The Type III Secretion System Effector SeoC of Salmonella enterica subsp. salamae and S. enterica subsp. arizonae ADP-Ribosylates Src and Inhibits Opsonophagocytosis.

Salmonella species utilize type III secretion systems (T3SSs) to translocate effectors into the cytosol of mammalian host cells, subverting cell signaling and facilitating the onset of gastroenteritis. In this study, we compared a draft genome assembly of Salmonella enterica subsp. salamae strain 3588/07 against the genomes of S. enterica subsp. enterica serovar Typhimurium strain LT2 and Salmonella bongori strain 12419. S. enterica subsp. salamae encodes the Salmonella pathogenicity island 1 (SPI-1), SPI-2, and the locus of enterocyte effacement (LEE) T3SSs. Though several key S Typhimurium effector genes are missing (e.g., avrA, sopB, and sseL), S. enterica subsp. salamae invades HeLa cells and contains homologues of S. bongori sboK and sboC, which we named seoC SboC and SeoC are homologues of EspJ from enteropathogenic and enterohemorrhagic Escherichia coli (EPEC and EHEC, respectively), which inhibit Src kinase-dependent phagocytosis by ADP-ribosylation. By screening 73 clinical and environmental Salmonella isolates, we identified EspJ homologues in S. bongori, S. enterica subsp. salamae, and Salmonella enterica subsp. arizonae The ß-lactamase TEM-1 reporter system showed that SeoC is translocated by the SPI-1 T3SS. All the Salmonella SeoC/SboC homologues ADP-ribosylate Src E310 in vitro Ectopic expression of SeoC/SboC inhibited phagocytosis of IgG-opsonized beads into Cos-7 cells stably expressing green fluorescent protein (GFP)-FcγRIIa. Concurrently, S. enterica subsp. salamae infection of J774.A1 macrophages inhibited phagocytosis of beads, in a seoC-dependent manner. These results show that S. bongori, S. enterica subsp. salamae, and S. enterica subsp. arizonae share features of the infection strategy of extracellular pathogens EPEC and EHEC and shed light on the complexities of the T3SS effector repertoires of Enterobacteriaceae.

Genetic evolution of the Spanish multidrug-resistant Salmonella enterica 4,5,12:i:- monophasic variant.

We analyzed a collection of 60 Salmonella enterica 4,5,12:i:- phage type U302 multidrug-resistant monophasic variant strains, isolated in Spain between 2000 and 2007. Most strains showed resistance to ampicillin (A), chloramphenicol (C), sulfamethoxazole (Su), gentamicin (G), streptomycin (S), tetracycline (T), and co-trimoxazole (SxT) (an ACSuGSTSxT resistance pattern). Only one pulsed-field gel electrophoresis (PFGE) type was detected, with 19 subtypes (Simpson's index of diversity [SID]=0.89). Multiple-locus variable-number tandem-repeat analysis (MLVA) showed more variability, with 32 profiles (SID=0.97), but only showed diversity at the STTR5 and STTR6 loci. PCR and sequencing demonstrated all strains contained the same allantoin-glyoxylate pathway deletion. Four types of deletions were detected in the fljAB operon, all starting at the same position, at the STM2758 gene, and followed by an IS26 insertion. Furthermore, a representative set of strains of the four deletion types harbored plasmids with IS26. We propose that a Salmonella enterica serotype Typhimurium U302 multidrug-resistant (ACSuGSTSxT) strain, defective for the allantoin-glyoxylate pathway and containing IS26 at plasmid pU302L, could be the ancestor of the variant in Spain.

Antimicrobial drug resistance in human nontyphoidal Salmonella isolates in Europe 2000-2004: a report from the Enter-net International Surveillance Network.

