Piperacillin/tazobactam Susceptibility Test Interpretive Criteria for Enterobacterales: Recommendations from the United States Committee on Antimicrobial Susceptibility Testing.

The in vitro susceptibility testing interpretive criteria (STIC) for TZP against Enterobacterales were recently updated by the Food and Drug Administration (FDA), Clinical & Laboratory Standards Institute (CLSI), and European Committee on Antimicrobial Susceptibility Testing (EUCAST). The United States Committee on Antimicrobial Susceptibility Testing (USCAST) also recently reviewed TZP STIC for Enterobacterales and arrived at different STIC for Enterobacterales and herein we explain our recommendations and rationale behind them. Based on our review of the available data, USCAST does not recommend TZP STIC for certain Enterobacterales species that have a moderate to high likelihood of clinically significant AmpC production (E. cloacae, C. freundii, and K. aerogenes only) or for third-generation cephalosporin-non-susceptible (3GC-NS) Enterobacterales. USCAST recommends a TZP susceptibility breakpoint of ≤ 16/4 mg/L for third-generation cephalosporin-susceptible (3GC-S) Enterobacterales but only endorses the use of extended infusion TZP regimens for patients with infections due to these pathogens.

Respiratory carriage of hypervirulent Klebsiella pneumoniae by indigenous populations of Malaysia.

Klebsiella pneumoniae is a Gram-negative Enterobacteriaceae that is classified by the World Health Organisation (WHO) as a Priority One ESKAPE pathogen. South and Southeast Asian countries are regions where both healthcare associated infections (HAI) and community acquired infections (CAI) due to extended-spectrum ß-lactamase (ESBL)-producing and carbapenem-resistant K. pneumoniae (CRKp) are of concern. As K. pneumoniae can also exist as a harmless commensal, the spread of resistance genotypes requires epidemiological vigilance. However there has been no significant study of carriage isolates from healthy individuals, particularly in Southeast Asia, and specially Malaysia. Here we describe the genomic analysis of respiratory isolates of K. pneumoniae obtained from Orang Ulu and Orang Asli communities in Malaysian Borneo and Peninsular Malaysia respectively. The majority of isolates were K. pneumoniae species complex (KpSC) 1 K. pneumoniae (n = 53, 89.8%). Four Klebsiella variicola subsp. variicola (KpSC3) and two Klebsiella quasipneumoniae subsp. similipneumoniae (KpSC4) were also found. It was discovered that 30.2% (n = 16) of the KpSC1 isolates were ST23, 11.3% (n = 6) were of ST65, 7.5% (n = 4) were ST13, and 13.2% (n = 7) were ST86. Only eight of the KpSC1 isolates encoded ESBL, but importantly not carbapenemase. Thirteen of the KpSC1 isolates carried yersiniabactin, colibactin and aerobactin, all of which harboured the rmpADC locus and are therefore characterised as hypervirulent. Co-carriage of multiple strains was minimal. In conclusion, most isolates were KpSC1, ST23, one of the most common sequence types and previously found in cases of K. pneumoniae infection. A proportion were hypervirulent (hvKp) however antibiotic resistance was low.

Uropathogenic E. coli and Hybrid Pathotypes in Mexican Women with Urinary Tract Infections: A Comprehensive Molecular and Phenotypic Overview.

Uropathogenic Escherichia coli (UPEC) is the main cause of urinary tract infections (UTIs) and carries virulence and resistance factors often found in mobilizable genetic elements, such as plasmids or pathogenicity islands (PAIs). UPEC is part of the extraintestinal pathogenic E. coli (ExPEC), but hybrid strains possessing both diarrheagenic E. coli (DEC) and ExPEC traits, termed "hypervirulent", present a significant health threat. This study assessed the prevalence of UPEC PAIs, ExPEC sequence types (ST), DEC genes, carbapenemase and extended-spectrum ß-lactamase (ESBL) phenotypes, resistance genotypes, and plasmids in 40 clinical isolates of UPEC. Results showed that 72.5% of isolates had PAIs, mainly PAI IV536 (53%). ESBL phenotypes were found in 65% of ß-lactam-resistant isolates, with 100% of carbapenem-resistant isolates producing carbapenemase. The predominant ESBL gene was blaCTX-M-2 (60%), and the most common resistance gene in fluoroquinolone and aminoglycoside-resistant isolates was aac(6')Ib (93%). Plasmids were present in 57% of isolates, and 70% belonged to the ST131 clonal group. Molecular markers for DEC pathotypes were detected in 20 isolates, with 60% classified as hybrid pathotypes. These findings indicate significant pathogenic potential and the presence of hybrid pathotypes in E. coli UTI clinical isolates in the Mexican population.

