Thirteen-Year Evolution of Azole Resistance in Yeast Isolates and Prevalence of Resistant Strains Carried by Cancer Patients at a Large Medical Center

Drug resistance is emerging in many important microbial pathogens, including Candida albicans. We performed fungal susceptibility tests with archived isolates obtained from 1984 through 1993 and fresh clinical isolates obtained from 1994 through 1997 by testing their susceptibilities to fluconazole, ketoconazole, and miconazole and compared the results to the rate of fluconazole use. All isolates recovered prior to 1993 were susceptible to fluconazole. Within 3 years of widespread azole use, we detected resistance to all agents in this class. In order to assess the current prevalence of resistant isolates in our hematologic malignancy and transplant patients, we obtained rectal swabs from hospitalized, non-AIDS, immunocompromised patients between June 1995 and January 1996. The swabs were inoculated onto sheep’s blood agar plates containing 10 μg of vancomycin and 20 μg of gentamicin/ml of agar. One hundred one yeasts were recovered from 97 patients and were tested for their susceptibilities to amphotericin B, fluconazole, flucytosine, ketoconazole, and miconazole. The susceptibility pattern was then compared to those for all clinical isolates obtained throughout the medical center. The antifungal drug histories for each patient were also assessed. The yeasts from this surveillance study were at least as susceptible as the overall hospital strains. There did not appear to be a direct linkage between prior receipt of antifungal agent therapy and carriage of a new, drug-resistant isolate. Increased resistance to newer antifungal agents has occurred at our medical center, but it is not focal to any high-risk patient population that we studied. Monitoring of susceptibility to antifungal agents appears to be necessary for optimizing clinical therapeutic decision making.Institutions across the United States have reported an increase in their rate of nosocomial fungal infections (3). In the 1980s Candida species were responsible for approximately three-quarters of these fungal infections (3, 24), with Candida albicans being the most commonly isolated (59.7%) species (3). The greatest increase has been noted in bloodstream infections (2, 3), focused primarily in critical care units (3, 35). The rise in fungemia has been striking, ranging from 75% in small (≤200 beds) nonteaching hospitals to 487% in large (>500 beds) teaching hospitals (2). High rates of morbidity and mortality are associated with candidal infections in immunocompromised patients (3, 15, 23, 24, 35, 37, 38), and early diagnosis can be difficult. With Candida bloodstream infections accounting for the highest rates of mortality, Pittet and colleagues found that even single positive cultures could not be ignored (26). Mindful of these factors, the use of antifungal agents for prophylaxis and therapy has grown (1, 28). Recent reports have also suggested an increasing prevalence of yeast isolates resistant to the newer azole class of antifungal agents (27, 29), implying that future antifungal treatment and prophylaxis may be more difficult.We undertook the current study to (i) determine the prevalence of resistance in yeast isolates colonizing non-AIDS, immunocompromised patients at Northwestern Memorial Hospital (NMH) and (ii) to assess any association of increased use of imidazoles, particularly fluconazole, to the increasing rate of resistance of yeasts to these agents.     

Kenyan Plasmodium falciparum Field Isolates: Correlation between Pyrimethamine and Chlorcycloguanil Activity In Vitro and Point Mutations in the Dihydrofolate Reductase Domain

Sixty-nine Kenyan Plasmodium falciparum field isolates were tested in vitro against pyrimethamine (PM), chlorcycloguanil (CCG), sulfadoxine (SD), and dapsone (DDS), and their dihydrofolate reductase (DHFR) genotypes were determined. The in vitro data show that CCG is more potent than PM and that DDS is more potent than SD. DHFR genotype is correlated with PM and CCG drug response. Isolates can be classified into three distinct groups based on their 50% inhibitory concentrations (IC₅₀s) for PM and CCG (P < 0.01) and their DHFR genotypes. The first group consists of wild-type isolates with mean PM and CCG IC₅₀s of 3.71 ± 6.94 and 0.24 ± 0.21 nM, respectively. The second group includes parasites which all have mutations at codon 108 alone or also at codons 51 or 59 and represents one homogeneous group for which 25- and 6-fold increases in PM and CCG IC₅₀s, respectively, are observed. Parasites with mutations at codons 108, 51, and 59 (triple mutants) form a third distinct group for which nine- and eightfold increases in IC₅₀s, respectively, of PM and CCG compared to the second group are observed. Surprisingly, there is a significant decrease (P < 0.01) of SD and DDS susceptibility in these triple mutants. Our data show that more than 92% of Kenyan field isolates have undergone at least one point mutation associated with a decrease in PM activity. These findings are of great concern because they may indicate imminent PM-SD failure, and there is no affordable antimalarial drug to replace PM-SD (Fansidar).The spread of chloroquine (CQ)-resistant Plasmodium falciparum populations in areas in Africa where malaria is endemic (5, 23, 48) has required that other drugs be introduced for malaria treatment. Pyrimethamine (PM) and proguanil (Paludrine) are specific competitive inhibitors of dihydrofolate reductase (DHFR), a key enzyme in nucleotide biosynthesis (13). Sulfadoxine (SD) is thought to inhibit dihydropteroate synthase (DHPS), an enzyme that catalyzes a reaction in the synthesis of folate in P. falciparum (13). Proguanil is metabolized to its active form, cycloguanil (CG), and has been used primarily for chemoprophylaxis. A combination of PM and SD (Fansidar) acts synergistically to inhibit the folate pathway (9).Genetic analysis of P. falciparum isolates has demonstrated that antifolate resistance in P. falciparum is caused by point mutations in the gene that encodes the protein target of PM, DHFR, leading to amino acid changes in the active site of the enzyme. The amino acid serine at position 108 (Ser-108) is linked to sensitivity to both PM and CG. The Ser-108-to-Asn-108 mutation confers resistance to PM, and Thr-108 is associated with resistance to CG (paired with a mutation of Ala-16 to Val-16). Subsequent mutations of Asn-51 to Ile-51, Cys-59 to Arg-59, and Ile-164 to Leu-164 enhance the resistance of the isolates to PM. These findings were based on the determination of the complete DNA sequence of the DHFR-coding region in a series of P. falciparum isolates whose sensitivities to PM and CG had been previously determined by in vitro testing (10, 14, 24, 25, 55). Isolates from some sub-Saharan and Southeast Asian countries have been analyzed by DNA sequencing (3, 4). This technique is reliable but expensive and time-consuming, which limits its use in large-scale epidemiological studies in areas of endemicity. Alternatively, PCR can be adapted for the rapid detection of sequences differing by a single base pair (8, 17, 21, 32, 35). Specific primers for allele-specific PCR (ASPCR) have been designed for the detection of mutations at codons 16, 108, and 164 in the P. falciparum DHFR gene (15, 26, 27). This approach has been used to detect mutations at codon 108 in epidemiological investigations of Brazilian (26) and Malian (28) P. falciparum isolates and in a comparative study on isolates from East and West Africa and South America (29). Restriction enzyme digestion of PCR products can also be used to identify some of these point mutations in the P. falciparum DHFR gene (12, 53).PM-SD is cheap and well tolerated and is increasingly being used as a first-line treatment against uncomplicated malaria in Kenya. As a result, there is a significant reduction in in vitro chemosensitivity to PM in Kenyan parasites. In the 1980s, an average of 20% of the samples tested were classed as PM resistant (37, 44, 46), but that average rose to about 90% between 1993 and 1995 (18). We report here the application of ASPCR and enzyme digestion methods for the detection of point mutations at codons 51, 59, 108, and 164 of the DHFR gene in in vitro-adapted Kenyan P. falciparum field isolates. The goal was to determine whether particular point mutations in the DHFR-coding region are correlated in each isolate with the isolates’ chemosensitivities to the DHFR inhibitors PM and CCG (the active metabolite of chlorproguanil [CPG] [Lapudrine]) or to the DHPS inhibitors SD and dapsone (DDS). CCG and DDS were included in the study because this combination has proved to be particularly potent against P. falciparum isolates in vitro (50) and in vivo (1).     

New Trimethoprim-Resistant Dihydrofolate Reductase Cassette,dfrXV, Inserted in a Class 1 Integron

The nucleotide sequence of a plasmid-borne trimethoprim resistance gene from a commensal fecal Escherichia coli isolate revealed a new dihydrofolate reductase gene, dfrXV, which occurred as a gene cassette integrated in a site-specific manner in a class 1 integron. The new gene shows 84% nucleotide identity and the predicted protein shows 90% amino acid identity with dfrI and DHFR type I, respectively. Genes for spectinomycin resistance, aadA1 [ant (3′′)-Ia], and sulfonamide resistance, sulI, were located downstream of dfrXV in a manner identical to that in pLMO229.Trimethoprim is an antimicrobial agent used on its own or in combination with sulfamethoxazole in the treatment of infections caused by gram-negative organisms. Trimethoprim selectively inhibits the bacterial dihydrofolate reductase (DHFR), thus preventing the reduction of dihydrofolate to tetrahydrofolate (8). The most common mechanism of resistance to trimethoprim in enterobacteria is the production of an additional plasmid-mediated DHFR which, unlike the chromosomal enzyme, is less sensitive to inhibition by trimethoprim (5). Sixteen trimethoprim resistance enzymes have been identified in enterobacteria and have been characterized and grouped on the basis of their nucleotide sequences and kinetic properties. The largest of these groups and by far the most prevalent are the type I-like enzymes, which include dfrI, dfrIb, dfrV, dfrVI, and dfrVII (14). This enzyme group is characterized by an open reading frame (ORF) of 157 amino acid residues, and the members of this group share between 64 and 88% amino acid sequence identity in this ORF (14). The majority of these enzymes have been found as gene cassettes inserted into the recombinationally active sites of integrons (22). In a survey of trimethoprim resistance in South Africa, 357 isolates of gram-negative, aerobic, commensal fecal flora were probed with oligonucleotide probes to determine the prevalence of DHFR resistance genes within the population (2, 3). Hybridization experiments revealed that contrary to all previous data, the most prevalent DHFR was type Ib (21.8%), followed by types VII (18.8%), I (14.6%), VIII (12.9%), XIII (12.3%), V (7.8%), and XII (0.3%) (1, 3). Forty-six of 357 isolates did not hybridize to any of the DHFR probes. One of these isolates, Escherichia coli UI14, which is highly resistant to trimethoprim (MIC, >2,048 μg/ml), was shown to transfer a 101-kb plasmid (pUK2317) which confers resistance to trimethoprim, spectinomycin, tetracycline, and sulfonamides to a recipient strain, E. coli J62-2, by conjugation (4).     

