AmpC Beta-lactamase Research Articles

Plasmid-Encoded AmpC and Extended-Spectrum Beta-Lactamases in Multidrug-Resistant Escherichia coli Isolated from Piglets in Portugal.

Considering the concept of "One Health," the aim of this study was to determine susceptibility profiles of Escherichia coli in piglets' intestinal microbiota from different farms in Portugal. Beyond antimicrobial susceptibility, the occurrence of multiple antibiotic resistance and detection of phenotypic/genotypic extended-spectrum beta-lactamases (ESBLs) and plasmid mediated AmpC beta-lactamases (pAmpC) were done. From 10 different pig farms, 340 E. coli isolates were obtained from 75 feces samples. Susceptibility to amoxicillin-clavulanic acid (AMC), piperacillin (PIP), cefoxitin (FOX), ceftazidime (CAZ), cefepime (FEP), aztreonam (AZT), imipenem (IP), amikacin (AK), ciprofloxacin (CIP), and trimethoprim-sulfamethoxazole (SXT) was determined. Five-gene panel for amplification of bla genes was used for ESBL (TEM, SHV, CTX-M) and pAmpC (CMY-2, ACC). Among E. coli isolates, 209 were distributed in three resistance profiles: 57.7% MDR, 3.5% extensively drug-resistance (XDR) (resistant to CIP, SXT, and beta-lactams, except IP, with variability to AK) and 0.3% pandrug-resistance (PDR) (resistant to all antibiotics used). pAmpC and/or ESBLs genes were presented in 65% of the isolates. Presence of different associations of bla genes in the same isolate was the most observed (31%), and the most common were an ESBL (TEM) and a pAmpC (CMY-2). Presence of three or four bla genes in various associations were detected. These isolates were very resistant, especially those with four genes, which were resistant to beta-lactams (except IP), CIP, and SXT. This study showed a surprisingly high rate of MDR E. coli isolated in Portuguese piglets, with enzymes that impair activity of the most used antibiotics in human therapeutic.

Clonal Relationship and Resistance Profiles Among ESBL-Producing Escherichia coli.

AmpC β-lactamases hydrolyze all β-lactams except cefepime and carbapenems. The study of AmpC-producing E. coli has high priority for the infection control committee. This research is aimed to investigate the resistant urinary AmpC-generating E. coli isolates and identify their genetic variety. Some 230 E. coli isolates from patients suffering urinary tract infection symptoms were studied in 2017–2018 to assess their susceptibility toward antimicrobial agents. AmpC gene was evaluated by PCR and molecular typing using the 10-loci MLVA method. MLVA images were examined by BioNumerics 6.6 software through the use of the UPGMA algorithms. Thirty-eight AmpC-generating E. coli isolates were detected. The most abundant determinant was blaCIT and blaEBC, blaFOX, and blaDHA had the next ranks, respectively. Six major clusters and a singleton were identified by MLVA. AmpC beta-lactamases in urinary isolates of E. coli in the hospital under study and high rate of additional resistance to gentamicin, cotrimoxazole and ciprofloxacin. The most frequent gene determinant of AmpC beta-lactamase was blaCIT and vary depending on time and geographical location.

Exploring antimicrobial resistance to beta-lactams, aminoglycosides and fluoroquinolones in E. coli and K. pneumoniae using proteogenomics

