Identification of Major Sequence Types among Multidrug-Resistant Staphylococcus epidermidis Strains Isolated from Infected Eyes and Healthy Conjunctiva.

We examined the presence of virulence and antibiotic resistance genes, SCCmec types and determined the genomic diversity among ocular S. epidermidis isolates (patients-23, healthy controls-29). PCR determined the presence of antibiotic resistance genes, virulence genes and SCCmec types among all isolates. MLST and PFGE determined the genomic relatedness among them. All isolates of S. epidermidis showed resistance to at least one class of antibiotics of which 48 isolates were multidrug resistant and carried ARGs. Thirty-five isolates were methicillin resistant and carried mecA gene. Majority of the isolates were resistant to fluoroquinolones and showed mutation in gyrA, parC, and parE genes, however, few isolates showed additional novel mutations in parC gene. Of the MRSE strains, 17 strains carried SCCmec type IV, four type V, two type II, and two UT4. Seven strains carried novel combination of ccr complex and SCCmercury element, not reported earlier. All the S. epidermidis strains harbored icaA and icaD genes, 47 carried ACME operon, and 50 contained IS256. A noteworthy finding was the presence of ST179 among 43% of infected eye isolates an observation rarely reported among S. epidermidis. PFGE and MLST analysis showed genomic diversity among them. Statistical analysis suggests that few healthy conjunctiva isolates had characteristics similar to infected eye isolates. S. epidermidis strains carrying mecA gene are multidrug resistant, virulent and diverse irrespective of sources of isolation. IS256 cannot be used as marker to differentiate isolates of infected eye from healthy conjunctiva.

Multidrug-resistant Staphylococcus haemolyticus isolates from infected eyes and healthy conjunctivae in India.

This study aimed to determine the presence of antibiotic resistance genes (ARGs), SCCmec elements and genetic relatedness among Staphylococcus haemolyticus isolated from patients with a variety of eye infections (n=11) and from healthy conjunctiva (n=7). Minimum inhibitory concentrations were determined for 14 antimicrobials according to BSAC guidelines. PCR was used to identify the presence of mecA, mecC, SCCmec type and ARGs. Sequencing was used to determine mutations in gyrA, gyrB, topoisomerase IVA and IVB genes. Genetic relatedness was determined by PFGE. Of the 18 isolates, 17 showed resistance to at least one antibiotic, but none showed resistance to vancomycin or rifampicin. Ten isolates were oxacillin-resistant and carried the mecA gene, eight of which belonged to SCCmec type V. The presence of non-mec SCC elements in two meticillin-susceptible isolates and untypeable SCC elements in meticillin-resistant isolates suggests the involvement of S. haemolyticus in the diversification of SCC elements. Sequence analysis revealed point mutations in gyrA (Ser-84→Leu) and topoisomerase IVA genes (Ser-80→Leu) in 13 isolates, and additional variation in the QRDR (Asp-84→Asn) of two isolates, showing good correlation between mutations in gyrA and topoisomerase IV genes and the level of resistance to fluoroquinolones. PFGE analysis showed distinct pulsotypes forming two major clusters, indicating the existence of diversity among isolates, irrespective of the source of isolation. This study suggests that S. haemolyticus isolates from infected eyes and healthy conjunctivae invariably carried ARGs and SCCmec elements and showed diversity in their genomic content, irrespective of the source of isolation.

Whole-Genome Sequence of Vibrio alginolyticus Isolated from the Mucus of the Coral Fungia danai in the Andaman Sea, India.

Vibrio alginolyticus, a halophilic Gram-negative bacterium, which is found in temperate marine and estuarine environments, is known to cause infections in humans and other organisms. We sequenced the genome of sulfamethoxazole-trimthoprim-positive V. alginolyticus strain 4-19 isolated from the mucus of the coral Fungia danai in the Andaman Sea, India.

