IN SILICO AND IN VITRO ANALYSIS OF BLASHV AND BLATEM GENE PRIMERS

Extended-spectrum beta-lactamases (ESBLs) are bacterial enzymes capable of hydrolyzing beta-lactams. The aim of this study was to describe the prevalence of TEM- and SHV-type ESBL-producing Klebsiella pneumoniae strains in Zahedan, Southeast Iran. A total of 170 non-repetitive K. pneumoniae strains were collected from patients referred to three teaching hospitals of Zahedan. Antibiotic susceptibility testing was determined for 17 antibiotics using the Kirby-Bauer disc diffusion method. The frequency of ESBL-producing strains was calculated, and minimum inhibitory concentrations of ESBL-producing strains were determined for cefotaxime, ceftazidime, ceftriaxone, and cefpodoxime. The presence of bla TEM and bla SHV genes was tested in all ESBL-producing strains using polymerase chain reaction and DNA sequencing. Among the 170 K. pneumoniae clinical isolates, 55 (32.4%) were ESBL producers; 92.7% (n=51) and 72.7% (n=40) of the isolates carried the bla SHV and bla TEM genes, respectively, and 67.3% (n=37) carried both genes. The sequencing results showed that all bla TEM types were bla TEM-1, except for two isolates that were bla TEM-104. The bla SHV types were bla SHV-1, bla SHV-11, bla SHV-12, bla SHV-99, bla SHV-108, and bla SHV-110. The percentage of bla TEM and bla SHV among ESBL-producing K. pneumoniae isolates from Zahedan is relatively high, indicating the need for further surveillance and consideration in antibiotic use. To the best of our knowledge, this is the first report of TEM-104-, SHV-99-, SHV-108-, and SHV-110-type ESBLs among clinical isolates of K. pneumoniae from Iran, and TEM-1, SHV-1, SHV-11, and SHV-12 appear to be the dominant ESBLs in this region.

Objective: Toll-like receptors (TLRs) are key pattern recognition receptors involved in tumorigenesis, apoptosis, and metastasis. Triple-negative breast cancer (TNBC) is a highly aggressive malignancy with a poor prognosis. Although the role of TLRs in breast cancer remains underexplored, recent studies suggest targeting TLRs in TNBC could be beneficial. In this study Thiostrepton, an antibiotic and novel inhibitor of TLR7-9 in psoriatic inflammation, was investigated for its effects on TLR3, TLR4, and TLR9 expression in TNBC cells (MDA-MB-231). Materials and Methods: The cytotoxicity of thiostrepton was assessed using the MTT assay. RT-PCR was used to measure gene expression levels of TLR3, TLR4, TLR9, Bax, Bcl-2, Nf-κB, and E-cadherin. Cell morphology changes were analyzed with Acridine Orange/Ethidium Bromide (AO/EtBr) staining. Molecular docking and dynamics simulations examined interactions between thiostrepton and the TLR4-MD-2 complex. Results: Thiostrepton led to a concentration- and time-dependent decrease in cell viability. It significantly inhibited TLR4, Bcl-2 gene expression and increased TLR3, Bax, and Nf-κB levels. The changes in Bax and Bcl-2 gene expression, along with alterations in cell morphology, demonstrated that thiostrepton promoted apoptosis in MDA-MB-231 cells. While TLR9 expression reduction was not significant, thiostrepton notably increased TLR3 expression and decreased TLR4 expression. The three independent molecular dynamics simulations demonstrated that thiostrepton binds stably to the TLR4-MD2 domain, exhibiting a high binding affinity as indicated by the binding free energy calculations. Conclusion: Thiostrepton effectively induces apoptosis and reduces cell viability in TNBC cells. In silico analysis suggest thiostrepton could modulate TLR4, highlighting its potential as a candidate for further research and therapeutic development.

