Resistance to antibiotics can occur either by mutation or by acquisition of resistance conferring genes via horizontal gene transfer (HGT), of which the latter is considered to be the most important factor in the current pandemic of antimicrobial resistance genes1. Pathogenic Staphylococcus aureus is an adept bacteria that becomes more dangerous with a strain’s procurement of the SCCmec complex. This provides multiple antibiotic resistance features rendering methicillin and most other beta-lactams useless in the fight against this pathogen. Utilizing computational methods, this study investigates methicillin resistant and methicillin susceptible bacteremia to elucidate the relationship between frequencies of recombination events and horizontally acquired antibiotic resistant genes. We hypothesized that methicillin resistant (R) strains will experience homologous recombination more frequent than methicillin susceptible (S) strains and therefore have a positive correlation with the number of antibiotic resistant genes present in the genome. Using a collection of patient blood samples, diagnosed with Staphylococcus aureus bacteremia, and computational biology to infer parameters of recombination. We examined the genomes for antibiotic resistance genes known to be gained through recombination. In clusters that were analyzed, R strains showed that sample diversity of genomes are greater than S strains and that a greater percentage of the genome is from recombination, respectively. Phylogenetic sequence cluster’s (SC) R genomes had more median recombination divergence than the SC S genomes, (SC5R = 0.15 nt, SC5S = 0.025 nt) and (SC8R = 0.048 nt SC8S = 0.044 nt) per locus. These same methicillin resistant SC genomes contained two and a half times and two times as many antibiotic resistant genes than its methicillin susceptible SC genomes, on average, (SC5R = 12, SC5S =5) and (SC8R =10, SC8S = 6).

1. Delineating Recombination Frequency Between Methicillin
Resistant and Susceptible Homologous Strains and the
Relationship with Levels of Antibiotic Resistance Genes in
Staphylococcus aureus
By
J. R. Matthews
July 27, 2021
A Thesis
Submitted to the University at Albany, State University of New York
In Partial Fulfillment of
the Requirements for the Degree of
Master of Science

2. Sections
•Introduction
•Phylogeny
•Recombination
• Parameters
• Summary
•Antibiotic Resistance
• Summary
•Conclusions
•Unknowns

3. INTRODUCTION
• Transfer of DNA
• Three ways to obtain foreign genes.
• Homologous Recombination
• Relationship Among Recombination
& Antibiotic Resistance
• Two concepts
• SCCmec Complex
• Rationale
• Hypothesis
• Goal of this Study
• Two Questions to be Answered

4. • Many bacterial species
have the unique ability to
acquire DNA from their
neighbors, regardless of
whether they are related
or not.
• HGT (Horizontal Gene
Transfer)
• The processes that
enable bacteria to
directly obtain foreign
DNA are:
Soucy et al. 2015
Conjugation:
transfer of DNA
through direct cell-
to-cell contact
Transduction:
phages carry DNA
from cell to cell
Transformation:
uptake of naked
DNA

5. Homologous recombination: Genetic material that is exchanged
between two strands of DNA containing stretches of similar
base sequences.
Incoming DNA
Host genome
Recombinant host
genome
Gene A
Gene A
Gene A
Gene C
Gene C
Gene C
Gene B’
Gene B
Gene B’
Incoming DNA
Recombinant
host genome
Host genome

6. Homologous recombination
• Results in a mosaic chromosome consisting of DNA segments from
different sources
• Recombination does not occur similarly among strains of the same
species.
• Some strains are hyper-recombinants  they are able to acquire foreign DNA
more often than others
• This process has the potential to provide different allelic variants
which may introduce new phenotypes, such as virulence or antibiotic
resistance, more rapidly than by mutation alone.

7. 2 scenarios to explain the association between
antibiotic resistance and recombination:
1. Different strains have different resistance levels because they are
able to acquire resistance genes at different recombination rates
(Hanage et al. 2009)
2. The distribution of resistance genes among strains is the result of
these genes conferring different fitness benefits on different strains
and not because of recombination (Lehtinen et al. 2020)
• The two scenarios have been investigated in the bacterial pathogen
Streptococcus pneumoniae.

8. Staphylococcal Chromosomal Cassette (SCCmec)
• Mobile genetic element (MGE) that carries the mecA gene
• confers resistance to most beta-lactams, including methicillin
Lakhundi and Zhang. 2018 Clin Microbiol Rev
mec complex ccr complex

9. Rationale
• Methicillin resistance is acquired through a mobile genetic element
(SCCmec) that can move from cell to cell.
• Major event in the evolution of S. aureus.
• Independent acquisition by several multi-drug resistant strains in 1960’s
(penicillin, streptomycin, tetracycline, and erythromycin) (Crisostomo, M. I. et al., 2001)
• When SCCmec is acquired, other DNA segments may also be
integrated into the recipient chromosome at the same time.
• (Hypothesis) It is likely then, that methicillin resistant strains will have
more recombined DNA.
• Therefore, they will have greater number of antibiotic resistant genes.

