"Association of periodontopathic anaerobic bacterial co-occurrence to atherosclerosis" - A cross-sectional study.

BACKGROUND: Epidemiological studies have shown a link between periodontitis and atherosclerosis. Hence the present study was chosen to assess the presence of eight anaerobic periodontal pathogens and their virulence genes in subgingival plaque (SGP) and atheromatous plaque (AP) of patients with Ischaemic heart disease. METHODS: SGP and AP collected from 65 Ischaemic heart disease patients were screened for the presence of periodontal bacterial pathogens by Polymerase chain reaction. The samples positive for Porphyromonas gingivalis, Treponema denticola and Tannerella forsythia were screened for virulence genes. Chronic periodontitis patients (ChP) without any systemic disease (n = 59) and healthy subjects free of both periodontitis and systemic disease were included as control groups (n = 100). RESULTS AND DISCUSSION: Statistical significance was observed for the prevalence of 16S rRNA of P. gingivalis, T. forsythia, T. denticola and P. nigrescens both in SGP and AP. Nine different periodontal bacterial co-occurrences were observed in SGP and AP of Ischaemic heart disease patients. Besides, the prevalence of these nine different bacterial co-occurrence was high in SGP OF Ischaemic heart disease patients compared to ChP without systemic disease. Among the nine different bacterial co-occurrence, only four were observed in SGP of ChP without systemic disease in spite of high prevalence of these anaerobic bacterial species. While, bacterial co-occurrences was completely absent among healthy subjects. Significant odds and risk ratio to atherosclerosis were observed for P. gingivalis, T. forsythia, T. denticola and P. nigrescens. Among the virulence genes, significance to atherosclerosis was observed for P. gingivalis type II fimA and T. forsythia bspA. CONCLUSION: The results of this study strongly correlate periodontal bacterial co-occurrence and periodontal bacterial adhesion factor to atherosclerosis.

Distribution and Relationships of Antimicrobial Resistance Determinants among Extended-Spectrum-Cephalosporin-Resistant or Carbapenem-Resistant Escherichia coli Isolates from Rivers and Sewage Treatment Plants in India.

To determine the distribution and relationship of antimicrobial resistance determinants among extended-spectrum-cephalosporin (ESC)-resistant or carbapenem-resistant Escherichia coli isolates from the aquatic environment in India, water samples were collected from rivers or sewage treatment plants in five Indian states. A total of 446 E. coli isolates were randomly obtained. Resistance to ESC and/or carbapenem was observed in 169 (37.9%) E. coli isolates, which were further analyzed. These isolates showed resistance to numerous antimicrobials; more than half of the isolates exhibited resistance to eight or more antimicrobials. The blaNDM gene was detected in 14/21 carbapenem-resistant E. coli isolates: blaNDM-1 in 2 isolates, blaNDM-5 in 7 isolates, and blaNDM-7 in 5 isolates. The blaCTX-M gene was detected in 112 isolates (66.3%): blaCTX-M-15 in 108 isolates and blaCTX-M-55 in 4 isolates. We extracted 49 plasmids from selected isolates, and their whole-genome sequences were determined. Fifty resistance genes were detected, and 11 different combinations of replicon types were observed among the 49 plasmids. The network analysis results suggested that the plasmids sharing replicon types tended to form a community, which is based on the predicted gene similarity among the plasmids. Four communities each containing from 4 to 17 plasmids were observed. Three of the four communities contained plasmids detected in different Indian states, suggesting that the interstate dissemination of ancestor plasmids has already occurred. Comparison of the DNA sequences of the blaNDM-positive plasmids detected in this study with known sequences of related plasmids suggested that various mutation events facilitated the evolution of the plasmids and that plasmids with similar genetic backgrounds have widely disseminated in India.

High Prevalence of Exfoliative Toxins Among Carrier Isolates of Staphylococcus aureus from Healthy Individuals from Various Communities in Chennai, South India.

Staphylococcus aureus causes infections both in community and hospital settings, nasal carriage is the important source of these infections. A total of 103 carrier isolates of S. aureus from 352 asymptomatic individuals were screened for methicillin-resistant S. aureus (MRSA) and exfoliative toxins (A, B and D) by two sets of multiplex PCRs. The overall nasal carriage of MRSA was found to be 13/352 (3.7 %), of which 4 were found to be positive for Panton valentine leucocidin (PVL). Twelve (11.65 %) strains were found to carry exfoliative toxins and belonged to one of the following spa types t159, t209 and t1515. High prevalence of exfoliative toxins, pvl and MRSA pose a major threat to public health, since the isolates were from the healthy in various community settings.

