Antibiotic resistance and molecular epidemiology of the beta‐lactamase‐producing Haemophilus influenzae isolated in Chongqing,China

This study aimed to determine the antibiotic resistance and molecular epidemiology of Haemophilus influenzae isolated from children with acute respiratory infection in Chongqing, China. To this end, 1967 H. influenzae isolates from 2006 to 2009 were analysed regarding β‐lactamase production and antibiotic resistance. Ninety‐nine β‐lactamase‐producing H. influenzae isolates from 2010 were analysed for antibiotic resistance and promoter regions of bla_TEM‐1. β‐lactamase production was found in 35.8% (705/1967) of the strains. All ninety‐nine β‐lactamase‐producing strains from 2010 were of the TEM‐1 type as determined by PCR but did not produce the predicted 1075 bp product. According to PCR‐SSCP and DNA sequencing, the promoter regions of bla_TEM‐1 were categorized into 6 genotypes as SSCP1 (Pdel), SSCP2 (Pa/Pb), SSCP3 (P4), SSCP4 (Prpt.b), SSCP5 (2Prpt) and SSCP6 (P3.b). The Pdel, Pa/Pb and Prpt.b were common promoters of bla_TEM‐1 for H. influenzae isolated from children in Chongqing. Strains with Prpt.b were more resistant to ampicillin (AMP) than strains with Pdel, Pa/Pb and P4 (p < 0.05). Therefore, bla_TEM‐1 β‐lactamase is the main mechanism for resistance of H. influenzae to ampicillin in Chongqing. Furthermore, the Prpt.b promoters may be related to the high resistance of H. influenzae to AMP.     

High level of sequence diversity in the 16S rRNA genes of Haemophilus influenzae isolates is useful for molecular subtyping

A molecular typing method based on the 16S rRNA sequence diversity was developed for Haemophilus influenzae isolates. A total of 330 H. influenzae isolates were analyzed, representing a diverse collection of U.S. isolates. We found a high level of 16S rRNA sequence heterogeneity (up to 2.73%) and observed an exclusive correlation between 16S types and serotypes (a to f); no 16S type was found in more than one serotype. Similarly, no multilocus sequence typing (MLST) sequence type (ST) was found in more than one serotype. Our 16S typing and MLST results are in agreement with those of previous studies showing that serotypable H. influenzae isolates behave as highly clonal populations and emphasize the lack of clonality of nontypable (NT) H. influenzae isolates. There was not a 1:1 correlation between 16S types and STs, but all H. influenzae serotypable isolates clustered similarly. This correlation was not observed for NT H. influenzae; the two methods clustered NT H. influenzae isolates differently. 16S rRNA gene sequencing alone provides a level of discrimination similar to that obtained with the analysis of seven genes for MLST. We demonstrated that 16S typing is an additional and complementary approach to MLST, particularly for NT H. influenzae isolates, and is potentially useful for outbreak investigation.     

Dominance of International High-Risk Clones in Carbapenemase-Producing Pseudomonas aeruginosa: Multicentric Molecular Epidemiology Report from India

