OXA-1 Beta-lactamase Research Articles

668. Presence of the Narrow-Spectrum OXA-1 Beta-Lactamase Enzyme is not Associated with Elevated Ceftolozane-Tazobactam MIC Values among ESBL-Producing Escherichia coli Clinical Isolates (CANWARD, 2011-2018)

Abstract Background Beta-lactam-beta-lactamase inhibitor combinations have been proposed as an alternative to carbapenems for the treatment of infections caused by extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli as an antimicrobial stewardship initiative. However, in a recent randomized trial evaluating patients with bacteremia, presence of the narrow spectrum OXA-1 beta-lactamase enzyme among ESBL-producing Enterobacterales was associated with higher piperacillin-tazobactam MICs, and in turn excess mortality among patients treated with piperacillin-tazobactam relative to meropenem. The purpose of this study was to determine whether the in vitro activity of ceftolozane-tazobactam versus ESBL-producing E. coli is similarly compromised by the presence of OXA-1. Methods E. coli clinical isolates were obtained from patients evaluated at hospitals across Canada (January 2011 to December 2018) as part of an ongoing national surveillance study (CANWARD). ESBL production was confirmed using the Clinical and Laboratory Standards Institute phenotypic method. Susceptibility testing was carried out using custom broth microdilution panels, and all isolates underwent whole genome sequencing for beta-lactamase gene detection. Results In total, 485 ESBL-producing E. coli identified as part of the CANWARD study were included. The majority of isolates (91.3%; 443/485) harbored a CTX-M ESBL enzyme. OXA-1 was present in 39.6% (192/485) of isolates. OXA-1 was detected in 62.5% (187/299) of isolates with a CTX-M-15 ESBL enzyme versus only 2.7% (5/186) of isolates with other ESBL enzyme types. Ceftolozane-tazobactam MIC50 and MIC90 values were identical (0.25 µg/mL and 1 µg/mL, respectively) for the isolate subsets with and without OXA-1. Overall, 97.4% and 96.9% of isolates with and without OXA-1 remained susceptible (CLSI breakpoint) to ceftolozane-tazobactam. Conclusion The presence of OXA-1 among ESBL-producing E. coli clinical isolates was not associated with an elevation in MIC values for ceftolozane-tazobactam. These data support further evaluation of ceftolozane-tazobactam as an alternative to carbapenems for the treatment of infections caused by ESBL-producing E. coli, as is being done with the MERINO-3 trial. Disclosures George Zhanel, PhD, Merck: Grant/Research Support.

Evaluation of Piperacillin-Tazobactam ETEST for the Detection of OXA-1 Resistance Mechanism among Escherichia coli and Klebsiella pneumoniae.

Globally, piperacillin-tazobactam resistance among Escherichia coli and Klebsiella pneumoniae is driven by OXA-1 beta-lactamases. Expression of blaOXA-1 yields piperacillin-tazobactam MICs of 8 to 16 μg/mL, which straddle the susceptible/susceptible-dose dependent breakpoint set by the Clinical and Laboratory Standards Institute in 2022. Variability of the reference broth microdilution method (BMD) was evaluated by manufacturing BMD panels using 2 brands of piperacillin, 2 brands of tazobactam and 2 brands of cation-adjusted Mueller-Hinton broth. In addition, ETEST, which harbors an intermediate dilution of 12 μg/mL was evaluated for the ability to differentiate isolates with and without blaOXA-1. A collection of 200 E. coli and K. pneumoniae, of which 82 harbored a blaOXA-1 gene, were tested. BMD variability was on average 1.3-fold, within the accepted 2-fold variability of MICs. However, categorical agreement (CA) between BMD reads was 74.0% for all isolates and 63.4% for those with a blaOXA-1 gene and 81.3% for those without blaOXA-1 detected (P = 0.004, Pearson's Chi Square). ETEST overall CA with the BMD mode was 68.0% and essential agreement (EA) was 80.5%. For isolates with blaOXA-1, CA was 50.0% and EA was 69.5%, versus 80.5% and 88.1%, respectively, for isolates without blaOXA-1 (P < 0.0001 for both comparisons). All ETEST errors were major errors (false resistance) compared to BMD mode. However, the negative predictive value of the ETEST for the presence of blaOXA-1 was 94.1%, compared to only 74.2% negative predictive value for BMD. Clinicians and microbiologists should be aware of the challenges associated with testing piperacillin-tazobactam in regions where blaOXA-1 is prevalent.

