Aminoglycoside Acetyltransferase Research Articles

Correlation between the Antibiotic Resistance Genes and Susceptibility to Antibiotics among the Carbapenem-Resistant Gram-Negative Pathogens.

In this study, the correlation between the antibiotic resistance genes and antibiotic susceptibility among the carbapenem-resistant Gram-negative pathogens (CRGNPs) recovered from patients diagnosed with acute pneumonia in Egypt was found. A total of 194 isolates including Klebsiella pneumoniae (89; 46%), Escherichia coli (47; 24%) and Pseudomonas aeruginosa (58; 30%) were recovered. Of these, 34 (18%) isolates were multiple drug resistant (MDR) and carbapenem resistant. For the K. pneumoniae MDR isolates (n = 22), blaNDM (14; 64%) was the most prevalent carbapenemase, followed by blaOXA-48 (11; 50%) and blaVIM (4; 18%). A significant association (p value < 0.05) was observed between the multidrug efflux pump (AcrA) and resistance to β-lactams and the aminoglycoside acetyl transferase gene (aac-6’-Ib) gene and resistance to ciprofloxacin, azithromycin and β-lactams (except for aztreonam). For P. aeruginosa, a significant association was noticed between the presence of the blaSHV gene and the multidrug efflux pump (MexA) and resistance to fluoroquinolones, amikacin, tobramycin, co-trimoxazole and β-lactams and between the aac-6’-Ib gene and resistance to aminoglycosides. All P. aeruginosa isolates (100%) harbored the MexAB-OprM multidrug efflux pump while 86% of the K. pneumoniae isolates harbored the AcrAB-TolC pump. Our results are of great medical importance for the guidance of healthcare practitioners for effective antibiotic prescription.

Emergence of the Novel Aminoglycoside Acetyltransferase Variant aac(6')-Ib-D179Y and Acquisition of Colistin Heteroresistance in Carbapenem-Resistant Klebsiella pneumoniae Due to a Disrupting Mutation in the DNA Repair Enzyme MutS.

ABSTRACTAmikacin and colistin are effective against carbapenem-resistant Klebsiella pneumoniae. In 2017, we successively isolated three carbapenem-resistant K. pneumoniae isolates (ST967) from a patient with chronic renal failure in Japan. The first (SMKP01, sputum, day 0) and second (SMKP02, blood, day 14) strains were resistant to most antimicrobials tested but still susceptible to amikacin (MICs of 4 and 0.5 mg/liter, respectively) and colistin (MIC of 0.5 mg/liter for both). The third strain (SMKP03, blood, day 51) was not susceptible to amikacin (MIC, 32 mg/liter), and its MIC for colistin varied (0.5 to 8 mg/liter). Whole-genome sequencing of SMKP01 revealed that 17 of 20 antimicrobial resistance genes, including qnrB91 (a novel qnrB2 variant) and aac(6′)-Ib-cr, were located on an 86.9-kb IncFII-IncQ plasmid. The qnrB91 conferred greater fluoroquinolone resistance than qnrB2. SMKP03 aac(6′)-Ib-cr that possessed a gene mutation that resulted in an R102W substitution, namely, aac(6′)-Ib-D179Y, made a greater contribution to amikacin resistance than did aac(6′)-Ib-cr. SMKP03 harbored a nonsense mutation in mutS, which encodes a DNA repair enzyme. Introduction of this mutation into SMKP01 (SMKP01mutSA307T) resulted in a dramatic increase (>58-fold) in the frequency of spontaneous amikacin-resistant mutants relative to SMKP01, and the substantial mutants possessed aac(6′)-Ib-D179Y. SMKP01mutSA307T exhibited an unstable MIC for colistin (0.5 to 8 mg/liter). The results demonstrate that a disruptive mutation in MutS, arising during the clinical course of an infection, created a platform for the acquisition of amikacin nonsusceptibility and colistin heteroresistance in multidrug-resistant K. pneumoniae, mediated by the elevated frequency of spontaneous mutations.

