Sequence of Closely Related Plasmids Encoding bla NDM-1 in Two Unrelated Klebsiella pneumoniae Isolates in Singapore

Background

Spread of the bla _NDM-1 gene that encodes the New Delhi metallo-β-lactamase (NDM-1) in Enterobacteriaceae is a major global health problem. Plasmids carrying bla _NDM-1 from two different multi-drug resistant Klebsiella pneumonia isolates collected in Singapore were completely sequenced and compared to known plasmids carrying bla _NDM-1.

Methodology/Principal Findings

The two plasmids, pTR3 and pTR4, were transferred to Escherichia coli recipient strain J53 and completely sequenced by a shotgun approach using 3-kb paired-end libraries on 454. Although the K. pneumoniae strains were unrelated by molecular typing using PFGE and MLST, complete sequencing revealed that pTR3 and pTR4 are identical. The plasmid sequence is similar to the E. coli NDM-1-encoding plasmid p271A, which was isolated in Australia from a patient returning from Bangladesh. The immediate regions of the bla _NDM-1 gene in pTR3/4 are identical to that of p271A, but the backbone of our plasmid is much more similar to another IncN2 plasmid reported recently, pJIE137, which contained an additional 5.2-kb CUP (conserved upstream repeat) regulon region in comparison to p271A. A 257-bp element bounded by imperfect 39-bp inverted repeats (IR) and an incomplete version of this element flanking the 3.6-kb NDM-1-encoding region were identified in these plasmids and are likely to be the vestiges of an unknown IS.

Conclusions

Although the hosts are not epidemiologically linked, we found that the plasmids bearing the bla _NDM-1 gene are identical. Comparative analyses of the conserved NDM-1-encoding region among different plasmids from K. pneumoniae and E. coli suggested that the transposable elements and the two unknown IR-associated elements flanking the NDM-1-encoding region might have aided the spreading of this worrisome resistance determinant.     

Characterization of an IncFII plasmid encoding NDM-1 from Escherichia coli ST131

Background

The current spread of the gene encoding the metallo-ß-lactamase NDM-1 in Enterobacteriaceae is linked to a variety of surrounding genetic structures and plasmid scaffolds.

Methodology

The whole sequence of plasmid pGUE-NDM carrying the bla _NDM-1 gene was determined by high-density pyrosequencing and a genomic comparative analysis with other bla _NDM-1-negative IncFII was performed.

Principal Findings

Plasmid pGUE-NDM replicating in Escherichia coli confers resistance to many antibiotic molecules including β-lactams, aminoglycosides, trimethoprim, and sulfonamides. It is 87,022 bp in-size and carries the two β-lactamase genes bla _NDM-1 and bla _OXA-1, together with three aminoglycoside resistance genes aacA4, aadA2, and aacC2. Comparative analysis of the multidrug resistance locus contained a module encompassing the bla _NDM-1 gene that is actually conserved among different structures identified in other enterobacterial isolates. This module was constituted by the bla _NDM-1 gene, a fragment of insertion sequence ISAba125 and a bleomycin resistance encoding gene.

Significance

This is the first characterized bla _NDM-1-carrying IncFII-type plasmid. Such association between the bla _NDM-1 gene and an IncFII-type plasmid backbone is extremely worrisome considering that this plasmid type is known to spread efficiently, as examplified with the worldwide dissemination of bla _CTX-M-15-borne IncFII plasmids.     

First Identification of Novel NDM Carbapenemase,NDM-7, in Escherichia coli in France

Background

The NDM-1 carbapenemase has been identified in 2008 in Enterobacteriaceae. Since then, several reports have emphasized its rapid dissemination throughout the world. The spread of NDM carbapenemases involve several bla _NDM gene variants associated with various plasmids among several Gram negative species.

Methodology

A multidrug-resistant E. coli isolate recovered from urine of a patient who had travelled to Burma has been characterized genetically and biochemically.

Principal Findings

E. coli COU was resistant to all antibiotics tested except amikacin, tigecycline, fosfomycin, and chloramphenicol. Analysis of the antibiotic resistance traits identified a metallo-ß-lactamase, a novel NDM variant, NDM-7. It differs from NDM-4 by a single amino acid substitution sharing an identical extended spectrum profile towards carbapenems. The bla _NDM-7 gene was located on an untypeable conjugative plasmid and associated with a close genetic background similar to those described among the bla _NDM-1 genes. The isolate also harbours bla _CTXM-15 and bla _OXA-1 genes and belonged to ST167.

Significance

This study highlights that spread of NDM producers correspond to spread of multiple bla _NDM genes and clones and therefore will be difficult to control.     

Complete sequencing of pNDM-HK encoding NDM-1 carbapenemase from a multidrug-resistant Escherichia coli strain isolated in Hong Kong

Background

The emergence of plasmid-mediated carbapenemases, such as NDM-1 in Enterobacteriaceae is a major public health issue. Since they mediate resistance to virtually all β-lactam antibiotics and there is often co-resistance to other antibiotic classes, the therapeutic options for infections caused by these organisms are very limited.

