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Iranian J Publ Health, Vol. 37, No.1, 2008, pp.103-107
Iranian J Publ
Health, Vol
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107 Original Article
High Level Resistance of Enterococcus faecium and E. faecalis
Isolates from Municipal Sewage Treatment Plants to
M Saifi 1,2, *MM Soltan Dallal 1, MR Pourshafie 2, MR Eshraghian 3, MR Pourmand 1, MH
Salari 1, MH Shirazi 1
1Dept. of Bacteriology, School of Public Health, Medical Sciences/University of Tehran, Iran 2 Dept. of Microbiology, Pasteur Institute of Iran, Tehran 3 Dept. of Biostatistics, School of Public Health, Medical Sciences/University of Tehran, Iran (Received 13 Jun 2007; accepted 17 Dec 2007)

Enterococci are members of the normal gut flora and released into the environment via sewage outlets,
where they can survive for long times. Infections with high-level gentamicin resistant (HLGR) enterococci are emerg-
ing worldwide. HLGR enterococci have developed a resistance to most antibiotics commonly used for enterococcal in-
fections therefore; treatment of infections caused by HLGR enterococci is difficult. The present study investigated the
distribution and antibiotic resistance of HLGR Enterococcus faecium and E. faecalis isolates from raw wastewater sam-
ples in Tehran.
Methods: Raw wastewater samples were collected during the period from November 2006 to May 2007 at 3 sewage
treatment plants located in different parts of Tehran. All 90 HLGR enterococcal isolates were identified to the species
level by biochemical and PCR assays and subjected to antibiotic susceptibility testing.
Results: Sixty four percent (58 of 90) of isolates were E. faecium and 29%(26 of 90) of them were E. faecalis. The
highest level of antibiotic resistance was observed with erythromycin (63%), co-trimoxazole (69%) and tetracycline
(92%) for E.faecalis and with erythromycin (97%), ciprofloxacin (47%), co-trimoxazole (45.5%) and tetracycline
(47%) for E. faecium. Multiresistance against 3 to 4 antimicrobial was present in 27.5% and 15.5% of the isolates, re-
Conclusion: HLGR E. faecium were more commonly found than E. faecalis. Species identification of HLGR entero-
cocci enables us to assess species-specific antibiotic susceptibility patterns in our area. The present study reviled that
HLGR E. faecalis remained more susceptible than E. faecium against the usual first-line and alternative treatments.
Keywords: E. faecalis, Enterococci, gentamicin resistance, Iran
The prevalence, severity and antibiotic resistance Enterococci are normal inhabitants of the intes- of and the mortality rate from enterococcal in- tinal floras of human and warm-blooded ani- fections are often species dependent (5). mals. They are released into the environment The emergence of high-level gentamicin resis- through feces (1). Enterococci have the ability tance (HLGR) is of serious worldwide due to to survive adverse environmental conditions (2). their multi-resistant (6). According to a study (7), The enterococci may be superior indicator or- 52% of enterococcal clinical isolates in Tehran ganisms than fecal coliforms as a water qual- ity indicator (1). They have emerged as impor- Nevertheless, based on data in the literatures, tant human pathogens that are responsible for 90-99% of multiple resistant E. faecium and nosocomial infections and with the emergence of E. faecalis found in sewage actually stem from aminoglycoside and glycopeptides resistance (3). the human population and several cycles of con- Of 14 or more, exist enterococcal species, only tinuation and recontamination with them ap- E. faecalis and E. faecium commonly colo- pears to exist between animals, humans and nize and infect humans in detectable numbers (3). They can cause urinary tract and wound in- This sampling was aimed at obtaining informa- fections, septicemia and endocarditis (4). tion on prevalence of two most clinically impor- *Corresponding author: Tel: +98 21 66465404, E-mail: [email protected],
M Saifi et al: High Level Resistance of .