Molecular Characterization of Ciprofloxacin Resistant Escherichia coli from Ghana

Aim: This study aimed to characterize ciprofloxacin-resistance genes in clinical Escherichia coli isolates obtained from a six-month antimicrobial resistance (AMR) surveillance from Ghana.&#x0D; Methods: Eighty-three of 440 archived E. coli isolates were confirmed by biochemical reactions and resistance profiles by the disc diffusion method. These isolates were cultured from urine (42), stool (23), vaginal swabs (12), wounds (5) and heart valve (1) during AMR surveillance. Minimum Inhibition Concentration (MIC) by E-test method was performed on all E. coli isolates that were resistant to ciprofloxacin by the disc diffusion method. Additionally, all isolates with reduced MIC to ciprofloxacin (&gt;32 µg/ml) were selected for molecular assays. Three chromosomal and nine plasmid-mediated resistance genes were screened in all Ciprofloxacin resistant E. coli (CRE) by polymerase chain reaction (PCR). Randomly selected amplified genes were commercially sequenced and analyzed.&#x0D; Results: In total, 47/83 (56.6%) E. coli isolates were resistant to ciprofloxacin and 29 (61.7%) had MIC values greater than 32 µg/ml. Chromosomal mediated genes (gyrA, gyrB and parC) were present in all 29 CRE isolates (100%). Distribution of the plasmid-mediated genes were as follows; qnrA 16/29 (55.1%), qnrB 16/29(55.1%), qnrC 22/29(75.8%), qnrS 26/29(89.6%), qepA 5/29(17.2%) and oqxB 19/29(65.5%). Genes encoding for altered aminoglycoside acetyltransferase [aac(6’)1bcr] were also present in all 29 CRE isolates. The majority (72.4%) of the CRE isolates had gyrA mutations at codons 83 and 87. In parC, the mutations were at codons 71 and 80. Five isolates had mutations at codon 56 and four each had mutations at positions 79 and 80.&#x0D; Conclusion: In this study, fluoroquinolone resistance genes were identified in all CRE isolates, mostly with putative mutations in the Quinolone Resistance Determining Region (QRDR). These chromosomal and plasmid-mediated genes may be widespread in Ghana and associated with CRE from the AMR surveillance. Although new mutations points were identified in parC, they may not be linked to the CRE.

Aminoglycoside riboswitch control of the expression of integron associated aminoglycoside resistance adenyltransferases

ABSTRACT The proliferation of antibiotic resistance has its origins in horizontal gene transfer. The class 1 integrons mediate gene transfer by assimilating antibiotic-resistance genes through site-specific recombination. For the class 1 integrons the first assimilated gene normally encodes an aminoglycoside antibiotic resistance protein which is either an aminoglycoside acetyltransferase (AAC), nucleotidyltransferase – (ANT), or adenyl transferase (AAD). An aminoglycoside-sensing riboswitch RNA in the leader RNA of AAC/AAD that controls the expression of aminoglycoside resistance genes has been previously described. Here we explore the relationship between the recombinant products of integron recombination and a series of candidate riboswitch RNAs in the 5ʹ UTR of aad (aminoglycoside adenyltransferases) genes. The RNA sequences from the 5ʹ UTR of the aad genes from pathogenic strains that are the products of site-specific DNA recombination by class 1 integrons were investigated. Reporter assays, MicroScale Thermophoresis (MST) and covariance analysis revealed that a functional aminoglycoside-sensing riboswitch was selected at the DNA level through integron-mediated site-specific recombination. This study explains the close association between integron recombination and the aminoglycoside-sensing riboswitch RNA.

Structural analysis of a novel substrate-free form of the aminoglycoside 6'-N-acetyltransferase from Enterococcus faecium.

Aminoglycoside acetyltransferases (AACs) catalyze the transfer of an acetyl group between acetyl-CoA and an aminoglycoside, producing CoA and an acetylated aminoglycoside. AAC(6')-Ii enzymes target the amino group linked to the 6' C atom in an aminoglycoside. Several structures of the AAC(6')-Ii from Enterococcus faecium [Ef-AAC(6')-Ii] have been reported to date. However, the detailed mechanism of its enzymatic function remains elusive. In this study, the crystal structure of Ef-AAC(6')-Ii was determined in a novel substrate-free form. Based on structural analysis, it is proposed that Ef-AAC(6')-Ii sequentially undergoes conformational selection and induced fit for substrate binding. These results therefore provide a novel viewpoint on the mechanism of action of Ef-AAC(6')-Ii.