An Extended Reservoir of Class-D Beta-Lactamases in Non-Clinical Bacterial Strains.

ABSTRACTBacterial genes coding for antibiotic resistance represent a major issue in the fight against bacterial pathogens. Among those, genes encoding beta-lactamases target penicillin and related compounds such as carbapenems, which are critical for human health. Beta-lactamases are classified into classes A, B, C, and D, based on their amino acid sequence. Class D enzymes are also known as OXA beta-lactamases, due to the ability of the first enzymes described in this class to hydrolyze oxacillin. While hundreds of class D beta-lactamases with different activity profiles have been isolated from clinical strains, their nomenclature remains very uninformative. In this work, we have carried out a comprehensive survey of a reference database of 80,490 genomes and identified 24,916 OXA-domain containing proteins. These were deduplicated and their representative sequences clustered into 45 non-singleton groups derived from a phylogenetic tree of 1,413 OXA-domain sequences, including five clusters that include the C-terminal domain of the BlaR membrane receptors. Interestingly, 801 known class D beta-lactamases fell into only 18 clusters. To probe the unknown diversity of the class, we selected 10 protein sequences in 10 uncharacterized clusters and studied the activity profile of the corresponding enzymes. A beta-lactamase activity could be detected for seven of them. Three enzymes (OXA-1089, OXA-1090 and OXA-1091) were active against oxacillin and two against imipenem. These results indicate that, as already reported, environmental bacteria constitute a large reservoir of resistance genes that can be transferred to clinical strains, whether through plasmid exchange or hitchhiking with the help of transposase genes.IMPORTANCE The transmission of genes coding for resistance factors from environmental to nosocomial strains is a major component in the development of bacterial resistance toward antibiotics. Our survey of class D beta-lactamase genes in genomic databases highlighted the high sequence diversity of the enzymes that are able to recognize and/or hydrolyze beta-lactam antibiotics. Among those, we could also identify new beta-lactamases that are able to hydrolyze carbapenems, one of the last resort antibiotic families used in human antimicrobial chemotherapy. Therefore, it can be expected that the use of this antibiotic family will fuel the emergence of new beta-lactamases into clinically relevant strains.

Dynamic Gene Clusters Mediating Carbapenem-Resistant Acinetobacter baumannii Clinical Isolates.

Acinetobacter baumanni (A. baumannii), a nonfermenting Gram-negative bacterium, has recently been associated with a broad range of nosocomial infections. To gain more meaningful insight into the problem of nosocomial illnesses caused by the multidrug-resistant (MDR) A. baumannii, as well as the factors that increase the risk of catching these infections, this investigation included a total of 86 clinical A. baumannii infections. Repetitive extragenic palindromic (REP)-PCR was used to investigate imipenem-resistant A. baumannii isolates for dynamic gene clusters causing carbapenem resistance. Four distinct A. baumannii lineages were found in the REP-PCR-DNA fingerprints of all isolates, with 95% of the samples coming from two dominant lineages. Imipenem, amikacin, and ciprofloxacin were less effective against genotype (A) isolates because of enhanced antibiotic tolerance. Lastly, to gain more insight into the mode of action of imipenem, we explored the binding affinity of imipenem toward different Acinetobacter baumannii OXA beta-lactamase class enzymes.

1216. Presence of the Narrow-Spectrum OXA-1 Beta-lactamase Enzyme Is Associated with Elevated Piperacillin-Tazobactam MIC Values Among ESBL-producing Escherichia coli Clinical Isolates (CANWARD, 2007-2018)

