Extended-spectrum beta-lactamase (ESBL) produced by Escherichia coli and Klebsiella pneumoniae isolated from Teikyo University Hospital--the second report

We studied the high-level resistant to cefotaxime (CTX, MIC > or = 512 micrograms/ml) clinical isolates of Escherichia coli and Klebsiella pneumoniae in Teikyo University Hospital. The CTX-resistance could be transferred to E. coli K-12 chi 1037 or ML4903 strains from 30 of the 33 isolates by conjugation at a frequency of 10(-4). When the hydrolysis rate of benzylpenicillin was 100%, the beta-lactamases which were extracted from the transconjugants hydrolyzed CTX, CAZ and AZT at the rate of 38-95%, 0-8.6% and 0-56%, respectively. These results demonstrate that these enzymes should be categorized into ESBL. The nucleotide sequence of CTX-resistant gene was identified to that of the CTX-M2 gene which was first described in Argentina. It was found to have 99.9% homology to Toho-1 gene in Japan and 99.6% homology to CMY-2 gene. Using a PCR methods for the detection of one of ESBL gene such as CTX-M2, Toho-1 or CMY-2, the DNA was amplified from all strains (11 isolates of E. coli and 21 isolates of K. pneumoniae).     

Extended-spectrum beta-lactamase (ESBL) produced by Escherichia coli and Klebsiella pneumoniae isolated from Teikyou University Hospital--the first report

We studied the cefotaxime (CTX)-resistant (MIC > or = 32 micrograms/ml) clinical isolates of E. coli and K. pneumoniae in Teikyo University Hospital from 1990 to 1996. The incidence of CTX-resistant isolates was 0.4% (6/1,282) in E. coli and 0.6% (7/1,044) in K. pneumoniae, in 1990. In 1995, the incidence of CTX-resistance increased to 1.7% (50/2,910) in E. coli (p = 0.0013) and 7.2% (144/1,996) in K. pneumoniae (p < 0.0001). These species have been detected in the stool (86 isolates), urine (59 isolates), sputum (15 isolates), pus (15 isolates), throat (10 isolates) and others (12 isolates) in 1995. MIC50 of ampicillin (ABPC), ABPC with clavlanic acid (CVA) 5 micrograms/ml, piperacillin (PIPC), PIPC with CVA 5 micrograms/ml, ceftazidime, CTX, ceftizoxime, cefpodoxime, cefepime, aztreonam, cefmetazole, latamoxef, and imipenem used against 33 isolates (11 isolates of E. coli, 22 isolates of K. pneumoniae), which were detected in 1996-1997, was > 512 micrograms/ml, 8 micrograms/ml, > 512 micrograms/ml, 8 micrograms/ml, 4 micrograms/ml, > 512 micrograms/ml, 16 micrograms/ml, > 512 micrograms/ml, 256 micrograms/ml, 32 micrograms/ml, 2 micrograms/ml, 0.25 microgram/ml and 0.25 microgram/ml, respectively. This susceptibility pattern were very similar to the Toho-1 type beta-lactamases producing strains.     

Analysis of extended-spectrum CTX-M-14 beta-lactamase (ESBLs) producing Escherichia coli isolates in the same ward over the long term

We bacteriologically and genetically analysed 30 cephalosporin-resistant Escherichia coli strains isolated from specimens from 19 neurology-ward inpatients at our hospital over the 3 years from April 2006 to March 2009, surveying and comparing subjects' backgrounds. Of the 30, 19 (63%) were urine, 6 (20%) sputum, and 3 (10%) blood. We tested extended-spectrum beta-lactamase (ESBLs) production, found in all samples. PCR and gene sequencing showed that 25 strains (83%) were CTX-M-14 and 5 (17%) CTX-M-2. Among CTX-M-14 strains, two cluster groups I and II, were obtained using pulsed-field gel electrophoresis (PFGE). Cluster group I in particular, continued to be detected for 18 months in the same hospital room. The detection rate was high at 13 (68%) in subjects with urinary catheters and morbidity was high in those with a history of cerebrovascular disease, diabetes, and hypertension. Our findings suggest that genetically identical strains may become established and spread in hospitals possibly due to inadequate contact prevention, subjects' immune status, and risk factor existence.     

