Showing papers in "Antimicrobial Agents and Chemotherapy in 2004"

Growing group of extended-spectrum beta-lactamases: the CTX-M enzymes.

Abstract: The production of β-lactamases is the predominant cause of resistance to β-lactam antibiotics in gram-negative bacteria. These enzymes cleave the amide bond in the β-lactam ring, rendering β-lactam antibiotics harmless to bacteria. They are classified according to the scheme of Ambler et al. (2) into four classes, designated classes A to D, on the basis of their amino acid sequences, with classes A and C being the most frequently occurring among bacteria. Oxyimino-cephalosporins such as cefotaxime and ceftazidime, which are inherently less susceptible to β-lactamases, were introduced in the early 1980s to treat infections caused by gram-negative bacilli that were resistant to established β-lactams and that produced class A, C, and D β-lactamases. Their repetitive and increased use induced the appearance of resistant strains, which overproduced class C enzymes (42, 72) and/or which produced extended-spectrum β-lactamases (ESBLs), mainly those of class A but also those of class D (19, 61). Class A ESBLs hydrolyze oxyimino-cephalosporins and aztreonam but not 7-α-substituted β-lactams. They are generally susceptible to β-lactamase inhibitors (clavulanate, sulbactam, tazobactam). According to the functional classification scheme of Bush et al. (23), class A ESBLs are therefore clustered in group 2be, which can be subdivided on the basis of their activities against ceftazidime and cefotaxime as ceftazidimases (higher levels of hydrolytic activity against ceftazidime than against cefotaxime) and cefotaximases (higher levels of hydrolytic activity against cefotaxime than against ceftazidime), respectively (48). However, class A ESBLs form a heterogeneous molecular cluster comprising β-lactamases sharing 20 to >99% identity. The earliest class A ESBLs, which were reported from 1985 to 1987, differed from widespread plasmid-mediated TEM-1/2 and SHV-1 penicillinases by one to four point mutations, which extend their hydrolytic spectra (51, 90, 91). TEM and SVH ESBLs now comprise at least 130 members and have a worldwide distribution. Most of them are ceftazidimases, and only a few are cefotaximases. More recently, non-TEM and non-SHV plasmid-mediated class A ESBLs have been reported: ceftazidimases of the PER, VEB, TLA-1, and GES/IBC types and cefotaximases of the SFO-1, BES-1, and CTX-M types (8, 12, 13, 16, 31, 58, 62, 75, 76, 79, 81, 88, 96). The CTX-M β-lactamases are the most widespread enzymes. They were initially reported in the second half of the 1980s, and their rate of dissemination among bacteria and in most parts of the world has increased dramatically since 1995. This review focuses on the origin, epidemiology, clinical impact, enzymatic properties, and structural relationships of the CTX-M-type ESBLs.

Simple and Inexpensive Fluorescence-Based Technique for High-Throughput Antimalarial Drug Screening

Abstract: Radioisotopic assays involve expense, multistep protocols, equipment, and radioactivity safety requirements which are problematic in high-throughput drug testing. This study reports an alternative, simple, robust, inexpensive, one-step fluorescence assay for use in antimalarial drug screening. Parasite growth is determined by using SYBR Green I, a dye with marked fluorescence enhancement upon contact with Plasmodium DNA. A side-by-side comparison of this fluorescence assay and a standard radioisotopic method was performed by testing known antimalarial agents against Plasmodium falciparum strain D6. Both assay methods were used to determine the effective concentration of drug that resulted in a 50% reduction in the observed counts (EC(50)) after 48 h of parasite growth in the presence of each drug. The EC(50)s of chloroquine, quinine, mefloquine, artemisinin, and 3,6-bis-epsilon-(N,N-diethylamino)-amyloxyxanthone were similar or identical by both techniques. The results obtained with this new fluorescence assay suggest that it may be an ideal method for high-throughput antimalarial drug screening.

Emergence of Oxacillinase-Mediated Resistance to Imipenem in Klebsiella pneumoniae

Abstract: Klebsiella pneumoniae strain 11978 was isolated in Turkey in 2001 and was found to be resistant to all beta-lactams, including carbapenems. Cloning and expression in Escherichia coli identified five beta-lactamases, including two novel oxacillinases. The beta-lactamase OXA-48 hydrolyzed imipenem at a high level and was remotely related (less than 46% amino acid identity) to the other oxacillinases. It hydrolyzed penicillins and imipenem but not expanded-spectrum cephalosporins. The bla(OXA-48) gene was plasmid encoded and not associated with an integron, in contrast to most of the oxacillinase genes. An insertion sequence, IS1999, was found immediately upstream of bla(OXA-48). Another plasmid that encoded a second oxacillinase gene, bla(OXA-47), located inside a class 1 integron was identified in K. pneumoniae 11978. OXA-47 had a narrow spectrum of hydrolysis activity and did not hydrolyze ceftazidime or imipenem, as is found for the beta-lactamase (OXA-1) to which it is related. In addition, beta-lactamases TEM-1 and SHV-2a were expressed from the same K. pneumoniae isolate. Analysis of the outer membrane proteins of this isolate revealed that it lacked a porin of ca. 36 kDa. Thus, the high-level resistance to beta-lactams of this clinical isolate resulted from peculiar beta-lactamases and modification of outer membrane proteins.

Novel Type V Staphylococcal Cassette Chromosome mec Driven by a Novel Cassette Chromosome Recombinase, ccrC

Abstract: Staphylococcal cassette chromosome mec (SCCmec) is a mobile genetic element composed of the mec gene complex, which encodes methicillin resistance, and the ccr gene complex, which encodes the recombinases responsible for its mobility. The mec gene complex has been classified into four classes, and the ccr gene complex has been classified into three allotypes. Different combinations of mec gene complex classes and ccr gene complex types have so far defined four types of SCCmec elements. Now we introduce the fifth allotype of SCCmec, which was found on the chromosome of a community-acquired methicillin-resistant Staphylococcus aureus strain (strain WIS [WBG8318]) isolated in Australia. The element shared the same chromosomal integration site with the four extant types of SCCmec and the characteristic nucleotide sequences at the chromosome-SCCmec junction regions. The novel SCCmec carried mecA bracketed by IS431 (IS431-mecA-ΔmecR1-IS431), which is designated the class C2 mec gene complex; and instead of ccrA and ccrB genes, it carried a single copy of a gene homologue that encoded cassette chromosome recombinase. Since the open reading frame (ORF) was found to encode an enzyme which catalyzes the precise excision as well as site- and orientation-specific integration of the element, we designated the ORF cassette chromosome recombinase C (ccrC), and we designated the element type V SCCmec. Type V SCCmec is a small SCCmec element (28 kb) and does not carry any antibiotic resistance genes besides mecA. Unlike the extant SCCmec types, it carries a set of foreign genes encoding a restriction-modification system that might play a role in the stabilization of the element on the chromosome.

