The epidemiology of p-lactamases

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1 Journal of Antimicrobial Chemotherapy (1989) 24, Suppl. B, 1-22 The epidemiology of p-lactamases B. Wiedemann, C. KHebe and M. Kresken Pharmazeutische Mikrobiologie der Universitdt Bonn, An der Immenburg 4, 5300 Bonn 1, FRG Chromosomally mediated species-specific /j-lactamases, as well as plasmid-mediated P-lactamases, contribute to bacterial resistance to 0-lactam antibiotics. Chromosomally mediated enzymes confer primary resistance to some drugs and secondary resistance, by mutation to over-production of the enzyme. By far the most prevalent and most important of the, more than thirty, described plasmid-mediated filactamases are those of the TEM group. They can be found in nearly all Gramnegative bacterial species of clinical importance. Furthermore these enzymes have changed their specificity by mutation so that recently described TEM enzymes hydrolyse even third generation cephalosporins. Although there is no change in the quantity of these enzymes, as far as the percentage of producer strains in any species is concerned, there has been a change in quality. The enzymes are further distributed to new species and hydrolyse more so-called 'stable' /Mactam compounds. Introduction Despite other mechanisms described, the /Mactamases, enzymes that bind and destroy /Mactam antibiotics, are the main reason for the resistance of bacteria to this group of drugs. The development of new /Mactam drugs is closely related to the evolution of new enzymes. This evolution can be divided into four periods; (1) 1942 Development of penicillin. Increase of penicillin-resistant Staphylococcus aureus strains due to plasmid-mediated penicillinases. Development of isoxazolyl penicillins, e.g. methicillin and oxacillin. (2) 1960 Development of aminopenicillins, e.g., ampicillin. Increase of ampicillinresistant Escherichia coli, Proteus mirabilis and Salmonella strains due to plasmid-coded penicillinases like TEM-1. Development of first-generation cephalosporins, e.g. cefazolin. (3) 1972 Development of second generation cephalosporins, e.g., cefuroxime and cefoxitin. Selection of mutants that overproduce chromosomally mediated cephalosporinases. Induction of chromosomally mediated enzymes. (4) 1980 Development of third generation cephalosporins e.g., cefotaxime, cefmenoxime and ceftazidime. Development of mutated plasmid-mediated /Mactamases, such as TEM-3 to TEM-7, and SHV-2 and SHV-3, plasmid-coded broad-spectrum /Mactamases. The existence of a /Mactamase, however, does not imply its importance in resistance manifestation. For this the interplay with other bacterial features such as penetration of the drug into the periplasmic space and the affinity for the penicillin-binding proteins /89/24B J02.00/ The British Society for Antimicrobial Chemotherapy

2 2 B. Wkdemam et al (PBPs) is relevant. Even more important is the incidence of strains producing efficient enzymes and the spread of the particular gene into other species. The TEM enzymes are by far the most widespread and their real origin is unknown. They were first identified in Enterobacteriaceae such as Klebsiella spp. and Esch. coli. About ten years later they appeared in Neisseria gonorrhoeae and Haemophilus influenzae; further spread to other species has occurred and now they even exist in N. meningitidis strains. Another aspect of epidemiology is the development of TEM mutants which have changed the specificity of the TEM enzyme from a penicillinase to a cephalosporinase or even a broad spectrum enzyme. In this paper we review the epidemiology of /Mactamases, including information on the incidence and importance of strains that produce these enzymes. Prevalence of pathogens causing bacterial infections The epidemiology of /Mactamases in clinically relevant species can only be assessed in the light of aetiological aspects of bacterial infections. In order to convey the incidence of different pathogens, O'Brien etal. (1987) subdivided these organisms into three groups; I. Those causing epidemic enteric infections. II. Those causing community-acquired infections. III. Those causing nosocomial infections. Table I shows important, frequently isolated, bacteria in each of these three groups. In the countries of the Third World pathogens belonging to group I are of greatest concern in antimicrobial treatment, while group II and group III pathogens are of major importance in industrialised areas. Pneumonia can be taken as an example to illustrate differences between pathogens causing community-acquired and nosocomial infections, (Table II). Among pathogens causing community-acquired pneumonia pneumococci are the predominant organisms, followed by Legionella spp. and H. influenzae. However, in many instances a pathogen cannot be isolated from this group of patients (MacFarlane etal., 1982; Friis-M0ller etal., 1986; Aubertin etal., 1987; Woodhead et al., 1987; Ruf et al., 1988). In nosocomial pneumonia, Staph. aureus and Gram-negative rods are predominant (Brown, Ryczak & Sands, 1986). Table L Organisms associated with epidemic enteric infections, community-acquired infections and nosocomial infections (O'Brien et al., 1987) Epidemic enteric infections Salmonella typhi paralyphi Shigella sonnei flexneri dysenteriae V. cholerae Communityacquired infections Streptococcus pneumoniae group A H. influenzae N. gonorrhoeae Esch. coli Staph. aureus Nosocomial infections Staph. aureus Esch. coli Klebsiella spp. Proteus spp. Enterobacter spp. Serratia spp. Enterococci Ps. aeruginosa

3 Species Epidemiology of fhactamases Table II. Aetiology of community-acquired and nosocomial pneumonia Community acquired Streptococcus pneumoniae H. influenzas Legionella spp. Mycoplasma pneumoniae Chlamydia psittaci Staph. aureus Other Gram-negative bacteria Others Influenza A and B No pathogen isolated infections' % Species Nosocomial Staph. aureus Klebsiella spp. Ps. aeruginosa Esch. coli Enterobacter spp. H. influenzae Proteus spp. Streptococci group A Serratia spp. Enterococci Streptococci group B Staph. epidermidis Others Polymicrobial Non-bacterial infections* % < MacFarlaue et al. (1982); Frii»-M0Der et al. (1986); Aubertin tl al. (1987); Woodhead el al. (1987); 'Ruf el al. (1988). *Brown et al. (1986). There are also differences between pathogens responsible for community-acquired and nosocomial urinary tract infections. Table III features the results of a study conducted in the London area between 1971 and 1982 (Gruneberg, 1984). In 70-75% Esch. coli was responsible for urinary tract infections in community-acquired infections but only in 55-60% in hospitalized patients. Nosocomial urinary tract infections are more often caused by Klebsiella spp., Enterobacter spp., enterococci and Pseudomonas aeruginosa. Septicaemia is typically a severe infection with a high risk of fatal outcome. Knowledge about responsible pathogens is of the utmost importance. Reviewing 8999 septicaemia isolates from eleven centres from West Germany, Berlin (West) and Austria, over a two-year period, Rosenthal (1986) found that Gram-negative pathogens were isolated in 49% and Gram-positive cocci in 45% of the samples, Staph. aureus and Esch. coli being predominant (Table IV). When assessing the epidemiology of /Mactamases, non-pathogens (or, better, 'less pathogenic' bacteria), accompanying pathogens in mixed infections must be considered Table HI. Aetiology of community-acquired and nosocomial urinary tract infections (data from Gruneberg, 1984) Community acquired infections Nosocomial infections Species % Species % Esch. coli Esch. coli Staphylococci Klebsiella I Enterobacter spp Prot. mirabilis Prot. mirabilis Klebsiella /Enterobacter spp Staphylococci Enterococci Others Enterococci Ps. aeruginosa Others

