OBJECTIVES: Despite clinical relevance, commercially available molecular tools for accurate β-lactamase detection are limited. In this study, we evaluated the performance of the ARM-D® β-lactamase Kit, a commercially available multiplex PCR assay designed to detect nine β-lactamase genes, including the five major plasmid-mediated carbapenemases, ESBL, or AmpC genes circulating in the United States. METHODS: A diverse collection of 113 Gram-negative isolates, including 42 with multiple β-lactamases was selected from the U.S. Centers for Disease Control and Prevention (CDC) & Food and Drug Administration (FDA) Antibiotic Resistance Isolate Bank, to represent the most frequently detected bacterial species carrying plasmid-mediated β-lactam resistance genes. RESULTS: Results were compared with whole genome sequence data. Of 164 β-lactamase gene targets with 49 unique variants, all were detected correctly without any cross-reactivity. The sensitivity and specificity were 100% (164/164) and 99.9% (852/853), respectively. CONCLUSION: The ARM-D® β-lactamase Kit detected a wide range of β-lactamase genotypes at a low upfront cost. The Streck assay represents a suitable, comprehensive tool for the detection of key β-lactamase resistance genes of public health concern in the United States.

Chlorhexidine bathing to prevent transmission of multidrug-resistant organisms has been adopted by many U.S. hospitals, but increasing chlorhexidine use has raised concerns about possible emergence of resistance. We sought to establish a broth microdilution method for determining chlorhexidine MICs and then used the method to evaluate chlorhexidine MICs for bacteria that can cause health care-associated infections. We adapted a broth microdilution method for determining chlorhexidine MICs, poured panels, established quality control ranges, and tested Staphylococcus aureus, Escherichia coli, Klebsiella pneumoniae, and Enterobacter cloacae complex isolates collected at three U.S. sites. Chlorhexidine MICs were determined for 535 isolates including 129 S. aureus, 156 E. coli, 142 K. pneumoniae, and 108 E. cloacae complex isolates. The respective MIC distributions for each species ranged from 1 to 8 mg/L (MIC(50) = 2 mg/L and MIC(90) = 4 mg/L), 1 to 64 mg/L (MIC(50) = 2 mg/L and MIC(90) = 4 mg/L), 4 to 64 mg/L (MIC(50) = 16 mg/L and MIC(90) = 32 mg/L), and 1 to >64 mg/L (MIC(50) = 16 mg/L and MIC(90) = 64 mg/L). We successfully adapted a broth microdilution procedure that several laboratories were able to use to determine the chlorhexidine MICs of bacterial isolates. This method could be used to investigate whether chlorhexidine MICs are increasing. IMPORTANCE Chlorhexidine bathing to prevent transmission of multidrug-resistant organisms and reduce health care-associated infections has been adopted by many hospitals. There is concern about the possible unintended consequences of using this agent widely. One possible unintended consequence is decreased susceptibility to chlorhexidine, but there are not readily available methods to perform this evaluation. We developed a method for chlorhexidine MIC testing that can be used to evaluate for possible unintended consequences.

Listeria monocytogenes is a foodborne pathogen and the causative agent of listeriosis, a disease associated with high fatality (20-30%) and hospitalization rates (>95%). ATP-Binding Cassette (ABC) transporters have been demonstrated to be involved in the general stress response. In previous studies, in-frame deletion mutants of the ABC transporter genes, LMOf2365_1875 and LMOf2365_1877, were constructed and analyzed; however, additional work is needed to investigate the virulence potential of these deletion mutants. In this study, two in vitro methods and one in vivo model were used to investigate the virulence potential of in-frame deletion mutants of ABC transporter genes. First, the invasion efficiency in host cells was measured using the HT-29 human cell line. Second, cell-to-cell spread activity was measured using a plaque forming assay. Lastly, virulence potential of the mutants was tested in the Galleria mellonella wax moth model. Our results demonstrated that the deletion mutant, ⊿LMOf2365_1875, displayed decreased invasion and cell-to-cell spread efficiency in comparison to the wild-type, LMOf2365, indicating that LMOf2365_1875 may be required for virulence. Furthermore, the reduced virulence of these mutants was confirmed using the Galleria mellonella wax moth model. In addition, the expression levels of 15 virulence and stress-related genes were analyzed by RT-PCR assays using stationary phase cells. Our results showed that virulence-related gene expression levels from the deletion mutants were elevated (15/15 genes from ⊿LMOf2365_1877 and 7/15 genes from ⊿LMOf2365_1875) compared to the wild type LMOf2365, suggesting that ABC transporters may negatively regulate virulence gene expression under specific conditions. The expression level of the stress-related gene, clpE, also was increased in both deletion mutants, indicating the involvement of ABC transporters in the stress response. Taken together, our findings suggest that ABC transporters may be used as potential targets to develop new therapeutic strategies to control L. monocytogenes.

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.

Four Enterobacteriaceae clinical isolates bearing mcr-1 gene-harboring plasmids were characterized. All isolates demonstrated the ability to transfer colistin resistance to E. coli; plasmids were stable in conjugants after multiple passages on non-selective media. mcr-1 was located on an IncX4 (n=3) or IncN (n=1) plasmid. The IncN plasmid harbored 13 additional antimicrobial resistance genes. Results indicate the mcr-1-bearing plasmids in this study are highly transferable in vitro and stable in the recipients.

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.

Listeria monocytogenes is a foodborne pathogen that causes listeriosis, which is a major public health concern due to the high fatality rate. LMOf2365_0442, 0443, and 0444 encode for fructose-specific EIIABC components of phosphotransferase transport system (PTS) permease that is responsible for sugar transport. In previous studies, in-frame deletion mutants of a putative fructose-specific PTS permease (LMOf2365_0442, 0443, and 0444) were constructed and analyzed. However, the virulence potential of these deletion mutants has not been studied. In this study, two in vitro methods were used to analyze the virulence potential of these L. monocytogenes deletion mutants. First, invasion assays were used to measure the invasion efficiencies to host cells using the human HT-29 cell line. Second, plaque forming assays were used to measure cell-to-cell spread in host cells. Our results showed that the deletion mutant DeltaLMOf2365_0442 had reduced invasion and cell-to-cell spread efficiencies in human cell line compared to the parental strain LMOf2365, indicating that LMOf2365_0442 encoding for a fructose specific PTS permease IIA may be required for virulence in L. monocytogenes strain F2365. In addition, the gene expression levels of 15 virulence and stress-related genes were analyzed in the stationary phase cells of the deletion mutants using RT-PCR assays. Virulence-related gene expression levels were elevated in the deletion mutants DeltaLMOf2365_0442-0444 compared to the wild type parental strain LMOf2365, indicating the down-regulation of virulence genes by this PTS permease in L. monocytogenes. Finally, stress-related gene clpC expression levels were also increased in all of the deletion mutants, suggesting the involvement of this PTS permease in stress response. Furthermore, these deletion mutants displayed the same pressure tolerance and the same capacity for biofilm formation compared to the wild-type parental strain LMOf2365. In summary, our findings suggest that the LMOf2365_0442 gene can be used as a potential target to develop inhibitors for new therapeutic and pathogen control strategies for public health.