The current study sheds a light on microbiome of male and female cancer patients. The study conducted through biopsy samples suggested by Wolfe et al., 2012 was to present the actual picture of the microbiome. Therefore, 10 (BLC8- female sample excluded, since it failed to meet set criteria of sequencing) heterogeneous subjects were enrolled, including one control male sample. Accordingly, in data analysis, there were no statistically prominent differences observed in alpha and beta diversity compared on the basis of gender division in the samples. However, female samples have shown more number of OTUs as compared to male samples.
Presently, the composition of cancer patient’s bladder microbiome is studied through biopsy samples. While, previous studies have been conducted using urine (“clean-catch, urethral swab sample and midstream voided urine samples”) samples of healthy versus diseased heterogeneous population [20,21,22, 27]. Investigation on the urinary microbiome of subjects with different urological conditions such as urgency urinary incontinence, neurogenic bladder dysfunction, sexually transmitted infections had a high variation between samples based on their gender, health and different medical conditions [22, 33, 34, 35]. Similarly, studies on urinary microbiome have helped in better understanding of its association with diseases and it has been reported to have helped in the cure of diseases as well. For example, the recurrence of bladder cancer can be delayed with the help of Lactobacillus casei together with epirubicin, by reducing chronic inflammation . In concordance of such finding the present study was designed to detect the microbiome of heterogeneous populations with bladder cancer.
Urothelial bladder cancer is the major type of the urinary tract malignancy which accounts for 145,000 deaths annually. Smoking is the main cause of bladder cancer. Additionally, a diet rich in fat has also found to increase the chances of developing bladder cancer . The microbiome association and incidence of cancer has not clearly been known at present, but a recent report on bladder cancer has highlighted microbiome of cancer patients .
In current study, the main phyla observed were Firmicutes, Bacteroidetes, Proteobacteria, Fusobacteria , Actinobacteria, TM7 and Tenericutes which have been confirmed by earlier studies as well [20,21,38]. Control sample (BLS1) and the rest of the diseased samples showed the marked differences at phylum level, e.g. in all cancer patients, except BLC5, phylum Firmicutes was found to be dominant than Proteobacteria with exception in BLC5 (youngest patient). Since the only control sample is not enough to draw significant conclusions yet the data for diseased sample suggested that it might be a possibility that Firmicutes are increased in cancer state [38, 27]. It has been reported that in female samples, the abundance of Actinobacteria and Bacteroidetes was higher than males, the same trend observed in the current study with a few variations [20,21]. In general, “intra individual variations” were detected at the phylum level studies  which have been demonstrated in previous studies as well. This poses a problem in defining the core microbiome of the bladder.
The occurrence of Alphaproteobacteria, Betaproteobacteria, Gammaproteobacteria, Bacilli and Clostridia bacterial classes mentioned here have been reported previously with variable quantity based on overall health or disease state of subjects studied [22,39]. Moreover, the presence of order Clostridiales has been attributed to healthy vaginal flora where they are of clinical significance for their use in the diagnosis of bacterial vaginosis .
Bacilli were more abundant in tumor samples, while Clostridia in non-tumor samples , and with the same correlation were observed in the current study. Of note, it was observed that the Pseudomonas or Anerococcus was found to be more abundant in samples where Streptococcus is low and vice versa. The same trend was observed in the current study wherein female sample BLC6, Anerococcus have shown a maximum abundance (19.6%) while Streptococcus showed the lowest abundance (0.1%), and inverse case was observed in male sample BLC3 in which Anerococcus was 0.2 % and Streptococcus was 6.1% . In various studies, the presence of Streptococcus associated with commensal flora of healthy males [23, 33]. However, it has found in cancerous and non-cancerous samples in the present study (except BLC2) and Xu et al . It was suggested that the cancer might have a role to play in altering the composition of urinary microbiome . Moreover, Anaerococcus has shown an unusual pattern of occurrence in male samples as it was absent in most of the males in this study while in BLC 10 (18.8%) its abundance is next to BLC6 female sample.
In our study Lactobacillus was found to be present in all samples, except BLC6 and 10, ranging between 0.1- 3.3%. The presence of Lactobacillus has been reported in earlier studies carried on healthy and one undergo urogynecological surgery females, and in a male study [41, 21]; these studies have confirmed the abundance of Lactobacillus more in healthy female than males [42, 43, 17]. Also, the abundance of Lactobacillus was found to decrease in disease state like in patients with interstitial cystitis as compared to healthy subjects [21,33]. In this study the Lactobacillus was found to be more abundant in two females than male, but was absent in two diseased samples including one female (BLC6 and BLC10).