Mosquito Larvicidal Activity of Compounds from Unripe Fruit Peel of Avocado (Persea americana Mill.).

Mosquitoes are important vectors responsible for spreading a number of diseases affecting both humans and animals. Many diseases as dengue, chikungunya, yellow fever, malaria, filariasis and Japanese encephalitis are spread by mosquitoes. There are many reports of plant extracts and their active constituents showing anti-mosquito activities as larvicidal, pupicidal, ovicidal and adulticidal activities. Persea americana Mill. (Lauraceae), known as avocado, has been reported to show many pharmacological and antimicrobial activities. In this communication, the mosquito larvicidal activities of the three-active constituents, avocadene, avocadyne and avocadenol-A, from the methanolic extract of the unripe fruit peel are presented. The three mosquito species studied were Aedes aegypti, Culex quinquefasciatus and Anopheles stephensi. All three compounds showed the highest larvicidal activity against An. stephensi, LC50 values being 2.80ppm for avocadene, 2.33ppm for avocadyne and 2.07ppm for avocadenol-A. Avocadene showed larvicidal activity of 3.73ppm against Ae. aegypti and 5.96ppm against Cx. quinquefasciatus. The LC50 value of avocadyne was 5.35ppm against Ae. aegypti and 3.98ppm against Cx. quinquefasciatus. Similarly, avocadenol-A showed 6.56ppm against Ae. aegypti and 2.35ppm against Cx. quinquefasciatus. The active constituents were isolated by bioactivity-guided fractionation by silica gel column chromatography and RP HPLC. The compounds were identified by physical and spectroscopic data and compared with literature values already reported.

AmpC ß-lactamases in nosocomial isolates of Klebsiella pneumoniae from India.

BACKGROUND & OBJECTIVES: AmpC ß-lactamases are clinically significant since these confer resistance to cephalosporins in the oxyimino group, 7-α methoxycephalosporins and are not affected by available ß-lactamase inhibitors. In this study we looked for both extended spectrum ß-lactamases (ESBL) and AmpC ß-lactamases in Klebsiella pneumoniae clinical isolates. METHODS: One hundred consecutive, non-duplicate clinical isolates of K. pneumoniae collected over a period of one year (June 2008 - June 2009) were included in the study. An antibiotic susceptibility method was used with 10 antibiotics for Gram-negative infections which helped in screening for ESBL and AmpC ß-lactamases and also in confirmation of ESBL production. The detection of AmpC ß-lactamases was done based on screening and confirmatory tests. For screening, disc diffusion zones of cefoxitin <18 mm was taken as cefoxitin resistant. All cefoxitin resistant isolates were tested further by AmpC disk test and modified three dimensional test. Multiplex-PCR was performed for screening the presence of plasmid-mediated AmpC genes. RESULTS: Of the 100 isolates of K. pneumoniae studied, 48 were resistant to cefoxitin on screening. AmpC disk test was positive in 32 (32%) isolates. This was also confirmed with modified three dimensional test. Indentation indicating strong AmpC producer was observed in 25 isolates whereas little distortion (weak AmpC) was observed in 7 isolates. ESBL detection was confirmed by a modification of double disk synergy test in 56 isolates. Cefepime was the best cephalosporin in synergy with tazobactam for detecting ESBL production in isolates co-producing AmpC ß-lactamases. The subsets of isolates phenotypically AmpC ß-lactamase positive were subjected to amplification of six different families of AmpC gene using multiplex PCR. The sequence analysis revealed 12 CMY-2 and eight DHA-1 types. INTERPRETATION & CONCLUSIONS: Tazobactam was the best ß-lactamase inhibitor for detecting ESBL in presence of AmpC ß-lactamase as this is a very poor inducer of AmpC gene. Amongst cephalosporins, cefepime was the best cephalosporin in detecting ESBL in presence of AmpC ß-lactamase as it is least hydrolyzed by AmpC enzymes. Cefepime-tazobactam combination disk test would be a simple and best method in detection of ESBLs in Enterobacteriaceae co-producing AmpC ß-lactamase in the routine diagnostic microbiology laboratories.

Intestinal carriage of colistin resistant Enterobacteriaceae in hospitalized patients from an Indian center.

