Occurrence of false positive results for the detection of carbapenemases in carbapenemase-negative Escherichia coli and Klebsiella pneumoniae isolates.

Adequate detection of the production of carbapenemase in Enterobacteriaceae isolates is crucial for infection control measures and the appropriate choice of antimicrobial therapy. In this study, we investigated the frequency of false positive results for the detection of carbapenemases in carbapenemase-negative Escherichia coli and Klebsiella pneumoniae clinical isolates by the modified Hodge test (MHT). Three hundred and one E. coli and K. pneumoniae clinical isolates were investigated. All produced extended spectrum ß-lactamases (ESBLs) but were susceptible to carbapenems. Antimicrobial susceptibility testing was performed by the disk diffusion and agar dilution methods. The MHT was performed using the standard inoculum of test organisms recommended by the CLSI. Genes that encoded ESBLs and carbapenemases were identified by PCR and DNA sequencing. Among the 301 clinical isolates, none of the isolates conformed to the criteria for carbapenemase screening recommended by the CLSI. The susceptibility rates for imipenem, meropenem, and ertapenem all were 100.0%, 100.0%, and 100.0%, respectively. Of the 301 E. coli and K. pneumoniae isolates, none produced carbapenemase. The MHT gave a positive result for 3.3% (10/301) of the isolates. False positive results can occur when the MHT is used to detect carbapenemase in ESBL-producing isolates and clinical laboratories must be aware of this fact.

Prevalence of plasmid-mediated quinolone resistance determinants in Citrobacter freundii isolates from Anhui province, PR China.

This study was conducted to detect and analyse the presence of plasmid-mediated quinolone resistance (PMQR) determinants [qnr, aac(6')-Ib-cr and qepA] among Citrobacter freundii isolates from patients in Anhui province, PR China. During 2009-2010, 31 C. freundii strains were collected from various hospital units and patient specimens. Using PCR, qnr genes were detected in eight isolates, but aac(6')-Ib-cr and qepA genes were not found. The genes qnrA1, qnrB1, qnrB2, qnrB4, qnrB10 and qnrB24 were present in 6.5, 3.2, 6.5, 3.2, 3.2 and 3.2% of C. freundii isolates, respectively. A new subgene of qnrB variant (qnrB24) was found and identified for what we believe to be the first time. PFGE after XbaI digestion of genomic DNA indicated that qnr-positive strains were not clonally related. Conjugation experiments were conducted to determine whether the qnr-carrying plasmids were self-transferable, and plasmids of transconjugants were extracted and analysed. The qnr genes were transferred from three clinical isolates to their transconjugants. Two qnrA1 genes transferred quinolone resistance with a plasmid of ~11 kb, whilst the size of the plasmid carrying the qnrB4 gene was ~64 kb. The susceptibility of positive isolates and transconjugants was tested using an agar dilution method according to Clinical and Laboratory Standards Institute guidelines, and the MICs of ciprofloxacin and levofloxacin were determined using Etest strips. Most isolates with qnr genes were resistant to fluoroquinolones and other antimicrobial agents. The MICs of transconjugants showed reduced susceptibility to fluoroquinolones.

Susceptibility of extended-spectrum-beta-lactamase-producing Enterobacteriaceae according to the new CLSI breakpoints.

In 2010 the Clinical and Laboratory Standards Institute (CLSI) lowered the susceptibility breakpoints of some cephalosporins and aztreonam for Enterobacteriaceae and eliminated the need to perform screening for extended-spectrum ß-lactamases (ESBLs) and confirmatory tests. The aim of this study was to determine how many ESBL-producing strains of three common species of Enterobacteriaceae test susceptible using the new breakpoints. As determined with the CLSI screening and confirmatory tests, 382 consecutive ESBL-producing strains were collected at Huashan Hospital between 2007 and 2008, including 158 strains of Escherichia coli, 164 of Klebsiella pneumoniae, and 60 of Proteus mirabilis. Susceptibility was determined by the CLSI agar dilution method. CTX-M-, TEM-, and SHV-specific genes were determined by PCR amplification and sequencing. bla(CTX-M) genes alone or in combination with bla(SHV) were present in 92.7% (354/382) of these ESBL-producing strains. Forty-two (25.6%) strains of K. pneumoniae harbored SHV-type ESBLs alone or in combination. No TEM ESBLs were found. Utilizing the new breakpoints, all 382 strains were resistant to cefazolin, cefotaxime, and ceftriaxone, while 85.0 to 96.7% of P. mirabilis strains tested susceptible to ceftazidime, cefepime, and aztreonam, 41.8 to 45.6% of E. coli strains appeared to be susceptible to ceftazidime and cefepime, and 20.1% of K. pneumoniae were susceptible to cefepime. In conclusion, all ESBL-producing strains of Enterobacteriaceae would be reported to be resistant to cefazolin, cefotaxime, and ceftriaxone by using the new CLSI breakpoints, but a substantial number of ESBL-containing P. mirabilis and E. coli strains would be reported to be susceptible to ceftazidime, cefepime, and aztreonam, which is likely due to the high prevalence of CTX-M type ESBLs.

