Rapid detection of cfiA metallo-ß-lactamase-producing Bacteroides fragilis by the combination of MALDI-TOF MS and CarbaNP.

AIMS: Carbapenem resistance in Bacteroides fragilis is emerging and is mainly attributed to insertion sequence (IS)-mediated activation of the carbapenemase gene cfiA. We investigated the use of matrix-assisted laser desorption ionisation time-of-flight mass spectrometry (MALDI-TOF MS) and the CarbaNP assay for the rapid identification of these strains. METHODS: This study used the Bruker MALDI Biotype system and the mass spectra models generated by 20 B. fragilis reference strains (10 cfiA-positive and 10 cfiA-negative) in the ClinProTools software to identify 404 B. fragilis (71 cfiA-positive and 333 cfiA-negative) clinical isolates. The ability of the CarbaNP assay to detect IS-mediated activation of the cfiA gene was assessed and the results obtained by molecular analysis were used as reference methods. RESULTS: The support vector machine model generated by ClinProTools was found to be the most reliable algorithm for differentiation of cfiA-positive and cfiA-negative B. fragilis subgroups. Using the direct transfer method, all but one cfiA-negative isolates were correctly identified to the two subgroups by the model. The correct identification of the cfiA-negative isolate was obtained upon retesting by the extraction method. Of the 81 cfiA-positive isolates, PCR and sequencing showed that 30 had an IS element providing the promoter for activation of cfiA. With regard to the presence of the IS element, the CarbaNP test in the cfiA upstream region had 100% sensitivity, 80.4% specificity, 75.0% positive predictive value and 100% negative predictive value. CONCLUSIONS: The combination of MALDI-TOF MS and the CarbaNP assay can be applied in diagnostic clinical laboratory for rapid identification of B. fragilis with IS element-activated cfiA gene.

Prevalence of aminoglycoside modifying enzyme and 16S ribosomal RNA methylase genes among aminoglycoside-resistant Escherichia coli isolates.

Aminoglycoside resistance determinants among 188 aminoglycoside-resistant blood culture Escherichia coli isolates from a tertiary hospital in Hong Kong, from 2004 to 2010 were investigated. Overall, 91% had aac(3)-II, 12.2% had aac(6')-Ib/Ib-cr, and 5.4% had the methylase genes (rmtB, armA). Aminoglycoside-resistant isolates with aac(')-Ib/Ib-cr, rmtB, and armA often had coresistance to multiple other antibiotics.

Clonal diversity of CTX-M-producing, multidrug-resistant Escherichia coli from rodents.

This territory-wide study investigated the occurrence of faecal carriage of extended-spectrum ß-lactamase (ESBL)-producing Escherichia coli among wild rodents from the 18 districts in Hong Kong. Individual rectal swabs were obtained from the trapped animals and cultured in plain and selective media. A total of 965 wild rodents [148 chestnut spiny rats (Niviventer fulvescens), 326 Indo-Chinese forest rats (Rattus andamanensis), 452 brown rats (Rattus norvegicus) and 39 black rats (Rattus rattus)] were sampled. ESBL carriage was 0 % in chestnut spiny rats, 0.6 % in Indo-Chinese forest rats, 7.7 % in black rats and 13.9 % in brown rats. Among brown rats, the prevalence of ESBL carriage differed markedly by geographical location: absent in two districts, low (7-10 %) in six districts, moderate (11-19 %) in seven districts and high (21-50 %) in three districts. Nonetheless, there was no correlation between the prevalence of ESBL in brown rats and human population density in the 18 districts. CTX-M-type enzymes were detected in 92.0 % of the ESBL-producing isolates, of which 83.1 % were resistant to three or more non-ß-lactam drugs. The CTX-M producing isolates were genetically diverse but a large proportion (47.8 %) were included in six successful clones that are strongly associated with human diseases and CTX-M dissemination, viz. sequence type complex [STC]10/phylogroup A, STC23/phylogroup B1, STC38/phylogroup D, STC155/phylogroup B1, ST405/phylogroup D and ST131/phylogroup B2. In conclusion, our results show that brown rats often carry potentially zoonotic clones of CTX-M producing, multidrug-resistant E. coli. The potential for rats to be a source of CTX-M producing E. coli for humans deserves further consideration.

