Molecular Characterization and Antibiogram Profiling of Multidrug Resistant Staphylococcus haemolyticus Isolated from Patients with Urinary Tract Infection in Bangladesh

Research Article

J Bacteriol Mycol. 2021; 8(2): 1166.

Molecular Characterization and Antibiogram Profiling of Multidrug Resistant Staphylococcus haemolyticus Isolated from Patients with Urinary Tract Infection in Bangladesh

Haque MH1*, Miah ML1, Sarker S2, Shamsuzzaman M3 and Shiddiky MJA4

1Department of Veterinary and Animal Sciences, Faculty of Agriculture, Rajshahi University, Rajshahi-6205, Bangladesh

2Department of Physiology, Anatomy and Microbiology, School of Life Sciences, La Trobe University, Melbourne VIC 3086, Australia

3Specialty doctor in Acute Medicine, Leighton Hospital, Middlewich Rd, United Kingdom

4School of Environment and Science (ESC), and Queensland Micro- and Nanotechnology Centre (QMNC) Griffith University, Nathan Campus, 170 Kessels Road, QLD 4111, Australia

*Corresponding author: HaqueMd. Hakimul, Department of Veterinary and Animal Sciences, Faculty of Agriculture, Rajshahi University, Rajshahi-6205, Bangladesh

Received: January 25, 2021; Accepted: March 09, 2021 Published: March 16, 2021


The emergence of antibiotic-resistant bacteria in human is a potential global public health concern. Profiling of antibiotic-resistant bacteria with their antimicrobial susceptibility patterns from Urinary Tract Infections (UTIs) is crucial to guide antibiotic therapy. Herein we report a detailed bacteriological and molecular analysis of Staphylococcus haemolyticus and their antibiogram typing from UTIs. A total of 100 human urine samples of patients with UTIs were collected between January and December 2019 and were subjected to the conventional characterization of bacteria using the standard protocol. Molecular characterization was performed via sequencing followed by phylogenetic analysis. All bacterial strains were examined against thirteen commonly used antibiotics for susceptibility using the Kirby-Bauer disk diffusion method. The overall prevalence of S. haemolyticus in UTI was 26% with female patients having a higher prevalence of UTI (21 out of 26 or 80.76%) than male patients (5 out of 26 or 19.24%). The isolated S. haemolyticus showed 100%, 100%, 88.46%%, 76.93%, 73.08% and 65.39% resistant to penicillin, ampicillin, amoxicillin, erythromycin, ciprofloxacin and tetracycline, respectively. Importantly, S. haemolyticus demonstrated the highest sensitivity to vancomycin (100%), followed by azithromycin (80.76%), amikacin (84.61%), gentamycin (69.23%), levofloxacin (73.08%), ceftriaxone (80.76%) and doxycycline (61.54%). Overall, six variations were noted in S. haemolyticus in which most (5/6) modifications were substitutions and one (1/6) was deletion. These findings imply that mutations in the 16S rRNA gene sequence are the dominant source for species identification and variation in the drug sensitivity pattern against the S. haemolyticus. Phylogenetic analysis of the resultant 16S rRNA indicated that the isolated S. haemolyticus in this study belonged to genus Staphylococcus, but was different from the rest of the available S. haemolyticus isolates in other countries. Multidrug-resistant pathogenic S. hemolyticus is commonly found in urine samples of UTI in human in Bangladesh, which warrants a one-health approach for controlling this emerging ailment.

Keywords: Multidrug-resistance; Staphylococcus hemolyticus; Urinary tract infection; Antibiotic-resistant bacteria; Antimicrobial susceptibility


Antimicrobial Resistance (AMR) is a global public, animal and poultry health ailment, which results from injudicious use of antimicrobial agents in all sectors since the early 1940s [1]. Globally, increasing incidence rate of AMR towards several antimicrobial groups has been reported in various bacterial pathogens [2]. Indeed, Urinary Tract Infection (UTI) is a chronic health problem caused by a variety of bacterial species affecting millions of individuals worldwide, including Bangladesh [3]. UTIs affect people in all age groups and sex, and are diagnosed in both hospitalized and unhospitalized patients. UTIs easily influence women than men due to the anatomical structure of their genitourinary tract. One-third of all women suffer from UTI at some point during their lifetime [4]. This type of infection causes a severe socioeconomic burden on affected individuals and leads a high rate of all types of antibacterial drug usages [5]. Importantly, the risk factors in the pathogenicity of UTI varies among countries owing to geography variation and antibiotic use. Furthermore, the rapid onset of antibiotic therapy with broad-spectrum antibiotics is crucial to treatment achievement. Still, the repeated antibiotic use often ensues in the emergence of antibiotic-resistant bacteria. The rate of antimicrobial resistance to antibiotics among community-acquired UTIs is increasing and shows significant geographical variations [6]. Deep understanding of the diverse etiology of UTIs and the resistance pattern against antibiotics of the causative organisms is crucial to physicians while managing such patients.

