Molecular characterization & epidemiology of carbapenem-resistant Acinetobacter baumannii collected across India

Background & objectives: Acinetobacter baumannii is an opportunistic pathogen responsible for causing nosocomial infections. A. baumannii develops resistance to various antimicrobial agents including carbapenems, thereby complicating the treatment. This study was performed to characterize the isolates for the presence of various ?-lactamases encoding genes and to type the isolates to compare our clones with the existing international clones across five centres in India. Methods: A total 75 non-repetitive clinical isolates of A. baumannii from five different centres were included in this study. All the isolates were confirmed as A. baumannii by bl aOXA-51-likePCR. Multiplex PCR was performed to identify the presence of extended spectrum ?-lactamases (ESBL) and carbapenemases. Multilocus sequence typing was performed to find the sequence type (ST) of the isolates. e-BURST analysis was done to assign each ST into respective clonal complex. Results: blaOXA-51-likewas present in all the 75 isolates. The predominant Class D carbapenemase was blaOXA-23-likefollowed by Class B carbapenemase, blaNDM-like. Class A carbapenemase was not observed. blaPER-likewas the predominant extended spectrum ?-lactamase. ST-848, ST-451 and ST-195 were the most common STs. Eight-novel STs were identified. e-BURST analysis showed that the 75 A. baumannii isolates were clustered into seven clonal complexes and four singletons, of which, clonal complex 208 was the largest. Interpretation & conclusions: Most of the isolates were grouped under clonal complex 208 which belongs to the international clonal lineage 2. High occurrence of ST-848 carrying blaOXA-23-likegene suggested that ST-848 could be an emerging lineage spreading carbapenem resistance in India.

Molecular epidemiology of sequence type 33 of Shiga toxin-producing Escherichia coli O91:H14 isolates from human patients and retail meats in Korea

Sequence type (ST) 33 of Shiga toxin-producing Escherichia coli (STEC) strain O91:H14 has been proposed as a potential domestic clone of STEC in Korea because of its high prevalence among human patients with mild diarrhea or asymptomatic carriers. Herein, the clonal diversity of 17 STEC O91:H14 isolates of ST33 during 2003 to 2014 was analyzed by pulsed-field gel electrophoresis, including 14 isolates from human patients and 3 from retail meats. Their virulence characteristics, acid resistance, and antimicrobial susceptibility were also determined. Our results showed that all isolates were clustered mainly into three different pulsotypes and were likely low pathogenic without antimicrobial resistance.

Antimicrobial susceptibility and fluctuations in clonal complexes of serogroup 6 Streptococcus pneumoniae isolates collected from children in Beijing, China, between 1997 and 2016

ABSTRACT This study examined the antimicrobial susceptibility patterns and clonal complex (CC) characteristics of serogroup 6 Streptococcus pneumoniae isolates collected from children in Beijing, China, between 1997 and 2016. Serotypes were determined using the Quellung reaction, and the antimicrobial susceptibility profiles of the isolates were determined using the disc-diffusion method or by E-test. Sequence types (STs) were assigned based on multilocus sequence typing. A total of 250 isolates were examined, with 55.2%, 30.0%, 12.8%, and 2.0% of isolates identified as serotypes 6A, 6B, 6C, and 6D, respectively. All of the isolates were susceptible to levofloxacin and vancomycin, and the non-suceptibitility rate to penicillin was 41.6%. Eighty-two distinct STs, assigned to 13 CCs and 28 singletons, were identified. CC982 was the most prevalent CC amongst serotype 6A isolates (34%), followed by CC9789 and CC3173. Amongst serotype 6B isolates, CC90 and CC4542 were the most common, accounting for 25.3% and 14.7% of isolates respectively. Over the study period, the prevalence of CC982, CC4542, and CC4536 isolates showing susceptibility to penicillin and cefuroxime decreased, and the proportion of CC3173, CC9789, CC855, and CC902 isolates showing non-susceptibility to these two antibiotics increased.