Molecular mechanisms associated with quinolone resistance in Enterobacteriaceae: review and update.

Quinolones are broad-spectrum antibiotics, which are used for the treatment of different infectious diseases associated with Enterobacteriaceae. During recent decades, the wide use as well as overuse of quinolones against diverse infections has led to the emergence of quinolone-resistant bacterial strains. Herein, we present the development of quinolone antibiotics, their function and also the different quinolone resistance mechanisms in Enterobacteriaceae by reviewing recent literature. All data were extracted from Google Scholar search engine and PubMed site, using keywords; quinolone resistance, Enterobacteriaceae, plasmid-mediated quinolone resistance, etc. The acquisition of resistance to quinolones is a complex and multifactorial process. The main resistance mechanisms consist of one or a combination of target-site gene mutations altering the drug-binding affinity of target enzymes. Other mechanisms of quinolone resistance are overexpression of AcrAB-tolC multidrug-resistant efflux pumps and downexpression of porins as well as plasmid-encoded resistance proteins including Qnr protection proteins, aminoglycoside acetyltransferase (AAC(6')-Ib-cr) and plasmid-encoded active efflux pumps such as OqxAB and QepA. The elucidation of resistance mechanisms will help researchers to explore new drugs against the resistant strains.

Development of 6'-N-Acylated Isepamicin Analogs with Improved Antibacterial Activity Against Isepamicin-Resistant Pathogens.

The development of new aminoglycoside (AG) antibiotics has been required to overcome the resistance mechanism of AG-modifying enzymes (AMEs) of AG-resistant pathogens. The AG acetyltransferase, AAC(6′)-APH(2″), one of the most typical AMEs, exhibiting substrate promiscuity towards a variety of AGs and acyl-CoAs, was employed to enzymatically synthesize new 6′-N-acylated isepamicin (ISP) analogs, 6′-N-acetyl/-propionyl/-malonyl ISPs. They were all active against the ISP-resistant Gram-negative bacteria tested, and the 6′-N-acetyl ISP displayed reduced toxicity compared to ISP in vitro. This study demonstrated the importance of the modification of the 6′-amino group in circumventing AG-resistance and the potential of regioselective enzymatic modification of AG scaffolds for the development of more robust AG antibiotics.

Detection of 5 Kinds of Genes Related to Plasmid-Mediated Quinolone Resistance in Four Species of Nonfermenting Bacteria with 2 Drug Resistant Phenotypes.

Objective This study aimed to detect 5 kinds of genes related to plasmid-mediated quinolone resistance in four species of nonfermenting bacteria with 2 drug resistance phenotypes (multidrug resistance and pandrug resistance), which were Acinetobacter baumannii (Ab), Pseudomonas aeruginosa (Pa), Stenotrophomonas maltophilia (Sm), and Elizabethkingia meningoseptica (Em). Methods The Phoenix NMIC/ID-109 panel and API 20NE panel were applied to 19 isolated strains, including 6 Ab strains (2 strains with multidrug resistance and 4 strains with pandrug resistance), 6 Pa strains (3 strains with multidrug resistance and 3 strains with pandrug resistance), 4 Sm strains (2 strains with multidrug resistance and 2 strains with pandrug resistance), and 3 Cm strains (2 strains with multidrug resistance and 1 strain with pandrug resistance). After strain identification and drug susceptibility test, PCR was applied to detect 5 genes related to plasmid-mediated quinolone resistance. The genes detected were quinolone resistance A (qnrA), aminoglycoside acetyltransferase ciprofloxacin resistance variant, acc(6′)-Ib-cr, and 3 integrons (intI1, intI2, and intI3). The amplified products were analyzed by 1% agarose gel electrophoresis and sequenced. Sequence alignment was carried out using the bioinformatics technique. Results Of 19 strains tested, 8 strains carried acc(6′)-Ib-cr and 6 of them were of pandrug resistance phenotype (3 Ab strains, 2 Pa strains, and 1 Sm strain). The carrying rate of acc(6′)-Ib-cr was 60.0% for strains of pandrug resistance (6/10). Two strains were of multidrug resistance (1 Ab strain and 1 Pa strain), and the carrying rate of acc(6′)-Ib-cr was 22.0% (2/9). The carrying rate was significantly different between strains of multidrug resistance and pandrug resistance (P < 0.05). The class 1 integron was detected in 11 strains, among which 6 strains were of pandrug resistance (3 Ab strains, 2 Pa strains, and 1 Sm strain). The carrying rate of the class 1 integron was 60.0% (6/10). Five strains were of multidrug resistance (3 Pa strains, 1 Ab strain, and 1 Em strain), and the carrying rate was 55.6% (5/9). The carrying rate of the class 1 integron was not significantly different between strains of multidrug resistance and pandrug resistance (P > 0.05). Both acc(6′)-Ib-cr and intI1 were detected in 6 strains, which were negative for qnrA, intI2, and intI3. Conclusion Quinolone resistance of isolated strains was related to acc(6′)-Ib-cr and intI1 but not to qnrA, intI2, or intI3. The carrying rate of acc(6′)-Ib-cr among the strains of pandrug resistance was much higher than that among the strains of multidrug resistance. But, the strains of two drug resistant phenotypes were not significantly different in the carrying rate of intI1. The detection rates of the two genes were high and similar in Ab and Pa strains. 1 Em strain carried the class 1 integron.

