Contribution of beta-lactamases to bacterial resistance and mechanisms to inhibit beta-lactamases

Contribution of Beta-Lactamases to Bacterial Resistance and Mechanisms To Inhibit Beta-Lactamases

Resistance of bacteria to beta-lactam antibiotics has become a serious problem in the past several decades. Virtually all Staphylococcus aureus, and many Hemophilus influenxae, Branhamella catarrhalis, Nelsseria gonorrhoeae, Enterobacteriaceae, and Bacteroldes species possess beta-lactamases that hydiolyxe penicillins and cephalosporins. The most common plasmld-mediated beta-lactamase is the TEM enzyme (Richmond-Sykes type Illa), which Is present in Hemophilus, Neisseria, and Enterobacteriaceae. One technique to overcome bacterial resistance has been the development of beta-lactamase inhibitors. Clavulanic acid is a beta-lactamase inhibitor that inhibits the beta-lactamases of S. aureus, Hemophllus, NC@seria, Branhamella, Eschericia coli, Klebsiella, and Bacteroides. Clavulanate acts as a “suicide” inhibitor, forming a stable enzyme complex that binds to serine at the active site of the enzyme. Clavulanate readily crosses the outer cell wall of most Enterobacteriaceae to interact with beta-lactamases in the periplasmic space. Clavulanate does not inhibit beta-lactamases such as the Richmond-Sykes type I enzymes found in Pseudomonas aeruginosa, Enterobacter, and Citrobacter species, which are inducible enzymes that function primarily as cephalosporinases.

HAROLD C. NEU, M.D. New

York, New

York

From the Division of Infectious Diseases and Epidemiology, Department of Medicine, College- of Physicians and Surgeons of Columbia University, New York, New York. Requests for reprints should

be addressedto Dr. HaroldC. Neu, Depattmentof Medicine,Collegeof Physiciansand Surgeonsof Columbia University, 630 West York, New York 10032.

2

168th

Street,

New

Resistance of many bacterial species to beta-lactam antibiotics was known before penicillin G became a widely used drug. Abraham and Chain [l] in 1940 reported in a note in Nature that they found an organism, then called Bacillus coli, now Escherichia coli, that produced an enzyme that inactivated penicillin. In the 1940% Kirby [2] stated in Science that an extract of staphylococcus was able to inactivate penicillin. By 1947, the majority of hospital isolates of staphylococci were resistant to penicillin G. Worldwide resistance of Staphyloccus aureus to penicillin followed quickly after the introduction of penicillin G for general use. Currently, more than 95 percent of the hospital isolates of S. aureus and 85 percent of the community isolates of S. aureus produce beta-lactamases that confer resistance to penicillin, ampicillin, and ticarcillin [3]. Although other classes of antibiotics such as etythromycins, tetracyclines, and chloramphenicol were developed, staphylococci quickly developed resistance to these agents [4]. The synthesis of semi-synthetic penicillins from 6-amino penicillanic acid, achieved by Beecham [5] in 1959, produced methicillin, which was beta-lactamase stable and, therefore, inhibited the growth of S. aureus resistant to penicillin. Ampicillin was also produced at that time. This provided antibiotics to inhibit not only staphylococci, but also E. coli, Hemophilus influenzae, and major enteric pathogens such as Salmonella and Shigella.

November 29,1955 The AmericanJournal of Medlclne Volume 79 (suppl5B)

SYMPOSIUM

In 1960, analysis of the organisms producing hospital infections showed that there had been a shift away from gram-positive species such as Streptococcus pneumoniae, S. pyogenes (group A), and S. aureus to enteric species such as E. coli, Klebsiella pneumoniae, and Proteus mirabilis [S]. Although these species were initially inhibited by ampicillin, outbreaks of ampicillin-resistant Klebsiella were soon reported from surgical services [7]. In the latter part of the 196Os, resistance of E. coli to ampicillin and other aminopenicillins was recognized as an increasing problem in isolates from hospitals. In 1974, ampicillin resistance appeared in H. influenzae [8], and ampicillin could no longer be used as single therapy for meningitis due to this organism. Later in the 1970s penicillinase-producing Neisseria gonorrhoeae [9] was discovered to be a worldwide problem. The development of resistance in other organisms such as Bacteroides and, more recently, Branhamella to beta-lactams was also recognized to be a significant problem during the 1970s and 1980s. Two approaches to the problem of beta-lactamases have been developed. One approach has been to modify the beta-lactam nucleus to yield beta-lactamase-stable beta-lactams. This approach has been remarkably successful [lo]. The other approach has been to find substances that can act as beta-lactamase inhibitors. Clavulanic acid was the first successful beta-lactamase inhibitor [11,12]. ACTION

