Everybody Nose: Molecular and Clinical Characteristics of Nasal Colonization During Active Methicillin-Resistant Staphylococcus Aureus Bloodstream Infection

Background. Healthcare-associated infections pose a potentially fatal threat to patients worldwide and Staphylococcus aureus is one of the most common causes of healthcare-associated infections. S. aureus is a common commensal pathogen and a frequent cause of bacteremia, with studies demonstrating that nasal and blood isolates from single patients match more than 80% of the time. Here we report on a contemporary collection of colonizing isolates from those with methicillin-resistant S. aureus (MRSA) bloodstream infections to evaluate the diversity within hosts, and detail the clinical features associated with concomitant nasal colonization.Methods. Swabs of the bilateral anterior nares were obtained from patients diagnosed with MRSA bacteremia. A single colony culture from the blood and an average of 6 colonies from the nares were evaluated for MRSA growth. For the nares cultures, we typed multiple isolates for staphylococcal protein A (spa) and derived the clonal complexes. Demographic and clinical data were obtained retrospectively from the electronic medical record system and analysed using univariate and multivariable regression models.Results. Over an 11-month period, 68 patients were diagnosed with MRSA bloodstream infection, 53 were swabbed, and 37 (70%) were colonized with MRSA in the anterior nares. We performed molecular typing on 210 nasal colonies. Spa types and clonal complexes found in the blood were also detected in the nares in 95% of the cases. We also found that 11% of patients carried more than one clone of MRSA in the nares. Male sex and history of prior hospitalization within the past 90 days increased odds for MRSA colonization. Conclusion. The molecular epidemiological landscape of colonization in the setting of invasive disease is diverse and defining the interplay between colonization and invasive disease is critical to combating invasive MRSA disease.

Whole-Genome Epidemiology and Characterization of Methicillin-Susceptible Staphylococcus aureus ST398 From Retail Pork and Bulk Tank Milk in Shandong, China

Staphylococcus aureus (S. aureus) is now regarded as a zoonotic agent. Methicillin-susceptible S. aureus (MSSA) ST398 is a livestock-associated bacterium that is most prevalent in China, but there are currently no data available for Shandong. Therefore, the aim of this study was to investigate the epidemiology and characterization of MSSA ST398 from retail pork and bulk tank milk (BTM) in Shandong. A total of 67 S. aureus isolates were collected from retail pork between November 2017 and June 2018. Among the isolates, high antimicrobial resistance rates were observed for penicillin (97.0%), and 92.5% of the isolates were multi-drug resistant (MDR). Eight sequence types (STs) were identified in the retail pork isolates, and the predominant type was ST15 (n=26), which was followed by ST398 (n=14). Staphylococcal protein A gene (spa) typing identified spa types t034 and t1255 in MSSA ST398 from retail pork. Using whole-genome sequencing analysis, we described the phylogeny of 29 MSSA ST398 isolates that were obtained from retail pork (n=14) and BTM (n=15). The phylogenetic tree showed that the MSSA ST398 isolates from different sources had the same lineage. Among the 29 MSSA ST398 isolates, five resistance genes were detected, and all isolates carried DHA-1. Fifteen toxin genes were detected, and all isolates carried eta, hla, and hlb. In conclusion, this study found that a high risk for MSSA ST398 was present in retail pork and BTM. These findings have major implications for how investigations of MSSA ST398 outbreaks should be conducted in the One-Health context.

More than meets the Kappa for Antibody Superantigen Protein L (PpL)

Immunoglobulin superantigens play an important role in the affinity purification of antibodies and underlie the microbiota-immune axis at mucosal areas Focussing on the Staphylococcal Protein A (SpA), Streptococcal Protein G (SpG), and the Finegoldia Protein L (PpL) that were previously thought to bind to only specific regions of human antibodies, a systematic and holistic analysis of the antibody regions using 63 antibody permutations involving six Vκ and seven VH region IgG1 revealed showed novel PpL-antibody interactions. While SpA and SpG showed relatively consistent interactions with the antibodies, our findings showed PpL binding to certain VH-Vκ2, 5 and 6 interactions had contribution by other antibody regions. The findings of this have implications on PpL-based affinity antibody purifications and antibody design as well as provides novel insights to PpL-based microbiota-immune axis effects.

