MicroScan Panels Research Articles

Identification of Diverse Multiple Drugs Resistant Bacterial Pathogens using Microscan Panel

Bacterial isolates from clinical sources have increased resistance to antimicrobial agents available and routinely used in developing countries like Ethiopia. One of the control measures of antimicrobial resistance is to know the susceptibility of pathogenic bacteria from clinical specimens and treat patients accordingly. Therefore the objective of the present study is to isolate bacterial pathogens from different clinical specimens and determine their antimicrobial susceptibility patterns. Clinical samples (urine, blood, pus and discharges from different body sites were cultured and isolation of bacterial pathogens were done following standard bacteriological methods using media recommended by Cheesbrough. Identification of bacterial pathogens and antimicrobial susceptibility tests were done using Micro Scan Identification Panel method. The Panels were read by Micro Scan Auto Scan 4 reader after incubating for 18 to 24 hours at 35o c aerobically. The retrospective data of microbiological culture and antimicrobial susceptibility test results were analysed. A total of 995 clinical specimens were cultured at Bethzatha Bacteriology Lab., Bethzatha Hospital, from May 2021 to February 2022. The most frequent specimens were, urine 87 (32%), blood 77(28%), pus and discharges from different body sites 65(24%). Out of these, 273(27%) yielded different bacterial pathogens. The most dominant gram negative bacterial isolates from urine samples 43/87(49%), 6/87(7%), 7/87(8%) were E. coli, Acinetobacter and Klebsiella spp, respectively in that order. Klebsiella pneumoniae 12/77 (16%) were most frequently isolated from blood culture followed by diverse coagulase negative Staphylococci (CoNS) 26/77 (33.7%) and S. aureus 15/77(19.5%). The most frequent 20/65(31%) isolates from pus and discharges were S. aureus followed by 12/65(18.5%) CoNS. In the present study, most bacterial isolates from different clinical samples were multiply resistant to ampicillin, amoxicillin, trimethoprim sulphametoaxzole, levofloxacillin and ampicillin-sulbactam. On the other hand the most frequent Gram negative bacteria, E.coli and Klebsiella pneumonia were most susceptible to amikacin and ertapenem, and the gram positive isolates, S. aureus were most susceptible to levofloxacillin and gentamicin; whereas coagulase negative staphylococci (CoNS) were most susceptible to gentamicin, tecioplanin, rifampicin, vancomycin, daptomycin. Therefore clinicians should be guided by antimicrobial susceptibility test. In the absence of antimicrobial susceptibility test we suggest that the above mentioned drugs to be most appropriate for empirical treatment in the study hospitals and health settings in Addis Ababa. Furthermore critical measures need to be taken to curb the increasing spread of AMR bacteria.

Isolation and Identification of Methicillin Resistant Staphylococci at Bethzatha Advanced Laboratory, Addis Ababa, Ethiopia

Methicillin resistant Staphylococcus aureus and coagulase negative staphylococci (CoNS) are widely spreading. So identification and antimicrobial susceptibility tests of methicillin resistant S. aureus and coagulase negative staphylococcus (CoNS) is critical to appropriately treat patients and control the increasing spread of these pathogens. Therefore the aim of the present work is to determine methicillin resistant S.aureus and CoNS from clinical samples sent to BethZatha Advanced Laboratory from different health institutions and Bethzatha Hospital, Addis Ababa, for bacterial isolation, identification and antimicrobial susceptibility. Bacterial isolation, identification and antimicrobial susceptibility testing were done using Microscan panel methods (Beckman Coulter, Brea, CA, USA) from May 2021 to February 2022 at Bethzatha advanced Medical Laboratory. The retrospective data was analysed to determine methicillin susceptibility of S.aureus and Coagulase negative staphylococci (CoNS). The finding shows 35% (40/114) S. aureus and 65.8% (75/114) coagulase negative staphylococci (CoNS) were isolated from different clinical specimens (urine, blood culture, pus and discharges). Out of 40 S. aureus isolates 30% (12/40) were methicillin resistant. Similarly, out of 75 CoNS, 52% (39/75) were methicillin resistant. Almost all methicillin resistant S. aureus and most CoNS isolates were multiply resistant to amoxicillin clavulnate, azithromycin, penicillin and ampicillin. Periodic surveillance and appropriate control measures and safety precautions of nosocomial and community acquired methicillin resistant multidrug resistant, staphylococcus aureus, and CoNS must be given prior attention.

