Finding, characterizing, and monitoring reservoirs for antimicrobial resistance (AMR) is vital to protecting public health. Hybridization capture baits are an accurate, sensitive, and cost-effective technique used to enrich and characterize DNA sequences of interest, including antimicrobial resistance genes (ARGs), in complex environmental samples. We demonstrate the continued utility of a set of 19,933 hybridization capture baits designed from the Comprehensive Antibiotic Resistance Database (CARD)v1.1.2 and Pathogenicity Island Database (PAIDB)v2.0, targeting 3,565 unique nucleotide sequences that confer resistance. We demonstrate the efficiency of our bait set on a custom-made resistance mock community and complex environmental samples to increase the proportion of on-target reads as much as >200-fold. However, keeping pace with newly discovered ARGs poses a challenge when studying AMR, because novel ARGs are continually being identified and would not be included in bait sets designed prior to discovery. We provide imperative information on how our bait set performs against CARDv3.3.1, as well as a generalizable approach for deciding when and how to update hybridization capture bait sets. This research encapsulates the full life cycle of baits for hybridization capture of the resistome from design and validation (both in silico and in vitro) to utilization and forecasting updates and retirement.Originality-Significance Statement This work is applicable to a wide range of research. It helps to define conditions under which hybridization capture is useful regarding not only antimicrobial resistance specifically, but also more generally how to assess the ongoing utility of existing bait sets - giving objective criteria for when and by what strategies baits should be updated. We also provide a method for quantifying and comparing antimicrobial resistance genes (ARGs) similar to what is used for RNAseq experiments. This approach improves comparison of ARGs across environments. Thus, the work provides an improved foundation for ARG future studies, while cutting across traditional areas of microbiology and extending beyond.Competing Interest StatementThe EHS DNA lab provides oligonucleotide aliquots and library preparation services at cost, including some oligonucleotides and services used in this manuscript (baddna.uga.edu). JE and AD were employed by, and thereby have financial interest in, Daicel Arbor Biosciences, who provided the in-solution capture reagents used in this work. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

BACKGROUND: Sink drains in healthcare facilities may provide an environment for antimicrobial-resistant microorganisms, including carbapenemase-producing Klebsiella pneumoniae (CPKP). METHODS: We investigated the colonization of a biofilm consortia by CPKP in a model system simulating a sink-drain P-trap. Centers for Disease Control (CDC) biofilm reactors (CBRs) were inoculated with microbial consortia originally recovered from 2 P-traps collected from separate patient rooms (designated rooms A and B) in a hospital. Biofilms were grown on stainless steel (SS) or polyvinyl chloride (PVC) coupons in autoclaved municipal drinking water (ATW) for 7 or 28 days. RESULTS: Microbial communities in model systems (designated CBR-A or CBR-B) were less diverse than communities in respective P-traps A and B, and they were primarily composed of β and γ Proteobacteria, as determined using 16S rRNA community analysis. Following biofilm development CBRs were inoculated with either K. pneumoniae ST45 (ie, strain CAV1016) or K. pneumoniae ST258 KPC+ (ie, strain 258), and samples were collected over 21 days. Under most conditions tested (CBR-A: SS, 7-day biofilm; CBR-A: PVC, 28-day biofilm; CBR-B: SS, 7-day and 28-day biofilm; CBR-B: PVC, 28-day biofilm) significantly higher numbers of CAV1016 were observed compared to 258. CAV1016 showed no significant difference in quantity or persistence based on biofilm age (7 days vs 28 days) or substratum type (SS vs PVC). However, counts of 258 were significantly higher on 28-day biofilms and on SS. CONCLUSIONS: These results suggest that CPKP persistence in P-trap biofilms may be strain specific or may be related to the type of P-trap material or age of the biofilm.

A surgical heater-cooler unit has been implicated as the source for Mycobacterium chimaera infections among cardiac surgery patients in several countries. We isolated M. chimaera from heater-cooler units and patient infections in the United States. Whole-genome sequencing corroborated a risk for these units acting as a reservoir for this pathogen.

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.

In this pilot study, traditional culture and PCR methods were compared to the Cepheid GeneXpert IV molecular diagnostic system with the Xpert Carba-R assay (Carba-R assay) for detection of carbapenem resistance genes in primary environmental samples collected during a health care-related outbreak. Overall, traditional culture-dependent PCR and the Carba-R assay demonstrated 75% agreement. The Carba-R assay detected carbapenemase genes in five additional samples and in two samples that had additional genes when compared to culture-dependent PCR. The Carba-R assay could be useful for prioritizing further testing of environmental samples during health care-related outbreaks.IMPORTANCE Use of the Carba-R assay for detection of carbapenem-resistant Gram-negative organisms (CROs) can provide data for implementation of a rapid infection control response to minimize the spread of CROs in the health care setting.

