Phenotypic and genotypic evaluation of antibiotic resistance and molecular characterization of Streptococcus species isolated from hospital cockroaches

The present investigation aimed to assess the antibiotic resistance properties and distribution of virulence factors in the Streptococcus spp. isolated from hospital cockroaches.


Abstract Background
The present investigation aimed to assess the antibiotic resistance properties and distribution of virulence factors in the Streptococcus spp. isolated from hospital cockroaches.

Methods
Six-hundred and sixty cockroach samples were collected. Cockroaches were vigorously washed with normal saline, and the achieved saline was used for bacterial culture. Isolated Streptococcus spp. were subjected to disk diffusion as well as PCR ampli cation of virulence factors and antibiotic resistance genes.

Conclusion
To the best of our knowledge, this is the rst prevalence report of virulence factors and antibiotic resistance genes in the Streptococcal spp. isolated from American, German and oriental hospital cockroaches. Findings recommended a certain role for cockroaches in the transmission of nosocomial infections and particularly those caused by virulent and resistant Streptococcus spp. in the hospital environment.

Background
Cockroaches are considered among the most common pests in numerous homes and public places such as hospitals, hotels, bughouses, boarding schools, barracks, kindergartens and dorms. Pest cockroaches are in close contact with human [1].Originally, they are tropical; however, in the temperate zones, most species live in parts of houses and other places where moisture, warmth, and food are adequate. Among over 3,500 recognized species, only few ones are important to human, including Blattella germanica (German cockroach), Periplaneta americana (American cockroach) and Blatta orientalis (Oriental cockroach) [2][3][4][5][6].
Cockroaches easily move from buildings, gardens, drains, sewers and latrines to human habitations. Since they feed on human food and feces, they can spread several types of pathogenic microorganisms. Likewise, several epidemiological investigations indicated that cockroaches were one of the main sources of different types of dangerous bacteria such as Shigella dysenteriae, Salmonella typhi, Streptococcus species (spp.), Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa [2][3][4][5][6].
According to the high presence of cockroaches in the hospital environment as well as their signi cant importance as risk factors for maintenance and transmission of pathogenic bacteria, the present investigation was conducted to study the prevalence rate, distribution of virulence factors and antimicrobial resistance properties of S. pneumoniae, S. pyogenes and S. agalactiae strains in German, American and Oriental cockroaches of Iranian hospitals. Table 2 presents the total distribution of Streptococcus spp. isolated from different types of hospital cockroaches. Prevalence of S. pyogenes, S. agalactiae and S. pneumonia strains among all the studied samples was 4.82%, 1.66% and 6.96%, respectively. The highest prevalence of S. pyogenes, S. agalactiae and S. pneumonia strains was found in oriental cockroaches (5.71%), oriental cockroaches (2.85%) and American cockroaches (7.71%), respectively. Statistical signi cant difference was found between types of cockroaches and prevalence of Streptococcus spp. (P < 0.05). Table 3 shows the distribution of streptococcal putative virulence factors in different types of studied cockroaches. We found that cfb (53.93%), cyl (52.8%), scaa (51.68%) and glna (50.56%) were the most commonly detected streptococcal virulence factors. The highest prevalence of Streptococcal spp. was found in German cockroaches. Statistical signi cant difference was found between types of cockroaches and prevalence of virulence factors (P < 0.05). Table 4 depicts the distribution of penicillin, macrolides, tetracyclines and streptogramins antibiotic resistance genes in the Streptococcus spp. isolated from different types of hospital cockroaches. We found that pbp2b (71.91%), pbp2 × (58.42%), mefA (46.06%), ermB (46.06%) and tetM (46.06%) were the most commonly detected antibiotic resistance genes in the Streptococcal spp. isolated from different types of hospital cockroaches. TetO (6.74%), DF-L22 (21.34%) and DF-L4 (24.71%) had the lowest prevalence in the studied antibiotic resistance genes. Statistical signi cant difference was found between types of cockroaches and prevalence of antibiotic resistance genes (P < 0.05).