Introducción. Las betalactamasas de espectro extendido (BLEE) son un grupo de enzimas que confieren resistencia bacteriana a un amplio espectro de antibióticos betalactámicos codificados en plásmidos y que deben estudiarse como herramientas de vigilancia del comportamiento de microorganismos frente al uso de antibióticos.
 Objetivo. Determinar los genes que codifican la resistencia en bacilos gramnegativos con fenotipo BLEE aislados de urocultivos, en una institución prestadora de servicios de salud del departamento de Boyacá.
 Métodos. Se llevó a cabo un estudio observacional, descriptivo y de corte transversal. Se identificaron 19 cepas resistentes con fenotipo BLEE, siguiendo los lineamientos del Clinical and Laboratory Standards Institute (CLSI M100-S23), y se estableció el índice de concordancia para la identificación microbiológica. Por medio de la estandarización de la técnica de reacción en cadena de la polimerasa (PCR), se determinó la presencia de los genes blaTEM, blaSHV, blaCTX y Amp-C en estas 19 cepas.
 Resultados. Se encontró que las 19 cepas con fenotipo BLEE (100 %) presentaban resistencia a la ampicilina; 12 (63,2 %) a la ampicilina-sulbactam; 17 (89,5 %) a la cefalotina; 16 (84,2 %) a la cefuroxima; 17 (89,5%) a la cefotaxima; 17 (89,5 %) a la ceftriaxona y 14 (73,7%) al cefepime. En 18 aislamientos se hizo amplificación de los genes, de los cuales 12 (61,11 %) posiblemente presentaron el gen blaCTX; 10 (55,6 %) el gen AmpC; 9 (50 %) el gen blaSHV y 7 (38,88 %) el gen blaTEM.
 Conclusión. La presencia de los genes blaCTX, AmpC, blaSHV y blaTEM presenta importancia epide-miológica, probablemente por la capacidad para movilizar la información genética de la resistencia en el ambiente hospitalario, donde tienen un claro potencial epidémico.
 Palabras clave: antibiótico, farmacorresistencia bacteriana, betalactamasa.

The objective was to genotypically determine by PCRc, the presence of the blaSHV, blaTEM and blaCTX-M genes, in K. pneumoniae producer of ESBL, of the ICU service of two hospitals in Cajamarca. In this study, different clinical biological samples were used, from which 59 strains of K. pneumoniae ESBL + were isolated, of which 37 (62.7%) strains correspond to the Regional Hospital of Cajamarca and 22 (37.3%) to the Hospital II Essalud Cajamarca. With respect to the production of resistance genes, in the Regional Hospital of Cajamarca 37/37 (100%) strains presented the blaSHV gene, 33/37 (89%) the blaTEM gene and 36/37 (97%) the blaCTX- M. In the Essalud Cajamarca II hospital, the presence of the blaSHV gene was 17/22 (77%) strains, blaTEM 20/22 (91%) and blaCTX-M 20/22 (91%). Performing the total statistical analysis at the level of hospitals of Cajamarca, the blaSHV gene is present in 54/59 (92%) of strains, the blaTEM gene in 53/59 (90%) and the blaCTX-M gene in 56/59 (95%) strains. It is therefore concluded that, in both hospitals of Cajamarca, antimicrobial resistance is high, which is mainly produced by the genes under study, especially for K. pneumoniae ESBL +, which encode extended spectrum beta-lactamases

Background: Recently, extended spectrum β-lactamase (ESβL) genes including blaTEM, blaCTX-M and blaSHV genes have shown rapid spread among Enterobacteriaceae and they become the most prevalent genes in many parts of the world. Methods: This study is aimed to detect ESβL producers among Escherichia coli isolates from urine specimens. Sixty-six uopathogenic E. coli (UPEC) strains were isolated at the National Center for Teaching Laboratories of Medical City in Baghdad. The phenotypic detection of ESβL was achieved by Vitek-2 system with AST-N204 card and the results revealed that among the 66 uropathogenic E. coli strains, 48 (72.7%) were ESβL producing and 18 (27.3%) were ESβL non-producing (significant difference at P < 0.05). Results: The presence of genes encoding TEM, CTX-M and SHV was tested by all ESβL producing uropathogenic E. coli phenotypically by conventional PCR. The result showed that blaTEM, blaCTX-M and blaSHV genes were 46 (95.83%), 42 (87.5%) and 39 (81.25%), respectively. In addition, the data showed the coexistence of blaTEM, blaCTX-M and blaSHV genes in 44 out of 48 strains (91.67%) while only 4 (8.33%) of UPEC strains harbored a single ESβL gene (significant difference at P < 0.05). Conclusions: This study showed that blaTEM was the widespread gene in ESβLs-producing uropathogenic E. coli strains in Baghdad/Iraq and there was a high percentage of blaTEM, blaCTX-M and blaSHV genes distribution among local strains under study and this may attribute to the indiscriminate use of antibiotics. Phylogenetic analyses based on the sequences of blaTEM, blaCTX-M and blaSHV genes were done with closely related blaTEM, blaCTX-M and blaSHV genes in the GenBank by using MEGA6 software.

