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The aim of this study was isolation and identification of the high-level aminoglycoside-resistant (HLAR) enterococci in raw milk and dairy products and to analyze their antibiotic resistance and the presence of aminoglycoside-modifying enzyme (AME) genes. A total of 59 HLAR enterococci were isolated from raw milk and traditional cheese samples. Thirty-nine of the 59 HLAR enterococci were isolated on streptomycin-containing agar medium, while the other 20 HLAR strains were isolated on gentamicin containing agar medium. The 59 HLAR enterococci were identified as 26 E. faecalis (44.07%), 18 E. faecium (30.51%), 13 E. durans (22.03%), and two E. gallinarum (3.39%) by species-specific PCR. Disk diffusion tests showed that teicoplanin were the most effective antibiotics used in this study, while 89.83% of isolates were found to be resistant to tetracycline. High rates of multiple antibiotic resistance were detected in HLAR isolates. Minimum inhibitory concentration (MIC) values of HLAR enterococci against streptomycin and gentamicin were found in the range of 64 to > 4096 µg/mL. Forty-seven (79.66%) of the 59 HLAR enterococci were found to be both high-level streptomycin-resistant (HLSR) and high-level gentamicin-resistant (HLGR) by MIC tests. However, no correlation was found between the results of the disk diffusion and MIC tests for gentamicin and streptomycin in some HLAR strains. The aph(3')-IIIa (94.92%) was found to be most prevalent AME gene followed by ant(4')-Ia (45.76%), ant(6')-Ia (20.34%) and aph(2'')-Ic (10.17%). None of the isolates contained the aac(6')-Ie-aph(2'')-Ia, aph(2'')-Ib or aph(2'')-Id genes. None of the AME-encoding genes were identified in E. durans RG20.1, E. faecalis RG22.4, or RG26.1. In conclusion, HLAR enterococci strains isolated in this study may act as reservoirs in the dissemination of antibiotic resistance genes.

Bacterial resistance towards aminoglycoside antibiotics mainly occurs because of aminoglycoside phosphotransferases (APHs). It is thus necessary to provide a rationale for focusing inhibitor development against APHs. The nucleotide triphosphate (NTP) binding site of eukaryotic protein kinases (ePKs) is structurally conserved with APHs. However, ePK inhibitors cannot be used against APHs due to cross reactivity. Thus, understanding bacterial resistance at the atomic level could be useful to design new inhibitors against such resistant pathogens. Hence, we carried out in vitro studies of APH from newly deposited multidrug-resistant organism Bacillus subtilis subsp. subtilis strain RK. Enzymatic modification studies of different aminoglycoside antibiotics along with purification and characterization revealed a novel class of APH, i.e., APH(5), with molecular weight 27 kDa approximately. Biochemical analysis of virtually screened inhibitor ZINC71575479 by coupled spectrophotometric assay showed complete enzymatic inhibition of purified APH(5). In silico toxicity study comparison of ZINC71575479 with known inhibitor of APH, i.e., tyrphostin AG1478, predicted its acceptable values for 96 h fathead minnow LC 50 , 48 h Tetrahymena pyriformis IGC 50 , oral rat LD 50 , and developmental toxicity using different QSAR methodologies. Thus, the present study gives novel insight into the aminoglycoside resistance and inhibition mechanism of APH(5) by applying experimental and computational techniques synergistically.

Nine aph genes, including aph(2″)-Ib , aph(2″)-Ic , aph(2″)-Ig , aph(2″)-If , aph(2″)-If1 , aph(2″)-If3 , aph(2″)-Ih , aac(6')-Ie - aph(2″)-Ia , and aac(6')-Ie - aph(2″)-If2 , were previously identified in Campylobacter To measure the contribution of these alleles to aminoglycoside resistance, we cloned nine genes into the pBluescript and expressed them in Escherichia coli DH5α. The nine aph expressed in E. coli showed various levels of resistance to gentamicin, kanamycin, and tobramycin. Three genes, aac(6″)-Ie - aph(2″)-Ia , aph2″-If1 , and aph2″-Ig , showed increased MICs to amikacin, and five aph genes were transferrable., (This is a work of the U.S. Government and is not subject to copyright protection in the United States. Foreign copyrights may apply.)

