Lipid A modification of colistin-resistant Klebsiella pneumoniae does not alter innate immune response in a mouse model of pneumonia.

Polymyxin resistance in carbapenem-resistant Klebsiella pneumoniae bacteria is associated with high morbidity and mortality in vulnerable populations throughout the world. Ineffective antimicrobial activity by these last resort therapeutics can occur by transfer of mcr-1, a plasmid-mediated resistance gene, causing modification of the lipid A portion of lipopolysaccharide (LPS) and disruption of the interactions between polymyxins and lipid A. Whether this modification alters the innate host immune response or carries a high fitness cost in the bacteria is not well established. To investigate this, we studied infection with K. pneumoniae (KP) ATCC 13883 harboring either the mcr-1 plasmid (pmcr-1) or the vector control (pBCSK) ATCC 13883. Bacterial fitness characteristics of mcr-1 acquisition were evaluated. Differentiated human monocytes (THP-1s) were stimulated with KP bacterial strains or purified LPS from both parent isolates and isolates harboring mcr-1. Cell culture supernatants were analyzed for cytokine production. A bacterial pneumonia model in WT C57/BL6J mice was used to monitor immune cell recruitment, cytokine induction, and bacterial clearance in the bronchoalveolar lavage fluid (BALF). Isolates harboring mcr-1 had increased colistin MIC compared to the parent isolates but did not alter bacterial fitness. Few differences in cytokines were observed with purified LPS from mcr-1 expressing bacteria in vitro. However, in a mouse pneumonia model, no bacterial clearance defect was observed between pmcr-1-harboring KP and parent isolates. Consistently, no differences in cytokine production or immune cell recruitment in the BALF were observed, suggesting that other mechanisms outweigh the effect of these lipid A mutations in LPS.

Structural basis of lipopolysaccharide maturation by the O-antigen ligase.

The outer membrane of Gram-negative bacteria has an external leaflet that is largely composed of lipopolysaccharide, which provides a selective permeation barrier, particularly against antimicrobials1. The final and crucial step in the biosynthesis of lipopolysaccharide is the addition of a species-dependent O-antigen to the lipid A core oligosaccharide, which is catalysed by the O-antigen ligase WaaL2. Here we present structures of WaaL from Cupriavidus metallidurans, both in the apo state and in complex with its lipid carrier undecaprenyl pyrophosphate, determined by single-particle cryo-electron microscopy. The structures reveal that WaaL comprises 12 transmembrane helices and a predominantly α-helical periplasmic region, which we show contains many of the conserved residues that are required for catalysis. We observe a conserved fold within the GT-C family of glycosyltransferases and hypothesize that they have a common mechanism for shuttling the undecaprenyl-based carrier to and from the active site. The structures, combined with genetic, biochemical, bioinformatics and molecular dynamics simulation experiments, offer molecular details on how the ligands come in apposition, and allows us to propose a mechanistic model for catalysis. Together, our work provides a structural basis for lipopolysaccharide maturation in a member of the GT-C superfamily of glycosyltransferases.

Klebsiella pneumoniae induces host metabolic stress that promotes tolerance to pulmonary infection.

K. pneumoniae sequence type 258 (Kp ST258) is a major cause of healthcare-associated pneumonia. However, it remains unclear how it causes protracted courses of infection in spite of its expression of immunostimulatory lipopolysaccharide, which should activate a brisk inflammatory response and bacterial clearance. We predicted that the metabolic stress induced by the bacteria in the host cells shapes an immune response that tolerates infection. We combined in situ metabolic imaging and transcriptional analyses to demonstrate that Kp ST258 activates host glutaminolysis and fatty acid oxidation. This response creates an oxidant-rich microenvironment conducive to the accumulation of anti-inflammatory myeloid cells. In this setting, metabolically active Kp ST258 elicits a disease-tolerant immune response. The bacteria, in turn, adapt to airway oxidants by upregulating the type VI secretion system, which is highly conserved across ST258 strains worldwide. Thus, much of the global success of Kp ST258 in hospital settings can be explained by the metabolic activity provoked in the host that promotes disease tolerance.

In Vitro Cytotoxicity and Clinical Correlates of MRSA Bacteremia.

