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Identification of genetic loci required for capsular expression in Vibrio vulnificus.

Transposon mutagenesis of an encapsulated, virulent strain of Vibrio vulnificus 1003(O) led to the identification of four genetic regions that are essential to capsular polysaccharide (CPS) expression and virulence. Of the four regions, three are believed to be part of a capsule gene locus comprised of biosynthesis, polymerization, and transport genes clustered on a single chromosomal fragment. Genes indicating a Wzy-dependent system of polymerization and transmembrane export are present, suggesting that the CPS of V. vulnificus is lipid linked. The fourth region, while it contains a gene essential for CPS expression, is characteristic of an integron-gene cassette region, similar to the super integron of V. cholerae. It is not believed to be part of a CPS gene locus and is located in a region of the chromosome separate from the putative CPS loci. It is comprised of open reading frames (ORFs) carrying genes of unknown function surrounded by direct repeats. This region also contains IS492, an insertion sequence located numerous times throughout a region of the genome, demonstrating a restriction fragment length polymorphism among an encapsulated and nonencapsulated morphotype of V. vulnificus. Collectively, 22 ORFs were recognized: 13 capsule synthesis genes, 4 insertion sequences, 1 truncated biosynthesis gene, and 4 genes of unknown function. This study has led to the identification of previously unrecognized genetic loci that may help to increase the understanding of capsular genetics and antigenic diversity among V. vulnificus strains.

Animals↗

PCR detection of metallo-beta-lactamase gene (blaIMP) in gram-negative rods resistant to broad-spectrum beta-lactams.

We applied PCR to the rapid detection of the metallo-beta-lactamase gene, blaIMP, in clinically isolated gram-negative rods. A total of 54 high-level ceftazidime-resistant strains (MICs, > 128 micrograms/ml) were subjected to PCR analyses with the blaIMP-specific primers, since the blaIMP-bearing clinical isolates tested in our previous study always demonstrated high-level resistance to ceftazidime. Twenty-two blaIMP-positive strains including 9 Pseudomonas aeruginosa, 9 Serratia marcescens, 2 Alcaligenes xylosoxidans, 1 Pseudomonas putida, and 1 Klebsiella pneumoniae strains were newly identified from 18 different hospitals in Japan. These strains were mostly isolated from urine samples and showed high-level resistance to almost every cephem, while their levels of resistance to carbapenems were diverse. The PCR analyses with novel integrase gene-specific (intI3) and acc(6')-Ib gene-specific primers suggested that the integron structure found in a large plasmid harbored by S. marcescens AK9373 was also well conserved among blaIMP-positive strains. These results imply that the blaIMP gene cassettes have been dispersing into various gram-negative rods with the help of the newly identified integron element. Thus, the PCR-aided rapid detection will be helpful for the early recognition of emerging blaIMP-positive clinical isolates which demonstrate consistent resistance to beta-lactams.

Alcaligenes↗

Typing and characterization of mechanisms of resistance of Shigella spp. isolated from feces of children under 5 years of age from Ifakara, Tanzania.

Eighty-six strains of Shigella spp. were isolated during the dry season from stool samples of children under 5 years of age in Ifakara, Tanzania. The epidemiological relationship as well as the antimicrobial susceptibility and mechanisms of resistance to ampicillin, chloramphenicol, and co-trimoxazole were investigated. Four different epidemiological tools, pulsed-field gel electrophoresis (PFGE), repetitive extragenic palindromic (REP)-PCR, plasmid analysis, and antibiogram, were compared for typing Shigella strains. Seventy-eight (90%) strains were Shigella flexneri and were distributed into four groups, by either PFGE or REP-PCR, with 51, 17, 7, and 3 strains. The four strains of Shigella dysenteriae belonged to the same group, and the four strains of Shigella sonnei were distributed in two groups with three and one strain each. Plasmid analysis showed a high level of heterogeneity among strains belonging to the same PFGE group, while the antibiogram was less discriminative. REP-PCR provided an alternative, rapid, powerful genotyping method for Shigella spp. Overall, antimicrobial susceptibility testing showed a high level of resistance to ampicillin (81.8%), chloramphenicol (72.7%), tetracycline (96.9%), and co-trimoxazole (87.9%). Ampicillin resistance was related to an integron-borne OXA-1-type beta-lactamase in 85.1% of the cases and to a TEM-1-type beta-lactamase in the remaining 14.8%. Resistance to co-trimoxazole was due to the presence of a dhfr Ia gene in all groups except one of S. flexneri, where a dhfr VII gene was found within an integron. Chloramphenicol resistance was associated in every case with positive chloramphenicol acetyltransferase activity. All strains were susceptible to nalidixic acid, ciprofloxacin, ceftazidime, cefotaxime, and cefoxitin. Therefore, these antimicrobial agents may be good alternatives for the treatment of diarrhea caused by Shigella in Tanzania.

