Enzymes In Clinical Isolates Research Articles

Identification of cell-associated and secreted serine-type peptidases in multidrug-resistant emergent pathogens belonging to the Candida haemulonii complex.

The Candida haemulonii complex (Candida haemulonii, Candida haemulonii var. vulnera, and Candida duobushaemulonii) comprises emerging opportunistic human fungal pathogens with recognized multidrug-resistance profiles. Little is known about the virulence markers produced by this fungal complex. However, it is recognized that Candida spp. express a large array of peptidases, which play multiple roles in different aspects of fungal-host interactions. In the present study, we have identified proteolytic enzymes in clinical isolates of the C. haemulonii complex using zymographic assays. Peptidases able to hydrolyze gelatin, casein, albumin, hemoglobin, and immunoglobulin G were detected in cell-free supernatants and cellular extracts taken from the three species forming the C. haemulonii complex. Overall, peptidases were preferentially evidenced at physiological pH and temperatures of 37-42°C, with molar masses between 35 and 85kDa. Peptidase profiles of C. haemulonii and C. haemulonii var. vulnera isolates were quite similar, contrasting to the peptidases produced by C. duobushaemulonii. Almost all peptidases were inhibited by phenylmethanesulfonyl fluoride (PMSF), thus classifying them as serine-type peptidases. Additionally, proteolytic cleavage of soluble azoalbumin was blocked by PMSF (65-95% inhibition depending on the fungal isolate). These unprecedented results have demonstrated the capability of the C. haemulonii complex to produce serine-type peptidases with an ability to cleave a broad spectrum of proteins, including key host components.

Different classes of hydrolytic enzymes produced by multidrug-resistant yeasts comprising the Candida haemulonii complex.

The production of enzymes in clinical isolates of Candida haemulonii (Ch, n = 5), Candida duobushaemulonii (Cd, n = 4) and Candida haemulonii var. vulnera (Chv, n = 3) was identified by agar plate. Aspartic protease, phytase, caseinolytic and hemolytic activities were detected in all the isolates. A distinct scenario was evidenced regarding the production of lipases. In this way, 80%, 50% and 100% of Ch, Cd and Chv, respectively, were phospholipase producers. Regarding esterase activity, 100%, 50% and 66.7% of Ch, Cd and Chv, respectively, were positive isolates. Esterase activity was significantly higher in isolates recovered from cutaneous candidiasis compared with those recovered from body fluids.

Determination of antibiotic resistance in clinical isolates of coagulase negative Staphylococci from hospitalized patients in selected hospitals of Tehran

To investigate the prevalence of methicillin and aminoglycoside resistance and gene encoding staphylococcal cassette chromosome mec (SCC mec and aminoglycoside modifying enzymes in clinical isolates of coagulase negative staphylococci(CoNS) from hospitalized patients. One hundred and three isolates of coagulase negative staphylococci(CoNS) were recovered from various clinical samples from August 2013 to November 2014. All the specimens were identified by conventional microbiological methods. These tests were contained colony morphology, gram stain, catalase, slide and tube coagulase. To determine the sensibility of CoNS to antimicrobial components including Cefoxitin(30µg), Tobramycin(10µg), Kanamycin(30µg), Amikacin(30µg) and Gentamicin(10µg), disk diffusion method was performed by Kirby Bauer antibiotic testing. In order to show the presence of methicillin and aminoglycoside resistant CoNS genes, PCR were demonstrated. In our study the rate of resistance to Cefoxitin, Kanamycin, Gentamicin, Tobramycin and Amikacin were 74(71.8%), 54(52,4%), 51(49.5%), 45(43.7%) and 16(15.5%), respectively. Some strains of CoNS have been detected with intermediate resistance to Kanamycin 4(3.9%), Tobramycin and Amikacin 2(1.9%). In our study, the distribution of mec A gene among clinical isolates of CoNS was 89(86.4%). The prevalence of aminoglycoside resistance genes like ant (4 ')-Ia, aac (6 ')/ aph (2 ) and aph (3 ')-IIIa were 89(86.4%), 87(84.5%) and 68(66%), respectively. The rate of coexistence of aac (6’)-Ie- aph (2’’) with aph (3’)-IIIa and aac (6’)-Ie- aph (2’’) with ant (4’)-Ia was 65(63%) and 77(74%), respectively. Resistance to aminoglycosides, develops quickly in coagulase-negative staphylococci from clinical areas where these antimicrobial agents are widely used. Therefore, higher investments should be directed towards identifying coagulase-negative staphylococcus species in healthcare institutions and in the community. Overall, Knowing the epidemiology and antibiotic resistance of CoNS is essential to implement the prevention strategies and reducing antibiotic consumptions.