High level aminoglycoside resistance (HLAR) in the lactic acid bacteria (LAB) derived from food animals is detrimental. The aim of this study was to investigate the localization and conjugal transfer of aminoglycoside resistance genes, aac(6')Ie-aph(2″)Ia and aph(3')IIIa in different Enterococcus species. The cross resistance patterns in Enterococcus faecalis MCC3063 to clinically important aminoglycosides by real time PCR were also studied. Southern hybridization experiments revealed the presence of aac(6')Ie-aph(2 ″ )Ia and aph(3')IIIa genes conferring HLAR in high molecular weight plasmids except in Lactobacillus plantarum. The plasmid encoded bifunctional aac(6')Ie-aph(2″)Ia gene was transferable from Enterococcus avium (n = 2), E. cecorum (n = 1), E. faecalis (n = 1) and Pediococcus lolii (n = 1) species into the recipient strain; E. faecalis JH2-2 by filter mating experiments thus indicating the possible risks of gene transfer into pathogenic strains. Molecular analysis of cross resistance patterns in native isolate of E. faecalis MCC3063 carrying aac(6')Ie-aph(2″)Ia and aph(3')IIIa gene was displayed by quantification of the mRNA levels in this study. For this, the culture was induced with increasing concentrations of gentamicin, kanamycin and streptomycin (2048, 4096, 8192, 16384 μg/mL) individually. The increasing concentrations of gentamicin and kanamycin induced the expression of the aac(6')Ie-aph(2″)Ia and aph(3')IIIa resistance genes, respectively. Interestingly, it was observed that induction with streptomycin triggered a significant fold increase in the expression of the aph(3')IIIa gene which otherwise was not known to modify the aminoglycoside. This is noteworthy as streptomycin was found to confer cross resistance to structurally unrelated kanamycin. Also, expression of the aph(3')IIIa gene when induced with streptomycin, revealed that bacteria harbouring this gene will be able to overcome streptomycin bactericidal action at specific concentrations. HLAR in E. faecalis MCC3063 may be due to the combined expression of both the aac(6')Ie-aph(2″)Ia and aph(3')IIIa genes which could be therapeutically challenging. A combined expression of both the genes in E. faecalis MCC3063 may yield HLAR which could be therapeutically challenging. The study highlights the significant alterations in the mRNA expression levels of aac(6')Ie-aph(2″)Ia and aph(3')IIIa in resistant pathogens, upon exposure to clinically vital aminoglycosides., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

The advent of metagenomics has greatly facilitated the discovery of enzymes with useful biochemical characteristics for industrial and biomedical applications, from environmental niches. In this study, we used sequence-based metagenomics to identify two antibiotic resistance enzymes from the secluded, lower convective layer of Atlantis II Deep Red Sea brine pool (68°C, ~2200 m depth and 250‰ salinity). We assembled > 4 000 000 metagenomic reads, producing 43 555 contigs. Open reading frames (ORFs) called from these contigs were aligned to polypeptides from the Comprehensive Antibiotic Resistance Database using BLASTX. Two ORFs were selected for further analysis. The ORFs putatively coded for 3'-aminoglycoside phosphotransferase [APH(3')] and a class A beta-lactamase (ABL). Both genes were cloned, expressed and characterized for activity and thermal stability. Both enzymes were active in vitro, while only APH(3') was active in vivo. Interestingly, APH(3') proved to be thermostable (T m  = 61.7°C and ~40% residual activity after 30 min of incubation at 65°C). On the other hand, ABL was not as thermostable, with a T m  = 43.3°C. In conclusion, we have discovered two novel AR enzymes with potential application as thermophilic selection markers., (© 2016 The Authors. Microbial Biotechnology published by John Wiley & Sons Ltd and Society for Applied Microbiology.)

