Range Expansion and the Origin of USA300 North American Epidemic Methicillin-Resistant Staphylococcus aureus.

The USA300 North American epidemic (USA300-NAE) clone of methicillin-resistant Staphylococcus aureus has caused a wave of severe skin and soft tissue infections in the United States since it emerged in the early 2000s, but its geographic origin is obscure. Here we use the population genomic signatures expected from the serial founder effects of a geographic range expansion to infer the origin of USA300-NAE and identify polymorphisms associated with its spread. Genome sequences from 357 isolates from 22 U.S. states and territories and seven other countries are compared. We observe two significant signatures of range expansion, including decreases in genetic diversity and increases in derived allele frequency with geographic distance from the Pennsylvania region. These signatures account for approximately half of the core nucleotide variation of this clone, occur genome wide, and are robust to heterogeneity in temporal sampling of isolates, human population density, and recombination detection methods. The potential for positive selection of a gyrA fluoroquinolone resistance allele and several intergenic regions, along with a 2.4 times higher recombination rate in a resistant subclade, is noted. These results are the first to show a pattern of genetic variation that is consistent with a range expansion of an epidemic bacterial clone, and they highlight a rarely considered but potentially common mechanism by which genetic drift may profoundly influence bacterial genetic variation.IMPORTANCE The process of geographic spread of an origin population by a series of smaller populations can result in distinctive patterns of genetic variation. We detect these patterns for the first time with an epidemic bacterial clone and use them to uncover the clone's geographic origin and variants associated with its spread. We study the USA300 clone of methicillin-resistant Staphylococcus aureus, which was first noticed in the early 2000s and subsequently became the leading cause of skin and soft tissue infections in the United States. The eastern United States is the most likely origin of epidemic USA300. Relatively few variants, which include an antibiotic resistance mutation, have persisted during this clone's spread. Our study suggests that an early chapter in the genetic history of this epidemic bacterial clone was greatly influenced by random subsampling of isolates during the clone's geographic spread.

Draft Genome Sequence of a Multidrug-Resistant Klebsiella pneumoniae Carbapenemase-Producing Acinetobacter baumannii Sequence Type 2 Isolate from Puerto Rico.

We report here the draft genome sequence of Acinetobacter baumannii strain M3AC14-8, sequence type 2 (ST2), carrying a chromosomally carried blaKPC-2 gene. The draft genome consists of a total length of 4.11 Mbp and a G+C content of 39.25%.

Genetic environment of the KPC gene in Acinetobacter baumannii ST2 clone from Puerto Rico and genomic insights into its drug resistance.

Carbapenems are considered the last-resort antibiotics to treat infections caused by multidrug-resistant Gram-negative bacilli. The Klebsiella pneumoniae carbapenemase (KPC) enzyme hydrolyses ß-lactam antibiotics including the carbapenems. KPC has been detected worldwide in Enterobacteriaceae and Pseudomonas aeruginosa isolates associated with transposon Tn4401 commonly located in plasmids. Acinetobacter baumannii has become an important multidrug-resistant nosocomial pathogen. KPC-producing A. baumannii has been reported to date only in Puerto Rico. The objective of this study was to determine the whole genomic sequence of a KPC-producing A. baumannii in order to (i) define its allelic diversity, (ii) identify the location and genetic environment of the blaKPC and (iii) detect additional mechanisms of antimicrobial resistance. Next-generation sequencing, Southern blot, PFGE, multilocus sequence typing and bioinformatics analysis were performed. The organism was assigned to the international ST2 clone. The blaKPC-2 was identified on a novel truncated version of Tn4401e (tentatively named Tn4401h), located in the chromosome within an IncA/C plasmid fragment derived from an Enterobacteriaceae, probably owing to insertion sequence IS26. A chromosomally located truncated Tn1 transposon harbouring a blaTEM-1 was found in a novel genetic environment within an antimicrobial resistance cluster. Additional resistance mechanisms included efflux pumps, non-ß-lactam antibiotic inactivating enzymes within and outside a resistance island, two class 1 integrons, In439 and the novel In1252, as well as mutations in the topoisomerase and DNA gyrase genes which confer resistance to quinolones. The presence of the blaKPC in an already globally disseminated A. baumannii ST2 presents a serious threat of further dissemination.

