identification of antibacterial resistance mechanisms:

ANNLDO 4(7) 57-68, 1987 ISSN 0738-1751 VOLUME 4, NUMBER 7, JULY 1987

EDITORIAL BOARD

Editor

DANIEL AMSTERDAM, PhD, State University of New York at Buffalo and Erie County Medical Center Buffalo, New York

Associate Editors

RONALD N. JONES, MD, Clinical Microbiology Institute Tualatin, Oregon

HAROLD C. NEU, MD, College of Physicians and Surgeons, Columbia University New York, New York

CLYDE THORNSBERRY, PhD, Center for Infectious Diseases Centers for Disease Control Atlanta, Georgia

LOWELL S. YOUNG, MD, Kuzell Institute for Arthritis and Infectious Diseases Medical Research Institute of San Francisco Pacific Presbyterian Medical Center San Francisco, California

EDITOR'S NOTE 57 D. AMSTERDAM

Identification of Antibacterial Resistance Mechanisms: Advances in Laboratory Assays 57

R. C. COOKSEY

L. W. MAYER

Antibiotics: Resistance Trends and Nontherapeutic Applications---Reports From the Literature 67

D. A M S T E R D A M

IDENTIFICATION OF ANTIBACTERIAL RESISTANCE MECHANISMS: A d v a n c e s in Laboratory A s s a y s

ROBERT C. COOKSEY L E O N A R D W. MAYER Antimicrobics Investigations Branch Hospital Infections Program and Molecular Biology Laboratory Division of Bacterial Diseases Centers for Disease Control Public Health Service Department of Health and Human Services Atlanta, Georgia In vitro susceptibility testing to antimicrobial agents is a generally straightforward approach to the control of bacterial infections. Sus-

ceptibility patterns offer obvious taxonomic, epidemiologic, and therapeutic benefits, and have become a familiar component of the ultimate profile of a particular bacterial pathogen. The techniques continue to improve in terms of interpretative standards and, as a result of automation, are becoming easier to perform. This improved state of affairs makes antibiograms the initial, and often the most important, step in characterizing anti-

EDITOR'S NOTE

The techniques described in this issue by Cooksey and Mayer, in "Identification of Antibacterial Re- sistance Mechanisms," are the same procedures that enabled Dr. Tom O'Brien and his Task Force to reach the conclusions summarized in their report on "Resistance of Bacteria to Antibacterial Agents" (see, "Reports from the Literature" in this issue). Drs. Cooksey and Mayer describe how convenient assays for the heterologous isoenzymes that are associated with both the aminoglycoside-modifying enzymes and the beta-lactamases can

be used as epidemiologic indicators to track the geographic spread of resistant strains. Moreover, they discuss the application of DNA probes to determine the potential dissemination of resistance markers associated with antibiotic-containing livestock feed to the human population. While DNA probes seem to be about the most elegant and intensive of methods for identifying resistance, it needs to be underscored that the microor- ganism probed may not phenotypi- cally express the particular resistance that can be detected by the target probe. In the authors' laboratory, the routine characterization of the beta-lactamases in-

dudes six tests, which, in addition to susceptibility testing, nitrocefin hydrolysis, and DNA probing, include isoelectric focusing, substrate profiling, and competitive substrate inhibition. How commonplace will some of these assays become in the diagnostic clinical microbiology laboratory? Nitrocefin hydrolysis and the chloramphenicol acetyltransferase (CAT) are routine. Clearly, as certain other procedures become more convenient, they must be considered for implemen- tation in the routine laboratory set- ting, as they will have a positive impact on therapeutic decisions and infection control procedures.

E L S E V I E R 0"8-1751/87/$0.00 + 2.20

58 THE ANTIMICROBIC NEWSLETTER, VOLUME 4, NUMBER 7, JULY 1987

bacterial resistance mechanisms. Adequate knowledge of the literature coupled to a properly determined resistance phenotype may be all that is required to correctly ascribe a mechanism to a particular organism. For example, one may safely assume (for the moment, anyway) that the mechanism un- derlying a spectinomycin minimal inhibitory concentration (MIC) of >2000 ~g/mL for a gonococcal isolate is a chromosomal mutation af- fecting 30S ribosomal protein(s), a so-called altered target mechanism of resistance. This same level of resistance for gentamicin in an en- terococcal isolate, however, would suggest a drug inactivation mechanism as a result of adenylation or phosphorylation of the compound by a plasmid-encoded gene product.

The goal of this article is not to describe the genetics or biochem- istry of resistance mechanisms nor to describe in detail the sophisticated methodology that pioneering basic scientists used in laying the foundations of our understanding of such mechanisms, but rather to offer an overview, with references, for techniques that may be useful for clinical and public health laboratories. Using the above ex- amples, assays for altered ribosomal proteins require purification and electrophoretic steps that are currently too cumbersome for such laboratories. Techniques for measuring modification of aminoglycosides, however, are becoming more convenient, and so are at least worth considering. DNA probes, as will be discussed in more detail below, have already been proven to be useful adjuncts to the latter assay and may offer a future shortcut in the characterization of altered ribosomal proteins as well. Resistance gene probes were recently reviewed by Ten- over. 105

Several obvious benefits are in- herent to the streamlined identification of resistance mechanisms. Especially for aminoglycoside-modifying enzymes (AMEs) and ~-lactamases (each with more than thirty heterologous isoenzymes), convenient assays for their identification could be used as routine epidemiologic typing tools. This ability, as exemplified below, may be useful in tracking the geographic spread of resistant strains. Con- cerned with the impact of antibiotics in livestock feed on the dissemination of resistant organisms to the human population, Marshall et a171 used DNA probes to survey tetracycline resistance mechanisms in lactose-positive coliforms. The finding that most (73.3%) of the 225 isolates tested harbored a class B tetracycline effiux mechanism, one that is encoded on a promiscuous transposon capable of relatively fast horizontal transfer, provided strong evidence regarding the impact of nonhuman reservoirs on resistance problems. 71 Conventional methods to identify the class B mechanism on a similarly large sample of isolates, eg, by characterizing specific efflux proteins, would most likely have been so ex- pensive and time-consuming that the study would have been unfea- sible. So, we extended Marshall's resistance epidemiology to human urine isolates of Escherichia coli, in which class B tetracycline resistance also predominates. 3°

Monitoring specific mechanisms may also aid therapeutic decisions, eg, those made by hospital infection control committees. Although 3'-aminoglycoside phosphorylating enzymes [APH(3')] do not recog- nize tobramycin, gentamicin, or netilmicin as substrates, an increase in isoenzymes of 3'-phosphotransferase that modify amikacin, such as those produced by some coagu-

lase-negative staphylococci, might sound the alert to hold this compound in even tighter reserve.