Whole-Genome Sequences of Staphylococcus haemolyticus Isolated from Infected Eyes and Healthy Conjunctiva in Bhubaneswar, India.

Staphylococcus haemolyticus, an opportunistic pathogen, is known to exhibit multidrug resistance and produce biofilm. We sequenced the genome of four multidrug resistant, biofilm forming isolates from infected eyes and asymptomatic healthy conjunctiva.

Development and evaluation of hexaplex PCR for rapid detection of methicillin, cadmium/zinc and antiseptic-resistant staphylococci, with simultaneous identification of PVL-positive and -negative Staphylococcus aureus and coagulase negative staphylococci.

We developed a multiplex PCR to detect the presence of methicillin- (mecA), cadmium/zinc-(czrC) and antiseptic-resistant (qacA/B) staphylococci and to identify Panton-Valentine leukocidin (PVL)-positive and -negative Staphylococcus aureus and coagulase-negative staphylococci (CoNS) from infected and healthy eyes. The assay was validated on 177 staphylococci comprising of 55 each of S. aureus and CoNS isolated from infected eyes and five S. aureus and 62 CoNS isolated from healthy eyes and nine direct ocular samples. Nine direct ocular samples for in situ testing consisted of corneal scrapings (4), conjunctiva swabs (2) and others (3). Multiplex PCR result was correlated with genotype data obtained with single PCR and dot-blot assay. The control strains that were positive in multiplex PCR for 16S rRNA, nuc, mecA, pvl, czrC and qacA/B genes were also positive in the dot-blot assay. The specificity of amplified genes obtained with reference strains was further confirmed by DNA sequencing. The single step-hexaplex PCR method can be used for rapid detection of mecA, nuc, pvl, czrC and qacA/B genes in staphylococci with simultaneous identification of PVL-positive and -negative S. aureus and CoNS from a variety of ocular samples.

Characterization of Listeria monocytogenes isolated from Ganges water, human clinical and milk samples at Varanasi, India.

Listeria monocytogenes isolated from Ganges water, human clinical and milk samples were characterized by antibiotic susceptibility, serotype identification, detection of virulence genes and ERIC- and REP-PCR fingerprint analyses. All isolates were uniformly resistant to ampicillin, except two isolates, and showed variable resistance to gentamicin, cotrimoxazole, ofloxacin, rifampicin and tetracycline. Of the 20 isolates found positive for pathogens, seven (four human and three water isolates) belong to serogroups 4b, 4d and 4e; six (one human and five water isolates) belong to serogroups 1/2c and 3c; four milk isolates belong to serogroups 1/2b and 3b; and three milk isolates belong to serogroups 1/2a and 3a. Two water isolates, all human isolates, except one (Pb1) lacking inlJ gene, and three milk isolates possess inlA, inlC, plcA, prfA, actA, hlyA and iap genes. The remaining water and milk isolates showed variable presence of inlJ, plcA, prfA, and iap genes. ERIC- and REP-PCR based analyses collectively indicated that isolates of human clinical samples belong to identical or similar clone and isolates of water and milk samples belong to different clones. Overall study demonstrates the prevalence of pathogenic L. monocytogenes species in the environmental and clinical samples. Most of the isolates were resistant to commonly used antibiotics.

Comparison of Type of Species and Antibacterial Susceptibility Profile of Staphylococci Isolated From Normal Healthy Conjunctiva and Ocular Infections.