Pathogenic and phylogenetic characteristics of non-O157 Shiga toxin-producing Escherichia coli isolates from retail meats in South Korea

Herein, we report the pathogenic and phylogenetic characteristics of seven Shiga toxin (Stx)-producing Escherichia coli (STEC) isolates from 434 retail meats collected in Korea during 2006 to 2012. The experimental analyses revealed that all isolates (i) were identified as non-O157 STEC, including O91:H14 (3 isolates), O121:H10 (2 isolates), O91:H21 (1 isolate), and O18:H20 (1 isolate), (ii) carried diverse Stx subtype genes (stx₁, stx(2c), stx(2e), or stx₁ + stx(2b)) whose expression levels varied strain by strain, and (iii) lacked the locus of enterocyte effacement (LEE) pathogenicity island, a major virulence factor of STEC, but they possessed one or more alternative virulence genes encoding cytotoxins (Cdt and SubAB) and/or adhesins (Saa, Iha, and EcpA). Notably, a significant heterogeneity in glutamate-induced acid resistance was observed among the STEC isolates (p < 0.05). In addition, phylogenetic analyses demonstrated that all three STEC O91:H14 isolates were categorized into sequence type (ST) 33, of which two beef isolates were identical in their pulsotypes. Similar results were observed with two O121:H10 pork isolates (ST641; 88.2% similarity). Interestingly, 96.0% of the 100 human STEC isolates collected in Korea during 2003 to 2014 were serotyped as O91:H14, and the ST33 lineage was confirmed in approximately 72.2% (13/18 isolates) of human STEC O91:H14 isolates from diarrheal patients.

Analysis on resistance genes and homology of carbapenem-resistant Klebsiella pneumoniae / 临床检验杂志

Objective To understand the prevalence of resistance gene and homology of carbapenem-resistant Klebsiella pneumoniae (CRKP) isolated from ICU of emergency.Methods A total of 19 CRKP isolates were obtained from emergency ICU of the Affiliated Hospital of Xuzhou Medical University from July 2015 to August 2016.PCR was performed to screen the genes encoding carbapenemase,extended spectrum beta-lactamase (ESBL) and AmpC.Pulsed field gel electrophoresis (PFGE) and multi-locus sequence typing (MLST) were used for molecular typing of these bacterial strains.Results Among the 19 CRKP,carbapenemase-resistant genes were detectable in 18 CRKP isolates,including 17 isolates of harboring blaKPC gene and 1 strain of harboring blaNDM gene.All the 18 strains carried ESBLs genes which were identified as 8 blaSHV-12,3 blaSHV-11,5 blaSHV-2a,15 blaTEM-1,10 blaCTX-M-65,3 blaCTX-M-15,1 blaCTX-M-14 and 1 blaCTX-M-27.The 13 strains harboring cephalosporin-resistant genes were all identified as blaDHA-1.PFGE results revealed that the 19 CRKP strains were grouped into 4 types (A,B,C and D) and 4 subtypes(A1,2,3 and 4):A1 (n =12),A2(n =1),A3 (n=1),A4(n=1),B(n=2),C(n=1) and D(n=1).MLST showed that ST11 was the predominant sequence type (n=15) among the 19 CRKP strains,and ST48 (n =2),ST37 (n =1) and untyped (n =1) were also identified.The 15 blaKPC-2-producing CRKP ST11 clone shared the A type of PFGE pattern.Conclusion The report on CRKP suggested the dissemination of blaKPC-2-producing ST11 clone was existed in the ICU of emergency department in this hospital.The surveillance for drug-resistance and effective disinfectant quarantine measures should be strengthened.

The Usefulness of Active Surveillance Culture of Extended-Spectrum β-Lactamase-Producing Escherichia coli in ICU Settings without Outbreak in the Situation of Wide Spread of Sequence Type 131 ESBL-Producing E. coli in Community / 대한임상미생물학회지

