Detection of rare carbapenemases in Enterobacterales-comparison of two colorimetric and three CIM-based carbapenemase assays.

Rapid and reliable detection of carbapenemase-producing Enterobacterales (CPE) is crucial for prompt treatment and infection control. Most assays target the primary four enzymes (KPC, OXA-48-like, VIM, and NDM), often missing less common variants (e.g., GES, IMI, OXA-23, and OXA-58). Therefore, assays based on the hydrolysis of carbapenems are recommended in addition to differentiation tests such as PCR or immunochromatographic assays. The aim of this study was to compare the currently Clinical and Laboratory Standards Institute (CLSI)-recommended tests mCIM (modified carbapenem inactivation method) and Carba NP with new colorimetric tests (NitroSpeed-Carba NP) and novel variations of the carbapenem inactivation method (CIM) such as simplified CIM (sCIM) or modified zinc-supplemented CIM (mzCIM). The challenge collection included 205 clinical isolates, 139 CPE vs 66 non-CPE. Among all 205 isolates, the sensitivity/specificity of mCIM was 81.3%/98.5%, Carba NP 76.3%/100%, NitroSpeed-Carba NP 86.3%/78.8%, sCIM 100%/94%, and mzCIM 97.8%/98.5%. For rare carbapenemases (n = 48), the sensitivity of mzCIM (98.3%) and sCIM (100%) was higher than that of mCIM (60.4%), Carba NP (50%), or NitroSpeed-Carba NP (70.2%). Most indeterminate results occurred for mCIM (14.4%), Carba NP (8.2%), and sCIM (6.3%). The detection of rare carbapenemases remains challenging with the currently recommended assays. The CIM-based tests demonstrated superior sensitivity, with sCIM and mzCIM outperforming the currently recommended mCIM and Carba NP, especially among isolates with weakly hydrolyzing carbapenemases (e.g., OXA-23 and OXA-58). Although colorimetric assays provide more rapid results, laboratories have to be aware of the low sensitivity for rare carbapenemases. Both sCIM and the new mzCIM performed well, are cost-effective, and can easily be implemented in any laboratory.IMPORTANCEDetection of so-called rare carbapenemases (e.g., GES, IMI, OXA-23, and OXA-58) in Enterobacterales is challenging, and data on the performance of currently available assays are scarce. This study systematically assessed the performance of currently recommended and novel hydrolysis-based assays on a set of molecularly characterized isolates. It demonstrates that the currently recommended assays mCIM and Carba NP perform well on isolates producing common carbapenemases such as KPC, VIM, NDM, and OXA-48, but have only a moderate sensitivity in the detection of rare carbapenemases. In contrast, the newer CIM-based variants, sCIM and mzCIM, are equally capable of detecting frequent and uncommon carbapenemases. These assays could potentially help to improve our knowledge on the epidemiology of these "rare" enzymes.

Proteus mirabilis - analysis of a concealed source of carbapenemases and development of a diagnostic algorithm for detection.

OBJECTIVES: To analyse carbapenemases in Proteus mirabilis and assess the performance of carbapenemase detection assays. METHODS: Eighty-one clinical P. mirabilis isolates with high-level resistance at least to ampicillin (>32 mg/L) or previous detection of carbapenemases were selected and investigated by three susceptibility testing methods (microdilution, automated susceptibility testing, and disk diffusion), six phenotypic carbapenemase assays (CARBA NP, modified carbapenemase inactivation method [CIM], modified zinc-supplemented CIM, simplified CIM, faropenem, and carbapenem-containing agar), two immunochromatographic assays, and whole-genome sequencing. RESULTS: Carbapenemases were detected in 43 of 81 isolates (OXA-48-like [n = 13]; OXA-23 [n = 12]; OXA-58 [n = 12]; New Delhi metallo-ß-lactamase (NDM) [n = 2]; Verona integron-encoded metallo-ß-lactamase (VIM) [n = 2]; Imipenemase (IMP) [n = 1]; Klebsiella pneumoniae carbapenemase (KPC) [n = 1]). Carbapenemase-producing Proteus were frequently susceptible to ertapenem (26/43; 60%), meropenem (28/43; 65%), ceftazidime (33/43; 77%), and some even to piperacillin-tazobactam (9/43; 21%). Sensitivity/specificity of phenotypic tests were 30% (CI: 17-46%)/89% (CI: 75-97%) for CARBA NP, 74% (CI: 60-85%)/82% (CI: 67-91%) for faropenem, 91% (CI: 78-97%)/82% (CI: 66-92%) for simplified CIM, and 93% (CI: 81-99%)/100% (CI: 91-100%) for modified zinc-supplemented CIM. An algorithm for improved detection was developed, which demonstrated sensitivity/specificity of 100% (CI: 92-100%)/100% (CI: 91-100%) on the 81 isolates, and 100% (CI: 29-100%)/100% (CI: 96-100%) in a prospective analysis of additional 91 isolates. Interestingly, several OXA-23-producing isolates belonged to the same clonal lineage reported previously from France. DISCUSSION: Current susceptibility testing methods and phenotypic tests frequently fail to detect carbapenemases in P. mirabilis, which could result in inadequate antibiotic treatment. In addition, the non-inclusion of blaOXA-23/OXA-58 in many molecular carbapenemase assays further impedes their detection. Therefore, the prevalence of carbapenemases in P. mirabilis is likely underestimated. With the herein proposed algorithm, carbapenemase-producing Proteus can be easily identified.

