Drug sensitivity, conjugative R plasmids and plasmid profiles of Salmonella isolated from humans with infectious enteritis

Using 92 Salmonella strains isolated from patients suspected of having infectious diseases of the intestinal tract who visited 13 hospitals in Japan during the six years between 1991 and 1996, we investigated the drug susceptibility, prevalence of conjugative R plasmid, and the plasmid profiles. 1) Of the bacterial isolates tested, 52.2% showed drug-resistance. Regarding the drug-resistance patterns, 70.8% of the isolates were resistant to a single drug, while 29.2% were multi drug-resistant. 2) Dividing the resistance patterns by the serotypes, among Salmonella Enteritidis isolates, single-drug resistance to SM was the most frequent, being detected in 27 isolates. Single-drug resistance to NA and two-drug resistance to SM/TC were the second-most frequent, each being detected in isolates. Among Salmonella Hadar isolates, four isolates showed two-drug resistance to SM/TC, and one isolate showed single-drug resistance to TC. Among Salmonella Typhimurium isolates, one isolate each showed three-drug resistance to ABPC/CER/KM and KM/TC/CP. Among Salmonella Agona isolates, one isolate each showed two-drug resistance to SM/TC and single-drug resistance to SM. Among Salmonella Derby isolates, two isolates showed single-drug resistance to SM. 3) The prevalence of conjugative R plasmid was investigated in 48 drug-resistant isolates, and six isolates (12.5%) contained the plasmid. 4) The prevalence of the plasmid was investigated in 29 drug-resistant S. Enteritidis isolates, and 22 isolates (75.9%) contained the plasmid. These isolated were classified by the plasmid profiles into types H1 to H7. 5) Regarding the plasmid profiles of the S. Enteritidis isolates, a position corresponding to 60 Kbp was the most frequently detected in 90.5%.     

Outbreak case caused by different colicin type of Shigella sonnei in a day nursery in Tokyo (1998)

From October through December, 1998, person to person infection caused by Shigella sonnei had occurred in the day nursery in Hachioji-city Tokyo, and a total of 41 patients including 3 suspected cases and 3 carriers had been confirmed. Although one case was imported case which is one-year-old kindergarten child, the remaining was domestic case. Of patients, 33 cases (80.5%) were a three-year-old kindergarten children and their families, accounting for 20 and 13 cases, respectively. From the data of symptom onset of patients, epidemic could distinguish to the former part and the later part. The recur or reinfection, and re-detection case was observed in the later part. Clinical symptom of the patients was diarrhea (100%), fever (80%) and abdominal cramps (70%), and LVFX for adult or FOM for child was used for the therapy. In the colicin typing test and the antibiotic susceptibility test for 9 reagents, almost isolates in the former part were type 0 and susceptible, and MBC for FOM ranged from 6.25 to 100 micrograms/ml, whereas those in the later part was type 2 and resistant for the TC, and showed highly MBC for FOM with 50 or 100 micrograms/ml. However, both isolates showed same patterns in plasmid profiles and DNA fingerprints by RAPD analysis. On the other hand, the strain from imported case was also colicin 0, but it was different as regards resistant for ABPC and ST and two genetic analysis.     

Atypical enteropathogenic Escherichia coli strains: phenotypic and genetic profiling reveals a strong association between enteroaggregative E. coli heat-stable enterotoxin and diarrhea

The virulence profiles of most atypical enteropathogenic Escherichia coli (EPEC) strains are unknown. A total of 118 typical and atypical strains of EPEC serotypes and non-EPEC serogroups isolated from children with or without acute diarrhea who were from different cities in Brazil were examined for virulence-associated markers and adherence to HEp-2 cells, and also had random amplified polymorphic DNA (RAPD) analysis performed. Atypical strains were identical to typical strains with regard to the virulence factors encoded on the locus of enterocyte effacement (LEE). In contrast with typical EPEC strains, none of the atypical strains reacted with the bfpA probe, and half of the strains hybridized with the perA probe. Most atypical strains presented Tir sequences that correlated with enteropathogenic or enterohemorrhagic E. coli (98%), had LEE inserted in either selC or pheU (88%), and presented a typeable intimin (52%). Eighteen new serotypes were found in the EPEC strains. Atypical and typical EPEC strains belonged to different RAPD clusters. Most atypical strains showed a localized-like adherence pattern (61.5%). Of the non-LEE-encoded virulence factors, enteroaggregative E. coli heat-stable enterotoxin was noted most frequently (45%) and was significantly associated with diarrhea (P=.01). Thus, this virulence marker may be used as an additional tool for the diagnosis of truly atypical pathogenic strains.     