Methodology

We characterized the first NDM-1 producing E. coli isolate recovered in Hong Kong. The plasmid encoding the metallo-β-lactamase gene was sequenced.

Principal Findings

The plasmid, pNDM-HK readily transferred to E. coli J53 at high frequencies. It belongs to the broad host range IncL/M incompatibility group and is 88803 bp in size. Sequence alignment showed that pNDM-HK has a 55 kb backbone which shared 97% homology with pEL60 originating from the plant pathogen, Erwina amylovora in Lebanon and a 28.9 kb variable region. The plasmid backbone includes the mucAB genes mediating ultraviolet light resistance. The 28.9 kb region has a composite transposon-like structure which includes intact or truncated genes associated with resistance to β-lactams (bla _TEM-1, bla _NDM-1, Δbla _DHA-1), aminoglycosides (aacC2, armA), sulphonamides (sul1) and macrolides (mel, mph2). It also harbors the following mobile elements: IS26, ISCR1, tnpU, tnpAcp2, tnpD, ΔtnpATn1 and insL. Certain blocks within the 28.9 kb variable region had homology with the corresponding sequences in the widely disseminated plasmids, pCTX-M3, pMUR050 and pKP048 originating from bacteria in Poland in 1996, in Spain in 2002 and in China in 2006, respectively.

Significance

The genetic support of NDM-1 gene suggests that it has evolved through complex pathways. The association with broad host range plasmid and multiple mobile genetic elements explain its observed horizontal mobility in multiple bacterial taxa.     

Epidemiological Characteristics of bla NDM-1 in Enterobacteriaceae and the Acinetobacter calcoaceticus-Acinetobacter baumannii Complex in China from 2011 to 2012

Objectives

The study aimed to investigate the prevalence and epidemiological characteristics of bla _NDM-1 (encoding New Delhi metallo-β-lactamase 1) in Enterobacteriaceae and the Acinetobacter calcoaceticus-Acinetobacter baumannii complex (ABC) in China from July 2011 to June 2012.

Methods

PCR was used to screen for the presence of bla _NDM-1 in all organisms studied. For bla _NDM-1-positive strains, 16S rRNA analysis and Analytical Profile Index (API) strips were used to identify the bacterial genus and species. The ABCs were reconfirmed by PCR detection of bla _OXA-51-like. Antibiotic susceptibilities of the bacteria were assessed by determining minimum inhibitory concentration (MIC) of them using two-fold agar dilution test, as recommended by the Clinical and Laboratory Standards Institute (CLSI). Molecular typing was performed using pulsed-field gel electrophoresis (PFGE). S1 nuclease-pulsed-field gel electrophoresis (S1-PFGE) and Southern blot hybridization were conducted to ascertain the gene location of bla _NDM-1. Conjugation experiments were conducted to determine the transmission of bla _NDM-1-positive strains.

Results

Among 2,170 Enterobacteriaceae and 600 ABCs, seven Enterobacteriaceae strains and two A. calcoaceticus isolates from five different cities carried the bla _NDM-1 gene. The seven Enterobacteriaceae strains comprised four Klebsiella pneumoniae, one Enterobacter cloacae, one Enterobacter aerogen and one Citrobacter freundii. All seven were non-susceptible to imipenem, meropenem or ertapenem. Two A. calcoaceticus species were resistant to imipenem and meropenem. Three K. pneumoniae showed the same PFGE profiles. The bla _NDM-1 genes of eight strains were localized on plasmids, while one was chromosomal.

Conclusions

Compared with previous reports, the numbers and species containing the bla _NDM-1 in Enterobacteriaceae have significantly increased in China. Most of them are able to disseminate the gene, which is cause for concern. Consecutive surveillance should be implemented and should also focus on the dissemination of bla _NDM-1 among gram-negative clinical isolates.     

National Surveillance Study on Carbapenem Non-Susceptible Klebsiella pneumoniae in Taiwan: The Emergence and Rapid Dissemination of KPC-2 Carbapenemase

Objectives

The global spread and increasing incidence of carbapenem non-susceptible Klebsiella pneumoniae (CnSKP) has made its treatment difficult, increasing the mortality. To establish nationwide data on CnSKP spread and carbapenem-resistance mechanisms, we conducted a national surveillance study in Taiwanese hospitals.

Methods

We collected 100 and 247 CnSKP isolates in 2010 and 2012, respectively. The tests performed included antibiotic susceptibility tests; detection of carbapenemase, extended-spectrum β-lactamases (ESBL), and AmpC β-lactamases genes; outer membrane porin profiles; and genetic relationship with pulsed-field gel electrophoresis and multilocus sequence type.