A third potential benefit bestowed by the ability to at least screen resistance mechanisms is the early-on recognition of novel mechanisms. Nonexistent ex- amples (now and, we hope, for- ever) include plasmid-borne gonococcal genes that encode a spectinomycin-inactivating enzyme. Pressure for the emergence of such a malady is great, considering the widespread use of this compound in areas where penicil- linase-producing gonococci are en- demic. Laboratories in these areas perhaps should be prepared not only to perform MIC testing but also AME screens. The detection of a suspected novel mechanism should be followed up, however, by more in-depth genetic and bio- chemical characterizations, such as molecular weight and kinetic studies for inactivating enzymes.

AMINOGLYCOSIDES

To exert their bactericidal effects at the bacterial ribosome, aminoglycosides (AGs) must move into the cell through a multiphasic pro- cess. 18,82 As with other classes, resistance to AGs therefore may be the result of exclusion or impermeability. This may be an intrinsic mechanism, as in anaerobic bacteria that lack an energized membrane, 17 or it may be a mutation, as is most often observed in resistant pseudomonads lacking AMEs. Such strains generally exhibit cross-resistance to a broader range of AGs than is bestowed by AMEs and at l ower MICs. 16,19,37,53,99 In addition, permeability mutants usually exhibit diminished colonial mor- phology on agar surfaces and fail to grow on a minimal medium containing sodium succinate. 36 De- creased uptake or surface binding

The Antimicrobic Newsletter (ISSN 0738-1751) is issued monthly in one indexed volume per year by Elsevier Science Publishing Co., Inc., 52 Vanderbilt Avenue, New York, NY 10017. Printed in USA at (25 Sand Creek Rd., Albany, NY 12205). Subscription price per year: $55.00. For air mail to Europe, add $21.00; for air mail elsewhere, add $24.00. Second-class postage pending at New York, NY, and at additional mailing offices. Postmaster: Send address changes to The Antimicrobic Newsletter, Elsevier Science Publishing Co., Inc., 52 Vanderbilt Avenue, New York, NY 10017.

© 1987 BY ELSEVIER SCIENCE PUBLISHING CO., INC. 0738-1751/87/$0.00 + 2.20

THE ANTIMICROBIC NEWSLI~I ikK, VOLUME 4, NUMBER 7, JULY 1987

of isotopically labeled aminoglycosides (as measured by increased radioactivity in spent media as compared to controls) may provide the necessary confirmation of the above phenotypic tests for permeability mutants.

Enzymatic modification is the most problematic mechanism of resistance to aminoglycosides, especially among nosocomially acquired bacteria.19 As with f~-lactamases, there are currently more than 30 known isoenzymes of AMEs; but, unlike the former, there are no rapid qualitative tests for their detection. Disk inactivation tests (as have been described for ~-lactamase; see Fig 1) have the potential for AME detection, but have not been widely used, possibly because lysis procedures, such as sonication or enzymatic cell wall degradation, are required (personal observa- tion). In one disk assay, cellular lysates are combined with various AGs and necessary cofactors and precursors, incubated and spotted to 6-mm paper disks, which are then placed on a lawn of a sensitive (indicator) organism such as Sarcina lutea strain ATCC9341. A zone with inhibition that appears decreased when compared to a control disk that contains no lysate suggests inactivation of a particular AG. An alternate technique, which in our (RCC) laboratory is less sensitive, is to place a disk with the

above reaction mixture, but without the AG, near a standard AG-containing disk on a lawn of the indicator strain. In this instance, if inactivation occurs, the zone of inhibition is truncated only between the two disks. The most convenient AME screen is the proper in- terpretation of susceptibility data, as was first described in 1978 by Drasar. 4° Shimuzi et a199 have described a similar phenotypic test to classify acetyltransferases and nucleotidyl transferases and to detect permeability mutants. One limita- tion of their scheme, however, is that it requires less available compounds, such as 6'-netilmicin. Disk susceptibility testing to classify AMEs was described by van de Klundert et a1112 using six aminoglycoside disks (gentamicin, siso- micin, tobramycin, kanamycin, netilmicin, and neomycin). Using a stepwise (flow chart) cluster analysis, they were able to detect up to 13 of the more common AMEs correctly in 97% of clinical strains evaluated. H2 The range of enzymes might have been extended (depending on species) to as many as 17 by including streptomycin and spectinomycin disks to screen for enzymes that acetylate or ade- nylate these drugs. All of the above assays are useful only for enzymes having aminoglycoside substrates, the activity of which are lowered as a result of modifica-

FIGURE 1. Inactivation of ~-lactam compounds by TEM (Class III) ~-lactamase. Indicator strain was Sarcina lutea ATCC9341 and dilutions are TEM ~-lactamase sonicates. Drug disks contained 10 p,g of penicillin ("P10") and cephaloridine ("CD10"). Extent of inactivation (eg, truncation of zone of inhibition around drug disk) was directly porportional to dilution of enzyme.

59

tion. Multiple enzymes that share the same AG substrate are difficult to discern with susceptibility and disk screens. In our laboratory, susceptibility screens are routinely followed up by the phosphocellulose paper-binding assay (PBA), which will detect modification and the presence of >1 AME class. 9,35,51 Although this assay requires radioisotopes and relatively complex methodology, it is quantitative and perhaps the most reliable means to characterize AMEs. The technique requires cell-free lysates of the organism (usually prepared by sonication or osmotic shock with or without a required pretreatment to degrade the cell wall), which are combined with a battery of AG- substrates, cofactors, and radiola- beled precursors. These reactions are then spotted to phosphocellulose, which electrostatically bind the negatively charged AGs. Scin- tillation counts on the paper will be higher (~5 times a control containing no AG is the interpretative standard we use) if an enzyme capable of transferring an isotopic sidegroup to the AG is present. We 31 have recently modified the performance of this assay by the use of microdilution plates and multichannel pipeting and by de- scribing a means to screen for all three AME classes by testing only the minimum number of substrates necessary to detect all enzymes within a class. Another method, recently described by Loevering et al, 6a is the measurement of AG acetylase and phosphotransferase reaction products using high performance liquid chromatography (HPLC). If an HPLC apparatus is accessible, this assay may be more convenient to implement than the PBA, especially since there are no isotope requirements. HPLC may offer the additional advantage, as shown by the Loevering study, of being more sensitive in detecting the presence of multiple enzymes within a given class, which is cer- tainly important when testing nos- ocomial isolates.

-738-1751/87/$0.00 + 2.20 © 1987 BY ELSEVIER SCIENCE PUBLISHING CO., INC.

6O

As with other drug classes, nucleic acid probes for AME-encoding genes have boosted the capabilities in both tracking AMEs among clinical bacteria and in localizing their genes, for example, to potentially promiscuous plasmids. Table 1 shows subclasses of AMEs for which probes have been described (at the time of writing). The list includes therapeutically important staphylococcal enzymes as well as four important Gram-negative types. Others are under development, but none are commercially available yet. Unless an interested research group has a source of a probe in ready-to-use form, they must be prepared to isolate restriction endonuclease fragments from appropriate plasmids, usually isolated from cesium-chloride/ ethidium bromide centrifugal gradients.