Staphylococcus has been reported in previous studies conducted on both females (with and without overactive bladder)  and male (with a sexually transmitted infection) . In current finding Staphylococcus ranged between 0.1-2.4% in all subjects except BLC6 and 10. Peptostreptococcus, commonly found in human commensal flora has shown the highest abundance in BLC10 (22.2%) while zero abundance in the control sample. The same trend has been reported in a former study . Another study conducted on a heterogeneous population confirmed its presence in females between 50-69 years of age, while in the case of males, it was found to be present at age 70 or above . The same trend was observed for females in the present study: BLC6 (21.4%) abundance observed, followed by BLC4 (0.1%) while BLC5 the youngest female has shown zero abundance for Peptostreptococcus. On the other side, in male samples maximum enrichment of 22.2% found in BLC10 (53 years) and 0.1% in BLC3 (50 years) which is not in accordance with Lewis (2013) studies except sample BLC2 (0.1%) of 72 years .
In the present study sequences belonging to different genera: unclassified_Enterobacteria, Acinetobacter, Methylotenera, Rolstonia, Paracoccus, Burkholderia, other_Enterobacteriaceae, Pseudomonas and unclassified_Methylobacteriaceae. The presence of these genera has been reported in previous studies in bladder through different sampling procedures [32, 42, 39].
A recent report in Nature Journal, Fusobacterium, Actinobaculum, Facklamia and Campylobacer were abundant in male samples while Veillonella, Streptococcus and Corynebacterium in healthy samples. Conversely, in this study, these genera were absent, only Fusobacterium was found in two male samples (BLC3 and 9)  whereas only Corynebacterium detected in control sample among the rest of genera mentioned in a report of Nature. Currently, the sequences belonging to Acinetobacter were detected in all samples (except BLC6 and 10) ranging from 0.3 to 22.6%. The Acinetobacter is declared to be a most abundant genus in healthy and urothelial carcinoma subjects in a previous study . The sequences belonging to unclassified_Enterobacteriaceae present in all samples have also been reported by another study on a heterogeneous population (Order: Enterobacteriales) as the most abundant taxa .
Paracoccus in the present study was detected in all subjects except BLC7, ranging as 0.1-3.6%. Its presence has been reported in the human skin microbiome . Pseudomonas was detected in our study in all samples except these: BLC6, 7 and 10. Its presence has been reported in an urothelial carcinoma study earlier  and in overactive bladder female samples . Methylotenera detected in all samples in the present study ranged between 0.1- 3.3% had been previously detected in a study on peritoneal tumors in humans along with multiple other genera .
The sequences belonging to genus Ralstonia were found in all samples except BLC10, ranging from 0.1- 13.9%. Rolstonia are categorized as human opportunistic pathogens that play vital role in degradation of xenobiotics and recalcitrant compounds . The occurrence of Ralstonia in the bladder has been reported previously [32, 21]. Presently, Burkholderia was detected in all samples except in BLC6, 7 and 10, ranging between 0.1- 1%. The presence of Burkholderia in the current study is in accordance with previous study where it occurred as a low abundant genus .
The sequences belonging to genus Meiothermus were detected in all samples ranging from 0.3-22%. The member of this genus was previously placed in Thermus. In 1996, new genus named Meiothermus was introduced. They belong from Gram-negative aerobic microbes that are able to make short filaments and normally found in thermal vents [47, 48]. It is noteworthy that Meiothermus has not been previously reported in the urine sample, but it was detected in all samples in the present study.
Additionally, Ureaplasma were found to be present in BLC6 (11.8%) and BLC10 (10.9%) only and other_Bacillaceae were detected in a range of 0.1-1.1% in all samples except BLC10. While Lysobacter were found to be present in all female samples and a male sample (BLC9), ranging from 0.1-0.5%. The presence of anaerobic and uncultivated fastidious bacteria, such as Ureaplasma has been confirmed in different studies on both, male and female bladder studies [25,21,43,32]. The presence of genus Corynebacterium found in the present study was low, ranging from 0.1-1.3%. Its presence has been reported in healthy male samples in different studies, confirming the current findings, e.g., high range (1.3%) was observed in the male control sample (BLS1). Previous findings suggested that Corynebacterium is expected to have a role in “the healthy urine microbiome” [21, 33].
In our study, the OTUs observed in both sexes through non-parametric estimation used to make rarefaction curves have shown more richness and evenness of bacterial diversity in females as compared to males (Figs. 5). There is no statistically significant differences were observed in the microbial profile of both sexes in current study, which is in concordance with previous studies . Overall, beta diversity calculations showed insignificant differences. However, based on clustering together, it was observed that BLS1 (control sample) and BLC5, and BLC3 and BLC4 were clustered with each other and showed similarity among themselves. Likewise, BLC6 and BLC10 have depicted close proximity and same number of genera as well. While BLC2, BLC7 and BLC9 were not clustered with any other sample and represents dissimilarity with the rest of the samples. Variations between samples on the basis of gender have shown that BLC2 and BLC7 have marked variation with female samples as they were not clustered with any female sample.