There is limited data on the gut colonization rate of colistin resistant (Col-R) bacteria in patients and healthy volunteers in India. Aim of this study was to investigate the stool carriage rate of Col-R in hospitalized patients. Stool samples were inoculated in Eosin Methylene Blue agar plates supplemented with colistin. Colistin minimum inhibitory concentrations (MICs) were determined by the broth microdilution method. PCR for the mcr-1 was performed on Col-R Enterobacteriaceae isolates. Mutations in the mgrB gene were analyzed in K. pneumoniae isolates. Mcr-1 positive E. coli was subjected to whole-genome sequencing. Out of 65 stool samples screened, 33 (51%) samples carried Col-R bacteria. Majority (76.7%) of the isolates were sensitive to carbapenem.

Clonal clusters and virulence factors of methicillin-resistant Staphylococcus Aureus: Evidence for community-acquired methicillin-resistant Staphylococcus Aureus infiltration into hospital settings in Chennai, South India.

Background and Objective: Staphylococcus aureus is one of the major pathogens of nosocomial infections as wells as community-acquired (CA) infections worldwide. So far, large-scale comprehensive molecular and epidemiological characterisation of S. aureus from very diverse settings has not been carried out in India. The objective of this study is to evaluate the molecular, epidemiological and virulence characteristics of S. aureus in both community and hospital settings in Chennai, southern India. Methods: S. aureus isolates were obtained from four different groups (a) healthy individuals from closed community settings, (b) inpatients from hospitals, (c) outpatients from hospitals, representing isolates of hospital-community interface and (d) HIV-infected patients to define isolates associated with the immunocompromised. Antibiotic susceptibility testing, multiplex polymerase chain reactions for detection of virulence and resistance determinants, molecular typing including Staphylococcal cassette chromosome mec (SCCmec) and agr typing, were carried out. Sequencing-based typing was done using spa and multilocus sequence typing (MLST) methods. Clonal complexes (CC) of hospital and CA methicillin-resistant S. aureus (MRSA) were identified and compared for virulence and resistance. Results and Conclusion: A total of 769 isolates of S. aureus isolates were studied. The prevalence of MRSA was found to be 7.17%, 81.67%, 58.33% and 22.85% for groups a, b, c and d, respectively. Of the four SCCmec types (I, III, IV and V) detected, SCCmec V was found to be predominant. Panton-Valentine leucocidin toxin genes were detected among MRSA isolates harbouring SCCmec IV and V. A total of 78 spa types were detected, t657 being the most prevalent. 13 MLST types belonging to 9 CC were detected. CC1 (ST-772, ST-1) and CC8 (ST238, ST368 and ST1208) were found to be predominant among MRSA. CA-MRSA isolates with SCCmec IV and V were isolated from all study groups including hospitalised patients and were found to be similar by molecular tools. This shows that CA MRSA has probably infiltrated into the hospital settings.

Detection of Tannerella forsythia bspA and prtH genotypes among periodontitis patients and healthy subjects-A case-Control study.

BACKGROUND: T. forsythia a gram negative, anaerobe inhabits the mature biofilm present at sites expressing progressive periodontitis. It is a part of "red complex" group which contributes to the pathogenesis of periodontitis. The BspA protein and prtH gene encoded cysteine protease play a vital role in the virulence of T. forsythia. The present study aims to detect the two genotypes (bspA and prtH) in periodontitis and healthy subjects. MATERIALS & METHOD: Subgingival plaque samples were collected from periodontitis patients and healthy subjects (Chronic Periodontitis n = 128, Aggressive Periodontitis n = 72, healthy subjects n = 200). The samples were screened for the presence of T. forsythia 16S rRNA, bspA and prtH genotypes by Polymerase Chain Reaction. The prevalence of the genotypes between periodontitis patients and healthy subjects was compared with Pearson's Chi-square test. A P value of < 0.05 was considered to be statistically significant. RESULTS: The prevalence for T. forsythia in Chronic Periodontitis (n = 128), Aggressive Periodontitis (n = 72) and health (n = 200) was 73.4%, 59.7% and 10.5% respectively. The prevalence of T.forsythia bspA/prtH genotypes was 81.90%/43.60%, 88.40%/53.50% and 33.30%/14.3% in Chronic Periodontitis, aggressive Periodontitis and health respectively. Compared to healthy subjects, the odds of detecting T.forsythia 16S rRNA was 18.53 times high in individuals with periodontitis (P = 0.0001). CONCLUSION: The high odds ratio of T.forsythia 16S rRNA among periodontitis strongly suggests its role in periodontitis. In addition, the high prevalence of T. forsythia bspA genotype among Chronic Periodontitis signifies it as a useful marker for chronic periodontitis.

