agar and Gram-Positive-Bacterial-Infections

Spectra™ VRE agar (Remel, Lenexa, KS) is a chromogenic agar that is FDA approved for screening patients for VRE colonization. The package insert recommends confirming isolates with identification and susceptibility testing, but confirming every culture delays time to result. Given the agar's historic high specificity for E. faecium isolates, we theorized the agar could be utilized as a stand-alone screening to minimize reagents and time.. Our laboratory sought to develop a workflow to optimize the use of the medium.. We plated 3,815 rectal swabs to the Spectra VRE agar and compared results to traditional identification and susceptibility testing.. Dark blue or purple colonies on the agar demonstrated a sensitivity of 98% and specificity of 85% for detection of VRE faecium, but light blue colonies were significantly less specific for E. faecalis.. We streamlined our workflow to accept dark blue or purple colonies as VRE faecium and plan to perform additional testing only on light blue colonies. Interestingly, higher quantity of growth increased the accuracy of the agar. In the future, growth quantity may be used to further streamline the workflow once more data is obtained.

Topics: Agar; Anti-Bacterial Agents; Enterococcus faecalis; Enterococcus faecium; Gram-Positive Bacterial Infections; Humans; Vancomycin; Vancomycin Resistance; Vancomycin-Resistant Enterococci; Workflow

E. faecium and E. faecalis are the common species of Enterococcus responsible for the majority of infections. Earlier, species other than the common ones were usually unidentified and reported as Enterococcus species. However, modern equipment, like MALDI-TOF and VITEK2, have been utilitarian, helping us to identify the previously unidentified species. E. hirae is an organism seldom reported to cause human infections. Here, we report a case of a biliary tract infection in a female patient with cholangiocarcinoma caused by E. hirae.. A 56-year-old female presented with fever and abdominal pain. Bile aspirated during the ERCP was received in our laboratory. The gram stain of the bile sample revealed abundant polymorphonuclear leucocytes along with gram-positive diplococci. The organism failed to grow on MacConkey agar. On blood agar, non-hemolytic colonies grew. The organism was identified as E. hirae by MALDI-TOF MS. The antibiotic susceptibility performed using VITEK2 revealed it to be resistant to high-level gentamicin and susceptible to all remaining drugs. She was successfully treated with oral ciprofloxacin for the infection.. Bile is colonized with bacteria due to obstruction in the biliary tree, leading to cholangitis. This causes bacterial proliferation and translocation of bacteria into the systemic circulation. Our case was resistant to high-level gentamicin, while all previously reported cases were susceptible. The resistant isolates of E. hirae being isolated from cattle and their surroundings amidst the rampant use of antibiotics in livestock can pose a difficult situation for humans. Thus, there should be regulations on antibiotic usage in livestock. Cases like these should be reported and recognized for their potential to cause outbreaks if they remain unreported.. Thus, E. hirae, when encountered, should not be ignored but considered a pathogen and reported. The presence of drug-resistant organisms in cattle and their surroundings, their zoonotic potential to cause infections in humans, and the uncontrolled usage of antibiotics in livestock are causes for concern. Thus, we need to be more vigilant regarding it in the future.

Topics: Agar; Animals; Anti-Bacterial Agents; Bacteria; Biliary Tract; Cattle; Cholangiocarcinoma; Drug Resistance, Bacterial; Enterococcus; Female; Gentamicins; Gram-Positive Bacterial Infections; Humans; Microbial Sensitivity Tests; Middle Aged

From 1 January to 31 December 2020, forty-nine institutions around Australia participated in the Australian Enterococcal Sepsis Outcome Programme (AESOP). The aims of AESOP 2020 were to determine the proportion of enterococcal bacteraemia isolates in Australia that were antimicrobial-resistant, and to characterise the molecular epidemiology of the E. faecium isolates. Of the 1,230 unique episodes of enterococcal bacteraemia investigated, 93.9% were caused by either E. faecalis (54.2%) or E. faecium (39.7%). Ampicillin resistance was not detected in E. faecalis but was detected in 88.2% of E. faecium . Vancomycin non-susceptibility was detected in 0.2% of E. faecalis and 32.6% of E. faecium . Overall, 35.2% of E. faecium harboured vanA and/or vanB genes. For the vanA/B positive E. faecium isolates, 38.8% harboured the vanA gene, 60.6% the vanB gene, and 0.6% harboured both vanA and vanB . Although the percentage of E. faecium bacteraemia isolates was significantly lower than that detected in the 2019 AESOP (presumably due to the COVID-19 elective surgery restrictions placed on hospitals), it remains substantially higher than that recorded in most European countries. The E. faecium isolates detected consisted of 71 multilocus sequence types (STs), with 81.7% of these isolates classified into eight major STs each containing ten or more isolates. All major STs belonged to clonal cluster 17 (CC17), a major hospital-adapted polyclonal E. faecium cluster. The major STs (ST17, ST1424, ST80, ST796, ST78, ST1421, ST555 and ST117) were found across most regions of Australia. The predominant clone was ST17, which was identified in all regions except the Northern Territory. Overall, 40.9% of isolates belonging to the eight major STs harboured the vanA or vanB gene. The AESOP 2020 has shown enterococcal bacteraemia episodes in Australia are frequently caused by polyclonal ampicillin-resistant high-level gentamicin-resistant vanA - or vanB -positive E. faecium which have limited treatment options.