QF BETA-LACTAY

ANTIBIOTICS

To understand the mechanism of inhibition of beta-lactamases, it is necessary to know how beta-lactam antibiotics interact with their targets in the bacterial cell, since the action of beta-lactamases with beta-lactams is quite similar to the interaction with enzymes that produce the cell wall. The macromolecular “net” surrounding bacteria is called peptidoglycan. Peptidoglycan consists of short strands of peptides that cross-link a long polysaccharide polymer consisting of alternating units of N-acetylglucosamine and N-acetylmuramic acid. The peptide component consists of D and L amino acids, which vary in gram-positive species but which are cross-linked via mesodiaminopimelic in gram-negative bacteria [13]. In gram-positive bacteria, peptidoglycan is directly linked by covalent bonds to teichoic acid or teichuronic acid. In gram-negative bacteria, the peptidoglycan is covalently attached to an outer-membrane lipoprotein. Peptidoglycan synthesis occurs in stages, and it is only the last stage of cross-linking that occurs outside the membrane. A new cell wall is made by cross-linking adjacent peptides and adding new units to the growing peptidoglycan strand. Transpeptidation is thought to be the site of action of penicillin and other beta-lactams. Tipper and Strominger [14] proposed in 1965 that penicillin acted as a steric analog of the terminal D-Ala-D-Ala

November

29,1995

ON BETA-LACTAMASE

INHI8ITION-NEU

portion of the pentapeptide chain as the peptidoglycan grows. Binding of penicillin by a covalent bond resulted in transpeptidase inactivation. In the last decade, this hypothesis has been shown, for the most part, to be correct. The enzymes performing the reactions to produce the cell wall have, in recent years, generally been referred to as penicillin-binding proteins. This is because their presence can be demonstrated by their binding to radioactive 3H- or 14C- labeled penicillin G. The formation of an acyl-enzyme complex also occurs when beta-lactamases interact with penicillins. Beta-lactamases may indeed have evolved from peptidoglycan-synthesizing enzymes since beta-lactamases and transpeptidases have major similarities in their amino acid sequences. BETA-LACTAMASE

REACTIONS

The basic reaction of beta-lactamase with a penicillin is to hydrolyze the cyclic amide bond of the penicillin nucleus (Figure l), leading to production of antibacterially inactive penicilloates. When a cephalosporin is hydrolyzed, the presence of the unsaturated bond between carbons 3 and 4 of the dihydrothiazine ring causes the structure to fragment, yielding unstable products. There are a number of ways in which beta-lactamase activity can be detected. As shown in Figure 2, penicillins or cephalosporins yield acid when incubated with beta-lactamases. This can be detected by use of indicator dyes or starch iodine. However, the availability of a chromogenic substrate, nitrocefin [15], which changes from yellow to red in the presence of a beta-lactamase has made it possible to rapidly screen clinical isolates for beta-lactamase. This is particularly important for H. influenzae and N. gonorrhoeae. CHARACTERIZATION

OF BETA-LACTAMASES

Beta-lactamases have been described by many investigators and a number of different classifications of the enzymes are possible. A classification based upon amino acid sequence has been proposed by Ambler [16] (Table I). This classification contains three classes called A, B, and C. Class A consists of four important beta-lactamases: the S. aureus enzyme, Bacillus licheniformis enzyme, B. cereus I enzyme, and E. coli TEM and pBR 322 enzymes. The key amino acid of the beta-lactamases of these species is the serine numbered as serine 70 or serine 44. These beta-lactamases form an acyl-enzyme complex that results in hydrolysis of the beta-lactam. Adjacent to the serine in this class of enzymes, two amino acids away, is a lysine that is also present in the class C enzymes having a serine at the critical position. Class C enzymes are chromosomally mediated and include those present in P. aeruginosa or those in E. coli that are chromosomally mediated by the amp C gene. Class B enzymes are metallo-enzymes, which are unique since they destroy compounds containing a 7-methoxy group as well

The

American

Journal

of Medicine

Volume

79

(ruppl5B)

3

SYMPOSIUM

ON BETA-IACTAMASE

INHIBITION-NEU

I Cl%

CH.

8-Lactamase H,R’ COT I

Figure 1. Action of beta-lactamase penicillins and cephaiosporins.

Cephalosporin

upon

NBturBl SubBtrats (Penicillin, Chyhaiosporin) B-Lactemase 8 Penidlkk Acid 4-

Chromogenic (NiNi,P~;

Substrate

+

Figure 2. Methods mases.

classification with a sixth class added for Sacteroides. This classification of beta-lactamases is based upon substrate and inhibition profiles, molecular weight, and particularly isoelectric points. Substrate profiles of beta-lactamases may not be completely helpful in classifying enzymes since they are often calculated using crude extracts of enzymes incubated with compounds at varying concentrations. The rate of destruction of a compound is compared with that of penicillin G for the enzymes that function primarily as penicillinases and to cephaloridine

as carbapenems having their acyl side chain in a trans configuration. To date, few clinical isolates possessing this latter type of enzyme have been found. This is important since the metallo-enzymes are not inhibited by any inhibitors except chelating agents that bind zinc, which is necessary for activity. Other classifications of enzymes that have proved clinically useful are the ones originally developed by Fiichmond and Sykes in 1973 [17] and the classification of Mitsuhashi [18]. Table II shows the Richmond-Sykes

