Rosco Diagnostica Research Articles

A Five-Year Analysis of Antibiotic Resistance Trends among Bacteria Identified in Positive Urine Samples in a Tertiary Care Hospital from Bucharest, Romania.

The rise of multidrug-resistant bacteria (MDR) has resulted in limited treatment options and poorer outcomes for patients. The objective of this study was to analyze the overall antibiotic resistance trends and distribution for pathogens identified in urine samples at the National Institute of Infectious Diseases "Prof. Dr. Matei Balș" from Bucharest, Romania, over a 5-year period. Antibiotic susceptibility testing was performed using automatic systems and the disk diffusion method. ESBL- and carbapenemases-producing strains were identified using immunochromatography tests, and ROSCO Diagnostica kits were used for definitive confirmation. All results were interpreted according to EUCAST clinical breakpoints. Gram-negative rods (GNR) had overall resistance rates higher than 50% for penicillin and 40% for 3rd- and 4th-generation cephalosporins. Escherichia coli resistance to fosfomycin (3%) and nitrofurantoin (2%) remains low, and 33.30% of E. coli, 48% of Klebsiella spp., and 37% of Pseudomonas aeruginosa isolates were multidrug-resistant (MDR). All Acinetobacter baumannii isolates were MDR by the last year of the study. For Gram-positive cocci (GPC), 37% of all Enterococcus faecium strains and 2% of Enterococcus faecalis were vancomycin-resistant (VRE). E. coli's incidence in UTIs' etiology is on a downward trend. The incidence of Klebsiella spp. and GPCs is rising. Antibiotic stewardship strategies should be implemented after carefully considering regional variations in etiology and resistance trends.

Prevalence of extended-spectrum β-lactamase and carbapenem-resistant Klebsiella pneumoniae in clinical samples.

To assess the prevalence of these resistant strains in the overall isolates of Klebsiella pneumoniae (K. pneumoniae) in hospital settings. This retrospective study was conducted from November 2020 to November 2021. The identification and antibiotic susceptibility testing were performed using standard laboratory methods according to the EUCAST standards. The detection of ESBL and carbapenemase production was performed using phenotypic methods such as E-test, combined-disk test with various inhibitors (ROSCO Diagnostica A/S), chromogenic medium for the detection of ESBL/carbapenemase-producing Enterobacteriaceae (CPE) isolates, and the VITEK 2 Compact system (BioMerieux). 944 isolates of K. pneumoniae were detected in various clinical specimens. Among these, ESBL-producing strains were detected in 349/944 (37%), whereas carbapenem- resistant strains in 188/944 (20%) of the isolates. The remaining isolates (407/944 [43%]) belonged to the wild type. ESBL isolates were the most common in wound swabs (138 [39.5%]), whereas CRKP isolates in screening samples (110 [58.5%]). The majority of ESBL isolates were detected in surgical departments (105 [30.1%]), whereas CRKP isolates in adult intensive care unit departments (79 [42.%]). Our results show an increasing frequency of CRKP strains. This presents a significant issue in terms of infection prevention and control in hospital settings.

Multidrug-Resistant and Extended-Spectrum β-Lactamase (ESBL) - Producing Enterobacterales Isolated from Carriage Samples among HIV Infected Women in Yaoundé, Cameroon.

The exacerbation of antimicrobial resistance (AMR) is a major public health threat worldwide. In sub-Saharan Africa, there is a scarcity of data regarding multidrug-resistant (resistance to at least one antibiotic of three or more families of antibiotics) as well as extended spectrum β-lactamase-producing Enterobacterales (ESBL-PE), isolated among clinical and asymptomatically healthy patients, especially in women living with HIV (WLHIV) despite their immunocompromised status. The overarching aim of this study was set to determine the prevalence and characterize genotypically multi-drug resistant Enterobacterales (MDR-E) and ESBL- PE isolated from vaginal swabs of WLHIV attending the Yaoundé Central Hospital, Yaoundé, Cameroon. A cross-sectional study was conducted among WLHIV during a four-month periods from 1 February to 31 May 2021. A total of 175 WLHIV, of childbearing age and under antiretroviral treatment were contacted. One hundred and twenty participants (120) were recruited and vaginal swabs were collected from them. After culture on Eosine-Methylen Blue (EMB) agar, the identification of Enterobacterales was performed using API 20E kit. A double-screening of ESBL-PE was performed using a combined disc diffusion method and ROSCO Diagnostica kits. An antibiotic susceptibility test was carried out by disc diffusion as per the Kirby-Bauer method and the β-lactamase resistance genes, blaCTX-M, blaCTX-M-group1-2-9, blaTEM were molecularly characterized using a conventional Polymerase Chain Reaction (PCR). Overall, 30.83% (37/120) of the included WLHIV were colonized with Enterobacterales and the prevalence of vaginal carriage of MDR Enterobacterales among them was 62.16% (23/37). Among MDR-E isolates, the most prevalent species were E. coli (56.0%; 14/25) and K. pneumoniae (20.0%; 5/25). High rates of resistance to trimethoprim-sulfamethoxazole (96.0%; 24/25), amoxicillin-clavulanic acid (88.0%; 22/25) and gentamicin (72%; 18/25) were observed. The resistance mechanisms detected among these isolates were ESBL (48.0%; 12/25), ESBL+ porin loss (8.0%; 2/25), ESBL+AmpC (24%; 6/25), with blaCTX-M, blaCTX-M-group-1,2,9 being identified at 48.0% (12/25) for each of them and blaTEM at 72.0% (18/25). Our findings confirm the high-prevalence of MDR as well as ESBL-PE isolated in WLHIV, and suggest that a real time monitoring system of antimicrobial resistant bacteria coupled with the reinforcement of infection prevention control (IPC) strategies are needed to sustainably contain these life-threatening pathogens especially in the most vulnerable populations.

