Role of Bioaerosols on the Short-Distance Transmission of Multidrug-Resistant Methicillin-Resistant Staphylococcus aureus (MRSA) in a Chicken Farm Environment

Methicillin-resistant Staphylococcus aureus (MRSA) is a dynamic and tenacious pathogenic bacterium which is prevalent in livestock farming environments. This study investigated the possibility of MRSA spread via bioaerosol transmission from an indoor chicken farm environment to outdoors downwind (up to 50 m). The concentration of total airborne bacteria colony formation units (CFUs) was decreased with increasing sampling distance ranging from 9.18 × 101 to 3.67 × 103 per air volume (m3). Among the 21 MRSA isolates, 15 were isolated from indoor chicken sheds and exposure square areas, whereas 6 were isolated from downwind bioaerosol samples. Molecular characterization revealed that all of them carried the staphylococcal cassette chromosome mec (SCCmec) VIII, and they were remarkably linked with the hospital-associated MRSA group. Spa typing analysis determined that all MRSA isolates belonged to spa type t002. Virulence analysis showed that 100% of total isolates possessed exfoliative toxin A (eta), whereas 38.09% and 23.80% strains carried exfoliative toxin B (etb) and enterotoxin A (entA). Additionally, all of these MRSA isolates carried multidrug resistance properties and showed their resistance against chloramphenicol, ciprofloxacin, clindamycin, tetracycline, and erythromycin. In addition, chi-squared statistical analysis displayed a significant distributional relationship of gene phenotypes between MRSA isolates from chicken farm indoor and downwind bioaerosol samples. The results of this study revealed that chicken farm indoor air might act as a hotspot of MRSA local community-level outbreak, wherein the short-distance dispersal of MRSA could be supported by bioaerosols.

Isolation and Genetic Characterization of Vancomycin-resistant and mecC+ Methicillin-resistant Staphylococcus aureus Strains in Clinical Samples of Bojnurd, Northeastern Iran

Background: The emergence of antibiotic-resistant Staphylococcus aureus strains is one of the major concerns about the various staphylococcal infections. Vancomycin is one the most important effective antibiotics on staphylococcal lethal infections. To date, vancomycin-resistant strains are increasingly isolated in different parts of the world, and it is alerting. Objectives: The current study was designed to evaluate the prevalence, and antibiotic susceptibility pattern of methicillin-resistant S. aureus (MRSA) and vancomycin-resistant S. aureus (VRSA) isolates in the main tertiary hospital of Bojnurd, Iran. Methods: S. aureus isolates were collected from different clinical samples in Imam Reza Hospital of Bojnurd. After identification of isolates through using conventional methods, they were evaluated by agar screening, disk diffusion, and minimum inhibitory concentration (MIC) methods to determine resistance to vancomycin and methicillin. We also performed polymerase chain reaction (PCR) for the detection of mecA, mecC, vanA, and vanB genes. After confirmation of vancomycin resistance, genetic analysis was performed using SCCmec, agr, and spa typing, and multilocus sequence typing (MLST) methods on VRSA isolates. Results: We found four vancomycin-resistant isolates (1.29%). Also, 75% of isolates were resistant to cefoxitin. Using the PCR method, mecA was found in 73%, mecC in 0.64%, and vanA in 1.29% of isolates. Interestingly, we found two mecC positive isolates in MRSA isolates. The alpha-hemolysin (81.81%) and enterotoxin C (27%) had the highest and lowest toxins percentage, respectively. Among mecA positive isolates, SCCmecIV (37%), SCCmecIII (31.27%), SCCmecI (14%), SCCmecII (11%), and SCCmecV (5.7%) were the most prevalent SCCmec types, respectively. It should be noted that the two mecC positive isolates belonged to SCCmecXI. AgrI (76.29%) was the highest agr type. We recognized t037 as the dominant spa type, and ST239, ST6, ST97, and ST8 were found in VRSA isolates. Conclusions: In our study, the frequency of mecA genes in MRSA isolates was very high. It seems that the resistant isolates belonged to endemic clones of Iran.

Distribution and Clonal Diversity of Staphylococcus aureus and Other Staphylococci in Surface Waters: Detection of ST425-t742 and ST130-t843 mecC-Positive MRSA Strains

Natural aquatic environments represent one of the most important vehicles of bacterial dissemination. Therefore, we aimed to isolate staphylococci from surface waters and to investigate the presence of antimicrobial resistance genes and virulence factors as well as the genetic lineages of all Staphylococcus aureus isolates. Staphylococci were recovered from water samples collected from 78 surface waters, including rivers, streams, irrigation ditches, dams, lakes, and fountains. The presence of antimicrobial resistance genes and virulence factors was investigated by PCR. Multilocus sequence typing and spa-typing were performed in all S. aureus isolates. From the 78 water samples, 33 S. aureus, one S. pseudintermedius, and 51 coagulase-negative staphylococci (CoNS) were identified. Among the S. aureus isolates, four MRSA were identified, and all harbored the mecC gene. Fourteen S. aureus were susceptible to all antimicrobials tested and the remaining showed resistance to penicillin, erythromycin and/or tetracycline encoded by the blaZ, ermT, msr(A/B), tetL, and vgaA genes. Regarding the clonal lineages, one mecC-MRSA isolate belonged to spa-type t843 and sequence type (ST) 130 and the other three to t742 and ST425. The remaining S. aureus were ascribed 14 spa-types and 17 sequence types. Eleven species of CoNS were isolated: S. sciuri, S. lentus, S. xylosus, S. epidermidis, S. cohnii spp. urealyticus, S. vitulinus, S. caprae, S. carnosus spp. Carnosus, S. equorum, S. simulans, and S. succinus. Thirteen CoNS isolates had a multidrug resistance profile and carried the following genes: mecA, msr(A/B), mph(C), aph(3′)-IIIa, aac(6′)-Ie–aph(2′’)-Ia, dfrA, fusB, catpC221, and tetK. A high diversity of staphylococci was isolated from surface waters including mecCMRSA strains and isolates presenting multidrug-resistance profiles. Studies on the prevalence of antibiotic-resistant staphylococci in surface waters are still very scarce but extremely important to estimate the contribution of the aquatic environment in the spread of these bacteria.

