Comparative study of apramycin-resistant microorganisms isolated from man and animals

Apramycin-modifying strains isolated from pigs with coli bacteriosis, from humans and hospital environment were studied comparatively. Production of enzymes modifying the aminoglycoside was estimated with the radioactive cofactor procedure. E. coli isolates from the animals were phenotypically resistant to apramycin and a number of other aminoglycosides. They produced acetyltransferase AAC(3)IV, phosphotransferase APH(3')(5"), APH(3") and other enzymes. Resistance of the strains to gentamicin was also conditioned by AAC(3)IV since these strains did not produce AAD(2") and AAC(6'). In the resistant strains of E. coli and their transconjugates there were detected plasmids with a relative molecular weight of 60-80 MD. Some of the belonged to the compatibility group I1, the others belonged to the compatibility group H1. Strains of S. marcescens, K. pneumoniae. K. oxytoca and S. aureus isolated from humans and hospital environment were sensitive to apramycin. Only isolates of P. aeruginosa were resistant to this antibiotic. However, all the isolates produced AAC(3)IV. Some of them additionally produced AAC(6'), an enzyme modifying amikacin, kanamycin and other antibiotics and not acetylating apramycin. Almost all the strains produced kanamycin- and streptomycin phosphotransferases. Possible coselection of strains resistant to apramycin and gentamicin using one of these aminoglycosides is discussed.     

Genes of resistance to aminoglycoside antibiotics in clinical strains of Serratia marcescens and the enzyme encoded by them]

The patterns of aminoglycoside inactivating enzymes were determined by AGRP in 31 clinical isolated of Serratia marcescens. The results were compared with the data on identification of the aminoglycoside resistance genes by the specific DNA probes. It was shown that all the isolates of Serratia marcescens contained the AAC(6')-Ic gene which was not expressed in some isolates. The other detected aminoglycoside inactivating enzymes were the following: AAC(3)-V in 17 isolates, ANT(2') in 7 isolates, AAC(3)-I in 4 isolates and APH(3')-I in 13 isolates. Reliability of the methods of AGRP and DNA-DNA hybridization was estimated in the assay of the aminoglycoside resistant clinical strains of Serratia marcescens.     

Occurrence of aminoglycoside-modifying-enzyme genesaac(6′)-aph(2″), aph(3′), ant(4′) andant(6) in clinical isolates ofEnterococcus faecalis resistant to high-level of gentamicin and amikacin

The genes coding for 4 aminoglycoside-modifying enzymes AAC(6')-APH(2"), APH(3'), ANT(4') and ANT(6) were determined in 44 Slovak clinical isolates of Enterococcus faecalis with high-level resistance to gentamicin (HLGR, collection 1) and 48 E. faecalis isolates with resistance to amikacin (AR, collection 2). The occurrence of spotted genes was (collection 1 vs. collection 2): aac(6)-aph(2") 81.8 vs. 8.3 %, ant(4') 52.3 vs. 81.3 %, aph(3') 50 vs. 56.3 % and ant(6) 6.8 vs. 4.2 %, the most frequent combinations of genes in the HLGR collection were aac(6')-aph(2") + ant(4') and aac(6')-aph(2") + aph(3). In contrast, the aph(3') + ant(4') gene profile was predominant in AR isolates. None of the isolates contained all four AGME genes simultaneously.     

Prevalence of AA(6')-APH(2")Ia gene among high-level aminoglycoside resistant Enterococci and Staphylococci

According to new reports the AAC (6')-APH (2")Ia gene is no longer the only gene encoding resistance to gentamycin in Gram-positive cocci and therefore the current method for predicting synergism aminoglycosides with bacterial cell wall active agents in this bacteria may need revision. To further our knowledge of aminoglycoside resistance mechanism in Gram-positive cocci in Gdańsk region we tested presence of AAC (6')-APH (2")Ia gene among 22 enterococcal (E. faecalis) and 41 staphylococcal (S. haemolyticus, S. aureus, S. epidermidis) gentamycin-resistant isolates. Presence of AAC (6')-APH (2")Ia gene varied from 50% (n = 6) in gentamycin-resistant S. epidermidis, 80% (n = 10) in gentamycin resistant S. haemolyticus 88% in methicillin-resistant Staphylococcus aureus (MRSA) (n = 25). In Enterococcus faecalis this gene was noticed only in 59% (n = 22) of gentamycin-resistant isolates. These results suggest that spread of resistance gene among different species is limited and AAC (6')-APH (2")Ia mediated gentamycin-resistance mechanism is more common among MRSA and Staphylococcus haemolyticus.     