Clinical Emergence of Entecavir-Resistant Hepatitis B Virus Requires Additional Substitutions in Virus Already Resistant to Lamivudine

Abstract: Entecavir (ETV) exhibits potent antiviral activity in patients chronically infected with wild-type or lamivudine (3TC)-resistant (3TC r ) hepatitis B virus (HBV). Among the patients treated in phase II ETV clinical trials, two patients for whom previous therapies had failed exhibited virologic breakthrough while on ETV. Isolates from these patients (arbitrarily designated patients A and B) were analyzed genotypically for emergent substitutions in HBV reverse transcriptase (RT) and phenotypically for reduced susceptibility in cultures and in HBV polymerase assays. After 54 weeks of 3TC therapy, patient A (AI463901-A) received 0.5 mg of ETV for 52 weeks followed by a combination of ETV and 100 mg of 3TC for 89 weeks. Viral rebound occurred at 133 weeks after ETV was started. The 3TC r RT substitutions rtV173L, rtL180M, and rtM204V were present at study entry, and the additional substitutions rtI169T and rtM250V emerged during ETV-3TC combination treatment. Reduced ETV susceptibility in vitro required the rtM250V substitution in addition to the 3TC r substitutions. For liver transplant patient B (AI463015-B), previous famciclovir, ganciclovir, foscarnet, and 3TC therapies had failed, and RT changes rtS78S/T, rtV173L, rtL180M, rtT184S, and rtM204V were present at study entry. Viral rebound occurred after 76 weeks of therapy with ETV at 1.0 mg, with the emergence of rtT184G, rtI169T, and rtS202I substitutions within the preexisting 3TC r background. Reduced susceptibility in vitro was highest when both the rtT184G and the rtS202I changes were combined with the 3TC r substitutions. In summary, infrequent ETV resistance can emerge during prolonged therapy, with selection of additional RT substitutions within a 3TC r HBV background, leading to reduced ETV susceptibility and treatment failure.

Combination Antifungal Therapy

Abstract: Invasive fungal infections continue to cause significant morbidity and mortality among hospitalized patients. In particular, recent studies indicate an increase in the incidence of mould infections among transplant recipients, and Candida species have risen to be the third most common pathogen isolated among intensive care unit patients [1, 2]. Advances in modern medical treatment have led to growth in the at-risk population for fungal infections [3]. For example, Cryptococcus neoformans has re-emerged as a growing cause of invasive fungal disease due in large part to the development of novel immune therapy for malignancies, rheumatologic disorders, and management of rejection in transplant populations [4]. Unfortunately, these infections are associated with failures and high rates of relapse even when patients receive recommended therapy [5, 6]. Treatment of invasive mycoses continues to be challenging and complicated by the net state of immunosuppression among infected hosts combined with relative lack of efficacy, significant toxicity, drug–drug interactions, and drug resistance associated with available antifungal agents.

Degradation of Human Antimicrobial Peptide LL-37 by Staphylococcus aureus-Derived Proteinases

Abstract: Cathelicidin LL-37 is one of the few human bactericidal peptides with potent antistaphylococcal activity. In this study we examined the susceptibility of LL-37 to proteolytic degradation by two major proteinases produced by Staphylococcus aureus, a metalloproteinase (aureolysin) and a glutamylendopeptidase (V8 protease). We found that aureolysin cleaved and inactivated LL-37 in a time- and concentration-dependent manner. Analysis of the generated fragments by mass spectroscopy revealed that the initial cleavage of LL-37 by aureolysin occurred between the Arg19-Ile20, Arg23-Ile24, and Leu31-Val32 peptide bonds, instantly annihilating the antibacterial activity of LL-37. In contrast, the V8 proteinase hydrolyzed efficiently only the Glu16-Phe17 peptide bond, rendering the C-terminal fragment refractory to further degradation. This fragment (termed LL-17-37) displayed antibacterial activity against S. aureus at a molar level similar to that of the full-length LL-37 peptide, indicating that the antibacterial activity of LL-37 resides in the C-terminal region. In keeping with LL-37 degradation by aureolysin, S. aureus strains that produce significant amounts of this metalloprotease were found to be less susceptible to LL-17-37 than strains expressing no aureolysin activity. Taken together, these data suggest that aureolysin production by S. aureus contributes to the resistance of this pathogen to the innate immune system of humans mediated by LL-37.

Outbreak of Klebsiella pneumoniae Producing a New Carbapenem-Hydrolyzing Class A β-Lactamase, KPC-3, in a New York Medical Center

Abstract: From April 2000 to April 2001, 24 patients in intensive care units at Tisch Hospital, New York, N.Y., were infected or colonized by carbapenem-resistant Klebsiella pneumoniae. Pulsed-field gel electrophoresis identified a predominant outbreak strain, but other resistant strains were also recovered. Three representatives of the outbreak strain from separate patients were studied in detail. All were resistant or had reduced susceptibility to imipenem, meropenem, ceftazidime, piperacillin-tazobactam, and gentamicin but remained fully susceptible to tetracycline. PCR amplified a blaKPC allele encoding a novel variant, KPC-3, with a His(272)-->Tyr substitution not found in KPC-2; other carbapenemase genes were absent. In the outbreak strain, KPC-3 was encoded by a 75-kb plasmid, which was transferred in vitro by electroporation and conjugation. The isolates lacked the OmpK35 porin but expressed OmpK36, implying reduced permeability as a cofactor in resistance. This is the third KPC carbapenem-hydrolyzing beta-lactamase variant to have been reported in members of the Enterobacteriaceae, with others reported from the East Coast of the United States. Although producers of these enzymes remain rare, the progress of this enzyme group merits monitoring.

