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The human society faces a serious problem due to the widespread resistance to antibiotics in clinical practice. Most antibiotic biosynthesis gene clusters in actinobacteria contain genes for intrinsic self-resistance to the produced antibiotics, and it has been proposed that the antibiotic resistance genes in pathogenic bacteria originated in antibiotic-producing microorganisms. The model actinobacteria Streptomyces clavuligerus produces the β-lactam antibiotic cephamycin C, a class A β-lactamase, and the β lactamases inhibitor clavulanic acid, all of which are encoded in a gene supercluster; in addition, it synthesizes the β-lactamase inhibitory protein BLIP. The secreted clavulanic acid has a synergistic effect with the cephamycin produced by the same strain in the fight against competing microorganisms in its natural habitat. High levels of resistance to cephamycin/cephalosporin in actinobacteria are due to the presence (in their β-lactam clusters) of genes encoding PBPs which bind penicillins but not cephalosporins. We have revised the previously reported cephamycin C and clavulanic acid gene clusters and, in addition, we have searched for novel β-lactam gene clusters in protein databases. Notably, in S. clavuligerus and Nocardia lactamdurans , the β-lactamases are retained in the cell wall and do not affect the intracellular formation of isopenicillin N/penicillin N. The activity of the β-lactamase in S. clavuligerus may be modulated by the β-lactamase inhibitory protein BLIP at the cell-wall level. Analysis of the β-lactam cluster in actinobacteria suggests that these clusters have been moved by horizontal gene transfer between different actinobacteria and have culminated in S. clavuligerus with the organization of an elaborated set of genes designed for fine tuning of antibiotic resistance and cell wall remodeling for the survival of this Streptomyces species. This article is focused specifically on the enigmatic connection between β-lactam biosynthesis and β-lactam resistance mechanisms in the producer actinobacteria.

Thienamycin, the first representative of carbapenem antibiotics was discovered in the mid-1970s from soil microorganism, Streptomyces cattleya, during the race to discover inhibitors of bacterial peptidoglycan synthesis. Chemically modified into imipenem (N-formimidoyl thienamycin), now one of the most clinically important antibiotics, thienamycin is encoded by a thienamycin gene cluster composed of 22 genes (thnA to thnV) from S. cattleya NRRL 8057 genome. Interestingly, the role of all thn-genes has been experimentally demonstrated in the thienamycin biosynthesis, except thnS, despite its annotation as putative β-lactamase. Here, we expressed thnS gene and investigated its activities against various substrates. Our analyses revealed that ThnS belonged to the superfamily of metallo-β-lactamase fold proteins. Compared to known β-lactamases such as OXA-48 and NDM-1, ThnS exhibited a lower affinity and less efficiency toward penicillin G and cefotaxime, while imipenem is more actively hydrolysed. Moreover, like most MBL fold enzymes, additional enzymatic activities of ThnS were detected such as hydrolysis of ascorbic acid, single strand DNA, and ribosomal RNA. ThnS appears as a MBL enzyme with multiple activities including a specialised β-lactamase activity toward imipenem. Thus, like toxin/antitoxin systems, the role of thnS gene within the thienamycin gene cluster appears as an antidote against the produced thienamycin.

Spore-forming bacteria encompass a diverse range of genera and species, including important human and animal pathogens, and food contaminants. Clostridioides difficile is one such bacterium and is a global health threat because it is the leading cause of antibiotic-associated diarrhoea in hospitals. A crucial mediator of C. difficile disease initiation, dissemination and re-infection is the formation of spores that are resistant to current therapeutics, which do not target sporulation. Here, we show that cephamycin antibiotics inhibit C. difficile sporulation by targeting spore-specific penicillin-binding proteins. Using a mouse disease model, we show that combined treatment with the current standard-of-care antibiotic, vancomycin, and a cephamycin prevents disease recurrence. Cephamycins were found to have broad applicability as an anti-sporulation strategy, as they inhibited sporulation in other spore-forming pathogens, including the food contaminant Bacillus cereus. This study could directly and immediately affect treatment of C. difficile infection and advance drug development to control other important spore-forming bacteria that are problematic in the food industry (B. cereus), are potential bioterrorism agents (Bacillus anthracis) and cause other animal and human infections.

A clinical isolate of Citrobacter freundii (JA99) obtained from a bile culture of a Taiwanese patient was found to produce a plasmid-encoded β-lactamase conferring resistance to oxyimino-cephalosporins and cephamycins. Resistance arising from production of the β-lactamase could be transferred by conjugation with an IncW plasmid (pJA99) into Escherichia coli J53. The substrate and inhibition profiles of this enzyme resembled that of an AmpC β-lactamase. The resistance gene of pJA99, cloned and expressed in E. coli DH5α, was shown to contain an open reading frame showing 92% amino acid identity with the plasmid-encoded enzyme CFE-1 of E. coli KU6400. DNA sequence analysis also identified a gene upstream of ampC in pJA99 whose sequence was 95.0% identical to the ampR gene from E. coli KU6400. In addition, orf1, the fumarate operon (frdABCD), blc, lolB and repB surrounding the ampR-ampC genes in C. freundii were identified. This DNA fragment was absent in other Citrobacter spp. Therefore, we describe a new plasmid-encoded AmpC β-lactamase, named CFE-2. This study highlights the emergence of broad-spectrum cephalosporin resistance in C. freundii owing to a new type of AmpC β-lactamase., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

The continued rise in infections caused by extended-spectrum β-lactamase (ESBL)-producing pathogens is recognized globally as one of the most pressing concerns facing the healthcare community. Carbapenems are widely regarded as the antibiotics of choice for the treatment of ESBL-producing infections, even when in vitro activity to other β-lactams has been demonstrated. However, indiscriminant carbapenem use is not without consequence, and carbapenem overuse has contributed to the emergence of carbapenem-resistant Enterobacteriaceae. The use of non-carbapenem β-lactams for the treatment of ESBL infections has yielded conflicting results. In this review, we discuss the available data for the use of cephamycins, cefepime, piperacillin-tazobactam, ceftolozane-tazobactam, and ceftazidime-avibactam for the treatment of ESBL infections., (© The Author 2017. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.)

