Gentamicin Resistance Determinant Research Articles

Mechanisms of Gentamicin Resistance in Listeria Monocytogenes: A Mini Review

Listeria monocytogenes is a Gram-positive foodborne pathogen capable of causing a foodborne infectionknown as listeriosis. There are two main types of listeriosis: a non-invasive gastroenteritis and an invasive infection which is often associated with a high mortality and hospitalization rate among susceptible individuals. Gentamicin, used as an adjunct therapy with ampicillin, remains the treatment of choice for the life-threatening invasive listeriosis. Nevertheless, there is little data on gentamicin resistance determinants in L. monocytogenes. Several well-controlled studies have reported that mechanisms of gentamicin resistance in this organism involve active efflux and genetic determinants that affect the uptake of the antibiotic through altered membrane potential. This mini review summarises current knowledge of genetic determinants of gentamicin resistance in L. monocytogenes, with the aim of contributing information that could facilitate the discovery of new therapeutic approaches to overcome, delay or avoid developments of drug resistance in this foodborne pathogen.

Mutations in atpG2 may confer resistance to gentamicin in Listeria monocytogenes.

Introduction Listeriosis, a foodborne infection caused by Listeria monocytogenes, could lead to febrile listerial gastroenteritis and a more invasive form which is often associated with a high mortality and hospitalisation rate. Gentamicin, used as an adjunct therapy with ampicillin, remains the treatment of choice for this life-threatening and invasive infection.Gap statement Nevertheless, there is little data on gentamicin resistance determinants in L. monocytogenes.Aim In this study, we selected and characterised B2b, a gentamicin-resistant mutant derived from L. monocytogenes ATCC 19115 to determine the target(s) of resistance in L. monocytogenes after exposure to gentamicin.Methodology Whole-genome sequencing was carried out to identify the mutation site(s) and possible mechanism(s) of resistance. The mutant was characterised using antimicrobial susceptibility testing and PCR. For biological verifications, complementation and allelic exchange mutagenesis were carried out.Results We found that the gentamicin resistance in B2b was caused by a 10 bp deletion in atpG2 which encodes a gamma subunit of the ATP synthase in L. monocytogenes. Using atpG2 PCR, various other mutations were identified in other gentamicin resistant mutants derived from ATCC 19115. In addition, the mutation from B2b, when introduced into L. ivanovii, also caused gentamicin resistance in this Listeria species.Conclusion Hence, atpG2 mutations appear to be important determinants of gentamicin resistance not only in L. monocytogenes but possibly also in other Listeria species.

508. Gentamicin Non-susceptibility is Associated with Persistence of Carbapenem-Resistant Klebsiella pneumoniae in the Urinary Tract

BackgroundUrinary tract infection (UTI) is the most common clinical manifestation of carbapenem-resistant Klebsiella pneumoniae (CRKp). Persistent CRKp bacteriuria is associated with the spread of CRKp and antibiotic overuse. Risk factors for persistent CRKp bacteriuria are uncertain.MethodsCRACKLE-1 was a multicenter, prospective study that included 960 patients with at least one carbapenem-resistant Enterobacteriaceae (CRE)-positive culture from December 2011 to June 2016 collected from 18 hospitals encompassing 8 healthcare systems in the Midwestern US and North Carolina. Patients with CRKp bacteriuria who were discharged alive from index hospitalization were included in the current study, and sporadic (single positive CRKp urine culture) and persistent (≥2 CRKp urine cultures during independent hospital admissions occurring at least 2 days apart) cases were compared. Antibiotic susceptibility testing was performed by local laboratories. Amikacin, gentamicin (GENT), and trimethoprim/sulfamethoxazole were included in the analysis based on variance and frequency of testing. The CDC/National Healthcare Safety Network criteria for UTI were used.ResultsCRKp was the most common CRE isolate (n = 869, prevalence 91%). In patients with CRKp, 527 had CRKp isolated from the urine (prevalence 61%, 95% CI 0.57, 0.64). Of these, 486 patients, of whom 129 (27%) were diagnosed with a UTI, were discharged alive. Notably, 135/486 (28%) patients with CRKp bacteriuria were readmitted and yielded a second urine culture of CRKp. Most patients with persistent bacteriuria, 99/135 (73%), were asymptomatic at initial admission. Of these patients, 20/99 (20%) were diagnosed with a UTI at second admission. In multivariable analysis, only GENT non-susceptibility was associated with an increased risk (adjusted OR 1.66, 95% CI 1.10–2.49) of persistent bacteriuria. Persistent bacteriuria was independent of GENT treatment during index hospitalization (GENT was used in 15% of patients).ConclusionBacteriuria with GENT non-susceptible CRKp strains was associated with persistent bacteriuria. As this was independent of GENT treatment, GENT resistance determinants may be co-transmitted along with traits that promote bacterial persistence in CRKp. Disclosures All authors: No reported disclosures.