BackgroundThe clinical outcome of patients with bacteremia due to an extended-spectrum beta-lactamase (ESBL)-producing member of the family Enterobacteriaceae who are treated with piperacillin-tazobactam appears to depend, at least in part, on the piperacillin-tazobactam MIC. The purpose of this study was to determine whether there is any association between the MIC of piperacillin-tazobactam and presence of the narrow spectrum OXA-1 beta-lactamase enzyme among ESBL-producing Escherichia coli.Methods E. coli clinical isolates were obtained from patients evaluated at hospitals across Canada (January 2007 to December 2018) as part of an ongoing national surveillance study (CANWARD). ESBL production was confirmed using the Clinical and Laboratory Standards Institute phenotypic method. Susceptibility testing was carried out using custom broth microdilution panels, and all isolates underwent whole genome sequencing for beta-lactamase gene detection.ResultsIn total, 671 ESBL-producing E. coli were identified as part of the CANWARD study. The majority of isolates (92.0%; 617/671) harbored a CTX-M ESBL enzyme. CTX-M-15 (62.3%; 418/671), CTX-M-27 (13.9%; 93/671), and CTX-M-14 (13.4%; 90/671) were the most common variants identified. The narrow spectrum OXA-1 beta-lactamase enzyme was present in 42.6% (286/671) of isolates. OXA-1 was detected in 66.3% (277/418) of isolates with a CTX-M-15 ESBL enzyme versus only 3.6% (9/253) of isolates with other ESBL enzyme types. The piperacillin-tazobactam MIC50 and MIC90 values were 8 µg/mL and 32 µg/mL for isolates that possessed the OXA-1 enzyme versus 2 μg/mL and 8 µg/mL for those that did not. The percentage of ESBL-producing E. coli isolates that were inhibited by a piperacillin-tazobactam MIC of ≤8 μg/mL was 68.5% for isolates that were OXA-1 positive and 93.8% for isolates that were OXA-1 negative.ConclusionThe MIC50 and MIC90 values of piperacillin-tazobactam among ESBL-producing E. coli were higher for the subset of isolates that harbored a narrow spectrum OXA-1 beta-lactamase enzyme relative to the subset that did not. This association was primarily observed among ESBL-producers with the CTX-M-15 enzyme variant. OXA-1 was infrequently detected among isolates with other ESBL enzyme types.Disclosures George Zhanel, PhD, AVIR (Grant/Research Support)Iterum (Grant/Research Support)Merck (Grant/Research Support)Pfizer (Grant/Research Support)Sandoz (Grant/Research Support)Sunovion (Grant/Research Support)Venatorx (Grant/Research Support)Verity (Grant/Research Support)

An Update on Eight "New" Antibiotics against Multidrug-Resistant Gram-Negative Bacteria.

Infections in the ICU are often caused by Gram-negative bacteria. When these microorganisms are resistant to third-generation cephalosporines (due to extended-spectrum (ESBL) or AmpC beta-lactamases) or to carbapenems (for example carbapenem producing Enterobacteriales (CPE)), the treatment options become limited. In the last six years, fortunately, there have been new antibiotics approved by the U.S. Food and Drug Administration (FDA) with predominant activities against Gram-negative bacteria. We aimed to review these antibiotics: plazomicin, eravacycline, temocillin, cefiderocol, ceftazidime/avibactam, ceftolozane/tazobactam, meropenem/vaborbactam, and imipenem/relebactam. Temocillin is an antibiotic that was only approved in Belgium and the UK several decades ago. We reviewed the in vitro activities of these new antibiotics, especially against ESBL and CPE microorganisms, potential side effects, and clinical studies in complicated urinary tract infections (cUTI), intra-abdominal infections (cIAI), and hospital-acquired pneumonia/ventilator-associatedpneumonia (HAP/VAP). All of these new antibiotics are active against ESBL, and almost all of them are active against CPE caused by KPC beta-lactamase, but only some of them are active against CPE due to MBL or OXA beta-lactamases. At present, all of these new antibiotics are approved by the U.S. Food and Drug Administration for cUTI (except eravacycline) and most of them for cIAI (eravacycline, ceftazidime/avibactam, ceftolozane/tazobactam, and imipenem/relebactam) and for HAP or VAP (cefiderocol, ceftazidime/avibactam, ceftolozane/tazobactam, and imipenem/relebactam).

Cross-Sectional Study of Extended Spectrum β-Lactamase Genes (SHV, TEM, CTX-M and OXA) in Klebsiella Species from Clinical Specimens, in Port Harcourt, Nigeria

