AmpC β-Lactamases

Summary: AmpC β-lactamases are clinically important cephalosporinases encoded on the chromosomes of many of the Enterobacteriaceae and a few other organisms, where they mediate resistance to cephalothin, cefazolin, cefoxitin, most penicillins, and β-lactamase inhibitor-β-lactam combinations. In many bacteria, AmpC enzymes are inducible and can be expressed at high levels by mutation. Overexpression confers resistance to broad-spectrum cephalosporins including cefotaxime, ceftazidime, and ceftriaxone and is a problem especially in infections due to Enterobacter aerogenes and Enterobacter cloacae, where an isolate initially susceptible to these agents may become resistant upon therapy. Transmissible plasmids have acquired genes for AmpC enzymes, which consequently can now appear in bacteria lacking or poorly expressing a chromosomal bla_AmpC gene, such as Escherichia coli, Klebsiella pneumoniae, and Proteus mirabilis. Resistance due to plasmid-mediated AmpC enzymes is less common than extended-spectrum β-lactamase production in most parts of the world but may be both harder to detect and broader in spectrum. AmpC enzymes encoded by both chromosomal and plasmid genes are also evolving to hydrolyze broad-spectrum cephalosporins more efficiently. Techniques to identify AmpC β-lactamase-producing isolates are available but are still evolving and are not yet optimized for the clinical laboratory, which probably now underestimates this resistance mechanism. Carbapenems can usually be used to treat infections due to AmpC-producing bacteria, but carbapenem resistance can arise in some organisms by mutations that reduce influx (outer membrane porin loss) or enhance efflux (efflux pump activation).     

Prevalence of CTX-M β-Lactamases in Philadelphia,Pennsylvania

CTX-M β-lactamases were thought to be rare in the United States, but a recent study in Texas showed that up to 70% of extended-spectrum β-lactamase (ESBL)-containing members of the Enterobacteriaceae family were CTX-M positive (J. S. Lewis, M. Herrera, B. Wickes, J. E. Patterson, and J. H. Jorgensen, Antimicrob. Agents Chemother. 51:4015-4021, 2007). We used PCR to detect CTX-M in all 291 extended-spectrum cephalosporin-resistant gram-negative bacteria isolated in our laboratory during 2007. Thirty (48%) Escherichia coli isolates, 6 (3%) Klebsiella sp. isolates, and 7 (100%) Proteus mirabilis isolates tested were CTX-M positive, with 15% of all Enterobacteriaceae tested being positive. The E. coli CTX-M groups were I (57%), IV (37%), II (3%), and not groupable (3%); three of the group IV isolates were positive for CTX-M-18, and three of the group I isolates were positive for CTX-M-15. One of seven positive P. mirabilis isolates was in group II, with the remainder being positive for a CTX-M-25-like β-lactamase; and 33% of the Klebsiella sp. isolates were in group I or IV, with the remainder not being in groups I to IV. CTX-M-producing bacteria were isolated from urine (n = 13), blood (n = 13), wounds (n = 12), and the respiratory tract (n = 4). All 31 CTX-M-positive isolates tested for the presence of ESBL were confirmed to produce ESBLs by the use of tests recommended by the CLSI. Pulsed-field gel electrophoresis of the CTX-M-positive isolates showed that six P. mirabilis isolates were clonal and that there were seven different E. coli clusters. Five of seven P. mirabilis isolates were from blood cultures. The CLSI tests for the confirmation of ESBL production reliably detect these isolates if both cefotaxime and ceftazidime are tested, but only about half would be classified as a possible CTX-M producers on the basis of the antibiogram alone. A new panprimer set increases the ability to detect CTX-M-producing strains. CTX-M-positive bacteria are common in our geographic region, are often invasive, and, with the exception of P. mirabilis, are multiclonal.CTX-M β-lactamases were thought to be uncommon in North America, although they are common on many other continents (1, 8, 10). CTX-M β-lactamases are extended-spectrum β-lactamases (ESBLs) that mainly inactivate cefotaxime and ceftriaxone and that have less activity against ceftazidime (8). As opposed to the usual epidemiology of other ESBL-positive members of the Enterobacteriaceae family, which are mainly nosocomial, CTX-M β-lactamase producers often appear to be community acquired, albeit in debilitated people with prior antibiotic exposure (8). The recognition of this group of resistant bacteria could have importance for decisions on empirical antibiotic therapy and could influence the accuracy of current procedures for screening for ESBLs. On the basis of the findings of a recent study from Texas showing that 70% of recent ESBL-producing isolates contained CTX-M β-lactamases (7), we tested all extended-spectrum cephalosporin-resistant Enterobacteriaceae isolated in the Clinical Microbiology Laboratory at the Hospital of the University of Pennsylvania (HUP) in 2007, as well as all multidrug-resistant Acinetobacter baumannii isolates recovered during the same time period. We show that CTX-M β-lactamases are common among extended-spectrum cephalosporin-resistant Escherichia coli and Proteus mirabilis isolates recovered from patients in hospitals in Philadelphia, PA.(This study was presented in abstract form at the 48th Interscience Conference on Antimicrobial Agents and Chemotherapy, Washington, DC, 2008.)     

