Types Of Β-lactamases Research Articles

Prevalence and molecular characterization of plasmidmediated beta-lactamase genes among nosocomial <i>Staphylococcus aureus</i> isolated in Taiwan

Purpose: To analyze the drug susceptibility phenotypes and the patterns of plasmid-mediated β- lactamase genes among nosocomial Staphylococcus aureus drug resistance isolates in Taiwan. Methods: The antibiotic susceptibilities of 617 clinical Staphylococcus aureus isolates collected from 2005 - 2009 from Chiayi Christian Hospital (Chiayi, Taiwan) were examined in vitro against 8 antimicrobial agents using agar diffusion method. Among the clinical isolates, 114 strains of methicillinsensitive Staphylococcus aureus and 45 strains of methicillin-resistant Staphylococcus aureus (MRSA) isolates were selected for plasmid profile analysis. The patterns of β-lactamase genes presented in plasmids were investigated by polymerase chain reaction analysis. Results: Most test strains were resistant to multiple antibiotics, particularly for the traditional agents such as ampicillin, penicillin, cephalexin and kanamycin. Plasmid profile analysis revealed that up to 36 % of the clinical strains harbored plasmids and were able to develop multi-drug resistant. Among them, most of the isolates harbored at least one plasmid (range 1 – 7) with a size range of 2.3 to 23 Kb. Among the several types of β-lactamases, blaTEM was the most prevalent. Conclusion: The results obtained from this study can serve as a valuable reference for the future control for clinical antibiotic resistant strains and more thorough discussions on resistance mechanisms. Keywords: Staphylococcus aureus , Antibiotic susceptibility, Nosocomial pathogens, Plasmid profile, β- lactamases

Synthesis, SAR and molecular docking study of novel non-β-lactam inhibitors of TEM type β-lactamase

The novel classes of acylated phenoxyanilide and thiourea compounds were investigated for their ability to inhibit TEM type β-lactamase enzyme. Two compounds 4g and 5c reveal the inhibition potency in micromolar range and show their action by non-covalent binding in the vicinity of the TEM-171 active site. The structure activity relationship around carbon chain length and different substituents in ortho- and para-positions of acylated phenoxyanilide as well as molecular modelling study has been performed.

Structural insight into mode of binding of Meropenem to CTX-M-15 type β-lactamase

Among Enterobacteriaceae, CTX-M type extended spectrum beta lactamase confers potent hydrolytic activity against cephalosporin group of antibiotics. Strains producing CTX-M type beta lactamase enzymes, show high level of resistance against cefotaxime. Therefore carbapenem antibiotics are used against beta lactamase producing strains. Hence, this study was designed to understand an insight of molecular basis of CTX-M-15 interaction with meropenem, and its effect on CTX-M-15 efficiency. Clinical strain of Enterobacter cloacae (EC-15) was used to clone blaCTX-M-15 gene in E.coli BL21cells. The protein was then expressed and purified. Results showed that CTX-M-15 producing strains are susceptible to meropenem. It quenches the fluorescence of CTX-M-15 spontaneously with binding constant of the order of 103M−1. Meropenem binds on the active site of CTX-M-15, hydrogen bonded with four common amino acid residues of cefotaxime binding site, as revealed by molecular docking studies. Conformational change in the structure of CTX-M-15 was observed upon meropenem binding by CD spectroscopy. The catalytic efficiency of CTX-M-15 was decreased up to 4 times upon meropenem binding. Docking study shows that few amino acids of active site of enzyme are also involved in meropenem binding, hence substrate is difficult to bind on active site properly and does not get hydrolysed. Moreover, meropenem binding induces structural changes in CTX-M-15, making the enzyme less efficient.