Detection and molecular characterization of Vibrio vulnificus from coastal waters of Malaysia.

A total of 57 Vibrio vulnificus isolates from coastal water were characterized for their antimicrobial resistance, plasmid profiles and were typed by the PCR-based techniques: a random amplification of polymorphic DNA (RAPD) method and the enterobacterial repetitive intergenic consensus sequence (ERIC) method. All isolates were susceptible to chloramphenicol, nalidixic acid, tetracycline and trimethoprim-sulfamethoxazole. Fifty-one isolates were resistant to one or more of the other antibiotics tested. Plasmid analysis indicated that only 18 isolates carried small plasmids of 1.6 to 16 megadaltons. Analysis of the RAPD and ERIC DNA fingerprints of the V. vulnificus isolates with Gel Compare and cluster analysis software revealed significant genetic heterogeneity among these isolates. The combination of RAPD and ERIC analysis allowed us to distinguish all isolates. Thus, the combination of the two techniques is recommended for epidemiological investigation.     

Antimicrobial resistance and conjugative R plasmids in Escherichia coli strains isolated from animals in Peninsular Malaysia

Fifteen independent E. coli strains of avian, bovine and porcine origin in Peninsular Malaysia were tested for antibiotic resistance and conjugative R plasmids. Eight (53%) isolates were found to be antibiotic resistant. Among them, 37.5% were mono-resistant and 62.5% were resistant to three or more antibiotics, i.e., multi-resistant. All of them were resistant to Tc and sensitive to Gm and Nx. Three of the eight antibiotic resistant strains were able to transfer all or part of their resistance to an E. coli K12 recipient by conjugation. The transfer frequencies of Km, Sm and Tc resistance of the three donors varied between 4.5 X 10(-8) to 6.8 X 10(-7). Analysis of the plasmid profiles of all the three donors and their respective transconjugants after agarose gel electrophoresis provided conclusive evidence that the transferable resistance traits were plasmid-mediated.     

Drug resistance of enterohemorrhagic Escherichia coli O157 and a possible relation of plasmids to the drug-resistance

Antimicrobial susceptibility was examined using 89 enterohemorrhagic Escherichia coli O157 isolates obtained from diarrhea patients in Aichi Prefecture, Japan between June 1996 and June 1997. Among the 89 isolates, 15 (16.9%) were found to be resistant to 6 of 9 antibiotics examined. These 6 antibiotics were ampicillin (ABPC), cefaloridine (CER), chloramphenicol (CP), kanamycin (KM), streptomycin (SM), and tetracycline (TC). Among the 15 drug-resistant isolates, 7 were resistant to 4 drugs (ABPC, CER, SM, TC), 3 were resistant to 3 (ABPC and 2 of CER, SM, TC), 2 were resistant to 2 (SM, TC), one each to KM or SM. Another isolate showed resistance to 5 drugs (ABPC, CP, KM, SM, TC). Selected 13 drug-sensitive and selected 12 multi-drug resistant isolates were tested for the presence of plasmids. All of the drug-sensitive isolates had 54 MDa plasmid and the majority (8/13) had 2.0 MDa plasmids, whereas; all of the drug-resistant isolates except one (1/12) had 54 MDa plasmid and the majority had 8.0 MDa (9/12) and 4.2 MDa (11/12) plasmids. The first transformation test revealed that plasmids of 8.0 MDa (3/4) and 46 MDa (1/4) were transferred to a donor cell with ABPC resistance. 54 MDa plasmid was transferred to a donor cell with both of ABPC and TC resistance. In the second transformation test, only the 8.0 MDa plasmid was confirmed to be transferred to a donor cell with ABPC resistance. Accordingly, it was indicated that the ABPC resistant gene was carried on 8.0 MDa plasmid, and it was suggested that resistant genes for ABPC and TC, and ABPC were carried on 54 MDa, and on 46 MDa plasmids, respectively.     