Insights into the activity of cefiderocol against PER-2 producing Enterobacterales.

The PER-2 ß-lactamase is a unique class A enzyme conferring broad spectrum cephalosporin resistance. In this study, we explored the stability of cefiderocol (FDC) against PER-2 ß-lactamase to gain insights into structure activity relationships (SAR) of this synthetic siderophore-conjugated antibiotic. Herein, we show that the MICs of FDC for PER-2 producing isolates and transformants ranged between 0.125 and 64 µg/mL; diazabicyclooctanes (DBOs) reduced the MIC values. In PER-2 mutants, MIC values decreased up to 10-12 dilutions in agreement with previous observations especially in the case of Arg220 substitutions. Catalytic efficiency for PER-2 was 0.072 µM-1 s-1, comparable with PER-1 (0.046 µM-1 s-1) and NDM-1 (0.067 µM-1 s-1). In silico models revealed that FDC within the active site of PER-2 demonstrates unique interactions as a result of the inverted Ω loop fold and extension of the ß3-ß4 connecting loop.

Crystal structure of the class A extended-spectrum ß-lactamase CTX-M-96 in complex with relebactam at 1.03 Angstrom resolution.

The use of ß-lactam/ß-lactamase inhibitors constitutes an important strategy to counteract ß-lactamases in multidrug-resistant (MDR) Gram-negative bacteria. Recent reports have described ceftazidime-/avibactam-resistant isolates producing CTX-M variants with different amino acid substitutions (e.g., P167S, L169Q, and S130G). Relebactam (REL) combined with imipenem has proved very effective against Enterobacterales producing ESBLs, serine-carbapenemases, and AmpCs. Herein, we evaluated the inhibitory efficacy of REL against CTX-M-96, a CTX-M-15-type variant. The CTX-M-96 structure was obtained in complex with REL at 1.03 Å resolution (PDB 8EHH). REL was covalently bound to the S70-Oγ atom upon cleavage of the C7-N6 bond. Compared with apo CTX-M-96, binding of REL forces a slight displacement of the deacylating water inwards the active site (0.81 Å), making the E166 and N170 side chains shift to create a proper hydrogen bonding network. Binding of REL also disturbs the hydrophobic patch formed by Y105, P107, and Y129, likely due to the piperidine ring of REL that creates clashes with these residues. Also, a remarkable change in the positioning of the N104 sidechain is also affected by the piperidine ring. Therefore, differences in the kinetic behavior of REL against class A ß-lactamases seem to rely, at least in part, on differences in the residues being involved in the association and stabilization of the inhibitor before hydrolysis. Our data provide the biochemical and structural basis for REL effectiveness against CTX-M-producing Gram-negative pathogens and essential details for further DBO design. Imipenem/REL remains an important choice for dealing with isolates co-producing CTX-M with other ß-lactamases.

Multicenter study to assess the use of BL-DetecTool for the detection of CTX-M-type ESBLs and carbapenemases directly from clinical specimens.

Antimicrobial resistance (AMR) is one of the major public health problems worldwide. Multiple strategies have been put in place to address this problem. One of them is the rapid detection of the mechanisms of resistance, such as extended-spectrum beta-lactamases (ESBLs) and/or carbapenemases. We conducted a multicenter study that included nine European centers for the assessment of prototypes of a novel lateral flow immunoassay-based device (BL-DetecTool) for a rapid detection of ESBL (NG-Test CTX-M-MULTI DetecTool) and/or carbapenemases (NG-Test CARBA 5 DetecTool) from Enterobacterales and Pseudomonas aeruginosa in positive urine, positive blood cultures, and rectal swabs. We performed a prospective analysis between January 2021 and June 2022, including overall 22,010 samples. Based on each hospital information, the sensitivity to detect CTX-M was 84%-100%, 90.9%-100%, and 75%-100% for urine, positive blood cultures, and enriched rectal swabs, respectively. On the other hand, the sensitivity to detect carbapenemases was 42.8%-100%, 75%-100%, and 66.6%-100% for urine, positive blood cultures, and enriched rectal swab, respectively. BL-DetecTool allows a rapid and reliable detection of ESBL and carbapenemases directly from urine, positive blood cultures, or enriched rectal swabs, being an easy technique to implement in the workflow of clinical microbiology laboratories. IMPORTANCE: The assessed rapid assay to detect CTX-M beta-lactamases and carbapenemases directly from clinical samples can favor in the rapid detection of these mechanisms of resistance and hence the administration of a more adequate antimicrobial treatment.

