Colistin Resistance in Escherichia coli and Salmonella enterica Strains Isolated from Swine in Brazil

Reports about acquired resistance to colistin in different bacteria species are increasing, including E. coli of animal origin, but reports of resistance in wild S. enterica of different serotypes from swine are not found in the literature. Results obtained with one hundred and twenty-six E. coli strains from diseased swine and one hundred and twenty-four S. enterica strains from diseased and carrier swine showed a frequency of 6.3% and 21% of colistin-resistant strains, respectively. When comparing the disk diffusion test with the agar dilution test to evaluate the strains, it was confirmed that the disk diffusion test is not recommended to evaluate colistin resistance as described previously. The colistin MIC 90 and MIC 50 values obtained to E. coli were 0.25 mu g/mL and 0.5 mu g/mL, the MIC 90 and MIC 50 to S. enterica were 1 mu g/mL and 8 mu g/mL. Considering the importance of colistin in control of nosocomial human infections with Gram-negative multiresistant bacteria, and the large use of this drug in animal production, the colistin resistance prevalence in enterobacteriaceae of animal origin must be monitored more closely.

Study of thiabendazole resistance and volatile organic compounds production of Penicillium expansum strains

The years of excessive use of thiabendazole to control Penicillium expansum has induced the development of resistance. Sensitivity of fourty eight strains collected from orchards and packinghouses in Emilia Romagna to pure and commercial TBZ was determined in vitro on TBZ amended medium (400μg/mL). Out of 48 strains, 35 were thiabendazole-sensitive (S) and 13 were thiabendazole-resistant (R). Microtiter assay adapted to P. expansum, showed EC50 values ranging from 54 to 320 μg/mL for ten TBZ-resistant strains. At the highest dose (50 μg/mL), resistant strains growth was not inhibited and the reported MICs value were >1000 μg/mL. Therefore, preliminary screening combined with microtiter assay, can be a good strategy to test susceptibility to TBZ. Mutations in the β-tubulin gene were studied on amino acid sequences from residue 167 to residue 357 of 10 P. expansum strains. Mutation at codon 198 was associated with TBZ-resistance. However, its absence in 3 resistant strains can be explained by the involvement of other mechanisms. Moreover, a P. expansum strain LB8/99 showed good antifungal effect against some fungal pathogens through double petri dish assay. It inhibited both mycelium growth and conidia germination of B. cinerea, C. acutatum, and M. laxa, and reduced significantly by 53% and 18% respectively P. expansum. Three major VOCS: geosmin, phenethyl alcolhol (PEA) and an unknown substance were identified by GC-MS analysis. Consistent fumigation of fungal pathogens with PEA (1230 mg/mL), inhibited both conidia germination and mycelium growth of all pathogens, except conidia germination of P. expansum that was reduced by 90% with respect to control. While, the concentration of PEA produced naturally by LB8/99 was ineffective in controlling the pathogens and seemed to have a synergic or additive effect with the other VOCS. Investigations to study the biofumigant effect of LB8/99 on other commodities like seeds and seedlings are in progress.

Characterization of Giardia lamblia WB C6 clones resistant to nitazoxanide and to metronidazole

OBJECTIVES: The characterization of Giardia lamblia WB C6 strains resistant to metronidazole and to the nitro-thiazole nitazoxanide [2-acetolyloxy-N-(5-nitro 2-thiazolyl) benzamide] as the parent compound of thiazolides, a novel class of anti-infective drugs with a broad spectrum of activities against a wide variety of helminths, protozoa and enteric bacteria. METHODS: Issuing from G. lamblia WB C6, we have generated two strains exhibiting resistance to nitazoxanide (strain C4) and to metronidazole (strain C5) and determined their susceptibilities to both drugs. Using quantitative RT-PCR, we have analysed the expression of genes that are potentially involved in resistance formation, namely genes encoding pyruvate oxidoreductases (POR1 and POR2), nitroreductase (NR), protein disulphide isomerases (PDI2 and PDI4) and variant surface proteins (VSPs; TSA417). We have cloned and expressed PDI2 and PDI4 in Escherichia coli. Using an enzyme assay based on the polymerization of insulin, we have determined the activities of both enzymes in the presence and absence of nitazoxanide. RESULTS: Whereas C4 was cross-resistant to nitazoxanide and to metronidazole, C5 was resistant only to metronidazole. Transcript levels of the potential targets for nitro-drugs POR1, POR2 and NR were only slightly modified, PDI2 transcript levels were increased in both resistant strains and PDI4 levels in C4. This correlated with the findings that the functional activities of recombinant PDI2 and PDI4 were inhibited by nitazoxanide. Moreover, drastic changes were observed in VSP gene expression. CONCLUSIONS: These results suggest that resistance formation in Giardia against nitazoxanide and metronidazole is linked, and possibly mediated by, altered gene expression in drug-resistant strains compared with non-resistant strains of Giardia.

