Extensively Drug-Resistant Shigella flexneri 2a, California, USA, 2022.

In Los Angeles, California, USA, persistent, refractory shigellosis was diagnosed in an immunocompetent man who has sex with men. Whole-genome sequencing augmented phenotypic antimicrobial susceptibility testing to comprehensively profile bacterial drug resistance and appropriately guide therapy and clear the infection.

Catheter-Related Bloodstream Infection Caused by Mycolicibacterium iranicum, California, USA.

We describe a case of catheter-related bacteremia caused by Mycolicibacterium iranicum in the United States. The case highlights the value of using next-generation sequencing to identify infrequent and emerging pathogens and the challenges associated with choosing appropriate treatments because of limited knowledge of drug resistance mechanisms in those emerging pathogens.

Domestically Acquired NDM-1-Producing Pseudomonas aeruginosa, Southern California, USA, 2023.

We describe a case of New Delhi metallo-ß-lactamase 1-producing carbapenem-resistant Pseudomonas aeruginosa (CRPA) in a transplant patient with multiple hospitalizations in California, USA. Whole-genome sequencing revealed the isolate was genetically distinctive, despite ≈95% similarity to other global strains. The patient's lack of international travel suggests this CRPA was acquired domestically.

Retrospective Detection of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) in Symptomatic Patients Prior to Widespread Diagnostic Testing in Southern California.

BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused one of the worst pandemics in recent history. Few reports have revealed that SARS-CoV-2 was spreading in the United States as early as the end of January. In this study, we aimed to determine if SARS-CoV-2 had been circulating in the Los Angeles (LA) area at a time when access to diagnostic testing for coronavirus disease 2019 (COVID-19) was severely limited. METHODS: We used a pooling strategy to look for SARS-CoV-2 in remnant respiratory samples submitted for regular respiratory pathogen testing from symptomatic patients from November 2019 to early March 2020. We then performed sequencing on the positive samples. RESULTS: We detected SARS-CoV-2 in 7 specimens from 6 patients, dating back to mid-January. The earliest positive patient, with a sample collected on January 13, 2020 had no relevant travel history but did have a sibling with similar symptoms. Sequencing of these SARS-CoV-2 genomes revealed that the virus was introduced into the LA area from both domestic and international sources as early as January. CONCLUSIONS: We present strong evidence of community spread of SARS-CoV-2 in the LA area well before widespread diagnostic testing was being performed in early 2020. These genomic data demonstrate that SARS-CoV-2 was being introduced into Los Angeles County from both international and domestic sources in January 2020.

Genomic epidemiology of the Los Angeles COVID-19 outbreak and the early history of the B.1.43 strain in the USA.

BACKGROUND: The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused global disruption of human health and activity. Being able to trace the early outbreak of SARS-CoV-2 within a locality can inform public health measures and provide insights to contain or prevent viral transmission. Investigation of the transmission history requires efficient sequencing methods and analytic strategies, which can be generally useful in the study of viral outbreaks. METHODS: The County of Los Angeles (hereafter, LA County) sustained a large outbreak of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). To learn about the transmission history, we carried out surveillance viral genome sequencing to determine 142 viral genomes from unique patients seeking care at the University of California, Los Angeles (UCLA) Health System. 86 of these genomes were from samples collected before April 19, 2020. RESULTS: We found that the early outbreak in LA County, as in other international air travel hubs, was seeded by multiple introductions of strains from Asia and Europe. We identified a USA-specific strain, B.1.43, which was found predominantly in California and Washington State. While samples from LA County carried the ancestral B.1.43 genome, viral genomes from neighboring counties in California and from counties in Washington State carried additional mutations, suggesting a potential origin of B.1.43 in Southern California. We quantified the transmission rate of SARS-CoV-2 over time, and found evidence that the public health measures put in place in LA County to control the virus were effective at preventing transmission, but might have been undermined by the many introductions of SARS-CoV-2 into the region. CONCLUSION: Our work demonstrates that genome sequencing can be a powerful tool for investigating outbreaks and informing the public health response. Our results reinforce the critical need for the USA to have coordinated inter-state responses to the pandemic.

Endofungal Mycetohabitans rhizoxinica Bacteremia Associated with Rhizopus microsporus Respiratory Tract Infection.

