Opening of a cryptic pocket in ß-lactamase increases penicillinase activity.

Understanding the functional role of protein-excited states has important implications in protein design and drug discovery. However, because these states are difficult to find and study, it is still unclear if excited states simply result from thermal fluctuations and generally detract from function or if these states can actually enhance protein function. To investigate this question, we consider excited states in ß-lactamases and particularly a subset of states containing a cryptic pocket which forms under the Ω-loop. Given the known importance of the Ω-loop and the presence of this pocket in at least two homologs, we hypothesized that these excited states enhance enzyme activity. Using thiol-labeling assays to probe Ω-loop pocket dynamics and kinetic assays to probe activity, we find that while this pocket is not completely conserved across ß-lactamase homologs, those with the Ω-loop pocket have a higher activity against the substrate benzylpenicillin. We also find that this is true for TEM ß-lactamase variants with greater open Ω-loop pocket populations. We further investigate the open population using a combination of NMR chemical exchange saturation transfer experiments and molecular dynamics simulations. To test our understanding of the Ω-loop pocket's functional role, we designed mutations to enhance/suppress pocket opening and observed that benzylpenicillin activity is proportional to the probability of pocket opening in our designed variants. The work described here suggests that excited states containing cryptic pockets can be advantageous for function and may be favored by natural selection, increasing the potential utility of such cryptic pockets as drug targets.

Temoneira-1 ß-lactamase is not a metalloenzyme, but its native metal ion binding sites allow for purification by immobilized metal ion affinity chromatography.

ß-lactamases protect bacteria from ß-lactam antibiotics. Temoneira (TEM) is a class A serine ß-lactamase and its coding sequence is designed into DNA vectors, such as pET-21a (+), to provide antibiotic resistance. TEM-1 ß-lactamase was overexpressed efficiently from this vector upon inducing protein expression by IPTG in BL21(DE3) cells. Immobilized metal ion affinity chromatography (IMAC) was used based on the three native putative metal ion binding sites of TEM-1 ß-lactamase, each consisting of a pair of histidine sidechains. Elution was achieved at low concentrations of imidazole (∼15-200 mM). Two steps of IMAC and a subsequent anion exchange purification produced highly pure TEM-1 ß-lactamase with a yield of 1.9 mg/g of wet bacterial pellet weight. Mass spectrometry revealed that the mature form of ß-lactamase (without the signal sequence) was obtained. The secondary structure composition, calculated from the circular dichroism spectrum, showed that the target protein was folded similar to the published crystal structure. Ni(II) binding to the enzyme was also investigated. Increasing amounts of Ni(II) ions had only a small effect on the protein structure. Mass spectrometry detected up to three bound metal ions at 10:1 Ni(II):protein molar ratio, but the major peak was assigned to the monometallated ß-lactamase indicating the presence of a paramount metal ion binding site formed by the H151/H156 pair.

A two-dimensional photonic crystal hydrogel biosensor for colorimetric detection of penicillin G and penicillinase inhibitors.

A simple penicillinase functionalized two-dimensional photonic crystal hydrogel (2DPPCH) biosensor was developed for colorimetric detection of penicillin G and penicillinase inhibitors. The penicillinase can specifically recognize penicillin G and catalyze it to produce penicilloic acid, which decreases the pH of the hydrogel microenvironment and shrinks the pH-sensitive hydrogel. The particle spacing decrease of the 2D photonic crystal array induced by the hydrogel shrinkage further causes a blue-shift in the diffraction wavelength. While the hydrolysis reaction is repressed upon treatment with clavulanate potassium (a kind of penicillinase inhibitor), no significant change in the diffraction wavelength is found. The detection of targets can be achieved by measuring the Debye diffraction ring diameter or observing the structural color change in the visible region. The lowest detectable concentrations for penicillin G and clavulanate potassium are 1 µM and 0.1 µM, respectively. Moreover, the 2DPPCH is proved to exhibit high selectivity and an excellent regeneration property, and it shows satisfactory performance for penicillin G analysis in real water samples.

Performance of penicillinase detection tests in Staphylococcus epidermidis: comparison of different phenotypic methods.

