Ambient science, 2016; vol. 0.

Ambient Science, 2016: Vol. 03(2); Online Published by: National Cave Research and Protection Organization, India Year 2016
Fecal Carriage of ES L
b types TEM, SHV, CTX Producing Genera
Proteus, Morganella, Providencia in Patientsof Iran
Mohammad Taghi Akhi ,
Pourya Gholizadeh ,
Reza Ghotaslu ,
Mohammad Asgharzadeh ,
Diseases like urinary tract infection, wound infections, Mohammad Hossein Sourush ,
bacteremia and other infections are mainly caused by the Naghilli1, Peyman Gholmohammadi ,
members of the genus Proteus, Morganella and Providencia which are mainly either found freely in the environment orin the gastrointestinal tract of humans. We studied Fecal 1Immunology research center, Tabriz University of Medical Sciences, Tabriz, Iran.
carriage of ES L producing species in carrier patients.Stoolsamples obtained from outpatients and inpatients not 2Department of Microbiology, School of Medicine, Tabriz suffering from diarrhea and were cultured in CTX-MC- University of Medical Sciences, Tabriz, Iran.
Conkey agar. Lactose negative and cefotaxime resistant 1Tabriz Universityof Medical Sciences, Tabriz, IR Iran bacteria were identif ied by biochemical tests and ES L- Study Area: Tabriz, East Azarbaijan, Iran producing isolates were detected using Combined Test.
TEM, SHV and CTX genes were investigated by PCR.Total 15 Coordinates: 38°09'N 46°27'E (7.35%) isolates of 204 stool samples were identif ied as Key words: Healthy carriers, Cephalosporins ESBL producing Proteus spp. (n=4, 1.96%), Morganella spp.
resistance, Extended spectrum bete-lactamase (n=5, 2.45%) and Providencia spp. (n=6, 2.94%). Further,amongst or of the 15 ES L b producing strains, blaTEM was the commonest genotype (86.66%), followed by blaSHV(26.66%) and blaCTX-M (20%). All isolates were resistantto ampicillin, and cefotaxime whereas all Providencia andMorganella spp. were found to resist ceftazidime. Althoughthe number of ES L- b producing Proteus, Morganella and Providencia isolates from fecal carriers were low, but still,they can be considered as a reservoir of TEM, SHV and CTXgenes and capable to transfer these resistant bacteria tohospitals.
classif ication, the class A includes three important genes Species of the Proteus, Morganella and Providencia genus of SHV, TEM, CTX-M; which are commonly found in the are facultative anaerobic gram-negative bacilli that Enterobacteriaceae family (Lagacé-Wiens et al., 2007).
belong to the Enterobacteriaceae family and are found in The f irst plasmid beta-lactamase in Gram-negative the open environment, waste water and gastrointestinal bacteria was TEM- 1 that identif ied in the early 1960s tract of mammals, humans and animals (O'Hara et al., (Bradford, 2001). CTX-M and TEM are common ESbLs that 2000). These organisms are one of the main causes of are found in isolates of Proteus, Morganella and infections such as UTI (urinary tract infection), Providencia (Tumbarello et al., 2004) and in recent years, respiratory tract, wounds, bacteremia and other SHV has been reported in Iran (Malekjamshidi et al., opportunistic infections and perhaps act as infection 2010). All the three bacteria are normal flora of the sources for extended-spectrum beta-lactamases (ESbLs) gastrointestinal and transferred through endogenous or productions in both society and hospitals. Some studies spreading f rom person to person, especially in have shown that the ESbL and AmpC producing isolates of hospitalized patients. The epidemiological analysis P. mirabilis can be a cause of clonal spread in the hospital, suggests that ESbL-producing Enterobacteriaceae species regional and continent-wide outbreak (Nakano, et al., such as Proteus, Morganella and Providencia could be 2012). Providencia stuartii has been reported to contain isolated in different environments of hospitals, human ESBL enzymes such as TEM, SHV or CTX-M (Aubert et al., feces, infectious and healthy carriers, uncooked foods, and 2005; Franceschini et al., 1998). Based on Ambler human sewage (Mesa et al., 2006). Fecal carriage of ESbL- ISSN- 2348 5191 (Print) & 2348 8980 (Electronic)

Ambient Science, 2016: Vol. 03(2); Online producing strains has not been studied enough in most of Detection of TEM, SHV, CTX gene all bacterial isolates
the Asian countries, including Iran. The aim of this study were grown for 24 hours at 37°C in Lauria-Bertani (LB) was to isolate and determine the types of ESbL (TEM, SHV, broth. DNA of isolates were extracted by sodium dodecyl and CTX) produced by Proteus, Morganella,Providencia sulphate-proteinase K modif ied with N, N, Ntrimethyl isolated from patients fecal carriers of teaching and ammonium bromide (Ranjbar et al., 2007). The PCR treatment hospital of Tabriz.