Antimicrobial resistance is mostly studied by means of phenotypic growth inhibition determinations, in combination with PCR confirmations or further characterization by means of whole genome sequencing (WGS). However, the actual proteins that cause resistance such as enzymes and a lack of porins cannot be detected by these methods. Improvements in liquid chromatography (LC) and mass spectrometry (MS) enabled easier and more comprehensive proteome analysis. In the current study, susceptibility testing, WGS and MS are combined into a multi-omics approach to analyze resistance against frequently used antibiotics within the beta-lactam, aminoglycoside and fluoroquinolone group in E. coli and K. pneumoniae. Our aim was to study which currently known mechanisms of resistance can be detected at the protein level using liquid chromatography–mass spectrometry (LC–MS/MS) and to assess whether these could explain beta-lactam, aminoglycoside, and fluoroquinolone resistance in the studied isolates. Furthermore, we aimed to identify significant protein to resistance correlations which have not yet been described before and to correlate the abundance of different porins in relation to resistance to different classes of antibiotics. Whole genome sequencing, high-resolution LC–MS/MS and antimicrobial susceptibility testing by broth microdilution were performed for 187 clinical E. coli and K. pneumoniae isolates. Resistance genes and proteins were identified using the Comprehensive Antibiotic Resistance Database (CARD). All proteins were annotated using the NCBI RefSeq database and Prokka. Proteins of small spectrum beta-lactamases, extended spectrum beta-lactamases, AmpC beta-lactamases, carbapenemases, and proteins of 16S ribosomal RNA methyltransferases and aminoglycoside acetyltransferases can be detected in E. coli and K. pneumoniae by LC–MS/MS. The detected mechanisms matched with the phenotype in the majority of isolates. Differences in the abundance and the primary structure of other proteins such as porins also correlated with resistance. LC–MS/MS is a different and complementary method which can be used to characterize antimicrobial resistance in detail as not only the primary resistance causing mechanisms are detected, but also secondary enhancing resistance mechanisms.

Emergence and Spread of Cephalosporinases in Wildlife: A Review.

Simple SummaryAntimicrobial resistance (AMR) is one of the global public health challenges nowadays. AMR threatens the effective prevention and treatment of an ever-increasing range of infections, being present in healthcare settings but also detected across the whole ecosystem, including wildlife. This work compiles the available information about an important resistance mechanism that gives bacteria the ability to inactivate cephalosporin antibiotics, the cephalosporinases (extended-spectrum beta-lactamase (ESBL) and AmpC beta-lactamase), in wildlife. Through a rigorous systematic literature review in the Web of Science database, the available publications on this topic in the wildlife sphere were analysed. The emergence and spread of cephalosporinases in wildlife has been reported in 46 countries from all continents (52% in Europe), with descriptions mainly in birds and mammals. The most widely disseminated cephalosporinases in human-related settings (e.g. CTX-M-1, CTX-M-14, CTX-M-15 and CMY-2) are also the most reported in wildlife, suggesting that anthropogenic pressure upon natural environments have a strong impact on antimicrobial resistance spread, including the dissemination of genes encoding these enzymes. Our work highlights the urgence and importance of public and ecosystem health policies, including improved surveillance and control strategies that breakdown AMR transmission chains across wildlife, as part of an integrated strategy of the One Health approach.In the last decade, detection of antibiotic resistant bacteria from wildlife has received increasing interest, due to the potential risk posed by those bacteria to wild animals, livestock or humans at the interface with wildlife, and due to the ensuing contamination of the environment. According to World Health Organization, cephalosporins are critically important antibiotics to human health. However, acquired resistance to β-lactams is widely distributed and is mainly mediated by extended-spectrum beta-lactamase and AmpC beta-lactamases, such as cephalosporinases. This work thus aimed to compile and analyse the information available on the emergence and dissemination of cephalosporinases in wildlife worldwide. Results suggest a serious scenario, with reporting of cephalosporinases in 46 countries from all continents (52% in Europe), across 188 host species, mainly birds and mammals, especially gulls and ungulates. The most widely reported cephalosporinases, CTX-M-1, CTX-M-14, CTX-M-15 and CMY-2, were also the most common in wild animals, in agreement with their ubiquity in human settings, including their association to high-risk clones of Escherichia coli (E. coli), such as the worldwide distributed CTX-M-15/ST131 E. coli. Altogether, our findings show that anthropogenic activities affect the whole ecosystem and that public policies promoting animal and environmental surveillance, as well as mitigation measures to avoid antimicrobial misuse and AMR spread, are urgently needed to be out in practise.

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.

Molecular Characterization of Antimicrobial Resistance and Virulence Genes of Bacterial Pathogens from Bovine and Caprine Mastitis in Northern Lebanon.

