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 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.     

Genetic Environment of Plasmid Mediated CTX-M-15 Extended Spectrum Beta-Lactamases from Clinical and Food Borne Bacteria in North-Eastern India

Background

The study investigated the presence of CTX-M-15 type extended spectrum beta-lactamases (ESBL), compared their genetic arrangements and plasmid types in gram negative isolates of hospital and food origin in north-east India. From September 2013 to April 2014, a total of 252 consecutive, non-duplicate clinical isolates and 88 gram negative food isolates were selected. Phenotypic and molecular characterization of ESBL genes was performed. Presence of integrons and gene cassettes were analyzed by integrase and 59 base-element PCR respectively. The molecular environments surrounding bla _CTX-M and plasmid types were investigated by PCR and PCR-based replicon typing respectively. Transformation was carried out to assess plasmid transfer. Southern blotting was conducted to localize the bla _CTX-M-15 genes. DNA fingerprinting was performed by ERIC-PCR.

Results

Prevalence of ESBL was found to be 40.8% (103/252) in clinical and 31.8% (28/88) in food-borne isolates. Molecular characterization revealed the presence of 56.3% (58/103) and 53.5% (15/28) bla _CTX-M-15 in clinical and food isolates respectively. Strains of clinical and food origin were non-clonal. Replicon typing revealed that IncI1 and IncFII plasmid were carrying bla _CTX-M-15 in clinical and food isolates and were horizontally transferable. The ISEcp1 element was associated with bla _CTX-M-15 in both clinical and food isolates.

Conclusions

The simultaneous presence of resistance determinants in non-clonal isolates of two different groups thus suggests that the microbiota of common food products consumed may serve as a reservoir for some of the drug resistance genes prevalent in human pathogens.     

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.     

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.     

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.     

Comparative genomic analysis of Acinetobacter baumannii clinical isolates reveals extensive genomic variation and diverse antibiotic resistance determinants

Background

Acinetobacter baumannii is an important nosocomial pathogen that poses a serious health threat to immune-compromised patients. Due to its rapid ability to develop multidrug resistance (MDR), A. baumannii has increasingly become a focus of attention worldwide. To better understand the genetic variation and antibiotic resistance mechanisms of this bacterium at the genomic level, we reported high-quality draft genome sequences of 8 clinical isolates with various sequence types and drug susceptibility profiles.

Results

We sequenced 7 MDR and 1 drug-sensitive clinical A. baumannii isolates and performed comparative genomic analysis of these draft genomes with 16 A. baumannii complete genomes from GenBank. We found a high degree of variation in A. baumannii, including single nucleotide polymorphisms (SNPs) and large DNA fragment variations in the AbaR-like resistance island (RI) regions, the prophage and the type VI secretion system (T6SS). In addition, we found several new AbaR-like RI regions with highly variable structures in our MDR strains. Interestingly, we found a novel genomic island (designated as GI_BJ4) in the drug-sensitive strain BJ4 carrying metal resistance genes instead of antibiotic resistance genes inserted into the position where AbaR-like RIs commonly reside in other A. baumannii strains. Furthermore, we showed that diverse antibiotic resistance determinants are present outside the RIs in A. baumannii, including antibiotic resistance-gene bearing integrons, the bla_OXA-23-containing transposon Tn2009, and chromosomal intrinsic antibiotic resistance genes.

Conclusions

Our comparative genomic analysis revealed that extensive genomic variation exists in the A. baumannii genome. Transposons, genomic islands and point mutations are the main contributors to the plasticity of the A. baumannii genome and play critical roles in facilitating the development of antibiotic resistance in the clinical isolates.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-1163) contains supplementary material, which is available to authorized users.     

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.     

Closely Related NDM-1-Encoding Plasmids from Escherichia coli and Klebsiella pneumoniae in Taiwan

Objective

Two plasmids carrying bla _NDM-1 isolated from carbapenem-resistant Klebsiella pneumoniae (CR-KP) and carbapenem-resistant Escherichia coli (CR-EC) were sequenced. CR-KP and CR-EC were isolated from two Taiwanese patients without travel histories.

Methods

Complete sequencing of the plasmids (pLK75 and pLK78) was conducted using a shotgun approach. Annotation of the contigs was performed using the RAST Server, followed by manual inspection and correction.

