Macrolide resistance genotypes and phenotypes among erythromycin-resistant clinical isolates of Staphylococcus aureus and coagulase-negative staphylococci, Italy

One hundred macrolide-resistant staphylococcal isolates from clinically relevant infections in Italy during a 19-month period were studied. Four distinct resistance phenotypes were observed using the triple-disk induction test (erythromycin, clindamycin, telithromycin): the cMLS_B phenotype (24 isolates); the iMLS_B phenotype (41 isolates); the MS phenotype (three isolates); and the iMTS phenotype (erythromycin-induced telithromycin resistance) (32 isolates). ermC and ermA genes predominated within erythromycin-resistant Staphylococcus aureus isolates with iMLS_B phenotype and cMLS_B phenotype, respectively. Among erythromycin-resistant CoNS isolates, half of the strains showed the iMTS or MS/ msrA association, and ermC gene predominated among isolates with MLS_B phenotype. By pulsed-field gel electrophoresis, high genetic heterogeneity was observed among the isolates studied. Both independent acquisition of macrolide resistance genes and spread of specific resistant clones were observed. Association between certain clonal types and specific types of infection could be detected. To our knowledge, this is the first report on characterization of erythromycin-resistant staphylococci in Italy.     

Antimicrobial susceptibility patterns,emm type distribution and genetic diversity of Streptococcus pyogenes recovered in Brazil

Streptococcus pyogenes is responsible for a variety of infectious diseases and immunological complications. In this study, 91 isolates of S. pyogenes recovered from oropharynx secretions were submitted to antimicrobial susceptibility testing, emm typing and pulsed-field gel electrophoresis (PFGE) analysis. All isolates were susceptible to ceftriaxone, levofloxacin, penicillin G and vancomycin. Resistance to erythromycin and clindamycin was 15.4%, which is higher than previous reports from this area, while 20.9% of the isolates were not susceptible to tetracycline. The macrolide resistance phenotypes were cMLSB (10) and iMLSB (4). The ermB gene was predominant, followed by the ermA gene. Thirty-two emm types and subtypes were found, but five (emm1, emm4, emm12, emm22, emm81) were detected in 48% of the isolates. Three new emm subtypes were identified (emm1.74, emm58.14, emm76.7). There was a strong association between emm type and PFGE clustering. A variety of PFGE profiles as well as emm types were found among tetracycline and erythromycin-resistant isolates, demonstrating that antimicrobial resistant strains do not result from the expansion of one or a few clones. This study provides epidemiological data that contribute to the development of suitable strategies for the prevention and treatment of such infections in a poorly studied area.     

MALDI-TOF mass spectrometry as a tool for differentiation of invasive and noninvasive Streptococcus pyogenes isolates

A novel mass spectral fingerprinting and proteomics approach using MALDI-TOF MS was applied to detect and identify protein biomarkers of group A Streptococcus (GAS) strains. Streptococcus pyogenes ATCC 700294 genome strain was compared with eight GAS clinical isolates to explore the ability of MALDI-TOF MS to differentiate isolates. Reference strains of other bacterial species were also analyzed and compared with the GAS isolates. MALDI preparations were optimized by varying solvents, matrices, plating techniques, and mass ranges for S. pyogenes ATCC 700294. Spectral variability was tested. A subset of common, characteristic, and reproducible biomarkers in the range of 2000-14 000 Da were detected, and they appeared to be independent of the culture media. Statistical analysis confirmed method reproducibility. Random Forest analysis of all selected GAS isolates revealed differences among most of them, and summed spectra were used for hierarchical cluster analysis. Specific biomarkers were found for each strain, and invasive GAS isolates could be differentiated. GAS isolates from cases of necrotizing fasciitis were clustered together and were distinct from isolates associated with noninvasive infections, despite their sharing the same emm type. Almost 30% of the biomarkers detected were tentatively identified as ribosomal proteins.     

Prevalence of a Streptococcal Inhibitor of a Complement-Mediated Cell lysis-like Gene (sicG) in Streptococcus Dysgalactiae subsp. Equisimilis

Streptococcus dysgalactiae subsp. equisimilis isolates (n = 110) were analyzed by PCR to determine whether the gene encoding SICG, a homolog of Streptococcus pyogenes SIC, was present. Nineteen strains (17%) had this gene of which 11 (55%) were isolated from patients with invasive disease. All 19 strains possessed group G carbohydrate. Molecular characterization of emm type revealed that the majority of emm sequences were stG643 and stG2078. Only the N-terminal sequence of SICG was similar to that of SIC in S. pyogenes. Although we found no significant relationship between pathogenic severity and sicG possession, further investigation into the mechanism of SICG may elucidate the virulence in S. dysgalactiae subsp. equisimilis infection.     