Detection of Aggregatibacter actinomycetemcomitans leukotoxin and fimbria-associated protein gene genotypes among periodontitis patients and healthy controls: A case-control study.

BACKGROUND: Aggregatibacter actinomycetemcomitans has been reported in higher proportions in subgingival microbiota of individuals with aggressive periodontitis (AgP) compared with those with chronic periodontitis (ChP) and healthy controls. The major virulence factors are the ones that help in colonization and evasion of host's defenses. Hence, this study was aimed to assess the prevalence of A. actinomycetemcomitans 16S rRNA and its virulent genotypes (leukotoxin [lktA] and fimbria-associated protein [fap]). MATERIALS AND METHODS: In this case- control study We performed periodontal examination and measured probing depth and clinical attachment level (CAL). Subgingival plaque samples from 200 (ChP: n = 128 and AgP: n = 72) periodontitis patients and 200 healthy controls were screened for the presence of A. actinomycetemcomitans 16S rRNA, lktA, and fap genotypes by polymerase chain reaction. The prevalence of genotypes between periodontitis patients and healthy controls was compared with Pearson's Chi-square test. P < 0.05 was considered statistically significant. RESULTS: Mean pocket probing depth and CAL were high as compared to the healthy controls. The prevalence of A. actinomycetemcomitans in ChP (n = 128), AgP (n = 72), and healthy individuals (n = 200) was 32.0%, 61.1%, and 2.5%, respectively. A. actinomycetemcomitans lktA genotype prevalence was 71.8% among periodontitis patients, while A. actinomycetemcomitans fap genotype showed 31.8% prevalence. The prevalence of these genotypes was insignificant in healthy controls. CONCLUSION: The high odds ratio for A. actinomycetemcomitans prevalence suggests its strong link to periodontitis. Detection of A. actinomycetemcomitans lktA + genotype may be a useful marker for AgP as its prevalence was found to be high in AgP.

Role of insertion sequence element is256 as a virulence marker and its association with biofilm formation among methicillin-resistant Staphylococcus epidermidis from hospital and community settings in Chennai, South India.

The objective of this study was to detect the association of biofilm formation with IS256 among clinical and carrier isolates of methicillin-resistant Staphylococcus epidermidis (MRSE). A total of 71 MRSE isolates were included in this study. Phenotypic detection of biofilm formation was done by Congo red agar method. Detection of genes associated with biofilm formation (icaAD, aap and atlE) and insertion sequence IS256 was done by polymerase chain reaction. Of the 71 MRSE isolates,19/40 (47.5%) clinical isolates from hospital settings and 11/31 (35.5%) carrier isolates from community settings respectively were found to be positive for all the three genes tested, namely, icaAD+, aap+ and atlE+ genes. Nearly 80% of clinical isolates were found to harbour IS256, whereas only 13% of community isolates harboured IS256.

Epidemiology of diabetic foot infections in a reference tertiary hospital in India.

INTRODUCTION: The present study attempts to examine the microbial profile and antibiotic susceptibility of diabetic foot infections in the intensive care unit of a tertiary referral centre for diabetic foot. As part of the study, we also attempted to find the prevalence of blaNDM-like gene among carbapenem-resistant gram negative infections. METHODOLOGY: A prospective study of 261 patients with diabetic foot infections was performed during the period between January 2014 and June 2014. RESULTS: A total of 289 isolates were obtained from 178 tissue samples from 261 patients, 156 (59.7%) males and 105 (40.2%) females, with a mean age of 58 years (-15 years), having diabetic foot infection. No growth was seen in thirty eight (17.6%) tissue samples. Out of the total samples, 44.3% were monomicrobial and 55.7% were polymicrobial. Gram negative pathogens were predominant (58.5%). Seven of the total isolates were fungal; 0.7% showed pure fungal growth and 1.7% were mixed, grown along with some bacteria. The most frequently isolated bacteria were Staphylococcus aureus (26.9%), followed by Pseudomonas aeruginosa (20.9%). Of the 58.5% gram negative pathogens, 16.5% were Enterobacteriaceae resistant to carbapenems. Among these isolates, 4 (25%) were positive for blaNDM-like gene. Among the rest, 18.6% were carbapenem-resistant Pseudomonas, among which 4 (36.3%) were blaNDM. Among the Staphylococci, 23.7% were methicillin-resistant Staphylococcus aureus. CONCLUSIONS: Our results support the recent view that gram negative organisms, depending on the geographical location, may be predominant in DFIs. There is an increase in multidrug-resistant pathogens, especially carbapenem resistance and this is creeping rapidly. We need to be more judicious while using empiric antibiotics.