Antimicrobial drug resistance has become prominent as a universal health threat. This has been studied not only in humans but food animals as well. Many genes located on the chromosomal DNA of bacteria have been linked with drug resistance. It is therefore crucial that its occurrence in abattoirs where these animals are slaughtered be studied. This study was thus aimed at identifying some resistance genes in microbes isolated from abattoir ecologies. One hundred and eighty (180) samples consisting of service water, waste blood, wastewater, soil and faecal matter collected from Iwofe, Rumuodumaya and Trans-Amadi abattoirs within a period of one year were used in this study. Processing of the samples was done using standard microbiological protocols and the antibiotics sensitivity profile of the isolated bacteria determined using popularly consumed antibiotics. The presence of Beta-lactamase genes was checked for in the multidrug resistant isolates after they had been identified using molecular biological technique. Two strains of Escherichia coli had the blaCTX-M gene, Pseudomonas sp. strain 6174 had the blaSHV and blaTEM genes, Bacillus amyloliquefaciens had the blaSHV gene, Bacillus flexus had the blaTEM genes, Staphylococcus aureus had blaSHV and blaTEM genes. blaSHV, blaCTX-M and blaTEM genes were found in most of the Gram negative bacteria as Klebsiella sp. strain EIKU11 showed prominence of all three β-lactamase genes. This brings to light that some microbes in abattoir environments possess the β-lactamase genes which enable these microorganisms exhibit multidrug resistance, thereby making treatment resulting from them difficult. Adequate sanitary measures should thus be carried out to reduce the spread of these organisms to humans.

IntroductionThe aim of this study was to investigate the antimicrobial susceptibility pattern and the presence of ESBLs among the uropathogenic Escherichia coli (UPEC) isolated from kidney transplant patients (KTP) and community-acquired urinary tract infections (UTIs) using phenotypic and molecular methods.Materials and MethodsA total of 111 pure cultures of UPEC isolates were collected from 65 and 46 of non-KTP and KTPs with UTIs. The pattern and ESBL production of the strains were evaluated. PCR reaction to detect the presence of blaSHV, blaTEM, and blaCTX-M genes was performed.ResultsThe results revealed that most of UPEC isolates obtained from KTPs and control group were resistant to trimethoprim/sulfamethoxazole (84.8% vs 46.2%), while carbapenems (100% sensitivity) were the most effective against UPEC isolates. ESBL-producing strains were significantly more frequent in KTPs compared with control group (43.5% vs 23.1%, P = 0.021). The molecular results revealed that 53.2% (59/111), 45% (50/111), and 5.4% (6/111) of isolates harbored blaCTX-M, blaTEM, and blaSHV genes, respectively. Of the genes investigated, blaCTX-M and blaTEM genes were significantly higher among KTP than the control group.ConclusionOur results showed a high proportion of multidrug-resistant and ESBL-producing isolates, which most of them harbor blaCTX-M. A significant high co-resistance to different classes of antibiotics was reported from ESBL-producing UPEC from KTPs, which remains a serious clinical challenge.

