J Arthropod-Borne Dis, June 2014, 8(1): 53–59 B Vazirianzadeh et al.: The First Report of … 53 Original Article The First Report of Drug Resistant Bacteria Isolated from the Brown-Banded Cockroach, Supella longipalpa, in Ahvaz, South-western Iran Babak Vazirianzadeh 1, *Rouhullah Dehghani 2, Manijeh Mehdinejad 3, Mona Sharififard 4, Nersi Nasirabadi 3 1Department of Medical Entomology, College of Health and Infectious and Tropical disease Research Centre, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran 2Department of Environmental Health, College of Health and Social Determinants of Health (SDH),Research Center, Kashan University of Medical Sciences, Kashan, Iran 3Departments of Medical Microbiology, College of Medicine, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran 4Departments of Medical Entomology, College of Health, Ahvaz Jundishapur University of Medical Sci- ences, Ahvaz, Iran (Received 7 July 2012; accepted 30 June 2013) Abstract Background: The brown-banded cockroach, Supella longipalpa is known as a carrier of pathogenic bacteria in ur- ban environments, but its role is not well documented regarding the carriage of antibiotic-resistant pathogenic bacte- ria in Iran. The aim of this study was to determine the resistance bacteria isolated from the brown-banded cockroach in Ahvaz, south west of Iran. Methods: Totally 39 cockroaches were collected from kitchen area of houses and identified. All specimens were cultured to isolate the bacterial agents on blood agar and MacConky agar media. The microorganisms were identified using necessary differential and biochemical tests. Antimicrobial susceptibility tests were performed for isolated or- ganisms by Kirby-Bauer’s disk diffusion according to NCLI guideline, using 18 antibiotics. Results: From the 39 collected S. langipalpa, 179 bacterial agents were isolated, 92 of alimentary ducts and 87 of external body surfaces. Isolated bacteria from cockroaches were identified as Enterobacter spp., Klebsiella spp., Citrobacter spp., Escherichia coli, Salmonella spp., Proteus spp., coagulase negative staphylococci, Serratia marcescens, Staphylococcus aureus, and Bacillus species. The pattern resistance rates were determined for gram negative bacilli and gram positive cocci regarding 18 antibiotics. Conclusion: The brown-banded cockroach can be involved in the spread of drug resistant bacteria and increases the possibility of contacting human environment to drug resistant bacteria. Therefore, the potential of removing this in- sect should be improved. This is the first original report of drug resistant bacteria isolated from the brown-banded cockroach of Iran. Keywords: Supella longipalpa, Drug Resistant Bacteria, Iran Introduction Cockroaches are the most abundant and important pest insects that inhabit various public places such as hospitals, food manu- facturing sites and kitchens (Ebelling 1978, Burgess and Chetwyn 1979). Cockroaches have been considered as transmitters and spreaders of pathogenic bacteria in hospitals and house- holds or residential areas (Lamiaa Bouamama 2005). Cockroaches feed indiscriminately on garbage and sewage and so have copious op- portunity to disseminate human pathogens. In addition, their nocturnal and filthy habits make them ideal carriers of various patho- genic microorganisms (Allen 1978, Graczyk et al. 2005, Pai et al. 2005). So far numerous pathogenic bacteria, in- *Corresponding author: Dr Rouhullah Dehghani, E- mail: dehghani37@yahoo.com http://jad.tums.ac.ir Published Online: December 18, 2013 J Arthropod-Borne Dis, June 2014, 8(1): 53–59 B Vazirianzadeh et al.: The First Report of … 54 cluding Salmonella spp., Shigella spp., Cam- pylobacter spp., Pseudomonas aeruginosa and K. pneumonia have been isolated from cock- roaches (Cotton et al. 2000). Antibiotic resistance is a serious public- health problem, reduced effectiveness of an- tibiotics results in greater patient mortality rates, prolonged hospitalization and increased healthcare costs. The economic impact of an- tibiotic resistance has been estimated be- tween $5 and $24 billion annually in the United States alone (Hall 2004). Insects as- sociated with food animals, especially