Genetic Diversity of Staphylococcus aureus Strains from a Tertiary Care Hospital in Rawalpindi, Pakistan

Staphylococcus aureus is an important healthcare-associated bacterium that causes a multitude of infections in humans such as superficial skin and soft tissue infections, necrotizing pneumonia, foodborne illnesses and postsurgical infections. Treatment of S. aureus infections has become more complicated due to the emergence of Methicillin-Resistant Staphylococcus aureus (MRSA), some of which are multidrug resistant. The present study aimed to characterize S. aureus isolates from a tertiary care hospital in the Rawalpindi district of Pakistan. Staphylococci were isolated from 300 clinical samples collected from January 2018 to January 2019 and S. aureus isolates were tested for antimicrobial susceptibility and analyzed using Pulsed-Field Gel Electrophoresis (PFGE), Multi-Locus Sequence Typing (MLST), staphylococcal cassette chromosome mec (SCCmec) and spa typing. Approximately 25.3% (76/300) of the clinical samples were positive for S. aureus; of those, 88.2% (67/76) were mecA+ (MRSA). In addition to the β-lactam antibiotics, high levels of resistance were also found to the fluoroquinolones (ciprofloxacin, gatifloxacin and levofloxacin (73.7% each)). Of the 23 different spa types identified, the majority of isolates belonged to spa type t632 and t657 (9/66; 13.6% each spa type). ST772-t657 (Bengal Bay clone) was the most commonly identified clone in this study although other clones circulating around different regions of the world were also found indicating the diversity in MRSA isolates from this area of Pakistan. This study emphasizes the need to monitor MRSA in the clinical setting for improved infection control and treatment options.

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)

First Report of Antimicrobial Susceptibility and Virulence Gene Characterization Associated with Staphylococcus aureus Carriage in Healthy Camels from Tunisia

A total of 318 nasal and rectal swabs were collected from 159 apparently healthy camels (Camelus dromedarius) randomly selected from five regions in southern and central Tunisia and screened for Staphylococcus aureus carriage. Staphylococcus spp. were recovered from 152 of 159 camels studied (95.6%) and in total 258 swabs (81%) were positive. Among these isolates, 16 were coagulase positive Staphylococcus (CoPS) (6.2%) and were characterized by biochemical and molecular tests as S. aureus. These were isolated from 14 camels (8.8%) with co-carriage in nasal and rectal mucosa by two camels. All S. aureus isolates recovered were methicillin-susceptible Staphylococcus aureus (MSSA) and were characterized by spa typing and PFGE. Three different spa types were recovered: t729, t4013 and a spa type newly registered as t19687, which was the most common. PFGE analysis revealed seven different patterns and these were characterized by MLST, which revealed five different sequence types (ST6, ST88, ST3583 and two new sequences, ST6504 and ST6506). All isolates harbored different virulence genes, including hld, encoding delta hemolysin; lukE–lukD, encoding bicomponent leukotoxin LukE–LukD; the clfB gene, encoding clumping factor B; the laminin gene, encoding laminin-binding protein; and cap8, encoding capsule type 8. Fifteen isolates harbored hemolysin beta (hlb) and fourteen encoded hemolysin alpha (hla) and hemolysin G2 (hlgv). Adhesin factors, including clfA and fnbB, were detected in five and four isolates respectively. Binding proteins, including collagen (cbp) and elastin-binding protein (ebp), were detected in two S. aureus isolates while fibrinogen-binding protein (fib) was identified in four isolates. This study provides the first set of genotyping data on the population structure and presence of toxin genes of S. aureus strains in Tunisian camels.

Staphylococcus aureus and Cystic Fibrosis—A Close Relationship. What Can We Learn from Sequencing Studies?

Staphylococcus aureus is next to Pseudomonas aeruginosa the most isolated pathogen from the airways of cystic fibrosis (CF) patients, who are often infected by a dominant S. aureus clone for extended periods. To be able to persist, the pathogen has to adapt to the hostile niche of the airways to counteract host defence, antibiotic therapy and the competition with coinfecting pathogens. S. aureus is equipped with many virulence factors including adhesins, toxins that are localized on the chromosome, on plasmids or are phage-related. S. aureus is especially versatile and adaptation and evolution of the pathogen occurs by the acquisition of new genes by horizontal gene transfer (HGT), changes in nucleotides (single nucleotide variations, SNVs) that can cause a selective advantage for the bacteria and become fixed in subpopulations. Methicillin-resistant S. aureus are a special threat to CF patients due to the more severe lung disease occurring in infected patients. Today, with decreasing costs for sequencing, more and more studies using S. aureus isolates cultured from CF patients are being published, which use whole genome sequencing (WGS), multilocus sequence typing (MLST) or spa-sequence typing (spa-typing) to follow the population dynamics of S. aureus, elucidate the underlying mechanisms of phenotypic variants, newly acquired resistance or adaptation to the host response in this particular niche. In the first part of this review, an introduction to the genetic make-up and the pathogenesis of S. aureus with respect to CF is provided. The second part presents an overview of recent studies and their findings using genotypic methods such as single or multilocus sequencing and whole genome sequencing, which identify factors contributing to the adaptation of S. aureus and its evolution in the airways of individuals with CF.