Drug-resistant Isolates Research Articles

Molecular Epidemiology and Polymorphism Analysis in Drug-Resistant Genes in M. tuberculosis Clinical Isolates from Western and Northern India.

IntroductionThe mechanistic details of first line drug (FLD) resistance have been thoroughly explored but the genetic resistance mechanisms of second line injectables, which form the backbone of the combinatorial drug resistant tuberculosis therapy, are partially identified. This study aims to highlight the genetic and spoligotypic differences in the second line drug (SLD) resistant and sensitive Mycobacterium tuberculosis (Mtb) clinical isolates from Mumbai (Western India) and Lucknow (Northern India).MethodsThe rrs, eis, whiB7, tlyA, gyrA and gyrB target loci were screened in 126 isolates and spoligotyped.ResultsThe novel mutations were observed in whiB7 loci (A43T, C44A, C47A, G48T, G59A and T152G in 5’-UTR; A42C, C253T and T270G in gene), tlyA (+CG200, G165A, C415G, and +G543) and gyrB (+G1359 and +A1429). Altogether, the rrs, eis, and whiB7 loci harbored mutations in ~86% and ~47% kanamycin resistant isolates from Mumbai and Lucknow, respectively. Mumbai strains displayed higher prevalence of mutations in gyrA (~85%) and gyrB loci (~13%) as compared to those from Lucknow (~69% and ~3.0%, respectively). Further, spoligotyping revealed that Beijing lineage is distributed equally amongst the drug resistant strains of Mumbai and Lucknow, but EAI-5 is existed at a higher level only in Mumbai. The lineages Manu2, CAS1-Delhi and T1 are more prevalent in Lucknow.ConclusionBesides identifying novel mutations in whiB7, tlyA and gyrB target loci, our analyses unveiled a potential polymorphic and phylogeographical demarcation among two distinct regions.

QSAR based on hybrid optimal descriptors as a tool to predict antibacterial activity against Staphylococcus aureus.

Staphylococcus aureus bacterial infections are still a serious health care problem. Therefore, the development of new drugs for these infections is a constant requirement. Quantitative structure-activity relationship (QSAR) methods can assist this development. The study included 151 structurally diverse compounds with antibacterial activity against S. aureus ATCC 25923 (Endpoint 1) or the drug-resistant clinical isolate of S. aureus (Endpoint 2). QSARs based on hybrid optimal descriptors were used. The predictive potential of developed models has been checked with three random splits into training, passive training, calibration, and validation sets. The proposed models give satisfactory predictive models for both endpoints examined. The results of the study show the possibility of SMILES-based QSAR in the evaluation of the antibacterial activity of structurally diverse compounds for both endpoints. Although the developed models give satisfactory predictive models for both endpoints examined, splitting has an apparent influence on the statistical quality of the models.

Comparing Pili Producing Gene (mtp-flp) in Susceptible and Resistant Dormant Mycobacterium Tuberculosis Strains with Active Clinical Isolates

Background: Pili producing genes in different life cycles of Mycobacterium tuberculosis (M. tuberculosis) were assessed. M. tuberculosis has two life cycles: dormant and active states. We aimed to assess the pili producing genes such as curli pili of M. tuberculosis (mtp) encoded by the mtp gene (Rv3312A) and fimbrial low-molecular-weight protein encoded by flp gene (Rv3656c) which were compared and analyzed. Methods: Two hundred M. tuberculosis isolates were investigated both at active and dormant states for production and expression of pili. The dormant M. tuberculosis was achieved by incubation in a sealed tube (modified Wayne method). The susceptibility of M. tuberculosis was evaluated on genes, rpob, inh, katg, and gyra by using multiplex polymerase chain reaction (PCR) and single-strand conformational polymorphism methods. The PCR–restriction fragment length polymorphism was used to express pili genes mtp and flp and then the PCR products was digested using restriction enzyme Fnu4HI, XmaI, and MspJI and AciI, TagII, and HaeII, respectively. The transmission electron microscopy was also used to detect pili in different isolates. The result was compared and analyzed using H37RV as a gold standard. Results: The mtp and flp PCR products were 263 and 122 bp in the studied strains irrespective of M. tuberculosis different life cycles, respectively. The PCR products were analyzed on 8% Polyacrylamide gel electrophoresis (PAGE), and in the 180/200 (20%), producing five fragments of 25,40,45,63,90 bp with the Fun4HI and two fragments of 126,138 bp with the XmaI and uncut with the MspJI for mtp gen were obtained at the dormant and active states of M. tuberculosis (P < 0.05). Similarly in flp gene producing three fragments of 22,35,65 bp with AciI and two fragments of 35.87 bp with TagII and two fragments of 38.84 bp with HaeII were obtained (P < 0.05). In contrast to genotyping analysis, the electron microscopy examination showed protruding of pili from M. tuberculosis, especially in dormant mycobacterium (15/100; 15%), that was multidrug resistance and extensive drug resistance isolates (P > 0.05). Conclusion: Pili were shown by electron microscopy, although at the gene expression, the insignificant difference was observed at the dormant strains in comparison to active states. Therefore, we may conclude that other genes might be involved in pili production of M. tuberculosis that needs further investigation. Although, the resistance phenomena might influence the pili producing gene expression that showed in our results.

