Identification of TEM-135 β-Lactamase in Neisseria gonorrhoeae Strains Carrying African and Toronto Plasmids in Argentina

One hundred forty-three penicillinase-producing Neisseria gonorrhoeae (PPNG) isolates obtained in Argentina from 2008 and 2012 were examined to detect bla_TEM-135 genes and to investigate plasmid profiles and multiantigen sequence types. Forty-two PPNG isolates were found to carry TEM-135, and two contained a new TEM derivative characterized as TEM-220. The bla_TEM-135 allele was carried by the Toronto/Rio and African plasmids. Molecular epidemiology revealed that two bla_TEM-135 isolates were related to previously described isolates from Thailand and China, indicating a common evolutionary origin.     

Characterization of azithromycin-resistant Neisseria gonorrhoeae isolated in Tokyo in 2005–2011

A total of 122 Neisseria gonorrhoeae isolated in the Tokyo metropolitan area in 2005–2011 were collected and analyzed by N. gonorrhoeae multiantigen sequence typing (NG-MAST) and for their susceptibility to azithromycin and ceftriaxone. All 122 strains were susceptible to ceftriaxone, but 8 strains were azithromycin-resistant, defined as an azithromycin MIC ≥ 1 μg/ml. The 8 azithromycin-resistant strains were in 6 NG-MAST types, 3 strains in NG-MAST type 1407 and each of the other 5 strains in a different NG-MAST type. NG-MAST type 1407 strains are multidrug-resistant and are disseminated worldwide.     

Emergence of cephem- and aztreonam-high-resistant Neisseria gonorrhoeae that does not produce β-lactamase

Regarding Neisseria gonorrhoeae, the National Committee for Clinical Laboratory Standards (NCCLS) has not defined the breakpoint minimum inhibitory concentration (MIC) for expanded spectrum cephems such as cefpodoxime and ceftizoxime, because of the absence of resistant strains to these antibiotics. To date, in gonococcal urethritis, after treatment with third generation cephems and aztreonam, clinical failures caused by resistant N. gonorrhoeae strains have not been reported. However, we experienced two clinical failures in patients with gonococcal urethritis treated with cefdinir and aztreonam. N. gonorrhoeae isolates from these two patients showed high-level MICs to these agents. The MIC of cefdinir was 1 μg/ml for both strains and that of aztreonam was 8 μg/ml for both strains, while the MICs of other β-lactams were also higher than the NCCLS value, except for ceftriaxone, for which the MIC was 0.125 μg/ml for both strains. Moreover, the MICs of fluoroquinolones, tetracyclines, and erythromycin against these two isolates were higher than the NCCLS susceptibility value. These isolates were susceptible to spectinomycin. In N. gonorrhoeae, the emergence of these β-lactam-resistant isolates is of serious concern. However, a more serious problem is that these isolates were already resistant to non-β-lactam antimicrobials. In Japan, ceftriaxone has not been permitted for clinical use against gonococcal infections. Therefore, in Japan, patients with gonococcal urethritis caused by these resistant N. gonorrhoeae strains should be treated with cefodizime or spectinomycin. Received: October 30, 2000 / Accepted: November 30, 2000     

Antimicrobial Resistance Testing of Verocytotoxin-Producing Escherichia coli and First Description of TEM-52 Extended-Spectrum β-Lactamase in Serogroup O26