Molecular epidemiology and the clinical impact of carbapenemase-producing Enterobacterales isolates among adult patients: aspects from a Romanian non-teaching hospital

Abstract Introduction: A dramatic increase of infections induced by carbapenemase-producing Enterobacterales (CPE) has been registered worldwide. The aim of this study was to evaluate the molecular epidemiology and the clinical impact of CPE strains isolated from adult inpatients. Material and methods: A one-year, single-center, retrospective observational study including 34 consecutive patients with 37 non-duplicate CPE strains recovered from clinical specimens was accomplished. The Vitek 2 Compact, M.I.C.Evaluator strips, the modified carbapenem inactivation method (mCIM), and the combination disks test (KPC, MBL, OXA-48 Confirm kit, Rosco Diagnostica) were applied as phenotypic tests. A multiplex polymerase chain reaction (PCR) assay was used for detection of blaKPC, blaNDM, and blaOXA-48-like genes. The clonality was assessed with pulsed-field gel electrophoresis (PFGE). Results: Klebsiella pneumoniae (n=25) was the most frequent CPE encountered. The carbapenemase types were NDM (n=13), KPC (n=12), and OXA-48-like (n=12). Two distinct clonal clusters were identified among the 12 KPC positive strains. All CPE isolates exhibited non-susceptibility to carbapenems, cephalosporins, ciprofloxacin. Respiratory tract infections (n=16) and hospitalization in the intensive care unit (ICU) (n=14) were dominant. The most common comorbidity was congestive heart failure (n=11). Monotherapy was the main strategy adopted (n=15). Death occurred in 18 patients. Conclusions: Our analysis underscores the scarcity of antibiotic solutions and high mortality. Monotherapy for urinary tract infections (UTIs) is beneficial. Inter- or intrahospital dissemination of successful epidemic clones is proved. The adequate CPE infections control programs and antimicrobial policies are essential..

2289. Accuracy of a Rapid Multiplex PCR Plus a Chromogenic Phenotypic Test Algorithm for the Detection of ESBL and Carbapenemase-Producing Gram Negatives Directly From Blood Cultures

BackgroundWe studied the multiplex PCR panel (BioFire Blood Culture ID panel, “BCID”) with phenotypic testing using the Rosco Diagnostica Rapid ESBL Screen kit 98022 (RE) and the Neo-Rapid CARB kit 98024 (RC) for extended-spectrum β-lactamase (ESBL)/carbapenemase producing Gram negative bacilli (CPGNB) detection directly from blood culture bottles, in patients with Gram negative bacteremia.MethodsThe RE and RC kits were evaluated in a verification phase with 98 blood cultures, comprising 43 spiked with GNB: 23 Escherichia coli, 9 Klebsiella pneumoniae, 7 Enterobacter cloacae, 2 Serratia marcescens, one Pseudomonas aeruginosa, one Acinetobacter baumanii complex with varying resistance genotypes (11 CTX-M-15, 5 CTX-M9, one SHV-18, one SHV-3, one TEM-10, 3 IMI, 4 IMP, 4 KPC, 2 NDM, one OXA-23+OXA-51-like, 3 OXA-232, one OXA-48, one SME-1, 2 VIM-1, 2 AmpC from reference and clinical isolate banks, and ATCC 25922), and 54 clinical blood cultures with GNB (5 phenotypic ESBL-positive, one KPC, 48 no known β-lactamase). In a prospective phase, a further 123 clinical blood cultures positive for GNB were tested simultaneously with the BCID, RE and RC kits.ResultsIn the verification phase, the RE kit detected 24/25 of ESBL-positive samples (sensitivity 96%, specificity 99%). The RE kit did not detect the 2 AmpC-producers, and was positive for a K. oxytoca isolate, which are known to produce chromosomally encoded β-lactamases. The RC kit detected 11/22 of CPGNB (sensitivity 50%, specificity 100%). It missed IMI, OXA-23+OXA-51-like, OXA-232, OXA-48, SME-1 and VIM CPGNB (weak carbapenemases), but detected NDM, KPC, IMP. In the prospective phase, the RE kit detected 20/20 ESBL-positive blood culture samples (sensitivity 100%). The single OXA-48 positive sample was detected by both the RE and RC kits. The 123 blood cultures had a total of 125 panel-represented targets detectable by BCID. The BCID detected 124 /125 (missed one K. pneumoniae in a polymicrobial bacteremia), and there were 2 Proteus false-positives (sensitivity 99%, specificity 98%). No KPC-positive samples were detected by BCID.ConclusionAn algorithm comprising the BCID and the RE/RC kits applied to positive blood cultures allows both rapid and accurate pathogen identification and detection of ESBLs and some carbapenemases (e.g., KPC, NDM, IMP). This may allow the institution of timelier, directed therapy.Disclosures S. Vasoo, bioMerieux: Grant Investigator, Research support. Rosco Diagnostica: In-kind support, Research support. R. Banerjee, Accelerate Diagnostics, Biomerieux, BioFire: Grant Investigator, Research grant and Research support. R. Patel, CD Diagnostics, BioFire, Curetis, Merck, Hutchison Biofilm Medical Solutions, Accelerate Diagnostics, Allergan, and The Medicines Company: Grant Investigator, Research grant - monies paid to Mayo Clinic. Curetis, Specific Technologies, Selux Dx, GenMark Diagnostics, PathoQuest and Genentech: Consultant and Scientific Advisor, Consulting fee - monies paid to Mayo Clinic. ASM and IDSA: Travel reimbursement and editor’s stipends, Travel reimbursement and editor’s stipends. NBME, Up-to-Date and the Infectious Diseases Board Review Course: Varies, Honoraria. Mayo Clinic: Employee, Salary.