Small Changes of Active Site Residues of an Acinetobacter Aminoglycoside Acetyltransferase Affect Antibiotic Susceptibility

Antibiotics are drugs used to treat bacterial infections by inhibiting growth and proliferation of microorganisms. Commonly prescribed broad‐spectrum antibiotics include aminoglycosides, but many bacteria have developed resistance to these drugs by acquiring enzymes that acetylate them. We previously reported the structural and kinetic characterization of two of these enzymes from Acinetobacter. However, several outstanding questions regarding the role of specific residues in the active site of these proteins remain. In particular, we observed an active site residue that was different between these two enzymes and appeared to alter the aminoglycoside binding pocket. It has been previously shown that active site mutations within aminoglycoside acetyltransferase enzymes can adjust their ability to modify aminoglycosides and therefore their susceptibility. Since we cannot predict which point mutations will ultimately enhance their activity and therefore resistance to aminoglycosides, we performed saturation mutagenesis of this residue in the Acinetobacter haemolyticus AAC(6′)‐Ig enzyme and tested the antibiotic susceptibility of Escherichia coli to four different aminoglycosides when these mutants were overexpressed. We found the identity of the amino acid at this position was critical for altering the susceptibility of E. coli to these antibiotics. Additionally, these mutants exhibited different susceptibility patterns depending upon the identity of aminoglycoside tested. Our results show how small changes of active site residues of aminoglycoside acetyltransferase can drastically affect bacterial susceptibility to aminoglycosides, and may provide insight to which antibiotics could be effective for treating Acinetobacter infections.Support or Funding InformationResearch reported in this work was supported by the National Institute of General Medical Sciences of the National Institutes of Health under Award Number R35GM133506 (to MLK).

Importance of Selected Acinetobacter AAC(6′)‐Ig Active Site Residues on Escherichia coli Susceptibility to Aminoglycosides

Aminoglycosides are broad‐spectrum antibiotics commonly used to treat infections caused by Gram‐negative pathogens. However, bacteria become resistant to these antibiotics by acquiring or utilizing innate aminoglycoside acetyltransferases (AACs) that modify the drug. For instance, Acinetobacter haemolyticus has acquired the AAC(6′)‐Ig enzyme and is resistant to multiple aminoglycosides. However, our current understanding of the key residues of the active site that are critical for enzymatic activity of this enzyme is limited. Therefore, we selected two residues in the active site of the protein that formed either H‐bonds or exhibited pi‐pi stacking with tobramycin in the 3D structure of the enzyme and mutated them to bulky or charged amino acids. Next, we examined the susceptibility of Escherichia coli in the presence and absence of these over‐expressed Acinetobacter proteins toward four aminoglycosides. We found the identity of the residue substituted at these positions altered their susceptibility to these antibiotics, which provides insight into how this enzyme recognizes and modifies its diverse substrates.Support or Funding InformationResearch reported in this work was supported by the National Institute of General Medical Sciences of the National Institutes of Health under Award Number R35GM133506 (to MLK).