By whatever means a restriction fragment is obtained, it must be labeled with a detectable tag in order to become a probe. The most common isotopic label is 32p, even though it is unstable (13-day half- life) and a rather dangerous emitter of beta radiation. Nucleotides labeled with 32p or other detectable groups, such as biotin or antigens, are most often incorporated into the duplex linear restriction fragment using convenient nick translation reaction kits. As nonradioactive labels, biotinylated nucleotides are convenient to use and detect with commercial kits. Background problems most likely associated with detection proteins (eg, strepavidin) may soon be eliminated. Another intriguing DNA-labeling kit enables immune detection of antigenic sulfone groups incorporated into the probe molecule (ChemiProbe; Orgenics, Ltd., Yavne, Israel) and may be useful in detecting resistance genes. Target sequences are most often bound to filters, the most popular being nylon and nitrocel- lulose. Thirty or more isolates may be examined by dot hybridization on a filter that is no more than 10

THE ANTIMICROBIC NEWSLETTER, VOLUME 4, NUMBER 7, JULY 1987

cm 2. These may be crude lysates obtained by growing cells at "spots" on the filter in contact with an a'gar plate surface and lysing them by alkaline treatment. 81 We have found, however, that more highly purified preparations of nucleic acids, such as Birnboim-Doly plasmid lysates for Gram-negatives or Goering-Ruff preps for staphylococci, deproteinized by either phenol or some other appropriate treatment, yield more interpretable results in dot blotting. 11,46 By whatever methods used, before in- cubation both probe and target sequences must be denatured to single strands, eg, by boiling. After electrophoresis, plasmids and chromosomes may likewise be blotted to filters using either capil- lary or electrophoretic blotting techniques and hybridized with resistance probes to localize homolo- gous sequences. Hybridization with a resistance or any other kind of probe is useful as an epidemiologic or typing screen, but does not conclusively prove the presence of a functional gene. Even among sensitive isolates, homology may be occasionally observed, depending upon hybridization stringency, the presence of extraneous probe sequences, defective yet conserved target regions, and so on. Con- trols, both positive (unit gene copy) and negative, are essential, since results are rarely "black or white" and require cautious interpreta- tion. Laboratorians interested in implementing probe technology should consult manuals or reviews, such as those by Maniatis et al, 7° Zwadyk and Cooksey, TM Meinkoth and Wahl, 77 or Minson and Darby. 79

[~-LACTAMS Just as ~-lactams (penicillins and cephalosporins) are the largest and most widely used antimicrobial class, ~-lactamase inactivation is perhaps the most widespread, problematic, and therefore studied mechanism of resistance. Al- though more than 30 isoenzymes of

f~-lactamase have been described, only a few are responsible for most of the clinical resistance to ~- lactams. Unlike AMEs, which modify a variety of aminoglycoside molecular constituents, and which have rather complex requirements for in vitro detection, ~-lactamases share a common site of action, the ~-lactam ring, and lend themselves to convenient qualitative detection. Two excellent ~-lactamase methodology reviews are those by Neu 84 and by Sykes. TM Despite the usefulness of earlier methods described in these reviews and elsewhere, including the iodomet- ric; 34,6° acidimetric, 13 and hydrox- ylamine 95 tests, the most popular assay currently is the nitrocefin chromogenic cephalosporin test available in disk form from BBL Microbiology Systems. 86 Although all ~-lactamases are detectable with this assay, some may need to be induced (producing organism is grown in the presence of other ~- lactam compounds), as is the case with some staphylococcal penicil- linases and most type I Gram-negative cephalosporinases. At least one other ~-lactamase, ROB-1 (found in Haemophilus influenzae), is constitutive, but it is nonetheless slower in its action upon nitrocefin. Other chromogenic compounds are available that accurately detect some types of ~-lactamase, but may give false negative results with other types.

Among schemes to classify ~-lactamases, the most universal is that described by Richmond and Sykes 93 and currently contains six classes, separated essentially on the basis of substrate profiles and enzyme inhibitors. 84 As for the aminoglycosides, ~-lactamases need not be highly purified to measure rates of substrate hydrolysis. For most Gram-negative organisms, crude cellular sonicates may be used and additional purification or concentration, where needed, has become easier with prepacked filtration columns, concentrators, and the like. Where accurate kinetic data


THE ANTIMICROBIC NEWSLETI'ER, VOLUME 4, NUMBER 7, JULY 1987 61

T A B L E 1. Antibacterial Resistance Gene Probes

Mechanism or Plasmid Drug class encoding gene Organisms example(s) References

Aminoglycosides Modifying enzymes 4'-Adenyltransferase I Staphylococci pUBH2 38,74 3'-Phosphotransferase IV Staphylococci pAT48 38,47 3'-Phosphotransferase I Enterics pA043 88 3'-Phosphotransferase II Enterics pSAY16 113 2"-Phosphotransferase-

6'-acetyltransferase Gram + Cocci pi l l3 38,42 2"-Adenyltransferase Gram - rods pFCT3103 106 3"-Adenyltransferase Enterics pHP45, pGB2 91,28

~-Lactamases TEM Gram - rods and cocci pBR322 a 12,29,65 OXA-1 Gram - rods pMON30P 65 OXA-2 Gram - rods pMON20 ~ 65 ROB-1 Gram - rods pMON401 65 PSE-1 Gram - rods pMON810 65 PSE-2 Gram - rods pMON230 65 PSE-4 Gram - rods pMON705 65 AmpC Gram - rods pNU81* 59 CARB-3 Gram - rods pMON41 65 CARB-4 Gram - rods pMON1025 65 AER-1 Gram - rods pMON510 65 SA1 Staphylococci pii147 85

Ribosome modification ermA Staphylococci pEM9503 83 ermB Staphylococci pi258 85 ermC Staphylococci pE194 100 erred Bacillus pBD90 49 MLS Streptococci pDL315 94 ermAM Enterics piP1527 3 erxA E. coli pAT75 4

inactivation ereA Enterics pAT63 90,4 ereB Enterics pAT72 4

Efflux/impermeability Class A Enterics pSL107 71 Class B Enterics pKT007 55 Class C Enterics pBR322 71 Class D Enterics pSL106 71 Class E Aeromonas pSL1504 72 Bacteroides Bacteroides pDP3 50 Class L Streptococci pVBA15 21 Class M Gram + cocci, gonococci pI13 21

Acetyl transferase CAT I Gram - rods pBR325, pACYC184 6,25 CAT C Staphylococci pC221 14 Active site CAT Gram + & - a 10

Dihydrofolate reductase DHFR I 20 DHFR II 20 DHFR III 44 DHFR SA1 32

[3-Lactams

Macrolides

Tetracycline

Chloramphenicol

Trimethoprim Gram - rods pFE872 Gram - rods pFE700 Gram - rods pFE1242 Gram - rods pG018

a Synthetic oligomers also described.

0738-1751/87/$0.00 + 2.20 © 1987 BY ELSEVIER SCIENCE PUBLISHING CO., INC.


