Wai-U Lo

Fecal carriage of CTXM type extended-spectrum beta-lactamase-producing organisms by children and their household contacts.

OBJECTIVES To investigate the epidemiology of fecal carriage of CTX-M type extended-spectrum beta-lactamases (ESBL)-producing organisms among children and their household contacts. METHODS Fecal carriage with CTX-M-producing organisms was studied in 53 children and 172 household members. Molecular methods were used to characterize the isolates. RESULTS The children were mostly healthy and hospitalized for relatively mild febrile illnesses. Overall, the prevalence of fecal carriage of CTX-M-producing bacteria was 43.5% (admission children, 37.7%; household children, 20.7% and household adults, 50.3%). Household colonization index (defined by number of household carriers/total number of members) was significantly higher among families with at least one individual having a history of prolonged (>3 months) out-of-town residence in the previous year (mean+/-standard deviation; yes group, 0.67+/-0.36 vs. no group, 0.39+/-0.28, P=0.009) and was inversely correlated with the living space per person (R-square=0.139, P=0.006). Among 29 households with at least two carriers of CTX-M-producing enterobacteria, six clusters of clonally related strains were shared by 15 individuals from seven households; with both intra- and inter-household transmission. CONCLUSION CTX-M beta-lactamases may spread extensively amongst family members in the home.

Prevalence and molecular epidemiology of plasmid-mediated fosfomycin resistance genes among blood and urinary Escherichia coli isolates

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 1996-2008, were used to investigate the prevalence and molecular epidemiology of plasmid-mediated 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 (n = 2), ST118 (n = 1), ST131 (n = 1), ST617 (n = 1), ST648 (n = 1), ST1488 (n = 1) and ST2847 (n = 1). In the isolates, fosA3 and blaCTX-M genes were co-harboured on conjugative plasmids with F2:A-:B- (n = 2), N (n = 1), F-:A-:B1 and N (n = 1) and untypable (n = 2) replicons. Both fosKP96-carrying plasmids belonged to replicon N. RFLP analysis showed that the two F2:A-:B- plasmids carrying fosA3 and blaCTX-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 blaCTX-M-carrying plasmid types, of which F2:A-:B- plasmids closely related to pHK23a were shared by isolates from human and animal sources.

Dissemination of plasmid‐mediated fosfomycin resistance fosA3 among multidrug‐resistant Escherichia coli from livestock and other animals

To investigate plasmid‐mediated fosfomycin resistance related to fosA3 in Escherichia coli isolates collected from different animals in Hong Kong, China, 2008–2010.

Dissemination of pHK01-like incompatibility group IncFII plasmids encoding CTX-M-14 in Escherichia coli from human and animal sources

Few studies have compared CTX-M encoding plasmids identified in different ecological sources. This study aimed to analyze and compare the molecular epidemiology of plasmids encoding CTX-M-14 among strains from humans and animals. The CTX-M-14 encoding plasmids in 160 Escherichia coli isolates from animal faecal (14 pigs, 16 chickens, 12 cats, 8 cattle, 5 dogs and 3 rodents), human faecal (45 adults and 20 children) and human urine (37 adults) sources in 2002-2010 were characterized by molecular methods. The replicon types of the CTX-M-14 encoding plasmids were IncFII (n=61), I1-Iγ (n=24), other F types (n=23), B/O (n=10), K (n=6), N (n=3), A/C (n=1), HI1 (n=1), HI2 (n=1) and nontypeable (n=30). The genetic environment, ISEcp1 -bla(CTX-M-14) - IS903 was found in 89.7% (52/58), 87.7% (57/65) and 86.5% (32/37) of the animal faecal, human faecal and human urine isolates, respectively. Subtyping of the 61 IncFII incompatibility group plasmids by replicon sequence typing, plasmid PCR-restriction fragment length polymorphism and marker genes (yac, malB, eitA/eitC and parB/A) profiles showed that 31% (18/58), 30.6% (20/65) and 37.8% (14/37) of the plasmids originating from animal faecal, human faecal and human urine isolates, respectively, were pHK01-like. These 52 pHK01-like plasmids originated from diverse human (20 faecal isolates from 2002, 2007 to 2008, 14 urinary isolates from 2004) and animal (all faecal, 1 cattle, 1 chicken, 5 pigs, 9 cats, 1 dog, 1 rodent from 2008 to 2010) sources. In conclusion, this study highlights the importance of the IncFII group, pHK01-like plasmids in the dissemination of CTX-M-14 among isolates from diverse sources.

Highly conjugative IncX4 plasmids carrying blaCTX-M in Escherichia coli from humans and food animals.

