Διερεύνηση νέων β-λακταμασών σε στελέχη gram-αρνητικών βακτηρίων στην κοινότητα

The aim of the study was to investigate the dissemination in the community of carbapenem-resistant gram-negative bacteria and the mechanisms of acquired resistance.Patients who were referred to the outpatient department of Serres General Hospital with community-onset infections due to carbapenem-resistant gram-negative bacteria during a 3 year period (2005-2008) were included in this study. The selected isolates were tested by determination of agar dilution MICs, phenotypic carbapenemase testing and molecular typing methods. PCR and sequencing analyses were employed for identification of bla genes and mapping of the integron carrying the metallo-β-lactamase (MBL) gene. The location of the MBL allele was investigated by mating experiments, plasmid analysis, and hybridization of the Southern-blotted plasmid extract with a blaVIM probe. The demographic and clinical characteristics of the outpatients were prospectively collected.During the study period 12 Proteus mirabilis, 97 Pseudomonas aeruginosa and 24 Klebsiella pneumoniae isolates with reduced susceptibility to imipenem and/or meropenem were recovered from urinary tract infections of 12, 45 and 12 outpatients, respectively. As many as 64 of the outpatients had a history of previous hospitalization or visit to the healthcare facilities in the preceding year while the remaining 5 outpatients with urinary tract infections due to P. aeruginosa carbapenem-resistant isolates had not been hospital admission in the preceding year.In 18 outpatients infected with P. aeruginosa and 6 outpatients infected with K. pneumoniae the carbapenem-resistant organisms caused recurrent community-onset infections, while in three outpatients P. aeruginosa isolates were also implicated in community-onset bacteremic episodes. Diabetes mellitus, prostatic hyperplasia and infection with an MBL-producing strain during a previous hospitalization were significantly associated with recurrent infections in the community setting.Recurrent infections were not detected among patients infected with MBL-producing P. mirabilis isolates. Among P. mirabilis isolates imipenem, meropenem and ertapenem MICs ranged from 32 to >128 mg/L, 1 to 8 mg/L and 0.5 to 4 mg/L, respectively. The isolates originated from the same clonal strain and harbored a blaVIM-1 gene in a common integron structure. Conjugation experiments, plasmid analysis and hybridization assays indicated the chromosomal location of blaVIM-1 gene. All 45 single-patient P. aeruginosa isolates harbored the blaVIM-2 MBL gene in a common class 1 integron structure. They belonged to one predominant pulsed-field gel electrophoresis type and three sporadically detected types; two of the sporadic clonal types were identified among outpatients without previous exposure to health care facilities, while the predominant clonal type was also identified to cause infections in hospitalized patient. The integron carrying the MBL gene was located on the bacterial chromosome.For the K. pneumoniae isolates imipenem, meropenem and ertapenem MICs ranged from 8 to 64mg/L, 4 to 32mg/L and 8 to 128mg/L, respectively. All studied isolates as well as those two recovered during previous hospitalization belonged to a single PFGE clone. They harbored a plasmid-mediated blaVIM-1 gene in an integron structure that has been previously described among clinical isolates from Greek hospitals.This is the first study to document the dissemination of MBL-producing P. mirabilis, P. aeruginosa and K. pneumoniae isolates in the community. Present clinical and molecular data provided evidence that MBL-producing strains could easily disseminated in the community from patients colonized during a previous hospitalization. Increased awareness and intensified infection control practices in the hospital as well as the community setting are the keys to curtailing the spread of these alarming carbapenem resistant pathogens

Evaluation of a New Phenotypic OXA-48 Disk Test for Differentiation of OXA-48 Carbapenemase-Producing Enterobacteriaceae Clinical Isolates.

The current phenotypic methods for detecting carbapenemase-producing Enterobacteriaceae (CPE) allow differentiation between class A and B carbapenemases, but they cannot confirm in a single test class D OXA-48 carbapenemase producers. In this study, we evaluated a new phenotypic test, the OXA-48 disk test, which is based on an imipenem disk and two blank disks adjacent to the imipenem disk, loaded with the tested strain and impregnated with EDTA and EDTA plus phenyl boronic acid (PBA), respectively. The evaluation of the OXA-48 disk test was performed with 81 genotypically confirmed OXA-48-type-producing Enterobacteriaceae isolates (41 extended-spectrum β-lactamase [ESBL] producers, 3 AmpC producers, and 37 non-ESBL, non-AmpC producers). To measure the specificity of the test, 173 genotypically confirmed OXA-48-negative Enterobacteriaceae isolates (57 Klebsiella pneumoniae carbapenemase [KPC] producers, 34 VIM producers, 23 KPC/VIM producers, 22 NDM producers, and 37 AmpC or ESBL producers and porin deficient) that were nonsusceptible to at least one carbapenem were chosen for testing. Using the imipenem disk and the distortion of the inhibition halo around both blank disks containing EDTA and EDTA/PBA, the test differentiated all but 3 of the 81 OXA-48 producers (sensitivity of 96.3%). The test was negative for OXA-48 production in all but 4 of the 173 carbapenem-nonsusceptible isolates producing other carbapenemases, AmpCs, or ESBLs (specificity of 97.7%). This evaluation shows that the OXA-48 disk test is an accurate phenotypic method for the direct differentiation of OXA-48-producing Enterobacteriaceae. Its use along with combined disk tests employing inhibitor-supplemented carbapenem disks might allow the differentiation of the currently known carbapenemase types in Enterobacteriaceae species and provide important infection control information