BACKGROUND: Staphylococcus epidermidis is the leading coagulase negative staphylococci (CoNS) species associated with healthcare associated infections. In order to de-escalate antimicrobial therapy, isolates of S. epidermidis lacking the blaZ gene should be eligible for targeted antimicrobial therapy. However, testing the susceptibility of coagulase negative staphylococci (CoNS) to penicillin G is no longer recommended by EUCAST, given the low performances for penicillinase detection in CoNS. The objective of this work was to determine a phenotypic method with high performance for detecting penicillinase production in S. epidermidis. RESULTS: Four techniques for the detection of penicillinase production (disk diffusion, zone edge test, nitrocefin test, Minimal Inhibitory Concentration (MIC) by automated system Vitek2®) were evaluated on 182 S. epidermidis isolates, using identification of blaZ gene by PCR as the reference method. The performance of the methods for penicillinase detection was compared by the sensitivity, the specificity, the negative predictive value and the positive predictive value, and with Cohen's kappa statistical test. Among the 182 S. epidermidis included in this study, 55 carried the blaZ gene. The nitrocefin test, characterized by a poor sensitivity (91%), was therefore excluded from S. epidermidis penicillinase detection. The algorithm proposed here for the penicillinase detection in S. epidermidis involved two common antimicrobial susceptibility techniques: disk diffusion method and MIC by Vitek2® system. Disk diffusion method, interpreted with a 26 mm breakpoint for penicillin G, was associated with a high sensitivity (98%) and specificity (100%). This method was completed with zone edge test for S. epidermidis with penicillin G diameter from 26 to 35 mm (sensitivity of 98%). The Vitek2® system is associated with a low sensitivity (93%) and a high specificity (99%) This low sensitivity is associated with false negative results, in isolates with 0.12 mg/L Penicillin G MIC values and blaZ positive. Thus for penicillin G MIC of 0.06 mg/L or 0.12 mg/L, a second step with disc diffusion method is suggested. CONCLUSIONS: According to our results, the strategy proposed here allows the interpretation of penicillin G susceptibility in S. epidermidis isolates, with an efficient detection of penicillin G resistance.

Fiber Mach-Zehnder-interferometer-based liquid crystal biosensor for detecting enzymatic reactions of penicillinase.

A novel, to the best of our knowledge, liquid crystal (LC) biosensor, based on an optical fiber Mach-Zehnder interferometer (MZI), is proposed. The proposed optical fiber MZI consists of two single-mode fibers and a tapered photonic crystal fiber (PCF). The PCF is coated with 4'-pentyl-biphenyl-4-carboxylic acid (PBA)-doped 4-cyano-4'-pentylbiphenyl (5CB). Being a pH-sensitive material, PBA can manipulate LC molecules to different orientations according to their pH values. When the orientation of LC molecules changes with varying pH, the effective refractive index of the cladding modes also is accordingly affected. Enzymatic reactions of penicillinase can release H+, which causes the decrease of the pH. Therefore, the enzymatic reactions of penicillinase can be sensed by monitoring the peak shift in the interference spectrum. The effects of the tapered diameter on the sensitivity of the sensor were experimentally investigated as well.

Past and Present Perspectives on ß-Lactamases.

ß-Lactamases, the major resistance determinant for ß-lactam antibiotics in Gram-negative bacteria, are ancient enzymes whose origins can be traced back millions of years ago. These well-studied enzymes, currently numbering almost 2,800 unique proteins, initially emerged from environmental sources, most likely to protect a producing bacterium from attack by naturally occurring ß-lactams. Their ancestors were presumably penicillin-binding proteins that share sequence homology with ß-lactamases possessing an active-site serine. Metallo-ß-lactamases also exist, with one or two catalytically functional zinc ions. Although penicillinases in Gram-positive bacteria were reported shortly after penicillin was introduced clinically, transmissible ß-lactamases that could hydrolyze recently approved cephalosporins, monobactams, and carbapenems later became important in Gram-negative pathogens. Nomenclature is based on one of two major systems. Originally, functional classifications were used, based on substrate and inhibitor profiles. A later scheme classifies ß-lactamases according to amino acid sequences, resulting in class A, B, C, and D enzymes. A more recent nomenclature combines the molecular and biochemical classifications into 17 functional groups that describe most ß-lactamases. Some of the most problematic enzymes in the clinical community include extended-spectrum ß-lactamases (ESBLs) and the serine and metallo-carbapenemases, all of which are at least partially addressed with new ß-lactamase inhibitor combinations. New enzyme variants continue to be described, partly because of the ease of obtaining sequence data from whole-genome sequencing studies. Often, these new enzymes are devoid of any phenotypic descriptions, making it more difficult for clinicians and antibiotic researchers to address new challenges that may be posed by unusual ß-lactamases.

