Elective distribution of resistance to beta-lactams among Enterobacter cloacae genetic clusters.

OBJECTIVES: The Enterobacter cloacae complex (Ecc), routinely referred to as "E. cloacae" in clinical microbiology, encompasses several species with 12 genetic clusters and one sequence crowd that can be identified based on hsp60 sequencing. Little is known about the pathogenicity and distribution of resistance to antibiotics among the Ecc. METHODS AND RESULTS: In this prospective multicentre study, a total of 193 Ecc clinical isolates were collected from 10 academic hospitals distributed nationally across France and identified at the genetic cluster level on the basis of hsp60 sequencing. E. hormaechei isolates, which belong to clusters VI-VIII, were the largest group (53%), followed by cluster III that accounted for 28% of clinical isolates. All other Ecc clusters were present except cluster VII (E. hormaechei subsp. hormaechei). Cephalosporinase overproduction and ESBL were significantly more present in E. hormaechei (33% and 20%) than in other clusters (19% and 3%, respectively). CONCLUSIONS: These results suggest that rapid identification of "E. cloacae" at the genetic cluster level could improve adequacy of empirical antibiotic treatment and reduce the unnecessary use of broad spectrum antibiotics.

[Fecal carriage of third-generation cephalosporins-resistant Enterobacteriaceae in asymptomatic young adults: evolution between 1999 and 2009]. / Portage digestif d'entérobactéries résistantes aux céphalosporines de troisième génération dans une population d'adultes jeunes asymptomatiques : évolution entre 1999 et 2009.

OBJECTIVES: The aim of this work was to evaluate the fecal carriage of third generation cephalosporins resistant Enterobacteriaceae in nonhospitalized asymptomatic young adults. METHODS: A total of 517 normal fecal samples were spread onto plates agar containing cefotaxime. Isolated strains were identified and studied with agar disk diffusion antibiogram, minimal inhibition concentration in liquid medium and phenotypic and molecular study. Data were compared with a previous study realised in the same conditions in 1999. RESULTS: In 2009, the prevalence of cefotaxime resistant enterobacteria was 4.2%. Of these 22 Enterobacteriaceae, 11 harboured overexpressed cephalosporinase and 11 produced extended-spectrum-betalactamase (ESBL). Among ESBL, six E. coli produced CTX-M from group 1 (n=6), group 2 (n=1), group 9 (n=2), one E. coli produced SHV-12 and one Klebsiella pneumoniae produced CTX-M from group 1. All ESBL were multiresistant. In 1999, all the CTX resistant isolates recovered produced a cephalosporinase and no ESBL was found. CONCLUSIONS: This study highlights the increasing prevalence of fecal carriage of ESBL-producing enterobacteria in asymptomatic young patients in the community (0% in 1999 versus 2.1% in 2009; P<0.001). E. Coli with CTX-M from group 1 was the most frequent ESBL identified, while fecal carriage of Enterobacteteriaceae overproducing cephalosporinase was similar (2.1%).

Rapid detection of multidrug resistant Gram-negative bacilli by Cica-Beta-Test strips.

AIM OF THE STUDY: This study aimed to evaluate the sensitivity and specificity of a new system (Cica-Beta-Test, Kanto Chemical, Japan) for rapid detection of AmpC-derepressed, extended-spectrum ß-lactamases (ESßL) and metallo-ß-lactamases (MßL). METHODS: Two hundred Multi-Drug Resistant (MDR)-Gram-negative bacilli were studied: 170 Enterobacteriaceae and 30 Gram-negative non-fermentative bacteria. One hundred and eighteen strains produced an ESßL, seven MßL and 75 derepressed cephalosporinases. One drop of substrate was dispensed onto the filter pad of the Cica-Beta-Test strip. The bacterial colonies were spread on the filter pad of strip. The reading was performed after 2 to 15 min: turning chromogenic indicated the positive test. Three tests were used: Cica-Beta I for detection of MDR bacteria; Cica-Beta MßL for detection of MßL-producing bacteria and Cica-Beta CVA, which distinguish ESßL and AmpC-derepressed producers. Results were compared with molecular assays. RESULTS: Cica-Beta-Test I has detected 194 MDR (sensitivity 97%), Cica-Beta-Test MßL has shown the presence of six MßL tested (sensitivity 85.7%). Five strains were non-MßL false positive (specificity 97.3%). Cica-Beta-Test CVA allowed the differentiation of ESßL-producing strains (109/115) and AmpC-derepressed strains (56/67) (sensitivity 94.8%, specificity 83.8%). CONCLUSIONS: Because of their epidemic nature, the MDR strains are screened and require strict hygienic measures patients. The simultaneous use of three strips can quickly determine the presence of MDR including ESßL and MßL. Rapid screening of MDR avoids transmission and limits the use of broad-spectrum antibiotics.