4

November29,1999

The

Amerlcan Journal

of Medicine

to detect beta-lacta-

Volume

79 (suppl5B)

SYMPOSIUM

TABLE

for those enzymes that function primarily as cephalosporinases. The chromogenic cephalosporin, nitrocefin, can also be used to determine relative rates. Isoelectric points are determined by analytic isoelectric focusing of enzymes in acrylamide gels [19]. Nitrocefin is overlaid on the gel, making it possible to determine if an organism contains several enzymes, which is often the case. Genetically, beta-lactams can be of either chromosomal or plasmid origin (Figure 3). Some beta-lactamases are inducible, and others are constitutively produced. Plasmid beta-lactamases of S. aureus are inducible enzymes as are the chromosomally mediated beta-lactamases of

TABLE

II

Richmond-Sykes

Classification

I

ON BETA-IACTAMASE

INHIBITION-NEU

Classification of Beta-Lactamases on Amino Acid Sequences

Based

Class A S. aureus, 8. licheniformis, B. cereus, E. coli TEM Key amino acid: serine Class B Metallo-enzymes; zinc; inactivate moxalactam, imipenem, cefoxitin Occurs in B. cereus Class C Chromosomally encoded amp C gene of E. coli K12; P. aeruginosa Key amino acid: serine

of Beta-Lactamases

with a Sixth Class Added

and

for Bacteroides

TVPE I Enzymes sulbactam

active

Molecular

principally

weight:

against

24,000

cephalosporins,

- 46,000

Profile:* Pen 100, Amp 100, Carb Trivial names: P99 of Enterobacter; Found

in: Enterobacter,

inhibited

Genetic

origin:

by isoxazoyl

Proteus

vulgaris,

and partially

bycarbenicillin,

but not byclavulanic

acid,

Chromosomal

5, Clox 0, CER 150 - 6000 Sabath-Abraham enzyme

Morganella,

penicillins

P. aeruginosa

Providencia,

Pseudomonas,

Klebsiella,

Serratia,

Citrobacter

inhibited

by clavulanic

TYPE II Enzymes

active

Molecular

against

weight:

penicillins

25,000

inhibited

by isoxazoyl

penicillins

but not by carbenicillin,

acid

- 30,000

Profile:* Pen 100, Amp 150, Carb 40, Clox Found in: Proteus mirabilis, E. co/i

0. CER 10

TVPE III Enzymes inhibited

that equally by clavulanic

hydrolyze penicillins acid, sulbactam

and cephalosporins,

Genetic origin: Molecular weight: 17,000 - 29,000 Profile:* Pen 100, Amp 110, Carb 10, Clox 0, CER 75 Trivial

names:

Found

in: E. cofi, Haemophilus,

TEM-1,

TEM-2,

SHV-1,

inhibited

by pCMB,

isoxazoyl

penicillins,

low activity

against

carbenicillin,

Plasmid

HMS

Neisseria.

Salmonella,

Shigella,

Pseudomonas.

Most

common

enzyme

worldwide

TYPE IV Enzymes inhibited Molecular Profile:*

equally hydrolyze by clavulanic acid weight:

16,000

Pen 100, Amp

penicillins and cephalosporins, and sulbactam - 25,000

150, Carb

Genetic 50, Clox

resistant

origin:

to inhibition

by isoxazoyl

penicillins

and carbenicillin,

cephalosporins,

inhibited

pCMB

but

Chromosomal

20. CER 70

Trivial name: K-l Found in: Klebsiella

TVPE V Equally hydrolyze Molecular weight:

penicillins and cephalosporins, 12,000 - 32,000 Genetic

Profile:* Pen 100, Amp 200, Carb, 90 - 250, Trivial names: OXA 1, 2. 3; PSE 1, 2, 3, 4 Found

in: E. co/i, Pseudomonas,

Clox

hydrolyze isoxaxoyl origin: Plasmid 200, CER

penicillins

better

than

by clavulanic

acid

10 - 50

Serratia

TVPE VI Hydrolyze cephalosporins better than penicillins. cefoxitin Profile:* Pen 3, Amp 1.5. Carb 0.5, CER 100 Found in: Bacteroides

*Pen = penicillin, Amp = ampicillin, Rates are relative.