Investigation of Clofazimine Resistance and Genetic Mutations in Drug-Resistant Mycobacterium tuberculosis Isolates.

Recently, as clofazimine (CFZ) showed a good therapeutic effect in treating multi-drug-resistant tuberculosis (MDR-TB), the anti-tuberculosis activity and resistance were re-focused. Here, we investigated the CFZ resistance and genetic mutations of drug-resistant Mycobacterium tuberculosis (DR-Mtb) isolates to improve the diagnosis and treatment of drug-resistant TB patients. The minimal inhibitory concentration (MIC) of CFZ was examined by resazurin microtiter assay (REMA) with two reference strains and 122 clinical isolates from Korea. The cause of CFZ resistance was investigated in relation to the therapeutic history of patients. Mutations of Rv0678, Rv1979c and pepQ of CFZ resistant isolates were analyzed by PCR and DNA sequencing. The rate of CFZ resistance with MIC > 1 mg/L was 4.1% in drug-resistant Mtb isolates. The cause of CFZ resistance was not related to treatment with CFZ or bedaquiline. A CFZ susceptibility test should be conducted regardless of dugs use history. The four novel mutation sites were identified in the Rv0678 and pepQ genes related to CFZ resistance in this study.

Rescue of susceptibility to second-line drugs in resistant clinical isolates of Mycobacterium tuberculosis.

Aim: Antibiotic resistance is one of the biggest threats to global health, and this study aimed better understand how the efflux pumps are related to this process in tuberculosis clinical isolates. Results: The combination of antibiotics plus efflux pumps (EP) inhibitors was able to restore the susceptibility of clinical isolates in 100% of aminoglycosides resistance and 33.3% of the fluoroquinolones resistance. The relative expression of EPgenes in pre-extensively drug-resistant isolates showed an increase of up to 1000-times. Conclusion: The rescue of susceptibility in the presence of EPinhibitors, the increased of activity and expression of the EPgenes alert that the inhibition of EPcan reduce the selection of resistant strains and improvetreatment.

Sporotrichosis: a Comprehensive Review on Recent Drug-Based Therapeutics and Management.

Purpose of ReviewOver a period of time, sporotrichosis has arisen as one of the leading fungal infections not only in animals but humans also. Several possible reasons that contribute to its emergence include change in epidemiology and distribution, evolutionary changes in taxonomy, and several outbreaks. World Health Organization has identified sporotrichosis as one of the major neglected tropical diseases (NTD) for 2021–2030 under the category of fungal NTDs. Several factors are contributing to increases in morbidity due to sporotrichosis such as delayed diagnosis and unavailability of appropriate antifungal therapy, which lead to redundant and inappropriate treatment with associate costs and adverse effects.Recent FindingsThe potassium iodide is the first line of treatment for cutaneous forms while amphotericin B is used for the most severe cases of the disease. The limited medication arsenal, side effects, failure of therapy, and the advent of drug-resistant isolates emphasize the need for the development of new therapeutic options. Several studies are focusing on the development of the new drugs which either used alone or in combination with already available treatment. Along with this, several new antigens have been identified as possible targets for its vaccine development.SummaryThe early diagnosis is required for selecting the best possible treatment strategy. The researchers should focus on developing new diagnostic methods and treatment options as well as vaccine development for the better management of sporotrichosis. In the long run, patient education for preventative features to reduce risk and counselling for prolonged therapy will be beneficial.