Presence of hypervirulence-associated determinants in Klebsiella pneumoniae from hospitalised patients in Germany.

BACKGROUND: Klebsiella (K.) pneumoniae is a ubiquitous Gram-negative bacterium and a common coloniser of animals and humans. Today, K. pneumoniae is one of the most persistent nosocomial pathogens worldwide and poses a severe threat/burden to public health by causing urinary tract infections, pneumonia and bloodstream infections. Infections mainly affect immunocompromised individuals and hospitalised patients. In recent years, a new type of K. pneumoniae has emerged associated with community-acquired infections such as pyogenic liver abscess in otherwise healthy individuals and is therefore termed hypervirulent K. pneumoniae (hvKp). The aim of this study was the characterisation of K. pneumoniae isolates with properties of hypervirulence from Germany. METHODS: A set of 62 potentially hypervirulent K. pneumoniae isolates from human patients was compiled. Inclusion criteria were the presence of at least one determinant that has been previously associated with hypervirulence: (I) clinical manifestation, (II) a positive string test as a marker for hypermucoviscosity, and (III) presence of virulence associated genes rmpA and/or rmpA2 and/or magA. Phenotypic characterisation of the isolates included antimicrobial resistance testing by broth microdilution. Whole genome sequencing (WGS) was performed using Illumina® MiSeq/NextSeq to investigate the genetic repertoire such as multi-locus sequence types (ST), capsule types (K), further virulence associated genes and resistance genes of the collected isolates. For selected isolates long-read sequencing was applied and plasmid sequences with resistance and virulence determinants were compared. RESULTS: WGS analyses confirmed presence of several signature genes for hvKp. Among them, the most prevalent were the siderophore loci iuc and ybt and the capsule regulator genes rmpA and rmpA2. The most dominant ST among the hvKp isolates were ST395 capsule type K2 and ST395 capsule type K5; both have been described previously and were confirmed by our data as multidrug-resistant (MDR) isolates. ST23 capsule type K1 was the second most abundant ST in this study; this ST has been described as commonly associated with hypervirulence. In general, resistance to beta-lactams caused by the production of extended-spectrum beta-lactamases (ESBL) and carbapenemases was observed frequently in our isolates, confirming the threatening rise of MDR-hvKp strains. CONCLUSIONS: Our study results show that K. pneumoniae strains that carry several determinants of hypervirulence are present for many years in Germany. The detection of carbapenemase genes and hypervirulence associated genes on the same plasmid is highly problematic and requires intensified screening and molecular surveillance. However, the non-uniform definition of hvKp complicates their detection. Testing for hypermucoviscosity alone is not specific enough to identify hvKp. Thus, we suggest that the classification of hvKp should be applied to isolates that not only fulfil phenotypical criteria (severe clinical manifestations, hypermucoviscosity) but also (I) the presence of at least two virulence loci e.g. iuc and ybt, and (II) the presence of rmpA and/or rmpA2.

Comparison of methods proposed for monitoring cefotaxime-resistant Escherichia coli in the water environment.

Escherichia coli is a promising subject for globally coordinated surveillance of antimicrobial resistance (AMR) in water environments due to its clinical relevance and widespread use as an indicator of fecal contamination. Cefotaxime-resistant E. coli was recently evaluated favorably for this purpose by the World Health Organization TriCycle Protocol, which specifies tryptone bile x-glucuronide (TBX) medium and incubation at 35°C. We assessed comparability with the U.S. Environmental Protection Agency-approved method for E. coli quantification, which uses membrane-thermotolerant E. coli (mTEC) agar and incubation at 44.5°C, in terms of recovery of E. coli and cefotaxime-resistant E. coli from wastewater influent and surface waters. Total E. coli concentrations in wastewater influent were 106-108 CFU/100 mL, while cefotaxime-resistant E. coli were ~100-fold lower. Total E. coli in surface waters were ~102 CFU/100 mL, and cefotaxime-resistant isolates were near the limit of detection (0.4 CFU/100 mL). Total and putative cefotaxime-resistant E. coli concentrations did not differ significantly between media or by incubation method; however, colonies isolated on mTEC were more frequently confirmed to species (97.1%) compared to those from TBX (92.5%). Incubation in a water bath at 44.5°C significantly decreased non-specific background growth and improved confirmation frequency on both media (97.4%) compared to incubation at 35°C (92.3%). This study helps to advance globally coordinated AMR in water environments and suggests that the TriCycle Protocol is adaptable to other standard methods that may be required in different locales, while also offering a means to improve specificity by decreasing the frequency of false-positive identification of cefotaxime-resistant E. coli by modifying incubation conditions.IMPORTANCEAs antibiotic-resistant bacteria in water environments are increasingly recognized as contributors to the global antibiotic resistance crisis, the need for a monitoring subject that captures antibiotic resistance trends on a global scale increases. The World Health Organization TriCycle Protocol proposes the use of cefotaxime-resistant Escherichia coli isolated on tryptone bile x-glucuronide agar. The U.S. Environmental Protection Agency (USEPA) criteria for safe recreational waters also use E. coli as an indicator but specify the use of mTEC agar at a higher incubation temperature (44.5°C vs 35°C). We assessed the comparability of these methods for isolating total and cefotaxime-resistant E. coli, finding overall good agreement and performance, but significantly higher specificity toward E. coli selection with the use of the USEPA incubation protocol and mTEC agar. This study is the first to directly compare these methods and provides evidence that the methods may be used interchangeably for global surveillance of antibiotic resistance in the environment.