Broth Microdilution Method To Detect Extended-Spectrum β-Lactamases and AmpC β-Lactamases in Enterobacteriaceae Isolates by Use of Clavulanic Acid and Boronic Acid as Inhibitors

This study was designed to evaluate the performance of the broth microdilution (BMD) method to detect production of extended-spectrum β-lactamases (ESBLs) and AmpC β-lactamases in Enterobacteriaceae by using clavulanic acid (CA) and boronic acid (BA) as ESBL and AmpC β-lactamase inhibitors, respectively. A total of 100 clinical isolates of Enterobacteriaceae were analyzed. Mueller-Hinton broth containing serial twofold dilutions of cefotaxime (CTX), ceftazidime (CAZ), aztreonam (ATM), or cefepime (FEP) with or without either or both CA and BA was prepared. An eightfold or greater decrease in the MIC of CTX, CAZ, ATM, or FEP in the presence of CA and BA was considered a positive result for ESBL and plasmid-mediated AmpC β-lactamase (PABL), respectively. In tests with CA, expanded-spectrum β-lactams containing BA (CTX-BA, CAZ-BA, ATM-BA, and FEP-BA) showed higher positive rates in detecting ESBL producers than those without BA. The combination of CTX- and CAZ-based BMD tests with CA and BA showed sensitivity and specificity of 100% for the detection of ESBLs and PABLs. The BMD testing could be applicable for routine use in commercially available semiautomated systems for the detection of ESBLs and PABLs in Enterobacteriaceae.The rapid global dissemination of Enterobacteriaceae harboring plasmid-borne extended-spectrum β-lactamases (ESBLs) and plasmid-mediated AmpC β-lactamases (PABLs) represents a significant clinical threat (2, 11). ESBLs can confer resistance to penicillins, oxyimino-cephalosporins, and monobactams to bacterial hosts, and their hydrolytic activities are usually inhibited by clavulanic acid (CA) (15). PABLs also confer resistance to expanded-spectrum β-lactams, but not carbapenems, but their hydrolytic activities are poorly inhibited by CA (16). The rapid and accurate detection of ESBLs and PABLs in Enterobacteriaceae is important to guide proper antimicrobial therapy and appropriate infection control measures. Many methods for the detection of ESBLs and PABLs have been proposed, but some procedures are difficult to perform in practice, time-consuming, and hard to interpret (7, 9). Dissemination of class A carbapenemases, including KPC enzymes, makes the detection more difficult (3, 17).The Clinical and Laboratory Standards Institute (CLSI) has described standard broth microdilution (BMD) and disk susceptibility test methods to screen and confirm ESBL production in Escherichia coli, Klebsiella pneumoniae, Klebsiella oxytoca, and Proteus mirabilis, but there are currently no available guidelines for other genera (5). Several semiautomated antimicrobial susceptibility testing systems, which infer ESBL production by inhibitory effects of CA, are now commercially available. However, they are available only for K. pneumoniae, K. oxytoca, and E. coli.No standardized method is recognized for screening and confirmation of the presence of AmpC enzymes. Resistance to a cephamycin is suggestive of the presence of an AmpC enzyme, but this can be mimicked by porin loss (8, 12). Recently, a test for AmpC β-lactamases that involves augmentation of the inhibition zones around cefotaxime (CTX), ceftazidime (CAZ), and cefotetan disks by an AmpC inhibitor, a boronic acid (BA) compound, has been proposed (6, 24). BA testing cannot distinguish between plasmid-borne and chromosome-borne AmpC β-lactamases. We recently evaluated the diagnostic utility of the BA disk test for the detection of strains producing ESBLs and PABLs, which is similar to the CLSI disk confirmatory test for ESBLs. We found that the BA disk test was an accurate and simple tool for the detection of ESBLs and PABLs in Enterobacteriaceae (10, 20, 21).The aim of the present study was to evaluate the performance of the BMD method, which is appropriate for routine use in commercially available semiautomated systems, for the detection of ESBLs and PABLs, using CA and BA to inhibit ESBLs and PABLs, respectively.     