Direct inoculation method for identification and antimicrobial susceptibility testing using matrix-assisted laser desorption ionization-time of flight mass spectrometry and both the Vitek 2 and MicroScan Walkaway 96 Plus systems

The aim of our study was to evaluate a protocol utilizing serum separator tubes (SST) to facilitate a faster, cost-effective, direct method for rapid sensitivity testing and identification of positive blood cultures. Spiked cultures were inoculated into either Becton Dickinson (BD) BACTECTM Aerobic Plus or Anaerobic/F bottles containing sterile human blood. Bottles were immediately processed when positive. A parallel study using patient isolates was used in which bacteria were pelleted by SST from positive blood cultures. For identification, a portion of the pellet was tested by matrix-assisted laser desorption/ionization as described by the manufacturer. MicroScan panels and Vitek 2 results were compared. Categorical agreement was used as comparison to standard subculture and/or polymerase chain reaction methods. No discordant identifications were observed, and 86% generated a successful identification when compared to subculture methods. For the Vitek 2, we observed a 99% essential agreement when compared to the subculture method. For the MicroScan Walkaway, we observed 94.9%, 97.4%, and 100% categorical agreement for MIC panels 53, 38, and MICroSTREP Plus 2, respectively. Turnaround times were reduced from 4 hours for identification and 11 hours for antimicrobial sensitivity testing. We conclude that the SST method results in timelier, actionable results for antimicrobial stewardship initiatives.

Phenotypic and genotypic characterization of oxacillin-susceptible and mecA positive Staphylococcus aureus strains isolated in Uruguay

The aim of this study was to characterize phenotypically and genotypically 27 mecA positive Staphylococcus aureus strains with oxacillin MICs of ≤2μg/ml by Vitek 2, isolated in different regions of Uruguay. Susceptibility to oxacillin and cefoxitin was studied by gradient diffusion, disk diffusion to cefoxitin, and Phoenix and MicroScan systems. PBP2a was determined. SCCmec typing was performed and the isolates were compared by PFGE. Twenty-six isolates were susceptible to oxacillin; one strain was susceptible to cefoxitin by disk diffusion and 3 strains by gradient diffusion. Phoenix and MicroScan panels detected methicillin resistance in 25 and 27 strains, respectively. Twenty-six strains tested positive for PBP2a. Twenty-six strains carried SCCmec V and 24 belonged to pulsotype A. One strain carried SCCmec IV and did not belong to pulsotype A. Cefoxitin disk diffusion test and PBP2a detection correctly identified 26 of these 27 strains as MRSA. PFGE results suggest the dissemination of a cluster of MRSA carrying SCCmec V.

Predictors of mortality and clinical characteristics among carbapenem-resistant or carbapenemase-producing Enterobacteriaceae bloodstream infections in Spanish children.

Carbapenem-resistant Enterobacteriaceae (CRE) are an emerging problem in the paediatric population worldwide with high mortality rates in bloodstream infection (BSI). To evaluate predictors of 30 day mortality in CRE BSI in a paediatric cohort. A retrospective observational single-centre study (December 2005-August 2018) was conducted. Cases of CRE BSI in children 0 to 16 years were included. Microbiological identification (MALDI Biotyper) and antimicrobial susceptibility testing (Vitek2® and MicroScan panel NBC44) according to EUCAST breakpoints were performed. PCR OXVIKP® was used to confirm carbapenemase genes (OXA-48, VIM, KPC, NDM). Demographic characteristics, underlying diseases, source of bacteraemia, antimicrobial therapy and outcomes were collected from medical records. Survival analysis to establish predictors of 30 day mortality was performed. Thirty-eight cases were included; 76.3% were hospital-acquired infections and 23.7% related to healthcare. All patients had at least one underlying comorbidity and 52.6% were recipients of an organ transplant. VIM carbapenemase was the predominant mechanism (92.1%). Previous CRE colonization or infection rate was 52.6%. Intestinal tract (26.3%) and vascular catheter (21.1%) were the most common sources of infection. Crude mortality within 30 days was 18.4% (7/38); directly related 30 day mortality was 10.5%. Conditions associated with an increment in 30 day mortality were intensive care admission and inadequate empirical therapy (P < 0.05). Combination-antibiotic targeted treatment and a low meropenem MIC were not related to improved survival. CRE BSI mortality rate is high. The most important factor related to 30 day survival in our CRE BSI cohort in children was empirical treatment that included at least one active antibiotic.