Phenotypic and molecular identification of carbapenemase-producing Enterobacteriaceae - challenges in diagnosis and treatment

Abstract Introduction: Infections due to carbapenemase-producing carbapenem-resistant Enterobacteriaceae (CPCRE) are an emerging global public health threat. The purpose of this study was to investigate phenotypic and genotypic features of CP-CRE strains isolated from hospitalized patients. Material and methods: Between 1st of January - 1st of July 2017, in the Department of Microbiology, “Dr. Constantin Opriş” County Emergency Hospital Baia Mare, Romania, 1110 strains of Enterobacteriaceae were isolated from bronchial secretions, urine, wounds and blood cultures. Bacterial identification and antimicrobial susceptibility tests were performed by conventional methods, Vitek 2 Compact and M.I.C.E. strips. We analysed all Enterobacteriaceae strains non-susceptible to carbapenems according to CLSI 2017 criteria. The modified Hodge test (MHT), the modified carbapenem inactivation method (mCIM) and the combination disks test (KPC, MBL, OXA-48 Confirm kit, Rosco Diagnostica) were used for phenotypic confirmation, whereas a multiplex PCR assay for genes blaKPC, blaNDM and blaOXA-48 was used for genetic confirmation. Results: 19 non-duplicate strains isolated from 16 patients were phenotypically identified as CP-CRE: Klebsiella pneumoniae (n=14), Escherichia coli (n=2), Providencia stuartii (n=2) and Serratia marcescens (n=1). Most strains were isolated from bronchial secretions (n=9). The carbapenem-hydrolizing enzymes were identified by the combination disks test as: KPC (n=9), OXA-48-like (n=5) and MBL (n=5). Molecular confirmation was performed in 18 phenotypically positive isolates with 100% concordant results with mCIM and combination disks test. Discrepant results were noticed with the MHT in case of 4 NDM-producers confirmed by PCR. All CP-CRE strains were resistant to all tested cephems. Three out of 9 K. pneumoniae strains tested against colistin were found resistant. Conclusions: The most common carbapenemase detected was KPC. Therapeutic options were limited in all positive cases. Rapid and reliable detection of CP-CRE is critical for preventing the spread of these pathogens

Sensitive and specific detection of carbapenemase-producing carbapenem resistant Enterobacteriaceae in the routine microbiology laboratory with modified Hodge test and CarbaNP test

Carbapenemase-producing carbapenem resistant Enterobacteriaceae (CP-CRE) are of major clinical and public health concerns due to the highly transmissible nature and the lack of therapeutic options. Rapid and reliable detection of CP-CRE in the routine clinical microbiology laboratory is critical for the timely optimisation of therapy and implementation of infection control measures. This study aims to evaluate the performance of the various phenotypic methods that are available in our laboratory. 312 carbapenem intermediate or resistant Enterobacteriaceae isolates were tested for carbapenemase production using the modified Hodge test (MHT), the CarbaNP test, the MAST Carbapenemase Detection Test (MASTDISCS D70C) and the KPC/MBL and OXA-48 Kit (ROSCO Diagnostica). The phenotypic test results were compared against molecular detection of the carbapenemase genes via polymerase chain reaction (PCR). In our laboratory, the MHT had a sensitivity of 95.8% and a specificity of 87.7%. The CarbaNP test had a sensitivity of 96.6% and a specificity of 93.7%. Combining the MHT and CarbaNP test improved the sensitivity to 100%, with a positive predictive value of 97% and a negative predictive value of 100%. In conclusion, the combination of MHT and CarbaNP test represents the most sensitive and cost effective methods of CP-CRE phenotypic detection in our laboratory.