Results

The resistance rate of CnSKP isolates to cefazolin, cefotaxime, cefoxitin, ceftazidime, and ciprofloxacin was over 90%. Susceptibility rate to tigecycline and colistin in 2010 was 91.0% and 83.0%, respectively; in 2012, it was 91.9% and 87.9%, respectively. In 2010, carbapenemase genes were detected in only 6.0% of isolates (4 bla _IMP-8 and 2 bla _VIM-1). In 2012, carbapenemase genes were detected in 22.3% of isolates (41 bla _KPC-2, 7 bla _VIM-1, 6 bla _IMP-8, and 1 bla _NDM-1). More than 95% of isolates exhibited either OmpK35 or OmpK36 porin loss or both. Impermeability due to porin mutation coupled with AmpC β-lactamases or ESBLs were major carbapenem-resistance mechanisms. Among 41 KPC-2-producing K. pneumoniae isolates, all were ST11 with 1 major pulsotype.

Conclusions

In 2010 and 2012, the major mechanisms of CnSKP in Taiwan were the concomitance of AmpC with OmpK35/36 loss. KPC-2-KP dissemination with the same ST11 were observed in 2012. The emergence and rapid spread of KPC-2-KP is becoming an endemic problem in Taiwan. The identification of NDM-1 K. pneumoniae case is alarming.     

Characterization of a Novel Plasmid Type and Various Genetic Contexts of bla OXA-58 in Acinetobacter spp. from Multiple Cities in China

Background/Objective

Several studies have described the epidemiological distribution of bla _OXA-58-harboring Acinetobacter baumannii in China. However, there is limited data concerning the replicon types of bla _OXA-58-carrying plasmids and the genetic context surrounding bla _OXA-58 in Acinetobacter spp. in China.

Methodology/Principal Findings

Twelve non-duplicated bla _OXA-58-harboring Acinetobacter spp. isolates were collected from six hospitals in five different cities between 2005 and 2010. The molecular epidemiology of the isolates was carried out using PFGE and multilocus sequence typing. Carbapenemase-encoding genes and plasmid replicase genes were identified by PCR. The genetic location of bla _OXA-58 was analyzed using S1-nuclease method. Plasmid conjugation and electrotransformation were performed to evaluate the transferability of bla _OXA-58-harboring plasmids. The genetic structure surrounding bla _OXA-58 was determined by cloning experiments. The twelve isolates included two Acinetobacter pittii isolates (belong to one pulsotype), three Acinetobacter nosocomialis isolates (belong to two pulsotypes) and seven Acinetobacter baumannii isolates (belong to two pulsotypes/sequence types). A. baumannii ST91 was found to be a potential multidrug resistant risk clone carrying both bla _OXA-58 and bla _OXA-23. bla _OXA-58 located on plasmids varied from ca. 52 kb to ca. 143 kb. All plasmids can be electrotransformed to A. baumannii recipient, but were untypeable by the current replicon typing scheme. A novel plasmid replicase named repAci10 was identified in bla _OXA-58-harboring plasmids of two A. pittii isolates, three A. nosocomialis isolates and two A. baumannii isolates. Four kinds of genetic contexts of bla _OXA-58 were identified. The transformants of plasmids with structure of IS6 family insertion sequence (ISOur1, IS1008 or IS15)-ΔISAba3-like element-bla _OXA-58 displayed carbapenem nonsusceptible, while others with structure of intact ISAba3-like element-bla _OXA-58 were carbapenem susceptible.

Conclusion

The study revealed the unique features of bla _OXA-58-carrying plasmids in Acinetobacter spp. in China, which were different from that of Acinetobacter spp. found in European countries. The diversity of the genetic contexts of bla _OXA-58 contributed to various antibiotics resistance profiles.     

Carbapenem-Resistant Bacteria Recovered from Faeces of Dairy Cattle in the High Plains Region of the USA

Objective

A study was conducted to recover carbapenem-resistant bacteria from the faeces of dairy cattle and identify the underlying genetic mechanisms associated with reduced phenotypic susceptibility to carbapenems.

Methods

One hundred and fifty-nine faecal samples from dairy cattle were screened for carbapenem-resistant bacteria. Phenotypic screening was conducted on two media containing ertapenem. The isolates from the screening step were characterised via disk diffusion, Modified Hodge, and Carba NP assays. Carbapenem-resistant bacteria and carbapenemase-producing isolates were subjected to Gram staining and biochemical testing to include Gram-negative bacilli. Whole genome sequencing was performed on bacteria that exhibited either a carbapenemase-producing phenotype or were not susceptible to ertapenem and were presumptively Enterobacteriaceae.

Results

Of 323 isolates collected from the screening media, 28 were selected for WGS; 21 of which were based on a carbapenemase-producing phenotype and 7 were presumptively Enterobacteriaceae and not susceptible to ertapenem. Based on analysis of WGS data, isolates included: 3 Escherichia coli harbouring bla_CMY-2 and truncated ompF genes; 8 Aeromonas harbouring bla_cphA-like genes; 1 Acinetobacter baumannii harbouring a novel bla_OXA gene (bla_OXA-497); and 6 Pseudomonas with conserved domains of various carbapenemase-producing genes.

