Emergence of Polymyxin Resistance in Clinical Klebsiella pneumoniae Through Diverse Genetic Adaptations: A Genomic, Retrospective Cohort Study.

BACKGROUND: Polymyxins are antimicrobials of last resort for the treatment of carbapenem-resistant Enterobacteriaceae, but resistance in 5% to >40% isolates has been reported. We conducted a genomic survey of clinical polymyxin-resistant (PR) Klebsiella pneumoniae to determine the molecular mechanisms of PR and the role of polymyxin exposure versus transmission in PR emergence. METHODS: We included 88 patients with PR K. pneumoniae from 2011-2018 and collected demographic, antimicrobial exposure, and infection data. Whole-genome sequencing was performed on 388 isolates, including 164 PR isolates. Variant calling and insertion sequence detection were performed, focusing on key genes associated with PR (mgrB, crrAB, phoPQ, and pmrAB). We conducted phylogenetic analyses of key K. pneumoniae multi-locus sequence types (ST258, ST17, ST307, and ST392). RESULTS: Polymyxin exposure was documented in 53/88 (60%) patients prior to PR detection. Through an analysis of key PR genes, we detected 129 individual variants and 72 unique variant combinations in PR isolates. This included multiple, distinct changes in 36% of patients with serial PR isolates. Insertion sequence disruption was limited to mgrB (P < .001). Polymyxin minimum inhibitory concentrations showed stepwise increases with the number of PR genes affected (P < .001). When clusters containing PR isolates in ≥2 patients were analyzed, 10/14 had multiple genetic events leading to PR. CONCLUSIONS: Molecular mechanisms leading to PR in clinical K. pneumoniae isolates are remarkably heterogenous, even within clusters or individual patients. Polymyxin exposure with de novo PR emergence led to PR in the majority of patients, rather than transmission. Optimizing polymyxin use should be a key strategy in stopping the spread of PR.

Evaluation of Calcium-Enhanced Media for Colistin Susceptibility Testing by Gradient Agar Diffusion and Broth Microdilution.

Despite the increasing reliance on polymyxin antibiotics (polymyxin B and colistin) for treatment of multidrug-resistant Gram-negative infections, many clinical laboratories are unable to perform susceptibility testing due to the lack of accurate and reliable methods. Although gradient agar diffusion is commonly performed for other antimicrobials, its use for polymyxins is discouraged due to poor performance characteristics. Performing gradient agar diffusion with calcium enhancement of susceptibility testing media has been shown to improve the identification of polymyxin-resistant isolates with plasmid-mediated resistance (mcr-1). We therefore sought to evaluate the broad clinical applicability of this approach for colistin susceptibility testing by assessing a large and diverse collection of resistant and susceptible patient isolates collected from multiple U.S. medical centers. Among 217 isolates, the overall categorical and essential agreement for calcium-enhanced gradient agar diffusion were 73.7% and 65.5%, respectively, compared to the results for reference broth microdilution. Performance varied significantly by organism group, with agreement being highest for Enterobacterales and lowest for Pseudomonas aeruginosa Nevertheless, even for Enterobacterales, there was a high rate of very major errors (9.2%). Performance was similarly poor for calcium-enhanced broth microdilution. While calcium enhancement did allow for more accurate categorization of mcr-1-resistant isolates, there were unacceptably high rates of errors for both susceptible and non-mcr-1-resistant isolates, raising serious doubts about the suitability of these calcium-enhanced methods for routine colistin susceptibility testing in clinical laboratories.

The effect of short-course antibiotics on the resistance profile of colonizing gut bacteria in the ICU: a prospective cohort study.