We report Mycetohabitans rhizoxinica bacteremia in a 65-year-old woman in California, USA, who was undergoing chimeric antigen receptor T-cell therapy for multiple myeloma. Acute brain infarction and pneumonia developed; Rhizopus microsporus mold was isolated from tracheal suction. Whole-genome sequencing confirmed bacteria in blood as genetically identical to endofungal bacteria inside the mold.

Mycoplasma hominis infections in solid organ transplant recipients: Clinical characteristics, treatment outcomes, and comparison of phenotypic and genotypic susceptibility profiles.

BACKGROUND: Mycoplasma hominis can cause significant infections after solid organ transplantation (SOT). Treatment should be guided by susceptibility testing, but conventional lab methods are laborious with prolonged turnaround time (TAT). This case series compares the phenotypic and genotypic susceptibility profiles of M. hominis isolates identified from SOT patients. METHODS: This is a single-center retrospective study evaluating SOT recipients with confirmed M. hominis infections. Patients' demographic, clinical, microbiological, and radiographic data were collected. Culture of M. hominis isolates was performed according to current Clinical and Laboratory Standards Institute guidelines. Phenotypic susceptibility testing was performed by University of Alabama Diagnostic Mycoplasma Laboratory. Whole genome sequencing (WGS) was performed followed by bioinformatic analysis of known genetic determinants of resistance. RESULTS: Seven SOT recipients with M. hominis infections were identified. Two out of seven (28.5%) patients had resistance detected by phenotypic susceptibility testing (Case 5 to levofloxacin and Case 7 to tetracycline). Genomic analyses confirmed the presence of mutations in the parC and parE topoisomerase genes at positions conferring to fluoroquinolone resistance in the isolate from Case 5, while the tetracycline-resistant isolate from Case 7 harbored the tetM gene. The median TAT from the date of specimen collection was 24 days for phenotypic susceptibility testing and 14 days for genotypic susceptibility testing. All seven patients received antimicrobials directed toward M. hominis and recovered with complete resolution of infection. CONCLUSIONS: WGS may offer a novel and more rapid methodology for M. hominis susceptibility testing to help optimize antimicrobial usage, but more data are needed.

Clinical Impact of Metagenomic Next-Generation Sequencing of Plasma Cell-Free DNA for the Diagnosis of Infectious Diseases: A Multicenter Retrospective Cohort Study.

BACKGROUND: Metagenomic next-generation sequencing (mNGS) of plasma cell-free DNA has emerged as an attractive diagnostic modality allowing broad-range pathogen detection, noninvasive sampling, and earlier diagnosis. However, little is known about its real-world clinical impact as used in routine practice. METHODS: We performed a retrospective cohort study of all patients for whom plasma mNGS (Karius test) was performed for all indications at 5 United States institutions over 1.5 years. Comprehensive records review was performed, and standardized assessment of clinical impact of the mNGS based on the treating team's interpretation of Karius results and patient management was established. RESULTS: A total of 82 Karius tests were evaluated from 39 (47.6%) adults and 43 (52.4%) children and a total of 53 (64.6%) immunocompromised patients. Karius positivity rate was 50 of 82 (61.0%), with 25 (50.0%) showing 2 or more organisms (range, 2-8). The Karius test results led to positive impact in 6 (7.3%), negative impact in 3 (3.7%), and no impact in 71 (86.6%), and was indeterminate in 2 (2.4%). Cases with positive Karius result and clinical impact involved bacteria and/or fungi but not DNA viruses or parasites. In 10 patients who underwent 16 additional repeated tests, only 1 was associated with clinical impact. CONCLUSIONS: The real-world impact of the Karius test as currently used in routine clinical practice is limited. Further studies are needed to identify high-yield patient populations, define the complementary role of mNGS to conventional microbiological methods, and discern how best to integrate mNGS into current testing algorithms.

Endogenous Endophthalmitis Caused by ST66-K2 Hypervirulent Klebsiella pneumoniae, United States.

We describe a case of endogenous endophthalmitis caused by sequence type 66-K2 hypervirulent Klebsiella pneumoniae in a diabetic patient with no travel history outside the United States. Genomic analysis showed the pathogen has remained highly conserved, retaining >98% genetic similarity to the original strain described in Indonesia in 1935.

