Dissemination of an extended spectrum beta-lactamase producing Klebsiella pneumoniae strains in a Tunisian university hospital center

On a period of four years (january 1999-december 2002), 49 strains (non redundant) of extended spectrum beta-lactamase-producing Klebsiella pneumoniae from 43 hospitalised patients and three strains from the environment hospital were collected at Mongi Slim University Hospital Center. The objectives of our work were to investigate for clonality of strains, to clarify transmission fashions and reservoirs of the infection by reviewing clinical records and typing strains. Antibiotic susceptibility testing, plasmid analysis and Random Amplified Polymorphic DNA (RAPD) have been done. 84% of the patients were hospitalized in the intensive care and pediatric units. Urinary infections and septicaemias were the more frequent infections (74.2%). The 49 isolats have been classified in 13 antibiotypes. The plasmid analysis showed 16 patterns. The RAPD revealed 28 patterns and variation within patterns in five cases. Our results showed a diversity of the strains suggesting endemicity, possible transmission of plasmids and persistence of some clones which circulated between services. The used markers permitted the evaluation of a long-term strategy of prevention requiring a strict observance of hygiene rules and rational use of antibiotics.     

VIM-1 Metallo-beta-lactamase-producing Klebsiella pneumoniae strains in Greek hospitals

Seventeen Klebsiella pneumoniae clinical isolates carrying the bla(VIM-1) metallo-beta-lactamase gene were collected in the intensive care units of three hospitals in Athens, Greece, in 2002. They exhibited various carbapenem resistance levels (Etest MICs of imipenem ranged from 4 to 32 microg/ml). All isolates gave positive results by the imipenem-EDTA synergy Etest. The isolates were classified into four main types by pulsed-field gel electrophoresis; the majority of the isolates (5 and 10 isolates) belonged to two types. The bla(VIM-1) gene cassette was part of the variable region of a class 1 integron that also included aac6, dhfrI, and aadA. This structure was carried by transferable plasmids.     

Spread of integron-associated VIM-type metallo-beta-lactamase genes among imipenem-nonsusceptible Pseudomonas aeruginosa strains in Greek hospitals

Fifty-eight imipenem-nonsusceptible (MIC >or= 8 microg/ml) Pseudomonas aeruginosa strains isolated during May 2001 in 15 Greek hospitals were studied. Thirty-six isolates derived from nine hospitals carried VIM-type metallo-beta-lactamase genes, as found by PCR. In 34 isolates, bla(VIM) was associated with class 1 integrons of various sizes. DNA sequencing indicated the presence of bla(VIM-2) gene cassettes in a variety of integron structures. Random amplified polymorphic DNA typing suggested diversity of the bla(VIM)-positive strains. Synergy between 2-mercaptoacetic acid and imipenem indicated carbapenemase activity in 26 bla(VIM)-positive strains.     

Molecular characterization of nosocomial CTX-M type beta-lactamase producing Enterobacteriaceae from a tertiary care hospital in south India

CTX-M group of extended spectrum beta lactamases (ESBLs) represents a rapidly emerging problem in many countries. The prevalence of nosocomial bla CTX-M-1 producing Enterobacteriaceae strains has not been reported earlier in Indian hospitals. This study describes molecular subtyping of nosocomial bla CTX-M producing strains of Enterobacteriaceae . Polymerase chain reaction with primers specific for bla CTX-M-1 coding genes was used to identify 95 Enterobacteriaceae strains producing bla CTX-M positive isolates. Of the 95 bla CTX-M producing isolates, 45 strains were positive for bla CTX-M-1 . bla CTX-M-1 was found to be most prevalent in Klebsiella strains.     

Application of minimal sequence quality values prevents misidentification of the blaSHV type in single bacterial isolates carrying different SHV extended-spectrum beta-lactamase genes

Nucleotide sequencing is the standard molecular method for determination of the beta-lactamase gene present in an isolate. Using minimal sequence quality values prevents misidentification of bla(SHV) genes, as illustrated by three strains of three different species that each contained two different bla(SHV) alleles, SHV-2 and SHV-12.     