Assessment of 16srRNA Methylase Genes Among Non-ESBL and ESBL-Producing Klebsiella pneumoniae Isolates

: The aim of our study was to investigate mechanisms of aminoglycoside resistance in extended-spectrum beta-lactamases (ESBL)-producing Klebsiella pneumoniae (K. pneumoniae) isolates from Iran. To this end, 154 clinical isolates of K. pneumoniae were collected from two hospitals in Ilam city, Iran. The Kirby-Bauer (agar diffusion) antibiotic testing method was used to determine the susceptibility pattern of the isolates against kanamycin, gentamicin, tobramycin, netilmicin and amikacin. Aminoglycoside acetyltransferases (aac(3)-IIa, aac(6’)-Ib, and aac(3)-Ia), 16SrRNA methylase genes (armA and rmtB) and ESBL genes (blaTEM, blaSHV, and blaCTX-M) were detected by PCR amplification. 59.1% (n = 91) of K. pneumoniae isolates were detected ESBL producers with the phenotypic test. Moreover, blaTEM, blaSHV and blaCTX-M were detected in 83.5% (n=76), 52.7% (n=48) and 26.4% (n=24) of the ESBL-producing isolates, respectively. Among 52 resistant or intermediate isolates against aminoglycosides, the aac(3)-IIa, aac(6’)-Ib and rmtB genes were detected in 55.8% (n = 29), 80.8% (n = 42) and 1.9% (n = 1) of the isolates, respectively; none of the isolates, however, had the aac(3)-Ia and armA genes. Therefore, the results showed the high prevalence of aminoglycosides resistance in the K. pneumoniae isolates. As observed, the acetyltransferase modifying enzymes (aac genes) played major roles in determining this resistance. However, the rate of 16srRNA methylase genes was extremely low in K. pneumoniae.

Antibiotic Susceptibility and Molecular Detection of Pseudomonas aeruginosa Isolated from Bovine Mastitis

This study aimed to isolate Pseudomonas aeruginosa from cattle (bovine) milk with mastitis to characterize its antimicrobial susceptibility against some antibiotics, and to identify aminoglycoside acetyltransferase (aac-3-Ib) gene.A total of 100 bovine milk samples were collected randomly from different local cow farms at districts of Wasit governorate, Iraq. Six P. aeruginosa isolates were obtained using bacterial culture method and further identified by Analytical Profile Index (API-20E). The antibiotic sensitivity test was performed by disc diffusion methods. Among the 5 antibiotics used, the highest resistance (100%) was found with Nalidixic acid andtetracycline, follow by gentamicin (50%) and the lowest resistance rate (16.6%, and 33.3%) was to the ciprofloxacin and cephalothin respectively. PCR was performed for all the gentamicin resistant isolates that be among the aminoglycoside family. The where the frequency of aac(3)-Ib gene have product (530bp) was to 3 of P. aeruginosa isolates. From the finding of present study we concluded that P.aeruginosa thatisolated from mastitic bovinehave been developed resistance against aminoglycosides through presence of aac(3)-Ibgene and the ciprofloxacin and cephalothin can be taken as good choice of treatment

Apralogs: Apramycin 5-O-Glycosides and Ethers with Improved Antibacterial Activity and Ribosomal Selectivity and Reduced Susceptibility to the Aminoacyltranserferase (3)-IV Resistance Determinant.

Apramycin is a structurally unique member of the 2-deoxystreptamine class of aminoglycoside antibiotics characterized by a monosubstituted 2-deoxystreptamine ring that carries an unusual bicyclic eight-carbon dialdose moiety. Because of its unusual structure, apramycin is not susceptible to the most prevalent mechanisms of aminoglycoside resistance including the aminoglycoside-modifying enzymes and the ribosomal methyltransferases whose widespread presence severely compromises all aminoglycosides in current clinical practice. These attributes coupled with minimal ototoxocity in animal models combine to make apramycin an excellent starting point for the development of next-generation aminoglycoside antibiotics for the treatment of multidrug-resistant bacterial infections, particularly the ESKAPE pathogens. With this in mind, we describe the design, synthesis, and evaluation of three series of apramycin derivatives, all functionalized at the 5-position, with the goals of increasing the antibacterial potency without sacrificing selectivity between bacterial and eukaryotic ribosomes and of overcoming the rare aminoglycoside acetyltransferase (3)-IV class of aminoglycoside-modifying enzymes that constitutes the only documented mechanism of antimicrobial resistance to apramycin. We show that several apramycin-5-O-β-d-ribofuranosides, 5-O-β-d-eryrthofuranosides, and even simple 5-O-aminoalkyl ethers are effective in this respect through the use of cell-free translation assays with wild-type bacterial and humanized bacterial ribosomes and of extensive antibacterial assays with wild-type and resistant Gram negative bacteria carrying either single or multiple resistance determinants. Ex vivo studies with mouse cochlear explants confirm the low levels of ototoxicity predicted on the basis of selectivity at the target level, while the mouse thigh infection model was used to demonstrate the superiority of an apramycin-5-O-glycoside in reducing the bacterial burden in vivo.