Drift in Susceptibility of Neisseria gonorrhoeae to Ciprofloxacin and Emergence of Therapeutic Failure

Ciprofloxacin, 500 mg, was introduced as the first-line therapy for gonorrhea at St. Mary’s Hospital, London, in 1989, when a surveillance program was initiated to detect the emergence of resistance. Isolates of Neisseria gonorrhoeae from consecutive patients attending the Jefferiss Wing, Genitourinary Medicine Clinic at St. Mary’s Hospital, between 1989 and 1997 have been tested for susceptibility to ciprofloxacin by using an agar dilution breakpoint technique. Isolates considered potentially resistant (MIC, >0.12 μg/ml) were further characterized by determination of the MICs of ciprofloxacin, nalidixic acid, and penicillin, auxotyped and serotyped, and screened for mutations in the DNA gyrase gene, gyrA, and the topoisomerase IV gene, parC. A total of 4,875 isolates were tested. While the majority of isolates were highly susceptible (MIC, ≤0.008 μg of ciprofloxacin/ml), there was a drift toward reduced susceptibility in N. gonorrhoeae isolated between 1993 and 1996 (P < 0.001). In 1997 this drift was reduced but remained above pre-1993 levels. Isolates from 18 patients were classed as potentially resistant (MIC, >0.12 μg/ml); all of these belonged to serogroup B, and NR/IB-1 was the most common auxotype/serovar class. The infections in 14 of the 18 patients were known to be acquired abroad, and 5 were known to result in therapeutic failure. The surveillance program has established that ciprofloxacin is still a highly effective antibiotic against N. gonorrhoeae in this population. However, it has identified a drift in susceptibility which may have resulted from increased usage of ciprofloxacin. High-level resistance has now emerged, although treatment failure is still uncommon.Ciprofloxacin is a fluoroquinolone that is highly effective against Neisseria gonorrhoeae. The spread of isolates of N. gonorrhoeae exhibiting chromosomal and/or plasmid-mediated resistance to penicillin in the last decade has resulted in increased usage of ciprofloxacin for the therapy of gonorrhea. Ciprofloxacin has the advantage of being administered as a single oral dose of either 250 or 500 mg (11). The relationship between dosage, MIC of ciprofloxacin for the infecting isolate, and therapeutic failure was unknown when ciprofloxacin was introduced as the first-line therapy for gonorrhea. Therapeutic failure was undocumented, and resistance mechanisms, attributed to mutations in DNA gyrase genes and reduced permeability of the cell membrane in other organisms, were unknown for N. gonorrhoeae. Subsequently, low-level resistance was reported for N. gonorrhoeae isolates for which MICs were 0.06 to 0.5 mg/liter (8, 14, 18, 21–23, 25, 27, 29, 30), followed by reports of high-level resistance in isolates for which MICs were >1 mg/liter (3, 4, 6, 8, 22, 28–30). Mutations in the DNA gyrase gene, gyrA, have been found in these isolates (6, 7, 9, 26), and high-level resistance has been associated with additional mutations in the topoisomerase gene, parC, in some isolates (8, 10).Ciprofloxacin as a single 500-mg dose was introduced as the first-line herapy for gonorrhea at St. Mary’s Hospital in 1989. At the same time we initiated a surveillance program to monitor the susceptibility of all isolates to ciprofloxacin and to detect the emergence of resistance and hence the efficacy of ciprofloxacin as a first-line therapy. We report the results of 9 years of surveillance.     

OXA-16, a Further Extended-Spectrum Variant of OXA-10 β-Lactamase,from Two Pseudomonas aeruginosa Isolates

Two extended-spectrum mutants of the class D β-lactamase OXA-10 (PSE-2) from Pseudomonas aeruginosa isolates obtained in Ankara, Turkey, were described previously and were designated OXA-11 and -14. P. aeruginosa 906 and 961, isolated at the same hospital, were highly resistant to ceftazidime (MIC ≥ 128 μg/ml) and produced a β-lactamase with a pI of 6.2. The MICs of ceftriaxone, cefoperazone, cefsulodin, and cefepime were 4- to 16-fold above the typical values for P. aeruginosa, whereas the MICs of penicillins and cefotaxime were raised only marginally. Ceftazidime MICs were not significantly reduced by clavulanate or tazobactam at 4 μg/ml. Ceftazidime resistance did not transfer conjugatively but was mobilized to P. aeruginosa PU21 by plasmid pUZ8. Both isolates gave similar DNA restriction patterns, suggesting that they were replicates; moreover, they yielded identically sized BamHI fragments that hybridized with a bla_OXA-10 probe. DNA sequencing revealed that both isolates had the same new β-lactamase, designated OXA-16, which differed from OXA-10 in having threonine instead of alanine at position 124 and aspartate instead of glycine at position 157. The latter change is also present in OXA-11 and -14 and seems critical to ceftazidime resistance. Kinetic parameters showed that OXA-16 enzyme was very active against penicillins, cephaloridine, cefotaxime, and ceftriaxone, but hydrolysis of ceftazidime was not detected despite the ability of the enzyme to confer resistance.Resistance to oxyimino cephalosporins in enterobacteria is often associated with extended-spectrum β-lactamases (ESBLs), most of which are mutants of molecular class A β-lactamases, specifically TEM-1 and SHV-1 (2, 19). In Pseudomonas aeruginosa, on the other hand, the most frequent mechanisms of resistance to the oxyimino cephalosporins are derepression of the AmpC chromosomal enzyme and up-regulation of multi-drug efflux (3, 4), and only one extended-spectrum TEM mutant (TEM-42) has been reported (24). Nevertheless, P. aeruginosa has been a major source of unusual ESBLs. Examples include IMP-I, the first plasmidic zinc β-lactamase (32); PER-1, a class A enzyme now widespread in Turkey, though not elsewhere (6, 26, 27, 30); OXA-15, an ESBL mutant of OXA-2 (7); and OXA-11 and -14, which are ESBL mutants of OXA-10 enzyme (5, 11). OXA-11 β-lactamase has two mutations compared with OXA-10, whereas OXA-14 has only one of them. OXA-11 and -14 were produced by isolates from Hacettepe University Hospital in Ankara, Turkey. Total-DNA restriction profiles of these two isolates were identical, and both carried plasmids that gave identical restriction fragments on digestion with EcoRI (5). It is likely that the producer isolates differed only in the presence or absence of the second mutation in the β-lactamase gene (5).In the present study, we describe the discovery and characterization of a further ESBL mutant of OXA-10, also from P. aeruginosa isolates collected from Hacettepe University Hospital.     

Echinocandin Susceptibility Testing of Candida Species: Comparison of EUCAST EDef 7.1, CLSI M27-A3, Etest,Disk Diffusion,and Agar Dilution Methods with RPMI and IsoSensitest Media