Chromosome-Encoded Broad-Spectrum Ambler Class A ß-Lactamase RUB-1 from Serratia rubidaea.

Whole-genome sequencing of Serratia rubidaea CIP 103234T revealed a chromosomally located Ambler class A ß-lactamase gene. The gene was cloned, and the ß-lactamase, RUB-1, was characterized. RUB-1 displayed 74% and 73% amino acid sequence identity with the GIL-1 and TEM-1 penicillinases, respectively, and its substrate profile was similar to that of the latter ß-lactamases. Analysis by 5' rapid amplification of cDNA ends revealed promoter sequences highly divergent from the Escherichia coli σ70 consensus sequence. This work further illustrates the heterogeneity of ß-lactamases among Serratia spp.

Poly(2-oxazoline)-Antibiotic Conjugates with Penicillins.

The conjugation of antibiotics with polymers is rarely done, but it might be a promising alternative to low-molecular-weight derivatization. The two penicillins penicillin G (PenG) and penicillin V (PenV) were attached to the end groups of different water-soluble poly(2-oxazoline)s (POx) via their carboxylic acid function. This ester group was shown to be more stable against hydrolysis than the ß-lactam ring of the penicillins. The conjugates are still antimicrobially active and up to 20 times more stable against penicillinase catalyzed hydrolysis. The antibiotic activity of the conjugates against Staphylococcus aureus in the presence of penicillinase is up to 350 times higher compared with the free antibiotics. Conjugates with a second antimicrobial function, a dodecyltrimethylammonium group (DDA-X), at the starting end of the PenG and PenV POx conjugates are more antimicrobially active than the conjugates without DDA-X and show high activity in the presence of penicillinase. For example, the conjugates DDA-X-PEtOx-PenG and DDA-X-PEtOx-PenV are 200 to 350 times more active against S. aureus in the presence of penicillinase and almost as effective as the penicillinase stable cloxacollin (Clox) under these conditions. These conjugates show even greater activity compared to cloxacollin without this enzyme present. Further, both conjugates kill Escherichia coli more effectively than PenG and Clox.

Enantio convergent biotransformation of O,O-dimethyl-4-oxoazetidin-2-ylphosphonate using fungal cells of Penicillium minioluteum and purified enzymes.

This report presents the bioconversion of O,O-dimethyl-4-oxoazetidin-2-ylphosphonate 1 performed in two ways: with the enzymatic system of P. minioluteum and with the application of purified enzymes: penicillinase and two proteases of different origin. Recorded NMR spectra allowed confirming the reaction progress and also postulating possible mechanism of conversion. The path of bioconversion was defined as enantio convergent process for both modes of applied biocatalysts. This means that kinetically driven resolution of racemic mixture of the substrate leads to the one enantiomer of the product. The bioconversion started from ester bond hydrolysis (equally in both enantiomers) with the conversion degree from 30% (whole-cell) to 35% (isolated enzymes) and with the production of optically pure monoester (compound 2; 100% of e.e). For whole-cell bioprocess it was the initiative step for the enantioselective amide bond hydrolysis, what resulted in synthesis of desired product 3-amino-3-phosphonopropanoic acid 4. However, the most effective enzymatic hydrolysis of ester bond performed with penicillinase from Enterobacter cloacae led only to the monoester product 2.

Label-Free Photonic Crystal-Based ß-Lactamase Biosensor for ß-Lactam Antibiotic and ß-Lactamase Inhibitor.

