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Isolation of bacteria with antibiotic resistance from household cockroaches (Periplaneta americana and Blattella germanica)
Acta Tropica 93 (2005) 259–265
Isolation of bacteria with antibiotic resistance from household cockroaches (Periplaneta americana and Blattella germanica) Hsiu-Hua Pai∗ , Wei-Chen Chen, Chien-Fang Peng Graduate Institute of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan, ROC Received 10 October 2003; received in revised form 25 October 2004; accepted 11 November 2004 Available online 1 February 2005
Abstract Cockroaches may harbor and disseminate microorganisms to the environment. In this study, Periplaneta americana and Blattella germanica were collected from 40 households in Kaohsiung City and Kaohsiung County, Taiwan. Cockroach infestation was found in 50% of the studied households and 226 cockroaches (123 P. americana and 103 B. germanica) collected by trapping. P. americana was more often found in the kitchen (70.7%) whereas B. germanica in the storage room (51.5%) and kitchen (36.9%). There was no significant difference between the percentages of P. americana (99.9%) and B. germanica (98.0%) carrying bacteria. A total of 25 species of bacteria was isolated from P. americana and only 21 from B. germanica. Antibiotic resistance was found in Staphylococcus aureus, Enterococcus species, Pseudomonas aeruginosa, Klebsiella pneumoniae, Escherichia coli, Serratia marcescens, and Proteus species isolated from the cockroaches. These findings suggest a potential role of cockroaches in the transmission of pathogenic bacteria with antibiotic resistance in households. © 2005 Elsevier B.V. All rights reserved. Keywords: Bacteria; Blattella germanica; Cockroaches; Antibiotic resistance; Vectors; Periplaneta americana
1. Introduction Owing to their omnivorous habit of feeding and indiscriminate deposition of fecal materials, cockroaches are well-known agents for harboring and transmission of microorganisms (Roth and Willis, 1957; Cloarec et al., 1992; Rivault et al., 1993). Over ∗ Corresponding author. Tel.: +886 7 312 1101x2141; fax: +886 7 322 2362. E-mail address: [email protected] (H.-H. Pai).
0001-706X/$ – see front matter © 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.actatropica.2004.11.006
100 species of bacteria have been isolated from or passed through cockroaches (Cruden and Markovetz, 1987). In addition to these bacteria, we have recently isolated non-tuberculous mycobacteria from hospital cockroaches (Pai et al., 2003a). Although studies with Periplaneta americana showed that Salmonella species do not readily become established in the intestinal tract of the insect (Klowden and Greenberg, 1976), Pseudomonas aeruginosa has been demonstrated to multiply in the gut of Blattela germanica and excretion of the bacteria continued up to 114
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days (Fotedar et al., 1993). Therefore, the cockroach may serve as a reservoir for multiplication of pathogens. Although important pathogenic microorganisms such as P. aeruginosa, Staphylococcus aureus, and Streptococcus faecalis have been isolated from B. germanica collected in hospitals, these species were not found in the corresponding residential areas (Fotedar et al., 1989.). Moreover, cockroaches in hospitals may harbor bacteria with resistance to antibiotics (Fotedar et al., 1991a) and those carrying Klebsiella species with antibiotic resistance has been suggested to play a role in nosocomial infections (Fotedar et al., 1991b). Recently, cockroaches have been suggested to be possible vectors for an outbreak of nosocomial beta-lactamase-producing Klebsiella pneumoniae infections in a neonatal unit with heavy cockroach infestation (Cotton et al., 2000). The subtropical climate in Taiwan is favorable to the growth and development of populations of cockroaches throughout the island (Hsu, 1990). Moreover, isolates of pathogenic bacteria from medical centers have been reported to have high prevalence of resistance to commonly used antimicrobial agents (Chang et al., 2001). It is possible for bacteria with antibiotic resistance to occur in household cockroaches. In this study, we collected P. americana and B. germanica from households in Kaohsiung City and Kaohsiung County. After isolating bacteria from the trapped insects, we determined whether antibiotic resistance exists in the potentially pathogenic ones.
2. Materials and methods 2.1. Collection and identification of cockroaches In March 2000, cockroaches were collected from 40 households in Kaohsiung City and Kaohsiung County. These households were randomly selected and at least 5 km from hospitals. In addition, the occupants were not working in health facilities and did not receive antibiotic therapy in the recent. Traps were manufactured according to the design of Reierson and Rust (1997). The traps were placed on the floor under beds, cupboards, wooden racks, and/or benches, for two consecutive days. Each trapped cockroach was placed in a sterile test tube before sending to our laboratory.
After immobilization by freezing at 0 ◦ C for 5 min, the species of cockroaches were identified under a dissecting microscope according to Harwood and James (1979). 2.2. Isolation and identification of microorganisms After species identification, 2 ml of sterile normal saline was added to the tube before thorough shaking for 2 min. Aliquots (0.01 ml) of the washings were then separately cultured on plates of tryptic soy agar blood agar (Difco, Detroit, USA), eosin methylene blue agar (Difco), and xylose lysine desoxycholate agar (Difco) at 35 ◦ C overnight. Bacterial colonies were identified by macroscopic examination, gram staining, and biochemical tests according to Murray (1999). After isolating microorganisms from the external surface, the cockroaches were washed in 70% ethyl alcohol for 5 min and allowed to dry at room temperature under sterile conditions. The insects were then washed with sterile normal saline for 3 min. The alimentary tract was dissected out and aseptically macerated in a sterile pestle and mortar with 2 ml of sterile normal saline. The macerate was then cultured on the same types of agar plates at 35 ◦ C overnight before specific identification of the colonies. 2.3. Antibiotic susceptibility tests The antibiotic susceptibility of S. aureus, Enterococcus species, P. aeruginosa, K. pneumoniae, Escherichia coli, Serratia marcescens, Proteus species to 17 antibiotics were tested by the Kirby-Bauer disk diffusion method according to the recommendations of the National Committee for Clinical Laboratory Standards (1993). These antibiotics (Becton Dickinson, Cockeysville, MD, USA) included ampicillin (10 g/disk), gentamicin (10 g/disk), ciprofloxacin (5 g/disk), ofloxacin (5 g/disk), chloramphenicol (30 g/disk), tetracycline (30 g/disk), sulfamethoxazole/trimethoprim (25 g/disk), penicillin (10 U/disk), streptomycin (10 g/disk), erythromycin (15 g/disk), oxacillin (1 g/disk), vancomycin (30 g/disk), cephalothin (30 g/disk), ceftazidime (30 g/disk), imipenem (10 g/disk), piperacillin (100 g/disk), and cefoperazone (75 g/disk).
