blaB Gene Research Articles

The individual contributions of blaB, blaGOB and blaCME on MICs of β-lactams in Elizabethkingia anophelis.

The blaB, blaGOB and blaCME genes are thought to confer β-lactam resistance to Elizabethkingia anophelis, based on experiments conducted primarily on Escherichia coli. To determine the individual contributions of β-lactamase genes to increased MICs in E. anophelis and to assess their impact on the in vivo efficacy of carbapenem therapy. Scarless gene deletion of one or more β-lactamase gene(s) was performed in three clinical E. anophelis isolates. MICs were determined by broth microdilution. Hydrolytic activity and expressions of β-lactamase genes were measured by an enzymatic assay and quantitative RT-PCR, respectively. In vivo efficacy was determined using Galleria mellonella and murine thigh infection models. The presence of blaB resulted in >16-fold increases, while blaGOB caused 4-16-fold increases of carbapenem MICs. Hydrolysis of carbapenems was highest in lysates of blaB-positive strains, possibly due to the constitutionally higher expression of blaB. Imipenem was ineffective against blaB-positive isolates in vivo in terms of improvement of the survival of wax moth larvae and reduction of murine bacterial load. The deletion of blaB restored the efficacy of imipenem. The blaB gene was also responsible for a >4-fold increase of ampicillin/sulbactam and piperacillin/tazobactam MICs. The presence of blaCME, but not blaB or blaGOB, increased the MICs of ceftazidime and cefepime by 8-16- and 4-8-fold, respectively. The constitutionally and highly expressed blaB gene in E. anophelis was responsible for increased MICs of carbapenems and led to their poor in vivo efficacy. blaCME increased the MICs of ceftazidime and cefepime.

Molecular characterization of the multi-drug resistant Myroides odoratimimus isolates: a whole genome sequence-based study to confirm carbapenem resistance.

The bacteria belonging to the Myroides genus are opportunistic pathogens causing community or hospital-acquired infections that result in treatment failure due to antibiotic resistance. This study aimed to investigate molecular mechanisms of antibiotic resistance, clonal relatedness, and the biofilm forming capacity of the 51 multi-drug resistant Myroides odoratimimus. All isolates were screened for blaKPC, blaOXA, blaVIM, blaIMP, blaMUS, blaTUS, blaNDM, and blaB genes by using PCR amplification. Whole genome sequencing (WGS) was applied on three randomly selected isolates for further investigation of antibiotic resistance mechanisms. Clonal relatedness was analyzed by Pulsed-field gel electrophoresis (PFGE) and the microtiter plate method was used to demonstrate biofilm formation. All isolates were positive for biofilm formation. PCR analysis resulted in a positive for only the blaMUS-1 gene. WGS identified blaMUS-1, erm(F), ere(D), tet(X), and sul2 genes in all strains tested. Moreover, the genomic analyses of three strains revealed that genomes contained a large number of virulence factors (VFs). PFGE yielded a clustering rate of 96%. High clonal relatedness, biofilm formation, and multi-drug resistance properties may lead to the predominance of these opportunistic pathogens in hospital environments and make them cause nosocomial infections.

Expression of blaA and blaB and Susceptibility to Penicillins and Cephalosporins in Yersinia enterocolitica from Different Foods

Objective: Yersinia enterocolitica which is an important foodborne pathogen causing illness in humans is an extremely heterogeneous species consisting of different subtypes. It has the intrinsic resistance to β-lactam antibiotics because of the production of β-lactamases, BlaA and BlaB. Y. enterocolitica exhibits variable susceptibilities to β-lactams. Materials and Methods: The expression of the blaA and blaB genes by polymerase chain reaction, and the susceptibility to some β-lactams including penicillins and cephalosporins using the broth microdilution and disk diffusion methods were determined. A total of 18 Y. enterocolitica isolates were examined. Results: Overall, 33.3% of these isolates carried the blaA and blaB genes, all of which were recovered from chicken meat. The wide range of MIC for ampicillin (≤2-128 µg/mL) and ceftazidime (≤0.0625-2 µg/mL) was also observed. Of the Y. enterocolitica isolates, 55.6% were resistant to ampicillin (≥32 µg/mL) while the remaining isolates (44.4%) were susceptible to ampicillin (≤ 8 µg/mL). All isolates were susceptible to ceftazidime at the concentration tested. According to the disk diffusion test, 55.6% and 33.3% of the isolates were resistant to ticarcillin and cefoxitin, respectively. No resistance to piperacillin and ceftriaxone was found. Conclusion: The results showed that the presence of the blaA and blaB genes and intrinsic resistance against penicillins and cephalosporins were variable among Y. enterocolitica food isolates. Furthermore, the blaA and blaB genes were expressed in most of the resistant isolates to β-lactams, which may indicate the contribution of the genes to the drug resistance.