Bacterial Typing Techniques↗

Molecular characterization of cephalosporin-resistant Salmonella enterica serotype Newport isolates from animals in Pennsylvania.

Multidrug-resistant (MDR) strains of Salmonella enterica serotype Newport have been described for many years. However, the recognition of Newport strains with resistance to cephalosporin antibiotics is more recent. Plasmid-mediated CMY-2 AmpC beta-lactamases have been identified in Salmonella in the United States, and the bla(CMY-2) gene has been shown to be present in Salmonella serotype Newport. This organism is currently undergoing epidemic spread in both animals and humans in the United States, and this is to our knowledge the first description of the molecular epidemiology of this Salmonella strain in animals. Forty-two isolates were included in this study. All isolates were characterized by pulsed-field gel electrophoresis, plasmid analysis, and antibiogram. Four pulsed-field profiles with XbaI were observed. Plasmid analyses showed that although the majority of isolates harbored a single plasmid of 140 kb, this plasmid was not identical in all strains. All isolates showed the presence of the bla(CMY) gene by PCR. Integrons were detected in 16 of the 42 isolates; a fragment of approximately 1,000 bp, amplified with the intI-F and aadAI-R primers, confirmed the presence of the aadAI gene cassette within an integron in these 16 isolates. The potential for coselection of the bla(CMY) gene, if located on an MDR replicon, may not be dependent on any particular antibiotic but rather may be the result of more general antimicrobial use. If this replicon is mobile, it is to be expected that similar MDR strains of additional Salmonella serotypes will be recognized in due course.

Animals↗

Heterogeneity among virulence and antimicrobial resistance gene profiles of extraintestinal Escherichia coli isolates of animal and human origin.

Extraintestinal pathogenic Escherichia coli (ExPEC) isolates collected from different infected animals and from human patients with extraintestinal infections in 2001 were characterized for their phenotypic and genotypic antimicrobial resistance profiles, genotypes, and key virulence factors. Among the 10 antimicrobial agents tested, resistance to ampicillin, tetracycline, and sulfonamides was most frequent. Multiresistant strains were found in both the animal and the human groups of isolates. Resistance gene distribution was assessed by colony hybridization. Similar antibiotic resistance patterns could be observed in the animal and the human isolates. Although some resistance genes, such as bla(TEM), sulI, and sulII, were equally represented in the animal and human ExPEC isolates, differences in the distributions of tetracycline [tet(D)], chloramphenicol (catI, catIII, and floR), and trimethoprim (dhfrI, dhfrV, dhfrVII, and dhfrXIII) resistance genes were observed between the animal and the human isolates. Approximately one-third of the ExPEC isolates possessed a class 1 integron. The four major different variable regions of the class 1 integron contained aminoglycoside (aadA1, aadA2, aadA5, and aadA6) and/or trimethoprim (dhfrIb, dhfrXII, and dhfrXVII) resistance genes. The ExPEC strains belonged to different phylogenetic groups, depending on their host origin. Strains isolated from animal tissues belonged to either a commensal group (group A or B1) or a virulent group (group B2 or D), while the majority of the human isolates belonged to a virulent group (group B2 or D). Although the limited number of isolates evaluated in the present study prevents firm epidemiological conclusions from being made, on a more global scale, these data demonstrate that extraintestinal isolates of E. coli can possess relatively distinct intra- and intergroup resistance gene profiles, with animal isolates presenting a more heterogeneous group than human isolates.