Molecular identification of aminoglycoside-modifying enzymes in clinical isolates of Escherichia coli resistant to amoxicillin/clavulanic acid isolated in Spain

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.

Novel Aminoglycoside 2″-Phosphotransferase Identified in a Gram-Negative Pathogen

Aminoglycoside 2″-phosphotransferases are the major aminoglycoside-modifying enzymes in clinical isolates of enterococci and staphylococci. We describe a novel aminoglycoside 2″-phosphotransferase from the Gram-negative pathogen Campylobacter jejuni, which shares 78% amino acid sequence identity with the APH(2″)-Ia domain of the bifunctional aminoglycoside-modifying enzyme aminoglycoside (6') acetyltransferase-Ie/aminoglycoside 2″-phosphotransferase-Ia or AAC(6')-Ie/APH(2″)-Ia from Gram-positive cocci, which we called APH(2″)-If. This enzyme confers resistance to the 4,6-disubstituted aminoglycosides kanamycin, tobramycin, dibekacin, gentamicin, and sisomicin, but not to arbekacin, amikacin, isepamicin, or netilmicin, but not to any of the 4,5-disubstituted antibiotics tested. Steady-state kinetic studies demonstrated that GTP, and not ATP, is the preferred cosubstrate for APH(2″)-If. The enzyme phosphorylates the majority of 4,6-disubstituted aminoglycosides with high catalytic efficiencies (k(cat)/K(m) = 10(5) to 10(7) M(-1) s(-1)), while the catalytic efficiencies against the 4,6-disubstituted antibiotics amikacin and isepamicin are 1 to 2 orders of magnitude lower, due mainly to the low apparent affinities of these substrates for the enzyme. Both 4,5-disubstituted antibiotics and the atypical aminoglycoside neamine are not substrates of APH(2″)-If, but are inhibitors. The antibiotic susceptibility and substrate profiles of APH(2″)-If are very similar to those of the APH(2″)-Ia phosphotransferase domain of the bifunctional AAC(6')-Ie/APH(2″)-Ia enzyme.

Burden of different beta-lactamase classes among clinical isolates of AmpC-producing Pseudomonas aeruginosa in burn patients: A prospective study

Background:Pseudomonas aeruginosa is one of the most common pathogens causing infections in burns, and shows increasing resistance to β-lactam antibiotics by producing different classes of beta-lactamases. It is also not unusual to find a single isolate that expresses multiple β-lactamase enzymes, further complicating the treatment options. Thus, in this study, we aimed to determine the coexistence of different beta-lactamase enzymes in clinical isolates of P. aeruginosa in the burn ward.Materials and Methods:A total of 101 clinical isolates of P. aeruginosa from the burn ward were identified and tested for the presence of different beta-lactamase enzymes (extended spectrum beta lactamase (ESBL), Amp C and metallo β-lactamases (MBL) from October 2006 to May 2009. In vitro susceptibility pattern of antipseudomonal antibiotics was done by the Kirby Bauer disc diffusion method.Results:A total of 33 (32.7%) isolates were confirmed to be positive for AmpC beta-lactamase. Co-production of AmpC along with ESBL and MBL was reported in 24.5% and 45.5% isolates, respectively. A total of 12 (11.9%) isolates were resistant to three or more antibiotic classes (multidrug resistance). Imipenem and piperacillin/tazobactum showed high sensitivity, with 86.1% and 82.2%, respectively.Conclusion:This study reveals the high prevalence of multidrug- resistant P. aeruginosa producing beta-lactamase enzymes of different mechanisms in this region from burn patients. The emerging antimicrobial resistance in burn wound pathogens poses serious therapeutic challenge. Thus proper antibiotic policy and measures to restrict the indiscriminate use of cephalosporins and carbapenems should be taken to minimize the emergence of this multiple beta -lactamase producing pathogen.