First report of Myroides phaeus bacteraemia identified by Polymerase chain reaction and genetic sequencing

We report the first case of Myroides phaeus isolated from blood, causing bacteremia in an immunocompromised patient using the automated MicroScan Walk Away 96 system, followed by bacterial identification by amplification-sequencing of the 16S rDNA. The sequences obtained were compared with the reference sequence of the BLAST ® platform - National Library of Medicine, USA, and the isolation was identified as Myroides phaeus strain with 99.67 % identity in Blast report. In the literature we did not find previous reported cases of infections by this bacterium, however its pathogenic role is still controversial; therefore, this isolation alerts us to carry out an exhaustive surveillance of other possible acquisition routes.

Staphylococcus aureus carriage in older populations in community residential care homes: Prevalence and molecular characterization of MRSA isolates

IntroductionThe epidemiology of S. aureus depends on conditions in specific populations. Few studies of S. aureus colonization in the older population have been performed in Spain. The aim of this study was to determine the prevalence of methicillin-resistant S. aureus (MRSA) colonization and its molecular epidemiological characteristics in an institutionalized population in community residential care homes in Cadiz, Spain. MethodsA cross-sectional epidemiological study was conducted in three residential care homes for older people. Axilla and nostril samples were tested. Identification of S. aureus and antimicrobial susceptibility testing were by MALDI-TOF and MicroScan panels. MRSA strains were subjected to SCCmec typing, multilocus sequence typing (MLST) and pulsed-field gel electrophoresis (PFGE). The presence of Panton–Valentine leukocidin (PVL) genes was determined by PCR in all S. aureus strains. ResultsA total of 293 residents were included. Fifty-one residents (17.4%) were colonized with methicillin-sensitive S. aureus (MSSA) and 11 (3.8%) with MRSA. Resistance to at least two aminoglycosides was observed in 25.4% of MSSA and 90.9% and of MRSA isolates, and resistance to levofloxacin in 80.3% of MSSA and 100% of MRSA isolates. SCCmecIV was detected in all isolates and all except one (ST-125) were ST-8. None of the S. aureus isolates were positive for PVL. ConclusionsA low rate of S. aureus carriage was detected and the prevalence of MRSA was very low. ST8-MRSA-IVc was the dominant clone, and only one strain belonged to ST125-MRSA-IVc. We found MRSA transmission within the residential care homes and a very high rate of quinolone resistance in MSSA and MRSA.

Staphylococcus aureus carriage in older populations in community residential care homes: Prevalence and molecular characterization of MRSA isolates

IntroductionThe epidemiology of S. aureus depends on conditions in specific populations. Few studies of S. aureus colonization in the older population have been performed in Spain. The aim of this study was to determine the prevalence of methicillin-resistant S. aureus (MRSA) colonization and its molecular epidemiological characteristics in an institutionalized population in community residential care homes in Cadiz, Spain. MethodsA cross-sectional epidemiological study was conducted in three residential care homes for older people. Axilla and nostril samples were tested. Identification of S. aureus and antimicrobial susceptibility testing were by MALDI-TOF and MicroScan panels. MRSA strains were subjected to SCCmec typing, multilocus sequence typing (MLST) and pulsed-field gel electrophoresis (PFGE). The presence of Panton–Valentine leukocidin (PVL) genes was determined by PCR in all S. aureus strains. ResultsA total of 293 residents were included. Fifty-one residents (17.4%) were colonized with methicillin-sensitive S. aureus (MSSA) and 11 (3.8%) with MRSA. Resistance to at least two aminoglycosides was observed in 25.4% of MSSA and 90.9% and of MRSA isolates, and resistance to levofloxacin in 80.3% of MSSA and 100% of MRSA isolates. SCCmecIV was detected in all isolates and all except one (ST-125) were ST-8. None of the S. aureus isolates were positive for PVL. ConclusionsA low rate of S. aureus carriage was detected and the prevalence of MRSA was very low. ST8-MRSA-IVc was the dominant clone, and only one strain belonged to ST125-MRSA-IVc. We found MRSA transmission within the residential care homes and a very high rate of quinolone resistance in MSSA and MRSA.

