Detection of significant bacteriuria by use of the iQ200 automated urine microscope.

In the microbiology laboratory, there is an augmented need for rapid screening methods for the detection of bacteria in urine samples, since about two-thirds of these samples will not yield any bacteria or will yield insignificant growth when cultured. Thus, a reliable screening method can free up laboratory resources and can speed up the reporting of a negative urine result. In this study, we have evaluated the detection of leukocytes, bacteria, and a new sediment indicator, the "all small particles" (ASP), by an automated instrument, the iQ200 urine analyzer, to detect negative urine samples that can be excluded from culture. A coupled automated strip reader (iChem Velocity), enabling the detection of nitrite and leukocyte esterase, was tested in parallel. In total, 963 urine samples were processed through both conventional urine culture and the iQ200/iChem Velocity workstation. Using the data, a multivariate regression model was established, and the predicted specificity and the possible reduction in urine cultures were calculated for the indicators and their respective combinations (leukocytes plus bacteria plus ASP and leukocyte esterase plus nitrite). Among all options, diagnostic performance was best using the whole microscopic content of the sample (leukocytes plus bacteria plus ASP). By using a cutoff value of ≥ 10(4) CFU/ml for defining a positive culture, a given sensitivity of 95% resulted in a specificity of 61% and a reduction in urine cultures of 35%. By considering the indicators alone, specificity and the culture savings were both much less satisfactory. The regression model was also used to determine possible cutoff values for running the instrument as part of daily routine. By using a graphical representation of all combinations possible, we derived cutoff values for leukocyte, bacterial, and ASP count, which should enable the iQ200 microscope to screen out approximately one-third of the urine samples, significantly reducing the workload in the microbiology laboratory.

Radiography-based score indicative for the pathogenicity of bacteria in odontogenic infections.

OBJECTIVE: To develop a new radiography-based score to assess the potential of bacteria to cause odontogenic infections derived from the occurrence of bacteria at small or large radiographical lesions. MATERIALS AND METHODS: The patients analyzed were a sub-population from a large randomized clinical trial comparing moxifloxacin and clindamycin in the treatment of inflammatory infiltrates and odontogenic abscesses. Routine radiographs were used to analyze the area of the periapical radiolucent lesions. Lesions were stratified by their radiographically measured area as large (>9 mm(2)) or small (≤9 mm(2)). A risk ratio was calculated for each species from the frequency of their occurrence in large vs in small lesions. RESULTS: Fifty-one patients, 19 with abscesses and 32 with infiltrates, were evaluated. Overall, the radiographical lesion areas ranged from 0.4-46.2 mm(2) (median = 9 mm(2)). An increased risk (risk ratio >1) to occur at large abscess lesions was observed for Prevotella (P.) oralis, P. buccae, P. oris, P. intermedia, Fusobacterium nucleatum and Streptococcus (Strep.) anginosus group. An increased risk to occur at large infiltrate lesions was found for Strep. salivarius, Strep. parasanguis, Strep. anginosus group, Capnocytophaga spp., Neisseria (N.) sicca, Neisseria spp., Staphylococcus (Staph.) aureus, P. intermedia, P. buccae, Prevotella spp. and P. melaninogenica. CONCLUSIONS: The radiography-based score suggests that certain Prevotella spp., F. nucleatum and Strep. anginosus groups play a crucial role in the pathogenesis of odontogenic abscesses, and that various streptococci, Neisseria spp., Capnocytophaga spp., Staph. aureus and Prevotella spp. are involved in the pathogenesis of odontogenic infiltrates.

Evaluation of a new screen agar plate for detection and presumptive identification of Enterobacteriaceae producing extended-spectrum beta-lactamases.