Extended-Spectrum β-Lactamases (ESBLs): Characterization,Epidemiology and Detection

β-lactamases of Enterobacteriaceae are the most important mechanism of resistance against β-lactam drugs. Two types of β-lactamases can confer resistance against 3rd generation cephalosporins. Chromosomally mediated β-lactamases are inducible and are not inhibited by clavulanic acid. Resistance due to these enzymes is non-transferable. The 2nd type of enzyme is plasmid-mediated β-lactamases, which are inhibited by clavulanic acid. These enzymes are more important clinically as these can be transferred between various species of Enterobacteria ceae. These enzymes are called extended-spectrum β-lactamases (ESBLs). ESBL-producing Enterobacteriaceae have been responsible for numerous outbreaks of infection throughout the world and pose challenging infection control issues. Antibacterial choice is often complicated by multi-resistance. ESBLs can confer resistance against all β-lactam drugs except carbapenems and cephamycins. Nursing home patients may be an important reservoir of ESBL-containing multiple antibiotic-resistant organisms. Use of broad-spectrum oral antibiotics and probably poor infection control practices may facilitate spread of this plasmid-mediated resistance. In addition to known populations at risk, ambulatory patients with chronic conditions represent another patient population that may harbor ESBL-producing organisms. Various methods can be used for detection of ESBLs in the laboratory. These tests include double disc diffusion test, Vitek ESBL test, E Tests, MIC Determination, genetic method, and isoelectric focusing (IEF).     

Laboratory Surveillance for Prospective Plasmid-Mediated AmpC β-Lactamases in the Kinki Region of Japan

Extended-spectrum β-lactamases, plasmid-mediated AmpC β-lactamases (PABLs), and plasmid-mediated metallo-β-lactamases confer resistance to many β-lactams. In Japan, although several reports exist on the prevalence of extended-spectrum β-lactamases and metallo-β-lactamases, the prevalence and characteristics of PABLs remain unknown. To investigate the production of PABLs, a total of 22,869 strains of 4 enterobacterial species, Escherichia coli, Klebsiella pneumoniae, Klebsiella oxytoca, and Proteus mirabilis, were collected during six 6-month periods from 17 clinical laboratories in the Kinki region of Japan. PABLs were detected in 29 (0.13%) of 22,869 isolates by the 3-dimensional test, PCR analysis, and DNA sequencing analysis. PABL-positive isolates were detected among isolates from 13 laboratories. Seventeen of 13,995 (0.12%) E. coli isolates, 8 of 5,970 (0.13%) K. pneumoniae isolates, 3 of 1,722 (0.17%) K. oxytoca isolates, and 1 of 1,182 (0.08%) P. mirabilis isolates were positive for PABLs. Of these 29 PABL-positive strains, 20 (69.0%), 6 (20.7%), 2 (6.9%), and 1 (3.4%) carried the genes for CMY-2, DHA-1, CMY-8, and MOX-1 PABLs, respectively. Pattern analysis of randomly amplified polymorphic DNA and pulsed-field gel electrophoretic analysis revealed that the prevalence of CMY-2-producing E. coli strains was not due to epidemic strains and that 3 DHA-1-producing K. pneumoniae strains were identical, suggesting their clonal relatedness. In conclusion, the DHA-1 PABLs were predominantly present in K. pneumoniae strains, but CMY-2 PABLs were predominantly present in E. coli strains. The present findings will provide significant information to assist in preventing the emergence and further spread of PABL-producing bacteria.β-Lactamase production is the most important factor for β-lactam resistance in Gram-negative rods (16). Plasmid-mediated β-lactamases, such as extended-spectrum β-lactamases (ESBLs), plasmid-mediated AmpC β-lactamases (PABLs), and plasmid-mediated metallo-β-lactamases (MBLs), hydrolyze broad-spectrum β-lactams. Detection of these plasmid-mediated β-lactamase-producing isolates is important for epidemiological studies and hospital infection control, because plasmid-mediated genes can spread to other organisms.The Study of Bacterial Resistance in the Kinki Region of Japan (SBRK) Antimicrobial Surveillance Program was established in 1997 to monitor the predominant pathogens and antimicrobial resistance patterns of nosocomial and community-acquired infections via a broad network of clinical laboratories differing in geographic location and size. Our previously reported survey data from the Kinki region of Japan revealed the prevalence of ESBLs and plasmid-mediated MBLs (21, 30); however, the epidemiology of PABLs remains unknown. For this reason, a laboratory-based surveillance study was conducted to determine the presence and prevalence of PABLs among members of the family Enterobacteriaceae.PABL CMY-1 was first found in a Klebsiella pneumoniae isolate in South Korea in 1989 (4, 5). Since then, additional organisms producing PABLs have been reported worldwide (25). PABLs are a heterogeneous group of enzymes that originated from the chromosomal genes of Enterobacter spp. (ACT-1/MIR-1 type), Citrobacter freundii (CMY/LAT type), Morganella morganii (DHA type), Hafnia alvei (ACC-1), and Aeromonas spp. (CMY/MOX type and FOX type). The most prevalent and most widely distributed PABLs are the CMY/LAT-type enzymes (25). In addition to these enzyme types, DHA-type enzymes have been identified in Taiwan (31) and China (15). In Korea (14, 26), DHA-, CMY/MOX-, and ACT-1/MIR-1-type enzymes have also been identified, while in the United States (1, 17), in addition to the types mentioned above, DHA-, ACT-1/MIR-1-, and FOX-type enzymes have been identified. To date, in Japan, MOX-1 (11), CMY-9 (9, 28), CMY-19 (28), CFE-1 (19), CMY-2 (18), and DHA-1 (18) have been found in clinical isolates. Muratani et al. (18) reported PABL producers among cephem-resistant K. pneumoniae isolates, but this report did not indicate the rate of occurrence of PABLs.For the present study, we collected 22,869 isolates from 17 clinical laboratories in the Kinki region of Japan, and we assessed the prevalence and types of PABL-positive bacteria.     