Results

These similar plasmids were obtained from two patients with overlapping stays at the same hospital. The pLK75 and pLK78 plasmids were 56,489-bp and 56,072-bp in length, respectively. Plasmid annotation revealed a common backbone similar to the IncN plasmid pR46. The regions flanking the bla _NDM-1 genes in these plasmids were very similar to plasmid pNDM-HU01 in Japan, which contains a complex class 1 integron located next to an ISCR1 element. The ISCR1 element has been suggested to provide a powerful mechanism for mobilising antibiotic resistance genes.

Conclusion

Two indigenous NDM-1-producing Enterobacteriaceae cases were identified for the first time in Taiwan, highlighting the alarming introduction of NDM-1-producing Enterobacteriaceae in this region.     

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.     

Stress tolerant virulent strains of Cronobacter sakazakii from food

Background

Cronobacter sakazakii is considered as an emerging foodborne pathogen. The aim of this study was to isolate and characterize virulent strains of Cronobacter sakazakii from food samples of Bangladesh.

Result

Six (6) Cronobacter sakazakii was isolated and identified from 54 food samples on the basis of biochemical characteristics, sugar fermentation, SDS-PAGE of whole cell protein, plasmid profile and PCR of Cronobacter spp. specific genes (esak, gluA, zpx, ompA, ERIC, BOX-AIR) and sequencing. These strains were found to have moderately high antibiotic resistance against common antibiotics and some are ESBL producer. Most of the C. sakazakii isolates were capable of producing biofilm (strong biofilm producer), extracellular protease and siderophores, curli expression, haemolysin, haemagglutinin, mannose resistant haemagglutinin, had high cell surface hydrophobicity, significant resistance to human serum, can tolerate high concentration of salt, bile and DNase production. Most of them produced enterotoxins of different molecular weight. The isolates pose significant serological cross-reactivity with other gram negative pathogens such as serotypes of Salmonella spp., Shigella boydii, Shigella sonnei, Shigella flexneri and Vibrio cholerae. They had significant tolerance to high temperature, low pH, dryness and osmotic stress.

Conclusion

Special attention should be given in ensuring hygiene in production and post-processing to prevent contamination of food with such stress-tolerant virulent Cronobacter sakazakii.

Electronic supplementary material

The online version of this article (doi:10.1186/0717-6287-47-63) contains supplementary material, which is available to authorized users.     

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.     

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.     

A Long-Term Low-Frequency Hospital Outbreak of KPC-Producing Klebsiella pneumoniae Involving Intergenus Plasmid Diffusion and a Persisting Environmental Reservoir

Background

To study the molecular characteristics of a long-term, low frequency outbreak of bla _KPC-2 in a low prevalence setting involving the hospital environment.

Methodology/Principal Findings

KPC-producing bacteria were screened by selective chromogenic agar and Real-Time PCR. The presence of antibiotic resistance genes was ascribed by PCRs and subsequent sequencing, and the KPC-producing isolates were phylogenetically typed using PFGE and multi-locus sequence typing. Bla _KPC-2-plasmids were identified and analysed by S1-nuclease-PFGE hybridization and PCR based replicon typing. A ∼97 kb IncFII plasmid was seen to carry bla _KPC-2 in all of the clinical isolates, in one of the isolates recovered from screened patients (1/136), and in the Klebsiella pneumoniae and Enterobacter asburiae isolates recovered from the environment (sinks) in one intensive care unit. The K. pneumoniae strain ST258 was identified in 6 out of 7 patients. An intergenus spread to E. asburiae and an interspecies spread to two different K. pneumoniae clones (ST27 and ST461) of the bla _KPC-2 plasmid was discovered. K. pneumoniae ST258 and genetically related E. asburiae strains were found in isolates of both human and environmental origins.

Conclusions/Significance

We document a clonal transmission of the K. pneumoniae ST258 strain, and an intergenus plasmid diffusion of the IncFII plasmid carrying bla _KPC-2 in this outbreak. A major reservoir in the patient population could not be unveiled. However, the identification of a persisting environmental reservoir of strains with molecular determinants linked to human isolates, suggests a possible role of the environment in the maintenance of this long-term outbreak.     

Antimicrobial Resistance in Invasive Non-typhoid Salmonella from the Democratic Republic of the Congo: Emergence of Decreased Fluoroquinolone Susceptibility and Extended-spectrum Beta Lactamases

Background

Co-resistance against the first-line antibiotics ampicillin, chloramphenicol and trimethoprim/sulphamethoxazole or multidrug resistance (MDR) is common in non typhoid Salmonella (NTS). Use of alternative antibiotics, such as fluoroquinolones or third generation cephalosporins is threatened by increasing resistance, but remains poorly documented in Central-Africa.

