Extended-spectrumβ-lactamases (ESBLs) are enzymes that compromise the efficacy of all β-lactams, apart from cephamycins and carbapenems, by hydrolysis of the β-lactam ring, and are inhibited by β-lactamase inhibitors (Coque et al., 2008). The most common cause of resistance to expanded- spectrum cephalosporins in Escherichiacoli is the production of ESBLs (Paterson, 2006) and ESBL-producing E. coli have rapidly spread worldwide with pose a serious hazard for health care-associated (HA) infection. ESBLs have been reported from all parts of the world. However, prevalence varies widely even in closely related regions. Most of the clavulanic acid-inhibited ESBLs are either derivatives of narrow-spectrum TEM and SHV-type β-lactamases or CTX- M, PER, VEB, and GES/IBC-type β-lactamases (Bauernfeind et al., 1996; Nordmann, 1998; Pitout et al., 2005). ESBL producing organisms are often resistant to several other classes of antibiotics, as the plasmids with the gene encoding ESBLs often carry other resistance determinants. Initially ESBL producing organisms were isolatedfrom nosocomial infections but these organisms are now also being isolatedfrom community (Pitout and Laupland 2008). The TEM-1 enzyme was first reported from an E.coli isolate in 1965 and is now the most common β-lactemase found in Enterobactereceae (Fonze et al., 1995). The CTX-M family, first described in 1992 (Bauernfeind et al., 1992), is known to be the most dominant non-TEM, non-SHV ESBL among Enterobacteriaceae and is recognized as a rapidly growing family of ESBLs that selectively prefer to hydrolyze cefotaxime rather than ceftazidime (Bonnet, 2004). CTX-M group The latter is a small, but growing, family of plasmid encoded ESBLs that hydrolyze cefotaxime and although 20 CTX-M enzymes have been described between 1989 and 2001 in various enterobacterial species but mostly clinical isolates of Salmonella typhimurium, Escherichiacoli and Klebsiella pneumonia carry CTX-M-1 (Barthelemy et al., 1992; Bauernfeind et al., 1996). VEB-1 ESBL was identified among Enterobacteriaceae and Pseudomonas aeruginosa isolates and was previously reported from Thailand (Girlich et al., 2001; Girlich et al., 2002). The widespread incidence of VEB-1 in Enterobacteriaceae and Pseudomonas aeruginosa suggests that this ESBL gene is prevalent in numerous gram-negative species (Girlich et al., 2001; Girlich et al., 2002). The PER family, first identified in P. aeruginosa, is also
The MLST scheme by Wirth et al.  was applied for analysing seven gene fragments according to the protocols found at http:// mlst.ucc.ie/mlst/dbs/Ecoli/documents/primersColi_html as pre- viously described . The MLST allele designations were determined via the online MLST database and all novel sequence alleles and ST designations discovered during this study were provided by the curator of the database (http://mlst.ucc.ie/mlst/ dbs/Ecoli). Each new sequence allele was independently verified by the curator of the database by evaluation of a minimum of two nucleotide sequence traces submitted by one of the authors (S.G) along with the information on the new reference strain for the public database. The allele data was analysed by the minimum spanning tree algorithm implemented in Bionumerics v.5.1 (Applied Maths NV) with priority rules set at first link genotypes that have maximum numbers of single-locus variants and then maximal numbers of single-locus variants and double-locus variants. Concatenated nucleotide datasets of the seven genes were used for constructing a neighbour-joining tree based on the number of pair- wise differences among strains using the Pearson coefficient. Forty- seven strainsof the ECOR collection previously characterised by MLST were used as phylogroup references [23,24] The E. coli strain Z205/ST125  was included to root the inferred phylogeny. Simpsons indices of diversity were calculated using the on-line tool at the website http://darwin.phyloviz.net/ComparingPartitions/ index.php?link = Home
to 1991 indicated 5% resistance to ceftazidime. Although these strains were initially only found on the east and west coasts, and in the Chicago region, K. pneumoniae ESBL producingstrains are now widespread all over the United States (20,21). One thousand two hundred and ten clinical isolates ofEscherichiacoli, collected from a University hospital in Southern Taiwan in 1999, were screened for ESBL-producingstrains; the rate of prevalence in that study was 1.6%, lower than that reported in Japan (2.9 to 8.%) .
