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Here we report the isolation of extended-spectrum β-lactamase (ESBL)- and carbapenemase-producing Enterobacteriaceae from German cockroaches caught in the burn unit of Batna University Hospital in Algeria. Nine of 12 isolates harbored the blaCTX-M-15 ESBL gene. One Enterobacter cloacae isolate belonging to sequence type 528 coexpressed the blaOXA-48, blaCTX-M-15, and blaTEM genes. Our findings indicate that cockroaches may be one of the most dangerous reservoirs for ESBL and carbapenemase producers in hospitals.
The emergence of multidrug-resistant Enterobacteriaceae (MDRE) and their dissemination in hospitals have become a major health concern worldwide (1). The production of β-lactamases such as extended-spectrum β-lactamase (ESBL) and carbapenemase remains the main mechanism of resistance in Enterobacteriaceae (2). Algeria is now feaures among the Mediterranean countries known to be affected by the spread of CTX-M-15 type ESBL and the emergence of the phantom menace OXA-48 type carbapenemase (3, 4). Indeed, the transmission and dissemination of MDRE in hospitals are usually related to hand contact (5) and poorly sterilized instruments (6). However, cockroaches which colonize these environments may also act as potential mechanical vectors of antibiotic-resistant bacteria (7). Cockroaches are among the most prevalent pests in hospitals, where they are attracted by moisture, food, and suitable temperatures (8, 9). The medical significance of cockroaches has been largely overlooked, but it has been proven that they can harbor a number of pathogenic microorganisms with different levels of antibiotic resistance (10). Blattella germanica is the most abundant cockroach species (11), and the external surfaces and excrement of these cockroaches can contaminate food, human habitats, and hospital equipment; therefore, these insects can present a threat to human health (12).
The aim of our study was to screen for broad-spectrum-cephalosporin- and carbapenem-resistant Enterobacteriaceae in B. germanica cockroaches collected from the burn unit of Batna University Hospital in Algeria and then to investigate the molecular support of ESBL and carbapenemase production.
In March 2015, 10 cockroaches were randomly captured from different parts of the burn unit, including the kitchen and the guard, treatment, and patient rooms, directly in sterile containers. Samples were immobilized by freezing at 0°C for 10 min (8). Each cockroach was soaked in 5 ml of Tween 80 solution at 0.05% and vortexed vigorously for 2 min. The resulting wash was used as an external-body-homogenate sample. To remove the external body contamination, cockroaches were immersed in bleach for 2 min, in sterile physiological saline for 2 min, and then in 70% ethanol for 5 min (12). Then, each sample was washed with sterile physiological saline. Subsequently, the insect was soaked in a sterile bottle containing 5 ml of Tween 80 solution at 0.05% and then crushed inside using a sterile pestle. The triturate was then vortexed vigorously for 2 min. The resulting suspension was used as an internal-body-homogenate sample. Each sample (5 ml) was preenriched for 4 h in 5 ml of a double concentration of brain heart infusion (BHI) (BD: Becton, Dickinson and Company, France). Subsequently, the screening for β-lactam-resistant Enterobacteriaceae began with a selective enrichment step using two selective media: BHI supplemented with 64 mg/liter of vancomycin and 1 mg/liter of ertapenem and BHI supplemented with 64 mg/liter of vancomycin and 2 mg/liter of cefotaxime. After overnight incubation at 37°C, cultures were inoculated by streaking 100 μl of the growth onto MacConkey agar plates (BD) with 64 mg/liter of vancomycin supplemented with 1 mg/liter of ertapenem or 2 mg/liter of cefotaxime, respectively. The obtained Enterobacteriaceae isolates were identified using an API 20E system (bioMérieux, France) and confirmed by matrix-assisted laser desorption ionization–time of flight mass spectrometry (13). Susceptibility testing was performed using a standard disk diffusion technique according to the recommendations of the Antibiotic Committee of the French Society for Microbiology (http://www.sfm-microbiologie.org/UserFiles/files/casfm/CASFM_EUCAST_V1_2015.pdf). ESBL detection was performed using the double-disk synergy test (DDST) as previously described (14). The modified Hodge test (MHT) and the modified Carba NP (MCNP) test were used to screen for carbapenemase activity (15, 16). All strains were screened using real-time PCR for the presence of the following β-lactamase-encoding genes: blaKPC, blaNDM, blaVIM, blaIMP, blaOXA-48, blaCTX-M, blaTEM, and blaSHV (17,–19). Strains positive by real-time PCR were subjected to standard PCR and sequencing (18, 20). Multilocus sequence typing (MLST) was carried out for carbapenemase-producing strains with the PubMLST scheme (http://pubmlst.org/ecloacae/).
Twelve Enterobacteriaceae isolates were obtained from 12 representative colonies, and strains of the following species were identified: Klebsiella oxytoca, Citrobacter freundii, Enterobacter cloacae, and Citrobacter amalonaticus (Table 1). The isolation from hospital cockroaches of such Enterobacteriaceae species with different levels of antimicrobial resistance has already been described (21). Additionally, in this study, we report for the first time the identification of the Enterobacteriaceae species Citrobacter farmeri, Citrobacter koseri, and Enterobacter kobei in German cockroaches. The majority of strains were isolated from the internal organs (Table 1), which could be explained by the great adaptation of these organisms in the intestinal environment (22). The antimicrobial susceptibility test results are presented in Table 1. The DDST was positive for 11 of 12 isolates. The MHT and the MCNP test were positive for only one E. cloacae isolate. Eleven of the 12 obtained strains harbored blaTEM, and 75% (n = 9) of all isolates coharbored the blaCTX-M-15 ESBL gene. One E. cloacae isolate belonging to sequence type 528 (ST528) coharbored the blaOXA-48, blaCTX-M-15, and blaTEM genes. According to the antibiogram results, some of the strains could express other β-lactamases not sought in this study. To our knowledge, this is the first description of CTX-M-15-producing Enterobacteriaceae isolated from B. germanica cockroaches. Few reports describing the presence of ESBL-producing Enterobactericeae in hospital cockroaches have been published (23,–26). The most interesting finding in the present study was the first detection of OXA-48-producing E. cloacae in B. germanica. Since its first description in Turkey (27), numerous reports have indicated the spread of OXA-48 type carbapenemase in Mediterranean countries (28,–30), where it represents the most common carbapenemase type (31). Subsequently, several studies have reported the isolation of OXA-48-producing Enterobacteriaceae from different samples (32, 33). Prior to its first description in Algeria, Poirel et al. suggested that OXA-48 may be endemic in this country (34). The huge spread of CTX-M-15 ESBL-producing Enterobacteriaceae and the emergence of OXA-48-type carbapenemase-producing Enterobacteriaceae in Algerian hospitals may explain the carriage of such drug-resistant bacteria by hospital cockroaches through their particular feeding habits (3, 4, 35).
The important role of cockroaches as a potential reservoir of multidrug-resistant bacteria demonstrated in this study and in earlier investigations with their presence in hospital environments, which has already been reported in Algeria (36) and other countries such as Turkey (9), Japan (37), and Cuba (38), urgently call for regulations regarding their control and elimination from clinical areas (11).
We are very grateful to Linda Hadjadj for technical assistance and to Mohamed Islam Hatcha for his contribution.
This work was partly funded by the Centre National de la Recherche Scientifique (CNRS 7278) and IHU Méditérranée Infection.
We declare no conflicts of interest.