J Arthropod-Borne Dis, March 2023, 17(1): 63–71 B Davari et al.: Bacterial Contamination of … 63 http://jad.tums.ac.ir Published Online: March 31, 2023 Original Article Bacterial Contamination of Collected Cockroaches and Determination their Antibiotic Susceptibility in Khorramabad City, Iran Behroz Davari1, Ali Ezat Hassanvand1, Aref Salehzadeh1, Mohammad Yousef Alikhani2, *Seyed Mostafa Hosseini2,3 1Department of Medical Entomology, Hamadan University of Medical Sciences, Hamadan, Iran 2Infectious Disease Research Center, Hamadan University of Medical Science, Hamadan, Iran 3Department of Microbiology, Hamadan University of Medical Sciences, Hamadan, Iran *Corresponding author: Dr Seyed Mostafa Hosseini, E-mail: Smhoseiny88@yahoo.com (Received 18 Dec 2021; accepted 05 Feb 2023) Abstract Background: Cockroaches are one of the most important carriers of pathogenic microorganisms. Therefore, the presence of cockroaches in public places, especially in hospitals, homes, and restaurants, is dangerous, and threatens the health of society, people, and the environment. The aim of this study was evaluation of bacterial contamination of cockroaches and the sensitivity of these bacteria to various antibiotics, captured from Khorramabad City, Iran Methods: This descriptive cross-sectional study was performed on 150 cockroaches collected from hospital environ- ments, homes, and restaurants in Khorramabad. The outer surface of the cockroaches was washed with physiological sa- line. The suspension was centrifuged for 5 minutes at 2000rpm. Isolation and identification of bacteria was performed using phenotypic methods. Antibiotic susceptibility testing was performed by disk diffusion method according to Clini- cal and Laboratory Standard Institute (CLSI) guideline. Results: A total of 100 American cockroaches (66.66%), 28 B. germanica (18.66%) and 22 Blatta orientalis (14.66%) were identified. In total, 97.33% of the collected cockroaches were infected with bacteria. The most bacterial infection of the cockroaches was Escherichia coli, coagulase-negative Staphylococci and Bacillus respectively. The overall re- sults of the antibiogram test showed that the identified bacteria were resistant to cephalothin, ampicillin, cefotaxime, and kanamycin antibiotics, semi-sensitive to ciprofloxacin and sensitive to tetracycline, gentamicin, nitrofurantoin, Tri- methoprim/sulfamethoxazole, and Chloramphenicol. Conclusion: Infection of cockroaches with pathogenic bacterial agents in hospital, residential, and restaurant environ- ments, as well as the observation of bacterial resistance to some common antibiotics is worrying. Keywords: Cockroaches; Bacteria; Antibiotic Introduction Insects are one of the most populous classes of arthropods, which are important in terms of economic, health, and medical (1). Cockroaches belong to the order Blattaria that so far about 4,000 species of cockroaches have been iden- tified, less than 1% of which are considered pests. Cockroaches live in warehouses, baker- ies, baths, hospitals, and even ships and harbors, hiding in dark, narrow crevices and crevices during the day and leaving their shelters at night to feed (2). Today, perhaps few places can be found that are safe from the presence of cock- roaches, and therefore increases their potential as a harmful health factor (3). This insect is important in medicine due to its habit of vomit (puking) a portion of the food eaten, defecating in the living environment, hav- ing weak bites, especially between the fingers, and producing a foul odor, particularly in the American cockroach, which is potentially dan- gerous to human health. Also, secretions and scabs from changing their skin contain aller- Copyright © 2023 The Authors. Published by Tehran University of Medical Sciences. This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International license (https://creativecommons.org/licenses/by- nc/4.0/). Non-commercial uses of the work are permitted, provided the original work is properly cited. http://jad.tums.ac.ir/ mailto:Smhoseiny88@yahoo.com https://creativecommons.org/licenses/by-nc/4.0/ https://creativecommons.org/licenses/by-nc/4.0/ J Arthropod-Borne Dis, March 2023, 17(1): 63–71 B Davari et al.: Bacterial Contamination of … 64 http://jad.tums.ac.ir Published Online: March 31, 2023 gens that dermatitis, itching, and many acute respiratory diseases are their complications (4, 5). Laboratory studies by using serological meth- ods on cockroach secretions and fecal particles have isolated and identified several allergens from them. Cockroaches are the second im- portant cause of asthma after dust allergies (6, 7). The ability of cockroaches to quickly run and move increase their ability to escape the control and transmission methods of pathogens (8). Cockroach feces contains compounds such as xanthurenic acid and quinolinic acid, all of which are derivatives of tryptophan and have mutagenic and carcinogenic properties (9). The importance of cockroaches in the spread of nos- ocomial bacteria and the incidence of nosocom- ial infections in patients is greater than previous- ly thought. Cockroaches are naturally infected with the pathogenic bacteria that cause lepro- sy, plague, bloody diarrhea, urinary tract in- fections, pimples, abscesses, and food poison- ing. Cockroaches are infected with approximate- ly 150 species of bacteria, 60 species of yeast, 90 species of protozoa, 45 species of ringworm, 12 species of pathogen fungi, and several hook- worms and whipworms. The most important and prominent carriers of these bacteria are Blat- tella germanica, Periplaneta americana, and Blatta orientalis (2). In Iran twelve species of this family have already been recognized. In terms of the num- ber of species, the family Ectobiidae (Blattelli- dae) is the largest order of cockroaches in the world and eight species of this family have been reported from the country. The German roach, B. germanica, the major pest of residential dwellings, belongs to family Blattellidae as well (10). Cockroaches are omnivores and due to feeding on a variety of foods and even human waste, they can spread diseases such as typhoid, leprosy, dysentery, etc. by transmitting large numbers of harmful microorganisms. These mi- croorganisms live for a long time in the intes- tines of cockroaches and are eventually trans- ferred to another organism (11-13). One of the most important challenges that the world is fac- ing in the fight against bacteria is their increas- ing antibiotic resistance. The resistance of mi- croorganisms to antibiotics is coded by differ- ent genes that are also capable of horizontal transfer between bacteria. Cockroaches can me- chanically transfer antibiotic-resistant bacteria from hospital environments to non-hospital ar- eas and vice versa, causing serious problems for public health (14). Due to the abundance of cockroaches and their role in the mechanical transmission of var- ious pathogens, and insufficient knowledge of the level of bacterial contamination on their ex- ternal surface and prevention of transmission of various infections, and raising the level of public health, this research aimed to evaluate of bacterial contamination of cockroaches and the sensitivity of these bacteria to various an- tibiotics, captured from Khorramabad City, Iran. Materials and Methods Sampling This is an analytical descriptive cross-sec- tional study. The study was conducted from March 2019 to March 2020 in Khoramabad, Iran. The study sites included two hospitals: Shohadaye Ashayer and Shahid Madani; and public places (including homes, and restaurants). In this study, 150 cockroaches were captured by hand catch method. After observing the cock- roaches, they were transported to the entomol- ogy