Background: Pseudomonas aeruginosa is one of the most common opportunistic pathogens that cause severe infections in humans. The burden of carbapenem resistance is particularly high and is on the rise. Very little information is available on the molecular mechanisms and its clonal types of carbapenem-resistant P. aeruginosa seen in Indian hospitals. This study was undertaken to monitor the β-lactamase profile and to investigate the genetic relatedness of the carbapenemase-producing (CP) P. aeruginosa collected across different hospitals from India. Materials and Methods: A total of 507 non-duplicate, carbapenem-resistant P. aeruginosa isolated from various clinical specimens collected during 2014–2017 across seven Indian hospitals were included. Conventional multiplex polymerase chain reaction for the genes encoding beta-lactamases such as extended-spectrum beta-lactamase (ESBL) and carbapenemase were screened. A subset of isolates (n = 133) of CP P. aeruginosa were genotyped by multilocus sequence typing (MLST) scheme. Results: Of the total 507 isolates, 15%, 40% and 20% were positive for genes encoding ESBLs, carbapenemases and ESBLs + carbapenemases, respectively, whilst 25% were negative for the β-lactamases screened. Amongst the ESBL genes, bla_VEB is the most predominant, followed by bla_PER and bla_TEM, whilst bla_VIM and bla_NDM were the most predominant carbapenemases seen. However, regional differences were noted in the β-lactamases profile across the study sites. Genotyping by MLST revealed 54 different sequence types (STs). The most common are ST357, ST235, ST233 and ST244. Six clonal complexes were found (CC357, CC235, CC244, CC1047, CC664 and CC308). About 24% of total STs are of novel types and these were found to emerge from the high-risk clones. Conclusion: This is the first large study from India to report the baseline data on the molecular resistance mechanisms and its association with genetic relatedness of CP P. aeruginosa circulating in Indian hospitals. bla_VIM- and bla_NDM-producing P. aeruginosa is the most prevalent carbapenemase seen in India. Majority of the isolates belongs to the high-risk international clones ST235, ST357 and ST664 which is a concern.     

Phenotypic and molecular characterization of beta-lactam resistance and capsular typing of colonizing Haemophilus influenzae strains isolated from neutropenic patients in Tunisia

Objective

The aim of this study was to determine the overall percentage of β-lactams susceptibility, beta-lactamase production, penicillin binding protein (PBP) modification and serotypes of colonizing Haemophilus influenzae strains.

Design

A total of 50 isolates of colonized H. influenzae, isolated from neutropenic patients. The prevalence of β-lactams resistance and β-lactamase production were recorded for each strains using E-test strips and chromogenic cephalosporin test, then were determined their resistance genes (bla_TEM and bla_ROB) by PCR as well as their capsular types by standard slide agglutination serotyping (SAST) and capsular genes amplification.

Results

Thirty-two percent of the 50 strains were amoxicillin resistant, among these, 20% were resistant by beta-lactamase production, and they produced all type TEM β-lactamase. Four percent of the isolates had PBP modification and three strains (6%) associated the two resistance mechanisms. Slide agglutination serotyping showed that 95.8% of the strains were unencapsulated, and 4.1% were of serogroup b. The result was confirmed by PCR capsular typing.

Conclusion

By the light of these results, our findings suggest that it becomes important to follow the evolution of the resistance background of our strains, and that the majority of colonizing H. influenzae strains isolated in our center are unencapsulated.     

Characteristics of Haemophilus influenzae invasive isolates from Portugal following routine childhood vaccination against H. influenzae serotype b (2002–2010)

We aimed to characterize Haemophilus influenzae invasive isolates recovered in Portugal over a 9-year period (2002–2010) following the inclusion of H. influenzae serotype b (Hib) conjugate vaccination in the National Immunization Program (NIP) in the year 2000 and compare the results with those obtained in a similar study from the pre-vaccination era (1989–2001) previously described by us. As part of a laboratory-based passive surveillance system, 144 invasive isolates obtained in 28 Portuguese hospitals were received at the National Reference Laboratory for Bacterial Respiratory Infections and were characterized. Capsular types and antibiotic susceptibility patterns were determined. The ftsI gene encoding PBP3 was sequenced for β-lactamase-negative ampicillin-resistant (BLNAR) isolates. Genetic relatedness among isolates was examined by multilocus sequencing typing (MLST). Most isolates (77.1 %) were non-capsulated, a significant increase compared to the pre-vaccination era (19.0 %, p?<?0.001). Serotype b strains decreased significantly (from 81.0 to 13.2 %, p?<?0.001) and serotype f increased significantly (from 0.8 to 6.9 %, p?=?0.03). Ten percent of the isolates were β-lactamase producers, a value lower than that previously observed (26.9 %, p?=?0.005). Eight percent of all isolates were BLNAR. A high genetic diversity among non-capsulated isolates was found. By contrast, capsulated isolates were clonal. The implementation of Hib vaccination has resulted in a significant decline in the proportion of serotype b H. influenzae invasive disease isolates. Most episodes of invasive disease occurring in Portugal are now due to fully susceptible, highly diverse, non-capsulated strains. Given the evolving dynamics of this pathogen and the increase in non-type b capsulated isolates, continuous surveillance is needed.     

