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.

In August of 2018, the United States Food and Drug Administration (FDA) announced a Class I recall associated with a methicillin-resistant Staphylococcus aureus (MRSA) Safety Alert.

A three-center study was performed to see if Etest gradient diffusion minimum inhibitory concentration (MIC) methodology correlated with reference broth microdilution (BMD) for antimicrobial susceptibility testing of Burkholderia pseudomallei against six antimicrobial agents known to be usually effective against B. pseudomallei. This study was performed to assist in the decision-making process for possible deployment of the Etest method for antimicrobial susceptibility testing of B. pseudomallei into several regional public health laboratories in the United States. Three laboratories each tested a challenge set of 30 genotypically diverse isolates collected from 15 different countries. MICs were performed by both Etest gradient diffusion and reference BMD for amoxicillin/clavulanate, ceftazidime, doxycycline, imipenem, tetracycline, and trimethoprim/sulfamethoxazole. Etest results for amoxicillin/clavulanate, ceftazidime, doxycycline, and imipenem correlated well with reference BMD by both category interpretation (>/=97%) and essential agreement of MIC (>/=93%). Tetracycline and trimethoprim/sulfamethoxazole Etests yielded poor correlation with BMD by category interpretation (80%) and essential agreement (70%), respectively. In conclusion, Etest gradient diffusion represents a valid option for antimicrobial susceptibility testing of B. pseudomallei against amoxicillin/clavulanate, ceftazidime, doxycycline, and imipenem. Tetracycline and trimethoprim/sulfamethoxazole Etest results showed some concerning lack of correlation with the corresponding reference BMD results.

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 beta-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 beta-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 DeltaISEcp1. In all isolates with blaOXA-48, the gene was located on a TN1999 transposon; these isolates also carried IncL/M plasmids.

Antimicrobial resistance is a threat to public health globally and leads to an estimated 23,000 deaths annually in the United States alone. Here, we report the genomic characterization of an unusual Klebsiella pneumoniae, nonsusceptible to all 26 antibiotics tested, that was isolated from a U.S. PATIENT: The isolate harbored four known beta-lactamase genes, including plasmid-mediated blaNDM-1 and blaCMY-6, as well as chromosomal blaCTX-M-15 and blaSHV-28, which accounted for resistance to all beta-lactams tested. In addition, sequence analysis identified mechanisms that could explain all other reported nonsusceptibility results, including nonsusceptibility to colistin, tigecycline, and chloramphenicol. Two plasmids, IncA/C2 and IncFIB, were closely related to mobile elements described previously and isolated from Gram-negative bacteria from China, Nepal, India, the United States, and Kenya, suggesting possible origins of the isolate and plasmids. This is one of the first K. pneumoniae isolates in the United States to have been reported to the Centers for Disease Control and Prevention (CDC) as nonsusceptible to all drugs tested, including all beta-lactams, colistin, and tigecycline.IMPORTANCE Antimicrobial resistance is a major public health threat worldwide. Bacteria that are nonsusceptible or resistant to all antimicrobials available are of major concern to patients and the public because of lack of treatment options and potential for spread. A Klebsiella pneumoniae strain that was nonsusceptible to all tested antibiotics was isolated from a U.S. PATIENT: Mechanisms that could explain all observed phenotypic antimicrobial resistance phenotypes, including resistance to colistin and beta-lactams, were identified through whole-genome sequencing. The large variety of resistance determinants identified demonstrates the usefulness of whole-genome sequencing for detecting these genes in an outbreak response. Sequencing of isolates with rare and unusual phenotypes can provide information on how these extremely resistant isolates develop, including whether resistance is acquired on mobile elements or accumulated through chromosomal mutations. Moreover, this provides further insight into not only detecting these highly resistant organisms but also preventing their spread.

The purpose of this study was to develop the modified Carbapenem Inactivation Method (mCIM) for the detection of carbapenemase-producing (CP) Pseudomonas aeruginosa (PA) and Acinetobacter baumannii (AB) and perform a multicenter evaluation of the mCIM and Carba NP tests for these non-fermenters. Thirty P. aeruginosa and 30 A. baumannii isolates previously characterized by whole genome sequencing from the CDC-FDA Antibiotic Resistance Isolate Bank were evaluated, including carbapenemase-producers (CP; Ambler Class A, B, and D), non-carbapenemase-producing (non-CP) carbapenem-resistant isolates, and carbapenem-susceptible isolates. Initial comparison of a 1 microl versus 10 microl loop inoculum for the mCIM was performed by two testing sites and showed that 10 microl was required for reliable detection of carbapenemase production among PA and AB. Ten testing sites then evaluated the mCIM using a 10 microl loop inoculum. Overall, the mean sensitivity and specificity of the mCIM for detection of CP-PA across all ten sites were 98.0% (95% CI: 94.3-99.6; range: 86.7-100) and 95% (95% CI: 89.8-97.7; range: 93.3-100), whereas the mean sensitivity and specificity among CP-AB were 79.8% (95% CI: 74.0-84.9; range: 36.3-95.7) and 52.9% (95% CI: 40.6- 64.9; range: 28.6-100), respectively. At three sites that evaluated the performance of the Carba NP using the same set of isolates, the mean sensitivity and specificity of the Carba NP were 97.8% (95% CI: 88.2-99.9; range: 93.3-100) and 97.8% (95% CI: 88.2-99.9; range: 93.3-100) for PA and 18.8% (95%CI: 10.4-30.1; range: 8.7-26.1) and 100% (95% CI: 83.9-100; range: 100) for AB. Overall, we found both the mCIM and the Carba NP to be accurate for detection of carbapenemases among PA and less reliable for use with AB isolates.