Carb

Inhibited

= carbenicillin,

November

by cloxacillin

or carbenicillin:

Clox = cloxacillin,

29, 1995

The

hydrolyze

cefamandole,

cefuroxime,

cefotaxime,

but not

CER = cephaloridine

American

Journal

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5B)

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SYMPOSIUM

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INHIBITION-NEU

Microbial

Gram-Posltiva Chromosomal Bacillus

p-lactamares

I NOcardla

Mycobacteria

Backma

I Chromosomal

PlasmId GramNegajive

s. aureus s. ep&mnd”s

I Chromosomal

Bacteria

I

Plasmid I

I

Broad Spectrum. but Pr~mmly Penicillinases TEM-I. TEM-2 0X41.2.3 PSE 1, 2. 3. 4 SW. HMS-1 LCR-1 OXA 4. 5, 6, 7 AER-1 CEPEnterobacterraceae Pseudomonas Achromobacter

Pseudomonas, whereas the plasmid-mediated beta-lactamase of H. infiuenzae is constitutive. All gram-negative species have beta-lactamases in the periplasmic space [20]. Table III illustrates the basis of activity of the betalactamases of clinically important bacteria. BETA-LACTAMASES

OF GRAM-POSITIVE

strain of S. aureus that has a minimal inhibitory concentration.greater than 0.25 w/ml for penicillin G should be considered resistant to this drug, since it will hydrolyze penicillin. Some strains of S. aureus have poor results on the gitrocefin test and might be mistakenly thought to be susceptible to penicillin. Conversely, some of the strains of S. aureus produce large amounts of beta-lactamase constitutively [21). Serologically, there are four types of S. aureus beta-lactamases, although they are microbiologically similar. The staphylococcal beta-lactamase is a single polypeptlde of 29,000 molecular weight with an isoelectric point of 8.9. Staphylococcal beta-lactamases hydrolyze penicillin G, aminopenicillins such as amoxicillin and ampicillin, and

BACtERlA

S. aureus and S. epidermidis contain. beta-lactamases that are exoenzymes (Figure 3). Although the synthesis of the enzyme is plasmid-mediated, resistance is not transferable in the sense that there is transfer of beta-lactamases by gram-negative bacteria. In S. aureus, the enzyme is ,inducible [21]. This means that only a small amount of enzyme is produced in some situations. A

TABLE

III =

Basis of Activity

of Clinically

Bacteria

Rlcbm,ndSykar Chl

6mullc mr

-

Plasmid

Exoenzyme

Inducible

15.co/i H. intluenzae N. gonorrhoeae Salmonella Shigella

Ill

Plasmid

Periplasmic

Constitutive

Klebsie)la

IV

Chromosomal

Periplasmic

Enterobacter &trobacter

la

Chromosomal

Periolasmic

Constitutive Inducible or Constitutive

Id

Chromosomal

Periplasmic

Inducible

I

Chromosome1 Plasmid

Periplasmic Periplasmic

Inducible Constitutive

Chromosomal Plasmid

Periplasmic Periplasmic

Constitutive Constitutive

Or(sl:m S. aureus S. epidermidis

Pseudomonas aeiuginosa Pseudomonas cepacia Bacteroides tragilis Branhamella

6

Important

Figure 3. Various types of betq-lactamases and their disiribution in nature.

NoVember 29,1985

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The American Joumel of MedIcMe

Volume 79 (ruppl58)

LOCltl8n

Pmducn8n

SYMPOSIUM

carboxypenicillins and ureidopenicillins. lsoxazolyl penicillins, such as oxacillin and cloxacillin, or compounds such as nafcillin are not inactivated. Most cephalosporins, with the exception of cephaloridine, are poorly hydrolyzed by staphylococcal beta-lactamases. Cephaloridine’s activity against staphylococci is the result of its affinity for penicillin-binding proteins. Clavulanate, sulbactam, and halopenicillinates inhibit staphylococcal beta-lactamases [12,22,23]. The beta-lactamases of coagulase-negative staphylococci such as S. epidermidis are similar to the beta-lactamases of S. aureus. Since these enzymes are exoenzymes, it is possible that the beta-lactamases of S. epidermidis may contribute to the inactivation of penicillins used to treat non-beta-lactamase-producing organisms that are responsible for wound or tissue infebtions. PLASMID-MEDIATED BETA-LACTAMASES OF GRAMNEGATIVE BACTERIA Anderson and Datta [24] found in 1965 that ampicillin resistance in Salmonella typhimurium could be transferred to recipient E. coli. Subsequently, Datta and Kontomichalou [25] repotted the isolation of an ampicillin-resistant E. coli strain from a young girl in Athens whose first name was Temoniera. Both ampicillin resistance and beta-lactamase production could be transferred to a recipient E. coli strain, which indicated that an R-factor (resistance factor) was involved. This first beta-lactamase was called TEM, a name that has persisted. In 1970, Sykes and Richmond [26] described an enzyme in P. aeruginosa that had a substrate profile similar to the TEM enzyme but which was different electrophoretically. This enzyme is now called TEM-2. TEL&type enzymes have been shown to be present in virtually all gram-negative bacteria (Table IV). The most serious occurrence was finding the TEM enzyme in H. influenzae in 1974 and in N. gonorrhoeae in 1977. In 1963, the enzyme was found in N. meningitidis. Studies by Matthew [27] in 1979 and by R?y et al [26].in 1963 have shown (Table V) that the TEM beta-lactamases are the most common enzymes found in clinical isolates, with 75 percent of beta-lactamases being the TEM-1 type. Simpson et al [29] also found that the TEM beta-lactamase was the most common beta-lactamase in bacterig causing urinary tract infections. Medeiros [30] reported that TEM-1 was found in 72 percent of 484 E. coli obtained from different parts of the world. The frequency of TEM-1 ranged from 66 to 93 percent. Other beta-lactamases of plasmid origin are known and,’ in 1965, Datta and Kontomichalou [25] also found an enzyme in E. koli that hydrolyzed cloxacillin, oxacillin, and other similar compounds. These enzymes were referred to as the OXA enzymes. There are seven distinct OXA enzymes on the basis of isoelectric focusing and substrate studies. It was postulated that the rarity of the OXA beta-lactamases was a result of the limited transfer ca-

