Identification of an unusual Brucella strain (BO2) from a lung biopsy in a 52 year-old patient with chronic destructive pneumonia

<p>Abstract</p> <p>Background</p> <p>Brucellosis is primarily a zoonotic disease caused by <it>Brucella </it>species. There are currently ten <it>Brucella </it>spp. including the recently identified novel <it>B. inopinata </it>sp. isolated from a wound associated with a breast implant infection. In this study we report on the identification of an unusual <it>Brucella</it>-like strain (BO2) isolated from a lung biopsy in a 52-year-old patient in Australia with a clinical history of chronic destructive pneumonia.</p> <p>Results</p> <p>Standard biochemical profiles confirmed that the unusual strain was a member of the <it>Brucella </it>genus and the full-length 16S rRNA gene sequence was 100% identical to the recently identified <it>B. inopinata </it>sp. nov. (type strain BO1^T). Additional sequence analysis of the <it>recA, omp2a </it>and <it>2b </it>genes; and multiple locus sequence analysis (MLSA) demonstrated that strain BO2 exhibited significant similarity to the <it>B. inopinata </it>sp. compared to any of the other <it>Brucella </it>or <it>Ochrobactrum </it>species. Genotyping based on multiple-locus variable-number tandem repeat analysis (MLVA) established that the BO2 and BO1^Tstrains form a distinct phylogenetic cluster separate from the other <it>Brucella </it>spp.</p> <p>Conclusion</p> <p>Based on these molecular and microbiological characterizations, we propose that the BO2 strain is a novel lineage of the newly described <it>B. inopinata </it>species.</p

Carbapenem Resistance in Klebsiella pneumoniae Not Detected by Automated Susceptibility Testing

Detecting β-lactamase–mediated carbapenem resistance among Klebsiella pneumoniae isolates and other Enterobacteriaceae is an emerging problem. In this study, 15 blaKPC-positive Klebsiella pneumoniae that showed discrepant results for imipenem and meropenem from 4 New York City hospitals were characterized by isoelectric focusing; broth microdilution (BMD); disk diffusion (DD); and MicroScan, Phoenix, Sensititre, VITEK, and VITEK 2 automated systems. All 15 isolates were either intermediate or resistant to imipenem and meropenem by BMD; 1 was susceptible to imipenem by DD. MicroScan and Phoenix reported 1 (6.7%) and 2 (13.3%) isolates, respectively, as imipenem susceptible. VITEK and VITEK 2 reported 10 (67%) and 5 (33%) isolates, respectively, as imipenem susceptible. By Sensititre, 13 (87%) isolates were susceptible to imipenem, and 12 (80%) were susceptible to meropenem. The VITEK 2 Advanced Expert System changed 2 imipenem MIC results from >16 μg/mL to <2 μg/mL but kept the interpretation as resistant. The recognition of carbapenem-resistant K. pneumoniae continues to challenge automated susceptibility systems

Faculty Publications for Academic Year 2018-19

Faculty publications of School of Architecture for the academic year 2018- 201

Evaluation of methods for detection of β-lactamase production in MSSA

OBJECTIVES: Correct determination of penicillin susceptibility is pivotal for using penicillin in the treatment of Staphylococcus aureus infections. This study examines the performance of MIC determination, disc diffusion and a range of confirmatory tests for detection of penicillin susceptibility in S. aureus. METHODS: A total of 286 consecutive penicillin-susceptible S. aureus blood culture isolates as well as a challenge set of 62 MSSA isolates were investigated for the presence of the blaZ gene by PCR and subjected to penicillin-susceptibility testing using broth microdilution MIC determination, disc diffusion including reading of the zone edge, two nitrocefin tests and the cloverleaf test. RESULTS: Using PCR-based detection of blaZ as the gold standard, both broth microdilution MIC testing and disc diffusion testing resulted in a relatively low accuracy (82%–93%) with a sensitivity ranging from 49%–93%. Among the confirmatory tests, the cloverleaf test performed with 100% accuracy, while zone edge interpretation and nitrocefin-based tests increased the sensitivity of β-lactamase detection to 96%–98% and 82%–96% when using MIC determination or disc diffusion as primary test, respectively. CONCLUSIONS: This investigation showed that reliable and accurate detection of β-lactamase production in S. aureus can be obtained by MIC determination or penicillin disc diffusion followed by interpretation of the zone edge as a confirmatory test for apparently penicillin-susceptible isolates. The more cumbersome cloverleaf test can also be used. Nitrocefin-based tests should not be used as the only test for confirmation of a presumptive β-lactamase-negative isolate

