Occurrence of K1 and K2 serotypes and genotypic characteristics of extended spectrumβ-lactamases-producing Klebsiella pneumoniae isolated from selected hospitals in Malaysia

Objective:To determine the distribution,phenotypic and genetic background of extended spectrumβ-lactamases(ESBL)-producing Klebsiella(K.)pneumoniae clinical isolates associated with K1 and K2 serotypes in two selected hospitals in Malaysia.Methods:A total of 192 K.pneumoniae isolates were collected and subjected to antibiotic susceptibility,hypermucoviscosity test and multiplex PCR to detect the presence of K1-and K2-serotype associated genes.Multilocus sequence typing(MLST)was performed on ESBL-producing K.pneumoniae isolates presented with K1 and K2 serotypes,followed by phylogenetic analysis.Results:A total of 87 out of 192(45.3%)of the K.pneumoniae isolates collected were ESBL producers.However,only 8.3%(16/192)and 10.9%(21/192)of the total isolates were detected to carry K1-and K2-serotype associated genes,respectively.Statistical analysis showed that K1 and K2 capsular serotypes were not significantly associated with ESBL phenotype(P=0.196).However,they were significantly associated with hypervirulent,as demonstrated by the positive string test(P<0.001).MLST analysis revealed that ST23 as the predominant sequence type(ST)in the K1 serotype,while the ST in the K2 serotype is more diverse.Conclusions:Although the occurrence of ESBL-producing isolates among the hypervirulent strains was low,their coexistence warrants the need for continuous surveillance.MLST showed that these isolates were genetically heterogeneous.

Evaluation of Disk Potentiation Test (DPT) and Double Disk Synergy Test (DDST) for The Detection of Metallo-β-Lactamases (MBLs) in Clinical Isolates of Bangladesh

Objective: Increasing the emergence of Metallo-β-lactamase (MBL) producing gram-negative bacteria and their dexterous horizontal transmission demands rapid and accurate detection. This study was conducted to determine a suitable method to promptly detect MBL-producing gram-negative bacteria. Methods: A total of 103 gram-negative bacteria were identified from various clinical samples at a tertiary care hospital in Dhaka city. MBL producers were detected by two phenotypic methods, the Disk Potentiation Test (DPT) and the Double Disk Synergy Test (DDST) based on β-lactam chelator combinations where EDTA/SMA has been used as an inhibitor and Imipenem, Ceftazidime as substrates. Results: 103 isolates which were identified as Escherichia coli spp, Klebsiella spp, Pseudomonas spp, Acinetobacter spp, Proteus spp, Providencia spp were found to be multidrug-resistant in antibiogram test. Isolates showed complete resistance (100%) to Imipenem, Meropenem, and Amoxiclav. The highest carbapenem-resistant etiological agents were Acinetobacter spp 40 (38.8%) followed by Pseudomonas spp 27 (26.2%), Klebsiella spp 26 (25.2%), Escherichia coli 8 (7.8%), Proteus spp 1 (1%) and Providencia spp 1 (1%). DPT method detected significantly (p = 0.000009) a higher number of MBL-producers (Imipenem with 0.5 M EDTA n = 61, 59.2% & Ceftazidime with 0.5 M EDTA n = 56, 54.4%) compared to the DDST method (Imipenem -0.5 M EDTA n = 43, 41.7%, Imipenem – SMA n = 38, 36.9% & Ceftazidime -0.5 M EDTA n = 15, 14.6%). Conclusion: Pieces of evidence suggest that DPT is a more sensitive method than DDST and could be recommended for identifying MBL-producing bacteria in Bangladeshi hospitals for the proper management of patients, to reduce time constraints and treatment costs.