Relationship between Resistance to Ethambutol and Rifampin and Clinical Outcomes in Mycobacterium avium Complex Pulmonary Disease.

We evaluated the associations between the in vitro activities of ethambutol and rifampin and clinical outcomes of Mycobacterium avium complex (MAC) pulmonary disease (PD). Among 158 patients with MAC-PD, there was no relationship between high MICs for ethambutol and/or rifampin and treatment failure for MAC-PD. Ethambutol and rifampin resistance was common among MAC isolates (rates of 87% and 59%, respectively), but mutations in embB, rpoB, and rpoC were rare, with detection in only 4% of the drug-resistant MAC isolates.

Pattern of Drug Resistance in Isolates of Staphylococcus Aureus from Hospitalized Patients

Staphylococcus aureus is a non motile, non spore- forming gram positive bacterium that causes a wide range of infections such as endocardatis, skin infections, bacteraemia, sepsis and food poisoning. The objective of this study is to evaluate the pattern of drug resistance of staphylococcus aureus in different type of samples that were taken from different wards (emergency and OPD) of the hospital. In this study we analyzed 40 isolates were obtained on blood agar and tryptic soy agar. Identification of the isolates was carried out by catalase test as well as methicillin resistance. Confirmed staphylococcus aureus isolates were analyzed for antimicrobial susceptibility testing. In our study the isolates from patients, particularly are frequently resistance to multiple antimicrobial agents more than 90% of staphylococcus aureus isolates were resistance to cefalothin, erythromycin and 80% are resistance to vancomycin. Our findings showed that MRSA isolates were coming from the community as indicated by hospital ward types, that even with no prolonged hospital stays the patients coming from the community were carrying MRSA strains.

Antibacterial Activities of Some Medicinal Plants on Common Multidrug-Resistant Bacterial Isolates from Some Patients Attending Federal Medical Center, Birnin Kebbi, Nigeria

Bacterial infections are among the major causes of morbidity and mortality worldwide. The development of drug resistance to most antibiotics by bacterial species are pandemic, these necessitate the urgent need to search for new antibacterial drugs from the natural sources. Hence this research was aimed at evaluating the antibacterial activity of some medicinal plants on the most commonly drug resistant bacterial isolates from some patients attending Federal Medical Center (FMC) Birnin Kebbi, Nigeria. An ethno-botanical survey was carried out within Zuru Emirate and three (3) plant samples (Lonchocarpus laxiflorus root, Mitragyna inermis root and Lawsonia inermis root) were selected based on their number of citations and limited number of reported researches on the antibacterial activities of their roots. The multidrug resistant bacterial strains (Staplococcus aureus, Escherichia coli and Psuedomonas aeruginosa) obtained from FMC, Birnin Kebbi, were taken from nutrient agar slant and sub-cultured in nutrient agar plates. The antibacterial activities of the root methanol extracts of the plants were evaluated using agar-well diffusion method. Tube dilution method was used to determine the minimum inhibitory concentration of each plant extract. The preliminary phytochemical screening of the extracts was conducted using standard methods. Alkaloids, Flavaniods, Steriods, Tannins, saponnins and Phenols, were detected in L. laxiflorus root methanol extract, Flavaniods were absent in L. inermis and Alkaloids in M. inermis root methanol extracts. The plant extracts and their combinations showed varying degrees of antibacterial activities on the test isolates. The root methanol extracts of L. laxiflorus, L. inermis, L. laxiflorus/L. inermis, L. laxiflorus/M. inermis and L. inermis/M. inermis roots presented significant increase (p<0.05) in the zone of inhibition against S. aureus. Extracts of L. laxiflorus root, L. inermis root and combination of M. inermis root were able to suppress the growth of P. aeruginosa at lower dose of 25mg/ml. The antibacterial activity revealed that, the extracts exhibited dose dependent effect. L. inermis root had the highest activity and least MIC value of 12.5mg/ml against all the tested isolates. Thus, this research justified the use of these plants in traditional medicine for the treatment of bacterial infections and can also be considered as potential sources for development of new antibacterial agents that may be more effective, safe and readily accessible than the current antibiotics.

Genomic features of an isolate of Empedobacter falsenii harbouring a novel variant of metallo-β-lactamase, blaEBR-4 gene.