This study compared nine susceptibility testing methods and 12 endpoints for anidulafungin, caspofungin, and micafungin with the same collection of blinded FKS hot spot mutant (n = 29) and wild-type isolates (n = 94). The susceptibility tests included EUCAST Edef 7.1, agar dilution, Etest, and disk diffusion with RPMI-1640 plus 2% glucose (2G) and IsoSensitest-2G media and CLSI M27A-3. Microdilution plates were read after 24 and 48 h. The following test parameters were evaluated: fks hot spot mutants overlapping the wild-type distribution, distance between the two populations, number of very major errors (VMEs; fks mutants misclassified as susceptible), and major errors (MEs; wild-type isolates classified as resistant) using a wild-type-upper-limit value (WT-UL) (two twofold-dilutions higher than the MIC₅₀) as the susceptibility breakpoint. The methods with the lowest number of errors (given as VMEs/MEs) across the three echinocandins were CLSI (12%/1%), agar dilution with RPMI-2G medium (14%/0%), and Etest with RPMI-2G medium (8%/3%). The fewest errors overall were observed for anidulafungin (4%/1% for EUCAST, 4%/3% for CLSI, and 3%/9% for Etest with RPMI-2G). For micafungin, VME rates of 10 to 71% were observed. For caspofungin, agar dilution with either medium was superior (VMEs/MEs of 0%/1%), while CLSI, EUCAST with IsoSensitest-2G medium, and Etest were less optimal (VMEs of 7%, 10%, and 10%, respectively). Applying the CLSI breakpoint (S ≤ 2 μg/ml) for CLSI results, 89.2% fks hot spot mutants were classified as anidulafungin susceptible, 60.7% as caspofungin susceptible, and 92.9% as micafungin susceptible. In conclusion, no test was perfect, but anidulafungin susceptibility testing using the WT-UL to define susceptibility reliably identified fks hot spot mutants.Three echinocandin class drugs, anidulafungin, caspofungin, and micafungin, are licensed for the treatment of invasive candidiasis. They are among the preferred agents for invasive candidiasis, as a number of recent fungemia surveys have reported a considerable proportion of cases involving species with reduced susceptibility to fluconazole (3, 4, 24, 28, 31, 37, 44). Additionally, anidulafungin has been associated with an improved success rate, even in cases involving fluconazole-susceptible species (39). Following increased use, sporadic cases of failures associated with elevated MICs have been reported. In the majority of cases, these failures have been associated with mutations in two hot spot regions of FKS genes, which encode the target and major subunit of the 1,3-ß-d-glucan synthase complex (5, 7, 22, 25, 26, 33, 34). Consequently, close monitoring and robust susceptibility testing methods have become increasingly important.EUCAST and CLSI have developed standard methods based on broth dilution for the susceptibility testing of yeasts (9, 41). Methodological differences include glucose concentration, inoculum size, shape of microtiter wells (flat or round), and end-point reading (visual or spectrophotometric), but the methods are more alike than different and in general generate similar results (11, 42). Recently, CLSI proposed an S value of ≤2 μg/ml as a tentative susceptibility breakpoint for caspofungin, micafungin, and anidulafungin for Candida spp., taking into account analysis of mechanisms of resistance, an epidemiological MIC population distribution, parameters associated with success in pharmacodynamic models, and results of clinical efficacy studies (9, 38). As no significant differences in clinical response were noted among the various species, results for all species were merged, and a susceptibility breakpoint of 2 μg/ml was found to encompass the vast majority of isolates, while not bisecting the population of Candida parapsilosis. The crucial issue is whether current susceptibility testing methods and breakpoints clearly and reliably identify isolates with resistance mechanisms associated with treatment failures (5, 7, 8, 13, 14, 16, 18, 22, 25, 26, 33, 40). Not only have cases involving isolates classified as susceptible using the reference methods been shown to contain resistance mutations (5, 7, 13, 14, 22, 25), but also recent studies suggest that a breakpoint of an S value of ≤2 μg/ml may be too high for anidulafungin and micafungin, considering the 1,3-ß-d-glucan synthase kinetic inhibition data of wild-type and mutant enzymes from resistant strains (17, 18). Finally, we recently reported a resistant Candida albicans isolate that failed to be identified as resistant when the reference methodologies were used, while Etest, agar dilution, and disk diffusion methods correctly identified it (5).We therefore undertook a comparative study of the two references methods, a modified EUCAST microdilution method using IsoSensitest medium, agar dilution, and disk and Etest diffusion using RPMI-1640 as well as IsoSensitest medium to evaluate their ability to reliably discriminate between a well-characterized panel of wild-type and fks hot spot mutant Candida isolates. The semisynthetic IsoSensitest medium was chosen as an alternative medium due to this medium having previously been shown to be appropriate for amphotericin B MIC testing (10).     

In Vitro and In Vivo Antifungal Susceptibilities of the Mucoralean Fungus Cunninghamella

We have determined the in vitro activities of amphotericin B (AMB), voriconazole, posaconazole (PSC), itraconazole (ITC), ravuconazole, terbinafine, and caspofungin against five strains of Cunninghamella bertholletiae and four of Cunninghamella echinulata. The best activity was shown by terbinafine against both species (MIC range = 0.3 to 0.6 μg/ml) and PSC against Cunninghamella bertholletiae (MIC = 0.5 μg/ml). We have also evaluated the efficacies of PSC, ITC, and AMB in neutropenic and diabetic murine models of disseminated infection by Cunninghamella bertholletiae. PSC at 40, 60, or 80 mg/kg of body weight/day was as effective as AMB at 0.8 mg/kg/day in prolonging survival and reducing the fungal tissue burden in neutropenic mice. PSC at 80 mg/kg/day was more effective than AMB at 0.8 mg/kg/day in reducing the fungal load in brain and lung of diabetic mice. Histological studies revealed an absence of fungal elements in organs of mice treated with either AMB at 0.8 mg/kg/day or PSC at 60 or 80 mg/kg/day in both models. ITC showed limited efficacy in both models. PSC could be a therapeutic option for the treatment of systemic infections caused by Cunninghamella bertholletiae.The genus Cunninghamella in the order Mucorales encompasses filamentous fungi that are inhabitants of soil and other environments and are common laboratory contaminants. Cunninghamella bertholletiae is the only member of the genus documented to cause human infections (8), although recently the species C. echinulata was also found in clinical samples (3). Infections caused by Cunninghamella are less frequent than those produced by other genera of Mucorales, e.g., Rhizopus and Mucor, but the mortality rate is higher (76%) (5, 19). In general, infections caused by the members of Mucorales are life-threatening and devastating, requiring prompt and aggressive treatment. There are several simultaneous approaches recommended for patient management, including surgical debridement, antifungal therapy, and correction of the underlying predisposing factors, when possible (5, 20).Amphotericin B (AMB) is the drug of choice, although very few data exist on the activity of this drug against some of the less frequent genera of Mucorales, such as Cunninghamella (1, 2). In a recent study in which a large set of clinical isolates of Mucorales was tested, C. bertholletiae demonstrated the highest resistance to AMB in vitro, with only 63% of the isolates tested showing MIC values under the working interpretative breakpoints described by the CLSI (2, 6). In addition, the failure of AMB therapy has been reported in clinical cases caused by Cunninghamella (4, 15, 18, 19). Posaconazole (PSC) appears to be a promising drug for the treatment of zygomycosis, having been successfully used as salvage therapy for patients who are refractory to or intolerant of AMB (10, 23). PSC is safer than AMB and shows good in vitro activity and in vivo efficacy against some zygomycetes (1, 2, 7, 22). There are only two clinical reports on the use of PSC for the management of Cunninghamella infection (9, 16), and there are no data on the use of this drug in animal models of C. bertholletiae infections. The aim of the present study, therefore, was to evaluate the in vitro activities of seven drugs against isolates of both C. bertholletiae and C. echinulata and to assess the in vivo efficacies of AMB, itraconazole (ITC), and PSC in neutropenic and diabetic murine models of disseminated infection by five strains of C. bertholletiae.     

Recombinant Expression and Characterization of the Major β-Lactamase of Mycobacterium tuberculosis

New antibiotic regimens are needed for the treatment of multidrug-resistant tuberculosis. Mycobacterium tuberculosis has a thick peptidoglycan layer, and the penicillin-binding proteins involved in its biosynthesis are inhibited by clinically relevant concentrations of β-lactam antibiotics. β-Lactamase production appears to be the major mechanism by which M. tuberculosis expresses β-lactam resistance. β-Lactamases from the broth supernatant of 3- to 4-week-old cultures of M. tuberculosis H37Ra were partially purified by sequential gel filtration chromatography and chromatofocusing. Three peaks of β-lactamase activity with pI values of 5.1, 4.9, and 4.5, respectively, and which accounted for 10, 78, and 12% of the total postchromatofocusing β-lactamase activity, respectively, were identified. The β-lactamases with pI values of 5.1 and 4.9 were kinetically indistinguishable and exhibited predominant penicillinase activity. In contrast, the β-lactamase with a pI value of 4.5 showed relatively greater cephalosporinase activity. An open reading frame in cosmid Y49 of the DNA library of M. tuberculosis H37Rv with homology to known class A β-lactamases was amplified from chromosomal DNA of M. tuberculosis H37Ra by PCR and was overexpressed in Escherichia coli. The recombinant enzyme was kinetically similar to the pI 5.1 and 4.9 enzymes purified directly from M. tuberculosis. It exhibited predominant penicillinase activity and was especially active against azlocillin. It was inhibited by clavulanic acid and m-aminophenylboronic acid but not by EDTA. We conclude that the major β-lactamase of M. tuberculosis is a class A β-lactamase with predominant penicillinase activity. A second, minor β-lactamase with relatively greater cephalosporinase activity is also present.Tuberculosis causes 3 million deaths annually, more than any other single infectious agent (2, 19, 35). Multidrug resistance is a growing clinical problem, with strains of Mycobacterium tuberculosis exhibiting resistance to 11 or more antimicrobial agents having been described (25). Although it was shown in the 1940s that under certain culture conditions penicillin inhibits the growth of M. tuberculosis (9, 10, 18, 31), the availability of other effective antimicrobial agents limited efforts to determine whether tuberculosis might respond to treatment with β-lactams. However, the recent rise in infections caused by multidrug-resistant strains has made it necessary to identify alternative treatment regimens, including the determination of whether some older classes of antibiotics such as the β-lactams might be effective in the clinical setting.The cell wall structure of M. tuberculosis contains a thick peptidoglycan layer. Cycloserine, a second-line drug in the treatment of tuberculosis, is a d-alanine analog that interferes with peptidoglycan synthesis (37). Recently, it has been shown that M. tuberculosis makes at least four penicillin-binding proteins (PBPs) that bind ampicillin and other β-lactams at clinically relevant antibiotic concentrations (3). The affinities of these agents for their PBP targets are of the magnitude seen for β-lactams that can be effectively used for the treatment of infections caused by other microbes. Also, the outer cellular structures of tubercle bacilli do not represent a major permeability barrier for β-lactams (3, 22). Therefore, the production of β-lactamase by M. tuberculosis appears to be its major mechanism of resistance to β-lactams.Most and possibly all isolates of M. tuberculosis produce β-lactamase (12, 13, 15, 42); however, data regarding its nature are limited. Opinions differ as to whether it is secreted, cytoplasmic, or bound to the cell membrane and as to whether its production is inducible or constitutive (10, 14, 15, 32, 42). Zhang et al. (42) have reported that isoelectric focusing of Triton X-100 extracts of acetone-precipitated cell pellets of strains of M. tuberculosis reveals two bands exhibiting β-lactamase activity with pI values of 4.9 and 5.1.Most of the information on the kinetic properties of M. tuberculosis β-lactamase comes from studies with relatively impure preparations of enzyme or has been inferred indirectly via the results of susceptibility tests involving β-lactams and β-lactam–β-lactamase inhibitor combinations. However, greater penicillinase activity than cephalosporinase activity is consistently reported (15, 20, 22, 42). M. tuberculosis β-lactamase is inhibited competitively by antistaphylococcal penicillins (13–15, 21, 22, 32) and by conventional β-lactamase inhibitors including clavulanic acid, sulbactam, and tazobactam (5, 8, 33, 38, 41, 42). β-Lactamase inhibitors improve the activities of some penicillins against M. tuberculosis in vitro (5, 8, 14, 33) and in vivo (13). In addition, some cephalosporins including ceforanide and cephapirin as well as carbapenems such as imipenem exhibit potent in vitro activities (23, 30, 36).Because a better understanding of the mechanisms by which M. tuberculosis expresses resistance to β-lactams might ultimately lead to strategies in which these agents could be used in the treatment of tuberculosis, we have worked to characterize its β-lactamase(s). In this report, we describe the isolation of three enzymes with distinct pI values directly from M. tuberculosis and the recombinant expression and kinetic characterization of the major enzyme.(Results of this study were presented in part at the 36th Interscience Conference on Antimicrobial Agents and Chemotherapy, New Orleans, La., 15 to 18 September 1996, and at the 32nd U.S.-Japan Conference of Tuberculosis/Leprosy, Cleveland, Ohio, 21 to 23 July 1997.)     