BACKGROUND: In the present study, the prevalence and risk factors for acquisition of extended-spectrum β-lactamase (ESBL)-producing Escherichia coli in intensive care unit (ICU) settings without outbreak in the situation of widespread sequence type (ST) 131 ESBL-producing E. coli in a Korean community was investigated. METHODS: Consecutive and prospective screening of ESBL-producing E. coli colonization was performed in all patients admitted to surgical or medical ICUs within 48 hours for two months. ESBL genotype was determined based on PCR and sequencing. PCR for O16-ST131/O25-ST131 was performed for all ESBL producers. Clinical information was obtained from a review of electronic medical record to determine the risk factors for ESBL-producing E. coli colonization. RESULTS: The colonization rate of ESBL-producing E. coli at ICU admission was 14.9% (42/281). CTX-M-15 (N=15), CTX-M-14 (N=12), and CTX-M-27 (N=10) were commonly detected using PCR of ESBL genes. Approximately half (45.2%, 19/42) of ESBL producers were ST131 clone with 14 ST131-O25 and 5 ST131-O16. In univariate analysis, independent risk factor for acquisition of ESBL-producing E. coli compared with controls was ICU type (odds ratio, 2.05; P < 0.032); however, site of acquisition, previous antibiotic use, and hospital stay were not significant risk factors. CONCLUSION: In this study, the colonization of ESBL-producing E. coli at ICU admission without outbreak was frequent and it could be an infection source, regardless of acquisition site. We recommend routine use of ASC to control endemic ESBL-producing E. coli considering the wide distribution of ST131-ESBL-producing E. coli in the Korean community.

First isolation of Laribacter hongkongensis from stool samples of Rattus norvegicus / 中国人兽共患病学报

In order to investigate whether Laribacter hongkongensis could be detected in stool samples of Rattus norvegicus in the wild,Rattus norvegicus were trapped alive in an urban community of Guangzhou,China over a period of one year from June 2015 to May 2016,and their stool samples were examined for the presence of L.hongkongensis strains.Isolates were identified based on phenotypic characteristics and 16S rRNA sequence analysis,and were examined for their susceptibility to 19 antimicrobial agents.Further typing of the isolates was performed using multi-loci sequence typing (MLST) analysis.A total of 191 R.norvegicus were trapped alive.L.hongkongensis was identified and successfully isolated from two samples,representing a prevalence of 1.05 ％.Although the two isolates possessed similar phenotypic characteristics and have no base difference of 16S rRNA gene,they constituted two new distinct sequence types (STs),ST-163 and ST-164.This is the first report that L.hongkongensis can be detected in the intestinal tract of R.norvegicus.Results suggest that R.norvegicus could serve as carriers of L.hongkongensis and therefore could be another potential source of infection.

Clonal and Virulence Distribution of Uropathogenic Escherichia coli Isolated from Children in Korea

Urinary tract infections (UTI) are one of the most frequent infectious diseases. Uropathogenic Escherichia coli (UPEC) are among major pathogens causing UTI. A variety of virulence genes are mainly responsible for the severity of these emerging infection. This study investigate the influences of virulence properties of UPEC isolates with reference to multi-locus sequence typing (MLST). The aim of this study was targeted that investigation of the bacterial pathogenicity associated with UTI in children. A total of 58 UPEC isolates were collected from urine samples from patients with clinical diagnosis of uncomplicated UTI. The MLST of UPEC strains were assessed by methods based on polymerase chain reaction. Motility was evaluated using soft-agar plates. Biofilm formation was analyzed in microtiter dish biofilm formation assay. Cell death assay was analyzed by Annexin V/Phosphatidylserine staining and DNA fragmentation assay. According the result, the predominant sequence type (ST) was ST95 (24.1%) and ST73 (17.2%). There were some difference in virulence gene and antibiotics resistance between ST95 and ST73. The number of 11 (18.9%) isolates were strongly adherent. Based on the detected biofilm formation, these strongly adherent are almost ST73. The ST95 was higher than ST73 in population, but ST95 was lower than ST73 in motility and cell death induction. This study indicated that the UPEC molecular strains are related to some virulence traits. Furthermore, the virulence factors carried by ST73 strains contribute to their abilities to colonize the host and cause disease.