BACKGROUND: The need for early antibiotics in the intensive care unit (ICU) is often balanced against the goal of antibiotic stewardship. Long-course antibiotics increase the burden of antimicrobial resistance within colonizing gut bacteria, but the dynamics of this process are not fully understood. We sought to determine how short-course antibiotics affect the antimicrobial resistance phenotype and genotype of colonizing gut bacteria in the ICU by performing a prospective cohort study with assessments of resistance at ICU admission and exactly 72 h later. METHODS: Deep rectal swabs were performed on 48 adults at the time of ICU admission and exactly 72 h later, including patients who did and did not receive antibiotics. To determine resistance phenotype, rectal swabs were cultured for methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococcus (VRE). In addition, Gram-negative bacterial isolates were cultured against relevant antibiotics. To determine resistance genotype, quantitative PCR (qPCR) was performed from rectal swabs for 87 established resistance genes. Within-individual changes in antimicrobial resistance were calculated based on culture and qPCR results and correlated with exposure to relevant antibiotics (e.g., did ß-lactam antibiotic exposure associate with a detectable change in ß-lactam resistance over this 72-h period?). RESULTS: Of 48 ICU patients, 41 (85%) received antibiotics. Overall, there was no increase in the antimicrobial resistance profile of colonizing gut bacteria during the 72-h study period. There was also no increase in antimicrobial resistance after stratification by receipt of antibiotics (i.e., no detectable increase in ß-lactam, vancomycin, or macrolide resistance regardless of whether patients received those same antibiotics). This was true for both culture and PCR. Antimicrobial resistance pattern at ICU admission strongly predicted resistance pattern after 72 h. CONCLUSIONS: Short-course ICU antibiotics made little detectable difference in the antimicrobial resistance pattern of colonizing gut bacteria over 72 h in the ICU. This provides an improved understanding of the dynamics of antimicrobial resistance in the ICU and some reassurance that short-course antibiotics may not adversely impact the stewardship goal of reducing antimicrobial resistance.

Multicenter Evaluation of Colistin Broth Disk Elution and Colistin Agar Test: a Report from the Clinical and Laboratory Standards Institute.

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). The methods were evaluated using a collection of 270 isolates of Enterobacterales, 122 Pseudomonas aeruginosa isolates, and 106 Acinetobacter spp. isolates. Overall, 94.4% of CBDE results were in essential agreement and 97.9% in categorical agreement (CA) with reference broth microdilution MICs. Nine very major errors (VME; 3.2%) and 3 major errors (ME; 0.9%) were observed. With the CBDE, 98.6% CA was observed for Enterobacterales (2.5% VME, 0% ME), 99.3% CA was observed for P. aeruginosa (0% VME, 0.7% ME), and 93.1% CA was observed for Acinetobacter spp. (5.6% VME, 3.3% ME). Overall, CA was 94.9% with 6.8% VME using CAT-1 and improved to 98.3% with 3.9% VME using CAT-10. No ME were observed using either CAT-1 or CAT-10. Using the CAT-1/CAT-10, the CA observed was 99.4%/99.7% for Enterobacterales (1%/0.5% VME), 98.7%/100% for P. aeruginosa (8.3%/0% VME), and 88.5%/92.3% for Acinetobacter spp. (21.4%/14.3% VME). Based on these data, the CLSI antimicrobial susceptibility testing (AST) subcommittee endorsed the CBDE and CAT-10 methods for colistin testing of Enterobacterales and P. aeruginosa.

Clonal Background, Resistance Gene Profile, and Porin Gene Mutations Modulate In Vitro Susceptibility to Imipenem-Relebactam in Diverse Enterobacteriaceae.

Treatment options for carbapenem-resistant Enterobacteriaceae (CRE) are limited. While Klebsiella pneumoniae strains harboring blaKPC account for most CRE, recent evidence points to increasing diversification of CRE. We determined whether the CRE species and antibiotic resistance genotype influence the response to relebactam (REL), a novel beta-lactamase inhibitor with class A/C activity, combined with imipenem-cilastatin (IMI). We carried out broth microdilution testing with IMI alone or in the presence of 4 µg/ml REL against 154 clinical isolates collected at a New York City hospital with a high prevalence of organisms carrying blaKPC, including Enterobacter spp. (n = 96), K. pneumoniae (n = 44), Escherichia coli (n = 1), Serratia marcescens (n = 9), and Citrobacter spp. (n = 4). Resistance gene profiles and the presence of major porin gene disruptions were ascertained by whole-genome sequencing. Addition of REL decreased the IMI MIC to the susceptible range (≤1 µg/ml) against 88% of isolates. However, S. marcescens IMI-REL MICs were 4- to 8-fold higher than those for other organisms. Most blaKPC-positive isolates had IMI-REL MICs of ≤1 µg/ml (88%), including isolates of Enterobacter cloacae ST171 (93%) and K. pneumoniae ST258 (82%). Nineteen isolates had IMI-REL MICs of ≥2 µg/ml, among which 84% harbored blaKPC and one was blaNDM-1 positive. Isolates with IMI-REL MICs of ≥2 µg/ml versus those with MICs of ≤1 µg/ml were significantly more likely to demonstrate disruption of at least one porin gene (42% versus 19%; P = 0.04), although most S. marcescens isolates (67%) had intact porin genes. In conclusion, while REL reduced IMI MICs in a majority of diverse CRE isolates, including high-risk clones, chromosomal factors had an impact on IMI-REL susceptibilities and may contribute to elevated MICs for S. marcescens.