Conclusions

Carbapenem resistant bacteria appear to be rare in cattle. Nonetheless, carbapenem-resistant bacteria were detected across various genera and were found to harbour a variety of mechanisms conferring reduced susceptibility. The development and dissemination of carbapenem-resistant bacteria in livestock would have grave implications for therapeutic treatment options in human medicine; thus, continued monitoring of carbapenem susceptibility among enteric bacteria of livestock is warranted.     

Dissemination of NDM-1-Producing Enterobacteriaceae Mediated by the IncX3-Type Plasmid

The emergence and spread of NDM-1-producing Enterobacteriaceae have resulted in a worldwide public health risk that has affected some provinces of China. China is an exceptionally large country, and there is a crucial need to investigate the epidemic of bla _NDM-1-positive Enterobacteriaceae in our province. A total of 186 carbapenem-resistant Enterobacteriaceae isolates (CRE) were collected in a grade-3 hospital in Zhejiang province. Carbapenem-resistant genes, including bla _KPC, bla _IMP, bla _VIM, bla _OXA-48 and bla _NDM-1 were screened and sequenced. Ninety isolates were identified as harboring the bla _KPC-2 genes, and five bla _NDM-1-positive isolates were uncovered. XbaI-PFGE revealed that three bla _NDM-1-positive K. pneumoniae isolates belonged to two different clones. S1-PFGE and southern blot suggested that the bla _NDM-1 genes were located on IncX3-type plasmids with two different sizes ranging from 33.3 to 54.7 kb (n=4) and 104.5 to 138.9 kb (n=1), respectively, all of which could easily transfer to Escherichia coli by conjugation and electrotransformation. The high-throughput sequencing of two plasmids was performed leading to the identification of a smaller 54-kb plasmid, which had high sequence similarity with a previously reported pCFNDM-CN, and a larger plasmid in which only a 7.8-kb sequence of a common gene environment around bla _NDM-1 (bla _NDM-1-trpF- dsbC-cutA1-groEL-ΔInsE,) was detected. PCR mapping and sequencing demonstrated that four smaller bla _NDM-1 plasmids contained a common gene environment around bla _NDM-1 (IS5-bla _NDM-1-trpF- dsbC-cutA1-groEL). We monitored the CRE epidemic in our hospital and determined that KPC-2 carbapenemase was a major risk to patient health and the IncX3-type plasmid played a vital role in the spread of the bla _NDM-1 gene among the CRE.     

Complete Nucleotide Sequence of CTX-M-15-Plasmids from Clinical Escherichia coli Isolates: Insertional Events of Transposons and Insertion Sequences

Background

CTX-M-producing Escherichia coli strains are regarded as major global pathogens.

Methodology/Principal Findings

The nucleotide sequence of three plasmids (pEC_B24: 73801-bp; pEC_L8: 118525-bp and pEC_L46: 144871-bp) from Escherichia coli isolates obtained from patients with urinary tract infections and one plasmid (pEC_Bactec: 92970-bp) from an Escherichia coli strain isolated from the joint of a horse with arthritis were determined. Plasmid pEC_Bactec belongs to the IncI1 group and carries two resistance genes: bla _TEM-1 and bla _CTX-M-15. It shares more than 90% homology with a previously published bla _CTX-M-plasmid from E. coli of human origin. Plasmid pEC_B24 belongs to the IncFII group whereas plasmids pEC_L8 and pEC_L46 represent a fusion of two replicons of type FII and FIA. On the pEC_B24 backbone, two resistance genes, bla _TEM-1 and bla _CTX-M-15, were found. Six resistance genes, bla _TEM-1, bla _CTX-M-15, bla _OXA-1, aac6''-lb-cr, tetA and catB4, were detected on the pEC_L8 backbone. The same antimicrobial drug resistance genes, with the exception of tetA, were also identified on the pEC_L46 backbone. Genome analysis of all 4 plasmids studied provides evidence of a seemingly frequent transposition event of the bla _CTX-M-15-ISEcp1 element. This element seems to have a preferred insertion site at the tnpA gene of a bla _TEM-carrying Tn3-like transposon, the latter itself being inserted by a transposition event. The IS26-composite transposon, which contains the bla _OXA-1, aac6''-lb-cr and catB4 genes, was inserted into plasmids pEC_L8 and pEC_L46 by homologous recombination rather than a transposition event. Results obtained for pEC_L46 indicated that IS26 also plays an important role in structural rearrangements of the plasmid backbone and seems to facilitate the mobilisation of fragments from other plasmids.

Conclusions

Collectively, these data suggests that IS26 together with ISEcp1 could play a critical role in the evolution of diverse multiresistant plasmids found in clinical Enterobacteriaceae.     

The Sudden Dominance of bla CTX–M Harbouring Plasmids in Shigella spp. Circulating in Southern Vietnam

Background

Plasmid mediated antimicrobial resistance in the Enterobacteriaceae is a global problem. The rise of CTX-M class extended spectrum beta lactamases (ESBLs) has been well documented in industrialized countries. Vietnam is representative of a typical transitional middle income country where the spectrum of infectious diseases combined with the spread of drug resistance is shifting and bringing new healthcare challenges.