Although cefoperazone is the most commonly used antibiotic in Campylobacter-selective media, the distribution of cefoperazone-resistant bacteria such as extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli is increasing. Here we evaluated the potential of cephamycins for use as supplements to improve modified charcoal-cefoperazone-deoxycholate agar (mCCDA) by replacing cefoperazone with the same concentrations (32 mg/L) of cefotetan (modified charcoal-cefotetan-deoxycholate agar, mCCtDA) and cefoxitin (modified charcoal-cefoxitin-deoxycholate agar, mCCxDA). In chicken carcass rinse samples, the number of mCCDA plates detecting for Campylobacter (18/70, 26%) was significantly lower than that of mCCtDA (42/70, 60%) or mCCxDA plates (40/70, 57%). The number of mCCDA plates (70/70, 100%) that were contaminated with non-Campylobacter species was significantly higher than that of mCCtDA (20/70, 29%) or mCCxDA plates (21/70, 30%). The most common competing species identified using mCCDA was ESBL-producing E. coli, while Pseudomonas species frequently appeared on mCCtDA and mCCxDA., (Copyright © 2016. Published by Elsevier B.V.)

Several antimicrobial agents are being investigated as alternatives to carbapenems in the treatment of infections caused by ESBL-producing Enterobacteriaceae, which may be useful in avoiding overuse of carbapenems in the context of recent global spread of carbapenem-resistant Enterobacteriaceae. The most promising candidates for invasive infections so far are β-lactam/β-lactamase inhibitor combinations and cephamycins., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

The Streptomyces clavuligerus cephamycin C gene cluster has been subcloned in a SuperCos-derived cosmid and introduced in Streptomyces flavogriseus ATCC 33331, Streptomyces coelicolor M1146 and Streptomyces albus J1074. The exconjugant strains were supplemented with an additional copy of the S. clavuligerus cephamycin regulatory activator gene, ccaRC, expressed from the constitutive Pfur promoter. Only S. flavogriseus-derived exconjugants produced a compound active against Escherichia coli ESS22-31 that was characterized by HPLC-MS as cephamycin C. The presence of an additional ccaR copy resulted in about 40-fold increase in cephamycin C production. Optimal heterologous cephamycin C production was in the order of 9% in relation to that of S. clavuligerus ATCC 27064. RT-qPCR studies indicated that ccaRC expression in S. flavogriseus::[SCos-CF] was 7% of that in S. clavuligerus and increased to 47% when supplemented with a copy of Pfur-ccaR. The effect on cephamycin biosynthesis gene expression was thus improved but not in an uniform manner for every gene. In heterologous strains, integration of the cephamycin cluster results in a ccaR-independent increased resistance to penicillin, cephalosporin and cefoxitin, what corresponds well to the strong expression of the pcbR and pbpA genes in S. flavogriseus-derived strains., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Objectives: To determine the in vitro activity of antibiotics, including arbekacin, cefminox, fosfomycin and biapenem which are all still unavailable in India, against Gram-negative clinical isolates., Methods: We prospectively collected and tested all consecutive isolates of Escherichia coli, Klebsiella spp., Pseudomonas aeruginosa and Acinetobacter spp. from blood, urine and sputum samples between March and November 2012. The minimum inhibition concentration (MIC) of 16 antibiotics was determined by the broth micro-dilution method., Results: Overall 925 isolates were included; 211 E. coli, 207 Klebsiella spp., 153 P. aeruginosa, and 354 Acinetobacter spp. The MIC50 and MIC90 were high for cefminox, biapenem and arbekacin for all pathogens but interpretative criteria were not available. The MIC50 was categorized as susceptible for a couple of antibiotics, including piperacillin/tazobactam, carbapenems and amikacin, for E. coli, Klebsiella spp. and P. aeruginosa. However, for Acinetobacter spp., the MIC50 was categorized as susceptible only for colistin. On the other hand, fosfomycin was the only antibiotic that inhibited 90% of E. coli and Klebsiella spp. isolates, while 90% of P. aeruginosa isolates were inhibited only by colistin. Finally, 90% of Acinetobacter spp. isolates were not inhibited by any antibiotic tested., Conclusion: Fosfomycin and colistin might be promising antibiotics for the treatment of infections due to E. coli or Klebsiella spp. and P. aeruginosa, respectively, in India; however, clinical trials should first corroborate the in vitro findings. The activity of tigecycline should be evaluated, as this is commonly used as last-resort option for the treatment of multidrug-resistant Acinetobacter infections.

We performed extended-spectrum beta-lactamase (ESBL) phenotypic testing and molecular characterization of three ESBL genes (TEM, SHV and CTX-M) and susceptibility testing by Clinical Laboratory Standards Institute (CLSI) disk diffusion method against three cephalosporins (ceftriaxone, ceftazidime, cefepime) and a cephamycin (cefoxitin) among 128 Thai Escherichia coli and 84 Thai Klebsiella pneumoniae clinical isolates. ESBL production was discovered in 62% of E. coli and 43% of K. pneumoniae isolates. All isolates susceptible to ceftriaxone were ESBL-negative. Nearly all isolates non-susceptible to ceftriaxone, ceftazidime and cefepime produced ESBL; the presence of CTX-M genes in the isolates correlated with a ceftriaxone non-susceptible phenotype. Thirty-nine of 83 isolates (47%) of ceftazidime-susceptible E. coli and 50 of 99 isolates (50.5%) of cefepime-susceptible E. coli were ESBL-producing. SHV-type beta-lactamase genes were more prevalent among K. pneumoniae than E. coli isolates. CTX-M was the major ESBL gene harbored by ESBL-producers in both E. coli and K. pneumoniae isolates. Non-CTX-M ESBL-producers were found only among K. pneumoniae isolates. This study reveals an increase in ESBL-producing Enterobacteriaceae among Thai isolates and demonstrates gaps in the current CLSI disk diffusion susceptibility guidelines; it indicates the results of ceftazidime and cefepime disk diffusion susceptibility testing using CLSI criteria should be interpreted with caution.