Molecular and in silico analysis of a new plasmid-mediated AmpC β-lactamase (CMH-2) in clinical isolates of Klebsiella pneumoniae

Two Klebsiella strains isolated from urine samples were positive for blaAmpC by PCR and showed sequence similarity with CMH-1 (98.6%) after sequencing. It also shares 82% similarity with ACT-1, 85% with MIR-1 and 81% with the chromosomal AmpC gene of Enterobacter cloacae. This gene was associated with the plasmid of IncK type. It has an open reading frame of 381 amino acid with four amino acid substitutions at position D144A, C189R, Q192E, and A195T as compared to CMH-1. When expressed in E.coli DH5α and E.coli strain B, this β-lactamase conferred resistance to cefotaxime, ceftriaxone and ceftazidime. In addition, both in vitro and in silico analysis revealed that this cephalosporinase was inhibited by cefepime and carbapenem group of drugs. Therefore, this new plasmid-encoded AmpC type β-lactamase gene was designated as CMH-2.

Ceftazidime-avibactam for the treatment of complicated intra-abdominal infections

ABSTRACTIntroduction: The treatment of complicated intra-abdominal infections (cIAI) is increasingly challenging due to increased resistance of Gram-negative organisms. These multidrug resistant organisms lead to an increase in morbidity and mortality. This has led to renewed interest in use of older β-lactam antibiotics in combination with newer β-lactamase inhibitors. Ceftazidime-avibactam is one of the newest such combination antibiotics, which has been released for treatment of complicated intra-abdominal infections in combination with metronidazole.Areas covered: In this drug evaluation manuscript cIAI along with the chemistry, pharmacodynamics, pharmacokinetics, metabolism and clinical study results of ceftazidime-avibactam are reviewed.Expert opinion: The role of ceftazidime-avibactam in combination with metronidazole in the treatment of cIAI is still to be defined. Patients with cIAI known to be infected with Klebsiella pneumoniae carbapenemase-producing Enterobacteriaceae would be clear candidates for treatment with this agent, as would patients infected with more common types of extended-spectrum β-lactamase producing Gram-negative pathogens if a carbapenem alternative were desired. At present, it is difficult to establish a clear group of patients with cIAI for whom initial empiric therapy with this agent would be warranted.

Evaluation of meat, fruit and vegetables from retail stores in five United Kingdom regions as sources of extended-spectrum beta-lactamase (ESBL)-producing and carbapenem-resistant Escherichia coli

We determined the prevalence and types of extended-spectrum β-lactamase (ESBL)-producing and carbapenem-resistant Escherichia coli in raw retail beef, chicken, pork, fruit and vegetables in five UK regions in 2013–14. Raw meat (n=397), and fruit and vegetable samples (n=400) were purchased from retail stores in London, East Anglia, North West England, Scotland and Wales. Samples were tested for the presence of ESBL-producing E. coli by plating enriched samples on CHROMagar CTX and CHROMagar ESBL, for AmpC-type E. coli by plating on “CHROMagar FOX” (CHROMagar ECC+16mg/L cefoxitin), and for carbapenem-resistant E. coli by plating on CHROMagar KPC. Additionally, pre-enrichment counts were performed on the above agars, and on CHROMagar ECC. Isolates of interest were characterised by MALDI-ToF to confirm identification, by PCR for blaCIT,blaCTX-M,blaOXA, blaSHV and blaTEM genes; ESBL or blaCIT genes were sequenced. Only 1.9% and 2.5% of beef and pork samples, respectively were positive for ESBL-producing E. coli after enrichment compared with 65.4% of chicken samples. 85.6% positive samples from chicken meat carried blaCTX-M-1; blaCTX-M-15 was not detected. None of the fruits or vegetables yielded ESBL-producing E. coli and none of the meat, fruit or vegetable samples yielded carbapenem-resistant E. coli. Retail chicken was more frequently a source of ESBL-producing E. coli than were beef, pork, fruit or vegetables. None of the foodstuffs yielded E. coli with CTX-M-15 ESBL, which dominates in human clinical isolates in the UK, and none yielded carbapenem-resistant E. coli.

