Aminoglycoside-inactivating Enzymes Research Articles

Whole-Genome Sequencing for Investigating a Health Care-Associated Outbreak of Carbapenem-Resistant Acinetobacter baumannii.

Carbapenem-resistant Acinetobacter baumannii (CRAB) outbreaks in hospital settings challenge the treatment of patients and infection control. Understanding the relatedness of clinical isolates is important in distinguishing outbreak isolates from sporadic cases. This study investigated 11 CRAB isolates from a hospital outbreak by whole-genome sequencing (WGS), utilizing various bioinformatics tools for outbreak analysis. The results of multilocus sequence typing (MLST), single nucleotide polymorphism (SNP) analysis, and phylogenetic tree analysis by WGS through web-based tools were compared, and repetitive element polymerase chain reaction (rep-PCR) typing was performed. Through the WGS of 11 A. baumannii isolates, three clonal lineages were identified from the outbreak. The coexistence of blaOXA-23, blaOXA-66, blaADC-25, and armA with additional aminoglycoside-inactivating enzymes, predicted to confer multidrug resistance, was identified in all isolates. The MLST Oxford scheme identified three types (ST191, ST369, and ST451), and, through whole-genome MLST and whole-genome SNP analyses, different clones were found to exist within the MLST types. wgSNP showed the highest discriminatory power with the lowest similarities among the isolates. Using the various bioinformatics tools for WGS, CRAB outbreak analysis was applicable and identified three discrete clusters differentiating the separate epidemiologic relationships among the isolates.

Importance of Core Genome Functions for an Extreme Antibiotic Resistance Trait.

ABSTRACTExtreme antibiotic resistance in bacteria is associated with the expression of powerful inactivating enzymes and other functions encoded in accessory genomic elements. The contribution of core genome processes to high-level resistance in such bacteria has been unclear. In the work reported here, we evaluated the relative importance of core and accessory functions for high-level resistance to the aminoglycoside tobramycin in the nosocomial pathogen Acinetobacter baumannii. Three lines of evidence establish the primacy of core functions in this resistance. First, in a genome scale mutant analysis using transposon sequencing and validation with 594 individual mutants, nearly all mutations reducing tobramycin resistance inactivated core genes, some with stronger phenotypes than those caused by the elimination of aminoglycoside-inactivating enzymes. Second, the core functions mediating resistance were nearly identical in the wild type and a deletion mutant lacking a genome resistance island that encodes the inactivating enzymes. Thus, most or all of the core resistance determinants important in the absence of the enzymes are also important in their presence. Third, reductions in tobramycin resistance caused by different core mutations were additive, and highly sensitive double and triple mutants (with 250-fold reductions in the MIC) that retained accessory resistance genes could be constructed. Core processes that contribute most strongly to intrinsic tobramycin resistance include phospholipid biosynthesis, phosphate regulation, and envelope homeostasis.

Transcriptional and Mutational Profiling of an Aminoglycoside-Resistant Pseudomonas aeruginosa Small-Colony Variant.

Pseudomonas aeruginosa is a major causative agent of both acute and chronic infections. Although aminoglycoside antibiotics are very potent drugs against such infections, antibiotic failure is steadily increasing mainly because of increasing resistance of the bacteria. Many molecular mechanisms that determine resistance, such as acquisition of genes encoding aminoglycoside-inactivating enzymes or overexpression of efflux pumps, have been elucidated. However, there are additional, less well-described mechanisms of aminoglycoside resistance. In this study, we profiled a clinical tobramycin-resistant P. aeruginosa strain that exhibited a small-colony variant (SCV) phenotype. Both the resistance and colony morphology phenotypes were lost upon passage of the isolate under rich medium conditions. Transcriptional and mutational profiling revealed that the SCV harbored activating mutations in the two-component systems AmgRS and PmrAB. Introduction of these mutations individually into type strain PA14 conferred tobramycin and colistin resistance, respectively. However, their combined introduction had an additive effect on the tobramycin resistance phenotype. Activation of the AmgRS system slightly reduced the colony size of wild-type PA14, whereas the simultaneous overexpression of gacA, the response regulator of the GacSA two-component system, further reduced colony size. In conclusion, we uncovered combinatorial influences of two-component systems on clinically relevant phenotypes such as resistance and the expression of the SCV phenotype. Our results clearly demonstrate that the combined activation of P. aeruginosa two-component systems has pleiotropic effects with unforeseen consequences.