4 B. Wfedanann et al Table IV. Aetiology of septicaemia (data from Rosenthai, 1986) Species n % Esch. coli Staph. aureus Staph. epidermidis Klebsiella spp. Enterococci Ps. aeruginosa Enterobacter spp. Non-haemolytic streptococci Haemolytic streptococci Anaerobes Pneumococci Prot. mirabilis Candida spp. Acinetobacter spp. Pseudomonas spp. Serratia spp. Enteritis Salmonella spp. Indole-positive Proteus Citrobacter spp. H. influenzae Salm. typhi/paratyphi Corynebacterium spp. N. meningitidis Listeria monocytogenes Others as well. Bacteria which might be involved in this process are Bacteroides spp. and Branhamella catarrhalis. They might destroy /Mactamase-unstable penicillins by producing /J-lactamases and thus counteract the drugs' activity against primary pathogens. This phenomenon is called 'indirect pathogenicity' (Maddocks, 1980). Resistance to p-lactam antibiotics Although 0-lactamases undoubtedly play the major role in resistance to /Mactam antibiotics, the other mechanisms conferring resistance to these compounds need brief mention. It has to be emphasized that the reduction of the activity of a /Mactam drug in a resistant cell, even though the /Mactamase is most important, is the result of many factors: (1) the sensitivity of the drug to /Mactamases; (2) the penetration through the outer membrane; (3) the affinity for the target (PBPs); (4) the amount of /Mactamase and (5) the affinity of the drug for the /Mactamase (Wiedemann & Tolxdorff-Neutzling, 1985). Clinical strains with reduced or modified affinity for the targets of the drugs, the penicillin-binding proteins, are being isolated with increasing frequency (Table V) (Bryan, 1988). Staph. aureus strains, resistant to methicillin because of low affinity of a PBP for the antibiotic, have caused important outbreaks of infections (Schaefer et al., 1984). Reduced permeability of the outer-membrane of Gram-negative bacteria can lead to resistance to third-generation cephalosporins in Enterobacter cloacae (Bush etal., 1985) or Serratia marcescens (Goldstein etal., 1983).

5 Epidemiology of p-lactamaaes Table V. Clinical isolates with 0-lactam resistance due to altered penicillin-binding proteins (PBPs) (adapted from Bryan, 1988) Bacteria PBPs affected Antibiotics resisted Streptococcus pneumoniae Multiple penicillin Staph. aureus K methicillin Group D streptococci slow reacting penicillin Viridans streptococci multiple ' penicillin N. gonorrhoeae PBPs 1, 2 penicillin H. influenzae PBPs 3a, 3b broad-spectrum /?-lactams Ps. aeruginosa multiple 3rd-generation cephalosporins penicillins Ser. marcescens? 3rd-generation cephalosporins penicillins (J-Lactamase classification systems /?-Lactamases are produced by many Gram-positive and Gram-negative bacteria. They differ in substrate-profile, molecular weight, isoelectric point, genetic determination, and susceptibility to inhibition by several inhibitors. For Gram-negative bacteria, at least three classification systems have been proposed (Table VI) but none has been totally satisfactory and accepted. The system proposed by Richmond & Sykes (1973) subdivided the enzymes into five classes. Class I enzymes are predominantly cephalosporinases; class II are penicillinases; class III enzymes show broad-spectrum activity and are sensitive to inhibition by cloxacillin but resistant to />-chloromercuribenzoate (pcmb); class FV are also broadspectrum enzymes but resistant to inhibition by cloxacillin and sensitive to pcmb; finally, class V enzymes are penicillinases able to hydrolyse cloxacillin and resistant to pcmb inhibition. Sykes & Matthew (1976) proposed two major groups, classes A and B, which are subdivided into three subclasses: class A enzymes are chromosomally mediated and subclassified into (a) penicillinases, (b) cephalosporinases, and (c) broad-spectrum filactamases. Class B enzymes are determined by R-plasmids and subclassification leads to (a) isoxazolyl-non-hydrolysing, (b) isoxazolyl-hydrolysing, and (c) other /Mactamases. The classification system proposed by Ambler (1980), extended by Jaurin & Grundstrom (1981) and Bush (1988) includes /Mactamases from Gram-positive and Gramnegative bacteria and is based on the amino acid sequence of the active site of the enzymes. Three classes, A, B, and C exist. Class B contains only /Mactamases from Bacillus cereus and related bacilli which require bivalent metal cations such as Zn 2+. Plasmid-coded /Mactamases can be distinguished by isoelectric focusing (Matthew etal., 1975), and on the basis of the isoelectric point (IP) more than 30 different enzymes have been described (Matthew, Hedges & Smith, 1979; Foster, 1983; Medeiros, 1984). Table VII shows the plasmid-mediated /Mactamases classified on the basis of substrate profile. Homology of fmactamase genes Recently, molecular studies on the genetic homology between plasmid-coded /Mactamases of Gram-negative bacteria, by using specific DNA probes for several enzymes,

6 Table VI. Classifications of /Mactamases Richmond & Sykes (1973) Sylces & Matthew (1976) Qass I (a) inducible cephalosporinases Enterobacter, Pseudomonas, Serratia indole-positive Proteus (b) constitutive cephalosporinases Esch. coli Gass A chromosomally mediated (a) peniciliinases rare (b) cephalosporinases Esch. coli, Enterobacter, Citrobacter, Pseudomonas, Providencia, indole-positive Proteus Bad. fragilis, Acinetobacter, Yersinia (c) broad-spectrum /Mactamases Klebsiella Ambler (1980) Jaurin & Grundstrom (1981) Class A* Staph. aureus, Bacillus cereus I, Bacillus licheniformis, TEM, OXA-2, Streptomyces, Klebsiella Class II peniciliinases rare Class III broad-spectrum /Mactamases* pcmb r, clox 1 TEM-1, TEM-2 Class IV broad-spectrum /Mactamases* pcmb', clox r Klebsiella Class B plasmid-mediated (a) isoxazolyl-non-hydrolysing TEM, SHV, etc (b) isoxazolyl-hydrolysing OXA-enzymes (c) others Qass B* metalloenzymes Bacillus cereus U Class C Esch. coli, Pseudomonas, Enterobacter, Citrobacter Serratia Class V peniciliinases*, pcmb', clox r OXA-enzymes, CARB-enzymes Amino acid sequence homology. *clox: cloxadqin, pcmb: p-chkjromercuribenzoate, I, sensitive to inhibition; r, resistant to inhibition.

