First report in Africa of two clinical isolates of Proteus mirabilis carrying Salmonella genomic island (SGI1) variants,SGI1-PmABB and SGI1-W

Two Proteus mirabilis strains, designated PmTAN59 and PmKAF126, were isolated from two different Egyptian cities in 2014 and 2015, respectively. PmTAN59 was isolated from a sputum swab from a pneumonia patient in Tanta University Teaching Hospital. PmKAF126 was isolated from a patient with a diabetic foot infection in a hospital in the city of Kafr El-Sheikh. The two isolates were identified with bacterial small ribosomal RNA (16S rRNA) gene amplification and sequencing and tested for antimicrobial sensitivity with a Kirby–Bauer disk diffusion assay. The two strains were resistant to amoxicillin/clavulante, ampicillin, cefotaxime, cefoxitin, ceftriaxone, chloramphenicol, ciprofloxacin, colistin, gentamicin, kanamycin, nalidixic acid, spectinomycin, streptomycin, sulfamethoxazole/trimethoprime, and tetracycline, but sensitive to aztreonam, imipenem, and meropenem. Molecular characterization was used to map the entire backbone, including the multiple antibiotic resistance (MDR) region, of Salmonella genomic island 1 (SGI1). Both isolates carried a structure similar to SGI1, with two different MDR regions corresponding to SGI1-PmABB in PmTAN59 and SGI1-W in PmKAF126. SGI1-PmABB carried an integron of ~ 1.5 kb with a two-gene cassette, aacCA5–aadA7, which confers resistance to gentamicin, streptomycin, and spectinomycin, whereas SGI1-W carried an integron of ~ 1.9 kb containing aadA2–lnuF, which confers resistance to spectinomycin, streptomycin, and lincosamides. PmKAF126 carried the entire SGI1 sequence, however PmTAN59 carried a SGI1 structure with a deletion in the region from ORF S005 to ORF S009 and accompanied by insertion of IS1359 (1258 bp). Furthermore, PmTAN59 carried class 2 integron of ~ 2.2 kb containing dfrA1-sat2-aadA1. An ERIC-PCR analysis detected no clonal relationship between the two strains. Molecular screening for other antimicrobial resistance genes and a plasmid analysis indicated that PmTAN59 carried an IncFIB plasmid type. This strain also carried bla_TEM-1 and the plasmid-mediated quinolone-resistance gene qnrA1. However, PmKAF126 carried no plasmids and no resistance gene other than that contained in the MDR region of SGI1 and floR gene conferring resistance to florfenicol. To the best of our knowledge, this is the first report of an SGI1-positive P. mirabilis strain in Egypt or on the entire African continent.     

Occurrence and distribution of various genetic structures of class 1 and class 2 integrons in Salmonella enterica isolates from foodborne disease patients in Korea for 16 years

We have investigated the distribution of integrons among 752 multidrug-resistant Salmonella isolates from human febrile and/or diarrheal patients during 1992-2007 and analyzed their genetic characteristics. Here, we report extensive integron analysis results within human isolates during the last 16 years. The gene or gene cassette(s) in the class 1 integrons found in the isolates were dfrA7, dfrA12-orfF-aadA2, aadA2, bla(PSE1), dfrA1-aadA1, dfrA17-aadA5, bla(OXA1)-aadA1, aadB-aadA1, aadA22, aadA1, and aac6'Ib-bla(OXA1)-aadA2. Class 2 integrons harboring dfrA1-sat2-aadA1 gene cassette were also found in four isolates. Twenty-nine isolates including one Salmonella Schleissheim isolate had two integrons harboring aadA2 and bla(PSE1) in their variable regions of 1.0 and 1.2?kb amplicons, respectively, which have been also found in Salmonella genomic island 1 (SGI1) of multidrug-resistant Salmonella Typhimurium DT104. The presence of SGI1 in Salmonella Schleissheim isolate was proved by SGI1-specific polymerase chain reaction. We first report a Salmonella Schleissheim having SGI1, Salmonella Typhimurium and Salmonella Heidelberg having the class 2 integron with dfrA1-sat2-aadA1 cassettes, Salmonella London with the aac6'Ib-bla(OXA1)-aadA2 gene cassette, Salmonella Chailey with the gene cassette of aadA22, and coexistence of two class 1 integrons carrying aadA22 and dfrA12-orfF-aadA2 in Salmonella Typhimurium.     