High Prevalence and Predominance of the aph(2″)-If Gene Conferring Aminoglycoside Resistance in Campylobacter.

Campylobacter is a major foodborne pathogen, and previous studies revealed that Campylobacter isolates from food-producing animals are increasingly resistant to gentamicin in China. The molecular epidemiology and genetic mechanisms responsible for gentamicin resistance in China have not been well understood. In this study, 607 Campylobacter isolates of chicken and swine origins collected in 2014 were analyzed, revealing that 15.6% (25/160) of the Campylobacter jejuni isolates and 79.9% (357/447) of the Campylobacter coli isolates were resistant to gentamicin. PCR detection of the gentamicin resistance genes indicated that aph(2″)-If was more prevalent than the previously identified aacA/aphD gene and has become the dominant gentamicin resistance determinant in Campylobacter Transformation and whole-genome sequencing as well as long-range PCR discovered that aph(2″)-If was located on a chromosomal segment inserted between two conserved genes, Cj0299 and panB Cloning of aph(2″)-If into gentamicin-susceptible C. jejuni NCTC 11168 confirmed its function in conferring high-level resistance to gentamicin and kanamycin. Molecular typing by pulsed-field gel electrophoresis suggested that both regional expansion of a particular clone and horizontal transmission were involved in the dissemination of the aph(2″)-If gene in Campylobacter To our knowledge, this is the first report describing the high prevalence of a chromosomally encoded aph(2″)-If gene in Campylobacter The high prevalence and predominance of this gene might be driven by the use of aminoglycoside antibiotics in food animal production in China and potentially compromise the usefulness of gentamicin as a therapeutic agent for Campylobacter-associated systemic infection.

Determinants of virulence and of resistance to ceftiofur, gentamicin, and spectinomycin in clinical Escherichia coli from broiler chickens in Québec, Canada

Antimicrobials are frequently used for the prevention of avian colibacillosis, with gentamicin used for this purpose in Québec until 2003. Ceftiofur was also used similarly, but voluntarily withdrawn in 2005 due to increasing resistance. Spectinomycin-lincomycin was employed as a replacement, but ceftiofur use was partially reinstated in 2007 until its definitive ban by the poultry industry in 2014.Gentamicin resistance frequency increased during the past decade in clinical Escherichia coli isolates from broiler chickens in Québec, despite this antimicrobial no longer being used. Since this increase coincided with the use of spectinomycin-lincomycin, co-selection of gentamicin resistance through spectinomycin was suspected. Therefore, relationships between spectinomycin, gentamicin, and ceftiofur resistance determinants were investigated here. The distribution of 13 avian pathogenic E. coli virulence-associated genes and their association with spectinomycin resistance were also assessed.A sample of 586 E. coli isolates from chickens with colibacillosis in Québec between 2009 and 2013 was used. The major genes identified for resistance to ceftiofur, gentamicin, and spectinomycin were blaCMY, aac(3)-VI, and aadA, respectively. The aadA and aac(3)-VI genes were strongly associated and shown to be located on a modified class 1 integron. The aadA and blaCMY genes were negatively associated, but when present together, were generally located on the same plasmids. No statistical positive association was observed between aadA and virulence genes, and virulence genes were only rarely detected on plasmids encoding spectinomycin resistance. Thus, the use of spectinomycin-lincomycin may likely select for gentamicin but not ceftiofur resistance, nor for any of the virulence-associated genes investigated.

Revisiting the Nucleotide and Aminoglycoside Substrate Specificity of the Bifunctional Aminoglycoside Acetyltransferase(6′)-Ie/Aminoglycoside Phosphotransferase(2″)-Ia Enzyme