Successive Emergence of Ceftazidime-Avibactam Resistance through Distinct Genomic Adaptations in blaKPC-2-Harboring Klebsiella pneumoniae Sequence Type 307 Isolates.

Ceftazidime-avibactam (CAZ-AVI) is a promising novel treatment for infections caused by carbapenem-resistant Enterobacteriaceae (CRE). Despite improved treatment outcomes compared to those achieved with aminoglycoside- and colistin-based regimens, the rapid evolution of CAZ-AVI resistance during treatment has previously been reported in Klebsiella pneumoniae sequence type 258 (ST258) blaKPC-3-harboring isolates. Here, we report the stepwise evolution and isolation of two phenotypically distinct CAZ-AVI-resistant Klebsiella pneumoniae isolates from a patient with pancreatitis. All susceptible (n = 3) and resistant (n = 5) isolates were of the ST307 clonal background, a rapidly emerging clone. Taking advantage of short-read Illumina and long-read Oxford Nanopore sequencing and full-length assembly of the core chromosome and plasmids, we demonstrate that CAZ-AVI resistance first occurred through a 532G → T blaKPC-2 point mutation in blaKPC-2 (D179Y protein substitution) following only 12 days of CAZ-AVI exposure. While subsequent isolates exhibited substantially decreased meropenem (MEM) MICs (≤2 µg/ml), later cultures demonstrated a second CAZ-AVI resistance phenotype with a lower CAZ-AVI MIC (12 µg/ml) but also MEM resistance (MIC > 128 µg/ml). These CAZ-AVI- and MEM-resistant isolates showed evidence of multiple genomic adaptations, mainly through insertions and deletions. This included amplification and transposition of wild-type blaKPC-2 into a novel plasmid, an IS1 insertion upstream of ompK36, and disruption of the rfb gene locus in these isolates. Our findings illustrate the potential of CAZ-AVI resistance to emerge in non-K. pneumoniae ST258 clonal backgrounds and alternative blaKPC variants. These results raise concerns about the strong selective pressures incurred by novel carbapenemase inhibitors, such as avibactam, on isolates previously considered invulnerable to CAZ-AVI resistance. There is an urgent need to further characterize non-KPC-mediated modes of carbapenem resistance and the intrinsic bacterial factors that facilitate the rapid emergence of resistance during treatment.

Reduced Vancomycin Susceptibility of Methicillin-Susceptible Staphylococcus aureus Has No Significant Impact on Mortality but Results in an Increase in Complicated Infection.

Methicillin-susceptible Staphylococcus aureus (MSSA) bloodstream infections (BSIs) often lead to severe complications despite the availability of effective antibiotics. It remains unclear whether elevated vancomycin MICs are associated with worse outcomes. We conducted a 2-year retrospective cohort study (n = 252) of patients with MSSA BSIs at a tertiary care hospital. We defined reduced vancomycin susceptibility (RVS) as a Microscan MIC of 2 mg/liter. All strains were genotyped (spa) and assessed for agr functionality. Multivariable logistic regression models were used to examine the impact of RVS phenotype and strain genotype on 30-day all-cause mortality and complicated bacteremia (metastatic spread, endovascular infection, or duration ≥3 days). One-third of patients (84/252) were infected with RVS isolates. RVS Infections were more frequently associated with metastatic or embolic sites of infection (36% versus 17%, P < 0.001), and endovascular infection (26% versus 12%, P = 0.004). These infections occurred more often in patients with fewer underlying comorbidities (Charlson comorbidity index of ≥3 [73% versus 88%, P = 0.002]). Genotyping identified 127 spa-types and 14 Spa-clonal complexes (Spa-CCs). Spa-CC002 and Spa-CC008 were more likely to exhibit the RVS phenotype versus other Spa-CCs (OR = 2.2, P < 0.01). The RVS phenotype was not significantly associated with 30-day mortality; however, it was associated with complicated bacteremia (adjusted odds ratio of 2.35 [range, 1.26 to 4.37]; P = 0.007) in adjusted analyses. The association of RVS strains with complicated infection and fewer underlying comorbidities suggests the phenotype as a potential marker of strain virulence in MSSA BSIs. The RVS phenotype itself was not a significant predictor of mortality in this patient cohort. Further studies are necessary to explore this host-pathogen relationship.