Increase in the nasopharyngeal carriage of non-vaccine serogroup 15 Streptococcus pneumoniae after introduction of children pneumococcal conjugate vaccination in Hong Kong.

This study assessed pneumococcal carriage in the early periods after routine use of 13-valent pneumococcal conjugate vaccine (PCV13) in Hong Kong. Nasopharyngeal swabs were obtained from 1110 children (<5 years) admitted with acute illness during September 2010-August 2013. Pneumococcal carriage rate was 13.5% in unvaccinated children, 14.1% in children who had ≥1 PCV dose and 15.3% in children who had ≥3 PCV doses. Nonv-PCV13 serotypes comprised 56.4% of all isolates. The most common serogroup/types were 15 (15A, 5.1%; 15B, 10.3%; 15C, 9.6%; 15F, 0.6%), 19F (17.9%), 6A (7.1%) and 6C (7.1%). Carriage of serogroup 15 was more common among vaccinated children (4.1% versus 0.6%, P = 0.033). Molecular typing revealed that expansion of several clones (clonal complex, CC63, CC199, CC1262, CC3397) was responsible for the increase in serogroup 15. Almost all CC63 and CC3397 isolates were nonsusceptible to both penicillin and erythromycin. The finding highlights the emergence of serogroup 15 following PCV13 use.

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.

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.

Clonality and antimicrobial susceptibility of Staphylococcus aureus and methicillin-resistant S. aureus isolates from food animals and other animals.

Out of 3,081 animals studied, 24.9% of pigs, 4.7% of chickens, 6.3% of dogs, 10.5% of cats, and 7.1% of rodents were Staphylococcus aureus positive. Prevalence of methicillin-resistant S. aureus (MRSA) was high in pigs (animals, 21.3%; batches, 46.5%), with all MRSA isolates and most methicillin-sensitive S. aureus isolates belonging to clonal complex 9 (CC9) and being multidrug resistant. The predominant S. aureus CCs among dog and cat isolates were similar. Among rodent isolates, CC398 predominated, with spa t034 the most frequent spa type detected.

Predominance of pHK01-like incompatibility group FII plasmids encoding CTX-M-14 among extended-spectrum beta-lactamase-producing Escherichia coli in Hong Kong, 1996-2008.

This study assessed the temporal changes in the molecular epidemiology of bacteremic Escherichia coli isolates producing CTX-M-14 in Hong Kong. Blood isolates from 1996 to 1998 (period 1, n = 50) and 2007 to 2008 (period 2, n = 117) were investigated by molecular methods. CTX-M-type ESBL was carried by 98.2% (164/167) of the isolates. In both periods, the CTX-M-9 group and CTX-M-14 allele were the predominant ESBL type. The major clones were found to change from ST68 and ST405 in period 1 to ST131, ST69, and ST12 in period 2. Among 65 CTX-M-14-producing plasmids investigated further, 54 had the FII replicon. Replicon sequence typing and plasmid polymerase chain reaction-restriction fragment length polymorphism showed that 79.6% (43/54) of the FII plasmid subset was similar to the completely sequenced plasmid, pHK01 (human urine, Hong Kong, 2004). These pHK01-like plasmids were found to have spread to the major clones (ST68, ST405, and ST131) and multiple singleton isolates of all 4 phylogenetic groups.

Molecular epidemiology and nasal carriage of Staphylococcus aureus and methicillin-resistant S. aureus among young children attending day care centers and kindergartens in Hong Kong.