Elucidation of role of an acetyltransferase like protein in paromomycin resistance in Leishmania donovani using in silico and in vitro approaches

Paromomycin, an aminoglycoside antibiotic, is an effective treatment for VL (visceral leishmaniasis) in India. The modification of aminoglycoside antibiotics by enzymes such as aminoglycoside acetyltransferases is the predominant mechanism of resistance to antibiotics in bacterial system. In the present study, we identified and characterized LdATLP (an acetyltransferase-like protein) and elucidated its role in paromomycin resistance in Leishmania donovani. Gene encoding LdATLP was consistently up-regulated (>2fold) in three distinct paromomycin resistant in comparison with sensitive parasites, although the gene sequence was identical in the two. In silico analysis revealed that LdATLP consisted of conserved GNAT (GCN5-related N-Acetyltransferase) domain which is characteristic of aminoglycoside N-acetyltransferases. Evolutionary relationship among LdATLP of Leishmania and aminoglycoside acetyltransferases of bacteria was established by phylogenetic analysis. The 3D structure of LdATLP, predicted by ab-initio modeling, constituted 6 α-helices and 6 β-sheets. A few residues, such as R175, R177, E196, R197, V198, V200, K202, R205, C206, D208, G210, R211, R215, A234, S237, S238, K239, D240, F241 and Y242 of GNAT domain were predicted to be present at active site. Molecular docking of LdATLP with paromomycin or indolicidin (broad spectrum inhibitor of aminoglycoside modifying enzymes), followed by molecular dynamics simulation of docked complex suggested that both paromomycin and indolicidin bind to LdATLP with comparable free energy of binding. In vitro studies revealed that in the presence of indolicidin, paromomycin resistant parasites exhibited reversion of phenotype into sensitive parasites with marked increase in paromomycin susceptibility, suggesting the role of LdATLP in paromomycin resistance.Communicated by Ramaswamy H. Sarma

Comparison of Antibiotic Resistance Mechanisms in Antibiotic-Producing and Pathogenic Bacteria.

Antibiotic resistance poses a tremendous threat to human health. To overcome this problem, it is essential to know the mechanism of antibiotic resistance in antibiotic-producing and pathogenic bacteria. This paper deals with this problem from four points of view. First, the antibiotic resistance genes in producers are discussed related to their biosynthesis. Most resistance genes are present within the biosynthetic gene clusters, but some genes such as paromomycin acetyltransferases are located far outside the gene cluster. Second, when the antibiotic resistance genes in pathogens are compared with those in the producers, resistance mechanisms have dependency on antibiotic classes, and, in addition, new types of resistance mechanisms such as Eis aminoglycoside acetyltransferase and self-sacrifice proteins in enediyne antibiotics emerge in pathogens. Third, the relationships of the resistance genes between producers and pathogens are reevaluated at their amino acid sequence as well as nucleotide sequence levels. Pathogenic bacteria possess other resistance mechanisms than those in antibiotic producers. In addition, resistance mechanisms are little different between early stage of antibiotic use and the present time, e.g., β-lactam resistance in Staphylococcus aureus. Lastly, guanine + cytosine (GC) barrier in gene transfer to pathogenic bacteria is considered. Now, the resistance genes constitute resistome composed of complicated mixture from divergent environments.

Differential thermal stability, conformational stability and unfolding behavior of Eis proteins from Mycobacterium smegmatis and Mycobacterium tuberculosis.

