Tetracycline resistance transmission in Campylobacter is promoted at temperatures resembling the avian reservoir.

Campylobacter is the causal agent of campylobacteriosis in humans, a self-limiting gastroenteritis. Campylobacteriosis is a zoonosis, commonly transmitted from contaminated chicken meat by either direct consumption or cross contamination during food manipulation. Presence of plasmids encoding for resistance to antibiotics such as tetracycline is common among Campylobacter isolates. In this report, we studied the effect of the temperature in the conjugation frequency of several tet(O) carrying plasmids, providing tetracycline resistance to the recipient cells. The conjugation frequency from donor cells carrying three previously characterized plasmids (pCjA13, pCjA9 and pTet) and from two clinical isolates was determined. Two temperatures, 37 and 42 °C, mimicking the conditions encountered by C. jejuni in the human and broiler chicken gastrointestinal tracts, respectively, were assessed. Our results clearly indicate that the conjugation process is promoted at high temperature. Accordingly, the transcriptional expression of some putative conjugative apparatus genes is thermoregulated, being induced at 42 °C. The two plasmids present in the clinical isolates were sequenced and assembled. Both plasmids are highly related among them and to the pTet plasmid. The high identity of the genes putatively involved in the conjugation process among the plasmids is in agreement with the similar behavior regarding the temperature dependency of the conjugative process. This report suggest that conjugation of plasmids carrying antibiotic resistance genes occurs preferentially at temperatures that resemble the gastrointestinal tract of birds, the main reservoir of C. jejuni.

TrhR, TrhY and HtdA, a novel regulatory circuit that modulates conjugation of the IncHI plasmids.

Bacterial conjugation promotes horizontal gene transfer and, consequently, the acquisition of new capabilities such as resistance to antimicrobial compounds and virulence related traits. Conjugative plasmids belonging to the incompatibility group HI are associated with multidrug resistance in Gram-negative pathogens. IncHI plasmid conjugation is thermodependent and all transfer-related genes are encoded in six operons (tra operons). Using R27, the prototype of IncHI1 plasmids, we reported that the plasmid-encoded factor HtdA represses four of the six tra operons. Moreover, our results indicated that other R27 factors were required for appropriate expression of the tra genes. In this report, using R27 libraries and random mutagenesis assays, two genes - trhR and trhY - have been identified as essential for the transcriptional expression of four tra operons and, accordingly, for the R27 conjugation. TrhR and TrhY are required simultaneously and their stimulatory activity is counteracted by HtdA. Functional and physical interactions between TrhR, TrhY and HtdA suggest that they form a three-element regulatory circuit that controls conjugation of IncHI plasmids. Expression studies suggest that H-NS represses conjugation at high temperature by repressing trhR expression. Remarkably, we show that this regulatory circuit is highly conserved among the IncHI plasmids.

An improved and versatile methodology to quantify biofilms formed on solid surfaces and exposed to the air-liquid interphase.

To study pellicle formation, a new method has been developed to quantify biofilm formed on solid surfaces and exposed to air-liquid interphase. It is a versatile system since different adherent material surfaces might be tested. The methodology is a robust and reproducible approach to quantify biofilm.

Biofilm infections, their resilience to therapy and innovative treatment strategies.

Biofilm formation of microorganisms causes persistent tissue and foreign body infections resistant to treatment with antimicrobial agents. Up to 80% of human bacterial infections are biofilm associated; such infections are most frequently caused by Staphylococcus epidermidis, Pseudomonas aeruginosa, Staphylococcus aureus and Enterobacteria such as Escherichia coli. The accurate diagnosis of biofilm infections is often difficult, which prevents the appropriate choice of treatment. As biofilm infections significantly contribute to patient morbidity and substantial healthcare costs, novel strategies to treat these infections are urgently required. Nucleotide second messengers, c-di-GMP, (p)ppGpp and potentially c-di-AMP, are major regulators of biofilm formation and associated antibiotic tolerance. Consequently, different components of these signalling networks might be appropriate targets for antibiofilm therapy in combination with antibiotic treatment strategies. In addition, cyclic di-nucleotides are microbial-associated molecular patterns with an almost universal presence. Their conserved structures sensed by the eukaryotic host have a widespread effect on the immune system. Thus, cyclic di-nucleotides are also potential immunotherapeutic agents to treat antibiotic-resistant bacterial infections.

