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Abstract : Infertility is a serious emotional and social issue that affects people all over the world. Bacteriospermia is one of the possible causes of poor sperm quality. There is, however, no complete agreement on the premises' negative role in bacterial semen infection and male infertility. The study's goal was to examine semen specimens using the Vitek 2 Compact system after isolation of bacteria by the culture method, followed by confirmation utilizing molecular biology techniques such as polymerase chain reaction (PCR) to discover 16sr RNA gene bacteria in the semen and gene sequence. A total of 100 semen samples were collected from infertile men attending Kamal Al-Sammrai Hospital in Baghdad during the period from May 25, 2020, to October 15, 2020. Seminal bacterial diagnosis was done by the Vitek 2 Compact system after isolation of bacteria by the culture method, followed by molecular investigations including bacterial DNA extraction from more dominant pathogenic causes. Gene amplification was done for gene amplification, and gene sequencing was applied to confirm the presence of specific 16s rRNA gene amplification. The current study showed that 43 isolates were sterile and 57 cultures had bacterial growth isolates. A high percentage of positive bacterial cultures of infertile men occurred at age (20–30) and (30–40) at an equal percentage (40%). The positive bacterial culture group (57.0%) has (43.0% primary infertility and 14.0% secondary infertility), whereas the negative bacterial culture group (43.0%) has (32.0% primary infertility and 11.0% secondary infertility). There were 38 Gram positive cocci (38.00%) and 19 (19.00%) were Gram negative bacilli. The commonest isolates were Escherichia coli (19.78%), followed by Staphylococcus warneri (17.54%), Staphylococcus aureus (16.02%), Staphylococcus haemolyticus (12.28%), Staphylococcus saprophyticus (10.52%), and 3.50% each for Enterococcus faecalis, Enterobacter cloacae, and Klebsiella pneumonia. Finally, Streptococcus agalactiae, Raoultella ornithinolytica, and Pseudomonas aeruginosa each received 1.75%. Molecular analysis revealed that of the 16 isolated, 8 (50%) were compatible with bacterial isolates isolated by the Vitek 2 Compact system and 8 (50%) were different in bacterial species that were diagnosed by the Vitek 2 Compact system. The bacteria Citrobacter freundii strain bright 16S ribosomal RNA gene, partial sequence (Accession no. OM538425.1) isolated from infertile patients' sperm was recorded for the first time in GenBank. Escherichia coli were isolated to be the commonest organisms followed by Staphylococcus warneri and Staphylococcus aureus and molecular biology methods (PCR) aid in confirming the detection and identification of bacteria in semen specimens.

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