Dissemination of transferable AmpC-type beta-lactamase (CMY-10) in a Korean hospital

To determine dissemination and genotype of AmpC beta-lactamases and an extended-spectrum beta-lactamase among clinical isolates of Enterobacteriaceae, we performed antibiotic susceptibility testing, pI determination, induction test, plasmid profiles, transconjugation test, enterobacterial repetitive consensus (ERIC)-PCR, and DNA sequencing. Among the 51 clinical isolates collected from a university hospital in Korea, six isolates were resistant to cephamycins. All six isolates produced a plasmid-encoded AmpC-type beta-lactamase, CMY-10. Five strains also produced one or more other beta-lactamases: SHV-12, an extended-spectrum beta-lactamase (five isolates); TEM-1, a class A beta-lactamase (two isolates); and a chromosomal AmpC beta-lactamase (one isolate, a strain of Enterobacter aerogenes, which produced all four of the beta-lactamases that were identified). One of six isolates produced only CMY-10. ERIC-PCR analysis revealed that dissemination of CMY-10 and SHV-12 was due to a clonal outbreak of a resistant strain and to the interspecies spread of resistance to cephamycins and broad-spectrum beta-lactams in Korea. CMY-10 beta-lactamase genes that are responsible for the resistance to cephamycins (cefoxitin and cefotetan), amoxicillin, cephalothin, and amoxicillin-clavulanic acid were cloned and characterized from six clinical isolates. A sequence identical to the common regions in In6, In7, and a novel integron from pSAL-1 was found upstream from blaCMY-10 gene at nucleotides 1-71. A total of 15 nucleotides (I-15) or 18 nucleotides (I-18) between position 71 and 72 were inserted into the blaCMY-10 gene. The blaCMY-10 gene might be inserted into a sul1-type complex integron by I-15 or I-18.     

对亚胺培南耐药粘质沙雷菌中质粒介导KPC-2型碳青霉烯酶的研究分析

目的研究粘质沙雷菌对亚胺培南耐药的分子机制。方法临床分离到1株亚胺培南耐药的粘质沙雷菌[最小抑菌浓度（MIC）为64μg／m1]。将粘质沙雷菌和大肠杆菌进行接合试验，采用琼脂稀释法检测粘质沙雷菌和接合前后大肠杆菌对药物的MIC；提取粘质沙雷菌和接合后大肠杆菌的酶粗提液进行等电聚焦电泳和三维试验；特异性PCR扩增和DNA序列分析确认引起粘质沙雷菌对亚胺培南耐药的β-内酰胺酶的基因型。结果除碳青霉烯类抗生素外，粘质沙雷菌还对青霉素类、头孢菌素类和单酰胺类等抗生素耐药，对喹诺酮类和氨基糖甙类抗生素敏感；接合试验可使大肠杆菌获得与粘质沙雷菌相似的耐药谱；等电聚焦电泳显示粘质沙雷菌中存在等电点（P1）分别为6．5和6．7的两种β内酰胺酶，接合后的大肠杆菌存在PI为6．7的β-内酰胺酶；特异性PCR扩增和DNA序列分析证实PI为6．7的β-内酰胺酶为碳青霉烯酶KPC-2，其核苷酸及氨基酸序列已递交到GenBank，PI为6．5的酶有待进一步研究；在三维试验中，克拉维酸、乙二胺四乙酸（EDTA）和氯唑西林均不能抑制KPG2酶对亚胺培南的水解活性。结论首次在粘质沙雷菌中发现碳青霉烯酶KPG2，该酶是引起粘质沙雷菌对亚胺培南耐药的主要原因。     

Plasmid-related beta-lactamase production in Bacteroides fragilis strains

Twenty Bacteroides fragilis group species isolated from children with and without diarrhea were analyzed. Antibiotic susceptibility was performed using an agar dilution method; beta-lactamase production was determined using a nitrocefin method, and plasmids were extracted using a commercial Miniprep System. MIC values ranged from 16 to 256 microg/ml for penicillin, 4-128 microg/ml for amoxicillin/clavulanic acid, 0.25-256 microg/ml for clindamycin, and 16-256 microg/ml for penicillin. beta-Lactamase was detected in all isolates. Only five isolates harbored plasmids varying from 7.8 to 1.8 kb. Loss of 6.4- and 3.8-kb plasmids in B. fragilis C68c was related to antibiotic resistance. Low molecular weight plasmids of 2.8-1.8 kb were stable. PCR amplification of cfiA and cepA genes was observed using total DNA, and the cfiA gene was also amplified from the 6.4-kb plasmid.     