In this study, a total of 68 raw milk samples were used to investigate the prevalence of Enterobacteriaceae in milk samples obtained from different dairy and supermarket in the province of Amasya (Turkey), as well as to determine the antibiotic resistance profile, the presence of Int1, blaTEM and blaSHV gene. In this study, isolates were obtained using classical culture technique. Then, detection of antibiotic resistance profile was carried out using disc diffusion methods. 12 different antibiotics were used as antibiotics including meropenem, cefotaxime, nalidixic acid, ceftriaxone, chloramphenicol, ceftazidime, streptomycin, ampicillin, gentamicin, tetracycline, levofloxacin and trimethoprim-sulfamethoxazole. Final, single strain PCR was created for the detection of ESBLs. For the aims, blaTEM and blaSHV genes (for determination of extended-spectrum beta-lactamase) were demonstrated by PCR assay. Then, for determination of Int1 was determined by using PCR assay. As a result, 50 isolates belonging to the Enterobacteiaceae family were obtained. Isolates against 41 (82%) ampicillin, 38 (76%) trimethoprim-sulfamethoxol, 7 (14%) ceftazidime, 6 (12%) cefotaxime, 2 (4%) meropenem and nalidixic acid and 1 (2%) ceftriaxone and streptomycin was determined as resistant. In addition, isolates were found to be 49 (98%), 50 (100%) 49 (98%) and 50 (100%) sensitive to chloramphenicol, gentamicin tetracycline and levofloxacin antibiotics, respectively. Among Enterobacteriaceae isolates, 22 (44%), 6 (12%) and 2 (4%) rates of strains were carrying Int1, blaSHV and blaTEM gene, respectively. In conclusion, the resistance of Enterobacteriaceae isolates isolated from milk samples to many antibiotics poses a potential danger in terms of public health

Simple and reliable multiplex PCR assay for detection of blaTEM, blaSHV and blaOXA-1 genes in Enterobacteriaceae

Aims: The aim of this work was to determine the resistance profile and to investigate the production of extended spectrum β-lactamases (ESBL) by clinically relevant Gram-negative Bacillus (GNB) strains.
 Methodology: About 191 strains were isolated from 1823 samples collected at the HKM National Hospital and University Center of Cotonou (Benin). Species identification was done with the Api 20th gallery. Two methods were used to search for β-lactamase production: the liquid acidimetric test for penicillinases and double halo method for ESBL. The susceptibility to conventional antibiotic molecules was investigated by the disk diffusion method. Polymerase Chain Reaction (PCR) was used to identify blaTEM and blaSHV genes in the β-lactamases.
 Results: A prevalence of 10.48% of GNB was recorded. Among the isolated strains, 51.31% came from samples collected from in-patients and 48.69% from out-patients’ samples. The most contaminated samples were urine (43.98%), pus (34.58%) and blood (9.42%). Majority of the isolated species included: Klebsiella pneumoniae (28.27%), Acinetobacter spp. (18.32%), Pseudomonas aeruginosa (15.72%), Escherichia coli (14.15%) and Enterobacter cloacae (12.04%). More than the half (57.07%) of the strains produced penicillinases; whereas 16.76% were ESBL-producers and these occurred only among Klebsiella pneumoniae, Enterobacter cloacae, Escherichia coli and Enterobacter agglomerans. The ESBL-producing strains were cross-resistant to beta-lactams. Imipenem is the most effective antibiotic on all isolated strains. ESBL-producing GNB strains possessed both the blaTEM gene and the blaSHV gene in a proportion of 25%; 37.5% of the strains had only the blaTEM gene and 12.5% of the strains had only the blaSHV gene.
 Conclusion: ESBL-producing strains of K. pneumonia in the hospital environment were the major carriers of blaTEM and blaSHV. Given this situation, it is necessary to continue research to identify resistance genes.