Draft Genome Sequence of Klebsiella pneumoniae Carbapenemase-Producing Acinetobacter baumannii Strain M3AC9-7, Isolated from Puerto Rico.

We report the draft genome of a multidrug resistant, Klebsiella pneumoniae carbapenemase (KPC)-producing Acinetobacter baumannii strain M3AC9-7 that belongs to the novel sequence type, ST250. The draft genome consists of a total length of 4.09 Mbp and a G+C content of 38.95%.

ISEcp1-mediated transposition of blaKPC into the chromosome of a clinical isolate of Acinetobacter baumannii from Puerto Rico.

Carbapenems are the last-resort antibiotics for the treatment of infections caused by multidrug-resistant Gram-negative bacilli. Klebsiella pneumoniae carbapenemase (KPC) hydrolyses ß-lactam antibiotics including the carbapenems. KPCs have been detected in Enterobacteriaceae and Pseudomonas aeruginosa isolates worldwide associated with transposon Tn4401 commonly located in plasmids. Acinetobacter baumannii has become an important multidrug-resistant nosocomial pathogen capable of hydrolysing the carbapenem antibiotics. KPC-producing A. baumannii has so far only been reported in Puerto Rico. During a surveillance study, four KPC-producing A. baumannii with identical pulse type were identified in a single institution. The objectives of this study were to characterize the KPC genetic background and the allelic diversity of one of the isolates. Next-generation sequencing and multilocus sequence typing (MLST) were performed. Molecular characterization of the isolate demonstrated blaKPC in Tn4401b located in the bacterial chromosome within a 26.5 kb DNA fragment, which included a KQ-like element (18.9 kb) very similar to that described previously in a K. pneumoniae plasmid and a 7.6 kb DNA fragment with 98 % homology to a putative plasmid from Yersinia pestis strain PY-95. Our data suggested that the 26.5 kb DNA fragment harbouring blaKPC was integrated in the chromosome by a transposition event mediated by the transposase of ISEcp1 found in the KQ-like element. MLST showed a novel sequence type, ST250. To our knowledge, this is the first report of the identification of the genetic background of blaKPC in A. baumannii.

Direct Detection and Quantification of Bacterial Genes Associated with Inflammation in DNA Isolated from Stool.

Although predominantly associated with health benefits, the gut microbiota has also been shown to harbor genes that promote inflammation. In this work, we report a method for the direct detection and quantification of these pro-inflammatory bacterial genes by PCR and qPCR in DNA extracted from human stool samples. PCR reactions were performed to detect (i) the pks island genes, (ii) tcpC, which is present in some strains of Escherichia coli and (iii) gelE presented in some strains of Enterococcus faecalis. Additionally, we screened for the presence of the following genes encoding cyclomodulins that disrupted mammalian cell division: (iv) cdt (which encodes the cytolethal distending toxin) and (v) cnf-1 (which encodes the cytotoxic necrotizing factor-1). Our results show that 20% of the samples (N = 41) tested positive for detectable amounts of pks island genes, whereas 10% of individuals were positive for tcpC or gelE and only one individual was found to harbor the cnf-1 gene. Of the 13 individuals that were positive for at least one of the pro-inflammatory genes, 5 were found to harbor more than one. A quantitative version of the assay, which used real-time PCR, revealed the pro-inflammatory genes to be in high copy numbers: up to 1.3 million copies per mg of feces for the pks island genes. Direct detection of specific genes in stool could prove useful toward screening for the presence of pro-inflammatory bacterial genes in individuals with inflammatory bowel diseases or colorectal cancer.

KPC screening by updated BD Phoenix and Vitek 2 automated systems.

Current BD Phoenix and Vitek 2 methodologies were assessed as screens for KPC ß-lactamases. Using carbapenem MICs or expert system interpretations as screens, both systems exhibited high (97%) sensitivity in tests with 103 well-characterized Gram-negative isolates, 77 of which were KPC producers.

Detection of the KPC gene in Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Acinetobacter baumannii during a PCR-based nosocomial surveillance study in Puerto Rico.