The bifunctional aminoglycoside-modifying enzyme aminoglycoside acetyltransferase(6')-Ie/aminoglycoside phosphotransferase(2″)-Ia, or AAC(6')-Ie/APH(2″)-Ia, is the major source of aminoglycoside resistance in gram-positive bacterial pathogens. In previous studies, using ATP as the cosubstrate, it was reported that the APH(2″)-Ia domain of this enzyme is unique among aminoglycoside phosphotransferases, having the ability to inactivate an unusually broad spectrum of aminoglycosides, including 4,6- and 4,5-disubstituted and atypical. We recently demonstrated that GTP, and not ATP, is the preferred cosubstrate of this enzyme. We now show, using competition assays between ATP and GTP, that GTP is the exclusive phosphate donor at intracellular nucleotide levels. In light of these findings, we reevaluated the substrate profile of the phosphotransferase domain of this clinically important enzyme. Steady-state kinetic characterization using the phosphate donor GTP demonstrates that AAC(6')-Ie/APH(2″)-Ia phosphorylates 4,6-disubstituted aminoglycosides with high efficiency (k(cat)/K(m) = 10(5)-10(7) M(-1) s(-1)). Despite this proficiency, no resistance is conferred to some of these antibiotics by the enzyme in vivo. We now show that phosphorylation of 4,5-disubstituted and atypical aminoglycosides are negligible and thus these antibiotics are not substrates. Instead, these aminoglycosides tend to stimulate an intrinsic GTPase activity of the enzyme. Taken together, our data show that the bifunctional enzyme efficiently phosphorylates only 4,6-disubstituted antibiotics; however, phosphorylation does not necessarily result in bacterial resistance. Hence, the APH(2″)-Ia domain of the bifunctional AAC(6')-Ie/APH(2″)-Ia enzyme is a bona fide GTP-dependent kinase with a narrow substrate profile, including only 4,6-disubstituted aminoglycosides.

Cotransfer of Antibiotic Resistance Genes and a hyl Efm -Containing Virulence Plasmid in Enterococcus faecium

The hyl(Efm) gene (encoding a putative hyaluronidase) has been found almost exclusively in Enterococcus faecium clinical isolates, and recently, it was shown to be on a plasmid which increased the ability of E. faecium strains to colonize the gastrointestinal tract. In this work, the results of mating experiments between hyl(Efm)-containing strains of E. faecium belonging to clonal cluster 17 and isolated in the United States and Colombia indicated that the hyl(Efm) gene of these strains is also carried on large plasmids (>145 kb) which we showed transfer readily from clinical strains to E. faecium hosts. Cotransfer of resistance to vancomycin and high-level resistance (HLR) to aminoglycosides (gentamicin and streptomycin) and erythromycin was also observed. The vanA gene cluster and gentamicin resistance determinants were genetically linked to hyl(Efm), whereas erm(B) and ant(6)-I, conferring macrolide-lincosamide-streptogramin B resistance and HLR to streptomycin, respectively, were not. A hyl(Efm)-positive transconjugant resulting from a mating between a well-characterized endocarditis strain [TX0016 (DO)] and a derivative of a fecal strain of E. faecium from a healthy human volunteer (TX1330RF) exhibited increased virulence in a mouse peritonitis model. These results indicate that E. faecium strains use a strategy which involves the recruitment into the same genetic unit of antibiotic resistance genes and determinants that increase the ability to produce disease. Our findings indicate that the acquisition of the hyl(Efm) plasmids may explain, at least in part, the recent successful emergence of some E. faecium strains as nosocomial pathogens.

Plasmid-Mediated High-Level Gentamicin Resistance among Enteric Bacteria Isolated from Pet Turtles in Louisiana

The sale of small turtles is banned by the Food and Drug Administration from the U.S. market due to concerns about their excretion of Salmonella spp. To produce a safe pet for the export market, the Louisiana pet turtle industry uses gentamicin sulfate baths (1,000 microg/ml) to eradicate Salmonella spp. from turtle eggs. In 1999, we analyzed bacterial samples recovered from turtle farms and found that strains of Salmonella enterica subsp. arizonae and other bacteria, such as Enterobacter cloacae, Citrobacter freundii, Pseudomonas aeruginosa, and Stenotrophomonas maltophilia, were resistant to high concentrations of gentamicin (>2,000 microg/ml) and to other aminoglycosides. The goal of this study was to identify the gene(s) which contributes to the high-level gentamicin resistance phenotype observed in bacteria from environmental samples with turtle farming activity, particularly the salmonellae, and to estimate the incidence of such genes in these bacteria. R plasmids from gentamicin-resistant strains were transferred by conjugation and transformation to naive Escherichia coli cells. Cloning and sequencing of the gentamicin resistance determinants on these plasmids revealed the presence of the aminoglycoside acetyltransferase genes aac(3)-IIa and aac(3)-VIa; the latter was present as a gene cassette of a class 1 integron. Multiplex PCR assays showed that every gentamicin-resistant isolate carried one of these acetyltransferase genes. Pulsed-field gel electrophoresis and restriction enzyme digestion analysis of R plasmids carrying these genes revealed different restriction profiles and sizes, indicating a dissemination of the gentamicin resistance genes through mobile molecular elements. The data presented highlight the need to develop an alternate method for the eradication of Salmonella spp. from turtle eggs.