Carbapenemase-producing Gram-negative bacteria in hospital wastewater, wastewater treatment plants and surface waters in a metropolitan area in Germany, 2020.

Carbapenemase-producing bacteria (CPB) such as Klebsiella pneumoniae and Escherichia coli are causing hospital outbreaks worldwide. An important transfer route into the aquatic environment is the urban water cycle. We aimed to determine the presence of CPB in hospital wastewater, wastewater treatment plants (WWTPs) and surface waters in a German metropolitan area and to characterise these bacteria by whole-genome comparisons. During two periods in 2020, 366 samples were collected and cultivated on chromogenic screening media. Bacterial colonies were selected for species identification and PCR-based carbapenemase gene screening. Genomes of all detected CPB were sequenced and analysed for resistance gene content, followed by multilocus sequence typing (MLST) and core genome MLST (cgMLST) for K. pneumoniae and E. coli isolates. Carbapenemase genes were detected in 243 isolates, most of which belonged to genera/species Citrobacter spp. (n = 70), Klebsiella spp. (n = 57), Enterobacter spp. (n = 52) and E. coli (n = 42). Genes encoding KPC-2 carbapenemase were detected in 124 of 243 isolates. K. pneumoniae produced mainly KPC-2 and OXA-232 whereas E. coli harboured various enzymes (KPC-2, VIM-1, OXA-48, NDM-5, KPC-2 + OXA-232, GES-5, GES-5 + VIM-1, IMP-8 + OXA-48). Eight and twelve sequence types (STs) were identified for K. pneumoniae and E. coli, respectively, forming different clusters. The detection of numerous CPB species in hospital wastewater, WWTPs and river water is of concern. Genome data highlight a hospital-specific presence of distinct carbapenemase-producing K. pneumoniae and E. coli strains belonging to "global epidemic clones" in wastewater samples representing local epidemiology. The various detected CPB species including E. coli ST635, which is not known to cause human infections, could serve as reservoirs/vectors for the spread of carbapenemase genes in the environment. Therefore, effective pretreatment of hospital wastewater prior to discharge into the municipal wastewater system may be required, although swimming lakes do not appear to be a relevant risk factor for CPB ingestion and infection.

Pentacyclic triterpenes from the leaves extract of Sandoricum koetjape.

Three new pentacyclic triterpenes, trivially named sandkoetjapic acids A-C (1-3), have been isolated from the leaves extract of Sandoricum koetjape, along with the known triterpenes 3-oxo-olean-12-en-29-oic (4), bryonolic (5), and bryononic (6) acids. The structures of the new triterpenes were determined mainly by NMR spectroscopic and mass spectroscopic data. The isolation of these pentacyclic triterpenes in the plant's leaves is for the first time. Preliminary biological evaluation of 1-6 was done against eight receptor tyrosine kinases (RTKs), including EGFR, HER2, HER4 (epidermal growth factor receptor), IGF1R, InsR (insulin receptor), KDR (kinase insert domain receptor), and PDGFRα/-ß (platelet-derived growth factor receptor), and their inhibitory properties against ß-lactamase. The results showed that none of them were active both as the inhibitors of these RTKs and ß-lactamase.

Comparison of five methods for detection of carbapenemases in Enterobacterales with proposal of a new algorithm.

