Molecular characterization and antibiotic sensitivity testing of bacteria in blood cultures of Hepatitis B virus infected subjects

Introduction: Hepatitis B virus is one of the most common infections worldwide. Many infected people are at risk of developing liver complications. Screening for common pathogenic bacterial infections that could contribute to complications is important for early diagnosis and appropriate management. Methods: A cross sectional study was carried out on subjects aged 20-75 years for a period of 6 months (November 2016 to April 2017). Blood cultures and HBsAg rapid tests were performed on all 122 blood samples collected in Ilorin Metropolis. The screening was carried out on 92 HbsAg positive patients who presented with fever, and 30 apparently healthy HbsAg positive donors from the blood bank. Results: Of 92 symptomatic patients, 44 (47.8%) had postive blood cultures and of the 30 HBV positive blood donors, 9 (30%) had positive blood cultures. The prevalence of Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa and Klebsiella pneumoniae in Hepatitis B positive subjects was 5.7% (n=7), 5.7% (n=7), 23.8% (n=29) and 9.8% (n=12) respectively. In the apparently healthy HbsAg positive blood donor group, only 9 samples showed positive bacterial growth of P. aeruginosa. All the bacterial isolates were resistant to amoxycillin-clavulanic acid, erythromycin, ceftriaxone, ceftazidime, cefuroxime, and ciprofloxacin. On PCR, Nuc, Stx2, Pf and PSUE genes were demostrated in E. coli, S. aureus, P. aeruginosa and K. pneumoniae respectively. Conclusion: This study showed a high percentage (45.1%) of bacteraemia in HBV infection. Early screening and treatment of HBV infection and concomitant bacterial infection is recommended to prevent complications.


Introduction
Many important questions still remain unanswered for many pathogen combinations. Co-infected individuals may be in worse health than those with single infections 1 and may also pose the biggest risk of transmission to others. 2 It is important to note that hepatitis is an occasional feature of the clinical symptoms induced by many agents of both living and non-living origin.
Hepatitis B virus (HBV) infection is an infection of major public health significance, being the 10 th leading cause of death globally. HBV infection accounts for 500,000 to 1.2 million deaths globally each year. 3 HBV infection can cause acute hepatitis, acute liver failure, chronic hepatitis, or can cause an asymptomatic infection and approximately 15-40% of infected patients will develop cirrhosis, liver failure or hepatocellular carcinoma. 4 Numerous studies have suggested that the genetic constitution of the host is a critical factor in determining the outcomes of HBV infection. Despite the effectiveness of the current vaccination policy, the prevalence of Hepatitis B infection remains high, and the burden for health services is considerable. 5 Blood stream infection by Gram negative bacteria is a common complication in patients with cirrhosis. Patients with cirrhosis and ascites showed a higher susceptibility to bacterial infections because of their inadequate defence mechanisms. 6 Very little is known about the correlation between HBV and different bacterial infections due to lack of diagnostic capability. The most common pathogenic agents, which enter the liver by vascular routes, are E. coli, K. pneumoniae, Salmonella Typhi, Proteus vulgaris, Streptococcus spp, and Staphylococcus spp, but anaerobes may also be present. 7 S. aureus, P. aeruginosa, and K. pneumoniae have been implicated in liver abscess. 8 P. aeruginosa is one of the most common causes of bacteraemia in liver transplant recipients. K. pneumoniae of high virulence (hvKP) has also been reported to lead to liver abscess in apparently healthy young adults. 9 Among cases of pyogenic liver abscesses, Staphylococcus aureus has been established as the leading cause in most series. 10 Uropathogenic E. coli (UPEC) has been reported to play a significant role in development of primary biliary cirrhosis (PBS). 6

Collection of samples and data
The study protocol was reviewed by the University Ethics Review Board of the Faculty of Life Sciences, University of Ilorin and approval to carry out research was obtained from the Ethics Review Committee of the State Ministry of Health. All subjects gave written informed consent and were assured that all information was confidential. The target population (with or without fever) were confirmed as HBsAg positive at the Kwara State Civil Service Hospital, Specialist Hospital Sobi, General Hospital Ilorin and Blood Banks Ilorin, Nigeria from November 2016 to May 2017. They were above 18 years and the subjects' socio-demographic information were collected by administering a questionnaire.
A total of 122 HBsAg positive blood samples from patients attending Kwara State Civil Service Hospital, Sobi Specialist Hospital, Ilorin General Hospital, and the Blood Bank, Ilorin, Nigeria were confirmed between November 2016 -May 2017 Ninety-two (92) of the blood samples were collected from HBsAg positive patients of Kwara State Civil Service Hospital, Sobi Specialist Hospital and Ilorin General Hospital. Thirty blood samples (30) were collected from apparently healthy HBsAg positive donors from the blood bank.
Seven ml of venous blood was taken aseptically by needle and syringe with 2ml dispensed in 18ml of Thioglycollate medium and 5ml into a plain bottle. The samples in plain bottles were left at room temperature for 20-30 minutes to clot, centrifuged at 3000rpm for 5 minutes, and the resultant clear serum samples were packaged and transported to the laboratory in the Department of Microbiology, University of Ilorin for serological analysis of HBV using HBsAg and HBcAg (LifeSpan BioSciences, inc) rapid testing kits. All samples were tansported in a cold box at +2 °C to +8 °C and kept at -20 °C in the laboratory for 18-24 hours.