7 Epidemiology of P-lactamaaes Table VII. Plasmid-coded /?-lactamases in Gram-positive and Gram-negative bacteria I TEM-type la penicfllinase Ib broadspectrum /J-lactamase TEM-1 TEM-2 TLE-1 SHV-1 LCR-1 HMS-1 ROB-1 OHIO-1 BRO-1 TEM-3 TEM-4 TEM-5 TEM-6 TEM-7 SHV-2 SHV-3 II OXA-type (oxacillin-hydrolysing /}-lactamases) OXA-1 OXA-2 OXA-3 OXA-4 OXA-5 OXA-6 OXA-7 "OXA-4"' (PSE-2) III CARB-{PSE-) type (carbenicillin-hydrolysing 0-lactamases) CARB-1 (PSE-4) CARB-2 (PSE-1) CARB-3 CARB-4 PSE-3 AER-1 SAR-1 IV others CEP-2 NPS-1 Staph. aureus A, B, C, D cefoxitin-hydrolysing enzyme from Bact. fragilu "Name given by Phillipon (1983); not identical with OXA-4, named by Medeiros el al. (1985). were carried out (Table VIII). In the group of TEM-type enzymes homology between TEM-1, TEM-2, TEM-3 to TEM-7 and TLE-1 was observed, whereas no hybridization could be demonstrated between TEM-1 and SHV-1, SHV-2, LCR-1, OHIO-1, and ROB-1, which belong to the TEM-type enzymes as well. Also in the PSE-(CARB-) and OXA-group homology could be detected only for a few genes. These results indicate that classification based on substrate profile alone is not satisfactory. Therefore, from these data and results of furthet studies on genetic homology of plasmid-mediated /?- lactamases, a classification system which is based on phylogenetic relations should be developed.

8 />-Lactamase TEM-1 TEM-1 TEM-1 OXA-1 OXA-1 OXA-2 PSE-1 PSE-2 PSE-4 ROB-1 SHV-1 SHV-1 B. Wkdemann a al Table VIIL DNA-homology of plasmid-mediated /Mactamase genes Homology with* Brackets indicate weak homology. TEM-1, TLE-1 TEM-2 TEM-3, TEM-4, TEM-5, TEM-6, TEM-7 (OXA-2) OXA-4 (OXA-3) PSE-4, CARB-3 OXA-6, (OXA-5) CARB-3 (LCR-1), (OXA-2), (CEP-1) SHV-2 Reference Levesque, Medeiros & Jacoby (1987) Cooksey, Clark & Thornsberry (1985) Sougakoff et al. (1988) OueUette & Roy (1986) Huovincn, Huovinen & Jacoby (1988) Boissinot, Mercier & Levesque (1987) Levesque et al. (1987) Huovinen et al. (1988) Huovinen et al. (1988) Levesque et al. (1987) Biscssar & James (1988) Huovinen et al. (1988) P-Lactamases in Gram-positive bacteria /?-Lactamases in Gram-positive bacteria differ from those of Gram-negative organisms in an important characteristic. In contrast to these enzymes from the Gram-negatives, which are located in the periplasmic space, the /Mactamases of Gram-positive bacteria are excreted into the medium and, therefore, destroy the antibiotics extracellularly. Chromosomal P-lactamases In staphylococci, streptococci and enterococci no chromosomal /Mactamases have been detected. Chromosomally mediated resistance to /Mactam antibiotics is due to other mechanisms such as alteration of PBPs (Brown & Reynolds, 1980; Hakenbeck, Tarpay & Tomasz, 1980; Williamson etal., 1983). Members of the Bacillus genus such as Bacillus cereus and Bacillus licheniformis produce inducible chromosomal /Mactamases, predominantly penicillinases (Abraham & Waley, 1979; Collins, 1979). In the genus Clostridium, /Mactamases (which were mainly inducible penicillinases) could be detected in only three species [Cl. butyricum, Cl. clostridiiforme, and Cl. ramosum). In Cl. difficile and Cl. perfringens, which are clinically more important, no /Mactamases have been found (Nord & Olsson-Liljequist, 1984). Plasmid-mediated P-lactamases Plasmid-coded /Mactamases are predominant in Staph. aureus, which is one of the most commonly isolated species not only in community but also in hospital-acquired infections. About 70% of all strains show resistance to /Mactam antibiotics (Kresken & Wiedemann, 1987). There are four immunologically distinct enzymes named A, B, C, and D (Richmond, 1965) which can be located on different transposons and therefore can be integrated in the bacterial chromosome (Lyon & Skurray, 1987). Coagulasenegative staphylococci also produce plasmid-coded /Mactamases in nearly 100% of resistant strains (Rosdahl, Jarloev & Knudsen, 1986). Recently, a plasmid-mediated /Mactamase has been isolated in strains of Enterococcusfaecalis (Murray & Mederiski-Samoraj, 1983). Hybridization analysis demonstrated that this gene was closely related to those of Staph. aureus (Murray et al., 1986).

9 Epidemiology of p-uctanunes 9 Nevertheless, this enzyme is relatively rare and has been found in only a few strains of Enterococcus faecalis (Murray & Patterson, 1988; Patterson, Masecar & Zervos, 1988). Chromosomal P-lactamases p-lactamases in Gram-negative bacteria Chromosomally mediated /Mactamases are easily detected in Gram-negative bacteria such as the Enterobacteriaceae, Ps. aeruginosa (Richmond & Sykes, 1973; Sykes & Matthew, 1976), Legionella, Campylobacter, Acinetobacter, Bacteroides, Aeromonas, and Bran, catarrhalis strains (Sawai etal., 1976; Olsson, Dornbusch & Nord, 1977; Fu & Neu, 1979; Fleming et al., 1982; Phillipon et al., 1986a; Joly-Guillo et al., 1988). They are so-called species-specific /Mactamases (Matthew etal., 1975). The enzymes of Enterobacter, Citrobacter, indole-positive Proteus, Providencia, Serratia, Morganella and Ps. aeruginosa are induciblc cephalosporinases (Sykes & Smith, 1979). For these strains it is possible to select mutants even during clinical therapy (Sanders & Sanders, 1988) which overproduce the enzyme and mediate resistance to third-generation cephalosporins (Seeberg, Toxdorff-Neutzling & Wiedemann, 1983; Gootz, Jackson & Sherris, 1984; Livermore & Yang, 1987). In the case of Enterobacter cloacae about 20% of all strains overproduce the enzyme (Kresken & Wiedemann, 1987). The mechanism for this overproduction is the inactivation of the ampd gene which usually negatively controls the expression of the /Mactamase-gene (Lindberg, Lindquist & Normark, 1987; Korfmann & Wiedemann, 1988). The species-specific /Mactamases of Esch. coli, Shigella and Bact. fragilis are mainly cephalosporinases which are not inducible but are produced constitutively in small amounts in nearly all strains (Olsson etal., 1977; Sykes & Smith, 1979; Wiedemann etal., 1985). Overproduction of the chromosomal gene of Esch.coli is caused by promoter mutations, attenuator mutations or gene amplification (Normark etal., 1983). Another /Mactamase in Esch. coli which is able to hydrolyse third-generation cephalosporins was described by Seibert & Limbert (1982). This enzyme has an altered activity against /Mactam antibiotics. Broad-spectrum /Mactamases which axe produced constitutively can be isolated from Klebsiella strains. These enzymes are the reason for the species-specific resistance to ampicillin in Klebsiella. /?-Lactamase-producing bacteria not belonging to Enterobacteriaceae or Ps. aeruginosa, and the prevalence of the enzymes in these strains, are listed in Table DC. Leg. pneumophila /J-lactamases have been described as cephalosporinases which, in contrast to the cephalosporinases of Enterobacteriaceae and Pseudomonas, are inhibited by clavulanic acid (Fu & Neu, 1979). However, Marre, Medeiros & Pasculle (1982) observed that Leg. pneumophila enzymes are primarily penicillinases. These differences are probably due to variations in the test conditions. In a survey of a hundred clinical isolates of Acin. calcoaceticus, 41% produced a chromosomalry mediated cephalosporinase but induribility was not demonstrated clearly (Joly-Guillo et al., 1988). On the basis of isoelectric focusing at least five different /Mactamases have been described in Bran, catarrhalis. These enzymes are mainly penicillinases and inhibited by clavulanic acid. Attempts to isolate extra-chromosomal DNA failed, indicating a location on the chromosome (Stobberingh, Da vies & van Boven, 1984). The enzymes are distributed in 39% (Stobberingh et al., 1986) to about 80% (Davies & Maesen, 1986; Phillipon etal., 1986<J) of all strains. Resistance of Achromobacter spp.