OBJECTIVES: The aim of this study was to evaluate the performance of five different carbapenemase tests and to develop an algorithm which will permit the detection of most common and rare carbapenemases in routine microbiology laboratories. METHODS: The immunochromatographic tests CARBA-5 (NG), RESIST-4 O.K.N.V. (Coris), the colorimetric ß-CARBA (BioRad), a newly developed carbapenem-inactivation method (CIM) supplemented with zinc (zCIM), and the Xpert Carba-R (Cepheid) were challenged with a collection of 189 molecularly characterized Enterobacterales isolates, including 146 carbapenemase producers (CPE): VIM (n = 48), OXA-48-like (n = 40), NDM (n = 29), KPC (n = 13), IMI (n = 9), IMP (n = 9), OXA-58 (n = 2), and GES (n = 2). RESULTS: The overall sensitivity/specificity values for the five carbapenemase detection tests were 84.2% (CI 77.6-89.2%)/100% (CI 91.8-100%) for RESIST-4, 88.2% (CI 82.1-92.4%)/100% (CI 91.8-100%) for CARBA-5, 88.2% (CI 82.1-92.4%)/100% (CI 91.8-100%) for Xpert Carba-R, 73.7% (CI 66.2-80.0%)/100% (CI 93.4-99.0%) for ß-CARBA, and 97.4% (CI 87.9-99.6%)/97.7% (CI 87.9-99.6%) for zCIM. The four common carbapenemases (KPC, OXA-48-like, NDM, and VIM) were detected with ≥97.6% sensitivity by all tests except for ß-CARBA (76.6% (CI 68.4-83.2%)). IMI and GES were only detected by zCIM (sensitivity 90.9% (CI 62.3-98.4%)). Based on these results a new algorithm was developed, consisting of an immunochromatographic assay as the first test followed by zCIM, which allows detection of 99.3% of all carbapenemases assessed. CONCLUSIONS: Except for ß-CARBA, all methods showed excellent sensitivity/specificity for the detection of the four most frequent carbapenemases. With the new algorithm, rare variants can also be detected. It is rapid, simple, and inexpensive and can be performed in any microbiology laboratory, as no PCR equipment is required.

Rapid detection of OXA-48-like, KPC, NDM, and VIM carbapenemases in Enterobacterales by a new multiplex immunochromatographic test.

The rapid detection of carbapenemase-producing Gram-negative bacteria is indispensable to optimize treatment and avoid the further spread of these organisms. While phenotypic tests are time-consuming and PCR is expensive and not available in many routine laboratories, immunochromatographic tests (ICT) can provide rapid results at moderate cost. The aim of this study was to determine the performance of the new ICT RESIST-4 O.K.N.V. K-SeT (Coris BioConcept, Gembloux, Belgium) which can detect the four most prevalent carbapenemases: OXA-48-like, KPC, NDM, and VIM. Additionally, we analyzed the impact of different culture conditions on the sensitivity. The new ICT was challenged with 169 carbapenem-resistant isolates. Of these, 125 were carbapenemase producers: 43 OXA-48-like, 15 KPC, 29 NDM, and 43 VIM. The ICT correctly detected 129 of the 130 carbapenemases resulting in a sensitivity of 99.2% and specificity of 100% when tested from Mueller-Hinton agar (MHA). The sensitivity of the assay increased to 100% when performed from zinc-supplemented MHA and sheep blood agar (SBA) or when the inoculum was harvested from the inhibition zone of an ertapenem disk. All carbapenemase-negative carbapenem-resistant bacteria tested negative and no cross-reaction was observed. The new ICT is an excellent test for rapid diagnostic of carbapenemase-producing Gram-negatives in the routine laboratory. It is easy to handle and provides rapid results with a high sensitivity. For best results, we recommend to obtain the inoculum from a medium with sufficient zinc or from the inhibition zone of an ertapenem disk.

Rapid detection of carbapenemases directly from positive blood cultures by the ß-CARBA test.

The rapid detection of blood stream infections (BSI) by carbapenemase-producing Enterobacterales (CPE) is indispensable to early optimize antibiotic treatment and to improve survival. While phenotypic tests are time-consuming and PCR is expensive and not available in many routine laboratories, colorimetric tests (e.g., Carba NP test) can provide rapid results at moderate cost. However, up to now, the detection of CPE-BSI requires a further 3-h incubation in broth supplemented with zinc sulfate and imipenem after a blood culture has become positive, thereby causing delay and additional hands-on time. The purpose of this study was to develop and evaluate a new method for the detection of CPE directly from positive blood culture without the need for incubation in broth, based on the commercially available colorimetric ß-CARBA test. For the evaluation, blood cultures spiked with 140 different Enterobacterales isolates producing diverse beta-lactamases were tested with the new method. Of these, 70 were CPE (OXA-48-like, NDM, KPC, VIM, and GIM). After blood cultures turned positive, blood culture fluid was drawn, and erythrocytes were hemolyzed with SDS, washed, and equilibrated before the ß-CARBA was performed on the bacterial pellet. All carbapenemases were reliably detected, including weak carbapenemases of the OXA-48 group. The sensitivity was 100% (95% CI 94.9-100) and the specificity 94.3% (95% CI 89.2-99.4). The time to result was 20 to 45 min. Carbapenemases can rapidly and reliably be detected directly from blood cultures using the new method, which could help to improve the outcome of these difficult-to-treat infections.