Characterization of high-level aminoglycoside-resistant enterococci in Kuwait hospitals.

This study investigated the distribution of genes for aminoglycoside-modifying enzymes (AME) and the genetic relatedness of high-level aminoglycoside-resistant enterococci isolated in Kuwait hospitals. A total of 117 enterococci, consisting of 109 Enterococcus faecalis, seven Enterococcus faecium, and one Enterococcus casseliflavus were studied. The MICs of gentamicin, kanamycin, amikacin, tobramycin, and streptomycin were determined by agar dilution and the genes encoding the AAC(6')- APH(2"), ANT(4'), APH(3'), APH (2")-Ib, APH (2")-Ic, APH (2")-Id, and ANT(6) enzymes were amplified by PCR. They were typed by pulsed-field gel electrophoresis (PFGE). Filter mating was used to transfer gentamicin resistance determinants. They were all resistant to kanamycin (MIC 2000 mg/L). Fifty-five isolates were resistant to gentamicin (MIC 500 mg/L), 72 were resistant to tobramycin (MIC 64 mg/L), 115 were resistant to amikacin (MIC 64 mg/L), and 97 were resistant to streptomycin (MIC 1000 mg/L). The aac(6')-Ie-aph(2")-Ia was detected in all isolates with gentamicin MIC 500 mg/L and in 15 isolates with gentamicin MIC 256 mg/L. The aph(3')-IIIa gene was detected in 101 isolates, whereas the ant(6')-Ia gene was detected in 85 of the 97 streptomycin-resistant isolates with MIC 1000 mg/L. The aac(6')-Ii gene was detected only in the seven E. faecium isolates. None of them contained ant(4')-Ia, aph(2")-Ib, aph(2")-Ic and aph(2")-Id. PFGE revealed heterogeneous patterns with no dominant clone. The results demonstrated that AME are common in aminoglycoside-resistant enterococci isolated in Kuwait. However, the absence of a dominant clone suggests that they acquired high-level aminoglycoside independently.

Class 1 integron containing a new gene cassette, aadA10, associated with Tn1404 from R151.

The carbenicillin, gentamicin, kanamycin, streptomycin, spectinomycin, sulfonamide, and tobramycin resistance determinants found on Pseudomonas aeruginosa plasmid R151 have previously been shown to translocate to another plasmid, R388, and it was inferred that a transposon, Tn1404, carried the resistance determinants. Sequencing of the cassette array from the plasmid known as R388::Tn1404 revealed two known gene cassettes, oxa10 and aadB, and a previously unidentified cassette determining resistance to streptomycin and spectinomycin, here designated aadA10, in the order oxa10-aadB-aadA10. These cassettes replaced the dfrB2-orfA cassette array of R388, indicating that movement of the resistance determinants from R151 to R388 resulted from recombinational exchange between two class 1 integrons rather than transposition. The AadA10 protein is most closely related to AadA6 (85% identical) and AadA7 (80% identical). The aadA10 cassette found here has only a simple site containing a 7-bp spacer derived from attI1 in place of a 59-base element and is likely to represent a derivative of the complete cassette. IntI1-mediated deletion of the aadA10 cassette was not detected, indicating that this single simple site is either inactive or only weakly active.

Construction and use of derivatives of transposon Tn4001 that function in Mycoplasma pulmonis and Mycoplasma arthritidis.

Previous attempts to introduce transposon Tn4001 into Mycoplasma pulmonis and Mycoplasma arthritidis have not been successful, possibly due to functional failure of the transposon's gentamicin resistance determinant. Tn4001C and Tn4001T were constructed, respectively, by insertion of a chloramphenicol acetyltransferase gene and the tetM tetracycline resistance determinant into Tn4001. Both Tn4001C and Tn4001T transposed in M. pulmonis, and Tn4001T transposed in M. arthritidis. The incorporation of a Tn4001T derivative that contained lacZ into either Mycoplasma species resulted in transformants with readily detectable LacZ activity. Tn4001T may be of general utility for use as a mycoplasma cloning vehicle because tetM functions in all species of Mycoplasma examined thus far.