A Comparative Study on Antibiotic Resistance of Klebsiella Strains from Surgical and Intensive Care Wards.

ABSTRACT: Introduction. Klebsiella infections are common in Intensive Care Unit (ICU) and surgical wards. In order to establish the prophylaxis protocols, we must know the prevalence of infections and the antibiotic resistance profiles. Material and methods. This cross-sectional study included isolates from patients in County Clinical Emergency County Hospital, Craiova, Romania: 1254 isolates from the ICU and 1040 isolates from surgical wards. We used an automated method (Phoenix analyzer, Becton-Dickinson, USA) with antimicrobial testing according to CLSI 2014. We tested by disc diffusion the ESBL and carbapenemases production, using kits ESBL Confirm ID and KPC/Metallo-beta-lactamase/OXA-48 Confirm (ROSCO Diagnostica, Denmark). The patients in ICU were also screened at admission for carbapenemase producting strains by PCR (GeneXpert® II, Cepheid, Sunnyvale, CA, USA) for the carbapenemases: KPC, IMP-1, VIM-1, NDM, OXA-48. Results. Klebsiella strains were more prevalent in ICU (20.81%) vs. surgical wards (16.34%) and they were resistant in high percentages at: cefuroxime (95.81% vs. 87.21%),ceftazidime (91.70% vs. 84.71%), cefepime (84.2% vs. 69.82%). The highest differences in resistance were observed for Tygecycline (Risk Ratio (RR) = 7.69), Imipenem/Cilastatine (RR=3.36), Cefoperazone with sulbactam (RR=2.58), Ciprofloxacine (RR=2.11), Gentamycin (RR=2.05) and Ertapenem (RR=1.93). The ICU strains showed MDR in 48.57% of cases vs. 23.57% in surgery strains. The prevalence of ESBL production was 82.4% in ICU vs. 32.3% in surgical wards. The prevalence of carbapenemase producing strains was 43.68% in ICU vs. 23.53% in surgical wards. Conclusions. The infections with Klebsiella spp. are more frequent in ICU compared with surgical wards and their antibiotic resistance is greater.

Emergence of Carbapenemase-producing Enterobacteriaceae, a Public Health Threat: a Romanian Infectious Disease Hospital Based Study / Emergenţa Enterobacteriaceaelor producătoare de carbapenemaze, o ameninţare pentru sănătatea publică: un studiu realizat într-un spital romanesc de boli infectioase

Abstract Introduction: Hospital-acquired infections caused by Enterobacteriaceae producing different types of carbapenem- hydrolizing enzymes are now commonly observed and represent a great limitation for antimicrobial therapy. The purpose of the study was to evaluate the emergence of carbapenem-resistant Enterobaceriaceae among the strains isolated from hospitalized patients to the National Institute of Infectious Diseases, Bucharest (NIID) and the identification of different types of carbapenemases, using phenotypic methods. Materials and methods: Between January - June 2014, 587 strains of Klebsiella pneumoniae, Enterobacter species and E.coli were isolated from various clinical specimens. We were included all non-susceptible strains to carbapenems, according to EUCAST 2014 clinical breakpoints, as determined by using microdilution MicroScan Panels (Siemens Healthcare Diagnostics). The modified Hodge test (MHT) was performed as phenotypic confirmatory test for carbapenemase production according to CLSI guidelines and the combination disk test (KPC, MBL , OXA-48 Confirm kit, Rosco Diagnostica) according to EUCAST guidelines. Results: A total of 45 non-repeat Enterobaceriaceae (32 strains Klebsiella pneumoniae, 5 strains E.coli, 8 strains Enterobacter spp) were identified as non-susceptibile to one or more carbapenems (93,33% ertapenem, 53,33% meropenem, 48,88% imipenem). Most strains were isolated from urine (75,55%). MHT was positive in 55,6% (25/45) of carbapenem-resistant strains; in 24 cases the carbapenem-hydrolizing enzyme was identified as: OXA-48-like (n=16), KPC (n=4), MBL (n=1), KPC + MBL (n=2) and MBL + OXA-48-like (n=1). All carbapenemase- positive strains were 100% resistant to 3rd and 4th generation cephalosporins, showing less resistance to tigecycline (12,5% resistant and 25% intermediate), colistin (37,5%) and fosfomycin (41,6%). Conclusion: During 6 months period, there were isolated 7,66% (45/587) carbapenem-resistant Enterobacteriaceae (K. pneumoniae 21,47%, E. coli 1,23%). Twenty four strains were carbapenemase-producers. The most frequent carbapenemase isolated in our study was OXA-48-like.