10 10 B. Wiedenuum et al Species Table DC. Prevalence of /Mactamases in bacteria not mentioned in Tables X and XI Bact. fragilis Leg. pneumophila Camp, jejuni Fus. nucleatum Aer. hydrophila Flav. odoratum Achromobacter spp. Bran, catarrhalis Acin. calcoaceticus Staph. aweus Enterococcus faecalis H. tnfluenzae N. gonorrhoeae N. meningitidis Pastewella multocida Type of /Mactamase chromosomal chromosomal chromosomal chromosomal chromosomal chromosomal chromosomal plasmid (CEP-2) chromosomal plasmid (BRO-1) chromosomal plasmid (TEM-1) plasmid plasmid plasmid (TEM-1) (TEM-1, ROB-1) plasmid (TEM-1) plasmid (TEM-1) plasmid (ROB-1) Prevalence" -87 7* most (-41) (71) (92, 8) -1 Reference Olsson et al. (1977) Fu & Neu (1979) Marre et al. (1982) Fleming et al. (1982) Tuner & Nord (1986) Sawai et al. (1976) Sato et al. (1985) Levesque et al. (1983) Levesque et al. (1982) Phillipon et al. (1986a) Eliasson & Kamme (1985) Joly-GuiUo et al. (1988) Richmond (1965) Kresken & Wiedemann (1987) Murray & Mederisky- Somoraj (1983) Patterson et al. {1988) Machka et al. (1988) Dawn et al. (1988) van Klingeren et al. (1985) Dillon et al. (1983) Livrclli et al. (1988) Data indicate per cent of /Mactamases; data in parenthesis indicate per cent of /Mactamues in resistant strains only. *No data available. to /J-lactam antibiotics is due to the production of chromosomally-mediated cephalosporinases which are expressed constitutively. There exist three different types which exhibit some notable differences from the Richmond & Sykes class I enzymes (Levesque, Letarde & Pechere, 1983). The /Mactamase produced by Flavobacterium odoratum shows a broad substrate profile by hydrolysing penicillins, cephalosporins and even oxyiminocephalosporins, imipenem, and cephamycins. The enzyme requires bivalent cations such as Zn 2+ and Fe* +. and therefore can be classified as a metalloenzyme of Ambler class B (Sato etal., 1985). /?-Lactamases isolated from Fusobacterium nucleatum are mainly penicillinases (Tuner, Lindquist & Nord, 1985a), which are inhibited by clavulanic acid but not by pcmb (Tuner, Lindquist & Nord, 19856). Recently, an increase in /Mactamase-producing Fusobacterium strains during therapy with phenoxymethylpenicillin has been described. The mechanism of this increase remains unclear (Tuner & Nord, 1986). Approximately 15% of clinical isolates of Camp, jejuni produce /Mactamases which can be divided into four different classes. The enzymes seem to be chromosomally mediated (Fleming etal., 1982; Taylor & Courvalinain, 1988). Plasmid-medJated fi-lactamases in Gram-negative bacteria More than thirty different plasmid-coded enzymes have been isolated to date. (Table VTI). The dissemination of some of these enzymes in Enterobacteriaceae and

11 EpManlology of fuactaumnes 11 Ps. aeruginosa is shown in Tables X and XI. The most prevalent enzyme is TEM-1. In Esch. coli, which shows resistance to ampicillin in about 25% of all strains (Kresken & Wiedemann, 1987); in 70% to almost 100% the resistance is due to the production of TEM-1 or TEM-2 (see Tables X and XI). Also in Prot. mirabilis and Salmonella the main reason for ampicillin-rcsistance is the formation of TEM-1 (Table X and XI). TEM-1 has been found in all Enterobacteriaceae but also in strains of Yersinia enterocolitica, Vibrio cholerae, Acinetobacter spp., H. influenzae and N. gonorrhoeae, and now also in N.meningitidis (Roberts, Elwell & Falkow, 1977; Matthew, 1979; Dillon, Pauze & Yeung, 1983; Joly-Guillo etal., 1988). In H. influenzae, up to 60% of all strains, depending on the country of origin, possess the TEM-enzyme (Machka etal., 1988). Similarly, the incidence of TEM-producing N.gonorrhoeae strains is variable for different countries. In the Netherlands about 10% of all strains are peniciuinase producers (van Klingeren etal., 1985). The ampicillin resistance of Acin. calcoaceticus is caused in 71% by TEM-1 (Joly-Guillo etal., 1988). The broad-spectrum 0-lactamases TEM-3, TEM-4, TEM-5, TEM-6, and TEM-7 are able to hydrolyse third-generation cephalosporins. These enzymes, which originate from TEM-2 by mutation (Sougakoff etal., 1988), have been found with high frequency in strains of Esch. coli, Serratia, Enterobacter, and Klebsiella, only in French hospitals (Sirot etal., 1988). SHV-1 has been detected mainly in Klebsiella. Between 33% and 94% of ampicillinresistant strains produce the enzyme; the genetic determination, however, is often unclear (Table X and XI). The broad-spectrum enzyme SHV-2 has been derived by a point-mutation from SHV-1 (Kliebc etal., 1985; Barthelemy etal., 1988). This /?- lactamase, first isolated from Klebsiella, has now been found also in Esch. coli and Salmonella, but is still infrequent (Ben Redjeb etal., 1988). The recently detected /J-lactamases LCR-1, TLE-1, and HMS-1 are very rare enzymes (Matthew etal., 1979; Simpson etal., 1983; Medeiros, Cohenford & Jacoby, 1985). OHIO-1, an enzyme isolated from Enterobacter and Serratia (Shlaes etal., 1986), has been disseminated also in strains of Citrobacter, Morganella, Providencia, Klebsiella, and Esch. coli, all isolated in hospitals in Ohio (Kron etal., 1987). From ampicillin-resistant H. influenzae strains another plasmid-mediated /Mactamase named ROB-1 could be isolated (Rubin etal., 1981). This enzyme could also be detected in animal pathogens such as H.plewopneumoniae and Pasteurella multocida (Medeiros, Levesque & Jacoby, 1986; Livrelli etal., 1988). In a survey of 161 ampicillin-resistant H. influenzae strains, 8% of the strains produced the ROB-enzyme whereas the remainder elaborated the TEM-1 enzyme (Daum etal., 1988). BRO-l isolated from Bran, catarrhalis but also detected in Moraxella nonliquefaciens seems to be the most common /Mactamase in Bran, catarrhalis (Kamme, Vang & Stahl, 1983; Eliasson & Kamme, 1985). In the group of OXA-enzymes the most common is OXA-1 (Tables X and XT) which is distributed in many Enterobacteriaceae (Matthew, 1979). In Esch. coli between 3 and 23% of ampicillin-resistant strains possess this enzyme (Tables X and XI). OXA-2 could be isolated from Esch. coli, Prot. mirabilis, Serratia, Salmonella and even Bordetella bronchiseptica (Matthew, 1979). In resistant Serratia the enzyme is detected with a frequency of 9% and 25%, respectively (Roy etal., 1983; Huovinen, Huovinen & Jacoby, 1988). OXA-3 has been found mainly in Klebsiella (Simpson, Harper & O'Callaghan, 1980; Roy etal., 1983) but also, although not very frequently, in

