Modification of outer membrane permeability and alteration of LPS in veterinary enterotoxigenic Escherichia coli.

Enterotoxigenic Escherichia coli (ETEC) is a worrying cause of diarrhoea in calves and the drug multiresistance phenotype concerning various antibiotic families are of concern. Resistance mechanisms associated with envelope changes (porin expression, efflux pump overexpression, lipolysaccahride (LPS) modification) were studied in 14 ETEC isolates selected for their resistance. We performed determinations of (i) antimicrobials Minimal Inhibitory Concentrations with or without the efflux pump inhibitor phenylalanine arginine ß-naphthylamide; (ii) colistin and polymyxin MICs with and without EDTA, (iii) intracellular accumulation of chloramphenicol in presence of an energy uncoupler of pump energy, (iv) and immunodetection of porins and evaluation of porin trimers thermostability. Results indicated that 9 strains presented significant efflux mechanisms overexpression, among them 8 were resistant to colistin and polymyxin B due to a modification of LPS structure as evidenced by EDTA effect and silver staining electrophoresis. The high resistant strains to colistin and polymyxin exhibited identical LPS patterns. Studies of E. coli porins indicated that the majority of strains didn't show modification in their amount, however analysis of porin thermostability showed that porin trimers of some resistant strains were relatively heat-labile, suggesting a misassembly of the functional trimer. The multidrug resistance (MDR) phenotypes detected in these selected ETEC corresponded to association of LPS modifications, abordive assembly of porin trimers and active efflux which drastically alter the antibiotic activity currently used to combat enteric infections caused by this pathogen.

Mycobacterium species related to M. leprae and M. lepromatosis from cows with bovine nodular thelitis.

Bovine nodular thelitis is a granulomatous dermatitis associated with infection with acid-fast bacteria. To identify the mycobacterium responsible for this infection, we conducted phylogenetic investigations based on partial sequencing of 6 genes. These bacteria were identified as an undescribed Mycobacterium species that was phylogenetically related to M. leprae and M. lepromatosis.

Mycobacterium bourgelatii sp. nov., a rapidly growing, non-chromogenic species isolated from the lymph nodes of cattle.

Three independent strains of a rapidly growing, non-chromogenic member of the genus Mycobacterium were isolated from lymph nodes of French cattle. Identification of the isolates was carried out using a polyphasic approach. The nearly complete SSU rRNA gene sequences (>1200 bp) of the strains MLB-A23, MLB-A30 and MLB-A84(T) were identical. A phylogenetic analysis of these unique SSU rRNA gene sequences showed that these strains were most closely related to Mycobacterium intermedium. Further phylogenetic analysis based on concatenated sequences (2854 bp) of four housekeeping genes (hsp65, rpoB, sodA and tuf), the transfer-messenger RNA (tmRNA) and SSU rRNA genes indicated that these three strains represented a distinct species that shares a common ancestor with M. intermedium. Phylogenetic and phenotypic data strongly indicate that the strains MLB-A23, MLB-A30 and MLB-A84(T) belong to a novel mycobacterial species for which the name Mycobacterium bourgelatii sp. nov. is proposed. The type strain is MLB-A84(T) ( = CIP 110557(T) = DSM 45746(T)).

Emergence of macrolide resistance gene mph(B) in Streptococcus uberis and cooperative effects with rdmC-like gene.

Streptococcus uberis UCN60 was resistant to spiramycin (MIC = 8 microg/ml) but susceptible to erythromycin (MIC = 0.06 microg/ml), azithromycin (MIC = 0.12 microg/ml), josamycin (MIC = 0.25 microg/ml), and tylosin (MIC = 0.5 microg/ml). A 2.5-kb HindIII fragment was cloned from S. uberis UCN60 DNA on plasmid pUC18 and introduced into Escherichia coli AG100A, where it conferred resistance to spiramycin by inactivation. The sequence analysis of the fragment showed the presence of an rdmC-like gene that putatively encoded a protein belonging to the alpha/beta hydrolase family and of the first 196 nucleotides of the mph(B) gene putatively encoding a phosphotransferase known to inactivate 14-, 15-, and 16-membered macrolides in E. coli. The entire mph(B) gene was then identified in S. uberis UCN60. The two genes were expressed alone or in combination in E. coli, Staphylococcus aureus, and Enterococcus faecalis. Analysis of MICs revealed that rdmC-like alone did not confer resistance to erythromycin, tylosin, and josamycin in those three hosts. It conferred resistance to spiramycin in E. coli and E. faecalis but not in S. aureus. mph(B) conferred resistance in E. coli to erythromycin, tylosin, josamycin, and spiramycin but only low levels of resistance in E. faecalis and S. aureus to spiramycin (MIC = 8 microg/ml). The combination of mph(B) and rdmC-like genes resulted in a resistance to spiramycin and tylosin in the three hosts that significantly exceeded the mere addition of the resistance levels conferred by each resistance mechanism alone.

Lincomycin resistance gene lnu(D) in Streptococcus uberis.

Streptococcus uberis UCN 42, isolated from a case of bovine mastitis, was intermediately resistant to lincomycin (MIC = 2 microg/ml) while remaining susceptible to clindamycin (MIC = 0.06 microg/ml) and erythromycin. A 1.1-kb SacI fragment was cloned from S. uberis UCN 42 total DNA on plasmid pUC 18 and introduced into Escherichia coli AG100A, where it conferred resistance to both clindamycin and lincomycin. The sequence analysis of the fragment showed the presence of a new gene, named lnu(D), that encoded a 164-amino-acid protein with 53% identity with Lnu(C) previously reported to occur in Streptococcus agalactiae. Crude lysates of E. coli AG100A containing the cloned lnu(D) gene inactivated lincomycin and clindamycin in the presence of ATP and MgCl(2). Mass spectrometry experiments demonstrated that the lnu(D) enzyme catalyzed adenylylation of clindamycin. A domain conserved in deduced sequences of lincosamide O-nucleotidyltransferases Lnu(A), Lnu(C), LinA(N2), and Lin(D) and in the aminoglycoside nucleotidyltransferase ANT(2'') was identified.

Antimicrobial susceptibility of Streptococcus species isolated from clinical mastitis in dairy cows.

The antimicrobial susceptibility was determined for 50 Streptococcus uberis, 42 S. dysgalactiae subsp. dysgalactiae and eight S. agalactiae strains isolated from cow mastitis. Only 27% of the strains were susceptible to all antimicrobial compounds tested. Resistance to tetracycline was most frequent (particularly for S. dysgalactiae strains), then macrolide and/or lincomycin resistance. High level resistance to streptomycin and kanamycin was detected. All S. dysgalactiae and S. agalactiae strains were susceptible to beta-lactams but 44% of the S. uberis strains showed an elevated penicillin G MIC. All strains were susceptible to chloramphenicol and rifampicin.