郑州市2006-2011年腹泻病人中沙门菌血清型和耐药谱分析

摘要：【目的】了解郑州市感染性腹泻病人中沙门病原菌血清型分布和耐药谱,为沙门菌防治提供基础数据。【方法】从2006-2011年腹泻病监测点感染性腹泻病例的粪便标本中进行沙门菌分离培养、血清学分型及药敏试验,用WHONET5.4软件分析药敏试验结果。【结果】2006-2011年共采集的3266份腹泻病人标本中共检出196株沙门菌,检出率为6.0%,分布于10个血清群,37个血清型;以B群和D1群为主,各占33.67%（66/196）和32.14%(63/196);血清型以肠炎沙门菌和鼠伤寒沙门菌为主,各占29.59%（58株）和19.90%（39株）。药敏试验结果提示对氨苄西林、萘啶酸、复方新诺明和四环素的耐药率分别达到44.90%、42.34%、34.18%和32.14%。【结论】沙门菌血清型和耐药谱监测对感染性腹泻疫情防控意义重大。     

Phage-associated cytotoxin production by and enteroadhesiveness of enteropathogenic Escherichia coli isolated from infants with diarrhea in West Germany

We assessed the frequency of isolating enteropathogenic Escherichia coli (EPEC) from the stools of infants with diarrhea, the enteroadhesiveness of the EPEC, their production of cytotoxin, and the association of cytotoxin synthesis with lysogenic phages. One hundred twenty-five isolates of EPEC obtained from 1,674 children with diarrhea; three were isolated from 868 controls. Thirty EPEC made elevated levels (greater than or equal to 10(4) 50% cytotoxic doses/mg of cell lysate protein) of a cytotoxin for HeLa cells, and cell-associated cytotoxicity for 27 of these isolates was neutralized by antibody to Shiga toxin. Cell lysates of these isolates were paralytic and lethal for mice. Phages from cytotoxin-producing strains were tested for toxin-converting capacity. Fifteen of 30 such strains harbored toxin-converting phages, and the cytotoxicity of 12 isolates of E. coli K12 transduced with these phages was neutralized by antibody to Shiga toxin. Fifty-seven EPEC exhibited either localized or diffuse adherence to HEp-2 cells, but only nine producers of elevated levels of cytotoxin were adherent.     

Functional characterization of a multiple-antibiotic resistant plasmid from clinical isolates of methicillin-resistant Staphylococcus aureus]

During surveillance done as part of the investigation of nosocominal infections due to methicillin-resistant Staphylococcus aureus (MRSA), we have been aware of the close relationship between the presence of certain plasmids and the characteristic patterns of antibiotic resistance. The hypothesis that a mechanism for the rapid and widespread dissemination of resistance to multiple aminoglycosides in clinical isolates of MRSA at our university hospital was the result of transfer of a single plasmid among these strains was examined. About 45% of the total isolates of MRSA (91/200 isolates) carried a 35.5 kb plasmid (designated pCL4) and these isolates always showed resistance to gentamicin, tobramycin, kanamycin, amikacin, astromicin, and arbekacin. Mating experiments between a susceptible strain and resistant MRSA isolates carrying pCL4 showed high frequency transfer of the plasmid. The aminoglycoside resistance patterns of all the transconjugants obtained corresponded well with those of parental strains. However, the plasmid could not necessarily be detected in the transconjugants, whereas the transformants obtained by means of electroporation usually possessed the plasmid. The plasmid-encoded aminoglycoside-resistance determinant, which has been identified as the gene aacA/aphD that encodes the bifunctional enzyme AAC (6')/APH(2"), either in the transconjugants and transformants could be transposed to their chromosomes in the absence of whole-plasmid integration resulting from a recombination event. Southern hybridization analysis using an aacA/aphD specific probe demonstrated that there are multiple sites of the insertions indistinguishable among the chromosomes of plasmid-free transconjugants and transformants. These results indicate that rapid dissemination of multiple-aminoglycoside-resistance in nosocominal strains of MRSA resulted from transfer of a conjugative plasmid and has been facilitated by translocations of the resistance gene to the chromosome.     