Correlation of Penicillin Binding Protein 2a Detection with Oxacillin Resistance in Staphylococcus aureus and Discovery of a Novel Penicillin Binding Protein 2a Mutation

We compared a rapid slide latex agglutination test (LAT; Oxoid, Basingstoke, United Kingdom) that detects penicillin binding protein 2a (PBP2a) with MicroScan conventional panels (Dade Behring, West Sacramento, CA) for detection of oxacillin resistance in Staphylococcus aureus. The PBP2a LAT demonstrated 99% agreement with MicroScan oxacillin MIC results for 388 isolates of S. aureus. All 249 oxacillin-resistant isolates gave strong positive reactions in the LAT (100% sensitivity). Three of the 139 oxacillin-susceptible isolates were also strongly positive and one was weakly positive in the LAT (97.1% specificity). The three oxacillin-susceptible isolates with strongly positive reactions were further characterized. The mecA gene was detected in all three by PCR; one isolate was determined to be resistant to oxacillin by reference broth microdilution testing (MIC, 8 μg/ml), one isolate was inducibly resistant to oxacillin (MIC of 16 μg/ml after overnight induction), and one isolate remained susceptible regardless of the method used for testing. Sequence analysis of a 2.1-kb gene fragment of the mecA gene from the susceptible isolate revealed a one-base substitution at nucleotide position 1449 which results in a Met-to-Ile change for amino acid residue 483. This amino acid substitution has not been previously reported and may be associated with a change in the function of PBP2a resulting in oxacillin susceptibility. An additional 487 isolates were tested in parallel with the both the LAT and MicroScan panels using criteria in which only strong (3 to 4+) or repeatedly weak (1 to 2+) LAT reactions were considered positive, and the results showed 99.4% agreement. The PBP2a LAT provided rapid and reliable detection of oxacillin resistance and proved a useful adjunct to the phenotypic method. Both methods provided reliable detection of oxacillin-resistant S. aureus and facilitated the discovery of a novel, functionally impaired form of PBP2a

Characterization of Staphylococcus aureus Isolates from Nasal Cultures Collected from Individuals in the United States in 2001 to 2004▿

This study characterizes methicillin-susceptible Staphylococcus aureus (MSSA) and methicillin-resistant S. aureus (MRSA) isolates recovered from nasal cultures of noninstitutionalized individuals in the United States obtained in 2001 to 2004 as part of the National Health and Nutrition Examination Survey. Every tenth MSSA isolate and all MRSA isolates were typed by pulsed-field gel electrophoresis (PFGE), screened for multiple toxin genes, and tested for susceptibility to 14 antimicrobial agents. USA200, USA600, and USA900 were the predominant PFGE types among MSSA isolates in both the 2001 to 2002 and the 2003 to 2004 time periods, although they accounted for only 51.3% of 316 MSSA isolates typed in 2001 and 2002 and only 43.4% of 237 MSSA isolates typed in 2003 and 2004. In contrast, USA100, USA800, and USA700 accounted for 80.0% of the 75 MRSA isolates typed in 2001 and 2002, while USA100, USA800, and USA300 accounted for 78.4% of 134 MRSA isolates typed in 2003 and 2004. The proportion of MRSA isolates that were USA300 increased significantly from the first to the second time period (P = 0.03). Most USA200 isolates (both MSSA and MRSA) carried the gene for toxic shock syndrome toxin; however, carriage of the genes encoding Panton-Valentine leukocidin, while common among MRSA of PFGE type USA300, was rare among MSSA USA300 in both time periods. Most MSSA isolates remained susceptible to all antimicrobial agents except erythromycin (79.1 and 76.0% susceptibilities in the 2001 to 2002 and the 2003 to 2004 periods, respectively). In contrast, the proportions of MRSA isolates that were susceptible to chloramphenicol, clindamycin, and erythromycin were lower in 2003 and 2004 than in 2001 and 2002, although none of these differences was statistically significant