Antibiotic therapy in hematologic patients, often weak and susceptible to a wide range of infections, particularly nosocomial infections derived from long hospitalization periods, is a challenging issue. This paper presents ESBL-producing strains isolated from such hematologic patients treated at the Amazon Hematology and Hemotherapy Foundation (HEMOAM) in the Brazilian Amazon Region to identify the ESBL genes carried by them as well as the susceptibility to 11 antimicrobial agents using the E-test method. A total of 146 clinical samples were obtained from July 2007 to August 2008, when 17 gram-negative strains were isolated in our institution. The most frequent isolates confirmed by biochemical tests and 16S rRNA sequencing were E. coli (8/17), Serratia spp. (3/17) and B.cepacia (2/17).All gram-negative strains were tested for extended-spectrum-beta-lactamases (ESBLs), where: (12/17) strains carried ESBL; among these, (8/12) isolates carried blaTEM, blaCTX-M, blaOXA , blaSHV genes, (1/12) blaTEM gene and (3/12) blaTEM, blaCTX-M, blaOXA genes. Antibiotic resistance was found in (15/17) of the isolates for tetracycline, (12/17) for ciprofloxacin, (1/17) resistance for cefoxitin and chloramphenicol, (1/17) for amikacin and (3/17) cefepime. This research showed the presence of gram-negative ESBL-producing bacteria infecting hematologic patients in HEMOAM. These strains carried the blaTEM, blaSHV, blaCTX-M and blaOXA genes and were resistant to different antibiotics used in the treatment. This finding was based on a period of 13 months, during which clinical samples from specific populations were obtained. Therefore, caution is required when generalizing the results that must be based on posological orientations and new breakpoints for disk diffusion and microdilution published by CLSI 2010.

This study aimed to identify extended-spectrum β-lactamase (ESBL)-producing Escherichia coli in chicken, beef, and pork meats sold in Recife, Pernambuco, Brazil. A total of 120 meat samples (40 of each type) were collected from supermarkets, butcher shops, and open-air markets across the city’s eight health districts, using convenience sampling. The samples were processed in a microbiology laboratory, and E. coli was identified through selective isolation on eosin-methylene blue agar and biochemical tests. Phenotypic resistance was assessed using disk diffusion tests on Müller–Hinton agar with cefotaxime, ceftazidime, and ceftriaxone disks, followed by the double-disk synergy test to confirm ESBL production. Genotypic analysis was conducted by polymerase chain reaction to detect the blaTEM and blaSHV genes. Of the 40 E. coli isolates obtained, 34 (85%) exhibited phenotypic resistance, while 21 (52.5%) and 23 (57.5%) tested positive for the blaSHV and blaTEM genes, respectively. A higher prevalence of blaSHV was observed in pork samples (73.3%, 11/15), whereas blaTEM was more prevalent in beef (70%, 7/10). The presence of resistant bacteria in commercial meats highlights contamination risks in the production chain and underscores the need for surveillance and public awareness to protect human health.

Infectious illnesses are a serious health concern in Indonesia. Widespread use of self-medication by the community increases the risk of developing multi-drug resistant (MDR) bacteria. This study assessed the potential of sappan wood as an inhibitor of extended-spectrum beta-lactamase (ESBL) encoded by blaSHV, blaTEM and blaCTX-M genes. In silico testing was conducted to develop an effective and economical starting strategy. Thereby, this study significantly advances the development of novel treatments to combat antibiotic resistance. Using clavulanic acid as the benchmark medicine, the potency of the beta-lactamase inhibitor brazilein was predicted. Using the Molegro Virtual Docker computer tool, docking was performed to estimate the chemical and physical properties of the compounds, as well as the biological activity of brazilein toward the required receptor. The receptors used were SHV-1 beta-lactamase, PDB code: 2H0T; TEM-1 beta-lactamase, PDB code: 4OQG and CTX-M-14 beta-lactamase, PDB code: 6VHS. Data analysis was performed by comparing the binding energies of the docking results between the ligands and the target receptor. The more stable the bond that formed between the ligand and the target receptor, the lower the bond energy. The in silico test results on the blaSHV gene were as follows: binding energy of ligand MA4_400[A] = -100.699, brazilein = -82.206, clavulanic acid = -79.3704; in the blaTEM gene: ligand bond energy 2UL_301[B] = -107.681, brazilein = -82.0296, clavulanic acid = -103.3; in the blaCTX-M gene: X57_301[A] ligand bond energy = -86.6197, and brazilein = -88.1586, clavulanic acid = -101.933. The findings of this study demonstrate the significant potential of brazilein sappan wood to block the beta-lactamase activity of blaCTX-M.