10. The aim of my study
• Analyze the frequency of recombination and mutation in MRSA and
MSSA isolates.
• Does MRSA genomes experience recombination more frequent than MSSA
genomes.
• Analyze the distribution of antibiotic resistance (ABR) genes within
MRSA and MSSA isolates.
• Does MRSA genomes posses a greater amount of ABR genes than MSSA
genomes.

11. Phylogeny
• Dataset
• Phylogenetic Tree
• Methicillin Resistance
• MLST
• Clades
• Sequence Clusters (SC)
• Pan-genome
• Isolates
• SC contents

12. • There is 323 Staphylococcus aureus genomes obtained from bacteremia
infected patients at Dartmouth-Hitchcock Med Center.
• Isolates were collected from 2010-2018.
• They consists of a mix of methicillin resistant (MRSA) and methicillin
susceptible (MSSA) strains.
• Selected the largest phylogenetic clusters, two consisting of a mix of
MRSA and MSSA strains, and one solely made up of MSSA strains.
Data Set to be Used

13. • Phylogenetic tree showing the
phenotypic results of MRSA
and MSSA (outer ring).
• Multi-locus sequence typing
(MLST) shows that sequence
types (ST) (inner ring) were
mixed with each cluster having
at least two dominant STs.
• Eight clades considered closely
related genomes were found
(indicated by the colors on the
tree branches).

14. • Divided the dataset into
sequence clusters (SC1-8),
which consists of closely
related genomes.
• MRSA strains are mostly
found in SC5 and SC8.
• The analysis is predominantly
on these two dominant SCs.
• SC3 has only MSSA strains
and used for additional
comparative analysis for ABR
gene levels.

15. Pan Genome
ML phylogenetic tree used only those genes that are common to all
strains (called core genes).
Shared Gene Percentage
Total pan
genome #
SC3 pan
genome #
SC5 pan
genome #
SC8 pan
genome #
(99% <= strains <= 100%) Core genes 1692Core genes 1971Core genes 1909Core genes 2012
(95% <= strains < 99%) Soft core genes 151Soft core genes 121
Soft core
genes 297Soft core genes 236
(15% <= strains < 95%) Shell genes 1381Shell genes 791Shell genes 594Shell genes 525
(0% <= strains < 15%) Cloud genes 5180Cloud genes 1367Cloud genes 2279Cloud genes 1710
(0% <= strains <= 100%) Total genes 8404Total genes 4250Total genes 5079Total genes 4483

16. Breakdown of Isolates
• Measure & compare different recombination parameters between:
• MRSA (n = 50 genomes) and MSSA (n = 39 genomes) in SC5
• MRSA (n = 52 genomes) and MSSA (n = 24 genomes) in SC8
• Determine & analyze the level of antimicrobial resistance between:
• MRSA (n = 50 genomes) and MSSA (n = 39 genomes) in SC5
• MRSA (n = 52 genomes) and MSSA (n = 24 genomes) in SC8
• MRSA (n = 0 genomes) and MSSA (n = 41 genomes) in SC3

17. Recombination
• Parameters based on evolutionary
divergence of loci
• Five factors to look at
• Summary

18. MRSA strains have significantly higher sequence diversity
than MSSA in both clusters. (p < 0.001, SC5 & SC8)
MRSA in SC5 MSSA in SC5 MRSA in SC8 MSSA in SC8
*** ***
0.004
0.0025
0.0025 0.0011

19. Mean number of mutations per locus differed significantly
between MRSA and MSSA in both clusters. (p < 0.001, SC5 & SC8)
MRSA in SC5 MSSA in SC5 MRSA in SC8 MSSA in SC8
*** ***
0.074
0.062
0.057
0.042

20. *** ***
Significantly greater mean number of recombination events
per locus occurs in MRSA than in MSSA. (p < 0.001, SC5 & SC8)
MRSA in SC5 MSSA in SC5 MRSA in SC8 MSSA in SC8
0.043
0.049
0.136
0.031

21. *** ***
Contrasting results in the relative rate of recombination (ϕ/θ) to
mutation between SC5 & SC8. (p < 0.001, SC5 & SC8)
MRSA in SC5 MSSA in SC5 MRSA in SC8 MSSA in SC8
1.84
0.492
0.042
0.043

22. *** ***
The fraction of the genome acquired through recombination was
significantly larger in MRSA than in MSSA. (p < 0.001, SC5 & SC8)
MRSA in SC5 MSSA in SC5 MRSA in SC8 MSSA in SC8
4.5% of sites in any one
genome were consequent
of recombination
2.6% of sites in any one
genome were derived
from recombination
4.3% of sites in any one
genome were derived
from recombination
5.8% of sites in any one
genome were consequent
of recombination

23. Summary Results of Recombination
• Recombination provides a wider range of genomic sample diversity in MRSA than in
MSSA in both SC5 and SC8.
• Mutational divergence is very pronounced in methicillin resistant genomes compared
to methicillin susceptible genomes.
• More mutations take place per locus in MRSA genomes than that of MSSA genomes
within its associated SC.
• Greater occurrences of recombinational divergence in MRSA genomes than its MSSA
counterpart.
• Recombination events occur significantly more within MRSA genomes than MSSA
genomes within both SC5 and SC8.
• Relative rate of recombination to mutation (ratio) displays opposing results.
• SC5 demonstrated that MRSA genomes experience greater recombination events
than mutation events significantly more then MSSA genomes.
• SC8 shows contrasting results that the MRSA genomes have experienced less
recombination to mutation events since divergence of LCA than MSSA genomes.