Plasmid mediated antibiotic resistance of Vibrio cholerae O1 biotype El Tor serotype Ogawa associated with an outbreak in Kolkata,India

ObjectiveTo determine the antibiotic resistance of Vibrio cholerae (V. cholerae) O1 biotype El Tor serotype Ogawa isolates involved in an outbreak of watery diarrhea in Kolkata, and to explore the role of plasmid in mediating antibiotic resistance.MethodsAntibiotic susceptibility and minimum inhibitory concentration (MIC) values of antibiotics for the isolated V. cholerae O1 Ogawa (n=12) were determined by disk diffusion and agar dilution methods, respectively, using ampicillin (Am), chloramphenicol (C), trimethoprim (Tm), tetracycline (T), erythromycine (Er), nalidixic acid (Nx), ciprofloxacin (Cp), amikacin (Ak) and cefotaxime (Cf). Plasmid curing of multidrug resistant (MDR) V. cholerae O1 Ogawa strains was done following ethidium bromide treatment. Following electrophoresis, the plasmid DNAs, extracted from the isolated MDR V. cholerae O1 Ogawa strains and their cured derivatives, were visualized and documented in ‘gel doc’ system.ResultsThe outbreak causing V. cholerae O1 Ogawa isolates were MDR as determined by disk diffusion susceptibility test, and MIC determination. The isolates showed three different drug resistance patterns: AmTmTErNx (for 6 isolates), TmTErCp (for 5 isolates), and AmTmNx (for one isolate), and showed uniform sensitivity to C, Ak and Cf. The loss of plasmids with the concomitant loss of resistance to Am, Tm, T and Er of the isolates occurred following ethidium bromide treatment.ConclusionsThe current findings suggest that the V. cholerae O1 Ogawa associated with the cholera outbreak were MDR, and resistance to Am, Tm, T and Er among the isolates were plasmid mediated.     

Resistance to antimicrobial agents, hemolytic activity and plasmids in Aeromonas species

A total of 174 Aeromonas isolates consisting of 100 strains from patients with diarrhea being mainly overseas travellers nd healthy subjects, and 74 strains from environmental sources including foods, fish, fresh water, sea water and river soil collected in the area of Tokyo Metropolis and Kanagawa Prefecture was examined for the antimicrobial resistance, presence of plasmids and hemolytic activity. Almost all the isolates (99.4%) were resistant to aminobenzyl penicillin. The isolation frequency of chloramphenicol- or tetracycline-resistant strain was low. Most environmental isolates of A. hydrophila were resistant to multiple antimicrobial agents. Thirty-seven percent of environmental isolates and 39% of human fecal ones carried plasmids. In environmental isolates, seven A. hydrophila and three A. sobria strains carried 63- to 150-kilobase pair (kb) conjugative R plasmids. Two A. hydrophila strains from both the healthy subject and domestic case with diarrhea carried 58- to 90-kb conjugative R plasmids, respectively. None of the isolates from the feces of overseas traveller's diarrhea carried the plasmid. Irrespective of the sources. A. hydrophila showed the highest hemolytic activity among three Aeromonas species. Eighty percent or more of A. hydrophila isolates were of hemolysin positive. The hemolytic titer of A. hydrophila strains from human feces was higher than that of the strains from environmental sources.     

The molecular epidemiological analysis of Shigella sonnei isolates from outbreak cases in Yokohama]

We conducted a molecular epidemiological analysis to evaluate the epidemiologic patterns of Shigella sonnei isolates from outbreak cases in Yokohama to clarify the epidemiologic linkages by contact tracing and sources of infection. In the first case (case A), all of the 6 isolates were the colicin 0 type and resistant to both streptomycin (SM) and trimethoprim/sulfamethoxazole (ST). The 5 isolates have plasmid of 230 kb. By RAPD analysis with 2 kinds of primers specific for Shigella, every 6 isolates showed the same pattern. But the DNA fingerprint analysis by PFGE that was performed according to 2 standardized restriction endonucleases revealed a discriminative pattern. However, the resemblance of all isolates, which was calculated by the UPGMA methods, was 0.90 or higher. In the second case (case B), all of the 14 isolates were the colicin 6 type and sensitive to 16 drugs. The serotype of 13 isolates was phase I. The 11 isolates have plasmids of 230 kb and 3 kb. The resemblance of all isolates, which was calculated by the UPGMA methods, was 0.89 or higher. The analysis with a combination of the plasmid, RAPD analysis and PFGE profiles may be effective in investigating detailed epidemiological features of isolates.     