Nucleoid proteins stimulate stringently controlled bacterial promoters: a link between the cAMP-CRP and the (p)ppGpp regulons in Escherichia coli.

We report that the H-NS nucleoid protein plays a positive role in the expression of stringently regulated genes in Escherichia coli. Bacteria lacking both H-NS and the paralog StpA show reduced growth rate. Colonies displaying an increased growth rate were isolated, and mapping of a suppressor mutation revealed a base pair substitution in the spoT gene. The spoT(A404E) mutant showed low ppGpp synthesizing ability. The crp gene, which encodes the global regulator CRP, was subject to negative stringent regulation. The stable RNA/protein ratio in an hns, stpA strain was decreased, whereas it was restored in the suppressor strain. Our findings provide evidence of a direct link between the cAMP-CRP modulon and the stringent response.

Expression of the hemolysin operon in Escherichia coli is modulated by a nucleoid-protein complex that includes the proteins Hha and H-NS.

The Escherichia coli protein Hha is a temperature- and osmolarity-dependent modulator of the expression of the hemolysin operon. The Hha protein was purified and its DNA-binding properties analyzed. Hha binds in a non-specific manner throughout the upstream regulatory region of the hemolysin operon in the recombinant hemolytic plasmid pANN202-312. A search for interacting proteins revealed that Hha interacts with H-NS. DNA-binding studies showed that, in vitro, Hha and H-NS together form a complex with DNA that differs from those formed with either protein alone. These data, together with the effects of hha and hns mutations on the expression of the hemolysin genes, suggest that in vivo H-NS and Hha form a nucleoid-protein complex that accounts for the thermo-osmotic regulation of the hemolysin operon in E. coli.

Alterations in protein expression caused by the hha mutation in Escherichia coli: influence of growth medium osmolarity.

The Hha protein belongs to a new family of regulators involved in the environmental regulation of virulence factors. The aim of this work was to study the effect of the hha mutation on the overall protein pattern of Escherichia coli cells by two-dimensional polyacrylamide gel electrophoresis. The growth medium osmolarity clearly influenced the effect of the hha mutation. The number of proteins whose expression was altered in hha cells, compared with wild-type cells, was three times larger at a high osmolarity than at a low osmolarity. Among the proteins whose expression was modified by the hha allele, both OmpA and protein IIAGlc of the phosphotransferase system could be identified. As this latter enzyme participates in the regulation of the synthesis of cyclic AMP and hence influences the catabolite repression system, we tested whether the expression of the lacZ gene was also modified in hha mutants. This was the case, suggesting that at least some of the pleiotropic effects of the hha mutation could be caused by its effect on the catabolite repression system.

Osmolarity modulates the expression of the Hha protein from Escherichia coli.

The effect of the osmolarity of the culture medium on the expression of the hha gene of Escherichia coli was investigated. When cells were grown in LB medium, expression reached a maximum in the exponential phase of growth and decreased in the stationary phase. Increasing the osmolarity of the LB medium had no significant effect on the expression of the hha gene, but depletion of NaCl led to a significant decrease in expression. Expression of the hha gene is thus sensitive to the osmolarity of the growth medium. High levels of expression of the hha gene when cells are grown at medium to high osmolarity are consistent with the finding that the Hha protein appears to play its main modulatory role when cells grow under these conditions.

Construction of a double hha hns mutant of Escherichia coli: effect on DNA supercoiling and alpha-haemolysin production.

