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ESBL-positive enterobacteria isolates in drinking water.

To the Editor: Extended-spectrum [beta]-lactamase (ESBL)-producing members of the family Enterobacteriaceae (enterobacteria) are a worldwide problem (1), but little data are available from central Africa (2). In recent years, ESBL-producing Enterobacteriaceae isolates have shifted from the hospital to the community and the environment (1). The aim of this study was to assess the presence of ESBL-producing Enterobacteriaceae isolates in sachet-packaged water bags sold as drinking water in the streets of Kinshasa, the capital of Democratic Republic of the Congo.

In November 2009 and June 2010, a total of 101 sachet-packaged water bags were bought from street vendors in 9 of 24 municipalities (covering residential areas and slums) of the city of Kinshasa. The bags were transported in ice coolers and processed within 4 hours of collection. We filtered 100 mL of each sample through 0.45-[micro]m pore size filters (Sartorius, Goettingen, Germany). The filters were then transferred to an agar plate containing mEndoLES agar (Difco, Franklin Lakes, NJ, USA) and incubated at 35[degrees]C for 24 hours.

Growing colonies were subcultured on Kligler iron agar (Oxoid, Cambridge, UK), and gramnegative glucose-fermenting isolates were identified to the species level and assessed for antimicrobial drug susceptibility with Microscan NBC42 panels (Siemens Healthcare Diagnostics Inc., West Sacramento, CA, USA). Isolates labeled by Microscan as ESBL producers were confirmed by the double-disk method, which compared 1 disk containing cefotaxime with 1 disk containing cefotaxime and clavulanic acid and 1 disk containing ceftazidime with 1 disk containing ceftazidime and clavulanic acid (Rosco Diagnostica, Taastrup, Denmark), according to the Clinical and Laboratory Standards Institute guidelines (3). We used Escherichia coli ATCC 25922 and Klebsiella pneumoniae ATCC 700603 as control strains. Detection and identification of ESBL-producing bla genes were carried out by using a commercial multiplex ligation PCR microarray CT 101 (Check-Points Health BV, Wageningen, the Netherlands) (4).

A total of 101 sachet-packaged water bags were purchased at random from different vendors: 88 sealed and branded bags (of 68 different brands) and 13 hand-tied unbranded bags. The precise origin of the water could not be determined because vendors are unlicensed resellers and most producers are not registered. Water bags lacked essential information such as contact addresses, batch number, and production and expiration dates. Because we also observed empty branded sheets of bags for sale on the market, we assumed that many of the branded bags are simply filled with tap water or water from other supplies without any prior treatment. None of the water bags tested for chlorine (36 of 101) contained free chlorine levels >0.1 mg/L. Nearly one third of the water bags were contaminated with Enterobacteriaceae isolates (22/88 branded bags and 9/13 hand tied bags). The bags were obtained in townships and residential quarters.

Overall, 150 nonduplicate Enterobacteriaceae isolates were recovered. The main species were K. pneumoniae (56.0% of isolates found in 23/101 of water bags) and Enterobacter spp. (30.6%, in 20/101 water bags); Citrobacter spp. accounted for 4.7% of isolates and E. coli for 3.3%. Eight isolates (5.3 %) were confirmed as ESBL producers by antimicrobial drug susceptibility tests, and they were recovered from 2 branded and 2 hand-tied bags. The species, microarray results, and the associated drug resistance are listed in the Table. Five isolates carried [bla.sub.CTX-M] genes belonging to CTX-M1 group, and 3 isolates carried blaSHV variants. No TEM-ESBL genes were detected. On the basis of checkpoint results and previously validated data, we further categorized the SHV G238S mutation as SHV-2-like and the double SHV G238A + SHV E240K mutation as SHV-18 (5).

ESBL-producing Enterobacteriaceae isolates constitute a major public health concern in industrialized and resource-poor settings. Few reports are available from Africa, although hospital-associated ESBL producers have been described in Cameroon and the Central African Republic (6,7). ESBL-producing bacteria have been recovered from different sources in the community, including food and companion animals (8,9), and 1 recent study from India reported that a substantial number of tap water samples were contaminated with carbapenemase [bla.sub.NDM-1] producing organisms (10).

Kinshasa is the second-largest city in sub-Saharan Africa. In 2008, of its estimated 8.7 million inhabitants, only 46%had access to safe drinking water, and 23% had access to improved sanitation facilities according to the World Bank. Opportunistic pathogens in drinking water and poor sanitary conditions may increase the risk of developing infectious enterocolitis for consumers, especially for those who are immunocompromised. It can eventually lead to chronic intestinal carriage of multi-drug-resistant organisms. The presence of ESBL producers in the intestinal flora could also lead to horizontal transfer of drug resistance genes from commensal flora to enteric pathogens. This emergence of ESBL-producing bacteria and further community-associated infections poses a public threat, especially in low-resource countries where surveillance is suboptimal and empiric treatment of invasive infections often includes third-generation cephalosporins.