Pneumonia due to resistant Streptococcus pneumoniae

In the past 10 to 20 years the pneumococcus, the most common pathogen of community-acquired pneumonia, has developed resistance to most antibiotics used for its treatment. Classes with important resistance problems include the beta-lactams, the macrolides and lincosamides, trimethoprim-sulfamethoxazole, and the tetracyclines. Unfortunately, resistance to more than one class of antibiotics is common in pneumococci, and their treatment is thus becoming more difficult. Patients likely to harbour resistant organisms include young children, particularly those attending day care, older patients, and subjects who have received recent antibiotic therapy, suffer from underlying diseases including HIV, or have nosocomial or polymicrobial pneumonia. The consequences of resistance development are different for different classes of antibiotics. With beta-lactams, the increase in minimal inhibitory concentrations is usually moderate in resistant strains, and because of the high concentrations that can be achieved with this class of drugs resistance does not usually lead to treatment failure. Thus, beta-lactams continue to be important drugs for the treatment of pneumococcal pneumonia, even if the organism is resistant. In contrast, resistance to other classes of antibiotics must be assumed to render these drugs ineffective. Newer quinolones represent valuable alternatives for the treatment of pneumococcal pneumonia, since their efficacy is not affected by resistance to other classes of antibiotics and they cover almost all pathogens of community-acquired pneumonia, including the atypical pathogens. However, they should be used with restraint in order to preserve this valuable class of drugs.

Effects of EDP-420 on penicillin-resistant and quinolone- and penicillin-resistant pneumococci in the rabbit meningitis model

OBJECTIVES: To test the efficacy of EDP-420, a new ketolide, in experimental pneumococcal meningitis and to determine its penetration into the CSF. METHODS: The experimental rabbit model was used in this study and EDP-420 was tested against a penicillin-resistant and a penicillin- and quinolone-resistant mutant. EDP-420 was also tested against both strains in time-killing assays over 8 h in vitro. RESULTS: In experimental meningitis, EDP-420 produced a bactericidal activity comparable to the standard regimen based on a combination of vancomycin with ceftriaxone against a penicillin-resistant Streptococcus pneumoniae and a penicillin- and quinolone-resistant S. pneumoniae isolate. The penetration of EDP-420 into inflamed meninges was 38% after an i.v. injection of 10 mg/kg. The bactericidal activity of EDP-420 was also confirmed in in vitro time-killing assays. CONCLUSIONS: EDP-420 is an efficacious alternative treatment in pneumococcal meningitis, especially when resistant strains are suspected.

Prevalence and risk factors for carriage of multi-drug resistant Staphylococci in healthy cats and dogs

We investigated the distribution of commensal staphylococcal species and determined the prevalence of multi-drug resistance in healthy cats and dogs. Risk factors associated with the carriage of multi-drug resistant strains were explored. Isolates from 256 dogs and 277 cats were identified at the species level using matrix-assisted laser desorption ionisation-time of flight mass spectrometry. The diversity of coagulase-negative Staphylococci (CNS) was high, with 22 species in dogs and 24 in cats. Multi-drug resistance was frequent (17%) and not always associated with the presence of the mecA gene. A stay in a veterinary clinic in the last year was associated with an increased risk of colonisation by multi-drug resistant Staphylococci (OR = 2.4, 95% CI: 1.1˜5.2, p value LRT = 0.04). When identifying efficient control strategies against antibiotic resistance, the presence of mechanisms other than methicillin resistance and the possible role of CNS in the spread of resistance determinants should be considered.