Methicillin-resistant Staphylococcus aureus (MRSA) bloodstream infections are associated with significant morbidity and mortality. MRSA secretes a number of virulence factors and pore-forming toxins that enable tissue invasion. Prior studies have found associations between decreased toxin production and poor outcomes in invasive MRSA infection, particularly in pneumonia. In this retrospective observational cohort study of MRSA bacteremia in adult patients from 2007 to 2015, we examined whether cytotoxicity was associated with 30-day mortality. Isolates were obtained from 776 patients and screened for cytotoxicity in a human HL-60 cell model, antimicrobial susceptibility, and spa type, and clinical data were abstracted from charts. We did not find an association between low cytotoxic activity and 30-day mortality in univariate logistic regression analyses. There was a difference in distribution of the genotypes across cytotoxicity phenotypes, with spa-CC008 accounting for a larger proportion of isolates in the high cytotoxicity group. Isolates with a skin and soft tissue primary infective site had a higher median cytotoxicity. There was no association between cytotoxicity and host factors such as age or comorbidity burden. The isolates in our study came from heterogeneous primary sites of infection and were predominantly from spa-CC002 and spa-CC008 lineages, so it is possible that findings in prior studies reflect a different distribution in genotypes and clinical syndromes. Overall, in this large study of cytotoxicity of MRSA bloodstream isolates, we did not find the low cytotoxicity phenotype to be predictive of poor outcomes in MRSA bacteremia.

An acquired acyltransferase promotes Klebsiella pneumoniae ST258 respiratory infection.

Klebsiella pneumoniae ST258 is a human pathogen associated with poor outcomes worldwide. We identify a member of the acyltransferase superfamily 3 (atf3), enriched within the ST258 clade, that provides a major competitive advantage for the proliferation of these organisms in vivo. Comparison of a wild-type ST258 strain (KP35) and a Δatf3 isogenic mutant generated by CRISPR-Cas9 targeting reveals greater NADH:ubiquinone oxidoreductase transcription and ATP generation, fueled by increased glycolysis. The acquisition of atf3 induces changes in the bacterial acetylome, promoting lysine acetylation of multiple proteins involved in central metabolism, specifically Zwf (glucose-6 phosphate dehydrogenase). The atf3-mediated metabolic boost leads to greater consumption of glucose in the host airway and increased bacterial burden in the lung, independent of cytokine levels and immune cell recruitment. Acquisition of this acyltransferase enhances fitness of a K. pneumoniae ST258 isolate and may contribute to the success of this clonal complex as a healthcare-associated pathogen.

Characterization of Bacterial and Fungal Infections in Hospitalized Patients With Coronavirus Disease 2019 and Factors Associated With Health Care-Associated Infections.

BACKGROUND: Patients hospitalized with coronavirus disease 2019 (COVID-19) are at increased risk of health care-associated infections (HAIs), especially with prolonged hospital stays. We sought to identify incidence, antimicrobial susceptibilities, and outcomes associated with bacterial/fungal secondary infections in a large cohort of patients with COVID-19. METHODS: We evaluated adult patients diagnosed with COVID-19 between 2 March and 31 May 2020 and hospitalized >24 hours. Data extracted from medical records included diagnoses, vital signs, laboratory results, microbiological data, and antibiotic use. Microbiologically confirmed bacterial and fungal pathogens from clinical cultures were evaluated to characterize community- and health care-associated infections, including describing temporal changes in predominant organisms on presentation and throughout hospitalization. Univariable and multivariable logistic regression analyses were performed to investigate risk factors for HAIs. RESULTS: A total of 3028 patients were included and accounted for 899 positive clinical cultures. Overall, 516 (17%) patients with positive cultures met criteria for infection. Community-associated coinfections were identified in 183 (6%) patients, whereas HAIs occurred in 350 (12%) patients. Fifty-seven percent of HAIs were caused by gram-negative bacteria and 19% by fungi. Antibiotic resistance increased with longer hospital stays, with incremental increases in the proportion of vancomycin resistance among enterococci and ceftriaxone and carbapenem resistance among Enterobacterales. Intensive care unit stay, invasive mechanical ventilation, and steroids were associated with HAIs. CONCLUSIONS: HAIs occur in a small proportion of patients hospitalized with COVID-19 and are most often caused by gram-negative and fungal pathogens. Antibiotic resistance is more prevalent with prolonged hospital stays. Antimicrobial stewardship is imperative in this population to minimize unnecessary broad-spectrum antibiotic use.

What about tocilizumab? A retrospective study from a NYC Hospital during the COVID-19 outbreak.