Aim: Culture remains important for the detection and typing of Clostridium difficile. Culture of C. difficile spores can be enhanced on media supplemented with a germinant. Despite this, unsupplemented media continues to be used in some laboratories. The aim of this study was to quantify the effect of the known germinant sodium taurocholate on recovery of C. difficile spores and to determine if the supplement impacts on the recovery of vegetative C. difficile., Methods: The recovery on cycloserine-cefoxitin-fructose agar (CCFA) with and without taurocholate, of spore, vegetative, and total cell fractions of broth cultures of eight C. difficile isolates was compared., Results: Taurocholate in CCFA did not inhibit growth of vegetative C. difficile and significantly increased recovery of spores (p = 0.04)., Conclusions: The routine incorporation of taurocholate in CCFA is recommended for improved sensitivity in C. difficile culture from specimens.

A study was made for purification of cephamycin C from fermentation of Streptomyces clavuligerus. Initially, the culture broth was clarified by microfiltration and ultrafiltration, after which the resulting permeates were subjected to nonspecific adsorption and ion-exchange chromatography on resin columns. The antibiotic activity was measured by the biological method at each stage by assaying its activity against the Escherichia coli ESS, super sensitive to β-lactam antibiotic. The purification processes were assessed in relation to the variables affecting each step. The purification efficiency by nonspecific adsorption was monitored by UV spectrophotometry, while the ion-exchange adsorption fractions were assessed by NMR spectroscopy. Some of the fractions obtained during purification were also analyzed by mass spectrometry (LC/MS and LC/MS/MS) to identify the cephamycin C molecule. These preliminary results proved the process feasibility.

We collected 97 non-repetitive carbapenemases-sensitive clinical isolates of Pseudomonas aeruginosa in Human Province, China, during the period of October 2006 to January 2007. From these isolates, we identified two novel oxacillin-hydrolysing (OXA) type extended-spectrum-β-lactamases (ESBLs): bla OXA-128 and bla OXA-129, which contain the mutations of I89V from bla OXA-56 and K134N from bla OXA-10, respectively. Clinical isolates containing either bla OXA-128 or bla OXA-129 show resistance to cephamycin-class antibiotics but sensitive to carbapenem-class antibiotics. The occurrence of novel OXA-type lactamases suggests a regional prevalent pattern of ESBLs Pseudomonas aeruginosa in this area.

We report the multiple detection of Proteus mirabilis isolates, from 4 different long-term care and rehabilitation facilities (LTCRFs) of Northern Italy, resistant to expanded-spectrum cephalosporins and cephamycins and producing an acquired ampC-like beta-lactamase, named CMY-16. Genotyping by PFGE showed that isolates were clonally related to each other, although not identical. In all isolates the bla(CMY16) gene was not transferable by conjugation and was found to be carried on the chromosome. These results revealed multifocal spreading of a CMY-16 producing P. mirabilis clone in Northern Italy and emphasize the emergence of similar acquired resistance determinants in the LTCRFs setting.

Forty-six cephamycin-resistant Escherichia coli isolates from patients diagnosed with community-acquired urinary tract infection were selected in order to study their resistance mechanism. With the exception of one isolate producing CMY-4, all isolates produced a CMY-2 beta-lactamase. Molecular typing showed that the CMY-2-producing isolates were not related. Cephamycin resistance was plasmid encoded and conjugatively transferred. Plasmid digest profiles suggested that the plasmids were different. Thirty-nine of the 45 CMY-2-producing isolates harboured a plasmid containing a specific DNA fragment, ISEcp1-bla(CMY-2)-blc-sugE, which was identical to those previously published in CMY-2-producing Salmonella enterica serotype Choleraesuis (SCB67) and Salmonella enterica serotype Typhimurium (pNF1358) from Taiwan and the USA, respectively. Among the remaining six isolates, insertion of IS1294 and IS1 at different positions was observed in one and five isolates, respectively. The regions surrounding bla(CMY-2) of the six isolates were identical to the other 39 isolates as well as to SCB67 and pNF1358. Since the present identical transmissible bla(CMY-2)-carrying element was observed in food animal sources both in the USA and Taiwan, its possible transmission to humans, as revealed in this study, is of great concern. Awareness of this mobile resistance element is required to prevent introduction into hospitals and to reduce the spread of this emerging resistance within the community.

The transcriptional and translational control of the biosynthesis of the beta-lactamase inhibitor clavulanic acid is a subject of great scientific and industrial interest. To study the role of the ribosomal protein L11 on control of clavulanic acid gene transcription, the DNA region aspC-tRNA(trp)-secE-rplK-rplA-rplJ-rplL of Streptomyces clavuligerus was cloned and characterized. An S. clavuligerus rplK(DeltaPALG) mutant, with an internal 12 nucleotides in-frame deletion in the rplK gene, encoding the L11 (RplK) ribosomal protein lacking amino acids (29)PALG(32), was constructed by gene replacement. This deletion alters the L11 N-terminal domain that interacts with the RelA and class I releasing factors-mediated translational termination. The mutant grew well, showed threefold higher resistance to thiostrepton, did not form spores and lacked diffusible brown pigments, as compared with the wild-type strain. The wild-type phenotype was recovered by complementation with the native rplK gene. S. clavuligerus rplK(DeltaPALG) produced reduced levels of clavulanic acid (15-26% as compared with the wild type) and cephamycin C (40-50%) in cultures grown in defined SA and complex TSB media. The decreased yields resulted from an impaired transcription of the regulatory genes ccaR and claR and the cefD and ceaS2 genes for cephamycin and clavulanic acid biosynthesis respectively. Expression of ceaS2 encoding carboxyethylarginine synthase (CEAS), the precursor-committing enzyme for clavulanic acid biosynthesis, was particularly affected in this mutant. In the wild-type strain polyphosphorylated nucleotides peaked at 36-48 h of growth in SA cultures whereas expression of the cephamycin and clavulanic acid genes occurred 12-24 h earlier than the increase in ppGpp indicating that there is no strict correlation between the peak of ppGpp and the onset of transcription of the clavulanic acid and cephamycin C biosynthesis. The drastic effect of the rplK(DeltaPALG) mutation on the onset of expression of the ceaS2 and the regulatory ccaR and claR genes and the lack of correlation with ppGpp levels suggest that the onset of transcription of these genes is modulated by the conformational alteration of the N-terminal region of L11 probably by interaction with the nascent peptide releasing factors and with RelA.