H.-H. Pai et al. / Acta Tropica 93 (2005) 259–265
2.4. Statistical analysis Rates were compared using the χ2 test. P < 0.05 was considered to be statistically significant.
3. Results 3.1. Cockroach infestation Of the 40 households studied, 20 (50%) were found to have cockroach infestation: 11 (55%) with P. americana, 3 (15%) with B. germanica, and 6 (30%) with both species. A total of 226 adult cockroaches were collected: 123 P. americana and 103 B. germanica. Most of the P. americana (70.7%) were collected from kitchen while the higher collection rates of B. germanica was obtained in the storage room (51.5%) and kitchen (36.9%). The percentages of P. americana collected in the sleeping room, living room, storage room, and dining room were 12.2, 10.6, 3.3, and 3.3%, respectively. The collection rates of B. germanica obtained in living room and dining room were 8.7 and 2.9%, respectively. 3.2. Bacterial isolation Among the 226 cockroaches collected, 222 (98.2%) were found to carry one or more species of bacteria
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on the external surface or in the alimentary tract. Although there was no significant difference between the overall positive rates in P. americana (99.9%) and B. germanica (98.0%) (P > 0.05), the positive rate of the alimentary tract (97.3%) was significantly higher than that of the external surface (88.1%) (P < 0.05). Six species of gram-positive bacteria were isolated from the two species of cockroaches. There was no significant difference between positive rates in the alimentary tract (P. americana 69.9%, B. germanica 57.3%) and those on the external surface (P. americana 70.7%, B. germanica 56.3%) (P > 0.05). However, the positive rate was found to be significantly higher in P. americana (86.2%) than in B. germanica (72.8%) (P < 0.05) (Table 1). Fourteen species of gram-negative Enterobacteriaceae species were isolated from P. americana and only 11 from B. germanica. Significantly higher positive rates were found in the alimentary tract (P. americana 52.8% and B. germanica 63.1%) than on the external surface (P. americana 25.2% and B. germanica 39.8%) (P < 0.05). However, there was no significant difference between the positive rates in P. americana (57.5%) and B. germanica (65.1%) (P > 0.05) (Table 2). Five species of glucose non-fermenter bacilli were isolated from P. americana and four from B. germanica. There were no significant differences
Table 1 Gram-positive bacteria isolated from Periplaneta americana (n = 123) and Blatella germanica (n = 103) collected from households Bacteria
Periplaneta americana Alimentary tract
Streptococcus species (non-Enterococcus) Bacillus species (except B. subtilis) Bacillus subtilis Staphylococcus aureus Staphylococcus epidermidis Enterococcus species Overalld a b c d
Blatella germanica
External surface
Totala No.
Alimentary tract
External surface
Total
No.
%
No.
%
%
No.
%
No.
%
No.
%
46
37.4
50
40.7
78
62.6
36
35.0
32
31.1
55
53.4
33
26.8b
17
13.8
44
35.8
17
16.5
16
16.5
28
27.2
36 2 16 12
29.3b 1.6 13.0 9.8
14 2 18 6
11.4 1.6 14.6 4.9
44 3 27 14
35.8c 2.4 22.0 11.4c
8 1 18 6
7.8 1.0 17.5 5.8
7 0 10 2
6.8 0 9.7 1.9
14 1 27 6
13.6 1.0 26.2 5.8
86
69.9
87
70.7
104
86.2c
59
57.3
58
56.3
75
72.8
One cockroach may simultaneously harbor the same bacterial species on the external surface and in the alimentary tract. Alimentary tract vs. external surface: P < 0.05. P. americana vs. B. germanica: P < 0.05. One cockroach may simultaneously harbor more than one species of bacteria.
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Table 2 Gram-negative Enterobacteriaceae spp. isolated from Periplaneta americana (n = 123) and Blatella germanica (n = 103) collected from households Bacteria
Periplaneta americana Alimentary tract
External surface No.
Blatella germanica Totala
External surface
Total
No.
%
No.
%
No.
%
No.
%
No.
%
Klebsiella oxytoca Klebsiella pneumoniae Escherichia coli Enterobacter agglomerans Enterobacter aerogenes Enterobacter cloacae Enterobacter gergoviae Enterobacter dissolvens Serratia marcescens Serratia odorifera Serratia rubidaea Citrobacter freundii Proteus mirabilis Hafnia alvei
6 1 5 21 5 7 1 3 5 4 1 11 4 8
4.9 0.8 4.1b 17.1b 4.1 5.7b 0.8 2.4 4.1 3.3 0.8 8.9 3.3 6.5
4 1 0 8 4 2 0 0 2 2 0 6 1 3
3.3 0.8 0 6.5 3.3 1.6 0 0 1.6 1.6 0 4.9 0.8 2.4
7 1 5 14 7 8 1 3 6 5 1 12 4 10
5.7 0.8 4.1 17.1 5.7 6.5 0.8 2.4 4.9 4.1 0.8 9.8 3.2 8.1c
5 0 2 6 5 12 15 0 2 16 1 4 6 0
4.9 0 1.9 5.8 4.9 11.7 14.6 0 1.9 15.5 1.0 3.9 5.8 0
1 0 3 2 4 6 13 0 3 8 0 2 4 0
1.0 0 2.9 1.9 3.9 5.8 12.6 0 2.9 7.8 0 1.9 3.9 0
6 0 3 6 6 14 19 0 4 17 1 4 8 0
5.8 0 2.9 5.8 5.8 13.6 18.4 0 3.9 16.5c 1.0 3.8 7.7 0
Overalld
65
52.8b
31
25.2
71
57.7
65
63.1b
41
39.8
67
65.1
a b c d
%
Alimentary tract
One cockroach may simultaneously harbor the same bacterial species on the external surface and in the alimentary tract. Alimentary tract vs. external surface: P < 0.05. P. americana vs. B. germanica: P < 0.05. One cockroach may simultaneously harbor more than one species of bacteria.
in the positive rates between the two species of cockroaches (P. americana 12.2% and B. germanica 8.8%) and between the alimentary tract (P. americana 8.9% and B. germanica 7.8%) and external surface (P. americana 6.5% and B. germanica 4.9%) (P > 0.05) (Table 3).