Emergence of Methicillin-Resistant Staphylococcus aureus ST239/241 SCCmec-III Mercury in Eastern Algeria

In this paper, we investigate the epidemiology of infections-associated Staphylococcusaureus (S. aureus) from the Medical Intensive Care Unit (MICU) at University Hospital Center of Constantine (UHCC) in Algeria, with a special emphasis on methicillin-resistant strains (MRSA) revealed by cefoxitin disks (30 μg), then confirmed by penicillin-binding protein (PBP2a) agglutination and real-time polymerase chain reaction (RT-PCR) targeting mecA and mecC genes. Staphylococcal Cassette Chromosome mec (SCCmec type), staphylococcal protein A (spa-type), multilocus sequence type (MLST), Panton–Valentine Leucocidin (PVL), and toxic shock syndrome toxin-1 (TSST-1) were further investigated in all isolates, and whole genome sequencing was performed for a selected subset of three hospital-acquired MRSA (HA-MRSA) isolates. A measurement of 80% out of the 50 S. aureus isolates were identified as HA-MRSA harbouring the mecA gene, and 72.5% of them were multidrug resistant (MDR). Twelve STs, four different SCCmec cassettes, fourteen spa types, ten isolates Panton–Valentine Leukocidin (PVL)-positive, and three isolates TSST-1 were identified. Interestingly, there was a high prevalence (n = 29; 72.5%) of a worrisome emerging clone: the HA-MRSA ST239/241 SCCmec-III mercury with PVL negative, resistant to β-lactams, aminoglycosides, quinolones, and tetracyclines. Other clones of HA-MRSA isolates were also identified, including PVL-positive ST80 SCCmec-IV/SCCmec-unknown (22.5%), ST34 SCCmec-V with TSST-1 positive (2.5%), and PVL-negative ST72 SCCmec-II (2.5%). Genome analysis enables us to describe the first detection of both PVL-negative HA-MRSA ST239/241 SCCmec-III mercury carrying ccrC, as well as SCCmec-V cassette, which dramatically changes the epidemiology of S. aureus infections in one of the hospitals in eastern Algeria.

1168. Are the Staphylococcus aureus Isolates that Recolonize Infants in a NICU Following Successful Decolonization the Same or Different from the Initial Colonizing Isolate?

Background Staphylococcus aureus is an important pathogen of infants in a neonatal intensive care unit (NICU). Colonization precedes infection and decolonization may prevent infection. The origin of colonizing organisms may be the NICU environment or personnel or visitors. We have observed infants who became recolonized after successful decolonization. The purpose of this study was to determine the proportion of infants who become recolonized with the same strain or a different strain. Methods Eligible infants were consecutive infants who 1. were colonized with methicillin-susceptible S. aureus and were successfully decolonized with topical mupirocin ointment (nares and umbilicus) as evidenced by 2 or more consecutive negative weekly surveillance cultures (in the absence of a course of systemic antibiotics with activity against MSSA), 2. subsequently became recolonized, and 3. the pair of isolates was available for analysis. Isolates were analyzed by staphylococcal protein A (spa) typing and pairs with concordant spa types were subjected to whole genome sequencing (WGS; Illumina MiSeq) and phylogenetic analyses. Pairs of isolates with fewer than 25 single nucleotide polymorphism differences were considered closely related. Results There were 19 occurrences of MSSA recolonization in 17 infants following 2-6 (median, 2) negative weekly intervening surveillance cultures. Based upon spa typing (that identified 19 spa types), in 11 (58%) there was a concordant spa type and in 8 (42%) there was a discordant spa type. Of the 11 pairs of isolates with concordant spa types that were compared after WGS, 10 were closely related resulting overall in recolonization with a closely related strain in 53% of episodes. Conclusion Among MSSA colonized infants who become recolonized after successful decolonization, the recolonizing strain is the same as the original strain in over half of cases. In such cases the source is more likely to be a visitor than the NICU environment or staff. The possibility that some cases classified as recolonization were in fact persistent low level colonization or carriage in another body site not detected by surveillance cultures cannot be excluded. Disclosures Anne-Catrin Uhlemann, MD, PhD, Merck (Grant/Research Support)