Empedobacter falsenii is an emerging opportunistic pathogen that has been occasionally implicated in various human infections. In this study, we described the genomic features of a multidrug resistant E. falsenii Q1655 obtained from a patient attending a public hospital in Sokoto, northwest Nigeria. The isolate, E. falsenii Q1655, was isolated from the stool sample of a patient in Sokoto, Nigeria. The identity of the isolate was confirmed by MALDITOF-MS. The disc diffusion test and modified Carba-NP test were used for phenotypic antibiotic susceptibility test and carbapenemase enzyme production test, respectively. The whole genome of the strain was sequenced using the Illumina MiSeq technique. Resistome analysis was done by annotation of the WGS against the ARG-ANNOT database. The isolate was resistant to all β-lactam antibiotics with the exception of cefepime. The MICs of imipenem and ertapenem as determined by E-test were 12μg/ml and 2μg/ml, respectively. Modified Carba NP test showed that the strain was carbapenemase producing. Resistome analysis revealed the presence of a novel metallo-β-lactamase, a chromosomal blaEBR-4, which exhibited 94.92% and 97.02% nucleotide and protein sequence identities respectively with blaEBR-3 gene of E. falsenii 174,820. Seven and eight amino-acid substitutions were observed with the blaEBR-1 and blaEBR-2, respectively. We reported the first isolation and genomic description of an extensively drug resistant isolate of Empedobacter falsenii in Nigeria. This report broadens our knowledge of carbapenem resistance in E. falsenii and it will serve as a useful guide in the development of antibiotic use policy.

Retrospective analysis of Vitek®2 performance compared to manual broth micro-dilution for colistin susceptibility testing of Acinetobacter baumanniicomplex isolates in South Africa.

The manual broth micro-dilution (mBMD) is the recommended reference method for colistin minimum inhibitory concentration determination; however, it is not as readily available in South Africa as the Vitek®2. This retrospective study compared the performance of Vitek®2 against mBMD in determining the colistin minimum inhibitory concentration of 337 extensively drug-resistant Acinetobacter baumannii complex isolates. Vitek®2 yielded a categorical agreement of 89%, an essential agreement of 56%, a major error rate of 8% and a very major error rate of 55%. The Vitek®2 is not an alternative to mBMD for colistin susceptibility testing.

Variants associated with Bedaquiline (BDQ) resistance identified in Rv0678 and efflux pump genes in Mycobacterium tuberculosis isolates from BDQ na\xefve TB patients in Pakistan

BackgroundMutations in the Rv0678, pepQ and atpE genes of Mycobacterium tuberculosis (MTB) have been reported to be associated with reduced antimycobacterial susceptibility to bedaquiline (BDQ). Resistance conferring mutations in treatment naïve MTB strains is likely to have implications for BDQ based new drug regimen that aim to shorten treatment duration. We therefore investigated the genetic basis of resistance to BDQ in MTB clinical isolates from BDQ naïve TB patients from Pakistan. In addition, mutations in genes associated with efflux pumps were investigated as an alternate mechanism of resistance.MethodsBased on convenience sampling, we studied 48 MTB clinical isolates from BDQ naïve TB patients. These isolates (from our strain bank) included 38 MDR/pre-XDR/XDR (10 BDQ resistant, 8 BDQ intermediate and 20 BDQ susceptible) and 10 pan drug susceptible MTB isolates. All strains were subjected to whole genome sequencing and genomes were analysed to identify variants in Rv0678, pepQ, atpE, Rv1979c, mmpLS and mmpL5 and drug resistance associated efflux pump genes.ResultsOf the BDQ resistant and intermediate strains 44% (8/18) had variants in Rv0678 including; two reported mutations S63R/G, six previously unreported variants; L40F, R50Q and R107C and three frameshift mutations; G25fs, D64fs and D109fs.Variants in efflux pumps; Rv1273c (G462K), Rv0507c (R426H) and Rv1634c (E198R) were found to be present in drug resistant isolates including BDQ resistant and intermediate isolates. E198R in efflux pump gene Rv1634c was the most frequently occurring variant in BDQ resistant and intermediate isolates (n = 10).ConclusionWe found RAVs in Rv0678 to be commonly associated with BDQ resistance. Further confirmation of the role of variants in efflux pump genes in resistance is required so that they may be incorporated in genome-based diagnostics for drug resistant MTB.

