Campylobacter Jejuni Isolates Research Articles

Low-Level Fluoroquinolone Resistance among Campylobacter jejuni Isolates in Australia

Ciprofloxacin-resistant Campylobacter jejuni isolates obtained from infected patients in Australia have not been detected in studies of isolates from specific geographic areas. The Australian government has prohibited the use of fluoroquinolone in food-producing animals. To assess the impact of this policy, we have examined the antimicrobial susceptibility of isolates from 5 Australian states. We conducted a period-prevalence survey of the susceptibility of C. jejuni isolates to 10 antimicrobial agents. C. jejuni isolates obtained from 585 patients from 5 Australian states (Queensland, South Australia, Tasmania, Victoria, and Western Australia) were identified by means of notifiable disease databases and were systematically selected from September 2001 to August 2002. Among locally acquired infections, only 2% of isolates (range, 0%-8% in different states) were resistant to ciprofloxacin. The locally acquired isolates also exhibited resistance to sulfisoxazole (55%), ampicillin (46%), roxithromycin (38%), tetracycline (7%), nalidixic acid (6%), chloramphenicol (3%), erythromycin (3%), gentamicin (2%), and kanamycin (0.2%). Treatment with antimicrobial agents in the 4 weeks before onset was not associated with ciprofloxacin resistance. The very low level of ciprofloxacin resistance in C. jejuni isolates likely reflects the success of Australia's policy of restricting use of fluoroquinolones in food-producing animals.

Amplified Fragment Length Polymorphism, Serotyping, and Quinolone Resistance of Campylobacter jejuni and Campylobacter coli Strains from Chicken-Related Samples and Humans in Taiwan

The high-resolution genotyping method of amplified fragment length polymorphism (AFLP) analysis was used to study the genetic relationships between Campylobacter jejuni isolates from chicken-related samples (n = 32) and humans (n = 27) as well as between Campylobacter coli isolates from chicken-related samples (n = 27) and humans (n = 5). These isolates were collected between 1994 and 2003 in Taiwan. All C. jejuni and C. coli isolates showed highly heterogeneous fingerprints. C. jejuni isolates were separated in two distinct genetic clusters (A and B) at 40% genetic similarity and 42 different AFLP types at 90% similarity. However, three clusters at 40% genetic similarity and 33 different AFLP types at 90% similarity were observed in C. coli isolates. These results showed that AFLP analysis could be used to identify individual isolates of two Campylobacter species. Among C. jejuni isolates, the predominant AFLP type 1 was observed in five (7.9%) isolates, and types 5 and 12 in four (6.3%) isolates each. Cluster B consisted of 10 isolates, while the majority of isolates (n = 53) belonged to cluster A. In some AFLP types (1, 5, 12, 14 and 31), AFLP fingerprints of chicken-related isolates were closely related genetically to those of isolates from humans with gastroenteritis. The predominant serotypes in C. jejuni isolates were B:2 and Y:37. All isolates belonging to serotype O:19 grouped into one single AFLP type. Some chicken samples yielded multiple isolates of Campylobacter harboring simultaneously quinolone-resistant and quinolone-sensitive isolates attributed to the same species, or harboring C. jejuni and C. coli that have the characteristics of quinolone resistance.

Prevalence of Thermophilic Campylobacter Infections in Humans, Chickens and Crows in Morogoro, Tanzania

Prevalence of thermophilic Campylobacter infections in humans, chickens and crows was determined in a cross-sectional study that was carried out in urban and rural areas of Morogoro region, Tanzania during the period of January 2003 to December 2004. A total of 632 human stool samples, 536 cloacal swabs from local and broiler chickens and 22 intestinal contents from crows were screened for presence of thermophilic campylobacters using Skirrow's protocol. Representative Campylobacter jejuni isolates from human and chicken samples were also analysed by polymerase chain reaction (PCR) as a definitive identification method. The overall prevalence of thermophilic campylobacters was 9.3% (95% CI: 7.2-11.9), 69.8% (95% CI: 65.7-73.6) and 72.7% (95% CI: 49.8-89.3) in humans, chickens and crows respectively. In humans, 59 thermophilic campylobacters were isolated of which 96.6% were C. jejuni and 3.4%Campylobacter coli. There was a significantly (P<0.001) higher prevalence in young individuals (16%) than in adults (7%). Of 341 isolates from chickens, 91.2% were C. jejuni and 8.8% were C. coli. A significantly (P<0.05) higher infection rate was observed in rural local chicken (76%) than in broilers (60%). In crows, of 16 isolates, 93.8% were C. jejuni and 6.2% were C. coli. Definitive identification of C. jejuni by PCR revealed positive results in 74.1% of 243 analysed isolates. Findings in this study indicate high prevalence of thermophilic campylobacters in humans, chickens and crows in Morogoro, and a higher infection rate of C. jejuni than that of C. coli in different animal species. Age of humans and location of chickens were identified as risk factors for thermophilic Campylobacter infections. Positive isolates to biochemical tests that indicated negative results on PCR indicates the additional value of PCR for definitive diagnosis of C. jejuni.