Prevalence of plasmid-mediated quinolone-resistance determinants in Shigella flexneri isolates from Anhui Province, China.

Quinolones are used extensively to treat Shigella flexneri infection, and plasmid-mediated quinolone resistance (PMQR) determinants were recently reported. A total of 26 S. flexneri isolates collected from Anhui province, China, in 2005 were screened for PMQR determinants, bla gene, gyrA and parC genes, by PCR and sequencing. PMQR determinants were present in 53.8% (14 of 26) of isolates and qnrA(1), qnrS(1), qnrS(2), aac(6')-Ib-cr and qepA were present in 30.8, 11.5, 3.8, 19.2 and 11.5% of those isolates, respectively. All PMQR determinants' positive isolates exhibiting 8 different genetic clones had mutations in gyrA and parC genes, and 11 carried bla genes, including 7 bla(CTX-M) with resistance to cefotaxime. These isolates were resistant to nalidixic acid and 57.1% (8 of 14) of them were resistant to ciprofloxacin and levofloxacin. Our data show that there was a high prevalence of PMQR determinants in S. flexneri isolates from China.

Protective effects of total flavones of rhododendra against global cerebral ischemia reperfusion injury.

The purpose of this research was to demonstrate the protective effects and possible mechanisms of total flavones of rhododendra (TFR) against global cerebral ischemia reperfusion injury in rats. Global cerebral ischemia/reperfusion injury was caused by four vessel occlusion (bilateral vertebral arteries and bilateral carotid arteries, 4-VO). The electroencephalographic (EEG) changes were recorded. The EEG, brain water content, levels of malondialdehyde (MDA) and lactate dehydrogenase (LDH) activity in plasma, aggregation of platelets induced by ADP, and the resting and CaCl(2)-induced increase of free intracellular calcium concentration ([Ca(2+)](i)), were also evaluated. TFR dramatically elevated EEG amplitude, reduced the brain water content and the resting cytoplasmic free calcium concentration, inhibited the increase of [Ca(2+)](i) induced by CaCl(2) and had an inhibitory effect on platelet aggregation. The LDH activity and the MDA content in plasma were also decreased. These results indicate that TFR has protective effects against cerebral injury in rats, which might be associated with its antioxidant properties, antiplatelet effects and possible inhibition of Cal(2+) influx to reduce [Ca(2+)](i).

Investigation of qnr and aac(6')-Ib-cr in Enterobacter cloacae isolates from Anhui Province, China.

Eighty-one Enterobacter cloacae isolates collected from 20 hospitals in Anhui Province, China, from January to August 2005 were screened for plasmid-mediated quinolone resistance determinants by polymerase chain reaction and sequencing. qnrA1, qnrB4, qnrS1, and aac(6')-Ib-cr were present in 6.2% (5/81), 6.2% (5/81), 8.6% (7/81), and 3.7% (3/81) of those isolates, respectively.

[Detection of the new type of aminoglycoside acetyltransferase gene in Enterobacter cloacae isolates].

OBJECTIVE: To investigate the prevalence of aac(6')-Ib-cr gene in Enterobacter cloacae isolates. METHODS: PCR and sequencing were performed on 81 strains of Enterobacter cloacae isolated clinically in Anhui province to identify aac (6')-IB-cr gene. Disk diffusion method was used to test the susceptibility of the Enterobacter cloacae isolates with aac (6')-Ib-cr gene to fluoroquinolones, aminoglycosides, and other antimicrobial agents. AmpC and extended-spectrum beta-lactamases (ESBLs) were detected by modified three-dimensional extract test, and the molecular typing was analyzed by ERIC-PCR. RESULTS: Aac (6')-Ib-cr gene was identified in 3 (3.7%) of the 81 Enterobacter cloacae isolates with different ERIC-PCR patterns. The isolates with aac (6')-Ib-cr gene were resistant to fluoroquinolones, aminoglycosides, chloramphenicol, ampicillin, cefoxitin, and second and third generation cephalosporins. Two of the 3 isolates produced AmpC and ESBLs, and 1 isolate produced only ESBLs. CONCLUSION: Aac (6')-Ib-cr gene is prevalent in Enterobacter cloacae isolates with resistance to most of antimicrobial agents and no clone spread in found in them.