头孢西丁诱导产ACT-1型AmpC酶大肠埃希菌的比较蛋白质组学研究

目的 研究产AmpC酶大肠埃希菌在头孢西丁诱导前后的蛋白质谱差异,筛选有价值的靶位.方法 提取产ACT-1 AmpC酶大肠埃希菌在头孢西丁诱导前后的全菌蛋白,应用双向凝胶电泳技术分析和Blue silver法染色.凝胶图像分析后,对差异蛋白质进行MALDI-TOF质谱分析.结果 在所鉴定的8个差异蛋白当中,AmpC酶、外膜蛋白Ⅱ和转座酶表达显著上调,而磷酸转移酶系统的葡萄糖特异性组分ⅡA蛋白、抗碲酸盐蛋白、丙三醇磷酰基二酯酶、硫醇过氧化物酶及细菌分化蛋白FtsZ表达下调.结论 所鉴定的这些差异蛋白将为阐明产AmpC酶大肠埃希菌耐药产生的机制提供线索,也为筛选具有潜在价值的靶位提供理论依据.     

头孢西丁诱导产ACT-1型AmpC酶大肠埃希菌的比较蛋白质组学研究

目的研究产AmpC酶大肠埃希菌在头孢西丁诱导前后的蛋白质谱差异,筛选有价值的靶位。方法提取产ACT-1AmpC酶大肠埃希菌在头孢西丁诱导前后的全菌蛋白,应用双向凝胶电泳技术分析和Blue silver法染色。凝胶图像分析后,对差异蛋白质进行MALDI-TOF质谱分析。结果在所鉴定的8个差异蛋白当中,AmpC酶、外膜蛋白II和转座酶表达显著上调,而磷酸转移酶系统的葡萄糖特异性组分IIA蛋白、抗碲酸盐蛋白、丙三醇磷酰基二酯酶、硫醇过氧化物酶及细菌分化蛋白FtsZ表达下调。结论所鉴定的这些差异蛋白将为阐明产AmpC酶大肠埃希菌耐药产生的机制提供线索,也为筛选具有潜在价值的靶位提供理论依据。     

Identification of a red-pigmented bacterium producing a potent anti-tumor N-alkylated prodigiosin as Serratia marcescens

A bacterial strain producing a novel prodigiosin analogue 2,2'-[3-methoxy-1'amyl-5'-methyl-4-(1'-pyrryl)] dipyrrylmethene (MAMPDM) possessing potent cytotoxic activity towards cancer cells was isolated and identified. The bacterial cells were spherical and occurred singly, and some of the biochemical tests matched with Micrococcus. Therefore, the isolate was earlier tentatively reported to be Micrococcus sp. In the present studies, analytical profile index (API) suggested this organism to be Klebsiella. However, Klebsiella is not known to produce the red pigment prodigiosin, which is produced by Serratia species and some other bacteria. Based on other biochemical characteristics, particularly DNase, gelatinase, lipase, ornithine decarboxylase, presence of a cell-associated N-alkylated prodigiosin (MAMPDM) and organic solvent tolerance, the strain has now been identified as a variant of Serratia marcescens. 16S rRNA gene analysis conclusively established this organism as S. marcescens ost3. The red pigment (MAMPDM) of this organism showed selective cytotoxic activity in cancer cell lines of different origin (LS-A and U937) and reduced toxicity to non-malignant cells. The LC50 of MAMPDM was 1.59 microM and 0.176 microM for U937 and LS-A cells, respectively, while there was no effect on the viability of L929, a non-malignant cell line, at these concentrations. Thus, S. marcescens ost3 may serve as a source of a new anti-cancer compound.     