reacted with specif ic primers for amplif ication of 569bpand 293 bp and 403 bp fragments (Bali et al., 2010): Methods and Materials:
Bacterial isolates: in between November 2014 to
R:5´-GGCTGGGTGAAGTAAGTGAC-3´ (569 bp) February 2015, we collected 204 stool samples from non- hospitalized (n=100) and hospitalized patients (after 48 R: 5´-CGAGTAGTCCACCAGATCCT-3´ (293 bp) hours of admission; n=104). Patients suffering from F: 5´-TTTCGTGTCGCCCTTATTCC-3´R: 5´-ATCGTTGTCAGAAGTAAGTTGG-3´(403 bp) gastrointestinal illness and diarrhea were excluded fromthe study. Stool samples obtained were cultured in Mac- The amplif ication was done in a DNA thermal cycler Conkey agar contain 2mg/L cefotaxime (CTX-Mac- (Eppendorf master cycler gradient, Germany), Conkey) and were incubated at 37°C for 24 hours. Lactose- programmed for a primary denaturation, (95°C, for 3 negative isolates were collected and were identif ied by minutes), followed by 35 cycles of denaturation (94°C, routine biochemical tests such as motility, urea hydrolysis, 45s), 30 seconds for annealing (60°C for SHV and CTX-M, citrate utilization, phenylalanine deaminase, arginine 55°C for TEM), elongation (72°C, one minute), and then decarboxylase and other necessary tests. Identif ied exten¬sion (72°C, 10 minutes). A negative control without isolates were stored at -20°Cin trypticase soy broth tem¬plate was included in each PCR run. The amplif ied containing 12% glycerol (Luvsansharav et al., 2012).
products were visualized by electrophoresis on 1.2%agarose gel in 1x TBE buffer (1 M Tris, 0.9 M boric acid, 0.01 Antibiogram and detection of ESbL producing
M EDTA, pH = 8.4), at 80 V, for two hours. A 100-bp DNA isolates: disk diffusion tests on isolates were carried out
ladder was used as a molecular mass marker. The gels were using Mueller-Hinton agar plates and antibiotic discs stained with ethidium bromide (0.5 µg mL-1) and including ampicillin (10 µg), cefotaxime (30 µg) photographed on a gel documentation system (UVP, USA) ciprofloxacin (5 µg), gentamycin (10 µg), ceftazidime (30 for the analysis of the bands (all the PCR materials µg ), cefoxitin (30 µg) and cefepime (30 µg). All the plates including primers were provided by CinnaGen; Nedayeh were incubated at 37°C for 24 hours.Zones of inhibition Fan Co., Iran). The total volume of PCR mix was 25 µl, around the disc were recorded using Clinical Laboratory including sterile redistilled H2O 17.05µl, 10X PCR buffer Standards Institute instruction (CLSI, 2014).All isolates 2.5µl, dNTP mix (10mM) 0.5µl, MgCl2 (50mM) 0.75µl, resistant to ampicillin, ceftazidime and cefotaxime were forward primer (25µM) 0.5µl and reverse primer (25µM) evaluated to conf irm ESBL production by combined disc 0.5µl for each gene, Taq DNA polymerase (5U/µl) 0.2µl, test (CDT). Ceftazidime (30 µg), ceftazidime+ clavulanic template DNA 3µl. Negative controls contained all acid (30+10 µg), were placed on Muller medium and after components except template DNA. Primers and other 24 hours of incubation at 37°C, increasing more than 5mm reagents were prepared according to the manufacturer's inhibition zone around the disk containing clavulanic acid recommendation (Akhi et al., 2015; Sharma et al., 2013).