Distribution of ESBLs, AmpC β-lactamases and carbapenemases among Enterobacteriaceae isolates causing intra-abdominal and urinary tract infections in the Asia-Pacific region during 2008-14: results from the Study for Monitoring Antimicrobial Resistance Trends (SMART).

To investigate the antimicrobial resistance and assess the molecular characteristics of β-lactamases (ESBLs, AmpC β-lactamases and carbapenemases) among Enterobacteriaceae isolates that caused intra-abdominal infections (IAIs) in patients hospitalized in the Asia-Pacific region during 2008-14. Multiplex PCR was used to detect the specific types of β-lactamase in 2893 isolates with MICs of ertapenem >0.5 mg/L. In-hospital acquisition times for most isolates were also delineated. Among 2728 (94.3%) isolates proven with β-lactamase production, the rates of non-susceptibility to imipenem were low (average = 7.9%) among IAI Enterobacteriaceae isolates from all Asia-Pacific countries except Vietnam (17.7%) and the Philippines (10.2%). A stepwise and significant increase in annual rates of carbapenemase production among these isolates was noted. CTX-M-15 and CTX-M-14 were the dominant ESBL variants in most IAI Enterobacteriaceae species. The most abundant AmpC β-lactamase variants were blaCMY-2 among isolates of Escherichia coli and blaDHA-1 among isolates of Klebsiella pneumoniae. In addition, the IAI Enterobacteriaceae isolates harbouring a blaCMY-2 or blaDHA-1 allele were associated with high community-acquired rates (38.0% and 42.6%, respectively). AmpC ACT and MIR variants were mostly detected in Enterobacter species. The blaNDM-1,4,5,7-harbouring isolates of E. coli, K. pneumoniae and Enterobacter cloacae were most commonly identified among IAI isolates from Vietnam and the Philippines. Also of note, blaOXA-48-harbouring IAI Enterobacteriaceae isolates were detected exclusively in Vietnam. The high resistance burden in Vietnam and the Philippines warrants aggressive control policies to combat the worsening trend in antimicrobial resistance among Enterobacteriaceae species causing IAIs.

Molecular characterization of extended-spectrum β-lactamase, plasmid-mediated AmpC cephalosporinase and carbapenemase genes among Enterobacteriaceae isolates in five medical centres of East and West Azerbaijan, Iran

Very little is known about the occurrence and various types of extended-spectrum β-lactamase (ESBL), AmpC and carbapenemase in Iran. The aims of this study were to determine the prevalence of ESBLs, AmpCs and carbapenemase genes among Enterobacteriaceae in Azerbaijan and to characterize the genetic composition of the detected genes. A total of 307 Enterobacteriaceae isolates, recovered from five medical centres, were screened for ESBL, AmpC and carbapenemase activities by the disc diffusion method and phenotypic confirmatory tests. The 162 selected strains (third-generation cephalosporins, cefoxitin- or carbapenem-resistant strains with positive or negative phenotypic confirmatory tests) were selected for multiplex PCR screening for β-lactamase genes, and detected genes were confirmed by sequencing. Of 162 isolates, 156 harboured 1 to 6 β-lactamase genes of 41 types. The most prevalent genes were blaTEM-1 (29.9 %), followed by blaCTX-M-15 (25.7 %). Plasmid-mediated AmpC was detected in 66 strains (21.5 %) alone or in combination with other genes. Carbapenemase-encoding genes were detected in 18 strains (5.8 %) of 27 carbapenem-non-susceptible isolates including 11, 7, 3 and 1 cases of blaOXA-48, blaNDM-1, blaKPC-2 and blaKPC-3 genes, respectively. Interestingly, 148 (94.8 %) of 156 strains with any β-lactamase gene were found to have a multidrug-resistant pattern. The rate of resistance to β-lactams and multidrug-resistant Enterobacteriaceae is high in Azerbaijan. All positive strains for carbapenemase genes were resistant to all β-lactams. The present study reveals the high occurrence of CTX-M-type ESBLs followed by TEM and SHV variants among Enterobacteriaceae isolates. East Azerbaijan seems to be an alarming focus for OXA-48, NDM-1 and KPC dissemination.