A simple, label-free, and visual photonic crystal-based ß-lactamase biosensor was developed for ß-lactam antibiotic and ß-lactamase inhibitor in which the penicillinase (a ß-lactamase) was immobilized on the pH-sensitive colloidal crystal hydrogel (CCH) film to form penicillinase colloidal crystal hydrogel (PCCH) biosensing film. The hydrolysis of penicillin G (a ß-lactam antibiotic) can be catalyzed by penicillinase to produce penicilloic acid, leading to a pH decrease in the microenvironment of PCCH film, which causes the shrink of pH-sensitive CCH film and triggers a blue-shift of the diffraction wavelength. Upon the addition of ß-lactamase inhibitor, the hydrolysis reaction is suppressed and no clear blue-shift is observed. The concentrations of ß-lactam antibiotic and ß-lactamase inhibitor can be sensitively evaluated by measuring the diffraction shifts. The minimum detectable concentrations for penicillin G and clavulanate potassium (a ß-lactamase inhibitor) can reach 1 and 0.1 µM, respectively. Furthermore, the proposed method is highly reversible and selective, and it allows determination of penicillin G in fish pond water samples.

Identification of peptide inhibitors of penicillinase using a phage display library.

There is a constant need to identify novel inhibitors to combat ß-lactamase-mediated antibiotic resistance. In this study, we identify three penicillinase-binding peptides, P1 (DHIHRSYRGEFD), P2 (NIYTTPWGSNWS), and P3 (SHSLPASADLRR), using a phage display library. Surface plasmon resonance (SPR) is utilized for quantitative determination and comparison of the binding specificity of selected peptides to penicillinase. An SPR biosensor functionalized with P3-GGGC (SHSLPASADLRRGGGC) is developed for detection of penicillinase with excellent sensitivity (15.8 RU nM(-1)) and binding affinity (KD = 0.56 nM). To determine if peptides can be good inhibitors for penicillinase, these peptides are mixed with penicillinase and their inhibition efficiency is determined by measuring the hydrolysis of substrate penicillin G using UV-vis spectrophotometry. Peptide P2 (NIYTTPWGSNWS) is found to be a promising penicillinase inhibitor with a Ki of 9.22 µM and a Ki' of 33.12 µM, suggesting that the inhibition mechanism is a mixed pattern. This peptide inhibitor (P2) can be used as a lead compound to identify more potent small molecule inhibitors for penicillinase. This study offers a potential approach to both detection of ß-lactamases and development of novel inhibitors of ß-lactamases.

Synergy between baicalein and penicillins against penicillinase-producing Staphylococcus aureus.

The combination of baicalein (the active constituent of Scutellaria baicalensis) with penicillin G/amoxicillin showed potent synergy against 20 clinical penicillinase-producing Staphylococcus aureus strains including 10 isolates that were additionally methicillin-resistant (MRSA). The fractional inhibitory concentration (FIC) indices of penicillins+baiclein ranged from 0.14 to 0.38. Baicalein protected penicillins (penicillin G and amoxicillin) from penicillinase and increased the susceptibility of penicillinase-supplemented S. aureus ATCC 29213 in a dose-dependent manner. The inhibition of penicillinase activity by baicalein should be responsible for the synergism and protective effect. These findings offer us good evidence that the penicillins combined with baicalein showed potent synergistic activity against penicillinase-producing S. aureus and penicillinase-producing MRSA in vitro and might provide promising implications for clinical treatment of these bacterial infections.

Prevalence of bla TEM-220 gene in Penicillinase-producing Neisseria gonorrhoeae strains carrying Toronto/Rio plasmid in Argentina, 2002 - 2011.