Metallo-β-Lactamases:A Major Threat to Human Health

Antibiotic resistance is one of the most significant challenges facing global healthcare. Since the 1940s, antibiotics have been used to fight infections, initially with penicillin and subsequently with various derivatives including cephalosporins, carbapenams and monobactams. A common characteristic of these antibiotics is the four-memberedβ-lactam ring. Alarmingly, in recent years an increasing number of bacteria have become resistant to these antibiotics. A major strategy employed by these pathogens is to use Zn(II)-dependent enzymes, the metallo-β-lactamases (MBLs), which hydrolyse theβ-lactam ring. Clinically useful MBL inhibitors are not yet available. Consequently, MBLs remain a major threat to human health. In this review biochemical properties of MBLs are discussed, focusing in particular on the interactions between the enzymes and the functionally essential metal ions. The precise role(s) of these metal ions is still debated and may differ between different MBLs. However, since they are required for catalysis, their binding site may present an alternative target for inhibitor design.

Computational Calculations of Molecular Properties and Molecular Docking of New and Reference Cephalosporins on Penicillin Binding Proteins and Various β-Lactamases

An approach of using molinspiration calculations and molecular docking on PBPs （penicillin-binding proteins） and certain β-lactamases is employed to predict the molecular properties, bioactivity and resistance of newer and reference cephalosporins. The previously synthesized cephalosporins 1-8 and reference cephalosporins were subjected to extensive evaluations by calculating the molecular properties, drug-likeness scores on the bases of Lipinski＇s rule and bioactivity prediction using the method of molinspiration web-based software. The TPSA （topological polar surface area）, OH-NH interactions, n-violation and the molinspiration Log partition coefficient （miLogP） values were also calculated. The investigated cephalosporins were subjected to molecular docking study on PBPs （lpyy） and on β-lactamases produced by S. aureus, K. pneumonia, E. coil and P. auroginosa using 1-click-docking website. Molecular properties of 1-8 recorded higher ＂FPSA than cephalexin and were lower than the reference cephalosporins and do not fulfill the requirements for Lipinski＇s rule. Bioactivities of 1-8 were predicted to be less and their docking scores on PBPs were comparable to those of the reference cephalosporins, particularly ceftobiprole. The references recorded various docking scores on the above β-lactamases and as expected, cefiobiprole recorded the lowest scores on all β-lactarnases. Cephalosporins 1-8 recorded various docking scores on β-lactamases. Molecular docking studies on PBPs and β-lactamases are considered as very useful, reliable and practical approach for predicting the bioactivity scores and to afford some information about the stability and selectivity of the newly proposed cephalosporins against β-lactamases of certain pathogenic microbes, such as P. auroginosa and MRSA, by recording the relative docking scores in comparison with those of reference cephalosporins.

Identification and preliminary characterization of novel B3-type metallo-β-lactamases

Antibiotic resistance has emerged as a major global threat to human health. Among the strategies employed by pathogens to acquire resistance the use of metallo-β-lactamases (MBLs), a family of dinuclear metalloenzymes, is among the most potent. MBLs are subdivided into three groups (i.e. B1, B2 and B3) with most of the virulence factors belonging to the B1 group. The recent discovery of AIM-1, a B3-type MBL, however, has illustrated the potential health threat of this group of MBLs. Here, we employed a bioinformatics approach to identify and characterize novel B3-type MBLs from Novosphingobium pentaromativorans and Simiduia agarivorans. These enzymes may not yet pose a direct risk to human health, but their structures and function may provide important insight into the design and synthesis of a still elusive universal MBL inhibitor.

Unusual metallo-β-lactamases may constitute a new subgroup in this family of enzymes

Metallo-β-lactamases (MBLs) are a family of Zn2+-dependent enzymes that have contributed strongly to the emergence and spread of antibiotic resistance. Novel members as well as variants of existing members of this family are discovered continuously, compounding their threat to global health care. MBLs are divided into three subgroups, i.e. B1, B2 and B3. The recent discovery of an unusual MBL from Serratia proteamaculans (SPR-1) suggests the presence of an additional subgroup, i.e. B4. A database search reveals that SPR-1 has only one homologue from Cronobacter sakazakii, CSA-1.These two MBLs have a unique active site and may employ a mechanism distinct from other MBLs, but reminiscent of some organophosphate-degrading hydrolases.