Biofilm formation capacity of Pseudomonas aeruginosa is significantly enhanced by sub-inhibitory concentrations of Tomatidine

BackgroundBiofilm associated infections of Pseudomonas aeruginosa are regarded as a huge problem in eradication antibiotic therapy. In this regard, other alternatives such as anti-biofilm or anti-virulence candidates are needed. This study was performed to investigate the effect of sub-inhibitory concentrations of Tomatidine on biofilm formation capacity and the expression of biofilm associated genes in some local isolates of P. aeruginosa. MethodsPAO1 strain and 20 local clinical isolates of P. aeruginosa were used in all assessments. Microtiter plate assay was used in phenotypic detection of biofilm formation. RNA was extracted from all bacteria and expression level of lasI, lasR, rhlI, rhlR and algD genes was investigated. All experiments were done in untreated condition and after treatment with 32 and 512 mg/L of Tomatidine. ResultsFifty-five percent of isolates were weak biofilm producers and 45% were strong biofilm producers. Majority of multiple drug resistant and extensively drug resistant isolates were weak biofilm producers. Biofilm formation and expression of all investigated genes were significantly increased by a dose dependent manner after treatment with two sub-inhibitory concentrations of Tomatidine. The lasI and rhlR were the most and the least Tomatidine affected genes, respectively. Among the five investigated genes only rhlR was expressed significantly more in weak biofilm producers in comparison to strong biofilm producers. ConclusionsSub-inhibitory concentrations of Tomatidine significantly induce biofilm formation and also expression of QS associated genes of P. aeruginosa. Therefore, many antibiotic agents and natural bioactive agents are regarded as double edged swords in the battle against the bacterial infections. We believe that the revision of empirical therapy regiments especially in Gram negative biofilm associated infections can be an important approach that has to be considered in development of future therapeutic guidelines.

New Insights on Drug-Resistant Clinical Isolates of Leishmania infantum-Infected Human Macrophages as Determined by Comparative Transcriptome Analyses.

Leishmaniasis is the second most important neglected tropical parasitic disease after malaria. This disease is distributed worldwide and can be present in a variety of clinical forms, depending on the parasite species and host's genetic background. As chemotherapy is the only effective weapon whose effectiveness is limited by the frequent appearance of drug resistance and therapeutic failure, new therapeutic strategies are required. To better understand the factors that contribute to therapeutic failure and drug resistance in leishmaniasis, we studied the transcriptomic changes in host THP-1 cells after infection with clinical Leishmania infantum isolates with different susceptibilities to antileishmanial drugs by RNA-seq. Analysis of the differentially expressed genes (DEGs) in infected host cells revealed variations in DEG numbers in the THP-1-infected cells depending on the Leishmania line. A key conclusion of this study is that the modulation of host cells is Leishmania line dependent. Gene ontology enrichment analyses of DEGs indicated that certain biological processes were modulated in the infected host cells, specifically related to cellular metabolism, immune response, defense response, signaling pathways, and cell proliferation and apoptosis. Furthermore, this study provides new potential therapeutic markers and insights into the THP-1 host transcriptomic changes that occur after late infection with drug-resistant L. infantum clinical isolates.

Anti-proliferative and antibacterial activity of oleo-gum-resin of Boswellia serrata extract and its isolate 3-hydroxy-11-keto-β-boswellic acid

Since time immemorial Boswellia serrata has been a useful traditional plant and its oleo-gum-resin is regularly used as an important constituent in herbal medicines for inflammation, cancer sufferers and cosmetics. This study focuses on the antibacterial and anticancer activities of B. serrata oleo-gum-resin extract, fractions and isolates. Time kill kinetic assay, combination study and ethidium bromide efflux pump inhibitory assay of the leading molecule 3-hydroxy-11-keto-β-boswellic acid (KBA) have been evaluated. The extract, fractions and the isolated molecule have shown anti-proliferative activity in different cancer cell lines, and the inhibition ranged from 13% to 93%. KBA caused anti-proliferative dose-dependent impacts on all human malignant cells (IC50 4.10–37.9 µg/ml). In antibacterial study of the extract, fractions and isolated molecules have shown minimum inhibitory concentration MIC range from 31.25 to 1000 μg/ml against 8 different Gram-positive and Gram-negative bacterial strains. KBA exhibited synergistic effect with a oxacillin against different drug resistant clinical isolates with fractional inhibitory concentration index (FICI) in the range of 0.325–0.450 µg/ml. KBA exhibited bacteriostatic killing mechanism and could also modulate ethidium bromide efflux pump to find its potential in combination therapy against multi-drug resistant Staphylococcal infections. KBA in the extract was quantified using HPLC-PDA and its structure was validated for the first time by X-ray crystallography. This study reinforced the promising treatment of KBA for cancer and S. aureus infections. KBA can be further explored to develop potential lead combinations of anti-cancer and anti-Gram-positive bacterial herbal formulations.