Erythromycin resistance genes in Streptococcus pyogenes isolates in Kanagawa, Japan

The susceptibility of 224 Streptococcus pyogenes isolates obtained from children in Japan from 1981 to 1997 to treatment with erythromycin was determined by the agar dilution method. A total of 17 isolates belonging to serotype M12T12 were resistant (MICs>1 microg/ml). Fourteen of the 17 resistant strains obtained from 1982 to 1985 harbored ermB and showed an identical pulsed-field gel electrophoresis pattern, indicating the spread of a single clone. Two ermTR-containing isolates were obtained in 1983. mefA gene was found in a strain obtained in 1994 in the present study, although this gene is predominantly associated with recent erythromycin resistance among S. pyogenes strains in many countries.     

Limited repertoire of the C-terminal region of the M protein in Streptococcus pyogenes

We have amplified genomic sequences (emm) that may encode M protein from strains of Streptococcus pyogenes using the polymerase chain reaction (PCR). Genomic DNA from 22 isolates representing 14 M serotypes was selected for the study. Primers which corresponded to the observed N-terminal signal sequence and the variable C-terminal sequences of emm6, emm49 and ennX were used. PCR products using emm6 and emm49 oligonucleotides were classified into two mutually exclusive groups which correspond to the presence or absence of serum opacity factor. These findings support the concept of limited heterogeneity in the C-terminal sequences of the M protein.     

emm Typing of group A streptococcus clinical isolates: identification of dominant types for throat and skin isolates

T and emm types were determined for group A streptococci isolated from patients with various infections during 1990-1999 in Toyama Prefecture, Japan. Out of 906 isolates, 872 isolates were divided into 20 T serotypes, and 34 isoltes were T nontypeable (TNT). T12, T1, and T4 were dominant among 699 throat isolates; on the other hand, T11, T28, TB3264, and TNT were dominant among 80 skin isolates. The emm types of 190 isolates were determined following specific PCR amplification and sequencing of the products. Twenty T serotypes were divided into 34 T type/emm type combinations. Thirty-four TNT isolates were divided into 14 emm types, in which emm58 was the most common (38%). Among 82 throat isolates randomly selected, predominant T types T12, T1, and T4 isolates were of the respective same numbers in emm type. T11/emm89, T28/emm28, TB3264/emm13w, and TNT/emm58 were predominant among 80 skin isolates. emm-type distribution observed in the present study was that usually reported in the western world. To our knowledge, 3 T/emm is a novel combination. These results show that emm typing allows the characterization of group A streptococci from various sources.     

Characterization of Staphylococcus aureus Isolates from Buffalo, Bovine, Ovine, and Caprine Milk Samples Collected in Rio de Janeiro State, Brazil

Eighty-four staphylococcal isolates were obtained from milk samples from cows, sheep, goats, and buffalo with subclinical mastitis and from colonization samples from ostriches. The animals were hosted in 18 small dairy herds and an ostrich breeding located in 10 municipalities of the state of Rio de Janeiro, Brazil. Thirty isolates were identified as Staphylococcus aureus by biochemical and molecular techniques and were comparatively characterized by phenotypic and genotypic methods. The molecular characterization by pulsed-field gel electrophoresis (PFGE), spa typing, and multilocus sequence typing (MLST) revealed five clonal types (PFGE A, spa type t359, sequence type 747 [ST747]; PFGE B, spa type t1180, ST750; PFGE C, spa type t605, ST126; PFGE D, spa type t127, ST751; and PFGE F, spa type t002, ST5). None of the isolates harbored the Panton-Valentine leukocidin or exfoliative toxin D gene. The detection of major clone A (in 63% of the isolates) in different herds, among all animal species studied, and in infection and colonization samples evidenced its geographical spread among Rio de Janeiro State and no host preference among the animal species. Comparison with S. aureus from a human origin suggested that all but one clone found in the present study might be animal specific.     

