Sulfhydryl Variable Research Articles

Diversity, functional classification and genotyping of SHV β-lactamases in Klebsiella pneumoniae.

Interpreting the phenotypes of bla SHV alleles in Klebsiella pneumoniae genomes is complex. Whilst all strains are expected to carry a chromosomal copy conferring resistance to ampicillin, they may also carry mutations in chromosomal bla SHV alleles or additional plasmid-borne bla SHV alleles that have extended-spectrum β-lactamase (ESBL) activity and/or β-lactamase inhibitor (BLI) resistance activity. In addition, the role of individual mutations/a changes is not completely documented or understood. This has led to confusion in the literature and in antimicrobial resistance (AMR) gene databases [e.g. the National Center for Biotechnology Information (NCBI) Reference Gene Catalog and the β-lactamase database (BLDB)] over the specific functionality of individual sulfhydryl variable (SHV) protein variants. Therefore, the identification of ESBL-producing strains from K. pneumoniae genome data is complicated. Here, we reviewed the experimental evidence for the expansion of SHV enzyme function associated with specific aa substitutions. We then systematically assigned SHV alleles to functional classes (WT, ESBL and BLI resistant) based on the presence of these mutations. This resulted in the re-classification of 37 SHV alleles compared with the current assignments in the NCBI's Reference Gene Catalog and/or BLDB (21 to WT, 12 to ESBL and 4 to BLI resistant). Phylogenetic and comparative genomic analyses support that (i) SHV-1 (encoded by bla SHV-1) is the ancestral chromosomal variant, (ii) ESBL- and BLI-resistant variants have evolved multiple times through parallel substitution mutations, (iii) ESBL variants are mostly mobilized to plasmids and (iv) BLI-resistant variants mostly result from mutations in chromosomal bla SHV. We used matched genome-phenotype data from the KlebNET-GSP AMR Genotype-Phenotype Group to identify 3999 K. pneumoniae isolates carrying one or more bla SHV alleles but no other acquired β-lactamases to assess genotype-phenotype relationships for bla SHV. This collection includes human, animal and environmental isolates collected between 2001 and 2021 from 24 countries. Our analysis supports that mutations at Ambler sites 238 and 179 confer ESBL activity, whilst most omega-loop substitutions do not. Our data also provide support for the WT assignment of 67 protein variants, including 8 that were noted in public databases as ESBL. These eight variants were reclassified as WT because they lack ESBL-associated mutations, and our phenotype data support susceptibility to third-generation cephalosporins (SHV-27, SHV-38, SHV-40, SHV-41, SHV-42, SHV-65, SHV-164 and SHV-187). The approach and results outlined here have been implemented in Kleborate v2.4.1 (a software tool for genotyping K. pneumoniae), whereby known and novel bla SHV alleles are classified based on causative mutations. Kleborate v2.4.1 was updated to include ten novel protein variants from the KlebNET-GSP dataset and all alleles in public databases as of November 2023. This study demonstrates the power of sharing AMR phenotypes alongside genome data to improve the understanding of resistance mechanisms.

Exploring Natural Compounds Targeting the Bacterial SHV Protein to Combat Antibiotic Resistance: A Biocomputational Study

Background: Antibiotic-resistant (AR) bacteria are rapidly spreading worldwide, posing a serious threat to antibiotic efficacy. Bacterial infections have emerged as a persistent threat following decades of antibiotic use. Sulfhydryl variable (SHV) is a well-known bacterial enzyme linked to AR. SHV has a high degree of genetic diversity, resulting in the existence of numerous distinct variants. Methods: The PyRx AutoDock VINA was used to conduct in-silico screening of a natural compound library to assess their interaction with the SHV-1 protein. SwissADME web tools were used to predict the physicochemical, drug-likeness, and ADMET properties of the selected compounds.Result: The compounds PSCdb00708, PSCdb00149, PSCdb00698, and PSCdb00175 bind strongly to the SHV-1 protein and interact strongly with the SHV-1 active site residues, as well as having several amino acid residue interactions in common with avibactam. These compounds exhibited higher binding affinity values than avibactam. Furthermore, these compounds demonstrated no violation of drug-likeness.Conclusion: The compounds PSCdb00708, PSCdb00149, PSCdb00698, and PSCdb00175 can be employed as SHV-1 inhibitors in the management of AR. However, experimental validation is required to optimize them as SHV-1 inhibitors.Keywords: Antibiotic-resistant; Antibiotic; Virtual screening; AutoDock; SHV-1 protein

Identification ofantibiotic resistance genes inEscherichia coli from subclinical mastitis milk indairy cows and goats, East Java Province.

