Insights into the molecular basis of reduced vancomycin susceptibility among three prominent Staphylococcus aureus clonal complexes.

Staphylococcus aureus is a leading cause of healthcare-associated infections globally. Vancomycin-resistant S. aureus (VRSA), those with high-level resistance [minimum inhibitory concentration (MIC) of 16-32 µg/mL vancomycin], are uncommon, whereas vancomycin-intermediate S. aureus (VISA; MIC of 4-8 µg/mL), are isolated more frequently and develop during long-term and/or repeated use of the antibiotic. VISA can be difficult to eradicate and infections may persist. Our knowledge of mechanisms that underlie the development of VISA is incomplete. We used a genomics approach to investigate the VISA phenotype in three prominent S. aureus lineages. All VISA clinical isolates tested had increased cell wall thickness compared with vancomycin-susceptible S. aureus strains. Growth rates of clonal complex (CC) 5, CC8, and CC45 clinical isolates were reduced in 2 µg/mL vancomycin compared to media alone. Culture in 2 and 4 µg/mL vancomycin sequentially for two weeks reduced susceptibility to daptomycin, televancin, tigecycline, and vancomycin in a majority of CC5, CC8, and CC45 isolates tested. We identified alleles reported previously to contribute to the VISA phenotype, but unexpectedly, these alleles were unique to each CC. A subtherapeutic concentration of vancomycin elicited changes in the VISA transcriptome-common and unique-among the three CCs tested. Multiple genes, including those encoding a glycerate kinase, an M50 family metallopeptidase, and an uncharacterized membrane protein, were upregulated among all three lineages and not reported previously as associated with VISA. Although there are lineage-specific changes in DNA sequence, our findings suggest changes in the VISA transcriptome constitute a general response to stress that confers reduced susceptibility to multiple antibiotics. IMPORTANCE: Our understanding of the mechanisms that underlie the development of vancomycin-intermediate Staphylococcus aureus (VISA) is incomplete. To provide a more comprehensive view of this process, we compared genome sequences of clonal complex (CC) 5, CC8, and CC45 VISA clinical isolates and measured changes in the transcriptomes of these isolates during culture with a subtherapeutic concentration of vancomycin. Notably, we identified differentially expressed genes that were lineage-specific or common to the lineages tested, including genes that have not been previously reported to contribute to a VISA phenotype. Changes in gene expression were accompanied by reduced growth rate, increased cell wall thickness, and reduced susceptibility to daptomycin, televancin, tigecycline, and vancomycin. Our results provide support to the idea that changes in gene expression contribute to the development of VISA among three CCs that are a prominent cause of human infections.

In vitro activity of taurolidine against clinical Candida auris isolates: relevance to catheter-related bloodstream infections.

Candida auris is an evolving and concerning global threat. Of particular concern are bloodstream infections related to central venous catheters. We evaluated the activity of taurolidine, a broad-spectrum antimicrobial in catheter lock solutions, against 106 C. auris isolates. Taurolidine was highly active with a MIC50/MIC90 of 512/512 mg/L, over 20-fold lower than lock solution concentrations of ≥13,500 mg/L. Our data demonstrate a theoretical basis for taurolidine-based lock solutions for prevention of C. auris catheter-associated infections.

Impact of a Rapid Molecular Test for Klebsiella pneumoniae Carbapenemase and Ceftazidime-Avibactam Use on Outcomes After Bacteremia Caused by Carbapenem-Resistant Enterobacterales.