PURPOSE: To compare the prevalence of various staphylococcal species in ocular infections [OIs (n = 105)] and in normal healthy conjunctiva [NC (n = 51)]. Antibacterial susceptibility profile of the isolates and prevalence of mecA gene among the isolates were also compared. DESIGN: A prospective, comparative, experimental study. METHODS: Antibiotic susceptibility was determined against chloramphenicol, ciprofloxacin, gatifloxacin, moxifloxacin, ofloxacin, cefazolin, vancomycin and cefoxitin by disc diffusion technique as per Clinical and Laboratory Standards Institute guidelines. Methicillin resistance was confirmed by detection of mecA gene by polymerase chain reaction. RESULTS: The prevalence of Staphylococcus aureus was significantly higher (P < 0.0001) in OIs compared with NC (48.6%, 9.8%), whereas the prevalence of Staphylococcus epidermidis was higher (P = 0.02) in NC (22.9%, 41.2%). Overall methicillin resistance was higher in S. epidermidis (66.7% each in OIs and NC) compared with S. aureus (OIs: 7.8%; NC: 0%), which was statistically significant (P < 0.0001). Methicillin resistance was also high among other coagulase-negative staphylococci in both groups [OIs: 40% (12/30); NC: 28% (7/25)]. Vancomycin was effective against all the isolates from both groups. Cefazolin was equally effective. CONCLUSIONS: This study found S. aureus to be a major pathogen in OIs, although it is not common conjunctival flora. The data caution that prevalence of methicillin resistance in coagulase-negative staphylococci is more than S. aureus in OIs and must be considered in their treatment. Despite methicillin resistance, staphylococci from OIs and NC remain sensitive to vancomycin and cefazolin.

Vibrio cholerae infection, novel drug targets and phage therapy.

Vibrio cholerae is the causative agent of the diarrheal disease cholera. Although antibiotic therapy shortens the duration of diarrhea, excessive use has contributed to the emergence of antibiotic resistance in V. cholerae. Mobile genetic elements have been shown to be largely responsible for the shift of drug resistance genes in bacteria, including some V. cholerae strains. Quorum sensing communication systems are used for interaction among bacteria and for sensing environmental signals. Sequence analysis of the ctxB gene of toxigenic V. cholerae strains demonstrated its presence in multiple cholera toxin genotypes. Moreover, bacteriophage that lyse the bacterium have been reported to modulate epidemics by decreasing the required infectious dose of the bacterium. In this article, we will briefly discuss the disease, its clinical manifestation, antimicrobial resistance and the novel approaches to locate drug targets to treat cholera.

Vibrio cholerae O1 biotype El Tor strains isolated in 1992 from Varanasi, India harboured El Tor CTXΦ and classical ctxB on the chromosome-I and classical CTXΦ and classical ctxB on the chromosome-II.

In this study, we report the presence of the El Tor CTXΦ and classical CTXΦ in Vibrio cholerae O1 strains isolated from Varanasi, India. Polymerase chain reaction, DNA sequencing and restriction fragment length polymorphism revealed that, although ctx-positive strains isolated after 1990 contain CTXΦ harbouring El Tor type of rstR and classical ctxB, strains isolated before 1990 contain El Tor type of rstR and El Tor ctxB. Two V. cholerae O1 strains (VC104 and VC106) represent an altered/hybrid strain containing the RS1 element followed by CTXΦ prophage harbouring El Tor type rstR and classical ctxB on the chromosome-I and RS2 element followed by second copy of CTXΦ prophage harbouring classical type rstR and classical ctxB on the chromosome-II. This is the first report of occurrence of El Tor CTXΦ harbouring classical ctxB and classical CTXΦ harbouring classical ctxB in chromosome-I and -II, respectively in diarrhoeal isolates of V. cholerae O1 El Tor strains from Varanasi, India, and that had been isolated in 1992.

Effect of storage and sodium chloride on excision of CTXPhi or pre-CTXPhi and CTXPhi from Vibrio cholerae O139 strains.

We examined the effect of storage and sodium chloride on excision of CTXPhi or pre-CTXPhi and CTXPhi from Vibrio cholerae O139 strains. We found that one strain of V. cholerae O139 VO146P showed loss of the complete phage array, and other strain VO170P showed partial loss of the phage array giving rise to altered strains designated as VO146N and VO170N. Results of PCR and RFLP analysis revealed that both strains (VO146P and VO170P) possessed a single copy of pre-CTX(ET)Phi and two copies of CTXPhi comprising CTX(Class)Phi and CTX(Calc)Phi arranged in tandem, and integrated in the large chromosome. The presence of classical ctxB was detected in CTX(Calc)Phi of both V. cholerae O139 strains. Nucleotide sequencing of three housekeeping genes showed no difference between parent and altered strains of V. cholerae O139.