Recommendations for Treatment of Human Infections Caused by Bartonella Species

Abstract: Members of the genus Bartonella are facultative intracellular bacteria belonging to the alpha 2 subgroup of the class Proteobacteria and are phylogenetically closely related to Brucella species (15, 73). Until 1993, only three diseases were known to be caused by Bartonella species: Carrion's disease (Bartonella bacilliformis), trench fever (Bartonella quintana), and cat scratch disease (CSD; Bartonella henselae). The genus now comprises B. bacilliformis, species of the former genera Rochalimea and Grahamella (14, 18), and additional, recently described species (Table ​(Table1).1). In mammals, each Bartonella species is highly adapted to its reservoir host; the bacteria can persist in the bloodstream of the host as the result of intraerythrocytic parasitism (49). Intraerythrocytic localization of B. henselae has been demonstrated in cat erythrocytes (88), and B. bacilliformis bacilli have been observed within erythrocytes during the acute phase of Carrion's disease (Oroya fever) (88). Bartonellae also have a tropism for endothelial cells, and intracellular B. henselae can be identified in endothelial cells infected in vitro (28), although intraendothelial cell bacilli have not been identified in vivo. TABLE 1. Epidemiological and clinical data for species of the genus Bartonella Bartonella species cause long-recognized diseases, such as Carrion's disease, trench fever, and CSD, and more recently recognized diseases, such as bacillary angiomatosis (BA), peliosis hepatis (PH), chronic bacteremia, endocarditis, chronic lymphadenopathy, and neurological disorders (Table ​(Table2)2) (73). A remarkable feature of the genus Bartonella is the ability of a single species to cause either acute or chronic infection and either vascular proliferative or suppurative manifestations. TABLE 2. Human diseases caused by Bartonella spp. The pathological response to infection with Bartonella spp. varies substantially with the status of the host immune system. Indeed, infection with the same Bartonella species (e.g., B. henselae) can result in a focal suppurative reaction (CSD in immunocompetent patients), a multifocal angioproliferative response (BA in immunocompromised patients), endovascular multiplication of the bacteria (endocarditis), or an exaggerated inflammatory response without evidence of bacteria in patient tissues (meningoencephalitis) (86). Some of the diseases due to Bartonella species can resolve spontaneously without treatment, but in other cases, the disease is fatal without antibiotic treatment and/or surgery. The clinical situations are so different that a single treatment for all Bartonella-related diseases has not been identified, and the approach to treatment must be adapted to each species and clinical situation (49). Moreover, the database of clinical studies with a standard case definition, culture confirmation, rigidly defined disease outcomes, and patients with similar host defenses is very limited. Thus, case reports with a very limited number of subjects often serve to dictate therapy. The objective of this minireview is to summarize the antibiotic treatment recommendations for the different infections caused by Bartonella species. We have compiled the in vitro antibiotic susceptibility data and our knowledge of the in vivo efficacies of antibiotics for each clinical manifestation, and finally, we have summarized and ranked our treatment recommendations according to the Infectious Diseases Society of America (IDSA) practice guidelines (see Table ​Table5)5) (51). TABLE 5. System for ranking recommendations in clinical guidelines recommended by IDSAa

Hazard ratio in clinical trials.

Abstract: Time-to-event curves analyzed by Cox proportional hazards regression are commonly used to describe the outcome of drug studies. This methodology has the advantage of using all available information, including patients who fail to complete the trial, such as in cancer chemotherapy or human immunodeficiency virus antiviral treatment studies. The goal of treatment in such studies may be to prevent the development of a complication, for example, Pneumocystis carinii pneumonia, and to describe the likelihood of this complication's developing in the treatment group compared to the control group. The hazard ratio describes the relative risk of the complication based on comparison of event rates.

Oxygen Limitation Contributes to Antibiotic Tolerance of Pseudomonas aeruginosa in Biofilms

Abstract: The role of oxygen limitation in protecting Pseudomonas aeruginosa strains growing in biofilms from killing by antibiotics was investigated in vitro. Bacteria in mature (48-h-old) colony biofilms were poorly killed when they were exposed to tobramycin, ciprofloxacin, carbenicillin, ceftazidime, chloramphenicol, or tetracycline for 12 h. It was shown with oxygen microelectrodes that these biofilms contain large anoxic regions. Oxygen penetrated about 50 μm into the biofilms, which averaged 210 μm thick. The region of active protein synthesis was visualized by using an inducible green fluorescent protein. This zone was also limited to a narrow band , approximately 30 μm wide, adjacent to the air interface of the biofilm. The bacteria in mature biofilms exhibited a specific growth rate of only 0.02 h−1. These results show that 48-h-old colony biofilms are physiologically heterogeneous and that most of the cells in the biofilm occupy an oxygen-limited, stationary-phase state. In contrast, bacteria in 4-h-old colony biofilms were still growing, active, and susceptible to antibiotics when they were challenged in air. When 4-h-old colony biofilms were challenged under anaerobic conditions, the level of killing by antibiotics was reduced compared to that for the controls grown aerobically. Oxygen limitation could explain 70% or more of the protection afforded to 48-h-old colony biofilms for all antibiotics tested. Nitrate amendment stimulated the growth of untreated control P. aeruginosa isolates grown under anaerobic conditions but decreased the susceptibilities of the organisms to antibiotics. Local oxygen limitation and the presence of nitrate may contribute to the reduced susceptibilities of P. aeruginosa biofilms causing infections in vivo.

Mechanisms of Resistance in Multiple-Antibiotic-Resistant Escherichia coli Strains of Human, Animal, and Food Origins

Abstract: Seventeen multiple-antibiotic-resistant nonpathogenic Escherichia coli strains of human, animal, and food origins showed a wide variety of antibiotic resistance genes, many of them carried by class 1 and class 2 integrons. Amino acid changes in MarR and mutations in marO were identified for 15 and 14 E. coli strains, respectively.

Vancomycin-Resistant Staphylococcus aureus Isolate from a Patient in Pennsylvania

Abstract: A vancomycin-resistant Staphylococcus aureus (VRSA) isolate was obtained from a patient in Pennsylvania in September 2002. Species identification was confirmed by standard biochemical tests and analysis of 16S ribosomal DNA, gyrA, and gyrB sequences; all of the results were consistent with the S. aureus identification. The MICs of a variety of antimicrobial agents were determined by broth microdilution and macrodilution methods following National Committee for Clinical Laboratory Standards (NCCLS) guidelines. The isolate was resistant to vancomycin (MIC = 32 μg/ml), aminoglycosides, β-lactams, fluoroquinolones, macrolides, and tetracycline, but it was susceptible to linezolid, minocycline, quinupristin-dalfopristin, rifampin, teicoplanin, and trimethoprim-sulfamethoxazole. The isolate, which was originally detected by using disk diffusion and a vancomycin agar screen plate, was vancomycin susceptible by automated susceptibility testing methods. Pulsed-field gel electrophoresis (PFGE) of SmaI-digested genomic DNA indicated that the isolate belonged to the USA100 lineage (also known as the New York/Japan clone), the most common staphylococcal PFGE type found in hospitals in the United States. The VRSA isolate contained two plasmids of 120 and 4 kb and was positive for mecA and vanA by PCR amplification. The vanA sequence was identical to the vanA sequence present in Tn1546. A DNA probe for vanA hybridized to the 120-kb plasmid. This is the second VRSA isolate reported in the United States.