Animals↗

Nosocomial outbreak caused by multidrug-resistant Pseudomonas aeruginosa producing IMP-13 metallo-beta-lactamase.

An outbreak of Pseudomonas aeruginosa showing a multidrug-resistant (MDR) phenotype (including carbapenems, ceftazidime, cefepime, gentamicin, tobramycin, and fluoroquinolones) was observed, during a 5-month period, in a general intensive care unit of a large tertiary care and clinical research hospital in southern Italy. The outbreak involved 15 patients, with a total of 87 isolates, mostly from lower respiratory tract specimens. Analysis of isolates involved in the outbreak revealed production of metallo-beta-lactamase (MBL) activity, and genotyping by pulsed-field gel electrophoresis of genomic DNA digested by SpeI revealed clonal relatedness among isolates. Molecular analysis of the MBL determinant showed the presence of a bla(IMP-13) gene carried on a gene cassette inserted in a class 1 integron which also contained an aacA4 aminoglycoside resistance cassette encoding an AAC(6')-Ib enzyme. The bla(IMP-13)-containing integron and its genetic environment appeared to be similar to those found in P. aeruginosa isolates producing IMP-13 from a hospital in Rome. The bla(IMP-13) gene was not transferable by conjugation and was apparently carried on the chromosome. The outbreak was coincidental with a shortage of nursing personnel, and resolution was apparently associated with reinstatement of nursing personnel and reinforcement of general infection control practices within the intensive care unit. To our best knowledge this is the first description of a nosocomial outbreak of relatively large size caused by an IMP-producing gram-negative pathogen in Europe.

Cross Infection↗

Integronlike structures in Campylobacter spp. of human and animal origin.

Resistance to antimicrobial agents used to treat severe Campylobacter spp. gastroenteritis is increasing worldwide. We assessed the antimicrobial resistance patterns of Campylobacter spp. isolates of human and animal origin. More than half (n = 32) were resistant to sulphonamide, a feature known to be associated with the presence of integrons. Analysis of these integrons will further our understanding of Campylobacter spp. epidemiology.

Animals↗

Salmonella enterica serotype Typhimurium DT104 isolated from humans, United States, 1985, 1990, and 1995.

First isolated from an ill person in 1985, multidrug-resistant Salmonella enterica serotype Typhimurium DT104 emerged in the mid-1990s as a strain of Salmonella frequently isolated from humans in the United States. We compared the integron content, plasmid profile, and XbaI pulsed-field gel electrophoresis (PFGE) patterns of multidrug-resistant S. Typhimurium DT104 (MR-DT104) isolated from humans in the United States in 1985, 1990, and 1995. All isolates contained a 60-mDa plasmid and had indistinguishable PFGE and integron profiles, supporting the idea of a clonal relationship between recent and historical isolates. The data suggest that the widespread emergence of MR-DT104 in humans and animals in the 1990s may have been due to the dissemination of a strain already present in the United States rather than the introduction of a new strain.

Drug Resistance, Multiple, Bacterial↗

Variant Salmonella genomic island 1 antibiotic resistance gene cluster in Salmonella enterica serovar Albany.

Salmonella genomic island 1 (SGI1) contains an antibiotic resistance gene cluster and has been previously identified in multidrug-resistant Salmonella enterica serovars Typhimurium DT104, Agona, and Paratyphi B. We identified a variant SGI1 antibiotic-resistance gene cluster in a multidrug-resistant strain of S. enterica serovar Albany isolated from food fish from Thailand and imported to France. In this strain, the streptomycin resistance aadA2 gene cassette in one of the SGI1 integrons was replaced by a dfrA1 gene cassette, conferring resistance to trimethoprim and an open reading frame of unknown function. Thus, this serovar Albany strain represents the fourth S. enterica serovar in which SGI1 has been identified and the first SGI1 example where gene cassette replacement took place in one of its integron structures. The antibiotic resistance gene cluster of serovar Albany strain 7205.00 constitutes a new SGI1 variant; we propose a name of SGI1-F.

Anti-Bacterial Agents↗

Deciphering the genomic landscape of novel Acinetobacter non-baumannii lineages causing neonatal septicemia: carbapenem resistance and virulence.