November 29,1999

TABLE IV

ON BETA-IACTAMASE

INHIBITION-NEU

Bacterial Species in which the TEM-1 (Richmond-Sykes Illa) Beta-Lactamase Is Found

Acinetobacfer

caicoaceticus

Aeromonas

Providencia Proteus

Alcaligenes

rettgeri vulgaris

Providencia

Citrobacler

freundii

stuarti

Pseudomonas

Enterobacter

aerogenes

Pseudomonas

Enrerobacter

cloacae

Salmonella

Escherichia

co/i

Haemophilus Morganella

morganii

Neisseria Neisseria Proteus

Salmonella

intluenzae gonorrhoeae meningitidis

aeruginosa pufida typhi species

Shigella

sonnei

Serratia

marcescens

Vibrio cholerae Yersinia enterocofitica

mirabilis

TABLE V

Frequency of Beta-Lactamases in Clinical Isolates of Enterobacterlaceae

TEM

1

75

TEM

2

3

OXA 1 OXA 2 2

OXA 3 SHV-1

=;-,-I

3

Other Mixture another

of TEM enzyme

and

5

pacity of plasmids. These enzymes have been found in transposons. In the late 1970% plasmid beta-lactamases that were inhibited by sulfhydro reagents were discoveied. The first was SHV-1, by Pitton [31] and later by Petrocheilou [32]. This enzyme has been found ptimarily in Klebsiella. The HMS-1 enzyme was reported by Matthew et al [19], but as Table IV shows these enzymes are much less frequently found in clinical isolates. In the 197Os, Hedges and Matthew [33] described a series of bdii-lactamases in Pseudomonas that were pl+n$d determined. TheseenzymesPSE-1, PSE-2, PSE-3, and PSE4-were originally thought to be restricted to P. aeruginosa, but they have recently been’ foupd in enteric species as well. The PSE-4 enzyme is also known as the Dalgleish beta-lactamase. PSE-4 is a carbenicillin-hydrolyzing enzyme that will also

The American Journal of Medlclne

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SYMPOSIUM

ON BETA-LACTAMASE

TABLE VI

INHIBITION-NEU

Nomenclature of Plasmid BetaLactamases of Gram-Negative Bacteria RichmondSykes

Matthew TEM-1 (Temoniera) TEM-2 SHV-1 (sulphydryl variable) HMS-1 (Hedges, Matthew, and Smith) OXA(oxacillinhydrolyzing) OXAPSE-1 (Pseudomonas specific) PSE-2 PSE-3 PSE4 (Dalgleish)

Mitsuhashi

llla

Type

la

llla Ill

Type

lb

Va

Type

11

Vb V

Type Ill Type IV

described a plasmid-determined beta-lactamase from Achromobacter, with an isoelectric point of 8.1, that preferentially hydrolyzes cephalosporins. Bauerfeind (personal communication) recently found a plasmid-mediated cephalosporinase in E. coli that will hydrolyze aminothiazolyl iminomethoxy cephalosporins. It is called CEPQ. The importance of these newer beta-lactamases is difficult to assess. They occur in bacteria from various parts of the world, but they have not been the cause of resistance in major outbreaks as had been noted for the TEM-1 enzyme. Since the amino acid composition of these recently described enzymes is unknown, it is conceivable that they are variants of the more common plasmid beta-lactamases created by mutation due to alteration in amino acid sequence, as occurs with hemoglobins.