The activity of eight aminoglycosides (amikacin, apramycin, arbekacin, gentamicin, kanamycin, neomycin, netilmicin and tobramycin) against a collection of 257 amoxicillin/clavulanic acid (AMC)-resistant Escherichia coli isolates was determined by microdilution. Aminoglycoside resistance rates, the prevalence of aminoglycoside-modifying enzyme (AME) genes, the relationship between AME gene detection and resistance phenotype to aminoglycosides, and the association of AME genes with mechanisms of AMC resistance in E. coli isolates in Spain were investigated. Aminoglycoside-resistant isolates were screened for the presence of genes encoding common AMEs [aac(3)-Ia, aac(3)-IIa, aac(3)-IVa, aac(6')-Ib, ant(2″)-Ia, ant(4')-IIa and aph(3')-Ia] or 16S rRNA methylases (armA, rmtB, rmtC and npmA). In total, 105 isolates (40.9%) were resistant to at least one of the aminoglycosides tested. Amikacin, apramycin and arbekacin showed better activity, with MIC90 values of 2mg/L (arbekacin) and 8mg/L (amikacin and apramycin). Kanamycin presented the highest MIC90 (128mg/L). The most common AME gene was aac(6')-Ib (36 strains; 34.3%), followed by aph(3')-Ia (31 strains; 29.5%), ant(2″)-Ia (29 strains; 27.6%) and aac(3)-IIa (23 strains; 21.9%). aac(3)-Ia, aac(3)-IVa, ant(4')-IIa and the four methylases were not detected. The ant(2″)-Ia gene was usually associated with OXA-1 [21/30; 70%], whilst 23/25 (92%) strains producing CTX-M-15 had the aac(6')-Ib gene. The most prevalent AME gene was aac(6')-Ib (18/41; 44%) in nosocomial isolates, whilst ant(2″)-Ia and aph(3')-Ia genes (20/64; 31%) were more frequent in strains of community origin. In 64.6% isolates the phenotypic profile correlated with the presence of commonly encountered AMEs., (Copyright © 2015 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved.)

The aminoglycoside phosphotransferase aph(3')-IIa primarily inactivates kanamycin and neomycin, whilst aph(3')-IIIa also inactivates amikacin. The aim of this study was to determine the frequency of both resistance genes in major human pathogens to obtain their baseline prevalence in the gene pool of these bacterial populations in Austria. In total, 10 541 Escherichia coli, Enterococcus faecalis, Enterococcus faecium, Pseudomonas aeruginosa, Salmonella enterica subsp. enterica and Staphylococcus aureus isolates were collected representatively without selection bias between 2008 and 2011. Isolates were analysed by aph(3')-IIIa/nptIII- and aph(3')-IIa/nptII-specific TaqMan real-time PCR. For positive strains, MICs using Etests were performed and resistance gene sequences were determined. The overall prevalence of aph(3')-IIIa/nptIII was 1.62 % (95 % confidence interval: 1.38-1.88 %). In Escherichia coli, enterococci, Staphylococcus aureus, P. aeruginosa and Salmonella spp., the aph(3')-IIIa/nptIII prevalence was 0.47 % (0-1.47 %), 37.53 % (32.84-42.40 %), 2.90 % (1.51-5.02 %), 0 % (0-0.32 %) and 0 % (0-0.037 %), respectively. Eleven of a total of 169 carriers showed single-nucleotide polymorphisms in the resistance allele. The overall prevalence of aph(3')-IIa/nptII was 0.0096 % (0-0.046 %). Escherichia coli (0-0.70 %), enterococci (0-0.75 %), Staphylococcus aureus (0-0.73 %) and P. aeruginosa (0-0.32 %) did not carry aph(3')-IIa. A single Salmonella isolate was positive, resulting in an aph(3')-IIa prevalence of 0.013 % (0-0.058 %). aph(3')-IIIa/nptIII carriers were moderately prevalent in the strains tested except for in enterococci, which appeared to be an important reservoir for aph(3')-IIIa. aph(3')-IIa/nptII genes were detected at clinically irrelevant frequencies and played no significant role in the aminoglycoside resistance gene pool during the observation period.

Activity of the aminoglycoside phosphotransferase APH(3')-Ia leads to resistance to aminoglycoside antibiotics in pathogenic Gram-negative bacteria, and contributes to the clinical obsolescence of this class of antibiotics. One strategy to rescue compromised antibiotics such as aminoglycosides is targeting the enzymes that confer resistance with small molecules. We demonstrated previously that ePK (eukaryotic protein kinase) inhibitors could inhibit APH enzymes, owing to the structural similarity between these two enzyme families. However, limited structural information of enzyme-inhibitor complexes hindered interpretation of the results. In addition, cross-reactivity of compounds between APHs and ePKs represents an obstacle to their use as aminoglycoside adjuvants to rescue aminoglycoside antibiotic activity. In the present study, we structurally and functionally characterize inhibition of APH(3')-Ia by three diverse chemical scaffolds, anthrapyrazolone, 4-anilinoquinazoline and PP (pyrazolopyrimidine), and reveal distinctions in the binding mode of anthrapyrazolone and PP compounds to APH(3')-Ia compared with ePKs. Using this observation, we identify PP derivatives that select against ePKs, attenuate APH(3')-Ia activity and rescue aminoglycoside antibiotic activity against a resistant Escherichia coli strain. The structures described in the present paper and the inhibition studies provide an important opportunity for structure-based design of compounds to target aminoglycoside phosphotransferases for inhibition, potentially overcoming this form of antibiotic resistance.