Forty-six cephem-resistant Klebsiella pneumoniae strains with minimum inhibitory concentrations>8 microg/mL for cefpodoxime and cefmetazole were selected from clinical isolates obtained between 2000 and 2002 from eight hospitals on Northern Kyushu Island, Japan. We investigated the mechanisms of resistance to cephems in these 46 K. pneumoniae isolates. The results of isoelectric focusing of beta-lactamases produced by these isolates, polymerase chain reaction for detection of various Class A, Class B and Class C beta-lactamases, and determination of the sequence of the beta-lactamase structural gene showed that most of these isolates had various types of broad-spectrum beta-lactamases. Of the 46 isolates, 2 were CMY-2 beta-lactamase producers and 41 were DHA-1 beta-lactamase producers. Forty of the 41 DHA-1 beta-lactamase producers simultaneously produced SHV-12 extended-spectrum beta-lactamase (ESBL), and the remaining isolate simultaneously produced SHV-27. Furthermore, one DHA-1 and SHV-12 beta-lactamase producer also produced IMP-1 beta-lactamase. The only broad-spectrum beta-lactamase with another isolate was IMP-1. Chromosomal DNA restriction fragment analysis using XbaI suggested that nosocomial infection due to DHA-1 and SHV-12 beta-lactamase producers had occurred at two centres. This is the first report of nosocomial infection due to DHA-1 beta-lactamase-producing K. pneumoniae including other plasmid-encoded AmpC beta-lactamases in Japan. The mechanisms of resistance of 44 of the 46 isolates to cephalosporins and cephamycins were ESBL production and/or plasmid-encoded AmpC beta-lactamase and/or IMP-1 beta-lactamase production. For two isolates, the mechanism of resistance to could not be identified. These results show that it is necessary to minimise the prevalence of these resistant strains as it will be a very serious problem if organisms producing these broad-spectrum beta-lactamases increase in clinical situations. It is important to detect these strains sooner and to perform rigorous infection control earlier.

On the basis of molecular dynamics simulations, we investigate the dynamic properties of the carbapenem- and cephamycin-resistant dinuclear zinc metallo-beta-lactamase from Bacteroides fragilis and its complex with a biphenyl tetrazole inhibitor, 2-butyl-6-hydroxy-3-[2'-(1H-tetrazol-5-yl)biphenyl-4-ylmethyl]-3H-quinazolin-4-one 1 (L-159061). The results obtained with the newly developed force field parameters for the coordination environment of the catalytic zinc ions show that the active site gorge comprising major and minor loops gets deeper and narrower upon binding of the inhibitor, which supports the previous experimental implication that the structural flexibility of the loop structures plays a significant role in enzymatic action. In the presence of the inhibitor, the Trp32 side chain at the apex of the major loop covers the entrance of active site channel, thereby contributing to the stabilization of the enzyme-inhibitor complex. In addition to a direct coordination of the inhibitor tetrazole ring to the second zinc ion in the active site, the hydrogen bonding of Lys167 to the inhibitor carbonyl group and hydrophobic interactions between the inhibitor and side chains of loop residues prove to be significant binding forces of the enzyme-inhibitor complex.

Among 99 clinical Klebsiella pneumoniae isolates resistant to cefoxitin and extended-spectrum cephalosporins, coexistence of AmpC (DHA-1, CMY-2, or CMY-8) and extended-spectrum beta-lactamases (CTX-M and/or SHV) was detected in a total of 35. The remainder produced AmpC (n = 42), extended-spectrum beta-lactamases (n = 9), metallo-beta-lactamases (n = 2), or none of these enzymes (n = 11). Phenotypic characteristics of these isolates were demonstrated.

Objectives: To develop an easy, rapid and reproducible spot-inoculation method for phenotypic detection of extended-spectrum beta-lactamases (ESBLs) and AmpC beta-lactamases and to make the existing three-dimensional extract test more convenient for use in routine diagnostic laboratories., Methods: ESBL and AmpC producing and non-producing isolates of Escherichia coli, Klebsiella pneumoniae and Pseudomonas aeruginosa, as identified by the conventional three-dimensional extract test, were used to evaluate the modified procedures. Whole bacterial cells and freeze-thaw preparations, as beta-lactamase sources, were strategically applied to culture plates near ceftazidime and cefoxitin discs on a lawn inoculum of E. coli ATCC 25922. Technical variations of the test included placing the beta-lactamase-containing inoculum into slits, wells and trenches, or onto the surface as spots at varying distances from the discs, and adding clavulanate or cloxacillin to the three-dimensional inoculum to confirm the presence of ESBLs and AmpC beta-lactamases, respectively., Results: All the methods adopted for ESBL and AmpC detection by using the whole bacterial cells gave positive results. However, the best results were given by the spot-inoculation method. In modifications using the enzymic extracts, the enhanced growth of surface organisms was better appreciated in the designed modifications compared with the conventional methods., Conclusions: The method described here is simple and cost-effective. Furthermore, up to 16 isolates may be tested on a single culture plate, thus it is a less labour-intensive and more economic technique than other reported phenotypic methods.