This study investigated the prevalence of IncX plasmid subtypes in commensal and pathogenic Escherichia coli isolates and the biological features of the IncX4 subtype. Two hundred and twenty-five E. coli isolates from multiple sources (47 chickens, 41 pigs, 30 cattle and 107 humans) obtained during the period 2006-2012 were tested for the presence of IncX1 to IncX5. Overall, the prevalence of IncX plasmids in chicken, pig, cattle and human isolates were 21.2 % (10/47), 19.5 % (8/41), 3.3 % (1/30) and 4.8 % (5/107), respectively. IncX4 was the most common subtype, followed by IncX1 and IncX3, while no IncX2 or IncX5 were found. Seven out of 16 (43.8 %) IncX4 plasmids were found to carry blaCTX-M genes and six of them originating from different host sources (four chickens, one pig and one human) had identical or highly similar RFLP patterns. Three IncX4 plasmids carrying blaCTX-M from different host sources were investigated further. It was found that the IncX4 plasmids had little effect on bacterial host growth parameters after their introduction to J53 recipients. Conjugation experiments demonstrated that the IncX4 plasmids could be efficiently transferred at 30-42 °C at rates which were generally 10(2)-10(5)-fold higher than those for the epidemic IncFII plasmid carrying blaCTX-M (pHK01). In conclusion, the IncX plasmids are more common than previously recognized. The efficient transfer of IncX4 plasmid at different temperatures and the lack of fitness burden on bacterial hosts highlight the ability of this plasmid replicon to be an important vehicle for dissemination of antimicrobial resistance.

Complete sequencing of the FII plasmid pHK01, encoding CTX-M-14, and molecular analysis of its variants among Escherichia coli from Hong Kong

OBJECTIVES We characterized plasmids encoding CTX-M-14 β-lactamase originating from Escherichia coli isolates recovered from patients with uncomplicated cystitis or individuals with faecal colonization in Hong Kong from 2002 to 2004. METHODS Plasmids carrying CTX-M-14 were studied by conjugation, replicon typing, S1 nuclease-PFGE and plasmid PCR-restriction fragment length polymorphism (RFLP). The complete sequence of pHK01, a 70 kb plasmid encoding CTX-M-14 from an E. coli strain, was determined and the results compared with reference plasmids and aligned with GenBank data. RESULTS The blaCTX-M-14 plasmids could be transferred in 23 of 44 E. coli strains tested. Among the 23 transconjugants, the replicon types of the CTX-M-14-encoding plasmid were FII (n=13), I1-Iγ (n=4), F1B (n=2), FII and I1-Iγ (n=1), K (80 kb, n=1) and undetermined (n=2). Plasmid pHK01 (FII replicon) shares a high degree of homology with R100 except mainly for a 11 kb variable region containing blaCTX-M-14 (with an upstream ISEcp1 and a downstream truncated IS903), an iron transport system, an outer membrane protein (malB, maltoporin) and a putative toxin-antitoxin plasmid stability system (yacABC). It was highly related to blaCTX-M-14 (pKF3-70) and blaCTX-M-24 (pEG356) plasmids reported from mainland China in 2006 and Vietnam in 2007, respectively. Subtyping by a plasmid PCR-RFLP scheme showed that 10 of the 13 FII plasmids originating from isolates collected by multiple laboratories exhibited either identical or highly similar profiles. CONCLUSIONS This study showed that narrow host-range FII plasmids play important roles in the dissemination of CTX-M-14. FII plasmids closely related to pHK01 have disseminated widely in the Hong Kong community.

Plasmid-mediated fosfomycin resistance in Escherichia coli isolated from pig.

Previous studies have reported plasmid-mediated fosA3 among Escherichia coli originating from human and companion animals. In this study, the plasmid, designated pHK23a originating from a multidrug-resistant E. coli isolate recovered from a slaughter pig in December 2008 in Hong Kong, China was sequenced. In conjugation, the plasmid readily transferred to E. coli J53 at high frequencies. It belongs to the narrow host range IncFII incompatibility group and is 73,607 bp in length. Sequence alignment showed that pHK23a has a 59.1 kb backbone which shares high homology with the prototype R100 plasmid and a 14.5 kb variable region. The variable region includes three genes mediating antimicrobial resistance (fosA3, Δbla(TEM-1), bla(CTX-M-3)), ten mobile genetic elements (four copies of IS26, insA, ΔinsB, ΔTn2, IS1, ΔISEcp1, Δintl1), the tir transfer inhibition protein, the pemI/pemK addiction system and eight ORFs of unknown functions (orf1, orf2, Δorf3, orf20, orf23, orf24, ycdA and ycdB). The three resistance genes were organized in a novel IS26-composite transposon-like structure. In conclusion, this is the first report of fosA3 containing plasmid in an isolate of pig origin. Since IncFII plasmids spread efficiently in Enterobacteriaceae, the detection of fosA3 with bla(CTX-M) is worrisome and might become a public health concern.

Clinical outcome of extended-spectrum beta-lactamase-producing Escherichia coli bacteremia in an area with high endemicity.