Analysis of clonally related environmental Vibrio cholerae O1 El Tor isolated before 1992 from Varanasi, India reveals origin of SXT-ICEs belonging to O139 and O1 serogroups.

In this study, we report the presence of SXT in environmental Vibrio cholerae O1 El Tor strains isolated before 1992 from Varanasi, India. All isolates, except one, were resistant to Tm, and/or Sul, Sm, Fr, Na and Am. None contained plasmids. PCR and DNA sequencing revealed the presence of SXT containing dfrA1 and/or sulII, strAB in six isolates and dfr18, sulII and strAB in five isolates. Three clinical V. cholerae O1 isolated during 1992 contained the antibiotic resistance gene cassette aadA1 in the class 1 integron. Conjugation experiments, followed by PCR analysis of transconjugants, provided evidence of the transferable nature of SXT and associated antibiotic resistance genes, and its integration into the prfC site. Results of phylogenetic analysis of the intSXT gene of clonally similar V. cholerae showed a clear difference between dfr18(+) and dfrA1(+) V. cholerae O1 isolates. This is the first report of the occurrence of SXT harbouring sulII, strAB, dfr18 and/or dfrA1 genes in environmental V. cholerae O1 isolated prior to 1992 from Varanasi, India, and suggests emergence of SXT(+) antibiotic-resistant V. cholerae O139 and O1 from an environmental V. cholerae progenitor by acquisition of SXT and antibiotic-resistant gene clusters.

Sequence analysis of Vibrio cholerae orfU and zot from pre-CTXΦ and CTXΦ reveals multiple origin of pre-CTXΦ and CTXΦ.

A multiplex PCR was developed to detect pre-CTXΦ and CTXΦ in Vibrio cholerae. A total of 115 V. cholerae were tested, of which 42 V. cholerae O1 and 18 V. cholerae O139 contained CTXΦ. Six V. cholerae O139 contained only pre-CTXΦ and three V. cholerae O1 and 23 V. cholerae O139 contained both pre-CTXΦ and CTXΦ. None of the V. cholerae non-O1 and non-O139 that were tested had pre-CTXΦ or CTXΦ. Results of Restriction Fragment Length Polymorphism (RFLP) analysis revealed the V. cholerae isolates possessed single or multiple copies of pre-CTXΦ and CTXΦ, always proceeded by a tandemly arranged RS1 element. Comparative nucleotide sequence analyses of the core region genes, orfU and zot, of 15 V. cholerae showed pre-CTX(ET) Φ and CTX(ET) Φ lineage with V. cholerae El Tor and pre-CTX(Class) Φ, pre-CTX(Calc) Φ, and CTX(Calc) Φ with classical V. cholerae O1 and O139. Two distinct types of ctxB were detected in V. cholerae O139. Multi-locus Sequence Typing (MLST) of seven V. cholerae housekeeping genes indicated clonal origin, irrespective of the presence of pre-CTXΦ and/or CTXΦ.

A unique methodology for detecting the spread of chloroquine-resistant strains of Plasmodium falciparum, in previously unreported areas, by analyzing anophelines of malaria endemic zones of Orissa, India.