3.3. Antibiotic resistance Of the two gram-positive bacteria studied, Enterococcus species had resistance to 8 of 12 antibiotics tested and S. aureus to 6 (Table 4). Among the gramnegative bacteria, E. coli and Proteus species had
Table 3 Glucose non- fermenter bacilli isolated from Periplaneta americana (n = 123) and Blatella germanica (n = 103) collected from households Bacteria
Pseudomonas aeruginosa Pseudomonas mallei Pseudomonas cepacia Acinetobacter species Oligella urethralis Shingobacterium mizutae Overallb a b
Periplaneta americana
Blatella germanica
Alimentary tract
External surface
Totala
No.
%
No.
%
No.
%
No.
%
No.
%
No.
%
0 1 4 3 1 3
0 0.8 3.3 2.4 0.8 2.4
0 2 3 1 2 0
0 1.6 2.4 0.8 1.6 0
0 3 6 3 2 3
0 2.4 4.9 2.4 1.6 2.4
1 0 6 1 0 1
1.0 0 5.8 1.0 0 1.0
1 0 3 0 0 1
1.0 0 2.9 0 0 1.0
1 0 7 1 0 1
1.0 0 6.8 1.0 0 1.0
11
8.9
8
6.5
15
12.2
8
7.8
5
4.9
9
8.8
Alimentary tract
External surface
One cockroach may simultaneously harbor the same bacterial species on the external surface and in the alimentary tract. One cockroach may simultaneously harbor more than one species of bacteria.
Total
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Table 4 Antimicrobial resistance of gram-positive bacteria isolated from cockroaches collected in households Bacteria
Staphylococcus aureus (n = 5) Enterococcus species (n = 26)
Antimicrobial resistance (%) GM
TE
SXT
SM
EM
OX
OFX
CRP
AMP
CIP
VA
PC
20.0 7.7
60.0 3.8
20.0 11.5
20.0 23.1
60.0 11.5
0 0
0 11.5
0 3.8
20.0 11.5
0 0
0 0
0 0
Abbreviations: GM: gentamicin; TE: tetracycline; SXT: sulfamethoxazole/trimethoprim; SM: streptomycin; EM: erythromycin; OX: oxacillin; OFX: ofloxacin; CRP: chloramphenicol; AMP: ampicillin; CIP: ciprofloxacin; VA: vancomycin; PC: penicillin.
resistance to 6 of 12 antibiotics tested, S. marcescens and P. aeruginosa to 5, and K. pneumoniae to 3 (Table 5). For individual strains of bacteria, E. coli was found to have multi-resistance to five antibiotics, Enterococcus species to four, P. aeruginosa, S. marcescens and Proteus species to three, and K. pneumoniae and S. aureus to two.
4. Discussion In this study, we isolated 26 species of bacteria from P. americana and B. germanica. From an urban area of France, 56 species of bacteria have been isolated from cockroaches and 14 species were pathogenic or potentially pathogenic for man and animals (Rivault et al., 1993). In an urban area of Malaysia, 17 species of bacteria were isolated from six species of cockroaches and E. coli and K. pneumoniae were the most important ones (Vythilingam et al., 1997). Although more bacterial species were isolated from P. americana in this study, no significant difference was observed in the bacterial positive rates in the two species of cockroaches. However, the distribution of the collection sites was significantly different between the two species: P. americana mainly in kitchen whereas
B. germanica in kitchen and storage room. It has been reported that cockroaches may play a role as vectors of microorganisms involved in food poisoning (Rueger and Olson, 1969). Moreover, association of cockroaches with an outbreak of dysentery has also been established (Burgess and Chetwyn, 1981). These findings and their special habitats suggest the importance of these two species of cockroaches in the transmission of causative agents for food poisoning. Although P. americana has been considered to be important as a spreader of Salmonella (Devi and Murray, 1991; Orlandella et al., 1994), we did not isolated any bacteria of this genus from the cockroaches in this study. Although we have found that P. americana harbored more species of bacteria than B. germanica, there was no significant difference between the positive rates of microorganisms between these two species of cockroaches. However, we have found that P. americana had a significantly higher positive rate for non-tuberculous mycobacteria than B. germanica in hospitals (Pai et al., 2003a). Although P. americana is three to four folds larger than B. germanica in length, the capability of harboring microorganisms in cockroaches is not only related to their sizes but may also depend on the sanitation conditions of the environment.
Table 5 Antimicrobial resistance of gram-negative bacteria isolated from cockroaches collected in households Bacteria
Pseudomonas aeruginosa (n = 2) Klebsiella pneumoniae (n = 2) Escherichia coli (n = 10) Serratia marcescens (n = 12) Proteus mirabilis (n = 15)
Antimicrobial resistance (%) AMP
PIP
CP
CRP
TE
SXT
CFP
CAZ
IMP
GM
CIP
OFX
100.0 100.0 60.0 50.0 73.3
0 0 10.0 16.6 20.0
50.0 0 50.0 58.3 26.6
50.0 0 10.0 0 13.3
50.0 50.0 80.0 0 26.7
100.0 100.0 20.0 33.3 40.0
0 0 0 7.7 0
0 0 0 0 0
0 0 0 0 0
0 0 0 0 0
0 0 0 0 0
0 0 0 0 0
Abbreviations: AMP: ampicillin; PIP: piperacillin; CP: cephalothin; CRP: chloramphenicol; TE: tetracycline; SXT: sulfamethoxazole/trimethoprim; CFP: cefoperazone; CAZ: ceftazidime; IMP: imipenem; GM: gentamicin; CIP: ciprofloxacin; OFX: ofloxacin.