OBJECTIVES This study assessed the impact of discordant empirical antibiotic therapy on the outcome of bacteremia caused by extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli. METHODS The clinical features and outcomes of a cohort of patients hospitalized with ESBL E. coli bacteremia between 2007 and 2008 were retrospectively reviewed. The effect of different antimicrobial regimens on patient outcomes was analyzed. RESULTS ESBL E. coli accounted for 24.2% (207/857) of E. coli bacteremia cases. The urinary tract (43.6%) was the most common source of infection, followed by the hepatobiliary tract (23.0%). Discordant empirical antibiotic therapy was given to 52.0% patients. Admission to the intensive care unit was associated with the use of a carbapenem as empirical antibiotic therapy (p<0.001). Univariate analysis revealed no significant differences in 30-day mortality rates between patients receiving concordant and discordant empirical antibiotic therapy (23.5% vs. 19.8%, p=0.526), carbapenem and non-carbapenem empirical antibiotic therapy (29.8% vs. 19.1%, p=0.118), beta-lactam/beta-lactam inhibitor combinations (BLBLIs) and non-BLBLIs empirical antibiotic therapy (20.3% vs. 22.3%, p=0.734), cephalosporin and non-cephalosporin empirical antibiotic therapy (19.7% vs. 22.6%, p=0.639), and fluoroquinolone and non-fluoroquinolone empirical antibiotic therapy (8.3% vs. 22.4%, p=0.251). The findings were confirmed by multivariate analysis. CONCLUSIONS Despite a high proportion of discordant empirical antibiotic therapy, ESBL production had little effect on 30-day mortality. Whether the observation can be applied to different ESBL types is unknown and warrants further study.

Complete Sequence of an IncN Plasmid, pIMP-HZ1, Carrying blaIMP-4 in a Klebsiella pneumoniae Strain Associated with Medical Travel to China

In China, IMP-4 is a major plasmid-mediated carbapenemase among multidrug-resistant Enterobacteriaceae, but little is known about the plasmid scaffolds involved in the dissemination of blaIMP-4 (1, 2). In 2010, we identified a Klebsiella pneumoniae CRE1 strain carrying blaIMP-4 from the penile swab of a 49-yearold man who was repatriated from Huizhou, China, where he had been hospitalized for intracranial hemorrhage. The isolate was resistant to all -lactams, including imipenem, ertapenem, and meropenem, and multiple non-lactam antibiotics (chloramphenicol, co-trimoxazole, nitrofurantoin) (3). Combined disc testing (4) showed that carbapenem resistance can be reversed by EDTA but not boronic acid. Multilocus sequence typing (MLST) (5) identified CRE1 as sequence type (ST) 11, which is a major lineage associated with blaKPC dissemination in China (6). Conjugation was carried out in filters with Escherichia coli J53 as the recipient and transconjugants selected with sodium azide (100 g/ml) and meropenem (1 g/ml) (7). The blaIMP-4-carrying plasmid was able to be transferred at a frequency of 1.2 10 4 per donor cell. Plasmid DNA from the transconjugant was extracted, amplified, and sequenced using a GS-FLX system (Roche, Mannheim, Germany) as described previously (7). The assembled plasmid pIMP-HZ1 has 50,775 bp (GenBank accession no. JX457479). pIMP-HN1 has a plasmid scaffold typical for the IncN plasmids in general (8, 9). The backbone regions shared by pIMP-HZ1 and the reference IncN plasmid R46 include the replicon repA, the mucAmucB genes (encoding UV protection), the stbABC operon (for plasmid stability), the ardA-ardB and ardK-ardR genes (with antirestriction function), the ccg genes (ccgEIII-ccgD-ccgC-ccgAIccgAII, providing protection from the type I restriction system), and the two transfer gene clusters (locus tra1 and locus tra2, en-

Plasmid-Mediated OqxAB Is an Important Mechanism for Nitrofurantoin Resistance in Escherichia coli

ABSTRACT Increasing consumption of nitrofurantoin (NIT) for treatment of acute uncomplicated urinary tract infections (UTI) highlights the need to monitor emerging NIT resistance mechanisms. This study investigated the molecular epidemiology of the multidrug-resistant efflux gene oqxAB and its contribution to nitrofurantoin resistance by using Escherichia coli isolates originating from patients with UTI (n = 205; collected in 2004 to 2013) and food-producing animals (n = 136; collected in 2012 to 2013) in Hong Kong. The oqxAB gene was highly prevalent among NIT-intermediate (11.5% to 45.5%) and -resistant (39.2% to 65.5%) isolates but rare (0% to 1.7%) among NIT-susceptible (NIT-S) isolates. In our isolates, the oqxAB gene was associated with IS26 and was carried by plasmids of diverse replicon types. Multilocus sequence typing revealed that the clones of oqxAB-positive E. coli were diverse. The combination of oqxAB and nfsA mutations was found to be sufficient for high-level NIT resistance. Curing of oqxAB-carrying plasmids from 20 NIT-intermediate/resistant UTI isolates markedly reduced the geometric mean MIC of NIT from 168.9 μg/ml to 34.3 μg/ml. In the plasmid-cured variants, 20% (1/5) of isolates with nfsA mutations were NIT-S, while 80% (12/15) of isolates without nfsA mutations were NIT-S (P = 0.015). The presence of plasmid-based oqxAB increased the mutation prevention concentration of NIT from 128 μg/ml to 256 μg/ml and facilitated the development of clinically important levels of nitrofurantoin resistance. In conclusion, plasmid-mediated oqxAB is an important nitrofurantoin resistance mechanism. There is a great need to monitor the dissemination of this transferable multidrug-resistant efflux pump.