Fluoroquinolone resistance in carbapenem-resistant Elizabethkingia anophelis: phenotypic and genotypic characteristics of clinical isolates with topoisomerase mutations and comparative genomic analysis.

MDR Elizabethkingia anophelis strains are implicated in an increasing number of healthcare-associated infections worldwide, including a recent cluster of E. anophelis infections in the Midwestern USA associated with significant morbidity and mortality. However, there is minimal information on the antimicrobial susceptibilities of E. anophelis strains or their antimicrobial resistance to carbapenems and fluoroquinolones. Our aim was to examine the susceptibilities and genetic profiles of clinical isolates of E. anophelis from our hospital, characterize their carbapenemase genes and production of MBLs, and determine the mechanism of fluoroquinolone resistance. A total of 115 non-duplicated isolates of E. anophelis were examined. MICs of antimicrobial agents were determined using the Sensititre 96-well broth microdilution panel method. QRDR mutations and MBL genes were identified using PCR. MBL production was screened for using a combined disc test. All E. anophelis isolates harboured the blaGOB and blaB genes with resistance to carbapenems. Antibiotic susceptibility testing indicated different resistance patterns to ciprofloxacin and levofloxacin in most isolates. Sequencing analysis confirmed that a concurrent GyrA amino acid substitution (Ser83Ile or Ser83Arg) in the hotspots of respective QRDRs was primarily responsible for high-level ciprofloxacin/levofloxacin resistance. Only one isolate had no mutation but a high fluoroquinolone MIC. Our study identified a strong correlation between antibiotic susceptibility profiles and mechanisms of fluoroquinolone resistance among carbapenem-resistant E. anophelis isolates, providing an important foundation for continued surveillance and epidemiological analyses of emerging E. anophelis opportunistic infections. Minocycline or ciprofloxacin has the potential for treatment of severe E. anophelis infections.

Molecular Characteristics of "BlaB-Like" Chromosomal Inducible Cephalosporinase of Yersinia enterocolitica Biotype 1A Strains.

Yersinia enterocolitica biotype 1A strains are emerging pathogens, frequently isolated from clinical samples across the globe. Y. enterocolitica strains produce two chromosomal β-lactamases, BlaA and BlaB. BlaA is a constitutively expressed, Ambler class A, penicillinase, whereas BlaB is Ambler class C-type, inducible cephalosporinase. An earlier study from our laboratory indicated that instead of BlaB, Y. enterocolitica biotype 1A produced a "BlaB-like" enzyme. The objective of this work was to study the molecular characteristics of "Bla-B like" β-lactamases produced by biotype 1A strains to discern their similarity with AmpC-type β-lactamases and the basis of varied levels of minimum inhibitory concentration (MIC) to cefotaxime. Thus, the promoters and blaB genes were investigated in four strains of biotype 1A. Three-dimensional structures of the "BlaB-like" enzymes were modeled, and docked in silico with cefotaxime to understand how specific substitutions in gene sequences affect antibiotic susceptibilities. Our results indicated that all the reported key catalytic residues were present in variants of "Bla-B-like" enzymes, discerned in biotype 1A strains, but at different positions. Molecular docking of enzyme variants with cefotaxime revealed that lesser was the number of the H-binding residues with cefotaxime in a strain, lower was the MIC of cefotaxime in that strain. To the best of our knowledge, this is the first study in which the molecular characteristics and enzymatic interactions of "BlaB-like" cephalosporinases of Y. enterocolitica biotype 1A strains have been reported.