The prevalence of antibiotic resistance and the implicated mechanisms of resistance were evaluated in Enterococcus spp. and Escherichia coli, isolated from a total of 250 faecal samples of echinoderms collected from Azorean waters (Portugal). A total of 144 enterococci (120 Enterococcus faecium, 14 E. hirae, 8 E. faecalis, 2 E. gallinarum) and 10 E. coli were recovered. High percentages of resistance in enterococci were found for erythromycin, ampicillin, tetracyclin and ciprofloxacin. The erm(A) or erm(B), tet(M) and/or tet(L), vat(D), aac(6')-aph(2″) and aph(3')-IIIa genes were found in isolates resistant to erythromycin, tetracycline, quinupristin/dalfopristin, high-level gentamicin and high-level kanamycin, respectively. Resistance in E. coli isolates was detected for streptomycin, amikacin, tetracycline and tobramycin. The aadA gene was found in streptomycin-resistant isolates and tet(A)+tet(B) genes in tetracycline-resistant isolates. The data recovered are essential to improve knowledge about the dissemination of resistant strains through marine ecosystems and the possible implications involved in transferring these resistances either to other animals or to humans., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Introduction: Pseudomonas aeruginosa (P. aeruginosa) is one of the primary opportunistic pathogens responsible for nosocomial infections. Aminoglycosides are an import ant component of antipseudomonal chemotherapy. The inactivation of drugs by modifying enzymes is the most common mechanism of aminoglycoside resistance., Objectives: The inactivation of aminoglycosides by modifying enzymes is the primary resistance mechanism employed by P. aeruginosa. The aim of the present study was to investigate the occurrence of aminoglycoside resistance and the prevalence of four import ant modifying enzyme genes (aac (6')-I, aac (6')-II, ant (2")-I, aph (3')-VI) in P. aeruginosa in Iran., Methods: A total of 250 clinical isolates of P. aeruginosa were collected from several hospitals in seven cities in Iran. Antimicrobial susceptibility tests (using the disk diffusion method and E-tests) were performed for all 250 isolates. In addition, all isolates were screened for the presence of modifying enzyme genes by polymerase chain reaction., Results: The resistance rates, as determined by the disk diffusion method, were as follows: gentamicin 43%, tobramycin 38%, and amikacin 24%. Of the genes examined, aac (6')-II (36%) was the most frequently identified gene in phenotypic resist ant isolates, followed by ant (2")-I, aph (3')-VI, and aac (6')-I., Conclusions: Aminoglycoside resistance in P. aeruginosa remains a significant problem in Iran. Therefore, there is considerable local surveillance of aminoglycoside resistance.

Capuramycin-related compounds, including A-500359s and A-503083s, are nucleoside antibiotics that inhibit the enzyme bacterial translocase I involved in peptidoglycan cell wall biosynthesis. Within the biosynthetic gene cluster for the A-500359s exists a gene encoding a putative aminoglycoside 3-phosphotransferase that was previously demonstrated to be highly expressed during the production of A-500359s and confers selective resistance to capuramycins when expressed in heterologous hosts. A similar gene (capP) was identified within the biosynthetic gene cluster for the A-503083s, and CapP is now shown to similarly confer selective resistance to capuramycins. Recombinant CapP was produced and purified from Escherichia coli, and the function of CapP is established as an ATP-dependent capuramycin phosphotransferase that regio-specifically transfers the gamma-phosphate to the 3''-hydroxyl of the unsaturated hexuronic acid moiety of A-503083 B. Kinetic analysis with the three major A-503083 congeners suggests that CapP preferentially phosphorylates A-503083s containing an aminocaprolactam moiety attached to the hexuronic acid, and bi-substrate kinetic analysis was consistent with CapP employing a sequential kinetic mechanism similar to most known aminoglycoside 3-phosphotransferases. The purified CapP product lost its antibiotic activity against Mycobacterium smegmatis, and this loss in bioactivity is primarily due to a 272-fold increase in the IC(50) in the bacterial translocase I-catalyzed reaction. The results establish CapP-mediated phosphorylation as a mechanism of resistance to capuramycins and now set the stage to explore this strategy of resistance as a potential mechanism inherent to pathogens and provide the impetus for preparing second generation analogues as a preemptive strike to such resistance strategies.