compared to without clavulanic acid conf irmed as ESBL producing isolates (CLSI, 2014) (Figure 1).
Figure 1: Phenotypic detection of ESbL-producing isolates; Figure 2: Genotypic detection of ESbL producing isolates. Ladder, increasing more than 5mm inhibition zone around the disc positive control for three genes, representative of SHV (383 containing cefotaxime + clavulanic acid (CEC, 30/10µg) bp), TEM (495 bp), CTX (560 bp) positive bacteria, negative compared to cefotaxime (CTX, 30µg) are conf irmed as ESBL control 1, negative control 2 and ladder left to right are shown producing isolates.
Ambient Science (2016) Ambient Science, 2016: Vol. 03(2); Online Statistical analysis: the study data was analyzed by using
descriptive statistics (frequency - percent) and using the ESbL production by nosocomial pathogens is a major software spss-17. Chi-square test were applied to evaluated challenge for infection control committees in hospitals all the incidence of genes with antibiotic susceptibility, to over the world. ESbL-producing strains and their observe the correlation between the prevalence of ESbL encoding genes can stay permanently in hospitals, causing genes and antibiotic susceptibility, Signif icance of results colonization and outbreaks. In recent years, antibiotic- were calculated at 95% conf idence level (p<0.05).
resistant ESbL producing bacteria in hospitalized patients has increased all over the world (Bradford, 2001; Denton, Total 15 (7.35%) isolates from204 stool samples were 2006; Gupta et al., 2003).
identif ied as Proteus (n=4, 1.96%), Morganella (n=5, Till date not suff icient reports available about the 2.45%) and Providencia (n=6, 2.94%). Ratio between distribution and source of infectious of these bacteria.In women and men were 39.1% and 60.9% respectively. The addition, how many healthy people are carrying ESbL- age ranges of the patients were 15-83 year; distribution of producing bacteria or are transmitted by inpatient or age according to Kolmogorov - Smirnov test was normal outpatient to the hospital are not known Asymptomatic colonization of the intestinal compartment with ESbL- Antibiogram and detection of ESbL producing
producing Enterobacteriaceaeisolates has already beenreported (Miró et al., 2005; Valverde et al., 2004). Higher isolates: all strains of Proteus, Morganella and
prevalence of ESbL-producing E. coli, klebsiella and other Providencia were found to be resistant to ampicillin.
Entero-bacteriaceae of fecal carriage has been reported in Proteus spp. showed resistance to cefotaxime, cefoxitin the nosocomial setting than in any community (Chong et and susceptible to other antibiotic. Morganella spp.
al., 2013). Nevertheless, little information is available showed resistance to amoxicillin + clavulanic acid, about healthy carrier of Proteus, Morganella and cefotaxime and ceftazidime while Providencia spp. was resistance to cefotaxime and ceftazidime. Antibiogram result of each genus has been shown in the table-1 ES L producing Proteus, Morganella and Providence causes clinically signif icant hospital associated infectionsand are also known to cause community-acquired Table 1: The frequency of resistance strains of species (Spp.) infections due to selective pressure owing to widespread 1.-Proteus, 2.-Morganella and 3.-Providencia use of third generation cephalosporin (Chong et al., 2013; Spp. Amp Amc Cec Ctx Cip Cac Gen Caz x Mahrouki et al., 2014; Poirel et al., 1999).