Expression of the Pseudomonas aeruginosa gentamicin resistance gene aacC3 in Escherichia coli.

The Pseudomonas aeruginosa aacC3 gene was expressed in Escherichia coli after cloning of the single gene behind the strong tac promoter. In the original Pseudomonas strain, aacC3 is preceded by cysC; together they form a single transcription unit. The ribosome-binding site and start codon of aacC3 are involved in a putative intercistronic hairpin, the stability of which interfered with the aminoglycoside resistance level. In Northern blots, full-length transcripts comprising both cysC and aacC3 could not be detected either in the original Pseudomonas strain or in E. coli harboring a plasmid with the cloned operon. In contrast, cysC transcripts were abundant. Cloning of the operon between the tac promoter and a transcription termination signal resulted in higher mRNA levels and phenotypic expression in E. coli. The absence of a transcription termination signal in the wild-type cysC-aacC3 sequence is associated with transcripts of heterogeneous size that were undetected in Northern blots. Our results shed more light on the expression of this gentamicin resistance determinant, although the discrepancies between its expression in E. coli and Pseudomonas are not fully solved.

Tn924, a chromosome-borne transposon encoding high-level gentamicin resistance in Enterococcus faecalis.

Enterococcus faecalis SF350 is a clinical isolate from Winnipeg, Canada, with high-level (MIC > 2,000 micrograms/ml) gentamicin resistance. The genetic determinant for gentamicin resistance was located on the chromosome of SF350 and could be mobilized by a coresident conjugative plasmid, pYN120. Genetic and physical analyses showed that the gentamicin resistance determinant was located on a 27-kb transposable element which was designated Tn924.

Nucleotide Sequence and Expression of a Gentamicin Resistance Gene Isolated from the R Plasmid in Serratia marcescens

The 1.0 kb fragment carrying a gentamicin resistance determinant was subcloned from the R plasmid in clinical isolates of Serratia marcescens and its nucleotide sequence was determined. It contains an open reading frame composed of 858 nucleotides, which shows 97% homology to the aacC2 gene in nucleotide sequence. Minicells carrying the 1.0 kb insert DNA on the plasmids produced a 30 kd protein that was consistent with the expected size of the deduced amino acid sequence from the open reading frame. A search for the promoter revealed a putative promoter of which the −35 region was provided by the right end of Tn3, suggesting that the expression of the gentamicin resistance gene is dependent on the Tn3 sequence.

Development of a specific probe for the aminoglycoside-(3)-N-acetyltransferase resistance gene.

Aminoglycoside-(3)-N-acetyltransferase isoenzyme V (AAC(3)V) is produced by > 90% of aminoglycoside-resistant Escherichia coli isolates from Hong Kong. A gentamicin resistance determinant from a conjugative plasmid carried by one of these E. coli strains producing AAC(3)V was cloned as a 6-kb fragment in the PstI site of the Bluescript II KS-phagemid vector. A 0.8-kb SphI-SalI fragment from the cloned insert was used as a probe in an epidemiological study. The specificity of this probe was evaluated by colony hybridization with 17 control strains producing different known aminoglycoside resistance enzymes. Hybridization was observed only with strains producing AAC(3)V. The probe demonstrated the presence of the AAC(3)V gene in 95.5% to 133 gentamicin-resistant E. coli isolates. This result agreed with the earlier data on the distribution of this enzyme that were based on the susceptibility profile.

Molecular characterization of highly gentamicin-resistant Enterococcus faecalis isolates lacking high-level streptomycin resistance.

Antimicrobial susceptibilities and DNA contents were analyzed for six clinical isolates of Enterococcus faecalis that had high-level resistance to gentamicin (MIC > 2,000 micrograms/ml) but not streptomycin and were obtained from patients in diverse geographic areas. Contour-clamped homogeneous electric field electrophoresis of genomic DNA showed all isolates to be different strains. Gentamicin resistance was transferred from four isolates to plasmid-free enterococcal recipients in filter matings. Restriction enzyme analysis of transconjugants showed distinct gentamicin resistance plasmids. A probe specific for the gentamicin resistance determinant hybridized to the plasmids of four isolates and to the chromosomes of two isolates. These findings suggest that clonal dissemination is not responsible for the spread of these resistant strains, that resistance determinants occur on different plasmids as well as on the chromosome of E. faecalis, and that the genetic determinants of resistance are related.