A double hha hns Escherichia coli mutant was constructed. The effect of the single hns mutation and of the double hha hns mutation on the expression of the alpha-haemolysin determinant of plasmid pANN202-312 was assessed. Whereas the hns mutant moderately increased expression of the toxin, the double hha hns mutant strongly enhanced transcription of the hly operon and hence expression of the toxin. This suggests that both Hha and H-NS proteins participate in the modulation of the expression of the toxin. The enhancement of haemolysin expression in the double mutant could not be correlated to a global alteration of DNA topology: DNA preparations of a reporter plasmid isolated from this mutant gave a topoisomer distribution similar to that of the parental strain.

Distribution and characterization of faecal verotoxin-producing Escherichia coli (VTEC) isolated from healthy cattle.

Faecal swabs obtained from a random sample of 268 cows and 90 calves on 19 Lugo farms were examined for verotoxin-producing Escherichia coli (VTEC). We found VTEC on 95% of the farms. The prevalence rates of VTEC infection in asymptomatic cows and calves were estimated to be 35 and 37%, respectively. The proportion of animals infected on each farm ranged from 0 to 100%. VTEC strains isolated from healthy cattle belonged to 27 O serogroups; however, 57% (85 of 149) were of one of 8 serogroups (O2, O8, O22, O77, O82, O105, O113 and O171). Nearly 60% of the bovine VTEC strains belonged to serogroups that cause haemorrhagic colitis and haemolytic uraemic syndrome in humans. The VTEC were all non-O157:H7; 91% were eae-negative and 86% produced VT2 or VT1 and VT2. These characteristics are different from those of VTEC isolated from calves with diarrhoea.

Prevalence and characteristics of enteropathogenic Escherichia coli with the eae gene in diarrhoeic rabbits.

A field study was carried out with the objective of investigating the prevalence of enteropathogenic Escherichia coli (EPEC) with the eae gene in diarrhoeic rabbits. EPEC eae+ were isolated from 60 (74%) of 81 diarrhoeic rabbits sampled in 30 industrial fattening farms localized in the four provinces of Galicia (northwestern Spain). Attaching and effacing lesions were found in 44 of 50 animals processed for histology. The 111 E. coli strains identified belonged to 19 different O serogroups and 13 biotypes. However, 53 (48%) of the strains belonged to serogroup O103 and 36 (32%) showed the serobiotype O103:B14. The eae gene was significantly more frequent (100%; 47 of 47) among the highly pathogenic rhamnose-negative strains of serobiotypes O103:B6 and O103:B14 than among the E. coli strains belonging to other serobiotypes (36%; 23 of 64) (P < 0.001). In this first report about the prevalence of EPEC with the eae gene in rabbits, we conclude that the class of E. coli strains observed is a common cause of diarrhoea in Galician rabbit farms, and that highly pathogenic rhamnose-negative strains of serotype O103:K-:H2 and biotype B14 are specially predominant.

Detection of pap, sfa and afa adhesin-encoding operons in uropathogenic Escherichia coli strains: relationship with expression of adhesins and production of toxins.

A total of 243 Escherichia coli strains isolated from patients with urinary tract infections (UTI) were investigated for the presence of pap, sfa and afa adhesin-encoding operons by using the polymerase chain reaction. It was found that 54%, 53% and 2% of the strains exhibited the pap, sfa and afa genotypes, respectively. Pap+ and/or sfa+ strains were more frequent in cases of acute pyelonephritis (94%) than in cases of cystitis (67%) (P < 0.001) and asymptomatic bacteriuria (57%) (P < 0.001). The pap and/or sfa operons were found in 90% of strains expressing mannose-resistant haemagglutination (MRHA) versus 37% of MRHA-negative strains (P < 0.001). The presence of pap and sfa operons was especially significant in strains belonging to MRHA types III (100%) (without P adhesins) and IVa (97%) (expressing the specific Gal-Gal binding typical of P adhesins). Both pap and sfa operons were closely associated with toxigenic E. coli producing alpha-haemolysin (Hly+) and/or the cytotoxic necrotizing factor type 1. There was an apparent correlation between the pap and sfa operons and the O serogroups of the strains. Thus, 93% of strains belonging to O1, O2, O4, O6, O7, O14, O15, O18, O22, O75 and O83 possessed pap and/or sfa operons, versus only 32% of strains belonging to other serogroups (P < 0.001). The results obtained in this study confirm the usefulness of our MRHA typing system for presumptive identification of pathogenic E. coli exhibiting different virulence factors. Thus, 85% of strains that possessed both pap and sfa adhesin-encoding operons showed MRHA types III or IVa previously associated with virulence of E. coli strains that cause UTI and bacteraemia.