BACKGROUND: Patients with bacteremia due to carbapenem-resistant Enterobacterales (CRE) experience delays until appropriate therapy and high mortality rates. Rapid molecular diagnostics for carbapenemases and new ß-lactam/ß-lactamase inhibitors may improve outcomes. METHODS: We conducted an observational study of patients with CRE bacteremia from 2016 to 2018 at 8 New York and New Jersey medical centers and assessed center-specific clinical microbiology practices. We compared time to receipt of active antimicrobial therapy and mortality between patients whose positive blood cultures underwent rapid molecular testing for the Klebsiella pneumoniae carbapenemase (KPC) gene (blaKPC) and patients whose cultures did not undergo this test. CRE isolates underwent antimicrobial susceptibility testing by broth microdilution and carbapenemase profiling by whole-genome sequencing. We also assessed outcomes when ceftazidime-avibactam and polymyxins were used as targeted therapies. RESULTS: Of 137 patients with CRE bacteremia, 89 (65%) had a KPC-producing organism. Patients whose blood cultures underwent blaKPC PCR testing (n = 51) had shorter time until receipt of active therapy (median: 24 vs 50 hours; P = .009) compared with other patients (n = 86) and decreased 14-day (16% vs 37%; P = .007) and 30-day (24% vs 47%; P = .007) mortality. blaKPC PCR testing was associated with decreased 30-day mortality (adjusted odds ratio: .37; 95% CI: .16-.84) in an adjusted model. The 30-day mortality rate was 10% with ceftazidime-avibactam monotherapy and 31% with polymyxin monotherapy (P = .08). CONCLUSIONS: In a KPC-endemic area, blaKPC PCR testing of positive blood cultures was associated with decreased time until appropriate therapy and decreased mortality for CRE bacteremia, and ceftazidime-avibactam is a reasonable first-line therapy for these infections.

Real-world implementation and impact of a rapid carbapenemase detection test in an area endemic for carbapenem-resistant Enterobacterales.

A retrospective study was conducted to describe the impact of a molecular assay to detect the most common carbapenemase genes in carbapenem-resistant Enterobacterales isolates recovered in culture. Carbapenemases were detected in 69% of isolates, and assay results guided treatment modifications or epidemiologic investigation in 20% and 4% of cases, respectively.

Colonization With Fluoroquinolone-Resistant Enterobacterales Decreases the Effectiveness of Fluoroquinolone Prophylaxis in Hematopoietic Cell Transplant Recipients.

BACKGROUND: Levofloxacin prophylaxis is recommended to prevent gram-negative bloodstream infections (BSIs) in patients with prolonged chemotherapy-induced neutropenia. However, increasing fluoroquinolone resistance may decrease the effectiveness of this approach. METHODS: We assessed the prevalence of colonization with fluoroquinolone-resistant Enterobacterales (FQRE) among patients admitted for hematopoietic cell transplantation (HCT) from November 2016 to August 2019 and compared the risk of gram-negative BSI between FQRE-colonized and noncolonized patients. All patients received levofloxacin prophylaxis during neutropenia. Stool samples were collected upon admission for HCT and weekly thereafter until recovery from neutropenia, and underwent selective culture for FQRE. All isolates were identified and underwent antimicrobial susceptibility testing by broth microdilution. FQRE isolates also underwent whole-genome sequencing. RESULTS: Fifty-four of 234 (23%) patients were colonized with FQRE prior to HCT, including 30 of 119 (25%) allogeneic and 24 of 115 (21%) autologous HCT recipients. Recent antibacterial use was associated with FQRE colonization (P = .048). Ninety-one percent of colonizing FQRE isolates were Escherichia coli and 29% produced extended-spectrum ß-lactamases. Seventeen (31%) FQRE-colonized patients developed gram-negative BSI despite levofloxacin prophylaxis, compared to only 2 of 180 (1.1%) patients who were not colonized with FQRE on admission (P < .001). Of the 17 gram-negative BSIs in FQRE-colonized patients, 15 (88%) were caused by FQRE isolates that were genetically identical to the colonizing strain. CONCLUSIONS: Nearly one-third of HCT recipients with pretransplant FQRE colonization developed gram-negative BSI while receiving levofloxacin prophylaxis, and infections were typically caused by their colonizing strains. In contrast, levofloxacin prophylaxis was highly effective in patients not initially colonized with FQRE.

Analysis of Multidrug Resistance in Staphylococcus aureus with a Machine Learning-Generated Antibiogram.