Structure of CARB-4 and AER-1 CarbenicillinHydrolyzing β-Lactamases

We determined the nucleotide sequences of bla_CARB-4 encoding CARB-4 and deduced a polypeptide of 288 amino acids. The gene was characterized as a variant of group 2c carbenicillin-hydrolyzing β-lactamases such as PSE-4, PSE-1, and CARB-3. The level of DNA homology between the bla genes for these β-lactamases varied from 98.7 to 99.9%, while that between these genes and bla_CARB-4 encoding CARB-4 was 86.3%. The bla_CARB-4 gene was acquired from some other source because it has a G+C content of 39.1%, compared to a G+C content of 67% for typical Pseudomonas aeruginosa genes. DNA sequencing revealed that bla_AER-1 shared 60.8% DNA identity with bla_PSE-3 encoding PSE-3. The deduced AER-1 β-lactamase peptide was compared to class A, B, C, and D enzymes and had 57.6% identity with PSE-3, including an STHK tetrad at the active site. For CARB-4 and AER-1, conserved canonical amino acid boxes typical of class A β-lactamases were identified in a multiple alignment. Analysis of the DNA sequences flanking bla_CARB-4 and bla_AER-1 confirmed the importance of gene cassettes acquired via integrons in bla gene distribution.Penicilloyl serine transferases, routinely called β-lactamases, cleave the cyclic amide bond of β-lactam antibiotics via the formation of a serine ester-linked penicilloyl enzyme giving a product devoid of antibacterial activity (46). A close inspection of databases indicated that in the last 3 years, a collection of at least 150 DNA sequences from plasmid-mediated and chromosomal bla genes has been acquired. Analysis of deduced peptides confirmed that most have conserved motifs typical of serine active-site enzymes that are divided into three major classes (classes A, C, and D) on the basis of a level of amino acid sequence identity of more than 20% between members in each class (10).In 1969, a β-lactamase was found in Pseudomonas aeruginosa Dalgleish, it was noticed to be “markedly active against carbenicillin,” and the enzyme was named PSE-4 (13, 32). As other β-lactamase enzymes were found, it was noticed that some β-lactamases have better activities than others against carbenicillin. All β-lactamases except class C enzymes hydrolyze carbenicillin at very different levels; class C enzymes hydrolyse it poorly. Genes encoding enzymes similar to PSE-4 were subsequently discovered in other bacterial species and are now known to be part of multidrug resistance transposons (24). In addition to the four original β-lactamases called PSE-1, PSE-2, PSE-3, and PSE-4, a plethora of plasmid-mediated enzymes capable of hydrolyzing carbenicillin at a high rate, such as LCR-1 (10), AER-1 (16), CARB-3 (22), NPS-1 (26), CARB-5 (35), and CARB-4 (36), were identified; but these enzymes have subtle differences in their biochemical properties and in their substrate profiles (7). The amino acid sequences of PSE-1 (17), PSE-2 (18), PSE-3 (8), PSE-4 (4), and CARB-3 (23) have been compared to those of other class A and class D enzymes, and it has been confirmed that PSE-2 (OXA-10) is a class D enzyme (10).The relationship of CARB-4 to other plasmid-mediated β-lactamases has been tested by determining the neutralization of benzylpenicillin-hydrolyzing activity with antisera prepared against purified the TEM-1, OXA-4, and CARB-3 β-lactamases (36). Antisera prepared with CARB-3 antigen inactivated the CARB-4 β-lactamase as well as the PSE-1, PSE-4, and CARB-3 enzymes (22, 36). The bla_CARB-3 and bla_CARB-4 genes are localized within transposons Tn1413 (7 kb) and Tn1408 (25 kb), respectively; these mobile elements were from plasmids isolated from bacterial strains of distinct origins (24, 27, 47).Unusual β-lactamases such as a metalloenzyme have been reported in Aeromonas hydrophila, a water-borne, gram-negative rod known to be highly resistant to β-lactam antibiotics, including carbenicillin (42). A carbenicillin-hydrolyzing β-lactamase has been discovered in an isolate of A. hydrophila from blood (16). The substrate profile of the AER-1 enzyme resembled those of plasmid-mediated carbenicillin-hydrolyzing enzymes, but it had a different isoelectric point (pI 5.9) and molecular mass (29 kDa); these values are reminiscent of those for BRO-1 (pI 5.45), PSE-1 (pI 5.7), CARB-3 (pI 5.75), and CARB-5 (pI 5.35). The gene coding for AER-1 is part of the Ω7711 unit which is IncP mobilizable but RecA dependent and which inserts only between purC and guaB at a specific site in the Escherichia coli chromosome (16). The Ω7711 unit cotransfers resistance to the antibiotics chloramphenicol, streptomycin, and sulfonamide; the transfer of multidrug resistance and insertion at a unique site are properties analogous to those of Tn7.In the study described in this report, we have focused on a carbenicillin-hydrolyzing enzyme identified from a clinical isolate, P. aeruginosa p83372, containing the pUD12 plasmid and producing CARB-4. This enzyme has an acidic isoelectric point (pI 4.3) and hydrolyzes carbenicillin very efficiently (36). We also present the nucleotide sequences of bla_AER-1 and bla_CARB-4, including flanking sequences containing integrons that explain their distribution and presence in different mobile genetic elements (15). We compared the deduced AER-1 and CARB-4 polypeptides with those of other group 2c enzymes (7) via a multiple alignment.     

Antimicrobial Susceptibility and Mechanisms of Resistance in Shigella and Salmonella Isolates from Children under Five Years of Age with Diarrhea in Rural Mozambique

The antimicrobial susceptibility and mechanisms of resistance of 109 Shigella and 40 Salmonella isolates from children with diarrhea in southern Mozambique were assessed. The susceptibility to seven antimicrobial agents was tested by disk diffusion, and mechanisms of resistance were searched by PCR or colorimetric method. A high proportion of Shigella isolates were resistant to chloramphenicol (Chl) (52%), ampicillin (Amp) (56%), tetracycline (Tet) (66%), and trimethoprim-sulfamethoxazole (Sxt) (84%). Sixty-five percent of the isolates were multidrug resistant. Shigella flexneri isolates were more resistant than those of Shigella sonnei to Amp (66% versus 0.0%, P < 0.001) and Chl (61% versus 0.0%, P < 0.001), whereas S. sonnei isolates presented higher resistance to Tet than S. flexneri isolates (93% versus 64%, P = 0.02). Resistance among Salmonella isolates was as follows: Tet and Chl, 15% each; Sxt, 18%; and Amp, 25%. Only 3% of Salmonella isolates were resistant to nalidixic acid (Nal), and none to ciprofloxacin or ceftriaxone (Cro). Among Salmonella isolates, multiresistance was found in 23%. Among Shigella isolates, antibiotic resistance was related mainly to the presence of oxa-1-like β-lactamases for Amp, dfrA1 genes for Sxt, tetB genes for Tet, and Chl acetyltransferase (CAT) activity for Chl. Among Salmonella isolates, resistance was conferred by tem-like β-lactamases for Amp, floR genes and CAT activity for Chl, tetA genes for Tet, and dfrA1 genes for Sxt. Our data show that Shigella isolates are resistant mostly to the most available, inexpensive antibiotics by various molecular mechanisms but remain susceptible to ciprofloxacin, Cro, and Nal, which is the first line for empirical treatment of shigellosis in the country.Diarrhea disease is one of the main causes of childhood mortality worldwide. It is estimated that 17% of the 10.8 million deaths of children under 5 years of age worldwide is due to diarrhea, with developing countries being the most affected (6). Diarrhea can be caused by different agents, such as bacteria, parasites, and virus (1, 24, 35). The severity of the illness is mediated by different factors related to both the patient (nutritional status, presence of concomitant illness, and human immunodeficiency virus status) and the etiological agent (specific bacterial virulence and antimicrobial resistance). Salmonella spp. and Shigella spp. remain among the bacteria most frequently isolated from stool samples obtained from diarrhea patients, especially in rural areas from developing countries (24, 35). Salmonella spp. usually produce a self-limited illness, whereas infections due to Shigella are likely to be more severe.The management of acute diarrhea is based on replacement of fluids (8). However, antibiotics might be required for management of the most severe cases or cases involving malnourished children. With shigellosis, appropriate antimicrobial therapy can reduce the duration of fever and the period of shedding of the pathogens (26), which is relevant to transmission of the pathogen to susceptible contacts. At the Manhiça District Hospital (MDH), the combination of ampicillin (Amp) plus gentamicin used with children younger than 2 months of age and chloramphenicol (Chl) used alone with older children are the most available therapeutic options for treating bacterial infections (32), while nalidixic acid (Nal) is recommended for cases of dysentery.Until recently, Salmonella spp. were highly susceptible to the most commonly used antibiotics (34). However, in the last decade, the emergence of multidrug-resistant nontyphoidal Salmonella strains, including isolates resistant to quinolones, has been described worldwide (14, 33, 36). Also, the increase in the number of Shigella isolates resistant to most of the antibiotics available in countries where the choice of treatment is limited (14, 16, 21, 25, 36) represents an important health problem.The mechanisms of antimicrobial resistance are associated with intrinsic resistance, point mutations, and acquired or extrachromosomal resistance (31). A wide range of molecular mechanisms, such as the presence of β-lactamases, dihydrofolate reductase, Chl acetyltransferase (CAT) enzymes and many others (7, 10, 11, 22, 27), have been described. However, few studies have investigated molecular mechanisms of antimicrobial resistance among isolates from sub-Saharan Africa, due mainly to the limited number of laboratories and research facilities with an adequate infrastructure available on the continent (10, 21).Currently, data about antimicrobial resistance among diarrheagenic bacteria in Mozambique are scarce. This study describes the antimicrobial susceptibility and the molecular mechanisms of resistance in Salmonella and Shigella isolates from children with diarrhea in a rural hospital in Mozambique.     

