Absence of vancomycin-resistant enterococci among highly ESBL-positive crows (Corvus splendens) foraging on hospital waste in Bangladesh.

BACKGROUND: Vancomycin-resistant enterococci (VRE) have emerged as a growing problem in hospitals; however, domesticated animals, poultry, and wild birds are acting as potential reservoirs. There is a knowledge gap in the Epidemiology of VRE from Bangladesh.
METHODS: To study the prevalence of VRE and the mechanisms of resistance implicated among wild birds, 238 fecal samples were collected in 2010 from house crows (Corvus splendens) foraging on hospital waste in Bangladesh. Fecal samples were screened by analyzing color change in broth and screening for vanA and vanB resistant genes by PCR.
RESULTS: Neither vanA nor vanB genes were detected from the fecal samples. The house crow does not seem to constitute a reservoir for VRE.
CONCLUSION: The zero prevalence is an indication that foraging on hospital waste does not constitute a major risk of VRE carriage in house crows and this is the first study to focus on the prevalence of VRE from wild birds in Bangladesh.

Different enterococci species are normally found as normal flora in human, mammals, birds, reptiles, and insects (1). The emergence and rapid spread of antibiotic resistance in enterococcus has become a serious public health concern. Vancomycin is a glycopeptide antibiotic, which is considered as the last lines of defense against many multiresistant Gram-positive cocci. Vancomycin-resistant enterococci (VRE) are one of the organisms responsible for hospital-acquired infections with a high morbidity and mortality in humans (2, 3). Resistance to vancomycin is caused by a series of genes (classified by the prefix van) that encode an enzyme important for the cell wall. There have been nine genes described in different species of enterococci: vanA, vanB, vanC, vanD, vanE, vanG, vanL, vanM, and vanN (4–7). However, E. faecium and E. faecalis carrying vanA or vanB gene have received the most attention in relation to human health care because of their clinical importance, transferability potential, and abundance in clinical isolates (8–10).

VRE have been reported in the hospitals of many countries throughout the world and spread is associated with poor hospital hygiene practice (8, 11). Different livestock species in different countries have been reported as potential reservoirs for vancomycin-resistant determinants (12, 13). VRE carrying the vanA gene were found in wild birds, including glaucous gulls (Larus hyperboreus) and wintering rooks (Corvus frugilegus) (14, 15). Different livestock species and/or humans were thought to be sources of VRE in free-living wild bird populations (15–17). Previously, we reported the house crow (Corvus splendens) and brown-headed gull (Chroicocephalus brunnicephalus) living close to human activities and hospital areas as potential carriers of clinically associated extended spectrum beta-lactamases (ESBL)-producing bacteria in Bangladesh (18, 19). There is no information regarding carriage of VRE among these wild birds that were reported to be carriers of ESBL producers.

The aim of this study was to determine the prevalence of VRE in crows foraging in hospital areas of Bangladesh and to investigate whether this bird species constitutes a potential reservoir for VRE.

Materials and methods

In total, 238 fresh fecal samples from house crows (C. frugilegus) were collected during February 2010 from the ground areas of Rajshahi Medical College Hospital (n=200) and Chittagong Medical College Hospital of Bangladesh (n=38). Fresh fecal droppings were collected by sterile cotton swabs, which after collection were immediately stored at −80°C in bacterial freeze media containing Luria–Bertani broth (Becton, Dickinson and Company, Sparks, MD), phosphate-buffered saline, and 4.4% glycerol. Samples were shipped to Sweden for analysis and cold chain logistics were used for shipment. Fecal samples in bacterial freeze media were inoculated by sterile swabs. The swabs were immediately inoculated with the sample into 1 mL bile azide esculin broth and stored at −70°C for analysis.