A voltammetric test for the rapid discrimination of ß-lactamase-producing Enterobacteriaceae in blood cultures.

The accurate identification of ß-lactamases produced by Enterobacteriaceae is a major challenge in clinical laboratories in order to optimize antimicrobial treatment and patient care. We describe here a rapid voltammetric-based method to detect and to discriminate ß-lactamase activity in Enterobacteriaceae i.e., penicillinase, cephalosporinase (inducible or overproduced), extended-spectrum beta-lactamase and carbapenemase producers. After a 2-h growth step of the sample under three separate conditions: 1) LB (Luria-Bertani) medium, 2) LB supplemented with 4 µg/mL cefotaxime and 3) LB supplemented with 4 µg/mL cefotaxime and 100 µg/mL potassium clavulanate, the ß-lactamase activity was measured by incubating a 0.5 mM nitrocefin solution for 15 min followed by the voltammetric detection of the hydrolyzed nitrocefin with disposable carbon screen-printed sensors. The development and the calibration of the method were carried out by analyzing pure cultures of fifty-seven strains with well characterized ß-lactam-resistance phenotypes. Thanks to the combination of the three currents (i1, i2, i3) recorded for each tested bacteria, the proposed procedure allowed to distinguish the different classes of ß-lactamase producers. In the second part of the study, the method was applied to the analysis of one hundred and fifteen samples Enterobacteriaceae-positive blood culture samples of bacteraemic patients. Overall data showed that the voltammetric method offered a sensitivity of 100% and a specificity of 80%. Interestingly, all of sixteen samples infected by a third-generation cephalosporins-resistant bacteria (i.e. ESBL and overproduced cephalosporinase producers) were detected. This study clearly demonstrated that the voltammetric assay is an efficient alternative technique for the rapid discrimination of ß-lactamases-producing Enterobacteriaceae in blood culture. In contrast to the approved routine assays, the electrochemical test did not require isolated colonies to be performed and was thus carried out in less than 3 h which could allow early administration of an appropriate antibiotic therapy.

Multiplex Immunochromatographic Detection of OXA-48, KPC, and NDM Carbapenemases: Impact of Inoculum, Antibiotics, and Agar.

For the rapid detection of carbapenemase-producing Enterobacteriaceae (CPE), immunochromatographic lateral flow tests (ICT) have recently been developed. The aim of this study was to assess the new multiplex ICT Resist-3 O.K.N. and to investigate if it can be performed directly from susceptibility testing plates. Additionally, the impact of the inoculum and carbapenem disks on sensitivity and specificity was evaluated. The new ICT was challenged using 63 carbapenem-resistant Enterobacteriaceae (CRE) isolates, including 51 carbapenemase producers. It was assessed under five different conditions directly from Mueller-Hinton agar (MHA): 1 µl or 10 µl of inoculum harvested in the absence of antibiotic pressure or 1 µl taken from the inhibition zone of either an ertapenem, imipenem, or meropenem disk. The sensitivity of the ICT was 100% for OXA-48-like and KPC carbapenemases and 94.4% for the NDM carbapenemase with the 1-µl inoculum. When harvested adjacent to a carbapenem disk, the sensitivity increased to 100%. Additionally, with zinc-supplemented MHA, both the sensitivity increased and the NDM band became visible faster (mean time, 8 ± 3.9 min for MHA compared to 1.9 ± 1.5 min for MHA plus zinc; P = 0.0016). The specificity of the ICT was 100%. The Resist-3 O.K.N. ICT is a sensitive and rapid test for the detection of three highly prevalent carbapenemases. However, false-negative results for NDM can occur. We recommend an inoculum of 1 µl that is harvested adjacent to an ertapenem or meropenem disk and the use of agars with sufficient zinc content to achieve the best performance.

Findings from an outbreak of carbapenem-resistant Klebsiella pneumoniae emphasize the role of antibiotic treatment for cross transmission.