High frequency of antimicrobial resistance in Salmonella and Escherichia coli causing diarrheal diseases at the Yirimadio community health facility, Mali.

BACKGROUND: Diarrhoea is a public health problem, especially in developing countries where it is the second leading cause of child mortality. In Low Income Countries like in Mali, self-medication and inappropriate use of antibiotics due to the scarcity of complementary diagnostic systems can lead to the development of multidrug-resistant bacteria causing diarrhoea. The objective of this work was to determine the microorganisms responsible for diarrhoea in children under 15 years of age and to characterize their sensitivity to a panel of antibiotics used in a peri-urban community in Mali. The study involved outpatient children visiting the Yirimadio Community Health Centre and diagnosed with diarrhoea. Stool samples from those patients were collected and analysed by conventional stools culture and the susceptibility to antibiotics of detected bacteria was determined by the disc diffusion method in an agar medium. RESULT: Overall, 554 patients were included. Children under the age of 3 years accounted for 88.8% (492 of 554) of our study population. Two bacterial species were isolated in this study, Escherichia coli 31.8% (176 of 554) and Salmonella 2.9% (16 of 554). In the 176, E. coli strains resistance to amoxicillin and to cotrimoxazole was seen in 93.8% (165 of 176) and 92.6% ( 163 of 176), respectively. The ESBL resistance phenotype accounted for 39,8% (70 of 176) of E. coli. Sixteen (16) strains of Salmonella were found, of which one strain (6.3%) was resistant to amoxicillin and to amoxicillin + clavulanic acid. Another one was resistant to chloramphenicol (6.3%). Two strains of Salmonella were resistant to cotrimoxazole (12.5%) and two others were resistant to cefoxitin (12.5%). CONCLUSIONS: The data suggest that E. coli is frequently involved in diarrhoea in children under 3 years of age in this peri-urban setting of Bamako, Mali, with a high rate of resistance to amoxicillin and cotrimoxazole, the most widely used antibiotics in the management of diarrhoea in this setting.

High prevalence of multidrug-resistant Enterobacterales carrying extended-spectrum beta-lactamase and AmpC genes isolated from neonatal sepsis in Ahvaz, Iran.

OBJECTIVES: In the recent years, multidrug resistant (MDR) neonatal septicemia-causing Enterobacterales has been dramatically increased due to the extended-spectrum beta-lactamases (ESBLs) and AmpC enzymes. This study aimed to assess the antibiotic resistance pattern, prevalence of ESBLs/AmpC beta-lactamase genes, and Enterobacterial Repetitive Intergenic Consensus Polymerase Chain Reaction (ERIC-PCR) fingerprints in Enterobacterales isolated from neonatal sepsis. RESULTS: In total, 59 Enterobacterales isolates including 41 (69.5%) Enterobacter species, 15 (25.4%) Klebsiella pneumoniae and 3 (5.1%) Escherichia coli were isolated respectively. Resistance to ceftazidime and cefotaxime was seen in all of isolates. Furthermore, all of them were multidrug-resistant (resistant to three different antibiotic categories). The phenotypic tests showed that 100% of isolates were ESBL-positive. Moreover, AmpC production was observed in 84.7% (n = 50/59) of isolates. Among 59 ESBL-positive isolates, the highest percentage belonged to blaCTX-M-15 gene (66.1%) followed by blaCTX-M (45.8%), blaCTX-M-14 (30.5%), blaSHV (28.8%), and blaTEM (13.6%). The frequency of blaDHA, blaEBC, blaMOX and blaCIT genes were 24%, 24%, 4%, and 2% respectively. ERIC-PCR analysis revealed that Enterobacterales isolates were genetically diverse. The remarkable prevalence of MDR Enterobacterales isolates carrying ESBL and AmpC beta-lactamase genes emphasizes that efficient surveillance measures are essential to avoid the more expansion of drug resistance amongst isolates.