A new agar screen plate for extended-spectrum beta-lactamase (ESBL) detection was evaluated with 50 clinical isolates of ESBL-producing Enterobacteriaceae species: Enterobacter cloacae (n = 10), Escherichia coli (n = 10), Klebsiella oxytoca (n = 3), Klebsiella pneumoniae (n = 25), and Proteus mirabilis (n = 2). Fecal samples were artificially inoculated with 2 concentrations (25 and 250 colony forming units [CFU]/plate) of the test strains and then applied to the new agar screen plates. By this approach, the new agar formula detected growth that was suggestive of ESBL activity in 44 of 50 (88%) and 50 of 50 (100%) of ESBL strains with 25 and 250 CFU/plate, respectively. A limitation of the agar screen plates was a lack of some specificity. Among 15 strains with resistant phenotypes other than ESBL (K1 producers of K. oxytoca, 6 strains; 9 strains with AmpC phenotype), growth was recorded in 7 (25 CFU/plate) and 11 (250 CFU/plate) of 15 strains. In conclusion, the new agar screen plate is a sensitive and convenient method to directly screen for ESBL organisms in rectal swabs or stool samples, with the potential for incorporation into routine clinical laboratory service.

Comparison of BDPhoenix and VITEK2 automated antimicrobial susceptibility test systems for extended-spectrum beta-lactamase detection in Escherichia coli and Klebsiella species clinical isolates.

The present study compares the ability to detect extended-spectrum beta-lactamases (ESBL) among a collection of 34 ESBL producing clinical isolates belonging to Escherichia coli and Klebsiella species with two new rapid susceptibility and identification instruments-VITEK2 (bioMérieux, Marcy l'Etoile, France) vs. BDPhoenix (BD Biosciences, Sparks, MD). ESBL content in these isolates was previously characterized on the basis of PCR amplification and sequencing results which were used as the reference method in our evaluation. BDPhoenix correctly determined the ESBL outcome for all strains tested (100% detection rate), whereas VITEK2 was not able to detect the ESBL status in 5 isolates (85% detection rate). Detailed analysis revealed that the discrepancies were mainly observed with 'difficult-to-detect' strains. Misidentification was either due to low oximino cephalosporin MIC in these strains or was associated with pronounced 'cefotaximase' or 'ceftazidimase' phenotypes. Klebsiella oxytoca chromosomal beta-lactamase (K1) is phenotypically quite similar to ESBL enzymes. In order to evaluate whether the K1 and ESBL enzymes could be discriminated, we expanded our analysis by 8 clinical K. oxytoca strains with K1 phenotypes. VITEK2 gave excellent identification of these strains whereas 7 out of 8 were falsely labeled ESBL-positive by the BDPhoenix system.

Extended-spectrum beta-lactamases: implications for the clinical microbiology laboratory, therapy, and infection control.

Extended-spectrum beta-lactamase (ESBL) producing gram-negative bacilli are a growing concern in human medicine today. When producing these enzymes, organisms (mostly K. pneumoniae and E. coli) become highly efficient at inactivating the newer third-generation cephaloporins (such as cefotaxime, ceftazidime, and ceftriaxone). In addition, ESBL-producing bacteria are frequently resistant to many classes of non-beta-lactam antibiotics, resulting in difficult-to-treat infections. This review gives an introduction into the topic and is focused on various aspects of ESBLs; it covers the current epidemiology, the problems of ESBL detection and the clinical relevance of infections caused by ESBL-producing organisms. Therapeutic options and potential strategies for dealing with this growing problem are also discussed in this article.

Rapid detection of methicillin-resistant Staphylococcus aureus directly from clinical samples: methods, effectiveness and cost considerations.

Methicillin-resistant Staphylococcus aureus (MRSA) isolates is a serious public health problem whose ever-increasing rate is commensurate with the pressure it is exerting on the healthcare system. At present, more than 20% of clinical S. aureus isolates in German hospitals are methicillin resistant. Strategies from low-prevalence countries show that this development is not necessarily inevitable. In the Scandinavian countries and the Netherlands, thanks to a rigorous prevention programme, MRSA prevalence has been kept at an acceptably low level (<1-3%). Central to these 'search and destroy' control strategies is an admission screening using several MRSA swabs taken from mucocutaneous colonisation sites of high-risk patients ('MRSA surveillance').It has also been reported that the speed with which MRSA carriage is detected has an important role to play, as it is a key component of any effective strategy to prevent the pathogen from spreading. Since MRSA culturing involves a 2-3 day delay before the final results are available, rapid detection techniques (commonly referred to as 'MRSA rapid tests') using PCR methods and, most recently, rapid culturing methods have been developed. The implementation of rapid tests reduces the time of detection of MRSA carriers from 48-72 to 2-5 h. Clinical evaluation data have shown that MRSA can thus be detected with very high sensitivity. Specificity however is sometimes impaired due to false-positive PCR signals occurring in mixed flora specimens. In order to rule out any false-positive PCR results, a culture screen must always be carried out simultaneously. The data provide preliminary evidence that a PCR assay can reduce nosocomial MRSA transmission in high-risk patients or high-risk areas, whereas an approach that screens all patients admitted to the hospital is probably not effective. Information concerning the cost-effectiveness of rapid MRSA tests is still sparse and thus the issue remains debated.