Mastitis is an infectious disease encountered in dairy animals worldwide that is currently a growing concern in Lebanon. This study aimed at investigating the etiology of the main mastitis-causing pathogens in Northern Lebanon, determining their antimicrobial susceptibility profiles, and identifying their antimicrobial resistance (AMR) genes. A total of 101 quarter milk samples were collected from 77 cows and 11 goats presenting symptoms of mastitis on 45 dairy farms. Bacterial identification was carried out through matrix-assisted laser desorption/ionization-time of flight mass spectrometry. Antimicrobial susceptibility was tested by disc diffusion and broth microdilution methods. Molecular characterization included polymerase chain reaction (PCR) screening for genes encoding extended-spectrum beta-lactamases (ESBLs) and plasmid-mediated AmpC among Enterobacterales isolates, and virulence factors among Staphylococcus isolates. Escherichia coli isolates were subjected to phylogenetic typing by a quadruplex PCR method. The most frequently identified species were Streptococcus uberis (19.2%), Streptococcus agalactiae (15.1%), E. coli (12.3%), and Staphylococcus aureus (10.96%). Gram-positive bacteria were resistant to macrolides and tetracycline, whereas gram-negative bacteria displayed resistance to ampicillin and tetracycline. Two ESBL genes, blaTEM (83.3%) and blaOXA (16.7%), and one AmpC beta-lactamase gene, blaCMY-II (16.7%), were detected among six E. coli isolates, which mainly belonged to phylogenetic group B1. Among Staphylococcus spp., the mecA gene was present in three isolates. Furthermore, four isolates contained at least one toxin gene, and all S. aureus isolates carried the ica operon. These findings revealed the alarming risk of AMR in the Lebanese dairy chain and the importance of monitoring antimicrobial usage.

Are There Effective Intervention Measures in Broiler Production against the ESBL/AmpC Producer Escherichia coli?

Extended-spectrum beta-lactamase (ESBL) and AmpC beta-lactamase (AmpC) producing Enterobacteriaceae occur frequently in livestock animals and the subsequent stages of the meat production chain and are therefore considered a risk for human health. Strict biosecurity measures and optimal farm management should reduce or even prevent poultry flock colonization at farm level. This review summarizes and evaluates published information on the effectiveness of specific intervention measures and farm management factors aiming to reduce the occurrence and spread of ESBL/AmpC producing or commensal or pathogenic E. coli in broiler chicken farms. In this systematic literature review, a total of 643 publications were analyzed, and 14 studies with significant outcome about the effectiveness of specific measures against E. coli and ESBL/AmpC producing E. coli in broiler chicken farms were found. Different feed additives seem to have an impact on the occurrence of those microorganisms. The measures ‘cleaning and disinfection’ and ‘competitive exclusion’ showed strong effects in prevention in some studies. In summary, some intervention measures showed potential to protect against or eliminate ESBL/AmpC-producing, commensal or pathogenic E. coli at farm level. Due to the high variability in the outcome of the studies, more specific, detailed investigations are needed to assess the potential of the individual intervention measures.

Review of AmpC Beta-Lactamases in the Enterobacterales—Answers to Common Questions

AmpC beta-lactamases confer resistance to a broad range of beta-lactams, including penicillins, oxyimino-beta-cephalosporins, cephamycins, and aztreonam. Susceptibility to cefepime, cefpirome, and the carbapenems is generally not affected. AmpC beta-lactamases can be found in a wide variety of Gram-negative organisms and are encoded, on transmissible plasmids and/or on the chromosome. In addition, the AmpC beta-lactamase can be expressed in several different ways which have some interesting implications for susceptibility testing and treatment. Most notably, AmpC beta-lactamases can be produced constitutively (i.e. always expressed), can be induced, or mutants can evolve to hyper-produce the enzyme. This article briefly reviews AmpC production in the Enterobacterales and attempts to answer questions that are commonly asked of the clinical microbiology laboratory.

Structure‐Based Drug Design (SBDD) and In Silico Pharmacophore Screening Enabled the Discovery of Small Organic Molecules and Peptides Modulators of Bla C, TEM‐1 and Amp C Beta Lactamases

