Characterization of Extensively Drug-Resistant or Pandrug-Resistant Sequence Type 147 and 101 OXA-48-Producing Klebsiella pneumoniae Causing Bloodstream Infections in Patients in an Intensive Care Unit.

Carbapenem-resistant Klebsiella pneumoniae causes important health care-associated infections worldwide. An outbreak of sequence type 11 (ST11) OXA-48-producing K. pneumoniae (OXA-48-Kp) isolates occurred in Tzaneio Hospital in 2012 and was contained until 2014, when OXA-48-Kp reemerged. The present study involved 19 bloodstream infection (BSI) OXA-48-Kp isolates recovered from 19 intensive care unit (ICU) patients hospitalized between August 2014 and July 2016. MICs were determined by broth microdilution. Beta-lactamase genes were detected by PCR. All isolates were typed by pulsed-field gel electrophoresis/multilocus sequence typing (PFGE/MLST), and 10 representative isolates were typed by next-generation sequencing (NGS). Of the 19 study patients, 9 had previous hospitalizations, and 10 carried OXA-48-Kp prior to BSI isolation; median time from ICU admission to BSI was 29 days. Four OXA-48-Kp isolates belonged to PFGE profile A (ST147) and were pandrug resistant (PDR), while 15 isolates exhibited PFGE profile B (ST101) and were extensively drug resistant. Genes detected via NGS resistome analysis accounted for most of the resistance phenotypes, except for tigecycline and fosfomycin. Insertional inactivation of mgrB (distinct per clone) conferred colistin resistance in all 19 isolates. NGS single nucleotide polymorphism (SNP) analysis validated the clonal relatedness of the ST147 and ST101 strains and revealed the possible presence of two index ST147 strains and the microevolution of ST101 strains. Distinct, but highly related, IncL OXA-48-encoding plasmid lineages were identified; plasmids of the ST147 strains were identical with the plasmid of ST11 OXA-48-Kp which caused the 2012 outbreak. In conclusion, biclonal circulation of OXA-48-Kp and, alarmingly, emergence of a PDR clone are reported. These observations, along with the challenging phenotypic detection of OXA-48 producers and the high reported transmissibility of blaOXA-48, necessitate intensive efforts to prevent their further spread.

Performance of the ß LACTA™ test for rapid detection of expanded-spectrum cephalosporin-non-susceptible Enterobacteriaceae.

OBJECTIVES: The aim of this study was to examine the performance of the chromogenic ß LACTA™ test for the rapid detection of expanded-spectrum cephalosporin (ESC) non-susceptibility among Enterobacteriaceae in a region endemic for potent ß-lactamases. METHODS: The ß LACTA™ test was applied prospectively on 235 consecutive Enterobacteriaceae clinical isolates and 163 previously characterised ESC-non-susceptible Enterobacteriaceae producing a range of ß-lactamases. RESULTS: The ß LACTA™ test exhibited excellent sensitivity (96.1%) and specificity (98.5%) for the detection of ESC non-susceptibility in the 235 clinical isolates, which harboured mainly extended-spectrum ß-lactamase (ESBL) and KPC- and NDM-type enzymes. Among the 163 challenged archived isolates, some false-negative or uninterpretable results were detected, mostly among ESC-non-susceptible isolates with AmpC/VIM/OXA-48-like enzymes. CONCLUSION: ß LACTA™ may be effectively applied in regions where ESC non-susceptibility among Enterobacteriaceae is mainly due to ESBL, KPC or NDM ß-lactamases.

Wide dissemination of linezolid-resistant Staphylococcus epidermidis in Greece is associated with a linezolid-dependent ST22 clone.

OBJECTIVES: Dependence on linezolid was recently described as significant growth acceleration of linezolid-resistant Staphylococcus epidermidis (LRSE) isolates upon linezolid exposure. We investigated the possible contribution of linezolid dependence to LRSE dissemination in Greece. METHODS: Linezolid resistance rates were estimated in six tertiary hospitals located throughout Greece between 2011 and 2013. Sixty-three randomly selected LRSE recovered in these hospitals during this period were studied. Growth curve analysis was conducted with and without linezolid. Clonality of the isolates was investigated by PFGE and MLST. RESULTS: During the study period, the LRSE rate in the participating hospitals rose significantly from 6.9% to 9% (P = 0.006); the increase was more prominent in ICUs (from 15.1% to 20.9%; P = 0.005). Forty-seven (74.6%) of the 63 LRSE, derived from all study hospitals, clearly exhibited linezolid dependence, growing significantly faster in the presence of 16 and 32 mg/L linezolid. Of note, 61 (96.8%) LRSE exhibited a single macrorestriction pattern and belonged to ST22, which included all linezolid-dependent LRSE. The remaining two LRSE belonged to unique STs. Five of six linezolid-dependent isolates tested also exhibited linezolid dependence upon exposure to 8 mg/L linezolid. Interestingly, five of six ST22 linezolid-non-dependent isolates tested developed linezolid dependence when linezolid exposure preceded growth analysis. CONCLUSIONS: The rapid LRSE dissemination in Greek hospitals threatens linezolid activity. The observation that most LRSE belonged to ST22 and expressed dependence on linezolid clearly implies that the spread of linezolid resistance should have been driven by this trait, which provided the LRSE with a selective advantage under linezolid pressure.

