Bianca Paglietti

Evolution and Population Structure of Salmonella enterica Serovar Newport

Salmonellosis caused by Salmonella enterica serovar Newport is a major global public health concern, particularly because S. Newport isolates that are resistant to multiple drugs (MDR), including third-generation cephalosporins (MDR-AmpC phenotype), have been commonly isolated from food animals. We analyzed 384 S. Newport isolates from various sources by a multilocus sequence typing (MLST) scheme to study the evolution and population structure of the serovar. These were compared to the population structure of S. enterica serovars Enteritidis, Kentucky, Paratyphi B, and Typhimurium. Our S. Newport collection fell into three lineages, Newport-I, Newport-II, and Newport-III, each of which contained multiple sequence types (STs). Newport-I has only a few STs, unlike Newport-II or Newport-III, and has possibly emerged recently. Newport-I is more prevalent among humans in Europe than in North America, whereas Newport-II is preferentially associated with animals. Two STs of Newport-II encompassed all MDR-AmpC isolates, suggesting recent global spread after the acquisition of the bla(CMY-2) gene. In contrast, most Newport-III isolates were from humans in North America and were pansusceptible to antibiotics. Newport was intermediate in population structure to the other serovars, which varied from a single monophyletic lineage in S. Enteritidis or S. Typhimurium to four discrete lineages within S. Paratyphi B. Both mutation and homologous recombination are responsible for diversification within each of these lineages, but the relative frequencies differed with the lineage. We conclude that serovars of S. enterica provide a variety of different population structures.

Lethal and sublethal effects of Brevibacillus laterosporus on the housefly ( Musca domestica )

Sporulated cultures of an isolate of the entomopathogenic bacterium Brevibacillus laterosporus (Laubach) from soil in Sardinia (Italy) were highly toxic by ingestion to both adults and juveniles of the housefly, Musca domestica L. (Diptera: Muscidae). Toxicity was associated with spores as the culture supernatant was not toxic and no parasporal inclusions were observed. The level of toxicity varied with the concentration of spores, the time of exposure to the treated diet, and the stage of insect development. Comparative LC50 values were 0.72 × 108 and 1.75 × 108 spores per gram of diet for 1st and 2nd instar larvae, respectively, and 3.84 × 108 spores per gram for adults. A significant increase in larval development time and reductions in pupal weight and emergence rate were observed when larvae were fed on diets containing sublethal concentrations of spores. Adults surviving after feeding on a sublethal diet showed a significant reduction in fecundity and longevity. Similarly, adults from treated larvae exhibited a lower fecundity, although their longevity was not influenced. A relationship between pupal weight and adult fecundity was found. Pupal stage duration and egg eclosion were not significantly affected.

High rate of levofloxacin resistance in a background of clarithromycin- and metronidazole-resistant Helicobacter pylori in Vietnam

Antimicrobial resistance in Helicobacter pylori has increased worldwide and has become a major cause of treatment failure in many countries, including Vietnam. It is advisable to perform an antibiogram to provide optimal regimens for H. pylori eradication. This study evaluated the rate of antibiotic resistance to the four commonly used antibiotics against H. pylori at a tertiary care hospital in Central Vietnam and analysed point mutations in genes related to clarithromycin (CLA) and levofloxacin (LFX) resistance. A total of 92 H. pylori strains from gastric biopsy specimens were tested; 42.4% were resistant to CLA (primary, 34.2%; secondary, 73.7%), 41.3% to LFX (primary, 35.6%; secondary, 63.2%), 76.1% to metronidazole (MTZ) and 1.1% to amoxicillin. Multidrug resistance was observed in 56.5% (primary, 50.7%; secondary, 78.9%) of isolates (P<0.05). The rate of resistance to LFX was significantly higher in females than males (P<0.05). Most of the CLA- and LFX-resistant strains harboured resistance-associated mutations, with common positions at A2143G and T2182C in the 23S rRNA gene and at Asn-87 or Asp-91 in GyrA. Minimum inhibitory concentrations (MICs) increased in strains carrying quadruple mutations in their 23S rRNA gene and in strains with Asn-87 GyrA mutation (P<0.05). One high-level LFX-resistant strain (MIC=32mg/L) had new mutations with a combination of N87A, A88N and V65I. High resistance rates to CLA, MTZ and LFX discourage standard and LFX-based triple therapies as first-line treatment in Vietnam.