Diversity of Escherichia coli Strains Producing Extended-Spectrum β-Lactamases in Spain: Second Nationwide Study

The prevalence of extended-spectrum β-lactamase (ESBL)-producing Escherichia coli (ESBLEC) in Spain increased 8-fold from 2000 to 2006. ESBL type, clonal relationship, antimicrobial susceptibility, and clinical data about infections caused by ESBLEC are evaluated in a second nationwide study developed in 2006. From 1008 clinical isolates obtained over 2 months from 44 hospitals, 254 were used for further analysis. ESBL production was evaluated by synergy testing, PCR, and sequencing. Antimicrobial activity was evaluated by microdilution. The clonal relationship was evaluated by repetitive extragenic palindromic-PCR (REP-PCR). The O25b subtype and the new afa operon {"type":"entrez-nucleotide","attrs":{"text":"FM955459","term_id":"218921801","term_text":"FM955459"}}FM955459 were determined by triplex PCR in isolates producing CTX-M-15. Multilocus sequence typing was performed on these isolates. A total of 72% of all ESBLs were of the CTX-M type, 26.8% were of the SHV type, and 1.2% were of the TEM type. The most prevalent ESBLs were CTX-M-14 (119 isolates), SHV-12 (68 isolates), CTX-M-15 (37 isolates), and CTX-M-9 (21 isolates). By REP-PCR, 214 clones were detected. All but five CTX-M-15 ESBLEC isolates corresponded to the international O25b/ST131 clone. This clone had not been detected in the first study (published in 2000). Epidemiological and clinical features were studied in 304 representative patients. A total of 60% of the patients were older than 60 and had nonfatal underlying diseases, and 55% had recently received antibiotics. Urinary tract infections accounted for 71% of cases, and 9% were bacteremic. There has been a significant increase in the prevalence of ESBLEC in Spain, with most of these strains being CTX-M-producing isolates, including the pandemic O25b-ST131. SHV-12-producing E. coli remains an important cause of community-acquired infection.Extended-spectrum β-lactamase (ESBL)-producing Escherichia coli (ESBLEC) has emerged worldwide as a significant cause of both community and healthcare-associated infections (13). Moreover, the role of this microorganism as a cause of nosocomial infection is also increasing (15). The type of ESBL expressed by this microorganism has changed in recent years. The classic SHV and TEM types have often been substituted by members of the CTX-M family (3).The epidemiology of ESBLEC is a complex and evolving phenomenon. A few years ago most ESBLEC strains were clonally unrelated, and the rapid emergence of ESBL was related to the dissemination of mobile genetic elements (14). Nevertheless, both plasmid and bacterial transmission between humans has been demonstrated (17). Recently, the international spread of the O25b-ST131 clone producing CTX-M-15 and other β-lactamases has been described (6, 11). For these reasons, the development of studies directed at discovering the epidemiology of ESBLs in a specific area is recommended.In 2000, the first nationwide study of ESBLEC was developed in Spain (GEIH-BLEE 2000) (8). The prevalence of ESBL production among E. coli isolates was determined to be 0.5%, with CTX-M-9, SHV-12, and CTX-M-14 being the most commonly found ESBLs. No CTX-M-15-producing E. coli strain was isolated. A nationwide study designed along similar lines was developed in 2006 (GEIH-BLEE 2006) because of perceived important changes in the epidemiology of ESBLEC. In 6 years, the prevalence of ESBLEC increased to 4.04% (range, 0.4 to 20.3%) in Spain (7). The distributions of origins of infection between community-acquired, healthcare-associated, and nosocomial strains were 32, 36, and 30%, respectively. The changes in ESBL type, clonal relationship, susceptibility to antimicrobial agents, and relevant clinical data pertaining to ESBLEC in Spain are discussed here.     