BACKGROUND: Penicillinase-producing Neisseria gonorroheae (PPNG) was first isolated in 1976. PPNG strains carrying bla TEM-1 and bla TEM-135 gene have been described in different countries. Recently, a novel bla TEM-220 allele was detected in PPNG isolates carrying Toronto/Rio plasmid. The prevalence and characteristics of TEM-220 strains worldwide are unknown, and therefore, it needs to be studied. The purpose of this study was to detect bla TEM-220 gene in PPNG strains possessing Toronto/Rio plasmid over a period of ten years in Argentina, and to evaluate the proportion of isolates producing non-TEM-220 containing the T539C substitution in the bla TEM allele. METHODS: One hundred and fifty one PPNG isolates carrying Toronto/Rio plasmid were studied between 2002 and 2011. A mismatch amplification mutation assay (MAMA) PCR was used to identify the T539C substitution in the bla TEM allele and a MAMA-PCR protocol was developed to detect the G547A substitution in the bla TEM-220. The reference agar dilution method of the Clinical and Laboratory Standard Institute (CLSI) was used for susceptibility testing to five ß-lactams antibiotics, ciprofloxacin, tetracycline and azithromycin. In all TEM-220-producing isolates, the whole bla TEM gene was sequenced and the isolates were typed using N. gonorroheae multiantigen sequence typing (NG-MAST). RESULTS: MAMA PCR successfully identified the G547A substitution in the bla TEM-220 allele. The proportion of isolates that possessed the bla TEM-220 allele was 2.6 %, and 93.2 % MAMA TEM-220 PCR-negative isolates showed the T539C substitution in the bla TEM gene. No differences in the susceptibility to five beta-lactam antibiotics tested were observed in PPNG isolates TEM-220-producing and PPNG isolates carrying the T539C substitution in the bla TEM gene. All TEM-220 isolates were indistinguishable by NG-MAST. CONCLUSION: This is the first study which shows the prevalence of bla TEM-220 in N. gonorrhoeae isolates carrying Toronto/Rio plasmid in Argentina. Although the bla TEM-220 allele does not appear to be associated with an extended spectrum beta-lactamase (ESBL) phenotype of resistance, a single nucleotide polymorphism added to the bla TEM-220 or bla TEM containing the T539C substitution could lead to the emergence of ESBL. Thus, it is imperative to investigate in surveillance programs, not only the plasmid type in PPNG isolates and the bla TEM allele associated, but phenotypical characteristics and geographical distribution of isolates.

The prevalence and epidemiology of plasmid-mediated penicillin and tetracycline resistance among Neisseria gonorrhoeae isolates in Guangzhou, China, 2002-2012.

BACKGROUND: Gonococcal antimicrobial resistance is a global problem. Different resistance plasmids have emerged and spread among the isolates of Neisseria gonorrhoeae worldwide and in China. We conducted this study to monitor the plasmid-mediated penicillin and tetracycline resistance among N. gonorrhoeae isolates in Guangzhou from 2002 to 2012. METHODS: Consecutive isolates of N. gonorrhoeae were collected from outpatients with gonorrhea attending the STD clinic in Guangdong Provincial Centre for Skin Diseases and STIs Control and Prevention. Penicillinase-producing N. gonorrhoeae (PPNG) isolates were analyzed by the paper acidometric method. Plasmid-mediated resistance to tetracycline in N. gonorrhoeae (TRNG) isolates was screened by the agar plate dilution method. Plasmid types were determined for TRNG and PPNG isolates using polymerase chain reaction (PCR). Minimum inhibitory concentrations (MICs) to penicillin and tetracycline were detected by the agar plate dilution. RESULTS: Of 1378 consecutive N. gonorrhoeae isolates, 429 PPNG and 639 TRNG isolates were identified. The prevalence of PPNG, TRNG, and PPNG/TRNG increased from 18.3 to 47.1 % (χ (2) = 31.57, p < 0.001), from 29.4 to 52.1 % (χ (2) = 16.28, p < 0.001) and from 10.0 to 26.2 % (χ (2) = 10.46, p < 0.001) between 2002 and 2012, respectively. Genotyping of plasmids among PPNGs showed that the majority (93.7 %) of the isolates were the Asian type plasmids, while the African type plasmid emerged in 2008 and rapidly increased to 14.0 % in 2012 (χ (2) = 25.03, p < 0.001). For TRNGs, all 639 isolates carried the Dutch type plasmid. MICs of penicillin G and tetracycline persisted at high levels and the MIC90s were 32-fold higher than the resistant cutoff point over 11 years. The prevalence rates of penicillin- and tetracycline-resistant N. gonorrhoeae varied from 90.9 to 91.1 % and from 88.3 to 89.3 % during 2002 to 2012, respectively. CONCLUSIONS: Resistance to penicillin and tetracycline among N. gonorrhoeae isolates remained at high levels in Guangzhou. The Asian type PPNG continued to spread and Dutch type TRNG was still the dominant strain. The African type PPNG has emerged and is spreading rapidly.