Evolution of the environmental microbiota of a new neonatal intensive care unit (NICU) and implications for infection prevention and control.

OBJECTIVE: To describe changes in the environmental microbiota of a new neonatal intensive care unit (NICU) and potential implications for infection prevention and control (IPC) efforts. DESIGN: Prospective observational study. SETTING: A newly constructed level IV neonatal cardiac intensive care unit (NCICU) before and after patient introduction and the original NICU prior to patient transfer. METHODS: Environmental samples were obtained from the original NICU prior to patient transfer to a new NCICU. Serial sampling of patient rooms and provider areas of the new NICU was conducted immediately prior to patient introduction and over an 11-month study period. Microbiota at each sampling point were characterized using Illumina sequencing of the V3/V4 region of the 16S rRNA gene. Microbiota characteristics (α and ß diversity and differential abundance) were compared based on time, location, and clinical factors (room-level antibiotic use and patient turnover). RESULTS: An immediate increase in the environmental differential abundance of gut anaerobes were seen after patient introduction. There was an increase in the relative abundance of Staphylococcus spp, Klebsiella spp, Pseudomonas spp, and Streptococcus spp over time. The new NCICU consistently showed more diverse microbiota and remained distinct from the original NICU. The microbiota of the provider areas of the NCICU eventually formed a cluster separate from the patient rooms. Patient turnover increased room-level microbiota diversity. CONCLUSION: Microbiota characteristics of the new NICU were distinct from the original ICU despite housing similar patients. Patient and provider areas developed distinct microbiota profiles. Non-culture-based methods may be a useful adjunct to current IPC practice.

Relationship of the Esophageal Microbiome and Tissue Gene Expression and Links to the Oral Microbiome: A Randomized Clinical Trial.

INTRODUCTION: Although the microbiome is altered in various esophageal diseases, there is no direct evidence for a link between the oral or esophageal microbiome and underlying esophageal tissue. Here, we aimed to address these gaps through use of an antimicrobial mouth rinse to modify the esophageal microbiome and tissue gene expression. METHODS: In this randomized controlled trial, patients scheduled to undergo endoscopy for clinical indications used chlorhexidine mouth rinse or no treatment for 2 weeks before endoscopy. Oral swabs and saliva were collected at baseline and at follow-up, and the esophagus was sampled on the day of endoscopy. The microbiome was analyzed by 16S rRNA gene sequencing, and esophageal tissue gene expression was ascertained by RNA-Seq. RESULTS: Twenty subjects were enrolled and included in the analyses. Within individuals, the oral and esophageal microbiome composition was significantly correlated. Chlorhexidine treatment associated with significant alterations to the relative abundance of several esophageal bacterial taxa, and to expression of genes in the esophagus including reductions in periostin, claudin-18, chemokines CXCL1 and CXCL13, and several members of the tumor necrosis factor receptor superfamily. A taxon in genus Haemophilus in the esophagus also associated with significant changes in tissue gene expression. DISCUSSION: The oral and esophageal microbiomes are closely related within individuals, and esophageal microbiome alterations correlate with tissue gene expression changes. The esophageal microbiome may act as an important cofactor that influences pathogenesis and outcomes of diseases such as eosinophilic esophagitis, gastroesophageal reflux, and Barrett's esophagus.

Gut microbiota, endotoxemia, inflammation, and oxidative stress in patients with heart failure, left ventricular assist device, and transplant.

