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

The New Delhi metallo-β-lactamase (NDM-1) is one of the most important resistance traits in Enterobacteriaceae. We characterized nine blaNDM-1 producing Enterobacteriaceae recovered from seven patients who have recently travelled or been treated in India (n=1) or mainland China (n=6) during December 2010–May 2012. All the China-linked patients had no links to the Indian subcontinent. The blaNDM-1 carrying plasmids belonged to the novel IncX3 (∼50 kb, in seven isolates including two Escherichia coli, two Klebsiella pneumoniae, one Citrobacter freundii, one Enterobacter aerogenes and one E. cloacae), IncA/C2 (∼140 kb, in one E. coli) or FII-F1B groups (∼110 kb, in one E. coli). Restriction fragment length polymorphism analysis of the seven IncX3 plasmids revealed identical pattern in six and two bands difference in the remaining one. The IncX3 plasmids carrying blaNDM-1 were epidemiologically linked to Guangzhou (n=1), Hunan (n=4), Haifeng (n=1) and Dongguan (n=1) in mainland China. Complete sequencing of the IncX3 plasmid pNDM-HN380 revealed that it was 54 035 bp long and encoded 52 open reading frames. The blaNDM-1 gene was found in a transposon-like structure flanked by ISAba125 and IS26, inserted into the plasmid genetic load region. The sequences of the blaNDM-1 containing module within the two IS elements were identical to those previously described for blaNDM-1-positive Tn125 in the plasmids or chromosome of Acinetobacter isolates. In summary, this is the first description of IncX3 plasmids carrying blaNDM-1. The findings indicate the worrisome involvement of an epidemic plasmid in the dissemination of NDM-1 in China.

This study shows that food animals are a major reservoir of E. coli with multidrug resistance to many antibiotics that are ranked as critically important in human medicine.

Aims: To investigate plasmid-mediated fosfomycin resistance related to fosA3 in Escherichia coli isolates collected from different animals in Hong Kong, China, 2008. Methods and Results: In total, 2106 faecal specimens from 210 cattle, 214 pigs, 460 chickens, 398 stray cats, 368 stray dogs and 456 wild rodents were cultured. The faecal colonization rates of fosfomycin-resistant E. coli were as follows: 11Á2% in pigs, 8Á6% in cattle, 7Á3% in chickens, 2Á4% in dogs, 0Á8% in cats and 1Á5% in rodents. The cultures yielded 1693 isolates of which 831 were extended-spectrum b-lactamases (ESBL) producers. Fosfomycin-resistant isolates were more likely than fosfomycin-susceptible isolates to be producers of ESBL and to have resistance to chloramphenicol, ciprofloxacin, cotrimoxazole, gentamicin and tetracycline. Of the 101 fosfomycin-resistant isolates, 97 (96Á0%) isolates were fosA3 positive and 94 (93Á1%) were bla CTX-M positive. PCR mapping showed that the fosA3-containing regions were flanked by IS26, both upstream and downstream in 81 (83Á5%) isolates, and by an upstream bla CTX-M-14 -containing transposon-like structure (DISEcp1-bla CTX-M-14 -DIS903

A total of 1878 non-duplicate clinical Escherichia coli isolates (comprising 1711 urinary isolates and 167 blood-culture isolates), which were collected from multiple centres in Hong Kong during 1996Kong during -2008, were used to investigate the prevalence and molecular epidemiology of plasmidmediated fosfomycin (fos) resistance genes. Eighteen of the 1878 clinical E. coli isolates were fosfomycin resistant, of which six were fosA3 positive and two were positive for another fosA variant (designated fosKP96). No isolates had the fosC2 gene. The clones of the eight isolates were diverse: sequence type (ST) 95 (n52), ST118 (n51), ST131 (n51), ST617 (n51), ST648 (n51), ST1488 (n51) and ST2847 (n51). In the isolates, fosA3 and bla CTX-M genes were coharboured on conjugative plasmids with F2:A":B" (n52), N (n51), F-:A":B1 and N (n51) and untypable (n52) replicons. Both fosKP96-carrying plasmids belonged to replicon N. RFLP analysis showed that the two F2:A":B" plasmids carrying fosA3 and bla CTX-M-3 genes shared the same pattern. Complete sequencing of one of the two F2:A":B" plasmids, pFOS-HK151325 (69 768 bp) demonstrated it to be .99 % identical to the previously sequenced plasmid pHK23a originating from a pig E. coli isolate in the same region. This study demonstrated the dissemination of fosA3 genes in diverse E. coli clones on multiple bla CTX-M -carrying plasmid types, of which F2:A":B" plasmids closely related to pHK23a were shared by isolates from human and animal sources.