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High prevalence of antibiotic resistance of common pathogenic bacteria occurs in medical centers in Taiwan. Over 30% of S. aureus, Strept. pneumoniae, Enterobacteriaceae species, P. aeruginosa, Acinetobacter baumannii, Haemophilus influenzae, coagulase-negative staphyloccocci, -hemolytic streptococci, viridans streptococci and enterococci have been found to have resistance to commonly used antibiotics (Chang et al., 2001). Recently, we reported resistance to ampicillin (13.7–100%), chloramphenicol (14.3–71.4%), tetracycline (14.3–73.3%), and sulfamethoxazole/trimethoprim (14.3–57.1%) in two gram-positive and five gram-negative pathogenic bacteria isolated from P. americana and B. germanica collected from hospitals of the same study areas (Pai et al., 2004). In this study, we found pathogenic bacteria such as S. aureus, Enterococcus species, P. aeruginosa, K. pneumoniae, E. coli, S. marcescens, and Proteus species isolated from household cockroaches with resistance to antibiotics. This may be the first time for the isolation of bacteria with antibiotic resistance from household cockroaches. Moreover, individual strains of E. coli were found to have multi-resistance to five antibiotics. Existence of bacteria with increased antibiotic resistance in household cockroaches is a sign of the spread of these bacteria also outside hospitals in Taiwan. Based on the results of this study, cockroach infestation occurs in 50% of the households studied. A considerable number of bacteria were isolated from nearly all cockroaches collected from these households. Moreover, potentially pathogenic bacteria isolated from these insects were also found to have an increased resistance to antibiotics. Recently, we have also determined that P. americana and B. germanica may play a role as potential mechanical disseminators of Entamoeba histolytica (Pai et al., 2003b). These insects may also play some role for the transmission of a wide range of bacteria with antibiotic resistance in the households of the tropical and subtropical regions.
Acknowledgements This study was supported in part by a grant (NSC902320-B-037-031) from the National Science Council, Executive Yuan, ROC.
References Burgess, N.R.H., Chetwyn, K.N., 1981. Association of cockroaches with an outbreak of dysentery. Trans. R. Soc. Trop. Med. Hyg. 75, 323–333. Chang, S.C., Hsieh, W.C., Liu, C.Y., The Antibiotic Resistance Study Group of the Infectious Disease Society of the Republic of China, 2001. High prevalence of antibiotic resistance of common pathogenic bacteria in Taiwan. Diag. Microbiol. Infect. Dis. 36, 107–112. Cloarec, A., Rivault, C., Fontaine, F., Leguyader, A., 1992. Cockroaches as carriers of bacteria in multi-family dwellings. Epidemiol. Infect. 109, 483–490. Cotton, M.F., Wasserman, E., Pieper, C.H., Theron, D.C., van Tubbergh, D., Campbell, G., Fang, F.C., Barnes, J., 2000. Invasive disease due to extended spectrum beta-lactamase-producing Klebsiella pneumoniae in a neonatal unit: the possible role of cockroaches. J. Hosp. Infect. 44, 13–17. Cruden, D.L., Markovetz, A.J., 1987. Microbial ecology of the cockroach gut. Ann. Rev. Microbiol. 41, 617–643. Devi, S.J., Murray, C.J., 1991. Cockroaches (Blatta and Periplaneta species) as reservoirs of drug-resistant salmonellas. Epidemiol. Infect. 107, 357–361. Fotedar, R., Nayar, E., Samantray, J.C., Shriniwas, U.B., Dogra, V., Kumar, A., 1989. Cockroaches as vectors of pathogenic bacteria. J. Commun. Dis. 21, 318–322. Fotedar, R., Shriniwas, U.B., Verma, A., 1991a. Cockroaches (Blattella germanica) as carriers of microorganisms of medical importance in hospitals. Epidemiol. Infect. 107, 181– 187. Fotedar, R., Shriniwas, U.B., Banerjee, U., Samantray, J.C., Nayar, E., Verma, A., 1991b. Nosocomial infections: cockroaches as possible vectors of drug-resistant Klebsiella. J. Hosp. Infect. 18, 155–159. Fotedar, R., Banerjee, U., Shriniwas, U.B., 1993. Vector potential of the German cockroach in dissemination of Pseudomonas aeruginosa. J. Hosp. Infect. 23, 55–59. Harwood, R.F., James, M.T., 1979. Entomology in Human and Animal Health, seventh ed. Macmillan Publishing, New York, NY. Hsu, E.L., 1990. Etiology and control of some common cockroaches in Taiwan. In: Proceedings of the 1990 Vector Control Workshop. Taiwan Provincial Association of Environmental Sanitation, Taipei, pp. 45–64. Klowden, M.J., Greenberg, B., 1976. Salmonella in the American cockroach: Evaluation of vector potential through dosed feeding experiments. J. Hyg. 77, 105–111. Murray, P.R., 1999. Manual of Clinical Microbiology, seventh ed. ASM Press, Washington, DC. National Committee for Clinical Laboratory Standards (NCCLS), 1993. Performance Standards for Antimicrobial Disk Susceptibility Tests, Approved Standard, fifth ed. NCCLS, Villanova, PA. Orlandella, B.M., Foti, M., Orlandella, V., Daidone, A., 1994. Environmental pollution and infectious diseases. I. Isolation of a new serovar of the Salmonella genus, S. V 13. 22:r: from a Periplaneta americana cockroach. G. Batteriol. Virol. Immunol. 86, 101–115.
H.-H. Pai et al. / Acta Tropica 93 (2005) 259–265 Pai, H.H., Chen, W.C., Peng, C.F., 2003a. Isolation of nontuberculous mycobacteria from nosocomial cockroaches. J. Hosp. Infect. 53, 224–228. Pai, H.H., Ko, Y.C., Chen, E.R., 2003b. Cockroaches (Periplaneta americana and Blattella germanica) as potential mechanical disseminators of Entamoeba histolytica. Acta Trop. 87, 355–359. Pai, H.H., Chen, W.C., Peng, C.F., 2004. Cockroaches as potential vectors of nosocomial infections. Infect. Contrl. Hosp. Epidemiol 25, 979–984. Reierson, D.A., Rust, M.K., 1997. Trapping, flushing, counting German roaches. Pest Control 40, 42–44. Rivault, C., Cloarec, A., Le Guyader, A., 1993. Bacterial load of cockroaches in relation to urban environment. Epidemiol. Infect. 110, 317–325.
265
Roth, L.M., Willis, E.R., 1957. The Medical and Veterinary Importance of Cockroaches. Smithson. Miscellaneous Collect. 134, 1–147. Rueger, M.E., Olson, T.A., 1969. Cockroaches (Blattaria) as vectors of food poisoning and food infection organisms. J. Med. Entomol. 6, 185–189. Vythilingam, I., Jeffery, J., Oothuman, P., Abdul Razak, A.R., Sulaiman, A., 1997. Cockroaches from urban human dwellings: Isolation of bacterial pathogens and control. Southeast Asian J. Trop. Med. Public Health 28, 218–222.