Stenotrophomonas maltophilia drug resistance

Stenotrophomonas maltophilia has emerged in recent years as a paradigm of an intrinsically resistant, opportunistic bacterial pathogen with an environmental origin. The recent publication of the sequences of two S. maltophilia genomes has shown that this bacterium contains a large repertoire of antibiotic resistance determinants, probably contributing to its characteristic susceptibility to antibiotics. Among those determinants, the best characterized are a number of multidrug efflux pumps, beta-lactamases and aminoglycoside-inactivating enzymes. Recently, the presence of a gene coding for a Qnr determinant in the genome of S. maltophilia has also been described. Together, these elements confer resistance to several of the drugs currently used for treating infections. Besides these chromosomally encoded determinants, which evolved in S. maltophilia long before the recent human use of antibiotics, this bacterial species is acquiring novel resistance genes by horizontal gene transfer, thereby increasing its resistance. Future studies are required to fully understand the mechanisms of resistance, their regulation and potential crosstalk with S. maltophilia virulence, as well as the population dynamics of the different isolates of this bacterial species.

The increasing role of Acinetobacter species as nosocomial pathogens

Among gram-negative organisms playing a significant role in nosocomial infections, Acinetobacter species have attracted increasing attention in intensive care units during the past two decades. Acinetobacter species are implicated in a wide spectrum of infections (eg, bacteremia, nosocomial pneumonia, urinary tract infections, secondary meningitis, superinfections in burn patients). One of the most striking features of Acinetobacter species is their extraordinary ability to develop multiple resistance mechanisms against major antibiotic classes. They have become resistant to broad-spectrum β-lactams (third-generation cephalosporins, carboxypenicillins, and increasingly to carbapenems); they produce a wide range of aminoglycoside-inactivating enzymes; and most strains are resistant to fluoroquinolones. In Acinetobacter nosocomial infections, the major problems confronting clinicians in intensive care units are related to the severity of Acinetobacter nosocomial infections and to resistance to major antibiotic classes of these organisms.

The increasing role of Acinetobacter species as nosocomial pathogens

Among gram-negative organisms playing a significant role in nosocomial infections, Acinetobacter species have attracted increasing attention in intensive care units during the past two decades. Acinetobacter species are implicated in a wide spectrum of infections (eg, bacteremia, nosocomial pneumonia, urinary tract infections, secondary meningitis, superinfections in burn patients). One of the most striking features of Acinetobacter species is their extraordinary ability to develop multiple resistance mechanisms against major antibiotic classes. They have become resistant to broad-spectrum beta-lactams (third-generation cephalosporins, carboxypenicillins, and increasingly to carbapenems); they produce a wide range of aminoglycoside-inactivating enzymes; and most strains are resistant to fluoroquinolones. In Acinetobacter nosocomial infections, the major problems confronting clinicians in intensive care units are related to the severity of Acinetobacter nosocomial infections and to resistance to major antibiotic classes of these organisms.

Gli Aminoglicosidi nel Trattamento delle Infezioni Pediatriche

A large proportion of aerobic Gram-negative organisms, which are responsible for a variety of serious infections especially in hospitalised patients, are susceptible to aminoglycosides. In paediatrics, the clinical situations in which these agents are useful include sepsis and pneumonia in neonates, sepsis in neutropenic patients, respiratory tract infections in patients with cystic fibrosis, endocarditis and urinary tract infections. Isepamicin is a new aminoglycoside antibiotic, which, compared with currently available agents of this class, demonstrates superior stability in the presence of aminoglycoside-inactivating enzymes.

New derivatives of kanamycin B obtained by combined modifications in positions 1 and 6". Synthesis, microbiological properties, and in vitro and computer-aided toxicological evaluation