Carbapenem-resistant Enterobacteriaceae represents a major public health issue. This study investigated the clonality and resistance mechanisms of 92 carbapenem-resistant E. coli (n = 21) and K. pneumoniae (n = 71) isolates collected consecutively from clinical specimens and patients at high risk of carriage between 2010 and 2012 in a healthcare region in Hong Kong. Combined disk tests (CDTs) and the Carba NP test were used for phenotypic detection of carbapenemases. PCR assays were used to detect carbapenemase genes. All isolates were intermediate or resistant to at least one carbapenem. Nine (9.8 %) isolates were genotypic carbapenemase producers and included six K. pneumoniae (one ST1306/bla IMP-4, one ST889/bla IMP-4, two ST11/bla KPC-2, one ST258/bla KPC-2, one ST483/bla NDM-1) and three E. coli (one ST131/bla IMP-4, two ST744/ bla NDM-1) isolates. All nine isolates carrying carbapenemase genes could be detected by the CDTs and the Carba NP test. PCR identified bla CTX-M and bla AmpC alone or in combination in 77.8 % (7/9) and 96.4 % (80/83) of the carbapenemase-producers and non-producers, respectively. Porin loss was detected in 22.2 % (2/9) and 59.0 % (49/83) of the carbapenemase-producers and non-producers, respectively. Overall, the E. coli clones were diverse (14 different STs), but 36.6 % (26/71) of the K. pneumoniae isolates belonged to ST11. In conclusion, the prevalence of carbapenemases among carbapenem-nonsusceptible E. coli and K. pneumoniae remained low in Hong Kong. Porin loss combined with AmpC and/or CTX-M type ESBL was the major mechanism of carbapenem resistance in the study population.

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We characterized two new gene cassettes in an Acinetobacter isolate: one harbored the metallo-␤-lactamase (IMP-4) gene bla IMP-4 , the other harbored the rifampin ADP-ribosyltransferase (ARR-2) gene arr-2, and both arrayed with the aminoglycoside acetyltransferase [AAC(6)-Ib 7 ] gene cassette aacA4 in two separate class 1 integrons. The epidemiology of these gene cassettes in isolates from blood cultures obtained from 1997 to 2000 was studied. Isolates bearing either the bla IMP-4 or the arr-2 gene cassette or both represented 17.5% (10 of 57) of isolates in 1997, 16.1% (10 of 62) in 1998, 2.5% (1 of 40) in 1999, and 0% (0 of 58) in 2000. These two gene cassettes, probably borne on two separate integrons, were found in at least three genomic DNA groups, with evidence of clonal dissemination in the intensive care unit during 1997 to 1998. Seventeen of the 52 Acinetobacter baumannii (genomic DNA group 2) isolates from 1997 to 2000 harbored intI1, but only one was positive for these gene cassettes, whereas 20 of the 21 intI1-positive isolates of all other genomic DNA groups were positive for either or both of them. Reduced susceptibility to imipenem and rifampin was seen only in isolates harboring the bla IMP-4 and arr-2 cassettes, respectively. The aminoglycoside phosphotransferase [APH(3)-VIa] gene aph(3)-VIa was detected in all 21 isolates for which the MIC of amikacin was >8 g/ml, with or without aacA4, whereas aacA4 alone was found in isolates for which the MIC of amikacin was 0.5 to 2 g/ml. Significant differences between the 17 intI1-positive and 47 intI1-negative isolates belonging to genomic DNA group 3 from 1997 to 1998 in the MICs of amikacin, gentamicin, imipenem, sulfamethoxazole, and ceftazidime were observed (Mann-Whitney test, P < 0.001 to 0.01).In the past decade, there has been an explosion of interest in the integron, a genetic element which facilitates gene dissemination among bacteria of different species and perhaps genera, thus establishing the possibility of a wide dispersal of antimicrobial resistance. These genetic elements are marked by the presence of two conserved regions between which gene cassettes are integrated at a recombination site known as attI. The 5Ј-conserved region of an integron contains an integrase gene, intI, and a promoter (P c ) which drives the expression of any integrated gene cassettes. Individual gene cassettes, existing in a free circularized form when not integrated, are comprised of an open reading frame (ORF) and a 59-base element (59-be) at the 3Ј end. The 59-be is a family of recombination sites that act as substrates to integrase-mediated recombination (43). To date, over 70 resistance cassettes have been described (32, 45). According to the 3Ј-conserved sequence (3Ј-CS), integrons are divided into three classes. The majority of the resistance cassette-bearing integrons belong to class 1, and these carry, at the 3Ј-CS, the truncated quaternary ammonium compound resistance gene qacE⌬1, the sul1 sulfonamide resistance gene, and orf5, of unknown function (20).A...