Characterization and Molecular Epidemiology of Extended-Spectrum β-Lactamases in Clinical Isolates of Enterobacteriaceae in a Tunisian University Hospital

One hundred extended-spectrum beta-lactamase (ESBL)-producing Enterobacteriaceae were recovered from the intensive care unit and the urology ward of the University Hospital of Sahloul in Tunisia between May 2005 and May 2006. The majority of strains showed a high level of resistance to cefotaxime and ceftazidime. Double-disk synergy test and E-test strips were used to confirm production of ESBLs. The molecular analysis revealed that the majority of strains (91%) carried genes encoding CTX-M-15. SHV-12 and SHV-2a were produced, respectively, by 9% and 3% of the strains. Pulsed-field gel electrophoresis of ESBL-producing Klebsiella pneumoniae isolates revealed four different clonal groups and three for Escherichia coli, showing the absence of spread of any epidemic clone. The CTX-M-15 ESBL-producing E. coli of the major clonal group belong to the B2 phylogenetic group, to the sequence type 131, and has a high virulence potential. In conclusion, CTX-M-15 ESBLs accounted for the overwhelming majority of ESBL types among Enterobacteriaceae from our hospital. This study confirms the high rate of ESBLs in Tunisia and further demonstrates the worldwide spread of genes coding for CTX-M-15 enzymes in clinical isolates.

Presence of different beta-lactamase classes among clinical isolates of Pseudomonas aeruginosa expressing AmpC beta-lactamase enzyme

Infections caused by Pseudomonas aeruginosa are difficult to treat as the majority of isolates exhibit varying degrees of beta-lactamase mediated resistance to most of the beta-lactam antibiotics. It is also not unusual to find a single isolate that expresses multiple beta-lactamase enzymes, further complicating the treatment options. Thus the present study was designed to investigate the coexistence of different beta-lactamase enzymes in clinical isolates of P. aeruginosa. A total of 202 clinical isolates of P. aeruginosa were tested for the presence of AmpC beta-lactamase, extended spectrum beta-lactamase (ESBL) and metallo beta-lactamase (MBL) enzyme. Detection of AmpC beta-lactamase was performed by disk antagonism test and a modified three-dimensional method, whereas detection of ESBL was done by the combined disk diffusion method per Clinical and Laboratory Standards Institute (CLSI) guidelines and MBL were detected by the Imipenem EDTA disk potentiation test. A total of 120 (59.4%) isolates were confirmed to be positive for AmpC beta-lactamase. Among them, 14 strains (7%) were inducible AmpC producers. Co-production of AmpC along with extended spectrum beta-lactamase and metallo beta-lactamase was reported in 3.3% and 46.6% isolates respectively. The study emphasizes the high prevalence of multidrug resistant P. aeruginosa producing beta-lactamase enzymes of diverse mechanisms. Thus proper antibiotic policy and measures to restrict the indiscriminative use of cephalosporins and carbapenems should be taken to minimize the emergence of this multiple beta-lactamase producing pathogens.

Prevalence and molecular characterization of extended-spectrum β-lactamase-producing Klebsiella pneumoniae in Riyadh, Saudi Arabia

BACKGROUND AND OBJECTIVES:Reports on extended-spectrum β-lactamases (ESBL) production by Enterobacteriaceae, and especially in Klebsiella pneumoniae, are few in Saudi Arabia. Therefore, we determined the prevalence of ESBL in K pneumoniae from Riyadh and characterized the predominant β-lactamase gene in these isolates.METHODS:A total of 400 K pneumoniae samples were isolated from two hospitals in Riyadh during 2007 and screened for production of ESBL using ESBL-E-strips and combined disk methods. PCR assay was used to detect blaTEM, blaSHV, and blaCTX-M genes.RESULTS:Phenotypic characterization identified a high ESBL rate of 55% of K pneumoniae isolates. ESBL-producing K pneumoniae were PCR positive for SHV, TEM and CTX-M β-lactamase genes with prevalences 97.3%, 84.1% and 34.1%, respectively. Within the CTX-M family, two groups of enzymes, CTX-M-1 and CTX-M-9-like genes were found with prevalences of 60% and 40%, respectively.CONCLUSIONS:This study confirms the high rate of ESBL in K pneumoniae clinical isolates in hospitals in Riyadh. This study demonstrates the worldwide spread of blaCTX-M genes. This first report of the presence of the blaCTX-M gene in clincial isolates in Saudi Arabia is evidence of the continuing worldwide spread of this gene.

