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Vaginal microbiota affects urinary tract infection risk in women with systemic lupus erythematosus: a pilot cross-sectional study from Thailand

Objective The risk factors associated with urinary tract infections (UTIs) in patients with SLE remain uncertain. We evaluated the vaginal microbiota pattern and its potential UTI-associated risk factors. Methods A pilot cross-sectional study of patients with SLE was conducted at Ramathibodi Hospital, Bangkok, Thailand, during 2019–2020. Patients’ demographic data and relevant information were collected. Vaginal microbiota was assessed in all patients and in 10 healthy volunteers. Results Fifty-two patients were enrolled (mean age: 46.1 years). All patients had SLE that was in low disease activity. As per the Simpson_e index, the within-group alpha diversity of the vaginal microbiota was low in the SLE with UTI and SLE receiving trimethoprim-sulfamethoxazole (TMP-SMX) prophylaxis groups. Multivariate logistic regression analysis revealed that TMP-SMX prophylaxis (adjusted OR (AOR), 30.96; 95% CI 3.63 to 264.11; p=0.002), elevated C3 levels (AOR, 35.33; 95% CI 1.33 to 936.67; p=0.033) and presence of Veillonella dispar in the vaginal microbiota (AOR, 6.68; 95% CI 1.27 to 35.07; p=0.025) were associated with UTI. Conclusions The vaginal microbiota diversity differed between patients with lupus with and without UTI, and unnecessary administration of TMP-SMX prophylaxis may affect the alpha diversity of the vaginal microbiota.


INTRODUCTION
SLE is a chronic autoimmune disease involving multiple organ systems. 1 Infections can trigger the body's immune response, which aggravate the SLE disease activity and result in morbidity and mortality in these patients. 2 Urinary tract infections (UTIs) are among the most common infections in patients with SLE and have a 36% prevalence compared with an approximately 12% prevalence among the general population. 3 4 Several risk factors can lead to UTI development, including defects in the host immune response either secondary to immunosuppressive drugs or patients' medical conditions; uncontrolled diabetes; anatomical abnormalities of the genitourinary system; imbalances of specific cytokines, such as interleukin 1β and interleukin 1β antagonists, which are reported to play roles in controlling Escherichia coli colonisation in the genitourinary tract; and altered vaginal microbiota. [5][6][7] Kirjavainen et al 8 found that the vaginal microbiota in UTI-prone women exhibited diminished Lactobacillus compositions compared with those in the control group (p=0.003).
One of the first large-scale, non-culturedependent studies of vaginal microbial communities assessed approximately 400 asymptomatic North American women and revealed that most vaginal microbial flora Lupus Science & Medicine (73%) were dominated by one or more species of Lactobacillus. 9 Proposed mechanisms by which Lactobacillus may prevent UTI include preventing E. coli from colonising the vagina via competitive mechanisms in which Lactobacillus spp adhere to uroepithelial cells, lowering the vaginal pH by producing lactic acid, and producing bacteriocins, surfactants and hydrogen peroxide. 10 Lactic acid concentrations are high in a Lactobacillus-dominated vaginal microbiota, and these conditions produce a potently antimicrobial environment in vitro. 11 Many factors affect the vaginal microbiota, including a natural or treatment-associated lack of oestrogen, especially during menopause, receiving antimicrobial treatment or contraceptive pills, and having bacterial vaginosis, especially when lacking Lactobacillus spp. 10 Women lacking vaginal Lactobacillus are at an increased risk of various urogenital disease conditions, including bacterial vaginosis, HIV infection, Neisseria gonorrhoeae infections and vaginal colonisation by E. coli. 12 13 Nielsen et al 14 compared the E. coli faecal flora characteristics in patients with UTI with those of healthy controls who had never had a UTI and discovered that 87% of patients with UTI carried the infecting strain in their faecal flora. Additionally, faecal clones causing the UTI were more often dominant in the faecal flora. These findings support the UTI pathogenesis in which the gut microbiota colonising the vaginal introitus and periurethra may cause an ascending infection via the urethra to the bladder and sometimes the kidneys. 10 Thus, many patients with SLE may be at risk of UTI after receiving immunosuppressive therapy, treatments affecting normal ovarian function, or antibiotics or if they have altered vaginal microbiota patterns either from impaired ovarian function or imbalanced glucose homeostasis. However, knowledge is lacking regarding patients with SLE and the associated risk factors for UTI considering that these individuals are immunocompromised. Moreover, the vaginal microbiota compositions in women with UTI-associated SLE are unreported. Here, we assessed the associated risk factors for UTI in this population.