BACKGROUND: Outbreaks of health care-associated infections (HAIs) caused by Burkholderia cepacia complex (Bcc) have been associated with medical devices and water-based products. Water is the most common raw ingredient in nonsterile liquid drugs, and the significance of organisms recovered from microbiologic testing during manufacturing is assessed using a risk-based approach. This incident demonstrates that lapses in manufacturing practices and quality control of nonsterile liquid drugs can have serious unintended consequences. METHODS: An epidemiologic and laboratory investigation of clusters of Bcc HAIs that occurred among critically ill, hospitalized, adult and pediatric patients was performed between January 1, 2016, and October 31, 2016. RESULTS: One hundred and eight case patients with Bcc infections at a variety of body sites were identified in 12 states. Two distinct strains of Bcc were obtained from patient clinical cultures. These strains were found to be indistinguishable or closely related to 2 strains of Bcc obtained from cultures of water used in the production of liquid docusate, and product that had been released to the market by manufacturer X. CONCLUSIONS: This investigation highlights the ability of bacteria present in nonsterile, liquid drugs to cause infections or colonization among susceptible patients. Prompt reporting and thorough investigation of potentially related infections may assist public health officials in identifying and removing contaminated products from the market when lapses in manufacturing occur.

Mycobacterium chimaera is a nontuberculous mycobacterium species commonly found in the environment. Here, we report the first complete genome sequence of a strain from the investigation of invasive infections following open-heart surgeries that used contaminated LivaNova Sorin Stockert 3T heater-cooler devices.

Mycobacterium wolinskyi is a nonpigmented, rapidly growing nontuberculous mycobacterium species that is associated with bacteremia, peritonitis, infections associated with implants/prostheses, and skin and soft tissue infections often following surgical procedures in humans. Here, we report the first functionally annotated draft genome sequence of M. wolinskyi CDC_01.

As annual influenza epidemics continue to cause significant morbidity and economic burden, an understanding of viral persistence and transmission is critical for public health officials and healthcare workers to better protect patients and their family members from infection. The infectivity and persistence of two influenza A (H1N1) strains (A/New Caledonia/20/1999 and A/Brisbane/59/2007) were evaluated on stainless steel (SS) surfaces using three different surfaces matrices (2% fetal bovine serum, 5 mg/mL mucin, and viral medium) at varying absolute humidity conditions (4.1 x 105 mPa, 6.5 x 105 mPa, 7.1 x 105 mPa, 11.4 x 105 mPa, 11.2 x 105 mPa, and 17.9 x 105 mPa) for up to seven days. Influenza virus was deposited onto SS coupons (7.07 cm2) and recovered by agitation and sonicating in viral medium. Viral persistence was quantified using a tissue culture based enzyme-linked immunosorbent assay (ELISA) to determine the median tissue culture infective dose (TCID50) of infectious virus per coupon. Overall, both strains of influenza A virus remained infectious on SS coupons with an approximate 2 log10 loss over seven days. Factors that influenced viral persistence included absolute humidity, strain/absolute humidity interaction, and time (P ≤ 0.01). Further studies into hand transfer of influenza A virus from fomites and the impact of inanimate surface contamination in transmission should be investigated as this study demonstrates prolonged persistence on non-porous surfaces. IMPORTANCE: The study tested the ability of two influenza A H1N1 strains to persist and remain infectious on stainless steel surfaces in varying environmental conditions. It is demonstrated that influenza A H1N1 virus can persist and remain infectious on stainless steel surfaces for 7 days. This raises the question of what role contaminated surfaces play in the transmission of influenza A virus and that additional studies should be conducted to assess this.

BACKGROUND: Composition and diversity of intestinal microbial communities (microbiota) are generally accepted as a risk factor for poor outcomes; however, we cannot yet use this information to prevent adverse outcomes. METHODS: Stool was collected from 8 long-term acute care hospital patients experiencing diarrhea and 2 fecal microbiota transplant donors; 16S rDNA V1-V2 hypervariable regions were sequenced. Composition and diversity of each sample were described. Stool was also tested for Clostridium difficile, vancomycin-resistant enterococci (VRE), and carbapenem-resistant Enterobacteriaceae. Associations between microbiota diversity and demographic and clinical characteristics, including antibiotic use, were analyzed. RESULTS: Antibiotic exposure and Charlson Comorbidity Index were inversely correlated with diversity (Spearman = -0.7). Two patients were positive for VRE; both had microbiomes dominated by Enterococcus faecium, accounting for 67%-84% of their microbiome. CONCLUSIONS: Antibiotic exposure correlated with diversity; however, other environmental and host factors not easily obtainable in a clinical setting are also known to impact the microbiota. Therefore, direct measurement of microbiome disruption by sequencing, rather than reliance on surrogate markers, might be most predictive of adverse outcomes. If and when microbiome characterization becomes a standard diagnostic test, improving our understanding of microbiome dynamics will allow for interpretation of results to improve patient outcomes.