laboratory of Hamadan University of Med- ical Sciences using sterile gloves. For prevent- ing (cockroaches) mixing contamination with each other, only one cockroach was used in each container. Then each sample was placed at 4 °C for 5 minutes for anesthesia. Species of cock- roaches were determined according to the iden- tification key (14, 15). Bacteria Isolation Firstly, in the laboratory, the outer surface of the cockroaches was washed with 5mL ster- http://jad.tums.ac.ir/ J Arthropod-Borne Dis, March 2023, 17(1): 63–71 B Davari et al.: Bacterial Contamination of … 65 http://jad.tums.ac.ir Published Online: March 31, 2023 ile normal saline. Then the solution was cen- trifuged for 5 minutes at 2000g. The sediments that remained at the bottom of the centrifuge tubes were cultured on the various mediums such as Eosin Methylene Blue (EMB) and blood agar (Merck, Germany) (16, 17). Cultivated mediums were incubated in 37 oC for 24 hours. In the next step, for each grown colony, all phenotypic differential tests were performed to isolate the bacteria into the genus or species level. For identification of Gram-neg- ative bacteria Gram stain, fermentation of sugars, motility test, Triple Sugar Iron (TSI) (Merck), Sulfide Indole Motility (SIM) (Merck, Germa- ny), and Simmon citrate (Merck) tests were ap- plied. For the identification of Gram-positive bacteria, Gram stain, catalase test, oxidase test, fermentation of sugars (mannitol), and sensi- tivity to antibiotics were employed (18). Antibiogram The resistance of bacterial strains isolated from the cockroaches was investigated using the Kirby-Bauer disk diffusion method (16, 19). A control strain of Pseudomonas aeruginosa ATCC 27853 was used for quality control of susceptibility testing. From the tested organisms, a suspension with turbidity equal to 0.5 McFarland stand- ards was provided for each bacterial strain and transferred to Muller-Hinton agar medium (Merck), and then antibiotic discs including: ampicillin (30µg), tetracycline (30µg), kana- mycin (30µg), chloramphenicol (30µg), trime- thoprim-sulfamethoxazole (23.75–1.25µg), gen- tamicin (30µg), ciprofloxacin (5µg), nitrofu- rantoin (300µg), cephalothin (30µg) and cefo- taxime (30µg) that all antibiotic discs were pur- chased from Mast Group Ltd., (England) and in- cubated for 24 hours at 37 °C. After 24 hours, the diameter of the growth inhibition zone was measured. The diameter of the growth inhibi- tion zone of the microorganisms for each an- tibiotic was divided into sensitive, semi-sensi- tive, or resistant according to the Clinical and Laboratory Standards Institute (CLSI) guide- lines (20). Finally, data were analyzed using SPSS version 20 software package developed by IBM cooperation, using chi-square tests. P< 0.05 was regarded as significant. The amount of bacterial contamination as well as the pat- tern of their resistance to antibiotics is checked with the type and location of collection of cock- roaches. The relationship between the place of isolation of cockroaches (home, hospital, and restaurant) with the Frequency of cockroaches and also the dominant species was determined. Results The Captured cockroaches and Extracted Bacterial Strains 100, 28, and 22 cockroaches were caught from hospitals, homes, and restaurants, respec- tively. A total of 146 bacterial colonies or strains were isolated from the 150 caught cockroach- es. Table 1 shows the Frequency of bacteria in each location. Out of 150 cockroaches, 100 (66.6%) were P. americana, 28 (18.6%) B. germanica, and 22 (14.66%) Blatta orientalis. In total, 97.33% of the collected cockroaches were infected with bacteria. The highest bacterial infection of cock- roaches was E. coli, and then coagulase-nega- tive Staphylococci and Bacillus species were the most common bacterial infections. Statis- tical analysis showed that there is a statistical- ly significant