Kohner, Peggy C.

Susceptibility testing of the polymyxins (colistin and polymyxin B) is challenging for clinical laboratories. The Clinical and Laboratory Standards Institute (CLSI) Antimicrobial Susceptibility Testing Subcommittee evaluated two methods to enable accurate testing of these agents. These methods were a colistin broth disk elution (CBDE) and a colistin agar test (CAT), the latter of which was evaluated using two inoculum volumes, 1 μl (CAT-1) and 10 μl (CAT-10).

ABSTRACT We studied the DNA sequence variation of van genes of 34 isolates of Enterococcus spp. The isolates containing the vanB gene exhibited between 0 and 41 base pair changes per 801 bp studied when the vanB sequences were compared to that of the reference strain Enterococcus faecalis V583. The isolates carrying the vanC-2 gene exhibited between 0 and 23 base pair changes per 346 bp studied when the vanC-2 sequences were compared to that of the reference strain E. casseliflavus ATCC 25788. Little variation was noted in the vanA and vanC-1 genes.

ABSTRACT Failure to eradicate Helicobacter pylori infection is often a result of antimicrobial resistance, which for clarithromycin is typically mediated by specific point mutations in the 23S rRNA gene. The purpose of this study was to define current patterns of antimicrobial susceptibility in H. pylori isolates derived primarily from the United States and to survey them for the presence of point mutations in the 23S rRNA gene and assess the ability of these mutations to predict phenotypic clarithromycin susceptibility. Antimicrobial susceptibility testing was performed using agar dilution on 413 H. pylori isolates submitted to Mayo Medical Laboratories for susceptibility testing. For a subset of these isolates, a 150-bp segment of the 23S rRNA gene was sequenced. A total of 1,970 MICs were reported over the 4-year study period. The rate of clarithromycin resistance was high (70.4%), and elevated MICs were frequently observed for metronidazole (82.4% of isolates had an MIC of >8 μg/ml) and ciprofloxacin (53.5% of isolates had an MIC of >1 μg/ml). A total of 111 archived H. pylori isolates underwent 23S rRNA gene sequencing; we found 95% concordance between genotypes and phenotypes ( P = 0.9802). Resistance to clarithromycin was most commonly due to an A2143G mutation (82%), followed by A2142G (14%) and A2142C (4%) mutations. Clinical H. pylori isolates derived primarily from the United States demonstrated a high rate of clarithromycin resistance and elevated metronidazole and ciprofloxacin MICs. The relative distribution of point mutations at positions 2143 and 2142 in the 23S rRNA gene in clarithromycin-resistant H. pylori was similar to that reported from other parts of the world; these mutations predict phenotypic resistance to clarithromycin.

ABSTRACT Phenotypic variants of Staphylococcus aureus that display small colonies, reduced pigmentation, and decreased hemolysis and/or coagulase activity are periodically isolated by the clinical laboratory. Antimicrobial susceptibility testing (AST) of these isolates is complicated, because many do not grow on routine AST media, including Mueller-Hinton agar (MHA) and cation-adjusted Mueller-Hinton broth. This multicenter study evaluated cefoxitin disk diffusion for 37 atypical S. aureus isolates (156 readings) with MHA supplemented with 5% sheep's blood (BMHA), using mecA PCR as the reference standard. The correlation of two commercial PBP2a assays with mecA PCR was also assessed. Ten isolates were negative and 27 positive for mecA . No major errors for cefoxitin were observed, but 19.5% very major errors (VMEs) were observed at 24 h of incubation, and 17.2% VMEs were observed at 48 h. The proportions of VMEs ranged from 14.7 to 23.0% at 24 h, and from 13.3 to 17.6% at 48 h, across three testing laboratories. PBP2a tests were performed from growth on BMHA and blood agar plates (BAP), with and without cefoxitin disk induction. The Alere PBP2a SA culture colony test sensitivities for mecA were 90.0% with uninduced growth and 97.4% with induced growth from BMHA. On BAP, sensitivity was 96.0% with induced growth. The sensitivities of the Oxoid PBP2′ latex agglutination test were 85.7% with uninduced growth and 93.9% with induced growth from BMHA and 95.9% with induced growth on BAP. On the basis of these data, we recommend that laboratories perform only mecA PCR and/or PBP2a tests when requested to perform AST on atypical isolates of S. aureus .