Aminoglycoside 2''-phosphotransferases are clinically important enzymes that cause high levels of resistance to aminoglycoside antibiotics by the organisms that harbor them. These enzymes phosphorylate aminoglycosides, and the modified antibiotics show significant reduction in the binding ability to target the bacterial ribosome. This report presents a detailed characterization of the antibiotic resistance profile and the aminoglycoside and nucleotide triphosphate substrate profiles of four common aminoglycoside 2''-phosphotransferases widely distributed in clinically important Gram-positive microorganisms. Although the antibiotic resistance phenotypes exhibited by these enzymes are similar, their aminoglycoside and nucleotide triphosphate substrate profiles are distinctive. Contrary to the dogma that these enzymes use ATP as the source of phosphate in their reactions, two of the four aminoglycoside 2'-phosphotransferases utilize GTP as the phosphate donor. Of the other two enzymes, one exhibits preference for ATP, and the other can utilize either ATP or GTP as nucleotide triphosphate substrate. A new nomenclature for these enzymes is put forth that takes into account the differences among these enzymes based on their respective substrate preferences. These nucleotide triphosphate preferences should have ramifications for understanding of the evolution, selection, and dissemination of the genes for these important resistance enzymes.

The level of resistance to antibiotics of various chemical structure in actinobacteria of the genus Streptomyces is shown to be regulated by Ca2+ ions. The inhibitors of Ca2+/calmodulin and Ca2+/phospholipid-dependent serine/threonine protein kinases (STPK) are found to reduce antibiotic resistance of actinobacteria. The effect of Ca2+ -dependent phosphorylation on the activity of the enzymatic aminoglycoside phosphotransferase system protecting actinobacteria from aminoglycoside antibiotics was studied. It is shown that inhibitors of Ca2+/calmodulin and Ca2+/phospholipid-dependent STPK reduced the Ca2+ -induced kanamycin resistance in Streptomyces lividans cells transformed by a hybrid plasmid which contained the aminoglycoside phosphotransferase VIII (APHVIII) gene. In S. coelicolor A3(2) cells, the protein kinase PK25 responsible for APHVIII phosphorylation in vitro was identified. It is suggested that STPK play a major role in the regulation of antibiotic resistance in actinobacteria.

The prevalence of glycopeptides, aminoglycosides and erythromycin resistance among Enterococcus faecalis and Enterococcus faecium was investigated. The susceptibility of 326 enterococcal hospital isolates to amikacin, kanamycin, netilmicin and tobramycin were determined using disk diffusion method. The minimum inhibitory concentration (MIC) of vancomycin, teicoplanin, gentamicin, streptomycin, and erythromycin were determined by microbroth dilution method. The genes encoding aminoglycoside modifying enzymes described as AMEs genes, erythromycin-resistant methylase (erm) and vancomycin-resistant were targeted by multiplex-PCR reaction. High level resistance (HLR) to gentamicin and streptomycin among enterococci isolates were 52% and 72% respectively. The most prevalent of AMEs genes were aac (6')-Ie aph (2") (63%) followed by aph (3')-IIIa (37%). The erythromycin resistance was 45% and 41% of isolates were positive for ermB gene. The ermA gene was found in 5% of isolates whereas the ermC gene was not detected in any isolates. The prevalence of vancomycin resistant enterococci (VRE) was 12% consisting of E. faecalis (6%) and E. faecium (22%) and all of them were VanA Phenotype. The results demonstrated that AMEs, erm and van genes are common in enterococci isolated in Tehran. Furthermore our results show an increase in the rate of vancomycin resistance among enterococci isolates in Iran.

A collection of bacterial antibiotic resistance strains isolated from arctic permafrost subsoil sediments of various age and genesis was created. The collection included approximately 100 strains of Gram-positive (Firmicutes, Arthrobacter) and Gram-negative bacteria (Bacteroidetes, gamma-Proteobacteria, and alpha-Proteobacteria) resistant to aminoglycoside antibiotics (gentamycin, kanamycin, and streptomycin), chloramphenicol and tetracycline. Antibiotic resistance spectra were shown to differ in Gram-positive and Gram-negative bacteria. Multidrug resistance strains were found for the first time in ancient bacteria. In studies of the molecular nature of determinants for streptomycin resistance, determinants of the two types were detected: strA-strB genes coding for aminoglycoside phosphotransferases and genes aadA encoding aminoglycoside adenylyltransferases. These genes proved to be highly homologous to those of contemporary bacteria.