Prevalence of ESBL phenotype, blaCTX-M-1, blaSHV and blaTEM genes among uropathogenic Escherichia coli isolates from 3 military hospitals of Tehran, Iran

Objective: To determine the extended-spectrum beta-lactamase (ESBL) production and prevalence of blaCTX-M-1, blaSHV and blaTEM genes among uropathogenic Escherichia coli (UPEC) isolates from 3 military hospitals of Tehran during 2015-2016. Methods: One-hundred and eleven isolates were adopted. The antibiotic susceptibility testing was conducted according to Clinical and Laboratory Standards Institute guidelines. The combine disk was used for phenotypic ESBL production. The ceftazidime MIC was conducted with the micro-broth dilution test. The PCR assay was used to detect the blaCTX-M-1, blaSHV and blaTEM genes. Results: In the broth microdilution method, 103 (92.7%) isolates showed minimal inhibitory concentration (MIC) ≥ 1 µg/mL, and also in the combined disk method, 89 (80.1% of all) were ESBL positive. On the other hand, among 91 ceftazidime resistant isolates, 86 (77.4% of all) were ESBL positive. The difference between the two methods for ESBL confirmation was not significant. The result of MIC was similar to the disk diffusion method in the detection of phenotypic ESBL production. Among ESBL producer isolates, the prevalence of blaCTX-M-1, blaSHV and blaTEM was 77.4% (n = 86), 47.4% (n = 53) and 2.4% (n = 2), respectively. These genes were amplified in a wide range MIC of ceftazidime. Conclusions: The prevalence of multi-drug resistant UPEC and ESBL positive isolates was high in military hospitals. The majority of UPEC isolates amplified blaCTX-M-I and blaSHV type β-lactamase genes. One-third of isolates were positive in presence of both these genes. There was no relation between ceftazidime MIC and presence of beta-lactamase genes.

Molecular signature of extended spectrum β-lactamase producing Klebsiella pneumoniae isolated from bovine milk in eastern and north-eastern India

The present study reports on 23 extended spectrum β-lactamase producing Klebsiella pneumoniae (KP), isolated from milk samples (n=340) of healthy cows (n=129) and cows with subclinical (n=159) and clinical (n=52) mastitis, from three different states of India viz. West Bengal, Jharkhand and Mizoram. Seven of them were AmpC type β-lactamase producers, as well. The ESBL producing KP were significantly (P=0.006, χ2=10.04, df=2) and more frequently detected in milk samples of mastitic cows than healthy ones. The β-lactamase genes - blaCTX-M, blaTEM and blaSHV were detected in 19, 8 and 3 isolates, respectively. In all but one CTX-M positive isolates, the genetic platform - ISEcp1-blaCTX-M-orf477 was detected. Ten of the isolates carried plasmid mediated quinolone resistance gene - qnrS and 1 isolate possessed qnrB. Again 11 of them were found to have sulfonamide resistance gene - sul1 and 12 possessed class I integron. Sequencing of the class 1 integron revealed the presence of dfrA12/dfrA17 and aadA2/aadA5 gene cassettes conferring resistance to trimethoprim and aminoglycosides, respectively. All the isolates, characterized by enterobacterial repetitive intergenic consensus (ERIC) PCR, yielded distinct fingerprinting profile. However, most of the isolates from Jharkhand were clustered along with two isolates each from West Bengal and Mizoram indicating their clonal relatedness even though isolated from geographically different areas. Isolation of ESBL producing KP from bovine milk samples implies its public health significance; as such pathogens may enter the human food chain causing severe health hazards.

Analysis of β-Lactamase Resistance Determinants in Enterobacteriaceae from Chicago Children: a Multicenter Survey.