Hilde De Boeck, Berthe Miwanda, Octavie Lunguya-Metila, Jean-Jacques Muyembe-Tamfum, Ellen Stobberingh, Youri Glupczynski, and Jan Jacobs

Author affiliations: Institute of Tropical Medicine, Antwerp, Belgium (H. De Boeck, J. Jacobs); Institut National de Recherche Biomedicale, Kinshasa, Democratic Republic of the Congo (B. Miwanda, O. LunguyaMetila, J.J. Muyembe-Tamfum); Maastricht University Medical Center, Maastricht, the Netherlands (E. Stobberingh); and Cliniques Universitaires Universite Catholique de Louvain de Mont-Godinne, Yvoir, Belgium (Y. Glupczynski)

DOI: http://dx.doi.org/10.3201/eid1806.111214

References

(1.) Okeke IN, Laxminarayan R, Bhutta ZA, Duse AG, Jenkins P, O'Brien TF, et al. Antimicrobial resistance in developing countries. Part I: recent trends and current status. Lancet Infect Dis. 2005;5:481-93. http://dx.doi.org/10.1016/S1473-3099 (05)70189-4

(2.) Vlieghe E, Phoba MF, Tamfun JJ, Jacobs J. Antibiotic resistance among bacterial pathogens in central Africa: a review of the published literature between 1955 and 2008. Int J Antimicrob Agents. 2009;34:295-303. http://dx.doi. org/10.1016/j.ijantimicag.2009.04.015

(3.) Clinical and Laboratory Standards Institute. Performance standards for antimicrobial susceptibility testing: 21st informational supplement. CLSI document M100-S21. Wayne (PA): The Institute; 2011.

(4.) Endimiani A, Hujer AM, Hujer KM, Gatta JA, Schriver AC, Jacobs MR, et al. Evaluation of a commercial microarray system for detection of SHV-, TEM-, CTX-M-, and KPC-type -lactamase genes in gram-negative isolates. J Clin Micro biol. 2010;48:2618-22. http://dx.doi. org/10.1128/JCM.00568-10

(5.) Bradford PA. Extended-spectrum [beta]-lactamases in the 21st century: characterization, epidemiology, and detection of this important resistance threat. Clin Microbiol Rev. 2001;14:933-51. http://dx.doi.org/10.1128/CMR.14.4.933951.2001

(6.) Gangoue-Pieboji J, Bedenic B, Koulla-Shiro S, Randegger C, Adiogo D, Ngassam P, et al. Extended-spectrum-[beta]-lactamase-producing Enterobacteriaceae in Yaounde, Cameroon. J Clin Microbiol. 2005;43:3273-7. http://dx.doi. org/10.1128/JCM.43.7.3273-3277.2005

(7.) Frank T, Arlet G, Gautier V, Talar min A, Bercion R. Extended-spectrum [beta]-lactamase-producing Enterobacteriaceae, Central African Republic. Emerg Infect Dis. 2006;12:863. http://dx.doi. org/10.3201/eid1205.050951

(8.) Ewers C, Grobbel M, Stamm I, Kopp PA, Diehl I, Semmler T, et al. Emergence of human pandemic O25:H4-ST131 CTX M-15 extended-spectrum-[beta]-lactamase-producing Escherichia coli among companion animals. J Antimicrob Che mother. 2010;65:651-60. http://dx.doi. org/10.1093/jac/dkq004

(9.) Warren RE, Ensor VM, O'Neill P, Butler V, Taylor J, Nye K, et al. Imported chicken meat as a potential source of quinolone-resistant Escherichia coli producing extended-spectrum [beta]-lactamases in the UK. J Antimicrob Chemother. 2008;61:504-8. http://dx.doi.org/10.1093/jac/dkm517

(10.) Walsh TR, Weeks J, Livermore DM, Toleman MA. Dissemination of NDM-1 positive bacteria in the New Delhi environment and its implications for human health: an environmental point prevalence study. Lancet Infect Dis. 2011;11:355-62. http://dx.doi.org/10.1016/S1473-3099 (11)70059-7

Address for correspondence: Hilde De Boeck, Institute of Tropical Medicine, Nationalestraat 155, Antwerp, Belgium; email: hdeboeck@itg.be
Table. ESBL-producing Enterobacteriaceae recovered from sachet-
packaged water bags in Kinshasa, Democratic Republic of the Congo *

Isolate
no.               Species           Microarray CT101 result

44          Citrobacter freundii         CTX-M1 group
48          Citrobacter freundii         CTX-M1 group
152         Enterobacter cloacae    SHV G238S (SHV-2 like)
154        Klebsiella pneumoniae         CTX-M1 group
163         Citrobacter freundii         CTX-M1 group
165        Klebsiella pneumoniae         CTX-M1 group
170        Klebsiella pneumoniae    SHV G238A+E240K(SHV-18)
171           Escherichia coli      SHV G238A+E240K(SHV-18)

                  Associated resistance ([dagger])
Isolate
no.        Aminoglycosides    Fluoroquinolones    Folate PI

44          AMK, GEN, TOB    CIP, LEV, MXF, NXN      T/S
48          AMK, GEN, TOB    CIP, LEV, MXF, NXN      T/S
152              NA                  NA              T/S
154           GEN, TOB            CIP, MXF           T/S
163              NA                 MXF              T/S
165           GEN, TOB               NA              T/S
170           GEN, TOB              MXF              NA
171           GEN, TOB               NA              NA

* ESBL, extended/spectrum [beta]-lactamase; PI, pathway inhibitors; AMK,
amikacin; GEN, gentamicin; TOB, tobramycin; CIP, ciprofloxacin; LEV,
levofloxacin; MXF, moxifloxacin; NXN, norfloxacin; T/S,
trimethoprim/sulfamethoxazole; NA, not applicable.

([dagger]) Based on interpretive breakpoints as indicated in
Clinical and Laboratory Standards Institute guidelines M100-S18,
published January 2008.
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Title Annotation:Extended-spectrum [beta]-lactamase
Author:De Boeck, Hilde; Miwanda, Berthe; Lunguya-Metila, Octavie; Muyembe-Tamfum, Jean-Jacques; Stobberingh
Publication:Emerging Infectious Diseases
Article Type:Letter to the editor
Geographic Code:4EUBL
Date:Jun 1, 2012
Words:1485
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