Substitution of the C-1 atom in the 2-deoxystreptamine moiety of gentamicin C2, a broad-spectrum aminoglycoside antibiotic, by an axial hydroxymethyl group has been reported to confer protection against most clinically important bacterial enzymes inactivating aminoglycosides, while simultaneously reducing the nephrotoxic potential of this drug. We report here on a similar modification of kanamycin B. Microbiological evaluation, however, revealed no useful protection, as established by the almost complete lack of activity of 1-C-(hydroxymethyl)kanamycin B against an array of organisms producing defined types of aminoglycoside-inactivating enzymes and against which 1-C-(hydroxymethyl)gentamicin C2 and amikacin (1-N-[(S)-2-hydroxy-4-aminobutyryl]kanamycin A) are active. Moreover, toxicological evaluation, based on the in vitro measurement of the drug inhibitory potential toward lysosomal phospholipases, a predictive test of the intrinsic nephrotoxic potential of aminoglycosides, showed not decreased but rather increased toxicity. Comparative conformational analysis of the interactions of the drug with a phosphatidylinositol monolayer explained the lack of protective effect, since no significant change of the mode of insertion of the derivative in this monolayer was detected compared to that of kanamycin B. Combination of a 1-C-(hydroxymethyl) substituent with a 6"-chloro, 6"-acetamido substituent resulted in a partial improvement of the toxicological behavior with no loss of activity for the 6"-chloro and the 6"-azido derivatives, but not to the extent of obtaining better derivatives than kanamycin B itself. We, therefore, suggest that the advantages of an axial hydroxymethyl substituent at C-1 are probably restricted to the gentamicin family and do not extend to kanamycins. It might be concluded that the structural differences between gentamicins and kanamycins play an important, still undescribed role both in their effective recognition by aminoglycoside-inactivating enzymes, which are responsible for most of the clinically important cases of resistance to aminoglycosides, and also in the interactions with phospholipids, which in turn cause nephrotoxicity.

Mechanism of aminoglycoside resistance in Danish Staphvlococcus aureus strains during the years 1979–1987

A total of 49 gentamicin-resistant strains of Staphylococcus aureus isolated from blood (n = 26) or from other sites (n = 23) during the years 1979 to 1987 were evaluated for the presence of aminoglycoside-inactivating enzymes on the basis of minimum inhibitory concentrations measured by agar dilution as well as inhibition zone diameters determined by disc diffusion. Enzymatic activity was caused by AAC (6')III/APH (2") in 45 strains, and by AAC (6')III/APH (2") + APH (3') in four strains. No changes in the distribution of enzymatic activity were observed during the eight years.

Analysis of Amikacin-Resistant Pseudomonas aeruginosa Developing in Patients Receiving Amikacin

During a 36-month period, 28 patients treated for infections due to amikacin-susceptible Pseudomonas aeruginosa subsequently developed infections or colonization with amikacin-resistant P aeruginosa at the same site. Eleven amikacin-susceptible/-resistant pairs of isolates were analyzed for aminoglycoside-inactivating enzymes, plasmid profiles, cellular proteins, outer membrane proteins (OMPs), lipopolysaccharide (LPS) profiles, and amikacin uptake. While clearly distinct from isolates of other patients, sensitive and resistant isolates from the same patients were indistinguishable in plasmid profile, LPS profiles, and OMPs. These results suggest that the resistant P aeruginosa isolates were derived from the sensitive isolates. None of the resistant isolates produced enzymes known to inactivate amikacin. In nine of 11 resistant isolates tested, transport of amikacin into P aeruginosa was reduced. A major mechanism of in vivo development of amikacin resistance in P aeruginosa is alteration in permeability to amikacin, but the aquisition of plasmids or changes in OMPs or LPS profile may not account for this phenomenon.

Identification of individual aminoglycoside-inactivating enzymes in a mixture by HPLC determination of reaction products.

A method is described for the detection and identification of two aminoglycoside-acetylating (AAC(2') or AAC(3) or AAC(6')) or -phosphorylating (APH(2'') or APH(3')) enzymes when these are present in the same crude enzyme extract. Crude enzyme extracts were prepared from reference strains of bacteria producing AAC(2'), AAC(3), AAC(6'), APH(2'') or APH(3') enzymes and mixed to give the following paired enzyme combinations: AAC(2') + AAC(3), AAC(2') + AAC(6'), AAC(3) + AAC(6') and APH(2") + APH(3'). These enzyme mixtures were examined by the radioenzymatic profile method for the identification of aminoglycoside-modifying enzymes and their reaction products with butirosin, lividomycin and kanamycin (AAC) or lividomycin and kanamycin (APH) characterized by high performance liquid chromatography (HPLC). Only two of the eight enzymes present were identified by the radioenzymatic method, but all eight were correctly identified by the HPLC method.