1164. Acquisition of Methicillin-Susceptible Staphylococcus aureus in the Neonatal Intensive Care Unit: Molecular Comparison of Relatedness of Isolates

Background In the NICU MSSA infections occur frequently and cause morbidity and mortality. Colonization is a risk factor for infection. Optimal infection prevention strategies await a more complete understanding of acquisition and transmission. To investigate possible transmission, we studied whether newly MSSA colonized infants share a strain with another contemporaneously colonized infant. Methods This is a prospective observational study in a level IV NICU from April through November 2019. Infants had weekly MSSA nasal surveillance cultures. Isolates from newly MSSA colonized infants and other infants colonized with MSSA during the same/previous week were subjected to staphylococcal protein A (spa) typing; most pairs with a concordant (CC) spa type were analyzed by whole genome sequencing (WGS; Illumina MiSeq). Pairs of isolates with a CC spa type and < 25 single nucleotide polymorphism differences on WGS were considered closely related (CC pairs). A control group consisted of pairs of isolates from a newly colonized infant with one randomly chosen colonized infant with a discordant (DC) spa type during the same/previous week. The medical records were reviewed for staff member (SM) and room assignment. Fischer’s exact test was used to compare proportions. Results Isolates from 60/68 consecutive newly MSSA colonized infants and 111/133 comparison infants were available for spa typing. Of these 60 infants, 23 (38 %) had a CC spa type with another infant colonized during the same/previous week. Of 18 isolate pairs from infants with a CC spa type that were subjected to WGS, 12 (67%) pairs of isolates were closely related. 7/12 (58 %) of CC pairs had a SM in common compared to 2/13 (15 %) in the DC pair groups, p=0.04. 2/12 (17 %) of CC pairs shared a room compared to 2/13 (15 %) pairs in the DC group, p=1.0. Conclusion Among newly MSSA colonized infants at least 25% are colonized with an isolate closely related to that of another colonized infant indicating likely infant to infant transmission. WGS is more discriminatory than spa typing for MSSA. Given the lack of commonality of room assignment and the commonality of SM assignment, a possible role of healthcare personnel in MSSA transmission should be further investigated. Disclosures Anne-Catrin Uhlemann, MD, PhD, Merck (Grant/Research Support)

Clonality, virulence genes, and antibiotic resistance of Staphylococcus aureus isolated from blood in Shandong, China