We have investigated the antimicrobial resistance of verocytotoxin-producing Escherichia coli (VTEC) strains isolated from humans, animals, food, and the environment in Belgium. Resistance was more frequent in non-O157 strains from humans than in O157 strains from humans or other sources, and among non-O157 VTEC strains, intimin-positive strains were more resistant than intimin-negative strains. We also report the first VTEC strain producing an IncI1 extended-spectrum β-lactamase encoded by plasmid-borne bla_TEM-52; this β-lactamase was previously associated with Salmonella enterica and E. coli isolates from different origins.Verocytotoxin-producing Escherichia coli (VTEC) is associated with gastrointestinal illness, and especially strains belonging to serogroups O157, O26, O103, O111, and O145 may cause hemolytic-uremic syndrome (HUS) (4). The cardinal virulence trait involved in HUS development is the production of one or more verocytotoxin types encoded by genes located on temperate lambdoid bacteriophages (14). Although most postdiarrheal HUS cases are linked to VTEC infection, antibiotic therapy has not demonstrated beneficial effects after HUS development (11, 14, 17). The underlying mechanism is unknown, but bacterial lysis could increase the amount of verocytotoxin released into systemic circulation and/or induce verocytotoxin-containing bacteriophages.Antimicrobials are widely used for disease prevention and growth promotion in cattle and other farm animals identified as important VTEC reservoirs [8; European Medicines Agency, Joint opinion on antimicrobial resistance (AMR) focused on zoonotic infections, 2009 (http://www.ema.europa.eu/pdfs/vet/sagam/44725909en.pdf)]. Consequently, resistance may be promoted in VTEC commensally present in the intestinal tracts of these animals. Recent reports indicate that antimicrobial resistance of VTEC is rising (15). Mora et al. reported that bovine VTEC O157:H7 strains were significantly more resistant to streptomycin, tetracycline, and sulfisoxazole than those from humans, whereas non-O157 VTEC strains isolated from humans and beef were more resistant than bovine non-O157 strains (8). Most non-O157 strains showing multidrug resistance belonged to HUS-associated serotypes. Although the overall frequency of β-lactamases in E. coli isolated from humans and farm animals is increasing (13, 16; European Antimicrobial Resistance Surveillance System, Susceptibility results for E. coli in Belgium, 2010 [http://www.rivm.nl/earss/database/]), only a few extended-spectrum β-lactamase (ESBL)-producing VTEC strains have been reported (3, 6, 10).We evaluated the antimicrobial resistance of VTEC strains isolated in Belgium from different origins in relation to established virulence factors and report the first TEM-52-producing VTEC isolate.(Part of this research has been presented at the Pathogenic Escherichia coli Network Conference Control and Management of Pathogenic Escherichia coli, Dublin, Ireland, 17 and 18 September 2009.)A total of 302 unduplicated, consecutive VTEC strains isolated in Belgium from humans, as well as from animals (n = 48), food (n = 21), and the environment (n = 1), referred to our laboratory between 2004 and 2009 by several Belgian laboratories were investigated. Among strains from humans, 153 belonged to serogroup O157 and 149 to non-O157 serogroups, whereas all strains of nonhuman origin (n = 70) belonged to serogroup O157. These strains were isolated from cattle (n = 47), a dog (n = 1), ground beef (n = 20), cheese (n = 1), and dust (n = 1). Four established virulence genes, the verocytotoxin 1 and 2 genes (vtx1 and vtx2), the intimin gene (eaeA), and the enterohemolysin gene (ehxA) were searched for by PCR (9). The flagellar type (fliC) was determined by PCR-restriction fragment length polymorphism (RFLP) (7). In vitro susceptibility tests were performed by the disk diffusion method for the antimicrobials listed in Table ​Table11 using the EUCAST and CLSI potency Neo-Sensitabs tablets (Rosco, Taastrup, Denmark), with interpretation of zones according to CLSI, as described by the manufacturer (Rosco Diagnostica A/S; Neo-Sensitabs user''s guide, document 3.1.0, 2010 [http://www.rosco.dk/]). In addition, cefotaxime plus clavulanate and ceftazidime plus clavulanate were systematically tested, and an ESBL was considered to be present if the inhibition zone increased by ≥5 mm in comparison with that for the antibiotic alone.

TABLE 1.

Antimicrobial resistance and correlation with the presence or absence of the intimin gene (eaeA) in VTEC isolated from humans and other sources in Belgium

Identification of Novel VEB β-Lactamase Enzymes and Their Impact on Avibactam Inhibition

Ceftazidime-avibactam has activity against Pseudomonas aeruginosa and Enterobacteriaceae expressing numerous class A and class C β-lactamases, although the ability to inhibit many minor enzyme variants has not been established. Novel VEB class A β-lactamases were identified during characterization of surveillance isolates. The cloned novel VEB β-lactamases possessed an extended-spectrum β-lactamase phenotype and were inhibited by avibactam in a concentration-dependent manner. The residues that comprised the avibactam binding pocket were either identical or functionally conserved. These data demonstrate that avibactam can inhibit VEB β-lactamases.     