Methodology/Principal findings

As part of a microbiological surveillance study in DR Congo, blood cultures were collected between 2007 and 2011. Isolated NTS were assessed for serotype and antimicrobial resistance including decreased ciprofloxacin susceptibility and extended-spectrum beta-lactamase (ESBL) production. In total, 233 NTS isolates (representing 23.6% of clinically significant organisms) were collected, mainly consisting of Salmonella Typhimurium (79%) and Salmonella Enteritidis (18%). The majority of NTS were isolated in the rainy season, and recovered from children ≤2 years old. MDR, decreased ciprofloxacin susceptibility, azithromycin and cefotaxime resistance were 80.7%, 4.3%, 3.0% and 2.1% respectively. ESBL production was noted in three (1.3%) isolates. Decreased ciprofloxacin susceptibility was associated with mutations in codon 87 of the gyrA gene, while ESBLs all belonged to the SHV-2a type.

Conclusions/Significance

Presence of almost full MDR among NTS isolates from blood cultures in Central Africa was confirmed. Resistance to fluoroquinolones, azithromycin and third generation cephalosporins is still low, but emerging. Increased microbiological surveillance in DR Congo is crucial for adapted antibiotic therapy and the development of treatment guidelines.     

Development of a real-time SYBRGreen PCR assay for rapid detection of acquired AmpC in Enterobacteriaceae

Introduction

Acquired AmpC enzymes, classified as miscellaneous extended-spectrum β-lactamase (ESBL_M) enzymes according to a recently proposed β-lactamase classification, are increasing according to several publications. Simple and rapid methods for detection of ESBL_M are needed for appropriate infection control. A gel-based multiplex PCR method for acquired bla_AmpC detection and subtype classification has been available for several years. Here, we describe a modification of the protocol to suit real-time PCR platforms and to include novel genotypes.

Material and methods

Clinical isolates with clavulanic acid non-reversible non-susceptibility to extended-spectrum cephalosporins were subjected to combination disk testing with cefoxitin +/− cloxacillin at Malmö University Hospital. Phenotypical AmpC production was defined as cloxacillin reversible cefoxitin resistance. In this study 51 phenotypical AmpC-producing isolates, were subjected to the acquired bla_AmpC real-time PCR assay. The acquired blaAmpC positive isolates were further characterized by DNA sequencing of the acquired AmpC encoding gene, Pulsed-Field Gel Electrophoresis (PFGE) and PCR-based replicon typing.

Results and discussion

The real-time PCR assay was able to detect and sub-classify all acquired bla_AmpC genes described to date. The assay can be performed in less than 3 h, including pre-PCR preparations. Analysis of the isolate collection resulted in 18 of 51 phenotypical AmpC-producing isolates being positive in the acquired bla_AmpC real-time multiplex PCR assay; 17 of subtype CIT and one DHA. Sequence analysis identified 16 isolates as blaCMY-2, one as blaCMY-16 and one as blaDHA-1. Detected plasmid replicon types were I1 and B/O. Two of the E. coli isolates were identical according to PFGE and the others were unrelated.     

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.     

Characterization of the MLO gene family in Rosaceae and gene expression analysis in Malus domestica

Background

Powdery mildew (PM) is a major fungal disease of thousands of plant species, including many cultivated Rosaceae. PM pathogenesis is associated with up-regulation of MLO genes during early stages of infection, causing down-regulation of plant defense pathways. Specific members of the MLO gene family act as PM-susceptibility genes, as their loss-of-function mutations grant durable and broad-spectrum resistance.

Results

We carried out a genome-wide characterization of the MLO gene family in apple, peach and strawberry, and we isolated apricot MLO homologs through a PCR-approach. Evolutionary relationships between MLO homologs were studied and syntenic blocks constructed. Homologs that are candidates for being PM susceptibility genes were inferred by phylogenetic relationships with functionally characterized MLO genes and, in apple, by monitoring their expression following inoculation with the PM causal pathogen Podosphaera leucotricha.

Conclusions

Genomic tools available for Rosaceae were exploited in order to characterize the MLO gene family. Candidate MLO susceptibility genes were identified. In follow-up studies it can be investigated whether silencing or a loss-of-function mutations in one or more of these candidate genes leads to PM resistance.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-618) contains supplementary material, which is available to authorized users.     

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.