Detección de carbapenemasas en pseudomonas aeruginosa mediante espectometría de masas MALDI-TOF MS

Introduction: Gram-negative bacteria especially Pseudomonas are resistance to multiple antibiotics including carbapenems. Carbapanemes resistance has increased in recent years caused by alterations of membrane or the production of carbapenemases. Objective: Assess the use of MALDI-TOF MS® mass spectrometry for the detection of carbapenemases class A or B in Pseudomonas aeruginosa. Material and methods: From isolated from Pseudomonas aeruginosa producing carbapenemases 12 class A or B identified by diffusion method disco-plate, classified using disks: meropenem 10μg, meropenem 10μg + boronic acid, meropenem 10μg + cloxacillin and meropenem 10μg + acid dipicolinic (Rosco Diagnostica), we analyzed possible hydrolysis of meropenem peaks after the action of the carbapenemases by MALDI-TOF MS® mass spectrometry. As negative controls were used 25 strains of Pseudomonas aeruginosa sensitive to meropenem and 8 strains of Pseudomonas aeruginosa with waterproof membrane, not detectable by the methodology used. Results: Of the 12 strains producing carbapenemases class A or B, (2/12 class A, 10/12 class B), MALDI-TOF MS® mass spectrometry technique detected peaks of degradation of the antibiotic in study to the presence of carbapenemases in 11/12 cases (94.4%). The strains used as controls negative, MALDI-TOF MS® mass spectrometry indicated the absence of carbapenemases class A or B at 31/33 cases (93.9%). Conclusion: MALDI-TOF MS® mass spectrometry can be a useful tool for the confirmation of carbapenemases class A and B in Pseudomonas aeruginosa.

Isolation of multiple carbapenemase-producing Gram-negative bacilli from a patient recently hospitalized in Nigeria

A 29-year-old female transferred to a tertiary care hospital in Winnipeg, Canada, after a prolonged period of hospitalization in Nigeria was found to be colonized with a VIM-2–producing Pseudomonas aeruginosa, a NDM-1–producing Klebsiella pneumoniae, and an OXA-181–producing Escherichia coli. Detection of carbapenemase-producing organisms from a rectal swab was accomplished by screening with chromogenic media, followed by confirmation with the Rapid CARB Screen kit (Rosco Diagnostica, Taastrup, Denmark). This case illustrates the need for clinical microbiology laboratories to have a protocol in place to screen patients for carbapenemase producers, even in countries where these organisms are rarely encountered.

Formation of a carbapenemase resistance detection algorithm for use in the routine laboratory

ABSTRACTThe increasing prevalence of carbapenem non-susceptible Gram negative organisms demands prompt and accurate identification of resistance mechanisms to limit their transmission. The aim of this study is to evaluate the rapid CARB screen (Rosco Diagnostica, Denmark), the ‘KPC/MBL in P. aeruginosa/Acinetobacter Confirm Kit’ (Rosco Diagnostica), Check-MDR Carba and Check-Direct CPE kits (Check-Points, The Netherlands). The purpose of this study is the formation of a carbapenemase resistance detection algorithm that can be used in the routine laboratory. Results of the rapid CARB screen kit were improved when isolates were tested from Muller Hinton agar with a meropenem 10 μg disc instead of blood agar. The rapid CARB screen (performed in 2–3 h) demonstrated overall 98.7% sensitivity and 87.7% specificity (n=133). The KPC/MBL in P. aeruginosa/Acinetobacter Confirm Kit (which requires overnight incubation) demonstrated a high number of false-positive results giving 38.6% specificity and 100% sensitivity (n=44). The Check-MDR Carba (performed in 5 h), detecting carbapenemase presence, provides a cabapenemase-positive or -negative result demonstrated 96.7% specificity and 98.6% sensitivity (n = 132). The Check-Direct CPE (performed in 3 h), which identifies KPC, NDM/VIM or OXA-48 type carbapenemases, demonstrated 96.5% specificity and 97.1% sensitivity (n=97). The Check-Direct CPE, however, failed to detect dual carbapenemase genes present in two out of four isolates. The principal conclusion is the recommendation of the rapid CARB screen and Check-MDR Carba for incorporation into a carbapenemase detection algorithm which, when used in combination, will yield results with 97.3% sensitivity and 99.6% specificity.

Evaluation of five chromogenic agar media and the Rosco Rapid Carb screen kit for detection and confirmation of carbapenemase production in Gram-negative bacilli.