12 Species Esch. coli Prot. vulgaris TaMe X. Prevalence of plasmid-mediated /Mactamases in Gram-negative bacteria in different studies. Number Number of of resistant strains strains TEM SHV OXA i i 20(74) (94) (69) (62) 45 (81) (85) 4(25) (25) 27 1(4) (2) 0-4 (2) (2) (8) 2(8) (3) (3) (11) 1 (6) (11) /?-Lac tarn ases* PSE-1 PSE-2 PSE-4 others Reference (0-8) (25) own data Roy et al. (1983) Roy et al. (1985) Simpson et al. (1986) (4) Huovinen et al. (1988) own data Roy et al. (1983) Roy et al. (1985) Prot. mirabilis (70) (85) (66) (75) 18 (100) 2(20) 1 (10) 1 (10) own data Roy et al. (1983) Roy et al. (1985) Huovinen et al. (1988) Morg. morganii (22) (100) (100) own data Providencia spp (100) own data Serratia spp (64) (59) 55 (50) (4) own data (9) Roy et al. (1983) Roy et al. (1985) (25) Huovinen et al. (1988)

13 Salmonella spp (100) (87) 21 (100) own data Roy et al. (1983) Roy et al. (1985) Simpson et al. (1986) Enterobacter spp (46) (13) (8) 35 (15) (43) 4 (8) (14) own data Roy et al. (1983) Roy et al. (1985) Simpson et al. (1986) Huovinen et al. (1988) Citrobacter spp. K. pneumoniae Klebsiella spp (13) (50) 40 (27) (40) 16 (28) (10) (25) (66) (46) 24 54(94) (76) (79) (33) (61) 70 (63) (4) own data Roy et al. (1983) Roy et al. (1985) Simpson et al. (1986) Huovinen et al. (1988) own data (4) Huovinen et al. (1988) (6) Roy et al. (1983) Roy et al. (1985) Simpson et al. (1986) i. " Q a. J i K. oxytoca K. ozaenae Ps. aeruginosa (8) 25 (100) 2(9) (20) 2(3) (10) Data indicate Da cent of B-toctsimiittex. data in Darenthesis indicate per cent ol own data own data 8(35) 2(9) own data (40) (10) Huovinen et al. (1988) (50) (7) Jouvenot et al. (1983) 0-lactamases in resistant strains only.

14 14 B. Wfedenuum et al Table XI. Prevalence of other plasmid-mediated /Mactamases Species /?-Lactamases" Reference Esch. coli OXA-2 (4) Huovinen et al. (1988) Prot. vulgaris PSE-2 (25) Roy et al. (1983) Prot. mirabilis OXA-2 1 (10) own data Prot. mirabuis OXA-2 1 Roy et al. (1985) Serratia spp. OXA-2 (9) Roy et al. (1983) Serratia spp. OXA-2 (25) Huovinen et al. (1988) K. pneumoniae OXA-3 (4) Klebsiella spp. OXA-3 (2) PSE-3 (4) Roy et al. (1983) 'Data indicate per cent of /7-lactamases. Data in parenthesis indicate per cent /Mactamases in resistant strains. Esch. coli and Ps. aeruginosa (Matthew etal., 1979). The other OXA-enzymes, OXA-4, OXA-5, OXA-6, and OXA-7 are relatively rare enzymes. OXA-4 and OXA-7 have been isolated from 196 ampicillin-resistant Esch. coli strains and OXA-5 and OXA-6 from 60 carbenicillin-resistant strains of Ps. aeruginosa (Medeiros etal., 1985). PSE-2 which belongs to the OXA-enzymes since this /Mactamase is able to hydrolyse oxacillin in large amounts (Phillipon, 1983) has been detected in Esch. coli, Enterobacter cloacae, K. pneumoniae, Prov. stuartii, and Ps. aeruginosa (Livermore, Maskell & Williams, 1984). The PSE- (CARB-) enzymes are mainly distributed in Ps. aeruginosa. PSE-1 (CARB- 2) has been found frequently (50% of 71 carbenicillin-resistant Ps. aeruginosa strains) whereas PSE-4 (CARB-1) (12% of these strains) and PSE-3 (4% of 24 carbenicillinresistant strains of Ps. aeruginosa) have not been isolated very often (Jacoby & Matthew, 1979; Jouvenot, Bonin & Michel-Briand, 1983). In contrast to the report of Hedges & Matthew (1979), who found the PSE enzymes to be pseudomonas-specific, Reid etal. (1988) isolated PSE-4 from K.pneumoniae and Enterobacter cloacae. PSE-1 (CARB-2) has been detected also in Esch. coli (Levy etal., 1985; Simpson etal., 1986) and in Salmonella (Medeiros, Hedges & Jacoby, 1982). The recently described enzymes CARB-3 and CARB-4 have been found only in two strains of Ps. aeruginosa (Labia, Guionie & Barthelemy, 1981; Phillipon et al., 19866). The /Mactamases AER-1, NPS-1, and SAR-1 are rare enzymes isolated from Aer. hydrophila (Hedges etal., 1985), Ps.aeruginosa (Livermore & Jones, 1986), and V. cholerae (Reid & Amyes, 1986). From Achromobacter a plasmid-mediated cephalosporinase CEP-2 has been isolated but is also infrequent (Levesque etal., 1982). A cefoxitin-hydrolysing /Mactamase has recently been described in one strain of Bact.fragilis (Cuchural etal., 1986). Development of pmactamase-mediated resistance From Tables I to IV it is clear that the most common bacteria causing community- and hospital-acquired infections are Streptococcus pneumoniae, H. influenzae, Esch. coli, Staph. aureus, Enterobacter spp. and Ps. aeruginosa. With the exception of Streptococcus pneumoniae all these bacteria have acquired /Mactam resistance by /Mactamases. The development of resistance seems to be a continuous process if one looks at the description of recently isolated /Mactamases. However, the analysis of resistance data