To determine dissemination and genotype of AmpC beta-lactamases and an extended-spectrum beta-lactamase among clinical isolates of Enterobacteriaceae, we performed antibiotic susceptibility testing, pI determination, induction test, plasmid profiles, transconjugation test, enterobacterial repetitive consensus (ERIC)-PCR, and DNA sequencing. Among the 51 clinical isolates collected from a university hospital in Korea, six isolates were resistant to cephamycins. All six isolates produced a plasmid-encoded AmpC-type beta-lactamase, CMY-10. Five strains also produced one or more other beta-lactamases: SHV-12, an extended-spectrum beta-lactamase (five isolates); TEM-1, a class A beta-lactamase (two isolates); and a chromosomal AmpC beta-lactamase (one isolate, a strain of Enterobacter aerogenes, which produced all four of the beta-lactamases that were identified). One of six isolates produced only CMY-10. ERIC-PCR analysis revealed that dissemination of CMY-10 and SHV-12 was due to a clonal outbreak of a resistant strain and to the interspecies spread of resistance to cephamycins and broad-spectrum beta-lactams in Korea. CMY-10 beta-lactamase genes that are responsible for the resistance to cephamycins (cefoxitin and cefotetan), amoxicillin, cephalothin, and amoxicillin-clavulanic acid were cloned and characterized from six clinical isolates. A sequence identical to the common regions in In6, In7, and a novel integron from pSAL-1 was found upstream from blaCMY-10 gene at nucleotides 1-71. A total of 15 nucleotides (I-15) or 18 nucleotides (I-18) between position 71 and 72 were inserted into the blaCMY-10 gene. The blaCMY-10 gene might be inserted into a sul1-type complex integron by I-15 or I-18., (Copyright Mary Ann Liebert, Inc.)

The nosocomial spread of six genetically related Klebsiella pneumoniae strains producing GES-type beta-lactamases was found in a neonatal intensive care unit, and we previously reported that one of the six strains, strain KG525, produced a new beta-lactamase, GES-3. In the present study, the molecular mechanism of cephamycin resistance observed in strain KG502, one of the six strains described above, was investigated. This strain was found to produce a variant of GES-3, namely, GES-4, which was responsible for resistance to both cephamycins (cefoxitin MIC, >128 microg/ml) and beta-lactamase inhibitors (50% inhibitory concentration of clavulanic acid, 15.2 +/- 1.7 microM). The GES-4 enzyme had a single G170S substitution in the Omega-loop region compared with the GES-3 sequence. This single amino acid substitution was closely involved with the augmented hydrolysis of cephamycins and carbapenems and the decreased affinities of beta-lactamase inhibitors to GES-4. A cloning experiment and sequencing analysis revealed that strain KG502 possesses duplicate bla(GES-4) genes mediated by two distinct class 1 integrons with similar gene cassette configurations. Moreover, the genetic environments of the bla(GES-4) genes found in strain KG502 were almost identical to that of bla(GES-3) in strain KG525. From these findings, these two phenotypically different strains were suggested to belong to a clonal lineage. The bla(GES-4) gene found in strain KG502 might well emerge from a point mutation in the bla(GES-3) gene harbored by its ancestor strains, such as strain KG525, under heavy antibiotic stress in order to acquire extended properties of resistance to cephamycins and carbapenems.

Escherichia coli HKY28, a ceftazidime-resistant strain isolated from a urine specimen in Japan, produced an inhibitor-sensitive AmpC beta-lactamase variant. The deduced amino acid sequence of the enzyme contained a number of substitutions and a tripeptide deletion (Gly286-Ser287-Asp288) compared with the sequence of native AmpC of E. coli. When the deletion was reverted by a 9-base insertion at the relevant site of ampC in the clone, the typical inhibitor-resistant phenotype of AmpC was restored, while at the same time the levels of resistance to ceftazidime, cefpirome, and cefepime were reduced eightfold or more. Molecular modeling studies indicated that a structural change took place in the H-10 helix as a result of the deletion, and this change caused an alteration of the substrate binding site, leading to a unique phenotype analogous to that of inhibitor-sensitive class A extended-spectrum beta-lactamases. The degree of inhibition was greater with sulbactam and tazobactam than with clavulanic acid. To our knowledge, this is the first report to have characterized an E. coli ampC that encodes chromosomal AmpC beta-lactamase sensitive to the available beta-lactamase inhibitors.

Objectives: Isolates previously identified as Citrobacter diversus are now known as Citrobacter koseri. We measured sequence variation at the beta-lactamase structural gene among a group of clinical isolates originally identified as C. diversus by API 20E profiling., Methods: beta-Lactamase and 16S rRNA genes were amplified by PCR and sequenced by standard methods. beta-Lactamase induction was attempted in liquid-grown cultures using cefoxitin. Nitrocefin hydrolysis assays were performed using a spectrophotometer., Results: Analysis of 16S rRNA gene sequences showed that Citrobacter spp. isolates with an inducible beta-lactamase gene, cdiA, closely related to 'C. koseri ' NF85 and ULA27 are actually Citrobacter amalonaticus. C. koseri isolates, whose identities were confirmed by 16S rRNA sequencing, produce a class A beta-lactamase, Cko, constitutively at low levels. The cko and cdiA beta-lactamase genes share <45% identity., Conclusions: We have confirmed that cko is a beta-lactamase gene carried by C. koseri, and that isolates previously identified as 'C. koseri ', but carrying the cdiA beta-lactamase gene are C. amalonaticus. Thus, beta-lactamase-gene-specific PCR may provide a valuable tool to differentiate these biochemically homogeneous Citrobacter species.

In Escherichia coli infection, the implications of fluoroquinolone (FQ) and extended-spectrum cephalosporin plus cephamycin (AmpC) resistance for phylogenetic origin and virulence potential are undefined, as is the influence of ecological context on these associations. Accordingly, 106 E. coli isolates exhibiting FQ and/or AmpC resistance and 98 susceptible isolates were compared with regard to phylogenetic background and virulence profiles, stratified by host group (104 predominantly extraintestinal human isolates and 100 predominantly intestinal cattle and swine isolates). Although resistant isolates exhibited significant shifts in phylogenetic distribution and virulence profiles, human and animal isolates exhibited different phylogenetic shifts, and only among human isolates did resistance predict reduced virulence. Evidence for similar strains being resistant versus susceptible was scant. The O15:K52:H1 clonal group and the closely related "clonal group A" featured prominently among resistant and susceptible human isolates, respectively. Thus, in E. coli, antibiotic resistance predicts phylogenetic background and virulence potential in a complex, context-dependent fashion.