Multidrug resistance (MDR) surveillance consists of reporting MDR prevalence and MDR phenotypes. Detailed knowledge of the specific associations underlying MDR patterns can allow antimicrobial stewardship programs to accurately identify clinically relevant resistance patterns. We applied machine learning and graphical networks to quantify and visualize associations between resistance traits in a set of 1,091 Staphylococcus aureus isolates collected from one New York hospital between 2008 and 2018. Antimicrobial susceptibility testing was performed using reference broth microdilution. The isolates were analyzed by year, methicillin susceptibility, and infection site. Association mining was used to identify resistance patterns that consisted of two or more individual antimicrobial resistance (AMR) traits and quantify the association among the individual resistance traits in each pattern. The resistance patterns captured the majority of the most common MDR phenotypes and reflected previously identified pairwise relationships between AMR traits in S. aureus Associations between ß-lactams and other antimicrobial classes (macrolides, lincosamides, and fluoroquinolones) were common, although the strength of the association among these antimicrobial classes varied by infection site and by methicillin susceptibility. Association mining identified associations between clinically important AMR traits, which could be further investigated for evidence of resistance coselection. For example, in skin and skin structure infections, clindamycin and tetracycline resistance occurred together 1.5 times more often than would be expected if they were independent from one another. Association mining efficiently discovered and quantified associations among resistance traits, allowing these associations to be compared between relevant subsets of isolates to identify and track clinically relevant MDR.

Implementation of infectious diseases rapid molecular diagnostic tests and antimicrobial stewardship program involvement in acute-care hospitals.

A survey of acute-care hospitals found that rapid molecular diagnostic tests (RMDTs) have been widely adopted. Although many hospitals use their antimicrobial stewardship team and/or guidelines to help clinicians interpret results and optimize treatment, opportunities to more fully achieve the potential benefits of RMDTs remain.

Piperacillin-Tazobactam-Resistant/Third-Generation Cephalosporin-Susceptible Escherichia coli and Klebsiella pneumoniae Isolates: Resistance Mechanisms and In vitro-In vivo Discordance.

We previously reported the detection of Escherichia coli and Klebsiella pneumoniae that displayed in vitro piperacillin-tazobactam (TZP) resistance but were susceptible to third-generation cephalosporins (TZP-R/Ceph3-S). In this study, we assessed the phenotypic and genotypic profiles of 12 clinical non-clonal TZP-R/Ceph3-S E. coli and K. pneumoniae isolates derived from bloodstream infections. Whole-genome sequencing revealed that most of the TZP-R/Ceph3-S E. coli and K. pneumoniae isolates examined harbored blaTEM-1 and blaSHV-1 genes, respectively, but none harbored extended-spectrum ß-lactamase, AmpC ß-lactamase or carbapenemase genes. Increasing the tazobactam concentration from 4 mg/L to 16 mg/L restored TZP in vitro susceptibility among E. coli isolates expressing TEM-1, but had minimal impact on the susceptibility of K. pneumoniae to TZP. Real-time qPCR analysis showed that blaTEM-1 expression was amplified in TZP-R E. coli upon incubation with sub-inhibitory TZP concentrations. Using an immunocompetent murine septicemia model, the efficacy of TZP against TZP-R/Ceph3-S isolates was assessed using TZP doses that mimicked human plasma exposures following intravenous (IV) administration of TZP 4.5 g q6h over 0.5 h for 24 h. Efficacy was assessed by survival through 96 h. There was high mortality in untreated control mice for all tested isolates. Compared with controls, TZP human-simulated exposure significantly improved survival for all TZP-R/Ceph3-S E. coli and K. pneumoniae isolates examined (P < 0.05). Thus, TZP was associated with remarkable in vivo activity against TZP-R/Ceph3-S E. coli and K. pneumoniae despite the observed resistance in vitro.

Impact of a Multiplexed Polymerase Chain Reaction Panel on Identifying Diarrheal Pathogens in Hematopoietic Cell Transplant Recipients.