Characteristics of Meropenem Heteroresistance in Klebsiella pneumoniae Carbapenemase (KPC)-Producing Clinical Isolates of K. pneumoniae▿

Meropenem heteroresistance was investigated in six apparently meropenem-susceptible, Klebsiella pneumoniae carbapenemase (KPC)-producing K. pneumoniae (KPC-KP) clinical isolates, compared with that in carbapenemase-negative, meropenem-susceptible controls. In population analyses, the KPC-KP isolates grew at meropenem concentrations of 64 to 256 μg/ml. Heteroresistant colonies had significantly elevated expression of the blaKPC gene compared with the native populations but did not retain heteroresistance when subcultured in drug-free media. Time-kill assays indicated that meropenem alone was not bactericidal against KPC-KP but efficiently killed the control strains

Large Dissemination of VIM-2-Metallo-β-Lactamase-Producing Pseudomonas aeruginosa Strains Causing Health Care-Associated Community-Onset Infections ▿

During a 3-year period (May 2005 to April 2008), a series of 45 outpatients presented with community-onset urinary tract infections due to carbapenem-resistant Pseudomonas aeruginosa isolates. Forty of them had a history of previous hospitalization or exposure to healthcare facilities, while the remaining five had not been previously admitted to our healthcare facilities or elsewhere within the preceding 12 months. In 18 outpatients, the carbapenem-resistant organisms caused recurrent community-onset urinary tract infections, while in three outpatients the organisms were also implicated in bacteremic episodes. All 45 single-patient P. aeruginosa isolates harbored the blaVIM-2 metallo-β-lactamase (MBL) gene in a common class 1 integron structure. They belonged to one predominant pulsed-field gel electrophoresis type and three sporadically detected types; two of the sporadic clonal types were identified among outpatients without previous exposure to healthcare facilities, while the predominant clonal type was also identified to cause infections in hospitalized patients. This is the first study documenting that MBL-producing P. aeruginosa isolates cause community-onset infections that are related or not with exposure to healthcare facilities. Community-onset infections in our patients most likely resulted from the nosocomial acquisition of MBL producers, followed by a prolonged digestive carriage. The high rate of recurrent infections in the community underlies the difficulty of constraining infections caused by such microorganisms in the extrahospital setting

A Combined Disk Test for Direct Differentiation of Carbapenemase-Producing Enterobacteriaceae in Surveillance Rectal Swabs

Carbapenemase-producing Enterobacteriaceae (CPE) are rapidly spreading worldwide. Early detection of fecal CPE carriers is essential for effective infection control. Here, we evaluated the performance of a meropenem combined disk test (CDT) for rapidly differentiating CPE isolates directly from rectal swabs. The screening method was applied for 189 rectal swabs from hospitalized patients at high risk for CPE carriage. Swabs were suspended in 1 ml saline and cultured for confluent growth onto a MacConkey agar plate with a meropenem (MER) disk alone, a MER disk plus phenyl boronic acid (PBA), a MER disk plus EDTA, and a MER disk plus PBA and EDTA. An inhibition zone of <= 25 mm around the MER disk alone indicated carriage of carbapenem-resistant organisms. Furthermore, >= 5-mm differences in the inhibition zone between MER disks without and with the inhibitors (PBA, EDTA, or both) were considered positive results for detecting Klebsiella pneumoniae carbapenemase (KPC), metallo-beta-lactamase (MBL), or both carbapenemases, respectively. For comparison, rectal suspensions were tested using MacConkey plates with ertapenem (MacERT) disks and PCR (PCR-S) for carbapenemase genes. Of the 189 samples, 97 were genotypically confirmed as CPE positive by one of the three protocols tested. The CDT, MacERT disks, and PCR-S assays exhibited sensitivities of 94.8%, 96.9%, and 94.8% and specificities of 100%, 98.9%, and 100%, respectively, for detecting CPE-positive swabs. Moreover, the CDT correctly differentiated the production of KPC, MBL, or both carbapenemases in 78 of the 97 (80.4%) CPE-positive rectal swabs. Our results demonstrate that the CDT may provide a simple and inexpensive method for detecting and differentiating the carbapenemase type within a single day without requiring further testing and additional delay, supporting the timely implementation of infection control measures