labia

Ill

Carb-2

CHROMOSOMALLY MEDIATED BETA-LACTAMASES V V V

Inducible Beta-Lactamases. Chromosomally mediated beta-lactamases are extremely common enzymes found in nonfermenting bacterial species such as Acinetobacter calcoaceticus, members of the Enterobacteriaceae such as Enterobacter and Serratia species, and Pseudomonads such as P. aeruginosa. These enzymes differ by genus, species, and subspecies. Although chromosomal beta-lactamases have been classified as RichmondSykes type la, lb, Ic, and Id enzymes [17] (Table I) or as cephalosporinases or cefuroximases by Mitsuhashi [18], there are marked differences among the enzymes. In general, these enzymes hydrolyze cephalosporins at rates that are five to 100 times faster than the rate at which they hydrolyze benzylpenicillin. These enzymes are susceptible to inhibition by cloxacillin, by 7-methoxy cephalosporins or 6-methoxy penicillins, and by the iminomethoxy cephalosporins, but generally not by clavulanic acid. Some penicillanic acid derivatives will inhibit these betalactamases, but sulbactam does not inhibit most Richmond-Sykes type I enzymes at clinically useful concentrations. Compounds such as cefoxitin induce these betalactamases, as do the penems and carbapenems. Clavulanate induces these beta-lactamases in some species. Constltutlve Beta-Lsctsmases. E. coli, Salmonella species, and Shigella species produce a low level of chromosomally mediated beta-lactamases. These species may also contain plasmid-mediated beta-lactamases. Some E. cloacae and E. aerogenes strains produce large amounts of chromosomal beta-lactamase. Other bacteria that contain beta-lactamases of this type are Yersinia enterocolitica. Most (90 percent) strains of B. fragilis produce constitutive beta-lactamases, which are primarily cephalosporinases. All other Bacteroides species probably produce beta-lactamases, but controversy exists concerning whether the enzymes are predominantly penicillinases or cephalosporinases [35]. B. melaninogenicus produces beta-lactamases that act primarily as penicillinases, as do

Garb-3 Carb-1

hydrolyze ticarcillin, piperacillin, cefoperazone, and cefsulodin. These PSE beta-lactamases are able to hydrolyze carbenicillin as rapidly as they hydrolyze benzyl penicillin. They have been divided into four types on the basis of molecular weight, isoelectric point, and inhibition by beta-lactamase inhibitors. PSE-1 has been found in E. coli from South America. PSE-2 has a limited range of bacteria in which it can exist because of the plasmid, R151, which specifies it. This is fortunate, since PSEQ can hydrolyze a number of compounds that are beta-lactamase stable, such as the aminothiazolyl iminomethoxy cephalosporins. PSE-2 resembles OXA enzymes and probably is the same enzyme. Interestingly, OXA-1, PSE-1, and PSE-2 will hydrolyze iminomethoxy cephalosporins, and PSEQ and PSE-3 will partially hydrolyze 7-methoxy cephalosporins and OXA-cephems. PSE-3 and PSE-4 are generally not self-transmissible, even among Pseudomonas. Table VI provides the nomenclature of plasmid betalactamases. A number of novel, plasmid-mediated beta-lactamases have been described in the last several years [30]. An enzyme similar to TEM-1 in substrate profile and molecular weight but differing in isoelectric point has been reported in an E. coli strain isolated in Brazil. A beta-lactamase, ROB-l, has been found in H. influenzae that may be membrane bound. There have also been a number of other OXA enzymes-OXA 4, OXA 5, OXA 6, and OXA 7-found on plasmids that also mediate resistance to sulfonamides, streptomycin, and mercurials. These enzymes have been found in E. coli and P. aeruginosa. All have distinct isoelectric points. Levesque and colleagues [34] 8

November

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Journal

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79 (suppl

5B)

SYMPOSIUM

Gram-Positive

ON BETA-LACTAMASE

INHIBITION-NEU

Bacteria

Peptidoglycan

Figure 4. Interaction of beta-lactam biotic with gram-positive bacteria.

anti-

elactam Destroyed

C--

p-lactamase

Gram-Negative

Bacteria

Penicilhn-Inhibition Binding Proteins

f%lactam -Outer-PeriplasmCell Wall \

of-Cell Peptidoglycan Synthesis

1 p-lactam Destroyed

Figure 5. Interaction of beta-lactam biotic with gram-negative bacteria.

Death

Cell *Survives

P-lactamase / (Present or Induced)

anti-

some B. bivius and 6. disciens strains. Clavulanic acid inhibits the beta-lactamases of most Bacteroides species. The most important chromosomally mediated beta-lactamases are those found in Klebsiella species. Their substrate profile, primarily for penicillins, is the same in various strains of K. pneumoniae and K. oxytoca, but there are markedly different isoelectric points among the enzymes, indicating different amino acid compositions. These enzymes are referred to as class IV in the Richmond-Sykes classification. Some K. oxytoca strains produce a beta-lactamase that will hydrolyze iminomethoxy cephalosporins and some monobactams. Beta-lactamases have been found in all serogroups of Legionella pneumophila [36]. These enzymes hydrolyze some cephalosporins and penicillins, are not inducible, and are inhibited by clavulanic acid. Beta-lactamases are found in species such as Nocardia, and mycobacteria possess beta-lactamases. The precise nature of these enzymes or the role of beta-lactamases in the resistance of these species to beta-lactam antibiotics has not been adequately established. PHYSIOLOGIC CONSEQUENCES OF BETALACTAYASE ACTIVITY Beta-lactamases are not the only means by which bacteria can be or can become resistant to beta-lactam compounds. Beta-lactam activity is due to three factors: ability to penetrate the wall of the bacterium and thereby reach a receptor: beta-lactamase stability; and affinity for the enzymes involved in cell wall synthesis, referred to as peniNovember