Coexistence of plasmid-mediated quinolone resistance (PMQR) and extended-spectrum beta-lactamase (ESBL) genes among clinical Pseudomonas aeruginosa isolates in Egypt.

BACKGROUND: Data about the prevalence of plasmid-mediated quinolone resistance (PMQR) and extended-spectrum beta-lactamase (ESBL) production in P. aeruginosa compared to the Enterobacteriaceae family is limited. The availability of limited therapeutic options raises alarming concerns about the treatment of multidrug-resistant P. aeruginosa. This study aimed to assess the presence of PMQR and ESBL genes among P. aeruginosa strains. METHODS: Fifty-six P. aeruginosa strains were isolated from 330 patients with different clinical infections. Phenotypically fluoroquinolone-resistant isolates were tested by PCR for the presence of six PMQR genes. Then, blaTEM, blaSHV, and blaCTX-M type ESBL genes were screened to study the co-existence of different resistance determinants. RESULTS: Overall, 22/56 (39.3%) of the studied P. aeruginosa isolates were phenotypically resistant to fluoroquinolones. PMQR-producing P. aeruginosa isolates were identified in 20 isolates (90.9%). The acc(6')-Ib-cr was the most prevalent PMQR gene (77.3%). The qnr genes occurred in 72.7%, with the predominance of the qnrA gene at 54.5%, followed by the qnrS gene at 27.3%, then qnrB and qnrC at 22.7%. The qepA was not detected in any isolate. The acc(6')-Ib-cr was associated with qnr genes in 65% of positive PMQR isolates. Significant differences between the fluoroquinolone-resistant and fluoroquinolone-susceptible isolates in terms of the antibiotic resistance rates of amikacin, imipenem, and cefepime (P value < 0.0001) were found. The ESBL genes were detected in 52% of cephalosporin-resistant P. aeruginosa isolates. The most frequent ESBL gene was blaCTX-M (76.9%), followed by blaTEM (46.2%). No isolates carried the blaSHV gene. The acc(6')-Ib-cr gene showed the highest association with ESBL genes, followed by the qnrA gene. The correlation matrix of the detected PMQR and ESBL genes indicated overall positive correlations. The strongest and most highly significant correlation was between qnrA and acc(6')-Ib-cr (r = 0.602) and between qnrA and blaCTX-M (r = 0.519). CONCLUSION: A high prevalence of PMQR genes among the phenotypic fluoroquinolone-resistant P. aeruginosa isolates was detected, with the co-carriage of different PMQR genes. The most frequent PMQR was the acc(6')-Ib-cr gene. Co-existence between PMQR and ESBL genes was found, with 75% of PMQR-positive isolates carrying at least one ESBL gene. A high and significant correlation between the ESBL and PMQR genes was detected.

Survival of highly related ESBL- and pAmpC- producing Escherichia coli in broiler farms identified before and after cleaning and disinfection using cgMLST.

BACKGROUND: Broiler chickens are frequently colonized with Extended-Spectrum Beta-Lactamase- (ESBL-) and plasmid mediated AmpC Beta-Lactamase- (pAmpC-) producing Enterobacterales, and we are confronted with the potential spread of these resistant bacteria in the food chain, in the environment, and to humans. Research focused on identifying of transmission routes and investigating potential intervention measures against ESBL- and pAmpC- producing bacteria in the broiler production chain. However, few data are available on the effects of cleaning and disinfection (C&D) procedures in broiler stables on ESBL- and pAmpC- producing bacteria. RESULTS: We systematically investigated five broiler stables before and after C&D and identified potential ESBL- and pAmpC- colonization sites after C&D in the broiler stables, including the anteroom and the nearby surrounding environment of the broiler stables. Phenotypically resistant E. coli isolates grown on MacConkey agar with cefotaxime were further analyzed for their beta-lactam resistance genes and phylogenetic groups, as well as the relation of isolates from the investigated stables before and after C&D by whole genome sequencing. Survival of ESBL- and pAmpC- producing E. coli is highly likely at sites where C&D was not performed or where insufficient cleaning was performed prior to disinfection. For the first time, we showed highly related ESBL-/pAmpC- producing E. coli isolates detected before and after C&D in four of five broiler stables examined with cgMLST. Survival of resistant isolates in investigated broiler stables as well as transmission of resistant isolates from broiler stables to the anteroom and surrounding environment and between broiler farms was shown. In addition, enterococci (frequently utilized to detect fecal contamination and for C&D control) can be used as an indicator bacterium for the detection of ESBL-/pAmpC- E. coli after C&D. CONCLUSION: We conclude that C&D can reduce ESBL-/pAmpC- producing E. coli in conventional broiler stables, but complete ESBL- and pAmpC- elimination does not seem to be possible in practice as several factors influence the C&D outcome (e.g. broiler stable condition, ESBL-/pAmpC- status prior to C&D, C&D procedures used, and biosecurity measures on the farm). A multifactorial approach, combining various hygiene- and management measures, is needed to reduce ESBL-/pAmpC- E. coli in broiler farms.