BACKGROUND: Tocilizumab, an interleukin-6 receptor blocker, has been used in the inflammatory phase of COVID-19, but its impact independent of corticosteroids remains unclear in patients with severe disease. METHODS: In this retrospective analysis of patients with COVID-19 admitted between March 2 and April 14, 2020 to a large academic medical center in New York City, we describe outcomes associated with tocilizumab 400 mg (without methylprednisolone) compared to a propensity-matched control. The primary endpoints were change in a 7-point ordinal scale of oxygenation and ventilator free survival, both at days 14 and 28. Secondary endpoints include incidence of bacterial superinfections and gastrointestinal perforation. Primary outcomes were evaluated using t-test. RESULTS: We identified 33 patients who received tocilizumab and matched 74 controls based on demographics and health measures upon admission. After adjusting for illness severity and baseline ordinal scale, we failed to find evidence of an improvement in hypoxemia based on an ordinal scale at hospital day 14 in the tocilizumab group (OR 2.2; 95% CI, 0.7-6.5; p = 0.157) or day 28 (OR 1.1; 95% CI, 0.4-3.6; p = 0.82). There also was no evidence of an improvement in ventilator-free survival at day 14 (OR 0.8; 95% CI, 0.18-3.5; p = 0.75) or day 28 (OR 1.1; 95% CI, 0.1-1.8; p = 0.23). There was no increase in secondary bacterial infection rates in the tocilizumab group compared to controls (OR 0.37; 95% CI, 0.09-1.53; p = 0.168). CONCLUSIONS: There was no evidence to support an improvement in hypoxemia or ventilator-free survival with use of tocilizumab 400 mg in the absence of corticosteroids. No increase in secondary bacterial infections was observed in the group receiving tocilizumab.

An efficient and versatile CRISPR-Cas9 system for genetic manipulation of multi-drug resistant Klebsiella pneumoniae.

Multi-drug resistant (MDR) Klebsiella pneumoniae remains an urgent public health threat. While whole-genome sequencing has helped identify genetic changes underlying resistance, functional validation remains difficult due to a lack of genetic manipulation systems for MDR K. pneumoniae. CRISPR-Cas9 has revolutionized molecular biology, but its use was only recently adapted in bacteria by overcoming the lack of genetic repair systems. We describe a CRISPR-Cas9/lambda recombineering system utilizing a zeocin resistance cassette allowing efficient and versatile genetic manipulation of K. pneumoniae. For complete details on the use and execution of this protocol, please refer to McConville et al. (2020).

Carbapenemase-producing Enterobacterales causing secondary infections during the COVID-19 crisis at a New York City hospital.

BACKGROUND: Patients with COVID-19 may be at increased risk for secondary bacterial infections with MDR pathogens, including carbapenemase-producing Enterobacterales (CPE). OBJECTIVES: We sought to rapidly investigate the clinical characteristics, population structure and mechanisms of resistance of CPE causing secondary infections in patients with COVID-19. METHODS: We retrospectively identified CPE clinical isolates collected from patients testing positive for SARS-CoV-2 between March and April 2020 at our medical centre in New York City. Available isolates underwent nanopore sequencing for rapid genotyping, antibiotic resistance gene detection and phylogenetic analysis. RESULTS: We identified 31 CPE isolates from 13 patients, including 27 Klebsiella pneumoniae and 4 Enterobacter cloacae complex isolates. Most patients (11/13) had a positive respiratory culture and 7/13 developed bacteraemia; treatment failure was common. Twenty isolates were available for WGS. Most K. pneumoniae (16/17) belonged to ST258 and encoded KPC (15 KPC-2; 1 KPC-3); one ST70 isolate encoded KPC-2. E. cloacae isolates belonged to ST270 and encoded NDM-1. Nanopore sequencing enabled identification of at least four distinct ST258 lineages in COVID-19 patients, which were validated by Illumina sequencing data. CONCLUSIONS: While CPE prevalence has declined substantially in New York City in recent years, increased detection in patients with COVID-19 may signal a re-emergence of these highly resistant pathogens in the wake of the global pandemic. Increased surveillance and antimicrobial stewardship efforts, as well as identification of optimal treatment approaches for CPE, will be needed to mitigate their future impact.

CrrB Positively Regulates High-Level Polymyxin Resistance and Virulence in Klebsiella pneumoniae.

Polymyxin resistance (PR) threatens the treatment of carbapenem-resistant Klebsiella pneumoniae (CRKP) infections. PR frequently arises through chemical modification of the lipid A portion of lipopolysaccharide. Various mutations are implicated in PR, including in three two-component systems-CrrA/B, PmrA/B, and PhoP/Q-and the negative regulator MgrB. Few have been functionally validated. Therefore, here we adapt a CRISPR-Cas9 system to CRKP to elucidate how mutations in clinical CRKP isolates induce PR. We demonstrate that CrrB is a positive regulator of PR, and common clinical mutations lead to the addition of both 4-amino-4-deoxy-L-arabinose (L-Ara4N) and phosophethanolamine (pEtN) to lipid A, inducing notably higher polymyxin minimum inhibitory concentrations than mgrB disruption. Additionally, crrB mutations cause a significant virulence increase at a fitness cost, partially from activation of the pentose phosphate pathway. Our data demonstrate the importance of CrrB in high-level PR and establish important differences across crrB alleles in balancing resistance with fitness and virulence.