Tuberculosis (TB) is caused by Mycobacterium tuberculosis, and remains a major, worldwide health concern. The emergence of M. tuberculosis strains that are resistant to front line antimycobacterial drugs such as isoniazid and rifampicin have greatly complicated efforts to control the spread of TB. Of the various resistance mechanisms, the most effective involves the production of β-lactamases that hydrolyze β-lactam antibiotics. The intrinsic resistance to β-lactam antibiotics was due mainly to the presence of a chromosomally encoded gene (blaC) in M. tuberculosis for a Class A, Ambler β-lactamase (BlaC). The BlaC enzyme has already been validated as one of the leading targets of tuberculosis therapy. In the search for new β-lactamase inhibitors we employed structure-based in silico screening of commercially available libraries of chemical compounds by using the on-line server Pharmit (pharmit.csb.pitt.edu). We aimed to discover novel 3-D pharmacophores features and further performed molecular docking of selected compounds provided by ChEMBL/MolPort/ZINC databases using Autodock Vina package and M. tuberculosis β-lactamases BlaC 3M6B.pdb, 1FSY.pdb (AMP-C), and 1PZP.pdb (TEM-1) as target proteins. The combination of 3-D pharmacophore screening and molecular docking lead to the discovery of natural products, and novel, small molecules potential inhibitors of targeted β-lactamases. We further complemented the “in silico” screening with additional molecular docking experiments employing scoring functions provided by “LeDock”, “ICM Docking” and SeeSAR (BiosolveIT (Germany, UK)) which provided important structural insights into the main pharmacophore features with significant contribution to the free energy of interaction between the hit ligands and the targeted proteins. The binding affinities of the highest scoring hits were measured in vitro revealing low μM inhibitory activity toward BlaC while showing an “activator” effect towards AmpC and TEM-1. Additionally, the in vitro screening platform, using kinetics of β-lactamase inhibition with nitrocefin substrate, enabled the discovery of other potential inhibitors of BlaC β-lactamase exhibiting inhibitory constants, Ki, in the low mM range. We also present the expansion of a peptide-pharmacophore space which lead to the discovery of cyclo [RRXY] tetrapeptides analogues that had at least a six-fold higher affinity for the BlaC β-lactamase than its linear sequence, RRHY. Moreover, we further discovered novel dipeptide-pharmacophores derived from tetrahydroharman acid, D-cyclohexylalanine and fluoro-phenylglycine exhibiting promising inhibitory activity against BlaC and AmpC beta lactamases, having Ki in the lowest tens of μM range. We further tested the lead compounds on the in vitro growth of mc26230 and found that the lowest concentration of beta lactamase inhibitors that prevented the bacterial growth was in the order of 100-500 μM for selected dipeptide and tetrapeptides compounds.

THE PREVALENCE OF EXTENDED-SPECTRUM BETA-LACTAMASES AND AMBLER CLASS C BETA-LACTAMASES IN ENUGU AND EBONYI STATES OF NIGERIA

Over the years, antibiotics have been among the primary drugs used to treat or prevent infections in animals and humans. Over the years, ESBL and AmpC producing isolates have become common microorganisms in medical institutions. They have contributed immensely to the clinical challenges caused by nosocomial infections with minimal therapeutic options.The present study investigated the prevalence of extended-spectrum beta-lactamases and Ambler class C beta-lactamases in the Enugu and Ebonyi States of Nigeria. A total of fifty isolates comprising 27 clinical and 23 environmental isolates were screened and observed for the presence of ESBL and AmpC producing isolates. The production of either of the two enzymes was detected by observing zones of inhibition in diameter. The findings showed the presence of ESBL and AmpC producing isolates in the clinical and environmental samples tested. The study concludes that ESBL and AmpC beta-lactamases production by resistant bacterial isolates (especially the Gram-negatives) remain the prevalent mechanism in bacterial resistance to beta-lactam drugs. The study recommends that adequate and appropriate detection and monitoring of ESBL and AmpC producing enzymes within the communities.

Evaluation of the presence of AmpC (FOX) beta-lactamase gene in clinical strains of Escherichia coli isolated from hospitalized patients in Tabriz, Iran