Método rápido para la detección de la sensibilidad a cefotaxima en enterobacterias

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.

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.

Systematic Evaluation of Commercial Susceptibility Testing Methods for Determining the In Vitro Activity of Daptomycin versus Staphylococcus aureus and Enterococci

We systematically evaluated 5 methods for testing daptomycin versus 48 Enterococcus faecalis, 51 Enterococcus faecium, and 50 Staphylococcus aureus isolates using (i and ii) broth microdilution (BMD) with 50-mg/liter calcium medium supplementation (reference method) and 30-mg/liter calcium medium supplementation (BMD30 method), (iii) Etest, and (iv and v) MicroScan panel 33 using 2 methods to prepare the bacterial inoculum (MicroScan turbidity and MicroScan Prompt). Isolates were categorized as susceptible (S) or nonsusceptible (NS) based on measured MICs. Essential (± 1 dilution) agreement (EA) and categorical (S/NS) agreement (CA) for each method were compared to the reference method. For E. faecium, categorical agreement was poor between the reference method and BMD30 as well as with the three commercial methods, with frequent false-NS results (30 for BMD30, 18 for Etest, 22 for MicroScan Prompt, and 25 for MicroScan turbidity). All E. faecalis isolates were judged to be S by the reference method; two of these isolates were categorized as NS using the BMD30 method, and one was categorized as NS by all three commercial methods. All S. aureus isolates were judged to be S using all five methods. MIC values determined by the comparator methods tended to be higher than those for the reference method, especially for E. faecium isolates. EAs between the reference BMD and BMD30, Etest, MicroScan Prompt, and MicroScan turbidity were 63%, 63%, 63%, and 56%, respectively, for E. faecium, 87%, 83%, 98%, and 80%, respectively, for E. faecalis, and all 100% for S. aureus.

Detection of bla SHV and bla CTX-M genes in ESBL producing Klebsiella pneumoniae isolated from Egyptian patients with suspected nosocomial infections

The correct identification of the genes involved in ESBL mediated resistance is necessary for the surveillance and epidemiological studies of their transmission in hospitals. The aim of the present study was to find the prevalence of ESBL producing Klebsiella pneumoniae among K. pneumoniae isolates separated from Egyptian patients with suspected nosocomial infections, to detect their drug resistance pattern and to look for bla SHV and bla CTX-M genes in such organisms. Subjects and methods: 138 K. pneumoniae isolates from Egyptian patients with suspected nosocomial infections were screened for ESBL production by the pattern of antimicrobial susceptibilities. Phenotypic identification for ESBL production was confirmed by double disc synergy test, phenotypic confirmatory double disc test and by MicroScan panel system. bla SHV and bla CTX-M genes in ESBL producing K. pneumoniae were detected using multiplex PCR. Results: The prevalence of ESBL producing K. pneumoniae was 21% (30/138). Their pattern of antimicrobial susceptibility showed that 90% was resistant to (Sulphamethoxazole/Trimethoprim), 70% was resistant to (Amoxicillin/Clavulanate), 63.3% was resistant to Cefotaxime and Ceftazidime, 60% was resistant to Amikacin, 46.7% was resistant to Doxycycline, Cefoxitin, Ceftriaxone and Levofloxacin, 40% was resistant to Cefepime, 20% was resistant to Ertapenem and (Sulbactam/Cefoperazone), 13.3% was resistant to (Piperacillin/Tazobactam), 10% was resistant to (Imipenem/Cilastatin) and Gentamycin and 6.7% was resistant to Meropenem and Ciprofloxacin. Among the ESBL producing K. pneumoniae, three out of 30 (10%) and 16 out of 30 (53.3%) were positive for bla SHV and bla CTX-M genes respectively.It could be concluded that ESBL producing isolates of K. pneumoniae have been increasingly recognized in the hospital settings in Egypt and are associated with multiple drug resistance. Thus, molecular identification of the genes encoding beta lactamases would be essential for a reliable epidemiological investigation of their transmission in hospitals and antimicrobial resistance.