Multidrug-resistant (MDR) Enterobacteriaceae infections are increasing in U.S. children; however, there is a paucity of multicentered analyses of antibiotic resistance genes responsible for MDR phenotypes among pediatric Enterobacteriaceae isolates. In this study, 225 isolates phenotypically identified as extended-spectrum β-lactamase (ESBL) or carbapenemase producers, recovered from children ages 0 to 18 years hospitalized between January 2011 and April 2015 at three Chicago area hospitals, were analyzed. We used DNA microarray platforms to detect ESBL, plasmid-mediated AmpC (pAmpC), and carbapenemase type β-lactamase (bla) genes. Repetitive-sequence-based PCR and multilocus sequence typing (MLST) were performed to assess isolate similarity. Plasmid replicon typing was conducted to classify plasmids. The median patient age was 4.2 years, 56% were female, and 44% presented in the outpatient setting. The majority (60.9%) of isolates were Escherichia coli and from urinary sources (69.8%). Of 225 isolates exhibiting ESBL- or carbapenemase-producing phenotypes, 90.7% contained a bla gene. The most common genotype was the blaCTX-M-1 group (49.8%); 1.8% were carbapenem-resistant Enterobacteriaceae (three blaKPC and one blaIMP). Overall, pAmpC (blaACT/MIR and blaCMY) were present in 14.2%. The predominant E. coli phylogenetic group was the virulent B2 group (67.6%) associated with ST43/ST131 (Pasteur/Achtman MLST scheme) containing the blaCTX-M-1 group (84%), and plasmid replicon types FIA, FII, and FIB. K. pneumoniae harboring blaKPC were non-ST258 with replicon types I1 and A/C. Enterobacter spp. carrying blaACT/MIR contained plasmid replicon FIIA. We found that β-lactam resistance in children is diverse and that certain resistance mechanisms differ from known circulating genotypes in adults in an endemic area. The potential impact of complex molecular types and the silent dissemination of MDR Enterobacteriaceae in a vulnerable population needs to be studied further.

Prevalence of CTX-M-Type and PER Extended-Spectrum β-Lactamases Among Klebsiella spp. Isolated From Clinical Specimens in the Teaching Hospital of Kashan, Iran.

Background:Extended-spectrum β-lactamases (ESBLs) is one of the most important mechanisms of resistance to β-lactams especially among Enterobacteriaceae family including Klebsiella spp. Different types of extended-spectrum β-lactamases including CTX-M-type and PER enzymes are identified among gram negative bacteria.Objectives:The current study aimed to determine the prevalence of CTX-M-type and PER extended-spectrum β-lactamases among Klebsiella spp. isolated from clinical specimens in the teaching hospital of Kashan, Iran.Patients and Methods:One hundred Klebsiella spp. were isolated from clinical specimens of hospitalized patients at Shahid-Beheshti hospital from December 2012 to November 2013. Disk diffusion method was used to determine the susceptibility of these isolates to 14 different antimicrobial agents; disks were purchased from MAST company (United Kingdom). The phenotypic double disk synergy confirmatory test was used to screen the isolates to produce extended-spectrum β-lactamase. DNAs of isolates were extracted using boiling method and PCR assay was used to characterize the blaCTX-M type and blaPER genes. The purified PCR products were sent to Macrogen research company (Korea) for sequencing.Results:Of the total 100 Klebsiella isolates, %93 was susceptible to imipenem. Resistance to ampicillin, ceftazidime, ceftriaxone, aztreonam and cefotaxime was (92%), (67%), (65%), (64%) and (59%), respectively. The phenotypic confirmatory test (PCT) confirmed that 35% (n = 35) of the isolates were ESBL-producing Klebsiella strains. The prevalence of blaCTX-M type and blaRER genes among Klebsiella isolates were 28% (n = 28) and 9% (n = 9), respectively.Conclusions:The prevalence of ESBL-producing Klebsiella strains in Shahid-Beheshti hospital in Kashan has increased. The study concluded that there was a high prevalence of the blaCTX-M type gene among ESBL positive isolates.