Complete Nucleotide Sequence of a 92-Kilobase Plasmid Harboring the CTX-M-15 Extended-Spectrum Beta-Lactamase Involved in an Outbreak in Long-Term-Care Facilities in Toronto, Canada

Abstract: A major outbreak involving an Escherichia coli strain that was resistant to expanded-spectrum cephalosporins occurred in Toronto and surrounding regions in 2000 to 2002. We report the complete sequence of a plasmid, pC15-1a, that was found associated with the outbreak strain. Plasmid pC15-1a is a circular molecule of 92,353 bp consisting of two distinct regions. The first is a 64-kb region that is essentially homologous to the non-R-determinant region of plasmid R100 except for several point mutations, a few small insertions and deletions, and the absence of Tn 10 . The second is a 28.4-kb multidrug resistance region (MDR) that has replaced the R-determinant region of the R100 progenitor and consists mostly of transposons or partial transposons and five copies of the insertion element IS 26 . All drug resistance genes found in pC15-1a, including the beta-lactamase genes bla CTX-M-15 , bla OXA-1 , and bla TEM-1 , the tetracycline resistance gene tetA , and aminoglycoside resistance genes aac ( 6 ′)- Ib and aac ( 3 ) -II , are located in the MDR. The bla CTX-M-15 gene was found downstream of IS Ecp1 as part of a transposition unit, as determined from the surrounding sequence. Examination of the plasmids from CTX-M-15-harboring strains isolated from hospitals across Canada showed that pC15-1a was found in several strains isolated from a site in western Canada. Comparison of pC15-1a and pCTX15, found in an E. coli strain isolated in India in 1999, revealed that the plasmids had several features in common, including an R100 backbone and several of the resistance genes, including bla CTX-M-15 , bla TEM-1 , bla OXA-1 , tetA , and aac ( 6 ′) -Ib .

High prevalence of antimicrobial resistance among clinical Streptococcus pneumoniae isolates in Asia (an ANSORP study).

Abstract: A total of 685 clinical Streptococcus pneumoniae isolates from patients with pneumococcal diseases were collected from 14 centers in 11 Asian countries from January 2000 to June 2001. The in vitro susceptibilities of the isolates to 14 antimicrobial agents were determined by the broth microdilution test. Among the isolates tested, 483 (52.4%) were not susceptible to penicillin, 23% were intermediate, and 29.4% were penicillin resistant (MICs ≥ 2 mg/liter). Isolates from Vietnam showed the highest prevalence of penicillin resistance (71.4%), followed by those from Korea (54.8%), Hong Kong (43.2%), and Taiwan (38.6%). The penicillin MICs at which 90% of isolates are inhibited (MIC90s) were 4 mg/liter among isolates from Vietnam, Hong Kong, Korea, and Taiwan. The prevalence of erythromycin resistance was also very high in Vietnam (92.1%), Taiwan (86%), Korea (80.6%), Hong Kong (76.8%), and China (73.9%). The MIC90s of erythromycin were >32 mg/liter among isolates from Korea, Vietnam, China, Taiwan, Singapore, Malaysia, and Hong Kong. Isolates from Hong Kong showed the highest rate of ciprofloxacin resistance (11.8%), followed by isolates from Sri Lanka (9.5%), the Philippines (9.1%), and Korea (6.5%). Multilocus sequence typing showed that the spread of the Taiwan19F clone and the Spain23F clone could be one of the major reasons for the rapid increases in antimicrobial resistance among S. pneumoniae isolates in Asia. Data from the multinational surveillance study clearly documented distinctive increases in the prevalence rates and the levels of antimicrobial resistance among S. pneumoniae isolates in many Asian countries, which are among the highest in the world published to date.

Expression of the RND-Type Efflux Pump AdeABC in Acinetobacter baumannii Is Regulated by the AdeRS Two-Component System

Abstract: The AdeABC pump of Acinetobacter baumannii BM4454, which confers resistance to various antibiotic classes including aminoglycosides, is composed of the AdeA, AdeB, and AdeC proteins; AdeB is a member of the RND superfamily. The adeA, adeB, and adeC genes are contiguous and adjacent to adeS and adeR, which are transcribed in the opposite direction and which specify proteins homologous to sensors and regulators of two-component systems, respectively (S. Magnet, P. Courvalin, and T. Lambert, Antimicrob. Agents Chemother. 45:3375-3380, 2001). Analysis by Northern hybridization indicated that the three genes were cotranscribed, although mRNAs corresponding to adeAB and adeC were also present. Cotranscription of the two regulatory genes was demonstrated by reverse transcription-PCR. Inactivation of adeS led to aminoglycoside susceptibility. Transcripts corresponding to adeAB were not detected in susceptible A. baumannii CIP 70-10 but were present in spontaneous gentamicin-resistant mutants obtained in vitro. Analysis of these mutants revealed the substitutions Thr153-->Met in AdeS downstream from the putative His-149 site of autophosphorylation, which is presumably responsible for the loss of phosphorylase activity by the sensor, and Pro116-->Leu in AdeR at the first residue of the alpha(5) helix of the receiver domain, which is involved in interactions that control the output domain of response regulators. These mutations led to constitutive expression of the pump and, thus, to antibiotic resistance. These data indicate that the AdeABC pump is cryptic in wild A. baumannii due to stringent control by the AdeRS two-component system.

Chloroquine Resistance in Plasmodium vivax

Abstract: Emerging resistance to chloroquine (CQ) by Plasmodium vivax threatens the health of the hundreds of millions of people routinely exposed to the risk of infection with this organism. CQ has been the first-line therapy for vivax malaria since 1946 (32, 115). Plasmodium falciparum developed resistance to CQ in the 1950s (110), and today it occurs globally (91). Resistance by P. vivax was unknown until 1989, when Australians repatriated from Papua New Guinea failed routine treatment (94). Subsequent reports affirmed that finding, and CQ-resistant P. vivax (CRPV) was reported from Indonesia (8, 35, 99, 100, 111). Reports from Myanmar (76, 82) and India (56, 107) followed. CRPV appeared in travelers from Guyana, South America (88). However, studies in Thailand (38, 72, 103), the Philippines (10), and Vietnam (105) revealed only CQ-sensitive P. vivax. Surveys in Indonesia revealed a low frequency of CRPV in the west (∼10%) (15, 16, 49, 50, 51, 53, 75) and a higher risk in the east (∼45%) (9, 18, 52, 81, 102, 106). This minireview summarizes the present state of knowledge of CRPV as a scientific, clinical, and public health problem. It examines the genesis of CQ therapy for P. vivax and the laboratory and clinical data underpinning the diagnosis of CRPV. The available data showing the global distribution of CRPV are listed. Finally, the clinical data on alternative therapies against CRPV are reviewed.