The ability of clinical microbiology laboratories to reliably detect carbapenemase-producing carbapenem-resistant Enterobacteriaceae (CP-CRE) is an important element of the effort to prevent and contain the spread of these pathogens and an integral part of antimicrobial stewardship. Existing methods each have limitations. A new, straightforward, inexpensive, and specific phenotypic method for the detection of carbapenemase production, the carbapenem inactivation method (CIM), was recently described. Here we describe a two-stage evaluation of a modified carbapenem inactivation method (mCIM), in which tryptic soy broth was substituted for water during the inactivation step and the length of this incubation was extended. A validation study was performed in a single clinical laboratory to determine the accuracy of the mCIM, followed by a nine-laboratory study to verify the reproducibility of these results and define the zone size cut-off that best discriminated between CP-CRE and Enterobacteriaceae that do not produce carbapenemases. Bacterial isolates previously characterized through whole genome sequencing or targeted PCR as to the presence or absence of carbapenemase genes were tested for carbapenemase production using the mCIM; isolates with Ambler class A, B, and D carbapenemases, non-CP-CRE isolates, and carbapenem-susceptible isolates were included. The sensitivity of the mCIM observed in the validation study was 99% (95% confidence interval [CI], 93 to 100) and the specificity was 100% (95% CI, 82 to 100). In the second stage of the study, the range of sensitivities observed across nine laboratories was 93% to 100%, with a mean of 97%; the range of specificities was 97% to 100%, with a mean of 99%. The mCIM was easy to perform and interpret for Enterobacteriaceae, with results in less than 24 hours and excellent reproducibility across laboratories.

In 2015, scientists reported the emergence of the plasmid-encoded mcr-1 gene conferring bacterial resistance to the antibiotic colistin (1), signaling potential emergence of a pandrug-resistant bacterium. In May 2016, mcr-1-positive Escherichia coli was first isolated from a specimen from a U.S. patient (2) when a Pennsylvania woman was evaluated for a urinary tract infection. The urine culture and subsequent testing identified the gene in an extended-spectrum beta-lactamase (ESBL)-producing E. coli with reduced susceptibility to colistin. The patient had no international travel for approximately 1 year, no livestock exposure, and a limited role in meal preparation with store-bought groceries; however, she had multiple and repeated admissions to four medical facilities during 2016.

The recent publication by Pickering et al [1] described a collection of methicillin-susceptible Staphylococcus aureus (MSSA) that displayed elevated ceftriaxone minimum inhibitory concentrations (MICs) when tested by Etest (bioMerieux, Durham, North Carolina) gradient diffusion and would have been called “Resistant” to ceftriaxone based on previous Clinical and Laboratory Standards Institute (CLSI) interpretive guidance. The authors reported that approximately 60% of MSSA tested at their institution would have been misclassified based on the current CLSI guidance, which recommends testing staphylococci only against penicillin and oxacillin or cefoxitin in order to infer susceptibility or resistance to other β-lactam agents. This article was available electronically ahead of print for several months. Although it was subsequently retracted as “an honest error in interpretation,” we believe a fuller explanation of the findings could improve understanding among Clinical Infectious Diseases readership. | We investigated the accuracy of the initial report by performing reference broth microdilution (BMD), disk diffusion, and Etest [both low (0.002–32 µg/mL) and high (0.016–256 µg/mL) range ceftriaxone Etest products] antimicrobial susceptibility testing on 8 pulsed field gel electrophoresis (PFGE)-matched pairs of MSSA from the Pickering study [1] reported to have mismatched ceftriaxone susceptibility. All 16 isolates were confirmed as oxacillin, cefoxitin, and ceftriaxone susceptible [2, 3] with BMD and disk methods. Ceftriaxone MICs obtained by both Etest products were typically higher than those obtained with BMD but were still in the susceptible range for 100% of isolates using the high concentration ceftriaxone Etest, and for 93.8% of isolates using the low concentration ceftriaxone Etest (1 isolate tested as intermediate). In addition, 30 consecutive, unique MSSA isolated from blood cultures during 2 months at a single hospital were tested against ceftriaxone byBMD, disk diffusion, and Etest using a single 0.5 McFarland inoculum. All isolates tested ceftriaxone susceptible by disk diffusion and BMD; 13 (43%) isolates tested nonsusceptible with Etest (Table 1). We also note that the Etest ceftriaxone package inserts do not list staphylococci as an organism group for which testing has been cleared [4, 5].

Utility of Etest for antimicrobial susceptibility testing of Yersinia pestis was evaluated in comparison with broth microdilution and disk diffusion for eight agents. Four laboratories tested 26 diverse strains and found Etest to be reliable for testing antimicrobial agents used to treat Y. pestis, except for chloramphenicol and trimethoprim-sulfamethoxazole. Disk diffusion testing is not recommended.

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

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.

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, <or=1 microg/ml [n = 60 strains], 2 microg/ml [n = 24], 4 microg/ml [n = 36], or 8 microg/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 <or= 2 microg/ml) grew on the VScr, but all strains for which the VA MICs were 8 microg/ml grew on the VScr. Only 12 (33.3%) strains for which the VA MICs were 4 microg/ml grew on VScr. The differentiation of isolates for which the VA MICs were 2 or 4 microg/ml was difficult for most systems and methods, including the reference methods.