Emergence of Panton-Valentine Leukocidin-Positive ST80 Clone of Community-Associated Methicillin-Resistant Staphylococcus aureus in Busan, Korea

Community-associated methicillin resistant Staphylococcus aureus (CA-MRSA) has become widespread in the community and healthcare settings, and a number of clonal lineages emerged on every country. Sequence type (ST) 80 clone of CA-MRSA was dominant in Europe and has increasingly been isolated from the Middle East but so far never found in Korea. In this study, 48 MRSA isolates recovered from ear infections were characterized by multilocus sequence typing (MLST), staphylococcal cassette chromosome mec (SCCmec) typing, staphylocoagulase (SC) genotyping, staphylococcal protein A gene (spa) typing, accessory gene regulator (agr) typing, and virulence gene profiling. Most MRSA strains belonged to three major clones: ST5-SCCmec II-SC type II (n=19, 39.6%), ST239-SCCmec III-SC type IV (n=15, 31.2%), and ST72-SCCmec IV-SC type Vb (n=11, 22.9%). Among the isolates, one strain was Panton- Valentine leukocidin (PVL)-positive ST80-SCCmec IV-SC type XIa - spa type t044-agr group III, and exfoliative toxin D-positive. This strain was susceptible to most antibiotics, but resistant to tetracycline and fusidic acid. This is the first report on the emergence of European ST80 CA-MRSA clone in Korea.

Prevalence and Molecular Characteristics of Methicillin-resistant Staphylococcus aureus Isolates in a Neonatal Intensive Care Unit

The molecular characteristics of methicillin-resistant Staphylococcus aureus (MRSA) isolated from neonates in a neonatal intensive care unit (NICU) were investigated by multilocus sequence typing (MLST), staphylocoagulase (SC) genotyping, staphylococcal cassette chromosome mec (SCCmec) typing, accessory gene regulator (agr) typing, and the presence of Panton-Valentine leukocidin (PVL). Among the 44 S. aureus isolates from nares in neonates between March and June 2014 at hospital in Busan, 27 (61.4%) were MRSA and 17 (38.6%) were methicillin-susceptible S. aureus (MSSA). The most prevalent clone in MRSA isolates was ST72-SC type Vb-SCCmec IV-agr I (n=26) and the remaining one was ST89-SC type I-SCCmec II-agr II. In MSSA isolates, the prevalent clone was ST121-SC type Va-agr IV (n=13), followed by ST72-SC type Vb-agr I (n=2), ST8-SC type III-agr I (n=1) and ST15-SC type X-agr II (n=1). All isolates did not possess the PVL. The data showed that the neonates in NICU carried high prevalence of ST72 MRSA and remarkably different clones with SC diversity between MRSA and MSSA isolates.

Application of Matrix-Assisted Laser Desorption Ionization Time-of-Flight Mass Spectrometry to Screen the Extended-Spectrum β-Lactamase-Producing ST131 Escherichia coli Strains / 대한임상미생물학회지

BACKGROUND: Sequence type 131 (ST131) O25b serogroup Escherichia coli, producing CTX-M type extended-spectrum β-lactamase (ESBL), is a major clone involved in worldwide pandemic spread in both community- and healthcare-associated infections. Recently, matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) has become a routine tool for the identification of bacteria in many laboratories. This study aimed to assess the performance of MALDI-TOF MS for the screening of ESBL-producing E. coli ST131 in a rapid, inexpensive, and simple way. METHODS: A total 26 clinical E. coli, isolated from blood between 2013 and 2014, were used. The characteristics are ST131-O25b ESBL producers (n=6), ST131-O16 ESBL producers (n=4), non-ST131 ESBL producers (n=11), and non-ST131 non-ESBL producers (n=5). Specific biomarker peaks to distinguish the ST131 clonal group from others were investigated by MicroIDSys (ASTA, South Korea) and ASTA Tinkerbell 2.0 software. RESULTS: A peak at 9,713 m/z peak is useful to screen for ST131 E. coli, regardless of serogroup O25 or O16, showing a sensitivity of 100%, specificity of 56.2%, positive predictive value of 58.8%, and negative predictive value of 100% when using a relative intensity cutoff of 15%. CONCLUSION: We can screen for ST131 E. coli using MicroIDSys (ASTA), MALDI-TOF MS in a rapid, inexpensive, and simple way. However, other confirmatory tests are needed to confirm ST131 E. coli due to the low specificity of this method.