In-silico insights of ESBL variants and tracking the probable sources of ESBL-producing Escherichia coli in a small-scale poultry farm.

Commercial broiler farms face challenges of extended spectrum beta-lactamase (ESBL)-producing Escherichia coli transmitted from both vertical and horizontal routes. Understanding the dynamics of ESBL-E. coli transmission in compromised biosecurity settings of small-scale rural poultry farms is essential. This study aimed to elucidate the probable transmission pathways of ESBL-E. coli in such settings, employing phylogenetic analysis and molecular docking simulations to explore the catalytic properties of ß-lactamase variants. Sampling was conducted on a small-scale poultry farm in West Bengal, India, collecting 120 samples at three intervals during the broiler production cycle. E. coli isolates underwent resistance testing against eight antimicrobials, with confirmation of ESBL production. Genotypic analysis of ESBL genes and sequencing were performed, alongside molecular docking analyses and phylogenetic comparisons with publicly available sequences. Among 173 E. coli isolates, varying resistance profiles were observed, with complete resistance to cefixime and high resistance to amoxicillin and tetracycline. The incidence of ESBL-E. coli fluctuated over the production cycle, with dynamic changes in the prevalence of blaCTX-M-type and blaSHV-type genes. Phylogenetic analysis indicated partial clonal relationships with human clinical strains and poultry strains from the Indian subcontinent. Molecular docking confirmed the catalytic efficiencies of these ESBL variants. The study highlights probable vertical transmission of ESBL-E. coli and emphasizes drinking water as a potential source of horizontal transmission in small-scale poultry farms. Strict biosecurity measures could prevent the spread of antimicrobial-resistant bacteria in birds and their products in a small scale poultry farm.

Antibiofilm activity of promethazine against ESBL-producing strains of Escherichia coli in urinary catheters.

The bacterium Escherichia coli is one of the main causes of urinary tract infections. The formation of bacterial biofilms, especially associated with the use of urinary catheters, contributes to the establishment of recurrent infections and the development of resistance to treatment. Strains of E. coli that produce extended-spectrum beta-lactamases (ESBL) have a greater ability to form biofilms. In addition, there is a lack of drugs available in the market with antibiofilm activity. Promethazine (PMZ) is an antihistamine known to have antimicrobial activity against different pathogens, including in the form of biofilms, but there are still few studies of its activity against ESBL E. coli biofilms. The aim of this study was to evaluate the antimicrobial activity of PMZ against ESBL E. coli biofilms, as well as to assess the application of this drug as a biofilm prevention agent in urinary catheters. To this end, the minimum inhibitory concentration and minimum bactericidal concentration of PMZ in ESBL E. coli strains were determined using the broth microdilution assay and tolerance level measurement. The activity of PMZ against the cell viability of the in vitro biofilm formation of ESBL E. coli was analyzed by the MTT colorimetric assay and its ability to prevent biofilm formation when impregnated in a urinary catheter was investigated by counting colony-forming units (CFU) and confirmed by scanning electron microscopy (SEM). PMZ showed bactericidal activity and significantly reduced (p < 0.05) the viability of the biofilm being formed by ESBL E. coli at concentrations of 256 and 512 µg/ml, as well as preventing the formation of biofilm on urinary catheters at concentrations starting at 512 µg/ml by reducing the number of CFUs, as also observed by SEM. Thus, PMZ is a promising candidate to prevent the formation of ESBL E. coli biofilms on abiotic surfaces.

Pre-transplant multidrug-resistant infections in liver transplant recipients-epidemiology and impact on transplantation outcome.