PURPOSE: In January 2015, we noticed by rectal swab analyses that seven of 23 patients at an early rehabilitation ward had been colonized with carbapenem-resistant Klebsiella pneumoniae (CKP). Here, we describe risk factors for CKP acquisition. METHODS: In the present study, the outbreak is described and risk factors for CKP acquisition are examined, e.g., antibiotic treatment. Microbiological analyses including corresponding results were examined to study when colonization with CKP occurred and whether patients had suffered from diarrhea. To examine whether spread of bacteria was clonal, multi-locus sequence typing as well as Xbal macrorestriction and pulsed-field gel electrophoresis was performed. The presence of carbapenmase was examined by PCR analysis. Through univariate analysis of risk factors in the small study sample, the role of antibiotic consumption, isolation procedures, patient's age, gender, and Barthel index on colonization was elucidated. RESULTS: Clonal spread of the novel sequence type (ST)2255 was identified. Additionally, one patient was colonized with Escherichia coli and Serratia marcescens, both resistant to carbapenems, while a further patient carried another carbapenem-resistant E. coli strain. In all isolates, carbapenemase gene bla OXA-48 was found to be located on a conjugative plasmid (60 kb), suggesting in vivo transmission from CKP to E. coli and S. marcescens. Univariate tests indicated that antibiotic treatment was the only risk factor showing a significant association with being colonized by CKP. In addition, the likelihood of diarrhea appeared to be higher in this group. Antibiotic treatment was associated with CKP colonization, whereas patients´ age, gender, Barthel index at admission, and residence with a CKP-colonized roommate were not. Diarrhea also seemed to support to distribution of CKP. CONCLUSIONS: In this small outbreak, antibiotic treatment seemed to be the predominant risk factor for monoclonal transmission of bla OXA-48 positive CKP.

An Improved Extended-Spectrum-ß-Lactamase Detection Test Utilizing Aztreonam plus Clavulanate.

Clinical laboratories test for extended-spectrum ß-lactamases (ESBLs) for epidemiological and infection control purposes and also for the potential of cephalosporins to cause therapeutic failures. Testing can be problematic, because the CLSI does not recommend the testing of all producers of ESBLs and also falsely negative results may occur with isolates that coproduce AmpC. Boronic acid-supplemented tests can enhance ESBL detection in AmpC producers. Because aztreonam inhibits AmpCs, a study was designed to compare ESBL detection by the CLSI disk test (CLSI), a boronic acid-supplemented CLSI disk test (CLSI plus BA), and an aztreonam plus clavulanate disk test (ATM plus CA). The study tested 100 well-characterized Enterobacteriaceae, Acinetobacter baumannii, and Pseudomonas aeruginosa isolates. Seventy produced TEM, SHV, or CTX-M ESBLs, with 15 coproducing an AmpC and 11 coproducing a metallo-ß-lactamase. Thirty ESBL-negative isolates were also tested. Tests were inoculated by CLSI methodology and interpreted as positive if an inhibitor caused a zone diameter increase of ≥5 mm. The percentages of ESBL producers detected were as follows: ATM plus CA, 95.7%; CLSI plus BA, 88.6%; and CLSI, 78.6%. When AmpC was coproduced, the sensitivities of the tests were as follows: ATM plus CA, 100%; CLSI plus BA, 93.3%; and CLSI, 60%. ATM plus CA also detected an ESBL in 90.1% of isolates that coproduced a metallo-ß-lactamase. Falsely positive tests occurred only with the CLSI and CLSI plus BA tests. Overall, the ATM plus CA test detected ESBLs more accurately than the CLSI and CLSI plus BA tests, especially with isolates coproducing an AmpC or metallo-ß-lactamase.

Virtual screening and repositioning of inconclusive molecules of beta-lactamase Bioassays-A data mining approach.

This study focuses on the best possible way forward in utilizing inconclusive molecules of PubChem bioassays AID 1332, AID 434987 and AID 434955, which are related to beta-lactamase inhibitors of Mycobacterium tuberculosis (Mtb). The inadequacy in the experimental methods that were observed during the invitro screening resulted in an inconclusive dataset. This could be due to certain moieties present within the molecules. In order to reconsider such molecules, insilico methods can be suggested in place of invitro methods For instance, datamining and medicinal chemistry methods: have been adopted to prioritise the inconclusive dataset into active or inactive molecules. These include the Random Forest algorithm for dataminning, Lilly MedChem rules for virtually screening out the promiscuity, and Self Organizing Maps (SOM) for clustering the active molecules and enlisting them for repositioning through the use of artificial neural networks. These repositioned molecules could then be prioritized for downstream drug discovery analysis.