Topics: Agar; Anti-Bacterial Agents; Bacteremia; COVID-19; Drug Resistance, Bacterial; Enterococcus; Gram-Positive Bacterial Infections; Humans; Northern Territory; Sepsis

Colonized surfaces, equipment, individuals, and infected patients can be sources for the hospital spread of vancomycin-resistant enterococci (VRE), which is one of the important nosocomial pathogens. The basic epidemiological tools for the prevention and control of hospital-acquired infections are the typing methods. Pulsed-field gel electrophoresis (PFGE) is a highly discriminating method used frequently to detect clonal associations in epidemics and hospital-acquired VRE infections. This study aimed to investigate the presence of cross-contamination, which service or services come to the forefront in case of possible cross-contamination and clonal relationship between VRE strains isolated from rectal swab, clinical and environmental swab samples taken in two different periods in various clinics of Istanbul University Istanbul Medical Faculty Hospital by PFGE method. A total of 125 VREs isolated in two different periods were included in the study. Rectal and environmental swab samples were inoculated on Enterococcosel agar and sodium azide broth, urine samples were inoculated on chromogenic agar, and other clinical samples were inoculated on 5% sheep blood agar and incubated for 18-24 hours. VITEK 2 Compact automation system GP panel (bioMerieux, MarcyL'Etoile, France) was used for the species identification of catalase-negative, gram-positive colonies and disc diffusion and minimum inhibitory concentration (MIC) gradient tests were used to determine vancomycin susceptibility. Multiplex polymerase chain reaction was used to search for vanA and vanB resistance genes in isolates identified as VRE, and PFGE was used to determine clonal association. All isolates were identified as Enterococcus faecium with the vanA resistance gene. It was shown that PFGE clones were divided into six types with 65% similarity (A-F), and in this polyclonal spread, the major type was type A, which contained 108 isolates in 37 subtypes existed in the hospital for years. In patients from whom similar isolates were obtained, the high rate of hospitalizations in the same emergency room or in different emergency services in the same building drew attention. Our results showed that the presence of VRE was established in our hospital, new isolates were added from time to time, and there was a cross-contamination. It was observed that emergency services, where infection control measures were neglected due to working conditions, were among the high-risk areas for VRE contamination. In o

Topics: Agar; Bacterial Proteins; Cross Infection; Electrophoresis, Gel, Pulsed-Field; Emergency Service, Hospital; Enterococcus faecium; Gram-Positive Bacterial Infections; Humans; Vancomycin-Resistant Enterococci

From 1 January to 31 December 2021, forty-eight institutions around Australia participated in the Australian Enterococcal Surveillance Outcome Programme (AESOP). The aim of AESOP 2021 was to determine the proportion of enterococcal bacteraemia isolates in Australia that were antimicrobial resistant, and to characterise the molecular epidemiology of the Enterococcus faecium isolates. Of the 1,297 unique episodes of enterococcal bacteraemia investigated, 94.4% were caused by either E. faecalis (54.1%) or E. faecium (40.3%). Ampicillin resistance was detected in one E. faecalis isolate and in 89.3% of E. faecium isolates. Vancomycin non-susceptibility was not detected in E. faecalis but was detected in 37.9% of E. faecium. Overall, 39.6% of E. faecium harboured the vanA and/or vanB genes. For the vanA/vanB positive E. faecium isolates, 35.8% harboured the vanA gene and 64.2% the vanB gene. Although the percentage of vancomycin-resistant E. faecium bacteraemia isolates was significantly lower than that reported in the 2020 AESOP report (presumably due to the COVID-19 elective surgery restrictions placed on hospitals), it remains substantially higher than that recorded in most European countries. Isolates of E. faecium consisted of 73 multi-locus sequence types (STs); 77.2% of isolates were classified into seven major STs each containing more than ten isolates. All major STs belonged to clonal cluster (CC) 17, a major hospital-adapted polyclonal E. faecium cluster. The major STs (ST17, ST1424, ST796, ST78, ST80, ST1421 and ST555) were found across most regions of Australia. The predominant ST was ST17 which was identified in all regions except the Northern Territory. Overall, 46.5% of isolates belonging to the seven major STs harboured the vanA or vanB gene. The AESOP 2021 has shown that enterococcal bacteraemia episodes in Australia are frequently caused by polyclonal ampicillin-resistant high-level gentamicin resistant vanA- or vanB-positive E. faecium which have limited treatment options.

Topics: Agar; Anti-Bacterial Agents; Bacteremia; COVID-19; Drug Resistance, Bacterial; Enterococcus; Gram-Positive Bacterial Infections; Humans; Microbial Sensitivity Tests; Northern Territory; Vancomycin

Enterococcus faecalis and Enterococcus faecium can cause community-acquired and nosocomial infections. Combination antibiotic therapies have a distinct advantage over monotherapies in terms of their synergistic effect. In the study, it was aimed to investigate in vitro activity of vancomycin combined with fosfomycin by agar dilution method against VRE strains.. A total of 30 clinical VRE strains were included in the study. Bacterial identifications of the strains were undertaken using conventional routine methods. The resistance to vancomycin was investigated using the broth microdilution method compared to fosfomycin by agar dilution method, and the results were interpreted in accordance with the CLSI guidelines. All experiments contained 25 mg/mL glucose-6-phosphate for fosfomycin. The fractional inhibitory concentration (FIC) indexes (FICI) were interpreted as synergism, FICI ≤ 0.5. Additionally, two strains in 30 VRE were studied to determine the time-kill curves to verify the synergistic results. Both antibiotics were studied at 1 x MIC in the tests. Viable counts were determined at 0, 4, 8, 12, and 24-hour intervals. Time-kill curves were constructed by plotting mean colony forming unit counts versus time.. Susceptibility rate to fosfomycin was found at 16.6% (5/30). The MIC50,90 and MICrange values of antimi-crobials were 512, 512, and 512 - 1,024 mg/L for vancomycin, and 128, 128, and 64 - 160 mg/L for fosfomycin. The rate of synergism was found as 100%.. The result shows that the combination of vancomycin with fosfomycin gives hope that it may be an option in the treatment of infections caused by VRE.

Topics: Agar; Anti-Bacterial Agents; Enterococcus faecium; Fosfomycin; Gram-Positive Bacterial Infections; Humans; Microbial Sensitivity Tests; Vancomycin

Linezolid is an alternative treatment option for infections with multidrug-resistant Gram-positive bacteria including vancomycin-resistant enterococci. Some countries report an increasing number of isolates with resistance to linezolid. The recent publication of the Commission for Hospital Hygiene in Germany on enterococci/VRE recommends screening for linezolid-resistant enterococci (LRE). However, a suitable selective medium or a genetic test is not available. Our aim was to establish a selective screening agar for LRE detection and validate its application with a comprehensive collection of clinical LRE and linezolid-susceptible enterococci.. We decided to combine the selective power of an enterococcal screening agar with a supplementation of linezolid. Several rounds of analyses with reference, control and test strains and under varying linezolid concentrations of a wider and a smaller range were investigated and assessed. The collection of linezolid-resistant enterococcal control strains included isolates with different resistance mechanisms (23S rDNA mutations, cfr(B), optrA, poxtA). Finally, we validated our LRE screening agar with 400 samples sent to our National Reference Centre in 2019.. Several rounds of pre-tests and confirmatory analyses favored Enterococcosel® Agar supplemented with a concentration of 2 mg/L linezolid. A 48 h incubation period was essential for accurate identification of LRE strains. Performance of the LRE screening agar revealed a sensitivity of 96.6% and a specificity of 94.4%.. Here we describe preparation of a suitable screening agar and a procedure to identify LRE isolates with high accuracy.