relationship between different plac- es of cockroach collection and the rate of bac- terial infection (p< 0.05). The results of investigating the relationship between bacterial isolates and cockroaches are shown in Figure 1. Out of 33 E. coli, 25 strains were isolated from the P. americana, and 15 strains out of 19 Enterococcus spp. were iso- lated from the P. americana, which was statis- tically significant (p <0.05). The distribution of other bacteria in cockroaches was uniform. Sensitivity of Bacteria to Antibiotics According to the results, it was found that the bacteria isolated from the hospital had a http://jad.tums.ac.ir/ J Arthropod-Borne Dis, March 2023, 17(1): 63–71 B Davari et al.: Bacterial Contamination of … 66 http://jad.tums.ac.ir Published Online: March 31, 2023 higher resistance to antibiotics than the bacte- ria isolated from homes and restaurants (table 2), which is a statistically significant differ- ence (P< 0.05). Table 1. Frequency of bacteria isolated from three cockroach species (P. americana, B. germaniaca, B. orientalis) in different parts of Khorramabad, 2019-2020 Bacteria Collection place Total Hospital No (%) * Home No (%) Restaurant No (%) Escherichia coli 21 (63.6) 9 (27.3) 3 (9.1) 33 (100) Bacillus spp. 8 (34.8) 4 (17.4) 11 (47.8) 23 (100) Klebsiella spp. 8 (80) 1 (10) 1 (10) 10 (100) Enterococcus spp. 16 (84.2) 2 (10.5) 1 (5.2) 19 (100) Acinetobacter spp. 1 (33.3) 1 (33.3) 1 (33.3) 3 (100) Coagulase negative Staphylococci 21 (67.7) 7 (22.6) 3 (9.7) 31 (100) Proteus spp. 7 (87.5) 1 (12.5) 0 (0.0) 8 (100) Enterobacter aerogenes 9 (100) 0 (0.0) 0 (0.0) 9 (100) Pseudomonas aeruginosa 7 (70.0) 2 (20.0) 1 (10.0) 10 (100) Total 98 (67.1) 27 (18.5) 21 (14.4) 146 (100) *(p< 0.05) E sc h er ic h ia c ol i B ac il lu s sp p K le bs ie ll a sp p E n te ro co cc u s sp p A ci n et ob ac te r sp p C O N s P ro te u s sp p E n te ro ba ct er a er og en es P se u do m on as a er u gi n os a 0 5 10 15 20 25 30 N u m b e r o f b a c te r ia Periplaneta americana Blattella germanica Blattella orientalis * * Fig. 1. Relationship between bacterial isolates and cockroach species, *(p< 0.05) http://jad.tums.ac.ir/ J Arthropod-Borne Dis, March 2023, 17(1): 63–71 B Davari et al.: Bacterial Contamination of … 67 http://jad.tums.ac.ir Published Online: March 31, 2023 Table 2. Antibiotic resistance pattern of bacteria isolated from cockroaches; the number of sensitive, resistant, and in- termediate resistant bacteria to different antibiotics are listed as numbers for each bacterium Antibiotics Response Bacteria Total E . c o li B a c il lu s K le b si e ll a E n te ro b a c te r sp p A c in e to b a c te r sp p C o n s1 P ro te u s sp p S . a u re u s P se u d o m o n a s sp p Gentamicin S I R 25 7 1 16 6 1 7 2 1 15 3 1 2 1 0 25 3 2 6 2 0 7 2 0 9 1 0 112* 27 6 Kanamycin S I R 7 16 11 3 13 7 5 3 2 2 13 4 0 2 1 6 14 10 4 2 2 5 1 3 3 5 2 35 69 42 Cefotaxime S I R 2 2 29 3 2 18 0 1 9 1 1 17 0 2 1 3 2 25 1 0 7 0 0 9 1 1 8 11 11 123* Cephalothin S I R 3 1 29 2 2 19 2 4 4 0 2 17 3 0 0 4 3 23 2 0 6 3 0 6 1 1 8 20 13 112* Ampicillin S I R 2 6 25 2 3 18 1 2 7 2 4 13 2 1 0 6 4 20 2 2 4 2 1 6 2 1 7 21 24 100* Tetracycline S I R 27 4 2 14 7 2 9 0 1 13 3 3 1 0 2 27 3 0 5 3 0 8 1 0 8 1 1 112* 22 11 Chloramphenicol S I R 20 11 2 7 9 5 3 3 4 9 3 7 1 1 1 19 6 5 3 3 2 2 4 3 6 2 2 70 42 31 Ciprofloxacin S I R 7 14 12 9 7 5 8 2 0 9 5 5 1 1 1 8 14 8 4 2 2 3 2 4 0 9 1 49 56 38 Nitrofurantoin S I R 10 10 13 6 10 7 9 0 1 7 6 6 0 1 2 6 16 8 4 1 3 5 1 3 2 2 6 49 47 49 Cotrimoxazole S I R 17 4 12 10 2 11 4 2 4 8 3 8 1 1 1 15 2 13 5 3 0 1 2 6 7 2 1 68 21 56 1. Coagulase-negative Staphylococci S: susceptible, I: intermediate, R: resistant, *(p< 0.05) Discussion In the present study, we identified three spe- cies of P. americana, B. germanica and B. ori- entalis that the most common of them were American cockroaches, which included 100 