Clinical and molecular features of methicillin-resistant, coagulase-negative staphylococci of pets and horses

OBJECTIVES To determine the antibiotic resistance and fingerprint profiles of methicillin-resistant coagulase-negative staphylococci (MRCoNS) from animal infections among different practices and examine the history of antibiotic treatment. METHODS Isolates were identified by mass spectrometry and tested for antimicrobial resistance by broth dilution, microarrays and sequence analysis of the topoisomerases. Diversity was assessed by PFGE, icaA PCR and staphylococcal cassette chromosome mec (SCCmec), arginine catabolic mobile element (ACME) and multilocus sequence typing. Clinical records were examined retrospectively. RESULTS MRCoNS were identified as Staphylococcus epidermidis (n=20), Staphylococcus haemolyticus (n=17), Staphylococcus hominis (n=3), Staphylococcus capitis (n=1), Staphylococcus cohnii (n=1) and Staphylococcus warneri (n=1). PFGE identified one clonal lineage in S. hominis isolates and several in S. haemolyticus and S. epidermidis. Fourteen sequence types were identified in S. epidermidis, with sequence type 2 (ST2) and ST5 being predominant. Ten isolates contained SCCmec IV, seven contained SCCmec V and the others were non-typeable. ACMEs were detected in 11 S. epidermidis isolates. One S. hominis and 10 S. epidermidis isolates were icaA positive. In addition to mecA-mediated β-lactam resistance, the most frequent resistance was to gentamicin/kanamycin [aac(6')-Ie-aph(2')-Ia, aph(3')-III] (n=34), macrolides/lincosamides [erm(C), erm(A), msr, lnu(A)] (n=31), tetracycline [tet(K)] (n=22), streptomycin [str, ant(6)-Ia] (n=20), trimethoprim [dfr(A), dfr(G)] (n=17), sulfamethoxazole (n = 34) and fluoroquinolones [amino acid substitutions in GyrA and GrlA] (n=30). Clinical data suggest selection through multiple antibiotic courses and emphasize the importance of accurate diagnosis and antibiograms. CONCLUSIONS MRCoNS from animal infection sites are genetically heterogeneous multidrug-resistant strains that represent a new challenge in the prevention and therapy of infections in veterinary clinics.

Does prior receipt of a rifamycin antibiotic increase the risk of acquiring an infection due to a rifamycin-resistant strain of Clostridium difficile?

Objectives. To examine the association between prior rifamycin exposure and later development of C. difficile infection (CDI) caused by a rifamycin-resistant strain of C. difficile , and to compare patient characteristics between rifamycin-resistant strains of C. difficile infection and rifamycin-susceptible strains of C. difficile infection. ^ Methods. A case-control study was performed in a large university-affiliated hospital in Houston, Texas. Study subjects were patients with C. difficile infection acquired at the hospital with culture-positive isolates of C. difficile with which in vitro rifaximin and rifampin susceptibility has been tested. Prior use of rifamycin, demographic and clinical characteristics was compared between case and control groups using univariate statistics. ^ Results. A total of 49 C. difficile strains met the study inclusion criteria for rifamycin-resistant case isolates, and a total of 98 rifamycin-susceptible C. difficile strains were matched to case isolates. Of 49 case isolates, 12 (4%) were resistant to rifampin alone, 12 (4%) were resistant to rifaximin alone, and 25 (9%) were resistant to both rifampin and rifaximin. There was no significant association between prior rifamycin use and rifamycin-resistant CDI. Cases and controls did not differ according to demographic characteristics, length of hospital stay, known risk factors of CDI, type of CDI-onset, and pre-infection medical co-morbidities. Our results on 37 rifaximin-resistant isolates (MIC ≥32 &mgr;g/ml) showed more than half of isolates had a rifaximin MIC ≥256 &mgr;g/ml, and out of these isolates, 19 isolates had MICs ≥1024 &mgr;g/ml. ^ Conclusions. Using a large series of rifamycin-non-susceptible isolates, no patient characteristics were independently associated with rifamycin-resistant CDI. This data suggests that factors beyond previous use of rifamycin antibiotics are primary risk factors for rifamycin-resistant C. difficile. ^

Lith1, a major gene affecting cholesterol gallstone formation among inbred strains of mice.