Beta-lactamases are the most common cause of bacterial resistance to beta-lactam antibiotics. AmpC-type beta-lactamases hydrolyze cephalosporins, penicillins, and cephamycins. Therefore, the study aims was to determine antibiotic resistance and to investigate the presence of AmpC beta-lactamase gene in clinical strains of Escherichia coli isolated from hospitalized patients in Tabriz. In this cross-sectional descriptive study, 289 E. coli specimens were collected from clinical specimens. Disk diffusion method and combined disk method were used to determine the phenotype of extended spectrum β-Lactamase producing (ESBLs) strains. Then PCR was used to evaluate the presence of AmpC (FOX) beta-lactamase gene in the strains confirmed in phenotypic tests. Antibiotic resistance was also determined using disk diffusion by the Kibry-Bauer method. A total of 121 isolates were identified as generators of beta-lactamase genes. 72 (59.5 %) isolates producing ESBL and 49 (40.5 %) isolates were identified as AmpC generators. In the PCR test, 31 isolates contained the FOX gene. The highest resistance was related to the antibiotics amoxicillin (76.12%), ceftazidime (70.24%) and nalidixic acid (65.05%). The results indicate an increase in the prevalence of beta-lactamase genes and increased resistance to beta-lactam antibiotics, which can be the result of improper use of antibiotics and not using antibiotic susceptibility tests before starting treatment. Also, using phenotypic and molecular diagnostic methods such as PCR together can be very useful.

#65: Characterization of Resistance Patterns Among Isolates of E. coli and Klebsiella sp. from Wastewater, and Clinical Samples in a Tertiary Care Children’s Hospital in Lima, Peru

Abstract Background Environmental monitoring of enterobacteria in hospital wastewater could be a useful tool to understand the composition of the microbiota in patients, their visits, and healthcare personnel, but it also may be useful for monitoring antimicrobial resistance among healthcare-associated infections in hospitalized patients. The aim of this study was to characterize and describe the phenotypic and genotypic antimicrobial resistance of Escherichia coli and Klebsiella sp. strains from wastewater and from clinical isolates in a tertiary care children’s hospital in Lima, Perú. Methods We systematically collected 70 isolates of Escherichia coli and Klebsiella sp. from wastewater and 24 isolates of the same enterobacteria from blood and urine cultures at Instituto Nacional de Salud del Niño San Borja (INSN-SB) in Lima from December 2018 to May 2019. Susceptibility profiles were evaluated following CLSI criteria. We used the Jarlier method for the detection of extended-spectrum beta-lactamases (ESBL). For detection of AmpC beta-lactamases, we used discs of cefoxitin, ceftazidime, and ceftriaxone combined with cloxacillin and cefotaxime. For the detection of carbapenemase production, we used EDTA and phenylboronic acid inhibitors between meropenem and imipenem disks. We performed genomic sequencing for the detection of resistance genes and to perform a phylogenetic cluster analysis. Results We collected a total of 94 isolates (70 from wastewater and 24 from clinical samples). Among the total isolates, 19 (20.2%) were ESBL producers. The frequency of ESBL producers in wastewater was 7.1% (5/24), whereas the frequency of ESBL producers in clinical samples was 58.3% (14/70). The most frequent resistance genes were tet (variant 34 and A) and blaTEM-1. The frequency of tet(34) and tet(A) were 10% and 7%, respectively, in isolates from wastewater, whereas the frequency of these genes were 0% and 6%, respectively, in clinical isolates. The gene blaCTX-M-15 was present in isolates from wastewater (1%) and clinical samples (2%). Phylogenetic cluster analysis found no similarities between isolates from wastewater and to those from clinical samples, suggesting that the population of these enterobacteria were different in wastewater compared with clinical samples. Conclusions Enterobacteria from hospital wastewater may not reflect the profile of infections caused by these microorganisms. However, they may reflect the microbiological microbiota among patients, their visits, and hospital healthcare personnel. Further studies that compare the phenotypic and genotypic characteristics among isolates from wastewater and the enteric microbiota from these individuals would be necessary to assess this hypothesis.