Diagnostic microbiology laboratory susceptibility test results discriminate distinctive antibiotic resistance plasmids

The sizes of the zones of inhibition around routinely tested antibiotic disks classified gentamicin-resistant isolates of Klebsiella pneumoniae from ohe hospital into the four major antibiotype classes. From each isolate of the prevalent class (A1), two plasmids could be transferred conjugally. One carried genes for resistance to tetracycline, sulfonamines, and chlorampheicol, and for the SHV β lactamase. The other carried genes for two aminoglycoside-inactivating enzymes, APH (3′)-I and AAC (3)-III, for the TEM 1 β lactamase, and for resistance to sulfonamides. Transconjugants of either plasmid from any A1 isolate yielded the same DNA fragments after restriction endonuclease digestion, but the two plasmids had no fragments in common. Fragments or genes from either plasmid were variously combined or lacking in plasmids from variant isolates (A2,A3, and A4). Plasmids transferable from isolates of classes B and C shared no common DNA restriction fragments with each ther or with either plasmid from Class A. Fragments and genes of the plasmids from C isolates, however, were identical with those of a plasmid endemic in a nearby hospital. Routine monitoring by diagnostic microbiology laboratories of distinctive antibiotypes and of the plasmids that produced them would aid infection control and antibiotic usage policy.

In vitro and in vivo antibacterial activities of K-4619, a new semisynthetic aminoglycoside.

The antibacterial activities of K-4619 (3-de-O-methylsporaricin A sulfate) were compared with those of sporaricin A, amikacin, dibekacin, and gentamicin. K-4619 exhibited a high order of activity against gram-positive and gram-negative bacteria, including Pseudomonas aeruginosa. Its activity against Providencia species and Serratia marcescens was the highest of all drugs tested. K-4619 was highly effective against bacteria that produce various aminoglycoside-inactivating enzymes, except for 3-acetyltransferase I. The bactericidal activity of K-4619 was somewhat greater than that of amikacin. The activity of K-4619 against gram-negative bacteria increased at alkaline pH and was hardly affected by inoculum size, addition of horse serum, and composition of the medium. The in vivo protective effect of K-4619 against infections with Klebsiella pneumoniae, S. marcescens, and P. aeruginosa in mice was greater than that of sporaricin A. K-4619 was also active in mice infected with gentamicin- or amikacin-resistant strains bearing some of the aminoglycoside-inactivating enzymes.

The emergence of bacterial resistance and its influence on empiric therapy.

The discovery of antimicrobial agents had a major impact on the rate of survival from infections. However, the changing patterns of antimicrobial resistance caused a demand for new antibacterial agents. Within a few years of the introduction of penicillin, the majority of staphylococci were resistant to that drug. In the 1960s the production of the semisynthetic penicillins provided an answer to the problem of staphylococcal resistance. In the early 1960s most Escherichia coli were susceptible to the new beta-lactam antibiotic ampicillin; by the end of that decade, plasmid-mediated beta-lactamase resistance was found in 30%-50% of hospital-acquired E. coli. Use of certain agents resulted in the selection of bacteria, such as Klebsiella, that are intrinsically resistant to ampicillin. The original cephalosporins were stable to beta-lactamase, but the use of these agents was in part responsible for the appearance of infections due to Enterobacter species, Citrobacter species, and Pseudomonas aeruginosa. These bacteria, as well as Serratia, were resistant to many of the available beta-lactam agents. Aminoglycosides initially provided excellent activity against most of the facultative gram-negative bacteria. However, the widespread dissemination of the genes that cause production of the aminoglycoside-inactivating enzymes altered the use of those agents. Clearly, the evolution of bacterial resistance has altered the prescribing patterns for antimicrobial agents. Knowledge that beta-lactam resistance to ampicillin or cephalothin is prevalent is causing physicians to select as empiric therapy either a combination of two or more agents or agents to which resistance is uncommon. The new cephalosporins offer a broad spectrum of anti-bacterial activity coupled with low toxicity. However, physicians must closely follow the changing ecology of bacteria when these agents are used, because cephalosporins can also select bacteria resistant to themselves and thereby abolish their value as empiric therapy.

Role of the 1-amino group in aminocyclitol antibiotics: synthesis of 1-deaminogentamicin C2.

The synthesis of 1-deaminogentamicin C2 described here, uses 3,2',6',3"-tetrakis-N-tert-butoxycarbonylgentamicin C2 (2) as intermediate. N-Formylation of 2 followed by per-O-acetylation and dehydration furnished the isocyanide 5. Radical-induced deamination of the latter using tri-n-butylstannane and removal of the protecting groups afforded the target 1-deaminogentamicin C2 (7). Its in vitro antibacterial activity is less than that of the parent gentamicin C2. The behaviour of 7 towards aminoglycoside-inactivating enzymes was also examined; interestingly, it was found to be neither substrate nor inhibitor for such enzymes. These results strongly suggest that the substitution pattern of the 1-position determines the biological properties of the aminoglycoside antibiotics.