The prevalence of cholesterol gallstones differs among inbred strains of mice fed a diet containing 15% (wt/wt) dairy fat, 1% (wt/wt) cholesterol, and 0.5% (wt/wt) cholic acid. Strains C57L, SWR, and A were notable for a high prevalence of cholelithiasis; strains C57BL/6, C3H, and SJL had an intermediate prevalence; and strains SM, AKR, and DBA/2 exhibited no cholelithiasis after consuming the diet for 18 weeks. Genetic analysis of the difference in gallstone prevalence rates between strains AKR and C57L was carried out by using the AKXL recombinant inbred strain set and (AKR x C57L)F1 x AKR backcross mice. Susceptibility to gallstone formation was found to be a dominant trait determined by at least two genes. A major gene, named Lith1, mapped to mouse chromosome 2. When examined after 6 weeks on the lithogenic diet, the activity of hepatic 3-hydroxy-3-methylglutaryl-CoA reductase (EC 1.1.1.88) was downregulated as expected in the gallstone-resistant strains, AKR and SJL, but this enzyme failed to downregulate in C57L and SWR, the gallstone-susceptible strains. This suggests that regulation of the rate-limiting enzyme in cholesterol biosynthesis may be pivotal in determining the occurrence and severity of cholesterol hypersecretion and hence lithogenicity of gallbladder bile. These studies indicate that genetic factors are critical in determining gallstone formation and that the genetic resources of the mouse model may permit these factors to be identified.

The prevalence of genotypes that determine resistance to macrolides, lincosamides, and streptogramins B compared with spiramycin susceptibility among erythromycin-resistant Staphylococcus epidermidis

Coagulase-negative staphylococci, particularly Staphylococcus epidermidis , can be regarded as potential reservoirs of resistance genes for pathogenic strains, e.g., Staphylococcus aureus . The aim of this study was to assess the prevalence of different resistance phenotypes to macrolide, lincosamide, and streptogramins B (MLSB) antibiotics among erythromycin-resistant S. epidermidis, together with the evaluation of genes promoting the following different types of MLSB resistance: ermA, ermB, ermC, msrA, mphC, and linA/A’. Susceptibility to spiramycin was also examined. Among 75 erythromycin-resistant S. epidermidis isolates, the most frequent phenotypes were macrolides and streptogramins B (MSB) and constitutive MLSB (cMLSB). Moreover, all strains with the cMLSB phenotype and the majority of inducible MLSB (iMLSB) isolates were resistant to spiramycin, whereas strains with the MSB phenotype were sensitive to this antibiotic. The D-shape zone of inhibition around the clindamycin disc near the spiramycin disc was found for some spiramycin-resistant strains with the iMLSB phenotype, suggesting an induction of resistance to clindamycin by this 16-membered macrolide. The most frequently isolated gene was ermC, irrespective of the MLSB resistance phenotype, whereas the most often noted gene combination was ermC, mphC, linA/A’. The results obtained showed that the genes responsible for different mechanisms of MLSB resistance in S. epidermidis generally coexist, often without the phenotypic expression of each of them.

Multi-drug resistant Staphylococcus aureus isolated from emergency ward of an Iranian hospital

Purpose: To study the prevalence of resistant strains of Staphylococcus aureus isolated from surfaces, beds and various equipment of an Iranian hospital emergency ward. Methods: Two hundred swab samples were collected from the surfaces, beds, trolleys, surgical equipment and diagnostic medical devices in emergency ward. Samples were cultured and those that were S. aureus-positive were confirmed using polymerase chain reaction (PCR). Antimicrobial resistance pattern was analyzed using disk diffusion method. Results: Nine of 200 samples (4.5 %) collected were positive for S. aureus. Surfaces (8.8 %), beds (5 %) and trolleys (5 %) were the most commonly contaminated. S. aureus isolates exhibited varying levels of resistance against antibiotics with the following being the highest: tetracycline (88.8 %), penicillin (88.8 %) and ampicillin (77.7 %). The prevalence of resistance against methicillin, oxacillin and azithromycin were 44.4, 33.3 and 33.3 %, respectively. There was no pattern of resistance against imipenem. Conclusion: Efficient disinfection of surfaces, beds, trolleys and surgical instruments should be performed periodically to reduce colonization of resistant strains of S. aureus in various areas of emergency health care centers.