TMC125, a Novel Next-Generation Nonnucleoside Reverse Transcriptase Inhibitor Active against Nonnucleoside Reverse Transcriptase Inhibitor-Resistant Human Immunodeficiency Virus Type 1

Abstract: Nonnucleoside reverse transcriptase inhibitors (NNRTIs) are potent inhibitors of human immunodeficiency virus type 1 (HIV-1); however, currently marketed NNRTIs rapidly select resistant virus, and cross-resistance within the class is extensive. A parallel screening strategy was applied to test candidates from a series of diarylpyrimidines against wild-type and resistant HIV strains carrying clinically relevant mutations. Serum protein binding and metabolic stability were addressed early in the selection process. The emerging clinical candidate, TMC125, was highly active against wild-type HIV-1 (50% effective concentration [EC50] = 1.4 to 4.8 nM) and showed some activity against HIV-2 (EC50 = 3.5 microM). TMC125 also inhibited a series of HIV-1 group M subtypes and circulating recombinant forms and a group O virus. Incubation of TMC125 with human liver microsomal fractions suggested good metabolic stability (15% decrease in drug concentration and 7% decrease in antiviral activity after 120 min). Although TMC125 is highly protein bound, its antiviral effect was not reduced by the presence of 45 mg of human serum albumin/ml, 1 mg of alpha1-acid glycoprotein/ml, or 50% human serum. In an initial screen for activity against a panel of 25 viruses carrying single and double reverse transcriptase amino acid substitutions associated with NNRTI resistance, the EC50 of TMC125 was <5 nM for 19 viruses, including the double mutants K101E+K103N and K103N+Y181C. TMC125 also retained activity (EC50 < 100 nM) against 97% of 1,081 recent clinically derived recombinant viruses resistant to at least one of the currently marketed NNRTIs. TMC125 is a potent next generation NNRTI, with the potential for use in individuals infected with NNRTI-resistant virus.

In Vivo Pharmacodynamic Activity of Daptomycin

Abstract: Daptomycin is a lipopeptide antibiotic with activity against a wide range of gram-positive bacteria. We used the neutropenic murine thigh model to characterize the pharmacodynamics of daptomycin. ICR/Swiss mice were rendered neutropenic with cyclophosphamide; and the thigh muscles of the mice were infected with strains of Staphylococcus aureus, Streptococcus pneumoniae, and Enterococcus faecium. Animals were treated by subcutaneous injection of daptomycin at doses of 0.20 to 400 mg/kg of body weight/day divided into one, two, four, or eight doses over 24 h. Daptomycin exhibited linear pharmacokinetics, with an area under the concentration-time curve (AUC) from time zero to infinity/dose of 9.4 and a half-life of 0.9 to 1.4 h. The level of protein binding was 90%. Free daptomycin exhibited concentration-dependent killing and produced in vivo postantibiotic effects (PAEs) of 4.8 to 10.8 h. Nonlinear regression analysis was used to determine which pharmacokinetic (PK) or pharmacodynamic (PD) parameter was important for efficacy by using free drug concentrations. The peak concentration/MIC (peak/MIC) ratio and 24-h AUC/MIC ratio were the PK and PD parameters that best correlated with in vivo efficacy (R2 = 83 to 87% for peak/MIC and R2 = 86% for the AUC/MIC ratio, whereas R2 = 47 to 50% for the time that the concentration was greater than the MIC) against standard strains of S. aureus and S. pneumoniae. The peak/MIC ratios required for a bacteriostatic effect ranged from 12 to 36 for S. pneumoniae, 59 to 94 for S. aureus, and 0.14 to 0.25 for E. faecium. The AUC/MIC ratios needed for a bacteriostatic effect ranged from 75 to 237 for S. pneumoniae, 388 to 537 for S. aureus, and 0.94 to 1.67 for E. faecium. The free daptomycin concentrations needed to average from one to two times the MIC over 24 h to produce a bacteriostatic effect and two to four times the MIC over 24 h to produce greater than 99% killing. The long PAE and potent bactericidal activity make daptomycin an attractive option for the treatment of infections caused by gram-positive bacteria.

Miltefosine Induces Apoptosis-Like Death in Leishmania donovani Promastigotes

Abstract: Miltefosine (hexadecylphosphocholine [HePC]) has proved to be a potent oral treatment for human visceral leishmaniasis due to Leishmania donovani. The molecular mechanisms that contribute to the antileishmanial activity of HePC are still unknown. We report that in wild-type promastigotes of Leishmania donovani HePC is able to induce a cell death process with numerous cytoplasmic, nuclear, and membrane features of metazoan apoptosis, including cell shrinkage, DNA fragmentation into oligonucleosome-sized fragments, and phosphatidylserine exposure. None of these changes were detected in an HePC-resistant clone treated with the same drug concentration. Therefore, HePC does not appear to kill L. donovani promastigotes by a direct toxic mechanism but, rather, kills the promastigotes by an indirect one. Pretreatment of wild-type promastigotes with two broad caspase inhibitors, z-Val-Ala-DL-Asp(methoxy)-fluoromethylketone and Boc-Asp(methoxy)-fluoromethylketone, as well as a broad protease inhibitor, calpain inhibitor I, prior to drug exposure interfered with DNA fragmentation but did not prevent cell shrinkage or phosphatidylserine externalization. These data suggest that at least part of the apoptotic machinery operating in wild-type promastigotes involves proteases. Identification of the death-signaling pathways activated in HePC-sensitive parasites appears to be essential for a better understanding of the molecular mechanisms of action and resistance in these parasites.

Enzymatic Detachment of Staphylococcus epidermidis Biofilms

Abstract: The gram-positive bacterium Staphylococcus epidermidis is the most common cause of infections associated with catheters and other indwelling medical devices. S. epidermidis produces an extracellular slime that enables it to form adherent biofilms on plastic surfaces. We found that a biofilm-releasing enzyme produced by the gram-negative periodontal pathogen Actinobacillus actinomycetemcomitans rapidly and efficiently removed S. epidermidis biofilms from plastic surfaces. The enzyme worked by releasing extracellular slime from S. epidermidis cells. Precoating surfaces with the enzyme prevented S. epidermidis biofilm formation. Our findings demonstrate that biofilm-releasing enzymes can exhibit broad-spectrum activity and that these enzymes may be useful as antibiofilm agents.