An integrated microfluidic chip-mass spectrometry system for rapid antimicrobial resistance analysis of bacteria producingβ-lactamases

Bacteria producingβ-lactamases have become a major issue in the global public health field.To restrain the development of drug resistance and reduce the abuse of antibiotics,it is very important to rapidly identify bacteria producingβ-lactamases and put forward a reasonable treatment plan.Here,an integrated microfluidic chip-mass spectrometry system was proposed for rapid screening ofβ-lactamaseproducing bacteria and optimization ofβ-lactamase inhibitor dosing concentration.The concentration gradient generator followed by an array of bacterial culture chambers,as well as micro-solid-phase extraction columns was designed for sample pretreatment before mass analysis.By using the combination system,the process of the hydrolysis of antibiotics byβ-lactamase-producing bacteria could be analyzed.To validate the feasibility,four antibiotics and two antibiotic inhibitors were investigated using three strains including negative control,SHV-1 and TEM-1 strains.SHV-1 and TEM-1 strains were successfully distinguished as theβ-lactamase producing strains.And the acquired optimal concentrations ofβ-lactamase inhibitors were in accordance with the results by that obtained from the traditional microdilution broth method.The total analysis time only needed around 2 h,which was faster than conventional methods that require a few days.The technique presented herein provides an easy and rapid protocol forβ-lactamase resistance related studies,which is important for the inhibition of antimicrobial resistance development and the reduction of antibiotics abuse.

Characteristics of β-Lactamase Synthesis in E. coli and K. pneumanie Strains in Nosocomial Infections

Background: Recently micro-organisms that synthesize extended-spectrum β-lactamase (ESBLs) were increased. The peculiarities of ESBL synthesis of Escherichia coli and Klebsiella pneumoniae strains that cause nosocomial urinary tract infections, surgical site infections and pneumonia in surgical clinic were studied. ESBL synthesis were observed 38.9% of E. coli strains obtained from urine, 92.3% of strains obtained from surgical site infections, and 50% of strains obtained from sputum. ESBL synthesis were observed 37.5% of K. pneumoniae strains obtained from urine, 85.7% of strains obtained from surgical site infections, and 60% of strains obtained from sputum. Different levels of ESBL synthesize of E. coli and K. pneumoniae strains isolated from different pattern is discussed. Conclusion. ESBL synthesis is common in E. coli and K. pneumoniae strains, which cause nosocomial infections. The frequency of occurrence of ESBL s synthesis among of these strains depends on clinical forms of nosocomial infections.

Antimicrobial resistance, genotypic characterization and pulsed-field gel electrophoresis typing of extended spectrum β-lactamases-producing clinical Escherichia coli strains in Macao, China

Background The rise of the production of CTX-M class extended spectrum β-lactamases （ESBLs） has been well documented in traveling countries but no data are found for Macao, an international travel city. The objectives of this study were to identify the antimicrobial resistance pattern, and determine the prevalence, genotype and clonal relationship of ESBLs in 209 clinical Escherichia coli strains from Macao, China. Methods Antimicrobial susceptibility test was performed to determine the resistance patterns of the isolates using the disk diffusion method with 17 antimicrobial agents. Phenotypic detection was screened and confirmed according to the Clinical and Laboratory Standards Institute. Genotypic characterization was detected by isoelectric focusing analysis, polymerase chain reaction and sequencing. The clonal relationship between the different ESBL isolates was studied by pulsed-field gel electrophoresis （PFGE）. Results Imipenem and meropenem exhibited 100% susceptible among 209 strains. Overall, 82.3%, 67.3%, 52.9%, 51.2% and 51.0% of the isolates displayed resistance to ampicillin, tetracylcline, ciprofloxacin, sulfamethoxazole trimethoprin and gentamycin. The prevalence rate of ESBLs was 30.1%. Antibiotic resistances were found to be significantly higher among the ESBL producing group compared to non-ESBL producing group. We detected CTX-M-14 to be the major genotypic characterization of ESBLs （76.2%）. Two strains showed indistinguishable patterns by PFGE. Conclusions The prevalence of antimicrobial resistance is alarming high in Macao. Antimicrobial resistance is significantly higher among the ESBL producing group. This study documented CTX-M-14 as the predominant ESBL type. Although indistinguishable pattern was found between two strains, it was too small to decide whether any of the investigated strains was epidemic. Our findings may be also pertinent for other geographic areas undergoing similar travel characteristics to understand the corresponding effects on bacterial populations.