Antibiotics are still used totreat mastitis indairy cows inIndonesia. This study aimed toanalyse antibiotic resistance genes inEscherichia coli (E.coli) from subclinical mastitis milk inEast Java Province, Indonesia. Thesamples consisted ofsubclinical mastitis milk from cows and goats. Atotal of592-quarter cow's milk and 71goat's milk samples from both halves ofthe udder were collected from 67farms inLumajang, Banyuwangi, Malang, Sidoarjo, Jember, Pasuruan, Probolinggo, and Mojokerto. Subclinical mastitis samples were screened using the California mastitis test (CMT). E.coli was identified byphenotypic and genotypic methods. E.coli wasconfirmed with aprimer specific tothe polymerase chain reaction (PCR) technique. Gene resistance ofE.coli was tested using the multiplex-PCR (mPCR) technique with primers encoding the genes temoneira enzyme (TEM), oxacillinase (OXA), sulfhydryl variable (SHV), and cefotaximase-munich IV (CTX-M IV). These genes were chosen because mastitis treatment generally uses oxacilline and β-lactam antibiotics. All data obtained were analysed descriptively. Theresults show that six isolates ofE.coli (46.15%) carried asingle resistance gene (TEM orSHV) and two isolates (33.33%) were confirmed asmultiple drug-resistant organisms (MDROs) (TEM and SHV). Theresistance genes were found insamples originating from Blitar, Banyuwangi, Lumajang, and Pasuruan Regencies. This research implies that antibiotic-resistance genes found inE.coli oncertain farms are dangerous and may allow gene transmission toother bacteria that make treatment for mastitis orother bacterial infections ineffective.

Distribution of Class A Extended-Spectrum β-Lactamases Among Pseudomonas aeruginosa Clinical Strains Isolated from Ardabil Hospitals

Background: Currently, the emergence of extended-spectrum β-lactamase (ESBL)-producing bacteria is becoming a major threat to patients in the hospital and community. Such enzymes have been recently detected in Pseudomonas aeruginosa, but there is no epidemiological data on the prevalence of ESBL-producing clinical isolates in the hospitals of Ardabil City (Iran). Objectives: This study aimed to determine the phenotypic and genotypic prevalence of class A ESBL-producing P. aeruginosa strains in Ardabil City. Methods: A total of 120 clinical isolates of P. aeruginosa collected from Ardabil hospitals were used in this study. Phenotypic detection of class A ESBL-producing P. aeruginosa isolates was performed using a double-disk synergy test. In addition, the detection of class A ESBL-encoding genes, including Pseudomonas extended resistant (PER), Vietnamese extended-spectrum β-lactamase (VEB), temoniera (TEM), sulfhydryl variable (SHV), cefotaximase (CTX-M), guyana extended-spectrum β-lactamase (GES), and Pseudomonas-specific enzyme (PSE), was performed using the polymerase chain reaction (PCR). Results: The prevalence of class A ESBL-producing P. aeruginosa strains was 8.3% (10 out of 120) based on the double-disk synergy test. However, 40% (48 out of 120) of these isolates were found to carry genes encoding class A ESBLs based on PCR. Among 48 class A ESBL-positive strains, the prevalence of PSE, TEM, VEB, CTX-M, and PER genes were 64.6% (31/48), 25% (12/48), 4.2% (2/48), 4.2% (2/48), and 2% (1/48), respectively. However, the frequency of other class A ESBL genes (SHV and GES genes) was 0%. Conclusions: Our results confirmed the presence of class A ESBL-producing P. aeruginosa strains in the hospital environment of Ardabil. On the other hand, the use of molecular tests can be a more precise and reliable method than phenotypic ones to identify these resistant strains and prevent the emergence of antibiotic resistance and ensuing treatment failure.

ANALISIS POTENSI DAUN TORBANGUN (Plectranthus amboinicus) SEBAGAI ANTIBAKTERI TERHADAP Klebsiella pneumoniae SECARA IN SILICO

Antibiotic-resistant is a global issue. Klebsiella pneumoniae has high priority to find its antibiotic because it has extended-spectrum beta-lactamase (ESBL) enzyme that makes the bacteria easy to resist the antibiotic. The study of phytochemicals in medicinal plants is one of the efforts to find antibiotics. Indrulizia is one of the empirical medicinal plants containing flavonoids used to treat disease. The aim of this study was to analyze the potential of borage as an antibacterial agent against Klebsiella pneumoniae in silico. Docking is the method for this study using AutoDock Vina. This is an application for docking the enzyme ESBL from K. pneumoniae, referred to as SHV (sulfhydryl variable), and the metabolite second from plants. The parameters analyzed were binding free energies, inhibition constants and visualization in 3D and 2D. The free energy of binding and the inhibition constant of the native ligand are -6.7 kcal/mol and 12.118 μM. The binding free energy values ​​of apigenin 7-glucuronide and rosmarinic acid show higher potencies than the native ligand of SHV-1 (1,4-thiazepine).