A fosfomycin-gentamicin combination in the treatment of experimental endocarditis caused by Klebsiella pneumoniae producing type TEM-3 beta-lactamase

The authors studied the activity of fosfomycin (FOS) and/or gentamicin (GEN) against a Klebsiella pneumoniae strain resistant to all beta-lactams--except cephamycins and imipenem--by production of a plasmid mediated extended broad-spectrum beta-lactamase-TEM-3, to all aminoglycosides--except gentamicin--by production of a plasmid mediated 6' aminoglycoside acetyltransferase IV, to sulfonamides and to tetracyclines. In vitro, the combination FOS (MIC = MBC = 32 mg/l) + GEN (MIC = MBC = 2) appeared indifferent (FIC = 0.75; FBC = 1). In vivo, on experimental endocarditis in rabbits, FOS alone was ineffective, GEN alone was active but only at high dose regimen, FOS - GEN combination was active as compared with controls. Fosfomycin - gentamicin combination may be an alternative in the therapy of severe infections due to multiresistant Enterobacteriacae.     

Molecular epidemiology of Klebsiella pneumoniae strains that produce SHV-4 beta-lactamase and which were isolated in 14 French hospitals.

Preliminary results suggested that the diffusion in France of the SHV-4 extended-spectrum beta-lactamase was probably due to the spread of one single epidemic strain of Klebsiella pneumoniae. In this study, we tested various phenotypic and genotypic markers to compare K. pneumoniae strains producing this enzyme isolated in 14 French hospitals between 1987 and 1989. All of the strains were of the same capsule serotype, K25. Twelve of them were of the same biotype: weak urease activity and no sucrose fermentation. Among the six plasmid profiles observed, one accounted for eight strains. Large plasmids of 170 kb encoding SHV-4 beta-lactamase were present in all strains of K. pneumoniae and could be transferred by conjugation with high frequency to Escherichia coli J53-2 or HB101 from all except one strain. Plasmid EcoRI restriction patterns suggested that these plasmids were closely related and similar to pUD18 encoding SHV-3 beta-lactamase, originally described in France and differing from SHV-4 by one amino acid substitution. Ribotyping with EcoRI and HindIII and genomic fingerprinting with XbaI by pulsed-field gel electrophoresis were concordant and suggested that 12 of the isolates recovered from the 14 hospitals were probably the same strain. Dissemination in France of the SHV-4 extended-spectrum beta-lactamase was thus essentially due to the diffusion of a single K. pneumoniae clone.     

Dissemination of Clonally Unrelated Erythromycin- and Glycopeptide-Resistant Enterococcus faecium Isolates in a Tertiary Greek Hospital

Between September 1999 to February 2001, 25 glycopeptide-resistant Enterococcus faecium (GRE) isolates were recovered from a Greek hospital. The isolates exhibited 13 distinct chromosomal macrorestriction types by pulsed-field gel electrophoresis, and all were erythromycin and vancomycin resistant, carrying the genes vanA and ermB. Vancomycin resistance, always linked with erythromycin resistance, was transferable from 17 isolates. The dissemination of erythromycin-resistant GRE strains may, at least in part, reflect the extensive use of macrolides in husbandry in Greece.     

Methicillin-resistant Staphylococcus aureus strains in New York City hospitals: inter-hospital spread of resistant strains of type 88.

A survey of methicillin-resistant strains of Staphylococcus aureus received for phage typing indicated a marked increase of resistant strains received in 1982 and 1983. Of 62 hospitals in New York City which sent strains for phage typing, 35 had methicillin-resistant isolates. A significant development was the presence of strains of the same phage type at several hospitals, indicating a possible inter-hospital spread of these strains. Among strains present at several hospitals, the largest group was of experimental phage type 88. Strains of type 88 were received from 23 hospitals, representing 56% of all methicillin-resistant strains received from New York City hospitals. Strains of type 88 were resistant to all antistaphylococcal antibiotics, with the exception of vancomycin, and represented a major source of nosocomial infections at 13 hospitals. As experimental phage 88 is not routinely used for typing in U.S. laboratories, the nationwide distribution of strains of type 88 is difficult to assess.