Detection of CTX-M-14 extended-spectrum beta-lactamase in Shigella sonnei isolates from China.

Shigellosis is an important cause of acute diarrheal disease and multidrug-resistant phenotype has been reported in S. sonnei. In this study, we investigate the resistance and identify extended-spectrum beta-lactamases (ESBLs) gene in 37 S. sonnei isolates by agar dilution procedure and the modified three-dimensional test, respectively. The bla genes of ESBL-producing isolates were detected by polymerase chain reaction (PCR) and sequencing. More than 50% of these strains were resistant to tetracycline, sulfamethoxazole-trimethoprim, ampicillin, ampicillin-sulbactam, or gentamicin. However, they were still susceptible to third generation cephalosporins, fluoroquinolones, and chloramphenicol. A total of 8.1% (3/37) of the isolates with intermediate susceptibility to ceftriaxone and cefotaxime were ESBL-producers, which produced CTX-M-14 ESBLs and TEM-1 beta-lactamases. This is the first report of CTX-M-14 in S. sonnei isolates from China and it is important to closely monitor such strains.

[Identification and expression of blaCTX-M-14 and blaCTX-M-24].

OBJECTIVE: To identify the ESBL gene and the prevalence in Escherichia coli and Klebsiella pneumoniae strain isolated from Huashan Hospital, Shanghai. METHODS: Isolates were confirmed as an ESBL producing strain by double-disk synergy test and NCCLS Confirmatory Test. Antibiotic susceptibilities were determined by standard agar dilution procedure on Mueller-Hinton agar. To determine whether the resistance was transferable, the conjugation experiment was performed; plasmids were isolated from clinical isolates and transcojugants. The partial bla(gene) of ESBL producing isolates and their transcojugants were detected by PCR using universal primers for TEM, SHV, CTX-M-1group, Toho-1group, CTX-M-13group respectively. The entire bla(CTX-M-13) group were amplified by PCR using the primers outside the Open Reading Frame (ORF) of CTX-M-13group beta-lactamases; the PCR products of entire bla(CTX-M-13)group were cloned into vector and the recombinant plasmids were transformed into the recipient strain for expression; the PCR products were also directly sequenced and analyzed; the clinical isolates of ESBL producers were detected by PFGE. RESULTS: ESBL producers were resistant to most beta-lactams and non-beta-lactams. Most transconjugants were obtained at frequency of 10(-4) approximately 10(-5) and resistance to non-beta-lactams was cotransferred with the ESBL activity to the transconjugant. A plasmid of about > 23.1 kb was obtained from each tansconjugant by plasmid extraction. Partial gene amplification products of CTX-M-13 group gene were obtained from isolates and their transconjugants. The bla(CTX-M-13)group from 4 transconjugants were identified as bla(CTX-M-14), and other six were bla(CTX-M-24); those ESBLs were mediated by plasmids (> 23.1 kb); the transformants producing CTX-M-14 or CTX-M-24 were resistant to most beta-lactams, which were much more resistant to cefotaxime than to ceftazidine; PFGE patterns of those isolates were different. CONCLUSION: clinical isolate of Escherichia coli and Klebsiella pneumoniae isolated from Huashan Hospital, Shanhai produced CTX-M-14 or CTX-M-24, which caused the isolate resistant to most beta-lactams; no clone spread in those isolates was found.

A Klebsiella pneumoniae producing three kinds of class A beta-lactamases encoded by one single plasmid isolated from a patient in Huashan Hospital, Shanghai, China.