Eis (Enhanced Intracellular Survival) is an important aminoglycoside N-acetyltransferase enzyme contributing to kanamycin resistance in Mtb clinical isolates. Eis proteins from M. tuberculosis (RvEis) and M. smegmatis (MsEis) have 58% identical and 69% similar amino acid sequences and acetylate aminoglycosides at multiple amines. Both the Eis proteins are hexameric and composed of two symmetric trimers. RvEis has remarkable structural stability and heat-stable aminoglycoside acetyltransferase activity. Although the structure and biochemical properties of MsEis have been studied earlier, the detailed characterization of its acetyltransferase activity and structural stability is lacking. In this study, we have performed comparative analysis of structural stability and aminoglycoside acetyltransferase activity of RvEis and MsEis proteins. Unlike RvEis, MsEis undergoes a three-state unfolding induced by heat or chemical denaturants and involves self-association of partially unfolded oligomers to form high molecular weight soluble aggregates. MsEis is highly susceptible to chemical denaturants and unfolds completely at lower concentrations of GdmCl and urea when compared to RvEis. In contrast to RvEis, the oligomeric forms of MsEis are SDS sensitive. However, SDS treatment resulted in increased helix formation in MsEis than RvEis. MsEis shows lesser thermostable activity with a decreased efficiency of kanamycin acetylation in comparison to RvEis. Furthermore, overexpression of MsEis does not provide thermal resistance to M. smegmatis unlike RvEis. Collectively, this study reveals that homologous proteins from pathogenic and nonpathogenic mycobacteria follow different modes of unfolding and demonstrate differential structural stability and activity despite highly similar sequences and oligomeric organization.

CHARACTERIZATION OF CLASS 1 INTEGRONS AND SOME ANTI-MICROBIAL RESISTANCE GENES IN Salmonella SPECIES ISOLAT-ED FROM POULTRY IN EGYPT

Salmonella enterica includes serotypes that were implicated as a food borne pathogens crucially affecting public health and the economic organization. This study was directed to isolate and identify of Salmonella strains from 222 different species and ages of poultry (broiler, chick, ducks, pigeon, quails) from Kafr El Sheikh governorate. The Salmonella isolation rate was (4.5%) as (0.9%) from apparently healthy, (3.6%) from diseased birds. The outer membrane protein F gene was used as promising tool for detection of Genus Salmonella, after that four isolates were identified serologically as two Salmonella enterica serovar Enteritidis and two Salmonella enterica serovar Typhimurium. The resistance pattern of positive Salmonella isolates showed multidrug resistance phenotypes and qnrS for quinolone resistant genes was recorded in one isolate while blaTEM for β-lactam resistant isolates, aacC for aminoglycosides were recorded in all four Salmonella isolates using PCR technique. Also, Class 1 integrons detected with a percentage of (100%) in examined isolates. Sequencing of the class 1 integrons cassettes showed genes encoding resistance specified to streptothricin acetyltransferase (sat) gene, aminoglycoside acetyltransferase (aac3-Id) and aminoglycoside adenyltransferase (aadA7). Class 1 integrons harbored gene encoding domains unfunction protein (duf) in one S. enterica serovar Typhimurium isolate. This study spotlights the significant role of the drug–resistance genes and Class 1 integrons in Salmonella as zoonotically important pathogens of public health importance.Key words: poultry; Salmonella; drug- resistance genes; integrons gene cassettes

Mycobacterial Aminoglycoside Acetyltransferases: A Little of Drug Resistance, and a Lot of Other Roles.