A 5-year survey, from 2000 to 2004, of results of antimicrobial susceptibility testing for 11 antimicrobials for 134,310 isolates of nontyphoidal salmonellas from cases of human infection in 10 European countries has demonstrated an overall increase in the occurrence of resistance, from 57% to 66% over the period of study. In contrast, multiple resistance (to four or more antimicrobial drugs) has declined from 18% to 15%. The most significant increase in resistance has been to nalidixic acid (14% to 20%), particularly in Salmonella enterica serovar Enteritidis (10% to 26%), the most common serovar. For England and Wales this increase has for the most part been attributed to infections linked to contaminated eggs originating outside the United Kingdom. For Salmonella Typhimurium, the second most prevalent serovar, there has been an overall decline in the occurrence of resistance to ampicillin, chloramphenicol, and tetracyclines, attributed to a decline in the occurrence of multiresistant Salmonella Typhimurium DT 104. For Salmonella Virchow, a serotype with a predilection for invasive disease, there has been a substantive increase in resistance to most antimicrobials, attributed to the spread of drug-resistant strains associated with poultry. Because of the widespread importation of foods, it is important that controls to reduce the emergence and spread of drug-resistant strains of Salmonella are internationally implemented.

Class 1 integrons in multidrug-resistant non-typhoidal Salmonella enterica isolated in Spain between 2002 and 2004.

In this study, 119 multidrug-resistant isolates of non-typhoidal Salmonella enterica serovars collected in Spain (2002-2004) were screened for integrons. Among the isolates, 73.1% contained class 1 integrons, however classes 2 and 3 were not detected. Integrons containing gene cassettes were found in S. Enteritidis (16/32), S. Typhimurium biphasic (18/32) and monophasic [4,5,12:i:-] (11/19), S. Virchow (17/18) and S. Brandenburg (8/8), but not in S. Hadar (0/10). Ten complete and four incomplete gene cassettes, combined in 10 variable regions, were identified, one of which (2100 bp/dfrA1-597 bp-aadA24) was a new description. Most integrons mapped on plasmids of ca. 40-340 kb. Exceptions were 1000 bp/aadA2 and 1200 bp/bla PSE-1 found on the chromosome of biphasic S. Typhimurium, probably as part of SGI1-like structures.

Serotypes, virulence genes and intimin types of Shiga toxin (verocytotoxin)-producing Escherichia coli isolates from minced beef in Lugo (Spain) from 1995 through 2003.

BACKGROUND: Shiga toxin-producing Escherichia coli (STEC) have emerged as pathogens that can cause food-borne infections and severe and potentially fatal illnesses in humans, such as haemorrhagic colitis (HC) and haemolytic uraemic syndrome (HUS). In Spain, like in many other countries, STEC strains have been frequently isolated from ruminants, and represent a significant cause of sporadic cases of human infection. In view of the lack of data on STEC isolated from food in Spain, the objectives of this study were to determine the level of microbiological contamination and the prevalence of STEC O157:H7 and non-O157 in a large sampling of minced beef collected from 30 local stores in Lugo city between 1995 and 2003. Also to establish if those STEC isolated from food possessed the same virulence profiles as STEC strains causing human infections. RESULTS: STEC were detected in 95 (12%) of the 785 minced beef samples tested. STEC O157:H7 was isolated from eight (1.0%) samples and non-O157 STEC from 90 (11%) samples. Ninety-six STEC isolates were further characterized by PCR and serotyping. PCR showed that 28 (29%) isolates carried stx1 genes, 49 (51%) possessed stx2 genes, and 19 (20%) both stx1 and stx2. Enterohemolysin (ehxA) and intimin (eae) virulence genes were detected in 43 (45%) and in 25 (26%) of the isolates, respectively. Typing of the eae variants detected four types: gamma1 (nine isolates), beta1 (eight isolates), epsilon1 (three isolates), and theta (two isolates). The majority (68%) of STEC isolates belonged to serotypes previously detected in human STEC and 38% to serotypes associated with STEC isolated from patients with HUS. Ten new serotypes not previously described in raw beef products were also detected. The highly virulent seropathotypes O26:H11 stx1 eae-beta1, O157:H7 stx1stx2 eae-gamma1 and O157:H7 stx2eae-gamma1, which are the most frequently observed among STEC causing human infections in Spain, were detected in 10 of the 96 STEC isolates. Furthermore, phage typing of STEC O157:H7 isolates showed that the majority (seven of eight isolates) belonged to the main phage types previously detected in STEC O157:H7 strains associated with severe human illnesses. CONCLUSION: The results of this study do not differ greatly from those reported in other countries with regard to prevalence of O157 and non-O157 STEC in minced beef. As we suspected, serotypes different from O157:H7 also play an important role in food contamination in Spain, including the highly virulent seropathotype O26:H11 stx1 eae-beta1. Thus, our data confirm minced beef in the city of Lugo as vehicles of highly pathogenic STEC. This requires that control measures to be introduced and implemented to increase the safety of minced beef.