FEATURES OF BETA-LACTAMASE ACTIVITY IN FRANCISELLA TULARENSIS subsp. MEDIASIATICA

Small Gram-negative bacteria Francisella tularensis is the tularemia causative agent. This species subdivides on four subspecies — ssp. tularensis, holarctica, mediasiatica and novicida, which have some differences in their distribution areas, pathogenicity and epidemical potencial. Until recently only subspecies holarctica was found on the territory of the Russian Federation, but in 2013 a natural focus of tularemia in which circulates F. tularensis subsp. mediasiatica was found on the Altai. Till now this subspecies was found only in Central Asia. The data of laboratory studies indicate the ability of strains of this subspecies to cause infection in rabbits and mice which is comparable in severity to infection caused by subsp. holarctica strains. However, the virulence of F. tularensis subsp. mediasiatica for humans and its epidemical potential are still unclear, since no cases of human infection caused by the strains of this subspecies have been recorded, probably due to the geographical aspects — mountainous Altai and Central Asia are extremely sparsely populated regions. The main phenotypic feature of this subspecies is the lack of activity of β-lactamase, which is responsible for the natural resistance to β-lactam antibiotics (penicillins, cephalosporins and carbapenems). Despite the absence of detectable enzymatic activity, subsp. mediasiatica strains are resistant to these antibiotics. In this article we report that subsp. mediasiatica strains have β-lactamase activity despite to current opinion, but the of β-lactams hydrolysis rate is much more lower in comparison with reaction rate of subs. holarctica strains. In addition, in case of a decrease of the microbial cells number in the nutrient medium, antibiotic susceptibility appears. We identified a single specific for subsp. mediasiatica nucleotide substitution G/A at the 290 position of the blaB gene, which encodes the active serine β-lactamase. This substitution leads to the amino acid substitution Gly/Arg at the 97 position of the protein BlaB. We assume, that enzymatic activity decreasing is the most likely caused by this substitution), for example it may cause some conformational changes leading either to enzyme — substrate affinity decreasing or to in the lifetime of the enzyme-substrate complex increasing. On the basis of the found nucleotide substitution, we developed an allele-specific PCR test that makes it possible to determine whether the studied strain F. tularensis belongs to the subspecies mediasiatica.

Presence of β-lactamase encoding genes in clinical isolates of Y. enterocolitica bioserotype 1B/O8mm isolated in Poland in 2009

Introduction: The intrinsic resistance to β-lactam antibiotics of Y. enterocolitica is mainly due to activity of two type β - lactamases, BlaA and BlaB, which are encoding by genes located on chromosome, blaA and ampC, respectively. High-pathogenicity bioserotype 1B/O8 of Y. enterocolitica has been isolated from clinical samples since 2004 year in Poland. The study shown that clinical isolates of Y. enterocolitica 1B/O8 collected from 2004 to 2009 in Poland constituted the same sensu stricto strain. The aim of present study was to determine the occurrence of genes encoding β-lactamases BlaA and BlaB with the MIC value characterization for selected antibiotics in clinical isolates of Yersinia enterocolitica bioserotype 1B/O isolated in Poland in 2009. Methods: Ampicilin and ticarcillin minimum inhibitory concentrations (MICs) for 64 clinical Y. enterocolitica bioserotype 1B/O8 isolates were determined by Etest and broth microdilution respectively. The blaA i blaB genes were detected by PCR. Results: All investigated isolates were resistant to ticarcillin (MIC 16 – 128 mg/ml), whereas 9,5% to ampicilin (MIC 12 mg/l) according to EUCAST interpretive criteria. All isolates yielded the expected amplicons of blaA and ampC genes. Conclusions: Observed differences in the resistance to ampicillin among analyzed isolates belonging to the epidemic sensu stricto strain can indicate the variable activity of genetic determinants of resistance to β-lactam antibiotics of Y. enterocolitica bioserotype 1B/O8.

Antimicrobial Susceptibility and Distribution of β-Lactamase A (blaA) and β-Lactamase B (blaB) Genes in Enteropathogenic Yersinia Species