The Hha protein as a modulator of expression of virulence factors in Escherichia coli.

We constructed hha derivatives from both a clinical uropathogenic Escherichia coli isolate (strain FVL4) and a wild E. coli strain causing bovine diarrhea (strain CCB21) and analyzed the effect of the hha allele on the expression of the different virulence factors exhibited by these strains. Expression of hemolysin and of the Vir antigen was altered in hha mutants. Whereas production of hemolysin by strain FVL4 was repressed both at a low temperature and at high osmolarity, the hha allele accounted for a significant increase of hemolysin production under these conditions. Also, the low temperature-sensitive expression of the Vir adhesin was modified in hha mutants, which were able to express this adhesin at a low temperature. Expression of other virulence factors, such as cytotoxic necrotizing factor type 1 and 2 toxins, remained unmodified in hha derivatives of strains FVL4 and CCB21.

Complementation of the hha mutation in Escherichia coli by the ymoA gene from Yersinia enterocolitica: dependence on the gene dosage.

The Hha protein from Escherichia coli is highly similar (82%) to the YmoA protein from Yersinia enterocolitica. Both are members of a new class of proteins that modulates gene expression, probably by influencing DNA topology. In this paper, complementation of the hha mutation in E. coli by the ymoA gene from Y. enterocolitica has been studied. We show that the ymoA gene complements one of the phenotypic properties of hha mutants (high level of haemolysin production when they carry the recombinant plasmid pANN202-312) when cloned in a medium-copy-number plasmid but not when carried in a low-copy-number plasmid. Western blot analysis of the expression of YmoA in E. coli rules out inefficient expression of the protein. Surprisingly, the hha gene itself fails to complement the hha mutation when cloned in a medium-copy-number vector and causes genetic rearrangements of the E. coli chromosome as a consequence of insertion sequences mobilization.

Environmental regulation of alpha-haemolysin expression in Escherichia coli.

The effect of osmolarity, temperature and anaerobiosis on the expression of Escherichia coli alpha-haemolysin was investigated. Low osmolarity of the culture medium, growth at high temperature (37 degrees C) and anaerobiosis increase haemolysin production in E. coli cells harbouring the haemolytic plasmid pHly152 as well as in haemolytic strains isolated from urinary-tract infections.

Escherichia coli hha mutants, DNA supercoiling and expression of the haemolysin genes from the recombinant plasmid pANN202-312.

The hha gene of Escherichia coli was identified as modulating the expression of the haemolysin (hly) genes encoded by the recombinant plasmid pANN202-312. hha mutants harbouring plasmid pANN202-312 showed increased haemolysin production. The product of the hha gene, the Hha protein, shows strong homology to the YmoA protein of Yersinia enterocolitica, which plays a role in the thermoregulation of various Y. enterocolitica virulence genes. We show in this study that the Hha protein modulates the expression of haemolysin at the transcriptional level in cells harbouring plasmid pANN202-312. In addition, hha mutants show alterations in the level of plasmid DNA supercoiling. This suggests that the hha mutation increases haemolysin expression through changes in the DNA topology. This hypothesis is supported by our finding that gyr mutations, inhibitors of DNA gyrase such as novobiocin, or growth in conditions reported to reduce levels of negative supercoiling, such as low osmolarity medium, increase haemolysin production.