Biochemical determinants of aminoglycoside resistance in Gr- microorganisms isolated from urocultures

The investigation was focused on 60 strains of Gr- microorganisms isolated from urocultures and resistant to gentamicin and/or amikacin. Resistance evaluation by the method of Bauer--Kirby with respect to 7 aminoglycoside aminocyclitols (streptomycin, spectinomycin, kanamycin, gentamicin, tobramycin, sisomicin, netilmicin and amikacin) as well as determination of minimal inhibitory concentrations revealed that the most frequently occurring resistance phenotype was streptomycin kanamycin gentamicin sisomicin tobramycin (91.66% tested microorganisms). Approximately 50% of all tested organisms were found to be susceptible to netilmicin. Assays for aminoglycoside-modifying enzymes using 32P ATP and 14C ATP confirmed APH(3')(5")--I and AAD(2") as resistance determinants regarding 4,6-substituted deoxystreptamines. Acetyltransferase determination by the method of Shannon and Phillips and that by van de Klundert et al. most frequently assumes for the formation of AAC(3)-II and AAC(3)-I. Assays utilizing radioactive labels in amikacin-resistant strains determine the enzymes APH(3') and AAD(2")-II.     

Nucleotide sequence analysis of the gene specifying the bifunctional 6''-aminoglycoside acetyltransferase 2"-aminoglycoside phosphotransferase enzyme in Streptococcus faecalis and identification and cloning of gene regions specifying the two activities.

The gene specifying the bifunctional 6'-aminoglycoside acetyltransferase [AAC(6')] 2"-aminoglycoside phosphotransferase [APH(2")] enzyme from the Streptococcus faecalis plasmid pIP800 was cloned in Escherichia coli. A single protein with an apparent molecular weight of 56,000 was specified by this cloned determinant as detected in minicell experiments. Nucleotide sequence analysis revealed the presence of an open reading frame capable of specifying a protein of 479 amino acids and with a molecular weight of 56,850. The deduced amino acid sequence of the bifunctional AAC(6')-APH(2") gene product possessed two regions of homology with other sequenced resistance proteins. The N-terminal region contained a sequence that was homologous to the chloramphenicol acetyltransferase of Bacillus pumilus, and the C-terminal region contained a sequence homologous to the aminoglycoside phosphotransferase of Streptomyces fradiae. Subcloning experiments were performed with the AAC(6')-APH(2") resistance determinant, and it was possible to obtain gene segments independently specifying the acetyltransferase and phosphotransferase activities. These data suggest that the gene specifying the AAC(6')-APH(2") resistance enzyme arose as a result of a gene fusion.     

Aminoglycoside resistance in gramnegative pathogens of nosocomial infections in Russia]

The study of the mechanisms of aminoglycoside resistance in gramnegative pathogens of nosocomial infections in 14 hospitals of Russia showed that the basic mechanism was production of aminoglycoside modifying enzymes, mainly adenylyl transferase ANT(2"), acetyl transferases AAC(3)-V and ACC(6)-I, and phosphotransferases APH(3')-I and APH(3')-VI. In all the hospitals enzymes modifying gentamicin and tobramycin were wide spread while the resistance phenotypes to aminoglycosides were different in separate hospitals. Isepamycin proved to be the most active aminoglycoside. Recommendations for the use of antibiotics in hospital formulas and empiric therapy should be developed on the basis of the local specific features of the resistance in nosocomial pathogens to aminoglycosides.     

Aminoglycosides resistance in clinical isolates of Staphylococcus aureus from a University Hospital in Bialystok, Poland

Staphylococcus aureus obtained from a University Hospital in Poland were characterized in relation to resistance to aminoglycoside antibiotics and the distribution of the genes encoding the most clinically relevant aminoglycoside modifying enzymes (AMEs). Of a total of 118 S. aureus, 45 (38.1%) isolates were found to be resistant to at least one of the tested antibiotics. All aminoglycoside resistant isolates except one 44 (97.8%) were resistant to kanamycin. The majority of strains 37 (82.2%) and 32 (71.1%) expressed resistance to neomycin and tobramycin, respectively. Eleven strains (24.4%) were resistant to gentamicin or amikacin. All S. aureus strains were sensitive to netilmicin. The most prevalent resistance gene was aac(6')-Ie+aph(2') found in 13 (28.9%) strains and 12 (26.7%) isolates carried ant(4')-Ia gene, whilst aph(3')-IIIa gene was detected in only 7 (15.6%) isolates. Additionally, the ant(6)-Ia and str genes were detected in 14 (31.1%) and 2 (4.4%) strains, respectively. Ten (22.2%) strains resistant to amikacin, tobramycin, kanamycin or neomycin did not harbor any of the above-noted genes.     