Conventional and molecular approaches to isolates of Salmonella hadar from sporadic and epidemic cases.

In September 1994 an outbreak of gastroenteritis occurred in 437 people who had consumed lunch in the canteen of a factory in Central Italy. Salmonella sp. was isolated from stools of 99 patients and in 73 of them Salmonella hadar was identified. This is the first outbreak caused by this serotype described in Italy. In order to examine the genotypic basis of the epidemic strains, molecular typing was applied to sporadic strains isolated before and after the outbreak episode. For this purpose phage type, resistance to antibiotics, DNA plasmid profile and sites of insertion of the mobile element of IS200 were determined. The epidemic strains were genetically distinct from the non‐epidemic isolates; they were shown to be phage type 26, harbouring four small plasmids, were resistant to nalidixic acid and showed a unique characteristic IS200 fingerprint. The typing methods used in this study allowed the identification and discrimination of the outbreak strains from related isolates. They can thus be considered as a tool for epidemiological purposes. In addition we should point out the emerging resistance to nalidixic acid, largely used in veterinary medicine, in Salm. hadar.

Synergistic antimicrobial activity of Camellia sinensis and Juglans regia against multidrug-resistant bacteria.

Synergistic combinations of antimicrobial agents with different mechanisms of action have been introduced as more successful strategies to combat infections involving multidrug resistant (MDR) bacteria. In this study, we investigated synergistic antimicrobial activity of Camellia sinensis and Juglans regia which are commonly used plants with different antimicrobial agents. Antimicrobial susceptibility of 350 Gram-positive and Gram-negative strains belonging to 10 different bacterial species, was tested against Camellia sinensis and Juglans regia extracts. Minimum inhibitory concentrations (MICs) were determined by agar dilution and microbroth dilution assays. Plant extracts were tested for synergistic antimicrobial activity with different antimicrobial agents by checkerboard titration, Etest/agar incorporation assays, and time kill kinetics. Extract treated and untreated bacteria were subjected to transmission electron microscopy to see the effect on bacterial cell morphology. Camellia sinensis extract showed higher antibacterial activity against MDR S. Typhi, alone and in combination with nalidixic acid, than to susceptible isolates.” We further explore anti-staphylococcal activity of Juglans regia that lead to the changes in bacterial cell morphology indicating the cell wall of Gram-positive bacteria as possible target of action. The synergistic combination of Juglans regia and oxacillin reverted oxacillin resistance of methicillin resistant Staphylococcus aureus (MRSA) strains in vitro. This study provides novel information about antimicrobial and synergistic activity of Camellia sinensis and Juglans regia against MDR pathogens

IS200 fingerprint of Salmonella enterica serotype Typhimurium human strains isolated in Sardinia

A collection of Salmonella enterica serotype Typhimurium human strains isolated in Northern Sardinia (Italy) was examined for the insertion sequence IS200, phage type, antibiotic profile, ribotyping polymorphisms and plasmid profile. All clinical isolates studied contained from 4 to 10 copies of the IS200 element. IS200 permitted to discriminate Typhimurium strains and to identify five IS200 types, some of them circulating in Sardinia at least since 1900. Strains belonging to phage DT104 predominated and correlated with a specific IS200 pattern.

Prevalence, diversity and disease association of Helicobacter pylori in dyspeptic patients from Pakistan

INTRODUCTION The etiological association of Helicobacter pylori with gastric ulcer (GU), gastric cancer (GC), and duodenal ulcer (DU) is well-known. Understanding the epidemiology of H. pylori facilitates the estimation of disease burden in a certain population. This study presents the diversity of H. pylori genotypes and their association with different clinical outcomes among dyspeptic patients in Pakistan over a period of four years. METHODOLOGY Gastric biopsy samples from a total of 450 dyspeptic individualswere subjected to PCR, genotypingand histology. RESULTS A total of 201 (45%) cases were found positive for H. pylori. The detection rate was high in GU (91%), DU (86%) and GC (83%) cases compared with those cases who had intact gastric mucosa (18%). Histology revealed the presence of infection in 68% of cases of mild/chronic nonspecific gastritis with others belonging to the GU sequel. cagA gene carriage was observed in 104 (51%) cases or mostly from DU, GU and GC groups, of which 97 were Western type strains while 3 were East-Asian type strains that are rarely observed in South Asia. vacA allelic variant s1am1 was most commonly observed, followed by s1am2, and s1bm1, with direct correlation in diseased cases (gastritis, GU, DU and GC). Prevalent genotypic combinations were s1am1/cagA- in gastritis and s1am1/cagA+ in DU, GU, and GC. CONCLUSIONS Our study indicates the predominant circulation of Western type cagA and vacAs1am1 type H. pylori strains in Pakistan.