The main antibiotics for treatment of UTIs are beta- lactame antibiotics (penicillins, second and third generation cephalosporins) and fluoroquinolone, but Enterobacte- riaceae can efficiently produce extended-spectrum beta- lactamases (ESBLs) to hydrolyze the β-lactame ring of a significant part of beta-lactames. ESBL-producing bacteria are mainly Escherichiacoli (considered as major producer) and Klebsiella spp. (Klebsilla pneumoniaå). These types of multidrug resistant bacteria are usually selected in hospital environment and respectively provoke nosocomial infec- tions. However, strainsof ESBL-producing bacteria are pro- gressively isolated in community-acquired infections and currently are a global public health problem .
The profiles of VF genes were extremely diverse: the 191 strains showed 159 different profiles, of these 134 were unique, 21 profiles appeared twice, 2 appeared three times, one profile was repeated four times and another one appeared five times. Such diversity prompted us to classify them in clusters; 29 clusters were found using a 70% similarity threshold; 6 clusters arbitrarily named C1– C6 grouped 128 isolates (67%). PGs, ESBLs, antimicrobial resistance, and epidemiological data according to cluster are shown in Table 3; distribution of VF among the clusters are shown in Figure 1. In summary, isolates from C1 caused infections in younger patients; those in C2 were associated with higher frequency of urinary tract source; C4 isolates showed higher frequency of CTX-M-1 group of ESBLs, resistance to amoxicillin/ clavulanic acid and tobramycin, and bacteremia from sources other than urinary or biliary tracts; those in C5 had the lower frequency of local predisposing factors; and C6 isolates showed less frequent resistance for ciprofloxacin. Sixteen of the 22 isolates from C4 (73%) belonged to ST131; also, 76% isolates from ST131 belonged to C4.
All isolates of type ST131 shared the same GECM-10 profile in all but the CVN014 and CVN016 loci. The isolates not belonging to E. coli O25b-ST131 differed by a marked number of repeats in locus CNV014, i.e., 11, as compared to five or six repeats observed for isolates of the O25b-ST131 type. Furthermore, the two E. coli O25b-ST131 type isolates not belonging to cluster A by PFGE also could be differentiated by GECM-10. CVN016 could be amplified in both isolates, with a PCR-product of 475 bp, although the repeat number was not able to be determined and, thus, was designated as ‘0’ (i.e., ‘null’), whereas no amplifications of the CVN016 locus (N) were observed in isolates of PFGE cluster A (Table 1, Figure 1). The isolate of PFGE type J that was more distantly related to the outbreak isolates of cluster A, according to PFGE profiling, also lacked one repeat in locus CVN014, compared to the type A and I isolates. Both of these isolates were fluoroquinolone resistant, as compared to the type A isolates. Interestingly, this resistance type has been reported previously to exhibit distinct PFGE-profiles within the 131 sequence type . Notably, locus CVN014 is known to be the most variable locus in the GECM-10 assay [27,28]. CVN016 also has been reported to exhibit variation among ESBL-E. coli isolates . Considering the recognized heterogeneity in E. coli O25b-ST131 , it is not unlikely that the number of tandem repeats may change over time in isolates of this type, particularly in the loci that are known to vary. Variation at a single locus, e.g., through homoplasy, may result in an underestimation of the actual genotypic variation between strains. However, the efficacy of the generic E. coli GECM-10 assay is based upon the combinations of highly variable and conserved loci, rather than only single loci. It has the ability to apply the more variable loci, such as CVN014 and CVN016, for high-resolution sub-typing of closely related isolates, such as those in an
was performed in a total volume of 50µL containing 1µL of each primer (10pM), 25µL of DNA Polymerase Master Mix RED (Ampliqon, Co., Denmark), 2µL of DNA, and 21µL of DNase and RNase free water in a FlexCycler PCR Thermal Cycler (Analytik Jena, Germany) under the following cycling conditions: an initial denaturation step at 95°C for 5min followed by 30 cycles of denaturation at 95 °C for 1min, annealing at 55-61°C for 1min (Table 1), extension at 72°C for 1min, and a final extension step at 72°C for 5min. The PCR products were separated by electrophoresis in a 1.5% agarose gel in 0.5 Tris, EDTA, Boric acid (TBE) buffer. For each ESBL group, amplicons from randomized selected strains were sequenced (Macrogen, South Korea). The obtained nucleotide sequences were compared with those in the GenBank database for homologous nucleotide sequences by BLAST (www.ncbi.nih.gov/BLAST program).