A simple phenotypic method for differentiation between acquired and chromosomal AmpC β-lactamases in Escherichia coli

Screening methods for the detection of plasmid-mediated AmpC beta-lactamases are technically demanding. The purpose of this study was to assess screening methods for the detection of these enzymes in clinical isolates of Escherichia coli, Klebsiella pneumoniae and Proteus mirabilis. Isolates were selected according to a resistance phenotype consistent with production of an AmpC-type beta-lactamase. Detection of acquired ampC genes was done with a multiplex ampC-PCR and sequencing. The phenotypic detection methods evaluated included visual examination of antibiogram plates to identify the presence of scattered colonies located near the edge of the inhibition halo of cefoxitin, cefotaxime, ceftazidime and aztreonam, and a double-disc synergy test using cloxacillin (500 mg) to inhibit AmpC enzymes. Seventy-seven isolates were selected from among 6,209 isolates recovered. Acquired ampC genes (blaCMY-2, blaDHA-1, blaCMY-4 and blaACC-1) were found in 19 (24.7%) of these isolates, including 14 E. coli, two K. pneumoniae and three P. mirabilis isolates. The differential trait for the presence of colonies in the inhibition halo was 100% sensitive and specific. Similar results were obtained for the cloxacillin test, except for the E. coli isolates in which specificity was 10.3%. The phenotypic trait described here can be considered useful for suspecting the presence of these enzymes. The cloxacillin test was only useful in isolates lacking a natural AmpC beta-lactamase.

First description of CTX-M β-lactamase-producing clinical Escherichia coli isolates from Egypt

We studied the presence of β-lactamases with an extended spectrum of activity in clinical Escherichia coli isolates from Cairo, Egypt. Forty-six E. coli isolates were collected from patients with urinary tract infections at a university hospital in 2001. Phenotypic characterisation identified a very high extended-spectrum β-lactamase (ESBL) rate of 60.9%. Pulsed-field gel electrophoresis and plasmid profiles revealed eight different clonal groups. All ESBL producers were polymerase chain reaction-positive for bla TEM and bla CTX-M genes. Within the CTX-M family, three different enzymes, CTX-M-14, CTX-M-15 and CTX-M-27, were found. The ESBL producers carried multiple plasmids and further plasmid-encoded resistances. In several strains, genes for up to six aminoglycoside-modifying enzymes were detected. A linkage to fluoroquinolone resistance was not observed. This study confirms the high rate of ESBLs in Egypt and further demonstrates the worldwide spread of genes coding for CTX-M enzymes in clinical isolates.

Occurrence of extended spectrum β-lactamase enzymes in clinical isolates of Klebsiella species from Harar region, eastern Ethiopia