PREVALENCE OF MULTIDRUG RESISTANT ESCHERICHIA COLI AND KLEBSIELLA PNEUMONIAE SPECIES ISOLATED FROM URINE SAMPLES OF PATIENTS AT ALEX EKWUEME FEDERAL UNIVERSITY TEACHING HOSPITAL ABAKALIKI, EBONYI STATE, NIGERIA

Aim and Objective: In hospitals and community, morbidity and mortality are attributed to urinary tract infections (UTIs). This study investigated the prevalence of multi-drug resistant isolates of Escherichia coli and Klebsiella species from urine samples of patients visiting Alex Ekwueme Federal University Teaching Hospital Abakaliki (AE-FUTHA), Ebonyi State. Method: With the use of standard microbiological and biochemical techniques for analysis, a total of 300 mid-stream samples of urine were collected in sterile bottles. Phynotype screening for extended spectrum beta lactamase (ESBL) production was achieved by double disc synergy test (DDST). Disc diffusion method was used to check for ESBL producing bacteria susceptible to antibiotics. With specific primers, the presence of temoniera (TEM) and sulfhydryl variable (SHV) beta lactamases genes was determined using polymerase chain reaction (PCR). Results: 88 isolates of E coli and Klebsiella species were isolated from the urine samples; 29 were E coli and 59 were K. pneumonia species. 47 ESBL positive isolates were identified with E. coli, 19 (40%) and Klebsiella, 28 (60%). Escherichia coli isolated from GOPD patients, gynecology, and men's surgery ward were 100% resistant to ofloxacin, ceftazidime, amoxicillin, nitrofurantoin, chloramphenicol, and aztreonam, while Klebsiella species isolated from gynecology department, maternity ward, and psychiatric ward were 100% resistant to ofloxacin, Nitrofurantoin and chloramphenicol. Multiple antibiotics resistance index (MARI) of E. coli and Klebsiella species isolated recorded an average of 0.64 for E. coli and 0.41 for Klebsiella species.The molecular analysis revealed that 44.4% of SHV beta lactamase and 65.6% TEM-type β-lactamase genes were present in ESBL producing E. coli while 54.5% of SHV beta lactamase gene and 45.5% TEM-type β-lactamase genes were present in ESBL producing Klebsiella species. Conlusion: In conclusion, TEM and SHV-type β-lactamase genes are the primary cause of β-lactam antibiotic resistance in E. Coli and Klebsiella species resulting in increased infections caused by organisms harboring the ESBL gene. Nitrofurantoin, ceftazidime, cefalexin, and cefotaxime may be the antibiotic of choice in the treatment of UTIs. Peer Review History: Received: 1 January 2023; Revised: 14 February; Accepted: 7 March 2023, Available online: 15 March 2023 Academic Editor: Dr. Amany Mohamed Alboghdadly, Princess Nourah bint abdulrahman university, Riyadh, amalbgadley@pnu.edu.sa Received file: Reviewer's Comments: Average Peer review marks at initial stage: 5.0/10 Average Peer review marks at publication stage: 7.0/10 Reviewers: Prof. Hassan A.H. Al-Shamahy, Sana'a University, Yemen, shmahe@yemen.net.ye Dr. Tamer Elhabibi, Suez Canal University, Egypt, tamer_hassan@pharm.suez.edu.eg Similar Articles: ANALYSIS OF THE ANTIBIOGRAM PROFILES OF BIOFILM FORMING STAPHYLOCOCCUS AUREUS AND ESCHERICHIA COLI

Molecular interaction of a putative inhibitor with bacterial SHV, an enzyme associated with antibiotic resistance.

Tackling the ever-looming threat of antibiotic resistance remains a challenge for clinicians and microbiologists across the globe. Sulfhydryl variable (SHV) is a known bacterial enzyme associated with antibiotic resistance. The SHV enzyme has many variants. The present article describes identification and molecular interaction of a putative inhibitor with the bacterial SHV enzyme as a step towards novel antibacterial drug discovery. The MCULE-platform was used for screening a collection of 5 000 000 ligand molecules to evaluate their binding potential to the bacterial SHV-1 enzyme. Estimation of pharmacokinetic features was realized with the aid of the 'SWISS ADME' tool. Toxicity-checks were also performed. The docked complex of 'the top screened out ligand' and 'the bacterial SHV-1 protein' was subjected to molecular dynamics simulation of 101 ns. The obtained ligand molecule, 1,1'-(4H,8H-Bis[1,2,5]oxadiazolo[3,4-b:3',4'-e]pyrazine-4,8-diyl)diethanone, displayed the most favourable binding interactions with bacterial SHV-1. A total of 15 amino acid residues were found to hold the ligand in the binding site of SHV-1. Noticeably, 12 of the 15 residues were found as common to the binding residues of the reference (PDB ID: 4ZAM). The RMSD values plotted against the simulation time showed that nearby 11 ns, equilibrium was reached and, thenceforth, the 'SHV-1-Top ligand' complex remained typically stable. Starting from around 11 ns and straight to 101 ns, the backbone RMSD fluctuations were found to be confined inside a range of 1.0-1.6 Å. The ligand, 1,1'-(4H,8H-Bis[1,2,5]oxadiazolo[3,4-b:3',4'-e]pyrazine-4,8-diyl)diethanone, satisfied ADMET criteria. Furthermore, the practicability of the described 'SHV-1-Top ligand' complex was reinforced by a comprehensive molecular dynamics simulation of 101 ns. This ligand hence can be considered a promising lead for antibiotic design against SHV-1 producing resistant bacteria, and thus warrants wet laboratory evaluation.