(eg, K m or V ~ ) are required, substrate profiles are routinely per- formed either acidimetrically 62 or spectrophotometrically, 87 the latter being more popular because of greater sensitivity. The introduc- tion of sophisticated spectropho- tometers that offer enzyme kinetics enables quick determinations of reaction rates. These rates should be obtained over a range of substrate concentrations 23 from which K m and Vma × values may now be more conveniently calculated with the aid of computer software. Qualita- tive substrate profiles may be obtained using either the cloverleaf method of McGhie et a176 or a disk substrate method originally described by Masuda et al, 73 which is similar to that described above for aminoglycosides. We have modified the latter technique by spotting sonicates of [3-1actamase-producing organisms to paper disks and placing them at a distance approximately equal to the normal inhibition radius of the substrate [3-1actam disk on freshly seeded MueUer-Hinton agar overlays of S. lutea (inoculum is approximately 5 x 105 cfu/mL). Up to four soni- cate-impregnated disks may be equally spaced around a drug disk, and, using 245-cm 2 plastic tissue culture plates (Vangard Interna- tional), the profiles of four soni- cated organisms against 10 or more substrates may be determined (Fig 1). Susceptibility to inhibitors, for example clavulanic acid or p-chloro- mercuribenzoate, simply requires the additional step of preincubating enzyme preps with inhibitor before assaying substrate hydrolysis.

Within our laboratory, the routine characterization of [3-1acta- mases includes six tests: susceptibility testing, nitrocefin hydrolysis, isoelectric focusing, substrate profiling, competitive substrate inhibition, and DNA probing. All are rarely required, especially when the enzyme is one common for a given species. Following susceptibility and nitrocefin testing, the enzyme's net charge

(pI) is usually determined before substrate profiling. Within each Richmond-Sykes class, the pI is regarded as the most definitive character of an enzyme, since isoenzymes do not share the same pI. Isoelectric focusing to evaluate pI's of ~-lactamases was originally described by Matthew et al 7s and has evolved primarily through advances in electrophoresis equip- ment and commercial precast acrylamide gels containing ampho- lytes for measuring pIs in various ranges. We routinely perform horizontal electrofocusing in a range of pH 3.5 to 9.5, using sonicates that occasionally require desalting and/ or concentration. Using a surface electrode, pH measurements are recorded at 1-cm intervals and the location of the ~-lactamase is determined by overlaying the gel with agarose containing nitrocefin, as recently described by Sanders et a l . 96 When multiple (satelliting) bands appear, pI(s) of the most prominent is (are) recorded, and where warranted, the preps are re- focused on narrower-range gels. Results of isoelectric focusing are usually followed by competitive substrate inhibition profiling, described by Gerlach et al in 1983. 45 Lysates are first titered to obtain the most dilute enzyme concentration capable of hydrolyzing nitrocefin. Fifty microliters of this diluted lysate is then combined in microdilution plate wells with se- lected ~-lactams, followed by nitrocefin (50 ~L of both components usually in stock concentrations of 400 ~g/mL). Depending on which ~-lactams competitively inhibit hydrolysis (indicated by yellow color persisting in the well), a profile is established that correlates with enzyme type. A refined variation of this method, the relative substrate affinity index, was described by Eliasson and Kamme in 198541 and is a quantitative assay in which 50% inhibition of nitrocefin hydrolysis is measured spectrophotometrically.

Several [3-1actamase gene probes

have been described, as have their applications to screening of f~-lactamase distribution in epidemiologic studies. 12,29,61,65,66,89,1°1 Three of these probes for ~qactamase types --OXA-1, OXA-2, and TEM-1-- represent the first synthetic nucleotidyl oligomers described for use in studies of resistance gene distribution. 12,89 Synthetic probes for other classes, most notably class I cephalosporinases (eg, E. coli ampC gene product), should be available soon, especially considering the advanced knowledge regarding the organization of their coding regions. Results from [3- lactamase gene probing of dot blots are currently considered tentative, subject to confirmation by one or more of the other tests, usually pI determinations.

Evidence is strong that high-level (intrinsic) methicillin resistance in staphylococci is mediated by an altered target mechanism, given that a novel penicillin-binding protein (PBP 2a or 2') with low methicillin affinity is produced in resistant strains. 11°,1n Electrophoretic assays to detect this protein, as reviewed by Malouin and Bryan 69 and by Chambers, 24 may be too cumbersome for routine performance in clinical laboratories. The most promising assays to detect methicillin-resistant staphylococci diag- nostically include antibodies (polyclonal or monoclonal) to PBP 2a or a nucleic acid probe for its encoding nucleotidyl sequences. Although advances in laboratory assays for staphylococcal ~-lactamases have, for the most part, not kept up with those for Gram-negative enzymes, improved procedures for extracting and characterizing these enzymes should provide a better understanding of the role that [3-1actamase plays in borderline methicillin resistance. Phenotypic testing, recommendations for performance of which were reviewed by Thornsberry, 1°9 remain the best tool for identifying methicillin-resistant staphylococci in clinical laboratories.


THE ANTIMICROBIC NEWSLETTER, VOLUME 4, NUMBER 7, JULY 1987 63

MACROLIDES Erythromycin, which is one of the macrolide-lincosamide-streptogramin type B, or MLS, group of antimi- crobials, is one of the most widely used antibacterials worldwide. 27 At least partially related to this use may be the diversity of genetic de- terminants that code for the same MLS resistance mechanism, N6-di - methylation of 23S ribosomal RNA in the 50S ribosomal subunit. 33 Three of these genes, ermA, ermB, and ermC, occur in staphylococci and are best identified with restriction fragment probes (Table 1). 1°7 Because of sequence conservation, however, hybridization and subse- quent filter washes should be per- formed under high stringency (eg, salt and formamide concentrations and hybridization temperature equivalent to 10°C below the melting temperature of strands ho- mologous to the probe and washes at 65°C). Even at high stringency, some subjectivity in the interpreta- tion of the autoradiographs may be unavoidable. Additional evidence for the presence of a functional staphylococcal ermA or ermC gene is readily obtainable through an antimicrobial disk induction assay. Since lincosamide resistance is inducible in such strains, demonstra- tion of a truncated zone of inhibition around a clindamycin disk placed approximately 8 mm from an erythromycin disk on a seeded Mueller-Hinton plate suggests the presence of at least one of these loci. A fourth methylase gene, erreD, originated in Bacillus licheniformis and bears no homology with the other three. 49 Among some species of streptococci, such as S. pneumoniae, S. faecalis, S. pyo- genes, and S. sanguis, erm deter- minants have been found to be highly related genetically not only intragenerically but also with the staphylococcal ermB locus, which suggests either extensive genetic exchange between members of these genera or a high degree of gene conservation.

In addition to altered target re-

sistance, impermeability and drug inactivation genes have been recently demonstrated on plasmids in coagulase-negative staphylococci and show promise as future probes. 63,64 In E. coli the production of erythromycin esterase was mapped to the ereA gene, which has already proven useful as a probe to study the distribution of this mechanism among erythromycin-resistant enteric organisms. 3,8,9° A convenient cross-streak test, described by Andremont to detect erythromycin- inactivating enzymes in E. coli should be applicable to screening other genera as well. 2 Since the description of ereA, heterogenous loci in E. coli encoding macrolide inactivation, ie, ereB, and ribosomal modification, ie, erxA, have been investigated. 4 With the addition of these, an extensive battery of macrolide resistance gene probes should soon be available.