Aminoglycoside-modifying enzymes associated with hospital isolates of Gram-negative rods.

Multi-resistant Enterobacteriaceae and Pseudomonas isolated from clinical specimens at RNS were characterized for resistance phenotype and transferred resistant phenotype. These isolates were obtained over a two year period from different specimens and from patients with varying clinical syndromes. Twenty-six of these isolates were further characterized with respect to their aminoglycoside modifying enzymes. Only three enzymes were detected: AAC(3)-I, APH(3')-I and AAD(2")-II. The substrate range of these enzymes was investigated and not always found to coincide with the transferred phenotype. AAD(2")-II was found only in association with APH(3')-I and not alone. The substrate range of AAC(3)-I from Pseudomonas was different from AAC(3)-I from Enterobacteriaceae but not sufficiently dissimilar to classify them as separate isoenzymes. The relatively few aminoglycoside-inactivating enzymes suggest that within this hospital there may be a limited gene pool for these enzymes.

Identification of R plasmids mediating gentamicin resistance from Escherichia coli strains in Japan.

Eight gentamicin-resistant Escherichia coli were identified from among 630 E. coli strans isolated from parenteral infections. All eight strains were multiply resistant and harbored R plasmids mediating gentamicin resistance. The R plasmids specified the formation of two types of aminoglycoside-inactivating enzymes AAD (2") and APH (3').

Gentamicin, tobramycin, and sisomicin disc susceptibility tests. Revised zone standards for interpretation.

Studies were undertaken to reevaluate interpretive zone standards for gentamicin and tobramycin disc tests. Disc tests with an investigational aminoglycoside, sisomicin, were also evaluated. The data suggest modification of zone standards for gentamicin disc tests to R (resistant) less than or equal to 12 mm and S (susceptible) greater than or equal to 16 mm. Currently recommended standards for tobramycin disc tests (R less than or equal to 12 mm and S greater than or equal to 15 mm) were found to be satisfactory. For 10-microgram sisomicin discs, zone standards of R less than or equal to 12 mm and S greater than or equal to 15 mm were also appropriate. Although gentamicin is structurally similar to sisomicin, it was less active against Pseudomonas aeruginosa. The activity spectrum of sisomicin against the collection of 470 bacterial isolates studied more nearly resembled that of tobramycin. Data analysis suggested that tobramycin disc tests might be used to predict susceptibility to sisomicin. However, these two drugs differ in their susceptibilities to aminoglycoside-inactivating enzymes produced by some resistant strains. In those institutions where such strains are endemic, the class concept of disc testing may not be applicable.

In vitro and in vivo antibacterial activity of KW1070, a new aminoglycoside antibiotic.

KW1070, a new aminoglycoside antibiotic with the novel aminocyclitol, fortamine, has a broad spectrum of activity in vitro and in vivo against gram-positive and gram-negative bacteria. The minimum inhibitory concentrations of KW1070 are similar to those of kanamycin against aminoglycoside-susceptible strains, slightly less than those of gentamicin or 3',4'-dideoxykanamycin B. Minimal bactericidal concentrations were found to be near minimal inhibitory concentrations. KW1070 was active in vitro against many aminoglycoside-resistant bacterial strains that possess aminoglycoside-inactivating enzymes, particularly AAC(6'), AAC(2'), AAD(2''), and APH(3'). The activities of KW1070 in mice infected with Staphylococcus aureus, Escherichia coli, Proteus sp., and Serratia marcescens compared favorably with the activities of amikacin and kanamycin; KW1070 was also singificantly active in mice infected with resistant strains bearing the aminoglycoside-inactivating enzymes listed above.

Production of a new aminoglycoside antibiotic by a mutant of Bacillus circulans.

A new aminoglycoside antibiotic, S-11-A, was isolated from the fermentation broth of the 2-deoxystreptamine negative (DOS-) mutant of Bacillus circulans S-11. The structure of S-11-A was elucidated as 1-deamino-1-hydroxyxylostasin, which contains an intermediate of DOS biosynthesis (S-11-P) and has resistance to some aminoglycoside-inactivating enzymes. This is the first finding of antibiotic production by a DOS- mutant without any supplementation of DOS or a DOS analog, and could be described as a novel method of getting a new aminoglycoside antibiotic.