BACKGROUND: Acinetobacter non-baumannii (Anb) species are reported worldwide to cause infections in both adults and neonates, although less frequently than Acinetobacter baumannii. However, limited information is available on their genomic diversity, resistance mechanisms, and virulence potential. This study investigates novel Anb isolates causing neonatal septicemia in India to characterize their resistance and pathogenic traits. METHODS: Anb isolates from neonatal blood cultures (2007-2025) were identified by VITEK2 Compact system, MALDI-TOF MS, and Whole-genome sequencing (WGS). Antimicrobial susceptibility was tested by VITEK2. Genomic analysis included MLST, resistome, virulome, plasmid typing, integrons, and core-genome phylogeny analysis. In vitro and in vivo studies assessed pathogenic potential of Anb species. RESULTS: Anb infections were low (11%) among the neonates during the study period. WGS revealed 11 novel Sequence Types (STs) which include A. indicus, A. variabilis, A. schindleri, and A. bereziniae. Six out of these eleven Anbs harbored carbapenemases such as bla NDM-1 and/or bla OXA-58-like genes (bla OXA-58, bla OXA-420). bla NDM-1 was acquired via Tn125 transposon. ISAba125 was located upstream of bla NDM-1, and a conserved structure extending to IS91 family transposase was detected in bla NDM-1-harboring genomes. bla OXA-58-like genes were found to be associated with ISAba3. Most carbapenemases were likely located on chromosome. Class 1 integrons carrying multiple antimicrobial resistance genes (ARGs) and diverse plasmid replicase families were detected in Anbs. Core genome phylogeny showed that the study Anbs were not closely related to the global Anbs. In vitro virulence-associated assays (biofilm formation, surface motility, adherence/invasion, apoptosis) and in vivo lethality in murine infection model showed reduced pathogenicity, reinforcing earlier observations that Anb species are generally less virulent than A. baumannii. Several virulence factors (VFs) were detected; however, no clear correlation was observed between virulence genes, in vitro pathogenicity, and in vivo lethality. CONCLUSION: These results indicate the multifactorial nature of Anb pathogenicity and the current limitations of knowledge of its VFs. However, the presence of numerous VFs suggests a capacity to cause disease, particularly in vulnerable host populations such as neonates. Furthermore, the presence of multiple ARGs indicates a strong potential for persistence and dissemination in hospital environments with high antibiotic pressure. Overall, these findings underscore the importance of continued AMR surveillance, genome characterization and further investigations into Anb pathogenicity.

Acinetobacter non-baumannii↗

Characterization of antimicrobial-resistant Salmonella isolated from imported foods.

Two-hundred eight Salmonella isolates recovered from over 5,000 imported foods entering the United States in 2001 were tested for antimicrobial susceptibilities and further characterized for quinolone resistance mechanisms, integron carriage, and genetic relatedness. Salmonella Weltevreden (20%), Salmonella Newport (6%), Salmonella Lexington (5%), and Salmonella Thompson (4%) were the four most common serotypes recovered. Twenty-three (11%) isolates were resistant to at least one antimicrobial, and seven (3.4%) to three or more antimicrobials. Resistance was most often observed to tetracycline (9%), followed by sulfamethoxazole (5%), streptomycin (4%), nalidixic acid (3%), and trimethoprim/sulfamethoxazole (2%). One Salmonella Schwarzengrund isolate recovered from squid imported from Taiwan exhibited resistance to eight antimicrobials, including ampicillin, chloramphenicol, gentamicin, kanamycin, nalidixic acid, sulfamethoxazole, tetracycline, and trimethoprim/sulfamethoxazole. Six isolates (Salmonella Bareilly, Salmonella Derby, Salmonella Ohio and three Salmonella Schwarzengrund) contained class 1 integrons, which carried several resistance genes including dhfrI/dhfrXII, aadA, pse-1, and sat1, conferring resistance to trimethoprim/sulfamethoxazole, streptomycin, ampicillin, and streptothricin, respectively. Five of six nalidixic acid-resistant isolates possessed DNA point mutations at either Ser83 or Asp87 in DNA gyrase. One ciprofloxacin-resistant isolate possessed double mutations in DNA gyrase at positions Ser83 and Asp87 as well as a single mutation at Ser80 in parC. The top three serotypes identified, Salmonella Weltevreden (n = 41), Salmonella Newport (n = 13), and Salmonella Lexington (n = 11), were further characterized for genetic relatedness by pulsed-field gel electrophoresis. Fifty-five distinct pulsed-field gel electrophoresis patterns were observed among the 65 isolates, indicating extensive genetic diversity among these Salmonella serotypes contaminating imported foods.