MurA, uhpT, glpT and fosA Genes of Fosfomycin Resistance in Multi-drug Resistant E. coli Isolated from Hematological Malignancies' Patients with Blood Stream Infection

Aim: To estimate the prevalence of murA, uhpT, glpT and fosA resistance genes of fosfomycin in multidrug resistant Escherichia coli isolated from patients suffering from hematological malignancies with on top blood stream infection together with correlating this distribution to the rate of expression of AmpC, ESBLs and MBLs in such isolates.&#x0D; Methods: 205 blood samples collected from patients with underlying hematological malignancies were cultured to isolate E. coli strains. Multidrug resistance was detected. PCR was done to determine fosfomycin resistance genes; murA, uhpT, glpT and fosA.&#x0D; Results: A total of 83 (40.5%) E. coli strains were isolated from blood samples. 58 (69.9%) were found to be multidrug resistant. AmpC beta-lactamase production was deduced in 15 (25.8%) isolates. 43 (74.1%) isolates were ESBLs producers whereas 9 (15.5%) were MBL class A carbapene-mases producers. Depending on PCR results, murA gene was detected in one isolate (1.7%), uhpT gene in 3 (5.2%), glpT gene in 4 (6.9%) whereas fosA gene was found in 7 E. coli strains (12.1%).&#x0D; Conclusion: Fosfomycin is a promising antibiotic with limited degree of prevalence of its resistant genes; however disseminating resistance might increase in multidrug resistant E. coli

An Update on Eight "New" Antibiotics against Multidrug-Resistant Gram-Negative Bacteria.

Infections in the ICU are often caused by Gram-negative bacteria. When these microorganisms are resistant to third-generation cephalosporines (due to extended-spectrum (ESBL) or AmpC beta-lactamases) or to carbapenems (for example carbapenem producing Enterobacteriales (CPE)), the treatment options become limited. In the last six years, fortunately, there have been new antibiotics approved by the U.S. Food and Drug Administration (FDA) with predominant activities against Gram-negative bacteria. We aimed to review these antibiotics: plazomicin, eravacycline, temocillin, cefiderocol, ceftazidime/avibactam, ceftolozane/tazobactam, meropenem/vaborbactam, and imipenem/relebactam. Temocillin is an antibiotic that was only approved in Belgium and the UK several decades ago. We reviewed the in vitro activities of these new antibiotics, especially against ESBL and CPE microorganisms, potential side effects, and clinical studies in complicated urinary tract infections (cUTI), intra-abdominal infections (cIAI), and hospital-acquired pneumonia/ventilator-associatedpneumonia (HAP/VAP). All of these new antibiotics are active against ESBL, and almost all of them are active against CPE caused by KPC beta-lactamase, but only some of them are active against CPE due to MBL or OXA beta-lactamases. At present, all of these new antibiotics are approved by the U.S. Food and Drug Administration for cUTI (except eravacycline) and most of them for cIAI (eravacycline, ceftazidime/avibactam, ceftolozane/tazobactam, and imipenem/relebactam) and for HAP or VAP (cefiderocol, ceftazidime/avibactam, ceftolozane/tazobactam, and imipenem/relebactam).

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.

Characterisation of AmpC and extended-spectrum beta-lactamase producing E. coli from New Zealand dairy farms

Dairy farms worldwide have been identified as a source of extended-spectrum β-lactamase (ESBL) and plasmid-mediated AmpC β-lactamase (pACBL) producing Enterobacteriaceae. There is limited data on the prevalence of these antimicrobial resistant bacteria from pasture-based farming systems. We carried out a regional cross-sectional study to examine the prevalence of ESBL and ACBL producing E. coli and characterised the genes responsible for antimicrobial resistance. Paddock faecal swabs were collected from fifteen dairy farms and screened for ESBL- and ACBL-producing E. coli. One farm was positive for ESBL-producing E. coli and three farms were positive for overexpressed chromosomal ampC. The three ESBL-producing E. coli isolates identified were further characterised using Illumina short-read and Oxford Nanopore long-read sequencing technologies. These three E. coli isolates were ST405 and contained an IncFII type plasmid with a blaCTX-M-14 gene. These results suggest the prevalence of ESBL- and pACBL-producing E. coli on pasture-based farms is low.