International high-risk clone of fluoroquinolone-resistant Escherichia coli O15:H1-D-ST393 in remote communities of Brazilian Amazon

The global dissemination of multidrug-resistant Escherichia coli lineages belonging to high- risk clones poses a significant public health threat. Herein we report the identification and genomic profiling of two multidrug-resistant E. coli strains [BL-II-03(2) and BL-II-11(3)] belonging to the O15:H1-D-ST393 (clonal complex 31) worldwide spread clone, isolated from fecal samples of indigenous peoples belonging to two different ethnic groups of remote communities of Brazilian Amazon. Genomic analysis revealed genes and mutations conferring resistance to β-lactams [bla_TEM-1], aminoglycosides [aadA5, aph(3″)-Ib, aph(6)-Id], tetracyclines [tetB], sulfamethoxazole/trimethoprim [sul1, sul2, dfrA17], and fluoroquinolones [gyrA (D87N, S83L), parC (S80I, S57T), parE (L416F)]; and presence of IncQ1, IncFIA, and IncFIB(pB171) plasmids. On the other hand, phylogenomics of globally reported E. coli ST393 assigned E. coli strains BL-II-03(2) and BL-II-11(3) to a cluster comprising human isolates from Australia, Canada, China, Sweden, and United States of America. These results might provide valuable information for understanding dissemination of intercontinental multidrug-resistant clones in remote communities with low levels of antibiotic exposure.     

The plasmidome of multidrug-resistant emergent Salmonella serovars isolated from poultry

The rapid emergence of resistant bacteria is occurring worldwide. The understanding of the dissemination of antimicrobial resistance using high-throughput sequencing and bioinformatics approaches is providing valuable insights into the genetic basis of the horizontal gene transfer and the emergence of the antibiotic resistance threat. This ultimately can offer vital clues to the development of coordinated efforts to implement new policies to continue fighting against bacterial infections. The poultry microbiota is characterized as a potential reservoir of resistance genes, mostly derived from the Enterobacteriaceae which have become increasingly important in human and animal infections. In this work, complete genome sequences were achieved for four multidrug-resistant Salmonella spp. isolated from poultry from different farms in Brazil. We identified highly similar IncHI2-ST2 megaplasmids (larger than 275.000 bp) in all Salmonella isolates studied. These megaplasmids carry a resistome comprised of eleven different resistance genes (aac(6′)-Iaa, aadA1b, aph(4)-Ia, aph(6)-Id, aph(3″)-Ib, aph(3′)-Ia, aac(3)-Iva, sul1, tetA, tetB and dfrA1b) and four heavy metal tolerance operons (telluride, mercury, silver and copper). In conclusion, the multidrug-resistant plasmids identified in S. enterica serovar Schwarzengrund and Newport isolated from poultry show a variety of antibiotic resistance and heavy metal tolerance genes, providing advantages for the bacteria to survive under extremely unfavorable conditions.     