Antibiotic therapy in hematologic patients, often weak and susceptible to a wide range of infections, particularly nosocomial infections derived from long hospitalization periods, is a challenging issue. This paper presents ESBL-producing strains isolated from such hematologic patients treated at the Amazon Hematology and Hemotherapy Foundation (HEMOAM) in the Brazilian Amazon Region to identify the ESBL genes carried by them as well as the susceptibility to 11 antimicrobial agents using the E-test method. A total of 146 clinical samples were obtained from July 2007 to August 2008, when 17 gram-negative strains were isolated in our institution. The most frequent isolates confirmed by biochemical tests and 16S rRNA sequencing were E. coli (8/17), Serratia spp. (3/17) and B.cepacia (2/17). All gram-negative strains were tested for extended-spectrum-beta-lactamases (ESBLs), where: (12/17) strains carried ESBL; among these, (8/12) isolates carried bla TEM, bla CTX-M, bla OXA , bla SHV genes, (1/12) bla TEM gene and (3/12) bla TEM, bla CTX-M, bla OXA genes. Antibiotic resistance was found in (15/17) of the isolates for tetracycline, (12/17) for ciprofloxacin, (1/17) resistance for cefoxitin and chloramphenicol, (1/17) for amikacin and (3/17) cefepime. This research showed the presence of gram-negative ESBL-producing bacteria infecting hematologic patients in HEMOAM. These strains carried the bla TEM, bla SHV, bla CTX-M and bla OXA genes and were resistant to different antibiotics used in the treatment. This finding was based on a period of 13 months, during which clinical samples from specific populations were obtained. Therefore, caution is required when generalizing the results that must be based on posological orientations and new breakpoints for disk diffusion and microdilution published by CLSI 2010.

BackgroundThe indiscriminate use and the similarity of prescribed antibiotics especially beta-lactams in human and small animal medicine, along with the close communication between pets and humans, increases the risk of the transfer of antibiotic-resistant bacteria and/or resistance elements especially integrons, between them. Therefore, we aimed to compare the frequencies of extended spectrum beta-lactamase (ESBL)-producing strains, major ESBL genes, classes 1 and 2 integrons, and antibiotic resistance patterns of fecal Escherichia coli (E. coli) isolates from dogs and their owners.MethodsThe present study was conducted on 144 commensal E. coli isolates from the feces of 28 healthy dog-owner pairs and 16 healthy humans who did not own pets. Phenotypic confirmatory test was used to identify the frequencies of ESBL-producing E. coli. Frequencies of blaCTX-M, blaSHV, and blaTEM genes, and also classes 1 and 2 integrons were determined by polymerase chain reaction. Resistance against 16 conventional antibiotics was determined by disk diffusion technique.ResultsESBL-production status was similar between the E. coli isolates of 71.4% of dog-owner pairs. The E. coli isolates of 75, 60.7, and 85.7% of dog-owner pairs were similar in terms of the presence or absence of blaCTX-M, blaTEM, and blaSHV genes, respectively. The presence or absence of class 1 and class 2 integrons was the same in E. coli isolates of 57.1% of dog-owner pairs. Prevalence of resistance to chloramphenicol and tetracycline was significantly higher in E. coli isolates of dogs than owners, but for other 10 (83.3%) tested antibiotics, no statistically significant difference was found in prevalence of antibiotic resistance between dogs and owners isolates. Furthermore, the antibiotic-resistance profile was the same in the E. coli isolates of 14.3% of dog-owner pairs.ConclusionsThe results of current research highlight the seriousness of the drug-resistance problem and the need to prevent further increases and spread of antibiotic-resistance to reduce treatment failure. Moreover, relatively similar characteristics of the E. coli isolates of dogs and their owners can show the risk of sharing resistant bacteria and/or resistance elements between them.