Topics: Agar; Anti-Bacterial Agents; Drug Resistance, Multiple, Bacterial; Feasibility Studies; Germany; Gram-Positive Bacterial Infections; Humans; Linezolid; Mass Screening; Microbial Sensitivity Tests; Vancomycin-Resistant Enterococci

Topics: Abiotrophia; Agar; Culture Media; Gram-Positive Bacterial Infections; Staphylococcus aureus

The main objective of the present study is to analyze different genotypic and phenotypic traits related to virulence in Enterococcus faecalis, as well as evaluated the agar invasion phenotype in a collection of isolates with different clinical origins.. Seventy-nine E. faecalis isolates, with invasive and non-invasive clinical origins, have been used in this work. Presence of cytolysin activator (cylA), gelatinase (gelE), surface protein (esp), aggregation substance (asa1), endocarditis antigen (efaA), and collagen-binding protein (ace) have been analyzed by PCR. Phenotypic characterization included gelatinase activity, haemolysin production, biofilm formation and agar invasion.. All the isolates tested harboured at least one of the virulence determinants. The 95.5% of isolates from haematologic samples were positive for agar invasion test, significantly higher than isolates from non-invasive diseases. A significant reduction in relative invasion area was observed in three selected agar-invasive strains after 15 serial passages.. It has been observed a significant high prevalence of agar-invasion positive isolates among strains belonged to haematological samples. Agar invasiveness is reduced after adaptation of clinical isolates to laboratory conditions, showing that agar invasion phenotype can be modulate by culture conditions as other virulence factors observed in different bacterial species.

Topics: Agar; Bacteremia; Bacterial Proteins; Biofilms; Enterococcus faecalis; Gelatinases; Genes, Bacterial; Genotype; Gram-Positive Bacterial Infections; Hemolysin Proteins; Humans; Microbial Sensitivity Tests; Phenotype; Virulence Factors

Accurate detection of vancomycin-resistant enterococci (VRE) is essential in preventing transmission in health care settings. Chromogenic media are widely used for screening VRE because of fast turnaround times (TAT) and high sensitivity. We report an outbreak of Enterococcus faecium bearing vanA yet susceptible to vancomycin (vancomycin-variable Enterococcus [VVE]). Between October 2009 to March 2011, clinical and screening specimens (n=14,747) were screened for VRE using VRE-selective medium and/or PCR. VVE isolates were genotyped to determine relatedness. Plasmids from these isolates were characterized by sequencing. Overall, 52 VVE isolates were identified, comprising 15% of all VRE isolates identified. Isolates demonstrated growth on Brilliance VRE agar (Oxoid) at 24 h of incubation but did not grow on brain heart infusion agar with 6 μg/ml vancomycin (Oxoid) or bile esculin azide agar with 6 μg/ml vancomycin (Oxoid) and were susceptible to vancomycin. Genotyping of 20 randomly selected VVE isolates revealed that 15/20 were identical, while 5 were highly related. PCR of the VVE transposon confirmed the presence of vanHAXY gene cluster; however, vanS (sensor) and vanR (regulator) genes were absent. The outbreak was controlled through routine infection control measures. We report an emergence of a fit strain of E. faecium containing vanA yet susceptible to vancomycin. Whether this new strain represents VRE has yet to be determined; however, unique testing procedures are required for reliable identification of VVE.

Topics: Agar; Aged; Anti-Bacterial Agents; Bacterial Proteins; Culture Media; Disease Outbreaks; Enterococcus faecium; Female; Genes, Bacterial; Genotype; Gram-Positive Bacterial Infections; Humans; Male; Microbial Sensitivity Tests; Plasmids; Polymerase Chain Reaction; Vancomycin; Vancomycin Resistance

Different antimicrobial susceptibility testing methods to detect low-level vancomycin resistance in enterococci were evaluated in a Scandinavian multicenter study (n=28). A phenotypically and genotypically well-characterized diverse collection of Enterococcus faecalis (n=12) and Enterococcus faecium (n=18) strains with and without nonsusceptibility to vancomycin was examined blindly in Danish (n=5), Norwegian (n=13), and Swedish (n=10) laboratories using the EUCAST disk diffusion method (n=28) and the CLSI agar screen (n=18) or the Vitek 2 system (bioMérieux) (n=5). The EUCAST disk diffusion method (very major error [VME] rate, 7.0%; sensitivity, 0.93; major error [ME] rate, 2.4%; specificity, 0.98) and CLSI agar screen (VME rate, 6.6%; sensitivity, 0.93; ME rate, 5.6%; specificity, 0.94) performed significantly better (P=0.02) than the Vitek 2 system (VME rate, 13%; sensitivity, 0.87; ME rate, 0%; specificity, 1). The performance of the EUCAST disk diffusion method was challenged by differences in vancomycin inhibition zone sizes as well as the experience of the personnel in interpreting fuzzy zone edges as an indication of vancomycin resistance. Laboratories using Oxoid agar (P<0.0001) or Merck Mueller-Hinton (MH) agar (P=0.027) for the disk diffusion assay performed significantly better than did laboratories using BBL MH II medium. Laboratories using Difco brain heart infusion (BHI) agar for the CLSI agar screen performed significantly better (P=0.017) than did those using Oxoid BHI agar. In conclusion, both the EUCAST disk diffusion and CLSI agar screening methods performed acceptably (sensitivity, 0.93; specificity, 0.94 to 0.98) in the detection of VanB-type vancomycin-resistant enterococci with low-level resistance. Importantly, use of the CLSI agar screen requires careful monitoring of the vancomycin concentration in the plates. Moreover, disk diffusion methodology requires that personnel be trained in interpreting zone edges.