Multilaboratory Study of the Biomic Automated Well-Reading Instrument versus MicroScan WalkAway for Reading MicroScan Antimicrobial Susceptibility and Identification Panels

This study compared the Biomic automated well reader results to the MicroScan WalkAway results for reading MicroScan antimicrobial susceptibility and identification panels at four different sites. Routine fresh clinical isolates and quality control (QC) organisms were tested at each study site. A total of 46,176 MicroScan panel drug-organism combinations were read. The Biomic category agreement for 3,117 Gram-negative bacteria was 98.4%, with 1.4% minor and 0.2% major discrepancies. The Biomic category agreement for 5,233 Gram-positive bacteria was 98.7%, with 0.9% minor, 0.3% major, and 0.1% very major errors. Essential agreement, defined as Biomic results that were within ±1 2-fold dilution of the MicroScan results, was 99.3% for Gram-negative bacteria and 98.3% for Gram-positive bacteria. Biomic reading of MicroScan identification panels provided an overall agreement (first- and second-choice organism match) of 99.5% with 846 Gram-negative isolates and 99.5% with 430 Gram-positive isolates. These results suggest that the Biomic automated reader can provide accurate reading of MicroScan panels and has the capability of a visual panel read for manual adjustment of results.

False Daptomycin-Nonsusceptible MIC Results by Microscan Panel PC 29 Relative to Etest Results for Staphylococcus aureus and Enterococci

This study correlated the daptomycin MIC results obtained by Microscan and by Etest in Staphylococcus aureus and enterococci and found that the Microscan panel GP 29 had a high rate of false nonsusceptible results. Of the isolates interpreted as nonsusceptible by Microscan, 87% of Staphylococcus aureus, 90% of Enterococcus faecalis, and 88% of Enterococcus faecium isolates were interpreted as susceptible by Etest. In turn, Etest also has false nonsusceptible results compared to reference methods.

Colistin resistance in KPC-producing Klebsiella pneumoniae strains from a high specialization rehabilitation facility

The worldwide rapid spread of KPC carbapenemase-producing Klebsiella pneumoniae (KPC-Kp) represents an increasing problem in clinical settings. Reports on KPC-Kp epidemic spread in Italian hospitals began to appear since 2010; colistin (COL) represents one of the few remaining therapeutic options available for the treatment of such multi drug-resistant (MDR) pathogens. Here we report the presence and diffusion of COL resistant KPC-Kp isolates from a High Specialization Rehabilitation Facility located in Northern Italy. Species identification and antimicrobial susceptibilities were obtained by NBC46/NM40 Microscan panels (Siemens); imipenem, meropenem and ertapenem MICs were also evaluated by Etest and broth microdilution method; blaKPC-like genes PCR were performed. PFGE (XbaI) was used to investigate clonal relatedness; epidemiological data were collected from the hospital database. Seventy-five carbapenem-resistant K. pneumoniae isolates were collected from the Fondazione S. Maugeri hospital during the period January-June 2011. Seven out of 75 MDR KPC-Kp isolates by Microscan System showed COL resistance (MIC &gt;2 mg/L). Among them, 5/7 were collected from coma and 2/7 from cardiology and rehabilitation cardiology wards. Most of these strains were from urine (5/7); the remaining 2/7 were from blood and bronco-alveolar lavage. The 85.7% of the strains showed susceptibility to tigecycline and fosfomycin; 71.4% only to gentamicina, 28.5% to trimethoprim/sulfamethoxazole and 14.2% to amikacin. The PFGE profiles obtained analyzing 5/7 isolates from patients hospitalized from almost 10 days, showed clonal relatedness between 4/5 isolates, thus confirming the high epidemic potential of almost one KPC-Kp clinical strain collected from 4 different wards.The emergence of COL resistance in KPC-Kp, dramatically reduces the available therapeutic options. These results underline the ability of a COL resistant KPC-producing clone to rapidly spread within this Rehabilitation Facility.