Inhibitor Resistance in the KPC-2 β-Lactamase,a Preeminent Property of This Class A β-Lactamase

As resistance determinants, KPC β-lactamases demonstrate a wide substrate spectrum that includes carbapenems, oxyimino-cephalosporins, and cephamycins. In addition, clinical strains harboring KPC-type β-lactamases are often identified as resistant to standard β-lactam-β-lactamase inhibitor combinations in susceptibility testing. The KPC-2 carbapenemase presents a significant clinical challenge, as the mechanistic bases for KPC-2-associated phenotypes remain elusive. Here, we demonstrate resistance by KPC-2 to β-lactamase inhibitors by determining that clavulanic acid, sulbactam, and tazobactam are hydrolyzed by KPC-2 with partition ratios (k_cat/k_inact ratios, where k_inact is the rate constant of enzyme inactivation) of 2,500, 1,000, and 500, respectively. Methylidene penems that contain an sp²-hybridized C₃ carboxylate and a bicyclic R1 side chain (dihydropyrazolo[1,5-c][1,3]thiazole [penem 1] and dihydropyrazolo[5,1-c][1,4]thiazine [penem 2]) are potent inhibitors: K_m of penem 1, 0.06 ± 0.01 μM, and K_m of penem 2, 0.006 ± 0.001 μM. We also demonstrate that penems 1 and 2 are mechanism-based inactivators, having partition ratios (k_cat/k_inact ratios) of 250 and 50, respectively. To understand the mechanism of inhibition by these penems, we generated molecular representations of both inhibitors in the active site of KPC-2. These models (i) suggest that penem 1 and penem 2 interact differently with active site residues, with the carbonyl of penem 2 being positioned outside the oxyanion hole and in a less favorable position for hydrolysis than that of penem 1, and (ii) support the kinetic observations that penem 2 is the better inhibitor (k_inact/K_m = 6.5 ± 0.6 μM⁻¹ s⁻¹). We conclude that KPC-2 is unique among class A β-lactamases in being able to readily hydrolyze clavulanic acid, sulbactam, and tazobactam. In contrast, penem-type β-lactamase inhibitors, by exhibiting unique active site chemistry, may serve as an important scaffold for future development and offer an attractive alternative to our current β-lactamase inhibitors.In Klebsiella pneumoniae, β-lactam resistance is mediated predominantly by class A SHV, TEM, and CTX-M β-lactamases (7, 35). Single amino acid substitutions in the SHV and TEM β-lactamases can drastically alter the substrate profiles of the enzymes and confer resistance to extended-spectrum cephalosporins and β-lactamase inhibitors (5, 12, 34, 36). β-Lactamases with altered substrate profiles (i.e., extended-spectrum or inhibitor-resistant β-lactamases) have significantly challenged the clinician''s approach to the treatment of serious infectious diseases (36). Thus, the search for effective mechanism-based inhibitors of novel β-lactamases merits significant effort (8, 9, 32).First identified in K. pneumoniae, KPC class A β-lactamases threaten the use of all current β-lactam antibiotics (57). These β-lactamase enzymes are present in an increasing number of bacterial genera, becoming the major carbapenemase expressed by Gram-negative pathogens (e.g., Enterobacter spp., Escherichia coli, Citrobacter freundii, Pseudomonas spp., Serratia marcescens, Proteus mirabilis, and Salmonella enterica) in the United States (3, 10, 11, 16, 17, 25, 37, 45, 49, 53, 59). Moreover, KPC β-lactamases are becoming geographically widespread (having been detected, e.g., in the United States, China, France, Colombia, Greece, Sweden, Norway, Argentina, the United Kingdom, Israel, Brazil, Puerto Rico, Canada, Ireland, Trinidad and Tobago, Poland, Italy, and Finland) (1, 2, 15, 23, 24, 29-31, 33, 38, 39, 42, 50, 51, 53, 55, 57). Evidence suggests that many K. pneumoniae strains in the United States harboring KPCs are genetically related (19).Why are KPC β-lactamases so problematic? KPC-2 has an overall structure similar to those of other class A enzymes, and interestingly, this β-lactamase has only 50% protein sequence conservation compared to CTX-M-1, 39% compared to SHV-1, and 35% compared to TEM-1. KPC-2 is more like other class A carbapenemases, having 55% identity to NmcA and Imi-1, 63% identity to Sfc-1, and 57% identity to Sme-1. The KPC-2 β-lactamase possesses a large and shallow active site, allowing it to accommodate “bulkier” β-lactams (26). As a result of these structural characteristics, KPC-2 is regarded as a versatile β-lactamase (37); it is a penicillinase, carbapenemase, and cephamycinase and an extended-spectrum β-lactamase (57, 58). Microbiologists and clinicians have observed that many bla_KPC-2-containing strains are resistant to β-lactam-β-lactamase inhibitor combinations (6, 19, 50, 54, 55, 59). According to Clinical and Laboratory Standards Institute (CLSI) breakpoints, bla_KPC-2-carrying clinical strains for which the MICs of amoxicillin-clavulanic acid are ≥32/16 mg/liter and those of piperacillin-tazobactam are ≥128/4 mg/liter are resistant (14, 58). These observations led us to examine the kinetic properties of the KPC-2 β-lactamase tested against commercially available and novel inhibitors.A β-lactamase inhibitor demonstrating an affinity in the nanomolar range for KPC-2 and other class A carbapenemases would be an important addition to our therapeutic armamentarium. Thus, we wondered if penem inhibitors that possess an sp²-hybridized C₃ carboxylate (a property resembling a characteristic of carbapenems), a complex and reactive R1 side chain, and inactivation chemistry different from that of clavulanic acid could be exploited to inhibit KPC enzymes (41). The methylidene inhibitors penem 1 and penem 2 have dihydropyrazolo[1,5-c][1,3]thiazole and dihydropyrazolo[5,1-c][1,4]thiazine moieties, respectively (see Fig. ​Fig.1).1). These penems demonstrate similar levels of in vivo efficacy in mice and have been shown to be effective inhibitors of several class A, C, and D β-lactamases (4, 43, 46-48, 52).Open in a separate windowFIG. 1.Chemical structures of the classical β-lactamase inhibitors, the novel penem β-lactamase inhibitors, cefotaxime, and imipenem.In this paper, we show why K. pneumoniae containing bla_KPC-2 and an E. coli laboratory strain harboring bla_KPC-2 are not susceptible to the commercially available β-lactamase inhibitors. Our results demonstrated that clavulanic acid, sulbactam, and tazobactam are hydrolyzed by the KPC-2 β-lactamase. 6-Methylidene penems with complex fused bicyclic R1 side chains are better inhibitors because they possess greater affinity for the active site, have low K_ms, and act as mechanism-based inactivators.     