OBJECTIVES: To investigate the prevalence and molecular epidemiology of Staphylococcus aureus and methicillin-resistant S. aureus (MRSA) nasal carriage in children. METHODS: We collected nasal and nasopharyngeal swabs from 2211 children aged 2-5 years attending 79 day care centers (DCCs) and 113 kindergartens (KGs) in all 18 geographical districts in Hong Kong. RESULTS: The overall carriage rates of S. aureus and MRSA were 27.6% (95% confidence interval [CI], 24.8-28.5%) and 1.3% (95% CI, 0.8-1.8%), respectively. Molecular typing (staphylococcal cassette chromosome mec [SCCmec], sequence type [ST], clonal cluster [CC]) showed that all the 28 MRSA isolates had SCCmec IV (n = 13) or V (n = 15) including 12 isolates with community-associated-MRSA genotypes (ST59-IV/V, ST30-IV and ST88-V), 10 isolates with healthcare-associated-MRSA genotypes (ST45-IV/V, CC5-IV and ST630-V) and six isolates with novel genotypes (ST10-V and CC1-IV). Spa typing indicated that there was some within and between DCCs/KGs transmission of certain MRSA and methicillin-sensitive S. aureus strains but this was not extensive. CONCLUSION: Our findings indicate the potential for DCCs to be a reservoir for emerging MRSA genotypes and highlight the need to enhance education and infection control measures to reduce their cross-transmission in this population.

Extensive dissemination of CTX-M-producing Escherichia coli with multidrug resistance to 'critically important' antibiotics among food animals in Hong Kong, 2008-10.

OBJECTIVES: To assess the occurrence of faecal carriage of Escherichia coli with resistance to 'critically important' antibiotics in various animals. METHODS: Rectal or cloacal swabs were obtained weekly from cattle, pigs, chickens, cats, dogs and wild rodents over a 2 year period. Plain and antibiotic-containing medium was used for bacterial isolation. Selected isolates were characterized by molecular methods. RESULTS: In total, 2106 faecal specimens from 398 cats, 460 chickens, 368 dogs, 210 cattle, 214 pigs and 456 rodents were cultured. The faecal carriage rate of extended-spectrum ß-lactamase (ESBL)-producing E. coli was highest in pigs (63.6%, 136/214) and lowest in rodents (4.2%, 19/456). The faecal ESBL-producing E. coli carriage rate for food-producing animals (53.6%, 474/884) was significantly higher than that for cats/dogs (14.0%, 107/766; P<0.01) and wild rodents (4.2%, 19/456; P<0.01). ESBL-producing isolates from food animals often (33%-81%) had multidrug (≥4) resistance to amikacin, chloramphenicol, ciprofloxacin, co-trimoxazole, gentamicin, nalidixic acid, netilmicin, nitrofurantoin and tetracycline. Most (91.2%) of the ESBL-producing isolates had CTX-M-type enzymes. A total of 10 alleles (3, 13, 14, 15, 24, 27, 28, 55, 65 and 98) from two CTX-M families (M1 and M9) were found. PFGE showed that the CTX-M-producing isolates were genetically diverse. CONCLUSIONS: 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.

The prevalence and characteristics of Streptococcus pneumoniae isolates expressing serotypes 6C and 6D in Hong Kong prior to the introduction of the 7-valent pneumococcal conjugate vaccine.

A study was conducted to determine the prevalence of the 2 newly described types, 6C and 6D, among pneumococcal isolates collected in Hong Kong before availability of the 7-valent pneumococcal conjugate vaccine. A total of 154 serogroup 6 isolates obtained from nasopharynx (n = 106), blood (n = 22), respiratory (n = 24), and cerebrospinal fluid (CSF) (n = 2) during 1995 to 2001 were analyzed by polymerase chain reaction typing. Five nasopharyngeal and 2 sputum isolates were found to belong to 6C and 6D, respectively. The isolates were genetically diverse, but one 6C and two 6D isolates exhibited some clonal relationship. Phylogenetic analysis of the wchA-wciN(ß)-wciO nucleotide sequences showed that the Hong Kong 6C/6D isolates had 2 allelic profiles, which were more closely related to 6C/6D isolates from Fijian and Korea than were those from Brazil and the United States. However, all of the wciP gene sequences for both Hong Kong and non-Hong Kong isolates clustered together: 6C isolates with the wciP-9 allele and 6D isolates with the wciP-5 allele. In conclusion, the prevalence of the 2 newly described serotypes was low before the era of the pneumococcal conjugate vaccine. Nonetheless, results from the molecular studies indicated that the evolution of the capsular genes have involved complex pathways.

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.