Resistance to gentamicin and related aminoglycosides in Staphylococcus aureus isolated in Brazil

Isolates of Staphylococcus aureus obtained from a Brazilian university hospital were characterized in relation to resistance to gentamicin and related aminoglycosides. Thirty-six isolates were susceptible to methicillin (MSSA) and 14 were resistant (MRSA). All isolates were sensitive to nucleic acid-binding compounds. All MRSA isolates and one MSSA isolate were demonstrated to be resistant to gentamicin and were coincidentally resistant to amikacin, kanamycin, neomycin and tobramycin. Among the gentamicin sensitive MSSA isolates, five isolates were found to be resistant only to kanamycin/neomycin. The resistance to gentamicin (and related aminoglycosides: kanamycin and tobramycin) must be due to AAC(6')-APH(2") activity. As these isolates also showed resistance to neomycin, they must carry an additional genetic element, probably the one responsible for APH(3')III activity, which accounts for the high level of resistance to kanamycin and to amikacin. The resistance to kanamycin/neomycin in the gentamicin sensitive isolates could not be attributed to the AAD(4')(4") activity because of the tobramycin sensitivity, and so could be ascribed to the APH(3')III activity. Curing and transfer experiments, as well as electrophoresis procedures, indicate that gentamicin resistance in Staph. aureus strains here studied has, characteristically, chromosomal localization.     

Aminoglycoside-inactivating enzymes in strains of Salmonella and genes encoding them

Aminoglycoside resistance patterns of 56 strains isolated from man, cattle and environment were determined. 34 out of 42 gentamicin-resistant strains were shown to produce AAC(3)-II and 7 strains produced ANT(2"). All the 48 kanamycin resistant strains produced APH(3')-I. Spot hybridization of the 42 gentamicin resistant strains with the inner fragment of the aacC2 gene revealed positive signals for all the strains. Hybridization of the 48 kanamycin-resistant strains with the aphA1 gene probe provided positive results in all the strains. The AAC(3)-IV encoding gene was not detected by DNA-DNA hybridization in the strains studied.     

Molecular cloning and analysis of Staphylococcus aureus chromosomal aminoglycoside resistance genes

Most of the aminoglycoside resistant Staphylococcus aureus strains isolated in France are resistant to all the antibiotics belonging to this family. Two aminoglycoside-modifying enzymes were detected in the wild-type strains studied: an APH3'III and an AAC6'-APH2". These strains also carry two types of streptomycin resistance: high-level resistance due to chromosomal mutation(s) affecting ribosome affinity and low-level resistance, the mechanism of which was not characterized. All the aminoglycoside resistance genes were located on the chromosome. DNA fragments of 1.5 and 1.95 kb carrying the aphA and aacA genes, respectively, were isolated, by cloning, from the cellular DNA of a clinical isolate. When these genes were introduced into Escherichia coli and Bacillus subtilis strains, the enzymes synthesized were indistinguishable from those produced by the S. aureus strains. When the cellular DNAs of wild-type and resistant strains were hybridized with the cloned fragments, sequences homologous to the fragment carrying the aphA gene were found to be located at the same chromosomal site, while those hybridizing with the fragment carrying the aacA gene were at different chromosomal sites.     

Survey of modifying enzymes and plasmids in amikacin-resistant Serratia marcescens

Forty amikacin-resistant strains of Serratia marcescens isolated from four different hospitals (A, B, C, and D) were examined for modifying enzymes and plasmids. Twenty-one of the isolates produced acetyltransferase that modified amikacin. Eighteen of the 21 acetyltransferase-bearing isolates were from different inpatients in hospital A and the other three were from hospital C. Amikacin resistance was mediated by conjugative plasmid of 24 megadaltons in 15 of the 18 acetyltransferase-bearing isolates of hospital A and by nonconjugative plasmids, derivatives of the 24-megadalton plasmids, in the remaining three isolates of the same hospital. The 24-megadalton plasmid determined aminoglycoside acetyltransferase (6') IV. This plasmid-borne enzyme conferred amikacin resistance on S. marcescens but not on Escherichia coli K12. The frequency of transfer of the 24-megadalton plasmid from the S. marcescens isolate to E. coli K12 by conjugation was approximately 10(-7) (transconjugants/donors) and was 0.1% of that between E. coli strains. In acetyltransferase-bearing isolates from hospital C, the enzyme was mediated by a nonconjugative plasmid in one case and could not be associated with a plasmid in the remaining two cases. Neither enzymes nor plasmids could be associated with amikacin resistance of the isolates of the other two hospitals.     