Molecular Typing and Resistance Profiles of Vancomycin-Intermediate Staphylococcus aureus in Korea: Results from a National Surveillance Study, 2007-2013 / 대한임상미생물학회지

BACKGROUND: To investigate the national molecular epidemiology and resistance profiles of vancomycin-intermediate Staphylococcus aureus (VISA), we analyzed the characteristics of methicillin-resistant Staphylococcus aureus (MRSA) collected from clinical samples at tertiary or general hospitals participating in a nationwide surveillance program for VISA and vancomycin-resistant Staphylococcus aureus (VRSA) in Korea during an 12-week period in each year from 2007 to 2013. METHODS: VISA was defined by agar dilution, broth dilution and E-test methods with vancomycin minimum inhibitory concentrations of >2 μg/mL. All VISA isolates were characterized by multilocus sequence typing, staphylococcal cassette chromosome mec typing, spa typing, accessory gene regulator typing, Diversilab analysis, and antibiogram analysis. RESULTS: Of 109,345 MRSA isolates, 87,354 were screened and 426 isolates were identified as positive on brain heart infusion agar containing 4 μg/mL vancomycin (BHI-V4). Of 426 isolates, 76 isolates were identified as VISA. No VRSA isolates were detected among the isolates. Overall, a total of 6 genotypes were identified among VISA strains and the predominant clones were ST5-II-t2460, ST72-IV-t324, and ST239-III-t037 (44.7%, 15.8%, and 10.5%, respectively). Of note, ST72-IV-t324 clones are known to be a typical community-associated MRSA. ST239-III-t037 strains were more resistant to trimethoprim-sulfamethoxazole than any other type of strain. ST72-IV-t324 strains were susceptible to all of the antimicrobial agents tested except erythromycin and daptomycin. All of the VISA isolates were susceptible to linezolid and quinupristin-dalfopristin. CONCLUSION: Although VRSA is still rare, continuous monitoring of VRSA occurrence is needed, as well as VISA prevalence, epidemic clonal shift, and antimicrobial resistance.

Molecular Characterization of Community-Associated Methicillin-Resistant and Methicillin-Susceptible Staphylococcus aureus Isolates from Children with Skin Infections in Busan, Korea

The prevalence and molecular characteristics of community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) and methicillin-susceptible S. aureus (CA-MSSA) from children with skin infection were investigated by staphylocoagulase (SC) typing, multilocus sequence typing (MLST), SCCmec typing and virulent toxins, including Panton-Valentine leucocidin (PVL), and exfoliative toxins (ET). Among 69 cases of CA-S. aureus for a 3 month period from March to June, 2014 at hospital in Busan, 28 (40.6%) were MRSA and 41 (59.4%) were MSSA. Of the 28 CA-MRSA isolates, two major clones were identified as SC type Vb-ST72-SCCmec type IV (53.6%) and SC type l-ST89-SCCmec type II variant (42.8%), and the remaining one (3.6%) was SC type lll-ST8-SCCmec type IV. In CA-MSSA, the prevalent clone was SC type Vb-ST72 (29.3%), followed by SC type Vb-ST188 (21.9%), SC type Va-ST121 (19.5%) and SC type lV-ST30 (9.6%). None was positive for PVL gene, and all of the SC type l-ST89-SCCmec type II variant clones were ETB gene positive. The data suggest that there are significant clonal relatedness with specific SC types, and genetic diversities in both community strains isolated from children with skin infections.

Comparison of pulsed-field gel electrophoresis & repetitive sequence-based PCR methods for molecular epidemiological studies of Escherichia coli clinical isolates.

Background & objectives: PFGE, rep-PCR, and MLST are widely used to identify related bacterial isolates and determine epidemiologic associations during outbreaks. This study was performed to compare the ability of repetitive sequence-based PCR (rep-PCR) and pulsed-field gel electrophoresis (PFGE) to determine the genetic relationships among Escherichia coli isolates assigned to various sequence types (STs) by two multilocus sequence typing (MLST) schemes. Methods: A total of 41 extended-spectrum β-lactamase- (ESBL-) and/or AmpC β-lactamase-producing E. coli clinical isolates were included in this study. MLST experiments were performed following the Achtman’s MLST scheme and the Whittam’s MLST scheme, respectively. Rep-PCR experiments were performed using the DiversiLab system. PFGE experiments were also performed. Results: A comparison of the two MLST methods demonstrated that these two schemes yielded compatible results. PFGE correctly segregated E. coli isolates belonging to different STs as different types, but did not group E. coli isolates belonging to the same ST in the same group. Rep-PCR accurately grouped E. coli isolates belonging to the same ST together, but this method demonstrated limited ability to discriminate between E. coli isolates belonging to different STs. Interpretation & conclusions: These results suggest that PFGE would be more effective when investigating outbreaks in a limited space, such as a specialty hospital or an intensive care unit, whereas rep-PCR should be used for nationwide or worldwide epidemiology studies.