Combinatorial Pharmacodynamics of Ceftolozane-Tazobactam against Genotypically Defined β-Lactamase-Producing Escherichia coli: Insights into the Pharmacokinetics/Pharmacodynamics of β-Lactam-β-Lactamase Inhibitor Combinations.

Despite a dearth of new agents currently being developed to combat multidrug-resistant Gram-negative pathogens, the combination of ceftolozane and tazobactam was recently approved by the Food and Drug Administration to treat complicated intra-abdominal and urinary tract infections. To characterize the activity of the combination product, time-kill studies were conducted against 4 strains ofEscherichia colithat differed in the type of β-lactamase they expressed. The four investigational strains included 2805 (no β-lactamase), 2890 (AmpC β-lactamase), 2842 (CMY-10 β-lactamase), and 2807 (CTX-M-15 β-lactamase), with MICs to ceftolozane of 0.25, 4, 8, and >128 mg/liter with no tazobactam, and MICs of 0.25, 1, 4, and 8 mg/liter with 4 mg/liter tazobactam, respectively. All four strains were exposed to a 6 by 5 array of ceftolozane (0, 1, 4, 16, 64, and 256 mg/liter) and tazobactam (0, 1, 4, 16, and 64 mg/liter) over 48 h using starting inocula of 10(6)and 10(8)CFU/ml. While ceftolozane-tazobactam achieved bactericidal activity against all 4 strains, the concentrations of ceftolozane and tazobactam required for a ≥3-log reduction varied between the two starting inocula and the 4 strains. At both inocula, the Hill plots (R(2)> 0.882) of ceftolozane revealed significantly higher 50% effective concentrations (EC50s) at tazobactam concentrations of ≤4 mg/liter than those at concentrations of ≥16 mg/liter (P< 0.01). Moreover, the EC50s at 10(8)CFU/ml were 2.81 to 66.5 times greater than the EC50s at 10(6)CFU/ml (median, 10.7-fold increase;P= 0.002). These promising results indicate that ceftolozane-tazobactam achieves bactericidal activity against a wide range of β-lactamase-producingE. colistrains.

Detection of Extended-spectrum β-lactamase and AmpC β-lactamase Producing Escherichia coli Isolates from Chickens

The aim of this study was to investigate the presence of Extended-spectrum β-lactamase (ESBL) and AmpC β-lactamase producing Escherichia coli strains isolated from meat and faecal samples of chicken. Faecal samples (n: 384) were collected from broiler and egg-type healthy chickens in 43 flocks and 384 fresh chicken meat samples from local markets, supermarkets and slaughterhouses. E. coli isolates were tested phenotypically according to the guidelines of CLSI (2013) for ESBL production. Phenotypic detection of AmpC production was carried out by determination of resistance to cefoxitin and susceptibility to cefepime. Also, presence of genes encoding different types of β-lactamases (blaCTX-M, blaTEM, blaSHV and the mutation in the promoter of AmpC) in all phenotypically ESBL or AmpC producing E. coli was investigated by PCR assays. In this study, blaCTX-M (95.4%), blaCTX-M-1 (81.8%), blaCTX-M-8 (4.5%), blaTEM (45.4%) and blaSHV (2.2%) genes were detected from faecal samples and blaCTX-M (100%), blaCTX-M-1 (28.5%) and blaTEM (7.1%) from meat samples. The only plasmidic AmpC β-lactamase found was the CIT type.