A 6-month, PCR-based, island-wide hospital surveillance study of beta-lactam resistance in Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Acinetobacter baumannii was conducted in Puerto Rico. Of 10,507 isolates, 1,239 (12%) unique, multi-beta-lactam-resistant isolates from all geographical regions were identified. The KPC gene was detected in 61 E. coli, 333 K. pneumoniae, 99 P. aeruginosa, and 41 A. baumannii isolates, indicating the widespread dissemination of the KPC gene in clinically significant nosocomial isolates.

Outbreak of carbapenem-resistant Klebsiella pneumoniae in Puerto Rico associated with a novel carbapenemase variant.

BACKGROUND: Carbapenem-resistant Klebsiella pneumoniae (CRKP) is resistant to almost all antimicrobial agents, and CRKP infections are associated with substantial morbidity and mortality. OBJECTIVE: To describe an outbreak of CRKP in Puerto Rico, determine risk factors for CRKP acquisition, and detail the successful measures taken to control the outbreak. DESIGN: Two case-control studies. SETTING: A 328-bed tertiary care teaching hospital. PATIENTS: Twenty-six CRKP case patients identified during the outbreak period of February through September 2008, 26 randomly selected uninfected control patients, and 26 randomly selected control patients with carbapenem-susceptible K. pneumoniae (CSKP) hospitalized during the same period. METHODS: We performed active case finding, including retrospective review of the hospital's microbiology database and prospective perirectal surveillance culture sampling in high-risk units. Case patients were compared with each control group while controlling for time at risk. We sequenced the bla(KPC) gene with polymerase chain reaction for 7 outbreak isolates and subtyped these isolates with pulsed-field gel electrophoresis. RESULTS: In matched, multivariable analysis, the presence of wounds (hazard ratio, 19.0 [95% confidence interval {CI}, 2.5-142.0]) was associated with CRKP compared with no K. pneumoniae. Transfer between units (adjusted odds ratio [OR], 7.5 [95% CI, 1.8-31.1]), surgery (adjusted OR, 4.0 [95% CI, 1.0-15.7]), and wounds (adjusted OR, 4.9 [95% CI, 1.1-21.8]) were independent risk factors for CRKP compared to CSKP. A novel K. pneumoniae carbapenemase variant (KPC-8) was present in 5 isolates. Implementation of active surveillance for CRKP colonization and cohorting of CRKP patients rapidly controlled the outbreak. CONCLUSIONS: Enhanced surveillance for CRKP colonization and intensified infection control measures that include limiting the physical distribution of patients can reduce CRKP transmission during an outbreak.

Detection of KPC in Acinetobacter spp. in Puerto Rico.

During an island-wide PCR-based surveillance study of beta-lactam resistance in multidrug-resistant (MDR) Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Acinetobacter calcoaceticus-baumannii complex isolates obtained from 17 different hospitals, 10 KPC-positive Acinetobacter isolates were identified. DNA sequencing of the bla(KPC) gene identified KPC-2, -3, and -4 and a novel variant, KPC-10. This is the first report of a KPC-type beta-lactamase identified in Acinetobacter species.

Surveillance of carbapenem-resistant Pseudomonas aeruginosa isolates from Puerto Rican Medical Center Hospitals: dissemination of KPC and IMP-18 beta-lactamases.

During a 6-month period, 37/513 (7.2%) Pseudomonas aeruginosa isolates belonging to 13 pulsed-field gel electrophoresis (PFGE) groups from Puerto Rican hospitals were carbapenem nonsusceptible. Seven of 37 isolates from four PFGE groups carried bla(IMP-18), and 25/37 isolates from seven PFGE groups carried bla(KPC). The results indicated the clonal spread of bla(KPC)-positive P. aeruginosa isolates into several Puerto Rican hospitals and the dissemination of bla(IMP-18) and bla(KPC) into genetically unrelated isolates.

The microbial etiologies of diarrhea in hospitalized patients from the Puerto Rico Medical Center Hospitals.

The development of diarrhea in hospitalized patients is a frequently encountered clinical problem, which may be due to infectious or non-infectious causes. The purpose of this study was to identify which common community enteric pathogens, if any, are responsible for diarrheal episodes in hospitalized patients. Stool samples from 76 consecutive, hospitalized patients were analyzed utilizing routine bacterial cultures, smears for identification of ova and parasites and Enzyme-Link Immunoadsorbent Assay (ELISA) for enteric bacteria, parasites and viruses. The results obtained demonstrated that the usual community enteric pathogens were not identified as a major cause of nosocomial diarrhea. In hospital-acquired diarrhea, Clostridium difficile toxins assay was the only clinically significant test in the evaluation of these patients. As a result of this study a guideline for the management of this condition in hospitalized patients is presented.