MICs of b-lactams for the CTX-M-94- and -100-producing isolates are shown in Table 1. The CTX-M-94 harbouring isolate exhibited four- to approximately 200-fold higher MICs of amoxicillin/clavulanate, cefoxitin, cefotaxime, ceftriaxone, ceftaz- idime (alone and with clavulanate), cefepime and aztreonam, when compared to the CTX-M-100 producer. According to the current CLSI guidelines , both isolates were highly resistant to amoxicillin, ampicillin, piperacillin, cefotaxime and ceftriaxone, and susceptible to piperacillin/tazobactam, cefepime and imipen- em. The resistance pattern of the CTX-M-100-producing isolate was similar to that of the previously described CTX-M-39- producing E. colifrom the same hospital , and matched well the typical CTX-M-associated phenotype, with asymmetry in MICs of cefotaxime and ceftazidime and good activity of inhibitor combinations [1,5,26]. In contrast, resistance of the CTX-M-94 producer was largely influenced by CMY-2, both in level and type. AmpC b-lactamases like CMY-2 are known to hydrolyze cephamycins and are much less inhibited by class A enzyme inhibitors compared to ESBLs , as exemplified by high MICs of cefoxitin and clavulanate combinations, respectively (Table 1). Furthermore, AmpC b-lactamases are also capable of hydrolyzing oxyiminocephalosporins and monobactams, as exemplified by elevated MICs of cefotaxime, ceftazidime, ceftriaxone and aztreonam (Table 1). Finally, the influence of CMY-2 on the phenotype exhibited by the wild-type CTX-M-94-harbouring isolate was confirmed by the decreased resistance pattern of pCR- CTX-M-94, a recombinant bla CTX-M-94 -harbouring strain that
With respect to PMQR genes, Seventy-five out of the 202 (37.1%) isolates possessed them, qnrS and aac(69)-Ib-cr were dominant while other PMQR genes were not detected in this study. A study by Tong et al. showed that the prevalence of PMQR among isolates from chickens were 42.4% during 2004 to 2011 in China . However, PMQR genes were detected in 5 (5.3%) of 94 E. coli chicken isolates in Turkey , and in 15 (15.6%)of 96 E. coli in 2006 in Nigeria . Plasmid mediated quinolone resistances (qnrS) were found in 2(1.3%) of 151 E. coli chicken strains in Slovakia . The frequency of PMQR genotypes in our study(37.1%) is lower than the report in China  and higher than that in Turkey,Nigeria and Slovakia. Typically, qnrB was considered to be the most prevalent PMQR gene in Enterobacteriaceae isolates in 2009 . In the present study, the aac(69)-Ib-cr enzyme was the most prevalent plasmid- mediated mechanism of quinolone resistance, as has been noted elsewhere . The prevalence of this gene is higher than that reported by older surveys [10,23–25]. Because of the high prevalence of PMQR genes and the relatively high levels of resistance to fluoroquinolones, it is important to intensify fluoroquinolone surveillance.
The β-lactam antibiotics have been amongst the most successful drugs for the treatment of bac- terial infections for the past 60 years . They are arguably the most important and widely used antimicrobial class for treating bacterial infections in both human and veterinary medi- cine, because of their excellent safety profile, broad antimicrobial spectrum, availability of orally bioavailable formulations, and the low cost of many products . More than half of all currently used antibiotics belong to the β-lactam group, but their clinical effectiveness is severely limited by the emergence ofβ-lactam resistant bacteria . The resistance to β-lactam antibiotics occurs as a result of drug inactivation by β-lactamases, target site (penicillin-binding proteins) alterations, diminished permeability and efflux . In Gram negative pathogens, β-lactamases are the major determinant of this resistance . Extended-spectrumβ-lactamases (ESBLs) are a rapidly evolving group ofβ-lactamases which hydrolyze third-generation cepha- losporins and aztreonam but not carbapenems . Extended-spectrumβ-lactamaseproducing Enterobacteriaceae (ESBL-E) are prevalent worldwide . Chicken meat has been proposed to constitute a source for ESBL-E that colonize and infect humans . Close genetic similarities among extended-spectrumβ-lactamase-producingEscherichiacoli (ESBL-EC) isolatedfrom chicken meat and humans together with the concurrent presence of CTX-M-1 and TEM-52 genes on similar plasmids ofEscherichiacoliisolatedfrom both sources support the occurrence of food-borne transmission of ESBL genes [9,10]. Furthermore, ESBL-EC isolatedfrom chicken meat was documented as a source of ESBL-EC in humans . Previous studies reported high ESBL contamination rates of chicken meat in the Netherlands [12,13], Sweden  and recently in Germany [8,15]. A recent study demonstrated the presence of carbapenemase-pro- ducing Enterobacteriaceae (CPE) in Broiler Chicken Fattening Farms  but there are no reports on acquired carbapenemase producers from retail chicken meat .