Gentamicin resistance in clinical strains ofEnterobacteriaceae associated with reduced gentamicin uptake

Seven strains ofEnterobacteriaceae resistant to gentamicin obtained as representatives of the predominant resistance profiles in the clinical laboratories ofRafeidia and Al-Watani Hospitals in Nablus (Palestine) were included. Five strains showed a broad aminoglycoside resistance profile but contained no evidence of gentamicin acetylation, adenylation, or phosphorylation. Gentamicin uptake in two tested strains was significantly reduced, compared to that of gentamicin-sensitiveE. coli (MIC, 0.5 μg/mL.) These strains are likely resistant due to a relative reduction of the amount of gentamicin and other aminoglycosides entering the bacterial cell. Two strains showed evidence of adenyltransferase ANT (2")-I activity.     

Determining the limits of the evolutionary potential of an antibiotic resistance gene

The AAC(6') enzymes inactivate aminoglycoside antibiotics by acetylating their substrates at the 6' position. Based on functional similarity and size similarity, the AAC(6') enzymes have been considered to be members of a single family. Our phylogenetic analysis shows that the AAC(6') enzymes instead belong to three unrelated families that we now designate as [A], [B], and [C] and that aminoglycoside acetylation at the 6' position has evolved independently at least three times. AAC(6')-Iaa is a typical member of the [A] family in that it acetylates tobramycin, kanamycin, and amikacin effectively but acetylates gentamicin ineffectively. The potential of the aac(6')-Iaa gene to increase resistance to tobramycin, kanamycin, or amikacin or to acquire resistance to gentamicin was assessed by in vitro evolution. Libraries of PCR mutagenized alleles were screened for increased resistance to tobramycin, kanamycin, and amikacin, but no isolates that conferred more resistance than the wild-type gene were recovered. The library sizes were sufficient to conclude with 99.9% confidence that no single amino acid substitution or combination of two amino acid substitutions in aac(6')-Iaa is capable of increasing resistance to the antibiotics used. It is therefore very unlikely that aac(6')-Iaa of S. typhimurium LT2 has the potential to evolve increased aminoglycoside resistance in nature. The practical implications of being able to determine the evolutionary limits for other antibiotic resistance genes are discussed.     

An aminoglycoside sensing riboswitch controls the expression of aminoglycoside resistance acetyltransferase and adenyltransferases

The emergence of antibiotic resistance in human pathogens is an increasing threat to public health. The fundamental mechanisms that control the high levels of expression of antibiotic resistance genes are not yet completely understood. The aminoglycosides are one of the earliest classes of antibiotics that were introduced in the 1940s. In the clinic aminoglycoside resistance is conferred most commonly through enzymatic modification of the drug although resistance through enzymatic modification of the target rRNA through methylation or the overexpression of efflux pumps is also appearing. An aminoglycoside sensing riboswitch has been identified that controls expression of the aminoglycoside resistance genes that encode the aminoglycoside acetyltransferase (AAC) and aminoglycoside nucleotidyltransferase (ANT) (adenyltransferase (AAD)) enzymes. AAC and ANT cause resistance to aminoglycoside antibiotics through modification of the drugs. Expression of the AAC and ANT resistance genes is regulated by aminoglycoside binding to the 5′ leader RNA of the aac/aad genes. The aminoglycoside sensing RNA is also associated with the integron cassette system that captures antibiotic resistance genes. Specific aminoglycoside binding to the leader RNA induces a structural transition in the leader RNA, and consequently induction of resistance protein expression. Reporter gene expression, direct measurements of drug RNA binding, chemical probing and UV cross-linking combined with mutational analysis demonstrated that the leader RNA functioned as an aminoglycoside sensing riboswitch in which drug binding to the leader RNA leads to the induction of aminoglycoside antibiotic resistance. This article is part of a Special Issue entitled: Riboswitches.     

The role of aminoglycoside--modifying enzymes in resistance of Gram-negative rods

The aim of the study was to evaluate the aminoglycoside resistance of Gram-negative bacilli isolated from patients. To the examination 35 strains of Enterobacteriaceae and 18 of non-fermentative bacteria were included. Resistance to aminoglycosides (gentamicin (G), netilmicin (Nt), tobramycin (T), amikacin (A), kanamycin (K), neomycin (N)) was established by disk diffusion method. Interpretation of enzymatic mechanisms was performed by Livermore. The most common enzymes AAC(6')I were found in Enterobacteriaceae group (mostly in E. cloaceae and P. mirabilis) and AAC(3') and in non-fermentative bacteria: AAC(6')I in P. aeruginosa and APH(3')VI and AAC(3')I in A. baumanii. The most frequent phenotype was resistance to six antibiotics (G, Nt, T, A, K, N) Resistance rates were high for gentamicin (>70 %) in both groups and amikacin (88,89 %) in non-fermentatives.     