15 Epidemiology of p-lactumnes * Figure 1. Development of resistance to /7-lacUm antibiotics in Staph. aureus from , Penicillin G, number of strains (») 5664, mean value of resistance (5) = 71-0%; D. oxacillin, n > 5511, i 3-4% * 1986 piperaauin instead of penicillin G. shows that in multicentre-studies no change in the amount of resistance occurs nowadays. The increase in the number of resistant strains usually occurred shortly after the introduction of the respective drug into therapy. This holds true not only for epidemic enteritic infections but for most community-acquired and nosocomial infections. N. gonorrhoeae and H. influenzae seem to be a exception to this rule. A good example of early development of resistance is penicillin resistance in Staph. aureus. In our study the percentage of penicillin-resistant strains of Staph. aureus has not changed between 1975 and the present time. The average over the years is about 70% (Figure 1) which has been noted already since 1953 (Kirby & Ahern, 1953). The reason for this resistance is the production of plasmid-mediated /Mactamases A, B I97S Figure 2. Development of resistance to /Mactam antibiotics in Esch. coli from ^. > Ampkallin, number of strains (n) = 9530, mean value of resistance (X) %; Q, cefoxitm, n = 6726,» 2-4%, #, cefotaxime, n = 3345, i =» 05%.* 1986 ceftazidime instead of cefotaxime.

16 16 B. Wkdemaim tt al Tabk XIL Incidence of /Mactamases in Gram-negative bacteria as calculated from resistance data (Kresken & Wiedemann, 1987) and from results from Tables X and XI Bacteria Esch. coli Prot. vulgaris Prot. mirabilis Morg. morganii Ser. marcescens Enterobacter cloacae Citrobacter spp. K. pneumoniae K. oxytoca Salmonella spp. Ps. aeruginosa Number of strains Resistant (%) Chromosomal /?-Lactamases (%) TEM SHV OXA others or C (Richmond, 1965) which had already established themselves between 1942 and The number of ampicillin resistant strains of Esch. coli did not change significantly between 1975 and During the same period, resistance to the newly developed /?- lactam antibiotics cefoxitin, and later cefotaxime, did not change either (Figure 2). The reason for this resistance (Table XII) is the production of a TEM /Mactamase in 15% of all strains. Other plasmid-mediated /Mactamases are rare. As mentioned in theintroduction this increase happened during the second period of /Mactamase development after the introduction of aminopenicillins (Anderson & Datta, 1965). Ampicillin resistance in Klebsiella is a species-specific resistance (Figure 3). It is probably due to the production of SHV-1 enzyme which may be genetically localized on the chromosome or on a plasmid. In some of the mezlocillin-resistant strains 100 \ I 60 TO I SO 1 «> 3O o I986 # Figure 3. Development of resistance to /Mactam antibiotics in Klebsiella spp. from Ampicillin, number of strains (n) 2620, mean value of resistance (i)» 89-9%; D, cefoxitin, n = 2030, x = 3-3%,, cefotaxime, n " 1498, i - 1-3% ceftazidime instead of cefotaxime.

17 Epidemiology of P-Uctamases I9BI * Flgan 4. Development of reautasce to /J-lactam antibiotics in Enterobacter ipp. from , Ampkinin, number of ttraini (n) = 1565, mean value of resitance (i) %; D, cefoxitin, n = 755, x «* 67K)Vo,, cefotaxime, n = 862, S 209% ceftazklime instead of cefotaxime. (23-3%, see Table XII) the same enzyme is probably produced in higher amounts. In addition some of these strains produce a TEM /J-lactamase. Again, other enzymes are extremely rare (Table XII). The ampicillin resistance in Enterobacter spp., like the cefoxitin resistance, is mainly regarded as species-specific resistance. The reason for the variation in the number of resistant strains in Figure 4 is the fact that the break-points for resistance are very close to the naturally occurring MICs, and slight variations in the method result in relatively great changes in the number of strains deemed resistant. Cefotaxime resistance in these strains is due to the overproduction of the chromosomally mediated /Mactamase. It is surprising that the percentage of resistance has not increased since the introduction of cefotaxime in Other species behave in a similar manner to those mentioned (Table XII) (Kresken & Wiedemann, 1987). Despite the fact that the percentage of resistant strains has not increased, / lactamase production in bacteria is of great importance. However, this value does not reflect the actual number of /Mactamase-producing strains. The development, however, is not yet finished. It may well be that, for example, the TEM-enzyme or others will continue to be as efficient and as able to spread as they have been until now. The TEM-/Mactamase exemplifies slow but efficient spread of a resistance gene in the environment. References Abraham, E. P. & Waley, S. G. (1979). /J-Lactamases from Bacillus cereus. In Beta-lactamases (Hamilton-Miller, J. M.T. & Smith, J.T., Eds), pp Academic Press, London. Ambler, R. P. (1980). The structure of /(-lactamases. Philosophical Transactions of the Royal Society of London B 289, Anderson, E. S. & Datta, N. (1965). Resistance to penicillins and its transfer into Enterobacteriaceae. Lancet i, Aubertin, J., Dabis, F., Fleurette, J., Bomstein, N., Salomon, R., Brottier, R. etal. (1987). Prevalence of legionellosis among adults: a study of community-acquired pneumonia in France. Infection 15, Barthclemy, M., Peduzzi, J., Yaghlane, H. B. & Labia, R. (1988). Single amino acid substitution