Whole genome sequence analysis of NDM-1, CMY-4, and SHV-12 coproducing Salmonella enterica serovar Typhimurium isolated from a case of fatal burn wound infection.

Salmonella species are frequently associated with gastrointestinal infections such as diarrhea. However, extraintestinal Salmonella infections, including burn infections, have been described. Here, we report the first case of a carbapenem-resistant and metallo-ß-lactamase (New Delhi metallo-ß-lactamase), extended-spectrum ß-lactamase (SHV-12), and AmpC ß-lactamase (CMY-4) coproducing Salmonella Typhimurium isolated from a fatal case of burn wound infection. The publication highlights the necessity for the rational use of antibiotics (particularly the rational use of last-resort antibiotics such as carbapenems) in hospitals and burn units, as well as the need for systematic screening of Salmonella spp. (including Salmonella enterica serovar Typhimurium) for resistance to carbapenem antibiotics.

Draft genome sequence of an O25:H4-ST131 Escherichia coli harbouring blaNDM-1 on an IncHI3 plasmid: A first report.

Carbapenem resistance conferred by New Delhi metallo-ß-lactamases is mediated by plasmids of diverse incompatibility types harboured by different lineages of Enterobacteriaceae. In this study, we report the draft genome sequence of a ST131 Escherichia coli harbouring the blaNDM gene on an IncHI3 plasmid.

Detection of Porphyromonas gingivalis fimA Type I Genotype in Gingivitis by Real-Time PCR-A Pilot Study.

INTRODUCTION: Published literature till date reveals a high prevalence of Porphyromonas gingivalis fimA type I genotype among healthy subjects. Quite a few studies have reported its prevalence also in periodontitis patients. Nevertheless incidence of this genotype in gingivitis is lacking in adult population. AIM: The present study was chosen to detect P. gingivalis fimA type I genotype among chronic gingivitis patients. MATERIALS AND METHODS: A total of 46 subgingival plaque samples collected from chronic marginal gingivitis (n=23) and chronic periodontitis subjects (control group) (n=23) were subjected to Real-Time Polymerase Chain Reaction to detect the P. gingivalis fimA type I gene. Statistical analysis was performed using chi-square test. RESULTS: Prevalence of P. gingivalis fimA type I gene among chronic periodontitis and chronic gingivitis patients were 8.7% and 30.4% respectively. P. gingivalis fimA type I genotype prevalence was found to be statistically insignificant between the two study groups (p=0.135). CONCLUSION: The avirulent P. gingivalis fimA type I genotype, occurred in high prevalence among chronic gingivitis patients, while its presence was low in chronic periodontitis patients. Presence of this avirulent genotype in chronic marginal gingivitis signifies its reversible condition.

Analysis of Antibiotic Resistance Genes and its Associated SCCmec Types among Nasal Carriage of Methicillin Resistant Coagulase Negative Staphylococci from Community Settings, Chennai, Southern India.

OBJECTIVE: The study was designed to find the distribution of SCCmec types and the various antibiotic resistance genes amongst MR-CoNS isolates from asymptomatic individuals. MATERIALS AND METHODS: A total of 145 nasal swabs were collected from asymptomatic healthy individuals from community settings. Identification and speciation of CoNS were done by standard biochemical methods. Screening of methicillin resistance (mecA gene) and detection of various antibiotic resistant genes were done using multiplex PCR method. SCCmec types (I - V) were determined using multiplex PCR. RESULTS: 50 (44.6%) isolates were found to be methicillin resistant both by cefoxitin method and multiplex PCR. S. epidermidis (40%) was the predominant species followed by S. haemolyticus (28%), S. hominis (20%) and S. warneri (12%). Highest resistance was shown for cotrimoxazole (26%), followed by ciprofloxacin (24%), tetracycline (20%), erythromycin (18%), fusidic acid (10%) and mupirocin (6%). Among SCCmec types, 44 isolates showed single type, including type I (30%), type IV (24%), type II (18%), type V (14%) and type III (2%). 6 isolates showed two types, III+IV (n= 2), II+V (n=2), IV+V (n=1) and type I+V (n=1). CONCLUSION: In conclusion, to the best of our knowledge, this is the first study in India to study the distribution of antibiotic resistant genes and SCCmec types among MR-CoNS from community settings. This study highlights high prevalence of MR-CoNS in community and its role in harbouring genetically diverse SCCmec elements as antibiotic resistance determinant.