Antibiotic susceptibility profiles and detection of genes mediating extended-spectrum β-lactamase (Esbl) production in escherichia coli isolates from National Hospital, Abuja.

Extended-spectrum beta-lactamases (ESBL) are the most widespread of the new beta-lactamases and are a significant threat to patient care in the hospital and the community. Aim: The study assessed the prevalence, antibiotic susceptibility profile, and major ESBL encoding genes among Escherichia coli isolated from clinical specimens of patients in the National Hospital, Abuja (NHA). E. coli isolated from diverse clinical specimens obtained from clinically proven cases of infection managed at the NHA were included in the study. The antimicrobial susceptibility was performed by the Kirby-Bauer method and E-test was used to confirm the ESBL phenotype. Multiplex polymerase chain reaction (PCR) was used to detect the genes mediating ESBL production. Meropenem, fosfomycin, and tigecycline demonstrated excellent activities against all isolates: of the 400 isolates, 392 (98%), 386 (96.5%), and 362 (90.5%) were susceptible, respectively. Similarly, 358 (89.5%) were susceptible to amikacin, 323 (80.3%) nitrofurantoin, 281 (70.3%) ceftazidime, and 279 (69.8%) cefotaxime. A total of 271 (67.8%), 219 (54.8%), and 208 (52.0%) were resistant to amoxicillin-clavulanate, ciprofloxacillin, and gentamicin, respectively. However, all the isolates were resistant to ampicillin. There was a significantly higher proportion of multidrug resistance among ESBL-producing isolates compared to non-ESBL-producing isolates (P = 0.0001). Of the 121 phenotypically detected ESBL isolates, 119 (98.3%) harbored genes mediating the production of Cefotaximase- Munich (CTX-M), Temoniera (TEM) or Sulfhydryl Variable (SHV) enzymes. The prevalence of ESBLs among E. coli was relatively high, at 30.2%. About 81% of all blood isolates were ESBL-producers. blaCTX-M is the predominant type of ESBL gene among E. coli. A high proportion of the ESBL-producing isolates expressed a combination of two or three genes together.

Simple and Rapid Detection of ESBL blaSHV gene from an Urban River in Tokyo by Loop-Mediated Isothermal Amplification.

Extended-spectrum β-lactamases (ESBLs) are produced mainly by gram-negative bacteria in Enterobacteriaceae. One of the major types of ESBLs is sulfhydryl variable (SHV) -type ESBL. Herein, we attempted to develop a simple and rapid method for the detection of the ESBL blaSHV gene by loop-mediated isothermal amplification (LAMP) . The five-primer set designed could amplify blaSHV gene at an isothermal temperature of 65℃. The detection limit of the LAMP method with the LF loop primer was 1 copy/tube, which was 10,000-fold more sensitive than that of the conventional PCR. The LAMP assay could also detect the direct amplification of blaSHV gene from a single river water sample in Tokyo. The LAMP method has great potential for applications in hospital, soil and water environment, food, and livestock.

Prevalence of Fecal Carriage of Extended-Spectrum β-Lactamase-Producing Enterobacteriaceae Among School Children in South Sulawesi, Indonesia

Background: Fecal carriage of extended-spectrum β-lactamase (ESBL)-producing bacteria is a potential risk for transmission and infection. Genotypes known to be associated with ESBL-producing Enterobacteriaceae are cefotaximases (CTX-M), temoniera (TEM), and sulfhydryl variable (SHV). Unfortunately, data on ESBL prevalence in Indonesia, especially in South Sulawesi, is still limited, so further research on the community is needed. Objectives: This study aimed to investigate the prevalence of fecal carriage of ESBL-producing Enterobacteriaceae among school children in South Sulawesi, Indonesia. Methods: The research was conducted among school-children in South Sulawesi, Indonesia. Detection of ESBL gene using polymerase chain reaction (PCR) methods. A total of 245 stool samples were collected. Results: The prevalence of fecal carriage of ESBL-producing Enterobacteriaceae among school children was 76.7% (188/245). Genotyping of EBSL-producing Enterobacteriaceae encoding genes using PCR found that TEM genes, 92.6% (174/188), were higher than SHV genes, 38.8% (73/188) and CTX-M genes, 4.3% (8/188). It was also revealed a combination of ESBL-producing genes of Enterobacteriaceae. The most combination genes were found in TEM + SHV genes, including 55 of 188 (29.26%). Conclusions: The presence of ESBL gene careers suggests that antibiotic resistance has spread in the community, which needs to be of concern since it can be an ESBL gene reservoir that can be transmitted to many pathogenic bacteria.