A comparison of the antimicrobial resistance patterns of gram-negative bacilli isolated from community-private and university-affiliated hospitals from Puerto Rico.

Few studies have been performed in Puerto Rico concerning the antimicrobial resistance pattern of clinically significant Gram-negative bacilli. The antimicrobial resistance patterns of 5,590 Gram-negative bacteria obtained from three Community-Private Hospitals (CPH) and three University-Affiliated Hospitals (UAH) were evaluated utilizing the institutions' antimicrobial susceptibility reports for the year 2000. The objectives of this study were: to retrospectively evaluate the reported in vitro resistance of clinical isolates of E. coli, K. pneumoniae, E. cloacae, S. marcescens, P. aeruginosa and A. baumannii to selected standard antibiotics and to compare the antimicrobial resistance patterns between Community-Private (CPH) and University Affiliated hospitals (UAH). E. coli was the most common Gram-negative enteric bacilli in both CPH and UAH. In UAH, E. coli demonstrated a statistically significant higher resistance to the selected beta lactams and amikacin antibiotics but not to ciprofloxacin or gentamicin. For K. pneumoniae, the antimicrobial resistant pattern showed that UAH isolates were significantly more resistant to the tested antibiotics with the exception of ceftriaxone. In CPH, E. cloacae isolates were significantly more resistant to piperacillin-tazobactam, ciprofloxacin and gentamicin, while in UAH this organism was more resistant to amikacin. In UAH, S. marcescens isolates demonstrated a statistically significant higher resistance to all tested antibiotics with the exception of imipenem, which was similar in both hospitals group. Pseudomonas aeruginosa demonstrated a statistically significant higher resistance in UAH to all selected antibiotics with the exception of ciprofloxacin and gentamicin, which was similar in both hospitals group. Acinetobacter baumannii was the most resistant organisms in both hospitals group. UAH isolates were significantly more resistant than CPH isolates for all tested antibiotics. When compare with other large-scale antimicrobial resistance studies, the present study results suggest an apparent higher resistance in the Puerto Rican isolates. The high numbers of antimicrobial resistant Gram-negative bacilli in our study strongly suggest multiple mechanisms of antimicrobial resistance including the presence of extended spectrum and chromosomally derepressed beta-lactamases.

A comparison of the antimicrobial resistance patterns of gram-positive cocci isolated from community-private and university-affiliated hospitals from Puerto Rico.

The antimicrobial resistance patterns of 2,462 selected Gram-positive cocci obtained from three Community-Private Hospitals (CPH) and three University-Affiliated Hospitals (UAH) were evaluated utilizing the institutions' antimicrobial susceptibility reports for the year 2000. The objectives of this study were: 1) to evaluate the in vitro resistance to selected standard antibiotics of Staphylococcus aureus, Enterococcus faecalis, Enterococcus faecium and Streptococcus pneumoniae clinical isolates, and 2) to compare the antimicrobial resistance patterns between community-private (CPH) and university-affiliated hospitals (UAH). Staphylococcus aureus was the most common Gram-positive isolated organism in CPH (63.3%) followed by E. faecalis (31.0%). In UAH, the most prevalent cocci were E. faecalis (51.7%) followed by S. aureus (43.9%). Enterococcus faecium represented 2.3% and 4.4% of CPH and UAH isolates, respectively. Streptococcus pneumoniae represented 3.4% of the total Gram-positive isolates from CPH, no S. pneumoniae was reported in UAH. The antimicrobial susceptibility results showed that for Staphylococcus aureus there was a statistically significant higher resistance to methicillin and thrimethoprim sulfamethoxazole in UAH, while resistance to erythromycin was significantly higher in CPH. There was no difference in the resistance of S. aureus to other antimicrobial agents between hospitals groups. A statistically significant resistant to vancomycin was found between enterococcal isolates from UAH (43%) and CPH (12.7%). High-level aminoglycoside resistance (HLAR) was observed among UAH enterococcal isolates with E. faecium showing a higher resistance than E. faecalis, no data for HLAR in CPH could be obtained. For pneumococci 46% of CPH isolates were resistant to penicillin. In summary, there are important differences in the prevalence and antimicrobial resistance between the Gram-positive bacteria isolated from community and teaching hospitals.