Diversity of β-Lactamases Produced by Ceftazidime-Resistant Pseudomonas aeruginosa Isolates Causing Bloodstream Infections in Brazil

A retrospective survey was conducted to characterize β-lactamases in a collection of 43 ceftazidime-resistant Pseudomonas aeruginosa isolates recovered from patients with bloodstream infections hospitalized at a Brazilian teaching hospital between January and December 2005. Resistance rates for carbapenems, aminoglycosides, and quinolones were over 80%, with only colistin remaining active against all isolates. Pulsed-field gel electrophoresis analysis identified seven different genotypes. AmpC overproduction was found to be the sole β-lactamase-mediated mechanism responsible for ceftazidime resistance in four isolates (9.3%). Nine isolates (20.9%) produced an extended-spectrum β-lactamase (ESBL), either GES-1 (n = 7, 16.3%) or CTX-M-2 (n = 2, 4.6%). Carbapenemase activity was detected in 30 (70%) additional isolates. Among those isolates, two isolates (4.6%) produced the ESBL GES-5, possessing the ability to hydrolyze imipenem; a single isolate (2.3%) produced the metallo-β-lactamase (MBL) IMP-1; and 27 isolates produced the MBL SPM-1 (62.8%). None of the isolates coproduced both ESBL and MBL. Insertion sequence elements ISCR4 and ISCR1 were associated with bla_SPM-1 and bla_CTX-M-2 genes, respectively, whereas the bla_GES-1 and bla_GES-5 genes were part of class 1 integron structures. This study underlines the spread of MBL- and ESBL-producing P. aeruginosa isolates as an important source of ceftazidime resistance in Brazil.Pseudomonas aeruginosa is a leading cause of hospital- acquired infections. Acquisition of β-lactamases, such as class A extended-spectrum β-lactamases (ESBLs) and class B metallo-β-lactamases (MBLs), by P. aeruginosa nosocomial isolates is detrimental to antimicrobial therapy in hospitalized patients (19).The ESBLs reported for P. aeruginosa are SHV, TEM, PER, VEB, BEL, GES, and, more recently, CTX-M types (1, 7, 8, 16, 20, 23, 29). The GES-type enzymes are unusual since point amino acid changes in their active sites may extend their hydrolytic activity to carbapenems (31, 39, 40). ESBL production in P. aeruginosa has been documented in Brazil (2, 5, 21), but its prevalence remains unknown.Five types of acquired MBLs have been identified in P. aeruginosa: IMP, VIM, SPM, GIM, and AIM (41, 42). In Brazil, IMP-, VIM-, and SPM-producing P. aeruginosa clinical isolates have been identified (35). In addition, SPM producers have been reported as endemic in Brazilian territory due to dissemination of a single clone (10).The aim of this study was to investigate the diversity and frequency of both ESBL and MBL production and to characterize the genetic support of those acquired β-lactamase genes in a collection of ceftazidime-resistant P. aeruginosa clinical isolates from Brazil, taken as a model of a developing country.     

Sordarins: In Vitro Activities of New Antifungal Derivatives against Pathogenic Yeasts,Pneumocystis carinii,and Filamentous Fungi

GM 193663, GM 211676, GM 222712, and GM 237354 are new semisynthetic derivatives of the sordarin class. The in vitro antifungal activities of GM 193663, GM 211676, GM 222712, and GM 237354 against 111 clinical yeast isolates of Candida albicans, Candida kefyr, Candida glabrata, Candida parapsilosis, Candida krusei, and Cryptococcus neoformans were compared. The in vitro activities of some of these compounds against Pneumocystis carinii, 20 isolates each of Aspergillus fumigatus and Aspergillus flavus, and 30 isolates of emerging less-common mold pathogens and dermatophytes were also compared. The MICs of GM 193663, GM 211676, GM 222712, and GM 237354 at which 90% of the isolates were inhibited (MIC₉₀s) were 0.03, 0.03, 0.004, and 0.015 μg/ml, respectively, for C. albicans, including strains with decreased susceptibility to fluconazole; 0.5, 0.5, 0.06, and 0.12 μg/ml, respectively, for C. tropicalis; and 0.004, 0.015, 0.008, and 0.03 μg/ml, respectively, for C. kefyr. GM 222712 and GM 237354 were the most active compounds against C. glabrata, C. parapsilosis, and Cryptococcus neoformans. Against C. glabrata and C. parapsilosis, the MIC₉₀s of GM 222712 and GM 237354 were 0.5 and 4 μg/ml and 1 and 16 μg/ml, respectively. The MIC₉₀s of GM 222712 and GM 237354 against Cryptococcus neoformans were 0.5 and 0.25 μg/ml, respectively. GM 193663, GM 211676, GM 222712, and GM 237354 were extremely active against P. carinii. The efficacies of sordarin derivatives against this organism were determined by measuring the inhibition of the uptake and incorporation of radiolabelled methionine into newly synthesized proteins. All compounds tested showed 50% inhibitory concentrations of <0.008 μg/ml. Against A. flavus and A. fumigatus, the MIC₉₀s of GM 222712 and GM 237354 were 1 and 32 μg/ml and 32 and >64 μg/ml, respectively. In addition, GM 237354 was tested against the most important emerging fungal pathogens which affect immunocompromised patients. Cladosporium carrioni, Pseudallescheria boydii, and the yeast-like fungi Blastoschizomyces capitatus and Geotrichum clavatum were the most susceptible of the fungi to GM 237354, with MICs ranging from ≤0.25 to 2 μg/ml. The MICs of GM 237354 against Trichosporon beigelii and the zygomycetes Absidia corymbifera, Cunninghamella bertholletiae, and Rhizopus arrhizus ranged from ≤0.25 to 8 μg/ml. Against dermatophytes, GM 237354 MICs were ≥2 μg/ml. In summary, we concluded that some sordarin derivatives, such as GM 222712 and GM 237354, showed excellent in vitro activities against a wide range of pathogenic fungi, including Candida spp., Cryptococcus neoformans, P. carinii, and some filamentous fungi and emerging invasive fungal pathogens.During the past two decades, the incidence of infections caused by opportunistic fungal pathogens in immunocompromised patients has increased substantially (1, 11, 30, 31, 36). Candida albicans is the major opportunistic pathogen, although the incidence of fungal infections caused by non-C. albicans species is increasing (37). Pneumocystis carinii remains an important pathogen in AIDS patients and other immunocompromised individuals (17), and invasive pulmonary aspergillosis remains a frequently fatal complication of bone marrow transplantation and of cancer chemotherapy in patients with hematologic neoplasms (23, 26, 29). Although there has been an expansion in the number of antifungal drugs available (8–10), in many cases, treatment of fungal diseases remains unsatisfactory. This situation has led to an ongoing search for fungicidal agents with different modes of action and fewer side effects and which can be administered both orally and parenterally.One of the major challenges to finding a potent yet safe antifungal agent is the similarity between fungal and mammalian cells. Like mammalian cells, fungi are eukaryotic, so they have many of the same structures and metabolic pathways as mammalian cells, making it more difficult to find targets of differential toxicity. Although protein synthesis is a universal process in living cells, it has always been considered as one of the more attractive targets for the development of antimicrobial agents (8, 12, 36). It is known that fungal protein has exploitable differences relative to its mammalian counterpart, e.g., the two soluble protein factors elongation factor 3 (EF-3) (19, 34) which is absent from mammalian cells, EF-2, which is functionally distinct from its mammalian counterpart (5, 6). On the basis of these differences, a target-based screening program was established, with the objective of isolating selective protein synthesis inhibitors of the fungal machinery (2). As part of this screening program, a novel antifungal compound, GR 135402, was isolated from fermentation broth of Graphium putredinis and characterized (20). This new compound is the first natural product described to date which possesses antifungal activity through inhibition of fungal but not mammalian protein synthesis (20). GR 135402 belongs to the sordarin class (13), and although it has some structural similarity to zofimarin (27), sordarin (33), and sordarin derivatives (3, 28), no mode of action was described for the antifungal activity of these compounds. A synthetic chemical program was initiated to improve the biological properties of GR 135402, and four compounds, designated GM 193663, GM 211676, GM 222712, and GM 237354, were selected for evaluation.In this study, we analyze the in vitro antifungal activities of these four new sordarin derivatives against several groups of clinical isolates.(This work was presented in part at the 37th Interscience Conference on Antimicrobial Agents and Chemotherapy, Toronto, Ontario, Canada, 28 September to 1 October 1997 [15, 16].)     