In Vivo Acquisition of a Plasmid-Mediated blaKPC-2 Gene among Clonal Isolates of Serratia marcescens▿

Three patients admitted to a Greek hospital were infected with Serratia marcescens isolates that exhibited reduced susceptibility to carbapenems and harbored Klebsiella pneumoniae carbapenemase (KPC) enzymes. In two of these cases, the patients were initially infected by carbapenem-susceptible S. marcescens isolates. Molecular typing and plasmid analysis suggested that all three patients had clonally indistinguishable isolates of S. marcescens that acquired a plasmid-mediated blaKPC-2 gene during the hospitalization

Evaluation of Boronic Acid Disk Tests for Differentiating KPC-Possessing Klebsiella pneumoniae Isolates in the Clinical Laboratory▿

The worldwide increase in the occurrence and dissemination of KPC β-lactamases among gram-negative pathogens makes critical the early detection of these enzymes. Boronic acid disk tests using different antibiotic substrates were evaluated for detection of KPC-possessing Klebsiella pneumoniae isolates. A total of 57 genotypically confirmed KPC-possessing K. pneumoniae isolates with varying carbapenem MICs were examined. To measure the specificity of the tests, 106 non-KPC-possessing isolates (89 K. pneumoniae and 17 Escherichia coli isolates) were randomly selected among those exhibiting reduced susceptibility to cefoxitin, expanded-spectrum cephalosporins, or carbapenems. As many as 56, 53, and 40 of the non-KPC-possessing isolates harbored extended-spectrum β-lactamases, metallo-β-lactamases, and plasmid-mediated AmpC β-lactamases, respectively. By use of CLSI methodology and disks containing imipenem, meropenem, or cefepime, either alone or in combination with 400 μg of boronic acid, all 57 KPC producers gave positive results (sensitivity, 100%) whereas all 106 non-KPC producers were negative (specificity, 100%). The meropenem duplicate disk with or without boronic acid demonstrated the largest differences in inhibition zone diameters between KPC producers and non-KPC producers. By use of disks containing ertapenem, all isolates were correctly differentiated except for five AmpC producers that gave false-positive results (sensitivity, 100%; specificity, 95.3%). These practical and simple boronic acid disk tests promise to be very helpful for the accurate differentiation of KPC-possessing K. pneumoniae isolates, even in regions where different broad-spectrum β-lactamases are widespread

Use of Boronic Acid Disk Tests To Detect Extended- Spectrum β-Lactamases in Clinical Isolates of KPC Carbapenemase-Possessing Enterobacteriaceae▿

We evaluated boronic acid (BA)-based methods for their ability to detect extended-spectrum β-lactamases (ESBLs) among clinical isolates of KPC-producing members of the Enterobacteriaceae family. A total of 155 isolates of Klebsiella pneumoniae (n = 141), Escherichia coli (n = 6), Enterobacter aerogenes (n = 6), and Klebsiella oxytoca (n = 2) genotypically confirmed to be KPC producers were analyzed. As many as 118 isolates harbored ESBLs (103 harbored SHV-type ESBLs, 13 harbored CTX-M-type ESBLs, and 2 harbored both SHV- and CTX-M-type ESBLs); the remaining 37 isolates were genotypically negative for ESBL production. The CLSI ESBL confirmatory test was positive for 79 of the 118 ESBL producers (sensitivity, 66.9%), while all 37 non-ESBL producers were negative (specificity, 100%). When a ≥5-mm increase in the zone diameter of either the cefotaxime (CTX)-clavulanate (CA) or the ceftazidime (CAZ)-CA disks containing BA compared with the zone diameter of the CTX or CAZ disks containing BA was considered to be a positive result for ESBL production, the method detected all 118 ESBL producers (sensitivity, 100%) and showed no false-positive results for non-ESBL producers (specificity, 100%). Double-disk synergy tests, in which disks of CTX, CAZ, aztreonam, or cefepime in combination with BA were placed at distances of 20, 25, and 30 mm (center to center) from a disk containing amoxicillin (amoxicilline)-clavulanate-BA, were able to detect 116 (98.3%), 101 (85.6%), and 28 (23.7%) of the ESBL-positive isolates, respectively; no false-positive results for non-ESBL-producing isolates were detected. Our results demonstrate that the modified CLSI ESBL confirmatory test with antibiotic disks containing BA is the most accurate phenotypic method for the detection of ESBLs in Enterobacteriaceae producing KPC carbapenemases