Identification and characterization of bacteria isolated from patients with cancer at Enugu State Teaching Hospital Parklane,

Cancer affects millions of people worldwide and contributes to the highest percentage of global deaths compared to other ailments. Most cancer sites are vulnerable to infection by a vast number of opportunistic pathogens . Data from several surveillance reports have revealed several opportunistic pathogens responsible for infections in cancer patients. The present study investigated the spectrum of bacteria isolated from acute cancer patients. Samples were recovered from urine, vaginal swab, and breast swab . Identification and characterization of the isolates were performed using standard microbiological methods. A total of 130 bacteria comprising 78(60%) gram-positive and 52(40%) gram-negative were recovered. A statistically significant difference (P<0.05) was observed between the two groups. The most prevalent organism was Staphylococcus spp. (42.3%) followed by Escherichia coli (36.2%), Lactobacillus spp. (8.5%), Micrococcus spp. (6.2%), Streptococcus spp. (3.1%), Klebsiella spp. (1.5%), Proteus spp. (1.5%) and Pseudomonas spp . (0.8%). Our findings showed the predominance of gram-positive bacteria in infections among cancer patients. However, Enterobacteriaceae ( E. coli ) was the most frequently isolated among the gram-negative. This study indicates that cancer patients may be infected by several opportunistic pathogens, highlighting an ongoing trend toward gram-positive organisms causing infection in cancer patients. Therefore, it underscores the importance of constant monitoring at regional levels as surveillance efforts are important to provide the clinicians with the appropriate information in choosing treatment regimens and implement a proper policy for infection control guidelines.


INTRODUCTION
Cancer is a lethal disease that is noncommunicable but can spread within the organs of the af f ected patients (Bernstein et al., 2013). Cancer is currently a major global public health threat and contributes to the highest percentage of global deaths compared to other ailments (Mof id et al., 2016). Currently, cancer is one of the highest causes of morbidity and mortality worldwide. It accounts f or the deaths of millions of people each year. As of 2018, the reported new cases and deaths as a result of cancer were 18.1 and 9.6 million, respectively (Bray et al., 2018). The most common cancers are those af f ecting the breast, lung, bronchus, prostate, rectum, melanoma, liver, cervical, and prostate (Jemal et al., 2011;Magalaes, 2013).
During aggressive therapy, inf ections due to bacteria and f ungi are very common. Currently, gram-negative and gram-positive bacteria are f ound to be opportunistic in cancer cells. This is broadly due to the use of immunosuppressive agents (chemotherapy and radiotherapy) that increase cancer patients' susceptibility to opportunistic inf ection (Rezaei-Tavirani et al., 2018). The current therapeutic strategies have helped increase survival rates (Sedighi et al, 2019). However, despite the availability of several antimicrobial agents (Kramer et al., 2018;Sedighi et al., 2019), bacterial inf ections in cancer patients persist as a critical challenge in medicine. In f act, inf ection due to multi-drug resistant bacteria (MDR) is currently a serious threat to global health (Cornejo-Juarez et al., 2015;Russell et al., 2018).
Generally, inf ection occurs in cancerous cells when there is a change in the microbiota of the cells. It can also occur when pathogens are introduced to the site. In addition, toxins and other damaging substances are of ten released into the sites. The release of these materials can enhanc e the growth of cancerous cells and f urther lead to a slow response to cancer treatment. Most inf ections in cancer patients are due to the invasion and prolif eration of f ungi ( In the present study, we sought to identif y and characterize the bacteria isolated f rom cancer patients. The goal was to understand the distribution and f requency of occurrence of dif f erent bacteria implicated in inf ection in cancer patients.

Ethical statement
This study was approved and carried out f ollowing the recommendations by the oncology unit of Enugu State University Teaching Hospital Parklane, Enugu, and the Faculty of Biological Science Ethical Review Board Committee, University of Nigeria, Nsukka. There was written inf ormed consent f rom all subjects.

Recruitment of cancer patients and sample collection
This study recruited dif f erent cancer patients (≥18 years old) prospectively. The patients consisted of those with the f ollowing cancer types: breast cancer, cervical cancer, placenta cancer, prostate cancer, uterine cancer, and vaginal cancer). Bef ore the commencement of the study, inf ormed consent was obtained f rom all patients. Clinical vaginal swab, breast swab, cervical swab, and urine samples were collected f rom all patients at the acute stage of cancer at the Oncology Unit of Enugu State University Teaching Hospital Parklane, Enugu. Thes e samples were collected by the hospital' laboratory technician and properly distinguished using standard operating procedures. Bef ore the sample collection, the vagina, vulva, and cervix of the uterus were thoroughly sterilized. Then, decidual tissue samples were collected by curettage with vacuum aspiration. Thereaf ter, blood clots were removed. The vagina isolates were collected by swabbing the vagina of the cancer patients. All the collected samples were immediately transported to the laboratory for f urther analysis.