Isolation of bacteria with antibiotic resistance from household cockroaches (Periplaneta americana and Blattella germanica) Hsiu-Hua Pai∗ , Wei-Chen Chen, Chien-Fang Peng Graduate Institute of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan, ROC Received 10 October 2003; received in revised form 25 October 2004; accepted 11 November 2004 Available online 1 February 2005
Abstract Cockroaches may harbor and disseminate microorganisms to the environment. In this study, Periplaneta americana and Blattella germanica were collected from 40 households in Kaohsiung City and Kaohsiung County, Taiwan. Cockroach infestation was found in 50% of the studied households and 226 cockroaches (123 P. americana and 103 B. germanica) collected by trapping. P. americana was more often found in the kitchen (70.7%) whereas B. germanica in the storage room (51.5%) and kitchen (36.9%). There was no significant difference between the percentages of P. americana (99.9%) and B. germanica (98.0%) carrying bacteria. A total of 25 species of bacteria was isolated from P. americana and only 21 from B. germanica. Antibiotic resistance was found in Staphylococcus aureus, Enterococcus species, Pseudomonas aeruginosa, Klebsiella pneumoniae, Escherichia coli, Serratia marcescens, and Proteus species isolated from the cockroaches. These findings suggest a potential role of cockroaches in the transmission of pathogenic bacteria with antibiotic resistance in households. © 2005 Elsevier B.V. All rights reserved. Keywords: Bacteria; Blattella germanica; Cockroaches; Antibiotic resistance; Vectors; Periplaneta americana
1. Introduction Owing to their omnivorous habit of feeding and indiscriminate deposition of fecal materials, cockroaches are well-known agents for harboring and transmission of microorganisms (Roth and Willis, 1957; Cloarec et al., 1992; Rivault et al., 1993). Over ∗ Corresponding author. Tel.: +886 7 312 1101x2141; fax: +886 7 322 2362. E-mail address: [email protected] (H.-H. Pai).
0001-706X/$ – see front matter © 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.actatropica.2004.11.006
100 species of bacteria have been isolated from or passed through cockroaches (Cruden and Markovetz, 1987). In addition to these bacteria, we have recently isolated non-tuberculous mycobacteria from hospital cockroaches (Pai et al., 2003a). Although studies with Periplaneta americana showed that Salmonella species do not readily become established in the intestinal tract of the insect (Klowden and Greenberg, 1976), Pseudomonas aeruginosa has been demonstrated to multiply in the gut of Blattela germanica and excretion of the bacteria continued up to 114
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days (Fotedar et al., 1993). Therefore, the cockroach may serve as a reservoir for multiplication of pathogens. Although important pathogenic microorganisms such as P. aeruginosa, Staphylococcus aureus, and Streptococcus faecalis have been isolated from B. germanica collected in hospitals, these species were not found in the corresponding residential areas (Fotedar et al., 1989.). Moreover, cockroaches in hospitals may harbor bacteria with resistance to antibiotics (Fotedar et al., 1991a) and those carrying Klebsiella species with antibiotic resistance has been suggested to play a role in nosocomial infections (Fotedar et al., 1991b). Recently, cockroaches have been suggested to be possible vectors for an outbreak of nosocomial beta-lactamase-producing Klebsiella pneumoniae infections in a neonatal unit with heavy cockroach infestation (Cotton et al., 2000). The subtropical climate in Taiwan is favorable to the growth and development of populations of cockroaches throughout the island (Hsu, 1990). Moreover, isolates of pathogenic bacteria from medical centers have been reported to have high prevalence of resistance to commonly used antimicrobial agents (Chang et al., 2001). It is possible for bacteria with antibiotic resistance to occur in household cockroaches. In this study, we collected P. americana and B. germanica from households in Kaohsiung City and Kaohsiung County. After isolating bacteria from the trapped insects, we determined whether antibiotic resistance exists in the potentially pathogenic ones.
2. Materials and methods 2.1. Collection and identification of cockroaches In March 2000, cockroaches were collected from 40 households in Kaohsiung City and Kaohsiung County. These households were randomly selected and at least 5 km from hospitals. In addition, the occupants were not working in health facilities and did not receive antibiotic therapy in the recent. Traps were manufactured according to the design of Reierson and Rust (1997). The traps were placed on the floor under beds, cupboards, wooden racks, and/or benches, for two consecutive days. Each trapped cockroach was placed in a sterile test tube before sending to our laboratory.
After immobilization by freezing at 0 ◦ C for 5 min, the species of cockroaches were identified under a dissecting microscope according to Harwood and James (1979). 2.2. Isolation and identification of microorganisms After species identification, 2 ml of sterile normal saline was added to the tube before thorough shaking for 2 min. Aliquots (0.01 ml) of the washings were then separately cultured on plates of tryptic soy agar blood agar (Difco, Detroit, USA), eosin methylene blue agar (Difco), and xylose lysine desoxycholate agar (Difco) at 35 ◦ C overnight. Bacterial colonies were identified by macroscopic examination, gram staining, and biochemical tests according to Murray (1999). After isolating microorganisms from the external surface, the cockroaches were washed in 70% ethyl alcohol for 5 min and allowed to dry at room temperature under sterile conditions. The insects were then washed with sterile normal saline for 3 min. The alimentary tract was dissected out and aseptically macerated in a sterile pestle and mortar with 2 ml of sterile normal saline. The macerate was then cultured on the same types of agar plates at 35 ◦ C overnight before specific identification of the colonies. 2.3. Antibiotic susceptibility tests The antibiotic susceptibility of S. aureus, Enterococcus species, P. aeruginosa, K. pneumoniae, Escherichia coli, Serratia marcescens, Proteus species to 17 antibiotics were tested by the Kirby-Bauer disk diffusion method according to the recommendations of the National Committee for Clinical Laboratory Standards (1993). These antibiotics (Becton Dickinson, Cockeysville, MD, USA) included ampicillin (10 g/disk), gentamicin (10 g/disk), ciprofloxacin (5 g/disk), ofloxacin (5 g/disk), chloramphenicol (30 g/disk), tetracycline (30 g/disk), sulfamethoxazole/trimethoprim (25 g/disk), penicillin (10 U/disk), streptomycin (10 g/disk), erythromycin (15 g/disk), oxacillin (1 g/disk), vancomycin (30 g/disk), cephalothin (30 g/disk), ceftazidime (30 g/disk), imipenem (10 g/disk), piperacillin (100 g/disk), and cefoperazone (75 g/disk).