BACKGROUND: Diarrhea is common and associated with substantial morbidity among hematopoietic cell transplant (HCT) recipients, but the etiology is often not identified. Multiplexed polymerase chain reaction (PCR) assays increase the detection of diarrheal pathogens, but the impact of this technology in this population has not been evaluated. METHODS: Our center replaced stool cultures and other conventional microbiologic methods with the FilmArray Gastrointestinal Panel (GI PCR) in June 2016. We reviewed all adult patients who received an HCT from June 2014-May 2015 (pre-GI PCR, n = 163) and from June 2016-May 2017 (post-GI PCR, n = 182) and followed them for 1 year after transplantation. Clostridioides difficile infection was diagnosed by an independent PCR test in both cohorts. RESULTS: The proportion of patients with ≥1 identified infectious diarrheal pathogen increased from 25% to 37% after implementation of GI PCR (P = .01). Eight patients (5%) in the pre-GI PCR cohort tested positive for a pathogen other than C. difficile versus 49 patients (27%) in the post-GI PCR cohort (P < .001). The most common non-C. difficile diarrheal pathogens in the post-GI PCR cohort were enteropathogenic Escherichia coli (n = 14, 8%), norovirus (n = 14, 8%), and Yersinia enterocolitica (n = 7, 4%). The percentage of diarrheal episodes with an identified infectious etiology increased from 14% to 23% (P = .001). Median total costs of stool testing per patient did not increase (pre: $473; post: $425; P = .25). CONCLUSIONS: Infectious etiologies of diarrhea were identified in a higher proportion of HCT recipients after replacing conventional stool testing with a multiplexed PCR assay, without an increase in testing costs.

First Report of blaVIM-4- and mcr-9-Coharboring Enterobacter Species Isolated from a Pediatric Patient.

An Enterobacter hormaechei isolate harboring blaVIM-4 and mcr-9 was recovered from a pediatric patient in a U.S. hospital. The blaVIM-4 and mcr-9 genes are carried on the same IncH12 plasmid, pME-1a. The isolate tested susceptible to colistin, without observed induction of colistin resistance. The mcr-9 gene is located between two insertion elements, IS903 and IS1, but lacks the downstream regulatory genes (qseC and qseB) found in other isolates that harbor mcr-9IMPORTANCE We describe the complete genome assembly and sequence of a clinical Enterobacter isolate harboring both blaVIM-4 and mcr-9 recovered from a pediatric patient in the United States with a history of travel to Egypt. Moreover, to the best of our knowledge, this is the first report of an Enterobacter isolate harboring both blaVIM-4 and mcr-9 from the United States. The blaVIM-4 and mcr-9 genes are carried on the same IncH12 plasmid, pME-1a. The isolate tested susceptible to colistin, without observed induction of colistin resistance. The mcr-9 gene is located between two insertion elements, IS903 and IS1, but lacks the downstream regulatory genes (qseC and qseB) found in other isolates that harbor mcr-9.

Activity of Imipenem-Relebactam and Comparator Agents against Genetically Characterized Isolates of Carbapenem-Resistant Enterobacteriaceae.

Carbapenem-resistant Enterobacteriaceae (CRE) strains are an urgent public health threat. We evaluated the in vitro activities of 19 antimicrobial agents, including imipenem-relebactam, against (i) 106 CRE bloodstream isolates that primarily expressed Klebsiella pneumoniae carbapenemase (KPC) and (ii) 20 OXA-48-like-expressing CRE isolates. Ninety-five percent of CRE bloodstream isolates were susceptible to imipenem-relebactam. In contrast to their comparable activities against KPC-producing CRE strains, ceftazidime-avibactam was more active in vitro against OXA-48-like CRE strains than was imipenem-relebactam (90% susceptible versus 15% susceptible).

Microbial Composition of the Human Nasopharynx Varies According to Influenza Virus Type and Vaccination Status.

Factors that contribute to enhanced susceptibility to severe bacterial disease after influenza virus infection are not well defined but likely include the microbiome of the respiratory tract. Vaccination against influenza, while having variable effectiveness, could also play a role in microbial community stability. We collected nasopharyngeal samples from 215 individuals infected with influenza A/H3N2 or influenza B virus and profiled the microbiota by target sequencing of the 16S rRNA gene. We identified signature taxonomic groups by performing linear discriminant analysis and effective size comparisons (LEfSe) and defined bacterial community types using Dirichlet multinomial mixture (DMM) models. Influenza infection was shown to be significantly associated with microbial composition of the nasopharynx according to the virus type and the vaccination status of the patient. We identified four microbial community types across the combined cohort of influenza patients and healthy individuals with one community type most representative of the influenza virus-infected group. We also identified microbial taxa for which relative abundance was significantly higher in the unvaccinated elderly group; these taxa include species known to be associated with pneumonia.IMPORTANCE Our results suggest that there is a significant association between the composition of the microbiota in the nasopharynx and the influenza virus type causing the infection. We observe that vaccination status, especially in more senior individuals, also has an association with the microbial community profile. This indicates that vaccination against influenza, even when ineffective to prevent disease, could play a role in controlling secondary bacterial complications.