Burden of Multidrug Resistant Escherichia coli among Patients with Urinary Tract Infections in a Tertiary Care Centre- A Retrospective Analysis

Introduction: Multidrug Resistant (MDR) Escherichia coli isolates causing Urinary Tract Infections (UTIs), capable of producing Extended Spectrum Beta Lactamase (ESBL), AmpC beta lactamase and resistance to carbapenems pose serious challenges to the clinicians causing worse clinical scenarios. The detection of these isolates has prime importance in infection control and improving therapeuticmanagementofpatients,astheirprevalenceisincreasing worldwide. Injudicious use of antibiotics has led to selective pressure, resulting in the emergence of antibiotic resistance in gram negative bacteria globally. This rise in antimicrobial resistance has decreased the options for empirical therapy, causing a global health concern in infections associated with ESBL producing and AmpC producing isolates. Aim: To investigate the burden of ESBL producing, AmpC beta-lactamase producing, carbapenem resistant, and MDR Escherichia coli isolated from urine samples in a tertiary care centre. Materials and Methods: The present retrospective study conducted from January to December 2019. A total of 10,535 urine samples were received during the study period, to find out the burden of drug resistant Escherichia coli isolates in urine samples, analyse their antibiogram and patients’ socio-demographic information. Records of microscopic observations to antibiogram of each isolate was noted down from the register for urine samples, and further analysed. The data was coded, verified, entered and analysed using Statistical Package for Social Sciences (SPSS) version 18.0. Results: Among the total urine samples (10,535), 1434 (13.6%) Escherichia coli isolates were identified, of which 553 (38.6%) were found to be ESBL producing, 497 (34.6%) AmpC beta- lactamase producing, 172 (12%) Carbapenem resistant, and 765 (53.3%) MDR. Majority of the isolates were from the age group of 51-60 years 288 (20.08%). The least susceptibility of the isolates was detected against penicillin G 1410 (98.3%), followed by ampicillin 1160 (80.9%) and cephazolin 74 (67.9%). Among the MDR isolates, the maximum drug resistance (398, 52%) was seen in penicillin, cephems, quinolones and Folate pathway antagonist group of antimicrobials. Conclusion: Identification of the drug resistant isolates is of high priority and crucial for therapeutic management of the patients, and infection control. Stringent antimicrobial stewardship policies and judicious use of antimicrobials can decrease the spread of antimicrobial resistant genes in the hospital environment.

Microbiological Profile of Orthopaedic Implant Associated Infections: A Prospective Study

Introduction: The incidence of orthopaedic implantation and implantation associated infections were correspondingly increasing among the elderly and trauma patients. The resistance among the pathogens pose a unique challenge to the clinicians in the management of the infection. Extended-Spectrum Beta-Lactamase (ESBL), AmpC beta-lactamases (AmpC), Metallo Beta-Lactamases (MBL) and Methicillin-Resistant Staphylococcus aureus (MRSA) producing bacterial pathogens are responsible for a high rate of relapsing infections and outbreaks of nosocomial infections. Aim: To focus on accounting the incidence of ESBL, AmpC, MBL and MRSA and its antibiogram for effective management of implant associated infection. Materials and Methods: The present study was a prospective observational study which was conducted using various samples like pus, wound swab, serous discharge. These samples were collected under aseptic precautions in sterile containers for the period of one year (February 2015-February 2016). A total of 200 samples were inoculated on Blood agar and McConkey agar and identified. The isolates were tested for detection of ESBL, AmpC, MBL and MRSA as per standard protocol. The statistical analysis was performed using Statistical Package for the Social Sciences (SPSS) software version 20.0 Chicago, USA. Results: Most commonly used implants were intramedullary interlocking nail 138 (65.7%) followed by dynamic compression plates 23 (11.0%) and anatomical plates 18 (8.6%). Of the total of 200 samples, 190 (95%) samples yielded monomicrobial isolates and 10 (5%) samples yielded polymicrobial isolate. gram negative isolates 108 (51.4%) were marginally higher than gram positive isolates 102 (48.6%). The predominant isolate was S. aureus 81 (38.5%) followed by Klebsiella spp. 28 (13.3%) and Pseudomonas aeruginosa 21 (10.0%). In gram negative isolates, 76 (70.4%) were ESBL and/or AmpC producers. Of which, 48 (44.4%) were ESBL and AmpC co-producers. There were no MBL producing isolates. In gram positive isolates, majority were S. aureus 81 (79.4%) followed by Coagulase-negative Staphylococcus 21 (20.6%). Out of 81 S. aureus isolates, 46 (56.8%) were MRSA. Out of 21 CoNS, majority 17 (81%) isolates were methicillin-resistant. Implant failures were observed in 28 (14%) cases. Out of 28, majority 12 (42.8%) of the implant failures were MRSA. Conclusion: High rates of ESBL, AmpC and MRSA infections associated with implant surgeries indicate the necessity to formulate antibiotic policies and control measures. ESBL and AmpC producing strains were found to show higher rates of resistance to the various class of antibiotics when compared to non-ESBL and non-AmpC producers. MRSA isolates were found to show higher rates of resistance to various classes of antibiotics when compared to MSSA