29,1995

cillin-binding proteins [37]. Beta-lactamases vary in their location, concentration, physiologic efficiency, and inducibility [36,39]. Resistance of Bacteria Due to Decreased Entry of Beta-Lactams. Decreased entry of penicillins through the bacterial wall has been an important form of resistance when considering why beta-lactamase-stable penicillins fail to inhibit some bacteria. An example of such resistance is seen with oxacillin, which inhibits organisms such as streptococci at concentrations of 0.1 pg/ml. However, concentrations in excess of 1,000 pg/ml are needed to inhibit strains of E. coli, which lack beta-lactamases. Alteration of cell wall porin structure by treatment with ethylenediaminetetraacetic acid, which removes some of the surface lipopolysaccharide, will reduce the minimal inhibitory concentrations of oxacillin for E. coli to 5 pg/ml [36]. It has been possible to select mutants of E. coli that are highly susceptible to penicillins because of altered cell walls. These permeability strains are inhibited by the isoxazolyl penicillins at concentrations several hundredfold lower than the concentrations needed to inhibit the parent strain. The ability of beta-lactams to reach penicillin-binding proteins is less of a problem in gram-positive species, since the cell wall of these species is a much looser net that allows free passage of many molecules to the site of peptidoglycan synthesis (Figure 4). Any inhibitor of gram-negative beta-lactamases would have to reach the molecule it was inhibiting (Figure 5). This explains why early experiments on beta-lactamase inhibition employing methicillin, cloxacillin, or dicloxacillin The

American

Journal

of Medlclne

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79

(suppl 58)

9

SYMPOSIUM

ON BETA-LACTAMASE

INHIBITION-NEU

p-lactamase

ClPYUlanlC complex

BCld

lnachaled Cla”“ianlC and enzyme

were only successful at high concentrations [38]. Such concentrations are not achieved under physiologic conditions except in the urine. Inducible Beta-Lactamases. There has been considerable interest in the role of beta-lactams as inducers of beta-lactamases and the impact that this may have with regard to the selection of resistant bacteria by the betalactamase-stable cephalosporins [39]. Initially, it was suggested that resistance of species such as Enterobacter to cephalosporins such as cefotaxime or ceftriaxone was caused by “trapping” of the enzyme [40]. It is now clear that the enzyme’s affinity for the iminomethoxy cephalosporins, combined with the low concentration of antibiotic in the bacteria, produces a situation in which the enzyme actually hydrolyzes the compound. In these bacteria, the decreased entry of the beta-lactam, combined with hydro-

TABLE VII j3- lutnrw

Inhibition

BETA-LACTAMASE INHIBITORS The antistaphylococcal penicillins are inhibitors of betalactamases. However, even though they act synergistically with beta-lactamase-susceptible penicillins, these agents are of limited clinical usefulness as beta-lactamase inhibitors. In contrast, the discovery of clavulanic acid provided a novel compound that interacts in a different manner with beta-lactamases. The interaction of beta-lactamase with beta-lactam antibiotics can be described as follows:

Id

Pseudomonas Morganella

morganii co/i

II

Escherichia

la

Enterobacter

IC

Proteus

IC

Pseudomonas

Inhibitors

Cl8UCOll~

la

aeruginosa

cloacae

vulgaris cepacia

II

Proteus

III (TEM)

Escherichia

Ill (TEM)

Pseudomonasaeruginosa

Cl~wbrlc

Acid

SMlhClll

tlll~D~lclllln~t~:

S

R

R

R

S

R

R

S

S

S

S

S

S

R

R

S

S

S

S

S

S

S

S

S S

mirabilis

S

S

S

co/i

S

S

S

S

R

S

R

R

Ill (TEM)

Haemophilus

Ill (SHV)

Klebsiella

pneumoniae

IV

Klebsiella

pneumoniae

IV

Eranhamella

influenzae

catarrhalis

S

S

S

S

R

S

S

S

R

S

S

S

S

S

S

R

V (OXA-2)

Escherichia

R

R

R

R

V (PSE 4) -

Pseudomonasaeruginosa

R

S

S

S

Staphylococcus

S

S

S

S

-

Bacillus

cereus-/

R

S

S

S

-

Bacillus

cereus-/l

R

R

R

R

*Based

on Richmond-Sykes

S = susceptible,

10

Orplb

Figure 6. Interaction of clavulanic acid with beta-lactamases showing lethal action of clavulanic acid.

lysis, allows the organisms to survive. A single beta-lactamase molecule can inactivate large numbers of beta-lactam molecules in minutes. If few beta-lactam molecules are present, these will *be inactivated in the periplasmic space before they can bind to penicillin-binding proteins.