Trends in antibiotic sensitivity pattern and molecular detection of tet(O)-mediated tetracycline resistance in campylobacter jejuni isolates from human and poultry sources

This study was conducted to determine the trends in Campylobacter antibiotic resistance occurring in our setting and to assess the differences in the isolates using patterns of plasmid profiles. One hundred Campylobacter jejuni strains of human and poultry origin isolated in 2002-2003 (phase A) and 2005-2006 (phase B) in the Kingdom of Bahrain were evaluated. Susceptibility to erythromycin, ciprofloxacin and tetracycline was determined, and plasmid extraction and polymerase chain reaction detection of the tet(O) gene was carried out. A single erythromycin-resistant isolate was identified, in sharp contrast to the high ciprofloxacin resistance which also showed an increment in phase B. Tetracycline resistance was higher in chicken (80.9%) compared to human (41.3%) isolates (P<0.01). Most isolates harbored two plasmids (23 kb and 35 kb) with significant correlation between tetracycline resistance and plasmid carriage in chicken isolates. The findings show continued effectiveness of erythromycin for campylobacteriosis but an increasing trend of high ciprofloxacin and tetracycline resistance. Tetracycline resistance is most likely due to the transfer of plasmids carrying the tet(O) gene between isolates.     

Determining of antibiotic resistance profile in Staphylococcus aureus isolates

ObjectiveTo determine the pattern of antibiotic resistance among Staphylococcus aureus (S. aureus) isolates from clinical specimens and to identify community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA) in specimens that have been collected from patients referring to one of the hospitals of Ahvaz.MethodsS. aureus isolates from a hospital in Ahvaz were screened for resistance to various antibiotics including methicillin. The susceptibility of the isolates was determined by Kirby-Bauer disc diffusion method. The MRSA was also treated with ethidium bromide to find the origin of resistance.ResultsAmong the bacterial isolates, all of 11 S. aureus were resistant to methicillin and cefixime, 2 were resistant to ciprofloxacine, 6 were resistant to tetracycline and the reminder were sensitive or intermediate to other antibiotics. The treated isolates were reminded resistant to methicillin and this suggested that the plasmid was not the origin of resistance in these isolates.ConclusionsThese results showed that infection due to MRSA is widespread in Ahvaz and with respect to the spread of vancomycin resistance among MRSA and appearance of overwhelming infections. It is necessary to identify continuously the profile of antibiotic resistance among S. aureus isolates in other regions and finding appropriate antibiotic for infection control and eradication.     

Analysis of amikacin-resistant Pseudomonas aeruginosa developing in patients receiving amikacin

During a 36-month period, 28 patients treated for infections due to amikacin-susceptible Pseudomonas aeruginosa subsequently developed infections or colonization with amikacin-resistant P aeruginosa at the same site. Eleven amikacin-susceptible/-resistant pairs of isolates were analyzed for aminoglycoside-inactivating enzymes, plasmid profiles, cellular proteins, outer membrane proteins (OMPs), lipopolysaccharide (LPS) profiles, and amikacin uptake. While clearly distinct from isolates of other patients, sensitive and resistant isolates from the same patients were indistinguishable in plasmid profile, LPS profiles, and OMPs. These results suggest that the resistant P aeruginosa isolates were derived from the sensitive isolates. None of the resistant isolates produced enzymes known to inactivate amikacin. In nine of 11 resistant isolates tested, transport of amikacin into P aeruginosa was reduced. A major mechanism of in vivo development of amikacin resistance in P aeruginosa is alteration in permeability to amikacin, but the aquisition of plasmids or changes in OMPs or LPS profile may not account for this phenomenon.     