Using Molecular Techniques for the Characterization of Escherichia coli Isolated from Creek Road/Bonny Estuary in Rivers State, Nigeria

This study characterised Escherichia coli isolated from Creek Road/Bonny Estuary using molecular techniques. One hundred and twenty (120) water samples were investigated for the presence of E. coli in Creek Road/Bonny Estuary. Most Probable Number (MPN) Technique, Eijkman test and molecular techniques were used for the isolation and detection of E. coli. The analysis was carried out periodically (morning and evening) and seasonally (end of rainy season (November), the start of rainy season (April) and in the middle of rainy season (July)). All E. coli isolates were screened for resistance genes in the pathotypes of E. coli (EHEC, EPEC, EAEC, ETEC and EIEC), including Sulfhydryl Variable (SHV), CTX-M, Temoniera (TEM) and MCR genes using the Polymerase chain reaction Technique. The results showed that 100% of the water samples were positive for coliforms and all the water samples harboured E. coli. The highest total coliforms count recorded was in the morning of April (48 MPN/100 ml). The distribution of individual genes in the E. coli isolates are: SHV (5.0), CTX-M (5.8), TEM (4.2), MCR (0.0%), stx1 and stx2 (4.2), esV and bfA (1.7), aaiC (4.2), elt (3.3) and invE (2.5). The CTX-M was the most found of all the resistance genes.

Broad-Spectrum Cephalosporin-Resistant Klebsiella spp. Isolated from Diseased Horses in Austria.

Simple SummaryBroad-spectrum cephalosporin-resistant Klebsiella pneumoniae is considered as a serious problem for public human health. To date, only a few broad-spectrum cephalosporin-resistant Klebsiella have been isolated from horses. Considering the zoonotic potential of the Klebsiella spp., and the close relationship between man and horse, this study intended to generate data on the genetic background of broad-spectrum cephalosporin-resistant Klebsiella spp. isolated from horses in Austria. Overall, samples isolated between 2012 and 2019 from 1541 horses underwent bacteriological testing, resulting in 51 specimens tested positive for Klebsiella ssp. Antimicrobial susceptibility tests revealed that seven Klebsiella ssp. isolates were not only cefotaxime-resistant but also showed resistance against other classes of antibiotics so that they were considered to be multidrug-resistant. Data from whole genome sequencing and mating experiments strongly suggest that the majority of antibiotic resistance genes is encoded on plasmids in these seven multidrug-resistant Klebsiella ssp. Considering the potential threat when commensal Klebsiella inhabiting a healthy human gut acquire new antibiotic resistances due to the exchange of plasmids with multidrug-resistant Klebsiella ssp. from horses, further monitoring of horses and other domestic animals for the presence of broad-spectrum cephalosporin-resistant Klebsiella, not only in Austria but worldwide is therefore advisable.The aim of the present study was to investigate the diversity of broad-spectrum cephalosporin-resistant Klebsiella spp. isolated from horses in Austria that originated from diseased horses. A total of seven non-repetitive cefotaxime-resistant Klebsiella sp. isolates were obtained during diagnostic activities from autumn 2012 to October 2019. Antimicrobial susceptibility testing was performed. The isolates were genotyped by whole-genome sequencing (WGS). Four out of seven Klebsiella isolates were identified as K. pneumoniae, two as K. michiganensis and one as K. oxytoca. All isolates displayed a multi-drug resistant phenotype. The detection of resistance genes reflected well the phenotypic resistance profiles of the respective isolates. All but one isolate displayed the extended-spectrum β-lactamases (ESBL) phenotype and carried CTX-M cefotaximases, whereas one isolate displayed an ESBL and AmpC phenotype and carried cephamycinase (CMY)-2 and sulfhydryl variable (SHV)-type b and Temoniera (TEM) β-lactamases. Among Klebsiella pneumoniae isolates, for different sequence types (ST) could be detected (ST147, ST307, ST1228, and a new ST4848). Besides resistance genes, a variety of virulence genes, including genes coding for yersiniabactin were detected. Considering the high proximity between horses and humans, our results undoubtedly identified a public health issue. This deserves to be also monitored in the years to come.