Methodology

We collected hospital admission data from the pediatric population attending the hospital for tropical diseases in Ho Chi Minh City with Shigella infections. Organisms were cultured from all enrolled patients and subjected to antimicrobial susceptibility testing. Those that were ESBL positive were subjected to further investigation. These investigations included PCR amplification for common ESBL genes, plasmid investigation, conjugation, microarray hybridization and DNA sequencing of a bla _CTX–M encoding plasmid.

Principal Findings

We show that two different bla _CTX-M genes are circulating in this bacterial population in this location. Sequence of one of the ESBL plasmids shows that rather than the gene being integrated into a preexisting MDR plasmid, the bla _CTX-M gene is located on relatively simple conjugative plasmid. The sequenced plasmid (pEG356) carried the bla _CTX-M-24 gene on an ISEcp1 element and demonstrated considerable sequence homology with other IncFI plasmids.

Significance

The rapid dissemination, spread of antimicrobial resistance and changing population of Shigella spp. concurrent with economic growth are pertinent to many other countries undergoing similar development. Third generation cephalosporins are commonly used empiric antibiotics in Ho Chi Minh City. We recommend that these agents should not be considered for therapy of dysentery in this setting.     

Characterization of Two New CTX-M-25-Group Extended-Spectrum β-Lactamase Variants Identified in Escherichia coli Isolates from Israel

Objectives

We characterized two new CTX-M-type extended-spectrum β-lactamase (ESBL) variants in Escherichia coli isolates from stool samples of two elderly patients admitted at the Tel Aviv Sourasky Medical Center, Israel. Both patients underwent treatment with cephalosporins prior to isolation of the E. coli strains.

Methods

ESBLs were detected by the double-disk synergy test and PCR-sequencing of β-lactamase genes. The bla _CTX-M genes were cloned into the pCR-BluntII-TOPO vector in E. coli TOP10. The role of amino-acid substitutions V77A and D240G was analyzed by site-directed mutagenesis of the bla _CTX-M-94 and bla _CTX-M-100 genes and comparative characterization of the resulting E. coli recombinants. MICs of β-lactams were determined by Etest. Plasmid profiling, mating experiments, replicon typing and sequencing of bla _CTX-M flanking regions were performed to identify the genetic background of the new CTX-M variants.

Results

The novel CTX-M β-lactamases, CTX-M-94 and -100, belonged to the CTX-M-25-group. Both variants differed from CTX-M-25 by the substitution V77A, and from CTX-M-39 by D240G. CTX-M-94 differed from all CTX-M-25-group enzymes by the substitution F119L. Glycine-240 was associated with reduced susceptibility to ceftazidime and leucine-119 with increased resistance to ceftriaxone. bla _CTX-M-94 and bla _CTX-M-100 were located within ISEcp1 transposition units inserted into ∼93 kb non-conjugative IncFI and ∼130 kb conjugative IncA/C plasmids, respectively. The plasmids carried also different class 1 integrons.

Conclusions

This is the first report on CTX-M-94 and -100 ESBLs, novel members of the CTX-M-25-group.     

Dissemination of IncF-type plasmids in multiresistant CTX-M-15-producing Enterobacteriaceae isolates from surgical-site infections in Bangui,Central African Republic

Background

Surgical-site infection is the most frequent health care-associated infection in the developing world, with a strikingly higher prevalence than in developed countries We studied the prevalence of resistance to antibiotics in Enterobacteriaceae isolates from surgical-site infections collected in three major tertiary care centres in Bangui, Central African Republic. We also studied the genetic basis for antibiotic resistance and the genetic background of third-generation cephalosporin-resistant (3GC-R) Enterobacteriaceae.

Results

Between April 2011 and April 2012, 195 patients with nosocomial surgical-site infections were consecutively recruited into the study at five surgical departments in three major tertiary care centres. Of the 165 bacterial isolates collected, most were Enterobacteriaceae (102/165, 61.8%). Of these, 65/102 (63.7%) were 3GC-R, which were characterized for resistance gene determinants and genetic background. The bla_CTX-M-15 and aac(6′)-Ib-cr genes were detected in all strains, usually associated with qnr genes (98.5%). Escherichia coli, the most commonly recovered species (33/65, 50.8%), occurred in six different sequence types, including the pandemic B2-O25b-ST131 group (12/33, 36.4%). Resistance transfer was studied in one representative strain of the resistance gene content in each repetitive extragenic palindromic and enterobacterial repetitive intergenic consensus sequence-PCR banding pattern. Plasmids were characterized by PCR-based replicon typing and sub-typing schemes. In most isolates (18/27, 66.7%), bla_CTX-M-15 genes were found in incompatibility groups F/F31:A4:B1 and F/F36:A4:B1 conjugative plasmids. Horizontal transfer of both plasmids is probably an important mechanism for the spread of bla_CTX-M-15 among Enterobacteriaceae species and hospitals. The presence of sets of antibiotic resistance genes in these two plasmids indicates their capacity for gene rearrangement and their evolution into new variants.