Caracterización molecular de aislados clínicos de Pseudomonas aeruginosa productores de la metalobetalactamasa VIM-2 en Cantabria, España / Molecular characterization of Pseudomonas aeruginosa isolates in Cantabria, Spain, producing VIM-2 metallo-beta-lactamase

Introducción En España el aislamiento de cepas de Pseudomonas aeruginosa productoras de metalobetalactamasas (MBL) es poco frecuente. En este artículo se describe la caracterización de 9 aislados clínicos de P. aeruginosa multirresistentes clonalmente relacionados, aislados en Cantabria (España) que poseen el gen (..) (AU)

Introduction Pseudomonas aeruginosa strains producing (..) (AU)

Identification of a novel cephalosporinase (DHA-3) in Klebsiella pneumoniae isolated in Taiwan.

A strain of Klebsiella pneumoniae resistant to cefoxitin and oxyimino-cephalosporins, but susceptible to cefepime, was isolated from an adult patient hospitalised in Taichung, Taiwan. Isoelectric focusing revealed three beta-lactamases with isoelectric points of 5.4, 8.2 and 7.9, respectively. Following PCR with plasmid DNA templates and gene sequencing, these enzymes were shown to correspond to TEM-1, SHV-5 and a novel DHA-1-like enzyme (designated DHA-3). The bla genes for TEM-1 and SHV-5 were transferable, but the bla(DHA-3) gene was non-self-transferable in conjugation experiments. All three bla genes were successfully introduced by electrotransformation into an Escherichia coli recipient (DH5alpha), resulting in a similar resistance profile to that observed in the original donor strain. Other K. pneumoniae strains producing DHA-1-like enzymes have been identified previously in Taiwan, and this report suggests that DHA-type beta-lactamases are continuing to emerge in this country.

Una síntesis versátil y eficiente de nuevas cefalosporinas «caballo de Troya» / An efficient and versatile synthesis of new Trojan-horse cephalosporins

Se describe un procedimiento útil para la síntesis de nuevas cefalosporinas de doble acción. Estas moléculas podrían representar una herramienta fascinante para el tratamiento de enfermedades infeccio- infecciosas sas bacterianas, ya que presentan una posible actividad inhibidora hacia las bacterias que Expresan la â-lactamasa. La principal ventaja de este enfoque sintético de tres pasos reside en su versatilidad, que permite la preparación sistemática de un amplio conjunto de nuevas moléculas

A useful synthesis of new dual-action cephalosporins is reported. These molecules could represent a fascinating tool for treatment of bacterial infectious diseases, since they display a possible inhibitor activity towards â-lactamase expressing bacteria. The major advantage of this 3-step synthetic approach lies in its versatility, which allows the systematic preparation of a wide pool of new molecules

Molecular epidemiology of multi-resistant Escherichia coli.