Class 1 Integron-Borne Multiple-Antibiotic Resistance Carried by IncFI and IncL/M Plasmids in Salmonella enterica Serotype Typhimurium

The presence and genetic content of integrons were investigated for 37 epidemiologically unrelated multiple-drug-resistant strains of Salmonella enterica serotype Typhimurium from humans. All isolates were resistant to ampicillin, chloramphenicol, kanamycin, streptomycin, sulfonamides, and trimethoprim, as well as to tetracycline and/or nalidixic acid; 20% of them were also resistant to gentamicin and amikacin. Three different class 1 integrons (In-t1, In-t2, and In-t3) were identified by Southern blot hybridization, PCR, and DNA sequencing, and these integrons were found to carry the aadB, catB3, oxa1, aadA1a, aacA4, and aacC1 gene cassettes. Integrons In-t1 (aadB and catB3) and In-t2 (oxa1 and aadA1a) were both located on a conjugative IncFI plasmid of 140 kb. In-t3 (aacA4, aacC1, and aadAIa) was located on an IncL/M plasmid of 100 kb which was present, in association with the IncFI plasmid, in gentamicin- and amikacin-resistant isolates. Despite the extensive similarity at the level of the antibiotic resistance phenotype, integrons were not found on the prototypic IncFI plasmids carried by epidemic Salmonella strains isolated during the late 1970s. The recent appearance and the coexistence of multiple integrons on two conjugative plasmids in the same Salmonella isolate are examples of how mobile gene cassettes may contribute to the acquisition and dissemination of antibiotic resistance.Bacterial resistance to antimicrobial agents is a serious problem worldwide, and understanding of the molecular basis of how resistance genes are acquired and transmitted may contribute to the creation of new antimicrobial strategies (47). One efficient mechanism for the acquisition and dissemination of resistance determinants is their transmission through mobile genetic elements. It has been proposed that promiscuous plasmids, conjugative transposons, and transposons carried by conjugative plasmids are responsible for the horizontal spread of resistance genes throughout bacteria (9). Recently, naturally occurring gene expression elements called “integrons” have been described as vehicles for the acquisition of resistance genes carried by mobile elements (14, 26, 46). These structures have also been found to be involved in the genetic reassortment of resistance determinants frequently observed in multiple-antibiotic-resistant bacterial pathogens (5).Three classes of integrons have been identified. Class 1 integrons are prevalent among clinical isolates and are composed of two conserved regions, the 5′CS and the 3′CS regions, surrounding an interposed variable region (14, 16). The variable region contains gene cassettes for antibiotic resistance integrated, all in the same orientation, at the attI site. Coordinate expression of the gene cassettes is driven by the tandemly arranged P_ant and P2 promoters (39). The 5′CS region of class 1 integrons contains the intI1 gene, which encodes the type 1 integrase protein, which is responsible for site-specific insertion and excision of gene cassettes (5). As a consequence of this activity, integrons exist in a large variety of forms with respect to the number, type, and order of the inserted genes. Each gene cassette includes an open reading frame and a recombination site known as the “59-base element” located downstream of each coding sequence (15). The 3′CS contains the sul1 and the qacEΔ1 genes, which confer resistance to sulfonamides and to quaternary ammonium compounds, respectively (33, 36). Gene cassette arrays similar to those of the class 1 integrons were observed for the transposon Tn7 and its close relatives, forming a second class of integrons. A putative defective integrase gene (intI2), whose product is 40% identical to that of intI1, is located in the distal portion of Tn7 (11). Recently, a third class of integron has been identified, and the putative integrase (intI3), located at the 5′ of the bla_IMP cassette, is 61% identical to the intI1 integrase (31).The occurrence of integrons among clinical bacterial isolates is under investigation, and recent studies report a widespread distribution of these elements among multiple-antibiotic-resistant nosocomial bacteria (18, 20, 34).Salmonella enterica serotype Typhimurium is one of the more frequent agents of bacterial enteritis worldwide (29). This serotype has often been described as being resistant to multiple drugs, most commonly showing resistance to ampicillin (Ap), chloramphenicol (Cm), streptomycin (Sm), sulfonamides (Su), and tetracycline (Te). The R types R-ApCmSmSuTe and R-ApSmSuTe have been associated with the phage types DT104 and DT193, respectively (17, 23, 48).In the study described in this report, we investigated the genetic determinants for antibiotic resistance of S. enterica serotype Typhimurium strains isolated from epidemiologically unrelated pediatric patients with gastroenteritis. Our results indicate that the multiple-drug resistance phenotype is determined by integrons carrying an unusually wide repertoire of resistance gene cassettes. Up to three types of integrons located on two conjugative plasmids were identified. The quality, number, and organization of the resistance genes were then determined.     

Foreign Travel Is a Major Risk Factor for Colonization with Escherichia coli Producing CTX-M-Type Extended-Spectrum β-Lactamases: a Prospective Study with Swedish Volunteers

Foreign travel has been suggested to be a risk factor for the acquisition of extended-spectrum beta-lactamase (ESBL)-producing Enterobacteriaceae. To our knowledge, this has not previously been demonstrated in a prospective study. Healthy volunteers traveling outside Northern Europe were enrolled. Rectal swabs and data on potential travel-associated risk factors were collected before and after traveling. A total of 105 volunteers were enrolled. Four of them did not complete the study, and one participant carried ESBL-producing Escherichia coli before travel. Twenty-four of 100 participants with negative pretravel samples were colonized with ESBL-producing Escherichia coli after the trip. All strains produced CTX-M enzymes, mostly CTX-M-15, and some coproduced TEM or SHV enzymes. Coresistance to several antibiotic subclasses was common. Travel to India was associated with the highest risk for the acquisition of ESBLs (88%; n = 7). Gastroenteritis during the trip was an additional risk factor (P = 0.003). Five of 21 volunteers who completed the follow-up after 6 months had persistent colonization with ESBLs. This is the first prospective study demonstrating that international travel is a major risk factor for colonization with ESBL-producing Enterobacteriaceae. Considering the high acquisition rate of 24%, it is obvious that global efforts are needed to meet the emergence and spread of CTX-M enzymes and other antimicrobial resistances.The prevalence of extended-spectrum beta-lactamase (ESBL)-producing Enterobacteriaceae is increasing globally, and community-onset infections with ESBL-producing Escherichia coli are a major clinical concern in many countries (5, 12, 16, 24). Known risk factors for infection with ESBL-producing bacteria include recent hospitalization or antibiotic use, urinary tract catheters, older age, and diabetes (5, 24, 25).There are marked geographical differences in the proportions of ESBL production among clinical isolates of Klebsiella pneumoniae and E. coli. A high prevalence of ESBL production has been reported for South America (>40% of K. pneumoniae and 5 to 10% of E. coli isolates) and Asia (20 to 30% and 15 to 20%, respectively) (2, 11, 32). The highest rates so far were reported from a multicenter survey study in India (>55% and >60%, respectively) (18). Significantly lower prevalences of ESBL phenotypes have been reported for Europe (10 to 15% and 5 to 10%, respectively) and North America (5 to 10% and <5%, respectively) (2, 9, 20). In Europe, the highest rates of ESBL-producing clinical isolates have been reported for Southern and Eastern European countries (15 to 30%) (7). In contrast, the rate of infections by ESBL-producing K. pneumoniae and E. coli is still very low in Sweden (<3%) (27).Data on fecal carriage with ESBLs in healthy individuals are lacking for most countries, including Sweden, but the rate of fecal carriage has been estimated to be 10% in Asia and was reported to be 5.5% and 13.2% in Spain and Saudi Arabia, respectively (10, 14, 30). Most likely, however, the geographical differences in prevalences of ESBL phenotypes in clinical cultures covary with the proportion of healthy individuals colonized with isolates producing ESBLs.Based on these geographical differences and a few retrospective studies of patients infected with bacteria producing ESBLs, foreign travel to countries with higher prevalences of ESBL-producing Enterobacteriaceae was suggested to be a risk factor for the acquisition of ESBLs (8, 15). A recent Swedish study of patients with traveler''s diarrhea reported an increased prevalence of fecal colonization with ESBL-producing bacteria for patients who had traveled outside Europe compared with the prevalence for those returning from European countries (29). However, no prospective study confirming this finding has previously been reported. The primary objective of our study was to prospectively study the fecal acquisition of ESBL-producing Enterobacteriaceae during foreign travel and the persistence rate of acquired ESBL-producing isolates after 6 months. Our secondary objective was to assess potential travel-associated risk factors for the acquisition of ESBL-producing bacteria, such as destination, gastroenteritis, and antibiotic use during travel.(The results of this study were in part presented at the 48th Interscience Conference on Antimicrobial Agents and Chemotherapy, Washington, DC, 2008.)     