Phenon cluster analysis as a method to investigate epidemiological relatedness between sources of Campylobacter jejuni

To develop a method for assessing the relative epidemiological significance of possible infection sources for human campylobacteriosis. Using fluorescent amplified fragment length polymorphism (AFLP), 243 apparently epidemiologically unrelated Campylobacter jejuni isolates were genotyped (77 human, 46 cattle, 49 pet and 71 poultry isolates). In total 136 different phena were identified, of which 48 were clusters grouping at least two isolates. Isolates from different sources were frequently clustered together, underlining the high degree of source mixing and the lack of host specificity of C. jejuni. The phena were classified into different phenon types according to the sources of the isolates they contained. The occurrence of these phenon types was analysed using an area-proportional Euler diagram to describe epidemiological relatedness among C. jejuni isolates. Group separation statistics revealed that 43% of analysed human isolates expressed maximum similarity to other human isolates, 9% to cattle isolates, 21% to pet isolates and 27% to poultry isolates; these results were in accordance with the pattern observed in the phenon cluster analysis. Based on the grouping of strains into molecular similarity clusters, ecological patterns between sources can be investigated. This approach is a new methodological contribution to establish the relative epidemiological significance of concurrent infection sources.

PCR detection of virulence-associated genes in Campylobacter jejuni strains with differential ability to invade Caco-2 cells and to colonize the chick gut

In this study, the presence of 20 putative virulence genes was examined in 11 Campylobacter jejuni isolates with different colonization and invasion abilities as determined in a chick colonization model and on Caco-2 cells, respectively. The majority of the genes were detected in all strains. Among them, there were genes of the flagellar secretion apparatus like flhA, flhB, flgB, flgE2, the flagellin genes flaA and flaB, invasion-associated genes like ciaB and iamA, the cytotoxin genes cdtA-C, the adhesion related gene cadF, and some genes involved in the colonization process (docA, docB). The plasmid gene virB11 could not be detected in any strain. Specific differences between the isolates were observed only in genes cgtB and wlaN involved in lipo-oligosaccharide (LOS) biosynthesis. The gene cgtB was only detectable in three of five strains with strong colonization and invasion abilities. Probably, wlaN can overcome the lack of cgtB in the two cgtB- isolates.

Comparison of Campylobacter isolated from humans and food-producing animals in Japan

To compare the antimicrobial resistance, serotypes and flagellin gene types of Campylobacter isolated from humans and food-producing animals and thereby facilitate elucidation of the origin of Campylobacter causing human infection in Japan. The MIC values of ampicillin, dihydrostreptomycin, gentamicin, erythromycin, oxytetracycline, nalidixic acid and enrofloxacin for Campylobacter isolated from humans (134 isolates), cattle (38 isolates), pigs (69 isolates), layers (84 isolates) and broilers (51 isolates) were compared. The MIC(90) values of ampicillin for Campylobacter jejuni isolates from poultry were higher than those from humans and cattle. Campylobacter coli that was resistant to dihydrostreptomycin and erythromycin was observed at a higher frequency in humans and pigs than in poultry. The restriction fragment profiles of flaA of human, bovine and broiler isolates were analysed by clustering, and the isolates were classified into five clusters. Cluster I contained only human and bovine isolates. Clusters III, IV and V contained human, bovine and broiler isolates. Campylobacter isolates from humans included isolates that exhibited characteristics identical to those of the bovine, porcine and poultry isolates. In addition to poultry, cattle and pigs are believed to be sources of campylobacteriosis in Japan.

