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)

Findings and measures to eradicate methicillin resistant Staphylococcus aureus clonal complex 7 spa-type t091 in two Norwegian pig farms: a case report

Background The Norwegian LA-MRSA surveillance and control strategy in pig farms has been largely successful in preventing the establishment of MRSA in the pig population by identifying positive pig herds and eradicating MRSA from these. It can, however, be challenging to determine whether a particular type of MRSA is livestock-associated, particularly in cases where there is little evidence available to aid in classification. Case presentation In two Norwegian pig farms linked by trade of live pigs, MRSA CC7 t091 was found in samples from pigs and their environment. Longitudinal sampling, with a time interval of 25 days, in one farm demonstrated an increase in samples positive for MRSA CC7 t091, supporting a classification of the finding as livestock associated. Measures to eradicate MRSA from both farms were imposed by the National Food Safety Authority. Different measures of MRSA sanitation were applied in the two farms, and MRSA was successfully eradicated from both farms. Conclusions A high-cost, labor intensive and a lower-cost, less labor intensive MRSA eradication protocol, both including total depopulation and repopulation were successful in eradicating MRSA CC7 t091 from two case farms.

Characterisation of Staphylococcus argenteus carried by healthy Royal Marines: a molecular epidemiology case-study

Objectives: During a prospective study of S. aureus carriage in Royal Marines (RM) recruits, six S. argenteus strains were identified in four recruits undertaking military training together. As S. argenteus sepsis leads to mortality similar to S. aureus, we determined the potential for person-to-person transmission, to evaluate future outbreak risk. Methods: We used whole-genome sequencing to characterise S. argenteus and investigate phylogenetic relationships between isolates. Participant colonisation with S. aureus and skin and soft tissue infection acquisition were recorded. Results: All six S. argenteus strains were spa-type t5078, ST2250. Strains were detected in 4/40 recruits in the same troop (training cohort) in weeks 1, 6 or 15 of training. No mec, tsst or LukPV genes were detected. We identified differences of 10-35 core SNPs between S. argenteus from different recruits. In two recruits, two S. argenteus strains were isolated; these could be distinguished by 3 and 15 core SNPs in each case. S. argenteus was not identified in any one of the other 21 participating troops (1,012 recruits). Conclusions: The identification of S. argenteus within a single troop from the total recruit population supports a common source for transmission, supported by SNP analysis. The high number of SNPs between some isolates may indicate a common source of diverse isolates or a high level of S. argenteus mutation in carriage. S. argenteus ST2250 is a newly recognised lineage; a better understanding of the frequency of genetic changes during transmission and transition from asymptomatic carriage to disease is required.

spa diversity of methicillin-resistant and -susceptible Staphylococcus aureus in clinical strains from Malaysia: a high prevalence of invasive European spa-type t032

Background Staphylococcus aureus is one of the important pathogens causing nosocomial infection. spa typing allows identification of S. aureus clones in hospital isolates and is useful for epidemiological studies and nosocomial infection control. This study aims to investigate the spa types in Malaysian S. aureus isolates obtained from various clinical specimens. Method A total of 89 methicillin-resistant S. aureus (MRSA) [pus (n = 55), blood (n = 27), respiratory (n = 5), eye (n = 2)] isolates and 109 methicillin-susceptible S. aureus (MSSA) [pus (n = 79), blood (n = 24), respiratory (n = 3), eye (n = 2) and urine (n = 1)] isolates were subjected to spa typing with sequences analysed using BioNumerics version 7. Results The spa sequence was successfully amplified from 77.8% of the strains (154/198) and 47 known spa types were detected. The distribution of known spa types in MRSA (36.2%, 17/47) was less diverse than in MSSA (70.2%, 33/47). The most predominant spa types were t032 (50%) in MRSA, and t127 (19%) and t091 (16.7%) in MSSA, respectively. spa type t091 in MSSA was significantly associated with skin and soft tissue infections (p = 0.0199). Conclusion The previously uncommon spa type t032 was detected in the Malaysian MRSA strains, which also corresponded to the most common spa type in Europe and Australia, and has replaced the dominant spa type t037 which was reported in Malaysia in 2010.

Occurrence and Characteristics of Staphylococcus aureus in a Hungarian Dairy Farm during a Control Program

In this research, our aim was to assess the occurrence of Staphylococcus aureus in a Hungarian large-scale dairy farm during the S. aureus control program conducted in the course of our studies. Furthermore, the phenotypic and genotypic properties of the isolates (type of haemolysis, antibiotic susceptibility, staphylococcal enterotoxin (SE) gene carrying ability and spa type) were determined. S. aureus was detected in all bulk tank milk samples collected during this study. Two different spa types were identified among the 17 strains isolated in the farm. A total of 14 of the 17 studied strains (82%) showed β-haemolysis on blood agar, 2/17 strains (12%) expressed double zone and 1/17 strains (6%) showed weak β-haemolysis. All strains were susceptible to most antibiotics tested (cefoxitin, chloramphenicol, clindamycin, erythromycin, gentamicin, tetracycline and trimethoprim/sulphamethoxazole), but all strains were resistant to penicillin G. A total of 11 of the 17 strains (65%) were found to harbour seg, sei, selm, seln, selo genes; 4/17 strains (24%) harboured sei, selm, seln, selo genes and 2/17 strains (11%) harboured sei gene. Since the new SEs/SEls can also cause foodborne outbreaks potentially and all strains were found to be resistant to penicillin G, it is essential to decrease and keep the prevalence of S. aureus low in the dairy farm and the implementation of the S. aureus control program is also highly justified. The results showed that the S. aureus count decreased by the end of our studies, so the control program was proved to be effective.

Findings and measures to eradicate methicillin resistant Staphylococcus aureus clonal complex 7 spa-type t091 in two Norwegian pig farms: a case report

BackgroundThe Norwegian LA-MRSA surveillance and control strategy in pig farms has been largely successful in preventing the establishment of MRSA in the pig population by identifying MRSA positive pig herds and eradicating MRSA from these. It can, however, be challenging to determine whether a particular type of MRSA is livestock-associated, particularly in cases where there is little evidence available to aid in classification.Case presentationIn two Norwegian pig farms linked by trade of live pigs, MRSA CC7 t091 was found in samples from pigs and their environment. Longitudinal sampling, with a time interval of 25 days, in one farm demonstrated an increase in samples positive for MRSA CC7 t091, supporting a classification of the finding as livestock associated. Measures to eradicate MRSA from both farms were imposed by the National Food Safety Authority. Different measures of MRSA sanitation were applied in the two farms, and MRSA was successfully eradicated from both farms.ConclusionsA high-cost, labor intensive and a lower-cost, less labor intensive MRSA eradication protocol, both including total depopulation and repopulation were successful in eradicating MRSA CC7 t091 from two case farms.

Complete Genome Sequences of Methicillin-Resistant Staphylococcus aureus Strains 110900 and 128254, Two Representatives of the CRISPR-Cas-Carrying Sequence Type 630/spa Type t4549 Lineage

ABSTRACT Methicillin-resistant Staphylococcus aureus (MRSA) sequence type 630 (ST630) and spa type t4549 is an emerging lineage in Nordic countries, and some representatives carry the CRISPR-Cas system. Here, the complete genome sequences of two isolates from this lineage are presented, comprising chromosomes of 2,918,239 and 2,877,083 nucleotides, respectively, and a 2,473-nucleotide plasmid carrying erm(C).