Pseudomonas aeruginosa Biofilms Exposed to Imipenem Exhibit Changes in Global Gene Expression and β-Lactamase and Alginate Production

Abstract: The lungs of cystic fibrosis (CF) patients are commonly colonized with Pseudomonas aeruginosa biofilms. Chronic endobronchial P. aeruginosa infections are impossible to eradicate with antibiotics, but intensive suppressive antibiotic therapy is essential to maintain the lung function of CF patients. The treatment often includes beta-lactam antibiotics. How these antibiotics influence gene expression in the surviving biofilm population of P. aeruginosa is not clear. Thus, we used the microarray technology to study the effects of subinhibitory concentrations of a beta-lactam antibiotic, imipenem, on gene expression in biofilm populations. Many genes showed small but statistically significant differential expression in response to imipenem. We identified 34 genes that were induced or repressed in biofilms exposed to imipenem more than fivefold compared to the levels of induction or repression for the controls. As expected, the most strongly induced gene was ampC, which codes for chromosomal beta-lactamase. We also found that genes coding for alginate biosynthesis were induced by exposure to imipenem. Alginate production is correlated to the development of impaired lung function, and P. aeruginosa strains isolated from chronically colonized lungs of CF patients are nearly always mucoid due to the overproduction of alginate. Exposure to subinhibitory concentrations of imipenem caused structural changes in the biofilm, e.g., an increased biofilm volume. Increased levels of alginate production may be an unintended adverse consequence of imipenem treatment in CF patients.

National Surveillance of Antimicrobial Resistance in Pseudomonas aeruginosa Isolates Obtained from Intensive Care Unit Patients from 1993 to 2002

Abstract: Nosocomial infections caused by Pseudomonas aeruginosa in critically ill patients are often difficult to treat due to resistance to multiple antimicrobials. The purpose of this study was to evaluate antimicrobial resistance among P. aeruginosa isolates from intensive care unit patients in the United States from 1993 to 2002 by using the Intensive Care Unit Surveillance Study database. Over the 10-year period, susceptibility of 13,999 nonduplicate isolates of P. aeruginosa was analyzed. From 1993 to 2002, nationwide increases in antimicrobial resistance were greatest for ciprofloxacin, imipenem, tobramycin, and aztreonam. Rates of multidrug resistance (resistance to ≥3 of the following drugs: ceftazidime, ciprofloxacin, tobramycin, and imipenem) increased from 4% in 1993 to 14% in 2002. The lowest dual resistance rates were observed between aminoglycosides or fluoroquinolones with piperacillin-tazobactam while the highest were for those that included β-lactams and ciprofloxacin. Ongoing surveillance studies are crucial in monitoring antimicrobial susceptibility patterns and selecting empirical treatment regimens.

Antimicrobial Peptides from Marine Invertebrates

Abstract: Marine invertebrates lack an acquired, memory-type immunity based on T-lymphocyte subsets and clonally derived immunoglobulins (72). This differs from the vertebrate immune system, which is characterized by somatic gene rearrangement, clonal selection, and expansion and a discriminative ability that includes lymphocytes, among other factors, which impart specificity and memory (71). Marine invertebrates rely solely on innate immune mechanisms that include both humoral and cellular responses. Humoral immunity in marine invertebrates is characterized by antimicrobial agents present in the blood cells and plasma (92), along with reactions such as hemolymph coagulation or melanization (79, 85). Cellular immunity in marine invertebrates is based on cell defense reactions, including encapsulation, nodule formation, and phagocytosis (92). The cellular component of marine invertebrate immunity is mediated by hemocytes, motile cells that phagocytize microbes and secrete soluble antimicrobial and cytotoxic substances into the hemolymph (53). This differs from insects, especially Drosophila melanogaster, which rely largely on the challenge-induced synthesis of antimicrobial peptides by the fat body (30, 88) and use exclusion, via a tough exoskeleton, as their major antimicrobial defense. The circulating hemolymph in marine invertebrates contains biologically active substances such as complement, lectins, clotting factors, and antimicrobial peptides (57). All of these factors contribute to a self-defense system in marine invertebrates against invading microorganisms, which can number up to 106 bacteria/ml and 109 virus/ml of seawater (2). The survival of marine invertebrates in this environment suggests that their innate immune system is effective and robust (52). Antimicrobial peptides are a major component of the innate immune defense system in marine invertebrates. They are defined as molecules less than 10 kDa in mass which show antimicrobial properties (12) and provide an immediate and rapid response to invading microorganisms (8). The major classes of antimicrobial peptides include (i) α-helices, (ii) β-sheet and small proteins, (iii) peptides with thio-ether rings, (iv) peptides with an overrepresentation of one or two amino acids, (v) lipopeptides, and (vi) macrocyclic cystine knot peptides (24). There is evidence that antimicrobial peptides are widespread in invertebrates (15), especially in tissues such as the gut and respiratory organs in marine invertebrates, where exposure to pathogenic microorganisms is likely. In spite of variations in structure and size, the majority of antimicrobial peptides are amphiphilic, displaying both hydrophilic and hydrophobic surfaces. These peptides generally act by forming pores in microbial membranes or otherwise disrupting membrane integrity (82), which is facilitated by their amphiphilic structure. This mode of action is unlikely to lead to the development of resistance (9, 58), although it must be noted that this presumption is debatable (10). Recently, cationic antimicrobial peptides have been reported to be involved in many aspects of innate host defenses, associated with processes such as acute inflammation (25). The value of antimicrobial peptides in innate immunity lies in their ability to function without either high specificity or memory, and their small size makes them easy to synthesize (72). In addition, many antibacterial peptides show remarkable specificity for prokaryotes with low toxicity for eukaryotic cells (97). This is a characteristic that has favored their investigation and exploitation as potential new antibiotics (97). The recent appearance of a growing number of bacteria resistant to conventional antibiotics has become a serious medical problem. To overcome this resistance, the development of antibiotics with novel mechanisms of action is a pressing issue (48). Endogenous antimicrobial peptides are exciting candidates as new antibacterial agents due to their broad antimicrobial spectra, highly selective toxicities, and the difficulty for bacteria to develop resistance to these peptides (11, 26, 47). The ocean covers 71% of the surface of the earth and contains approximately half of the total global biodiversity, with estimates ranging between 3 and 500 × 106 different species (28). Marine macrofauna alone comprise 0.5 to 10 × 106 species (23). Therefore, the marine environment, especially marine invertebrates that rely solely on innate immune mechanisms for host defense, is a spectacular resource for the development of new antimicrobial compounds. This minireview will encompass what is known about gene-encoded antimicrobial peptides from marine invertebrates, covering the phyla Arthropoda, Chordata, and Mollusca (Table ​(Table11). TABLE 1. Antimicrobial peptides from marine invertebrates

Molecular characterization of a beta-lactamase gene, blaGIM-1, encoding a new subclass of metallo-beta-lactamase.