CHLORAMPHENICOL

Chloramphenicol resistance is common in several groups of bacteria, including the Enterobacteria- ceae, Staphylococcus sp, H. influenzae, and others.I°2 Most chloramphenicol-resistant bacteria elaborate chloramphenicol acetyl-transferase (CAT), an enzyme that inactivates the antibiotic.

The CAT enzymes from various bacteria share enough properties to be referred to as the family of CAT enzymes, which has more than 15 variants. 98 Most of the CAT proteins have monomer molecular weights of 22,500, but exist in the cytoplasm as a tetramer. Their classification into the various types has been based on molecular weight, isoelectric point, relative binding of chloramphenicol, inhibitor studies, inducibility, and im- munochemical reactivity. The major division between the types has generally followed whether their source was a Gram-positive or -negative organism. Active hybrid tetramers can be made by mixing monomers of types I, II, and III

(Gram-negatives) with each other, or similarly by mixing monomers of types A through D (Gram-posi- tives), but not by mixing between the groups. 98

CAT-producing bacteria generally have higher MICs than those that are resistant by other mechanisms. Those bacteria that produce type I CAT (often plasmid-encoded and seen in enterics) have the highest MICs. Colonies of CAT-producing bacteria bind rosanilin (and pararo- sanilin) dyes. 92 The amount of staining indicates the relative amount of CAT produced, and may suggest a high or low copy number plasmid. Since the mechanism of resistance is by inactivation of the antibiotic, CAT can be demonstrated by cross-streaking CAT- producing organisms with a highly sensitive organism, such as S. lutea, on media that contain chloramphenicol.

A simple colorimetric assay for CAT has been described and a kit is commercially available. 5 Due to the ease of detecting enzymatic activity, the CAT gene is used as a selective marker in several cloning vectors, and in expression studies in a variety of cells.

Both the DNA sequence of the CAT gene,1 and the amino acid sequence of the protein 97 have been completely determined for the type I CAT. The complete DNA sequences have been determined for the CAT genes of staphylococcal plasmids pC22114 and pC194, s6,57 as well as CAT genes of Proteus mirabilis and Bacillus pumilis. 26,54 Partial amino acid sequences have been determined for some other CAT proteins. 43,98 The N-terminal amino acid sequences show a high degree of homology, with near identity when comparing Staphylo- coccus aureus types A through D. Comparison of Gram-positive and -negative N-terminal sequences shows slightly less homology, but many of the differences could be caused by only a single base change. There is also a high degree of homology at the active site


64

of the protein, with seven of eight amino acids identical, when comparing the type I and type C sequences. 43 This active site homology has been exploited by Shaw's group to construct a synthetic oligonucleotide specific for the active site region (Table 1). This oligonucleotide probe has been used to clone the gene for a chromosomal gene for CAT from Flavobacterium. lo

Other mechanisms besides CAT have been investigated, including an often plasmid-mediated de- crease in uptake seen in Pseudo- monas aeruginosa and H. influenzae. 22 These include the transposons Tn169622 and Tn2001.58 The DNA sequence of a gene with similar function from plasmid R26 has been completely determined. 39 It is not known if these are related genes.

Although a wide variety of potential probes, including cloned fragments and synthetic oligonucleotides, are available, the ease and rapidity of the kit test for CAT activity has made this test the most widely used when studying the mechanism of chloramphenicol resistance.

TETRACYCLINE

While all the details of the mechanism of resistance to tetracyclines (Tc) are not known, some general- izations can be made. Low level resistance can be due to ribosomal mutation, porin deficiencies (such as ompF mutants), or mutations af- fecting lipopolysaccharide structure. 67 High level Tc resistance is often plasmid encoded and the result of lower intracellular concentration either through decreased uptake or by effiux of the drug. 67

Typing of Tc resistance deter- minants is based on relative susceptibility to Tc analogs and DNA hybridization. 78 Most Tc resistance is inducible, but class B confers constitutive Tc resistance in Haemo- philus while being inducible in various enteric organisms tested. The types of Tc resistance deter-

THE ANTIMICROBIC NEWSLETTER, VOLUME 4, NUMBER 7, JULY 1987

minants (Table 1) have been di- vided according to their source from Gram-negative (type A through E), or Gram-positive (type L and M), but Neisseria gonorrhoeae have been observed with the class M determinant originally seen in streptococci. 8°

SULFONAMIDES AND TRIMETHOPRIM

Almost every mechanism of resistance ever proposed for any antibiotic has been proposed as a mechanism of resistance to sulfonamides (Su) and trimethoprim (Tp) (for review, see reference 103). Very few of these possible mechanisms have been well studied. Re- sistance among Pseudomonads is due to poor intracellular penetra- tion of the compound. Neisseria sp and anaerobes are resistant to Tp because they lack a sensitive target (dihydrofolate reductase or DHFR) for drug action. Most other ex- amples of resistance to Su or Tp are acquired resistance, s2

The Su are para-amino-benzoic acid (PABA) analogs, and inhibit dihydropteroate synthetase (DPS). An altered (insensitive) DPS, which can be chromosomal or plasmid- mediated, is the most common type of resistance. Over-production of PABA has also been observed in Su-resistant bacteria. Other mechanisms have also been seen, including two or more mechanisms at the same time. 108 Al- though potential DNA probes exist, the widespread resistance to Su has made their use (alone) undesirable, and as a result there has been lim- ited research on Su resistance.

Trimethroprim inhibits DHFR. Mutations that give an insensitive enzyme can occur in the chromosomal genes. Plasmid-mediated insensitive DHFRs occur on plasmids of many incompatibility groups and in many species of bacteria. 7,48,1°8 Types of DHFRs, originally distinguished by their different isoelectric points, 15 which have DNA probes available 2°,44 are listed in Table 1. An additional

type observed in Sri Lanka which is not related to types ! through lII has been described. 1°3

REFERENCES

1. Alton NK et al: Nucleotide sequence analysis of the chloramphenicol resistance transposon Tn9. Nature (Lond) 282:864-869, 1979.

2. Andremont A et al: Plasmid-mediated high-level resistance to erythromycin in Escherichia coli. Antimicrob Agents Chemother 29:515-518, 1986.

3. Arthur Met al: Distribution of erythromycin esterase and rRNA methylase genes in members of the family Enterobacteriaceae highly resistant to erythromycin. Antimi- crob Agents Chemother 31:404- 409, 1987.

4. Arthur Met al: Contribution of two different mechanisms to erythromycin resistance in Esche- richia coli. Antimicrob Agents Chemother 30:694-700, 1986.

5. Azeman Pet al: Rapid detection of chloramphenicol resistance in Haemophilus influenzae. Antimicrob Agents Chemother 20:168-170, 1981.