First Detection of Plasmid-Encoded blaOXY β-Lactamase

Three Klebsiella oxytoca isolates and one Klebsiella pneumoniae isolate from three children admitted to the Hematology Unit of Hospital Vall d''Hebron (Barcelona, Spain) exhibited a susceptibility pattern suggesting OXY β-lactamase hyperproduction. All the isolates contained a 95-kb plasmid that harbored bla_OXY-1, which was transferred by electrotransformation but could not be self-transferred by conjugation. A qnrS1 gene was also harbored in the bla_OXY-1-carrying plasmid. This is the first report of a plasmid-encoded OXY β-lactamase.Klebsiella oxytoca is a member of the Enterobacteriaceae with broad environmental distribution; it also colonizes the human gut and is able to produce severe opportunistic infections, particularly in neonates (2, 9). This species possesses a chromosomally encoded class A β-lactamase (bla_OXY) which is constitutively expressed at low levels, conferring resistance to aminopenicillins and carboxypenicillins. However, such strains remain susceptible to other β-lactams such as cephalosporins, monobactams, and the β-lactamase inhibitor combination agents. However, hyperproduction of OXY, which generally is due to mutations in the promoter region of the gene, confers resistance to all penicillins (except temocillin) and to the combination of amoxicillin (amoxicilline) plus clavulanic acid, narrow- and expanded-spectrum cephalosporins, and aztreonam, plus a variable level of reduced susceptibility to cefotaxime and ceftriaxone, without affecting susceptibility to ceftazidime (1, 6, 7, 13).Six bla_OXY groups (bla_OXY-1 to bla_OXY-6) have been described on the basis of the gene nucleotide sequence (5). The different bla_OXY groups have similar profiles of activity against β-lactams; however, three bla_OXY-2 derivatives have been described which confer resistance to β-lactamase inhibitor combination agents or to ceftazidime by point amino acid substitutions in different positions (14, 18, 20).In January 2008, a Klebsiella oxytoca isolate (KO279) was obtained from the urine of a 3-year-old boy who was undergoing bone marrow transplantation. The isolate had a phenotype of resistance to β-lactams compatible with the presence of bla_OXY hyperproduction (Table ​(Table1).1). Simultaneously, a Klebsiella pneumoniae isolate (KP278) with a pattern of susceptibility to β-lactams similar to that of K. oxytoca KO279 was obtained from the urine of a 4-year-old girl, admitted in the same nursing unit also for bone marrow transplantation.