An efficient workflow to screen for and confirm the presence of carbapenemase-producing Gram-negative bacilli was developed by evaluating five chromogenic screening agar media and two confirmatory assays, the Rapid Carb screen test (Rosco Diagnostica A/S, Taastrup, Denmark) and the modified Hodge test. A panel of 150 isolates was used, including 49 carbapenemase-producing isolates representing a variety of β-lactamase enzyme classes. An evaluation of analytical performance, assay cost, and turnaround time indicated that the preferred workflow (screening test followed by confirmatory testing) was the chromID Carba agar medium (bioMérieux, Marcy l'Étoile, France), followed by the Rapid Carb screen test, yielding a combined sensitivity of 89.8% and a specificity of 100%. As an optional component of the workflow, a determination of carbapenemase gene class via molecular means could be performed subsequent to confirmatory testing.

ENTEROBACTERIACAE, PRODUCING ESBLS AND METALLO-β-LACTAMASE NDM-1, ISOLATED IN HOSPITALS OF BALTIC REGION COUNTRIES

Abstract. We studied the prevalence of K. pneumoniae and E. coli resistance to extended spectrum cephalosporins and carbapenems, isolated from patients of eight hospitals in St-Petersburg from January to May, 2012. Prevalence of cephalosporin resistant isolates varied in different hospitals: E. coli – from 7,8 to 50%, K. pneumoniae – from 25,4 to 88,4%. Isolates produced extended spectrum beta-lactamases СТХ-М, mainly СТХ-М-1, also СТХ-М-2 and СТХ-М-9. Twenty two carbapenem-resistant K. pneumoniae strains (also resistant to other antimicrobials) were isolated in three hospitals. MALDI-TOF MS showed that carbapenem resistance was caused by carbapenemase. Carbapenemases of all isolates belonged to metallo-β-lactamases according to results of the ROSCO Diagnostica tests. The gene coding production of New Delhi metallo-β-lactamase (blaNDM-1) were detected in all strains. Our data confirmed that the main cephalosporin resistance mechanism of E. coli и K. pneumoniae in Baltic region (including Russia, St-Petersburg) is CTX-M-1 production. For the first time in Russia K. pneumoniae strains producing metallo-β-lactamases NDM-1 were isolated in several hospitals of St-Petersburg.

Comparison of phenotypic methods for the detection of carbapenem non-susceptible Enterobacteriaceae.

BackgroundMultidrug resistance and, in particular, carbapenem resistance is spreading worldwide at an alarming rate, comprehending a variety of bacterial species and causing both nosocomial and community acquired outbursts. Early and efficient detection of infected patients or colonized carriers are mandatory steps in infection control and prevention of multidrug resistance diffusion. The latest EUCAST guidelines for detection of carbapenemase-producing Enterobacteriaceae have set low clinical breakpoints to ensure the maximum detection sensitivity of positive samples. Current workflows involve an initial screening step for species and resistance pattern detection, followed by phenotypic and/or genotypic confirmation. The aim of the present study was to assess the efficiency of six widely used and validated phenotypic assays for the detection of carbapenemases/AmpC in Enterobacteriaceae, to estimate the best workflow in the routine characterization of Enterobacteriaceae isolates.MethodsA panel of 108 non-repetitive Enterobacteriaceae isolates with reduced susceptibility to carbapenems was analyzed by means of 1) Modified Hodge Test, 2) Metallo Beta Lactamase Etest, 3) Double disk test with EDTA, 4) Rosco Diagnostica KPC and MBL confirm kit (RDCK™), 5) AmpC Etest and 6) Cloxacillin inhibition test. Confirmation and validation of results was achieved by genotypic analysis.ResultsThe most accurate identification of resistance determinants was obtained with the combined disc test (Rosco Diagnostica KPC and MBL confirm kit) which had to be coupled with the cloxacillin inhibition test for correct detection of AmpC enzymes. However, in general, phenotypic tests failed to characterize isolates harboring multiple carbapenem resistance determinants, which were successfully assessed only by PCR-based analysis.ConclusionsTo detect and control the spread of pathogens with complicated resistance patterns, both optimized phenotypic analysis (i.e. Rosco Diagnostica KPC and MBL confirm kit coupled with the cloxacillin inhibition test) and genotypic assays are recommended in the routine diagnostic of clinical laboratories.

Evaluation of phenotypic detection methods for metallo-β-lactamases (MBLs) in clinical isolates of Pseudomonas aeruginosa