Discussion
Medically, cockroaches are much more important than generally realized as they have been demonstrated to harbor some pathogenic and non-pathogenic microorganisms. Since various workers have reported the isolation of various human pathogens from these insects, cockroaches are known vectors of human enteropathogens. Their lthy and nocturnal habits cause them to be ideal carriers for transmitting numerous pathogenic microorganisms. Klebsiella spp., E. coli, P. aeruginosa, Streptococcus spp. and some other potential pathogens have been isolated from cockroaches gathered from hospitals [3][4][5][6].
The present investigation was conducted to assess the prevalence rate, antibiotic resistance pattern and genotyping evaluation of antibiotic resistance and virulence factor of the Streptococcus spp. isolated from American, German, Oriental and other species of hospital cockroaches. We found that S. pneumoniae had the highest prevalence among the studied cockroaches (6.96%). Oriental cockroaches had the highest prevalence of S. pyogenes (5.71%) and S. agalactiae (2.85%), while German cockroaches had the highest prevalence of S. pneumoniae (6.83%). Presence of hospital cockroaches in different parts of hospitals, and sewage system caused high prevalence of Streptococcus spp. Although American cockroaches harbored considerable prevalence of bacteria, this species was more often gathered in kitchens, restaurants and supply rooms. In the hospital environment, this distribution, as a vector for nosocomial infections, may reduce its potential. German cockroaches can be a more signi cant potential vector for nosocomial infections, as they were more commonly found in nursing stations, outpatient rooms, registration rooms, wards, medical record storage rooms as well as drug storage rooms. Various studies have been carried out in this eld. For example, Fotedar et al. (1991) [18] indicated that one hundred and fty-eight out of 159 (99 − 4%) cockroaches gathered from hospital (test) and 113 out of 120 (94 − 2%) cockroaches gathered from residential areas (control) carried medically signi cant microorganisms. They indicated that 10-20% of cockroaches harbored Streptococcus spp.. Kassiri et al. (2014) [19] disclosed that culturing outer surface wash of cockroaches resulted in the isolation of Klebsiella, Pseudomonas, E. coli, Staphylococcus, Proteus and Streptococcus. The main common bacteria were Klebsiella (35%) and Pseudomonas (30%). Elgderi et al. (2006) [20] indicated that 27 and 25 species of potential pathogen were isolated from the hospital and household cockroaches, respectively, with Klebsiella, Enterobacter, Serratia and Streptococcus being predominant. Salehzadeh et al. (2007) [21] demonstrated that 130 out of 133 (98%) German cockroaches had contamination with high bacterial load (more than 1 × 10 3 ). Enterobacter (22.60%), Klebsiela (21%), Enterococcus (17.30%), Staphylococcus (16.50%), E. coli and Streptococcus (8.3%), Pseudomonas (3%), as well as Shigella, Haemophilus and group A ß-hemolytic Streptococcus (less than 1%) were the most commonly detected bacteria. Pai et al. (2004) [5] revealed that the prevalence of Streptococcus spp. in the intestinal content and surface of American and German cockroaches were 38.10% and 38.80% and 32.80% and 17.20%, respectively. Similar ndings were achieved in the studies conducted in Iran [22,23], Thailand [24] and Brazil [25].
Results of our investigation indicated that the Streptococcus spp. strains isolated from cockroaches harbored the high prevalence of resistance against commonly used antibiotic, particularly tetracycline, trimethoprim, enro oxacin, erythromycin, lincomycin and penicillin. The ndings demonstrate the antibiotic resistance seriousness of the common pathogenic bacteria in Iran. A boost prevalence of antibiotic resistance was also reported in the pathogenic bacteria in the hospitals of Taiwan [26]. More than 30% of S. pneumoniae, S. aureus, Enterobacteriaceae, P. aeruginosa, Acinetobacter baumannii, Haemophilus in uenzae, coagulasenegative staphylococci, beta-hemolytic streptococci, viridans streptococci, and enterococcal isolates of Taiwanese hospitals were resistant to different antibiotics [26]. Pai et al. (2004) [5] reported that all of the species of common pathogenic bacteria (Streptococcus spp. S. aureus and P. aeruginosa) isolated from cockroaches harbored resistance against ampicillin, chloramphenicol, tetracycline, trimethoprim and sulfamethoxazole. Bouamama et al. (2010) [27] reported that pathogenic bacterial strains isolated from American cockroaches in Spain harbored the high prevalence of resistance against ampicillin, amoxicillinclavulanate, cefoxitin; gentamicin, cotrimoxazole and cipro oxacin antibiotics. Hammad and Mahdy (2012) [28] reported the high prevalence of antibiotic resistance of Streptococcus spp. isolated from cockroaches against ampicillin, cephalothin, chloramphenicol, cipro oxacin, gentamycin, nalidixic acid, tetracycline, trimethoprim and sulfamethoxazole. Different patterns of antibiotic resistance of pathogenic bacterial strains isolated from cockroaches have been reported from Bangladesh [29], Nigeria [30], and India [31]. Such differences in the prevalence of antibiotic resistance reported in different study may be due to the differences in the idea of medical practitioners in antibiotic prescription, availability and expense of antibiotics and nally laws of various countries for antibiotic prescription. Furthermore, high prevalence of antibiotic resistance reported in the present study may be due to the irregular and unauthorized prescription of antibiotics. Phenotypic pattern of antibiotic resistance was supported by the genotypic pro le of antibiotic resistance genes. We found that the genes encoding resistance against penicllins (pbp), tetracyclines (tetK, tetM, tetO and tetL), macrolides (erm and mef), streptogramins A and B (rplV), and the lytA gene had