One hundred eighty-six strains of enteropathogenic Yersinia (Y.) enterocolitica of bioserotypes 2/O:5,27, 2/O:9, 3/O:3, and 4/O:3 and 12 strains of Yersinia pseudotuberculosis of bioserotypes 1/O:1, 1/O:2, and 2/O:1 from different human (feces) and nonhuman (pig, pork, wild boar, monkey, chinchilla, mara, capybara, salad) sources collected in the years 1995-2009 were examined. Antimicrobial resistance patterns for 12 antimicrobial agents were generated using broth microdilution. The presence and characterization of the β-lactamase genes blaA and blaB were studied using polymerase chain reaction (PCR) and PCR-restriction fragment length polymorphism (RFLP), respectively. The expression of β-lactamase BlaA and BlaB was detected using double-disc diffusion. Y. enterocolitica strains showed resistance to ampicillin (92%), streptomycin (13%), and sulfamethoxazole (2%). Intermediate susceptibility to tetracycline was shown by two Y. enterocolitica strains. All Y. pseudotuberculosis strains were susceptible to all tested antimicrobial agents. Most (99%) of the Y. enterocolitica strains carried both β-lactamase genes. One strain of bioserotype 3/O:3 lacked both genes. In contrast, all Y. pseudotuberculosis strains carried neither of the β-lactamase genes. Homogeneity was detected in all blaA and blaB genes of Y. enterocolitica using PCR-RFLP. The majority (89%) of Y. enterocolitica strains expressed both β-lactamase enzymes, whereas none of the Y. pseudotuberculosis strains showed expression of either enzyme. Also, it seems that the resistance of Y. enterocolitica has not changed during the last years.

Genetic diversity of chromosomal metallo-β-lactamase genes in clinical isolates of Elizabethkingia meningoseptica from Korea

This study was performed to characterize the chromosomal metallo-beta-lactamases (MBLs) of Elizabethkingia meningoseptica isolated from Korea and to propose a clustering method of BlaB and GOB MBLs based on their amino acid similarities. Chromosomal MBL genes were amplified by PCR from 31 clinical isolates of E. meningoseptica. These PCR products were then cloned into a vector and electrotransformed into E. coli DH5 alpha. Nucleotide sequencing was performed by the dideoxy chain termination method using PCR products or cloned DNA fragments. Antimicrobial susceptibilities were determined by the agar dilution method. PCR experiments showed that all 31 E. meningoseptica isolates contained both the blaB and the bla (GOB) genes. DNA sequence analysis revealed that E. meningoseptica isolates possessed seven types of blaB gene, including five novel variants (blaB-9 to blaB-13) and 11 types of bla (GOB) gene, including 10 novel variants (bla (GOB-8) to bla (GOB-17)). The most common combination of MBL was BlaB-12 plus GOB-17 (n=19). Minimum inhibitory concentrations of imipenem and meropenem for the electrotransformants harboring novel BlaB and GOB MBLs were two- or four-fold higher than those for the recipient E. coli DH5 alpha. BlaB and GOB MBLs were grouped in three and six clusters including fifteen novel variants, respectively, based on amino acid similarities.

Single-copy chromosomal integration systems for Francisella tularensis

Francisella tularensis is a fastidious Gram-negative bacterium responsible for the zoonotic disease tularemia. Investigation of the biology and molecular pathogenesis of F. tularensis has been limited by the difficulties in manipulating such a highly pathogenic organism and by a lack of genetic tools. However, recent advances have substantially improved the ability of researchers to genetically manipulate this organism. To expand the molecular toolbox we have developed two systems to stably integrate genetic elements in single-copy into the F. tularensis genome. The first system is based upon the ability of transposon Tn7 to insert in both a site- and orientation-specific manner at high frequency into the attTn7 site located downstream of the highly conserved glmS gene. The second system consists of a sacB-based suicide plasmid used for allelic exchange of unmarked elements with the blaB gene, encoding a beta-lactamase, resulting in the replacement of blaB with the element and the loss of ampicillin resistance. To test these new tools we used them to complement a novel d-glutamate auxotroph of F. tularensis LVS, created using an improved sacB-based allelic exchange plasmid. These new systems will be helpful for the genetic manipulation of F. tularensis in studies of tularemia biology, especially where the use of multi-copy plasmids or antibiotic markers may not be suitable.