Real-time single-nucleotide polymorphism profiling using Taqman technology for rapid recognition of Campylobacter jejuni clonal complexes

The rapid identification of Campylobacter jejuni isolates to strain level would significantly inform the public health investigation of C. jejuni infection. Conceptual advances provided by multilocus sequence typing (MLST) have established the clonal complex as an important epidemiological group at the strain level, enabling accurate and phylogenetically valid strain identification for C. jejuni. The development of real-time PCR assays for allelic discrimination of strain-associated single-nucleotide polymorphisms (SNPs) based upon MLST locus alleles offers one possible approach for rapid strain detection. SNPs defining key alleles diagnostic for the most prevalent clonal complexes were identified following a detailed analysis of the available MLST data. Real-time Taqman allelic discrimination assays designed to detect the SNPs specific for six major clonal complexes, ST-21, ST-45, ST-48, ST-61, ST-206 and ST-257, were developed, allowing the rapid detection of C. jejuni isolates and preliminary strain identification. This will provide an important complementary technique to sequence typing for rapid detection and strain characterization to inform in real-time the public health management and investigation of C. jejuni infections.

Genetic Diversity of Campylobacter sp. Isolates from Retail Chicken Products and Humans with Gastroenteritis in Central Michigan

Multilocus sequencing was used to compare Campylobacter sp. strains isolated from retail chicken products and humans with gastroenteritis in central Michigan. Sequence comparisons demonstrated overlapping diversity between chicken and human isolates. Campylobacter jejuni isolates from clinical sources had a greater diversity of flagellin alleles and a higher rate of quinolone resistance than isolates from retail chicken products.

A newly constructed primer pair for the PCR amplification, cloningand sequencing of the flagellin (flaA) gene from isolatesof urease-negative Campylobacter lari

A newly constructed primer pair (lari-Af/lari-Ar) designed to generate a product of the flagellin (flaA) gene for urease-negative Campylobacter lari produced a PCR amplicon of about 1700 bp for 16 isolates from 7 seagulls, 5 humans, 3 food animals and one mussel in Japan and Northern Ireland. Nucleotide sequencing and alignments of the flaA amplicons from these isolates demonstrated that the deduced amino acid sequences of the possible open reading frame were 564-572 amino acid residues in length with calculated molecular weights of 58,804 to 59,463. The deduced amino acid sequence similarity analysis strongly suggested that the ORF of the flaA from the 16 isolates showed 70-75% sequence similarities to those of Campylobacter jejuni isolates. The approximate Mr of the flagellin purified from some of the isolates of urease-negative C. lari was estimated to range from 59.6 to 61.8 kDa. Thus, flagellin from the isolates of urease-negative C. lari was shown for the first time to have a molecular size similar to those of C. jejuni and Campylobacter coli isolates, but to be different from the shorter flaA and smaller flagellin of urease-positive thermophilic Campylobacter (UPTC) isolates. Flagellins from C. lari spp., consisting of the two representative taxa of urease-negative C. lari and UPTC, thus show genotypic and phenotypic diversity.

Prevalence of potential virulence markers in Polish Campylobacter jejuni and Campylobacter coli isolates obtained from hospitalized children and from chicken carcasses

The pathogenicity of thermotolerant Campylobacter species, common food-borne pathogens, depends on certain factors unevenly distributed among strains of different origin. The prevalence of such markers has never been examined in a population of Polish Campylobacter strains of human and poultry origin. Therefore, we analysed the presence of the cadF, cdtA, cdtB and cdtC genes and the iam sequence in Campylobacter jejuni (n = 115) and Campylobacter coli (n = 57) isolates from children with diarrhoea and from chicken carcasses. The cadF gene was present in nearly 100% of Campylobacter isolates tested, regardless of their origin or species. In contrast, the iam region was found in 83.3% and 100% of C. coli isolates from children and chickens, respectively, but in only 1.6% and 54.7%, respectively, of C. jejuni isolates. Similarly, the detection rates of cdt genes varied between human and chicken isolates. All three cdt genes were found in nearly all C. jejuni isolates from both children and chickens, but in only 5.6% of human C. coli isolates as compared to 87.2% of chicken C. coli isolates. This different distribution of genetic markers between human and chicken isolates indicates that some Campylobacter infections in children may have additional sources other than contaminated chicken meat.