Microbiologic and Clinical Comparison of Patients Harboring <i>Escherichia coli</i>Blood Isolates with and without Extended-Spectrum <i>β</i>-Lactamases

The clinical and microbiologic characteristics of 34 patients with extended-spectrum β-lactamase (ESBL) positive E. coli isolated from blood were compared to 66 bacteremic patients with ESBL negative E. coli, from January 2007 through December 2009. Of the 21 ESBL positive isolates available for PCR analysis, 13 were positive for CTX-M, 8 for TEM, 4 for SHV β-lactamases, with 6 possessing multiple enzymes. Twenty of 34 (59%) ESBL-positive and 41 of 66 (62%) ESBL-negative blood isolates were considered community-associated. All but one isolate in both groups had MICs of ≤1.0 μg/ml to meropenem. However, when compared to ESBL-negative isolates, ESBL-positive isolates were more frequently resistant to levofloxacin, trimethoprim/sulfamethoxazole and had higher MICs to gentamicin, tobramycin and piperacillin/tazobactam. The use of intravenous and urinary catheters was strongly associated with the isolation of E. coli bloodstream isolates in both groups of patients. Although hospital stay was similar in both groups, appropriate therapy was given in 87% of patients with ESBL positive vs. 98% of patients with ESBL negative isolates and mortality was greater for patients with ESBL positive isolates (26% vs. 17%). Since a large proportion of E. coli blood isolates were ESBL-positive and community-associated, carbapenems should be considered as initial empiric therapy for such infections in our locale.

Prevalence and Risk Factors of Penicillinase-Type β-Lactamase Producing Neisseria gonorrhoeae Isolated from Patients Attending Health-Facilities in Yaounde, Cameroon

Background and Objectives: Mitigation of antibiotic resistant Neisseria gonorrhoeae has become a priority due to considerable health and economical disabilities it generates. In order to tackle the emergence of resistant Neisseria gonorrhoeae, this study aimed to determine the prevalence and risk factors of penicillinase type β-lactamase-producing Neisseria gonorrheae among patients consulting for genital infectious disorders in two health-facilities in Yaounde, Cameroon. Materials and Method: A cross-sectional descriptive and analytical study was conducted over a 3-month period, from July 2nd to October 2nd, 2022. Vaginal and urethral secretions were collected. Biochemical identification tests were performed on colonies grown on chocolate agar + polyvitex using the Api NH gallery. The detection of penicillinases was equally performed using the API NH gallery and confirmed using the antimicrobial susceptibility testing. The Minimum Inhibitory Concentrations of some antibiotics were determined using the E-Test. Results: The results showed that out of the 198 patients sampled, 16 (8.08%) were positive for Neisseria gonorrhoeae, among which 13/16 (81.25%) were penicillinase-type β-lactamase producers. Antimicrobial susceptibility testing results showed high co-resistances to antibiotics, mainly ciprofloxacin (100%), nalidixic acid (92.31%) and azithromycin (84.62%). Moreover, high Minimum Inhibitory Concentrations of ceftriaxone (ranging from 6 to 24 mg/L) was observed toward Neisseria gonorrhoeae isolates. The risk factors of the carriage of penicillinase-type β-lactamase producing Neisseria gonorrhoeae identified were: a history of Sexually Transmitted infections (p = 0.01) and unprotected sexual intercourse (p = 0.01). Conclusion: The emergence of penicillinase-type β-lactamase producing Neisseria gonorrhoeae is increasing and the situation is becoming worrisome. The identified risk factors can constitute a basic outlook to tackle resistant Neisseria gonorrhoeae, and therefore sustain antibiotic stewardship.