Nucleotide sequence of the aacC3 gene, a gentamicin resistance determinant encoding aminoglycoside-(3)-N-acetyltransferase III expressed in Pseudomonas aeruginosa but not in Escherichia coli

A chromosomal gentamicin resistance determinant from Pseudomonas aeruginosa was cloned on a 2.4-kb fragment in the broad-host-range vector pLAFR3. Substrate profiles for eight aminoglycosides at three concentrations showed that resistance was due to aminoglycoside-(3)-N-acetyltransferase III. This enzyme was produced in Pseudomonas strains but not in an Escherichia coli strain bearing the aacC3 gene. Nucleotide sequencing revealed two contiguous open reading frames (ORFs) preceded by a potential promoter and a ribosome-binding site. ORF-1 was 642 bp in length and encoded a protein of unknown function with a molecular mass of 23.9 kDa. ORF-2 was 813 bp in length and encoded a protein of 29.6 kDa. From deletion mutagenesis, in vitro transcription-translation data, and protein analysis of bacterial lysates, it was inferred that this 29.6-kDa protein represents the aminoglycoside-(3)-N-acetyltransferase III enzyme. A polymerase chain reaction with two specific intragenic 20-mer primers was developed to detect the aacC3 gene. A BstEII restriction site in the amplified DNA region was used to demonstrate the specificity of the reaction. Tests of 23 reference strains, which produced 12 different aminoglycoside-modifying enzymes, confirmed the specificities of the primers. The gene proved to be absent from a collection of 50 gentamicin-resistant P. aeruginosa strains selected at random in The Netherlands.

High-level chromosomal gentamicin resistance in Streptococcus agalactiae (group B)

This is the first report of high-level gentamicin resistance in a group B streptococcus. Strain B128 of serotype II was isolated from an infected leg wound in 1987. B128 was resistant to high levels of gentamicin as well as of all other available aminoglycosides and was also resistant to tetracyclines. No bactericidal synergism was found between ampicillin or vancomycin and any of these aminoglycosides. Gentamicin, kanamycin, streptomycin, and tetracycline resistance determinants transferred by conjugation into a plasmid-free group B streptococcus recipient at a frequency of 10(-8) to 10(-9) transconjugants per donor cell. No transconjugants were detected when streptococci of groups A, C, and G, Streptococcus sanguis, or Enterococcus faecalis was used as a recipient. No plasmids were detected in B128 or in any of the four transconjugants tested. By DNA-DNA hybridization, homology was detected between gene aac6/aph2, of E. faecalis origin, and a 2.4-kilobase HindIII chromosomal fragment of B128; homology to the genes aph3 and aadE, of E. faecalis origin, was found with HindIII chromosomal fragments of the same size (3.0 kilobases). Strains like B128, which potentially can be responsible for severe neonatal infections, are of great clinical concern, since there are to date no antibiotic combinations exhibiting bactericidal synergism against them.

Nucleotide sequence of the aacC2 gene, a gentamicin resistance determinant involved in a hospital epidemic of multiply resistant members of the family Enterobacteriaceae

A gentamicin resistance determinant of a conjugative plasmid from Enterobacter cloacae was cloned on a 3.2-kilobase fragment in the PstI site of pBR322. Substrate profiles for eight aminoglycosides at three concentrations showed that the resistance was due to aminoglycoside-(3)-N-acetyltransferase isoenzyme II. Insertion mapping by the gamma-delta transposon revealed that the size of the gene was approximately 1 kilobase. Nucleotide sequencing of the aacC2 gene identified an open reading frame of 858 base pairs, preceded by a promoter and a ribosome-binding site. From these data the molecular mass of the protein was calculated to be 30.6 kilodaltons. A comparison of the nucleotide sequence of the aacC2 gene with those published for the aacC3 and aacC4 genes showed complete homology of the aacC2 gene and the presumed aacC3 gene. An internal restriction fragment of the gene used as a probe in colony hybridization demonstrated the presence of the aacC2 gene in 86% of 86 multiply resistant isolates of the family Enterobacteriaceae obtained during an 18-month hospital epidemic. This corroborates our earlier data on the enzyme identification by the susceptibility profiling method.

Structure and mobilization of an ampicillin and gentamicin resistance determinant.

A DNA segment originally found in an epidemic plasmid of Escherichia coli encoding an aminoglycoside-(3)-N-acetyltransferase gene (aacC5) and a TEM-type beta-lactamase gene was characterized. The two genes were adjacent and constituted a single transcriptional unit. In addition, these genes were simultaneously mobilized through the action of an insertion sequence related to IS26, IS140, and IS15-delta. This DNA segment is a composite transposon which has been called Tn2922.