Extended Spectrum β-Lactamases (ESBLs) producer and multidrug resistant Klebsiella spp. are becoming a major nosocomial pathogen globally. There are no documented reports yet on the occurrence of ESBL enzymes in Klebsiella spp. species from Ethiopia. This study was undertaken to isolate and determine the occurrence of ESBLs and multi-drug resistant Klebsiella spp. in different clinical samples obtained from patients. A cross-sectional survey was conducted in four different hospitals of Harar region (Hiwot Fana, Misrak-Arbegnoch, Police and Army) from December 2003 to February 2004. Three hundred and eighty four clinical specimens (202 sputum, 164 urine and 18 pus) were collected from patients admitted in different wards. Antimicrobial susceptibilities were performed on 57 clinical isolates by standard disk diffusion procedures against eight antimicrobial agents. The ESBLs detection was made by using cefotaxime and ceftazidime alone and in combination with clavulanate. A total of 57 (15%) Klebsiella spp. were isolated from 384 patients. Of the 57 isolates, 33 (58%) were from sputum, 18 (31.5%) from urine and 6 (10.5%) from pus. Of the 57 Klebsiella spp., 54 (94.7%) were identified as K. pneumoniae and 3 (5.3%) as K. oxytoca. Resistance was found against cephalosporins [cefotaxime (39.0%), cefoxitin (39.0%), ceftazidime (40.0%), ceftriaxone (40.0%), cephalothin (42.0%)], chloramphenicol (70.0%), gentamicin (61.0%) and trimethoprim–sulphamethoxazole (65.0%). Analyzed Klebsiella isolates were characterized also by a high degree of multi-resistance (67.0%). In 19/57 (33.3%) of the Klebsiella isolates, ESBL production was detected. Rates of detection of ESBL producers were 42.1, 26.3, 26.3 and 5.3% in Hiwot-Fana, Misrak-Arbegnoch, Police and Army hospitals, respectively. Multi-drug resistant isolates were more prevalent among the ESBLs producers (95.0%) than non-producers (53.0%) ( p = 0.24). In conclusion, our results show that awareness of ESBL production by Klebsiella spp. is clinically important. In the absence of infection control measures, ESBL producing organisms readily pass horizontally from patient to patient. These strains also transiently colonize the hands of hospital staff members, thereby facilitating patient-to-patient transmission of the organism.

CTX-M and SHV-12 β-lactamases are the most common extended-spectrum enzymes in clinical isolates of Escherichia coli and Klebsiella pneumoniae collected from 3 university hospitals within Korea

Among the 443 clinical isolates of Escherichia coli and Klebsiella spp. collected between June and November 2003 from 3 university hospitals in Korea, 62 isolates were confirmed as extended-spectrum β-lactamase (ESBL)- or plasmid-mediated AmpC β-lactamase-producers by double disk synergy test, PCR and sequencing for β-lactamase genes. The most frequently identified ESBL gene among E. coli and K. pneumoniae isolates was bla SHV-12 and bla CTX-M ( bla CTX-M-9, bla CTX-M-14, bla CTX-M-3, and bla CTX-M-15). Four kinds of plasmid-mediated AmpC β-lactamases, ACT-1, CMY-1, CMY-2, and DHA-1, were detected. ESBL production was associated with high levels of resistance to tetracycline, sulfisoxazole, streptomycin, kanamycin, gentamicin and tobramycin when compared to non-ESBL producing isolates. Conclusively, this study suggests that the CTX-M β-lactamases are prevalent and various kinds of plasmid-mediated AmpC enzymes are distributed in clinical isolates of E. coli and Klebsiella spp. in Korea.

Identification of an atypical form of 30 kDa SOD—a possible virulence factor in clinical isolates of Shigella spp.

Reviews on the pathogenic mechanisms of Shigella species show a lacunae in the understanding of the bacterial antioxidant defense system and its regulations. This study was done to investigate the regulation of expression of antioxidant enzymes in clinical isolates of Shigella species, under various growth conditions. The in vitro expression of superoxide dismutase in the clinical isolates of Shigella spp., is modulated by both endogenous and exogenous factors. During aerobic and iron repleted growth conditions, the expression of the MnSOD and FeSOD enzymes were higher, and an atypical SOD was also expressed. However, under anaerobic growth conditions and in plasmid-cured strains, the antioxidant enzyme activities were decreased and the atypical SOD was not expressed. Absence of the atypical form of SOD may be due to the low oxygen environment. Plasmid-encoded factors may also play a role in the expression of this SOD, which had a molecular weight of approximately 30 kDa. In the rat ileal loop ligation assay, mild lesions were observed only in the intestinal microvilli of rats injected with plasmid-cured strains of Shigella spp., suggesting that plasmid-encoded factors, including those that regulate the expression of the atypical SOD, are essential for the virulence of Shigella spp.