Conclusions

Diverse modes are involved in transmission of resistance, plasmid dissemination probably playing a major role.     

New Delhi Metallo-β-Lactamase 1(NDM-1), the Dominant Carbapenemase Detected in Carbapenem-Resistant Enterobacter cloacae from Henan Province,China

The emergence of New Delhi metallo-β-lactamase 1 (NDM-1) has become established as a major public health threat and represents a new challenge in the treatment of infectious diseases. In this study, we report a high incidence and endemic spread of NDM-1-producing carbapenem-resistant Enterobacter cloacae isolates in Henan province, China. Eight (72.7%) out of eleven non-duplicated carbapenem-resistant E. cloacae isolates collected between June 2011 and May 2013 were identified as NDM-1 positive. The bla _NDM-1 gene surrounded by an entire ISAba125 element and a bleomycin resistance gene ble _MBL in these isolates were carried by diverse conjugatable plasmids (IncA/C, IncN, IncHI2 and untypeable) ranging from ~55 to ~360 kb. Molecular epidemiology analysis revealed that three NDM-1-producing E. cloacae belonged to the same multilocus sequence type (ST), ST120, two of which were classified as extensively drug-resistant (XDR) isolates susceptible only to tigecycline and colistin. The two XDR ST120 E. cloacae isolates co-harbored bla _NDM-1, armA and fosA3 genes and could transfer resistance to carbapenems, fosfomycin and aminoglycosides simultaneously via a conjugation experiment. Our study demonstrated NDM-1 was the most prevalent metallo-β-lactamase (MBL) among carbapenem-resistant E.cloacae isolates and identified a potential endemic clone of ST120 in Henan province. These findings highlight the need for enhanced efforts to monitor the further spread of NDM-1 and XDR ST120 E. cloacae in this region.     

Transmission and characterization of <Emphasis Type="Italic">bla</Emphasis><Subscript>NDM-1</Subscript> in <Emphasis Type="Italic">Enterobacter cloacae</Emphasis> at a teaching hospital in Yunnan,China

Background

In recent years, New Delhi metallo-beta-lactamases 1 (bla _NDM-1) has been reported with increasing frequency and become prevalent. The present study was undertaken to investigate the epidemiological dissemination of the bla _NDM-1 gene in Enterobacter cloacae isolates at a teaching hospital in Yunnan, China.

Methods

Antimicrobial susceptibility testing was performed using VITEK 2 system and E test gradient strips. The presence of integrons and insertion sequence common region 1 were examined by PCR and sequencing. Clonal relatedness was assessed by pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing. Conjugation experiments and Southern blot hybridization were performed to determine the transferability of plasmids.

Results

Ten E. cloacae isolates and their Escherichia coli transconjugants were exhibited similar resistant patterns to carbapenems, cephalosporins and penicillins. 8 (80%) of E. cloacae isolates carried class 1 integron and 1 (12.5%) carried class 2 integron. Integron variable regions harbored the genes which encoded resistance to aminoglycosides (aadA1, aadA2, aadA5, aadB, aac(6′)-Ib-cr), sulfamethoxazole/trimethoprim (dfrA17, dfrA12, dfrA15) and Streptozotocin (sat2). Six E. cloacae isolates belonged to ST74 and exhibited highly similar PFGE patterns. Each isolate shared an identical plasmid with ~33.3 kb size that carried the bla _NDM-1 gene, except T3 strain, of which the bla _NDM-1 gene was located on a ~50 kb plasmid.

Conclusions

Our findings suggested that plasmid was able to contribute to the dissemination of bla _NDM-1. Hence, more attention should be devoted to monitor the dissemination of the bla _NDM-1 gene due to its horizontal transfer via plasmid. In addition, nosocomial surveillance system should actively monitor the potential endemic clone of ST74 to prevent their further spread.

     

Epidemic Plasmid Carrying bla CTX-M-15 in Klebsiella penumoniae in China

Objective

To investigate the local epidemiology of Klebsiella penumoniae carrying bla _CTX-M-15 in southern China and to characterize the genetic environment of bla _CTX-M-15.

Methods

PCR and DNA sequencing were used to detect and characterize the genetic contexts of bla _CTX-M-15. The clonal relatedness of isolates carrying bla _CTX-M-15 was determined by pulse-field gel electrophoresis. Conjugative plasmids carrying bla _CTX-M-15 were obtained by mating and were further subject to restriction analysis and replicon typing.

Results

A total of 47CTX-M-15 ESBL-producing isolates of K. pneumoniae were collected from nine hospitals in China from October 2007 to October 2008. Isolates were clustered into various clonal groups. The local spread of bla _CTX-M-15 was mainly mediated by one major conjugative plasmid as determined by S1-PFGE and restriction analysis. A 90-kb plasmid belonging to incompatible group FII was the major carrier of bla _CTX-M-15 in K. pneumoniae. Except bla _TEM-1, the resistance genes such as bla _SHV, bla _DHA-1, bla _OXA-1, qnrB, qnrS, aac(3)-II, and aac(6′)-Ib were not found in the plasmid. In the comparing of conjugative gene sequence, it is 100% identical with the plasmid pKF3–94, which was found in K. pneumonia from Zhejiang province of china previously.