Abstract: As part of the SENTRY Antimicrobial Surveillance Program in 2002, five multidrug-resistant Pseudomonas aeruginosa clinical isolates were detected with metallo-β-lactamase (MβL) activity. The isolates were recovered from different patients in a medical center located in Dusseldorf, Germany. The resistant determinant was isolated amplifying the region between the integrase and the aacA4 gene cassette. Sequencing revealed a novel MβL gene, designated blaGIM-1. Additional analysis showed that GIM-1, comprising 250 amino acids and with a pI value of 5.4, differs in its primary sequence from that described for IMP, VIM, and SPM-1 enzymes by 39 to 43%, 28 to 31%, and 28%, respectively. The enzyme possesses unique amino acids within the major consensus sequence (HXHXD) of the MβL family. Kinetics analysis revealed that GIM-1 has no clear preference for any substrate and did not hydrolyze azlocillin, aztreonam, and the serine-β-lactamase inhibitors. blaGIM-1 was found on a 22-kb nontransferable plasmid. The new MβL gene was embedded in the first position of a 6-kb class 1 integron, In77, with distinct features, including an aacA4 cassette downstream of the MβL gene that appeared to be truncated with blaGIM-1. The aacA4 was followed by an aadA1 gene cassette that was interrupted by a copy of the IS1394. This integron also carried an oxacillinase gene, blaOXA-2, before the 3′-CS region. GIM-1 appears to be a unique MβL, which is located in a distinct integron structure, and represents the fourth subclass of mobile MβL enzymes to be characterized.

Pharmacokinetics and Safety of Intravenous Voriconazole in Children after Single- or Multiple-Dose Administration

Abstract: We conducted a multicenter study of the safety, tolerability, and plasma pharmacokinetics of the parenteral formulation of voriconazole in immunocompromised pediatric patients (2 to 11 years old). Single doses of 3 or 4 mg/kg of body weight were administered to six and five children, respectively. In the multiple-dose study, 28 patients received loading doses of 6 mg/kg every 12 h on day 1, followed by 3 mg/kg every 12 h on day 2 to day 4 and 4 mg/kg every 12 h on day 4 to day 8. Standard population pharmacokinetic approaches and generalized additive modeling were used to construct the structural pharmacokinetic and covariate models used in this analysis. In contrast to that in adult healthy volunteers, elimination of voriconazole was linear in children following doses of 3 and 4 mg/kg every 12 h. Body weight was more influential than age in accounting for the observed variability in voriconazole pharmacokinetics. Elimination capacity correlated with the CYP2C19 genotype. Exposures were similar at 4 mg/kg every 12 h in children (median area under the concentration-time curve (AUC), 14,227 ng. h/ml) and 3 mg/kg in adults (median AUC, 13,855 ng. h/ml). Visual disturbances occurred in 5 (12.8%) of the 39 patients and were the only drug-related adverse events that occurred more than once. No withdrawals from the study were related to voriconazole. We conclude that pediatric patients have a higher capacity for elimination of voriconazole per kilogram of body weight than do adult healthy volunteers and that dosages of 4 mg/kg may be required in children to achieve exposures consistent with those in adults following dosages of 3 mg/kg.

Issues in Pharmacokinetics and Pharmacodynamics of Anti-Infective Agents: Kill Curves versus MIC

Abstract: The success of antimicrobial therapy is determined by complex interactions between an administered drug, a host, and an infecting agent. In a clinical situation, the complexity of these interactions is usually reflected by a high variability in the dose-response relationship. Therefore, to minimize the dose-response variability, key characteristics of the drug, the infecting agent and the host have to be taken into account for selecting an appropriate antibiotic and an appropriate dose. Failure to do so may result in either therapeutic failure or emergence of resistant strains. To date, dose and drug selection is mostly based on a static in vitro parameter, the MIC and on the drug′s serum concentration as a pharmacokinetic parameter. In practice, however, a pharmacodynamic effect in vivo is rather the result of a dynamic exposure of the infective agent to the unbound antibiotic drug fraction at the relevant effect site. Thus, static conditions in an in vitro setting hardly reflect a dynamic situation in a target organ under in vivo conditions. Furthermore, serum concentrations do not reflect the unbound concentrations at the target site. In recent years substantial efforts were devoted to systematically elucidate the dynamic relationship between pharmacokinetic and pharmacodynamic variables. The main concept of this pharmacokinetic-pharmacodynamic approach is to use the concentration-effect relationship of the drug of interest in dosage adjustment and product development in a logical way and minimize trial-and-error approaches (29, 80). This approach can potentially result in substantial savings of time and expenses and may help to avoid unnecessary and, hence, unethical clinical studies (97). Thus, dosages and dosing intervals of antimicrobial agents should be designed with reference to dynamic pharmacokinetic and pharmacodynamic parameters. Accordingly, several efficacy indices or surrogate markers that take into account both pharmacokinetic and pharmacodynamic information have been defined and used by different authors to describe the antibacterial activity of various classes of antimicrobial agents (100, 106, 59). Currently there are two main trends for antibiotic pharmacokinetic-pharmacodynamic models; those based on the MIC and those based on a kill-curve approach, both of which will be described in detail in the following.

Impact of High-Inoculum Staphylococcus aureus on the Activities of Nafcillin, Vancomycin, Linezolid, and Daptomycin, Alone and in Combination with Gentamicin, in an In Vitro Pharmacodynamic Model

Abstract: We evaluated the impact of high (9.5 log10 CFU/g) and moderate (5.5 log10 CFU/g) inocula of methicillin-susceptible and -resistant Staphylococcus aureus (MSSA and MRSA, respectively) on the activities of nafcillin, linezolid, vancomycin, and daptomycin, alone and in combination with gentamicin in an in vitro pharmacodynamic model with simulated endocardial vegetations over 72 h. Human therapeutic dosing regimens for nafcillin, daptomycin, vancomycin, linezolid, and gentamicin were simulated. At a moderate inoculum, nafcillin (MSSA only), vancomycin, and daptomycin demonstrated equivalent and significant (P < 0.01) bactericidal (99.9% kill) activities (decreases of 3.34 +/- 1.1, 3.28 +/- 0.4, and 3.34 +/- 0.8 log10 CFU/g, respectively). Bactericidal activity was demonstrated at 4 h for nafcillin and daptomycin and at 32 h for vancomycin. Linezolid demonstrated bacteriostatic activity over the course of the study period. At a high inoculum, daptomycin exhibited bactericidal activity against both MSSA and MRSA by 24 h (decrease of 5.51 to 6.31 +/- 0.10 log10 CFU/g). Nafcillin (versus MSSA), vancomycin, and linezolid (MSSA and MRSA) did not achieve bactericidal activity throughout the 72-h experiment. The addition of gentamicin increased the rate of 99.9% kill to 8 h for daptomycin (P < 0.01) and 48 h for nafcillin (MSSA only) (P = 0.01). The addition of gentamicin did not improve the activity of vancomycin or linezolid for either isolate for the 72-h period. Overall, high-inoculum Staphylococcus aureus had a significant impact on the activities of nafcillin and vancomycin. In contrast, daptomycin was affected minimally and linezolid was not affected by inoculum.