In this case-control study multi-resistant Escherichia coli isolates were characterized on a molecular level and risk factors for their development were identified. Thirty-two multi-resistant E. coli strains were isolated from the urine of 13 patients attending a renal clinic for chronic urinary tract infection (UTI) and from different sites of 11 terminally ill patients with nosocomial infections hospitalized on five different wards. All 32 isolates were resistant to ciprofloxacin, cotrimoxazole and produced beta-lactamase. All strains contained plasmids of 2-110 MDa of which a 50 MDa and a 100 MDa plasmid were present in 81% of the strains. Pulse-field gel electrophoresis (PFGE) analysis demonstrated 17 genotypes among 32 strains which indicates a polyclonal outbreak with some geographic clustering. Monitoring of patients over the study period showed that either the resident genotype remained the same and that these retained strains underwent changes in their plasmid contents, or that they were replaced by a different genotype after several months of therapy for chronic UTI. Univariate analysis indicated that multi-resistant E. coli develop in the presence of long-term selective ciprofloxacin pressure at a dosing regimen of 250 mg bid for more than 20 days and that treatment with a broad spectrum antimicrobial for more than three days favours the selection of multi-resistant E. coli in the flora of terminally ill patients with multiple disorders.

Carbapenems as inhibitors of OXA-13, a novel, integron-encoded beta-lactamase in Pseudomonas aeruginosa.

A clinical Pseudomonas aeruginosa strain, PAe391, was found to be resistant to a number of antibiotics including ticarcillin, piperacillin, cefsulodin and amikacin, and a disk diffusion assay showed evidence of pronounced synergy between imipenem and various beta-lactam antibiotics. Cloning and nucleotide sequence analysis revealed the dicistronic arrangement of an aac(6')-Ib variant and a novel blaOXA-type gene between the intI and qacE delta 1 genes typical of integrons, in PAe391, this integron was apparently chromosome-borne. The beta-lactamase, named OXA-13, displayed nine amino acid changes with respect to OXA-10:I in position 10 of OXA-10 to T (I10T), G20S, D55N, N73S, T107S, Y174F, E229G, S245N and E259A, OXA-13 (pIapp = 8.0) showed poor catalytic activity against penicillins as well as cephalosporins, but was efficient in hydrolysing some penicillinase-resistant beta-lactams, such as cefotaxime and aztreonam. It was efficiently inhibited by imipenem (KIapp = 11 nM), and formed a stable complex. While the KIapp value of meropenem was similar (16 nM), the corresponding complex was less stable.

Molecular and genetic analysis of the Bacteroides uniformis cephalosporinase gene, cblA, encoding the species-specific beta-lactamase.

The gene, cblA, encoding the species-specific, clavulanate-susceptible, endogenous cephalosporinase was cloned from Bacteroides uniformis WAL-7088. The nucleotide sequence was determined, and the cblA structural gene was found to be 891 nucleotides, with a 48% G+C composition, which is similar to that of the B. uniformis genome. The cblA open reading frame encoded an Ambler class A beta-lactamase polypeptide precursor of 296 amino acid residues with a predicted molecular weight of 33,450. A beta-lactamase-deficient B. uniformis mutant with increased beta-lactam susceptibility was constructed by insertional inactivation of the chromosomal gene. This mutant was complemented by plasmids bearing the cblA gene, and the resulting strains were resistant to cephaloridine and had a beta-lactamase that comigrated with the parental beta-lactamase on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (30,500 Da) and in isoelectric focusing gels (pI 4.6), confirming a role for this beta-lactamase in resistance.

Fluorescent spot test method for specific detection of beta-lactamases.

A simple, sensitive fluorescent spot test method for specific detection of microbial beta-lactamases has been developed, based on the modification of a previously disclosed method (K. C. S. Chen, October 23, 1990, U.S. Patent 4,965,193). The new fluorescence developer used in the present study consisted of 0.5 mM HgCl2, in 0.5 M sodium citrate buffer, pH 4.5, prepared in 0.5% formaldehyde aqueous solution. A beta-lactam substrate solution consisting of a beta-lactam antibiotic with an acyl side chain containing an alpha-amino group and an alpha-phenyl group, or its derivatives, was incubated with a beta-lactamase-producing organism. One volume of the fluorescence developer was added to 4 vol of the incubated beta-lactam substrate solution, followed by heating the mixture at 45 degrees C for 10 min. The mixture was spotted on filter paper. Production of fluorophore indicated beta-lactamase activity. Each fluorophore was analyzed by TLC and its chemical identity was determined. Using ampicillin as the penicillinase substrate and cephalexin as the cephalosporinase substrate, the new method can be conveniently carried out by using dropping bottles for storing and dispensing the substrate solutions and the fluorescence developer. This modified method also provided more favorable conditions for the penicillinases to remain active during fluorescence development. Therefore, the sensitivity of the test was increased.