Objectives
The primary objective of this work was to study the potential characteristics of the vaginal microbiota associated with UTI in women with SLE. The secondary objectives were to evaluate the associated risk factors for UTI in women with SLE and explore whether SLE-related treatment affects UTI development in this population.  19 20 Alpha diversity Alpha diversity represents bacterial richness, evenness or both within a single sample. 21

Simpson_e index
Simpson_e index is one of the most commonly used alpha diversity measures. 22 Data and sample collection Patients' demographic data, menstrual status, cause of menopause, vaginal pH, coexisting medical conditions, modified SLEDAI-2K scores, use of trimethoprimsulfamethoxazole (TMP-SMX) and/or acyclovir as prophylactic agents for opportunistic infections, complete blood count, glycosylated haemoglobin (HbA1C), and fasting plasma glucose, C3 and C4 levels were collected at enrolment. The immunosuppressive drug type and data on the 3-month cumulative dose of each drug were recorded.
On the day of enrolment, vaginal swabs were taken at the posterior fornix to assess the vaginal microbiota and urinalyses were performed on all participants. Five healthy menopausal and five healthy non-menopausal women agreed to participate in the study and were used as controls to compare with the enrolled study population on their menstrual status. All healthy controls were screened for the same inclusion/exclusion criteria as were the patients with SLE. The vaginal swabs were stored at −80°C until DNA extraction. Blank swabs and blank buffer controls were also included.
Sample processing and DNA extraction Total DNA was extracted using the GenUP gDNA Extraction Kit (Biotechrabbit, Berlin, Germany) with additional mechanical lysis steps. First, 1 mL of swab suspension was transferred to a 2 mL lysis tube and centrifuged at 2000 revolutions per minute (rpm) for 5 min to pellet the bacterial cells. After removing the supernatant, the sample was added to 200 µL of lysis buffer from the extraction kit and lysed using a TissueLyser LT with a 5 mm stainless steel bead (Qiagen, Hilden, Germany) at 50 Hz for 5 min. The unlysed residue was pelleted again by centrifuging at 12 000 rpm for 2 min. The lysate was then processed following the standard protocol from the extraction kit.

16S rDNA amplification
The amplification method for next-generation sequencing was modified from a previous publication. 23 Briefly, the V4 region of the bacterial 16S rDNA was amplified using four phasing primer sets (spacers 1-4) containing the target primer sequences 515F: 5′-GTGC-CAGCMGCCGCGGTAA-3′ and 806R: 5′-GGACTACH-VGGGTWTCTAAT-3′. Each  paired-end reads were merged and trimmed based on quality score (<Q30). Joined reads were deduplicated and clustered with 97% similarity using VSEARCH. 25 Chimeric sequences were also filtered out by the UCHIME algorithm. 26 The filtered reads were classified based on 99% operational taxonomic units clustered in the 16S Greengene database (V.2013.8) 27 using the VSEARCH algorithm.

Statistical analyses
Categorical variables are presented as frequencies and percentages. Continuous variables are presented as mean and SD or median and IQR for data with and without a normal distribution, respectively. Characteristics between patients with and without UTI were compared via χ 2 or Fisher's exact tests for categorical variables and Mann-Whitney U test or Student's t-test for continuous variables. ORs and their 95% CIs were calculated. Two-tailed tests were used to determine statistical significance, and p<0.05 was considered significant. All variables with p<0.05 in the univariate analysis were considered for inclusion in the multivariate analysis. All statistical analyses were performed using SPSS V.17.0 statistical software.