A reliable combination method to identification and typing of epidemic and endemic clones among clinical isolates of Acinetobacter baumannii

The multi-drug resistant (MDR) Acinetobacter baumannii as an important nosocomial pathogen has emerged a global health concern in recent years. In this study, we applied three easier, faster, and cost-effective methods including PCR-based open reading frames (ORFs) typing, sequence typing of bla_OXA-51-like and RAPD-PCR method to rapid typing of A. baumannii strains. Taken together in the present study the results of ORFs typing, PCR-sequencing of bla_OXA-51-like genes and MLST sequence typing revealed there was a high prevalence (62%, 35/57) of ST2 as international and successful clone which detected among clinical isolates of multi-drug resistant A. baumannii with ORF pattern B and bla_OXA-66 gene. Only 7% (4/57) of MDR isolates belonged to ST1 with ORF pattern A and bla_OXA-69 gene. Interestingly, we detected singleton ST513 (32%, 18/57) that encoded bla_OXA-90 and showed the ORF pattern H as previously isolated in Middle East. Moreover, our data showed RAPD-PCR method can detect divergent strains of the STs. The Cl-1, Cl-2, Cl-3, Cl-4, Cl-10, Cl-11, Cl-12, Cl-13 and Cl-14 belonged to ST2. While the Cl-6, Cl-7, Cl-8 and Cl-9 belonged to ST513. Only Cl-5 belonged to ST1. It seems that the combination of these methods have more discriminatory than any method separately and could be effectively applied to rapid detection of the clonal complex (CC) of A. baumannii strains without performing of MLST or PFGE.     

Emergence of carbapenem resistant Escherichia coli isolates producing blaNDM and blaOXA-48-like carried on IncA/C and IncL/M plasmids at two Iranian university hospitals

The emergence of carbapenem resistance among Escherichia coli is a serious threat to public health. The objective of this study was to investigate resistance genes and clonality of carbapenem resistant E. coli in Iran. Between February 2015 and July 2016, a total of 32 non-duplicate E. coli isolates that were ertapenem resistant or intermediate (R/I-ETP) were collected from patient clinical or surveillance cultures (rectal swabs) at two university hospitals. Resistance genes were identified by PCR and sequencing. Conjugation experiments, PCR-based replicon typing, PFGE and multilocus sequence typing (MLST) were performed. PCR assays showed, among the 32 isolates, twenty-nine strains produced carbapenemase genes. The predominant carbapenemase was bla_OXA-48 (82.8%), followed by bla_NDM-1 (31%), bla_NDM-7 (6.9%) and bla_OXA-181 (3.4%). Seven of the bla_NDM positive isolates co-harbored bla_OXA-48 carbapenemases. The bla_NDM and bla_OXA-48 were found in IncA/C and IncL/M conjugative plasmids, respectively. The bla_CTX-M-15, qnrA and intI1 genes were also present in most isolates. The PFGE revealed genetic diversity among the 28 E. coli isolates, which belonged to six minor PFGE clusters and 14 isolates were singletons. The 26 isolates were distributed into 18 STs, of which two were dominant (ST648 and ST167). We identified one bla_NDM-1-positive ST131 E. coli isolates that harbor the bla_CTX-M-15 and bla_TEM genes. Horizontal transfer of IncA/C and IncL/M plasmids has likely facilitated the spread of the bla_OXA-48 and bla_NDM genes among E. coli. Their clonal diversity and the presence of faecal carriers in isolates suggest an endemic spread of OXA-48 and NDM. Therefore, it emphasizes the critical importance of monitoring and controlling the spread of carbapenem resistant E. coli.     

Worldwide Dissemination of the blaOXA-23 Carbapenemase Gene of Acinetobacter baumannii1

To assess dissemination of OXA-23–producing strains of Acinetobacter baumannii, we obtained 20 carbapenem-resistant, OXA-23–producing isolates from different regions. Their clonal relationship was assessed by pulsed-field gel electrophoresis and multilocus sequence typing. We identified 8 sequence types, including 4 novel types. All except 2 strains belonged to 2 main European clonal lineages. The bla_OXA-23 gene was either located on the chromosome or on plasmids and associated with 4 genetic structures.     