cock- roaches not only are important nuisance pests but also potential vectors of animal and human pathogens. Organic waste in and around animal production facilities provide excellent habi- tats for the growth and development of these insects. Because of their habitat preferences, unrestricted movement, mode of feeding, and attraction to residential areas, cockroaches have a great potential to disseminate fecal bacteria, including human and animal path- ogens and antibiotic resistant strains (Zurek and Gorham 2008, Graczyk et al. 2001). The brown-banded cockroach, Supella longipalpa, is a small species of cockroach, measuring about 10 to 14,mm long. Cock- roaches like S. longipalpa which live in drier parts of houses can harbor and transmit the different bacteria within and between prem- ises because of small bodies. This leads to spread the bacteria which may be pathogenic and antibiotic resistance, potentially (Man- weiler 1998, Gibson and Hunter 2009). There are insufficient data on the poten- tial health impact of this species in the resi- dent areas of Iran. More infestation with this species has recently been reported from ur- ban environments of Iran, but its role is not well documented regarding the carriage of antibiotic-resistant pathogenic bacteria in Iran. This is the first original report of drug re- sistant bacteria isolated from the brown- banded cockroach of Ahvaz and Iran. The aim of this study was to determine the resistance bacteria isolated from external surfaces and digestive tract of the brown- banded cockroaches (S. longipalpa) which were collected from kitchen area of houses of Ahvaz, south west of Iran. Materials and Methods A total of 39 cockroaches were collected from kitchen area of houses, Ahvaz SW Iran. The samples were transported to the medical entomology laboratory of Ahvaz Jundyshapur University of Medical Sciences (AJUMS) and placed in the sterile dishes in freeze-temper- ature for 10–15min to anaesthetize them. The cockroaches were identified after immo- bilization by freezing, using keys of Depart- ment of Entomology and Nematology Univer- sity of Floridaas and Cohran (Cochran 1999). Afterward, two samples were taken from each cockroach, one of external surface body with sterile cotton swabs and the other one directly from alimentary duct after dissecting the insects. All specimens were cultured to isolate the bacterial agents on blood agar and Mac Conky agar media (Himedia India and Merck Germany). The microorganisms were identified using necessary differential and bi- ochemical tests. Cultured media were incubated at 37 ˚C for 24 hours. The various bacteria growth on the agar media were identified by colonial morphology on blood agar plates, Gram stains characteristics. In addition, other nec- essary biochemical tests including: oxidase, catalase, motility, coagulase, indole, methylred, voges proskaeur (MRVP), gelatine hydroly- sis, gas from glucose, H2S production and acid produced from various sugar according to standard microbiological procedures were also used for identification (Forbes et al. 2002, Jorgensen et al. 2005). Antimicrobial susceptibility testing was performed for isolated organisms by Kirby- Bauer’s disk diffusion according to Clinical and http://jad.tums.ac.ir Published Online: December 18, 2013 J Arthropod-Borne Dis, June 2014, 8(1): 53–59 B Vazirianzadeh et al.: The First Report of … 55 Laboratory Standards Institute (CLSI) guide- line. Antibiotic disks were used and the sus- ceptibility pattern of isolated from samples to commonly used antibiotics was then report- ed. The antibiotic disks (padten Teb, Tehran, Iran) comprised ampicillin (10μg), cephalothin (30μg), ceftriaxone (30μg), ciproflexoxacin (5μg), chloramphenicol (30μg), gentamicin (10μg), tetracycline (30μg), trimethoprim- sulfamethoxazole (25μg), ceftazidime (10μg), amikacin (30μg), nitrofurantoin (300μg), na- lidixic acid (30μg), cefalexin (10μg), imipenem (10μg), vancomycin (30μg), cefotaxime (10μg), kanamycin (30μg), and penicillin (10μg) guidelines to determine susceptibility of UTIs agents (CLSI 2002, Mpuchane 2006). Results From 39 collected S. langipalpa, 179 bac- terial agents were isolated, 92 of