Detection of SHV and TEM-type Extended spectrum β-lactamase in bacterial isolates recovered from clinical samples of patients attending military hospitals

BackgroundMulti-drug resistant bacterial strains have been increasingly implicated in clinical infections worldwide and beta-lactamase production is one of the commonest mechanisms of resistance in these strains. This study investigated the prevalence of extended spectrum â-lactamase (ESBL)-producing isolates and determined the temoneira (TEM) and sulfhydryl variable (SHV) types implicated in two military hospitals in South-South Nigeria. MethodsThree-hundred and eighty (380) consecutive non-duplicate bacterial isolates (Gram negative bacilli) recovered from clinical samples were identified following standard techniques. Antimicrobial susceptibility tests were performed for each isolate following the Clinical Laboratory Standards Institute guidelines. Bacterial isolates recovered which comprised Escherichia coli, Klebsiella spp, Proteus spp and Pseudomonas aeruginosa were screened for ESBL using a phenotypic method (double disc synergy test). All positive isolates were screened for TEM and SHV genes by PCR method. ResultsSixty-five isolates (17.1%) were ESBL producing using phenotypic method, E. coli showed the highest ESBL prevalence (24.3%). One isolate was SHV positive (1.5%), 8 (12.3%) were TEM positive while 3 (4.6%) isolates harbored both SHV and TEM genes. Fluoroquinolone - ofloxacin showed marked activity against ESBL-producing isolates (90.8%) while the least active were ceftriaxone (9.2%), ceftazidime (3.1%) and ampicillin (1.5%). ConclusionThis study demonstrated that 17.1% of Gram-negative bacilli were ESBL producers. Screening of clinical isolates for ESBL should be implemented. The findings of this study suggest the need for caution in the use of antimicrobial agents in order to curb the incidence of antimicrobial resistance.

First Detection of Carbapenem-Resistant Escherichia fergusonii Strains Harbouring Beta-Lactamase Genes from Clinical Samples.

Recently discovered extraintestinal Escherichia fergusonii obtained from non-clinical samples has exhibited the potential for acquiring multiple beta-lactamase genes, just like many extraintestinal Escherichia coli strains. Albeit, they are often omitted or classified as E. coli. This study aimed to, therefore, identify carbapenem-resistant extended-spectrum beta-lactamase (ESBL) producing E. fergusonii isolates from clinical samples, determine their evolutionary relatedness using 16S rRNA sequencing analysis and screen for beta-lactamase genes. A total of 135 septic wound samples were obtained from patients on referral at a General Hospital in Lagos, Nigeria. For the phenotypic identification of isolates from culture-positive samples, morphological, and physiological tests were carried out. Identities of the isolates harbouring beta-lactamase genes were assigned to their genus strains using the 16S rRNA sequencing. The Kirby Bauer disc diffusion technique and double-disc synergy test were used to screen isolates for multidrug resistance and ESBL production. Carbapenem-resistant ESBL producing isolates were screened for beta-lactamase genes in a polymerase chain reaction. Three E. fergusonii isolates (CR11, CR35 and CR49) were obtained during this study. E. fergusonii strains were motile, non-lactose and non-sorbitol fermenting but positive for cellobiose and adonitol fermentation. The I6S rRNA assigned the phenotypically identified isolates to E. fergusonii species. All three isolates were multidrug-resistant, carbapenem-resistant and ESBL producers. Isolates CR11 and CR35 harboured cefotaximase (CTX-M) and temoniera (TEM) beta-lactamase genes while CR49 harboured sulfhydryl variable (SHV) beta-lactamase gene. We herein report the detection of multiple beta-lactamase genes in carbapenem-resistant ESBL producing E. fergusonii from clinical samples.

In vitro Detection of Antibacterial Activity of Glycyrrhizic Acid Nanoparticle against ESBL Producing Klebsiella pneumoniae Strains

EXTENDED-spectrum β-lactamase (ESBL) producing Klebsiella pneumoniae strains can present resistance to many antibiotic groups due to resistant genes. This study conducted to detect and identify multi-drug resistant (MDR), ESBL producing K. pneumoniae strains from different clinical samples with detection and sequencing of both Temoneira (TEM) and sulfhydryl variable (SHV) genes and using Glycyrrhizic acid nanoparticle as an antimicrobial agent for ESBL producing K. pneumoniae strains. One hundred and fifty clinical specimens were processed. ESBL producing K. pneumoniae strains were detected by double disk synergy test. TEM and SHV genes responsible for MDR in K. pneumoniae were detected by polymerase chain reaction (PCR) and sequence alignment was done using DNA sequencing. The effect of different concentrations of Nano Glycyrrhizic acid was determined. K. pneumoniae was detected in 53.3% of the total collected samples (80/150). Seventy one percent (57/80) of them were found to be multi-drug resistant strains and 63% (36/57) also found to contain the ESBL enzymes. Males were highly infected than females. TEM gene was detected in 52.8% of the ESBL isolates while SHV gene was detected in 72.2%. Twenty Five percent of the ESBL producing K. pneumoniae was found to contain both TEM and SHV genes. Nucleic acid sequence alignment of both genes showed some mutations. Chloramphenicol was found to be the drug of choice to overcome ESBL producing K. pneumoniae with inhibition of 97.2%. The antibacterial activity of Nano Glycyrrhizic acid revealed that 10μg/ml was found to be the minimum bactericidal concentration (MBC) against ESBL producing K. pneumoniae isolates.