First report of OXA-48-producing Klebsiella pneumoniae strains in Iran

Carbapenem-resistant Enterobacteriaceae are increasingly reported worldwide and cause therapeutic problem in health care facilities. In this study 28 imipenem-resistant K. pneumoniae were examined for expression of carbapenemases by phenotypic and genotypic methods. Modified Hodge Test (MHT), CarbaNP test were used for phenotypic detection, and PCR using specific primers for the detection of bla_OXA-48-, bla_KPC-, bla_NDM- and bla_VIM-type carbapenemases with specific primers were performed. MHT and CarbaNP tests were positive for all of imipenem-resistant K. pneumoniae. The bla_OXA-48 gene was detected in 27/28 isolates. One isolate was positive for the presence of the bla_VIM-4 gene. According to our results NP test and MHT have high sensitivity and specificity for detection of those carbapenemases. This study reports the first cases of OXA-48-producing K. pneumoniae in Iran.     

Worldwide Diversity of Klebsiella pneumoniae That Produce ��-Lactamase blaKPC-2 Gene

Klebsiella pneumoniae isolates that produce carbapenemases (KPCs) are rapidly disseminating worldwide. To determine their genetic background, we investigated 16 bla_KPC-2-harboring K. pneumoniae isolates from 5 countries. The isolates were multidrug resistant, possessed the bla_KPC-2 gene, and differed by additional β-lactamase content. They harbored a naturally chromosome-encoded bla gene (bla_SHV-1 [12.5%], bla_SHV-11 [68.7%], or bla_OKP-A/B [18.8%]) and several acquired and plasmid-encoded genes (bla_TEM-1 [81.3%], bla_CTX-M-2 [31.3%], bla_CTX-M-12 [12.5%], bla_CTX-M-15 [18.7%], and bla_OXA-9 [37.5%]). The bla_KPC-2 gene was always associated with 1 of the Tn4401 isoforms (a, b, or c). Tn4401 was inserted on different-sized plasmids that belonged to different incompatibility groups. Several bla_KPC-containing K. pneumoniae clones were found: 9 different pulsotypes with 1 major (sequence type 258) and 7 minor distinct allelic profiles. Different clones harboring different plasmids but having identical genetic structure, Tn4401, could be at the origin of the worldwide spread of this emerging resistance gene.     

Nosocomial spread of carbapenem-resistant Acinetobacter baumannii (types ST75 and ST137) carrying blaOXA-23-like gene with an upstream ISAba1 in a Chinese hospital

The most widespread type of carbapenemases among carbapenem-resistant Acinetobacter baumannii (CRAB) belongs to the oxacillinase (OXA) group. A total of 57 CRAB isolates and 20 non-CRAB isolates (i.e., A. baumannii susceptible to carbapenems) were studied to investigate the molecular epidemiology of CRAB isolates and to identify the OXAs responsible for resistance to imipenem. The ISAba1-bla_OXA-23-like gene was detected in all 57 CRAB isolates but was detected in none of the non-CRAB isolates. Pulsed-field gel electrophoresis (PFGE) revealed that clones A and B were the dominant genotypes, and all bla_OXA-23-like gene positive strains were classified as either clone A or B strains. ST75 and ST137 were the most prominent sequence types (STs). Finally, the A. baumannii isolates of clone A, C and F were all demonstrated to be genetically similar to the previously identified European clone II. In conclusion, ST75- and ST137-type CRAB isolates that produced the bla_OXA-23-like gene with an upstream ISAba1 contributed to the nosocomial outbreaks studied in this work.     