Topics: Agar; Anti-Bacterial Agents; Bacterial Proteins; Culture Media; Disk Diffusion Antimicrobial Tests; Enterococcus faecalis; Enterococcus faecium; Genotype; Gram-Positive Bacterial Infections; Humans; Microbial Sensitivity Tests; Sensitivity and Specificity; Vancomycin; Vancomycin Resistance

A chromogenic medium for identification of vancomycin-resistant Enterococcus faecalis and Enterococcus faecium, VRESelect, was compared to bile esculin azide agar with 6 μg/ml vancomycin (BEAV) for the isolation of vancomycin-resistant enterococci (VRE) from stool specimens. At 24 to 28 h, VRESelect demonstrated 98.7% (confidence interval [CI], 96.1 to 99.7%) sensitivity and 99.0% (CI, 98.0 to 99.6%) specificity versus 85.1% (CI, 79.8 to 89.5%) and 90.1% (CI, 79.8 to 89.5%) sensitivity and specificity, respectively, for BEAV.

Topics: Agar; Bacteriological Techniques; Chromogenic Compounds; Culture Media; Enterococcus faecalis; Enterococcus faecium; Feces; Gram-Positive Bacterial Infections; Humans; Prospective Studies; Sensitivity and Specificity; Time Factors; Vancomycin Resistance

An oleate-dependent Enterococcus faecalis isolate representing small-colony variants (SCVs) was isolated from the umbilical exudate of a 31-month-old Japanese male patient in Nagano Children's Hospital, Azumino, Japan. The patient had been suffering from recurrent omphalitis since early infancy. The initial E. faecalis SCV isolate formed small colonies on sheep blood agar plates and tiny colonies on chocolate and modified Drigalski agar, although no visible growth was observed in HK-semi solid medium after 48 h incubation in ambient air. Moreover, the SCV isolate, the colonial morphology of which was reminiscent of Streptococcus species, could not be identified using the MicroScan WalkAway-40 and API 20 Strep systems, both of which yielded profile numbers that did not correspond to any bacterial species, probably as a result of insufficient growth of the isolate. The SCV isolate was subsequently identified as E. faecalis based on its morphological, cultural and biochemical properties, and this was confirmed by sequencing the 16S rRNA gene of the organism. Investigations revealed that the addition of oleate, an unsaturated fatty acid, enabled the isolate to grow on every medium with normal-sized colony morphology. Although it has long been known that long-chain fatty acids, especially unsaturated oleic acid, have a major inhibitory effect on the growth of a variety of microorganisms, including not only mycobacteria but also streptococci, this is, to the best of our knowledge, the first clinical isolation of an oleate-dependent E. faecalis SCV isolate. In addition, oleic acid might be considered to affect the cell membrane permeability of carbohydrates or antimicrobial agents such as β-lactams.

Topics: Agar; Animals; Asian People; Bacterial Proteins; Child, Preschool; Culture Media; DNA, Bacterial; Enterococcus faecalis; Fatty Acids; Gram-Positive Bacterial Infections; Humans; Male; Multienzyme Complexes; Oleic Acid; RNA, Ribosomal, 16S; Sheep; Umbilical Cord

Topics: Agar; Bacteriological Techniques; Culture Media; Enterococcus; Gram-Positive Bacterial Infections; Humans; Rectum; Sensitivity and Specificity; Vancomycin Resistance

Frequencies of vanB-type Enterococcus faecium increased in Europe during the last years. VanB enterococci show various levels of vancomycin MICs even below the susceptible breakpoint challenging a reliable diagnostics. The performance of 3 chromogenic vancomycin-resistant enterococci (VRE) screening agars, 2 Etest® vancomycin protocols, and different microdilution methods to detect 129 clinical vanB E. faecium strains was investigated. Altogether, 112 (87%) were correctly identified as VanB-type Enterococcus by microdilution MICs. An Etest® macromethod protocol was more sensitive than the standard protocol while keeping sufficient specificity in identifying 15 vanA/vanB-negative strains. Three chromogenic VRE agars performed similarly with 121 (94%), 123 (95%), and 124 (96%) vanB isolates that grew on Brilliance™ VRE Agar, CHROMagar™ VRE, and chromID™ VRE agar, respectively. Using identical media and conditions, we did not identify different growth behaviour on agar and in broth. A few vanB strains showed growth of microcolonies inside the Etest® vancomycin inhibition zones, suggesting a VanB heteroresistance phenotype.

Topics: Agar; Bacterial Proteins; Chromogenic Compounds; Culture Media; Enterococcus faecium; Europe; Genotype; Gram-Positive Bacterial Infections; Humans; Mass Screening; Microbial Sensitivity Tests; Sensitivity and Specificity; Vancomycin Resistance

Rapid detection of vancomycin-resistant enterococci (VRE) infection is very important for control and prevention of nosocomial spread of these bacteria. A multiplex PCR method for rapid screening of VRE has recently been developed. We performed a prospective study of VRE screening tests to compare the performance of PCR to that of a chromogenic agar-based culture method. From January to December 2009, a total of 8815 rectal swab specimens were tested simultaneously for VRE by VRE selective culture and by PCR. The specimens were inoculated onto ChromID VRE agar containing 8 µg vancomycin ml⁻¹ and examined after 24 and 48 h of incubation. Identification and antibiotic susceptibility tests were performed using the automated VITEK-2 system and a supplementary E-test and disk diffusion test. Detection of the vanA and vanB genes was performed with the Seeplex VRE detection kit. Specimens were inoculated in enterococcosel broth for 16-24 h before PCR for enrichment of VRE. VRE were isolated from 741 of the 8815 specimens by chromogenic agar-based culture (8.4 %). vanA and vanB genotypes were detected in 758 (8.6 %) and 3 (0.03 %) specimens, respectively, by multiplex PCR. Sensitivity, specificity, positive predictive value and negative predictive value of PCR for detection of VRE were 98.2 %, 99.6 %, 95.7 %, and 99.8 %. No VRE were isolated from vanB-positive specimens. The overall performance of PCR is comparable to that of a chromogenic agar-based culture method for screening of VRE, so PCR could be an alternative or supportive method for effective control of nosocomial VRE infection.