CTX-M-15 is Established in Most Multidrug-Resistant Uropathogenic Enterobacteriaceae and Pseudomonaceae from Hospitals in Nigeria.

β-Lactam antibiotics are widely used to treat urinary tract infections in Nigeria. This study aimed to determine the presence and characteristics of extended spectrum β-lactamases in commonly isolated uropathogenic Gram-negative bacteria (GNB) in Nigeria.Fifty non-duplicate GNB isolates consisting of Escherichia coli, 19; Klebsiella pneumoniae, 21; and Pseudomonas aeruginosa, 10 were obtained from three tertiary hospitals in Nigeria. The antibiotic susceptibility testing of all isolates to a panel of antibiotics including minimum inhibitory concentrations (MICs) and extended spectrum β-lactamases was determined. Polymerase chain reactions and sequencing were used to detect β-lactam genes.Polymerase chain reactions and sequencing identified varying extended spectrum β-lactamases (ESBLs) encoding genes for 24 isolates (48.0%). Cefotaximase-Munich (CTX-M) 15 was the dominant gene with 20/24 of the isolates positive at 83.3%; multiple genes (2 to 6 ESBL genes) were found in 20 of the isolates. The isolates encoded other genes such as CTX-M-14, 33.3%; sulfhydryl variable (SHV) variants, 58.3%; oxacillinase (OXA) variants, 70.8%; OXA-10, 29.2%; and Vietnamese extended β-lactamase (VEB) 1, 25.0%. There was no difference between the MIC50 and MIC90 of all the isolates.The high-level multidrug resistance of uropathogens to third generation cephalosporins including other antibiotics used in this study is strongly associated with carriage of ESBLs, predominantly CTX-M-15, as well as CTX-X-M-14, OXA-10, and VEB-1.

Antimicrobial susceptibility, risk factors and prevalence of bla cefotaximase, temoneira, and sulfhydryl variable genes among Escherichia coli in community-acquired pediatric urinary tract infection.

INTRODUCTION:The emergence of extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli has become an important challenge among pediatric patients with community-acquired urinary tract infection (UTI).OBJECTIVES:The aim of this study was to assess the antimicrobial susceptibility patterns, associated risk factors and to survey the frequency of bla cefotaximase (CTX-M), bla temoneira (TEM), and bla sulfhydryl variable (SHV) genotypes in ESBL-producing E. coli isolated from children with community-acquired UTI.METHODS:This was a prospective study conducted from November 2012 to March 2016 in a tertiary care center. E. coli isolated in urine cultures from children aged ≤18 years was identified and confirmed for ESBL production. ESBL-positive strains were screened for ESBL encoding genes. Chi-square test and Fisher's exact test were used to compare the difference in antibiotic susceptibility with respect to ESBL positive and negative, and binary logistic regression was used to identify the risk factors associated with ESBL production.RESULTS:Among 523 E. coli isolates, 196 (37.5%) were ESBL positive, >90% were resistant to cephalosporins, and 56% were resistant to fluoroquinolones. Least resistance was observed for imipenem, netilmicin, and nitrofurantoin (2%, 8.6%, 15.3%). Association between ESBL production and drug resistance was significant for ceftazidime (P < 0.001), cefixime (P < 0.001), cefotaxime (P = 0.010), ceftazidime-clavulanic acid (P < 0.001), levofloxacin (P = 0.037), and gentamicin (P = 0.047) compared to non-ESBL E. coli. CTX-M gene was the most prevalent (87.5%), followed by TEM (68.4%) and SHV (3.1%). Previous history of UTI and intake of antibiotics were the common risk factors.CONCLUSION:ESBL-producing E. coli from community-acquired pediatric UTI carries more than one type of beta-lactamase coding genes correlating their increased antibiotic resistance. Aggressive infection control policy, routine screening for detecting ESBL isolates in clinical samples, and antimicrobial stewardship are the keys to prevent their dissemination in community settings.

Phenotypic and molecular characterization of cefotaximases, temoniera, and sulfhydryl variable β-lactamases in Pseudomonas and Acinetobacter isolates in an Indian tertiary health-care center.