Plasmid Derived AmpC Genotypes among the Multidrug Resistant Enterobacteriaceae Strains Isolated from Urine Samples in Southern India

Introduction: The most common pathogens causing Urinary Tract Infections (UTI) in community and hospital settings are Enterobacteriaceae. Antibiotic resistance is a major problem worldwide because of an increase in the use of antibiotics. Production of Extended Spectrum Beta-Lactamases (ESBLs) and AmpC beta-lactamases is the most common cause of resistance among Enterobacteriaceae (AmpC). Initially, AmpC β-lactamases received less attention globally, but now it has become a rising problem. Detection of AmpC β-lactamases expressing microbes is a requirement for addressing surveillance, for problems of hospital infection control, and for choosing optimal antimicrobial therapy. Aim: To study the genotype distribution of plasmid mediated AmpC β-lactamase produced in Enterobacteriaceaestrains isolated from urine samples. Materials and Methods: A cross-sectional study based on clinical laboratory surveillance was conducted from July 2019 to February 2020. Sixty Enterobacteriaceae isolates were identified by standard biochemical reactions. AmpC screening were done by cefoxitin disk diffusion and confirmed by an inhibitor-based assay using boronic acid. The presence of six plasmid mediated AmpC genes was determined by multiplex Polymerase Chain Reaction (PCR). Statistical Package for the Social Science (SPSS) version 20.0 was used to obtain descriptive data. Results: Among 60 Enterobacteriaceae isolates, 23 (38.3%) were cefoxitin-resistant isolates which contain Escherichia colistrain (n=17) while the remaining samples consist ofKlebsiella pneumoniae (n=5) and Proteus mirabilis strains (n=1). AmpC β-lactamase production was phenotypically confirmed in 12(20%) isolates and genotypically confirmed by PCR analysis in 16(26.6%) of all the urine isolates. In the present study, 3(13%), 2 (8.6%) of cefoxitin resistant isolates harboured the DHA, EBC gene and 1(4.3%) each harboured FOX and CIT gene, and 9(39.1%) harboured a combination of the genes. Conclusion: The present study suggested the predominant existence of plasmid mediated AmpC producers in Multi-Drug Resistant (MDR) Escherichia coli and Klebsiella pneumoniae. We suggest continuous surveillance is important to effectively control the spread of these strains and for optimal clinical outcome.

Phenotypic and Molecular Characterization of AmpC β-Lactamases among Escherichia coli a Prospective Study in Southeast Iran.

Escherichia coli (E. coli) causes serious health problems due to the high rate of its resistance to common antibiotics. AmpC β-lactamases are important cephalosporinases, encoded by E. coli chromosome, that cause resistance to cefoxitin, cephalothin, most penicillins, cefazolin, and beta-lactamase inhibitor-beta-lactams. This study aimed to detect AmpC β-lactamases among E. coli strains at three educational hospitals in Iran. Two hundred and thirty samples were recovered from the three educational hospitals in Zahedan City in southeast Iran. Sixty E. coli strains were identified by biochemical and differential tests. E. coli strains with extended-spectrum β-lactamases (ESBLs) were selected by disk-diffusion agar method and combined diffuse disc test. Finally, selected isolates were screened by polymerase chain reaction (PCR) for the common AmpC genotypes MOX, FOX, CIT, DHA, EBC, and ACC. Out of 60 E. coli strains, 13 isolates (21.7%) were ESBL positive and 7 strains (11.6%) were phenotypically AmpC beta-lactamase producers. Three isolates had CIT (23.1%), 5 had DHA (38.5%), 4 had EBC (30.8%), and 1 had ACC (7.7%) genotypes. The high prevalence of AmpC-producing E. coli strains in hospital settings is an important challenge for health care systems. Data about beta-lactamase producers that are crucial for controlling bacterial resistance should be upgraded.