Infections caused by Klebsiella sp. are increasingly becoming difficult to treat partly due to the rise in trend of extended-spectrum beta-lactamase (ESBL)-producing Klebsiella sp. This study aimed to detect the prevalence of ESBL-producing Klebsiella sp. and SHV, TEM, CTX-M, and OXA beta-lactamase encoding genes in isolates of Klebsiella sp. from two healthcare facilities in Port Harcourt. A cross-sectional study of 146 clinical specimens was analyzed using standard bacteriological identification techniques. ESBL-producing Klebsiella sp. was identified using clinical laboratory standard institute standards. Beta-lactamase genes SHV, TEM, OXA, and CTXM were extracted and amplified. Of the 146 clinical specimens; 47 (32.2%) were from males and 99 (67.8%) females. The number of Klebsiella sp. isolated from the female samples 11 (73.3%) was more than 2-fold higher than those isolated from male samples 4 (26.7%). Eighty different bacterial isolates were obtained from which 15 (18.8%) were Klebsiella sp. while 12 (80%) of the identified Klebsiella sp. were β-lactamase producing Klebsiella sp. The prevalence of the Klebsiella sp. according to the different samples were 8 (53.3%), 1 (6.7%), 2 (13.3%), 2 (13.3%), 1 (6.7%), 1 (6.7%) were obtained from urine, endocervical, sputum, high vaginal, throat, hospital environment samples respectively. Isolates 2, 4-6, and 9-11 were positive for SHV gene (293 bp); all the Klebsiella isolates were positive for TEM gene (840 bp); isolates 4-8 showed positive bands for CTX-M gene (550 bp); and isolates 2, 3, 6, 8, and 12 showed positive bands for OXA gene (908 bp). The urine sample numbered 106, 42, and 55 exhibited complete resistance to all the antibiotics used and showed different types of ESBL genes: SHV; SHV, TEM, CTX-M and OXA; and OXA, and TEM respectively. This study shows that β-lactamase genes were differentially expressed in the various types of samples collected. Again, total resistance to beta-lactam drugs may not be completely dependent on the constitutive expression of several ESBL genes but TEM is present in all the sample that showed complete resistance.

Hospital wastewater effluent: hot spot for antibiotic resistant bacteria

AbstractHospital wastewaters are highly complex effluents acting as a hotspot for antibiotic resistant bacteria. Especially, Gram-negative bacteria bearing multiple antibiotic resistant genes are increasingly found in hospital wastewaters. The aim of this study was to evaluate the presence of extended spectrum beta-lactamases (ESBL) and carbapenemase producing Enterobacteriaceae in hospital wastewaters from one Slovenian and two Austrian hospitals, as well as the occurrence of antibiotic resistant genes encoding for VIM, KPC, NDM, CTXM and OXA beta-lactamases in isolates from hospital wastewater. The results indicated high levels of ESBL producing Enterobacteriaceae in ranges up to 107 cfu/mL. Carbapenemase producing Enterobacteriaceae and OXA 48-type CPE were present in ranges up to 105 cfu/mL. Out of 89 multiplied polymerase chain reaction (PCR) amplicons, only 36 were positive for different β-lactamase gene families, among those only three isolates were identified as multiresistant. The dominant ESBL family was CTXM in 19 different isolates. This was followed by 10 OXA-48 positive isolates and 10 VIM positive isolates. KPC or NDM carbapenemases were not identified with PCR screening of the isolates. Hospital wastewaters serve as a reservoir for nearly all clinically important antibiotic resistances. The importance of evaluating such potential environmental reservoirs is especially evident when outbreak cases could not be linked to an epidemiological source.

Antibiotic resistance pattern of Acinetobacter baumannii from burns patients: increase in prevalence of blaOXA-24-like and blaOXA-58-like genes

Notwithstanding the increased prevalence of Acinetobacter baumannii drug-resistant isolates, treatment options are progressively limiting. This study aims to provide a recent report on antibiotic susceptibility in burn wound isolates of A. baumannii, and the importance of OXA beta-lactamases in carbapenem resistance. The susceptibility levels to different antimicrobial categories were determined among 84 A. baumannii isolates from burn wound infection between 2016 and 2018. Multiplex PCR was used to detect OXA beta-lactamases genes, including bla OXA-51, bla OXA-23, bla OXA-24 and bla OXA-58. ISAba-1 association with bla OXA-51, bla OXA-23 and bla OXA-58 was detected by PCR mapping. All the isolates were determined as multidrug-resistant (MDR) and 69% as extensively drug-resistant (XDR). Different carbapenems MIC ranges (MIC50 and MIC90) were observed among the isolates harboring bla OXA-like genes and isolates with the OXA-24-like enzyme showed higher carbapenems MIC ranges. The prevalence of bla OXA-51-like, bla OXA-23-like, bla OXA-24-like and bla OXA-58-like were 100%, 53.57%, 41.66% and 30.95%, respectively. ISAba-1 insertion sequence was found to be upstream to bla OXA-23-like and bla OXA-58-like genes in 23 out of 45 (71.1%) bla OXA-23-like-positive and 4 out of 23 (15.3) bla OXA-58-like-positive isolates, respectively. Resistance to carbapenems as the last resort for treatment of A. baumannii infections is growing. This study, for the first time in Iran, has observed the increased frequency of bla OXA-24-like and bla OXA-58-like genes and found an association between ISAba-1 and bla OXA-58-like gene, which signifies the possible risk of increased diversity in OXA beta-lactamases and growth in carbapenem resistance.