H.-H. Pai et al. / Acta Tropica 93 (2005) 259–265
2.4. Statistical analysis Rates were compared using the χ2 test. P < 0.05 was considered to be statistically significant.
3. Results 3.1. Cockroach infestation Of the 40 households studied, 20 (50%) were found to have cockroach infestation: 11 (55%) with P. americana, 3 (15%) with B. germanica, and 6 (30%) with both species. A total of 226 adult cockroaches were collected: 123 P. americana and 103 B. germanica. Most of the P. americana (70.7%) were collected from kitchen while the higher collection rates of B. germanica was obtained in the storage room (51.5%) and kitchen (36.9%). The percentages of P. americana collected in the sleeping room, living room, storage room, and dining room were 12.2, 10.6, 3.3, and 3.3%, respectively. The collection rates of B. germanica obtained in living room and dining room were 8.7 and 2.9%, respectively. 3.2. Bacterial isolation Among the 226 cockroaches collected, 222 (98.2%) were found to carry one or more species of bacteria
261
on the external surface or in the alimentary tract. Although there was no significant difference between the overall positive rates in P. americana (99.9%) and B. germanica (98.0%) (P > 0.05), the positive rate of the alimentary tract (97.3%) was significantly higher than that of the external surface (88.1%) (P < 0.05). Six species of gram-positive bacteria were isolated from the two species of cockroaches. There was no significant difference between positive rates in the alimentary tract (P. americana 69.9%, B. germanica 57.3%) and those on the external surface (P. americana 70.7%, B. germanica 56.3%) (P > 0.05). However, the positive rate was found to be significantly higher in P. americana (86.2%) than in B. germanica (72.8%) (P < 0.05) (Table 1). Fourteen species of gram-negative Enterobacteriaceae species were isolated from P. americana and only 11 from B. germanica. Significantly higher positive rates were found in the alimentary tract (P. americana 52.8% and B. germanica 63.1%) than on the external surface (P. americana 25.2% and B. germanica 39.8%) (P < 0.05). However, there was no significant difference between the positive rates in P. americana (57.5%) and B. germanica (65.1%) (P > 0.05) (Table 2). Five species of glucose non-fermenter bacilli were isolated from P. americana and four from B. germanica. There were no significant differences
Table 1 Gram-positive bacteria isolated from Periplaneta americana (n = 123) and Blatella germanica (n = 103) collected from households Bacteria
Periplaneta americana Alimentary tract
Streptococcus species (non-Enterococcus) Bacillus species (except B. subtilis) Bacillus subtilis Staphylococcus aureus Staphylococcus epidermidis Enterococcus species Overalld a b c d
Blatella germanica
External surface
Totala No.
Alimentary tract
External surface
Total
No.
%
No.
%
%
No.
%
No.
%
No.
%
46
37.4
50
40.7
78
62.6
36
35.0
32
31.1
55
53.4
33
26.8b
17
13.8
44
35.8
17
16.5
16
16.5
28
27.2
36 2 16 12
29.3b 1.6 13.0 9.8
14 2 18 6
11.4 1.6 14.6 4.9
44 3 27 14
35.8c 2.4 22.0 11.4c
8 1 18 6
7.8 1.0 17.5 5.8
7 0 10 2
6.8 0 9.7 1.9
14 1 27 6
13.6 1.0 26.2 5.8
86
69.9
87
70.7
104
86.2c
59
57.3
58
56.3
75
72.8
One cockroach may simultaneously harbor the same bacterial species on the external surface and in the alimentary tract. Alimentary tract vs. external surface: P < 0.05. P. americana vs. B. germanica: P < 0.05. One cockroach may simultaneously harbor more than one species of bacteria.
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Table 2 Gram-negative Enterobacteriaceae spp. isolated from Periplaneta americana (n = 123) and Blatella germanica (n = 103) collected from households Bacteria
Periplaneta americana Alimentary tract
External surface No.
Blatella germanica Totala
External surface
Total
No.
%
No.
%
No.
%
No.
%
No.
%
Klebsiella oxytoca Klebsiella pneumoniae Escherichia coli Enterobacter agglomerans Enterobacter aerogenes Enterobacter cloacae Enterobacter gergoviae Enterobacter dissolvens Serratia marcescens Serratia odorifera Serratia rubidaea Citrobacter freundii Proteus mirabilis Hafnia alvei
6 1 5 21 5 7 1 3 5 4 1 11 4 8
4.9 0.8 4.1b 17.1b 4.1 5.7b 0.8 2.4 4.1 3.3 0.8 8.9 3.3 6.5
4 1 0 8 4 2 0 0 2 2 0 6 1 3
3.3 0.8 0 6.5 3.3 1.6 0 0 1.6 1.6 0 4.9 0.8 2.4
7 1 5 14 7 8 1 3 6 5 1 12 4 10
5.7 0.8 4.1 17.1 5.7 6.5 0.8 2.4 4.9 4.1 0.8 9.8 3.2 8.1c
5 0 2 6 5 12 15 0 2 16 1 4 6 0
4.9 0 1.9 5.8 4.9 11.7 14.6 0 1.9 15.5 1.0 3.9 5.8 0
1 0 3 2 4 6 13 0 3 8 0 2 4 0
1.0 0 2.9 1.9 3.9 5.8 12.6 0 2.9 7.8 0 1.9 3.9 0
6 0 3 6 6 14 19 0 4 17 1 4 8 0
5.8 0 2.9 5.8 5.8 13.6 18.4 0 3.9 16.5c 1.0 3.8 7.7 0
Overalld
65
52.8b
31
25.2
71
57.7
65
63.1b
41
39.8
67
65.1
a b c d
%
Alimentary tract
One cockroach may simultaneously harbor the same bacterial species on the external surface and in the alimentary tract. Alimentary tract vs. external surface: P < 0.05. P. americana vs. B. germanica: P < 0.05. One cockroach may simultaneously harbor more than one species of bacteria.
in the positive rates between the two species of cockroaches (P. americana 12.2% and B. germanica 8.8%) and between the alimentary tract (P. americana 8.9% and B. germanica 7.8%) and external surface (P. americana 6.5% and B. germanica 4.9%) (P > 0.05) (Table 3).