BACKGROUND: Gut microbial imbalance may contribute to endotoxemia, inflammation, and oxidative stress in heart failure (HF). Changes occurring in the intestinal microbiota and inflammatory/oxidative milieu during HF progression and following left ventricular assist device (LVAD) or heart transplantation (HT) are unknown. We aimed to investigate variation in gut microbiota and circulating biomarkers of endotoxemia, inflammation, and oxidative stress in patients with HF (New York Heart Association, Class I-IV), LVAD, and HT. METHODS: We enrolled 452 patients. Biomarkers of endotoxemia (lipopolysaccharide and soluble [sCD14]), inflammation (C-reactive protein, interleukin-6, tumor necrosis factor-α, and endothelin-1 adiponectin), and oxidative stress (isoprostane) were measured in 644 blood samples. A total of 304 stool samples were analyzed using 16S rRNA sequencing. RESULTS: Gut microbial community measures of alpha diversity were progressively lower across worsening HF class and were similarly reduced in patients with LVAD and HT (p < 0.05). Inflammation and oxidative stress were elevated in patients with Class IV HF vs all other groups (all p < 0.05). Lipopolysaccharide was elevated in patients with Class IV HF (vs Class I-III) as well as in patients with LVAD and HT (p < 0.05). sCD14 was elevated in patients with Class IV HF and LVAD (vs Class I-III, p < 0.05) but not in patients with HT. CONCLUSIONS: Reduced gut microbial diversity and increased endotoxemia, inflammation, and oxidative stress are present in patients with Class IV HF. Inflammation and oxidative stress are lower among patients with LVAD and HT relative to patients with Class IV HF, whereas reduced gut diversity and endotoxemia persist in LVAD and HT.

Alterations to the Esophageal Microbiome Associated with Progression from Barrett's Esophagus to Esophageal Adenocarcinoma.

BACKGROUND: The incidence of esophageal adenocarcinoma has risen dramatically over the past half century, and the underlying reasons are incompletely understood. Broad shifts to the upper gastrointestinal microbiome may be partly responsible. The goal of this study was to describe alterations in the esophageal microbiome that occur with progression from Barrett's esophagus to esophageal adenocarcinoma. METHODS: A case-control study was performed of patients with and without Barrett's esophagus who were scheduled to undergo upper endoscopy. Demographic, clinical, and dietary intake data were collected, and esophageal brushings were collected during the endoscopy. 16S rRNA gene sequencing was performed to characterize the microbiome. RESULTS: A total of 45 patients were enrolled and included in the analyses [16 controls; 14 Barrett's esophagus without dysplasia (NDBE); 6 low-grade dysplasia (LGD); 5 high-grade dysplasia (HGD); and 4 esophageal adenocarcinoma]. There was no difference in alpha diversity between non-Barrett's esophagus and Barrett's esophagus, but there was evidence of decreased diversity in patients with esophageal adenocarcinoma as assessed by Simpson index. There was an apparent shift in composition at the transition from LGD to HGD, and patients with HGD and esophageal adenocarcinoma had decreased Firmicutes and increased Proteobacteria. In addition, patients with HGD or esophageal adenocarcinoma had increased Enterobacteriaceae and Akkermansia muciniphila and reduced Veillonella. In the study population, patients taking proton pump inhibitors had increased Streptococcus and decreased Gram-negative bacteria overall. CONCLUSIONS: Shifts in the Barrett's esophagus-associated microbiome were observed in patients with HGD and esophageal adenocarcinoma, with increases in certain potentially pathogenic bacteria. IMPACT: The microbiome may play a role in esophageal carcinogenesis.

Colonizing multidrug-resistant bacteria and the longitudinal evolution of the intestinal microbiome after liver transplantation.

Infections by multidrug-resistant bacteria (MDRB) remain a leading cause of morbidity and mortality after liver transplantation (LT). Gut dysbiosis characteristic of end-stage liver disease may predispose patients to intestinal MDRB colonization and infection, in turn exacerbating dysbiosis. However, relationships between MDRB colonization and dysbiosis after LT remain unclear. We prospectively recruited 177 adult patients undergoing LT at a single tertiary care center. 16 S V3-V4 rRNA sequencing was performed on 723 fecal samples collected pre-LT and periodically until one-year post-LT to test whether MDRB colonization was associated with decreased microbiome diversity. In multivariate linear mixed-effect models, MDRB colonization predicts reduced Shannon α-diversity, after controlling for underlying liver disease, antibiotic exposures, and clinical complications. Importantly, pre-LT microbial markers predict subsequent colonization by MDRB. Our results suggest MDRB colonization as a major, previously unrecognized, marker of persistent dysbiosis. Therapeutic approaches accounting for microbial and clinical factors are needed to address post-transplant microbiome health.