Generally, clinical data is referred to study drug-resistance patterns of Plasmodium falciparum in an area. This is only possible after a clear manifestation of drug-resistance parasites inside the human host, and thereafter detection by healthcare persons. The detection of spread of drug-resistant P. falciparum in a population, before any pathological symptoms detected in humans is possible by analyzing the anopheline vectors, transmitting malaria. In the present study we implemented a new strategy to detect the spread of chloroquine-resistant (CQR) strains of P. falciparum by the major malaria vectors prevalent in selected endemic regions of Orissa, India. We screened P. falciparum positive vectors by using polymerase chain reaction (PCR)-based assay and thereafter detected K76T mutation in the Pfcrt gene, the chloroquine-resistance marker, of parasites present within the vectors. This study showed higher transmission rate of chloroquine-resistant P. falciparum parasites by Anopheles culicifacies and Anopheles fluviatilis. This study will help in assigning chloroquine-resistant P. falciparum sporozoite transmission potential of malaria vectors and suggest that by adopting the mentioned methodologies, we can detect the spreading of the drug-resistant P. falciparum in its transmission. This approach of studying the anophelines during regular vector collection and epidemiological analysis will give the knowledge of chloroquine-resistance pattern of P. falciparum of an area and help in devising effective malaria control strategy.

Determination of relationships among non-toxigenic Vibrio cholerae O1 biotype El Tor strains from housekeeping gene sequences and ribotype patterns.

Sequencing of three housekeeping genes, mdh, dnaE and recA, and ribotyping for seven non-toxigenic Vibrio cholerae O1 strains isolated from different geographic sources indicate a phylogenetic relationship among the strains. Results of MLST and ribotyping indicate a clear difference between three toxigenic strains (N16961, O395, and 569B) and three non-toxigenic strains from India (GS1, GS2, and GW87) and one Guam strain (X392), the latter of which were similar in both MLST and ribotyping, while two other non-toxigenic strains from the USA and India (2740-80 and OR69) appeared to be more closely related to toxigenic strains than to non-toxigenic strains, although this was not supported by ribotyping. These results provide clues to the emergence of toxigenic strains from a non-toxigenic progenitor by acquisition of virulence gene clusters. Results of split decomposition analysis suggest that widespread recombination occurs among the three housekeeping genes and that recombination plays an important role in the emergence of toxigenic strains of V. cholerae O1.

Vibrio cholerae non-O1, non-O139 strains isolated before 1992 from Varanasi, India are multiple drug resistant, contain intSXT, dfr18 and aadA5 genes.

In this study, we report the presence of the SXT element and Class I integron in Vibrio cholerae non-O1, non-O139 strains isolated from Varanasi, India. Isolates were resistant to cotrimoxazole, trimethoprim and/or streptomycin, furazolidone and ampicillin. None contained plasmids. Polymerase chain reaction (PCR) and DNA sequencing revealed the presence of antibiotic resistance gene cassettes, aadA1, aadA2, aadA5 and dfrA15, in the Class I integron and SXT, an integrative conjugative element containing dfr18, sulII and strAB, in three and six of the isolates respectively. Conjugation experiments, followed by PCR analysis of transconjugants, provided evidence for the transferable nature of intSXT and associated antibiotic resistance gene cassettes. This is the first report of the occurrence of SXT ICE, dfr18, sulII, strAB and aadA5 genes in environmental V. cholerae non-O1, non-O139 strains from Varanasi, India, that had been isolated before 1992.

Application of DNA-based methods in typing Vibrio cholerae strains.

Molecular biology-based techniques based on microbial genotype or DNA sequence have emerged as a basic tool in biological research and in the establishment of large databases of characterized organisms. Genotyping methods have the potential to provide information on subtypes of the organism and their source and/or origin of infection, and to recognize particularly virulent strains of the organism and monitor vaccination programs. Pulsed-field gel electrophoresis, ribotyping, CTX typing, amplified fragment length polymorphism, enterobacterial intergenic consensus sequence-PCR, multilocus sequence typing and microarray methods are more often used for the determination of genetic changes of toxigenic and nontoxigenic Vibrio cholerae strains, origin of infection and relationship between clinical and environmental strains, with the simultaneous detection of the number of copies and types of CTX prophages and genes required for persistence in diverse aquatic environments. This review will discuss DNA-based techniques for the molecular analysis of V. cholerae, its application and future directions.