Variant Salmonella genomic island 1 antibiotic resistance gene cluster in Salmonella enterica serovar Albany

Salmonella genomic island 1 (SGI1) contains an antibiotic resistance gene cluster and has been previously identified in multidrug-resistant Salmonella enterica serovars Typhimurium DT104, Agona, and Paratyphi B. We identified a variant SGI1 antibiotic-resistance gene cluster in a multidrug-resistant strain of S. enterica serovar Albany isolated from food fish from Thailand and imported to France. In this strain, the streptomycin resistance aadA2 gene cassette in one of the SGI1 integrons was replaced by a dfrA1 gene cassette, conferring resistance to trimethoprim and an open reading frame of unknown function. Thus, this serovar Albany strain represents the fourth S. enterica serovar in which SGI1 has been identified and the first SGI1 example where gene cassette replacement took place in one of its integron structures. The antibiotic resistance gene cluster of serovar Albany strain 7205.00 constitutes a new SGI1 variant; we propose a name of SGI1-F.     

Extensively drug-resistant IMP-16-producing Pseudomonas monteilii isolated from cerebrospinal fluid

IMP-1-producing Pseudomonas aeruginosa was first reported in Japan and since then, bacteria with this metallo-β-lactamase have been detected worldwide. Pseudomonas monteilii (part of P. putida group) were considered an environmental pathogen with low virulence potential; however, multidrug-resistant and carbapenem-resistant P. monteilii have emerged. The present study reports the draft sequence of an extensively drug-resistant IMP-16-producing P. monteilii 597/14 isolated from cerebrospinal fluid in 2014. The sequencing data revealed bla_IMP-16 as a gene cassette on class 1 integron, In1738 characterized in this study. Furthermore, the resistome of Pm597/14 consisted of 7 resistance genes (aadA1b, strA, strB, aacA4, bla_IMP-16, bla_OXA-2, sul1) and diverse virulence determinants involved in the adherence, LPS, antiphagocytosis, iron uptake and mercuric resistance. Although different virulence determinants were found in this study, using Galleria mellonella infection model, Pm597/14 did not kill any larvae between 7 days post-infection. P. monteilii isolates have been reported from clinical and environmental sources, carrying different MBL genes showing its potential role as their reservoir.     

Resistance phenotype-genotype correlation and molecular epidemiology of Citrobacter, Enterobacter, Proteus, Providencia, Salmonella and Serratia that carry extended-spectrum β-lactamases with or without plasmid-mediated AmpC β-lactamase genes in Thailand

Extended-spectrum β-lactamases (ESBLs) and plasmid-mediated AmpC β-lactamases (pAmpCs) have been increasingly reported among less commonly encountered genera of Enterobacteriaceae. However, little is known regarding the genetic characteristics of resistance genes and epidemiology of these genera. Lack of accurate ESBL and pAmpC detection may adversely affect therapeutic outcomes. This study investigated resistance phenotype-genotype correlation and molecular epidemiology among six genera of Enterobacteriaceae (Citrobacter, Enterobacter, Proteus, Providencia, Salmonella and Serratia) that carried ESBL with or without pAmpC genes at a university hospital in Thailand. From a total of 562 isolates, 105 isolates (18.7%) had ESBL-positive phenotype whilst 140 isolates (24.9%) harboured one or more ESBL genes. CTX-M and TEM were common ESBL-related bla genes among these isolates. The sensitivity and specificity of ESBL phenotypic detection as opposed to ESBL gene detection were 70.7% and 98.6%, respectively. pAmpC genes were detected in 96 ESBL gene-carrying isolates (68.6%) and significantly caused false negative detection of ESBL. Molecular typing based on pulsed-field gel electrophoresis revealed several clones that may be endemic in this hospital. This study indicated a high prevalence of ESBLs and pAmpCs among less common members of the family Enterobacteriaceae in Thailand and these resistant bacteria need to be monitored.     