Epidemiological characterization and distribution of carbapenem-resistant Acinetobacter baumannii clinical isolates in Italy

This study was aimed at tracing the molecular characteristics of carbapenem-resistant Acinetobacter baumannii (CRAB) clinical isolates in Italy with both pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). Two hundred and two CRAB isolates were collected during 2004–2009, in two different surveillance periods, from 22 Italian hospitals that were representative for both distribution and infection. PFGE was performed, and the MLST scheme used was based on the gene sequence as published on the MLST Pasteur website http://www.pasteur.fr/mlst. Representatives of the major European clones I (RUH 875) and II (RUH 134) were used as controls. The two groups of isolates were characterized for their carbapenem resistance genes: 154 of 202 carried bla_OXA-58 alone, 21 of 202 also carried bla_OXA-23, and 27 of 202 carried bla_OXA-23 alone. No isolates were positive for bla_OXA-24. Genotype analysis of all isolates identified four distinct patterns by PFGE, which correlated with four distinct sequence types (STs) by MLST. The distribution of these four clusters in Italy confirmed the propensity of A. baumannii for nosocomial cross-transmission in a vast geographical area. We observed that clones A and B had similarities with European clone II and I respectively. By MLST, clone A was ST2, like European clone II, and clone B was ST1, like European clone I. PFGE and MLST showed the same discriminatory power and reproducibility. In addition, the two methods were concordant in defining CRAB Italian clones and in correlating them with the two pan-European clones.     

Similar Frequencies of Pseudomonas aeruginosa Isolates Producing KPC and VIM Carbapenemases in Diverse Genetic Clones at Tertiary-Care Hospitals in Medellín,Colombia

Carbapenem-resistant Pseudomonas aeruginosa has become a serious health threat worldwide due to the limited options available for its treatment. Understanding its epidemiology contributes to the control of antibiotic resistance. The aim of this study was to describe the clinical and molecular characteristics of infections caused by carbapenem-resistant P. aeruginosa isolates in five tertiary-care hospitals in Medellín, Colombia. A cross-sectional study was conducted in five tertiary-care hospitals from June 2012 to March 2014. All hospitalized patients infected by carbapenem-resistant P. aeruginosa were included. Clinical information was obtained from medical records. Molecular analyses included PCR for detection of bla_VIM, bla_IMP, bla_NDM, bla_OXA-48, and bla_KPC genes plus pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST) for molecular typing. A total of 235 patients were enrolled: 91.1% of them were adults (n = 214), 88.1% (n = 207) had prior antibiotic use, and 14.9% (n = 35) had urinary tract infections. The bla_VIM-2 and bla_KPC-2 genes were detected in 13.6% (n = 32) and 11.5% (n = 27), respectively, of all isolates. Two isolates harbored both genes simultaneously. For KPC-producing isolates, PFGE revealed closely related strains within each hospital, and sequence types (STs) ST362 and ST235 and two new STs were found by MLST. With PFGE, VIM-producing isolates appeared highly diverse, and MLST revealed ST111 in four hospitals and five new STs. These results show that KPC-producing P. aeruginosa is currently disseminating rapidly and occurring at a frequency similar to that of VIM-producing P. aeruginosa isolates (approximately 1:1 ratio) in Medellín, Colombia. Diverse genetic backgrounds among resistant strains suggest an excessive antibiotic pressure resulting in the selection of resistant strains.     