Multiplex PCR assay for the detection of Anopheles fluviatilis species complex, human host preference, and Plasmodium falciparum sporozoite presence, using a unique mosquito processing method.

A multiplex PCR assay has been developed for detection of Anopheles fluviatilis cryptic species, their human host preference, and Plasmodium falciparum presence in the mosquito. PCR conditions were optimized using primer sets specific for A. fluviatilis cryptic species, Homo sapiens, and P. falciparum and evaluated with field-collected mosquitoes. A unique mosquito processing method was used for screening P. falciparum carrying capacity and human host preference of A. fluviatilis mosquitoes in first-round multiplex PCR. The vectorial status of the mosquito for P. falciparum parasite was confirmed in second-round PCR. Of the 121 collected mosquitoes, 92 were of S type, 26 of T type, and 3 were of other types. Human host preference was dominant in S type, of which 4% were P. falciparum sporozoite positive. This assay and processing method can also be used to evaluate vector competence of other anophelines.

Multiplex PCR for detection of antibiotic resistance genes and the SXT element: application in the characterization of Vibrio cholerae.

This study describes a multiplex PCR assay for the detection of antibiotic resistance genes and the SXT element in Vibrio cholerae. Conditions were optimized to amplify fragments of sulII (encoding sulfamethoxazole resistance), dfrA1 (O1-specific trimethoprim resistance), dfr18 (O139-specific trimethoprim resistance), strB (streptomycin B resistance) and the SXT element simultaneously in one PCR. This multiplex PCR was evaluated on 142 V. cholerae isolates and the results correlated with the phenotypic antibiotic data obtained using a disc diffusion assay and a colony blot assay. Thus this one-step PCR can be used as a simple, rapid and accurate method for identification of antibiotic resistance profiles and could be used for the surveillance of the spread of antibiotic resistance determinants in epidemiological and environmental studies.

Characterization of the genetic background of Vibrio cholerae O1 biotype El Tor serotype Inaba strains isolated in Trivandrum, southern India.

Isolates of Vibrio cholerae O1 biotype El Tor serotype Inaba associated with an outbreak of cholera in Trivandrum, southern India, were characterized. PCR testing revealed that all five isolates examined carried the TCP pathogenicity island, the CTX genetic element and the RTX toxin, and produced cholera toxin (CT). RFLP analysis revealed that these Inaba isolates possessed a single copy of the CTX element flanked by two tandemly arranged copies of the RS element upstream of the core region. The isolates were resistant to ampicillin, nalidixic acid, trimethoprim, sulfamethoxazole, streptomycin and the vibriostatic agent 2,4-diamino-6,7-diisopropylpteridine (O/129). Ribotyping of these Inaba isolates revealed a hybridization profile similar to a strain of serotype Ogawa prevalent in southern India.

Septaplex PCR assay for rapid identification of Vibrio cholerae including detection of virulence and int SXT genes.

In this study, we describe a septaplex PCR assay for rapid identification of Vibrio cholerae including detection of the virulence and intsxt genes. Conditions were optimized to amplify fragments of ISRrRNA (encoding for 16S-23S rRNA gene, Intergenic spacer regions), O1rfb (O1 serogroup specific rfb), O139rfb (O139 serogroup specific rfb), ctxA (cholera toxin subunit A), tcpA (toxin coregulated pilus), and intsxt (sxt integron) simultaneously in a single PCR. The septaplex PCR was evaluated using 211 strains of V. cholerae and six water samples for in situ testing. PCR results were correlated with genotype data obtained by individual PCR and slot-blot assays. The one-step PCR described here can be used to identify V. cholerae accurately and rapidly. Also, the virulence and intsxt genes can be simultaneously detected, providing a useful method for monitoring pathogenic, intsxt-positive and nonpathogenic, intsxt-negative V. cholerae serogroups both in the environment and clinical settings.