3.3. Antibiotic resistance Of the two gram-positive bacteria studied, Enterococcus species had resistance to 8 of 12 antibiotics tested and S. aureus to 6 (Table 4). Among the gramnegative bacteria, E. coli and Proteus species had
Table 3 Glucose non- fermenter bacilli isolated from Periplaneta americana (n = 123) and Blatella germanica (n = 103) collected from households Bacteria
Pseudomonas aeruginosa Pseudomonas mallei Pseudomonas cepacia Acinetobacter species Oligella urethralis Shingobacterium mizutae Overallb a b
Periplaneta americana
Blatella germanica
Alimentary tract
External surface
Totala
No.
%
No.
%
No.
%
No.
%
No.
%
No.
%
0 1 4 3 1 3
0 0.8 3.3 2.4 0.8 2.4
0 2 3 1 2 0
0 1.6 2.4 0.8 1.6 0
0 3 6 3 2 3
0 2.4 4.9 2.4 1.6 2.4
1 0 6 1 0 1
1.0 0 5.8 1.0 0 1.0
1 0 3 0 0 1
1.0 0 2.9 0 0 1.0
1 0 7 1 0 1
1.0 0 6.8 1.0 0 1.0
11
8.9
8
6.5
15
12.2
8
7.8
5
4.9
9
8.8
Alimentary tract
External surface
One cockroach may simultaneously harbor the same bacterial species on the external surface and in the alimentary tract. One cockroach may simultaneously harbor more than one species of bacteria.
Total
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Table 4 Antimicrobial resistance of gram-positive bacteria isolated from cockroaches collected in households Bacteria
Staphylococcus aureus (n = 5) Enterococcus species (n = 26)
Antimicrobial resistance (%) GM
TE
SXT
SM
EM
OX
OFX
CRP
AMP
CIP
VA
PC
20.0 7.7
60.0 3.8
20.0 11.5
20.0 23.1
60.0 11.5
0 0
0 11.5
0 3.8
20.0 11.5
0 0
0 0
0 0
Abbreviations: GM: gentamicin; TE: tetracycline; SXT: sulfamethoxazole/trimethoprim; SM: streptomycin; EM: erythromycin; OX: oxacillin; OFX: ofloxacin; CRP: chloramphenicol; AMP: ampicillin; CIP: ciprofloxacin; VA: vancomycin; PC: penicillin.
resistance to 6 of 12 antibiotics tested, S. marcescens and P. aeruginosa to 5, and K. pneumoniae to 3 (Table 5). For individual strains of bacteria, E. coli was found to have multi-resistance to five antibiotics, Enterococcus species to four, P. aeruginosa, S. marcescens and Proteus species to three, and K. pneumoniae and S. aureus to two.
4. Discussion In this study, we isolated 26 species of bacteria from P. americana and B. germanica. From an urban area of France, 56 species of bacteria have been isolated from cockroaches and 14 species were pathogenic or potentially pathogenic for man and animals (Rivault et al., 1993). In an urban area of Malaysia, 17 species of bacteria were isolated from six species of cockroaches and E. coli and K. pneumoniae were the most important ones (Vythilingam et al., 1997). Although more bacterial species were isolated from P. americana in this study, no significant difference was observed in the bacterial positive rates in the two species of cockroaches. However, the distribution of the collection sites was significantly different between the two species: P. americana mainly in kitchen whereas
B. germanica in kitchen and storage room. It has been reported that cockroaches may play a role as vectors of microorganisms involved in food poisoning (Rueger and Olson, 1969). Moreover, association of cockroaches with an outbreak of dysentery has also been established (Burgess and Chetwyn, 1981). These findings and their special habitats suggest the importance of these two species of cockroaches in the transmission of causative agents for food poisoning. Although P. americana has been considered to be important as a spreader of Salmonella (Devi and Murray, 1991; Orlandella et al., 1994), we did not isolated any bacteria of this genus from the cockroaches in this study. Although we have found that P. americana harbored more species of bacteria than B. germanica, there was no significant difference between the positive rates of microorganisms between these two species of cockroaches. However, we have found that P. americana had a significantly higher positive rate for non-tuberculous mycobacteria than B. germanica in hospitals (Pai et al., 2003a). Although P. americana is three to four folds larger than B. germanica in length, the capability of harboring microorganisms in cockroaches is not only related to their sizes but may also depend on the sanitation conditions of the environment.
Table 5 Antimicrobial resistance of gram-negative bacteria isolated from cockroaches collected in households Bacteria
Pseudomonas aeruginosa (n = 2) Klebsiella pneumoniae (n = 2) Escherichia coli (n = 10) Serratia marcescens (n = 12) Proteus mirabilis (n = 15)
Antimicrobial resistance (%) AMP
PIP
CP
CRP
TE
SXT
CFP
CAZ
IMP
GM
CIP
OFX
100.0 100.0 60.0 50.0 73.3
0 0 10.0 16.6 20.0
50.0 0 50.0 58.3 26.6
50.0 0 10.0 0 13.3
50.0 50.0 80.0 0 26.7
100.0 100.0 20.0 33.3 40.0
0 0 0 7.7 0
0 0 0 0 0
0 0 0 0 0
0 0 0 0 0
0 0 0 0 0
0 0 0 0 0
Abbreviations: AMP: ampicillin; PIP: piperacillin; CP: cephalothin; CRP: chloramphenicol; TE: tetracycline; SXT: sulfamethoxazole/trimethoprim; CFP: cefoperazone; CAZ: ceftazidime; IMP: imipenem; GM: gentamicin; CIP: ciprofloxacin; OFX: ofloxacin.