Haemophilus influenzae in children with cystic fibrosis: antimicrobial susceptibility, molecular epidemiology, distribution of adhesins and biofilm formation

Haemophilus influenzae commonly infects the respiratory tract of patients with cystic fibrosis (CF), early in childhood. In this investigation, 79 H. influenzae isolates were recovered from the respiratory secretions of 64 CF patients (median age: 5 years) included in a 5-year follow-up study. Fifteen of the 64 patients contributed two or more H. influenzae isolates overtime. Serotyping, antibiotic susceptibility testing, genotyping, detection of both hmwA and hia adhesin genes and hypermutable strains was carried out. Biofilm formation ability was investigated. Most strains (72/79, 91.2%) were nonencapsulated or nontypeable (NTHi). Resistance to ampicillin (13.9%) and imipenem (17.7%) was the most detected. Few isolates (2.5%) exhibited the hypermutable phenotype. The NTHi strains showed 55 different genotypes, but 19 clusters of closely related strains were identified. Nine clusters included strains that cross-colonised several patients over a long-time period (mean: 3.7 years). Most patients with sequential isolates harboured strains genetically unrelated, but persistent colonisation with the same clone was observed in 37.5% of patients. Over 45% of NTHi strains contained hmwA-related sequences, 26.3%, hia, 8.3% both hmwA and hia, while 19.4% lacked both. A significant association was found between occurrence of an adhesive gene (irrespective of which) and both persistence (P < 0.0001) and long-term cross-colonisation (P < 0.0001). Mean biofilm level formed by the persistent strains was found significantly increased compared to non-persistent ones (P < 0.0001). Hia-positive strains produced significantly more biofilm than hmwA-carrying strains (P < 0.01). Although a high turnover of NTHi strains in FC patients was observed, distinct clones with increased capacity of persistence or cross-colonisation occurred.     

Genotyping,local prevalence and international dissemination of β-lactamase-producing Kingella kingae strains

β-lactamase production has been sporadically reported in the emerging Kingella kingae pathogen but the phenomenon has not been studied in-depth. We investigated the prevalence of β-lactamase production among K. kingae isolates from different geographical origins and genetically characterized β-lactamase-producing strains. Seven hundred and seventy-eight isolates from Iceland, the USA, France, Israel, Spain and Canada were screened for β-lactamase production and, if positive, were characterized by PFGE and MLST genotyping, as well as rtxA, por, bla_TEM and 16S rRNA sequencing. β-lactamase was identified in invasive strains from Iceland (n = 4/14, 28.6%), the USA (n = 3/15, 20.0%) and Israel (n = 2/190, 1.1%) and in carriage strains in the USA (n = 5/17, 29.4%) and Israel (n = 66/429, 15.4%). No French, Spanish or Canadian isolates were β-lactamase producers. Among β-lactamase producers, a perfect congruency between the different typing methods was observed. Surprisingly, all US and Icelandic β-lactamase-producing isolates were almost indistinguishable, belonged to the major international invasive PFGE clone K/MLST ST-6, but differed from the four genetically unrelated Israeli β-lactamase-producing clones. Representative strains of different genotypes produced the TEM-1 enzyme. K. kingae β-lactamase producers exhibit a clear clonal distribution and have dissimilar invasive potential. The presence of the enzyme in isolates belonging to the major worldwide invasive clone K/ST-6 highlights the possible spread of β-lactam resistance, and emphasizes the importance of routine testing of all K. kingae clinical isolates for β-lactamase production.     

Low Genetic Diversity of Haemophilus influenzae Type b Compared to Nonencapsulated H. influenzae in a Population in Which H. influenzae Is Highly Endemic

Immunization with Haemophilus influenzae type b (Hib) conjugate polysaccharide vaccines has dramatically reduced Hib disease worldwide. As in other populations, nasopharyngeal carriage of Hib declined markedly in Aboriginal infants following vaccination, although carriage has not been entirely eliminated. In this study, we describe the genetic characteristics and the carriage dynamics of longitudinal isolates of Hib, characterized by using several typing methods. In addition, carriage rates of nonencapsulated H. influenzae (NCHi) are high, and concurrent colonization with Hib and NCHi is common; we also observed NCHi isolates which were genetically similar to Hib. There is a continuing need to promote Hib immunization and monitor H. influenzae carriage in populations in which the organism is highly endemic, not least because of the possibility of genetic exchange between Hib and NCHi strains in such populations.     