Escherichia coli 83972 expressing a P fimbriae oligosaccharide receptor mimic impairs adhesion of Uropathogenic E. coli

Urinary tract infections (UTIs) caused by uropathogenic Escherichia coli (UPEC) are a significant health concern, exacerbated by the rapid emergence of multidrug resistant strains refractory to antibiotic treatment. P fimbriae are strongly associated with upper urinary tract colonization due to specific binding to α-D-galactopyranosyl-(1-4)-β-D-galactopyranoside receptors in the kidneys. Thus, inhibiting P-fimbrial adhesion may reduce the incidence of UPEC-mediated UTI. E. coli 83972 is an asymptomatic bacteriuria isolate successfully used as a prophylactic agent to prevent UTI in human studies. We constructed a recombinant E. coli 83972 strain displaying a surface-located oligosaccharide P fimbriae receptor mimic that bound to P-fimbriated E. coli producing any of the 3 PapG adhesin variants. The recombinant strain, E. coli 83972:: lgtCE, impaired P fimbriae–mediated adhesion to human erythrocytes and kidney epithelial cells. Additionally, E. coli 83972::lgtCE impaired urine colonization by UPEC in a mouse UTI model, demonstrating its potential as a prophylactic agent to prevent UTI.

Characteristics of microbial drug resistance and its correlates in chronic diabetic foot ulcer infections

While virulence factors and the biofilm-forming capabilities of microbes are the key regulators of the wound healing process, the host immune response may also contribute in the events following wound closure or exacerbation of non-closure. We examined samples from diabetic and non-diabetic foot ulcers/wounds for microbial association and tested the microbes for their antibiotic susceptibility and ability to produce biofilms. A total of 1074 bacterial strains were obtained with staphylococci, Pseudomonas, Citrobacter and enterococci as major colonizers in diabetic samples. Though non-diabetic samples had a similar assemblage, the frequency of occurrence of different groups of bacteria was different. Gram-negative bacteria were found to be more prevalent in the diabetic wound environment while Gram-positive bacteria were predominant in non-diabetic ulcers. A higher frequency of monomicrobial infection was observed in samples from non-diabetic individuals when compared to samples from diabetic patients. The prevalence of different groups of bacteria varied when the samples were stratified according to age and sex of the individuals. Several multidrug-resistant strains were observed among the samples tested and most of these strains produced moderate to high levels of biofilms. The weakened immune response in diabetic individuals and synergism among pathogenic micro-organisms may be the critical factors that determine the delicate balance of the wound healing process.

Identification and preliminary evaluation of viriditoxin, a metabolite of Paecilomyces varioti, as an insecticide for sheep blowfly, Lucilia cuprina (Wied.)

Solvent extracts of cultures of the fungus Paecilomyces varioti are toxic to sheep blowfly, Lucilia cuprina (Wiedemann) (Diptera: Calliphoridae). Different components of the culture extracts were isolated and bioassayed with L. cuprina. The component with most toxicity was purified and identified from its proton magnetic resonance spectrum as viriditoxin, a known antibiotic metabolite of the fungus. The insecticidal properties of viriditoxin were then evaluated. Mean LCso values for first instar larvae of organophosphate susceptible and resistant strains of L. cuprina were 7.5 and 8.4 ppm respectively. Pilot implant trials in sheep demonstrated that the compound provided protection for 9-17 weeks against both strains of L. cuprina. No adverse effects on the trial sheep were detected.

Potential of the neonicotinoid imidacloprid and the oxadiazine indoxacarb for controlling five coleopteran pests of stored grain.

The potential for using imidacloprid (a neonicotinoid) and indoxacarb (an oxadiazine) as grain protectants was investigated in bioassays against resistant strains of five stored grain beetles. The species investigated were Rhyzopertha dominica (F.) (the lesser grain borer), Sitophilus oryzae (L.) (the rice weevil), Tribolium castaneum (Herbst) (the rust-red flour beetle), Oryzaephilus surinamensis (L.) (the saw tooth flour beetle), and Cryptolestes ferrugineus (Stephens) (the flat grain beetle). Each of these species has developed resistance to one or more protectants, including organophosphorus insecticides, synthetic pyrethroids and the juvenile hormone analogue methoprene. Mortality and reproduction after a 2-week exposure of adults to treated wheat depended on species, dose and insecticide. Imidacloprid had no effect on S. oryzae at any dose, but none of the other species produced any live progeny at 10 mg/kg. Indoxacarb had no effect on T. castaneum at any dose, but none of the other species produced any live progeny at 5 mg/kg. The results show that although both imidacloprid and indoxacarb can control at least four of the five key pests tested at doses comparable to those used for organophosphorus protectants, more potent neonicotinoid or oxadiazine insecticides would be needed than either of these to provide broad spectrum protection of stored grain.