tant species E. faecalis and E. faecium HLGR heim, Germany), 2.5 pM of each primers, 2.5 enterococci in sewage treatment plants in Tehran, µl of 10X PCR buffer, 1.5 mM MgCl2 and DNA template 5 µl. DNA amplification was carried out with the following thermal cycling profile. Materials and Methods
Initial denaturation at 94 ºC for 5 min, 30 cy-cles of amplification (denaturation at 94 ºC for Sample collection: Inflow raw wastewater sam- 1 min ,annealing at 54 ºC for 1 min and ex- ples were collected from three sewage treat- tension at 72 ºC for 1 min ), followed by a fi- ment plants located in the different parts of Te- hran during the period from November 2006 Antimicrobial susceptibility testing: The suscep- to May 2007. Five hundred milliliter of each tibility to antibiotics was tested by the agar disk sample was collected in a sterile container trans- diffusion method according to CLSI guidelines ported to the laboratory and analyzed within a (13). The antimicrobial disks were used as fol- lows: vancomycin (30µg), ciprofloxacin (5µg), Isolation of HLGR enterococci: Samples were ampicillin (10µg), high content gentamicin (120 subjected to serial 10-fold dilutions with normal µg), trimethoprim/sulfametoxazole (1.25: 22.75), saline, before filtration. The membrane filters tetracycline (30µg), erythromycin (30µg) and were put on brain heart infusion agar (Becton nitrofurantoin (300µg) were purchased from BBL Dickinson and Co., Sparks, MD, USA) plates and preincubated for 2 h at 37 ºC (9). The mem- dalfopristin (15 µg) and linezolid (30 µg) from branes were transferred to m-Enterococcus Mast Diagnostics Ltd. (Bootle, Mersey Side, agar (Becton Dickinson and Co., Sparks, MD, UK) and teicoplanin (30 µg) from BR (BioRad, USA) supplemented with 64 mg of gentamicin Hercules, CA, USA). Isolates with intermediate per liter. Agar plates were incubated for 48 h levels of susceptibility were classified as resistant. at 37 ºC. Distinct colony growth was transferred to blood agar plates for following investigations. Biochemical identification of species: All of the enterococcal isolates were tested for phenotypic Ninety HLGR enterococcal isolates were re- characteristics by conventional methods, based covered from three sewage treatment plant on the following criteria: growth on Bile Es- samples. Among HLGR isolates, the follow- culin agar and in 6.5% NaCl broth, absence of ing species were identified; 58 isolates of E. catalase and presence of pyrolidonyl arylami- faecium (64%), 26 isolates of E. faecalis dase (PYR test). Species- level identification (29%) and six (7%) other enterococcal spe- was performed by biochemical tests including cies. Amplification of genus, E. faecals- spe- acid fermentation of manitol, sorbitol, sucrose, cific and E. faecium- specific targets produced arabinose and raffinose, motility and arginine 320 bp, 941 bp and 658 bp bands, respectively (Fig. 1). Species distribution by PCR method Detection of genus and species by PCR. All was the same as detected by biochemical tests. strains were confirmed for genus and species Data obtained from antimicrobial susceptibility by PCR with three different primer sets. Am- testing summarized in Table 2. Among 58 E. plification of both species specific, and genus faecium and 26 E. faecalis isolates with HLGR (rrs) targets produced bands corresponding to phenotype, resistance percents to erythromicin, their respective molecular size (Table 1). For ciprofloxacin, co-trimoxazole, tetracycline, am- DNA extraction, one isolated colony was sus- picillin, nitrofurantoin and synercid were 97% pended in 200 µl distilled water and boiled at versus 63%, 47% versus 19%, 45.5% versus 100 ºC for 10 min (11). After centrifugation, 69%, 47% versus 92%, 18% versus 7.5%, 17% 10 µl of supernatant was used as the DNA versus 0% and 3.5% versus 100%, respectively. template. PCR reaction were performed in a Simultaneously resistance to ampicillin, ciproflox- total volume of 25 µl containing 0.8 mM dNTP, acin, tetracycline and co-trimoxazole were 15.5% which was more detected in E. faecium. Iranian J Publ Health, Vol. 37, No.1, 2008, pp.103-107
Only 19% of E. faecalis isolates were resistant to comycin, teicoplanin and linezolid was detected ciprofloxacin, which differs markedly from 47% obtained by E. faecium. No resistance to van- Table 1: Nucleotides sequences of primer sets
Type of primers
Product size(bp)
Table 2: Antibiotic resistance among HLGR strains isolated from sewage treatment plants in Tehran. The
abbreviations are; V: vancomycin, Te: tetracycline, Am: ampicillin, E: erythromycin, Cip: ciprofloxacin, Tei: teicoplanin, Fm: nitrofurantoin, Syn: quinupristin-dalfopristin (Synercid), Lin:linezolid, SXT: co-trimoxazole. Number of
Number (%) of HLGR isolates resistant to:
V Te Am E Cip Tei Fm Syn
26(29) (0) 23(92) 2(7.5) 16(63) 5(19) (0) 58(64) (0) 27(47) 10(18) 56(97) 27(47) (0) 10(17) 2(3.5) (0) 25(43)
Fig. 1: A: PCR for Enterococcus faecium, B: PCR for Enterococcus faecalis, C: PCR for Genus of Enterococci
with nosocomial infections (14). HLGR in E. In the present study HLGR enterococci were faecalis is now a very common feature world- found in sewage along with high resistance to wide and the reported prevalence exceeds 50% other antimicrobial agents especially among E. of the E. faecalis strains isolated in some hos- faecium isolates. HLGR multiresistant entero- pitals (6). According to an area specific sur- cocci have been identified as a major issue of vey in Tehran the prevalence of HLGR E. fae- concern, as they have been found associated calis isolates reported 30% with high degree pp
M Saifi et al: High Level Resistance of .