Metallo-beta-lactamase (MBL) production in Pseudomonas aeruginosa is a growing issue across the globe. Fast and reliable diagnostic tools are needed for appropriate implementation of infection control measures. In this study we evaluated the performance of three commercial combined disk tests, two EDTA based in-house combined disk tests and the Carba NP test in comparison to molecular detection of MBL genes on 133 meropenem non-susceptible non-duplicate P. aeruginosa clinical isolates. The meropenem/DPA based commercial KPC + MBL-confirm ID kit (Rosco Diagnostica, Denmark) and the MASTDISCS™ ID carbapenemase (Enterobacteriaceae) detection disc set (MAST Diagnostics, UK) showed sensitivities of 31.1 % and 28.8 % and specificities of 69.3 % and 79.6 %, respectively. The total MBL confirm kit (Rosco Diagnostica, Denmark) contains imipenem/DPA and imipenem/EDTA combination disks. Evaluation of the single disk combinations revealed 84.4 % sensitivity and 81.8 % specificity for the imipenem/DPA assay and 86.7 % sensitivity and 51.1 % specificity for the imipenem/EDTA test. Applying both tests simultaneously resulted in a slightly higher sensitivity of 88.9 % but a lower specificity of 48.9 % when compared to the single tests alone. The Carba NP test showed 93.3 % sensitivity and 96.6 % specificity. All phenotypic combined disk tests lacked either sensitivity or specificity for the detection of MBL in P. aeruginosa. The Carba NP test showed excellent test properties, but suffers from drawbacks in handling and high costs. The optimal diagnostic approach needs to be chosen depending on the epidemiological situation, laboratory resources and availability of molecular confirmation tests.

Investigations on the efficacy of routinely used phenotypic methods compared to genotypic approaches for the identification of staphylococcal species isolated from companion animals in Irish veterinary hospitals

BackgroundIdentification of Staphylococci to species level in veterinary microbiology is important to inform therapeutic intervention and management. We report on the efficacy of three routinely used commercial phenotypic methods for staphylococcal species identification, namely API Staph 32 (bioMérieux), RapID (Remel) and Staph-Zym (Rosco Diagnostica) compared to genotyping as a reference method to identify 52 staphylococcal clinical isolates (23 coagulase positive; 29 coagulase negative) from companion animals in Irish veterinary hospitals.ResultsGenotyping of a 412 bp fragment of the staphylococcal tuf gene and coagulase testing were carried out on all 52 veterinary samples along with 7 reference strains. In addition, genotyping of the staphylococcal rpoB gene, as well as PCR-RFLP of the pta gene, were performed to definitively identify members of the Staphylococcus intermedius group (SIG). The API Staph 32 correctly identified all S. aureus isolates (11/11), 83% (10/12) of the SIG species, and 66% (19/29) of the coagulase negative species. RapID and Staph-Zym correctly identified 61% (14/23) and 0% (0/23) respectively of the coagulase-positives, and 10% (3/29) and 3% (1/29) respectively of the coagulase-negative species.ConclusionsCommercially available phenotypic species identification tests are inadequate for the correct identification of both coagulase negative and coagulase positive staphylococcal species from companion animals. Genotyping using the tuf gene sequence is superior to phenotyping for identification of staphylococcal species of animal origin. However, use of PCR-RFLP of pta gene or rpoB sequencing is recommended as a confirmatory method for discriminating between SIG isolates.

Laboratory Detection of Enterobacteriaceae That Produce Carbapenemases

A study was designed to evaluate the modified Hodge test (MHT), Mastdiscs ID inhibitor combination disks (MDI), Rosco Diagnostica Neo-Sensitabs (RDS), metallo-β-lactamase (MBL) Etest, and in-house multiplex PCR for the detection of well-characterized carbapenemase-producing Enterobacteriaceae. One hundred forty-two nonrepeat clinical isolates of carbapenemase-producing Enterobacteriaceae (including Klebsiella spp., Escherichia coli, Citrobacter freundii, and Enterobacter spp.) obtained from the SMART worldwide surveillance program during 2008 to 2009 were included. These included 49 KPC-, 27 NDM-, 19 VIM-, 14 OXA-48-like enzyme-, and 5 IMP-producing isolates and 28 carbapenem-resistant, carbapenemase-negative isolates. The manufacturer's instructions were followed for MDI, RDS, and MBL Etest and CLSI guidelines for MHT. A multiplex PCR was designed to detect KPC, NDM, VIM, IMP, and OXA-48-like carbapenemases. Overall, the sensitivity and specificity were 78% and 93% for MDI, 80% and 93% for RDS, 58% and 93% for MHT, and 55% and 100% for MBL Etest, respectively. The PCR had 100% sensitivity and specificity. MDI and RDS performed well for the detection of KPCs and NDMs but poorly for VIMs, IMPs, and OXA-48-like enzymes. MHT performed well for KPCs and OXA-48-like enzymes but poorly for NDMs, VIMs, and IMPs. MDI and RDS were easy to perform and interpret but lacked sensitivity for OXA-48-like enzymes, VIMs, and IMPs. MHT and MBL Etest were often difficult to interpret. We recommend using molecular tests for the optimal detection of carbapenemase-producing Enterobacteriaceae.