Epidemiology of Staphylococcus aureus in neonates on admission to a Chinese neonatal intensive care unit.

PURPOSE: Little is known about the molecular epidemiology of Staphylococcus aureus in Chinese neonatal intensive care units (NICUs). We describe the molecular epidemiology of S. aureus isolated from neonates on admission to Beijing Children's Hospital. METHODS: From May 2015-March 2016, nasal swabs were obtained on admission from 536 neonates. Cultures were also obtained from body sites with suspected infections. S. aureus isolates were characterized by staphylococcal chromosomal cassette (SCCmec) type, staphylococcal protein A (spa) type, multilocus sequence type (MLST), sasX gene, antimicrobial susceptibility and cytotoxicity. Logistic regression assessed risk factors for colonization. RESULTS: Overall, 92 (17%) infants were colonized with S. aureus and 20 (3.7%) were diagnosed with culture-positive S. aureus infection. Of the colonized infants, 70% (64/92) harbored methicillin-susceptible S. aureus (MSSA), 30% (28/92) harbored methicillin-resistant S. aureus (MRSA) while 70% (14/20) of infected infants were culture-positive for MRSA, 30% (6/20) were culture-positive for MSSA. Risk factors for colonization included female sex, age 7-28 days, higher birthweight (3270 IQR [2020-3655] grams) and vaginal delivery (p<0.05). The most common MRSA and MSSA clones were community-associated ST59-SCCmecIVa-t437 (60%) and ST188-t189 (15%), respectively. The sasX gene was not detected. Some MSSA isolates (16%) were penicillin-susceptible and some MRSA isolates (18%) were oxacillin-susceptible. MRSA and MSSA had similar cytotoxicity, but colonizing strains were less cytotoxic than strains associated with infections. CONCLUSIONS: S. aureus colonization was common in infants admitted to our NICU and two community-associated clones predominated. Several non-modifiable risk factors for S. aureus colonization were identified. These results suggest that screening infants for S. aureus upon admission and targeting decolonization of high-risk infants and/or those colonized with high-risk clones could be useful to prevent transmission.

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.

Sino-Orbital Fungal Infection by Tilletiopsis minor, a Rare Human Pathogen, Diagnosed by Internal Transcribed Spacer Sequencing.

Orbital infection can be caused by numerous pathogens, and accurate diagnosis informs appropriate therapy. The authors report a case of a 78-year-old man with well-controlled diabetes mellitus and recurrent sino-orbital infection following multiple surgical procedures with negative microbiologic results. This case presented a diagnostic and treatment challenge and was aided by the use of internal transcribed spacer sequencing for pathogen identification. The fungal pathogen, Tilletiopsis minor, has not previously been described as a human pathogen in the sinus and orbit. This report describes a novel orbital pathogen and highlights the importance of diagnostic diligence and utilizing internal transcribed spacer sequencing in the workup of atypical orbital infection.

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.

Staphylococcus epidermidis Protection Against Staphylococcus aureus Colonization in People Living With Human Immunodeficiency Virus in an Inner-City Outpatient Population: A Cross-Sectional Study.

BACKGROUND: People living with human immunodeficiency virus (PLWH) have been disproportionally affected by methicillin-resistant Staphylococcus aureus (MRSA) colonization and infection, in particular by clones USA300 and USA500. However, the contribution of epidemiological, bacterial, and immunological risk factors to the excess of S aureus in PLWH remain incompletely understood. METHODS: In this cross-sectional study, we determined the prevalence and molecular epidemiology of S aureus colonization in 93 PLWH attending an urban human immunodeficiency virus (HIV) clinic. Participants completed a structured interview assessing demographic information and risk factors for MRSA. Swabs were obtained from the nose, throat, and groin and cultured for S aureus and Staphylococcus epidermidis. RESULTS: Most participants had well controlled HIV infection (89, 96% CD4 >200). Thirty-six (39%) individuals were colonized with S aureus at 1 or more body sites, including 6 (6%) with MRSA. Regular gym use was a risk factor for S aureus but not MRSA carriage. In contrast, S epidermidis was present in almost all individuals (n = 84, 90%), predominantly in the nares (n = 66, 71%). Using generalized estimating equation models, we observed that the odds of S aureus colonization were significantly and drastically reduced when S epidermidis was detected (P = .0001). After controlling for site, gender, and age, we identified that the odds of S aureus colonization were 80% less if S epidermidis was present (adjusted odds ratio, 0.20; 95% confidence interval, .09-.45; P < .0001). CONCLUSIONS: Taken together, we observed a lower prevalence of S aureus and MRSA colonization than has been previously reported in PLWH. In this cohort, colonization with S epidermidis was protective against S aureus colonization.