Topics: Agar; Anti-Bacterial Agents; Bacteriological Techniques; Chromogenic Compounds; Enterococcus; Gram-Positive Bacterial Infections; Humans; Polymerase Chain Reaction; Prospective Studies; Vancomycin; Vancomycin Resistance

A total of 142 stool specimens were evaluated for vancomycin-resistant enterococcus (VRE). Twenty-four-hour sensitivities and specificities, respectively, were 98% and 95% for Spectra VRE chromogenic agar (Remel, Lenexa, KS), 86% and 92% for bile esculin azide with vancomycin (BEAV; Remel), and 96.5% and 92% for Campylobacter agar (CAMPY; Remel). Spectra VRE and CAMPY are significantly more sensitive at 24 h than BEAV.

Topics: Agar; Anti-Bacterial Agents; Azides; Bacteriological Techniques; Bile; Chromogenic Compounds; Culture Media; Enterococcus; Esculin; Feces; Gram-Positive Bacterial Infections; Humans; Sensitivity and Specificity; Vancomycin; Vancomycin Resistance

BBL CHROMagar VanRE (CVRE) was compared with bile esculin azide agar plus vancomycin to screen for vancomycin-resistant enterococcus (VRE) colonization. CVRE distinguishes Enterococcus faecalis (green colonies) from Enterococcus faecium (mauve colonies) on the basis of chromogenic substrate use. CVRE sensitivity and specificity were 98.6% and 99.1%. Positive and negative predictive values were 95.9% and 99.7%.

Topics: Agar; Bacteriological Techniques; Carrier State; Chromogenic Compounds; Culture Media; Enterococcus faecalis; Enterococcus faecium; Gram-Positive Bacterial Infections; Humans; Predictive Value of Tests; Sensitivity and Specificity; Vancomycin Resistance

The incidence of Bacillus cereus bacteremia is increasing, but the identification of Bacillus species remains difficult. Brilliance Bacillus cereus agar (BBC agar; Oxoid, UK) is a new CHROMagar medium that allows selective isolation and identification of B. cereus; however, its clinical usefulness is seldom studied. We evaluated the usefulness of BBC agar to identify B. cereus isolates recovered from blood cultures.. We analyzed a total of 53 blood isolates that showed a Bacillus-like morphology on Gram staining. All isolates were identified by using both the API Coryne (bioMérieux, France) and API 50CH/B (bioMérieux) systems. They were subsequently subcultured on BBC agar, incubated for 24 hr, and then examined for characteristic blue-green colonies. The clinical characteristics of patients whose isolates were identified as B. cereus were assessed.. Of the 53 isolates, 18 were identified as B. cereus by API 50CH/B. With the API 50CH/B system used as gold standard, the sensitivity and specificity for the identification of B. cereus were 100% (18/18) and 100% (35/35), respectively, using BBC agar, and 67% (12/18) and 100% (35/35), respectively, using the API Coryne system. Of the 18 patients with B. cereus bacteremia, 15 showed infectious signs, and 3 had more than 2 blood cultures positive for B. cereus on separate days.. Our study shows, for the first time, that BBC agar, with its good agreement and ease of use, is a valuable alternative to the API 50CH/B system for the presumptive identification of B. cereus isolates from blood cultures.

Topics: Adolescent; Adult; Agar; Aged; Bacillus cereus; Bacteremia; Child; Child, Preschool; Chromogenic Compounds; Culture Media; Female; Gram-Positive Bacterial Infections; Humans; Infant; Infant, Newborn; Male; Middle Aged; Reagent Kits, Diagnostic; Sensitivity and Specificity

Spectra VRE (Remel, Lenexa, KS) is a chromogenic medium designed to recover and differentiate vancomycin-resistant Enterococcus faecium and Enterococcus faecalis (VRE). This medium was compared to bile esculin azide agar (BEAV) and was 98.2% sensitive and 99.3% specific compared to BEAV, which was 87.6% sensitive and 87.1% specific at 24 h.

Topics: Agar; Bacteriological Techniques; Chromogenic Compounds; Culture Media; Enterococcus faecalis; Enterococcus faecium; Gram-Positive Bacterial Infections; Humans; Sensitivity and Specificity; Vancomycin Resistance

The findings from a preliminary assessment of a new instrument designed for the inoculation and spreading of specimens for microbiological analysis onto agar plates are described. The study found that the instrument was able to select full or biplates from a number of input cassettes, each containing different agar types. Samples were then inoculated by the instrument onto the agar surfaces and spread by a novel plastic applicator. Following this, the instrument labeled the plates and sorted them into a number of specified output stations. It was found that the instrument was able to inoculate and spread samples over a greater proportion of the agar plate surface than the manual loop-to-plate method. As a consequence, up to 44% more usable colonies were produced per plate from clinical specimens and standard cultures. Viable counts showed that the instrument was able to detect as few as 10(2) CFU/ml in fluids and also facilitated the enumeration of organisms, particularly in specimens such as urine.

Topics: Agar; Automation; Bacteriological Techniques; Blood; Colony Count, Microbial; Culture Media; Gram-Negative Bacteria; Gram-Negative Bacterial Infections; Gram-Positive Bacteria; Gram-Positive Bacterial Infections; Humans; Specimen Handling; Urine

Human blood agar (HuBA) is widely used in developing countries for the isolation of bacteria from clinical specimens. This study compared citrated sheep blood agar (CSBA) and HuBA with defibrinated horse blood agar and defibrinated sheep blood agar (DSBA) for the isolation and antibiotic susceptibility testing of reference and clinical strains of Streptococcus pneumoniae, Streptococcus pyogenes, and Staphylococcus aureus. Reference and clinical strains of all organisms were diluted in brain heart infusion and a clinical specimen of cerebrospinal fluid and cultured on all agars. Viable counts, colony morphology, and colony size were recorded. Susceptibility testing for S. pneumoniae and S. pyogenes was performed on defibrinated sheep blood Mueller-Hinton agar, citrated sheep blood Mueller-Hinton agar (CSB MHA), and human blood Mueller-Hinton agar plates. For all organisms, the colony numbers were similar on all agars. Substantially smaller colony sizes and absent or minimal hemolysis were noted on HuBA for all organisms. Antibiotic susceptibility results for S. pneumoniae were similar for the two sheep blood agars; however, larger zone sizes were displayed on HuBA, and quality control for the reference strain failed on HuBA. For S. pyogenes, larger zone sizes were demonstrated on HuBA and CSBA than on DSBA. Poor hemolysis made interpretation of the zone sizes difficult on HuBA. CSBA is an acceptable alternative for the isolation of these organisms. The characteristic morphology is not evident, and hemolysis is poor on HuBA; and so HuBA is not recommended for use for the isolation or the susceptibility testing of any of these organisms. CSB MHA may be suitable for use for the susceptibility testing of S. pneumoniae.