Cefotaximases (CTX-M), temoniera (TEM), and sulfhydryl variable (SHV) constitute a rapidly growing cluster of enzymes that have disseminated geographically. They are spreading to species other than Enterobacteriaceae and might be responsible for the presence of blaCTX-M,blaTEM, and blaSHVgenes in Pseudomonas and Acinetobacter spp. The present study was designed to characterize CTX-M, TEM, and SHV phenotypically and genotypically in Pseudomonas and Acinetobacter spp. A total of 90 isolates (73 Pseudomonas and 17 Acinetobacter spp.), resistant to any of the third-generation cephalosporins, were randomly selected from clinical samples. Of 90 isolates, 64 (71.11%) were tested positive for extended-spectrum β-lactamase (ESBL) production. Among phenotypically tested ESBL producers, forty isolates were randomly selected for molecular characterization. The prevalence of CTX-M, TEM, and SHV was found to be 57.5%, 15%, and 75%, respectively. Multiplex polymerase chain reaction assay categorized blaCTX-Mgenes into Groups 1 and 26 where Group 1 was present in only 5 isolates and Group 25 was present in rest of the 18 isolates. This is among the premier systemic reports from India documenting phenotypic and molecular characterization of CTX-M, TEM, and SHV β-lactamases in Pseudomonas and Acinetobacter spp. With judicious use of antibiotics and strict infection control procedures, it may be possible to limit the effects of these newer β-lactamases.

OCCURRENCE OF CARBAPENEMASES AND EXTENDED-SPECTRUM ΒETA-LACTAMASES IN UROPATHOGENIC ENTEROBACTERIACEAE ISOLATED FROM A COMMUNITY SETTING, SETTAT, MOROCCO

ABSTRACTObjective: Urinary tract infections (UTIs) are still commonly diagnosed in outpatients as well as in hospitalized patients. In this study, weinvestigated the prevalence and performed molecular characterization of extended-spectrum-β-lactamases (ESBL) and carbapenemases produced byEnterobacteriaceae isolates that cause community UTIs in Settat city, Morocco.Methods: From January 2012 to December 2013, all uropathogenic community Enterobacteriaceae isolates were collected from the microbiologylaboratory of Hassan II Hospital, Settat, Morocco. Antibiotic susceptibility testing was performed as recommended by Clinical and LaboratoryStandard Institute. Phenotypic identification of ESBL and carbapenemase producer isolates was confirmed by the double-disk synergy test and themodified Hodge test, respectively. Molecular characterization of β-lactamase genes was performed using polymerase chain reaction (PCR), followedby sequencing of the obtained products.Results: Among 153 isolates, 31 (20.26%) were multi-drug resistant (MDR). Nine strains (5.88%) were ESBL producers, of which Klebsiellapneumoniae (n=5; 20.83%), Escherichia coli (n=3; 3.15%), and Enterobacter cloacae (n=1; 9.09%) species were identified. The results of ESBLencodinggenedetectionby sequencing revealed the presence of CTX-M-15 (n=9) in association with other β-lactamase genes such as temoneira 1(n=8) and sulfhydryl variable 1 (n=5). According to the modified Hodge test and PCR, three isolates (1.96%) were positive carry the blaConclusion: The emergence of MDR uropathogenic Enterobacteriaceae isolates in our community is highly alarming. Strict measures will be requiredto control the further spread of these uropathogenic isolates.Keywords: Carbapenemase, Enterobacteriaceae, Extended-spectrum-β-lactamases, Moroccan community.OXA-48 gene.

Design and exploration of novel boronic acid inhibitors reveals important interactions with a clavulanic acid-resistant sulfhydryl-variable (SHV) β-lactamase.

Inhibitor resistant (IR) class A β-lactamases pose a significant threat to many current antibiotic combinations. The K234R substitution in the SHV β-lactamase, from Klebsiella pneumoniae , results in resistance to ampicillin/clavulanate. After site-saturation mutagenesis of Lys-234 in SHV, microbiological and biochemical characterization of the resulting β-lactamases revealed that only -Arg conferred resistance to ampicillin/clavulanate. X-ray crystallography revealed two conformations of Arg-234 and Ser-130 in SHV K234R. The movement of Ser-130 is the principal cause of the observed clavulanate resistance. A panel of boronic acid inhibitors was designed and tested against SHV-1 and SHV K234R. A chiral ampicillin analogue was discovered to have a 2.4 ± 0.2 nM K(i) for SHV K234R; the chiral ampicillin analogue formed a more complex hydrogen-bonding network in SHV K234R vs SHV-1. Consideration of the spatial position of Ser-130 and Lys-234 and this hydrogen-bonding network will be important in the design of novel antibiotics targeting IR β-lactamases.