The incidence and the valuation of eaeA gene of enteropathogenic Escherichia coli (EPEC) or aggR gene of enteroaggregative E. coli (EAggEC) in the strains isolated from patients in sporadic diarrhea cases

To clarify the pathogenic role of enteropathogenic Escherichia coli (EPEC) or enteroaggregative E. coli (EAggEC), the possession of eaeA gene of EPEC or aggR gene of EAggEC in the strains isolated from 525 patients in sporadic diarrhea cases during 3 years (1998-2000) in Tama, Tokyo was investigated by a PCR method. The eaeA-positive E. coli strains were confirmed from 23 cases including 5 cases detected verotoxin-producing E. coli (VTEC), and those except VTEC strains (18 cases, 3.4%) were the 5th predominant enteropathogen following rotavirus, Campylobacter, adenovirus, and Salmonella. By age, 17 eaeA-positive cases were from children < 10 years of age, and noticeably, of which 9 were from infants < 24 months of age. On the other hand, although aggR-positive E. coli strains were detected from 11 cases (2.1%), of which 6 also were from infants < 24 months of age. Clinical symptoms of patients whom eaeA or aggR gene-positive E.coli was isolated as the only potential enteric pathogen were similar, showing a mild gastroenteritic features. Only one strain of eaeA-positive E. coli and 4 of aggR-strains were typed with the commercial O-antisera, which were O55, and O86, O111 or O126. In antibiotic sensitivity tests for 9 agents, 22% of eaeA-strains and 91% of aggR-strains showed resistant, especially 10 aggR-strains had resistant to ABPC. These findings suggest that these organisms are a significant causative agents of infantile diarrhea and the PCR method is a useful procedure for the diagnosis of EPEC or EAggEC infectious disease.     

Penicillin-binding protein families: evidence for the clonal nature of penicillin resistance in clinical isolates of pneumococci

In view of the worldwide emergence of penicillin-resistant pneumococci among clinical isolates it was of importance to examine a large number of strains to test the uniformity of the resistance mechanism. Among 160 clinical isolates of pneumococci (minimum inhibitory concentration [MIC], 0.005-16 micrograms/mL), susceptible strains showed a common pattern of five penicillin-binding proteins (PBPs) with high penicillin affinities (PBP 3 greater than 1A greater than or equal to 2A greater than 1B greater than 2B). PBPs 1A, 2A, and 2B (but not PBP 3) each showed distinct stepwise decreases in penicillin affinities parallel with increasing levels of antibiotic resistance. The number and molecular sizes of PBPs became variable in strains with MIC values greater than 1.0 microgram/mL; among 39 strains with a MIC of greater than or equal to 1.0 microgram/mL, 11 distinct and stable PBP patterns could be identified. Using PBP profiles, serotypes, and antibiotic resistance patterns, as well as data on isolation dates and sites, we identified at least three groups of resistant strains that showed clear indication of clonal origin.     

Plasmids in Salmonella typhi in Lima, Peru, 1987-1988: epidemiology and lack of association with severity of illness or clinical complications.

A prospective study was conducted to better characterize the epidemiology of plasmid-bearing strains of Salmonella typhi in an endemic area of Lima, Peru, and to determine if plasmids were associated with specific manifestations of typhoid fever. Of 228 S. typhi isolated from patients at Cayetano Heredia University Hospital in Lima during 1987-1988, 76 (33%) contained plasmids. Ten different plasmid profiles were identified, with ten distinct plasmids present within these profiles. There was significant temporal clustering of isolates having common plasmid profiles. Two plasmids (both from the same isolate) carried antibiotic resistance genes. Two cryptic plasmids with approximate sizes of 55 and 65 kilobases (kb) appeared to be closely related, based on restriction endonuclease digestions and Southern blot analysis. An ampicillin resistance plasmid from a 1989 patient isolate differed by only a single restriction fragment from the cryptic 65-kb plasmid. No association was found between any plasmid or plasmid profile and severity or clinical manifestations of disease.     

Enterotoxins,colonization factors,serotypes and antimicrobial resistance of enterotoxigenic Escherichia coli (ETEC) strains isolated from hospitalized children with diarrhea in Bolivia