Genomic Characterizations of Clade III Lineage of Candida auris, California, USA.

Candida auris is an emerging multidrug-resistant yeast. We describe an ongoing C. auris outbreak that began in October 2019 in Los Angeles, California, USA. We used genomic analysis to determine that isolates from 5 of 6 patients belonged to clade III; 4 isolates were closely related.

Catabacter hongkongensis bacteremia identified by direct metagenomic sequencing of positive blood culture fluid, first case report in the US.

Catabacter hongkongensis, an increasingly recognized bacteria in clinical samples, was identified by direct metagenomic sequencing of positive blood culture fluid from a 55-year-old patient with colonic perforation. The bacteremia was cleared by both antibiotic treatment and surgical intervention. This is the first case report of C. hongkongensis infection in the US.

Seawater-Associated Highly Pathogenic Francisella hispaniensis Infections Causing Multiple Organ Failure.

A rare case of Francisella hispaniensis infection associated with seawater exposure occurred in a deep-sea diving fisherman in Zhejiang, China. He had skin and soft tissue infection that progressed to bacteremia and multiple organ failure. Moxifloxacin treatment cleared the infections, but the patient suffered a sequela of heart damage.

Coinfections of Two Strains of NDM-1- and OXA-232-Coproducing Klebsiella pneumoniae in a Kidney Transplant Patient.

We report here a fatal case of carbapenem-resistant Klebsiella pneumoniae (CRKP) infections in a renal transplant patient without a travel history in the prior year, from whom 2 genetically different CRKP (sequence type 14 [ST14] and ST2497) strains carrying the same plasmids and antimicrobial resistance genes, including blaNDM-1, blaOXA-232, blaCTX-M-15, armA, and tet(D), were isolated from blood and the abdominal cavity. The isolates were susceptible to colistin, tigecycline, eravacycline, and cefiderocol, which was used to treat the CRKP in combination with ceftazidime-avibactam and polymyxin B and resulted in bacterial clearance. Despite the aggressive treatment, the patient died of ischemic colitis and multiorgan failure.

Diversity of upper respiratory tract infections and prevalence of Streptococcus pneumoniae colonization among patients with fever and flu-like symptoms.

BACKGROUND: Many upper respiratory pathogens cause similar symptoms. In China, routine molecular tests for upper respiratory pathogens are not widely performed and antibiotics abuse in treating upper respiratory tract infections (URTIs) is a major public health concern. METHODS: We performed qualitative real-time PCR tests to detect common upper respiratory tract pathogens including 9 viruses and 3 bacteria in 1221 nasopharyngeal swabs from patients with fever and influenza-like symptoms in a Chinese city. A quantitative real-time PCR was also performed to measure the bacterial density of the colonizing Streptococcus pneumoniae in these samples. RESULTS: We found very diverse pathogens including 81.7% viruses, 11.6% bacteria and 6.7% mixed viruses and bacteria. S. pneumoniae colonization was found in 8.0% of the cases but most of them had low bacterial density (Mean = 3.9 log cfu/ml). We also discovered an increase of S. pneumoniae colonization frequency (but not the density) in patients with detectable upper respiratory tract pathogens, in a pathogen variety-dependent manner. CONCLUSIONS: Our study provided strong evidence against empiric antibiotic use for treating URTIs, and highlighted a strong need for improving the diagnostic capacity for URTIs by using more molecular testing in China.

Selection of hyperproduction of AmpC and SME-1 in a carbapenem-resistant Serratia marcescens isolate during antibiotic therapy.

Objectives: Antibiotic selective pressure may result in changes to antimicrobial susceptibility throughout the course of infection, especially for organisms that harbour chromosomally encoded AmpC ß-lactamases, notably Enterobacter spp., in which hyperexpression of ampC may be induced following treatment with cephalosporins. In this study, we document a case of bacteraemia caused by a blaSME-1-harbouring Serratia marcescens that subsequently developed resistance to expanded-spectrum cephalosporins, piperacillin/tazobactam and fluoroquinolones, over the course of several months of treatment with piperacillin/tazobactam and ciprofloxacin. Methods: Susceptibility testing and WGS were performed on three S. marcescens isolates from the patient. ß-Lactamase activity in the presence or absence of induction by imipenem was measured by nitrocefin hydrolysis assays. Expression of ampC and blaSME-1 under the same conditions was determined by real-time PCR. Results: WGS demonstrated accumulation of missense and nonsense mutations in ampD associated with stable derepression of AmpC. Gene expression and ß-lactamase activity of both AmpC and SME-1 were inducible in the initial susceptible isolate, but were constitutively high in the resistant isolate, in which total ß-lactamase activity was increased by 128-fold. Conclusions: Although development of such in vitro resistance due to selective pressure imposed by antibiotics is reportedly low in S. marcescens, our findings highlight the need to evaluate isolates on a regular basis during long-term antibiotic therapy.