Highly Conserved Neisseria meningitidis Surface Protein Confers Protection against Experimental Infection

A new surface protein, named NspA, which is distinct from the previously described Neisseria meningitidis outer membrane proteins was identified. An NspA-specific mAb, named Me-1, reacted with 99% of the meningococcal strains tested indicating that the epitope recognized by this particular mAb is widely distributed and highly conserved. Western immunoblotting experiments indicated that mAb Me-1 is directed against a protein band with an approximate molecular mass of 22,000, but also recognized a minor protein band with an approximate molecular mass of 18,000. This mAb exhibited bactericidal activity against four meningococcal strains, two isolates of serogroup B, and one isolate from each serogroup A and C, and passively protected mice against an experimental infection. To further characterize the NspA protein and to evaluate the protective potential of recombinant NspA protein, the nspA gene was identified and cloned into a low copy expression vector. Nucleotide sequencing of the meningococcal insert revealed an ORF of 525 nucleotides coding for a polypeptide of 174 amino acid residues, with a predicted molecular weight of 18,404 and a isoelectric point of 9.93. Three injections of either 10 or 20 μg of the affinity-purified recombinant NspA protein efficiently protected 80% of the mice against a meningococcal deadly challenge comparatively to the 20% observed in the control groups. The fact that the NspA protein can elicit the production of bactericidal and protective antibodies emphasize its potential as a vaccine candidate. N eisseria meningitidis causes both endemic and epidemic diseases, principally meningitidis and meningococcemia (1, 2). This pathogenic bacteria primarily affects young children between 6 mo and 2 yr of age, but often infects teenagers (1). The incidence per year of meningococcal diseases during endemic periods is normally ∼1–3 cases per 100,000 in developed countries, but it can be as high as 500 per 100,000 during epidemics (2, 3). N. meningitidis is classified into 12 serogroups based on the immunological characteristics of the capsular polysaccharides found at their surface. Within serogroups, different serotypes, subtypes, and immunotypes can be identified based on the antigenic specificity of the major outer membrane (OM)¹ proteins and LPS (4). Approximately 90% of all meningococcal diseases worldwide are caused by isolates of serogroups A, B, and C (5). Vaccines based on the capsular polysaccharides of serogroups A, C, W-135, and Y were developed and proved efficient to control outbreaks and epidemics of meningococcal diseases (6). However, these vaccines are poorly immunogenic in very young children. Moreover, they do not induce immunological memory and the duration of the protection they provide is relatively short (5, 7–11). Recently, it was demonstrated that conjugation of capsular polysaccharides of serogroups A and C to carrier proteins resulted in a better immunogenicity and a longer persistence of specific antibodies against isolates of these serogroups (12–16). Attempts to develop an efficient vaccine against serogroup B isolates, which are responsible for 50–70% of the meningococcal disease in the developed countries were unsuccessful because the group B capsular polysaccharide is not a good immunogen in human, inducing only a poor IgM response of low specificity which is not protective (17–19). Furthermore, the presence of closely similar, cross-reactive structures in the glycoproteins of neonatal human brain tissue might discourage attempts to improve the immunogenicity of serogroup B polysaccharide (10).To develop a vaccine effective against meningococci of serogroup B several non-capsular surface structures are under investigation (6, 10). Importantly, the presence of bactericidal antibodies against N. meningitidis have been strongly correlated with human immunity and protection (20–22). For that reason, it is believed that non-capsular surface antigens shown to stimulate bactericidal antibodies should be considered as the prime vaccines candidates (6). Early studies using sera of immunized volunteers and convalescent patients indicated that certain meningococcal surface proteins such as the ones responsible for serotype specificity and LPS could induce bactericidal antibodies and be involved in protection (23, 24). mAbs were then used to clearly establish the protective potential of certain meningococcal major surface proteins such as the PorA (class 1), PorB (class 2/3), and Opc (class 5C) (25–28).Different vaccines based on OM proteins were recently evaluated in clinical trials and efficiency between 50 and 80% were recorded (6, 10). These first generation OM proteins vaccines often induced protection against a limited number of strains. Thus, these vaccines could be used during meningococcal epidemics when the antigenic variation of the meningococci causing diseases is relatively low. The specificity of the bactericidal antibodies induced by these vaccines was determined to be directed mainly against PorA and Opc proteins (29, 30). However, the PorA-specific bactericidal antibodies were found to be directed against epitopes located in surface-exposed highly variable regions (31). Moreover, the Opc protein was shown to be produced by only 60% of strains of different serogroups (32), and by ∼20% of serogroup B isolates (33). To improve the protection conferred by the PorA protein, strategies such as multivalent PorA vaccines or the incorporation of additional epitopes on PorA protein are presently under study (34–36). Proteins induced by iron limitation such as FrpB and Tbp-2 are also likely vaccine candidates, but they also show type specificity with respect to the induction of bactericidal antibodies (37–40). Antigenically conserved proteins such as the Lip (or H.8) (41, 42) and the Rmp (or class 4) (43) proteins were identified in the meningococcal OM, but antibodies directed against these proteins were found to be nonbactericidal. Moreover, high concentrations of antibodies to the Rmp protein were also reported to block the bactericidal activity of antibodies directed against PorA protein and could prevent the efficient killing of meningococcal cells (43).In the present report, we described a new highly conserved protein, called NspA for Neisserial surface protein A, which was shown to be present in the OM of all N. meningitidis strains tested. An mAb, named Me-1, which is directed against the NspA protein was found to be bactericidal and to passively protect BALB/c mice against experimental infections. To further characterize this protein and to clearly establish its protective potential, the nspA gene was identified, sequenced and cloned into a low copy expression vector to obtain large quantities of the recombinant protein. In addition, we present data that demonstrate that the injection of purified recombinant NspA protein efficiently protect BALB/c mice against a bacterial challenge with a lethal dose of a meningococcal strain of serogroup B.     

β-Lactamase Inhibitors Are Substrates for the Multidrug Efflux Pumps of Pseudomonas aeruginosa

The MexAB-OprM multidrug efflux system exports a number of antimicrobial compounds, including β-lactams. In an attempt to define more fully the range of antimicrobial compounds exported by this system, and, in particular, to determine whether β-lactamase inhibitors were also accommodated by the MexAB-OprM pump, the influence of pump status (its presence or absence) on the intrinsic antibacterial activities of these compounds and on their abilities to enhance β-lactam susceptibility in intact cells was assessed. MIC determinations clearly demonstrated that all three compounds tested, clavulanate, cloxacillin, and BRL42715, were accommodated by the pump. Moreover, by using β-lactams which were readily hydrolyzed by the Pseudomonas aeruginosa class C chromosomal β-lactamase, it was demonstrated that elimination of the mexAB-oprM-encoded efflux system greatly enhanced the abilities of cloxacillin and BRL42715 (but not clavulanate) to increase β-lactam susceptibility. With β-lactams which were poorly hydrolyzed, however, the inhibitors failed to enhance β-lactam susceptibility in MexAB-OprM⁺ strains, although BRL42715 did enhance β-lactam susceptibility in MexAB-OprM⁻ strains, suggesting that even with poorly hydrolyzed β-lactams this inhibitor was effective when it was not subjected to efflux. MexEF-OprN-overexpressing strains, but not MexCD-OprJ-overexpressing strains, also facilitated resistance to β-lactamase inhibitors, indicating that these compounds are also substrates for the MexEF-OprN pump. These data indicate that an ability to inactivate MexAB-OprM (and like efflux systems in other bacteria) will markedly enhance the efficacies of β-lactam–β-lactamase inhibitor combinations in treating bacterial infections.Pseudomonas aeruginosa is an opportunistic human pathogen characterized by an innate resistance to a variety of antimicrobial agents. Previously attributed to a highly impermeable outer membrane (22), this resistance is now recognized to result from the synergy between broadly specific drug efflux pumps and low outer membrane permeability (16). One such efflux system, encoded by the mexAB-oprM operon (8, 28, 29), effluxes a range of antibiotics, including tetracycline, chloramphenicol, quinolones, β-lactams, novobiocin, macrolides, and trimethoprim (8, 9, 12, 29). Expressed constitutively in wild-type cells, where it contributes to intrinsic drug resistance (5, 12, 29), the operon is hyperexpressed in nalB mutants (30), producing elevated levels of resistance to substrate antibiotics (8, 9, 12, 29). Homologous efflux systems encoded by the mexC-mexD-oprJ (27) and mexE-mexF-oprN (10) operons have also been described. Apparently not expressed during growth under normal laboratory conditions, these systems are expressed in nfxB (27) and nfxC (10) multidrug-resistant mutants, respectively. nfxB strains are resistant to chloramphenicol, tetracycline, quinolones, macrolides, novobiocin, and newer cephalosporins such as cefepime and cefpirome but display hypersusceptibility to most β-lactam antibiotics (18). nfxC strains exhibit resistance to chloramphenicol, trimethoprim, quinolones, and carbapenems, including imipenem, although the resistance to imipenem results from the loss of the porin protein OprD in these mutants and not from the overexpression of MexEF-OprN (6, 10).The tripartite efflux pumps consist of an inner membrane component (MexB, MexD, and MexF) which functions as a resistance-nodulation-division family H⁺ antiport exporter (21, 31), an outer membrane, a presumed channel-forming component (OprM, OprJ, and OprN) (16, 23), and a so-called membrane fusion protein predicted to link the membrane-associated efflux components (MexA, MexC, and MexE) (16, 23). Recent data suggest that the operation of MexAB-OprM (and by analogy the remaining efflux systems) is at least partially dependent upon the TonB energy-coupling protein implicated in the opening of outer membrane gated channels responsible for iron-siderophore uptake across the P. aeruginosa outer membrane (36). Thus, the outer membrane components of these efflux pumps may be gated channels.In an effort to further define the range of antibiotic compounds which are accommodated by the known P. aeruginosa efflux systems, we examined β-lactamase inhibitors as possible pump substrates by assessing the influence of pump status (its presence or absence) on the intrinsic antibacterial activities of these compounds and on their abilities to enhance the efficacies of β-lactam compounds.     