Novel, Rapid, and Inexpensive Cell-Based Quantification of Antimalarial Drug Efficacy

Abstract: We report on the development of a new SYBR Green I-based plate assay for analyzing the activities of antimalarial drugs against intraerythrocytic Plasmodium falciparum. This assay is considerably faster, less labor-intensive, and less expensive than conventional radiotracer (e.g., [3H]hypoxanthine and [3H]ethanolamine)-based assays or P. falciparum lactate dehydrogenase activity-based assays. The assay significantly improves the pace at which antimalarial drug discovery efforts may proceed.

Update of the Standard Numbering Scheme for Class B β-Lactamases

Abstract: β-Lactamases represent the major cause of bacterial resistance against β-lactam antibiotics, and they have been divided into four classes (A to D) on the basis of their amino acid sequences (21). The class B enzymes have no sequence or structural similarity to the active-site serine enzymes of classes A, C, and D (6); require a bivalent metal ion (Zn2+) for activity; and constitute group 3 in the Bush-Jacoby-Medeiros functional classification (2). The identification of Zn-β-lactamase-producing pathogenic strains of Aeromonas, Bacteroides, Flavobacterium, Legionella, Serratia, and Stenotrophomonas has greatly increased interest in this class of enzymes (2). The fact that they hydrolyze almost all β-lactam antibiotics, including carbapenems, underlines their clinical relevance. In consequence, the potential spreading of these enzymes among pathogenic bacteria is a frightening possibility, which emphasizes the importance of understanding their properties. On the basis of the sequences, three subclasses of class B β-lactamases (B1 to B3) were identified, and a standard numbering scheme (BBL numbering) was proposed (13) by analogy to the ABL numbering scheme which has been widely used for class A β-lactamases. Due to the general low degree of identity between subclass sequences (<20%), classical alignment programs produce unreliable results. The proposed alignment (13) was facilitated by the availability of X-ray structures for B1 and B3 enzymes. Crystallographic structures have been described for several B1 enzymes: Bacillus cereus BcII (4, 11), Bacteroides fragilis CcrA (5, 8), Pseudomonas aeruginosa IMP-1 (7) and VIM-2 (unpublished data), and Chryseobacterium meningosepticum BlaB (14). Structural data are also available for two B3 enzymes: Stenotroptromonas maltophilia L1 (28) and Legionella gormanii FEZ-1 (15). Recently, we solved the first X-ray structure of a subclass B2 enzyme (CphA) produced by various species of Aeromonas (G. Garau, C. Bebrone, C. Anne, M. Galleni, J.-M. Frere, and O. Dideberg, unpublished data). Using all available three-dimensional structures, it is now possible to propose a bonafide structural alignment of the class B β-lactamases, and accordingly, to update the first proposed BBL scheme (Fig. ​(Fig.11). FIG. 1. Structural alignment of eight class B β-lactamases with known X-ray structures. The sequences are referred to by their familiar names. BCII, B. cereus 569H (16); IMP-1, P. aeruginosa 101/477 (17); CcrA, B. fragilis TAL3636 (25); VIM-2, P. aeruginosa ... For the three-dimensional structure comparison of the eight available structures, we used the program TOP (18) with the new option MAPS, allowing multiple alignments of protein structures. In addition, the program produces two ranking scores: the sequence identities of aligned residues and the structural diversity. The structural-diversity score was defined as RMS/(Nmatch/N0)3/2, where RMS is the root mean square deviation of the distances between matched Cα atoms, Nmatch is the number of matching residues, and (Nmatch/N0) is the matching fraction of two compared structures. N0 = (N1 + N2/2), where N1 and N2 are the numbers of amino acids in the two compared proteins. This score estimates the evolutionary distance between proteins. These two scores are shown in Table ​Table11 for all known X-ray structures. TABLE 1. Sequence identities of aligned residues and structure diversity among proteins Figure ​Figure11 displays the proposed alignment and numbering. Interestingly, the numbering of the important class B residues is conserved between old and new alignments. Improvements in the alignment concern mainly N and C termini and small shifts along the sequences. The main result of the new alignment is the identification of 14 sequence fragments of structurally conserved positions, which cover the entirety of all sequences (Fig. ​(Fig.1);1); they belong mainly to secondary-structure elements (α helices or β sheets). Notably, all Zn ligands are structurally aligned. The following comments can be made. (i) Only sequences of proteins of known structures are shown. (ii) For residues in lightface, the fact that they have the same number does not imply that they are structurally equivalent. (iii) For newly discovered enzymes, any insertion departing from the present numbering can be characterized by lowercase letters following the number of the last residue of the consensus sequence. Table ​Table22 shows the numbering of the putative zinc ligands. Not all proteins of known sequence are shown. Only enzymes with <50% sequence identity compared to the first reported sequence are included in the table. TABLE 2. Numbering of important class B residues In 1997, Neuwald et al. (23) detected a few proteins that have sequence similarities to (and may have given rise to) Zn-β-lactamases. They include enzymes with large variations in function (sulfatase; DNA cross-link repair enzyme) and which are encoded by yeast, plant, or bacterial open reading frames. Human glyoxalase II was also shown to belong to the superfamily. More recently, 17 groups with known functions were identified (9). In order to evaluate the structural diversity of the Zn-β-lactamase superfamily, human glyoxalase II (3) and rubredoxin oxygen-oxidoreductase from Desulfovibrio gigas (12) were also aligned using TOP, along with one member of each subclass. Table ​Table33 shows the sequence identities and structural diversity of the two proteins and BCII, CphA, and FEZ-1. As expected, low sequence identity corresponds to a high structural-diversity score. The structural-diversity scores for proteins belonging to a superfamily range from 1.4 to 2, in contrast to 3.5 to 4 for proteins with different folds (18). Interestingly and surprisingly, FEZ-1 is closer to glyoxalase II and rubredoxin oxygen-oxidoreductase than to BcII or CphA. TABLE 3. Sequence identities of aligned residues and structural diversity among proteins In the structural alignment, a large number of amino acid changes and insertions-deletions are observed. One hypothesis is that an ancient protein gave rise to the different subclasses of Zn-β-lactamases. A few candidates for the ancient protein are those related to essential biological functions within the cell, such as DNA or RNA processing or DNA repair (9). Nature used a limited number of scaffolds to generate a large variety of biological functions. Zn-β-lactamases are good examples of such a selection.