Investigation on the cause of recurrent tuberculosis in a rural area in China using whole-genome sequencing: A retrospective cohort study

Recurrent tuberculosis (TB) in China has been studied in urban areas but not in rural settings. We conducted a retrospective cohort study on all successfully treated bacteriologically confirmed TB patients between 2009 and 2020 in Wusheng County, Sichuan Province using whole-genome sequencing (WGS). Isolates differing by ≤ 12 single nucleotide polymorphisms (SNPs) were considered relapse, otherwise were considered exogenous reinfection, and molecular drug susceptibility results were also compared. In total, 4.9% (75/1532) of successfully treated cases had subsequent bouts of TB, with 4 patients having two subsequent bouts, yielding a total of 79 events and a rate of 8.56 (95% CI 6.86–10.67) cases per 1000 person years, 8.9 times higher than in the general population. Most recurrences (74.4%, 58/79) occurred within three years. The risk for recurrent TB was increased for men and patients with drug-resistant isolates. Among the 44 recurrent events with genotyped paired isolates, 39 were classified as relapse and 5 as exogenous reinfection. Ten (25%, 10/40) recurrent patients acquired new resistance during treatment. Recurrent TB in rural Wusheng was mostly relapse and associated with drug resistance, suggesting the need to improve patient management, resistance testing and patient follow-up for at least 3 years after completing treatment.

Occurrence Of Multi Drug Resistant Bacteria From Raw Chicken Meat Of South India Retail Markets.

Antibiotic resistance and its dissemination from poultry food to human beings further sequentially to the environment is becoming an ever-increasing distress worldwide. Nowadays a small ratio of meat sample from the rural area itself may be found positive with Multi Drug Resistant (MDR) isolates. This study is aimed to enumerate the prevalence of antibiotic resistant isolates in raw chicken meat collected from retail shops (fresh meat) and supermarkets (frozen meat) situated in different areas of Coimbatore, South India. Also, the study focused to exhibit the severity of consuming poultry food with the antibiotic residues unknowingly. Resistant isolates from raw chicken meats was collected during the period of December 2020 to February 2021 and screened for resistant attributes against antibiotics as per clinical settings using VITEK® 2 Compact. The identified MDR isolates was submitted to the National Database of Antibiotic Resistant Organisms (NDARO). Out of 22 chicken meat samples collected, a total of 50 pathogens were isolated. Among them, 2 frozen chicken meats showed no bacterial growth, and other chicken meat samples was found to have resistant bacteria like MDR E.coli, Salmonella spp. and Staphylococcus spp. This study confirmed the presence of MDR isolates in raw chicken meat and it is the first study from South India to evidence the presence of MDR in raw chicken samples. This study also suggests that antimicrobial stewardship programs and monitoring practices needs more concentration in the veterinary antibiotics usage in poultry and Livestock. Hence it is an alarm of poultry stock which leads to risk on public health and ecological system of South India.

Multiplex PCR: A Molecular Modality for Rapid Detection of CTX-M-15, AAD A1 and QNR S1 Genes among Drug Resistant Clinical Isolates of Enterobacteriaceae in Kanchipuram, South India