6. Balbas Pet al: Plasmid vector pBR322 and its special purpose derivatives--a review. Gene 50:3-40, 1986.

7. Barth PT et al: Transposition of a deoxyribonucleic acid sequence encoding trimethoprim and streptomycin resistances from R483 to other replicons. J Bacteriol 125:800-810, 1976.

8. Barthelemy Pet al: Enzymatic hydrolysis of erythromycin by a strain of Escherichia coli: A new mechanism of resistance. J Anti- biot 37:1692-1696, 1984.

9. Benveniste R et al: R-Factor mediated gentamicin resistance: A new enzyme which modifies aminoglycoside antibiotics. FEBS Lett 14:293-296, 1971.

10. Beschle HG et al: Use of a synthetic oligonucleotide to identify a chromosomal gene for chloramphenicol acetyltransferase in a plasmid bearing flavobacterium. J Gen Microbiol 130:3335-3338, 1984.

11. Birnboim HC et al: A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res 7:1513- 1523, 1979.

© 1987 BY ELSEVIER SCIENCE PUBLISHING CO., INC, 0738-1751/87/$0.00 + 2.2o

THE ANTIMICROB1C NEWSLETTER, VOLUME 4, NUMBER 7, JULY 1987 65

12. Boissinot M et al: Development of natural and synthetic probes for OXA-2 and TEM-1 13-1actamases. Antimicrob Agents Chemother 31:728-734, 1987.

13. Boxer GE et al: Colorimetric determination of benzyl penicillin. Anal Chem 21:670-673, 1949.

14. Brenner DG et al: The use of synthetic oligonucleotides with universal templates for rapid DNA sequencing: Results with staphylococcal replicon pC221. Eur Mol Biol Organ J 4:561-568, 1985.

15. Broad DF et al: Classification of trimethoprim resistant dihydrofolate reductases mediated by R- plasmids using isoelectric focusing. Eur J Biochem 125:617- 622, 1982.

16. Bryan LE et al: Gentamicin resistance in clinical isolates of Pseudo- monas aeruginosa associated with diminished gentamicin accumula- tion and no detectable enzymatic modification. J Antibiot 29:743- 753, 1976.

17. Bryan LEet al: Mechanism of aminoglycoside antibiotic resistance in anaerobic bacteria. Anti- microb Agents Chemother 15:7-13, 1979.

18. Bryan LE et ah Gentamicin accu- mulation by sensitive strains of Escherichia coli and Pseudomonas aeruginosa. J Anfibiot 696-703, 1975.

19. Bryan LE: Aminoglycoside resistance. In: Bryan LE, ed. Antimi- crobial Drug Resistance. Orlando, FL, Academic Press, 1984, pp. 242-277.

20. Burchall JJ et al: Molecular mechanisms of resistance to trimethoprim. Rev Infect Dis 4:246-254, 1982.

21. Burdett V et al: Heterogeneity of tetracycline resistance deter- minants in Streptococcus. J Bacteriol 149:995-1004, 1982.

22. Burns JL et al: Cloning and expression in Escherichia coli of a gene encoding nonenzymatic chloramphenicol resistance from Pseudomonas aeruginosa. Antimi- crob Agents Chemother 29:445- 450, 1986.

23. Bush K et al: Methodology for the study of 13-1actamases. Antimicrob Agents Chemother 30:6-10, 1986.

24. Chambers HF: Understanding penicillin-binding proteins. Anti- microb Newsl 3:47-54, 1986.

25. Chang ACY et al: Construction and characterization of amplifiable multicopy DNA cloning vehicles

derived from the P15A cryptic miniplasmid. J Bacteriol 134:1141- 1146, 1978.

26. Charles IG et ah Nucleotide sequence analysis of the cat gene of Proteus mirabilis: Comparison with the Type I (Tn9) cat gene. J Bac- teriol 164:123-129, 1985.

27. Chin N et al: Activity of A-56268 compared with that of erythromycin and other oral agents against aerobic and anaerobic bacteria. Antimicrob Agents Che- mother 31:463-466, 1987.

28. Churchward G et al: A pSC101- derived plasmid which shows no sequence homology to other com- monly used cloning vectors. Gene 31:165-171, 1984.

29. Cooksey RC et al: A gene probe for TEM type ~-lactamases. Anti- microb Agents Chemother 28:154- 156, 1985.

30. Cooksey RC et ah DNA hybridization studies of resistance to tetracycline (Tc) and trimethoprim (Tmp) among ampicillin-resistant (Apr) Escherichia coll. Abstr Ann Meeting American Society for Mi- crobiology, 1987.

31. Cooksey RC et al: Microplate phosphocellulose binding assay for aminoglycoside-modifying enzymes. Antimicrob Agents Che- mother 30:883-887, 1986.

32. Coughter JP et al: Characterization of the staphylococcal trimethoprim resistance gene product. Abst Intersc Conf Antimicrob Agents Chemother, 1986.

33. Courvalin P e t al: Multiplicity of macrolide-lincosamide-streptogramin antibiotic resistance deter- minants. J Antimicrob Chemother 16:91-100, 1985.

34. Dale JW et al: The purification and properties of the ~-lactamase specified by the resistance factor R-1818 in Escherichia coli and Proteus mirabilis. Biochem J 123:493-500, 1971.

35. Davies J: Aminoglycoside-amino- cyclitol antibiotics and their modifying enzymes. In: Lorian V, ed. Antibiotics in Laboratory Medi- cine. Baltimore, Williams and Wilkins, 1986, pp. 790-809.

36. Davies J: Life among the aminoglycosides. ASM News 52:620-624, 1986.

37. Davies JE: Resistance to aminoglycosides. Mechanisms and frequency. Rev Infect Dis 5:$261- $267, 1983.

38. Dickgiesser N et ah Determination of aminoglycoside resistance in

Staphylococcus aureus by DNA hybridization. Antimicrob Agents Chemother 29:930-932, 1986.

39. Dormon CJ et al: Nucleotide sequence of the R26 chloramphenicol resistance determinant and identification of its gene product. Gene 41:349-353, 1986.

40. Drasar FA: Detection of aminoglycoside degrading enzymes. In: Reeves DS, Phillips I, Williams JD, Wise R, eds. Laboratory Methods in Antimicrobial Chemotherapy. Edinburgh, Scotland, Churchill Livingstone, 1978, pp. 70-75.

41. Eliasson I e t al: Characterization of the plasmid-mediated f~-lactamase in Branhamella catarrhalis with special reference to substrate affinity. J Antimicrob Chemother 15:139- 149, 1985.

42. Ferretti JJ et ah 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. J Bacteriol 167:631-638, 1986.

43. Fitton JE et al: Plasmids and the evolution of chloramphenicol resistance. In: Schlessinger D, ed. Microbiology 1978. Washington DC, American Society for Microbi- ology 1978, pp 249-252.

44. Fling ME et al: Monitoring of plasmid-encoded trimethoprim-resistant dihydrofolate-reductase genes: Detection of a new resistant enzyme. Antimicrobial Agents Chemother 22:882-888, 1982.