Método rápido para la detección de la sensibilidad a cefotaxima en enterobacterias / Rapid test for detection of susceptibility to cefotaxime in Enterobacteriaceae

En este trabajo se evalúa una prueba rápida in house para la detección de enterobacterias sensibles a cefotaxima, basada en el cambio de pH del rojo fenol debido a la hidrólisis de este antibiótico. Las cepas de enterobacterias procedentes de 1.947 urocultivos se evaluaron mediante los paneles MicroScan y esta prueba in house. Mediante los paneles de MicroScan se estudiaron 499 aislados de enterobacterias, entre los cuales había 27 aislados de Escherichia coli productora de β-lactamasa de espectro extendido (BLEE), 16 de Klebsiella pneumoniae BLEE y una de Klebsiella oxytoca BLEE. La prueba in house mostró una sensibilidad del 98% y una especificidad del 97%, con un valor predictivo negativo del 100% y un valor predictivo positivo del 78%. La prueba in house basada en el cambio de pH es útil en nuestro medio para detectar presuntivamente de forma rápida cepas de enterobacterias con cierta resistencia a cefotaxima.

In this work an "in house" rapid test based on the change in pH that is due to hydrolysis for detecting Enterobacteriaceae susceptible to cefotaxime is evaluated. The strains of Enterobacteriaceae from 1947 urine cultures were assessed using MicroScan panels and the "in house" test. This rapid test includes red phenol solution and cefotaxime. Using MicroScan panels, 499 Enterobacteriaceae isolates were evaluated, which included 27 isolates of Escherichia coli producing extended-spectrum beta-lactamases (ESBL), 16 isolates of Klebsiella pneumoniae ESBL and 1 isolate of Klebsiella oxytoca ESBL. The "in house" test offers the following values: sensitivity 98% and specificity 97%, with negative predictive value 100% and positive predictive value 78%. The "in house" test based on the change of pH is useful in our area for detecting presumptively cefotaxime-resistant Enterobacteriaceae strains.

Amoxicillin concentrations in relation to beta-lactamase activity in sputum during exacerbations of chronic obstructive pulmonary disease.

BACKGROUND: Acute exacerbations of chronic obstructive pulmonary disease (COPD) are often treated with antibiotics. Theoretically, to be maximally effective, the antibiotic concentration at sites of infection should exceed the minimum inhibitory concentration at which 90% of the growth of potential pathogens is inhibited (MIC90). A previous study showed that most hospitalized COPD patients had sputum amoxicillin concentrations

Drug susceptibility and treatment response of common urinary tract infection pathogens in children.

BACKGROUND/PURPOSE: To document the trends of sensitivity and to find whether it is necessary to change antibiotics in selected patients according to the sensitivity test results in our clinical practice. METHODS: We collected urine culture results from 0-18-year-old patients in the National Taiwan University Hospital from January 1, 2003 to October 31, 2012. Their medical chart was reviewed to identify true pathogens responsible for their urinary tract infection (UTI). We checked the percentage of susceptibility of these pathogens to ampicillin, amoxicillin-clavulanate (AMC), cefazolin, cefmetazole, ceftriaxone, gentamicin, and trimethoprim-sulfamethoxazole (TMP-SMX) according to the Clinical and Laboratory Standards Institute (CLSI) guideline. The extended-spectrum-beta-lactamases (ESBLs) rate was also checked. In addition, we reviewed the treatment response of different antibiotics. Defervescence within 48 hours after initial antibiotics use was considered responsive. RESULTS: A total of 7758 urine cultures positive for Escherichia coli infection were collected during the 10-year period. The E. coli cefazolin susceptibility rate was 62-73% during 2003-2010, but it dropped to 23% in 2011 and 28% in 2012 after the new CLSI guideline (M100-S21) was released. However, other antibiotics did not show a significant difference. In UTI caused by E. coli, on average, the sensitivity rates for various antibiotics were as follows: cefmetazole, 90%; ceftriaxone, 85%; gentamicin, 77%; AMC, 61%; TMP-SMX, 47%; and ampicillin, 20%. The ESBL rate was also found to increase (2-11%; p < 0.01). The overall response rate of UTI caused by E. coli to first-line antibiotics such as first-generation cephalosporin and/or gentamicin was 78%. CONCLUSION: The susceptibility of common urinary tract pathogens to cefazolin has decreased dramatically since 2010. This trend may be due to the change in the CLSI guideline. Although the susceptibility rate to first-line empirical antibiotics shows a decreasing trend, we found that the clinical response was acceptable for our first-line empirical antibiotics.