Phage types, virulence genes and PFGE profiles of Shiga toxin-producing Escherichia coli O157:H7 isolated from raw beef, soft cheese and vegetables in Lima (Peru).

The present study was conducted in Lima Metropolitana to evaluate the prevalence of Shiga toxin-producing Escherichia coli (STEC) O157:H7 in raw beef, raw ground beef, soft cheese and fresh vegetables, sampled at different markets in the city. Between October 2000 and February 2001, 407 food samples were collected from different markets in the 42 districts of Lima Metropolitana. Samples were assayed for E. coli O157 by selective enrichment in modified Tryptic Soy Broth containing novobiocin, followed by immunomagnetic separation (IMS) and plating onto sorbitol MacConkey agar supplemented with cefixime and potassium tellurite. Fifty (12.3%) of 407 food samples resulted positive for E. coli O157 isolation (23 of 102 ground beef; 15 of 102 beef meat; eight of 102 soft cheese and four of 101 fresh vegetables). Thirty-five E. coli O157 isolates were further analysed for the presence of virulence genes. All 35 were positive by PCR for O157 rfbE, fliCh7, eae-gamma1 and ehxA genes. In addition, genes encoding Shiga toxins were detected in 33 of 35 isolates, five isolates (14%) encoded stx(1), stx(2), and 28 (80%) stx2 only. The isolates were of seven different phage types (PT4, PT8, PT14, PT21, PT34, PT54, and PT87) with three phage types accounting for 80% of isolates: PT4 (15 isolates), PT14 (8 isolates), and PT21 (5 isolates). Interestingly, the majority (31 of 35; 89%) of E. coli O157:H7 isolates characterized in this study belonged mainly to the phage types previously found in STEC O157:H7 strains associated with severe human disease in Europe and Canada. Pulsed-field gel electrophoresis (PFGE) of 32 isolates revealed 14 XbaI-PFGE groups (I to XIV) of similarity >85%, with 23 (72%) isolates grouped in five clusters. Some isolates from different districts presented a high clonal relatedness. Thus, PFGE group VIII clustered eleven strains from nine different districts. The broad range of PFGE subtypes found in this study demonstrates the natural occurrence of many genetic variants among STEC O157:H7 spread in Lima.

Dissemination of Salmonella enterica serotype agona and multidrug-resistant Salmonella enterica serotype typhimurium in Cuba.

The molecular epidemiology, antimicrobial susceptibility, and mechanisms of resistance of 34 Salmonella spp. strains causing acute gastroenteritis, isolated from different provinces in Cuba, were determined. Sixty-four percent of the strains showed multiresistance. Salmonella typhimurium was the most frequent with 15 strains (44%), 13 of which belonged to phagotype 104 and presented similar genetic profiles of pulsed field gel electrophoresis. High levels of resistance to tetracycline (53%), spectinomycin (50%), ampicillin (44%), and chloramphenicol (41%) were found. Resistance to tetracycline was associated with the tet G and tet A genes. Resistance to ampicillin was caused by the presence of beta-lactamases, mainly the CARB type. The floR gene was the main mechanism of resistance to chloramphenicol. Our results showed an antimicrobial susceptible clone of Salmonella enterica serotype Agona in two separate regions. This is the first report of the widespread dissemination of a multiresistant clone of S. enterica serotype Typhimurium definitive phage type 104 in Cuba.

Phenotypic and genotypic characterization of Salmonella enterica serotype paratyphi B isolates from environmental and human sources in Galicia, Spain.