ABSTRACT MICs of 25 Abiotrophia defectiva and 109 Granulicatella adiacens isolates were determined by broth microdilution. Using CLSI breakpoints, the susceptibilities of A. defectiva and G. adiacens isolates were, respectively, 24% and 34% to penicillin, 92% and 22% to ceftriaxone, 48% and 3% to cefepime, 72% and 87% to meropenem, 92% and 10% to cefotaxime, 100% and 97% to levofloxacin, 92% and 80% to clindamycin, and 24% and 50% to erythromycin. All isolates were susceptible to vancomycin. In the penicillin-susceptible subgroup, all A. defectiva isolates were susceptible to ceftriaxone; however, 62% of G. adiacens isolates were ceftriaxone nonsusceptible.

ABSTRACT Among 177 carbapenemase-producing Gram-negative bacilli (108 KPC, 32 NDM, 11 IMP, 8 OXA-48, 4 OXA-181, 2 OXA-232, 5 IMI, 4 VIM, and 3 SME producers), aztreonam-avibactam was active against all isolates except two NDM producers with elevated MICs of 8/4 and 16/4 mg/liter; ceftazidime-avibactam was active against all KPC-, IMI-, SME-, and most OXA-48 group-producing isolates (93%) but not metallo-β-lactamase producers. Among older and contemporary antimicrobials, the most active were colistin, tigecycline, and fosfomycin, with overall susceptibilities of 88%, 79%, and 78%, respectively.

ABSTRACT We assessed the performance of a duplex real-time PCR assay for bla KPC and bla NDM performed directly (D-PCR) on perianal and perirectal swabs and stool. Spiked specimens and 126 clinical surveillance swabs (comprising a sensitivity panel of 46 perirectal double swabs previously determined to be culture positive for bla KPC -PCR-positive Enterobacteriaceae and a specificity panel of 80 perianal swabs from patients at risk of carbapenemase-producing Enterobacteriaceae [CPE] colonization) were studied. For the surveillance swabs, D-PCR was compared to PCR after broth enrichment (BE-PCR) and two culture-based methods: the HardyCHROM ESBL agar (HC-A) and the CDC screening (CDC-A) methods. PCR was performed on morphologically distinct colonies that were isolated by culture. All of the initial PCR testing was done without extraction using a simple lysis procedure. The analytical sensitivities of D-PCR for bla KPC were 9 CFU/μl (for swabs) and 90 CFU/μl (for stool), and for bla NDM , it was 1.9 CFU/μl (for both swabs and stool). In the clinical sensitivity panel, D-PCR and BE-PCR were initially positive for bla KPC in 41/46 (89.1%) and 43/46 (93.5%) swabs, respectively. The swabs that were initially negative by D-PCR ( n = 5) and BE-PCR ( n = 3) were visibly stool soiled; all swabs were bla KPC positive upon repeat testing after lysate extraction. The CDC-A and HC-A yielded bla KPC -positive Enterobacteriaceae from 36/46 (78.3%) and 35/46 (76.1%) swabs, respectively (sensitivities of D-PCR/BE-PCR postextraction of soiled specimens versus HC-A, P = 0.0009, and versus CDC-A, P = 0.0016). All swabs in the specificity panel were negative for CPE by all four methods. D-PCR allows for the timely detection of bla KPC and bla NDM carriage with excellent sensitivity when specimens visibly soiled with stool undergo preparatory extraction.

ABSTRACT We compared carbapenemase detection among 271 Gram-negative bacilli (of which 131 were carbapenemase producers) using a novel chromogenic rapid test—the Carba NP test (CNP)—and the modified Hodge test (MHT). Sensitivities were comparable (CNP, 100%, versus MHT, 98%; P = 0.08), but CNP was more specific (100% versus 80%; P < 0.0001) and faster.