Molecular Epidemiology of Extended-Spectrum β-Lactamases from Klebsiella pneumoniae Strains Isolated in Zagreb, Croatia

 In order to assess the molecular epidemiology of 40 previously identified extended-spectrum β-lactamase (ESBL)-producing clinical isolates of Klebsiella pneumoniae, gene sequencing was performed. While the previous examination of these isolates revealed one TEM producer, the sequencing procedure performed in this study identified 13 additional TEM producers, and all of the sequenced genes reflected production of nonESBL TEM-1. All 38 suspected SHV producers were confirmed to be carriers of bla _SHV-ESBL genes using the PCR/NheI test and sequencing. Among them, types SHV-2, SHV-5, and SHV-12 were found in 20, 10, and 7 isolates, respectively, and SHV-2a was identified in 1. SHV-5 and SHV-12 conferred higher resistance to ceftazidime and cefepime, while SHV-2 and SHV-2a raised the minimum inhibitory concentrations of cefotaxime and cefpirome. Fourth-generation cephalosporins were found to be more active against the isolates than third-generation cephalosporins.     

Modified CLSI Extended-Spectrum β-Lactamase (ESBL) Confirmatory Test for Phenotypic Detection of ESBLs among Enterobacteriaceae Producing Various β-Lactamases

The worldwide dissemination of Enterobacteriaceae producing AmpC β-lactamases and carbapenemases makes difficult the phenotypic detection of extended-spectrum β-lactamases (ESBLs), as they may be masked by these additional enzymes. A modification of the CLSI ESBL confirmatory test was developed and evaluated in a comparative study for its ability to successfully detect ESBLs among Enterobacteriaceae producing various carbapenemases (Klebsiella pneumoniae carbapenemase [KPC], VIM, NDM, and OXA-48) and plasmidic or derepressed AmpCs. The modified CLSI ESBL confirmatory test was performed with cefotaxime and ceftazidime disks with and without clavulanate, on which both boronic acid (BA) and EDTA were dispensed. A total of 162 genotypically confirmed ESBL-positive Enterobacteriaceae isolates (83 carbapenemase/ESBL producers, 25 AmpC/ESBL producers, and 54 ESBL-only producers) were examined. For comparison, 139 genotypically confirmed ESBL-negative Enterobacteriaceae isolates (94 of them possessed carbapenemases and 20 possessed AmpCs) were also tested. The standard CLSI ESBL confirmatory test was positive for 106 of the 162 ESBL producers (sensitivity, 65.4%) and showed false-positive results for 4 of the 139 non-ESBL producers (specificity, 97.1%). The modified CLSI ESBL confirmatory test detected 158 of 162 ESBL producers (sensitivity, 97.5%) and showed no false-positive results for non-ESBL producers (specificity, 100%). The findings of the study demonstrate that the modified CLSI ESBL confirmatory test using antibiotic disks containing both BA and EDTA accurately detects ESBLs in Enterobacteriaceae regardless of the coexistence of additional β-lactam resistance mechanisms.     

International Clones of Klebsiella pneumoniae and Escherichia coli with Extended-Spectrum β-Lactamases in a Czech Hospital