Salmonella serotype Paratyphi B isolates obtained from shellfish and human infections in Galicia (northwest Spain) from 1998 were investigated by different phenotypic and genetic methods to evaluate their systemic or enteric nature. Isolates were investigated for D-tartrate fermentation, presence of genes encoding the effector proteins sopE1 and avrA, pulsed-field gel electrophoresis profile, and antimicrobial susceptibility. Systemic variant strains (dT-) were the dominant among the marine environment isolates. All dT- isolates were sopE1 positive and avrA negative, presented an indistinguishable electrophoresis profile, and were grouped in a single cluster. More electrophoresis heterogeneity was observed among dT+ isolates. Only two isolates showed resistance to any of the 16 antibiotics included in our panel. The present study identified the marine environment as a potential natural source of systemic variant isolates of Salmonella Paratyphi B. The presence of systemic variant isolates of Salmonella Paratyphi B in the marine environment is of notable public health significance as a result of the potential risk of acquiring enteric fever linked to the consumption of raw shellfish.

Molecular characterization of a new serovar of Salmonella bongori 13,22:z39:- isolated from a lizard.

Three Salmonella strains isolated from a lizard (Gallotia simoni) in the "Isla del Hierro" (Canary Islands, Spain) were serotyped as Salmonella bongori serotype 13,22:z39:-, which has not been described in the Kauffmann-White scheme of Salmonella serovars. In order to shed light on the assignment of those strains to the S. bongori species, several genes were amplified and/or sequenced. The iroB gene has been reported to be present only in S. enterica, while the invA gene has been described as being a helpful tool in distinguishing Salmonella from other bacterial species. Both genes were amplified and, as expected, only invA could be amplified. The fliC gene, encoding the phase 1 flagellin fljB gene, encoding phase 2 flagellin, and the gapA gene, which is believed to present polymorphic alleles among different subspecies, were amplified and sequenced. The sequence obtained from fliC(z39) matched with the sequences fliC(z39) obtained from other serovars. The sequence obtained from gapA clustered into the S. bongori group when it was compared to others previously described. We conclude that these three isolates are members of the S. bongori species representing a new serovar that will be described in the next supplement to the Kauffmann-White scheme.

Antimicrobial resistance of Shiga toxin (verotoxin)-producing Escherichia coli O157:H7 and non-O157 strains isolated from humans, cattle, sheep and food in Spain.

A total of 722 Shiga toxin-producing Escherichia coli (STEC) isolates recovered from humans, cattle, ovines and food during the period from 1992 to 1999 in Spain were examined to determine antimicrobial resistance profiles and their association with serotypes, phage types and virulence genes. Fifty-eight (41%) out of 141 STEC O157:H7 strains and 240 (41%) out of 581 non-O157 STEC strains showed resistance to at least one of the 26 antimicrobial agents tested. STEC O157:H7 showed a higher percentage of resistant strains recovered from bovine (53%) and beef meat (57%) than from human (23%) and ovine (20%) sources, whereas the highest prevalence of antimicrobial resistance in non-O157 STEC was found among isolates recovered from beef meat (55%) and human patients (47%). Sulfisoxazole (36%) had the most common antimicrobial resistance, followed by tetracycline (32%), streptomycin (29%), ampicillin (10%), trimethoprim (8%), cotrimoxazole (8%), chloramphenicol (7%), kanamycin (7%), piperacillin (6%), and neomycin (5%). The multiple resistance pattern most often observed was that of streptomycin, sulfisoxazole, and tetracycline. Ten (7%) STEC O157:H7 and 71 (12%) non-O157 strains were resistant to five or more antimicrobial agents. Most strains showing resistance to five or more antimicrobial agents belonged to serotypes O4:H4 (4 strains), O8:H21 (3 strains), O20:H19 (6 strains), O26:H11 (8 strains eae-beta1), O111:H- (3 strains eae-gamma2), O118:H- (2 strains eae-beta1), O118:H16 (5 strains eae-beta1), O128:H- (2 strains), O145:H8 or O145:H- (2 strains eae-gamma1), O157:H7 (10 strains eae-gamma1), O171:H25 (3 strains), O177:H11 (5 strains eae-beta1), ONT:H- (3 strains/1 eae-beta1) and ONT:H21 (2 strains). Interestingly, most of these serotypes, i.e., those indicated in bold) were found among human STEC strains isolated from patients with hemolytic uremic-syndrome (HUS) reported in previous studies. We also detected, among non-O157 strains, an association between a higher level of multiple resistance to antibiotics and the presence of the virulence genes eae and stx(1). Moreover, STEC O157:H7, showed an association between certain phage types, PT21/28 (90%), PT23 (75%), PT34 (75%), and PT2 (54%), with a higher number of resistant strains. We conclude that the high prevalence of antimicrobial resistance detected in our study is a source of concern, and cautious use of antibiotics in animals is highly recommended.