Localization of cephalosporinase in Enterobacter cloacae by immunocytochemical examination.

Enterobacter cloacae NUH10 was isolated at Nagasaki University Hospital in 1987. E. cloacae NUH10 is a mutant strain which produces high levels of cephalosporinase. E. cloacae ATCC 23355 is known to be sensitive to so-called third generation cephems and produces an inducible cephalosporinase. The polyclonal antibody to cephalosporinase extracted from E. cloacae NUH10 was utilized in post-embedding immunogold labeling in order to localize this protein in E. cloacae ATCC 23355 and E. cloacae NUH10. Immunocytochemical localization of the cephalosporinase in both strains was observed with and without incubation with an inducer. Cephalosporinase was detected in both the cytoplasm and periplasmic space of E. cloacae ATCC 23355 and E. cloacae NUH10 incubated in medium including cefoxitin as an inducer. In the case of incubation without the inducer, a small quantity of cephalosporinase was located in the periplasmic space in either strain of bacteria. Western blot analysis showed that cephalosporinase was predominantly localized in the periplasmic space rather than in the cytoplasmic space.

[In vitro activity of tazobactam and piperacillin combination against 224 strains of Pseudomonas aeruginosa according to the production of beta-lactamase]. / Activité in vitro de l'association pipéracilline-tazobactam sur 224 souches de Pseudomonas aeruginosa en fonction de la production de bêtalactamase.

Piperacillin (PIP) alone and combined with 4 mg/l, 8 mg/l of tazobactam (TAZ) were tested by MIC determination on Mueller-Hinton agar against 224 clinical strains of P. aeruginosa: "wild type" (BLA-), 32 producing a constitutive cephalosporinase (CEP), 41 producing the PSE-1 type beta-lactamase, 7 PSE-2, 8 PSE-3, 9 PSE-4, 13 TEM-1, 24 TEM-2, 13 OXA-1, 22 OXA-2, 5 OXA-3. The combination with 8 mg/l was more effective than that one with 4 mg/l. Combinations of PIP-TAZ 8 mg/l reduced the MICs of PIP for the resistant strains (MICs greater than 64 mg/l) to the susceptible ot the moderately susceptible range (MICs less than or equal to 64 mg/l) for 31% of the CEP producing strains, 63% of the PSE-1, 15% of the PSE-2, none of the PSE-3, 34% of the PSE-4, 39% of the TEM-1, 30% of the TEM-2, 23% of the OXA-1, 14% of the OXA-2, 27% of the OXA-3, TAZ is the first beta-lactamase inhibitor effective against the constitutive cephalosporinase of P. aeruginosa; it is also very effective against the most frequently found PSE-1 beta-lactamase in P. aeruginosa.

In vitro and in vivo transferrable beta-lactam resistance due to a new plasmid-mediated oxyiminocephalosporinase from a clinical isolate of Proteus mirabilis.

A new plasmid-mediated beta-lactamase (FPM-1) with an isoelectric point of 7.2 and a molecular weight of 26,000 was found in a cefuroxime-resistant clinical isolate of Proteus mirabilis strain 6003. FPM-1 can be classified as a type I oxyimino-cephalosporinase on the basis of its substrate specificity and inhibition pattern by clavulanic acid etc., and its conferred resistance on both the strain and transconjugants against most oxyme-type cephalosporins as well as the older ones but not against cefamycins and a few exceptional oxyme-type cephalosporins such as ceftizoxime, ceftazidime and cefixime. In a murine systemic infection model, only these FPM-1-stable drugs exhibited protective activity against the FPM-1-producing P. mirabilis 6003 similar to that against a nonproducing derivative strain. The FPM-1-mediated cefuroxime resistance in P. mirabilis 6003 was transferred to co-infected Escherichia coli 7004 at frequencies between 3.8 x 10(-3) and 4.0 x 10(-2) in a murine ascending urinary tract infection model. In the same infection model due to the FPM-1-producing E. coli transconjugant, FPM-1-stable cefixime was significantly more effective than FPM-1-labile cefteram pivoxil, although both drugs had similar therapeutic effect against its FPM-1-nonproducing counterpart strain.