TABLE 1.

MICs of antimicrobial agents for the studied strains

Membrane-Bound PenA β-Lactamase of Burkholderia pseudomallei

Burkholderia pseudomallei is the etiologic agent of melioidosis, a difficult-to-treat disease with diverse clinical manifestations. β-Lactam antibiotics such as ceftazidime are crucial to the success of melioidosis therapy. Ceftazidime-resistant clinical isolates have been described, and the most common mechanism is point mutations affecting expression or critical amino acid residues of the chromosomally encoded class A PenA β-lactamase. We previously showed that PenA was exported via the twin arginine translocase system and associated with the spheroplast fraction. We now show that PenA is a membrane-bound lipoprotein. The protein and accompanying β-lactamase activity are found in the membrane fraction and can be extracted with Triton X-114. Treatment with globomycin of B. pseudomallei cells expressing PenA results in accumulation of the prolipoprotein. Mass spectrometric analysis of extracted membrane proteins reveals a protein peak whose mass is consistent with a triacylated PenA protein. Mutation of a crucial lipobox cysteine at position 23 to a serine residue results in loss of β-lactamase activity and absence of detectable PenA_C23S protein. A concomitant isoleucine-to-alanine change at position 20 in the signal peptide processing site in the PenA_C23S mutant results in a nonlipidated protein (PenA_{I20A C23S}) that is processed by signal peptidase I and exhibits β-lactamase activity. The resistance profile of a B. pseudomallei strain expressing this protein is indistinguishable from the profile of the isogenic strain expressing wild-type PenA. The data show that PenA membrane association is not required for resistance and must serve another purpose.     

Crystal Structure of the Extended-Spectrum β-Lactamase PER-2 and Insights into the Role of Specific Residues in the Interaction with β-Lactams and β-Lactamase Inhibitors

PER-2 belongs to a small (7 members to date) group of extended-spectrum β-lactamases. It has 88% amino acid identity with PER-1 and both display high catalytic efficiencies toward most β-lactams. In this study, we determined the X-ray structure of PER-2 at 2.20 Å and evaluated the possible role of several residues in the structure and activity toward β-lactams and mechanism-based inhibitors. PER-2 is defined by the presence of a singular trans bond between residues 166 to 167, which generates an inverted Ω loop, an expanded fold of this domain that results in a wide active site cavity that allows for efficient hydrolysis of antibiotics like the oxyimino-cephalosporins, and a series of exclusive interactions between residues not frequently involved in the stabilization of the active site in other class A β-lactamases. PER β-lactamases might be included within a cluster of evolutionarily related enzymes harboring the conserved residues Asp136 and Asn179. Other signature residues that define these enzymes seem to be Gln69, Arg220, Thr237, and probably Arg/Lys240A (“A” indicates an insertion according to Ambler''s scheme for residue numbering in PER β-lactamases), with structurally important roles in the stabilization of the active site and proper orientation of catalytic water molecules, among others. We propose, supported by simulated models of PER-2 in combination with different β-lactams, the presence of a hydrogen-bond network connecting Ser70-Gln69-water-Thr237-Arg220 that might be important for the proper activity and inhibition of the enzyme. Therefore, we expect that mutations occurring in these positions will have impacts on the overall hydrolytic behavior.     