Point-of-care testing in microbiology: the advantages and disadvantages of immunochromatographic test strips.

BACKGROUND: Point-of-care testing (POCT) for the demonstration of pathogens was introduced several years ago. The present study describes the current technical status of POCT, giving some examples, and summarizes the specific advantages and disadvantages of the POCT approach in microbiology. METHODS: Selective review of the literature found in medical databases under consideration of current German and international guidelines. RESULTS/CONCLUSIONS: The test systems available today are technically mature and offer good to very good performance. For HIV, malaria, group A streptococci, and legionellae, POCT testing, when indicated, is on a par with conventional procedures. The information yielded by rapid tests for pneumococci and for influenza tends to be supplementary in nature. The rapid test for group B streptococci is unsuitable for routine use because its sensitivity is still too low compared with bacterial culture. POCT can be successful only if the tests are performed correctly by trained personnel, quality management procedures are followed, and the severity of illness and the epidemiological circumstances are taken into account when interpreting the results.

Detection of extended-spectrum beta-lactamases among Enterobacteriaceae by use of semiautomated microbiology systems and manual detection procedures.

Three commercially available microbiology identification and susceptibility testing systems were compared with regard to their ability to detect extended-spectrum beta-lactamase (ESBL) production in Enterobacteriaceae, i.e., the Phoenix Automated Microbiology System (BD Diagnostic Systems, Sparks, MD), the VITEK 2 System (bioMérieux, Marcy l'Etoile, France), and the MicroScan WalkAway-96 System (Dade Behring, Inc., West Sacramento, CA), using routine testing panels. One hundred fifty putative ESBL producers were distributed blindly to three participating laboratories. Conventional phenotypic confirmatory tests such as the disk approximation method, the CLSI double-disk synergy test, and the Etest ESBL were also evaluated. Biochemical and molecular characterization of beta-lactamases performed at an independent laboratory was used as the reference method. One hundred forty-seven isolates of Escherichia coli, Klebsiella pneumoniae, Klebsiella oxytoca, Enterobacter cloacae, Enterobacter aerogenes, Citrobacter freundii, Serratia marcescens, Proteus mirabilis, Proteus vulgaris, and Morganella morganii were investigated. Of these isolates, 85 were identified as ESBL producers by the reference method. The remaining isolates were identified as non-ESBL producers; they were either hyperproducers of their chromosomal AmpC, Koxy, or SHV enzymes or lacked any detectable beta-lactamase activity. The system with the highest sensitivity for the detection of ESBLs was the Phoenix (99%), followed by the VITEK 2 (86%) and the MicroScan (84%); however, specificity was more variable, ranging from 52% (Phoenix) to 78% (VITEK 2). The performance of the semiautomated systems differed widely with the species investigated. The sensitivities of the conventional test methods ranged from 93 to 94%. The double-disk synergy test showed the highest specificity and positive predictive value among all test methods, i.e., 97% and 98%, respectively.

Detection and genotyping of SHV beta-lactamase variants by mass spectrometry after base-specific cleavage of in vitro-generated RNA transcripts.

Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) after base-specific cleavage of PCR-amplified and in vitro-transcribed bla(SHV) genes was used for the identification and genotyping of SHV beta-lactamases. For evaluation, bla(SHV) stretches of 21 clinical Enterobacteriaceae isolates were PCR amplified using T7 promoter-tagged forward and reverse primers, respectively. In vitro transcripts were generated with T7 RNA and DNA polymerase in the presence of modified analogues replacing either CTP or UTP. Using RNase A, the in vitro transcripts were base-specifically cleaved at every "T" or "C" position. Resulting cleavage products were analyzed by MALDI-TOF MS, generating a characteristic signal pattern based on the fragment masses. All 21 individual SHV genes were identified unambiguously using reference sequences, and the results were in perfect concordance with those obtained by fluorescent dideoxy sequencing, which represents the current standard method. As multiple point mutations can be detected in a single assay and newly emerged mutations which are not yet described in public databases can be identified too, MALDI-TOF MS appears to be an ideal tool for analysis of sequence polymorphisms in resistance-associated gene loci.

A novel extended-spectrum beta-lactamase CTX-M-23 with a P167T substitution in the active-site omega loop associated with ceftazidime resistance.

OBJECTIVE: In recent years, cefotaximases of the CTX-M type have become a predominant cause of resistance to extended-spectrum cephalosporins in Gram-negative bacteria. Although most enzymes provide higher levels of resistance to cefotaxime than to ceftazidime, mutants with enhanced catalytic efficiency against ceftazidime have recently been described. This report identifies another ceftazidime-resistant mutant of the CTX-M class of enzymes. METHODS: Two ceftazidime-resistant strains, Escherichia coli IFI-1 and Klebsiella pneumoniae IFI-2, were isolated from a 46-year-old man during treatment of postoperative peritonitis with ceftazidime. Susceptibility testing, mating-out assays, isoelectric focusing as well as PCR and sequencing techniques were carried out to investigate the underlying mechanism of resistance. RESULTS: E. coli IFI-1 and K. pneumoniae IFI-2 exhibited a clavulanic acid-inhibited substrate profile that included extended-spectrum cephalosporins. Notably, both strains had up to a 32-fold higher level of resistance to ceftazidime than to cefotaxime. Further characterization revealed that a novel bla(CTX-M) gene encoding a beta-lactamase with a pI of 8.9 was implicated in this resistance: CTX-M-23. Along with the substitutions D114N and S140A, CTX-M-23 differed from CTX-M-1, the most closely related enzyme, by a P167T replacement in the active-site omega loop, which has not previously been observed in other CTX-M enzymes. By analogy with what was observed with certain TEM/PSE/BPS-type beta-lactamases, the amino acid substitution in the omega loop may explain ceftazidime resistance, which has only rarely been reported for other CTX-M enzymes. CONCLUSION: The emergence of a new ceftazidime-resistant CTX-M-type mutant provides evidence that these enzymes are able to broaden their substrate spectrum towards ceftazidime, probably due to substitutions in the active-site omega loop.

Cloning and sequencing of Enterobacter aerogenes OmpC-type osmoporin linked to carbapenem resistance.

Using outbreak-related strains of Enterobacter aerogenes, we cloned and sequenced ompK39, the structural gene coding for outer membrane protein OmpK39. Its lack of expression was closely associated with a phenotype exhibiting low-level carbapenem resistance. Detailed alignment of the predicted amino acid sequence revealed that OmpK39 is a member of the OmpC subclass of enterobacterial porins, with the highest degree of homology to Klebsiella pneumoniae OmpK36. Based on a computerized alignment including Escherichia coli PhoE and OmpF, the 3D structures of which are known from X-ray studies, OmpK39 can be assumed to form the typical beta-barrel structure which is common to all enterobacterial porins. Since no inhibitory DNA sequences could be detected in ompk39 in the resistant strains, porin deficiency leading to carbapenem resistance seems to involve alterations in key regulatory genes and/or the promotor sequence rather than a direct mutation in the structural gene.

Evaluation of the MicroScan ESBL plus confirmation panel for detection of extended-spectrum beta-lactamases in clinical isolates of oxyimino-cephalosporin-resistant Gram-negative bacteria.