Changing antimicrobial resistance profiles among Neisseria gonorrhoeae isolates in Italy, 2003 to 2012.

The emergence of Neisseria gonorrhoeae isolates displaying resistance to antimicrobial agents is a major public health concern and a serious issue related to the occurrence of further untreatable gonorrhea infections. A retrospective analysis on 1,430 N. gonorrhoeae isolates, collected from 2003 through 2012, for antimicrobial susceptibility by Etest and molecular characterization by Neisseria gonorrhoeae multiantigen sequence typing (NG-MAST) was carried out in Italy. Azithromycin-resistant gonococci decreased from 14% in 2007 to 2.2% in 2012. Similarly, isolates with high MICs to cefixime (>0.125 mg/liter) decreased from 11% in 2008 to 3.3% in 2012. The ciprofloxacin resistance rate remains quite stable, following an increasing trend up to 64% in 2012. The percentage of penicillinase-producing N. gonorrhoeae (PPNG) significantly declined from 77% in 2003 to 7% in 2012. A total of 81 multidrug-resistant (MDR) gonococci were identified, showing 11 different antimicrobial resistance patterns. These were isolated from men who have sex with men (MSM) and from heterosexual patients. Two sequence types (STs), ST661 and ST1407, were the most common. Genogroup 1407, which included cefixime-, ciprofloxacin-, and azithromycin-resistant isolates, was found. In conclusion, a change in the antimicrobial resistance profiles among gonococci was identified in Italy together with a percentage of MDR isolates.

Antibiotic sensitivity and resistance patterns in pediatric staphylococcal scalded skin syndrome.

Historical resistance patterns often guide empiric antibiotic choices in staphylococcal scalded skin syndrome (SSSS), but little is known about the difference in susceptibility between SSSS and other childhood staphylococcal infections. A retrospective chart review of culture-confirmed cases of SSSS seen in the inpatient dermatology consultation service at the Children's Hospital of Philadelphia between 2005 and 2011 was performed. Most cases of SSSS at our institution are due to oxacillin-susceptible Staphylococcus aureus, and approximately half of the cases are due to clindamycin-resistant strains. Clindamycin and a penicillinase-resistant penicillin are suggested as empiric treatment for SSSS until culture susceptibility data are available to guide therapy.

Effect of different combinations of antibodies and enzyme labels on ELISA of progesterone.

In steroid immunoassays, selection of right combination of antibody and enzyme-labeled antigen determine the sensitivity and specificity of ELISA. Antibodies raised against different positions of progesterone adopting heterologous systems were reported to provide better assays for progesterone. Four different antibodies developed against progesterone-11α-hemiglutarate-BSA (P-11-HG-BSA), progesterone-11α-hemisuccinate-BSA (P-11-HS-BSA), progesterone-3-O-carboxymethyloxime-BSA (P-3-CMO-BSA), and progesterone-3-O-carboxymethyloxime-ovalbumin (P-3-CMO-ova) were tested in combination with enzyme-labeled P-11-HG, P-11-HS, progesterone-11α-carboxymethyl ether (P-11-CME), P-3-CMO, 17-hydroxyprogesterone-3-O-carboxymethyl oxime (17-P-3-CMO), and progesterone-4-carboxymethyl thioether (P-4-CMTE). These were variously labeled with penicillinase, alkaline phosphatase (ALP), and horseradish peroxidase (HRP). When antibody developed against P-11-HS-BSA was tested with P-3-CMO labeled separately with penicillinase, ALP, and HRP, the type of enzyme used had no effect on the performance of the assay. It was found that a homologous assay using P-3-CMO-ova as immunogen and P-3-CMO-HRP as label, as well as a heterologous ELISA with antibody raised against P-11-HS-BSA in combination with P-3-CMO-HRP, provided sensitive assays for progesterone. The use of 17α-hydroxy progesterone-3-O-carboxymethyl oxime-HRP with the same antibodies against P-3-CMO-BSA and P-11-HS-BSA also proved to be better than P-3-CMO-HRP. These findings implied that the sensitivity and specificity of ELISA to a great extent depended on the nature of the antibody produced, while the choice of enzyme labels could be manipulated.