Multi-locus sequence typing (MLST) of non-fermentative Gram-negative bacilli isolated from bloodstream infections in southern Poland

Non-fermentative Gram-negative bacilli are now one of the most important causes of severe infections in Polish hospitals. Acinetobacter species are serious concern because of the high prevalence of multi-drug resistance among strains. Resistance profiles for 53 Gram-negative non-fermentative blood isolates were done. MLST was carried out using 44 strains representing the most commonly isolated species: A. baumannii, P. aeruginosa, and S. maltophilia. MLST revealed that all 22 A. baumannii belonged to sequence type (ST) 2. The P. aeruginosa isolates belonged to 10 different STs. Four S. maltophilia isolates matched STs present in the database (ST4, ST15, ST116, ST142), seven isolates showing novel sequence types. Among P. aeruginosa and S. maltophilia PFGE confirmed the genetical variety of strains.     

Characterization of Staphylococcus aureus isolates from buffalo, bovine, ovine, and caprine milk samples collected in Rio de Janeiro State, Brazil

Eighty-four staphylococcal isolates were obtained from milk samples from cows, sheep, goats, and buffalo with subclinical mastitis and from colonization samples from ostriches. The animals were hosted in 18 small dairy herds and an ostrich breeding located in 10 municipalities of the state of Rio de Janeiro, Brazil. Thirty isolates were identified as Staphylococcus aureus by biochemical and molecular techniques and were comparatively characterized by phenotypic and genotypic methods. The molecular characterization by pulsed-field gel electrophoresis (PFGE), spa typing, and multilocus sequence typing (MLST) revealed five clonal types (PFGE A, spa type t359, sequence type 747 [ST747]; PFGE B, spa type t1180, ST750; PFGE C, spa type t605, ST126; PFGE D, spa type t127, ST751; and PFGE F, spa type t002, ST5). None of the isolates harbored the Panton-Valentine leukocidin or exfoliative toxin D gene. The detection of major clone A (in 63% of the isolates) in different herds, among all animal species studied, and in infection and colonization samples evidenced its geographical spread among Rio de Janeiro State and no host preference among the animal species. Comparison with S. aureus from a human origin suggested that all but one clone found in the present study might be animal specific.     

Insights into a multidrug resistant Escherichia coli pathogen of the globally disseminated ST131 lineage: genome analysis and virulence mechanisms

Escherichia coli strains causing urinary tract infection (UTI) are increasingly recognized as belonging to specific clones. E. coli clone O25b:H4-ST131 has recently emerged globally as a leading multi-drug resistant pathogen causing urinary tract and bloodstream infections in hospitals and the community. While most molecular studies to date examine the mechanisms conferring multi-drug resistance in E. coli ST131, relatively little is known about their virulence potential. Here we examined E. coli ST131 clinical isolates from two geographically diverse collections, one representing the major pathogenic lineages causing UTI across the United Kingdom and a second representing UTI isolates from patients presenting at two large hospitals in Australia. We determined a draft genome sequence for one representative isolate, E. coli EC958, which produced CTX-M-15 extended-spectrum β-lactamase, CMY-23 type AmpC cephalosporinase and was resistant to ciprofloxacin. Comparative genome analysis indicated that EC958 encodes virulence genes commonly associated with uropathogenic E. coli (UPEC). The genome sequence of EC958 revealed a transposon insertion in the fimB gene encoding the activator of type 1 fimbriae, an important UPEC bladder colonization factor. We identified the same fimB transposon insertion in 59% of the ST131 UK isolates, as well as 71% of ST131 isolates from Australia, suggesting this mutation is common among E. coli ST131 strains. Insertional inactivation of fimB resulted in a phenotype resembling a slower off-to-on switching for type 1 fimbriae. Type 1 fimbriae expression could still be induced in fimB-null isolates; this correlated strongly with adherence to and invasion of human bladder cells and bladder colonisation in a mouse UTI model. We conclude that E. coli ST131 is a geographically widespread, antibiotic resistant clone that has the capacity to produce numerous virulence factors associated with UTI.     

Role of M3 protein in the adherence and internalization of an invasive Streptococcus pyogenes strain by epithelial cells

Streptococcus pyogenes utilizes multiple mechanisms for adherence to and internalization by epithelial cells. One of the molecules suggested of being involved in adherence and internalization is the M protein. Although strains of the M3 serotype form the second largest group isolated from patients with severe invasive diseases and fatal infections, not much information is known regarding the interactions of M3 protein with mammalian cells. In this study we have constructed an emm3 mutant of an invasive M3 serotype (SP268), and demonstrated that the M3 protein is involved in both adherence to and internalization by HEp-2 cells. Fibronectin promoted both adherence and internalization of SP268 in an M3-independent pathway. Utilizing speB and speB/emm3 double mutants, it was found that M3 protein is not essential for the maturation of SpeB, as was reported for the M1 protein. Increased internalization efficiency observed in both the speB and emm3/speB mutants suggested that inhibition of S. pyogenes internalization by SpeB is not related to the presence of an intact M3 protein. Thus, other proteins in SP268, which serve as targets for SpeB activity, have a prominent role in the internalization process.     