High prevalence of TEM, VIM, and OXA-2 beta-lactamases and clonal diversity among Acinetobacter baumannii isolates in Turkey.

Acinetobacter baumannii is an opportunistic pathogen that causes nosocomial infections with high mortality. Treatment options are limited owing to its resistance to numerous antibiotics. Here, we sought to determine the antibiotic susceptibilities of A. baumannii isolates, investigate clonal relationship among the strains, and determine the frequency of beta-lactamase resistance genes. The identification and antibiotic susceptibilities of 69 A. baumannii strains were determined using a BD-Phoenix automated system. The presence of blaOXA-2, blaOXA-10, blaOXA-23, blaOXA-24/40, blaOXA-51, blaOXA-58, blaTEM, blaSHV, blaIMP, blaVIM, and blaGIM genes were investigated using polymerase chain reaction (PCR), and clonal relatioships among the isolates were determined using pulsed-field gel electrophoresis (PFGE). All strains were resistant to ampicillin-sulbactam, gentamicin, cefepime, ciprofloxacin, and ceftriaxone. While 65 of the 69 strains (94.2%) were resistant to piperacillin-tazobactam, amikacin, imipenem, and meropenem, all strains were susceptible to tigecycline and colistin. The frequencies of blaOXA-51, blaOXA-23, blaTEM, blaOXA-2, blaVIM, and blaSHV were 100%, 94.2%, 53.6%, 21.7%, 14.5%, and 2.9%, respectively. Based on PFGE results, 56 of the 69 strains were clonally related, and the clustering rate was 81.2%. No common outbreak isolate was detected. The most prevalent OXA genes were blaOXA-51, blaOXA-23, and blaOXA-2. Furthermore, blaTEM, blaSHV, and blaVIM, which are common in Enterobacterales and Pseudomonas spp, were detected, suggesting horizontal gene transfer had occurred between bacteria. No single clone outbreak was detected by PFGE. However, multiclonal spread and the high clustering rate suggest cross-contamination. Therefore, in future, more effective infection control measures must be implemented.

Detection of OXA Beta Lactamases Among Clinical Isolates of Acinetobacter baumannii Isolated from Tehran Hospitals, Iran

Background and Objective:Acinetobacter baumanniiis a non-motile Gram-negative bacterial pathogen with the history of vast resistant to antibiotics. The aim of this study was to determine the possibility of existence of OXAs genes among clinical isolates ofA. baumanniiobtained from Tehran hospitals.Materials and Methods:A total of 101 isolates were identified asA. baumanniiby common biochemical and molecular tests. The susceptibility to different antibiotics was assessed with Kirby-Bauer disk diffusion method. Phenotypic Detection of MBLs was performed with CDT test and PCR assay was also performed for detection ofblaOXA-23-like,blaOXA-24-like,blaOXA-40-like,blaOXA-51-like,blaOXA-58-likeandblaOXA-143-likegenesResults:All isolates ofA. baumanniishowed high-level of resistance to all antibiotics except for Polymyxin B. TheblaOXA-51 likegenes was found in all of the isolates and the prevalence ofblaOXA-143like,blaOXA-23like,blaOXA-40likeandblaOXA-24likewere 56%, 45.45%, 33% and 11.8%, respectively.Conclusion:TheblaOXA-51-likewas the predominant mechanism of resistance to imipenem inA. baumanniiand therefore, early recognition of carbapenem-resistantA. baumanniiisolates is a useful tools to prevent their spreading within the hospital environment.

Antimicrobial susceptibility profile of class D Oxa B-lactamases producing bacteria in Kano state, Nigeria