Objective: To determine the antibiotics resistance, aminoglycoside-modifying enzymes and homology of high-level gentamycin resistant enterococcus in clinical specimens., Methods: The high-level gentamicin resistant (HLGR) isolates were screened by the agar method and the resistance of 14 antimicrobial agents was determined by K-B method. The aminoglycoside-modifying enzyme genes were detected by polymerase chain reaction (PCR). Pulsed-field gel electrophoresis (PFGE) was used to analyze the homology of HLGR isolates., Results: The ratio of HLGR was 64.2% (68/106). Among the HLGR,there were no isolates resistant to linezolid, vancomycin and tecoplanin, and Enterococcus faecium was more resistant to beta-lactam antibiotics and quinolone than Enterococcus faecalis. The positive rate of aac(6')-Ie-aph(2')-Ia was 92.6% and 3 isolates had the resistance gene mostly similar to aph(2')-Id. And among 51 HLGR isolates from the hospitalized patients, PFGE grouped 17 E. faecalis isolates into 4 clusters (A-D), and 33 E. faecium isolates into 8 clusters (A-H) with A cluster as predominant., Conclusion: HLGR has become the important antibiotic resistance bacteria which results in nosocomial infection; and aac(6')-Ie-aph(2')-Ia is the main aminoglycoside-modifying enzyme gene which causes HLGR.

In Alcaligenes faecalis FL-424/98, a clinical isolate that produces the PER-1 extended-spectrum beta-lactamase, the bla(PER-1) gene was found to be carried on a 44-kb nonconjugative plasmid, named pFL424, that was transferred to Escherichia coli by electroporation. Investigation of the genetic context of the bla(PER-1) gene in pFL424 by means of a combined cloning and PCR mapping approach revealed that the gene is associated with a transposonlike element of the Tn3 family. This 14-kb element is a Tn5393 derivative of original structure, named Tn5393d, which contains the transposition module and the strAB genes typical of other members of the Tn5393 lineage plus additional resistance determinants, including the bla(PER-1) gene and a new allelic variant of the aphA6 aminoglycoside phosphotransferase gene, named aphA6b, whose product is active against kanamycin, streptomycin, and amikacin. Tn5393d apparently originated from the consecutive insertion of two composite transposons into a Tn5393 backbone carrying the aphA6b and the bla(PER-1) genes, respectively. The putative composite transposon carrying bla(PER-1), named Tn4176, is made of two original and nonidentical insertion sequences of the IS4 family, named IS1387a and IS1387b, of which one is interrupted by the insertion of an original insertion sequence of the IS30 family, named IS1066. In pFL424, Tn5393d is inserted into a Tn501-like mercury resistance transposon. Transposition of Tn5393d or modules thereof containing the bla(PER-1) gene from pFL424 to small multicopy plasmids or to a bacterial artificial chromosome was not detected in an E. coli host harboring both replicons.

The conserved nature of the genes that code for actinomycete secondary metabolite biosynthetic pathways suggests a common evolutionary ancestor and incidences of lateral gene transfer. Resistance genes associated with these biosynthetic pathways also display a high degree of similarity. Actinomycete aminoglycoside phosphotransferase antibiotic resistance enzymes (APH) are coded for by such genes and are therefore good targets for evaluating the bioactive potential of actinomycetes. A set of universal PCR primers for APH encoding genes was used to probe genomic DNA from three collections of actinomycetes to determine the utility of molecular screening. An additional monitoring of populations for the predominance of specific classes of enzymes to predict the potential of environmental sites for providing isolates with interesting metabolic profiles. Approximately one-fifth of all isolates screened gave a positive result by PCR. The PCR products obtained were sequenced and compared to existing APH family members. Sequence analysis resolved the family into nine groups of which six had recognizable phenotypes: 6'-phosphotransferase (APH(6)), 3'-phosphotransferase (APH(3)), hydroxyurea phosphotransferase (HUR), peptide phosphotransferase, hygromycin B phosphotransferase (APH(7")) and oxidoreductase. The actinomycetes screened fell into seven groups, including three novel groups with unknown phenotypes. The strains clustered according to the environmental site from where they were obtained, providing evidence for the movement of these genes within populations. The value of this as a method for obtaining novel compounds and the significance to the ecology of antibiotic biosynthesis are discussed.