Phenotypic and Genotypic Characterization of Enterobacteriaceae Producing Oxacillinase-48–Like Carbapenemases, United States

Oxacillinase (OXA)–48–like carbapenemases remain relatively uncommon in the United States. We performed phenotypic and genotypic characterization of 30 Enterobacteriaceae producing OXA-48–like carbapenemases that were recovered from patients during 2010–2014. Isolates were collected from 12 states and not associated with outbreaks, although we could not exclude limited local transmission. The alleles β-lactamase OXA-181 (blaOXA-181) (43%), blaOXA-232 (33%), and blaOXA-48 (23%) were found. All isolates were resistant to ertapenem and showed positive results for the ertapenem and meropenem modified Hodge test and the modified carbapenem inactivation method; 73% showed a positive result for the Carba Nordmann–Poirel test. Whole-genome sequencing identified extended-spectrum β-lactamase genes in 93% of isolates. In all blaOXA-232 isolates, the gene was on a ColKP3 plasmid. A total of 12 of 13 isolates harboring blaOXA-181 contained the insertion sequence ΔISEcp1. In all isolates with blaOXA-48, the gene was located on a TN1999 transposon; these isolates also carried IncL/M plasmids

Accuracy of Commercial and Reference Susceptibility Testing Methods for Detecting Vancomycin-Intermediate Staphylococcus aureus▿

We compared the results obtained with six commercial MIC test systems (Etest, MicroScan, Phoenix, Sensititre, Vitek Legacy, and Vitek 2 systems) and three reference methods (agar dilution, disk diffusion, and vancomycin [VA] agar screen [VScr]) with the results obtained by the Clinical and Laboratory Standards Institute broth microdilution (BMD) reference method for the detection of VA-intermediate Staphylococcus aureus (VISA). A total of 129 S. aureus isolates (VA MICs by previous BMD tests, ≤1 μg/ml [n = 60 strains], 2 μg/ml [n = 24], 4 μg/ml [n = 36], or 8 μg/ml [n = 9]) were selected from the Centers for Disease Control and Prevention strain collection. The results of BMD with Difco Mueller-Hinton broth were used as the standard for data analysis. Essential agreement (percent ±1 dilution) ranged from 98 to 100% for all methods except the method with the Vitek Legacy system, for which it was 90.6%. Of the six commercial MIC systems tested, the Sensititre, Vitek Legacy, and Vitek 2 systems tended to categorize VISA strains as susceptible (i.e., they undercalled resistance); the MicroScan and Phoenix systems and Etest tended to categorize susceptible strains as VISA; and the Vitek Legacy system tended to categorize VISA strains as resistant (i.e., it overcalled resistance). Disk diffusion categorized all VISA strains as susceptible. No susceptible strains (MICs ≤ 2 μg/ml) grew on the VScr, but all strains for which the VA MICs were 8 μg/ml grew on the VScr. Only 12 (33.3%) strains for which the VA MICs were 4 μg/ml grew on VScr. The differentiation of isolates for which the VA MICs were 2 or 4 μg/ml was difficult for most systems and methods, including the reference methods

Effect of Carbon Dioxide on Broth Microdilution Susceptibility Testing of Brucella spp. ▿

Since some strains of Brucella species may require carbon dioxide for growth, a multilaboratory study was conducted to compare broth microdilution susceptibility results using ambient air (AA) and 5% CO2 incubation conditions. Six antimicrobial agents were tested against 39 Brucella isolates. Aminoglycoside MICs tended to be 1 log2 dilution higher in CO2 than in AA; tetracycline-class MICs to be 1 log2 dilution lower in CO2