Urinary tract infections in human are caused by a wide range of Gram-negative and Gram-positive bacterial species, but most commonly by Escherichia coli, Klebsiella pneumoniae, Pseudomonas spp., Proteus mirabilis, Citrobacter spp., Enterococcus faecalis, and coagulase-negative staphylococci [7,8]. Staphylococus haemolyticus is a coagulase-negative bacterium and a member of the genus Staphylococcus. Although coagulase-negative staphylococci represent significant causative microorganisms in nosocomial infections, they typically account for fewer than 10% of all UTIs [9]. Most of these coagulase-negative staphylococcal UTIs comprise of the three species, namely Staphylococcus epidermidis, Staphylococcus haemolyticus and Staphylococcus saprophyticus. S. haemolyticus is commonly present on the human skin and can be isolated from axillae, perineum, and inguinal areas of humans and is dismissed as culture contaminants, for example in urine cultures [10]. This opportunistic bacterium species colonize the urethra or periurethral of males and females. S. haemolyticus with other coagulase-negative staphylococcal species often exceed those of Gram-negative bacilli, which cause 80% of all UTIs [11]. Staphylococcus haemolyticus present an average of 10% of clinical coagulase-negative staphylococcal isolates in infections [12]. Strains of S. haemolyticus produce a haemolysin, cytolysin, and enterotoxin, and are frequently resistant to antibiotic [13]. Species determinations of coagulase-negative staphylococci are not typically done in most clinical microbiology laboratories. Therefore, the mechanisms of antimicrobial resistance in S. haemolyticus and the extent to which it shares resistance genes with other staphylococci are also unknown. The possibility of unique arrangements of resistance in this poorly understood species led us to investigate its characteristics further.

Besides, in developing countries, antibiotics can be bought by the public from various sources including hospitals and pharmacies, licensed medicine stalls, and drugstores, roadside stalls and peddlers. Despite restrictive laws, antibiotics can be purchased by people without a prescription. This widespread availability has resulted in inappropriate use of antibiotics by patients, pharmacists, health-care providers and public themselves. Furthermore, antibiotic therapy is mainly empirical due to the relative lack of appropriate laboratory facilities for culture and susceptibility testing of bacteria. The heavy use of antibiotics and the absence of susceptibility testing have led to a steady increase in antimicrobial drug resistance. In addition, humans may be exposed to antibiotics indirectly, i.e. through food, water and herbal medicines. The antimicrobial agents are also used in livestock to treat, prevent, and control diseases and to enhance feed efficiency and weight gain. Like human medicine, the use of antimicrobial agents in veterinary medicine creates selective pressure for the emergence and dissemination of antimicrobial-resistant bacteria in livestock. These resistant bacteria can usually be transmitted to humans either by direct contact with animals or through the food. In this way, Staphylococcus haemolyticus could receive various resistance genes through the same species of staphylococci or other bacteria. However, people in developing countries are more prone to be affected due to the inappropriate use of antimicrobial agents, nonhuman antibiotic use, poor quality of drugs, inadequate surveillance, and factors associated with individual and national poverty including poor health-care standards, malnutrition, chronic and repeated infections, and unaffordability of more effective and costly drugs [14].

Increasing antimicrobial resistance poses a regional and global threat to the developing country of South Asia, including Bangladesh. It was previously reported that the widespread availability of antimicrobials without prescription in Bangladesh is responsible for the habit of self-medication among patients, and the indiscriminate utilization of antibiotics in humans and food animals and fisheries, and consequently for spreading resistant strains through environmental contaminations [5]. In addition, very few studies investigated coagulase-negative staphylococci bacteria associated with UTI in Bangladesh. However, no study has investigated the isolation, molecular detection, and antibiogram profiling of multidrug-resistant S. haemolyticas isolated from human urine in Bangladesh. The present study was therefore conducted for the first time to isolate, characterize and determine antimicrobial resistant of S. haemolyticas isolated from the urine of UTI in Bangladesh using molecular techniques.