Surveys of beta-lactamases in different parts of the world show an important increase in class C beta-lactamases, thus the study of these enzymes is becoming an important issue. We created an overproduction system for Mox-1, a plasmid class C beta-lactamase, by cloning the gene encoding this enzyme, and placing it under the control of a T7 promoter, using vector pET 28a. The enzyme, purified by ion exchange chromatography, was used to obtain the molecular mass (38246), the N-terminal sequence (GEASPVDPLRPVV), and pI (8.9), and to perform a detailed kinetic study. Cephalotin was used as reporter substrate in the case of poor substrates. The kinetic study showed that benzylpenicillin, cephalotin, cefcapene and moxalactam were good substrates for Mox-1 (k(cat)/K(m) values >2.5 x 10(6) M(-1) s(-1)). On the other hand, ceftazidime and cefepime were poor substrates for this enzyme (K(m) values >200 microM). Clavulanic acid had no inhibitory effect on Mox-1 (K(m)=30.2 mM), however aztreonam behaved as an inhibitor of Mox-1 (K(i)=2.85 microM).

AmpC enzymes can be differentiated from other extended-spectrum beta-lactamases by their ability to hydrolyse cephamycins as well as other extended-spectrum cephalosporins. The present study was designed to determine the occurrence of AmpC enzyme-harbouring Gram-negative clinical isolates in a tertiary care hospital in Delhi, India. Among the 135 clinical isolates of Gram-negative bacilli tested, 20.7% were found to harbour AmpC enzymes using a modified three-dimensional test. Inhibition of zone distortion in the presence of cloxacillin confirmed AmpC-harbouring isolates. Maximal incidence of AmpC producers was found among Acinetobacter spp. (42.8%) followed by Klebsiella pneumoniae isolates (33.3%). No AmpC-harbouring isolates revealed decreased susceptibility to cefoxitin. Therefore, Gram-negative bacilli showing resistance to any cephalosporin or aztreonam irrespective of cefoxitin susceptibility should be screened for the AmpC enzyme. The modified three-dimensional test is easy to carry out and can be applied as a phenotypic screening method for detection of AmpC-harbouring Gram-negative organisms. This is the first study to determine the occurrence of AmpC beta-lactamases from India.

The beta-lactam antibiotics are the most widely used of all the groups of antimicrobials, but beta-lactam resistance is increasingly common among members of the Bacteroides fragilis group. Three major mechanisms are involved in beta-lactam resistance, and they act together in certain instances. In the present study, 2 resistant mutants (238m and 1186m) of Bacteroides thetaiotaomicron, obtained from clinical isolates (238 and 1186) by selection with increasing concentrations of cefoxitin, showed decreased susceptibilities to cefoxitin and other beta-lactam antibiotics. Alterations in both penicillin-binding proteins (PBPs) and outer-membrane proteins (OMPs) were observed in the mutants in comparison with their parent strains. The similar alteration in OMPs was also observed in clinical isolates. In conclusion, the beta-lactam-resistant mutants of B. thetaiotaomicron with deficiency in both PBPs and OMPs can be selected for by exposure to cefoxitin, and several mechanisms are involved in the beta-lactam resistance in the strains investigated.

Susceptibilities to 16 antimicrobial agents were determined by measurement of MICs for 344 isolates of anaerobic bacteria recovered from patients with significant infections. Resistance rates varied among antimicrobial agents and the species tested. The beta-lactams were more active in gram-positive than in gram-negative anaerobes. Resistance to meropenem was low (<1%). For beta-lactam-beta-lactamase inhibitors, piperacillin-tazobactam was most active for all species (resistance, <6%). The rates of resistance to cefoxitin (31 to 65%) and clindamycin (50 to 70%) for non-Bacteroides fragilis species of the B. fragilis group were higher than those for B. fragilis (4% resistant to cefoxitin and 33% resistant to clindamycin). Among members of B. fragilis group, Bacteroides thetaiotaomicron was the most resistant to clindamycin (70%) and cefoxitin (65%). Rates of susceptibility to imipenem and metronidazole for B. fragilis continue to be high compared to those from a previous study 10 years ago. However, resistance to metronidazole was found recently in five strains of B. fragilis. We analyzed the genetic relationships among the metronidazole-resistant B. fragilis strains by pulsed-field gel electrophoresis. The metronidazole-resistant B. fragilis strains showed genotypic heterogeneity, excluding the dissemination of a single clone.

Investigation of a Philippine specimen of the red alga Ceratodictyon spongiosum and its sponge symbiont Haliclona cymaeformis led to the isolation of p-sulfooxyphenylpyruvic acid, whose structure was elucidated using spectroscopic methods, with the Z-enol geometry determined through analysis of (3)J(C,H) coupling constants. The metabolite was tested for tyrosine kinase inhibition using a (3)H-thymidine incorporation assay, but was found inactive.

A group-specific bioluminescent Escherichia coli strain for studying the action of beta-lactam antibiotics is described. The strain contains a plasmid, pBlaLux1, in which the luciferase genes from Photorhabdus luminescens are inserted under the control of the beta-lactam-responsive element ampR/ampC from Citrobacter freundii. In the presence of beta-lactams, the bacterial cells are induced to express the luciferase enzyme and three additional enzymes generating the substrate for the luciferase reaction. This biosensor for beta-lactams does not need any substrate or cofactor additions, and the bioluminescence can be measured very sensitively in real time by using a luminometer. Basic parameters affecting the light production and induction in the gram-negative model organism E. coli SNO301/pBlaLux1 by various beta-lactams were studied. The dose-response curves were bell shaped, indicating toxic effects for the sensor strain at high concentrations of beta-lactams. Various beta-lactams had fairly different assay ranges: ampicillin, 0.05-1.0 microg/ml; piperacillin, 0.0025-25 microg/ml; imipenem, 0.0025-0.25 microg/ml; cephapirin, 0.025-2.5 microg/ml; cefoxitin, 0.0025-1.5 microg/ml; and oxacillin, 25-500 microg/ml. Also, the induction coefficients (signal over background noninduced control) varied considerably from 3 to 158 in a 2-hour assay. Different non-beta-lactam antibiotics did not cause induction. Because the assay can be automated using microplate technologies, the approach may be suitable for higher throughput analysis of beta-lactam action.