Emergence and spread of carbapenem-resistant Acinetobacter baumannii sequence type 191 in a Korean hospital

Emergence and spread of specific carbapenem-resistant Acinetobacter baumannii (CRAB) clones cause a serious therapeutic problem. This study was aimed to investigate the clonal diversity and genetic basis of antimicrobial resistance among the 69 CRAB isolates from 2009 to 2010 in a Korean hospital. All CRAB isolates were found to be sequence type (ST) 2 using the Institute Pasteur’s multilocus sequence typing (MLST) scheme, but classified into two sequence groups and nine pulsotypes. Fifty-six CRAB isolates belonging to two main pulsotypes were found to be ST191 using the Bartual’s MLST scheme. All CRAB isolates showed an extensively drug-resistant phenotype. The bla_OXA-51/bla_OXA-23, bla_AmpC/bla_PER-1 and armA genes were largely responsible for resistance to carbapenems, extended-spectrum β-lactams and aminoglycosides, respectively. The first CRAB strains identified in 2005 in this hospital were found to be ST2 using the Institute Pasteur’s MLST scheme, but showed ST353 using the Bartual’s MLST scheme and different pulsotypes from the CRAB isolates from 2009 to 2010. In conclusion, this is the first report of emergence and spread of A. baumannii ST191 in Korea, as well of the genetic basis of its antimicrobial resistance.     

Multidrug resistance IncC plasmid carrying blaCMY-97 in Shiga toxin-producing Escherichia coli ST215-H54 of ovine origin

CMY-type β-lactamases are the most reported plasmid-mediated AmpC (pAmpC), with the CMY-2-like group being the most clinically relevant described in Escherichia coli at human-animal-environment interface. Shiga toxin-producing E. coli (STEC) lineages are zoonotic pathogens commonly reported causing serious clinical conditions in humans, including severe diarrheagenic diseases. Therefore, this study aimed to investigate a multidrug-resistant (MDR) STEC isolate (A313) recovered from a healthy sheep and carrying mobile bla_CMY-97, that encodes a pAmpC belonging to the CMY-2-like group. The A313 isolate exhibited a MDR profile to clinically relevant antimicrobials (i.e., cephalosporins, aminoglycosides, and fluoroquinolones), but reduced susceptibility to extended-spectrum cephalosporins and aztreonam. Besides, virulence genes (stx2, gad and iutA) were detected in A313, which belonged to ST215/CC10 and phylogenetic group A, whereas the fimH54 was identified. The bla_CMY-97 gene and other antimicrobial resistance determinants [aph(6)-Id, aph(3″)-Ib, aac(3)-IId, aadA5, floR, tetA, sul1, and sul2], as well as genes encoding tolerance to mercury (merRTPCADE), were harbored by an IncC plasmid (named pA313-CMY-97, ~ 176 kb). A novel genetic context of bla_CMY-2-like, in which a 208-bp ISEcp1 was truncated by an IS26 in the opposite orientation upstream of the bla_CMY-97 gene (IS26-∆ISEcp1-bla_CMY-97-blc-sugE-encR), was also identified in pA313-CMY-97. To the best of our knowledge, this is the first report on the acquisition of bla_CMY-97 into a plasmid. Therefore, we reported ovine as reservoir of clinically relevant MDR bacteria carrying mobile bla_CMY-97 with potential for zoonotic transmission.     

A new trilocus sequence-based multiplex-PCR to detect major Acinetobacter baumannii clones

A collection of 163 Acinetobacter baumannii isolates detected in a large Brazilian hospital, was potentially related with the dissemination of four clonal complexes (CC): 113/79, 103/15, 109/1 and 110/25, defined by University of Oxford/Institut Pasteur multilocus sequence typing (MLST) schemes. The urge of a simple multiplex-PCR scheme to specify these clones has motivated the present study. The established trilocus sequence-based typing (3LST, for ompA, csuE and bla_OXA-51-like genes) multiplex-PCR rapidly identifies international clones I (CC109/1), II (CC118/2) and III (CC187/3). Thus, the system detects only one (CC109/1) out of four main CC in Brazil. We aimed to develop an alternative multiplex-PCR scheme to detect these clones, known to be present additionally in Africa, Asia, Europe, USA and South America. MLST, performed in the present study to complement typing our whole collection of isolates, confirmed that all isolates belonged to the same four CC detected previously. When typed by 3LST-based multiplex-PCR, only 12% of the 163 isolates were classified into groups. By comparative sequence analysis of ompA, csuE and bla_OXA-51-like genes, a set of eight primers was designed for an alternative multiplex-PCR to distinguish the five CC 113/79, 103/15, 109/1, 110/25 and 118/2. Study isolates and one CC118/2 isolate were blind-tested with the new alternative PCR scheme; all were correctly clustered in groups of the corresponding CC. The new multiplex-PCR, with the advantage of fitting in a single reaction, detects five leading A. baumannii clones and could help preventing the spread in healthcare settings.     