Isolated microorganisms in plastic biliary stents placed for benign and malignant diseases.

BACKGROUND: Biliary stenting is a well-established method to treat patients with malignant and benign biliary diseases. However, occlusion of plastic biliary stents is considered as a drawback and bacterial colonization seems to be the key factor in this process. METHODS: During a 3-year period, 51 plastic biliary stents were extracted from 42 patients. Twenty three stents were inserted for treating malignant and 28 for benign diseases. Stent samples were taken under a strict protocol, and were immediately sent to microbiological laboratory for culturing. RESULTS: A polymicrobial growth was present in nearly all stents. The most frequently isolated organisms were Enterococcus spp (74%), Escherichia coli (E. coli) (62%), and Klebsiella spp (58%). E. coli was more frequently encountered in benign vs. malignant disease (78% vs. 43%, P<0.05). Klebsiella spp, Pseudomonas spp, and Candida spp were more frequently isolated in occluded vs. non-occluded stents, 68% vs. 37%, 22% vs. 0 and 40% vs. 6% respectively (P<0.05). E. coli and Pseudomonas spp had 34% and 50% resistance rate to quinolones respectively. Enterobacter spp expressed Amp-C derepression in 35%. Enterococcus spp, Klebsiella spp and Pseudomonas spp had a low resistance rate. CONCLUSION: Enterococcus spp, E. coli and Klebsiella spp are the most frequently associated organisms in plastic biliary stents. In occluded stents Pseudomonas spp and Candida spp should be taken into account. Quinolones may not be adequate for the treatment of cholangitis associated with stent occlusion. In patients under chemotherapy for malignancy and stent occlusion-related biliary sepsis, antifungal and enterococcal covering should be considered.

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-ß-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.

Linezolid dependence in Staphylococcus epidermidis bloodstream isolates.

We document linezolid dependence among 5 highly linezolid-resistant (LRSE) Staphylococcus epidermidis bloodstream isolates that grew substantially faster at 32 µg/mL linezolid presence. These isolates carried the mutations T2504A and C2534T in multiple 23S rRNA copies and 2 mutations leading to relevant amino acid substitutions in L3 protein. Linezolid dependence could account for increasing LRSE emergence.

Outbreak of OXA-48 carbapenemase-producing Klebsiella pneumoniae in Greece involving an ST11 clone.

OBJECTIVES: First detected in Enterobacteriaceae isolates in Turkey, the OXA-48 carbapenemase has gradually disseminated in the wider Mediterranean area and Europe. Despite reports from other European regions, until now no such isolates have been detected in Greece. We describe the characteristics of the first outbreak caused by OXA-48-producing Klebsiella pneumoniae in Greece. METHODS: From December 2011 to March 2012, 13 ertapenem-resistant K. pneumoniae isolates, which were positive by the modified Hodge test while remaining negative by phenotypic screening for metallo-ß-lactamase (MBL) and KPC production, were recovered from nine patients. Patient records were retrieved to access patterns of acquisition. Resistance genes were identified by PCR and sequencing. ompK35, ompK36 and the genetic environment of the bla(OXA-48) gene were investigated. Plasmid profiling, conjugation experiments, PFGE and multilocus sequence typing (MLST) were performed. RESULTS: All isolates harboured the bla(OXA-48) gene along with the bla(CTX-M-15) and bla(OXA-1) genes. The bla(OXA-48) gene was located on a self-transferable IncL/M-type plasmid of ~62 kb, which harboured no other resistance genes. IS1999 was located upstream of the bla(OXA-48) gene. Genetic disruptions of the ompK35 and ompK36 genes were not detected. The isolates belonged to a unique PFGE clone and MLST assigned them to sequence type ST11. All cases were characterized as hospital acquired and none of them was linked to immigration or history of travel in endemic areas. CONCLUSIONS: Carbapenem resistance due to MBL and KPC carbapenemases is currently on an endemic scale in Greece and this report highlights the wider undetected dissemination of yet another carbapenemase in this region.

Comparative evaluation of tigecycline susceptibility testing methods for expanded-spectrum cephalosporin- and carbapenem-resistant gram-negative pathogens.

We evaluated the Vitek2, Etest, and MIC Test Strip (MTS) methods of tigecycline susceptibility testing with 241 expanded-spectrum cephalosporin-resistant and/or carbapenem-resistant Enterobacteriaceae and Acinetobacter baumannii clinical isolates by using dry-form broth microdilution (BMD) as the reference method. The MIC(50/90)s were as follows: BMD, 1/4 µg/ml; Vitek2, 4/≥8 µg/ml; Etest, 2/4 µg/ml; MTS, 0.5/2 µg/ml. Vitek2 produced 9.1/21.2% major errors, Etest produced 0.4/0.8% major errors, and MTS produced no major errors but 0.4/3.3% very major errors (FDA/EUCAST breakpoints). Vitek2 tigecycline results require confirmation by BMD or Etest for multidrug-resistant pathogens.