Molecular characterization and antimicrobial susceptibility of nasal Staphylococcus aureus isolates from a Chinese medical college campus

Staphylococcus aureus colonization and infection occur more commonly among persons living or working in crowded conditions, but characterization of S. aureus colonization within medical communities in China is lacking. A total of 144 (15.4%, 144/935) S. aureus isolates, including 28 (3.0%, 28/935) MRSA isolates, were recovered from the nares of 935 healthy human volunteers residing on a Chinese medical college campus. All S. aureus isolates were susceptible to vancomycin, quinupristin/dalfopristin and linezolid but the majority were resistant to penicillin (96.5%), ampicillin/sulbactam (83.3%) and trimethoprim/sulfamethoxazole (93.1%). 82%, (23/28) of the MRSA isolates and 66% (77/116) of the MSSA isolates were resistant to multiple antibiotics, and 3 MRSA isolates were resistant to mupirocin--an agent commonly used for nasal decolonization. 16 different sequence types (STs), as well as SCCmec genes II, III, IVd, and V, were represented among MRSA isolates. We also identified, for the first time, two novel STs (ST1778 and ST1779) and 5 novel spa types for MRSA. MRSA isolates were distributed in different sporadic clones, and ST59-MRSA-VId- t437 was found within 3 MRSA isolates. Moreover, one isolate with multidrug resistance belonging to ST398-MRSA-V- t571 associated with animal infections was identified, and 3 isolates distributed in three different clones harbored PVL genes. Collectively, these data indicate a high prevalence of nasal MRSA carriage and molecular heterogeneity of S. aureus isolates among persons residing on a Chinese medical college campus. Identification of epidemic MRSA clones associated with community infection supports the need for more effective infection control measures to reduce nasal carriage and prevent dissemination of MRSA to hospitalized patients and health care workers in this community.     

A new rapid and cost-effective method for detection of phages, ICEs and virulence factors encoded by Streptococcus pyogenes

Streptococcus pyogenes (group A Streptococcus, GAS) is a human pathogen that causes diseases of various intensity, from mild strep throat to life threatening invasive infections and postinfectional sequelae. S. pyogenes encodes multiple, often phage encoded, virulence factors and their presence is related to severity of the disease. Acquisition of mobile genetic elements, carrying virulence factors, as phages or ICEs (integrative and cojugative elements) has been shown previously to promote selection of virulent clones. We designed the system of eight low volume multi- and one singleplex PCR reactions to detect genes encoding twenty virulence factors (spd3, sdc, sdaB, sdaD, speB, spyCEP, scpA, mac, sic, speL, K, M, C, I, A, H, G, J, smeZ and ssa) and twenty one phage and ICE integration sites described so far for S. pyogenes. Classification of strains based on the phage and virulence factors absence or presence, correlates with PFGE MLST and emm typing results. We developed a novel, fast and cost effective system that can be used to detect GAS virulence factors. Moreover, this system may become an alternative and effective system to differentiate between GAS strains.     

Characteristics of CTX-M Extended-Spectrum β-Lactamase-Producing Escherichia coli Strains Isolated from Multiple Rivers in Southern Taiwan

Extended-spectrum β-lactamase (ESBL)-producing Escherichia coli sequence type ST131 has emerged as the leading cause of community-acquired urinary tract infections and bacteremia worldwide. Whether environmental water is a potential reservoir of these strains remains unclear. River water samples were collected from 40 stations in southern Taiwan from February to August 2014. PCR assay and multilocus sequence typing (MLST) analysis were conducted to determine the CTX-M group and sequence type, respectively. In addition, we identified the seasonal frequency of ESBL-producing E. coli strains and their geographical relationship with runoffs from livestock and poultry farms between February and August 2014. ESBL-producing E. coli accounted for 30% of the 621 E. coli strains isolated from river water in southern Taiwan. ESBL-producing E. coli ST131 was not detected among the isolates. The most commonly detected strain was E. coli CTX-M group 9. Among the 92 isolates selected for MLST analysis, the most common ESBL-producing clonal complexes were ST10 and ST58. The proportion of ESBL-producing E. coli was significantly higher in areas with a lower river pollution index (P = 0.025) and regions with a large number of chickens being raised (P = 0.013). ESBL-producing E. coli strains were commonly isolated from river waters in southern Taiwan. The most commonly isolated ESBL-producing clonal complexes were ST10 and ST58, which were geographically related to chicken farms. ESBL-producing E. coli ST131, the major clone causing community-acquired infections in Taiwan and worldwide, was not detected in river waters.     