Aminoglycoside acetyltransferases are important determinants of resistance to aminoglycoside antibiotics in most bacterial genera. In mycobacteria, however, aminoglycoside acetyltransferases contribute only partially to aminoglycoside susceptibility since they are related with low level resistance to these antibiotics (while high level aminoglycoside resistance is due to mutations in the ribosome). Instead, aminoglycoside acetyltransferases contribute to other bacterial functions, and this can explain its widespread presence along species of genus Mycobacterium. This review is focused on two mycobacterial aminoglycoside acetyltransferase enzymes. First, the aminoglycoside 2′-N-acetyltransferase [AAC(2′)], which was identified as a determinant of weak aminoglycoside resistance in M. fortuitum, and later found to be widespread in most mycobacterial species; AAC(2′) enzymes have been associated with resistance to cell wall degradative enzymes, and bactericidal mode of action of aminoglycosides. Second, the Eis aminoglycoside acetyltransferase, which was identified originally as a virulence determinant in M. tuberculosis (enhanced intracellular survival); Eis protein in fact controls production of pro-inflammatory cytokines and other pathways. The relation of Eis with aminoglycoside susceptibility was found after the years, and reaches clinical significance only in M. tuberculosis isolates resistant to the second-line drug kanamycin. Given the role of AAC(2′) and Eis proteins in mycobacterial biology, inhibitory molecules have been identified, more abundantly in case of Eis. In conclusion, AAC(2′) and Eis have evolved from a marginal role as potential drug resistance mechanisms into a promising future as drug targets.

Encoding of Promiscuity in an Aminoglycoside Acetyltransferase.

Aminoglycoside antibiotics are a large family of antibiotics that can be divided into two distinct classes on the basis of the substitution pattern of the central deoxystreptamine ring. Although aminoglycosides are chemically, structurally, and topologically diverse, some aminoglycoside-modifying enzymes (AGMEs) are able to inactivate as many as 15 aminoglycosides from the two main classes, the kanamycin- and neomycin-based antibiotics. Here, we present the crystal structure of a promiscuous AGME, aminoglycoside- N3-acetyltransferase-IIIb (AAC-IIIb), in the apo form, in binary drug (sisomicin, neomycin, and paromomycin) and coenzyme A (CoASH) complexes, and in the ternary neomycin-CoASH complex. These data provide a structural framework for interpretation of the thermodynamics of enzyme-ligand interactions and the role of solvent in the recognition of ligands. In combination with the recent structure of an AGME that does not have broad substrate specificity, these structures allow for the direct determination of how antibiotic promiscuity is encoded in some AGMEs.

The Role of Antibiotic-Target-Modifying and Antibiotic-Modifying Enzymes in Mycobacterium abscessus Drug Resistance.

The incidence and prevalence of non-tuberculous mycobacterial (NTM) infections have been increasing worldwide and lately led to an emerging public health problem. Among rapidly growing NTM, Mycobacterium abscessus is the most pathogenic and drug resistant opportunistic germ, responsible for disease manifestations ranging from “curable” skin infections to only “manageable” pulmonary disease. Challenges in M. abscessus treatment stem from the bacteria’s high-level innate resistance and comprise long, costly and non-standardized administration of antimicrobial agents, poor treatment outcomes often related to adverse effects and drug toxicities, and high relapse rates. Drug resistance in M. abscessus is conferred by an assortment of mechanisms. Clinically acquired drug resistance is normally conferred by mutations in the target genes. Intrinsic resistance is attributed to low permeability of M. abscessus cell envelope as well as to (multi)drug export systems. However, expression of numerous enzymes by M. abscessus, which can modify either the drug-target or the drug itself, is the key factor for the pathogen’s phenomenal resistance to most classes of antibiotics used for treatment of other moderate to severe infectious diseases, like macrolides, aminoglycosides, rifamycins, β-lactams and tetracyclines. In 2009, when M. abscessus genome sequence became available, several research groups worldwide started studying M. abscessus antibiotic resistance mechanisms. At first, lack of tools for M. abscessus genetic manipulation severely delayed research endeavors. Nevertheless, the last 5 years, significant progress has been made towards the development of conditional expression and homologous recombination systems for M. abscessus. As a result of recent research efforts, an erythromycin ribosome methyltransferase, two aminoglycoside acetyltransferases, an aminoglycoside phosphotransferase, a rifamycin ADP-ribosyltransferase, a β-lactamase and a monooxygenase were identified to frame the complex and multifaceted intrinsic resistome of M. abscessus, which clearly contributes to complications in treatment of this highly resistant pathogen. Better knowledge of the underlying mechanisms of drug resistance in M. abscessus could improve selection of more effective chemotherapeutic regimen and promote development of novel antimicrobials which can overwhelm the existing resistance mechanisms. This article reviews the currently elucidated molecular mechanisms of antibiotic resistance in M. abscessus, with a focus on its drug-target-modifying and drug-modifying enzymes.