Diversity among human non-typhoidal salmonellae isolates from Zimbabwe

BACKGROUND Non-typhoidal Salmonella infections are an important public health problem in sub-Saharan Africa, especially among children and HIV-seropositive patients in whom they may cause invasive disease. METHODS In order to better understand the epidemiology of Salmonella infections in southern Africa we typed, using serotyping, phage typing and multilocus sequence typing, 167 non-typhoidal Salmonella strains isolated from human clinical specimens during 1995-2000. RESULTS The most common serovars were Salmonella Typhimurium DT56/ST313, Salmonella Enteritidis PT4 and Salmonella Isangi ST216. Isolates of Salmonella Isangi showed a multidrug-resistant phenotype that was resistant to extended-spectrum cephalosporins. Twelve new sequence types and six new serotypes of Salmonella were identified. CONCLUSIONS Given the diversity detected in the study it seems likely that many new variants of S. enterica are extant in Zimbabwe and by implication across sub-Saharan Africa. We have demonstrated the presence in Zimbabwe of a multidrug-resistant strain of the serovar Salmonella Isangi and demonstrated the diversity of Salmonella circulating in one sub-Saharan African country. Further studies on the characteristics of Salmonella Isangi isolates from Zimbabwe, including plasmid typing and genotyping, are essential if effective control of the spread of this potential pathogen in sub-Saharan Africa is to be achieved.

Antibiotic resistance determinants and genetic analysis of Salmonella enterica isolated from food in Morocco.

Antimicrobial-resistant non-typhoidal Salmonella (NTS) are an important cause of infection in Africa, but there is a lack of information on their molecular mechanisms of resistance and epidemiology. This study contributes to fill this gap through the characterization by pulsed-field gel electrophoresis (PFGE), multilocus sequence typing (MLST), plasmid profiling and analysis of antibiotic-resistance determinants of 94 Salmonella enterica strains isolated from food in Morocco. PFGE revealed considerable heterogeneity among the strains, showing 32 pulsotypes. MLST of strains representative of the different serovars evidenced 13 sequence types (STs), three of which were newly identified (ST1694, ST1768 and ST1818) and nine not previously reported in Morocco. Thirty-four strains harbored from one to four plasmids, of IncI1 group in S. Mbandaka, IncFIIA in S. Typhimurium, IncL/M in S. Hadar and S. Blockley. For the first time in Morocco an intact Salmonella Genomic Island 1 (SGI1) carrying the resistance genes aadA2, floR, tetG, blaPSE-1 and sul1 was detected in S. Typhimurium DT104. In serovar Hadar resistance to ampicillin, tetracycline and streptomycin was associated to blaTEM-1, tetA and strA genes respectively, whereas one mutation in gyrA (Asp87Asn) and one in parC (Thr54Ser) genes conferred resistance to nalidixic acid. These findings improve the information on foodborne Salmonella in Morocco, evidencing the presence of MDR strains potentially dangerous to humans, and provide useful data for future studies.

A novel broadly applicable PCR-RFLP method for rapid identification and subtyping of H58 Salmonella Typhi

Salmonella Typhi (S. Typhi), the human-adapted agent of typhoid fever, is genetically monomorphic. SNPs accumulation divided the S. Typhi population in 85 haplotypes (H) of which one, H58, has undergone a clonal expansion. The surveillance of H58 S. Typhi is particularly important, especially in areas where typhoid fever is endemic. We developed a simple PCR and PCR-RFLP method to detect and subtype H58 S. Typhi based on the presence of genomic deletion and specific SNPs. The method was validated against 39 S. Typhi isolates of known haplotype, showing 100% of specificity and high sensitivity, and then used to screen a collection of 99 S. Typhi from Asia, demonstrating a high incidence of H58 S. Typhi in Jordan and India. Our method is designed to be applied in all laboratories with basic molecular biology equipment and few financial resources and allows the surveillance of H58 S. Typhi in resource poor settings.