Anti-Bacterial Agents↗

[Gram-positive bacterial infections resistant to antibiotic treatment].

Antibiotic resistance has become a major problem in the treatment of gram-positive bacterial infections. These organisms are able to escape antibiotic activity through several mechanisms including beta-lactamase production, altered penicillin-binding proteins, aminoglycoside-modifying enzymes, modification of the target site of the antibiotic, and active efflux. Resistant determinants may be carried on the chromosome, and are transmitted vertically by clonal dissemination, or on mobile elements such as plasmids, transposons or integrons capable of horizontal transfer both within and between species. Several resistant genes can be inserted in a given integron, resulting in multi-drug resistance. Antimicrobial resistance mechanisms can be caused or induced following the widespread use of antibiotics. The most important gram-positive resistant organisms include penicillin-resistant Streptococcus pneumoniae, methicillin-resistant Staphylococcus aureus and coagulase-negative staphylococci, methicillin-resistant Staphylococcus aureus with intermediate resistance to vancomycin, and enterococcal strains that express high-level resistance to aminoglycosides and/or resistance to vancomycin. In several instances, these strains show multi-drug resistance and cannot be treated with currently available agents. Therapeutic strategies include the use of a higher antibiotic dosage, the use of alternative, non-conventional drugs, alone or in combination, after demonstration of their efficacy in vitro and in experimental animal models, and the development of new drugs. New agents available include quinupristin/dalfopristin and line-zolid with activity against most resistant gram-positive bacteria. Promising new drugs that may reach the market in the near future include daptomycin and oritavancin. Careful use of antibiotics and adherence to infection control standards are crucial in preventing the development and the spread of resistant organisms.

Drug Resistance, Microbial↗

The role of aquatic ecosystems as reservoirs of antibiotic resistant bacteria and antibiotic resistance genes.

The widespread and indiscriminate use of antibiotics has led to the development of antibiotic resistance in pathogenic, as well as commensal, microorganisms. Resistance genes may be horizontally or vertically transferred between bacterial communities in the environment. The recipient bacterial communities may then act as a reservoir of these resistance genes. In this study, we report the incidence of antibiotic resistance in enteric bacteria isolated from the Mhlathuze River and the distribution of genetic elements that may be responsible for the observed antibiotic resistance. The resistance of the enteric bacteria isolated over a period of one year showed that resistance to the older classes of antibiotics was high (94.7% resistance to one antibiotic and 80.8% resistance to two antibiotics). Furthermore, antibiotic resistance data of the environmental isolates showed a strong correlation (r = 0.97) with data obtained from diarrhoea patients. PCR based methods demonstrated that class 1 integrons were present in >50% of the environmental bacterial isolates that were resistant to multiple antibiotics. This class of integrons is capable of transferring genes responsible for resistance to beta-lactam, aminoglycoside, sulfonamide and quaternary ammonium antimicrobial agents. Conjugate plasmids were also isolated, but from a small percentage of isolates. This study showed that the Mhlathuze River (a) is a medium for the spread of bacterial antibiotic resistance genes, (b) acts as a reservoir for these genes and (c) due to socio-economic pressures, may play a role in the development and evolution of these genes along this river system.

DNA, Bacterial↗

[Mechanism of acquiring drug-resistance genes in pathogenic bacteria].