Although antimicrobial therapy is an impor- tant tool for infection treatment, antimicrobial resistance may become a major problem in veterinary medicine as a consequence of the intensive use or misuse of antimicrobial drugs (Monroe and Polk, 2000). Susceptibility patterns of indicator bacteria obtained from healthy an- imals have been suggested as good predictors of resistance situation in a bacterial population as a whole (Van den Bogaard and Stobberingh, 2000). During the processing of carcasses, fecal contamination or transfer of bacteria from the animal’s hide to the carcass can promote trans- mission of pathogenic E. coli to food supplies (Bell, 1997; Barkocy-Gallagher et al., 2001). An- timicrobial drug resistance data of fecal E. colistrainsfrom animals were difficult to find in the literature from Brazil, most of them showing high levels of resistance against several antimi- crobial agents from commensal E. coliisolatedfrom diarrheic calves (Rigobelo et al., 2006) as well as from STEC strainsisolatedfrom meat (Rodolpho and Marin, 2007).
Screening of bacteriocin producer and its characterization Bacteriocin producing bacteria were screened as previously described (12). The culture of bacteriocin producers was identified according to their morphological, and biochemical properties. The used tests were: Gram reaction; growth at 15, 25 and 45°C; production of catalase, cytochrome oxidase; acid and gas production from glucose; acid production from carbohydrates (1% w/v) lactose, sucrose, mannitol, ribose, sorbitol and mannose in MRS broth devoid of glucose and beef extract with phenol red as indicator.
Forty unrelated family dairy farms in Hulunbeier area (Inner Mongolia, Northern China), were randomly selected for test. The population in this area is predominantly engaged in animal husbandry. Stool specimens were collected from the rectums of 212 apparently healthy dairy cows (20 % of the herd or a minimum of 5 cows from each farm). The specimens were transported on ice to the laboratory. Swabs of stool were inoculated onto the surface of eosin–methylene blue agars (EMB) and streaked for isolated colonies. After incubation for 24 h at 37 °C, three or four colonies with typical E. coli morphology and one of another morphological type were
DNA analysis: Selected multi-resistant isolates were tested by pulsed-field gel electrophoresis (PFGE) in order to evaluate the method of dissemination of ESBL-producingstrains in hospital B in Goiânia. Macrorestriction analysis of chromossomal DNA was performed by PFGE in accordance to the published procedures (19) with Xba I (New England Biolabs, Boston, Mass.). PFGE was run in a CHEF-DR II apparatus (Bio-Rad Laboratories, Richmond, California) with pulses ranging from 5 to 60 seconds at a voltage of 6V/cm for 23 hours at 14ºC for 20 hours. Lambda ladder (48.5 Kb, Sigma) was used as molecular weight marker. Fragments were stained with ethidium bromide (Sigma) and photographed. Visual comparisons were made and the criteria of Pfaller (19) were used to establish the relationship among the isolates.
ampliﬁ cations, 2µL of DNA was added to a 50-µL solution containing 1µL of deoxynucleotide triphosphates (dNTPs), 1.5µL Mgcl 2 , 2µL of each primer, and 0.3µL of Taq polymerase (CinnaGen, Iran) in 1x PCR buffer. An Eppendorf Thermocycler (Germany) was used for ampliﬁ cation, with cycling parameters comprising an initial denaturation at 95°C for 10 min followed by 35 cycles of denaturation at 95°C for 30s, annealing at 51°C for 40s, ampliﬁ cation elongation(extension) at 72°C for 30s, and a ﬁ nal extension at 72°C for 3 min.
there is clonal spread of resistant strains, transfer of resistance genes between human and animal bacteria and also the sharing of phylogenetic and genotypic similarities (Van Den Bogaard and Stobberingh, 2000). Extended-spectrum b-lactamases (ESBLs) have contributed to the recently noted large increase in resistance to b-lactams. Many of the ESBLs arise from point mutations in plasmid-mediated genes that encode narrow- spectrum b-lactamases such as TEM and SHV (Bradford, 2001). However, members of a newly emerging ESBL group, CTX-M, derived from class A chromosomal b-lactamases have been identified (Bonnet, 2004). In Japan, Kon et al. (2005) described an STEC O26:H11 strain resistant to cefotaxime and cefpo- doxime (but not ceftazidime), producing CTX-M-3 type ESBL. Despite the fact that antimicrobial resistance in STEC has been the subject of several studies, information on the molecular basis of antimicrobial resistance among this zoonotic group of bacteria is still limited (Guerra et al., 2006; Lee, 2009; Nagachinta and Chen, 2009; Srinivasan et al., 2007; Zhao et al., 2001). The aims of this study were to further charac- terize the antimicrobial resistance displayed by various STEC strainsisolatedfrom human infections and the animal reservoir in Brazil, and evaluate their genetic relatedness.