DISCUSSION
Several factors affect UTI development in women. 28 The unidirectional urine flow resulting from ureteric and bladder peristalsis helps reduce bacterial adhesion to epithelial cells. 29 Another local mechanism is glycosaminoglycan secretion by bladder transitional cells, which acts as an antiadherence factor and traps bacteria. 30 If the innate immune system fails to respond to the invading bacteria, the complement system and other complex mechanisms will occur next. C3 is one of the essential factors that promote opsonisation to speed up bacterial  levels. In our study, the mean serum C3 levels were higher in the SLE with UTI group than in the SLE without UTI group, possibly because C3 acts as an acute-phase protein and may be elevated during infectious episodes such as UTIs. 33 However, determining the serum C3 level at one time point may not explain the entire clinical course but may be used to monitor dynamic changes in the complement. The vaginal microbiota is no less critical than the intact host immune responses. These microbes help maintain a healthy environment in the birth canal by hindering excessive growth of colonising uropathogens, thus reducing the risk of UTI. 10 34-37 The vaginal microbiota responds to uropathogens by competitive exclusion.
Lactobacillus adheres to uroepithelial cells and produces lactic acid and other substances that alter the vaginal canal environment for excessive growth of specific pathogens. [38][39][40][41][42][43][44] Prolonged antibiotic use may affect the vaginal microbiota, mostly reducing the microbial diversity. This effect triggers abnormal multiplication of the uropathogens, eventually altering the vaginal colonisation pattern. One animal study supported this theory: the researchers flushed cefadroxil into monkeys' vaginas and found predominant E. coli colonisation in 9 of 10 monkeys. Conversely, another group found no E. coli colonisation in the vaginal canals despite flushing them with P-fimbriated E. coli. 45 46 Consistently, in the current study, Enterococcus spp, Peptostreptococcus spp and V. dispar proportions were higher in the SLE with UTI group than in the SLE without UTI group (p=0.038, p=0.046 and p=0.015, respectively). Additionally, the alpha diversity was low in the SLE with UTI group and in those who received TMP-SMX prophylaxis. Thus, prolonged use of a TMP-SMX prophylaxis may affect the vaginal microbiota, but consequent UTI development requires additional clinical information to conclude.
Smith et al 46 examined whether prior antimicrobial use influenced the subsequent relative risk of UTI in premenopausal women. These authors concluded that previous antibiotic use within 28 days as well as TMP-SMX were associated with an increased risk of UTI (relative risk: 5.83, 95% CI 3.17 to 10.70 and relative risk: 4.56, 95% CI 2.10 to 9.87, respectively). Another retrospective study conducted at the University Student Health Center of the University of Florida between 2006 and 2014 revealed that TMP-SMX use as an initial agent in cystitis increased the risk of subsequent UTI events (AOR: 2.14, 95% CI 1.23 to 4.09). 47 This finding may be explained in that increasingly resistant bacteria caused the UTI, which could only be partially treated with TMP-SMX. Unknown effects on the vaginal microbiota may also cause recurrent infections. In our study, TMP-SMX prophylaxis was a potent associated risk factor of UTI (AOR: 30.96, 95% CI 3.63 to 264.11; p=0.002). Thus, prolonged use of TMP-SMX prophylaxis may cause unrecognised harm in patients with SLE.

Co-morbidities
TMP-SMX is considered an effective treatment for uncomplicated UTIs; therefore, some clinicians acknowledge that patients may benefit from reduced risk of UTI, pneumocystis pneumonia and toxoplasmosis. However, one extensive clinical survey conducted at 28 hospitals across Thailand from 2000 to 2005, focusing on the susceptibility patterns of E. coli to TMP-SMX among non-critically ill individuals, demonstrated that 62.6% of the isolates were resistant to TMP-SMX. 48 Therefore, routinely prescribing TMP-SMX to prevent UTI should be discouraged in Thailand.
TMP-SMX is indicated for opportunistic infection prophylaxis when CD4 counts are low or the patient is receiving high-dose corticosteroid therapy. 49 However, to date, no consensus has been reached regarding when TMP-SMX should be provided to patients with SLE. Therefore, TMP-SMX for opportunistic infection prophylaxis as part of SLE treatment is solely individualised. Interestingly, most patients in our study were treated with only low-level immunosuppressive drugs, but a large proportion of them received TMP-SMX prophylaxis. Our data may reflect unnecessary treatment and provide evidence to support discontinuing TMP-SMX prophylaxis in patients with SLE who are receiving low-level immunosuppressive agents.
To further analyse whether TMP-SMX affects Lactobacillus proportions, Melkumyan et al 50 conducted a clinical study assessing the sensitivity patterns of 123 vaginal Lactobacillus strains to a variety of antibiotic types and noticed that all strains were resistant to TMP-SMX. Hence,   Our study had several limitations. First, the study population was small, and other essential risk factors of UTI may have been missed. Second, the nature of the crosssectional study did not allow comparing the vaginal microbiota patterns among individuals prior to and after TMP-SMX prophylaxis and prior to and after UTI events. Third, the results may not apply to active patients with SLE requiring a higher level of immunosuppressive therapy. A final limitation was that we lacked patients' histories of sexual activity. However, this study highlighted some significant findings as follows. First, vaginal microbiota diversity differs between patients with SLE with and without UTI. Second, unnecessary use of TMP-SMX prophylaxis may affect the alpha diversity of the vaginal microbiota.
In conclusion, UTI in patients with SLE remains a critical infectious complication that can increase patients' morbidity and mortality. In addition to comprehensively assessing the anatomical abnormalities of the genitourinary system, prophylactic agents must be carefully prescribed to help maintain a healthy and diverse vaginal microbiota and help prevent future UTI events. Further studies on a larger scale and with higher modified SLEDAI-2K scores are encouraged.