Therefore, in this study, we have cultured gastrointestinal patient's stool samples that attended to Amp (Ampicillin, 10 µg), Amc (Amoxicillin + clavulanic acid, 30 the hospital and examined ESbL producing, antibiotic µg), Cec (cefotaxime + clavulanic acid, 40 µg), Ctx (cefotaxime, 30 susceptibility pattern and the presence of TEM, SHV and µg), Cip (ciprofloxacin, 5 µg), Cac (ceftazidime + clavulanic acid, CTX genes in Proteus, Morganella and Providencia 40 µg), Gen (gentamycin, 10 µg), Caz (ceftazidime, 30 µg), Cx isolates. The rate of ESbL producing isolates in our study (cefoxitine, 30 µg), Cpm (cefepime, 30 µg), A/S (ampicillin + was 7.35%, including Proteus (n=4, 1.96%), Morganella sulbactam, 20 µg) (n=5, 2.45%) and Providencia (n=6, 2.94%) which are Detection of TEM, SHV, CTX gene: of the 15 (7.35%)
higher than studies carried on Enterobacteriaceae in ESBL producing strains, blaTEM was the most common different parts of the world such as Switzerland (5.8%), genotype (86.66%), followed byblaSHV (26.66%) and Sweden(3%), Spain(5.5%), India (10%) and Saudi Arabia blaCTX-M (20%). Most of the Proteus spp. wereTEM and (13.2%) (Geser et al., 2011; Tängdén et al., 2010) fortunately SHV genes positive whereas all of the Morganella spp were no report available on ESbL producing Proteus, TEM gene positive and SHV gene negative and most of the Morganella and Providencia. Albeitall of these studies Providencia spp. was TEM gene positive. The results of were of different genus and species of Enterobacteriaceae genes detection in each genus has been shown in the table 2 but most of them isolated only ESbL producing E.coli and Klebsiella spp. Our research was the f irst report about Table 2: The frequency of genes detectation in strains of healthy ESbL carrier of Proteus, Morganella and species (Spp.) 1.-Proteus, 2.-Morganella and 3.-Providencia Providencia in Tabriz. The main reason for a high prevalence rate of ESbL producers in our city could be the lack of strict policy for an antibiotic prescription and also the excessive use of these antibiotics could explain the higher prevalence of fecal carriage of ESbL-producing % 83.3(5) % 16.7(1) % 16.7(1) 0 Ambient Science (2016) Ambient Science, 2016: Vol. 03(2); Online organisms in the hospital, compared with the rate in the Although the number of ESbL-producing Proteus, Morganella and Providencia isolates from fecal carriers are In contrast to our results Kader et al. (2007) reported low, but still, they can be considered as a reservoir of TEM, that the rate of fecal carriage of ESbL-producing organisms SHV and CTX genes; thus carrier are also able to transfer among inpatients (26.1%) was higher than that among these resistant bacteria to hospitals.
Saharman & Lestari (2013) reported 8.04% of ESbL This study was f inancially supported by the Immunology Research producing Proteus mirabilis isolated from ICU patients Center, Tabriz University of Medical Sciences, Tabriz, Iran and the which is much higher than our results (1.96%) indicating manuscript was written based on a dataset of MSc thesis of that there is much difference between isolates of healthy PouryaGholizadeh registered at Tabriz University of Medical carriers and those isolated from patients.
Sciences. The authors would like to thank all Microbiology lab staff Our results showed that blaTEM was the commonest of ghazitabatabaei and Imam Reza teaching and treatment center genotype (86.66%), followed by blaSHV (26.66%) and of Tabriz for their collaborations and helps. This study was blaCTX-M (20%), which corresponds to the results approved by the ethical committee of regional Medical Research ofTabriz University of Medical Science and all patients provided obtained by Bali et al. (2010) in turkey for E. coli and written informed consent for this research (TBZMED. REC.
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Ambient Science (2016)

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