Prediction of BK viremia by urine viral load in renal transplant patients: An analysis of BK viral load results in paired urine and plasma samples.

BK virus (BKPyV)-associated nephropathy (BKPyVAN) may affect up to 10% of renal transplant recipients, causing graft failure in the absence of intervention. The dilemma in monitoring BKPyVAN in renal transplant patients has been that only testing urine BK viral load represents higher sensitivity (earlier detection) but lower specificity, while testing plasma BK viral load represents lower sensitivity (later detection) but higher specificity. However, blindly testing both urine and plasma inevitably contributes to unnecessary medical cost. We analyzed 1030 paired urine and plasma BKPyV viral load results and identified a reliable urine BKPyV viral load cutoff (4.0 log IU/mL) that can predict BKPyV viremia with 99.7% negative predictive value (NPV). We propose a cost-effective screening algorithm to first only monitor the urine BKPyV levels until the viral load reaches 4.0 log IU/mL, and then only monitor plasma with higher frequency. This approach ensures 98.7% sensitivity of catching the earliest BKPyV viremia onset, and 100% sensitivity of detecting the critical BKPyV viremia. In addition, we identified a urine BKPyV viral load cutoff of 6.7 log IU/mL as predictive of critical BKPyV viremia (defined as plasma viral load >4.0 log IU/mL) with 100% sensitivity and 100% NPV.

Evolution and Transmission of Carbapenem-Resistant Klebsiella pneumoniae Expressing the blaOXA-232 Gene During an Institutional Outbreak Associated With Endoscopic Retrograde Cholangiopancreatography.

BACKGROUND: Whole-genome sequencing (WGS) is an emerging and powerful technique by which to perform epidemiological studies in outbreak situations. METHODS: WGS was used to identify and evaluate an outbreak of OXA-232-expressing carbapenem-resistant Klebsiella pneumoniae (CRKP) transmitted to 16 patients over the course of 40 weeks via endoscopic retrograde cholangiopancreatography procedures at a single institution. WGS was performed on 32 OXA-232 CRKP isolates (1-7 per patient) and single-nucleotide variants (SNVs) were analyzed, with reference to the index patient's isolate. RESULTS: Interhost genetic diversity of isolates was between 0 and 15 SNVs during the outbreak; molecular clock calculations estimated 12.31 substitutions per genome per year (95% credibility interval, 7.81-17.05). Both intra- and interpatient diversification at the plasmid and transposon level was observed, significantly impacting the antibiogram of outbreak isolates. The majority of isolates evaluated (n = 27) harbored a blaCTX-M-15 gene, but some (n = 5) lacked the transposon carrying this gene, which resulted in susceptibility to aztreonam and third- and fourth-generation cephalosporins. Similarly, an isolate from a colonized patient lacked the transposon carrying rmtF and aac(6')lb genes, resulting in susceptibility to aminoglycosides. CONCLUSIONS: This study broadens the understanding of how bacteria diversify at the genomic level over the course of a defined outbreak and provides reference for future outbreak investigations.

Resistance to Ceftazidime-Avibactam Is Due to Transposition of KPC in a Porin-Deficient Strain of Klebsiella pneumoniae with Increased Efflux Activity.