Genome sequence of a nephritogenic and highly transformable M49 strain of Streptococcus pyogenes

The 1,815,783-bp genome of a serotype M49 strain of Streptococcus pyogenes (group A streptococcus [GAS]), strain NZ131, has been determined. This GAS strain (FCT type 3; emm pattern E), originally isolated from a case of acute post-streptococcal glomerulonephritis, is unusually competent for electrotransformation and has been used extensively as a model organism for both basic genetic and pathogenesis investigations. As with the previously sequenced S. pyogenes genomes, three unique prophages are a major source of genetic diversity. Two clustered regularly interspaced short palindromic repeat (CRISPR) regions were present in the genome, providing genetic information on previous prophage encounters. A unique cluster of genes was found in the pathogenicity island-like emm region that included a novel Nudix hydrolase, and, further, this cluster appears to be specific for serotype M49 and M82 strains. Nudix hydrolases eliminate potentially hazardous materials or prevent the unbalanced accumulation of normal metabolites; in bacteria, these enzymes may play a role in host cell invasion. Since M49 S. pyogenes strains have been known to be associated with skin infections, the Nudix hydrolase and its associated genes may have a role in facilitating survival in an environment that is more variable and unpredictable than the uniform warmth and moisture of the throat. The genome of NZ131 continues to shed light upon the evolutionary history of this human pathogen. Apparent horizontal transfer of genetic material has led to the existence of highly variable virulence-associated regions that are marked by multiple rearrangements and genetic diversification while other regions, even those associated with virulence, vary little between genomes. The genome regions that encode surface gene products that will interact with host targets or aid in immune avoidance are the ones that display the most sequence diversity. Thus, while natural selection favors stability in much of the genome, it favors diversity in these regions.     

苯唑西林敏感、mecA基因阳性的猪源金黄色葡萄球菌的流行、分子分型及耐药性研究

[目的] 为了解我国猪源苯唑西林敏感-mecA阳性金黄色葡萄球菌（Oxacillin-susceptible,mecA-positive Staphylococcus aureus,OS-MRSA）的流行情况、菌株分子特征及耐药性,本研究对我国中西部4个省份（甘肃、陕西、河南和广西）的9个规模化养猪场进行鼻腔拭子样本采集。[方法] 运用PCR扩增nuc和mecA基因及苯唑西林耐药性检测对OS-MRSA菌株进行分离鉴定。然后对分离所得的OS-MRSA菌株进行26种毒素编码基因、16种抗生素耐药性以及spa、MLST和SCCmec分型检测。[结果] 结果表明,采集的884份样本中,67份样本7.6%（67/884）分离到金黄色葡萄球菌,包括50株甲氧西林敏感菌株（Methicillin-sensitive Staphylococcus aureus,MSSA）、8株苯唑西林耐受-mecA阳性金黄色葡萄球菌（Oxacillin-resistant mecA-positive,OR-MRSA）和9株OS-MRSA菌株。26种被检毒素编码基因中有9种毒素编码基因被检出,其中hla基因检出率最高,其次为hld、hlb、hlg、sei、sem、seg、sen和seo。此外,67株分离株中仅有16株携带肠毒素编码基因,其中OR-MRSA和OS-MRSA菌株分别占37.5%（6/16）和50.0%（8/16）,且携带毒素编码基因的菌株克隆型均为ST9-t899。16种所测试抗生素中,菌株对12种抗生素表现为耐药,其中MSSA、OR-MRSA和OS-MRSA分离株分别主要对1-8、10-12和7-11种抗生素耐药。所有分离株共有4种克隆型ST398-t571、ST9-t899、ST398-t034和t11241,其中ST9-t899为MRSA菌株唯一克隆型和ST398-t571为MSSA优势克隆型。除4株分离株未检测到SCCmec分型外,IV_b（76.5%,13/17）是MRSA分离株的唯一分型。[结论] 结果表明,我国猪源MRSA分离株对苯唑西林药物敏感性发生了改变,出现了较多的苯唑西林敏感菌株。此外,MSSA和MRSA分离株优势克隆型分别为ST398-t571和ST9-IVb-t899。研究还发现,克隆型与毒素编码基因有显著相关性,携带毒素编码基因的菌株克隆型均为ST9-t899。通过了解我国猪源MSSA、OR-MRSA和OS-MRSA的流行、分子特征和耐药性,可以为我国猪源金黄色葡萄球菌的防控提供数据支持。     

