Penicillin-Binding Proteins, ß-Lactamases, and ß-Lactamase Inhibitors in ß-Lactam-Producing Actinobacteria: Self-Resistance Mechanisms.

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 ß-lactam antibiotic cephamycin C, a class A ß-lactamase, and the ß lactamases inhibitor clavulanic acid, all of which are encoded in a gene supercluster; in addition, it synthesizes the ß-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 ß-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 ß-lactam gene clusters in protein databases. Notably, in S. clavuligerus and Nocardia lactamdurans, the ß-lactamases are retained in the cell wall and do not affect the intracellular formation of isopenicillin N/penicillin N. The activity of the ß-lactamase in S. clavuligerus may be modulated by the ß-lactamase inhibitory protein BLIP at the cell-wall level. Analysis of the ß-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 ß-lactam biosynthesis and ß-lactam resistance mechanisms in the producer actinobacteria.

A metallo-ß-lactamase enzyme for internal detoxification of the antibiotic thienamycin.

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 ß-lactamase. Here, we expressed thnS gene and investigated its activities against various substrates. Our analyses revealed that ThnS belonged to the superfamily of metallo-ß-lactamase fold proteins. Compared to known ß-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 ß-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.

Cephamycins inhibit pathogen sporulation and effectively treat recurrent Clostridioides difficile infection.

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.

Identification of CFE-2, a new plasmid-encoded AmpC ß-lactamase from a clinical isolate of Citrobacter freundii.

A clinical isolate of Citrobacter freundii (JA99) obtained from a bile culture of a Taiwanese patient was found to produce a plasmid-encoded ß-lactamase conferring resistance to oxyimino-cephalosporins and cephamycins. Resistance arising from production of the ß-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 ß-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 ß-lactamase, named CFE-2. This study highlights the emergence of broad-spectrum cephalosporin resistance in C. freundii owing to a new type of AmpC ß-lactamase.

The Use of Noncarbapenem ß-Lactams for the Treatment of Extended-Spectrum ß-Lactamase Infections.

The continued rise in infections caused by extended-spectrum ß-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 ß-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 ß-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.

Evaluation of cephamycins as supplements to selective agar for detecting Campylobacter spp. in chicken carcass rinses.

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.

The times they are a-changin': carbapenems for extended-spectrum-ß-lactamase-producing bacteria.

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 ß-lactam/ß-lactamase inhibitor combinations and cephamycins.

Determination of MIC distribution of arbekacin, cefminox, fosfomycin, biapenem and other antibiotics against gram-negative clinical isolates in South India: a prospective study.

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.

Heterologous expression of Streptomyces clavuligerus ATCC 27064 cephamycin C gene cluster.

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.

Improved recovery of Clostridium difficile spores with the incorporation of synthetic taurocholate in cycloserine-cefoxitin-fructose agar (CCFA).

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.

Molecular characterization of extended-spectrum beta-lactamases and its correlation with clinical laboratory standards institute interpretive criteria for disk diffusion susceptibility testing in enterobacteriaceae isolates in Thaialnd.

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.

Preliminary studies for cephamycin C purification technique.

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 ß-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.

Identification of blaOXA-128 and blaOXA-129, two novel OXA-type extended-spectrum-ß-lactamases in Pseudomonas aeruginosa, in Hunan Province, China.

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-ß-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.

Acquired AmpC type beta-lactamases: an emerging problem in Italian long-term care and rehabilitation facilities.

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.

CMY-2 beta-lactamase-carrying community-acquired urinary tract Escherichia coli: genetic correlation with Salmonella enterica serotypes Choleraesuis and Typhimurium.

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.

An rplKDelta29-PALG-32 mutation leads to reduced expression of the regulatory genes ccaR and claR and very low transcription of the ceaS2 gene for clavulanic acid biosynthesis in Streptomyces clavuligerus.

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.

Emergence and prevalence of beta-lactamase-producing Klebsiella pneumoniae resistant to cephems in Japan.

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.