http://jad.tums.ac.ir/ J Arthropod-Borne Dis, March 2023, 17(1): 63–71 B Davari et al.: Bacterial Contamination of … 68 http://jad.tums.ac.ir Published Online: March 31, 2023 cockroaches (67%), followed by German cock- roaches 28 (19.4%) and B. orientalis 22(14/8%). In a study conducted by Zarchi et al. in Teh- ran hospitals, they showed that 65.6% of cock- roaches were American, 12.1% German, and 22.3% Oriental, which is statistically consistent with the number of American cockroaches in the present study (21). Also in other similar re- searches in the provinces of Iran (Zanjan, Ma- zandaran, and Esfahan), P. americana and Ger- man cockroaches were identified as two dom- inant species (22, 23). The results of Pai et al. studies showed that in clinical settings (environ- ment) most cockroaches are German type and in non-clinical places, most cockroaches are American type but this difference was not sta- tistically significant (24). The most common in hospital samples were coagulase-negative Staphylococci and E. coli. The most bacteria found in the house samples were E. coli and coagulase-negative Staphylo- cocci. Bacillus spp. was the most common bac- terium found in restaurant samples. This study showed that the prevalence of bacterial infec- tion in cockroaches caught in hospitals, houses, and restaurants in Khorramabad were 97.33%. As in some reports, the role and importance of cockroaches as carriers of bacteria, fungi, and pathogens in humans has been confirmed (25). This study also showed that cockroaches can transmit a variety of bacteria. In the study of Chitsazi et al. in Mashhad during the years 2010‒2011, the most isolated bacteria were re- lated to Enterococcus from cockroaches in Mashhad hospital, and the most frequent of cockroaches caught from student dormitories were related to Enterobacter aerogenes and most abundant of isolated cockroaches from houses are related to Klebsiella oxytoca (26). In Bangkok, several species of bacteria were also isolated from cockroaches in residential and hospital areas. Escherichia coli and Klebsiella pneumoniae were most common in the home, and E. coli and Enterobacter cloacae were most common in the hospital, which was consistent with our study of the prevalence of E. coli (27). In a study by Lin et al, the most isolated bac- teria from cockroaches were E. coli, Pseudo- monas aeruginosa, Salmonella spp., and Staph- ylococcus aurous (28). In another study by Chaichanawongsaroj et al. E. coli and Klebsiella spp. were commonly isolated from nosocomi- al cockroaches and showed that the insect, as a carrier of this bacterium, was involved in the transmission of these bacteria (27). The study by Fakoorziba et al. in 2010 on P. americana and B. germanica showed the high potential of these organisms to carry pathogenic bacte- ria, which isolated 25 different species of im- portant medical bacteria that 22 species were gram-negative bacteria. In this study, bacteria from the family Enterobacteriaceae of the gen- era Proteus, Citrobacter, Aerobacter, Klebsiel- la and Bacillus were isolated from the outer surface of cockroaches (29). Akbari and col- leagues conducted a study to determine aero- bic bacterial community of American cock- roach guts and found 11 bacterial species in- cluding: E. coli, Shigella flexineri, Citrobacter freundii, E. vulneris, Enterobacter cloacae, Yer- sinia pseudotuberculosis, Y. intermedia, Lecler- icia adecarboxylata, Klebsiella oxytoca, K. planticola, and Rahnella aquatilis. Some of the isolated bacteria of that study were consistent with the present study (13). Also in the present study, Staphylococcus was the second bacterium that was abundantly identified in the studied cockroaches, this bac- terium causes a wide range of infections from simple skin infections (Such as decoctions, boils, scabs, eyelashes, and abscesses) to life-threat- ening diseases such as