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19 Epidemiology of (Uactamases 19 in penicillin-resistant clinical isolates of Streptococcus pneumoniae. Antimicrobial Agents and Chemotherapy 18, Hedges, R. W. & Matthew, M. (1979). Acquisition by Escherichia coli of plasmid-borne /Jlactamases normally confined to Pseudomonas spp. Plasmid 2, Hedges, R. W., Medeiros, A. A., Cohenford, M. & Jacoby, G. A. (1985). Genetic and biochemical properties of AER-1, a novel carbenicillin-nydrolyzing /Mactamase from Aeromonas hydrophila. Antimicrobial Agents and Chemotherapy 27, Huovinen, S., Huovinen, P. & Jacoby, G. A. (1988). Detection of plasmid-mediated /Mactamases with DNA probes. Antimicrobial Agents and Chemotherapy 32, Jacoby, G. A. & Matthew, M. (1979). The distribution of /Mactamases genes on plasmids found in Pseudomonas. Plasmid 2, Jaurin, B. & GrundstrSm, T. (1981). ampc cephalosporinase of Escherichia coli K-12 has a different evolutionary origin from that of /Mactamases of the penicillinase type. Proceedings of the National Academy of Sciences, USA 78, Joly-Guillo, M. L., Valle, E., Bergogne-Berezin, E. & Phillipon, A. (1988). Distribution of /Jlactamases and phenotype analysis in clinical strains of Acinetobacter calcoaceticus. Journal of Antimicrobial Chemotherapy 22, Jouvenot, M., Bonin, P. & Michel-Briand, Y. (1983). Frequency of /Mactamases that are markedly active against carbenicillin in the Pseudomonas aeruginosa strains isolated in a Medical School Hospital. Journal of Antimicrobial Chemotherapy 12, Kamme, C, Vang, M. & Stahl, S. (1984). Transfer of beta-lactamase production in Branhamella catarrhalis. Scandinavian Journal of Infectious Diseases 15, Kirby, W. M. M. & Ahem, J. J. (1953). Changing patterns of resistance of staphylococci to antibiotics. Antimicrobial Agents and Chemotherapy 3, Kliebe, C, Nies, B. A., Meyer, J. F., Tolxdorff-Neutzling, R. M. & Wiedemann, B. (1985). Evolution of plasmid-coded resistance to broad-spectrum cephalosporins. Antimicrobial Agents and Chemotherapy 28, Korfmann, G. & Wiedemann, B. (1988). Genetic control of /Mactamase production in Enterobacter cloacae. Reviews of Infectious Diseases 10, Kresken, M. & Wiedemann, B. (1987). Die Epidemiologie der Resistenz bei Bakterien und ihre Bedeutung fur die Wirksamkeit von Chemotherapeutika. Fortschritte der Antimikrobiellen, Antineoplastischen Chemotherapie 6, Kron, M. A., Shlaes, D. M., Currie-McCumber, C. & Medeiros, A. A. (1987). Molecular epidemiology of OHIO-1 /Mactamase. Antimicrobial Agents and Chemotherapy 31, Labia, R. Guionie, M. & Barthelemy, M. (1981). Properties of three carbeniciuin-hydrolysing /Jlactamases (CARB) from Pseudomonas aeruginosa: identification of a new enzyme. Journal of Antimicrobial Chemotherapy 7, Levesque, R., Letarde, R. & Pechere, J.-C. (1983). Comparative study of the bcta-lactamase activity found in Achromobacter. Canadian Journal of Microbiology 29, Levesque, R. C, Medeiros, A. A. & Jacoby, G. A. (1987). Molecular cloning and DNA homology of plasmid-mediated /Mactamase genes. Molecular and General Genetics 206, Levesque, R., Roy, P. H., Letarde, R. & Pechere, J.-C. (1982). Novel plasmid-mediated /?- lactamase from Achromobacter species. Journal of Infectious Diseases 145, Levy, S. B., Hedges, R. W., Sullivan, F., Medeiros, A. A. & Sosroseputro, H. (1985). Multiple antibiotic resistance plasmids in Enterobacteriaceae isolated from diarrhocal specimens of hospitalized children in Indonesia. Journal of Antimicrobial Chemotherapy 16, lindberg, F., Lindquist, S. & Normark, S. (1987). Inactivation of the ampc gene causes semiconstitutive overproduction of the inducible Citrobacter freundii /Mactamases. Journal of Bacteriology 169, Livrelli, V.-O., Darfeuille-Richaud, A., Rich, C. D., Joly, B. H. & Martel, J. L. (1988). Genetic determination of the ROB-1 /Mactamase in bovine and porcine Pasteurella strains. Antimicrobial Agents and Chemotherapy 32, Livermore, D. M. & Jones, C. S. (1986). Characterization of NPS-1, a novel plasmid-mediated /}- lactamase, from two Pseudomonas aeruginosa isolates. Antimicrobial Agents and Chemotherapy 29, Livermore, D. M., Maskell, J. P. & Williams, J. D. (1984). Detection of PSE-2 /Mactamase in Enterobacteria. Antimicrobial Agents and Chemotherapy 25,

20 20 B. Wiedemann et al Livermore, D. M. & Yang, Y.-J. (1987). 0-Lactamase lability and inducer power of newer /?- lactam antibiotics in relation to their activity against ^-lactamasc-inducibility mutants of Pseudomonas aeruginosa. Journal of Infectious Diseases 155, Lyon, B. R. & Skurray, R. (1987). Antimicrobial resistance of Staphylococcus aureus. Microbiological Reviews 51, MacFarlane, J. T., Finch, R. G., Ward, M. J. & MacRae, A. (1982). Hospital study of adult community-acquired pneumonia. Lancet ii, Machka, K., Braveny, I., Dabernat, H., Dornbusch, K., Van Dycke, E., Kayser, F. H. etal. (1988). Distribution and resistance patterns of Haemophilia influenzae: a European cooperative study. European Journal of Clinical Microbiology and Infectious Diseases 7, Maddocks, J. L. (1980). Indirect pathogenicity. Journal of Antimicrobial Chemotherapy 6, Marre, R., Medeiros, A. A. & Pasculle, W. A. (1982). Characterization of the beta-lactamascs of six species of Legionella. Journal of Bacteriology 151, Matthew, M. (1979). Plasmid-mediated /Mactamases of Gram-negative bacteria: properties and distribution. Journal of Antimicrobial Chemotherapy 5, Matthew, M. & Harris, A. M. (1976). Identification of /J-lactamases by analytical isoelectric focusing: correlation with bacterial taxonomy. Journal of General Microbiology 94, Matthew, M., Harris, A. M., Marshall, M. J. & Ross, G. W. (1975). The use of analytical isoelectric focusing for detection and identification of /Mactamases. Journal of General Microbiology 125, Matthew, M., Hedges, R. W. & Smith, J. T. (1979). Types of 0-lactamase determined by plasmids in gram-negative bacteria. Journal of Bacteriology 138, Medeiros, A. A. (1984). /?-Lactamases. British Medical Bulletin 40, Medeiros, A. A., Cohenford, M. & Jacoby, G. A. (1985). Five novel plasmid-determined /Jlactamases. Antimicrobial Agents and Chemotherapy 27, Medeiros, A. A., Hedges, R. W. & Jacoby, G. A. (1982). Spread of a 'Pseudomonas''-specific p- lactamase to plasmids of Enterobacteria. Journal of Bacteriology 149, Medeiros, A. A., Levesque, R. & Jacoby, G. A. (1986). An animal source of the ROB-1 0- lactamases of Haemophilus influenzae type b. Antimicrobial Agents and Chemotherapy 29, Murray, B. E. & Mederiski-Samoraj, B. D. (1983). Transferable beta-lactamase: a new mechanism for in vivo penicillin resistance in Streptococcus faecalis. Journal of Clinical Investigation Tt, Murray, B. E., Church, D. A., Wanger, A., Zscheck, K., Levison, M. E., Ingermann, M. J. et al. (1986). Comparison of two /Mactamase-producing strains of Streptococcus faecalis. Antimicrobial Agents and Chemotherapy 30, Murray, B. E. & Patterson, J. E. (1988). Penicillinases in enterococci. The Alliance for the Prudent Use of Antibiotics Newsletter W, 5-7. Nord, C. E. & Olsson-Liljequist. (1984). Anaerobic bacteria and beta-lactam antibiotics. Scandinavian Journal of Infectious Diseases, Suppl. 42, Normark, S., Bergstrom, S., Edlind, T., Grundstrom, T., Jaurin, B., Lindberg, F. P. etal. (1983). Overlapping genes. Annual Review of Genetics 17, O'Brien, T. F. & the members of Task Force 2 (1987). Resistance of bacteria to antimicrobial agents; report of Task Force 2. Reviews of Infectious Diseases 9, Suppl. 3, Olsson, B., Dornbusch, K. & Nord, C. E. (1977). Susceptibility to beta-lactam antibiotics and production of beta-lactamases in Bacteroides fragilis. Medical Microbiology and Immunology 163, Ouellette, M. & Roy, P. H. (1986). Analysis by using DNA probes of the OXA-1 0-lactamase gene and its transposon. Antimicrobial Agents and Chemotherapy 30, Patterson, J. E., Masecar, B. L. & Zervos, M. L. (1988). Characterization of two penirillinaseproducing strains of Streptococcus (Enterococcus) faecalis. Antimicrobial Agents and Chemotherapy 32, Phillipon, A. (1983). The /Mactamases. Technologie, Biologie 2, Phillipon, A., Riou, J. Y., Guibourdenche, M. & Sotolongo, F. (1986a)- Detection, distribution and inhibition of Branhamella catarrhalis /Mactamases. Drugs 31, Suppl. 3, Phillipon, A. M. Paul, G. C, Thabaut, A. P. & Jacoby, G. A. (1986*). Properties of a novel