Dissemination of carbapenem-non-susceptible Acinetobacter baumannii isolates collected from educational hospitals in Qazvin province of Iran

Background:Acinetobacter baumannii(A.baumannii)is known as an opportunistic pathogen related to health-care-associated infection that has a high antibiotic resistance potential,notably against carbapenems that are widely used to combat A.baumannii infections.This study aimed to detect oxacillin-hydrolyzing(OXA)carbapenemases and metallo-β-lactamases(MBL)among carbapenem-resistant A.baumannii isolated strains and to determine their clonal relationship by repetitive extragenic palindromic PCR(rep-PCR).Methods:In the present study,a total of 211 non-repetitive isolates of A.baumannii were collected from Qazvin educational hospitals(2016–2017).The disk diffusion method was used to investigate the antibiotic susceptibility of studied strains,followed by the detection of MBL and OXA-type genes using polymerase chain reaction(PCR)and sequencing methods.The rep-PCR method assessed the clonal relationship of carbapenem-non-susceptible A.baumannii isolates.Result:The obtained results showed that 87.2%and 86.7%of isolates were non-susceptible to imipenem and meropenem.The blaOXA-24(93.5%)was the most frequent gene,followed by the blaOXA-23(4.34%),blaIMP-1(1.63%),and blaVIM-1(0.54%).Meanwhile,blaOXA-58 and blaOXA-143 genes were not found.81.5%and 66.1%of isolates contained ISAba1 upstream of the blaOXA-23 and blaOXA-58 genes,respectively.Rep-PCR results revealed the carbapenem non-susceptible isolates belonged to three distinct clones:A 171(81%),B 34(16.1%),and C 6(2.8%).Conclusions:The results indicated a high prevalence of carbapenem-non-susceptible A.baumannii,with the emergence of the blaOXA-24 gene as the most common gene and the notable prevalence of MBL genes.These results revealed the need for appropriate therapeutic and infection control strategies and monitoring susceptibility patterns for controlling A.baumannii infections.

Metallo-β-lactamase producing nonfermentative gram-negative bacteria:An increasing clinical threat among hospitalized patients

Objective:To detect and evaluate the various methods for metallo-β-lactamases(MBL) production in Pseudomonas aeruginosa(P.aeruginosa) and Acinetobacter species.Methods:A total of 109 P.aeruginosa and 85 Acinetobacter species were screened for imipenem resistance by Kirby- Bauer disc diffusion methods.Detection of MBL production was(lone by imipenem-EDTA combined disc test,double disc synerygy test(DDST) and imipenem-EDTA MBL E test.Results: A total of 63(57.8%) strains of P.aeruginosa and 46(54.1%) strains of Acinetobacter spp.were found to be resistant to imipenem.Of the 63 imipenem resistant P.aeruginosa tested for MBL production.44(69.89;) were found to be positive and among 46 imipenem resistant Acinetobacter. 19(41.3%) were shown to be the MBL producers.Conclusions:Imipenem-EDTA combined disc test and MBL E test are equally effective for MBL detection in both P.aeruginosa and Acinetobacter spp.,but given the cost-constraints,combined disc can be used as a convenient screening method in the clinical microbiology laboratory.

Prevalence and Potential Risk Factors of Hospital Acquired Extended-Spectrum Beta-Lactamase—Producing <i>Proteus</i>Species