Haemophilus influenzae type b carriage and novel bacterial population structure among children in urban Kathmandu, Nepal

Haemophilus influenzae type b (Hib) is a major cause of invasive bacterial infection in children that can be prevented by a vaccine, but there is still uncertainty about its relative importance in Asia. This study investigated the age-specific prevalence of Hib carriage and its molecular epidemiology in carriage and disease in Nepal. Oropharyngeal swabs were collected from children in Kathmandu, Nepal, from 3 different settings: a hospital outpatient department (OPD), schools, and children's homes. Hib was isolated using Hib antiserum agar plates, and serotyping was performed with latex agglutination. Hib isolates from children with invasive disease were obtained during active microbiological surveillance at Patan Hospital, Kathmandu, Nepal. Genotyping of disease and carriage isolates was undertaken using multilocus sequence typing (MLST). Swabs were taken from 2,195 children, including 1,311 children at an OPD, 647 children attending schools, and 237 children in homes. Overall, Hib was identified in 5.0% (110/2,195; 95% confidence interval [95% CI], 3.9% to 6.4%). MLST was performed on 108 Hib isolates from children carrying Hib isolates and 15 isolates from children with invasive disease. Thirty-one sequence types (STs) were identified, and 20 of these were novel STs. The most common ST isolates were sequence type 6 (ST6) and the novel ST722. There was marked heterogeneity among the STs from children with disease and children carrying Hib. STs identified from invasive infections were those commonly identified in carriage. This study provides evidence of Hib carriage among children in urban Nepal with genetically diverse strains prior to introduction of universal vaccination. The Hib carriage rate in Nepal was similar to the rates observed in other populations with documented high disease rates prior to vaccination, supporting implementation of Hib vaccine in Nepal in 2009.     

Single-Locus-Sequence-Based Typing of blaOXA-51-like Genes for Rapid Assignment of Acinetobacter baumannii Clinical Isolates to International Clonal Lineages

Single-locus bla_OXA-51-like sequence-based typing (SBT) was evaluated for its ability to determine correctly sequence types (STs) in Acinetobacter baumannii clinical isolates, in comparison with the Pasteur''s multilocus sequence typing (MLST) reference method and 3-locus sequence typing (3-LST). The comparative study was performed in 585 multidrug-resistant (MDR) A. baumannii clinical isolates recovered from 21 hospitals located throughout Greece, Italy, Lebanon, and Turkey. The isolates belonged to nine clonal complexes (CCs) that correspond to 12 distinct sequence types (STs) and to one singleton ST. These clonal lineages predominate worldwide among nosocomial MDR A. baumannii strains. The most common clone was CC2 (ST2 and ST45; n = 278 isolates) followed by CC1 (ST1 and ST20; n = 155), CC25 (n = 65), ST78 (n = 62), CC15 (ST15 and ST84; n = 9), CC10 (n = 4), CC3 (n = 4), CC6 (n = 3), CC54 (n = 3), and CC83 (n = 2). Using the bla_OXA-51-like SBT method, all 585 isolates of the study were typed and assigned correctly to the nine CCs and the singleton ST78. The 3-LST method was not able to classify isolates belonging to CC6, CC10, CC54, and CC83, which are not yet characterized in its database. The low-cost and convenient bla_OXA-51-like SBT method, compared with 3-LST and MLST, discriminated all epidemic and sporadic lineages of our collection and could be effectively applied to type rapidly A. baumannii strains.     