Prospective study using standardized methodology for antimicrobial susceptibility of gram-positive cocci isolated from the Puerto Rico Medical Center.

The Gram-positive cocci (GPC), Staphylococcus aureus, Streptococcus pneumoniae, Enterococcus faecalis and Enterococcus faecium, have become important causes of community and nosocomial-acquired infections. The prevalence of multiple resistant isolates to standard antimicrobial drugs has significantly increased over the past decades. Few prospective studies have been performed in Puerto Rico (PR) concerning the GPC antimicrobial susceptibilities pattern. The purpose of this study was to evaluate the in vitro susceptibility of GPC clinical isolates from PR to selected standard antibiotics and to the new antimicrobial agents, linezolid (LZ), quinupristin/dalfopristin (Q/D) and gemifloxacin (GM). The in vitro susceptibility utilizing disk diffusion and Etest methods to selected antibiotics was determined for a total of 429 isolates obtained during a period of 5 months from the Puerto Rico Medical Center Bacteriology Laboratory. The distribution of GPC collected was as follows: 213 S. aureus isolates, 162 E. faecalis, 16 E. faecium and 38 S. pneumoniae. The results of the susceptibility test demonstrated: 1) that in S. aureus, 100% of the isolates were susceptible to vancomycin (VAN), LZ and Q/D; 93% to GM; and 61% to methicillin/oxacillin; 2) in S. pneumoniae, 100% were susceptible to LN and GM; 87% to Q/D; and 53% to penicillin; 3) in E. faecalis, 99% were susceptible to ampicillin; 93% to LZ; 79% to GM; 78.6% to VAN; and 0% to Q/D. Sixty eight and 66% of the E. faecalis isolates were susceptible to gentamicin and streptomycin respectively; and 4) in E. faecium, 100% were susceptible to LZ; 94% to Q/D; 69% to GM; 37.5% to VAN and 20% to ampicillin. In E. faecium isolates, 50% and 31% were susceptible to gentamicin and streptomycin, respectively. Of the vancomycin resistant enterococci, 88.9% and 21% of E. faecium and faecalis showed VanA phenotypic resistance, respectively. These results show that there is a significant degree of antimicrobial resistance in GPC, including 38% methicillin resistance in S. aureus, a near 50% penicillin resistant S. pneumoniae, and a significant resistance of enterococcal species to VAN. The new agents, LZ, Q/D and GM, proved to be effective against both, S. aureus and S. pneumoniae. For E. faecium, both, LZ and Q/D were active, while for E. faecalis, only LZ showed consistent activity.

Endemic plants of Puerto Rico: brine shrimp lethality and antibacterial activity

A total of 49 endemic plants of Puerto Rico were evaluated for their toxicological and antibacterial activities. The toxicological analysis was conducted with brine shrimp (Artemia salina Leach) lethality bioassay and the antibacterial screening was carried out by means of the agar diffusion test. In the toxicological bioassay, six plant extracts showed LC-50 values below 200 g/ml., indicating the potential presence of bioactive compounds. In the antibacterial screening, over 80 per cent of the plant extracts displayed activity against gram positive bacteria, whereas only 6 per cent of the extracts inhibited the growth of the gram negative bacteria

Effect of various microbial preparations on P-388 mouse lymphocytic leukemia

Four bacteria-derived immunopotentiators were tested for their protective effect on a P-388 mouse lymphocytic leukemia model. The microbial test products were prepared from the following bacterial strains: ATCC 35983 Staphylococcus epidermidis isolated from a patient with IV catheter; ATCC 31874, a patented strain listed as Staphylococcus epidermidis isolated from the urine of a cancer patient; ATCC 25615 Staphylococcus hominis obtained from a child with lymphocytic leukemia, and ATCC 25614 Staphylococcus warneri, an isolate from a patient with adenocarcinoma of the breast. A limited degree of protection and prolongation in survival time was observed in the animal group treated with the bacterial strain ATCC 31874