Topics: Agar; Animals; Anti-Bacterial Agents; Bacteriological Techniques; Blood; Clinical Laboratory Techniques; Culture Media; Developing Countries; Gram-Positive Bacterial Infections; Gram-Positive Cocci; Horses; Humans; Microbial Sensitivity Tests; Sheep

Colony morphology on kanamycin esculin azide agar was investigated as a means of selecting different species and strains of enterococci from clinical specimens. Four representative colonies of each morphotype were indistinguishable by pulsed-field gel electrophoresis, biotype, and antibiogram analysis. The optimum time for identification of different colony morphotypes was 72 h.

Topics: Agar; Anti-Bacterial Agents; Azides; Bacterial Typing Techniques; Culture Media; Electrophoresis, Gel, Pulsed-Field; Enterococcus; Esculin; Gram-Positive Bacterial Infections; Humans; Kanamycin; Species Specificity

To establish an efficient and sensitive technique for recovering vancomycin-resistant enterococci (VRE) from perianal and environmental samples collected during implementation of control measures for an outbreak of VRE.. Perianal and environmental samples were collected in triplicate on sterile swabs. One swab was used to inoculate a selective broth medium containing 6 pg of vancomycin and 8 pg of ciprofloxacin per mL, one to inoculate Campylobacter agar containing 10 microg/mL of vancomycin, and one to inoculate Enterococcosel agar containing 8 microg/mL of vancomycin.. Samples were collected in the intensive care units of a 600-bed university hospital over a period of 2 months. SAMPLE SELECTION: Patients and their immediate environment were sampled if they resided in a ward with a patient known to be colonized or infected with VRE.. Of the 88 perianal samples obtained from 63 patients, 37 were positive for VRE by broth culture, with 36 also recovered on both types of solid media (sensitivity, 97.3%; negative predictive value, 98.1%). Of the initial samples collected from each of the 63 patients, 20 were positive for VRE by all methods. Of the 500 environmental samples cultured, 139 were positive for VRE in broth, with only 33 recovered on Campylobacter agar (sensitivity, 23.7%; negative predictive value, 77.2%) and 22 on Enterococcosel agar (sensitivity, 15.8%; negative predictive value, 75.2%).. Our data indicate that, when performing surveillance cultures during an outbreak of VRE, use of an enrichment broth medium is required to recover VRE contaminating environmental surfaces; however, direct inoculation to selective solid medium is adequate to recover VRE in patient perianal specimens.

Topics: Agar; Anal Canal; Colony Count, Microbial; Cross Infection; Disease Outbreaks; Enterococcus; Gram-Positive Bacterial Infections; Humans; Infection Control; Sensitivity and Specificity; Specimen Handling; Vancomycin Resistance

Prospective laboratory methodology study.. Certain tissues, by virtue of their shape and extreme thinness or pliability, are difficult to position correctly during routine paraffin embedding to provide the optimal orientation for histopathologic studies. Biopsy specimens from temporal arteries must be sampled at different points along the length of the artery. Other tissues such as subfoveal neovascular membranes and fragments of lens capsule lack the thickness and rigidity to be positioned on edge to yield cross-sectional views. The authors' technique improves the orientation and thereby maximizes the histologic information obtained from such specimens.. From January 1, 1990, to April 30, 1999, the authors studied 500 consecutive temporal artery biopsy specimens and 200 successive subfoveal neovascular membranes.. Cutting a 20-mm cylindrical fragment of temporal artery at 1- to 1.5-mm intervals yielded approximately 13 to 20 cross-sections along the length of the artery. When the specimens were positioned together and embedded in agar, the pathologist could easily study multiple cross-sections of the artery. Additionally, using the agar technique, the authors were able to obtain cross-sections of other specimens submitted, such as subfoveal neovascular membranes, and studied each of the different layers to evaluate the disease process. By the same method, the authors placed small fragments of lens capsule with underlying cortex on edge and readily identified short, gram-positive coccobacilli consistent with Propionibacterium acnes endophthalmitis.. The agar technique can greatly improve the quality of diagnostic information gleaned from temporal artery biopsy specimens and other small tissue samples.

Topics: Agar; Biopsy; Endophthalmitis; Epiretinal Membrane; Eye Infections, Bacterial; Giant Cell Arteritis; Gram-Positive Bacterial Infections; Humans; Lens Capsule, Crystalline; Ophthalmology; Pathology, Surgical; Propionibacterium acnes; Prospective Studies; Specimen Handling; Temporal Arteries

Lactobacilli produce many antimicrobial substances including organic acids, hydrogen peroxide and bacteriocins. Antagonistic activity of lactobacilli is an important factor in the protection of the vagina of fertile women against infection by certain pathogens. In the present study, we investigated 17 strains of lactobacilli, including 11 strains of vaginal origin. The aim was to investigate in more detail the antibacterial activity of lactobacilli and to attempt to assess substances responsible for inhibition. The investigated lactobacilli inhibited some strains of Escherichia coli, Serratia marcescens, Shigella boydii, Listeria monocytogenes, Listeria ivanovii, Listeria innocua and Staphylococcus aureus. We have provided evidence that inhibition is due mainly to organic acids and to a lesser extent, to bacteriocins.