BACKGROUND: Cirrhotic patients are highly exposed to healthcare services and antibiotics. Although pre-liver transplantation (LT) infections are directly related to the worsening of liver function, the impact of these infections on LT outcomes is still unclear. This study aimed to identify the effect of multidrug-resistant microorganism (MDRO) infections before LT on survival after LT. METHODS: Retrospective study that included patients who underwent LT between 2010 and 2019. Variables analyzed were related to patients' comorbidities, underlying diseases, time on the waiting list, antibiotic use, LT surgery, and occurrences post-LT. Multivariate analyses were performed using logistic regression, and Cox regression for survival analysis. RESULTS: A total of 865 patients were included; 351 infections were identified in 259 (30%) patients, of whom 75 (29%) had ≥1 pre-LT MDRO infection. The most common infection was spontaneous bacterial peritonitis (34%). The agent was identified in 249(71%), 53(15%) were polymicrobial. The most common microorganism was Klebsiella pneumoniae (18%); the most common MDRO was ESBL-producing Enterobacterales (16%), and carbapenem-resistant (CR) Enterobacterales (10%). Factors associated with MDRO infections before LT were previous use of therapeutic cephalosporin (p = .001) and fluoroquinolone (p = .001), SBP prophylaxis (p = .03), ACLF before LT (p = .03), and days of hospital stay pre-LT (p < .001); HCC diagnosis was protective (p = .01). Factors associated with 90-day mortality after LT were higher MELD on inclusion to the waiting list (p = .02), pre-LT MDRO infection (p = .04), dialysis after LT (p < .001), prolonged duration of LT surgery (p < .001), post-LT CR-Gram-negative bacteria infection (p < .001), and early retransplantation (p = .004). CONCLUSION: MDRO infections before LT have an important impact on survival after LT.

Genomic insights into Leminorella grimontii and its chromosomal class A GRI ß-lactamase.

Leminorella grimontii strain LG-KP-E1-2-T0 was isolated from Zophobas morio larvae. It showed a susceptibility phenotype compatible with the expression of an inducible extended-spectrum ß-lactamase. The presence of a chromosomal bla gene encoding for the class A GRI-1 ß-lactamase was revealed by whole-genome sequencing. GRI-1 shared the highest amino acid identity with RIC-1 and OXY-type ß-lactamases (76-80%). Analysis of six further publicly-available L. grimontii draft genomes deposited in NCBI revealed that blaGRI-1 was always present. Core-genome analysis indicated that LG-KP-E1-2-T0 was unique from other strains. We provided the first complete genome of L. grimontii and new insights on its chromosomal ß-lactamases.

MAST® D72C test: a novel option for ESBL, AmpC and carbapenemase detection.

PURPOSE: The MAST® D72C test is a phenotypical test which can detect ESBL and AmpC production in Enterobacterales. It can also identify the suspected presence of carbapenemase. The aim of the present study was to assess the sensitivity and specificity of this test and to discuss its usefulness in laboratories, especially those that use only an automated AST system. METHOD: The performance of the MAST® D72C test was assessed against a collection of 119 non-redundant Enterobacterales isolates characterized for their content in ß-lactamases, and compared with that of the reference double disk synergy test. ß-lactamase content was established from phenotypic and genotypic analyses to collect a broad diversity of resistance mechanisms and bacterial strains, including 30 ESBL-producing strains, 32 strains overproducing chromosomal AmpC, 10 strains producing plasmid-encoded AmpC, 12 carbapenemase-producing strains, 13 strains combining the production of several ß-lactamases, and 22 strains that produced other ß-lactamases. RESULTS: The sensitivity and specificity for ESBL-detection were comparable with those of the synergy test, 75 versus 72.5%, and 94.9 versus 93.7%, respectively. The sensitivity and specificity for AmpC-detection were 71.7% and 100%, respectively, and sensitivity reached 78.7% if we excluded carbapenem-resistant isolates. Carbapenemase-detection sensitivity was 90%. CONCLUSION: These results show that the MAST® D72C test can be a useful tool for the detection of ESBL- and AmpC-production in clinical laboratories.

Real-world experience with meropenem/vaborbactam for the treatment of infections caused by ESBL-producing Enterobacterales and carbapenem-resistant Klebsiella pneumoniae.