45. Gerlach EH et al: Distribution of f~-lactamase types among clinical isolates as determined by enzyme inhibition patterns. Abst Ann Meeting American Society for Mi- crobiology, 1983.

46. Goering RV et al: Comparative analysis of conjugative plasmids mediating gentamicin resistance in Staphylococcus aureus. Antimicrob Agents Chemother 24:450-452, 1983.

47. Gray GS et al: Evolution of antibiotic resistance genes: The DNA sequence of a kanamycin resistance gene from Staphylococcus aureus. Mol Biol Evol 1:57-66, 1983.

48. Grey D et al: Incidence and mechanisms of resistance to trimethoprim in dinically isolated Gram-negative bacteria. Chemo- therapy 25:147-156, 1979.

49. Gryczan TJ, Israeli-Reches M, Del



Bue M, et al: DNA sequence and regulation of erreD, macrolide-lincosamide-streptogramin B resistance element from Bacillus licheniformis. Mol Gen Genet 194:349-356, 1984.

50. Guiney DG Jr et al: Expression in Escherichia coli of cryptic tetracycline resistance genes from Bacter- oides R plasmids. Plasmid 11:248-252, 1984.

51. Haas MJ et al: Aminoglycoside- modifying enzymes. Methods En- zymol 43:611-628, 1975.

52. Hamilton-Miller JMT: Resistance to antibacterial agents acting on antifolate metabolism: In: Bryan LE, ed. Antimicrobial Drug Resis- tance. Orlando, FL, Academic Press, 1984, pp 173-190.

53. Hare RS et al: Mechanisms of aminoglycoside resistance. Anti- microb Newsl 1:77-84, 1984.

54. Harwood CR et al: Nucleotide sequence of a Bacillus pumilus gene specifying chloramphenicol acetyltransferase. Gene 24:163-169, 1983.

55. Hillen W, Schollmeier K: Nucleo- tide sequence of the Tn encoded tetracycline resistance gene. Nu- cleic Acids Res 11:525-539, 1983.

56. Horinouchi Se t al: Nucleotide sequence and map of pE194 a plasmid that specifies inducible resistance to macrolide, lincosamide, and streptogramin type B antibiotics. J Bacteriol 150:804-814, 1982.

57. Horinouchi Se t al: Nucleotide sequence and functional map of pC194, a plasmid that specifies inducible chloramphenicol resistance. J Bacteriol 150:815-825, 1982.

58. Iyobe Set al: Tn2001, a transposon encoding chloramphenicol resistance in Pseudomonas aeruginosa. Antimicrobial Agents Che- mother 29:445-450, 1986.

59. Jaurin Bet al: Sequence elements determining ampC promoter strength in E. coll. EMBO J 1:875- 881, 1982.

60. Jorgensen JH et al: Rapid penicil- linase strip test for detection of [3- lactamase producing Haemophilus influenzae and Neisseria gonorrhoeae. Antimicrob Agents Chemother 11:1087-1088, 1977.

61. Jouvenot M e t al: Molecular hybridization versus isoelectric focusing to determine TEM-type ~-lactamases in gram-negative bacteria. Antimicrob Agents Che- mother 31:300-305, 1987.

62. Labia R et al: Computerized mi- croacidimetric determination of a

[3-1actamase Michaelis-Menten constant. FEBS Lett 33:42-44, 1973.

63. Lampson BC et al: Novel mechanism for plasmid-mediated erythromycin resistance by pNE24 from Staphylococcus epidermidis streptogramin antibiotic resistance deter- minants. J Antimicrob Chemother 16:91-100, 1985.

64. Leclerq R et al: Plasmid-mediated resistance to lincomycin by inactivation in Staphylococcus haemoly- ticus. Antirnicrob Agents Chemother 28:421-424, 1985.

65. Levesque RC et al: Molecular cloning and DNA homology of plasmid-mediated [3-1actamases. Mol Gen Genet 206:252-258, 1987.

66. Levesque RC et al: Molecular cloning and construction of specific DNA probes for the SHV-1, PSE-2, and CARB-4 [3-1actamase genes. Abst Interscience Conf An- timicrob Agents Chemother, 1986.

67. Levy SB: Resistance to tetracyclines: In: Bryan LE, Ed. Antimi- crobial Drug Resistance. Orlando, FL, Academic Press, 1984, pp 191-240.

68. Loevering AM et al: Identification of individual aminoglycoside-inactivating enzymes in a mixture by HPLC determination of reaction products. J Antimicrob Chemother 18:139-144, 1986.

69. Malouin F et al: Modification of penicillin-binding proteins as mechanisms of [3-1actam resistance. Antimicrob Agents Che- mother 30:1-5, 1986.

70. Maniatis T et al: Molecular cloning: A laboratory manual. Cold Spring Harbor: Cold Spring Harbor Laboratory, 1982.

71. Marshall Bet al: Frequency of tetracycline resistance determinant classes among lactose-fermenting coliforms. Antimicrob Agents Chemother 24:835-840, 1983.

72. Marshall Bet al: A new tetracycline-resistance determinant, class E, isolated from Enterobacteria- ceae. Gene 50:111-117, 1986.

73. Masuda G e t al: Detection of 13- lactarnase production by gram- negative bacteria. J Antibiot 24:662-664, 1976.

74. Matsumura M e t al: Enzymatic and nucleotide sequence studies of a kanamycin-inactivating enzyme encoded by a plasmid from ther- mophilic bacilli in comparison with that encoded by plasmid pUB110. J Bacteriol 160:413-420, 1984.

75. Matthew M e t al: The use of ana- lytical isoelectric focusing for de-

tection and identification of [3-1actamases. J Gen Microbiol 88:169-178, 1975.

76. McGhie D et al: Detection of ~- lactamase activity of Haemophilus influenzae. J Clin Pathol 30:585-587, 1977.

77. Meinkoth Je t al: Hybridization of nucleic acids immobilized on solid supports. Anal Biochem 138:267- 284, 1984.

78. Mendez Bet al: Heterogeneity of tetracycline resistance deter- minants. Plasmid 3:99-108, 1980.

79. Minson AC et al: Hybridization techniques. In: Laboratory and Research Methods in Biology and Medicine. New York, AR Liss, 1982, pp 185-229.

80. Morse SA et al: High level tetracycline resistance in Neisseria gonorrhoeae is the result of acquisition of streptococcal tetM determinant. Antimicrobial Agents Chemother 30:664-670, 1986.

81. Moseley SL et al: Identification of enterotoxigenic Escherichia coli by colony hybridization using three enterotoxin gene probes. J Infect Dis 145:863-869, 1982.

82. Mumford LM et al: Bacterial resistance to aminoglycosides. Clin Microbiol Newsl 2:1-4, 1980.

83. Murphy E: Inhibition of Tn554 transposition: Deletion analysis. Pasmid 10:260-269, 1983.

84. Neu HC: Antibiotic inactivating enzymes and bacterial resistance. In: Lorian V, ed. Antibiotics in Laboratory Medicine. Baltimore: Williams and Wilkins, 1986, pp 757-789.

