM-198/214. This study highlights the potential role of sheep and goats in disseminating AMR determinants and/or resistant bacteria to humans. The study regenerates interest for the development of a One Health approach to combat this formidable problem.

Relating the prevalence of plasmid-mediated AmpC beta-lactamase genes to aquatic environmental factors.

It is important that environmental parameters that may affect the prevalence of AmpC beta-lactamase genes are investigated to devise frameworks for their surveillance, management and prevention. The aim of this study was thus to determine which environmental parameters are associated with the prevalence of clinically relevant AmpC beta-lactamase genes in aquatic systems. River water was sampled from seven sites in the Crocodile West River, South Africa. Physical-chemical parameters, metal levels and beta-lactam levels were measured. Environmental DNA was extracted from the water samples and six AmpC beta-lactamase gene groups (ACC, ACT/MIR, BIL/LAT/CMY, DHA, FOX, MOX/CMY) were quantified using quantitative PCR. Additionally, 16S rRNA gene metabarcoding analyses were performed on eDNA for each site and metabolic pathways were predicted using PICRUST2. Network analysis was performed to establish co-occurrences of AmpC genes with environmental factors. Quantification results indicated that AmpC gene copy numbers were significantly high (Kruskal Wallis H Test, p < 0.05) at Sites 1-3 of the Crocodile West River. In contrast, no significant changes regarding environmental factors were observed across the seven sites. Results of network analysis indicated that the AmpC gene groups had limited associations with all the environmental parameters, except for some key bacterial families, specifically Pseudomonadaceae, Aeromonadaceae and Enterobacteriaceae. A significant positive correlation between population density and AmpC genes suggested that in more densely populated areas more faecal pollution will be prevalent which is associated with high AmpC gene levels. Areas such as these are also likely to be linked with more antibiotic use which supports the notion that pre-selection of AmpC genes occurs before entering the aquatic environment. Moreover, it was demonstrated that prevalent selectors of AmpC genes do not ensure that continuous selection occurs in an aquatic environment. This information could be vital in future detection and management of AmpC genes in aquatic systems.

Ceftolozane-tazobactam versus meropenem for definitive treatment of bloodstream infection due to extended-spectrum beta-lactamase (ESBL) and AmpC-producing Enterobacterales ("MERINO-3"): study protocol for a multicentre, open-label randomised non-inferiority trial.

BACKGROUND: Extended-spectrum beta-lactamase (ESBL) and AmpC-producing Enterobacterales are common causes of bloodstream infection. ESBL-producing bacteria are typically resistant to third-generation cephalosporins and result in a sizeable economic and public health burden. AmpC-producing Enterobacterales may develop third-generation cephalosporin resistance through enzyme hyper-expression. In no observational study has the outcome of treatment of these infections been surpassed by carbapenems. Widespread use of carbapenems may drive the development of carbapenem-resistant Gram-negative bacilli. METHODS: This study will use a multicentre, parallel group open-label non-inferiority trial design comparing ceftolozane-tazobactam and meropenem in adult patients with bloodstream infection caused by ESBL or AmpC-producing Enterobacterales. Trial recruitment will occur in up to 40 sites in six countries (Australia, Singapore, Italy, Spain, Saudi Arabia and Lebanon). The sample size is determined by a predefined quantity of ceftolozane-tazobactam to be supplied by Merck, Sharpe and Dohme (MSD). We anticipate that a trial with 600 patients contributing to the primary outcome analysis would have 80% power to declare non-inferiority with a 5% non-inferiority margin, assuming a 30-day mortality of 5% in both randomised groups. Once randomised, definitive treatment will be for a minimum of 5 days and a maximum of 14 days with the total duration determined by treating clinicians. Data describing demographic information, risk factors, concomitant antibiotics, illness scores, microbiology, multidrug-resistant organism screening, discharge and mortality will be collected. DISCUSSION: Participants will have bloodstream infection due to third-generation cephalosporin non-susceptible E. coli and Klebsiella spp. or Enterobacter spp., Citrobacter freundii, Morganella morganii, Providencia spp. or Serratia marcescens. They will be randomised 1:1 to ceftolozane-tazobactam 3 g versus meropenem 1 g, both every 8 h. Secondary outcomes will be a comparison of 14-day all-cause mortality, clinical and microbiological success at day 5, functional bacteraemia score, microbiological relapse, new bloodstream infection, length of hospital stay, serious adverse events, C. difficile infection, multidrug-resistant organism colonisation. The estimated trial completion date is December 2024. TRIAL REGISTRATION: The MERINO-3 trial is registered under the US National Institute of Health ClinicalTrials.gov register, reference number: NCT04238390 . Registered on 23 January 2020.

In Vitro Activity of Cefotetan against ESBL-Producing Escherichia coli and Klebsiella pneumoniae Bloodstream Isolates from the MERINO Trial.

Extended-spectrum-beta-lactamase (ESBL)-producing Enterobacterales continue to pose a major threat to human health worldwide. Given the limited therapeutic options available to treat infections caused by these pathogens, identifying additional effective antimicrobials or revisiting existing drugs is important. Ceftriaxone-resistant Escherichia coli and Klebsiella pneumoniae containing CTX-M-type ESBLs or AmpC, in addition to narrow-spectrum OXA and SHV enzymes, were selected from blood culture isolates obtained from the MERINO trial. Isolates had previously undergone whole-genome sequencing (WGS) to identify antimicrobial resistance genes. Cefotetan MICs were determined by broth microdilution (BMD) testing with a concentration range of 0.125 to 64 mg/liter; CLSI breakpoints were used for susceptibility interpretation. BMD was performed using an automated digital antibiotic dispensing platform (Tecan D300e). One hundred ten E. coli and 40 K. pneumoniae isolates were used. CTX-M-15 and CTX-M-27 were the most common beta-lactamases present; only 7 isolates had coexistent ampC genes. Overall, 98.7% of isolates were susceptible, with MIC50s and MIC90s of 0.25 mg/liter and 2 mg/liter (range, ≤0.125 to 64 mg/liter), respectively. MICs appeared higher among isolates with ampC genes present, with an MIC50 of 16 mg/liter, than among those containing CTX-M-15, which had an MIC50 of only 0.5 mg/liter. Isolates with an ampC gene exhibited an overall susceptibility of 85%. Presence of a narrow-spectrum OXA beta-lactamase did not appear to alter the cefotetan MIC distribution. Cefotetan demonstrated favorable in vitro efficacy against ESBL-producing E. coli and K. pneumoniae bloodstream isolates. IMPORTANCE Carbapenem antibiotics remain the treatment of choice for severe infection due to ESBL- and AmpC-producing Enterobacterales. The use of carbapenems is a major driver of the emergence of carbapenem-resistant Gram-negative bacilli, which are often resistant to most available antimicrobials. Cefotetan is a cephamycin antibiotic developed in the 1980s that demonstrates enhanced resistance to beta-lactamases and has a broad spectrum of activity against Gram-negative bacteria. Cefotetan holds potential to be a carbapenem-sparing treatment option. Data on the in vitro activity of cefotetan against ESBL-producing Enterobacterales remain scarce. Our study assessed the in vitro activity of cefotetan against ceftriaxone-nonsusceptible blood culture isolates obtained from patients enrolled in the MERINO trial.