Background Bloodstream infection (BSI) caused by Staphylococcus aureus (S. aureus) can be life-threatening and pose a great challenge to infection control and clinical treatment. However, little information exists regarding the characterization of S. aureus in BSI patients in Shandong, China. To identify the clonality, virulence genes, and antibiotic resistance of S. aureus in blood, a total of 101 nonrepetitive blood isolates were collected. The antibiotic resistance phenotypes were determined, and virulence genes were analyzed with polymerase chain reaction (PCR). Finally, the genetic relatedness was investigated with Staphylococcus chromosomal cassette mec (SCCmec) typing for methicillin-resistant S. aureus (MRSA) isolates, Staphylococcal protein A (spa), and multilocus sequence typing (MLST) for all of 101 isolates. Results Of the 101 S. aureus isolates, 24 MRSA isolates and 77 methicillin-susceptible S. aureus (MSSA) isolates were identified. Overall, MRSA isolates had higher resistance rates than MSSA isolates when exposed to any of the 15 antibiotics tested in this study except for trimethoprim/sulfamethoxazole. Among the 17 virulence genes tested in this study, hla, hld, and hlg could be detected in all isolates. MRSA isolates were more likely to carry seb and hlb genes, while MSSA isolates were more likely to carry seg and sei genes. Thirty-five sequence types (STs) and 49 spa types were identified, of which ST59-t437 and ST398-t571 were the most abundant. These two genotypes were also the most abundant ST-spa types in MRSA and MSSA isolates, but their abundances shifted over time, with ST398-t571 being the predominant genotype from 2016 to 2017, and ST59-t437 from 2018 to 2020. Besides, all the ST59-t437 isolates harbored hlgb gene, whereas most (88.9%) ST398-t571 did not. In addition, twenty-four MRSA isolates were subject to SCCmec typing. SCCmec IVa was the most prevalent SCCmec type, and all the ST59-t437 MRSA isolates were SCCmec IVa. We also observed 15 new STs, and some of them were MRSA. Conclusion These findings provide additional observations and epidemiological data for blood S. aureus isolates, which can improve future infection-control measures and aid in potential clinical treatments in hospitals and other clinical settings.

Development of a Double Nanobody-Based Sandwich Immunoassay for the Detecting Staphylococcal Enterotoxin C in Dairy Products

Staphylococcal enterotoxins (SEs) represent the leading reason for staphylococcal food poisoning (SFP) and various other diseases. Reports often indicate Staphylococcal enterotoxin C (SEC) as the most frequently found enterotoxin in dairy products. To minimize consumer exposure to SEC, this paper aimed to create a sandwich enzyme-linked immunosorbent assay (ELISA) based on nanobodies (sandwich Nbs-ELISA) to accurately detect SEC in dairy products without the influence of staphylococcal protein A (SpA). Therefore, after inoculating a Bactrian camel with SEC, a phage display Nb library was created. Eleven Nbs against SEC were identified in three biopanning steps. Based on their affinity and pairing level, a sandwich Nbs-ELISA was developed using the C6 anti-SEC Nb as the capture antibody, while the detection antibody was represented by the C11 phage display anti-SEC Nb. In optimal conditions, the quantitative range of the present sandwich ELISA was 4-250 ng/mL with a detection limit (LOD) of 2.47 ng/mL, obtained according to the blank value plus three standard deviations. The developed technique was subjected to specific measurements, revealing minimal cross-reactivity with Staphylococcus aureus (S. aureus), Staphylococcal enterotoxin A (SEA), Staphylococcal enterotoxin B (SEB), and SpA. The proposed method exhibited high specificity and an excellent recovery rate of 84.52~108.06% in dairy products. Therefore, the sandwich Nbs-ELISA showed significant potential for developing a specific, sensitive technique for SEC detection in dairy products.

The Synergistic Effect of Nicotine and Staphylococcus aureus on Peri-Implant Infections

Smoking is considered a key risk factor for implant survival; however, how it interacts with the pathogens in peri-implant infections is not clear. Here, we identified that nicotine, the key component of cigarette smoking, can interact with Staphylococcus aureus and synergistically induce peri-implant infections in a rat osteolysis model. The nicotine–S. aureus combination group increased the gross bone pathology, osteolysis, periosteal reactions, and bone resorption compared to the nicotine or S. aureus single treated group (p < 0.05). Nicotine did not promote the proliferation of S. aureus both in vitro and in vivo, but it can significantly upregulate the expression of staphylococcal protein A (SpA), a key virulence factor of S. aureus. The nicotine–S. aureus combination also synergistically activated the expression of RANKL (receptor activator of nuclear factor-kappa B ligand, p < 0.05) to promote the development of peri-implant infections. The synergistic effects between nicotine and S. aureus infection can be a new target to reduce the peri-implant infections.