Homology Analysis of Pathogenic Yersinia Species Yersinia enterocolitica,Yersinia pseudotuberculosis,and Yersinia pestis Based on Multilocus Sequence Typing

We developed a multilocus sequence typing (MLST) scheme and used it to study the population structure and evolutionary relationships of three pathogenic Yersinia species. MLST of these three Yersinia species showed a complex of two clusters, one composed of Yersinia pseudotuberculosis and Yersinia pestis and the other composed of Yersinia enterocolitica. Within the first cluster, the predominant Y. pestis sequence type 90 (ST90) was linked to Y. pseudotuberculosis ST43 by one locus difference, and 81.25% of the ST43 strains were from serotype O:1b, supporting the hypothesis that Y. pestis descended from the O:1b serotype of Y. pseudotuberculosis. We also found that the worldwide-prevalent serotypes O:1a, O:1b, and O:3 were predominated by specific STs. The second cluster consisted of pathogenic and nonpathogenic Y. enterocolitica strains, two of which may not have identical STs. The pathogenic Y. enterocolitica strains formed a relatively conserved group; most strains clustered within ST186 and ST187. Serotypes O:3, O:8, and O:9 were separated into three distinct blocks. Nonpathogenic Y. enterocolitica STs were more heterogeneous, reflecting genetic diversity through evolution. By providing a better and effective MLST procedure for use with the Yersinia community, valuable information and insights into the genetic evolutionary differences of these pathogens were obtained.     

Characterisation of invasive Haemophilus influenzae isolates in Slovenia, 1993–2008

The objectives of our study were to describe the epidemiology of invasive Haemophilus influenzae disease from 1993 to 2008 in Slovenia, a country with routine H. influenzae serotype b (Hib) conjugate vaccination since the year 2000. A total of 292 isolates of H. influenzae, recovered from a normally sterile site, were collected in the study period. The isolates were serotyped by slide agglutination and antibiotic susceptibility was determined. One hundred and eight isolates received after the year 2000 were serotyped by slide agglutination and by polymerase chain reaction (PCR) capsule typing, and both methods were compared. After the introduction of the routine Hib vaccination, the incidence of H. influenzae disease in children under the age of 5 years has decreased by 87.6% and type b was replaced by non-typeable H. influenzae as the predominant serotype. The proportion of serotype b decreased from 85.3% in the pre-vaccination period to 13.0% in the vaccination period and the proportion of non-capsulated isolates increased from 12.0 to 85.2%. The study of genetic relatedness by pulsed-field gel electrophoresis (PFGE) demonstrated that the isolates of serotypes b and f were genetically homogeneous within the serotype. The results of our national surveillance showed that the vaccine has been very efficient in preventing Hib invasive disease in Slovenia. Nevertheless, we see a need for further monitoring of invasive H. influenzae infections at a national level.     

Characteristics of Haemophilus influenzae type b responsible for meningitis in Poland from 1997 to 2004

Two hundred forty-five H. influenzae isolates responsible for meningitis in Poland from 1997 to 2004 were studied. Among these, 233 (95.1%) belonged to serotype b (Hib), 2 belonged to serotype f, and 10 were noncapsulated. The relatedness of all isolates was evaluated by pulsed-field gel electrophoresis (PFGE), and selected representatives were evaluated by multilocus sequence typing. Resistance to ampicillin was identified in 34 (14.6%) of the Hib isolates and was associated with the production of beta-lactamase only. Except for four isolates nonsusceptible to chloramphenicol, all isolates were susceptible to cefotaxime, ciprofloxacin, and rifampin. The PFGE analysis divided the Hib isolates into five PFGE types; however, all of them were possibly related. The most common PFGE type, with 25 subtypes, was characteristic for 97.4% of the isolates. The most prevalent PFGE subtype found in our study was also the most common among the Hib isolates responsible for invasive disease in Italy and the Czech Republic and was found among isolates causing lower respiratory tract infections in Poland. The most prevalent sequence types (STs) in the studied group were ST6 and ST92. Four new STs were found: ST188, ST189, ST190, and ST268. Results of this study support the evidence that the genetic structure of encapsulated H. influenzae is clonal. The continuing high number of meningitis cases due to Hib in Poland underlines the need for mass vaccination against Hib in Poland.     