High β-Lactamase Levels Change the Pharmacodynamics of β-Lactam Antibiotics in Pseudomonas aeruginosa Biofilms

Resistance to β-lactam antibiotics is a frequent problem in Pseudomonas aeruginosa lung infection of cystic fibrosis (CF) patients. This resistance is mainly due to the hyperproduction of chromosomally encoded β-lactamase and biofilm formation. The purpose of this study was to investigate the role of β-lactamase in the pharmacokinetics (PK) and pharmacodynamics (PD) of ceftazidime and imipenem on P. aeruginosa biofilms. P. aeruginosa PAO1 and its corresponding β-lactamase-overproducing mutant, PAΔDDh2Dh3, were used in this study. Biofilms of these two strains in flow chambers, microtiter plates, and on alginate beads were treated with different concentrations of ceftazidime and imipenem. The kinetics of antibiotics on the biofilms was investigated in vitro by time-kill methods. Time-dependent killing of ceftazidime was observed in PAO1 biofilms, but concentration-dependent killing activity of ceftazidime was observed for β-lactamase-overproducing biofilms of P. aeruginosa in all three models. Ceftazidime showed time-dependent killing on planktonic PAO1 and PAΔDDh2Dh3. This difference is probably due to the special distribution and accumulation in the biofilm matrix of β-lactamase, which can hydrolyze the β-lactam antibiotics. The PK/PD indices of the AUC/MBIC and C_max/MBIC (AUC is the area under concentration-time curve, MBIC is the minimal biofilm-inhibitory concentration, and C_max is the maximum concentration of drug in serum) are probably the best parameters to describe the effect of ceftazidime in β-lactamase-overproducing P. aeruginosa biofilms. Meanwhile, imipenem showed time-dependent killing on both PAO1 and PAΔDDh2Dh3 biofilms. An inoculum effect of β-lactams was found for both planktonic and biofilm P. aeruginosa cells. The inoculum effect of ceftazidime for the β-lactamase-overproducing mutant PAΔDDh2Dh3 biofilms was more obvious than for PAO1 biofilms, with a requirement of higher antibiotic concentration and a longer period of treatment.     

Carriage of Escherichia coli Producing CTX-M-Type Extended-Spectrum β-Lactamase in Healthy Vietnamese Individuals

Healthy carriage of CTX-M-type extended-spectrum β-lactamase (ESBL)-producing Escherichia coli was examined by thrice collecting fecal samples from the same 199 healthy Vietnamese subjects every 6 months. Using pulsed-field gel electrophoresis (PFGE), identical PFGE patterns throughout the three samplings were not observed, although prevalence of E. coli in the subjects was around 50% in the three samplings. Our results suggested a short carriage period of the CTX-M-type ESBL-producing E. coli in healthy Vietnamese subjects.     

In Vitro Activity of β-Lactams in Combination with β-Lactamase Inhibitors against Multidrug-Resistant Mycobacterium tuberculosis Isolates

The combination of β-lactams and β-lactamase inhibitors has been shown to have potent in vitro activity against multidrug-resistant tuberculosis (MDR-TB) isolates. In order to identify the most potent β-lactam–β-lactamase inhibitor combination against MDR-TB, we selected nine β-lactams and three β-lactamase inhibitors, which belong to different subgroups. A total of 121 MDR-TB strains were included in this study. Out of the β-lactams used herein, biapenem was the most effective against MDR-TB and had an MIC₅₀ value of 8 μg/ml. However, after the addition of clavulanate or sulbactam, meropenem exhibited the most potent anti-MDR-TB activity with an MIC₅₀ value of 4 μg/ml. For meropenem, 76 (62.8%), 41 (33.9%), and 22 (18.2%) of the 121 MDR-TB strains were subjected to a synergistic effect when the drug was combined with sulbactam, tazobactam, or clavulanate, respectively. Further statistical analysis revealed that significantly more strains experienced a synergistic effect when exposed to the combination of meropenem with sulbactam than when exposed to meropenem in combination with tazobactam or clavulanate, respectively (P < 0.01). In addition, a total of 10.7% (13/121) of isolates harbored mutations in the blaC gene, with two different nucleotide substitutions: AGT333AGG and ATC786ATT. For the strains with a Ser111Arg substitution in BlaC, a better synergistic effect was observed in the meropenem-clavulanate and in the amoxicillin-clavulanate combinations than that in a synonymous single nucleotide polymorphism (SNP) group. In conclusion, our findings demonstrate that the combination of meropenem and sulbactam shows the most potent activity against MDR-TB isolates. In addition, the Ser111Arg substitution of BlaC may be associated with an increased susceptibility of MDR-TB isolates to meropenem and amoxicillin in the presence of clavulanate.