considerable prevalence in the Streptococcus spp. strains isolated from hospital cockroaches. To the best of our knowledge, there existed no previously published data in this led all around the world. High prevalence of pbp, tetK, tetM, tetO, tetL, erm, mef, rplV and lytA antibiotic resistance genes was reported in the Streptococcus spp. strains isolated from different hospital infections [32][33][34]. Kargar et al. (2012) [35] reported the high prevalence of ermB, mefA, pbp1a, pbp2b and pbp2x genes in the S. pneumonia strains isolated from different types of the hospital infections of hospitalized patients in Intensive Care Unit (ICU) centers. Presence of these genes in the Streptococcus spp. caused their severe resistance against some speci c antibiotics. Our ndings were also disclosed higher incidence of phenotypic pro le of resistance to some antibiotic agents than genotypic pro le. This nding is maybe owing to the fact that presence of antibiotic resistance genes is one of the known procedures for occurrence of antibiotic resistance in bacteria. In the other hand, higher incidence of phenotypic resistance toward antibiotics may support by procedures other than presence of antibiotic resistance genes.
The nal part of the present research was focused on detecting putative virulence genes in the Streptococcus spp. strains isolated from different types of hospital cockroaches. We found that bac, cyl, glnA, cfb, hylB, scaA, bca, scpB and lmb had considerable prevalence in the Streptococcus. Spp. strains isolated from hospital cockroaches. To the best of our knowledge, there existed no previously published data in this led all around the world. The α-protein of protein C was encoded by bac and bca genes. This gene group helps bacteria to enter the host cells. Genes bac and bca were detected in 1.12% and 6.74% of bacteria, respectively. Eskandarian et al. (2015) [36] reported the bca and bac genes were found in 14.6% and 9.7% of Streptococcus isolates of hospital infections. Lower prevalence of the bac gene was reported from the United States, New Zealand and Europe [37][38][39]. We found that the prevalence of cyl, lmb, and scpB genes was 52.80%, 22.47% and 7.86%, respectively. Duarte et al. (2005) [40] reported that the prevalence of lmb and scpB genes in the Streptococcus spp. strains isolated from clinical samples was 97.30% and 96.70%, respectively, which was higher than our ndings. Sterile normal saline (0.9%) (5 mL) (Merck, Germany) was added to each test tube, and the cockroaches were vigorously washed and transferred to the secondary sterile test tubes using the sterile forceps. A loop full of each suspension was cultured on streptococcal selection broth (BD Biosciences, USA) and incubated at 37 °C for 6 h with 5% CO 2 . After enrichment, the samples were streaked onto 5% sheep blood agar and incubated at 37 °C for 24-48 h with 5% CO 2 . The suspected streptococcal colonies were puri ed on BHI agar (Merck, Germany).The cultures puri ed were tentatively identi ed on the basis of Gram's staining and biochemical tests, including bile esculin hydrolysis, catalase, and oxidase. Species identi cation was carried out using the certain biochemical tests, including hemolysis activity (S. pneumonia (alpha), S. pyogenes (beta) and S. agalactiae (beta)), resistance to bacitracin (S. pneumonia (resistant/sensitive), S. pyogenes (sensitive) and S. agalactiae (resistant)), resistance to sulfamethoxazole (S. pneumonia (-), S. pyogenes (resistant) and S. agalactiae (resistant)), resistance to optochin (S. pneumonia (sensitive), S. pyogenes (resistant) and S. agalactiae (resistant)), bile:asculin activity (S. pneumonia (-/-), S. pyogenes (-/-) and S. agalactiae (-/+ and -/+)) and growth on 6.5% NaCl (S. pneumonia (-), S. pyogenes (-) and S. agalactiae (-)). Con rmation of the species was carried out using the speci c Polymerase Chain Reaction (PCR). Genomic DNA was extracted from bacterial colonies using the DNA extraction kit (Fermentas, Germany) according to the manufacturer's instruction. DNA quality and concentration were examined using the spectrophotometer. Table 1  PCR-based detection of virulence factors Table 1 represents the primer sequence and PCR conditions used to detect putative virulence factors in the Streptococcus spp. isolated from different types of hospital cockroaches. Each PCR reaction contained 5 µL of 10X PCR ampli cation buffer, 2 µL of extracted DNA, 1 U of Taq DNA polymerase, 1 µM of each primer, 2 mM MgCl2, 200 µM dNTPs, and double-distilled water was added to a nal volume of 25µL.
PCR-based detection of antibiotic resistance genes Table 1 represents the primer sequence and PCR conditions used to detect penicillin, macrolide, streptogramin and tetracycline antibiotic resistance genes in the Streptococcus spp. isolated from different types of hospital cockroaches. Each PCR reaction contained 3 µL of extracted template DNA, 1U of Taq DNA polymerase, 1 µM of each primer, 2 mM MgCl2, 5 µL of 10X PCR buffer, 200 µM dNTPs, and double-distilled water was added to a nal volume of 25 µL.

Agarose gel electrophoresis
The PCR ampli ed products (10µL) were subjected to electrophoresis on a 1.5% agarose gel in 1X TBE buffer at 80 V for 30-40 min stained with a solution of Ethidium Bromide (Fermentas, Germany) and examined under Ultra Violet illumination (Uvitec, UK).

Statistical analysis
The data obtained from all the tests were entered the Microsoft Excel spreadsheet (Microsoft Corp., Redmond, WA) to be analyzed. All the data were rst presented to Kolmogorov-Smirnov test in order to study their distribution. In this respect, the statistical analysis was then conducted using SPSS/20. The datasets used and/or analyzed during the current study available from the corresponding author on reasonable request.

-Competing interests
The authors declare that they have no competing interests.

-Funding
The authors declare that no funding was received for the research.