Conclusions

bla _CTX-M-15 was prevalent in K. pneumonia of southern China. The dissemination of bla _CTX-M-15 appeared to be due to the horizontal transfer of a 90-kb epidemic plasmid.     

Molecular Characteristics of Extended-Spectrum Cephalosporin-Resistant Enterobacteriaceae from Humans in the Community

Objective

To investigate the molecular characteristics of extended-spectrum cephalosporin (ESC)-resistant Enterobacteriaceae collected during a cross-sectional study examining the prevalence and risk factors for faecal carriage of extended-spectrum β-lactamase (ESBL)-producing Enterobacteriaceae in humans living in areas with high or low broiler density.

Methods

ESC-resistant Enterobacteriaceae were identified by combination disc-diffusion test. ESBL/AmpC/carbapenemase genes were analysed using PCR and sequencing. For E. coli, phylogenetic groups and MLST were determined. Plasmids were characterized by transformation and PCR-based replicon typing. Subtyping of plasmids was done by plasmid multilocus sequence typing.

Results

175 ESC-resistant Enterobacteriaceae were cultured from 165/1,033 individuals. The isolates were Escherichia coli(n=65), Citrobacter freundii (n=52), Enterobacter cloacae (n=38), Morganella morganii (n=5), Enterobacter aerogenes (n=4), Klebsiella pneumoniae (n=3), Hafnia alvei (n=2), Shigella spp. (n=2), Citrobacter amalonaticus (n=1), Escherichia hermannii (n=1), Kluyvera cryocrescens (n=1), and Pantoea agglomerans (n=1). The following ESBL genes were recovered in 55 isolates originating from 49 of 1,033 (4.7 %) persons: bla _CTX-M-1 (n=17), bla _CTX-M-15 (n=16), bla _CTX-M-14 (n=9), bla _CTX-M-2 (n=3), bla _CTX-M-3 (n=2), bla _CTX-M-24 (n=2), bla _CTX-M-27 (n=1), bla _CTX-M-32 (n=1), bla _SHV-12 (n=2), bla _SHV-65 (n=1) and bla _TEM-52 (n=1). Plasmidic AmpC (pAmpC) genes were discovered in 6 out of 1,033 (0.6 %) persons. One person carried two different E. coli isolates, one with bla _CTX-M-1 and the other with bla _CMY-2 and therefore the prevalence of persons carrying Enterobacteriaceae harboring ESBL and/or pAmpC genes was 5.2 %. In eight E. coli isolates the AmpC phenotype was caused by mutations in the AmpC promoter region. No carbapenemase genes were identified. A large variety of E. coli genotypes was found, ST131 and ST10 being most common.

Conclusions

ESBL/pAmpC genes resembled those from patients in Dutch hospitals, indicating that healthy humans form a reservoir for transmission of these determinants to vulnerable people. The role of poultry in the transmission to humans in the community remains to be elucidated.     

Transition of bla OXA-58-like to bla OXA-23-like in Acinetobacter baumannii Clinical Isolates in Southern China: An 8-Year Study

Background

The prevalence of carbapenem-resistant Acinetobacter baumannii in hospitals has been increasing worldwide. This study aims to investigate the carbapenemase genes and the clonal relatedness among A. baumannii clinical isolates in a Chinese hospital.

Methods

Carbapenemase genes and the upstream locations of insertion sequences were detected by polymerase chain reaction (PCR), and the clonal relatedness of isolates was determined by pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing.

Results

A total of 231 nonduplicate carbapenemase gene-harboring A. baumannii clinical isolates recovered from Shenzhen People’s Hospital, were investigated between 2002 and 2009. bla _OXA-23-like, bla _OXA-58-like, bla _OXA-40-like, and ISAba1-bla _OXA-51-like were identified in 119, 107, 1, and 4 isolates, respectively. IS1008-ΔISAba3, ISAba3, and ISAba1 were detected upstream of the bla _OXA-58-like gene in 69, 35, and 3 isolates, respectively. All bla _OXA-23-like genes but one had an upstream insertion of ISAba1. bla _OXA-58-like was the most common carbapenemase gene in A.baumannii before 2008, thereafter bla _OXA-23-like became rapidly prevalent and replaced bla _OXA-58-like in 2009. The majority of bla _OXA-58-like-carrying isolates showed lower level of resistance to imipenem and meropenem (minimum inhibitory concentrations (MICs), 1 μg/ml to 16 μg/ml), compared with the majority of bla _OXA-23-like-carrying isolates (MICs, 16 μg/ml to 64 μg/ml for both imipenem and meropenem). All 231 bla _OXA carbapenemase gene-harboring isolates belonged to 14 PFGE types (A–N), and three dominant clones A, J, and H accounted for 43.3%, 42.0%, and 8.2% of the tested isolates, respectively. Clone A (sequence type ST92/ST208) with bla _OXA-58-like was the most prevalent before 2008. Clone H (ST229) with bla _OXA-23-like became striking between 2007 and 2008. Clone J (ST381) with bla _OXA-23-like rapidly spread and replaced clones A and H in 2009.