Emergence of NDM-producing non-baumannii Acinetobacter spp. isolated from China

One hundred and thirty-six bla _OXA-51-negative strains were identified from 1,067 Acinetobacter calcoaceticus–A. baumannii complex (ACB complex) isolates, which were collected during October 2010 to March 2013 from 15 general hospitals in 10 cities throughout Zhejiang Province, China. Seven of the 136 bla _OXA-51-negative ACB complex isolates were New Delhi metallo-β-lactamase-1 (NDM-1)-positive, among which three were identified as A. nosocomialis and four were identified as A. pittii strains using 16S–23S rRNA gene intergenic spacer (ITS) sequencing and partial RNA polymerase β-subunit (rpoB) sequencing. Pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST) analysis showed that the seven NDM-positive isolates belonged to three clonal strains with three novel sequence types (STs). Polymerase chain reaction (PCR) assays and DNA sequence analysis of the carbapenemase and other β-lactamase genes indicated that all the isolates harbored the bla _NDM-1 gene, and that only one strain of A. nosocomialis isolates harbored both bla _NDM-1 and bla _OXA-23. All of them were positive for bla _ADC, from which three novel bla _ADC genes (designated as bla _ADC-69, bla _ADC-70, and bla _ADC-71) were detected for the first time. The presence of ISAba125 upstream of bla _NDM-1 was identified through genetic environment analysis. Carbapenem resistance can be transferred from A. nosocomialis and A. pittii to Escherichia coli EC600 by the conjugation experiment. Plasmid analysis, DNA hybridization, and extraction experiments indicated that bla _NDM-1 was located on a plasmid of approximately 50 kb. In conclusion, we characterized the dissemination of NDM-1-positive A. pittii strains in Zhejiang Province, China, and reported the NDM-producing A. nosocomialis for the first time.     

Epidemiological characterisation of Streptococcus pneumoniae from India using multilocus sequence typing

Objective: The aim of this study was to utilize the multilocus sequence typing (MLST) technique to characterise Streptococcus pneumoniae among clinical isolates in India. MLST was used to determine clonality, to establish genetic relatedness, to check for correlation between serotypes and sequence types (STs) and its relevance associated with antibiotic resistance. Methods: Forty consecutive invasive S. pneumoniae isolates in children <5 years were characterised. Preliminary identification of serotype and antibiotic susceptible profile was followed with MLST technique to identify the STs of the isolates. STs were then analysed for clonality using an eBURST algorithm and genetic relatedness using Sequence Type Analysis and Recombinational Tests version 2 software. Results: The most common ST was ST63. Among the forty isolates, we identified nine novel STs, six of which had known alleles but in new combinations, three of which had new alleles in their sequence profile. The new STs assigned were 8501–8509. One clonal complex was found among the 40 strains characterised. The most common serotypes in this study were serotype 19F, 14 and 5. Non-susceptibility to penicillin and erythromycin was observed in 2.5% and 30% of the isolates, respectively. Conclusion: This study shows a significant number of novel STs among the 40 isolates characterised (9/40, 22.5%), however, internationally recognised strains were also circulating in India, indicating, there could be greater geographical variation in pneumococcal STs in India. Molecular epidemiology data is essential to understand the population dynamics of S. pneumoniae in India before the introduction of pneumococcal vaccines in NIP in India.     