Antimicrobial resistance patterns of Campylobacter from feedlot cattle*

This study examined 448 Campylobacter strains isolated in 1999 and 2000 from US feedlot cattle for resistance to 12 antimicrobials. Isolates were tested for antimicrobial susceptibility using the E-test method. Approximately 60% (n = 267) were resistant to one or more antimicrobials, and 19.6% (n = 88) were resistant to two or more antimicrobials. Of the Campylobacter jejuni isolates, 49.1% (n = 187) were resistant to tetracycline, 10.2% (n = 39) were resistant to nalidixic acid, 8.4% were resistant to trimethoprim/sulfamethoxazole, and 1.8% (n = 7) were resistant to ciprofloxacin. Resistance to any of the other eight antimicrobials was 1.3% or less, but 14.4% (n = 55) were resistant to two or more antimicrobials. In the Campylobacter coli group, 65.7% (n = 44) were resistant to tetracycline, 52.2% (n = 35) were resistant to trimethoprim/sulfamethoxazole, 22.4% (n = 15) were resistant to nalidixic acid, and 9.0% (n = 6) were resistant to ciprofloxacin. Resistance to any of the remaining eight antimicrobials was 3.0% or less, although 49.3% (n = 33) were resistant to two or more antimicrobials. Although antimicrobials are widely used in US feedlot cattle production, our results demonstrate generally low levels of resistance to a broad range of commonly used antimicrobials relative to other recent studies. Resistance data on Campylobacter isolated from this major US livestock commodity is lacking. This overview enhances current knowledge and provides a basis for further studies.

Genotyping of Campylobacter jejuni strains from Danish broiler chickens by restriction fragment length polymorphism of the LPS gene cluster

To apply and evaluate LG (LPS genes) genotyping, which is a genotyping method based on a cluster of genes involved in the synthesis of surface lipopolysaccharides (LPS) in Campylobacter species, for typing of Campylobacter jejuni isolates obtained from Danish broiler chickens. Furthermore, the LG genotyping method was used to study the genetic stability of four C. jejuni strains after gastrointestinal passage through experimentally infected chickens. In the present study, the LG genotyping method was modified with respect to the restriction enzymes used. To validate the method, 63 Penner serotype reference strains and 107 C. jejuni chicken isolates, representing the most common Penner serotypes of C. jejuni in Danish poultry, were selected for typing. The method was successfully used for typing all isolates and the LG genotype profiles were reproducible. There were no changes in the LG genotype of the C. jejuni strains obtained after experimental passage through chickens. All C. jejuni strains obtained from broiler chickens were typeable by the LG genotyping method. Application of the RsaI restriction enzyme improved the method in terms of ease and consistency of analyses and increase of discriminatory power. The LG genotyping method is a valuable tool for typing C. jejuni isolates obtained from poultry. However, the association between Penner serotyping based on passive haemagglutination of heat-stable antigens and LG genotyping was low when applied to poultry isolates. This is in contrast to previous studies on isolates of human origin that reported a high correlation between results obtained by the two typing methods (Shi et al. 2002).

Genotype dynamics of Campylobacter jejuni in a broiler flock

We investigated the genotype diversity and dynamics of Campylobacter in a commercial broiler flock during rearing and slaughter. In total, 220 Campylobacter jejuni isolates collected on four sampling occasions during rearing and from routine sampling during slaughter were subtyped by SmaI macrorestriction and pulsed-field gel electrophoresis, PFGE. Eight different SmaI types were found. During rearing, a subsequent addition of genotypes occurred, with two SmaI types found at 2 weeks of age and six types on the day before slaughter. All types that were detected in more than one isolate were also found on all succeeding sampling occasions, including the slaughter sampling. Two new types were found in the slaughter samples. In two-thirds of the individual birds sampled the day before slaughter, more than one SmaI type were found, although there was a clear tendency for dominance of one type in individual birds. Our results show that multiple genotypes of C. jejuni may be present in a commercial broiler flock during rearing and even in gastrointestinal tracts of individual birds. Both recurring environmental exposure and genetic changes within the population may explain the genotype diversity. Although the distribution of genotypes varied between different sampling occasions, we found no indication that any subtype excluded another during the rearing of the broiler flock.