OBJECTIVE: We aimed to assess the performance of the MicroScan ESBL plus confirmation panel using a series of 87 oxyimino-cephalosporin-resistant Gram-negative bacilli of various species. METHODS: Organisms tested included 57 extended-spectrum beta-lactamase (ESBL) strains comprising Enterobacter aerogenes (3), Enterobacter cloacae (10), Escherichia coli (11), Klebsiella pneumoniae (26), Klebsiella oxytoca (3) and Proteus mirabilis (4). Also included were 30 strains resistant to oxyimino cephalosporins but lacking ESBLs, which were characterized with other resistance mechanisms, such as inherent clavulanate susceptibility in Acinetobacter spp. (4), hyperproduction of AmpC enzyme in Citrobacter freundii (2), E. aerogenes (3), E. cloacae (3), E. coli (4), Hafnia alvei (1) and Morganella morganii (1), production of plasmid-mediated AmpC beta-lactamase in K. pneumoniae (3) and E. coli (3) or hyperproduction of K1 enzyme in K. oxytoca (6). RESULTS: The MicroScan MIC-based clavulanate synergy correctly classified 50 of 57 ESBL strains as ESBL-positive and 23 of 30 non-ESBL strains as ESBL-negative (yielding a sensitivity of 88% and a specificity of 76.7%, respectively). False negatives among ESBL producers were highest with Enterobacter spp. due to masking interactions between ESBL and AmpC beta-lactamases. False-positive classifications occurred in two Acinetobacter spp., one E. coli producing plasmid-mediated AmpC beta-lactamase and two K. oxytoca hyperproducing their chromosomal K1 beta-lactamase. CONCLUSION: The MicroScan clavulanate synergy test proved to be a valuable tool for ESBL confirmation. However, this test has limitations in detecting ESBLs in Enterobacter spp. and in discriminating ESBL-related resistance from the K1 enzyme and from inherent clavulanate susceptibility in Acinetobacter spp.

Evaluation of a new cefepime-clavulanate ESBL Etest to detect extended-spectrum beta-lactamases in an Enterobacteriaceae strain collection.

OBJECTIVES: In this study, we evaluated the performance of a new ESBL Etest configuration based on clavulanate synergy with cefepime compared with cefotaxime-clavulanate and ceftazidime-clavulanate ESBL Etest strips for the detection of extended-spectrum beta-lactamases (ESBL) in an Enterobacteriaceae strain collection, with special focus on Enterobacter spp. METHODS: Overall, a total of 54 clinical isolates of ESBL-producing Enterobacteriaceae species were evaluated: Enterobacter aerogenes (n=3), Enterobacter cloacae (n=10), Escherichia coli (n=10), Klebsiella oxytoca (n=3), Klebsiella pneumoniae (n=25) and Proteus mirabilis (n=3). To check Etest behaviour with resistance phenotypes similar to ESBL, our panel was expanded by six clinical isolates of K. oxytoca that were identified as putative producers of their chromosomal K1 beta-lactamase. RESULTS: With this panel, ESBL Etest was 98% sensitive with cefepime-clavulanate, 83% with cefotaxime-clavulanate, and 74% with ceftazidime-clavulanate strips. Concentrating on Enterobacter spp., reliable ESBL detection could only be achieved by the new cefepime-clavulanate strip since it confirmed ESBL production in all strains (100% sensitivity) whereas only 4/13 (31%) of Enterobacter strains were positive using cefotaxime-clavulanate or ceftazidime-clavulanate strips. A limitation of using the new cefepime strip was less than optimal specificity with K1 phenotypes of K. oxytoca: among six strains, four isolates were scored false-positive by Etest strips containing cefepime-clavulanate. CONCLUSION: The new Etest ESBL strip containing cefepime-clavulanate is a valuable supplement to current methods for detection of ESBLs. In our study collection, the cefepime-clavulanate strip was the best configuration for detection of ESBLs, particularly in Enterobacter spp.

In vitro activity of moxifloxacin against bacteria isolated from odontogenic abscesses.

We evaluated the antimicrobial susceptibility of 87 pathogens isolated from 37 patients with odontogenic abscesses. The most prevalent bacteria were viridans group streptococci and Prevotella species. Considering all bacterial isolates, 100% were susceptible to amoxicillin-clavulanic acid, 98% were susceptible to moxifloxacin and to levofloxacin, 76% were susceptible to doxycycline, 75% were susceptible to clindamycin, and 69% were susceptible to penicillin.