Assay procedure for concomitant bacteria
Thioglycollate media samples were incubated for 7 days with intermittent sub-culturing on Blood Agar at 37 °C for 24hrs and examined for growth. Positive blood cultures were identified and subcultured on Blood Agar, MacConkey, Salmonella-Shigella Agar and Mannitol Salt Agar. Colonial and cellular morphology of the culture plates were observed, and necessary biochemical tests were carried out. 11

Antibiotics susceptibility test and ESBL screening
This was done according to Clinical Laboratory Standard Institute (CLSI) Guidelines. 12 Standard antimicrobial discs for Gram negative and Gram positive organisms was placed onto the surface of the inoculated agar plates accordingly and incubated for 24 hours. The zones of inhibition were measured and interpreted using the CLSI guidelines. ESBL screening was also performed by the disk synergy test. 13

DNA extraction and amplification of genes
Crude method was used for DNA extraction of the bacterial isolates and polymerase chain reaction (PCR) was carried out to detect Universal genes Nuc, Stx2, Pf and PSUE in isolated S. aureus, E.coli, Klebsiella spp. and Pseudomonas spp. respectively using the primers and cycling parameters listed in Table 1. PCR amplification of genes was carried out for each gene singly using geneAmp PCR system 9700 thermal cycler (Applied Biosystems). All PCR assays were performed directly from bacterial suspensions obtained after rapid DNA extraction method. For the amplification master mix, an aliquot of 2µl of the bacterial suspension was added to 23µl of PCR mixture containing 50mM KCl, 10mM Tris-HCl (pH 8.6), 1.5mM MgCl2, 5% glycerol, 0.08% NP-40, 0.05% Tween-20, 0.2mM of each deoxynucleoside triphosphate (dATP, dTTP, dGTP, and dCTP), 10µM of respective primers and 25 units/ml of Taq DNA polymerase. All PCR assay runs incorporated a reagent control (without template DNA).

Discussion
The severity of liver infection is likely to contribute to susceptibility to co-infection and bacterial infections can also contribute to the chronicity of disease in HBV patients. Bacterial infections are common in patients suffering with viral hepatitis and are critical for prognosis. 14 Other studies also have confirmed the association between bacterial infections and HBV and also bacterial infections in cirrhotic patients as an important cause of morbidity and mortality. 15 It is important to note that most of the patients in the current study were in their acute stage although liver function tests were not determined. In this study, the prevalence of bacterial infections in patients with HBsAg was determined as 45.1%.
The organisms isolated were P. aeruginosa, E. coli, K. pneumoniae, and S. aureus. P. aeruginosa was isolated most often in both patients and blood donors in this study which was similar to a study conducted in Korea. 16   Antibiotic susceptibility of E. coli isolates in the current study were similar to those previously reported. Kibret et al reported E. coli resistant to ciprofloxacin (79.6%) and gentamicin (71.4%) in a study in Ethiopia. 19 All E. coli isolates in the current study were resistant to ciprofloxacin, similar to the results of Cheong et al. 15 Ciprofloxacin resistance of S. aureus in the current study was 100%, similar to a study conducted in Korea which demonstrated very high resistance to ciprofloxacin and oxacillin. 11 Of the E. coli isolated in the current study, 42.9% were ESBL producers. Similar results have been reported from Pakistan (56.9%) 20 and India (40%). 13 Of K. pneumoniae isolated in the current study, 25% were ESBL producers. A similar result (33%) was obtained in a study in Iran 21 in contrast to an African study which reported 71.7%. 22 Of P. aeruginosa isolated in the current study, 24%were ESBL producers. These results were similar to other studies which reported 22% 11 and 13.79% 23 ESBL production. The ESBL producing P. aeruginosa isolates exhibited co-resistance against most of the antibiotics tested, consistent with results of other recent studies. 8,18 The high incidence of ESBL producers among the isolates in the current study has considerable health implications as shown in recent studies, where infection with ESBL producing Gram negative bacilli resulted in significantly higher fatality rates than those with non-ESBL isolates. 8

Conclusion
Bacteraemia with or without symptoms appear to occur in those demonstrated to be HBV positive. Further studies to follow up such patients and assess the significance of these bacteraemic episodes would be useful, particularly as a significant number of the isolated organisms showed antibiotic resistance to commonly used antibiotics.