Detection of Seven Virulence and Toxin Genes of Campylobacter jejuni Isolates from Danish Turkeys by PCR and Cytolethal Distending Toxin Production of the Isolates

A total of 117 Campylobacter jejuni isolates from Danish turkeys were tested for the presence of seven virulence and toxin genes by PCR. One hundred seventeen (100%) isolates were positive for flaA, cadF, and ceuE gene primers. One hundred three (88%) isolates were positive for cdt gene cluster PCR detection (cdt gene cluster–PCR), whereas 101 (86.3%), 102 (87.2%), and 110 (94%) isolates were positive for cdtA-, cdtB-, and cdtC-PCR, respectively. Only 39 (33.3%) isolates were positive for virB11. Of 117 isolates, 114 (97.4%) produced cytolethal distending toxin (CDT) in Vero cell assays, 105 (89.7%) in Colon 205 assays, and 109 (93.2%) in chicken embryo cell assays. The CDT titers were determined in Vero cell assays. Of 117 isolates, 50 (42.7%) produced a CDT titer of 1:100, 29 (24.8%) of 1:50, and 27 (23%) of 1:5 to 1:10; 8 (6.8%) produced a CDT titer at undiluted supernatants and 3 (2.6%) produced no toxin. Twenty-nine C. jejuni isolates that were PCR negative for one or more individual cdt toxin genes also produced low or no CDT toxin. The high prevalence of the seven virulence and toxin genes demonstrates that these putative pathogenic determinants are widespread among Campylobacter isolates from turkeys and calls for further investigation for the elimination of Campylobacter infection in industrial turkey production and in industrial food chains.

Salmonella Amager, Campylobacter jejuni, and urease-positive thermophilic Campylobacter found in free-flying peregrine falcons (Falco peregrinus) in Sweden.

Rare species with small population sizes are vulnerable to perturbations such as disease, inbreeding, or random events. The threat arising from microbial pathogens could be large and other species could act as reservoirs for pathogens. We report finding three enteric bacterial species, Salmonella Amager, Campylobacter jejuni, and urease-positive thermophilic Campylobacter, in nestling free-flying peregrine falcons (Falco peregrinus) in Sweden in 2000. Campylobacter jejuni isolates exhibited marked genetic similarities to an isolate from a human, providing a possible association between a human-associated strain of this bacterium and peregrine falcons.

Genetic and epidemiological relationships among Campylobacter isolates from humans.

84 Campylobacter jejuni isolates from Swedish patients with domestic infection were characterized with pulsed-field gel electrophoresis (PFGE), and the subtype information considered in relation to epidemiological data. Based on pattern combinations from restriction cleavage with SmaI and SalI, 52 different PFGE types were identified. Types with an average pattern similarity of at least 82% and 63% were assembled in groups and clusters, respectively. The 2 largest clusters included 71% of the isolates. The distribution in time varied between different groups and clusters, where some were isolated sporadically during the whole period and others appeared more concentrated in time. Types in 1 cluster were significantly more often isolated in summer than other types in the study. Isolates from children showed lower pattern similarity to other isolates than isolates from adults. Sets of type and time related cases, possibly representing small outbreaks, were identified when indistinguishable PFGE patterns were found in isolates from temporally related cases. Our results indicate that although a large number of genotypes may be found among C. jejuni strains infecting humans, a large proportion of these may be genetically related, and that different genotypes may appear during different seasons and infect individuals of different ages.

Determination of toxicity of Campylobacter jejuni isolated from humans and from poultry carcasses acquired at various stages of production.

The research focused on the determination of the toxicity variation associated with Campylobacter jejuni isolated from humans and chickens. Campylobacter jejuni isolates were obtained from chicken carcasses and from humans exhibiting symptoms of campylobacteriosis. Using HeLa cells as the in vitro model, toxicity was determined for each isolate. The mean toxicity level of the chicken isolates was significantly lower than that of the human isolates (P < 0.001). There was a wide range of toxicity in C. jejuni isolated from chickens and the percentage of isolates exhibiting low toxicity remaining relatively constant. All C. jejuni isolates from humans possessed either medium or high levels of toxicity. All wildtype C. jejuni isolates obtained from poultry carcasses may not be equally important as a human foodborne pathogen. Campylobacter jejuni remains a primary foodborne pathogen and increased efforts are needed to determine the impact of wildtype isolates in causing human illness. The present research indicates that all isolates may not be equally important in regards to disease potential. The information found should be included in efforts to develop C. jejuni detection, control and infection modelling.