Screening for VRE was performed in a selective bile azide esculin broth supplemented with 4 mg/L vancomycin and 60 mg/L aztreonam (ICN Biomedicals, Inc., Aurora, OH), and PCR for vanA and vanB, according to the methods described previously (20, 21). Briefly, this real-time PCR protocol was used to detect the vanA and vanB gene. Amplification was conducted in a total volume of 20 µL containing 12.1 µL of PCR water, 4 µL of LC480 M-Mix (2×), 0.4 µL of vanA F (10 µM) (5′-CGGCAAGACAATATGACAGCAA-3′), 0.4 µL of vanA R (10 µM) (5′-TCAGTACAATGCGGCCGTTA-3′), 0.4 µL of vanB F (10 µM) (5′-GGGAGGATGGTGCGATACA-3′), 0.4 µL of vanB R (10 µM) (5′-CCGAAATCGCTTGCTCAA-3′), 0.15 µL of vanA prob (10 µM) (5′-HEX-CAGTTATAACCGTTCCCGCAGACCTT-BHQ1-3′), 0.15 µL of vanB prob (10 µM) (5′-FAM-CTTTGTGAAGCCGGCACGGTCAGGTT-BHQ1-3′), and 2 µL of the samples. The following cycling parameters were used in the PCR run: 95°C for 5 min (95°C for 10 s, 60°C for 30 s) for 45 cycles. Finally, the amplified PCR product was analyzed.

Results, discussion, and conclusion

All together 238 fecal samples were analyzed. Surprisingly, none of them demonstrated a color change after testing in the bile azide esculin broth meaning no growth of enterococci was demonstrated. Neither vanA nor vanB were confirmed through PCR screening. These results indicate that there was no VRE carriage by the house crows in Bangladesh. As the testing in bile azide esculin broth did not demonstrate growth of any enterococci, the findings may be interpreted as a low propensity of crows to carry enterococci in general, and thus also to carry VRE. However, previous findings of VRE among birds of the genus Corvus (15, 17) indicate that carriage of VRE is indeed possible, and that crows therefore can be used as sentinels. VRE also reported in different wild bird species like European robins (Erithacus rubecula), quail (Coturnix coturnix conturbans), Common chaffinch (Fringilla coelebs), Blackcap (Sylvia atricapilla) and buzzards (Buteo buteo) (16, 22). More interestingly, wild birds very close to contact with human activities, for example crows and gulls, are potential carriers of clinically important VRE (14, 17). A study from Bangladeshi hospitals showed a low prevalence (3%) of E. faecalis among patients with urinary tract infections (23). Another study from Bangladesh showed that E. faecalis is the dominant species among patients with Enterococcus infection and all E. faecalis and E. facium strains isolated from patients were resistant to vancomycin (24). Thus, VRE is a serious challenge for hospitals; however, there are no reports about the prevalence of VRE from livestock, poultry, or wild birds in Bangladesh.

The house crow (C. splendens) has a widespread distribution in South Asian countries like Bangladesh and India, and it lives close to humans. It occupies different ecological niches, including household areas, city dumps, hospital dumps, and water sources like lakes, ponds, and rivers. The house crow has been shown to carry several human pathogenic bacteria in its intestines (25, 26). A study on these samples demonstrated that 59% of the crows living on hospital waste were potential carriers of clinically relevant ESBL-producing E. coli and Klebsiella (19), and hospital waste has been reported as a potential source of antibiotic-resistant bacteria (19, 27). These crows were reported as potential carriers of clinical relevant human-associated ESBL-producing E. coli ST13-O25b clones, likely because of their foraging behaviors in hospital waste dumps (28). It has also been documented that wild birds can carry VRE and ESBL-producing bacteria simultaneously (16). Thus, the high prevalence of ESBL-producing bacteria among crows living in hospital areas has increased our suspicion about the possible carriage and spread of VRE as well, like in Azores, Portugal (16).

This study indicates the absence of VRE harboring vanA and vanB genes among crows foraging on hospital wastes even though they were potential carriers of ESBL-producing Enterobacteriaceae (28). Thus, spread through hospital waste via house crows does not seem to be a major mechanism for dissemination of VRE in Bangladesh. However, proper steps should be taken to stop the possible future environmental spread of VRE, as well as other resistant microorganisms, through hospital waste and from hospitals settings in Bangladesh.