Multifocal diffusion of KPC-3-producing ST512 Klebsiella pneumoniae in Italy

Introduction. The dissemination of carbapenemase-producing Klebsiella pneumoniae strains is an increasing problem worldwide. KPC ß-lactamases are Ambler class A enzymes mostly plasmid-encoded; their global spread represents a threat to clinical patients care and public health. Multi locus sequence type (ST)258 is currently the most spread K. pneumoniae clone associated with KPC enzymes. Here we report the first identification and multifocal spread of KPC-3 producing K. pneumoniae clinical strains belonging to ST512 in Italy. Materials and Methods. Fifty six carbapenem-resistant K. pneumoniae isolates were collected from 7 Italian hospitals during the period June 2009-May 2011. Isolates were obtained from different wards (spinal unit, medicine, hematology, etc.) and biological samples (mostly rectal swabs, urine and blood). Species identification and antimicrobial susceptibilities were obtained by NBC46/NM40 Microscan panels (Siemens). MICs values were interpreted according to EUCAST 2011 breakpoints. Modified Hodge test and combined disk test with phenyl-boronic acid (BOR) and EDTA were performed.The presence of blaKPC genes were confirmed by PCR and sequencing. A complete characterization of the produced ß-lactamases (BLs) was obtained by IEF followed by PCR experiments using primers specific for the detection of blaCTX-M-, blaTEM- and blaSHV type genes. PFGE and multilocus sequence typing (MLST) were both used to investigate clonal isolates relatedness. Results. All 56 isolates resulted positive for the presence of KPC-type carbapenemases by both phenotypical and molecular analysis. Fifteen isolates, chosen as representative, were further investigated. Ten out of 15 isolates harboring the blaKPC-2 gene clustered with the known ST258, while the remaining 5/10 belonged to the newly described ST512 and harbored the blaKPC-3 gene. ST512 isolates, from 3/7 hospitals, were collected from rectal swabs (40%), blood (20%), endotracheal aspirate (20%) and urinary (20%) samples. Carbapenem MICs ranged from 2 mg/L to more than 16 mg/L; the isolates were characterized by the same multi-drug-resistant (MDR) profile. Only in 1/5 cases such antimicrobial resistance profile was extended also to colistin (&gt;2 mg/L).Three different PFGE clones were identified (A, B and C); one resulting characteristic of each hospital. Coherently with pIs obtained by IEF, the presence of additional BLs were investigated by PCR; clones B and C resulted positive for blaCTX-M and blaSHV genes, while blaSHV and blaTEM genes were found in clone A. Conclusion.Intra- and inter-hospital spread of KPC-positive strains was demonstrated.The heterogeneity of PFGE profiles showed the spread of different K. pneumoniae ST258 clones and the emergence of the new ST512 coupled with blaKPC-3 genes in Italian clinical settings. Strategies for carbapenemase detection and appropriate hospital hygiene precautions are indicated to limit the spread of the here described ST512 clone appears essential.