PURPOSE: Real-world experience with meropenem/vaborbactam (M/V) is limited. Our aim is to report a clinical experience of M/V in the treatment of resistant Gram-negative bacilli. METHODS: This is a prospective observational study including patients hospitalized in the University Hospital of Pisa (March 2021-Jan 2023) with infections by both extended-spectrum ß-lactamases (ESBL)-producing Enterobacterales and carbapenem-resistant Klebsiella pneumoniae (Kp) treated with M/V. The primary outcome measure was clinical success, defined as a composite of survival, resolution of signs and symptoms and absence of microbiological failure at day 30 from infection onset. A multivariable regression analysis was performed to identify factors associated with clinical failure. Odds ratio (OR) with 95% confidence intervals (CI) was calculated. RESULTS: A total of 104 patients who received M/V were included: 24/104 (23.1%) infections were caused by ESBL non-hypervirulent Enterobacterales, 17/104 (16.3%) by ESBL-producing hypervirulent Klebsiella pneumoniae (hvKp) and 63/104 (60.6%) by CRE. The most common infections were bloodstream infections, followed by urinary tract infections, hospital-acquired pneumonia, intra-abdominal infections and others. Septic shock occurred in 16/104 (15.4%) patients. Clinical success was achieved in 77% of patients, and 30-day mortality rate was 15.4%. In patients with KPC-producing Kp infections, clinical success and 30-day mortality rates were 82% and 11.5%, respectively. On multivariable analysis, SOFA score (OR 1.32, 95% CI 1.02-1.7, p=0.032) was independently associated with clinical failure, while source control (OR 0.16, 95% CI 0.03-0.89, p=0.036) was protective. CONCLUSIONS: M/V is a promising therapeutic option against infections caused by difficult-to-treat ESBL-producing Enterobacterales and CR-Kp.

Effect of incremental tazobactam concentrations on the bactericidal activity of piperacillin against ESBL-producing enterobacterales clinical isolates causing bacteraemia.

We evaluated the activity of piperacillin in relation to INCREASING TAZOBACTAM CONCENTRATION against ESBL-producing Enterobacterales collected from patients with bacteraemia. Increasing tazobactam concentration (4, 12 or 24 mg/L) exerted a reduction of piperacillin MICs under the clinical breakpoint in a concentration-dependent manner (0%, 60% and 90% of clinical isolates). Also, activity of piperacillin/tazobactam based at higher achievable serum concentrations (123/14 mg/L) is needed to reduce the bacterial growth in 92% of ESBL-producers. CHANGES IN THE PIPERACILLIN MIC IN RELATION TO INCREASING TAZOBACTAM SUGGEST THAT REALTIME TDM COULD BE USED FOR DRIVEN ANTIMICROBIAL THERAPY WITH PIPERACILLIN/TAZOBACTAM IN BSI DUE TO ESBL STRAINS.

Antimicrobial activity of cefepime-tazobactam combination against extended spectrum beta-lactamase and/or AmpC beta-lactamase- producing gram-negative bacilli.

BACKGROUND: The problem of resistance to beta-lactam antibiotics, which is caused by ESBL and AmpC ß-lactamases, is getting worse globally. Infections caused by bacterial isolates harboring these enzymes are difficult to treat with carbapenems being the sole effective treatment option for such infections. The objective of this study was to determine the frequency of ESBLs and AmpC-producing Gram-negative bacilli isolated from clinical specimens and to evaluate the sensitivity of cefepime-tazobactam combination against them. METHODS: This is an observational cross-sectional study carried out on 100 Gram-negative bacilli at Theodor Bilharz Research Institute Hospital during the period from February 2015 to January 2016. ESBL production was screened by using the disc diffusion test followed by confirmation by the combined disc confirmatory test, the screening for AmpC production was conducted using the cefoxitin disc test, which was subsequently confirmed by the AmpC disc test. Isolates confirmed positive for ESBL and/ or AmpC production were investigated for their susceptibility to antibiotics. RESULTS: Among 100 Gram-negative bacilli, 44 isolates were confirmed as ESBL producers by the combined disc confirmatory test out of 56 isolates that tested positive for ESBL production through the disc diffusion test. The presence of AmpC production was assessed using the cefoxitin disc test, 32 isolates were screened to be AmpC producers, and the AmpC disc test confirmed AmpC production in 9 isolates of them. Using the Mast® D68C set, 32 isolates were ESBL producers, 3 were AmpC producers, and 4 isolates were ESBL/AmpC co-producers. The highest sensitivity was to cefepime-tazobactam (91.48%) followed by the carbapenems. CONCLUSION: Cefepime-tazobactam showed remarkable activity against ESBL and/or AmpC-producing Gram-negative bacilli and may be considered as a therapeutic alternative to carbapenems.