Topics: Agar; Antibiosis; Bacteriocins; Carboxylic Acids; Culture Media; Enterobacteriaceae; Enterobacteriaceae Infections; Female; Gram-Positive Bacteria; Gram-Positive Bacterial Infections; Humans; Hydrogen Peroxide; Lactobacillus; Vagina

An evaluation was undertaken to determine the optimal method for testing the susceptibilities of 100 clinical isolates and two reference strains of Enterococcus spp. to vancomycin in vitro. Six testing methods were studied by using the following media and incubation times: agar screen with the Synergy Quad Plate (Remel, Lenexa, Kans.), an in-house-prepared brain heart infusion (BHI) agar plate, and an in-house-prepared Mueller-Hinton (MH) agar plate, all incubated for 24 or 48 h; broth microdilution (Sensititre Just One Strip; AccuMed International, Inc., West Lake, Ohio) with BHI or cation-adjusted MH broth incubated for 24 or 48 h; agar dilution with BHI or MH agar incubated for 24 or 48 h; epsilometer test (E test; AB BioDisk, Solna, Sweden) with BHI or MH agar incubated for 24 or 48 h; disk diffusion with BHI or MH agar incubated for 24 or 48 h; and the automated Vitek method with the gram-positive susceptibility Staphylococcus aureus card and R02.03 software (bioMerieux, Inc., Hazelwood, Mo.). Growth failures occurred with MH media (n = 6) but not with BHI media. One growth failure occurred with the Vitek method. Results for each testing method for each Enterococcus strain were interpreted as susceptible, intermediate, or resistant according to current National Committee for Clinical Laboratory Standards (NCCLS) criteria and compared to the vancomycin resistance genotype (i.e., vanA, vanB, vanC-1, or vanC-2/3). For all methods, extension of the incubation time from 24 h to 48 h either produced no difference in the results or gave poorer results. The following methods produced no very major or major interpretive errors: broth microdilution with BHI media incubated for 24 h, agar dilution with BHI media incubated for 24 or 48 h, and E test with BHI media incubated for 24 or 48 h. Unacceptable frequencies of very major errors (> 1%) occurred with all methods for which MH media were used. Minor interpretive errors were frequent with all methods. These minor interpretive errors also occurred most frequently with Enterococcus strains with vanC genes, which encoded low-level vancomycin resistance (MIC < or = 8 microg/ml), as opposed to Enterococcus strains which possessed vanA or vanB genes, which encoded higher-level vancomycin resistance (MIC > or = 64 microg/ml). Modification of NCCLS breakpoints, especially for motile Enterococcus spp. (E. casseliflavus, E. flavescens, and E. gallinarum), may resolve this problem; however, in the current study, one E. f

Topics: Agar; Anti-Bacterial Agents; Culture Media; Drug Resistance, Microbial; Enterococcus; Enterococcus faecalis; Enterococcus faecium; Evaluation Studies as Topic; Genes, Bacterial; Genotype; Gram-Positive Bacterial Infections; Humans; Microbial Sensitivity Tests; Phenotype; Vancomycin

A new chromogenic plate medium, CHROMagar Orientation, was evaluated for use in the differentiation and presumptive identification of gram-negative bacilli and Enterococcus species by a multipoint inoculation (replicator) technique. In this study, 1,404 gram-negative bacilli and 74 enterococcal isolates were tested on CHROMagar Orientation. Six control American Type Culture Collection strains were also included with the testing to ensure quality control of the media. Of the Escherichia coli isolates (n = 588) tested, 99.3% produced a pink-to-red color. Only in four isolates that were O-nitrophenyl-beta-D-galactopyranoside (ONPG) negative did this result differ. Proteus mirabilis and P. vulgaris were well differentiated on this medium. P. mirabilis (n = 184) produced a clear colony with diffusible brown pigment around the periphery. By contrast, 15 of 16 P. vulgaris isolates produced bluish-green colonies with a slight brown background. All Aeromonas hydrophila isolates (n = 26) tested produced clear to pink colonies at 35 to 37 degrees C. This colony color changed to blue after 2 to 3 h of incubation at room temperature. A. hydrophila exhibited stronger color and better growth at 30 degrees C. Serratia marcescens (n = 29) demonstrated an aqua blue color that deepened to a darker blue when exposed to room temperature. All enterococcal isolates (n = 74) resulted in a blue color and gave pinpoint colonies on purity subcultures at 35 to 37 degrees C after 18 h of incubation. Similarity in color resulted in failure to discriminate accurately between Klebsiella, Enterobacter, and Citrobacter species. However, these species could be readily differentiated from other members of the family Enterobacteriaceae. Pseudomonas aeruginosa (n = 151) was easily differentiated from members of the Enterobacteriaceae but was less easily distinguishable from other gram-negative nonmembers of the Enterobacteriaceae. The medium was found to facilitate easy visual detection of mixed bacterial isolates in culture. When used in a replicator system, it easily detected mixed growths of organisms which may have otherwise led to false antibiotic susceptibility results. These mixed growths were not obvious on the routine susceptibility testing medium (Isosensitest).

Topics: Agar; Bacterial Typing Techniques; Bacteriological Techniques; Chromogenic Compounds; Color; Culture Media; Enterococcus; Evaluation Studies as Topic; Gram-Negative Bacteria; Gram-Negative Bacterial Infections; Gram-Positive Bacterial Infections; Humans; Species Specificity

Propionibacterium acnes has been identified as a significant agent of nosocomial infections, including endophthalmitis. Data concerning susceptibility of P. acnes to newer beta-lactam antibiotics and fluoroquinolones are limited. Recent reports suggest that quinolones have activity against these organisms sufficient to warrant further study. We undertook a study to select appropriate antimicrobial agents for use in a rabbit model of P. acnes endophthalmitis. We compared the antibiotic susceptibilities of P. acnes by using the National Committee for Clinical Laboratory Standards method of agar dilution with the E test. Thirteen clinical isolates obtained from eye specimens and three American Type Culture Collection control strains were tested against 14 antibiotics. All the clinical isolates were susceptible by both methods to piperacillin, piperacillin-tazobactam, ampicillin-sulbactam, ticarcillin-clavulanate, cefotaxime, cefotetan, ceftriaxone, cefoxitin, and imipenem in addition to clindamycin but were resistant to metronidazole. The clinical P. acnes isolates also displayed high-level susceptibility to ciprofloxacin, sparfloxacin, and ofloxacin. Almost all the P. acnes strains demonstrated E-test MICs within 2 dilutions of the MICs observed by the agar dilution method. Those few strains for which discrepancies were noted exhibited E-test susceptibilities three- to fivefold dilutions lower than the agar dilution method susceptibilities but only with ampicillin-sulbactam, ticarcillin-clavulanate, and/or clindamycin. On the basis of our study, all of clinical eye isolates were susceptible to these newer antimicrobial agents and the two methods demonstrated similar susceptibility patterns.