Multiple locus sequence typing of Salmonella Typhi, isolated in north India - a preliminary study.

Background & objectives: In India enteric fever is a major public health problem and Salmonella Typhi is the most common aetiologic agent. Any control strategy for such infections depends to a large extent on the understanding of the disease and relatedness of strains across the world. Multi locus sequence typing (MLST) is one such method of genotyping of bacteria based upon housekeeping genes of known function and chromosome position. MLST data of pathogens are important to determine the molecular evolution by a stable and reproducible method. This study was undertaken to determine the sequence types of representatives S. Typhi isolates obtained from enteric fever patients in a tertiary care centre in north India, over a period of 20 years (1990-2010). Methods: A total of 30 representative isolates of S. Typhi identified by biochemical and serological tests were subjected to multi locus sequence typing (MLST). Seven housekeeping genes of known function and chromosome position were used for the typing by MLST. Sequencing was carried out by using an automated DNA sequencer and results were analyzed to generate phylogenetic tree. Results: MLST pattern grouped S. Typhi into two sequence types- ST1 and ST2. ST1 was predominantly present followed by ST2. Interpretation & conclusions: By MLST the presence of both sequence types, ST1 and ST2, was found in S. Typhi isolates in our region. Predominately ST1 was present followed by ST2. These preliminary results corroborate the global distribution of both sequence types of S. Typhi and also emphasize for the continuous screening of S. Typhi.

Multi locus sequence type comparison of invasive and commensal Haemophilus influenzae isolates from Delhi.

Haemophilus influenzae is a major public health concern in the developing world. The most virulent strain is H. influenzae Type b (Hib). Hib also constitutes a major portion of nasopharyngeal commensal flora in otherwise healthy individuals. Through dendogram based on composite gene sequences of seven multi locus sequence type genes, it was observed that invasive and commensal isolates made two completely separate clusters which are indicative of independent evolution of these two groups of H. influenzae in the Indian subcontinent.

Clonal Types and Antimicrobial Susceptibilitiy Patterns in Methicillin-Resistant Staphylococcus aureus Isolates from a Korean Hospital

A total of 54 non-duplicate methicillin-resistant Staphylococcus aureus (MRSA) isolates from clinical specimens and 3 MRSA isolates from healthy medical staffs were obtained from Kyungpook National University Hospital. They were analyzed for clonal types by multilocus sequence typing, protein A gene (spaA) typing, the staphylococcal cassette chromosome mec (SCCmec) typing, and pulsed-field gel electrophoresis (PFGE). The MRSA isolates were examined for antimicrobial susceptibility. Clinical MRSA isolates were classified into 4 clonal complexes, 4 sequence types (STs), 5 spaA types, 4 PFGE patterns, and 3 SCCmec types with variants. On the basis of ST, ST239 (n=25) and ST5 (n=24) were the most frequently encountered. MRSA isolates belonging to ST239 were genotypically homogenous, while those belonging to ST5 showed variations in spaA and SCCmec types. Of the 3 MRSA isolates from healthy medical staffs, one was genotypically identical to MRSA isolates belonging to ST5 and the other two ST239. All MRSA isolates were susceptible to vancomycin and teicoplain. Only 4% of isolates were resistant to rifampin, while 91% of isolates were resistant to ciprofloxacin. The resistance rate of MRSA isolates belonging to ST239 against trimethoprim-sulfamethoxazole (SXT) was significantly higher than that of the isolates belonging to ST5 (76% vs 0%, p<0.001). In summary, ST239 and ST5 were responsible for most MRSA infections and healthy medical staffs also carried these MRSA strains. The susceptibility of the ST239 clone against SXT, which was commonly used for oral therapy to treat MRSA infection, was significantly different from the ST5 clone.