Comparative evaluation of combined-disk tests using different boronic acid compounds for detection of klebsiella pneumoniae carbapenemase-producing enterobacteriaceae clinical isolates.

The accurate phenotypic detection of Klebsiella pneumoniae carbapenemase (KPC)-producing Enterobacteriaceae is an increasing necessity worldwide. We evaluated the performance of boronic acid combined-disk tests using as substrate imipenem or meropenem and as inhibitor of KPC production 300 µg aminophenylboronic acid (APBA), 600 µg APBA, or 400 µg phenylboronic acid (PBA). Tests were considered positive when an increase in the growth-inhibitory zone around a carbapenem disk with KPC inhibitor was 5 mm or greater of the growth-inhibitory zone diameter around the disk containing carbapenem alone. The comparison of the combined-disk tests was performed with 112 genotypically confirmed KPC-possessing Enterobacteriaceae isolates. To measure the specificity of the tests, 127 genotypically confirmed KPC-negative Enterobacteriaceae isolates that were nonsusceptible to at least one carbapenem were chosen for testing. Using disks containing imipenem without and with 300 µg APBA, 600 µg APBA, or 400 µg PBA, 72, 92, and 112 of the KPC producers, respectively, gave positive results (sensitivities, 64.3%, 82.1%, and 100%, respectively). Using disks containing meropenem without and with 300 µg APBA, 600 µg APBA, or 400 µg PBA, 87, 108, and 112 of the KPC producers, respectively, gave positive results (sensitivities, 77.7%, 96.4%, and 100%, respectively). Among KPC producers, the disk potentiation tests using meropenem and PBA demonstrated the largest differences in inhibition zones (P < 0.001). All combined-disk tests correctly identified 124 of the 127 non-KPC producers (specificity, 97.6%). This comparative study showed that PBA is the most effective inhibitor of KPC enzymes, and its use in combined-disk tests with meropenem may give the most easily interpreted results.

A simple phenotypic method for the differentiation of metallo-beta-lactamases and class A KPC carbapenemases in Enterobacteriaceae clinical isolates.

BACKGROUND: The increasing frequency of class A KPC enzymes and class B metallo-beta-lactamases (MBLs) among Enterobacteriaceae as well as their possible co-production makes their early differentiation urgent. METHODS: A simple phenotypic algorithm employing three combined-disc tests consisting of meropenem alone and with phenylboronic acid (PBA), EDTA, or both PBA and EDTA was designed for the differentiation of KPC and MBL enzymes. Augmentation of the zone of inhibition by >or=5 mm was considered a positive combined-disc test result. A total of 141 genotypically confirmed carbapenemase-positive Enterobacteriaceae clinical isolates (63 KPC producers, 47 MBL producers, and 31 KPC and MBL producers) with various carbapenem MICs were examined. For comparison, 84 genotypically confirmed carbapenemase-negative Enterobacteriaceae clinical isolates [39 extended-spectrum beta-lactamase (ESBL) producers, 22 AmpC producers, and 23 ESBL and AmpC producers] were also tested. RESULTS: The phenotypic algorithm was able to differentiate MBL from KPC producers as well as to detect the possible co-production of both carbapenemases (positive result only with the combined-disc test using meropenem alone and with both PBA and EDTA). The method detected all KPC or MBL producers (sensitivity 100%) as well as 30 of the KPC and MBL producers (sensitivity 96.8%). All three combined-disc tests were negative for non-carbapenemase-producing isolates, except two ESBL and AmpC producers that gave positive combined-disc tests using meropenem alone and with PBA and both PBA and EDTA (specificity for KPC detection 98.8%). CONCLUSIONS: This phenotypic method is very helpful to detect carbapenemase production and provides a simple algorithm for the differentiation of KPC and MBL enzymes, especially in regions where KPC- and MBL-possessing Enterobacteriaceae are highly prevalent.

Risk factors and outcomes associated with acquisition of colistin-resistant KPC-producing Klebsiella pneumoniae: a matched case-control study.

A matched 1:3 case-control study investigated factors predicting colistin-resistant versus colistin-susceptible KPC-producing Klebsiella pneumoniae acquisition and its impact on patient outcomes. Case patients were more often admitted from other institutions (P = 0.019) and had longer therapy with beta-lactam/beta-lactamase inhibitors (P = 0.002) and higher overall mortality (P = 0.05). All 52 study isolates were clonally related, suggesting horizontal dissemination. None of these parameters independently predicted colistin resistance, which probably occurred in a susceptible KPC-KP strain that was subsequently disseminated horizontally.

Use of boronic acid disk tests to detect extended- spectrum beta-lactamases in clinical isolates of KPC carbapenemase-possessing enterobacteriaceae.

We evaluated boronic acid (BA)-based methods for their ability to detect extended-spectrum beta-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 > or =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.

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 beta-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 beta-lactamases, metallo-beta-lactamases, and plasmid-mediated AmpC beta-lactamases, respectively. By use of CLSI methodology and disks containing imipenem, meropenem, or cefepime, either alone or in combination with 400 microg 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 beta-lactamases are widespread.