A 2-month survey of extended-spectrum β-lactamase (ESBL) producers was performed in a Czech hospital. Klebsiella pneumoniae produced SHV-2, -5, or -12, Escherichia coli produced CTX-M-9 or -15, and other species produced TEM-92 or -132. All K. pneumoniae and E. coli isolates belonged to sequence types (STs) or clonal complexes (CCs) spread across the world (K. pneumoniae clonal complex 11 [CC11], CC14, and sequence type 101 [ST101] and E. coli CC31, CC73, CC131, and CC405) and carried various plasmids (mainly with A/C- and FII-type replicons).Plasmid-encoded extended-spectrum β-lactamases (ESBLs) hydrolyzing penicillins, cephalosporins, and monobactams are a major reason behind the resistance of members of the family Enterobacteriaceae to antimicrobials (4, 21, 35). Organisms with these enzymes, especially Klebsiella pneumoniae and Escherichia coli, have been disseminating worldwide over the last two decades, reaching alarming prevalence rates in some countries or larger geographic areas (5, 43). From among several ESBL types identified, enzymes of the TEM, SHV, and more recently, CTX-M families have been playing prominent roles (30, 39). Growing evidence indicates that global spread of some ESBL variants greatly depends on particular clones of the producer species and on specific types of plasmids carrying their genes (11, 12, 24, 31, 33).In the Czech Republic, the data on ESBLs have been scarce (25, 27). However, in 2007, the rates of ESBL-producing K. pneumoniae and E. coli nosocomial invasive isolates were as high as 30.8% and 6.2%, respectively (P. Urbášková, unpublished results). This work aimed to produce a detailed analysis of ESBL-producing organisms from a large Czech hospital.In June and July 2006, a 2-month survey was performed in the University Hospital in Plzeň, Czech Republic (1,800 beds). All nonrepeated Enterobacteriaceae isolates identified as ESBL producers were collected. Species identification was carried out by Entero test 24 (Pliva Lachema Diagnostika, Brno, Czech Republic), and ESBLs were detected by the double-disk test with modifications for the species with natural AmpC β-lactamases (15). Twenty-four isolates were identified, including 10 K. pneumoniae isolates, 9 E. coli isolates, 3 Providencia stuartii isolates, 1 Enterobacter cloacae isolate, and 1 Proteus mirabilis isolate (Table ​(Table1).1). All these isolates represented nosocomial contamination. Several isolates had no or unclear clinical significance (four K. pneumoniae and E. coli isolates from throat swabs that were surveillance cultures and the K. pneumoniae 1838/06 isolate from the sputum of a geriatric patient that represented colonization of the respiratory tract). Of the total number of 1,710 enterobacterial isolates recovered in the hospital over the study period (including surveillance cultures), ESBL producers represented 1.4% of the isolates, including 3.4% of K. pneumoniae isolates (293 isolates in total) and 1.2% of E. coli isolates (768 isolates in total).

TABLE 1.

Clinical data, typing, β-lactamase content, and plasmid characteristics of the study isolates

Trends in Human Fecal Carriage of Extended-Spectrum β-Lactamases in the Community: Toward the Globalization of CTX-M

SUMMARY

In the last 10 years, extended-spectrum β-lactamase-producing enterobacteria (ESBL-E) have become one of the main challenges for antibiotic treatment of enterobacterial infections, largely because of the current CTX-M enzyme pandemic. However, most studies have focused on hospitalized patients, though today it appears that the community is strongly affected as well. We therefore decided to devote our investigation to trends in ESBL-E fecal carriage rates and comprehensively reviewed data from studies conducted on healthy populations in various parts of the world. We show that (i) community ESBL-E fecal carriage, which was unknown before the turn of the millennium, has since increased significantly everywhere, with developing countries being the most affected; (ii) intercontinental travel may have emphasized and globalized the issue; and (iii) CTX-M enzymes, especially CTX-M-15, are the dominant type of ESBL. Altogether, these results suggest that CTX-M carriage is evolving toward a global pandemic but is still insufficiently described. Only a better knowledge of its dynamics and biology will lead to further development of appropriate control measures.     

Use of Boronic Acid Disk Tests To Detect Extended- Spectrum β-Lactamases in Clinical Isolates of KPC Carbapenemase-Possessing Enterobacteriaceae