Aumento significativo de la resistencia a fosfomicina en cepas de Escherichia coli productoras de Beta-lactamasas de espectro extendido (BLEE) aisladas de urocultivos (2005-2009-2011) / Increasing prevalence of fosfomycin resistance in extended-spectrum-beta-lactamaseproducing Escherichia coli urinary isolates (2005-2009-2011)

Introducción. Escherichia coli es el principal uropatógeno. La aparición de cepas productoras de Beta-lactamasas de espectro extendido (BLEE), que con frecuencia presentan multirresistencia, deja pocas opciones terapéuticas, y es necesario realizar un seguimiento de su sensibilidad a lo largo del tiempo. En el presente trabajo se presentan los porcentajes de aislados urinarios de E.coli productores de BLEE durante 2005, 2009 y 2011 y se comparan los resultados de la determinación de su sensibilidad a antibióticos de diferentes grupos, fosfomicina entre ellos. Métodos. Se analizaron 5.053, 6.324 y 6.644 aislados urinarios de E. coli en 2005, 2009 y 2011 respectivamente. Se excluyeron duplicados. La sensibilidad se determinó por microdilución con el sistema Wider (Soria Melguizo S.A.) y se seleccionó el fenotipo que indicaba producción de BLEE (CLSI 2009). Resultados. El 3,9% de las cepas (198) resultó productor de BLEE en 2005, el 7,3% (463) en 2009 y el 8,7% (584) en 2011. Se detectó resistencia a carbapenemicos en 2009, aunque continúan con un 95% de sensibilidad. Entre los no-Betalactámicos, colistina fue el más activo, seguido de nitrofurantoina. Ciprofloxacino y sulfametoxazol-trimetoprim presentaron un 80% y 60% de resistencia, respectivamente. Se observó una tendencia al aumento de la resistencia en fosfomicina, desde 0% a 9,3 llegando al 14,4% en 2011. Conclusiones. Se observó una creciente prevalencia de cepas de E. coli productoras de BLEE aisladas de urocultivos, alcanzando el 8,7% en 2011. Los carbapenemicos siguen siendo los antibióticos más activos frente a este tipo de cepas. El aumento de resistencia a fosfomicina fue significativo(AU)

Introduction. Escherichia coli is the most important uropathogen. The appearance of extended- spectrum beta-lactamase (ESBL)-producing E.coli in urinary tract infections (UTI) constitutes an important therapeutic challenge that requires the study of its evolution throughout time in order to establish a suitable empirical treatment. Our aim was to determine the prevalence of ESBL-producing E. coli urinary isolates in 2005, 2009 and 2011. We also determined the antimicrobial coresistance to several agents, including fosfomycin. Methods. We analyzed 5053, 6324 and 6644 E. coli isolates obtained from urine cultures in 2005, 2009 and 2011 respectively. Duplicate isolates were excluded. Antimicrobial susceptibility was determined by the Wider microdilution system (Soria Melguizo S.A.) and the phenotypic pattern of resistance that indicated a BLEE-producing E.coli was selected (CLSI 2009). Results. 3.9% of strains (198) were ESBL producers in 2005, 7.3% (463) in 2009 and 8.7% (584) in 2011. Resistance to carbapenems was detected in 2009, they inhibited more than the 95% of strains in 2011. Among the non-beta-lactams, colistin was the most active antibiotic followed by nitrofurantoin. Ciprofloxacin and sulfamethoxazole-trimethoprim were not effective with 80% and 60% resistant isolates, respectively. An increasing resistance trend, from 0% to 9.3% in 2009 and 14.4% in 2011 was observed for fosfomycin. Conclusions. From 2005 our institution had an increasing prevalence of ESBL-producing E. coli rising to 8.7% in 2011. Carbapenems are still the most active agents. The increase of resistance was significant for fosfomycin(AU)

Alternatives to carbapenems in ESBL-producing Escherichia coli infections.

OBJECTIVES: The authors had for objective to assess the activity of a wide panel of antibiotics on extended-spectrum-ß-lactamase producing Escherichia coli isolates (ESBL-Ec), because of the sharp increase of their frequency, leading to an increased use of carbapenems. MATERIAL AND METHODS: We selected 100 ESBL-Ec in which ESBLs were identified by PCR and sequencing, between 2009 and 2010. We determined the MICs of amoxicillin-clavulanate, piperacillin-tazobactam, temocillin, mecillinam, cefoxitin, cefotaxime, ceftazidime, aztreonam, tigecycline, nitrofurantoin, and fosfomycin using reference methods. The susceptibility profiles were defined according to EUCAST 2012 recommendations. RESULTS: Fosfomycin, nitrofurantoin, and pivmecillinam were active against more than 90% of isolates and remain excellent choices for the oral treatment of urinary tract infections (UTIs). Temocillin and piperacillin-tazobactam are also good candidates for the treatment of pyelonephritis or bloodstream infections. Only 27, 23, and 8% of isolates were susceptible to ceftazidime, cefepime, and cefotaxime, respectively. CONCLUSION: Our study results prove that in many cases, there are non-carbapenem alternatives for the treatment of ESBL-Ec infections.

New Delhi Metallo-beta lactamase (NDM-1) in Enterobacteriaceae: treatment options with carbapenems compromised.