English

Staphylococcus lugdunensis can cause virulent infections in immunosuppressed individuals. Here we describe a retrospective analysis of hemodialysis patients with Staphylococcus lugdunensis bacteremia admitted to a New York City hospital from January 2005 to December 2010. Six cases of S. lugdunensis bacteremia were identified. The majority had received antibiotic therapy for at least several weeks. None of them developed endocarditis or other serious complications. Four had their tunneled permanent dialysis catheters changed or removed. This pathogen is rare, but could often fail medical therapy alone and may require surgical intervention. This can be avoided with surveillance and early intervention in patients with high risk for infection. Key wordsStaphylococcus lugdunensis, Staphylococcus, bacteremia, infection, hemodialysis. International Journal of Microbiology Research ISSN: 0975-5276 & E-ISSN:0975-9174, Volume 4, Issue 2, 2012 Introduction S. lugdunensis is coagulase negative staphylococcus described by Fleurette et al in 1989 [1] but has been recently described as an emerging human pathogen [2]. While characterized as coagulase negative, because it does not produce coagulase, there is a membrane bound form of the enzyme in some isolates that can cause it to be misidentified as S. aureus [3]. Although it is considered a skin normal flora, it can cause virulent infections usually in immunosuppressed patients with implanted medical devices including bone replacements and cardiac valve replacements [4]. We report cases of S. lugdunensis bacteremia in hemodialysis patients with permanent tunneled cuffed catheters admitted to a New York City hospital. Methods At least one set of blood cultures were taken from each patient. Blood culture was collected in an adult Bac T/ALERT blood culture set, and upon receipt by the Microbiology laboratory, it was incubated in Bac T/ALERT instrument. Positive blood cultures were then Gram stained and sub cultured on TSA, MacConkey and Chocolate plated media. Next Day Positive cultures on the plate were identified using Walk Away MicroScan panels. Results Patients on hemodialysis who were infected with S. lugdunensis as a cause of bacteremia were identified. As shown in Table 1, all patients were African American and represented both genders equally. The average patient age was 53.8 years and the majority (83%) had hypertension among other co-morbidities. Half of the patients had their catheter placement at the NYC hospitals or its affiliates and 75% of patients had their hemodialysis treatment site elsewhere. The majority of patients were on dialysis for 2 years or less and none of the patients suffered any complications at time of discharge. Antibiotic sensitivities of S. lugdunensis cultured from infected patients are shown in Table 2. Isolates obtained from all patients were sensitive to ceftriaxone, clindamycin, oxacillin, rifampin, trimethoprim/sulfamethoxisole and vancomycin and resistant to penicillin. Of those tested, one of six patients was separately resistant to gentamycin and tetracycline. A brief summary of each of the patients infected with S. lugdunensis follows: Citation: Mallappallil M.C., et al. (2012) Staphylococcus lugdunensis bacteremia in hemodialysis patients. International Journal of Microbiology Research, ISSN: 0975-5276 & E-ISSN:0975-9174, Volume 4, Issue 2, pp.-178-181. Copyright: Copyright©2012 Mallappallil M.C., et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Usefulness of Microscan System panels with EUCAST clinical breakpoints to evaluate the antimicrobial susceptibility of ß-lactamase producing- Gram negative isolates

The study aimed to evaluate the ability of NBC45, NBC46 and NB40 Microscan (MS) panels, updated to 2010 EUCAST breakpoints, to identify at species level and to correctly define the susceptibility to s-lactams of 61 s-lactamases (BLs) producing Gram-negative isolates. A collection of 73 fully identified strains was analyzed: 21 Klebsiella spp., 17 E. coli, 15 P. mirabilis, 9 A. baumannii (Ab), 7 P. aeruginosa and 4 Enterobacter spp.. 61/73 were BLs and/or carbapenemases producers: 15 were CTX-M-1/-2/-14/-15 positive, and among them two were also VIM-1 positive. Four were TEM-52/-92, 3 PER-1, 2 SHV-12/-18 and 6 CMY-16 producers, while 11 were KPC-2/-3, 9 OXA-51/-58/-23, 8 VIM-1 and 2 IMP-13 positive. One K. oxytoca K-1 iper-producer, 11 non-BL producers/ATCC control strains and a OprD2 porin lacking P. aeruginosa were also included. All isolates were identified by Api-20E and VITEK-2 System and antibiotic susceptibilities were obtained by broth microdilution method. Resistance genes were identified by PCR and sequencing. All 73 isolates were correctly identified and a complete agreement for susceptibility patterns was observed for both ATCC control strains and non-BL clinical isolates. MS failed to detect a BL/Extended-Spectrum-s-Lactamase (ESâL) production in 5/61 cases: any ESsL alert was detected using NBC46 panel for 3/15 CTX-M positive strains and 2 VIM-1/CTX-M-15 producing K. pneumoniae isolates. Intermediate resistance to cefoxitin (MIC 16 mg/L), susceptibility to cefepime (MIC 8 mg/L for ertapenem (ETP), according to previously results. All VIM-1 producers resulted intermediate/resistant to imipenem (IP) and meropenem (MP); decreased MIC values were observed in 2/8 cases. Carbapenem MICs >8 mg/L were detected for IP-13 P. aeruginosa producers; 6/9 OXA carbapenemases- producing Ab showed IP MIC >8 mg/L and 3/6 MP MIC >8 mg/L. 3/9 Ab OXA-58/-51 producers, tested using NB40 panel, were intermediate or resistant to doripenem and meropenem. Regarding the detection of BLs overall agreement between MS and reference methods was 91.9%. Carbapenems MIC values resulted a fold lower than previously determined. Nevertheless using 2010 EUCAST breakpoints for ETP, MP and IP was possible to detect all carbapenemases- producers. MS System represents a useful tool to perform identification of BL- producing Gram negative bacteria.

