Background. Data on the use of ceftolozane-tazobactam and emergence of ceftolozane-tazobactam resistance during multidrug resistant (MDR)-Pseudomonas aeruginosa infections are limited.Methods. We performed a retrospective study of 21 patients treated with ceftolozane-tazobactam for MDR-P. aeruginosa infections. Whole genome sequencing and quantitative real-time polymerase chain reaction were performed on longitudinal isolates.Results. Median age was 58 years; 9 patients (43%) were transplant recipients. Median simplified acute physiology score-II (SAPS-II) was 26. Eighteen (86%) patients were treated for respiratory tract infections; others were treated for bloodstream, complicated intraabdominal infections, or complicated urinary tract infections. Ceftolozane-tazobactam was discontinued in 1 patient (rash). Thirty-day all-cause and attributable mortality rates were 10% (2/21) and 5% (1/21), respectively; corresponding 90-day mortality rates were 48% (10/21) and 19% (4/21). The ceftolozane-tazobactam failure rate was 29% (6/21). SAPS-II score was the sole predictor of failure. Ceftolozane-tazobactam resistance emerged in 3 (14%) patients. Resistance was associated with de novo mutations, rather than acquisition of resistant nosocomial isolates. ampC overexpression and mutations were identified as potential resistance determinants.Conclusions. In this small study, ceftolozane-tazobactam was successful in treating 71% of patients with MDR-P. aeruginosa infections, most of whom had pneumonia. The emergence of ceftolozane-tazobactam resistance in 3 patients is worrisome and may be mediated in part by AmpC-related mechanisms. More research on treatment responses and resistance during various types of MDR-P. aeruginosa infections is needed to define ceftolozane-tazobactam's place in the armamentarium.

Staphylococcus aureus is capable of infecting nearly every organ in the human body. In order to infiltrate and thrive in such diverse host tissues, staphylococci must possess remarkable flexibility in both metabolic and virulence programs. To investigate the genetic requirements for bacterial survival during invasive infection, we performed a transposon sequencing (TnSeq) analysis of S. aureus during experimental osteomyelitis. TnSeq identified 65 genes essential for staphylococcal survival in infected bone and an additional 148 mutants with compromised fitness in vivo. Among the loci essential for in vivo survival was SrrAB, a staphylococcal two-component system previously reported to coordinate hypoxic and nitrosative stress responses in vitro. Healthy bone is intrinsically hypoxic, and intravital oxygen monitoring revealed further decreases in skeletal oxygen concentrations upon S. aureus infection. The fitness of an srrAB mutant during osteomyelitis was significantly increased by depletion of neutrophils, suggesting that neutrophils impose hypoxic and/or nitrosative stresses on invading bacteria. To more globally evaluate staphylococcal responses to changing oxygenation, we examined quorum sensing and virulence factor production in staphylococci grown under aerobic or hypoxic conditions. Hypoxic growth resulted in a profound increase in quorum sensing-dependent toxin production, and a concomitant increase in cytotoxicity toward mammalian cells. Moreover, aerobic growth limited quorum sensing and cytotoxicity in an SrrAB-dependent manner, suggesting a mechanism by which S. aureus modulates quorum sensing and toxin production in response to environmental oxygenation. Collectively, our results demonstrate that bacterial hypoxic responses are key determinants of the staphylococcal-host interaction.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein (S) plays critical roles in host cell entry. Non-synonymous substitutions affecting S are not uncommon and have become fixed in a number of SARS-CoV-2 lineages. A subset of such mutations enable escape from neutralizing antibodies or are thought to enhance transmission through mechanisms such as increased affinity for the cell entry receptor, angiotensin-converting enzyme 2 (ACE2). Independent genomic surveillance programs based in New Mexico and Louisiana contemporaneously detected the rapid rise of numerous clade 20G (lineage B.1.2) infections carrying a Q677P substitution in S. The variant was first detected in the US on October 23, yet between 01 Dec 2020 and 19 Jan 2021 it rose to represent 27.8% and 11.3% of all SARS-CoV-2 genomes sequenced from Louisiana and New Mexico, respectively. Q677P cases have been detected predominantly in the south central and southwest United States; as of 03 Feb 2021, GISAID data show 499 viral sequences of this variant from the USA. Phylogenetic analyses revealed the independent evolution and spread of at least six distinct Q677H sub-lineages, with first collection dates ranging from mid-August to late November 2020. Four 677H clades from clade 20G (B.1.2), 20A (B.1.234), and 20B (B.1.1.220, and B.1.1.222) each contain roughly 100 or fewer sequenced cases, while a distinct pair of clade 20G clusters are represented by 754 and 298 cases, respectively. Although sampling bias and founder effects may have contributed to the rise of S:677 polymorphic variants, the proximity of this position to the polybasic cleavage site at the S1/S2 boundary are consistent with its potential functional relevance during cell entry, suggesting parallel evolution of a trait that may confer an advantage in spread or transmission. Taken together, our findings demonstrate simultaneous convergent evolution, thus providing an impetus to further evaluate S:677 polymorphisms for effects on proteolytic processing, cell tropism, and transmissibility.

Bacterial populations vary in their stress tolerance and population structure depending upon whether growth occurs in well-mixed or structured environments. We hypothesized that evolution in biofilms would generate greater genetic diversity than well-mixed environments and lead to different pathways of antibiotic resistance. We used experimental evolution and whole genome sequencing to test how the biofilm lifestyle influenced the rate, genetic mechanisms, and pleiotropic effects of resistance to ciprofloxacin in Acinetobacter baumannii populations. Both evolutionary dynamics and the identities of mutations differed between lifestyle. Planktonic populations experienced selective sweeps of mutations including the primary topoisomerase drug targets, whereas biofilm-adapted populations acquired mutations in regulators of efflux pumps. An overall trade-off between fitness and resistance level emerged, wherein biofilm-adapted clones were less resistant than planktonic but more fit in the absence of drug. However, biofilm populations developed collateral sensitivity to cephalosporins, demonstrating the clinical relevance of lifestyle on the evolution of resistance.

Background Most hospitals use traditional infection prevention (IP) methods for outbreak detection. We developed the Enhanced Detection System for Healthcare-Associated Transmission (EDS-HAT), which combines whole-genome sequencing (WGS) surveillance and machine learning (ML) of the electronic health record (EHR) to identify undetected outbreaks and the responsible transmission routes, respectively. Methods We performed WGS surveillance of healthcare-associated bacterial pathogens from November 2016 to November 2018. EHR ML was used to identify the transmission routes for WGS-detected outbreaks, which were investigated by an IP expert. Potential infections prevented were estimated and compared with traditional IP practice during the same period. Results Of 3165 isolates, there were 2752 unique patient isolates in 99 clusters involving 297 (10.8%) patient isolates identified by WGS; clusters ranged from 2–14 patients. At least 1 transmission route was detected for 65.7% of clusters. During the same time, traditional IP investigation prompted WGS for 15 suspected outbreaks involving 133 patients, for which transmission events were identified for 5 (3.8%). If EDS-HAT had been running in real time, 25–63 transmissions could have been prevented. EDS-HAT was found to be cost-saving and more effective than traditional IP practice, with overall savings of $192 408–$692 532. Conclusions EDS-HAT detected multiple outbreaks not identified using traditional IP methods, correctly identified the transmission routes for most outbreaks, and would save the hospital substantial costs. Traditional IP practice misidentified outbreaks for which transmission did not occur. WGS surveillance combined with EHR ML has the potential to save costs and enhance patient safety.