24. Antibiotic Resistance
• Comparison of MRSA vs.
MSSA isolates within SCs
• MRSA vs. MRSA isolates
between SCs
• MSSA vs. MSSA isolates
between SCs
• SCs average & maximum
antibiotic resistance genes
• Each resistant & susceptible
group
• Genes Present
• Similar & different ABR genes
• Summary

25. Antibacterial Resistance Genes

26. SC3 (MSSA) has a mean of 5.85 ABR genes per genome and 1 isolate has 8 ABR genes, the
most of any within SC3.
SC5 (MRSA) has a mean of more than 10.88 ABR genes per genome and 1 isolate has a total
of 18 associated ABR genes in SC5 resistant strains.
SC5 (MSSA) possess a mean of 4.95 ABR genes per genome and 2 isolates possess 12 ABR
genes, the maximum within SC5 susceptible strains.
SC8 (MRSA) has a mean of 9.62 ABR genes per genome and a total of 5 isolates have a
maximum of 12 ABR genes in SC8 MRSA.
SC8 (MSSA) has a mean of 6.29 ABR genes per genome, with only one isolate having a
maximum of 11 ABR genes in SC8 MSSA.
Amount of ABR Genes

27. Genes Present
fosB – protein coding gene which makes
a fosfomycin-inactivating enzyme.
• Catalyzes l-cys or BSH (bacillithiol) to the
antibiotic by nucleophilic addition.
• All isolates
tet(31) – codes for an energy-
dependent efflux protein.
• A membrane assoc. protein which
exports tetracycline out of the bacterial
cell, thus protecting the ribosomes
• All isolates
bleO – codes for a high affinity
bleomycin binding protein.
• Potentially conferred in niche
environments.
• (56% SC5 MRSA & 5% MSSA) ( 0% all
SC8 isolates).
tet(K) – another gene which codes an
efflux protein.
• SC5 MSSA 3%, MRSA 0%
• SC8 MSSA 4%, MRSA 0%

28. Summary of Antibiotic Resistance results
SC5 & SC8 showed that MRSA genomes have, on average, a significantly greater
number of ABR genes compared to MSSA genomes.
• There was more than a 2-fold difference in the number of ABR genes present in MRSA
isolates than MSSA isolates in SC5.
• More than a 1.5-fold difference exists between the number of ABR genes in resistant
strains than in susceptible strains in SC8.
There is a slight difference among MSSA genomes between SC3 & SC5 (p = 0.046),
reject null hypothesis if p-value < 0.05.
• MSSA SC5 v. SC8 no significant difference, null accepted.
• MSSA SC3 v. SC8 no significant difference, null accepted.
SC5 MRSA and SC8 MRSA have no difference of significance when comparing the
amount of ABR genes.
Though some ABR genes in MSSA strains only, majority of genes present in only
MRSA strains or in both MRSA & MSSA, w/more MRSA strains possessing the genes.

29. Conclusions
• The characteristics of homologous recombination has been defined for two
major lineages of S. aureus.
• Genomes that were resistant to methicillin had more point mutations when
compared to the susceptible genomes within its cluster.
• Both SC5 and SC8 had more instances of recombination in methicillin resistant
isolates than the susceptible counterparts within the clusters.
• MRSA strains have a larger percentage of its genome that consists of foreign
DNA than that of MSSA strains from recombination.
• Results demonstrate that MRSA strains possessed greater numbers of HGT
antibiotic resistance genes, which may in part be due to those strains
experiencing more recombination events than the MSSA strains.

30. • The mecA gene being acquired by recombination is not the determining factor
for having more ABR genes.
• But it has been shown in other papers to be a co-contributor for Staphylococcus
aureus obtaining a particular antibiotic resistant designation (beta-lactum tolerant).
• Incorporation of the mecA gene typically is by acquisition of the SCCmec
complex, which potentially brings numerous resistance determinants in a single
instance​.
• This could be ABR genes, virulence factors, associated transcription proteins, and
mechanisms pertaining to the proper function of the related determinants.
• The presence of mecA seems to positively correlate with greater recombination
frequencies​
• Recombinational divergence is continuously greater within strains that are
methicillin resistant.
Factoring in the SCCmec

31. Outstanding Questions
• What factors can explain the contrasting results in the relative rates of recombination
to mutation:
• Environment which SC8 MRSA or MSSA infection was acquired?
• Length of time either strain type infection was present?
• Are the genes that frequently recombine similar between SC5 and SC8?
• What percentage of core genes overlap? Accessory genes?
• Are the genes that frequently recombine similar between MSSA and MRSA?
• How many genes are shared in the pan-genome of resistant & susceptible strains?
• Does the amount of virulence factors present in isolates follow the same trend as
antibiotic gene enumeration?
• With over 64 known virulent factors in S. aureus, does MRSA posses more than
MSSA?

32. END