Prevalence of extended-spectrum beta-lactamase and class 1 integron integrase gene intl1 in Escherichia coli from Thai patients and healthy adults

Among 120 Escherichia coli isolates from Thai patients, 37 and 9 isolates were extended-spectrum beta-lactamase (ESBL) and suspected ESBL producers respectively while 5 E. coli isolates from 120 Thai healthy adults were suspected ESBL producers. Integrase (intl1) gene was detected in 99% of the clinical and 87% of the non-clinical isolates. Among 37 ESBL producers, percent recovery of bla(TEM), bla(CTX-M), bla(SHV) and bla(VEB) was 78%, 78%, 8% and 8%, respectively. Twenty-five isolates of ESBL producers carried both bla(TEM) and bla(CTX-M), 2 isolates carried 3 genes (bla(TEM), blac(CTX-M), and bla(SHV)) and 3 showed no detectable bla gene. Among the 14 suspected ESBL producers, intl1 and bla(TEM) were detected in 13 isolates. ESBL producers from clinical samples were resistant to most of the tested antimicrobial agents compared to non-ESBL producers and isolates from healthy adults with about half of the latter susceptible to all tested antimicrobial agents. Only one clinical isolate was resistant to imipenem. Susceptibility to trimethoprim/sulfamethoxazole among the clinical isolates in ESBL producer group (27%) and non-producer group (33%) were comparable, whereas the percent susceptibility of the non-clinical isolates was about twice that of the clinical isolates.     

A case of sepsis caused by Enterobacter cloacae producing both type SHV-12 and type CTX-M-14 ESBLs in a post bone-marrow transplantation patient

Third-generation cephalosporin-resistant Enterobacter cloacae was isolated from the blood culture of a 31-year-old woman after bone-marrow transplantation. Since this strain was resistant to third-generation cephalosporins, the production of extended-spectrum beta-lactamases (ESBLs) was suspected. PCR and a sequence analysis confirmed two ESBL genes, bla(SHV-12) and bla(CTX-M-14). No bacteria were detected after meropenem was administered, and symptoms abated. This is, to our knowledge, the first report in Japan of E. cloacae clinical isolates simultaneously producing both SHV-12 and CTX-M-14 ESBL. In cases where chromosomal AmpC over-production of E. cloacae concomitantly produces ESBL, caution should be exercised due to the potential development of resistance against extended-spectrum beta-lactam agents.     

SHV-type extended-spectrum beta-lactamase (ESBL) are encoded in related plasmids from enterobacteria clinical isolates from Mexico

OBJECTIVE: In this work we report the molecular characterization of beta-lactam antibiotics resistance conferred by genes contained in plasmids from enterobacteria producing extended-spectrum beta-lactamases (ESBL). MATERIAL AND METHODS: Fourteen enterobacterial clinical isolates selected from a group of strains obtained from seven different hospitals in Mexico during 1990-1992 and 1996-1998 were analyzed at the Bacterial Resistance Laboratory (National Institute Public Health, Cuernavaca). Molecular characterization included PFGE, IEF of beta-lactamases, bacterial conjugation, PCR amplification and DNA sequencing, plasmid extraction and restriction. RESULTS: Isolates were genetically unrelated. ESBL identified were SHV-2 (5/14) and SHV-5 (9/14) type. Cephalosporin-resistance was transferable in 9 of 14 (64%) clinical isolates with only one conjugative plasmid, DNA finger printing showed a similar band pattern in plasmids. CONCLUSIONS: The dissemination of cephalosporin resistance was due to related plasmids carrying the ESBL genes.     

Prophage (phi 80) induction in Escherichia coli K-12 by specific deoxyoligonucleotides.