This study was carried out to determine the antibiotic susceptibility profile of Gram negative bacteria obtained from three different hospitals for class D Oxa β-lactamases in Kano metropolis. The clinical isolates include: Escherichia coli, Klebsiella y pneumoniae, Pseudomonas aeruginosa, Proteus sp and Salmonella spp. A total of 500 clinical isolates were screened phenotyphically using double discs synergy test. Confirmatory tests were performed among the suspeced isolates according to the Clinical and Laboratory Standard Institutes guidelines (CLSI). A total of 13 antibiotic discs were used for sensitivity test including: Amoxicillin/clavulanic acid, ceffazidime, cefepime, cepatozime, cefuruxime, imipenem, meropenem, ciprofloxacin, genetamicin, levoflaxacin, nitrofuranton, tigercyline and ofloxacin. Nine antibiotic discs were used on lactose fermenters and seven antibiotics were used on non–lactose fermenters. The result of the prevalence of class D oxa β lactamases producing bacteria among the clinical isolates revealed that, 26.40% of the clinical isolates were confirmed positive for class D oxa Beta lactamases production with the highest prevalence in E. coli (37.88%) and absent in K. oxytoca. The isolates producing this enzyme were sourced mainly from urine (55.17%) or wound swabs (26.44%). The antibiotic susceptibility profile using class D blaOxa showed that E. coli has overall resistance to clavulanic acid and third generation cephalosporins, with high susceptibility profile to tigercycline. (52.0%) and nitrofuranton (49.00%). K. pneumoniae has the highest susceptibility with tigercyclin and Nitrofuranton (56.7% and 56.4% respectively). The most active agents against non–lactose fermenters were meropenem, tigercycline and levofloxacin. The data highlighted the widespread of antibiotic resistance associated with bla OXA among the Gram –negative bacterial isolates in hospitals from Kano metropolis. The attention of the authorities and healthcare sectors is needed urgently towards the rising spate and widespread resistance due to class D oxa βlactamases so as to device a method to curb this threatening trend. Key words : Antibiotic susceptibility, resistance, lactose fermenters, non–lactose fermenters.

Extensively drug-resistant (XDR) Pseudomonas aeruginosa identified in Lima, Peru co-expressing a VIM-2 metallo-β-lactamase, OXA-1 β-lactamase and GES-1 extended-spectrum β-lactamase

IntroductionPseudomonas aeruginosa has the ability to acquire plasmids and other mobile genetic elements that confer resistance to antibiotics. Bacterial genes encoding different β-lactamases (bla), such as metallo-β-lactamases (MBLs) and extended-spectrum β-lactamases (ESBL), can confer resistance to multiple classes of β-lactam antibiotics.Case presentationAn 83 year old female was admitted in 2012 to the Peruvian Naval Hospital, Centro Médico Naval ‘Cirujano Mayor Santiago Távara’ (CEMENA), in Lima, Peru. A midstream urine sample was collected and sent to the local CEMENA laboratory for routine urine culture. P. aeruginosa was isolated and initial antibiotic susceptibility testing showed it to be sensitive to imipenem. The clinicians started a course of meropenem, but the patient did not improve. After 5 days, a second urine culture was performed and a P. aeruginosa was isolated again, but this time the strain showed resistance to imipenem. The treatment course was changed to fosfomycin and the patient improved. Phenotypic and molecular laboratory testing to characterize the antibiotic resistance were performed, demonstrating the presence of both MBL and ESBL genes.ConclusionTo our knowledge, this is the first report of a P. aeruginosa XDR clinical isolate that co-expresses an MBL (VIM-2), OXA-1 beta-lactamase and the ESBL (GES-1) in Peru. It is also the first report of a VIM carbapenemase in Peru.

Involvement of AmpG in mediating a dynamic relationship between serine beta-lactamase induction and biofilm-forming ability of Escherichia coli.

AmpG permease is implicated both in beta-lactamase induction and peptidoglycan recycling in enterobacterial isolates. Here, physiological studies using molecular genetics show that deletion of AmpG permease dramatically increases beta-lactam susceptibility even in the presence of AmpC, TEM-1 and OXA beta-lactamases. Also, there is an appreciable decrease in the biofilm-forming ability of strains lacking this protein. Expression of this permease in excess probably compromises the integrity of the bacterial cells, leading to cell lysis. Based on these results, we propose that AmpG permease may be used as a potential antibiotic target and its suppression could efficiently inhibit both beta-lactamase induction and biofilm formation.

Reduced Susceptibility to Cefepime in Clinical Isolates of Enterobacteriaceae Producing OXA-1 Beta-Lactamase.