Detection of integrons and antibiotic-resistance genes in Salmonella enterica serovar Typhimurium isolates with resistance to ampicillin and variable susceptibility to amoxicillin-clavulanate.

We characterized 29 antimicrobial-resistant Salmonella enterica serovar Typhimurium strains, including four belonging to the monophasic variant 4,5,12:i:-, mostly isolated from infants. They were selected from 3230 strains isolated in the years 1990-2001 on the basis of resistance to ampicillin and variable susceptibility to the amoxicillin-clavulanate combination. Twenty-three strains were resistant to more than four antibiotics. All the strains carried the bla(TEM) gene and most were able to transfer this gene by conjugation. Sequencing of the gene from one of the amoxicillin-clavulanate-resistant strains allowed identification of the encoded beta-lactamase as TEM-1; all of these strains carried a second gene encoding beta-lactamase production, either pse-1 or oxa1. However, the association of bla(TEM) plus pse-1 genes did not always confer resistance to amoxicillin-clavulanate. The pse-1 gene, found in 17 strains, was located in the Salmonella Genomic Island-1 (SGI1), which carries two integrons and encodes multiple drug-resistance. None of the oxa1-bearing strains had the SGI1, yet this gene was found as part of an integron that also carried the aadA1 gene and was not plasmid-associated. Thirteen of the strains harbouring SGI1 belonged to the definitive phage type (DT) 104, and most of those remaining to DT104b and U302; particularly, strains carrying the oxa1-aadA1 integron belonged to the last two phage types. Pulsed field electrophoresis confirmed the clonal organization of DT104 strains, whereas U302 strains fell into different groups, depending on their resistance determinants.

Prevalence and antimicrobial susceptibility of Salmonella infections in free-range pigs.

The prevalence of Salmonella spp. infection was determined in 67 free-range pig herds in southern Spain. Microbiological assessment was performed on ileocolic lymph nodes collected at slaughter according to ISO 6579:2002 procedures. Overall, 33% of herds were infected and the prevalence of infection was 5.3%. Salmonella spp. serovars most frequently isolated were Anatum and Typhimurium, although uncommon serovars such as Hessarek and Mikawasima were also detected. Isolates were tested against 16 antimicrobial agents and exhibited resistance to streptomycin (46%), tetracycline (30%), sulphonamides (25%) and ampicillin (23%) by the break-point method. Multi-drug resistance, defined as resistance to ≥ 4 antimicrobials, was 36%.

Surveillance of antibiotic resistance evolution and detection of class 1 and 2 integrons in human isolates of multi-resistant Salmonella Typhimurium obtained in Uruguay between 1976 and 2000.

OBJECTIVES: To study the evolution of antibiotic resistance in isolates of Salmonella enterica subspecies enterica serovar Typhimurium (Salmonella Typhimurium) obtained in Uruguay between the years 1976 and 2000, and to determine the incidence of class 1 and 2 integrons in the multi-resistant isolates. METHODS: We studied 258 strains of Salmonella Typhimurium from various sources, isolated between 1976 and 2000. We determined the evolution of antibiotic resistance and the distribution of class 1 and 2 integrons in all isolates by means of disk diffusion assays and PCR. RESULTS: During the period 1989-2000 resistance to streptomycin was 56.8%, tetracycline 13.6%, sulfonamides 11.2%, and ampicillin 7.2%. Resistance to gentamicin, kanamycin, chloramphenicol, and nalidixic acid were lower than 5%; no resistance was detected to fluoroquinolones, oxyiminocephalosporins, and amikacin. These results show a dramatic decrease with respect to values found in the period 1976-1988. In this period, resistance to streptomycin was 63.2%, tetracycline 36.8%, sulfonamides 32.3%, and ampicillin 27.8%. Throughout the two periods, 29 multi-resistant Salmonella Typhimurium strains were isolated harboring some class of integron: 15 strains had only intI2, 11 strains presented both intI1 and intI2, and three isolates only intI1. CONCLUSIONS: Our results show a marked decrease in resistance throughout these years, along with a correlation between resistance to different antibiotics and the presence of integrons.