Extended-Spectrum β-Lactamase Enzymes (ESBLs) Produced by Escherichia coli Urinary Pathogens at Riyadh, Saudi Arabia

Background: This study aim was to determine the probable type of β-lactamase gene which is responsible for resistance. It was found that OXA (701 bp) was the main type of β-lactamase (35.7%), CTX-M (569 bp) was second (28.9%), TEM (403 bp) was third (20.5%) and SHV (293 bp) (14.9%) was fourth. The aim and objectives of this study were to investigate the prevalence of ESBLs producing in these bacteria isolated from uropathogenic out-patients and to look for the presence of TEM or SHV, CTX and OXA genes in E. coli. Results: The present study was carried out from the Central Laboratory of Riyadh Hospital in Saudi Arabia from January 2014 to June 2015. Total 116 urine samples were tested bacteriologically and for antibiotic susceptibility using standard procedures, Detection of extended-spectrum β-lactamases and determination of the genotype of β- lactamase of 75 E. coli isolates by PCR: It was found that OXA (701 bp) was the main type of β-lactamase (35.7%), CTX-M (569 bp) was second (28.9%), TEM (403 bp) was third (20.5%) and SHV (293 bp) (14.9%) was fourth. Conclusions: This study showed that the ESBL producing isolates detected PCR with oligonucleotide primers of TEM, SHV, and CTX-M and OXA genes and were carried out on E. coli DNA of 75 isolates. PCR, incorporating the primers for commonly prevalent ESBLs may be a valuable clinical and research tool for characterization of ESBLs

Prevalence of Inhibitor Resistant Beta Lactamase Producing E. coli in Human and Poultry Origin of Bangladesh

Human clinical specimens (n=48) and poultry fecal specimens (n=40) were collected from a sub district (Savar) of Bangladesh. Total 25 E. coli were isolated from these specimens. These E. coli were tested for their antibiotic sensitivity against commonly used antibiotics. In addition, to detect the ESBL (extended spectrum beta lactamases) producers, double disk synergy (DDS) test and PCR method were used. Twenty three E. coli were multidrug resistant, i.e. resistant against at least three different groups of antibiotics. DDS method showed all E. coli from poultry specimens and 78% of E. coli from clinical specimens were resistant to amoxicillin-clavulanic acid combination. Thus they are phenotypically confirmed as inhibitor resistant beta lactamase producers. PCR result showed only blaTEMgene in E. coli from poultry specimens. Thus inhibitor resistant type β-lactamase was found to be dominant in E. coli from both types of specimens.

Exploring the Mechanism of β-Lactam Ring Protonation in the Class A β-lactamase Acylation Mechanism Using Neutron and X-ray Crystallography

The catalytic mechanism of class A β-lactamases is often debated due in part to the large number of amino acids that interact with bound β-lactam substrates. The role and function of the conserved residue Lys 73 in the catalytic mechanism of class A type β-lactamase enzymes is still not well understood after decades of scientific research. To better elucidate the functions of this vital residue, we used both neutron and high-resolution X-ray diffraction to examine both the structures of the ligand free protein and the acyl-enzyme complex of perdeuterated E166A Toho-1 β-lactamase with the antibiotic cefotaxime. The E166A mutant lacks a critical glutamate residue that has a key role in the deacylation step of the catalytic mechanism, allowing the acyl-enzyme adduct to be captured for study. In our ligand free structures, Lys 73 is present in a single conformation, however in all of our acyl-enzyme structures, Lys 73 is present in two different conformations, in which one conformer is closer to Ser 70 while the other conformer is positioned closer to Ser 130, which supports the existence of a possible pathway by which proton transfer from Lys 73 to Ser 130 can occur. This and further clarifications of the role of Lys 73 in the acylation mechanism may facilitate the design of inhibitors that capitalize on the enzyme's native machinery.

Methicillin-susceptible strains responsible for postoperative orthopedic infection are not selected by the use of cefazolin in prophylaxis

Comparison of methicillin-susceptible Staphylococcus aureus (MSSA) isolates responsible for bone and joint infection (BJI, n=73) and nasal colonization (n=57) revealed similar prevalence of β-lactamase (blaZ) type A production, associated with cefazolin hydrolysis, suggesting that blaZ type A–carrying MSSA isolates implicated in postoperative BJI are not selected by cefazolin prophylaxis.