of Beta-Lactamases by Beta-L&am

Typ’

aad

November

co/i aureus

classification

R = resistant

29,1@85

The American

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Volume

79 (ruppl5B)

SYMPOSIUM

KI E+P

K2

TABLE

K3

K

m

=

MK-l/k) Kz +

K3

If k > >K2, then K, = K-,/K, and becomes an equilibrium constant. For example, the TEM beta-lactamase can hydrolyze penicillin at K, values of 0.02 to 0.6 mM and K3 values from 0.004 to 2OOOs-‘, with most penicillins being hydrolyzed at the higher rates [41]. The mechanism of the interaction of clavulanic acid with beta-lactamases has been studied extensively by Knowles’ group [42]. They primarily studied the interaction with the TEM enzyme, although the S. aureus beta-lactamase and the B. cereus I enzyme have also been studied. Clavulanate interacts with serine, and the molecule then binds to the lysine two amino acids away, resulting in a stable enzyme-molecule complex. These enzymes are inhibited progressively, but the rate and amount of inhibition varies for each enzyme. Incubation of the TEM betalactamase, which is the most important beta-lactamase clinically, results in inactivation of clavulanic acid, formulation of an inactive clavulanic-enzyme complex for a transient period and, finally, formation of a stable inactive clavulanic-beta-lactamase complex (Figure 6). Since the transient complex eventually decomposes, all of the enzyme will be bound and inactivated. Clavulanic acid is a true “suicide” inhibitor. Furthermore, it binds to the active site of the beta-lactamase, since some substrates afford slight protection against inactivation if preincubated with enzyme before addition of clavulanic acid. Other beta-lactamase inhibitors such as the penicillanic acid sulfones and sulbactam have a mechanism of betalactamase inactivation similar to that of clavulanic acid. Halopenicillanic acids such as 6-beta-bromo and iodopenicillanic acids also appear to be progressive and irreversible beta-lactamase inhibitors that bind to the serine residue at the active site of the enzyme. Clavulanic acid inhibits beta-lactamases from S.

November

29,1955

INHIBITION-NEU

Penetration of Beta-Lactamase Inhibitors into Escherichia coli

VIII

=EP-EE-P-E+X K-1

The penicillin (P) binds to the enzyme (E) forming a noncovalent complex E P. This acylates the beta-lactamase, forming a covalent complex E-P, and the penicillin is finally released as an inactive substance, X. If the constants K-,/K, and K3 are of low order while the constant K2 is of high order, the penicillin acts as an inhibitor. If, however, K-,/K, is of low order and K2 and K3 are of very high order, the penicillin is a substrate and is inactivated. For beta-lactamases, the interaction of the penicillins and/ or cephalosporins with the enzymes are reported as K,,,, or Vmax,and the ratio of V,,/K, is often referred to as the physiologic efficiency of the enzyme.

ON BETA-LACTAMASE

ten-ba Inhlblter

Exlrect

lnlxct Cella

Index Permeablllty

Clavulanate

0.056

Sulbactam

0.3

23

0.69

77

p-bromopenicillanate

0.45

34

76

Iso = concentration at which there of the hydrolysis of nitrocefin.

is 50%

12.3

inhibition 1

aureus, B. cereus, and other Bacillus species, and also the beta-lactamases of species such as L. pneumophila, B. catarrhalis, and Bacteroides species (Table VII). It inhibits plasmid beta-lactamases of the Richmond-Sykes class III type, chromosomal beta-lactamases of class II and IV types such as those present in some P. mirabilis, and the common beta-lactamase enzyme found in Klebsiella species. One important aspect of the action of beta-lactamase inhibitors such as clavulanic acid is their ability to enter the bacterial cell wall. There appears to be no major problem with the ability of clavulanic acid to enter the periplasmic space, where it interacts with the beta-lactamase. This is not true for the penicillanic acid sulfones (Table VIII). The outer membrane can limit the rate at which these compounds inactivate periplasmic beta-lactamases. Obviously, the type of beta-lactamase, the affinities of both the penicillin and the beta-lactamase inhibitor, the location of the beta-lactamase, and the beta-lactamaseinducing properties of the beta-lactarri and of the beta-lactamase inhibitor are all important aspects that will determine whether a particular penicillin and a beta-lactamase inhibitor will function synergistically. COMMENTS This has been a review of beta-lactamases with an attempt to place in perspective the role these enzymes play in bacterial resistance to beta-lactam antibiotics. The distribution of these enzymes in nature, as well as their interaction with penicillins and cephalosporins, is so great that the problem of beta-lactamases will not vanish. Beta-lactamase inhibition of the type provided by clavulanatenoncompetitive, irreversible inactivation of the enzymeis an approach to the treatment of infections caused by beta-lactamase-producing organisms. Beta-lactamase inhibition increases the activity of older antibiotics and permits a single agent, albeit a combination, to be used in selected clinical situations.

The American

Journal

of Medicine

Volume

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SYMPOSIUM

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INHIBITION-NEU

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