Transduction of staphylococcal cassette chromosome mec elements between strains of Staphylococcus aureus.

Methicillin-resistant Staphylococcus aureus (MRSA) is a well-known public health concern. However, the means by which methicillin resistance genes are transferred among staphylococci in nature remains unknown. Older scientific literature suggests transduction as a means of mecA transfer, but the optimal conditions are reported to require plasmids and potentially a lysogenic phage. These reports preceded discovery of the staphylococcal cassette chromosome mec (SCCmec) elements. We undertook studies to confirm and clarify the conditions promoting transduction of SCCmec in S. aureus populations using well-characterized donor and recipient strains primarily of the USA300 lineage. Both bacteriophages 80α and 29 were capable of transducing SCCmec type IV and SCCmec type I to recipient strains of S. aureus. Pulsed-field gel electrophoresis and mec-associated dru typing were used to confirm the identity of the transductants. Transfer of mecA via transduction occurred at low frequency and required extended selection times for mecA gene expression and the presence of a penicillinase plasmid in the recipient. However, interference with the process by clavulanic acid and the necessity of lysogeny with 11 in the recipient or the presence of a small (4-kb) tetracycline resistance plasmid, as previously reported, were not confirmed. SCCmec transduction was occasionally associated with substantial deletions or truncation of SCCmec and the arginine catabolic metabolic element in USA300 recipients. Overall, these data clarify the conditions required for SCCmec transduction and document that rearrangements may occur during the process.

Inhibition of the class C beta-lactamase from Acinetobacter spp.: insights into effective inhibitor design.

The need to develop beta-lactamase inhibitors against class C cephalosporinases of Gram-negative pathogens represents an urgent clinical priority. To respond to this challenge, five boronic acid derivatives, including a new cefoperazone analogue, were synthesized and tested against the class C cephalosporinase of Acinetobacter baumannii [Acinetobacter-derived cephalosporinase (ADC)]. The commercially available carbapenem antibiotics were also assayed. In the boronic acid series, a chiral cephalothin analogue with a meta-carboxyphenyl moiety corresponding to the C(3)/C(4) carboxylate of beta-lactams showed the lowest K(i) (11 +/- 1 nM). In antimicrobial susceptibility tests, this cephalothin analogue lowered the ceftazidime and cefotaxime minimum inhibitory concentrations (MICs) of Escherichia coli DH10B cells carrying bla(ADC) from 16 to 4 microg/mL and from 8 to 1 microg/mL, respectively. On the other hand, each carbapenem exhibited a K(i) of <20 microM, and timed electrospray ionization mass spectrometry (ESI-MS) demonstrated the formation of adducts corresponding to acyl-enzyme intermediates with both intact carbapenem and carbapenem lacking the C(6) hydroxyethyl group. To improve our understanding of the interactions between the beta-lactamase and the inhibitors, we constructed models of ADC as an acyl-enzyme intermediate with (i) the meta-carboxyphenyl cephalothin analogue and (ii) the carbapenems, imipenem and meropenem. Our first model suggests that this chiral cephalothin analogue adopts a novel conformation in the beta-lactamase active site. Further, the addition of the substituent mimicking the cephalosporin dihydrothiazine ring may significantly improve affinity for the ADC beta-lactamase. In contrast, the ADC-carbapenem models offer a novel role for the R(2) side group and also suggest that elimination of the C(6) hydroxyethyl group by retroaldolic reaction leads to a significant conformational change in the acyl-enzyme intermediate. Lessons from the diverse mechanisms and structures of the boronic acid derivatives and carbapenems provide insights for the development of new beta-lactamase inhibitors against these critical drug resistance targets.