Characteristics of β-lactamases and their genes (blaA and blaB) in Yersinia intermedia and Y. frederiksenii

BackgroundThe presence of β-lactamases in Y. enterocolitica has been reported to vary with serovars, biovars and geographical origin of the isolates. An understanding of the β-lactamases in other related species is important for an overall perception of antibiotic resistance in yersiniae. The objective of this work was to study the characteristics of β-lactamases and their genes in strains of Y. intermedia and Y. frederiksenii, isolated from clinical and non-clinical sources in India.ResultsThe enzymes, Bla-A (a constitutive class A penicillinase) and Bla-B (an inducible class C cephalosporinase) were found to be present in all the clinical and non-clinical strains of Y. intermedia and Y. frederiksenii by double disc diffusion method. The results showed differential expression of Bla-A as indicated by presence/absence of synergy whereas expression of Bla-B was quite consistent. The presence of these enzymes was also reflected in the high minimum inhibitory concentrations, MIC50 (126–1024 mg/L) and MIC90 (256–1024 mg/L) of β-lactam antibiotics against these species. Restriction fragment length polymorphism (RFLP) revealed heterogeneity in both blaA and blaB genes of Y. intermedia and Y. frederiksenii. The blaA gene of Y. intermedia shared significant sequence identity (87–96%) with blaA of Y. enterocolitica biovars 1A, 1B and 4. The sequence identity of blaA of Y. frederiksenii with these biovars was 77–79%. The sequence identity of blaB gene of Y. intermedia and Y. frederiksenii was more (85%) with that of Y. enterocolitica biovars 1A, 1B and 2 compared to other species viz., Y. bercovieri, Y. aldovae and Y. ruckeri. Isoelectric focusing data further revealed that both Y. intermedia and Y. frederiksenii produced Bla-A (pI 8.7) and "Bla-B like" (pI 5.5–7.1) enzymes.ConclusionBoth Y. intermedia and Y. frederiksenii showed presence of blaA and blaB genes and unequivocal expression of the two β-lactamases. Limited heterogeneity was detected in blaA and blaB genes as judged by PCR-RFLP. Phylogenetic relationships showed that the two species shared a high degree of identity in their bla genes. This is the first study reporting characteristics of β-lactamases and their genes in strains of Y. intermedia and Y. frederiksenii isolated from Asian region.

Characterization and sequence of the Chryseobacterium (Flavobacterium) meningosepticum carbapenemase: a new molecular class B beta-lactamase showing a broad substrate profile.

The metallo-beta-lactamase produced by Chryseobacterium (formerly Flavobacterium) meningosepticum, which is the flavobacterial species of greatest clinical relevance, was purified and characterized. The enzyme, named BlaB, contains a polypeptide with an apparent Mr of 26000, and has a pI of 8.5. It hydrolyses penicillins, cephalosporins (including cefoxitin), carbapenems and 6-beta-iodopenicillanate, a mechanism-based inactivator of active-site serine beta-lactamases. The enzyme was inhibited by EDTA, 1-10 phenanthroline and pyridine-2,6-dicarboxylic acid, with different inactivation parameters for each chelating agent. The C. meningosepticum blaB gene was cloned and sequenced. According to the G+C content and codon usage, the blaB gene appeared to be endogenous to the species. The BlaB enzyme showed significant sequence similarity to other class B beta-lactamases, being overall more similar to members of subclass B1, which includes the metallo-enzymes of Bacillus cereus (Bc-II) and Bacteroides fragilis (CcrA) and the IMP-1 enzyme found in various microbial species, and more distantly related to the metallo-beta-lactamases of Aeromonas spp. (CphA, CphA2 and ImiS) and of Stenotrophomonas maltophilia (L1).

Purification and characterization of the Proteus vulgaris BlaA protein, the activator of the beta-lactamase gene.

Induction of the expression of the beta-lactamase gene, blaB, is regulated by the blaA gene located just upstream of blaB in the opposite direction in Proteus vulgaris. The expression of the blaA gene is negatively autoregulated by its own product BlaA, the activator of the blaB gene. The P. vulgaris BlaA protein shares high amino acid homology with the LysR family members, which are prokaryotic transcriptional activators that possess a putative helix-turn-helix DNA binding motif. To characterize its function, we purified the BlaA protein to homogeneity from Escherichia coli carrying the expression plasmid of the blaA gene driven by the tac promoter. The gel shift assay and DNaseI footprinting showed that purified BlaA specifically bound to the blaA promoter region, which resides immediately upstream of that of blaB. The binding region contained an inverted repeat, including a T-N11-T sequence which is similar to the LysR motif (T-N11-A) that is conserved in some LysR family members [Goethals et al. (1992) Proc. Natl. Acad. Sci. USA 89, 1646-1650]. We also showed that the BlaA protein forms a dimer in solution, using glycerol gradient centrifugation and glutaraldehyde crosslinking.