Detection of Salmonella enterica serotype typhimurium DT104 in Mozambique.

The spread of Salmonella enterica serotype Typhimurium definitive phage type DT104 in sub-Saharan Africa is a public health concern. We obtained two isolates of S. typhimurium DT104 from blood cultures of infants with malaria in Mozambique. Both isolates contained Salmonella genomic island 1A and had the same pulsed-field gel electrophoresis PulseNet pattern (STYMXB.0005). Results showed the need for continuous surveillance of Salmonella spp. serotypes circulating in this area.

Fecal carriage of Escherichia coli O157:H7 and carcass contamination in cattle at slaughter in northern Italy.

Feedlot cattle slaughtered at a large abattoir in northern Italy during 2002 were examined for intestinal carriage and carcass contamination with Escherichia coli O157:H7. Carcass samples were taken following the excision method described in the Decision 471/2001/EC, and fecal material was taken from the colon of the calves after evisceration. Bacteria were isolated and identified according to the MFLP-80 and MFLP-90 procedures (Food Directorate's Health Canada's). Eighty-eight non-sorbitol-fermenting E. coli O157:H7 isolates were obtained from 12 of the 45 calves examined. In particular, E. coli O157:H7 isolates were found in 11 (24%) fecal and five (11%) carcass samples. PCR analysis showed that all 11 fecal samples and five carcass samples carried eae-gamma1-positive E. coli O157:H7 isolates. In addition, genes encoding Shigatoxins were detected in O157:H7 isolates from nine and two of those 11 fecal and five carcasses, respectively. A representative group of 32 E. coli O157:H7 isolates was analyzed by phage typing and DNA macrorestriction fragment analysis (PFGE). Five phage types (PT8, PT32v, PT32, PT54, and PT not typable) and seven (I-VII) distinct restriction patterns of similarity >85% were detected. Up to three different O157:H7 strains in an individual fecal sample and up to four from the same animal could be isolated. These findings provide evidence of the epidemiological importance of subtyping more than one isolate from the same sample. Phage typing together with PFGE proved to be very useful tools to detect cross-contamination among carcasses and should therefore be included in HACCP programs at abattoirs. The results showed that the same PFGE-phage type E. coli O157:H7 profile was detected in the fecal and carcass samples from an animal, and also in two more carcasses corresponding to two animals slaughtered the same day.

Antimicrobial resistance in non-typhoidal Salmonella from human sources, Spain, 2001-2003.

OBJECTIVES: To determine the current state of antimicrobial resistance among non-typhoidal Salmonella strains isolated from humans in Spain. METHODS: All strains of Salmonella from human sources received in the reference laboratory from 2001 to 2003 were serotyped and phage types were determined in the most common serovars. A systematic sampling procedure was carried out in order to obtain a random sample for susceptibility testing. The selected strains were tested for susceptibility to 12 different antimicrobial agents by a disc diffusion method using Mueller-Hinton agar. Results were scored as susceptible, moderately susceptible or resistant, according to CLSI criteria. RESULTS: From 2001 to 2003, 5777 strains of Salmonella were tested for susceptibility. Fifty per cent of strains of Salmonella Enteritidis were resistant to nalidixic acid. This was the most frequent resistance pattern of this serovar and it was characteristic of PT1, the most frequent phage type of Salmonella Enteritidis in Spain. Seventy-four per cent of Salmonella Typhimurium strains were resistant to four antibiotics or more. Resistance to ampicillin, chloramphenicol, streptomycin, sulphonamide and tetracycline was the most frequent resistance pattern of Salmonella Typhimurium and it was characteristic of DT104, the most frequent phage type in Spain. Sixty-nine per cent of Salmonella Hadar strains were resistant to at least four antibiotics. CONCLUSIONS: The results of our study showed both a worrying percentage of strains of Salmonella Enteritidis resistant to nalidixic acid and of strains of Salmonella Typhimurium with a pattern of resistance to four antibiotics or more. Surveillance of antimicrobial resistance should carry on and improve in order to be able to evaluate the control measures carried out for decreasing resistance in Salmonella, specifically that addressed to the prudent use of antimicrobial agents by farmers and veterinarians.