Multidrug resistance is a potential threat to the mankind. The prevalence rate of multidrug resistant bacteria is worrisome and has gained significant attention globally. Hence it becomes imperative for clinical laboratories to identify these multidrug resistant pathogens for appropriate therapy. It is equally important to detect the resistance mechanisms which would aid in understanding and development of new therapeutic options. This study was carried out to identify the prevalence of multidrug resistant Enterobacteriaceae from clinical samples recovered from Kanchipuram district. The study was also implicated in designing of novel primers using primer3 tool and standardizing a multiplex PCR targeting ctxm- 15, aadA1, qnrS1 genes in the isolates. Of the 60 clinical isolates studied, 14, 13 and 12 isolates harbored ctx-m-15, qnrS1 gene and aadA1genes respectively. Among these, 2 isolates carried both ctx-m-15 and qnrS1 genes and 3 isolates were found to carry both ctx-m-15 and aadA1 genes. 1 isolate was found to be positive for both aadA1 and qnrS1 genes. None of these isolates harbored all the three targeted genes qnrS1 and aadA1, ctx-m-15. The present study will aid in detecting drug resistant genes like ctx-m-15, aadA1, qnrS1 in clinical isolates with higher sensitivity and specificity and will also reduce the cost of reaction than that required for uniplex PCR. The prevalence rate of drug resistant genes among Kanchipuram district alerts us for the measures needed to be undertaken to control the spread of drug resistant genes in community.

Synthetic antimicrobial peptides as enhancers of the bacteriolytic action of staphylococcal phage endolysins

Bacteriophage endolysins degrade the bacterial cell wall and are therefore considered promising antimicrobial alternatives to fight pathogens resistant to conventional antibiotics. Gram-positive bacteria are usually considered easy targets to exogenously added endolysins, since their cell walls are not shielded by an outer membrane. However, in nutrient rich environments these bacteria can also tolerate endolysin attack if they keep an energized cytoplasmic membrane. Hence, we have hypothesized that the membrane depolarizing action of antimicrobial peptides (AMPs), another attractive class of alternative antibacterials, could be explored to overcome bacterial tolerance to endolysins and consequently improve their antibacterial potential. Accordingly, we show that under conditions supporting bacterial growth, Staphylococcus aureus becomes much more susceptible to the bacteriolytic action of endolysins if an AMP is also present. The bactericidal gain resulting from the AMP/endolysin combined action ranged from 1 to 3 logs for different S. aureus strains, which included drug-resistant clinical isolates. In presence of an AMP, as with a reduced content of cell wall teichoic acids, higher endolysin binding to cells is observed. However, our results indicate that this higher endolysin binding alone does not fully explain the higher susceptibility of S. aureus to lysis in these conditions. Other factors possibly contributing to the increased endolysin susceptibility in presence of an AMP are discussed.

Antimicrobial activities of aztreonam-avibactam and comparator agents tested against Enterobacterales from European hospitals analysed by geographic region and infection type (2019-2020).

The purpose of this study is to evaluate the activities of aztreonam-avibactam and comparator agents againstEnterobacterales isolates from European medical centres as well as the occurrence of carbapenemases (CPEs). A total of 11,655 Enterobacterales isolates were collected consecutively in 2019-2020 from 38 medical centres located in Western Europe (W-EU; n = 8,784; 25 centres in 10 countries) and the Eastern European and Mediterranean region (E-EU; n = 2,871; 13 centres in 10 countries). Isolates were susceptibility tested by broth microdilution methods in a monitoring laboratory. The antimicrobial susceptibility and frequency of key resistance phenotypes were assessed and stratified by geographic region and infection type. Isolates that showed resistance to carbapenems (CRE) and/or elevated MICs (> 8mg/L) for aztreonam-avibactam were screened for β-lactamase-encoding genes by whole-genome sequencing. Aztreonam-avibactam inhibited 99.9% of Enterobacterales at ≤ 8mg/L (MIC50/90, ≤ 0.03/0.12mg/L) and retained potent activity against CRE (MIC50/90, 0.25/0.5mg/L), multidrug-resistant isolates (MDR; MIC50/90, 0.12/0.5mg/L), and extensively drug-resistant (XDR) isolates (MIC50/90, 0.25/0.5mg/L). Susceptibility to comparator agents was consistently lower among isolates from E-EU compared to W-EU for all infection types evaluated. CRE rates varied from 0.6% (urinary tract infection [UTI]) to 2.3% (bloodstream infection) in W-EU, and from 6.1% (UTI) to 17.0% (pneumonia) in E-EU. A CPE-encoding gene was identified in 360 of 424 (84.9%) CRE isolates, and the most common CPEs were blaKPC (36.3% of CRE), blaOXA-48 type (27.1% of CRE), and the MBLs (25.7% of CRE). All CPE producers were inhibited at an aztreonam-avibactam concentration of ≤ 8mg/L. Aztreonam-avibactam demonstrated potent activity across the evaluated geographic regions and infection types.