Aims: Isolates from various samples obtained during 1998 and 1999 were identified and their susceptibility to third-generation cephalosporins, monobactams and/or cephamycins studied along with any production of ESBLs., Methods and Results: Of these samples, bacteria most frequently isolated by the conventional techniques and Vitek GNI card were Escherichia coli (37%), Klebsiella pneumoniae (27%) and Enterobacter cloacae (16%). Using disk diffusion and double-disk synergy tests, we found that 71% strains produced ESBLs and 18% strains produced ESBLs and cephamycinases. Banding patterns of PCR amplification with the designed primers showed that 57% strains were capable of harbouring bla(SHV) genes. The bla(TEM), bla(CMY) and bla(AmpC) genes were harboured by 55%, 31% and 12% strains, respectively. Forty-five percent of strains contained more than two types of beta-lactamase genes. In particular, one strain contained bla(TEM), bla(SHV), bla(CMY) and bla(AmpC) genes., Conclusions: The percentage of ESBL-producing strains was high. The most prevalent beta-lactamase gene was bla(SHV) gene. The bla(CMY) genes have been prevalent in cephamycin-resistant strains. The multidrug-resistant strains resistant to third-generation cephalosporins and cephamycins were detected in high percentage., Significance and Impact of the Study: Resistance mechanisms to beta-lactams, comprising mostly extended-spectrum beta-lactamase (ESBL) production, lead to the resistance against even recently developed beta-lactams in enterobacteria, which is now a serious threat to antibiotic therapy. The high prevalence of bla(CMY) genes and multidrug-resistant genes may also cause therapeutic failure and lack of eradication of these strains by third-generation cephalosporins or cephamycins.

Escherichia coli may become resistant to cephamycines and oxyimino cephalosporins by virtue of promotor and attenuator mutations or because they have acquired mobilized beta-lactamases from other gram-negative bacilli. This study examined Canadian strains to determine how often promotor and/or attenuator mutations account for this mechanism of resistance and the extent to which clonal spread of these organisms has occurred. We sequenced the promotor and attenuator region of 30 strains resistant to cefoxitin. Twenty-two strains had promotor mutations, 26 had attenuator mutations. Most promotor mutations resulted either in a change in the -35 promotor region towards the E. coli sigma 70 consensus sequence or in the creation of a new consensus hexamer upstream. Eight strains had mutations that increased the typical ampC 16-nucleotide spacer region to the consensus 17- or an 18-nucleotide sequence. Of the attenuator mutations, most did not substantially affect the attenuator loop. Several of the mutations have previously been described in South Africa, Scandinavia, and France. There was evidence that strains bearing certain mutations were clonally disseminated; however, the 11 strains bearing a complex set of attenuator mutations were not. The majority of cephamycin resistant E. coli strains in Toronto have attenuator and/or promotor mutations upstream of the chromosomal ampC gene.

Signaling patterns measured in large cell populations are the sum of differing signals from separate cells, and thus, the detailed kinetics of Ca(2+) pulses can often be masked. In an effort to evaluate whether the cytosolic Ca(2+) pulses previously reported in populations of elicitor- and stress-stimulated tobacco cells accurately represent the pulses that occur in individual cells, a study of single cell Ca(2+) fluxes in stress-stimulated tobacco cells was undertaken. Individual aequorin-transformed cells were isolated from a tobacco suspension culture and placed directly on a sensitive photo-multiplier tube mounted in a dark chamber. Ca(2+)-dependent luminescence was then monitored after stimulation with hypo- or hyper-osmotic shock, cold shock, or defense elicitors (oligogalacturonic acid and harpin). Hypo-osmotic shock induced a biphasic Ca(2+) transient in 67% of the single cells tested that exhibited similar kinetics to the biphasic pulses measured repeatedly in 1ml cell suspensions. In contrast, 33% of the stimulated cells displayed Ca(2+) flux patterns that were not previously seen in cell suspension studies. Additionally, because only 29% of the cells tested responded with measurable Ca(2+) pulses to oligogalacturonic acid and 33% to the harpin protein, we conclude that not all cells in a suspension are simultaneously sensitive to stimulation with defense elicitors. In contrast, all cells tested responded with an immediate Ca(2+) influx after cold or hyperosmotic shock. We conclude that in many cases the Ca(2+) signaling patterns of single cells are accurately represented in the signaling patterns of large populations, but that single cell measurements are still required to characterize the Ca(2+) fluxes of the less prominent cell populations., (Copyright 2001 Harcourt Publishers Ltd.)

Incubation of moxalactam and cefoxitin with the Aeromonas hydrophila metallo-beta-lactamase CphA leads to enzyme-catalyzed hydrolysis of both compounds and to irreversible inactivation of the enzyme by the reaction products. As shown by electrospray mass spectrometry, the inactivation of CphA by cefoxitin and moxalactam is accompanied by the formation of stable adducts with mass increases of 445 and 111 Da, respectively. The single thiol group of the inactivated enzyme is no longer titrable, and dithiothreitol treatment of the complexes partially restores the catalytic activity. The mechanism of inactivation by moxalactam was studied in detail. Hydrolysis of moxalactam is followed by elimination of the 3' leaving group (5-mercapto-1-methyltetrazole), which forms a disulfide bond with the cysteine residue of CphA located in the active site. Interestingly, this reaction is catalyzed by cacodylate.

The gene encoding BLIP, a beta-lactamase-inhibitory protein, was disrupted in wild-type Streptomyces clavuligerus and in a clavulanic acid non-producing mutant. The resulting BLIP mutant and BLIP/clavulanic acid double mutant showed no residual proteinaceous beta-lactamase-inhibitory activity, indicating that only a single beta-lactamase-inhibitory protein exists in S. clavuligerus. The lack of any proteinaceous beta-lactamase-inhibitory activity in the bli and bli/claR mutants also indicates that BLP, the BLIP-like protein, encoded by S. clavuligerus does not possess beta-lactamase-inhibitory activity despite its similarity to BLIP. The bli mutant and the bli/claR double mutant did not show any aberrant growth morphology, sporulation defects, or alterations in cephamycin C production or penicillin G resistance when compared to wild-type S. clavuligerus or to the claR single mutant. Mutants bearing the bli gene disruption did show an elevated level of production of clavam-2-carboxylate and hydroxymethyl clavam as well as clavulanic acid. This phenomenon was observed in the middle stages of production of these clavams but was not detected during maximum production. The production of BLIP was also determined to be down-regulated in a ccaR mutant, lacking the pathway-specific transcriptional regulator required for production of cephamycin C and clavulanic acid. Sequencing of the regions flanking the bli gene showed the presence of a partial open reading frame that encodes a DNA-binding protein, and several open reading frames apparently involved in the production of an ABC transporter.