Multiclonal epidemic of Klebsiella pneumoniae isolates producing DHA-1 in a Spanish hospital

Between June 2007 and January 2008, 26 Klebsiella pneumoniae isolates carrying bla_DHA-1 on an IncL/M plasmid were obtained from clinical samples at Granollers Hospital, Barcelona, Spain. Three of the isolates also carried a bla_CTX-M-15 gene. The 26 isolates showed 11 pulsed-field gel electrophoresis (PFGE) patterns. Multilocus sequence typing showed that PFGE patterns A, B and C belonged to sequence type (ST)17, D to ST13, E to ST427, F and G to ST416, H to ST37, I to ST440, J to ST326, and K to ST428. Results demonstrated the effectiveness of the infection control programme in place at the centre. This study reports the first characterization of STs for bla_DHA-1-producing K. pneumoniae isolates.     

Copy Number of Pilus Gene Clusters in Haemophilus influenzae and Variation in the hifE Pilin Gene

Brazilian purpuric fever (BPF)-associated Haemophilus influenzae biogroup aegyptius strain F3031 contains two identical copies of a five gene cluster (hifA to hifE) encoding pili similar to well-characterized Hif fimbriae of H. influenzae type b. HifE, the putative pilus tip adhesin of F3031, shares only 40% amino acid sequence similarity with the same molecule from type b strains, whereas the other four proteins have 75 to 95% identity. To determine whether pilus cluster duplication and the hifE_F3031 allele were special features of BPF-associated bacteria, we analyzed a collection of H. influenzae strains by PCR with hifA- and hifE-specific oligonucleotides, by Southern hybridization with a hifC gene probe, and by nucleotide sequencing. The presence of two pilus clusters was limited to some H. influenzae biogroup aegyptius strains. The hifE_F3031 allele was limited to H. influenzae biogroup aegyptius. Two strains contained one copy of hifE_F3031 and one copy of a variant hifE allele. We determined the nucleotide sequences of four hifE genes from H. influenzae biogroup aegyptius and H. influenzae capsule serotypes a and c. The predicted proteins produced by these genes demonstrated only 35 to 70% identity to the three published HifE proteins from nontypeable H. influenzae, serotype b, and BPF strains. The C-terminal third of the molecules implicated in chaperone binding was the most highly conserved region. Three conserved domains in the otherwise highly variable N-terminal putative receptor-binding region of HifE were similar to conserved portions in the N terminus of Neisseria pilus adhesin PilC. We concluded that two pilus clusters and hifE_F3031 were not specific for BPF-causing H. influenzae, and we also identified portions of HifE possibly involved in binding mammalian cell receptors.     

Immunologic and Structural Relationships of the Minor Pilus Subunits among Haemophilus influenzae Isolates

Two proteins, HifD and HifE, have been identified as structural components of Haemophilus influenzae pili. Both are localized at the pilus tip, and HifE appears to mediate pilus adherence to host cells. In this study we examined the immunologic and structural diversity of these pilus subunits among type b H. influenzae (Hib) and nontypeable H. influenzae (NTHI) strains. Western immunoblot analysis revealed that antibodies directed against the C terminus of HifD and HifE from Hib strain Eagan bound to HifD and HifE proteins, respectively, of all piliated Hib and NTHI strains tested. Whole-cell enzyme-linked immunosorbent assays showed that antibodies specific for native HifD or HifE of strain Eagan also bound to all piliated Hib strains but did not bind to the piliated NTHI strains. Antibodies against HifE of strain Eagan inhibited the binding of Hib to human erythrocytes but did not inhibit the binding of NTHI strains. Restriction fragment length polymorphism (RFLP) analysis was used to determine strain-to-strain structural differences within hifD and hifE genes, either by PCR or by nucleotide sequence analysis. DNA and derived amino acid sequence analyses of HifD and HifE confirmed the uniqueness of the RFLP types. The hifD and hifE genes of all type b strains showed identical restriction patterns. Analysis of hifD and hifE genes from the NTHI strains, however, revealed seven unique RFLP patterns, suggesting that these genes encode proteins with diverse primary structures. These results indicate that HifD and HifE are immunologically and structurally similar among the Hib strains but vary among the NTHI strains.