We evaluated boronic acid (BA)-based methods for their ability to detect extended-spectrum β-lactamases (ESBLs) among clinical isolates of KPC-producing members of the Enterobacteriaceae family. A total of 155 isolates of Klebsiella pneumoniae (n = 141), Escherichia coli (n = 6), Enterobacter aerogenes (n = 6), and Klebsiella oxytoca (n = 2) genotypically confirmed to be KPC producers were analyzed. As many as 118 isolates harbored ESBLs (103 harbored SHV-type ESBLs, 13 harbored CTX-M-type ESBLs, and 2 harbored both SHV- and CTX-M-type ESBLs); the remaining 37 isolates were genotypically negative for ESBL production. The CLSI ESBL confirmatory test was positive for 79 of the 118 ESBL producers (sensitivity, 66.9%), while all 37 non-ESBL producers were negative (specificity, 100%). When a ≥5-mm increase in the zone diameter of either the cefotaxime (CTX)-clavulanate (CA) or the ceftazidime (CAZ)-CA disks containing BA compared with the zone diameter of the CTX or CAZ disks containing BA was considered to be a positive result for ESBL production, the method detected all 118 ESBL producers (sensitivity, 100%) and showed no false-positive results for non-ESBL producers (specificity, 100%). Double-disk synergy tests, in which disks of CTX, CAZ, aztreonam, or cefepime in combination with BA were placed at distances of 20, 25, and 30 mm (center to center) from a disk containing amoxicillin (amoxicilline)-clavulanate-BA, were able to detect 116 (98.3%), 101 (85.6%), and 28 (23.7%) of the ESBL-positive isolates, respectively; no false-positive results for non-ESBL-producing isolates were detected. Our results demonstrate that the modified CLSI ESBL confirmatory test with antibiotic disks containing BA is the most accurate phenotypic method for the detection of ESBLs in Enterobacteriaceae producing KPC carbapenemases.During the last decade, carbapenem resistance has emerged among clinical isolates of the Enterobacteriaceae family, and this is increasingly attributed to the production of β-lactamases capable of hydrolyzing carbapenems (23). Among these enzymes, a new type of Ambler class A β-lactamase, the Klebsiella pneumoniae carbapenemase (KPC), has been rapidly spreading among K. pneumoniae isolates and other Enterobacteriaceae in the northeastern regions of the United States and has now spread to several regions of North and South America, as well as in Israel, China, and Greece (2, 13, 16, 21).The current spread of KPC enzymes makes them a potential threat to currently available antibiotic-based treatments. These enzymes confer various levels of resistance to all β-lactams, including carbapenems, even though cefamycins and ceftazidime are only weakly hydrolyzed (15, 18). KPC-possessing strains frequently carry extended-spectrum β-lactamase (ESBL) genes (1, 3, 8, 13, 24), which could possibly contribute to the expression and dissemination of the β-lactam resistance trait (8, 18, 21). It should be also noted that KPCs and ESBLs are mostly plasmid-encoded determinants that can easily disseminate to other enterobacterial strains (3, 7, 15, 18, 26). Therefore, the phenotypic detection of ESBLs in KPC-producing isolates of the Enterobacteriaceae is of potential interest for epidemiological purposes as well as for limiting the spread of the underlying resistance mechanisms.The CLSI recommends a phenotypic confirmatory test for ESBL production that consists of measuring the growth-inhibitory zones around both cefotaxime (CTX) and ceftazidime (CAZ) disks with or without clavulanate (CA) for K. pneumoniae, Klebsiella oxytoca, Escherichia coli, and Proteus mirabilis (4). Different double-disk synergy tests (DDSTs) based on the synergy of amoxicillin (amoxicilline)-clavulanate (AMC) with extended-spectrum cephalosporins and aztreonam have also been extensively used for the detection of ESBLs (7). However, strategies for the laboratory identification of ESBLs need to be reviewed and adjusted as additional mechanisms of resistance to β-lactams coexist in enterobacterial strains (7). KPCs hydrolyze several β-lactam antibiotics, and hence, the presence of an ESBL can be masked by the expression of a KPC. Moreover, the weak inhibition of KPCs by the β-lactam inhibitors (15, 18, 30) may interfere with the interpretation of ESBL detection methods and KPC enzymes may be mistaken for ESBLs. Thus, there is a need to accurately detect ESBLs in the presence of coexisting KPC expression.Boronic acid (BA) compounds were recently reported to be reversible inhibitors of KPCs (6, 16, 27). In particular, we have shown that BA disk assays are considered positive for the detection of the KPC enzyme when the growth-inhibitory zone diameter around a meropenem, imipenem, or cefepime disk with phenylboronic acid is 5 mm or greater of the growth-inhibitory zone diameter around the disk containing meropenem or cefepime alone (27). The results of this study also showed that BA affected the activity of CAZ in ESBL-negative KPC-producing isolates but not in SHV ESBL-positive KPC-producing isolates, most likely due to the presence of the SHV ESBL, which is not restrained by BA (27). BA-based tests with disks of CAZ and CTX have also been successfully employed for the identification of ESBLs in AmpC producers (11, 25). These observations led us to design a modified CLSI ESBL confirmatory test using antibiotic disks containing BA as well as different DDSTs employing BA for the accurate detection of ESBLs in KPC-producing enterobacterial isolates.     

Simultaneous Identification of Multiple β-Lactamases in Acinetobacter baumannii in Relation to Carbapenem and Ceftazidime Resistance,Using Liquid Chromatography-Tandem Mass Spectrometry

Shotgun proteomics using liquid chromatography-tandem mass spectrometry (LC-MS/MS) was applied to detect β-lactamases in clinical Acinetobacter baumannii isolates. The correlation of the detection of β-lactamase proteins (rather than PCR detection of the corresponding genes) with the resistance phenotypes demonstrated an added value for LC-MS/MS in antimicrobial susceptibility testing.     

Metallo-β-Lactamases: the Quiet before the Storm?