Identification and characterization
The isolates were identif ied via microscopic examination, cultural characteristics, and biochemical f eatures. The appearance, microscopic and chemical evaluation of the urine were specif ically carried out. The isolates were cultivated on nutrient agar, blood agar (Oxoid, Basingstoke, UK), MacConkey agar (Biozyme Laboratories Ltd, San Diego, USA), chocolate agar (Oxoid, Hampshire, UK), and potato dextrose agar (PDA) (HiMedia, Mumbai, India) and incubated at 37 O C f or 24 h. The morphology (color, elevation, edge, and density) of the colonies was thoroughly evaluated. The isolates were f urther dif f erentiated into gram-positive and gram-negative through gram staining. Standard biochemical characterization (citrate utilization test, catalase test, urease test, and sugar f ermentation test) as described in the manual of clinical microbiology (Cheesbrough 2005;Ezeonu et al., 2011) was used in the f inal identif ication of the bacteria.

Statistical analysis
Chi-square test was used to determine whether there was a statistically signif icant dif ference in the distribution of the isolated bacterial species. Results with P< 0.05 were considered signif icant. The statistical analysis was done using the SPSS sof tware version 23.0 (SPSS Inc., Chicago, IL, USA).

RESULTS
All our samples were collected f rom patients who had cancer (breast cancer, cervical cancer, placenta cancer, prostate cancer, uterine cancer, and vaginal cancer) and admitted in the hospital during the time of the study. The presence of a high number of pus cells in the urine was indicative of bacterial inf ection. Budding yeast cells (small oval budding cells) were also observed. A total of 130 bacteria species comprising 78(60%) gram-positive and 52(40% ) gram-negative were recovered. We observed a statistically signif icant dif f erence (P<0.05) in the distribution of the gram-positive and gramnegative isolates. The recovered groups of organisms include Streptococcus spp, Staphylococcusspp., Klebsiella spp., Proteus spp, Escherichia coli, Pseudomonas spp, Lactobacillus spp, and Micrococcus spp. Staphylococcus spp (42%) was the most predominant bacteria in our study, f ollowed by Escherichia coli (36%) ( Table 1). Table 2 shows the distribution of gram-positive and gramnegative bacteria.
It is important to emphasize that microbes can produce toxigenic substances and even carcinogenic metabolites. These substances can trigger cancer-promoting inf lammation. They can also enhance the resistance of tumors to chemotherapeutic drugs and decreases the immune cells responsible f or f ighting against cancer. Aymeric et al. (2017) showed that bacteria could produce a specif ic bacteriocin. This toxin-like substance creates a suitable environment that promotes cancer. Thus, most bacteria associated with cancer are not necessarily the principal cause of cancer, but they are, however, auxiliary f actors enhancing the development of cancer. Thus, bacteria may be one of the key f actors responsible f or the development of cancer. The development of methods f or the detection of cancer-associated bacteria will be of help to oncologists (Bullman et al., 2017;Yu et al., 2017). Studies comparing healthy individuals and cancer patients are needed to conclude that these bacteria are involved in the initiation and development of cancer.
The inability to identif y the bacteria to their species level and the small sample size are major limitations in our study. This drawback makes our method likely unsuitable f or comparison of our results with those obtained f rom other researchers working on the association between bacteria and cancer. However, the reliability of our study is supported by the f act that our f indings are consistent with previous investigations reporting the characterization of bacteria isolated f rom cancer patients (Raad 2017;Fan et al., 2017;Seif u and Gubissa 2018;MergaDuf f a et al., 2018).

Conclusion
Inf ection due to bacteria is a common problem and one of the contributing f actors in deaths among cancer patients. These inf ections are caused by a variety of bacteria (gram-positive and gram-negative). The gram negatives in our study were mainly Enterobacteriaceae. It is possible these organisms are contributing to diseases seen in cancer patients. Theref ore, the signif icance of these organisms emphasizes the need to reduce bacterial inf ection and improve the health of cancer patients. Future work can look at comparing healthy individuals and cancer patients using larger sample size and also comparing the results with patients' demographic inf ormation. Finally, molecular characterization of the bacterial isolates f rom cancer patients is also needed f or a more accurate def inition of bacterial burden in cancer patients.