The emergence of extended-spectrum beta-lactamase (ESBL)- producing Klebsiella pneumoniae and other ESBL-producing Enterobacteriaceae is an increasing problem and presents a dilemma for clinicians. The problem is not only detection of this resistance in the microbiology laboratory, but also a therapeutic dilemma due to multiple drug resistance to extended-spectrum beta-lactams and other agents, including fluoroquinolones, trimethoprim-sulfamethoxazole, and gentamicin. Currently, imipenem appears to be the drug of choice for serious infections due to these isolates, based on accumulating clinical experience. Because options for therapy are limited, control and prevention measures are particularly important. These should include not only traditional infection control measures such as contact precautions, but also antibiotic utilization measures, such as widespread empiric use of ceftazidime.

The AmpC beta-lactamase gene and a small portion of the regulatory ampR sequence of Enterobacter aerogenes 97B were cloned and sequenced. The beta-lactamase had an isoelectric point of 8 and conferred cephalosporin and cephamycin resistance on the host. The sequence of the cloned gene is most closely related to those of the ampC genes of E. cloacae and C. freundii.

Fifty-two strains of Klebsiella pneumoniae producing an AmpC-type plasmid-mediated beta-lactamase were isolated from 13 patients in the same intensive care unit between March 1998 and February 1999. These strains were resistant to ceftazidime, cefotaxime and ceftriaxone, but susceptible to cefoxitin, cefepime and aztreonam. Plasmid content and genomic DNA restriction pattern analysis suggested dissemination of a single clone. Two beta-lactamases were identified, TEM-1 and ACC-1. We used internal bla(ACC-1) primers, to sequence PCR products obtained from two unrelated strains of Hafnia alvei. Our results show that the ACC-1 beta-lactamase was derived from the chromosome-encoded AmpC-type enzyme of H. alvei.

Aeromonas spp. are increasingly being recognized as human pathogens. The presence of metallo-beta-lactamases in these organisms represents a potential problem in antimicrobial therapy. Mechanism-based inactivators of beta-lactamases are used to overcome the resistance of clinical pathogens to beta-lactam antibiotics, but no clinical useful inhibitors of the metallo-beta-lactamases are presently known. Studying the interaction between cefotetan and Aeromonas spp. producing metallo-beta-lactamase activity, we observed that cefotetan behaved as a transient inactivator for both the crude extracts of Aeromonas strains and the purified enzymes from Aeromonas hydrophila AE036 and Aeromonas schubertii MNSA20. The direct hydrolysis of cefotetan showed that it was a poor substrate for both purified enzymes. In view of the minimum inhibitory concentrations, cefotetan shows to be a useful antimicrobial agent against Aeromonas spp., (Copyright 2000 S. Karger AG, Basel.)

In vitro synthesis of cefminox by cell-free extracts of Streptomyces clavuligerus was investigated using alpha-ketoglutarate, L-ascorbic acid, FeSO(4), S-adenosyl-L-methionine, and 7alpha-demethoxycefminox as the substrates. The formation of cefminox was detected both by a biological assay with Proteus vulgaris GN 76/C-1 and by high performance liquid chromatography. Although the conversion rate of 7alpha-demethoxycefminox to cefminox was observed to be quite low, it still demonstrated the potential for an enzymatic process to replace the chemical steps which are currently in use for the production of cefminox.

Mycobacterium fortuitum is an uncommon, but well-recognized, pathogen in immunocompromised hosts, with special predilection for bone and soft tissue infections in solid organ transplant recipients. We describe a case of osteomyelitis due to this pathogen in a peripheral blood stem cell transplant recipient.

Escherichia coli strains showing an increased resistance to oxyiminocephalosporins without an extended spectrum beta-lactamase production have been screened for mutations in their ampC beta-lactamase gene promoter. Mutations were found by direct sequencing of seven promoters at positions -42, -32 (box -35), -18, -1, +5, +24 (attenuator), +31 (attenuator) and +58. By using rapid and simple methods, three of these mutations (-42, -32 and +24), which could enhance transcription, were searched in 37 resistant and 25 sensitive isolates. The -42 mutation was present in 33 of the 37 promoters from the resistant isolates. The -32 and +24 mutations were present only in three and two promoters, respectively, and they were combined in the most resistant strain of the study. The +24 mutation was detected in another strain associated with a 1-bp insertion between the -35 and -10 conserved sequences. None of these mutations was detected in the ampC beta-lactamase from the sensitive isolates.

We have demonstrated by using an in vitro approach that interruption of the OmpK36 porin gene by insertion sequences (ISs) is a common type of mutation that causes loss of porin expression and increased resistance to cefoxitin in Klebsiella pneumoniae. This mechanism also operates in vivo: of 13 porin-deficient cefoxitin-resistant clinical isolates of K. pneumoniae, 4 presented ISs in their ompK36 gene.

A strain of Proteus mirabilis resistant to beta-lactams, including cefoxitin, was isolated from the urine of a woman from Tunisia. Its antibiotic susceptibility pattern and that of the Escherichia coli transconjugant suggested the presence of an AmpC-type beta-lactamase. Two bands of beta-lactamase activity (pI 5.4 and 9.2) were detected by isoelectric focusing. The nucleotide sequence of the gene encoding the AmpC-type enzyme was determined. The deduced amino acid sequence was 98-99% identical to CMY-3 and to those of the plasmid-mediated AmpC-type beta-lactamases originated from Citrobacter freundii and 97% identical to the chromosome-encoded beta-lactamase of a Tunisian clinical isolate of C. freundii. This enzyme differs from CMY-2 by one substitution (Arg for Trp at position 221) and from CMY-3 by two substitutions (Glu for Gly at position 42 and Ser for Asn at position 363) and we propose the denomination CMY-4.