The ascendancy of metallo-beta-lactamases within the clinical sector, while not ubiquitous, has nonetheless been dramatic; some reports indicate that nearly 30% of imipenem-resistant Pseudomonas aeruginosa strains possess a metallo-beta-lactamase. Acquisition of a metallo-beta-lactamase gene will invariably mediate broad-spectrum beta-lactam resistance in P. aeruginosa, but the level of in vitro resistance in Acinetobacter spp. and Enterobacteriaceae is less dependable. Their clinical significance is further embellished by their ability to hydrolyze all beta-lactams and by the fact that there is currently no clinical inhibitor, nor is there likely to be for the foreseeable future. The genes encoding metallo-beta-lactamases are often procured by class 1 (sometimes class 3) integrons, which, in turn, are embedded in transposons, resulting in a highly transmissible genetic apparatus. Moreover, other gene cassettes within the integrons often confer resistance to aminoglycosides, precluding their use as an alternative treatment. Thus far, the metallo-beta-lactamases encoded on transferable genes include IMP, VIM, SPM, and GIM and have been reported from 28 countries. Their rapid dissemination is worrisome and necessitates the implementation of not just surveillance studies but also metallo-beta-lactamase inhibitor studies securing the longevity of important anti-infectives.     

In Vitro Activity of Ceftolozane-Tazobactam as Determined by Broth Dilution and Agar Diffusion Assays against Recent U.S. Escherichia coli Isolates from 2010 to 2011 Carrying CTX-M-Type Extended-Spectrum β-Lactamases

Ceftolozane MIC₅₀/MIC₉₀s were 4/8 μg/ml when tested against 26 CTX-M-14-type-producing isolates and 64/>64 μg/ml against 219 CTX-M-15-type-producing isolates. The addition of 4 μg/ml tazobactam lowered the ceftolozane MIC₅₀/MIC₉₀s to ≤0.25/0.5 μg/ml by broth microdilution and Etest. The zone diameters for the ceftolozane-tazobactam disks were 23 to 29 mm for 92.2% of the isolates.     

Increased Expressions of Integrin Subunit β1, β2 and β3 in Patients with Acute Infection

Objective: Our previous studies have shown that integrin subunits β1, β2 and β3 were the core proteins of venous thrombi and potential useful biomarker of venous thromboembolism (VTE). Patients with acute infection have a high risk of VTE. In this study we explored that is there any relevance between core proteins and acute infection.Methods: A total of 230 patients (112 females) with clinically proven acute infection in the emergency unit were recruited into this study, meanwhile 230 patients without acute infection matched in sex and age were recruited as control group. Flow cytometry was done to measure the expressions of blood integrin β1, β2, β3 and cellular immunity (CD3, CD4, CD8, CD4/CD8, CD16CD56 and CD19). The association degree between increased core proteins and acute infection was analyzed by calculating the relative risk (RR).Results: The expression of integrin β1, β2 and β3 was markedly increased in patients with acute infection (P=0.000, 0.000 and 0.015, respectively). The relative risk ratio (RR) of increased integrin β1, β2 and β3 in acute infection patients was 1.424 (95%CI: 1.156-1.755, P=0.001), 1.535 (95%CI: 1.263-1.865, P=0.000) and 1.20 (95%CI: 0.947-1.521, P=0.148), respectively. Combined integrin β1, β2 and β3 analysis showed that the relative risk ratio (RR) of increased in patients with acute infection was 2.962 (95%CI: 1.621-5.410, P=0.001), and this relative risk (RR) rise to 3.176 (95%CI: 1.730-5.829, P=0.000) in patients with respiratory tract infection (RTI).Conclusion: As the core proteins of venous thrombi, integrinβ1, β2 and β3 were markedly increased expression in patients with acute infection, which maybe explain the increased risk of VTE in acute infection patients. A weakened immune system could be the basic condition of VTE occurrence.     

Thiamine deficiency increases β-secretase activity and accumulation of β-amyloid peptides

Thiamine pyrophosphate (TPP) and the activities of thiamine-dependent enzymes are reduced in Alzheimer's disease (AD) patients. In this study, we analyzed the relationship between thiamine deficiency (TD) and amyloid precursor protein (APP) processing in both cellular and animal models of TD. In SH-SY5Y neuroblastoma cells overexpressing APP, TD promoted maturation of β-site APP cleaving enzyme 1 (BACE1) and increased β-secretase activity which resulted in elevated levels of β-amyloid (Aβ) as well as β-secretase cleaved C-terminal fragment (β-CTF). An inhibitor of β-secretase efficiently reduced TD-induced up-regulation of Aβ and β-CTF. Importantly, thiamine supplementation reversed the TD-induced alterations. Furthermore, TD treatment caused a significant accumulation of reactive oxygen species (ROS); antioxidants suppressed ROS production and maturation of BACE1, as well as TD-induced Aβ accumulation. On the other hand, exogenous Aβ_1-40 enhanced TD-induced production of ROS. A study on mice indicated that TD also caused Aβ accumulation in the brain, which was reversed by thiamine supplementation. Taken together, our study suggests that TD could enhance Aβ generation by promoting β-secretase activity, and the accumulation of Aβ subsequently exacerbated TD-induced oxidative stress.