Pathogenic bacteria acquire resistance to chemotherapeutic agents by mutational events in the intrinsic genes or by incorporating foreign resistance genes. The resistance genes to various drugs were transferred among bacteria by transformation with free DNA, phage-mediated transduction, or cell to cell conjugation. Plasmids are capable of self-replication and self-transfer by conjugation. Drug-resistance genes are incorporated into plasmids by transposable elements, transposons. Several kinds of transposons carrying resistance genes to various drugs, or some transposons carrying multiresistance genes, are mobile among genetic elements and confer multiresistance on pathogenic bacteria. There is a specific element, integron, on transposons or plasmids. The integron has the gene and the site for incorporating resistance genes as cassettes and allows expression of the genes.

Bacteria↗

Association of different mobile elements to generate novel integrative elements.

Among the more important problems in modern hospitals is the prevalence of bacterial pathogens expressing resistance to multiple antimicrobial agents. The frequency of multiresistance suggests mechanisms by which bacterial species can concentrate and efficiently exchange a variety of resistance determinants. Mechanisms by which this occurs include insertion of transposons within transposons, coalescence through the activity of insertion sequences and the employment of integrons. In some instances, more than one of these mechanisms is involved in creating large multiresistance genetic elements. The association of the elements with transferable elements or transposons may promote rapid dissemination among clinical strains, and create further opportunities for inclusion of additional resistance determinants.

Bacteria↗

Phylogenetic analysis of bacterial isolates from man-made high-pH, high-salt environments and identification of gene-cassette-associated open reading frames.

Environmental samples were collected from high-pH sites in Pakistan, including a uranium heap set up for carbonate leaching, the lime unit of a tannery, and the Khewra salt mine. Another sample was collected from a hot spring on the shore of the soda lake, Magadi, in Kenya. Microbial cultures were enriched from Pakistani samples. Phylogenetic analysis of isolates was carried out by sequencing 16S rRNA genes. Genomic DNA was amplified by polymerase chain reaction using integron gene-cassette-specific primers. Different gene-cassette-linked genes were recovered from the cultured strains related to Halomonas magadiensis, Virgibacillus halodenitrificans, and Yania flava and from the uncultured environmental DNA sample. The usefulness of this technique as a tool for gene mining is indicated.

Alkalies↗

High prevalence of multidrug resistance and random distribution of mobile genetic elements among uropathogenic Escherichia coli (UPEC) of the four major phylogenetic groups.

One hundred and ten UTI Escherichia coli strains, from Ljubljana, Slovenia, were analyzed for antibiotic resistances, mobile DNA elements, serotype, and phylogenetic origin. A high prevalence of drug resistance and multidrug resistance was found. Twenty-six percent of the isolates harbored a class 1 integron, while a majority of the strains (56%) harbored rep sequences characteristic of F-like plasmids. int as well as rep sequences were found to be distributed in a random manner among strains of the four major phylogenetic groups indicating that all groups have a similar tendency to acquire and maintain mobile genetic elements frequently associated with resistance determinants.

Conjugation, Genetic↗

The chloramphenicol resistance gene cmlA is disseminated on transferable plasmids that confer multiple-drug resistance in swine Escherichia coli.

A recent study of beta-hemolytic Escherichia coli isolated from diarrheic swine found that 53% were resistant to chloramphenicol, a drug that has been prohibited from use in food animals in the US since the mid-1980s. To identify the factors governing the persistence of chloramphenicol resistance in the absence of specific selection pressure, the location of the chloramphenicol resistance gene cmlA and its linkage to other resistance determinants were investigated. Southern blot analysis of plasmid DNA from 46 swine E. coli isolates indicated that cmlA was present on large plasmids greater than 100 kbp. Fifty-two percent of the isolates were able to transfer chloramphenicol resistance to an E. coli recipient at conjugation frequencies ranging from 10(-3) to 10(-8) per recipient. Antimicrobial susceptibility tests on transconjugant strains demonstrated that resistance to sulfamethoxazole, tetracycline, and kanamycin frequently transferred along with chloramphenicol resistance. The transconjugant strains possessed at least two distinct class 1 integrons that linked cmlA to both aminoglycoside resistance genes aadA1 and aadA2 and either to sul1 or to sul3 sulphonamide resistance genes. These results suggest that in the absence of specific chloramphenicol selection pressure, the cmlA gene is maintained by virtue of gene linkage to genes encoding resistance to antimicrobials that are currently approved for use in food animals.

Animals↗