pneumonia, meningitis, osteomyelitis, endocarditis, toxic shock syn- drome, and septicemia. Staphylococcus aureus is one of the five most common causes of nos- ocomial infections, especially post-surgical wound infections. Each year, 500,000 people in hospitals in the United States become infect- ed with S. aureus (30‒32). The results of the antibiogram test showed that the identified bacteria were resistant to the antibiotics Cephalothin, Ampicillin, Cefotaxime, http://jad.tums.ac.ir/ J Arthropod-Borne Dis, March 2023, 17(1): 63–71 B Davari et al.: Bacterial Contamination of … 69 http://jad.tums.ac.ir Published Online: March 31, 2023 and Kanamycin that this bacterial resistance was worrying, and they were also relatively resistant to the antibiotics Nitrofurantoin and Co-trimox- azole. The cultured bacteria were also semi-sen- sitive to the antibiotics kanamycin and ciprof- loxacin and were sensitive to the tetracycline, gentamycin, and chloramphenicol antibiotics. In a study by Stypułkowska-Misiurewicz et al. and colleagues, they examined cockroaches in nine Warsaw hospitals and found that the bacteria carried by their bodies were the same ones (species) responsible for nosocomial in- fections. Therefore, they stated that there was a microbiological risk of cockroaches in the hospital environment and it was proved that some bacteria in the body of cockroaches are re- sponsible for nosocomial infections, in which there is also antibiotic resistance (5). Davari et al. conducted a study with the aim of investi- gating the frequency of resistant and sensitive bacteria isolated from houseflies, their findings showed, K. pneumonia had the highest bacte- rial count with 43% and P. aeruginosa 37%, Proteus mirabilis 29.1%, and Citrobacter freun- dii were reported 4.28%. Among all hospital samples, resistance to cephalexin, chloramphen- icol, ampicillin, and tetracycline was high at 32.5%. The result was that gentamicin showed the highest sensitivity among all hospital speci- mens (33). In our study, the highest sensitivity to tetracycline, gentamicin, and chloramphen- icol antibiotics was reported. Considering that many diagnostic tests were needed to determine the bacterial strain, and this required a lot of financial resources, so we de- cided to stop the differentiation tests at the species level in some isolates. Conclusions According to the results of this study, it was found that cockroaches can be very effective in transmitting pathogenic bacteria and they can also mechanically transfer bacteria from hos- pital environments to the outside of the hospi- tal and vice versa, which is very important. In addition, according to the findings of this re- search, it can be assumed that cockroaches play a major role in the spread of antibiotic resistant bacteria. The next point is that cockroaches in hospital environments were more contaminat- ed with bacteria, one of the reasons for this is that people who visit hospital centers are usu- ally sick and carry various pathogens. There- fore, raising the level of health and combating cockroaches seems very necessary. Acknowledgments The study was funded by Vice-chancellor for Research and Technology, Hamadan Uni- versity of Medical Sciences (No: 9404302417). Ethical considerations Since human samples and animal studies were not used in this study, there were no eth- ical restrictions. Conflict of Interest statement The authors declare there is no conflict of interests. References 1. Donkor ES (2019) Nosocomial pathogens: an in-depth analysis of the vectorial po- tential of cockroaches. Infect Dis Trop Med. 4(1): 14. 2. Spagna JC, Goldman DI, Lin PC, Ko- ditschek DE, Full RJ (2007) Distributed mechanical feedback in arthropods and ro- bots simplifies control of rapid running on challenging terrain. Bioinspir. Biomim. 2 (1): 239‒256. 3. 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