Bacteriological analysis of Periplaneta americana L. (Dictyoptera; Blattidae) and Musca domestica L. (Diptera; Muscidae) in ten districts of Tangier, Morocco

In this study, Periplaneta americana and Musca domestica were collected from ten districts in Tangier, to isolate and identify some bacteria from their body using selective media. The results indicate that the amounts of bacteria were different between the districts. Moreover, the bacteria isolated from body of American cockroaches differed from those of the houseflies. These findings show that American cockroaches and houseflies may carry pathogenic bacteria in the urban areas of Tangier.


INTRODUCTION
American cockroaches (Periplaneta americana L.) and houseflies (Musca domestica L.) are often found in close association with humans, living in homes, businesses and health care facilities.The intimate association of these insects with humans has resulted in their relegation to pest status (Rau, 1924;Ebeling, 1978;Graczyk et al., 2005).Both American cockroaches and houseflies are abundant in urban and rural areas where unsanitary conditions prevail; they are usually scarce when sanitary conditions are enforced (Greenberg, 1973;Graczyk et al., 2001Graczyk et al., , 2005)).Moreover, because of their association with human environments, both of them may incidentally contact and acquire human pathogens.Pathogens may subsequently be mechanically transmitted to humans or susceptible artefacts or surfaces which may then function as vehicles of indirect transmission (Roth and Willis, 1957).
The American cockroaches are the potential vectors for bacteria (over 100 species of bacteria have been isolated from or passed through cockroaches), fungi and para-*Corresponding author.E-mail: lamiaab1@yahoo.fr.Phone: +212 66375295.Fax: +212 39393953.
The city of Tangier records a high demographic growth and a persistent rhythm of urbanisation which create a formation of insalubrious and under-equipped districts.Moreover, in its various districts, there are noticeable differences in density, town planning and social level.The objective of this study was to confirm that P. americana and M. domestica carry pathogenic bacteria in some districts of the city by bacteriological analysis of these two insects.

Collection and identification of cockroaches and flies
Fifty adult of P. americana (five per site) and seventy M. domestica (seven per site) were collected from houses of the ten selected sites.Flies were caught with sterilised nets from kitchens, living rooms and bathrooms.Each trapped insect (cockroach or fly) was placed in a sterile test tube and was subsequently taken to the laboratory.The insects were immobilised by freezing at 0°C for 5 min.Identification was made by examining the insect under a low power microscope and following standard taxonomic keys.

Isolation and identification of bacteria from external surfaces of insects
Two millilitres of sterile normal saline (0.9%) was added to test tube containing one fly or cockroach, and the tubes were thoroughly shaken for 2 min to isolate micro-organisms from the external surface.Aliquots (0.01 ml) of the washing were then separately inoculated onto the surface of agar plates such as Chapman, Litsky, MacConkey, and Hektoen.Selenite and Roth broths were used as enrichment media successively for Salmonella and Streptococcus, before culturing.The enrichment broths and agar plates were incubated at 37°C for 24 h.The bacteria growth on the agar media were identified by colonial morphology, Gram staining, and biochemical tests such as oxidase, catalase, motility, coagulase, indole, methyl red, voges proskaeur (MRVP), gelatine hydrolysis, glucose, lactose, mannitol, and gas and H2S production (Murray 1999).

Statistical analysis
Rates were compared using the ANOVA/MANOVA test.P<0.05 was considered to be statistically significant.