Enterotoxigenic Escherichia coli (ETEC) is recognized as the main cause of bacterial diarrhoea among children in Asia, Africa and Latin America but less investigated in Bolivia. Objective: To determine the relation between enterotoxins, CFs and serotypes as well as the antimicrobial resistance patterns in a set of ETEC isolates collected from hospitalized children with acute diarrhea. In the present study we characterized 43 ETEC strains isolated from 2002 to 2006 from hospitalized children (0–5 years) with acute diarrhea in Bolivia. The strains were analyzed for heat-labile (LT) and heat-stable (ST) enterotoxins and colonization factor (CF) profiles, as well as for serogroups and antimicrobial resistance using phenotypic (ELISA, dot blot, slide agglutination and disc diffusion) and genotypic (Multiplex PCR) methods. Among the ETEC isolates tested, 30 were positive for LT, 3 for STh and 10 for LT/STh. Sixty-five percent (28/43) of the strains expressed one or more CF. The most common CFs were CS17 (n = 8) and CFA/I (n = 8). The phenotypical and genotypical results for toxins and CFs were congruent except for CS21 that was amplified in 10 of the strains by multiplex PCR, but CS21 pili was only detected phenotypically in four of these strains. The ETEC strains had diverse O and H antigens and the most common types were O8:H9 LT CS17 (n = 6; 14%) and O78:HNM LT-ST CFA/I (n = 4; 9%). The analysis of antibiotic resistance showed that 67% (n = 29/43) of the strains were resistant to one or several of the antimicrobial agents tested. Presence of CFs was associated with antibiotic resistance. Conclusion: The most common toxin profile was LT 70%, LT/STh 23% and STh 7%. High antimicrobial resistance to ampicillin among serogroups O6, O8 and O78 were the most common.     

Prevalent phenotypes and antibiotic resistance in Escherichia coli and Klebsiella pneumoniae at an Indian tertiary care hospital: plasmid-mediated cefoxitin resistance.

BACKGROUND: The beta-lactam antibiotics, in combination with aminoglycosides, are among the most widely prescribed antibiotics. However, because of extensive and unnecessary use, resistance to these drugs continues to increase. In recent years, resistance in the Indian bacterial population has increased markedly, the majority showing complex mechanisms. Due to increased transcontinental movement of the human population, it would be wise to know the prevalence and resistance complexity of these strains, well in advance, in order to formulate a policy for empirical therapy. METHODS: One hundred and eighty-one isolates of Escherichia coli and 61 isolates of Klebsiella pneumoniae obtained from 2655 non-repeat samples of pus (912) and urine (1743) were studied, and their resistance rates and patterns were noted. The isolates were analyzed for prevalent aminoglycoside and cephalosporin resistance phenotypes and for the presence of extended spectrum beta-lactamase (ESBL) and AmpC enzymes by spot-inoculation and modified three-dimensional tests developed in our laboratory. Fourteen isolates of E. coli and six of K. pneumoniae, resistant to all of the antibiotics tested, were selected for plasmid screening, curing, and transconjugation experiments, and for comparative evaluation of the double disk synergy test (DDST) and modified three-dimensional test (TDT) for detection of beta-lactamases. RESULTS: Urinary E. coli isolates showed maximum susceptibility to amikacin (57.1%), followed by tobramycin (38.5%) and gentamicin (31.9%). Eighteen (19.8%) isolates were susceptible to cefotaxime, whereas 11 (12.1%) were susceptible to ceftriaxone. The K. pneumoniae isolates from urine samples showed maximum susceptibility to tobramycin (63.6%) followed by amikacin (54.5%). Of the K. pneumoniae isolates, 31.8% were susceptible to cefotaxime and 13.6% were susceptible to ceftriaxone. A more or less similar trend of antibiotic susceptibility was noted in E. coli and K. pneumoniae isolates from pus samples. Twenty-six (14.4%) E. coli and 15 (24.6%) K. pneumoniae isolates were found to be ESBL-producers by NCCLS-ESBL phenotypic confirmatory test. Eighteen (9.9%) E. coli and 19 (31.1%) K. pneumoniae isolates were found to be AmpC enzyme-producers by our modified TDT. The simultaneous occurrence of ESBL and AmpC enzymes was noted in 7.7% and 9.8% isolates of E. coli and K. pneumoniae, respectively. CONCLUSIONS: The prevalence of multidrug-resistant bacterial isolates is quite high in our bacterial population. On comparative evaluation of DDST and TDT in resistant isolates, TDT was found to be the better method, detecting ESBLs in 80% of isolates compared to 15% with DDST. A 19.9-kb plasmid was consistently present in all the screened isolates of E. coli and K. pneumoniae, and was inferred to encode cefoxitin and tetracycline resistance based on curing and transconjugation experiments.