alimentary ducts and 87 of external body surfaces (Ta- ble 1). Isolated bacteria from cockroaches were identified as Enterobacter spp., Klebsiella spp., Citrobacter spp., E. coli, Salmonella spp., Proteus spp., coagulase negative staph- ylococci, S. marcescens, Staphylococcus aureus, and Bacillus species. The pattern resistance rates were deter- mined for gram negative bacilli and gram pos- itive cocci regarding 18 antibiotics. Among all the isolates Gram negative bacilli, from kitchen area of houses, ampicillin, cephalothin, ceftazidime, nitrofurantoin, nalidixic acid, trimethoprim-sulfamethoxazole, cefalexin and tetracycline, resistance rates were above 52.4% and cefotaxime expressed the highest sus- ceptibility among all the isolates from the kitchen area of houses. Among all the iso- lates Gram positive cocci, from kitchen area of houses, ampicillin, amikacin penicillin, ceftazidime, nitrofurantoin, nalidixic acid, trimethoprim- sulfamethoxazole, cefalexin, cefotaxime and tetracycline, resistance rates were above 53.8% and ciproflexoxacin ex- pressed the highest susceptibility among all the isolates from the kitchen area of houses (Table 2). Table 1. Frequency of isolated bacteria from Cockroaches Total No. (%)External Surface No. (%)Alimentary Tract No. (%)Isolated Bacteria from Cockroaches 5(2.79) 6(3.35) 18(10.10) 29(16.20) 2(2.30) 3(3.45) 9(10.35) 14(16.10) 3(3.26) 3(3.26) 9(9.78) 15(16.30) Enterobacter aerogenes Enterobacter cloacae Enterobacter agglomerans total 25(14.00) 3(1.70) 28(15.64) 13(14.94) 1(1.15) 14(16.10) 12(13.04) 2 (2.17) 14(16.30) Klebsiella pneumonia Klebsiella oxytoca total 16(8.94)7(8.05)9(9.78)Citrobacter freund 24(13.41)11(12.64)13(14.13)Escherichia coli 5(2.79)2(2.30)3(3.26)Salmonella para A 8(4.50)4(4.60)4(4.35)Serratia marcescens 11(6.2) 3(1.70) 14(7.82) 6(7.00) 1(1.15) 7(8.10) 5(5.43) 2(2.17) 7(7.61) Proteus mirabilis Proteus vulgaris total 20(11.17)9(10.3511 (11.96)coagulase Negative Staphylococci 7(3.91)4(4.60)3 (3.26)Staphylococcus aureus 19(10.61) 7(3.91) 26(14.53) 10(11.50) 4(4.60) 14(16.10) 9(9.78) 3(3.26) 12(13.04) Bacillus cereus Bacillus subtilis total 179(100)87(47.43)92(52.57)Total http://jad.tums.ac.ir Published Online: December 18, 2013 J Arthropod-Borne Dis, June 2014, 8(1): 53–59 B Vazirianzadeh et al.: The First Report of … 56 Table 2. Percentages of resistant of isolated bacteria from Cockroaches Gram positive cocciGram negative bacilliAntibiotics Resistance 10086.8Ampicillin 44.369.5Cephalothin 45.542.4Ceftriaxone 19.624.7Ciproflexoxacin -47.3Chloramphenicol 38.844.2Gentamicin 63.459.4Tetracycline 57.153.6Trimethoprim-sulfamethoxazole 85.3100Ceftazidime 53.825.9Amikacin -70.1Nitrofurantoin 74.952.4Nalidixic acid 63.267.9Cefalexin --Imipenem --Vancomycin 85.313.5Cefotaxime -36.5kanamycin 100-Penicillin Discussion The biology and ecology of S. longipalpa make it an ideal mechanical vector of bacte- ria to animal and human. Brown-banded cockroaches are found in homes, apartments, hotels, and hospitals. They are less frequent- ly found in stores restaurants and kitchens. They are frequently transported in furniture and will spread rapidly through an entire building. Brown-banded cockroaches are gen- erally found on ceilings, high on walls, be- hind picture frames, and near motors of re- frigerators and other appliances. The results of this study indicated that Supella longi- palpa could play a great role as a mechanical carrier of bacteria. In this study, all 39 cock- roaches were found to carry the 9 genera of medically important bacteria. The most frequent bacteria isolated from S. angipalpa coming from external bodies of this cockroaches was K. pneumonia fol- lowed by E. coli and E. coli was the most frequent coming of alimentary tract of S. langipalpa followed by K. pneumonia in the present study was. This is very similar to the studies of Vahabi et al. in Iran and Bouamama et al. in Morocco (Bouamama et al. 2010, Va- habi et al. 2011). However, the most biodiverse isolated bacteria during the current investi- gation was totally related to Enterobacter sp.: E. aerogenes, E. cloacae and E. agglomerans, with 16.20% of all species. This is important when Enterobacter spp. are known as