A cell preparation that is permeable to proteins and oligonucleotides yet produces infectious phage particles after induction treatments was obtained by plasmolysis of Escherichia coli cells lysogenic for phi 80. When the permeabilized cells were exposed to specific oligo(deoxynucleotides), prophage (phi 80) was induced during further incubation. Of the dinucleotides tested, only d(A-G), d(G-G), and d(I-G) induced prophage. The essential base sequence of the deoxydinucleotides for the induction was determined to be deoxy(purine-G). Among oligo(deoxynucleotides) with unique base composition examined, only oligo(deoxyguanylates) exhibited the inducing activity. Although this specific oligo(deoxynucleotide)-triggered induction occurred in recB- cells, the induction was not detected in recA- cells or in the cells lysogenic for induction-negative phi 80(ind-). Possible biological significance of the oligo(deoxynucleotide)-triggered prophage induction is discussed.     

Bloodstream infection due to extended-spectrum beta-lactamase (ESBL)-positive K. pneumoniae and E. coli: an analysis of the disease burden in a large cohort

Purpose

The burden of extended-spectrum beta-lactamase (ESBL)-positive Enterobacteriaceae (ESBL-E) is growing worldwide. We aimed to determine the financial disease burden attributable to ESBL-positive species in cases of bloodstream infection (BSI) due to K. pneumoniae and E. coli.

Methods

We conducted a cohort study on patients with BSI due to K. pneumoniae or E. coli between 2008 and 2011 in our institution. Data were collected on true hospital costs, length of stay (LOS), basic demographic parameters, underlying diseases as Charlson comorbidity index (CCI) and ESBL positivity of the pathogens. Multivariable regression analysis on hospital costs and length of stay was performed.

Results

Overall we found 1,851 consecutive cases of ESBL-E BSI, 352 (19.0 %) cases of K. pneumoniae BSI and 1,499 (81.0 %) cases of E. coli BSI. Sixty-six of E. coli BSI (18.8 %) and 178 of K. pneumoniae BSI (11.9 %) cases were due to ESBL-positive isolates, respectively (p = 0.001). 830 (44.8 %) cases were hospital-onset, 215 (61.1 %) of the K. pneumoniae and 615 (41.0 %) of the E. coli cases (p < 0.001). In-hospital mortality was overall 19.8, 25.0 % in K. pneumoniae cases and 18.5 % in E. coli cases (p = 0.006). Increased hospital costs and length of stay were significantly associated to BSI with ESBL-positive K. pneumoniae.

Conclusion

In contrast to BSI due to ESBL-positive E. coli, cases of ESBL-positive K. pneumoniae BSI were associated with significantly increased costs and length of stay. Infection prevention measures should differentiate between both pathogens.     

Colonic hemorrhage produced in mice by a unique vero cell cytotoxin from an Escherichia coli strain that causes hemorrhagic colitis

A Vero cell cytotoxin that produces colonic lesions and subsequent colonic hemorrhage in mice has been purified from a strain of Escherichia coli O157:H7 that causes hemorrhagic colitis in humans. This toxin is different in physicochemical properties from the Shiga-like toxin previously associated with this organism and may be responsible for the unique diffuse mucosal hemorrhage in the colon of individuals with E. coli O157:H7 infections.     

Detection of a novel variant bla(CTX-M-3) extended spectrum beta-lactamase gene in a community-acquired Escherichia coli isolate

A highly cefotaxime- and cefepime-resistant but ceftazidime-sensitive Escherichia coli isolate was recovered from a community-acquired urinary infection of a Greek patient. Susceptibility testing, transfer assays, plasmid analysis as well as PCR and sequencing techniques were used to investigate the underlying mechanism of resistance. The isolate carried a new variant of the bla(CTX-M-3) gene that possessed a T instead of A at nt position 663. Cefotaxime resistance was transferable and carried on a 60 kb plasmid. The bla(CTX-M-3) variant was located downstream of an ISEcp1B element. The emergence of this new derivative indicates further evolution of the worldwide-distributed bla(CTX-M-3) gene.     