Identification of Haemophilus haemolyticus in clinical samples and characterization of their mechanisms of antimicrobial resistance.

The objectives of this study were to establish the frequency of Haemophilus haemolyticus in clinical samples, to determine the antimicrobial resistance rate and to identify the mechanisms of resistance to β-lactams and quinolones. An updated database was used to differentiate between MALDI-TOF MS results for Haemophilus influenzae and H. haemolyticus. Antimicrobial susceptibility was studied by microdilution, following EUCAST criteria. The β-lactamase types were identified by PCR analysis of isolates that tested positive for nitrocefin hydrolysis. Mutations in the ftsI gene were identified in isolates with ampicillin MICs ≥0.25 mg/L. Mutations in the quinolone resistance-determining region (QRDR) were identified in isolates with ciprofloxacin MICs ≥0.5 mg/L. Overall, we identified 69 H. haemolyticus isolates from 1706 clinical isolates of Haemophilus spp. from respiratory, genital, invasive, and other infection sources. The frequency of H. haemolyticus was low in respiratory samples compared with that of H. influenzae, but in genital-related samples, the frequency was similar to that of H. influenzae. We found low antimicrobial resistance rates among H. haemolyticus isolates, with 8.7% for ampicillin, 8.7% for co-trimoxazole, 7.2% for tetracycline and 4.3% for ciprofloxacin. Mutations in the ftsI gene classified the isolates into four groups, including the newly described Group Hhae IV, which presents mutations in the ftsI gene not identified in H. influenzae and H. haemolyticus type strains. Three ciprofloxacin-resistant H. haemolyticus isolates with mutations affecting GyrA and ParC were identified. The frequency of H. haemolyticus was low, especially in respiratory samples, where H. influenzae is the main pathogen of this genus. Although antimicrobial resistance rates were low, three ciprofloxacin-resistant H. haemolyticus clinical isolates have been identified for the first time.

Phylogenetic lineages, clones and β-lactamases in an international collection of Klebsiella oxytoca isolates non-susceptible to expanded-spectrum cephalosporins.

The objective of this study was to examine Klebsiella oxytoca clonal and phylogenetic diversity, based on an international collection of carriage isolates non-susceptible to expanded-spectrum cephalosporins (ESCs). The study material comprised 68 rectal carriage K. oxytoca isolates non-susceptible to ESCs recovered in 2008-11 from patients in 14 hospitals across Europe and Israel. ESC resistance was tested phenotypically; genes encoding ESBLs, AmpC cephalosporinases and carbapenemases were amplified and sequenced. The isolates were typed by PFGE and MLST, followed by sequencing of blaOXY genes. MLST and PFGE distinguished 34 STs and 47 pulsotypes among the isolates, respectively. Six STs were split into several pulsotypes each. Five STs were more prevalent (n = 2-9) and occurred in several countries each, including ST2, ST9 and ST141, which belong to a growing international clonal complex (CC), CC2. Four phylogenetic lineages were distinguished, each with another type of chromosomal OXY-type β-lactamase. Three of these, with OXY-1/-5, OXY-2 types and OXY-4, corresponded to previously described phylogroups KoI, KoII and KoIV, respectively. A single isolate from Israel represented a distinct lineage with a newly defined OXY-7 type. The phylogroups showed interesting differences in mechanisms of ESC resistance; KoI strains rarely overexpressed the OXY enzymes but commonly produced ESBLs, whereas KoII strains often were OXY hyperproducers and carried ESBLs much less frequently. AmpCs (DHA-1) and carbapenemases (VIM-1) occurred sporadically. The study confirmed the high genetic diversity of the collection of K. oxytoca ESC-non-susceptible isolates, composed of phylogroups with distinct types of OXY-type β-lactamases, and revealed some STs of broad geographical distribution.