Molecular characterization of a carbapenem-resistant Enterobacter hormaechei ssp. xiangfangensis co-harbouring blaNDM-1 and a chromosomally encoded phage-linked blaCTX-M-15 genes

Enterobacter cloacae complex (ECC) members are rapidly emerging as successful nosocomial pathogens, especially, with the emergence of carbapenem-resistant clones. In this study, we performed a comprehensive molecular characterization of a carbapenem-resistant E. hormaechei ssp. xiangfangensis LAU_ENC1. hsp60 and average nucleotide identity (ANI) were used for its identification. The repertoire of resistance genes and phage content were analyzed. Plasmid sequences were extracted and compared to closest references. The isolate LAU_ENC1 was identified as an E. hormaechei ssp. xiangfangensis and belonged to ST-114A sub-cluster. bla_NDM-1, bla_CTX-M-15, bla_OXA-1, and bla_ACT-16 genes were detected as β-lactam resistance determinants. A chromosomal hybrid intact phage, Enterobacter phage LAU1, with bla_CTX-M-15 integrated in its direct vicinity within an ISEcp1 - bla_CTX-M-15 - wbuC - ∆Tn2 rare cassette was detected. bla_NDM-1 was integrated within a novel IncFII conjugative plasmid, pLAU_ENC1, through an IS3000- ΔISAba125-bla_NDM-1-ble_MBL−//−Tn5403 cassette. To our knowledge, this is the first report of a multi-drug resistant (MDR) E. hormaechei ssp. xiangfangensis carrying a bla_CTX-M-15 integrated within the proximity of a provirus chromosomal region. Treatment options for MDR ECC members are becoming scarce, thus warranting an increased monitoring of the dissemination of these pathogens in clinical settings.     

Evaluation of carbapenem-resistant Enterobacteriaceae in an Italian setting: Report from the trench

The spread of carbapenem resistant Enterobacteriaceae (CRE) has recently become a matter of concern in public health, mainly due to the wide distribution of carbapenemase genes. Italy is a country considered endemic for the spread of bla_KPC Klebsiella pneumoniae (KP). The aim of this study was to depict the epidemiological trend of CRE in one Italian hospital over a long period (3 years surveillance, from May 2011 to April 2014). Based on defined MIC cut-off for specific carbapenems, 164 strains isolated from 146 different patients were analyzed both phenotypically and genotypically to establish the resistance genes. Molecular typing was performed using the RAPD technique. 77 strains were demonstrated to harbor the bla_KPC gene (73 KP, 4 Escherichia coli – EC), 51 strains the bla_VIM gene (44 KP, 3 EC, 2 Enterobacter cloacae and 2 Klebsiella oxytoca), 8 the bla_NDM gene (3 KP, 4 EC and one Providencia stuartii), 3 the bla_OXA-48 gene (2 KP, 1 EC), whereas 25 out of the 164 isolates (of different genera and species) had a negative multiplex-PCR amplification for all the targets tested. 39 out of the 164 strains analyzed (23.8%) revealed discrepancies between the MICs obtained with automated instrument and gradient MICs of more than two logs of difference; the broth microdilution provided a better agreement with the results obtained with the gradient MIC. The use of RAPD allowed to distinguish different clusters, closely related, both for bla_KPC and for bla_VIM KP.