A chronic myeloid leukemia-like syndrome case with del (12) (p12) in a Li-Fraumeni syndrome family

Li-Fraumeni syndrome is a familial cancer syndrome characterized by different tumors and hereditary p53 mutations. Here, a chronic myeloid leukemia-like syndrome case in a Li-Fraumeni syndrome family with del (12) (p12) cytogenetic abnormality was presented. A hereditary p53 mutation (pro309ser) supported the Li-Fraumeni syndrome diagnosis in this family. This syndrome was characterized by the clonal myeloproliferative accumulation in bone marrow and peripheral blood with negative bcr/abl gene rearrangement finding. The etiology of this rare syndrome is still unclear. This is the only chronic myeloid leukemia-like syndrome case reported in a Li-Fraumeni syndrome family. Del (12)(p12) was observed in leukemias except chronic myeloid leukemia-like syndrome. The deletion in chromosome 12p12 with hereditary p53 mutation should have a critical role in chronic myeloid leukemia-like syndrome etiology in our case.     

Detection of rabies-specific antigens by egg yolk antibody (IgY) to the recombinant rabies virus proteins produced in Escherichia coli

We obtained rabies-specific egg yolk antibodies (IgY) by immunizing hens with recombinant His-tagged nucleoprotein and phosphoprotein (rN, rP) of the rabies virus (CVS-11 strain) expressed in Escherichia coli. The anti-rN and rP IgY were shown to bind specifically to the respective proteins of the CVS-11 strain of rabies virus by Western blotting, immune fluorescent assay and immunohistochemistry, indicating that IgY to rabies recombinant proteins could serve as a reagent for diagnosis of rabies virus infection.     

A case of fulminant amoebic colitis with an abscess in the abdominal cavity rescued by conservative management

A 75-year-old man was admitted because of watery diarrhea, hematochezia and right lower abdominal pain. Many deep undermining colonic ulcers were found by colonoscopy, and we detected trophozoite amoeba pathologically. Metronidazole was administered orally from 3 days after admission. However, since CT demonstrated a huge abscess in the abdominal cavity, we performed percutaneous drainage from 17 days after admission. On day 157, the patient was discharged, because the colonic ulcers had almost healed, and trophozoite amoebas were not recognized pathologically.     

Intervention with Shiga toxin (Stx) antibody after infection by Stx-producing Escherichia coli

Shiga toxins (Stxs) produced by Escherichia coli (STEC) cause systemic vascular damage, manifested as hemolytic uremic syndrome in humans and as edema disease in pigs. Edema disease, a naturally occurring disease of pigs, was used to determine whether Stx antibodies, administered after infection and after the onset of Stx production, could prevent the systemic vascular damage and clinical disease caused by Stxs. A total of 119 STEC-infected pigs were treated with low, medium, or high doses of Stx antibody or with placebo. After inoculation with STEC, antibodies or placebo was injected intraperitoneally at 2 days postinoculation (DPI; low dose) or 4 DPI (medium and high doses). Edema disease was prevented with the low- and high-dose Stx antibody treatments administered at 2 and 4 DPI, respectively. High-dose antibody treatment also reduced the incidence and extent of vascular lesions. The degree of protection depended on the dose of antibody and the time of administration.     

Isolation of enteroaggregative escherichia coli (EAggEC) from patients with diarrheal disease in an outbreak case of overseas tour

Twenty four patients out of 78 Cambodia tourists (31%) suffered from diarrhea and/or abdominal pain. Though the stools of 20 patients were examined in some local health centers and institutes, well-known pathogens were detected in only a low level and the cause of the outbreak remained unclear. We suspected the enteroaggregative Escherichia coli (EAggEC) as a cause of this outbreak. We examined E. coli strains isolated from stools of 8 patients (Okayama:7, Aichi:1) at first by the PCR method targeted both the aggR and the astA genes related to the virulence factors of EAggEC. As a result, the E. coli strains with positive aggR and/or astA genes were isolated from 8 patients. And the E. coli strains with positive both aggR gene and clump formation isolated from 3 patients adhered aggregatively to HEp-2 cells and accordingly identified as EAggEC. The plasmid profiles, PFGE patterns and drug resistance patterns of these EAggECs agreed completely. From these results, we concluded that at least 3 patients were infected with EAggEC of the same origin. Though we could not examine all samples from 20 patients, it is possible that the still uncommon EAggEC might be a cause of the outbreak. The E. coli strains with positive aggR gene did not always aggregatively adhered to HEp-2 cells. So we recommend to perform stepwise EAggEC screening tests by the PCR and the clump formation, and final confirmation test by the aggregative adhesion to HEp-2 cells.