Genetic analysis of bacterial acetyltransferases: identification of amino acids determining the specificities of the aminoglycoside 6'-N-acetyltransferase Ib and IIa proteins.

The aminoglycoside 6'-N-acetyltransferase [AAC(6')-I] and AAC(6')-II enzymes represent a class of bacterial proteins capable of acetylating tobramycin, netilmicin, and 2'-N-ethylnetilmicin. However, an important difference exists in their abilities to modify amikacin and gentamicin. The AAC(6')-I enzymes are capable of modifying amikacin. In contrast, the AAC(6')-II enzymes are capable of modifying gentamicin. Nucleotide sequence comparison of the aac(6')-Ib gene and the aac(6')-IIa gene showed 74% sequence identity (K. J. Shaw, C. A. Cramer, M. Rizzo, R. Mierzwa, K. Gewain, G. H. Miller, and R. S. Hare, Antimicrob. Agents Chemother. 33:2052-2062, 1989). Comparison of the deduced protein sequences showed 76% identity and 82% amino acid similarity. A genetic analysis of these two proteins was initiated to determine which amino acids were responsible for the differences in specificity. Results of domain exchanges, which created hybrid AAC(6') proteins, indicated that amino acids in the carboxy half of the proteins were largely responsible for determining specificity. Mutations shifting the specificity of the AAC(6')-Ib protein to that of the AAC(6')-IIa protein (i.e., gentamicin resistance and amikacin sensitivity) have been isolated. DNA sequence analysis of four independent isolates revealed base changes causing the same amino acid substitution, a leucine to serine, at position 119. Interestingly, this serine occurs naturally at the same position in the AAC(6')-IIa protein. Oligonucleotide-directed mutagenesis was used to construct the corresponding amino acid change, a serine to leucine, in the AAC(6')-IIa protein. This change resulted in the conversion of the AAC(6')-IIa substrate specificity to that of AAC(6')-Ib. Analysis of additional amino acid substitutions within this region of AAC(6')-Ib support the model that we have identified an aminoglycoside binding domain of these proteins.     

Prospects for using DNA probes for rapid diagnosis of antibiotic resistance in clinical strains of enteric bacteria

Aminoglucoside resistance patterns of clinical strains of enteric bacteria isolated from inpatients of Moscow clinics were determined. APH(3')-I and AAC(3)-II were shown to be the most frequent. The aphA1 and aacC2 genes encoding the enzymes were cloned from the R plasmid of the transconjugant of the E. coli clinical strains. DNA probes based on the determined nucleotide sequences of the cloned genes were constructed and used in DNA-DNA hybridization experiments. The results on the occurrence of APH(3')-I and AAC(3)-II in the strains tested were confirmed by the DNA-DNA hybridization. Prospects for developing a set of DNA probes for rapid diagnosis of antibiotic resistance are discussed.     

Transposon Tn5281 is the main distributor of the aminoglycoside modifying enzyme gene among isolates of Enterococcus faecalis in Tehran hospitals

Infections with high levels of gentamicin-resistant (HLGR) isolates of Enterococcus faecalis are common in Tehran hospitals. Genes encoding such resistance are transmissible by conjugation at high frequency. The purpose of this study was to determine the existence of Tn5281 and its flanking aminoglycoside modifying enzyme gene aac(6')-aph(2") among 102 HLGR isolates of E. faecalis cultured from patients at three hospitals in Tehran, Iran. These isolates were detected by disks containing 120 microg of gentamicin and made 65% of all E. faecalis during the study period. DNA was extracted from HLGR isolates and subjected to PCR assays targeting aac(6')-aph(2") and conjugative transposon Tn5281. The amplified aac(6')-aph(2") gene was labeled with digoxigenin and probed with Tn5281 amplicons in dot blot hybridization assays. The aac(6')-aph(2") gene was detected in 91%-92% (n = 93) of the HLGR isolates. All isolates containing aac(6')-aph(2") were positive in long-PCR targeting Tn5281 and the probe hybridized with Tn5281 amplicons. The number of HLGR isolates of E. faecalis has increased considerably in Tehran hospitals. Tn5281 is the main cause of transmission of aac(6')-aph(2") to different isolates of E. faecalis in the hospitals studied.     

Characterization of glycopeptides, aminoglycosides and macrolide resistance among Enterococcus faecalis and Enterococcus faecium isolates from hospitals in Tehran

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.