Isolation of antibiotic-resistant gram-negative organisms from donor respiratory culture does not impact non-lung solid organ recipient management.

BACKGROUND: Multidrug-resistant (MDR) and extensively drug-resistant (XDR) gram-negative bacteria may be transmitted from organ donors to solid organ transplant recipients and are associated with poor outcomes post-transplant. METHODS: We reported the prevalence of MDR/XDR gram-negative respiratory colonization among 702 deceased organ donors in the New York City area from 2011 to 2014 and performed chart reviews for a subset of recipients to determine whether donor respiratory culture results were predictive of subsequent recipient infection or used to guide post-transplant antimicrobial therapy. RESULTS: Fifty donors (7% of the cohort) had MDR or XDR gram-negative bacteria isolated from endotracheal aspirate or bronchoalveolar lavage culture. Organs from these 50 donors were transplanted into 120 recipients; chart review was performed for 89 of these recipients (38 kidney, 32 liver, 11 heart, 6 kidney/pancreas, 1 liver/kidney, 1 lung). None of the 89 recipients of organs from donors with MDR/XDR gram-negative respiratory colonization were reported to have a donor-derived infection post-transplant, and chart review for the 88 non-lung recipients indicated that peri-transplant antibiotics were not adjusted specifically for donor respiratory culture results. CONCLUSION: These results suggest that donor respiratory culture results are not predictive of post-transplant infection in non-lung recipients and are unlikely to impact post-transplant management.

In vitro activity of ampicillin and ceftriaxone against ampicillin-susceptible Enterococcus faecium.

OBJECTIVES: To assess activity of the combination of ceftriaxone and ampicillin against clinical isolates of ampicillin-susceptible Enterococcus faecium. METHODS: Ampicillin-susceptible E. faecium (n = 29) and Enterococcus faecalis (n = 10) collected from locations in the USA and France were used for this analysis. Susceptibility testing was performed by gradient diffusion strip (GDS) and broth microdilution (BMD). Synergy with the combination of ceftriaxone and ampicillin was assessed in all isolates using GDS crossing and double disc diffusion methods. Selected isolates (nine E. faecium and three E. faecalis) were assessed for synergy in time-kill studies using ampicillin alone and in combination with ceftriaxone. RESULTS: In isolates of E. faecium, the median (range) ampicillin MIC by BMD was 0.5 (0.25-4) mg/L and by GDS it was 2 (1-8) mg/L. In E. faecalis, the median (range) ampicillin MIC by BMD was 0.5 (0.5-1) mg/L and by GDS it was 2 (0.75-3) mg/L. A total of 24/29 (82.8%) isolates of E. faecium displayed synergy by GDS and 22/29 (75.9%) by double disc diffusion. Seven of 10 (70%) isolates of E. faecalis displayed synergy by GDS and 4/10 (40%) by double disc diffusion. Time-kill studies found synergy in 3/9 (33.3%) E. faecium and 3/3 (100%) E. faecalis. CONCLUSIONS: In contrast to the demonstrated synergy in time-kill models of ceftriaxone and ampicillin for E. faecalis, this combination does not appear to provide uniform synergy in E. faecium. Antagonism was not observed. Clinical correlation is necessary and caution should be used when considering ampicillin and ceftriaxone for the treatment of infections caused by ampicillin-susceptible E. faecium.

First reported human isolation of Staphylococcus delphini.

The Staphylococcus intermedius group is a collection of coagulase-positive staphylococci composed of 5 members, including Staphylococcus pseudintermedius, a zoonotic pathogen often associated with exposure to dogs, and Staphylococcus delphini, which has not previously been recovered from humans. Here, we describe the first human case of S. delphini infection.

Acinetobacter radioresistens infection with bacteremia and pneumonia.