A multidrug-resistant Escherichia coli isolate from an abdominal lesion displayed resistance to all beta-lactams tested, including carbapenems, in addition to macrolides, fluoroquinolones, and tetracycline. Sequence analyses demonstrated the presence of blaNDM-5 in addition to at least 13 genes and 6 efflux pumps associated with antibiotic resistance.

Research on influenza viruses regarding transmission and survival has surged in the recent years due to infectious emerging strains and outbreaks such as the 2009 Influenza A (H1N1) pandemic. MS2 coliphage has been applied as a surrogate for pathogenic respiratory viruses, such as influenza, as it's safe for personnel to handle and requires less time and labor to measure virus infectivity. However, direct comparisons to determine the effectiveness of coliphage as a surrogate for influenza virus regarding droplet persistence on personal protective equipment such as N95 filtering facepiece respirators (FFRs) are lacking. Persistence of viral droplets deposited on FFRs in healthcare settings is important to discern due to the potential risk of infection via indirect fomite transmission. The objective of this study was to determine if MS2 coliphage could be applied as a surrogate for influenza A viruses for studying persistence when applied to the FFRs as a droplet. The persistence of MS2 coliphage and 2009 Pandemic Influenza A (H1N1) Virus on FFR coupons in different matrices (viral media, 2% fetal bovine serum, and 5 mg ml(-1) mucin) were compared over time (4, 12, 24, 48, 72, and 144 hours) in typical absolute humidity conditions (4.1 × 10(5) mPa [18°C/20% relative humidity (RH)]). Data revealed significant differences in viral infectivity over the 6-day period (H1N1- P <0.0001; MS2 - P <0.005), although a significant correlation of viral log(10) reduction in 2% FBS (P <0.01) was illustrated. Overall, MS2 coliphage was not determined to be a sufficient surrogate for influenza A virus with respect to droplet persistence when applied to the N95 FFR as a droplet.

The effect of packaging, shipping temperatures and storage times on recovery of Bacillus anthracis. Sterne spores from swabs was investigated. Macrofoam swabs were pre-moistened, inoculated with Bacillus anthracis spores, and packaged in primary containment or secondary containment before storage at -15°C, 5°C, 21°C, or 35°C for 0-7 days. Swabs were processed according to validated Centers for Disease Control/Laboratory Response Network culture protocols, and the percent recovery relative to a reference sample (T(0)) was determined for each variable. No differences were observed in recovery between swabs held at -15° and 5°C, (p ≥ 0.23). These two temperatures provided significantly better recovery than swabs held at 21°C or 35°C (all 7 days pooled, p ≤ 0.04). The percent recovery at 5°C was not significantly different if processed on days 1, 2 or 4, but was significantly lower on day 7 (day 2 vs. 7, 5°C, 10(2), p=0.03). Secondary containment provided significantly better percent recovery than primary containment, regardless of storage time (5°C data, p ≤ 0.008). The integrity of environmental swab samples containing Bacillus anthracis spores shipped in secondary containment was maintained when stored at -15°C or 5°C and processed within 4 days to yield the optimum percent recovery of spores.

In the U.S., the 2009 pandemic influenza A (H1N1) virus (pH1N1) infected almost 20% of the population and caused >200,000 hospitalizations and >10,000 deaths during April 2009 to April 2010. On April 24, 2009, CDC posted interim guidance on infection control measures in healthcare settings specifically for pH1N1, which recommended filtering face respirators (FFRs) when in close contact with a suspected/confirmed infected individual and particularly when performing aerosol procedures. The persistence and infectivity of pH1N1 was evaluated on FFRs, specifically N95 respirators, under varying conditions of absolute humidity (AH; 4.1 millibar (mb), 6.5 mb, and 14.6 mb), sample matrices (2% fetal bovine serum (FBS), 5 mg/ml mucin, and viral media), and time (4, 12, 24, 48, 72, and 144 hours). pH1N1 was distributed onto N95 coupons (3.8 to 4.2 cm(2)), extracted by a vortex/centrifugation/filtration process, and the ability of remaining virus to replicate was quantified by ELISA to determine the log(10) concentration of infectious virus per coupon. Overall, pH1N1 remained infectious for 6 days with approximately 1 log(10) loss of viral concentrations over this time period. Time and AH both affected viral survival. We found significantly higher (p ≤0.01) reductions in virus concentrations at time-points beyond 24 to 72 hours (-0.52 log(10) reduction) and 144 hours (-0.74) at AHs of 6.5 mb (-0.53) and 14.6 mb (-0.47). This research supports the discarding of respirators after close contact with a person with suspected or confirmed influenza infection due to the virus's demonstrated ability to persist and remain infectious.