Ceftazidime-avibactam is an antibiotic with activity against serine beta-lactamases, including Klebsiella pneumoniae carbapenemase (KPC). Recently, reports have emerged of KPC-producing isolates resistant to this antibiotic, including a report of a wild-type KPC-3 producing sequence type 258 Klebsiella pneumoniae that was resistant to ceftazidime-avibactam. We describe a detailed analysis of this isolate, in the context of two other closely related KPC-3 producing isolates, recovered from the same patient. Both isolates encoded a nonfunctional OmpK35, whereas we demonstrate that a novel T333N mutation in OmpK36, present in the ceftazidime-avibactam resistant isolate, reduced the activity of this porin and impacted ceftazidime-avibactam susceptibility. In addition, we demonstrate that the increased expression of blaKPC-3 and blaSHV-12 observed in the ceftazidime-avibactam-resistant isolate was due to transposition of the Tn4401 transposon harboring blaKPC-3 into a second plasmid, pIncX3, which also harbored blaSHV-12, ultimately resulting in a higher copy number of blaKPC-3 in the resistant isolate. pIncX3 plasmid from the ceftazidime-avibactam resistant isolate, conjugated into a OmpK35/36-deficient K. pneumoniae background that harbored a mutation to the ramR regulator of the acrAB efflux operon recreated the ceftazidime-avibactam-resistant MIC of 32 µg/ml, confirming that this constellation of mutations is responsible for the resistance phenotype.

Risk factors associated with the transmission of carbapenem-resistant Enterobacteriaceae via contaminated duodenoscopes.

BACKGROUND AND AIMS: The duodenoscopes used to perform ERCP have been implicated in several outbreaks of carbapenem-resistant Enterobacteriaceae (CRE) infection. The risk factors for CRE transmission via contaminated duodenoscopes remain unclear. METHODS: In this retrospective, single-center, case-control study, all patients who underwent ERCP with either 1 of 2 contaminated duodenoscopes were evaluated. We compared the patients who acquired CRE (active infection or colonization) with those who did not. RESULTS: Between October 3, 2014, and January 28, 2015, a total of 125 procedures were performed on 115 patients by using either of the contaminated duodenoscopes. Culture data were available for 104 of the 115 exposed patients (90.4%). Among these patients, 15 (14.4%) became actively infected (n = 8, 7.7%) or colonized (n = 7, 6.7%) with CRE. On univariate analysis, recent antibiotic exposure (66.7% vs 37.1%; P = .046), active inpatient status (60.0% vs 28.1%; P = .034), and a history of cholangiocarcinoma (26.7% vs 3.4%; P = .008) were patient characteristics associated with an increased risk of CRE infection. Biliary stent placement (53.3% vs 22.5%; P = .024) during ERCP was a significant procedure-related risk factor. After adjusting for cholangiocarcinoma, biliary stent placement (odds ratio 3.62; 95% confidence interval, 1.12-11.67), and active inpatient status (odds ratio 3.74; 95% confidence interval, 1.15-12.12) remained independent risk factors for CRE transmission. CONCLUSIONS: In patients undergoing ERCP with a contaminated duodenoscope, biliary stent placement, a diagnosis of cholangiocarcinoma, and active inpatient status are associated with an increased risk of CRE transmission.

Development of a novel real-time PCR assay with high-resolution melt analysis to detect and differentiate OXA-48-Like ß-lactamases in carbapenem-resistant Enterobacteriaceae.

The rapid global spread of carbapenem-resistant Enterobacteriaceae (CRE) poses an urgent threat to public health. More than 250 class D ß-lactamases (OXAs) have been described in recent years, with variations in hydrolytic activity for ß-lactams. The plasmid-borne OXA-48 ß-lactamase and its variants are identified only sporadically in the United States but are common in Europe. Recognition of these OXA-48-like carbapenemases is vital in order to control their dissemination. We developed a real-time PCR assay based on high-resolution melt analysis, using bla OXA-48-like-specific primers coupled with an unlabeled 3'-phosphorylated oligonucleotide probe (LunaProbe) homologous to OXA-48-like carbapenemase genes. The assay was validated using genomic DNA from 48 clinical isolates carrying a variety of carbapenemase genes, including bla KPC, bla SME, bla IMP, bla NDM-1, bla VIM, bla OXA-48, bla OXA-162, bla OXA-181, bla OXA-204, bla OXA-244, bla OXA-245, and bla OXA-232. Our assay identified the presence of bla OXA-48-like ß-lactamase genes and clearly distinguished between bla OXA-48 and its variants in control strains, including between bla OXA-181 and bla OXA-232, which differ by only a single base pair in the assay target region. This approach has potential for use in epidemiological investigations and continuous surveillance to help control the spread of CRE strains producing OXA-48-like enzymes.