food spoilage bacteria and are considered as cock- roach transmitted bacteria in food poisoning. In this study, most of the bacteria isolated were medically important, including Enter- obacter spp., Klebsiella spp., Citrobacter spp., E. coli, Salmonella spp., Proteus spp., coag- ulase negative Staphylococci, S. marcescens, Staphylococcus aureus, and Bacillus species. These findings agree with the results of Chaichanawongsaroj et al. (2004) in Thai- land, which showed presence Enterobacter spp., Klebsiella spp., Citrobacter spp., E. coli, Salmonella spp., Proteus spp., Serratia spp., species on the Periplaneta americana and Blatta orientalis collected from Hospital, Food-handling establishments and human dwellings (Bennett 1993, Chaichanawongsaroj et al. 2004, Salehzadeh et al. 2007). The similar bacteria have been isolated from P. americana and B. germanica in an- http://jad.tums.ac.ir Published Online: December 18, 2013 J Arthropod-Borne Dis, June 2014, 8(1): 53–59 B Vazirianzadeh et al.: The First Report of … 57 other study of Botswana (Mpuchane 2006). However, they are known as pathogenic (Salmonella, Shigella, B. cereus), opportunistic pathogens (Pseudomonas, Klebsiella, Vibrio) and food spoilage species (Pseudomonas, Enterobacter, Escherichia, Erwinia) they belong to cockroach transmitted bacteria (Mpuchane 2006). Bacterial species, such as Klebsiella spp., E. coli, Staphylococcus spp., Enterobacter spp., Streptococcus spp., Pseudomonas spp., Pro- teus spp., Bacillus spp., Neisseria spp., Shig- ella spp., and Salmonella spp., have also been isolated from P. Americana and Poly- phaga aegyptica cockroaches by other re- searchers from Khuzestan (Vazirianzadeh et al. 2011, Kassiri and Kazemi 2012). It is im- portant to say that the above named bacteria have been isolated from housefly, Musca domestica in Khuzestan (Vazirianzadeh et al. 2008). The present study indicates that brown- banded cockroach posses a possible health risk to communities proved that the isolated strains of bacteria were resistant to various antibiotics. It is well-established fact that the resistance to various antimicrobials may be due to presence of some virulence gene, involve- ment of secretion machinery of multi drug ef- flux proteins, through mutations in bacterial genome or by gaining additional genes through horizontal gene transfer or by physiology dependent resistance (Mitchell et al. 2004). One of the most important problems fac- ing global public health today is antimicro- bial resistance. The problem is most horrible in developing countries, where the bacterial infections causing human disease are also those in which emerging antibiotic resistance is most evident (Shears 2000, Fathpour et al. 2003, Kalantar et al. 2008). Cockroaches are able to mechanically transfer infectious agents in unsanitary places. Some bacteria that exist in the digestive tract of cockroaches can be also found in environment, that most of them belong to Gram-negative bacilli of Enter- obacteriacea family (Cloarec and Rivault 1992) this organisms can cause urinary infections, poisoning, inflammation of the stomach and intestines, and abdominal cavityinfection, pneu- monia or wound infections (Le Guyader et al. 1989, Kopanic et al. 1994). Therefore, elim- ination of cockroaches from sensitive areas, such as hospitals, is essential. In food-han- dling establishments and human dwellings, cockroaches must also be controlled, to main- tain acceptable hygiene standards (Chai- chanawongsaroj et al. 2004). Conclusion Brown-banded cockroaches collected from kitchen area of houses may be involved in the spread of drug resistant bacteria and may increase the potential for human exposure to drug resistant bacteria. It is recommended that suitable steps must be taken to control the cockroaches and monitor the sensitivity pattern of the pathogens transmitted by the cockroaches. Therefore, the potential of removing this insect should be improved. Parallel to inte- grated pest management of cockroaches, we must go ahead to produce new antibiotic from natural resources like the filthy habitats of cockroaches which kills them (Lee 2012). 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