defined typing techniques that are currently recommended for use. Additionally, it is not clear how any results obtained should actually be interpreted. Therefore, in this publication, the authors developed their own definitions for ‘healthcare-related transmission event’ and ‘likeli- hood of healthcare-related transmission’ (Table 2). In a systematic review, Kramer et al. deter- mined how long E. coli can survive on inanimate surfaces, which differed from 1.5 hours up to 16 months . As this range is not practical, we selected the reference that was most applica- ble to the hospital setting when considering environmental contamination in our definitions of epidemiological relatedness (Table 2). However, Neely at al. found that most E. coli isolates had died in the environment only after 36 days . Therefore, we extended our time frame up to 2 months and incorporated this within the definitions ‘probable’ and ‘possible’ in Table 2. In case we still missed important links we extended the time frame used in model 3 from 2 to 3 months and used this time period in model 4 (Table 2). However, 33.9% of the 112 patients were considered as culture positive for ESBL-producing E. coli via a healthcare-related trans- mission event, but no healthcare-related transmission event was identified using our ‘likeli- hood’ definition. The question therefore arises if some of the 112 patients should actually be considered as ‘community acquired’, since there could still be some form of endogenous selec- tion because of antibiotic use. This however is a subject for future research.
O estudo de Baccaro et al. (2002), em amostras de E. coli isoladas de fezes de leitões de granjas no estado de São Paulo, revelou resistência de 87,4% das amostras a sulfazotrim, e 86,8% a ampicilina, dados semelhantes aos observados nesta pesquisa, porém o mesmo autor encontrou resistência de 92% à norfloxacina, enquanto neste estudo foi observada a resistência de apenas 15% a este antimicrobiano. A divergência de dados pode ser atribuída ao fato dos autores citados terem utilizado a concentração inibitória mínima (CIM) como metodologia na de- terminação da resistência, e também a variação regional.
method for AmpC β-lactamases with 100% sensitivity and 100% specificity compared to the cefoxitin screening method, which yielded results of 100% sensitivity but only 65% specificity. These results are contrary to other studies [35,45]. Polsfuss et al. (2011) reported that although the cefoxitin screening showed a lower specificity (78.7%), it evidenced a higher sensitivity (97.4%) than AmpC E-test (77.4%), proposing a flow chart for AmpC detection with cefoxitin resistance as a screening method and the cefoxitin/cloxacillin combination as a confirming phenotypic assay . The ambiguous results obtained may be due to the lower number of isolates evaluated in this study, requiring further investigation.
nificant differences were in respect to the frequency of the categories and the kind of serotypes. For example, the most frequent categories in their studies were a-EPEC, EAEC, and EHEC while in our studies the most frequent ones were t-EPEC and EAEC. Regarding serotypes, it was surprising the total absence in UK of the most frequent t- EPEC serotypes in our studies, namely O111:H2 and O55:H6. In the past these two serotypes were very fre- quent in UK as a cause of diarrhea outbreaks and spo- radic cases of infantile diarrhea. Comparison of the fre- quency of EPEC and EHEC along the years in UK (and others developed countries) and in Brazil offers some in- teresting aspects. In the past the EPEC was extremely fre- quent in UK (Smith et al. 1996) and others developed coun- tries and EHEC was practically inexistent. At present t- EPEC are very rare and the atypical EPEC and EHEC are frequents. In Brazil (São Paulo) we need to consider two periods. Until the end of the 1980s, t-EPEC was the pre- dominant agent of diarrhea and EHEC extremely rare (To- ledo et al. 1983, Gomes et al. 1989, 1991). In fact at that time we had isolated only three O111ac:H- strains (Stx+) and a very few strainsof others serotypes (Campos et al. 1994). At present this situation has changed substantially. It seens that we are approaching the present situation in developed countries which was started in the 1960s for t- EPEC and the 1980s for EHEC. Accordingly most of our EPEC isolates lack EAF and we have started to isolate O157:H7 and O26:H11 Stx+ strains. The reasons for this kind of events have not been established but they are not exclusive of this pathogen. They are similar to the events that have occurred with Salmonella serotypes: a dramatic fall in the frequency of S. Typhi and a vast increase in the frequency of S. Typhimurium and similar serotypes. S. Typhi as typical EPEC is specific human pathogens and S. Typhimurium as EHEC and atypical EPEC are pathogens associated with humans and animals. The reductions of the typical EPEC frequency in Brazil (São Paulo) may be related to the improvement of the living conditions in re- cent years.