Distribution of Serotypes,Genotypes, and Resistance Determinants among Macrolide-Resistant Streptococcus pneumoniae Isolates

Macrolide resistance in Streptococcus pneumoniae has emerged as an important clinical problem worldwide over the past decade. The aim of this study was to analyze the phenotypes (serotype and antibiotic susceptibility), genotypes (multilocus sequence type [MLST] and antibiotic resistance gene/transposon profiles) among the 31% (102/328) of invasive isolates from children in New South Wales, Australia, in 2005 that were resistant to erythromycin. Three serotypes—19F (47 isolates [46%]), 14 (27 isolates [26%]), and 6B (12 isolates [12%])—accounted for 86 (84%) of these 102 isolates. Seventy four (73%) isolates had the macrolide-lincosamide-streptogramin B (MLS_B) resistance phenotype and carried Tn916 transposons (most commonly Tn6002); of these, 73 (99%) contained the erythromycin ribosomal methylase gene [erm(B)], 34 (47%) also carried the macrolide efflux gene [mef(E)], and 41 (55%) belonged to serotype 19F. Of 28 (27%) isolates with the M phenotype, 22 (79%) carried mef(A), including 16 (57%) belonging to serotype 14, and only six (19%) carried Tn916 transposons. Most (84%) isolates which contained mef also contained one of the msr(A) homologues, mel or msr(D); 38 of 40 (95%) isolates with mef(E) (on mega) carried mel, and of 28 (39%) isolates with mef(A), 10 (39%) carried mel and another 11(39%) carried msr(D), on Tn1207.1. Two predominant macrolide-resistant S. pneumoniae clonal clusters (CCs) were identified in this population. CC-271 contained 44% of isolates, most of which belonged to serotype 19F, had the MLS_B phenotype, were multidrug resistant, and carried transposons of the Tn916 family; CC-15 contained 23% of isolates, most of which were serotype 14, had the M phenotype, and carried mef(A) on Tn1207.1. Erythromycin resistance among S. pneumoniae isolates in New South Wales is mainly due to the dissemination of multidrug-resistant S. pneumoniae strains or horizontal spread of the Tn916 family of transposons.Streptococcus pneumoniae is an important cause of respiratory tract infections, bacteremia, and meningitis, for which antibiotic treatment is often difficult because of resistance to penicillin and other antibiotics, especially macrolides. During the last decade, macrolide resistance among S. pneumoniae isolates has increased, with considerable geographical variation among the genotypes and phenotypes involved (3, 14, 17, 34, 35).Macrolide resistance in S. pneumoniae is mediated by two main mechanisms. Target modification due to a ribosomal methylase, encoded by erm(B) confers high-level resistance to macrolides, lincosamides, and streptogramin B (MLS_B phenotype). In S. pneumoniae and related Streptococcus spp., the frequent association of erythromycin and tetracycline resistance is often related to insertion of erm(B) into a conjugative transposon of the Tn916 family that harbors tet(M) and carries integrase (int) and excisase (xis) genes. Members of this family, which carry erm(B), include Tn6002, Tn1545 (which also carries the kanamycin resistance gene aphA3), and Tn3872 (which also carries transposase genes tnpA and tnpR) (2, 8).The second macrolide resistance mechanism is an efflux pump system encoded by mef which confers resistance to 14- and 15-member macrolides only (M phenotype) (22). The two main subclasses of mef in S. pneumoniae, mef(E) and mef(A), are carried on different, but related elements: mef(A) on the defective transposon Tn1207.1 (32, 33) or the closely related Tn1207.3 and mef(E) on an element named “macrolide efflux genetic assembly” (mega) (11, 19). Both of these elements carry an open reading frame downstream of mef, designated msr(D) (9, 10) or mel (1, 11, 19), which are homologues of msr(A), which codes for an ATP-dependent efflux pump in Staphylococcus spp. (31). msr(D) and mel are cotranscribed with mef and contribute to an erythromycin-inducible dual efflux system in S. pneumoniae (1, 9, 19). Different investigators have reported and named these msr(D) homologues separately, and it is not clear whether or not they are identical.The prevalence of isolates carrying both mef and erm(B) has reportedly increased as a result of the worldwide spread of a limited number of multidrug-resistant clonal complexes (CCs), of which the most prevalent is Taiwan^19F-14 (CC-271) (15). Recently, two new composite elements of the Tn916 family, containing tet(M) plus mega (Tn2009) and tet(M), erm(B), and mega (Tn2010), have been described (10, 12). The distribution of these transposons and the genes they carry also vary in different geographic regions (4, 9) and provide a clue to the origins of antibiotic-resistant strains of S. pneumoniae.This study is the first to analyze the distribution of antibiotic susceptibility patterns and phenotypic and genotypic characteristics of erythromycin-resistant invasive S. pneumoniae isolates in Australia, including the transposons on which resistance genes are carried. The isolates studied had been referred to the Pneumococcal Reference Laboratory at the Centre for Infectious Diseases and Microbiology, which receives all sterile-site isolates from patients with invasive pneumococcal disease, in New South Wales, for serotyping (30). The 7-valent pneumococcal conjugate vaccine (PCV7) first became available in Australia in 2001, with limited uptake. It was introduced into the routine infant immunization schedule in January 2005 as a 3-dose regimen given at 2, 4, and 6 months.     

Cloning and Sequencing of the Gene Encoding Toho-2, a Class A β-Lactamase Preferentially Inhibited by Tazobactam

Escherichia coli TUM1083, which is resistant to ampicillin, carbenicillin, cephaloridine, cephalothin, piperacillin, cefuzonam, and aztreonam while being sensitive to cefoxitin, moxalactam, cefmetazole, ceftazidime, and imipenem, was isolated from the urine of a patient treated with β-lactam antibiotics. The β-lactamase (Toho-2) purified from the bacteria hydrolyzed β-lactam antibiotics such as penicillin G, carbenicillin, cephaloridine, cefoxitin, cefotaxime, ceftazidime, and aztreonam and especially had increased relative hydrolysis rates for cephalothin, cephaloridine, cefotaxime, and ceftizoxime. Different from other extended-spectrum β-lactamases, Toho-2 was inhibited 16-fold better by the β-lactamase inhibitor tazobactam than by clavulanic acid. Resistance to β-lactams was transferred by conjugation from E. coli TUM1083 to E. coli ML4909, and the transferred plasmid was about 54.4 kbp, belonging to the incompatibility group IncFII. The cefotaxime resistance gene for Toho-2 was subcloned from the 54.4-kbp plasmid. The sequence of the gene was determined, and the open reading frame of the gene was found to consist of 981 bases. The nucleotide sequence of the gene (DDBJ accession no. {"type":"entrez-nucleotide","attrs":{"text":"D89862","term_id":"3142148","term_text":"D89862"}}D89862) designated as bla_toho was found to have 76.3% identity to class A β-lactamase CTX-M-2 and 76.2% identity to Toho-1. It has 55.9% identity to SHV-1 β-lactamase and 47.5% identity to TEM-1 β-lactamase. Therefore, the newly isolated β-lactamase designated as Toho-2 produced by E. coli TUM1083 is categorized as an enzyme similar to Toho-1 group β-lactamases rather than to mutants of TEM or SHV enzymes. According to the amino acid sequence deduced from the DNA sequence, the precursor consisted of 327 amino acid residues. Comparison of Toho-2 with other β-lactamase (non-Toho-1 group) suggests that the substitutions of threonine for Arg-244 and arginine for Asn-276 are important for the extension of the substrate specificity.β-Lactam antibiotics are widely used as front line agents in the clinical field. In the early 1980s, expanded-spectrum β-lactams, with stability for β-lactamase and good activity against gram-negative bacteria, were first used in the clinical setting. Not long after the beginning of wide use of the expanded-spectrum β-lactams, extended-spectrum β-lactamases were isolated in Europe and the United States and now have become a serious problem in the clinical field (29). In the late 1980s and early 1990s, those enzymes hydrolyzing the expanded-spectrum β-lactams were generally derived from TEM- or SHV-type β-lactamases through several mutations (6, 24). The mutations of Glu-104, Arg-164, and Glu-240 have been suggested to be important for the spectrum expansion (16, 24). In more recent years, non-TEM- or non-SHV-type β-lactamases such as Toho-1 (13), CTX-M-2 (5), and MEN-1 (3) have been identified. Those β-lactamases have high homology to the chromosomally encoded β-lactamase of Proteus vulgaris or Klebsiella oxytoca (2, 9, 23). In most cases, the β-lactamase-producing organisms show resistance to expanded-spectrum β-lactams such as cefotaxime and ceftazidime (6, 24). On the other hand, they are susceptible to carbapenems such as imipenem (6, 24). The main characteristic of those class A β-lactamases, except TEM-30 to TEM-40, is that they are sensitive to β-lactamase inhibitors such as clavulanic acid, sulbactam, and tazobactam (6). The reaction mechanism and the amino acid residues associated with the spectrum expansion of β-lactamases are still under investigation. Ishii et al. (13) proposed that mutations at positions 244 and 276 are important for the substrate extension after performing sequence alignment of Toho-1 and other β-lactamases.The expanded-spectrum β-lactam-resistant strains isolated from several hospitals were surveyed and collected. We investigated those strains by enzymological and molecular biological methods and focused upon Escherichia coli TUM1083, a cefotaxime-resistant clinical isolate.In this report, we discuss a correlation between the mutation and the substrate specificity of the β-lactamase from E. coli TUM1083 based on the sequence alignment and a three-dimensional structure of a related β-lactamase of Bacillus licheniformis (17).