Topics: Agar; Animals; Anti-Bacterial Agents; Anti-Infective Agents; Cross Infection; Disease Models, Animal; Endophthalmitis; Fluoroquinolones; Gram-Positive Bacterial Infections; Humans; Lactams; Microbial Sensitivity Tests; Propionibacterium acnes; Rabbits

The aim of the study was to evaluate the chromogenic agar plate CPS ID2 (bioMérieux) and determine its cost-benefit ratio.. A total of 2,193 urinary sediments were processed. The urine culture was carried out in CPS ID2 agar and in cystine-lactose electrolyte deficient (CLED) agar, when needed. Identification of the microorganisms was performed following standard microbiologic procedures through biochemical tests prepared in our laboratory. The identification, from CPS ID2 agar, by direct detection in medium of four metabolic activities: beta-glucuronidase, beta-glucosidase, deaminase, and indol production, was performed following to manufacturer's instructions.. A total of 289 urine cultures were positive, 18 were negative and 34 were contaminated samples. The identification, directly performed from the colonies detected in CPS ID2 agar, was correct in 96% of 166 Escherichia coli, in 92% of 24 Proteus mirabilis and in 97% of 38 enterococci. CPS ID2 agar exhibited 94% and 100% sensitivity and specificity, respectively in E. coli identification, 92% and 100% in P. mirabilis and 97% and 99% in Enterococcus. The use of this new media, CPS ID2, in our laboratory, implies a budgetary increment. However, if commercial galleries are used for routine identification, the cost will be reduced using this new media.. The CPS ID2 agar allows the isolation and direct identification of the most frequent urinary tract pathogens: E. coli, P. mirabilis and Enterococcus in primary isolation medium. Using this medium, bacteriologists will be able to save time and reagents when identifying the most common uropathogens. Furthermore, the use of this medium would reduce costs in some laboratories.

Topics: Agar; Amino Acid Oxidoreductases; Bacterial Proteins; Bacteriological Techniques; beta-Glucosidase; Candida albicans; Candidiasis; Chromogenic Compounds; Cost-Benefit Analysis; Culture Media; Enterobacteriaceae; Enterobacteriaceae Infections; Evaluation Studies as Topic; Glucuronidase; Gram-Negative Bacteria; Gram-Negative Bacterial Infections; Gram-Positive Bacteria; Gram-Positive Bacterial Infections; Humans; Indoles; L-Amino Acid Oxidase; Urinary Tract Infections; Urine

The E test and the reference agar dilution methods were compared for detecting high-level aminoglycoside resistance (HLAR) among 71 selected clinical isolates, including 62 Enterococcus faecalis and 9 Enterococcus faecium isolates. High-level gentamicin resistance alone was found in 11% (5 E. faecalis and 3 E. faecium strains) and high-level streptomycin resistance was found in 42% (28 E. faecalis, 2 E. faecium strains) of the strains tested, and 31% of the strains demonstrated high-level resistance to both antimicrobial agents (21 E. faecalis and 1 E. faecium strains). The E test detected all HLAR populations, but the streptomycin strip may require recalibration to achieve absolute MIC comparisons with the reference value (twofold less) or the use of an alternative interpretive resistance breakpoint, e.g., > 1,000 micrograms/ml. By the E test, MIC results indicate that ampicillin, imipenem, penicillin, piperacillin, and vancomycin remain active against the HLAR E. faecalis isolates; however, these tested drugs were less effective on the HLAR E. faecium isolates (< 50%).

Topics: Agar; Aminoglycosides; Anti-Bacterial Agents; Cross Infection; Drug Resistance, Microbial; Enterococcus faecalis; Enterococcus faecium; Evaluation Studies as Topic; Gram-Positive Bacterial Infections; Humans; Microbial Sensitivity Tests

Ceftazidime was compared with cefotaxime, ceftizoxime, cefmenoxime, cefotiam, cefoperazone, moxalactam, piperacillin, carbenicillin, mezlocillin, cefsulodin and the aminoglycoside antibiotic gentamicin in a series of mouse protection tests. Ceftazidime, together with the other cephalosporin antibiotics and moxalactam were equally effective against infections caused by Staphylococcus aureus with ED50 values ranging from 3.5 to 25 mg/kg. Gentamicin was the most active antibiotic with ED50 values of 0.4 and 1.6 mg/kg. Ceftazidime showed excellent activity against Enterobacteriaceae with ED50 values ranging from 0.2-0.9 mg/kg for Escherichia coli strains, 1.1-13.8 mg/kg for indole positive and negative Proteus spp, and 0.1-25 for Enterobacter cloacae, Klebsiella pneumoniae and Serratia spp. Similar activity against many of the test strains of Enterobacteriaceae was found for gentamicin, cefotaxime, ceftizoxime, cefmenoxime and moxalactam, although cefotiam and cefoperazone were significantly less active than ceftazidime. Ceftazidime was significantly more active than the other beta-lactam antibiotics tested against Pseudomonas aeruginosa infections with ED50 values ranging from 02-108 mg/kg. Only the aminoglycoside antibiotic gentamicin, with ED(50,S) 0.6-6.3 mg/kg, was as effective as ceftazidime. Very poor activity was found for moxalactam, cefoperazone, piperacillin, carbenicillin and mezlocillin against the majority of the test strains of Pseudomonas. The results of these in-vivo indicate that ceftazidime is a promising potential alternative to aminoglycoside antibiotics.

Topics: Agar; Animals; Anti-Bacterial Agents; Bacterial Infections; beta-Lactams; Ceftazidime; Cephalosporins; Colony Count, Microbial; Female; Gentamicins; Gram-Negative Bacteria; Gram-Negative Bacterial Infections; Gram-Positive Bacteria; Gram-Positive Bacterial Infections; Mice; Microbial Sensitivity Tests