An increase of Enterobacteriaceae isolates with reduced susceptibility to cefepime (FEP) and amoxicillin/clavulanate (AMC) has been observed in our area. The aim of this study was to characterize this antibiotic resistance phenotype and its molecular epidemiology. A total of 33 Enterobacteriaceae strains were studied. blaOXA-1 genes and their genetic environment were analyzed by polymerase chain reaction (PCR) and sequencing. Plasmids were transferred by conjugation and/or transformation and classified using PCR-based inc/rep typing and IncF subtyping. Escherichia coli isolates were typed by phylogroup, pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing. Outer membrane proteins were studied by sodium dodecylsulfate-polyacrylamide gel electrophoresis and expression of blaOXA-1 genes by reverse transcription-PCR. FEP minimum inhibitory concentration yielded values of 1-16 mg/L. Twenty-nine (87.9%) isolates produced OXA-1, of which 24 (82.7%) were located in class 1 integron, and 9 (27.3%) produced TEM-1. Among the 24 E. coli OXA-1-producers, PFGE revealed two main clusters: one belonged to C-ST88 and the other to B23-ST131. Thirteen plasmids containing blaOXA-1 were transferred, nine belonged to IncF replicon (4 F2:A1:B-, 2 F1:A1:B1, 1 F1:A2:B-, 1 F18:A2:B1, 1 F5:A-:B1) and four were nontypeable. In conclusion, reduced susceptibility to FEP was mostly due to OXA-1 beta-lactamase. In E. coli, this increase is mainly due to the dissemination of two clones, which have captured different IncF plasmids. Among non-E. coli strains, five isolates produced OXA-1 and one isolate produced only TEM-1.

Clonal spread of Klebsiella pneumoniae producing OXA-1 betalactamase in a Spanish hospital.

Multi-drug resistant Klebsiella pneumoniae isolates are associated with nosocomial infections, in which colonized patients act as a reservoir and source of cross-infection for other patients. In this study, the antimicrobial susceptibility of K. pneumoniae was tested by microdilution using the commercial method MicroScan (Siemens). The genetic relatedness of K. pneumoniae strains was determined by pulsed field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). PCR experiments were carried out to obtain primer sets and positive PCR products were purified and sequenced. From May 2007 until December 2009, 98 clonally related K. pneumoniae isolates were detected from clinical samples of 38 patients admitted to the University Hospital of Bellvitge, Barcelona, Spain, including 27 admitted to the intensive care unit (ICU). The most important sources of the isolates were: lower respiratory tract (n = 12), urine (n = 12), and blood (n = 11). The strains were resistant to amoxicillin/clavulanic acid, piperacillin/tazobactam, tobramycin, amikacin, and ciprofloxacin, and had diminished susceptibility to cefepime. All the isolates shared a common PFGE pattern related to sequence type 14 after MLST analysis. In K. pneumoniae isolates and their transconjugants, the bla(OXA-1) gene was located in the variable region of a class I integron that also contains the aac(6')Ib-cr gene. Sequencing of the quinolone resistance determinant regions of gyrA and parC revealed a S83F change in GyrA and no changes in ParC.

Carbapenem Susceptibility Testing Errors Using Three Automated Systems, Disk Diffusion, Etest, and Broth Microdilution and Carbapenem Resistance Genes in Isolates of Acinetobacter baumannii-calcoaceticus Complex

The Acinetobacter baumannii-calcoaceticus complex (ABC) is associated with increasing carbapenem resistance, necessitating accurate resistance testing to maximize therapeutic options. We determined the accuracy of carbapenem antimicrobial susceptibility tests for ABC isolates and surveyed them for genetic determinants of carbapenem resistance. A total of 107 single-patient ABC isolates from blood and wound infections from 2006 to 2008 were evaluated. MICs of imipenem, meropenem, and doripenem determined by broth microdilution (BMD) were compared to results obtained by disk diffusion, Etest, and automated methods (the MicroScan, Phoenix, and Vitek 2 systems). Discordant results were categorized as very major errors (VME), major errors (ME), and minor errors (mE). DNA sequences encoding OXA beta-lactamase enzymes (bla(OXA-23-like), bla(OXA-24-like), bla(OXA-58-like), and bla(OXA-51-like)) and metallo-β-lactamases (MBLs) (IMP, VIM, and SIM1) were identified by PCR, as was the KPC2 carbapenemase gene. Imipenem was more active than meropenem and doripenem. The percentage of susceptibility was 37.4% for imipenem, 35.5% for meropenem, and 3.7% for doripenem. Manual methods were more accurate than automated methods. bla(OXA-23-like) and bla(OXA-24-like) were the primary resistance genes found. bla(OXA-58-like), MBLs, and KPC2 were not present. Both automated testing and manual testing for susceptibility to doripenem were very inaccurate, with VME rates ranging between 2.8 and 30.8%. International variability in carbapenem breakpoints and the absence of CLSI breakpoints for doripenem present a challenge in susceptibility testing.