of multiresistance (15). This finding was alarm- resistant isolates was found. Co-resistance to four ing as infection due to HLGR isolates are dif- and five antibiotic simultaneously was reported Finding HLGR E. faecalis in sewage, which Similar results were obtained in other study, is much more common in human enterococcal which showed distribution of enterococci in infections, suggest a potential high risk for com- sewage (18). Out of 45 enterococcal isolates munity- acquired HLGR as well as the pos- from municipal wastewater, 32 isolates were sibility of transferring antibiotic resistance genes E. faeclais, 10 isolates belonged to E. faecium Resistance to multiple classes of antibiotics is According to de Costa’s report, among 983 common in enterococci as was seen in this enterococci, multidrug resistant isolates were study. We found that, resistance patterns dif- present in 49.4% of the isolates. Only 3.3% fered between species. Overall, E. faecium had and 0.6% of the investigated strains were re- a higher prevalence of resistance among the sistant to ampicillin and vancomycin. Resis- panel antibiotics, while E. faecalis isolates had a tance rates to tetracycline, erythromycin, nitrofu- relatively lower resistance to ampicillin, eryth- rantoin and ciprofloxacin were 34.6%, 24.8%, romycin and ciprofloxacin excluding their in- herent resistance to quinupristin/dalfopristin (syn- Ferreira in 2006 in Portugal studied the antibi- ercid). Resistance rate against synercid among otic resistance of enterococci and related bac- HLGR E. faecium isolates was 3.5%. Synercid teria in an urban wastewater treatment plant has a spectrum of activity against multi-resis- and demonstrated that the predominant species tant enterococci excluding E. faecalis and is were E. haire, E. faecium and E. faecalis. The available for the treatment of multiresistant E. percents of resistance observed to erythromy- cin, ciprofloxacin and tetracycline ranged be- E. faecalis resistance to ampicillin and vanco- tween 23% and 57%. Only two E. faecium of mycin is uncommon (17).However, in this study 27 enterococcal isolates were HLGR (20). E. faecalis resistance to ampicillin was found All the HLGR E. faecium isolates in our study in low degree (7.5%) and no resistance to van- were sensitive to linezolid, which was recently launched for the treatment of Gram-positive The resistance rate to co-trmoxazole and tetra- cycline among E. feacalis was more than E. Conclusively, our data indicate that the relax faecium. This may be because of widely usage usage of antimicrobials had created a large pool of these two antimicrobials in human and animal of resistance genes which may be disseminate infections and selective antibiotic pressure or sim- resistant bacteria into the environment and could ply transfer of resistance genes or combination potentially pose a health treat to human in the A high percentage of the E. faecium isolates were resistant to multiple drugs, contributes to Acknowledgements
the challenge of selecting therapeutic measures. This study was supported by the Deputy of Re- Distribution of enterococcal isolates was com- search, Medical Science/University of Tehran. parable to other studies. For example Cupakova The authors declare that they have no Conflict in Czech Republic during one year (2003) re- covered 100 enterococcal isolates from differ- ent wastewater samples including 60 isolates References
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18. Cupakova S, Lukasova J (2003). Agricultural age water treatment plants Water Res, and municipal waste water as a source of antibiotic-resistant enterococci. Acta Vet 20. Ferreira da Silva M, Tiago I, Verissimo A, 19. Martins da Costa P, Vaz-Pires P,Bernardo F (2006). Antimicrobial resistance in En- FEMS Microbiol Ecol, 55(2):322-29.



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