85. Novick RP et al: PeniciUinase plasmids of Staphylococcus aureus: Restriction-deletion maps. Plasmid 2:109-129, 1979.

86. O'Callaghan CH et al: Novel method for detection of [3-1acta- mase by using a chromogenic cephalosporin substrate. Antimi- crob Agents Chemother 1:283-288, 1972.

87. O'Callaghan CH et al: Effects of ~-lactamase from gram-negative organisms on cephalosporins and penicillins. Antimicrob Agents Chemother 1968:57-63, 1969.

88. Oka A e t al: Nucleotide sequence of the kanamycin resistance transposon Tn903. J Mol Biol 147:217- 226, 1981.

89. Ouellette M e t al: Analysis by using DNA probes of the OXA-1 ~-lactamase gene and its transposon. Antimicrob Agents Che- mother 30:46-51, 1986.

© 1987 BY ELSEVIER SCIENCE PUBLISHING CO,, INC. 0738-1751/87/$0.00 + 2.20

THE ANTIMICROBIC NEWSLETI'ER, VOLUME 4, NUMBER 7, JULY 1987

90. Ounissi H et al: Nucleotide sequence of the gene ereA encoding the erythromycin esterase in Esch- erichia coil Gene 35:271-278, 1985.

91. Prentki Pet al: In vitro insertional mutagenesis with a selectable DNA fragment. Gene 29:303-313, 1984.

92. Procter GN et al: Rosanilins: Indi- cator dyes for chloramphenicol-resistant enterobacteria containing chloramphenicol acetyltransferase. J Bacteriol 150:1375-1382, 1982.

93. Richmond MH et al: The ~-lactamases of gram-negative bacteria and their possible physiological role. Adv Microbiol Physiol 9:31-88, 1973.

94. Rollins LD et al: Evidence for a disseminated erythromycin resistance determinant mediated by Tn917-1ike sequences among group D streptococci isolated from pigs, chickens, and humans. Anti- microb Agents Chemother 27:439- 444, 1985.

95. Rubin FA et ah Characterization of R-factor [3-1actamase by the acidimetric method. Antimicrob Agents Chemother 3:68-73, 1973.

96. Sanders CC et al: Characterization of [3-1actamases in situ on poly- acrylamide gels. Antimicrob Agents Chemother 30:951-952, 1986.

97. Shaw WV et ah Primary structure of a chloramphenicol acetyltransferase specified by R plasmids. Nature (Lond) 282:870-872, 1979.

98. Shaw WV: Chloramphenicol Ace- tyltransferase: Enzymology and Molecular Biology. CRC Crit Rev Biochem 14:1-46, 1983.

99. Shimizu K et al: Comparison of aminoglycoside resistance patterns in Japan, Formosa, Korea, Chile, and the United States. Antimicrob Agents Chemother 28:282-288, 1985.

100. Shivakumar AG et al: Organiza- tion of the pE194 genome. Mol Gen Genet 179:241-252, 1980.

101. Shlaes DM et al: Novel plasmid- mediated f3-1actamase in members of the family enterobacteriaceae from Ohio. Antimicrob Agents Chemother 30:220-224, 1986.

102. Smith AL et ah Resistance to chloramphenicol and fusidic acid. In: Bryan LE (ed). Antimicrobial Drug Resistance. Orlando, FL, Academic Press, 1984, pp 293-315.

103. Sundstrom L et al: Novel type of plasmid-borne resistance to trimethoprim. Antimicrobial Agents Chemother 31:60-66, 1987.

104. Sykes RB: Methods for detecting ~-lactamases. In: Reeves, et al (eds). Laboratory Methods in An- timicrobial Chemotherapy. Edin- burgh, Scotland, Churchill Livingstone. 1978, pp 64-69.

105. Tenover FC: Studies of antimicrobial resistance genes using DNA probes. Antimicrob Agents Che- mother 29:721-725, 1986.

106. Tenover FC et al: Development of a DNA probe for the structural gene of the 2"-O-adenyltransferase aminoglycoside modifying enzyme. J Infect Dis 150:678-686, 1984.

107. Thakker-Varia Set ah Molecular epidemiology of macrolide-lincosamide-streptogramin B resistance

67

in Staphylococcus aureus and coagulase-negative staphylococci. Anti- microb Agents Chemother 31:735-743, 1987.

108. Then RL: Mechanisms of resistance to trimethoprim, the sulfonamides, and trimethoprim- sulfamethoxazole. Rev Infect Dis 4:261-269, 1982.

109. Thornsberry C: Methicillin-resis- rant (heteroresistant) staphylococci. Antimicrob Newsl 3:43-50, 1984.

110. Ubukata K et al: Occurrence of a f~-lactam-inducible penicillin- binding protein in a methicillin-resistant staphylococci. Antimicrob Agents Chemother 27:851-857, 1985.

111. Utsui Yet al: Role of an altered penicillin-binding protein in methicillin- and cephem-resistant Staphylococcus aureus. Antimicrob Agents Chemother 28:397-403, 1985.

112. Van de Klundert JAM et ah Simple method for the identification of aminoglycoside-modifying enzymes. J Antimicrob Chemother 14:339-348, 1986.

113. Young SA et al: Development of two DNA probes for differen- tiating the structural genes of subclasses I and II of the aminoglycoside-modifying enzyme 3'-aminoglycoside-phosphotransferase. Antimicrob Agents Che- mother 27:739-744, 1985.

114. Zwadyk P, Cooksey RC: Nucleic acid probes in clinical microbiology. CRC Crit Rev Clin Labor Sci 25:71-103, 1987.

REPORTS FROM THE LITERATURE

ANTIBIOTICS: Resistance Trends and Nontherapeutic Applications

Two issues that have occupied these pages since the inception of The Antimicrobic Newsletter have been, first, gauging and evaluating the increasing resistance to antimicrobial agents and, second, the potential threat (if any) played by antibiotics as food additives. 1-3 Both of these concerns were ad- dressed in different ways in the

May/June 1987 publication of Re- views of Infectious Diseases. In the journal's supplemental volume is a report of the study sponsored by the Fogarty International Center of the National Institutes of Health entitled, "Antibiotic Use and Anti- biotic Resistance Worldwide. ''4 It contains the summary reports from the Six Task Force Chairmen and encompasses estimations of the worldwide usage of antimicrobial agents, and pertains to the regula- tory (policies and laws) as well as political, behavorial, and economic

considerations in the use of these agents. Our focus here is the Task Force 2 presentation by Thomas F. O'Brien, "Resistance of Bacteria to Antibacterial Agents."s Task Force members acknowledged that tabu- lating resistance patterns was difficult, as the drug-organism combinations that were tested varied, as did the test methods and quality control parameters. Addi- tionally, they noted that sample bias at the various test centers plays a role in statistical analysis. The task force studied resistance to


identification of antibacterial resistance mechanisms:

Documents