Acinetobacter species are non-fermentative Gram-negative coccobacilli that are ubiquitous in the environment. The archetype pathogen within the genus is Acinetobacter baumannii, however, other species have the potential to cause human infection, especially in the hospital setting. We describe a patient with infection due to Acinetobacter radioresistens, a rare agent of human disease, which is often misidentified using biochemical methods. Acinetobacter radioresistens is the source of the Class D OXA-23 carbapenemase that can confer carbapenem resistance in A. baumannii. Therefore, accurate identification of A. radioresistens is important for clinical management and to potentially prevent the spread of carbapenem resistance.

Improved Performance of a Rapid Immunochromatographic Assay for Detection of PBP2a in Non-Staphylococcus aureus Staphylococcal Species.

Non-Staphylococcus aureus staphylococcal species (non-SASS) are important pathogens in both animal and human populations. The development of ß-lactam resistance in non-SASS through acquisition and expression of penicillin-binding protein 2a (PBP2a) represents a significant clinical and public health threat. Here, we evaluated the diagnostic performance of two versions of a PBP2a immunochromatographic assay with non-SASS. Our data show that the revised version of the assay, the PBP2a SA culture colony test, has superior diagnostic sensitivity compared to the previous version of the assay, the PBP2a culture colony test, 100% (95% confidence interval [CI], 93.3 to 100%) versus 67.9% (95% CI, 53.7 to 80.1%), respectively, while both assays display a specificity of 100% (95% CI, 92.5 to 100%). Therefore, the PBP2a SA culture colony test offers a rapid, accurate, and relatively inexpensive method for detecting PBP2a-mediated ß-lactam resistance in clinically relevant non-SASS for the management of infections due to these organisms and for antimicrobial stewardship.

Epidemiology of Bloodstream Infections Caused by Escherichia coli and Klebsiella pneumoniae That Are Piperacillin-Tazobactam-Nonsusceptible but Ceftriaxone-Susceptible.

BACKGROUND: Piperacillin-tazobactam-nonsusceptible (TZP-NS) Enterobacteriaceae are typically also resistant to ceftriaxone. We recently encountered bacteremias due to Escherichia coli (Ec) and Klebsiella pneumoniae (Kp) that were TZP-NS but ceftriaxone-susceptible (CRO-S). METHODS: We reviewed all Ec and Kp bacteremias from 2011 to 2015 at our center and assessed the prevalence, antimicrobial susceptibilities, genetic profiles, patient characteristics, treatments, and outcomes of TZP-NS/CRO-S infections. We identified risk factors for TZP-NS/CRO-S infections compared with Ec and Kp bacteremias that were TZP-S and CRO-S (Control Group 1) and compared outcomes of patients with TZP-NS/CRO-S bacteremias, Control Group 1, and patients bacteremic with extended-spectrum ß-lactamase (ESBL)-producing Ec and Kp. RESULTS: There were 1857 Ec and Kp bacteremia episodes, of which 78 (4.2%) were TZP-NS/CRO-S (Ec: 50/1227 [4.1%]; Kp: 28/630 [4.4%]). All TZP-NS/CRO-S isolates were also ampicillin-sulbactam-NS. Of 32 TZP-NS/CRO-S isolates that were sequenced, 28 (88%) harbored bla TEM-1 or bla SHV-1, none had an ESBL or AmpC ß-lactamase gene, and many sequence types were represented. Independent risk factors for TZP-NS/CRO-S bacteremia were exposure to ß-lactam/ß-lactamase inhibitors (BL/BLIs; adjusted odds ratio [aOR], 5.5; P < .001) and cephalosporins (aOR, 3.0; P = .04). Thirty-day mortality after TZP-NS/CRO-S bacteremia was 25%, which was similar to control groups and was similar in patients treated empirically with BL/BLIs compared with those treated with cephalosporins or carbapenems. Targeted therapy with cephalosporins did not yield a higher 30-day mortality rate than carbapenem therapy. CONCLUSIONS: TZP-NS/CRO-S Ec and Kp are emerging causes of bacteremia, and further research is needed to better understand the epidemiology, resistance mechanisms, and clinical impact of these strains.