Mutation of the Active Site Carboxy-Lysine (K70) of OXA-1 β-Lactamase Results in a Deacylation-Deficient Enzyme

Class D beta-lactamases hydrolyze beta-lactam antibiotics by using an active site serine nucleophile to form a covalent acyl-enzyme intermediate and subsequently employ water to deacylate the beta-lactam and release product. Class D beta-lactamases are carboxylated on the epsilon-amino group of an active site lysine, with the resulting carbamate functional group serving as a general base. We discovered that substitutions of the active site serine and lysine in OXA-1 beta-lactamase, a monomeric class D enzyme, significantly disrupt catalytic turnover. Substitution of glycine for the nucleophilic serine (S67G) results in an enzyme that can still bind substrate but is unable to form a covalent acyl-enzyme intermediate. Substitution of the carboxylated lysine (K70), on the other hand, results in enzyme that can be acylated by substrate but is impaired with respect to deacylation. We employed the fluorescent penicillin BOCILLIN FL to show that three different substitutions for K70 (alanine, aspartate, and glutamate) lead to the accumulation of significant acyl-enzyme intermediate. Interestingly, BOCILLIN FL deacylation rates (t(1/2)) vary depending on the identity of the substituting residue, from approximately 60 min for K70A to undetectable deacylation for K70D. Tryptophan fluorescence spectroscopy was used to confirm that these results are applicable to natural (i.e., nonfluorescent) substrates. Deacylation by K70A, but not K70D or K70E, can be partially restored by the addition of short-chain carboxylic acid mimetics of the lysine carbamate. In conclusion, we establish the functional role of the carboxylated lysine in OXA-1 and highlight its specific role in acylation and deacylation.

Rapid and widespread dissemination of multidrug-resistant blaCMY-2Salmonella Typhimurium in Mexico

We describe the emergence and dissemination of multidrug-resistant (MDR) Salmonella Typhimurium in humans, retail meat and food animals from Yucatan, Mexico. Salmonella Typhimurium isolates were collected through an active surveillance system and tested for susceptibility to 12 antimicrobial agents. Isolates that were non-susceptible to ceftriaxone were tested with 10 additional antimicrobials and assayed by PCR for the presence of CMY, CTX-M, SHV, TEM and OXA beta-lactamase genes. Plasmid-borne phenotypes were identified by transfer to susceptible Escherichia coli. Isolates from humans, retail meat and food animals were compared by PFGE to determine genetic relatedness. MDR Salmonella Typhimurium containing a plasmid-mediated blaCMY-2 AmpC beta-lactamase rose from 0% (0/27) during 2000 and 2001 to 75% (63/84) in 2004 and 2005 (P<0.0001). MDR blaCMY-2 Salmonella Typhimurium (n=115) was most common in ill children (44.3%) and pork or swine intestine (36.5%). In several cities, MDR blaCMY-2 Salmonella Typhimurium from retail meat or swine intestine exhibited PFGE patterns and antibiograms indistinguishable from those in strains recovered from hospitalized children. The CMY gene was transferred to E. coli by electroporation, along with resistance to three to six other antimicrobials. Children with MDR blaCMY-2 Salmonella Typhimurium infection (n=39) had a higher frequency of systemic infection (13% versus 0%), mortality (8% versus 0%) and hospital re-admission due to protracted diarrhoea (28% versus 17%) than children with non-MDR-Salmonella Typhimurium (n=24), although the difference was not statistically significant. The rapid and widespread dissemination of MDR blaCMY-2 Salmonella Typhimurium in Mexico calls for urgent interventions to contain this potentially fatal pathogen.

Prevalence of extended-spectrum β-lactamases in isolates of the Enterobacter cloacae complex from German hospitals

In 2002, 119 isolates of the Enterobacter cloacae complex were collected randomly from 11 German laboratories nationwide. Antibiotic susceptibilities were tested by disk-diffusion tests according to CLSI guidelines, and MICs were determined using Etests. PCRs were performed to amplify all TEM and SHV, and most CTX-M and OXA beta-lactamase genes. PCR products were sequenced to identify the precise extended spectrum beta-lactamase (ESBL) types. Isoelectric focusing (IEF) and PM/PML Etests were used to confirm production of the respective ESBLs. According to susceptibility tests and CLSI criteria, 49 (40%) isolates were resistant to extended-spectrum cephalosporins. Seven (5.8%) isolates were positive in at least one of the PCR assays. Sequencing identified production of TEM-1 beta-lactamase genes by three (2.9%) isolates, and ESBL genes of the CTX-M and SHV beta-lactamase families by five (4.2%) isolates. IEF confirmed the production of beta-lactamases in the expected pI ranges of the respective ESBLs, and four of the five ESBL-producers were detected using the PM/PML Etest. All ESBL-producing isolates showed co-resistance to sulphonamides.