Carbapenemase-ProducingKlebsiella pneumoniaein Hospital, Singapore, 2011

Carbapenemase-Producing<i>Klebsiella pneumoniae</i>in Hospital, Singapore, 2011

ESBL-Positive Enterobacteria Isolates in Drinking Water

To the Editor: Extended-spectrum β-lactamase (ESBL)–producing members of the family Enterobacteriaceae (enterobacteria) are a worldwide problem (1), but little data are available from central Africa (2). In recent years, ESBL-producing Enterobacteriaceae isolates have shifted from the hospital to the community and the environment (1). The aim of this study was to assess the presence of ESBL-producing Enterobacteriaceae isolates in sachet-packaged water bags sold as drinking water in the streets of Kinshasa, the capital of Democratic Republic of the Congo. In November 2009 and June 2010, a total of 101 sachet-packaged water bags were bought from street vendors in 9 of 24 municipalities (covering residential areas and slums) of the city of Kinshasa. The bags were transported in ice coolers and processed within 4 hours of collection. We filtered 100 mL of each sample through 0.45-µm pore size filters (Sartorius, Goettingen, Germany). The filters were then transferred to an agar plate containing mEndoLES agar (Difco, Franklin Lakes, NJ, USA) and incubated at 35°C for 24 hours. Growing colonies were subcultured on Kligler iron agar (Oxoid, Cambridge, UK), and gram-negative glucose-fermenting isolates were identified to the species level and assessed for antimicrobial drug susceptibility with Microscan NBC42 panels (Siemens Healthcare Diagnostics Inc., West Sacramento, CA, USA). Isolates labeled by Microscan as ESBL producers were confirmed by the double-disk method, which compared 1 disk containing cefotaxime with 1 disk containing cefotaxime and clavulanic acid and 1 disk containing ceftazidime with 1 disk containing ceftazidime and clavulanic acid (Rosco Diagnostica, Taastrup, Denmark), according to the Clinical and Laboratory Standards Institute guidelines (3). We used Escherichia coli ATCC 25922 and Klebsiella pneumoniae ATCC 700603 as control strains. Detection and identification of ESBL-producing bla genes were carried out by using a commercial multiplex ligation PCR microarray CT 101 (Check-Points Health BV, Wageningen, the Netherlands) (4). A total of 101 sachet-packaged water bags were purchased at random from different vendors: 88 sealed and branded bags (of 68 different brands) and 13 hand-tied unbranded bags. The precise origin of the water could not be determined because vendors are unlicensed resellers and most producers are not registered. Water bags lacked essential information such as contact addresses, batch number, and production and expiration dates. Because we also observed empty branded sheets of bags for sale on the market, we assumed that many of the branded bags are simply filled with tap water or water from other supplies without any prior treatment. None of the water bags tested for chlorine (36 of 101) contained free chlorine levels >0.1 mg/L. Nearly one third of the water bags were contaminated with Enterobacteriaceae isolates (22/88 branded bags and 9/13 hand-tied bags). The bags were obtained in townships and residential quarters. Overall, 150 nonduplicate Enterobacteriaceae isolates were recovered. The main species were K. pneumoniae (56.0% of isolates found in 23/101 of water bags) and Enterobacter spp. (30.6%, in 20/101 water bags); Citrobacter spp. accounted for 4.7% of isolates and E. coli for 3.3%. Eight isolates (5.3 %) were confirmed as ESBL producers by antimicrobial drug susceptibility tests, and they were recovered from 2 branded and 2 hand-tied bags. The species, microarray results, and the associated drug resistance are listed in the Table. Five isolates carried blaCTX-M genes belonging to CTX-M1 group, and 3 isolates carried blaSHV variants. No TEM-ESBL genes were detected. On the basis of checkpoint results and previously validated data, we further categorized the SHV G238S mutation as SHV-2-like and the double SHV G238A + SHV E240K mutation as SHV-18 (5). Table ESBL-producing Enterobacteriaceae recovered from sachet-packaged water bags in Kinshasa, Democratic Republic of the Congo* ESBL-producing Enterobacteriaceae isolates constitute a major public health concern in industrialized and resource-poor settings. Few reports are available from Africa, although hospital-associated ESBL producers have been described in Cameroon and the Central African Republic (6,7). ESBL-producing bacteria have been recovered from different sources in the community, including food and companion animals (8,9), and 1 recent study from India reported that a substantial number of tap water samples were contaminated with carbapenemase blaNDM-1 producing organisms (10). Kinshasa is the second-largest city in sub-Saharan Africa. In 2008, of its estimated 8.7 million inhabitants, only 46%had access to safe drinking water, and 23% had access to improved sanitation facilities according to the World Bank. Opportunistic pathogens in drinking water and poor sanitary conditions may increase the risk of developing infectious enterocolitis for consumers, especially for those who are immunocompromised. It can eventually lead to chronic intestinal carriage of multidrug-resistant organisms. The presence of ESBL producers in the intestinal flora could also lead to horizontal transfer of drug resistance genes from commensal flora to enteric pathogens. This emergence of ESBL-producing bacteria and further community-associated infections poses a public threat, especially in low-resource countries where surveillance is suboptimal and empiric treatment of invasive infections often includes third-generation cephalosporins.