Conclusion

This study is the first to reveal that the distinct bla _OXA-23-like-carrying A. baumannii ST381 displaced the previously prevalent bla _OXA-58-like-carrying A. baumannii ST92/ST208, resulting in the rapidly increasing resistance to carbapenems in A. baumannii in Shenzhen People’s Hospital in 2009.     

Characteristics of plasmids in multi-drug-resistant Enterobacteriaceae isolated during prospective surveillance of a newly opened hospital in Iraq

Background

Gram-negative multidrug-resistant (MDR) bacteria are major causes of nosocomial infections, and antibiotic resistance in these organisms is often plasmid mediated. Data are scarce pertaining to molecular mechanisms of antibiotic resistance in resource constrained areas such as Iraq.

Methodology/Principal Findings

In this study, all MDR Enterobacteriaceae (n = 38) and randomly selected non-MDR counterparts (n = 41) isolated from patients, healthcare workers and environmental surfaces in a newly opened hospital in Iraq were investigated to characterize plasmids found in these isolates and determine their contribution to antibiotic resistance. Our results demonstrated that MDR E. coli and K. pneumoniae isolates harbored significantly more (≥3) plasmids compared to their non-MDR counterparts, which carried ≤2 plasmids (p<0.01). Various large plasmids (∼52 to 100 kb) from representative isolates were confirmed to contain multiple resistance genes by DNA microarray analysis. Aminoglycoside (acc, aadA, aph, strA/B, and ksgA), β-lactam (bla _TEM1, bla _AMPC, bla _CTX-M-15, bla _OXA-1, bla _VIM-2 and bla _SHV), sulfamethoxazole/trimethoprim (sul/dfr), tetracycline (tet) and chloramphenicol (cat) resistance genes were detected on these plasmids. Additionally, multiple plasmids carrying multiple antibiotic resistance genes were found in the same host strain. Genetic transfer-associated genes were identified on the plasmids from both MDR and non-MDR isolates. Seven plasmid replicon types (FII, FIA, FIB, B/O, K, I1 and N) were detected in the isolates, while globally disseminated IncA/C and IncHI1 plasmids were not detected in these isolates.

Conclusions/Significance

This is the first report of the characteristics of the plasmids found in Enterobacteriaceae isolated following the opening of a new hospital in Iraq. The information provided here furthers our understanding of the mechanisms of drug resistance in this specific region and their evolutionary relationship with other parts of world. The large plasmids, carrying resistance genes and transfer-associated genes, may be potential factors for regional dissemination of antibiotic resistance.     

Prevalence of Plasmid-Mediated Quinolone Resistance and Aminoglycoside Resistance Determinants among Carbapeneme Non-Susceptible Enterobacter cloacae

Background

Simultaneous resistance to aminoglycosides and fluoroquinolones in carbapeneme non-susceptible (CNS) isolates will inevitably create problems. The present study was performed to characterize the prevalence of the plasmid-mediated quinolone resistance determinants (QRDs) and aminoglycoside resistance determinants (ARDs) among the CNS Enterobacter cloacae (E. cloacae) isolates in a Chinese teaching hospital, and to acquire their molecular epidemiological characteristics.

Methods

The β-lactamases genes (including class A carbapenemase genes bla_KPC and bla_SME, metallo-β-lactamase genes (MBLs) bla_IMP, bla_VIM and bla_NDM, and extended spectrum β-lactamases (ESBLs),bla_CTX-M, bla_TEM and bla_SHV), QRDs (including qnrA, qnrB, qnrS and aac(6′)-Ib-cr) and ARDs (including aac(6′)-Ib, armA and rmtB) of these 35 isolates were determined by PCR and sequenced bidirectionally. The clonal relatedness was investigated by pulsed-field gel electrophoresis (PFGE).

Results

Of the 35 isolates, 9 (25.7%) harbored a carbapenemase gene; 23 (65.7%) carried ESBLs; 24 (68.6%) were QRD positive; and 27 (77.1%) were ARD positive. Among the 5 bla_IMP-8 positive strains, 4 (80%) contained both ESBL and QRD genes, and all the 5 (100%) harbored ARD genes. Of the 23 ESBLs positive isolates, 6 (26.1%) were carbapenemase positive, 14 (60.9%) were QRD positive, and 18 (78.3%) were ARD positive. PFGE revealed genetic diversity among the 35 isolates, indicating that the high prevalence of CNS E. cloacae isolates was not caused by clonal dissemination.

Conclusion

QRD and ARD genes were highly prevalent among the CNS E. cloacae isolates. Multiple resistant genes were co-expressed in the same isolates. The CNS E. cloacae isolate co-expressing bla_NDM-1, bla_IMP-26, qnrA1 and qnrS1 was first reported.