Origins of the aminoglycoside modifying enzymes

The aminoglycosides represent an important class of antibiotics for the treatment of bacterial infections. Interaction of the aminoglycosides with the bacterial ribosome inhibits protein synthesis, which is their primary mode of action. However, in gram negative bacteria, the ability of aminoglycosides to perturb the cell envelope is also an important mode of action. A common mechanism for aminoglycoside resistance involves modifying enzymes which acetylate, phosphorylate or adenylylate the aminoglycoside. There exists a staggering number of aminoglycoside modifying enzymes in clinical isolates of bacteria. This diversity suggests multiple origins for the present day enzymes. In this review, the possible origins of these modifying enzymes will be presented. Previous proposals describing the origins of these enzymes will be reviewed and potential mechanisms for the development of new aminoglycoside modifying enzymes will be discussed.

Plasmid-specified aminoglycoside-modifying enzymes in clinical isolates of Klebsiella pneumoniae.

In 106 clinical isolates of multiresistant Klebsiella pneumoniae strains, we found that aminoglycoside-resistance was due mostly to two adenylating enzymes: AAD (2") (56.6%), that modifies gentamicin, kanamycin, tobramycin and sissomicin, and AAD (3") 9 (56.6% + 19.8%) that modifies streptomycin and spectinomycin. The identification of these enzymes was possible by MICs determination against a set of aminoglycosides antibiotics. AAD (2") + AAD (3")9 were coded by conjugative plasmid of about 120 Md.

A simple method for the identification of aminoglycoside-modifying enzymes.

In order to study the correlation between the presence of aminoglycoside-modifying enzymes in bacteria and the susceptibility of these bacteria to aminoglycosides, 133 resistant strains were collected, representing the most frequently occurring modifying enzymes in clinical isolates today. Enzymes in these resistant strains were identified by the determination of substrate profiles for eight different aminoglycosides in vitro. Thirteen different enzymes or combinations of enzymes appeared to be present in this collection, whereas in seven cases the resistance appeared to be non-enzyme-mediated. The enzyme activities were not reflected in the bacterial susceptibility data for each antibiotic. Cluster analysis of the combined sensitivity data, with biochemical enzyme analysis as a guideline, proved to be unsatisfactory for the identification of aminoglycoside-modifying enzymes in clinical isolates. Much better results were obtained with a stepwise determination scheme. This method relies upon inhibition zone diameters around commercially available sensitivity discs for six aminoglycoside antibiotics. These zone diameters are compared with empirically established critical values, which are characteristic of an enzyme or group of enzymes. By this procedure proper identification of the enzyme(s) proved to be possible in 84% of the 133 resistant strains. For the evaluation of the method 100 consecutively isolated aminoglycoside resistant clinical isolates were analysed by means of the stepwise scheme and the biochemical method. Results were identical for 97 of the 100 strains.

Aminoglycoside-inactivating enzymes in clinical isolates of Streptococcus faecalis. An explanation for resistance to antibiotic synergism.

Clinical isolates of enterococci (Streptococcus faecalis) with high-level resistance to both streptomycin and kanamycin (minimal inhibitory concentration >2,000 mug/ml), and resistant to synergism with penicillin and streptomycin or kanamycin were examined for aminoglycoside-inactivating enzymes. All of the 10 strains studied had streptomycin adenylyltransferase and neomycin phosphotransferase activities; the latter enzyme phosphorylated amikacin as well as its normal substrates, such as kanamycin. Substrate profiles of the neomycin phosphotransferase activity suggested that phosphorylation occurred at the 3'-hydroxyl position, i.e., aminoglycoside 3'-phosphotransferase. A transconjugant strain, which acquired high-level aminoglycoside resistance and resistance to antibiotic synergism after mating with a resistant clinical isolate, also acquired both enzyme activities. Quantitative phosphorylation of amikacin in vitro by a sonicate of the transconjugant strain inactivated the antibiotic, as measured by bioassay, and the phosphorylated drug failed to produce synergism when combined with penicillin against a strain sensitive to penicillin-amikacin synergism.No differences were found in the sensitivity of ribosomes from a sensitive and resistant strain when examined in vitro using polyuridylic acid directed [(14)C]-phenylalanine incorporation in the presence of streptomycin, kanamycin, or amikacin. Therefore, we conclude that aminoglycoside-inactivating enzymes are responsible for the aminoglycoside resistance, and resistance to antibiotic synergism observed in these strains.