In vitro activity of cefpirome compared with other third generation cephalosporins against nosocomial isolates in Argentina.

The in vitro activity of cefpirome was evaluated against strains that showed conflicting results for third generation cephalosporins. Against isolates with derepressed inducible chromosomal cephalosporinase (n = 40) cefpirome was the sole cephalosporin with an MIC90 in the susceptible range; Klebsiella spp. with plasmid-mediated beta-lactamases (broad spectrum SHV-2 or SHV-2 type) (n = 40) remained most susceptible to ceftizoxime and cefpirome; against aminoglycoside-resistant Pseudomonas aeruginosa (n = 50), cefpirome was as active as ceftazidime and cefoperazone; against oxacillin-susceptible and oxacillin-resistant Staphylococcus spp., (n = 40), cefpirome was more active than other third generation cephalosporins but killing was inadequate against both oxacillin-resistant staphylococci and enterococci.

7 alpha-formylamino substituent confers beta-lactamase-inactivating potency on 1-oxacephalosporins.

7 alpha-Formylamino-1-oxacephalosporins 7 alpha-formylamino-7 beta-[2- (methylaminocarbonyl)amino-2-(2-thienyl)acetamido]-3-[(1-methyl-1H -tetra zol-5-yl)thiomethyl]-1-oxa-3-cephem-4-carboxylic acid (F1) and 7 alpha-formylamino-7 beta-(2-[(4-ethyl-2,3-dioxopiperazine-1- yl)carbonylamino]-2-phenylacetamido)-3-[(1-methyl-1H-tetr azol-5-yl) thiomethyl]-1-oxa-3-cephem-4-carboxylic acid (F2) were stable against penicillinases and, moreover, inactivated cephalosporinases of Pseudomonas aeruginosa, Citrobacter freundii, and Enterobacter cloacae. Extensive studies of the inactivation of cephalosporinase of P. aeruginosa showed that it resulted from the formation of a transiently stable enzyme-compound complex. The 7 alpha-formylamino substituent was involved in the enzyme inactivation, because 7 alpha-methoxy congeners did not inactivate the enzyme. The number of compound molecules required for inhibition of an enzyme molecule was found to be 36 for F1 and 5.5 for F2, which suggests that the pathway to the complex formation branched off the hydrolysis pathway. Half-lives of the complexes were 400 min for F1 and 260 min for F2. 7 alpha-Formylamino compounds F1 and F2 had antibacterial activities similar to those of 7 alpha-methoxy congeners against beta-lactamase-producing gram-negative bacteria, whereas they were less active against non-beta-lactamase-producing strains of Escherichia coli and Staphylococcus aureus. The conclusion was that the 7 alpha-formylamino substituent conferred the ability to inactivate cephalosporinase on the 1-oxacephalosporins tested, without much impairment of their antibacterial activity.

Cloning and sequencing of the metallothioprotein beta-lactamase II gene of Bacillus cereus 569/H in Escherichia coli.