Topoisomerase II and IV quinolone resistance-determining regions in Stenotrophomonas maltophilia clinical isolates with different levels of quinolone susceptibility.

The quinolone resistance-determining regions (QRDRs) of topoisomerase II and IV genes from Stenotrophomonas maltophilia ATCC 13637 were sequenced and compared with the corresponding regions of 32 unrelated S. maltophilia clinical strains for which ciprofloxacin MICs ranged from 0.1 to 64 microg/ml. GyrA (Leu-55 to Gln-155, Escherichia coli numbering), GyrB (Met-391 to Phe-513), ParC (Ile-34 to Arg-124), and ParE (Leu-396 to Leu-567) fragments from strain ATCC 13637 showed high degrees of identity to the corresponding regions from the phytopathogen Xylella fastidiosa, with the degrees of identity ranging from 85.0 to 93.5%. Lower degrees of identity to the corresponding regions from Pseudomonas aeruginosa (70.9 to 88.6%) and E. coli (73.0 to 88.6%) were observed. Amino acid changes were present in GyrA fragments from 9 of the 32 strains at positions 70, 85, 90, 103, 112, 113, 119, and 124; but there was no consistent relation to higher ciprofloxacin MICs. The absence of changes at positions 83 and 87, commonly involved in quinolone resistance in gram-negative bacteria, was unexpected. The GyrB sequences were identical in all strains, and only one strain (ciprofloxacin MIC, 16 microg/ml) showed a ParC amino acid change (Ser-80-->Arg). In contrast, a high frequency (16 of 32 strains) of amino acid replacements was present in ParE. The frequencies of alterations at positions 437, 465, 477, and 485 were higher (P < 0.05) in strains from cystic fibrosis patients, but these changes were not linked with high ciprofloxacin MICs. An efflux phenotype, screened by the detection of decreases of at least twofold doubling dilutions of the ciprofloxacin MIC in the presence of carbonyl cyanide m-chlorophenylhydrazone (0.5 microg/ml) or reserpine (10 microg/ml), was suspected in seven strains. These results suggest that topoisomerases II and IV may not be the primary targets involved in quinolone resistance in S. maltophilia.

DNA microarray-based typing of an atypical monophasic Salmonella enterica serovar.

A multidrug-resistant fljB-lacking Salmonella enterica serovar [4,5,12:i:-] emerged in Spain in 1997. We analyzed the genome from four strains of this serovar using a microarray containing almost all the predicted protein coding regions of serovar Typhimurium strain LT2, including the pSLT plasmid. Only a few differences from serovar Typhimurium LT2 were observed, suggesting the serovar to be Typhimurium as well. Six regions of interest were identified from the microarray data. Cluster I was a deletion of 13 genes, corresponding to part of the regulon responsible for the anaerobic assimilation of allantoin. Clusters II and IV were associated with the absence of the Fels-1 and Fels-2 prophage. Cluster III was a small group of Gifsy-1 prophage-related genes that appeared to be deleted or replaced. Cluster V was a deletion of 16 genes, including iroB and the operon fljAB, which is reflected in the serovar designation. Region VI was the gene STM2240, which appears to have an additional homologue in these strains. The regions spanning the deletions involving the allantoin operon and the fljAB operon were PCR amplified and sequenced. PCR across these regions may be an effective marker for this particular emergent serovar. While the microarray data for all isolates of the new serovar were essentially identical for all LT2 chromosomal genes, the isolates differed in their similarity to pSLT, consistent with the heterogeneity in plasmid content among isolates of the new serovar. Recent isolates have acquired a more-complete subset of homologues to this virulence plasmid. In general, microarrays can provide useful complementary data to other typing methods.