Background: Multidrug resistance and production of extended spectrum β-lactamases (ESBLs) by a large group of bacterial agents in hospitals are to be a matter of scientific concern. Objective: This cross-sectional study was aimed to investigate the prevalence of ESBL producing Proteus species and risk factors associated with hospital acquired infection in addition to study the antibiotics susceptibility patterns of all bacterial isolates from inpatients of four Yemeni general hospitals. Methods: A total of 740 consecutive non-repeat culture isolates were obtained from admitted patients of Al-Kuwait University Hospital, Al-Thowra General Hospital, Al-Jumhori Teaching Hospital, and Military General Hospitals Sana’a city. We used Kirby-Bauer disk diffusion method to detect antimicrobial susceptibility and establish the presence of ESBLs-producing bacteria according to the Clinical and Laboratory Standards Institute guidelines. Results: Out of 740 isolate, 233 (31.5%) were Escherichia coli followed by Staphylococcus aureus 188 (25.4%), Pseudomonas aeruginosa 149 (20.1%), Klebsiella sp. 107 (14.5%), Enterococcus faecalis 25 (3.4%) and Proteus spp. 38 (5.1%). The highest frequencies of ESBLs producing among Proteus sp. were Proteus mirabilis 26 out 38 (68.4%) and Proteus vulgaris 12 out 38 (31.6%). The most effective of antimicrobial susceptibility pattern among Proteus spp. were Imipenem (100%) followed by Pipracillin-Tazobactam (92.3%) for P. mirabilis and (83.3%) for P. vulgaris, while the Amikacin (80.8%) for P. mirabilis and P. vulgaris with (91.7%). Amoxicillin and Cefotaxime were the highest for both species (100%). Conclusion: The prevalence of ESBL-producing Proteus spp. detected in this study is of great concern for public health authorities and a strict adherence of infection control policies and procedures with continuous antibiotics resistance surveillance including antimicrobial management and routine detection of ESBL-producing isolates are very important to prevent nosocomial infections.

Phenotypic and Genotypic Characterization of Extended Spectrum β-Lactamase Klebsiella pneumoniae and Fluorescent Pseudomonas spp. Strains from Market Garden Products and Their Watering Water in Benin (West Africa)

Market garden products can carry several types of microorganisms, and their consumption is the source of many cases of food poisoning. This work aimed to improve food safety in Benin. In characterizing strains of K. pneumoniae and fluorescent Pseudomonas spp. at the biochemical and molecular level, the target was to identify contaminated watering water and garden products sold during Cotonou in both the dry and rainy seasons. A total of 164 samples of market garden products and 22 samples of watering water were investigated. The results showed that 5.91% of market garden products and watering water were contaminated by K. pneumoniae and 20.43% by fluorescent Pseudomonas spp. During the dry season, cabbage was most contaminated by fluorescent Pseudomonas spp. (50%). Pool water was more contaminated with K. pneumoniae (17%). All isolated strains were resistant to both amoxicillin and penicillin. All strains of K. pneumoniae and fluorescent Pseudomonas spp. were not resistant to imipenem, and 22% of them produced penicillinase. Among the 49 strains producing penicillinase isolated, 64.29% and 21.43% carried blaTEM and blaSHV respectively while 14.28% carried blaCTX-M genes. In light of the previously-developed results and considering the importance of horticultural products in Beninese food habits, we must improve national awareness of the risk for foodborne illness.

Multiple Antimicrobial Resistance of Extended Spectrum Beta-Lactamase-Producing Escherichia coli from Small-Scaled Poultry Farms and Retail Chicken

Antibiotics used for agricultural purpose has contributed to the increased prevalence of antibiotic-resistant bacteria. The goal of this study was to investigate the prevalence and antimicrobial resistance of ESBL-producing E. coli in small-scaled poultry farms and retail chicken. The cultured E. coli isolates were subjected to phenotypic tests, susceptibility tests, and the polymerase chain reaction for detection of blacTX-M, blasHv, and blaTEM genes. From 120 samples each of chicken feces, retail chicken, soil and chicken feed, ESBL-producing E. coli isolates were detected in 75.9%, 63.6%, 39.2%, and 13.3% of the samples, respectively. Minimum inhibitory concentration （MICs） values indicated that ESBL-producing E. coli were resistance to ampicillin （MIC 〉 32 μg/mL）, gentamicin （M1C ≥ 16 μg/mL）, cefotaxime （MIC 〉 4 μg/mL） and cefhiaxone （MIC 〉 4 gg/mL）, respectively. The total resistance for imipenem was also observed at 1.0% （MIC ≥ 4 gg/mL） and none of the isolates were resistant to ceftazidime （MIC 〉 16 μg/mL）. ESBL-producing E. coli from chicken feces and retail chicken carried blasHv gene at a rate of 6.8% and 5.7%, respectively and blaCTX-M gene was also revealed at 2.9% in retail chicken. Moreover, ESBL-producing E. coli isolated from soil harbored blasnv and blaCTX-M genes at 5%. None of the feed samples yielded ESBLs genes. Twenty three resistance patterns were observed for multi-resistant ESBL-producing E. coli. This study highlights the prevalence of multi-antimicrobial resistant ESBL-producing E. coli in small-scaledpoultry farms and retail chicken, hence the need to review poultry management practices to minimize the occurrence.