RESULTS
All the cockroaches and flies were found to carry several species of bacteria on the external surface.The bacteria isolated from the insects were Gram-positive bacteria (particularly Staphylococcus and Streptococcus) and Gram-negative belonging to Enterobacteriacae.Morever, most of these bacteria are pathogenic.1).
Although, there was no significant difference between the means of Gram-positive bacteria in P. americana and M. domestica (p>0.05) in all the districts, the mean in P. americana (93.33%) was significantly higher than that in M. domestica coming from Moughogha (61.90%) (p=0.0003<0.05)Tables 2 and 3.In the same way, there was no significant difference between the means of Gram-negative bacteria in P. americana and M. domestica (p>0.05),but it was significantly higher in P. americana coming from Bnimakada and Tanjabalya (57.50 and 47.50%) than that in M. domestica coming from these two sites (28.57and 17.86%) (p = 0.033 and 0.042).
Different species of Gram-positive bacteria were isolated from the two species of insects in different districts.There was no significant difference between the amounts of these bacteria in the different districts (Tables 2 and 3).Eight species of gram-negative Enterobacteriaceae were isolated from P. americana and M. domestica in these districts (Tables 4 and 5).Moreover, there was a highly significant difference between the amounts of these bacteria in all districts; significantly higher means were found in Bendiban, Benkiran, Bnimakada and student's residences (M.domestica 33.93% and P. americana 62.5%).The minimum amounts were often observed in Boughaz and Hayidari (M.domestica 12.50% and P. americana 25%) (Figures 1 and 2).

DISCUSSION
This study showed that eleven species of bacteria were isolated from M. domestica and P. americana (Tables 3  and 4).Our results are in accordance with other reports which highlight the importance of houseflies and American cockroaches in carrying various enteropathogens (Greeberg, 1973, Echeverria et al., 1983;Fotedar et al., 1992b;Grubel et al., 1997;Kobayashi et al., 1999;Koura and Kamel, 1990;Fotedar et al., 1992a;Rivault et al., 1993;Pai et al., 2003).In this study, more bacterial species were recovered from P. americana than those from M. domestica.In similar studies, 56 species of bacteria were isolated from cockroaches in France and 14 species were found to be pathogenic or potentially pathogenic for man and animal, with E. coli and K. pneumoniae being the most important (Rivault et al., 1993;Vythilingam et al., 1997).The role of the cockroaches and houseflies in the transmission of pathogens and gastrointestinal diseases had already been established (Greenberg, 1971;Rueger and Olsen, 1969).Moreover, these two species of insects are the most common ones in Africa because of the favourable environmental and climatic conditions (Boulesteix et al., 2005).These findings and their special habits suggest the importance   3 42,86 2 28,57 1 14,29 1 14,29 4 57,14 1 14,29 2 28,57 2 28,57 1 14,29 3 42,86
The houseflies and cockroaches caught in Benkiran (Bk), Bendeban (Bd), Bnimakada (Bm), Moghogha (Mg) and Tanja Balya (Tb) carried pathogenic bacteria more often than those caught in Hay Idari (Hi), Boughaz (Bz) and Boukhachkhach (Bkh) (Figures 1 and 2).These results could be attributed to social-economic factors related to each district (kind of population, urbanism and social level) and the insalubrious conditions in some of these districts.The number of areas containing waste re- fuse and excreta of humans and domestic animals might have provided ideal conditions for the breeding and multiplication of the synanthropic insects (insects associated with the human habitat) as this fly and cockroach species are known to feed and breed most successfully on filth (Fotedar, 2001).This study has established that P. americana and M. domestica carry pathogenic bacteria in ten districts of Tangier.

FrequencyFigure 2 .
Figure 2. Frequency of Gram -ve bacteria in P. Americana and M. domestica in various neighbourhoods of Tangier.
Klebsiella spp., Serratia spp., Proteus vulgaris, Proteus spp., Shigella spp., and Salmonella spp.The most frequent bacteria isolated from the houseflies Bouamama et al. 2039 and the American cockroaches coming from 10 districts of the city are Streptocoques, S. epidermidis and E. coli, while the least present are P. vulgaris and Klebsiella spp.(Table

Table 2 :
Gram-positive bacteria isolated from Musca domestica (n= 7 per district) collected from houses.
n: one insect may simultaneously harbour more than one species of bacteria.

Table 3 :
Gram-positive bacteria isolated from Periplaneta americana (n= 5 per district) collected from houses.
n: one insect may simultaneously harbour more than one species of bacteria.

Table 5 :
Gram-negative bacteria isolated from Periplaneta americana (n= 5 per district) collected from houses.
n: one insect may simultaneously harbour more than one species of bacteria.Figure 1.Frequency of Gram +ve bacteria in P. Americana and M. domestica in various neighbourhoods of Tangier.