BACKGROUND: Carbapenems are among the few useful antibiotics against multidrug resistant gram negative bacteria particularly those with extended spectrum beta lactamase. However resistance to carbapenems occurs and is mediated by mechanisms like loss of outer membrane proteins and production of beta lactamase that is capable of hydrolyzing carbapenems. An alert issued in the UK in 2009 warned of an increasing number of carbapenem resistant Enterobacteriaceae strains identified in UK hospital patients. Many of them were recently hospitalized in India and Pakistan and had new type of metallo beta lactamase designated as New Delhi Metallo-1 (NDM-1). OBJECTIVE: To assess the production of NDM-1 type Metallo beta lactamase enzyme in Enterobacteriaceae at a tertiary care centre in Mumbai. MATERIALS AND METHODS: Consecutive carbapenem resistant Enterobacteriaceae isolates were collected from August 2009 to November 2009. Susceptibility testing for carbapenems was performed by the disc diffusion method. Carbapenemase production was confirmed by Modified Hodge test. These strains were then subjected to single target PCR. A 475bp product was amplified by the NDM primers and visualized on 3% agarose gel. RESULTS AND CONCLUSIONS: Modified Hodge test was positive for all carbapenem resistant isolates. Of 24 carbapenem resistant Enterobacteriaceae 22 were NDM producers while 2 were NDM non producers. Amongst the 22 NDM producing organisms 10 were Klebsiella spp, 9 were Escherichia coli, 2 were Enterobacter spp and 1 was Morganella morganii. This high number in a relatively short span is a worrisome trend that compromises the treatment options with the carbapenems.

[In vitro activity of fosfomycin trometamol in the treatment of Escherichia coli related uncomplicated urinary tract infections]. / Escherichia coli nedenli komplike olmamis üriner sistem enfeksiyonlarinda fosfomisin trometamolün in vitro etkinligi.

Although in certain countries in Europe fosfomycin trometamol (FT) has been used for many years, in Turkey FT has become available in recent years. FT has a broad-spectrum activity against most of gram-positive and gram-negative bacteria. In this study, we aimed to evaluate the effect of FT, a new alternative antimicrobial agent in the treatment of patients with Escherichia coli related uncomplicated lower urinary tract infection (UTI). For this purpose, between May 2007-July 2008, FT susceptibility of 771 nonduplicate E. coli strains, isolated from urine samples of patients with uncomplicated lower UTI (bacteria > or = 10(5) cfu/mL), was determined by disk diffusion method according to Clinical and Laboratory Standarts Institute (CLSI) criteria. Simultaneously, extended-spectrum beta-lactamase (ESBL) detection was performed by double disk synergy test in all isolates. Among all E. coli isolates, FT resistance rate was 0.4% (3/771) and ESBL positivity was 19.5% (150/771). The rates of ESBL producing strains isolated from inpatients and outpatients were 34.1% (70/205) and 14.1% (80/566), respectively, and the difference was found statistically significant (p = 0.0001). Although resistance to FT was not detected in non-ESBL producing E. coli isolates (n = 621), FT resistance rate was 2% (3/150) in ESBL producers. As far as the current literature was concerned this was the largest scale study investigating the activity of FT in Turkey. Resistance to antimicrobials that had been used frequently as therapeutic options for the treatment of E. coli related UTIs, has been increasing. In the present study high susceptibility rates to FT was determined for urinary E. coli isolates. In conclusion, these data suggest that FT may be a good alternative for the treatment of uncomplicated UTIs as a first line antimicrobial agent.

Development of a method for the detection of beta-lactamases in milk samples.

With the rapid growth of the dairy industry and the establishment of strict antimicrobial residue limits in the People's Republic of China's (PRC) milk supply, a beta-lactamase product known as "antimicrobial destroyer" was introduced into dairy production without regulatory review. We developed a method for detecting this product in milk samples based on a modified cylinder plate method. The presence of beta-lactamase is defined as a difference between the inhibitory zones of the test samples (supplemented with 25 microg/mL sulbactam plus 0.5 microg/mL penicillin G) and control samples (supplemented only with 0.5 microg/mL penicillin G) > or = 3 mm. Using this method, 77 individually packaged milk samples were randomly collected from 5 retail stores in 3 cities over a 4-month period (May to August 2006). Of the 77 samples, 49 were found to be beta-lactamase-positive. In 2 undiluted milk samples showing extremely high beta-lactamase activity, 25 microg/mL sulbactam could not inhibit penicillin G activity. Because there is a lack of safety data on beta-lactamases in milk products, these data indicated a potentially serious safety concern for the dairy industry in the PRC.