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High prevalence of antibiotic resistance of common pathogenic bacteria occurs in medical centers in Taiwan. Over 30% of S. aureus, Strept. pneumoniae, Enterobacteriaceae species, P. aeruginosa, Acinetobacter baumannii, Haemophilus influenzae, coagulase-negative staphyloccocci, -hemolytic streptococci, viridans streptococci and enterococci have been found to have resistance to commonly used antibiotics (Chang et al., 2001). Recently, we reported resistance to ampicillin (13.7–100%), chloramphenicol (14.3–71.4%), tetracycline (14.3–73.3%), and sulfamethoxazole/trimethoprim (14.3–57.1%) in two gram-positive and five gram-negative pathogenic bacteria isolated from P. americana and B. germanica collected from hospitals of the same study areas (Pai et al., 2004). In this study, we found pathogenic bacteria such as S. aureus, Enterococcus species, P. aeruginosa, K. pneumoniae, E. coli, S. marcescens, and Proteus species isolated from household cockroaches with resistance to antibiotics. This may be the first time for the isolation of bacteria with antibiotic resistance from household cockroaches. Moreover, individual strains of E. coli were found to have multi-resistance to five antibiotics. Existence of bacteria with increased antibiotic resistance in household cockroaches is a sign of the spread of these bacteria also outside hospitals in Taiwan. Based on the results of this study, cockroach infestation occurs in 50% of the households studied. A considerable number of bacteria were isolated from nearly all cockroaches collected from these households. Moreover, potentially pathogenic bacteria isolated from these insects were also found to have an increased resistance to antibiotics. Recently, we have also determined that P. americana and B. germanica may play a role as potential mechanical disseminators of Entamoeba histolytica (Pai et al., 2003b). These insects may also play some role for the transmission of a wide range of bacteria with antibiotic resistance in the households of the tropical and subtropical regions.
Acknowledgements This study was supported in part by a grant (NSC902320-B-037-031) from the National Science Council, Executive Yuan, ROC.
References Burgess, N.R.H., Chetwyn, K.N., 1981. Association of cockroaches with an outbreak of dysentery. Trans. R. Soc. Trop. Med. Hyg. 75, 323–333. Chang, S.C., Hsieh, W.C., Liu, C.Y., The Antibiotic Resistance Study Group of the Infectious Disease Society of the Republic of China, 2001. High prevalence of antibiotic resistance of common pathogenic bacteria in Taiwan. Diag. Microbiol. Infect. Dis. 36, 107–112. Cloarec, A., Rivault, C., Fontaine, F., Leguyader, A., 1992. Cockroaches as carriers of bacteria in multi-family dwellings. Epidemiol. Infect. 109, 483–490. Cotton, M.F., Wasserman, E., Pieper, C.H., Theron, D.C., van Tubbergh, D., Campbell, G., Fang, F.C., Barnes, J., 2000. Invasive disease due to extended spectrum beta-lactamase-producing Klebsiella pneumoniae in a neonatal unit: the possible role of cockroaches. J. Hosp. Infect. 44, 13–17. Cruden, D.L., Markovetz, A.J., 1987. Microbial ecology of the cockroach gut. Ann. Rev. Microbiol. 41, 617–643. Devi, S.J., Murray, C.J., 1991. Cockroaches (Blatta and Periplaneta species) as reservoirs of drug-resistant salmonellas. Epidemiol. Infect. 107, 357–361. Fotedar, R., Nayar, E., Samantray, J.C., Shriniwas, U.B., Dogra, V., Kumar, A., 1989. Cockroaches as vectors of pathogenic bacteria. J. Commun. Dis. 21, 318–322. Fotedar, R., Shriniwas, U.B., Verma, A., 1991a. Cockroaches (Blattella germanica) as carriers of microorganisms of medical importance in hospitals. Epidemiol. Infect. 107, 181– 187. Fotedar, R., Shriniwas, U.B., Banerjee, U., Samantray, J.C., Nayar, E., Verma, A., 1991b. Nosocomial infections: cockroaches as possible vectors of drug-resistant Klebsiella. J. Hosp. Infect. 18, 155–159. Fotedar, R., Banerjee, U., Shriniwas, U.B., 1993. Vector potential of the German cockroach in dissemination of Pseudomonas aeruginosa. J. Hosp. Infect. 23, 55–59. Harwood, R.F., James, M.T., 1979. Entomology in Human and Animal Health, seventh ed. Macmillan Publishing, New York, NY. Hsu, E.L., 1990. Etiology and control of some common cockroaches in Taiwan. In: Proceedings of the 1990 Vector Control Workshop. Taiwan Provincial Association of Environmental Sanitation, Taipei, pp. 45–64. Klowden, M.J., Greenberg, B., 1976. Salmonella in the American cockroach: Evaluation of vector potential through dosed feeding experiments. J. Hyg. 77, 105–111. Murray, P.R., 1999. Manual of Clinical Microbiology, seventh ed. ASM Press, Washington, DC. National Committee for Clinical Laboratory Standards (NCCLS), 1993. Performance Standards for Antimicrobial Disk Susceptibility Tests, Approved Standard, fifth ed. NCCLS, Villanova, PA. Orlandella, B.M., Foti, M., Orlandella, V., Daidone, A., 1994. Environmental pollution and infectious diseases. I. Isolation of a new serovar of the Salmonella genus, S. V 13. 22:r: from a Periplaneta americana cockroach. G. Batteriol. Virol. Immunol. 86, 101–115.
H.-H. Pai et al. / Acta Tropica 93 (2005) 259–265 Pai, H.H., Chen, W.C., Peng, C.F., 2003a. Isolation of nontuberculous mycobacteria from nosocomial cockroaches. J. Hosp. Infect. 53, 224–228. Pai, H.H., Ko, Y.C., Chen, E.R., 2003b. Cockroaches (Periplaneta americana and Blattella germanica) as potential mechanical disseminators of Entamoeba histolytica. Acta Trop. 87, 355–359. Pai, H.H., Chen, W.C., Peng, C.F., 2004. Cockroaches as potential vectors of nosocomial infections. Infect. Contrl. Hosp. Epidemiol 25, 979–984. Reierson, D.A., Rust, M.K., 1997. Trapping, flushing, counting German roaches. Pest Control 40, 42–44. Rivault, C., Cloarec, A., Le Guyader, A., 1993. Bacterial load of cockroaches in relation to urban environment. Epidemiol. Infect. 110, 317–325.
265
Roth, L.M., Willis, E.R., 1957. The Medical and Veterinary Importance of Cockroaches. Smithson. Miscellaneous Collect. 134, 1–147. Rueger, M.E., Olson, T.A., 1969. Cockroaches (Blattaria) as vectors of food poisoning and food infection organisms. J. Med. Entomol. 6, 185–189. Vythilingam, I., Jeffery, J., Oothuman, P., Abdul Razak, A.R., Sulaiman, A., 1997. Cockroaches from urban human dwellings: Isolation of bacterial pathogens and control. Southeast Asian J. Trop. Med. Public Health 28, 218–222.