The structural gene for beta-lactamase II (EC 3.5.2.6), a metallothioenzyme, from Bacillus cereus 569/H (constitutive for high production of the enzyme) was cloned in Escherichia coli, and the nucleotide sequence was determined. This is the first class B beta-lactamase whose primary structure has been reported. The amino acid sequence of the exoenzyme form, deduced from the DNA, indicates that beta-lactamase II, like other secreted proteins, is synthesized as a precursor with a 30-amino acid N-terminal signal peptide. The pre-beta-lactamase II (Mr, 28,060) is processed in E. coli and in B. cereus to a single mature protein (Mr, 24,932) which is totally secreted by B. cereus but in E. coli remains intracellular, probably in the periplasm. The expression of the gene in E. coli RR1 on the multicopy plasmid pRWHO12 was comparable to that in B. cereus, where it is presumably present as a single copy. The three histidine residues that are involved (along with the sole cysteine of the mature protein) in Zn(II) binding and hence in enzymatic activity against beta-lactams were identified. These findings will help to define the secondary structure, mechanism of action, and evolutionary lineage of B. cereus beta-lactamase II and other class B beta-lactamases.

The amino acid sequence of the zinc-requiring beta-lactamase II from the bacterium Bacillus cereus 569.

The amino acid sequence of the zinc-requiring beta-lactamase II from Bacillus cereus strain 569 has been determined. It consists of a single polypeptide chain of 227 residues. It is the only example so far fully characterized of a class B beta-lactamase, and is structurally and mechanistically distinct from both the widely distributed class A beta-lactamases (such as the Escherichia coli RTEM enzyme) and from the chromosomally encoded class C enzymes from Gram-negative bacteria.

[Isolation and properties of beta-lactamase of the cephalosporinase type from cells of Enterobacter aerogenes 6803]. / Vydelenie i svoistva beta-laktamazy tsefalosporinaznogo tipa iz kletok Enterobacter aerogenes 6803.

beta-Lastamase with the molecular weight of 32500 was isolated from the cells of clinical strain 6803 of Enterobacter aerogenes and purified. By the substrate profile determined microiodometrically beta-lactamase was classified as belonging to the cephalosporinase type. The activity of the electrophoretically homogenous enzyme was equal to 430 microM a minute per mg protein with respect to benzylpenicillin. The Km for benzylpenicillin, dicloxacillin, cephaloridin and cephalothin was 6.5410(-5), 3 X 10(-4), 2.1 X 10(-5) and 5.7 X 10(-5) M, respectively. The isoelectric point of the enzyme equal to 5.45 was estimated with the method of preparative isoelectrofocusing. The presence of the serine residue or residues was shown with the use of selective reagents applied to the functionally important groups. With the method of circular dichroism the ratio of alpha- and beta-structures in the enzyme molecule was determined, the slow hydrolysis of cephazolin was demonstrated and the values of Km and Kcat for this process were estimated.

Evaluation of rapid tests for the identification of mycobacteria.

The use of eight rapid tests for the identification of 1307 strains of mycobacteria belonging to 18 species was evaluated. The standard niacin, nitrate-reductase and catalase tests were supplemented by new tests for the detection of beta glucosidase, urease, penicillinase, trehalase and cephalosporinase. This combination of eight rapid tests was not able to replace more conventional procedures but in some cases was of value in discriminating between closely related species.

Cefoxitin resistance by a chromosomal cephalosporinase in Escherichia coli.

Cefoxitin resistance, an unique property found in clinical isolates of Escherichia coli was investigated. Cefoxitin resistant strains, 255 and GN206, produced cephalosporinase constitutively. The cephalosporinase was located in the periplasm, and its production was considered to be mediated by chromosomal gene(s). Cephalosporinase-less mutants from both strains were susceptible to cefoxitin as well as other beta-lactam antibiotics, suggesting that the cephalosporinase was responsible for the resistance to beta-lactam antibiotics including cefoxitin. The cephalosporinases from the E. coli strains were partially purified and their enzymological properties were compared with those of cephalosporinases of Citrobacter freundii and Proteus morganii. Although the cephalosporinases of E. coli, as well as other cephalosporinases, showed little activity for cefoxitin-hydrolysis, the E. coli cephalosporinases exhibited a significantly higher affinity for cefoxitin than other cephalosporinases. It was assumed that the E. coli enzyme located around the targets of cefoxitin protected the targets from the antibiotic by its high affinity for the antibiotic.