A Klebsiella pneumoniae strain was isolated from a sputum specimen of a patient in the intensive care unit in 1999 in Shanghai Huashan Hospital, China. The isolate was confirmed as an extended-spectrum beta-lactamase (ESBL) producing strain by double-disk synergy test. The results of susceptibility test showed that it was resistant to most beta-lactams (including third generation cephalosporins) and non-beta-lactam antimicrobial agents. Transconjugants were obtained at a frequency of 10(-4). A plasmid of about 60 kb was obtained from the transconjugant by plasmid extraction. Three major nitrocefin-hydrolysing bands with pIs of 5.4, 8.2 and 8.4, were shown in extracts of the transconjugant. Partial gene amplification products of bla(TEM), bla(SHV), and CTX-M-1 group gene were obtained from the isolate as well as its transconjugant. The entire bla(TEM), bla(SHV), and bla(CTX-M) in the transconjugant were amplified by PCR and the PCR products were cloned into a pHSG398 vector. Afterwards, the susceptibility of transformants and activities of beta-lactamases of transformants on antibiotics were tested. The PCR products were directly sequenced, analysed and identified as TEM-1, SHV-12, and CTX-M-3 genes. These results confirm that this strain of Klebsiella pneumoniae produces SHV-12, CTX-M-3 ESBLs and TEM-1 beta-lactamase, encoded by one single plasmid, which is responsible for the resistance of this strain to most beta-lactams.

Investigation of extended-spectrum beta-lactamase in Klebsiellae pneumoniae and Escherichia coli from China.

Extended-spectrum beta-lactamases (ESBLs) are an increasing cause of resistance to third-generation cephalosporins in Enterobacteriaceae. However, they have not been well studied in China. We investigated the prevalence, resistance, and probable gene type of ESBLs using MICs testing and polymerase chain reaction in 559 Klebsiellae pneumoniae and 427 Escherichia coli isolates collected from patients in Huashan Hospital from 1 January to 31 December 1999. The incidence of ESBL-producing strains was 51% among Klebsiellae pneumoniae (285/559) and 23.6% among Escherichia coli (101/427), most of which were collected from patients in intensive care unit and neurosurgical ward. PFGE showed that some epidemic ESBL-producing strains were present in the ICU, especially among ESBL-producing Klebsiellae pneumoniae. The major source of ESBL-producing Klebsiellae pneumoniae and Escherichia coli was sputum specimen (63.5%) and urine (64.3%), respectively. These strains were resistant to most beta-lactams (including the third-generation cephalosporins and monobactams) and non-beta-lactams (such as fluoroquinolones, aminoglycosides, tetracycline, and chloramphenicol), all or most ESBL producers were susceptible to imipenem, cefmetazole and beta-lactam/clavulanic acid. TEM was the main type of beta-lactamases and the CTX-M type of ESBLs was common in these isolates. Some ESBL-producing Escherichia coli and most ESBL-producing Klebsiellae pneumoniae produced more than one type of beta-lactamase. These data confirm that ESBL producers are common among hospital strains of Escherichia coli and Klebsiellae pneumoniae in China. It is important to monitor such strains closely and prevent their spread.

[Extended-spectrum beta-lactamase in Klebsiella pneumoniae and Escherichia coli isolates].

OBJECTIVE: To investigate the prevalence, drug resistance, gene typing, and epidemicity of extended-spectrum beta-lactamases (ESBL) Klebsiella pneumoniae and Escherichia coli isolates. METHODS: 559 strains of K. pneumoniae and 427 strains of E.coli were isolated form Huanshan Hospital from 1 January to 31 December 1999. The ESBL-producing strains were detected by double disc test and confirmed by minimal inhibitory concentration (MIC). The MIC of ESBL-producing strains was detected by agar dilution test. The beta-lactamase genes were detected by PCR. DNA fingerprinting was made by pulsed-field gel electrophoresis (PFGE). RESULTS: The incidence of ESBL-producing strains was 51% among the isolated K. pneumoniae (285/559) and 23.6% among the isolated E. coli (101/427), most of which were collected from the patients in the intensive care unit and neurosurgical ward. 63.5% of the ESBL-producing K. pneumoniae strains were collected from sputum specimens, and 64.3% of the ESBL-producing E. coli strains were collected from the urine specimens. Most ESBL-producing strains were resistant to most beta-lactam antibiotics, including the third-generation cephalosporins, and non- beta-lactam antimicrobial drugs, such as fluoroquinolones, aminoglycosides, tetracycline, and chloramphenicol. Most of the ESBL-producing strains were susceptible to imipenam, cefmetazole, and beta-lactam antibiotic/clavulanic acid. TEM type beta-lactamase was the main type among those EBSL-producing strains, followed by SHV type and CTX-M type. Some ESBL-producing E. coli and most ESBL-producing K. pneumoniae produced more than one type of beta-lactamase. CONCLUSION: ESBL-producing strains are common among hospital strains of E. coli and K. pneumoniae. Most of them are multidrug resistant. Prevalence and transmission of these strains exist in hospital.