Campylobacter infection in 682 bulgarian patients with acute enterocolitis, inflammatory bowel disease, and other chronic intestinal diseases

The aim of the study was to assess Campylobacter infections in 309 patients with acute enterocolitis, 272 patients with relapses of chronic enterocolitis, 70 patients with inflammatory bowel disease (involving Crohn's disease and ulcerative colitis) and 31 patients with other chronic intestinal illnesses. Isolation and identification were performed conventionally. Limited agar dilution method was used for susceptibility testing of the strains. Campylobacter species were isolated in patients with acute enterocolitis (7.8%), chronic enterocolitis (6.2%), Crohn's disease (6.2%), ulcerative colitis (3.7%), and irritable bowel syndrome (8.3%). Hippurate-positive Campylobacter jejuni isolates accounted for 62.2% of Campylobacter strains. One tetracycline resistant Campylobacter upsaliensis isolate was detected from a girl with acute enterocolitis. Resistance rates to erythromycin (31.1%) and clarithromycin (22.2%) were high, whereas those to amoxicillin/clavulanate (4.4%), ampicillin/sulbactam (13.3%), tetracycline (24.4%) and ciprofloxacin (22.2%) were relatively low. Resistance to erythromycin and either tetracycline or ciprofloxacin was detected in 8.9% and 6.7%. The involvement of Campylobacter infection in relapses of chronic intestinal disorders and the susceptibility patterns of the strains strongly emphasize the role of Campylobacter as a cause of infection in this group of patients.

Analysis of gyrA mutations in quinolone-resistant and -susceptible Campylobacter jejuni isolates from retail poultry and human clinical isolates by non-radioactive single-strand conformation polymorphism analysis and DNA sequencing

The aims of this study were to characterize the molecular variations in the quinolone resistance-determining region (QRDR) of gyrA among quinolone-resistant and -susceptible Campylobacter jejuni isolates originating from foods of animal origin and human infections and to evaluate the suitability of the single-strand conformation polymorphism (SSCP) method as a screening method for molecular characterization of fluoroquinolone resistance. Alterations in QRDR of gyrA from 182 C. jejuni isolates were determined by nonradioisotopic SSCP analysis and direct sequencing. A total of 13 types of nucleic acid sequence combinations within the QRDR of the gyrA gene resulted in 11 different SSCP patterns. All nalidixic acid resistant strains possessed nucleotide substitution at either codon Thr-86 or Asp-90. Silent mutations were detected additionally. Thr-86 to Ile mutation was detected in all 139 ciprofloxacin resistant strains, which showed cross-resistance to nalidixic acid. The SSCP method is suitable for a molecular screening of quinolone resistant C. jejuni isolates and in combination with DNA sequencing suitable to detect genetic variations of the QRDR of gyrA. This study provides data of the genetic variations of the QRDR of gyrA from C. jejuni isolates of foods and human beings.

Phenotypic characterisation of flagellin and flagella of urease-positive thermophilic campylobacters

In this study, flagellin is purified biochemically from eight urease-positive thermophilic camplylobacters (UPTC) isolated from river water, sea water and mussels, and purified also from two isolates of Campylobacter jejuni and C. coli and fractionated by sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE). Results showed that no flagellin components were detected in the two Japanese UPTC isolates (CF89-12 and CF89-14) and the two UPTC NCTC strains (NCTC12893 and NCTC12894). Flagellin components, each consisting of a single peptide, with a heterogeneous molecular mass of approximately 52–63 kDa were demonstrated in the other four UPTC isolates (NCTC12892, NCTC12895, NCTC12896 and NI15F [from Northern Ireland]) and the two Japanese isolates of C. jejuni (JCM2013 and C. coli 27). The approximate molecular mass of flagellin from the flagellin-positive UPTC isolates was smaller than those of C. jejuni and C. coli. Flagella were not detected by electron microscopy in the four flagellin-negative UPTC isolates but they were detected in the four flagellin-positive UPTC isolates and the two isolates of C. jejuni and C. coli. Thus, significant phenotypic diversity for flagellin, which must be due to genotypic variations, was demonstrated among the UPTC isolates.