Materials and Methods

Collection of samples

A total of 100 patients with urinary tract infection have been recruited randomly without any bias from a private diagnostic centre, Bangladesh during the period from January to December 2019. Informed consent was taken from all patients. Clean-catch midstream morning urine samples were collected in sterile widemouth glass containers using the standard protocol. The samples were then transported immediately to the Department of Veterinary and Animal Sciences, Rajshahi University, for bacteriological analysis while maintaining sterile and cold chain conditions. The time between sample collection and sample analysis did not go beyond one hour. Clinical and pathological parameters were documented for each patient, including age, sex, colour, and appearance of urine, presence of blood or pus, pH, marital status, pregnancy, residence, UTI acquired from a hospital or as an outpatient and season. Ethical permission was obtained from the Institutional Animal, Medical Ethics, Biosafety, and Biosecurity Committee (IAMEBBC) of Institute of Biological Science (IBSc), the University of Rajshahi for experimentations on the animal, human, Microbes and living natural sources (Memo no:144/320/I.A.M.E.B.B.C./IBSc). All methods were performed following relevant guidelines and regulations.

Isolation and identification of Staphylococcus spp

Initially, a sterile loopful sample was used to inoculate into the nutrient broth, followed by culturing on different selective media such as blood agar, soybean casein digest agar and mannitol salt agar to observe specific colony characteristics. The broth and agar plates were incubated overnight at 37°C aerobically. The colony was counted to measure the significant growth of bacteria. Gram staining was performed from discrete colonies, and further subculturing was done to obtain pure cultures. The morphological and biochemical characterization of Staphylococcus haemolyticus was done through carbohydrate fermentation test, catalase test, coagulase test, Methyl Red test, Voges-Proskauer test, indole test and reaction in TSI agar tests, as previously described [15,16].

Extraction of genomic DNA

Bacterial genomic DNA was extracted from pure cultures of Staphylococcus haemolyticus by the boiling method as described previously by Mahmud et al. [17]. Briefly, initially 200 μL deionized ultrapure water was added into an Eppendorf tube. A loop full pure bacterial colony of Staphylococcus haemolyticus from the overnight culture grown at 37°C on mannitol salt agar plate and mixed gently to make a homogenous cell suspension. Then the tube was transferred into boiling water and incubated for 10 min, transferred immediately into ice for a cold shock for about 10min. Finally, the tubes with bacterial cell suspension were centrifuged at 10,000 rpm for 10 min. 100 μL of supernatant containing bacterial DNA from each tube was collected. The quality and quantity of purified DNA were checked via Nanodrop Spectrophotometer (BioLab, Ipswich, MA, USA), and the DNA was stored at -20°C until use.

Polymerase Chain Reaction (PCR)

All isolates were confirmed as Staphylococcus haemolyticus using a previously established PCR method and a set of designed primers (Sense27F, 5'-AGAGTTTGATCMTGGCTCAG-3' and antisense1492R, 5'-CGGTTACCTTGTTACGACTT-3') targeted for 16S rRNA gene [18]. PCR reaction was achieved in a total volume of 20 μL reaction mixture comprising 10 μL of Hot Start Green Master Mix (Promega, USA), 1 μL of every ten picomoles/μL primer, 1 μL of bacterial genomic DNA at 50 ng/μL and 7 μL of Nuclease-free water. Thermal cycling consisted of initial denaturation at 95°C for 3 min followed by 35 cycles of 95°C for 30 seconds (denaturation), 48°C for 30 seconds (annealing), and 72°C for 90 seconds (extension) and a final extension at 72°C for 5 min. Amplified products were analyzed by electrophoresis in 1.5% agarose gel. Ethidium bromide was used, and PCR products were visualized under ultraviolet transilluminator (Biometra, Germany). The 1 KB DNA ladder (Thermo Fisher Scientific, MA, USA) was used as molecular weight markers.

Purification of PCR products and sequencing

Successfully amplified specific PCR bands were cut and purified according to the manufacturer’s protocols from the NucleoSpin Gel and PCR Clean-up kit (Macherey- Nagel, Bethlehem, PA, USA). The purified DNA was mixed with the primer (10-40 ng of DNA + 1 μL of 3.2 pmol primers in 10 μL of H2O) and sequenced by Sanger sequencing to further confirm the detected Staphylococcus haemolyticus. The Sanger sequencing was performed and analyzed using an ABI PRISM 3730×l Capillary sequencer (Applied Biosystems, USA) under standardized cycling PCR conditions. The sequences were trimmed for primers, aligned to construct contigs using a minimum overlap of 35 bp and a minimum match percentage of 95%, and the construction of consensus sequence was carried out in Geneious 10.2.2 (Biomatters, New Zealand).