Accuracy of Commercial Susceptibility Testing Method for Measuring Vancomycin MIC Against Methicillin-ResistantStaphylococcus aureus(MRSA)

Methods: We evaluated the vancomycin (VAN) minimum inhibitory concentrations (MIC) on methicillin-resistant Staphylococcus aureus (MRSA) strains using MicroScan panel (Pos combo 3.1J). We also used the following 2 methods of preparing the bacterial solution: MicroScan Prompt system (P method) and manual preparation with inoculums adjusted to match a 0.5 McFarland turbidity standard (M method). The Clinical and Laboratory Standards Institute (CLSI) method was used as a reference. Results: On comparing the distribution of VAN MICs, the CLSI and M methods showed a good correlation, while the P method results were higher. Colony counts indicated the final inoculum concentrations from the P method were higher than those from the M method, which were within the ideal inoculum concentration. Comparing the results between the M method with the W/A 96 System (18hour incubation period) and the M method with a 24-hour incubation period revealed a discrepancy in the detection of some strains with 2.0 µg/mL of VAN. Conclusion: The P method appears to be less reliable than the M method for measuring VAN MIC against MRSA. Hence, the M method with a 24-hour incubation period should be used instead.

Performance of VITEK-2 Compact and overnight MicroScan panels for direct identification and susceptibility testing of Gram-negative bacilli from positive FAN BacT/ALERT blood culture bottles

We describe the reliability of the VITEK-2 Compact and overnight MicroScan panels for direct identification and susceptibility testing from the BacT/ALERT blood culture system when using FAN (FA and FN) bottles. A simple procedure, in two centrifugation steps, was designed to remove the charcoal particles present in FA and FN bottles. A total of 113 positive blood cultures showing Gram-negative rods were investigated. Enterobacteriaceae were isolated in 104 cases, and Pseudomonas aeruginosa in nine. The MicroScan system correctly identified 106 (93.8%) of the 113 isolates. The seven identification errors included P. aeruginosa (three), Enterobacter cloacae (one), Escherichia coli (one), Klebsiella oxytoca (one), and Klebsiella pneumoniae (one). The VITEK-2 system correctly identified 109 (96.5%) of the 113 samples obtained directly from the blood culture bottles. The four unidentified isolates were Enterobacter cloacae (two), Escherichia coli (one), and P. aeruginosa (one). MicroScan yielded 4/779 (0.5%) very major errors and 28/2825 (0.9%) minor errors. VITEK-2 yielded 2/550 (0.36%) very major errors, 1/1718 (0.05%) major error, and 32/2373 (1.3%) minor errors. Both systems provided excellent identification (correlation of >90%) and susceptibility (correlation of >98%) results. The average times required to obtain identification and susceptibility results using the direct test applied to the VITEK-2 Compact system were 4.57 ± 1.37 h and 6.52 ± 1.64 h, respectively. The VITEK-2 compact system provided results on the same day that the blood culture was found to be positive.