L1 β-Lactamase催化反应机理研究

用混合量子力学和分子力学(QM/MM)方法和密度泛函理论讨论了L1β-Lactamase催化Nitrocefin水解的过程,研究结果表明,反应为多步反应:第一步亲核进攻反应为反应的决速步骤,并且伴随着酰胺键的断裂,第二步反应为质子迁移反应.同时讨论了金属锌在反应中的作用.

Prevalence and characteristics of extended spectrum β-lactamase-producing Escherichia coli from bovine mastitis cases in China

The aim of the study was to investigate the prevalence and characterization of extended-spectrum β-lactamase （ESBL）- producing Escherichia coli isolated from bovine mastitis cases in China. ChromID ESBL agar was used to confirm ESBL-producing E. coli. PCR and DNA sequencing were employed to characterize the genotype of ESBL-producers. Antimicrobial susceptibility was measured by disc diffusion. Overall, 73 of 318 E. coli isolates （22.96％） were identified as ESBL-producers. Of these ESBL-producing E. coli, the prevalence of blaCTX-M and blaTEM-1 was 97.26 and 71.23％, respectively. The predominant CTX-M-type ESBL was CTX-M-15 （65.75％）, followed by CTX-M-14 （10.96％）, CTX-M-55 （9.59％）, CTX-M-64 （5.48％）, CTX-M-65 （4.11％） and CTX-M-3 （1.37％）. This study is the first report of CTX-M-64 and CTX-M-65 in E. coli isolated from bovine mastitis. Furthermore, 72 ESBL-producing E. coli isolates （98.63％） were found to be multidrug-resistance. This study noted high prevalence and rates of antimicrobial resistance of ESBL-producing E. coli isolates from bovine mastitis cases in China.

Analysis of AmpC β-lactamase Gene in Pseudomonas aeruginosa

The gene and the amino acid sequence of the structural and regulatory region of the Pseudomonas aeruginosa with different resistance patterns were analyzed. Six strains with different resistance patterns were selected and the AmpC β-lactamase was identified. The objective gene fragment was amplified by colonies PCR. The sequences of the PCR-products were analyzed. The DNA sequence of the structural gene ampC and the regulatory genes ampR, ampD and ampE was detected. The 6 strains and the wild-type Pseudomonas aeruginosa are highly homogeneous in structural and regulatory region. Some new mutant points were found.

Antimicrobial Resistance and Genotype Analysis of Extended-Spectrum-β-Lactamase-Producing Proteus Mirabilis

To analyse the genotypes of clinical isolates of Extended-Spectrum-β-Lactamase-Producing (ESBL-producing) Proteus mirabilis (P. mirabilis) and the mechanisms of antimicrobial resistance, to guide reasonable use of antibiotics and to avoid nosocomial outbreak infections by ESBL-producing P. mirabilis. 125 clinical isolates of P. mirabilis were collected from the Drug-Resistant Bacteria Surveillance Center of Anhui Province (from Jan 2009 to May 2010). Searching for the genotypes of ESBLs was perfomed by PCR amplification and DNA sequencing, and performed conjugation test simultaneously. Among ESBL-producing strains, CTX-M was the major genotype (3 CTX-M-13 and 1 CTX-M-3). TEM-1b spectrum β-lactamase was also prevalence in P. mirabilis. The diversity of β-lactamases in P. mirabilis and the emergency of multi-drug-resistance clinical strains will present serious threat to clinical therapy and even will lead to outbreak of nosocomial infections. Our study emphasizes the need for enhanced supervision of ESBL-producing P. mirabilis. Timely and reasonable drug-resistance data are indispensable to clinical therapy.