Population structure of Staphylococcus aureus from remote African Babongo Pygmies

Background

Pandemic community-acquired methicillin-resistant Staphylococcus aureus isolates (CA-MRSA) predominantly encode the Panton-Valentine leukocidin (PVL), which can be associated with severe infections. Reports from non-indigenous Sub-Saharan African populations revealed a high prevalence of PVL-positive isolates. The objective of our study was to investigate the S. aureus carriage among a remote indigenous African population and to determine the molecular characteristics of the isolates, particularly those that were PVL-positive.

Methodology/Principal Findings

Nasal S. aureus carriage and risk factors of colonization were systematically assessed in remote Gabonese Babongo Pygmies. Susceptibility to antibiotics, possession of toxin-encoding genes (i.e., PVL, enterotoxins, and exfoliative toxins), S. aureus protein A (spa) types and multi-locus sequence types (MLST) were determined for each isolate. The carriage rate was 33%. No MRSA was detected, 61.8% of the isolates were susceptible to penicillin. Genes encoding PVL (55.9%), enterotoxin B (20.6%), exfoliative toxin D (11.7%) and the epidermal cell differentiation inhibitor B (11.7%) were highly prevalent. Thirteen spa types were detected and were associated with 10 STs predominated by ST15, ST30, ST72, ST80, and ST88.

Conclusions

The high prevalence of PVL-positive isolates among Babongo Pygmies demands our attention as PVL can be associated with necrotinzing infection and may increase the risk of severe infections in remote Pygmy populations. Many S. aureus isolates from Babongo Pygmies and pandemic CA-MRSA-clones have a common genetic background. Surveillance is needed to control the development of resistance to antibiotic drugs and to assess the impact of the high prevalence of PVL in indigenous populations.     

Genetic organisation of the M protein region in human isolates of group C and G streptococci: two types of multigene regulator-like (mgrC) regions

In addition to beta-haemolytic streptococci belonging to Lancefield group A (Streptococcus pyogenes, GAS), human isolates of group C (GCS) and group G (GGS) streptococci (S. dysgalactiae subsp. equisimilis) have been implicated as causative agents in outbreaks of purulent pharyngitis, of wound infections and recently also of streptococcal toxic shock-like syndrome. Very little is known about the organisation of the genomic region in which the emm gene of GCS and GGS is located. We have investigated the genome sequences flanking the emm gene in GCS by sequencing neighbouring fragments obtained by inverse PCR. Our sequence data for GCS strains 25287 and H46A revealed two types of arrangement in the emm region, which differ significantly from the known types of mga regulon in GAS. We named this segment of the genome mgrC (for multigene regulon-like segment in group C streptococci). In strains belonging to the first mgrC type (prototype strain 25287) the emm gene is flanked up-stream by mgc, a gene that is 61% identical to the mga gene of GAS. A phylogenetic analysis of the deduced protein sequences showed that Mgc is related to Mga proteins of various types of GAS but forms a distinct cluster. Downstream of emm, the mgrC sequence region is bordered by rel. This gene encodes a protein that functions in the synthesis and degradation of guanosine 3',5' bipyrophosphate (ppGpp) during the stringent regulatory response to amino acid deprivation. In the second mgrC type (prototype strain H46A), the genes mgc and emm are arranged as in type 1. But an additional ORF (orf) is inserted in opposite orientation between emm and rel. This orf shows sequence homology to cpdB, which is present in various microorganisms and encodes 2',3' cyclo-nucleotide 2'-phosphodiesterase. PCR analysis showed that these two mgrC arrangements also exist in GGS. Our sequence and PCR data further showed that both types of mgrC region in GCS and GGS are linked via rel to the streptokinase region characterised recently in strain H46A. A gene encoding C5a peptidase, which is present at the 3' end of the mga regulon in GAS, was not found in the mgrC region identified in the GCS and GGS strains investigated here.