Nucleotide Sequence Accession Numbers

The nucleotide base sequences of the gene 16S rRNA reported in this paper were submitted to the GenBank using the National Centre for Biotechnology Information (NCBI, Bethesda, /MD, USA) under the accession numbers MT622589 and MT622590, respectively.

Phylogenetic tree analysis

The base sequence of the PCR product was matched and aligned with known 16S ribosomal RNA gene sequences in the same genus, which was randomly selected from the GenBank database via multiple sequence alignment. Also, the 16S rRNA gene sequences of the isolates of the same species from 14 different countries, and at least two strains of each reference genera with their GenBank accession numbers were recorded for phylogenetic analysis including Escherichia, Shigella, Salmonella, Citrobacter, Yersinia, Acinetobacter, Pseudomonas, Legionella, Bartonella, Ochrobactrum, Streptomyces and Staphylococcus. The evolutionary relationship of the bacterial isolates analyzed was inferred using the neighbourjoining method [19]. The evolutionary distances were computed by the Maximum Composite Likelihood method as the number of base substitutions per site [20]. All steps involved in evolutionary analysis were conducted in MEGA6 software.

Antimicrobial Susceptibility Testing

The Kirby-Bauer disk diffusion method was used to determine the antibiogram phenotype against thirteen commonly used antibiotics classes, namelyfluoroquinolones (levofloxacin-5 μg), tetracycline (doxycycline-30 μg), aminoglycoside (Amikacin-30 μg), glycopeptides (Vancomycin-30 μg), cephalosporin (ceftriaxone-30 μg), macrolides (erythromycin-15 μg), macrolides (azithromycin-15 μg), aminoglycoside (gentamycin -10 μg), penicillin (amoxicillin -30 μg), penicillin (penicillin -30 μg), penicillin (ampicillin -25 μg), fluoroquinolones (ciprofloxacin-5 μg) and tetracycline (tetracycline-30 μg) (Hudzicki, 2009). The antimicrobial susceptibility test by the disk diffusion method was done on Mueller-Hinton agar (Hi-Media, India) plates with a concentration of bacteria equivalent to 0.5 McFarland standard and aerobic incubation at 37°C for 18- 24 h. Vancomycin screen agar (Mueller-Hinton agar with six μg/ ml of vancomycin) was used to determine its minimum inhibitory concentration. The results of the antimicrobial susceptibility profiling were documented as sensitive or resistant based on the diameters of the zones of inhibition as per the guidelines provided by the European Committee on Antimicrobial Susceptibility Testing (EUCAST) [21]. Staphylococcus haemolyticus isolates found to be resistant to at least three classes of antibiotics were categorized as multidrug resistance (MDR) [22].

Statistical analysis

All data were incorporated in the Excel sheet (Microsoft-2010) and analyzed by SPSS software version-24 (IBM Corp., Armonk, NY, USA). Descriptive analysis was performed to calculate prevalence. Chi-square (Χ2) test was done to evaluate the significant relationship of the clinicopathological parameter with the bacterial isolates and the difference among proportions of antibiotic resistance. A p-value <0.05 was considered significant.


Identification of Staphylococcus haemolyticus in human urine

Among the 100 samples bacteriologically analyzed, a total of 26 (26%) was found to be positive for the presence of Staphylococcus haemolyticus from human urine samples collected from patients with UTI. This is evidenced by the isolation on selective media, followed by identification via Gram staining, and biochemical tests. Cultural, microscopic, and various biochemical methods for positive growth of coagulase-negative S. haemolyticus are presented in Table S1 and Figures S1, S2. Of these 26 positive cases, 5 (19.24%) were from male and the rest 21 (80.76%) were from female. This result indicated that female patients had a higher prevalence of Staphylococcus haemolyticus in UTI than in males. As can be seen in Table S2, the most susceptible age group of patients to UTI was 31-45 years (42.30%) followed by 16-30 years (26.92%), 46-60 years (19.23%), > 60 years (7.70%) and 0-15 years (3.85%) (Figure 1). This study suggests that UTI is overall common in the age group between 16-60 years. The bacterial isolates of S. haemolyticus in UTI showed no significant statistical correlations with the clinicopathological parameters of the patients (p>0.05).