21 Epidemiology of p-lactamases 21 carbenicillin-hydrolyzing /Mactamase (CARB-4) specified by an IncP-2 plasmid from Pseudomonas aeruginosa. Antimicrobial Agents and Chemotherapy 29, Richmond, M. H. (1965). Wild-type variants of exopenicillinase from Staphylococcus aureus. Biochemical Journal 94, Richmond, M. H. & Sykes, R. B. (1973). The beta-lactamases of Gram-negative bacteria and their possible physiological role. Advances in Microbial Physiology 9, Reid, A. J. & Amyes, S. G. B. (1986). Plasmid penicillin resistance in Vibrio cholerae: identification of new /Mactamase SAR-1. Antimicrobial Agents and Chemotherapy 30, Reid, A. J., Simpson, I. N., Harper, P. B. & Amyes, S. G. B. (1988). The differential expression of genes for the PSE-4 /Mactamase in Pseudomonas aeruginosa and the Enterobacteriaceae. Journal of Antimicrobial Chemotherapy 21, Roberts, M., Elwell, L. P. & Falkow, S. (1977). Molecular characterization of two betalactamase-specifying plasmids isolated from Neisseria gonorrhoeae. Journal of Bacteriology 131, Rosdahl, V. T., Jarloev, J. O. & Knudscn, A. M. (1986). Beta-lactamase production in coagulasenegative Micrococcaceae. Ada Pathologica et Microbiologica Scandinavica B 94, Rosenthal, E. J. F. (1986). Septikamie-Erreger Deutsche Medhinische Wochenschrift 111, Roy, C, Foz, A., Segura, C, Tirado, M., Fuster, C. & Reig, R. (1983). Plasmid-detennined 0- lactamases identified in a group of 204 ampicillin-resistant Enterobacteriaceae. Journal of Antimicrobial Chemotherapy 12, Roy, C, Segura, C, Tirado, M., Reig, R., Hermida, M., Teruel, D. etal. (1985). Frequency of plasmid-detennined beta-lactamases in 680 consecutively isolated strains of Enterobacteriaceae. European Journal of Clinical Microbiology 4, Rubin, L. G., Medeiros, A. A., Yolken, R. H. & Moxon, E. (1981). Ampicillin treatment failure of apparently /Mactamase-negative Haemophilus influenzas type b meningitis due to a novel /Mactamase. Lancet i, Ruf, B., Schurmann, D., Horbach, I. & Pohle, H. D. (1988). Klinik und Epidemiologie der Legionella-Pneumonit. Atemwegs- und Lungenkrankheiten 14, Sanders, W. E. & Sanders, C. C. (1988). Inducible /Mactamases: clinical and epidemiological implications for use of newer cephalosporins. Reviews of Infectious Diseases 10, Sato, K., Fujii, T., Okamoto, R., Inoue, M. & Mitsuhashi, S. (1985). Biochemical properties of /Mactamase produced by Flavobacterium odoratum. Antimicrobial Agents and Chemotherapy 27, Sawai, T., Morioko, K., Ogawa, M. & Yamagishi, S. (1976). Inducible oxacillin-hydrolyzing penicillinase in Aeromonas hydrophila isolated from fish. Antimicrobial Agents and Chemotherapy 10, Schaefer, S., Jones, D., Perry, W., Baradet, T., Mayr, E. & Ramersad, C. (1984). Methicillinresistant Staphylococcus aureus strains in New York City hospitals: interhospital spread of resistant strains of type 88. Journal of Clinical Microbiology 20, Seeberg, A. H., Tolxdorff-Neutzling, R. M. & Wiedemann, B. (1983). Chromosomal /Mactamase of Enterobacter cloacae are responsible for resistance to third-generation cephalosporins. Antimicrobial Agents and Chemotherapy 23, Seibert, G. & Lambert, M. (1982). Purification and characterization of a cephalosporinase from Escherichia coli. Zentralblatt fur Bakteriologie und Hygiene 253, Shales, D. M., Medeiros, A. A., Kron, M. A., Currie-McCumber, C, Papa, E. & Vartian, C. V. (1986). Novel plasmid-mediated /Mactamase in members of the family Enterobacteriaceae from Ohio. Antimicrobial Agents and Chemotherapy 30, Simpson, I. N., Harper, P. B. & O'Callaghan, C. H. (1980). Principal /Mactamases responsible for resistance to /Mactam antibiotics in urinary tract infections. Antimicrobial Agents and Chemotherapy 17, Simpson, I. N., Plested, S. J., Budin-Jones, M. J., Lees, J. & Hedges, R.W. (1983). Characterisation of a novel plasmid-mediated /Mactamase and its contribution to resistance in Pseudomonas aeruginosa. Federation of European Microbiological Societies Microbiology Utters 19, Simpson, I. N., Knothe, H., Plested, S. J. & Harper, P. B. (1986). Qualitative and quantitative aspects of /Mactamase production as mechanisms of /Mactam resistance in a survey of clinical isolates from faecal samples. Journal of Antimicrobial Chemotherapy 17,